layout 1 [journal of entomological and acarological research 2013; 45:e13] [page 69] chironomid (diptera) species recorded from uk lakes as pupal exuviae l.p. ruse apem, aquatic ecology laboratories, uk abstract an inventory of chironomid species (diptera, chironomidae) data collected from 221 lake basins or reservoirs is detailed together with major physical and chemical characteristics of these waterbodies. aquatic species of chironomidae must rise to the water surface for adult emergence. floating exuviae are transported by wind and water currents to lakeshores. species data were obtained by collecting lake marginal floating pupal exuviae representing juvenile stages dwelling from across the lake. among the 450 species found, several were new records for the british isles. introduction from 1998 the author has gathered chironomid species and environmental data from inland waterbodies across england, wales and scotland for the purpose of developing methods to assess ecological status for the water framework directive (wfd) (european regulation, 1999). the chironomid pupal exuviae technique (cpet) (wilson & ruse, 2005) was used to obtain representative biotic samples from large waterbodies. cpet exploits the easy collection and identification of pupal exuviae (skins) discarded by emerging adults. pupae of all aquatic chironomid species rise to the water surface for the adults to emerge (langton, 1995). cpet proved to be a simple and effective sampling method for implementing wfd ecological assessment of lake anthropogenic nutrient impact (ruse, 2010) and acidification (ruse, 2011). the chironomidae are the most species-rich of aquatic macroinvertebrate families and provide an excellent surrogate for benthic invertebrates, representing all their major feeding modes (berg, 1995). from britain 615 chironomid species could be listed from pupal exuviae collections (wilson & ruse, 2005; langton & ruse, 2005a, 2005b, 2010a, 2010b) although 591 species can be identified using the adult key by langton & pinder (2007). in either case, this exceeds the combined number of species of non-dipteran aquatic macroinvertebrates found in the british isles. raunio et al. (2007) demonstrated that marginal cpet samples provided more profundal species than had profundal grab samples and was the only method found to detect land use impacts among mesohumic finnish lakes. the collection of floating chironomid pupal exuviae at the leeward shore of standing water bodies provides a simple and safe means of obtaining abundant macroinvertebrate data representative of at least a large part of the lake. the sample is passively collected by wind and water currents, integrating adult chironomid emergence over the previous day or two (coffmann, 1973; mcgill, 1980). there are none of the difficulties associated with separating benthic invertebrates from silt and macrophytes (moss et al., 1996) or requirement for numerous stratified samples (resh, 1979). ferrington et al. (1991) has quantitatively demonstrated the efficiency and economy of cpet compared with direct sampling of larvae. the ease of obtaining representative lake chironomid species by collecting their floating exuviae was first suggested by thienemann (1910) who began the trophic classification of lakes in europe from that period. there is a european standard guidance on sampling and processing chironomid pupal exuviae for ecological assessment (cen, 2006). up to 203 of the lake surveys provided in this paper were used to calculate the sensitivity of chironomid species to anthropogenic nutrient enrichment and acidification (ruse, 2010, 2011). the observed sensitivity score of a lake was compared with the lake’s reference score modeled from impact-independent characteristics of that lake. a modified observed to reference ratio provided the ecological quality ratio (eqr) required by the wfd (anonymous, 2003). the eqr was used to classify the ecological status of each lake as defined by the wfd (european regulation, 1999, annex v); boundaries were defined by the relative proportion of sensitive to tolerant species. the research of most of these data and the development of two wfd methods for assessing lake ecological status have already been published (ruse, 2010, 2011) so it is the purpose of this publication to provide the raw data as a resource for others to investigate further and to compare with, and augment, their own data. ultimately, the objective is to further improve our understanding of lake ecology and measurement of lake functioning. correspondence: leslie p. ruse, apem, aquatic ecology laboratories, fba east stoke, dorset bh20 6bb, uk. e-mail: les.ruse@roehampton.ac.uk key words: chironomidae species distribution, lake environmental data, pupal exuviae. acknowledgements: most of the data were gathered while in the employ of the former environment agency of england and wales. subsequent data were largely obtained during my current employment with apem limited who also supported the preparation and writing of this paper. i am indebted to nathaele rahmani in the remote sensing team of apem for producing the map of sampling sites. received for publication: 9 may 2013. revision received: 4 june 2013. accepted for publication: 4 june 2013. ©copyright l.p. ruse, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e13 doi:10.4081/jear.2013.e13 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e13 jear_2013_2:hrev_master 16/09/13 13.56 pagina 69 no nco mm er cia l ronmental data from inland waterbodies across england, wales and no nco mm er cia l ronmental data from inland waterbodies across england, wales and scotland for the purpose of developing methods to assess ecological no nco mm er cia l scotland for the purpose of developing methods to assess ecological status for the water framework directive (wfd) (european no nco mm er cia l status for the water framework directive (wfd) (european regulation, 1999). the chironomid pupal exuviae technique (cpet) no nco mm er cia l regulation, 1999). the chironomid pupal exuviae technique (cpet) (wilson & ruse, 2005) was used to obtain representative biotic samno nco mm er cia l (wilson & ruse, 2005) was used to obtain representative biotic samdemonstrated that marginal cpet samples provided more profundal no nco mm er cia l demonstrated that marginal cpet samples provided more profundalspecies than had profundal grab samples and was the only method no nco mm er cia l species than had profundal grab samples and was the only method no nco mm er cia l found to detect land use impacts among mesohumic finnish lakes. no nco mm er cia l found to detect land use impacts among mesohumic finnish lakes. the collection of floating chironomid pupal exuviae at the leeward no nco mm er cia l the collection of floating chironomid pupal exuviae at the leeward shore of standing water bodies provides a simple and safe means of no nco mm er cia l shore of standing water bodies provides a simple and safe means of no nco mm er cia l no nco mm er cia l correspondence: leslie p. ruse, apem, aquatic ecology laboratories, fba no nco mm er cia l correspondence: leslie p. ruse, apem, aquatic ecology laboratories, fba key words: chironomidae species distribution, lake environmental data, no nco mm er cia l key words: chironomidae species distribution, lake environmental data, acknowledgements: most of the data were gathered while in the employ ofno nco mm er cia l acknowledgements: most of the data were gathered while in the employ of the former environment agency of england and wales. subsequent datano nco mm er cia l the former environment agency of england and wales. subsequent data us e 2005a, 2005b, 2010a, 2010b) although 591 species can be identified us e 2005a, 2005b, 2010a, 2010b) although 591 species can be identified using the adult key by langton & pinder (2007). in either case, this us e using the adult key by langton & pinder (2007). in either case, thisexceeds the combined number of species of non-dipteran aquatic us e exceeds the combined number of species of non-dipteran aquatic macroinvertebrates found in the british isles. raunio us e macroinvertebrates found in the british isles. raunio demonstrated that marginal cpet samples provided more profundalus e demonstrated that marginal cpet samples provided more profundal on ly the chironomidae are the most species-rich of aquatic macroinon ly the chironomidae are the most species-rich of aquatic macroinvertebrate families and provide an excellent surrogate for benthic on lyvertebrate families and provide an excellent surrogate for benthicinvertebrates, representing all their major feeding modes (berg, on lyinvertebrates, representing all their major feeding modes (berg,1995). from britain 615 chironomid species could be listed from on ly1995). from britain 615 chironomid species could be listed from pupal exuviae collections (wilson & ruse, 2005; langton & ruse,on ly pupal exuviae collections (wilson & ruse, 2005; langton & ruse, 2005a, 2005b, 2010a, 2010b) although 591 species can be identifiedon ly 2005a, 2005b, 2010a, 2010b) although 591 species can be identified using the adult key by langton & pinder (2007). in either case, this on ly using the adult key by langton & pinder (2007). in either case, this [page 70] [journal of entomological and acarological research 2013; 45:e13] materials and methods floating debris containing chironomid pupal skins was collected at the lake shore, to which the wind blew, using a 250 μm mesh net attached to an extendable lightweight pole. the netted sample was passed through a 4 mm and a 250 μm mesh sieve, and then the residue of the coarse sieve was refloated and passed again through the sieve stack before repeating at least once more. finally, the residue on the fine sieve was preserved in a small pot by adding 100% denatured ethanol. in the laboratory, the residue was resuspended in water, stirred and small aliquots removed. a subsample was placed in a petri dish and all chironomid pupal skins removed while viewed through a low-power (x60–300) microscope. approximately 200 skins were subsampled randomly from each collection. this provided an unbiased, representative subsample of the whole collection (wilson & mcgill, 1979; ruse, 1993). occasionally it would be necessary to sort the entire sample to obtain sufficient skins. exuviae were mounted temporarily in 70% alcohol, or permanently in euparal, on a microscope slide and identified to species (langton & visser, 2003) under high magnification (x400). each survey comprised four cpet samples from different months between april and october. this strategy yielded 80-90% of the species obtained when sampling every month (ruse, 2002, 2006) comparable with the 91-100% found by rufer & ferrington (2008) taking four cpet samples. chironomid data from 243 lake surveys (221 different lakes or basins; figure 1) reported here were collected from 1998 to 2012 apart from three of these lakes, which were also surveyed during the 1980’s (shearwater, blagdon and chew valley). six lakes near maidenhead that were surveyed twice, in 2000 and 2001, were temporary gravel lakes created during the construction of a flood-relief anabranch of the river thames (ruse, 2012). all the existing waterbodies surveyed have a unique waterbody identification number (wbid) within the uk lakes database (http://www.uklakes.net). lake surface area and catchment area, if not already reported, were measured from 1:25,000 or larger scale maps using a planimeter. catchment area excluded lake surface area. mean depth was calculated from lake volume/surface area. mean retention time (days) was calculated from: [volume (106 m3)/catchment area (km2)]¥ [365/net precipitation (m yr�1)] net precipitation is mean annual rainfall – mean annual evaporation. the age of artificial lakes at the end of a survey was normally obtained from the managing authorities while natural lakes formed at the end of the last glacial period were given a default value of 9999 years. kenfig pool, little sea, slapton ley and loe pool have formed more recently through land/sea interactions. conductivity and ph were metered and supplemented by data from relevant regulatory agencies or water companies who also provided alkalinity and nutrient data. other sources are acknowledged in ruse (2002). for each lake survey, chemical data were averages of measurements taken over a minimum period of one year up to the date of the last cpet sample. for the following tables, lakes have been ordered in terms of their mean conductivity, from lowest to highest. in a canonical correspondence analysis, conductivity was the most influential supplied variable explaining chironomid species compositional turnover across the lake data set. for this dataset it was the best available surrogate for geological change across the britain. chironomid pupal exuviae were identified using the cd-rom key of langton & visser (2003) and followed its nomenclature unless subsequently changed and recorded in fauna europaea (version 2.6., 2013; http://www.faunaeur.org). the cd-rom version of the key is currently being replaced by an online next generation version (http://www.eti.uva.nl). chironomid data for each survey were amalgamated and species abundance recorded as a percentage of the total number of skins collected. species were ordered according to their tribe and subfamily. results species supplementary table 1 records the relative percentages of species found in each of the 243 surveys of lakes and reservoirs. the total count of individual exuviae identified from each survey is recorded at the bottom of each table. waterbodies have been ordered from lowest to highest mean conductivity. species author names may be abbreviated after first use. in the nomenclature of langton & visser (2003) pupal species without an associated adult are given a pe number. at that time they considered microtendipes chloris (meigen) and m. pedellus (de geer) as the same species. only after many of these surveys had been completed was it possible to distinguish two species and therefore the practice of continuing to name all of them as m. chloris was retained. on the contrary, tanytarsus lestagei goetghebuer and t. palmeni lindeberg are identified separately in langton & visser (2003) but have since been synonymized. environmental data compilation of environmental characteristics of each waterbody has been explained under methods. the recorded year is that of the final cpet sample in a survey. the wbid will uniquely identify the location of each waterbody from the uk lakes database. the maidenhead lakes no longer exist after becoming a flood-relief channel of the river thames. instead of wbid, their approximate location is provided within the table by a numerical ordinance survey grid reference. waterbodies are ordered by their mean conductivity, as in the species tables. alkalinity data were not available for elterwater. the same lake names are given in the supplementary tables 1 and 2, except that maidenhead lakes sampled in 2001 with the prefix ‘1’ are differentiated from the same maidenhead lakes sampled in 2000. remarks the provision of these species and environmental data can supplement other researchers’ data to improve understanding chironomid distribution and lake ecology. from 243 lake surveys 450 species of chironomidae were collected. several species were recorded for the first time in the british isles during these surveys and published in following papers or are in press. langton & ruse (2005a) reported the first british records of cryptochironomus defectus and paratanytarsus dimorphis. langton & ruse (2005b) refer to further records of the following species: chironomus crassimanus (now named acerbiphilus), c. entis, c. holomelas, c. carbonarius, cladopelma bicarinata, glyptotendipes salinus, rheotanytarsus rioensis, tanytarsus anderseni, t. mancospinosus and limnophyes spinigus as well as a first record of glyptotendipes signatus. langton & ruse (2010a) report the first record of hydrobaenus distylus while langton & ruse (2010b) record limnophyes angelicae occurring in britain. the previously circummediterranean distribution of chaetocladius algericus (moubayed, 1989) was greatly extended northwards by its occurrence at sowley pond in august 2010 subsequent to the lake’s original surveillance (ruse & moubayed-breil, 2013). article jear_2013_2:hrev_master 16/09/13 13.56 pagina 70 no nco mm er cia l have a unique waterbody identification number (wbid) within the no nco mm er cia l have a unique waterbody identification number (wbid) within the uk lakes database (http://www.uklakes.net). lake surface area and no nco mm er cia l uk lakes database (http://www.uklakes.net). lake surface area and catchment area, if not already reported, were measured from 1:25,000 no nco mm er cia l catchment area, if not already reported, were measured from 1:25,000 or larger scale maps using a planimeter. catchment area excluded no nco mm er cia l or larger scale maps using a planimeter. catchment area excluded lake surface area. mean depth was calculated from lake volume/surno nco mm er cia l lake surface area. mean depth was calculated from lake volume/surface area. mean retention time (days) was calculated from: no nco mm er cia l face area. mean retention time (days) was calculated from: )/catchment area (km no nco mm er cia l )/catchment area (km2 no nco mm er cia l 2)] no nco mm er cia l )]¥ no nco mm er cia l ¥ [365/net precipitation (m yr no nco mm er cia l [365/net precipitation (m yr�1 no nco mm er cia l �1[365/net precipitation (m yr�1[365/net precipitation (m yr no nco mm er cia l [365/net precipitation (m yr�1[365/net precipitation (m yr )] no nco mm er cia l )] net precipitation is mean annual rainfall – mean annual evaporation. no nco mm er cia l net precipitation is mean annual rainfall – mean annual evaporation. the age of artificial lakes at the end of a survey was normally no nco mm er cia l the age of artificial lakes at the end of a survey was normally obtained from the managing authorities while natural lakes formed atno nco mm er cia l obtained from the managing authorities while natural lakes formed at the end of the last glacial period were given a default value of 9999no nco mm er cia l the end of the last glacial period were given a default value of 9999 compilation of environmental characteristics of each waterbody no nco mm er cia l compilation of environmental characteristics of each waterbodyhas been explained under methods. the recorded year is that of the no nco mm er cia l has been explained under methods. the recorded year is that of the final cpet sample in a survey. the wbid will uniquely identify the no nco mm er cia l final cpet sample in a survey. the wbid will uniquely identify the location of each waterbody from the uk lakes database. the no nco mm er cia l location of each waterbody from the uk lakes database. the maidenhead lakes no longer exist after becoming a flood-relief channo nco mm er cia l maidenhead lakes no longer exist after becoming a flood-relief chanus e t. palmeni us e t. palmenilangton & visser (2003) but have since been synonymized. us e langton & visser (2003) but have since been synonymized. environmental dataus e environmental data compilation of environmental characteristics of each waterbodyus e compilation of environmental characteristics of each waterbody on ly microtendipes chloris on ly microtendipes chloris (de geer) as the same species. only after many of these suron ly(de geer) as the same species. only after many of these sur-veys had been completed was it possible to distinguish two species on lyveys had been completed was it possible to distinguish two speciesand therefore the practice of continuing to name all of them as on lyand therefore the practice of continuing to name all of them as was retained. on the contrary, on ly was retained. on the contrary, t. palmenion ly t. palmeni lindeberg are identified separately inon ly lindeberg are identified separately in [journal of entomological and acarological research 2013; 45:e13] [page 71] article figure 1. location of waterbodies indicated by dots. names abbreviated or missing due to congestion. jear_2013_2:hrev_master 16/09/13 13.56 pagina 71 no nco mm er cia l u se on ly [page 72] [journal of entomological and acarological research 2013; 45:e13] references anonymous, 2003 common implementation strategy for the water framework directive. guidance document 10: river and lakes typology, reference conditions and classification systems, 12 pp. available from: circa.europa.eu/public/irc/env/wfd/home berg m.b., 1995 larval food and feeding behaviour. in: armitage p.d., cranston p.s., pinder l.c.v. (eds.), the chironomidae. chapman and hall, london: 136-168. cen, 2006 water quality. guidance on sampling and processing of the pupal exuviae of chironomidae (order diptera) for ecological assessment. standard en15196. european committee for standardization, brussels: 9. coffmann w.p., 1973 energy flow in a woodland stream ecosystem: ii. the taxonomic composition and phenology of the chironomidae as determined by the collection of pupal exuviae. archiv. hydrobiol. 71: 281-322. european regulation, 1999 directive 9085/99 establishing a framework for community action in the field of water policy. env203, codec 336: 141. available from: http://register.consilium.europa.eu/pdf/en/99/st09/st09085.en99.pdf ferrington l.c., blackwood m.a., wright c.a., crisp n.h., kavanaugh j.l., schmidt f.j., 1991 a protocol for using surface-floating pupal exuviae of chironomidae for rapid bioassessment of changing water quality. in: peters n.e., walling d.e. (eds.), sediment and stream water quality in a changing environment: trends and explanations. publication no. 203 iahs publication, london: 181-190. langton p.h., 1995 the pupa and events leading to eclosion. in: armitage p.d., cranston p.s., pinder l.c.v. (eds.), the chironomidae. chapman and hall, london: 169-193. langton p.h., pinder l.c.h., 2007 keys to the adult male chironomidae of britain and ireland volume 1. publication no. 64. freshwater biological association scientific publ., windermere: 239 pp. langton p.h., ruse l.p., 2005a cryptochironomus defectus (kieffer, 1913) and paratanytarsus dimorphis reiss, 1965 (diptera, chironomidae) new to britain. dipterist’s digest. 12: 74. langton p.h., ruse l.p., 2005b further species of chironomidae (diptera) new to the british isles and data for species newly recorded in the 1998 checklist. dipterist’s digest. 12: 135-140. langton p.h., ruse l.p., 2010a hydrobaenus distylus (kieffer, 1915) (diptera, chironomidae) new to britain. dipterist’s digest. 17: 99-101. langton p.h., ruse l.p., 2010b mainland britain records for limnophyes angelicae sæther, 1990 and the deletion of pseudosmittia holsata thienemann & strenzke, 1940 (diptera, chironomidae) from the british list. dipterist’s digest. 17: 108. langton p.h., visser h., 2003 chironomid exuviae. a key to pupal exuviae of the west palaearctic region. eti, university of amsterdam, amsterdam: cd-rom. mcgill j.d., 1980 the distribution of chironomidae throughout the river chew drainage system, avon, england. phd thesis. university of bristol, bristol: 197 pp. moss b., johnes p., phillips g., 1996 the monitoring of ecological quality and the classification of standing waters in temperate regions: a review and proposal based on a worked scheme for british waters. biol. rev. 71: 301-339. moubayed j., 1989 description of chaetocladius algericus sp. n. and smittia durandae sp. n. (diptera, chironomidae, orthocladiinae). hydrobiologia. 185: 91-94. raunio j., ihaksi t., haapala a., muotka t., 2007 withinand among-lake variation in benthic macroinvertebrate communities comparison of profundal grab sampling and the chironomid pupal exuvial technique. j. north am. benthol. soc. 26: 708-718. resh v.h., 1979 sampling variability and life history features: basic considerations in the design of aquatic insect studies. j. fishery res. board can. 36: 290-311. rufer m.m., ferrington l.c., 2008 sampling frequency required for chironomid community resolution in urban lakes with contrasting trophic status. boletim do museu municipal do funchal. 13: 77-84. ruse l.p., 1993 chironomid distribution in the river pang in relation to environmental variables. phd thesis. university of bristol, bristol: 365 pp. ruse l.p., 2002 chironomid pupal exuviae as indicators of lake status. archiv. hydrobiol. 153: 367–390. ruse l.p., 2006 sampling efficiency using the chironomid pupal exuvial technique in a survey of cotswold water park lake 12. available from: http://www.freshwaterlife.org/ ruse l.p., 2010 classification of nutrient impact on lakes using the chironomid pupal exuvial technique. ecol. indic. 10: 594-601. ruse l.p., 2011 lake acidification assessed using chironomid pupal exuviae. fund. appl. limnol. 178: 267-286. ruse l.p., 2012 trait-based surveillance of flood channel effects on the river thames. fauna norv. 31: 109-116. ruse l.p., moubayed-breil j., 2013 record of chaetocladius algericus moubayed (chironomidae) from sowley pond, new forest, england. dipterist’s digest. [in press]. thienemann a., 1910 das sammeln von puppenhäuten der chironomiden. archiv. hydrobiol. 6: 213-214. wilson r.s., mcgill j.d., 1979 the use of chironomid pupal exuviae for biological surveillance of water quality. technical memorandum no. 18 department of the environment, water data unit, london: 20 pp. wilson r.s., ruse l.p., 2005 a guide to the identification of genera of chironomid pupal exuviae occurring in britain and ireland and their use in monitoring lotic and lentic freshwaters. special publication no.13. freshwater biological association scientific publ., windermere: 176 pp. article jear_2013_2:hrev_master 16/09/13 13.56 pagina 72 no nco mm er cia l langton p.h., pinder l.c.h., 2007 keys to the adult male no nco mm er cia l langton p.h., pinder l.c.h., 2007 keys to the adult male chironomidae of britain and ireland volume 1. publication no. no nco mm er cia l chironomidae of britain and ireland volume 1. publication no. 64. freshwater biological association scientific publ., no nco mm er cia l 64. freshwater biological association scientific publ., cryptochironomus defectus no nco mm er cia l cryptochironomus defectus (kieffer, no nco mm er cia l (kieffer, reiss, 1965 (diptera, no nco mm er cia l reiss, 1965 (diptera, chironomidae) new to britain. dipterist’s digest. 12: 74. no nco mm er cia l chironomidae) new to britain. dipterist’s digest. 12: 74. langton p.h., ruse l.p., 2005b further species of chironomidae no nco mm er cia l langton p.h., ruse l.p., 2005b further species of chironomidae (diptera) new to the british isles and data for species newly no nco mm er cia l (diptera) new to the british isles and data for species newly recorded in the 1998 checklist. dipterist’s digest. 12: 135-140. no nco mm er cia l recorded in the 1998 checklist. dipterist’s digest. 12: 135-140. langton p.h., ruse l.p., 2010a no nco mm er cia l langton p.h., ruse l.p., 2010a hydrobaenus distylus no nco mm er cia l hydrobaenus distylus 1915) (diptera, chironomidae) new to britain. dipterist’sno nco mm er cia l 1915) (diptera, chironomidae) new to britain. dipterist’s langton p.h., ruse l.p., 2010b mainland britain records forno nco mm er cia l langton p.h., ruse l.p., 2010b mainland britain records for ruse l.p., 2006 sampling efficiency using the chironomid pupal no nco mm er cia l ruse l.p., 2006 sampling efficiency using the chironomid pupalexuvial technique in a survey of cotswold water park lake 12. no nco mm er cia l exuvial technique in a survey of cotswold water park lake 12.available from: http://www.freshwaterlife.org/ no nco mm er cia l available from: http://www.freshwaterlife.org/ ruse l.p., 2010 classification of nutrient impact on lakes using the no nco mm er cia l ruse l.p., 2010 classification of nutrient impact on lakes using the chironomid pupal exuvial technique. ecol. indic. 10: 594-601. no nco mm er cia l chironomid pupal exuvial technique. ecol. indic. 10: 594-601. ruse l.p., 2011 lake acidification assessed using chironomid pupal no nco mm er cia l ruse l.p., 2011 lake acidification assessed using chironomid pupal us e tion to environmental variables. phd thesis. university of us e tion to environmental variables. phd thesis. university of bristol, bristol: 365 pp. us e bristol, bristol: 365 pp.ruse l.p., 2002 chironomid pupal exuviae as indicators of lake staus e ruse l.p., 2002 chironomid pupal exuviae as indicators of lake status. archiv. hydrobiol. 153: 367–390.us e tus. archiv. hydrobiol. 153: 367–390. ruse l.p., 2006 sampling efficiency using the chironomid pupalus e ruse l.p., 2006 sampling efficiency using the chironomid pupal on ly rufer m.m., ferrington l.c., 2008 sampling frequency required on ly rufer m.m., ferrington l.c., 2008 sampling frequency required for chironomid community resolution in urban lakes with conon lyfor chironomid community resolution in urban lakes with conon lytrasting trophic status. boletim do museu municipal do funchal. on lytrasting trophic status. boletim do museu municipal do funchal. ruse l.p., 1993 chironomid distribution in the river pang in rela-on ly ruse l.p., 1993 chironomid distribution in the river pang in relation to environmental variables. phd thesis. university ofon ly tion to environmental variables. phd thesis. university of jear2012 [journal of entomological and acarological research 2013; 45:e21] [page 117] first record of gonatocerus litoralis (haliday) (hymenoptera: mymaridae) from anoplotettix putoni ribaut (hemiptera: cicadellidae) g. viggiani laboratorio di entomologia dipartimento di agraria, università degli studi di napoli federico ii, portici (na), italy abstract the mymarid gonatocerus litoralis (haliday) is recorded for the first time as an egg parasitoid of the leafhopper anoplotettix putoni ribaut. a study of the main morphological characters shows unusual variation in distribution of the multiporous plate sensilla on the antenna and relative length of the ovipositor. the parasitoid overwinters as an immature within the host egg and the adult emerges from late april to early july, which coincides with oviposition by the leafhopper into grapevine bark. introduction during studies of the leafhopper anoplotettix putoni ribaut (hemiptera: cicadellidae) in vineyards of southern italy (di luca & viggiani, 2007; viggiani & rillo, 2007) an egg parasitoid was found. it was identified initially as belonging to the litoralis group of gonatocerus (hymenoptera: mymaridae) (viggiani et al., 2008). the genus gonatocerus nees includes 287 species worldwide (noyes, 2013), classified in several subgenera and species-groups. gonatocerus litoralis (haliday) is widely distributed in all biogeographical regions and belongs to the subgenus lymaenon walker (triapitsyn et al., 2010; triapitsyn, 2013). the species has been redescribed several times (debauche, 1948; matthews, 1986; baquero & jordana, 2002; triapitsyn et al., 2010; triapitsyn, 2013), but remains difficult to define because of substantial morphological variability. to date, this variability of g. litoralis has been evaluated using only specimens collected by sweeping or by trapping. triapitsyn (2013) reported the species in several locations in italy (campania, lazio, molise and sicily). in spite of its abundance and very wide distribution, the biology of g. litoralis still remains poorly known. known hosts are eggs of cicadula sexnotata (fallén), acocephalus sp. (matthews, 1986), neoaliturus (circulifer) tenellus (baker) (bayoun et al., 2008; triapitsyn, 2013) and zyginidia sohrab zachvatkin (hemiptera, cicadellidae) (fallahzadeh & huber, 2011). in the present paper the morphological variability of g. litoralis, reared from eggs of a single host (a. putoni), is analyzed and the life cycle of the parasitoid is outlined. materials and methods pieces of vine bark were randomly collected in some vineyards of the campania and basilicata regions of italy (taurasi, bn; rivello, pz), mostly during winter and spring 2005-2006 (viggiani et al., 2008). eggs of a. putoni were placed singly in small vials and maintained at room temperature (18-25°c). twenty-two gonatocerus specimens, all females, emerged from these eggs and were dissected and mounted on slides in canada-phenol balsam. specimens were measured by using a zeiss axiophot microscope. specimens are deposited in the collection of the entomological collection of dipartimento di agraria, università degli studi di napoli federico ii. results and discussion gonatocerus litoralis (haliday) measurements of the main morphological characters of the reared specimens are presented in tables 1 and 2. those concerning the antennal segments (figure 1a and table 1), forewing, mesotibia and ovipositor (table 2), may be compared in detail with the data reported by baquero & jordana (2002). except for the scape, all other antennal segments are relatively longer in our specimens. baquero & jordana (2002) give the following distribution of multiporous plate sensilla (mps) on the funicular segments: f5 (1), f7 (1 or 2), f8 (2). in contrast, the results here (table 3) show that the presence of mps on f5 is rare (9%) (figure 1d), but on f6 1or 2 mps are present rather frecorrespondence: gennaro viggiani, laboratorio di entomologia dipartimento di agraria, università degli studi di napoli federico ii, via università 133, 80055 portici (na), italy. tel.: +39.081.2539003 fax: +39.081.7755872. e-mail: genviggi@unina.it key words: parasitoid, variability, leafhopper, egg, grapevine. acknowledgments: the author is grateful to john noyes, natural history museum, london, for the improvements made to the paper. received for publication: 5 june 2013. revision received: 6 september 2013. accepted for publication: 24 september 2013. ©copyright g. viggiani, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e21 doi:10.4081/jear.2013.e21 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e21 no n c om me rci al us e o nly [page 118] [journal of entomological and acarological research 2013; 45:e21] article figure 1. female antenna of g. litoralis. a) antenna from distal end of scape to clava. b) antenna without multiporous plate sensilla on f5 and f6. c) antenna with multiporous plate sensilla on f6 and not on f5. d) antenna with multiporous plate sensilla on f5 and not on f6. table 1. measurements (mm) of the female antenna segments. antenna length width ratio l/w min.-max. min.-max. min.-max. (av± sd) (av±sd) (av±sd) radicle 0.060-0.110 0.015-0.022 3.25-5.6 0.087±0.0197 0.020±0.0014 4.383±0.8977 scape 0.125-0.175 0.035-0.050 3.11-3.66 0.148±0.0177 0.044±0.0051 3.333±0.2285 pedicel 0.040-0.065 0.020-0.045 1.33-2.00 0.058±0.0053 0.039±0.0049 1.508±0.1477 f1 0.030-0.045 0.017-0.025 0.8-2.25 0.038±0.0050 0.022±0.0026 1.730±0.3058 f2 0.025-0.045 0.015-0.027 1.25-2.33 0.036±0.0066 0.022±0.0030 1.645±0.2969 f3 0.030-0.055 0.020-0.027 1.55-2.00 0.040±0.0079 0.023±0.0024 1.728±0.1834 f4 0.040-0.060 0.020-0.035 1.66-2.25 0.044±0.0055 0.024±0.0035 1.944±0.1469 f5 0.045-0.065 0.020-0.035 1.71-2.75 0.054±0.0047 0.027±0.0043 2.074±0.2842 f6 0.045-0.065 0.025-0.037 1.63-2.16 0.056±0.0072 0.031±0.0040 1.825±0.1465 f7 0.045-0.065 0.025-0.040 1.5-2.00 0.058±0.0054 0.033±0.0055 1.755±0.1551 f8 0.040-0.065 0.030-0.050 1.2-1.85 0.055±0.0070 0.040±0.0051 1.382±0.1607 clava 0.160-0.215 0.050-0.070 2.66-3.72 0.187±0.0180 0.060±0.0071 3.100±0.2651 av, average; sd, standard deviation; ratio l/w, ratio length/width; f, funicular segment. no n c om me rci al us e o nly quently (59%) (figure 1c). several specimens (37%) (figure 1b) show any mps on f5 and f6. the ratio l/w of the forewing is similar, but the ratio mfl/fww is markedly different, as is the ratio ovl/mtl. the measurements and ranges of variation presented here fall within the broad concept of g. litoralis given by triapitsyn et al. (2010) and triapitsyn (2013). however, without additional evidence from more material reared from known hosts, combined with an analysis of molecular data, it is difficult to determine whether triapitsyn’s (2013) concept represents a species complex or a single variable species. biology in southern italy, a. putoni overwinters as an egg inserted into the rough bark of the grapevine. eggs hatch in march and april and the nymphs migrate to wild and cultivated plants (mainly fabaceae), where they feed and develop. adults appear in late may; their population increases during june and july and then progressively declines. there is one generation per year, similar to the allied species a. fuscovenosus (ferrari), a possible host of g. litoralis, distributed in central and northern italy (alma, 1995). g.litoralis overwinters as an immature in the host eggs and the adults emerge from late april to early july. the life cycle of the parasitoid seems to be synchronized with that of the host. as with other parasitoids associated with grapevine bark, it is likely that g. litoralis can also develop in the egg of scaphoideus titanus (ball) (arzone & alma, 1994). references alma r., 1995 ricerche bio-etologiche su anoplotettix fuscovenosus (ferrari) (cicadellidae deltocephalinae). boll. zool. ge. bach. milano ser. ii 1: 45-52. arzone a., alma a., 1994 indagini sui parassitoidi oofagi di scaphoideus titanus ball (auchenorrhyncha cicadellidae). maf convegno “lotta biologica”, acireale: 83-88. baquero e., jordana r., 2002 the genus gonatocerus nees (hymenoptera chalcidoidea mymaridae) in corn fields of navarra, north spain. redia 85: 1-19. bayoun i.m., walker g.p., triapitsyn s.v., 2008 parasitization of beet leafhopper eggs, circulifer tenellus, in california. j. appl. entomol. 132: 412-424. debauche h.r., 1948 étude sur les mymarommidae et les mymaridae de la belgique (hymenoptera-chalcidoidea). mem. mus. his. nat. belg. 108: 1-248. di luca a., viggiani g., 2007 indagine su anoplotettix (homoptera: cicadellidae) in vigneti campani.proc. xxi congr. naz. ital. ent., campobasso: 191. fallahzadeh m., huber j. t., 2011 the occurrence of gonatocerus litoralis (haliday, 1833) (chalcidoidea, mymaridae) in iran, with a new host record. munis entomol. zool. 6: 297-300. matthews m. j., 1986 the british species of gonatocerus nees (hymenoptera: mymaridae), egg parasitoids of homoptera. syst. entomol. 11: 213-229. noyes j.s., 2013 universal chalcidoidea database. available from: http://www.nhm.ac.uk/entomology/chalcidoids/index.html triapitsyn s., 2013 review of gonatocerus (hymenoptera: mymaridae) in the palaearctic region, with notes on extralimital distributions. zootaxa 3644: 1-178. triapitsyn s.v., huber j.t., logarzo g.a., berezovskiy v.v., aquino d.a., 2010 review of gonatocerus (hymenoptera: mymaridae) in the neotropical region, with description of eleven new species. zootaxa 2456: 1-243. viggiani g., di luca a., rillo l., 2008 ulteriori dati sull’anoplotettix putoni ribaut (homoptera: cicadellidae) e i suoi ooparassitoidi in campania. boll. lab. ent. agr. filippo silvestri 62: 11-17. viggiani g., rillo l., 2007 cicaline del vigneto e loro ooparassitoidi: nota preliminare su anoplotettix spp. (homoptera: cicadellidae). atti i conv. naz. viticoltura parte i, ancona, 21-23 giugno 2006. italus hortus 14: 230-231. [journal of entomological and acarological research 2013; 45:e21] [page 119] article table 2. measurements (mm) of the forewing, mesotibia and ovipositor. min.-max. av±sd fwl 0.920-1.080 1.013±0.0490 fww 0.250-0.350 0.314±0.0263 l/w 2.94-3.68 3.23±0.187 mfl 0.050-0.100 0.070±0.0104 mlf/fww 3.40-5.83 4.51±0.523 mtl 0.250-0.320 0.289±0.0173 ovl 0.480-0.550 0.519±0.0171 ovl/mtl 1.56-2.03 1.79±0.107 av, average; sd, standard deviation; fwl, forewing length; fww, forewing width; l/w, ratio forewing length/forewing width; mfl, maximum fringe length; mfl/fww, ratio maximum fringe length/maximum forewing width; mtl, mesotibia length; ovl, ovipositor length; ovl/mtl, ratio ovipositor length/mesotibia length. table 3. distribution of multiporous plate sensilla on the female antenna segments. sp. f5 f6 f7 f8 clava 1 0 0 2 2 10 2 0 1 2 2 10 3 0 0 1 1 10 4 0 1 1 2 10 5 0 1 0 2 10 6 0 0 2 2 10 7 0 1 1 2 10 8 1 1 1 2 10 9 0 0 1 2 10 10 0 1 1 2 10 11 0 1 1 2 10 12 0 0 1 1 10 13 0 1 1 2 10 14 0 1 1 2 10 15 0 0 1 2 10 16 0 1 1 2 10 17 0 0 2 3 10 18 0 2 2 3 10 19 1 0 2 2 10 20 0 0 1 2 10 21 0 2 2 2 10 22 0 2 2 2 10 sp., specimen; f, funicular segment. no n c om me rci al us e o nly j. entomol. acarol. res. ser. ii, 42 (1): 19-26 30 april 2010 p. trematerra lepidoptera tortricidae from se european russia with description of ceratoxanthis saratovica sp. n. abstract faunistic data of some lepidoptera tortricidae collected in southern  russian territory are reported; moreover, the new species ceratoxanthis saratovica sp. n., from saratov region is described. externally c. saratovica resembles to  ceratoxanthis argentomixtana (staudinger, 1870); in the male genitalia is closed  to ceratoxanthis iberica baixeras, 1992. riassunto lepidoptera tortricide dalla russia sud-orientale e descrizione di  ceratoxanthis saratovica sp. n. vengono riportati nuovi dati faunistici su alcuni lepidotteri tortricidi rinvenuti nei  territori della russia meridinale, inoltre viene descritta ceratoxanthis saratovica  sp. n. raccolta nella regione di saratov. per le caratteristiche morfologiche esterne la  nuova specie assomiglia a ceratoxanthis argentomixtana (staudinger, 1870); gli apparati genitali maschili sono simili a quelli di ceratoxanthis iberica baixeras, 1992. key words: lepidoptera tortricidae, se european russia, ceratoxanthis saratovica, new species. introduction the paper reports few notes on tortricids recognized in material collected in se  european russia, during different expeditions to territories of iuriuzan, e. samara, saratov, volga region, and volgograd, realized in 1998-1999 by povilas ivinskis, vidmantas  karalius and jan miatleuski (vilnius, lithuania). moreover, is described ceratoxanthis saratovica sp. n., externally the new species resembles to ceratoxanthis argentomixtana (staudinger, 1870), in male genitalia is similar to ceratoxanthis iberica baixeras, 1992  (kuznetsov, 1989; razowski, 2002 and 2003). ceratoxanthis razowski, 1960, is a west-palaearctic genus known from spain  and se europe, from caucasus to n syria and kazakhstan. in the genus are reported 4  species: c. externana (eversmann, 1844) from russia (s. ural) n syria, caucasus and  turkmenia, c. argentomixtana from se russia (volgograd, sarepta, lower volga, s.  ural), c. iberica from spain (guadalaviar, teruel) and iran (bäzargän, west azarbäijournal of entomological and acarological research, ser. ii, 42 (1), 201020 jän), c. rakosyella wies & huemer, 2000, from romania (mangalia, dobrogea), and c. adriatica elsner & jaros, 2003, from montenegro (buljarica) (baixeras, 1992; wieser &  huemer, 2000; elsner & jaros, 2003; brown, 2005; alipanah, 2009; razowski, 2009).  list of taxa lepidoptera tortricidae subfamily tortricinae tribe tortricini aleimma loeflingianum (linnaeus, 1758) material examined. 1 female, european p. of russia, se saratov env., 28.06.1999,  leg. miatleuski j., karalius v. tribe cochylini phtheochroa inopiana (haworth, 1811) material examined: 2 females, se russia, 150m, 70km e. samara, chernovka, 25.6.98. cochylimorpha asiana (kennel, 1899) material examined: 1 male, s. russia, volga reg., khalinsk, 24.6.98. cochylimorpha woliniana (schleich, 1868) material examined: 1 female, s. russia, volga reg., khalinsk, 24.6. 98. agapeta hamana (linnaeus, 1758) material examined: 2 females, se russia, 150m, 70km e. samara, chernovka, 25.6.98;  1 male and 1 female, s. russia, w. saratov, sosnovka, 23.6.98; 1 female, s. russia,  w. saratov, sosnovka, 23.6.98. ceratoxanthis saratovica sp. n. material examined: 1 male, holotypus, labelled as follows: european p. of russia  se, saratov env., 28.06.1999, leg. miatleuski j., karalius v.; 2 males, paratypus,  european p. of russia se, saratov env., 28.06.1999, leg. miatleuski j., karalius  v.; 2 males, paratypus, european p. of russia se, saratov env., 02.06.1999, leg.  miatleuski j., karalius v. adult. wing exspanse: male 21-24 mm (figs 1-2). antenna brown. labial palpus dark  yellow with a brownish hue. frons and vertex concolorous with palpus. thorax  21p. trematerra: ceratoxanthis saratovica from se european russia  and tegula yellowish-brown. forewing ground colour yellowish densely strigulated  with rust-brown in distal part, rust-brown along costa, markings almost completely  atrophied in form of weak spots at dorsum, median blotch and subtornal spot rustbrown. cilia concolorous with ground colour. hindwing white cream, distally pale  greyish; cilia white with pale brown sub-basal line. male genitalia: reported in figures 3-5. tegumen short and broad, socius broad, uncus  in form of a sub-triangular prominence. transtilla with lateral process on the valva  rounded and armed with 10 apical spines. valva broad, with distal margin more or  less sinuous, sacculus curved. lateral process of juxta long, curved downwards, provided with a row of spines extended from basal to terminal part, which the spines are  longer. aedeagus simple long and narrow with broad coecum penis; cornutus present. female genitalia. unknown. distribution: known only from the type locality: russia, se saratov region. host: unknown. biology: moths collected in june in open grassy areas. remarks: externally the new species resembles c. argentomixtana. the genitalia of c. saratovica are similar to those of c. argentomixtana, c. iberica and c. rakosyella.  however, they differ in the basal process, distal margin of the valvae, the sacculus,  transtilla and juxta. the basal process in c. saratovica is similar in shape to c. iberica, but provided with a long row of spines extended from basal to terminal  part; compared with c. rakosyella lateral process of juxta is shorter and curved  downwards. in c. saratovica sacculus is curved while in c. iberica sacculus is  straight. in c. saratovica lateral process of juxta approximately is 2 times longer  than aedeagus, while in c. iberica lateral process of juxta approximately is 1.5 times  longer than aedeagus. figs 1-2 - ceratoxanthis saratovica sp. n. adults: holotypus (1), european p. of russia se, saratov  env., 28.06.1999, leg. miatleuski j., karalius v.; paratypus (2), european p. of russia se, saratov  env., 02.06.1999, leg. miatleuski j., karalius v.  journal of entomological and acarological research, ser. ii, 42 (1), 201022 figs 3-5 - ceratoxanthis saratovica sp. n. genitalia: holopytus (3a, 4), european p. of russia se,  saratov env., 28.06.1999, leg. miatleuski j., karalius v.; paratypus (3b, 3c, 3d, 5), european p.  of russia se, saratov env., 02.06.1999, leg. miatleuski j., karalius v. 23p. trematerra: ceratoxanthis saratovica from se european russia  the type specimens of c. saratovica is deposited in the trematerra collection, university  of molise, campobasso, italy. etymology: the new species is named after the position of the type locality saratov  region. cochylis posterana zeller, 1847 material examined. 3 males, european p. of russia, se saratov env., 02.06.1999, leg.  miatleuski j., karalius v. tribe cnephasiini eana argentana (clerck, 1759) material examined. 1 female, s. ural, 15.7.98, iuriuzan, pervukha, 500m. eana incanana (stephens, 1852) material examined. 1 male, s. ural, 15.7.98, iuriuzan, pervukha, 500m. cnephasia asseclana (denis & shiffermüller, 1775) material examined. 1 female, european p. of russia, se saratov env., 28.06.1999,  leg. miatleuski j., karalius v. cnephasia stephensiana (doubleday, 1849) material examined. 1 female, s. russia, volga reg., khalinsk, 24.6.98. tribe archipini archips crataeganus (hübner, 1796-99) material examined. 1 female, se russia, 150m, 70km e. samara, chernovka, 25.6.98. archips podanus (scopoli, 1763) material examined. 1 male and 1 female, se russia, 150m, 70km e. samara, chernovka, 25.6.98; european p. of russia, se saratov env., 28.06.1999, leg. miatleuski  j., karalius v. archips rosanus (linnaeus, 1758) material examined. 1 male,  european p. of russia, se saratov env., 02.06.1999, leg.  miatleuski j., karalius v. choristoneura diversana (hübner, 1817) material examined. se russia, 150 m, 70km e. samara, chernovka, 25.6.98. journal of entomological and acarological research, ser. ii, 42 (1), 201024 choristoneura hebenstreitella (müller, 1764) material examined. 2 females, se russia, 150m, 70km e. samara, chernovka, 25.6.98. argyrotaenia ljungiana (thunberg, 1797) material examined. 1 male, european p. of russia, se saratov env., 28.06.1999, leg.  miatleuski j., karalius v. pandemis cerasana (hübner, 1796) material examined. 1 female, s. russia, w. saratov, sosnovka, 23.6.98. pandemis cinnamomeana (treitschke, 1830) material examined. s. ural, 15.7.98, iuriuzan, pervukha, 500m. syndemis musculana (hübner, 1796-99)  material examined. 1 male, european p. of russia, se saratov env., 02.06.1999, leg.  miatleuski j., karalius v. aphelia viburnana (denis & schiffermüller, 1775) material examined. 1 male, s. ural, 15.7.98, iuriuzan, pervukha, 500m. subfamily olethreutinae tribe olethreutini endothenia gentianaeana (hübner, 1796-99) material examined. 1 male, se russia, 150m, 70km e. samara, chernovka, 25.6.98. eudemis profundana (denis & schiffermüller, 1775) material examined. 1 male, european p. of russia, se saratov env., 28.06.1999, leg.  miatleuski j., karalius v. metendothenia atropunctana (zetterstedt, 1840) material examined. 1 female, s. russia, volga reg., khalinsk, 24.6.98. apotomis sororculana (zetterstedt, 1840) material examined. 1 male, s. ural, 15.7.98, iuriuzan, pervukha, 500m; 1 female, s.  ural, 28.6.98, iuriuzan, pervukha. celypha striana (denis & schiffermüller, 1775) material examined. 2 males, se russia, 150m, 70km e. samara, chernovka, 25.6.98. 25p. trematerra: ceratoxanthis saratovica from se european russia  syricoris rivulana (scopoli, 1763) material examined. 1 male, se russia, 150m, 70km, e. samara, chernovka, 25.6.98. tribe eucosmini eriopsela quadrana (hübner, 1811-13) material examined. 1 male, s. russia, w. saratov, sosnovka, 23.6.98. thiodia placidana staudinger, 1871 material examined. 3 males, european p. of russia, se saratov env., 28.06.1999, leg.  miatleuski j., karalius v. pelochrista hepatariana (herrich-schäffer, 1851) material examined. 1 male, european p. of russia, se saratov env., 28.06.1999, leg.  miatleuski j., karalius v. eucosma albidulana (herrich-schäffer, 1851) material examined. 1 male, s. russia, 250km, w. volgograd, morozovsk, 28.6.98;  1 male, european p. of russia, se saratov env., 28.06.1999, leg. miatleuski j.,  karalius v. eucosma aspidiscana (hübner, 1814-17) material examined. 1 male, s. ural, 26.6.98, iuriuzan, pervukha. eucosma metzneriana (treitschke, 1830) material examined. 1 male and 1 female, s. russia, 250km, w. volgograd, morozovsk,  28.6.98. eucosma pupillana (clerck, 1759) material examined. 2 males and 1 female, european p. of russia, se saratov env.,  28.06.1999, leg. miatleuski j., karalius v. epiblema foenellum (linnaeus, 1758) material examined. 1 male, s. russia, volga reg., khalinsk, 24.6.98; 1 female, se russia, 150m, 70km e. samara, chernovka, 25.6.98; 1 male, s. ural, 28.6.98, iuriuzan,  pervukha; 1 male, s. ural, 15.7.98, iuriuzan, pervukha, 500m. journal of entomological and acarological research, ser. ii, 42 (1), 201026 tribe grapholitini cydia pomonella (linnaeus, 1758) material examined. 1 male, european p. of russia, se saratov env., 2.06.1999, leg. miatleuski j., karalius v.; 1 male, european p. of russia, se saratov env., 28.06.1999,  leg. miatleuski j., karalius v. latronympha strigana (fabricius, 1775) material examined. 1 female, european p. of russia, se saratov env., 28.06.1999,  leg. miatleuski j., karalius v. acknowledgements i wish to express my sincere thanks to dr. povilas ivinskis (vilnius university,  lithuania) and dr. giuseppe spina (università degli studi del molise, italy).  references alipanah h., 2009 - synopsis of the cochylini (tortricidae: tortricinae: cochylini) of iran, with  the description of a new species. - zootaxa, 2245: 1-31. baixeras j., 1992 - a new species of ceratoxanthis razowski from spain (lepidoptera, tortricidae). - nota lepid., 14 (4): 294-296. brown j.w., 2005 - tortricidae (lepidoptera). in: world catalogue of insects 5. - apollo book  aps., stenstrup, denmark: 1-741. elsner g., jaros j., 2003 - a new species of ceratoxanthis razowski, and distribution records  for two species of aethes billberg from the balkan peninsula (tortricidae: cochylini). - nota  lepid., 25 (4): 221-225. kuznetsov v.i., 1989 - family tortricidae (olethreutidae, cochylidae) - tortricid moths. in:  medvedev g.s., keys to the insects of the european part of the ussr, volume iv, lepidoptera, part i. - e.j. brill, leiden: 279-991. razowski j., 2002 - tortricidae of europe. volume 1. tortricinae and chidanotinae. - frantisek  slamka, bratislava: 1-247. razowski j., 2003 - tortricidae of europe. volume 2. olethreutinae. - frantisek slamka, bratislava: 1-301. razowski j., 2009 - tortricidae of the palaearctic region, volume 2, cochylini. - frantisek  slamka, bratislava: 1-195. wieser c., huemer p., 2000 - ceratoxanthis rakosyella sp.n., eine bemerkenswerte neue schmetterlingsart aus rumänien (lepidoptera, tortricidae). - entomol. rom., 4: 5-9. prof. pasquale trematerra - università degli studi del molise, dipartimento di scienze animali,  vegetali e dell’ambiente, via de sanctis, i-86100 campobasso (italy). trema@unimol.it accepted 15 april 2010 layout 1 journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:xxxx [journal of entomological and acarological research 2014; 46:1828] [page 85] abstract research was conducted on the fauna of thysanoptera in the urban green spaces of hangzhou, zhejiang province, china, during 20082012. the thrips were collected in different plant communities (mainly in parks) in the city. a total of 26 species from 19 genera in three different families were collected, among them scolothrips latipennis priesner, which is newly recorded for the fauna of china. new distribution records of seven species in china are reported. results of the research indicate that the fauna of thrips of green areas of hangzhou was quite abundant and diversified, and the occurrence of selenothrips, scirtothrips, thrips, frankliniella and haplothrips species seems diverse and should be investigated further. introduction the urban environment is a complex of habitats developed by humans from natural sites or agricultural land. houses, villages, towns, cities, buildings, roads, and other features that characterize the urban environment have gradually and irreversibly changed the landscape of natural and agricultural areas. as a part of this change, some habitats and their associated plant and animal communities have been eliminated, while others have been expanded and new ones created. many of the new habitats were intentional parks, waterways, street trees, turfgrass, food stores but some were incidental; e.g., standing water in roadside ditches, garbage and landfill sites near residential neighborhoods, and the underground sewer and storm drain networks in urban and suburban areas. these all provide habitats for a select group of insects and other arthropods, some of which have attained pest status (robinson, 2005). vegetation plays a key role in urban environments by providing food, breeding sites and shelter for animals and plants, and also by modifying the microclimate. local conditions, climate, and available resources determine the distribution of some arthropods in the urban environment, and the abundance of some species is limited. other species are broadly adapted to the resources and harborages in and around buildings, and these are cosmopolitan in their distribution and pest status (rudd et al., 2002). thrips constitute the order thysanoptera, of which there are presently over 6000 known species. the order is divided into two suborders, tubulifera and terebrantia. tubulifera contains a single family, the phlaeothripidae, whereas there are eight recognized families in the terebrantia (mound & morris, 2007; mirab-balou et al., 2011a). thysanoptera comprise an order of minute insects of considerable scientific and economic importance. their habits range from forest and grasslands, to gardens and crops. members of many species are fungivorous, phytophagous or carnivorous, or are gall-makers or inquilines, and some are vectors of viral and bacterial diseases of plants, or pollinators of flowers (lewis, 1973; mound, 1997). a large number of thrips species are considered pests, because they feed on plants of commercial value. almost all species of pest thrips (>90%) are in the terebrantia family thripidae; i.e., thrips tabaci, frankliniella occidentalis, scirtothrips dorsalis, and thrips palmi (moritz et al., 2004; mound, 2005). only a few species of phlaeothripidae are considered to be pests. these include gynaikothrips ficorum (marchal) and gynaikothrips uzeli (zimmerman) as pests of ficus, and haplothrips spp., which include pests of grains in europe and central asia (reitz et al., 2011). thrips in the genera frankliniella (flower thrips) and thrips also spread plant diseases through the transmission of viruses, such as tospoviruses, tomato spotted wilt virus, and the impatiens necrotic spot viruses (lewis, 1973; mirab-balou & chen, 2011; mound, 2005; tong & lv, 2013). in china, about 566 species of thrips have been recorded (mirabbalou et al., 2011a), but a few of them, i.e., frankliniella occidentalis (pergande), scirtothrips dorsalis hood, thrips palmi karny, and thrips tabaci lindeman, are known as serious pests in this country (reitz et al., 2011; mirab-balou et al., 2012a, 2013). up to the present, only a few works have been conducted on thrips species associated with the urban environment. for example, correspondence: xue-xin chen, institute of insect sciences, zhejiang university, 866 yuhangtang road, hangzhou 310058, china. e-mail: xxchen@zju.edu.cn key words: thrips, park vegetation, predator, hangzhou, china. acknowledgements: we are grateful to prof. zhang wei-qiu of south china agricultural university for his useful advice and to the anonymous reviewers for their useful comments. received for publication: 15 july 2013. revision received: 30 march 2014. accepted for publication: 1 april 2014. ©copyright m. mirab-balou et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1828 doi:10.4081/jear.2014.1828 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2014; volume 46:1828 thrips species diversity in urban green spaces of hangzhou (zhejiang province), china m. mirab-balou,1 x.l. tong,2 x.x. chen3 1department of plant protection, college of agriculture, ilam university, iran; 2department of entomology, south china agricultural university, guangzhou, china; 3institute of insect sciences, zhejiang university, hangzhou, china jear_2014_3_hrev_master 16/12/14 10:38 pagina 85 no nco mm er cia l u se on ly thysanoptera in the city of lublin, poland (kucharczyk & seczkowska, 1990; czepiel, 2004) or a few common species of thrips in the globally urban environment were mentioned by robinson (2005). in china, data concerning the species composition, ecological connections and the number of thrips in urban areas are scarce; the present research is the first attempt to document the biodiversity of thrips species in the urban green spaces of hangzhou, china. materials and methods area of research hangzhou is the largest city of zhejiang province, and is located in northern zhejiang province, eastern china, at the southern end of the grand canal of china, on the plain of the mid-lower reaches of the yangtze river. the prefecture-level region of hangzhou extends west to the border with the hilly anhui province, and east to the flatland near hangzhou bay. the city center is built around the eastern and northern sides of west lake (xihu), just north of the qiantang river. hangzhou’s climate is humid subtropical with four distinctive seasons, characterised by long, very hot, humid summers and short, chilly, cloudy dry winters (with occasional snow). collection of specimens to establish the occurrence of thysanoptera species associated with urban green spaces of hangzhou, different sites (i.e., parks, street trees, turfgrass, etc.) (figure 1a-d) were randomly sampled from 2008-2012. thrips collection methods included sweep net, aspirator, and shaking plants into a white dish; all specimens were preserved in 70% ethanol. preparation of material for identification all collected material was macerated in 5% koh and subjected to dehydration in an ethanol series before being mounted onto glass slides in hoyer’s medium [see mirab-balou and chen (2010), for details on slide mounting]. all descriptions, measurements and photos were made with a leica dm irb microscope, and a leica mz apo microscope with a leica image 1000 system. identification of slide-mounted specimens slide-mounted specimens were identified using published keys (reyes, 1994; han, 1997; zur strassen, 2003; moritz et al., 2004; masumoto, 2010). species identity was confirmed by comparison with identified slide-mounted material held at the institute of insect sciences, zhejiang university, hangzhou, china (zjuh); the insect collection of department of entomology, south china agricultural university (scau); entomological museum, northwest a. & f. university, yangling, shaanxi province, china (nwafu); and national zoological museum of china, institute of zoology, chinese academy of sciences, beijing, china (ioz). depository for thrips specimens the specimens are deposited in the institute of insect sciences, zhejiang university, hangzhou, china (zjuh). results among the 26 species listed in table 1, some are graminicolous: anaphothrips obscurus, anaphothrips sudanensis and chirothrips manicatus; these species are also common in china; most of them are article [page 86] [journal of entomological and acarological research 2014; 46:1828] table 1. thrips species associated with urban green spaces of hangzhou (zhejiang province). family sub-family species thrips-associated plants aeolothripidae aeolothrips fasciatus (linnaeus)* different plants infested with mites and thrips thripidae dendrothripinae dendrothrips ornatus (jablonowski)* ligustrum sp., rhododendron simsii thripidae panchaetothripinae heliothrips haemorrhoidalis (bouche)* feeding on the leaves of a very wide range of trees and shrubs thripidae panchaetothripinae selenothrips rubrocinctus (giard) viburnum odoratissimum, rhododendron simsii thripidae sericothripinae sericothrips houji (chou & feng) clover thripidae thripinae anaphothrips obscurus müller grasses thripidae thripinae anaphothrips sudanensis trybom grasses thripidae thripinae chaetanaphothrips orchidii (moulton)* fatsia japonica thripidae thripinae chirothrips manicatus (haliday)* grasses thripidae thripinae frankliniella intonsa (trybom) highly polyphagous; flowers of different plants thripidae thripinae lefroyothrips lefroyi (bagnall) camellia sinensis thripidae thripinae megalurothrips distalis (karny) ophiopogon japonicus; on flowers of plants family fabaceae thripidae thripinae microcephalothrips abdominalis (crawford) various asteraceae thripidae thripinae mycterothrips glycines (okamoto) glycine max, alnus japonica thripidae thripinae scirtothrips dorsalis hood highly polyphagous thripidae thripinae scolothrips latipennis priesner** thuja sp. infested with mites thripidae thripinae scolothrips takahashii priesner thuja sp. infested with mites thripidae thripinae taeniothrips eucharii (whetzel) ophiopogon japonicus thripidae thripinae thrips flavus schrank highly polyphagous thripidae thripinae thrips hawaiiensis (morgan) highly polyphagous thripidae thripinae thrips palmi karny highly polyphagous thripidae thripinae thrips tabaci lindeman highly polyphagous phlaeothripidae phlaeothripinae bagnalliella yuccae (hind) yucca flower phlaeothripidae phlaeothripinae gynaikothrips ficorum (marchal)* ficus trees phlaeothripidae phlaeothripinae haplothrips (haplothrips) ganglbaueri schmutz grasses phlaeothripidae phlaeothripinae haplothrips (haplothrips) reuteri (karny)* flowers of various asteraceae *newly recorded from the zhejiang province; **newly recorded for fauna of china. jear_2014_3_hrev_master 16/12/14 10:38 pagina 86 no nco mm er cia l u se on ly article [journal of entomological and acarological research 2014; 46:1828] [page 87] figure 1. a-c) a view of some sites sampled in the urban environment of hangzhou; d) leaves of the gingko tree, infested by thrips; e-h) leaves damaged by selenothrips rubrocinctus. jear_2014_3_hrev_master 16/12/14 10:38 pagina 87 no nco mm er cia l u se on ly florivorous, such as frankliniella intonsa; a few of them, such as selenothrips rubrocinctus and scirtothrips dorsalis, are regarded as pests in this ecosystem, whereas scolothrips and aeolothrips species are predatory and feed on other small arthropods. predatory thrips three species, aeolothrips fasciatus (linnaeus), scolothrips latipennis priesner and scolothrips takahashii priesner, represent predators that play an important role in this ecosystem by feeding on other small arthropods. among them, s. latipennis priesner is a newly recorded species for china. scolothrips latipennis priesner (new record) scolothrips latipennis priesner, 1950: 54 material examined. 6♀2♂ (in zjuh), china: huajiachi campus at zhejiang university, hangzhou, zhejiang province, on thuja sp. (cupressaceae) (infested with tetranychid mites), 13.v.2011, coll. m. mirab-balou. remarks (figure 2a-d). the species of scolothrips are predators of tetranychid mites and other small arthropods; they can be easily recognized by the presence of six pairs of very long setae on the pronotum and fore wings with dark bands (masumoto, 2010). presently, five species of this genus has been recorded from china (mirab-balou et al., 2011a); s. lattipennis priesner is here recorded for the first time among the fauna of china. distribution. china (zhejiang province); iran, egypt, crimea, spain, morocco, canary islands (zur strassen, 2003), and australia (mound, 2011). phytophagous thrips the remaining 23 species listed in table 1 are phytophagous thrips that feed on different plant parts. the onion thrips, thrips tabaci, is a widespread pest around the world. it is highly phytophagous and is also widely distributed on agricultural crops, fruit trees, flowers and other plants in hangzhou. the chilli thrips or yellow tea thrips, scirtothrips dorsalis, is another species distributed in china, and is a common pest in southern china, in particular in guangdong, guangxi, hunan, jiangxi, fujian, anhui and yunnan provinces (han, 1997; mirab-balou et al., 2011a). due to s. dorsalis’ polyphagous behavior and very large host range, this species has the potential to cause significant economic damage. it has been reported as a serious pest of a diverse variety of commodities in several countries. this species is widely distributed in hangzhou, especially on ginkgo trees. recently, another species of scirtothrips, s. ginkgoe mirab-balou & chen article [page 88] [journal of entomological and acarological research 2014; 46:1828] figure 2. scolothrips latipennis (♀): a) antenna, b) head, c) pronotum, d) fore wing. jear_2014_3_hrev_master 16/12/14 10:38 pagina 88 no nco mm er cia l u se on ly (2012b), was also recorded as a new pest in the urban environment of hangzhou by mirab-balou et al. (2012b). the red-banded thrips, selenothrips rubrocinctus (figure 1f), is polyphagous (reyes, 1994) and recently noted to be widely distributed on different plants in hangzhou. in this investigation, we observed a high level of damage (figure 1e-h) caused by this species on plants near west lake (xihu), mostly in parks. symptoms of red-banded thrips injury to plants result from feeding by the adults and/or larvae on the leaves and pods. on leaves, the feeding punctures cause the development of chlorotic spots and premature leaf drop, while on the pods, they cause brown patches that coalesce in severe infestations to form a dark brown, corky layer of dead cells that makes the determination of pod ripeness virtually impossible. necrotic lesions are produced in the leaves and pods by adults and larvae, and in the flowers by adults. small brown patches of excretory droplets, typical of thrips infestation, are an obvious means of identifying damage (figure 1e-h). clover is cultivated in many regions in the environs of hangzhou; consequently, sericothrips houji (chou and feng) has become established on this plant at high populations (mirab-balou et al., 2011b), which indicates a need for studies of its biology and potential control methods. the japanese aralia, fatsia japonica (thunb.) decne. and planch. (araliaceae), has been cultivated as a popular ornamental flower in many regions of hangzhou, especially around west lake (xihu) and most roadsides, gardens and parks. chaetanaphothrips orchidii (moulton) is one of the important thrips species collected from flowers of japanese aralia, and is established on this plant within hangzhou’s landscape. aside from the above thrips species, other species such as bagnalliella yuccae (hind) are widely distributed on yucca flowers (mirab-balou et al., 2011a); frankliniella intonsa (trybom), lefroyothrips lefroyi (bagnall), haplothrips spp. and megalurothrips distalis (karny) are also found on different varieties of plants (mostly on flowers), and mycterothrips glycines (okamoto) is found on leaves of trees in hangzhou’s urban environment. discussion and conclusions thysanoptera have been used as indicators of changes in agroecosytems (lewis, 1973; vasiliu-oromulu, 2002), and as indicators of climatic changes (vasiliu-oromulu, 1995, 2002) and air pollution (vasiliu-oromulu et al., 2008, 2009). in this regard, some of the species listed here have the capability of serving as biomonitoring indices of polluted urban green spaces, such as bagnalliella yuccae, thrips tabaci, and frankliniella intonsa. references czepiel k., 2004 thrips (thysanoptera, insecta) collected in wooded areas of the city of lublin (south-eastern poland). acta phytopathol. entomol. hung. 39: 271-279. han y.f., 1997 thysanoptera. economic insect fauna of china. vol. 55. editorial committee of fauna sinica chinese academy of sciences, science press, beijing. [in chinese]. kucharczyk h., seczkowska k., 1990 thrips (thysanoptera) of grond plant community (tilio-carpinetum) in the “bachus” reserve (lublin upland). fragmenta faunistica, warszawa, 33: 349-360. lewis t., 1973 thrips their biology, ecology and economic importance. academic press, london, new york: 349 pp. masumoto m., 2010 key to genera of the subfamily thripinae (thysanoptera: thripidae) associated with japanese plant quarantine. res. bull. plant protect. japan 46: 25-59. mirab-balou m., chen x.x., 2010 a new method for preparing and mounting thrips for microscopic examination. j. environ. entomol. 32: 115-121. mirab-balou m., chen x.x., 2011 iranian thripinae with ctenidia laterally on the abdominal tergites (thysanoptera: thripidae). natura montenegrina: 10: 435-466. mirab-balou m., tong x.l., feng j.n., chen x.x., 2011a thrips (insecta: thysanoptera) of china. check list j. spec. lists distrib. 7: 720-744. mirab-balou m., hu q.l., feng j.n., chen x.x., 2011b a new species of sericothripinae from china (thysanoptera: thripidae), with two new synonyms and one new record. zootaxa 3009: 55-61. mirab-balou m., chen x.x., tong x.l., 2012a thrips (thysanoptera) species associated with tea (camellia sinensis) in hangzhou, china. persian gulf crop protect. 1: 28-34. mirab-balou m., tong x.l., chen x.x., 2012b scirtothrips ginkgoe sp. n. (thysanoptera: thripinae), a new species infesting ginkgo biloba in eastern china. j. insect sci. 12: 1-7. mirab-balou m., yang s.l., tong x.l., 2013 one new species, four new records and key to species of hydatothrips (thysanoptera: thripidae) from china (including taiwan). zootaxa 3641: 74-82. moritz g., mound l.a., morris d.c., goldarazena a., 2004 pest thrips of the world-visual and molecular identification of pest thrips. cd, centre for biological information technology, brisbane, australia. mound l.a., 1997 biological diversity. in: lewis t. (ed.), thrips as crop pests. cab international, new york: 197-215. mound l.a., 2005 thysanoptera: diversity and interactions. annu. rev. entomol. 50: 247-269. mound l.a., 2011 species recognition in the genus scolothrips (thysanoptera, thripidae, predators of leaf-feeding mites. zootaxa 2797: 45-53. mound l.a., morris d.c., 2007 the insect order thysanoptera: classification versus systematics. zootaxa 1668: 395-411. priesner h., 1950 studies on the genus scolothrips (thysanoptera). bull. soc. r. entomol. egypt. 34: 39-68. reitz s.r., gao y.l., lei z.r., 2011 thrips: pests of concern to china and the united states. agric. sci. china 10: 867-892. reyes c.p., 1994 thysanoptera (hexapoda) of the philippine islands. raffles bull. zool. 42: 107-507. robinson w.h., 2005 handbook of urban insects and arachnids. cambridge university press, cambridge: 481 pp. rudd h., vala j., schaefer v., 2002 importance of backyard habitat in a comprehensive biodiversity conservation strategy: a connectivity analysis of urban green spaces. restor. ecol. 10: 368-375. tong x.l., lv y.b., 2013 frankliniella cephalica (crawford) (thysanoptera, thripidae), a newly recorded exotic invasive species in mainland china. chinese j. appl. entomol. 50: 496-499. vasiliu-oromulu l., 1995 population diversity of thysanoptera in romanian meadows. in: parker b.l., skinner m., lewis t. (eds.), thrips biology and management. plenum press, new york, nato asi series, series a, life sciences, 276: 469-477. vasiliu-oromulu l., 2002 the temporal and spatial dynamics of the thrips populations from the mountainous meadows. in: marullo r. and mound l. (eds.), thrips and tospoviruses. australian national insect collection, canberra: 295-313. vasiliu-oromulu l., barbuceanu d., bianu e., 2009 thysanoptera capability for biomonitoring of urban polluted green spaces (insecta: thysanoptera). acta entomol. serb. 14: 185-194. vasiliu-oromulu l., jenser g., barbuceanu d., 2008 frankliniella intonsa (trybom, 1895) a very sensitive bioindicator for air pollution. acta phytopatol. entomol. hungarica 43: 401-408. zur strassen r., 2003 die terebranten thysanopteren europas und des mittelmeer-gebietes. die tierwelt, deutschlands, 74: 1-271. article [journal of entomological and acarological research 2014; 46:1828] [page 89] jear_2014_3_hrev_master 16/12/14 10:38 pagina 89 no nco mm er cia l u se on ly layout 1 [journal of entomological and acarological research 2014; 46:1868] [page 35] abstract the digestive proteolytic profile of apodiphus amygdali was determined by using several substrates and specific inhibitors. analysis of optimal ph and temperature showed the highest enzymatic activity at the ph range of 6-7 and temperature of 40°c when azocasein was used as a substrate. by using a negative control, the presence of several specific proteases were determined including tryspin-like, chymotrypsinlike, elastase, cathepsin b, cathepsin l, aminoand carboxypeptidases in the midgut content of a. amygdali, with the highest and the lowest activities of cathepsin l and carboxypeptidase, respectively. ph dependency of specific proteases revealed optimal phs of 9, 8 and 9 for trypsin-, chymotrypsin-like, 6 for cathepsins and 5-6 for carboxyand aminopeptidases, respectively. specific inhibitors, including phenylmethylsulfonyl fluoride, na-p-tosyl-l-lysine chloromethyl ketone, ntosyl-l-phenylalanine chloromethyl ketone, l-trans-epoxysuccinylleucylamido-(4-guanidino)-butane, phenanthroline and ethylendiamidetetraacetic acid, significantly decreased proteolytic activity, indicating the presence of different proteases in the midgut of a. amygdali. extracted inhibitors from the midgut demonstrated significant inhibition of specific proteolytic activities of a. amygdali except for cathepsin b and aminopeptidase. the results indicated that determination of digestive proteolytic activity could be helpful to clarify digestion process in insects. moreover, understanding the nature of digestive proteases might be used to develop several inhibitors for providing resistant crop varieties against pests. introduction apodiphus amygdali germar (hemiptera: pentatomidae) is a polyphagous and univoltine hemipteran distributed throughout europe and the middle east (schuh & slater 1995). it feeds on several fruit trees, especially plum, apricot, apple, olive, pear and pistachio as well as non-fruit trees like poplar, pine, plane-tree, elm and willow bark (muhammed & al-iraqi, 2010). nymphs and adults feed on leaves, fruits and flowers by injecting their salivary secretions into the plant tissues to liquefy nutrient materials. feeding on stems and fruits causes host weakness and attracts other insects to feed on host plants. feeding on fruits is the primary damage and causes complete degradation and yield loss (schuh & slater, 1995). digestive proteases are one of the crucial enzymes in the alimentary canals of insects since proteins are the key nutrients for growth, development and reproduction (nation, 2008). the enzymes are classified based on their activity against protein molecules. in fact, proteases that attack internal bonds are called endopeptidases (proteinases) but exopeptidases refers to proteases separating amino acids from nand c-terminal of proteins, known as aminopeptidases (ec 3.4.11.2) and carboxypeptidases (ec 3.4.11.7) (terra & ferreira, 2012). endopeptidases are divided into different subclasses based on their ph dependency and catalytic sites. serine proteases, including trypsin(ec 3.4.21.4), chymotrypsin-like (ec 3.4.21.1) and elastase (ec 3.4.21.36), are active in alkaline ph and they have serine, histidine, and aspartic acid residues in their catalytic sites (terra & ferreira, 2012). meanwhile, these enzymes have different structural features that are associated with their different substrate specificities (terra & ferreira, 2012). cysteine proteases are active at acidic ph, including cathepsins b (ec 3.4.22.1) and l (ec 3.4.22.15). cathepsin l (ec 3.4.22.15) is a true endopeptidase that preferentially cleaves peptide bonds in p2 against the hydrophobic amino acid residues, but cathepsin b prefers arginine at the same position (barrett et al., 1998). digestion of proteins is initiated by endopeptidases; due to the activity of endopeptidases, oligopeptidases are attacked from the nand c-terminal by aminoand carboxypeptidases, resulting in dipeptides that are hydrolyzed by dipeptidases (terra & ferreira, 2012). since synthetic chemicals cause severe negative effects on the environment, non-target organisms and induce resistance in pests, numerous studies have been conducted on the use of biocontrol agents and digestive enzyme inhibitors against agricultural pests. enzymatic inhibitors disrupt digestion process of insects and nutrient absorption, thereby decreasing reproduction and therefore pest populations. protease inhibitors are small proteins that are present in up to 100 plant correspondence: arash zibaee, department of plant protection, faculty of agricultural sciences, university of guilan, rasht, 416351314, iran. tel.: +98.0131.6690274 fax: +98.0131.6690281. e-mail: arash.zibaee@gmx.com ; arash.zibaee@guilan.ac.ir key words: apodiphus amygdali, digestive protease, inhibitor. acknowledgements: the authors want to thank dr. ali sarafrazi from iranian institute of plant protection for his contribution in identification of collected insects. received for publication: 7 august 2013. revision received: 19 november 2013. accepted for publication: 19 november 2013. ©copyright s. ramzi and a. zibaee, 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1868 doi:10.4081/jear.2014.1868 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:1868 digestive proteolytic activity in apodiphus amygdali germar (hemiptera: pentatomidae): effect of endogenous inhibitors s. ramzi, a. zibaee department of plant protection, university of guilan, rasht, iran no nco mm er cia l u se on ly [page 36] [journal of entomological and acarological research 2014; 46:1868] species and they are effective on serine and cysteine proteases (nation, 2008). although this approach may be suitable for decreasing economic losses caused by pests with the least environmental disruption, comprehensive experiments are required to achieve the final goal. determination of proteolytic profiles and their properties are the first and fundamental step in this approach. the objectives of the current study were to determine digestive proteolytic profiles in a. amygdali, the effect of synthetic inhibitors, and to extract endogenous inhibitors. materials and methods insect rearing fifth nymphal instars of a. amygdali were collected from elm trees in shiraz (fars province, iran) and transferred to the laboratory. the nymphs were reared on elm leaves at 28±1°c, 70% relative humidity and a 16l:8d of photoperiod. when adults emerged, they were allowed to feed on leaves for 48 h prior to being randomly selected for biochemical analysis. sample preparation based on the method of cohen (1993), adults of a. amygdali were randomly selected and their midgut removed by dissection in iced saline solution (nacl, 10 mm, 1.06404.1000, merck-chemicals, darmstadt, germany). integument and unneeded organs were removed and the midgut was gently separated and rinsed in 1 ml of iced distilled water. to obtain appropriate samples, five midguts were placed in one eppendorff tube (eppendorff, hamburg, germany) containing 1 ml of distilled water. tissues were ground using a homogenizer and centrifuged at 13,000 rpm for 20 min at 4°c (daika, rika kugyo co., tokyo, japan). the supernatant was carefully removed, transferred to new tubes and stored at –20°c for no more than one week until use in the experiments. general proteolytic assay azocasein (2%; a2765; sigma-aldrich co., st. louis, mo, usa) was used to find the general proteolytic activity in the midgut of a. amygdali adults. general proteolytic activity was measured by using azocasein 2% based on the method of elpidina et al. (2001). the reaction mixture consisted of 50 ml of appropriate buffer solutions (universal buffer, 0.02 m, containing succinate, glycine and 2-morpholinoethanesulfonic acid; ph range 3-12; frugoni, 1957), 20 ml azocasein and 20 ml of the enzyme solution. after incubation at 37°c for 60 min, proteolysis was stopped by adding 100 ml of 30% trichloroacetic acid (tca). precipitation was achieved by cooling at 4°c for 10 min; it was then centrifuged at 13,000 rpm for another 10 min. an equal volume of 2 m naoh was added to the supernatant and the absorbance was recorded at 450 nm (awareness technology inc., palm city, fl, usa). a blank solution consisted of all the above-mentioned portions except the for enzyme solution. optimal ph and temperature (°c) for general proteolytic activity the reaction mixture was the same as described above, but different ph ranges of universal buffer (from 3 to 12) and temperature (from 20 to 70°c) were used to determine optimal ph and temperature. for the optimal ph assays, 40 ml of buffer solution (at different phs) was incubated with azocasein as a substrate for 10 min at 30°c (terra & ferreira, 2012). then, 20 ml of sample was added and the experiment continued as above. for the optimal temperature assays, 40 ml of buffer solution (at the optimal ph found) was incubated with azocasein as a substrate for 10 min at different temperature regimes from 20 to 70°c. after adding 20 ml of the sample, the reaction mixture was incubated at each given temperature for 60 min. other steps were conducted as described above. specific protease assays serine proteases trypsin-, chymotrypsinand elastase-like activities (as three subclasses of serine proteases) were assayed using a 1 mm concentration of bapna (nabenzoyl-l-arginine-p-nitroanilide, sigma-aldrich, 19362), 1 mm saapppna (n-succinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide, sigma-adrich, s7388) and 1 mm saaapna (n-succinyl-alanine-alanine-alanine-p-nitroanilide, sigma-aldrich, s4760) as substrates, respectively. the reaction mixture consisted of 35 ml of universal buffer (ph 8, as the recommended ph for serines in the literature), 5 ml of each substrate, and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period of 0-10 min before adding 30% tca to terminate the reaction. the absorbance of the resulting mixture was then measured spectrophotometrically at 405 nm by p-nitroaniline release. to prove the specific proteolytic activity, a negative control for each substrate was provided separately containing all the above components except for enzyme pre-boiled at 100°c for 30 min (oppert et al., 1997). cysteine proteases cathepsins b and l activities (as three subclasses of cysteine proteases) were assayed using a 1 mm concentration of z-ala-arg-arg 4methoxy-b-naphtylamide acetate (sigma-aldrich, c8536) and nbenzoyl-phe-val-arg-p-nitroanilide hydrochloride (sigma-aldrich, b2133) as substrates, respectively. the reaction mixture consisted of 35 ml of universal buffer (ph 5 as the recommended ph for cysteines in the literature), 5 ml of each substrate and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period from 0-10 min before adding 30% tca to terminate the reaction and read at 405 nm. to prove the specific proteolytic activity, a negative control for each substrate was provided separately containing all the above components except for enzyme pre-boiled at 100°c for 30 min (oppert et al., 1997). exopeptidases the activities of two exopeptidases were obtained by using hippuryll-arginine (sigma-aldrich, h2508) and hippuryl-l-phenilalanine (sigma-aldrich, h6875) for aminoand carboxypeptidases in the midgut of a. spinidens. the reaction mixture consisted of 35 ml of universal buffer (ph 7 as the recommended ph in the literature for exopeptidases), 5 ml of each substrate and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period from 0-10 min before adding 30% tca to terminate the reaction and read at 340 nm. to prove the specific proteolytic activity, a negative control for each substrate was provided separately containing all the above components except for enzyme pre-boiled at 100°c for 30 min (oppert et al., 1997). optimal ph determination of specific proteases universal buffer was used to find the optimal ph of serine and cysteine proteases in addition to exopeptidases by using the specific substrates described. the reaction mixture and experiment procedure were the same as above. effect of specific inhibitors on proteolytic activity the following compounds were used to find any alteration of the proteolytic activity in the midgut of a. spinidens: phenylmethylsulfonyl fluoride (pmsf) (sigma-aldrich, p7626); trypsin inhibitor, na-p-tosyl-llysine chloromethyl ketone (tlck) (sigma-aldrich, t5012); chymotrypsin inhibitor, n-tosyl-l-phenylalanine chloromethyl ketone article no nco mm er cia l u se on ly (tpck) (sigma-aldrich, t7254); cysteine protease inhibitor, l-transepoxysuccinyl-leucylamido-(4-guanidino)-butane (e-64) (sigmaaldrich, e3132); cystatin (sigma-aldrich, c8917), metalloprotease inhibitors including phenanthroline (sigma-aldrich, 131377) and ethylendiamidetetraacetic acid (edta) (merck-chemicals). extraction of endogenous inhibitors endogenous inhibitors from the midgut of a. amygdali were extracted by the method of lwalaba et al. (2010) with slight modifications. midguts of adults (n=10) were incubated in 500 ml of low glucose ringer’s solution (121.5 mm nacl; 10 mm kcl; 2.1 mm nah2po4; 10 mm nahco3; 0.7 mm mgcl2; 2.2 mm cacl2; ph=6.8 and 0.01 g glucose) for 30 min at 30°c. the tissues were then ground using a glass homogenizer. these samples taken from different species were heated to 90°c for 30 min. samples were centrifuged at 13,000 rpm for 20 min at 4°c. the obtained supernatants were used as the source of inhibitors. effect of endogenous inhibitors on specific proteolytic activity the reaction mixture contained 50 ml of universal buffer, 20 ml of each substrate and 20 ml of endogenous inhibitor. after 5 min of incubation, 10 ml of the enzyme was added and proteolytic activity was read at 405 nm after 10 min. a blank contained all components except the enzyme. protein assay protein concentration was measured according to the method of lowry et al. (1951). in this method, peptide bonds are oxidized by folinciocalteu reagent. briefly, 20 ml of sample was added to 100 ml of reagent, which was incubated for 30 min prior to reading the absorbance at 545 nm (recommended by ziest chem. co., tehran, iran). statistical analysis the experimental design was based on a completely randomized design and tukey’s test was used to compare treatment means. statistical differences were considered at p≤0.05. results from the synthetic inhibitors trials were analyzed using polo-pc software to calculate inhibitory concentration (ic) values. results optimal ph and temperature of general proteolytic activity determination of the optimal ph for general proteolytic activity resulted in two peaks in the acid and alkaline ranges. the larger peak was observed at the ph range of 6-7 and the smaller peak was found at the ph range of 9-10 (figure 1a; f=40.99, pr<f: 0.0001). general proteolytic activity increased at temperatures from 25-40°c with the highest value at 40°c, and then sharply decreased at 45-60°c (figure 1b; f=32.81, pr<f: 0.0001). determination of specific proteases present activities of trypsin-, chymotrypsin-like, elastase, cathepsin b, l, aminoand carboxypeptidases were confirmed by using boiled midgut homogenate as a negative control (figure 2). chymotrypsin showed the highest activity among serine proteases and cathepsin l and aminopeptidase had the highest activity among cysteine proteases and exopeptidases, respectively (f=31.78, pr<f: 0.0001). taken collectively, the highest and the lowest enzymatic activities were observed for cathepsin l and carboxypeptidase, respectively (figure 2; f=31.78, pr<f: 0.0001). optimal ph determination of specific proteases figures 3-5 demonstrate the ph dependency of proteases with specific substrates. the highest activity of trypsin-like protease was found at a ph range of 7-9, with the highest activity at ph 9 (figure 3; f=27.53, pr<f: 0.0001). chymotrypsin-like and elastase proteases had the highest activity at phs of 8 and 9, respectively (figure 3b and c; f=9.21, pr<f: 0.0001; f=10.95, pr<f: 0.0001). the highest activity of cathepsin b was obtained at ph 6 (figure 4; f=17.17, pr<f: 0.0001) but cathepsin l showed the highest activity at a ph range of 4-6 (figure 4; f=17.75, [journal of entomological and acarological research 2014; 46:1868] [page 37] article figure 1. optimal ph (a) and temperature (b) for general proteolytic activity in the midgut of a. amygdali. letters show statistical differences among values (tukey’s test, p≤0.05). figure 2. presence of specific proteases in the midgut of a. amygdali by using specific substrates and negative control. letters show statistical differences among values (tukey’s test, p≤0.05). no nco mm er cia l u se on ly [page 38] [journal of entomological and acarological research 2014; 46:1868] pr<f: 0.0001). aminoand carboxypeptidases showed the highest activity at phs of 6 and 5, respectively, although there was a slight non-statistical difference between these values for aminopeptidase (figure 5a and b; f=28.08, pr<f: 0.0001; f=20.34, pr<f: 0.0001). effect of synthetic and endogenous inhibitors on proteolytic activity pmsf, tlck and tpck, as specific inhibitors of serines, trypsin and chymotrypsin proteases, significantly decreased proteolytic activity in the midgut of a. amygdali (figure 6a; pr<f: 0.0001, f=1547.84; pr<f: 0.0001, f=16.83; pr<f: 0.0001, f=54.56). among these inhibitors, pmsf had the most significant effect on proteolytic activity, with the result that the enzymatic activity sharply decreased to lower than 20% (figure 6a; pr<f: 0.0001, f=1547.84). proteolytic activity in the midgut of a. amygdali was significantly affected by cystatin and e-64, with cystatin decreasing the enzymatic activity to lower than 5% (figure 6b; pr<f: 0.0001, f=74.76; pr<f: 0.0001, f=54.36; pr<f: 0.0001, f=68.44). phenanthroline and edta also significantly decreased proteolytic activity to 20% (figure 6c; pr<f: 0.0001, f=25.58; pr<f: 0.0001, f=48.75). table 1 shows ic values of the above mentioned inhibitors in which pmsf had the lowest ic50 on proteolytic activity, while tlck, phenanthroline and edta had the highest value. extracted inhibitor from the midgut of a. amygdali significantly decreased specific proteolytic activity except for cathepsin b and aminopeptidase (table 2; f=35.64, pr>f: 0.0001). discussion and conclusions a critical role of proteases could be expected in the midgut of a. amygdali, as this is a polyphagous hemipteran that feeds on different trees. in the current study, several proteases were found in the midgut, although cysteine proteases, mainly cathepsin l, showed the highest activity among the proteases assayed. results of other studies partially correspond with our findings. stamopoulos et al. (1993) and bell et al. (2005) demonstrated trace activities of trypsin and chymotrypsin in the gut of podisus maculiventris say (hemiptera: pentatomidae). silva & terra (1994) reported the presence of cysteine proteases in the midgut of dysdercus peruvianus guérin-méneville (hemiptera: pyrrhocoridae). pascual-ruiz et al. (2009) reported serine, cysteine, aminopeptidase and carboxypeptidase as the main proteases involved in protein digestion of p. maculiventris, although their activities can differ due to variability of prey. zhu et al. (2003) and wright et al. (2006) found high activities of cysteine and serine proteases in the midgut of lygus lineolaris and l. hesperus knight (hemiptera: miridae). bigham & hosseininaveh (2010) found the presence of cathepsins b and l in the article figure 3. optimal ph for serine proteases (a-c) in the midgut of a. amygdali. letters show statistical differences among values (tukey’s test, p≤0.05). figure 4. optimal ph for cysteine proteases (a and b) in the midgut of a. amygdali. letters show statistical differences among values (tukey’s test, p≤0.05). no nco mm er cia l u se on ly midgut of brachynema germari kolenati (hemiptera: pentatomidae). fialho et al. (2012) obtained cathepsin l and aminopeptidase as the major proteases from the midgut of podisus nigrispinus dallas (hemiptera: pentatomidae). sorkhabi-abdolmaleki et al. (2013) reported both serine and cysteine proteases through the significant role of cysteines in the midgut content of andrallus spinidens fabricius (hemiptera: pentatomidae). the optimal ph for general proteolytic activity in the midgut of a. amygdali was represented as two peaks at both acidic and alkaline phs, indicating the presence of all the determined proteases (see above). zhu et al. (2003) found optimal proteolytic activity of lygus lineolaris knight (hemiptera: miridae) at ph 4.25 and 8.5. wright et al. (2006) found acidic and neutral phs for caseinolytic activity in l. lineolarius that might be attributed to cysteine and serine proteases. bigham & hosseininaveh (2010) observed the presence of general protease activity by acidic and alkaline optima in the midgut extract of b. germari using hemoglobin as a substrate. sorkhabi-abdolmaleki et al. (2013) found an alkaline ph for general proteolytic activity, serine proteases and two exopeptidases with another peak at ph 6, as with the cysteine proteases. the optimal ph of specific proteases corresponds with the expected values, as noted in the literature (terra & ferreira, 2012). a ph peak was observed for all specific proteases except for cathepsin l, for which the enzyme was active at a ph range of 4-6. this could be attributed to the presence of several isozymes and higher activity of cathepsin l. variations in optimal ph of general proteolytic activity in insects could be attributed to feeding habits as either monophagous or polyphagous. since polyphagous insects feed on various hosts, the presence of different proteases and the difference in general protease ph could be attributed to host materials or secondary metabolites of plants. an optimal temperature of 40°c was found for general proteolytic activity, although there was slightly lower but not statistically different activity at 35°c. generally, optimal temperature of an enzyme in in vitro assays reflects the temperature of the environment where the organism feeds on its hosts. increasing temperature can disrupt hydrogen bonds in the three-dimensional structure of the molecule, leading to denaturation of the protein (zeng et al., 2000). at the same time, biological reactions occur faster with increasing temperature, up to the point of enzyme denaturation, where the enzymatic activity and the rate of the reaction decrease sharply (zeng et al., 2000). swart et al. (2006) found 37°c as the optimal temperature for proteolytic activity in the salivary secretion of two belostomatid bugs. sorkhabi-abdolmaleki [journal of entomological and acarological research 2014; 46:1868] [page 39] article figure 6. effect of specific inhibitors on general proteolytic activity in the midgut of a. amygdali. a) specific inhibitors of serine proteases; b) specific inhibitors of cysteine proteases; c) specific inhibitors of metalloproteinase. letters show statistical differences among values (tukey’s test, p≤0.05). pmsf, phenylmethylsulfonyl fluoride; tlck, na-p-tosyl-l-lysine chloromethyl ketone; tpck, n-tosyl-l-phenylalanine chloromethyl ketone; e64, l-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane; edta, ethylendiamidetetraacetic acid. figure 5. optimal ph for exopeptidases (a and b) in the midgut of a. amygdali. letters show statistical differences among values (tukey’s test, p≤0.05). no nco mm er cia l u se on ly [page 40] [journal of entomological and acarological research 2014; 46:1868] & zibaee (2013) reported an optimal temperature of 25°c for proteolytic activity in the midgut of a. spinidens that corresponded with laboratory conditions of mass-rearing. our results demonstrated inhibitory effects of pmsf, tlck and tpck on the proteolytic activity in the midgut of a. amygdali. pmsf showed the highest inhibition of enzymatic activity with the lowest ic50 value (0.574 mm), in comparison with tlck (7.13) and tpck (5.19). these results corresponded with previous results on the presence of serine proteinases, especially chymotryptic activities, in the midgut of a. amygdali, which have been supported by using a negative control and specific substrates. zhu et al. (2003) found serine proteases as the major enzymes in the gut of l. lineolaris by tlck (zhu et al., 2003). bigham & hosseininaveh (2010) and sorkhabi et al. (2013) found significant inhibition of digestive proteases by pmsf, tlck and tpck in b. germary and a. spinidens. presence of cysteine proteases was confirmed by using specific substrates, cystatin, e-64 and dtt, although the experiment with a negative control showed cathepsin l as the major protease in the midgut of a. amygdali. houseman and dowe (1983) found cathepsin b and l activities in the posterior midgut of rodnius prolixus l. (hemiptera: reduviidae). overney et al. (1997) reported cysteines as the major proteases in the midgut of perillus bioculatus fabricius (hemiptera: pentatomidae), showing inhibition of 90% by e-64. proteolytic activity in the midgut of l. lineolaris was significantly decreased with e-64 (wright et al., 2006). bigham & hosseininaveh (2010) found negative effects of e-64 on the proteolytic activity of a. spinidens. sorkhabi-abdolmaleki et al. (2013) observed that e-64 and cystatin decreased proteolytic activity in the midgut of a. spinidens. edta as general chelating agent and phenanthroline as metalloproteinase inhibitor significantly decreased proteolytic activity in the midgut of a. amygdali, indicating the presence of metal ions in the active sites of aminopeptidase. aminopeptidases are divided into aminopeptidase a and n based on the presence of zn2+ and mn2+ in their active sites (terra & ferreira, 2012). there is little knowledge on the characterization of aminopeptidases’ active site. ferreira & terra (1986) employed multiple inhibition analysis to find the possible role of edta on inactivation of aminopeptidase in rhynchosciara americana wideman (diptera: sciaridae) larvae. the authors found two subsites in the active center of the enzyme: a hydrophobic subsite to which isoamyl alcohol binds, exposing the metal ion, and a polar subsite, to which hydroxylamine binds (ferreira & terra, 1986). exposure of the metal ion after isoamyl alcohol binding may be analogous to the situation when a part of the substrate occupies the hydrophobic subsite to cause conformational changes associated with the catalytic step (ferreira & terra, 1986). cristofoletti & terra (2000) found that the catalysis property of aminopeptidase in tenebrio molitor l. (coleoptera: tenebrionidae) depends on a metal ion, a carboxylate and a protonated imidazole group. detailed experiments revealed that the enzyme is a zinc metallopeptidase like mammalian aminopeptidase n, but it differs in some details (cristofoletti & terra, 2000). extraction of endogenous inhibitors from the midgut of a. amygdali revealed diverse effects on specific proteolytic activities. in serine proteases, inhibition was more significant in the case of trypsin and elastase than chymotrypsin. in the case of cysteine and exopeptidases, cathepsin b and aminopeptidase were not affected by the endogenous inhibitor. endogenous inhibitors may have several functions, such as protection of a-amylase from proteolytic activity (stein & fischer, 1958), attacking fungi and bacteria in gut (taranushenko et al., 2009) and protection of epithelial cells from digestive enzymes when food is not present in the midgut (lwalaba et al., 2010). engelmann & geraert (1980) reported extraction of the midgut lumen content in leucophaea madera fabricius (blattodea: oxyhaloidae) as an endogenous inhibitor. lwalaba et al. (2010) extracted an endogenous inhibitor from the midgut of spodoptera frugiperda hubner (lepidoptera: noctuidae) larvae. it was found that the ability of the endogenous inhibitor to decrease trypsin activity was similar to the results from ingestion of the exogenous inhibitor sbti. zibaee et al. (2012) and sorkhabi-abdolmaleki et al. (2013) extracted endogenous inhibitors from chilo suppressalis walker (lepidoptera: crambidae), naranga aenescens moore (lepidoptera: noctuidae), pieris brassicae l. (lepidoptera: pieridae), hyphantria cunea drury (lepidoptera: arctiidae) and ephestia kuhniella zeller (lepidoptera: pyralidae). the authors found different effects of inhibitors on salivary and midgut proteases of a. spinidens (zibaee et al., 2012; sorkhabi-abdolmaleki & zibaee, 2013). specifically, extracted inhibitors from c. suppressalis, n. aenescens and e. kuehniella signifiarticle table 1. determination of inhibitory concentrations (mm) of specific inhibitors on proteolytic activity in the midgut of a. amygdali. compound ic10 ic30 ic50 slope±sf χ2 pmsf 0.045e 0.574f 0.574e 1.16±0.27 0.568 tlck 3.57b 5.38a 7.13ab 4.27±0.40 25.27 tpck 3.29b 4.65b 5.91b 5.04±0.39 37.46 cystatin 1.99c 3.18c 4.41c 3.71±0.29 17.44 e-64 1.39cd 2.75cd 4.41c 2.55±0.25 0.293 dtt 0.59d 1.56e 3.07d 1.79±0.24 39.21 phenanthroline 3.44b 5.43a 7.46a 3.81±0.38 2.79 edta 4.33a 5.80a 7.10ab 5.95±0.52 21.49 ic, inhibitory concentrations; pmsf, phenylmethylsulfonyl fluoride; tlck, na-p-tosyl-l-lysine chloromethyl ketone; tpck, n-tosyl-l-phenylalanine chloromethyl ketone; e-64, l-trans-epoxysuccinyl-leucylamido-(4guanidino)-butane; ddt, dithiothreitol; edta, ethylendiamidetetraacetic acid. a,b,c,d,e,fletters show statistical differences among values (tukey’s test, p≤0.05). table 2. effect of endogenous inhibitor on specific proteolytic activity in the midgut of a. amygdali. tryspin chymotrypsin elastase cathepsin b cathepsin l aminopetidase carboxypeptidase control 1.59±0.02* 0.82±0.01* 1.10±0.11* 0.55±0.05 1.04±0.024* 0.094±0.009 0.16±0.003* treatment 0.15±0.025 0.23±0.06 0.046±0.003 0.65±0.05 0.015±0.012 0.17±0.001* 0.024±0.001 *asterisks show statistical differences among values (student’s t-test, p≤0.05). no nco mm er cia l u se on ly cantly decreased trypsin and chymotrypsin activities. all extracted inhibitors significantly decreased elastase and cathepsin activities of a. spinidens except for c. suppressslis (sorkhabi-abdolmaleki & zibaee, 2013). extracted inhibitors from all species significantly decreased cathepsins b and l activities, but this decrease was not significantly different in the case of c. suppressalis. in the case of exopeptidases, endogenous inhibitors from e. kuehniella and h. cunea on aminopeptidase and all inhibitors on carboxypeptidase significantly decreased enzymatic activities (sorkhabi-abdolmaleki & zibaee, 2013). in conclusion, the presence of different specific proteases involved in protein digestion was found in a. amygdali using specific substrates and inhibitors. these enzymes had different ph profiles and extracted endogenous inhibitors from the insect’s midgut significantly decreased the majority of the proteases. these results were expected, as a. amygdali feeds on several host plants. sometimes, a. amygdali is used as a rearing host for trissolcus spp. parasitoids (a biocontrol agent of wheat sunn pest), so these findings could be helpful for efficient mass-rearing. a. amygdali occasionally occurs as a secondary pest of agricultural products, so the possible use of protease inhibitors could be a promising method to avoid the use of pesticide sprays. references barrett a.j., rawlings n.d., wasner j.f., 1998 handbook of proteolytic enzymes. -academic press, london. bell h.a., down r.e., edwards j.p., gatehouse j.a., anghard m.r., gatehouse m.r., 2005digestive proteolytic activity in the gut and salivary glands of the predatory bug podisus maculiventris (heteroptera: pentatomidae); 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[journal of entomological and acarological research 2014; 46:1868] [page 41] article no nco mm er cia l u se on ly j. ent. acar. res. ser. ii, 43 (3): 301-313 30 december 2011 g. pellizzari two new species of scale insects (hemiptera, coccoidea) from sardinia (italy) with a check list of sardinian coccoidea abstract - two new species of scale insects collected in sardinia (italy) are described and illustrated: spinococcus giuliae sp. n. (pseudococcidae) off the roots  of umbilicus rupestris (crassulaceae) and micrococcus sardous sp. n. (micrococcidae) off the root of an undetermined grass (poaceae) growing near the sea. an  identification key to micrococcus species and a revised list of the scales presently  known in the island are also provided.  riassunto - due nuove specie di cocciniglie (hemiptera, coccoidea) della sardegna (italia) con una check list dei coccoidea sardi vengono descritte due nuove specie di cocciniglie, spinococcus giuliae sp. n.  (pseudococcidae) e micrococcus sardous sp. n.(micrococcidae), raccolte in sardegna. gli esemplari di s. giuliae sono stati raccolti su radici di umbilicus rupestris  (crassulaceae) sul monte albo (sassari), mentre m. sardous è stato raccolto su radici  di una graminacea indeterminata sulla spiaggia di capo ceraso (olbia). vengono  presentati una chiave di identificazione delle 8 specie di micrococcus finora note e  una check list degli hemiptera coccoidea di sardegna. key words: pseudococcidae, spinococcus giuliae n. sp., micrococcidae, micrococcus sardous n.sp., genus micrococcus identification key. introduction one hundred and one species of scale insects (hemiptera: coccoidea) are currently  known from sardinia, (pellizzari & russo, 2006), including alien invasive species,. when  one considers that almost 400 species of scale insect are known from mainland italy  (pellizzari, 2010), the few recorded from sardinia suggests that the scale insect fauna of  the island is still largely unknown. during a survey, carried out some years ago, mostly  in north-eastern sardinia, some apparently new scale insect species were collected.  their presence on the island was briefly commented on a previous paper (pellizzari &  fontana, 1996). in the present paper, two new species, one belonging to spinococcus  (pseudococcidae) and the other to micrococcus (micrococcidae) are described and illustrated. the opportunity is taken to also revise the list of sardinian scale insects based  on previous papers (pellizzari & fontana, 1996; pellizzari, 2003; pellizzari & russo,  2006) and on scalenet (ben-dov et al., 2011). journal of entomological and acarological research, ser. ii, 43 (3), 2011302 materials and methods specimens were slide mounted according to the procedures of kosztarab and kozár  (1988). measurements and frequencies are given as mean, followed by the ranges in  parentheses. terminology follows that of williams (1985) and miller & williams (1995)  respectively for pseudococcidae and micrococcidae. specimens depository: the scientific museums of the university of padova (italy),  department of environmental agronomy & crop production - entomology (deae); pseudococcidae spinococcus giuliae n. sp. adult female (fig. 1) material studied: holotype: adult female, mount albo, nuoro province, sardinia (italy), off roots of umbilicus rupestris (crassulaceae), 21 may 1995, deae, slide  n.651/3. paratypes: 4 adult females, same data as holotype. slides n.651/1, 651/2,  651/4, 651/5. living specimens: body broadly oval, convex. derm covered with white powdery wax,  body segmentation apparent. mounted specimen. body broadly oval, 2 (1.7-2.2) mm long, 1.4 (1-1.6) mm wide;  anal lobes poorly developed. venter. labium 3-segmented, with 2 pairs of setae on unsclerotised basal segment, one  pair on middle segment and 5 pairs on apical segment. stylet loop not quite reaching  level of second coxae antennae 9-segmented; total length of each antenna 343 (325370) µm. scape with 3 setae, 2nd segment with 2 setae and 1 sensory pore, 3rd segment  without setae, 4th segment with 2 setae, 5th segment with 1 fleshy seta, 6th segment with  1 fleshy seta + 2 setose setae, 7th segment with 3 fleshy setae + 7 flagellate or hair-like.  eyes near margin, protruding. legs well developed; hind coxa without translucent  pores; trochanter with 2 campaniform pores. measurements of metathoracic leg (in µm):  coxa 117 (100-125); trochanter + femur 216 (190-240); tibia 176 (150-200); tarsus 80  (70-90); tarsal digitules knobbed; claw digitules longer than claw, knobbed; claw about  18 µm long, with a small denticle. body setae: ventral setae hair-like, distributed in a  transvers single row on each abdominal segment, with the 2 medial setae usually longer;  other setae present near coxae, on thorax, and on head. minute hair-like setae sparse  on abdominal segments; minute spine-like setae, each about 4-6 µm long, distributed  on body submargin. trilocular pores, each 3–4 µm wide, numerous, forming transverse  bands on posterior abdominal segments, becoming less abundant anteriorly and sparse  on head and on medial and submarginal parts of thorax; also with 2–6 pores laterad to  each spiracle opening. tubular ducts absent. quinquelocular disc-pores and multilocular  303g. pellizzari: two new species of scale insects from sardinia (italy) fig. 1 - spinococcus giuliae sp. n.: adult female. journal of entomological and acarological research, ser. ii, 43 (3), 2011304 disc-pores very rare or absent: one of the five specimens had one quinquelocular discpore and another had one multilocular disc-pore (slides n.651/3 and n.651/4) near vulva.  circulus present between segment iii and iv, oval, poorly developed. anal lobes each  with one apical seta 117 (100-175) µm long and 2 subapical setae. spinulae present on  last abdominal segments. dorsum. cerarii numbering 18 pairs, each with 2 spinose setae, each about 10 µm  long, and 3-5 associated trilocular pores. on two specimens, some abdominal cerarii  were elevated from surrounding derm (slides n.651/2 and n.651/4), suggesting that all  cerarii could be elevated from surrounding derm on young females. anal lobe cerarii  with 3 spinose conical setae, longer than other cerarian setae, each about 16 µm long,  and 9-12 trilocular pores.  dorsal surface with spinose setae similar to cerarian setae, each 6-8 µm long, each often  associated with 2 or 3 trilocular pores, but sometimes with 2 setae close together, plus 5  or 6 trilocular pores. trilocular pores otherwise evenly distributed, each about 4 µm wide. anterior pair of ostioles membranous but posterior ostioles with inner edge of lips lightly  sclerotized; each lip with 5-8 trilocular pores. multilocular pores absent. tubular ducts  absent. anal ring with a double row of pores and 3 pairs of setae.  derivatio nominis. the species is named after my younger daughter giulia.  comments there is some disagreement about the status of spinococcus borchsenius, which was a  replacement name for acanthococcus kiritchenko (a homonym of acathococcus signoret). danzig (1980) synonymized spinococcus with peliococcus borchsenius. according  to williams (1962), kostzarab & kozár (1988) and tang (1992), the main morphological  characters of the genus spinococcus (presence of 17 or 18 pairs of cerarii, each consisting of two conical setae with some triloculars at their base, and presence of dorsal setae,  similar to cerarian setae, associated with triloculars) differ from peliococcus which is  characterized by dorsal clusters of multilocular disc-pores, each cluster with one or more  tubular ducts in centre (williams, 1962; kosztarab, 1996). the boundaries of the genus  peliococcus appear unclear since species previously placed in the genus spinococcus  (i.e. s. morrisoni kiritshenko, p. multispinus sirawa) were later included in peliococcus  (danzig, 1980). because of these differences, several authors (for instance, kostzarab  & kozár, 1988; tang, 1992; marotta & tranfaglia, 1995; lagowska, 2005) have recognised both spinococcus and peliococcus. indeed, danzig herself, in a subsequent paper  (2001), distinguishes two distinct forms in the genus peliococcus, named respectively  the “peliococcus type” and the “spinococcus type”. for these reasons. the new species  has been described in the genus spinococcus borchsenius. the most peculiar character of s. giuliae is the absence, or extreme scarcity, of disc-pores  and the absence of tubular ducts. many coccoidea have few or even no multilocular  disc-pores near their genital opening and this character is generally associated with  ovoviviparity. however, tubular ducts are usually present in pseudococcids, at least on  the venter. 305g. pellizzari: two new species of scale insects from sardinia (italy) in the mediterranean basin, the genus spinococcus previously included s. convolvuli  ezzat, recorded in egypt, s. mathisi (balachowsky) known from france and tunisia,  and s. multispinus (siraiwa) recorded from southern italy (marotta & tranfaglia, 1995).  micrococcidae micrococcus sardous n. sp. adult female (fig. 2) material studied: holotype: adult female, capo ceraso, olbia province, sardinia (italy), off roots of ammophila arenaria? (poaceae) growing near the beach, 24.v.1995,  deae, slide n.712/1. paratypes: 6 adult females, same data as holotype. slides n.712/2-7; 2 first instars,  slide n.712/8. living specimens. body shape broadly oval, very convex or almost spherical in reproductive females; red brown in colour. mounted specimen. body broadly oval in pre-reproductive female, almost round in  reproductive female, 3.12 (2.2-3.6) mm long, 2.9 (1.7-3.6) mm wide. venter. antennae 3-segmented, segment i: 65 (56-70) µm long, ii 46 (40-50) µm, iii  105 (100-110) µm. labium apparently of one segment, 110 µm long and 132 µm wide,  with 4 pairs of setae, each about 34 µm long. stylet loop reaching level of mid coxae.  legs well developed, femur enlarged, tarsus and tibia fused; trochanter + femur 216  (200-250) µm long, tibia + tarsus 233 (210-270) µm long, claw without denticle, 43 µm  long, tarsal digitules 40 µm long. spiracles well developed, 80 µm wide and 90 µm long, each spiracle surrounded by a  slightly sclerotised area; atrium of spiracle filled with about 50 intrastigmatic multilocular  pores. parastigmatic multilocular pores, each 8 µm wide with 7-20 loculi, distributed  irregularly in a submarginal band on thorax, most numerous near spiracles, and forming 2 or 3 loose groups on submargin of abdominal segments, plus a few laterad to anal  opening; rare on middle of abdominal segment v. tubular ducts present, of two sizes:  larger 20.4 (16-24) µm long, 6.4 µm wide, with thin inner filament about 16 µm long,  distributed on mid-abdominal segments; smaller type slender, 27 (24-32) µm long and  2.4-3.3 µm wide, with inner filament about 20 µm long, distributed medially on thorax  and head, but most abundant on apex of head. ventral setae sparse, small, about 11 µm  long. discoidal pores circular, with sclerotised rim, each about 5-6.4 µm wide, scattered. minute bilocular pores sparse, mainly on body submargin; larger bilocular pores  sparse on thorax.  dorsum. apparent anal lobes broad, with long and stout setae, number variable, from  4 to 8 on each lobe, each seta 380 µm (335-416) long. true anal lobes modified into  two crescentic anal plates surrounding anal opening, each bearing 4 setae, 2 on anterior  journal of entomological and acarological research, ser. ii, 43 (3), 2011306 fig. 2 - micrococcus sardous sp. n.: adult female 307g. pellizzari: two new species of scale insects from sardinia (italy) edge, about 16 µm, long, 1 on posterior edge, 65 µm long, plus 1 near apex of posterior  edge, about 10 µm.  anal opening situated away from abdominal margin. anal ring with 2 or 3 rows of cells  and with 7-10 long setae on each side, longest 160 (120-200) µm long, smallest 138  (128-144) µm. discoidal pores with sclerotised rim, circular, about 4.5-6.5 µm wide,  scattered over dorsum. short setae, each 6.5 µm long, scattered. small and minute  bilocular pores sparse throughout. derivatio nominis the species name sardous is the latin adjective, gender masculine, meaning “of sardinia,  pertinent to sardinia”, after the italian island of sardinia where this species was collected. comments the genus micrococcus was diagnosed by marotta et al. (1995) and miller et al. (2005).  m. sardous is near to m. baeticae matile-ferrero & williams, but differs mainly as follows (character-states on m. baeticae in brackets): 1) anal opening some distance from  apex of abdomen (very close to apex of abdomen) 2) absence or paucity of parastigmatic  multilocular pores on ventral abdominal segments (parastigmatic multilocular pores  forming bands across abdominal segments), and 3) presence of several discoidal pores  with a sclerotised rim around labium (absent on m. baeticae). on the other hand, the  morphology of the first-instar nymph of m. sardous (slides n.712/8 and in.712/2, inside  the female body) is close to that of m. bodenheimeri bytinski-salz (miller & williams,  1995): it has parastigmatic pores, each with 5-8 loculi) on each side of the body, in groups  of 3 - 4 near each spiracle and hind coxa, with one pore inside each spiracle, and there  are no pores on the body margin between the anterior and posterior spiracles.  the genus micrococcus is presently known only in countries surrounding the mediterranean basin (morocco, tunisia, algeria, spain, sardinia and italy mainland, croatia,  turkey, cyprus, israel) (marotta et al., 1995; miller et al., 2005; matile-ferrero &  williams, 2006; kaydan et al., 2007; masten milek & simala, 2008). all were collected off roots of wild or cultivated gramineae and are often associated with ants of  the genus tapinoma. with this new species, the known micrococcus species reaches 8.  in sardinia, 2 other micrococcus species are known, namely m. silvestrii leonardi and  m. similis leonardi.  key to adult female of micrococcus  (modified after miller & williams, 1995) 1  parastigmatic pores absent or restricted to thorax ....................................................2 –  parastigmatic pores present on thorax and abdomen ...............................................5 2   (1) parastigmatic pores absent; with fewer than 10 setae on each anal plate ...........3 –   parastigmatic pores present near each spiracular plate; with more than 10 setae on  each anal plate.  ................................................................................similis leonardi journal of entomological and acarological research, ser. ii, 43 (3), 2011308 3   (2) some marginal discoidal pores oval, with oval sclerotization in shape of an eye  (according to miller & williams, based on 2nd instar female .................... m. rungsi  balachowsky –   marginal discoidal pores all round, or if oval, without sclerotization in shape of an  eye ............................................................................................................................4 4    (3) antennae 3-segmented; longest seta on apparent anal lobes 237 (172-306) µm  long  ....................................................................... m. bodenheimeri bytinsky-salz –   antennae 2-segmented (segments iii and ii partially or completely fused); longest  seta on apparent anal lobes 90 (83-96) µm long ............. m. dumonti balachowsky. 5   (1) longest dorsal seta on metathorax longer than 100 µm; with 9 or more setae on  each hind femur  ................................................. m. longispinus miller & williams –   longest dorsal seta on metathorax shorter than 100 µm; with 8 or fewer setae on  each hind femur ........................................................................................................6 6    (5) tibia + tarsus more than 300 mm long; with more than 5 parastigmatic pores near  each lateral margin of abdominal segment iv .........................m. silvestrii leonardi  –    tibia + tarsus less than 300 mm long; parastigmatic pores absent on margin of abdominal segment i ....................................................................................................7 7    (6) parastigmatic multilocular pores present on margin of head, thorax and abdominal  segment i and ii ...................................................... m. confusus miller & williams –    parastigmatic multilocular pores present on margin of head and thorax and also  submarginally or medially on other abdominal segments  .......................................8 8    (7) anal ring situated on apex of abdomen. parastigmatic multilocular pores forming  wide bands across segments ii-v of abdomen  ......... m. baeticae matile ferrero &  williams –   anal ring situated far from apex of abdomen. parastigmatic multilocular pores forming loose groups on submargin of abdominal segments ii-viii  ....m. sardous n. sp. conclusion with the addition of the newly described species and the revision of the previous lists  (pellizzari & fontana, 1996; pellizzari & russo, 2005), 105 scale insect species have  now been recorded from sardinia. the revised list is shown in table 1. the previously  recorded mealybug chaetococcus sulcii (green) (ben-dov et al., 2011) proved to be an  erroneous record for sardinia. so far, c. sulcii is known in italy only in valle d’aosta  (north italy) (matile-ferrero & pellizzari, 2002). the diaspidid melanaspis inopinata  (leonardi) is added to the list of known species from the island (melis, 1930). the presence of the eriococcid acanthococcus devoniensis (green) in sardinia is regarded as  309g. pellizzari: two new species of scale insects from sardinia (italy) table 1 check-list of scale insects recorded in sardinia. an asterisk marks the alien introduced species. family species validation source aclerdidae aclerda berlesii buffa, 1897 pellizzari & russo, 2005  asterolecaniidae asterodiaspis bella (russell, 1941) pellizzari & fontana, 1996 » asterodiaspis ilicicola (targioni tozzetti, 1888) pellizzari & fontana, 1996 » planchonia arabidis signoret, 1876 pellizzari & fontana, 1996 » planchonia zanthenes (russell, 1941) pellizzari & russo, 2005  » pollinia pollini (costa, 1857) pellizzari & russo, 2005  coccidae ceroplastes rusci (linnaeus, 1758)* pellizzari & russo, 2005 » ceroplastes sinensis del guercio, 1900* pellizzari & russo, 2005  » coccus hesperidum linnaeus, 1758* pellizzari & russo, 2005  » eulecanium ericae (balachowsky, 1936) pellizzari & fontana, 1996 » eulecanium tiliae (linnaeus, 1758) pellizzari & russo, 2005  » filippia follicularis targioni tozzetti, 1867 pellizzari & russo, 2005  » lecanopsis myrmecophila leonardi, 1908 pellizzari & russo, 2005  » lichtensia viburni signoret, 1873 pellizzari & fontana, 1996 » parthenolecanium persicae (fabricius, 1776) pellizzari & russo, 2005  » pulvinaria floccifera (westwood, 1870)* pellizzari & russo, 2005  » pulvinaria vitis (linnaeus, 1758) pellizzari & russo, 2005  » pulvinariella mesembryanthemi (vallot, 1830)* pellizzari & russo, 2005  » rhizopulvinaria maritima canard, 1967 pellizzari & fontana, 1996 » saissetia coffeae (walker, 1852)* pellizzari & russo, 2005  » saissetia ficinum (paoli, 1915) pellizzari & russo, 2005  » saissetia oleae (olivier, 1791)* pellizzari & russo, 2005  » sphaerolecanium prunastri (fonscolombe, 1834) pellizzari & russo, 2005  » stotzia ephedrae (newstead,1901) pellizzari, 2003 diaspididae abgrallaspis cyanophylli (signoret, 1869) pellizzari & russo, 2005 » adiscodiaspis ericicola (marchal, 1909) pellizzari & russo, 2005  » aonidia lauri (bouché, 1833) pellizzari & russo, 2005  » aonidia mediterranea (lindinger, 1910) pellizzari & fontana, 1996 » aonidiella aurantii (maskell, 1879)* pellizzari & russo, 2005  » aspidiotus nerii bouché, 1933* pellizzari & russo, 2005  » aulacaspis rosae (bouché, 1833) pellizzari & russo, 2005  » carulaspis minima (signoret, 1869)  pellizzari & russo, 2005  » chionaspis etrusca leonardi, 1908 pellizzari & fontana, 1996 » chrysomphalus dictyospermi (morgan, 1889)* melis, 1930, longo et al.,  1995  » diaspidiotus bavaricus (lindinger, 1912) pellizzari & fontana, 1996 » diaspidiotus cecconii (leonardi, 1908) pellizzari & russo, 2005  » diaspidiotus labiatarum (marchall, 1909) pellizzari & fontana, 1996 journal of entomological and acarological research, ser. ii, 43 (3), 2011310 » diaspidiotus lenticularis (lindinger, 1912) pellizzari & fontana, 1996 » diaspidiotus ostreaeformis (curtis,1843) pellizzari & russo, 2005  » diaspidiotus perniciosus (comstock, 1881)* pellizzari & russo, 2005  » diaspis echinocacti (bouché, 1833)* pellizzari & russo, 2005  » duplachionaspis berlesii (leonardi, 1898) pellizzari & russo, 2005  » dynaspidiotus ephedrarum (lindinger, 1912) pellizzari & russo, 2005  » epidiaspis leperii (signoret, 1869) pellizzari & russo, 2005  » furchadaspis zamiae (morgan, 1890)* pellizzari & russo, 2005  » gonaspidiotus minimus (leonardi, 1896) pellizzari & fontana, 1996 » hemiberlesia lataniae (signoret, 1869)* pellizzari & fontana, 1996 » hemiberlesia rapax (comstock, 1881) pellizzari & russo, 2005  » lepidosaphes beckii (newmann, 1869)* pellizzari & russo, 2005  » lepidosaphes conchiformis (gmelin, 1789)  pellizzari & russo, 2005  » lepidosaphes flava (signoret, 1870)  pellizzari & fontana, 1996 » lepidosaphes gloverii (packard, 1869)* pellizzari & russo, 2005  » lepidosaphes ulmi (linnaeus, 1758) pellizzari & russo, 2005  » leucaspis pusilla loew, 1883 pellizzari & russo, 2005  » leucaspis signoreti targioni tozzetti, 1868 pellizzari & russo, 2005  » lineaspis striata (newstead, 1897) pellizzari & russo, 2005  » melanaspis inopinata (leonardi) melis 1930 » parlatoria oleae (colvée, 1880) pellizzari & russo, 2005  » parlatoria pergandii comstock, 1881 pellizzari & russo, 2005  » parlatoria proteus (curtis, 1843)* pellizzari & russo, 2005  » parlatoria ziziphi (lucas, 1853)* pellizzari & russo, 2005  » pseudaulacaspis pentagona (targioni tozzetti, 1886)* pellizzari & russo, 2005  » rungaspis capparidis (bodenheimer, 1929) pellizzari, 2003 » saharaspis ceardi (balachowsky, 1928) pellizzari & fontana, 1996 » targionia nigra signoret, 1870 pellizzari & fontana, 1996 » targionia vitis (signoret, 1876) pellizzari & russo, 2005  » unaspis euonymi (comstock, 1881)* pellizzari & russo, 2005  eriococcidae acanthococcus acutus (goux, 1938) pellizzari & fontana, 1996 » acanthoccus araucariae araucariae (maskell, 1879)* pellizzari & fontana, 1996 » acanthoccus devoniensis (green, 1896)?? hoy, 1963 longo et al.,  1995  » acanthococcus ericae signoret tranfaglia & esposito,  1985 » gossyparia spuria (modeer, 1778) pellizzari & russo, 2005  kermesidae kermes bacciformis leonardi, 1908 pellizzari & russo, 2005  » kermes ilicis (linnaeus, 1758) pellizzari & russo, 2005  » kermes vermilio planchon, 1864 pellizzari & russo, 2005  lecanodiaspididae lecanodiaspis sardoa targioni tozzetti, 1869 pellizzari & russo, 2005  311g. pellizzari: two new species of scale insects from sardinia (italy) monophlebidae gueriniella serratulae (fabricius, 1775) pellizzari & russo, 2005  » icerya purchasi maskell, 1879* pellizzari & russo, 2005  micrococcidae micrococcus sardous sp. n. present paper » micrococcus silvestrii leonardi, 1907 pellizzari & russo, 2005  » micrococcus similis leonardi, 1907 pellizzari & russo, 2005  pseudococcidae balanococcus orientalis dantsig & ivanova, 1976  pellizzari & russo, 2005  » chorizococcus rostellum (lobdell, 1930) pellizzari & fontana, 1996 » dysmicoccus kozari pellizzari & fontana, 1996 pellizzari & fontana, 1996 » dysmicoccus pietroi marotta, 1992 pellizzari & fontana, 1996 » euripersia inquilina (leonardi, 1908) pellizzari & russo, 2005  » euripersia sardiniae (leonardi, 1908) pellizzari & russo, 2005  » nipaecoccus delassusi (balachowsky, 1925) pellizzari & russo, 2005  » peliococcus manifectus borchsenius, 1949 pellizzari, 2003 » phenacoccus aceris (signoret, 1875) pellizzari & fontana, 1996 » phenacoccus asphodeli goux, 1942 pellizzari, 2003 » phenacoccus graminicola leonardi, 1908 pellizzari & russo, 2005  » phenacoccus incertus (kiritchenko, 1940) pellizzari & russo, 2005  » planococcus citri (risso, 1813) pellizzari & russo, 2005  » planococcus ficus (signoret, 1875) pellizzari & russo, 2005  » planococcus vovae (nassonov, 1908)  pellizzari & fontana, 1996 » pseudococcus calceolariae (maskell, 1878)° pellizzari & russo, 2005  » pseudococcus longispinus (targioni tozzetti, 1867)* pellizzari & russo, 2005  » pseudococcus viburni (signoret, 1875) * pellizzari & russo, 2005  » spinococcus giuliae sp. n. present paper » trabutina mannipara (hemprich & ehrenberg, 1829)  pellizzari & russo, 2005  » trionymus multivorus (kiritchenko, 1936) pellizzari, 2003 » trionymus myrmecarius (leonardi, 1908) pellizzari & russo, 2005  putoidae puto palinuri marotta e tranfaglia, 1993 pellizzari & fontana, 1996 » puto superbus (leonardi, 1907) pellizzari & russo, 2005  doubtful. it was first reported from sardinia by leonardi (1908, p.159). later, leonardi  himself (1920) placed this first record among the synonyms of a. ericae (signoret) and  clearly reported that the species was collected in sardinia. subsequent authors (paoli,  1916; hoy, 1963; pellizzari & russo, 2005) only referred to the first record of 1908.  tranfaglia & esposito (1985) redescribed a. ericae from old specimens preserved in  the portici collection, collected in sardinia possibly by leonardi, and labelled e. devoniensis. some specimens collected more recently in sardinia by pellizzari and fontana  and identified as a. devoniensis (pellizzari & fontana, 1996) have proved to be a misidentification of a. ericae (f. kozár, personal communication, 2011). the old record of  carulaspis visci (schrank) is regarded as a misidentification of c. minima or, likely, of  c.juniperi, with which. it was in the past confused, until the situation was clarified by  journal of entomological and acarological research, ser. ii, 43 (3), 2011312 baccetti (1960). this is strengthen by the fact that the only host plant of the true c. visci,  is viscum album, and this epiphytic plant is absent in sardinia (zuber, 2004). acknowledgements many thanks to ferenc kozár, plant protection department, hungarian academy  of sciences, budapest, for his useful remarks and observations and to c. hodgson, the  national museum of wales, cardiff, uk, who revised the manuscript and suggested  some improvements. thanks to paolo paolucci, dipartimento agronomia ambientale e  produzioni vegetali, university of padova, who kindly made the drawings. references baccetti b. 1960 - le cocciniglie italiane delle cupressaceae. - redia, 45: 23-111. ben-dov, y., miller, dr., gibson, g.a.p., 2011 - scalenet: a database of the scale insects  of the world. available in: http://www.sel.barc.usda.gov/scalenet/scalenet.htm (accessed  16.8.2011). danzig e.m., 1980 - coccoids of the far east ussr (homoptera, coccinea) with phylogenetic  analysis of scale insects fauna of the world. (in russian). nauka, leningrad. 367 pp. (english  translation in 1986 by amerind publishing co., new delhi, india. 450 pp.  danzig e.m., 2001 - mealybugs of the genera peliococcus and peliococcopsis from russia and  neighbouring countries (homoptera: coccinea: pseudococcidae). - zoosystematica rossica,  9(1): 123-154. hoy, j.m. 1963 - a catalogue of the eriococcidae (homoptera: coccoidea) of the world. - new  zealand department of scientific and industrial research bulletin 150, 260 pp. kaydan m.b., ülgentürk s., erkiliç l., 2007 - turkiye’nin gozden gecirilmis coccoidea (hemiptera) turleri listesi. [checklist of turkish coccoidea species (hemiptera)]. - yüzüncü yil  üniversitesi ziraat fakültesi, tarim bilimleri dergisi, 17(2): 89-106. kosztarab m., 1996 - scale insects of northeastern north america. identification, biology, and  distribution. - virginia museum of natural history, martinsburg, virginia. 650 pp. kosztarab m., kozár f., 1988 - scale insects of central europe. - akademiai kiado, budapest,  456 pp. lagowska b., 2005 - spinococcus morrisoni (kirichenko, 1936) (hemiptera: pseudococcidae)  new to the fauna of poland. - polskie pismo entomologiczne, 74(1): 39-42. leonardi g., 1908 - seconda contribuzione alla conoscenza della cocciniglie italiane. - bollettino  del regio laboratorio di entomologia agraria di portici, 3: 150-191. leonardi, g., 1920 - monografia delle cocciniglie italiane. - della torre, portici, 555 pp. marotta s., tranfaglia a., 1995 - nuovi pseudococcidi per la fauna italiana, con descrizione di  una nuova specie. - bollettino della società entomologica italiana. genova, 126(3): 269-276.  marotta s., spicciarelli r., tranfaglia a., 1995 (1993) - diagnosis of micrococcus leonardi,  redescription of type with discussion of the status of the family micrococcidae (homoptera  coccoidea). - bollettino del laboratorio di entomologia agraria ‘filippo silvestri’, 50:  175-198. 313g. pellizzari: two new species of scale insects from sardinia (italy) masten milek t., simala m., 2008 - list of the scale insects (hemiptera: coccoidea) of croatia.  105-119 in: branco m., franco j.c., hodgson c.j., (editors), proceedings of the xi international symposium on scale insect studies, oeiras, portugal, 24-27 september 2007. isa  press, lisbon, portugal, 322 pp. matile ferrero d., pellizzari g., 2002 -contribution to the knowledge of the scale insects  (hemiptera coccoidea) from the aosta valley (italy). - bollettino di zoologia agraria e di  bachicoltura ser.ii, 34 (3): 347-360. matile-ferrero d., williams d.j., 2006 - description of a new species of micrococcus leonardi from spain (hemiptera, coccoidea, micrococcidae). - revue française d’entomologie,  28(3): 125-128. melis a., 1930 - contribuzione alla conoscenza degli insetti dannosi alle piante agrarie e forestali  della sardegna. - redia, 18: 1-120. miller d.r., williams d.j., 1995 (1993) - systematic revision of the family micrococcidae  (homoptera: coccoidea), with a discussion of its relationships, and a description of a gynandromorph. - bollettino del laboratorio di entomologia agraria ‘filippo silvestri’. portici,  50: 199-247. miller d.r., gimpel m.e., rung a., 2005 - a systematic catalogue of the cerococcidae, halimococcidae, kermesidae, micrococcidae, ortheziidae, phenacoleachiidae, phoenicococcidae  and stictococcidae (hemiptera: coccoidea) of the world. - intercept limited, wimbourne,  uk, 554 pp. paoli, g. 1915 - contributo alla conoscenze della cocciniglie della sardegna. - redia, 11: 239-268. pellizzari g., 2003 - hemiptera coccoidea nuovi o poco noti per l’italia. - bollettino di zoologia  agraria e di bachicoltura, 35(2): 99-106. pellizzari g., 2010 - new data on the italian scale insects fauna. - acta phytopathologica et  entomologica hungarica, 45 (1): 89-93. pellizzari g., fontana p., 1996 - contribution to the knowledge of homoptera coccoidea of  sardinia with description of a new species. - bollettino di zoologia agraria e di bachicoltura,  28: 119-140. pellizzari g., russo a., 2005 - list of the scale insects (hemiptera, coccoidea) of italy. in:  erkiliç l. & kaydan m.b. (editors), proceedings of the x international symposium on scale  insect studies, adana/ turkey, 19-23 april 2004: 167-183. tang f.t., 1992 - the pseudococcidae of china. shanxi agricultural university, taigu, shanxi,  china. 768 pp (in chinese, summary in english). tranfaglia a., esposito a., 1985 - studi sugli homoptera coccoidea. vii. le specie italiane  del genere eriococcus targioni-tozzetti, 1869. - bollettino del laboratorio di entomologia  agraria ‘filippo silvestri’, 42: 113-134. williams d.j. 1962 - the british pseudococcidae (homoptera: coccoidea). - bulletin of the  british museum (natural history) entomology, 12: 1-79. zuber d., 2004 - biological flora of central europe: viscum album l.. – flora. morphology,  distribution, functional ecology of plants, 199 (39): 181-203. giuseppina pellizzari - università di padova, dipartimento agronomia ambientale e produzioni vegetali, viale dell’università 16, 35020 legnaro, italy. e-mail: giuseppina.pellizzari@ unipd.it accepted 10 november 2011 layout 1 abstract lobesia arzilae sp. n. and willibaldiana culatrae sp. n. (lepidoptera: tortricidae: olethreutinae) found in portugal are described. the new species were collected respectively in paúl de arzila, a nature reserve located in central-west portugal, close to the city of coimbra and in ilha da culatra, which lies in the parque natural da ria formosa, in the region of faro, situated in the extreme south of portugal. l. arzilae differs from other species of the genus lobesia by male genitalia; socius is lateral, developed extending upwards; sacculus has a group of long spines apically dentate and is armed with strong dorsal thorn. w. culatrae differs from other species of the genus willibaldiana by genitalia: in male valva is simple, in female cingulum is long and developed. images of the holotypes and the genitalia are provided. introduction after isotrias penedana trematerra, 2013, tortricidae of subfamily chlidanotinae tribe polyorthini, collected in serra da peneda, portugal (trematerra, 2013), two more new portuguese tortricids assigned to genus lobesia guenée, 1845 and willibaldiana knud larsen, 2013, are described: lobesia arzilae sp. n. (olethreutinae: olethreutini) from paúl de arzila, (coimbra, beira litoral), and willibaldiana culatrae sp. n. (olethreutinae: eucosmini) from algarve (ilha da culatra) (brown, 2005; razowski, 1989, 2003; larsen, 2013). paúl de arzila, a nature reserve in the lower mondego valley, is a freshwater marsh with an extensive reedbed, located in central-west portugal, close to the city of coimbra; ilha da culatra is a small island, forming part of the parque natural da ria formosa, in the region of faro, situated in the extreme south of portugal. lobesia arzilae sp. n. material examined: 1 male, holotypus, labelled as follows: p7482, portugal, paúl de arzila, coimbra, beira litoral, 24.ix.2004, m.f.v. corley. 1 male genitalia preparation (corley 2161) portugal, torre, taipal, montemor-o-velho, beira litoral, 7.iv.2004, p. pires (this specimen was in poor condition and is probably lost, but the genitalia are very clearly the same as the arzila specimen). adult. wingspan 9-12 mm (figure 1). antenna brownish. head light brown, palpi brownish light brown, inner part whitish. frons and vertex concolorous with palpus. thorax light brown, tegula brown with light brown edge. ground colour of forewing whitish, in distal part of wing slightly creamier or ochreous creamy. strigulae brownish. markings brown to blackish brown: basal blotch consisting of several stripes and spots; median fascia broad, rust-brown with blackish correspondence: pasquale trematerra, dipartimento di agricoltura, ambiente e alimenti, università degli studi del molise, via de sanctis, 86100 campobasso, italy. e-mail: trema@unimol.it key words: lobesia arzilae sp. n., willibaldiana culatrae sp. n., lepidoptera tortricidae, olethreutinae, new species, portugal. acknowledgements: the author would like to express his thanks to leif aarvik (norway), joaquin baixeras (spain), jozef razowski (poland), and martin corley (england) for comments and providing the study material. received for publication: 13 september 2013. revision received: 11 december 2013. accepted for publication: 29 january 2014. ©copyright p. trematerra, 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1923 doi:10.4081/jear.2014.1923 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. lobesia arzilae sp. n. and willibaldiana culatrae sp. n. new species from portugal (lepidoptera: tortricidae: olethreutinae) p. trematerra department of agriculture, environment and food sciences, university of molise, campobasso, italy [page 66] [journal of entomological and acarological research 2014; 46:1923] journal of entomological and acarological research 2014; volume 46:1923 figure 1. lobesia arzilae sp. n., adult. no nco mm er cia l u se on ly brown places, interrupted postmedially; terminal area brownish ferruginous, darker at apex. cilia pale cream suffused brown from apex of wing to middle of termen, concolorous with ground colour at tornus. hindwing light brownish grey; cilia grey or whitish grey. male genitalia (figures 2-4). top of tegumen rounded, uncus short; socius lateral, developed extending upward, provided with delicate bristles. valva elongate; basal cavity wide; sacculus with anterior group of long and slender bristles, angular group of large spines mixed with few dentate spines, a group of long dentate spines along median half ventrally; sacculus distinctly convex near middle ventrally, armed with a strong dorsal thorn; cucullus rounded distinct also spiny; ventral incision small. aedeagus moderately long. female genitalia. unknown. distribution. known only from two sites in the lower mondego valley, west of coimbra: paúl de arzila and montemor-o-velho, coimbra, beira litoral, central-west portugal. host. unknown. biology. the holotype male was trapped at light in late september, but the montemor-o-velho specimen was taken in april, so there must be two generations. the arzila site is a track separating the marsh, dominated by phragmites australis (cav.) trin. ex steud., with some marginal salix atrocinerea brot. and fraxinus angustifolia vahl, from drier sloping ground with crataegus monogyna jacq., rubus ulmifolius schott and quercus faginea lam. diagnosis. l. arzilae differs from other species of the genus lobesia by male genitalia. socius is lateral, developed extending upwards; sacculus has groups of long dentate spines along posterior half ventrally and is armed with a strong dorsal thorn. the type specimen and slide of male genitalia of l. dearzilae are deposited in the trematerra collection, university of molise, campobasso, italy. etymology. the new species is named after paúl de arzila (portugal), the area from which the holotype comes. wllibaldiana culatrae sp. n. material examined. 1 male, holotypus, labelled as follows: p10329, portugal, ilha da culatra, algarve, 18.viii.2008, p. pires. + 1 male with same data. paratypes, 2 males, 1 female portugal, ilha da culatra, 3.viii.2008, j. p. cardoso in coll. m. corley. adult (figure 5). wingspan 9-12 mm. antenna white-cream. head white-creamy, palpi concolorous sprinkled with light brown. frons and vertex concolorous with palpus. thorax and tegula light cream. abdomen yellowish-brown. ground colour of forewing white delicately suffused brownish; lines between markings and costal dividings darker; speculum white with brownish inner suffusion and brownish spots. [journal of entomological and acarological research 2014; 46:1923] [page 67] article figure 2. lobesia arzilae sp. n., male genitalia. figure 3. lobesia arzilae sp. n., male genitalia. uncus and socius. figure 4. lobesia arzilae sp. n., male genitalia. sacculus with thorn and groups of bristles and spines. figure 5. willibaldiana culatrae sp. n., adult. no nco mm er cia l u se on ly [page 68] [journal of entomological and acarological research 2014; 46:1923] marking brownish with lighter suffusions or dots along edges: dorsopostbasal fascia fusing with the subcostal marking; submedian interfascia with light brown stripes; median fascia interrupted in the middle; subterminal fascia and subapical markings fused. cilia light brown, brown at base. hindwing pale cream with the apex brownish; cilia concolorous with wing. male genitalia (figures 6-9). uncus minute; tegumen delicate; socius slender, hairy: tuba analis membranous. valva elongate, sacculus short and curved; ventral edge of sacculus less than half length of valva; pulvinus indistinct; neck of valva moderately broad; cucullus rounded, with slender spines. aedeagus long and slender, open dorso-posteriorly. female genitalia (figures 10 and 11). postostial part of sterigma provided with indistinct latero-posterior lobes, anteostial part large, expanding distally; sclerite of colliculum absent; cingulum long extending from base of bursa copulatrix to before end of ductus bursae; signa broad, rounded, unequally sized. article figure 9. willibaldiana culatrae sp. n., male genitalia. basal cavity of valva and pulvinus. figure 8. willibaldiana culatrae sp. n., male genitalia. valva. figure 7. willibaldiana culatrae sp. n., male genitalia. tegumen and aedeagus. figure 6. willibaldiana culatrae sp. n., male genitalia. figure 10. willibaldiana culatrae sp. n., female genitalia. no nco mm er cia l u se on ly distribution. known only from algarve, ilha da culatra, southern portugal. host. unknown. biology. adults were collected at light during august. ilha da culatra is low and windswept with sparse dune vegetation consisting of plants such as lotus creticus l., medicago marina l., otanthus maritimus (l.) hoffmanns & link, armeria maritima (mill.) willd., thymus carnosus boiss., helichrysum italicum (roth) g. don, ammophila arenaria (l.) link and eryngium maritimum l. the northern side of the island has salt marsh vegetation, but the collection site is in the sandy part. diagnosis. w. culatrae differs from w. paasi knud larsen, 2013, and w. schmitzi knud larsen, 2013, by light wings and by male and female genitalia. in male valva is simple, in female cingulum is long and more developed. the type specimens of w. culatrae are deposited in the trematerra collection, university of molise, campobasso, italy, and three paratypes are deposited in the private collection of martin corley, england. etymology. the new species is named after ilha da culatra (portugal), the island from which the type series comes. references brown j., 2005 world catalogue of insects. vol. 5. tortricidae (lepidoptera). apollo books, stenstrup: 1-741. larsen k., 2013 a new genus and two new species of tortricidae (lepidoptera) from the canary islands. phegea 41: 50-54. razowski j., 1989 the genera of tortricidae (lepidoptera). part ii: palaearctic olethreutinae. acta zool. cracov. 32: 107-328. razowski j., 2003 tortricidae of europe. vol. 2. olethreutinae. frantisek slamka, bratislava: 1-301. trematerra p., 2013 isotrias penedana sp. n. a new species of lepidoptera (tortricidae: chlidanotinae: polyorthini) from portugal. j. entomol. acarol. res. 45: 93-95. [journal of entomological and acarological research 2014; 46:1923] [page 69] article figure 11. willibaldiana culatrae sp. n., female genitalia. antrum, cingulum and signa. no nco mm er cia l u se on ly layout 1 abstract vector control is one of the most important components in combating vector-borne diseases throughout the world. application of insecticides is a widely known and popular vector control strategy. the objective of the present study was to evaluate the larvicidal activity of the hexane, diethyl ether, ethyl acetate and acetone extracts of abutilon indicum, hyptis suaveolens and leucas aspera against third-stage larvae of anopheles culicifiacies. the results clearly suggest that all three selected plant extracts exhibited moderate larvicidal activity after 24, 48 and 72 h at 250, 500, 750 and 1000 ppm; the lethal concentrations (lc) at 50% and 90% of a. indicum, h. suaveolens against third instar larvae at 24, 48 and 72 h (hexane, diethyl ether, ethyl acetate and acetone) were as follows: a. indicum, lc50=1031.65, 949.18, 833.58 and 673.68 ppm; lc90=2215.87, 2234.39, 2152.97 and 2455.10 ppm; h. suaveolens, lc50=423.00, 347.50, 236.58 and 217.24 ppm; lc90=1431.91, 1292.15, 1138.49 and 1049.27 ppm and l. aspera, lc50=559.77, 401.56, 299.71 and 263.01 ppm; lc90=1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively. overall, the highest larvicidal activity was observed with h. suaveolens extract followed by l. aspera and a. indicum at various concentrations at 48 and 72 h, respectively. the objective of this investigation was an attempt to search for a userand eco-friendly vector control agent. the study proved that the selected plant leaf extracts could serve as potent larvicidal agents against a. culicifacies in vector control programs. introduction malaria is a disease that inflicts a serious negative impact on public health and socio-economic development in resource-limited settings of the world. malaria directly or indirectly affects the health and wealth of individuals as well as nations. indeed, malaria is identified both as a disease associated with and a cause of poverty (karunamoorthi, 2012). currently, malaria control is hampered by many operational and technical problems. however, the development of insecticidal resistance in malaria vectors to existing conventional insecticides has made malaria vector control more challenging (sharma & saxena, 1996). anopheles stephensi and anopheles culicifacies are the two primary malarial vectors in india. in india, there are five sibling species reported, named a, b, c, d, and e; however, species a, c, d, and e are considered to be major vectors, while b is a minor vector (subbarao et al., 1999). amerasinghe et al. (1999) reported that a. culicifacies is the main vector and a. subpictus is a significant secondary malarial vector in sri lanka. in india, a. culicifacies is a primary vector in rural as well as periurban areas, which constitutes nearly 65% of all malaria cases. at the moment, dichlorodiphenyltrichloroethane (ddt) (organochlorine), malathion (organophosphorus) and �d-methrin, cyfluthrin, a-cypermethrin and l-cyhalothrin (synthetic pyrethroids) are the most commonly applied insecticides for vector control in the public health sector. insecticide susceptibility of a. culicifacies to ddt, malathion and �methrin was evaluated in 2009 in several districts of india by adopting the who standard protocol for adult susceptibility (who, 1998). it was found that a. culicifacies has developed resistance to all insecticides tested (mishra et al., 2012). currently, malaria control largely relies on a limited arsenal of materials; viz., artemisinin derivative drugs and pyrethroids. however, these products could also become ineffective due to the continuing evolution of resistance development. in this context, a new innovative userand eco-friendly alternative vector control strategy is mandatory. a search for powerful contextual communitybased vector control interventions is therefore warranted. correspondence: kalimuthu kovendan, division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore 641 046, india. tel.: +91.9962447932 fax: +91.422.2422387. e-mail: gokulsuryah@gmail.com key words: abutilon indicum, hyptis suaveolens, leucas aspera, anopheles culicifiacies, larvicidal activity. funding: the authors are thankful to science engineering research board (serb), department of science and technology (dst), govt. of india, new delhi (sr/ft/ls-156/2012) for providing financial support for the present work. acknowledgements: the authors are grateful to mr. m. munirathnam, (icmr, madurai), taxonomy and field station in coimbatore, tamil nadu, for helping in mosquito collection and identifying mosquito species of samples for the experiment work. received for publication: 13 june 2013. revision received: 18 april 2014. accepted for publication: 12 may 2014. ©copyright k. kovendan et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1747 doi:10.4081/jear.2014.1747 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. larvicidal activity of indigenous plant extracts on the rural malarial vector, anopheles culicifacies giles. (diptera: culicidae) k. kovendan,1 p. mahesh kumar,1 j. subramaniam,1 k. murugan,1 s. john william2 1division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore; 2p.g. research & department of advanced zoology and biotechnology, loyola college, nungambakkam, chennai, tamil nadu, india [page 90] [journal of entomological and acarological research 2014; 46:1747] journal of entomological and acarological research 2014; volume 46:1747 jear_2014_3_hrev_master 16/12/14 10:38 pagina 90 no nco mm er cia l u se on ly abutilon indicum (malvaceae), which is commonly known as thuthi (the vernacular name in tamil), is distributed throughout the driest areas in india (chopra et al., 1992). it is well known and reputable in the tamil traditional medicinal system called siddha as a phytotherapeutic agent against various illnesses such as jaundice, piles, ulcers and leprosy (yoganarasimhan, 2000). it is also reported to possess effective analgesic activity; a study by ahmed et al. (2000) indicated that an 80% ethanol root extract of a. indicum had a potential effect against a. aegypti and guppy fish (promsiri et al., 2006). the larvicidal activity of crude hexane, ethyl acetate, petroleum ether, acetone, and methanol extracts of a. indicum, aegle marmelos, euphorbia thymifolia, jatropha gossypifolia and solanum torvum were evaluated (rahuman, 2008). although hyptis suaveolens is a native plant of tropical america, it is also widespread in tropical africa, asia and australia. it grows under a wide range of soil and climatic conditions, mainly in warm areas of the country. h. suaveolens is also administered as a traditional medicine for treatment of various ailments, and its essential oil possesses insecticidal and larvicidal properties (peerzada, 1997; azevedo et al., 2001). in nigeria, ethanol extracts of orange peel (citrus sinensis) and bush tea leaves of h. suaveolens were compared for their toxicity against a. aegypti (amusan et al., 2005). it has been reported that h. suaveolens extract causes notable mortality of a. aegypti larvae because the extract also contained insecticidal compounds such as a-tepinoline, a monoterpene that is similar in action to d-limonone, which is present in c. sinensis. leucas aspera (labiatae) is a small herbaceous plant. it is commonly administered as an antipyretic herb in south india. the juice of its leaves is used for psoriasis and swellings as an external application. the plant extract mixed with honey is a good remedy for stomach pain and indigestion. preliminary chemical examination of l. aspera revealed the presence of triterpenoids (kamar & singh, 1994). the whole plant is reported to contain oleanolic acid, ursolic acid, and 3-itosterol (chaudhury & ghosh, 1969). aerial parts are reported to contain nicotine (mangathayaru et al., 2006). the flower is reported to contain ten compounds, among them amyl propionate (15.2%) and isoamyl propionate (14.4%), which were dominant (kalachaveedu et al., 2006). l. aspera leaves are used as an insecticide and mosquito repellent in rural areas (kiritikar and basu, 1990; sadhu et al., 2003). the hexane crude extracts of l. aspera showed high larvicidal activity against c. quinquefasciatus and a. aegypti (maheswaran et al., 2008; kovendan et al., 2012b). the aim of the present investigation was to determine the effect of a. indicum, h. suaveolens and l. aspera leaf extracts against third-stage larvae of a. culicifacies as a target species. materials and methods collection and rearing of mosquitoes larvae of a. culicifacies were collected from kallar village, near mettupalayam, tamil nadu, in different breeding habitats in using an o type brush. the mosquito larvae were fed with pedigree dog biscuits and yeast in a 3:1 ratio. feeding was continued until the larvae transformed into the pupal stage. the pupae were collected from the culture trays using a dipper and transferred to plastic containers (12×12 cm) containing 500 ml of water. the plastic jars were kept in a 90×90×90cm mosquito cage for adult emergence. mosquito larvae were maintained at 27+2°c, 75-85% relative humidity, under a photoperiod of 14:10 (light/dark). a 10% sugar solution was provided for a period of 3 days before blood feeding. the adult female mosquitoes were allowed to feed on the blood of a rabbit (one rabbit per day, exposed on the dorsal side) for 2 days to ensure adequate blood feeding for 5 days. after blood feeding, enamel trays with water from the culture trays were placed in the cage as oviposition substrates. collection of plants and preparation of plant extracts the selected medicinal plants were collected in and around maruthamalai hills and bharathiar university campus, coimbatore, tamil nadu. the fresh aerial part of a. indicum, and fresh leaves of h. suaveolens and l. aspera were washed thoroughly with tap water and shade dried at room temperature (28±2°c) for 5 to 12 days. the airdried materials were powdered separately using a commercial electric blender. from each plant, 300 g of powdered material was macerated with 1.0 l of hexane, diethyl ether, ethyl acetate and acetone, sequentially, for a period of 72 h each and filtered. the yield of the a. indicum, h. suaveolens and l. aspera crude extracts with hexane, diethyl ether, ethyl acetate and acetone, were: a. indicum 8.94, 10.15, 9.36 and 11.55 g and h. suaveolens 8.12, 9,66, 10.47, and 9.73 g and l. aspera 11.12, 8.29, 9.34 and 10.13 g, respectively. the extracts were concentrated at a reduced temperature on a rotary vacuum evaporator and stored at a temperature of 4°c. one gram of the plant residue was dissolved in 100 ml of acetone, which was considered as a 1% stock solution, from which concentrations were prepared ranging from 250, 500, 750 and 1000 ppm, respectively. larval toxicity test larvicidal activity was assessed using the procedure of who (1996) with slight modifications. a laboratory colony of a. culicifacies larvae was used for the larvicidal activity. twenty-five third-instar larvae of a. culicifiacies were kept in 250-ml glass containers, containing 200 ml of dechlorinated water. five replicates were set up for each concentration (250, 500, 750, 1000 ppm) and mixed with acetone and triton-80 (mixing solution). larval mortality was assessed at 24, 48 and 72 h. each experiment was replicated 3 times at room temperature (28±2°c) for three different plants. the control mortalities were corrected by using abbott’s formula (abbott, 1925). corrected mortality = observed mortality in treatment – observed mortality in control ¥ 100 100 – control mortality (1) percentage mortality = number of dead larvae ¥ 100 (2) number of larvae introduced the lethal concentrations (lc) at 50% and 90% were calculated from toxicity data using probit analysis (finney, 1971). statistical analysis the average larval mortality data were subjected to probit analysis for calculating lc50 and lc90, and other statistics at a 95% upper fiducidal limit and lower fiducidal limit, and chi-square values were calculated using spss 9.0 version (statistical software package; statacorp., college station, tx, usa). results at p<0.05 were considered to be statistically significant. results preliminary screening is a good means of evaluating the potential larvicidal activity of crude plant extracts, which is often assessed using different solvent extracts. mortality from the three plants tested is presented in tables 1-3. at 24 h, a. indicum demonstrated mortality levels in hexane, diethyl ether, ethyl acetate and actone extracts ranging from 22.18, 27.32, 32.55 and 41.23% at 24 h at 250 ppm to 51.65, 56.12, 61.55 and 64.35% at 1000 ppm, respectively (table 1). for h. suaveolens, mortality levels at 24 h from these extracts ranged from 41.15, 46.33, 50.60 and 52.15% at 250 ppm, to 77.18, 83.14, 88.15 and 90.77% at 1000 ppm, [journal of entomological and acarological research 2014; 46:1747] [page 91] article jear_2014_3_hrev_master 16/12/14 10:38 pagina 91 no nco mm er cia l u se on ly [page 92] [journal of entomological and acarological research 2014; 46:1747] respectively (table 2). finally, for l. aspera, mortality at 24 h from these same extracts ranged from 31.12, 44.50, 46.66, and 49.15% at 250 ppm, to 74.18, 75.27, 87.55 and 89.26% at 1000 ppm, respectively (table 3). we observed correspondingly higher larval mortality from these extracts at higher concentrations after 48 and 72 h, respectively (tables 2 and 3). the lc50 and lc90 values of hexane, diethyl ether, ethyl acetate and acetone extracts of a. indicum, h. suaveolens and l. aspera at 24 were as follows: a. indicum, lc50=1031.65, 949.18, 833.58 and 673.68 ppm; and lc90=2215.87, 2234.39, 2152.97 and 2455.10 ppm, respectively. for h. suaveolens, lc50=423.00, 347.50, 236.58 and 217.24 ppm; and lc90=1431.91, 1292.15, 1138.49 and 1049.27 ppm, respectively. finally, for l. aspera, lc50=559.77, 401.56, 299.71 and 263.01 ppm; and lc90=1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively (tables 1-3). discussion a. culicifacies is one of the major malaria vectors in the indian subcontinent and is generally regarded as intolerant to salinity, preferring to breed in newly-dug freshwater pits, domestic wells and sites used for plantings of coconuts and casurina trees in india (russell & rao, 1942; sabesan et al., 1986). ansari et al. (2000) suggested that the peppermint oil (mentha piperita) showed strong repellent action against adult mosquitoes when applied on human skin. the protection obtained against a. annularis, a. culicifacies, and c. quinquefasciatus was 100, 92.3, and 84.5%, respectively. the present investigation tested three plant leaf extracts in different solvents for their potential larvicidal activity against a. culicifacies. the biological activity of the experimental plant extracts varied, which may be due to the presence of various phytochemically active compounds in the plants, including phenolics, terpenoides, flavonoids and alkaloids. these active principles may have jointly or independently influenced or contributed to produce larvicidal effects against a. culicifacies. sharma & ansari (1994) demonstrated the protective effect of cyperus rotundus against a. culicifacies, a. stephensi and c. quinquefasciatus, and showed greater protection than neem oil (37.5%). sharma et al. (1995), reported that the crude extract of solanum nigram leaves showed significant larvicidal activity against a. culcifacies, c. quinquefasciatus and a. aegypti at a dose equivalent to the lc90, ranging from 0.18 to 0.21% (singh et al., 2002). the present results concur with some of the previous findings of a. indicum against the third instar larvae of a. culicifacies, with lc50 and lc90 values of hexane, diethyl ether, ethyl acetate and acetone extracts of (lc50) 1031.65, 949.18, 833.58 and 673.68 ppm, and (lc90) 2215.87, 2234.39, 2152.97 and 2455.10 ppm at 24 h, respectively. one study reported that the lethal concentration values of the aqueous extract of roots of h. abelmoschus against the larvae of a. culicifacies, a. stephensi, and c. quinquefasciatus were 52.3, 52.6, and 43.8 ppm, respectively (dua et al., 2006). the lc50 and lc90 values of s. indicus, c. collinus and m. koenigii against third-instar larvae at 24, 48 and 72 h (in hexane, chloroform and ethyl acetate extracts) were: for s. indicus, (lc50) 544.93, 377.86 and 274.79 ppm, and (lc90) 1325.32, 1572.55 and 1081.29 ppm at 24 h; for c. collinus, (lc50 ) 375.34, 318.29 and 226.10 ppm, and (lc90) 699.65, 1577.62 and 1024.92 ppm at 24 h; and, for m. koenigii, (lc50) 963.53, 924.85 and 857.62 ppm, and (lc90) 1665.12, 1624.68 and 1564.37 ppm at 24 h, respectively (kovendan et al., 2012a). article table 1. larvicidal activity of abutilon indicum against third instars larvae of anopheles culicifacies. exposure solvents percentage larval mortality±sd lc50 95% confidence limit x2 (time) concentration of a. indicum (ppm) (lc90) lfl ufl (df=4) 250 500 750 1000 lc50 (lc90) lc50 (lc90) hexane 22.18±0.86 26.31±1.12 34.56±0.98 51.65±1.50 1031.65 877.34 1360.29 1.39a (2215.87) (1733.59) (3414.23) diethyl ether 27.32±0.42 30.40±0.65 37.19±1.32 56.12±1.25 949.18 802.18 1260.00 2.41a (2234.39) (1722.60) (3594.44) 24 h ethyl acetate 32.55±1.23 34.12±1.45 40.66±0.44 61.55±1.18 833.58 700.82 1076.37 3.78a (2152.97) (1659.17) (3484.70) acetone 41.23±1.65 43.65±0.68 45.22±1.36 64.35±1.33 673.68 479.50 928.05 3.52a (2455.10) (1742.72) (5435.70) 655.69 561.41 757.04 hexane 30.63±0.54 38.50±1.69 55.60±1.74 68.92±1.95 (1586.80) (1333.98) (2079.68) 0.56a 592.82 487.55 690.49 diethyl ether 34.12±1.55 42.26±0.72 58.77±0.95 71.23±1.27 (1556.39) (1303.21) (2061.01) 0.48a 48 h 444.90 255.91 562.52 ethyl acetate 44.35±1.21 49.22±1.56 61.10±1.22 74.88±1.48 (1625.26) (1310.21) (2373.37) 1.10a 274.54 69.32 392.03 acetone 50.56±1.35 63.91±1.75 71.25±1.66 85.45±1.30 (1207.15) (1026.23) (1561.63) 0.76a hexane 48.61±1.52 65.22±1.88 84.14±1.47 91.35±1.32 273.83 140.65 362.35 0.46a (935.20) (831.15) (1100.14) diethyl ether 51.70±0.80 70.50±1.95 91.36±1.93 96.50±1.40 245.95 136.20 321.82 0.99a (764.39) (688.68) (873.81) 72 h ethyl acetate 58.44±1.65 79.20±1.20 93.18±1.65 98.42±1.91 230.45 130.83 299.81 0.14a (676.51) (611.13) (767.98) acetone 61.12±1.40 80.10±1.33 96.54±1.21 100.00±0.00 179.87 66.42 253.45 1.95a (594.49) (532.79) (681.76) control, nil mortality; sd, standard deviation; lc50, lc90, lethal concentration at 50% and 90%; lfl, lower fiducidal limit; ufl, upper fiducidal limit; x2, chi-square value; df, degrees of freedom. mean values of five replicates. asignificant at p<0.05 level. jear_2014_3_hrev_master 16/12/14 10:38 pagina 92 no nco mm er cia l u se on ly [journal of entomological and acarological research 2014; 46:1747] [page 93] article table 2. larvicidal activity of hyptis suaveolens against the third instars larvae of anopheles culicifacies. exposure solvents percentage larval mortality±sd lc50 95% confidence limit x2 (time) concentration of h. suaveolens (ppm) (lc90) lfl ufl (df=4) 250 500 750 1000 lc50 (lc90) lc50 (lc90) hexane 41.15±0.94 54.59±1.23 65.20±1.13 77.18±1.19 423.00 262.74 527.13 0.06a (1431.91) (1195.44) (1920.40) diethyl ether 46.33±1.89 57.16±0.88 68.15±0.92 83.14±1.93 347.50 171.86 454.55 0.72a (1292.15) (1094.04) (1682.95) 24 h ethyl acetate 50.60±1.51 67.24±0.85 72.13±1.80 88.15±1.37 236.58 22.09 357.75 1.83a (1138.49) (973.62) (1454.65) acetone 52.15±0.88 69.10±1.46 74.78±1.69 90.77±0.98 217.24 15.04 334.37 2.00a (1049.27) (907.98) (1305.85) 367.14 191.69 474.69 hexane 43.18±0.56 58.14±0.84 68.93±0.92 79.50±1.25 (1343.81) (1130.97) (1772.94) 0.08a 284.56 86.93 399.26 diethyl ether 48.50±1.16 63.20±0.91 70.18±0.79 86.00±1.77 (1207.01) (1027.61) (1555.88) 1.19a 48 h 219.38 9.19 339.44 ethyl acetate 51.65±1.27 68.15±1.85 75.21±1.54 89.14±0.69 (1081.48) (931.45) (1360.06) 1.00a 196.53 4.99 309.95 acetone 54.01±0.85 70.36±0.78 79.36±1.10 92.56±0.75 (962.17) (841.05) (1170.61) 0.91a hexane 47.10±1.27 65.48±1.35 77.23±1.16 84.78±1.68 268.78 80.81 380.42 0.51a (1134.84) (977.06) (1426.91) diethyl ether 57.03±0.78 79.85±1.18 85.55±0.77 98.32±0.88 164.33 7.30 262.41 3.56a (751.50) (668.80) (875.67) 72 h ethyl acetate 70.01±0.96 97.36±0.89 100.0±0.00 100.0±0.00 158.80 56.706 211.84 0.04a (382.51) (339.78) (452.69) acetone 75.13±0.70 98.56±0.00 100.00±0.00 100.00±0.00 138.30 6.89 195.95 0.01a (349.48) (307.90) (421.39) control, nil mortality; sd, standard deviation; lc50, lc90, lethal concentration at 50% and 90%; lfl, lower fiducidal limit; ufl, upper fiducidal limit; x2, chi-square value; df, degrees of freedom. mean values of five replicates. asignificant at p<0.05 level. table 3. larvicidal activity of leucas aspera against third instars larvae of anopheles culicifacies. exposure solvents percentage larval mortality±sd lc50 95% confidence limit x2 (time) concentration of l. aspera (ppm) (lc90) lfl ufl (df=4) 250 500 750 1000 lc50 (lc90) lc50 (lc90) hexane 31.12±1.17 47.12±1.45 62.14±1.88 74.18±1.25 559.77 464.21 643.56 0.09a (1400.80) (1203.49) (1758.85) diethyl ether 44.50±1.20 52.43±1.94 65.36±1.70 75.27±1.33 401.56 198.85 520.40 0.22a (1549.31) (1259.51) (2219.35) 24 h ethyl acetate 46.66±1.32 65.16±1.80 69.52±1.57 87.55±0.88 299.71 126.24 405.43 2.48a (1157.96) (996.97) (1455.63) acetone 49.15±1.50 66.98±1.57 71.55±1.36 89.26±1.44 263.01 77.46 373.42 2.56a (1108.72) (956.98) (1387.02) 426.33 279.40 524.96 hexane 37.92±0.95 54.35±1.65 68.12±1.30 77.10±1.65 (1377.58) (1163.25) (1800.20) 0.14a 312.92 131.45 421.96 diethyl ether 45.42±0.77 61.72±1.10 73.50±1.55 82.64±1.93 (1223.97) (1044.38) (1568.28) 0.16a 48 h 253.69 81.55 359.15 ethyl acetate 48.16±1.13 69.41±1.72 76.92±1.42 89.18±1.18 (1038.54) (906.06) (1269.33) 0.95a 183.68 22.88 284.62 acetone 51.75±1.95 78.56±1.23 87.30±1.22 93.48±1.35 (824.85) (732.59) (967.34) 2.14a hexane 45.60±1.26 61.54±0.90 73.81±1.18 87.36±1.44 325.57 178.16 420.93 0.38a (1119.70) (974.76) (1374.18) diethyl ether 52.76±1.17 79.30±1.29 91.10±1.94 94.75±1.20 183.77 39.44 276.83 2.18a (761.60) (680.09) (881.77) 72 h ethyl acetate 63.15±0.92 86.25±1.12 96.63±1.61 100.00±0.00 144.51 14.56 225.14 0.63a (560.83) (499.23) (646.87) acetone 88.80±1.13 93.24±1.50 100.00±0.00 100.00±0.00 138.24 11.798 211.30 2.54a (478.13) (423.46) (557.32) control, nil mortality; sd, standard deviation; lc50, lc90, lethal concentration at 50% and 90%; lfl, lower fiducidal limit; ufl, upper fiducidal limit; x2, chi-square value; df, degrees of freedom. mean values of five replicates. asignificant at p<0.05 level. jear_2014_3_hrev_master 16/12/14 10:38 pagina 93 no nco mm er cia l u se on ly [page 94] [journal of entomological and acarological research 2014; 46:1747] it is known that placing h. suaveolens branches or whole plants in and around houses is one of the most effective methods in western kenya, for repelling the malaria vector anopheles gambiae giles (seyoum et al., 2002). kovendan et al. (2012b) reported on the effects of hexane, chloroform, ethyl acetate and methanol extracts of j. curcas against third instar larvae of c. quinquefasciatus, with lc50 values of 230.32, 212.85, 192.07 and 113.23 ppm, respectively. for h. suaveolens, lc50 values of these extracts were 213.09, 217.64, 167.59 and 86.93 ppm, respectively. for a. indicum, the lc50 values were 204.18, 155.53, 166.32 and 111.58 ppm, respectively. finally, for l. aspera, lc50 values were 152.18, 118.29, 111.43 and 107.73 ppm, respectively. similarly, our results with h. suaveolens against the third-instar larvae of a. culicifacies showed lc50 and lc90 values of hexane, diethyl ether, ethyl acetate and acetone extracts of (lc50) 423.00, 347.50, 236.58 and 217.24 ppm, and (lc90) 1,431.91, 1292.15, 1138.49 and 1049.27 ppm at 24 h, respectively. previous studies have been conducted using a methanol extract of clerodendron inerme and acanthus ilicifolius at different concentrations (20, 40, 60, 80 and 100 ppm), with lc50 values against a. stephensi first to fourth-instar larvae and pupae of 55.04, 63.33, 73.05, 80.74 and 74.33 ppm, and 52.76, 57.76, 63.36, 70.18 and 62.78 ppm, respectively. corresponding lc90 values were 125.50, 137.16, 153.55, 156.93, 199.20 ppm, and 108.30, 115.83, 125.24, 131.28 and 141.03 ppm, respectively (kovendan & murugan, 2011). mahesh kumar et al. (2012) reported the lc50 and lc90 values of s. xanthocarpum against the first to fourth instar larvae and pupae of c. quinquefasciatus as 155.29, 198.32, 271.12, 377.44, and 448.41 ppm, and 687.14, 913.10, 1,011.89, 1,058.85, and 1,141.65 ppm, respectively. in the present results, the hexane, diethyl ether, ethyl acetate and acetone extracts of l. aspera against third-instar larvae of a. culicifacies had lc50 values of 559.77, 401.56, 299.71 and 263.01 ppm, and lc90 values of 1400.80, 1549.31, 1157.96 and 1108.72 ppm at 24 h, respectively. conclusions the larvicidal properties of crude extracts of a. indicum, h. suaveolens and l. aspera against a. culicifacies were studied under laboratory conditions. the results clearly demonstrated the highest mortality with h. suaveolens, followed by l. aspera and a. indicum. in addition, the study also showed that the solvents used for the extractions have some impact on the level of larval mortality. the highest mortality was seen with acetone extract followed by ethyl acetate, diethyl ether and hexane. this mortality profile demonstrates the extraction properties of different solvents from which the maximum effect was obtained. we conclude that botanicals could be a better alternative than relying on the most hazardous currently used synthetic chemical insecticides and bio-pesticides, and could contribute to a healthier environment. the use of plant-based products could be an ideal ecoand user-friendly vector control strategy for diminishing and eventual elimination of the malaria burden in the near future. references abbott w.s., 1925 a method of computing the effectiveness of insecticides. j. econ. entomol. 18: 267-269. ahmed m., amin s., islam m., takahashi m., okuyama e., hossain c.f., 2000 analgesic principle from abutilon indicum. pharmazie. 55: 314. amerasinghe p.h., amerasinghe f.p., konradsen f., fonseka k.t., wirtz r.a., 1999 malaria vectors in a traditional dry zone village in sri lanka. am. j. trop. med. hyg. 60: 421-9. amusan a.a., idowu a.b., arowolo f.s., 2005 comparative toxicity effect of bush tea leaves (hyptis suaveolens) and orange peel (citrus sinensis) oil extract on larvae of the yellow fever mosquito aedes aegypti. tanzan. health. res. bull. 7: 174-178. ansari m.a., vasudevan p., tandon m., razdan r.k., 2000 larvicidal and mosquito repellent action of peppermint (mentha piperita) oil. biores. technol. 71: 267-271. azevedo n.a., campos l.f.p., ferreira h.d., 2001 chemical variability in the essential oil of hyptis suaveolens. phytochemistry 57: 733-736. chaudhury a., ghosh d., 1969 insecticidal plants: chemical examination of leucas aspera. j. indian chem. soc. 46: 95. chopra r.n., nayer s.l., chopra i.c., 1992 glossary of indian medicinal plants, 3rd ed. council of scientific and industrial research, new delhi, pp. 7-246. dua v.k., pandey a.c., alam m.e., dash a.p., 2006 larvicidal activity of hibiscus abelmoschus linn. (malvaceae) against mosquitoes. j. am. mosq. control. assoc. 22: 155-157. finney d.j., 1971 probit analysis. cambridge university press, london, pp. 68-78. kalachaveedu m., ghosh a., ranjan r., vedam venkat k., 2006 volatile constituents of leucas aspera (wilid.). j. essent. oil. res. 18: 104-5. kamar m., singh t.p., 1994 preliminary chemical examination of some compounds in the different parts of the genus leucas. geobios. 21: 31-33. karunamoorthi k., 2012 global malaria burden: socialomics implications. j. socialomics 1: e108. kitikar k.r., basu b.d., 1990 indian medicinal plants. in: bidtter e., caaius j.f., mhaskar k.s. (eds.). periodical experts book company, new delhi: 2019. kovendan k., murugan k., 2011 effect of medicinal plants on the mosquito vectors from the different agro-climatic regions of tamil nadu, india. adv. environ. biol. 5: 335-344. kovendan k., arivoli s., maheshwaran r., baskar k., vincent s., 2012a larvicidal efficacy of sphaeranthus indicus, cleistanthus collinus and murraya koenigii leaf extracts against filarial vector, culex quinquefasciatus say (diptera: culicidae) parasitol. res. 111: 1025-1035. kovendan k., murugan k., panneerselvam c., mahesh kumar p., amerasan d., subramaniam j., vincent s., barnard d.r., 2012b laboratory and field evaluation of medicinal plant extracts against filarial vector, culex quinquefasciatus say (diptera: culicidae). parasitol. res. 110: 2105-2115. mahesh kumar p., murugan k., kovendan k., subramaniam j., amaresan d., 2012 mosquito larvicidal and pupicidal efficacy of solanum xanthocarpum (family: solanaceae) leaf extract and bacterial insecticide, bacillus thuringiensis, against culex quinquefasciatus say (diptera: culicidae). parasitol. res. 110: 2541-2550. maheswaran r., sathis s., ignacimuthu s., 2008 larvicidal activity of leucus aspera (willd.) against the larvae of culex quinquefasciatus say. and aedes aegypti. int. j. int. biol. 2: 214-217. mangathayaru k., thirumurugan d., patel p.s., pratap d.v., david d.j., karthikeyan j., 2006 isolation and identification of nicotine from leucas aspera (willd). ind. j. pharm. sci. 68: 88-90. mishra a.k., chand s.k., barik t.k., dua v.k., raghavendra k., 2012 insecticide resistance status in anopheles culicifacies in madhya pradesh, central india. j. vect. borne. dis. 49: 39-41. peerzada n., 1997 chemical composition of the essential oil of hyptis suaveolens. molecules. 2: 165-168. promsiri s., naksathit a., kruatrachue m., tharava u., 2006 evaluations of larvicidal activity of medicinal plant extracts to aedes aegypti (diptera: culicidae) and other effects on a non target fish. insect. sci. 13: 179-188. article jear_2014_3_hrev_master 16/12/14 10:38 pagina 94 no nco mm er cia l u se on ly rahuman a.a., gopalakrishnan g., venkatesan p., geetha k., bagavan a., 2008 mosquito larvicidal activity of isolated compounds from the rhizome of zingiber officinale. phytother. res. 22: 1035-1103. russell p.f., rao t.r., 1942 on the ecology of larvae of anopheles culicifacies giles, in borrow-pits. bull. entomol. res. 32: 341-361. sabesan s., krishnamoorthy k., jambulingam k., rajendran g., kumar p.n., rajagopalan p.k., 1986 breeding habitats of anopheles culicifacies in rameshwaram island. ind. j. med. res. 84: 44-52. sadhu s.k., okuyama e., fujimoto h., ishibashi m., 2003 separation of leucas aspera, a medicinal plant of bangladesh, guided by prostaglandin inhibitory and antioxidant activities. chem pharm bull (tokyo). 51: 595-598. seyoum a., pålsson k., kung’a s., kabiru e.w., lwande w., killeen g.f., hassanali a., knots b.g.j., 2002 traditional use of mosquitorepellent plants in western kenya and their evaluation in semi field experimental huts against anopheles gambiae: ethnobotanical studies and application by thermal expulsion and direct burning. trans. r. soc. trop. med. hyg. 96, 225-231. sharma v.p., ansari m.a., 1994 personal protection from mosquitoes (diptera: culicidae) by burning neem oil in kerosene repellent action of neem. j. med. entomol. 3: 95-102. sharma s.k., dua v.k., sharma v.p., 1995 field study on the mosquito repellent action of neem oil. south asian j. trop. med. pub. health. 26: 180-182. sharma m., saxena r.c., 1996 sphaeranthus indicus as a mosquito larvicidae. j. appl. zool. res. 7: 87-88. singh s.p., ragavendhira k., singh r.k., subbarao s.k., 2002 studies on larvicidal prosperities of leaf extract of solanum nigrum linn (family: solanaceae). curr. sci. 81: 1529. subbarao s.k., nanda n., raghavendra k., 1999 malariogenic stratification of india using anopheles culicifacies sibling species prevalence. icmr bulletin. 29: 75-80. who., 1996 report of the who informal consultation on the evaluation on the testing of insecticides. ctd/who pes/ic/96.1. world health organisation, geneva, p 69. who., 1998 test procedures for insecticide resistance monitoring in malaria vectors. bio-efficacy and persistence of insecticides on treated surfaces. report of the who informal consultation. geneva who/cdc/mal/ 98.12. world health organisation, geneva. yoganarasimhan s.w., 2000 medicinal plants of india. cyber media, bangalore, pp. 10-11. [journal of entomological and acarological research 2014; 46:1747] [page 95] article jear_2014_3_hrev_master 16/12/14 10:38 pagina 95 no nco mm er cia l u se on ly jear2012 abstract understanding the dispersion pattern of a species is an important pre-requisite for developing an effective pest management program. in this study, four hundred wheat plants were surveyed for sitobion avenae twice a week during 2010 and 2011 growing seasons in two fields of badjgah (fars province) in iran. in each field only one of the two cultivers of bahar or shiraz was planted. analysis of spatial distribution pattern using taylor’s power law and iwao’s regression model showed that s. avenae exhibited an aggregated distribution on wheat. taylor’s power law was estimated from 84 data sets and fitted the data better than iwao’s regression model. the optimal sample sizes needed for fixed precision levels of 0.25 and 0.30 were estimated using taylor’s regression coefficients, and the required sample sizes increased dramatically with increased levels of precision. therefore, the samplingplan we presented here should be used as a tool for an efficient estimation of s. avenae population density in wheat fields for pest management decision. introduction approximately 463,000 ha of winter wheat triticum aestivum l., are annually planted in fars province (iran) annually (kherad, 2013). the english grain aphid sitobion avenae (fabricius) (hemiptera: aphididae) is regarded as one of the most important aphids of cereals in this region (hodjat & azmayesh fard, 1986) and causes damage by sap feeding it is also a vector of barley yellow dwarf virus (bydv) which may result in significant yield losses (williams & wratten, 1987). feeding by adult and nymphs of s. avenae before the flowering stage can result in reduceing the number of grains in the ear. after flowering to the end of grain filling, it reducing directly the size of the grain (hodjat & azmayeshfarrd, 1986). this species is more cold hardy than r. padi, and thus has a more significant role in the secondary spread of bydv in winter cereals (williams & wratten, 1987). dispersion and abundance of organisms are the most important properties of insect population and essential ecological properties of species (siswanto et al., 2008). knowledge about dispersion pattern of an organism is required for understanding population biology, resource exploitation and dynamics of biological control agents (fauvergue & hopper, 1994). it provides a better understanding of the relationship that exists between organism and its environment which may be helpful in planning efficient sampling programs for population estimates, development of population models and pest management strategy (soemargono et al., 2008). there are many methods used to describe the dispersion of arthropod populations, but most estimates are based on sample means and variances (bisseleua et al., 2011), while the relationships between the variance and mean are used as indices of aggregation (arnaldo & torres, 2005). the models of taylor and iwao also depend on the relationship between the sample mean and the variance of insect numbers per sampling unit. the slope of the regression model is used as an index of aggregation. designing sampling plans based on these indicators has been reported to reduce sampling effort, cost and minimize variation of sampling precision (kuno, 1991; payandeh et al., 2010). despite the fact that fars province has the first rank of wheat production in iran (kherad, 2013), and economic importance of s. avenae to wheat growers, little is known about its dispersion in iran. thus, there is an urgent requirement for such information as it will provide wheat pest managers, researchers, and farmers with a cost-effective sampling method for s. avenae. therefore, this study was undertaken to determine dispersion pattern of s. avenae in order to develop a suitable sampling plan for the pest. correspondence: maryam aleosfoor, department of plant protection, college of agriculture, shiraz university, shiraz, iran. e-mail: aosfoor@shirazu.ac.ir key words: spatial distribution, sitobion avenae, taylor’s power law, sequential sampling, iwao’s regression model. acknowledgments: we would like to express our sincere gratitude to dr. mohiseni for generous assistance with various aspects of this project. this study was supported by the grants from shiraz university, iran. received for publication: 14 july 2013. revision received: 13 october 2013. accepted for publication: 16 october 2013. ©copyright v. soltani ghasemloo and m. aleosfoor, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e22 doi:10.4081/jear.2013.e22 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. dispersion pattern and fixed precision sequential sampling of sitobion avenae (fabricus) (hemiptera: aphididae) in wheat fields of badjgah (fars province) in iran v. soltani ghasemloo, m. aleosfoor department of plant protection, college of agriculture, shiraz university, shiraz, iran [page 120] [journal of entomological and acarological research 2013; 45:e22] journal of entomological and acarological research 2013; volume 45:e22 no n c om me rci al us e o nly materials and methods study site and population sampling the study was carried out from march 2010 to june 2011 at two pesticidefree rectangular wheat fields at badjgah region, fars province (n 52’42’’ e 29’50’’). each field has an area of 2 hectares. in each field, one of the two cultivars, shiraz and bahar, were planted separately and agronomic practices, such as application of manure, were given to wheat fields at regular intervals. the fields were sampled 2 days per week throughout the growing season (from initiation of tillering till grain ripening stage), unless rainfall increased intervals between sampling dates. tillers were collected by traveling a x-shaped procedure and the data from primary sampling were then used to develop sample size for the english grain aphid using formula (1) described by karandinos (1976): (1) where n is the number of samples (each sample contains 5 tillers), d is the precision level, zα/2 is the value of z distribution for the desired significance level (in our case α= 0.1), s2 and m are variance and mean respectively. determination of the appropriate sample unit then, the most appropriate sample unit was estimated by calculating the relative variation (rv) using formula (2): (2) sampling efficiency also was calculated as the relative net precision (rnp) using formula (3): (3) where rv, se, and cs are the relative variation, standard error of mean, and the cost in minutes to count aphid abundance on an individual sample unit, or mean search time (pedigo et al., 1972; karandinos 1976; zar, 2010 missing in ref list; hall et al., 1991; buntin, 1994). taylor’s power law taylor’s power law (tpl) discribes the regression between logarithm of population variance and logarithm of population mean according to the following equation: (4) where s2 is the population variance, is the population mean, α is the y-intercept and b is the slope of the regression line;. b<1, b=1 and b>1 indicate uniform, random and aggregated spatial patterns, respectively (southwood, 1978; taylor, 1984; davis, 1994). iwao’s method the iwao’s patchiness regression method quantifies the relationship between the mean crowding index (m*) and the mean (m) by the following formula: m* = α + βm (5) where m was determined as [m(s2/m-1)]. the intercept (α) is the index of the basic component of a population or basic contagion (where α<1, α=1, and α>1 represent regularity, randomness, and aggregation of populations in spatial patterns, respectively), and the slope (β) is the density contagiousness coefficient interpreted in the same manner as b of taylor’s regression (sule et al., 2012). test for significant difference between regression coefficients (b index) from 1 was calculated by the following formula: (6) where slope and se slope were taylor’s coefficient and its standard error in regression equations, respectively. the amount of calculated t was compared with t value given in the table, the degrees of freedom is (n–1). if the absolute value of calculated t was greater than the value given in the table, then the spatial distribution of the aphid was aggregation (feng & nowierski, 1992). presence or absence of difference between cultivars were calculated based on formula (7) with (n1+n2)-2 degrees of freedom (feng & nowierski, 1992): (7) where b1 and b2 were taylor’s coefficient of two cultivars and se1 and se2 were their standard errors. constructing fixed percision sampling schemes based on the sample counts, the optimal sample sizes (n) was calculated with a and b from taylor’s power law to develop the enumerative sampling plan by green (1970), with precision levels of 0.15, 0.25 and 0.3 for ecological and pest management purpose, as recommended by green (1970), using the following formula: (8) where n is the number of sample unit required to estimate the mean number of aphids, d is a desired precision and a and b are the taylor’s power law coefficients. the sampling stop line was calculated as suggested by elliott et al. (2003) using the following formula: (9) where tn is the cumulative number of aphids in a sample of n sample units and defines the sequential sampling stop line. sample size curves and sequential sampling stop lines were generated by a computer program in excel. the coefficients of taylor’s power law were estimated by linear least square regressions using proc reg (sas, 1999) on the linearized version of tpl. using green’s method, the resampling for validation of sampling plans (rvsp) program was used to validate the sequential sampling plans of s. avenae (naranjo & hutchison, 1997; o’rourke & hutchison, 2003). rvsp requires the use of independent data sets for validation. thus, 15 data sets representing a range of low, medium, and high densities were selected at random from both the 84 s. avenae data sets to serve as validation data sets. resampling was repeated 500 times for each data set, producing the average, minimum and maximum precision level and the average, minimum and maximum sample size (naranjo & hutchison, 1997). then, the numbers of samples in conventional method and green’s method were compared (shahrokhi & amir-maafi, 2011; mohiseni et al., 2009). wilson and room’s model to describe the relationship between the proportion (p) of sampling units (tillers) with >0 s. avenae individuals and the mean number of individuals per sampling unit, the equation of wilson & room (1983) was used: [journal of entomological and acarological research 2013; 45:e22] [page 121] article no n c om me rci al us e o nly [page 122] [journal of entomological and acarological research 2013; 45:e22] (10) where a and b are taylor’s estimates. this p(i) equation can be used for predicting the mean number of individuals of a given species per sampling unit ( ) from a simple count of the proportion of sampling units in which this species is present (p). results determination sample size and sample unit in all cases, levels of precision (d values) decrease as the mean increases. despite the fact that precision is improved with an increase in the sample sizes, gains in precision become minor at high sample sizes. since, the level of the precision needed is a choice made based on the purpose of a sampling plan, according to facilities, capabilities and time, in the precision levels of 0.25 and 0.3, one hundred plants (500 tillers) were sampled from each (diagonal) line of the fields (figure 1). the results of rv and rnp analyses indicated that the best sample unit was 4 or 5 tillers per wheat plant (table 1). according to rv analyses, there wasn’t any significant difference between 4 and 5 tillers. considering that the lower rv showed more precise and lower error, 4 stems was selected. distribution pattern the distribution patterns of s. avenae on t. aestivum were established in accordance with taylor’s and iwao’s indices of dispersion. the result of the current study reveals the dispersion patterned of s. avenae to be highly aggregated within t. aestivum (figures 2 and 3). taylor’s power law analysis appeared to illustrate the distribution of s. avenae well by showing highly significant relationships between the variance and mean of s. avenae population (figure 2). the slope values of taylor’s power law for this aphid was found to be significantly greater than 1 for shiraz (t=8.12, df=133, p<0.0001) and bahar cultivars (t=8.5, df=126, p<0.0001), indicating an aggregated or clumped distribution pattern for s. avenae on t. aestivum. on the contrary, iwao’s patchiness regression based on the same sampled tillers did not show high significant relationship between the mean crowding index (m*) and the mean (m) of s. avenae (figure 3). although, the constant α in the iowa’s model indicates the tendency to crowding when it is positive (+) or repulsion when it is negative (-) as it is the index of basic contagion defined by iwao (1968). based on the higher value of r2 made by taylor’s power law compared to iwao’s patchiness regression, it could be expressed that taylor’s model fitted the data better than iwao’s model. furthermore, taylor’s power law provides a more even distribution of the points along the line than iwao’s model. in spite of iwao’s model inability to fit the data very well, it could still give an insight into the interpretation of implication of ecological parameters (kuno, 1991). for instance, the positive value of α of iwao’s patchiness regression in the present study is indicative of a mutual attraction (positive interaction) between the individuals even at a low density. the heterogeneity of slopes regression model indicated that neither the slope nor the intercept of power law regressions differed signifiarticle figure 1. sample sizes with different precision levels for s. avenae in wheat fields of badjgah. figure 2. regression analysis of taylor’s power law for s. avenae populations on t. aestivum; a) shiraz cultivar, b) bahar cultivar. no n c om me rci al us e o nly cantly for the two wheat cultivars (slope, df=99, t=1.06, intercept, df=99, t=1.28). in spite of this observation, taylor’s indices for two wheat cultivars were calculated together. constructing fixed percision sampling schemes the relationship between the cumulative number of aphids and number of sample taken for the fixed precision levels of 0.25 and 0.30 and the stop lines for sequential sampling is showed in figure 4. since the variance mean regression in taylor’s model provided a good description of the data (figure 2), the regression variability would only have a minor effect at very low mean density. in order to achieve high fixed precision levels of 0.15 for precise number of sample taken, quite a large number of samples are required (figure 4). for example in 15 sample plants (with four tillers) in dexp=0.15, dexp=0.25 and dexp=0.3, 290, 118 and 46 aphids will be observed, respectively. validation of green’s model was evaluated using rvsp software. from the result of the present study, in precision level of 0.15, this program could not run. resampling analysis for s. avenae with precision set at 0.25 resulted in an average sample number of 111 plants, ranging from 359 (0.06 aphids per sample unit) to 24 (1.46 aphids per sample unit). in precision level of 0.3, the average number of 78 samples ranged from 253 (0.07 aphids per sample unit) to 17 (1.48 aphids per sample unit) (figure 5, table 2). comparing number of samples in conventional methods with green’s method indicated that in precision levels of 0.25 and 0.3 the number of needed samples in green’s method compared to conventional one was reduced by 79.5 and 66 percent, respectively (table 3). wilson and room’s model equations for the wilson and room model (based on a and b values calculated from taylor’s power law) are described by hyperbolic curves (figure 5). according to the p-x relation, when 50% of the sampling units (4 stems) contain aphids, the mean number of aphids/ sampling unit is approx. 1 (figure 6). as can be seen based on wilson and room’s model (1983), with the increase percentage of infected plants in the field, the number of required samples decreases to. for example, in s. avenae, when the proportion of infection was 0.5, in decision levels 0.15, 0.25 and 0.3 the sample’s number was 143, 29 and 20, respectively (figure 7). discussion and conclusions since evaluation of the spatial distribution pattern is a key element in pest management strategies, two methods of taylor and iwao were tested for s. avenae on t. aestivum. according to hutchison et al. (1988), both of these two regression models can estimate insect population distribution parameters. in this research, taylor’s power law [journal of entomological and acarological research 2013; 45:e22] [page 123] article figure 3. regression analysis of iwao’s mean crowding index (m*) on mean density (m) for s. avenae populations on t. aestivum; a) shiraz cultivar, b) bahar cultivar. table 1. results of relative variation and relative net precision analysis for s. avenae in wheat fields of badjgah. analysis cultivar 1 stems 2 stems 3 stems 4 stems 5 stems rv shiraz 55.69c 44bc 40.7c 35.1d 30.8d bahar 50.90a 38.7b 32.9c 29.7d 28.2d rnp shiraz 4.1a 3.6b 3.4bc 3.2cd 3d bahar 4.3a 4a 3.8c 3.5cd 3.3d a,b,c,dmeans within a row followed by the same letter are not significantly different at the 5% confidence level according to duncan’s studentized range test. rv, relative variation; rnp, relative net precision. no n c om me rci al us e o nly [page 124] [journal of entomological and acarological research 2013; 45:e22] article table 2. results of validation by resampling for validation of sampling plans software for d=0.25 and d=0.30 for s. avenae in wheat field of badjgah. number meanobs mean d (in simulation model) number of sample of data population mean higher lower mean higher lower 0.25 0.03 0.25 0.03 0.25 0.03 0.25 0.03 0.25 0.03 0.25 0.03 0.25 0.03 1 0.06 0.06 0.07 0.27 0.32 0.30 0.37 0.24 0.27 359 253 670 623 140 105 2 0.09 0.10 0.10 0.24 0.29 0.28 0.33 0.20 0.25 250 177 418 338 118 75 3 0.11 0.12 0.12 0.23 0.27 0.27 0.33 0.20 0.22 210 147 344 310 109 66 4 0.18 0.20 0.21 0.32 0.38 0.40 0.51 0.23 0.24 141 10 320 253 49 31 5 0.20 0.23 0.24 0.32 0.38 0.41 0.48 0.21 0.26 127 91 251 209 48 21 6 0.26 0.27 0.28 0.26 0.31 0.31 0.36 0.21 0.24 105 73 193 168 42 28 7 0.38 0.42 0.43 0.28 0.33 0.34 0.44 0.20 0.21 72 52 137 108 33 20 8 0.44 0.47 0.48 0.25 0.30 0.34 0.41 0.19 0.21 65 45 106 91 32 19 9 0.49 0.54 0.53 0.24 0.29 0.03 0.37 0.17 0.20 57 42 96 83 29 20 10 0.58 0.63 0.63 0.26 0.31 0.35 0.43 0.20 0.21 51 37 93 72 26 15 11 0.93 0.97 0.97 0.20 0.24 0.29 0.38 0.13 0.15 34 24 57 38 21 13 12 01.03 01.10 01.13 0.25 0.30 0.32 0.41 0.16 0.17 31 22 59 44 15 9 13 01.14 01.18 01.23 0.24 0.28 0.31 0.39 0.17 0.18 29 20 49 45 14 10 14 01.42 01.46 01.48 0.17 0.20 0.24 0.28 0.10 0.10 24 17 35 26 16 10 mean 0.52 0.55 0.55 0.25 0.29 0.31 0.38 0.18 0.20 111.7 78.56 202 171.99 49.4 31.57 table 3. number of samples of s. avenae using green’s method compared to conventional methods used in badjgah. precision level conventional method green reduction of sample number lower higher mean lower higher mean lower higher mean 0.25 73.1 6146 843 12 93 52 71.2 85.9 79.5 0.3 50.7 4268 585.4 9 83 46 0.55 88.4 66 figure 4. sampling stop line at a fixed precision level of 0.15, 0.25 and 0.30 for s. avenae on triticum aestivum. figure 5. summary of resembling validation analysis showing range of s. avenae densities over number of sample taken for green’s sequential sampling plan. no n c om me rci al us e o nly analysis appeared to illustrate the distribution of s. avenae better by showing highly significant relationships between the variance and mean of s. avenae population. this result corroborates the previous finding by eliot & kieckhefer (1987), who showed that taylor’s power law showed the spatial distribution of s. avenae, rhopalosiphum padi, r. maidis and schizaphis graminum better than iwao. in addition, other results similar to ours were reported by previous studies (dean & luring, 1970; feng & nowierski, 1992; burgio et al., 1995; elliot & kieckhefer, 1987; athanassiou et al., 2005; kavallieratos et al., 2002, 2005; fievet et al., 2007; tomanovic et al., 2008a; afshari & dastranj, 2010), on other aphid species, s. avenae, r. padi, r. maidis and schizaphis graminum, metopolophium dirhodum, d. noxia and myzus persicae. many authors have reported that an aggregated distribution pattern is a predominant form of arthropod distribution and regular distribution is rare and mainly found in the population where there is a strong competition among individuals (argov et al., 1999). the aggregated distribution pattern displayed by s. avenae in the present study might be attributed to food source, since s. avenae was reported to be more attracted to the ear and upper leaves of cereals for feeding (gianoli, 2000) and, or to some variations of the environment such as microclimate and natural enemies (gianoli, 2000; tomanovic et al., 2008b; elliott & kieckhefer, 2000). sequential sampling models, due to its high accuracy, lower costs and faster decisions, have a special importance in the study of insect populations (binns, 1994; pedigo & zeiss, 1996; young & young, 1998). comparing number of samples in conventional methods with green’s method indicated that in precision levels of 0.25 and 0.3, the number of needed samples in green’s method was reduced 79.5 and 66 percent, respectively compared to conventional one. this result is corroborated by the result of several authors (mohiseni et al., 2009; afshari, 2009; pieters & sterling, 1975; shahrokhi & amir-maafi, 2011). validation of green’s model was evaluated using rvsp software. similar density-based, fixedprecision sequential sampling plans have been developed and validated using the resembling approach (naranjo & hutchison, 1997) for several insect species, including: macrosteles quadrilineatus (o’rourke et al., 1998), cryptolestes ferrugineus (subramanyam et al., 1997), acaymma vittatum (burkness & hutchison, 1998), leptinotarsa decemlineata (hamilton et al., 1998), eurygaster integriceps (mohiseni et al., 2009) and schyzaphis graminum (afshari & dastranj, 2010). elliott et al. (2003) examined spatial distribution of s. avenae in south dakota in 1993. they stated the number of samples 40-250 in precision d=0.25 (elliott et al., 2003), while the results of this study showed 24-350 samples. this difference depends on the extent of the variation in relation to sampling scheme. this approach illustrates that, when adequate independent data set are used for validation, the final sequential sampling plans can be used with confidence to ensure that the desired fixedprecision levels are achieved. in our study, taylor’s slope values showed an aggregated distribution pattern among sampling units. this aggregation of high numbers of individuals in a relatively low number of sampling units reduces the precision obtained in estimating mean insect density. determining the proportion of leaves with >0 individuals can be considered as an alternative for estimating the mean number of aphid directly. thus, if a specific threshold is established, based on the given mean density value, this mean can be predicted by simple presence/absence characterization of the samples, without counting the individuals found. hence if this ratio can be accurately predicted from the prelation, insecticidal applications should be done when necessary (wilson & room, 1983). our data suggest that wilson and room’s model are useful and save time and cost. based on this model, by increasing the percentage of infected plants in the field, the number of required samples reduced. this result is in accordance with results of athanassiou et al. (2005) on myzus persicae and macrolophus costalis. a sampling based management strategy in wheat is essential under the establishment of certain thresholds, which can vary among countries, pest species, plant varieties and so forth. the determination of these thresholds would encourage wheat farmers or managers to follow a sampling-based control strategy, under the principles of integrated pest [journal of entomological and acarological research 2013; 45:e22] [page 125] article figure 6. relation between the proportion of sampling units (4 stems) that had one or more (i.e. >0) individuals of aphids, and the mean number of aphids per sample unit. figure 7. number of samples required for estimating the population density of s. avenae in precision levels of 0.15, 0.25 and 0.3 in the fields of badjgah based on wilson and room’s model. no n c om me rci al us e o nly [page 126] [journal of entomological and acarological research 2013; 45:e22] management. the findings of this study will go a long way in reducing the problem faced by farmers on decision-making with respect to pest. references afshari a., soleiman-negadian e., shishebor p., 2009 population density and spatial distribution of aphis gossypii glover (homoptera: aphididae) on cotton in gorgan, iran. j. agric. sci. technol. 11: 27-38. afshari a., dastranj m., 2010 density, spatial distribution and sequential sampling plans for cereal aphids infesting wheat spike in gorgan, northern iran. plant protection. sci. j. agric. 32: 89-102. argov y., rossler y., voet h., rose d., 1999 spatial dispersion and sampling of citrus whitefly, dialeurodes citri, for control decisions in citrus orchards. agric. for. entomol. 1: 305-318. arnaldo p.s., torres l.m., 2005 spatial distribution and sampling of thaumetopoea pityocampa (den. & schiff.) 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[journal of entomological and acarological research 2013; 45:e22] [page 127] article no n c om me rci al us e o nly jear2012 abstract the effects of tricyclazole treatments on benthic macroinvertebrates in the field and in laboratory were studied. in field conditions, low density of benthic populations was observed, both in treated and untreated plots, which was attributed to the short period of submersion of the rice field and high water temperature, fungicide treatments had no significant effect. both laboratory acute toxicity test and a test using a mesocosm suggested a low toxicity of tricyclazole on invertebrates. a reduction of the macroinvertebrate density was observed only when tricyclazole concentration was applied at concentrations 100 times the ones tested in the field, acute toxicity test gave an lc50 after 48 h of 26 mg*l–1, in agreement with data obtained for other species. introduction rice paddy fields, as man modified ecosystems derived from natural wetlands (odum & barret, 2005), are characterized by periodic floods and droughts and become an habitat suitable for some aquatic invertebrates. macroinvertebrates give a significant contribution to the ecosystem biodiversity and are a common component of rice field fauna (leitão et al., 2007). however, the species diversity in this human modified system and its reduction in relation to water management, addition of fertilizers and pesticides treatments has been poorly investigated. natural ecosystems evolve toward an increase in diversity accompanied by a decrease in production (odum & barret, 2005). this is exactly the opposite of agriculture ecosystems, where the aim is to enhance productivity with a consequent decrease in biodiversity. the management of water inflow and outflow has a huge effect on benthic macroinvertebrates, since many species cannot tolerate temporary absence of soil submersion, as well as herbicide, insecticide and fungicide treatments could cause density reduction or selective elimination of some sensitive species (stener et al., 2009). in italy, among chemicals employed in rice paddy fields, tricyclazole is widely used to manage rice blast epidemics, caused by the fungus magnaporthe oryzae (bertocchi et al., 2007; cortesi, 2011). at present the main effects of tricyclazole on rice blast are relatively well known (kunova et al., 2013), whereas the information about its side effects on benthic macroinvertebrates in rice fields is scarce, and only few studies focused on the impact of tricyclazole on aquatic macroinvertebrates (simpson & rogert, 1991; faria et al., 2007; suarez-serrano et al., 2010; hayasaka et al., 2012; rossaro et al., 2013; tsochatzis et al., 2013). little information about tricyclazole acute toxicity is also available (encarna et al., 2009; tsochatzis et al., 2013). the application of a fungicide could interfere with aquatic macroinvertebrate metabolism, for example with chitin synthesis (grigarick et al., 1990), or it could alter biological interactions between different components of the community (competitors, predators). the aim of the present research was to assess acute toxicity of tricyclazole on chironomus riparius and to compare the effect of tricyclazole in a mesocosm and in a rice field treated with different concentrations of tricyclazole. material and methods the rice paddy field, where the study was carried out, is located at poiago farm in carpiano (milan, italy). the experimental design consisted of 5 plots, each of 42 m2, inside a field with continuous water supply (figure 1a). each plot was delimited by soil dykes and had independent inlet water supply and no outlet to avoid cross contamination. plots were sown in dry soil, with oryza sativa subsp. japonica cv sirio, and the crop was submerged at 3 leaf-tillering phenological stage. fungicide treatments were randomly assigned to plots. tricyclazole was applied as commonly used to manage rice blast epidemics, i.e. two treatments at 300 g*ha–1 of the commercial fungicide beam das wp (lilly research lab., eli lilly and co., indianapolis, in, usa) (tricyclazole 75%) and as single treatments at 600 and 1200 g*ha–1 (table 1). correspondence: bruno rossaro, department of food, environmental and nutritional sciences, university of milan, via celoria 2, 20133 milan, italy. tel.+ +39.02.5031.6734 fax: +39.02.5031.6748. e-mail: bruno.rossaro@unimi.it key words: rice field, fungicide, benthic macroinvertebrates, tricyclazole, ecotoxicology, bioassay. acknowledgements: research supported in part by dow agrosciences italia s.r.l. received for publication: 24 june 2013. revision received: 22 august 2013. accepted for publication: 24 september 2013. ©copyright b. rossaro and p. cortesi, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e23 doi:10.4081/jear.2013.e23 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. the effects of tricyclazole treatment on aquatic macroinvertebrates in the field and in laboratory b. rossaro, p. cortesi department of food, environmental and nutritional sciences, university of milan, italy [page 128] [journal of entomological and acarological research 2013; 45:e23] journal of entomological and acarological research 2013; volume 45:e23 no n c om me rci al us e o nly fungicide aqueous solutions were sprayed on the crop at 500 l*ha–1 with the motorised backpack sprayer fox f320 with a hand-held 1.5 m boom, operating at 500 kpa. in 2011, benthic macroinvertebrates were sampled with a cylindrical core sampler, 7 cm in diameter. four random replicate samples were collected within each plot. plots 1 and 5 were used as controls and were sampled at 6 dates; two controls allow estimation of the variability between plots, not bound to treatment. treatments were applied to plots 2-3-4; sampling was done immediately before the fungicide treatment and the sampling in each treated plot continued weekly for 2 weeks in plot 3 and 4, while in plot 2 two treatments were applied on the 14th of july and on the 4th of august, and sampling continued for 5 weeks after the first treatment so that it finished 2 weeks after the second treatment (table 1). in 2012, the second year of study, the core sampler was replaced by a square hand net; 30 cm of size, with a net mesh of 300 micron, the net was drawn over the submerged soil, making a low pressure. this sampling technique is not as quantitative as the core sampler, but collects macroinvertebrates on a larger surface, allowing the capture of a larger numbers of specimens. because of the two different sampling tools, a comparison between the two years is biased, however, transformation of counts to in m–2 allowed a rough comparison between years. for each species, the number of specimens found in each sample was counted. the counts in the core sampler were then multiplied by, having the core 7 cm of diameter, whereas the counts of the samples collected with the hand net were multiplied by 3.33, assuming that 1/3 of square meter surface was sampled. a total of 134 samples were studied: 104 samples in 2011: 4 replicates * 3 plots * 2 dates + 4 replicates * 5 plots * 4 dates, and 30 samples in 2012: 5 plots at 6 sampling dates. the two untreated plots allowed an estimation of the variance not bound to treatment. in both years, samples were collected early in the morning, and immediately after brought to the laboratory. at the same day the samples were separated into different fractions with sieves of 10000, 1000, 500 and 300 micron mesh size. benthic macroinvertebrates obtained from each fraction were separated from detritus with the aid of a stereomicroscope, transferred into eppendorf tubes and fixed in alcohol 75° or in formol 4% (oligochaetes). because of the low numbers of specimens found, the whole sample was always examined without subsampling. the specimens were counted under the stereomicroscope at 30 x magnification and identified to species level using different identification keys (campaioli et al., 1994, 1999; timm, 2009; wiederholm, 1983); the species identification often required the preparation of permanent slides examined with the aid of an optical microscope at high magnification (1000 x). water temperature (°c) was measured in the field with a digital thermometer at sampling. water samples were collected in acidcleaned graduated bottles for chemical analysis by standard methods (apha, 2005). the following analyses were performed in laboratory on the same day or the day after: dissolved oxygen (mg l–1) determined with winkler method, conductivity (µscm–1), ph, alkalinity (mg l–1 of caco3)measured by titrating with hcl until color change of methyl red, bromocresol green, total phosphorous (tp) concentration was measured with the phosphomolybdic method. two tanks with a capacity of 20 l were used for the mesocosm test. the sediment for the mesocosm test was obtained adding about 2 kg (dry weight) of soil, collected from the carpiano rice field. the two tanks were prepared on 8/5/2012; one was used as control, the other for the experiment, where an aqueous suspension of tricyclazole was added on two dates, 8/5/2012 and 15/5/2012. the quantities added were 0.266 g of beam das at 8/5/2012 and 1.333 g at 15/5/2012, corresponding to 0.2 and 1 g of tricyclazole, to have a concentration in [journal of entomological and acarological research 2013; 45:e23] [page 129] article figure 1. a) spatial disposition of treatment plots, b) water temperature in the investigated periods. table 1. dates of treatment. 14/7/2011 1° sampling 1° treatment on plot 2 21/7/2011 2° sampling 28/7/2011 3° sampling 4/8/2011 4° sampling 2° treatment on plot 2, 1° treatment on plots 3, 4 11/8/2011 5° sampling 22/8/2011 6° sampling 11/7/2012 1° sampling 1° treatment on plot 2 19/7/2012 2° sampling 30/7/2012 3° sampling 2° treatment on plot 2 1° treatment on plots 3, 4 6/8/2012 4° sampling 10/8/2012 5° sampling 20/8/2012 6° sampling no n c om me rci al us e o nly [page 130] [journal of entomological and acarological research 2013; 45:e23] the water of 10 and 50 mg*l–1 respectively, estimated as 100 and 500 mg*kg–1 in the sediment. a sample of about 100 larvae of chironomus riparius coming from a rearing in our (defens) department was added on 8/5/2012 in each tank. in the same rearing two oligochaetes (dero digitata and limnodrilus hoffmeisteri), one copepod (mesocyclops leuckarti) and one cladocera (moina brachiata) were present. c. riparius larvae at the third and fourth instar were counted at the beginning of the experiment, after 7 and 14 days on 15/5 and 22/5/2012. tricyclazole concentration present in the soil of the rice field and in the mesocosm was measured by neotron spa using the method icms-qpos-a and the analytical technique lc ms/ms. tricyclazole residue was measured in the five plots investigated (figure 1a); samples were collected with the same hand net used to sample macroinvertebrates on 6/8/2012, a week after the first treatment (30/7/2012); the tricyclazole concentration in the mesocosm test was measured in a sample collected in the treated tank on 14/6/2012, about one month after the second tricyclazole addition. data analysis environmental data and species counts were summarized with microsoft excel and access and analyzed using matlab r2012b® and r 2.15.2® software. at first, a shannon diversity index was calculated according to the formula: where yij is the abundance of species j in the sampling unit (a plot in a sampling date) i, p is the number of species present in the unit i and hi is the shannon diversity index. using the shannon index as dependent variable, a factorial anova was carried out with sampling dates (between years and among weeks within the same year) and tricyclazole treatments as factors. tricyclazole treatments were coded as: i) 1 no treatment; ii) 2 two treatment with 300 g*ha–1; 3 one treatment with 600 g*ha–1; 4 one treatment with 1200 g*ha–1. correlations between environmental variables, species abundances and the diversity index were calculated. multivariate analysis of variance and covariance (mancova) was carried out using species matrix as dependent variable, tricyclazole concentration and sampling date as target variable and environmental data matrix as covariate matrix (water temperature, ph, dissolved oxygen, conductivity, alcalinity). species counts were log10(y+1) transformed before analysis. for each of the three target variables separated mancova were carried out. mancova allows extracting information from each species. uniand multivariate f tests were carried out to detect the significance of responses, the multivariate test considered the highest eigenvalue of the matrix product between the inverse of sum of squares (ssq) within groups (w) with the ssq between groups (b): w–1*b (morrison, 1967). a canonical correlation analysis was carried out to have a graphical joint representation of the relation between species, environmental variables and plots with different tricyclazole treatments. the analysis searches for linear combination of the environmental set that maximizes the correlation with a linear combination of species. an acute toxicity test was carried out at the following tricyclazole concentrations: 0, 25, 50, 100 mg l–1. the lc50 was calculated considering mortality at 24, 48, 72 h. the acute toxicity test was repeated in four dates (6/3/2012, 19/3/2012, 7/5/2012, 22/5/2012). the parameters of the logistic curve (ritz et al., 2006) were calculated with tricyclazole concentration as independent variable and survival fraction as dependent variable. a four-parameter curve was fitted, according to the following equation: which can be expressed in a logarithmic form: where: x=tricyclazole concentration in mg*l–1; y=% of survival after 48 h; d=upper survival limit; c=lower survival limit; e=ec50 (lc50 in the present case); b=slope of the curve around the point of inflection. results several different invertebrate species were found in the soil of rice field (table 2). in 2012, 24 taxa were identified, with a higher number of specimens in respect to 2011, when only 13 taxa were collected and in lower number. the differences in species number and in specimen abundances were probably bound to the different sampling tool used in the two years, but the different water temperature pattern measured in the two years should also be taken into account (figure 1b). the presence of high numbers of polypedilum nubifer in 2012 and its absence in 2011 is of special interest (figure 2a). on the opposite, branchyura sowerbyi was more abundant in 2011 (figure 2b): this is also a thermophilous species abundant in rice fields; a competition between these two species may be an explanation of the observed results. the different sampling technique must be in any case considered when these results are discussed. the tricyclazole concentration in the sediments was measured on the 6th august 2012, with the following results: i) plot 1 <0.2 g*kg–1; ii) plot 2 0.5+/-0.2 g*kg–1; iii) plot 3 0.9+/-0.2 g*kg–1; iv) plot 4 1.4+/-0.3 g*kg–1; v) plot 5 <0.2 g*kg–1. the ratio between the tricyclazole concentration in the treatment (performed at 30/7/2012) and its concentration in the soil a week after (6/8/2012) was calculated (table 3). for the calculation following considerations were taken: the sediment was sampled with a hand net over a surface of about 1/3 m2, sediment was collected until a depth of about 2 cm, the soil density was about 2.5 kg m–3. with these assumptions 16.665=2.5*1000*0.02*0.3333 g of substrate should have been sampled. if these assumptions are true it could be concluded that after a week, tricyclazole amount remaining in the soil varies from 1/3 to 1/5 of the tricyclazole sprayed. the approximate calculation is: shannon diversity index (mean values) in the control and in the treated plots (figure 2c) confirmed that no substantial differences were observed in relation to tricyclazole treatment. factorial anova with shannon diversity as dependent variable and tricyclazole concentrations, years and weeks as factors, emphasized highly significant differences between years and among weeks, whereas no significant difference was found in correlation to tricyclazole treatment (table 4), article no n c om me rci al us e o nly [journal of entomological and acarological research 2013; 45:e23] [page 131] article table 2. the species found. class order family taxon year oligochaetes naididae dero digitata 2011 2012 tubificidae branchyura sowerbyi 2011 2012 limnodrilus hoffmeisteri 2012 oligochaetes cocoon 2011 2012 hirudinea glossiphoniidae glossiphonia heteroclita 2011 2012 gasteropoda pulmonata planorbidae gyralus albus 2011 2012 prosobranchia physidae physa fontinalis 2011 2012 ostracoda 2011 2012 copepoda cyclopidae mesocyclops leuckarti 2011 2012 cladocera cladocera daphniidae moina brachiata 2011 2012 hemiptera notonectidae notonecta glauca 2012 neuroptera chrysopidae chrysopa sp. 2012 coleoptera gyrinidae 2011 2012 dityscidae dityscus marginalis 2012 hydrophilidae hydrophilus piceus 2012 elmintidae elmis maugetii 2012 hydrochidae 2012 diptera culicidae ochlerotatus caspius 2011 2012 ceratopogonidae 2012 stratiomyidae 2012 tabanidae tabanus sp. 2012 chironomidae tanypus punctipennis 2011 polypedilum nubeculosum 2011 tanytarsus fimbriatus 2012 polypedilum nubifer 2012 chironomus annularius 2012 figure 2. a) p. nubifer (ind*m–2) during the sampled period, b) b. sowerbyi (ind*m–2) during the sampled period, c) shannon diversity index at different tricyclazole concentrations (median values and 25 % and 75 % percentiles, d) bonferroni multiple comparisons. no n c om me rci al us e o nly [page 132] [journal of entomological and acarological research 2013; 45:e23] this was confirmed by a bonferroni multiple comparison test which never gave significant differences between pairs (figure 2d). a multivariate analysis of variance and covariance (mancova) allowed a more detailed analysis of obtained results. the species matrix with 21 species in 56 samples (26 in 2011 and 30 in 2012) was included as the dependent variables matrix, the matrix of environmental variables in the same 56 samples with water temperature, dissolved oxygen, ph, conductivity, and alkalinity, was treated as a covariate matrix, the target variables were treatment, the two years and a temporal trend, coded as an integer from 1 to 6 indicating the first and the 6th sampling week respectively; separate analyses were carried out for each target variable. manova without covariates and manova using the environmental variables as target were also carried out (table 5). a univariate f test was performed to analyze the contribution of each species to sum of squares (table 6). the multivariate test did not show any significant difference bound to treatment, but highlighted differences between years and among weeks. in the univariate tests, few species showed significant differences, but the species counts were not linearly decreasing with tricyclazole concentrations, therefore obtained results must be interpreted with caution. article table 3. ratio between tricyclazole concentration in the treated plots (performed at 30/7/2012) and its concentration in the soil a week after (6/8/2012). concentration in treatment (t) concentration in soil (c) ratio t/c 0 g*ha–1=0 mg*m–2 <0.2 g*kg–1 0.0 300 g*ha–1=30 mg*m–2 0.5 g*kg–1 3.6 600 g*ha–1=60 mg*m–2 0.9 g*kg–1 4.0 1200 g*ha–1=120 mg*m–2 1.4 g*kg–1 5.2 table 4. factorial anova results, with shannon diversity as dependent variable, tricyclazole, years and weeks as factors. source sum sq. d.f. mean sq. f prob>f among treatment 0.6955 3 0.2318 1.1444 0.3399 error 10.5350 52 0.2026 total 11.2305 55 treatment 0.6002 3 0.2001 1.1742 0.3287 between years 1.8458 1 1.8458 10.8338 0.0018** error 8.6892 51 0.1704 total 11.2305 55 treatment 0.2900 3 0.0967 1.1044 0.3583 among weeks 6.9468 11 0.6315 7.2159 0.0000** error 3.5882 41 0.0875 total 11.2305 55.0000 s.q., ; d.f., ; prob, probabilities. **p<0.01. table 5. multivariate analysis of variance and covariance. target variable target variables corrected target variables not corrected environmental for covariates for covariates variables alone (mancova) (manova) f test treatment 0.8175 2.5438 17.4968 year 5.7726 21.6395 27.1358 trend 16.4429 26.7760 24.5270 prob di f treatment 0.6986 0.0109 0.0000 year 0.0000** 0.0000 0.0000 trend 0.0000** 0.0000 0.0000 eigenvalue of w–1*b treatment 0.788 1.908 9.998 year 5.566 16.230 15.506 trend 15.856 20.082 14.015 prob, probabilities. *p<0.05; **p<0.01. no n c om me rci al us e o nly the two species giving significantly different response to treatment showed a clear drop in density only at concentrations increasing from 600 to 1200, well above the ones normally applied (table 7). the sum of squares bound to treatment was much lower than the ssq due to years and to weeks (figure 3, table 5). the error ssq was very high respect to all the other sources of ssq, the ssq trace of error sum of squares ranged from 947 to 1102, while the trace of the ssq due to tricyclazole treatment was 51.38, the one due to years was 207, the trace of ssq due to weeks was 91, a large trace of ssq was bound to covariates (temperature, conductivity, alkalinity, ph, tp) and was equal to 777. the canonical correlation analysis summarized the response of the community to environmental variables; it was evident that ph, conductivity and alcalinity were the most important contributors to the observed ssq along the first canonical axis, while water temperature, dissolved oxygen and total phosphorous contributed more to ssq in the second axis, the tricyclazole treatment was not able to order plots according to increasing tricyclazole concentration applied (figure 4). acute toxicity test results of the acute toxicity test are summarized in table 8 and figure 5. survival at 48 h was used to calculate the lc50. the fitted loglogistic curve with four parameters gave an estimated value of the tricyclazole lc50=26 mg*l–1, with a standard error of 3 mg*l–1. mesocosm test population dynamics of chironomus riparius in a mesocosm was analyzed in the presence of other species, the oligochaetes dero digitata and limnodrilus hoffmeisteri and the cladocera mesocyclops and moina brachiata in a tank treated with tricyclazole (see materials and methods section). on 14/6/2012 the content of tricyclazole in sediment was analyzed on 11/6/29012, giving a value of 96 mg kg–1. it can be concluded that 0.192=96/ (100+500) or approximately 16% of the tricyclazole added in the water could be found in the sediment one month after. a decrease in number of the larvae in the fourth stage of development was observed with increasing tricyclazole treatment. the larvae in the third stage showed an increase in number one week after the first treatment (15/5/2012), while on 22/5/2012 (week after the second [journal of entomological and acarological research 2013; 45:e23] [page 133] article table 6. univariate f test, with treatment as target and environmental variables (water temperature, oxygen, conductivity and alcalinity as covariates. treatment f prob p. nubifer 7.267 0.010** d. marginalis 6.730 0.013* n. glauca 4.938 0.031* ceratopogonidae 4.916 0.031* t. fimbriatus 4.723 0.035* stratiomyidae 4.154 0.047* hydrochidae 1.628 0.208 chrysopa sp. 1.399 0.243 chironomus annularius 1.316 0.257 oligochaetes cocoon 0.980 0.327 ostracoda 0.577 0.451 p. fontinalis 0.460 0.501 o. caspius 0.428 0.516 e. maugetii 0.207 0.651 g. albus 0.096 0.758 tabanus sp. 0.054 0.817 b. sowerbyi 0.029 0.866 oligochaetes imm 0.028 0.867 h. piceus 0.023 0.879 g. heteroclita 0.004 0.951 prob, probabilities. *p<0.05; **p< 0.01. table 7. densities of two species at different treatments. log10(ind/m2+1) p. nubifer d. marginalis control 2.300 1.96 300 g*ha–1 1.77 1.33 600 g*ha–1 2.02 1.05 1200 g*ha–1 0 0.45 figure 3. proportion of sum of square (ssq) due to different treatments; treat=ssq bound to tricyclazole concentration, trend=ssq bound to differences among weeks (temporal trend), year=ssq bound to differences between years. figure 4. canonical correlation analysis; species and environmental variables scores in the plot of the first two axes. a circle of different color maps the stations grouped according to tricyclazole treatment. no n c om me rci al us e o nly [page 134] [journal of entomological and acarological research 2013; 45:e23] treatment with the highest tricyclazole concentration, performed on 15/5/2012, 50 mg l–1=500 mg kg–1), evidenced a drop in numbers (figure 6). it must be emphasized that in this experiment benthic macroinvertebrates were in contact with tricyclazole concentrations much higher (500 mg kg–1) than the highest one measured in the field, which was equal to 1.4 mg kg–1 at most. conclusions the field and mesocosm experiments carried out in 2012, together with acute toxicity tests allowed a better description of tricyclazole effects on benthic macroinvertebrates, outlined in the field investigation carried out in 2011 (rossaro et al., 2012), in which period of soil submersion and temperature fluctuations were considered the most relevant confounding effects in detecting the influence of the fungicide on benthic macroinvertebrates. for this reason field study in 2012 was accompanied by laboratory and mesocosm tests. in 2011, only 4 species were collected in significant numbers, in particular 2 snails: gyraulus albus and physa fontinalis, 1 oligochaetes: branchyura sowerbyi and 1 leech: glossiphonia heteroclita. gyraulus albus, the most common species, rarely reached densities near to 10,000 ind m–2, while oligochaetes, probably all belonging to branchyura sowerbyi, rarely were estimated with densities above 1000 ind m–2. the short time of soil submersion (two months between rice field inundation and the first sampling date) did not probably allow the transformation of the mineral compact terrestrial soil in a soft substrate suitable to benthic macroinvertebrates colonization. this condition led us to modify the sampling technique in 2012. the hand net was preferred to core sampler, allowing sampling only the superficial substrate, richer in fauna, on a larger area. in this manner a qualitative research of the most represented species was preferred to a strictly quantitative sampling. the result was a larger number of species captured (24), compared to 13 species sampled in 2011. during 2011, beside snails, few coleoptera, two chironomid species tanypus punctipennis and polypedilum nubeculosum, and the mosquito ochlerotatus caspius were sampled. in 2012 the hand net allowed to sample many species of aquatic insects, hemiptera and coleoptera above all, chironomids were also collected in larger numbers with more species. the presence and abundance of p. nubifer in 2012, not captured in 2011, was the most striking result. this is an afrotropical species, strictly termophilous, which invaded other continents including europe and america and is characteristically abundant in rice fields. p. nubifer tolerates water temperatures around 30°c. the lower water temperature in 2011 may be a reason of its absence in this year. on the other hand, the higher hemiptera and coleoptera numbers captured in 2012 were probably related to sampling technique. no statistical analysis was able to detect any effect of tricyclazole treatment on fauna composition, while large differences were observed between the two years and among weeks. this conclusion was supported by a factorial anova followed by bonferroni multiple comparison, which was unable to emphasize an effect of tricyclazole on shannon diversity, whereas both seasonal and between-year-difference were significant. multivariate analysis of variance and covariance and canonical correlation analysis again emphasized the importance of seasonal succession bound to variation in water temperature and conductivity and the negligible influence of fungicide treatment on benthic invertebrates. tricyclazole was found to be degraded in water (dt50=16.4 days) but persistent in soil (dt50=197 days) (tsochatzis et al., 2013); our results do not allow to estimate the persistence of tricyclazole in soil, even if suggested at least in the mesocosm a shorter degradation time, neararticle figure 5. acute toxicity test: survival of chironomus riparius at different tricyclazole concentrations after 48 h treatment; 4 parameters log-logistic curve. figure 6. counts of chironomus riparius larvae at iii and iv instar a week after the application of different concentrations of tricyclazole. a0: untreated tank at the beginning of the experiment (no treatment), a1: untreated tank a week after the application of 100 mg l–1 of tricyclazole in tank b, a2: untreated tank a week after the application of 500 m gl–1 in tank b, b0 treated tank at the beginning of the experiment (no treatment), b1: a week after the application of 100 mg l–1 of tricyclazole, b2: a week after the application of 500 mg l–1 of tricyclazole. no n c om me rci al us e o nly er to the one measured in the water by tsochatzis et al. (2013). despite its possible persistence tricyclazole does not seem to have a detrimental effect on benthic macroinvertebrates. laboratory acute toxicity tests and mesocosm experiments emphasized that the effect can be detected only at concentrations much higher (10-100 times) than the ones measured in the field following treatment. in the mesocosm only concentrations well above 100 mg kg–1 showed an effect on fauna composition, in the field much lower concentrations (1.4 mg kg–1) were measured following the tricyclazole treatment applied at the highest concentrations. the acute toxicity tests carried out with a laboratory population of c. riparius gave an lc50 near 26 mgl–1after 48 hours; this value is in agreement with epa data, epa data give an lc50 after 3 h for daphnia magna equal to 94 mg l–1, or 34 mg l–1 after 48 h and an lc50 after 48 h for ciprinus carpio equal to 22 mg l–1 (table 9). the field, mesocosm and laboratory results confirm that the tricyclazole has a low toxicity to benthic macroinvertebrates; therefore it is possible to conclude that there is no concern about its application in nature at the concentrations recommended for control of fungal infection. the matter merits a deeper study in any case. one question is if different benthic macroinvertebrate taxa can express different tolerance to tricyclazole in relation to their particular metabolic pathways. in particular it was recently demonstrated the ability of chironomids to perform melanin synthesis, giving higher tolerance to toxic metals (loayza-muro et al., 2013), it is of particular interest in considering the interference of tricyclazole with melanin synthesis (kunova et al., 2013), this requires further study but can be a basis to explain a tolerance of some benthic macroinvertebrates to tricyclazole toxicity. references apha (american public health association), 2005 standard methods for the examination of water and wastewater, 21th ed. american public health association, american water works association, and water environment federation, washington, dc. bertocchi d., pizzatti c., cortesi p., 2007 epidemics and disease management of rice brown spot and rice blast in italy. in: bocchi s., ferrero a., porro a. (eds.), proc. 4th temperate rice conference, 25–28 june 2007, novara, italy: 344-345. campaioli s., ghetti p. f., minelli a., ruffo s., 1994 manuale per il riconoscimento dei macroinvertebrati delle acque dolci italiane. vol. i. provincia autonoma di trento: 1-357. campaioli s., ghetti p. f., minelli a., ruffo s., 1999 manuale per il riconoscimento dei macroinvertebrati delle acque dolci italiane. vol. ii. provincia autonoma di trento: 358.484. cortesi p., 2011. il brusone del riso: danni causati dalle epidemie e ottimizzazione della difesa con triciclazolo. in: il ruolo economico del triciclazolo nella risicoltura italiana. a.g.r.a. srl, roma: 87-140. encarna s., fernández-vega c., villarroel m. j., andreumoliner e., ferrando m. d., 2009 physiological effects of tricyclazole on zebrafish (danio rerio) and post-exposure recovery. comparat. biochem. physiol. part c 2009: 25-32. faria m.s., nogueira a.j.a., soares a.m.v.m., 2007 the use of chironomus riparius larvae to assess effects of pesticides from rice fields in adjacent freshwater ecosystems. ecotoxicol. environ. saf. 67: 218-226. grigarick a.a., webster r.k., meyer r.p., zalom f.g., smith k.a., 1990. effects of pesticide treatments on non-target organisms in california rice paddies. hilgardia 58: l-36. hayasaka d., korenaga t., sánchez-bayo f., goka k., 2012 differences in ecological impacts of systemic insecticides with different physicochemical properties on biocenosis of experimental paddy fields. ecotoxicology 2012: 191-201. kunova a., pizzatti c., cortesi p, 2013 impact of tricyclazole and azoxystrobin on growth, sporulation and secondary infection of the rice blast fungus, magnaporthe oryzae. pest manage. sci. 69: 278-284. leitão s., pinto p., pereira t., brito m. f., 2007 spatial and temporal variability of macroinvertebrate communities in two farmed mediterranean rice fields. aquat. ecol. 41: 373-386. loayza-muro r.a., marticorena-ruiz j.k., palomino e.j., [journal of entomological and acarological research 2013; 45:e23] [page 135] article table 8. acute toxicity test with c. riparius, log-logistic model with 4 parameters. parameter estimate std. error t-value p-value slope: (intercept) b 3.480 1.772 1.964 0.053 lower limit: (intercept) c 0.001 0.086 �0.012 0.991 upper limit: (intercept) d 0.952 0.069 13.798 0.000 ed50: (intercept) e 26.510 2.980 8.895 0.000 residual standard error 0.316 (80 d.f.) table 9. acute toxicity and no effect concentration of 75% wp, tricyclazole (http://www.dowagro.com ; http://sitem.herts.ac.uk/aeru/footprint/it/ reports/660.htm ; http://www.pesticideinfo.org/list_aquireall.jsp?rec_id=pc35823). common name effect time lc50 min max concentration units cyprinus carpio mortality 48 h 22.0 20 24 mg l–1 cyprinus carpio mortality 96 h 14.0 11 17 mg l–1 scapholeberis kingi mortality 3 h 94.0 82 110 mg l–1 daphnia magna mortality 48 h 34.0 mg l–1 chironomus riparius noec 28 days 2.2 mg l–1 noec, no effect concentration. no n c om me rci al us e o nly [page 136] [journal of entomological and acarological research 2013; 45:e23] merritt c., de baat m.l., van gemert m., et al., 2013 persistence of chironomids in metal polluted andean high altitude streams: does melanin play a role? environ. sci. technol. 47: 601-607. morrison df., 1967 multivariate statistical methods. mcgraw-hill co., new york: 343. odum e., barret, g.w. 2005 fundamental of ecology. thomson brook/cole, trad. ital.: piccin ed., padova: 594. ritz c., cedergreen n., jensen j.e., streibig j.c., 2006 relative potency in non similar dose-response curves. weed sci. 54: 407-412. rossaro b., marziali l., cortesi p., 2013 the effects of tricyclazole treatment on aquatic invertebrates in a rice paddy field. clean soil air water [epub ahead of print]. simpson i.c., roger p.a., 1991 the impact of pesticides on nontarget aquatic invertebrates in wetland ricefield. a review. in: prabhu pingali l., pierra roger a. (eds.), impact of pesticides on farmer health and the rice environment. kluweer academic publ., aa dordrecht, the netherlands: 249-270. stenert c., bacca r.c., maltchik l., rocha o., 2009 can hydrologic management practices of rice fields contribute to macroinvertebrate conservation in southern brazil wetlands? hydrobiologia 635: 339-350. suarez-serrano a., ibanez c., lacorte s., barata c. 2010 ecotoxicological effects of rice field waters on selected planktonic species: comparison between conventional and organic farming. ecotoxicology 19: 1523-1535. timm t., 2009 a guide to the freshwater oligochaeta and polychaeta of northern and central europe. lauterbornia 66: 1-235. tsochatzis e. d., tzimou-tsitouridou r., menkissoglu– spiroudi u., 2013 laboratory and field dissipation of penoxsulam, tricyclazole and profoxydim in rice paddy systems. chemosphere 91: 1049-1057. wiederholm t., 1983 chironomidae of the holoarctic region. keys and diagnoses. part. 1. larvae. entomol. scand. suppl. 19:1-457. article no n c om me rci al us e o nly 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res. ser. ii, 42 (2): 81-90 30 august 2010 d. lupi, m.l. giudici, c. cenghialta, b. villa, d. passoni, m. colombo    on the spatial spread of the rice water weevil, lissorhoptrus oryzophilus kuschel (coleoptera: erirhinidae), in italy abstract - a five year study has been made to establish the spread of the rice water weevil lissorhoptrus oryzophilus (coleoptera: erirhinidae) in northern italy.  data obtained with gps from 2005 throughout 2009 were first georeferenced with  sw arcgis® 9.2, then overlapped and compared to the map of the european environmental landscape based on the interpretation of satellite images (corine land  cover map) and to the hydrographic chart ct10 (technical regional map 10000).  the analysis of the radial rate of spread per year indicates a deceleration in the  expansion from 10.864 ± 6.801 km/year in 2005 to 5.318 ± 1.401 km/year in 2009.  in five years the weevil has expanded its distribution in nearly all rice paddies in  lombardy and piedmont, over an area of about 200,000 ha, which correspond to  86% of the total italian rice area. its expansion is thought to follow a type of stratified dispersal, due both to insect adult active dispersal and to accidental movements  caused by human transportation.  riassunto diffusione in italia del punteruolo acquatico del riso lissorhoptrus  oryzophilus kuschel (coleoptera: erirhinidae) si riportano i risultati di uno studio quinquennale sulla distribuzione spaziale  di lissorhoptrus oryzophilus, il punteruolo acquatico del riso, nel nord italia. i  dati registrati con gps dal 2005 al 2009 sono stati dapprima georeferenziati con  sw arcgis® 9.2, quindi sovrapposti alla mappa europea dell’uso dei suoli basata  sull’interpretazione di immagini satellitari (corine land cover map) e sul reticolo  idrografico ct10 (mappa tecnica regionale 10000). l’analisi ha evidenziato una  progressiva decelerazione nel raggio di espansione dai 10.864 ± 6.801 km/anno  nel 2005 ai 5.318 ± 1.401 km/anno nel 2009. in cinque anni il fitofago è riuscito a  colonizzare quasi tutta l’area risicola di lombardia e piemonte, su una superficie  di circa 200.000 ha, che corrispondono all’86% della superficie risicola italiana. si  pensa che la sua espansione abbia seguito una diffusione stratificata, dovuta allo  spostamento attivo dell’insetto e a quello accidentale determinato dai movimenti  antropici.   key words: italian rice growing areas; settlement; new records; expansion radius. journal of entomological and acarological research, ser. ii, 42 (2), 201082 introduction the rice water weevil (rww), lissorhoptrus oryzophilus kuschel, is a polyphagous phytophagous mainly feeding on gramineous and cyperaceous plants (tindall &  stout, 2003; lupi et al., 2009). it is considered one of the most important pests of rice  over the world. damage is caused mainly by larvae, whose activity occurs on rice roots.  crop damage, resulting from rww larval feeding, reduces plant growth, height, tillering, culm density, and ultimately yield. in extreme cases wind may cause the plant to  dislodge and to float (wu & wilson, 1997). adults are semiaquatic herbivores and they  leave longitudinal feeding scars on leaves. their feeding rarely has economic importance  but can be an indicator of the magnitude of consequent larval infestation and damage  (way & wallace, 1992). originally classified as lissorhoptrus simplex say (webb, 1914), it was described  as the new species l. oryzophilus in 1951 (kuskel, 1951). both species are native to the  united states where they originally fed on gramineous and cyperaceous plants. the collection records for l. oryzophilus given by kuschel were mostly from southern, central  and eastern united states. the rww was never reported from california before 1959,  when it was first discovered in sacramento valley (lange & grigarick, 1959). this species remained confined in north america until 1976 when it spread to  japan (saito et al., 2005). thereafter it was detected in china, in korea, and in india  (lee & uhm, 1992; hix et al., 2000; chen et al., 2004). the first detection in europe  was in italy (caldara et al., 2004). before this detection, no other species of the genus  lissorhoptrus had been ever detected in italy (caldara & diotti, 2005). only stenopelmus rufinasus gyllenhal, 1836, belonging to the same tribe of stenopelmini, imported  from america, was formerly present in italy (abbazzi & osella, 1992). as this finding  accidentally occurred during faunistic studies in the litter under woody trees in ticino  park (lombardy), it was impossible to acquire information on the pathway of introduction of the insect in italy.  rww detection in italy was really important because the ticino park is within a  really important rice growing area and italy is the largest rice producing country in the  european union. rice in italy is mainly cultivated in the north in the po valley (fig.1). about 52%  of the rice area is in piedmont, mostly in vercelli and novara provinces, and about 41%  in lombardy, for the most part in pavia and milan provinces, but rice is also grown in  veneto, emilia-romagna and sardinia.  the dimension of a typical rice farm in italy is about 48 ha and the average rice yield  ranges from 5.0 tons/ha to 6.9 tons/ha, according to the varieties. in 2008, 1,333,000 tons  of rice were produced on a surface of 224,198 ha (e.n.r. 2008). in italy rice is normally  water-seeded but in about 25% of the rice growing area it is dry-seeded and subsequently  flooded when seedlings are at the 3-4 leaf stage. in italy both the traditional italian type  (generally large or short sized, with a soft cooking grain) japonica varieties, and the  so-called indica type (long and with slender grains) varieties are grown. climatic conditions in italian rice plantations are generally inadequate for authentic indica varieties.  83d. lupi et al.: spatial spread of lissorhoptrus oryzophilus in italy fig. 1 - rice field distribution in italy (cartographic elaboration paolo molinari on istat, italian  statistic institute, data, 2004)  journal of entomological and acarological research, ser. ii, 42 (2), 201084 fig. 2 - sw arcgis® 9.2 representation of lissorhoptrus oryzophilus spread in rice growing area  in lombardy and piedmont [2004 (a); 2005 (b); 2006 (c); 2007 (d); 2008 (e); 2009 (f)] 85d. lupi et al.: spatial spread of lissorhoptrus oryzophilus in italy in some areas, particularly in vercelli province, rice is grown as a monoculture but can  also be grown in rotation with soybean, corn or other cereals. as rice is cultivated also in many other european countries (spain, greece, portugal, france, romania, bulgaria, hungary) l. oryzophilus was added to eppo alert list  (eppo, 2005) where it remained until 2009. the present survey aimed to establish the capability of settlement of rww in italy  and its spread.  materials and methods the survey began in march 2005 and was executed throughout 2009.  study area the area of study was the rice growing area in the po valley. the main four rice  producing provinces (novara, vercelli, pavia, and milan) are contiguous and are located  in this area. they are rich of rivers and canals and are characterized by a continuity  of rice fields. rice is normally water-seeded; except for half of the rice areas in pavia  province. rice is dry-seeded there to allow the growth of the old traditional varieties,  used to cook “risotto” whose plants are very tall (culm length of cv carnaroli is 120 cm)  and highly susceptible to lodging. rww monitoring procedures the methods used to detect the insect in the monitoring process were dependent  from the season and, consequently, from the biology of the insect (bowling, 1972).  overwintering sites were studied in february, in march, in september and in october,  collecting the soil and the litter at the base of trees near the rice fields. this material  was washed in water and only organic floating material was collected and placed in a  berlese-tullgren extractor to detect crawling adults. in early spring when the temperature caused the exit of the insect from the litter but rice has not been present yet, adults  were searched on weeds with an insect sweep net. after emergence, they were directly  searched on rice with direct visual observation from june until july. larval and pupal  presence was checked from the beginning of may until late august by taking root/soil  core samples with a metal sampler (10.0 cm in diameter by 10.0 cm deep). cores were  placed into basins with salt water, and soil was washed from roots. larvae were detected  as they floated to the water surface, pupae were searched within rice roots.  samples of rww adults, randomly collected in the localities where detected, were  carried to laboratory. specimens were then observed with a stereomicroscope and sexed  evaluating externally visible gender-specific characters (everett & newson, 1964). first detections in 2004 were localized across ticino river, about 30 km west of milan (45°24.17’n 8°55.56’e; 45°19.34’n 8°56.41’e; 45°20.85’n 8°55.46’e; 45°17.09’n  8°55.30’e; 45°15.26’n 9°01.27’e). these localities were at first visited in 2005, to realize if the insect has settled down and if it was present in rice fields. further sites were  randomly chosen in centrifugal-direction in rice area.  journal of entomological and acarological research, ser. ii, 42 (2), 201086 each year the localities visited in the previous year were checked again to evaluate  the status of the insect and its distribution. new ones were added as appropriate. the position of each site was georegistered with a gpsmap® 76s garmin (gps  coordinates - map datum wgs84 were used). information on the insect presence/absence was added. data obtained with gps from 2005 throughout 2009 of the rww presence in the  territory (positive or negative) of lissorhoptrus oryzophilus were georeferenced with  sw arcgis® 9.2. these data were interpolated in all the territory involved with inverse  distance weighted criteria and radius of influence equal to 5 km. moreover, these data  were overlapped and compared to the corine land cover map and hydrographic chart  ct10 (technical regional map 10000). ct10 is a geographical database built up from digitalization of the main element of  technical regional map at scale 1:10,000. the “corine land cover” (european environment agency 2009) is the map of  the european environmental landscape based on the interpretation of satellite images  scale of 1:100,000, in order to provide consistent localized geographical information  and make a representation of the territory as a paddy field. rww spread was estimated as an increase in range radius over time, where radial  rate of expansion (r) was calculated as follows where r = radius of expansion per year; d= distance among new match point in the year  (i) and the nearest point in which the insect was detected in the year (i-1); n= number  of localities with new detection.  correlation between radius of expansions and years were analyzed with spss 17®. pearson’s correlation coefficient (p < 0.01).  results a total of 309 localities were surveyed: 67 in 2005; 103 in 2006; 139 in 2007; 156  in 2008 and 309 in 2009 (table 1). in 2005, the insect expanded its radius of distribution moving to south and to west in  rice paddies, where rice is water seeded and maintained under permanent submersion. it  was found in 34 localities, 33 of which in the area surrounding the first detection. there  was only one isolated colony in vercelli province (45°11,10’n 8°18,31’e).  in 2006, the survey was extended to 103 localities and rww was detected in 51 sites.  it was possible to confirm rww settlement in all the places in which it was detected in  2005. the insect spreading was limited in the eastern areas, while it was greater in the  87d. lupi et al.: spatial spread of lissorhoptrus oryzophilus in italy western localities where rice is usually water seeded. two isolated flash points were also  detected (45°23.91’n 8°46.97’e; 45°25.00’n 8°46.77’e).  in 2007 and 2008, 139 and 156 localities were respectively visited. insect diffusion  continued westward in the rice areas. only few areas remained unsettled especially in  the south of milan where the major presence of dry seeded fields may have affected  and postponed the diffusion of the insect. however, four localities gave positive match  in this area in 2008. in 2009 rww was detected in 267 localities of the 309 visited. results showed that  insect colonization expanded in unsettled areas.  the analysis using pearson’s correlation coefficient indicates a statistically significant negative linear relationship between year and radius of expansions (r = -0.401,  p < 0.01). the radial rate of spread reached its maximum in 2005 when the mean was  10.864 ±6.801 km and the maximum was of 50 km. lower values were detected in 2009  when mean radial rate was 5.318 ±1.401 km and maximum radial rate of 15 km (tab. 1). the comparison of rww distribution with the corine land cover and ct10 maps  allowed the evaluation of the insect diffusion per year in function of the rice paddies and  the hydrographic reticulum. while insect presence was strictly related to rice paddies  (fig. 2), it was impossible to find any relationships with the presence of river and canals.  all the specimens collected were parthenogenetic female. no males were ever  detected during surveys. table 1 results of the surveys in the study area over the period of observation year visited locations positive match negative match new records radial rate of spread (km/year) (1) maximum distance (km2) 2005 67 34 33 29 10.864 ±6.801 40 2006 103 51 52 17 8.750 ±7.986 30 2007 139 131 8 80 8.532 ±5.250 30 2008 156 140 16 9 9.333 ±5.172 20 2009 309 267 42 127 5.318 ±1.401 15  (1) data are expressed as mean±stand. dev. discussion the present study documents the expansion of rww in one of the most important  rice producing areas in italy. until rww detection, there were few arthropod species  related to rice cultivation in italy with few outbreaks and, consequently, damage (suss  et al., 2008). when rww arrived in italy, it found an available niche, without competitors or predators. considering that three phases are generally recognized in all biological invasions  processes (arrival, establishment, and spread) (williamson, 1996; liebhold & tobin,  journal of entomological and acarological research, ser. ii, 42 (2), 201088 2006), we have to consider that the first one is still unknown for rww. besides, since  in 2005 the insect was detected in a wide area, we consider that probably rww had  arrived in italy before 2004. in fact there is typically a period of latency between the  arrival of an exotic species and the growth of the population until a level in which it is  easy to notice and to spread (sakai et al., 2001; liebhold & tobin, 2006). we hypothesize that in 2005 the insect has already overcome the initial colonization phase. in  fact in the following years the insect diffusions rapidly enlarged into the adjacent rice  growing areas. in five years, the weevil has expanded its distribution all over rice paddies in lombardy and piedmont, over an area which corresponds to 86% of the total  italian paddy fields area. rww diffusion was particularly favoured westwards by the  presence of water-seeded fields, and hindered eastwards by the adoption and frequency  of different cultivation strategies. we hypothesize that rww spread has been facilitated by the presence of a continuity of rice paddies near the area where it was first detected. in fact several theoretical  explorations indicate that spread rates are affected by habitat fragmentation (williamson,  1996; kamata et al., 2006).  rww expansion is thought to follow a type of stratified dispersal, in which short  range expansion is due to insect adult active dispersal flying, crawling and swimming,  while long range expansion is due to accidental movements caused by human transportation (lockwood et al., 2007). subsequently the comparison of rww distribution spreading with rivers and the human route did not allow finding any relationship.  however it is important to consider that the rice area in northern italy is characterized  by a dense network of secondary canals, and it is difficult to evaluate their influence on  insect movement.  the analysis of the radial rate of spread per year indicates a deceleration in the expansion. this occurrence is common in all exotic pests, in fact after a period of latency  there is a subsequent phase of rapid expansion and then a reduction. however rww  radial rate in italy is quite limited if compared with the insect diffusion in other countries  (liebhold & tobin, 2008).   notwithstanding the quickness of expansion and the area in which the insect had been  detected, economic damages had been rarely reported. in 2005 they were only reported  in the perimeter zone some paddies in pavia province (45°22.74’n; 8°50.14’e). in 2006  heavy damage was detected in various farms, dry and water seeded, at the beginning of  may 2005. in these farms, the leaf surface of plants localized in the most shaded places,  near levees with trees, was completely covered by scars. among these farms some were  dry seeded, but not yet flooded. although delayed flooding, according to many authors  (rice et al., 1999; stout et al., 2002) negatively influences and postpones the feeding  behaviour and the oviposition of rww, the particular location of these paddies and the  presence of humid areas favoured the insects’ settlement. in addition, in 2006, in various farms in the area near the paddies where only slight damage was detected in 2005  (45°22.73’n; 8°50.14’e), the yield production was compromised as a consequence of  a culm density reduction due to plants dislodging. from 2007 till 2009 damages were  limited, and especially in 2009 this was probably due to the adoption of a commercial  product (a.p. alfacipermetrine) that has received the extension label for few months. 89d. lupi et al.: spatial spread of lissorhoptrus oryzophilus in italy acknowledgments authors want to thank the technical assistance of ente nazionale risi who helped  in the monitoring process, and prof. larry godfrey (uc davis department of entomology) for his useful comments.  work published with a grant of lombardy region (italy) research projects: “entomological pests in rice fields: biology and control of the rice water weevil and other  pests” and “the rice water weevil: biology and biological control”  references abbazzi p., osella g., 1992 - elenco sistematico-faunistico degli anthribidae, rhinomaceridae,  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environ. entom. 26 (6): 1191-1201. daniela lupi, prof.mario colombo dipsa università degli studi di milano, via celoria, 2,  i-20133 milano, italy. e-mail: daniela.lupi@unimi.it giudici maria luisa, villa bruno, cenghialta cesare ente nazionale risi - centro ricerche sul riso, strada per ceretto 4 - i-27030 castello d’agogna (pv), italy. e-mail: crr. patologia@enterisi.it passoni daniele politecnico di milano, diiar-sez. rilevamento, p.za l. da vinci, 32  i-20133 milano. e-mail: daniele.passoni@polimi.it accepted 27 august 2010 jear2012 [journal of entomological and acarological research 2015; 47:1986] [page 31] bioefficacy of morinda tinctoria and pongamia glabra plant extracts against the malaria vector anopheles stephensi (diptera: culicidae) d. amerasan, k. murugan, c. panneerselvam, n. kanagaraju, k. kovendan, p. mahesh kumar department of zoology, bharathiar university, coimbatore, tamil nadu, india abstract mosquito-borne diseases have an economic impact, including loss in commercial and labour outputs, particularly in countries with tropical and subtropical climates; however, no part of the world is free from vector-borne diseases. the aim of the present study was to investigate the larvicidal, adulticidal and ovicidal activity of dried leaf chloroform, ethyl acetate, acetone, aqueous, and methanol extracts of morinda tinctoria and pongamia glabra against larvae of anopheles stephensi (diptera: culicidae). larvae were exposed to varying concentrations of plant extracts for 24 h. all extracts showed moderate larvicidal effects after 24 h of exposure; however, the highest larval mortality was found with the leaf methanol extracts of m. tinctoria and p. glabra against the larvae of a. stephensi lethal concentration (lc)50=136.24 and 141.05 ppm; lc90=342.67 and 368.89 ppm, respectively. the results of the adulticidal activity assays of chloroform, ethyl acetate, acetone, aqueous, and methanol extracts of m. tinctoria and p. glabra showed significant mortality against larvae of a. stephensi. the methanol extract showed maximum activity compared with the other extracts. the greatest effect on mean percentage hatch in the ovicidal assays was observed 48 h post-treatment. percent hatch was inversely proportional to the concentration of extract, and directly proportional to the number of eggs. a mortality of 100% was observed with 100-400 ppm methanol extracts and 200-400 ppm aqueous extracts of m. tinctoria, and 200-400 ppm aqueous and methanol extracts of p. glabra. this study provides the first report of the larvicidal, adulticidal and ovicidal activities of m. tinctoria and p. glabra plant extracts against the malaria vector, a. stephensi, representing an ideal eco-friendly approach for its control. introduction the prevalence of mosquito-borne diseases is one of the world’s most important health problems. mosquitoes are responsible for transmitting various infectious diseases; for this reason, the mosquito has been declared public enemy number one (world health organisation, 1996). mosquitoes belonging to the genera anopheles, culex and aedes are vectors for the pathogens of different diseases such as malaria, filariasis, japanese encephalitis, dengue and dengue haemorrhagic fever, yellow fever and chickungunya (rahuman et al., 2009; borah et al., 2010). they cause allergic responses, including local skin and systemic reactions such as angioedema and urticaria (peng et al., 1999). tropical areas are more vulnerable to parasitic diseases, and the risk of contracting arthropod-borne illnesses has increased due to climate change and intensifying globalisation (karunamoorthy et al., 2010). it is necessary to prevent mosquito-borne diseases and improve public health by controlling mosquitoes. malaria is an infectious disease that is prevalent in tropical and some temperate areas of the world. malaria is caused by a parasite that is transmitted from one human to another by the bite of infected anopheles stephensi. half of the world’s population is at risk from malaria. each year, almost 250 million cases occur, causing 860,000 deaths (world health organisation, 2010). in india, 2-3 million malaria cases and about 1000 deaths are reported every year (lal et al., 2010). currently, a resistant variety of the malarial parasite is commonly found in almost all parts of the world where malaria is endemic (cooper et al., 2005). the increased incidence of drug resistance continues to be a major issue, with ongoing problems related to drug quality, availability, and cost of treating the disease (garcia, 2010). for this reason, the transmission of malaria is best reduced by the control of the mosquito vector. botanical and microbial insecticides have been increasingly used for mosquito control because of their efficacy and documented non-toxic effects on non-target organisms (ascher et al., 1995). the highest number of malaria (plasmodium falciparum) cases and malaria-related deaths is recorded from the state of orissa, located in the eastern part of india (sharma et al., 2010). widely used chemical insecticides to control mosquitoes are often harmful to other beneficial organisms that prey on mosquito larvae, as well as to humans (amer & mehlhorn, 2006). alternative pest control strategies, especially those that are effective and low-cost, are therefore needed. a recent emphasis has been placed on plant materials correspondence: duraisamy amerasan, division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore 641 046, tamil nadu, india. tel.: +91.9443746799. e-mail: ezhil_amar@yahoo.co.in key words: morinda tinctoria; pongamia glabra; anopheles stephensi; larvicidal; adulticidal; ovicidal. received for publication: 22 october 2013. revision received: 24 november 2014. accepted for publication: 16 january 2015. ©copyright d. amerasan et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:1986 doi:10.4081/jear.2015.1986 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:1986 no nco mm er cia l u se on ly [page 32] [journal of entomological and acarological research 2015; 47:1986] that demonstrate larvicidal properties (kovendan et al., 2012b; mahesh kumar et al., 2012a; panneerselvam et al., 2013b). morinda tinctoria, which belongs to the family rubiaceae, grows wild and is distributed throughout southeast asia. commercially known as nunaa, it is indigenous to tropical countries and is considered an important traditional medicine, where its leaves and roots are used as an astringent and emmengogue, and to relieve pain caused by gout (thirupathy et al., 2009). there is a greater demand for fruit extracts of morinda species in treatments for arthritis, cancer, gastric ulcers and other heart diseases (mathivanan et al., 2006). anti-convulsant, analgesic, anti-inflammatory anti-oxidant activities of m. tinctoria leaves have been reported (jeyabalan & palayan, 2009; sreena et al., 2011). the major components that have been identified in the nunaa plant include octoanicacid, potassium, vitamin c, terpenoids, scopoletin, flavones, glycosides, linoleic acid, anthraquinones, morindone, rubiadin, and alizarin (moorthy & reddy, 1970; singh & tiwari, 1976; levand & larson, 1979; farine et al., 1996). deepti et al. (2011) studied the in vitro free radical-scavenging efficacy of methanolic, chloroform and ethyl acetate extracts of m. tinctoria, which can be recommended as potential antioxidants. pongamia glabra vent. (syn. pongamia pinnata), in the family fabaceae, commonly known as karanja, is a tree found all over india that bears imparipinnate leaves and pinkish-white flowers (kirtikar & basu, 1984). roots, bark, leaves, flowers and seeds of this plant also have medicinal properties and are traditionally used as medicinal treatments. all parts of the plant have been used as a crude drug for the treatment of tumours, piles, skin diseases, wounds and ulcers (tanaka et al., 1992). seeds contain karanjin, ponga-mol and glabrin. karanjin is reported to be an effective remedy for all skin diseases such as scabies, eczema, leprosy and ulcers (sivarajan & balachandran, 1999). the leaves are spicy, digestive, laxative, anthelmintic and cure piles, wounds and other inflammations. a hot infusion of leaves is used as a medicated bath for relieving rheumatic pains and for cleaning ulcers from gonorrhoea and scrofulous enlargement (chopra, 1933; satyavati et al., 1987). in addition, phytochemical examinations of this plant have indicated the presence of furanoflaovones, furanoflavonols, chromenoflavones, flavones, furanodiketones and flavonoid glucosides (rangaswami et al., 1942; murthy & seshadri, 1944; sharma et al., 1973; talapatara et al., 1980; pathak, 1983; tanaka et al., 1992; ahemed et al., 2004; yin et al., 2006). the acetone, chloroform, ethyl acetate, hexane, and methanol leaf extracts of acalypha indica, achyranthes aspera, leucas aspera, m. tinctoria, and ocimum sanctum were studied against the early fourthinstar larvae of aedes aegypti and culex quinquefasciatus (bagavan et al., 2008); the chloroform fruit extract of morinda pubescens (m. tinctoria) showed wound-healing properties in rats (mathivanan et al., 2006). larvicidal effects of neem (azadirachta indica) and karanja (p. glabra) oil cakes (separately and in combination) were studied against several mosquito species. the combination of neem and karanja oil cakes in equal proportion proved to be more effective than individual treatments against the mosquitoes c. quinquefasciatus, a. aegypti and a. stephensi, with lethal concentration (lc)95 values of 0.93, 0.54 and 0.77 ppm, respectively (shanmugasundaram et al., 2008). oviposition deterrent activity of the ethanolic extract of pongamia pinnata, coleus forskohlii, and datura stramonium leaves against a. aegypti and c. quinquefaciatus was reported by swathi et al. (2010). the individual and combined efficacy of annona squamosa and p. glabra extracts against three mosquito vectors, c. quinquefasciatus, a. stephensi, and a. aegypti, compared with that of a. indica was investigated by george & vincent (2005). leaf extracts of some common plants such as vitex negundo, gliricidia maculata, wedelia chinensis, m. tinctoria, and p. glabra, were evaluated for their acaricidal activity against the red spider mite, oligonychus coffeae, in the laboratory using a leaf disc method under controlled conditions. among them, the aqueous extract of m. tinctoria and p. glabra showed maximum ovicidal action, ovicidal deterrence and 100% adult mortality (vasanthakumar et al., 2012). in the present study, we report for the first time on larvicidal, adulticidal and ovicidal activities of different solvent extracts of m. tinctoria and p. glabra against the malarial mosquito vector, a. stephensi. materials and methods plant collection fully developed fresh leaves of m. tinctoria and p. glabra were collected from the maruthamalai hills, near the bharathiar university campus in coimbatore. identification was authenticated by a plant taxonomist from the department of botany, bharathiar university. a voucher specimen is deposited at the herbarium of the entomology division, bharathiar university. extraction the leaves were washed with tap water, shade-dried at room temperature (28±2°c) for 5-10 days. the air-dried materials were powdered individually using a commercial electric blender. the finely ground plant material (1000 g/solvent) was loaded into a soxhlet apparatus and extracted individually with five different solvents: chloroform, ethyl acetate, acetone, aqueous, and methanol. the solvent from the extract was removed using a rotary vacuum evaporator to collect the crude extract. the crude residue of these plants varies with the solvents used. the m. tinctoria and p. glabra with five different solvents yielded 58.20, 64.09, 54.11, 67.34, 88.05 g and 47.10, 53.31, 41.18, 54.30, 79.16 g of crude residue, respectively. standard stock solutions were prepared at 1% by dissolving the residues in acetone. from this stock solution, different concentrations were prepared, and these solutions were used for the larvicidal, adulticidal and ovicidal bioassays. insect rearing the eggs of a. stephensi were collected from different breeding sites (overhead tanks) in coimbatore district, tamil nadu, india. these were taken to the laboratory and transferred (in approximately the same aliquot numbers of eggs) to 18 cm l×13 cm w×4 cm d enamel trays containing 500 ml of water, where they were allowed to hatch. mosquito larvae were reared (and adult mosquitoes held) at 27°c±2°c and 75%-85% relative humidity in a 14:10 (l:d) photoperiod. larvae were fed 5 g of ground dog biscuit and brewer’s yeast daily in a 3:1 ratio. pupae were collected and transferred to plastic containers with 500 ml of water. the container was placed inside a screened cage (90 cm l×90 cm h×90 cm w) to retain emerging adults, for which 10% sucrose in water solution (v/v) was available ad libitum. on days 5 post-emergence, the mosquitoes were provided access to a rabbit host for blood feeding. the shaved dorsal side of the rabbit was positioned on the top of the mosquito cage in contact with the cage screen (using a cloth sling to hold the rabbit) and held in this position overnight. glass petri dishes lined with filter paper and containing 50 ml of water were subsequently placed inside the cage for oviposition by female mosquitoes. larvicidal bioassays a laboratory colony of a. stephensi larvae was used for the larvicidal activity. twenty-five individuals of early fourth-instar larvae were kept in a 500-ml glass beaker containing 249 ml of dechlorinated water, and 1 ml of the desired concentration of plant extracts were added. larval food was provided for the test larvae. at each tested concentration, two to five trials were made and each trial consisted of five replicates. the control was set up by mixing 1 ml of acetone with 249 ml of dechlorinated water. the larvae exposed to dechlorinated water without article no nco mm er cia l u se on ly acetone served as a control. the control mortalities were corrected using abbott’s formula (abbott, 1925). lc50 and lc90 were calculated from toxicity data using probit analysis (finney, 1971). corrected mortality = observed mortality in treatment�– observed mortality in control ¥ 100 100 – control mortality (1) percentage mortality = number of dead larvae ¥ 100 number of larvae introduced (2) adulticidal bioassays sugar-fed adult female mosquitoes (5-6 days old) were used. the m. tinctoria and p. glabra leaf extracts were diluted with acetone to make different concentrations. the diluted plant extracts were impregnated on filter papers (140×120 mm). a blank paper consisting of only ethanol was used as a control. the papers were left to dry overnight at room temperature to let the ethanol evaporate. impregnated papers were prepared fresh prior to testing. the bioassay was conducted in an experimental kit consisting of two cylindrical plastic tubes, both measuring 125×44 mm, following the method of world health organisation (1981). one tube served to expose the mosquitoes to the plant extract and the other tube was used to hold the mosquitoes before and after the exposure periods. the impregnated papers were rolled and placed in the exposure tube. each tube was closed at one end with a 16-mesh wire screen. sucrosefed and blood-starved mosquitoes (20) were released into the tube, and the mortality effects of the extracts were observed every 10 min for a 3-h exposure period. at the end of 1-, 2-, and 3-h exposure periods, the mosquitoes were placed in the holding tube. cotton pads soaked in 10% sugar solution with vitamin b complex were placed in the tube during the holding period for 24 h. mortality of the mosquitoes was recorded after 24 h. the above procedure was replicated three times using plant extracts of each concentration. ovicidal activity assays freshly laid eggs were collected by providing ovitraps in mosquito cages. ovitraps were kept in the cages 2 days after the female mosquitoes were given a blood meal. the eggs were laid on filter paper lining provided in the ovitrap. after scoring, 100 gravid females were placed in a screen cage where 10 oviposition cups were introduced for oviposition 30 min before the start of the dusk period. of these 10 cups, each nine were filled with test solution of 12.5, 25.0, 50.0, 100.0, 200.0, 400.0 ppm, respectively and one was filled with 100 ml of the water and polysorbate 80 that served as a control. the experiment was repeated trice with three replicates. a minimum of 100 eggs was used for each treatment, and the experiment was replicated five times. after treatment, the eggs were sieved through muslin cloth, thoroughly rinsed with tap water, and left in plastic cups filled with dechlorinated water for hatching assessment after counting the eggs under microscope (su & mulla, 1998). the percentage of egg mortality was calculated on the basis of non-hatch of eggs with unopened opercula (chenniappan & kadarkarai, 2008). the hatching rate of eggs was assessed after 98 h post-treatment, as per the method of rajkumar & jebanesan (2009). statistical analysis the average adult mortality data were subjected to probit analysis for calculating lc50, lc90, and other statistics at 95% upper and lower fiducial limits, and chi-square values were calculated by using the spss statistical software package, ver. 16.0. results with p<0.05 were considered to be statistically significant. results the present study explored the potential mosquitocidal properties of two plants, using different solvents for the crude extracts (table 1). chloroform, ethyl acetate, acetone, aqueous, and methanol leaf extracts of the plants m. tinctoria and p. glabra was studied for use as eco-friendly insecticides, as alternatives to potentially harmful synthetic insecticides. results of larvicidal and adulticidal assays with these leaf extracts (tables 2-5) confirm their potential ability to control adult and larval populations of the mosquito a. stephensi. all extracts showed moderate larvicidal effects; however, the highest larval mortality was found with the methanol extract of m. tinctoria and p. glabra against the fourth-instar larvae of a. stephensi (lc50=136.24 and 141.05 ppm; lc90=342.67 and 368.89 ppm, respectively) (figure 1). the chi-square values are significant at the p<0.05 level. the high chi-square values in the bioassays possibly indicate the heterogeneity of the test population. the 95% confidence limits for the lc50 lower/upper fiducidal limits (lcl-ucl) and lc90 (lcl-ucl) were also calculated. no mortality was recorded in the control. the results of the larvicidal assay clearly indicate that the percentage of mortality was directly proportional to concentration of the extract. after exposure to the test concentrations, the treated larvae exhibited restlessness, sluggishness, tremors, and convulsions, followed by paralysis. five different solvents were tested, and the highest adulticidal activity was observed with the methanol extract of m. tinctoria followed by p. glabra, with lc50 values of 194.78 and 198.65 ppm and lc90 values of 336.27 and 357.92 ppm, respectively (figure 2). at higher concentrations, the adults showed restless movement for some time, accompanied by abnormal wiggling movements, [journal of entomological and acarological research 2015; 47:1986] [page 33] article table 1. list of medicinal plants tested for bioactivity against eggs, larvae and adults of anopheles stephensi. botanical name common name family medicinal property plant parts (tamil) tested morinda tinctoria roxb. mannanunai rubiaceae leaves are useful as tonic, febrifuge and emmenagogue. leaves (coffee family) it is also used for curing dyspepsia, diarrhoea, ulceration, stomatitis, digestion, wound and fever. the poultice or the paste of its leaves is applied on the wounds and swellings for relief. the green fruit and leaves are used to treat menstrual cramps, bowel irregularities and urinary tract infections pongamia glabra vent. pungai fabaceae leaves of p. glabra have been known as a remedy for diarrhoea. leaves or leguminosae it is also used as a digestive and laxative and to treat inflammation and wounds. leaf juice aids in treatment of leprosy, gonorrhoea, flatulence, coughs, and colds. the leaf infusions and extracts alleviate rheumatism and itches, respectively no nco mm er cia l u se on ly [page 34] [journal of entomological and acarological research 2015; 47:1986] and death. the mean percentage egg hatch of a. stephensi was also tested against these solvents and leaf extracts; results are shown in table 6. the percentage hatch was inversely proportional to the concentration of extract, and directly proportional to the no. of eggs. of the extracts tested for ovicidal activity, the leaf methanol extract of m. tinctoria resulted in 100% mortality (no hatch) at both 100 and 400 ppm. the leaf extract of m. tinctoria was more effective than p. glabra against larvae and eggs of this mosquito vector. eggs in the control treatment had 100% hatch. discussion and conclusions insect pest control is often a complex, expensive task, frequently requiring the cooperative efforts of communities as well as such groups as industry, agriculture, state, and local governments. we must be concerned with the harmful effects of synthetic pesticides on the environment and people, and reports have emerged on the resurgence of several mosquito-borne diseases in the world as a consequence of the increasing resistance of mosquitoes to commercial insecticides (becker et al., 2003). this has necessitated the need for research and development of an environmentally safe, biodegradable and indigenous material for vector control. many herbal products were used as natural insecticides before the discovery of synthetic organic insecticides (mittal & subbarao, 2003). plant allelochemicals may be quite useful in increasing the efficacy of biological control agents, because plants produce a large variety of compounds that increase their resistance to insect attack (berenbaum, 1988; murugan et al., 1996; senthil nathan et al., 2005). in this study, good larvicidal activity against a. stephensi was achieved with different solvent extracts of m. tinctoria and p. glabra. the mode of action of these leaf extracts on mosquito larvae is not known, but previous studies have demonstrated that phytochemicals interfere with the proper functioning of mitochondria, more specifically at the proton transferring sites (usta et al., 2002). other studies by rey et al. (1999) and david et al. (2000) found that phytochemicals primarily affect the midgut epithe article figure 1. larvicidal activity of a) morinda tinctoria and b) pongamia glabra leaf extracts against anopheles stephensi with lethal concentration (lc)50 and lc90 values. figure 2. adulticidal activity of a) morinda tinctoria and b) pongamia glabra leaf extracts against anopheles stephensi with lethal concentration (lc)50 and lc90 values. no nco mm er cia l u se on ly [journal of entomological and acarological research 2015; 47:1986] [page 35] article table 2. larvicidal activity of different solvent extracts of morinda tinctoria against fourth instar larvar of anopheles stephensi. solvent concentration (ppm) % mortality±sd lc50, ppm lc90, ppm χ2 extracts (lfl-ufl) (lfl-ufl) chloroform control 0.0±0.0 80 26.36±1.62 160 45.44±1.43 191.938 (168.13-212.98) 416.188 (379.32-468.55) 3.751* 240 59.38±1.40 320 71.02±1.96 400 92.22±1.57 ethyl acetate control 0.0±0.0 80 29.48±1.48 160 48.34±1.59 240 62.24±1.52 177.080 (151.89-198.57) 400.763 (365.20-451.21) 3.295* 320 74.42±1.61 400 93.54±1.46 acetone control 0.0±0.0 80 32.82±1.04 160 49.52±1.51 165.573 (139.98-186.98) 382.190 (348.85-429.06) 4.702* 240 64.04±0.99 320 77.06±1.07 400 96.02±1.18 aqueous control 0.0±0.0 80 34.06±1.02 160 51.14±1.12 240 66.44±1.77 156.298 (91.6-198.70) 353.845 (297.79-470.72) 5.452* 320 81.84±1.23 400 98.34±1.31 methanol control 0.0±0.0 80 39.32±1.62 160 56.22±1.78 136.242 (48.50-184.70) 342.678 (282.75-480.98) 6.210* 240 70.06±1.20 320 83.24±1.64 400 99.04±1.16 sd, standard deviation; lc, lethal concentration; lfl, lower fiducidal limits; ufl, upper fiducidal limits; χ2 chi square value. *significant at p<0.05 level. table 3. larvicidal activity of different solvent extracts of pongamia glabra against fourth instar larvar of anopheles stephensi. solvent concentration (ppm) % mortality±sd lc50, ppm lc90, ppm χ2 extracts (lfl-ufl) (lfl-ufl) chloroform control 0.0±0.0 80 23.34±1.47 160 44.36±1.64 240 55.06±1.13 205.973 (182.63-227.268) 436.574 (397.02-493.32) 3.103* 320 69.04±1.18 400 89.22±1.35 ethyl acetate control 0.0±0.0 80 28.42±1.62 160 45.12±1.69 240 60.62±1.78 188.888 (163.09-211.26) 428.639 (388.43-487.00) 2.174* 320 71.02±1.26 400 90.12±1.10 acetone control 0.0±0.0 80 30.06±1.10 160 47.08±1.11 240 62.32±1.53 176.264 (151.03-197.77) 399.610 (364.25-449.68) 1.206* 320 77.06±1.07 400 92.34±1.59 aqueous control 0.0±0.0 80 32.12±1.13 160 49.52±1.35 240 63.08±1.22 166.882 (140.53-188.85) 390.575 (355.82-439.81) 1.264* 320 79.42±1.54 400 93.04±1.06 methanol control 0.0±0.0 80 37.06±1.24 160 56.46±1.67 240 68.38±1.54 141.058 (110.40-165.10) 368.890 (335.10-417.11) 1.965* 320 81.66±1.83 400 95.16±1.42 sd, standard deviation; lc, lethal concentration; lfl, lower fiducidal limits; ufl, upper fiducidal limits; χ2 chi square value. *significant at p<0.05 level. no nco mm er cia l u se on ly [page 36] [journal of entomological and acarological research 2015; 47:1986] lium and secondarily the gastric caeca and the malpighian tubules in mosquito larvae. furthermore, the crude extracts may be more effective than the individual active compounds, due to a natural synergism that discourages the development of resistance in the vectors (maurya et al., 2007). the present investigation revealed that the crude chloroform, ethyl acetate, acetone, aqueous, and methanol leaf extracts of the plants m. tinctoria and p. glabra have significant larvicidal, adulticidal as well as ovicidal activity. these results are comparable to earlier reports of panneerselvam et al. (2012), who observed larvicidal activity of artemisia nilagirica against a. stephensi. they reported lc50 values against the first instar of 272.50 ppm, second instar 311.40 ppm, third instar 361.51 ppm, and fourth instar 442.51 ppm; the corresponding lc90 values were: first instar, 590.07 ppm, second instar, 688.81 ppm, third instar, 789.34 ppm, and fourth instar, 901.59 ppm; the lc50 and lc90 values against the pupae were 477.23 and 959.30 ppm, respectively. the petroleum ether (pe) and methanol (meoh) extracts of rhinacanthus nasutus and derris elliptica exhibited larvicidal effects against a. aegypti, c. quinquefasciatus, anopheles dirus, and mansonia uniformis, with lc50 values between 3.9 and 11.5 mg/l, while the meoh extract gave lc50 values between 8.1 and 14.7 mg/l. d. elliptica pe extract showed lc50 values between 11.2 and 18.84 mg/l, and the meoh extract exhibited lc50 values between 13.2 and 45.2 mg/l, respectively (komalamisra et al., 2005); the n-hexane, ethyl acetate, and methanol extracts of cassia nigricans showed 100% larval mortality against ochlerotatus triseriatus (georges et al., 2008). the leaf hexane, chloroform, ethyl acetate, acetone and methanol extracts of acalypha alnifolia were tested for larvicidial activity against a. stephensi, a. aegypti and c. quinquefasciatus, with lc50 values for a. stephensi of 197.37, 178.75, 164.34, 149.90 and 125.73 ppm, respectively; for a. aegypti, 202.15, 182.58, 160.35, 146.07 and 128.55 ppm, respectively; and for c. quinquefasciatus, 198.79, 172.48, 151.06, 140.69 and 127.98 ppm, respectively (kovendan et al., 2012c). in our results, the larvicidal activity of chloroform, ethyl acetate, acetone, aqueous, and methanol extracts of m. tinctoria and p. glabra exhibited larvicidal effects against a. stephensi with lc50 values of (with m. tinctoria) 191.93, 177.08, 165.57, 156.29 and 136.24; and (with p. glabra) 205.97, 188.88, 176.26, 166.88 and 141.05, respectively. the leaf benzene, petroleum ether, ethyl acetate, and methanol extracts of citrullus vulgaris were previously tested for larvicidial activity against a. stephensi, with lc50 values of 18.56, 48.51, 49.57, and 50.32 ppm, respectively (mullai et al., 2008b). the insecticidal activity of zingiber officinale against third-instar larval maturation and adult emergence of anopheles pharoensis was evaluated at concentrations of 100, 70, 50, 25, 5, 2, 1, 0.9, 0.7, 0.5 and 0.3%, showing 100% larval mortality and, at 0.2% and 0.1%, mortality of 66.7%. the effects of the tested extracts on adult emergence and adulticidal activity against the mosquitoes are remarkably greater than those reported for other plant extracts in the literature. for example, at the highest concentration, 50% inhibition of adult emergence was observed from the ethyl acetate fractions of calophyllum inophyllum seed and leaf, solanum suratense and samadera indica leaf extracts, and the petrol ether fraction of rhinocanthus nasutus leaf extract for c. quinquefasciatus, a. stephensi and a. aegypti (muthukrishnan & puspalatha, 2001). similarly, 88% adult mortality was observed from pelargonium citrosa leaf extracts at 2% concentration against a. stephensi (jeyabalan et al., 2003). adult mortality was caused by the ethanol extract of citrus sinensis, with lc50 and lc90 values of 272.19 and 457.14 ppm, and for a. stephensi, 289.62 and 494.88 ppm, and a. aegypti, 320.38 and 524.57 ppm, respectively (murugan et al., 2012). these findings correspond with those of govindarajan & sivakumar article table 4. adulticidal activity of different solvent extracts of morinda tinctoria against anopheles stephensi. solvent concentration (ppm) % mortality±sd lc50, ppm lc90, ppm χ2 extracts (lfl-ufl) (lfl-ufl) chloroform control 0.0±0.0 160 26.44±1.65 220 37.28±1.62 280 60.14±1.99 253.101 (235.09-269.40) 429.981 (399.62-473.73) 0.864* 340 72.44±1.46 400 86.06±1.00 ethyl acetate control 0.0±0.0 160 27.44±1.45 220 43.36±1.80 280 62.54±1.73 240.968 (222.22-257.28) 414.465 (386.03-455.13) 0.098* 340 76.08±1.94 400 88.06±1.11 acetone control 0.0±0.0 160 33.12±0.97 220 47.02±1.28 280 68.18±1.11 220.466 (201.34-236.39) 378.697 (354.98-411.62) 0.594* 340 84.52±1.39 400 92.62±1.52 aqueous control 0.0±0.0 160 37.36±1.47 220 50.28±1.52 280 74.54±1.74 206.379 (185.55-223.09) 363.338 (340.67-394.75) 1.082* 340 86.06±1.01 400 94.44±1.77 methanol control 0.0±0.0 160 41.02±1.29 220 55.56±1.57 280 76.52±1.40 194.785 (174.49-210.88) 336.270 (316.49-363.13) 1.563* 340 90.54±1.75 400 98.02±1.27 sd, standard deviation; lc, lethal concentration; lfl, lower fiducidal limits; ufl, upper fiducidal limits; χ2 chi square value. *significant at p<0.05 level. no nco mm er cia l u se on ly (2012), who reported on the adulticidal activity of hexane, ethyl acetate, benzene, chloroform and methanol leaf extracts of cardiospermum halicacabum against c. quinquefasciatus, a. aegypti and a. stephensi. the plant extracts showed moderate toxic effects on the adult mosquitoes after an exposure period of 24 h. however, when compared with other solvents, the highest mortality was found with a methanol extract of c. halicacabum against all three species. among them, a. stephensi had the highest lc50 and lc90 values (186.00 and 346.06 ppm). nathan et al. (2005) considered pure limonoids from neem seed, testing for biological, larvicidal, pupicidal, adulticidal and antiovipositional activity. against a. stephensi, larval mortality was dose-dependent, with the highest dose of 1-ppm azadirachtin causing almost 100% mortality, exhibiting pupicidal and adulticidal activity and significantly decreased fecundity and longevity. in the present study, we found the methanol extract of m. tinctoria to have the highest adulticidal activity compared with p. glabra, with lc50 and lc90 values of 194.78, 198.65 ppm and 336.27, 357.92 ppm, respectively. similarly, the greatest adulticidal effect was seen from piper sarmentosum, followed by p. ribesoides and p. longum, with ld50 values of 0.14, 0.15 and 0.26 mg/female, respectively (choochote et al., 2006). adulticidal activity of the essential oil isolated from mentha longifolia was screened using a [journal of entomological and acarological research 2015; 47:1986] [page 37] article table 5. adulticidal activity of different solvent extracts of pongamia glabra against anopheles stephensi. solvent concentration (ppm) % mortality±sd lc50, ppm lc90, ppm χ2 extracts (lfl-ufl) (lfl-ufl) chloroform control 0.0±0.0 160 23.16±1.15 220 36.32±0.81 280 58.52±1.54 262.077 (244.43-278.53) 41.527 (409.68-487.70) 0.677* 340 70.54±1.45 400 83.06±1.14 ethyl acetate control 0.0±0.0 160 25.48±1.70 220 42.36±1.67 280 60.42±1.44 247.164 (228.86-263.40) 421.814 (392.58-463.72) 0.082* 340 75.32±1.64 400 86.38±1.56 acetone control 0.0±0.0 160 30.62±1.77 220 46.38±1.48 280 65.34±1.73 227.727 (208.47-243.94) 393.580 (367.89-429.71) 0.378* 340 82.54±1.71 400 90.02±1.01 aqueous control 0.0±0.0 160 36.08±1.08 220 48.04±1.28 280 72.38±1.81 212.097 (190.37-229.53) 381.306 (356.05-417.02) 1.537* 340 84.76±1.35 400 91.08±1.10 methanol control 0.0±0.0 160 39.24±1.70 220 54.34±1.76 280 74.28±1.64 198.657 (176.28-216.19) 357.921 (335.27-389.43) 0.586* 340 88.72±1.10 400 94.26±1.78 sd, standard deviation; lc, lethal concentration; lfl, lower fiducidal limits; ufl, upper fiducidal limits; χ2 chi square value. *significant at p<0.05 level. table 6. ovicidal activity of different plant leaf extracts against eggs of anopheles stephensi. plant species solvent percent egg hatch concentration (ppm) 12.5 25 50 100 200 400 control m. tinctoria chloroform 88.04±0.95 74.22±1.02 63.52±1.78 53.02±1.20 40.84±1.10 nh 100±0.0 ethyl acetate 81.08±0.94 67.12±0.97 55.46±1.73 44.42±1.35 33.54±1.54 nh 100±0.0 acetone 76.44±1.43 62.14±1.20 49.48±1.40 36.52±1.57 26.02±0.95 nh 100±0.0 aqueous 70.42±1.39 56.32±1.63 40.56±1.72 27.26±1.57 nh nh 100±0.0 methanol 63.36±1.36 51.44±1.77 38.56±1.63 nh nh nh 100±0.0 p. glabra chloroform 80.14±1.15 71.38±1.89 59.08±1.17 49.18±1.04 34.52±1.77 nh 100±0.0 ethyl acetate 75.36±1.57 62.44±1.52 48.98±1.86 38.52±1.07 29.38±1.42 nh 100±0.0 acetone 70.26±1.53 57.28±1.47 43.54±1.01 31.24±1.57 22.98±0.95 nh 100±0.0 aqueous 63.02±0.63 52.62±1.51 38.82±1.11 23.86±0.97 nh nh 100±0.0 methanol 59.32±1.40 46.04±1.31 32.34±1.65 25.54±1.42 nh nh 100±0.0 nh, no hatch. no nco mm er cia l u se on ly [page 38] [journal of entomological and acarological research 2015; 47:1986] fumigant toxicity assay against the house mosquito, culex pipiens l. (diptera: culicidae), by oz et al. (2007). the present study corresponds with the findings of amerasan et al. (2012) who reported the lc50 and lc90 values of cassia tora leaf extracts as adulticidal activity of hexane, chloroform, benzene, acetone, and methanol against c. quinquefasciatus, a. aegypti, and a. stephensi as the following: for c. quinquefasciatus, lc50 values were 338.81, 315.73, 296.13, 279.23, and 261.03 ppm, and lc90 values were 575.77, 539.31, 513.99, 497.06, and 476.03 ppm; for a. aegypti, lc50 values were 329.82, 307.3, and 252.03 ppm, and lc90 values were 563.24, 528.33, 36 496.92, 477.61, and 448.05 ppm; and for a. stephensi, lc50 values were 317.28, 300.30, 277.51, 263.35, and 251.43 ppm, and lc90 values were 538.22, 512.90, 483.78, 461.08, and 430.70 ppm, respectively. the adulticidal activity of the essential oil of lantana camara was evaluated against different mosquito species on 0.208 mg/cm2 impregnated papers, and the knockdown time (kdt)50 and kdt90 values of the essential oil were 20, 18, 15, 12 and 14 min and 35, 28, 25, 18 and 23 min against a. aegypti, c. quinquefasciatus, a. culicifacies, ancylus fluvialitis and a. stephensi, with corresponding percentage of mortalities of 93.3%, 95.2%, 100%, 100% and 100%, respectively (dua et al., 2010). the ovicidal efficacy in the current study compared well with an earlier report; the bioactive compound azadirachtin (a. indica) showed complete ovicidal activity against eggs of culex tarsalis and c. quinquefasciatus exposed to a 10-ppm concentration (su & mulla, 1998). the ovicidal activity of 21 hyphomycete fungi species against a. aegypti was reported. the tested fungi were paecilomyces carneus, paecilomyces marquandii, isaria fumosorosea, metarhizium anisopliae, penicillium sp., paecilomyces lilacinus, beauveria bassiana, and evlachovaea kintrischica. these are the first results to show the effects of entomopathogenic fungi against eggs of a. aegypti, and they suggest their potential as control agents of this vector (luz et al., 2007). assis et al. (2003) reported that egg hatching inhibition of ethyl acetate and methanol extracts of spigelia anthelmia ranged from 97.4-100%, respectively, at 50.0 mg ml−1. the oviposition deterrent properties against a. stephensi have been observed for various plant extracts, including the methanol extract of pelargonium citrosa, which exhibited 56% and 92% inhibition of oviposition at 1 and 4 ppm, respectively (jeyabalan et al., 2003). the benzene extracts of c. vulgaris caused 100% mortality (zero hatch) at 250 ppm, and at 200 ppm a very low hatch rate (11.8%), with complete ovicidal activity at 300 ppm. fraction i at 80 ppm caused a very low hatch rate of 3.2%, followed by fraction ii (6.9%), fraction iii (4.9%), and fraction iv (5.3%) against a. stephensi (mullai et al., 2008a). the leaf extracts of andrographis paniculata, cassia occidentalis, and euphorbia hirta with different solvents (i.e., hexane, ethyl acetate, benzene, aqueous, and methanol) was studied for adulticidal, repellent and ovicidal activity against a. stephensi. among the extracts tested for ovicidal activity against a. stephensi, the leaf methanol extract of a. paniculata caused 100% mortality (zero hatch) at 150 and 300 ppm, respectively (panneerselvam & murugan, 2013a). the leaf extract of cassia fistula in different solvents (methanol, benzene, and acetone) were studied for larvicidal, ovicidal, and repellent activity against a. aegypti (govindarajan, 2009). in the present work, the crude methanol and aqueous extracts of m. tinctoria resulted in zero hatch (100% mortality) at 100 and 200 ppm; followed by crude methanol. the aqueous extract of p. glabra caused zero hatch (100% mortality) at 200 ppm for a. stephensi. in the case of ovicidal activity, exposure to freshly laid eggs was more effective than with older eggs. it has been shown that the age of the embryos at the time of treatment plays a crucial role with regard to the effectiveness of the chitin synthesis inhibitor, dimilin, to c. quinquefasciatus (miura et al., 1976). malarvanan et al. (2009) reported that exposure of cipadessa baccifera, melia dubia, clausena dentata and dodonaea angustifolia to petroleum ether, hexane, chloroform, acetone and water extracts exhibited ovicidal activity against helicoverpa armigera, and maximum activity was observed with the hexane extract of clausena dentate. the leaf extract of solanum trilobatum reduced egg laying by gravid females of a. stephensi from 18% to 99%, compared with ethanol-treated controls at 0.01%, 0.025%, 0.05%, 0.075%, and 0.1% (rajkumar & jebanesan, 2005). recently, ovicidal, repellent, adulticidal and field evaluations of plant extracts were reported against dengue, malaria and filarial vectors (kovendan et al., 2012a). findings of the present investigation reveal that the leaf extracts of m. tinctoria and p. glabra possess remarkable larvicidal, adulticidal and ovicidal activity against this malarial vector. the extract could be used directly as a larvicidal, adulticidal and ovicidal agent in small-volume aquatic habitats or breeding sites of limited size around human dwellings. studies to confirm this hypothesis under field conditions are under way in our laboratory. the results suggest the possible utilisation of cheap and readily available medicinal plants for the possible control of mosquitoes as a part of an integrated vector management program. references abbott w.s., 1925 a method of computing the effectiveness of insecticides. j. econ. entomol. 18: 267-269. ahemed g., yadav p.p., mary r., 2004 furanoflavonoid glycosides from pongamia pinnata fruits. phytochem. 65: 921-924. amer a., mehlhorn h., 2006 larvicidal effects of various essential oils against aedes, anopheles, and culex larvae (diptera: culicidae). parasitol. res. 99: 466-472. amerasan d., murugan k., kovendan k., mahesh kumar p., panneerselvam c., subramaniam j., john william s., hwang j.s., 2012 adulticidal and repellent properties of cassia tora linn. 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amylolytic activities of glyphodes pyloalis walker (lepidoptera: pyralidae) were determined at 24±1°c, 75±5% rh and a photoperiod of 16:8 l:d. fifth instar larvae reared on shin ichinose showed the highest efficiency of conversion of digested food and efficiency of conversion of ingested food (3.82±0.16% and 3.11±0.07%, respectively). approximate digestibility values of the fourth instar larvae were highest (95.23±0.73%) and lowest (91.77±1.45%) on kenmochi and shin ichinose, respectively. the fifth instar larvae fed on kenmochi had the highest consumption index (4.6±0.73) and lowest relative growth rate (0.03±0.10), respectively. our results showed that the highest protease activity in optimal ph was on malalii variety (0.97 u/mg) and the lowest was on kenmochi (0.75 u/mg). in addition, the highest amylase activity in optimal ph was on mahalii (0.17 u/mg) and lowest on kenmochi (0.103 u/mg). specific proteolytic analysis showed that larvae feeding on mahalii had the highest activity of trypsin and elastase (2.30 and 2.13 u/mg, respectively). this research showed that plasticity in food utilization and enzyme activity is functionally relevant to host plant cultivars. the results of nutritional indices and activity of digestive enzymes indicated that kenmochi was an unsuitable host for feeding of glyphodes pyloalis. introduction mulberry is the sole host for silkworm (bombyx mori l) rearing and is also used for shade trees in cities (kumar et al., 2002). the lesser mulberry pyralid, glyphodes pyloalis walker (lepidoptera: pyralidae) is considered a serious pest of mulberry in india, china, korea, japan, malaysia, pakistan, uzbekistan and burma (madyarov et al., 2006). this pest has caused severe damage to mulberry plantings in northern iran and has turned into a serious concern for the silk industry (khosravi & sendi, 2010). larvae form threads on the outer part of mulberry leaves and feed on the mesophyll from under those threads, leaving only a network of epidermis (aruga, 1994). if leaves infected with excreta of larvae are fed to silkworms, they develop constipation and are unable to defecate (aruga, 1994). in addition, g. pyloalis larvae are considered alternate hosts of bombyx densoviruses and picornaviruses (watanabe et al., 1988) nutrition is the interaction of physiological processes and ecology, so it is directly associated with natural selection as well as competition for food (sheikher et al., 2001; zhu et al., 2005; xue et al., 2010). in numerous studies of the relationships between insect pests and plants, attempts have been made to quantify the efficiency with which insects use their food plants (david & gardiner, 1962; sheikher et al., 2001; xue et al., 2010). together with data on the rate of food ingestion and growth, food utilisation efficiency is an important component of herbivore performance (slansky & scriber, 1985). the quality and quantity of food consumed could affect the growth, development, and reproduction of insects (scriber & slansky, 1981). of the tools of pest management, host plant resistance is important in terms of being both economically and environmentally acceptable. therefore, as a method of controlling pest insects, host plant resistance is not only favourable to the environment, but also reduces expenses for growers (li et al., 2004). the factors determining nutrient availability for growth and maintenance over a given period of development are the amount and type of food consumed and the efficiency with which it is utilized (barton browme & raubenheimer, 2003). the study of host plant resistance can play an important role in identifying anti-digestive or antifeedant compounds and their further use in pest management strategies (lewis et al.,1997). a possible strategy to control insect pests is to produce crops with elevated levels of endogenous resistance. one such approach has been to overexpress plant proteins that are known to play a role in plant defence against herbivores (hosseininaveh et al., 2007). many plants respond to insect feeding by the synthesis of protease inhibitors (pis) (ryan, 1990). plant pis have been shown to inhibit gut proteases of insects and prevent larval growth and development when delivered in artificial diets (johnston et al., 1993) or when expressed in transgenic crops (hilder, 1987). efforts are being made to explore their use in developing insect resistance in susceptible crop plants (sharma et al., 2000). however, as a first step to achieving this goal, an understanding of how correspondence: jalal jalali sendi, department of plant protection, faculty of agricultural sciences, university of guilan, rasht, 416351314, iran. tel.: +981316690817 fax: +981316690281. e-mail: jjalali@guilan.ac.ir key words: glyphodes pyloalis, feeding indices, digestive enzymes. received for publication: 2 may 2013. revision received: 9 november 2013. accepted for publication: 25 november 2013. ©copyright m. oftadeh et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1633 doi:10.4081/jear.2014.1633 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. effect of four varieties of mulberry on biochemistry and nutritional physiology of mulberry pyralid, glyphodes pyloalis walker (lepidoptera: pyralidae) m. oftadeh, j.j. sendi, a. zibaee, b. valizadeh department of plant protection, university of guilan, rasht, iran [page 42] [journal of entomological and acarological research 2014; 46:1633] journal of entomological and acarological research 2014; volume 46:1633 no nco mm er cia l u se on ly digestive enzymes function is essential to plan strategies for successful and sustainable implementation of pi-based control methods (franco et al., 2002). insect digestive proteases catalyze the release of peptides and amino acids from dietary proteins in the insect digestive canal to meet its nutritional requirements (terra & ferreira, 1994). the lepidopteran larval midgut has been shown to have complex proteolytic activities including trypsins, chymotrypsins, elastases, cathepsin-b-like proteases, aminopeptidases, and carboxypeptidases that are all required in protein digestion. lepidopteran insects mainly depend on serine proteases for protein digestion (bown et al., 1997). the other important classes of digestive enzymes from these insects include a-amylases, which are also the main digestive enzymes of many other insects that feed absolutely on starchy seeds throughout their life (pereira et al., 1999). the a-amylases (a-1, four glucan four glucanohydrolases; ec 3.2.1.1) catalyze hydrolysis of a -d-(1, 4)-glucan linkage in starch components glycogen, and other different associated carbohydrates to supply an energy source (franco et al., 2000). in insects, the activity of digestive enzymes such as proteases and a-amylases depends on the nature of food sources or chemical compounds ingested (mendiola-olaya et al., 2000). protease and a-amylase activities in crude extracts of larval guts of different lepidopteran species have been described (zibaee et al., 2008). study of the insect digestive system is an important method for discovering new control techniques in integrated pest management programs (lawerence & koundal, 2002). many factors are involved in the host preference of insects. among the most important are plant species and regional plant diversity (davidson et al., 2001), chemical composition of leaves (foss & riseke, 2003), and leaf age, which itself is the cause of physical and chemical changes (meyer & montgomery, 2004). the goal of this research was to compare nutritional indices and activity of digestive enzymes in g. pyloalis larvae reared on different host plants, and to determine how these parameters change after an additional instar and in response to different hosts. materials and methods plant sources four host plants were used in this study, including kenmochi, ichinose, shin ichinose and mahalii. these varieties were selected because they are the most important economic varieties used in iran to rear silkworms. insect rearing g. pyloalis larvae were collected from mulberry orchards near the city of rasht in northern iran. they were reared on fresh mulberry leaves (different host plants) in the laboratory at 24±1°c, 75±5% rh and 16:8 (l:d) h photoperiod in transparent plastic boxes (18×15×7 cm) covered with muslin for aeration. as adults emerged, they were separated and placed in transparent plastic boxes (18×7 cm) with a 10% honey solution on cotton wool for feeding, and mulberry leaves were provided for oviposition. nutritional indices nutritional indices were determined using second to fifth instars, which were easier for measuring these indices than on the 1st instar. ten newly emerged second instar larvae that had been reared on each of the four varieties of mulberry as host plants were selected and provided with 10 fresh leaves from each of the four hosts; they were individually weighed and maintained in small plastic tubes (2×5 cm) until they stopped feeding before molting to the next instar. this method was used for other larval instars as well. the initial fresh food and the food and feces remaining at the end of each experiment were weighed daily. the quantity of food ingested was determined by subtracting the diet remaining at the end of each experiment from the total weight of diet provided. the weight of feces produced by the larvae fed on each mulberry variety was recorded daily. to find the dry weights of the pods, feces, and larval to adult stages, extra specimens (20 specimens for each) were weighed, oven-dried (48 h at 60°c), and then re-weighed to establish a percentage of their dry weight. food utilization rates were then calculated based on the following formulas of waldbauer (1968): i) consumption index (ci): calculated on the basis of the rate of intake relative to the mean weight of the larva during the feeding period, according to the following formula: ci=f/ta; ii) relative growth rate (rgr): calculated as: rgr=g/ta; iii) efficiency of conversion of ingested food (eci): the efficiency of conversion of ingested food to body substance is calculated as: eci=wg/fi×100; iv) approximate digestibility (ad): the approximate digestibility is calculated as: ad=fi-wf/fi×100; v) efficiency of conversion of digested food (ecd): the efficiency with which digested food is converted to body substance is calculated as: ecd=wg/fiwf×100. where is a=mean fresh weight of the larva during the feeding period, f=fresh weight of food eaten, t=duration of feeding period (days), g=fresh weight gain of the larva during the feeding period, wg=weight gained, fi=weight of food ingested, wf=weight of feces. biochemical assessments the guts of fifth instar larvae were used to measure proteolytic and amylolytic activities, and the whole body to measure other biochemical assessments as follows: fifth-instar larvae reared on different hosts were cold anesthetized and quickly dissected under a stereomicroscope. the midguts were then cleaned by deletion of unneeded tissues. the midguts, including contents, were collected into a known amount of distilled water and homogenized with a handheld glass grinder on ice, and the homogenates were centrifuged at 16,000× g for 10 min at 4°c. the resulting supernatant was stored at –20°c for later protease and amylase assays. each biochemical analysis was repeated 3 times. general proteolytic activity present in the midgut of g. pyloalis larvae fed on different hosts was determined using azocasein as a substrate at an optimum ph. a universal buffer system (50 mm sodium phosphate-borate) was used to determine the ph optimum of proteolytic activity over a ph range of 7-12. to determine the azocaseinolytic activity, the reaction mixture containing 80 ml of 1.5% azocasein solution in 50 mm universal buffer (ph 12) and 50 ml of crude enzyme was incubated at 37°c for 50 min. proteolysis was terminated by the addition of 100 ml of 30% trichloroacetic acid (tca) followed by cooling at 4 °c for 30 min and centrifugation at 16,000 × g for 10 min. an equal volume of 2 m naoh was added to the supernatant, and the absorbance was measured at 440 nm. appropriate blanks in which tca had been added prior to the substrate were prepared for each assay. unit activity was expressed as an increase in optical density mg−1 protein of the tissue min−1 due to azocasein proteolysis (vinokurou et al., 2007). digestive trypsin-, chymotrypsinand elastase-like activities of the larvae fed on either all varieties of mulberry were estimated using final concentrations of 1 mm bapna, 1mm saapfpna and 1 mm saaapna as substrates, respectively. a reaction mixture consisted of 20 ml of enzyme extract for trypsinand elastase-like activities and 10 ml of enzyme extract for chymotrypsin-like activity, 75 ml of universal buffer at the appropriate ph optimum (ph 10.5 for trypsinand chymotrypsinlike enzymes and ph 11 for elastase-like enzyme) and 5 ml of the above-mentioned substrate. absorbance was then measured at 405 nm for 40 min (at 2, 1 and 4 min time intervals respectively). all assays were carried out in triplicate against appropriate blanks. the a-amylase activity was measured using the procedure of bernfeld (1955), with 1% soluble starch as substrate. a quantity of 50 [journal of entomological and acarological research 2014; 46:1633] [page 43] article no nco mm er cia l u se on ly [page 44] [journal of entomological and acarological research 2014; 46:1633] ml of the enzyme was incubated with 250 ml of universal buffer (ph 10) and 20 ml of soluble starch for 30 min at 37°c. the reaction was terminated by addition of 50 ml dns and heating in boiling water for 10 min. the absorbance was then read at 540 nm after cooling on ice. one unit of amylase activity was defined as the amount of enzyme required to produce 1 mg of maltose in 30 min at 37°c under the given assay conditions. alanine aminotransferase (alt) and aspartate aminotransferase (ast) were measured using thomas’ (1998) procedure. this assay was done by ast and alt kit (biochem co., tehran, iran). the absorption was read at 340 nm. assays of estimation of acid (acp) and alkaline phosphatases (alp) were carried out as described by bessey et al. (1946). the buffered substrate (phosphate buffer, 0.02 m, ph 7.2) was incubated with the samples for 30 min. alkali was added to stop the reaction and to adjust the ph for the determination of concentration of the product formed. the spectral absorbance of p-nitrophenolate was maximal at 310 nm. the molar absorbance of p-nitrophenolate at 400 nm is about double that of p-nitrophenyl phosphate at 310 nm. on converting the p-nitrophenolate into pnitrophenol by acidification, the absorption maximum is shifted to about 320 nm with no detectable absorption at 400 nm. glucose was analyzed as described by sigert (1987). protein was measured based on bradford’s (1976) method and by utilizing a total protein assay kit (biochem co.). in this method, proteins made a complex purplish blue with an alkaline copper solution, for which the absorption value was read at 540 nm. to measure the total cholesterol of hemolymph, richmond’s (1973) method was conducted. the principles of this method are based on hydrolysis of cholesterol esters by cholesterol oxidase, cholesterol esterase and peroxidase. glycogen and trehalose content were assessed using the method of van handel (1965). this method separates glycogen and trehalose. trehalose and glycogen were similarly assayed using the anthrone-sulfuric acid method. chemical analysis of the leaves leaves from all four mulberry trees were collected during the experimental period in july 2012, dried in the shade, and prepared for chemical analysis. for phosphorous, calcium and potassium, dry ashing and mixing with hcl was first performed. total organic nitrogen content was determined by the micro-kjeldahl method, potassium content was measured by flame photometry, using a lithium internal standard, and phosphorus content was determined using the colorimetric method with blue coloured acid ascorbic and read at 880 nm. for measuring calcium, the compleximetric method was used. statistical analysis nutritional indices of g. pyloalis reared on different hosts were analyzed with one-way anova using the statistical software minitab ver. 14.0 (minitab inc., philadelphia, pa, usa. http://www.minitab.com; 1994) to determine similarities and significant differences. statistical differences among the means were assessed using an lsd test at a=0.01. data were tested for normality before analysis. the data from other experiments were subjected to analysis of variance (anova) using sas software. the least significant differences among treatments were compared using tukey’s multiple range test (sas institute, cary, nc, usa; 1997). differences among means were considered significant at p≤0.01. results nutritional indices the results of the nutritional indices of secondfifth larval instars of g. pyloalis are shown in tables 1-4. nutritional indices of the second instar larvae of g. pyloalis were significantly different for different host plants. the larvae reared on kenmochi showed the highest value of ecd (f=17.70; df=3; p<0.0007) (1.15±0.12%) and ci (f=143.38; df=3; p<0.0001) (7.14±0.9). however, the lowest value of ecd (f=0.91; d=3; p<0.0004) and ci (f=0.35; d=3; p<0.0007) was on ichinose (0.45±0.20% and 3.28±0.04, respectively). also, the highest value of eci (f=18.91; d=3; p<0.0005) (1.13±0.12%) was on kenmochi compared with the other hosts. however, the larvae reared on ichinose had the highest value of ad and the lowest value of ci (98.00±0.91 and 3.28±0.04, respectively). the highest and lowest value of rgr (f=18.6; article table 1. nutritional indices of second instar larvae of glyphodes pyloalis on different hosts (means±se). host ci ad eci ecd rgr ichinose 3.28±0.04d 98.00±0.91a 0.44±0.20c 0.45±0.20c 0.02±0.00d shin ichinose 4.96±0.07c 97.50±0.27a 0.64±0.26bc 0.66±0.03bc 0.03±0.00c kenmochi 7.14±0.09a 97.60±0.25a 1.13±0.12a 1.15±0.12a 0.05±0.01b mahalii 5.85±2.02b 96.99±0.72a 0.82±0.27b 0.85±0.28ab 0.07±0.00a ci, consumption index; ad, approximate digestibility; eci, efficiency of conversion of ingested food; ecd, efficiency of conversion of digested food; rgr, relative growth rate. a,b,c,dmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). table 2. nutritional indices of third instar larvae of glyphodes pyloalis on different hosts (means±se). host ci ad eci ecd rgr ichinose 5.20±0.10c 94.64±1.20a 1.51±0.16a 1.54±0.07a 0.04±0.01b shin ichinose 4.83±0.60c 91.69±0.89b 1.32±0.12b 1.45±0.13b 0.17±0.02a kenmochi 9.23±0.40a 95.12±0.55a 0.98±0.22c 1.05±0.24c 0.06±0.02b mahalii 7.88±0.26b 94.60±0.50a 1.55±0.12a 1.64±0.13a 0.08 ±0.01b ci, consumption index; ad, approximate digestibility; eci, efficiency of conversion of ingested food; ecd, efficiency of conversion of digested food; rgr, relative growth rate. a,b,cmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). no nco mm er cia l u se on ly d=3; p<0.0003) were on mahalii and ichinose (0.07±0.00 and 0.02±0.00, respectively) (table 1). the highest (95.12±0.55%) and lowest (91.69±0.89%) ad values (f=10.51; df=3; p<0.0038) of the third instar larvae of g. pyloalis were on kenmochi and shin ichinose, respectively. the shin ichinose and ichinose showed the highest and lowest values of rgr (f=51.57; df=3; p<0.0001) (0.17±0.02 and 0.04±0.01), respectively. the highest (9.23±0.40%) and lowest (4.83±0.60%) ci values (f=100.63; df=3; p<0.0001) were on kenmochi and shin ichinose, respectively (table 2). in the fourth instar, the highest (95.23±0.74) and lowest (91.77±1.46) values of ad (f=6.71; df=3; p<0.0141) were on kenmochi and shin ichinise. the larvae fed on shin ichinose had the highest eci (f=9.58; df=3; p<0.005) and ecd (f=16.69; df=3; p<0.0001) values (0.80±0.06 and 0.97±0.21, respectively). however, the lowest value of eci and ecd (0.40±0.03 and, 0.41±0. 03 respectively) was observed on kenmochi (table 3). it was observed that larvae fed on kenmochi in the fifth instar had the highest ci (f=26.84; df=3; p<0.0002) and ad (f=0.35; df=3; p<0.7885) values (4.60±0.73 and 89.22±5.60, respectively) and the larvae that fed on mahalii had the lowest values of ci and ad (1.86±0.06 and 77.54±5.63). the highest and lowest rgr values (f=29.67; df=3; p<0.0001) were observed on mahalii and kenmochi (0.10±0.00 and 0.04±0.10, respectively) (table 4). in most cases, the highest and lowest values of ad (f=7.48; df=2, 116; p<0.01) were on the second and fifth instars, respectively. the highest and lowest values of eci (f=8.75; df=2, 114; p<0.01) and ecd (f=15.56; df=2, 113; p<0.01) were on the fifth and fourth instars, respectively. biochemical assessments the results of biochemical assessments of g. pyloalis larvae fed on different host are shown in table 5. the larvae reared on mahalii (0.97±0.02 u/mg) showed the highest levels of proteolytic activity. however, protease activity was the lowest in midgut extracts from larvae fed on kenmochi (0.75±0.03 u/mg) (f=6.86, df=3, p<0.0013). it was observed that larvae fed on mahalii had the highest activity of trypsin (f=26.8, df=3, p<0.0001) and elastase (f=58.86, df=3, p<0.008), (2.30±0.0 and 2.13±0.00 u/mg, respectively) and the larvae that fed on kenmochi had the lowest activity of trypsin and elastase (0.54±0.00 and 1.36±0.02 u/mg, respectively). the larvae reared on kenmochi (1.31±0.01 u/mg) showed the highest activity of chymotrypsin (f=6.35, df=3, p<0.003). as for proteolytic activity, the highest levels of amylase activity (f=1.51, df=3, p<0.0028) were found on the larvae that fed on mahalii (0.17±0.10 u/mg). as illustrated in table 5, the highest activity level of alt (f=59.27, df=3, p<0.0001) and ast (f=3.59, df=3, p<0.006) were found on shin ichinose (8.92±0.69 and 15.65±2.44 u/mg, respectively) and the lowest on kenmochi. the larvae that fed on mahalii had the highest alp (f=3.55, df=3, p<0.006) and acp (f=8.49, df=3, p<0.003) values (0.09±0.05 and 0.11±0.07 u/mg, respectively) and were lowest on ichinose (0.02±0.01and 0.04±0.01, respectively). the highest total protein was observed on mahalii, while the lowest was on kenmochi (1.19±0.20, 0.89±0.12, respectively) (f=4.39, df=3, p<0.0019). it was observed that larvae fed on mahalii had the highest trehalose (f=3.43, df=3, p<0.003) and cholesterol (f=0.49, df=3, p<0.0009) values [21.14±2.53 (mg/dl) and 0.08±0.04 (mg/dl) respectively], and larvae reared on shin ichinose showed the highest level of glucose (f=10.45, df=3, p<0.003) 0.98±0.02 (mg/dl) (table 5). chemical analyses of the leaves the results of leaf analysis demonstrated that phosphorus content was highest in shin ichinose and lowest in ichinose (0.34±0.02 and 0.24±0.01), respectively. the other two varieties were not significantly different from each other (f=6.57; df=3; p>0.0150). potassium content was not significantly different among the varieties (f=2.76; df=3; p>0.1113), while the highest nitrogen content was observed in mahalii (3.07±0.24) and the lowest in kenmochi (2.07±0.68) (f=5.88; df=3; p>0.0202). similarly, protein content was highest in mahalii and lowest in kenmochi (19.18±0.52 and 12.93±2.67, respectively). however, calcium content was highest in shin ichinose and kenmochi and lowest in mahalii and ichinose (f=8.03; df=3; p>0.0085) (table 6). [journal of entomological and acarological research 2014; 46:1633] [page 45] article table 3. nutritional indices of fourth instar larvae of glyphodes pyloalis on different hosts (means±se). host ci ad eci ecd rgr ichinose 2.60±0.22bc 92.53±0.59b 0.76±0.25a 0.79±0.21ab 0.03±0.04b shin ichinose 2.23±0.18c 91.77±1.46b 0.80±0.06a 0.97±0.21a 0.02±0.00b kenmochi 4.62±0.10a 95.23±0.74a 0.40±0.03b 0.41±0.03b 0.02±0.01b mahalii 2.93±0.05b 91.90±1.29b 0.58±0.33ab 0.63±0.37ab 0.06±0.02a ci, consumption index; ad, approximate digestibility; eci, efficiency of conversion of ingested food; ecd, efficiency of conversion of digested food; rgr, relative growth rate. a,b,cmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). table 4. nutritional indices of fifth instar larvae of glyphodes pyloalis on different hosts (means±se). host ci ad eci ecd rgr ichinose 3.14±0.27b 81.80±3.89ab 2.64±0.13ab 2.82±0.24b 0.05±0.01bc shin ichinose 2.34±0.19bc 81.98±0.52ab 3.10±0.09a 3.82±0.16a 0.06±0.01b kenmochi 4.60±0.73a 89.22±5.60a 2.23±0.36b 2.81±0.50b 0.04±0.10c mahalii 1.86±0.06c 77.54±5.63b 2.68±0.15ab 3.20±0.29ab 0.10±0.00a ci, consumption index; ad, approximate digestibility; eci, efficiency of conversion of ingested food; ecd, efficiency of conversion of digested food; rgr, relative growth rate. a,b,cmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). no nco mm er cia l u se on ly [page 46] [journal of entomological and acarological research 2014; 46:1633] discussion and conclusions the use of resistant varieties is one of the core strategies of an integrated pest management program, and secondary substances of plants or allelochemicals play a major role in plant resistance to pests (wilson & huffaker, 1976). differences in allelochemical concentrations between host plant varieties can affect an insect’s performance as a larva (martin & pulin, 2004). it is generally accepted that low dietary protein can cause an increase in the rate at which larvae feed (rausher, 1981; slansky, 1993); conversely, a high protein diet can reduce feeding rates (mattson, 1980). in our study, the highest level of protein was recorded in leaves of the variety mahalii. however, the lowest ci was recorded in fifth instar g. pyloalis larvae fed on mahalii leaves. also, the highest ci was observed on the variety kenmochi, which had the lowest leaf protein content. hemati et al. (2011) recorded that h. armigera larvae had the highest ci values when they were fed on tomato leaves, for which kotkar et al. (2009) reported very low protein content. significant differences were found within the nutritional indices, especially eci and ecd values, of g. pyloalis reared on different mulberry varieties, suggesting that the varieties have different nutritional value. among nutritional indices, eci may vary with the digestibility of food and the proportional amount of the digestible portion of food that is converted to body mass and metabolized for energy needed for vital activity (abdel-rahman & al-mozini, 2007). eci is the insect’s ability to utilize the food ingested for growth and development, and ecd is a measure of the efficiency of conversion of digested food into growth (senthil-nathan et al., 2005). change in ecd also indicates the overall increase or decrease of the proportion of digested food metabolized for energy (koul et al., 2004). for the fifth larval instars, the highest eci and ecd values were on shin ichinose, suggesting that the larvae were more efficient at conversion of ingested and digested food to body biomass with a high increase in larval weight. despite kenmochi’s having the highest ci value, it also had the lowest values of eci and ecd (table 4), indicating that larvae feeding on this host were less effective in converting ingested and digested food into biomass. it is well known that the degree of food utilization depends on the digestibility of food, and the efficiency with which digested food is converted into biomass (batista pereira et al., 2002). the reduction in dietary utilization suggests that reduction in nutritional values may be a result of both behavioural and physiological effects (senthil-nathan et al., 2005). approximate digestibility (ad) and efficiency of conversion of digested food (ecd) are inversely related (waldbauer, 1968; scriber & slansky, 1981) and this is demonstrated in larvae reared on kenmochi, which had the highest rate of ad and lowest rate of ecd. reduced rgr due to decreased ecd, despite an observed increase in ad, was noticed in larvae of 4th and 5th instar reared on the variety kenmochi. growth reduction in response to a new environment (host) has been previously shown in phytophagous insects (grabstein & scriber, 1982; sheppard & friedman, 1990; lazarevic & pericmataruga, 2003). lepidopteran larvae fed on high-nutrient food have increased growth rates and a shorter developmental period than larvae fed on lownutrient food (hwang et al., 2008). our results showed that rgr values article table 5. comparison of biochemical compounds of glyphodes pyloalis on four varieties of mulberry. compounds ichinose shin ichinose kenmochi mahalii a-amylase (u/mg) 0.11±0.01b 0.10±0.05b 0.10±0.05b 0.17±0.10a ptotease (u/mg) 0.89±0.01a 0.80±0.04ab 0.75±0.03b 0.97±0.02a trypsin (u/mg) 2.14±0.00b 1.91±0.01c 0.54 ±0.00d 2.30±0.02a chymotrypsin (u/mg) 0.66±0.02b 0.83±0.01b 1.31±0.01a 0.65±0.01b elastase (u/mg) 1.75±0.01b 1.86±0.03a 1.36±0.02c 2.13±0.00a protein (mg/dl) 0.97±0.04ab 1.03±0.02ab 0.89±0.12b 1.19±0.20a glucose (mg/dl) 0.80±0.01b 0.98±0.02a 0.92±0.08a 0.95±0.01a cholesterol (mg/dl) 0.01±0.00b 0.02±0.01b 0.02±0.01b 0.08±0.04a glycogen (mg/dl) 0.02±0.00a 0.02±0.00a 0.02±0.00a 0.02±0.00a trehalose (mg/dl) 12.86±2.63c 18.45±0.66ab 17.53±5.57b 21.14±2.53a alkalinaminoteransferase (iu/l) 6.60±0.66b 8.92±0.69a 2.99±0.47c 6.03±0.27b aspartate aminoteransferase (iu/l) 13.35±2.57a 15.65±2.44a 3.99±0.58b 6.95±1.59b alkaline phosphatase (iu/l) 0.04±0.01b 0.04±0.01b 0.05±0.02b 0.09±0.05a acids phosphatase (iu/l) 0.02±0.01b 0.02±0.01b 0.04±0.01b 0.11±0.07a a,b,c,dmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). table 6. chemical analyses of leaves of four different varieties of mulberry (means±se; ingredient %d.m.). host p k n ca protein ichinose 0.24±0.01b 2.31±0.05a 2.62±0.11ab 2.59±1.14b 16.37±0.72ab shin ichinose 0.34±0.02a 2.44±0.02a 2.76±0.61ab 3.22±0.22a 17.25±1.60ab kenmochi 0.32±0.01ab 2.42±0.04a 2.07±0.68b 3.16±0.17a 12.93±2.67b mahalii 0.32±0.16ab 2.47±0.39a 3.07±0.24a 2.59±0.48b 19.18±0.52a a,bmeans followed by different letters in the same columns are significantly different (lsd, p<0.01). no nco mm er cia l u se on ly were highest on mahalii and lowest on kenmochi, indicating that kenmochi was a low-nutrient food for larvae, and a longer period of development was therefore needed by the immature stages. conversely, the mahalii plants were high-nutrient foods for larvae, and a shorter period of development was needed by the immature stages. the data on nutritional indices for the fourth and fifth instars of g. pyloalis are not consistent. this is because the nutritional requirements of the insect change through development, and such differences typically result in changes in food consumption and feeding behavior (barton browme, 1995). analysis of nutritional indices can lead to an understanding of the behavioural and physiological bases of insect response to host plants (lazarevic & peric-mataruga, 2003). the lower fitness of g. pyloalis on some hosts may be due to the presence of some secondary phytochemicals in these hosts, or the absence of primary nutrients necessary for growth and development. to obtain more applicable information for g. pyloalis control, more attention should be devoted to studying the demographic parameters of this pest under laboratory and field conditions, as well as to investigate its nutritional indices on different varieties of mulberry under field conditions. isolation and study of distinct proteases and amylases is of little aid in ascertaining the composition of the midgut or designing pi-based approaches for insect resistance, especially when the insect has the ability to modify midgut compounds within a single generation in order to deactivate the influence of pis (patankar et al., 2001; vinokurov et al., 2007). insects adjust to plant pis by producing inhibitor-insensitive, inhibitor-resistant and inhibitor-declining proteinases in their midgut to compensate for the influence of transgenic or dietary pis (jongsma et al., 1995; broadway, 1997; michaud, 1997; girard et al., 1998; giri & kachole, 1998). therefore, determination of the midgut protease and amylase activities induced upon intake of pis will be essential for selecting pis or a mixture of pis for expanding insect resistance (patankar et al., 2001). in the current research, higher protease activities in the mahalii-fed larvae may have been due to the high protein content of the diet or to the response of the insect to the dietary pis that partially inhibit midgut protease activity (patankar et al., 2001). additionally, hyperproduction of proteases in response to ingested pis leads to an extra load on the insect for energy and essential amino acids, resulting in a retardation of insect growth (broadway & duffy, 1986). the highest trypsinand elastase-like activities were also in mahalii-fed larvae compared with the other varieties, and proteases and trypsin activity in the larvae reared on kenmochi and shin ichinose were lowest. lectins are carbohydrate-binding proteins distributed in different species of plants (etler, 1986; ratanapo, 2005). they play different roles in plants; many of them have a direct inhibitory effect on some digestive enzymes of higher animals and insects, including a-amylases (thompson & gabon, 1986; fish & thompson, 1991), and esterases and proteases (belzunces et al., 1994; thompson et al., 1986). ratanapo et al. (2005) reported two mulberry leaf lectins, mll1 and mll2, that have an inhibitory effect on trypsin-like alkaline proteases purified from the digestive fluid of the fifth larval instar of the silkworm, bombyx mori. anti-proteolytic effect of lectins on the digestive proteases might occur prior to silkworm digestion of food protein. in this research, lower activity of protease and trypsin in shin ichinose than in ichinose may indicate the existence of lectin/s in this variety. however, the larvae fed on kenmochi exhibited the highest chymotrypsin activity compared with other varieties. the tentative answer to this phenomenon could be the over-expression of chymotrypsin-like enzymes in response to the trypsin inhibitors in this variety. trypsin and chymotrypsin occur as multiple isoforms in insects (lam et al., 1999, 2000; lopes et al., 2006; sato et al., 2008). the highest levels of amylase activity were found on the larvae that fed on mahalii. the reason could be that this host had the highest balance of carbohydrates. according to chemical analyses of leaves, we observed that the varieties mahalii and kenmochi had the highest and lowest levels of protein, respectively. hemati et al. (2011) showed that the larvae reared on the tomato cultivar dehghan had the highest level of proteolytic and amylolytic activity. the aminotransferases are enzymes that catalyze the reaction between an amino acid and a keto acid. this reaction removes the amino group from the amino acid, leaving a keto acid and converting it into an amino acid. these enzymes serve as a link between the carbohydrates and protein metabolism and are altered during various physiological processes (etebari et al., 2004; shekari et al., 2008). our results show that shin ichinose and kenmochi had the highest and lowest level of alt and ast, respectively. the reason may be that kenmochi had the lowest balance of protein. acp and alp are the hydrolase enzyme responsible for removing phosphate groups from many types of molecules, including nucleotides, proteins, and alkaloids in alkaline and acidic conditions, both of which were highest in mahalii. according to the results of chemical analyses of the leaves, mahalii leaves indeed had the highest level of protein (table 6). glycogen is a polymer of several glucose residues in a branched chain storage form (klowden, 2007; lide, 1998); both glucose and glycogen were highest in the larvae that were reared on shin ichinose. in this research, we observed that the larvae that were reared on mahalii and kenmochi had the highest and lowest total protein, respectively, which corresponds with other results showing that these varieties have the highest and lowest protein contents. in this study, we found that the chemical compounds present in the host plant can play an important role in the feeding activity, digestive enzyme activity and chemical compounds stored in the plant when fed upon by pests. based on our results, the variety mahalii had the highest nutritional indices and the highest activity of digestive enzymes to make it the best host for g. pyloalis. the variety kenmochi had the lowest activity of digestive enzymes and nutritional indices, identifying it as an unsuitable host. the low activity of digestive enzymes and nutritional indices of g. pyloalis on kenmochi could possibly indicate the presence of some enzyme inhibitors in this variety. however, the suitability of the mahalii and shin ichinose varieties could be due to the protein content and nutritional value of these hosts in comparison to other hosts in this study. the inappropriateness of some of the hosts for g. pyloalis may be due to some chemical secondary compounds in the hosts, or absence of an essential nutrient for the growth of g. pyloalis. references abdel-rahman h.r., al-mozini r.n., 2007 antifeedant and toxic activity of some plant extracts against larvae of cotton leafworm spodoptera littoralis (lepidoptera: noctuidae). -pak. j. biol. sci. 10: 4467-4472. aruga h., 1994 principles of sericulture, 1st ed. crc press, boca raton, fl: 79-80. barton browme l., 1995 ontogenetic changes in feeding behavior. in: chapman r.f., de boer g. 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(eds.), theory and practice of biological control. academic press, new york: 3-15. xue m., pang y.h., wang h.t., li q.l., liu t.x., 2010 effects of four host plants on biology and food utilization of the cutworm, spodoptera litura. j. insect. sci. 10: 22. zhu j.h., zhang f.p., ren h.g., 2005 development and nutrition of prodenia litura on four food plants. chinese bull. entomol. 42: 643-646. zibaee a., bandani a.a., kafil m., ramzi s., 2008 characterization of a-amylase in the midgut and the salivary glands of rice striped stem borer, chilo suppressalis walker (lepidoptera: pyralidae). j. asia-pacific. entomol. 11: 201-205. [journal of entomological and acarological research 2014; 46:1633] [page 49] article no nco mm er cia l u se on ly j. ent. acar. res. ser. ii, 43 (3): 333-336 30 december 2011 s. longo, p. suma first report of eurytoma plotnikovi nik. (hymenoptera, eurytomidae), a seed parasite of pistachio, in sicily (italy) abstract - the pistachio seed wasp, eurytoma plotnikovi nik.(hymenoptera, eurytomidae), is a new pest recently arrived in pistachio orchards in central-western  sicily (italy). information on the damaging effects of this seed wasp in the affected  areas is provided. riassunto - prima segnalazione di eurytoma plotnikovi nik. (hymenoptera, eurytomidae), dannoso per il seme di pistacchio in sicilia (italia) la presenza di eurytoma plotnikovi nik. (hymenoptera, eurytomidae) in alcuni pistacchieti della sicilia centro-occidentale viene segnalata per la prima volta. vengono  inoltre fornite informazioni sul danno prodotto dall’imenottero, nelle aree infestate. key words: pistachio seed wasp, new record, megastigmus pistaciae. edible pistachio (pistacia vera l.) nuts infested with larvae of an unidentified wasp  were collected during an insect survey conducted in spring 2011 in pistachio orchards  in central-western sicily (latitude 37°51’ 01” n; longitude 13°52’64” e). the wasp  larvae, reared under laboratory conditions, developed into adults that were identified as  eurytoma plotnikovi, an indigenous pest of inedible nuts of the ornamental pistachio (p. chinensis) in china (qin et al., 2007; tian et al., 1994). the occurrence of this pistachio  seed wasp in sicily is a new record for italy. in the surveyed orchards, e. plotnikovi was  associated with another wasp, the pistachio seed chalcid, megastigmus pistaciae walker  that is native to the mediterranean region. in addition to china, the pistachio seed wasp  is present in tunisia (jarraya & helali, 1978), iran (basirat & seyedoleslami, 2000),  israel (izhaki, 1998), turchia (doğanlar et al., 2009), greece (mourikis, et al., 1998)  and in other pistachio producing countries in the middle east. a total of 553 nuts were  collected in the infested sicilian pistachio orchards and dissected for wasp infestation.  all the nuts presenting the wasp larvae (254) were isolated under laboratory conditions  waiting for the eclosion of the adults. an association of 177 and 77 specimens of e. plotnikovi and m. pistaciae walker, respectively was produced. the damage induced  by e. plotnikovi in the surveyed orchards has been noticed since 2009 by the growers  journal of entomological and acarological research, ser. ii, 43 (3), 2011334 eurytoma plotnikovi nik.: lateral (top) and dorsal view (bottom) of adult.  335s. longo, p. suma: first report of eurytoma plotnikovi in sicily (italy) who confused it with that caused by m. pistaciae. according to the literature, this new  pistachio seed wasp completes one generation per year. the insect overwinters as a  full grown larva inside the infested pistachio nuts which remain on the tree or fall to  the ground. adult emerges from the seeds in late april early may. the newly emerged  females search for unripe pistachio nuts left on the trees or on the ground to deposit  their eggs. the hatched larvae feed on the nuts until all or almost all the seed embryo is  consumed and then enter diapause usually by july (braham, 2005). according to basirat  and seyedoleslami, (2000) each pistachio nut allows the development of only one wasp  specimen. however, studies conducted in iran and tunisia, where the two pistachio wasps  are present, indicate that e. plotnikovi is able to outperform m. pistaciae displacing it and  becoming the dominant species infesting pistachio orchards (basirat and seyedoleslami,  2000; braham, 2005). the early emergence (about one month earlier) of e. plotnikovi  adults, compared to m. pistaciae, favors its spread and rapid colonization of pistachio  nuts (jerraya and bernard, 1971; braham, 2005). poor management practices of the  wasp infestations may also influence the spread of e. plotnikovi in pistachio orchards  (wu et al., 2009). the results of chemical control trials reported in the literature indicate  that stem injections of neonicotinoids are the most effective measures for managing  infestations of e. plotnikovi, providing better results than spray and drench applications  of this product. taking into consideration the importance of the pistachio industry as an  economic resource in eastern sicily, the only italian region where pistachios are grown,  biological and ecological studies of e. plotnikovi in the environmental conditions of  sicily are needed to prevent the spread of this pest from west into east sicily (bronte  municipality) and, in general, all over the country. acknowledgements sincere thanks are expressed to prof. g. viggiani and prof. s. laudonia, naples  university, for confirming the identification of the insect. references basirat m., seyedoleslami h., 2000 - biology of pistachio seed wasp eurytoma plotnikovi  nikoloskaya (hym.: eurytomidae) in isfahan province, iran. in: j. sci. & tech. agric. &  nat. resour., vol 4, no. 1, p. 148 (abstract).  braham m., 2005 - management of the pistachio seed wasp eurytoma plotnikovi nikolskaya  (hymenoptera, eurytomidae) in tunisia: integration of pesticides sprays and other means of  control. in: international pest control, vol. 47(6), p. 319-324. doğanlar m., karadağ s., mendel, z., 2009 - notes on pistachio seed wasps from two locations in the east mediterranean. phytoparasitica, 37:147–151. izhaki i., 1998 - the relationships between fruit ripeness, wasp seed predation, and avian fruit  removal in pistacia palaestina. israel journal of plant sciences 46(4): 273-278. jarraya a.; helali t., 1978 - contribution to the study of the insect fauna of pistachio. on the  spatial distribution of megastigmus pistaciae walk. (hym. torymidae) and of eurytoma plotnikovi nik. (hym. eurytomidae) in tunisia. bulletin des recherches agronomiques de  gembloux 13(3): 215-252. journal of entomological and acarological research, ser. ii, 43 (3), 2011336 jerraya a., bernard j., 1971 - premières observations bioécologiques sur megastigmus pistaciae  en tunisie. in: annales de l’institut national de la recherche agronomique de tunisie, vol.  44(3), p. 1-26.  mourikis p. a., tsourgianni a., chitzanidis a., 1998 - pistachio nut insect pests and means of  control in greece. acta horticulturae, 470: 604-611. qin f., guo t., song m., liu z., 2007 - study on eurytoma plotnikovi nikolskaya. journal of  jiangsu forestry science & technology. doi: cnki:sun:jsly.0.2007-06-014. tian s.b., qin x.r., zhao x., 1994 - infestation charactersitics of the larvae of eurytoma plotnikovi and their control.plant protection, china 20(2):15-16. wu y., wen x., chen x., li w., zhang y., liu m., 2009 - eurytoma plotnikovi: incidence harms  and the management countermeasure. forest by-product and speciality in china. doi: cnki:sun:ctfl.0.2009-03-04. santi longo, pompeo suma - dipartimento di gestione dei sistemi agroalimentari e ambientali,  sezione entomologia agraria, università degli studi di catania, 100 via santa sofia, 95123  catania, italy. e-mail: longosan@unict.it accepted 12 december 2011 j. ent. acar. res. ser. ii, 43 (1): 7-22 30 april 2011 m. montagna, g.c. lozzia, c. andreis, a. giorgi & j. baumgärtner the beetle (coleoptera) and true bug (heteroptera) species pool of the alpine “pian di gembro” wetland (villa di tirano, italy) and its conservation abstract - the coleoptera and heteroptera species pool was investigated in the  “pian di gembro” wetland (villa di tirano, sondrio, italy). the wetland consists  of a bog and its surroundings, referred to as wetland components, that are both  subjected to a diversified intermediate management regime (dimr). the application  of the dimr for plant species conservation resulted in the establishment of 11  wetland zones with a characteristic vegetation. in a three year sampling program,  997 coleoptera and heteroptera representing 141 species from 14 families were  collected. among these species, 64 species share both wetland components, 11  are restricted to the bog and 63 were found in the surroundings only. among the  species pool there were 23 tyrphophile taxa and only one tyrphobiont. with the  exception of one zone, all zones are inhabited by zone-specific species. by taking  into account both the general species pool and the pool of species of particular  interest to conservationists, only one zone can be considered as redundant since  it is inhabited by species that occur also in other zones. hence, all the zones, with  one exception, are effective for species pool conservation. the existing dimr  implemented for plant species conservation is also effective for conserving the  species pool of coleoptera and heteroptera.  riassunto - nel presente studio sono state indagate le specie di coleotteri ed  eterotteri presenti nell’area umida di pian di gembro (villa di tirano, sondrio, italia),  costituita da una componente di torbiera e da ambienti ecotonali circostanti sottoposti  entrambi ad un regime di gestione intermedia diversificata (dimr). l’applicazione  di strategie dimr nella gestione e conservazione delle specie vegetali ha portato  alla determinazione di 11 zone umide con vegetazione caratteristica, all’interno  delle quali si sono svolti i campionamenti. il programma di campionamento, della  durata di tre anni, ha permesso di raccogliere 997 campioni di coleotteri ed eterotteri  appartenenti a 141 specie e 14 famiglie. tra queste specie, 64 sono state censite in  entrambe le componenti della zona umida, 11 sono ristrette alla torbiera e 63 agli  ecotoni circostanti. nel gruppo di specie censite sono presenti 23 taxa tirfofili e  uno solo tirfobionte. tutte le stazioni di raccolta, eccetto una, presentano specie  uniche. considerando quelle di interesse conservazionistico, solo una zona può dirsi  ridondante, in quanto le specie censitevi sono presenti in altre zone. concludendo,  10 delle 11 zone che compongono la zona umida di pian di gembro sono utili nella  journal of entomological and acarological research, ser. ii, 43 (1), 20118 conservazione dello “species pool”. la strategia di gestione dimr, utilizzata per  la conservazione delle specie vegetali risulta essere funzionale alla conservazione  delle specie di coleotteri ed eterotteri presenti. key words - bog, insect coenoses, species pool, conservation categories. introduction wetlands are ecologically sensitive adaptive systems and much attention has been  given to the design and implementation of adequate management strategies (turner et al. 2000). because of their capacity to conserve species of conservation interest and to  provide ecosystem services, wetlands are considered as natural capital and often assigned  protected status (spitzer & danks, 2006; fisher et al., 2009). the term “protected area” refers to any area of land or sea managed for the persistence  of biodiversity and other natural processes, achieved through constraints on incompatible  land uses (possingham et al., 2006). despite high levels of protection and adequate  management within their borders, many protected areas are not functioning as originally  envisioned. agriculture, settlements, and other human land uses in the unprotected part  of the ecosystem, as well as the lack of any management, may alter the flow of energy,  material and organisms across the ecosystems in ways that change ecological functioning  within protected areas (hansen & defries, 2007). wetlands are areas whose soil is  saturated with moisture either permanently or seasonally. nevertheless, the importance  of the surroundings for the integrity of the bog and the interest in conservation measures  motivated us to apply the term ‘wetland’ to a bog and its surroundings and refer to them  as ‘wetland components’.  in the wetland under study, both the bog and its surroundings are subjected to  diversified management procedures whose frequency and intensity change through  time in different zones. importantly, the diversification of these practices falls into  range that maintains the integrity of the wetland and prevents it from shifting into an  irreversible late successional state where the desirable wetland characteristics are lost  (andreis & rodondi, 1982). ecological theory predicts that species diversity is highest  under intermediate disturbance (smith & smith, 2001). a combination of diversified  and intermediate management regimes (dimr) holds to promise to conserve both the  species pool and a high species diversity (guo, 2003). specific adaptations may restrict  species to either the bog or its surroundings or limit their distribution to particular zones  resulting from the application of the dimr to the two wetland components. while  detailed information is available for northern wetlands (spitzer & danks, 2006), little is  known on the diversity and the pool of species inhabiting alpine wetlands. the limited  information is restricted to specific taxa such as odonata (marcuzzi, 1948; balestrazzi et al. 1983), heteroptera (rampazzi & dethier, 1997; montagna et al., 2008), coleoptera  (focarile, 1957) and trichoptera (cianficconi et al., 2005). a dimr is applied for plant species conservation to the ‘pian di gembro’ wetland  in the northern italian alps (andreis & rodondi, 1982; andreis & rodondi, 2005).  9m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland the application of the dimr produced 11 different ecological zones corresponding to  habitat typologies (carta habitat natura 2000 it2040025). this work deals with beetles  (coleopera) and true bugs (heteroptera) as important elements of the insect species pool. material and methods  study site the ‘pian di gembro’ wetland is located north of the aprica pass at 1350 m above  sea level in villa di tirano, sondrio (italy) (fig. 1). with other alpine wetlands, it shares  the features of raised and blanket bogs characterized by a mosaic of plant associations  (andreis & rodondi, 1982). since 1988, the wetland and the surrounding area has  become a regional reserve and site of community importance (sci it2040025) under  the habitat directive act (92/43/eec). the protected area covers 126.5 ha, and the  major and minor diameters measure 2000 m and 300 m, respectively. coniferous forests,  deciduous trees and shrubs, as well as pastures and meadows, surround the bog. though  no synthetic fertilizers are applied in the wetland, it receives nitrogen and sulphur from  the atmosphere (krupa, 2003; erisman et al., 2005). fig. 1 - the location of the ‘pian di gembro’  wetland in the northern italian alps; the map  shows 11 sampling zones (z1: sampling zone  1; z2: sampling zone 2; z3: sampling zone 3;  z4: sampling zone 4; z5: sampling zone 5; z6:  sampling zone 6; z7: sampling zone 7; z8:  sampling zone 8; z9: sampling zone 9; z10:  sampling zone 10; z11: sampling zone 11).  journal of entomological and acarological research, ser. ii, 43 (1), 201110 sampling the sampling program started in 2005 and continued until 2007. it was restricted  to the snow-free periods lasting from march to october. sampling was carried out in  the different zones corresponding to the habitat typologies specified in the habitat  directive 92/43/eec part. i and identified by a numeric code. for the protected wetland,  the carta habitat natura 2000 it2040025 establishes habitat typologies and allows the  identification of 11 zones (table 1). during 2005, one standard pitfall trap (mason et al., 2002; liu et al., 2007; uys et al., 2010) was deployed in each zone (table 1); to improve the sampling coverage, an  additional trap was put into each of the larger sampling zones 1, 2 and 4. the pitfall traps  were baited with different types of attractants such as meat, fish, beer, banana and a water  solution of nacl (mason et al., 2002). a total of 19 traps were deployed in the wetland  and visited every 10 days for 7 months per year. to improve the sampling efficiency,  the pitfall trap technique was complemented by other sampling techniques including the  use of sweep nets and entomological umbrellas (mason et al., 2002), sieves, and direct  observations of specimens in specific micro-habitats (e.g. tree trunks, underneath stones,  and at the bottom of carex spp.).  table 1 the sampling zones located within the “pian di gembro” wetland components, the description of the vegetation and the habitat code (carta habitat natura 2000 it2040025) (z1: sampling zone 1; z2: sampling zone 2; z3: sampling zone 3; z4: sampling zone 4; z5: sampling zone 5; z6: sampling zone 6; z7: sampling zone 7; z8: sampling zone 8; z9: sampling zone 9; z10: sampling zone 10; z11: sampling zone 11). wetland components zones description of the vegetation habitat code bog z1 pools with utricularia spp. community 3160 z2 patch vegetation with trichophorum spp. and  molinia spp. 7140 and 7150 z3 carex lasiocarpae community 7140 z4 trichophorum caespitosum community 7140 surroundings z5 calluna dry heaths 4030 z6 calluna dry heaths 4030 z7 mountain hay meadows 6520 drainage  channel z8 drainage channel vegetation surroundings z9 managed calluna dry heaths 4030 z10 calluna dry heaths 4030 z11 mountain hay meadows 6520 11m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland species pool and species of particular conservation interest the collected specimens were brought to the laboratory for identification at the  species level. the individuals were assigned to different families and sent to specialists  who identified the species.  first, the insects were grouped into three main coenoses: i) species occurring in  the bog, ii) species inhabiting the surroundings, and iii) species distributed over both  the bog and the surroundings. thereafter, species and families were listed according to  the different zones in which they were captured. subsequently, the species specific to a  zone were separated from the ones found in more than one zone. second, we assessed  the species pool by selecting species of particular interest to conservationists. the  scientific literature and experts were consulted to establish the categories of interest:  a) species of humid biotopes (tyrphophiles); b) bioindicator species; c) endemic alpine  taxa; d) species with boreo-alpine distribution; e) species of mountainous environments;  f) species of lowland environments; g) species with wide ecological tolerance to the  effect of abiotic factors. results species pool the sampling program made available 997 individuals representing 141 species  from 11 families of heteroptera (anthocoridae, nabidae, miridae, reduviidae, coreidae,  rhopalidae, lygaeidae, acanthosomatidae, pentatomidae, scutellaridae, thyreocoridae)  and three families of coleoptera (carabidae, chrysomelidae sensu latu, curculionidae  sensu latu). table 2 lists the 141 collected species, the number of specimens and the  sampling zones in which the specimens were collected.  species grouping the insects listed in table 2 belong to three main coenoses. the first coenosis is  composed of species exclusively found in the bog (zones 1, 2 or 3, table 3) and consists  of 11 species including 5 species of heteroptera, 2 species of carabidae, 3 species of  chrysomelidae and 1 species of curculionidae. of note, the tyrphophiles donacia obscura, limnobaris dolorosa and platysma oenotrium are members of this coenosis.  the second coenosis consists of 63 species exclusively found in the surroundings (zones  5, 6, 7, 8, 9 or 10, table 3). these insects belong to 21 species of heteroptera, 9 species  of carabidae, 21 of chrysomelidae and 12 of curculionidae. the most interesting  species of this coenosis are the four endemic alpine taxa cryptocephalus sericeus sp.  zambanellus, otiorhynchus scaber, o. frigidus and pterostichus dissimilis that are of  conservation interest (osella et al., 2005). the third coenosis is composed of 64 species  that are found in both the bog and the surroundings (table 4). the highest number of species specific to one zone were found in zones 10 (23  journal of entomological and acarological research, ser. ii, 43 (1), 201112 table 2 species list of beetles (coleoptera) and true bugs (heteroptera) sampled in the “pian di gembro” wetland. the number of indiviaduls is given for each sampling zone (z1: sampling zone 1; z2: sampling zone 2; z3: sampling zone 3; z4: sampling zone 4; z5: sampling zone 5; z6: sampling zone 6; z7: sampling zone 7; z8: sampling zone 8; z9: sampling zone 9; z10: sampling zone 10; z11: sampling zone 11). species z1 z2 z3 z4 z5 z6 z7 z8 z9 z10 z11 temnostethus pusillus (herrich-schäffer 1835) 0 0 0 0 1 0 0 0 0 0 0 tetraphleps bicuspis (herrich-schäffer 1835) 2 0 0 0 0 0 1 0 0 0 0 nabis flavomarginatus scholz 1847 0 0 0 0 0 0 0 0 0 0 1 nabis rugosus (linnaeus 1758) 0 0 0 0 4 0 0 0 0 6 0 alloeotomus gothicus (fallén 1807) 0 0 0 0 0 0 1 0 0 0 0 deraeocoris ruber (linnaeus 1758) 1 0 0 0 0 0 0 0 0 1 0 halticus apterus (linnaeus 1761) 1 0 0 0 0 0 1 0 0 2 0 adelphocoris seticornis (fabricius 1775) 0 0 0 0 1 0 1 0 0 0 0 camptozygum aequale (villers 1789) 2 0 0 0 0 0 0 0 0 0 0 capsus ater (linnaeus 1758) 2 0 0 0 2 0 0 1 0 3 0 charagochilus gyllenhalii (fallén 1807) 0 0 0 0 0 0 0 0 0 1 0 leptopterna dolabrata (linnaeus 1758) 0 0 0 0 2 0 1 0 2 2 2 lygus pratensis (linnaeus 1758) 0 0 0 0 1 6 0 0 0 3 0 lygus wagneri remane 1955 0 0 0 0 1 0 0 0 0 1 0 megaloceroea recticornis (geoffroy 1785) 0 0 0 0 0 0 0 0 1 1 0 notostira elongata (geoffroy 1785) 0 0 0 0 0 0 1 0 0 0 0 polymerus palustris (reuter 1905) 0 0 0 0 1 0 0 0 0 3 0 stenodema calcarata (fallén 1807) 2 2 0 0 0 0 0 0 0 4 0 stenodema holsata (fabricius 1787) 1 0 0 0 4 0 0 0 0 7 0 stenodema laevigata (linnaeus 1758) 0 0 0 0 0 0 0 0 0 1 0 heterocordylus genistae (scopoli 1763) 0 0 0 0 0 0 0 0 5 0 0 chlamydatus pulicarius (fallén 1807) 0 0 0 0 2 0 1 0 0 3 0 plagiognathus arbustorum (fabricius 1794) 0 0 0 0 0 0 0 0 0 1 0 plagiognathus chrysanthemi (wolff 1864) 0 0 0 0 2 0 0 0 0 0 0 coranus subapterus (de geer 1773) 0 3 0 0 0 0 0 0 0 0 0 rhynocoris annulatus (linnaeus 1758) 1 0 0 0 0 0 0 0 0 0 0 coreus marginatus (linnaeus 1758) 0 0 0 0 0 1 0 0 0 1 0 corizus hyoscyami (linnaeus 1758) 0 0 0 0 0 0 0 0 1 0 0 myrmus miriformis (fallén 1807) 22 22 0 1 6 0 0 2 1 5 0 rhopalus maculatus (fieber 1837) 0 1 2 0 3 1 1 0 1 6 0 rhopalus parumpuncactus schilling 1829 0 0 0 0 0 0 1 0 0 2 0 stictopleurus punctatonervosus (goeze 1778) 5 3 0 0 9 2 0 0 2 10 0 kleidocerys resedae (panzer 1797) 0 0 0 0 0 0 1 0 1 1 0 nithecus jacobaeae (schilling 1829) 2 0 0 0 13 1 2 0 1 8 0 eremocoris plebejus plebejus (fallén 1807) 1 0 0 0 0 0 0 0 0 0 0 13m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland gastrodes abietum bergroth 1914 0 0 0 0 0 0 3 0 3 0 1 pachybrachius luridus hahn 1826 0 0 0 0 0 0 0 0 0 1 0 peritrechus geniculatus (hahn 1832) 1 2 0 0 0 1 0 0 0 0 0 pterotmetus staphyliniformis (schilling 1829) 0 0 0 0 0 0 3 0 0 2 0 rhyparochromus pini (linnaeus 1758) 2 0 0 0 0 0 1 0 0 0 1 stygnocoris pygmaeus r.f. sahlberg 1848 0 0 1 0 0 0 0 0 0 0 0 stygnocoris sabulosus (schilling 1829) 0 0 0 0 8 0 1 0 1 11 0 trapezonotus desertus seidenstücker 1951 0 0 0 0 1 0 0 0 0 0 0 trapsonotus dispar stål 1872 0 0 0 0 0 0 2 0 0 0 0 cyphostethus tristriatus (fabricius 1787) 0 0 0 0 0 0 3 0 0 0 0 elasmostethus interstinctus (linnaeus 1758) 0 0 0 0 1 0 0 0 1 1 0 elasmucha grisea (linnaeus 1758) 0 0 0 0 1 0 0 0 1 0 0 picromerus bidens (linnaeus 1758) 0 0 0 0 0 0 0 0 0 1 0 zicrona caerulea (linnaeus 1758) 0 0 0 0 0 0 0 0 0 1 0 aelia acuminata (linnaeus 1758) 0 0 0 0 1 0 0 0 3 6 0 carpocoris purpureipennis (de geer 1773) 0 0 0 0 3 0 0 0 0 4 0 dolycoris baccarum (linnaeus 1758) 2 0 0 0 2 0 1 0 0 4 0 eurydema oleracea (linnaeus 1758) 0 0 0 0 0 0 0 0 0 5 0 holcostethus vernalis (wolff 1804) 0 0 0 0 3 0 0 0 0 0 0 neottiglossa bifida (a. costa 1847) 0 0 0 0 0 0 0 0 0 1 0 palomena prasina (linnaeus 1761) 0 0 0 0 0 0 0 0 0 2 0 pentatoma rufipes (linnaeus 1758) 0 0 0 1 1 0 0 0 0 1 0 eurygaster maura (linnaeus 1758) 0 0 0 0 0 0 0 0 1 0 0 eurygaster testudinaria (geoffroy 1785) 0 0 0 0 0 1 0 0 0 2 0 thyreocoris scarabaeoides (linnaeus 1758) 0 0 0 0 0 0 2 0 0 2 0 carabus germarii sturm 1815 0 0 0 0 0 0 1 0 0 0 0 agonum sexpunctatum (linne 1758) 0 0 0 0 2 0 0 0 0 0 0 platysma oenotrium (ravizza 1975) 0 2 0 0 0 0 0 0 0 0 0 pterostichus dissimilis (a. villa & g.b. villa 1833) 0 0 0 0 0 0 0 0 3 0 0 poecilus versicolor (sturm 1824) 0 0 0 0 1 0 0 0 0 0 0 phonias diligens (sturm 1824) 0 59 0 3 0 0 0 0 0 0 0 bothriopterus oblongopunctatus (fabricius 1787) 1 0 0 0 0 0 0 0 0 0 0 amara eurynota (panzer 1797) 0 0 0 0 2 0 0 0 0 0 0 amara lucida (duftschmid 1812) 0 0 0 0 0 0 0 0 1 0 0 amara cursitans (zimmermann 1832) 0 0 0 0 0 0 0 0 1 0 0 anisodactylus binotatus (fabricius 1787) 0 0 0 0 1 0 0 0 0 0 0 acupalpus flavicollis (sturm 1825) 0 0 0 0 0 0 0 0 0 1 0 lamprias cyanocephalus (linne 1758) 0 0 0 0 0 0 0 0 2 0 0 zeugophora flavicollis (marsham 1802) 0 0 0 0 1 0 0 0 0 0 0 donacia obscura gyllenhal 1813 2 0 0 0 0 0 0 0 0 0 0 journal of entomological and acarological research, ser. ii, 43 (1), 201114 gonioctena decemnotata (marsham 1802) 3 2 0 0 1 0 0 0 35 0 0 gonioctena quinquepunctata (fabricius 1787) 4 14 0 0 4 0 0 0 7 6 0 chrysolina fastuosa (scopoli 1763) 0 0 0 0 0 0 0 0 0 3 0 chrysolina marginata (linnaeus 1758) 0 0 0 0 0 0 0 0 0 3 0 chrysolina geminata (paykull 1799) 0 0 0 0 0 2 0 0 0 8 0 chrysomela populi linnaeus 1758 0 0 0 0 13 0 0 0 0 0 0 chrysomela tremulae fabricius 1787 0 0 0 0 59 0 0 0 0 0 0 chrysomela vigintipunctata scopoli 1763 0 0 0 0 1 0 0 0 0 0 0 lochmaea caprea (linnaeus 1758) 14 6 0 0 0 0 5 0 0 0 0 galeruca pomonae (scopoli 1763) 0 0 0 0 1 0 0 0 0 0 0 galeruca tanaceti (linnaeus 1758) 0 0 0 0 1 0 1 0 0 0 0 luperus flavipes (linnaeus 1767) 0 1 0 0 9 0 0 0 1 0 0 luperus longicornis (fabricius 1781) 0 0 0 0 0 0 0 0 1 0 0 luperus viridipennis germar 1824 0 0 0 0 22 0 1 4 2 0 0 aphthona herbigrada (curtis 1837) 0 0 0 0 0 0 0 1 2 0 0 aphthona venustula (kutschera 1861) 0 0 0 0 6 0 0 1 6 47 0 longitarsus lewisii (baly 1874) 0 0 0 0 0 0 0 0 1 0 0 longitarsus luridus (scopoli 1763) 0 0 0 0 0 0 1 0 0 3 0 longitarsus melanocephalus (de geer 1775) 0 0 0 0 0 0 0 0 4 0 0 longitarsus pratensis (panzer 1794) 0 0 0 0 0 0 0 0 2 0 0 altica oleracea (linnaeus 1758) 3 6 0 0 8 1 0 0 4 22 0 bathophila rubi (paykull 1799) 0 0 0 0 0 0 0 0 0 1 0 neocrepidodera peirolerii (kutschera 1860) 0 0 0 0 0 0 0 2 0 1 0 crepidodera lamina (bedel 1901) 0 0 0 0 1 0 0 0 0 0 0 chaetocnema concinna (marsham 1802) 0 0 0 0 0 0 0 0 0 2 0 chaetocnema picipes stephens 1831 0 0 0 0 0 0 0 0 0 1 0 chaetocnema hortensis (geoffroy 1758) 0 0 0 0 0 1 0 0 0 2 0 chaetocnema sahlbergi (gyllenhal 1827) 1 2 0 0 0 1 0 0 0 0 0 smaragdina affinis (illiger 1794) 3 4 0 0 0 0 0 0 7 0 0 smaragdina salicina (scopoli 1763) 0 0 0 0 2 1 0 0 0 0 0 cryptocephalus elegantulus gravenhorst 1807 1 0 0 0 0 0 0 0 0 0 0 cryptocephalus labiatus linnaeus 1761 1 0 0 0 1 0 2 0 4 0 0 cryptocephalus vittula suffrian 1848 0 0 0 0 0 0 0 0 0 1 0 cryptocephalus bipunctatus (linnaeus 1758) 1 0 0 0 0 0 0 0 0 0 0 cryptocephalus flavipes fabricius 1781 0 0 0 0 0 1 0 0 0 0 0 cryptocephalus moraei (linnaeus 1758) 0 0 0 0 0 4 0 0 0 0 0 cryptocephalus nitidus (linnaeus 1758) 0 0 0 0 1 0 0 0 1 0 0 cryptocephalus quadripustulatus gyllenhal 1813 0 0 0 0 0 0 0 0 0 1 0 cryptocephalus sericeus marseul 1875 0 0 0 0 0 2 0 0 0 0 0 cryptocephalus transiens franz 1949 0 0 0 0 0 9 0 0 6 1 0 15m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland bromius obscurus (linnaeus 1758) 0 0 0 0 6 0 0 0 6 11 0 cassida sanguinolenta o.f. müller 1776 0 0 0 0 0 0 0 0 1 0 0 otiorhynchus armadillo (rossi 1792) 0 0 0 0 0 0 0 0 0 2 0 otiorhynchus scaber (linnaeus 1758) 0 0 0 0 3 0 0 0 0 0 0 otiorhynchus frigidus (mulsant 1859) 0 0 0 0 1 0 0 0 0 0 0 otiorhynchus anthracinus (scopoli 1763) 0 0 0 0 0 0 0 0 0 1 0 phyllobius viridicollis (fabricius 1792) 0 0 0 0 1 0 0 0 0 0 0 phyllobius arborator (herbst 1797) 1 0 0 0 1 0 0 0 0 0 0 phyllobius pyri (linnaeus 1758) 0 1 0 0 0 0 0 0 1 1 0 polydrusus marginatus stephens 1831 0 0 0 0 0 0 0 0 0 1 0 polydrusus cervinus (linnaeus 1758) 0 0 0 0 0 0 0 1 0 0 0 sciaphilus asperatus (bonsdorff 1785) 0 0 0 0 3 0 0 0 0 1 0 strophosoma melanogrammum (forster 1771) 0 0 0 0 17 0 0 0 0 0 0 sitona nigriclavis stephens 1829 0 0 0 0 0 0 1 0 1 0 0 sitona sulcifrons (gyllenhal 1834) 0 0 0 0 2 0 9 0 37 8 0 lepyrus capucinus (schaller 1783) 0 0 0 0 0 0 0 0 0 1 0 hylobius abietis (linnaeus 1758) 0 0 0 0 0 0 0 0 1 0 0 magdalis memnonia (gyllenhal 1832) 0 0 0 0 1 0 0 0 1 0 0 ceutorhynchus erysimi (fabricius 1787) 0 0 0 0 1 0 0 0 2 1 0 nedyus quadrimaculatus (linnaeus 1758) 0 0 0 0 0 0 0 0 0 1 0 limnobaris dolorosa (goeze 1777) 0 1 0 0 0 0 0 0 0 0 0 limnobaris t-album (linnaeus 1758) 0 0 0 0 0 0 0 0 0 2 0 anthonomus rubi (herbst 1795) 0 0 0 0 0 0 4 0 0 0 0 tachyerges salicis (linnaeus 1758) 0 0 0 0 0 0 0 0 3 1 0 tachyerges stigma (germar 1821) 0 0 0 0 0 0 0 0 0 1 0 miarus campanulae linnaeus 1767 0 0 0 0 1 0 0 0 2 3 0 species) and 5 (19 species). the zone specificity could be explained by the presence of  particular host plants and abiotic ecological requirements that are present in a particular  zone. it is worth noting that there were no species specific to zone 4. all the other zones,  however, had a number of specific species ranging from 1 (zone 3, 8) to 18 (zone 5).  in general, there were more exclusive species in the surroundings (zones 5, 9, 10) than  in the bog area (zones 1, 2, 3, 8), with the exception of zone 1 which had 7 exclusive  species. from the systematic point of view, both heteroptera and coleoptera recorded  in this study are characterized by exclusive species, but the highest number of exclusive  species belongs to the heteroptera suborder (27 species) and to the chrysomelidae family  (24 species). in zone 6, we found exclusively chrysomelidae, while the 3 species found  in zone 2 belong to three different taxa. some of the species listed in table 2, such as the  aforementioned o. scaber and o. frigidus, have a restricted geographical distribution,  while others such as anthonomus rubi (osella et al., 2005) and longitarsus pratensis are common species with a wide geographical distribution (biondi, 2005). journal of entomological and acarological research, ser. ii, 43 (1), 201116 table 3 species exclusively found in one component of the “pian di gembro” wetland (lombardy, italy): black squares show species exclusively found in the bog; grey squares show species exclusively found in the surroundings (h: heteroptera; c: carabidae; ch: chrysomelidae; cu: curculionidae sensu lato; z1: sampling zone 1; z2: sampling zone 2; z3: sampling zone 3; z4: sampling zone 4; z5: sampling zone 5; z6: sampling zone 6; z7: sampling zone 7; z8: sampling zone 8; z9: sampling zone 9; z10: sampling zone 10; z11: sampling zone 11). h c ch h c cu h h c ch zo ne /s pe ci es rh yn oc or is an nu la tu s ca mp to zy gu m ae qu al e er em oc or is pl eb eju s bo th rio pt er us o bl un go pu nc ta tu s do na cia o bs cu ra cr yp to ce ph al us el eg an tu lu s cr yp to ce ph al us b ip un cta tu s co ra nu s s ub ap ter us pt er os tic hu s oe no tri um li mn ob ar is do lo ro sa st yg no co ris p yg ma eu s te mn os tet hu s p us ill us pl ag io gn at hu s c hr ys an th em i tr ap ez on ot us d es er tu s ho lco ste th us ve rn al is an iso da cty lu s b in ot at us ag on um se xp un cta tu m po ec ilu s v er sic ol or am ar a eu ry no ta ze ug of or a fla vic ol lis ch ry so me la p op ul i ch ry so me la tr em ul ae ch ry so me la vi gi nt ip un cta ta ga ler uc a po mo na e cr ep id od er a la mi na z1               z2       z3   z5                             cu ch h c cu h c ch zo ne /s pe ci es ot io rh yn ch us sc ab er ot io rh yn ch us fr ig id us ph yll ob iu s v iri di co lli s st ra ph os om a me la no gr am mu m cr yp to ce ph al us fl av ip es cr yp to ce ph al us m or ae i cr yp to ce ph al us se ric eu s cy ph os tet hu s t ris tri at us an th on om us ru bi al lo eo to mu s g ot hi cu s no to sti ra el on ga ta tr ap ez on ot us d isp ar ca ra bu s g er ma ri po lyd ru su s c er vin us co riz us h yo sc ya mi eu ry ga ste r m au ra he ter oc or dy lu s g en ist ae pt er os tic hu s d iss im ili s am ar a lu cid a am ar a cu rs ita ns la mp ria s c ya no ce ph al a lu pe ru s l on gi co rn is lo ng ita rs us le wi sii lo ng ita rs us m ela no ce ph al us lo ng ita rs us p ra ten sis z5         z6       z7             z8   z9                       ch cu h c ch cu zo ne /s pe ci es ca ss id a sa ng ui no len ta hy lo bi us a bi eti s ch ar ag oc hi lu s g yll en ha lii st en od em a la ev ig at um pa ch yb ra ch iu s l ur id us pi cr om er us b id en s zi cr on a ca er ul ea eu ry de ma o ler ac ea ne ot tig lo ss a bi fid a pa lo me na p ra sin a au cu pa lp us fl av ico lli s ba th op hi la ru bi ch ae to cn em a pi cip es ch ae to cn em a co nc in na ch ry so lin a ma rg in at a ch ry so lin a fa stu os a cr yp to ce ph al us q ua dr ip us tu la tu s cr yp to ce ph al us vi ttu la li mn ob ar is t-a lb um rh yn ca en us st ig ma ot io rh in ch us a nt hr ac in us po lyd ru su s m ar gi na tu s ot io rh in ch us a rm ad ill o le py ru s c ap uc in us ne dy us q ua dr im ac ul at us z9     z10                                               17m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland table 4 species found in at least two zones of pian di gembro wetland (lombardy, italy). grey squares show species widespread across surrounding zones (group a); black squares show species widespread across both the surrounding and the bog zones (group b) (z1: sampling zone 1; z2: sampling zone 2; z3: sampling zone 3; z4: sampling zone 4; z5: sampling zone 5; z6: sampling zone 6; z7: sampling zone 7; z8: sampling zone 8; z9: sampling zone 9; z10: sampling zone 10; z11: sampling zone 11). z o n e/ sp ec ie s sm ar ag di na sa lic in a sc ia ph ilu s a sp er at us lu pe ru s v iri di pe nn is ly gu s w ag ne ri ca rp oc or is pu pu re ip en ni s ly gu s p ra te ns is na bi s r ug os us st yg no co ris sa bu lo su s po ly m er us p al us tri s co re us m ar gi na tu s ch la m yd at us p ul ic ar iu s el as m uc ha g ris ea cr yp to ce ph al us n iti du s m ag da lis m em no ni a eu ry ga ste r t es tu di na ria ch ae to cn em a ho rte ns is el as m os te th us in te rs tin ct us ch ry so lin a ge m in at a br om iu s o bs cu ru s g al er uc a ta na ce ti ad el ph oc or is se tic or ni s z5                                   z6               z7           z8   z9               z10                             zo ne /s pe ci es ap ht on a ve nu stu la cr yp to ce ph al us se ric eu s m ia ru s c am pa nu la e ce ut or hy nc hu s e ry sim i ae lia a cu m in at a le pt op te rn a do lo br at a lo ng ita rs us lu rid us rh op al us p ar um pu nc at us ne oc re pi do de ra p ei ro le ri m eg al oc er oe a re ct ic or ni s th yr eo co ris sc ar ab ae oi de s po ly dr us us ce rv in us rh yn ca en us sa lic is kl ei do ce ry s r es ed ae pt er ot m et us st ap hy lin ifo rm is si to na su lc ifr on s ap ht on a he rb ig ra da g as tro de s a bi et um si to na n ic ric la vi s lu pe ru s fl av ip es ni th ec us ja co ba ea e z1   z2   z3 z4 z5                 z6     z7                     z8         z9                               z10                                 z11     zo n e/ sp ec ie s rh op al us m ac ul at us st en od em a ho lsa tu m ca ps us a te r d ol yc or is ba cc ar um cr yp to ce ph al us la bi at us al tic a ol er ac ea st ic to pl eu ru s p un ct at on er vo su s g on io ct en a de ce m no ta ta h al tic us a pt er us ph yl lo bi us a rb or at or pe nt at om a ru fip es d er ae oc or is ru be r ph yl lo bi us p yr i p yr i st en od em a ca lc ar at um te tra ph le ps b ic us pi s sm ar ag di na a ffi ni s g on io ct en a qu in qu ep un ct at a m yr m us m iri fo rm is lo ch m ae a ca pr ea e pe rit re ch us g en ic ul at us ch ae to cn em a sa hl be rg i ph on ia s d ili ge ns z1                                     z2                           z3   z4       z5                         z6             z7             z8     z9                   z10                           journal of entomological and acarological research, ser. ii, 43 (1), 201118 the 64 species (45% of species pool) that occur in more than one zone could  be considered as “generalists”, with a wider ecological tolerance than species found  in one zone only (table 3 and 4). we split these species into two groups: group a  contains “generalist” species widespread in surrounding zones, while group b contains  “generalist” species widespread in both surrounding and bog zones. as expected, the  outflow zone 8 is inhabited by species occurring in the bog and in the surrounding zones.  in group a, there are species of conservation interest and species with a wide geographical  distribution such as lygus pratensis and galeruca tanaceti, which has also been found  in nearby commercial yarrow fields (limonta et al., 2003; sassi, 2007) where penata  gama et al. (2010) studied the dynamics of aphid populations. within group b, there  are species of conservation interest and species with a wide geographical distribution,  such as gonioctena decemnotata and altica oleracea (sassi, 2007; montagna, 2009). species of particular conservation interest we found 39 species (28% of the species pool) of conservation interest (table 5).  pachybrachius luridus is the only species that could be considered a obligate bog or  tyrphobiont species linked to sphagnum spp. and other bog plants (e.g. carex spp., rhynchospora sp., trichophorum sp. and eriophorum sp.) (montagna et al., 2008). there  were 23 species considered tyrphophiles, including donacia obscura and chaetocnema sahlbergi that prefer humid biotopes or microenvironments with high humidity (doguet,  1994; montagna, 2009). five species, including d. obscura and pterostichus dissimilis,  are considered bioindicators of natural environments (giaccalone et al., 2002; casale et al., 2005; sassi, 2005; montagna et al., 2008; montagna, 2009). othiorrynchus scaber, o. frigidus, c. sericerus sp. zambanellus and p. dissimilis (2.8% of species pool) are  taxa endemic to the italian alpine region (casale et al., 2005; osella et al., 2005; sassi,  2005). three species of heteroptera (2.1% of species pool), including trapezonotus desertus, show a boreo-alpine distribution (pericart, 1987; pericart, 1998a, 1998b,  1998c). nine species of the ‘pian di gembro’ species pool are typical of mountainous  regions (e.g. c. sericeus and amara cursitans are typical alpine taxa), and three are  typical of lowland environments (magistretti, 1965; biondi, 2005; casale et al., 2005;  cianficconi, 2005; sassi, 2005; montagna et al., 2008). four species exhibit a wide  ecological tolerance (casale et al., 2005; montagna et al., 2008). it should also be noted  that even at the altitude of 1350 m above sea level and in the heart of alps, 2% of the  species are lowland species.  among the 39 species of conservation interest, there are 25 species that occur in  one zone only, 8 in two zones and 6 in more than 2 zones (table 5).  discussion the insect species pool of ‘pian di gembro’ wetland consists of 141 species collected  during three years of sampling. the evaluation of the species pool is difficult because  studies on alpine wetland insects are rare and usually restricted to particular taxa. boyse  (2004) studied the insect fauna of acid mires in england and found only 24 species  19m. montagna et al.: beetle and true bug of the alpine “pian di gembro” wetland table 5 species of pian di gembro wetland (lombardy, italy) that are considered of conservation interest. species zo ne s sp ec ie s o f h um id b io to pe bi on di ca to r a lp in e en de m ic sp ec ie s bo re oa lp in e di st ri bu tio n m ou nt ai ne ou s s pe ci es lo w la nd sp ec ie s w id e ec ol og ic al to le ra nc e species zo ne s sp ec ie s o f h um id b io to pe bi on di ca to r a lp in e en de m ic sp ec ie s bo re oa lp in e di st ri bu tio n m ou nt ai ne ou s s pe ci es lo w la nd sp ec ie s w id e ec ol og ic al to le ra nc e donacia obscura 1       limnobaris t-album 10         rhynocoris annulatus 1       cryptocephalus quadripustulatus 10     coranus subapterus 2       rhyncaenus stigma 10       platysma oenotrium 2       lepyrus capucinus 10         limnobaris dolorosa 2       nabis flavomarginatus 11       trapezonotus desertus 5     phonias diligens 2, 4         agonum sexpunctatum 5         neocrepidodera peiroleri 8, 9       anisodactylus binotatus 5         sciaphilus asperatus 5, 10       chrysomela vigintipunctata 5       polymerus palustris 5, 10       crepidodera lamina 5       eurygaster testudinaria 6, 10       otiorhynchus scaber 5         rhopalus parumpuncatus 7, 10       otiorhinchus frigidus 5       megaloceroea recticornis 9, 10       cryptocephalus sericeus ssp. zambanellus 6         rhyncaenus salicis 9, 10       anthonomus rubi 7       chaetocnema sahlbergi 1, 2, 6       pterostichus dissimilis 9       stenodema calcaratum 1, 2, 10           amara cursitans 9       phyllobius pyri pyri 2, 9, 10           longitarsus lewisii 9       sitona sulcifrons argutulus 5, 7, 9,  10           pachybrachius luridus 10             nithecus jacobaeae 1, 5, 6, 7, 9, 10       picromerus bidens 10       rhopalus maculatus 2, 3, 5,  6, 7, 9,  10       aucupalpus flavicollis 10       journal of entomological and acarological research, ser. ii, 43 (1), 201120 belonging to coleoptera (carabidae, chrysomelidae sensu latu, curculionidae sensu latu) and heteroptera. rampazzi & dethier (1997) found 105 species of heteroptera  in a bog located in southern switzerland, and in the zehlau bog in the western russia  entomologists collected 38 species of coleoptera (carabidae) (främbs et al., 2002).  the results confirm the important contribution of coleoptera and heteroptera to the  wetland inhabiting insect fauna. moreover, the studies indicate that the results depend  on sampling techniques and sampling efforts. this confirms the importance of building  the sampling program on different techniques and justifies their implementation over  an extended period of three years. the collected species grouped into three main coenoses show that 64 species share  both wetland components. nevertheless, the 11 species restricted to the bog and the 63  species found in the surroundings show that a clear difference should be made between  the bog and the surroundings. we found 23 tyrphophile insects, which exhibit preferences  for boggy areas, but the only obligate bog (tyrphobiont) species found was pachybrachius luridus. with the exception of zone 4, all zones are inhabited by zone-specific species  (table 1). hence, all zones except zone 4 are important in efforts aiming at conserving the  general pool of coleoptera and heteroptera species. from the standpoint of conserving  species of particular interest (table 5), 24 species are found in at least one out of 8 zones.  in the remaining zones, zone 4 and zone 8 share a single species with zone 2 and zone 9,  respectively, while only zone 3 shares the same species with 7 other zones. hence, the  zones 3, 4, 8 could be considered as redundant in efforts to conserve the pool of species  of particular interest. by taking into account both the general species pool (table 1) and  the pool of species of particular interest to conservationists (table 2), only zone 4 can be  considered as redundant since it is inhabited by species that occur also in other zones. in conclusion, with one exception, all the zones are important for conserving the  general species pool and the pool of particular interest in conservation programs. as  previously described, these zones result from the application of a dimr aiming at wetland  plant species conservation. from the standpoint of conserving the insect species pool,  the existing dimr is also effective for coleoptera and heteroptera. acknowledgements  we are grateful to dr. paride dioli, dr. maurizio pavesi, renato regalin, dr. davide  sassi for species identification. references andreis c., rodondi g., 1982 - la torbiera di pian di gembro (prov. di sondrio). - c.n.r. progr.  fin. “promozione della qualità dell’ambiente”, roma, aq/1/221: 1-41.  andreis c., rodondi 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università degli studi di milano, milano, italy; dipartimento di protezione dei sistemi  agroalimentare e urbano e valorizzazione delle biodiversità, università degli studi di milano,  milano, italy. e-mail: matteo.montagna@unimi.it. giuseppe carlo lozzia, johann baumgärtner, dipartimento di protezione dei sistemi  agroalimentare e urbano e valorizzazione delle biodiversità, università degli studi di  milano, milano. carlo andreis, dipartimento di biologia, sezione di botanica sistematica e geobotanica,  università degli studi di milano, milan, italy. anna giorgi, gesdimont, centro di studi applicati per la gestione sostenibile della montagna.  università degli studi di milano, edolo, italy. accepted 26 april 2011 jear2012 abstract a lectin was extracted from seeds of citrullus colocynthis (cucurbitaceae) by column chromatography using sepharose 4bgalactose and deae-cellulose fast flow. the inhibitory effects of the extracted lectin on digestive α-amylase of ectomyelois ceratoniae larvae were studied using ph, temperature, time of incubation and kinetic parameters. different concentrations of extracted lectin, citrullus colocynthis agglutinin (cca), inhibited digestive amylolytic activity by 22-49%. the highest inhibition was obtained at ph 8 and 9, which corresponds with the highest enzymatic activity in the control. the highest inhibition of e. ceratoniae α-amylase was found at 40°c, which corresponds with the optimal temperature for enzymatic activity. timecourse experiments revealed the highest amylolytic activity at 20-40 min post-incubation, while the highest inhibition was found after 2030 min. kinetic analysis showed that incubation of α-amylase with cca significantly decreased vmax, indicating non-competitive inhibition, but no statistical difference was found in the km value. our results indicated that cca significantly inhibited activity of digestive α-amylase in e. ceratoniae larvae, suggesting its possible application as a potential alternative control method against this pest. introduction ectomyelois ceratoniae zeller (lepidoptera: pyralidae) is the major pest of pomegranate and some dried fruits that annually causes 1590% damage (farazmand et al., 2008). adults are gray moths with a wingspan of 9-12 mm. larvae are pink, and hibernate in infested fruits on the soil surface. adults lay their eggs on the pomegranate crown, and larvae hatch and feed on tissues around the pomegranate grains (farazmand et al., 2008). different control tactics, such as collection of infested fruits, removal of pomegranate crowns, and release of biocontrol agents, have been used to decrease damage by e. ceratoniae larvae (farazmand et al., 2008). lectins are the heterogenous proteins that bind reversibly to monoor oligosaccharides (peumans & van damme, 1995). these molecules have been extracted from plants, fungi, bacteria and animals (komath et al., 2006). in plants, lectins have a critical role in plant-insect coevolution (chen, 2008). various lectins have been extracted from plants, such as asa i and asa ii from allium sativum l., rice, legumes and cucurbitaceae (peumans & van damme, 1995; van damme, 1998; zhu-salzman et al., 2002; jiang et al., 2006; clement et al., 2010; clement & venkatesh, 2010). several studies have confirmed lectins as being insecticidal, and transgenic crops expressing lectin genes have been introduced in many economically important crops (bell et al., 1999; de oliveira et al., 2001). for example, the efficiency of galanthus nivalis agglutinin (lectin) has been determined in potato (down et al., 1996; gatehouse et al., 1996), rice (foissac et al., 2000; nagadhara et al., 2004), maize (wang et al., 2005), tobacco (hilder et al., 1995), wheat (stoger et al., 1999), tomato (wu et al., 2000) and sugarcane (sétamou et al., 2002, 2003; li & romeis, 2009). citrullus colocynthis l. is a medicinal plant belonging to the cucurbitaceae family that is native to iran and which is found in the southern and eastern regions (tavakkol-afshari et al., 2005). fruits contain bitter glycosides that are used as drugs for gut and liver disorders. in addition to having anti-viral and anti-cancer properties, the crude fruit extract is effective in decreasing blood sugar (tavakkolafshari et al., 2005). α-amylase (α-1,4-glucan-4-glucanohydrolases; ec 3.2.1.1) is a hydrolytic enzyme that catalyzes the hydrolysis of endoα-d-(1,4)-glucan linkages in glycogen and other related carbohydrates (strobl et al., 1998; franco et al., 2000). there are six different classes of α-amylase inhibitors, which are known as lectin-like, knottin-like, cereal-type, kunitz-like, c-purothionin-like, and thaumatin-like, that may be useful in pest control (franco et al., 2002). these inhibitors show structural diversity leading to different modes of inhibition and different specificity against a diverse range of α-amylases (mehrabadi et al., 2010). although the effect of lectins on epithelial cells has been well elucidated, their inhibitory mechanisms on digestive enzymes remains correspondence: samar ramzi, department of plant protection, faculty of agriculture science, university of guilan, rasht, iran, 41637-1314. tel.: +98.0131.6690485 fax: +98.0131.6690281. e-mail: samarramzi@yahoo.com key words: lectin, citrullus colocynthis, ectomyelois ceratoniae, α-amylase. acknowledgements: this study was supported by a research grant behalf of research deputy in university of guilan. the authors would like to thank dr. arash zibaee for his assistance. received for publication: 9 may 2013. revision received: 21 august 2013. accepted for publication: 24 september 2013. ©copyright s. ramzi and a. sahragard, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e20 doi:10.4081/jear.2013.e20 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. a lectin extracted from citrullus colocynthis l. (cucurbitaceae) inhibits digestive α-amylase of ectomyelois ceratoniae zeller (lepidoptera: pyralidae) s. ramzi, a. sahragard department of plant protection, university of guilan, rasht, iran [page 110] [journal of entomological and acarological research 2013; 45:e20] journal of entomological and acarological research 2013; volume 45:e20 no n c om me rci al us e o nly unclear. therefore, this study was conducted to determine the effective concentration of c. colocynthis lectin, citrullus colocynthis agglutinin (cca) on digestive α-amylase of e. ceratoniae by considering ph, temperature, time, kinetic parameters and gel electrophoresis. material and methods insect rearing e. ceratoniae larvae were collected from pomegranate gardens in yazd and fed on artificial diet containing wheat bran (100 g), yeast (3 g), sugar (10 g), glycerine (40 ml) and water (40 ml). adults were allowed to lay eggs, and newly hatched larvae were reared on artificial diet to reach fourth larval instar. growth conditions were 28±2°c, 85% relative humidity and photoperiod of 16l:8d. preparation of sepharose 4b-galactose column to prepare the column, 20 ml of sepharose 4b was suspended in 40 ml of 0.5 m na2co2 (ph 11.0), then 2 ml of divinylsulphone was added to the suspension and the mixture was incubated for 70 min at room temperature with gentle agitation. after activation, 500 mg of galactose was added in 50 ml 0.5 m na2co2 (ph 11.0) and the suspension were re-incubated for an additional 12 h. the sorbent was washed with water; the unbound arm was blocked with b-mercaptoethanol-containing buffer, and then packed into a 1.5×30 cm column. the sorbent was equilibrated with tris-hcl (0.1 m) and used for the affinity purification of cca. purification of citrullus colocynthis agglutinin seeds of c. colocynthis were ground to fine powder using a mill. the dry powder was incubated in phosphate buffer (0.1 m ph 7.1) for approximately 20 h at 4°c. the mixture was then centrifuged at 4000 g for 20 min, and the remaining debris removed by passing the supernatant through filter paper (whatman no. 4) (hamshou et al., 2010). supernatant was precipitated by 0-60% concentrations of ammonium sulfate and centrifuged at 4000 g for 20 min. debris was eluted in trishcl buffer (0.1 m, ph 7) and dialyzed in the same buffer overnight (de oliveira et al., 2011). affinity chromatography was performed on a sepharose 4b-galactose column equilibrated with tris-hcl buffer (0.1 m, ph 7). after loading the extract, the affinity column was washed with buffer and the bound lectin was eluted with 20 mm of 1,3-diaminopropane (dap) (hamshou et al., 2010). fractions showing the highest protein content were pooled and used for the next step. the lectin fractions obtained after the first affinity chromatography were loaded on an anion exchange chromatography column of deae-cellulose fast flow, equilibrated with dap (hamshou et al., 2010). after washing with dap, the lectin was eluted using tris–hcl (0.1 m, ph 7.0) containing 0.5 m nacl. finally, the lectin fractions were dialyzed against water and lyophilized. the purity of the lectin was analyzed by sds-page. sample preparation e. ceratoniae larvae (4th instars) were randomly selected and dissected under a stereo-microscope in ice-cold saline solution (10 mm). larval bodies were cut separately using a scalpel and the midgut exposed by removal of fat bodies and other undesirable organs. the midgut was separated from the larval body and rinsed in ice-cold distilled water. the mixture was placed in a pre-cooled homogenizer and ground before centrifugation. equal portions of larval midgut and distilled water were used to obtain a desirable concentration of the enzyme (w/v). homogenates were separately transferred to 1.5-ml centrifuge tubes and centrifuged at 25,000 g for 20 min at 4°c. the supernatants were pooled and stored at −20�c for subsequent analyses. all the experiments were conducted immediately following sample preparation. α-amylase assay the method described by bernfeld (1955) was used to assay α-amylase activity. ten microliters of the homogenate was incubated for 30 min at 35°c with 50 �l of phosphate buffer (0.02 m, ph 7.1) and 20 �l of soluble starch (1%) as substrate. the reaction was stopped by addition of dinitrosalicylic acid (dns, 80 ml) and heated in boiling water for 10 min prior to reading the absorbance at 540 nm. one unit of α-amylase activity was defined as the amount of enzyme required to produce 1 mg maltose in 30 min at 35°c. the negative control contained all reaction mixtures with pre-boiled enzyme (for 15 min) to prove enzyme presence in the samples. inhibition of α-amylase by different concentrations of citrullus colocynthis agglutinin to find possible inhibition of the digestive α-amylase, 50 ml of pbs (0.02 m, ph 7.1), 20 ml of starch 1% and 20 ml of different concentrations of lectin (0, 0.1, 0.5, 1, 1.5 and 2 mg/ml) were incubated for 5 min. then, 10 ml of the enzyme was added and the reaction continued as described above. blanks were run containing pbs, starch 1% and each concentration of lectin. effect of ph on α-amylase inhibition by citrullus colocynthis agglutinin effect of ph on cca inhibition on α-amylase was determined at different ph values using tris-hcl buffer (20 mm) at ph levels of 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12. the enzyme activity was assayed after incubation of the reaction mixture containing tris-hcl buffer (at a given ph value), starch 1%, cca (2 mg/ml) and midgut homogenate. controls were run at each ph value with midgut α-amylase alone as a control. other steps were carried out as previously described. effect of temperature on α-amylase inhibition by citrullus colocynthis agglutinin to obtain the effect of temperature on α-amylase inhibition by cca, the reaction mixture containing tris-hcl (20 mm ph, 9), starch 1%, cca (2 mg/ml) and enzyme was incubated at different temperatures of 15, 20, 25, 30, 35, 40, 45, 50 and 60°c. a control was carried out without the inhibitor. other steps were carried out as previously described. time-course inhibition of α-amylase by citrullus colocynthis agglutinin time-course inhibition of α-amylase by cca was carried out by incubating the enzyme extract with cca and other reaction constituents in tris-hcl buffer (20 mm, ph 9) at 40°c for different time intervals of 10, 20, 30, 40, 50 and 60 min. other steps were carried out as previously described. kinetic studies kinetic parameters of inhibition and control were carried out with increasing concentrations of starch as the substrate (0.5-2.0%) in the presence of cca (2 mg/ml). lineweaver-burk plot analysis was done based on the data to find affinity of enzyme to substrate (km) and velocity of enzyme (vmax) values. inhibition in non-denaturing page enzyme extract was pre-incubated with different concentrations of cca for 30 min at 30°c, then the remaining α-amylase activity was determined by polyacrylamide gel electrophoresis. page was carried [journal of entomological and acarological research 2013; 45:e20] [page 111] article no n c om me rci al us e o nly [page 112] [journal of entomological and acarological research 2013; 45:e20] out using the procedures described by laemmli (1970). concentrations of resolving and stacking gel were 12 and 4%, respectively. electrophoresis was conducted at a voltage of 70 v until the blue dye reached the bottom of the slab gel. the gel was rinsed with distilled water and washed with 1% (v/v) of triton x-100. the gel was then immersed in a solution of pbs (0.02 m ph 7.1) containing 1% starch, 10 mm of nacl and 2 mm of cacl2. finally, it was stained with solutions of 1.3% i2, and 3% ki to obtain white bands with dark backgrounds. protein assay protein concentrations were assayed according to the method described by lowry et al. (1951). statistical analysis all data were compared by one-way analysis of variance (anova) followed by tukey’s test at the p≤0.05 level. results in the current study, a lectin with a molecular weight of 14.5 kda was extracted from seeds of c. colocynthis (figure 1), which significantly inhibited digestive α-amylase of e. ceratoniae by 22-49% (figure 2a). also, incubation of larval midgut homogenate with 2 mg/ml of cca decreased sharpness of amylolytic isozymes (figure 2b). the protein was able to inhibit 50% of total amylolytic activity both in assay conditions and with gel electrophoresis (figure 2). the effect of ph on α-amylase inhibition by cca is shown in figure 3a. there were significant differences among ph levels (f=5.43, p=0.004), with the highest inhibition at ph 8; there was slightly less inhibition, although not different, at ph 9 (figure 3a). the optimal ph of the α-amylase (control) was observed at ph 8 and 9 (figure 3b) (f=18.56, pr>f: 0.0001). in the case of temperature, the highest inhibition of α-amylase was found at 40°c, while the optimal temperature was observed to be between 20-45°c (figure 4; pr>f: 0.004, f=5.02; pr>f: 0.0001, f=21.21). results revealed the highest inhibition of the enzyme at 20-30 min of post-incubation (figure 5a). in the control, the highest enzymatic activity was observed at 20-40 min of post-incubation (figure 5b). there was a correlation between the times of the highest enzymatic activity and the highest inhibition. in this study, vmax and km values for the control were found to be 0.46 article figure 1. sds-page showing purity and molecular weight of the purified lectin. ccl, citrullus colocynthis lectin; mm, molecular weight. figure 2. inhibition of e. ceratoniae α-amylase by different concentrations (mg/ml) of c. colocynthis lectin in the enzymatic assay (a) and gel electrophoresis (b). reaction conditions were phosphate buffer (ph 7) at 30°c. (a) (b) no n c om me rci al us e o nly od/min and 1.08%, respectively (figure 6) while these parameters were 0.155 od/min and 0.96 % in the incubation of the enzyme with cca (figure 6). kinetic analysis showed that incubation of enzyme with inhibitor significantly decreased the vmax parameter, indicating non-competitive inhibition. although a change in km value was observed, it was not statistically different. discussion and conclusions one of the promising alternatives for insect control is the use of biotechnological processes to provide resistant varieties of host plants. there are several genes identified to do so, such as bt toxins, digestive enzyme inhibitors, chitinases and lectins (bishop et al., 2000; sales et al., 2000; carlini & grossi-de-sa, 2002; bertrand et al., 2003; bellincampi et al., 2004; haq et al., 2004; de azevedo pereira et al., 2006). several classes of plant proteins have been discovered and characterized, including lectins, ribosome-inactivating proteins, and protease and α-amylase inhibitors, which have shown insecticidal effects on different insect pests (ishimoto et al., 1989; ryan, 1990; chrispeels et al., 1998; gatehouse & gatehouse, 1998; ussuf et al., 2001). there are six different classes of α-amylase inhibitors: lectin-like, knottinlike, cereal-type, kunitz-like, c-purothionin-like, and thaumatin-like, which may be useful in pest control (franco et al., 2000; bonavides et al., 2007). suzuki et al. (1994) believed that these inhibitors had a high degree of sequence homology and specificity. different studies have also been carried out determining types of lectin-like inhibitors and their effects on α-amylases of insects (grossi-de-sá & chrispells, 1997; da silva et al., 2000; yamada et al., 2001). α-ai1 inhibited digestive αamylases from callosobruchus maculatus fabricius (coleoptera: bruchidae) and c. chinensis, but it had no inhibitory effect against zabrotes subfasciatus boheman (coleoptera: chrysomelliade) amylase. another inhibitor was α-ai2, which was not able to inhibit the first three α-amylases of assayed bruchids, but which inhibited α-amylase of z. subfaciatus (grossi-de-sá & chrispells, 1997; da silva et al., 2000; yamada et al., 2001). mirkov et al. (1994) believed that these types of inhibition indicated an evolutionary relationship with phyto-hemagglutinnins and arcelins. temperature and ph are the two critical factors in biochemical reactions that could affect both activity and inhibitory mechanisms. in the current study, the highest inhibition of α-amylase occurred at an alkaline ph, where the highest enzymatic activity was observed. however, in the case of eurygaster integriceps puton (hemiptera: scutelleridae), the highest inhibition by triticale extraction was observed at ph 5 and 6 for both salivary and midgut α-amylases (mehrabadi et al., 2010; mehrabadi et al., 2012). several authors have confirmed a ph-dependent interaction between amylases and inhibitors (powers & whitaker, 1977; valencia et al., 2000; mehrabadi et al., 2010, 2012). extracted αai from phaseolus vulgaris l. inhibited porcine pancreatic α-amylase at ph 5.5, an effect that varied at ph 4.5 to 5.5, depending on the strain of bean used (barbosa et al., 2010). since the gut lumen of insects is the place where the interaction between α-amylase and inhibitors occurs, the ph showing the highest inhibition may reflect the fact that the ph of insect midgut is alkaline (ranjbar et al., 2011). since α-amylase may have the highest activity under such conditions, it should be more inhibited if cca [journal of entomological and acarological research 2013; 45:e20] [page 113] article figure 3. a and b) ph dependency of e. ceratoniae α-amylase inhibition by c. colocynthis lectin (2 mg/ml) versus the control. reaction conditions were tris-hcl buffer (20 mm, phs 6-12) at 30°c. different letters indicate statistical difference among values, tukey’s test (p≤0.05). (a) (b) (a) (b) figure 4. a and b) effect of temperature on e. ceratoniae α-amylase inhibition by c. colocynthis lectin (2 mg/ml) versus control. reaction conditions were tris-hcl buffer (20 mm, ph 8) at different temperatures (°c). different letters indicate statistical difference among values, tukey’s test (p≤0.05). no n c om me rci al us e o nly [page 114] [journal of entomological and acarological research 2013; 45:e20] is available at an appropriate concentration. this phenomenon has been described in other studies (biggs & mcgregor, 1996; valencia et al., 2000; mehrabadi et al., 2010, 2012). the observed specific temperature for inhibition and activity of α-amylase could reflect the environmental temperature where e. ceratoniae, a poikilothermic organism, lives. regarding incubation time for inhibition, mehrabadi et al. (2010) demonstrated the maximum amylolytic inhibition in the midgut of e. integriceps by triticale extract after 20-30 min post-incubation. similar results were found by marshall & lauda (1975) and leberre-anton et al. (1997). lineweaver-burk analysis is used to indicate the behavior and inhibition mechanism of an enzyme. vmax and km are the two main parameters in these calculations showing the highest velocity of enzyme (vmax) and affinity of enzyme to substrate (km). additionally, km may show affinity of an inhibitor to enzyme or enzyme-substrate complex. higher vmax and lower km shows the desirable values for better performance of an enzyme. in the present study, incubation of the enzyme with inhibitor significantly decreased vmax value, indicating non-competitive inhibition. with this kind of inhibition, the inhibitor binds to a specific site of the enzyme and causes a type of conformation (eisenthal & cornishbowden, 1974). this conformation does not prevent substrate binding but prevents the enzyme from converting the bound substrate to product. this kind of inhibition has been reported by several authors (marshall & lauda, 1975; leberre-anton et al., 1997; mehrabadi et al., 2010). the present study uncovers a new lectin protein with α-amylase inhibitory properties that may be used in the genetic modification of crops by gene encoding to create transgenic plants showing resistance against insect pests. besides the in vivo effects of cca on the digestive physiology of e. ceratoniae, we found that cca significantly disrupts digestion of food in this insect. determination of a cca-encoding gene and its transferral to plants may therefore lead to a resistant variety of host plant. these findings could ultimately be used to design specific bio-insecticides for use against economically important pests. references barbosa a.e.a.d, albuquerque e.v.s., silva m.c.m., souza d.s.l., oliveira-neto o.b., valencia a.v., et al., 2010 rαeseaarcmhayrtilcalese inhibitor-1 gene from phaseolus vulgaris expressed in coffea arabica plants inhibits α-amylases from the coffee berry borer pest. bmc biotechnol. 10: 44. bell h.a., fitches e., down r.e., marris g.c., edwards j.p., gatehouse j.a. 1999 the effect of snowdrop lectin (gna) delivered via artificial diet and transgenic plants on eulophus pennicornis (hymenoptera: eulophidae), a parasitoid of the tomato moth lacanobia oleracea (lepidoptera: noctuidae). j. insect. physiol. 45: 983-991. bellincampi d., camardella l., delcour j.a., desseaux v., d’ovidio r., duranda e.g., gebruers k., giovane a., juge n., 2004 potential physiological role of plant glycosidase inhibitors. biochim. biophys. acta. 1696: 265-274. bernfeld p., 1955 amylases, α and �. meth. enzymol. 1:149-158. bertrand b., guyot b., anthony f., lashermes p., 2003 impact of the coffea canephora gene introgression? on beverage quality of c. arabica. theoret. appl. gen. 107: 387-394. biggs d.r., mcgregor p.g., 1996 gut ph and amylase and protease activity in larvae of the new zealand grass grub (costelyra zealandica; coleoptera: carabaeidae) as a basis for selecting inhibitors. insect. biochem. mol. biol. 26: 69-75. bishop j.g., dean a.m., mitchell-olds t., 2000 rapid evolution in plant chitinases: molecular targets of selection in plant-pathogen coevolution. pnas 97: 5322-5327. bonavides k.b., pelegrini p.b., laumann r.l., grossi-de-sá m.f., article figure 5. a and b) time course inhibition of e. ceratoniae α-amylase by c. colocynthis lectin (2 mg/ml). midgut samples were preincubated with inhibitor in 20 mm tris-hcl buffer (ph 8) at 30°c, after which enzyme was added and reaction was recorded at the given time intervals. different letters indicate statistical difference among values, tukey’s test (p≤0.05). 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horn et al., 1979) and at times may warrant control measures if only to enhance esthetics on their respective host plants. natural enemies, primarily an array of predators (e.g. lacewings, orius spp. minute pirate bugs, assassin bugs, and predacious mirid bugs), and unaccountable mortality of eggs are cited as factors contributing to reducing damage caused by lace bug species (connell & beacher, 1947; horn et al., 1983). sheeley & yonke (1977) studied seven species of lace bugs in missouri, usa, and found no evidence of parasitoids attacking any life stages of them. an exception was the egg parasitoid anagrus takeyanus gordh (hymenoptera, mymaridae) which parasitizes the azalea, stephanitis pyrioides (scott), and andromeda, s. takeyai drake & maa, lace bugs in connecticut, southeastern usa, and japan (balsdon et al., 1996; gordh & dunbar, 1977; tsukada, 1999). there are very few references on the species of egg parasitoids attacking lace bugs in the usa (gordh, 1979; huber, 1986). huber (1986) and triapitsyn (2003) reviewed the world literature on host associations of the egg parasitoids in the family mymaridae and the genus erythmelus enock, respectively, and found the only references pertaining to their relationships with lace bugs were occasional species in the genera anagrus haliday or erythmelus. these were recorded in all zoogeographical regions except the nearctic where there are no other described species of anagrus or erythmelus associated with lace bugs with the exception of the previously mentioned a. takeyanus (gordh & dunbar, 1977) and a. virginiae triapitsyn & puttler (puttler & triapitsyn, 2006). yet an erythmelus sp. was mentioned as being reared in the usa from the sycamore lace bug, corythucha ciliata (say), the hawthorn lace bug, c. cydoniae (fitch), and c. floridana heidemann (horn et al., 1979; horn et al., 1983; triapitsyn, 2003). on 25 july 2003 a lace bug determined (by b. puttler and corroborated by r.l. blinn) to be oak lace bug (olb), corythucha arcuata (say), was found infesting a bur oak (quercus macrocarpa michaux) (fagaceae) on the university of missouri – columbia (boone co.). eggs were readily observed on the underside of leaves and a serendipitous collection (ca. 10 egg clusters) was made from which 12 and 13 days correspondence: serguei v. triapitsyn, department of entomology, university of california, riverside, ca 92521, usa. tel.: +1.951.827.7817 fax: +1.951.827.3086. e-mail: serguei@ucr.edu key words: mymaridae, erythmelus klopomor, distribution, host association, lace bugs, corythucha, classical biological control. acknowledgments: appreciation is expressed to r.l. blinn, north carolina state university, raleigh for corroborating the identifications of the lace bug species. c.j. starbuck, university of missouri, horticulture department, columbia aided in determining the plant species mentioned in the study. m.w. gates, usda, ars, systematic entomology laboratory, washington, d.c. initially facilitated obtaining identification of the parasitoid by forwarding material to s.v. triapitsyn. thanks are also due to d.l. hostetter for review of an earlier draft of the manuscript. this study is a contribution from the missouri agricultural experiment station. received for publication: 31 july 2013. revision received: 1 november 2013. accepted for publication: 11 december 2013. ©copyright b. puttler et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1857 doi:10.4081/jear.2014.1857 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. notes on distribution, host associations, and bionomics of erythmelus klopomor triapitsyn (hymenoptera, mymaridae), an egg parasitoid of lace bugs in missouri, usa, with particular reference to its primary host corythucha arcuata (say) (hemiptera, tingidae) b. puttler,1 w.c. bailey,1 s.v. triapitsyn2 1division of plant sciences (entomology), university of missouri, columbia, mo; 2department of entomology, entomology research museum, university of california, riverside, ca, usa [page 30] [journal of entomological and acarological research 2014; 46:1857] journal of entomological and acarological research 2014; volume 46:1857 no nco mm er cia l u se on ly later (6 and 7 august) mymarid parasitoids emerged. an additional collection of olb eggs on 31 july 2003 also produced parasitoids as did a collection of olb eggs from howard co. on 6 august 2003 which yielded parasitoids four days later. one of the authors (s.v. triapitsyn) determined the parasitoid as probably a new, previously undescribed species of erythmelus, which somewhat resembled the neotropical tingid egg parasitoid e. tingitiphagus (soares), and subsequently described it as e. klopomor triapitsyn (triapitsyn et al., 2007). the missouri specimens are identical to specimens of e. klopomor from florida, north carolina, and maryland in the usa (triapitsyn et al., 2007). later, peña et al. (2009) reported e. klopomor also as an egg parasitoid of the avocado lace bug, pseudacysta perseae (heidemann), in florida. recoveries of the e. klopomor in missouri and illinois then represented new host records and distributions approximately 2000-2500 km from its other previously known locations (table 1). since a dearth of mymarid species host associations exists, an investigation of the parasitoid status in missouri and elsewhere was undertaken. materials and methods the university of missouri campus at columbia, missouri (umc) (boone co.) and its horticulture and agroforestry research center (harc) at new franklin in howard co., both in central missouri, were the major study sites. the umc site is typical of an urban environment with a landscape of trees, shrubs, flowering plants, and weeds scattered throughout the campus. the harc site is rural, consisting of an experimental plantation of a variety of trees and shrubs in the midst of an area of open fields, orchards, and pastures. both sites harboured lace bugs with eight species present, seven corythucha spp. and one gargaphia sp., on their respective host plants which are listed in table 2. in addition, the eggplant lace bug, gargaphia solani heidemann, was collected from horsenettle, solanum carolinense l. (solanaceae), at concordia, lafayette co., missouri. in 2004 observations for olb eggs located within the city of columbia and at the harc site began as trees leafed out in may and terminated by late september, when viable eggs were no longer present on host plants. in addition, one time surveys were conducted from june-september to determine distribution of the parasitoid e. klopomor from egg surveys collected from oak species (table 2) at selected locations from 51 missouri counties and 4 adjoining counties in illinois (table 1). eggs from other lace bug species also were collected to delineate the potential host range of the parasitoid occurring in the study areas. sampling was performed visually on the various host plants by searching leaves that showed typical lace bug feeding, e.g. discoloration and whitening of the upper leaf surface (connell & beacher, 1947). depending on the observed severity of the lace bug infestation various numbers of leaves were collected from one to five plants at each collection site, placed in sealed plastic bags and returned to the laboratory where they were examined under the microscope to reliably distinguish between hatched and unhatched eggs. two types of oviposition were characterized by lace bug species. the eggs of c. arcuata and c. cydoniae were laid in irregular clusters, readily visible, varying in numbers (ca. 10100+) and slightly embedded in leaf tissue, whereas, the eggs of c. ciliata and c. pergandei heidemann were deeply embedded in pubescent leaf tissue along the veins, midribs, and secondary vein junctions and laid singularly or in groups of <10 (barber & weiss, 1922). rearing of e. klopomor from samples collected at all locations and dates was accomplished by excising the leaf portion containing unhatched eggs and placing them on the surface of filter paper lined 100x150 mm petri dishes. the egg masses were monitored daily for 21 days for egg parasitoid emergence with most adults removed daily. these observations provided a means of estimating the parasitoids’ develop[journal of entomological and acarological research 2014; 46:1857] [page 31] article table 1. known distribution of the oak lace bug parasitoid erythmelus klopomor from recoveries in missouri and other states in the usa. state/county locality missouri adair kirksville audrain mexico barton lamar boone columbia callaway fulton cedar eldorado clark kahoka clay smithville cole jefferson city dade greenfield dallas buffalo franklin gray summit gasconade rosebud gentry albany henry clinton howard new franklin jackson blue springs jefferson eureka johnson kingsville knox edina lafayette concordia lawrence mt. vernon lewis lewiston livingston burger marion palmyra monroe madison pettis sedalia polk humansville ralls new london saline sweet springs st. charles weldon springs st. claire osceola st. louis st. louis vernon nevada warren warrenton worth grant city illinois henderson gladstone hancock nauvoo florida broward pompano bay palm beach west palm beach monroe islamorada okeechobee fort drum osceola canoe creek maryland prince george greenbelt north carolina wake raleigh no nco mm er cia l u se on ly [page 32] [journal of entomological and acarological research 2014; 46:1857] mental time from field collected eggs to adult emergence. a subsample of 15 emerged adults were placed in shell vials with and without a diluted honey food source to determine adult longevity under the rearing conditions described. in addition, a small number of adults were retained in petri dish samples and observed under the microscope for ovipositional behavior when exposed to unparasitised lace bug eggs. these eggs were further monitored to determine the parasitoid life cycle from time of oviposition to adult emergence. sample dishes were held in the laboratory where rearing conditions for both eggs and emerged adults ranged from 20-24°c with 8-10 h. of daily light exposure during the week and total darkness on weekends at ca. 40-50% r.h. voucher specimens of e. klopomor from this study are deposited in the collections of the entomology research museum, university of california at riverside, california, usa (ucrc) and w.r. enns entomological museum, university of missouri, columbia, missouri, usa (umrm) (triapitsyn et al., 2007), and those of its lace bug hosts in the umrm. results lace bug and plant host associations of erythmelus klopomor at the harc study site, c. arcuata eggs were first detected on bur oak on 10 may 2004. egg samples collected on this date and monitored for adult parasitoid emergence did not yield parasitoids. from the next sample on 18 may and weekly samples thereafter, e. klopomor adults emerged from egg collections from each sampling date. the first adult parasitoids appeared on 2 june and the last on 27 september; 15 and 6 days post collection, respectively. observations on bur oak at umc deviated slightly. c. arcuata eggs were first seen and sampled on 12 may with subsequent collections on 21 and 27 may. no e. klopomor was reared from these collections. they were reared from each egg collection at this site starting 2 june and weekly thereafter till 1 october. the first parasitoid adults emerged on 11 june and were again present in each sample with the last emergence on 6 october, 9 and 5 days post collection, respectively. in both localities, minimum/maximum emergence times ranged from 1-17 days as per other collection dates with all variations in between. in 2004 the last parasitoids to emerge from the umc bur oak samples of 1 and 3 october was 14 october. there were no apparent differences in the phenology of the olb as to when the eggs were first observed at the study sites (10 versus 12 may). yet there was a difference in first generation parasitoid emergence as e. klopomor appeared 9 days later at umc than at harc (11 versus 2 june). at both umc and harc e. klopomor was reared from miscellaneous collections of c. arcuata infesting other oak species (table 2). all of these oaks are referred to as white oak and the olb is apparently specific to these oak species. red oaks [e.g. q. rubra michaux (northern red oak), q. palustris muenchhausen (pin oak)] adjacent to and in close proximity to olb infestations were never infested. e. klopomor also was reared article table 2. host plants of lace bugs (tingidae) and those species serving as hosts for erythmelus klopomor* at the university of missouri study sites. host plants umc harc tingid species fagaceae quercus alba l. (white oak) q. alba corythucha arcuata (say)* (oak lace bug) q. macrocarpa michaux (bur oak) q. macrocarpa q. bicolor willdenow (swamp white oak) q. bicolor q. robur j.f. ehrhart (english oak) q. robur q. muehlenbergii engelman (chinkapin oak) q. muehlenbergii q. prinoides willdenow (dwarf chinkapin oak) q. acutissima carruhers (sawtooth oak) q. acutissima q. gambellii nuttall (hybrid oak) q. asjeses willdenow (hybrid oak) rosaceae aronia arbutifolia l. (persoon) (red chokeberry) a. arbutifolia c. cydoniae (fitch)* (hawthorn lace bug) crataegus viridis l. (winter king hawthorn) juglandaceae juglans nigra l. (black walnut) c. juglandis (fitch) (walnut lace bug) asteraceae aster novae-angeliae l. (new england aster) c. marmorata (uhler)* (chrysanthemum lace bug) solidago altissima l. (goldenrod) betulaceae betula nigra l. (river birch) b. nigra c. pergandei heidemann* plantaceae platanus occidentalis l. (sycamore) p. occidentalis c. ciliata (say)* ulmaceae ulmus americana l. (american elm) u. americana c. ulmi osborn & drake (elm lace bug) tiliaceae tilia americana l. (american linden/basswood) t. americana gargaphia tiliae (walsh) (linden lace bug) *denotes lace bug species serving as hosts for erythmelus klopomor. no nco mm er cia l u se on ly from c. cydoniae on aronia arbutifolia l. (rosaceae) at harc, but not from c. ulmi osborn & drake, c. ciliata, c. juglandis (fitch) nor from gargaphia tiliae (walsh) at this locality. in contrast, all lace bug species at umc except g. tiliae were hosts of e. klopomor (table 2). e. klopomor were reared from one-time collections of c. arcuata eggs in each of 36 missouri counties (figure 1, table 1). parasitoids were not detected in another 10 counties due to the absence of eggs and another 5 counties in which eggs yielded no parasitoids (figure 1). we did recover parasitoids in two of four illinois counties (hancock co. and henderson co.) from olb on bur oak and these represent new illinois state records as do our other samples for missouri. the known hosts of e. klopomor at the study sites in missouri are listed in table 2. biology of erythmelus klopomor based on our laboratory observations, e. klopomor is a solitary idiobiont parasitoid of lace bug eggs. it was found that unmated females produced females; therefore, the species apparently reproduces thelytokously even though occasional males are recorded (triapitsyn et al., 2007). newly emerged females do not undergo a preovipositional period and are capable of ovipositing on or shortly after emergence (proovigenic). the adults are readily attracted to egg clusters where the eggs are examined by antennal palpitation to determine their suitability for oviposition. oviposition is apparently deterred if eggs are too far along in development or previously parasitized. no host feeding was observed. acceptable eggs were mounted and the ovipositional act occurred via the ovipositor penetrating the egg through the rim of its operculum. the entire ovipositional procedure was completed in 3-5 min. the life cycle from egg to adult ranged from 11-17 days (n=20; mean of 14.15+0.4 s.e.) under our rearing conditions and was ca. half the time (26-36 days) it took a. takeyanus to develop under similar rearing conditions (balsdon et al., 1996). as per some other mymarid species, the parasitoid emerges by pushing out the operculum. it is short lived, with or without a food source. longevity was usually <48 h and was similar to that of a. takeyanus. discussion and conclusions five corythucha spp. in missouri (table 2) plus g. solani (a new host record; eggs of this host had been collected in the same state but outside of the study sites, and these were successfully parasitized by e. klopomor when exposed in the laboratory), as well as c. floridana (triapitsyn et al., 2007) and p. perseae (peña et al., 2009), both in florida, are recorded as hosts of e. klopomor. based on our observa[journal of entomological and acarological research 2014; 46:1857] [page 33] article figure 1. distribution of erythmelus klopomor in missouri, usa. no nco mm er cia l u se on ly [page 34] [journal of entomological and acarological research 2014; 46:1857] tions the parasitoid was most readily reared from c. arcuata because this species oviposits eggs that are more easily accessible to e. klopomor females (larger numbers/cluster) and not as deeply embedded in leaf tissue. next in preference was c. cydoniae with all other lace bugs serving as incidental hosts. the ease in which they were recovered from samples of <10-30 viable egg clusters per county is indicative of at least a somewhat wider distribution of e. klopomor within missouri and other states; however, the species is not yet known from the western usa. this assumption is based on the parasitoid’s known distribution within missouri (figure 1), illinois (a new state record), florida, maryland, and north carolina from eggs of c. ciliata and c. cydoniae (horn et al., 1979; horn et al., 1983; triapitsyn et al., 2007). we predict that e. klopomor will eventually be found coincident with its primary hosts of c. arcuata and c. cydoniae since their distribution encompasses most of the usa as do their known insect hosts (table 2) and related host plants (i.e. quercus spp.). since all the known lace bug hosts of e. klopomor overwinter as adults (barber & weiss, 1922) the question arises as how this short lived parasitoid survives year-to-year, from ca. october until the following may when no host eggs were found. we are of the opinion that it overwinters as a diapausing adult in secluded niches on its hosts’ host plants (cracks and crevices in trunk and branches) or in nearby environments. the latter unfortunately cannot be duplicated under artificial conditions. puttler et al. (1973) came to a similar conclusion with another mymarid anaphes nigrellus girault [as a. behmani girault (huber, 1992)] as did anderson & paschke (1968) for anaphes flavipes (foerster). in contrast, a. takeyanus has a known overwintering stage (eggs) to maintain the species (gordh & dunbar, 1977; tsukada, 1999). corythucha arcuata has recently become an invasive species in parts of europe and also in the asian part of turkey (mutun et al., 2009). it was first detected in northern italy in 2000 and subsequently found in switzerland in 2002, turkey in 2003, and the balkan peninsula (bulgaria) in 2012 (bernardinelli, 2000; mutun, 2003; forster et al., 2005; dobreva et al., 2013). should c. arcuata manifest itself as an oak pest in its present distribution and disperse throughout europe as c. ciliata did on sycamore over the same area (maceljski, 1986), e. klopomor could be useful as a candidate agent in a potential classical biological control program against this pest and also possibly against c. ciliata after an evaluation of the possibility that the parasitoid did not interfere with eu autochthonous lace bug species. the following considered parasitoid attributes could lend themselves to such a program: i) an apparently thelytokous reproduction; multivoltism, short life cycle (at least half that of its preferred host); ii) preference for c. arcuata; iii) occurs in temperate, mediterranean, and subtropical climates; and iv) parasitoid is readily collectable in its country of origin (usa). references anderson r.c., paschke j.d., 1968 the biology and ecology of anaphes flavipes (hymenoptera: mymaridae) an exotic parasite of the cereal leaf beetle. ann. entomol. soc. am. 61: 1-5. balsdon j.a., braman s.k, espelie k.e., 1996 biology and ecology of anagrus takeyanus (hymenoptera: mymaridae), an egg parasitoid of the azalea lace bug (heteroptera: tingidae). environ. entomol. 25: 383-389. barber h.b., weiss h.b., 1922 the lacebugs of new jersey. new jersey dept. agr. bur. stat. insp. circ. 54: 3-24. bernardinelli i., 2000 distribution of the oak lace bug corythucha arcuata (say) in northern italy (heteroptera tingidae). redia 83: 157-162. connell w.a., beacher j.h., 1947 life history and control of the oak lace bug. delaware agr. exp. sta. bull. 265: 5-28. dobreva m., simov n., georgiev g., mirchev p., georgieva m., 2013 first record of corythucha arcuata (say) (heterotera: tingidae) on the balkan peninsula. acta zool. bulg. 65: 409-412. forster b., giacalone i., moretti m., dioli p., wermelinger b., 2005 die amerikanische eichennetzwanze corythucha arcuata (say) (heteroptera, tingidae) hat die südschweiz erreicht. mitt. schweiz. entomol. ges. 78: 317-323. gordh g., 1979 superfamily chalcidoidea. in: krombein k.v., hurd jr. p.d., smith d.r., burks b.d. (eds), catalog of hymenoptera in america north of mexico, vol. 1, symphyta and apocrita (parasitica). smithsonian institution press, washington, d.c.: 743-1043. gordh g., dunbar d.m., 1977 a new anagrus important in the biological control of stephanitis takeyai and a key to the north american species. florida entomol. 60: 85-95. horn k.f., farrier m.h., wright c.g., 1983 some mortality factors affecting eggs of the sycamore lace bug, corythucha ciliata (say) (hemiptera: tingidae). ann. entomol. soc. am. 76: 262-265. horn k.f., wright c.g., farrier m.h., 1979 the lace bugs (hemiptera: tingidae) of north carolina and their hosts. north carolina agr. exp. sta. tech. bull. 257: 1-22. huber j.t., 1986 systematics, biology, and hosts of the mymaridae and mymarommatidae (insecta: hymenoptera) 1758-1984. entomography 4: 185-243. huber j.t., 1992 the subgenera, species groups, and synonyms of anaphes (hymenoptera: mymaridae) with a review of the described nearctic species of the fuscipennis group of anaphes s.s. and the described species of anaphes (yungaburra). proc. entomol. soc. ontario 123: 23-110. johnson w.t., lyon h.h., 1991 insects that feed on trees and shrubs (2nd ed.). cornell university press, ithaca, new york: 560. maceljski m., 1986 current status of corythucha ciliata in europe. bull. oepp/eppo 16: 621-624. mutun s., 2003 first report of the oak lace bug, corythucha arcuata (say, 1832) (heteroptera: tingidae) from bolu, turkey. israel j. zool. 49: 323-324. mutun s., ceyhan z., sözen c., 2009 invasion by the oak lace bug, corythucha arcuata (say) (heteroptera tingidae), in turkey. turk. j. zool. 33: 263-268. peña j.e., triapitsyn s.v., long d., evans g.a., roltsch w., 2009 first record of erythmelus klopomor (hymenoptera: mymaridae) as a parasitoid of the avocado lace bug, pseudacysta perseae (heteroptera: tingidae). florida entomol. 92: 394-395. puttler b., thewke s.e., warner r.e., 1973 bionomics of three nearctic species, one new, of hypera (coleoptera: curculionidae), and their parasitoids. ann. entomol. soc. am. 66: 1299-1306. puttler b., triapitsyn s.v., 2006 a new species of anagrus (hymenoptera: mymaridae) from missouri (u.s.a.), egg parasitoid of corythucha marmorata (hemiptera: tingidae) entomol. news 117: 25-30. sheeley r.d., yonke t.r., 1977 biological notes on seven species of missouri tingids (hemiptera: tingidae). j. kansas entomol. soc. 50: 342-356. triapitsyn s.v., 2003 review of the mymaridae (hymenoptera, chalcidoidea) of primorskii krai: genus erythmelus enock, with taxonomic notes of some extralimital species. far eastern entomol. 126: 1-44. triapitsyn s.v., berezovskiy v.v., hoddle m.s., morse j.g., 2007 a review of the nearctic species of erythmelus (hymenoptera: mymaridae), with a key and new additions to the new world fauna. zootaxa 1641: 1-64. tsukada m., 1999 interpopulation variation of hibernal-aestival-diapause in the egg parasitoid wasp anagrus takeyanus: adaptation to seasonal host-plant alteration of the tingid host, stephanitis takeyai. entomol. exp. appl. 92: 37-43. article no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res. ser. ii, 43 (1): 1-6 30 april 2011 f. m. iaccarino - r. jesu - r. giacometti  paraleyrodes minei iaccarino 1990 (homoptera: aleyrodidae), new specie for italy, on citrus aurantium l., 1758 abstract the nesting whitefly paraleyrodes minei iaccarino 1990 (homoptera:  aleyrodidae) was found on leaves of sour orange tree citrus aurantium l., 1758, in the gussone park of the faculty of agriculture, of the university of naples  “federico ii”, at portici, italy. riassunto paraleyrodes minei iaccarino 1990 (homoptera: aleyrodidae), una nuova specie per l’italia, su citrus aurantium linneo 1758, in italia. paraleyrodes minei iaccarino 1990 (homoptera: aleyrodidae) è stato ritrovato su  foglie di arancio amaro, citrus aurantium l., 1758, nel parco gussone della facoltà  di agraria, dell’università di napoli, in portici, italia.  key words: aedeagus, aleurodicinae, antenna, pores, western palaearctic.   introduction the nesting whitefly paraleyrodes minei iaccarino 1990 belongs to the subfamily  aleurodicinae quaintance et baker, characterized by adults with forewings displaying a  median and a forked radial vein and the legs with a tarsal paronychium like a spine. the  immature stages present compound-style pores in addition to simple ones, legs with a  terminal claw and the lingula extended beyond the posterior margin of the vasiform orifice  and with 2 apical setae pairs (quaintance et baker, 1913; bondar, 1923; evans et al.,  2006; evans, 2008). the operculum partially covers the lingula and the vasiform orifice. the genus paraleyrodes quaintance 1909 is distinguished from other genera of the  subfamily, to have the unforked radial sector and 4 antennal articles in the female and  3 in the male (quaintance, 1909; quaintance et baker, 1913; bondar, 1923; iaccarino,  1990; martin, 1999, 2004; evans et al., 2006, evans, 2008). the p. minei has been reported, for the western palaearctic region, in iran, israel,  lebanon, madeira, malaga, morocco, spain, syria(1) and turkey (iaccarino, 1990; garcia  (1) russell l.m. (personal correspondence, 1989) told that the species was certainly already present in  the usa, but it was not reported because confused with other species of paraleyrodes genus; in fact belows  journal of entomological and acarological research, ser. ii, 43 (1), 20112 garcia et al., 1992; llorens climent et garrido vivas, 1992; argov, 1994; aguiar et pita,  1996; martin et al., 2000, martin, 2004; ghahani et al., 2009).  material and methods the specimens, including several life stages, (some eggs and 2nd larval instars, two  3rd larval instars, 8 pupal cases, 1 male and 5 females,) were collected from leaves of  sour orange tree in the gussone park of the faculty of agriculture of the university of  naples “federico ii” at portici, italy at the end of 2010. this host plant was also infested  by aleurothrixus floccosus (maskell 1895) and the different type and form of wax  secreted by the 2 whiteflies has facilitated its recognition. the samples were placed in a  10% potassium hydroxide solution for the maceration of the internal organs and then in  glacial acetic acid to neutralize koh and remove any fat substances. the dehydration  was performed with a first step in 50% phenol and a second in 100% phenol. the canada  balsam-phenol solution was used for slides mounting. the dissection of the adults was  carried out during the transfer in pure phenol. results adult female: pale yellow in colour with some white powdery wax covering the body,  especially the wings; 4 antennal articles (fig. 1 a); sub-pyriform abdomen with 4 pairs  of ventral wax plates. male: similar to female; 3 antennal articles (fig. 1 b); abdomen with three pairs  of ventral wax plates; stumpy claspers ending in a sharp nail; aedeagus with the “cock  head” shaped apex, with 3 short appendixes located on upper and posterior surface, 2  long and thin appendixes below an anterior short appendix(2) (fig. 1 c) (iaccarino, 1990;  martin, 1996; evans et al, 2006).  immature stages egg: suboval, covered with powdery white waxy secretion, attached to leaf by long  pedicel, which gradually bending to set down the egg on the leaf surface. neanid i (crawler): subelliptic with a hyaline wax fringe along the marginal area,  where, as in all other stages, there are numerous simple pores, and a thin band of white  flocculent wax on the dorsum, from the thorax to the abdomen. neanid ii: suboval, with a fringe of short hyaline wax rods along the margin and  et al., 1998 reported that paraleyrodes minei was first noted in 1984 in san diego and the california plant  pest and disease report, 1991 and 1992, that it was first collected in san diego county in 1985 as reported  in cppdr , 1985 it was recorded as paraleyrodes sp. undescribed. (2) the reduced number of antennal articles, in both sexes, and the unforked radial sector of the wings are  two fundamental characters to separate this genus from the others; the typical shape of the aedeagus allows  for easy separation of this species from others. 3f.m. iaccarino et al.: paraleyrodes minei on citrus aurantium new species for italy with white wax rods arising from 2 pairs of compound pores, posterior one and the  anterior others. neanid iii: subelliptic, with marginal fringe and with 2 wax rods, produced by 2  pairs of compound pores located on the thorax (fig. 2 b). pupal case (fig. 2 a): subelliptic, marginally fringed with 5 compound pores pairs:  a single pair of cephalic and 4 pairs of abdominal pores, from 5th to 8th segment (fig. 2  d.i), each of them secreting single white wax rods upward that, then, break, falling all  around the pupal case, and, as for the other stages, with a crown shaped appearance(3).  two pairs of compound pores smaller are present on 3rd and 4th abdominal segments (fig.  2 d.ii). thorax with two pairs of “cicatrices” scars of compound pores found in the 3rd  instar (fig. 2 d.iii). a pair of hairs is located on anterior and another one on posterior  margin; a single cephalic pair of setae, and other two pairs, the former on 1st and the  latter on 8th abdominal segment on side of the vasiform depression. finally 14 setae, on  every side, alternate, irregularly, to the numerous simple wax pores are located on the  margin. the lingula, spatulate-globose, exserted, and its tip presents two pairs of long  setae (fig. 2 c) (iaccarino, 1990; martin, 1996; martin et al., 2000).  (3) for separating the p. minei pupal case from those ones of the major species that commonly infest  citrus in italy, it is useful to consider the presence and type of wax: long cylindrical rods in p. minei, abundant  and flocculent in aleurothrixus floccosus (maskell 1895), hyaline marginal band in parabemisia myricae  (kuwana 1927) (it can also be found on the upper leaves surface), without wax in bemisia afer (priesner et  honsy 1934) and in dialeurodes citri (asmead 1885) (it is often larger than pupal case of previous species). fig. 1 paraleyrodes minei, adulto: a, female antenna; b, male antenna; c, aedeagus. scale bar represents 63 _m in a, 79 _m in b and 45 _m in c. journal of entomological and acarological research, ser. ii, 43 (1), 20114 discussion the adult females lay eggs pale yellow usually surrounded , especially in heavy  infestation, by annular fluffy wax secreted by the adults and so they appear to be in a  white “nest” of woolly wax. the crawlers can remain in the nests, so the neanides of the  following stages will add the flocculent wax rods, or emerge from the nests and have  only the characteristic rods in a circular pattern (iaccarino, 1990).  in spain, in laboratory trials at room temperature, on lemon leaves, p. minei has  developed three generations per year (garcia garcia et al., 1992), while in california, in  field trials, on orange leaves, the generations were four per year. population development  fig. 2 - paraleyrodes minei, immature stages: a, pupal case; b, neanide iii; c, vasiform orifice;  d,i large compound pore, ii small compound pore, iii cicatrice. scale bar represents 59 _m in a,  45 _m in b, 45 _m in c, 50 _m in d.i, 29 _m in d.ii and 32 _m in d.iii. 5f.m. iaccarino et al.: paraleyrodes minei on citrus aurantium new species for italy is continuous throughout the year, albeit it has lower trophic activity in adverse time  (bellows et al., 1998). in the mediterranean basin the host plants belong to the genus citrus l. 1758  (iaccarino, 1990; martin, 1996; evans, 2007b; evans, 2008). in other areas (belize,  benin, bermuda, guatemala, honduras, hong kong, mexico, puerto rico and usa) p. minei is polyphagous and is found on anonaceae, apocynaceae, araceae, arecaceae,  asteraceae, compositae, ericaceae, euphorbiaceae, lauraceae, myrtaceae, piperaceae,  poligonaceae, rhizophoraceae, rubiaceae and rutaceae ( martin et al., 2000; evans,  2008). the natural enemies reported are the predatories clitostetus arcuatus (rossi 1794)  and serangium parcesetosum sicari 1929 (coccinellidae), and the parasitoids encarsia dominicana evans et serra 2002, e. parvella silvestri 1929, e. variegata howard 1908  and e. sp., (aphenidae) (evans, 2007a, evans, 2008). references aguiar a.m.f., pita m.t., 1966 - contribution the knowledge of the whiteflies (hom.:aleyr.) from madeira island.- boletim do museu municipal do funchal, supl. 4: 285-309. argov y., 1994 - the woolly, a new pest in israel. - alon hanotea, 48: 290-292. california plant pest and disease report - 1985 - department of food and agriculture 4 (4): 101118. http://www.cdfa.ca.gov/phpps/ppd/publications/cppdr.html california plant pest and disease report - 1991- department of food and agriculture 10 (1-2):  1-29. http://www.cdfa.ca.gov/phpps/ppd/publications/cppdr.html california plant pest and disease report - 1992 - department of food and agriculture 11(5-6):  59-93. http://www.cdfa.ca.gov/phpps/ppd/publications/cppdr.html bellows jr. t.s., meisenbacher c., headrick d.h., 1998 - population ecology field biology  of paraleyrodes minei (hom.:aleyr.) in southern california. - enviromental entomology,  27(2): 277-281. bondar g., 1923 - aleyrodideos do brasil. - official state publisher. bahia, pp. 182. evans g.a., 2007a - parasitoids (hym.) associated with whiteflies (aleyr.) of the world.-aphis  computer version, pp.173. http://www.sel.barc.usda.gov:591/1wf/parasitoidcatalog.pdf. evans g.a., 2007b - host plant list of the whiteflies (aleyr.) of the world. - aphis computer  version, pp. 290. http://www.sel.barc.usda.gov:591/1wf/whiteflyhost.pdf evans g.a., 2008 - the whiteflies (hem.:aleyr.) of the world and their host plants and natural  enemies.- aphis computer version, pp.703. http://www.sel.barc.usda.gov:591/1wf/worldwhitefly-catalog.pdf evans g.a., dooley j., gill r., 2006 - hawaiian whiteflies (aleyr.). - homoptera workshop.  unpublished, pp. 51. garcia garcia e.j., garijo alba c., garcia segua s., 1992 - presenza de paraleyrodes sp.  pr. citri (bondar, 1931) (insecta:hom.:aleyr.) en los cultivos de citricos de la provincia de  malaga (sue de espana): aspectos biologicos y ecologicos de la plaga. - boletin de sanidad vegetal, plagas, 18: 3-9. ghahari h., abd-rabour s., zahradnik j., ostovan h., 2009 - annotated catalogue of  whiteflies (hem.:stern.:aleyr.) from arasbaran, northwestern iran. - journal of entomology  and nematology, 1(1): 7-18.  iaccarino f.m., 1990 - descrizione di paraleyrodes minei n.sp. (hom.:aleyr.), nuovo aleirodide  journal of entomological and acarological research, ser. ii, 43 (1), 20116 degli agrumi, in siria. - bollettino del laboratorio di entomologia agraria “filippo silvestri” di portici, 46(1989): 131-149. llorens climent j.m., garrido vivas a., 1992 - homoptera iii. mosca blancas y sus controllo  biologico. pp. 203. alicante. pisa ediciones. martin j.h., 1996 - neotropical whiteflies of the subamily aleurodicinae estabilished in the  western paleartictc (hom.:aleyr.). - journal of natural history, 30: 1849-1859. martin j.h., 2004 - whiteflies of belize (hem.:aleyr.). part 1. introduction and account of the  subfamily aleurodicinae quaintance & baker. - zootaxa, 681: 1-199. martin j.h., mifsud d., rapisarda c., 2000. - the whiteflies (hem.:aleyr.) of europe and the  mediterranean basin. - bulletin of entomological research, 90: 407-448. quaintance a.l., 1909 - a new genus of aleyrodidae, with an aleyrodes nubifere berger and  aleyrodes citri riley and haward. - united states department of agriculture, bureau of  entomology, technical service, 12 (ix): 169-174. quaintance a.l., baker a.c., 1913 - classification of the aleyrodidae. part.1. united states  department of agriculture, bureau of entomology, technical service, 27: 1-93. ulusoy m.r., uygun n., 1996 - dogu akdeniz bolgesi turuncgillerinde potansiyel iki yeni zararli:  aleurothrixus floccosus (maskell) ve paraleyrodes minei iaccarino (hom.:aleyr.). - turkiye  entomoloji dergisi, 202 : 113-121. fabio m. iaccarino - dipartimento di entomologia e zoologia agraria “f.silvestri, università  degli studi di napoli “federico ii”, facoltà di agraria, via università 100 - 80055 portici  (napoli), italy. e-mail fiaccari@unina.it.  riccardo jesu, rosa giacometti - dipartimento di entomologia e zoologia agraria “f. silvestri,  università degli studi di napoli ”federico ii”, facoltà di agraria, via università, 100 - 80055  portici (napoli), italy. accepted 30 march 2011 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. layout 1 [journal of entomological and acarological research 2014; 46:1920] [page 59] abstract silver nanoparticles (agnps) were synthesized from the latex of the medicinally important plants euphorbia milii, euphorbia hirta, ficus racemosa and jatropha curcas. synthesized agnps were characterized by uv-vis spectrophotometry, scanning electron microscopy, energy dispersive x-ray analysis, x-ray diffraction, fourier transformed infrared spectroscopy, particle size, and zeta potential analysis. potency of latex and latex-synthesized agnps was evaluated against the 2nd and 4th instar larvae of aedes aegypti and anopheles stephensi. the lowest lethal concentration 50 (lc50) value among the different types of plant latex studied was observed for latex of e. milii (281.28±23.30 and 178.97±37.82 ppm, respectively) against 2nd instar larvae of ae. aegypti and an. stephensi. e. milii latex-synthesised agnps showed a high reduction in lc50 compared with its latex; i.e., 8.76±0.46 and 8.67±0.47 ppm, respectively, for 2nd instars of ae. aegypti and an. stephensi. lc50 values of agnps synthesized using the latex of e. hirta, f. racemosa and j. curcas were lower than those of the latex of the respective plants; i.e., 10.77±0.53, 9.81±0.52, 12.06±0.60 and 8.79±0.51, 9.83±0.52, 9.60±0.51 ppm, respectively, for 2nd instars of an. stephensi and ae. aegypti. similarly, as compared with the plant latex, lower lc50 values were reported for latex-synthesized agnps against 4th instars of ae. aegypt and an. stephensi. results showed that all the types of plant latex investigated have the potential to convert silver nitrate into agnps showing a spectrum of potent mosquito larvicidal effects, indicating the possibility of further exploration of the bioefficacy of latex and latex-synthesized agnps against vectors of public health concerns. introduction about 3.3 and 2.5 billion people, respectively, are at risk of malaria and dengue worldwide, with a higher frequency in the population of sub-saharan africa (ssa) (who, 2009, 2011). in india, 1.49 million cases of malaria, 28,292 cases of dengue, 767 and 108 deaths were reported from malaria and dengue in 2010 (nvbdcp, 2011). the above figures indicate the global impact of mosquito-transmitted diseases with respect to loss of national productivity due to mortality and morbidity. mosquito species such as anopheles stephensi, aedes aegypti and culex quinquefasciatus are widely distributed in the tropical and subtropical zones, acting as vectors of diseases like malaria, dengue, filariasis, japanese encephalitis, yellow fever, and chikungunya (who, 2009). to control the outbreak of mosquito-borne diseases, attention should be given to targeting the larval stage of mosquitoes, which are unable to fly and are present in the breeding habitat. devising a control methodology should therefore be relatively easy for the larval stage. during the past several decades, organophosphates such as temephos and fenthion, and insect growth regulators such as diflubenzuron and methoprene, have been used to control mosquito larvae (yang et al., 2002). insecticides of microbial origin, such as bacillus thuringiensis, have also been employed for larval control (raghvendra et al., 2011). however, continued and indiscriminate use of these insecticides creates problems such as insecticide resistance, environmental pollution and toxicity to human and non-target organisms (raghvendra et al., 2011). to combat these shortcomings of chemical insecticides, research has shifted toward products of biological origin (patil et al., 2012a; karunamoorthi, 2013). use of products of plant origin to control mosquito larvae has been shown to be an exciting alternative to traditional methods of larval management, as they are not associated with the problems noted above (shaalan et al., 2005, borase et al., 2013). for example, root and leaf extracts of plumbego zeylanica and cestrum nocturnum (patil et al., 2011b), leaf extracts of ocimum sanctum, phyllanthus emblica (murugan et al., 2012), and hydrodistillate extracts of mentha piperita, ocimum basilicum, zingiber officinale, and curcuma longa (kalaivani et al., 2012) have been used against mosquito larvae of an. stephensi, ae. aegypti and cu. quinquefasciatus. the use of phytosynthesized silver nanoparticles as a larvicidal correspondence: satish v. patil, school of life sciences, north maharashtra university, post box 80, jalgaon 425001, maharashtra, india. tel.: +91.257.2257421 fax: +91.257.2258403. e-mail: satish.patil7@gmail.com key words: plant latex, mosquito biolarvicidal, silver nanoparticles, anopheles stephensi, aedes aegypti. acknowledgements: hemant p. borase is dst-inspire fellow (grant file no. dst/inspire fellowship/2011[149].), chandrashekhar d. patil is thankful to csir (ref: 09/728 (0028)/2012emr-i) for the award of senior research fellowship. received for publication: 12 september 2013. revision received: 26 december 2013. accepted for publication: 17 january 2014. ©copyright h.p. borase et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1920 doi:10.4081/jear.2014.1920 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:1920 mosquito larvicidal and silver nanoparticles synthesis potential of plant latex h.p. borase,1 c.d. patil,1 r.b. salunkhe,1 c.p. narkhede,1 r.k. suryawanshi,1 b.k. salunke,1 s.v. patil1,2 1school of life sciences, north maharashtra university; 2north maharashtra microbial culture collection centre (nmcc), north maharashtra university, india no nco mm er cia l u se on ly [page 60] [journal of entomological and acarological research 2014; 46:1920] agent instead of chemical insecticides is gaining importance because of their safety to users as well as nontarget species, and the novelty of their mechanism of action (marimuthu et al., 2011; patil et al., 2012b). several plants have been screened successfully for silver nanoparticle synthesis, such as plumeria rubra. (patil et al., 2011a), pergularia daemia (patil et al., 2012a), acacia arabica (thakur et al., 2013), cadaba indica lam leaf extract (kalimuthu et al., 2013), euphorbia tirucalli, and alstonia macrophylla (borase et al., 2013), as described in a review by gan & li (2012). chemical and physical methods of nanosynthesis have shortcomings such as the use of toxic chemicals and high temperatures. to address these, the use of living organisms such as plants and microorganisms (bacteria and fungi) for nanoparticle synthesis is gaining momentum. latex is a milky to transparent sap produced in some plants and studied mostly with respect to rubber production, interactions with insects as a plant defense mechanism, and in explorations of different pharmacological activities (kekwick, 2007). the latex-producing plants e. milii, e. hirta, f. racemosa and j. curcas used in the present study are available in large quantities locally in india and have been reported in the literature for their medicinal applications as well as for their active biochemical constituents (table 1). for these reasons and because of the potent mosquito larvicidal activity showed by plant plumeria rubra and pergularia daemia and synthesized agnps in our earlier study (patil et al., 2011a; 2012a), we wanted to investigate the potential of other types of plant latex as eco-friendly mosquito larvicidal agents, and as precursors for environmentally benign silver nanoparticle synthesis. materials and methods plant material e. milii, e. hirta, f. racemosa and j. curcas growing in the vicinity of jalgaon, india, were used as sources of fresh latex. latex was collected in the early morning during march, 2013, by making a small incision near the youngest leaves and at the ends of branches. extruded latex was collected in sterile tubes (10 ml). tubes were kept at 4°c to stop coagulation until the time of the experiments. phytochemical characterisation of latex latex samples were subjected to qualitative tests for the presence of different metabolites as reported by kokate (1999) and patil et al. (2012b). synthesis of silver nanoparticles one ml of fresh latex was added to 100 ml of an aqueous solution of silver nitrate (100 ppm). the flask was incubated on a rotary shaker (28°c at 120 rpm). simultaneously, controls containing latex with milli-q deionized water and silver nitrate solution alone were maintained under the same conditions. solutions were observed periodically for any colour change. test organisms for the laboratory trials, locally collected early 2nd and 4th instar larvae of ae. aegypti and an. stephensi were used as experimental specimens. the larvae were kept in plastic enamel trays containing dechlorinated tap water, and were maintained as reported by kalimuthu et al. (2013). mosquito larvicidal bioassay different concentrations of latex and agnps were prepared in dechlorinated tap water. larvicidal activity was assessed using the procedure of who (1996) with some modifications and as per the methods of patil et al. (2011b, 2012b). twenty five 2nd and 4th instar larvae were taken in four batches in 249 ml of water, and 1.0 ml of the desired concentration of latex plus agnps were added. the control was set up with dechlorinated tap water. the numbers of dead larvae were counted after 24 h of exposure, and the percent mortality was recorded for the average of four replicates. the experimental media, in which 100% mortality of larvae occurred, was selected for the dose-response bioassay (data not shown). dose response bioassay based on the preliminary screening results, crude latex extract of the experimental plants plus synthesized agnps were subjected to a dose-response bioassay for larvicidal activity against the larvae of ae. aegypti and an. stephensi. different concentrations ranging from 62.25 article table 1. medicinal properties and chemical constituents of latex producing plants used for analysing larvicidal and silver nanoparticle synthesis potential. botanical name common name family medicinal chemical references (vernacular name) property constituents euphorbia milii milli crown of throrn euphorbiaceae molluscicidal miliin, serine proteas, yadav et al. (2006); (christ plant) flavons, triterpenoids, steroids, steroidal glycoside, alkaloids euphorbia hirta tawa-tawa euphorbiaceae antihelminthic, sterols, alkaloids, tannins, iwu (1993); (dudhi) repellent, antifeedant glycosides, triterpenoids, wei et al. (2005); and controlling plutella alkenes, phenolic acids, kumar et al. (2002); xylostella and nematicidal choline and shikimic acid parekh & chanda (2007); and against roundworm rajeh et al. (2012) like guinea worm ficus racemosa cluster fig tree moraceae anti-inflammatory, racemosic acid, khan & sultana (2005); (udumbara) antidiarrheal, clears horsevoice triterpenes li et al. (2004); and chemomodulatory, larvicides rahuman et al. (2008) jatropha curcus bagbherenda euphorbiaceae nematicidal, fungicidal, triglycerols, sterols, sharma & trivedi (2002); (jungli erand) mosquito (ochlerototatus oils, phorbal esters, gübitz et al. (1999) triseriatus) larvicidal, glucanase protein insecticidal activities no nco mm er cia l u se on ly to 2000 ppm (for the latex) and 0.625 to 20 ppm (for the synthesized agnps) were prepared, and numbers of dead larvae were counted after 24 h of exposure; percent mortality was reported from the average of four replicates. statistical analysis mortality was calculated using abbott’s formula (abbott, 1925). the dose-response data were subjected to probit regression analysis (finney, 1971). the lethal concentrations in parts per million (lc50, lc90) and the 95% confidence intervals of lc50 (upper confidence limit) and (lower confidence limit) were calculated. characterisation of silver nanoparticles agnps solutions were centrifuged at 10,000 rpm for 10 min (remi, cooling centrifuge, c-24 bl, india); the pellet obtained was resuspended in water and used to analyse surface plasmon resonance of the silver nanoparticles using a uv-vis spectrophotometer (shimadzu 1601, tokyo, japan) at the resolution of 1 nm from 200 to 800 nm. other techniques used for agnps characterization included fourier-transformed infrared spectroscopy (ft-ir) (shimadzu, prestige 21, tokyo, japan), scanning electron microscopy (sem), energy dispersive x-ray (edx) (hitachis4800, tokyo, japan), x-ray diffraction (xrd) (brucker d8 advance, karlsruhe, germany), particle size, and zeta potential analysis (zetasizer, malvern instrument ltd, westborough, ma, usa). results synthesis and characterisation of agnps transformations of agno3 to agnps were clearly indicated by a colour change of agno3 from colourless to yellowish brown, depleting all the plant latex within 5 to 20 min of latex addition, without agglomeration and indicating synthesis of stable agnps (figure 1a). latex of e. milii showed the fastest colour change among all types of latex tested (within 5 min). synthesized nanoparticles were characterized by uv-visible spectroscopy showing a surface plasmon resonance band at 410 to 450 nm, which arises due to the conduction of free electrons on the surface of agnps (figure 1b) (smitha et al., 2008). absorption maxima at 440, 433,419, and 444 nm were observed for agnps synthesized from e. milii, e. hirta, f. racemosa and j. curcas, respectively. similar results have been shown by borase et al. (2013) and thakur et al. (2013). absorbance of agnps synthesized from the latex of e. milii was found to be higher than the other types of plant latex under study. e. milii latex-fabricated agnps show a smaller size of 208 nm with a zeta potential of –9.19 mv (figure [journal of entomological and acarological research 2014; 46:1920] [page 61] article figure 1. a) tubes showing colour change of agnps: a. silver nitrate solution, b. e. milii, c. e. hirta, d. f. racemosa, and e. j. curcas. b) uv spectra of agnps. c and d) particle size and zeta potential analysis of agnps produced from e. milii latex. figure 2. a and b) scanning electron microscopy and energy dispersive x-ray image of agnps synthesized from e. milii. no nco mm er cia l u se on ly [page 62] [journal of entomological and acarological research 2014; 46:1920] 1c and d). agnps from e. hirta, f. racemosa and j. curcas showed larger size particles having low stability, as compared with e. milii-synthesized agnps (data not shown). sem imaging showed high density of spherical size, monodispersed agnps (figure 2a). edx spectra confirmed the presence of elemental silver in the samples, as there was a strong signal for the silver atom (figure 2b). fourier transformed infrared spectroscopy analysis showed the presence of different functional groups corresponding to proteins, alkaloids, tannins, saponins and other plant metabolites (figure 3a). a peak at 670.03 cm–1 was assigned to n-h wag of amines of proteins, 701.91 cm–1 as a c-h deformation in carbohydrates, 3439.96 cm–1 for aroh, o-h and n-h for phenols, alcohols and amides, and 2997.48 cm–1 for the c=o bond found in terpenoids and flavonoids. the remaining peaks also indicate the presence of proteins, flavonoids, saponins and other plant metabolites, as evidenced by qualitative phytochemical analysis (table 2). xrd analysis revealed the crystalline nature of agnps. other peaks in the xrd may arise due to biomolecules capped on the agnps surface (figure 3b). mosquito larvicidal bioassay the plant latex under study and agnps fabricated from the latex were used to analyse their potency against the 2nd and 4th instar larvae of ae. aegypti and an. stephensi. the results of larvicidal bioassays of the plant latex are presented in tables 3 and 4, and that of the plant latex-synthesized agnps are presented in tables 5 and 6. all tested plant latex and synthesized agnps showed larvicidal efficacy within 24 h of exposure. mortality rate (y) was positively related to the dose (x), indicating that mortality is dose-dependent. latex materials from all the plants tested were less toxic than the synthesized agnps to both mosquito species. among the agnps tested, the agnps synthesized from the latex of e. milii were highly effective against an. stephensi (lc50=8.76 ppm, lc90= 17.11 ppm), and the agnps from j. curcas was highly effective against ae. aegypti (lc50=9.43ppm, lc90=18.20 ppm). all the plants used in the present study showed lc50 values less than 13 ppm, which could be an important factor in determining a practical larvicidal dose. discussion latex producing plants secrete milky fluid from a network of laticifer cells, in which subcellular organelles intensively synthesize proteins and secondary metabolites (lopes et al., 2009). the biological importance of latex fluids is still unclear and knowledge of their physiological role is still limited (ramos et al., 2007). ramos et al. (2009) presented first evidence for the use of calotropis procerra (ait.) r.br.-secreted proteolytic enzymes as chemical agents against ae. aegypti larvae. plant latex has been reported to have a negative effect on several insect functions such as egg hatch, larval growth and survival (giridhar et al., 1984; morsy et al., 2001; ramos et al., 2006). the chemico-physical method of nanoparticle synthesis involves the use of toxic substances (sodium borohydrate, polyvinylpyrrolidone) that are harmful to the environment. our method of agnps synthesis using latex, which has an abundance of proteins, enzymes and secondary metabolites, is novel, eco-friendly, and does not require toxic chemicals. previous studies have demonstrated the involvement of proteins, polyphenols and carbohydrates in the synthesis of article table 2. phytochemical analysis of plant latex. sr. no. metabolites e. milii e. hirta f. racemosa j. curcas 1 protein + + + + 2 carbohydrates + – + – 3 terpenoids + + + + 4 alkaloids – + + + 5 phenolics + + + + 6 flavonoids + + + + 7 tannin + + – – 8 saponins + + + + 9 glycosides + – + – +=present; –= absent. sr., serial number. figure 3. a and b) fourier transformed infrared spectroscopy and x-ray diffraction spectrum of agnps synthesized from e. milii.no nco mm er cia l u se on ly metal nanoparticles (gan & li, 2012). nanoparticles produced using chemical methods are of a defined size and shape due to the use of a single reducing and capping agent. in biological synthesis, diverse particle size and shape is observed because of multiple reducing and capping agents. consequently, isolation, purification and scale-up of compounds responsible for nanoconversion of silver represent potentially valuable alternatives to chemical synthesis. duran et al. (2011) discussed involvement of the enzyme nadphdependent nitrate reductase in production of agnps, while vigneshwaran et al. (2006) showed the role of reducing sugars in agnps production, agnps synthesis were also reported from combination of reducing agents and terpenoids (shankar et al., 2004), polyols, eugenol, quinines and phyllanthin (jha et al., 2009; kasthuri et al., 2009; singh et al., 2010). the plant latex used in the present study also showed the presence of proteins and secondary metabolites (terpenoids, tannins, alkaloids and others), so we may preliminarily conclude there is an interaction of enzymatic and non-enzymatic compounds in agnps formation. corbel et al. (2007) showed that increased insecticide resistance in mosquitoes is due to increased activity of enzymes involved in insecticide metabolism (e.g., esterases, oxidases, glutathione-s-transferase) and mutation in the target sites of insecticide action. this can be corroborated with how agnps exhibit their larvicidal action. agnps have a high surface area-to-volume ratio, which imparts to them many types of biocidal and catalytic activities. also, in latex-mediated nanosynthesis, capping of latex metabolites on the surface of the agnps, in addition to imparting stability, also increases their larvicidal action. the higher mortality at lower doses is consistent with earlier reports of agnps produced from leaf extracts of nelumbo nucifera (lc50=0.69 ppm, lc90=2.15 ppm) against an. subpictus and cu. quinquefasciatus (lc50=1.10 ppm, lc90=3.59 ppm) thirunavukkarasu et al. (2010). marimuthu et al. (2011) reported bioactivity of mimosa pudica-synthesized agnps against the larvae of an. subpictus, cu. quinquefasciatus, and r. microplus (lc50=13.90, 11.73 and 8.98 ppm), respectively. agnps synthesized using tinospora cordifolia extract were tested against the larvae of an. subpictus (lc50=6.43 mg/l) and cu. quinquefasciatus (lc50=6.96 mg/l) (jayaseelan et al., 2011). shaalan et al. (2005) reported that varying results obtained in lethal concentration values can be due to differences in the levels of toxicity among the insecticidal components of different plants, and the effect of plant extracts can vary significantly depending on plant species, plant part, age of the plant part, extraction solvent, seasonal variation, and mosquito species in prokaryotic systems, agnps have multiple targets for biocidal effects by causing structural damage (kim et al., 2007), generation of reactive oxygen species, interfering with dna replication, and reacting with the thiol enzyme group (liau et al., 1997; feng et al., 2000). patil et al. (2012b) also pointed out the antagonistic effect of agnps on enzymes and proteins regardless of the gram characteristics in bacteria. the mechanism of larvicidal action of silver nanoparticles requires more detailed study. conclusions studies were conducted to evaluate the potential mosquito larvicidal activity of plant latex and latex-synthesized agnps. our results suggest the possibility of addressing the problem of emerging mosquito resistance to chemical insecticides by using latex, latex-synthesized agnps, or combinations of chemical insecticides with latex and agnps, which could be considered an alternative larval eradication tactic that could help reduce the burden of toxic chemical insecticides on the environment and non-target organisms. [journal of entomological and acarological research 2014; 46:1920] [page 63] article table 3. larvicidal activity of latex against 2nd instars larvae of aedes aegypti and anopheles stephensi. mosquito species plant latex lc50±se 95% fiducial limits lc90±se 95% fiducial limits regression (mg l–1) (lcl-ucl) (mg l–1) (lcl-ucl) equation aedes aegypti e. milii 281.28±23.30 234.87-327.91 752.27±51.56 665.59-874.59 y=9.58+0.00941x e. hirta 675.26±39.73 601.94-760.27 1422.69±88.19 1272.29-1626.91 y=33.63+0.0112x f. racemosa 726.69±42.33 647.66-815.91 1555.16±90.48 1399.16-1761.71 y=3.49+0.0111x j. carcus 746.98±48.52 655.56-848.55 1768.99±109.92 1580.74-2022.16 y=4.49+0.00993x anopheles stephensi e. milii 178.97±37.82 95.93-248.31 909.88±73.06 788.93-1087.58 y=11.9+0.00772x f. racemosa 549.52±54.24 441.85-658.39 1809.71±134.66 1584.20-2130.08 y=7.62+0.00820x e. hirta 568.74±46.84 477.61-664.21 1621.64±111.01 1433.66-1881.56 y=6.70+0.00916x j. carcus 755.70±49.04 294.70-391.19 1772.58±112.93 1579.869-2033.902 y=4.37+0.00985x lc50, 50% lethal concentration; se, standard error; lcl, lower confidence limit; ucl, upper confidence limit; lc90, 90% lethal concentration. table 4. larvicidal activity of latex against 4th instars larvae of aedes aegypti and anopheles stephensi. mosquito species plant latex lc50±se 95% fiducial limits lc90±se 95% fiducial limits regression (mg l–1) (lcl-ucl) (mg l–1) (lcl-ucl) equation aedes aegypti e. milii 638.11±36.53 571.00-716.69 1299.02±80.07 1162.58-1484.72 y=3.45+0.0116x e. hirta 683.69±39.32 611.31-768.07 1408.23±86.27 1260.96-1607.78 y=3.33+0.0114x f. racemosa 777.43±43.49 697.64-870.85 1563.74±93.01 1404.19-1777.41 y=2.55+0.0113x j. carcus 798.89±46.00 713.78-896.79 1678.54±99.45 1507.56-1906.32 y=3.00+0.0108x anopheles stephensi e. milii 761.11±43.43 680.80-853.64 1580.75±93.51 1420.08-1795.11 y=3.02+0.0111x e. hirta 783.42±42.89 704.43-875.06 1560.04±89.73 1405.42-1764.95 y=2.38+0.0115x f. racemosa 884.69±45.65 800.79-982.26 1681.22±92.00 1521.86-1889.82 y=1.43+0.0115x j. carcus 919.31±52.52 822.32-1031.26 1930.60±113.96 1734.54-2191.33 y=2.68+0.010x lc50, 50% lethal concentration; se, standard error; lcl, lower confidence limit; ucl, upper confidence limit; lc90, 90% lethal concentration. no nco mm er cia l u se on ly [page 64] [journal of entomological and acarological research 2014; 46:1920] references abbott w.s., 1925 a method of computing the effectiveness of an insecticide. j. ecol. entomol. 18: 265-266. borase h.p., patil c.d., salunkhe r.b., narkhede c.p., salunke b.k., patil s.v., 2013 phyto-synthesized silver nanoparticles: a potent mosquito biolarvicidal agent. j. nanomed. biother. discov. 3: 1-7. corbel v., guessan r.n., brengues c., chandre f., djogbenou l., martin t., akogbeto m., hougard j.m., rowland m., 2007 multiple insecticide resistance mechanisms in anopheles gambiae and culex quinquefasciatus from benin, west africa. acta tropica 101: 207-216. duran n., marcato p.d., duran m., yadav a., gade a., rai m., 2011 mechanistic aspects in the biogenic synthesis of extracellular metal nanoparticles by peptides, bacteria, fungi and plants. appl. microbiol. biotechnol. 90: 1609-1624. feng q.l., wu j., chen g.q., cui f.z., kim t.n., kim j.o., 2000 a mechanistic study of the antibacterial effect of silver ions on escherichia coli and staphylococcus aureus. j. biomed. mater. res. 52: 662-668. finney d.j., 1971 probit analysis. cambridge university press, cambridge: 76-80. gan p.p, li s.f.y., 2012potential of plant as biological factory to synthesize gold and silver nanoparticles and there applications. rev. environ. sci. biotechnol. 11:169-206. giridhar g., deval k., mittal p.k., vasudevan p., 1984 mosquito control by calotropis procera latex. pesticides 18: 26-9. gübitz g.m., mittelbach m., trabi m., 1999 exploitation of the tropical oil seed plant jatropha curcas l. bioresour. technol. 67: 73-82. iwu m.m., 1993 handbook of african medicinal plants. crc press, boca raton, fl: 24-33. jayaseelan c., rahuman a.a., rajakumar g., vishnu kirthi a., santhoshkumar t., marimuthu s., bagavan a., kamaraj c., zahir a.a., elango g., 2011 synthesis of pediculocidal and larvicidal silver nanoparticles by leaf extract from heartleaf moonseed plant, tinospora cordifolia miers. parasitol. res. 109: 185-94. jha a.k., prasad k., kulkarni a.r., 2009 plant system: nature’s nanofactory. colloids. surf. b. biointerf. 73: 219-223. kalaivani k., senthil-nathan s., murugesan a.g., 2012 biological activity of selected lamiaceae and zingiberaceae plant essential oils against the dengue vector aedes aegypti l. 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(ed.), handbook of plant science. john wiley and sons, uk: 1060-1074. khan n., sultana s., 2005 chemomodulatory effect of ficus racemosa extract against chemically induced renal carcinogenesis and oxidative damage response in wistar rats. life sci. 77: 1194-1205. kim j.s., kuk e., yu k.n., kim j.h., park s.j., lee h.j., kim s.h., park y.k., park y.h., hwang c.y., kim y.k., lee y.s., jeong article table 6. larvicidal activity of latex synthesized agnps against larvae of 4th instars of aedes aegypti and anopheles stephensi. mosquito species plant agnps lc50±se 95% fiducial limits lc90±se 95% fiducial limits regression (mg l–1) (lcl-ucl) (mg l–1) (lcl-ucl) equation aedes aegypti j. carcus 9.43±0.48 8.53-10.46 18.20±0.97 16.50-20.41 y=9.08+0.009x e. milii 9.49±0.48 8.61-10.53 17.60±0.96 15.93-19.79 y=0.884+1.14x e. hirta 10.67±0.54 9.57-11.85 20.00±1.10 18.08-22.51 y=0.808+1.06x f. racemosa 11.44±0.65 10.26-12.86 23.07±1.43 20.63-26.38 y=1.89+0.925x anopheles stephensi e. milii 9.95±0.49 9.05-11.02 18.13±0.97 16.45-20.33 y=0.515+1.14x j. carcus 10.01±0.51 9.07-1.10 19.01±1.02 17.24-21.33 y=1.13+1.10x f. racemosa 11.76±0.60 10.66-13.7 21.98±1.22 19.86-24.77 y=0.723+1.00x e. hirta 12.63±0.66 11.44-14.07 23.39±1.35 21.07-26.49 y=0.525+0.95x lc50, 50% lethal concentration; se, standard error; lcl, lower confidence limit; ucl, upper confidence limit; lc90, 90% lethal concentration. table 5. larvicidal activity of latex synthesized agnps against 2nd instars of aedes aegypti and anopheles stephensi. mosquito species plant agnps lc50±se 95% fiducial limits lc90±se 95% fiducial limits regression (mg l–1) (lcl-ucl) (mg l–1) (lcl-ucl) equation anopheles stephensi e. milii 8.76±0.46 7.91-9.74 17.11±0.94 15.48-19.24 y=1.82+1.13x e. hirta 10.77±0.53 9.78-11.91 20.11±1.06 18.27-22.5 y=0.84+1.07x f. racemosa 9.81±0.52 8.85-10.93 19.34±1.07 17.47-21.78 y=1.69+1.06x j. carcus 12.06±0.60 10.97-13.36 22.00±1.19 19.94-24.71 y=0.36+1.01x aedes aegypti e. milii 8.67±0.47 7.81-9.68 17.62±1.01 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[journal of entomological and acarological research 2014; 46:1920] [page 65] article no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. j. entomol. acarol. res. ser. ii, 42 (1): 39-45 30 april 2010 g.viggiani, f. nugnes description of the larval stages of dryokosmus kuriphilus yasumatsu (hymenoptera: cynipidae), with notes on their phenology abstract the larval stages of dryokosmus kuriphilus yasumatsu are described  and illustrated. the terminal-instar larva shows marked variation in the mandibular  asymmetry and in number and position of the respiratory spiracles. notes on the  larval phenology are given.  riassunto descrizione degli stadi larvali di dryokosmus kuriphilus yasumatsu  (hymenoptera: cynipidae), con note sulla loro fenologia. vengono descritti gli  stadi larvali di dryokosmus kuriphilus yasumastsu. la larva matura mostra marcate variazioni nell’asimmetria mandibolare, nonché nel numero e posizione degli  spiracoli tracheali. sono date notizie sulla fenologia larvale.  key words: variations, respiratory spiracle, asymmetric mandibles. introduction the gall wasp dryokosmus kuriphilus yasumatsu (hymenoptera: cynipidae) is a  worldwide chestnut pest, which reproduces by parthenogenesis and develops one generation a year. in recent years the species was recorded also in italy, where it quickly spread  from piedmont to the other main areas where chestnut is grown (graziosi & santi, 2008).  several studies have been carried out on the bio-ecology, natural enemies and control of  the cynipid (itô, 1967; aebi et al., 2007; ôtake, 1980; rieske, 2007, viggiani & voto,  2009), but the detailed morphology and phenology of the larva still remain unknown.  materials and methods during 2008-2009 the life-cycle of d. kuriphilus was studied in two campania  locations (calvanico, sa; teano, ce). samples of 100 galls were taken weekly from  june 2008 to september 2009. all types of larvae in the galls were recorded and, for  each sample, 20 larvae of d. kuriphilus were mounted on slide using a mixture of water  and glycerine. for each larval type 5 permanent slides were made using balsam-phenol.  journal of entomological and acarological research, ser. ii, 42 (1), 201040 observations and images of morphological and anatomical larval traits were taken by   axiophot microscope equipped with camera lucida and photocamera. for sem microscopy the traditional method of dehydration in alcohol, critical-point drying and goldsputter coating was utilized. before making preparations, all larvae were measured.  fifty couples of left and right mandibles were dissected and permanent slides were  made in order to study their anatomical variation. for the same purpose mandibles of  the terminal-instar larval skin were removed from the pupal site and examined. the variation in number and position of the respiratory spiracles was recorded in 127 terminalinstar larvae mounted in a mixture of water and glycerin. at present, at least in italy, no  inquiline cynipid was recorded in the galls of d. kuriphilus. the terminology follows  nieves-aldrey et al. (2005). results larval instars the present study shows that d. kuriphilus undergoes three instars in the larval  development.  first-instar larva (fig. 1). subglobular, with very small mandibles (fig. 2) which  are subtriangular and apparently unidentate. no tracheal system was detected by the  external and internal examination of the larva. length: 0.2-0.6 mm. this shape derives  from the typical cynipid egg, characterized by a suboval body and a long and thin stalk  (fig. 3). the first-instar larva develops slowly from the summer (july-august) to the  late march-april of the next year. intermediate instar larva (fig. 4). hymenopterifom, mandibles (fig. 5) distally with  two teeth, without tracheal system. length: 0.8-1.5 mm. the intermediate larva stage  appears in april-may and lasts less than one month.  terminal-instar larva (fig. 6). hymenopteriform, with asymmetric mandibles and  a number of variable teeth (fig. 7). length: in average 2.3 mm (n = 127; sd = 0.4). in  frontal view the well-developed head capsule (fig. 8) shows the antennal areas (fig.  8, aa), a subtriangular clypeus (fig. 8, c), followed by a transverse labrum. the mandibles (fig. 8, m; fig. 9) are only partially exposed from the mouth opening. maxillae  subtriangular (fig. 8, mx) with vestigial palpi (fig. 8, mp), as on labium, which shows  a marked salivary opening (fig. 8, la). respiratory system with 4-6 pairs of spiracles. the terminal-instar is the only larval stage present in the d.kuriphilus galls from  late april to end of may.  variation. remarkable variation in the morphology of mandibles and respiratory  system is recorded within the terminal-instar. mandibles. the distal parts of the left and right mandible of 50 specimens were  examined. the most common type (figs. 10-13) shows a strong external tooth, ventrally  concave, followed by an internal and laminar structure. this structure is differently  developed and a toothed margin is sometimes present. in other types (fig. 14-15) the  dental part is represented by 2 sclerotized teeth. 41g.viggiani, f. nugnes: larval stages of dryokosmus kuriphilus, with notes on their phenology figs. 1-7. dryokosmus kuriphilus: first-instar larva, dorsal view (fig. 1), mandible (fig. 2), egg  (fig. 3), intermediate larva, ventral view (fig. 4), mandible (fig. 5), terminal-instar larva, lateral  view (fig. 6), mandible (fig.7).  journal of entomological and acarological research, ser. ii, 42 (1), 201042 fig. 8-9. dryokosmus kuriphilus: terminal-instar larva. head capsule, frontal view (fig. 8), mouth  opening and mandible (fig. 9). aa = antennal areas; c = clypeus; l = labrum; la = labium; lp  = labial palpus; m = mandible; mp = maxillary palpus; mx = maxilla; so = salivary opening;  v = vertex.  43g.viggiani, f. nugnes: larval stages of dryokosmus kuriphilus, with notes on their phenology respiratory system. the variation in number and position of the respiratory spiracles have been studied in 127 larvae. the observations are reported in table 1. most of the larvae have 4 (44.9%) or 5 (33.8) pairs of spiracles, the first situated  on the mesothorax and the others starting from the second abdominal segment. the  extreme number of respiratory spiracles found were 3 and 6.  discussion and conclusion the cynipoidea (hymenoptera) include endoparasitoids and phytophagous species;  most of the latter group are gall makers. although they are distributed worldwide, and  have been subject to years of investigation, our knowledge of pre-imaginal morphology is fragmentary and far from complete (stojanova & draganov, 2008). particularly  lacking is the knowledge on the youngest larval stages. the shape of the first-instar  larva follows that of the egg. the egg of d. kuriphilus falls in the category with a long  and thin stalk and a stout body, typical of the cynipidae (vårdal et al., 2003). the firstinstar larva of this species has a globular shape and is macroscopically indistinguishable  fig. 10-15. terminal-instar larva of dryokosmus kuriphilus. main types of dental parts of the  mandibles (dorsal view).  journal of entomological and acarological research, ser. ii, 42 (1), 201044 from the body of the egg, after the loss of the peduncle, as reported by earlier authors  (itö, 1967). a very slight body segmentation and the presence of very small mandibles  characterize this instar.  the variation of the terminal-instar larva in the cynipoid is remarkable, even between sexes (stojanova & draganov, 2008). most of them concern the shape and the  asymmetry of the mandibles. in a comparative study of the terminal-instar larvae of  cynipoidea, nieves-aldrey et al. (2005) pointed out that head sclerites and mandibles  offer many characters of phylogenetic value. the general features of the terminal-instar  larva of d. kuriphilus are similar to those of other cynipini (cynipidae), but the notable  variation shown by the dental part of the mandibles, suggests a more extended study of  this trait in other species. the intraspecific variation in number and position of the respiratory spiracles found  in the terminal-instar larva of d. kuriphilus is certainly surprising and apparently unrecorded for other species. probably this trait is connected to the fluid content in each  loculus of the gall. in general, the maximum number of tracheal spiracles is linked to  a dry larval habitat. some terminal-instar larvae of parasitic hymenoptera in a host  containing body fluid show a reduced number of respiratory openings (viggiani, 1973;  viggiani, 1984).  acknowledgment  the author thanks dr. hege vårdal, department of systematic zoology, evolutionary biology centre, uppsala university, sweden, for reviewing the manuscript.  table 1. variation in number and position of the respiratory spiracles in the terminal-instar larva of dryokosmos kuriphilus.  n. spiracles position percentage 3 t2 a2 a3 0.8 3 t2 a2 a6 0.8 4 t2 a2 a3 a6 3.9 4 t2 a3 a5 a6 0.8 4 t2 a2 a3 a4 2.4 4 t2 a2 a4 a6 44.8 4 t2 a2 a4 a7 0.8 5 t2 a2 a3 a4 a6 33.8 5 t2 a2 a3 a5 a6 0.8 5 t2 a2 a4 a5 a6 6.3 5 t2 a2 a3 a4 a5  1.6 5 t3 a1 a2 a5 a6 0.8 6 t2 a2 a3 a4 a5 a6  2.4 legend: a1 = first abdominal segment; a2 = second abdominal segment; a3 = third abdominal segment; a4  = fourth abdominal segment; fifth abdominal segment; a6 = sixth abdominal segment; seventh abdominal  segment; t2 = mesothorax; t3 = metathorax. 45g.viggiani, f. nugnes: larval stages of dryokosmus kuriphilus, with notes on their phenology references aebi a., schönrogge k., melika g., quacchia a., alma a., stone g. n., 2007 - native and  introduced parasitoids attacking the invasive chestnut gall wasp dryocosmus kuriphilus.- bull.  oepp/eppo, 37: 166-171.  graziosi i., santi f., 2008 - chestnut wasp (dryocosmus kuriphilus): spreading in italy and new  records in bologna province. - bulletin of insectology, 61 (2): 343-348.  itô y., 1967 - population dynamics of the chestnut gall-wasp, dryokosmus kuriphilus yasumatsu  (hymenoptera: cynipidae) iv. further analyses of the distribution of eggs and young larvae  in buds using the truncated negative binomial series. - res. popul. ecol., 9: 177-191.  nieves-aldrey j. l., vårdal h., ronquist f., 2005 - comparative morphology of terminalinstar larvae of cynipoidea: phylogenetic implications. - zoologica scripta, 34 (1): 15-36.  ôtake a., 1980 - chestnut gall wasp, dryokosmus kuriphilus yasumatsu (hymenoptera: cynipidae): a preliminary study on the trend of adult emergence and some other ecological aspects  related to the final stage of its life cycle. - appl. ent. zool., 15(1): 96-105.  rieske j. k., 2007 - success of an exotic gallmaker, dryokosmus kuriphilus, on chestnut in the  usa: a historical account. - eppo bulletin, 37: 172-174.  stojanova a. m., draganov m. m., 2008. - life cycle of aylax hypecoi (insecta: hymenoptera: cynipidae), a gall inducer on hypecoum ssp. (papaveraceae). - cent. eur. j. biol., 3(2):  199-204. vårdal h., sahlen g., ronquist f., 2003 - morphology and evolution of the cynipoid egg  (hymenoptera). - zool. j. linn. soc. lond., 139: 247-260. viggiani g., 1973 - osservazioni morfo-biologiche sull’azotus pulcherrimus merc. (hymenoptera:  aphelinidae). ricerche sugli hymenoptera chalcidoidea. xl. - boll. lab. ent. agr. filippo  silvestri 30: 300-311. viggiani g., 1984 - bionomics of the aphelinidae. - ann. rev. entomol. 29: 257-276. viggiani g., voto a., 2009 - preliminary data on the phenology of dryocosmus kuriphilus yasumatsu and the beneficial arthropodfauna of the chestnut in campania. - castanea 2009 - 1st  european congress on chestnut - 5° convegno nazionale castagno. cuneo, italy, 13-16  october. abstracts: 67.  prof. gennaro viggiani, dipartimento di entomologia e zoologia agraria “filippo silvestri”,  via università, 100 -80055- portici (na). e-mail: genviggi@unina.it accepted 31 march 2010 j. entomol. acarol. res. ser. ii, 42 (1): 11-17 30 april 2010 m. moghaddam & m. alikhani two new species of mealybugs (hemiptera, coccoidea, pseudococcidae) from iran abstract - polystomophora arakensis moghaddam and phenacoccus salviacus moghaddam are described and illustrated in detail from iran. the species p. arakensis was collected from the roots of atraphaxis sp. (family: polygonaceae) and  p. salviacus was collected from the leaves of salvia bracteata (family: labiatae).  the genera phenacoccus and polystomophora are briefly reviewed and a key to the  iranian species of phenacoccus is provided. riassunto two new species of mealybugs (hemiptera, coccoidea, pseudococcidae) from iran. sono descritti polystomophora arakensis moghaddam and phenacoccus salviacus  moghaddam. p. arakensis è stato raccolto su radici di atraphaxis sp. (polygonaceae),  mentre p. salviacus è stato rinvenuto su foglie di salvia bracteata (labiatae). sono  brevemente rivisti i generi phenacoccus e polystomophora ed è stata predisposta  una chiave di classificazione delle specie di phenacoccus dell’iran. key words: scale-insects, mealybugs, new species, iran. introduction the genus polystomophora borchsenius is represented in the palaearctic region by  only 2 species, p. ostiapluriana (kiritshenko) and p. orientalis matesova (ter-grigorian).  the latter species, p. orientalis, has been recorded recently from iran (moghaddam et al., 2009). the genus phenacoccus is one of the largest genera of the family pseudococcidae  with about 180 species worldwide (ben-dov, 1994). moghaddam (2009) has recently  listed 3 species of phenacoccus from iran, and moghaddam & bagheri (2010) reported  p. solenopsis tinsley from the southern part of iran, and was probably introduced from  pakistan where is known as a serious pest of cotton. (hodgson et al. 2008). bodenheimer  (1944) recorded p. sherbinovskyi bodenheimer and kozár et al. (1996) mentioned two  more species of phenacoccus from iran.  journal of entomological and acarological research, ser. ii, 42 (1), 201012 methods the specimens were slide-mounted according to the method of williams & granara  de willink (1992) and terminology follows that of williams (2004). body measurements  are given in millimeters (mm) and measurements of the microscopic characters in microns (μm). the illustrations show the morphology of the dorsum on the left side and  the venter on the right, with enlargements of important characters. these enlargements  are not drawn to scale. the specimens are deposited at the iranian research institute of plant protection  (iripp), tehran, iran. polystomophora arakensis moghaddam sp. n. (fig. 1) habit. it occurs on the root of the host plant. diagnosis. appearance of adult female in life not recorded. mounted female broadly  oval, posterior end of body rounded, 2.4–2.6 mm long and 1.9–2.3 mm wide. eyes  30.0–32.5 µm wide. antennae 9 segmented, 330–340 µm long, apical segment 40-42  µm long, 30 µm wide. legs moderately developed; hind trochanter + femur 200–210  µm long, hind tibia + tarsus 230–240 µm long, hind claw 27–28 µm long, with a denticle; claw digitules longer than claws with a slightly expanded apex. ratio of lengths of  hind tibia + tarsus to hind trochanter + femur 1.04–1.06; ratio of lengths of hind tibia  to tarsus 2.42, ratio of length of hind trochanter + femur to greatest width of femur  about 3.3–3.5. trochanter with 2 campaniform sensilla on each surface and with one  long apical seta. translucent pores present on posterior surface of hind femur, and on  anterior and posterior surface of hind tibia. tibia cylindrical, with two apical spines.  circulus present, 105 µm wide, notched on each side, anterior margin slightly wider  than posterior margin. ostioles well developed, each lip with a few trilocular disc pores.  cerarii absent except anal lobe cerarii, each with 2 long slender setae, 35–55 µm long  and a few trilocular pores. anal ring rounded, about 75 µm wide, lying at some distance  from the apex, with two rows of pores, bearing 6 setae , each 30-35 µm long. anal lobes  inconspicuous, each lobe bearing an apical seta about 115 µm, a shorter seta about 72  µm, and 1-2 long slender setae 50 µm long.  dorsum. dorsal surface with long slender setae and flagellate setae. multilocualr disc  pores scattered across head and thoracic segments, and present on margin and midline  of abdominal segments v- vii, and located across abdominal segments i-iv in 2 rows.  quinquelocular disc pores few, occurring on midline thoracic segments. trilocular disc  pores distributed on dorsum. oral collar ducts present sparsely on dorsum. venter. ventral surface with flagellate setae. multilocular disc pores same as those  on dorsum, present across head and thoracic segments, distributed across posterior and  anterior edges of abdominal segments and posterior to vulva. quinquelocular disc pores  located in midline thoracic segments, present on anterior edges of abdominal segments  i-iii. trilocular disc pores scattered over entire body. oral collar tubular ducts dispersed  throughout body to lateral edges. 13m. moghaddam, m. alikhani: two new species of mealybugs from iran fig. 1 - polystomophora arakensis moghaddam, sp.n. journal of entomological and acarological research, ser. ii, 42 (1), 201014 comments the species p. arakensis differs from p. orientalis matesova in having translucent  pores on the hind femur and hind tibia; posterior end of body rounded and anal lobes  inconspicuous. the mealybug p. arakensis differs from p. ostiaplurima (kiritchenko)  in having multilocular disc pores on the head, thorax and abdominal segments and also  possessing one circulus. material examned holotype adult♀, iran. markazi province, arak, from the roots of atraphaxis sp.,  10.x.2009, leg. m. alikhani (iripp). paratypes, iran. same data as holotype, 1 adult female on the same slide as holotype and 14 adult females (iripp). etymology the name refers to arak, the collecting site for the species. phenacoccus salviacus moghaddam, sp. n. (fig. 2) habit. it occurs on the leaves of the host plant. diagnosis. appearance of adult female in life not recorded. body of adult female on  microscope slide elongate oval, 2.12–2.80 mm long. anal lobes moderately developed,  each ventral surface membranous, with an apical seta 95 µm long and an inner seta 40  µm long. antennae slender, each 415–440 µm long, 9 segmented. legs well developed;  hind trochanter + femur 270-300 µm long, hind tibia + tarsus 320-335 µm long, claw  about 45 µm long, with a denticle present. ratio of lengths of hind tibia + tarsus to hind  trochanter + femur 1.12–1.25. ratio of lengths of hind tibia to tarsus 2.4–2.6. translucent pores absent from hind legs. circulus present between abdominal segments iii  and iv, 150 µm wide. ostioles well developed, each lip with 4-5 trilocular pores and a  few setae. anal ring about 65 µm wide, bearing 6 setae, each about 110-118 µm long.  cerarii numbering 12 pairs. anal lobe cerarii with 2 conical setae, a few trilocular pores  and 2 shorter setae, all situated on a lightly sclerotized area. anterior cerarii each with  2 slightly shorter conical setae and 1 trilocular pore situated between setae. posterior  cerarii on abdominal segments v –vii, each with 2 setae and 2 trilocular pores, located  on a slightly sclerotized area protruding from surface of derm.  dorsum. dorsal surface with slender, conical setae, mostly each 17.5 µm long, some  minute setae, each about 5 µm long; some single setae with 1 trilocular pore at base of  setal collar present on median, submedian and submarginal areas of dorsal segments. 1  median cerarius containing 2 setae and 2 trilocular pores at base, present on abdominal  segment vii. multilocular disc pores few, each about 7.5 µm in diameter, present on  posterior edges of abdominal segment vii. oral collar tubular ducts, numbering 4-5,  present across head, thorax and abdominal segments.  15m. moghaddam, m. alikhani: two new species of mealybugs from iran fig. 2 - phenacoccus salviacus moghaddam sp.n. journal of entomological and acarological research, ser. ii, 42 (1), 201016 venter. ventral surface with long flagellate setae, except for some short conical  setae situated around margins. multilocular disc pores each about 7.5 µm in diameter,  present posterior to vulva, and on posterior edges of abdominal segments iv-vii, some  on anterior edge of segment vii; multilocular pores never reaching margins. trilocular  pores sparse, present on margins and submargins of head, thorax and abdominal segments. quinquelocular pores, each wider than a trilocular pore, present on midline and  near midline of head, thorax and abdominal segments i - vi, also present on anterior  edges of iv - vii abdominal segments. oral collar tubular ducts, present along margins  of thorax and anterior abdomen, single on thorax, and forming rows across abdominal  segments v- viii. comments the species p. salviacus differs from p. transcaucasicus hadzibejli in having a  few multilocular disc pores on abdominal segment vii; cerarii numbering 12 pairs, all  cerarii with 2 slender, conical setae and 1 or 2 trilocular pores; also p. salviacus differs in having 1 median cerarius on abdominal segment vii and the presence of dorsal setae  with 1 trilocular pore at the base of each setal collar, forming the rows across the dorsal  segments.  material examned holotype adult♀, iran. markazi province, arak, shazand, suraneh, on the leaves  of salvia bracteata, 10.x.2009, leg. m. alikhani (iripp). paratypes, iran. same data as holotype, 8 adult females (iripp). etymology the name is based on the host plant name salvia. key to iranian species of phenacoccus 1  circuli numbering 2 – 4 ................................................................ aceris (signoret) –   circuli numbering only 1 .......................................................................................2 2  cerarii numbering 18 pairs. quinquelocular pores absent from venter. dorsal setae  lack trilocular pores at base of setal collar. multilocular disc pores absent from  dorsum ....................................................................................................................3 –  cerarii numbering 12 pairs. multilocular present. quinquelocular pores present  on venter. dorsal setae with 1 trilocular pore at base of setal collar. at most 2 - 4  multilocular disc pores present on dorsal abdominal segment vii ..salviacus sp. n.   3  antennae normally 9-segmented. multilocular disc pores on venter usually present  only as far forward as segment vi, normally present at anterior edge of segment  vii. circulus often produced laterally, flaccid ............................solenopsis tinsley 17m. moghaddam, m. alikhani: two new species of mealybugs from iran –  antennae normally 8-segmented. multilocular disc pores on venter usually present  as far forward as segment iv, normally absent from anterior edge of segment vii.  circulus usualy small, slightly oval ..................................................... solani ferris acknowledgements we thank. dr. d. j. williams, the natural history museum, uk, for reading the  manuscript and for their valuable comments and suggestions and our thanks also go to  dr. m. parchami-araghi (iripp) for his useful suggestions on the developing manuscript. references ben-dov, y. (1994) a systematic catalogue of the mealybugs of the world (insecta: homoptera: coccoidea: pseudococcidae and putoidae) with data on geographical distribution, host plants, biology and economic importance. intercept limited, andover, uk. 686 pp. hodgson, c.j., abbas, g., arif, m.j., saeed, s. & karar, h. (2008) phenacoccus solenopsis  tinsley (sternorrhyncha: coccoidea: pseudococcidae), an invasive mealybug damaging cotton in pakistan and india, with a discussion on seasonal morphological variation. zootaxa  1913: 1-35. bodenheimer, f.s. (1944) note on the coccoidea of iran, with description of new species. bulletin de la société fouad 1er d’entomologie 28: 85-100. kozár, f., fowjhan, m.a. & zarrabi, m. (1996) check- list of coccoidea and aleyrodoidea  (homoptera) of afghanistan and iran, with additional data to the scale insects of fruit trees  in iran. acta phytopathologica et entomologica hungarica 31 (1-2): 61-74. moghaddam, m. (2009) insects of iran, the list of coccoidea in the hayk mirzayans insect museum (2) hemiptera: coccoidea. iranian research institute of plant protection, tehran,  iran. 37pp. moghaddam, m. & bagheri, m. (2010) a new mealybug pest in the south of iran, phenacoccus solenopsis tinsley (hemiptera: coccoidea: pseudococcidae). journal entomology society of iran (in press). moghaddam, m., ram, p., kamali, h. & bazoobandi, m. (2009) polystomophora orientalis  (hemiptera: coccoidea: pseudococcidae), a new genus and species record for iran. journal of entomological society of iran 29(1): 41. ter-grigorian, m.a., (1973) soft and armoured scales (coccoidea) mealybugs (pseudococcidae). fauna of armenian ssr. izvestiya akademii nauk armianskoi ssr. erevan, 1-246. williams, d.j. (2004) mealybugs of southern asia. the natural history museum, kuala lumpur:  southdene sdn. bhd. 896 pp. williams, d.j. & granara de willink, m.c. (1992) mealybugs of central and south america,  uk: cab international. 635pp. masumeh moghaddam - insect taxonomy research department, iranian research institute plant  of protection, p.o. box 19395-1454, tehran, iran. moghaddam@iripp.ir  mamud alikhani - azad university of arak, p.o.box 567- 38135, arak, iran. mahmud.alikhani@gmail.com accepted 15 april 2010 j. ent. acar. res. ser. ii, 43 (3): 291-300 30 december 2011 g. pellizzari, f. porcelli, g. seljak, f. kozár  some additions to the scale insect fauna (hemiptera: coccoidea) of crete with a check list of the species known from the island abstract - a list of the scale insects (homoptera: coccoidea) recorded by the authors  for the greek island of crete is reported. this includes twenty-seven species new  to the island the most interesting records are kermes palestiniensis balachowsky  (kermesidae), only recorded previously from israel, and getulaspis bupleuri (marchal) (diaspididae), only known previously from north africa and the middle east.  with the present additions, the number of scale insect species recorded on crete  has reached 82. a revised check list of the scales presently known from the island  is also provided. riassunto - nuovi reperti per la fauna di homoptera coccoidea di creta e check list delle specie note nell’isola viene riportato l’elenco di cocciniglie (hemiptera, coccoidea) raccolte dagli autori,  in periodi diversi, nell’isola di creta (grecia). di queste, 27 sono le specie non  ancora note per l’isola. di particolare interesse biogeografico è il reperimento di  kermes palestiniensis, balachowsky (kermesidae), noto finora solo per israele e di  getulaspis bupleuri (marchal) (diaspididae), nota per nordafrica e medio oriente.  viene presentata una check list delle cocciniglie finora note per l’isola. key words: greece, kermes palestiniensis, getulaspis bupleuri this paper deals with the scale insect species collected by the authors on crete at  different periods. these records stimulated a deeper study on the scale insect fauna of  this mediterranean island, as it was apparent that the present knowledge on this subject  was poor and largely incomplete. according to scalenet (ben-dov et al., 2010), there  are only 43 scale insect species recorded from crete. recently jansen et al. (2011) added  six species to this list, raising to 49 the total number of species known to occur on the  island. this number is much lower than expected. even very common, widely distributed  species in the mediterranean basin, and significant agricultural and forestry pest species  had not been recorded there, although they are generally listed for the greek fauna. several authors (podsiadlo, 1983; kozár et al., 1991; kozár & nagy, 1998) have  provided valuable contributions to the knowledge of greek scale insects, but many  journal of entomological and acarological research, ser. ii, 43 (3), 2011292 records from crete were either overlooked or had been included in the greek mainland  fauna (ben-dov et al., 2010). on the other hand, milonas et al. (2008) list 168 scale  insect species for the whole of greece. this list includes also species only known to  occur in crete. argyriou (1984), in her paper dealing with scales of agricultural plants in greece,  only mentions explicitly lichtensia viburni signoret, filippia follicularis targioni tozzetti, marchallina hellenica gennadius and pollinia pollini (a. costa) as being  present in crete. other common agricultural scale pests are reported as being “widely  distributed all over the country” (i.e. sphaerolecanium prunastri (fonscolombe) and  planococcus ficus (signoret)), possibly inferring that they are present also in crete. from a biogeographic point of view, the flora and fauna of the larger mediterranean  islands (corsica, sardinia, sicily, crete, cyprus) are kept in separated lists from their  mainland country of pertinence as they often host endemic species. due to the uncertainty of the occurrence of some species in crete, each bibliographic  source recorded in scalenet, as being present in crete, has been checked by reading the  original paper. this has led to the discovery of several trivial mistakes, so that species  clearly referred in the original papers as being present on the greek mainland only, have  been listed for crete, (i.e. eumyrmococcus corinthiacus williams (williams, 1993), eulecanium ciliatum douglas and scythia festuceti (šulc) (kozár et al., 1991)). in addition,  species recorded from crete only have been listed for mainland greece (i.e rhizococcus  agropyri borchsenius), whilst other species are not known from either mainland greece  or crete (i.e. atrococcus arakelianae (ter-grigorian) (kozár & nagy, 1998)). the present paper lists the species collected by the authors in crete, along with the  localities, collecting dates and host plants. species newly recorded from the island are  marked with an asterisk. a provisional check-list of the scale insects now known from  crete, along with bibliographic sources, is given in table 1. erroneous and uncertain  previous records have been omitted.  among the collected species, diaspidiotus lenticularis and lepidosaphes flava were  infected by septobasidium sp., a fungus often associated with armoured scale insects,  and which changes the scale morphology (couch, 1938). fam. ortheziidae *orthezia urticae (linnaeus): knossos, 25.ix.2009, undetermined plant (f. kozár). fam. monophlebidae gueriniella serratulae (fabricius): vai, 10.iv.2010, genista acanthoclada (g. pellizzari).  previously recorded in crete (stalis) by podsiadlo (1983). icerya purchasi maskell: iraklion, 21-26.ix.2009, citrus (f. kozár); chania, 8.iv.2010, pittosporum tobira (g. pellizzari). fam. marchalinidae marchalina hellenica gennadius: high infestations were observed at agios ioannis, 7.iv.2010, pinus brutia (g. seljak); anopoli, 7.iv.2010, cupressus sp., pinus halepensis  293g. pellizzari et al.: scale insect fauna of crete table 1 species recorded in crete and validation source fam. ortheziidae species validation source orthezia urticae (linnaeus) present paper fam. monophlebidae species validation source gueriniella serratulae (fabricius) podsiadlo, 1983; present paper icerya purchasi maskell ayoutantis, 1940; podsiadlo, 1983 fam. marchalinidae species validation source marchalina hellenica gennadius argyriou, 1984; hodgson & gounari,  2006 family matsucoccidae  species validation source matsucoccus josephi bodenheimer & harpaz mendel, 1998 fam. pseudococcidae species validation source atrococcus arakelianae (ter-grigorian) kozár & nagy, 1998 coccidohystrix sp. n.? present paper chorizococcus rostellum lobdell present paper heliococcus bohemicusšulc jansen et al., 2010 heterococcus nudus green present paper peliococcus kimmericus (kiritshenko) kozár et al., 1991 phenacoccus bicerarius borchsenius kozár et al., 1991 phenacoccus madeirensisgreen jansen et al., 2010 planococcus citri(risso) ayoutantis, 1940 planococcus ficus (signoret) argyriou, 1984,  planococcus vovae(nasonov) williams & moghaddam, 2000; present paper pseudococcus longispinus(targioni tozzetti) present paper spilococcus halli(mckenzie & williams) present paper trionymus aberrans goux kozár et al., 1991 trionymus multivorus present paper journal of entomological and acarological research, ser. ii, 43 (3), 2011294 fam. eriococcidae species validation source acanthococcus sp. n.,  present paper acanthococcus desertus (matesova) present paper acanthococcus munroi boratynski kozár et al., 1991 rhizococcus agropyri borchsenius kozár et al., 1991 rhizococcus cynodontis kiritchenko kozár et al., 1991 family kermesidae species validation source kermes palestiniensis balachowsky  present paper kermes vermilio planchon lindinger, 1912; hoy, 1963; present  paper fam. cerococcidae  species validation source cerococcus cistarum balachowsky kozár & nagy, 1998  fam. coccidae species validation source ceroplastes floridensis comstock present paper ceroplastes rusci (linnaeus) ayoutantis, 1940; podsiadlo, 1983; present paper ceroplastes sinensis del guercio present paper coccus hesperidum linnaeus ayoutantis, 1940 podsiadlo, 1983 filippia follicularis (targioni tozzetti) argyriou, 1984 lecanopsis formicarum (newstead) present paper lichtensia viburni signoret argyriou, 1984 parthenolecanium corni (bouché) kozár et al.,1991 poaspis intermedia (goux) kozár & nagy, 1998 protopulvinaria pyriformis (cockerell) jansen et al., 2010; present paper pulvinariella mesembryanthemi (vallot) kozàr et al.,1991 saissetia coffeae (walker) podsiadlo, 1983  saissetia oleae (olivier) ayoutantis, 1940; argyriou & michelakis, 1975 sphaerolecanium prunastri (boyer de fonscolombe) argyriou & paloukis, 1976;  podsiadlo, 1981; kozár et al.,1991;  present paper fam asterolecaniidae species validation source pollinia pollini (a. costa) alexandrakis, 1980a 295g. pellizzari et al.: scale insect fauna of crete fam. diaspididae  species validation source abgrallaspis cyanophylli (signoret) kozár et al.(1991); present paper acanthomytilus intermittens (hall) kozár et al., 1991 adiscodiaspis ericicola (marchal) present paper aonidia mediterranea (lindinger) present paper aonidia lauri (bouché) present paper aonidiella aurantii (maskell) ayoutantis, 1940; debach & argyriou, 1967; present paper aonidiella yehudithae ben-dov ben-dov, 2006; jansen et al., 2010 aspidiotus nerii bouché koronéos, 1934; ayoutantis, 1940 aulacaspis rosae (bouché) kozár et al., 1991 carulaspis minima (signoret) present paper chrysomphalus dictyospermi (morgan) ayoutantis, 1940 diaspidiotus lenticularis (lindinger) present paper diaspidiotus osborni (newell & cockerell) kozár et al. 1991 duplachionaspis berlesii leonardi present paper duplachionaspis natalensis (maskell) kozár et al., 1991 dynaspidiotus britannicus (newstead) koronéos, 1934 dynaspidiotus greeni balachowsky) kozár et al., 1991 getulaspis bupleuri (marchal) present paper gonaspidiotus minimus (leonardi) jansen et al., 2010; present paper hemiberlesia lataniae (signoret) rosen & debach, 1979; podsiadlo,1983  koroneaspis aegilopos (koroneos) present paper lepidosaphes beckii newman ayoutantis, 1940 lepidosaphes conchiformis (gmelin) kozár et al., 1991 lepidosaphes flava (signoret) present paper lepidosaphes ulmi (linnaeus) podsiadlo, 1983; present paper leucaspis löwi colvée kozár et al., 1991 leucaspis pusilla löw podsiadlo, 1983; jansen et al., 2010 leucaspis riccae targioni tozzetti lindinger, 1912; present paper lineaspis striata (newstead) panis, 1981; present paper mercetaspis halli (green) podsiadlo, 1983 oceanaspidiotus spinosus (comstock) present paper odonaspis ruthae kotinsky present paper parlatoria oleae (colvée) argyriou, 1967; present paper parlatoria parlatoriae (šulc) jansen et al., 2010  parlatoria ziziphi (lucas) ayoutantis, 1940 present paper;  pseudaulacaspis pentagona (targioni tozzetti) kozár et al., 1991; present paper prodiaspis tamaricicola (malenotti) present paper rhizaspidiotus donacis (leonardi) kozár et al., 1991 unaspis euonymi (comstock) present paper journal of entomological and acarological research, ser. ii, 43 (3), 2011296 (c.j. hodgson). its presence in the island was previously generally recorded by argyriou  (1984) and later confirmed by hodgson & gounari (2006).  fam. pseudococcidae *chorizococcus rostellum lobdell: iraklion, 21-26.ix.2009, sorghum halepense, unidentified asteracea; knossos, 25.ix.2009, piptatherum miliaceum (f. kozár). *coccidohystrix sp. n.: knossos, 25.ix.2009, rosmarinus sp. (f. kozár). *heterococcus nudus green: sorghum halepense, knossos, 25.9.2009 (f. kozár). planococcus citri (risso): iraklion, 21-26.ix.2009, parietaria sp., cynodon dactylon,  unidentified asteracea (f. kozár); collected also by pheromone traps in iraklion. planococcus vovae (nasonov): chania, 23.iii.1997, cupressus sp. (g. pellizzari). *pseudococcus longispinus (targioni tozzetti): chania, 8.iv.2010, pittosporum sp. (g.  pellizzari). rhizoecus albidus goux: iraklion, 21-26.ix.2009, cynodon dactylon (f. kozár). *spilococcus halli (mckenzie & williams): iraklion, 21-26.ix.2009, cynodon; knossos, 25.ix.2009, piptatherum miliaceum (f kozár). previously reported for the greece  mainland by kozár (1985) as chorizococcus viktorinae.  trionymus aberrans goux: knossos, 25.ix.2009, piptatherum miliaceum (f. kozár).  previously recorded in iraklion by kozár et al. (1991). *trionymus multivorus (kiritchenko): iraklion, 21-26.ix.2009, undetermined asteracea  (f. kozár).  fam. eriococcidae *acanthococcus sp. n.: knossos, 25.ix.2009, host plant unknown (f. kozár). * acanthococcus desertus (matesova): knossos, 25.ix.2009, piptatherum miliaceum (f. kozár) fam. kermesidae *kermes palestiniensis balachowsky: aradena, 35°13’22’’n 24°03’42’’s, 7.iv.2010;  04.vi.2011 quercus coccifera (g. pellizzari, f. porcelli, s. convertini). kermes vermilio planchon: aradena, 35°13’22’’n 24°03’42’’s, 04.vi.2011 quercus coccifera ( s. convertini). fam. coccidae *ceroplastes floridensis comstock; iraklion, 35°20’16”n, 25°08’06”e, 10.iv.2010,  citrus sp., laurus nobilis, ficus microcarpa (g. pellizzari, g. seljak, f. kozár). ceroplastes rusci (linnaeus): iraklion, 21-26.ix.2009, pittosporum sp. (f. kozár);  chania, 9.iv.2010, ficus carica, nerium oleander (g. pellizzari); aptera, 11.iv.2010,  nerium oleander (c.j. hodgson). 297g. pellizzari et al.: scale insect fauna of crete *ceroplastes sinensis del guercio: chania – maich, 9.iv.2010, myrtus communis (g.  pellizzari). coccus hesperidum linnaeus: iraklion, 21-26.ix.2009, citrus sp. (f. kozár); kissamu,  7.iv.2010, ficus microcarpa; iraklion 10.iv.2010, ficus sp., laurus nobilis (g. pellizzari). this very common species was first generically reported for crete by ayoutantis  (1940) and later recorded in knossos on iris craetensis by kozár et al. (1991).  *lecanopsis formicarum (newstead): iraklion, 23.ix.2009, koeleria sp. (f. kozár). lichtensia viburni signoret: episkopi, 3.iv.2010, olea europea (c.j. hodgson). protopulvinaria pyriformis (cockerell): chania, 35°31’00”n, 24°00’54”e, 8.iv.2010,  hedera helix ssp. canariensis; chania –maich 35°29’41”n, 24°03’01”e, 12.iv.2010,  hedera helix (g. seljak). pulvinariella mesembryanthemi (vallot): chania, 8.viii.1975, mesembryanthemum sp. (g. pellizzari). sphaerolecanium prunastri fonscolombe: chania, 9.iv.2010, ficus carica, (g. pellizzari); knossos, 35°17’50”n, 25°09’48”e, 10.iv.2010, prunus dulcis (g. seljak, f. porcelli). fam. diaspididae abgrallaspis cyanophylli (signoret): chania – maich 35°29’41”n, 24°03’01”e, 6.iv.2010, opuntia ficus-indica (g. seljak, g. pellizzari). previously recorded in iraklion,  on mesembryanthemum sp., by kozár et al. (1991). acanthomytilus intermittens (hall): iraklion, 21-26.ix.2009, cynodon dactylon, piptatherum miliaceum (f. kozár). previously recorded in iraklion and knossos on cynodon  dactylon and oryzopsis sp. by kozár et al. (1991). *adiscodiaspis ericicola (marchal): elafonisi, 10.viii.2000, erica manipuliflora, (f.  porcelli). *aonidia mediterranea (lindinger): chania, 23.iii.1997, cupressus sp., (g. pellizzari);  iraklion, cupressus sp., thuja sp., 21-26.9.2009 (f. kozár). *aonidia lauri (bouché): iraklion, 21-26.ix.2009, laurus nobilis (f. kozár). aonidiella aurantii (maskell): chania, 9.iv.2010, euonymus sp. (g. pellizzari) aspidiotus nerii bouché: aghia sophia cave - topolia gorge, 01.viii.2000, lavatera cretica & euphorbia sp. (woody srubs); agria gramvousa, 28.vii.2000, thymelaea hirsuta;  bali, 29.vii.2000, ceratonia siliqua (f. porcelli). iraklion, 21-26.ix.2009, pittosporum tobira, cercis sp., (f. kozár). previously recorded at iraklion and knossos, on capparis spinosa, nerium oleander, pittosporum tobira, sedum sp., agave americana, convolvolus sp., eucayptus sp., iris craetensis, malva silvestris, vitis sp. by kozàr et al. (1991). *carulaspis minima (signoret): iraklion, 21-26.ix.2009, cupressus sp., thuja sp. (f.  kozár). *diaspidiotus lenticularis (lindinger): potamida, 1.viii.2000, olea europea, associated  with and infected by septobasidium sp. (f. porcelli).  journal of entomological and acarological research, ser. ii, 43 (3), 2011298 *duplachionaspis berlesii leonardi: chania – maich, 9.iv.2010, asparagus acutifolius  (g. pellizzari). *getulaspis bupleuri (marchal): elafonisi, 15.vii.2000, juniperus oxycedrus (f. porcelli). gonaspidiotus minimus (leonardi in: berlese & leonardi): aradena, 35°13’22’’n  24°03’42’’s, 04.vi.2011 quercus coccifera (s. convertini). *koroneaspis aegilopos (koroneos): aradena, 7.iv.2010, quercus coccifera (g. pellizzari). *lepidosaphes flava (signoret): potamida, 1.viii.2000, olea europaea, associated with  and infected by septobasidium sp. (f. porcelli). lepidosaphes ulmi (linnaeus): agyos nicolaos, 8.viii.1975, ceratonia siliqua (g. pellizzari). elafonisi, 15.vii.2000, pistacia lentiscus (f. porcelli). *leucaspis riccae targioni tozzetti: aghia sophia cave - topolia gorge, 1.viii.2000, euphorbia sp. (woody shrub); agria gramvousa, 28.viii.2000, euphorbia dendroides; elafonisi, 01.viii.2000, erica manipuliflora; bali, 29.vii.2000, olea europaea (f. porcelli). leucaspis pusilla löw: chania, 9.iv.2010, pinus halepensis (g. pellizzari). lineaspis striata (newstead): agios nikolaos, 10.viii.1975, cupressus sempervirens  (g. pellizzari); iraklion, 21-26.9.2009, cupressus sp. (f. kozár). *oceanaspidiotus spinosus (comstock): iraklion, 29.vii.2000, opuntia sp. (f. porcelli). *odonaspis ruthae kotinsky: iraklion, 21-26.ix.2009, sorghum halepensis, cynodon dactylon (f. kozár).  parlatoria oleae colvée: iraklion, 21-26.ix.2009, pyrus sp. (f. kozár); chania, 9.iv.2010,  prunus sp. (g. pellizzari); bali, 29.vii.2000, pyrus pyraster (f. porcelli). parlatoria ziziphi (lucas): potamida, 1.viii.2000, citrus aurantium (f. porcelli); aptera,  12.iv.2010, citrus lemon (c. j. hodgson). pseudaulacaspis pentagona targioni tozzetti: chania, 9.iv.2010; chania- maich, by  pheromone traps (f. kozár); aptera, 12.iv.2010, morus sp. (c. j. hodgson). *prodiaspis tamaricicola (malenotti): iraklion, 30.vii.2000, elafonisi, 15.vii.2000,  tamarix gallica (f. porcelli). agios nektarios, 35°11’35”n, 24°12’56”e, 7.iv.2010,  tamarix gallica (g. seljak). *unaspis euonymi (comstock): iraklion, 01.viii.2000, euonymus japonicus (f. porcelli). according to the new collections and including the revised list from previous records,  the total number of species recorded from the island is now 82 (table 1). twenty-seven  of them are new to the crete fauna. some new records refer to species widely distributed in the mediterranean basin (i.e. aonidia lauri, carulaspis minima), whilst others  are common introduced pests (i.e. pseudococcus longispinus, ceroplastes floridensis,  unaspis euonymi), so that their presence on the island was predictable. significant  records of interest are kermes palestiniensis, previously only known to occur in israel  (balachowsky, 1953) and getulaspis bupleuri, previously known only from the canary  islands, north african countries (morocco, tunisia, algeria, lybia) and saudi arabia  299g. pellizzari et al.: scale insect fauna of crete (balachowsky, 1954; matile ferrero, 1984). the present list should be considered as a  starting point for future biogeographic studies on the scale insects fauna of crete. acknowledgements the authors wish to thank the university of padova (italy), for the scientific grant  given to last author for the cooperative work, the otka (hungarian national science  fund (grant no. 75889) for financial support for this project and chris hodgson, for  reviewing the manuscript and for allowing us to include his collecting data on marchalina hellenica, ceroplastes rusci, lichtensia viburni, parlatoria ziziphi, pseudaulacaspis pentagona.  references alexandrakis v., 1980 - essai d’appreciation des dégâts provoqués sur oranger en crète par la  presence d’aonidiella aurantii (mask.) (hom. diaspididae). fruits 35: 555-560.  alexandrakis v., 1980a - données bio-ecologiques sur pollinia pollini (hom. coccoidea, asterolecaniidae) sur olivier en crète. annales de la société entomologique de france 16: 9-17. 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journal of entomological and acarological research, ser. ii, 43 (3), 2011300 hoy j.m., 1963 - a catalogue of the eriococcidae (homoptera: coccoidea) of the world. new  zealand department of scientific and industrial research bulletin 150: 1-260.  koroneos j., 1934 - les coccidae de la grèce surtout du pélion (thessalie). i. diaspinae. athens,  95 pp. kozár f., 1985 - new data to the knowledge of scale-insects of bulgaria, greece, and rumania  (homoptera: coccoidea). acta phytopathologica academiae scientiarum hungaricae 20:  201-205. kozár f., paloukis s., papadopoulos n., 1991 - new scale insects (homoptera: coccoidea) in  the greek entomofauna. entomologia hellenica 9: 63-68. kozár f., nagy b., 1998 - new data to the distribution of some palaearctic scale insects (homoptera: coccoidea). folia entomologica hungarica 59: 53-56.  jansen g.m., ben-dov y., kaydan b., 2010 - new records of scale insects from crete island,  greece (hem., coccoidea). bulletin société entomologique de france 115: 483-484. matile-ferrero d., 1984 - insects of saudi arabia homoptera: subordo coccoidea. fauna of  saudi arabia 6: 219-228. mendel z., 1998 - biogeography of matsucoccus josephi (homoptera: matsucoccidae) as related  to host resistance in pinus brutia and pinus halepensis. canadian journal of forest research  28: 323-330. milonas p.g., kozár, f., kontodimas d.c., 2008 - list of scale insects of greece. 143-147 in:  branco, m., franco, j.c. & hodgson, c.j. (editors), proceedings of the xi international  symposium on scale insect studies, oeiras, portugal, 24-27 september 2007. isa press,  lisbon, portugal, 322 pp. panis a., 1981 - les cochenilles circum-méditerranéennes des arbres d’alignement et brise-vent  (homoptera, coccoidea). sixièmes journées phytiatrie and phytopharmacie circum-mediterranéennes: 1-11.  podsiadlo e., 1983 - notes on scale insects (homoptera, coccoidea) found in crete and their  parasites. fragmenta faunistica 27: 271-277.  rosen, d., debach, p. 1979 - in: species of aphytis of the world (hymenoptera: aphelinidae).  series entomologica: vol. 17) dr. w. junk, the hague, boston, london, 801 pp.  williams d.j., 1993 - a new species of mealybug from greece, the first from europe belonging  to the ant-attended genus eumyrmococcus silvestri (hemiptera: coccoidea: pseudococcidae).  entomologist’s gazette 44: 216-220. williams d.j., moghaddam m., 2000 (1999) - mealybug species of the genus planococcus ferris  in iran (homoptera: coccoidea: pseudococcidae) with a discussion of planococcus vovae  (nasonov). journal of entomological society of iran 18: 32-43.  giuseppina pellizzari - università di padova, dipartimento agronomia ambientale e produzioni  vegetali, viale dell’università 16, 35020 legnaro, italy. e-mail: giuseppina.pellizzari@ unipd.it francesco porcelli - università di bari aldo moro, dibca sez. entomologia e zoologia, via  amendola 165a, 70126 bari, italy. gabrijel seljak - agriculture and forestry service nova gorica, pri hastu 18, si-5000 nova  gorica, slovenia. ferenc kozár hungarian academy of sciences, plant protection institute, p.o. box 102, budapest, h 1525, hungary. accepted 15 september 2011 ������������������ ������������������������ ���������������� ��������������������������������������� ����������������������������������������������������������������������������� ��������������������������������� ������������������������������������������������������������������������������� ������������������������������������������������������������������������������������ ���������������������������������������������������������������������������������� �������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������ ������������������������������������� 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�������������������������������� ���������������������������������������������������������������������������������������������� ����������������������������������� �������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������� ������������������������������������������������������������� ������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������� ������������������������������� ������������������������������������������������������������������������������������������������� ��������������������������� ���������������������������������������������������������������������������������������������� ����������������������������������������������������������������������� ��������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������� ����������������������� 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract farmers in kenya continue to raise concerns of difficulty in managing tetranychus evansi, the most widespread pest species of tomato applying the most commonly used acaricides. this invasive pest species is not only found in kenya, but in eastern and southern africa, as well as parts of europe and asia. in the current study, populations of t. evansi were collected from farms in the four major tomato-growing areas of kenya (loitoktok, kibwezi, athi-river and subukia) and their susceptibility compared to a laboratory culture (icipe) that had been maintained for three years without exposure to acaricides. susceptibility of t. evansi eggs and adults (contact and residual) to brigade (bifenthrin), dimethoate (dimethoate), karate (lambdacyhalothrin), kelthane (dicofol), omite (propargite) and polytrin (profenofos+cypermethrin) was tested in the laboratory using respective manufacturer’s recommended concentrations. dimethoate resulted in variable ovicidal mortality while kelthane, brigade, karate, omite and polytrin had high mortality across all populations. similarly, adult contact and residual mortality was lower than that of the other chemicals when exposed to dimethoate regardless of the location. furthermore, it also had no residual effect on the mites from icipe and kibwezi. on the other hand, kelthane was most lethal against the mites from all locations followed by brigade and polytrin in that order. omite caused significantly lower mortality on mites from subukia while karate produced variable effects on mites from kibwezi, loitoktok and subukia. the implications of these findings are further discussed. introduction the tomato red spider mite, tetranychus evansi baker & pritchard, is an important invasive pest species of solanaceous plants not only in kenya and other parts of africa (varela et al., 2003; knapp et al., 2003), but in parts of europe (ferreira & carmona, 1995; ferragut & escudero, 1999; migeon, 2005; castagnoli et al., 2006; tsagkarakou et al., 2007) as well as asia (ho et al., 2004; gotoh et al., 2009). it is believed to have originated from south america (moutia, 1958) and was first reported in continental africa in 1979 on tobacco in zimbabwe (blair, 1983) from where it spread to other parts of the continent. in kenya, t. evansi was initially reported in 2001 on tomato (knapp et al., 2003) and has since been reported in several other solanaceous plants in many parts of the country (toroitich et al., 2009). besides its invasive nature, recent reports indicate that t. evansi may be displacing native spider mite species hence posing new pest management challenges (ferragut et al., 2013). while there are efforts to control t. evansi using cultural practices (saunyama & knapp, 2003), application of synthetic acaricides remains the method of choice in kenya. this, however, is faced by the risk of resistance development among pest populations due to prolonged exposure (cranham & helle, 1985; blair, 1989; tsagkarakou et al., 2002; nyoni et al., 2011) or even poor application techniques correspondence: faith jebet toroitich, department of plant science and crop protection, faculty of agriculture, university of nairobi, p.o box 29053, kangemi, 00625, kenya. tel.: +254.722.274454. e-mail: fjtoroitich@gmail.com; fjtoroitich@uonbi.ac.ke key words: tetranychus evansi, pesticide tolerance, acaricides, tomato. funding: this study was funded through icipe dissertation research internship programme (drip). contributions: fjt, mk, jhn, fmo designed the experiments; fjt carried out the experiments; fjt, mo prepared draft of the manuscript; mk, mo performed statistical analyses; mk, mo, jhn, fmo provided several editorial advice and reviews of the manuscript. acknowledgements: the authors wish to thank bernard muia for help during field sampling and icipe red spider mites project through the dissertation research internship program (drip) for providing the research funds. we appreciate two anonymous reviewers for their critical review of the manuscript. received for publication: 19 march 2013. revision received: 31 october 2013. accepted for publication: 19 november 2013. ©copyright f.j. toroitich et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1469 doi:10.4081/jear.2014.1469 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. susceptibility of geographically isolated populations of the tomato red spider mite (tetranychus evansi baker & pritchard) to commonly used acaricides on tomato crops in kenya f.j. toroitich,1,2 m. knapp,3 j.h. nderitu,1 f.m. olubayo,1 m. obonyo4 1department of plant sciences and crop protection, faculty of agriculture, university of nairobi, nairobi, kenya; 2african insect science for food and health (icipe), nairobi, kenya; 3koppert biological systems, postbus, berkel en rodenrijs, the netherlands; 4department of biochemistry, egerton university, njoro, kenya [page 18] [journal of entomological and acarological research 2014; 46:1469] journal of entomological and acarological research 2014; volume 46:1469 no nco mm er cia l u se on ly (sibanda et al., 2000). it has been observed that farmers in africa frequently apply lower than recommended tank concentrations of chemicals and even use sprayers with inappropriate or worn-out nozzles and do not spray carefully enough leading to poor crop coverage (sibanda et al., 2000; saunyama & knapp, 2003). as a result, farmers frequently complain of ineffective acaricides, as the pest populations appear to resurge immediately after application. it was hypothesised that geographically isolated t. evansi populations were likely to differ in their susceptibility to acaricides depending on their history of chemical exposure. a prerequisite was to conduct farmer interviews to establish the predominant chemical(s) used in each region and thereafter test the susceptibility of t. evansi populations to the reported chemicals at the respective manufacturer’s recommended concentrations. materials and methods the chemical compounds (and respective trade names) commonly used for red spider mite control in tomato production in kenya are listed below (table 1). spider mite collection spider mites were collected from tomato fields in athi-river division (machakos county), kibwezi division in makueni county, loitoktok division in kajiado county and subukia division in nakuru county, kenya. twenty to thirty infested tomato leaves were collected from each farm where mites were observed. mites from six farms in athi-river, four in loitoktok, two in kibwezi and six in subukia were collected between april and july 2004. leaf samples were put in paper bags, then placed in a cool box and transported to icipe laboratory for examination. in the laboratory, at least 20 males from each of the sampled sites were mounted for identification. at the same time, 60-100 live females were picked from the infested leaves collected per site (no more than one spider mite was taken from a single leaf) for rearing. the t. evansi populations from athi river, kibwezi, loitoktok and subukia were reared in water isolation cages designed after those described by dennehy and granett (1982). potted tomato plants (variety cal-j) were placed on top (bottom part) of inverted pots inside a basin (90 cm diameter) half-filled with water. a clear-sided perspex cage (30×30×90 cm) whose topside was covered with fine polyester lining (for ventilation) was placed over the potted plants to prevent escape of mites (figure 1). the spider mites were reared at same conditions of temperature (25±2°c), relative humidity (50-80%) (rh) and photoperiod (12l: 12d) h in the laboratory. an icipe laboratory culture previously collected from mwea (kirinyaga county) and maintained on tomatoes without exposure to acaricides for three years was used for comparison. commercial acaricides were used in the experiments as follows: polytrin 440 ec (profenofos q 400 g/l+cypermethrin 40) (syngenta crop protection, switzerland), brigade 025ec (bifenthrin 25 g/l) (fmc corporation, usa), karate 1.75 ec (lambda-cyhalothrin 17.5 g/l (syngenta crop protection), dimethoate 40% ec (dimethoate 400 g/l) (eastchem, china), omite 57 ec (propargite 57%, crompton corporation, usa) and kelthane ec (dicofol 18.5%) (rohm and haas company, usa). ovicidal tests for each pesticide, twenty t. evansi females from respective colonies were transferred onto tomato leaf discs (25 mm diameter) placed lower side up on moist cotton wool in petri dishes (9 cm diameter). the petri dishes were placed in rectangular plastic boxes (30×50×10 cm) and maintained at 25±2°c in an incubator. the females were allowed 24 h oviposition time after which, they were removed and the number of eggs adjusted to 20. pesticide solutions were prepared following respective manufacturer’s recommended rates as follows: polytrin (1.5 l/ha), brigade (2.5 l/ha), karate (2.5 l/ha), dimethoate (1 l/ha), omite (1 l/ha) and kelthane (2.5 l/ha). in addition, aquawet (nonylphenol ethoxylate, osho chemicals, kenya) was added as a sticker to all the chemical solutions and water at 1 l/ha. the leaf discs containing t. evansi eggs from respective locations were dipped in pesticide solutions for five seconds and placed lower side up in plastic petri dishes containing moist cotton wool. the num[journal of entomological and acarological research 2014; 46:1469] [page 19] article table 1. chemicals commonly used for red spider mite control in tomato production in kenya (icipe, 2003, unpublished survey data). compound trade name class % of farmers dicofol kelthane a 25.8 lambda-cyhalothrin karate i/a 24.7 dimethoate dimethoate i/a 22.7 cypermethrin+profenofos polytrin i/a 19.6 bifenthrin brigade i/a 13.4 propargite omite a 10.3 a, acaricide; i, insecticide. figure 1. sketch of the mites rearing cage (not drawn to scale). no nco mm er cia l u se on ly [page 20] [journal of entomological and acarological research 2014; 46:1469] ber of eggs was checked to ensure none were lost in the dipping process. the petri dishes were placed on a wire mesh inside plastic boxes (30×50×10 cm) and maintained under the same conditions earlier described. water and aquawet sticker was used as the negative control. the experiments were arranged in a completely randomized design and replicated six times for all treatments. the leaves were examined the first time after four days and subsequently daily for another five days for larval emergence. egg mortality was determined by comparing the number of unhatched eggs with the initial number of post treatment eggs (agnello et al., 1994). adulticidal tests the bioassays employed were similar to those described by kabir et al. (1993) with some modifications as outlined below. modified leaf disc direct method twenty, day-old adult t. evansi females from the colonies that had been reared for six weeks from the time of collection were transferred onto the lower side of tomato leaf discs (25 mm diameter). the leaf discs containing mites were carefully dipped in the acaricide solutions for 5 seconds before being placed lower side up in plastic petri dishes containing moist cotton wool. the petri dishes were left on the bench for one hour to allow the leaves to dry then introduced into plastic boxes (mentioned above) and maintained in an incubator as already described. similarly, the experiments were arranged in a completely randomized design and replicated six times. after 24 h the mites were observed and scored as dead, alive or escaped for those which were trapped in the cotton barrier (agnello et al., 1994). mites were considered dead when they did not react to gentle prodding with a camel hairbrush. escaped mites were excluded from the analyses. leaf disc residue – dipping method leaf disc residue – dipping method differs from leaf disc direct method (ldd) slightly in that the leaf discs were first dipped in acaricide solutions for five seconds, then placed in petri dishes and allowed to dry at room temperature for about 30 min. similar to the ldd, spider mites were transferred onto the discs and observed after 24 h and scored as dead, alive or escaped. escaped mites were excluded from the analyses. data analysis data on egg and adult mortalities were arcsine square root transformed then subjected to one-way analysis of variance (proc glm) and means separated by student-newman-keuls test (sas 9.1, 1990). however, data presented in the tables are actual percent mortalities. in addition, only for ovicidal tests, control mortality was corrected using abbott’s formula (abbott, 1925) while for adult mortalities actual uncorrected figures were used. results acaricides used for spider mites control in loitoktok, three of four sampled farmers used dimethoate while the remaining one used polytrin. on the other hand, in subukia three farmers used polytrin, two-used tata alpha (alpha-cypermethrin) and the remaining one used karate. in kibwezi, both farmers used karate but one alternated its use with brigade. in athi-river, four of six farmers used karate while each of the remaining ones used dimethoate and kelthane. the farmers in subukia complained of spider mites being a persistent problem in the area and explained their choice of polytrin as the most effective chemical against insects and spider mites. in kibwezi and athi river, spider mites were not cited as a major problem, although the farmers conventionally applied acaricides albeit infrequently. similarly, most farmers in loitoktok had scanty knowledge of spider mites hence they were not ranked as important pest of tomato. however, the farmers routinely applied dimethoate against all pest infestations. ovicidal tests compared to the other chemicals, dimethoate resulted in variable ovicidal mortality among the tested t. evansi populations. significantly lower mortality was observed in subukia and kibwezi populations while loitoktok, icipe and athi river ones had minimal susceptibility comparable to the negative control. on the other hand, treatments by kelthane, brigade, karate, omite and polytrin resulted in significantly (<0.0001) high (about 100%) egg mortality in all populations (table 2). adulticidal tests: contact mortality (leaf disc direct method) in a trend similar to the ovicidal test, mortality of adult mites was significantly lower (p=0.0099) than the other chemicals when exposed to dimethoate regardless of the location (table 3). however, with the exception of athi river where mortality did not differ significantly from the negative control, in the other locations some differences were reported. significant differences (p=0.0202) were observed among populations when karate was applied as follows: highest mortality attained was with icipe and kibwezi mites at 70% and as low as 43% for those from loitoktok. this is much lower than the response to article table 2. mortality of spider mite eggs following treatment by various acaricides (n=6 in all cases). treatment/location athi river icipe kibwezi loitoktok subukia p-values brigade 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a control 0.0±0**a 0.0±0**a 0.0±0***a 0.0±0**a 0.0±0***a dimethoate 0.0±0**c 8.7±3.4**c 33.3±7.6**b 13.0±4.3**c 54.05±10.9**a <0.0001 karate 98.8±1.17*a 100.0±0*a 96.7±2.1*a 99.0±1.0*a 93.5±6.5*a 0.6084 kelthane 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a omite 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a polytrin 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a 100.0±0*a p-values <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 *,**,***means±se down the columns are significantly different (p=0.05) [analysis of variance (anova) and student-newman-keuls (snk)]; a,b,cmeans±se followed by different letters across the rows are significantly different (p=0.05) (anova and snk). no nco mm er cia l u se on ly omite, brigade, polyrin and kelthane which caused nearly 100% mortality among all spider mite populations. residual effect of the acaricides on t. evansi (leaf disc residue – dipping method) the residual effect of various acaricides followed the pattern observed with contact mortality (table 4). compared to the other chemicals dimethoate had the least significant residual effect on t. evansi adults from subukia, loitoktok and athi river while it had no effect on those from icipe and kibwezi. on the other hand, kelthane was most lethal against the mites from all locations followed by brigade and polytrin in that order. omite caused significantly lower (p=0.0363) mortality on mites from subukia while karate produced variable effects on mites from kibwezi, loitoktok and subukia. discussion and conclusions farmers in the major tomato producing areas of kenya appear to prefer omite, polytrin, dimethoate and karate as acaricides. it is evident that most of these acaricides are still effective against t. evansi both as ovicides and adulticides. the current study shows that kelthane (dicofol) was the most effective acaricide. this corroborates earlier findings that reported high efficacy of dicofol against tetranychus urticae koch (wilson et al., 1995). dicofol, cyhexathin, fenbutatin oxide and propargite are among the few selective acaricides that have been successfully used in integrated pest management (ipm) of t. urticae (rizzieri et al., 1988; jacobson et al., 1999). they are considered useful in ipm due to the fact that they are only slightly-to-moderately toxic to phytoseiid mites that are used to control phytophagous mite species (van leeuwen et al., 2005). from the current findings, omite (propargite) was observed to be highly effective against t. evansi in the laboratory, although this contradicted complaints by farmers of poor control outcomes using this chemical in the field. it is highly likely that the varied results observed in the field could be caused by other factors commonly known to be behind pesticide abuse in africa among them: poor application techniques, low levels of education, inadequate public awareness and lack of understanding of pest behavior (schwab et al., 1995). on the other hand, polytrin (profenofos+cypermethrin) and brigade (bifenthrin) are broad spectrum chemicals also used as insecticides and have shown high efficacy against t. evansi. however, resistance to bifenthrin was reported in t. urticae after four seasons of continuous use in australian cotton fields (herron et al., 2001). as such, its effectiveness in the current study could be attributed to the fact that it was the least applied acaricide in the farms sampled in kenya. another interesting observation is that t. evansi from loitoktok appears tolerant to karate (lambda-cyhalothrin) yet the farmers from that region had not used it for spider mite control. this is very likely the outcome of two possibilities: i) the farmers could have been using karate to control other pests in which case t. evansi was not a target or ii) it could be a case of cross-resistance since most farmers used dimethoate. the possibility of cross-resistance between an organophosphate (dimethoate) and a pyrethroid (bifenthrin) has been reported before in t. urticae (yang et al., 2002). from the findings of the current study, tolerance to dimethoate appears to have been widespread across the different populations of [journal of entomological and acarological research 2014; 46:1469] [page 21] article table 3. mortality of adult mites due to contact with various acaricides (n=6 in all cases). treatment/location athi river icipe kibwezi loitoktok subukia p-values brigade 100±0*a 100±0*a 100±0*a 96.82±0.05*a 100±0*a 0.4261 control 4.46±0.05***a 0.0±0****a 7.01±0.05****a 2.55±0.04****a 3.38±0.05****a 0.4404 dimethoate 11.46±0.09***b 7.01±0.05***b 48.4±0.19***a 27.4±0.14***ab 27.39±0.09***ab 0.0099 karate 56.69±0.08**ab 71.34±0.05**a 70.1±0.11**a 43.95± 0.07**b 63.06±0.12**ab 0.0202 kelthane 100±0*a 100±0*a 100±0*a 100±0*a 100±0*a omite 100±0*a 97.45±0.04*a 100±0*a 100±0*a 100±0*a 0.4261 polytrin 97.45±0.04*a 100±0*a 100±0*a 100±0*a 100±0*a 0.4261 p-values <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 *,**,***,****means±se down the columns are significantly different (p=0.05) [analysis of variance (anova) and student-newman-keuls (snk)]; a,bmeans±se followed by different letters across the rows are significantly different (p=0.05) (anova and snk). table 4. mortality of adult mites due to residual effect of various acaricides (n=6 in all cases). treatment/location athi river icipe kibwezi loitoktok subukia p-values brigade 96.82±0.05*a 100.0±0*a 70.01±0.30*a 92.99±0.07*a 89.17±0.11*,**a 0.2202 control 7.01±0.05**a 0.0±0**a 0.0±0**a 5.73±0.06***a 5.73±0.06***a 0.2256 dimethoate 8.28±0.06**a 0.0±0**a 0.0±0**a 3.82±0.04***a 9.55±0.05***a 0.0403 karate 81.53±0.15*ab 95.54±0.07*a 22.29±0.26**c 50.32±0.18**bc 75.16±0.09**abc 0.0004 kelthane 100±0*a 100±0*a 100±0*a 100±0*a 100±0*a omite 80.25±0.12*ab 93.63±0.07*a 75.8±0.25*ab 97.45±0.05*a 60.51±0.07**a 0.0363 polytrin 100±0*a 78.98±0.21*a 85.99±0.14*a 88.54±0.18*a 87.26±0.15***a 0.6725 p-values <0.0001 <0.0001 <0.0001 <0.0001 <0.0001 *,**,***means±se down the columns are significantly different (p=0.05) [analysis of variance (anova) and student-newman-keuls (snk)]; a,b,cmeans±se followed by different letters across the rows are significantly different (p=0.05) (anova and snk). no nco mm er cia l u se on ly [page 22] [journal of entomological and acarological research 2014; 46:1469] spider mites. this corroborates earlier findings, which reported widespread tolerance of t. evansi to this chemical at levels as high as 1000fold in zimbabwe (blair, 1989). similarly, in other spider mite species, 289.2 fold and 104.7 fold decrease in susceptibility to dimethoate by oligonychus pratensis banks and t. urticae respectively after only 10 cycles of exposure was reported (yang et al., 2002). resistance to another organophosphate (chlorpyrifos) by t. evansi was also reported in mites from zimbabwe (blair, 1989), malawi and southern france (nyoni et al., 2011; carvalho et al., 2012). the spider mites which had been maintained in icipe laboratory free of any chemicals also showed high levels of tolerance to dimethoate. therefore, it is not clear whether resistance to some organophosphates is innate in this species or as a result of selection pressures. on the other hand, it is also possible that the icipe culture could have had prior exposure to chemicals before laboratory rearing. yang et al. (2002) observed that even after three months without pesticide exposure, there was reduced susceptibility to dimethoate in the two-spotted spider mites (t. urticae). however, it is unknown whether this would be the case even after three years of continuous rearing without pesticide exposure as was observed in the current study. this observation is important as it could have serious ramifications for future pest control programs; especially in areas like athi river and kibwezi where the farmers perceive that mites have not acquired pest status yet they continue to use broad spectrum chemicals for routine pest management. with the foregoing, it is possible to conclude, dimethoate should not be recommended for t. evansi control but instead, the specific acaricides omite (propargite) and kelthane (dicofol) should be used. from the findings of this study, farmer complaints of inability to control spider mites using the tested chemicals could be attributed to poor application methods due to insufficient knowledge of the pest behaviour. therefore, there is need for farmer sensitization on proper application methods and acaricide rotation. there are acaricides that have been recently developed with new and complex modes of action that could be used together with other management options or in acaricide rotation strategies in order to delay resistance development. gotoh et al. (2010) reported that bifenazate, cyenopyrafen, milbemectin, spirodiclofen and tebufenpyrad caused high toxicity to t. evansi from nine localities in the world. this means that these new products can also be incorporated into acaricide rotations taking into consideration their individual modes of action. the use of acaricides should be limited by adhering to ipm principles including cultural control practices to reduce t. evansi populations. in addition, other compatible strategies such as biological control using the phytoseiid mite phytoseiulus longipes evans (furtado et al., 2007; ferrero et al., 2007, 2011) as well as some strains of the entomopathogenic fungi metarhizium anisopliae metsch and beauveria bassiana balsamo (bugeme et al., 2008; maniania et al., 2008) could be considered as alternative control agents in order to minimize acaricide use. references abbott w.s., 1925 a method of computing the effectiveness of an insecticide. j. econ. entomol. 18: 265-267. agnello a.m., reissig w.h., harris t., 1994 management of summer populations of european red mite (acari: tetranychidae) on apple with horticultural oil. j. econ. entomol. 87: 148-161. blair b.w., 1983 tetranychus evansi baker and pritchard (acari; tetranychidae), a new pest of tobacco in zimbabwe. coresta. phytopathology and agronomy study group, bergerac, france: 1-6. blair b.w., 1989 laboratory screening of acaricides against tetranychus evansi baker & pritchard. crop prot. 8: 217-222. bugeme d.m., maniania n.k., knapp, m., boga h.i., 2008 effect of temperature on virulence of beauveria bassiana and metahizium anisopliae isolates to tetranychus evansi. exp. appl. acarol. 46: 275-285. carvalho r., yang y., field l.m., gorman k., moores g., williamson m.s., bass c., 2012 chlorpyrifos resistance is associated with mutation and amplification of the acetylcholinesterase1 gene in the tomato red spider mite, tetranychus evansi. pestic. biochem. phys. 104: 143-149. castagnoli m., nannelli r., simoni s., 2006 un nuovo temibile fitofago per la fauna italiana: tetranychus evansi baker & pritchard (acari: tetranychidae). infor. fitopat. 56: 50-53. cranham j.e., helle w., 1985 pesticide resistance in tetranychidae, pp 405-421. in: helle, w. and sabelis, m.w. 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[journal of entomological and acarological research 2014; 46:1469] [page 23] article no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2013; 45:e19] [page 105] regulation of the abundance of clover seed weevils, apion spp. (coleoptera: curculionidae) in a seed stand of red clover (trifolium pratense l.) p. kolařík, j. rotrekl agriculture research, ltd., department of plant protection, troubsko, czech republic abstract the clover seed weevils, apion trifolii and protapion apricans, members of the genus apion, are responsible for causing serious economic damage in clover. in 2010-2012, the effectiveness of some insecticides against clover seed weevils in the genus apion were tested in red clover stands. the efficacy of different products was evaluated on the basis of analyses of specimens trapped in the herb layer of red clover using a sweep net and red clover heads sampled in individual plots. over the course of these trials, the applications of the products tested resulted in a marked reduction in their numbers (particularly of adults and, to a lesser extent, also of larvae). the highest efficacy was observed with biscaya 240 (a.i. thiacloprid) and mospilan 20 sp (a.i. acetamiprid). results obtained in this study corroborated the low efficacy of the insecticide karate zeon technology 5 cs against seed weevils of the genus apion. introduction in the czech republic, red clover (trifolium pratense l.) and alfalfa (medicago sativa l.) are among the most frequently cultivated fodder crops. they are grown mainly for production of green fodder and seed material. in 2011, the total acreage of red clover stands approved for seed production was only 4491 ha; compared with the preceding year; this was a reduction of approximately 10% (rotrekl &, kolařík 2011). however, red clover, with its 57% share of fodder crops grown is a dominant species among other seed producing stands of forage legumes; e.g., alfalfa, sanfoin, crown vetch, etc. (kolařík & rotrekl, 2012a, 2012c). in the production of red clover, it is especially important to observe all technological measures concerning seed-producing stands, as this is the only way to obtain high yields of seeds (rotrekl, 2000). there are several general rules that should be followed; e.g., selection of a suitable seed material (variety) and a locality with good potential conditions for high yields; it is also necessary to minimize the damage caused by insect pests. many pesticides that were used earlier in legume and pulse crops have been gradually eliminated and their registrations are now cancelled. registration of new products occurs only sporadically (anonymous, 2008; kolařík & rotrekl, 2012a). the clover seed weevils apion trifolii (l.) and protapion apricans (herbst), members of the genus apion, are pests that damage most clover stands (hansen & boelt, 2008; lundin et al., 2012; rotrekl, 2000). these species are closely related, their bionomics is similar, and they co-occur on red clover (kolařík & rotrekl, 2012c). adults survive the winter in different sites, and in spring they migrate to clover stands, where they feed on leaves, into which they bore small circular or oval holes. in may, they begin to lay eggs into green but not flowering heads during the period when the first rosy petals start to appear. first-instar larvae feed on the basal parts of the flower; they damage ovaries and developing seeds and migrate (climb) to neighboring florets in the head. a developing larva can destroy an average of 7-11 florets. infested inflorescences are much smaller and develop into a hard, knotty mass. after finishing their development, larvae pupate and a new generation of beetles hatches in june and july (hansen & boelt, 2008; ma et al., 2012; lundin et al., 2012; rotrekl, 2000). larvae cause the most significant damage and significantly reduce seed yield (hansen & boelt, 2008; ma et al., 2012). to minimize damage and protect the crops, both agrotechnical (cultural) and chemical methods of control may be used (kolařík & rotrekl, 2012a, 2012c). agrotechnical measures involve selection of a locality suitable for production of seed material and also possessing a protective buffer zone. the stand for seed production should not be established near storehouses of dry clover or in the neighborhood of older clover stands, which should be plowed. after the first cutting, it is also appropriate to preserve a small part of the buffer zone, in which seed weevils can lay their eggs; after the end of flowering, this buffer stand can be mowed and the harvested forage destroyed so that the developing pests are killed. in addition, the blooming buffer stands can attract insects, which after the cutting can continue to visit and pollinate flowers of the second growth (kolařík & rotrekl, 2012c). to protect clover stands against seed weevils, it is necessary to monitor and predict their occurrence for an indication of a need for treatcorrespondence: pavel kolařík, agriculture research ltd., department of plant protection, zahradní 1, troubsko 664 41, czech republic. tel.: +420547138835 fax: +420547138800. e-mail: kolarik@vupt.cz key words: plant protection, insecticides, insect pests, red clover, clover seed weevils, apion spp. funding: this study was supported by financial resources granted by the research organization agriculture research, ltd., troubsko. received for publication: 13 march 2013. revision received: 24 june 2013. accepted for publication: 28 june 2013. ©copyright p. kolařík and j. rotrekl, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e19 doi:10.4081/jear.2013.e19 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e19 no n c om me rci al us e o nly [page 106] [journal of entomological and acarological research 2013; 45:e19] ment of the infested crops. pest incidence may not be harmful every year, but in some years (e.g., 2010) they can occur in very high numbers or even exhibit mass outbreaks. prognosis should be made before the first cutting, with the detection of 350 adults per 100 samples during the flowering period indicating a potentially harmful level of these pests. all stands with more than 200 adults per 100 samples at the beginning of the flowering period should be treated (anonymous, 2008; kolařík & rotrekl, 2012a, 2012c). currently, the product karate zeon technology 5cs at a rate of 0.2 l/ha is the only insecticide approved for protection of seed production stands against these pests in the czech republic (anonymous, 2008). materials and methods experimental sites experiments to assess the effectiveness of some selected insecticides against seed weevils in the genus apion were carried out during 2010-2012. in 2010, the experimental site was located near the village of litostrov (49°13’22.868’’ n; 16°20’11.868’’ e) and the individual treatments were: treatment 1 untreated control; treatment 2 mospilan 20 sp (a.i. acetamiprid 20%) at 150 g/ha; treatment 3 mavrik 2 f (a.i. tau-fluvalinate 240 g/l) at 0.2 l/ha; treatment 4 biscaya 240 od (a.i. thiacloprid 240 g/l) at 0.3 l/ha; and treatment 5 karate zeon technology 5 cs (a.i. lambda-cyhalothrin 50 g/l) at 0.2 l/ha. in 2011, the experimental site was near the village of lesní hluboké (49°16’13.167’’ n; 16°18’50.952’’ e) and the treatments tested were: treatment 1 untreated control; treatment 2 proteus 110 od (a.i. thiacloprid 100 g/l + deltamethrin 10 g/l) at 0.5 l/ha; treatment 3 mospilan 20 sp at 150 g/ha; treatment 4 biscaya od 240 at 0.3 l/ha; and treatment 5 karate zeon technology 5 cs at 0.2 l/ha. in 2012, the experimental site was again near the 2011 site (49°16’7.719’’ n; 16°18’40.217’’ e) and the treatments were the same as in 2011 (except for treatment 2, in which proteus 110 od was applied at 0.75 l/ha). in all years, the surfactant silwet star was applied with all insecticide treatments at a rate of 0.06 l/ha. evaluation of adults and larvae the total area of individual treatment plots ranged from 1-5 ha. based on the results of monitoring, application of the insecticides tested was made from immediately before flowering to the appearance of the first florets. immediately before treatment, samples of weevils present in the herb layer of clover stands were taken using a sweep net. samples comprised four replicates of 10 net sweeps per treatment. the collected insects were killed in the laboratory by fumigation withethyl acetate,and counted on filter paper under a stereoscope. a post-treatment evaluation of adults and larvae present in the plots was conducted using the same procedure. approximately one month after treatment, 100 clover heads were collected randomly in individual treatments and examined in the laboratory for the presence of seed weevil larvae. effectiveness of the different treatments was evaluated statistically using the formula of henderson & tilton (1955), and the numbers of adults were compared with the untreated control. average numbers of larvae per head were assessed in each treatment as well. after desiccation of the individual stands, plants from a 0.25-m2 area were collected in four different sites of each treatment, for a total plot area sampled of 1 m2. plant samples were processed in the laboratory and the seed material obtained was cleaned and weighed. projected seed yields per hectare were calculated (excluding field effects) for each treatment. we assessed the following parameters: thousand grain weight (tgw), germination energy, and germination rate of collected seed samples. tgw was calculated from the proportion of pure seeds in 4 reps of 1000 seeds sampled. germination energy and germination rate were calculated from 4 samples of 100 seeds taken 4 and 10 days after the establishment of the of germination tests. results 2010 trial in 2010, a high incidence of seed weevils in the genus apion was observed before the first application of insecticides (figure 1). on average, the abundance of seed weevils was approximately 1650 specimens per 100 sweep net samples. treatments were applied on 9 july, late in the afternoon. at the time of application, the weather was sunny and very hot (26.7°c). because the specific recommendations concerning the application of pyrethroid insecticides were not observed, their efficacy might have been negatively influenced. evaluation of the incidence of insects 3 d after insecticide application showed the highest abundance of clover seed weevils per 100 samples to be in treatment 5 (the standard, karate zeon technology 5 cs). in the other treatments, pest incidence was low, and ranged from zero in treatment 4 (biscaya 240 od) to 10 adults in the two remaining treatments. in treatments 2-4, a high level of efficacy was observed, ranging from 98.9 to 100% mortality (figure 2). on the final evaluation date, 19 july (10 days after application), the highest numbers of adults were found in treatment 5, which was the article figure 1. effect of individual insecticides on the abundance of clover seed weevils in 2010. figure 2. biological efficiency of the insecticides tested in 2010. no n c om me rci al us e o nly registered standard, karate zeon technology 5 cs (f=196.935). very low efficacy was also observed in treatment 3, the pyrethroid mavrik 2 f (28.5%). the lowest incidence of clover seed weevils was detected in treatment 4 (biscaya 240 od) and treatment 3 (mospilan 20 sp). in 2010, during which there were very high populations of seed weevils in clover stands, the efficacy of both products was excellent, ranging from 92.2 to 94.5% mortality. the difference between these treatments and both the standard (treatment 5) and the untreated control (treatment 1) was statistically highly significant (f=196.935). in evaluation of clover heads, the highest number of larvae per head (4.5) was found in the untreated control. among the treated plots, the highest numbers of larvae per clover head (3.6) were in treatments 5 and 2 (karate zeon technology 5 cs and mavrik 2 f, respectively). in the remaining treatments, the incidence of seed weevil larvae was low. the difference between these two treatments and other treatments tested was statistically highly significant (f=13.671). 2011 trial in 2011, the pre-treatment incidence of clover seed weevils was not as high as in 2010. the incidence of pests in the individual treatments averaged 518 per 100 samples; however, this number still exceeded the economic threshold for this pest in stands of red clover (figure 3). in the individual treatments, weevil samples were collected and analyzed 3 d after the insecticide applications. treatments were applied on 28 june. the greatest number of adults was recorded in the untreated check, while in all treated plots the pest incidence was very low. this was reflected in the observed efficacies of the individual insecticides, which ranged from 99.2% (biscaya 240 od) to 100% (proteus 110 od). in contrast to 2010, the only currently registered product, karate zeon technology 5 cs, showed high insecticidal activity (figure 4). ten days after application, there was a significant difference in numbers of adults between the untreated control and all treated plots (f=539.310). in these treatments, insecticide efficacy was very high, ranging from 92.6% (mospilan 20 sp) to 100% (biscaya 240 od). evaluation of larvae in the clover heads showed no statistically significant difference between the control and karate zeon technology 5 cs. this product did not have any effect on larval incidence in the clover heads, despite the fact that it killed the adults very effectively. in other treatments, numbers of larvae were very low, particularly in treatment 4 (mospilan 20 sp) and treatment 3 (biscaya 240 od). again, the difference between the unregistered products (treatments 2-4) and treatments 5 and 1 (the standard insecticide and the untreated control, respectively) was statistically highly significant. no significant differences were found among the remaining treatments (f=34.979). 2012 trial in 2012, the pre-treatment pest incidence averaged 659 specimens per 100 samples (figure 5). this high incidence could have been influenced by the fact that the trials were carried out in the same locality as in the preceding year, with the fields separated only by a rural road, so that the beetles could have migrated easily to the experimental plots. treatments were applied on 29 june. one day after application, a high efficacy of the products applied was observed in all treatments, with values ranging from 85.4% (treatment 5) to 98.6% mortality (treatment 2). five days after application, the number of clover seed weevils increased significantly in treatment 5, translating into an efficacy of only 28%. on the last assessment date (14 days after application), the highest efficacy was recorded in treatment 2 (98.2%) and treatment 3 (96.6%). very good efficacy was observed also in remaining treatments (figure 6). efficacy of karate zeon technology 5 cs was very low, with incidence of the seed weevils much higher than in the control. the difference between treatments 2-4 and karate was statistically highly significant (f=138.676). on 8 august, analysis of the clover heads revealed the highest number of larvae in karate zeon technology 5 cs. the lowest numbers of larvae were recorded in treatments 3 and 4. again, the difference between treatments 2-4 and treatments 1 and 5 was statistically highly significant (f=18,450). in 2012, seed yields were evaluated shortly before harvest of all treatments. the lowest yield was recorded in treatment 5 (karate zeon technology 5 cs) and the highest were in treatments 3 and 4 (table 1). differences between treatments 1 and 5 and the remaining test treatments (treatments 2-4) were statistically highly significant (f=106.489). the qualitative analysis of harvested seed material did not show any differences in tgw (f=2.437), germination energy (f=2.776) or germination rate (f=2.381) between treatments 2-4 collectively, and treatments 1 and 5. discussion and conclusions due to restrictions on the application of older insecticides containing less suitable active ingredients, the only approved applicable for[journal of entomological and acarological research 2013; 45:e19] [page 107] article figure 3. effect of application of insecticides on abundance of seed weevils in 2011. figure 4. biological efficiency of the insecticides tested in 2011. no n c om me rci al us e o nly [page 108] [journal of entomological and acarological research 2013; 45:e19] mulation was the single registered pyrethroid, karate zeon technology 5 sc (anonymous, 2008; kolařík & rotrekl, 2012a, 2012c). the effectiveness of this product can be especially influenced by weather conditions at the time of application, as it is known that efficacy of pyrethroids is markedly influenced by high temperatures and solar uv radiation (kolařík & rotrekl, 2012b; ma et al., 2012). these products can completely lose their efficacy, enabling insect pests to continue to survive and feed, so that a grower expecting such an application to prevent seed yield losses will not be successful if the application takes place on an unsuitable date. in stands of red clover, seed weevils of the genus apion represent the most important group of insect pests (hansen & boelt, 2008; kolařík & rotrekl, 2012a; rotrekl & kolařík, 2011; langer & rohde, 2008) and can markedly decrease yields of clover seed material (langer & rohde, 2008; lundin et al., 2012). over the course of these trials, application of the products tested resulted in a marked reduction of their numbers, particularly of adults, and to a lesser extent, also of larvae (figure 7). the highest efficacy was observed from biscaya 240 (thiacloprid) and mospilan 20 sp (acetamiprid). these products are relatively new insecticides that show both contact and systemic activity and belong to the class of neonicotinoids (cloyd & bethke, 2011). these insecticides’ efficacy is not dependent on weather conditions, and they show good residual activity on both adults and larvae, even at higher temperatures. pyrethroids are only contact insecticides, and their residual effect on adults is relatively short (kolařík & rotrekl, 2012b; lundin et al., 2012). our results corroborated the low efficacy of karate zeon technology 5 cs against seed weevils of the genus apion. there were no significant differences in seed yields between this treatment and the untreated control. compared with the control, plots treated with the neonicotinoid products showed seed yields equivalent to 300 kg per ha. the difference between these products and the pyrethroid was statistically highly significant as well. it should be noted that no negative effects of these treatments on plants, beneficial species or honey bees were observed during the experimental period, and that their numbers remained high after applications of the products tested. references anonymous, 2008. metodická příručka ochrany rostlin proti chorobám, škůdcům a plevelům. i. polní plodiny. česká společnost rostlinolékařská, praha: 504. cloyd r.a., bethke j.a., 2011. impact of neonicotinoid insecticides on natural enemies in greenhouse and interiorscape environments. pest. manag. sci. 67: 3-9. hansen l.m., boelt b., 2008. thresholds of economic damage by clover seed weevil (apion fulvipes geoff.) and lesser clover leaf weevil (hypera nigrirostris fab.) on white clover (trifolium repens l.) seed crops. grass forage sci. 63: 433-437. henderson c.f., tilton e. w., 1955. tests with acaricides against the brow wheat mite. j. econ. entomol. 48:157-161. article table 1. evaluation of yield 2012. variants energy of germination tgw yield total yield germination (%) (%) per 1 head (g) (kg/ha) check 69 70.8 1.7005 0.065 552.32 proteus 110 od 69 71 1.61475 0.086 733.4 biscaya 240 od 81.3 83.5 1.7068 0.101 863.1 mospilan 20 sp 62.3 63.8 1.5915 0.099 845.88 karate zeon technology 5 cs 75 77.3 1.5533 0.057 485.81 tgw, thousand grain weight. figure 5. effect of individual insecticides on the abundance of clover seed weevils in 2012. figure 7. average number of larvae per 1 head (2010-2012). figure 6. biological efficiency of the insecticides tested in 2012.no n c om me rci al us e o nly kolařík p., rotrekl j., 2012a. ochrana semenných porostů jetele lučního proti hmyzím škůdcům. agromanuál 6: 36-37. kolařík p., rotrekl j., 2012b. rezistence škodlivých organismů vůči pesticidům. -úroda 12, vědecká příloha: 43-48. kolařík p., rotrekl j., 2012c. výskyt nosatčíků rodu apion vjeteli lučním a nové možnosti jejich regulace. pícninářské listy 28: 36-38. langer v., rohde b., 2008. factors reducing yield of organic white clover seed production in denmark. grass forage sci. 60: 168-174. lundin o., rundlöf m., smith h.g., bommarco r., 2012. towards integrated pest management in red clover seed production. j. econ. entomol. 105: 1620-1628. ma y.h., gao z.l., dang z.h., li y.f., pan w.l., 2012. effect of temperature on the toxicity of several insecticides to apolygus lucorum (heteroptera: miridae). j. pest. sci. 37: 135-139. rotrekl j., 2000. zemědělská entomologie. mendelova zemědělská a lesnická univerzita vbrně: 84. rotrekl j., kolařík p., 2011. významní hmyzí škůdci vsemenných porostech jetele lučního. agromanuál 7: 32-33. [journal of entomological and acarological research 2013; 45:e19] [page 109] article no n c om me rci al us e o nly jear2012 abstract the water bugs diplonychus rusticus (fabricius) (heteroptera: belostomatidae) and anisops bouvieri (kirkaldy) (heteroptera: notonectidae) co-occur in wetlands sharing mosquito larvae as prey. as a consequence, an asymmetrical intraguild predation (igp) involving d. rusticus as ig predator and a. bouvieri as ig prey can be possible, the outcome of which may vary with the relative density of interacting species. based on this proposition density dependent effects on the ig prey and shared prey mortality were assessed in the laboratory using varying numbers of ig predator and shared prey (iv instar culex quinquefasciatus larva). in contrast to single predator system, mosquito larvae were proportionately less vulnerable to predation in igp, at low density of shared prey. an increase in density of mosquito decreased the mortality of ig prey (a. bouvieri), but the mean mortality of the ig prey increased with the density of ig predator, in igp system. increase in density of mosquito and d. rusticus enhanced risk to predation of mosquito while reducing the mortality of a. bouvieri. interaction between d. rusticus and a. bouvieri as a part of igp system provides a possible reason of coexistence of mosquito immature along with predators in wetlands. biological regulation of mosquitoes may be affected, if appropriate predator numbers are not available in the habitats. introduction coexistence of multiple insect predators of mosquitoes is observed in mosquito larval habitats like rice fields and similar wetlands (sunish & reuben, 2002; bambaradeniya et al., 2004; das et al., 2006; banerjee et al., 2010). in contrast to a single predator, the presence of multiple predators increases the possibility of sharing mosquito prey, thereby increasing the complexity of food web. resource sharing by predators implies a degree of competition that may manifest as intraguild predation (igp) system. in igp, the shared prey is linked to both the intraguild predator (ig predator) and the intraguild prey (ig prey). in isolation, both the ig predator and the ig prey impart a different level of regulation on the shared prey. thus igp system is a distinct phenomenon involving predators that compete with each other for shared resource. the outcome of igp on the shared prey vary with the identity (polis et al., 1989; arim & marquet, 2004) and relative numbers (denno et al., 2002; balfour et al., 2003; borer et al., 2003; walzer et al., 2004) of interacting taxa, signifying that igp influences stability and diversity of species ensembles (walls & williams, 2001; crumrine & crowley, 2003; rosenheim & corbett, 2003). considering biological control of mosquito, the influence of the top predator on the ig prey and mosquito larvae would determine the degree of regulation and efficacy of the biocontrol agent. prey consumption by multiple predators can be more than or less than expected based on the individual consumption by the predators (sih et al., 1998). if the expected prey consumption is greater than observed, it indicates a synergistic effect of predation (soluk & collins, 1988; soluk, 1993), augmenting the risk of predation on the target prey (crumrine & crawley, 2003; crumrine, 2005). in situations when shared prey consumption is less than expected, it can be due to the interference competition with the intraguild prey. the interactions between ig prey and ig predator can influence the population of the shared prey, since the ig prey become vulnerable to intraguild predation that would mean a reduced number of available predators and shifting of prey choice by the top predator (crumrine & crowley, 2003). however, in igp system, the consequence on shared prey is expected to vary with its own relative density in contrast to the ig prey and ig predators in the community. to test these propositions, the present study considered diplonychus rusticus fabricius, 1781 correspondence: goutam k. saha, department of zoology, university of calcutta, 35 ballygunge circular road, kolkata 700019, india tel.: +91.33.2461.5445 fax: +91.33.2461.4849. e-mail: gkszoo@rediffmail.com key words: heteroptera, diplonychus rusticus, anisops bouvieri, intraguild predation, density. acknowledgements: the authors acknowledge the respective heads, department of zoology, university of calcutta, kolkata and the university of burdwan, burdwan, india for the facilities provided, including dst-fist and ugc-drs sap-ii. funding: ga acknowledges the financial assitance of ugc through research award [sanction no f.30-90(sa-ii)/2009;17.09.2009] in carryng out the work. sb acknowledges the financial assistance of ugc-rfsms in carrying out this work. received for publication: 11 october 2013. revision received: 11 november 2013. accepted for publication: 19 november 2013. ©copyright s. brahma et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1977 doi:10.4081/jear.2014.1977 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. influence of density on intraguild predation of aquatic hemiptera (heteroptera): implications in biological control of mosquito s. brahma,1 g. aditya,1,2 d. sharma,1 n. saha,1 m. kundu,2 g.k. saha1 1department of zoology, university of calcutta, kolkata; 2department of zoology, the university of burdwan, golapbag, india [page 6] [journal of entomological and acarological research 2014; 46:1977] journal of entomological and acarological research 2014; volume 46:1977 no nco mm er cia l u se on ly (heteroptera: belostomatidae) as ig predator, anisops bouvieri kirkaldy, 1741 (heteroptera: notonectidae) as the ig prey and iv instar larvae of culex quinquefasciatus say, 1823 (diptera: culicidae) as shared prey. often the igp system is classified as symmetric or asymmetric based on the role of the constituent predator species as ig predator and ig prey. in symmetric igp system, the role of predators may change, based on ontogeny or biomass; while in asymmetric igp system, the role of the predator species remain unchanged as ig predator and ig prey. the ig predator d. rusticus, and the ig prey a. bouvieri, considered in the present study constitute an asymmetrical igp system. these predatory insects and the mosquito prey are common in many of the tropical wetlands including rice fields (sunish & reuben, 2002; bamabaradeniya et al., 2004; das et al., 2006; banerjee et al., 2010). the dietary choice of these predators includes mosquitoes as a component which makes them suitable as mosquito control agent (aditya et al., 2004, 2005; saha et al., 2007a, 2007b; banerjee et al., 2010). earlier studies indicate that the mosquito prey consumption of d. rusticus, and a. bouvieri vary with the relative density (aditya et al., 2005; saha et al., 2010), light availability (saha et al., 2008) and habitat complexity (saha et al., 2009). in view of the predatory behavior of these water bugs, relative density of prey and predators can be useful explanatory factors to infer about the effect of density on the mosquito larvae as shared prey in igp system. thus, density dependent effects in igp system involving the heteropteran predators and mosquito prey are being addressed in the present study. the results are expected to highlight the interaction between the insect predators and mosquito prey and indicate the biocontrol efficacy of these insect predators of mosquito. earlier studies have shown that the mosquito fish gambusia affinis could not reduce the mosquito population effectively in the rice fields owing to shifting in prey consumption that included beetles and bugs which are themselves predators of mosquito larvae (bence, 1988; blaustein, 1992). interactions among potential mosquito predators may be a reason favoring abundance of mosquitoes even in presence of multitude of predators in the mosquito larval habitats like rice fields and temporary pools. in the present instance the co-occurrence of the predators and the mosquito may possibly due to such interaction between the predators d. rusticus and a. bouvieri, which could be revealed through the assessment of density dependent effects on the shared prey and the ig prey. in igp system of larval odonates (crumrine & crowley, 2003; crumrine, 2005; flynn & moon, 2011; lupi et al., 2013), shared prey abundance influence consumption of both shared prey and ig prey mortality. assuming similar manifestations in heteropteran mosquito predator guild, effects of predator and shared prey density on ig prey and shared prey mortality were tested in the present study to deduce feasibility of biological control of wetland mosquitoes using insect predators. materials and methods the insect predators adult morphs of the water bugs a. bouvieri and d. rusticus were collected from the ponds behind ballygunge science college campus, university of calcutta, kolkata and from the wetlands along eastern metropolitan bypass, kolkata. an insect net of 200 µm mesh size fixed to a long handle was used for collection of d. rusticus and a. bouvieri from the ponds. the collected insects were brought to the laboratory and were kept separately in glass aquaria containing 35 l of tap water, with few macrophyte specimens such as chara sp. and vallisneria sp. to simulate natural conditions. mosquito larvae were provided as food every day. this setup was kept for at least seven days before commencement of the experiment. the body length of the predators was measured from tip of the rostrum to the end of the abdomen; the average body length of d. rusticus used in this experiments was 16.4 mm (range, 15-18 mm), the one of a. bouvieri was 6.32 mm (range, 5.8-7.2 mm). the prey the mosquito larvae c. quinquefasciatus were collected from the sewage drains in and around ballygunge science college campus, university of calcutta, kolkata. the collected larvae were brought to the laboratory and placed in an enamel tray (30¥20¥10 cm3) for segregation of the iv instar larvae (5.1-6.0 mm in length, iv instar; 1.9-2.1 mg in weight) to be used in the experiments. the collection of mosquito larvae was continued, as and when required, in course of the experiment. experimental design the experiments were conducted in glass aquaria (38¥36¥36 cm3) using 35 l of water (aged tap water and pond water in a ratio of 1:1; ph 7.9-8.1) under room temperature (27-30°c) in a light (l): dark (d) cycle of 13d:11l h. using four different densities of mosquito larvae viz. 50, 100, 200 and 400, and different predator combinations, three experiments were carried out in replicates. in the first experiment, d. rusticus was used as a predator in three different densities of 1, 2 and 4 individuals. for each density level of the predator, mosquito larvae were provided in four different densities, and the numbers of mosquito larvae consumed were recorded at the end of 24 h period. a total of 216 replicates was carried out in this experiment (3 predator density ¥4 mosquito prey density ¥18 trials=216 replicates). in the second experiment, a. bouvieri was used as a predator in a single density of 10 individuals. mosquito larvae were provided as prey in four different densities, and the numbers of mosquito larvae consumed were recorded at the end of 24 h period. a total of 72 replicates was carried out in this experiment (1 predator density ¥4 mosquito prey density ¥18 trials=72 replicates). in the third experiment, d. rusticus and a. bouvieri were considered as ig predator and ig prey respectively, in three different combinations viz. 1:10, 2:10 and 4:10 at each of the mosquito larval (shared prey) densities. the number of mosquito larvae (shared prey) and the ig prey (a. bouvieri) consumed at the end of 24 h period was recorded. this experiment constituted an igp system, and a total of 216 replicates was carried out (3 ig predator density ¥4 shared prey density ¥18 trials). in all the experiments, a predator was used in a single trial only. the experimental trials were carried out at different time interval such that each trial represents a true replicate (hurlbert, 1984). the data obtained on shared prey mortality and ig prey mortality were separately subjected to twoway factorial anova (zar, 1999) using prey density and predator combinations as explanatory factor for prey mortality. size effects of the predator and prey density in explaining the observed variation on the prey mortality was estimated through partial η2 (eta square) (zar, 1999). risk to predation analysis shared prey (i.e. mosquito larvae) vulnerability in the form of risk of predation to individual predator type and in intraguild predation system was assessed by using the formula of crumrine & crowley (2003) and crumrine (2005) to observe the effect of ig prey on the consumption of shared prey by ig predator. according to the formula the shared prey and ig prey mortality rate (ki) was determined for each replicate of treatment (i) over the trial duration t (=24 h). proportion of prey killed was assumed as pi and thus the proportion surviving is (1-pi): 1-pi=e-kit (1) or ki=-ln(1-pi)/t (2) [journal of entomological and acarological research 2014; 46:1977] [page 7] article no nco mm er cia l u se on ly [page 8] [journal of entomological and acarological research 2014; 46:1977] accordingly for a. bouvieri and d. rusticus predation on mosquito, if ka is assumed as shared prey mortality in the presence of ig prey a. bouvieri alone and kd is the shared prey mortality in the presence of d. rusticus alone, then the null hypothesis for predation when both are present is: ka+kd=ka+d (3) expected value for the joint predation can be generated by summation of proportional prey consumption by two predator species when present alone. when summation of individual effect is equal to combined effect of both predators i.e. ka+kd=ka+d, it may be assumed that igp is absent on the prey consumption by the top predator. if observed ka+d<expected ka+kd, this implies risk reduction in prey mortality and favors prey survival, but when observed ka+d> expected ka+kd, then it indicates higher number of prey mortality i.e. risk enhancement occurs. a twotailed t-test was applied to find out significant deviation of the ratio of observed ka+d/ expected ka+ kd from unity (zar, 1999). the term risk reduction and risk enhancement originates from the null additive models (soluk & collins, 1988) and explanation regarding multiple predator effects (sih et al., 1998). we assumed that interactions between the ig predator, ig prey and the shared prey as a multiple predator effect and used the model of crumrine & crowley (2003) and crumrine (2005) to analyze the risk reduction and risk enhancement on the shared prey. the effect of density of the shared prey and ig predator on the risk reduction and risk enhancement for the shared prey and the ig prey was assessed. results the mortality of the mosquito prey (c. quinquefasciatus iv instar larva) varied with the combinations and effective number of predators present. the prey mortality due to consumption by the ig predator d. rusticus and the ig prey a. bouvieri varied with the relative densities of the mosquito prey available when present separately as well as igp system. density impact of the ig predator on the mortality of the mosquito prey was also evident. however, at the highest density of mosquito, a fourfold increase in density of the ig predator resulted in 30 percent increment in number of prey consumed (figures 1 and 2). the number of mosquito prey consumed under these conditions is shown in figure 2. the two way factorial anova revealed significant differences in the prey mortality as a function of prey and ig predator relative densities (tables 1 and 2). the partial η2 was higher for prey density factor than predator density suggesting that the prey density contributed to higher variability of shared prey mortality. in igp system, the mortality of the ig prey a. bouvieri reduced with increasing mosquito prey density, although relative mortality of ig prey increased with the ig predator density (figure 3). the two-way anova results (tables 3 and 4) suggest significant effect of shared prey density on the mortality of ig prey. thus, two levels of density effect on the shared prey mortality were observed in all experimental trials: i) with increase in density the mortality of mosquito (iv instar c. quinquefasciatus larva) increased, both in single predator and igp system; ii) increase in density of mosquito (shared prey) resulted in decreased mortality of the ig prey (a. bouvieri), but the mean mortality of the ig prey increased with the density of ig predator (d. rusticus), in igp system. as a part of igp system, the risk to predation for mosquito larvae was higher at higher relative density, while at lower densities it was low (figure 4). for all the ig predator densities, the observed ka+d /expected ka+kd values were consistently less than 1 when the initial shared prey densities were 50 and 100, while the values were greater than 1 when the initial shared prey densities were 200 and 400. the results of the t-test suggest significant (p<0.001) deviations from the lower than expected values (<1) and greater than expected values (>1) (table 5). this suggests that the increase in relative density of mosquito larvae (shared prey) increases the risk of predation possibly as an additive effect while reducing the mortality of a. bouvieri (ig prey) simultaneously. discussion and conclusions the prey consumption pattern of the ig predator (d. rusticus) and ig prey (a. bouvieri) in single predator experiments remained similar to the observations made in earlier studies (saha et al., 2007a, 2007b, 2009, 2010). the predation on mosquito larvae by these heteropteran predators is density-dependent (aditya et al., 2004; saha et al., 2007a, 2007b) with switching to abundant prey size (aditya et al., 2005) and species (saha et al., 2009, 2010). this is reflected in the single predator experiments where the prey mortality increased with increased density of prey and ig predator. similar effect of density on mosquito mortality was observed in igp system. the mortality of the a. bouvieri (ig prey) decreased with increased shared prey density but the relative mortality increased with ig predator (d. rusticus) density levels. thus density of ig predator resulted in increased mortality in both shared prey and ig prey. low density of mosquito reduced its risk to predation and increased mortality of ig prey was observed. this is comparable to the observation on the density-dependent and species-specific mortality of larvae of mayfly subjected to individual and joint predation by sculpins and stoneflies. interference between predators resulted in reduced predation of baetis at moderate and high prey density while facilitation between predators increased mortality of ephemerella at low and moderate densities (soluk, 1993). in the present instance, the article figure 1. schematic representation of the trophic relations among the mosquito prey and heteropteran predators, considered in the study. the arrows are directed towards the predator and the thickness of the arrows are proportionate to the numbers consumed. single prey-predator interactions are shown by continuous arrows and inclusion of dashed arrow indicates the intraguild predation (igp) system. experiments were carried out using single predator and both predators (igp system) with the density levels stated in the right hand side. no nco mm er cia l u se on ly igp interaction between the predators favoured mosquitoes at lowshared prey density but the effect diminished at high shared prey density. we assume that at low shared prey density with random encounter of prey, the probability of encounter with a. bouvieri was higher contrast to the mosquito prey and this could account for reduced proportionate mosquito mortality. risk reduction is common when predatorpredator interactions are probable and more specifically among predators that vary in size (crumrine, 2005). in addition, density effects of the ig predator and the shared prey can modify the multiple predator effect and the shared prey and ig prey mortality. studies on amphibian igp demonstrate that increase in density of tadpole of wood frog rana sylvaticus (ig predator) resulted in increased egg predation and reduced growth of the spotted salamander ambystoma maculatum (ig prey) (burley et al., 2006). this interaction resulted in reduced hatchling number of a. maculatum but enhanced larval development. similarly in the present context, the relative risk of predation was low when the density of the mosquito prey was low but increased at higher density. this is significant in terms of the prey predator interaction in wetlands. in habitats like ricefields and allied wetlands, the mosquito prey densities vary with time and the colonization pattern follow a succession sequence (dale & knight, 2008), along with co-occuring macro invertebrates. in the initial stage of rice plantation, mosquito prey density is low followed by the concurrent invasion of the heteropteran predators (lawler & dritz, 2005). under such situation the mortality of ig prey can be higher to complement the dietary requirements of d. rusticus, provided the availability of other prey is low. with time, the complexity of the aquatic species assemblages in rice fields increase and the density of the mosquito prey increases too. risk enhancement of mosquitoes can be anticipated when the predation from both the predators increase. the results of the present study support this proposition. studies on the igp system on water scorpion laccotrephes japonensis as ig predator demonstrated that the smaller instar nymph of kirkaldyia deyrolli (ig prey) were consumed at a higher rate than the shared prey larger instar of appasus japonicas and tadpoles of hyla japonica (ohba & swart, 2009). field and laboratory observations reveal that predation pressure of l. japonensis on smaller instar of k. deyrolli reduces with increase in the density of the shared prey tadpoles (ohba & nakasuji, 2007), signifying density mediated effects on igp. in larval odonates, interactions between sympetrum vicinum (dragonfly) and enallagma civile (damselfly) varied with the shared prey abundance, prey identity and habitat complexity. when prey abundance was low, the wet mass of e. civille was affected in contrast to the conditions, when the shared prey was high (flynn & moon, 2011). preceding examples and observations on the wetland insect assemblages (bambaradeniya et al., 2004; banerjee et al., 2010) indicate that: i) igp system is common in heteropteran predators of wetlands; and ii) the prey preference is crucial in the outcome of such multiple prey-predator interactions. in the present instance, the mortality of the ig prey (a. bouvieri) was dependent on the relative density of ig [journal of entomological and acarological research 2014; 46:1977] [page 9] article figure 2. (a-c) the number (mean+se) of shared prey (mosquito larva) consumed by intraguild (ig) predator d. rusticus (dr) and ig prey a. bouvieri (ab) in isolation and in combinations at different initial prey densities and different predator combinations (a: ig predator 1: 10 ig prey; b: ig predator 2: 10 ig prey; and c: ig predator 4: 10 ig prey) (n=18 trials per combinations per prey density). table 1. results of two way factorial anova, using prey density and predator density as explanatory variables for the mosquito larval (shared prey) mortality in presence of both intraguild predator and guild predator prey. source df mean square f partial η2 prd 2 62,169.93 167.08* 0.443 pd 3 558,706.85 1501.52* 0.915 prd¥pd 6 10,440.17 28.06* 0.286 error 420 372.09 total 431 prd, predator density; pd, prey density. *f-values indicate significance at p<0.05 level. table 2. results of post hoc tukey test, using prey density and predator density as explanatory variables for the mosquito larval (shared prey) mortality in presence of both intraguild predator and ig prey. predator density: prey density: se=2.27; df –420.2 se=2.62; df –420.3 (i) (j) i-j (i) (j) i-j 1 2 –16.06* 50 100 –30.83* 1 4 –41.22* 50 200 –107.2* 2 4 –25.17* 50 400 –158.1* 100 200 –76.37* 100 400 –127.3* 200 400 –50.93* *f-values indicate significance at p<0.05 level. no nco mm er cia l u se on ly [page 10] [journal of entomological and acarological research 2014; 46:1977] predator (d. rusticus) and shared prey densities. we predict that the variations in the relative densities of the ig prey and shared prey determine the outcome of prey-predator interactions in the igp system. when the densities of the shared prey are high the prey consumption of the ig predator is highly inclined towards the shared prey. the additive effects of the two predators – ig predator and the ig prey – on the shared prey mortality reflects that the risk to predation increases with the increase in the density of the mosquito prey. increase in number of ig predator, not only increased the ratio of ig predator and ig prey but also increased the overall predator density. while interpreting the predator density effects on shared prey mortality, it seems that higher number of ig predators increased vulnerability of both shared prey and ig prey, proportionate to the availability of shared prey. in several studies demonstrating igp system, the resultant effect on the shared prey varied with the predator and prey densities (wissinger, 1992; wissinger & mcgrady, 1993; de clercq et al., 2003; okuyama & ruyle, 2003; holbrook & petranka, 2004; flynn & moon, 2011). the body size of the ig prey and the shared prey are highly disproportionate and article figure 4. the risk to predation index (mean+se) for mosquito larvae (shared prey) at different initial densities against different intraguild (ig) predator: ig prey ratio and numbers (a: 1:10; b: 2:10; c: 4:10). e=expected ka+kd, o=observed ka+d. irrespective of density of ig predator and shared prey, the observed and expected values were different significantly at p<0.001, marked as ***. figure 3. the number (mean+se) of a. bouvieri (ig prey) consumed as a function of initial shared prey density and intraguild (ig) predator density (1, 2 and 4). table 3. results of two way factorial anova, using shared prey density and intraguild (ig) predator density as explanatory variable for a. bouvieri (ig prey) mortality in presence of both ig predator and ig prey. source df mean square f partial η2 prd 2 8.39 18.18* 0.151 pd 3 7.34 15.89* 0.189 prd¥pd 6 0.23 0.49 0.014 error 204 0.47 total 215 prd, predator density; pd, prey density. *f-values indicate significance at p<0.05 level. table 4. results of post hoc tukey test, using shared prey density and intraguild (ig) predator density as explanatory variable for a. bouvieri (ig prey) mortality in presence of both ig predator and ig prey. predator density: prey density: se=0.11; df=204.2 se=0.13; df=204.2 (i) (j) i-j (i) (j) i-j 1 2 –0.292* 50 100 0.5185* 1 4 –0.681* 50 200 0.6852* 2 4 –0.389* 50 400 0.8519* 100 200 0.16667 100 400 0.33333 200 400 0.16667 *f-values indicate significance at p<0.05 level. table 5. the results of t-test to justify differences in the expected and observed values in the index of risk of predation. all t-values are significant at p<0.001 level (two-tailed), at df –17. ig predator density initial shared prey density (numbers) 50 100 200 400 1 t=614.39 t=206.13 t=22.29 t=15.31 2 t=127.37 t=65.12 t=35.32 t=15.016 4 t=214.54 t=278.09 t=27.97 t=17.37 ig, intraguild. no nco mm er cia l u se on ly inclined towards the ig prey inviting the ig predator to consume more of the ig prey than shared prey. however, this was not exhibited at higher densities of mosquito, providing a reasonable basis to conclude that mosquitoes are preferred than the backswimmers, although the energy returns are higher for the latter. this may be favourable for mosquito control using water bugs. in mosquito larval habitats both the ig predator and the ig prey co-occur with mosquito larvae (sunish & reuben, 2002; bambaradeniya et al., 2004; banerjee et al., 2010). therefore predatory interference between these insects is inevitable. we assume that the risk to predation of mosquito larvae (shared prey) will vary with its relative density and the relative densities of the ig predator. interactive effect of the ig predator and the ig prey insects will reduce mosquitoes at a higher proportion, when the density of the shared prey is high than when the density is low. from biological control viewpoint, igp system seems to be favourable if mosquito density is high along with higher relative densities of the predators. the mosquito larval habitats are heterogenous with the presence of multiple predators and physical structures including debris and detritus (saha et al., 2008; banerjee et al., 2010). ability to judge the presence of predators and quality of the habitat restrain mosquitoes from oviposition in larval habitats as evident from the studies on culex tritaeniorhynchus (ohba et al., 2012) and culiseta longiareolata (blaustein et al., 2004). avoidance of predators in natural situations may influence the outcome of the prey predator interactions as observed in the present instance. in many situations, the larval habitats like rice fields and allied wetlands forma mosaic space to sustain metacommunity. dispersal of the predatory insects is obvious in such situations as observed for the beetles graphoderus occidentalis and rhantus sericans (yee et al., 2009; yee, 2010). variation in abundance of the predatory insects and the avoidance of habitats by mosquito prey may influence outcome of the prey-predator interactions which need to be evaluated further to comment on the utility of the igp system under field conditions. habitat complexity influences the outcome of igp system as evidenced from the studies of larval odonata (flynn & moon, 2011). the identity of the ig predator and the ig prey may also be important factor since cannibalism and inter specific predation are common among the various heteropteran species common in the wetlands. while considering heteropteran species for biological control, predator species substitutability (sih et al., 1998; crumrine, 2005) may be important in successful regulation of mosquitoes. nonetheless, the present study reflects that the relative density of the ig predator and ig prey are important determinants in the outcome of the igp system and the resultant impact on the shared prey, mosquito larvae. in view of biological control of mosquitoes in wetland habitats, the insect predators d. rusticus and a. bouvieri should be present in suitable relative densities, to avoid the ig prey mortality and increase mortality of mosquito prey. references aditya g., bhattacharya s., kundu n., saha g.k., 2005 frequency dependent prey selection of predacious water-bugs on armigeris subalbatus immatures. j. vec. borne. dis. 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indirect effects and biological control of mosquitoes by mosquitofish. j. appl. ecol. 25: 505-521. blaustein l., 1992 larvivorous fishes fail to control mosquitoes in experimental plots. hydrobiologia 232: 219-232. blaustein l., kiflawi m., eitam a., mangel m., cohen j.e., 2004 oviposition habitat selection in response to risk of predation in temporary pools: mode of detection and consistency across experimental venue. oecologia. 138: 300-305. borer e.t., briggs c.j., murdoch w.w., swarbrick s.l., 2003 testing intraguild predation theory in a field system: does numerical dominance shift along a gradient of productivity? ecol. lett. 6: 929-935 burley l.a., moyer a.t., petranka j.w., 2006 density of an intraguild predator mediates feeding group size, intraguild egg predation and intra-and interspecific competition. oecologia. 148: 641-649. crumrine p.w., 2005 size structure and substitutability in an odonate intraguild predation system. oecologia. 145: 132-139. crumrine p.w., crowley p.h., 2003 partitioning components of risk reduction in a dragonfly-fish intraguild predation system. ecology. 84: 1588-1597. dale p.e.r., knight j.m., 2008 wetlands and mosquitoes: a review. wet. ecol. manage. 16: 255-276. das p.k., sivagnaname n., amalraj d.d., 2006 population interactions between culex vishnui mosquitoes and their natural enemies in pondicherry, india. j. vec. ecol. 31: 84-88. de clercq p., peeters i., vergauwe g., thas o., 2003 interaction between podisus maculiventris and harmonia axyridis, two predators used in augmentative biological control in greenhouse crops. biocontrol 48: 39-55. denno r.f., gratton c., peterson m.a., langellotto g.a., finke d.l., huberty a.f., 2002 bottomup forces mediate natural-enemy impact in a phytophagous insect community. ecology. 83: 1443-1458. flynn k.e., moon d.c., 2011 effects of habitat complexity, prey type, and abundance on intraguild predation between larval odonates. hydrobiologia 675: 97-104. hampton s.e., gilbert j.j., 2001 observations of insect predation on rotifers. hydrobiologia 446/447: 437-444. holbrook c.t., petranka j.w., 2004 ecological interactions between rana sylvatica and ambystoma maculatum: evidence of interspecific competition and facultative intraguild predation. copeia 2004: 932-939. hurlbert s.h., 1984 pseudoreplication and the design of ecological field experiments. ecol. monogr. 54:187-211. lawler s.p., dritz d.a., 2005 straw and winter flooding benefit mosquitoes and other insects in a rice agroecosystem. ecol. appl. 15: 2052-2059. lupi d., rocco a., rossaro b., 2013 benthic macroinvertebrates in italian rice fields. j. limnol. 72: 184-200. ohba s., nakasuji f., 2007 density-mediated indirect effects of a common prey, tadpole on interaction between two predatory bugs: kirkaldyia deyrolli and laccotrephes japonensis. popul. ecol. 49: 331-336. ohba s., swart c.c., 2009 intraguild predation of water scorpion laccotrephes japonensis (nepidae: heteroptera). ecol. res. 24:1207-1211. [journal of entomological and acarological research 2014; 46:1977] [page 11] article no nco mm er cia l u se on ly [page 12] [journal of entomological and acarological research 2014; 46:1977] okuyama t., ruyle r.l., 2003 analysis of adaptive foraging in an intraguild predation system. web. ecol. 4: 1-6. polis g.a., myers c.a., holt r.d., 1989 the ecology and evolution of intraguild predation: potential competitors that eat each other. annu. rev. ecol. syst. 20: 297-330. rosenheim j.a., corbett a., 2003 omnivory and the indeterminacy of predator function: can a knowledge of foraging behaviour help? ecology. 84: 2538-2548. saha n., aditya g., bal a., saha g.k., 2007a a comparative study of predation of three aquatic hemipteran bugs on culex quinquefasciatus larvae. limnology. 8: 73-80. saha n., aditya g., bal a., saha g.k., 2007b comparative study of functional response of common hemipteran bugs of east calcutta wetlands, india. int. rev. hydrobiol. 92: 242-257. saha n., aditya g., bal a., saha g.k., 2008 light and habitat structure influences predation of culex quinquefasciatus larvae by the water bugs (hemiptera: heteroptera). insect. sci. 15: 461-469. saha n., aditya g., saha g.k., 2009 habitat complexity reduces vulnerability of preys: an experimental analysis using aquatic insect predators and dipteran immatures. j. asia. pacific. entomol. 12: 233-239. saha n., aditya g., saha g.k., hampton s.e., 2010 opportunistic foraging by heteropteran mosquito predators. aquat. ecol. 44: 167-176. sih a., englund g., wooster d., 1998 emergent impacts of multiple predators on prey. trends. ecol. evol. 13: 350-355. soluk d.a., 1993 multiple predator effects: predicting combined functional response of stream fish and invertebrate predators. ecology. 74: 219-225. soluk d.a., collins n.c., 1988 synergistic interactions between fish and stoneflies-facilitation and interference among stream predators. oikos. 52: 94-100. sunish i.p., reuben r., 2002 factors influencing the abundance of japanese encephalitis vectors in rice fields in india-ii. biotic. med. vet. entomol. 16: 1-9. walls s.c., williams m.g., 2001the effect of community composition on persistence of prey with their predators in an assemblage of pond-breeding amphibians. oecologia. 128: 134-141. walzer a., paulus h.f., schausberger p., 2004 ontogenetic shifts in intraguild predation on thrips by phytoseiid mites: the relevance of body size and diet specialization. bull. entomol. res. 94: 577-584. wissinger s., mcgrady j., 1993 intraguild predation and competition between larval dragonflies: direct and indirect effects on shared prey. ecology. 74: 207-218. wissinger s.a., 1992 niche overlap and the potential for competition and intraguild predation between size-structured populations. ecology. 73: 1431-1444. yee d.a., 2010 behavior and aquatic plants as factors affecting predation in three species of larval predaceous diving beetles (coleoptera: dytiscidae). hydrobiologia. 637: 33-43. yee d.a., taylor s., vamosi s.m., 2009 beetle and plant density as cues initiating dispersal in two species of adult predaceous diving beetles. oecologia. 160: 25-36. zar j.h., 1999 biostatistical analysis. iv ed. pearson education (singapore) pte. ltd., (indian branch), new delhi: 663. article no nco mm er cia l u se on ly layout 1 [journal of entomological and acarological research 2013; 45:e12] [page 65] the grass-living thrips (insecta: thysanoptera) from iran with the first record of the genus arorathripsbhatti k. minaei, m. alichi department of plant protection, college of agriculture, shiraz university, shiraz, iran abstract a list of grass-dependent thysanoptera genera in iran is provided, including arorathrips with one species, a. mexicanus, a chirothripsrelated thripid genus as a new record for iranian fauna. the specimens of this species were collected from mixed grasses in the city of minab located in hormozgan province, south of iran. the importance of grasses as host plants for members of the family thripidae is briefly discussed. introduction the members of the insect order thysanoptera exhibit a wide range of bionomics. about 50% are fungivorous, feeding on the hyphae or spores of fungi (mound, 2003). of the remaining species, although a few are obligate predators on other small arthropods (palmer & mound, 1990), most of them are phytophagous, including several opportunist species considered as crop pests (lewis, 1997; moritz et al., 2004). nine families are recognized in the order thysanoptera (mound et al., 2013), of which five (aeolothripidae, stenurothripidae, melanthripidae phlaeothripidae, thripidae) have been recorded in iran so far (minaei & alichi, 2007). the objective of this paper is to provide a list of thysanoptera genera in which species breed on grasses (family poaceae) in iran, and to record the genus arorathrips bhatti as another grass-dwelling genus, for the first time in this country. illustrations and diagnostic characters are also included. full nomenclatural information about thysanoptera is available on the web (thripswiki, 2013). materials and methods the list of iranian thrips that are associated with grasses is extracted from the published literature. the species discussed here, a. mexicanus (crawford), was collected by beating mixed grasses (poaceae) onto a plastic tray. the specimens were removed with a fine brush into a collecting vial containing 90% ethyl alcohol. they were then mounted onto slides in canada balsam using the protocol of mound & kibby (1998). microphotographs were obtained using a dino-lite microscope, eyepiece camera. digital images were enhanced and plates prepared by adobe photoshop™ (adobe systems inc., san jose, ca, usa). the terminology used here follows minaei & mound (2010a) and hoddle et al. (2013). all specimens studied are deposited in the collection of the plant protection department, college of agriculture, shiraz university, shiraz, iran. results the only recorded member of the family stenurothripidae in iran, holartrothrips josephi bhatti, feeds on the pollen of date palm (bhatti, 1986). concomitantly, two genera of melanthripidae (ankothrips bagnall and melanthrips bagnall) include flower-feeding species in various plant families (minaei et al., 2012). grass-living thrips are distributed among another three families. among these, there are a few species that breed on grasses in aeolothripidae and phlaeothripidae, but most grass-living thysanoptera belong to the family thripidae, including arorathrips mexicanus, which is discussed below. arorathrips mexicanus (crawford) chirothrips mexicana d.l. crawford 1909: 114. arorathrips mexicanus (crawford); bhatti 1990: 196. the genus arorathrips was separated by bhatti (1990) from the genus chirothrips, and four species were placed in the new genus at that time. however, currently 15 species are placed in this genus, all of which are considered endemic to the new world and breed only in the flowers of grasses (mound & marullo, 1996; mound, 2011; nakahara & foottit, 2012). arorathrips mexicanus is recorded here from iran, and correspondence: kambiz minaei, department of plant protection, college of agriculture, shiraz university, shiraz, iran. e-mail: kminaei@shirazu.ac.ir key words: arorathrips mexicanus, hormozgan province, new record, poaceae. acknowledgements: the authors would like to thank agricultural research, education and extension organization of hormozgan for providing facilities during field work in hormozgan. the manuscript was improved through the advice kindly provided by two anonymous reviewers. received for publication: 15 march 2013. revision received: 1 june 2013. accepted for publication: 7 june 2013. ©copyright k. minaei and m. alichi, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e12 doi:10.4081/jear.2013.e12 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e12 jear_2013_2:hrev_master 16/09/13 13.56 pagina 65 no nco mm er cia l spores of fungi (mound, 2003). of the remaining species, although a no nco mm er cia l spores of fungi (mound, 2003). of the remaining species, although a few are obligate predators on other small arthropods (palmer & no nco mm er cia l few are obligate predators on other small arthropods (palmer & mound, 1990), most of them are phytophagous, including several no nco mm er cia l mound, 1990), most of them are phytophagous, including several opportunist species considered as crop pests (lewis, 1997; moritz no nco mm er cia l opportunist species considered as crop pests (lewis, 1997; moritz et no nco mm er cia l et ., 2004). nine families are recognized in the order thysanoptera no nco mm er cia l ., 2004). nine families are recognized in the order thysanoptera ., 2013), of which five (aeolothripidae, stenurothripidae, no nco mm er cia l ., 2013), of which five (aeolothripidae, stenurothripidae, ed from the published literature. the species discussed here, no nco mm er cia l ed from the published literature. the species discussed here, canus no nco mm er cia l canus no nco mm er cia l (crawford), was collected by beating mixed grasses (poaceae) no nco mm er cia l (crawford), was collected by beating mixed grasses (poaceae)onto a plastic tray. the specimens were removed with a fine brush into no nco mm er cia l onto a plastic tray. the specimens were removed with a fine brush into a collecting vial containing 90% ethyl alcohol. they were then mountno nco mm er cia l a collecting vial containing 90% ethyl alcohol. they were then mounted onto slides in canada balsam using the protocol of mound & kibby no nco mm er cia l ed onto slides in canada balsam using the protocol of mound & kibby no nco mm er cia l no nco mm er cia l correspondence: kambiz minaei, department of plant protection, college of no nco mm er cia l correspondence: kambiz minaei, department of plant protection, college of agriculture, shiraz university, shiraz, iran. no nco mm er cia l agriculture, shiraz university, shiraz, iran. arorathrips mexicanus no nco mm er cia l arorathrips mexicanus, hormozgan province, new record, no nco mm er cia l , hormozgan province, new record, us e materials and methods us e materials and methods the list of iranian thrips that are associated with grasses is extract-us e the list of iranian thrips that are associated with grasses is extracted from the published literature. the species discussed here, us e ed from the published literature. the species discussed here, (crawford), was collected by beating mixed grasses (poaceae)us e (crawford), was collected by beating mixed grasses (poaceae) on ly for the first time in this country. illustrations and diagnostic characon ly for the first time in this country. illustrations and diagnostic characters are also included. full nomenclatural information about on ly ters are also included. full nomenclatural information about thysanoptera is available on the web (thripswiki, 2013). on lythysanoptera is available on the web (thripswiki, 2013). on ly materials and methodson ly materials and methods [page 66] [journal of entomological and acarological research 2013; 45:e12] this is the first record of this genus and species in this country. the genus is distinguished from the closely related genus, chirothrips, in having the mesothoracic endofurca greatly reduced and fore tibia prolonged around the external margin of the fore tarsus. diagnosis: female fully winged. body color light brown, tarsi yellow, forewing and clavus shaded (figure 1). antennae 8-segmented, segment i with median dorsal setal pair wider apart than width of base of segment ii, segment ii distinctly produced at apex on outer margin with terminal sensorium, segments iii-iv with simple sensorium (figure 2). head small, with a distinct prolongation in front of eyes, vertex with three pairs of setae. pronotum trapezoidal, two pairs of prominent posteroangular setae present (figure 3). mesothoracic endofurca reduced (figure 4). fore tibia extending around external margin of fore tarsus (figure 5). tergites with transverse sculpture lines medially; antecostal ridge of tergites ii-v with row of small tubercles; campaniform sensilla anterior to median, its setae on tergites i-viii. sternites ii-iv medially with pattern of tubercles. male smaller, wingless, yellow (figure 6); sternites iii-vii medially with circular glandular area (figure 7). material examined: 5 females, 5 males, hormozgan, minab, from mixed grasses, 20.1.2009 (km 259). article figure 1. arorathrips mexicanus, female: adult. figure 2. arorathrips mexicanus, female: antennal segments ii-iv. figure 3. arorathrips mexicanus, female: head and pronotum. figure 4. arorathrips mexicanus, female: meso and metathorax furca reduced. figure 5. arorathrips mexicanus, female: foretibia and tarsus. figure 6. arorathrips mexicanus, male: adult. figure 7. arorathrips mexicanus, male: sternites vi-vii. jear_2013_2:hrev_master 16/09/13 13.56 pagina 66 no nco mm er cia l arorathrips mexicanus no nco mm er cia l arorathrips mexicanus, no nco mm er cia l , no nco mm er cia l no nco mm er cia l figure 3. no nco mm er cia l figure 3. arorathrips mexicanus no nco mm er cia l arorathrips mexicanus no nco mm er cia l female: head and pronotum. no nco mm er cia l female: head and pronotum. no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l u se us e us e us e us e us e us e o nlyon ly on ly arorathrips mexicanus on ly arorathrips mexicanus, female: adult. on ly , female: adult. discussion and conclusions about 40 thripidae genera are recorded from iran (bhatti et al., 2009), and a large proportion of these (about 40%) live on grasses (table 1). this is in accordance with the situation reported from australia (mound, 2011). grasses support a rich fauna of thrips (table 1), possibly due to the availability of a range of such plants in most areas (unpublished lecture by la mound at 20th iranian plant protection congress, shiraz, iran, august 2012). some of the species in the genera listed in table 1 are considered plant pests in other countries (lewis, 1997; moritz et al., 2004). however, no thripid pest is recorded on grasses in iran. in contrast, one of the species in the family phlaeothripidae, haplothrips tritici (kurdjumov) (minaei & mound, 2008, 2010b), is an important pest throughout iran on wheat. all species in the genus arorathrips have been considered endemic to the new world (mound & marullo, 1996; mound, 2011; nakahara & foottiit, 2012). however, the presence of a. mexicanus is not surprising in iran because this species is introduced around the world and is widely distributed in the tropics and subtropics in association with grasslands (mound & palmer, 1972; mound & marullo, 1996). references bhatti j.s., 1975 a revision of exothrips priesner and two related genera. orient. insects. 9: 43-90. bhatti j.s., 1986 a new species of holarthrothrips from iraq, with notes on host plants and key to species, along with clarification of the position of this genus among thysanoptera. zoology (jpaz). 1: 1-33. bhatti j.s., 1990 on some genera related to chirothrips (insecta: terebrantia: thripidae). zoology (jpaz). 2: 194-200. bhatti j.s., alavi j., zur strassen r., telmadarraiy z., 2009 thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 1-82. crawford d.l., 1909 some thysanoptera of mexico and the south. i. pomona coll. j. entomol. l: 109-119. hoddle m.s., mound l.a., paris d., 2013 thrips of california 2012. available from: http://keys.lucidcentral.org/keys/v3/thrips_of_california/thrips_of_california.html accessed: 15 march 2013. lewis t., 1997 pest thrips in prespective. in: lewis t. (ed.), thrips as crop pests. cab international, wallingford: 1-13. minaei k., alichi m., 2007 thysanoptera fauna of shiraz and vicinity. j. insect sci. 7: 22-23. minaei k., azmayeshfard p., mound l.a., 2007 the thrips genusgroup (thysanoptera: thripidae) in iran. j. entomol. soc. iran 27: 29-36. minaei k., haftbaradaran f., mound l.a., 2012a new ankothrips species (thysanoptera: melanthripidae) from iran with unusually short setae. – zootaxa. 3552: 37-42. minaei k., mound l.a., 2008 the thysanoptera haplothripini (phlaeothripidae) of iran. j. nat. his. 42: 2617-2658. minaei k., mound l.a., 2010a grass-flower thrips of the genus chirothrips (thysanoptera: thripidae), with a key to species from iran. zootaxa. 2411: 33-43. minaei k., mound l.a., 2010b taxonomic problems in character state interpretation: variation in the wheat thrips haplothrips tritici (kurdjumov) (thysanoptera: phlaeothripidae) in iran. dtsch. entomol. z. 57: 233-241. moritz g., mound l.a., morris d.c., goldarazena a., 2004 pest thrips of the world: an identification and information system using molecular and microscopical methods. cd-rom. cent. biol. inf. technol, brisbane. mound l.a., 2003 thysanoptera. in: resh v.h., carde r.t. (eds.), the encyclopedia of insects. academic press, st. louis: 1127-1132. mound l.a., 2011 grass-dependent thysanoptera of the family thripidae from australia. zootaxa 3064: 1-40. mound l.a., desley t., paris, d., 2013 oz thrips, thysanoptera in australia. available from: http://www.ozthrips.org/terebrantia/ aeolothripidae/ accessed: 15 march 2013. [journal of entomological and acarological research 2013; 45:e12] [page 67] article table 1. the genera of thysanoptera associated with grasses in iran. family genus reference aeolothripidae aeolothrips haliday* hoddle et al., 2013 rhipidothrips uzel mound et al., 1976 phlaeothripidae haplothrips amyot and serville* minaei & mound, 2008 cephalothrips uzel hoddle et al., 2013 thripidae anaphothrips uzel* mound & masumoto, 2009 aptinothrips haliday palmer, 1975 arorathrips bhatti nakahara & foottit, 2012 bregmatothrips hood zur strassen, 2003 caliothrips daniel wilson, 1975 chirothrips haliday minaei & mound, 2010a collembolothrips priesner zur strassen, 2003 eremiothrips priesner* ramezani et al., 2009 exothrips priesner bhatti, 1975 florithrips bhatti ramezani et al., 2012 frankliniella karny* mound et al., 1976 limothrips haliday zur strassen, 2003 sitothrips priesner zur strassen, 2003 sphaeropothrips priesner minaei et al., 2007 stenchaetothrips bagnall zur strassen, 2003 stenothrip uzel zur strassen, 2003 *not all species breeding on grasses. jear_2013_2:hrev_master 16/09/13 13.56 pagina 67 no nco mm er cia l ., 2009), no nco mm er cia l ., 2009), and a large proportion of these (about 40%) live on grasses (table 1). no nco mm er cia l and a large proportion of these (about 40%) live on grasses (table 1). this is in accordance with the situation reported from australia (mound, no nco mm er cia l this is in accordance with the situation reported from australia (mound, 2011). grasses support a rich fauna of thrips (table 1), possibly due to no nco mm er cia l 2011). grasses support a rich fauna of thrips (table 1), possibly due to the availability of a range of such plants in most areas (unpublished lecno nco mm er cia l the availability of a range of such plants in most areas (unpublished leciranian plant protection congress, shiraz, iran, no nco mm er cia l iranian plant protection congress, shiraz, iran, august 2012). some of the species in the genera listed in table 1 are conno nco mm er cia l august 2012). some of the species in the genera listed in table 1 are considered plant pests in other countries (lewis, 1997; moritz no nco mm er cia l sidered plant pests in other countries (lewis, 1997; moritz however, no thripid pest is recorded on grasses in iran. in contrast, one no nco mm er cia l however, no thripid pest is recorded on grasses in iran. in contrast, one of the species in the family phlaeothripidae, no nco mm er cia l of the species in the family phlaeothripidae, (kurdjumov) (minaei & mound, 2008, 2010b), is an important pest no nco mm er cia l (kurdjumov) (minaei & mound, 2008, 2010b), is an important pest arorathripsno nco mm er cia l arorathrips have been considered endemicno nco mm er cia l have been considered endemic to the new world (mound & marullo, 1996; mound, 2011; nakahara &no nco mm er cia l to the new world (mound & marullo, 1996; mound, 2011; nakahara & foottiit, 2012). however, the presence of n on -co mm er cia l foottiit, 2012). however, the presence of thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 1-82. no nco mm er cia l thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 1-82. crawford d.l., 1909 some thysanoptera of mexico and the south. no nco mm er cia l crawford d.l., 1909 some thysanoptera of mexico and the south. i. pomona coll. j. entomol. l: 109-119. no nco mm er cia l i. pomona coll. j. entomol. l: 109-119. hoddle m.s., mound l.a., paris d., 2013 thrips of california 2012. no nco mm er cia l hoddle m.s., mound l.a., paris d., 2013 thrips of california 2012. us e thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 1-82. us e thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 1-82. on ly zur strassen, 2003 on ly zur strassen, 2003 minaei on ly minaei zur strassen, 2003 on lyzur strassen, 2003 on lyzur strassen, 2003 on lyzur strassen, 2003 [page 68] [journal of entomological and acarological research 2013; 45:e12] mound l.a., kibby g., 1998 thysanoptera: an identification guide. second edition. cab international institute of entomology and british museum (natural history), london: 70 pp. mound l.a., marullo r., 1996 the thrips of central and south america: an introduction (insecta: thysanoptera). mem. entomol. int. 6: 1-488. mound l.a., masumoto m., 2009 australian thripinae of the anaphothrips genus-group (thysanoptera), with three new genera and thirty-three new species. zootaxa. 2042: 1-76. mound l.a., morison g.d., pitkin b.r., palmer j.m., 1976 thysanoptera. handbooks for the identification of british insects, vol. 1. royal entomological society of london (res), london: 1-79. mound l.a., palmer j.m., 1972 grass-flower infesting thrips of the genus chirothrips haliday in australia. j. aust. entomol. soc. 11: 332-339. nakahara s., foottit r.g., 2012 review of chirothrips and related genera (thysanoptera: thripidae) of the americas, with descriptions of one new genus and four new species. zootaxa. 3251: 1-29. palmer j.m., 1975 the grass-living genus aptinothrips (thysanoptera: thripidae). j. entomol. 44: 175-188. palmer j.m., mound l.a., 1990 thysanoptera in: rosen d. (ed.), the armoured scale insects, their biology, natural enemies and control. elsevier, amsterdam: 67-75. ramezani l., bhatti j.s., mossadegh m.s., soleimannejadian e., 2009 discovery of eremiothrips similis bhatti 1988 in iran (insecta: terebrantia: thripidae). thrips. 11: 1-18. ramezani l., mossadegh m.s., soleimannejadian e., bagheri s., minaei k., 2012 the first report of the genus and species of florithrips traegardhi (thysa.: thripidae) from iran. j. entomol. soc. iran 31: 101-103. [in persian]. thripswiki, 2013 thripswiki providing information on the world’s thrips. available from: thrips.info/wiki/ accessed: 1 june 2013. wilson t.h., 1975 a monograph of the subfamily panchaetothripinae (thysanoptera: thripidae). mem. am. entomol. inst. 23: 1-354. zur strassen r., 2003 die terebranten thysanopteren europas und des mittelmeer-gebietes. die tierwelt deutschlands 74: 1271. [in german]. article jear_2013_2:hrev_master 16/09/13 13.56 pagina 68 no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res. ser. ii, 42 (3): 117-124 30 december 2010 l. de marzo a further evaluation of the sperm length in aleocharines (coleoptera staphylinidae) abstract - observations on 8 species allowed to extend the known range: the new  minimum and maximum values are respectively 100 µm for aleochara intricata  mannerheim and 3.000 µm for heterota plumbea (waterhouse). evaluations for  most species were carried out either on spermatozoa separated from the spermatheca  or on bundles extracted from testes. sperm length of atheta inquinula was supposedly assumed as corresponding to that of the vasa deferentia. conclusive diagrams  show sperm length in some species to be disproportionate if compared with the  size of the receptacle. riassunto - ulteriore valutazione della lunghezza degli spermatozoi nelle aleocarine (coleoptera staphylinidae) valutata precedentemente su 7 specie, la gamma del carattere in questione era  150-2.300 µm. una valutazione su altre 8 specie ha fatto registrare un nuovo  valore minimo e un massimo, rispettivamente di 100 µm in aleochara intricata  mannerheim e 3.000 µm in heterota plumbea (waterhouse). come in precedenza,  le misurazioni sono state effettuate su singoli spermatozoi estratti dalla spermateca,  oppure su fascetti di spermatozoi estratti dai testicoli. nel caso di atheta inquinula  (gravenhorst), non è stato tecnicamente possibile ottenere una valutazione diretta,  e si suppone che la lunghezza degli spermatozoi sia di circa 1.500 µm, cioè corrispondente a quella dei dotti deferenti. come già visto in precedenza, gli spermatozoi  di una parte delle specie esaminate sono smodatamente lunghi in confronto con le  dimensioni del ricettacolo. key words: new range, exceeding sperm length. introduction a previous evaluation of the character “sperm length” in 7 species the subf.  aleocharinae allowed to register a range from 150 to 2.300 µm (de marzo, 2008). an  evaluation on further 8 species of the same subfamily allows to extend this range in both  its minimum and maximum values. most species have been identified thanks to dr. adriano zanetti (verona natural  history museum). journal of entomological and acarological research, ser. ii, 42 (3), 2010118 material and methods names of the examined species are listed in tab. a; they agree with the checklists  of ciceroni et al. (1995) and smetana (2004), except for atheta mucronata (kraatz),  which has been more recently revised by feldmann (2007). four specimens at least  were examined for females and males of each species; they were killed with ethyl acetate vapours and dissected in physiological salt solution (nacl 0,9%). measures were  taken with graduated eye-piece and 40x phase-contrast lens on spermathecae or testes  compressed on slides in the same solution. the first evaluation was attempted on spermatozoa isolated the mass extracted from compressed spermathecae. then, evaluation  was attempted on sperm bundles extracted from the testicular follicles. parts of the spermatheca are designated according to de marzo (2009a; 2009b). results spermathecal anatomy in the species examined here agrees with the rule in the  aleocharines, as spermatheca includes: (a) a receptacle sclerotized to some extent and  provided with a compressor muscle; (b) a long duct, which usually exhibits a lower  sclerotization degree; (c) a gland connected with the receptacle. usually, duct contains  only a very poor number of spermatozoa, as aleochara tristis (fig. 1.a); otherwise, it  is occluded by a spermatophore, as in aleochara intricata (fig. 1.b), and is therefore  filled with a dense mass of sperm. length of spermatozoa was found comparatively low in the nominal genus aleochara,  where it ranges from 100 to 1.000 µm (table a), and wasn’t therefore difficult to be  evaluate on female material. the same material was suitable also to measure the very short  spermatozoa of atheta coriaria (320 µm) and caloderina hierosolymitana (200 µm). tab. a sperm length (microns) in the examined aleocharines. evaluations were carried out either on spermatozoa isolated from the spermatheca (material from females) or on sperm bundles extracted from testis (material from males). in the case of atheta inquinula sperm length does supposedly correspond to that of the vasa deferenttia. species material from females material from males aleochara bipustulata (linnaeus) 1.000 aleochara intricata mannerheim 100 aleochara tristis gravenhorst 500 atheta coriaria (kraatz) 320 atheta mucronata (kraatz) 1.100 atheta inquinula (gravenhorst) 1.500 (supposed) caloderina hierosolymitana (saulcy) 200 heterota plumbea (g. waterhouse) 3.000 119l. de marzo: evaluation of the sperm length in aleocharines fig. 1 - spermathecal features suggesting of the two different insemination modalities known in  aleocharinae: a, duct adapted to a very long endophallic filament in aleochara tristis gravenhorst;  b, duct occluded by a spermatophore in aleochara intricata mannerheim. journal of entomological and acarological research, ser. ii, 42 (3), 2010120 fig. 2 - heterota plumbea (waterhouse): details of female/male internal genitalia. 121l. de marzo: evaluation of the sperm length in aleocharines fig. 3 - atheta inquinula (gravenhorst): details of male/female internal genitalia and supposed  sperm length compared at the same magnification with the size of the spermatheca. journal of entomological and acarological research, ser. ii, 42 (3), 2010122 fig. 4 - examined species of the genus aleochara: sperm length compared at the same magnification with the size of their receptacle. 123l. de marzo: evaluation of the sperm length in aleocharines fig. 5 - other examined aleocharines: sperm length compared at the same magnification with the  size of their receptacle. journal of entomological and acarological research, ser. ii, 42 (3), 2010124 higher values of sperm length were appreciated on male material, by isolating bundles from the testicular follicles of atheta mucronata (1.100 µm) and heterota plumbea  (3.000 µm). attempts to obtain separate spermatozoa of these species were unsuccessful,  as their spermathecae contained a very intricate mass of sperm. both female and male materials were not suitable to evaluate sperm length in the  case of atheta inquinula, as spermathecae provided only an intricate sperm mass and  testicular follicles didn’t contain clear sperm bundles. supposedly, the spermatozoa of  this species are 1.500 µm long as the vasa deferentia (fig. 3).   conclusive remarks this new evaluation does extended the known range of the character “sperm length”  in aleocharines at both its minimum and maximum values. these are respectively 100  and 3.000 µm. as elsewhere observed (de marzo, 2008), sperm length of some species looks  somewhat disproportionate if compared with the size of the receptacle. this is especially  evident for aleochara bipustulata, atheta mucronata and heterota plumbea (figs. 3-4).  because spermatozoa of this species do bend two or more times to enter receptacle, the  question “how these very long spermatozoa can go away from the receptacle to fertilise eggs?” comes again. bibliografia ciceroni a., puthz v., zanetti a., 1995 - coleoptera polyphaga iii. staphylinidae. - in: minelli  a., ruffo s., la posta s. (eds.), checklist delle specie della fauna italiana, calderini ed.,  bologna, fasc. 48, 65 pp. de marzo l., 2008 - lunghezza degli spermatozoi rilevata in alcune aleocarine (coleoptera  staphylinidae). - boll. zool. agr. bachic., milano, ser. ii, 40 (1): 1-8. de marzo l., 2009a - biodiversità della spermateca nei coleotteri. - atti accademia nazionale  italiana di entomologia, anno lvi (2008): 69-96. de marzo l., 2009b - aspetti morfologici della spermateca in diestota guadalupensis pace e  altre aleocharinae (coleoptera staphylinidae). - entomologica, bari, 40 (2006-2007): 57-73. feldmann b., 2007 - on the identity of atheta mucronata (kraatz 1859) (coleoptera: staphylinidae,  aleocharinae). - linzer biol. beitr., 39: 57-63. smetana a., 2004 - staphylinoidea. - in: löbl i. & smetana a. (eds), catalogue of palaearctic  coleoptera, vol. 2, apollo books, 942 pp. luigi de marzo, via f. turati 3, i-70016 noicàttaro (ba), e-mail: l.demarzo@alice.it accepted 22 october 2010 j. ent. acar. res. ser. ii, 42 (3): 143-145 30 december 2010 s. barjadze, i̇. karaca, b.yaşar, n. gratiashvili new evidence of parasitoids of pest aphids on roses and grapevine in turkey (hem., aphididae; hym., braconidae, aphidiinae) abstract - aphidius eglanteriae as a parasitoid of chaetosiphon tetrarhodum/ rosa is newly recorded for turkey. short information about its parasitism rate and  distribution in isparta province, turkey, is given. another new information pertains  to the association between aphidius matricariae aphis illinoisensis - vitis vinifera.  riassunto nuova segnalazione di parassitoidi di afidi su rose e vite in turchia (hem., aphididae; hym., braconidae, aphidiinae) viene segnalato per la prima volta in turchia aphidius eglanteriae haliday come  parassitoide di chaetosiphon tetrarhodum/rosa (walker). vengono fornite anche  indicazioni sulla percentuale di parassitizzazione e la sua distribuzione nella provincia di isparta. ulteriori informazioni riguardano l’associazione tra aphidius  matricariae aphis illinoisensis - vitis vinifera key words: aphidius eglanteriae, aphis illinoisensis, aphidius matricariae, chaetosiphon tetrarhodum, grapevine, roses roses turkey is one of the biggest producer of the rose oil and rose concrete in the world  (bayrak & akgül, 1994). only chemical control is used to decrease harmful aphids  quantity in rose (rosa damascena) plantations of isparta province. chaetosiphon tetrarhodum (walker), is one of main pest aphid of the damask rose in isparta province,  turkey. this aphid causes weakness of plant by sucking activities and problems for  producing great quantitities of honeydew, which is deposited on the leaves. both these  phenomena cause loss of yield of damask rose flowers at the harvest time. during our investigation we reared in the laboratory aphidius eglanteriae haliday,  (1834), from the field sampled mummified c. tetrarhodum on rosa damascena  (figure  1). it was found in three localities of isparta province: isparta, kuyucok and sorkuncak.  in july parasitism rate caused by attacking parasitic wasps to ch. tetrarhodum was  4.35% in kuyucok, 3.81% in isparta and 0.92% in sorkuncak. in september parasitism  rate was 6.5% in isparta. low parasitism rate probably was caused by high temperature. journal of entomological and acarological research, ser. ii, 42 (3), 2010144 this species is oligophagous and it attacks aphid species belonging to the genera  chaetosiphon and longicaudus (kavallieratos et al., 2004). it is distributed mainly in  europe (starý, 1976; kavallieratos et al., 2004). the association is new for turkey (elmali et al. 2004; aslan and karaca 2005; tomanović et al., 2008).  a possibility of mass-rearing and release of this parasitoids in the rose fields should  be further considered.  an over-all biodiversity of beneficial organisms in the biocenoses of oil-bearing  rose  was  presented in bulgaria (balevski et al., 2008). however, merely  aphidius ervi  hal., aphidius rosae hal. and ephedrus laevicollis (thoms.) were listed as participating  in the rose ecosystem. as no host aphid associations were included, we may presume  an association between  a. ervi and a. rosae to macrosiphum rosae and e. laevicollis  possibly to chaetosiphon or myzaphis (p. stary, pers. comm.). grapevine on 21 september 2010, we found aphis illinoisensis (shimer) on vitis vinifera in  isparta province for the first time (barjadze et al., unpublished). there were big colonies of the grapevine aphids on the shoots and leaves of the grape. through mechanical  injury and honeydew production this aphid represents possibly an important economic  threat to viticulture. fig. 1 - mumified chaetosiphon tetrarhodum on a leaf of rosa damascena 145s. barjadze et al.: new evidence of parasitoids of pest aphids one male specimen of aphidius matricariae haliday was reared from an aphid  colony on 21 september 2010. this parasitoid was recorded from different provinces  of turkey, including isparta province (el-mali et al., 2004; aslan and karaca, 2005)  but the presented association between aphidius matricariae aphis illinoisensis - vitis vinifera is recorded for the first time in the world. further investigations will show  whether a. matricariae is effective against grapevine aphids to use it in ipm. also, it is  recommendable to find further native enemies that attack a. illinoisensis or introduce  them for controlling this invasive exotic grapevine aphid in turkey.  acknowledgements we are grateful to dr. petr starý (institute of entomology, acad. sci. of the czech  republic) for help in parasitoid identification and valuable comments on our ms.  the project: “aphids and their natural enemies associated with oil-bearing rose  (rosa damascena) in isparta province (turkey)” has been fulfilled by the financial support of research fellowships for foreign citizens program (code-2216) of tubitak.  references aslan b., karaca i̇., 2005 - fruit tree aphids and their natural enemies in isparta region, turkey.  j. pest sci. 78: 227-229. balevski n., simova s., draganova s., 2008 - biodiversity of beneficial organisms (entomopathogens, predators and parasitoids) in  the biocenoses of oil-bearing rose (rosa damascena mill.)  in bulgaria. plant sci. 45:115-122. bayrak a., akgül a., 1994 - volatile oil composition of turkish rose (rosa damascena). j. sci.  food agr. 64: 441-448. el-mali m.u., starý p., sahbaz a., ozsermeci f., 2004 - a review of aphid parasitoids (hym.,  braconidae, aphidiinae) of turkey. egypt. j. biol. pest control 14: 355-370. kavallieratos n.g., tomanović ž., starý p., athanassiou ch.g., sarlis g.p., petrović o., niketić m., veroniki m.a., 2004 - a survey of aphids parasitoids (hymenoptera: braconidae: aphidiinae) of southeastern europe and their aphid-plant associations. appl. entomol.  zool. 39: 527-563. starý p., 1976 - aphid parasites (hymenoptera, aphidiidae) of the mediterranean area. trans.  czech. acad. sci., ser. math. natur. sci. 86: 1-95. tomanović ž., beyarslan a., erdoğan ö., žikić v., 2008 - new records of aphids parasitoids  (hymenoptera, braconidae, aphidiinae) from turkey. period. biol. 110: 335-338.  shalva barjadze, nana gratiashvili, entomology and biocontrol research centre of ilia state  university, chavchavadze av. 31, 0179, tbilisi, georgia e-mail: shalva1980@yahoo.com. i̇smail karaca, bülent yaşar, department of plant protection, faculty of agriculture, süleyman  demirel university, 32260, isparta, turkey accepted 20 december 2010 j. ent. acar. res. ser. ii, 42 (3): 153-160 30 december 2010 l. limonta, j. sulo, d.p. locatelli  temperature-dependent development and survivorship of idaea inquinata (scopoli) (lepidoptera geometridae) eggs at two humidity levels abstract idaea inquinata (scopoli) mainly feeds on dried plants, nevertheless, it  is also a potential pest of stored product as it is able to develop on cereal products.  the few references on the biology of this species do not deal with the influence of  temperature and relative humidity on egg hatching. to fill this gap, groups of 100  eggs, 24-48 hours old, were exposed to constant temperatures (13, 15, 36, and 38±1  °c), two relative humidities (35, 70±5%) and a photoperiod of 0:24 (light:dark);  eight tests were carried out. each test was replicated four times. the lowest proportion of hatched eggs was observed at 15 °c (9.5) and 36 °c (8.7) with 35±5% r.h. while at 13 and 38 °c eggs did not hatch. a non-linear function is used to represent  the developmental rates and survivorship of eggs at 35 and 70% r.h. between lower  and upper thresholds temperature.  riassunto sviluppo e sopravvivenza di uova di idaea inquinata (scopoli) (lepidoptera geometridae) a diverse temperature e umidità. idaea inquinata (scopoli) si sviluppa principalmente su piante essiccate, tuttavia  è un potenziale infestante di cereali e derivati. la biologia di questa specie è poco  conosciuta, in particolare non ci sono informazioni sull’influenza di temperatura e  umidità relativa sulla schiusura delle uova.  gruppi di 100 uova deposte da 24-48 ore sono state poste a temperature costanti (13,  15, 36, e 38±1 °c), due diversi valori di umidità relativa (35, 70±5%) e fotoperiodo  0:24 (luce:buio); sono state condotte otto prove, replicate quattro volte. il numero  più basso di uova schiuse è stato osservato a 15 °c (9,5) e 36 °c (8,7) con 35±5%  u.r. mentre a 13 e 38 °c le uova non sono schiuse. una funzione non lineare è stata  impiegata per rappresentare il tasso di sviluppo e la sopravvivenza delle uova alle  due umidità considerando il limite inferiore e superiore di temperatura. key words: eggs hatching, temperature, relative humidity, rusty wave moth introduction idaea inquinata (scopoli) can be a serious pest in warehouses where dehydrated  plants and cereals are stored; spices and medicinal plants can be heavily damaged and  made unsuitable to essential oil extraction (candura 1931a, b; locatelli et al., 2005). journal of entomological and acarological research, ser. ii, 42 (3), 2010154 there are few references to the biology of this species (candura 1931a; locatelli et al., op.cit.). according to candura, each female laid one hundred eggs within a week,  oviposition started on the 4th and lasted until the 11th day. eggs were laid singly or in  pairs, and hatching occurred between the 4th and the 15th day, according to the season  and to the weather, in a temperature range of 19-28 °c. limonta et al. (2010) studied  egg hatching at five temperature and two values of relative humidity, observing that  i. inquinata tolerates a low relative humidity and high temperatures; in fact, high percentages of eggs hatched even at 34 °c at both relative humidities tested (35 and 70%  r.h.). with 35% and 70% r.h., at 29 and 34 °c, the hatching period was the same but  was shorter at 17, 21, 26 °c. at 17 °c, both at 35 and 70% r.h., a lower egg hatch and  longer hatching period was observed. a significantly higher number of hatched eggs was  observed at 26 and 29 °c at 70% r.h. this paper deals with eggs development and survival at different temperatures and  two humidity levels, tests were carried out in order to identify the thermal thresholds. the results are a contribution to the development of an integrated pest management  system in warehouses. materials and methods idaea inquinata has been reared continuously for 6 years, on an artificial diet1 in a  thermostatic chamber at 26±1°c, 70±5% r.h. and photoperiod of 16:8 (light:dark).  ten newly formed couples were isolated and the number of eggs layed by each female  was recorded.  groups of 100 eggs, were put in petri dishes (diameter 6 cm) at different temperatures  and relative humidities. specifically, 24-48 hours old eggs were used as more tolerant  to cold than newly layed eggs (bell, 1975). tests were carried out at 13, 15, and 36,  38±1°c with two levels of relative humidity (35 and 70±5%) and a photoperiod of 0:24  (light:dark). each combination of temperature and humidity was replicated four times.  egg hatching was observed daily. eggs were considered hatched when the young larvae  successfully chewed emergence holes in the chorion and left the egg shell. observation  were carried out daily until eggs appeared not viable. for each level of relative humidity, the data of this and of a previous research  summarized in the introduction section and published by limonta et al. (2010) were  submitted to anova and duncan’s multiple range test (spss 17.0 for windows and  microsoft excel 2003).  the model of brière et al. (1999) was used to predict the curvilinear relationship in  the entire temperature range permitting development  r(t ) = α t (t − tmin) (tmax − t )0.5. (1) (1) ingredients: 114 g bran, 61 g corn flour, 55 g wheat flour, 17 g wheat germ, 14 g dried yeast, 85 g  glycerine, 67 g honey. the diet was stored in polyethylene bags at 6 °c.  155l. limonta et al.: development and survivorship of i. inquinata eggs  the parameters tmin, tmax, α were estimated on the basis of our observations on  individuals via non-linear least square regression techniques implemented in the spss  statistics 17.00 software.  the stage-specific survivorships (ε) between the upper tmax and the lower tmin  thresholds for development is based on the beta function ε(t ) = a (t − tmin)b (tmax − t )c. (2) the values of tmin, tmax have been obtained from equation 1, while the parameters  a, b, c were estimated on the basis of our observations on individuals via non-linear  least square regression techniques implemented in the spss statistics 17.00 software.  results idaea inquinata eggs are pearly colored, with a netlike pattern, maximum width is  5/7 of length. when eggs are just laid they adhere by the poles building a short lived  coil, after a while they split.  the mean number of eggs laid by a female was 154.3±46.21 (sd), with range 76211, and a mean hatching of 82. 8% (72.81-90.65). table 1 mean number (sd) of eggs of idaea inquinata (scopoli) hatched at 13, 15, 17, 21, 26, 29, 34, 36, 38 and 40 °c, 35 and 70 % r.h. °c % r.h. 35 70 mean (sd) min-max mean (sd) min-max 13 0 0 15   9.5(1.29)a   8-11 25.2(2.75)a 22-28 17* 64.7(7.85)b 57-72 61.5(11.12)b 51-73 21* 83.5(3.87)c 79-88 73.7(7.27)c 63-79 26* 77.5(3.87)c 72-81 91.5(2.38)d 89-94 29* 78.5(3.51)c 75-82 91.0(6.48)d 84-97 34* 79.7(4.27)c 74-83 81.7(5.91)cd 73-86 36   8.7(5.85)a   2-15 15.5(10.78)a   4-30 38 0 0 35% r.h.: f5,18=199.12 p<0.001; 70% r.h.: f5,18=70.01 p<0.001 *data from limonta et al., 2010. eggs hatched in the temperature range 15-36 °c with both the tested value of relative  humidity (tab. 1). at 36 °c a limited number of eggs hatched and newly born larvae  died within few hours and a third of the larvae died when hatching from the egg. at 13  and 38 °c egg hatching was not observed.  at 15 °c the mean number of hatched eggs was 9.5±1.29 (sd) with 35% r.h., and  25.2±2.75 with 70%; at 36 °c the mean number of hatched eggs was 8.7±5.85 (sd)  with 35% r.h., and 15.5±5.91 with 70%. journal of entomological and acarological research, ser. ii, 42 (3), 2010156 figure  1  -  the  observed  and  predicted  developmental  rates  of  eggs  of  idaea inquinata (scopoli) at different temperatures with 35 (a) and 70% r.h. (b). (■▲: our data,  used  to  parametrize  the  developmental  rate  functions.  the  rates  are  predicted  by  r(t ) = α t (t − tmin) (tmax − t )0.5 with the stage specific parameters (α, tmin, tmax) reported in  table 2). a b 157l. limonta et al.: development and survivorship of i. inquinata eggs  figure 2 - the observed and predicted stage specific survival of eggs of  idaea inquinata (scopoli) at different temperatures. with 35 (a) and 70% r.h. (b). (■▲: our data,  used  to  parametrize  the  developmental  rate  functions.  the  survival  is  predicted  by   ε(t ) = a (t − tmin)b (tmax − t )c  with the stage specific parameters (a, tmin, tmax, b, c) reported in  table 2). a 6 journal of entomological and acarological research, ser. ii, 42 (3), 2010158 in fig. 1 the observed and predicted developmental rates of eggs of i. inquinata were  reported and the curves for both the values of relative humidity are similar regarding  the upper temperature of development (table 2) and the higher developmental rate (32  °c), while the lower temperature of development is lower with 70% r.h. brière et al.  (1999) model satisfactorily represented the developmental rates. in fig. 2 the observed and the predicted egg survival at the two values of relative  humidity are depicted. by visual examination, the survival appears to be better represented by equation 2 in fig. 2b than in fig. 2a.  discussion a higher mean number of hatched eggs of idaea inquinata (scopoli) was observed  with 70% r.h. at the threshold temperatures of 15 and 36 °c. in the case of ephestia kuehniella zeller, jacob & cox (1977) found that “humidity has little influence on egg  development and developmental periods increase only at very low relative humidities”.  eggs of i. inquinata did not hatch at 13 °c, and this lower threshold is similar to the  one of plodia interpunctella (hübner), that is 13.5 °c (savov, 1973). in the range 15-36  °c i. inquinata eggs hatched, as in ephestia cautella (walker) that complete embryonic  development within 14-36 °c (nawrot, 1979). the thermal thresholds for ephestia figulilella gregson and e. calidella (guenee) are within 15 and 36 °c with 70% rh.  corcyra cephalonica (stainton) eggs cannot survive out of the range 17.5-32.5 °c (cox  et al., 1981), while sitotroga cerealella oliver eggs hatch at 35 °c with different values  of r.h. (maity et al., 1999). in i. inquinata at 36 °c, a limited number of eggs hatched, and larvae do not survived. at 38 °c eggs collapsed, as in galleria mellonella l. where at 40 °c eggs dried,  became brownish and did not hatch (kumar et al., 2009). results of this study suggest that storing at 13 or at 38 °c can prevent the development of the rusty wave moth. the use of high temperatures is not applicable as the  properties of medicinal plants could be destroyed.  the equation 1 proposed by briére et al. (1999) and equation 2 satisfactorily represent  the developmental rates and the survival of eggs of i. inquinata at different temperatures.  the same result has been obtained for other insects life stages reported in the literature  table 2 parameter estimates and standard errors (se) for the developmental rate (eq. 1) and surivival rate (eq. 2) for idaea inquinata (scopoli) eggs (tmin = minimum temperature for development and survival, tmax = maximum temperature for development and survival). % r.h. n tmin [°c] (se) tmax [°c] (se) α (se) a (se) b (se) c (se) 35 400 11.01 (0.165) 38.65 (0.104) 0.0096 (0.0000015) 0.65 (0.87) 1.634 (0.317) 1.043 (0.212) 70 400 8.86 (0.174) 38.10 (0.069) 0.00008888 (0.0000) 0.77 (0.71) 1.644 (1.043) 0.993 (0.142) 159l. limonta et al.: development and survivorship of i. inquinata eggs  (e.g. bell, 1975; maity et al., 1999). in this paper different parameters have been found  for the to humidity regimes under study. the predicted lower developmental threshold  is lower at higher humidity, whereas the upper threshold appears to be similar at both  relative humidities. this indicate that i. inquinata eggs find more suitable conditions at  higher humidity than at lower values. here we assumed that thresholds for survival are equal to the thresholds obtained  for the developmental rates. this assumption may be justified by the scope of this work,  but should be revised if a more precise representation of the survival at temperature  extremes is required. this paper allows to make some tentative raccomandations on the management  of this pest. first, in order to prevent development of i. inquinata on medicinal plants,  they must be stored at temperature below 13 °c, a method economically sustainable. it  is important to consider the distribution of interstitial space in the plant species and the  part of the plant stocked as they influence the time necessary to obtain a uniform temperature in the commodity. second the relationships between developmental rates and  temperatures as well as humidities can be used as a component in a forecasting system. references bell c.h., 1975 - effects of temperature and humidity on development of four pyralid moth pests  of stored products. journal of stored products research, 11 (3/4): 167-175. brière j.f., pracros p., le roux a.y., priere j.s. 1999 - a novel rate model of temperature  dependent development for arthropods. environmental entomology 28: 22-29. candura, g.s., 1931a - studio sulla tignola del fieno (ptychopoda herbariata). (observations  on the rusty wave moth (ptychopoda herbariata)). bollettino di zoologia generale e agraria  di portici 24: 233-266. candura, g.s., 1931b - ricerche sugli insetti e sui danni da essi causati ai prodotti dell’economia  rurale o delle industrie agrarie. 2° contributo - gli insetti della camomilla secca e di altre  erbe medicinali e industriali disseccate. (researches on the insects and on their damage to  agricultural and industrial products. 2° part - insects of desiccated chamomile and medicinal  and industrial plants). bollettino della società naturalistica di napoli 43: 343-350. cox p.d., 1974 - the influence of temperature and humidity on life-cycles of ephestia figulilella  gregson and e. calidella (guenee) (lepidoptera: phycitidae). journal of stored products  research, 10 (1): 43-55. cox p.d., crawford l.a., gjestrud g., bell c.h., bowley c.r., 1981 - the influence of temperature  and humidity on the life cycle of corcyra cephalonica (stainton) (lepidoptera: pyralidae).  bulletin of entomological research 71: 171-181. jacob, t.a., cox, p.d., 1977 - the influence of temperature and humidity on the life-cycle of  ephestia kuehniella zeller (lepidoptera: pyralidae). journal of stored product research 13:  107-118. kumar y., kumar k., kaushik h.d., 2009 - effect of different temperature, relative humidity  levels and diet on incubation period and hatchability of galleria mellonella linn. eggs. annals of agri-bio research, 14 (1): 53-58. limonta l., stampini m., locatelli d.p., 2010 - egg hatching at different temperatures and  relative humidities in idaea inquinata (scopoli) (lepidoptera geometridae). in: fields, journal of entomological and acarological research, ser. ii, 42 (3), 2010160 p.g., adler, c.s., arthur, f.h., athanassiou, c.g., campbell, j.f., carvalho, o.m, fleurat-lessard, f., flinn, p.w., hodges, r.j., isikber, a.a. navarro, s., noyes, r.t., riudavets, j., sinha, k.k., thorpe, g.r., timlick, b.h., trematerra, p., white, n.d.g. (eds.), proceedings of the tenth international working conference of stored product protection, 27 june-2 july 2010, estoril, portugal, julius-kãnhn institut, berlin, germany, 147-149. locatelli, d.p., di egidio, v., stampini, m., 2005 - observations of the development of idaea inquinata (scop.) (lepidoptera geometridae) on medicinal plants and other food substrates.  bollettino di zoologia agraria e bachicoltura, serie ii 37 (2): 123-132. maity b.k., tripathi m.k., panda h.k., 1999 - effect of temperature and relative humidity on the  life history of angoumois grain moth, sitotroga cerealella oliv., (gelechiidae: lepidoptera).  environment & ecology, 17 (2): 471-473. navrot j., 1979 - effect of temperature and relative humidity on population parameters for  almond moth (cadra cautella wlk.) (lep. phycitidae). prace naukowe instytutu ochrony  roslin, 21 (2): 41-52. savov d., 1973 - development of plodia interpunctella hb. (lepidoptera, pyralidae) in the  optimum temperature range. gradinarska i lozarska nauka, 10 (5): 33-40. lidia limonta, juljus sulo, daria patrizia locatelli - dipartimento di protezione dei sistemi  agroalimentare e urbano e valorizzazione delle biodiversità- dipsa, università degli studi  di milano, via celoria 2, 20133 milano - italy. e-mail: lidia.limonta@unimi.it accepted 20 december 2010 j. ent. acar. res. ser. ii, 42 (3): 185-188 30 december 2010 l. süss - m. costanzi presence of drosophila suzukii (matsumura, 1931) (diptera drosophilidae) in liguria (italy) abstract - the presence of drosophila suzukii in liguria (italy) on strawberries  and raspberries is reported.  riassunto - presenza di drosophila suzukii (matsumura, 1931) (diptera drosophilidae) in liguria (italia). viene segnalata la presenza di drosophila suzukii in liguria, su coltivazioni di  fragola e lampone. key words: drosophila suzukii, alien insects, strawberry pest, raspberry pest, new  presence, spotted wing drosophila. in fall 2010 was noticed a heavy infestation on strawberries in a greenhouse and on  raspberry in fields, near savona (liguria, italy); the attacks were produced by larvae of  a diptera, with activity in the fruits close to ripening. after the breeding in the laboratory, from the fruits came some adults of drosophila suzukii (matsumura, 1931), (spotted wing drosophila), a species from extreme orients,  but recently signaled both in north america and in europe as noxious on strawberries  (fragaria spp.), raspberries (rubus idaeus) and other rubus spp., blueberries (vaccinium  spp.), sweet cherries (prunus avium), plums (p. domestica) and many others fruit crops  (kiwis, persimmons, peaches, table and wine grapes) (eppo quarantine alert, 2010). in the area near savona some sporadic attacks were ascertained in the year 2009,  but for the farmer there weren’t important (pers. comm.). in the year 2010 the presence of this diptera increases, with 30%-40% damages on  strawberries until 80% in the period of the most important attack. the most infested cultivar was mara de bois, a strawberries very fragrant, but with  the increasing of the attack, the cultivars annabel, diamante and sant’andrea were  damaged too. the top of the infestation was in september; the presence of drosophila suzukii  was reduced in fall. the spotted wing drosophila (swd) belongs, at systematic level, to the melanogaster group, suzukii subgroup. journal of entomological and acarological research, ser. ii, 42 (3), 2010186 okada (1954) studied the general structures of the fallic organs of the melanogaster  group. in the case of d. suzukii the male genitalia are characterized by “aedeagus apparently bifid, pubescent ventroapically. anterior paramere (forcep) broad, abruptly narrowing near apex and with a few subapical sensilla. posterior paramere (stylus)  dorsobasally dilated and pubescent. ventral fragma triangular. novasternum (9th sternite, or hypandrium) with low lateral processes, paired fine spines and a small median projection instead of a medium notch” (figure 1). the female can put the eggs in the fruits, close to the ripening, because the ovipositor  is characterized on the very long valvae, by a series of strong stings and another inner  triple row of teeth (figure 2).  the damages on the fruits at the beginning appears as a light depression; the fruit  collapse around the feeding site; secondary fungal or bacterial infection may contribute  to the total destruction of the fruits. in italy, the swd was first found in the province of trento (grassi et al., 2009).  the authors provides detailed description of this pest, the world distribution, biology  and damages. consequently to the ascertained infestations in particular in usa, starting from  2008, and to the damages in trento province in 2009, this insect was added to the alert  list (eppo rs 2010/007). figure 1 - drosophila suzukii: male genitalia. 187l. süss, m. costanzi: drosophila suzukii in liguria figure 2 - drosophila suzukii: female genitalia. in 2010 it was found in tuscany region in occasion of faunistic studies without  causing damages (eppo rs 2010/112). the diffusion in trentino-alto adige region  was confirmed (eppo rs 2010/178); since 2010 the swd has been found in france,  in particular on cherry in corse, on strawberries in alps-maritimes and var and in other  regions, on various fruit crops (eppo rs 2010/111; eppo rs 2010/179). the short life cycle (8-15 days, depending of the climatic conditions), the high  number of generation per year (in japan this insect complete about 13 generations per  year) (grassi et al., 2009), the damages on fruits of particular value render very worried  the progressive diffusion of d. suzukii in italy. at the consequence, studies on the possibility to get ready suitable strategies of pest control are necessary. references okada t., 1954 - comparative morphology of the drosophilid flies i. kontyû, 22, nos.1/2: 36-48. grassi a., palmieri l., giongo l., 2009 - drosophila (sophophora) suzukii (matsumura). nuovo  fitofago per i piccoli frutti in trentino. terra trentina, 10/2009: 19-23. eppo quarantine alert, 2010. http://www.eppo.org/quarantine/alert_list/insects/drosophila_suzukii.htm journal of entomological and acarological research, ser. ii, 42 (3), 2010188 eppo rs n. 1, 2010/007. http://archives.eppo.org/epporeporting/reporting_archives.htm eppo rs n. 6, 2010/111. http://archives.eppo.org/epporeporting/reporting_archives.htm eppo rs n. 6, 2010/112. http://archives.eppo.org/epporeporting/reporting_archives.htm eppo rs n. 10, 2010/178. http://archives.eppo.org/epporeporting/reporting_archives.htm eppo rs n. 10, 2010/179. http://archives.eppo.org/epporeporting/reporting_archives.htm luciano süss - dipartimento di protezione dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (dipsa), università degli studi di milano, via g. celoria 2, i-20133,  milano. e-mail: luciano.suss@unimi.it mariella costanzi - istituto regionale per la floricoltura (irf), via carducci 12, i-18038,  sanremo (im). e- mail: costanzi@regflor.it  accepted 20 december 2010 j. ent. acar. res. ser. ii, 42 (2): 103-116 30 august 2010 z. p. gama, p. morlacchi, a. giorgi, g. c. lozzia,  j. baumgärtner towards a better understanding of the dynamics of aphis spiraecola patch (homoptera: aphididae) populations in commercial alpine yarrow fields  abstract - the spatial distribution of aphis spiraecola patch was studied in two  commercial yarrow fields located in the swiss and italian alps and represented by  taylor’s (1961) power law. the respective parameters indicate a highly aggregated  distribution and lead to a high optimum sample size of 400-500 plants in the design  of a sampling program. opportunities for reducing the sampling efforts are discussed.  the infestation patterns were studied on the basis of vansickle’s (1977) time varying  distributed delay adequate for modelling the dynamics of age-structured populations.  published literature data were used to parametrize the functions representing the  temperature-dependent duration and survival of the nymphal and adult stage.  likewise, literature data were available to obtain reliable estimates for the parameters  of the fecundity function comprising the reproductive profile and the number of  nymphs produced at different temperatures. the field data were used to parametrize  the functions for wing formation and a compound mortality compromising the effects  of plant senescence, stem cutting and natural enemies. the model satisfactorily  represented the observed infestation patterns. however, there are opportunities for  improving parameter estimation and validation. moreover, the separation of the  compound mortality into host plant and natural enemy effects would improve the  mechanistic basis of the model and lead towards a tool that could be used to study  bottom-up and top-down effects in the yarrow-aphid-natural enemy system. riassunto verso una migliore comprensione della dinamica di popolazioni di aphis spiraecola patch (homoptera: aphididae) in campi commerciali alpini di  achillea. è stata studiata la distribuzione spaziale di aphis spiraecola patch in due campi  coltivati di achillea situati sulle alpi italiane e svizzere; la distribuzione spaziale  della specie è stata descritta dalla legge della potenza di taylor (1961). i parametri  specifici indicano una distribuzione spaziale fortemente aggregata e nel contesto  di un programma di campionamento portano al calcolo di un’elevata dimensione  ottimale del campione, pari a 400-500 piante. le possibilità per una riduzione  dell’impegno per il campionamento vengono discusse. journal of entomological and acarological research, ser. ii, 42 (2), 2010104 gli schemi di infestazione sono stati studiati sulla base del modello a ritardo distribuito  a tempo variabile di vansickle (1977), adatto per le dinamiche di popolazioni  strutturate per età. i dati di letteratura sono stati usati per la parametrizzazione delle  funzioni che descrivono la durata e la sopravvivenza temperatura-specifiche degli  stadi di sviluppo preimmaginali e immaginali. allo stesso modo, i dati di letteratura  sono stati impiegati per ottenere stime affidabili per i parametri della funzione per la  riproduzione, che comprende il profilo riproduttivo e il numero di neanidi prodotte  a differenti temperature. i dati di campo sono stati utilizzati nella parametrizzazione  delle funzioni formazione degli individui alati e mortalità complessiva, composta  dai fattori senescenza della pianta, taglio del culmo e nemici naturali. il modello  descrive in modo soddisfacente gli schemi di infestazione osservati. tuttavia esistono  possibilità per il miglioramento della stima dei parametri e della validazione. inoltre,  la separazione della mortalità complessiva nelle componenti pianta ospite e nemici  naturali migliorerebbe la base meccanicistica del modello e indirizzerebbe verso uno  strumento che potrebbe essere usato nell’analisi degli effetti bottom-up e top-down  nel sistema achillea-afidi-nemici naturali. key words: aphis spiraecola, achillea collina, spatial distribution, sampling plan,  infestation pattern, delay model, parameter estimation, model validation  introduction yarrow (achillea collina becker ex rchb.) is cultivated for commercial purposes  in the european alps. of interest in human medicine is the high content of secondary  metabolites, i.e. organic compounds that are not directly involved in the normal growth,  development, or reproduction of organisms (fraenkel, 1959; wink, 2003; madeo et al.,  2009). the aqueous and alcoholic extracts have digestive, antiphlogistic, spasmolytic,  stomachic, carminative, and estrogenic properties (benedek et al., 2007). in two fields  located in the southern italian and swiss alps, morlacchi et al. (2010) studied crop  yield formation and recorded two insect communities of possible economic importance.  the first community, not studied in this paper, consists of three leaf eating chrysomelids  (galeruca tanaceti l., chrysolina marginata marginata l., cassida spp.) and their  natural enemies. the second community comprises three phloem feeding aphid species  (macrosiphoniella millefolli degeer, aphis spiraecola patch and coloradoa achilleae hille ris lambers), their parasites and the predator coccinella septempunctata l. the  populations of these aphids occurred in sufficiently high numbers as to possibly reduce the  yield in terms of biomass on one hand and increase the contents of secondary metabolites  on the other hand (madeo et al., 2009). an adequate knowledge on the spatio-temporal  dynamics of the aphid populations is indispensable for the design of an integrated pest  and crop management system (gutierrez and baumgärtner, 2007).  the spirea aphid a. spiraecola of interest in this paper is a polyphagous species  of far eastern origin with a worldwide distribution (wang and tsai, 2000). it is a pest  of citrus, apples and ornamentals, and transmits a number of plant viruses (wang and  105z. penata gama et al.: dynamics of a. spiraecola in commercial alpine yarrow fields  tsai, 2000). for supervised control purposes, hermoso de mendoza et al. (2006) defined  intervention thresholds in spanish citrus orchards. for rationalizing control, hong et al. (2003) identified the sex pheromone and studied the circadian rhythm in release.  the density-dependent effect of predators on a. spiraecola in apple orchards (brown,  2004) stimulated attempts to manage a. spiraecola populations by enhancing biological  control (brown and matthews, 2008). in a classical biological control effort, the aphelinid  aphelinus gossypii timberlake was introduced into florida to control a. spiraecola on  citrus (hoy and ru, 2008).  the purpose of this paper is to describe the spatial distributions, to design sampling  plans, and to analyze, via the development of mechanistic population models, the  temporal infestation patterns of. a. spiraecola in commercial alpine yarrow fields. the  model parameters are estimated on the basis of published life table data (wang and  tsai, 2000), the assumed formation of winged morphs (holst and ruggle, 1997) and the  observed natural enemy presence (morlacchi et al., 2010). the design of sampling plans  and the analysis of infestation patterns should provide indications for obtaining reliable  density estimates for model validation and population management purposes, and for  improving the population model with respect to its mechanistic basis.  material and methods study sites and population sampling for the study, we selected two commercial yarrow fields located in the southern  alps (poschiavo, canton of the grisons, switzerland, 1140 m asl, and dazio, sondrio  province, italy, 900 m asl). at both locations, the farmers planted the variety ‘spak’,  selected by valplantons bio (saillon, switzerland) for a high content of secondary  metabolites (morlacchi et al., 2010). at the time of the study (2007, 2008), the plants  were several years old and grown on black plastic mulch at a 0.5 m x 0.5 m spacing. at  poschiavo, the plants were harvested on july 25 (2007) and july 30 (2008), while the  dazio grower renounced on cutting the plants during the year of observation (2008).  this study deals with the dynamics of a. spiraecola populations inhabiting the two fields  between the beginnings of april to the ends of july.  the fields at poschiavo and dazio were divided into 9 and 7 strata, respectively. in  each of the strata, the beating tray method was applied to 3 randomly selected plants to  obtain the number of a. spiraecola mummies of parasitized a. spiraecola, and coccinellid  larvae and adults (morlacchi et al., 2010). in the relatively small commercial yarrow  fields, destructive sampling was not possible.  in 2007 and 2008, the poschiavo field was visited 8 times (april 24, may 9, may 25,  june 8, june 22, july 8, july 26, september 5) and twice (june 3, june 24), respectively.  in 2008, the dazio field was visited three times (june 3, june 24 and july 21). since  morlacchi et al. (2010) did not find a significant difference between strata, the samples  are treated as simple random samples taken from a homogenous sampling universe.  journal of entomological and acarological research, ser. ii, 42 (2), 2010106 spatial distributions and optimum sample size the sampling program provided the means, variances and standard errors of 13  samples. the ratio of the standard error to the mean is used to assess the reliability of  the estimates. the spatial distribution of a. spiraecola was described by taylor’s (1961) power  law that expresses the variance (s2) in relation to the mean (m) by .                [1] to obtain estimates for a and b through least square linear regression techniques, equation [1] was changed into ln(s2)=ln(a) +b ln(m). the optimum sample size is the smallest number n of sample units that satisfies the  objectives of the sampling program and achieves the desired precision of the estimate. for  calculating n, we defined the reliability in terms of formal probabilistic statements with  the length d of the confidence interval equal to a proportion of the mean (karandinos,  1976). the consideration of equation [1] yields the optimum sample size n 2 2 2/ −⎟ ⎠ ⎞ ⎜ ⎝ ⎛ = bma d z n α              [2] where zα/2 is the upper α/2 point of the standard normal distribution. the definition of the  optimum sample size n depends on the objective of the sampling program (karandinos,  1974). the values of zα/2= 1.65 and d = 0.3 reflect a high end of a range that is considered  reasonable for pest management purposes (hutchison et al., 1988).  basic model if the variability in developmental time is high relative the mean developmental time,  a stochastic model may be appropriate (di cola et al., 1999). in this work, we use the  time varying distributed delay of vansickle (1977) to model the development of both the  nymphal and adult cohorts. however, we limit the description of the model to the basic  elements only and refer the reader to the recent examples of gutierrez (1996), holst and  ruggle (1997), alilla et al., 2005, severini et al. (2009), gutierrez and baumgärtner  (2007) and limonta et al. (2009a;b) for additional explanations and applications. briefly,  ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ ++−= − dt tdeld k tdel tartrtr tdel k dt tdr ii i )()()(1)()( )( )( 1           [3a]   i=1,2….k  where t = time [days], ri(t) = the transition rate of the i-th sub-stage, k = number of delay  sub-stages, del(t) = time dependent developmental time [days] in absence of losses, and  ar(t) = time dependent proportional losses or attrition. the output rk(t) of the nymphal  107z. penata gama et al.: dynamics of a. spiraecola in commercial alpine yarrow fields  stage becomes the input x(t) into the adult stage. for constant temperatures and a cohort  input x(t) into the first sub-stage, vansickle (1997) describes the procedures for obtaining  estimates for the parameters k, del(k) and ar(t) as follows. 2 2 s k μ =     [3b] ⎟ ⎠ ⎞ ⎜ ⎝ ⎛ − = kdel 1 εμ [3c] ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ −= del kar 11 μ     [3d] where μ is the observed developmental time with s2 = variance, and ε = stage-specific  survival. the input into the larval stage corresponds to the below described fecundity  rate, while input into the adult stage corresponds to the output of the larval stage modified  by the proportion of emigrating winged aphids. the survival of larvae depends on  temperature and the compound effect of predation, parasitism, plant senescence and  cutting. these model components are described in the next section. for simulation  purposes, we select a time increment of 1h for which the mean temperature is calculated  on the basis of a cosine function fitted through the daily temperature maxima and minima  (bianchi et al., 1990). model components the developmental rate of nymphs and adults is represented by the model of brière  et al. (1999) ( )βαμ tttttt ul −−= )()( [4] where the tl and tu = the lower and upper thresholds for development, α and β =  parameters. while tu has been estimated from experiments and β has been set to 0.5  (see brière et al. 1999), tl and α have been estimated via linear least square regression  techniques applied to the data of wang and tsai (2000). the intrinsic survival e of larvae  only is represented on the basis of a beta function ( ) ( ) ( ) ςξ λε ttttt ul −−=     [5] whose parameters λ , ξ , and ς were estimated by applying linear least square regression  techniques to the data reported by wang and tsai (2000). the values are reported in tab.  1. following curry and feldman (1987), the reproduction is based on the reproductive  profile fi, i.e. the normalized age-specific fecundity rate in the i-th sub-stage, and the  temperature-dependent total fecundity f(t)  journal of entomological and acarological research, ser. ii, 42 (2), 2010108   [6]   [7] the parameters τ, υ ø φ and ι are estimated by applying linear least square regression  techniques to the data reported by wang and tsai (2000), while r corresponds to the  total per capita fecundity realized over the k sub-stages. the product of eqs. 6 and 7  multiplied by the i-th transition rate of equation 3a and the time step length (1/24) yields  the fecundity of the i-th age group per time step with temperature t. the reproduction  of the adults, becoming the input x(t) into sub-stage 1 of equation 3a, in all age groups,  is obtained by summing the fecundities realized in all sub-stages (i=1,2,..k). curry and  feldman (1987) provide further details on modelling reproduction under time-varying  temperature conditions. the proportion w(n) of winged aphid depending on aphid density (n) is derived  from holst and ruggle (1997) ( ) ων +−+ = ne nw ln1 1 )(   [8] according to the field observations reported in fig. 1, the proportion w(n) may be  low (0.05) and high (0.99) at densities n = 20 and n = 40, respectively. this tentative  interpretation allows the calculation of the parameters ν and ω reported in tab. 2. as  indicated above, the input x(t) into the adult stage is modified by (1-w(n)). the physiological time-dependent compound effect v(τ) of natural enemies, plant  senescence and cutting is represented by  ( ) ψτρτ +−+ = ln1 1 )( e v   [9] where τ = physiological time in day-degrees (dd) above the lower developmental  threshold tu.. the field observations reported in fig. 1 suggest a small value for v(τ) =  0.05 at τ = 1200 dd and high value v(τ) = 0.99 at τ =1600 dd. this tentative interpretation  allows the calculation of the parameters ρ and ψ reported in tab.1. the product of the  stage specific survival of nymphs (equation 5) and (1-v(τ)) for both nymphs and adults  is the basis for the calculation of attrition ar(t) according to equation 3d. model validation the validation procedures consist of testing the model capabilities with respect to  its intended use (rykiel, 1996) which is the mechanistic representation of infestation  patterns. the predictions of the model are compared to the observations made in 2007  109z. penata gama et al.: dynamics of a. spiraecola in commercial alpine yarrow fields  and 2008 in the poschiavo yarrow field. this field may receive higher radiation levels and  is located at a lower altitude than robbi-poschiavo where the temperature was recorded.  moreover, the field is situated near a lake with presumably mitigating effects on low  temperatures. to take into account these observations, the daily temperature maxima and  minima recorded at robbi-poschiavo were increased by 1.0 °c, and the resulting values  were used to calculate the hourly mean temperatures. taking into account the observed  infestations, the simulation tentatively starts with a density of 5 medium age nymphs in  sub-stage i = 35 on day 100 corresponding to 391 daydegrees after january 1st.  the infestation patterns was simulated first with intrinsic parameters only, i.e. in  absence of the effect of wing formation and compound mortalities. second, the infestation  pattern was simulated with intrinsic parameters and wing formation but absence of  compound mortality effects. third, the infestation patterns were simulated with intrinsic  parameters, wing formation and compound mortality.  results fig. 1 shows the logarithm of the variance plotted against the logarithm of the  mean density for each sample. the parameters a = exp(2.6552) and b = 1.926 reported  in tab. 1 indicate, for the sampling method used here, a highly aggregated distribution  of a. spiraecola. fig. 1. the relationship between ln (variance) and ln (mean) density of aphis spiraecola sampled  in two alpine yarrow fields. journal of entomological and acarological research, ser. ii, 42 (2), 2010110 fig. 2 shows the optimum sample size, i.e the number of pants to be sampled for  estimating the densities with zα/2 = 1.65 and d = 0.3. accordingly, to obtain reliable  density estimates for research purposes at low densities, the high number of about 500  plants needs to be sampled. this requires high investments into sampling studies and  relatively big fields to facilitate random sampling. a high proportion of samples may  require finite population corrections in statistical analyses (cochran, 1977). to obtain  reliable estimates for population management purposes, the optimum sample size can  be reduced to the still high number of 400 plants. fig. 2. the optimum number of plants required for estimating the density of aphis spiraecola with a predefined level of reliability (the standard normal variate zα/2 = 1.65, and the ratio of the  standard error to the mean d = 0.3). tab. 1 lists the parameter estimates for taylor’s (1961) spatial distribution model  (a,b, equation 1), for the order of the vansickle’s (1977) delay (k, equation 3), for the  developmental rate according to brière et al. (1999) (α, β, tl , tu, equation 4), and for  the stage-specific intrinsic survival (λ, ξ, ς, equation 5). while the spatial distribution  is described for the combined densities of adults and nymphs, the two life stages are  separated in the study on aphid infestations. consequently, the order of the delay and  the developmental rate parameters differ between nymphs and adults. only nymphs  suffer from intrinsic mortalities, while adult survivorship is controlled by the order of  the delay. the order k=13 satisfactorily yields the observed adult survivorship in the  data of wang and tsai (2000). noteworthy is the relatively low developmental threshold  obtained for both life stages. 111z. penata gama et al.: dynamics of a. spiraecola in commercial alpine yarrow fields  table 1. estimates for the parameters of taylor’s (1961) spatial distribution model (a,b, equation 1), for the order of the delay (k, equation 3), for the developmental rate (α, β, tl , tu, equation 4), and for the stage-specific intrinsic survival (λ, ξ, ς, equation 5) of aphis spiraecola.  life stages spatial distribution delay order developmental rates intrinsic survival a b k α β tl tu λ ξ ς nymphs 14.228 1.926 71 7.97e-05 0.5 2.3 35.0 0.015 0.782 0.792 adults 13 5.09e-04 0.5 2.3 35.0 tab. 2 lists the parameter estimates for the reproductive profile, the fecundity, the  wing formation and the compound mortality function. the data provided by wang and  tsai (2000) were sufficient to obtain satisfactory estimates for the former two functions,  while holst and ruggle (1997) provided only the basic model for density-dependent wing  formation. the parameters of this function as well as the parameters of the compound  mortality function were obtained by comparing model predictions with observed  infestation patterns. noteworthy, the compound effect depends on physiological time  rather than density. hence, the model disregards possible density-dependent effects of  natural enemies and the host plant on a. spiraecola populations. tab. 2. parameter estimates for the reproductive profile (τ, υ, r, equation 6), for the temperaturedependent fecundity (ø, φ, ι, equation 7), for the density dependent wing formation (ν, ω, equation 8), and for the physiological time-dependent stage-specific compound mortality of aphis spiraecola  (ρ, ψ, equation 9). life stages reproductive profile fecundity wing formation compound mortality τ υ r ø φ ι ν ω ρ ψ nymphs 15.8274 112.2685 adults 7.97 1.27 42.37 0.000556 2.335 1.694 6.3677 26.4214 15.8274 112.2685 the infestation patterns of a. spiraecola in the poschiavo yarrow field is depicted in  fig. 3. accordingly, a. spiraecola increases after the beginning of april until july and  decreases thereafter to low numbers. morlacchi et al. (2010) observed a second peak in  september which is not considered in this work. in all samples, the ratio of the standard  error to the mean high was (from 0.31 to 0.94) indicating a low level of reliability of  the density estimates. nevertheless, the low reliability is considered as sufficient for  validating the predicted infestation patterns. as expected, the disregard of wing formation and compound mortality predicts an ever  increasing population during the time under study (fig. 3). the slow increase is due to  the relatively low temperatures of the alpine environment. the density-dependent wing  formation stabilizes the population in summer. as previously mentioned, the population  suffers in summer from losses due to natural enemies, plant senescence and cutting  journal of entomological and acarological research, ser. ii, 42 (2), 2010112 (morlacchi et al., 2010). only the consideration of the physiological-time dependent  compound mortality reduces the population to low levels in mid summer (fig. 3).    fig. 3. simulated and observed infestation patterns of aphis spiraecola in the poschiavo yarrow  field (triangles for 2007 data, quadrats for 2008 data). the line indicates the simulated patterns  in absence of wing formation and compound mortalities, the dots indicate the simulated patterns  without compound effect of mortalities, the dashed line indicates the simulated pattern with wing  formation and compound mortalities (the density of 119.81 aphids on may 10, 2007, is omitted). discussion the parameters of taylor’s power law represented in equation 1 indicate a highly  aggregated distribution among plants. the relatively big physical size of the sampling  unit (plant) may have contributed to the relatively high sampling factor of a = 14.2278.  possibly, the selection of a whole plant sample unit rather than the consideration of plant  parts is responsible for this result (morlacchi et al., 2010). according to the enumerative sampling plan, the optimum sample size consists of  400 – 500 plans. this indicates that the sample size of 27 plants used in this work, albeit  satisfactory for monitoring infestation patterns, is too low for estimating population  densities in analyses of the population dynamics. the optimum sample size, resulting  from the high values of the parameters of taylor’s law (1961), requires considerable  investments in sampling activities. however, there are possibilities for reducing the efforts  even if non-destructive sampling is required. apart from a revision of the reliability levels,  there are three opportunities for making sampling more efficient. first, it may be possible  to rely on visual examinations rather than on the beating tray technique. second, during  the reproductive growth phase, the design of a two-stage sampling plan with plants as  113z. penata gama et al.: dynamics of a. spiraecola in commercial alpine yarrow fields  primary and stems as secondary sampling units may be feasible (e.g. cochran, 1977).  third, the substitution of enumerative by binomial sampling plans may also reduce the  investments into sampling activities (morlacchi et al., 2010). in absence of wing formation and compound effects of the host plant, cutting and  natural enemy activity, the model predicts slowly increasing aphid densities (fig. 3).  the slow increase may be due to the low temperatures in the alpine environment. at the  beginning of the infestation, higher temperatures as expected in apple and citrus growing  areas would produce a higher increase. the model parameters have been estimated from  life table data obtained on citrus with a florida aphid biotype (wang and tsai, 2000).  in a subsequent paper, tsai and wang (2001) demonstrated how different host plants  affect the life table statistics of a. spiraecola. to put parameter estimates on a more  solid ground than done in this paper, we recommend the construction of life tables for  the alpine biotype of a. spiraecola on the spak yarrow cultivar.   in absence of wing formation and compound effects of the host plant, cutting and  natural enemy activity, the model was parametrized with the data of wang and tsai  (2000) and visually compared with an independent data set. the consideration of wing  formation and compound mortality effects, however, was only possible by using the same  field data for both parameter estimation and model testing. to overcome this limitation,  more field data, preferably taken in different environments, should be collected and used  for model validation against independent data sets. additional field and laboratory data  are also required for creating a more solid ground for the formulation of wing formation  and for extending the model towards the development of other morphs than winged and  wingless individuals, and towards the development of overwintering eggs. this would  allow a more satisfactory initialization of the model than done here. the compound mortality consists of the combined effect of natural enemies, plant  senescence and cutting (morlacchi et al., 2010). the separation of the compound effect  into different components would undoubtedly improve the mechanistic basis of the  model. a comparison between the poschiavo field, where the plants have been harvested,  and the dazio field, where the grower renounced on harvesting in the year under study,  indicates that cutting strongly affects the aphid dynamics. additional field studies on  the effects of cutting on aphid survival and the on-going studies on the interactions  between aphids and the yarrow host plant (madeo et al., 2009), on one hand, hold the  promise for separating plant effects from predation in further model development. the  on-going studies on the interactions between aphids and natural enemies (morlacchi et al., 2010), on the other hand, could lead to a separation of biological control from plant  effects. brown and matthews (2008) remind us that natural enemy activity is important  in some but not all studies on aphid population dynamics. finally, the resulting model  could be used to study bottom-up and top-down effects in the yarrow-aphid-natural  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phytochemistry, 64: 3-19. zulfaidah penata gama, m.si., biology department, mipa faculty, brawijaya university, jl.  veteran, malang 65145, indonesia. pablo morlacchi, giuseppe carlo lozzia, johann baumgärtner, dipartimento di protezione  dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (d.i.p.s.a.),  università degli studi di milano, via celoria 2, 20133 milano, italy. anna giorgi, dipartimento di produzione vegetale (di.pro.ve.), università degli studi di milano,  via celoria 2, 20133 milano, italy. accepted 27 august 2010 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd f. palla, l. sineo, b. manachini bacteria, fungi and arthropod pests collected on modern human mummies abstract a survey of opportunistic biocenosis (macro and micro organisms) associated with a rest of human mummy samples was carried out to characterise the biocenosis and to detect the potential of biodeteriogens. the rests of the human modern mummies come from a hypogeic site. since mummies are relevant from a historic-artistic-scientific point of view, an aspect of this study was the identification and characterization of the biological systems related with biodeterioration of organic matter. in a first step, different sampling methods, according to the taxa, were applied. technological procedures were combined in order to have an interdisciplinary approach to the conservation actions for testing future restoration protocols. specimens were collected, identified and characterized by microscopy (light, sem, clsm) and molecular analyses (dna extraction, in vitro target sequence amplification, sequencing, sequence analysis). the results highlight a rather complex biocenonsis consisting of fungi, cyanobacteria, several insects and other arthropods. key words: biodeteriogens, biocenosis, conservation biology, hypogeic site. introduction in recent years, biomedical technologies have led to a great increase of knowledge concerning the study of human remain collections, as mummies, and their associated materials (sineo et al., 2008). both mummies and skeletons can yield a huge amount of biological information. since they represent cultural assets with relevant historicartistic-scientific interest (favole, 2003), an aspect of this study is the identification and characterization of the biological systems related with biodeterioration of organic matter. the detection of opportunistic biocenosis and the identification of macro and micro biodeteriogens are essential for an optimal conservation. biodeterioration involves different biological systems (especially microbial and insect species), representing a relevant problem in conservation of cultural assets (valentin, 2003a; valentin, 2003b; jurado et al., 2008). in this study we integrate the observations of the microbial composition with arthropods affecting the conservation of modern mummies. assessing the composition of microorganisms and of the fauna communities represents the first step in understanding and controlling colonization and infestation. fungi, bacteria, cyanobacteria and related microrganisms, can be found on and beneath surfaces of j. ent. acarol. res. ser. ii, 43 (2): 69-76 30 september 2011 cultural assets in indoor and outdoor environments in relation to environmental factors and food sources (palla et al., 2010). after colonization, microbes can initiate biodeterioration processes or they can continue to live at a low metabolic activity rate, in a biofilm. moreover, many insects and other animals can be found in association with human mummified bodies or conservation environments (smith, 1986; sukontason et al., 2006). for this reason we focus the attention on bacteria, fungi and entomofauna associated with the mummies inhabiting catacombs or other hypogeic sites. in the literature, many studies were conducted on the sequence of arthropods that colonize exposed corpses (bourel et al., 2004). however, some defined environmental conditions such as hypogeic site have been poorly studied. catacombs are particular hypogean environments which have a partial connection with the outdoor and the soil that may influence the characteristics of the biocenosis (saarela et al., 2004). our goal is to investigate the biocenosis associated with mummies and indicate the potential biodeteriogens currently or previously present. materials and methods sampling we pay attention on micro-organisms and macro-organisms found in hypogean environments, colonizing both mummy surfaces and aerosol. microbial biofilms were sampled by sterilized swabs while arthropod rests were collected by a rigger brush. identification identification was performed using different procedures according to the different taxa: light microscopy: samples were prepared for observation under dlmb leica (wetzlar, d, magnitude 400-1200 x). scanning electron microscopy (sem): samples were coated with approximately 13 nanometers of gold by agar-auto-spotter-coater (b7341) and observed under high vacuum leica cambridge-leo 420. confocal laser scanning microscopy (clsm): since some bacterial colonies, especially cyanobacteria, are autofluorescent we performed the observations by clsm (olympus fv 300) equipped with argon (488 nm) and helium-neon (543 nm) lasers, together with calibrated emission filters. molecular dna analysis: total microbial dna, directly extracted from surface and aerosol samples, was used as template in pcr reactions. the target dna sequences were localised both on specific 18s (fungi) or 16s (bacteria) rna locus and on 18s26s (fungi) or 16s-23s (bacteria) intergenic transcribed spacer (its). the sequence of amplified fragments were determined and analysed to elicit the prevalent microbial species in biofilm and bio-aerosol. comparison between fragment sequences and bacterial genomic dna deposited in genebank, was carried out by dedicated software (blast, clustalw, treew). journal of entomological and acarological research, ser. ii, 43 (2), 201170 results and discussion analysis of mummy and debris of the bonds showed different evidences of deterioration: huge microbiological colonization (bacteria, fungi), tiny holes in the bonds and brownish dusts (indicators of insect activity). carcasses and fecal pellets of the insects were mixed with dusts and debris. the presence of insects (fig. 1, a) and microorganism colonization can indicate also the environmental conditions of the process and the estate of the mummy. particularly, we focused on samples from crania that showed a quite complex biocenosis characterised by the presence of several species of insects. levinson & levinson (1994) reported that a complicate biocenosis was recorded in the ancient egypt tombs and mummies, but some of insects were associated with the food f. palla et al.: bacteria, fungi and arthropods pests collected on human mummies fig. 1 (a) rests of insects found in the crania debris of human mummy. (b) puparium of phoridae specimen with the typical respiratory horns. present in the tombs. in our study we recorded many taxa of insects (at least six were non accidental species). parts of adult arthropods (head, tibia, claw and pre-claw, thorax, femur) have been found, mainly belonging to coleoptera curculionidae, cleridae, latridiidae and ptinidae. also a golden brown chela of pseudoscorpion (pseudoscorpionida) was recorded. pseudoscorpions are found under rocks, or in caves, and use their pinchers to prey upon micro-arthropods. the arthropods collected are reported in tab. 1. different mites and dipteran puparia were also recorded (fig. 1, b). most of the puparia were empty: 4 entire and many rests. since anterior rupture, due to adult emergence, was circular they belong to diptera cyclorrhapha. a number of characters helped the identification: the overall structure of puparia is ovoidal, two big respiratory horns, with several holes and a trabecolar struca b 71 tab. 1 arthropods collected in 2.5 grams of human mummy cranial debris. n: certain number of individuals. ture are present on the dorsum, at the end of 5th segment. altogether, these morphological features allowed us to identify these specimens as belonging to the phoridae, the specific identification is possible only by the adults (liu & greenberg, 1989), as taxonomy is based upon the female ovipositor. nevertheless, phorid puparia morphology is rather uniform, so examination under sem (in progress) could reveal details useful for identification at specie level (sukontason et al., 2006). a complete identification could be important to define their role in mummy conservation. phorids are known as coffin flies, because they live in human corpses with such tenacity that they can even continue their life cycle within the wood of coffins (smith, 1986). larvae live in a numerous variety of locations such as dung, fungi, and decaying organic matter. they can be saprophagous, coprophagous, mycophagous, or parasitic. in nature, they are typically associated with dead animals and heavily decaying matter. their reproductive potential is tremendous. regarding microorganisms we found bacteria. the most abundant species were cyanobacteria (fig. 2a, b) revealed by molecular analysis through clsm, and fungi (fig. 3). considering the high number of specimens collected and the kind of taxa they belong to, we can conclude that the organisms found good conditions for their propagation and rapid build-up of dense pest populations. in fact, we can suppose that a complex biocenosis with different trophic levels (fig. 4) is present in the rest of this mummified body. however, bioceonosis can be associated to: i) the time of the dead to the body (what does this mean?); ii) different processes of mummification; iii) the decomposition levjournal of entomological and acarological research, ser. ii, 43 (2), 201172 f. palla et al.: bacteria, fungi and arthropods pests collected on human mummies el (?); iv) the eventual parasitism before dead; v) the environmental location of the mummy. thus the findings of dead insects and microbial colonization does not allow to date the time of infestation. nevertheless the presence of living organisms such as cyanobacteria (fig. 2a, b) highlights that colonization is going on. the study of insects and other animals associated to mummies or inhabiting catacombs is important also in relation to fungi. recently jurado et al. (2008) conclude that when fig. 2 – photographs (a,b) of bacteria and cyanobacteria at confocal laser scanning microscopy. fig. 3 pcr amplified dna fragments analysed by 2% agarose gel elctrophoresis. amplification reactions were performed by using its (fungal specific) primers. 73 investigating fungal colonisation in cultural properties a carefully studies on the arthropods is needed in order to know fungal dispersion patterns and propose countermeasures for the protection of cultural sites. the authors also recorded entomopathogenic fig. 4 trophic web recorded in the human modern mummies. fig. 5 fungi and bacteria on upper (a) and inner (b) elytra surfaces of a coleopteran specie collected in the human mummy cranial debris, photographed by scanning electron microscopy leica cambridge-leo 420. in b it is possible to notice the venations of the metathoracic wings. journal of entomological and acarological research, ser. ii, 43 (2), 201174 fungi on mural paintings and in catacombs that could be useful for the control of insect pests. in this study we also report several bacteria and fungi associated to the insect rests (fig. 5a, b). conclusions concerning microorganisms, as clsm observations were consistent with pcr results, we conclude that the combination of these approaches improved our understanding of microbial communities thriving on cultural assets, which is essential for the development of conservation and/or restoration strategies. moreover, using molecular techniques, the analyses are faster; as they are performed with microscopic amount of sample and increased accuracy. further studies are necessary to identify the biocoenosis present in the catacombs, possibly using different kind of traps, and to understand their relationships and their role in degradation processes. acknowledgements we would like to express our gratitude to father calogero peri, for granting us permission to collect mummy samples. a special thank is due to c. di liberto for sem and g. morici for clsm analyses. references bourel b., tournel g., hédouin v., gosset d., 2004 entomofauna of buried bodies in northern france. international journal of legal medicine, 118 : 215-220. favole a., 2003 resti di umanità. vita sociale del corpo dopo la morte. roma-bari, ed. editori laterza: 1-216. jurado v., sanchez-moral s., saiz-jimenez c., 2008 entomogenous fungi and the conservation of the cultural heritage: a review, 62 (4): 325-330. levinson h., levinson a., 1994 origin of grain storage and insect species consuming desiccated food. journal of pest science, 67 (3): 47-60. liu d., greenberg b., 1989 immature stage of some flies of forensic importance. annals of entomological society of america, 82: 80-93. palla f., billeci n., mancuso f.p., pellegrino l., 2010 microscopy and molecular biology techniques to study biocenosis diversity in semi-confined environment. conservation science in cultural heritage, 10: 185-194. saarela m., alakomi h.l., suihko m.l., maunuksela l., raaska l., mattila-sandholm t., 2004 heterotrophic microorganisms in air and biofilm samples from roman catacombs, with special emphasis on actinobacteria and fungi. intern. biodeter. & biodeg., 54: 27-37. sineo l., manachini b., carotenuto g., piombino-mascali d., zink a.r., palla f., 2008 -the palermo capuchin catacombs project: a multidisciplinary approach to the study of a modern mummy collection (ca 1600-1900). conservation science in cultural heritage, 8: 155165. f. palla et al.: bacteria, fungi and arthropods pests collected on human mummies 75 smith k.g.v., 1986 a manual of forensic entomology. cornell university press, ithaca, ny. sukontason k.l., boonsriwong w., siriwattanarungsee s., piangjai s., 2006 morphology of puparia of megaselia scalaris (diptera: phoridae), a fly species of medical and forensic importance. parasitology research, 98: 268-272. valentin n., 2003a insect infestation in museum collections: organic materials. in: molecular biology & cultural heritage. ed. c. saiz-jmenez, balkema, netherland: 225-230. valentin n., 2003b microbial contamination in museum collections: organic materials. in: molecular biology & cultural heritage ed. c. saiz-jmenez, balkema, netherland: 85-91. barbara manachini, department of environmental biology and biodiversity, university of palermo, via archirafi, 3890123 palermo, italy. e-mail: barbara.manachini@unipa.it journal of entomological and acarological research, ser. ii, 43 (2), 201176 layout 1 [journal of entomological and acarological research 2013; 45:e14] [page 73] response of chironomid species (diptera, chironomidae) to water temperature: effects on species distribution in specific habitats l. marziali,1 b. rossaro2 1cnr-irsa water research institute, u.o.s. brugherio, brugherio (mb); 2department of food, environmental and nutritional sciences (defens), university of milan, milan, italy abstract the response of 443 chironomid species to water temperature was analyzed, with the aim of defining their thermal optimum, tolerance limits and thermal habitat. the database included 4442 samples mainly from italian river catchments collected from the 1950s up to date. thermal preferences were calculated separately for larval and pupal specimens and for different habitats: high altitude and lowland lakes in the alpine ecoregion; lowland lakes in the mediterranean ecoregion; heavily modified water bodies; kryal, krenal, rhithral and potamal in running waters. optimum response was calculated as mean water temperature, weighted by species abundances; tolerance as weighted standard deviation; skewness and kurtosis as 3rd and 4th moment statistics. the responses were fitted to normal unior plurimodal gaussian models. cold stenothermal species showed: i) unimodal response, ii) tolerance for a narrow temperature range, iii) optima closed to their minimum temperature values, iv) leptokurtic response. thermophilous species showed: i) optima at different temperature values, ii) wider tolerance, iii) optima near their maximum temperature values, iv) platikurtic response, often fitting a plurimodal model. as expected, lower optima values and narrower tolerance were obtained for kryal and krenal, than for rhithral, potamal and lakes. thermal response curves were produced for each species and were discussed according to species distribution (i.e. altitudinal range in running water and water depth in lakes), voltinism and phylogeny. thermal optimum and tolerance limits and the definition of the thermal habitat of species can help predicting the impact of global warming on freshwater ecosystems. introduction global warming is affecting freshwater macroinvertebrate communities with alteration of species distribution and phenology. in particular, increased water temperature will induce a change in distribution of species, which will react following their thermal optimum along an altitudinal and/or latitudinal gradient (hughes, 2000; nyman et al., 2005; bonada et al., 2007; sheldon, 2012). according to species adaptations, each habitat will show different sensibility: in southern europe, kryal, krenal, high altitude lakes and ponds are supposed to be sensitive habitats, being characterized by stenotopic taxa directly influenced by water temperature (boggero et al., 2006; rossaro et al., 2006a; tixier et al., 2009; jacobsen et al., 2012; lencioni et al., 2012). a lot of species won’t probably survive global warming, since spatial isolation may give little opportunity to migrate elsewhere. on the contrary, the response of habitats at lower altitude is poorly understood, as species thermal optimum and tolerance are less known and other factors generally contribute in structuring biotic communities (jacobsen et al., 1997). moreover, some studies showed that local adaptations may induce different thermal sensibility of single species at different sites and habitats. in particular, acclimation temperature during lifetime was proved to affect tolerance of populations (dallas & rivers-moore, 2012). besides, microevolutionary dynamics at local scale may separate the response of populations, and consequently their fitness (hogg et al., 1998; van doorsalaen et al., 2009). therefore it is necessary to determine the extent to which thermal response of species varies among habitats, to determine which communities are more menaced by global warming. studies on aquatic organisms based on lethal or sub-lethal endpoints (e.g. death, ability to escape unfavourable conditions, growth, reproduction, etc.) were carried out in experimental mesocosms or lab tests to derive thermal performance curves that relate species response to water temperature (hester & doyle, 2011; dallas & riversmoore, 2012), with definition of critical thermal maxima or minima. this approach may be successful to detect biological or physiological processes mostly affected by altered temperature. nonetheless thermal history, acclimation, rate of temperature change, test duration, life stage have been shown to affect results. moreover, the difficulty of taxa identification may hinder test application at species level, and many correspondence: laura marziali, cnr-irsa water research institute, u.o.s. brugherio, via del mulino 19, 20861 brugherio (mb), italy. tel.: +39.039.21694207 fax: +39.039.2004692. e-mail: marziali@irsa.cnr.it key words: chironomidae, thermal tolerance, ecological traits, global warming. acknowledgements: data stored in the chirdb were collected within sampling surveys supported by different grants. details about the projects are in the publications of the first author quoted in the reference paragraph. received for publication: 4 april 2013. revision received: 29 april 2013. accepted for publication: 30 may 2013. ©copyright l. marziali and b. rossaro, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e14 doi:10.4081/jear.2013.e14 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e14 jear_2013_2:hrev_master 16/09/13 13.56 pagina 73 no nco mm er cia l response, ii) tolerance for a narrow temperature range, iii) optima no nco mm er cia l response, ii) tolerance for a narrow temperature range, iii) optima closed to their minimum temperature values, iv) leptokurtic response. no nco mm er cia l closed to their minimum temperature values, iv) leptokurtic response. thermophilous species showed: i) optima at different temperature valno nco mm er cia l thermophilous species showed: i) optima at different temperature values, ii) wider tolerance, iii) optima near their maximum temperature no nco mm er cia l ues, ii) wider tolerance, iii) optima near their maximum temperature values, iv) platikurtic response, often fitting a plurimodal model. as no nco mm er cia l values, iv) platikurtic response, often fitting a plurimodal model. as expected, lower optima values and narrower tolerance were obtained no nco mm er cia l expected, lower optima values and narrower tolerance were obtained for kryal and krenal, than for rhithral, potamal and lakes. thermal no nco mm er cia l for kryal and krenal, than for rhithral, potamal and lakes. thermal ular, increased water temperature will induce a change in distribution no nco mm er cia l ular, increased water temperature will induce a change in distributionof species, which will react following their thermal optimum along an no nco mm er cia l of species, which will react following their thermal optimum along analtitudinal and/or latitudinal gradient (hughes, 2000; nyman no nco mm er cia l altitudinal and/or latitudinal gradient (hughes, 2000; nyman 2005; bonada no nco mm er cia l 2005; bonada no nco mm er cia l no nco mm er cia l correspondence: laura marziali, cnr-irsa water research institute, u.o.s. no nco mm er cia l correspondence: laura marziali, cnr-irsa water research institute, u.o.s. brugherio, via del mulino 19, 20861 brugherio (mb), italy. no nco mm er cia l brugherio, via del mulino 19, 20861 brugherio (mb), italy. tel.: +39.039.21694207 fax: +39.039.2004692. no nco mm er cia l tel.: +39.039.21694207 fax: +39.039.2004692. key words: chironomidae, thermal tolerance, ecological traits, global warming.no nco mm er cia l key words: chironomidae, thermal tolerance, ecological traits, global warming. us e global warming is affecting freshwater macroinvertebrate commuus e global warming is affecting freshwater macroinvertebrate communities with alteration of species distribution and phenology. in partic-us e nities with alteration of species distribution and phenology. in particular, increased water temperature will induce a change in distributionus e ular, increased water temperature will induce a change in distribution on ly tat of species can help predicting the impact of global warming on on ly tat of species can help predicting the impact of global warming on on ly [page 74] [journal of entomological and acarological research 2013; 45:e14] studies considered genera, families or even orders (dallas & riversmoore, 2012). more realism could be achieved determining the temperature range that organisms experience in the field (rossaro, 1991a, 1991b, 1991c). data from different ecological surveys in freshwater ecosystems could be gained and specimens collected can be identified at species level. in this way a large amount of data for each species can be gathered. this approach could be successful to determine species thermal preferences and tolerance limits (i.e. temperature beyond which organisms avoid) in different habitats, seasons and life stages. in fact, empirical data may allow going beyond local adaptations of taxa and drawbacks of manipulation tests. this approach was recently adopted at european scale (aqem project) (hering et al., 2004) for many macroinvertebrate groups collecting published data to derive species’ ecological preferences (schmidt-kloiber & hering, 2012). nonetheless species responses have been expressed as qualitative rather than quantitative features, because most publications do not provide raw data. therefore much work is still needed to better quantify the response to natural and anthropogenic factors, as a valuable tool for biomonitoring. for what concerns water temperature, among macroinvertebrate taxa, insects were shown to be mainly responsive to this pressure (bonada et al., 2007; čiamporová-zat’ovičová et al., 2010; dallas & rivers-moore, 2012). in particular, chironomids are a suitable indicator group, being characterized by a large number of species with a wide range of responses to environmental factors (lindegaard et al., 1995). fossil remains of these dipterans in lake sediments have been used as proxy to reconstruct shifts in air and water temperature, since many species were shown to respond rapidly to climatic fluctuations (larocque et al., 2001; lotter et al., 2012). moreover, they have been used as indicators of oxygen concentration (rossaro et al., 2007b) and trophic levels in lakes (sæther 1979, rossaro et al., 2011) and as indicators of organic (raunio et al., 2007) and toxic (cortelezzi et al., 2011) pollution in rivers. nonetheless many studies showed that water temperature is one of the main factors determining taxa assemblages and species distribution (rossaro, 1991a, 1991b, 1991c; brooks & birks, 2000; medeiros & quinlan, 2011). lack of information could be possibly filled by biogeographic studies considering ecological equivalents in different regions (jacobsen et al., 1997, 2012; hamerlik & brodersen 2010; hamerlik et al., 2011), but species names are often not corresponding in different areas, since at large spatial scale biogeographic gradients may be present (catalan et al., 2009) or, at smaller scale, taxonomic determination by different experts often affects data comparability (kernan et al., 2009; heiri et al., 2011). therefore at present only data at regional scale can be likely compared. the present research aims at quantitatively determine the thermal response of chironomid species in different freshwater habitats in southern europe, following the empirical approach. at this purpose, chironomid samples collected in many surveys mostly from italy but also from other alpine and mediterranean countries are considered. species response to altitude, source distance in rivers and water depth in lakes is also determined. different life stages are analyzed. materials and methods to investigate the thermal response of chironomid species the chirdb database (rossaro et al., 2006b) was used. this database contains records about chironomid samples collected in freshwater ecosystems mainly in italy, but also in algeria, austria, france, switzerland and germany from the 1950s up to date (table 1). other data were derived from published papers (table 1). a map of the sampling sites is shown in figure 1. sampling sites were grouped into different habitats: – kryal=glacial streams above the tree line (rossaro et al., 2006b); note that this definition of kryal is more extended than the one given by milner & petts (1994) and water temperature can be much higher than 2°c – krenal=springs (vannote et al., 1980) – rhithral=mountain reach of rivers below the tree line (vannote et al., 1980) – potamal=lowland reach of rivers (vannote et al., 1980) – alpine lowland lakes=natural lakes within the alpine ecoregion (with latitude >44° 00’) with altitude below 800 m a.s.l. (tartari et al., 2006) – alpine high altitude lakes=natural lakes within the alpine ecoregion (with latitude >44° 00’) with altitude above 800 m a.s.l. (tartari et al., 2006) – mediterranean lakes=natural lowland lakes within the mediterranean ecoregion (with latitude <44° 00’), with altitude below 800 m a.s.l. (tartari et al., 2006) – heavily modified water bodies=reservoirs and artificial lakes – brackish ponds=ponds with high salinity (water conductivity >2500 µs cm–1 at 20°c) (tartari et al., 2006) sampling sites are summarized in table 2. samples are grouped into river catchments and the number of samples collected in each habitat is reported. the same site was generally sampled covering all seasons. chironomid samples were collected using different tools, according to the habitat: i) pond net collections of larvae from small water bodies (krenal, kryal, high altitude alpine lakes) (rossaro et al., 2006b); ii) surber net collections of larvae in stony bottom streams (rhithral) (rossaro, 1991b, 1991c, 1992, 1993; marziali et al., 2010a, 2010b); iii) ekman, petersen, ponar dredge samples of larvae from natural lowland lakes and heavily modified water bodies, brackish ponds and from large rivers (potamal) (rossaro, 1988; battegazzore et al., 1992; rossaro et al., 2006a, 2011); iv) drift samples of pupal exuviae using a brundin net (lakes, kryal, krenal, rhithral, potamal) (rossaro, 1991b, 1991c); v) adult captures collected with hand nets, emergence traps or malaise traps (rossaro, 1987); imagines were used for confirming species identifications, but were not considered for data analysis. for each sampling site latitude, longitude, altitude (m a.s.l.), distance from source (km) in running waters and sampling depth (m) in lakes were recorded in the field or were derived using geographic information system-based cartographic data (http://www.sinanet.isprambiente.it). water temperature (°c) was measured with a field multiprobe during the samplings. chironomid samples were slide mounted and identified to species using specialized keys (wiederholm 1980, 1983, 1986; ferrarese & rossaro, 1981; ferrarese, 1983; rossaro, 1982; nocentini, 1985; langton, 1991) and comparing different life stages (e.g. larval exuviae with pupae; pupal exuviae with imagines). in the present work, the abundances of 309 species as larvae (18,886 records) and 325 species as pupal exuviae (7619 records) from 4442 samples were considered. chironomid species nomenclature and systematics follow sæther (1977), rossaro (1991c), sæther (2000), cranston et al. (2012). data analysis data were stored in a microsoft access database (chirdb) (rossaro et al., 2006b). data on larval samples were expressed as specimens per square meter when collected with surber (rhithral) and dredge samples (lowland lakes, heavily modified water bodies, potamal, brackish ponds); and as number of specimens for unit of effort (about 15 min sampling) when collected with pond nets (high altitude lakes, kryal, krenal). data on pupal exuviae samples collected with a brundin net in all habitats were expressed as number of specimens per unit of effort (about 15 min sampling). records of species abundances matching water temperature measures were selected using ms-access queries and were imported into article jear_2013_2:hrev_master 16/09/13 13.56 pagina 74 no nco mm er cia l many studies showed that water temperature is one of the main factors no nco mm er cia l many studies showed that water temperature is one of the main factors determining taxa assemblages and species distribution (rossaro, 1991a, no nco mm er cia l determining taxa assemblages and species distribution (rossaro, 1991a, 1991b, 1991c; brooks & birks, 2000; medeiros & quinlan, 2011). lack of no nco mm er cia l 1991b, 1991c; brooks & birks, 2000; medeiros & quinlan, 2011). lack of information could be possibly filled by biogeographic studies considering no nco mm er cia l information could be possibly filled by biogeographic studies considering et al. no nco mm er cia l et al., 1997, 2012; no nco mm er cia l , 1997, 2012; , 2011), but species names no nco mm er cia l , 2011), but species names are often not corresponding in different areas, since at large spatial scale no nco mm er cia l are often not corresponding in different areas, since at large spatial scale biogeographic gradients may be present (catalan no nco mm er cia l biogeographic gradients may be present (catalan et al. no nco mm er cia l et al., 2009) or, at no nco mm er cia l , 2009) or, at smaller scale, taxonomic determination by different experts often affects no nco mm er cia l smaller scale, taxonomic determination by different experts often affects , 2009; heiri no nco mm er cia l , 2009; heiri et al. no nco mm er cia l et al., 2011). therefore at no nco mm er cia l , 2011). therefore at present only data at regional scale can be likely compared. no nco mm er cia l present only data at regional scale can be likely compared. the present research aims at quantitatively determine the thermal no nco mm er cia l the present research aims at quantitatively determine the thermal response of chironomid species in different freshwater habitats inno nco mm er cia l response of chironomid species in different freshwater habitats in southern europe, following the empirical approach. at this purpose,no nco mm er cia l southern europe, following the empirical approach. at this purpose, ekman, petersen, ponar dredge samples of larvae from natural lowland no nco mm er cia l ekman, petersen, ponar dredge samples of larvae from natural lowlandlakes and heavily modified water bodies, brackish ponds and from large no nco mm er cia l lakes and heavily modified water bodies, brackish ponds and from largerivers (potamal) (rossaro, 1988; battegazzore no nco mm er cia l rivers (potamal) (rossaro, 1988; battegazzore al. no nco mm er cia l al., 2006a, 2011); iv) drift samples of pupal exuviae using a brundin net no nco mm er cia l , 2006a, 2011); iv) drift samples of pupal exuviae using a brundin net (lakes, kryal, krenal, rhithral, potamal) (rossaro, 1991b, 1991c); v) no nco mm er cia l (lakes, kryal, krenal, rhithral, potamal) (rossaro, 1991b, 1991c); v) adult captures collected with hand nets, emergence traps or malaise no nco mm er cia l adult captures collected with hand nets, emergence traps or malaise traps (rossaro, 1987); imagines were used for confirming species no nco mm er cia l traps (rossaro, 1987); imagines were used for confirming species us e the habitat: i) pond net collections of larvae from small water bodies us e the habitat: i) pond net collections of larvae from small water bodies (krenal, kryal, high altitude alpine lakes) (rossaro us e (krenal, kryal, high altitude alpine lakes) (rossaro surber net collections of larvae in stony bottom streams (rhithral) us e surber net collections of larvae in stony bottom streams (rhithral) (rossaro, 1991b, 1991c, 1992, 1993; marziali us e (rossaro, 1991b, 1991c, 1992, 1993; marziali ekman, petersen, ponar dredge samples of larvae from natural lowlandus e ekman, petersen, ponar dredge samples of larvae from natural lowland lakes and heavily modified water bodies, brackish ponds and from largeus e lakes and heavily modified water bodies, brackish ponds and from large on ly sampling sites are summarized in table 2. samples are grouped into on ly sampling sites are summarized in table 2. samples are grouped into river catchments and the number of samples collected in each habitat on lyriver catchments and the number of samples collected in each habitatthe same site was generally sampled covering all seasons. on lythe same site was generally sampled covering all seasons. chironomid samples were collected using different tools, according toon ly chironomid samples were collected using different tools, according to the habitat: i) pond net collections of larvae from small water bodieson ly the habitat: i) pond net collections of larvae from small water bodies (krenal, kryal, high altitude alpine lakes) (rossaro on ly (krenal, kryal, high altitude alpine lakes) (rossaro matlab environment for statistical analyses. the moment statistics, used for describing probability distributions, were then calculated. the expected value of a random variable (the mean) is derived by the first moment, the variance by the second moment, the skewness (i.e. the asymmetry of the probability distribution) by the third moment, the kurtosis (i.e. the peakedness of the probability distribution) by the fourth moment (khurshid, 2007). the water temperature range experienced by each species was divided into 20 equally-ranged classes and the frequency of the species in each of the 20 classes was calculated. a thermal response curve was then produced for each species relating species abundance to water temperature. the formulae used to calculate the first (weighted average), second (weighted standard deviation), third (skewness=g1) and fourth (kurtosis=g2) central moments can be found in sokal & rohlf (1981). [journal of entomological and acarological research 2013; 45:e14] [page 75] article table 1. data stored in the chirdb database are derived from different surveys here summarized. country region river catchment sampling years references italy aosta valley dora baltea river 1995-98 rossaro et al., 2006b; unpublished data trentino-alto adige sarca, adige and noce rivers 1990, 1996-98, 2005 boggero et al., 2006; lencioni et al., 2007 lakes lases, lamar, caldonazzo 1996, 2000, 2004-07 lencioni et al., 2006 and tenno (brenta river) lombardy oglio and mincio rivers 1978-83, 2006 rossaro, 1991c lambro and olona rivers 1977-78, 1986-87, 2003 unpublished data brembo and serio rivers 1980-81, 2003 po river 1977-93 rossaro 1987, 1988; battegazzorre et al., 1992 adda river 1977, 1988-89, 2001-07 unpublished data ticino river 1979, 1985, 2001-04, 2009-10 berra et al., 2004 lake garda 1970-71, 1982, 2004, 2007, 2011 rossaro et al., 2006a, 2011; bonomi, 1974 lakes viverone and avigliana 2005-06 rossaro et al., 2006a, 2011 lake varese 1987, 1994-97, 2002-05 rossaro et al., 2006a, 2011 lake monate 1977, 2004-05 rossaro et al., 2006a, 2011; nocentini, 1979 lake como 1980-84, 2004-05, 2007 unpublished data lakes comabbio, alserio, pusiano and annone 1967, 1977, 2004-07 rossaro et al., 2006a, 2011 lake iseo 1967, 2003-04 unpublished data piedmont lake mergozzo 1963-64, 1971-72, 1975, 1994, 2010 rossaro et al., 2006a, 2011; nocentini, 1979 lake maggiore 1953-54, 1960-61, 1966-67, 1985-88, rossaro et al., 2006a, 2011; nocentini, 1963 1995-96, 2004, 2007, 2009-10 ticino river 1985-87, 1991-94, 2000, 2007 boggero et al., 2006; unpublished data dora baltea river 2005 boggero et al., 2006 agogna river 1976-77, 1981-82 rossaro, 1991c toce river 1991-94, 2000 unpublished data sesia river 1987 unpublished data lake lugano 2004-04 unpublished data po and tanaro rivers 1989-90 unpublished data lake orta 1976 unpublished data emilia romagna po and trebbia river 1977-83 rossaro 1987, 1988; battegazzore et al., 1992 taro river 2001-03 marziali et al., 2010b liguria danè river 1998-99 unpublished data toscana magra river 2001 unpublished data marche potenza river 1986 rossaro, 1988 abruzzo tordino, vomano and aterno rivers 1978, 1986-92, 1995, 2010 unpublished data lazio tevere and nera rivers 1989-90 unpublished data trasimeno river 2003 unpublished data lakes bolsena, bracciano and vico 1970-73 rossaro et al., 2006a, 2007a umbria tevere river 1977-03 campania sele river 2000-01 marziali et al., 2010a puglia ofanto river 1990 unpublished data sardinia cedrino and rio mannu rivers 1978, 1986 unpublished data lazio, abruzzo, heavily modified water bodies 1976-77, 1934-85, 1989, 1991 unpublished data basilicata, (fibreno, brasimone, scontrone, puglia, sicily pertusillo, occhito, dirillo) switzerland ticino river 2005 boggero et al., 2006 france garonna river 2004 unpublished data germany donau river 2006 free et al., 2009 austria donau river 2006 free et al., 2009 algeria algerian wadi 2007 zerguine et al., 2009; chaib et al., 2011 jear_2013_2:hrev_master 16/09/13 13.56 pagina 75 no nco mm er cia l lakes comabbio, alserio, pusiano and annone 1967, 1977, 2004-07 no nco mm er cia l lakes comabbio, alserio, pusiano and annone 1967, 1977, 2004-07 no nco mm er cia l 1967, 2003-04 no nco mm er cia l 1967, 2003-04 lake mergozzo 1963-64, 1971-72, 1975, 1994, 2010 rossaro no nco mm er cia l lake mergozzo 1963-64, 1971-72, 1975, 1994, 2010 rossaro lake maggiore 1953-54, 1960-61, 1966-67, 1985-88, rossaro no nco mm er cia l lake maggiore 1953-54, 1960-61, 1966-67, 1985-88, rossaro 1995-96, 2004, 2007, 2009-10 no nco mm er cia l 1995-96, 2004, 2007, 2009-10 1985-87, 1991-94, 2000, 2007 boggero no nco mm er cia l 1985-87, 1991-94, 2000, 2007 boggero agogna river no nco mm er cia l agogna river toce river no nco mm er cia l toce river no nco mm er cia l sesia river no nco mm er cia l sesia river lake lugano no nco mm er cia l lake lugano po and tanaro rivers no nco mm er cia l po and tanaro rivers no nco mm er cia l lake orta no nco mm er cia l lake orta po and trebbia river no nco mm er cia l po and trebbia river no nco mm er cia l taro river no nco mm er cia l taro river no nco mm er cia l no nco mm er cia l no nco mm er cia l u se 1970-71, 1982, 2004, 2007, 2011 rossaro us e 1970-71, 1982, 2004, 2007, 2011 rossaro 1987, 1994-97, 2002-05 us e 1987, 1994-97, 2002-051977, 2004-05 us e 1977, 2004-05 1980-84, 2004-05, 2007us e 1980-84, 2004-05, 2007 lakes comabbio, alserio, pusiano and annone 1967, 1977, 2004-07us e lakes comabbio, alserio, pusiano and annone 1967, 1977, 2004-07 on lyrossaro 1987, 1988; battegazzorre on lyrossaro 1987, 1988; battegazzorre 1970-71, 1982, 2004, 2007, 2011 rossaro on ly 1970-71, 1982, 2004, 2007, 2011 rossaro [page 76] [journal of entomological and acarological research 2013; 45:e14] article figure 1. map of the sampling sites. table 2. river catchments with mean latitude and longitude, and number of samples collected in each habitat. river catchment lat long kn kr rh pt al al me hm br garonna (france) 44°00’00’’ 02°00’00’’ 0 0 10 0 0 0 0 0 0 donau (germany) 47°41’19’’ 11°26’16’’ 0 0 0 0 50 0 0 0 0 donau (austria) 47°47’17’’ 13°20’17’’ 0 0 0 0 41 0 0 0 0 dora baltea 45°37’24’’ 07°35’14’’ 7 44 29 1 0 47 0 0 0 sesia 45°38’00’’ 07°55’00’’ 0 0 0 0 1 0 0 0 0 orta 45°49’00’’ 08°24’00’’ 0 0 0 0 1 0 0 0 0 agogna 45°36’02’’ 08°28’03’’ 17 0 107 0 0 0 0 0 0 ticino (ch) 46°24’33’’ 08°36’25’’ 0 0 4 0 0 14 0 0 0 ticino (no) 45°37’00’’ 08°38’00’’ 0 0 0 9 0 0 0 0 0 ticino (mi) 45°22’33’’ 09°24’28’’ 37 0 0 35 0 0 0 0 0 toce 46°15’35’’ 08°16’27’’ 0 0 11 0 19 0 0 0 0 maggiore (ch) 46°26’09’’ 08°48’11’’ 0 0 0 0 18 0 0 0 0 maggiore (vb) 45°48’21’’ 08°34’16’’ 0 0 0 0 303 0 0 0 0 maggiore (va) 45°51’12’’ 08°40’10’’ 0 0 0 0 78 0 0 0 0 mergozzo 45°57’21’’ 08°27’36’’ 0 0 0 0 162 0 0 0 0 varese 45°50’96’’ 08°43’73’’ 0 0 1 0 119 0 0 0 0 lugano 46°28’06’’ 09°38’12’’ 0 0 3 0 14 0 0 0 0 olona 45°30’11’’ 09°20’52’’ 0 0 0 43 0 0 0 0 0 lambro 45°48’37’’ 09°16’60’’ 0 0 0 1 163 0 0 0 0 adda (so) 46°19’02’’ 09°43’01’’ 1 24 0 0 0 0 0 0 0 to be continued on next page jear_2013_2:hrev_master 16/09/13 13.56 pagina 76 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l table 2. river catchments with mean latitude and longitude, and number of samples collected in each habitat. no nco mm er cia l table 2. river catchments with mean latitude and longitude, and number of samples collected in each habitat. no nco mm er cia l river catchment lat long kn kr rh pt al al me hm br no nco mm er cia l river catchment lat long kn kr rh pt al al me hm br garonna (france) 44°00’00’’ 02°00’00’’ no nco mm er cia l garonna (france) 44°00’00’’ 02°00’00’’ 0 0 no nco mm er cia l 0 0 no nco mm er cia l no nco mm er cia l donau (germany) 47°41’19’’ 11°26’16’’ no nco mm er cia l donau (germany) 47°41’19’’ 11°26’16’’ 0 0 0 0 no nco mm er cia l 0 0 0 0 donau (austria) 47°47’17’’ 13°20’17’’ no nco mm er cia l donau (austria) 47°47’17’’ 13°20’17’’ no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l dora baltea 45°37’24’’ 07°35’14’’ 7 44 29no nco mm er cia l dora baltea 45°37’24’’ 07°35’14’’ 7 44 29 45°38’00’’ 07°55’00’’no nco mm er cia l 45°38’00’’ 07°55’00’’ us e o nly the first central moment has the meaning of optimum response value, the second moment can be interpreted as a measure of tolerance (ter braak & prentice, 1988). a positive value of g1 means a response curve skewed to the right, i.e. the optimum value is closer to the minimum response value. a negative value of g1 means a response curve skewed to the left, i.e. optimum water temperature is closer to the maximum response value. a positive value of g2 is a measure of the peakedness of a curve. a curve with a high g2 (>3) is called leptokurtic and it has a defined peak, i.e. the species has a defined optimum temperature. a negative value of g2 means a platykurtic response or flat response, i.e. the species is present over a wide range of water temperature values. in general, a negative value of g2 suggests a bior plurimodal gaussian distribution (khurshid, 2007). moment calculations were performed converting in matlab® envi[journal of entomological and acarological research 2013; 45:e14] [page 77] article table 2. continued from previous page. river catchment lat long kn kr rh pt al al me hm br adda (lc) 45°48’16’’ 09°23’27’’ 0 0 3 0 21 0 0 0 0 adda (mi) 45°37’00’’ 09°29’97’’ 0 0 0 17 0 0 0 0 0 adda (lo) 45°16’02’’ 09°37’00’’ 0 0 1 19 4 0 0 0 0 adda (cr) 45°28’00’’ 09°31’00’’ 0 0 0 18 0 0 0 0 0 adda (bg) 46°07’00’’ 09°53’00’’ 13 0 0 1 0 0 0 0 0 sarca 46°08’02’’ 10°37’32’’ 87 206 115 0 0 15 0 0 0 noce 46°17’00’’ 10°40’00’’ 0 3 0 0 1 0 0 0 0 adige (bz) 46°02’41’’ 11°15’33’’ 0 0 0 0 4 0 0 0 0 adige (tn) 46°20’25’’ 10°29’21’’ 0 0 1 0 114 38 0 0 0 brenta 46°01’34’’ 11°19’39’’ 0 0 0 0 78 0 0 0 0 como 45°40’01’’ 09°17’02’’ 0 0 0 0 107 0 0 0 0 brembo 45°42’46’’ 09°38’39’’ 1 0 56 0 0 0 0 1 0 serio 45°30’10’’ 09°44’12’’ 1 0 36 0 0 0 0 0 0 iseo 45°40’24’’ 09°35’38’’ 0 0 0 0 28 0 0 0 0 oglio 45°35’17’’ 09°45’14’’ 2 4 25 2 51 0 0 0 0 mincio (mn) 45°33’32’’ 10°39’45’’ 0 0 0 0 6 0 0 0 0 garda(vr) 45°41’00’’ 10°41’01’’ 0 0 0 0 353 0 0 0 0 po (mi and pv) 45°41’05’’ 09°16’02’’ 0 0 216 103 46 0 0 0 0 po (pc) 45°07’00’’ 10°25’06’’ 0 0 0 427 0 0 0 0 0 po (fe) 44°10’00’’ 12°00’00’’ 0 0 0 1 0 0 0 0 0 tanaro 44°21’00’’ 08°11’04’’ 0 0 85 27 0 0 0 0 0 danè 44°16’00’’ 08°25’00’’ 0 0 95 0 0 0 0 0 0 trebbia 44°29’16’’ 09°21’18’’ 4 0 11 0 5 0 0 0 0 taro 44°35’30’’ 09°33’21’’ 2 0 31 28 0 0 0 0 0 magra 44°22’00’’ 09°53’00’’ 0 0 1 0 0 0 0 0 0 reno (brasimone) 44°08’00’’ 11°08’00’’ 0 0 0 0 0 0 0 1 0 potenza 43°19’00’’ 13°24’00’’ 0 0 10 10 0 0 0 0 0 tevere (pg) 43°18’00’’ 12°18’00’’ 0 0 0 3 0 0 0 0 0 trasimeno 43°10’00’’ 12°00’00’’ 0 0 0 0 0 0 2 0 0 bolsena 42°35’00’’ 11°55’00’’ 0 0 0 0 0 0 102 0 0 bracciano 42°07’00’’ 12°14’00’’ 0 0 0 0 0 0 59 0 0 vico 42°18’00’’ 12°10’00’’ 0 0 0 0 0 0 40 0 0 tordino-vomano 42°36’00’’ 13°38’00’’ 0 0 2 3 0 0 0 1 0 nera 42°25’00’’ 13°05’00’’ 0 0 2 0 0 0 0 0 0 aterno-pescara 42°26’00’’ 13°22’00’’ 12 0 4 0 0 0 0 1 0 sangro (scontrone) 41°34’00’’ 13°38’00’’ 1 0 2 1 2 0 0 4 0 fortore (occhito) 41°35’00’’ 14°57’00’’ 0 0 0 0 0 0 0 14 0 liri (fibreno) 41°38’00’’ 13°22’00’’ 0 0 0 0 0 0 1 0 0 ofanto 40°52’00’’ 15°05’00’’ 0 0 1 0 0 0 0 0 0 cedrino 40°35’00’’ 09°42’00’’ 1 0 0 0 0 0 0 0 0 sele 40°33’00’’ 15°19’00’’ 0 0 33 0 0 0 0 0 0 agri (pertusillo) 40°16’00’’ 15°56’00’’ 0 0 0 0 0 0 0 103 0 rio mannu 39°18’00’’ 09°08’00’’ 0 0 2 0 0 0 0 0 3 dirillo 37°08’00’’ 14°45’00’’ 0 0 0 0 0 0 0 4 0 kebir (algeria) 36°46’38’’ 08°19’31’’ 0 0 90 0 0 0 0 0 0 lat, latitude; long, longitude; kn, krenal; kr, kryal; rh, rhithral; pt, potamal; al, alpine ecoregion lowland lakes; al, alpine ecoregion high altitude lakes; me, mediterranean ecoregion lakes; hm, heavily modified water bodies; br, brackish ponds. abbreviations in brackets are italian provinces. jear_2013_2:hrev_master 16/09/13 13.56 pagina 77 no nco mm er cia l no nco mm er cia l no nco mm er cia l 31 28 no nco mm er cia l 31 280 0 1 0 0 0 0 0 0 no nco mm er cia l 0 0 1 0 0 0 0 0 0 no nco mm er cia l no nco mm er cia l 0 0 0 0 0 0 0 1 0 no nco mm er cia l 0 0 0 0 0 0 0 1 0 10 10 no nco mm er cia l 10 10 no nco mm er cia l no nco mm er cia l 0 0 0 3 0 0 0 0 0 no nco mm er cia l 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 2 0 0 no nco mm er cia l 0 0 0 0 0 0 2 0 0 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l 0 0 0 0 0 0 no nco mm er cia l 0 0 0 0 0 0 0 0 0 0 0 0 no nco mm er cia l 0 0 0 0 0 0 no nco mm er cia l no nco mm er cia l 0 0 0 0 0 0 no nco mm er cia l 0 0 0 0 0 0 0 0 2 3 0 0 0 1 0 no nco mm er cia l 0 0 2 3 0 0 0 1 0 no nco mm er cia l no nco mm er cia l 0 0 2 0 0 0 0 0 0 no nco mm er cia l 0 0 2 0 0 0 0 0 0 aterno-pescara 42°26’00’’ 13°22’00’’ 12 no nco mm er cia l aterno-pescara 42°26’00’’ 13°22’00’’ 12 no nco mm er cia l no nco mm er cia l sangro (scontrone) 41°34’00’’ 13°38’00’’ no nco mm er cia l sangro (scontrone) 41°34’00’’ 13°38’00’’ 1 0 2 1 2 0 0 4 0 no nco mm er cia l 1 0 2 1 2 0 0 4 0 fortore (occhito) 41°35’00’’ 14°57’00’’ no nco mm er cia l fortore (occhito) 41°35’00’’ 14°57’00’’ no nco mm er cia l no nco mm er cia l no nco mm er cia l liri (fibreno) 41°38’00’’ 13°22’00’’ no nco mm er cia l liri (fibreno) 41°38’00’’ 13°22’00’’ 40°52’00’’ 15°05’00’’no nco mm er cia l 40°52’00’’ 15°05’00’’no nco mm er cia l cedrino 40°35’00’’ 09°42’00’’no nco mm er cia l cedrino 40°35’00’’ 09°42’00’’ us e us e us e 0 0 0 0 0 us e 0 0 0 0 0 us e us e 0 0 0 0 0 0 us e 0 0 0 0 0 0 0 5 0 0 0 0us e 0 5 0 0 0 0us e us e o nlyon ly on ly 0 0 0 0 6 0 0 0 0 on ly 0 0 0 0 6 0 0 0 0 0 0 0 0 on ly0 0 0 0 on ly on ly0 0 0 0 on ly0 0 0 0 0 0 0 0 0on ly 0 0 0 0 0on ly on ly 0 0 0 1 0 0 0 0 0on ly 0 0 0 1 0 0 0 0 0 0 0 0 0 0 on ly 0 0 0 0 0 [page 78] [journal of entomological and acarological research 2013; 45:e14] ronment, version r2012a, some fortran programs, program 9 (davies, 1971) and program statfd (rohlf, 1987). the central moment calculation formulae were used also to analyze the response of species to altitude, water depth (for lacustrine species) and distance from source (for lotic species). regression between species optima for water temperature and standard deviation, g1 or g2 was also calculated to relate species optimum and tolerance characters. to represent graphically species response to water temperature the curve-fitting matlab® toolbox was used, fitting species abundances against water temperature values; the toolbox allows to fit many different models, in particular the one-, twoor n-term gaussian library model: y = a1 * e – ((x–m1)/s1) 2+... an * e – ((x–mn)/sn) 2 where 1 and n are the peaks to be fitted, a1 and an are the amplitude, m1 and mn the centroid (location), s1 and sn are coefficients related to the peak width. separate models were tested for each species collected as larvae and pupal exuviae in the different habitats. the fitted curves given in figures 2-11 are the ones giving the best fit (i.e. the lowest mean square error). models with more than three terms (see formula) were not considered to avoid overfitting. regression curves between optima for water temperature (as dependent variable) and optima for altitude, water depth, distance from source (as independent variables) were calculated. results of all available data, 281 samples were from kryal, 186 from krenal, 987 from rhithral, 749 from potamal, 1903 from lakes in the alpine ecoregion (i.e. 114 from high altitude lakes and 1789 from lowland lakes), 204 from natural lakes in the mediterranean ecoregion, 129 from heavily modified water bodies, 3 from brackish ponds (table 2). a total of 443 chironomid species were present in the sampling sites. water temperature thermal response was first calculated considering all data on larvae (i.e. joining all habitats) to generally characterize each species’ preferences for water temperature. results for the 55 species present in ≥100 records are given in table 3. for each species the number of samples used to calculate the weighted mean, standard deviation, skewness and kurtosis are reported. in general, species with preference for low temperature had a lower standard deviation than species with optima in warm waters. for this reason the former can be defined as cold stenothermal, the latter as warm eurithermal. in fact, the r2 value obtained regressing optimum water temperature of each species with its standard deviation was significant [r2=0.48, 53 degree of freedom (df), p<0.01]. the regression between optimum for water temperature (m°c) and skewness (g1) (table 3) gave an inverse relation (r2=0.34, 53 df, p<0.01). as well, optimum for water temperature (m°c) and kurtosis (g2) were inversely related (r2=0.22, 53 df, p<0.01). these relations suggest that cold stenothermal species generally show a response curve skewed to the right, with optimum value closed to minimum values, and leptokurtic (i.e. unimodal trend); whereas thermophilous species generally show a curve skewed to the left, with optimum value closed to maximum values, and platykurtic (i.e. bior plurimodal trend). thermal response was then calculated for each separate habitat to better characterize each species’ preferences (i.e. using data on larvae collected with the same sampling method) (appendix). the thermal response of some species is represented in figures 2-9. for example, thermal curves for conchapelopia pallidula are shown in figure 2. optimum response calculated from 615 records (all habitats pooled, figure 2a) was 13.54°c, with a standard deviation of 5.93°c, a small positive skewness of 0.34 and a negative kurtosis of �1.03 (table 3). the negative kurtosis suggested a trimodal response with three peaks at 8.13°c (main peak), 11.39°c and 22.42°c (secondary peaks). peaks were at 4.93°c (main peak), 7.45°c and 20.77°c considering only samples from alpine lowland lakes (figure 2b). optimum for rhithral samples was 13.9 °c (unimodal response) (figure 2c, appendix), while potamal samples gave a trimodal response with peaks at 11.5 °c, 18.64 °c and 23.89 °c (figure 2d). article figure 2. response of conchapelopia pallidula larvae (number of individuals m–2) to water temperature (°c) in all habitats (a), alpine ecoregion lowland lakes (b), rhithral (c) and potamal (d). figure 3. thermal response of diamesini larvae. response of diamesa bertrami (number of individuals m–2) to water temperature (°c) in all habitats (a), kyral (b), krenal (c) and rhithral (d). jear_2013_2:hrev_master 16/09/13 13.56 pagina 78 no nco mm er cia l pooled, figure 2a) was 13.54°c, with a standard deviation of 5.93°c, a no nco mm er cia l pooled, figure 2a) was 13.54°c, with a standard deviation of 5.93°c, a no nco mm er cia l 114 from high altitude lakes and 1789 from lowland no nco mm er cia l 114 from high altitude lakes and 1789 from lowland lakes), 204 from natural lakes in the mediterranean ecoregion, 129 no nco mm er cia l lakes), 204 from natural lakes in the mediterranean ecoregion, 129 from heavily modified water bodies, 3 from brackish ponds (table 2). a no nco mm er cia l from heavily modified water bodies, 3 from brackish ponds (table 2). a total of 443 chironomid species were present in the sampling sites. no nco mm er cia l total of 443 chironomid species were present in the sampling sites. small positive skewness of 0.34 and a negative kurtosis of �1.03 (table 3). no nco mm er cia l small positive skewness of 0.34 and a negative kurtosis of �1.03 (table 3).the negative kurtosis suggested a trimodal response with three peaks at no nco mm er cia l the negative kurtosis suggested a trimodal response with three peaks at 8.13°c (main peak), 11.39°c and 22.42°c (secondary peaks). peaks were no nco mm er cia l 8.13°c (main peak), 11.39°c and 22.42°c (secondary peaks). peaks were at 4.93°c (main peak), 7.45°c and 20.77°c considering only samples no nco mm er cia l at 4.93°c (main peak), 7.45°c and 20.77°c considering only samples from alpine lowland lakes (figure 2b). optimum for rhithral samples no nco mm er cia l from alpine lowland lakes (figure 2b). optimum for rhithral samples was 13.9 °c (unimodal response) (figure 2c, appendix), while potamal no nco mm er cia l was 13.9 °c (unimodal response) (figure 2c, appendix), while potamal no nco mm er cia l u se collected with the same sampling method) (appendix). us e collected with the same sampling method) (appendix). the thermal response of some species is represented in figures 2-9. us e the thermal response of some species is represented in figures 2-9.for example, thermal curves for us e for example, thermal curves for figure 2. optimum response calculated from 615 records (all habitats us e figure 2. optimum response calculated from 615 records (all habitats pooled, figure 2a) was 13.54°c, with a standard deviation of 5.93°c, aus e pooled, figure 2a) was 13.54°c, with a standard deviation of 5.93°c, a small positive skewness of 0.34 and a negative kurtosis of �1.03 (table 3).us e small positive skewness of 0.34 and a negative kurtosis of �1.03 (table 3). on ly unimodal trend); whereas thermophilous species generon ly unimodal trend); whereas thermophilous species generally show a curve skewed to the left, with optimum value closed to maxion ly ally show a curve skewed to the left, with optimum value closed to maxii.e. on lyi.e. bior plurimodal trend). on lybior plurimodal trend).thermal response was then calculated for each separate habitat to on lythermal response was then calculated for each separate habitat to better characterize each species’ preferences (on ly better characterize each species’ preferences ( collected with the same sampling method) (appendix).on ly collected with the same sampling method) (appendix). the thermal response of some species is represented in figures 2-9.on ly the thermal response of some species is represented in figures 2-9. [journal of entomological and acarological research 2013; 45:e14] [page 79] article table 3. thermal response (°c) of species (larvae) in all habitats: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance vs water temperature values. only the species with ≥100 records in the dataset are reported. species are in phylogenetic order. species n m (°c) sd (°c) g1 g2 procladius choreus 1018 13.39 5.7 0.63 −0.65 macropelopia nebulosa 127 10.5 5.17 0.47 −0.63 zavrelimyia barbatipes 128 11.58 4.11 −0.21 0.45 conchapelopia pallidula 615 13.54 5.93 0.34 −1.03 rheopelopia ornata 111 14.77 4.3 −0.19 −1.15 pseudodiamesa branickii 115 5.63 3.08 0.8 0.71 diamesa steinboecki 106 1.98 1.45 1.06 1.19 diamesa latitarsis 134 3.43 1.97 0.85 0.27 diamesa bertrami 200 2.68 1.96 1.16 0.79 diamesa tonsa 186 7.19 4.66 0.61 −0.18 diamesa zernyi 215 3.72 2.51 1.62 8.22 prodiamesa olivacea 246 9.48 4.33 1.79 3.56 brillia bifida 202 11.38 4.76 0.19 −0.69 tvetenia calvescens 537 11.08 5.81 0.06 −1.24 eukiefferiella brevicalcar 133 4.51 1.94 1.66 6.37 eukiefferiella claripennis 215 14.7 4.41 −0.49 −0.3 eukiefferiella minor 176 6.8 3.78 0.72 0.41 psectrocladius (psectrocladius) oxyura 283 12.17 6.22 0.43 −1.04 rheocricotopus effusus 124 13.15 5.83 −0.16 −0.49 rheocricotopus fuscipes 245 16.97 7.97 0.06 −1.49 synorthocladius semivirens 128 13.38 4.42 −0.16 −0.78 orthocladius (euorthocladius) rivicola 366 9.85 4.7 0.52 −0.01 orthocladius frigidus 261 6.17 3.72 1.25 1.4 orthocladius oblidens 138 9.18 5.5 1.16 0.21 orthocladius rhyacobius 212 12.14 4.02 −0.15 −0.24 orthocladius rubicundus 111 12.45 3.19 0.55 0.91 paratrichocladius rufiventris 253 17.33 6.32 0.17 −0.82 cricotopus annulator 161 14.24 4.79 0.09 0.16 cricotopus bicinctus 276 14.63 5.08 −0.23 −1.04 cricotopus (isocladius) sylvestris 183 11.19 5.08 0.82 −0.09 parametriocnemus stylatus 218 11.14 4.97 0.36 −0.83 parakiefferiella bathophila 117 5.89 3.69 3.66 12.52 thienemanniella partita 107 7.73 4.08 0.93 0.3 corynoneura scutellata 259 11.07 4.06 −0.5 −0.35 tanytarsus gregarius 421 11.11 6.8 0.72 −1.07 cladotanytarsus atridorsum 268 14.59 5.11 0.63 −1.05 paratanytarsus lauterborni 101 10.53 3.01 3.1 9.11 micropsectra atrofasciata 490 13.79 5.33 0.52 0.88 micropsectra pallidula 125 6.3 3.58 1.1 0.44 pagastiella orophila 115 8.12 4.63 1.43 0.75 pseudochironomus prasinatus 209 13.95 6.56 0.02 −1.37 paratendipes albimanus 351 12.22 4.43 1.35 0.65 microtendipes pedellus 394 12.29 2.73 0.6 1.06 polypedilum convictum 138 15.44 4.07 −0.61 0.44 polypedilum laetum 112 16.65 5.52 −0.14 −0.38 polypedilum nubeculosum 566 12.08 4.09 1.26 1.58 endochironomus tendens 106 12.51 3.91 0.8 0.08 dicrotendipes nervosus 276 10.08 5.24 0.86 0 glyptotendipes pallens 154 13.88 7.65 0.08 −1.25 chironomus anthracinus 525 13.54 6.35 0.5 −1.44 chironomus plumosus 571 11.19 6.1 0.67 −0.59 chironomus riparius 333 15.28 4.65 0.32 1.44 cladopelma viridulum 294 13.63 5.98 0.51 −0.7 cryptochironomus defectus 473 13.86 5.67 0.43 −0.74 demicryptochironomus vulneratus 143 12.96 7.28 0.44 −1.36 n, number of samples; m, weighted mean; sd, standard deviation; g1, skewness; g2, kurtosis. jear_2013_2:hrev_master 16/09/13 13.56 pagina 79 no nco mm er cia l no nco mm er cia l no nco mm er cia l 12.14 no nco mm er cia l 12.14 no nco mm er cia l no nco mm er cia l 12.45 no nco mm er cia l 12.45 17.33 no nco mm er cia l 17.33 no nco mm er cia l no nco mm er cia l 14.24 no nco mm er cia l 14.24 14.63 no nco mm er cia l 14.63 no nco mm er cia l no nco mm er cia l 218 no nco mm er cia l 218 no nco mm er cia l no nco mm er cia l no nco mm er cia l 117 no nco mm er cia l 117 107 no nco mm er cia l 107 no nco mm er cia l no nco mm er cia l no nco mm er cia l 259 no nco mm er cia l 259 421 no nco mm er cia l 421 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l u se us e us e 7.97 us e 7.974.42 us e 4.42 us e us e 4.7us e 4.7 3.72us e 3.72 on ly on ly on ly 1.66 on ly 1.66 on ly on ly−0.49 on ly−0.490.72 on ly0.72 on ly on ly on ly [page 80] [journal of entomological and acarological research 2013; 45:e14] many cold stenothermal species such as diamesa zernyi and pseudokiefferiella parva showed only one maximum, with a high g2, i.e. leptokurtic response (table 3, appendix). species with low temperature optimum (cold stenothermal) showed a response curve skewed to the right (g1>0). diamesa bertrami showed a moderately platykurtic response (g2=0.79), with a trimodal curve considering all habitats (figure 3a), a bimodal curve with main peak at 2.76°c in kryal samples (with a second peak at 0.93°c) (figure 3b), a unimodal response in krenal with peak at 3.90°c (figure 3c), a trimodal response in rhithral with peaks at 3.67°c, 6.79°c and 8.52°c (figure 3d). species with optimum at high temperatures (thermophilous species) showed a response curve skewed to the left (g1<0). for example, cricotopus (isocladius) sylvestris in potamal (figure 4c, appendix) showed optimum at 17.80°c and g1=2.13; paratanytarsus mediterraneus in potamal (figure 5d; appendix) had optimum at 19.42°c and a g1=1.59. tanytarsus brundini in rhithral with optimum at 14.37°c and a negative g1 (g1=0.29) is an example of a curve moderately skewed to the left (figure 5b; appendix). some exceptions were shown: paratrichocladius rufiventris (figure 4a) had optimum temperature value of 17.33°c and a response curve skewed to the right (g1>0, i.e. g1=0.17) (table 3). a negative value of g2 was an index of a bior plurimodal response; tanytarsus gregarius in alpine ecoregion lakes with a negative g2 (g2=1.09; appendix) had a bimodal response with two peaks at 5.68°c and 20.66°c (figure 5c); the very different optima suggest the presence of two populations, the former inhabiting high depth habitats (down to 350 m depth) characterized by low temperatures. similarly, it was possible to compare the response of polypedilum nubeculosum larvae in different habitats (figure 8). a plurimodal response was evident, with different peaks in different habitats. the response of the larval and pupal stages was compared in different habitats (figures 6-7, table 4). for example, larvae of micropsectra atrofasciata in rhithral showed peaks at 6.63°c, 11.83°c and 17.84°c (figure 6c), while pupal exuviae at 8.91°c, 12.65°c and 15.92°c (figure 7c); in potamal larvae had peaks at 6.26°c, 9.43°c and 17.95°c (figure 6d), while pupal exuviae at 9.40°c, 13.53°c and 18.39°c (figure 7d). the response of species belonging to the same genus was also analyzed (figures 7 and 9). chironomus anthracinus showed a bimodal response in alpine lowland lakes (figure 9a). chironomus plumosus had a trimodal response in alpine lowland lakes, and the main peak was at the lowest temperature (figure 9b); a similar response was observed in mediterranean lakes (figure 9c). chironomus riparius showed a unimodal response in the rhithral habitat (optimum at 15 °c) (figure 9d, appendix). altitude the response to altitude for the most frequently captured species is reported in table 5. all data on larvae were used (i.e. all habitats). the regression between optima for altitude and for water temperature was calculated selecting 78 species present in at least 66 samples, for which both altitude and water temperature values were available. this selection gave the highest r2. regression coefficient was negative (r2=0.60, 76 df, p<0.01, figure 10). at high altitudes, zavrelimyia barbatipes, corynoneura scutellata, paratanytarsus austriacus showed an optimum water temperature higher than predicted by altitude, whereas d. bertrami, paratrichocladius skirwithensis, orthocladius (eudactylocladius) fuscimanus had temperature optima lower than expected by altitude; at article figure 4. thermal response of orthocladiini larvae. response of paratrichocladius rufiventris (number of individuals m–2) to water temperature (°c) in all habitats (a) and rhithral (b); response of cricotopus (isocladius) sylvestris in potamal (c); response of corynoneura scutellata in alpine ecoregion high altitude lakes (d). table 4. thermal response (°c) of micropsectra atrofasciata (chironominae) in specific habitats at different life stages: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance vs water temperature values. life stage habitat n m (°c) sd (°c) g1 g2 larvae rhythral 363 14.20 6.17 0.42 −0.33 pupal exuviae rhythral 89 13.24 4.11 0.45 0.50 larvae potamal 37 13.50 5.48 −0.03 −1.02 pupal exuviae potamal 79 14.86 5.87 −0.06 −1.09 larvae alpine lakes 48 14.05 4.62 0.67 2.47 pupal exuviae alpine lakes 56 16.31 7.54 0.58 −1.38 n, number of samples; m, weighted mean; sd, standard deviation; g1, skewness; g2, kurtosis; alpine lakes, alpine ecoregion lowland lakes. figure 5. thermal response of tanytarsini larvae. response of tanytarsus brundini (number of individuals m–2) to water temperature (°c) in all habitats (a) and rhithral (b); response of tanytarsus gregarius in alpine ecoregion lowland lakes (c); response of paratanytarsus mediterraneus in potamal (d). jear_2013_2:hrev_master 16/09/13 13.56 pagina 80 no nco mm er cia l in rhithral showed peaks at 6.63°c, 11.83°c and 17.84°c (figure no nco mm er cia l in rhithral showed peaks at 6.63°c, 11.83°c and 17.84°c (figure 6c), while pupal exuviae at 8.91°c, 12.65°c and 15.92°c (figure 7c); in no nco mm er cia l 6c), while pupal exuviae at 8.91°c, 12.65°c and 15.92°c (figure 7c); in potamal larvae had peaks at 6.26°c, 9.43°c and 17.95°c (figure 6d), no nco mm er cia l potamal larvae had peaks at 6.26°c, 9.43°c and 17.95°c (figure 6d), while pupal exuviae at 9.40°c, 13.53°c and 18.39°c (figure 7d). no nco mm er cia l while pupal exuviae at 9.40°c, 13.53°c and 18.39°c (figure 7d). the response of species belonging to the same genus was also anano nco mm er cia l the response of species belonging to the same genus was also anashowed a bimodal no nco mm er cia l showed a bimodal no nco mm er cia l no nco mm er cia l no nco mm er cia l life stage habitat n m (°c) sd (°c) g1 g2 no nco mm er cia l life stage habitat n m (°c) sd (°c) g1 g2larvae rhythral 363 14.20 6.17 0.42 −0.33 no nco mm er cia l larvae rhythral 363 14.20 6.17 0.42 −0.33 no nco mm er cia l no nco mm er cia l pupal exuviae rhythral 89 13.24 4.11 0.45 0.50 no nco mm er cia l pupal exuviae rhythral 89 13.24 4.11 0.45 0.50 larvae potamal 37 13.50 5.48 −0.03 −1.02 no nco mm er cia l larvae potamal 37 13.50 5.48 −0.03 −1.02 no nco mm er cia l no nco mm er cia l pupal exuviae potamal 79 14.86 5.87 −0.06 −1.09 no nco mm er cia l pupal exuviae potamal 79 14.86 5.87 −0.06 −1.09 us e table 4. thermal response (°c) of us e table 4. thermal response (°c) of (chironominae) in specific habitats at different life stages: numus e (chironominae) in specific habitats at different life stages: num-ber of samples, weighted mean, standard deviation, skewness and us e ber of samples, weighted mean, standard deviation, skewness andkurtosis of species abundance vs water temperature values. us e kurtosis of species abundance vs water temperature values. us e us e life stage habitat n m (°c) sd (°c) g1 g2us e life stage habitat n m (°c) sd (°c) g1 g2 on ly had temperature optima lower than expected by altitude; at on ly had temperature optima lower than expected by altitude; at table 4. thermal response (°c) of on ly table 4. thermal response (°c) of (chironominae) in specific habitats at different life stages: numon ly (chironominae) in specific habitats at different life stages: num[journal of entomological and acarological research 2013; 45:e14] [page 81] article table 5. response of species (larvae) to altitude (m a.s.l.) in all habitats: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance vs site altitude values. only the species with ≥100 records in the dataset are reported. species are in phylogenetic order. species n m (m a.s.l.) sd (m a.s.l.) g1 g2 tanypus punctipennis 118 237 207 2.78 16.96 procladius choreus 1530 437 303 2.42 7.53 macropelopia nebulosa 274 1278 524 −0.95 −0.67 ablabesmyia monilis 143 662 513 1.97 3.43 zavrelimyia barbatipes 243 1961 540 −2.09 3.16 conchapelopia pallidula 1005 363 285 3.14 14.63 rheopelopia ornata 137 177 160 2.22 8.04 pseudodiamesa branickii 262 1913 611 −1.09 0.11 diamesa steinboecki 119 2559 221 −2.42 8.87 diamesa latitarsis 171 2213 572 −1.60 2.59 diamesa bertrami 277 1933 653 −0.86 0.04 diamesa tonsa 409 897 654 1.27 0.75 diamesa zernyi 353 2145 564 −1.14 1.04 pseudokiefferiella parva 119 2348 475 −1.52 2.49 prodiamesa olivacea 393 300 421 3.56 12.80 brillia longifurca 100 458 264 0.87 0.95 brillia bifida 413 434 298 1.76 6.13 cardiocladius fuscus 148 677 750 1.60 0.77 tvetenia calvescens 840 1281 945 0.14 −1.81 eukiefferiella brevicalcar 162 2013 461 −1.55 2.01 eukiefferiella claripennis 353 651 691 2.00 2.23 eukiefferiella minor 324 1489 772 −0.39 −1.52 psectrocladius (psectrocladius) oxyura 334 272 373 4.56 20.39 rheocricotopus chalybeatus 116 342 168 1.50 5.34 rheocricotopus effusus 205 866 743 1.17 −0.33 rheocricotopus fuscipes 515 361 242 3.10 17.76 synorthocladius semivirens 212 451 280 4.10 22.43 orthocladius (eudactylocladius) fuscimanus 124 1825 709 −1.25 −0.09 orthocladius (euorthocladius) rivicola 618 1052 902 0.66 −1.40 orthocladius excavatus 141 335 152 1.96 15.17 orthocladius frigidus 463 1767 743 −0.90 −0.49 orthocladius oblidens 179 305 188 1.73 2.60 orthocladius rhyacobius 312 422 228 0.79 1.82 orthocladius rubicundus 204 409 214 1.19 6.43 paratrichocladius rufiventris 456 737 610 0.81 −1.18 paratrichocladius skirwithensis 210 1849 538 −1.57 1.75 cricotopus annulator 245 412 335 3.81 17.08 cricotopus bicinctus 422 189 198 1.31 5.93 cricotopus fuscus 169 1067 624 0.17 −1.18 cricotopus tremulus 126 968 725 0.75 −0.27 cricotopus triannulatus 220 220 231 2.56 8.14 cricotopus (isocladius) sylvestris 276 322 593 2.89 6.69 metriocnemus hygropetricus 180 937 685 0.88 −0.59 chaetocladius laminatus 142 1628 913 −0.44 −1.62 paratrissocladius excerptus 114 434 242 −0.07 −0.01 heterotrissocladius marcidus 174 1936 595 −1.45 1.02 parametriocnemus stylatus 349 1137 878 0.51 −1.19 parakiefferiella bathophila 165 226 138 4.06 28.87 thienemanniella partita 173 1141 904 0.19 −1.69 corynoneura scutellata 395 2130 447 −3.37 11.70 stempellina bausei 115 426 209 0.00 −1.67 tanytarsus gregarius 652 561 577 1.21 −0.31 cladotanytarsus atridorsum 342 406 136 1.92 17.06 paratanytarsus austriacus 135 2087 311 −2.58 8.72 to be continued on next page jear_2013_2:hrev_master 16/09/13 13.56 pagina 81 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l 866 no nco mm er cia l 866 no nco mm er cia l no nco mm er cia l no nco mm er cia l 361 no nco mm er cia l 361 451 no nco mm er cia l 451 no nco mm er cia l no nco mm er cia l 1825 no nco mm er cia l 1825 no nco mm er cia l no nco mm er cia l 463 no nco mm er cia l 463 no nco mm er cia l no nco mm er cia l 179 no nco mm er cia l 179 312 no nco mm er cia l 312 no nco mm er cia l no nco mm er cia l 204 no nco mm er cia l 204 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l u se 945 us e 945 us e us e 461 us e 461691 us e 691 us e us e 772us e 772 on ly on ly on ly −1.52 on ly −1.52 3.56 on ly3.56 on ly on ly0.87 on ly0.87 1.76 on ly 1.76 on ly on ly [page 82] [journal of entomological and acarological research 2013; 45:e14] article table 5. continued from previous page. species n m (m a.s.l.) sd (m a.s.l.) g1 g2 paratanytarsus lauterborni 125 410 549 1.76 1.19 micropsectra atrofasciata 890 425 361 3.06 10.30 micropsectra contracta 386 402 114 8.52 93.32 micropsectra notescens 108 527 313 0.31 1.08 micropsectra pallidula 166 2184 293 −1.55 3.46 pagastiella orophila 127 575 245 −0.77 −0.90 pseudochironomus prasinatus 256 396 202 0.30 −1.74 paratendipes albimanus 464 308 172 2.83 17.00 microtendipes pedellus 510 204 213 3.86 18.41 polypedilum convictum 145 347 167 −0.32 −1.23 polypedilum laetum 199 340 294 3.06 15.31 polypedilum cultellatum 100 142 153 1.68 2.34 polypedilum nubeculosum 812 228 143 5.21 61.77 phaenopsectra flavipes 149 399 429 2.03 3.05 endochironomus tendens 140 148 198 6.14 57.78 stictochironomus pictulus 101 460 443 2.21 2.90 dicrotendipes nervosus 373 270 104 1.28 1.94 glyptotendipes pallens 237 241 67 1.56 18.49 chironomus anthracinus 751 482 356 1.79 3.65 chironomus plumosus 762 283 132 2.04 7.37 chironomus riparius 521 229 199 0.93 −0.24 cladopelma viridulum 390 238 133 6.26 70.75 parachironomus arcuatus 113 195 98 2.73 16.60 paracladopelma camptolabis 107 631 546 1.21 0.57 paracladopelma nigritulum 188 388 55 10.07 221.97 cryptochironomus defectus 606 305 156 0.93 0.25 demicryptochironomus vulneratus 163 226 88 3.18 12.09 n, number of samples; m, weighted mean; sd, standard deviation; g1, skewness; g2, kurtosis. figure 6. thermal response of polypedilum nubeculosum larvae (number of individuals m–2) to water temperature (°c) in alpine ecoregion lowland lakes (a), mediterranean ecoregion lakes (b), rhithral (c) and potamal (d). figure 7. thermal response of micropsectra spp. larvae. response of m. pallidula (number of individuals m–2) to water temperature (°c) in krenal (a); response of m. atrofasciata in alpine ecoregion lowland lakes (b), rhithral (c) and potamal (d). jear_2013_2:hrev_master 16/09/13 13.56 pagina 82 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l 226 no nco mm er cia l 226 no nco mm er cia l u se us e us e us e us e 133 us e 133 us e 98us e 98us e us e 546us e 546 55 us e 55 on ly on ly on ly 2.21 on ly 2.21 1.28 on ly1.28 on ly on ly1.56 on ly1.56 1.79on ly 1.79on ly on ly 2.04on ly 2.04 lower altitudes, the higher temperature optima were observed for p. mediterraneus, p. rufiventris and tanypus punctipennis and the lower for orthocladius oblidens, pagastiella orophila, parakiefferiella bathophila, prodiamesa olivacea, diamesa tonsa. depth response of lacustrine species (i.e. larvae in alpine ecoregion lowland lakes) to depth is summarized in table 6. only few species showed optimum at >40 m depth (micropsectra contracta, paracladopelma nigritulum), others had maxima at lower depth (e.g. at 20-25 m, procladius choreus, prodiamesa olivacea). response curves of some species are shown in figure 11. c. plumosus, c. anthracinus, demicryptochironomus vulneratus and t. gregarius showed a wide range of depth tolerance (table 6). source distance the optimum values for source distance were calculated for species (i.e. larvae in running water habitats) for which at least 100 samples were available (table 7). a relation between optimum for water temperature and for source distance was calculated for the 75 species present in ≥81 samples. the relation is shown in figure 12, with r2=0.33 (73 df, p<0.01) fitting a linear model. as expected, cold stenothermal species had optimum near the stream source (e.g. diamesa species) while eurithermal ones (endochironomus tendens, c. riparius, glyptotendipes pallens, c. i. sylvestris, cricotopus triannulatus, cricotopus bicinctus) showed optimum at high distance from source. discussion notwithstanding the approximation of joining data collected with different sampling methods in different habitats, some generalizations could be argued by the analysis of the dataset on larvae collections. thermophilous species often showed platikurtic responses, fitting plurimodal gaussian models, with: i) optima closed to their maximum temperature values, ii) wide tolerance, iii) negative skewness and negative kurtosis (rossaro, 1991a, 1991c). on the contrary, species restricted to few habitats, such as kryal (e.g. diamesa steinboecki, diamesa latitarsis) or krenal (e.g. chaetocladius laminatus, micropsectra pallidula), showed low optima for water temperature (cold stenothermal) and low tolerance (stenoecious). these species often showed: i) optima closed to their minimum temperature values, ii) tolerance for a narrow temperature range, iii) positive skewness and positive kurtosis (rossaro, 1991c). even if a bimodal response can be fitted, the two maxima are generally rather closed to each other (figure 3). these species could be thus more sensitive to an increasing trend of temperature (hester & doyle, 2011). for a better approximation of species preferences and tolerance, optima for water temperature were calculated for each species in different habitats, thus considering data collected with the same sampling strategy (appendix). as expected, lower values were obtained for kryal and krenal, and higher values for rhithral, potamal and lakes. most taxa showed different responses according to the habitat. when data are [journal of entomological and acarological research 2013; 45:e14] [page 83] article table 6. response of lacustrine species (larvae) to water depth (m depth) in alpine ecoregion lowland lakes: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance vs sampling depth values only the species with ≥100 records in the dataset are reported. species are in phylogenetic order. species n m (m depth) sd (m depth) g1 g2 procladius choreus 1046 21.02 23.02 3.17 23.24 conchapelopia pallidula 232 4.87 3.34 4.05 31.03 prodiamesa olivacea 179 21.70 17.71 1.08 1.21 psectrocladius (psectrocladius) oxyura 255 4.97 2.39 1.67 4.75 orthocladius oblidens 110 4.99 2.14 1.97 20.31 parakiefferiella bathophila 113 5.86 3.27 2.22 5.64 tanytarsus gregarius 459 10.15 32.08 4.62 23.67 cladotanytarsus atridorsum 253 3.62 2.38 2.65 17.66 micropsectra contracta 359 84.91 56.80 1.33 1.62 pagastiella orophila 116 7.10 2.77 2.69 13.03 pseudochironomus prasinatus 212 4.26 3.04 6.85 140.29 paratendipes albimanus 295 4.44 8.81 7.69 152.02 microtendipes pedellus 228 6.06 4.29 1.54 3.00 polypedilum nubeculosum 377 3.27 7.90 8.12 126.29 dicrotendipes nervosus 232 5.70 3.88 2.05 4.46 chironomus anthracinus 529 13.39 18.62 8.41 113.15 chironomus plumosus 480 9.73 49.52 7.03 51.04 cladopelma viridulum 270 8.34 16.38 10.19 163.69 paracladopelma nigritulum 171 73.31 41.44 0.88 −0.64 cryptochironomus defectus 423 6.51 4.75 3.70 52.20 demicryptochironomus vulneratus 144 4.06 24.24 11.46 138.48 n, number of samples; m, weighted mean; sd, standard deviation; g1, skewness; g2, kurtosis. jear_2013_2:hrev_master 16/09/13 13.56 pagina 83 no nco mm er cia l table 6. response of lacustrine species (larvae) to water depth (m depth) in alpine ecoregion lowland lakes: number of samples, weightno nco mm er cia l table 6. response of lacustrine species (larvae) to water depth (m depth) in alpine ecoregion lowland lakes: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance no nco mm er cia l ed mean, standard deviation, skewness and kurtosis of species abundance in the dataset are reported. species are in phylogenetic order. no nco mm er cia l in the dataset are reported. species are in phylogenetic order. no nco mm er cia l n m (m depth) sd (m depth) g1 no nco mm er cia l n m (m depth) sd (m depth) g1 1046 no nco mm er cia l 1046 no nco mm er cia l no nco mm er cia l no nco mm er cia l 232 no nco mm er cia l 232 no nco mm er cia l no nco mm er cia l psectrocladius (psectrocladius) oxyura no nco mm er cia l psectrocladius (psectrocladius) oxyura no nco mm er cia l no nco mm er cia l no nco mm er cia l u se strategy (appendix). as expected, lower values were obtained for kryal us e strategy (appendix). as expected, lower values were obtained for kryal and krenal, and higher values for rhithral, potamal and lakes. most taxa us e and krenal, and higher values for rhithral, potamal and lakes. most taxashowed different responses according to the habitat. when data are us e showed different responses according to the habitat. when data areo nly (figure 3). these species could be thus more sensitive to an increason ly (figure 3). these species could be thus more sensitive to an increasing trend of temperature (hester & doyle, 2011). on lying trend of temperature (hester & doyle, 2011).for a better approximation of species preferences and tolerance, on lyfor a better approximation of species preferences and tolerance,optima for water temperature were calculated for each species in difon lyoptima for water temperature were calculated for each species in different habitats, thus considering data collected with the same samplingon ly ferent habitats, thus considering data collected with the same sampling strategy (appendix). as expected, lower values were obtained for kryalon ly strategy (appendix). as expected, lower values were obtained for kryal and krenal, and higher values for rhithral, potamal and lakes. most taxa on ly and krenal, and higher values for rhithral, potamal and lakes. most taxa [page 84] [journal of entomological and acarological research 2013; 45:e14] available for the same species in different habitats, as for orthocladius (euorthocladius) rivicola, optimum values are lower in krenal (2.83°c) and kryal (5.23°c) than in rhithral (11.98°c), potamal or lakes. other species (e.g. m. atrofasciata) did not show significant differences between optima values in different habitats, but the response curves were very different (figures 7-8). these species are euryecious and eurythermal with more than one generation per year with different water temperature optimum for the different populations developing during the year. among stenothermal taxa, some species at lower altitude habitats (rhithral, potamal) showed restricted tolerance to temperature, being potentially good indicators of climate change. for example, microtendipes pedellus showed optimum for warm temperature (12.29°c), but a narrow range of tolerance (sd=2.73°c). for these taxa, the increasing temperature trend may induce a migration toward higher elevations, changing in some years the response curve to altitude (nyman et al., 2005; bonada et al., 2007) and increasing species diversity at high elevation sites (čiamporová-zat’ovičová et al., 2010; jacobsen et al., 2012). alternatively, species may adapt to higher temperature, showing altered thermal curves in some years (hogg et al., 1998; van doorsalaen et al., 2009). in the case of cold stenothermal or stenotopic species, a probable loss is expected (jacobsen et al., 2012), as was observed in some localities in the apennines for some species, such as diamesa insignipes (rossaro et al., 2006b). even if species response to altitude is surely influenced by water temperature, high elevations also imply different habitats and different ecological conditions. therefore species distribution could be constrained by other factors. for example, the chidb data showed that some species colonizing high altitude lakes such as zavrelimyia spp., heterotrissoclaius marcidus, c. scutellata and p. austriacus are more warm stenothermal than predicted by altitude, while species living in kryal, krenal or rhithral habitats such as diamesa spp., pseudodiamesa branickii and p. parva (rossaro, 2006b) are more cold stenothermal than expected. likewise, at lower altitude species living in the profundal zone of lakes, such as p. olivacea, p. bathophila, micropsectra radialis and c. plumosus as well as species living in lowland springs such as brillia bifida, chaetocladius perennis or in the interstitial habitats as hydrobaenus distylus are cold stenothermal. for what concerns lacustrine species, distribution could be affected by water depth beside water temperature (rossaro et al., 2006a; luoto, 2012). only few species showed an optimum depth below 20 m (e.g. m. contracta, p. nigritulum). their distribution plotted against depth showed that they have more than one maximum, often with the main peak at lower depth than the other peaks (figure 11). results suggest that possibly depth does not influence species distribution directly, but indirectly through temperature, dissolved oxygen or competition. different thermal optimum values were derived for different life stages (i.e. larvae vs pupal exuviae), probably due to species phenology. in particular, pupation in chironomids has a short duration, lasting at most 72 h (langton, 1995). therefore pupal exuviae are found in specific seasons and times. on the contrary, larval stage has a long duration, lasting most lifetime. according to species voltinism, more than one generation per year was often observed. this occurs both in lacustrine and in lotic species. this could explain bimodal or trimodal responses of species. lindegaard & mortensen (1988) observed that chironomids generally do not have more than four generations per year, but some species (e.g. c. riparius) have surely more than four generation per year in southern europe areas. thus, a plurimodal response could also be expected, but more data are needed to fit plurimodal models with a higher number of parameters. likewise, plurimodal response could be due to spatial distribution of species, which may show preferences for more than one specific habitat; local adaptations of single populations may as well be responsible for plurimodal trends of some species (dallas & rivers-moore, 2012). in fact, such curves were mostly achieved for eurythermal and euryecious species. sometimes curves with two peaks might suggest the presence of more than one species instead of more than one population. this is the case of taxa belonging to genera rich in species, which are not easily separated at the larval stage, such as diamesa [e.g. d. latitarsis/steinboecki (juvenilia), appendix] and tanytarsus spp. article figure 8. thermal response of micropsectra atrofasciata pupal exuviae (number of individuals m–2) to water temperature (°c) in all habitats (a), alpine ecoregion lowland lakes (b), rhithral (c) and potamal (d). figure 9. thermal response of chironomus spp. larvae. response of c. anthracinus (number of individuals m–2) to water temperature (°c) in alpine ecoregion lowland lakes (a); response of c. plumosus in alpine ecoregion lowland lakes (b), and mediterranean ecoregion lakes (c); response of c. riparius in rhithral (d). jear_2013_2:hrev_master 16/09/13 13.56 pagina 84 no nco mm er cia l tat; local adaptations of single populations may as well be responsible no nco mm er cia l tat; local adaptations of single populations may as well be responsiblefor plurimodal trends of some species (dallas & rivers-moore, 2012). no nco mm er cia l for plurimodal trends of some species (dallas & rivers-moore, 2012). no nco mm er cia l (rossaro, 2006b) are more cold stenothermal no nco mm er cia l (rossaro, 2006b) are more cold stenothermal likewise, at lower altitude species living in the profundal zone of no nco mm er cia l likewise, at lower altitude species living in the profundal zone of micropsectra radialis no nco mm er cia l micropsectra radialis and no nco mm er cia l and c. no nco mm er cia l c. in fact, such curves were mostly achieved for eurythermal and euryeno nco mm er cia l in fact, such curves were mostly achieved for eurythermal and euryecious species. sometimes curves with two peaks might suggest the no nco mm er cia l cious species. sometimes curves with two peaks might suggest the presence of more than one species instead of more than one populano nco mm er cia l presence of more than one species instead of more than one population. this is the case of taxa belonging to genera rich in species, which no nco mm er cia l tion. this is the case of taxa belonging to genera rich in species, which no nco mm er cia l u se expected, but more data are needed to fit plurimodal models with a us e expected, but more data are needed to fit plurimodal models with a higher number of parameters. us e higher number of parameters. likewise, plurimodal response could be due to spatial distribution of us e likewise, plurimodal response could be due to spatial distribution of species, which may show preferences for more than one specific habi-us e species, which may show preferences for more than one specific habitat; local adaptations of single populations may as well be responsibleus e tat; local adaptations of single populations may as well be responsible on ly this could explain bimodal or trimodal responses of species. on ly this could explain bimodal or trimodal responses of species. lindegaard & mortensen (1988) observed that chironomids generally on lylindegaard & mortensen (1988) observed that chironomids generallydo not have more than four generations per year, but some species ( on lydo not have more than four generations per year, but some species () have surely more than four generation per year in on ly) have surely more than four generation per year in southern europe areas. thus, a plurimodal response could also beon ly southern europe areas. thus, a plurimodal response could also be expected, but more data are needed to fit plurimodal models with aon ly expected, but more data are needed to fit plurimodal models with a [journal of entomological and acarological research 2013; 45:e14] [page 85] article table 7. response of lotic species (larvae) to distance from source in all riverine habitats: number of samples, weighted mean, standard deviation, skewness and kurtosis of species abundance vs distance from source values. only the species with ≥100 records in the dataset are reported. species are in phylogenetic order. species n m (km) sd (km) g1 g2 procladius choreus 497 84.56 83.31 1.27 0.06 zavrelimyia barbatipes 118 3.86 20.05 9.52 123.78 conchapelopia pallidula 663 81.40 134.03 3.35 10.99 pseudodiamesa branickii 173 15.96 33.89 2.17 4.00 diamesa steinboecki 108 0.69 7.32 15.03 226.29 diamesa latitarsis 123 4.26 13.38 5.16 29.23 diamesa bertrami 205 2.22 16.28 12.63 218.61 diamesa tonsa 324 12.20 61.51 23.06 817.69 diamesa zernyi 229 1.90 10.74 12.79 238.84 prodiamesa olivacea 207 128.57 96.06 0.14 −1.76 brillia bifida 302 19.64 31.95 3.35 19.85 cardiocladius fuscus 115 18.68 79.56 13.42 331.58 tvetenia calvescens 588 20.91 39.27 4.24 24.07 eukiefferiella brevicalcar 131 0.81 11.87 20.57 475.86 eukiefferiella claripennis 243 19.03 32.48 6.56 50.10 eukiefferiella minor 216 8.79 19.15 4.85 46.65 psectrocladius (psectrocladius) oxyura 162 60.00 16.03 0.20 52.13 rheocricotopus effusus 138 28.92 30.16 0.67 −0.12 rheocricotopus fuscipes 391 48.28 98.83 5.40 30.02 synorthocladius semivirens 163 22.23 40.18 3.74 24.97 orthocladius (euorthocladius) rivicola 457 28.15 66.53 6.22 42.93 orthocladius excavatus 109 31.70 87.00 7.89 133.38 orthocladius frigidus 322 6.39 52.28 33.79 1454.39 orthocladius oblidens 121 55.14 26.38 0.47 7.27 orthocladius rhyacobius 215 35.31 89.49 5.16 85.39 orthocladius rubicundus 106 57.75 43.71 0.40 −0.22 paratrichocladius rufiventris 317 3.76 35.13 29.05 1517.03 paratrichocladius skirwithensis 134 14.01 23.29 2.15 3.32 cricotopus annulator 176 34.10 68.19 7.05 60.01 cricotopus bicinctus 241 128.77 120.05 1.29 2.98 cricotopus triannulatus 197 131.92 128.18 1.72 5.47 cricotopus (isocladius) sylvestris 150 139.59 119.98 0.02 −0.86 metriocnemus hygropetricus 132 31.94 50.11 4.62 42.91 chaetocladius laminatus 117 13.86 30.01 5.65 45.96 parametriocnemus stylatus 241 16.18 27.90 4.51 33.89 parakiefferiella bathophila 101 63.12 1.86 −5.20 2484.04 thienemanniella partita 133 12.60 53.86 9.37 101.81 corynoneura scutellata 233 12.58 61.42 6.02 39.72 tanytarsus gregarius 238 67.50 30.09 10.27 181.68 cladotanytarsus atridorsum 104 57.74 17.82 7.56 111.14 micropsectra atrofasciata 529 35.41 66.56 8.86 269.38 micropsectra pallidula 120 1.54 2.29 2.80 15.69 pseudochironomus prasinatus 119 54.93 5.42 −1.21 11.16 paratendipes albimanus 130 32.79 28.69 5.33 95.49 microtendipes pedellus 235 53.65 38.30 2.77 11.10 polypedilum laetum 164 59.77 73.36 3.91 23.25 polypedilum nubeculosum 434 90.31 86.70 2.38 9.45 dicrotendipes nervosus 188 72.25 78.47 6.66 46.00 glyptotendipes pallens 138 152.20 113.95 0.91 2.80 chironomus anthracinus 273 57.22 19.98 −1.93 2.80 chironomus plumosus 282 26.89 44.06 9.88 134.42 chironomus riparius 227 213.81 69.27 −1.73 2.69 cladopelma viridulum 131 50.98 23.96 −1.54 0.53 cryptochironomus defectus 236 84.54 67.67 2.65 9.59 demicryptochironomus vulneratus 134 53.81 16.62 −2.59 5.90 n, number of samples; m, weighted mean; sd, standard deviation; g1, skewness; g2, kurtosis. jear_2013_2:hrev_master 16/09/13 13.56 pagina 85 no nco mm er cia l no nco mm er cia l no nco mm er cia l 35.31 no nco mm er cia l 35.31 no nco mm er cia l no nco mm er cia l 57.75 no nco mm er cia l 57.75 3.76 no nco mm er cia l 3.76 no nco mm er cia l no nco mm er cia l 14.01 no nco mm er cia l 14.01 34.10 no nco mm er cia l 34.10 no nco mm er cia l no nco mm er cia l 197 no nco mm er cia l 197 no nco mm er cia l no nco mm er cia l no nco mm er cia l 150 no nco mm er cia l 150 132 no nco mm er cia l 132 no nco mm er cia l no nco mm er cia l no nco mm er cia l 117 no nco mm er cia l 117 241 no nco mm er cia l 241 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l u se us e us e 40.18 us e 40.1866.53 us e 66.53 us e us e 87.00us e 87.00 52.28us e 52.28 on ly on ly on ly 6.56 on ly 6.56 on ly on ly4.85 on ly4.850.20 on ly0.20 on ly on ly on ly [page 86] [journal of entomological and acarological research 2013; 45:e14] conclusions chironomids are considered generalist, opportunistic, r-strategy organisms and their distribution is driven by environmental variables, such as water temperature (rempel & harrison, 1987), substrate composition (rae, 1985), current velocity (caspers, 1983) and other variables such as competition, parasitism, predation and other biological constraints (tokeshi, 1995; vodopich & cowell, 1984). water temperature has been often recognized as the factor that accounts for the largest percentage of variation in community composition (heiri et al., 2011). beyond direct effects caused by increased water temperature, such as distribution, phenology and adaptation, also indirect effects are expected, such as different balance of interand intra-specific relation, i.e. competition, predation and parasitism (tixier et al., 2009). these latter aspects still need to be investigated. some chironomid species showed unimodal response to water temperature (larocque et al., 2001), but bimodal and trimodal responses were also frequently found. the present data emphasized that standard deviation generally increased with optimum temperature, meaning that eurythermal species are often warm-water adapted, while coldwater dwellers are mostly stenothermal. nonetheless some warm stenothermal species were also found, being possibly good indicators of water temperature in lowland habitats (e.g. m. pedellus). aquatic insect ecology can be interpreted by an evolutionary perspective. entire orders of aquatic insects probably evolved in cool habitats. thus, groups inhabiting warmer waters are considered later descendants of cool-adapted ancestral lines (ward & stanford, 1982; ward, 1992). it is supposed that plesiomorphic species are cold stenothermal while apomorphic species are warm stenothermal or eurythermal. the chironomid ancestral habitat is supposed to be cool head-waters (brundin, 1966; cranston & oliver, 1987; cranston et al., 2012) and ecology and biogeography of diamesinae gives support to this statement (serra-tosio, 1973; rossaro, 1995). a phylogenetic trend from plesiomorphic cold-stenothermal species to apomorphic warm adapted species was then hypothesized (rossaro, 1991c), since a general trend toward increasing adaptation to warm habitats was observed from cold stenothermal diamesini to warm eurythermal chironomini (rossaro et al., 2007b). this was confirmed only in part, likely because: i) ecological data on species are incomplete, ii) the evolutionary tree of chironomids is not completely known (cranston et al., 2012), iii) the relation between thermal response and the position of a taxon in the phylogenetic tree may be observed at different taxonomic hierarchy, i.e. at the level of populations within the same species, of species within the same genus or of genus within the same tribe. in this paper emphasis is given to water temperature, with the aim of quantifying the responses of single species in different habitats and to describe the detailed pattern of response. the authors acknowledge that results may be biased, being a different number of data available for each species, with a different spatial and temporal resolution in different sites, and thus optimum values must be interpreted with caution. nevertheless it must be considered the difficulty of selecting a balanced database for a large number of species, some of which rare, living in specialized habitats, others common and widespread, living in different habitats. the data considered in the present paper are still fragmentary and will be revised in the future, as soon as new information will become available. at present, a comparison of quantitative results with other published papers is article figure 10. correlation between species optima for water temperature (°c) vs optima for altitude (m a.s.l.). figure 11. response of prodiamesa olivacea (a), micropsectra contracta (b), paracladopelma nigritulum (c), chironomus anthracinus (d) larvae (number of individuals m–2) to water depth (m) in alpine ecoregion lowland lakes. jear_2013_2:hrev_master 16/09/13 13.56 pagina 86 no nco mm er cia l ture has been often recognized as the factor that accounts for the no nco mm er cia l ture has been often recognized as the factor that accounts for the et al. no nco mm er cia l et al., no nco mm er cia l , 2011). beyond direct effects caused by increased water temperature, no nco mm er cia l 2011). beyond direct effects caused by increased water temperature, such as distribution, phenology and adaptation, also indirect effects are no nco mm er cia l such as distribution, phenology and adaptation, also indirect effects are expected, such as different balance of interand intra-specific relation, no nco mm er cia l expected, such as different balance of interand intra-specific relation, et al. no nco mm er cia l et al., 2009). these no nco mm er cia l , 2009). these some chironomid species showed unimodal response to water temno nco mm er cia l some chironomid species showed unimodal response to water tem, 2001), but bimodal and trimodal responses no nco mm er cia l , 2001), but bimodal and trimodal responses were also frequently found. the present data emphasized that standard no nco mm er cia l were also frequently found. the present data emphasized that standard deviation generally increased with optimum temperature, meaning no nco mm er cia l deviation generally increased with optimum temperature, meaning that eurythermal species are often warm-water adapted, while coldno nco mm er cia l that eurythermal species are often warm-water adapted, while coldwater dwellers are mostly stenothermal. nonetheless some warmno nco mm er cia l water dwellers are mostly stenothermal. nonetheless some warm stenothermal species were also found, being possibly good indicators ofno nco mm er cia l stenothermal species were also found, being possibly good indicators ofno nco mm er cia l u se on ly on ly on ly recommended. for example, a comparison could be achieved with estimated tolerance and optima for lacustrine species used as climate proxy in palaeolimnological studies (larocque et al., 2001; larocque-tobler et al., 2012), even if available data are mainly from northern areas. otherwise, a comparison could be carried out with sensitivity derived from specific studies on existing chironomid communities (tixier et al., 2009; čiamporová-zat’ovičová et al., 2010; hamerlik & jacobsen, 2012). knowledge on thermal tolerance of species is important for a longterm management and monitoring of aquatic ecosystems exposed to the effects of climate change. in fact, thermal curves can help anticipate impacts of climate change to various species by quantifying their thermal habitat (hester & doyle, 2011). species response under different global change scenarios can thus be predicted (bonada et al., 2007; sauer et al., 2011). to this purpose, more understanding into species adaptations by acclimation and genetics is also needed (hogg et al., 1998; van doorsalaen et al., 2009). references battegazzore m., petersen r.c., moretti g.p., rossaro b., 1992 an evaluation of the environmental quality of the river po using benthic macroinvertebrates. arch. hydrobiol. 125: 175-206. berra e., forcella m., giacchini r., marziali l., rossaro b., parenti p., 2004 evaluation of enzyme biomarkers in freshwater macroinvertebrates of taro and ticino river, italy. int. j. lim. 40: 169-180. boggero a., füreder l., lencioni v., simcic b., thaler b., ferrarese u., et al., 2006 littoral chironomid community of alpine lakes in relation to environmental factors. hydrobiologia. 562: 145-165. bonada n., dolédec s., statzner b., 2007 taxonomic and biological trait differences of stream macroinvertebrate communities between mediterranean and temperate regions: implications for future climatic scenarios. glob. change biol. 13: 1658-1671. bonomi g., 1974 benton profondo. indagini sul lago di garda. quaderni dell’istituto di ricerca sulle acque. 18: 211-223. brooks s.j., birks h.j.b., 2000 chironomid-inferred late-glacial and early holocene mean july air temperature from kråkanes lake, western norway. j. paleolimnol. 23: 77-89. brundin l., 1966 transantarctic relationships and their significance, as evidenced by chironomid midges, with a monograph of the subfamilies podonominae and aphroreniinae and the austral heptagyiae. kungliga svenska vetenskapsakadamiens handlingar, series 4. 11: 1-472. caspers n., 1983 chironomiden-emergenz zweier lunzer bäche, 1972. arch. hydrobiol. suppl. 65: 484-549. catalan j., curtis c.j., kernan m., 2009 remote european mountain lake ecosystems regionalisation and ecological status. freshwater biol. 54: 2419-2432. chaib n., samraoui b,. marziali l., rossaro b., 2011 chironomid taxocenosis in a south mediterranean wadi, the kebireast (algeria). studi trentini di scienze naturali. 89: 29-34. čiamporová-zat’ovičová z., hamerlík l., šporka f., bitušík p., 2010 littoral benthic macroinvertebrates of alpine lakes (tatra mts) along an altitudinal gradient: a basis for climate change assessment. hydrobiologia. 648: 19-34. cortelezzi a., paggi a.c., rodriguez m.a., rodriguez c., 2011 taxonomic and non-taxonomic responses to ecological changes in an urban lowland stream through the use of chironomidae (diptera) larvae. sci. total environ. 409: 1344-1350. cranston p., hardy n.b., morse g., 2012 a dated molecular phylogeny for the chironomidae (diptera). syst. entomol. 37: 172-188. cranston p.s., oliver d.r., 1987 problems in holarctic chironomid biogeography. entomol. scand. 29: 51-56. dallas h.f., rivers-moore n.a., 2012 critical thermal maxima of aquatic macroinvertebrates: towards identifying bioindicators of thermal alteration. hydrobiologia. 679: 61-76. davies r.g., 1971 computer programming in quantitative biology. academic press, london: 504 pp. ferrarese u., 1983 chironomidi, 3 (diptera: chironomidae: tanypodinae). guide per il riconoscimento delle specie animali delle acque interne italiane. consiglio nazionale delle ricerche: no. 26. aq/1/204. ferrarese u., rossaro b. 1981 chironomidi, 1 (diptera, chironomidae: generalità, diamesinae, prodiamesinae). guide per il riconoscimento delle specie animali delle acque interne italiane. consiglio nazionale delle ricerche: no. 12. aq/1/129. free g., solimini a., rossaro b., marziali l., giacchini r., paracchini b., et al., 2009 modelling lake macroinvertebrate species in the shallow sublittoral: relative roles of habitat, lake [journal of entomological and acarological research 2013; 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j. ent. acar. res..indd l. nilsen integrated pest management as european standard – is it possible? abstract as part of the work within the european committee for standardization (cen), standards for conservation of cultural property are being developed in cen/tc (technical committee) 346, conservation of cultural property. in working group 4 environment, a draft is being prepared to create a proposal for standardised integrated pest management. the author of this paper welcomes delegates to the meeting on cultural heritage pests in piacenza to contribute to the discussion regarding standardised methods for pest control in the cultural heritage sector. key words: ipm, integrated pest management, standardization. introduction standards are an indispensible part of daily life, though many of us may not be aware of it. standards are used in all levels of society – trade, construction, etc. there are standards for where to put the brake pedal in a car, how children’s’ playgrounds should be constructed to fulfil safety requirements, and there is even a standard for how glasses for wine-tasting should be designed in order to fulfil the requirements of the wine making industry. at present, standards are developed as management tools to help us taking care of our environment, and making it easier for companies to become more environmentally aware. the definition of a standard has been interpreted as a ‘voluntary common solution to recurring problems’. the official definition by the european committee for standardization is: a standard (french: norme, german: norm) is a technical document designed to be used as a rule, guideline or definition. it is a consensus-built, repeatable way of doing something. every european country has its own national standard institute that works with national standards, european standards (cen) and global standards (iso). anyone with an interest in creating standards can take part. to give an example, the swedish national mirror group for conservation of cultural property have members from the national heritage board, the royal library, the state property board, museums, universities, a showcase manufacturer and a transport company. european standards on conservation of cultural property in spring 2007 a business plan was formed to start working on european standards j. ent. acarol. res. ser. ii, 43 (2): 107-110 30 september 2011 for conservation of cultural property. technical committee cen/tc 346 conservation of cultural property was created. the business plan states: “a specific european standardisation activity in the field of conservation of cultural property is essential to acquire a common unified scientific approach to the problems relevant to the preservation and conservation of the cultural property.” 1 since the start, there have at times been heated discussions within the conservation community whether standards for conservation are the way forward. critics have said that it is impossible to create standards for conservation, where objects and collections are unique. others have replied that the standards will be of great help to museums, contractors and others who are in need of standardised methods to correctly measure temperature and relative humidity, transport and pack, define colour measurements, and similar. “if you think standards are complicated, you should try without!” is a quote sometimes heard at meetings when consensus is hard to reach. it is important to involve the conservation community as a whole in this work. therefore, cen/tc 346 is liaising with organisations such as icom-cc (international council of museums – committee for conservation), iic (international institute for conservation of historic and artistic works), and ecco (european confederation of conservator – restorers’ organisations). they thus have an opportunity to be of assistance with constructive criticism and add comments. cen/tc 346 is divided in five so called working groups, wgs. wg 4 is called environment, and works for example on standards for measurement of temperature and relative humidity, light and lighting, storage, showcases, heating and ventilation in historic buildings, energy efficiency and risk assessment. presentation of ipm standard in 2010, a new work item was adopted, integrated pest management (ipm). the aim for this proposal was to give a comprehensive standard method managing the pest problem for end users. the scope was to define methods for reducing pests and pest infestations, and managing pest infested objects and areas within the cultural property sector. it was clearly stated that ipm implies a holistic approach to the pest problem, including preventive measures and treatments, emphasizing on non-toxic methods. the justifications for creating a standard were the following: • this standard will not repeat work already done but will draw on and make reference to such work and combine it into one unified european document. • although ipm exists in other areas, the sensitivity and unique nature of cultural material requires a separate standard. • there is a eu requirement on sustainability and increased focus on dangerous substances, for example through the reach directive. since many traditional toxic substances used for insect eradication are being banned, there is a need for prevention rather than cure, and non-toxic methods. • rising temperatures caused by climatic changes may cause influx of insects pests not formerly known in many european countries. end users were identified from a broader basis than just cultural heritage institu 1 cen/tc 346 n. 51, 2007-03-29, see business plan on the official website. journal of entomological and acarological research, ser. ii, 43 (2), 2011108 tions (museums, archives and libraries and similar). in addition commercial companies such as auction houses, transport companies, banks (for storage in bank vaults) and private storage companies were included. it is important to state that there is no new research involved in creating a standard. existing methods and knowledge form the foundations of any standard. the aim with a standard for ipm is digesting and extracting the best available practice from literature into the format of a standard, containing standard methods and planning procedures. at the first task group meeting in florence in october 2010, it was decided that the standard would focus on collections, not building fabric or very large artefacts such as museum ships or similar. a widely accepted framework for preventive conservation developed by cci (canadian conservation institute) was to be followed to guide the work – avoid, block, detect, respond and treat/recover (michalski, 1994). current work two meetings per year are organised within wg 4. a first draft of the standard is being prepared to be presented at the next meeting in bilbao in october 2011. at present, countries that are represented in the task group working with the ipm standard are sweden, denmark, uk and germany. at the latest meeting in visby in april 2011, the following challenges were identified: • it is of great importance that experts from other countries in south and east europe join the group, considering the different regional variations in fauna and also national legislation on eradication methods. • any liaison from national groups working with ipm is welcome. • treatments methods are to be mentioned in the standard. however, they should be dealt with in more details in a part 2 of the standard. the task group is interested to know what kind of existing standards there are on pest management and pest eradication methods. discussion at piacenza meeting the meeting on cultural heritage pests in piacenza provides an excellent opportunity for experts in the field and cultural heritage professionals to give their views and input for a standard for ipm. the author welcomes this possibility, and hopes for a fruitful discussion. useful links: http://www.cen.eu/cen/sectors/technicalcommitteesworkshops/centechnicalcommittees/ pages/default.aspx?param=411453&title=conservation%20of%20cultural%20property (cen/tc 346 conservation of cultural property) http://ec.europa.eu/enterprise/sectors/chemicals/reach/index_en.htm (reach directive) http://europeanreach.net/ (reach directive) l. nilsen: integrated pest management as european standard – is it possible? 109 acknowledgements the organisers of this conference are gratefully thanked for putting this item on the programme. cen/tc 346 wg 4 secretary erling trudsø is thankfully acknowledged for support and assistance. references michalski s., 1994 a systematic approach to preservation: description and integration with other museum activities. in: preventive conservation practice, theory and research. preprints of the contributions to the ottawa congress, 12-16 september 1994, international institute for conservation of historic and artistic work. ed. roy & smith, pp. 8-11. lisa nilsen, conservator acr, lisa nilsen kulturvård (collection care), nytorgsgatan 17 a 116 22 stockolm, sweden. email: lisa@lisanilsenkulturvard.se journal of entomological and acarological research, ser. ii, 43 (2), 2011110 j. ent. acar. res..indd b. krüger-carstensen, r. plarre outdoor trapping and genetical characterization of populations of the webbing clothes moth tineola bisselliella (lepidoptera: tineidae) in the broader area of berlin abstract adapted and effective pest management strategies for the protection of irreplaceable culture heritage as well as for the prevention of damages in households and warehouses are based on reliable information about the presence and distribution of the pest organisms. monitoring the webbing clothes moth tineola bisselliella at thirteen outdoor stations in the broader area of berlin give a first idea of their occurrence in an urban area and the hinterlands. the results demonstrate a seasonal abundance in the city and a missing of this species in the countryside. data suggest a synanthropic occurrence of the webbing clothes moth rather than an invasion from natural reservoires. possible molecular examinations on the species and subspecies level are presented to analyze the gene flow between populations and give an impression of species mobility as well as pathways of infestation. key words: infestation, monitoring, pheromone lures, synanthropy. introduction the webbing clothes moth tineola bisselliella is an economical important cosmopolitan pest. their larvae feed on wool, feather, hair and pelt and effect damages in households, fabrics and on culture heritage (kemper, 1935; becker, 1960, pinniger, 2010). the geographical origin is assumed to be in south africa (meyrick, 1927) from where the webbing clothes moth is immigrated with the developing global trade in the modern age. in europe it was first mentioned in the 19th century (zeller, 1852). in the course of urbanization natural habitats are destroyed and a combination of physical factors is recreated (weidner, 1958). human homes with a balanced indoor climate, stored products and protection against environmental hazards offer a new diversity of food and habitats (laibach, 1970). this situation provides niches with the opportunity for the colonization of invasive species from warmer clime with the ecological potential to survive and reproduce among dry conditions. t. bisselliella has a wide tolerance for abiotic environmental factors and its resistance to a relative humidity about 30% suggests a synanthropic occurrence (griswold & crowell, 1936). identifications of the webbing clothes moth in natural reservoires like bird nests are rare in the literature and do not indicate for a sustained colonization of such habitats in a moderate temperature j. ent. acarol. res. ser. ii, 43 (2): 129-135 30 september 2011 zone (plarre, 2009, plarre & krüger-carstensen, 2011). although some nidicolous insect species of the keratinophagus guild are known as pest organisms in flats and warehouses an invasion of the webbing clothes moth from natural habitats seem to be improbable (abraham & peters, 2008). due to the little notion to fly of t. bisselliella, especially of the females, it is supposed that it can not overcome a distance about several meters with the exception of some cases or the utilization of favourable wind (kemper, 1935; weidner, 1970). in spite of numerous observations and monitoring studies of this pest organism the pathways of infestation remain obscure (back, 1931; child & pinniger, 1993, brand & wudtke, 1997). for the pest management today which is first concentrated on the prevention of pest outbreak, the success strongly depends on the localization of the origin of infestation. this paper shows results of an outdoor trapping of t. bisselliella in the broader area of berlin and discusses the possible pathways of infestation. materials and methods from the end of march 2010 up to november 2010 thirteen outdoor trapping locations in the broader area of berlin were selected for a monitoring study of t. bisselliella (fig. 1). sticky traps with pheromone lures from the company insect limited inc. (usa) were handed out to volunteers who put the traps on the balcony or in the fig. 1 twelve of the thirteen trapping locations (fl01 – fl23) in the broader area of berlin (missing trapping location fl26 is situated in the north of brandenburg and not included in this map). journal of entomological and acarological research, ser. ii, 43 (2), 2011130 garden of their dwelling house. the lure, a polyethylene dispenser, contains a synthetic pheromone with the components of the sex pheromone of t. bisselliella. both, lures and traps, were biweekly replaced and analyzed for the number of trapped organisms at each trapping location. since the pheromone lures only trap the males we also tested funnel traps filled with feather which were saturated with a 5% yeast dilution and replaced them every fourth week. the contents of each trap were incubated at 27°c and 68% relative humidity and checked biweekly for moth eclosion. results the funnel traps did not contain any moths and no moth eclosed within five month after incubation at 27°c and 68% r.h. the results from the outdoor trapping with sticky traps containing pheromone lures indicate a seasonal abundance of t. bisselliella in the city of berlin. starting with the moth fly in the end of march the number of trapped webbing clothes moths increase up to the midyear and drop about the summer with single last catches in autumn, the begin of october (fig. 2). in the hinterlands the number of trapped webbing clothes moths is low or absent and decrease with a growing distance to the city center (fig. 3). the trapping locations in the city also show some larger or smaller differences in the number of trapped moths of t. bisselliella but the occurrence does not base upon the city center distance (fig. 3) or the human population b. krüger-carstensen, r. plarre: outdoor trapping of tineola bisselliella fig. 2 biweekly results of a seven months outdoor trapping of t. bisselliella at eight trapping locations (fl03, fl04, fl08, fl09, fl10, fl12, fl14, fl23) in berlin. the observation was done from the 26th march up to the 5th november 2010. 131 fig. 3 trapping results over a period of seven months (26.03 – 05.11.2010) for each trapping location in several districts of berlin and in the hinterlands. (0 = no trapped webbing clothes moth) with increasing distance to the city center of berlin. density (data not shown). fig. 4 shows the results for each trapping locations separated into three groups concerning their disposition to the next dwelling house. every balcony and terrace trap catches a different number of moths from t. bisselliella. also, the traps in the gardens have different results of the number of trapped webbing clothes moths but their occurrence diminishes with prolonging distance of the trap to the next dwellings. discussion and conclusion this paper uses the well established monitoring method with pheromone lures for an outdoor trapping of t. bisselliella to get an impression of possible natural reservoirs and their appearance in our geographical area. whereas kemper (1935) suggests two generations with a fly period in the summer and autumn, the results from our first outdoor trapping of t. bisselliella in 2010 point out one generation per year, only (fig. 2). a seasonal abundance with a presence during the warm season from april up to november and a disappearance during the coolest months, from december up to march, was observed in warehouses by trematerra & fontana (1996), too. but they also detected responsiveness of the males during winter period if the temperature conditions were favourable. journal of entomological and acarological research, ser. ii, 43 (2), 2011132 the decreasing occurrence of the webbing clothes moth in the hinterlands compared to a higher number of trapped moths in the city leads to the question of their origin (fig. 3). a high-density housing in urban areas like berlin not only offers a higher probability for infestation around the trapping location than the countryside but also more beneficial climate due to the heat island effect (frankie & ehler, 1978; pickett et al., 2001; kuttler, 2004). if we have a look on the number of trapped webbing clothes moths in the city (fig. 4), differentiated into the traps on a balcony and terrace, the data fluctuated without any evident reason. but for the trapping locations in the gardens the catches decline with an increasing distance to the dwellings. therefore, the occurrence of webbing clothes moths in natural habitats is more and more unlikely. if we take into account that the adults of this species rather stay inside of the infested material and fly only over a short distance, the origin of the trapped organisms should be close to the trapping location (trematerra & fontana, 1996). natural reservoirs like bird nest are availible in the city as well as in the countryside and if the webbing clothes moth is a nidicolous living insect, we should also find it in the countryside. but no funnel trap containing feathers mixed with yeast caught any webbing clothes moth in the hinterfig. 4 trapping results over a period of seven months (26.03 – 05.11.2010) for each trapping location in the broader area of berlin (fl14, fl04, fl12, fl23, fl09, fl10, fl08, fl01, fl03, fl11, fl26, fl19, fl20). trapping locations are separated into three groups concerning their disposition to the dwellings. 133b. krüger-carstensen, r. plarre: outdoor trapping of tineola bisselliella lands although yeast odor is known to lure females ready for oviposition (traynier et al., 1994). if t. bisselliella represents an absolute synanthropic living insect and natural reservoirs are lacking, an infestation would mainly correlate with the dispersal of infested material and the population of each trapping location must then be distinguishable. a low mobility of populations is attended by the inhibition of the gene flow between them and become manifest in genetic differences over the time. large genetic divergences with a minimal morphological change are possible and result in so-called “cryptic species” (beebee & row, 2007). a powerful tool for studying population genetics is a mitochondrial genome (mtdna) analysis. because of its generally conserved gene complement and rapid rate of nucleotide substitution it provides an ideal system for comparative studies (hu et al., 2009). mainly the mtdna control region is the most variable segment and has a large non-coding region containing controlling elements for replication and transcription (boore, 1999). the molecular analysis of the trapped webbing clothes moths as well as further examinations about the occurrence of t. bisselliella at the urban area and the hinterlands are the next steps to be carried out to get definite information about the pathways of infestation. references abraham r., peters r. s., 2008 nistkästen als lebensraum für insekten, besonders fliegen und ihre schlupfwespen. vogelwarte, 46: 195-205. back e. a., 1931 the control of moths in upholstered furniture. u. s. department of agriculture, farmers’ bulletin, 1655. becker g., 1960 biologische untersuchungen an textilien. handbuch der werkstoffprüfung 2. auflage band 5, thieme verlag, berlin/ göttingen/ heidelberg 971-1007. beebee t., row g., 2007 an introduction to molecular ecology. chapter 1. a history of molecular ecology. ii. ed. oxford university press. boore j. l., 1999 animal mitochondrial genomes. nucleic acids research, 27 (8): 1767-1780. brand, j., wudtke, a., 1997 bekämpfung von textilschädlingen mit kohlenstoffdioxid. restauro 4, callwey verlag. child r. e., pinniger d. b., 1993 insect trapping in museums and historic houses. proceedings of the first international conference on urban pests, 1993. frankie g. w., ehler l. e., 1978 ecology of insects in urban environments. ann. rev. entomol., 23: 367-387. griswold g. h., crowell m. f., 1936 the effect of humidity on the development of the webbing clothes moth, tineola bisselliella. ecology, 17: 241-250. hu l., jianyu g., haiyu l., wanzhi c., 2009 progress in the researches on insect mitochondrial genome and analysis of gene order. science foundation in china, 17 (2): 39-45. kemper h., 1935 die pelzund textilschädlinge und ihre bekämpfung; hygienische zoologie, monographien zur biologie und bekämpfung der gesundheitsund wohnungsschädlinge. band 7, deutsche gesellschaft für kleintierund pelztierzucht gmbh & co, leipzig: 69. kuttler w., 2004 stadtklima teil 2: phänomene und wirkungen. zeitschrift für umweltchemie und ökotoxikologie, 16 (4): 263-274. journal of entomological and acarological research, ser. ii, 43 (2), 2011134 laibach e., 1970 textilien – nahrung und unterschlupf für insekten während des transportes, im lager und haushalt. zeitschrift für angewandte entomologie, 65 (4): 431-435. meyrick e., 1927 a revised handbook of british lepidoptera. watkins and doncaster, london, uk. pickett s.t.a., cadenasso m. l., grove j. m., nilon c. h., pouyat r. v., zipperer w. c., constanza r., 2001 urban ecological systems: linking terrestrial ecological, physical, and socioeconomic components of metropolitan areas. annu. rev. ecol. syst., 32: 127-157. pinniger d., 2010 saving our heritage pest management in museums and historic houses. outlooks on pest management, 21 (5): 239-241. plarre r., 2009 materialund vorratsschädlinge, ihre anpassung an den synanthropen lebensraum sowie ihre wirtschaftliche relevanz einschließlich ihrer bekämpfung. habilitationsschrift fachbereich biologie, pharmazie, chemie der freien universität berlin. plarre r., krüger-carstensen b., 2011 an attempt to reconstruct the natural and cultural history of the webbing clothes moth tineola bisselliella (lepidoptera: tineidae). journal of entomological and acarological research, ser. ii, 43 (2): 83-93. traynier r. m. m., schumacher r. k., lau d. m., 1994 oviposition site selection by tineola bisselliella, tinea spp (lepidoptera: tineidae) and anthrenus flavipes (coleoptera: dermestidae). journal of stored products research, 30 (4): 321-329. trematerra p., fontana f., 1996 monitoring of webbing clothes moth, tineola bisselliella (hummel), by sex pheromone. anz. schädlingskde. pflanzenschutz umweltschutz, 69: 119121. weidner h., 1958 die entstehung der hausinsekten. zeitschrift für angewandte entomologie, 42 (4): 429-447. weidner h., 1970 die kleidermotte, tineola bisselliella (hummel, 1823). der praktische schädlingsbekämpfer, 22(5): 70-76. zeller p.c., 1852 die schaben mit langen kieferntastern. linnaea entomologica, 6: 81-197. bianca krüger-carstensen, bam, unter den eichen 87, 12205 berlin, germany. e-mail: bianca.krueger-carstensen@bam.de rudy plarre, bam, unter den eichen 87, 12205 berlin, germany. e-mail: ruediger.plarre@bam.de 135b. krüger-carstensen, r. plarre: outdoor trapping of tineola bisselliella j. ent. acar. res..indd j. ent. acarol. res. ser. ii, 43 (2): 157-168 30 september 2011 m. schöller, s. prozell biological control of cultural heritage pest coleoptera and lepidoptera with the help of parasitoid hymenoptera abstract natural enemies are known from many cultural heritage pests, but their potential for biological control has been marginally exploited only. in this publication, examples of practical and commercial application of parasitoids of beetles and moths are compiled as well as laboratory research that contributes to the development of guidelines for parasitoid releases. one the one hand there are parasitoids found to occur simultaneously with the pests in buildings, on the other hand there are parasitoids that were never found to be associated with the respective pests but accept them if brought into the cultural heritage environments. an example for the latter is the egg parasitoid trichogramma evanescens euproctidis, a parasitoid of moth eggs including those of the cloth moth tineola bisselliella. in semi-field trials it was shown that inundative releases of the egg parasitoids are necessary and that effectiveness is reduced on thick cloth with long strand. trichogramma release units have to be placed directly on the cloth to be protected. a naturally occuring parasitoid of anobiid beetles is the pteromalid larval parasitoid lariophagus distinguendus. this parasitoid was applied against the drugstore beetle stegobium paniceum in historic libraries and against spider beetles (ptininae) in historic buildings. a simulation model for the population-dynamics of l. distinguendus and the golden spider beetle niptus hololeucus is presented. finally, monitoring of the braconid larval parasitoid spathius exarator used for indirect monitoring of the common furniture beetle anobium punctatum is described. the future potential of parasitoids to control cultural heritage pests is discussed. key words: museum pests, ptininae, natural enemies, monitoring, parasitoids. introduction biological control in a strict sense is the application of living organisms for the control of pest organisms. a further subdivision into micro-biological control applying smaller organisms like e.g. fungi or bacteria, and macro-biological control applying larger organisms like nematodes or insects is useful and relevant in the context of registration of biological control agents (franz & krieg, 1982). other biologically based controls, like bio-technological methods (e.g. bt-toxins), pheromones and other semiochemicals, insect growth regulators and phytochemicals (botanical insecticides) are not included here. this review focuses on commercially available parasitoids for journal of entomological and acarological research, ser. ii, 43 (2), 2011 the control of beetles and moths attacking cultural heritage. adult parasitoids search for hosts (i.e. pests) for oviposition, and the progeny of parasitoids typically need only one host individual to complete development (franz & krieg, 1982). consequently one advantage of parasitoids is the active search for pest individuals in hidden places and their ability to locate hosts also at low host-densities. parasitoids feed exclusively on host insects, frequently the adults do not need to feed at all and they do not damage artefacts. the target pests are synanthropic insects attacking wood or other materials used for museum items, or the buildings themselves. while a number of species can be found almost exclusively associated with these materials, there is another group of species that attack additionally stored products for human consumption. these stored-product pests may destroy materials as well, either by feeding on the materials or on their way to pupation sites. while little information is available on natural enemies of the more specific cultural heritage pests, and almost none on biological control, a lot of information is available on natural enemies and biological control of stored-product pests (schöller et al., 2006). looking at the parasitoids studied in this context, on the one hand there are species that are associated with human-based habitats and their stored-product insect hosts. on the other hand, there are parasitoid species that accept stored-product insects as hosts, but were transferred from agricultural ecosystems to indoor habitats. biological control of the cloth moth tineola bisselliella egg parasitoids in the genus trichogramma are applied for biological control of various pest lepidoptera in field crops like corn or apple. they are polyphagous and accept eggs of many lepidoptera (wajnberg & hassan, 1994). immature trichogramma sp. are glued on cardboard release units, the adults emerge continuously for several weeks. nowadays, release units are available that provide activity of t. evanescens euproctidis (girault, 1911) for three or even 4 weeks. several species of trichogramma have been shown to accept eggs of tineola bisselliella (hummel, 1823) including t. evanescens euproctidis, the species applied against stored-product moths in central europe (zimmermann et al., 2003). for trichogramma spp., the surface structure of textiles and carpests is comparable with hairy leaf surfaces. hairy leaf surfaces were shown to reduce the foraging activity of trichogramma sp., e.g. on tomato leaves (wührer, 1994). in order to test if a large surface will reduce the effectiveness of released parasitoids on cloth, zimmermann (2005) placed cloth (25 cm x 45 cm) in cages (100 cm x 50 cm x 65 cm) previously used for semi-field trials with trichogramma spp. on green plants. he compared 3 types of cloth: (1) finely woven cloth 1.5 mm in thickness without long distant strand (fibres) (2) medium-finely woven cloth ca. 3.0 mm in thickness with long distant strand (fibres) and (3) tanned sheepskin rug ca. 2.5 cm in thickness. five batches of eggs with 120 t. bisselliella eggs each were placed 10 cm, 20 cm, 30 cm and 40 cm from the release point as baits. fresh t. bisselliella-host eggs were provided on day 2, 3 and 5 after release of trichogramma individuals. the number of trichogramma individuals on the egg baits were recorded as well as parasitism by counting black host eggs. the number of female t. evanescens active on the cloth was increasing with an increasing number of parasitoids released (fig. 1). the number of 158 m. schöller, s. prozell: biological control of cultural heritage pest with parasitoids fig. 1 number of active female trichogramma evanescens on fi nely woven cloth depending on the number of parasitoids released. after data in zimmermann (2005). fig. 2 number of active female trichogramma piceum on cloth depending on the thickness of the cloth. after data in zimmermann (2005). 159 trichogramma-females active on the cloth was decreasing with increasing thickness of the cloth (fig. 2). trials with egg-baits on stuffed animals pointed in the same direction (prozell & schöller, unpubl.). the conclusions drawn for the release recommendations were: trichogramma spp. have to be applied inundatively, in most cases all year round, and the release units have to be placed directly on the cloth or shelf to be protected. currently 1000 t. evanescens per m2 and week are recommended. first applications of t. evanescens euproctidis were performed in a depot of an ethnographical museum in south-west germany, and in the jewish museum in berlin. in depots, the identification of infested items might be very time consuming, but the parasitoids actively search for moth eggs. in the jewish museum, the parasitoids helped to suppress a residual cloth moth population feeding on fluff balls formed by wear debris of the visitor’s wool clothes in cracks and crevices and was integrated with an improved cleaning procedure. for more recent releases in museum collections see biebl & querner (this volume). one of the problems associated with the release of trichogramma spp. for control of the cloth moth is the exclusive acceptance of the egg stage of the moth. the disruption of the developmental cycle is expected to be slow due to the slow developmental speed of the cloth moth. consequently the integration with larvicidal strategies is a future challenge. however, as a preventive control strategy at low densities of the cloth moth t. evanescens is already a valuable tool. considerable work has also been done to study the braconid wasp apanteles carpatus (say, 1836), however, this parasitoid is not commercially available yet. apanteles carpatus is a solitary koinobiont endoparasitoid of t. bisselliella and tinea pellionella l., 1758 larvae, i.e. one wasp develops per moth host larva and the moth larva is carrying the parasitoid larva for some time before being killed by the latter. a. carpatus is capable of complete development in all larval stages of t. bisselliella (plarre et al., 2000). for a field study in a heavily infested rug store, plarre et al. (1999) released laboratory reared a. carpatus monthly. the release of a. carpatus alone had no suppression effect on the cloth moth population, only the combination of a. carpatus-release with a sanitation program significantly reduced the pest. a number of other predators, parasitoids and pathogens of t. bisselliella are known (zacher, 1933; wudtke, 2002; zimmermann, 2005), but none of them was evaluated for biological control so far. biological control of the drugstore beetle stegobium paniceum stored-product pests may destroy materials as well, either on their way to pupation sites or because the materials contain ingredients suitable for development. in halle / saale, saxony-anhalt, germany, a historic library became infested by the drugstore beetle stegobium paniceum (l., 1758). the beetles were thriving both below the floorboard on wheat straw used as insulation, and in book covers. the books originated from the 16th to 18th century, when the book covers were filled with pulp made from linen scraps. stegobium paniceum developed in the pulp, produced the characteristic exit holes and therewith destroyed irreplaceable cultural heritage. the books were moved to a fumigation-chamber and treated with nitrogen. however, some re-infestation was detected after the books were moved back to the library, presumably originating from journal of entomological and acarological research, ser. ii, 43 (2), 2011160 the floorboard. the parasitoid lariophagus distinguendus (förster, 1841) was released on the shelves, 2000 in october and 2000 in june. the release was evaluated to have successfully suppressed the re-infestation of the library (schöller, 2010). another trial on host-finding in boxes containing books was carried out in an israeli library, where l. distinguendus was shown to find host larvae both between and inside infested books (wilamowski et al., 2008). a bibliography of the natural enemies of stegobium paniceum and the tobacco beetle lasioderma serricorne (f., 1792), a species with similar biology, is given in schöller (1998). tab.2 temperature and relative humidity conditions used in the model of biological control of niptus hololeucus by lariophagus distinguendus with sitophex software. tab.1 oviposition of niptus hololeucus at different temperatures, data from howe & burges (1952) used for simulation with sitophex. m. schöller, s. prozell: biological control of cultural heritage pest with parasitoids 161 fig. 3 simulation of biological control of niptus hololeucus by releases of lariophagus distinguendus with sitophex. arrows indicate 7 releases of 400 l. distinguendus each, black triangles = young n. hololeucus larvae, white triangles = old n. hololeucus larvae, other stages below 20 individuals. fig. 4 simulation of biological control of niptus hololeucus by releases of lariophagus distinguendus with sitophex. arrows indicate 4 releases of 400 l. distinguendus each, black triangles = young n. hololeucus larvae, white triangles = old n. hololeucus larvae, other stages below 20 individuals, oblique arrow (right) indicates increase in young larvae. fig. 5 simulation of biological control of niptus hololeucus by releases of lariophagus distinguendus with sitophex. arrows indicate 4 releases of 400 l. distinguendus each, black triangles = young n. hololeucus larvae, white triangles = old n. hololeucus larvae, other stages below 20 individuals. journal of entomological and acarological research, ser. ii, 43 (2), 2011162 biological control of spider beetles (anobiidae, ptininae) spider beetles are mainly scavengers feeding equally on plant or animal materials. beside their natural habitats, a number of species infest historic houses feeding on organic insulation materials and become a nuisance in residences (howe, 1959). moreover, spider beetles were found to infest historic books and herbaria (gamalie, 2006). a number of spider beetle species were found to be suitable hosts for l. distinguendus, such as ptinus fur l., 1758 (herold, 1933; hüsing, 1935), ptinus tectus boieldieu, 1856 (kaschef, 1955), gibbium psylloides (czenpinski, 1778) (kaschef, 1961) and niptus hololeucus (faldermann, 1835) (schöller, unpubl.). spider beetles are difficult to control in houses because the larvae develop hidden within walls and in dead floors, and no monitoring devices are available. in recent years, l. distinguendus was released against the hump beetle g. psylloides and the golden spider beetle n. hololeucus in germany by pest control companies and became a regularly applied control technique (kassel, 2008). however, due to the lack of appropriate monitoring techniques, the optimal timing of the parasitoid releases has still to be determined. in this case, modelling the pest and parasitoid’s population dynamics might be useful. as a first step, we used the modelling software “sitophex” (rossberg et al., 2004) originally programmed for the system sitophilus granarius – lariophagus distinguendus. we replaced biological data of the granary weevil s. granarius (l., 1758) by those of the golden spider beetle n. hololeucus. one of the major differences in biology of the granary weevil compared to the golden spider beetle is the low reproduction of the latter at temperatures higher than 25°c (tab. 1). the temperature and relative humidity conditions used in the model are given in tab. 2. fig. 3 shows a simulation of the current release strategy by pest control companies, i.e. monthly releases when temperature conditions are favourable. the susceptible old larval stages and the pupae are controlled within one month and the population is suppressed in a way that few or no adults enter the living rooms. if the number of releases is reduced to four, one in beginning of july, september, march and may, respectively, an increase in young larvae is predicted in june (fig. 4). however, if the timing of the four releases is changed to beginning of july, march, may and june, this increase in young larvae can be suppressed (fig. 5). this effect can be explained by the poor development of n. hololeucus during high temperatures in september, indicating the importance of population suppression early in spring in temperate climates. even though these models cannot be validated at present due to the lack of monitoring devices, simulation models are thought to give some decision-support for parasitoid releases. monitoring of parasitoids of anobium punctatum the study of natural enemies of cultural heritage pests might be useful not only for biological control, but also for monitoring. early detection of material destroying pests is essential to prevent damage, especially when irreplaceable objects of cultural heritage are concerned. however, monitoring of these pest species is often difficult. for example, pheromone traps for detection of anobium punctatum (degeer, 1774) resulted in very poor trap catches in field trials in germany. during the course of study m. schöller, s. prozell: biological control of cultural heritage pest with parasitoids 163 of natural enemies, it turned out that some natural enemies are more easily detectable and give indirect evidence of the presence of the pest. paul et al. (2007) studied a. punctatum and its natural enemies in a church closed for restoration in erfurt, germany. fig. 6 number of anobium punctatum caught in yellow dish traps in a church in erfurt, germany. after data in paul et al. (2007). fig. 7 number of spathius exarator caught in yellow dish traps in a church in erfurt, germany. after data in paul et al. (2007). journal of entomological and acarological research, ser. ii, 43 (2), 2011164 yellow dish traps, a monitoring technique used in outdoor ecological field studies was used here in the context of protection of museum artefacts and wood. yellow dishes are filled with water and a bit of detergents in order to attract flower-visiting insects. these traps are especially attractive for parasitoids that do no host-feeding and rely on nectar for adult nutrition. other arthropods are trapped by chance, the number caught in the trap is affected by the number of insects present and temperature. fig. 6 shows the number of a. punctatum and fig. 7 the number of the braconid parasitoid spathius exarator (l., 1758) trapped in yellow dish traps. few a. punctatum were trapped, mostly between mid of june and mid of july. s. exarator was trapped throughout the trapping season from mid of may to mid of july in relatively large numbers, the peak coinciding with that of a. punctatum. the presence of a. punctatum could therefore be proven before adult beetles became active. such relationships have to be worked out for each parasitoid-host combination, e.g. the death watch beetle xestobium rufovillosum degeer, 1774 was trapped on white sticky traps, but only small numbers of its predator korynetes caeruleus (degeer, 1775) and no parasitoids (belmain et al., 1999). conclusions and outlook the speculations about the potential of biological control of cultural heritage pests by pinniger (2001) did not fulfil 10 years later. pinniger (2001) reported one of the questions most frequently asked to him as a pest manager in museums, archives and historic houses was ‘can we use biological control for pests in museums?’ and his answer for all practical purposes was ‘no’. this view was based on the widespread assumption that a biological equilibrium is needed as a prerequisite for biological control. the result of a biological control would be again a biological equilibrium including the presence of a low level of pests to ensure the continuity of the system resulting in a failure to meet the requirement for zero tolerance of pests in museum environments. however, the inundative release strategy was developed already in the 1960’s to resolve this problem by the release of large numbers of laboratory-reared natural enemies in order to artificially augmenting the number of parasitoids compared to the hosts, i.e. the pests (debach & hagen, 1964). each parasitoid female is adapted to increase its own fitness, i.e. to produce as much offspring as possible, and this may lead even to a local extinction of the host in such an artificial system. no pests have to be released in collection areas in order to maintain the equilibrium as suggested by pinniger (2001) when the inundative control strategy is applied. it was the inundative release strategy that was applied both in stored-product protection and in the commercial applications of biological control in museums so far. the second assumption of pinniger (2001) was high cost of natural enemies. in the cases of t. evanescens euproctidis (3000 wasps cost 2 euros) and l. distinguendus (40 wasps cost 10 euros) this seems not to limit the application, however, this might be an issue for more specialised natural enemies or those that are difficult to mass-rear. m. schöller, s. prozell: biological control of cultural heritage pest with parasitoids 165 biological control of cultural heritage pests is in its very beginnings. several species of natural enemies were recorded to occur spontaneously in houses, but even the host-parasitoid or predator-prey relationships are not clarified yet for all cases (becker, 1954). their potential for biological control is far from been exploited. social insects like termites and ants developed multiple defence strategies possibly limiting biological control efforts in this area. the examples of practical application described here show that both the control strategies and the potential control success heavily depend on the artefacts to be protected and the biology of the pest species, i.e. every application has to be worked out in detail. for example, the population dynamics of l. distinguendus with niptus hololeucus as host will not apply for gibbium psylloides as host. in many field studies parasitoids have been shown to be effective at low pest numbers, and typically heavy infestations cannot be controlled by natural enemies. biological control of stored product pests is nowadays widely known by farmers and industry and is applied by pest control companies in central europe against moths and beetles. those parasitoids that attack both stored-product and cultural heritage pests will be commercially available for future field trials. the development of a commercial application of other species of natural enemies migth require 5 to 10 years, consequently a lot of work remains to be done for the commerzialisation of natural enemies of cultural heritage pests. references belmain s.r., simmonds m.s.j., blaney w.m., 1999 death watch beetle, xestobium rufovillosum, in historical buildings: monitoring the pest and its predators. entomologia experimentalis et applicata, 93: 97-104. debach p., hagen k.s., 1964 manipulation of entomophagous species. in: debach p., biological control of insect pests and weeds. chapman and hall, london: 429-455. becker g., 1954 räuber und parasiten holzzerstörender insekten in gebäuden. verhandlungen der deutschen gesellschaft für angewandte entomologie, 1954: 76-86. franz j.m., krieg a., 1982 biologische schädlingsbekämpfung. parey, berlin, 3. auflage, 252 pp. gamalie g., 2006 old books damaging ptinids (insecta, coleoptera, ptinidae) in romania. analele stiintifice ale universitatii “al. i. cuza” din iasi sectiunea biologie animala, 52: 137-146. howe r.w., burges h.d., 1952 studies on beetles of the family ptinidae. vii. the biology of five ptinid species found in stored products. bulletin of entomological research, 48 (1): 153-186. howe r.w., 1953 studies on beetles of the family ptinidae. viii. the intrinsic rate of increase of some ptinid beetles. annals of applied biology, 40 (1): 121-133. paul f., prozell s., schöller m., 2008 monitoring natürlicher feinde des gemeinen nagekäfers anobium punctatum (degeer, 1774) (coleoptera: anobiidae). mitteilungen der deutschen gesellschaft für allgemeine und angewandte entomologie, 16: 323-326. herold w., 1933 über einige wohnungsschädlinge (glyciphagus, lathridius, ptinus, tenebrio, und lariophagus). mitteilungen der gesellschaft für vorratsschutz, 9: 51-56. hüsing j., 1935 über einen parasiten, lariophagus distinguendus först. (hym., chalc.), an ptinus fur l. (col., ptin.). zoologischer anzeiger, 110: 324-326. journal of entomological and acarological research, ser. ii, 43 (2), 2011166 kassel a., 2008 im würgegriff. biologische schädlingsbekämpfung bei messingkäferund kugelkäfer-befall. b+b bauen im bestand, 31: 42-43. kaschef a.h., 1955 étude biologique du stegobium paniceum l. (col. anobiidae) et de son parasite lariophagus distinguendus först. (hym. pteromalidae). annales de la societé entomologique de france, 124: 1-88. kaschef a.h., 1961 gibbium psylloides czemp. (col., ptinidae) new host of lariophagus distinguendus först. (hym., pteromalidae). zeitschrift für parasitenkunde, 21: 65-70. pinniger d., 2001 pest management in museums, archives and historic houses. archetype publications, london: 115 pp. plarre r., lieber k., burkholder w., phillips j., 1999 host and host instar preference of apanteles carpatus (say) (hymenoptera: braconidae) a possible parasitoid for biological control of clothes moths (lepidoptera: tineidae). journal of stored products research, 35: 197213. plarre r., lieber k., burkholder w., 2000 host instar preference of apanteles carpatus (say) (hymenoptera: braconidae) a parasitoid of tineola bisselliella (hummel) (lepidoptera: tineidae). mitteilungen der deutschen gesellschaft für allgemeine und angewandte entomologie, 12: 499-504. rossberg d., prozell s., steidle j.l.m., schöller m., neukampf r., 2004 modeling software sitophex / kornkäfer. julius kühn-institut, berlin, germany. schöller m., 1998 biologische bekämpfung vorratsschädlicher arthropoden mit räubern und parasitoiden. sammelbericht und bibliographie. in: reichmuth ch., 100 jahre pflanzenschutzforschung. wichtige arbeitsschwerpunkte im vorratsschutz. mitteilungen aus der biologischen bundesanstalt für landund forstwirtschaft, parey, berlin, heft 342: 85-189. schöller m., flinn p.w., grieshop m.j., zdarkova e., 2006 biological control of stored product pests. in: heaps j.w., insect management for food storage and processing. second edition. aacc international, st. paul, minnesota: 67-87. schöller m., 2010 biological control of stored-product insects in commodities, food processing facilities and museums. julius-kühn-archiv, 425: 599-609. wajnberg e., hassan s.a., 1994 biological control with egg parasitoids. wallingford, uk: cab international on behalf of the international organization for biological control of noxious animals and plants: xiv + 286 pp. wilamowski a., kessler i., rabin i., prozell s., navarro s., 2008 biological control of anobium punctatum in infested books, using the parasitoid lariophagus distinguendus. ninth international conference on ndt of art, jerusalem israel, 25-30 may 2008, http://www.ndt. net/article/art2008/papers/232wilamowski.pdf wudtke a., 2002 mö glichkeiten des methodentransfers vom vorratsschutz zum materialschutz. bekä mpfung von museumsschä dlingen am beispiel der kleidermotte tineola bisselliella (hum. 1823), lepidoptera: tineidae mensch und buch verlag, berlin, at the same time dissertation humboldt-universität zu berlin: 130 pp. wührer b., 1994 auswahl wirksamer trichogramma-stämme zur bekämpfung von schadlepidopteren im tropischen gemüsebau. dissertation, university darmstadt: 156 pp. zacher f., 1933 haltung und züchtung von vorratsschädlingen. handbuch der biologischen arbeitsmethoden. parey verlag, hamburg: 92 pp. zimmermann o., schöller m., prozell s., 2003 the use of apanteles carpatus (hym.: braconidae) and trichogramma spp. (hym.: trichogrammatidae) for biological control of tineid moths (lepidoptera). in: credland p.f., armitage d.m., bell c.h., cogan p.m., highley e., advances in stored product protection. proceedings of the 8th international working conference on stored product protection, 22-26 july 2002 york, united kingdom: 319-321. m. schöller, s. prozell: biological control of cultural heritage pest with parasitoids 167 zimmermann o., 2005 untersuchungen zur biologischen bekämpfung der kleidermotte tineola bisselliella (hummel 1823) und anderer tineider textilschädlinge (lepidoptera: tineidae) mit parasitoiden hymenopteren. dissertation johannes-gutenberg-universität zu mainz: 202 pp. matthias schöller, sabine prozell, biologische beratung ltd., storkower str. 55, 10409 berlin, germany. e-mail: bip@biologische-beratung.de journal of entomological and acarological research, ser. ii, 43 (2), 2011168 j. ent. acar. res. ser. ii, 43 (1): 41-46 30 april 2011 gadi v.p. reddy, rosalie kikuchi, jenelyn e. remolona new mite species associated with certain plant species from guam abstract - several new mite species have been reported from certain plants from  guam. most remarkably, the spider mite, tetranychus marianae (prostigmata:  tetranychidae) and the predatory mite  phytoseius horridus (mesostigmata:  phytoseiidae) (solanum melongena) have been found on eggplant. the noneconomically important species of brevipalpus californicus(banks) prostigmata:  tenuipalpidae),eupodes sp. (acarina: eupodidae) and predator  cunaxa sp.  (prostigmata: cunaxidae) have been reported on guava (psidium guajava l.).  also, the non-economically important species brevipalpus californicusprostigmata:  tenuipalpidae), lepidoglyphus destructor (astigmata: glycyphagidae) and a predator  amblyseius obtusus, species groupamblyseius near lentiginosus (mesostigmata:  phytoseiidae), have been recorded on cycad (cycas micronesica). key words - tetranychus marianae, new mite species, eggplant, cycad, guava,  pepper, guam introduction spider mites are common pest problems on many plants around yards and gardens in  guam and other parts of the usa. guam has been a transportation hub for several major  airlines and shipping companies, with extensive connections to other pacific islands in  micronesia, asia, and the continental usa. there are numerous direct flights from guam  to cairns (australia), seoul (south korea), manila (philippines), osaka/tokyo (japan),  taipei (taiwan), hawaii, and to other micronesian islands, that raise concern about the  possibility of introducing mite species to guam from other areas of current infestation.  the introduction of mite species reported in the above asian countries to guam would  increase the probability of their introduction into other areas of the usa. perhaps of  most concern is the presence of various spider mites and broad mites in japan, taiwan  and korea, which greatly increases the probability of their introduction to guam, the  commonwealth of the northern mariana islands, and other islands of micronesia given  the high flow of civilian, military and cargo traffic between these areas by direct air and  sea routes. additionally, some of guam’s food supply is directly imported from asian  countries including, but not limited to the philippines, korea and japan. while no such  journal of entomological and acarological research, ser. ii, 43 (1), 201142 work on mites in guam has been done thus far, there is a possibility that mites may have  come in with such food shipments. local farmers and producers have already brought  to our knowledge information about the occurrence of mites on vegetables and other  such crops. because there have been no studies conducted on mites locally, we have no  knowledge as to what mites may be present in this region. consequently, surveillance of  phytophagous mites will allow local authorities in guam, the cnmi, and elsewhere in  non-infested areas of the pacific to take immediate action upon their discovery, thereby  lessening the chance of successful establishment and spread of the mites throughout the  region. therefore, surveillance work was initiated on guam to record the incidence of  mites on various crop plants.  material and methods the mites provided in vials were prepared by the following method. leaves were  collected from the various agricultural plants, and taken back to the laboratory where  they were examined under a microscope. all mites occurring on a leaf were collected  with a small paint brush moistened with 70% ethanol, placed in a vial containing 5 ml  of 70% ethanol, labeled, and prepared for shipping. the vials with mites were sent to  angela marmont centre for uk biodiversity, natural history museum, london for  identification. results several new mite species, both of economically, and non-economically important  plants have been identified, and the presence of a predatory mite species confirmed.  these are shown in table 1.  1. eggplant, (solanum melongena) specimen-1: the spider mite, tetranychus marianaemcgregor, 1950 (prostigmata:  tetranychidae) was the dominant mite species recorded from the samples collected from  eggplant. the form of the aedeagus is an important character for confirming species  identity. however, the females conform to the redescription of t. marianae given in  moraes et al. (1987).  distribution: widespread in the pacific islands, e.g., the marianas, from where it was  first described.  it has previously been recorded from eggplant on guam (de moraes et al., 1987) but disappeared until recently.  other distribution records include australia,  bahamas, central, south and north (mexico, florida) america, west indies and  southeast asia. 43g.v.p. reddy et al.: new mite from guam significance: tetranychus marianae is known as a pest of tomato and cotton, but also  occurs on a wide range of other plants, e.g., castor bean (ricinus communis), passion  flower (passiflora edulis), squash (cucurbita maxima), centrosema pubescens, sweet  potato (ipomoea batatas), merremia vitifolia and orchidaceae.  spider mites feed on  leaf cell contents and feeding activity is indicated by the appearance of pale areas on  the upper leaf surface as chlorophyll is removed.   table 1 mite species recorded on different host plants on guam scientific name associated with host plants / prey species pest or predatory location with gps points tetranychus marianae  mcgregor, 1950  (prostigmata:  tetranychidae) eggplant (solanum melongena l.) pest mangilao  (13.43°n,  144.80°e) phytoseius horridusribaga,  1904 (mesostigmata:  phytoseiidae) eggplant (solanum melongena l.) predatory mangilao  (13.43°n,  144.80°e) brevipalpus californicus(banks)  prostigmata: tenuipalpidae) guava (psidiumguajava l.) feed on higher  plants yigo (13.54°n,  144.89°e) cunaxa sp. (prostigmata:  cunaxidae) guava (psidium guajava l.) predatory yigo (13.54°n,  144.89°e) eupodes sp. (acarina:  eupodidae) guava (psidium guajava l.) many live in  soil and leaf  litter (not  regarded as  pests) yigo (13.54°n,  144.89°e) brevipalpus californicus(banks)  prostigmata: tenuipalpidae) cycad (cycas micronesica  k.d. hill) feed on higher  plants dededo  (13°30.700’ n,  144°51.173’ e) lepidoglyphus destructor (schrank) (astigmata:  glycyphagidae) cycad (cycas micronesica  k.d. hill) mites of  this genus  are common  inhabitants of  house dust and  stored food merizo  (13°15.058’ n,  144°43.071’ e) amblyseius obtusus species  group, amblyseius near  lentiginosusdenmark  &schicha (mesostigmata:  phytoseiidae) cycad (cycas micronesica  k.d. hill) predatory tamuning  (13°30.109’ n,  144°46.939’ e) journal of entomological and acarological research, ser. ii, 43 (1), 201144 biology: the egg to adult life span lasted 10.73 ± 0.18 days, with a 92% survival. the  sex ratio was 81% females. the mean female longevity was 24.53 days and the daily  mean oviposition was 3.69 eggs / female. the intrinsic rate of increase (rm) was 0.172;  the finite rate of increase (x) was 1.187 individuals / female / day; the mean time span  of one generation (t) was 22.81 days; and the net rate of reproduction (r0) was 50.14  (noronha, 2006). specimen-2: phytoseius horridus species group (mesostigmata: phytoseiidae): phytoseius horridus, most probably p. mayottae schicha, were identified from adult  females. phytoseiids are predominantly predators of other small arthropods, including pest species.   phytoseius mayottae was originally described from specimens collected in mayotte island  (near madagascar), but has also been recorded in sri lanka, the seychelles, vanuatu, new  caledonia and australia.  host plants include acalypha sp., curculigo sp., flemingia strobilifera, glycine javanica, macroptilium atropurpureum, psidium guajava, turnera  sp. and solanum melongena (de moraes et al., 2004).  no information is available on  its biology and feeding preferences. 2. guava (psidium guajava) spicemen-1: false spider mites, flat mites, brevipalpus californicus (banks) (prostigmata:  tenuipalpidae): description: tenuipalpids have been collected in most parts of the world.  they feed  on higher plants, usually on the under surface of the leaves.  they have long, needlelike cheliceral digits with which they pierce the epidermis and feed on the contents of  the underlying cells.  some species are of economic importance.  brevipalpus spp. have  been identified as pests of watermelons in guam (yudin, 1999). the submitted specimen  is morphologically close to b. californicus, a cosmopolitan species that is a pest of a  wide range of ornamental and agricultural crops, but differs slightly in the form of the  ventral ornamentation. spicemen-2: cunaxa sp. (prostigmata: cunaxidae): distribution:  fast moving mites, found in a wide variety of habitats, e.g., on plants  (including crop species), shrubs and trees, and in moss, bark, soil and food stores.  they  prey on small arthropods, other mites and nematodes, including pest species (smiley,  1992).   spicemen-3: eupodes sp. (order prostigmata) species of eupodes have a worldwide distribution.  many live in soil and leaf litter, but  some have been collected on grasses and other plants.  available evidence indicates that  their diet is comprised of algae and fungi, and they are not regarded as pests.   45g.v.p. reddy et al.: new mite from guam spicemen-3: bdellidae, larva (prostigmata: bdellidae) bdellids have a worldwide distribution.  their diet, habits and habitats are like those of  the family cunaxidae (smiley, 1992).  some species have been investigated as control  agents of pest mites, insects and nematodes (gerson et al., 2007). spicemen-4: ascinae adult female (mesostigmata: ascidae, subfamily ascinae) members of the predatory ascinae are very diverse in biology and habitat, occurring, for  example, on plants and at the sea shore, in salt marshes, soil, humus, stored foods, and  bird, mammal and arthropod nests.  species that live on plants prey on other invertebrates  or feed on fungi or pollen (halliday et al., 1998). 3. cycad (cycas micronesica k.d. hill) spicemen-1:lepidoglyphus destructor (schrank) (astigmata: glycyphagidae): glycyphagids and lepidoglyphus have a worldwide distribution.  mites of this genus  are common inhabitants of house dust and stored foods, e.g., oats, rye, linseed, rice and  dried fruit.  they also occur in outdoor habitats, e.g., grassland soil, and stacks of grain,  straw and hay.  they thrive in damp, humid conditions and feed on the microscopic fungi  that grow in such environments (knülle, 1987). spicemen-2: amblyseius obtusus species group, amblyseius near lentiginosus denmark  &schicha, adult female (mesostigmata: phytoseiidae) description: members of this species group were originally collected from apple  trees and have subsequently been recorded on other plants, e.g., grapevines, citrus sp.,  ficus sp., strawberry, mango, pinus radiata and quercus sp.  they are known to prey  on pest mites, e.g., panonychus citri (tetranychidae), and tegolophus australis and  phyllocoptruta oleivora (both eriophyidae), and have been evaluated as a biocontrol  agent (james et al., 2001)  acknowledgments this project was supported by fy 2010 united states department of agriculture  (usda), animal  and  plant  health  inspection  service  (aphis),  cooperative  agricultural pest survey (caps) program agreement#10-8510-1302-ca and fy  2009 u.s. environmental protection agencystrategic agriculture initiative / food  quality protection act (fqpa) grant agreement #x8-00t32301-0. the authors thank  yolisaishibashi, pest survey specialist, usda, aphis, ppq, honolulu, hi 96850 and  cindy wire, agriculture program, us epa, region 9, san francisco, ca 94105 for their  encouragement and support during all phases of the project. we also thank anne baker,  angela marmont centre for uk biodiversity, natural history museum, london for  journal of entomological and acarological research, ser. ii, 43 (1), 201146 identifying the species. in accordance with federal law and usda policy, this institution  is prohibited from discrimination on the basis of race, color, national origin, sex, age,  or disability.  references de moraes g.j., mcmurtry, j.a. & baker, e.a., 1987 - redescription and distribution of the  spider mites tetranychus evansi and t. marianae. acarologia, 28: 333-343.   de moraes g.j., lopes p.c., fernando, l.c.p., 2004 - phytoseiid mites (acari: phytoseiidae)  of coconut growing areas in sri lanka, with descriptions of three new species. journal of acarological society of japan, 13: 141-160. gerson u., smiley r. l., ochoa r., 2003 - mites (acari) for pest control. blackwell publishing,  oxford, uk. halliday r.b., walter d. e., lindquist e.e., 1998 - revision of the australian ascidae (acarina:  mesostigmata). invertebrate taxonomy, 12: 1-54.   james d.g., halliday r.b., walter d.e., proctor h.c., norton, r.a., colloff m.j., 2001  - history and perspectives of biological mite control in australian horticulture using exotic  and native phytoseiids. csiro publishing, collingwood, australia, acarology: proceedings  of the 10th international congress: 436-443.  knülle w., 1987 - genetic variability and ecological adaptability of hypopus formation in a stored  product mite. experimental and applied acarology, 3: 21-32. noronha a.c.s., 2006 - biological aspects of  tetranychus marianae mcgregor (acari,  tetranychidae) reared on yellow passion fruit (passiflora edulis sims f.flavicarpa deg.)  leaves. revistabrasileira de zoologia 23: 404-407. smiley r.l., 1992 - the predatory mite family cunaxidae (acari) of the world with a new  classification. west bloomfield, michigan, usa, indira publishing house 356 pp. yudin l.s., 1999 - crop profile for water melons in guam. www.ipmcenters.org/cropprofiles/ docs/guwatermelons.pdf.  gadi v.p. reddy, rosalie kikuchi, jenelyn e. remolona, western pacific tropical research  center, university of guam, mangilao, guam 96923, usa, e-mail: reddy@uguam.uog.edu accepted 21 april 2011 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd p. querner, m. morelli, e. oberthaler, m. strolz, k. schmitz von ledebur, i. zatschek, a. femi-mebarek, j. diehl, r. hölzl, i. engelhardt, h. krammer, s. fürnkranz ten years of integrated pest management (ipm) at the kunsthistorisches museum in wien abstract the kunsthistorisches museum wien is one of the largest fine arts collections worldwide, comprising the kunsthistorisches museum, the austrian theater museum, the museum of ethnology, all placed in vienna, and schloß ambras in tirol. we present results from up to 10 years of insect pest monitoring in different collections and the implementation of an integrated pest management (ipm) concept. the kunsthistorisches museum was the first museum in vienna to introduce such a concept. we also present specific insect pest problems such as a biscuit beetle (stegobium paniceum) infestation of paintings lined with starch paste backings (linings) or the webbing clothes moth (tineola bisselliella) infestation at the museum of carriages, both repeatedly occurring problems in the museum. with the help of the insect pest monitoring programs, these and other problems were found and the infested objects treated, usually with anoxia (nitrogen). key words: cultural heritage, insect pests, ipm concept, monitoring. introduction the concept of integrated pest management (ipm) was developed in the 1950s in the food industry and is since the 1980s successfully applied in museums (see for example albert & albert 1988; linnie 1987; story 1986). strategies for museums and ipm concepts were described by jessup (1998), kingsley et al. (2001), pinninger & winsor 2004, querner & morelli (2010a,b) and strang & kigawa (2006). the most recent and complete works on ipm in museums were written by pinniger (2004, 2008) and brokerhof et al. (2007). in ipm the prevention of an insect or fungi attack is an important part. this is implemented by sealing the building, regulating the climate, periodical general cleaning, introducing quarantine and regular monitoring the collection with traps. all these aspects have team up and one centralized person should be in charge of the ipm concept. if an infestation still occurs, unharmful treatment methods like freezing, heating or anoxia (nitrogen) are applied. today, there is hardly any museum in europe still using pesticides against insect pests. but pests like the webbing clothes moth tineola bisselliella (hummel, 1823), less frequently the case-bearing clothes moth tinea pellij. ent. acarol. res. ser. ii, 43 (2): 185-190 30 september 2011 onella linnaeus, 1758, the drugstore beetle stegobium paniceum (linnaeus, 1761), the common furniture beetle anobium punctatum (degeer, 1774), different carpet beetles (attagenus sp. or anthrenus sp.), silverfish lepisma saccharina linnaeus, 1758, mice, pigeons and mold regularly cause problems in european museums. in the present work we describe the ipm strategy applied in the kunsthistorisches museum wien and the experiences collected over the last ten years. the kunsthistorisches museum is one of the largest museums in europe with numerous exhibitions and storage rooms housed mainly in historic buildings and with a large variety of object types. formerly all kinds of chemicals were applied in the collections against insect pests or fungi, for example ddt, naphthalene, methyl bromide, lindan (up to 1982), pyrethroids (until 1998) or ethylene oxide. eulan was sprayed or objects submerged with until 1990, thymol applied to remove mold or xylamon was used to combat wood-destroying insects. in addition, natural crystalline camphor, patschuli, lavender flowers, essential oils like clover in alcohol or lemon grass were used to prevent infestations. from 1996 fumigations with nitrogen were tested in the picture gallery collection (ranacher, 1998) and the construction of the walk-in nitrogen chamber initiated for the whole museum. in 1998 this 32 m3 chamber was built and since then all infected objects from the museum, but also from other museums, institutions and private collections are successfully treated in a five-week rhythm. different pest species still occur from time to time in the collections (see querner 2009 for an overview). by now, most collections have set up an insect pest monitoring and no pesticides or other chemicals are applied. in 2011 a large part of the museum collection will move to a new high standard storage site. museum of carriages and department of court uniforms (wagenburg und monturdepot). museum of carriages is located in the park of the schönbrunn palace. the collection includes about 180 historic vehicles, sleighs and sedan chairs. there are currently about 60 carriages and sleighs displayed in the 1,300 m2 exhibition space, the remaining objects are stored in two depots also located in the historic buildings. these also house large parts of the historic horse harnesses collection. the badly insulated building (historic doors and windows, untight roof and a number of shafts) result in reoccurring pest infestations. for many years the major pest in the collection has been the tineola bisselliella, since many objects are made of wool, silk, feathers and the padding of the carriages and sleigh upholstery are filled with horse hair. other pests are stegobium paniceum that are infecting the starch paste of the horse harnesses and horse figurines. anthrenus (nathrenus) verbasci (linnaeus, 1767) and anthrenus (florilinus) scrophulariae (linnaeus, 1758) are also repeatedly found in the collection where they probably mainly feed on dead insects (querner, 2009). heavily infested objects are treated with nitrogen in the chamber of the museum and in especially constructed large tents on the storage sites. since 2000 a monitoring for webbing clothes moths is carried out and so the number of moths trapped could already be reduced over the years (tab. 1). in 2010 a powderpost beetle lyctus brunneus (stephens, journal of entomological and acarological research, ser. ii, 43 (2), 2011186 1830) infestation was discovered in the parquet floor in the exhibition space. it was first treated with microwaves (2010), but after beetles and exit holes repeatedly reoccurred, a heat treatment using heat blankets was applied against the beetles in 2011. picture gallery (gemäldegalerie) the kunsthistorisches museum has a large collection of historic paintings that are exhibited in the museum and stored in two locations. in the picture gallery stegobium paniceum are repeatedly attacking and damaging paintings lined with starch paste backings (also described by fohrer et al., 2006). objects are treated with anoxia and additionally in 2010 a mass-release of parasitoid wasps was tested in the storage depot (querner & biebl, 2011). many pictures were damaged by the beetles over the last decades. biscuit beetles were also found in the collection feeding on old poisonous mouse baits that were left in the storage depot by a pest control company. the removal of the old bait boxes resulted in an improvement of the situation. collection of sculpture and decorative arts (kunstkammer) in october 2004 an infestation of rhinoceros horn objects (goblets) was found in the storage room of the collection. feeding traces on the horns and some individuals of the two spotted fur beetle attagenus pellio (linnaeus, 1758) were found on and around the objects. all relevant objects were treated in the nitrogen chamber of the museum. in 2010 some of the 16th century tokens were damaged by lepisma saccharina in a further storage room. the miniature portraits were made of a protein and cellulose based substance. all infested objects were treated with anoxia and since then, monitoring was started with sticky blunder and pheromone traps for webbing clothes moths. treasury (schatzkammer) the treasury houses a large collection of precious royal and religious valuable gems and jewels, textiles and artifacts and is the collection with the highest percentage of visitors per year. after webbing clothes moths were discovered in the exhiquerner et al.: ten years of integrated pest management at the kunsthistorisches museum tab. 1 results from the webbing clothes moth monitoring the museum of carriages in the schönbrunn palace in vienna. 187 bition space in 2008, an insect monitoring was introduced with traps placed in and underneath the show cases and in the exhibition rooms. the monitoring showed that no objects like the royal coats or other precious textiles were infested, but it revealed that the moths were coming from spaces underneath the show cases that have not or could not be cleaned over the past years (querner & morelli, 2009). the austrian theatre museum (österreichisches theatermuseum) a systematic insect monitoring was introduced in the different storage sites of the austrian theater museum in 2010 as part of the forthcoming move to a newly built storage site. an active webbing clothes moth infestation was already known to the conservators, but its large extent could be shown by the monitoring in 2010. all textile objects from the infested storage site will be treated with nitrogen prior to the transfer to the new storage building. the museum of ethnology (museum für völkerkunde) the museum of ethnology in vienna is one of the most significant ethnological museums in the world. its collections comprise more than 200,000 ethnographic objects, 25,000 historical photographs, 136,000 printed works, and over 10,000 minutes of film on the history, culture, art and everyday life of predominantly non-european peoples. like in all ethnographic collections the use of pesticides was formerly very common in order to prevent pests. the museum of ethnology in vienna had also its own ethylene oxid fumigation chamber for treating infested objects. many pest problems were found related to climate, the characteristics of a historic building and neglected general housekeeping (cleaning). but with retrospective effect we can state, that only a small amount of ethnographic objects were infested by different insect pest species and mold. after the renovation of the storage rooms, infested objects were treated in the nitrogen chamber. in 2003 an intensive pest monitoring was started as part of a new developed ipm concept. now, after seven years of regular (partly weekly) checking with pheromone traps for webbing clothes moths and self-made sticky traps, pest outbreaks seldom have occurred and new infestations by tineola bisselliella or anthrenus sp. are quickly detected. the ipm in this collection with thousands of valuable materials and objects is very time consuming but the results show it is worth the effort. discussion and conclusions two aspects, the characteristics of historic buildings and the allocation of the multidisciplinary responsibilities, have been particularly problematic and difficult in the application of an ipm strategy in a large museum with many historic buildings and different collections like the kunsthistorisches museum wien. our experience showed that the transition to an ipm concept is a process taking several years. the old buildings are often difficult to seal and are repeatedly infested. renovating historic buildings to the standard of a modern museum is often too costly. the building and planned moving of large parts of the museum collection (the picture gallery for example) in the sumjournal of entomological and acarological research, ser. ii, 43 (2), 2011188 mer 2011 to a new storage site will be a big improvement as the new storage was built under modern and high conservation standards and will therefore better prevent pests infestations from outside. all infested and relevant collections (like the pictures) will be treated with nitrogen before the relocation. a new and larger nitrogen chamber will be built in the new storage site for the rising demands of the museum. currently, an external company (the first author) specialized in ipm in museums is responsible for the pest monitoring, coordination of various tasks and adjustment of the overall concept in most collections of the museum (all except for the museum of ethnology). he is cooperating with the conservators of the individual collections. acknowledgements we thank the kunsthistorische museum in vienna for supporting and funding an ipm program in the different collections the conservators ernst gregor and marianne novotny-kargl for their help and informations on past pesticide treatments in the collection and daniela sailer for comments on the manuscript. references albert g.d., albert l.m., 1988 integrated pest management: a program for museum environments, in: zycherman l.a., schrock j.r., (eds.) a guide to museum pest control. the foundation of the american institute for conservation of historic and artistic works and association of systematics collections, washington d.c.: 169-173. brokerhof a.w., van zanen b., van de watering k., porck h., 2007 buggy biz: integrated pest management in collections, netherlands institute for cultural heritage (icn), amsterdam: 79. fohrer f., baslé k., daniel f., 2006 problématique de l’infestation des colles de rentoilage des peintures de chevalet par le stegobium paniceum (l.), support tracé, 6: 78-85. jessup w.c., 1998 integrated pest management into operation. collections caretaker, 1 (3): 1-8. kingsley h., pinniger d., xavier-rowe a., winsor p., 2001 integrated pest management for collections, in: proceedings of 2001: a pest odyssey, james & james, london: 150. linnie m.j., 1987 pest control: a survey of natural history museums in great britain and ireland. the international journal of museum management and curatorship, 6: 277-290. pinniger d.b., 2004 pest management in museums, archives and historic houses, archetype publications, london: 115. pinniger d.b., 2008 pest management a practical guide. collection trust, cambridge: 52. pinninger d.b., winsor p., 2004 integrated pest management. a guide for museums, libraries and archives. museum libraries archives. querner p., 2009 museumsschädlinge und die umsetzung der integrierten schädling bekämpfung in wiener museen. ein erster überblick. mitteilungen der deutschen gesellschaft für allgemeine und angewandte entomologie, 17: 231-233. querner p., bielb s., 2011 using parasitoids wasps in integrated pest management in museums against biscuit beetles (stegobium paniceum) and webbing clothes moths (tineola bisselliella). j. ent. acar. res ser ii, 43 (2). querner et al.: ten years of integrated pest management at the kunsthistorisches museum 189 querner p., morelli m., 2009 nachweis von museumsschädlingen in schmutz. restauro, fachzeitschrift für kunsttechniken, restaurierung und museumsfragen, 2: 85. querner p., morelli m., 2010a integrierte schädlingsbekämpfung in museen erfahrungen einer umstellung. restauro, fachzeitschrift für kunsttechniken, restaurierung und museumsfragen, 4: 234-241. querner p., morelli m., 2010b leitfaden für eine einführung und umstellung zurintegrierten schädlingsbekämpfung (ipm). restauro, fachzeitschrift für kunsttechniken, restaurierung und museumsfragen, 5: 332-333. ranacher m., 1998 mikroorganismenund schadinsektenbefall im depot: ursachen, sanierung, hygiene und gesundheitsschutz. in: landesstelle für die nichtstaatlichen museen (eds.) das depot. der andere teil der sammlung, 9. bayerischer museumstag, münchen: 53-74. story k.o., 1986 approaches to pest management in museums. in: conservation analytical laboratory, smithsonian institution, washington d.c.: 85-101. strang t.j.k., kigawa r., 2006 levels of ipm control: matching conditions to performance and effort. collection forum, 21 (1-2): 96-116. pascal querner, university of natural resources and life sciences department of integrated biology and biodiversity research, institute of zoology, gregor-mendel-straße 33, a-1180 vienna, austria. e-mail: pascal.querner@boku.ac.at michaela morelli, kunsthistorisches museum mit mvk und ötm museum of carriages and department of court uniforms, textile conservation schloss schönbrunn, a-1130 vienna, austria. elke oberthaler, monica strolz, katja schmitz von ledebur, johanna diehl, kunsthistrisches museum mit mvk und ötm , picture gallery, maria theresien-platz, a-1010 vienna, austria. isabell zatschek, anna fermi-mebarek, austrian theatre museum lobkowitzplatz 2, 1010 vienna, austria. regina hölzl, irene engelhardt, kunsthistorisches museum mit mvk und ötm, egyptian and near eastern collection, maria theresien-platz, a-1010 vienna, austria. hugo krammer, sophie fürnkranz, kunsthistorisches museum mit mvk und ötm, the museum of ethnology, neue burg, a-1010 vienna, austria. journal of entomological and acarological research, ser. ii, 43 (2), 2011190 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. layout 1 [journal of entomological and acarological research 2014; 46:3787] [page 119] a six-arm olfactometer for analysing olfactory responses of goniozus legneri gordh (hymenoptera: bethylidae), the larval ectoparasitoid of carob moth m. aleosfoor,1 f. ehteshami,2 l. fekrat3 1department of plant protection, college of agriculture, shiraz university, shiraz; 2department of plant protection, college of agriculture, shiraz university, shiraz; 3department of plant protection, ferdowsi university of mashhad, mashhad, iran abstract the behavioural responses of goniozus legneri were investigated in a six-arm olfactometer. among the different odours examined, carob moth (ectomyelois ceratoniae zeller) frass elicited the highest olfactory responses, while ephestisa larvae, which were less suitable hosts, elicited the lowest response. the different preferences to various odours suggest that goniozus legneri can discriminate among suitable and less suitable insect hosts. introduction the carob moth, ectomyelois ceratoniae zeller (lep.: pyralidae), is a major pest of pomegranate in iran that causes quantitative and qualitative reduction of pomegranate yield in all cultivation regions of the country (mozaffarian et al., 2007; kishani farahani et al., 2011, 2012a). in some years, this pest can cause yield losses of up to 80%, and thus constitutes a major threat to the pomegranate industry in iran (shakeri, 2004; kishani farahani et al., 2011). larval development and feeding by this pest inside the fruits protects them from insecticides and makes chemical control inefficient (shakeri, 2004). in iran, collecting and destroying infected pomegranates at the end of the growth season is the most widely recommended control method for carob moth, as it eliminates overwintering sites (behdad 1991; mozaffarian et al., 2007). removing flags (shakeri, 2004), and staking the pomegranate fruit neck (mirkarimi, 1996) are other common methods for removing the hatch sites. currently, none of the conventional methods alone are able to control this pest (shakeri, 2004). due to the developmental biology and behaviour of carob moth larvae, natural enemies could play a fundamental role in reducing the damage caused by this pest, and there appears to be much potential for employing various parasitoid species in biological control programs targeting it. currently, several hymenopterous species have been reported as larval parasitoids of carob moth (gothilf, 1978; kishani farahani et al., 2011, 2012b). goniozus legneri gordh is one of the important natural enemies of lepidopteran larvae on several crops, as it can develop successfully in ectomyelois ceratonia larvae (ehteshami et al., 2013; legner et al., 1982; zaviezo et al., 2007). when considering the effectiveness of a natural enemy for pest population regulation, several attributes are crucially important, including the ability of a biological control agent to locate suitable host material. parasitoid searching and host location behaviour is influenced by several external stimuli such as olfactory, visual, and tactile cues (vinson, 1976, 1998; broad & quicke, 2000; graziosi & rieske, 2013). olfactory cues are used by many hymenopteran parasitoids to orient to a potential host habitat as well as to a host, so odours play an essential role throughout the life span of the parasitoids. olfactometers, in which the parasitoids can choose simultaneously among several different odours, are used to study and reveal responses of parasitoids to various odours (vet et al., 1983; vinson, 1981; weseloh, 1981). in this study, a sixarm olfactometer was used to examine simultaneous cues by which females of this parasitoid species could locate larvae of the carob moth. materials and methods insect rearing carob moth larvae were separated from infested fruits originally collected from several pomegranate orchards in fars province (shiraz, kavar, estahban, ij, seyyedan, faroogh) in the south of iran. ectomyelois ceratonia larvae were reared on artificial diet containing wheat flour (72%), honey (12%), glycerin (10%), yeast (1%), and distilled water, in transparent plastic jars (20×10×10 cm) at room temperature. the emerged adults from infested fruits were transferred into transparent jars (18×7 cm) and provided with cotton wool soaked in 10% honey as a food source. correspondence: lida fekrat, department of plant protection, faculty of agriculture, ferdowsi university of mashhad, mashhad, iran. e-mail: fekrat@ferdowsi.um.ac.ir key words: parasitoid, carob moth, ectomyelois ceratonia, pomegranate, olfactory responses. received for publication: 15 april 2014. revision received: 27 august 2014. accepted for publication: 4 september 2014. ©copyright m. aleosfoor et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:3787 doi:10.4081/jear.2014.3787 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:3787 jear_2014_3_hrev_master 16/12/14 10:38 pagina 119 no nco mm er cia l u se on ly [page 120] [journal of entomological and acarological research 2014; 46:3787] for all experiments, 2-3-day old female goniozus legneri adults were used. for rearing, ephestia kuehniella (lepidoptera: noctuidae) caterpillars (third instar) were offered to a single mated female (4-7 days old) for 12 h in a plastic box (diam. 8 cm, height 5 cm) (figure 1). the parasitised caterpillars were kept on a wheat germ-based artificial diet in an incubator (25°c, l:d 16:8 h) until cocoon formation. cocoons were kept in petri dishes until adult emergence. emerging adults were sexed and kept in cages (20×20×20 cm) in the same incubator at a sex ratio of 1: 2 (male:female), with moist cotton wool and honey as a food source. olfactometer design we designed a six-arm olfactometer for our experiments (figure 2), which consisted of a central petri dish with six arms extending from its sides. the arms had an internal diameter of 1 cm. at the distal end of each arm, there was a petri dish of 5 cm diameter. a small flow fan was connected to the petri dishes using a bended tube to provide uniform airflow. illumination was provided using two fluorescent lamps 40 cm above the olfactometer. olfactometer experiments the adult parasitoids were 24 and 72 h days old and were tested individually. all female parasitoids were provided only with water from the time of emergence for the behavioural test, and had no contact with the test odour sources before the experiment. a maximum of 10 min was allowed for each parasitoid to make its final choice. after testing five insects, the whole apparatus was rotated 90° and, after testing each parasitoid, cleaned with 95% ethanol. to start a measurement, one female parasitoid was transferred from the cage to the central petri dish using a 2.5-cm teflon tube closed on one end by a nylon mesh. carob moth frass, a healthy pomegranate, a carob moth larva, a carob moth larva+ artificial diet, and an ephestia larva were tested for their attractiveness to the female parasitoids. the small fraction of females that did not enter the side arms within five minutes were considered non-responders and removed from the experiment. to randomise possible side effects on the preferences of g. legneri, odour sources were renewed for each replicate and their positions alternated between replicates. five replicates were performed on a given experimental day. one of the six side-petri dishes was kept empty as a control in each replicate. an odour source was considered attractive when a parasitoid exhibited a preference for it. the time for a parasitoid to initially cross the junction line into one arm was used to test whether a preference could be detected as soon as the female entered an arm, or whether she needed some exploration before displaying a preference. the preference time was designated as the time it took for females to cross the junction line of each of the six lateral arms. females were considered to have a lasting preference for an odour source when they spent significantly more time in one lateral arm than in the others. statistics the data were normally distributed, so we performed one-way analysis of variance (anova), with treatment differences separated using fisher’s least significant difference test at 5% level of significance (spss statistics ver. 21.0. released 2012; ibm corp., armonk, ny, usa). results we evaluated the absolute attractiveness of different odours, with air as a neutral control; there were significant differences among selection of various odour sources by parasitoids (f=39.896, p=0.000). the parasitoids significantly more frequently selected the arm connected to frass of carob moth larvae compared with the other arms (figure 3). there were no significant differences in preference among the three other choices: pomegranates, larvae+ artificial diet, and article figure 1. goniozus legneri rearing on ephestia kuehniella caterpillars. figure 2. six-arm olfactometer designed for this experiment. figure 3. mean number of females choosing each odour arm (±standard error); different letters indicate significant differences at 5% significance level. jear_2014_3_hrev_master 16/12/14 10:38 pagina 120 no nco mm er cia l u se on ly carob moth larvae. the parasitoids selected the ephestia larvae and the control at lower frequencies; however, there were no significant differences between them (figure 3). there were significant differences among the preference times for the various odours (f=2.580; p=0.047). the preference time for ephestia larvae was significantly longer than those for other odours (table 1). however, the difference between preference times of carob moth larvae and ephestia larvae was not significant (table 1). discussion in spite of the breadth and complexity of the environment, parasitoids are able to find suitable hosts for their progeny (cusumano et al., 2010). a good cue for parasitoids to use in finding their hosts is undoubtedly the signals they receive from their surrounding environments, such as the presence and quality of a host, or a sign of host feeding, such as host frass, if it is detectable over an appropriate distance (vet & dicke, 1992; hilker & mcneil, 2007). several studies of behavioural responses of parasitoids using an olfactometer have been carried out to evaluate the attractiveness of different plant volatiles (belz et al., 2012), parasitoid hostinfested plants (castelo et al., 2009), or sex pheromones of parasitoid hosts (noldus, 1998). the aim of our study was to investigate the olfactory attractiveness of five different odour sources to g. legneri. this study demonstrated that g. legneri could show a behavioural response to the different odours. all of the tested odours except that of ephestia larvae were highly attractive when compared with air alone, both in terms of number of choices and preference time, although some were more attractive than others. carob moth larval frass was the most attractive odour to the parasitoids; pomegranate fruits, carob moth larvae + artificial diet, and carob moth larvae were equally, but less attractive, and all were more attractive than the ephestia larvae and the control. ephestia larvae were the least attractive. these findings highlight the fact that the presence of carob moth larvae and their feeding signs, such as their frass, are important cues for parasitoids in identifying pomegranates that harbour carob moth larvae. according to our data, g. legneri wasps rely mainly on direct host-related cues, such as the presence of carob moth larvae or their frass, to locate their hosts. it is likely that the chemicals released by the host and/or its frass could be the cue that signals the searching female that the host is suitable for parasitism. however, further studies are needed to identify the chemical compounds that may account for these interactions. based on our results, g. legneri did not exploit volatiles produced by the ephestia larvae, as no significant attraction was detected when these were tested. as the ephestia larvae are not hosts of g. legneri in nature, the low attractiveness of ephestia larvae compared with the other odours is not unexpected. this difference in preference suggests that g. legneri can discriminate among suitable and less suitable host insects by volatile signals, which would enhance their rate of successful parasitism and ensure a high survival ratio of their offspring. it is worth noting that the ability to find insect hosts using different signals implies a certain level of behavioural plasticity in the parasitoids. references behdad e., 1991 pests of fruit crops in iran. 2nd ed. markaz-e-nashre-bahman, tehran. belz e., k�lliker m., balmer o., 2012 olfactory attractiveness of flowering plants to the parasitoid microplitis mediator: potential implications for biological control. biocontrol. 58: 163-173. broad g.r., quicke d.i.j., 2000 the adaptive significance of host location by vibrational sounding in parasitoid wasps. proc. r. soc. lond. b biol. sci. 267: 2403-2409. castelo m.k., nouhuys s.v., corley j.c., 2009 olfactory attraction of the larval parasitoid, hyposoter horticola, to plants infested with eggs of the host butterfly, melitaea cinxia. j. insect sci. 10: 1-16. cusumano a., gonz�lez j.m., colazza s., vinson s.b., 2010 behavioral responses of the parasitoid melittobia digitata to volatiles emitted by its natural and laboratory hosts. entomol. exp. appl. 136: 301-307. ehteshami f., aleosfoor m., allahyari h., alichi m., 2013 first record of goniozus legneri (hym.: bethylidae), the larval ectoparasitoid of carob moth, in iran. j. entomol. soc. iran 33: 89. gothilf s., 1978 establishment of the imported parasite pentalitomastix plethoricus (hym.:encyrtidae, on ectomyelois ceratoniae (lep.: phycitidae) in israel. entomophaga. 23: 299-302. graziosi i., rieske l.k., 2013 response of torymus sinensis, a parasitoid of the gallforming dryocosmus kuriphilus, to olfactory and visual cues. biolog. control. 67: 137-142. hilker m., mcneil j.n., 2007 chemical and behavioral ecology in insect parasitoids: how to behave optimally in a complex odorous environment?. in: wajnberg e.c., bernstein c.b., van alphen j. (eds), behavioural ecology of insect parasitoids: from theoretical approaches to field applications. blackwell publishing, oxford: 92-112. kishani farahani h., bell h., goldansaz s.h., 2012a biology of apanteles myeloenta (hymenoptera: braconidae), a larval parasitoid of carob moth ectomyelais ceratoniae (lepidoptera: pyralidae). j. asia pacific entomol. 15: 607-610. kishani farahani h., goldansaz s.h., sabahi q., 2012b a survey on the over wintering larval parasitoids of ectomyelois ceratoniae in three regions in iran. crop prot. 36: 52-57. kishani farahani h., goldansaz s.h., sabahi q., shakeri m., 2011 a study on the larval parasitoids of carob moth, ectomyelois ceratoniae in varamin, qom and saveh. j. iranian plant prot. soci. 41: 337-344. legner e.f., gordh g., silveria-guido a., badgley m.e., 1982 new wasp may help control navel orangeworm. california agric. 36: 4-5. mirkarimi a., 1996 -the effect of stuffing pomegranate fruit neck (calyx) on reduction of pomegranate neck worm spectrobates (ectomyelois) ceratoniae zell. available from: http://iman.ut.ac.ir/ news/agr.htm mozaffarian f., sarafrazi a., nouri ganbalani g., 2007 host plant-associated population variation in the carob moth ectomyelois ceratoniae in iran: a geometric morphometric analysis suggests a nutritional basis. j. insect sci. 6: 1-11. noldus l.p.p.j.j., 1988 response of the egg parasitoid trichogramma [journal of entomological and acarological research 2014; 46:3787] [page 121] article table 1. mean preference time in parasitoid choice of each odour arm. frass healthy pomegranate larvae+artificial diet larvae ephestia larvae mean±se 4.919±0.508a 4.861±1.0571a 3.335±0.938a 5.902±1.098ab 8.412±0.571b se, standard error. a,bdifferent letters indicate significant differences at 5% significance level. jear_2014_3_hrev_master 16/12/14 10:38 pagina 121 no nco mm er cia l u se on ly [page 122] [journal of entomological and acarological research 2014; 46:3787] pretiosum to the sex pheromone of its host heliothis zea. entomol exp. appl. 48: 293-300. nakamuta k., van tol r.w.h., visser j.h., 2005 an olfactometer for analyzing olfactory responses of death-feigning insects. appl. entomol. zool. 40: 173-175. shakeri m., 2004 pests and diseases of pomegranate. tasbih publication. [in farsi]. vet l.e.m., dicke m., 1992 ecology of infochemical use by ntural enemies in a tritrophic context. annu. rev. entomol. 37: 141-172. vet l.e.m., van lanteren j. c., heymans m., meelis e., 1983 an airflow olfactometer for measuring olfactory responses of hymenopterous parasitoids and other small insects. physiol. entomol. 8: 97-106. vinson s.b., 1976 host selection by insect parasitoids. annu. rev. entomol. 21: 109-133. vinson s.b., 1981 habitat location. in: nordlund d.a., jones r.l., lewis w.j. (eds), semiochemicals, their role in pest control. wiley & sons, new york: 51-78. vinson s.b., 1998 the general host section behavior of parasitoid hymenoptera and a comparison of initial sterategies utilized by larvaphagous and oophagous species. biol. control. 11: 79-96. weseloh r.m., 1981 host location by parasitoids. in: nordlund d.a., jones r.l., lewis w.j. (eds), semiochemicals, their role in pest control. wiley & sons, new york: 79-95. zaviezo t., romero a., castro d., wagner a., 2007 first record of goniozus legneri (hymenoptera: bethylidae) in chile. cien. investig. agr. 34: 49-52. article jear_2014_3_hrev_master 16/12/14 10:38 pagina 122 no nco mm er cia l u se on ly j. ent. acar. res..indd e. chiappini, r. nicoli aldini morphological and physiological adaptations of wood-boring beetle larvae in timber abstract beetles which develop boring tunnels inside and feed on seasoned wood present morphological and physiological adaptations related to the specific activities of their larvae in such a peculiar substrate. as far as protection of antiquarian goods made of wood is concerned, we are dealing mainly with three coleoptera families, namely lyctidae, anobiidae, and cerambycidae, which include species with wood-boring larvae. the adaptation to wood-boring and wood-feeding activities in beetle larvae was reached independently by phyletic lines not closely related, as a convergent evolution due to feeding behaviour. among these adaptations, the following are examined with reference to the three families mentioned above. the conformation and activity of the larval mandibles and their possible correlations with the characteristics of the wood attacked are considered together with the presence of body structures for anchoring the larvae to the wood substrate inside the tunnel during the gnawing action. intracellular endosymbiosis (endocytobiosis) with yeasts or bacteria, capable of supplementing larval diets lacking in some essential nutrients, and its main features are summarized. last, structural and functional characteristics are discussed as regards tracheal spiracles, provided with filter devices important for preventing intrusion of wood powder into tracheae from larval tunnels as well as useful for avoiding dehydration. key words: coleoptera, lyctidae, anobiidae, cerambycidae, convergent evolution, wood-feeding, mouthparts, anchoring devices, endocytobiosis, spiracles. a unique environment and food wood-boring beetles, which develop by digging tunnels in timber, live in a rather unusual environment and substrate, passing their larval life sealed inside the wood mass, without communication with the external environment. only in the case of heavy or protracted infestations, when there is a higher possibility that new and old tunnels intersect, may they be in communication with the outside, through the exit holes of the previous generations. the larvae, which penetrate the timbered tissue directly, after having completed embryonic development and hatching, feed exclusively on wood (unless the timber is invaded by fungal mycelia), extracting from this substrate all the main nutrients j. ent. acarol. res. ser. ii, 43 (2): 47-59 30 september 2011 and water they need for their metabolism and development. being confined within the wood mass, they display adaptations to this kind of life and, at the same time, to the food available, i.e. to living within wood and to living off wood (cymorek, 1968). of course, these two aspects are closely interconnected. we first consider the type of environment determined by the xylophagous regime and the endophytic lifestyle. wood, and timber even more so, is a hard and resistant substrate, which generally is attacked from the inside, so as to have an attachment point. in fact, since larvae remove the wood particles with their mandibles, being able to fracture its surface without having an anchorage would be extremely difficult. as the endophytic larvae explicate their feeding activity, they gradually penetrate the wood mass, thus finding themselves trapped in the tunnel they are digging. actually, the tunnel is closed in front by sound wood and behind it is smaller than the larvae, because of their minor development during the period when it was dug. its diameter is usually approximately the same as that of the larva, the empty space around it being very reduced, in order to improve larval digging efficiency. therefore, the larvae spend their entire lives in a closed and dark environment, which provides food and protection. their movement is limited to the digging action, functional to feeding, and consequently to the progression of the tunnel, gradually lengthened forward. usually only at completion of preimaginal development, mature larvae slightly widen the tunnel in view of pupation. their tunnelling activity also causes the production of frass, which occupies the back of the gallery and differs in consistency and granulometry, depending on the wood-boring species. these are the conditions of preimaginal life shared by most wood-boring beetles which damage cultural artifacts and that determine their functional adaptations. other conditions, however, may vary; for example the type of wood, which may have a different composition and hardness, depending on the essence and the part of the trunk or branch from which the attacked timber derives (battisti, 2001). it is known that the duramen contains mainly cellulose, together with hemicellulose and lignin, and very little starch, while the sapwood still maintains a high percentage of starch. different food preferences, related to a different set of available digestive enzymes, orient the various wood-borers towards one or the other type of substrate. feeding and boring in wood, a convergent evolution although wood is a food poor in some essential nutrients, among hexapoda, xylophagy is a rather widespread feeding habit, common to orders, families and genera not closely related to each other. a number of exopterygote insects, such as some blattaria and all the isoptera, are xylophagous. for these, wood represents food not only during postembryonic development but also during adult life (chopard, 1949; grassé, 1949). a large number of endopterygote insects such as, for example, species of lepidoptera cossidae and sesiidae, hymenoptera siricidae and coleoptera scarabaeoidea, buprestidae, lyctidae, bostrichidae, anobiidae, lymexylonidae, oedemeridae, cerambycidae, curculionoidea (scolytidae, etc.), are xylophagous as well, but, in this journal of entomological and acarological research, ser. ii, 43 (2), 201148 case, wood-feeding (mostly wood-boring) is generally limited to the larval instars, with a variety of trophic specializations and different needs of ectoor endosymbiotic associations (jeannel & paulian, 1949; grandi, 1951; ebeling, 1978; borror et al., 1989). therefore, it is obvious that xylophagy represents a trophic specialization independently achieved many times in phylogenetically distant insect orders. quality and other characteristics of the wood attacked, as well as the mode of attack and nutrient mobilization, may vary not only from one systematic group to another, but also inside the same group of closely related taxa. certain species have a more ample range with regard to the conditions of the wood substrate, others a more restricted one; there are species which prefer healthy wood, others which feed on wood already damaged by biotic or abiotic factors, others which disseminate it with microscopic fungi in order to make it more suitable as food, others which need dead but still sound wood, others which, in contrast, require rotten wood (saproxylophagy), etc. (grandi, 1951; masutti, 2003). as far as spatial relationship is concerned, feeding and development can take place inside the wood mass (most frequently) or from the outside (e.g. in buried timber) or also through intermediate modalities. nevertheless, as the majority of xylophagous beetles do not attack dry, seasoned wood but develop in living wood (even if sometimes suffering or decaying), only a few families of wood-boring beetles, principally cerambycidae (long-horned beetles), lyctidae (powderpost beetles), and anobiidae, are of interest with regard to the protection of timber in various phases of its use, particularly wood structures of antiquarian, historical and artistic value (chiappini et al., 2001). apart from some more recent rearrangements by the subsequent authors, the main traits of the phylogeny of the coleoptera, according to current opinion (laurence & newton, 1982; grimaldi & engel, 2006), do not differ substantially from the arrangement outlined by crowson (1960) around the middle of the last century. crowson (1955; 1960), basing his conclusions also on the contributions of some previous authors, recognized four suborders in the coleoptera order. of these, two (archostemata, mixophaga) are very small and two (adephaga, polyphaga) very large. their adaptive radiation was partly due to the different feeding specializations and resulted in the very scarce and primitive archostemata (primarily endophytic xylophagous and mainly neotropical), the zoophagous adephaga, the initially mycetophagous and subcorticicole polyphaga. this is the most recent and largest coleoptera suborder, characterized by the greatest evolutionary success as well as by secondary differentiations in an extremely various diversity of trophic niches. the xylophagous beetles of applied interest belong to polyphaga; in this suborder wood-boring and wood-feeding habits were independently reached many times by single systematic groups pertaining to various lines. among them are found the series bostrichiformia, comprising the superfamily bostrichoidea (with lyctidae, bostrichidae, anobiidae) as well as cucujiformia comprising, among others, the superfamilies lymexylonoidea (with lymexylonidae), chrysomeloidea (with cerambycidae), and curculionoidea (with curculionidae) (fig. 1). in the same way as happened at insect order level, also among coleoptera, an e. chiappini, r. nicoli aldini: adaptations of wood-boring beetle larvae 49 journal of entomological and acarological research, ser. ii, 43 (2), 2011 extremely large order, xylophagy appears to be the result of a convergence of many phyletic lines more or less distant from each other. fig. 1 cladogram of phyletic relations among suborders and superfamilies of coleoptera (modified after tremblay, 2000). adaptations to feeding on seasoned wood as mentioned above, the nutrients present in the wood are basically starch, hemicellulose and cellulose; the latter, however, is not easy to digest. the larvae of some species of wood-boring beetles may be restricted to using starch alone, but others are able to digest cellulose. depending on whether the larva is able to use it or not, the feeding mode can vary, starting from the conformation and function of the mouth appendages, to producing and ingesting more or less fine wood particles. the attack on a substrate as hard as wood may also require that the larva has on its body surface some structures that enhance its grip on the wooden substrate while the action of the jaws is performed (cymorek, 1968). in addition, considering the wood as the food substrate, besides requiring that the larva have a set of enzymes for cellulose digestion, which not all wood-boring species 50 do, wood is also deficient in essential nutrients, such as vitamins, amino acids and lipids, that have to be found elsewhere, generally by means of symbiosis with microorganisms (nardon & grenier, 1989). the following notes refer to such features in the three main families of woodboring beetles in which we find the most common and important pests of seasoned wood and timber. biting off and type of wood attacked watanabe & tokuda (2010) state the importance of the digestive system morphology in order to understand cellulose digestion. the first step of digestion takes place at mouth level. in wood-boring coleoptera, both adults and larvae have chewing mouthparts. in the larvae of different taxa of wood-boring coleoptera, the general organization is the same: the labrum is subrectangular and bordered with setae on the distal margin; laterally and posteriorly to it mandibles are present and, behind these, the maxillae with galea, lacinia and palps; back and medially, the labium, with postmentum, prementum and palps (fig. 2). mandibles are the appendages used to break down wood. they are usually short and heavily sclerotized, especially along the medial margin, where the strength of the jaws seems to be due to the presence of zinc and manganese that have been found in larval mandibles of anobiids and long-horned beetles (hillerton et al., 1984, morgan et al., 2003). the overall shape of the mandibles of wood-boring larvae is very much the same; even if morphological differences are present between different taxonomic entities. in most anobiidae, the larvae exhibit toothed mandibles with a medial margin characterized by triangular teeth (fig. 2, a). this shape makes it possible to detach little wooden pieces from the timber mass, which are subsequently brushed into the cibarium by means of labrum, laciniae, and labium setae. lyctid larvae, such as those of lyctus linearis (goeze) (fig. 2, b), have “chiselshaped” mandibles, with the medial margin, perpendicular to its major axis, without teeth but linear and sharp. the same type of mandible is present in cerambycid larvae (haack & slansky, 1987) like, for example, those of hylotrupes bajulus (linnaeus) (schmidt & parameswaran, 1977) and those of trichoferus holosericeus (rossi) (fig. 2, c). mandibles shaped much like these are also found in ptilinus pectinicornis (linnaeus) (anobiidae), where the remains of a small tooth are present in the lower corner of the medial margin (fig. 2, d). being present in coleoptera families phylogenetically far from each other (anobiidae, cerambicidae, lyctidae) (fig. 1), contrary to what was stated by schmidt (1966), the peculiar shape of these jaws does not appear to be linked to the systematic group but could represent an evolutionary convergence due to the type and mode of feeding and digestion. watanabe & tokuda (2010) assumed that the reduction in size of the mandibles in wood-eating insects would confer an advantage by allowing finer wood powder to be produced, that would correspond to an increased “exposure of cellulose fibers buried e. chiappini, r. nicoli aldini: adaptations of wood-boring beetle larvae 51 in hemicellulose and lignin”, thus enhancing “the access of cellulolytic enzymes to cellulose”. nevertheless, they specifically state that “it has not been elucidated so far whether a reasonable correlation exists between designs and sizes of mandibles and the efficiency of wood crushing in coleopterans”. the study of the frass produced by these different types of larvae, considering the size of the individuals producing it, can help to elucidate larval habits (solomon, 1977). the species having toothed mandibles produce gritty frass consisting of fusiform faecal particles, thus indicating that all the dug wood passes through the alimentary canal. on the contrary, all the species considered that possess chisel-shaped mandibles produce a very fine frass, mixed with faeces, thus signifying that they dig more wood than they ingest. in addition, we observed that toothed mandibles are able to detach little pieces of wood from the mass, while chisel-shaped mandibles make it possible to pulverize the wood to very fine fragments. fig. 2 larval biting mouthparts of four species of wood-boring beetles: a. stegobium paniceum (linnaeus) with mandibles characterized by triangular teeth on their medial margin. b. lyctus linearis (goeze) with “chisel shaped” mandibles, with linear and sharp medial margin. c. trichoferus holosericeus (rossi) with chisel-shaped mandibles. d. ptilinus pectinicornis (linnaeus), with mandibles very similar to chisel-shaped ones but with remains of a small tooth in the lower corner of the medial margin. journal of entomological and acarological research, ser. ii, 43 (2), 201152 in agreement with watanabe & tokuda (2010), this could be functional to better digestion of cellulose but we hypothesize that the rupture of cell walls would expose the starch granules which should thus be available for digestion. therefore, these mandibles would be suited to larvae that feed mainly on starch and which, being unable or not very efficient in cellulose digestion, need to gain access to starch without necessitating the digestion of the cell walls. this interpretation is consistent with the kind of wood attacked by these different larvae. most anobiid species, which possess toothed mandibles, also attack the duramen or very seasoned wood, both very low in starch, because they feed also on cellulose and hemicellulose (eaton & hale, 1993). of the larvae that show chisel-shaped mandibles, lyctid larvae feed exclusively on starch and attack sapwood of “susceptible hardwood species which have sufficient starch (ca. >3%)” and they are called powderpost beetles because they “reduce the wood to flour-like powder” (eaton & hale, 1993). hylotrupes and trichoferus larvae generally attack sapwood, even if they can also tunnel in heartwood (eaton & hale, 1993) and their frass also is composed of extremely fine wood fragments mixed with faeces (chiappini et al., 2010). p. pectinicornis larva attacks sapwood and produces a fine, silky frass, densely packed (eaton & hale, 1993). it seem obvious that wood-boring coleoptera that cannot digest cellulose or that digest it at a low rate take advantage of the disruption of wood walls operated by chisel-shaped mandibles and, in search of starch, dig more than what they ingest, producing a fine frass, mixed with faeces. these type of mandibles could therefore represent an evolutionary convergence towards better starch utilization. body adaptations for anchorage the peculiar lifestyle of the larvae of wood-boring beetles, which have a minimal need of movement but necessitate a tight hold on the wood, essential for their gnawing, involves morphological adaptations which can differ according to the systematic groups and regarding the taking off the food and the correlative locomotion. a more or less evident reduction of the legs in the larvae, sometimes even their total absence, corresponds to the limited requirement of moving in a narrow space. in the oligopod larvae of the lyctidae (e.g. lyctus spp.) and of some anobiid species (e.g. in gen. anobium, stegobium, etc.) the legs, developed even if relatively short and tiny, seem to perform different functions besides moving (cymorek, 1968). on the other hand, cerambycid larvae are either apod or only keep vestigial rudiments of legs (e.g. gen. hylotrupes, trichoferus) (gardiner, 1960; peterson, 1960). the need to gnaw a compact, and often very hard substrate, such as wood is, in addition to the structure and constitution of the mandibles mentioned above, is therefore met by other devices such as rough areas on the thoracic and abdominal surfaces of the body, as well as rows of spicules, which can be useful also for movement, but which principally permit the anchorage necessary to the larva during its prolonged feeding activity. in cerambycid larvae, for example, besides the nearly prognathous head, immersed into the prothorax, and bearing short and extremely strong mandibles, the surface of thorax and abdomen often show ambulatorial areas consisting of dorsal and/or ventral integumental corrugated and thickened plates (grandi, 1951). without e. chiappini, r. nicoli aldini: adaptations of wood-boring beetle larvae 53 any doubt, the larva is helped in taking off the food also by the limited diameter of the tunnel and by filling it behind with compact frass and excrement. the anobiid larvae are generally scarabaeiform (c-shaped), with a hypognathous head (but sometimes nearly prognathous); therefore it is likely that their activity of excavation, alimentation and progression differs in comparison with that of cerambycids. in the majority of their larval forms, anobiids (e.g. anobium spp.) bear transversal rows of hooks on the anterior dorsal folds of most of the toracic and abdominal segments and on the sides of the ninth abdominal segment (parkin, 1933; böving, 1954). towards the end of the abdomen, these hooks can be larger, clearly curved. these larvae bend and push their distal end towards the tunnel wall; they subsequently straighten, stretching forwards. also in lyctids the larva is c-shaped, but the body surface (e.g. in the genus lyctus) is completely free of anchoring hooks, possibly because larval activity occurs in a relatively tender wood, that can be tunnelled more easily. endosymbiosis and nutrition the ability of many insects to use unpromising foodstuffs, as also wood is, is due to cryptic microbial assistance (dadd, 1985). besides ectosymbiosis with fungi in ambrosia beetles (scolytidae), and in other wood-boring beetles (e.g. anobium punctatum (degeer) and xestobium rufovillosum (degeer)) that develop preferably in rotten wood (bletchly, 1953), it is well known that two other types of symbiosis with microorganisms occur in wood-boring beetles, namely extracellular endosymbiosis, limited to intestinal lumen, and intracellular endosymbiosis (endocytobiosis) (nardon & grenier, 1989). both types are mutualistic symbioses, not excluding each other and with possible intermediate forms. in wood-boring beetles, the presence and the role of gut extracellular endosymbiosis is still very little known (vasanthakumar et al., 2007). the role of this type of endosymbiosis could be that of entirely or partly supplying enzymes, necessary for cellulose utilization (chapman, 1972). in fact, even if the formerly hypothesized acquisition of cellulases has been challenged by the discovery of endogenous enzymes in anobiids and cerambycids (parkin, 1940; martin, 1983), the microorganisms could play a role in freeing cellulose and hemicellulose from lignin (genta et al., 2005). the detoxification activity, that has been demonstrated for the anobiid lasioderma serricorne (fabricius) (shen & dowd, 1991), could also be proved for wood-boring species. endocytobiosis is the third type and best known symbiosis in wood-boring beetles infesting timber. it is a complex type of endosymbiosis which seems to have the main function of supplementing insect diets lacking in some essential nutrients, i.e. vitamins (especially of the b-group), lipids, sterols, amino acids and, maybe, other growth factors, increasing the fitness of the insect to the environment and substrate (nardon & grenier, 1989). in their turn, intracellular symbionts (endocytobiotes) take advantage of being protected from a dry environment and having availabile a substrate rich in carbon sources. there are, however, more complex metabolic interactions between host and endocytobiotes that behave as cellular organelles regulated by the host itself. in fact, they can modulate enzymatic activities, metabolism of certain amino acids, etc.. journal of entomological and acarological research, ser. ii, 43 (2), 201154 endocytobiotes are yeasts or bacteria included in specialized cells, mycetocytes and bacteriocytes respectively, lodged singly between the epithelial cells of the midgut, or grouped to form organs (symbiosomes), variously located as to the gut, named mycetomes and bacteriomes, respectively. according to the host species, symbiosomes are present only in the larvae, or both in larvae and adults. endocytobiotes are always maternally inherited, following two main routes, as mentioned later on. in powderpost beetles, for example in l. linearis, symbiotes are bacteria or bacterium-like organisms in bacteriomes located in the posterior third of the larva, close to the fat body; the matrifilial transmission is transovarial. in the adult female, symbiotes migrate into ovaries and infect ovocytes so that the intracellular condition of the symbiotes in the host is therefore almost permanent. also in bostrichidae (a family which also includes some species that damage seasoned wood), for example in the genus sinoxylon, endocytobiotes are bacteria or bacterium-like organisms which are transmitted with transovarial modality (nardon & grenier, 1989). in anobiids, endocytobiosis is widespread and relatively well-known; symbiotes are yeasts or yeast-like organisms, located more or less close to the midgut of the larva and present also in adults, in the same location. the matrifilial transmission is performed by the ovipositor, which is provided with intersegmental tubules and vaginal pouches from which the symbiotes, which secondarily transmigrated here, are expelled during the oviposition and fixed with secretions on the corion (egg smearing). the emerging larva is re-infected when it devours part of the eggshell. in stegobium paniceum (linnaeus), mycetocytes are located in ceca of the midgut, close to its junction with the foregut; in anobium spp. they are gathered similarly in blind sacs, not connected with the gut; in p. pectinicornis, instead, mycetomes are connected with the midgut by a narrow canal. numerous species of cerambycidae have been observed to be symbiotic. in cerambycid endocytobiosis, similarly to anobiid ones, symbiotes are yeasts or yeast-like microorganisms, mycetocytes are kept in evaginations located at the beginning of the larval midgut (described for example in tetropium), the matrifilial transmission occurs through deposition of yeasts on the egg surface. nevertheless, endocytobiosis is less widespread in cerambycidae than in anobiidae as in many long-horned beetles (h. bajulus among them) it has not been found. in larvae of cerambycidae having endocytobiotes, a constant elimination of mycetocytes in the lumen of the gut can be observed, and constant replacement by re-infection of gut cells by yeasts exists. therefore, as in this case symbiotes present an intracellular and an extracellular phase, they could represent an example of an intermediate condition between endocytobiosis and extracellular intestinal symbiosis (nardon & grenier, 1989). considering that the significance of endocytobiosis should be that of providing lacking nutrients to the larva and not enzymes for cellulose digestion, it is difficult to explain why it is absent in h. bajulus, which lives on seasoned wood. in fact, in lyctidae, which feed exclusively on starch, these symbioses are present all the same. not for this reason alone, it must be admitted that the biological significance of endocytobiosis in wood-boring beetles is still relatively unknown. e. chiappini, r. nicoli aldini: adaptations of wood-boring beetle larvae 55 peculiar features in tracheal spiracles undoubtedly, the presence of more or less fine frass inside the gallery and the fact that such an environment is often rather or very dry also affects the respiratory system. structural and functional adaptations, some evidence of which is found in the surface respiratory devices, the tracheal spiracles, are needed. in wood-boring larvae, the most obvious risk is that tiny wood fragments enter the tracheal lumen, compromising gas exchanges. indeed, when the trachea is in communication with the outside, and air penetrates as an effect of the depression, small wood particles could be sucked into the inner space of the tube and occlude it. at the same time, there is the need to minimize water vapour losses through spiracles, due to transpiration, as a consequence of the gradient existing between the lumen of tracheal ramifications and the dry air in the tunnel. spiracles with highly developed filter mechanisms in order to exclude extraneous particles and/or to reduce loss of humidity, are suited to preventing both risks (mill, 1985; 1998). nevertheless it is noteworthy that many other kinds of insects (beetles of other families, flies, etc.) living in heterogeneous habitat conditions, have, as berlese (1909) already noted, analogous exigencies and are provided with spiracular ‘filters’ morphologically varying but with a common structural feature that is the presence of fig. 3 spiracles of wood-boring beetles observed under sem. lyctus linearis (goeze): a. abdomen extremity of the larva with the last two spiracles; b. penultimate abdominal spiracle; c. adult abdominal spiracle; d. trichoferus holosericeus (rossi), larval thoracic spiracle. journal of entomological and acarological research, ser. ii, 43 (2), 201156 ramified and intersected hairs, in order to create a barrier at the level of spiracular entrance. the observation of spiracles of some of the wood-boring beetle larvae considered above shows clear morphological adaptations responding to the double requirement (fig. 3). the operating mode of the respiratory system in insects could also explain the function of the filtering spiracles. when air is sucked into the tracheal lumen by means of the depression existing between the outside and the inside, wood particles are stopped by the hairs, but when the air is actively pushed out, it cleans the spiracle sieve. the idea that this kind of spiracle represents an adaptation to the peculiar 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(2007) showed that food deprived anisops bouvieri fed on two to 34 mosquito larvae per day. another study noted rates of eight to 30 mosquito larvae per day for notonecta sellata (fischer et al., 2012). in that study, 30 was the maximum number of mosquito larvae in each experimental trial, thus predation rates could possibly have been higher. notonecta sellata fed on early mosquito instars at significantly higher rates than on late instars. a mean predation rate of 16 mosquito larvae per day was reported from australia for an anisops species (shaalan et al., 2007). here the predation rate for first instar mosquito larvae was 25 larvae per day, while fourth instars were preyed upon at rates of 13 larvae per day. the current study provides insights into the mosquito predation rates of anisops breddini, kirkaldy 1901, a backswimmer species common to southeast asia. this species is often found in ponds and canals (leong, 1962), but it was also frequently noticed in water storage tanks and other container-like ornaments. water-filled containers are a major source of vector mosquitoes such as aedes aegypti (clements, 1999). therefore, mosquito predators that inhabit these habitats are of particular interest for vector control. the effects of predator density, prey density and prey type on the predation rates were experimentally studied. it was hypothesized that with increasing predator densities the predation rates decrease. interfering behaviour such as social behaviour is known to reduce predation rates (beddington, 1975; crowley & martin, 1989). in backswimmers, cannibalism is not uncommon and can cause a decrease in active hunting behaviour (martín & lópez, 2004). prey density was expected to positively affect correspondence: robbie weterings, cat drop foundation, boorn 45, 9204 az, drachten, the netherlands. tel.: +66.890176087. e-mail: r.weterings@catdropfoundation.org key words: mosquito predation, anisops breddini, biological control, predator density, prey density, prey type. contributions: rw data collecting and manuscript writing; kcv statistical analyses; cu study design and manuscript reviewing. conflict of interests: the authors declare no potential conflict of interests. funding: the work was mutually funded by a scholarship from the faculty of agriculture natural resources and environment, naresuan university and the cat drop foundation. acknowledgements: the authors would like to thank the faculty of agriculture natural resources and environment of naresuan university and the cat drop foundation for financial support. we would also like to thank dr. hannah l. buckley for support, ideas and encouragement and mr. andrew hunt for proof reading the manuscript. received for publication: 25 april 2014. revision received: 5 july 2014. accepted for publication: 9 july 2014. ©copyright r. weterings et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:4036 doi:10.4081/jear.2014.4036 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:4036 jear_2014_3_hrev_master 16/12/14 10:38 pagina 107 no nco mm er cia l u se on ly [page 108] [journal of entomological and acarological research 2014; 46:4036] predation rates, subsequently showing a typical holling type ii functional response (holling, 1959). the predation rates decelerate when the rates reach a food saturation point and handling time increases with lower prey densities (holling, 1959; giller, 1980; gergs et al., 2010). also, prey size and type are known to affect the predation rates of backswimmers (murdoch et al., 1984; chesson, 1989). a backswimmer needs to consume smaller prey to gain the same amount of energy in comparison with a larger prey (gergs et al., 2010). therefore predation rates were expected to differ among different prey types. materials and methods an. breddini specimens were collected with a hand-held net in a temporary pond on october 22nd, 2012 in kamphaeng phet, thailand (latitude: 16° 29’ 34.551” n, longitude: 99° 30’ 53.0778” e). the specimens were kept in a bucket filled with water from the same pond. the specimens were collected one day prior to the experiment. mosquito larvae were collected from a roof drain in the same area and from water storage containers in the nearby village, nong pling. the specimens were then identified using pictures taken with a digital microscope and the keys provided by nieser (2004). on the day prior to the experiment, an. breddini specimens were divided into 25 1.5-l transparent plastic containers, each filled with 1 l of water from the pond where the backswimmers were collected. this water was filtered to remove other aquatic organisms and debris. backswimmers were then added to the containers in different densities ranging from one to five individuals per container. in total, there were five containers for every density treatment. however, a single backswimmer in one of the treatments with five backswimmers was found dead and was excluded from the study. a total of 70 backswimmers were used, which included both nymphs and adults with an average size of 3.7 mm (+/0.13). the smallest specimen was 1.57 mm and the largest was 6.67 mm. the mean body size for each predator density treatment is displayed in table 1. this study did not focus on adult specimens only, because this would inflate the predation rates. natural populations consist of a mix of different age classes; therefore, a range of developmental stages was used that was representative for the habitat from which we collected the backswimmers (table 2). a water plant (pistia stratiotes l.) was also added to each of the containers to simulate a more natural habitat and to provide a substrate for the backswimmers and refuge for the mosquito larvae. the backswimmers were kept in their experimental habitat for one day to acclimatise. the mosquito larvae were grouped per species (ar. moultoni and ae. aegypti) and were added to the containers in different densities, ranging from 5 to 30 mosquito larvae per container. mosquitoes were added one day after the backswimmers were added, which marked the start of the experiment. only 3rd and 4th instar mosquito larvae and pupae were used for this experiment. prey treatments consisted of ar. moultoni larvae, ae. aegypti larvae or pupae of both species. three containers were supplied with five prey items, five with ten prey items, five with 15 prey items, five with 20 prey items, four with 25 prey items, and two with 30 prey items. the mosquito larvae were added to the container in a way that maximized the number of unique treatment combinations (prey and predator densities), which increased the variation in the data. after 24 h, the number of remaining mosquitoes were counted. this number was then subtracted from the starting density to estimate the number of mosquito larvae preyed upon. after the experimental trials, the backswimmers were placed onto a ruler and photographed. the photographs were analysed on the computer with imagej 1.46a software (ferreira & rasband, 2012) to measure the length of every backswimmer. finally, the number of mosquitoes preyed upon were divided by the number of backswimmers in each container to gain an estimated predation rate (larvae/day/backswimmer). these predation rates were then analysed using linear regression models. a priori hypothesized models were developed following the model inference approach described by anderson (2008). the predation rates were (log+1)-transformed to achieve normality. nonlinear relationships were linearised using log transformations on the specific explanatory variables. all explanatory variables were standardised to a mean of zero and a standard deviation of one (zuur et al., 2009). the levene’s test for equal variances was used to check for any violation of the assumption of equal variances among the different prey types. the akaike information criterion (aic) scores were used to select the best model, the model with the lowest aicc score being the best model. aicc scores are similar to aic scores but contain an extra penalty for additional variables; therefore, the aicc score is more conservative (anderson, 2008). the model with the lowest aicc score was visualised using the visreg package (breheny & burchett, 2012) in rstudio, version 0.97.551 (rstudio, 2012), built on r version 3.0.2 (r development core team, 2013). eventually, model selection probabilities were calculated to indicate the likeliness of a specific model to be the best model. these model probabilities were used to calculate an average model based on all the tested models (anderson, 2008). results the regression models displayed a typical non-linear functional response curve for predator density but not for prey density (figure 1). prey type was the most important variable in our models, followed by predator density and prey density, respectively (table 3). the model with the lowest aicc score differed more than three points, which made calculation of an average model unnecessary (table 4). this is reflected in the small differences of the parameter estimates between the best and averaged model (table 3). the log-transformed predation rates were significantly different for the three different prey types (f=6.14, degree of freedom=21, p=0.008). tukey’s post hoc test showed that the transformed predation rates were different for ae. aegypti and pupae. there was no significant difference between ar. moultoni and ae. aegypti or ar. moultoni and article table 1. mean size of an. breddini for treatments with different predator densities. treatment mean predator size (mm) 1 predator 3.8 (±0.6) 2 predators 3.9 (±0.6) 3 predators 3.9 (±0.3) 4 predators 3.6 (±0.6) 5 predators 3.5 (±0.5) table 2. number of an. breddini in different size classes. size class (mm) number of individuals 0-2 4 2.1-3 15 3.1-4 20 4.1-5 19 greater than 5 7 jear_2014_3_hrev_master 16/12/14 10:38 pagina 108 no nco mm er cia l u se on ly pupae. the mean (±standard error) predation rate for ae. aegypti was 5.94 (±0.79) mosquito larvae per day for ar. moultoni, and for the pupae this was 3.78 (±2.2) and 1.23 (±0.42), respectively. the mean predator size was 3.71 (±0.14). discussion the results of this study suggest that an. breddini feeds on a moderate number of mosquito larvae. the predation rates are highly dependent on the type of prey, the predator density and the prey density. previous studies of other notonectidae species have shown similar results. the predation rates were generally lower than that of other notonectidae species. saha et al. (2007) found predation rates between 2-32 mosquito larvae per day, with a mean of 15 for an. bouvieri. these predators, with a mean size of 6.22 mm, were fed with culex quinquifasciatus, a slightly smaller species than those from the genus aedes (saha et al., 2007). moreover, their study did not include different predator densities (saha et al., 2007). when considering only the treatment with a single predator and the smallest prey item, the mean predation rate was 8.7. adult an. breddini and an. bouvieri are generally equal in size, with an approximate body length of 5.7-6.8 mm and 5.7-6.3 mm, respectively (nieser, 2004). nevertheless, the an. breddini specimens used in the current study were generally much smaller than an. bouvieri, which might explain the difference in predation rates. predator [journal of entomological and acarological research 2014; 46:4036] [page 109] article figure 1. visualisation of the best model based on back-transformed predation rates (larvae per day). the grey areas display confidence bands. the dots in the graphs are kept constant for the variables that are not displayed in each specific graph. a) predator density versus predation rates, b) prey density versus predation rates and c) prey type versus predation rates. table 3. model estimates of the best model and the averaged model for the regression models in which the predation rates were log+1 transformed. best model averaged model model parameter estimate standard error estimate standard error log(prey density) 0.33 0.07 0.33 0.07 log(predator density) 0.38 0.07 �0.38 0.07 type=aedes 1.83 0.09 1.83 0.09 type=armigeres 0.43 0.17 �0.43 0.17 type=pupae �1.15 0.14 �1.14 0.14 table 4. comparison of all linear regression models. model* k aicc δi wi adjusted r2 m+p+t 6 22.55 0.00 1.00 0.82 p+t 5 36.75 14.20 0.00 0.64 m+t 5 41.75 19.20 0.00 0.55 t 4 42.33 19.78 0.00 0.50 m+p 4 50.98 28.43 0.00 0.28 p 3 52.66 30.11 0.00 0.17 null 2 55.63 33.08 0.00 na m 3 57.39 34.84 0.00 -0.01 *the variables for each model are given in the first column. m, prey density; p, predator density; t, prey type; k, number of parametes; aicc, akaike information criterion; i, difference in aicc score in comparison with the best model; wi, the model weights. jear_2014_3_hrev_master 16/12/14 10:38 pagina 109 no nco mm er cia l u se on ly [page 110] [journal of entomological and acarological research 2014; 46:4036] and prey size are important factors that strongly influence handling time and capture rates (thompson, 1975; hewett, 1980; hirvonen & ranta, 1996). fischer et al. (2012) also found higher predation rates for notonecta sellata when feeding on culex pipiens. they found a mean predation rate of 22 for 4th instar mosquito larvae. these rates were slightly higher for 2nd and 3rd instar mosquito larvae (fischer et al., 2012). notonecta sellata is a relatively large species with a mean size of 8.7-9.6 mm (heckman, 2011). predator densities strongly affected the predation rates of an. breddini. predator density was also shown to affect predation rates in other studies on notonectidae (sih, 1981). in general, the mechanism behind this effect is that, with higher predator densities, the number of interactions between the predators increases (sih, 1982; crowley & martin, 1989). also, cannibalism is more likely to occur with increased predator densities; this specifically affects predation rates of the smaller specimen when encountering larger conspecifics (sih, 1982). our data did not show a typical functional response curve for prey densities. we predicted that increased prey densities would positively affect predation rates in a decelerating manner until it reaches a maximum. this effect is often caused by satiation (holling, 1959). if prey densities would have been further increased, this satiation level might have been reached, which also indicates that predation rates could potentially be higher with higher prey densities. predation rates differed significantly among different prey types. predation rates were much higher for ae. aegypti larvae in comparison to ar. moultoni larvae or pupae. although larval size was not measured, ae. aegypti are generally smaller than ar. moultoni larvae (clements, 1999). therefore, more prey items are needed to meet the same energy demands (stephens & krebs, 1986). prey size is an important driver in the predation rates of notonectidae, which affects both the handling time as well as the capture rates (murdoch et al., 1984). several studies have investigated the prey preference of different notonectidae species. when notonectidae are exposed to different prey items, they generally show a preference for mosquito larvae over three species of water fleas (cladocera) (murdoch et al., 1984). other studies, however, do not identify a preference for mosquito larvae (chesson, 1989). sih (1986) conducted an experiment on notonecta undulata in which he compared the predation on culex versus aedes larvae. this study showed that the behaviour of the mosquito larvae was an important factor in prey selection. aedes larvae generally did not display predator avoiding behaviour, while the culex larvae did. as a result, aedes larvae were preyed upon at much higher rates. this might partially explain the difference in predation rates, in particular the low predation rates for the pupae (pupae are generally less active). nevertheless, in the current study, prey behaviour was not observed, nor were the predators exposed to different prey types simultaneously. therefore, conclusions with regard to prey behaviour and predation rates cannot be drawn. the current study showed that an. breddini feeds on only a small number of mosquito larvae compared to other backswimmer species (saha et al., 2007; fischer et al., 2012). these relative low predation rates can partly be ascribed to the use of mixed predator instars. natural communities do not only consist of adult specimens, and these lower predation rates are therefore more realistic in comparison with predation rates based on adult specimens. in the current experiment, only third and fourth instar mosquito larvae were used. early instar mosquito larvae are generally preyed upon in much higher rates than third and fourth instar mosquito larvae (saha et al., 2007; fischer et al., 2012). predation rates are thus likely to be higher when an. breddini is exposed to first or second instar mosquito larvae. an. breddini is very common in thailand and other southeast asian countries (leong, 1962), which is beneficial for the control of vector mosquitoes. although other mosquito larvae predators might be more efficient in vector control, their wide distribution and abundance could potentially make an. breddini a valuable addition to existing biological vector control agents. not only does an. breddini feed on mosquito larvae, there might also be other mechanisms that can benefit mosquito control. other notonectidae species are known to affect the development of mosquito larvae into adults (fischer et al., 2012). in the presence of notonectidae, development of mosquito larvae can take longer and adults tend to be smaller (fischer et al., 2012). other species are known to repel certain mosquito species from ovipositing (blaustein et al., 2005; silberbush & blaustein, 2011). more research is needed that focus on these last two aspects for an. breddini, which might reveal all the vector control benefits of this common species. references anderson d.r., 2008 model based inference in the life sciences: a primer 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[journal of entomological and acarological research 2014; 46:4036] [page 111] article jear_2014_3_hrev_master 16/12/14 10:38 pagina 111 no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd l. maistrello, j. d’ilario, g. bicheron, c. bouleau damage by insect pests to the djingarey ber mosque in timbuktu: detection and control abstract the djingarey ber mosque in timbuktu (mali) is one of the most significant earthen construction in west africa. originally constructed in 1327, it was included in 1988 on the world heritage unesco list for its unique architecture and historical importance. during its restoration, recently undertaken by the aga khan trust for culture, the wooden parts of the roof and architraves showed clear signs of threatening insect presence. in order to identify the pests responsible of the damage, evaluate its extent and suggest a proper control strategy, a detailed survey was performed inside the mosque complex and in its immediate surroundings. the entomological inspection, performed in the dry-cold season, allowed to detect signs of insect damage in most of the wooden elements, even in the recently replaced beams, but also in walls, pillars and the precious decorated panels. damages in the wood elements could be attributed to amitermes evuncifer silvestri (termitidae), bostrychoplites zycheli marseuli (bostrichidae) and lyctus africanus lesne (lyctidae), which were collected alive on site. injures in the walls and decorated panels appeared to be performed by hymenopterans such as “plasterer bees” (colletidae) and sphecidae. from the evaluation of the type and extent of damage in relation to the architecture and materials used in its construction and decoration, the most serious pest and the worse threat for the mosque is represented by termites. control and preventive measures, in the view of a sustainable, long-lasting integrated management are suggested. key words: bostrichidae, earthen construction, entomological survey, termites, xylophagous pests. introduction known to be constructed in 1327 at the initiative of the king hadj moussa upon his return from pilgrimage to mekka, the djingarey ber mosque is one of the most significant earthen construction in west africa and the main congregational mosque of timbuktu. in 1988, the site was included in the world heritage unesco list for its unique architecture and historical importance. not only the mosque has experienced a number of modifications but the organic nature of earthen architecture and its vulnerability to weathering elements makes it subject to rapid changes and alterations. archaeological test pits, carried out in the main prayer hall, showed that at least 3 successive buildings have occupied the site. on the other hand, the main earthen ornaments on the qebbla j. ent. acarol. res. ser. ii, 43 (2): 99-106 30 september 2011 wall and some pillars may date back from the 16th century. the mosque, as it stands today, includes a large covered prayer hall supported by 25 rows of massive square pillars lined up in the direction east-west. this is where the imam preaches and the community gathers for friday prayers. on top of the flat roof, the square minaret (14 m height) is the most significant urban feature, visible from distance. both the minaret and the external walls of the mosque complex have “torons”, typical elements of this architecture, consisting in beams of marula wood (sclerocarya birrea (hochst.), anacardiaceae) partially inserted in the walls and jutting out of them. the eastern wall, including a niche, the mihrab, providing direction to mekka for the worship, is the oldest part of the mosque, which later developed to the west and north. two courtyards are part of the precinct: the big sandy western courtyard is an open air extension of the prayer hall designed to accommodate the large number of community members for friday prayers, while the northern courtyard is a more intimate one, separating the women praying area from the main praying hall. there are no foundations: the mosque is directly built on the sand with alhore stone blocks, a kind of tuff stone quarried in the nearby desert; the irregular stone blocks are assembled with mud mortar, the whole is then sealed with three layers of mud and straw plaster. the ceiling (about 460 cm height) is made of circular doum palm (hyphaene thebaica (l.) mart., arecaceae) beams (320 cm length, 20 cm ø) covered with panels of “golettes”, small wooden sticks (45 cm length, 2.5 cm ø) obtained from local shrub wood (boscia angustifolia guill. and perr., capparidaceae and salvadora persica wall., salvadoraceae), to support the various mud layers of water tightness. the architraves between the pillars are made of doum palm beams (220 m length, 20 cm ø); the 14 entrances have doors of marula wood. there are no windows, light and ventilation are provided by round holes on the roof, covered by special terracotta elements. between 2008 and 2010, the aga khan trust for culture (aktc), the cultural agency of the aga khan development network, undertook the restoration of the whole site. before restoration work commenced, the mosque was in poor condition: the roof was overloaded with hundreds of tons of accumulated mud and sand, creating damages on the carpentry palm tree beams and supportive pillars. moreover, the inappropriate slopes design of the roof created water ingress compromising the wall’s consistency and involving cavities. during the works, worrying signs of insect damage were discovered in the palm beams forming the roof and architraves. in order to evaluate the type and extent of the damage and to guarantee a proper and safe management of the problem, the aktc recognized the need of a professional entomologist to perform a thorough inspection of the mosque and its immediate surroundings. the present work shows the results of the entomological survey and provides suggestions for a sustainable integrated management strategy of the insect pests in the djingarey ber mosque. matherials and methods the entomological survey was performed on january 6-12, 2009 in the city of timbuktu (mali) during the cold-dry season. at that time, the mosque was in an interjournal of entomological and acarological research, ser. ii, 43 (2), 2011100 mediate phase of the restoration project, with areas already fully restored, areas under restoration (roof removed, walls with removed external layers) and areas to be restored (fig. 1). accurate monitoring of all different parts and structures of the mosque complex and its surroundings was performed by looking carefully for any type of signs of insect presence and for alive/dead insects or insect parts. some of the wooden poles (wood of eucalyptus sp.) inside the western courtyard were l. maistrello et al.: damage by insect pests to the djingarey ber mosque, detection and control fig. 1 map of the djingarey ber mosque. at the time of the entomological survey, areas 1-2a2b were already restored, areas 3a-3b were under restoration, and areas 4a-4b not yet restored. 101 extracted from the sand, opened, and alive insects were detected and collected. the survey was performed also in the mosque surroundings, that include: a) the external perimeter of the mosque complex and the streets leading to it, where heaps of old beams were found all over; b) the storage area, with piles of new and old beams and golettes, some of which were opened to collect alive insects; c) private houses in front of the mosque. observations were recorded and many pictures were taken by means of a digital camera (canon powershot a620). the following description refers to the situation at the time of inspection. alive specimens were collected in vials and brought to the laboratory for taking measurements and pictures under stereomicroscope, and for identification by means of specific keys. results the following is a description of the signs of insect presence discovered during the inspection, together with the identification of the causative agents when alive specimens were found. tab. 1 shows where the signs were detected in the mosque and its surroundings, and their extent. big round holes (3-5 mm ø): in wood elements, mostly on the beams, ranging from a few dozens to a few hundred on each beam. they represent either entrance holes of adult females or the exit holes of newly emerged false powderpost beetles. alive specimens collected from opened beams were identified as bostrychoplites zycheli marseul (coleoptera, bostrychidae). small round holes (1-2 mm ø): in wood elements, from a few dozens/golette to a few hundred/beam. they represent exit holes of newly emerged true powderpost beetles. alive specimens from opened beams/golettes were identified as lyctus africanus lesne (coleoptera, lyctidae). big irregular holes and corresponding galleries (7-12 mm ø): only in some of the beams exposed to open air, along the courts. they represent the nests of some local carpenter bee (hymenoptera, apidae, xylocopinae), which were seen actively flying around the beams, although not catched. partially/totally destroyed beams or poles: almost all discarded beams (those removed during restoration operations) and the beams inside the minaret were almost completely destroyed. the type of damage and the dark “replacement wood” inside indicate high termite activity. in the big sandy court, all the wooden poles were seriously attacked by termites, and alive specimens found inside were later identified as amitermes evuncifer silvestri (isoptera, termitidae, termitinae). journal of entomological and acarological research, ser. ii, 43 (2), 2011102 tab. 1 signs of insect presence inside the mosque complex and its surroundings (ar= area already restored, ur= area under restoration, nr= area not restored; extent of damage: – = absent, + = small, ++ = medium, +++ = high, n.d.= not detectable, √ = alive insects detected). l. maistrello et al.: damage by insect pests to the djingarey ber mosque, detection and control mud tubes mud coverings: found over roof beams, door architraves and other wood elements; inside private houses there were mud tubes up to 300 mm long hanging from the roof beams. they clearly indicate termite activity as they are typically made by termites as shelter structures during their foraging activities using saliva, excrements and soil. 103 dark galleries: discovered inside walls and decorated panels in the area under restoration, where the outer layers had been removed. although no living insects were detected, these structures are likely to represent galleries performed by the termites coming from the ground, digging their way between the clay-straw layer and the yellow plaster layer to reach the roof beams. the dark colour of these galleries is due to the fact that usually termites, in order to prevent desiccation, line their galleries with mud/nest material, mixed with saliva and faeces. mud nests (50-100 mm long, 20-50 mm wide): frequently found inside the mosque, attached to the walls or to the beams. while inspecting, no living insects were ever seen around them. they represent “paedotrophic nests” built by adult females of some predator hymenopterans (sphecidae) with mud collected from the surroundings and saliva, to provide shelter and food for their larvae. small holes in the outer coverings (1-2 mm ø): detected in groups of 20-40 on some pillars and decorated panels. although no living insects were ever detected, they could have been performed by some kind of local “plasterer bees” (hymenoptera, colletidae). discussion and conclusions from the evidence obtained during the inspection, the most serious problem for the djingarey ber mosque was represented by termites as structural pests of all wooden elements. although presence of other species is not excluded, the one found inside the buried wood poles is a. evuncifer, a wood feeding termite common in west africa, known to be a serious pest of buildings and wooden structures (omo-malaka & leuthold, 1986), that usually builds mounds in forests while in savannah is either subterranean or nesting in logs (wood et al., 1980). unfortunately the architecture and the materials used in this mosque are particularly favourable for termites: abundance of food sources (beams, golettes, rice straw inside walls) and walls/pillars directly in touch with the ground, whose structure allow termites to reach the food source on the roof using the “dark galleries” system, well sheltered inside the walls. moreover, abundance of wood elements both inside the mosque complex and in its immediate surroundings provides constant food supply for already established colonies and may attract new ones. xylophagous beetles also represent a serious threat to the mosque and the high numbers of holes observed in the beams even in the recently restored area possibly indicates considerable structural damages to the affected elements. lesne (1896) reports that b. zycheli was found in timbuktu developing in palm wood and is well known as a pest of timber in service, just like l. africanus, endemic in the oriental-african bioregions and presently worldwide distributed in all inter-tropical regions with dry climate (walker, 2005). from this survey it emerged also that termites, with their dark galleries inside the journal of entomological and acarological research, ser. ii, 43 (2), 2011104 wall coverings, could represent a serious damage for the precious decorated panels found on some of the pillars, together with the small holes probably dug by local plasterer bees. mud nests found all over inside the mosque could be considered just as a purely aesthetic damage. suggestions for a sustainable integrated management strategy aiming at reduction and prevention of attacks from wood pests include: a) wood treatment of all elements of the mosque complex and possibly also its surroundings using borate compounds which provide long-lasting efficacy against wood pests (gentz & grace, 2006) and are relatively safe for mammals and the environment (currie, 1996), b) removal of any non-treated wood/cellulosic material from the mosque and its surroundings to prevent re-infestation; c) in order the reduce wood pests in the mosque as well as inside private houses, it would be extremely useful to activate a basic education program on essential elements of wood protection, such as the prohibition to recycle old/infested wood, the basics of xylophagous insects biology and the use of borates instead of residual insecticides. acknowledgements the authors are grateful to dr. souleymane konate, universite d´abobo-adjame (abidjan, côte d´ivoire) for termite species determination. references currie w.e., 1996 the environmental advantages of using diffusible preservatives. in: second international conference on wood protection with diffusible preservatives and pesticides, ed. by williams l.h., pp 38-41. forest products society, madison, wi. gentz m.c., grace j.k., 2006 a review of boron toxicity in insects with an emphasis on termites. j. agr. urban entomol., 23: 201-207. lesne p., 1896 revision des coléoptères de la famille des bostrychides. société entomologique de france, paris, 65. omo malaka s.l., leuthold r.h., 1986 mechanisms of recruitment for the retrieval of food in amitermes evuncifer silvestri (isoptera: termitidae: termitinae). insect science and its application, 7 (6): 707-721. walker k., 2005 powderpost beetle (lyctus africanus). pest and diseases image library. updated on 9/2/2011 3:33 pm available online: padil http://www.padil.gov.au wood t.g., smith r.w., johnson r.a., komolafe p.o., 1980 termite damage and crop loss studies in nigeria pre-harvest losses to yams due to termites and other soil pests. tropical pest management, 26: 355-370. l. maistrello et al.: damage by insect pests to the djingarey ber mosque, detection and control 105 lara maistrello, dip. scienze agrarie e degli alimenti, univ. di modena e reggio emilia, via g. amendola 2, i-42122 reggio emilia, italy. e-mail: lara.maistrello@unimore.it josephine d’ilario, aga khan trust for culture, 1-3 avenue de la paix, 1202 geneva, switzerland. e-mail: josephine.dilario@akdn.org gautier bicheron, aga khan trust for culture, 1-3 avenue de la paix, 1202 geneva, switzerland. e-mail: bicheron.gautier@free.fr christophe bouleau, aga khan trust for culture, 1-3 avenue de la paix, 1202 geneva, switzerland. e-mail: christophe.bouleau@akdn.org journal of entomological and acarological research, ser. ii, 43 (2), 2011106 jear2012 abstract the mediterranean fruit fly, ceratitis capitata (wiedemann) is an economically important pest on fruits all over the world. the origin of this fly is thought to be from africa, but it has recently expanded its distribution in many geographic regions including iran. due to the wide spread of this pest in iran and its serious damage to fruit on trees, including citrus orchards of northern iran, the present study was conducted firstly to investigate genetic diversity within populations of c. capitata based on the sequences of three mitochondrial dna (mtdna) genes including cytochrome c oxidase i (coi), nahd dehydrogenase subunits 4 and 5 (nd4 and nd5) and secondly to compare the iranian haplotypes with those found in other countries. results of this study indicated low levels of genetic diversity (four, four and three haplotypes among different populations of this pest, respectively for the coi, nd4 and nd5 genes) in northern iranian populations. the genetic similarity and very low levels of genetic diversity of northern iranian populations suggest that the pest colonisation occurred relatively recently. in addition, haplotypes of mazandaran province are similar to haplotypes of those countries that have recently been infected by this pest. introduction the mediterranean fruit fly, ceratitis capitata (wiedemann) (diptera: tephritidae) is one of the world’s most economically important pest species of fruit trees (sheppard et al., 1992). this pest has spread rapidly from its putative source area in central africa to north and south africa in 1842 (hagen et al., 1981). the medfly is a major reason for direct economic losses in fruit production, and eradication programs are routinely carried out in several countries where this pest causes considerable damage (dowell & krass, 1992). for these reasons, several research projects have focused on the genetic diversity of the medfly populations in many regions worldwide (sheppard et al., 1992; elfekih et al., 2010a, 2010b). different kinds of genetic markers have been used for analysis of diversity in medfly populations (reyes & ochando, 2004). c. capitata was originally recorded from iran in 1958 (southern iran), and subsequently in 1980 (northern iran) (mirsardo et al., 2010). given the wide distribution, high invasiveness and serious economic impact of this pest on fruit production, it is crucial to conduct a survey of the genetic diversity of medfly populations where this pest occurs (elfekih et al., 2010b). previous studies used several markers including mitochondrial dna, which proved to be very useful given its haploid nature and maternal inheritance as well as its lack of recombination (hewitt, 2004). despite the extensive research work done on medfly genetic diversity in several areas worldwide (reyes & ochando, 2004; barr, 2009; elfekih et al., 2010b), very limited information is available on the genetic variability of medfly populations in iran. the main objective of the present study is to characterise the genetic diversity among medfly populations in iran using mitochondrial dna markers. the sequences of mtdnas including the cytochrome oxidase i (coi), nadh dehydrogenase subunits 4 and 5 (nadh4 and nadh5) genes are used for characterisation of genetic diversity in medfly populations in different areas of mazandaran province (northern iran). furthermore, the sequences of mitochondrial dna from other countries, recorded in national centre for biotechnology information (ncbi), were employed for comparisons. materials and methods adult specimens of c. capitata were collected from different areas of mazandaran province in northern iran (figure 1). the specimens correspondence: masoumeh shayanmehr, department of plant protection, faculty of crop sciences, sari university of agricultural sciences and natural resources, sari p.o. box: 578, mazandaran, iran. telefax: +981513822567. e-mail: shayanm30@yahoo.com key words: mediterranean fruit fly; mitochondrial dna; genetic variation; haplotypes; northern iran. acknowledgements: we would like to thank the sari agricultural sciences and natural resources university for financial support of this research. also we are appreciated to dr. antonio carapelli from italy for kind help during the work. received for publication: 16 may 2014. revision received: 22 october 2014. accepted for publication: 16 january 2015. ©copyright m. rajabiyan et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:4055 doi:10.4081/jear.2015.4055 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. the mediterranean fruit fly (ceratitis capitata) in iran: genetic diversity and comparison with other countries m. rajabiyan, m. shayanmehr, m. mohammadi sharif department of plant protection, sari university of agricultural sciences and natural resources, sari, mazandaran, iran [page 20] [journal of entomological and acarological research 2015; 47:4055] journal of entomological and acarological research 2015; volume 47:4055 no nco mm er cia l u se on ly were captured using pheromone traps (xlure-cec), placed in citrus orchards. all samples were preserved in 90% ethanol (table 1). total genomic dna was extracted from individual specimens using the dneasy blood and tissue dna extraction kit (qiagen, hilden, germany). dna was extracted by placing single individuals in an eppendorf tube and the wings were removed. the extracted dna from medfly specimens was amplified using three pair primers (table 2). the coi primer pair amplifies a 631 bp fragment of the coi gene. amplifications were performed in 25 µl microtubes containing 1 µl mgcl2, 1.5 µl primers (10 pm/µl), 0.5 µl dntps, 2.5 µl polymerase chain reaction (pcr) buffer, 0.2 taq polymerase, 16.8 µl ddh2o, and 1 µl dna. the amplification program has an initial denaturation step of 5 min at 94°c, followed by 35 cycles of 60 s in 94°c, 60 s in 43°c, 90 s at 72°c, and a final extension of 7 min at 72°c. the nd4 and nd5 primer pairs are used to amplify a 350 and 950 bp fragment of the nadh4 and nadh5 genes, respectively. the pcr mix for these primers contain 16.8 µl ddh2o, 2.5 µl pcr buffer, 3 µl mgcl2, 0.5 µl dntps, 0.5 µl primers, 1 µl dna and 0.2 µl taq [journal of entomological and acarological research 2015; 47:4055] [page 21] article figure 1. map of mazandaran province (northern iran) indicating the sampling localities. table 1. information on sampling sites in mazadaran province. sampling site coordinate number of individuals from each gene locality longitude (n) latitude (e) coi nd4 nd5 amol 36° 28’ 14” 52° 21’ 47” 1 2 3 behshahr 36° 41’ 39” 53° 32’ 33” 1 1 3 tonekabon 36° 48’ 31” 50° 52’ 54” 1 1 2 juybar 36° 38’ 17” 52° 54’ 42” 1 2 3 sari 36° 34’ 4” 53° 03’ 31” 1 2 3 qaemshahr 36° 27’ 47” 52° 51’ 36” 1 2 3 mahmudabad 36° 36’ 47” 53° 51’ 36” 0 1 3 neka 36° 39’ 5” 53° 17’ 48” 1 2 3 nur 36° 33’ 45” 52° 01’ 54” 1 2 3 table 2. name and sequence of mtdna primers used for polymerase chain reaction reaction. primer sequence of primer (5�-3�) gene size (bp) reference lco1490 ggtcaacaaatcataaagatattgg coi 360 folmer et al., 1994 hco2198 taaacttcagggtgaccaaaaaatc n-4-j-8883 taataatccatatcctccta n-4-n-9243 ttagttttaacatttagaag nd4 350 elfekih et al., 2010a n-5-j-7991 taataaactcattcaatcaa nd5 950 elfekih et al., 2010b n-4-n-8916 atagaagctcctgtatctgg no nco mm er cia l u se on ly [page 22] [journal of entomological and acarological research 2015; 47:4055] polymerase. the amplification program has an initial denaturation step of 2 min at 94°c, followed by 40 cycles of 30 s in 94°c, 30 s in 46°c, 2 min at 66°c, and a final extension of 2 min at 72°c (elfekih et al., 2010a). amplification products (5 µl) were visualised after electrophoresis in 2% agarose gels in tae buffer (24.2 gr tris acetic acid 71 ml edta 10 ml ddh2o 60 ml) with the 1-log ladder as a molecular weight marker. purification and sequencing of pcr products for forward primer from individual specimens performed by takapu-zist company. the accession numbers in genbank for coi sequences of c. capitata obtained here are km660641-km660652. a blast search of genbank sequences using the sequence obtained from the pcr product was conducted through the ncbi website (http://www.ncbi.nlm.gov). sequences were aligned using clustal x with defaults/parameters (thompson et al., 1997). phylogenetic trees were constructed using the maximum likelihood algorithm in mega5 (tamura et al., 2011). numbers of haplotypes and numbers of mutations were identified using dnasp (librado & rozas, 2009). results intraspecific genetic variation was investigated using molecular marker obtained from coi, nd4 and nd5 sequences in nine populations of the mediterranean fruit fly, c. capitata from northern iran. in sum, genetic relationships were constructed for mitochondrial dna sequences generated from 48 individuals. the total length of coi, nd4 and nd5 products were 650, 372 and 950 bp, respectively (figure 2). in order to eliminate the errors due to sequencing artifacts, we considered only fragments of 631 bp of coi, 350 bp of nd4 and 899 bp of nd5 for all sequences by deleting the beginning and end of the sequences. the number of haplotypes, number of mutations and average frequency of bases for the different markers are shown in table 3. the at rich sequences were observed in nucleotide composition, which is a pattern that has been repeatedly seen in the mtdna of dipteran species (bajpai & tewari, 2010). article figure 2. a) coi; b) nd4; c) nd5-dna fragment amplified of mediterranean fruit fly from different cities of mazandaran province. figure 3. maximum likelihood tree of 25 specimens of ceratitis capitata from mazandaran province and twelve other countries for coi, calculated in mega5. drosophila yakuba used as outgroup. numbers above the branches indicate the bootstrap values for nodes (1000 replications). no nco mm er cia l u se on ly genetic distances were calculated among the nine populations using the binary data obtained for each primer pair. very low genetic distance values were found, indicating a high genetic similarity among the derived populations in general. the largest genetic distance values are observed between neka1 and neka3 (0.008) for coi, behshahr and neka2 (0.022) for nd5, qaemshahr and sari (0.002) for nd5. phylogenetic trees were constructed using likelihood based (ml from 1000 bootstrap replicates) algorithms in mega5 (tamura et al., 2011). a set of medfly mitochondrial dna sequences, available on genbank, was used in order to compare the iranian populations with medfly populations from other geographical regions (figures 3-5). the species, drosophila yakuba (diptera: drosophilidae) was used as an out-group for construction of phylogenetic trees. additionally, statistical parsimony (templeton, 2004) implemented in the tcs software (clement et al., 2000) with a 150-step connection limit, was used to construct parsimonious networks of coi haplotypes for populations of c. capitata. the phylogenetic tree (figure 3) shows that different populations of the medfly are similar in coi gene sequences in that they are placed in one branch separated from the out-group. three populations, including neka-1, neka-3 and tonekabon-2, appear to be a little bit different from the others. the presence of four haplotypes among the nine populations of the medfly is confirmed by the tcs plot as well for coi gene (figure 6). these medfly populations have a bias toward haplotype a. the phylogenetic tree also affirms a very close genetic similarity among populations of medfly in northern iran and some other countries such spain, middle east, and venezuela. however, populations of south africa and kenya show less similarity with the population in northern iran. the phylogenetic tree constructed for the nd4 gene (figure 4) shows populations of medfly from different cities are genetically similar to each other. also the maximum likelihood tree shows the medfly populations come from middle east and tunisia are relatively close to iranian population. but in compare to the phylogenetic tree constructed by coi gene middle east population showed a bit different from iranian population. the phylogenetic tree constructed based on nd5 sequences (figure 5) also reveals genetic similarity among the populations of northern iran. populations of neka, behshahr, jouybar and tonekabon are mostly close to each other and form one haplotype. such genetic similarity is also repeated between populations of nur, sari and amol. the populations of the other countries (usa and argentina) are closely related to the populations of northern iran. however tunisia population showed again a bit deviation from others. [journal of entomological and acarological research 2015; 47:4055] [page 23] article figure 4. maximum likelihood tree of sequences of 15 specimens of ceratitis capitata from mazandaran province and two other countries for nd4, with drosophila yakuba as an out-group, calculated in mega5. numbers indicate bootstrap values for nodes (1000 replications). figure 5. maximum likelihood tree of sequences of 8 specimens of ceratitis capitata from mazandaran province and three other countries for nd5, with drosophila yakuba as an out-group, calculated in mega5. numbers indicate bootstrap values for nodes (1000 replications). table 3. the haplotypes of medfly populations according to sequences of the coi, nd4 and nd5 genes. no. of haplotypes no. of mutations average frequency of bases a (%) t (%) c (%) g (%) coi 4 6 28.7 39.3 16 16 nd4 4 10 47.1 33 10.9 8.9 nd5 3 3 44.2 31.9 16.1 7.8 no nco mm er cia l u se on ly [page 24] [journal of entomological and acarological research 2015; 47:4055] discussion and conclusions dna markers, such as those derived from nuclear genes, rapds and intron sequences, microsatellites, as well as those derived from mitochondrial dna, have been used previously to study mediterranean fruit fly populations (elfekih et al., 2010b). despite the wide spread of the c. capitata in northern iran, no research has been done on previously on genetic diversity and evolutionary relationships of different populations of this pest in iran. according to nei’s estimates (1975) of the genetic distance ranges for local race of a species, low genetic distances among populations of different areas show genetically close relationships among them (menezes, 1990). the low genetic distances found among populations of the medfly in mazandaran province in this study indicate that these populations are genetically similar and that colonisation in citrus orchards by this pest took place once and relatively recently. the similarity among the medfly populations can be for different reasons. one reason could be the close distance of cities in mazandaran province. secondly, the spaces between the sampling localities have been filled with vast citrus orchards. also the products of the orchards are exchanged between the cities, and transmission of infested fruit is one of the most important paths for distribution of the medfly in northern iran (mirsardo et al., 2010). high genetic diversity is generally used as evidence that a species is native, or that it has been established for a long period of time (solorzano et al., 2010). according to the haplotype diversity calculated in this study, it can be concluded that in northern iran the medfly has not been present for a long time. also, it is a new pest recorded for the first time in 1980 in northern iran. additionally, according to the assumptions of malacrida et al., c. capitata populations can be divided into three main categories according to their colonisation pattern: ancestral, ancient and new populations, corresponding to populations from africa, the mediterranean basin and america, respectively (reyes & ochando, 2004). finding of this study indicated there are few haplotypes in medfly population in mazndaran province, so the population of medfly in northern iran can be placed in the third category of malacrida’s classification. in other words, the colonisation of the pest has happened more recently and the pest has not had enough time for genetic divergence (reyes & ochando, 2004). the phylogenic trees for coi gene sequences also confirm malacrida’s classification very well. the population from africa (kenya and south africa) showed older colonisation than populations from europe such as spain and italy, middle east countries and even countries of the americas such as argentina and venezuela. the close relations of the population of iran with other countries show short history of existence of the medfly in these countries. the phylogenic tree for nd4 showed also the african population (tunisia) colonised older than middle east countries such as iran. low genetic diversity in populations from middle east (constructed by coi and nd4) indicated recent colonisation of pest in this area. the phylogenetic tree for nd5 also indicated older colonisation in tunisia (african population) than american and iranian populations. the results of this study indicate that genetic analyses based on the use mitochondrial genes can provide useful tools for unravelling genetic and phylogenetic relationships in c. capitata populations in northern iran. the variability identified by the direct analysis of such sequences will allow for answering a range of questions relevant to pest population dynamics such as the origin of new infestations, the relationships of existing populations and will allow pest management controls to be examined in greater detail than had been possible previously. references bajpai n., tewari r.r., 2010 mitochondrial dna sequence-based 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[journal of entomological and acarological research 2015; 47:4055] [page 25] article no nco mm er cia l u se on ly j. ent. acar. res. ser. ii, 42 (3): 147-151 30 december 2010 g. pellizzari first record and establishment of chionaspis wistariae cooley (hemiptera, diaspididae) in europe abstract - the occurrence of the asiatic diaspidid chionaspis wistariae cooley  in europe is reported. the scale was collected on the leaves of an old wisteria tree  growing outdoors in the university botanical garden of cluj napoca (romania).  riassunto - prima segnalazione e adattamento di chionaspis wistariae cooley (hemiptera, diaspididae) in europa viene segnalata la presenza nell’orto botanico di cluj napoca (romania) della  cocciniglia asiatica chionaspis wistariae cooley, finora mai segnalata in europa.  la specie è stata raccolta su una vecchia pianta di glicine (wisteria sp.) vegetante  all’aperto ed è stata probabilmente introdotta accidentalmente insieme alla sua  pianta ospite.  key words: wisteria sp., armoured scale insects, encarsia citrina (craw), romania introduction the botanical gardens are often a reservoir of alien pests due to the presence of exotic  plants introduced from different areas of the world. the introduction of living plants  can lead to the incidental introduction of their insect pests which, in some cases, may  persist on their host plant in the new environment. if they are small, concealed and at  low population levels, their presence can be undetected for years (pellizzari & danzig,  2007; pellizzari et al., 2010).  a visit to the university botanical garden of cluj napoca (romania) led to the  discovery of chionaspis wistariae cooley, an asiatic diaspidid associated with wisteria  plants, not yet recorded outdoors in europe. wisterias are popular ornamentals, largely  cultivated in europe since xix century when w. floribunda and w. sinensis were imported from japan and china. journal of entomological and acarological research, ser. ii, 42 (3), 2010148 chionaspis wistariae cooley, 1897 syn: phenacaspis fujicola kuwana, 1931  on august 21, 2010 several white covers were observed on the leaves and petioles  of an old wisteria sp. tree (fabaceae) growing outdoors, near the entrance to the greenhouses of the university botanical garden of cluj napoca (fig.1). once mounted on  slides the specimens were identified as chionaspis wistariae, an asiatic species so far  not recorded outdoors in europe.  at the collection date the following stages were present on the leaves: egg-laying  females, eggs and crawlers. several scales were holed by a parasitoids. empty male  puparia, white and tricarinate, were also observed. the scales were settled along the  main or the secondary veins of the leaflets, both on the upper and lower surface; their  number per leaflet did not exceed 25-30, apparently without any damage for the plant.  the scales were almost evenly distributed on the leaves of the old wisteria tree. this  fact, the host specificity of this species and the presence of holed covers indicate that its  incidental introduction probably occurred several years ago, together with its host plant. host plants and distribution c. wistariae develops on wisteria species (wisteria brachybotrys, w. floribunda,  w. multijuga, w. nankinensis, w. sinensis) (liu et al., 1989; malumphy, 2010, personal  fig. 1 - leaf of wisteria sp infested by chionaspis wistariae cooley. 149g. pellizzari: first record and establishment of c. wistariae in europe communication). it was described on specimens first collected in california off wisteria plants imported from japan (cooley, 1897). it is a supposed native of japan and is  present also in china. it has been intercepted several times in the u.s.a. on wisterias  imported from japan, and has become established in california and pennsylvania (liu  et al., 1989; ben-dov et al., 2010). according to miller et al. (2005), it is not considered  a pest over there.  with regard to europe, between 1981 and 1990, c. wistariae was officially detected  on six occasions at commercial plant nurseries in the uk (in the counties east sussex,  norfolk and surrey) on bonsai wisteria brachybotrys and wisteria floribunda imported  from japan (naka-ku, nagoya, aichi prefecture and yokohama, kanagawa prefecture).  in all cases, action was taken to eradicate the scale insects under the guidance of the u.k.  plant health and seeds inspectorate authorities and all infested plants were destroyed  (malumphy, 2010, personal communication). probably c. wistariae has been incidentally  introduced with its host plant several times from japan to european countries, even if  we are aware only of the above reported cases. notes on biology as with other chionaspis species, c. wistariae has a white, oystershell-shaped  cover, 1.5-2.5mm long (fig.2); the specimens settled on the petioles are distinctly longer  than specimens settled on the leaf. its biology is not known in detail but, as with other  fig. 2 - wisteria leaflet with the white, oyster shaped covers of c. wistariae. journal of entomological and acarological research, ser. ii, 42 (3), 2010150 chionaspis species, it develops two generations/year: a summer generation that develops  on the leaves and a second generation that settles on the bark and overwinters as adult  female (liu et al., 1989). the adult females of c. wistariae exhibit two morphological  forms, linked to the feeding site, differing in the shape of pygidial lobes: the leaf-infesting  form and the branch-infesting form. this host-site induced dimorphism is recorded also  in other diaspidids: the two morphological forms of the same species are so dissimilar  that, in the past, they were described and considered as separate species until the situation was clarified (lupo, 1943; takahashi, 1953; takagi, 1961). a detailed account on  this subject is reported by liu et al. (1989) and by takagi (1990).  a few parasitoids swarmed in laboratory from c. wistariae adult females when  in italy (august 28-30, 2010). they have been identified as encarsia citrina (craw)  (aphelinidae), a common solitary endoparasitoid of several armoured scale insects. c. wistariae is a new host of the above recorded aphelinid. the scale insects of wisteria sp. according to scalenet more than 20 different polyphagous scale insect species  have been recorded on wisteria trees, but none of these has proved to be a pest, with the  exception of eulecanium excrescens (ferris) (fam. coccidae). this asiatic soft scale,  which has recorded also in the u.s.a., was first detected in europe, london (uk), in  2001 and has since spread in south-east england, where outbreaks still occur (malumphy,  2005; salisbury et al., 2010). e. excrescens has also been recorded occasionally on other  ornamental and fruit trees, but apparently wisterias are the preferred host. according to  malumphy (2005), e. excrescens has one generation/year; the nymphs overwinter and  reach maturity in april. the adult females lay eggs in may; crawlers emerge in may-june  and settle on the leaves; in autumn, before the leaves fall, they move from the leaves  to the twigs to overwinter. damage consists in sap sucking, excretion of honeydew and  the development of black sooty mould which reduces the aesthetic value of the plant.  with regard to c. wistariae, as reported above, no damage to the infested plant has been  observed; this is in accordance with miller et al. (2005), who consider that c. wistariae  is not a pest of wisterias in the u.s.a., where the scale was introduced more than one  hundred years ago. the university botanical garden of cluj napoca is, so far, the only european  location where this species has been recorded outdoors; further investigations on old  wisterias trees growing outdoors will probably demonstrate a larger distribution of this  species in europe. aknowledgements many thanks are due to dr. c. malumphy (food and environment research agency, sand  hotton, york, uk) for his valuable and detailed information on chionaspis wistariae  interceptions, to prof. antonio garonna (university of naples, italy) for the parasitoid  identification and to c. hodgson (the national museum of wales, cardiff, uk) for  kindly revising the manuscript. 151g. pellizzari: first record and establishment of c. wistariae in europe references ben-dov y., miller d. r., gibson g. a. p., 2010 - scalenet: a database of the scale insects of  the world. available from: http://www.sel.barc.usda.gov/scalenet/scalenet.htm (accessed  september 2010). cooley r.a., 1897 - new species of chionaspis. - canadian entomologist 29: 278-282. kuwana s.i., 1931 -. the diaspine coccidae of japan. vi. genus phenacaspis. scientific bulletin  (ministry of agriculure and forestry, japan) 2: 1-14. liu t., kosztarab m., rhoades m., 1989 - biosystematics of the adult females of the genus  chionaspis (homoptera: coccoidea: diaspididae) of north america, with emphasis on  polymorphism. - virginia polytech. inst & state university, virginia agricultural experiment  station, blacksburg, bulletin 88-2:1-126 lupo v., 1943 - il mytilococcus ficifoliae (berlese) è una forma estiva del m. conchiformis (gmelin) - bollettino del r. laboratorio di entomologia agraria. portici 5: 196-205. malumphy c.p., 2005 - eulecanium excrescens (ferris) (hemiptera: coccidae), an asian pest  of woody ornamentals and fruit trees, new to britain.- british journal of entomology and  natural history 18(1): 45-49.  miller d.r., miller g.l., hodges g.s., davidson j.a., 2005 - introduced scale insects (hemiptera: coccoidea) of the united states and their impact on u.s.- agriculture. proceedings of  the entomological society of washington 107 (1): 123-158. pellizzari g., danzig e., 2007 - the bamboo mealybugs balanococcus kwoni n.sp. and palmicultor lumpurensis (takahashi) (hemiptera, pseudococcidae).- zootaxa 1583: 65-68. pellizzari g., cassina g., cappelletti e.m., frigimelica g., 2010 - the botanical garden of  padua (italy): a reservoir of alien insects and mites.- book of abstracts, 4th global botanic  gardens congress, 13th-18th june 2010, dublin, ireland: 132 (poster abstract ). salisbury a., halstead a.j., malumphy c., 2010 - wisteria scale, eulecanium excrescens  (hemiptera: coccidae), spreading in south east england.- british journal of entomology  and natural history, 23: 4.  takagi s., 1961 - a contribution to the knowledge of the diaspididini of japan (homoptera:  coccoidea) pt. ii.- insecta matsumurana 24: 4-42. takagi, s. 1990 - polymorphism. 59-64 in: rosen, d. (editor), armored scale insects, their  biology, natural enemies and control. world crop pests, vol. 4a, elsevier, amsterdam,  the netherlands: 384 pp. takahashi r., 1953. dimorphism in some species of chionaspis or phenacaspis. (diaspididae,  coccoidea, homoptera).- bollettino del laboratorio di zoologia generale e agraria “filippo  silvestri” 33: 48-56. giuseppina pellizzari - università di padova, dipartimento di agronomia ambientale e produzioni vegetali - viale dell’università 16, 35020 legnaro, italy. e-mail: giuseppina.pellizzari@ unipd.it accepted 30 november 2010 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd k. baslé, o. guillon, f. daniel, f. fohrer monitoring techniques: “stegogis: a geographical information system for knowing and preventing infestation risks in cultural heritage” abstract since 2004, the cicrp: “centre interrégional de conservation et restauration du patrimoine”, located in marseilles has been involved in an interdisciplinary research program dealing with infestation and re-infestation, on lining pastes used in painting conservation, by the stegobium paniceum through a gis system : a geographical information system called “stegogis”. the gis helps understand the insects ethology in its environmental context, mainly in flour and semolina millings in order to determine analysis criteria to prevent, mitigate and fight infestation in the cultural property environment. our approach is based on three main lines: 1a transverse approach of infestation in any type of cultural heritage institution: archives, libraries, museums, historic buildings where organic material collections and environment are attractive on an “insect point of view”. 2 –an ipm strategy (integrated pest management) including conservation and management of collections and buildings also based on an infestation survey with actual or potential risks. 3a “decisionmaking tool” in diagnosis, preventing methods and treatments for professional conservation staff. key words: cartography, ipm (integrated pest management), insects. introduction since 1990, marseilles museums have been facing a huge infestation problem. this phenomenon has been studied and surveyed from the 90’s and carried on a research program in 2004 at the cicrp (baslé et al., 2009). the infestation was found on more than one hundred paintings, mainly found in the art museum of marseilles. well known and high degradations were noted, exceeding the usual signs of woodworm exit holes (fig. 1). the first target consisted in identifying the insect and determining the reasons for its activity. the insect was the stegobium paniceum (kashef, 1955; delobel & tran, 1993; delvare & aberlenc, 1989; fabre, 1979; favral & d’aguilar, 2004) (fig. 2) particularly attracted by the starch paste cooked with flour and commonly used in relining techniques (bouillon & fohrer, 2008). j. ent. acarol. res. ser. ii, 43 (2): 117-127 30 september 2011 fig. 2 stegobium paniceum adult (left) and larva (right); pictures taken from a scanning electronic microscope jeol 6460 lv, (f. daniel, iramat-crp2a, université de bordeaux 3-michel de montaigne). fig. 1alteration on the painting media and lining support. journal of entomological and acarological research, ser. ii, 43 (2), 2011118 working from the archives of the art museum resulted in valuable data: the infested artefacts had been relined in marseilles from 1977 to 1986 by a conservator using glue-paste adhesives combining animal and flour glues (traditional latina method documented since 1660). through painting survey, it appeared that degradations were followed by a problem of reinfestation of the paintings which had been disinfested in 1993. a major infestation occurred, not only in the panel of the relined paintings but also on other paintings: we were facing a generalized and permanent infestation (fohrer & baslé, 2005a; fohrer & baslé, 2005b; fohrer & baslé, 2006; fohrer et al., 2006b). finally, the survey showed that visual examination was not a sufficient criteron to discover an infested or potentially infested state. in fact, the active character of this infestation was difficult to be determined. recognizing or identifying insect pests could be done through common signs of insect activity such as fresh frass (debris of food fragments or dry excreta) from woodworm exit holes or presence of insects adults (dead or alive) but several artefacts didn’t show any of these visible signs and, later, showed an infestation. these specific criteria of stegobium paniceum’s infestation led the cicrp to develop working processes related to the knowledge of the insect life cycle (paulian, 1988), conditions of reproduction and proliferation (fohrer et al., 2006a). it also led us to accurately analyse the various glue-paste adhesives components according to different countries’ recipes: coletta in italy, fish adhesive in russia etc.. all these approaches led to think about a preventive strategy. among the different assistance tools, we decided to choose a surveying cartography tool: a gis (geographical information system) that we called “stegogis”. matherials and methods definition and principles of the gis tool: what is a gis? a geographical information system is a computerized tool to represent and analyse some localized data from a geographical point of view which contribute to environmental management. gis offers opportunities for all kinds of data bases, such as query and statistical analysis, through a unique visualization of geographical maps. otherwise, and because of this specific capability, gis is a unique tool, accessible to a large audience and applying to a wide variety of applications. a gis deals with five functions: geographical data: digital data capture (data acquisition) database management (archive storage) geographical data processing (analysis) visualization (display) graphic representation (abstraction) a gis should reply to five questions: k. baslé et al.: a geographical information system for preventing infestations in cultural heritage 119 -where? localization of the study field and definition of its geographical extent -what? definition of the objects which can be represented in the geographical space -how? distribution of these objects and their relations between them (space analysis) -when? object or event description (time analysis)? -and then? what would happen if such event occurred? a gis is intended to structure the geographical information data which appear as “stackable” layers. stegogis gis helps us to understand the ethology of the stegobium paniceum and other insects, in their environmental context, found in flour and semolina millings. the method was adapted to the cultural property environment. our approach is based on three main lines: 1artefacts potential or actual infestation survey, artefacts being able to move from inside the building to the outside, in case of exhibits for instance, or artefacts moving from the outside to the inside in case of restoration or acquisition, or after building renovation… fig. 3 « bouches du rhône » map ign bd ortho (left) and map ign scan 25 (right). 2recognising pests and assessing the problem by inspection, looking for insects activities like woodworm exit holes on paintings. 3stegobium paniceum and other insect trapping survey inside the geo referenced places. materials the stegogis has been developed from map info 7.5 related to a microsoft access data base. we used airplane ortho-photos provided by the ign (national geographic institute, bd ortho) and scanned images (ign scan 25) (fig. 3). in order to process the working data, we chose a cartography raster (scale: 25/1000). the investigation was made in marseilles where there are several flour and semolina mills (4 mills) and collections too (13 museums, 2 places for archive collections, and several historic buildings and libraries) but the tool can be extended to a regional or national area. journal of entomological and acarological research, ser. ii, 43 (2), 2011120 results data and analysis data were first collected in flour mills and then the experience was extended to the fig. 5 insect identification: family-genus-species. fig. 4 trap localization. k. baslé et al.: a geographical information system for preventing infestations in cultural heritage 121 cultural heritage, based on the same approach concerning insects but of course different concerning the relevant information. the data are collected through the installation of ultra violet (uv) traps (fig. 4) with sticky boards and specific pheromones traps in order to record insects caught from traps (materials : date-label traps, brand traps) and characteristics. as for insects: identification, number, and classification according to the “linnaeus’s» (figs 5 and 6). traps fig. 6 insect identification in marseilles museum storage area. fig. 7 artists list, traps (stars) and painting localization (red squares). journal of entomological and acarological research, ser. ii, 43 (2), 2011122 are located with accuracy in the gis. we also record data on environmental conditions (relative humidity and temperature), of interest regarding the insect cycle and presence. suspended uv (ultra violet) traps were used both for monitoring and mitigation and gave us satisfactory results. in order to get the best results for trapping, it should be noted that the trap location is determined for maintenance reasons (changing uv tubes for which life is limited: from 6000 to 9000 hours). the best is to change them each, once a year, according mainly to uv tubes cost which has to be multiplied by the number of traps. we chose uv traps with a maximum of health and safety requirements: standard anti explosion, water repellent, certified atex dusts zone 22. the size of one trap was 690 x 205 x 400mm with four 20 w/5 tubes and a potential attraction of 350 square meters. disinsectisation treatments history was also integrated (type of treatment and date of application) in flour and semolina millings and cultural heritage places. this historical aspect is very useful especially for the ipm (insect pest management) in order to follow the re-infestation risks and also to establish a “decision-making tool” for people in charge of the collections, according to the treatment repeatability and according to the location of the infestation (generalized or partial). it is also valuable as a memory tool in relation to the “turn over” of the people in charge of the institutions. for cultural objects, different data are stored: information concerning the artefact itself (painter, date, techniques) (fig. 7). we also record information concerning the restoration and the conservators who have been working on paintings. conservation memory of the different operations from insects treatments to painting conservation is of high importance regarding the artefact’s history. fig. 8 structure and protocol. k. baslé et al.: a geographical information system for preventing infestations in cultural heritage 123 fig. 9 image analysis: descriptive method (picture f. baussan & p. glotain, cicrp). image analysis first we had to figure out a new structure to take pictures and a relevant protocol to reproduce pictures in the same conditions at different times (fig. 8). then we developed an “observation tool” through comparative image analysis. this “woodworm exit holes counting system” is based on a statistical comparing pictures protocol. this system provides us with a reproducible tool (figs 9 and 10). discussion and conclusion we started the gis in 2004, recording the data in the flour milling in marseilles then we added the semolina milling data in vitrolles and stopped to record the environmental data in 2010 in this field. it helped for a better understanding of the evolution in a general context according to insect pests which are present all around us in our everyday lives and can have effects on the cultural heritage present in the same areas. it was also useful for people in charge of the food processing industry and helped them to get better assessment of the infestation causes that are constantly faced. journal of entomological and acarological research, ser. ii, 43 (2), 2011124 fig. 10 image analysis system summary. according to the cultural heritage, the gis application makes slower progress for different reasons. first of all these people are not familiar with gis systems: these tools are commonly used by the archaeologists but not by the curators and conservators (question of training and references). but, as time goes on and when compared to our beginnings, things are getting better. these two worlds are completely different: food processing industry and cultural heritage field especially concerning the notion of time and the way the insect pests are dealt. meanwhile we learned a lot from each other and even if our purpose wasn’t to borrow their technology to apply it to our field, we appreciate their organization and their abilities to face insect pests. we learned a lot on the methodological point of view. we also exchanged a lot of data with the pest exterminators people dealing, every day, with all kinds of pests. gis is also a valuable tool for biologists, especially the entomologists, to store all the useful data. gis is a complex tool needing a suitable maintenance as far as computer and software systems are concerned. today we can get the maps information through google map which wasn’t possible at the time we started to work. as we develop the stegogis we extend the experimentation in the cultural heritage media through an extranet colk. baslé et al.: a geographical information system for preventing infestations in cultural heritage 125 laborative portal server: http://stegobium.cicrp.fr. the collaborative portal server intends to centralize the information and consolidates the data. in 2011 and 2012 a “national cartography survey in the french cultural heritage” (museums, archives, libraries and historic buildings) for a better knowledge of infestation risks and reality in the cultural heritage on our territory will be developed. acknowledgements we would like to thank the people of the food processing industry as m. mario cameli, mrs elisabeth martin, and m. rené devot, who let us investigate and collect data in our quest of a better understanding the insect pests phenomena. we would also like to thank the people of the extermination field; especially m. didier jehanno and m. jean françois delbonnel who gave us a lot of information concerning ipm and treatments in mills. we would like to thank the university of perpignan and especially the iut of carcassonne and the people who helped us, in providing their knowledge and their competences: m. jean pierre sorribas, m. pierre wolsztynski and all the team. related to the collaborative server we would like to thank apem society end especially m. laurent plainecassagne and m. thomas portier, with them, we will keep going on the project of the national cartography survey. references baslé k., bouillon n., fohrer f., guillon o., may r., 2009 pour une approche raisonnée des problématiques d’infestation en milieu patrimonial: le cas du stegobium paniceum, techné, 29: 109-114. bouillon n., fohrer f., 2008 study of pest infestation of glue paste lined easel paintings: a characterization of traditional glue paste recipes and their relevant volatils organic compounds by chromatography/mass spectrometry raster. icom-cc 5th triennial meeting, new delhi, 22-26 september 2008. poster. delobel a., tran m., 1993 les coléoptères des denrées alimentaires entreposées dans les régions chaudes, faune tropicale xxxii, paris, orstom / cta, 1993. delvare g., aberlenc h.p., 1989 les insectes d’afrique et d’amérique tropicale: clés pour la reconnaissance des familles, cirad, département gerdat, laboratoire de faunistique, 170-200. fabre j.h., 1979 souvenirs entomologiques: étude sur l’instinct et les mœurs des insectes (10 séries), delagrave editeurs 1921, réédition 1979. favral a., d’aguilar j., 2004 glossaire entomologique, delachaux et niestlé. fohrer f., baslé k., 2005a le stegobium paniceum: un amateur d’art indélicat (1ère partie). npi (nuisibles et parasites information), 41: 24-25. fohrer f., baslé k., 2005b le stegobium paniceum: un amateur d’art indélicat (2ère partie). npi (nuisibles et parasites information), 42: 20. fohrer f., baslé k., 2006 un amateur d’art démasqué. npi (nuisibles et parasites information), 49: 31-33. fohrer f., baslé k., daniel f., 2006a problématique de l’infestation des colles de rentoilage des peintures de chevalet par le stegobium paniceum (l.). support tracé n° 6, 78-85. journal of entomological and acarological research, ser. ii, 43 (2), 2011126 k. baslé et al.: a geographical information system for preventing infestations in cultural heritage fohrer f., baslé k., daniel f., 2006b l’affaire stegobium: mémogravure numéro 001. portefeuille pédagogique, 1-35. kashef a., h., 1995 etude biologique du stegobium paniceum l. (col. anobiidae) et de son parasite lariophagus distinguendus först. (hym. pteromalidae). annales de la société entomologique de france, 124: 1-88. paulian r., 1988 biologie des coléoptères, lechevalier ed. katia baslé, cicrp, 21 rue guibal 13003 marseille, france. e-mail: katia.basle@cicrp.fr odile guillon, cicrp, 21 rue guibal 13003 marseille, france. e-mail: odile.guillon@cicrp.fr fabien fohrer, cicrp, 21 rue guibal 13003 marseille, france. e-mail: fabien.fohrer@cicrp.fr floréal daniel, institut de recherche sur les archéomatériaux, umr cnrs 5060, centre de recherche en physique appliquée à l’archéologie (crpaa), université bordeaux 3, domaine universitaire maison de l’archéologie, esplanade des antilles, 33607 pessac, france. e-mail: fdaniel@u-bordeaux3.fr 127 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd l. maistrello, a. berzolla, i. macias-pavon, f. vignali, g. predieri, e. chiappini wood impregnated with metal chelates dissolved in organic media tested for termite resistance abstract wood manufactured products are subjected to biological decay due to fungi and insects. the use of copper chelates as biocides was proposed, due to their high stability which minimizes copper leaching into the environment. considering the remarkable effectiveness showed by copper chelates on brown rot fungi, zinc and copper salicylate complexes were prepared in order to have metal chelates soluble in organic media available. the present study aimed at evaluating these metal chelates complexes as preservative agents for wood treatment against termites. trials were performed on reticulitermes lucifugus (rossi) and kalotermes flavicollis (fabricius). results showed that in both termite species wood consumption was significantly lower on cu-chelates treated samples compared to untreated wood, whereas the wood slices impregnated with zn-chelates and the organic media alone gave an intermediate response. interestingly, in one case solvent-impregnated wood was significantly more attractive than untreated wood for both species and further investigations are being carried out to clarify this behaviour. key words: wood treatment, preservative agents, copper chelates, kalotermes flavicollis, reticulitermes lucifugus. introduction wood employed in manufactured products is exposed to physical-chemical degradation and to biological decay due to insects and fungi. chromate copper arsenate (cca) has been used for wood preservation for more than 30 years, but in the last decade the use of cca has been restricted in europe due to its toxicity against humans and animals. the new generation of copper based preservatives consists of copper complexes with amines; in particular ethanolamine is largely used owing to its copper fixative properties (humar & lesar, 2007). very recently, the use of copper chelates as biocides, in particular aminoacid derivatives such as copper glycinate, was proposed (palanti et al., 2008), taking advantage of the high stability of these compounds, which would minimize copper leaching into the environment. copper glycinate chelate showed remarkable effectiveness against brown rot coniophora puteana even without any association with other biocides such as boric acid (palanti et al., 2008). j. ent. acarol. res. ser. ii, 43 (2): 277-285 30 september 2011 zinc-based products have also been used as preservatives in the past, among them chromate and zinc chloride formulation and zinc naphtenate are well known; zinc mainly has an antibacterial activity (perelshtein, 2009) but it is also known to inhibit fungal growth at adequate concentration (schultz, 2008). zinc has also been employed against mould fungi, decay fungi, and eastern subterranean termites (kartal, 2009). the aim of the present study was to evaluate copper and zinc salicylates with a biocide action against fungi as preservative agents for wood treatment against termites, using different types of solvents. materials and methods the efficacy of the selected treatments as wood preservatives was tested on the termites kalotermes flavicollis (fabricius) and reticulitermes lucifugus (rossi), taken from naturally infested wood branches fallen on the ground in san rossore forest (pisa) in tuscany. pinus sylvestris l. wood slices, previously impregnated in the treatment solutions, were used. each pine slice (40 x 30 x 3 mm3 in size) was numbered, oven dried at 103 ± 2°c for 18 hours and weighed, than it was soaked for 8 h in one of the treatment solutions listed below, left 24 h to air dry at room temperature, oven dried at 103 ± 2°c for 18 hours and then re-weighed. wood slices impregnated with chelated copper (cu) or chelated zinc (zn) in different media were compared with slices left untreated or treated with solvent alone. in test 1, the following treatments were performed: wood not impregnated (control), wood impregnated with oily solvent (linseed oil), wood impregnated with cu-salicylate in oily solvent, wood impregnated with zn-salicylate in oily solvent, in test 2 the oily solvent was substituted with a very polar solvent (ethylene glycol) in order to increase chelates concentrations in the solutions and a treatment with copper glycinate, that proved to be effective against fungi, was added. therefore, the following treatments were performed: wood impregnated with water, wood impregnated with ethylene glycol, wood impregnated with cu-glycinate in water, wood impregnated with cu-salicylate in ethylene glycol, wood impregnated with zn-salicylate in ethylene glycol. the preservative formulations were prepared as follows. zinc and copper salicylates were directly synthesized starting from a salicylic acid and a metal oxide or salt according to a patent method (leonardi, 2011). in a typical experiment, the carboxylic acid was dissolved in oil at 50°c under stirring. when complete solution was achieved, zinc basic carbonate or copper oxide was added. the system was maintained under vigorous stirring at 60°c for about 30 minutes. a pale pink (zn-salicylate) or green (cu-salicylate) solution was obtained, which was then transferred in a large glass container in order to facilitate evaporation and subsequent formation of crystals. copper glycinate was prepared by reaction of copper acetate and glycine in water. journal of entomological and acarological research, ser. ii, 43 (2), 2011278 in test 1, oily solutions were obtained by solving the powdered metal complexes in oil at 60°c under stirring for 1 hour. a zn-salicylate 0.065m solution (pale orange) and a cu-salicylate 0.043m solution (dark green) in linseed oil were prepared under stirring at room temperature. the same solutions were also obtained by solving the carboxylic acid in oil (at 50°c for about 60 minutes) and allowing it to react with zinc or copper added in the form of oxides or salts directly in the oily solution. for test 2, copper chelates were directly solved in water or ethylene glycol at room temperature in order to obtain solutions with the following concentrations: 0.04m copper glycinate in water, 0.11m copper salicylate in ethylene glycol, 0.2m zinc salicylate in ethylene glycol. in both tests, wood treatment procedure consisted in the immersion of the conditioned wood slices into a volume of solution corresponding to 10/1 of the sum of wood sample volumes. in all cases wood samples were maintained bathed by means of tweezers. treated samples were employed in the tests of effectiveness against termites in the number of 5 replicates for each treatment. test chambers and procedures were different for the two species. considering r. lucifugus, for each replicate 50 workers plus 1 soldier were introduced in a sterilized cylindrical plastic container (3.5 cm height, 6 cm diameter) whose lid had a hole with an externally applied metallic mesh, together with 20 g of sterile sand, 2 ml micro-filtered water and a wood slice. all containers were kept inside a plastic box (30 x 19 x 7 cm) closed with a hermetic lid whose bottom had layers of absorbing paper completely soaked in water, so that inside each box r.h. = 95%. during the experiment the boxes and containers were regularly opened to add water in order to maintain proper moisture. both tests lasted 21 days. for k. flavicollis, sterilized unventilated glass petri dishes (10 cm diameter), with 35 g of sterile sand, 1 ml of micro-filtered water and the pine sample laying on the sand, were used. in each capsule, one soldier plus 28 workers in the first tests and 18 in the second one were introduced. tests 1 and 2 lasted respectively 60 and 95 days. for both species, the boxes were kept in a thermostatic chamber in the dark at t = 25 ± 2°c; during the trial period, test chambers were checked every 10 days to assess mortality. at the end of the experiments, live and dead specimens were counted. wood slices were removed, carefully brushed clean from debris and faeces, then oven-dried at 103 ± 2°c for 18 hours and weighed to assess wood consumption. statistical analyses were performed on angular transformations of the percentage data of wood consumption and mortality, however, untransformed data are shown in the figures. data were processed using parametric statistical analysis (anova) and tukey test to separate the media (p≤ 0.05). when error variances were not homogenous at the levene test for homogeneity of variance, non parametric kruskal-wallis analyses of variance was performed. l. maistrello et al.: wood impregnated with metal chelates tested for termite resistance 279 results r. lucifugus in test 1, considering wood consumption (fig. 1), significant differences were detected among treatments (h= 12.51, p= 0.006, kruskal-wallis test) and, in particular, between untreated wood (highest weight loss, about 1-2%) and the cu-salicylate impregnated wood (almost no consumption) (z= 2.47, p= 0.003, multiple comparisons after kruskal-wallis test). considering mortality, observations performed during the periodical water additions inside the containers allowed to detect a high number (no exact counts were performed to avoid disturbance) of dead termites in the groups with cu fig. 1 test 1, weight loss (%) consumption on differently treated wood slices, recorded at the end of the experiment due to the two species. r. lucifugus data (above) were processed using non parametric kruskal-wallis analyses of variance. k. flavicollis data (below) were processed using tukey test to separate the media (p≤ 0.05). columns indicated by different letters show significant differences. journal of entomological and acarological research, ser. ii, 43 (2), 2011280 fig. 2 test 2, weight loss (%) due to r. lucifugus (above) and k. flavicollis (below) consumption on differently treated wood slices, recorded at the end of the experiments. columns indicated by different letters show significant differences at the tukey test (p≤ 0.05). l. maistrello et al.: wood impregnated with metal chelates tested for termite resistance and zn-salicylate impregnated wood even after the first 5-10 days, whereas in the control termites appeared very active and performed galleries inside the sand. at the end of the experiment, which lasted 21 d, no significant differences were detected among treatments with the lowest mortality found in the control group (78.40 ± 19.60%) and the highest in the cu-chelate treated wood (100%). in test 2, considering wood consumption (fig. 2), highly significant differences were detected among treatments (f= 91.73, p< 0.0001): lowest consumption was detected for the wood treated with cu-chelate + water, whereas in presence of the solvent (with or without the metal chelates), wood consumption was always significantly high281 er than in the control group. regarding mortality, similar observations as those reported for test 1 were reported for copper glycinate in water and metal chelates treated wood. at the end of the experiment, after 21 d, no significant differences were detected among groups: all termites were dead in all groups except of those with the solvent treated wood (where mortality was 80.80 ± 11.83%). k. flavicollis in test 1, no mortality was observed during the trial period of sixty days. therefore, only wood consumption percentage was analyzed. the samples’ weight loss was significantly lower (f= 4.011, p= 0.015) in those impregnated with cu-chelates, especially compared to non treated ones, while those impregnated with zn-chelates and the oily solvent alone gave an intermediate response (fig. 1). in test 2, wood consumption was significantly lower (f= 6.297, p= 0.02) in the samples impregnated with cu-chelates in water and significantly higher in wood impregnated with ethylene glycol, while the other treatments gave an intermediate response (fig. 2). also for mortality significant difference were detected (f= 3.381, p= 0.029) and the situation was exactly the opposite (fig. 3). discussion and conclusions results of the present study show that wood impregnated with cu-chelates provides a better protection against both r. lucifugus and k. flavicollis than untreated fig. test 2, k. flavicollis average percentage of mortality in differently treated wood slices, recorded at the end of the experiment (90 d). columns indicated by different letters show significant differences at the tukey test (p≤ 0.05). journal of entomological and acarological research, ser. ii, 43 (2), 2011282 wood. results from the bioassays, despite being performed with different protocols in different laboratories, showed that wood consumption by both species of termites was significantly lower in presence of copper salicylate in oily solvent or copper glycinate in water. efficacy of these products is confirmed by the higher mortality observed in k. flavicollis on the same treatments. this is in agreement with bibliographic data. the efficacy of copper-based wood preservatives has been proved in formulations such as copper soaps (pizzi, 1993), with amine solvents such as acq (alkaline copper quat) and mcq (micronized copper quat) (cookson et al., 2010; tascioglu & tsunoda, 2010; lin et al., 2009), in association with tannins (yamaguchi et al., 2002) or with metaborates (furuno et al., 2006), as nanoparticles (kartal et al., 2009) and in combination with boron and hydroxylamine (köse et al., 2009). nevertheless, in our experiments, efficacy of the same cu-chelate in a different solvent (ethylene glycol) was not demonstrated. wood impregnated with copper chelate in ethylene glycol was not protected by the treatment. in addition, from this study it emerged that for both termite species ethylene glycol alone elicited a significantly higher wood consumption and resulted in lower mortality for k. flavicollis. therefore it seems that the use of ethylene glycol as solvent elicits a phago-stimulant effect which is able to contrast the negative effects of copper for both species. zinc action is not so clear. in test 1, when only oily solvent was used, wood treated with zn-chelate gave exactly the same results with both termites species. wood consumption was intermediate between control and cu-chelate, being slightly lower, although not significantly different than the control and higher than cu-chelate. in test 2, the results for zn-chelate in ethylene glycol are different for the two species. mortality and wood consumption in k. flavicollis were the same as those on wood treated with cu-chelate or water. in this case, the result could be due to the solvent, as hypothesized for cu-chelate. on the opposite, in r. lucifugus wood consumption in zn-chelate treatment was similar to cu-chelate treatment but significantly lower than in wood impregnated with ethylene glycol alone, showing that the attractive effect of the solvent is reduced by the presence of zn-chelate. the attractive action of ethylene glycol is recognized for some insect species, especially for fruit flies (diptera, tephritidae) (thomas et al., 2001; uchida et al., 2007), where it has been used in baited traps. use of this substance is known also for other insect species in pit-fall traps (koivula et al., 2003; greenslade & greenslade, 1971), acting as a preservative although it poses serious environmental hazards (ash & ash, 2004; beasley, 1985; barceloux et al., 1999; hall, 1991). further investigations are needed and will be carried out to clarify this behavior. references ash m., ash i., 2004 handbook of preservatives. synapse information resources endicott. new york. u.s.a. 850 pp. barceloux d. g., krenzalik e. p., olson k., watson w., 1999 guidelines on the treatment of ethylene glycol poisoning. clinical toxicology, 37: 537-560. l. maistrello et al.: wood impregnated with metal chelates tested for termite resistance 283 beasley v. r., 1985 diagnosis and management of ethylene glycol (antifreeze) poisoning. feline practice, 15: 41-46. cookson l.j., creffield j.w., mccarthy k.j., scown d.k., 2010 trials on the efficacy of micronized copper in australia. forest products journal, 60 (1): 6-12. furuno t., wada f., yusuf s., 2006 – biological resistance of wood treated with zinc and copper metaborates. holzforschung, 60 (1): 104-109. greenslade p., greenslade p. j., 1971 the use of baits and preservatives in pitfall traps. journal of the australian entomological society, 10: 253-260. hall d., 1991 the environmental hazard of ethylene glycol in insect pitfall traps. coleopterists bulletin, 45: 193-194. humar m., lesar b., 2007 fungicidal properties of individual components of copper ethanolamine-based wood preservatives. international biodeterioration & biodegradation, 62: 4650. kartal s.n., green f., clausen c.a., 2009 do the unique properties of nanometals affect leachability or efficacy against fungi and termites?. international biodeterioration & biodegradation, 63: 490-495. koivula m., kotze d. j., hiisivuori l., rita h., 2003 pitfall trap efficacy: do trap size, collecting fluid and vegetation structure matter. entomologica fennica, 14: 1-14. köse c., terzi e., kartal s.n., 2009 – evaluation of decay and termite resistance of wood treated with copper in combination with boron and n’-n-(1,8-naphthalyl) hydroxylamine (nha-na). international biodeterioration & biodegradation, 63: 727-731. leonardi g., 2011 italian patent application n° mi2011a001033. lin l.d., chen y.f., wang s.y., tsai m.j., 2009 leachability, metal corrosion, and termite resistance of wood treated with copper-based preservative. international biodeterioration & biodegradation, 63: 533-538. palanti s., predieri g., casoli a., vignali f., feci e., 2008 new preservatives based on copper chelates and copper complexes grafted to functionalized silica gel. proceedings of cost action e37 final conference, bordeaux (france), 29-30 september 2008: 23-29. pizzi a., 1993 a new approach to non-toxic, wide-spectrum, ground-contact wood preservatives. part ii. accelerated and long-term field tests. holzforschung, 47 (4): 343-348. tascioglu c., tsunoda k., 2010 laboratory evaluation of wood-based composites treated with alkaline copper quat against fungal and termite attacks. international biodeterioration & biodegradation, 64: 683-687. thomas d. b., holler t. c., heath r. r., salinas e. j., moses a. l., 2001 trap-lure combinations for surveillance of anastrepha fruit flies (diptera: tephritidae). florida entomologist, 84: 344-351. uchida g.k., mackey b.e., mcinnis d.o., vargas r.i., 2007 attraction of bactrocera dorsalis (diptera: tephritidae) and nontarget insects to methyl eugenol bucket traps with different preservative fluids on oahu island, hawaiian islands. j. econ. entomol., 100 (3): 723-729. yamaguchi h., yoshino k., kido a., 2002 termite resistance and wood-penetrability of chemically modified tannin and tannin-copper complexes as wood preservatives. j. wood sci., 48: 331-337. journal of entomological and acarological research, ser. ii, 43 (2), 2011284 l. maistrello et al.: wood impregnated with metal chelates tested for termite resistance lara maistrello, dip. scienze agrarie e degli alimenti, univ. di modena e reggio emilia, via g. amendola 2, i-42122 reggio emilia, italy. e-mail: lara.maistrello@unimore.it alessia berzolla, cpbc, università cattolica del sacro cuore, via emilia parmense 84, i-29122 piacenza, italy. e-mail: alessia.berzolla@unicatt.it irene macias-pavon, dip. scienze agrarie e degli alimenti, univ. di modena e reggio emilia, via g. amendola 2, i-42122 reggio emilia, italy. e-mail: irene_pasiempre@hotmail.com francesca vignali, dipartimento di chimica giaf, università di parma, viale g.p. usberti 17/a, i-43100 parma, italy. e-mail: francescavignali82@gmail.com giovanni predieri, dipartimento di chimica giaf, università di parma, viale g.p. usberti 17/a , i-43100 parma, italy. e-mail: giovanni.predieri@unipr.it elisabetta chiappini, cpbc, università cattolica del sacro cuore, via emilia parmense 84, i-29122 piacenza, italy. e-mail: elisabetta.chiappini@unicatt.it 285 jear2012 [journal of entomological and acarological research 2023; 55:10861] [page 1] abstract despite their potential as indicators of water quality and their key role in river ecosystems, chironomidae is still poorly studied in neotropical rivers. this lack of knowledge is especially relevant for rivers subjected to intense human activities, such as many rivers in mexico. the aim of this investigation is to contribute to the knowledge of the midges of the pesquería river (mexico) along its main courses and relate the composition and abundance to river health. thirty samples were collected during two different periods (august 2015 and february 2016) using a d-frame and kick sampling. thirty-five taxa were found in total, with four taxa found in more than 50% of the sites and 19 only found once. midges accounted for more than 50% of the total macroinvertebrate abundance. chironomus gr. plumosus, rheotanytarsus spp. and cricotopus gr. bicinctus were the most abundant species. collector-gatherers dominated in august (71% of individuals), whereas collector-filterers dominated in february (43,2%). the major factor explaining the midge distribution and abundance is pollution, while the structure of riparian area does not explain much of the midge richness. this is most likely related to the organic pollution coming from untreated or poorly treated sewage in the city of monterrey and its surroundings. three main sectors are distinguished along the river: i) the upper part section with higher biodiversity and presence of intolerant taxa; ii) the middle sewage polluted area with the presence of large red midges very tolerant to pollution (chironomus, dicrotendipes); iii) the lower section in the agricultural zone where the community is dominated by red, small midges (rheotanytarsus). overall, our study shows that chironomidae can be useful as better indicators of water quality when genera or species levels are used instead of family or subfamily, as is usually found in most papers on river pollution. introduction midges are an important component of river life. at some sites chironomidae larvae comprise an important percentage of the total density and biomass of aquatic macroinvertebrates and they can be an important component of the diet of fishes and other predators (armitage et al., 1995). they can even significantly contribute to the terrestrial organic matter budget of riparian areas by exporting flying adults (soininen et al., 2015). however, the taxonomic classification of midges is difficult and time-consuming (andersen et al., 2013). thereby, midges are usually identified only at the family level in ecological studies and water quality assessments and are considered to be pollution-resistant taxa and collector-gatherers as a trophic group (armitage et al., 1995). however, they comprise a great variety of species covering a wide range of pollution tolerance. thus, the taxonomic level at which midges are identified is very important (edward et al., 2000; molineri et al., 2020). the use of genera, species groups, or species (when possible) can help to reveal ecological patterns and journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2023; volume 55:10861 insect ecology chironomidae as indicators of water pollution in pesquería river (méxico) narcís prat, daniel castro-lópez f.e.m. research group (freshwater ecology management), departament de biología evolutiva, ecologia i ciències ambientals, sección ecología, universitat de barcelona, barcelona, spain correspondence: narcís prat, f.e.m. research group (freshwater ecology management), departament de biología evolutiva, ecologia i ciències ambientals. sección ecología. universitat de barcelona, diagonal 645, 08028, barcelona, spain. tel.: +34.934037135/607074784. e-mail: nprat@ub.edu key words: midges; pollution; biomonitoring; aquatic macroinvertebrates; wastewater; functional feeding groups. acknowledgments: the authors are grateful to the civil engineering institute and the international water centre of the universidad autónoma de nuevo léon for helping in field and laboratory work. thanks to miguel cañedo-argüelles for english improvement and comments to the text. contributions: np, conceived the paper, identified midges, wrote the paper; dcl, field work, sort and classification of macroinvertebrates until family and help in the writing. all the authors approved the final version to be published. conflict of interest: the authors declare no potential conflict of interest. funding: centro internacional del agua, facultad ingenieria civil, universidad autónoma de nuevo león. received: 25 october 2022. accepted: 22 march 2023. publisher’s note: all claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher. ©copyright: the author(s), 2023 licensee pagepress, italy journal of entomological and acarological research 2023; 55:10861 doi:10.4081/jear.2023.10861 this article is distributed under the terms of the creative commons attribution-noncommercial international license (cc by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly [page 2] [journal of entomological and acarological research 2023; 55:10861] processes (e.g., puntí et al., 2009; cañedo-argüelles et al., 2016; gonzález-trujillo et al., 2019). usually, environmental factors explain most of the differences in the chironomid assemblage composition (e.g., nicacio & juen, 2018), although dispersal limitations might be important depending on the network connectivity (cañedo-argüelles et al., 2015) and the temporal (cañedo-argüelles et al., 2020) and spatial scales considered (viana & chase, 2020). micro-scale study of substrate composition and temporal changes between seasons and years may explain the coexistence of many species in the same site, but species level is necessary to detect such differences (prat & garcia-roger, 2018). the chironomidae of mexico are still poorly known (bellogonzález et al., 2019); a taxa list is available by spies & reiss, 1996, mostly from old papers. as mexico shares part of its territory with neotropical and nearctic regions, the study of midges is more complex in this region. no specific keys exist for midges of mexico; larvae are classified using the keys of ferrington et al., (2008) and epler (2001) for nearctic fauna. for the neotropical region, the key of da silva et al., (2018) is useful. but there is still a long way to arrive at a good understanding of the taxonomy, biogeography, richness, and ecology of mexican chironomidae. mexican rivers are subjected to great pressure. despite that conabio (national commission for the understanding and usage of biodiversity) implemented the priority hydrological regions (rhp) program in 1998 (arriaga et al., 2009), their ecological degradation is increasing year after year. the program considers the pesquería river (nuevo léon, méxico) as a priority case for ecological evaluation. recent studies on this river showed that it is heavily impacted along most of its catchment, and a few sites are in good ecological status (castro-lópez et al., 2019a, b). despite recent efforts to consider biological communities (torres-olmera et al., 2018), water quality assessment currently relies exclusively on physicochemical parameters in mexico. however there are several macroinvertebrate-based biological indices in mexico (e.g., perez-munguia & pineda-lópez, 2005), but most of them use a limited taxonomical knowledge (family) (e.g., carmona-jiménez & caro-borrero, 2017), also in biomonitoring studies (castillo et al., 2018). only a few studies regarding paleolimnology (pérez et al., 2013) or focusing on epiphytic midges (caro-borrero, a. & carmona-jiménez, j. 2018) have explored the use of genera or species-level. the aim of this investigation was to contribute to the knowledge of midges in the pesquería river (mexico) along its main course and to assess their potential as indicators of river health. the specific objectives of the study were to: i) characterise the biodiversity of chironomidae in the pesquería river; ii) assess the relationship of chironomidae communities with river health; iii) discuss the potential of chironomidae to be used as bioindicators in mexican rivers and streams. materials and methods study area this study was conducted in the pesquería river located in northeastern mexico (san juan river basin, at the coordinates 26º38’24”-25º26’24” latitude n and 100º54’00”-98º56’24” longitude w). the main stem flows through monterrey city metropolitan area. the river has an average gradient of 0.4%, a length of 288.22 km and an area of 5255.56 km2, with an annual average flow of 2,04 m3/s (ferriño, 2016). the section studied consisted of the last 108 km of the main course and comprised altitudes ranging between 104 and 542 m. a.s.l. the climate in the area is semi-arid to arid. the wet season occurs during the months of may to october, while the dry season occurs during the remaining months (november to april) (castro-lópez et al., 2019) sampling we sampled chironomidae on two occasions (august 2015 and february 2016) at 15 sites. the location of stations along the river is in figure 1 from castro-lópez et al., (2019b). macroinvertebrate samples were taken using a d-frame net (250 um mesh), in a 100 m reach. 20 kick samples were taken proportionally to the habitats present and combined in a unique sample. we preserved samples in 10% formalin and transported them to the laboratory. initially, we identified macroinvertebrates at different taxonomic levels but midges only at the family level (castrolópez et al., 2019b). during the sorting, we pooled midges together in a tube and preserved them in 70% ethanol until the moment of their examination for taxonomical purposes. we studied a total number of 1670 and 1723 individuals for each season. the midges were classified using a two-step process. first, we classified midges into morphotypes under a stereoscope at x20. we used color, the presence of body setae, and others for distinguishing morphotypes following the criteria of prat et al. (2012) and rossaro et al., 2022a. with this approach, several genera o even species groups are easily recognized. when the morphotypes were not enough for identifying a genera or species group, we mounted 10 individuals of each morphotype to be identified using a microscope and following epler (2001). instead of euparal, we use dmxf as mounting media because no dehydration was necessary. we identified the larvae using the keys of epler (2001), andersen et al. (2013), ferrington et al. (2008), and prat et al. (2012). we used the material from the collection of the senior author (n. prat) for comparison. genera or species groups are the result of such work, which is enough for the purposes of this paper and relevant for ecological studies (molineri et al., 2020). we measured the status of riparian areas with the índex qbr adapted for the pesquería river (qbr-rnmx in castro-lópez et al., 2019a) from the original índex described in munné et al. (2003). data treatment the assignment of chironomidae to a trophic group was made according to the categories used in castro-lópez et al. (2019): predators, shredders, herbivores, collector-gatherers, and collectorfilterers. usually, midges were considered as collectors gatherers article figure 1. relationship between qbr and number of taxa of midges. no nco mm er cia l u se on ly in many studies. however, there are different feeding strategies within the chironomidae genera or species (serra et al., 2015, caleño-ruiz, 2015; rodriguez-lozano et al., 2015). we used our knowledge and personal observations on gut contents of midges from pesqueria, to assign to each genus a trophic category. results environmental data environmental data, the extent, and characteristics of the land cover are provided in previous papers (castro-lópez et al., 2019a, 2019b). we distinguish three areas along the river (table 1). the upper part area, before the urban metropolitan area of monterrey (sites 1-3). here, most of the basin cover is a mixed forest or shrubland that extends until the riparian area, while water is mesohaline according to the geology of the area. in the middle area, the urban sprawl of monterrey dominates, and the riparian area is mostly devoid of vegetation. in this section, six sewage plants and several unthreatened discharges discharge along the river, with a very large increase in values of conductivity and lower values of oxygen. finally, in the final part of the river, after monterrey, orchards dominated, both in the basin and in the riparian area. conductivity is lower, and oxygen recovers, but pollution from sewage plants remains. we will use this river delimitation, previously defined by castro-lópez et al., 2019a, to compare changes in the composition and abundance of midges. number of taxa 35 chironomidae taxa were found, 31 in august 2015 and 29 in february 2016 (supplementary material). the number of midge taxa per site ranged between 1 and 16 in august and between 2 and 14 in february. nineteen taxa in august and 17 in february were rare (recorded in less than 2 samples). only 7 taxa in august and 3 in february were present at more than 50% of the sites. seven of the taxa were found only in august, and four only in february. in august, the headwater sites (1-4) accounted for most of the species richness, whereas on february 6 sites captured most of the taxa (figure 2). composition and relative abundance chironomidae was present in all samples, and its total abundance was close to 50% of the macroinvertebrates found in both seasons (table 2). the most frequent and abundant taxa were the same on both dates (table 3): rheotanytarsus spp., chironomus gr. plumosus, cricotopus gr. bicintus and thienemannimyia sp. while ch. gr plumosus dominated in august (up to 50% of the individuals), rheotanytarsus (42%) was in february. [journal of entomological and acarological research 2023; 55:10861] [page 3] article figure 2. accumulated number of taxa of chironomidae in the two seasons studied in the pesquería river (mexico). table 1. riparian uses and values of conductivity in the three main sections of the pesqueria-river (data from castro-lópez et al., 2019a). % riparian use natural urban agricultural sites 1-3 73-100 0-27 0 sites 4-9 0 100 0 sites 10-16 0-39 0 41-100 conductivity 1758 (44) 5272 (1008) 2187 (116) table 2. most frequent and relatively abundant taxa of macroinvertebrates in pesquería river (méxico) (data from castro-lópez et al., 2019). august february macroinvertebrates sites (n=16) % sites (n=16) % chironomidae (cg) 16 48.38 15 54.53 hyalella (cg) 7 18.78 8 12.06 oligochaeta (cg) 14 14.71 16 15.96 notodromidae (cg) 10 6.13 15 3.87 smicridea (cf) 11 3.17 1 1.69 simulinii (cf) 6 0.27 11 4.06 nehalennia (p) 11 0.18 10 0.11 enallagama (p) 11 0.15 11 0.11 ceratopogonidae (p) 11 1.6 6 0.16 argia (p) 11 0.27 12 0.37 agraylea (h) 10 0.65 10 0.36 p, predator; h, herbivore; cg, collector gatherer; cf, collector-filterer. no nco mm er cia l u se on ly [page 4] [journal of entomological and acarological research 2023; 55:10861] in polluted parts (below sewage plants, sites 9 and 10) chironomus were present and abundant together with polypedilum gr. halterale (see annex 1). only in sites 1 to 3 (upstream) and 16 and 17 (downstream), the four most abundant taxa were not dominant in both sampling periods. most of the “rare species” are present in these 4 sites, while impacted sites usually have lower richness and higher abundances of common species with red color (with hemoglobin). however, large community differences exist between sites 1-3 (upstream) and 16-17. the last ones have dominance of some pollution-tolerant species (like dicrotendipes gr fumidus or rheotanytarsus), while these are scarce in less disturbed sites of the headwaters of the river. functional feeding groups according to the previous studies (castro-lópez et al., 2019a) and personal observations from gut contents, the most common midges in pesquería river are collector-gatherers and collector filterers (tables 3, 4). as we can see in table 3, the collector-gatherers dominate in the first sampling season and collector filterers in february. this is coincident with the dominance of the different subfamilies of chironomidae, as can be seen in table 5, where chironominae (chironomus and dicrotendipes) dominated in the first season and tanytarsinii (rheotanytarsus) in the second. the predators are members of the subfamily tanypodinae and represent close to 10% of the individuals usually. orthocladiinae are the more abundant subfamily within the herbivores. the importance of riparian area for midge composition and abundance in the upper part of the river, qbr-rnmx is high or very high (70-100). values may be very low (sites 4-5-6) or high (up to 70 in sites 7-8). in the most polluted sites, qbr is lower than 50 (sites 9-10), with some recovery afterward in the agricultural zone, where the index is closer to or higher than 70. more details about the qbr and its importance in the pesquería river are in castrolópez et al. (2019a). there is no direct relationship between the values of qbrrnmx and the number of taxa of midges (fig. 2). high values of both indexes upstream, while in the urban area taxa richness is low, but qbr may be variable because, although the river flows into article table 3. most common and abundant chironomidae of pesquería river in the two seasons studied. august february chironomidae sites (n=16) % sites (n=16) % larsia (p) 8 2.8 6 2.6 thienemanninyia (p) 9 4.6 10 5.3 thienemanniella (h) 6 1.9 5 1.6 cricotopus gr. bicinctus (h) 8 6.3 7 13 chironomus gr. plumosus (cg) 10 56 6 19 dicrotendipes gr. fumidus (cg) 9 5.7 5 7.4 polypedilum gr. halterale (cg) 9 8.9 3 0.8 rheotanytarsus (cf) 9 2.4 10 42 p, predator; h, herbivore; cg, collector gatherer; cf, collector-filterer. table 4. percentage of individuals of midges in each functional feeding group, in pesquería river (categories of each taxon in table 2). functional feeding groups august february predators 11.3 10 herbivores 12.2 15.8 collectors-gatherers 71.8 31 collectors-filterers 4.6 43.2 table 5. changes in the frequency of appearance and dominance in the subfamilies of chironomidae in the pesquería river in two different seasons. subfamilies august february of chironomidae sites % sites % tanypodinae 14 9.58 14 9.29 orthocladiinae 12 10.90 9 15.79 chironominae 15 79.52 15 74.93 chironominii 15 73.35 14 31.40 pseudochironominii 3 1.80 4 0.70 tanytarsinii 11 4.37 12 42.83 total 15 100 15 100 no nco mm er cia l u se on ly densely populated area, some stretches still have patches of remnant riparian forests, where qbr-rnmx may be high. in the agricultural zones (sites 13-15), the values of qbr recover (up to 85) but not the richness of midges. no relationships exist with the midge trophic group or the qbr-rnmx (data not shown). these results are in concordance with those of castro-lópez et al. (2019a), which also conclude that no relationships existed between the landscape, riparian characteristics, and freshwater macroinvertebrates. relationships with pollution we use the three-river sectors from table 1 to explore the relationships between pollution and midges. the upper less polluted part had lower values of conductivity (below 2000 us/cm), high oxygen content (>8mg/l), and no inputs from sewage plants or urban effluents and is the more biodiverse area. high-polluted sites are in the monterrey urban area, especially after the inputs of the 6 sewage plants, where oxygen can be extremely low, salinities higher, and plenty of organic matter. the river's lower part, surrounded by agricultural fields, has middle values of conductivity with variable oxygen contents but not direct sewage inputs. we have made some statistical treatment of the data (inval, for example), but no clear pattern emerged from this treatment due to the high variability between sites and the low number of taxa. any other statistical data treatment provides relevant information. discussion the limited information about midges and their importance for environmental studies there is a very limited information on midges in mexico. even less from the pesquería river where only unpublished degree thesis of the engineering faculty of the universidad nuevo león provide some information. thus bermejo (2003) reports some genera from two sites of pesquería river and torres (2013) studies the water quality and some biological indicators but identifies only chironomus gr. plumosus from all the midges collected and uses the family chironomidae in the biological indices he proposed. if we classify chironomidae (diptera) only at family level, their relative importance in taxa richness is not evident. in many cases, the larvae of midges are a major component in taxa number and abundance, especially when some nutrient enrichment or pollution is present. it is the case of pesquería river, where 1/3 of the taxa and close to 50% of the total individuals are chironomidae (castro-lópez et al., 2019b). the study of larvae of chironomidae is not easy, and it usually takes some time to identify genera or species groups, and the separation of species is not possible in many genera; this is the main reason why such studies are unusual. in addition, several species of the same genera may coexist in a site or even in the same stone (prat & garcia-roger, 2018). the coexistence of several species may be explained by differences in substrate composition or life-cycle characteristics as in prat & garciaroger, (loc. cit.), but this requires such a work that studies on this topic are the exception in the literature as are those related to functional feeding groups, and even more in the neotropical region (caleño-ruiz et al., 2018). for these reasons, it is usual that most of the studies in macroinvertebrates do not use midges at the genera level, and few ones use the subfamily level. the lack of keys to classify the midges made more difficult the study of midge larvae in mexico and latin america in general. even the most recent keys (da silva et al., 2018) are difficult for a non-specialist. our study reports the actual fauna of midges in the pesquería river and its relationship with major river basin changes. the results can be used as an indicator of recovery or degradation in further studies. few papers in latin america examined the importance of identifying midges at the genera level for a better interpretation of the biodiversity of streams and its relationships with environmental factors (gonzález-trujillo et al., 2019). the protection of headwaters may be the key to the recovery of downstream waters, due to the facility for the drift of insects, including the midges (gonzáleztrujillo, et al., loc. cit.). the use of midges in biomonitoring most of the literature on bioindicators uses midges at the family level (or at most at the subfamily). many researchers as edwards et al. (2000) and rossaro et al. (2022b), pointed out the need to use midges at the genera or species level for biomonitoring studies. the revision on the topic (chironomids as bioindicators) made by nicacio and juin (2015), does not seem to have contributed to an increase of taxonomic studies in midges related to biomonitoring in mexico or elsewhere in latin america (except one paper by molineri et al., 2020). many of the water quality metrics used for biomonitoring use family or even order as taxonomic level. this is for most of mexican rivers where people use as the main pollution index the bmwp system (e.g., pérez-munguia and lópez-pinedo, 2005), including the pesquería river (torres-muñiz, 2013; castro-lópez et al., 2019b). usually this may be enough to characterize the importance of pollution for macroinvertebrates, but doing this (and forgetting the chironomidae), some important information on richness, diversity, feeding strategies, or pollution tolerance of macroinvertebrate assemblages is missed. midges are not used at a low taxonomic level for biomonitoring to the difficulty and time-consuming task of classifying larvae at genera or species level (even more in mexico), unless molecular methods may be applied. for example, ekrem (2019) pointed out that the use of barcoding may improve the usefulness of midge species as bioindicators, but still much work is required to associate a large number of otus of midges found in streams to actual species (failla et al., 2015). for example, in spain, more than 50% of the taxa of midges present a lack of barcoding results (murria et al., 2020). the task appears even more difficult in latin america, where most of the chironomidae species remain undescribed. midges, pollution, and landscape the use of midges as indicators of water quality, including eutrophication (mostly in lakes), is common, mostly for their ability to cope with love oxygen water due to the presence of haemoglobin in their body (reviewed by nicacio & juen, 2015). in pesquería river, the red-blood midges are very abundant in both periods (66-71% of individuals). in august, the large chironomus plumosus (chironomini) are the dominant species. but this taxa is replaced in february by small rheotanytarsus;(tanytarsini), less suited for strong anoxia but still with haemoglobin. this indicated the predominance of organic matter in water and the pollution coming mainly from the non-effective sewage plants or raw sewage in the waters when the river encounters the metropolitan area. in the less polluted sites, several taxa from different subfamilies appear as representatives. our results also suggest that the use of subfamilies and tribes may be, sometimes, useful for biomonitoring purposes, as did molineri et al. (2020). we found that the riparian environment is not important, in pesquería river, for the composition of midges as was in caroborrero and carmona-jimenéz (2019), because no correlation [journal of entomological and acarological research 2023; 55:10861] [page 5] article no nco mm er cia l u se on ly [page 6] [journal of entomological and acarological research 2023; 55:10861] between the values of qbr and the species richness exists. as the river water pollution is so high, the contribution of riparian forests to increase the biodiversity (shade, leaf litter inputs, nutrient filtering…) is overpassed by the intensity of river pollution, which only diminishes at the very end of the river after several sites with moderate to the low influence of human population in the riparian quality (castro-lópez et al., 2019a). regarding the question of the relevance of landscape in midge composition, we found that only sites with non-modified uses have some characteristic midges, but no differences exist between agricultural and urban landscapes, probably because agricultural landscapes are downstream of the urban sites and part of the pollution coming from urban areas remains in the agricultural landscapes. the change is limited to dominant species, from large red chironomus to small red rheotanytarsus, a temporal change related to the season (august vs. february, dry vs. wet season). this paper is relevant because it characterizes the actual situation of the pesquería river and may be used as control when further implementation of water purification is done. references andersen t., cranston p., epler j.h., 2013 chironomidae of holartic region. keys and diagnoses. larvae. insects’ systematics and evolution. supplement 66, 573 pp. armitage p.d., cranston p.s., pinder l.c.v., 1995 (eds) the chironomidae: biology and ecology of non-biting midges. chapman and hall, london, 572 pp. arriaga-cabrera l., aguilar v., espinoza, j.m., 2009 regiones prioritarias y planeación para la 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[journal of entomological and acarological research 2023; 55:10861] [page 7] article online supplementary material: table 1. no nco mm er cia l u se on ly jear2012 journal of entomological and acarological research 2012; volume 44:e6 abstract we describe the larva of protanypus sp. a from the italian alps. all the larval characteristics fit the diagnosis of the genus, but it is impossible to assign the specimens examined to one of the known species. the low number of labral scales (12-14) and the serrated median lamellae of the medioventral appendix of the prementum exclude the identity of the species with p. morio or with the east palaearctic p. pseudomorio. the antennal ratio (2.3) excludes the identity with p. caudatus or p. forcipatus, which are the other two protanypus species known from the alpine region. in sæther’s key (1975) the larva fits with the nearctic p. ramosus, but identification of the species needs to be supported by pupal and adult material. in the southern alps, the genus is restricted to cold lakes at high altitude and is confirmed as an indicator of oligotrophic lakes. introduction larval material belonging to the genus protanypus was recently collected in the southern alps. the genus is considered to be an indicator of oligotrophic conditions in lakes (sæther, 1979). there are few records of the genus on the southern side of the alps and the material collected was never described. the genus is holarctic in distribution with three species known to occur in europe, p. caudatus edwards, 1924, p. forcipatus (egger, 1864) and p. morio (zetterstedt, 1838), two species from east palearctic, p. pseudomorio makarchenko, 1982, also captured in alaska (sæther and willassen, 1985), and p. tshereshnevi makarchenko, 1982, three species from north america, p. ramosus sæther, 1975, p. hamiltoni sæther, 1975 and p. sætheri wiederholm, 1975. two other species were described as larvae (sæther, 1975; ashe and o’connor, 2009). the genus is restricted to cold oligotrophic lakes and is considered an indicator of oligotrophy (sæther, 1979). the larva was described by sæther (1975), doughman (1983), wiederholm (1983) and makarchenko (2006). materials and methods larvae were fixed in 70% ethanol, body parts were dehydrated in acetic acid, butanol, phenol 3: xylene 1 (wirth and marston, 1968) and slide-mounted in canada balsam. the larval head capsule was dissected as described by wiederholm (1983). terminology and abbreviations used in the description follow sæther (1976, 1980). measurements are according to sæther (1976) and are given in μm, unless otherwise specified. results diagnosis the larvae do not exceed 10 mm in length. the postoccipital margin has only a small ventrolateral, posteriorly directed projection on each side (figure 1 poc). antennal ratio is over 2.0, there are 12-14 labral scales (figure 1 lab), the median tooth of mentum is very large (figure 1 mnt, prh), and the median lamellae of medio-ventral appendix of prementum serrated (figure 1 prh). procerci are approximately 100 μm long. description the description is based on 2 larvae at the 4th stage and 3 larvae at the 3rd stage, collected in lake palù (lombardy, italy). these are medium-sized, large larva in the 4th stage, approximately 10 mm long. the head capsule is covered with numerous small setae and there is a small ventrolateral projection on its occipital margin on each side. the antenna (figure 1 ant, figure 2 ant, figure 3, figure correspondence: bruno rossaro, dipartimento di protezione dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (di.p.s.a.), università degli studi di milano, italy. e-mail: bruno.rossaro @unimi.it key words: taxonomy, chironomidae, ecology. acknowledgements: the authors are grateful to arpa lombardia and cnr-ise for collecting the material, to the università degli studi, milan (defnd) and to the cnr-ise allowing us to examine the samples in their collections. our thanks also go to the reviewers for their invaluable suggestions. received for publication: 9 march 2012 revision received: 13 april 2012 accepted for publication: 13 april 2012 ©copyright b. rossaro et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e6 doi:10.4081/jear.2012.e6 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. description of the larva of protanypus sp. a (diptera, chironomidae) from the italian alps b. rossaro,1 a. boggero,2 f. buzzi,3 c. agostinelli,3 f. nastasi3 1dipartimento di protezione dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (di.p.s.a.), università degli studi di milano; 2cnr-ise istituto per lo studio degli ecosistemi, verbania pallanza; 3arpa lombardia, lecco, italy [page 28] [journal of entomological and acarological research 2012; 44:e6] no nco mm er cia l u se on ly 4) has 4 segments. antennal segments are 78.2, 21.7, 2.7, 6.5 μm long. antennal ratio (ar) is 2.26: 1st segment is very long, 2nd is shorter, 3rd segment is very short, 4th segment is much longer than the 3rd. the antennal blade is 26 μm long, accessory blade 23 μm, style 5.6 μm, lauterborn organ 8.9 μm. the ring organ near the base of antenna is only 13 μm from the base and approximately 62 μm from the distal margin of the first antennal segment. all s setae of the labrum (figure 1 lab, figure 5, figure 6) are short and simple. there are approximately 12-14 labral scales. these are broad, leaf-like, circular, apically serrated, overlapping, and disposed in a semicircular row. there are no lateral chaetae, pecten epipharyngis consists of 3 large scales, pointed at the apex, continuous with chaetulae laterals. there is a narrow premandible with a well-developed, but very slender inner tooth. the other 2 teeth are very small and difficult to see. the mandible (figure 1 mdb, figure 2 mdb) has 5 inner teeth and a very long slender apical tooth. the seta subdentalis is pointed and narrow. there are no seta interna. the mentum (figure 1 mnt, prh, figure 2 mnt, mapp, ph) has a very large transparent median tooth. the width of the whole mentum is 142 μm. two to three lateral teeth are restricted to the extreme lateral region (figure 2 ph). ventromental plates are present but not developed (figure 2 ph). the supporting endoskeleton is 190 μm wide. the medio-ventral appendix m (figure 2 mapp) is 12.6 μm at its narrowest point, 38.7 μm at its base, with median lamellae serrated at its apex. the ratio of supporting endoskeleton to median tooth of mentum is approximately 1.4. the ratio of the medio-ventral appendix of prementum is 3.1 at its base to its minimum width. the pecten hypopharyngis has well-developed scales (figure 1 prh, figure 2 ph). maxilla (figure 1 mxl, figure 2 mxl): palp is longer than it is wide, and there are numerous setae maxillaris. the anal tubules of the body are triangular (figure 1 atb) with long narrow claws on the anterior and posterior parapods. there are few claws with small denticles (figure 1 clw) on the posterior parapods. the procerci are approximately 100 μm long with 5 setae at the apex. the larvae described here were collected in lake palù, sondrio, lombardy, italy (lat. 46°17’59” 5127957.30 wgs/84 utm 32n; long. 9°52’06” 567045.75 wgs/84 utm 32n) on 2 august 2011 (depth 6m, water temperature 13°c, oxygen saturation 90%) and on 2 september 2011 at the same station. previous records of the genus from the southern side of the alps are from the lakes laiozza (19/9/1991) and zota (19/9/1991) in the lavizzara valley, river basin of maggia, one of the tributaries of the river ticino (switzerland), and from the lakes paione medio and superiore (13/9/2000) in the ossola valley, river basin of the bognanco (tributary of the river toce), verbania, piedmont, italy. the genus is also known from the northern side of the alps from lake constance (reiss, 1968), and from southern germany and the austrian lakes (faaker see, 11/6/2008 and vorderlang see, 25/6/2008) (free et al., 2009). discussion the species will be keyed to the nearctic p. ramosus using the key of sæther (1975). the previous reports of the genus protanypus in italy can be confirmed in the generic diagnosis, but the citation of p. caudatus (boorman et al., 1995) cannot be confirmed on the basis of larval material; the ar is over 2.0, and this value is only reported for article [journal of entomological and acarological research 2012; 44:e6] [page 29] figure 1. the larva of protanypus sp. a. ant, antenna; lab, labrum; poc, prolongation of occipital margin of head capsule; mnt, mentum; prh, prementum and hypopharinx; mdb, mandible; mxl, maxilla; atb, anal tubules; clw, claws of posterior prolegs. blue bar: 100 µm. figure 2. the larva of protanypus sp. a. ant, antenna; lab, labrum; mdb, mandible; mxl, maxilla; mnt, mentum; mapp, median appendage of prementum; ph, margin of mentum and pecten hypopharyngis. no nco mm er cia l u se on ly article [page 30] [journal of entomological and acarological research 2012; 44:e6] figure 3. antenna: the four antennal segments with antennal blade, accessory blade and ring organ. figure 4. antenna: abl, antennal blade; acb, accessory blade; ro, ring organ; 2nd, 3rd, 4th, second, third and fourth antennal segments. figure 5. labrum: labral scales, epipharynx, premandibles. figure 6. labrum: lb, labral scales; prm, premandibles; eph, pecten epipharyngis. no nco mm er cia l u se on ly article nearctic material. the genus is well described and documented in the adult and in the pupal stages (brundin, 1952; wiederholm, 1975; makarchenko, 1982; sæther and willassen, 1985). a short description of the genus in the larval stage has been given by wiederholm (1983). different species of the genus have been described by zavrel (1926), hirvenoja (1973), sæther (1975), doughman (1983) and makarchenko (2006). unfortunately, makarchenko’s description of p. caudatus (2006) does not agree with the description of sæther (1975) because the first author described the species as having 16-20 labral scales, while sæther reports that the species has no more than 12 labral scales. also other characteristics, such as the premandible, do not help identify the species; protanypus sp. a has a premandible with 3 teeth, like the p. caudatus (makarchenko, 2006). p. pseudomorio has a premandible with 5 teeth. the ratio between the medioventral appendix of the prementum, the minimum width at its base and between the supporting endoskeleton of the appendix, and the median tooth width of the mentum were used as characteristics to distinguish between species (sæther, 1975). however, these criteria are difficult to apply, and it must be emphasized that many morphometric characteristics change with larval size. conclusions the larvae of protanypus can easily be identified to genus but the species determination within the genus according to morphometric measurements is still highly problematic since these depend on larval size. in this case, unless pupae and adults are examined the species should not be named. attempts are now being made to identify larvae using molecular markers (willassen, 2011). preliminary results require confirmation because of the modest levels of phylogenetic signal obtained with low credibility of some nodes. from an ecological point of view, all samples of the genus captured come from high altitude oligotrophic lakes, over 1900 m. the larvae are free living and predatory. in the alps, they have been reported to be limited to the profundal zone, but our samples were taken at a depth of 6 m. references ashe p., o'connor j.p., 2009 a world catalogue of chironomidae (diptera) part 1. buchonomyiinae, chilenomyiinae, ponono minae, aphroteniinae, tanypodinae, usambaromyiinae, diame sinae, prodiamesinae and telmatogetoninae. the irish bio geographical society in association with the national museum of ireland: 445 pp. boorman j., coluzzi m., contini c., ferrarese u., rivosecchi l., rossaro b., et al., 1995 diptera culicomorpha (in minelli a., ruffo s., la posta s., (eds.), checklist delle specie della fauna italiana. calderini, bologna, italy: 65:1-32. brundin l., 1952 zur kenntnis der taxonomie and metamorphose der chironomidengattungen protanypus kieff., prodiamesa kieff. und monodiamesa kieff. rep. inst. freshwater res. drottnin gholm 33: 39-53. doughman j.s., 1983 a guide to the larvae of the nearctic diame sinae (diptera, chironomidae). the genera boreoheptagyia, protanypus, diamesa and pseudokiefferiella. u.s. ecological survey. water research investigation rep. 83-4006: 1-58. free g., solimini a., rossaro b., marziali l., giacchini r., paracchini b., et al., 2009 modelling lake macroinvertebrate species in the shallow sublittoral: relative roles of habitat, lake morphology, aquatic chemistry and sediment composition. hydrobiologia 633: 123-136. hirvenoja m., 1973 revision der gattung cricotopus van der wulp and ihrer verwandten (diptera, chironomidae). ann. zool. fenn. 10: 1-363. makarchenko e.a., 1982 khironomidy roda protanypus kieffer (diptera, chironomidae) dal'nego vostoka sssr, (in biologiya presnovodnykh zhivotiykh dal'nego vostoka.) akad. nauk sssr: 125-144. makarchenko, e.a,. 2006 34. chironomidae. in: key to the insects of russian far east. vol. vi. diptera and siphonaptera pt. 4: 204734. reiss, f., 1968 – ökologische und systematische untersuchungen an chironomiden (diptera) des bodensees. arch. hydrobiol. 64: 176-323. sæther o. a., 1975 two new species of protanypus kieffer, with keys to nearctic and palaearctic species of the genus (diptera: chironomidae). j. fish. res. bd can. 32: 367-388. sæther o.a., 1976 revision of hydrobaenus, trissocladius, zalutschia, paratrissocladius, and some related genera (diptera chironomidae). bull. fish. res. bd. canada. 195: 1-287. sæther o.a., 1979 chironomid communities as water quality indicators. holarctic ecol. 2: 65-74. sæther o. a., 1980 glossary of chironomid morphology terminology (diptera: chironomidae). entomol. scand. suppl. 14: 1-51. sæther o. a. and willassen e., 1985 the first record of protanypus pseudomorio makarchenko (diptera chironomidae) from the nearctic, with a description of the female and a revised key to males of the genus. aquat. insects 7: 141-148. wiederholm t., 1975 description of protanypus sætheri n.sp. from alaska (diptera: chironomidae). entomol. scand. 6: 224-228. wiederholm t., 1983 chironomidae of the holarctic region. part i larvae. entomol. scand. suppl. 19: 1-457. willassen e., 2011 phylogeny of diamesinae inferred from mtdna sequences. abstract volume. eighteenth international symposium on chironomidae, throndheim, norway, 4-6 july 2011: 49. wirth w.w. and marston n., 1968 a method for mounting small insects on microscope slides in canada balsam. ann. entomol. soc. am. 61: 783-784. zavrel j., 1926 chironomiden aus wigry-see. arch. hydrobiol. ichtyol. 1: 197-220. [journal of entomological and acarological research 2012; 44:e6] [page 31] no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract this study aimed to evaluate the entomopathogenic activity of fusarium avenaceum (strain 10a) against adults of sitophilus oryzae infesting wheat grain. bioassays were carried out to determine the adult mortality of s. oryzae when the conidial suspension of the fungus strain was applied using three types of fungus treatment. results obtained have indicated significant differences (p=0.05) in the mean percentage of adult mortality due to the treatment with the fungus compared to the control. the highest mean percentage of adult mortality was obtained by the direct spraying of s. oryzae adults with the fungus conidial suspension before introduction of the treated adults into pots containing wheat grain; the lowest mean percentage of adult mortality was obtained by spraying the inner surfaces of pots with the fungus conidial suspension before introducing the grain and insects. this study demonstrated the typical growth of f. avenaceum on the outer surfaces of the dead treated adults of s. oryzae. presence of the external fungus growth on the dead insects indicated that the death of s. oryzae adults was attributed to the fungus infection. results obtained in the present paper represent the first record of efficacy of f. avenaceum against a coleopteran stored-grain insect, mainly including s. oryzae. introduction rice weevil (sitophilus oryzae) is one of the most serious threats to stored grain. adults and larvae of this insect attack healthy grain, especially cereals, causing extensive damage, especially to those cereals stored under high temperatures and medium relative humidity (rh) (>25°c and 80%) (padin et al., 2002). during feeding, these insects consume all of the internal contents of the attacked grain leaving them as empty shells (dal bello et al., 2000). many types of control measures can be undertaken against s. oryzae. chemical and biological control measures are the most common (dal bello et al., 2000; lee et al., 2001; fang et al., 2002; pungitore et al., 2005; batta, 2008; govindan & nelson, 2009; yankanchi & gadache, 2010). biocontrol of s. oryzae using entomopathogenic fungi is still uncommon and very few species of these fungi (e.g., metarhizium anisopliae and beauveria bassiana) have been used to control s. oryzae (padin et al., 2002; batta, 2004). fusarium species are generally considered soil borne fungi because of their abundance in the soil and their frequent association with plant roots as parasites or saprophytes. these species have usually been reported as plant pathogens causing serious diseases on many plant species (e.g., vascular wilts on a wide range of horticultural crops, crown rot and root rot diseases on many crops, head blight on cereal grain and other plant species) (booth, 1971). however, some of these species may act as weak to virulent entomopathogens and may live as saprophyte on dead insects (teetor-barsch & roberts, 1983). as entomopathogens, some fusarium species have been reported to cause moderate to high levels of infection, principally against homopterous and dipterous insects. however, low to moderate levels of infections with these entomopathogens have been reported from insects of other orders (e.g., coleoptera and lepidoptera) (teetor-barsch & roberts, 1983). many species of entomopathogenic fusarium may kill their host insects through the activity of toxins produced by penetrating hyphae (gupta et al., 1991). fusarium avenaceum was one of the entomopathogenic species of fusarium that has been isolated from a number of insect species, and its pathogenicity to insects has been demonstrated in few species, such as greenhouse whitefly, trialeurodes vaporariorum (eleonorarojas et al., 2003), spruce budworm, choristoneura fumiferana, (strunz & strongman, 1988) and wheat stem sawfly, cephus cinctus, (sun, 2008). to the best of our knowledge, no reports are yet available on the entomopathogenic activity of f. avenaceum against s. oryzae or any other coleopteran insects of stored grain. therefore, the objectives of the present research were: i) to assess the efficacy of f. avenaceum (strain 10a) against s. oryzae adults by applying the fungus conidial suspension in different ways, then comparing the treatment effect by journal of entomological and acarological research 2012; volume 44:e11 correspondence: yacoub a. batta, department of plant production and protection, faculty of agriculture, an-najah national university, nablus, west bank, palestinian authority. e-mail: yabatta@najah.edu yabatta@windowslive.com key words: fusarium avenaceum, sitophilus oryzae, entomopathgenic effect, bioassays, direct application. received for publication: 26 april 2012. revision received: 8 september 2012. accepted for publication: 12 september 2012. ©copyright y.a. batta, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e11 doi:10.4081/jear.2012.e11 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. the first report on entomopathogenic effect of fusarium avenaceum (fries) saccardo (hypocreales, ascomycota) against rice weevil (sitophilus oryzae l.: curculionidae, coleoptera) y.a. batta department of plant production and protection, faculty of agriculture, an-najah national university, nablus, west bank, palestinian authority [journal of entomological and acarological research 2012; 44:e11] [page 51] no nco mm er cia l u se on ly calculating the adult mortality percentage in the different types of treatments; and ii) to demonstrate that infection with f. avenaceum (strain 10a) can kill the adults of s. oryzae by first colonizing the internal organs of treated insects and then by the growth of the internal hyphae of the fungus on the outer surfaces of dead insects. materials and methods insect rearing a local strain of sitophilus oryzae (so25) was used in the present bioassays. adults of this strain were reared on wheat grain (variety: anbar) stored in plastic pots (90 mm diameter and 140 mm deep). during rearing, each pot was covered with a piece of cheesecloth fastened with a rubber band on the top. the rearing pots containing the grain and insects were kept in an incubator with fixed conditions (20±1°c, 80±5% rh, 14/10 h light/dark (l/d) photoperiod). to exclude any possibility of insect mortality during bioassays due to aging, young adults of f1 generation were collected from rearing pots and then used for conducting bioassays. fungus strain strain (10a) of fusarium avenaceum was used in the present study. it was originally isolated by our laboratory from infected nymphs of citrus whitefly (dialeurodes citri ashmead, aleyrodidae, homoptera) on sabouraud dextrose agar (sda) culture medium amended with chloramphenicol (250 mg/l, w/v) then sub-cultured on sda medium for ten days before being stored at 4°c as a pure culture of the fungus. to conduct bioassays, the fungus obtained from the pure culture was sub-cultured on sda medium. morphological and molecular characteristics of the fungus species, such as fusarium avenaceum, were confirmed both by our laboratory and by the molecular biology laboratory at an-najah national university. these characteristics conform to those described by other investigators for the genus and species of the fungus (nelson et al., 1994; satyaprasad et al., 2000; waalwijk et al., 2004; leslie & summerell, 2006). a pure culture of the fungus strain grown for 15 days on the same medium was used to conduct bioassays. the fungus culture was characterized by an abundant cottony mycelial growth that varies from white to light yellow in color with pale orange sporodochia containing macroconidia, the typical conidia of the fungus species. these macroconidia are long and slender, straight to slightly curved in shape. the mature conidia are multicellular with 6 cells and usually have 5 internal septa (figure 1). the basal cell of these conidia may have a slight notch but the apical cell is long and tapering to a point; it may be bent (arrows in figure 1). neither microconidia (fusoid in shape with 1 or 2 septa) nor chlamydospores (single or clustered or chain) were observed in the fungus culture. therefore, the conidial suspension of the fungus that has been prepared from this culture and used for bioassays has contained only the typical macroconidia of the fungus strain. inoculation method a conidial suspension of f. avenaceum was prepared from a 15-day old pure culture of the fungus strain on sda medium. the concentration of conidia in the suspension was 4.0¥104 conidia/ml and the total volume of conidial suspension that has been used for inoculation was 2.0 ml per replicate. the technique of inoculation used during bioassays consisted of spraying the conidial suspension of the fungus onto: i) adult insects of s. oryzae before their introduction into grains placed in plastic pots; or ii) inner surfaces of plastic pots (treated before introduction of insects and wheat grains); or iii) grains already placed in plastic pots (treated before introduction of insects). the details of the technique will be described below. bioassays plastic pots (90 mm diameter, 140 mm deep) were used to carry out bioassays. each pot was covered with a piece of cheesecloth fastened with a rubber band after introduction of 10 g of wheat grain (variety: anbar) and 20 insects of s. oryzae adults (young f1 adults). four types of treatments were used during bioassays: i) treatment 1 represents spraying of the inner surfaces of each plastic pot with the conidial suspension before introduction of wheat grain and adult insects; ii) treatment 2 represents direct spraying of the adult insects with the conidial suspension before their introduction into each pot with wheat grain already introduced; iii) treatment 3 represents direct spraying of the grain with the conidial suspension after their introduction into each pot. the insects were introduced into grain after the treatment; and iv) the control which represents pots containing non-treated insects and non-treated grain with conidial suspension. spraying of the conidial suspension during bioassays was performed using a small-calibrated hand sprayer (1 l capacity). the sprayer was standardized to apply fine jetting sprays with 0.5 ml spray volume for each jetting spray. a total volume of 2.0 ml (4 jetting sprays) of the conidial suspension was applied per replicate and four replicates were used per treatment. the treatments were distributed in a completely randomized design and the experiment was repeated 3 times under the same conditions to confirm the results. an incubator with fixed conditions (20±1°c, 80±5% rh and 14/10 h, l/d photoperiod) was used during bioassays. results were the average of the 3 repeated experiments conducted under the same conditions. to ensure that the infection with the fungus was the cause of death of treated adult insects, the dead insects were incubated under humid conditions at 20±1°c for three days using tightly closed petri dishes with moistened filter papers. before introduction into dishes, the dead insects were surface sterilized using 2% sodium hypochlorite solution for 3 min then rinsed three times with sterile distilled water then placed on clean glass slides to avoid the dead insects getting too wet if placed directly in the bottom of dishes. assessment after ten days of applying the conidial suspension of the fungus in each type of the treatments, the dead adults of s. oryzae in each replicate were counted. the mean percentage of adult mortality was then calculated in each treatment type. this calculated mortality is called figure 1. typical macroconidia of fusarium avenaceum (strain 10a) formed in sporodochia observed in the fungus culture on sabouraud dextrose agar medium or borne by the mycelium of the fungus strain appeared on the external surfaces of dead sitophilus oryzaeadults due to infection with the fungus as shown in figure 2. each mature macroconidium consists of 6 cells (multicellular with 5 internal septa) and its apical cell is long and tapering to a point; it may be bent (arrows). average dimensions are 57.7 mm long and 4.8 mm wide (n=100 macroconidia; scale bar 20 mm; magnification: 950¥). article [page 52] [journal of entomological and acarological research 2012; 44:e11] no nco mm er cia l u se on ly observed mortality. corrected mortality using abbott’s formula for control mortality was calculated from observed mortality then used to assess the entomopathogenic effect of the fungus. the mycosis that appeared on the outer surfaces of the dead insects after incubation under humid conditions was microscopically examined to verify whether the fungus growth and the conidia produced on the outer surfaces conformed to those that characterize our fungus strain. statistical analysis the data obtained from bioassays on the mean percentage of corrected s. oryzae adult mortality were angularly transformed and then analyzed statistically using analysis of variance (anova). abbott’s formula for calculating the corrected mortality from observed mortality was used [abbott’s formula=(mortality observed-mortality control/100-mortality control)¥100]. the significant differences between the treatment means at p=0.05 were determined by f-test and the treatment means were separated using tukey’s hsd test. sas software version 8 (2000) was used for statistical analysis (institute inc., cary, nc, usa). results comparison of the treatment effect with f. avenaceum against s. oryzae adults has indicated that significant differences (p=0.05) were obtained in the mean percentage of corrected adult mortality in the different types of treatments used in bioassays (table 1). therefore, the highest mean percentage of corrected adult mortality (94.86%) was obtained with treatment 2 by direct spraying of insects with conidial suspension before their introduction into pots containing wheat grain, followed by the mean percentage of corrected adult mortality (68.88%) obtained in treatment 3 by direct spraying of the grain already introduced into pots before introduction of insects, and then by the mean percentage of corrected adult mortality (52.22%) obtained in treatment 1 by spraying inner surfaces of pots before introduction of the grain and insects. however, the lowest mean percentage of adult mortality (5%) was obtained in the control (table 1). therefore, the most effective treatment was the direct spraying of the adult insects with the conidial suspension of the fungus; this gave the highest mortality percentage in the treated adults. incubation of dead insects of s. oryzae obtained from bioassays, under humid conditions at 20±1°c for three days resulted in the appearance of white to light-yellow cottony mycelium growth, typical of f. avenaceum on the outer surfaces (figure 2). microscopic examination of this mycelium has indicated the presence of aerial multicellular conidia typical of f. avenaceum macrocodidia. these macroconidia were long and slender, straight to slightly curved in shape (figure 1). at maturity, they consist of 6 cells with 5 internal septa and the apical cell of these conidia is long and tapering to a point; it may be bent (arrows in figure 1). these conidia (n=100 conidia) were on average 57.7 mm long (48-63 mm) and 4.8 mm wide (4-6 mm). table 1. percentage of mortality (%) of sitophilus oryzae adults ten days after treatment with conidial suspension of fusareum avenaceum (strain 10a). incubation was carried out at 20±1°c, 80±5% rh and 14/10 h, l/d photoperiod. four replicates per treatment were used and 20 adults per replicate were introduced into each plastic pot with wheat grain. the experiment was repeated 3 times and average data were presented and then corrected according to abbott’s formula for corrected mortality. treatment type mortality % of s. oryzae adults rep i rep ii rep iii rep iv mean treatment 1 65 60 55 40 55.00 (65) (55.55) (55) (33.33) (52.22)a treatment 2 100 100 85 95 95.00 (100) (100) (85) (94.44) (94.86)c treatment 3 40 90 80 70 70.00 (40) (88.88) (80) (66.66) (68.88)b control 0 10 0 10 5.00 percentage of mortality between two bracts represent the corrected mortality according to abbott’s formula for control mortality. other values mortality (%) in the table represent the observed mortality. abbott’s formula for corrected mortality=(m observed–m control/100–m control)¥100. treatment means followed by the same letter are not significantly different at p=0.05 using anova and tukey’s hsd test. treatment 1 represents spraying inner surfaces of pots with the conidial suspension before introduction of the grain and the insects; treatment 2 represents direct spraying of the insects with the conidial suspension before introduction into pots with the grain; treatment 3 represents direct spraying of the grain with the conidial suspension introduced into pots before introduction of the insects; control represents non-treated pots or insects or grain. figure 2. typical mycosis observed on the external surfaces of dead sitophilus oryzae adults due to infection with fusarium avenaceum (strain 10a). a white cottony mycelium growth characteristic of f. avenaceum appeared on the external surfaces of dead adults after three days of incubation under humid conditions at 20±1°c. typical macroconidia formed by the fungus on the dead insects were microscopically observed and examined. article [journal of entomological and acarological research 2012; 44:e11] [page 53] no nco mm er cia l u se on ly article discussion it is well known that fusarium avenaceum is a soil borne fungus. it may act as a saprophyte in the soil or plant pathogen of roots and stems of plants. as a plant pathogen, it can cause serious diseases to the plants it attacks, such as seed borne diseases of legumes (kelloc et al., 1978), stem and root rot diseases in various pasture legumes (mcgee & kellock, 1974; lamprecht et al., 1988; satyaprasad et al., 2000), head blight of wheat (kang et al., 2005). however, the entomopathogenic effect of this fungus has been recently reported by a few investigators on certain insect species, such as the greenhouse whitefly, trialeurodes vaporariorum, spruce budworm, choristoneura fumiferana, and wheat stem sawfly, cephus cinctus, (strunz & strongman, 1988; eleonora-rojas et al., 2003; sun, 2008). our strain of f. avenaceum has demonstrated a high level of entomopathogenic effect against s. oryzae adults, and the direct spraying of the adult weevils with the fungus conidial suspension gave the highest mortality percentage compared to the other types of treatment or the control. it is worthy of note that the emtomopathogenic activity of the fungus against s. oryzae adults has been demonstrated and reported here for the first time. in addition, there have been no reports of the entomopathogenic effect of f. avenaceum against any other coleopteran insects of stored grain. the inoculation method used during bioassays through the direct spraying of the adult insects with the conidial suspension has been shown to be the most effective due to the direct contact of applied conidia with the treated insect’s cuticle, so that the direct germination of applied conidia and the subsequent penetration of the germ tube into the insect’s cuticle took place within a short time. this mode of action may explain the occurrence of the highest mortality percentage in the treated s. oryzae adults. it is known that the most common entomopathogenic fungi, such as beauveria bassiana and metarhizium anisopliae, can cause the highest mortalities in their host insects when applied directly to their host insects as biocontrol agents (hidalgo et al., 1998; moino et al., 1998; rice & cogburn, 1999; odour et al., 2000; toshio, 2000; sheeba et al., 2001). however, there are some limitations to this method of large-scale fungus inoculation to control s. oryzae infesting stored grain in large containers. for example, the relative increase in humidity in the vicinity of storage containers causes an increase in mold development that is not favorable for storage of stocked cereals. to reduce this effect, a certain number of fungus applications (e.g., 2 or 3 applications during the storage period) should be applied. the frequent application of entomopathogenic fungi as biocontrol agents of storage insects is not favorable for the stocked cereals due to the relative increase in the atmospheric humidity after application. however, the low number of applications for these biocontrol agents could be compensated by the horizontal transmission of these agents already introduced into storage containers during the earlier applications (furlong & pell, 2001; quesada-moraga et al., 2004). the dead insects infected with the fungus with sporulation on the outer surfaces constitute the inoculum that can be horizontally transmitted to new insect infestation occurring during the storage period. this low number of fungus applications could be integrated into programs of s. oryzae management. morphological characteristics of the fungus strain described in the present research, especially those relevant to macroconidia and mycelium growth obtained in the fungus culture on sda medium, conform to those described by other researchers. for example, leslie and summerell (2006) used carnation leaf pieces agar to cultivate the fungus but obtained similar effects. however, the culture of our fungus strain on sda medium did not produce either microconidia or chlamydopspores so that the conidial suspension prepared from this culture and used for conducting bioassays has only contained the typical macroconidia of the fungus. the lack of microconidia and chlamydospores was explained by nelson et al. (1994), leslie and summerell (2006), and latiffa et al. (2010) who stated that the isolates of f. avenaceum did not usually produce chlamydospores, whereas some of these isolates may produce microconidia. furthermore, they stated that if the microconidia are produced, they will be rare or only few in number, and have a fusoid shape with 1 or 2 septa. the shape of macroconidia is the principal characteristic used to distinguish f. avenaceum species from other fusarium species (leslie & summerell, 2006). accordingly, this characteristic is consistent and it can be applied if the cultures are carefully prepared and macroconidia are obtained from sporodochia; in this way, the shape of macroconidia can be used to identify the fungus species. recently, a more accurate molecular identification of f. avenaceum isolates from other isolates of fusarium species could be made, and more precisely, when primers and protocols for detecting f. avenaceum through real-time polymerase chain reaction assays are available (waalwijk et al., 2004). until now, it has not been known how f. avenaceum, as entomopathogen, can kill its host insects. however, it is well known how this fungus, as plant pathogen, can attack and kill its host plant. some explanation of the entomopathogenic effect of the fungus was based on production of toxins, such as beauvericin (logrieco et al., 2002), fusarin c (thrane, 1988; abbas et al., 1989), and moniliformin (rabie et al., 1982; abbas et al., 1989; chelkowski et al., 1990). some strains of f. avenaceum can synthesize enniatin cyclic peptides, primarily enniatins a, b and b1 under laboratory conditions (logrieco et al., 2002) but these enniatins may have a role in plant pathogenicity (haschek et al., 2001). in conclusion, overall results indicate the effectiveness of f. avenceum against s. oryzae adults when applied directly to the adults by the spraying method of fungus conidia. however, further experiments are recommended using different doses of the fungus under a wide range of storage conditions of grain (e.g., different levels of temperature and relative humidity). results obtained in the present research, in addition to those that could be obtained after conducting the recommended experiments, may be exploited in pest management strategies of s. oryzae on stored grain. this will be possible when virulent strains of the fungus with high entomopathogenic activity are available. formulation of these effective strains can increase their efficacy, since it has already been shown that the formulation of other entomopathogenic fungi, such as beauveria bassiana and metarhizium anisopliae, increased their effectiveness and kept them viable for a longer period of time (batta, 2004, 2008). references abbas h.k., mirocha c.j., gunther r., 1989 mycotoxins produced by toxic fusarium isolates obtained from agricultural and nonagricultural arctic areas of norway. mycopathologia 105: 143-152. batta y.a., 2004 control of rice weevil (sitophilus oryzael., coleoptera: curculionidae) with various formulations of metarhizium anisopliae. crop prot. 23: 103-108. batta y.a., 2008 control of main stored-grain insects with new formulations of entomopathogenic fungi in diatomaceous earth dusts. internat. j. food engin. 4: 1-16. available from: http://www.bepress. com/ijfe/vol4/iss1/art9 booth c., 1971 the genus fusarium. commonwealth mycological institute, kew, surrey, england: 237 pp. chelkowski j., zawadzki m., zajkowski p., logrieco a., bottalico a., 1990 moniliformin production by fusarium species. mycotoxin res. 6: 41-45. dal bello g., padin s., lopez-lastra c., fabrizio m., 2000 [page 54] [journal of entomological and acarological research 2012; 44:e11] no nco mm er cia l u se on ly article [journal of entomological and acarological research 2012; 44:e11] [page 55] laboratory evaluation of chemical-biological control of the rice weevil (sitophilus oryzae l.) in stored grains. j. stored products res. 37: 77-84. eleonora-rojas m., perea e.i., pineda y.a., 2003 fusarium species on trialeurodes vaporariorum from tobacco and kidney bean of garcia rovera, santander, colombia. revista colomb. entomol. 29: 1-10. fang l., subramanyam b., arthur f.h., 2002 effectiveness of spinosad on four classes of wheat against five stored-products insects. j. econ. ent. 95: 640-650. furlong m.j., pell j.k., 2001 horizontal transmission of entomopathogenic fungi by the diamondback moth. biol. cont. 22: 288-299. govindan k., nelson s.j., 2009 insecticidal activity of twenty plant powders on mortality, adult emergence of sitophilus oryzae l. and grain weight loss in paddy. j. biopesti. 2: 169-172. gupta s., krasnoff s.b., underwood n.l., renwick j.a.a., roberts d.w., 1991 isolation of beauvericin as an insect toxin from fusarium semitectum and fusarium moniliforme var. subglutinans. mycopathologia 115: 185-189. haschek w.m., gumprecht l.a., smith g., tumbleson m.e., constable p.d., 2001 fumonisin toxicosis in swine: an overview of porcine pulmonary edema and current perspectives. environ. health perspec. 109 (suppl 2): 251-257. hidalgo e., moore d., le patourel g., 1998 the effect of different formulations of beauveria bassiana on sitophilus zeamais in stored maize. j. stored products res. 34: 171-179. kang z., zingen-sell i., buchenauer h., 2005 infection of wheat spikes by fusarium avenaceum and alterations of cell wall components in the infected tissue. europ. j. plant pathol. 111: 19-28. kellock a.w., stubbs l.l., parbery d.g., 1978 seed-borne fusarium avenaceum on subterranean clover and other pasture legumes. austr. j. agric. res. 29: 975-982. lamprecht s.c., knox-davies p.s., marasas w.f.o., 1988 fungi associated with root rot of annual medicago spp. in south africa. phytophylactica 20: 281-286. latiffah z., nurul-izzati h., baharuddin s., 2010 fusarium species isolated from peat soil of pondok tanjung and sungai beriah, perak. malaysian j. microbiol. 6: 102-105. lee b., choi w., lee s., park b., 2001 fumigant toxicity of essential oils and their constituent compounds towards the rice weevil, sitophilus oryzae (l.). crop prot. 20: 317-320. leslie j.f., summerell b.a., 2006 the fusarium laboratory manual, 1st ed. blackwell publishing, ames, iowa, usa. logrieco a., rizzo a., ferracane r., ritieni a., 2002 occurrence of beauvericin and enniatins in wheat affected by fusarium avenaceum head blight. appl. environ. microbiol. 68: 82-85. mcgee d.c., kellock a.w., 1974 fusarium avenaceum, a seedborne pathogen of subterranean clover roots. aust. j. agric. res. 25: 549557. moino jr a., alves s.b., pereira r.m., 1998 efficacy of beauveria bassiana (bal.) vuilemin isolates for control of stored-grain pests. j. appl. entomol. 122: 201-205. nelson p.e, dignani m.c., anaissie e.j., 1994taxonomy, biology, and clinical aspects of fusarium species. clin. microbiol. rev. 7: 479-504. odour g.i., smith s.m., chandi e.a., karanja l.w., agano j.o., moore d., 2000 occurrence of beauveria bassiana on insect pests of stored maize in kenya. j. stored products res. 36: 177185. padin s., dal bello g., fabrizio m., 2002 grain loss caused by tribolium castaneum, sitophilus oryzae and acanthoscelides obtectus in stored durum wheat and beans treated with beauveria bassiana. j. stored products res. 38: 69-74. pungitore c.r., garcía m., gianello j.c., sosa m.e., tonn c.e., 2005 insecticidal and antifeedant effects of junellia aspera (verbenaceae) triterpenes and derivatives on sitophilus oryzae (coleoptera: curculionidae). j. stored products res. 41: 433-443. quesada-moraga e., samtos-quiros r., valverde-gracia p., santiago-alvarez c., 2004 virulence, horizontal transmission, and sub-lethal reproductive effects of metarhizium anisopliae (anamorphic fungi) on the german cockroach (blattodea: blattellidae). j. invertebr. pathol. 87: 51-58. rabie c.j., marasas w.f.o., thiel p.g., lübben a., vleggaar r., 1982moniliformin production and toxicity of different fusarium species from southern africa. appl. environ. microbiol. 43: 517521. rice w.c., cogburn r.r., 1999 activity of the entomopathogenic fungus beauveria bassiana (deuteromycotina: hyphomycetes) against three coleopteran pests of stored grains. j. econ. entomol. 92: 691-694. satyaprasad k., bateman g.l., ward e., 2000 comparisons of isolates of fusarium avenaceum from white lupine and other crops by pathogenicity tests, dna analyses, and vegetative compatibility tests. j. phytopathol. 148: 211-219. sheeba g., seshardi s., raja n., janarthana s., ignacinutha s., 2001 efficacy of beauveria bassiana for control of the rice weevil sitophilus oryzae l. (coleoptera: curculionidae). appl. entomol. zool. 36: 117-120. strunz g.m., strongman d.b., 1988 insecticidal metabolites from fusarium avenaceum, a fungus associated with foliage of abies balsamea infested by spruce budworm, choristoneura fumiferana. in: biotechnology for crop protection, chapter 8, acs symposium series, vol. 379. washington, dc: 110-116. sun z., 2008 the pathogenicity of fusarium spp. to wheat stem sawfly, cephus cinctus norton (hymenoptera: cephidae), ph. d thesis in plant sciences. montana state university, bozeman, montana, usa: 156 pp. teetor-barsch g.h., roberts d.w., 1983 entomogenous fusarium species. entomopathologica 84: 3-16. thrane u., 1988 screening for fusarin c production by european isolates of fusarium species. mycotoxin res. 4: 2-10. toshio m., 2000 microbial control of the diamondback moth, plutella xylostella, by an entomopathogenic fungus, beauveria bassiana. ii. effect of temperature on mycoses and conidial invasion time. japan. j. appl. entomol. zool. 44: 177-182. waalwijk c., van der heide r., de vries i., van der lee t., schoen c., costrel de corainville g., et al., 2004 quantitative detection of fusarium species in wheat using taqman. europ. j. plant pathol. 110: 481-494. yankanchi s.r., gadache a.h., 2010 grain protectant efficacy of certain plant extracts against rice weevil, sitophilus oryzae l. (coleoptera: curculionidae). j. biopesticides 3: 511-513. no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd b. bentivoglio-ravasio, d. dreossi, e. marconi, n. sodini, l. mancini, c. tonini, l. trotta, f. zanini synchrotron radiation microtomography of musical instruments: a non-destructive monitoring technique for insect infestations abstract x-ray computed tomography is becoming a common technique for the structural analysis of samples of cultural relevance, providing luthiers, art historians, conservators and restorators with a unique tool for the characterization of musical instruments. synchrotron-radiation phase-contrast microtomography is an ideal technique for the non-destructive 3d analysis of samples where small lowabsorbing details such as larvae and eggs can be detected. we report results from the first feasibility studies performed at the elettra synchrotron laboratory, where the 1494 organ by lorenzo gusnasco da pavia has been studied. together with important information about the structural conditions, the presence of xylophages could be detected and characterized. key words: cultural heritage, musical instruments, xylophages, x-ray imaging. introduction several types of insects infest wooden musical instruments, and damage inflicted by wood-destroying insects can occur over a long time before detection. while it is critical to assess the amount of actual and potential damage, it is even more important to recognize this kind of event at an early stage and, if possible, to identify the insects before taking appropriate control measures. in case of instruments of great historical, artistic and economic value, a monitoring technique must necessarily be non-invasive and compatible with their typical dimensions. clinical computed tomography has been applied successfully to evaluate both the normal structure and abnormal conditions that may affect an instrument (sirr & waddle, 1997; 1999; gattoni et al., 1999; iwamoto et al., 2002). the main limitation in the application of the technique, however, is related to the limited spatial resolution of commercial instruments, where the typical voxel size is of the order of 0.4x0.6x0.6 mm3. every defect with lateral dimensions smaller than this value cannot, therefore, be detected with state-of-the-art hospital instruments. the dynamical range of the detectors usually employed, moreover, are not compatible when metal parts such as strings in bowed instruments or keys in woodwind instruments are present and cannot be removed. additionally, conventional absorption radiology is not the ideal technique when soft matter details need to be detected inside harder matrices. j. ent. acarol. res. ser. ii, 43 (2): 149-155 30 september 2011 as a last consideration, it is not possible to adequately control environmental parameters such as temperature and humidity when delicate instruments need to be analysed. in this article we describe the phase-contrast x-ray microtomography (mancini et al., 2006; rigon et al., 2010) as a non-invasive tool for the analysis of insect infestation in wooden musical instruments. conservators and restorators can obtain digital threedimensional morphological information about the samples without the need of removing metal parts from their instruments and, thanks to the dimensions of the typical experimental setups, a precise monitoring and control of the environment can be achieved during the data acquisition. materials and methods the positive organ of lorenzo da pavia (1494) the only surviving paper-pipe organ belongs to the collection of musical instruments of the musei civici veneziani, and kept at the museo correr (fig. 1). the author of the instrument is lorenzo gusnasco da pavia, well known for his relationship with the most important families of his age (prizer, 1982). while the original destination of the organ, built in 1494, is still not clear (the previous identification with the instrument fig. 1 the 1494 positive organ of lorenzo gusnasco da pavia. journal of entomological and acarological research, ser. ii, 43 (2), 2011150 built for mattia corvino, king of hungary (sansovino, 1581; haraszty, 1940) is at least uncertain) we know that it has been bought by the museo correr around 1874 (barozzi, 1880). the documentation on the analysis and restoration of the organ is negligible, and the first complete study has been published in 1976 by marco tiella (tiella, 1976). the importance of the instrument, however, has always limited the kind of analysis performed, and the non-destructivity of the techniques has always been a fundamental requirement. phase-contrast radiology in conventional radiology the image formation relies on the x-ray absorption properties of the sample and can be expressed by means of geometrical optics. the image contrast is originated by a variation of density, composition or thickness of the sample and is based exclusively on the detection of amplitude variation of the transmitted xrays. information about the phase of x-rays is not taken into account. the main limitation of this technique is the poor inherent contrast in samples with low-z composition: indeed this is the case of “soft matter” which is considered, in the common sense, as transparent to x-rays. contrary to absorption radiography, “phase-contrast imaging techniques” are based on the observation of the phase shifts produced by the object on the incoming wave. they are described by means of wave optics. absorption and phase shifts are effects occurring to x-rays crossing any kind of materials. their relationships is considered in the definition of the material complex index of refraction n, that in the x-ray region, slightly differs from unity: n = 1 δ + iβ, where δ is related to the refractive properties and β determines the absorption. in the energy range between 15 and 25 kev, the phase shift term δ (of the order of 10-7) can be up to 1000 times greater than the absorption term β (of the order of 10-10), therefore it is possible to reveal phase effects even if the absorption is negligible (phase objects). the observation of the local variations in the optical path-length, determined by variations of δ, is related to fresnel diffraction. in general, phase information can be accessed if the x-ray source has a high spatial coherence as in the case of synchrotron light sources (snigirev et al., 1995; baruchel et al., 2000) like esrf in france or elettra in italy. several approaches for phase-contrast radiology have been reported (fitzgerald, 2000). among these, the phase-contrast (pc) radiography technique based on free space propagation can also be called in-line holography in analogy with optics and has a quite simple application: the pc set-up is the same of conventional radiography with the difference that the detector is positioned at a certain distance d from the sample. the x-rays exiting from the sample propagate in the free space until they reach the detector. free space propagation transforms phase modulation of the transmitted beam into amplitude modulation. contrast is originated from interference among parts of the wavefronts that have experienced different phase shifts. according to the choice of d with respect to the size of the feature to be identified perpendicularly to the beam direction, one may discriminate between two regimes: the edge detection regime (d << a2/λ, where λ is the x-ray wavelength) and the holography regime (d ≈ a2/λ). in the edge detection regime images can be used directly to extract morphological information. the produced diffraction pattern b. bentivoglio-ravasio et al.: a non-destructive monitoring technique for insect infestations 151 appears superimposed to the conventional absorption pattern (if any) on the detector and contributes mainly to enhance the visibility of the edges of the sample features. the microtomographic analysis in order to obtain more detailed information about the internal structure of the paper pipes, we used the syrmep beamline (abrami et al., 2005) and the tomolab facility of elettra, the italian synchrotron laboratory of trieste. the synchrotron images have been recorded by a water-cooled 12/16 bit, 4008 x 2672 full frame ccd camera with 4.5 micron pixel size and a field of view of 18 x 12 mm2, coupled to a gadox scintillator placed on a straight fiber optics coupler. during data acquisition, samples were rotated 180° around an axis perpendicular to the incident beam with a sampleto-detector distance of 20 cm and a beam energy of 20 kev. projection images were recorded at 0.125 degree rotation step for a total of 1440 projections for each sample. these parameters were optimized in a preliminary step by imaging several samples under different beam energy, phase contrast and resolution conditions. a typical reconstructed slice of a pipe at the level of the wooden foot is shown in fig. 2, where the experimental parameters have been optimized for the definition of the number and thickness of the paper layers, as well as the overall condition of the paper and wood foot structure. the final spatial resolution is about 10 microns. we can evaluate the present situation of the paper pipe (which appears to be more critical in comparison with other points of the same pipe), but we can also extract important information about the wooden part. the different kinds of wood used by lorenzo gusnasco fig. 2 left: virtual slice of a paper pipe at the wooden foot position taken at the syrmep beamline. right: inset showing the different wood qualities and the presence of larvae. journal of entomological and acarological research, ser. ii, 43 (2), 2011152 can be determined with great precision, and we also have a clear view of the presence of wormholes caused by the infestation of powderpost beetles. thanks to the phasecontrast approach we can also detect the presence of larvae inside the wooden foot. in order to compare the synchrotron results with a state-of-the-art conventional system, we repeated the same analysis at the tomolab facility (polacci et al., 2009). tomolab is a micro-ct system equipped with a sealed microfocus x-ray tube, which guarantees a focal spot size of 5 μm, in an energy range from 40 up to 130 kv, and a maximum current of 300 μa. a water cooled ccd camera providing a good combination between a large field of view (49.9 mm × 33.2 mm) and a small pixel size (12.5 × 12.5 μm2) is used as detector. thanks to the cone-beam geometry, it is possible to achieve a spatial resolution close to the focal spot size (feldkamp et al., 1984; kak & slaney, 1987), even if in this case spatial resolution has been partly sacrifice to achieve a wider field of view. thanks to the small size of the x-ray tube focal spot, a limited but clearly detectable phase contrast effect can be achieved (wilkins et al., 1996), especially when relatively low energies are involved and object-to-detector distances are in excess of 30 cm. thanks to its geometry, even this setup allows to perform phase-contrast measurements. the tomographic scan of the sample was performed with a pixel size of 20 μm2, with a tube voltage of 45 kv and a tube current of 177 μa. a total number of 1800 projections were collected (source-to-sample distance = 45 cm and source-todetector distance = 55 cm). in fig. 3 the paper pipe has been digitally removed and only the wooden part is shown, and we choice a different graphical representation in order to show the different ways that the data can be displayed. again, the womholes are well defined as well as the presence of the powderpost beetles larvae. fig. 3 3d rendering of a slice of a wooden foot taken at the tomolab facility. b. bentivoglio-ravasio et al.: a non-destructive monitoring technique for insect infestations 153 conclusions phase-contrast microtomography looks like a promising non-invasive technique for the monitoring and characterization of insect infestation in wooden musical instruments, but can be applied, even in a bidimensional configuration, to other samples of artistic and historical interest. during the morphological analysis of ancient violins of the cremonese school, the synchrotron radiation approach has not induced any degradation of the instrument. additional mesaurements could show if the typical exposure times of 30-60 minutes could have the additional effect of killing any powderpost beetles still present inside the samples. acknowledgements the authors would like to acknowledge marco merlini for his continuous encouragment and inspiration and marco fioravanti for useful discussions. references abrami a., arfelli f., barroso r.c., bergamaschi a., bille f., bregante p., brizzi f., casarina k., castelli e., chenda v., dalla palma l., dreossib d., fava c., longo r., mancini l., menk r.-h., montanari f., olivo1, a., pani s., pillona a., quai e., ren kaiser s., rigon l., rokvic t., tonutti m., tromba g., vascotto a., venanzi c., zanconati f., zanetti a., zanini f., 2005 medical applications of synchrotron radiation at the syrmep beamline at elettra. nuclear instruments and methods in physics research, section a, 548: 221227. barozzi n., 1880 doni fatti al museo correr fino al 1880 e cenni intorno al suo collocamento nel nuovo edificio. naratovich, venice. baruchel j., cloetens p., härtwig j., ludwig w., mancini l., pernot p., schlenker m., 2000 phase imaging using highly coherent x-rays: radiography, tomography, diffraction topography. journal of synchrotron radiation, 7: 196-201. feldkamp l.a., davis l.c., kress j.w., 1984 practical cone-beam algorithm. journal of the optical society of america, section a1: 612-619. fitzgerald r., 2000 phase sensitive x-ray imaging. physics today, 53: 23-36. gattoni f., melgara c., sicola c., uslenghi c.m., 1999 unusual application of computerized tomography: the study of musical instruments. radiol med, 97: 170-173. haraszty e., 1940 a propos de l’orgue de mathias corvin du musée correr à venise. l’orgue, 21: 7-17. iwamoto j., kenmochi y., kotani k., nagasawa i., 2002 extraction of a 3d graph structure of wormholes in a wooden statue of buddha by x-ray ct image analysis. proceedings of the fifth asian conference on computer vision, accv2002: 864-870. kak a.c., slaney m., 1987 priciples of computerized tomographic imaging. ieee press, new york. mancini l., tromba g., zanini f., 2006 structural microanalysis with synchrotron radiation: archaeometric applications at elettra. journal of neutron research, 14: 75-79. polacci, m., baker d. r., mancini l., favretto s., hill r. j., 2009 vesiculation in magmas from stromboli and implications for normal strombolian activity and paroxysmal explojournal of entomological and acarological research, ser. ii, 43 (2), 2011154 sions in basaltic systems. journal of geophysical research, 114: b01206-b01220. prizer w.f., 1982 isabella d’este and lorenzo da pavia, ‘master instrument-maker’. early music history, 2: 87-127. rigon l., vallazza e., arfelli f., longo r., dreossi d., bergamaschi a., schmitt b., chen r., cova m.a., perabò r., fioravanti m., mancini l., menk r.h., sodini n., tromba g., zanini f., 2010 synchrotron-radiation microtomography for the non-destructive structural evaluation of bowed stringed instruments. e-preservation science, 7: 71-77. sansovino m.f., 1581 venetia città nobilissima et singolare. curti, venice: 379-380. sirr s.r., waddle j.r., 1997 ct analysis of bowed stringed instruments. radiology, 203: 801805. sirr s.r., waddle j.r., 1999 use of ct in detection of internal damage and repair and determination of authenticity in high-quality bowed stringed instruments. radiographics, 19: 639-646. snigirev a., snigireva i., kohn v., kuznetsov s., schelokov i., 1995 on the possibilities of x-ray phase contrast microimaging by coherent high-energy synchrotron radiation. review of scientific instruments, 66: 5486-5492. tiella m., 1976 the positive organ of lorenzo da pavia. the organ yearbook, 4: 4-15. wilkins s.v., gureyev t.e., gao d., pogany a., stevenson a.w., 1996 phase-contrast imaging using polychromatic hard x-rays. nature, 384: 335-338. beatrice bentivoglio-ravasio, emanuele marconi, leonardo trotta, direzione regionale per i beni culturali e paesaggistici della lombardia. corso magenta 24 20123 milano, italy. e-mail: beatrice.bentivoglio@beniculturali.it diego dreossi, nicola sodini, lucia mancini, franco zanini, sincrotrone trieste, s.s. 14, km 163,5 34149 basovizza-trieste, italy. e-mail: franco.zanini@elettra.trieste.it camillo tonini, fondazione musei civici veneziani, piazza san marco 52 30124 venezia, italy. e-mail: camillo.tonini@comune.venezia.it b. bentivoglio-ravasio et al.: a non-destructive monitoring technique for insect infestations 155 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res..indd f. palla characterization of microbial communities in pest colonized books by molecular biology tools abstract this work presents the identification of bacteria and fungi colonies in insect infesting books, by cultural-independent methodologies based on molecular biology techniques. microbial genomic dna extraction, in vitro amplification of specific target sequences by polymerase chain reactions (pcr), sequencing and sequence analysis were performed. these procedures minimized the samples amount, optimized the diagnostic studies on bacteria and fungi colonization and allowed the identification of many species also in complex microbial consortia. the molecular techniques for sure accomplish and integrate the microbiological standard methods (in vitro culture) and morphological analyses (om, sem, clsm), in order to understand the role of microorganisms in bio-deterioration of cultural assets. this monitoring is also indispensable to shed light on the risk for visitors and/or professionals to contract potential illnesses within indoor environments. key words: genomic dna, paper biodeterioration, pcr, termites infestation. introduction insects and microorganisms play a significant role in the deterioration of organic substrates, their distribution and development is closely related to environmental parameters (temperature, relative humidity, water activity) and chemical-physical properties of materials (camuffo, 1998; valentin, 2003). particular environments, such as museum, archives, libraries, basements are very often colonized by insects and microorganisms due to the favorable micro-climate, characterized by water condensation phenomena and poor air exchange (singh et al., 1995; gallo et al., 1999; else et al., 2003; valentin, 2003). there is a huge literature on insects and microbial communities related to biodegradation of historic collections made by organic materials. specifically, insects as thysanura (lepismatidae), isoptera (rhinotermitidae, kalotermidae), coleoptera (anobiidae, cerambycidae, lictidae, dermestidae, ptnidae), bacteria and/ or fungal species belonging respectively to bacillus, micrococcus, streptomyces, actinomycetes, and/or penicillum, cladosporium, aspergillus, alternaria, trichoderma, rhizopus have been reported (florian, 1994; rust et al., 1996; valentin, 2003), but many others have still to be identified. in this study a wide microbial colonization was revealed on books pages, must j. ent. acarol. res. ser. ii, 43 (2): 61-67 30 september 2011 likely correlated to the termite infestation (reticulitermes lucifugus rossi) recognized not only on the collection but even in the wooden cabinets where the books were stored (not & guarneri, 2003). since termites metabolic products (tmp) represent an excellent substrate for microbial growth, we hypothesize a close relationship between the tmp and the complexity of microbial consortia and consequently the clear biodeterioration of books. moreover by trophallaxis termites can transfer symbiotic bacteria and protozoa in the surrounding environment, where are also able to release chemical compounds that causes physiological changes and behavior in other individuals, usually of the same species (chiappini et al., 2001). as previously described we applied different molecular technologies for the characterization of bacteria and cyanobacteria colonizing cultural assets (gargano et al., 2009; palla et al., 2010). results described the identification of bacterial species onto organic substrates. materials and methods sampling sampling on the surfaces of books pages was performed by fragments (20 x 40 mm) of sterile nylon membrane (hybond membrane, amersham-biosciences), as described by palla et al. (2006). this procedure allowed us to isolate single bacterial and fungal colonies. colonizing biocenosis were analyzed by scanning electron microscopy and bacteria through molecular protocols. microbial dna extraction bacteria colonies isolated on nutrient agar: each colony was dissolved in 20 μl of 1x t.e. (10 mm tris-hcl ph 7,5 / 1 mm edta) and lysed at 94°c per 2 min. aliquots (0.2 g) were utilized for dna extraction by using qiaamp dna stool kit (qiagen); the yield was approximately 500 ng dna per gram of sample. in vitro amplification of bacteria target sequences (pcr reactions) the ribosomal intergenic spacer was performed the polymerose chain reaction (its-pcr) using the oligonucleotides its1 = 5’-gtcgtaacaaggtagccgta-3’ and its2 = 5’-gccaaggcatccacc-3’, as primers (cardinale et al., 2004). genomic dna extracted from single bacterial colonies (2 μl) or directly from tmp (100 ng) was utilized as template in each reaction. reaction mixture (up to 50 μl) consisted of: 0.5 units of taq dna polymerase (invitrogen) in mgcl2 and salt conditions suggested by the company. the target sequence was amplified for 35 cycles as follows: denaturation for 1 min at 94°c; annealing for 1 min at 50°c; extension for 1 min at 72°c. a final extension step (7 min at 72°c) was added in order to allow the full-elongation of pcr products. pcr reaction products were resolved by electrophoresis on 2% agarose gel stained with sybr safe dna gel stain (invitrogen). journal of entomological and acarological research, ser. ii, 43 (2), 201162 sequencing of its amplified fragments its-pcr products were purified by qia-quick pcr purification kit (qiagen) and sequenced by mwg-biotech custom sequencing services (http://www.mwg-biotech. com). the research for homology (16s-23s-its rdna) was performed by nucleotidenucleotide blast analyzer (altschul et al., 1997; pearson et al., 1988). scanning electron microscopy samples from books or termites metabolic products (tmp) were coated with gold particles (thickness ≈ 13 nm) by agar-auto-spotter-coater (b7341) and observed under high vacuum leica cambridge-leo 420. results f. palla: characterization of microbial communities by molecular biology tools fig. 1 fragment of wood cabinet infested by r. lucifugus. fig. 2 bacteria colonies (a, b, c) growth on nylon membrane fragments on nutrien agar plate, after incubation at 30°c per 24 hours. a single fungal colony (f) is also recognizable. 63 fig. 3 sem micrographs of book fragments colonized by bacteria (left) and actinomyces (right). fig. 4 fragments of termites metabolic products (left) utilized for the extraction of microbial dna (right), analysed on 0.8% agarose gel, stained by sybr safe (invitrogen). m = λ hind iii dna (molecular weight marker). bar = 0.5 cm. journal of entomological and acarological research, ser. ii, 43 (2), 201164 biodeterioration by termites r. lucifugus was clearly shown in both wood cabinet (fig. 1) and in most of the books where a noticeable microbial colonization was also identified by a multidisciplinary approach. bacteria and fungi colonies were revealed by in vitro culture (nutrien agar, difco) incubating the petri dish for 24 hours at 30°c (fig. 2), and scanning electron microscopy (fig. 3). bacterial colonization was characterized by its-pcr reactions, using as template the genomic dna obtained from single colonies or extracted from tmp fragments (fig. 4). analysis of its-pcr products (fig. 5) allowed to identify arthrobacter nicotianae, micrococcus luteus, kocuria rosea and curtobacterium flaccumfaciens as predominant species. discussion and conclusion in this work we applied non-destructive sampling procedures, combining sem analysis with in vitro culture and molecular biology techniques in order to characterize microbial, and particularly bacteria colonizing historic book collections. furthermore, the study point out the potential relationship between termites infestation and microbial colonization. it is to be underlined that m. luteus and k. rosea are able to produce lytic enzymes and pigmented compounds (gallo et al., 1999; valentin, 2003; palla et al., 2007) and the high cellulose degradation activity of c. flaccumfaciens (lednicka et al., 2000) represents, for this typologies of organic substrates, an high potential risk of biodegradation. in this paper we set up protocols to revealing the presence of bacterial consortia over books surfaces and o tmp, which represent an extraordinary source for microorganisms. fig. 5 electrophoretic analysis of amplified its (16s-23s) bacterial region. m= 100 bp dna ladder (promega). f. palla: characterization of microbial communities by molecular biology tools 65 we want also point out that bacteria and fungi colonizing indoor environment are able to damage artifacts and release toxins detrimental to human health (peltola et al., 2001; nilsson et al., 2004). molecular technologies could be used for characterizing these dangerous microbes, in order to establish suitable strategies for conservation and fruition of cultural assets. finally, we are setting up molecular protocols for fungal identification by using oligonucleotide primers specific for its regions or tubulin gene (glass et al., 1995; palla et al., 2009). acknowledgements the author wishes to acknowledge the centro regionale progettazione e restauro della regione siciliana for the support throughout the project. the author acknowledge c. di liberto for sem analysis. references altschul s.f., madden t.l., schäffer a.a., zhang j., zhang z., miller w., lipman d.j., 1997 gapped blast and psi-blast: a new generation of protein database search programs. nucleic acids research, 25: 3389-3402. camuffo d., 1998 microclimate for cultural heritage. amsterdam and new york: elsevier. cardinale m., brusetti l., quatrini p., borin, s., puglia a. m., rizzi a., zanardini e., sorlini c., corselli c., daffonchio d., 2004 comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. applied environmental microbiology, 70: 6147-6156. chiappini e., liotta g., reguzzi m.c., battisti a., 2001 insetti e restauro. calderini ed. agricole, bologna. else t.a., pantle c.r., amy p.s., 2003 boundaries for biofilm formation: humidity and temperature. applied environmental microbiology, 69: 5006-5010. florian m.l.e., 1994 heritage eaters. insects & fungi in heritage collections. london: james & james. gallo f., maggi o., pasquariello g., persiani a.m., sclocchi m.c., scorrano m., valenti p., 1999 aerobiological researches in book and archival heritage and graphic arts. in p. tiano & c. feroci (eds). international conference on microbiology and conservation: of microbes and art. florence, 16-19 june 1999. cnr, florence, italy. gargano v., mancuso f.p., vitale f., reale s., caracappa s., palla f., 2009 la tecnologia del dna-microarray per l’identificazione di specie microbiche su superfici e nell’aerosol di ambienti confinati. in: sistemi biologici e beni culturali, convegno nazionale aiar, orto botanico palermo, 6-7 ottobre 2009, crpr sicilia university of palermo, italy. glass l.n., donaldson g.c., 1995 development of primer sets designed for use with pcr to amplify conserved genes from filamentous ascomycetes. applied environmental microbiology, 61: 1323-1330. lednicka d., mergaert j., cnockaert m.c., swings j., 2000 isolation and identification of cellulolytic bacteria involved in the degradation of natural cellulosic fibres. systematic applied microbiology, 23: 292-299. journal of entomological and acarological research, ser. ii, 43 (2), 201166 nilsson a., kihlstrom e., lagesson v., wessen b., larsson l., tagesson c., 2004 microorganism and volatile organic compound airborne dust from damp residences. indoor air, 14: 74-82. not r., guarneri e., 2003 infestazione termitica nella biblioteca comunale di taormina (me): proposte d’intervento. atti della giornata studio: l’intervento di deacidificazione a libro integro. taormina (me): 105-115. crpr assessorato dei bbcc, ambientali e p.i. regione siciliana. palla f., anello l., marineo s., lombardo g., 2006 characterization of bacterial community in indoor environment. in: fort r., alvarez de buergo m., gomez-heras m. vazquez-calvo. heritage, weathering and conservation. (1, pp. 361-365) taylor & francio, u.k. palla f., tartamella e., 2007 chromatic alteration on marble surfaces analysed by molecular biology tools. conservation sciences in cultural heritage, 7: 1-10. palla f., lanza a., mancuso f.p., lombardo g., saitta a., gargano m.l., 2009 definizione di un protocollo per la caratterizzazione morfologica e molecolare del genere daldinia ces. & de not. (ascomycota) in sicilia. congresso nazionale della società botanica italiana, 16-19 settembre 2009, university of molise, italy. palla f., billeci n., mancuso f.p., pellegrino l., 2010 microscopy and molecular biology techniques to study biocenosis diversity in semi-confined environment. conservation science in cultural heritage, 10: 185-194. pearson w.r., lipman d.j., 1988 improved tools for biological sequence comparison. proceedings of national academy of sciences usa, 85: 2444-2448. peltola j., andersson m.a., haahtela t., mussalo-rauhamaa h., rainey f.a., kroppenstedt m., samson r.a., salkinoja-salonen s., 2001 toxic-metabolite-producing bacteria and fungus in an indoor environment. applied environmental microbiology, 67: 3269-3274. rust m., vinod d., druzik j., presseur f., 1996 – the feasability of using modified atmosphere to control insect pests in museum. restaurator, 17: 43-60. singh a., ganguli m., sing a.b., 1995 fungal spores are an important component of library air. aerobiologia, 11: 231-237. valentin n., 2003 microbial contamination in museum collections: organic materials. in c. saiz-jimenez (ed.), molecular biology and cultural heritage: 85-91. lisse/abingdon/exton/tokyo. a.a. balkema publishers. franco palla, dipartimento di biologia ambientale e biodiversità, università degli studi di palermo, via archirafi 38, 90123 palermo, italy. e-mail: franco.palla@unipa.it f. palla: characterization of microbial communities by molecular biology tools 67 j. ent. acar. res..indd a. berzolla, m.c. reguzzi, e. chiappini controlled atmospheres against insect pests in museums: a review and some considerations abstract controlled atmospheres using nitrogen represent a safe and effective method for both objects and human health. the use of this technique against pests in museums has received an increasing amount of interest during the last twenty years. this paper looks at the researches into anoxic treatments that use nitrogen from the late ‘80s until now. at the moment, the recommended protocol suggests an oxygen percentage below 1% for at least three weeks. considering that the major practical problems of controlled atmospheres are connected to treatment time and low oxygen percentage, it is very important to develop more flexible protocols that consider higher oxygen percentages or shorter treatment times, exploiting temperature and/or relative humidity. at oxygen percentage higher than those commonly used, temperature and relative humidity are very critical to insects’ development and success. preliminary data (unpublished) show that it is possible to adapt the application of the controlled atmospheres to different situations, taking advantage of favorable conditions already present in the considered situation and at the same time to use the other parameters at more favorable levels. key words: cultural heritage, relative humidity, protocol. introduction control of insect pests in conservation institutions was based on pesticide applications at least until the eighties. of course the high use of chemicals has led to accumulations of residues in the environment (glastrup, 1987; koestler et al., 1993). reviews concerning problems caused by biocides used on cultural objects can be found in allsopp & seal (1985), story (1985), dawson (1988), caneva et al. (1991), and pinniger (1994). biocides are reactive products and many, if not all, can cause alterations in some kinds of works of art. an array of preventive and “non chemical” control methods should represent the rule to be adopted to solve pest problems (strang, 1998). controlled atmospheres using nitrogen represent a safe and effective method for both objects and human health. nitrogen is nontoxic, non-flammable, and non-reactive. this technique, in fact, is particularly useful for very vulnerable objects, which might be damaged by low or high j. ent. acarol. res. ser. ii, 43 (2): 197-204 30 september 2011 temperature treatments (pinniger, 2010). after 150 days of treatment, painting materials showed no visual effects as a consequence of exposure to nitrogen (koestler et al., 1993; koestler et al., 2004). despite the fact that an inert atmosphere “retards color fading” of most organic colorants (arney et al., 1979; burke, 1992), a possible negative effect, due to oxygen deprivation, is a change in specific pigments (e.g. prussian blue) (rowe, 2004). use of controlled atmospheres against pests in museums has received an increasing amount of interest during the last twenty years (gilberg, 1989; valentin, 1990; gilberg, 1991; daniel et al., 1993; hanlon et al., 1993; reichmuth et al., 1993; valentin, 1993; kaplan & schulte, 1996; newton et al., 1996; reirson et al., 1996; rust et al., 1996; grassi, 1997; nielse, 1998; selwitz and maekawa, 1998; kigawa et al., 2001; valentin et al., 2002; bergh et al., 2003; brandon & hanlon, 2003; child, 2007; child & pinniger, 2008). this research especially addressed two aspects of the method: the oxygen percentage (but always very low, between 0.03% and 1.0%) (child & pinniger, 2008) and the treatment time, which varied from a few days to weeks (gilberg, 1991; grassi, 1997; daniel et al., 1993; hanlon et al., 1993; pinniger & child, 1996; valentin et al., 2002; bergh et al., 2003). at the moment, the recommended protocol, which gilberg (1991) validated for tineola bisselliella (hummel), lasioderma serricorne (fabricius), stegobium paniceum (linnaeus), anthrenus vorax (linnaeus), recommends an oxygen percentage below 1% for at least three weeks. according to professionals, the most common problems are: 1) long treatment times that, together with low oxygen percentages, cause high costs and difficulties to the management of conservation institutions (selwitz & maekawa, 1998), 2) difficulty in achieving and maintaining such low oxygen concentrations; if the oxygen level increases during the treatment, it has to be repeated. regarding the first point, the total time required for treatment is the sum of the time necessary for the controlled atmosphere to permeate the substrate plus the time needed to obtain total mortality of the insects. therefore, the exposure time is affected by the oxygen drop time, not only in the “bubble” where the treatment is done, but especially within the object in which the insect lives. the oxygen desorption time of objects depends intrinsically on the nature of the materials, on the thickness of the objects to be treated (normally, wood needs more time for a uniform distribution than textiles) (reichmuth, 1993), on their volume and on the anoxia chamber volume. in addition, it varies as a function of the permeability of the materials to the gas mixture that replaces oxygen in the material. gunn et al. (2006) elaborated a modelling of nitrogen diffusion in porous objects that makes it possible to predict oxygen desorption time, in relation to the nature of the material and the size of the objects, and therefore to estimate achievement of anoxia at the core of a treated object. they concluded that the length of treatment depends closely on the desorption time and that the oxygen drop time is between 1 and 2 days for most objects. it is therefore advisable that this aspect is studied by both an entomologist and a physicist. with regard to the second point (use of very low oxygen concentrations), some considerations are necessary on the way oxygen deprivation acts on insects. the effects journal of entomological and acarological research, ser. ii, 43 (2), 2011198 of different oxygen percentages in the controlled atmosphere on insect metabolism are different. at oxygen concentrations below 3%, in order to meet their energy requirements, insects adopt an anaerobic metabolism (edwards, 1953; wegener, 1993; mitcham et al., 2006). with oxygen levels ranging from 3% to 5%, insects face the reduced availability of oxygen by reducing the oxidative metabolism. neither strategy produces enough energy to maintain a standard metabolic level, obliging the insects to reduce this and, as a consequence, their energy demand (hochachka et al., 1993; mitcham et al., 2006). an accumulation of toxic end products, together with a very slow metabolism, is the cause of stress that results in death (ofuya & reichmuth, 2002). at oxygen concentrations above 5%, the insects increase their respiratory rate in order to take in the same amount of oxygen. the increase in respiratory rate results in dehydration due to prolonged opening of the stigma, especially at high temperatures and low humidity (emekci et al., 2002; mitcham et al., 2006). jay et al. (1971) studied the relationship between mortality and r.h., by examining the death rate of red flour beetles tribolium castaneum (herbst) and confused flour beetles tribolium confusum jacquelin du val in a nitrogen atmosphere, containing between 0.8% and 1% oxygen, after 24 hours at r.h. of 9%, 33%, 54%, and 68%. both species showed a marked increase in mortality as the r.h. decreased, but they died also at high r.h. (68%). considering the possibility of using controlled atmospheres with higher oxygen percentages, anoxia treatments were shown to be effective at oxygen percentages from 3 to 10%, in at most one week, at temperatures between 20°c and 40°c (chiappini et al., 2009), taking advantage of the “positive influence of a temperature increase in order to compensate for the effects of the reduced anoxia”. in this case, the efficacy of treatments with oxygen concentrations higher than 5% was certainly due also to the low r.h. registered in the experiments, always lower than 15% (chiappini et al., 2009). humidity influences the survival of insects and dry conditions generally appear to be highly unfavourable to most insects (child, 2007). controlled atmospheres increase the action of low humidity, by prolonging opening of the spiracles, thereby permitting rapid loss of water. in the same way, high humidity suppresses the desiccation mechanism (gullan & cranston, 1994; emekci et al., 2002; mitcham et al., 2006). discussion and conclusions considering that the major practical problems of controlled atmospheres are connected to treatment time and low oxygen percentage, and that these parameters are strictly related to each other and to those of the microclimate (temperature and relative humidity), it is very important to develop more flexible protocols that consider higher oxygen percentages or shorter treatment times, exploiting temperature and/or relative humidity. the availability of these data also allows us to adapt to different situations, even to take advantage of favourable conditions already present and to use the other parameters at more favourable levels. a.berzolla et al.: controlled atmospheres against insects pests in museums 199 temperature and relative humidity are critical to insects’ developmental success. considering the positive correlation between metabolic rate and temperature in insects (calderwood, 1961), anoxic treatment is, of course, temperature-dependent to a high degree. it is generally agreed to be relatively ineffective at temperatures below15°c (child, 2007). the effect of high temperatures can be useful for speeding up the treatment time (navarro & calderon, 1980; chapman, 1998). valentin (1993) studied the possibility of shortening treatment time, rising the temperature, at a very low oxygen percentage (0.03%). complete eradication of anobium punctatum (degeer) was achieved after 5 days at 30°c (50% r.h.), l. serricorne, required 8 days at 25°c (50% r.h.), or 9 days at 20°c (40% r.h.), while for hylotrupes bajulus an exposure time of 10 days at 30°c (40% r.h.) or 20 days at 20°c was necessary (valentin, 1993), thus demonstrating the efficacy of exposure times much shorter than the standard of 21 days. in some cases, at low oxygen percentages, a total mortality was reported even at not very high temperatures. it was reached in less than 4 days at 25.5°c (55% r.h., < 0.1% oxygen percentage) for adults, larvae, and eggs of tineidae (rust et al., 1996) and in only 3 days at 25°c (55% r.h., 0.3% oxygen percentage) for six dermestidae species (bergh et al., 2003). if higher oxygen percentages are taken into account, at which, as we have already clarified, water loss becomes a key factor for mortality, relative humidity should also be considered. some preliminary results on adults of the food pest, t. confusum, are extremely promising. at 3 ± 0.1% oxygen, total mortality was obtained in 5 days of treatment at 32 ± 1°c and 30 ± 2% r.h., while lower mortality (86%) was registered at the same treatment time (5 days), oxygen percentage (3 ± 0.1%) and temperature (32 ± 1°c) but at higher relative humidity (50 ± 2% r.h.). a similar result was obtained also at a higher oxygen percentage; at 5 ± 0.1% oxygen total mortality was obtained in 4 days of treatment at 35 ± 1°c and 30 ± 2% r.h.. these data demonstrate the high correlation existing between temperature and relative humidity for controlled atmosphere efficacy (unpublished data), especially at higher oxygen percentages. in many conservation institutions all over the world, recommended temperature and relative humidity conditions (25°c, 45-50% r.h.) are not sustainable; moreover, many times a wide range of relative humidity could be suitable; for paper conservation a r.h. from 35% to 50% is acceptable, as pointed out by dean (www.nyalgro.org/ enemies%20of%20paper05.ppt). however, low humidity is preferable to high. miller in 1993 (http://www.artframe.us/article-enemiesofpaper.pdf), talking about the conservation of paper, underlines that 50% is the ideal value, but the most important factor is not 50% r.h., but that it remains constant. humidity changes are in fact the main cause of paper materials damage. from his experience michalski (1993) identifies real examples of incorrect relative humidity in museums, falling into one of four categories: “damp, above or below a critical humidity, any humidity over 0%, and humidity fluctuations”. also in particular situations, such as moving or building restoration, the “correct” journal of entomological and acarological research, ser. ii, 43 (2), 2011200 temperature or humidity could be temporarily lost. in addition some institutions (e.g. canadian libraries and archives) opt to achieve mass desiccation for free during the winter by using heating systems with no humidifiers (michalski, 1993). therefore, why not considering the opportunity of treatment under more favourable conditions? with respect to relative humidity and oxygen percentage also the technique chosen could acquire importance. a static approach can be optimal when the previous r.h. is low and therefore useful for enhancement of treatment efficacy while a dynamic approach, which requires humidification to avoid hygrometric shock to museum objects during treatment (child & pinniger, 2008), could be used when the r.h. is relatively high and does not have to be lowered. some additional considerations are necessary. considering that survival parameters for many insect pest species have been studied and are well understood and that for holometabolous insects the ability to live in hypoxia is stage-specific (hoback & stanley, 2001), a systematic study is necessary in which the museum insect mortality, of various species is determined following exposure to a range of oxygen concentrations for varying periods of time at different temperatures and relative humidity. furthermore, most of the tests on the mortality of the insects in cultural property in an anoxic environment have been done not on laboratory reared insects, but on “natural populations” in objects to be treated, not knowing their precise age or even their health. therefore, in literature it is often not basic research on the insects that is presented, but treatments to test the functionality of a specific instrument (gilberg, 1990; brandon & hanlon, 2003; valentin, 2002). information in literature on respiration rates of the various developmental stages of insects is very rare. gilberg (1991), hoback & stanley (2001), and child (2007), underline that it appears that investigation into insect adaptations to hypoxic and anoxic environments is an emerging field of inquiry, but these challenges are still evident today. references alsopp d., seal k. j., 1986 introduction to biodeterioration edward arnold ltd, 41 bedford square, london wc1b 3dq, 136 pp. arney j.s., jacobs a.j., newman r., 1979 influence of oxygen on the fading of organic colorants. journal of american institute for conservation, 18: 108-117. bergh j.e., stengard h.l., vagn jensen k.m., nielsen p.v., 2003 the effect of anoxic treatment on the larvae of six species of dermestids (coleoptera). journal of applied entomology, 127: 317-321. brandon j., hanlon g., 2003 a low tech method for insect eradication using ageless tm. wag postprints, arlington, virginia. burke j., 1992 vapor barrier films. waac (western association of art conservation) 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controlled atmospheres against insects pests in museums valentin n., bergh j.e., ortega r., åkerlund m., hallström a., jonsson k., 2002 evaluation of a portable equipment for large scale de-infestation in museum collections using a low oxygen environment. 13th triennial meeting. preprints of icom committee for conservation, 1: 96-101. valentin n., preusser f., 1990 insect control by inert gases in museums archives and archives. restaurator, 11: 22-33. wegener g., 1993 hypoxia and post hypoxic recovery in insects: physiological and metabolic aspects. in: hochachka p.w., lutz p.l., rosenthal m., sick t., van den tillarth g., surviving hypoxia mechanisms of control and adaptation, crc press, bocaraton, 4-32. www.nyalgro.org/enemies%20of%20paper05.ppt http://www.artframe.us/article-enemiesofpaper.pdf alessia berzolla, istituto di entomologia e patologia vegetale, facoltà di agraria, università cattolica del sacro cuore, via emilia parmense 84, i-29122 piacenza, italy. email: alessia.berzolla@unicatt.it maria cristina reguzzi, istituto di entomologia e patologia vegetale, facoltà di agraria, università cattolica del sacro cuore, via emilia parmense 84, i-29122 piacenza, italy. email: cristina.reguzzi@unicatt.it elisabetta chiappini, istituto di entomologia e patologia vegetale, facoltà di agraria, università cattolica del sacro cuore, via emilia parmense 84, i-29122 piacenza, italy. e-mail: elisabetta.chiappini@unicatt.it journal of entomological and acarological research, ser. ii, 43 (2), 2011204 429 too many requests you have sent too many requests in a given amount of time. j. ent. acar. res. ser. ii, 42 (3): 171-181 30 december 2010 i. tóbiás, f. kozár, b. m. kaydan, k. fetykó use of molecular tools for the identification of males of some scale insects (hemiptera: coccoidea), in pheromone traps used for monitoring and comparison with females abstract - species from pseudococcidae family were studied. it was determined  that the dry males of planococcus citri, and pseudococcus comstocki, collected by  pheromone traps could be useful for the molecular analyses too. the its-2 sequences  of males and females in case of pl.citri, planococcus ficus and ps. comstocki were  identical. this molecular method could differentiate the two mealybug species and  this method can be useful to have idea specimens collected by pheromone traps.  riassunto uso degli strumenti molecolari per l’identificazione di maschi di cocciniglie (hemiptera: coccoidea), in trappole a feromoni utilizzate per monitoraraggio e comparazione con le femmine. sono state prese in considerazione specie appartenenti alla famiglia degli pseudococcini. e’ stato visto che maschi essiccati di planococcus citri, e pseudococcus comstocki, raccolti da trappole a feromoni possono essere utilizzati anche per analisi  molecolari. la sequenza its-2 di maschi e femmine di p.citri, planococcus ficus e  p. comstocki è identica. questo metodo molecolare può differenziare le due specie  di cocciniglie e può essere utile per avere un’idea delle specie raccolte in trappole  a feromoni. key words: planococcus citri, planococcus ficus, pseudococcus comstocki, male,  pheromone trapping, climate change. the climate change already supported spread of new pest species, among them a  lot of scale insect species (pseudococcus comstocki (kuwana), pseudococcus viburni (signoret), planococcus citri (risso), planococcus ficus (signoret), etc, which are very  important virus vectors in different crops. these species shows a substantial northward  spread in different parts of europe especially in the last forty years (ben-dov et al.,  2009; kozár, 1997, 1998, 2005; kozár and nagy dávid, 1986; kozár and szentkirályi,  2005; pellizzari, 1991; godinho and franco, 2001; sentenac and kuntzmann, 2003;  boudon-padieu and maixner, 2007; sforza et al, 2005; etc).  the pheromone traps are useful and easy method to study on spread of these pests,  although they collect only males. the use of pheromone traps to monitor scale insect  journal of entomological and acarological research, ser. ii, 42 (3), 2010172 populations requires some basic knowledge about morphology of males, but the identification of scale insect males is a current problem because all descriptions and keys are  based only on females (kosztarab and kozár, 1988). kozár et al. (1996, 1997) prepared  morphological and biological keys for males collected by pheromone traps of diaspidiotus (quadraspidiotus) perniciosus (comstock) because it was found that this pheromone  compounds are not species specific. this result was verified by molecular method by  frey and frey (1995). from this point of view the identification problems of the males of  pl. citri, pl. ficus, and ps. comstocki taxa collected by pheromone traps could be solved  by using more detailed morphological analyses combined with molecular approaches. the aim of this work was (1) to present results of the monitoring of distribution three  mealybug species in different parts of europe, (2) to study the molecular differences of  scale insect males of two mealybug species collected by pheromone traps which can  occur together easily in the same pheromone traps, (3) to compare the sequences of  females and males in case of pl. citri, pl. ficus, and ps. comstocki. material and methods monitoring of mealybugs was conducted by nagykovácsi type tent trap (10x10 cm),  by using (pheromone ingredients for pl. citri; (+)-2,2-dimethyl-3-(1-methylethenyl)  cyclobutanemethanol acetate, for pl. ficus; (s)-lavandulyl senecioate-(s)-lavandulyl  isovalerate), for ps. comstocki; (2,6-dimethyl-l,5-heptadien-3-ol acetate) with soveurode /witasek pflanzenschutz gmbh, austria/ glue and biochemtech /biochemtech ltd.  kishinev, moldavia/ pheromone dispensers.  the traps were used in austria (wien), slovakia (bratislava), serbia (belgrade),  greece (athens, iraklion, and chania), macedonia, and in hungary in 2009 and 2010,  in different times and places (tab 1).  tab 1 number of mealybug males collected by pheromone traps in 2009*. country, locality time period planococcuscitri planococcus ficus pseudococcus comstocki hungary, budapest, ördögárok ú.  04.28-07.31.2009 0 0 0 hungary, budapest, ördögárok ú.  07.31-09.02.2009 8 2 1 hungary, budapest, ördögárok ú.  09.02-10.02.2009 0 0 hungary, budapest, ördögárok ú.  10.02-11.02.2009 3 2 hungary, budapest, m0,csepel 05.18-03.08.2009 0 0 0 hungary, budapest, m0,csepel 03.08-03.09.2009 2 0 0 hungary, budapest, m3, szilas 05.11-07.30.2009 0 0 0 173i. tóbiás et al.: molecular tools for the identification of males of scale insects hungary, budapest, m3, szilas 05.11-07.30.2009 0 0 0 hungary, budapest, m3, ecséd 05.11-07.30.2009 0 0 0 hungary, budapest, m3, ecséd 07.30-09.09.2009 1 0 0 hungary, budapest, m5, kecskemét 05.14-07.28.2009 0 0 0 hungary, budapest, m5, kecskemét 07.28-09.03.2009 0 0 0 hungary, budapest, m5, röszke 05.14-07.28.2009 0 0 0 hungary, budapest, m5, röszke 07.28-09.03.2009 0 0 0 hungary, budapest, m7, budaörs 05.06-07.30.2009 0 0 0 hungary, budapest, m7, budaörs 07.28-08.29.2009 1 0 0 hungary, budapest, m7, velence 05.06-07.24.2009 0 0 0 hungary, budapest, m7, velence 07.08-08.31.2009 2 1 1 hungary, budapest, m7, töreki 05.06-07.25.2009 0 0 0 hungary, budapest, m7, töreki 07.25-10.09.2009 3 1 2 hungary, budapest, m7, letenye 05.06-07.25.2009 0 0 0 hungary, budapest, m7, letenye 07.25-10.09.2009 0 0 hungary, nagykovácsi, around greenhouse 04.28-07.31.2009 22 0 0 hungary, nagykovácsi, around greenhouse 07.31-09.06.2009 95 1 2 hungary, nagykovácsi, in greenhouse 05.10-06.10.2009 8 0 0 austria, wien 04.03-07.29.2009 0 0 0 austria, wien 07.29-08.28.2009 3 0 0 greecex, olympus plaza 09.20-09.26.2009 0 0 greece, kifissia 09.20-09.26.2009 14 0 greece, iraklion 09.20-09.25.2009 48 41! makedonia,  vardar bridge 09.20-09.27.2009 0 0 slovakia, bratislava 07.29-08.28.2009 102 0 0 switzerland, lausane 06.16-06.28.2009 0 0 0 *  between 04.04-04.10, 2010 the traps in serbia (beograd), greece (gevgelija, thessaloniki, olympus plaza,  kifissia, chania), and in hungary (budapest) between 05.01-05.31, 2010, did not collected males of these  three species. journal of entomological and acarological research, ser. ii, 42 (3), 2010174 dna extraction, amplification, cloning and sequencing total genomic dna was extracted from single individuals (males and females)  using redextract-n-ampltm tissue pcr kit (sigma) according to manufacturer  instruction. females and males of p. citri, p. ficus and p. comstocki were tested (tab  2.). after preliminary experiments cas5p8sfc (sense) (5’-gcgaacatcgacaagtcgaacgcacat-3’) and cas28sb1d (antisense) (5’-ttgttttcctccgcttattaatatgcttaa-3’) primer pair  (kim and lee 2008) was selected for pcr. the primers amplified the its 2 sequence  of nuclear dna. pcr was performed using taq dna polymerase (fermentas) in  thermo-cycler (eppendorf mastercycler gradient) according to the following procedure: initial denaturation at 95 oc for 4 min, followed by 40 cycles of 95 oc for 30 sec,  annealing temperature 50 oc for 30 sec, extension at 72 oc for 60 sec; final extension  at 72 oc for 10 min. the pcr products were purified using gel/pcr dna fragments  extraction kit (geneaid). purified pcr products from 3 dry individuals of p. citri from pheromone traps, and three females living on ficus benjamina (budapest) and  3 females of p. comstocki from mass culture on potato (van), and three males from  ethanol from mass culture on potato (van). pl. ficus individuals (2 females and 2 males)  were from mass culture on potato (van). each were cloned into clonejet (fermentas)  vector and inserted into escherichia coli dh5α competent cells. all cloning steps  were based upon standard molecular biology protocols (sambrook et al. 1989). the  recombinant plasmids isolated from selected colonies were sequenced using pjet1.2  forward and reverse primers, the pcr products of two p. citri, and p. comstocki were  sequenced by cas5p8sfc and cas28sb1d primers by an automated dna sequencer  (applied biosystem gene analyzer 3100). sequence comparisons were performed  using wisconsin package version 10.0 genetic computer group (gcg) sequence  analysis software (devereux et al. 1984). results and discussion the pheromone trap catches show that only pl. citri males were collected (tab 1.)  and this species was caught only in greece (kifissia, iraklion) in field conditions. in other  places such as in hungary (nagykovácsi), or slovakia (bratislava) the insects caught on  outdoor plants probable originate from greenhouses plants. in these places, this species  probably could not overwinter, or may be only in mild winter. the males collected by ps. comstocki pheromone traps in greece (crete, iraklion)  belongs to pl. citri according to the molecular data, which was abundant in park on  catalpa sp., ficus sp., morus sp. and other plants, and their males could appear accidentally in ps. comstocki traps.  on the other hand pl. ficus and ps. comstocki were not found in studied places and  times. the one or two males caught by traps in some places probable belong to other  genera and species.  175i. tóbiás et al.: molecular tools for the identification of males of scale insects ta b 2 o ri gi n an d ty pi ng o f m ea ly bu gs s tu di ed h er e. sp ec ie s o ri gi n ty pe v oc he r no . g en eb an k ac ce si on n o. se qu en ci ng p se ud oc oc cu s co m st oc ki tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re fe m al e k oz ar f  n o.  9 25 4 hm 62 85 68 80 1  bp p se ud oc oc cu s co m st oc ki tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re fe m al e k oz ar f  n o.  9 25 4 hm 62 85 69 80 1  bp p se ud oc oc cu s co m st oc ki tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re m al e,  d ry k oz ar f  n o.  9 25 4 hm 62 85 70 80 1  bp p se ud oc oc cu s co m st oc ki tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re m al e,  in  e th an ol k oz ar f  n o.  9 25 4 hm 62 85 71 80 1  bp p se ud oc oc cu s co m st oc ki k or ea fj 43 01 47 13 57  b p p se ud oc oc cu s co m st oc ki fr an ce 09 00 00 67 gu 13 46 68 70 9  bp p se ud oc oc cu s co m st oc ki fr an ce g u1 34 66 9 70 9  bp p s. co m -p c r 1 tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re m al e,  e th an ol k oz ar f  n o.  9 25 4 73 9  bp p s. co m -p c r 2 tu rk ey  v an  u ni ve rs ity  la bo ra to ry  c ul tu re m al e,  d ry k oz ar f  n o.  9 25 4 74 1  bp p la no co cc us c itr i ir ak lio n,  c re te , p .c om st oc ki  fe ro m on e  tr ap m al e,  d ry k oz ar f  n o.  9 46 1 hm 62 85 72 77 3  bp p l.c itr i  p c r 1 h un ga ry m al e,  d ry k oz ar  f . n o  92 03 71 2  bp p l.c itr i p c r 2 h un ga ry m al e,  d ry k oz ar  f . n o  94 62 71 4  bp p la no co cc us c itr i h un ga ry ,  fe m al e k oz ar f  n o.  9 25 5 hm 62 85 73 77 3  bp p la no co cc us c itr i h un ga ry ,  fe m al e k oz ar f  n o.  9 25 5 hm 62 85 74 77 3  bp p la no co cc us c itr i ir ak lio n,  c re te , p .c om st oc ki  fe ro m on e  tr ap m al e,  d ry k oz ar f  n o.  9 46 1 hm 62 85 75 77 3  bp p la no co cc us c itr i h un ga ry ,  fe m al e k oz ar f  n o.  9 20 4 hm 62 85 76 77 3  bp p la no co cc us c itr i k or ea fj 43 01 45 13 32  b p p la no co cc us c itr i fr an ce 08 08 9i nr a gu 13 46 78  6 88  b p p la no co cc us c itr i fr an ce 08 00 2i nr a gu 13 46 75 68 8  bp p la no co cc us m in or fr an ce 08 03 22 3 gu 13 46 76 68 6  bp p la no co cc us fi cu s fr an ce 08 25 58 in ra gu 13 46 77 69 2  bp p la no co cc us fi cu s tu rk ey m al e k oz ar  n o9 58 7/ 1 77 0  bp p la no co cc us fi cu s tu rk ey fe m al e k oz ar  n o9 58 7/ 2 77 0  bp journal of entomological and acarological research, ser. ii, 42 (3), 2010176 characteristics of sequences it was determined that the used dna extraction procedure was suitable to handle  adequate quantity and quality dna from dry males from pheromone traps (6-7 months  kept at laboratory condition), and males from ethanol or from living males and females.  the pcr product size (without the primers) of its2 sequences were 801 bp for ps. comstocki, 773 bp for pl. citri and 770 bp for pl. ficus. the sequence variation among  ps. comstocki males and females were between 0.375 – 0.0 and among pl. citri between  0.26 and 0.00 (see tab 3. and 4.). the amplified its2 sequences were compared with  the similar sequences in the genebank. it is very interesting that ps. comstocki collected  and characterized in korea (1357 bp genebank accession no. fj430147), and france (709  bp genebank accession no.gu134668 and gu134669) showed very high identity with  species collected in turkey (in case of gu134668 and hm628568 there is 100 %) the  same tendency could be observed in the case of pl. citri and pl. ficus. pl. citri individuals  collected and characterized in france, korea or hungary showed 99.9 - 100 % identity.  its2 sequences of pl. ficus individuals also showed 99.9 – 100 % identity regardless of  their origin, but differed from pl. citri (90.4% identity). pl. minor were also included in  the comparison test and showed 99.7 % identity with pl. citri. considerable divergence  was observed between the ps. comstocki and pl. citri (53.3 % – 56.1% identity), which  give possibility to identify and distinguish the males of these species (fig. 1). the very low sequence variation of different individuals regardless their origin and  state (living, dry material or conserved in ethanol) indicate that its-2 molecular marker  is suitable method for reliable characterization and differentiation of pl. citri and ps. comstocki species. it was determined that males from the pheromone traps kept in 6-7  months in laboratory condition were suitable for molecular work. on basis of molecular  data it was possible to separate the males of ps. comstocki and pl. citri. the high divergence for the its 2 region suggest that it should be possible to design species-specific  primers and discriminate ps. comstocki and pl. citri males based on pcr method.  the molecular data could be a very important tool for the verification of the males  collected by pheromone traps, to exclude the accidentally present specimens in the trap,  and other species, for which the pheromone compound could serve as attractant. this  result is especially important in quarantine and ecological studies of distribution of  important pest species in new localities. acknowledgements the authors are grateful to the hungarian fund for research (otka) (no. 75889) for  financial support for this work, as well as to dr. panos milonas and zsuzsanna konczné  benedicty for help in trapping works in greece and in hungary. 177i. tóbiás et al.: molecular tools for the identification of males of scale insects ta b 3 i de nt ity o f i ts 2 se qu en ce s o f p s. c om st oc ki c ol le ct ed fr om k or ea (f j4 30 14 7) , f ra nc e (g u1 34 66 8 an d gu 13 46 69 ) a nd t ur ke y (h m 62 85 68 – hm 62 85 71 ). (p s. co m -p c r 1 k oz ár f . r ef . n o. 9 25 4 an d ps .c om -p c r 2 k oz ár f . r ef . n o. 9 25 4 ar e p c r p ro du ct s of s ep ar at e in di vi du al s) . gu 13 46 68 gu 13 46 69 hm 62 85 68 hm 62 85 69 hm 13 85 70 hm 13 85 71 p s. co m -p c r 1 p s. co m -p c r 2 fj 43 01 47 99 .3 0 99 .4 99 .2 99 .1 99 .1 99 99 .1 98 .9 gu 13 46 68   99 .9 10 0 99 .9 99 .7 99 .9 99 .9 99 .7 gu 13 46 69     99 .9 99 .7 99 .6 99 .7 99 .7 99 .6 hm 62 85 68       99 .8 99 .8 99 .9 99 .9 99 .7 hm 62 85 69         99 .6 99 .8 99 .7 99 .6 hm 13 85 70           99 .6 10 0 99 .6 hm 13 85 71             99 .6 10 0 p s. co m -p c r 1               99 .6 ta b 4 i de nt ity o f i ts 2 se qu en ce s of p l. ci tr i c ol le ct ed fr om k or ea (f j4 30 14 5) , f ra nc e (g u1 34 67 5 an d gu 13 46 78 ) a nd h un ga ry (h m 62 85 72 hm 62 85 75 ), pl . fi cu s an d ps . c om st oc ki ( h m 62 85 69 ), (p l.c itr ip c r 1 k oz ár f . r ef . n o. 9 20 3 an d pl .c itr ip c r 2 k oz ár f . r ef . n o. 9 46 2 ar e p c r p ro du ct s of s ep ar at e in di vi du al s) . gu 13 46 75 gu 13 46 78 hm 62 85 76 hm 62 85 75 hm 62 85 74 hm 62 85 73 hm 62 85 72 gu 13 46 76 gu 13 46 77 pl .ci tri pc r1 pl .ci tri pc r2 pl .fic us t57 9-l 1/1 pl .fic us t5 79 -l 3/1 hm 62 85 69 fj4 30 14 5 10 0 99 ,9 99 ,9 99 ,9 10 0 99 ,9 10 0 99 ,7 90 ,4 10 0 10 0 91 .4 91 .5 55 .6 gu 13 46 75   99 ,9 10 0 10 0 10 0 10 0 10 0 10 0 90 ,4 10 0 10 0 90 ,4 90 ,6 53 .3 gu 13 46 78     99 ,9 99 ,9 99 ,9 99 ,9 99 ,9 99 ,6 90 ,2 99 ,9 99 ,9 90 .3 90 .4 53 .5 hm 62 85 76       10 0 99 ,9 99 ,7 99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,4 91 ,5 56 .0 hm 62 85 75         99 ,9 99 ,7 99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,3 91 ,5 55 .9 hm 62 85 74           99 ,9 10 0 99 ,7 90 ,4 10 0 10 0 91 ,5 91 ,6 56 ,0 hm 62 85 73             99 ,9 99 ,7 90 ,4 10 0 10 0 91 ,4 91 ,5 56 ,0 hm 62 85 72               99 ,7 90 ,4 10 0 10 0 91 ,5 91 ,6 56 .1 gu 13 46 76                 90 ,4 99 ,7 99 ,7 90 ,4 90 ,5 49 .9 gu 13 46 77                   90 ,4 90 ,4 99 ,9 10 0 54 .3 pl .ci tri -p cr 1                     10 0 90 ,7 90 ,9 54 .3 pl .ci tri -p cr 2 90 ,8 90 ,9 54 .4 pl . fi cu s t 57 9l1 /1 99 ,9 57 .1 pl . fi cu s t 57 9l3 /1 57 .1 journal of entomological and acarological research, ser. ii, 42 (3), 2010178 fig. 1 comparison of its 2 sequences of ps. comstocki and pl. citri males. percent similarity: 55.906 percent identity: 55.906 match display thresholds for the alignment(s): | = identity : = 5 . = 1 pcomstockif.seg x planococcuscitria5.seg january 12, 2011 09:56 . . . . . 1 tgcggccctcgtg.accaaagagtcctgggccacgcctgtctgagggtcg 49 ||||||||||| | || ||||||||||||||||||||||||||||||||| 1 tgcggccctcgcgaacaaaagagtcctgggccacgcctgtctgagggtcg 50 . . . . . 50 gtcatacgtgtaacacgatggttgcgttctcgcgagtttcgcgctcgctc 99 || ||||||||||||||||||||||||||||||||| | ||| | 51 gttatacgtgtaacacgatggttgcgttctcgcgagcgcctcgcagtttt 100 . . . . . 100 cgcagcagaccagatcgtgcgcgctcgtcgcgcgcgtgtgtgtgaattca 149 | | || | || | | | | ||| || | 101 caccgcgaggttagctcgccccgtgacagaccagatcgagcgtgtgttta 150 . . . . . 150 cgcacgccgccgag...cgcgcgagagcgagtacgttgcgctttgcggcg 196 ||||| | ||| |||| | ||| || | || | 151 cgcacatgccagagcgacgcggttttcgaatcgcgtcgctcgaagctgac 200 . . . . . 197 acttcgtacgctcgaagctgacgattcgtttgcccgcgtgacttgtcgcg 246 |||| ||| | | | ||| | | ||| || || 201 gactcgttcgcgctgacgcgggccatcgcctccgtgcggtggttagcgtc 250 . . . . . 247 tcgacggcgaacgttcgtcgaaatattgcggttctcgcccgcatcgatag 296 | || ||||||||||| |||||| | | ||| || 251 gtg.cgtcgaacgttcgttgaaatacacggcgccgatgccgagaaacaag 299 . . . . . 297 ttcgttcacgaacaacaacgtgttcgacgaaagcgtgccgcgtgcagcgg 346 |||||||||||||| | | ||| || | ||| | 300 ttcgttcacgaacaccgtgttcgaaaacgttgccgcgtatagtggtagag 349 . . . . . 347 accatatgcgaacgcgatgcggtagatactcggagagtgcgttggccgtc 396 | | | ||| || || ||| || || | || 350 agcgaaggcggtagctatcgtagagagcgtccgatgaacgggcatccaga 399 . . . . . 397 ggatgtacgggcgtcgaaaattctctctcggggcgacggcgggtgacgcg 446 | | | | ||| || | | | |||| 400 gagaggtggtggcgagaagat..............atacggaggatcgcg 435 . . . . . 179i. tóbiás et al.: molecular tools for the identification of males of scale insects 447 cgtgttgtacgatcgcgcgttcc.......aacaccccctcgtcgcttga 489 |||||||||||||||||||| | || | | ||| | 436 cgtgttgtacgatcgcgcgtctccgaaaaaaaaagcttaacgttaccgcc 485 . . . . . 490 gaaaaaaattatacgacgcgaagacggttttctctttacacgccaaccgc 539 | || | |||||| ||||||||| | || || | || | 486 gtcgaatctcatacgatgcgaagacgtctctcgaaatagaggcgatagtt 535 . . . . . 540 gatcgtttcccgatcggactttgagtattttttccgcagttcgcccgcct 589 |||||| | ||| | || || || || || | 536 tgccgtttctctatccacgtacga..........cgttgtacgtgcgatt 575 . . . . . 590 gcacgacgacgttggcgcacgtacgcgcgcgcgttagcgtgtgttcgcgt 639 || || |||| ||| || | || | | ||||| 576 gcgcg....cgttcgcgtcgttattccggccgttgtcgacgaattcgctc 621 . . . . . 640 cgtcattccggccgttgtcgacgaaattcaataattcgaagacggcactc 689 | | | || ||| || | | | || || 622 acgcgtcatgtacggacgcgatgatgacgatcgagaacgggcacacattc 671 . . . . . 690 ggaaggcgatttcgcgactcgcgttacgtgcgacgcacgcgaacgtaccc 739 ||||| ||| | || | | | | ||| | ||| || | 672 ggaagacga.tgcgagcgtgcgataattcgcgttataagcgtactt.... 716 . . . . . 740 gacacacacgcacgcatacgatttcacgccgacctcagatcaggtaagac 789 | || | || | | ||||||||||||||||||||||||||| 717 ..cgtaccaggcttcactctgtatcacgccgacctcagatcaggtaagac 764 . 790 tacccgccgaat 801 ||||||||| 765 tacccgccg... 773 journal of entomological and acarological research, ser. ii, 42 (3), 2010180 references ben-dov y., miller d. r., gibson g. a. p., 2009 - scalenet: a database of the scale insects of  the world. source: http//www.sel.barc.usda.gov/scalenet/scalenet.htm, using - scales in a  region query results.  beuning l., murphy p., wu e., batchelor t., morris b., 1999 - molecular-based approach to  the differentiation of mealybug (hemiptera: pseudococcidae) species. -journal of economic  entomology, 92, 463-472. devereux j., haeberli p., smithis o., 1984 - a comprehensive set of sequence analysis program  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of mealybugs (hemiptera: pseudococcidae) found on korean pears. - journal of economic  entomology, 103, 25-33. pellizzari scaltriti g., 1991 - recent data on italian fauna of homoptera coccoidea. atti xvi  congresso nazionale italiano di entomologia, bari - martina franca (ta) 23/28 settembre  1991, 763-769.  rung a., scheffer s. j., evans g., miller d., 2008 - molecular identification of two closely  related species of mealybugs of the genus planococcus (homoptera: pseudococcidae). -  annals of the entomological society of america, 101, 525-532. sambrook j., fritsch e.f., maniatis t.a., 1989 - molecular cloning: a laboratory manual. - cold  spring harbor laboratory press, cold spring harbor, ny, 1659 pp. sentenac g., kuntzmann p., 2003 - etude des cochenilles et des antagonistes qui leur sont  associés dans des vignobles en bourgogne et en alsace de 2000 à 2002. - iobc wprs  bulletin, 26, 247-252. sforza r., boudon-padieu e., greif c. 2003 - new mealybug species vectoring grapevine  leafroll associated viruses 1 and 3 (glrav-1 and 3). - european journal of plant pathology,  109, 975-981. istván tóbiás - plant protection institute, hungarian academy of sciences, h-1525, p.o.box  102, budapest, hungary. tobias@julia-nki.hu ferenc kozár - plant protection institute, hungarian academy of sciences, h-1525, p.o.box  102, budapest, hungary. bora. m. kaydan - yüzüncü yıl üniversity, agriculture faculty, plant protection department,  65080 campus, van. kinga fetykó - plant protection institute, hungarian academy of sciences, h-1525, p.o.box  102, budapest, hungary. accepted 20 december 2010 j. ent. acar. res..indd s. ignatowicz, k. janczukowicz, p. olejarski integrated pest management (ipm) of the drug store beetle, stegobium paniceum (l.), a serious pest of old books abstract recently (january 2010), we have found that drugstore beetle, stegobium paniceum (l.), is a serious pest of old books in a religious library in cracow, poland. about 80% old books were found more or less damaged by this pest. as the result of larval activity, many holes and tunnels were formed in damaged books. some of them were old, some of them were new tunnels with live larvae and pupae. many characteristic emergence holes of the adults were found in the book bindings. around books there was an enormous amount of dust as a result of larval feeding activity. live and dead adults were found around old books and within the library room, especially near windows. the integrated pest management program was suggested to the owners and curator of the library to control infestation and to avoid the continuation of the damage. key words: drug store beetle, infestation, ipm, old books, stegobium paniceum. introduction museums contain a wide range of organic materials subject to insect attack. beetles belonging to the families anobiidae and dermestidae and moths belonging to the family tineidae are major pests of collections in art and natural history museums. a list of commonly damaged materials in museums and their associated pests is given by schrock (1988). old books are subject of attack by anobiids. in 2004, the common furniture beetle, anobium punctatum (degeer), was found to have caused a very serious infestation of books stored in one of the largest libraries in israel containing about 5 million books. of them, 7% of the rare and valuable books were infested or suspected of being infested and required treatment (wilamowski et al., 2008). recently (january 2010), we have found the similar infestation of old and rare books in a religious library in cracow, poland. however, not the common furniture beetle, but the drugstore beetle, stegobium paniceum (l.), was found as a serious pest of these books. about 80% old books in this library were found more or less damaged by this pest. the paper describes and discusses the damage of old books caused by the drugstore beetle, and lists the suitable treatments within the integrated pest management program j. ent. acarol. res. ser. ii, 43 (2): 177-183 30 september 2011 that were suggested to the owners and curator of the library to control infestation and to avoid the continuation of the damage. materials and methods a religious library in cracow, poland, comprises five big rooms, each with several thousands of books. in three halls there were books that were printed as long ago as the 17th century, and some of them are rare and irreplaceable printings (fig. 1). temperature and humidity were not recorded and controlled in the rooms with the book stacks. an entomological survey was performed in january 2010 aimed to collect developmental stages of the pest, to identify the infestation, and to estimate the extent of the infestation and the damage. information collected was needed to develop the integrated pest management program to control infestation and to avoid the continuation of the damage. results live and dead adults were found around old books, and the library room floor, and especially near a window-sill. larvae and pupae of the pest were found inside the books (fig. 2). all developmental stages of pest were collected, and the pest was identified in a laboratory as the drugstore beetle, stegobium paniceum (l.). fig. 1 old books in the library room. journal of entomological and acarological research, ser. ii, 43 (2), 2011178 a few larvae, their exuviae, and adults of the varied carpet beetle, anthrenus verbasci (l.), and some related species were also collected in the library. however, these beetles do not infest books. they are known to feed upon dead insects (golebiowska & nawrot, 1976), including the drugstore beetles. it was noted that larvae of s. paniceum fed on paper and starch glue as they borrowed in book. at the beginning young larvae produced narrow tunnels. with the time, these tunnels become larger as larvae grow. larvae were found to be specially very active near book binding and/or leather cover of book where they fed on starch glue. after four to six instars, the larvae pupated in large cells located near book binding. finally, many emergence holes of the adults appeared in book bindings. as the result of larval activity, many holes and tunnels were found in damaged books. some of them were old, some of them were new tunnels with live larvae and pupae. tunnels were of varying shape (fig. 3), and several dozens of book pages were fig. 2 larvae of s. paniceum feeding on paper burrow tunnels in book. s. ignatowicz et al.: integrated pest management of the drug store beetle stegobium paniceum usually destroyed when a single tunnel was fully formed. many characteristic emergence holes of the adults were found in the book bindings (fig. 4) when larvae completed their development. around books there was an enormous amount of dust (fig. 5) as a result of larval feeding activity. none of modern books kept in two rooms of the library was damaged by the drugstore beetle. only old books were subject to pest infestation. it was estimated that about 80% old books kept in three rooms of the library were found more or less damaged by this pest. 179 fig. 3 tunnels in the book become larger as larvae grow. fig. 4 emergence holes in book bindings. journal of entomological and acarological research, ser. ii, 43 (2), 2011180 discussion and conclusions the drugstore beetle infests nearly any dry plant or animal material, and it has been reported from stored grain, dry bread, macaroni, black and red pepper, various spices and herbs, botanical collections, drugs, tobacco products, dried fruits and vegetables, textiles, leather, and books (arbogast, 1991; golebiowska & nawrot, 1976). only larvae are responsible for damage of goods, as adults do not feed (lefkovitch, 1967). the drugstore beetle female lays about 75 eggs on the products (arbogast, 1991), such as books, or near them. on hatching, larvae of s. paniceum borrow into the book, feeding on paper as they go. often larvae feed on starch glue near book binding and/ or leather cover of book. after four to six instars, the larvae pupate in large cells near book binding. the damage signs caused to the old books by the drugstore beetle were found to be similar to those done by the common furniture beetle to books in israeli library (wilamowski, 2008). some live adults of the pest were found around old books. most tunnels in damaged books were old, but some of them were new tunnels with live larvae and pupae of the drugstore beetle, indicating the active infestation of books in the library. therefore, the following integrated pest management (ipm) program and recommendations were s. ignatowicz et al.: integrated pest management of the drug store beetle stegobium paniceum fig. 5 dust around books as a result of larval activity. 181 suggested to the owners and curator of the library to control infestation and to avoid the continuation of the damage: 1) keep the library room at the minimum temperature and humidity, and monitor the changes of climatic conditions there. 2) improve insect-tightness of the library room and adjacent rooms. 3) vacuuming the dust with live and dead insects that accumulated on the shelves to stop or limit the population growth of the pest as well the varied carpet beetle, anthrenus verbasci l., and related species that feed upon dead insects. 4) aerosol application (fogging) with dichlorvos (“winylotox 1000”) to reduce the exposed adult populations. 5) install 2-3 insect light traps per each library room. they are effective in controlling adults as beetles positively respond to the uv light. 6) apply the veloxy technique (very low oxygen) or try biological method involving repeated releases of the parasitoid lariophagus distinguendus (f.). 7) after these treatments install and maintain a monitoring system. use pheromone traps for anobiids with stegobinone as a pheromone. 8) damaged books, that are especially valuable, should be restored by a specialist. 9) received books check for any sign of infestation, and refuse them if infested by the pest. treatment of books by freezing to -30°c in order to kill developmental stages of the drugstore beetle (nesheim, 1984) was refused by the owners and curator of the library. they did not agree that the valuable books must leave the library rooms. studies have shown that gaseous chemicals can cause harm to their operators, destroy the environment, and may damage rare antiquities and artifacts (dawson, 1988; florian, 1988). therefore, fumigations with phosphine (ph3) were not suggested as the gas could have unexpected side-effects on ink and paintings within the old books. in museums, the future for fumigation with traditional chemicals seems to be uncertain. references arbogast r.t., 1991 beetles: coleoptera. in: gorham r.j., ecology and management of foodindustry pests. ed. fda technical bulletin, 4: 131-193. dawson j., 1988 the effects of insecticides on museum artifacts and materials. in: zycherman l.a. & schrock j.r. a guide to museum pest control. ed. assoc. of syst. collections, washington d. c.: 135-150. florian m.l., 1988 ethylene oxide fumigation: a literature review of the problems and interac tions with materials and substances in artifacts. in: zycherman l.a. & schrock j.r., a guide to museum pest control. ed. assoc. of syst. collections, washington d. c.: 151-158. golebiowska z., nawrot j., 1976 szkodniki magazynowe [stored product pests]. pwril, warszawa. lefkovitch l.p., 1967 a laboratory study of stegobium paniceum (l.) (coleoptera: anobiidae). j. stored prod. res., 3: 235-249. nesheim k., 1984 the yale non-toxic method of eradication book-eating insects by deep-freezing. restaurator, 1984: 147-164. journal of entomological and acarological research, ser. ii, 43 (2), 2011182 schrock j.r., 1988 list of insect pests by material or apparent damage. in: zycherman l.a. & schrock j.r., 1988 a guide to museum pest control. ed. assoc. of syst. collections, washington d. c.: 113-120. wilamowski a., schnur h., kessler i., navarro s., 2008 anobium punctatum (coleoptera, anobiidae), a new pest of books in israel. iobc/wprs bulletin, 40: 23-28. stanislaw ignatowicz, university of life sciences-sggw, department of applied entomology, ul. nowoursynowska 159, 02-787 warszawa, poland. e-mail: ignatowiczst@yahoo.com krystyna janczukowicz, pest control company “insectum-2”, ul. dobrego pasterza 67, 31-416 krakow, poland. e-mail: biuro@insektum2.krakow.pl pawel olejarski, plant protection institute, ul. w. wegorka 20, 60-318 poznan, poland. e-mail: op68@wp.pl s. ignatowicz et al.: integrated pest management of the drug store beetle stegobium paniceum 183 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract the paper deals with the development, parametrization and validation of a phenology model of the overwintering process of european grapevine moth lobesia botrana (denis & schiffermüller) populations in northern latitudes. the model is built on diapause and poikilothermic population development theories and represents the phenological events of entries into and emergence from pre-diapause, diapause and post-diapause phases. the rate sum models for pre-diapause and post-diapause development are based on published non-linear temperature dependent rate functions. the rate sum model for diapause, however, is negatively affected by the photoperiod during diapause and positively influenced by the photoperiod at the time of diapause entry. the diapause model is parametrized with 3-year data from 25 locations in europe and cyprus, and validated with 1-3 year observations from 18 locations in europe and california. despite restrictive assumptions and limitations imposed by weather data recorded at variable distances from the observation sites, and the variable qualities of observation data, the model’s predictive and explanatory capabilities are useful for adaptive pest management and assessments of the invasive potential. the need for controlled experiments is recognized and suggestions are made for improving the model. introduction the polyphagous european grapevine moth [lobesia botrana (denis & schiffermüller): tortricidae] is found in southern russia, japan, the middle east, the near east, and northern and western africa, and the mediterranean basin where it is considered the most important pest of grapes (savopoulou-soultani et al., 1990; venette et al., 2003; frolov and saulich, 2005; thiéry and moreau, 2005; de yong, 2010). in 2009, l. botrana was discovered in napa county, california (varela et al., 2010), and in their prospective analysis of the invasive potential in california, gutierrez et al. (2012) use the extensive european experimental and modeling literature. however, most of these studies focused on population development during the grape growing season and only a few dealt with aspects of diapause during overwintering (kharazinov et al., 1980; tzanakakis et al., 1988; roditakis and karandinos, 2001; andreadis et al., 2005). annual cycles in resources and unfavorable conditions characterize virtually all biological environments, and according to nechols et al. (1999), insects have developed a set of adaptations that leads to appropriate timing of recurring events in their life cycles. among them is diapause which is a hormonally mediated state of low metabolic activity associated with reduced morphogenesis, increased resistance to environmental extremes, and altered or reduced behavioral activity. diapausing stimuli are perceived only during species-specific, genetically determined life stages in response to token environmental cues that precede unfavorable conditions. the life stages with diapause in the life cycle may be different from those responding to diapausing stimuli. photoperiod and temperature are the most important stimuli. diapause development is mainly, but not exclusively, controlled by a combination of temperature and photoperiod (tauber and tauber, 1976; tauber et al. 1986; nechols et al., 1999). according to leather et al. (1993), one of the major functions of temperature is to maintain the condition by acting as a regulatory factor on the rate of diapause development. in many species, the temperature range over which diapause development occurs is different from that for non-diapause development; in such insects, the low optimum temperatures for diapause development ensure that warm autumn conditions do not result in the resumption of development. according to leather et al. (1993), the length of day at the time of induction has no effect on the maintenance or termination of diapause in many insects, while in others the correspondence: johann baumgärtner, dipartimento di protezione dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (di.p.s.a.), università degli studi di milano, via g. celoria 2, 20133, milano, italy. e-mail: j.baumgaertner@bluewin.ch key words: lobesia botrana phenology, pre-diapause, diapause, post-diapause, rate sum model, first flight. acknowledgments: are grateful to all colleagues listed in the text for providing information on the first flight and for critically reviewing an initial draft of the paper. we are also grateful to an unknown reviewer for corrections and suggestions that clarified the paper. received for publication: 2 march 2012. revision received: 23 march 2012. accepted for publication: 27 march 2012. ©copyright j. baumgärtner et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e2 doi:10.4081/jear.2012.e2 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. a model for the overwintering process of european grapevine moth lobesia botrana (denis & schiffermüller) (lepidoptera, tortricidae) populations j. baumgärtner,1 a.p. gutierrez,2,3 s. pesolillo,4 m. severini4 1dipartimento di protezione dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità (di.p.s.a.), università degli studi di milano, italy; 2division of ecosystem science, university of california, berkeley, ca, usa; 3centre for the analysis of sustainable agricultural systems (casas), kensington, ca, usa; 4università della tuscia, viterbo, italy [page 8] [journal of entomological and acarological research 2012; 44:e2] journal of entomological and acarological research 2012; volume 44:e2 no nco mm er cia l u se on ly conditions prevailing at this point have been shown to affect the duration of the diapause period. many insects appear to have evolved to take advantage of the seasonal progression of photoperiods during winter, and diapause development often involves responses to a series of photoperiods that exert their influence as diapause proceeds rather than to a single critical photoperiod (tauber et al., 1986; leather et al., 1993). the study is a phenology model for overwintering of l. botrana populations across a wide geographical range where the pest may occur. phenology deals with the timing of recurring biological events, the causes of their timing with regard to biotic and abiotic forces, and the interrelation among phases of the same or different species (lieth, 1976). the model should have satisfactory predictive and explanatory capabilities and be useful for further developing the population model (e.g. gutierrez et al. 2012) by explicitly dealing with overwintering in a wide range of latitudes. model development, parametrization and validation rely on the efficient use of available information within the conceptual framework provided by the theory of diapause (leather et al., 1993; nechols et al., 1999) and the rate sum approach to poikilothermic development formalized by stinner et al. (1974) and curry and feldman (1987). materials and methods model description and initialization overwintering process the l. botrana overwintering model starts with diapause induction and represents the development through pre-diapause (j=1), diapause (j=2) and post-diapause (j=3) phases that eventually lead to the emergence and the flight of the adults (figure 1). with a decrease in the length of day during late summer and fall, eggs and larvae respond increasingly to photoperiod and enter the pre-diapause phase. newly formed pupae pass first through diapause followed by a post-diapause phase (gutierrez et al., 2012). of particular interest in this paper are the first individuals (labeled with subscript b) and the last individuals (labeled with subscript e) stimulated to enter the winter diapause on days db and de, respectively (i.e. cohorts 1 and 2). as poikilotherms, they develop at temperature-dependent rates. in late summer and fall, both cohorts become overwintering diapause pupae on day dtb and dte with diapause terminated on days dpb and dpe, respectively. after passing through the post-diapause phase, the two cohorts emerge as adults on days dfb and dfe, respectively. also of interest is the size of the cohorts entering the pre-diapause phase during the period ddb-dde as these produce the flight patterns of adults during the dfb-dfe period. diapause induction riedl (1983) published data on life cycle of cydiapomonella that showed a linear dependence of the critical length of day (dlc) of diapause initiation on latitude l measured in decimal degrees in california. specifically, dlc for 50% of the larvae entering diapause is: dlc = 10.242 + 0.1226 l (1) roditakis and karandinos (2001) working at heraklion with a local l. botrana population showed that the diapause depends on length of day (dl). gutierrez et al. (2012) obtained dl values for the beginning and the ending of the diapause induction (dlb = 14.15 h, and dle = 11.98 h). assuming that riedl’s (1983) equation can also be applied to l. botrana overwintering at different latitudes (l), the length of day for the beginning and the ending of diapause entry (dlb, dle) is: dlb = ab + bb · l dle = ae + be · l (2) to apply (2) to the roditakis and karandinos (2001) data, we have a system of two equations with four unknowns (ab, bb, ae, be). in order to solve the system, we assume from riedl (1983) that bb = be = 0.1226. now, we are able to calculate the numerical values ab = 9.83 and ae = 7.66 and obtain two equations for determining the lengths of day (dlb, dle) at the beginning and the ending of diapause induction: dlb = 9.83 + 0.1226 · l dle = 7.66 + 0.1226 · l (3) once these latitude-dependent day lengths are known, the method of glarner (2010) can be used to calculate the dates ddb and dde, i.e. the latitude-specific julian days on that cohort 1 (ddb) and cohort 2 (dde) enter pre-diapause development. [journal of entomological and acarological research 2012; 44:e2] [page 9] article figure 1. the overwintering phases of l. botrana (pre-diapause, diapause, and post-diapause) for cohorts 1 and 2 responding to length of day on days ddb and dde, entering diapause on days dtb and dte, terminating diapause on days dpb and dpe and emerging as adults on days dfb and dfe. no nco mm er cia l u se on ly [page 10] [journal of entomological and acarological research 2012; 44:e2] overwintering model stinner et al. (1974) and curry and feldman (1987) represent the duration dj of a life stage j by the sum rsj of daily rates rj(d): (4) and state that the stage j is completed once the sum reaches unity (rsj(dj)=1). for poikilotherms, the developmental rates depend on daily temperatures where rj(d)=rj(td) where rj(td) is called rate function of the j-th stage. knowledge of the beginning of phase j, the temperature profile during phase j, and the rate function rj(td) allows the emergence on day dj to be predicted. here, this model is applied to the three overwintering phases of l. botrana (figure 1) and different rate functions are used as if these phases were life-stages. in addition, hourly (tnd) rather than daily temperatures are calculated. for n=24, the rate sum for the pre-diapause (j=1) and post-djapause (j=3) phase becomes: (5) for diapausing pupae, however, the development rate, summed up over 24 time increments per day, is modified by photoperiodic effects and the rate sum for the diapause phase (j=2) becomes: (6) where p0 = photoperiod at the time of entry into diapause (dtb, dte), pd= photoperiod on the d-th day, a and b = constants. as to the analytical form of rj(tnd) in eqs. 2 and 3, gutierrez et al. (2012) used a modified form of the brière and pracros (1998) model to represent article table 1. overwintering model for l. botrana: data set used for parameter estimation. ddb and dde are the calculated beginnings and endings of the entry into the pre-diapause phase; dfb and dfe are the observed beginnings and endings of the first flight; the temperatures for the different weather stations are from yang et al. (2010). region location ddb, dde flight data source with latitude year, station (dfb – dde) sachsen (d) 1 dresden-radebeul/coswig 203, 237 18.04-22.05 mrs. e. harbrecht 51°06’40” 2007-2009 07.05-18.06 sächsisches landesamt für umwelt, altenburg-nobitz* 12.05-22.06 landwirtschaft und geologie 2 dresden-pillnitz 203, 237 16.05-23.06 d 01326 dresden-pillnitz 51°00’31” 2003-2005 28.04-16.06 altenburgnobitz* 12.05-22.06 rheinland-pfalz (d) 3 ahr 202, 237 09.05-11.06 mr. fr.-j. treis 50°27’53” 2001-2003 26.04-03.06 dienstleistungszentrum ländlicher raum, mendig* 17.04-08.05 d 54470 bernkastel-kues 4 bernkastel 202, 238 10.05-22.06 49°54’40” 2001-2003 24.04-03.06 hahn* 17.04-26.05 5 piesport 202, 238 28.04-01.06 49°52’44” 2004-2006 01.05-02.06 hahn* 05.05-11.06 franken (d) 6 altmannsdorf 202, 238 29.04-10.05 http://www.lwg.bayern.de/weinbau/ (sonnenwinkel) 2003-2005 03.05-30.05 rebschutz_lebensraum_weinberg/34270/ 49°56’10” giebelstadt* 04.05-29.05 7 castell (kirchberg) 200, 238 24.04-27.05 49°44’34” 2002-2004 23.04-30.05 illesheim* 03.05-31.05 bratislava (sk) 8 modra-horné 200, 239 15.04-24.06 gabel and mocko (1984) 48°20’34” 1978-1980 29.04-24.06 bratislava* 23.04-25.06 northand 9 wädenswil 199, 241 21.04-18.06 dr. h. hoehn, southeastern 47°13’36” 2000-2002 30.04-08.06 agroscope, switzerland (ch) wädenswil° 10.04-10.06 ch 8820 wädenswil 10 maienfeld (malans) 199, 241 30.04-07.06 47°01’28” 2001-2003 24.04-01.06 chur 28.04-01.06 southern 11 carasso 198, 242 04.04-07.05 dr. m. jermini, agroscope, centro di ricerca switzerland (ch) 46°12’15” 2007-2009 23.04-16.05 di cadenazzo,ch 6594 contone mr. l. colombi, magadino 13.04-22.05 servizio fitosanitario cantonale ch 6500 bellinzona 12 mezzana 198, 242 12.04-09.05 45°51’11” 2007-2009 23.04-19.05 lugano 17.04-07.05 *temperatures corrected for altitude; °if not available, data from zurich. no nco mm er cia l u se on ly the developmental rates of eggs and larvae combined, and of non-diapausing pupae. the same model is applied here for simulating the three overwintering phases: (7) tnd indicates the ambient hourly temperature, and jtl and jtu are the phase-specific lower and upper temperature thresholds, respectively, αj and βj are phase-specific constants, and ξj is a factor changing the developmental rate of the combined egg and larval development (gutierrez et al., 2012) into the pre-diapause phase. the factor ξj is applied to phase j=1 and set to 1 for j≠1. model parametrization information available the beginning (dfb) and the end (dfe) of the first flight at 25 different locations in europe and cyprus were provided by extension services personnel, retrieved from the internet or obtained from the scientific literature (tables 1 and 2). the information consisted of verbal and written communications, published tables or graphics. all the latitudes were obtained from www.google.com using information provided by the data sources. when available, observations over three consecutive years were used (tables 1 and 2). for the overwintering periods of the two cohorts (i.e. ddb, dde), daily maximum and minimum temperature from nearby weather stations were retrieved from yang et al. (2010). the cosine intrapolation [journal of entomological and acarological research 2012; 44:e2] [page 11] article table 2. overwintering model for l. botrana: data set used for parameter estimation. ddb and dde are the calculated beginnings and endings of the entry into the pre-diapause phase; dfb and dfe are observed dates of the beginning and endings of the first flight; the temperatures for the different weather stations are from yang et al. (2010). region location ddb, dde flight data source with latitude years, station (dfb – dde) emilia-romagna (i) 13 carpi 197, 244 06.04-06.05 dr. alda butturini, dr. t. rocco, 44°46’59” 2007-2009 12.04-14.05 servizio fitosanitario regionale, parma° 08.04-16.05 i 40127 bologna 14 san lodovico di rio saliceto 197, 244 08.04-16.05 44°47’59” 2007-2009 06.04-28.05 parma° 09.04-19.05 aquitaine (f) 15 villenave d’ornon 197, 244 03.04-n.a. delbac (2010) 44°46’20” 2007-2009 09.04-n.a. agen 06.04-n.a. puglia (i) 16 ruvo 197, 250 09.04-23.05 moleas (1979) 41°07’06’’ 1976-1978 05.04-18.04 bari* 09.04-23.05 ribatejo (p) 17 lezirão 198, 255 28.03-26.04 gonçalves (1989) 39°14’10” 1985-1987 20.03-24.05 lisboa 27.03-20.05 extremadura (e) 18 san servàn 198, 256 28.03-28.05 martín-vertedor et al. (2010) 38°48’06” 2006, 2007 28.03-30.05 badajoz* attiki (gr) 19 spata 199, 258 29.03-n.a. moschos et al. (2004) 37°57’44” 1996-1998 05.04-n.a. lamia* 28.03-n.a. western and 20 marsala 199, 258 02.05-21.05 prof. gaetano siscaro, university of catania i 95123 catania central sicily (i) 37°47’57” 2008-2010 11.04-07.05 dr. luigi neri, assessorato regionale delle trapani n.a.-03.06 risorse agricole e alimentari i 93013 mazzarino 21 mazzarino 199, 260 05.05-29.05 37°18’20” 2008-2010 18.04-29.05 enna* 25.04-19.05 southeastern sicily (i) 22 ispica 200, 261 23.04-26.04 36°47’08” 2008-2010 29.04-22.05 gela* 26.04-21.05 23 licata 199, 263 16.05-30.06 37°06’08” 2008-2010 21.04-18.05 gela 11.05-28.05 andalucia (e) 24 jerez 200, 261 12.03-01.04 del tio et al. (2001) 36°41’12” 1990-1992 27.03-01.05 jerez# 07.03-28.04 vassilis (2009) limassol (cy) 25 pissouri 203, 268 14.03-9.05 34°40’00” 2005-2006 22.03-3.05 larrnaca* *temperatures corrected for altitude; °if not available, data from bologna; #temperature 1989. available from: http://www.tutiempo.net/en/climate/jerez_de_la_frontera_aeropuerto/84510.htm no nco mm er cia l u se on ly [page 12] [journal of entomological and acarological research 2012; 44:e2] method of bianchi et al. (1990) was used to compute hourly temperatures. at some locations (tables 1 and 2), temperature differences between phenological observation sites and corresponding weather stations were corrected for altitude using an environmental lapse rate of 0.7°c per 100 m, as used by the international civil aviation organization (aguado and burt, 2007). development of larvae stimulated to become diapause pupae based on twice weekly data, gutierrez et al. (2012) estimated that cohorts (e.g. ddb) completed 5/6 of the combined egg and larval development at the time of entry into diapause (dtb), and are presumed to emerge as the first adults on day dfb (figure 1). the same pattern is assumed for other cohorts during the period (ddbdde) (figure 1). this assumption is made because during fall, eggs and young larvae are unlikely to survive pre-diapause development. estimates of dfb and dfe, and on the duration of post-diapause development allows the calculation of dtb and dte. values for the parameters for the rate sum functions of pre-diapause and post-diapause phase obtained from gutierrez et al. (2012) are listed in table 3. diapause development the values for βj are given by gutierrez et al. (2012), while the article table 3. developmental rate parameter estimates for the model on overwintering l. botrana. parameter overwintering phases pre-diapause diapause post-diapause (mature larvae)* (j=1) (pupae) (j=2) (pupae)* (j=3) α�j 0.00225 3.02579e-04 0.00785 β�j 5 1.5 4.5 jtl 8.9 7.1 11.5 jtu 33.0 28.5 33.0 ξ j 6.0 1.0 1.0 a n.a. -3.0258e-06 n.a. b n.a. 2.42064e-04 n.a. *parameters provided by gutierrez et al. (2012); αj, βj and ξj are constants of the basic rate sum function; jtl, jtu are lower and upper temperature thresholds; a and b are constants for the linear latitude correction; n.a., not applicable. table 4. overwintering model for l. botrana: data set used for validation purposes. predictions of the beginning and the ending of the first flight. ddb and dde are the calculated beginnings and endings of the entry into pre-diapause; dfb and dfe are the observed beginnings and endings of the first flight; the temperatures for the different weather stations were obtained from yang et al. (2010). region location ddb, dde flight data source with latitude year, station (dfb – dde) franken (d) 26 hammelburg 202, 238 9.05-30.05 http://www.lwg.bayern.de/weinbau/ 50°06’55” 2005 rebschutz_lebensraum_weinberg/34270/ giebelstadt* rheingau (d) 27 eltville 202, 238 17.04° reineke (2008) 50°01’30” 2008 zweibrücken burgenland (a) 28 rust 200, 240 28.04-15.05 polesny et al.(2000) 47°48’09” 1998 eisenstadt moldava (r) 29 iaşi 199, 241 13.05-08.06 cazacu et al. (2009) 47°09’25” 2007 iaşi western switzerland (ch) 30 begnins 198, 242 9.04-20.06 charmillot et al. (1998) 46°26’31” 1997 genève* 31 venthône* 198, 242 2.04-15.06 46°18’23” 1997 sion* valtellina (i) 32 albosaggia 198, 242 19.04-22.05 pavese (1996) 46°08’55” 1995 sondrio* piemonte (i) 33 ghemme 198, 243 8.05-29.05 45°36’03” 1995 novara* veneto (i) 34 colli goriziano 198, 242 9.05-2.06 zangheri et al. (1987) 45°57’04” 1980 ronchi dei legionari* *temperatures corrected for altitudes; °only beginning of the first flight. no nco mm er cia l u se on ly parameters αj, jtl, jtu, a, b were estimated by simulating the overwintering process for the two cohorts at all the locations and years given in tables 1 and 2. the values for the parameters αj, βj, jtl, jtu a and b are obtained as follows. for varying parameter values, the diapause model, applied to the calculated diapause duration at the different locations (tables 1 and 2) for the two cohorts, yielded different mean rate sums with associated variances. the parameter values producing the smallest coefficient of variation were accepted as model parameter estimates. model validation the intended use of the model has improved understanding of the overwintering process for use in pest emergence forecasting (rykiel, 1996). implicitly, the models representing pre-diapause and post-diapause development have been examined by gutierrez et al. (2012), allowing us to focus here on the diapause process. for model validation, we make use of information on dfb and dfe at 17 different locations in europe and one location in california (tables 4 and 5) and calculate the observed date of diapause termination by means of the post-diapause function described by gutierrez et al. (2012). as in the aforementioned case of model parametrization, the information was provided by extension services personnel, retrieved from the internet or obtained from the scientific literature. likewise, an altitudedependent correction of some data was carried out. results the location-specific days for the beginning (ddb) and the end (dde) of diapause induction are reported in tables 1, 2, 4 and 5. across the latitudes, the earliest entries occur during a small time period delimitated by the central location 40 (ddb=196) and both the northernmost and southernmost locations (ddb=203). the latest entries occur in a longer period extending from the northernmost locations (dde= 237) to the southernmost location (dde= 268). table 3 lists the parameters for the overwintering model. the model for pre-diapause development (j=1) predicts the entry into diapause after about 1-3 weeks after ddb and dde at all 18 locations, depending on temperature (eq. 3). according to figure 2, diapause is terminated in cohort 1 on december 21 at location 42 in sicily and on february 23 at location 27 in germany’s rheingau. cohort 2 terminates diapause between march 19 at location 39 in portugal and on may 27 at location 29 in romania. hence, for cohort 1, the earliest and latest dates of diapause termination are in the southernmost and northernmost regions under study. however, the corresponding extreme dates for diapause termination in cohort 2 are in the westernmost and easternmost european regions under study. figure 2 shows the predicted and observed dates for diapause termination at the 18 locations listed in tables 4 and 5. the average difference between the predicted and the observed diapause duration is [journal of entomological and acarological research 2012; 44:e2] [page 13] article table 5. overwintering model for l. botrana: data set used for validation purposes. predictions of the beginning and the ending of the first flight. ddb and dde are the calculated beginnings and endings of the entry into pre-diapause; dfb and dfe are the observed beginnings and endings of the first flight; the temperatures for the different weather stations were obtained from yang et al. (2010). region location ddb, dde flight data source with latitude year, station (dfb – dde) aquitaine(f) 35 dordogne 198, 243 8.04-1.06 maille (2010) 45°08’49” 2009 bergerac acquitaine (f) 36 pessac 196, 243 28.04-9.06 fargeas (2005) 44°48’14” 2005 bordeaux aquitaine (f) 37 pont de la maye 197, 244 18.04-6.06 roehrich et al. (1976) 44°46’51” 1974 bordeaux lazio (i) 38 cerveteri 197, 249 7.05-2.06 cafarelli and di cicco (1983) 41°59’38” 1981 roma* northwestern 39 arcos de valdevez 197, 249 20.03-8.05 agular et al. (2008) portugal (p) 41°50’50” 1999 pedras rubras* macedonia (gr) 40 kavala 197, 251 20.04-01.06 stavraki et al (1987) 40°56’12” 1985 bitola* (mk) california (usa) 41 napa 198, 257 19.02.-30.05 gutierrez et al. (2012) 38°18’17” 2009 napa sicily (i) 42 camporeale 199, 258 30.04-26.05 prof. gaetano siscaro 37°53’67” 2010 university of catania i 95123 catania palermo* dr. luigi neri assessorato regionale delle risorse agricole e alimentari i 93013 mazzarino 43 noto 200, 261 26.04-21.05 36°53’30” 2010 gela* *temperatures corrected for altitudes. no nco mm er cia l u se on ly [page 14] [journal of entomological and acarological research 2012; 44:e2] 8.3 days for cohort 1 and 21.4 days for cohort 2, respectively. if the observation on day 45 is disregarded, the average difference among the data for cohort 1 is only 6.8 days. accordingly, eq. 3 is better able to predict diapause development in cohort 1 than in cohort 2. in general, the predicted number of days for cohort 1 are slightly higher than the observed number of days, while the corresponding numbers of days for cohort 2 are scattered around the line of correspondence (figure 2). discussion the model is based on the diapause theory which states that development is mainly but not exclusively controlled by a combination of temperature and photoperiod (tauber and tauber, 1976; tauber et al. 1986; nechols et al., 1999). driven by photoperiod and temperature, the model satisfactorily predicts the overwintering of l. botrana under the conditions considered in this study. since satisfactory forecasting on solid theoretical grounds is possible, the model exhibits adequate predictive and explanatory capacities. the favorable qualification of the model is possible in spite of shortcomings in the data used for model parametrization and validation. first, model development relied primarily on information on the beginnings and the endings of the first flight recorded by pheromone traps. the quality of this information, however, is limited, since pheromone trap catches are negatively affected by adverse weather conditions. pheromone trap catches represent activities of males and may, therefore, provide more reliable information on flight beginnings than endings. to some extent, this may explain the difference between the quality of the predictions for cohort 1 and cohort 2 (figure 2). moreover, the pheromone traps were deployed for supervised pest management rather than research purposes and hence, the observations focused on specific periods rather than on the entire flight period. in many cases, the time resolution of the observations was imprecise making it difficult to estimate the beginning and the end of flights. since vineyards are generally set up in environments favorable for grape production, we assume that the temperature experienced by l. botrana is higher than those recorded even after the altitude correction. the quality of the temperature data is further limited by the variable distances between the weather stations and vineyards being monitored. the correction of temperatures is particularly important since it may influence the temperature range for diapause development discussed below. biased temperature data may also explain the deviation of location 42 from the line of corresponding observations and predictions in figure 2. furthermore, the effects of adverse weather on flight activities, the quality of observations and the difference between vineyard and weather station temperatures may have varied through time. this would also jelp explain the difference in the quality of the predictions for the flights of the two cohorts (figure 2). the model has been developed on the basis of restrictive, albeit plausible assumptions. first, we assumed that the response seen in c. pomonella to latitude (riedl, 1983) can be used as a model for l. botrana. next, we assumed that both cohorts consist exclusively of mature larvae that do not suffer from overwintering mortality and successfully pass the pre-diapause, diapause and post-diapause phases. to be able to use the information available for modeling the population phenology, we had to assume that male trap catches were related to population densities. finally, the model for diapause development assumes only additive effects of temperature and photoperiods and disregards possible interactions. the northernmost and southernmost locations considered for model parametrization and validation are dresden-radebeul/coswig at 51°06’40” and pissouri at 34°40’00” (tables 1 and 2). the former location may be at the limit of the distribution in the north (frolov and saulich, 2005; de yong, 2010). portuguese locations in the west and cypriot locations in the east further delimitate the palaearctic area providing information for model parametrization and validation. the literature suggests, however, that the explicit consideration of other environmental factors may be needed when dealing with locations outside this area. for example, the distribution in the palaearctics extends further to the south and towards desert environments than taken into account in this paper (al-zyoud and elmosa, 2001; el-wakeil et al., 2009). for these areas, it may be necessary to build the effect of relative humidity explicitly into the overwintering model. in israel, rakefet et al. (2009) reported a significant effect of cultivars on the numbers of trapped males and a cultivar effect on female host choice. in this case, the explicit consideration of host plant effects may be necessary for obtaining satisfactory forecasts. sciarretta et al. (2008) studied the spatial distribution of pheromone trap catches in mediterranean landscape. since a time-varying part of the population inhabits areas outside vineyards, an explicit consideration of a wide range of plant species and movements between vineyards and their surroundings may improve predictive and explanatory model capabilities. the explanatory capabilities of the model allow us to tentatively assign l. botrana to an insect diapause type characterized by temperature and photoperiod influence on diapause development (leather et al., 1993). in comparison to preand post-diapausing individuals (table 3), the pattern of the diapause development rates occurs in a slightly lower temperature range (the lower threshold β2 is smaller than the thresholds β1 and β3, table 3) and the temperature allowing fastest development is shifted towards lower temperatures. a shift of the temperature range for diapause development has been recorded for other insects and may prevent individuals from resuming development under warm autumn conditions (nechols et al., 1999). the sensitivity to the photoperiod at the time of entry into diapause is known for other insects (leather et al. 1993). to verify these assumptions, to study alternative models for representing photoperiodic and temperature influences, and to explicitly include other environmental factors and ascertain the shift in the temperature response curve, observations from other sites may be useful. to overcome the limitations of observation and temperature data, specific measurements should be to provide more reliable data for model development. article figure 2. validation of the diapause model for l. botrana: observed and predicted days for diapause termination for the first (filled diamonds) and the last (empty triangles) cohort of pupae entering diapause at different geographical locations (the reference day is 1 january, the 45 degree line represents correspondence between observed and predicted values, observed days’ refer to the dates obtained by subtracting post-diapause development from observations on the beginning and the end of the first flight). no nco mm er cia l u se on ly more promising for model improvements, however, are experiments under controlled conditions, possibly complemented with gas exchange measurements (kharizanov et al., 1980). in conclusion, the conceptual framework provided by diapause theory (leather et al., 1993; tauber and tauber, 1976; tauber et al., 1986; nechols et al., 1999) and the rate sum approach to poikilothermic development formalized by stinner et al. (1974) and curry and feldman (1987) allowed efficient use of existing data and yielded a model with satisfactory explanatory and predictive capacities. the predictive capabilities are considered sufficient to allow the model to be used as part of an adaptive vineyard pest management system (rigamonti et al., 2011), while predictive and explicative capabilities are satisfactory for considering the model as a component in the ongoing prospective analysis of the invasive potential of this pest (gutierrez et al., 2012). references aguado e.a., burt j.e., 2007 understanding weather and climate 4th edition. prentice hall. agular a., aubyn st., mexia a., 2008 decision making on the control of the european grape berry moth lobesiabotrana in the ‘vinhosverdes’ region, in the northwest of portugal. international congress mountain steep slope viticulture, 2 march 2008, galicia. available from: http://www. repository.utl.pt/handle/10400.5/1054 accessed: on august 5, 2010. al-zyoud f., elmosa h., 2001 population dynamics of the grape berry moth, lobesia botrana schiff. 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pellizzari & simala, 2007; pellizzari & danzig, 2007; pellizzari & duso, 2009; faccoli et al., 2009; pellizzari et al., 2010). on may 30, 2012 several red-pinkish galls were observed on the leaves of two prunus subhirtella cv. pendula trees (rosaceae) (weeping higan cherry), about 40 years old, growing in the university botanical garden of padua (figure 1). the galls were formed on a portion of the leaf edge, rolled towards the underside and were almost evenly distributed on the tree canopy. at the collecting date, the following morphs were present inside the galls: few nymphs, nymphs with wing pads, alatae fundatrigeniae (emigrants) (figure 2). the species was identified as tuberocephalus (trichosiphoniella) higansakurae hainnevilleae remaudière and sorin, 1993, (hemiptera aphididae), an asiatic heteroecious species known so far in japan and china (su et al., 2010) and not yet recorded in italy. the first incidental introduction to europe of an oriental aphid of the genus tuberocephalus shinji occurred in 1991 in france [remaudière & sorin (1993), these authors identified the species as t. higansakurae (monzen, 1927) and described the new subspecies hainnevilleae remaudiére et sorin]. a few years later the same authors (sorin & remaudière, 1998) pointed out the possibility that t. higansakurae hainnevilleae, could be a synonym of t. (trichosiphoniella) tianmushanensis zang, 1980, due to their similarity. su et al. (2010) compared the types of t. (trichosiphoniella) tianmushanensis with the original description and drawings of t. (trichosiphoniella) higansakurae hainnevilleae by remaudière and sorin (1993) and confirmed the possible synonymy of the two species. the synonymy of the two species is apparently accepted in the aphid species file data base (favret, 2013). according to remaudière and sorin (1993), in autumn 1990, a few thousands of p. subhirtella pendula seedlings were imported from japan to france. in the following spring the trees were strongly infested by aphids forming galls on the leaves. the infested leaves were eliminated and all the young trees treated with an insecticide. an attempt to transfer emigrants to artemisia vulgaris, the secondary host plant, under controlled conditions, in order to obtain alienicolae (exules) failed. no galls were observed on the trees in the following spring, so that it was correspondence: giuseppina pellizzari, dipartimento di agronomia, animali, alimenti, risorse naturali e ambiente (dafnae), università di padova, viale dell’università 16, 35020 legnaro (pd), italy. e-mail: giuseppina.pellizzari@unipd.it key words: tuberocephalus spp, prunus subhirtella cv. pendula, weeping higan cherry, leaf galls, alien insects. acknowledgements: the authors are grateful to prof. s. barbagallo, university of catania, italy, for kindly confirming the identification of the species, for tracing fundamental references and for his valuable remarks and suggestions. many thanks to danièle matile-ferrero, muséum national d'histoire naturelle entomologie, paris, france for her help on providing reprints by g. remaudière and m. sorin. received for publication: 31 july 2013. revision received: 19 november 2013. accepted for publication: 19 november 2013. ©copyright g. pellizzari and g. frigimelica, 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:1855 doi:10.4081/jear.2014.1855 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. first record and establishment of tuberocephalus (trichosiphoniella) tianmushanensis zang, (hemiptera aphididae) on ornamental cherry trees in italy g. pellizzari, g. frigimelica dipartimento di agronomia, animali, alimenti, risorse naturali e ambiente (dafnae), università di padova, italy [page 24] [journal of entomological and acarological research 2014; 46:1855] journal of entomological and acarological research 2014; volume 46:1855 no nco mm er cia l u se on ly assumed that the species was eradicated (remaudière and sorin, 1993; sorin and remaudière, 1998). no other records of tuberocephalus species have been recorded in europe since (coeur d’acier et al., 2010). biological notes on the genus tuberocephalus about twelve tuberocephalus species are known in east asia (japan, korea, china, pakistan, india, malaysia and eastern russia) galling the leaves of prunus trees, the primary host (sorin & remaudière, 1998; blackman & eastop, 1994, 2006; holman, 2009). the secondary host plants, when known, are mostly asteraceae of the genus artemisia. the alienicolae of species belonging to the genus tuberocephalus s. str. colonize the underside of artemisia leaves, whereas species of the subgenus trichosiphoniella colonize artemisia roots or stolons. the life-cycle of the tuberocephalus species is quite similar and is known for some species, taking into account that they all overwinter in the egg stage, near the buds of prunus trees. the fundatrix of t. artemisiae shinij hatches in late march or late april and forms a gall on the leaf. the alatae fundatrigeniae migrate to the secondary host, a. vulgaris var. indica, in june. the alienicolae are apterous and feed on the underside of the leaves. the gynoparae appear in october and migrate to the primary host. males return to the primary host in november (sorin, 1994). t. sakurae (matsumura) has a similar cycle, but the migration to a. vulgaris var. indica lasts from may to august; moreover the apterous alienicolae feed on young shoots in the ground (sorin, 1993). the fundatrix of t. misakurae moritsu & hamasaki hatches in late march. each fundatrix produces many nymphs; apterous fundatrigeniae are rather rare, most of nymphs evolve into alates (moritsu & hamasaki, 1983). in may the alatae fundatrigeniae migrate to the secondary host dendranthema sinensis (=chrysanthemum morifolium). the apterous alienicolae feed on thin lateral roots through summer. gynoparae appear from late october to early december and migrate to the primary host; the males migrate from mid november to early december (sorin & imura, 1996). observations on t. (trichosiphoniella) tianmushanensis zang the primary host of t. (trichosiphoniella) tianmushanensis zang are prunus sp., p. donarium spontanea, p. itosakurae, p. pseudocerasus, p. pendula, p. subhirtella var. pendula, whereas the secondary host are a. vulgaris var. indica and artemisia spp. (sorin & remaudière, 1998; su et al., 2010). according to sorin and remaudière (1998), this species forms two types of galls on prunus leaves. the fundatrix hatches in late march, and produces a bag-shaped gall on the edge of tender leaves. the adult fundatrices appear in may and give birth to apterous and alatae fundatrigeniae. the alatae migrate to the secondary host whereas the apterous fundatrigeniae also produce a tube-like gall, curled and wrinkled, on the leaf edge and give birth to nymphs that become alatae emigrants in late may-june. on artemisia plants they give birth to nymphs that get into the ground and feed on the young shoots of the host plant. the alatae gynoparae emerge from late october to mid november and return to the primary host to produce the oviparous female. the overwintering eggs are laid near the buds. in the botanical garden of padua, the galls of t. (trichosiphoniella) tianmushanensis zang were first detected on may 30, 2012. at this date the most numerous morphs inside the galls were fundatrigeniae nymphs with wing pads and alatae fundatrigeniae (emigrants). during the first week of june the emigrants progressively left the galls and by june 11 all the galls resulted empty. after the first detection of the aphid, investigations were carried out from august to october 2012 on p. subhirtella pendula trees growing in public parks and avenues of the town of padua and in plant nurseries, located in a large area, eastern of the town; occasional observations occurred wherever p. subhirtella pendula trees were traced. besides the botanical garden of padua, galled leaves were detected in a plant nursery at saonara (padua district) on an old tree growing outdoors and on several potted trees grafted with scions taken from the old plant. another infested tree was detected in a garden at rossano veneto (vicenza district), about 40 km far away from the first detected infested trees in padua. as above reported, the secondary hosts of tuberocephalus species, when known, are mostly artemisia plants. in the botanical garden of padua the following species of artemisia are regularly cultivated: a. abrotanum, a. absinthium, a. annua, a. atrata, a. dracunculus, a. pontica, a. vulgaris. in the attempt to find the alienicolae on the secondary host, roots and stolons of the above recorded artemisia species were checked on june 18th, on july 16th and on august 18th without any result. artemisia vulgaris is a very common palaearctic perennial plant that grows on weedy and uncultivated areas, such as waste places, roadsides and riversides. many artemisia vulgaris plants were growing in the surroundings of galled p. hirsutella pendula trees detected in the plant nursery and in other locations. in august samples of a. vulgaris roots and stolons were collected and checked, but no aphids were observed. in [journal of entomological and acarological research 2014; 46:1855] [page 25] article figure 1. galls of tuberocephalus (trichosiphoniella) tianmushanensis on leaf of prunus subhirtella pendula. figure 2. alatae fundatrigeniae (emigrant). no nco mm er cia l u se on ly [page 26] [journal of entomological and acarological research 2014; 46:1855] october 2012 sampling of prunus subhirtella twigs were collected and checked but no overwintering eggs were detected on, whereas several overwintering eggs were definitely found in february 2013. in may 2013 many galled leaves with dark fundatrices inside (figures 3 and 4) were observed again on the same p. subhirtella cv. pendula trees growing both in the botanical garden and in the nursery. these records clearly indicate that many overwintering eggs were laid by oviparous females in the previous autumn and suggest that the aphid is established in the veneto region. references blackman r.l., eastop v.f., 1994 aphids on the world’s trees. an identification and information guide. cab international, wallingford: 986. blackman r.l., eastop v.f., 2006 aphids on the world’s herbaceous plants and shrubs. vol. 1. host lists and keys; vol. 2. the aphids. wiley & sons, ltd, hoboken: 1439. coeur d’acier a., pérez hidalgo n., petrovi-obradovi� o., 2010 chapter 9.2: aphids (hemiptera, aphididae). in: roques a., kenis m., lees d., lopez-vaamonde c., rabitsch w., rasplus j.y., roy d.b. (eds), alien terrestrial arthropods of europe biorisks. vol. 4. pensoft publishers, sofia: 435-474. faccoli m., frigimelica g., mori n., toffolo petrucco e., vettorazzo m., simonato m., 2009 first record of ambrosiodmus (hopkins, 1915) (coleoptera: curculionidae, scolytinae) in europe. zootaxa 2303: 57-60. favret c., 2013 aphid species file. version 5.0/5.0. available from: http://aphid.speciesfile.org accessed: march 2013. holman j., 2009 host plant catalog of aphids. palaearctic region. springer, berlin: 1216. moritsu m., hamasaki s., 1983 a new gall forming aphid of the genus tuberocephalus shinji (homoptera, aphididae) on cherry trees in japan. kontyû, tokyo 51: 221-227. pellizzari g., danzig e., 2007 the bamboo mealybugs balanococcus kwoni n.sp. and palmicultor lumpurensis (takahashi) (hemiptera, pseudococcidae). zootaxa 1583: 65-68. pellizzari g., duso c., 2009 occurrence of stigmaeopsis nanjingensis in europe. bull. insectol. 62: 149-151. pellizzari g., simala m., 2007 first record of aleuroclava guyavae (takahashi, 1932) (hemiptera, aleyrodidae) in europe. boll. zool. agr. bachic. ser. ii 39: 91-95. pellizzari g., cassina g., cappelletti e.m., frigimelica g., 2010 the botanical garden of padua (italy): a reservoir of alien insects and mites, (poster abstract). book of abstracts, 4th global botanic gardens congress, 13th-18th june 2010, dublin, ireland: 132. remaudière g., sorin m., 1993 two new aphids of the genus tuberocephalus shinji (homoptera, aphididae). japan. j. entomol. 61: 683-690. sorin m., 1993 the life cycle of japanese species of tuberocephalus. in: kindlman p., dixon a.f. (eds), critical issues in aphid biology. proc. 4th int. symp. on aphids, ceske budejovice: 123-126. sorin m., 1994 life history of tuberocephalus artemisiae (aphididae, homoptera). spec. bull. trans. essa entomol. soc. 2: 109-112. sorin m., imura h., 1996 life history of tuberocephalus misakurae moritsu et hamasaki (homoptera aphididae). rostria 45: 1-6. sorin m., remaudière g., 1998 a review of the genus tuberocephalus shinji (hemiptera, aphididae) with description of two new species from pakistan and japan. bull. faculty literature kogakkan univ. 37: 91-146. su x.m., jiang l.t., qiao g.x., 2010 tuberocephalus (hemiptera: aphididae) from china with description of a new species. oriental insects 44: 157-191. williams d.j., pellizzari g., 1997 two species of mealybugs (homoptera pseudococcidae) on the roots of aloaceae in greenhouses in england and italy. boll. zool. agr. bachic. ser. ii 29: 157-166. article figure 3. dark fundatrix inside a gall, with yellow nymphs. figure 4. two fundatrices in the same gall, with nymphs. no nco mm er cia l u se on ly layout 1 abstract a colony of the encyrtid wasp psyllaephagus euphyllurae (masi) (hymenoptera, encyrtidae) has been established in the quarantine laboratory at the university of california, riverside, california, usa as part of a classical biological control program against its invasive host, the olive psylla, euphyllura olivina (costa) (hemiptera, psylloidea, liviidae), an important pest of olives in some parts of the world. the colony originators were reared from the same host found on abandoned, commercial olives in catalonia, spain; additional collections were made in murcia. the parasitoid reproduces primarily by thelytoky; however, a few occasional males have been found in the field in spain, but not in colonies reared under quarantine or laboratory conditions. here, the female of p. euphyllurae is redescribed and its male is described and illustrated for the first time; the only previous mention of male p. euphyllurae was from tunisia, reared from the same psyllid host but without any details on its morphology. a lectotype is designated for encyrtus euphyllurae masi. information is given on the results of genetic matching between the two sexes of the parasitoid and also on the presence of the bacterial wolbachia symbiont that apparently is affecting reproduction of this species, including its sex ratio in the field. two species of hyperparasitoids have also emerged from the parasitized olive psylla nymphs from catalonia: numerous specimens of apocharips trapezoidea (hartig) (hymenoptera, figitidae) and one specimen of a pachyneuron sp. (hymenoptera, pteromalidae). introduction the olive psylla, euphyllura olivina (costa) (hemiptera, psylloidea, liviidae), was first reported in california, usa in 2007 infesting olive trees (olea europaea l.) in san diego and orange counties. it has now spread to riverside and los angeles counties and has been found on olive trees at one private residence in monterey county (pickett and rung, unpublished data, 2010). we anticipate it spreading north into commercial production regions of central and northern california. this pest naturally occurs throughout the mediterranean basin, both coastally and inland, and exclusively attacks the flower blossoms and growing tissue of olive (tzanakakis, 2006). the olive psylla is reproductively active during spring months when nymphal populations can cause significant reductions to the olive fruit set. spring infestations have been reported reducing fruit yields by up to 60% in some parts of the mediterranean basin (jardak et al., 1985; tzanakakis, 2006). the only known primary parasitoid attacking this pest in the western mediterranean basin, psyllaephagus euphyllurae (masi) (hymenoptera, encyrtidae) (mercet, 1921; aversenq et al., 2005), has correspondence: serguei v. triapitsyn, department of entomology, university of california, riverside, ca 92521, usa. tel.: +1.(951).827.7817 fax: +1.(951).827.3086. e-mail: serguei@ucr.edu key words: encyrtidae, psyllaephagus euphyllurae, host association, liviidae, olive psylla, euphyllura olivina, classical biological control, taxonomy, spain, apocharips trapezoidea, pachyneuron sp. acknowledgments: the first author thanks his father prof. vladimir a. trjapitzin (moscow, russia) and dr. andré panis (montauroux, france) for providing valuable information on p. euphyllurae, prof. gennaro viggiani (portici, na, italy) for the loan of its type material, mr. vladimir v. berezovskiy (irvine, ca, usa) for mounting the voucher specimens, dr. roger a. burks (university of california at riverside, usa – ucr) for confirmation of the identification of pachyneuron sp. and also for taking two digital images, and mr. ryan perry (ucr) for taking a photograph of the lectotype of encyrtus euphyllurae. the second author thanks dr. thomas a. miller for supervision of the project at ucr and also dr. genet m. tulgetske (ucr) for establishing a colony of the olive psylla in the quarantine. authors also thank drs. juan antonio sánchez and michelangelo la spina (imida, murcia, spain), dr. kim a. hoelmer (usda-ars, newark, de, usa), and ms. marie roche (usda-ars european biological control laboratory, montferrier-sur-lez, montpellier, france) for assistance with collecting. received for publication: 19 may 2014. revision received: 5 july 2014. accepted for publication: 9 july 2014. ©copyright s.v. triapitsyn et al., 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:4092 doi:10.4081/jear.2014.4092 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. description of the male of psyllaephagus euphyllurae (masi) (hymenoptera, encyrtidae), a parasitoid of the olive psylla, euphyllura olivina (costa) (hemiptera, liviidae), with notes on its reproductive traits and hyperparasitoids s.v. triapitsyn,1 j.m.l. jones,1 c.h. pickett,2 m.l. buffington,3 p.f. rugman-jones,1 k.m. daane4 1department of entomology, university of california, riverside, ca; 2biological control program, integrated pest control branch, plant health and pest prevention services, california department of food and agriculture, sacramento, ca; 3systematic entomology laboratory, usda-ars, c/o national museum of natural history, smithsonian institution, washington, dc; 4department of environmental science, policy and management, university of california, berkeley, ca, usa [page 112] [journal of entomological and acarological research 2014; 46:4092] journal of entomological and acarological research 2014; volume 46:4092 jear_2014_3_hrev_master 16/12/14 10:38 pagina 112 no nco mm er cia l u se on ly been collected as part of an importation effort to introduce into california host specific natural enemies to control olive psylla. specimens were collected in may 2013 from both the provinces of catalonia and murcia, spain, and colonies of this species have been established at the university of california, riverside, california, usa (hereafter ucr) quarantine facility. currently cultures of both this parasitoid and olive psylla are being maintained at this laboratory to conduct studies on the host specificity of p. euphyllurae, needed for a field release permit from the united states federal government. our long-term goal will be to establish permanent populations of p. euphyllurae in southern california, before the olive psylla spreads farther north into the major commercial olive production areas. psyllaephagus euphyllurae is primarily a thelytokous endoparasitoid. since the first collections made in 2009, we learned that it is active during spring months; it stops reproducing in july and aestivates as a preadult (inside its host’s mummy) until the following spring (pickett, unpublished data, 2012-2014). as a result, culturing quantities of adults needed for host range testing is difficult or impossible. adequate numbers were obtained by collecting large numbers of parasitized olive psylla nymphs during may 2013 in spain on abandoned, commercial olives; these were then hand-carried to the ucr quarantine. approximately 500 adults were produced for the host range testing studies conducted in 2013. from this large field collection, three males were discovered from the populations of p. euphyllurae in spain. although there is one published report on a male associated with this species in tunisia (ferrière, 1961), there are no published descriptions of the male. herein we provide such a description using both traditional morphological features and molecular genetics. materials and methods parasitized nymphs of e. olivina were brought in may 2013 from spain, under the appropriate federal permit, into the quarantine laboratory at ucr where the emerging parasitoids were then collected. the emerged females of the encyrtid wasp p. euphyllurae were exposed to the different instars of the olive psylla, a colony of which had been established earlier on small olive plants in ucr quarantine under the appropriate state permit. a colony of the parasitoid has been successfully established and maintained ever since; the continuing colony originators all came from the specimens collected in catalonia. all the original parasitoids that emerged in ucr quarantine in the course of this study were preserved in 95% ethanol as voucher specimens (both p. euphyllurae and the hyperparasitoids) and then labeled and deposited in the collection of the entomology research museum, university of california at riverside, california, usa (ucrc) except for some specimens of the hyperparasitoid apocharips trapezoidea (hartig) (hymenoptera, figitidae, charipinae), as indicated in the material examined section for this species, which are deposited in the national museum of natural history, washington, district of columbia, usa (usnm) along with the specimens of a. trapezoidea from france. additional specimens of p. euphyllurae collected in france, greece and spain in 2009, determined by john s. noyes (the natural history museum, london, england, uk), are deposited in the california state collection of arthropods, california department of food and agriculture, sacramento, california, usa (csca). the syntype specimens of p. euphyllurae were received on loan from dipartimento di entomologia e zoologia agraria filippo silvestri, università degli studi di napoli federico ii, portici, napoli, italy (deza). all other identifications were made by s.v. triapitsyn except for that of a. trapezoidea which was made by m.l. buffington. selected specimens were critical point dried from ethanol and then point-mounted, of which one female and one male of p. euphyllurae were dissected and slide-mounted in canada balsam, examined under a zeiss axioskop 2 plus compound microscope using nomarski differential interference contrast optics, and photographed using the auto-montage® system; the photographs were then retouched where necessary using adobe photoshop®. terms used for morphological features are those of gibson (1997). abbreviations used are: f=antennal funicle segment; mps=multiporous plate sensillum or sensilla on the antennal flagellar segments (=longitudinal sensillum or sensilla or sensory ridge(s) of authors). dna extraction, sequencing and assessment of wolbachia infection status. two male (from the field) and eleven female (4 from the field, 7 from the laboratory) p. euphyllurae were killed in 95% ethanol and subjected to individual non-destructive dna extraction using the edna hispex tissue kit (saturn biotech, perth, australia) and the manufacturer’s protocol for extraction from 1 mm3 of tissue. the same methods were also used to extract the dna from a single female pachyneuron sp. for post-extraction, each specimen carcass was retrieved, stored in 70% ethanol, and deposited in ucrc. molecular confirmation that males and females of p. euphyllurae indeed represented the same species was sought by sequencing the ribosomal internal transcribed spacer 2 (its2) gene of one male and two female specimens (denoted by an * in the material examined section for p. euphyllurae below). the its2 was amplified via the polymerase chain reaction (pcr) using the primers 58sf (5’-gtgaactgcaggacacatgaac-3’; porter & collins, 1991) and its4 (5’-tcctccgcttattgatatgc-3’; white et al., 1990). reactions were performed in 25 ml volumes containing, 1x thermopol buffer (new england biolabs, ipswich, ma, usa), 200 mm each dntp, 200 nm each primer, 1 u taq dna polymerase (neb), an additional 1 mm mgcl2, and 2.0 ml dna. the pcr was performed on a mastercycler® gradient thermocycler (eppendorf ag, hauppauge, ny, usa) programmed for an initial 95°c for 3 min; followed by 36 cycles of 94°c for 45 s, 58°c for 45 s and 72°c for 1 min 30 s; and concluding with 3 min at 72°c. amplification was verified by electrophoresis in a 0.9% agarose gel stained with ethidium bromide and resulting pcr products were purified using a geneclean® spin kit (mp biomedicals, llc, santa ana, ca, usa). purified dna was sequenced in both directions at the institute for integrated genome biology, ucr. sequences were aligned and compared by eye in bioedit v. 7.0.5.3 (hall, 1999). resulting sequences were deposited in genbank (benson et al., 2014). males and females were checked for infection with wolbachia using a diagnostic pcr that targets the 16s region of the bacterium (werren & windsor, 2000). pcr was performed in 25 �l reactions containing 2 �l of dna template, 1×thermopol pcr buffer (neb), 200 �m each dntp, 1 u taq polymerase (neb) and 0.8 �m each of the primers w-specf and wspecr (werren &windsor, 2000). the pcr cycling consisted of one cycle of 94°c for 2 min; followed by 40 cycles of 94°c for 30 s, 55°c for 45 s and 72°c for 1 min 30 s; and concluding with 10 min at 72°c. amplification was checked by electrophoresis in a 0.9% agarose gel. wolbachia infection status of each specimen was assigned based on the presence/absence of a 438 bp diagnostic band (werren & windsor, 2000). results and conclusions taxonomy primary parasitoid the primary parasitoid, p. euphyllurae, had been identified using the keys to the palaearctic species of the genus psyllaephagus ashmead by trjapitzin (1967, 1982, 1989), the original description (masi, 1911) and the redescription of the female by mercet (1921), and then compared with the original syntypes of this species. a male of this species from [journal of entomological and acarological research 2014; 46:4092] [page 113] article jear_2014_3_hrev_master 16/12/14 10:38 pagina 113 no nco mm er cia l u se on ly [page 114] [journal of entomological and acarological research 2014; 46:4092] sfax, tunisia, reared from e. olivina, was mentioned by ferrière (1961) who, however, did not provide any other details. therefore, the male of p. euphyllurae is described here for the first time while the female is redescribed and illustrated to facilitate its recognition. psyllaephagus euphyllurae (masi, 1911) figures 1-4. encyrtus euphyllurae masi, 1911: 169-171 (as “encyrtus euphyllurae silv.[estri] in litt.”). original type localities: bevagna, perugia, italy and catania, sicily, italy; of the lectotype designated here: catania. psyllaephagus euphyllurae (masi): mercet, 1921: 700-702 (as “p. euphyllurae “(silvestri)”: redescription, distribution, illustration of female); gahan & waterston, 1926: 375 (as p. euphyllurae “(silv.[estri])”: host association, distribution); ferrière, 1961: 46 (distribution, host), 48 (key); trjapitzin, 1967: 194 (key, distribution, host); trjapitzin, 1982: 398 (key, distribution, host); trjapitzin, 1986: 58 (comparison with p. hyperboreus trjapitzin, type information); trjapitzin, 1989: 262 (key, distribution, host); evans & abd-rabou, 2013: 125 (list, distribution, hosts). type material examined: lectotype female (figure 1) (deza), here designated to avoid any possible confusion regarding the status of the type specimens of this taxon or its identity, labeled: 1. “catania euphyll. 9-vii-909”. the lectotype (mounted on the same card together with 12 other original syntype females) is a laterally-mounted specimen on the left at the apex of the card (figure 1); it is in fair condition, complete. paralectotypes: the aforementioned 12 females; also 10 females (deza) on another card labeled: “catania euphyll. 28-vi-909”. some of the paralectotype specimens are incomplete, lacking either the heads or only the antennae. the species was described from an unspecified number of syntype females. according to trjapitzin (1986), 2 females of p. euphyllurae from sicily labeled “encyrtus euphyllurae silv. catania” were sent to him (as an exchange) by gennaro viggiani from deza to zin (zoological institute, now russian academy of sciences, saint petersburg, russia); these without any doubt also are the original syntypes of this species and thus are designated here as paralectotypes. mercet (1921) mentioned a female specimen (as forma italiana or forma típica) of p. euphyllurae from italy sent to him by filippo silvestri, but without indicating its type status or providing any label details. material examined (all emerged in ucr quarantine, except when indicated otherwise, from parasitized nymphs of e. olivina on olive, olea europaea l.): spain: catalonia: near la jana, 40.49635°n 00.23692°e, 313 m, 13.v.2013, c.h. pickett (emerged 26.v.2013, s&r 1353-03), 3 females. near les cases d’alcanar: 40.56043°n 00.53147°e, 14 m, c.h. pickett: 11.v.2013, 1 female (emerged 21.v.2013, s&r 13-5303); 12.v.2013, 1 female (emerged 26.v.2013, s&r 13-53-03); 13.v.2013, 3 females (emerged 23.v.2013*, 24.v.2013, and 28.v.2013, s&r 13-5303) and 1 male* (same data except emerged 24.v.2013); 40.57948°n 00.55229°e, 11 m, 13.v.2013, c.h. pickett (emerged 31.v.2013, s&r 1353-03), 1 female; 40.56314°n 00.53565°e, 6 m, 8.v.2013, c.h. pickett, k. hoelmer, 2 females (emerged 25.v.2013 and 26.v.2013, s&r 13-53-01). near sant carles de la ràpita: 40.63309°n 00.59722°e, 13 m, 8.v.2013, c.h. pickett, k. hoelmer, 3 females (emerged 23.v.2013*, 27.v.2013, and 29.v.2013, s&r 13-53-01) and 1 male (emerged 30.v.2013 in california department of food and agriculture (cdfa) quarantine laboratory, sacramento, california, usa, coll. c.h. pickett). murcia: near jumilla, 38.39974°n 1.38686°w, 411 m, 10.v.2013, c.h. pickett, j. a. sánchez, 2 females (emerged 26.v.2013, s&r 13-53-02) and 1 male (same data except emerged v.2013 in cdfa quarantine laboratory, coll. c.h. pickett). usa, california, riverside co., riverside, ucr quarantine laboratory: 23.vii.2013, j.m.l. jones, 1 female (first generation progeny from colony on e. olivina on olive), originally from: spain, catalonia, near sant carles de la ràpita, 40.63309°n 0.59722°e, 13 m, 8.v.2013, c.h. pickett, k. hoelmer (emerged 25.v.2013 in ucr quarantine, s&r 13-53-01, coll. by j.m.l. jones); also 1 female (same data except second generation progeny, collected from colony 13.ix.2013). 16.vi.2013, j.m.l. jones, 1 female (first generation progeny from colony on e. olivina on olive), originally from: spain, catalonia, near les cases d’alcanar, 40.56043°n 0.53147°e, 14 m, 11.v.2013, c.h. pickett (emerged 21.v.2013 in ucr quarantine, s&r 13-53-03, coll. by j.m.l. jones); also 1 female (same data except second generation progeny, collected from colony 12.viii.2013), 2 females (same data except third generation progeny, collected from colony 11.ix.2013 and 18.ix.2012), and 1 female (same data except fourth generation progeny, collected from colony 13.x.2013. additional material examined: france, hérault, near puéchabon, 43.71127°n 3.62394°e, 192 m, 14.v.2009, c.h. pickett, k.m. daane (from euphyllura sp. on olive), 2 females. greece, central macedonia, thessaloniki, thermaikos, near epanomi, 40.38841°n 22.96091°e, 20 m, 10.v.2009, c.h. pickett, k.m. daane (from euphyllura sp. on olive), 1 female. spain: catalonia, near amposta and sant carles de la ràpita, 40.66133°n 0.58362°e, 11 m, 5.v.2009, c.h. pickett, k.m. daane (from euphyllura sp. on olive), 2 females. murcia, near jumilla, 38.39973°n 1.38706°w, 410 m, 4.v.2009, c.m. pickett, k.m. daane (from euphyllura sp. on olive), 2 females. redescription (female, specimens from spain): body length 1.2-1.8 mm (critical point-dried specimens). body (figure 2b) mostly dark brown except sometimes gaster brown; head with strong bluish-violet metallic tinge, mesoscutum, scutellum, and first gastral tergite with a strong greenish metallic tinge; scape and pedicel dark brown, flagellum brown; coxae dark brown, femora and tibiae light to dark brown, tarsi light brown to brown. head, dorsal part of pronotum, mesoscutum, axilla, and scutellum with distinct mesh-like sculpture; sculpture on scutellum more reticulate but with smaller cells than that on mesoscutum. pronotum, mesoscutum, axilla, and scutellum with short, dusky setae. propodeum laterally with inconspicuous whitish pubescence. head a little wider than high. ocelli in right triangle. mandible with 1 tooth and a wide truncation; labial palpus 3-segmented, maxillary palpus 4-segmented. antenna (figure 2a) inserted at lower eye margin level. radicle about 0.2x total scape length, rest of scape slender, about 3.7x as long as wide, a little wider closer to apex, with reticulate sculpture. pedicel about 1.6x as long as wide, longer than any funicle segment. funicle segments about as long as wide, f6 the widest; f1 without mps or with article figure 1. psyllaephagus euphyllurae, female (lectotype of encyrtus euphyllurae): habitus (an arrow points to its position on the card). jear_2014_3_hrev_master 16/12/14 10:38 pagina 114 no nco mm er cia l u se on ly 1 mps, f2-f6 each with several short mps. clava 3-segmented, 2.1-2.2x as long as wide and as long as combined length of 3 preceding funicle segments (f4-f6); each claval segment with many mps. mesosoma shorter than metasoma. pronotum very narrow. mesoscutum about 1.5x as wide as long. scutellum slightly wider than long, almost as long as mesoscutum; scutellar placoid sensilla a little closer to posterior margin of scutellum than to its anterior margin and close to each other. wings not abbreviated, fore wing extending far beyond apex of gaster. fore wing (figure 2c) 2.2-2.3x as long as wide; disc hyaline throughout, without an infuscation at base of stigmal vein; postmarginal vein about 0.6x length of stigmal vein; marginal setae very short, longest at most 0.03x maximum wing width. hind wing about 3.6x as long as wide, with disc hyaline; longest marginal seta 0.12-0.13x maximum wing width. mesotibial spur about 0.6x length of mesobasitarsus. ovipositor occupying 0.6-0.7x length of gaster, exserted slightly beyond gastral apex; ovipositor length:metatibia length ratio about 0.6:1. measurements of the type specimens (all air-dried): body length of the lectotype 1.189 mm, of the paralectotypes 1.123-1.486 mm. description (male, specimens from spain): body length about 1.1 mm (one air-dried specimen with a collapsed head). body dark brown to black (head and mesosoma with faint greenish metallic tinge), antenna brown, legs brown to dark brown. head (figure 3a) with vertex in frontal view about 0.5x head width. antenna (figure 3b) with scape minus short radicle about 1.9x as long as wide; pedicel much shorter than any flagellar segment; flagellum with very short setae; all funicle segments longer than wide and more or less subequal in length (f6 slightly shorter), f2 and f3 the widest, each funicle segment with several short mps (all of them apical); clava entire, about 2.7x as long as wide, a little narrower than f1-f4, as wide as f5, and slightly wider than f6, with several short mps (all of them in the middle and at apex). mesosoma a little longer than metasoma (figure 3c). fore wing (figure 4a) about 2.2x as long as wide, with disc hyaline; marginal setae very short as in female. hind wing (figure 4a) hyaline, about 3.3x as long as wide; marginal setae short. mesotibial spur a little more than 0.8x length of mesobasitarsus. genitalia (figure 4b) elongate, occupying about 0.6x length of gaster; digitus with 2 short spines. distribution: egypt (japoshvili & noyes, 2005b), france (panis, 1998; aversenq et al., 2005); greece (stavraki, 1980), italy (masi, 1911), portugal (mercet, 1921), spain (trjapitzin, 1967, 1982, 1989), tunisia (anonymous, 1957; ferrière, 1961; arambourg, 1964), turkey (trjapitzin, 1989; dirlik & öncüer, 1990; öncüer, 1991; trjapitzin & doğanlar, 1997; japoshvili & noyes, 2005a). the previous distribution records of this species from spain by trjapitzin (1967, 1982, 1989), however, did not mention any specimens examined. hosts: euphyllura olivina (costa) (masi, 1911 [as “euphyllura oleae”]; mercet, 1921; anonymous, 1957; ferrière, 1961; arambourg, 1964; chermiti et al., 1987 [as “p. euphyllurae silv.”]), e. phillyreae foerster (dirlik & öncüer, 1990; öncüer, 1991; trjapitzin & do�anlar, 1997), and e. straminea loginova (japoshvili & noyes, 2005b) (hemiptera, liviidae). according to dr. andré panis (panis, personal communication, 2014), in provence, france p. euphyllurae parasitizes e. phillyreae on phillyrea angustifolia l., and in béja and sousse governorates of [journal of entomological and acarological research 2014; 46:4092] [page 115] article figure 2. psyllaephagus euphyllurae, female (catalonia, spain): a) antenna; b) body; c) fore wing. figure 3. psyllaephagus euphyllurae, male (catalonia, spain): a) head (frontal view); b) antenna; c) mesosoma and metasoma. jear_2014_3_hrev_master 16/12/14 10:38 pagina 115 no nco mm er cia l u se on ly [page 116] [journal of entomological and acarological research 2014; 46:4092] tunisia it attacks both e. olivina and e. phillyreae on olive trees. biology: some bio-ecological aspects and developmental characteristics of p. euphyllurae parasitizing e. olivina were investigated by chermiti (1983) and chermiti et al. (1987) who unfortunately did not mention where the studies were conducted in tunisia according to dr. andré panis (panis, personal communication, 2014). hyperparasitoids two species of hyperparasitoids have also emerged from the parasitized olive psylla nymphs collected in catalonia (but none from murcia): numerous specimens of a. trapezoidea and one specimen of pachyneuron sp. (hymenoptera, pteromalidae, pteromalinae). among the 221 wasps that emerged from the samples from catalonia, 86 (ca. 39%) were a. trapezoidea and 134 (ca. 61%) were the primary parasitoid p. euphyllurae. even though we, unfortunately, had no capacity to conduct experimental work in quarantine to prove that a. trapezoidea attacks p. euphyllurae in parasitized nymphs of e. olivina, there is no doubt whatsoever that this is indeed the case because this encyrtid was the only primary parasitoid of the olive psylla found in spain, and species of the charipine genus apocharips fergusson are known to be hyperparasitoids of psyllids through the encyrtidae (ferrer-suay et al., 2013). apocharips trapezoidea (hartig, 1841) figures 5-6. material examined (all emerged in ucr quarantine from parasitized nymphs of e. olivina on olive): spain, catalonia: near les cases d’alcanar: 40.56043°n 00.53147°e, 14 m, 13.v.2013, c.h. pickett (emerged 30.v.2013, s&r 13-53-03), 1 female (usnm) and 1 male (usnm) (same data except collected 12.v.2013); 40.57948°n 00.55229°e, 11 m, 13.v.2013, c.h. pickett (emerged 23.v.2013, s&r 1353-03), 1 female and 1 male (same data except emerged 29.v.2013); 40.56314°n 00.53565°e, 6 m, 8.v.2013, c.h. pickett, k. hoelmer (emerged 28.v.2013, s&r 13-53-01), 1 male. near sant carles de la ràpita: 40.63309°n 00.59722°e, 13 m, 8.v.2013, c.h. pickett, k. hoelmer (emerged 23.v.2013, s&r 13-53-01), 1 female and 1 male (same data except emerged 24.v.2013); near sant carles de la ràpita, 40.59252°n 00.55462°e, 49 m, 8.v.2013, c.h. pickett, k. hoelmer (emerged 31.v.2013, s&r 13-53-01), 1 female. additional material examined: france, hérault, near puéchabon, 43.71127°n 3.62394°e, 192 m, 14.v.2009, c.h. pickett, k.m. daane (from euphyllura sp. on olive), 3 females and 1 male. comments: a female (figure 5a) and a male (figure 6a) of a. trapezoidea are illustrated to facilitate their recognition; the female antenna (figure 5b) has 11 flagellomeres whereas the male antenna (figure 6b) has 12 flagellomeres. previously, silvestri (1915) described (in a footnote, p. 274) a. eleaphila (silvestri) [as “alloxista eleaphila silv.”] from italy as a parasitoid of article figure 4. psyllaephagus euphyllurae, male (catalonia, spain): a) a pair of wings; b) genitalia. figure 6. apocharips trapezoidea, male (catalonia, spain): a) habitus; b) antenna. figure 5. apocharips trapezoidea, female (catalonia, spain): a) habitus; b) antenna. jear_2014_3_hrev_master 16/12/14 10:38 pagina 116 no nco mm er cia l u se on ly e. olivina; this charipine species was recently synonymized under a. trapezoidea by ferrer-suay et al. (2013) who also designated a lectotype of silvestri’s species from specimens collected in vittoria, sicily. based on the condition of the lectotypes of both a. eleaphila and a. peraperta (silvestri) (the latter was described from eritrea simultaneously with a. eleaphila), the fourth author here questions the synonymy of these species with a. trapezoidea. both lectotypes (received on loan from deza with prof. gennaro viggiani’s kind help) are slide-mounted, crudely dissected, and in relatively poor curatorial condition overall. despite these facts, ferrer-suay et al. (2013) provide evidence for their synonymy [based on the shape of the radial cell, proportional (length) of the flagellomeres, and the shape of the propodeal carinae] but ignore the rampant morphological plasticity inherent in hyperparasitoid morphology, especially within charipinae. this phenomenon is readily apparent in the silvestri types, where the relative lengths of flagellomeres are clearly different between two specimens on the same slide, and the marginal cell veins run from parallel to convergent. an additional complication can be found with the propodeal carinae, in that among the type specimens, they are only visible on a single specimen. the host data is another problem here: the only host data for a. trapezoidea prior to the synonymization of the two silvestri species was that of a “psylla sp.” (fergusson, 1986); since very little is known regarding host specificity within apocharips, it’s currently impossible to determine whether the host records reported here for a. trapezoidea do indeed reflect a broad host range for the species, or, in fact, the a. trapezoidea reported here are actually a. eleaphila synonymized by ferrer-suay et al. (2013). while we take no nomenclatural action at the present time, we do suggest additional collecting from the type localities of the silvestri species, confirming host species identifications, and generating contemporary collections for more advanced morphological and molecular diagnostics research. in the end, it is quite possible that a. trapezoidea, here recorded from spain, may turn out to be conspecific with the specimens reared by f. silvestri from the same psyllid host in sicily. pachyneuron sp. material examined: spain, catalonia, near les cases d’alcanar, 40.57948°n 0.55229°e, 11 m, 13.v.2013, c.h. pickett (emerged 29.v.2013 in ucr quarantine from a parasitized nymph of e. olivina on olive, s&r 13-53-03), 1 female. molecular matching of the sexes of psyllaephagus euphyllurae and presence of bacterial wolbachia symbiont the its2 of two females and one male of p. euphyllurae were identical (genbank accession numbers kj747349, kj747350 and kj747351) thus providing a proof of their conspecificity. in our diagnostic pcr, ten of the eleven p. euphyllurae females (both from the field in spain and from the colony at ucr) tested were infected with wolbachia, revealed by the amplification of a pcr product of approximately 438 bp. of the two males tested, one was infected (producing a faint diagnostic pcr product) but the other was uninfected. under laboratory conditions, the parasitoid reproduces exclusively by thelytoky; the role of the few occasional males that have been found in the field in spain is not fully understood. the single female specimen of pachyneuron sp. was also infected with wolbachia. references anonymous, 1957 liste d’identification no 2 (présentée par le secrétatiat du service d’identification des entomophages). entomophaga 2: 313-332. arambourg y., 1964 characteristics of the insect population of the olive tree in the sahel at sfax. ann. inst. nat. rech. agron. tunisie 37: 1-137. aversenq s., gratraud c., pinatel c., 2005 ravageurs et auxiliaires des oliviers. synthèse de trois ans d’observations dans le sudest de la france. phytoma 586: 32-36. benson d.a., cavanaugh m., clark k., karsch-mizrachi i., lipman d.j., ostell j., sayers e.w.m., 2014 genbank. nucl. acids res. 42: d32-d37. chermiti b., 1983 contribution à l’étude bio-écologique du psylle de l’olivier, euphyllura olivina costa (homoptera: psyllidae) et de son endoparasite, psyllaephagus euphyllurae silv. (hymenoptera: encyrtidae). thèse docteur ingénieur, université d’aix-marseille. chermiti b., hawlitzky n., boulay c., onillon j.c., 1987 quelques caractéristiques du développement de l’endoparasite psyllaephagus euphyllurae [hymenoptere (sic), encyrtidae] et exploitation de son hôte euphyllura olivina [homoptere (sic), psyllidae]. entomophaga 31: 351-361. dirlik s., öncüer c., 1990 investigations of the species of the family encyrtidae (hymenoptera) in turkey, their distribution hosts and activity. ege üniv. fen bil. enst. derg. 1: 79-85. evans g.a., abd-rabou s., 2013 an annotated list of the encyrtids of egypt (hymenoptera: chalcidoidea: encyrtidae). acta phytopath. entomol. hung. 48: 107-128. fergusson n.d.m., 1986 charipidae, ibaliidae & figitidae: hymenoptera: cynipoidea. handbooks ident. british ins. 8: 1-55. ferrer-suay m., paretas-martínez j., pujade-villar j., 2013 revision of apocharips fergusson (hymenoptera: figitidae: charipinae) with description of three new species from colombia. zootaxa 3646: 487-500. ferrière c., 1961 encyrtides palearctiques parasites de psylles. entomophaga 6: 39-51. gahan a.b., waterston j., 1926 notes on encyrtidae (hym.chalcidoidea) bred from psyllids, with description of a new species. bull. entomol. res. 16: 373-375. gibson g.a.p., 1997 chapter 2. morphology and terminology. in: gibson g.a.p., huber j.t., woolley j.b., annotated keys to the genera of nearctic chalcidoidea (hymenoptera). nrc research press, ottawa: 16-44. hall t.a., 1999 bioedit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/nt. nucl. acids symp. ser. 41: 95-98. japoshvili g., noyes j.s., 2005a checklist and new data on encyrtidae of transcaucasia and turkey (hymenoptera: chalcidoidea). zoosyst. ross. 14: 135-145. japoshvili g.o., noyes j.s., 2005b new record of encyrtidae (hymenoptera: chalcidoidea). caucasian entomol. bull. 1: 159-160. jardak t., smiri h., moalla m., khalfallah h., 1985 tests to assess the damage caused by the olive psyllid euphyllura olivina costa (homoptera, psyllidae): preliminary data on the harmfulness threshold. in: cavalloro r., crovetti a., integrated pest control in olive-groves. proceedings of the cec/fao/iobc international joint meeting, pisa, 3-6 april 1984, commission of the european communities, a.a. balkema, rotterdam-boston: 270-284. masi l., 1911 contribuzioni alla conoscenza dei calcididi italiani. (parte iv). boll. lab. zool. gen. agr. r. scuola super. agric. portici 5: 140-171. mercet r.g., 1921 fauna ibérica. himenópteros fam. encírtidos. museo nacional de ciencias naturales, madrid: i-xi + 732 pp. öncüer c., 1991 a catalogue of the parasites and predators of insect pests of turkey. ege üniv. zir. fak. yay. 503: i-vi + 354 pp. panis a., 1998 evaluation d’hyménoptères en tant qu’indicateurs de la qualité biologique d’un milieu agricole. cahiers aidec (cahiers de l’association internationale des entretiens écologiques) 36: 55-73. [journal of entomological and acarological research 2014; 46:4092] [page 117] article jear_2014_3_hrev_master 16/12/14 10:38 pagina 117 no nco mm er cia l u se on ly [page 118] [journal of entomological and acarological research 2014; 46:4092] porter c.h., collins f.h., 1991 species-diagnostic differences in a ribosomal dna internal transcribed spacer from the sibling species anopheles freeborni and anopheles hermsi (diptera: culicidae). am. j. trop. med. hyg. 45: 271-279. silvestri f., 1915 contributo alla conoscenza degli insetti dell’ olivo dell’ eritrea e dell’africa meridionale. boll. lab. zool. gen. agr. r. scuola super. agric. portici 9: 240-334. stavraki h.g., 1980 biology of euphyllura sp. (homoptera: psyllidae) in an olive grove in attiki (greece). med. fac. landbouwwetenschappen, rijksuniv. gent 45: 603-611. trjapitzin v.a., 1967 [encyrtids (hymenoptera, encyrtidae) of the maritime territory]. trudy zoologicheskogo instituta, akademiya nauk sssr [proc. zool. inst., leningrad] 41: 173-221. [in russian]. trjapitzin v.a., 1982 key to palaearctic species of the genus psyllaephagus [hym.: encyrtidae]. entomophaga 26: 395-399. trjapitzin v.a., 1986 [new palaearctic species of the genus psyllaephagus ashmead (hymenoptera, encyrtidae)]. trudy zoologicheskogo instituta, akademiya nauk sssr [proc. zool. inst., leningrad] 159: 57-63. [in russian]. trjapitzin v.a., 1989. [parasitic hymenoptera of the fam. encyrtidae of palaearctics]. nauka, leningrad division, leningrad. [in russian]. trjapitzin v.a., doğanlar m., 1997 [a review of encyrtids (hymenoptera, encyrtidae) of turkey]. entomol. obozr. 76: 213222. [in russian]. tzanakakis m.e., 2006 insects and mites feeding on olive: distribution, importance, habits, seasonal development and dormancy. koninklijke brill nv, leiden, the netherlands. werren j.h., windsor d.m., 2000 wolbachia infection frequencies in insects: evidence of a global equilibrium? proc. r. soc. london ser. b 267: 1277-1285. white t.j., burns t., lee s., taylor t.j., 1990 amplification and direct sequencing of fungal ribosomal rna genes for phylogenetics. in: innis m.a., gelfand d.h., sninsky j.j., white t.j., pcr protocols: a guide to methods and applications. academic press, burlington: 315-322. article jear_2014_3_hrev_master 16/12/14 10:38 pagina 118 no nco mm er cia l u se on ly jear2012 journal of entomological and acarological research 2012; volume 44:e5 abstract some vine mealybug, planococcus ficus (signoret) populations in tunisian vineyards have been morphologically and genetically characterized. the morphological examination was based on the main distinctive characteristics of species of planococcus, namely the number and distribution of the multilocular disc pores and tubular ducts on the adult female. this showed the existence of two different vine mealybug populations in tunisia. likewise, in the molecular analyses, two separate clades were revealed in the neighbour-joining phylogenetic tree, supporting the morphological studies and suggesting that there are two distinct populations of p. ficus on grapevine in tunisia. introduction the coccoidea (hemiptera: sternorrhyncha) contains nearly 8000 species of plant-feeding scale insects in up to 32 families (gullan and cook, 2007). the most common families are those with the most species, namely the diaspididae, pseudococcidae and coccidae (kondo et al., 2008). pseudococcidae (mealybugs) is the second most speciesrich family with 2291 species described (ben-dov et al., 2011). it is found worldwide and is considered the most economically important scale insect group infesting grapevine (vitis vinifera l.) (dalla montà et al., 2001; daane et al., 2006; walton et al., 2009; charles et al., 2010). more specifically, planococcus ficus (signoret) is considered to be the most serious mealybug threat to grapevine in many grape-growing areas around the world, e.g. italy, iran, california (usa), argentina and south africa (trjapitzin and trjapitzin, 1999; dalla montà et al., 2001; daane et al., 2006; moghaddam, 2006; walton et al., 2009). due to its phloem-feeding habit, p. ficus eliminates honeydew, which supports the growth of sooty mold fungi on leaf and fruit surfaces, thus inhibiting photosynthesis. furthermore, p. ficus has been shown to transmit grapevine viruses (mahfoudhi et al., 2009; tsai et al., 2010). mealybugs are often impossible to identify in the field and have, therefore, been preserved in alcohol and then mounted on microscope slides for identification (williams and watson, 1988). some species are morphologically very similar (e.g. p. ficus and p. citri, risso) and identification using morphological characteristics can be difficult and/or inaccurate. for these reasons, molecular studies have recently been introduced to better discriminate and characterize some closely related mealybug species (cavalieri et al., 2008; hardy et al., 2008; malausa et al., 2011). such new molecular tools can complement and confirm the morphological studies. in tunisia, p. ficus and p. citri have been shown to coexist on grapevines within the same vineyard (mansour et al., 2009) and p. ficus is now considered to be the most common mealybug species in vineyards in tunisia (mansour et al., 2011). however, there is still no published information on combined morphological and molecular studies of vine mealybug populations from tunisia. in this context, the present study was undertaken to characterize some vine mealybug populations to be found in tunisia to verify whether morphologically and genetically different populations of this insect occuring in some tunisian grape-growing areas. materials and methods sampling of mealybugs sampling was carried out during the summer 2009 in 11 tablegrape vineyards belonging to 8 grape growing sites (table 1). most of the vineyards investigated were planted with the variety muscat of italy, a variety that is pergola trained and drip irrigated. adult females were collected mainly from vine trunks and then preserved correspondence: ramzi mansour, department of plant protection and postharvest diseases, laboratory of entomology, national agronomic institute of tunisia, university of carthage, 43 charles nicolle avenue, 1082 cité mahrajène, tunis, tunisia. e-mail: ramzi_mansour82@yahoo.co.uk key words: planococcus ficus, morphological variation, genetic diversity, vitis vinifera, mediterranean basin. acknowledgements: we would like to thank everyone from the crda, ctv and cra (tunisian ministry of agriculture and environment) and grapegrowers in the sites investigated in this study for their valuable collaboration. r.m. received a study grant from the averroes program (erasmus mundus, european commission). received for publication: 20 december 2011. revision received: 20 march 2012. accepted for publication: 18 april 2012. ©copyright r. mansour et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e5 doi:10.4081/jear.2012.e5 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. a morphological and molecular characterization of vine mealybug populations (hemiptera, pseudococcidae) from tunisia r. mansour,1,2 v. cavalieri,1 g. mazzeo,1 k. grissa lebdi,2 a. russo1 1dipartimento di gestione dei sistemi agroalimentari e ambientali, sezione entomologia agraria, università degli studi di catania, italy; 2department of plant protection and post-harvest diseases, laboratory of entomology, national agronomic institute of tunisia, university of carthage, tunis, tunisia [page 24] [journal of entomological and acarological research 2012; 44:e5] no nco mm er cia l u se on ly in small vials containing 70% ethanol until morphological or molecular characterization was carried out. morphological characterization the mealybugs were prepared on microscope slides according to the method described by williams and watson (1988). for each sample vineyard, 20 slide mounted specimens (20 replicates) were selected for morphological characterization. the identification of mealybug species was performed using the keys of tranfaglia and tremblay (1982), cox (1989) and williams and granara de willink (1992). the main characteristics studied were those for discriminating between species in the genus planococcus ferris (tranfaglia and tremblay, 1982; cox, 1989; williams and granara de willink, 1992): the total number of multilocular disc pores on pro-, mesoand metathorax, and the number of tubular ducts on the head and near cerarii viii (counting from the head). thus, for each site, the population was characterized by the mean (n=20) of each of the five characteristics mentioned above in order to highlight any morphological differences between mealybug populations belonging to different grape-growing sites. to compare the same characteristic between the different sites, data were analyzed using one-way anova and the means separated using the least significant difference (lsd)-test at p=0.05. all statistical analyses were performed using statistica 7.0 (statsoft inc., 2004). molecular characterization single mealybug individuals were subjected to total dna extraction using chelex 100 resin (biorad), following the protocol described by walsh et al. (1991) and modified by de barro and driver (1997). each sample was homogenized in 40 μl chelex 5% and then incubated at 54°c for 15 min and at 98.8°c for 7 min, before being cooled at -20°c for 5 min and then centrifuged at 13,200 rpm for 10 min. the supernatant containing dna was recovered and was stored at -20°c to be used for polymerase chain reaction (pcr) assay. the relationship between different populations of p. ficus was investigated by amplification and sequencing of ribosomal its1 region (internal transcribed spacer 1). dna amplification was conducted using tw81 forward primer: 5’-gtttccgtaggtgaacctgc-3’ (brust et al., 1998) and reverse primer 5.8r 5’-atccgcgagccgagtgatcc-3’ (de barro et al., 2000). pcr reactions were conducted in 21 μl volumes with 8.5 μl buffer premix 2x f (failsafe™ pcr premix selection kit, epicentre technologies), 1 μl of each primer (10 μm), 0.5 μl taq polymerase (invitrogen) and 2 μl dna template. the cycle parameters were: 94°c for 5 min, then 30 cycles at 94°c for 1 min, 52°c for 1 min, 72°c for 1.5 min. pcr products were run in 1.6% agarose gel and highlighted with sybr® safe dna gel stain (invitrogen). pcr products of approximately 500 bp were sequenced by bmr genomics sequencing service (padua, italy) and analyzed through neighbor-joining (saitou and nei, 1987) and tamura-nei distances as implemented in mega5 (tamura et al., 2011). the citrus mealybug p. citri was used as the outgroup. results and discussion statistical analyses did not reveal any differences between vine mealybug populations in either the number of tubular ducts on head (f5, 114=1.22; p=0.3) or near cerarii viii (f5, 114=1.95; p=0.08) (table 2). by contrast, significant differences were found in the total number of multilocular disc pores on the prothorax (f5, 114=4.26; p=0.01), on the mesothorax (f5, 114 = 5.41; p=0.0001) and on the metathorax (f5, 114=10.74; p<0.0001). overall, the number of multilocular disc pores on p. ficus was significantly lower in populations article [journal of entomological and acarological research 2012; 44:e5] [page 25] table 1. tunisian table-grape growing areas sampled (summer 2009). grape-growing geographic n sampling site coordinates vineyards date mornag 36°41’7.04”n, 10°17’17.01”e 3 16/6; 2/7; 28/8 takelsa 36°47’24”n, 10°37’48”e 1 27/6 le kef 36°10’45.38”n, 8°42’50.9”e 1 30/6 sidi thabet 36°54’50”n, 10°2’10”e 1 22/7 rafraf 37°11’50.53”n, 10°10’43.43”e 2 7/7 testour 36°33’17.38”n, 9°26’41.49”e 1 11/6 bousalem 36°36’0”n, 8°41’60”e 1 11/6 table 2. mean number of tubular ducts on adult female vine mealybugs (n = 20) from six vine growing areas in tunisia. grape-growing mean n. (±se) of mean n. (±se) of site tubular ducts tubular ducts near on the head cerarii viii le kef (nw) 0.15±0.08 1.5±0.32 mornag (ne) 0.2±0.09 0.75±0.26 takelsa (ne) 0.15±0.08 0.8±0.22 testour (nw) 0.3±0.10 1.7±0.40 rafraf (en) 0.45±0.20 1.45±0.28 bousalem (nw) 0.1±0.07 0.85±0.27 within columns, there is no significant difference in the means at 5% level of significance (p>0.05). nw, north-west tunisia; ne, north-east tunisia; en, extreme north of tunisia. se, standard error. table 3. mean number of multilocular disc pores on adult female vine mealybugs (n=20) from six vine growing areas in tunisia. grape-growing mean n. mean n. mean n. site (±se) of (±se) of (±se) of multilocular multilocular multilocular disc pores on disc pores on disc pores on prothorax mesothorax metathorax le kef (nw) 4.5±0.55a 0.9±0.23a 1.95±0.37a mornag (ne) 5.05±0.89ab 1.8±0.37ab 3.7±0.67a takelsa (ne) 6.75±0.59bc 4.5±0.83d 6.7±0.7b testour (nw) 7±0.68c 3.4±0.61cd 8.15±0.94b rafraf(en) 7.85±0.65c 2.9±0.49bc 7±0.59b bousalem (nw) 7.9±0.71c 2.7±0.45bc 6.35±0.83b within columns, there is no significant difference in means followed by the same letter at 5% level of significance (lsd test). nw, north-west tunisia; ne, north-east tunisia; en, extreme north of tunisia. se, standard error. figure 1. molecular characterization. neighbour-joining phylogenetic tree of vine mealybug planococcus ficus populations from six vine areas in tunisia. p. citri, was used as the outgroup. no nco mm er cia l u se on ly from the le kef and mornag sites compared to populations from the sites at takelsa, testour, rafraf and bousalem (table 3). more specifically, the most significant difference between these populations were found in the number of multilocular disc pores on the metathorax (table 3). these morphological results were supported by our molecular analysis. the neighbour-joining phylogenetic tree (figure 1) shows that the p. ficus populations are clearly separated into two clades, suggesting that p. ficus is comprised of two genetically different populations on grapevine in tunisia. indeed, the mealybugs from the sites of le kef and mornag (the pairwise genetic distances between these populations and the other four populations ranged from 0.005 to 0.023) were genetically different from populations collected from the sites at rafraf, takelsa, sidi thabet and testour (pairwise genetic distances between these populations ranged from 0 to 0.002) (table 4). into this clade the genetic distances of the four population are very low. using the internal transcribed spacer 1 (its1) region, no genetic differences were found between the rafraf and testour populations or between the takelsa and sidi thabet populations, respectively. further studies are needed to confirm these results and facilitate recognition of the vine mealybug populations in tunisia. these results sugest two possible hypotheses. first, that the populations from the mornag and le kef sites are f1 hybrids from either the cross p. ficus females x p. citri males or the cross p. citri females x p. ficus males. in fact, to support this possible hypothesis, both male and female p. ficus and p. citri have been recorded from the same vineyard in tunisia (mansour, 2008; mansour et al., 2009). if such hybrids were present on grapevines, then identification based solely on morphological characters would probably be difficult and might inaccurate identification, making it necessary to carry out molecular analyses for more accurate discrimination (characterization). consequently, if further study does confirm the presence of two distinct morphs, then combining morphological and molecular analyses could be important. the second hypothesis might be that the two populations of p. ficus represent two different biotypes. in this context, performing biological crosses between males and females from each population would be useful to determine whether or not they are reproductively incompatible. understanding how these morphological and molecular differences in p. ficus populations could influence bioecological traits is clearly important with regard to integrated pest management. furthermore, differences in their ability to transmit grapevine viruses need to be evaluated. further studies should be focused on carefully investigating other vine mealybug populations from vineyards throughout the mediterranean basin, as this is the area of origin of this pseudococcid species. these would widen our understanding of the phylogenetic (evolutionary) relationships among vine mealybug populations from different mediterranean countries. references ben dov y., miller d.r., gibson g.a.b., 2011 scalenet. a database of the scale insects of the world. available from: http://www. sel.barc.usda.gov/scalecgi/chklist.exe?family=pseudococcidae&g enus= brust r.a., ballard j.w.o., driver f., hartley d.m., galway n.j., curran j., 1998 molecular systematic, morphological analysis and hybrid crossing identity a third taxon, aedes (halaedes) australis species-group (diptera: culicidae). can. j. zoolog. 76: 1236-1246. cavalieri v., mazzeo g., tropea garzia g., buonocore e., russo a., 2008 identification of planococcus ficus and planococcus citri (hemiptera: pseudococcidae) by pcr-rflp of coi gene. zootaxa 1816: 65-68. charles j.g., bell v.a., lo p.l., cole l.m., chhagan a., 2010 mealybugs (hemiptera: pseudococcidae) and their natural enemies in new zealand vineyards from 1993-2009. new zeal. entomol. 33: 84-91. cox j.m., 1989 the mealybug genus planococcus (homoptera: pseudococcidae). bull. br. mus. (nat. hist.) entomol. 58: 1-78. daane k.m., bentley w.j., walton v.m., malakar-kuenen r., yokota g.y., millar j.g., et al., 2006 new controls investigated for vine mealybug. calif. agr. 60: 31-38. dalla montà l., duso c., malagnini v., 2001 current status of scale insects (hemiptera: coccoidea) in the italian vineyards. boll. zool. agrar. bachic. 33: 343-350. de barro j.p., driver f., 1997 use of rapd pcr to distinguish the b biotype from others biotypes of bemisia tabaci (gennadius) (hemiptera: aleyrodidae). aust. j. entomol. 36: 149-152. de barro j.p., driver f., trueman j.w.h., curran j., 2000 phylogenetic relationship of world population of bemisia tabaci (gennadius) using ribosomal its1. mol. phylogenet. evol. 16: 29-36. gullan p.j., cook l.g., 2007 phylogeny and higher classification of the scale insects (hemiptera: sternorrhyncha: coccoidea). zootaxa 1668: 413-425. hardy n.b., gullan p.j., hodgson c.j., 2008 a subfamily-level classification of mealybugs (hemiptera: pseudococcidae) based on integrated molecular and morphological data. syst. entomol. 33: 51-71. kondo t., gullan p.j., williams d.j., 2008 coccidology: the study of scale insects (hemiptera: sternorrhyncha: coccoidea). rev. cor. cienc. tecnol. agrop. 9: 55-61. mahfoudhi n., digiaro m., dhouibi m.h., 2009 transmission of grapevine leafroll viruses by planococcus ficus (hemiptera: pseudococcidae) and ceroplastes rusci (hemiptera: coccidae). plant dis. 93: 999-1002. article [page 26] [journal of entomological and acarological research 2012; 44:e5] table 4. pairwise genetic distances between planococcus ficus populations belonging to different sites. the citrus mealybug planococcus citri was used as the outgroup. specimens p. citri p. ficus p. ficus p. ficus p. ficus p. ficus p. ficus (italy) (le kef) (mornag) (sidi thabet) (takelsa) (rafraf) (testour) p. citri (italy) 0.000 0.011 0.012 0.010 0.010 0.010 0.010 p. ficus (le kef) 0.048 0.000 0.007 0.003 0.003 0.004 0.004 p. ficus (mornag) 0.061 0.021 0.000 0.007 0.007 0.007 0.007 p. ficus (sidi thabet) 0.043 0.005 0.021 0.000 0.000 0.002 0.002 p. ficus (takelsa) 0.043 0.005 0.021 0.000 0.000 0.002 0.002 p. ficus (rafraf) 0.046 0.007 0.023 0.002 0.002 0.000 0.000 p. ficus (testour) 0.046 0.007 0.023 0.002 0.002 0.000 0.000 no nco mm er cia l u se on ly article malausa t., fenis a., warot s., germain j-f., ris n., prado e., botton m., vanlerberghe-masutti f., sforza r., cruaud c., couloux a., kreiter p., 2011 dna markers to disentangle complexes of cryptic taxa in mealybugs (hemiptera: pseudococcidae). j. appl. entomol. 135: 142-155. mansour r., 2008 etude des cochenilles farineuses planococcus citri (risso) et planococcus ficus (signoret) en viticulture: systématique, biologie et lutte intégrée. ms thesis in biological control and integrated pest management. national agronomic institute of tunisia, university of carthage, tunis, tunisia. mansour r., grissa lebdi k., la torre i., zappalà l., russo a., 2009 preliminary study on mealybugs in two vineyards of the cap-bon region (tunisia). tunisian j. plant. protec. 4:185-196. mansour r., mazzeo g., la pergola a., grissa lebdi k., russo a., 2011 a survey of scale insects (hemiptera: coccoidea) and tending ants in tunisian vineyards. j. plant. protec. res. 51: 197203. moghaddam m., 2006 the mealybugs of southern iran (hem.: coccoidea: pseudococcidae). j. entomol. soc. iran 26: 1-11. saitou n., nei m., 1987 the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol. biol. evol. 4: 406-425. statsoft inc., 2004 statistica (data analysis software system) version 7. statsoft, inc., tulsa, oklahoma, usa. tamura k., peterson d., peterson n., stecher g., nei m., kumar s., 2011 mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. mol. biol. evol. 28: 2731-2739. tranfaglia a., tremblay e., 1982 a morphological comparison between planococcus citri (ris.), planococcus ficus (sign.) and their f1 hybrids. entomotaxonomia 4: 1-5. trjapitzin s.v., trjapitzin v.a., 1999 parasites of mealybugs (homoptera, pseudococcidae) on cultivated grapes in argentina, with description of a new species of the genus aenasius walker (hymenoptera, encyrtidae). entomol. rev. 79: 386-390. tsai c.w., rowhani a., golino d.a., daane k.m., almeida r.p.p., 2010 mealybug transmission of grapevine leafroll viruses: an analysis of virus-vector specificity. phytopathology 100: 830-834. walsh p.s., metzquer d.a., higuchi r., 1991 chelex 100 as a medium for single extraction of dna for pcr-based typing from forensic material. biotechniques 10: 506-512. walton v.m., krüger k., saccaggi d.l., millar i.m., 2009 a survey of scale insects (sternorryncha: coccoidea) occurring on table grapes in south africa. j. insect sci. 9: 47. williams d.j., watson g.w., 1988 the scale insects of the tropical south pacific region, part 2: the mealybugs (pseudococcidae). cab international institute of entomology, london, u.k. williams d.j., granara de willink m.c., 1992 mealybugs of central and south america, second part. cab international institute of entomology, london, u.k. [journal of entomological and acarological research 2012; 44:e5] [page 27] no nco mm er cia l u se on ly j. ent. acar. res..indd k. murugan, c. vasugi combined effect of azadirachta indica and the entomopathogenic nematode steinernema glaseri against subterranean termite, reticulitermes flavipes abstract laboratory study has been conducted on the bioactivities of entomopathogenic nematodes and neem seed kernel extract (nske) against worker termites of reticulitermes flavipes. neem at various concentrations did not affect the survivability of nematodes, whereas neem had considerable impact on the survivability of worker termites and this may be due to the presence of active neem compounds (azadirachtin, salanin etc.). mortality was 40% on 4th day at lower concentration of 1.0% nske treatment; whereas mortality has been increased to 70% at higher concentration (4.0%) on 4th day. there was 100% mortality after the combined treatment with 4.0% nske + 600 infective juvenile steinernema glaseri, even at the first day of the experiment. in the present experiment, neem extract does not affected the survival of the nematodes. hence, nematode and neem extract can be used for soil-insect control particularly for the subterranean termites. key words: infectivity, neem, reticulitermes flavipes, steinernema glaseri. introduction subterranean termites are economically important pests of dwellings and other human structures where they feed on wood. because of human health and environmental concerns, organochlorine and organophosphate insecticides are not used in dwellings. accordingly, there has been great interest in finding alternative biological approaches to control termites (grace, 1997). in addition, more environmentally friendly insecticides such as imidacloprid are being employed for termite control (shelton & grace, 2003). however, imidacloprid has a delayed mode of action and the termites continue to feed. steinernematids and heterorhabditids are obligate insect parasites (poinar, 1979) with associated bacterial symbionts, xenorhabdus spp. and photorhabdus spp., respectively (akhurst & boemare, 1990). the infective juvenile (ij) stage of the nematode remains in the soil until it can invade the body of the susceptible insect after infection, release symbiotic bacteria into the insect hemocoel causing septicemia and death (kaya & gaugler, 1993). entomopathogenic nematodes have been tried against termites, but a high concentration of 400 nematodes / termite is needed to get some degree of control (wang et al., 2003). hence, the present paper is to study the control of the subterranean termite, reticulitermes flavipes (kollar, 1837), a major pest of dwellings in urban j. ent. acarol. res. ser. ii, 43 (2): 253-259 30 september 2011 and suburban areas of tamil nadu using a combination of biological control agents (entomopathogenic nematodes) and environmentally friendly (neem) insecticides. the principles learned from this research can be adapted to other insect pests and will be useful in india where alternative control approaches are needed to replace the more toxic insecticides that are currently in use. matherials and methods collection and maintenance of termites termites (reticulitermes flavipes) were collected directly from their nests by placing wood bait buried near trees in and around the university campus, coimbatore, india. they were kept in cylindrical plastic containers with 1-2 cm deep vermiculite and sand (1:1 by volume). corrugated wood blocks were added as diet. one hundred to 2,000 individuals were collected from each termite colony. the termite colonies were kept for a maximum period of 120 days from field collection date at room temperature (21-25°c) in the laboratory. nematode cultures and extraction the greater wax moth galleria mellonella (pyralidae, lepidoptera) larva was used for the nematode baiting and multiplication. cultured nematodes were used within one month of collection from the host cadaver. twenty moth larvae were placed on the artificial diet in cylindrical plastic containers. the containers were kept at 25-28°c with 72-75% relative humidity to avoid fungal contamination (dutky et al., 1964). a representative sample of 250 ml was placed in a plastic box with ten last instar larvae of g. mellonella by baiting technique (white, 1927). preparation of neem seed kernel extract neem seed kernels were collected from the bharathiar university campus, coimbatore-641 046, tamil nadu, india. fifty grams of seed kernels of a. indica were washed and oven dried to constant weight at 55°c and the dried seeds were pulverized into fine powders and 1.0 g of the neem was stirred in 100 ml of distilled water. after 24 hours, the water extracts were filtered and used for the experiments. mortality bioassay the wooden blocks were immersed with different concentrations of neem seed kernel extract ranging from 1% to 4% and steinernema glaseri (150 to 600 ijs). control wooden blocks were treated with distilled water only and the wooden blocks were allowed to dry at room temperature for ten minutes and then placed in 15 cm in diameter petri dishes on moist filter paper discs. newly emerged 24 h starved late instar worker termites were segregated into groups and each group was individually fed with treated and untreated wooden blocks. each experiment was carried out for 1 d, 2 d, 3 d or 4 d, respectively, with hundred workers per concentration and replicated five times. after 2 journal of entomological and acarological research, ser. ii, 43 (2), 2011254 d, the termites were transferred to fresh untreated wooden blocks and maintained until they died. the total number of normal workers that survived was noted. the workers were observed for mortality and morphological changes associated with growth disrupting effects. survivability test effect of nske on nematode survivability was studied in the laboratory at 28 ± 1°c in the dark. standard petri dish bioassays were conducted to evaluate the effect of nske on mortality of nematodes. different concentrations of neem extract were taken in separate petri dishes and about 1000 juveniles were introduced into each concentration. water was taken in control petri dishes. every two days of exposure, live nematode juveniles were collected and observed under a binocular microscope. infectivity test the nematode infectivity rate after the neem treatment of r. flavipes workers was observed under laboratory conditions. the r. flavipes workers were kept in a cylindrical plastic container with 1-2 cm deep vermiculite and sand (1:1 by volume). corrugated cardboard and wood blocks were added as food treated in different concentrations of neem seed kernel extract. after 24 h, the ijs of s. glaseri were then released into the treated container. after 24 h the infectivity and mortality of neem treated worker termites were observed. the number of nematodes in the whole termite cadaver was noted by dissecting the termite and by counting the number of nematodes in the insect body with the help of a dissecting microscope. similarly, the infectivity rate of nematodes without neem treatment on workers of r. flavipes was also studied in separate experiment. statistical analysis for the mortality bioassay, the per cent mortality data after corrections were subjected to probit analysis for calculating mean lethal concentrations (lc50, lc90) (finney, 1971). results were corrected for control mortality by using abbott’s (1925) formula. all other percent mortality, infectivity and percent survivability data were subjected to analysis of variance (anova) and the means were separated using duncan’s multiple range tests (alder & rossler, 1977). results effect of nske and s. glaseri on mortality of r. flavipes workers considerable mortality of termites was evident after the treatment with neem seed kernel extract and it was dose dependent. lethal doses (lc50 and lc90) were also worked out (tab. 1). the lc50 values after 1.0%, 2.0%, 3.0% and 4.0% nske concentration treatment were 4.602, 3.689, 3.164, and 2.606, respectively. similarly, the lc50 values after treatment with 150 ijs, 300 ijs, 450 ijs and 600 ijs entomopathogenic nematodes was 694.713, 577.59, 458.15, and 408.58, respectively. the lc50 values after combined k. murugan, c. vasugi: azadirachta indica against subterranean termites 255 tab. 1 – mortality of reticulitermes flavipes workers after treatment with neem seed kernel extracts and steinernema glaseri. within a column, means followed by the same letter(s) are not significantly different at 5% level by dmrt. treatment of nske + s. glaseri with 1.0 % + 150 ijs, 2.0 % + 300 ijs, 3.0 % + 450 ijs and 4.0 % + 600 ijs was 165.906, 55.840, 57.343, and 19.799, respectively (tab. 1). side-effect of nske on the survivability of s. glaseri survivability of s. glaseri after treatment with nske, even at the higher concentrations, was not affected. at 4.0% nske, the percentage survivability was 98% in the 4th week. at the higher concentration of nske (8%), 98% survival was noted in the 2nd week and the average survival of s. glaseri was 95.5% (tab. 2). journal of entomological and acarological research, ser. ii, 43 (2), 2011256 tab. 2 efficacy of neem seed kernel extract on the percentage survivability of s. glaseri. within a column means followed by the same letter (s) are not significantly different at 5% level by dmrt. tab. 3 infectivity of s. glaseri on r. flavipes worker communities after the treatment with 1% nske, or untreated. within a column means followed by the same letter (s) are not significantly different at 5% level by dmrt. infectivity of treated and untreated s. glaseri on worker communities of r. flavipes. the infectivity of nematodes on nske-treated and untreated workers of r. flavipes is given in tab. 3. the percentage of s. glaseri-infected workers was considerably higher in nsketreated r. flavipes compared to untreated termites. there was 100% infectivity after the combined treatment of nske (1%) and nematodes (3000 ijs), and the percentage of infectivity was lower (81%) in the nske-untreated control (tab. 3). k. murugan, c. vasugi: azadirachta indica against subterranean termites 257 discussion and conclusion plants are the store-house of bio-active chemicals that have antifeedant, antiovipositional, growth disrupting and fecundity reducing properties towards different insects (mordue (luntz) & blackwell, 1993). in the present study the neem seed kernel extract (nske) in combination with entomopathogenic nematodes have shown toxicity against subterranean termites. earlier studies demonstrated that, to enhance nematode infection against these soil insects, combinations of nematodes with other control agents can be synergistic and provide better control than each agent alone (koppenhöfer & kaya, 1998; koppenhöfer et al., 2000; 2003). thus, koppenhöfer & kaya (1998) demonstrated that the combination of a low concentration of a neonicotinoid (i.e., imidacloprid) insecticide and a low concentration of entomopathogenic nematodes provided excellent control of scarab larvae. the neonicotinoid insecticides are considered to be more environmentally friendly, have low vertebrate toxicity, low application rates, and longer persistence (elbert et al., 1991) than the more toxic, persistent organochlorine or organophosphate insecticides. in the present study, also after application of nske, a higher infectivity of nematodes on termites was shown. previous studies combining bt and a nematode species against a pest showed that their combination was better than the use of nematodes alone (bari & kaya, 1984). hence, neem can be used to enhance the activity of nematodes for infectivity on termites. extracts of a. indica and many other plants are known to exert multiple, acute and chronic effects such as growth regulatory and antifeedants, etc., on the same or different insects (jacobson, 1988; saxena, 1989; schmutterer, 1990). murugan & vanithakumari (2009) studied the bioactivities of neem products against insects. moreover, in the present study infectivity by nematodes was higher in the nsketreatment compared to the untreated control, and at the same time the nske treatment did not affect the survival and infectivity of the entomopathogenic nematode, s. glaseri. soil insect pests like termites have their own behavioral mechanism to prevent entry of nematodes into the body by grooming activity. in the present study, the application of neem seed kernel extract affected the physiological activity and thereby arrested the grooming activity and it facilitated the easy entry of nematodes and their infectivity. the principles learned from this research can be adapted to other insect pests and will be useful in india where alternative control approaches are needed to replace the more toxic insecticides that are currently in use. references abbot w.s., 1925 a method for computing the effectiveness of an insecticide. j. econ. ent., 18: 264-265. akhrst r.j., boemare n.e., 1988 a numerical taxonomic study of the genus xenorhabdus (enterobacteriaceae) and proposed elevation of the subspecies of x. nematophilus to species. journal of general microbiology, 134: 1835-1845. alder h.l., rossler e.b., 1977 introduction to probability and statistics (sixth edition). w.h. freeman company, san francisco, 246 pp. journal of entomological and acarological research, ser. ii, 43 (2), 2011258 bari m.a., kaya h.k., 1984 evaluation of the entomogenous nematode neoaplectana carpocapsae weiser and the bacterium bacillus thuringiensis berliner var. kurstaki for suppression of the artieboke plume moth. journal of economic entomology, 77: 225-229. dutky s.r., thompson j.v., cantwell g.e., 1964 a technique for the mass propagation of the dd-136 nematode. journal of insect pathology, 6: 417-422. elbert a., becker b., hartwig j., erdelen c., 1991 imidacloprid – a new systemic insecticide. pflanzenschutz nachrichten bayer, 44(2): 113-136. finney d.j., 1971 probit analysis. cambridge university press, p. 333. grace j.k., 1997 biological control strategies for suppression of termites. journal of agricultural entomology, 14: 281-289. jacobsob m., 1989 botanical pesticides; past, present and future. in: arnason, philogene, morand (eds.) insecticides of plant origin, acs symposium series, 387: 1-10. kaya h.k., gaugler r., 1993 entomopathogenic nematodes. annual review entomology, 38: 181-206. koppenhöfer a.m., choo h.y., kaya h.k., lee d.w., gelernter w.d., 1999 increased field and greenhouse efficacy against scarab grubs with a combination of an entomopathogenic nematode and bacillus thuringiensis. biological control, 14: 37-44. koppenhöfer a.m., kaya h.k., 1997 additive and synergistic interactions between bacillus thuringiensis buibui strain and entomopathogenic nematodes. biological control, 8: 131137. koppenhöfer a.m., brown i.m., gaugler r., grewal p.s., kaya h.k., klein m.g., 2000 synergism of entomopathogenic nematodes and imdacloprid against white grubs: greenhouse and field evaluation. biological control, 19: 245-251. mordue (luntz) a.j., blackwell a., 1993 azadirachtin: an update. journal of insect physiology, 39: 903-924. murugan k., vanithakumari g., 2009 integration of botanical and microbial pesticides for the sustainable management of insect pests. in: neem a treatise. singh k.k., suman phogat, alka tomar dhillon r.s. (eds); i.k. international publising house pvt. ltd., new delhi, india, pp. 299-315. poinar g.o., 1979 nematodes for biological control of insects. crc, boca raton, fl. saxena r.c., 1989 insecticides from neem. in: acs symp.ser., 387 (eds. arnason j.t., philogene b.j.r., morand o.), american chemical society. washington, d.c. pp. 110-135. schmutterer h., 1990 properties and potential of natural pesticides from the neem tree, azadirachta indica. annual review of entomology, 35: 271-297. shelton t.g., grace j.k., 2003 effects of exposure duration on transfer on non-repellant termiticides among workers of coptotermes formosanus shiraki (isoptera: rhinotermitidae). journal of economic entomology, 96: 456-460. wang c., powell j.e., 2003 isolation and evaluation of beauveria bassiana for control of coptotermes formosanus and reticulitermes flavipes (isoptera: rhinotermitidae). sociobiology 41: 369-381. white g.f., 1927 a method for obtaining infective nematode larvae from cultures. science, 66: 302-303. kadarkarai murugan, department of zoology, bharathiar university coimbatore, india. e-mail: kmvvk@yahoo.com. chellamuthu vasugi, department of zoology, bharathiar university coimbatore, india. e-mail: vasugi.research@gmail.com k. murugan, c. vasugi: azadirachta indica against subterranean termites 259 jear2012 [journal of entomological and acarological research 2015; 47:4705] [page 41] orgya antiqua (linnaeus) (lepidoptera: lymantriidae): an occasional pest on pelargonium g. viggiani laboratorio di lotta biologica, dipartimento di agraria, università degli studi di napoli federico ii, italy abstract the larval development of orgya antiqua on pelargonium has been reported for the first time. short paper in july 2014 plants of pelargonium located in rivello (potenza), south italy, basilicata, in a house at the border of a woody land with castanea sativa, quercus spp. and many other broadleaved trees and shrubs, resulted damaged (figure 1). their leaves were progressively eroded by caterpillars identified as belonging to the vapourer orgya antiqua (linnaeus). most of the damage was caused by the last instar larvae. some of these caterpillars were collected, kept in a box and fed with pelargonium leaves. they completed their development and pupated in a transparent web cocoon. the adults emerged around mid august. during july-august the species developed one generation, probably the second of the year. the recorded infestation of pelargonium was most likely caused by young caterpillars passively transported by wind. o. antiqua is a sexual dimorphic species; the female (figure 2a) is light grey, covered with greyish white scales and apterous; the male (figure 2b) has comb-like antennae, fore wings rusty-brown, with white tornal spot in lower part at external margin (wingspan to 30 mm). the eggs are greyish (figure 2c) with a brown micropylar area. the full grown larva (figure 2d) is grey, with reddish lateral and dorsal spots, tufts of long blackish setae on each side of the prothorax directed forward and two on the eighth abdominal segment in the opposite direction; four yellow, dense tufts of setae are present on the urotergites 1-4; tufts of setae are present at the sides of third and, sometime, second urite. o. antiqua is widely distributed in western europe, the original area, and in many other countries of the palaearctic and nearctic regions (carter, 2004). the number of generations developed in a year varies from 1 to 4 (tremblay, 1986; wagner, 2005). the polyphagous caterpillars are able to damage fruit and landscape cultures, such as apple, pear, cherry, bird cherry, roses, mountain ash etc., and also forest trees, preferring willow, poplar, birch, larch. they also cause damage to currant, wild growing rosaceous, maple, linden, cowberry, and bilberry. insignificant damage is reported on clover, soya, castor oil plant and other herbaceous plants (carter, 1984; tremblay, 1986; porter, 1997; pollini, 1998). no records have been found in literature concerning pelargonium spp. a female lays 100-500 eggs in a batch frequently on the surface of the own pupa cocoon. the larval feeding period lasts 25-55 days for each generation, depending on the environment. in the geographic areas where the species develops four generation a year the young caterpillars emerge from the overwintering eggs in april-may, and their development is completed in june. the subsequent generations, partially overlapping, take place respectively around july, august and october (tremblay, 1986). correspondence: gennaro viggiani, laboratorio di lotta biologica, dipartimento di agraria, università degli studi di napoli federico ii, via università 133, 80055 portici (na), italy. tel.: +39.081.2539003 fax: +39.081.7755872. e-mail: genviggi@unina.it key words: geraniums; vapourer; italy. received for publication: 12 september 2014. revision received: 23 december 2014. accepted for publication: 16 january 2015. ©copyright g. viggiani, 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:4705 doi:10.4081/jear.2015.4705 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:4705 figure 1. pelargonium infested by caterpillars of orgya antiqua. no nco mm er cia l u se on ly [page 42] [journal of entomological and acarological research 2015; 47:4705] control measures, if needed, include biological control with formulations of bacillus thuringiensis applied when the very young caterpillars appear. it is suggested to avoid the use of synthetic insecticides in household environment. references carter d.j., 1984 pest lepidoptera of europe with special reference to british isles. dr w. junk publishers, dordrecht: 431 pp. carter n.e., 2004 status of forest pests in new brunswick in 2003. department of natural resources, fredericton, new brunswick: 7-8. pollini a., 1998 manuale di entomologia applicata. edagricole, bologna: 1462 pp. porter j., 1997 the colour identification guide to caterpillars of the british isles. viking, london: 275 pp. tremblay e., 1986 entomologia applicata. editore liguori, napoli: 381 pp. wagner d.m., 2005 caterpillars of eastern north america. princeton university press: 512 pp. short paper figure 2. orgya antiqua: a. female; b, male; c, egg batch; d, larva. no nco mm er cia l u se on ly j. ent. acar. res. ser. ii, 43 (1): 33-38 30 april 2011 l. limonta, m.c. morosini, d.p. locatelli development of rhyzopertha dominica (f.) (coleoptera bostrichidae) on durum wheat kernels and semolina abstract - the time necessary to larvae of rhyzopertha dominica to drill kernels  with or without dusts (semolina or debris from adults), and the possibility of  development on semolina were evaluated. tests were carried out on durum wheat  kernels (triticum durum desfontaines), debris deriving from rearing, and semolina.  development was observed also on 0.5 and 6 mm of semolina and of debris. thirty  replicates were carried out for each test.  the number of first instar larvae, that successfully drill sound kernels within 10 days,  was higher by adding semolina or debris. when only kernels were provided, the  time needed to larvae to drill increased. development and the number of emerging  adults were not significantly influenced by the addition of semolina to the kernels.  larvae couldn’t develop on 0.5 mm, while an equal number of individuals completed  the development to adult in 6 mm of semolina as in the tests with kernels. when  development was on debris, a lower number of emerged adults was observed.  riassunto sviluppo di rhyzopertha dominica (f.) (coleoptera bostrichidae) su cariossidi e farina di grano duro è stato valutato il tempo necessario alle larve di prima età di rhyzopertha dominica  per penetrare nelle cariossidi di grano duro (triticum durum desfontaines) in  presenza o in assenza di semola o rosura dell’insetto e la possibilità di completare  il ciclo su spessori di 0,5 e 6 mm di semola e rosura. per ogni prova sono stati  osservati trenta individui singolarmente.  il numero di larve che penetra nelle cariossidi integre entro dieci giorni è maggiore  in presenza di semola o rosura; in assenza di polveri aumenta il tempo necessario. il  tempo di sviluppo e il numero di adulti sfarfallato su cariossidi non varia in presenza  o assenza di semola o rosura. le larve non riescono a svilupparsi su un substrato  con spessore di 0,5 mm, mentre con 6 mm di semola il numero di adulti sfarfallati  è analogo a quello osservato su cariossidi in assenza o presenza di semola; nel caso  della rosura, si osserva un numero minore di adulti sfarfallati. key words: lesser grain borer, cereal grain, flour, debris. journal of entomological and acarological research, ser. ii, 43 (1), 201134 introduction rhyzopertha dominica (f.) is one of the most economically important beetles  infesting kernels. newly born larvae feed on dusts, produced by adults or deriving  from food processing; then they drill kernels, where they complete the life cycle  (golebiowska, 1969). larvae enter kernels within the first instar, a short time after egg  hatching (crombie, 1944), preferring breaks or the germ area where the covering testa is  loose (birch, 1945). according to özkaya et al. (2009), larvae feed on both endosperm  and germ. osuji (1982) observed a faster development in larvae that entered the germ  compared to the ones that entered through the endosperm. the development from egg to adult is possible, when wheat kernels are damaged,  with a 9% moisture content and temperatures between 26 and 36 °c (birch, 1945). in  sound kernels, with the same moisture content, development is possibile at 34 °c, while  with a moisture content below 9% the lesser grain borer does not survive. the highest  egg survival was observed at 30 °c and 75% r.h. and the lowest at 40 °c and 65%  r.h. (kumawat, 2007). in mills and pasta factories, due to poor cleaning of machines and of processing  departments, dusts, kernels, and pasta debris can accumulate and become breeding  ground of pests. in this research the time necessary to larvae to drill kernels with or  without dusts (semolina or debris from adults), and the possibility of development on  semolina were evaluated. materials and methods laboratory cultures of rhyzopertha dominica kept in a rearing room at 28±1 °c,  70±5% u.r. and photoperiod of 16:8 (light:dark) were used for these tests. eggs were  collected according to elek (1994) and incubated 7-8 days, in order to obtain first instar  larvae, 0-24 hours old.  tests were carried out on durum wheat kernels (triticum durum desfontaines),  with debris deriving from rearing, and semolina. debris were collected from rearing  every two weeks, with a 30 mesh sieve and divided with 30, 40, 50, and 70 mesh sieves.  debris presented 98,8% of particles size lower than 70 mesh1. semolina was sieved2  with the same procedure. food substrate were kept at -18 °c for 20 days, to remove  possible infestations. the substrate and a first instar larva were placed in a polypropylen cylinder (ø: 14  mm, h: 11 mm). thirty replicates were carried out for each test. (1) percentage of particle size obtained from debris with sieves: 30 mesh 0.04%; 40 mesh 0.24%; 50  mesh 0.92%; 70 mesh 98.8%. (2) percentage of particle size obtained from semolina with sieves: 30 mesh 0.1%; 40 mesh 24.9%; 50  mesh 37.5%; 70 mesh 37.5%. 35l. limonta, m.c. morosini, d.p. locatelli: r. dominica on durum wheat kernels and semolina development on durum wheat kernels with semolina or debris in each cylinder, ten sound durum wheat kernels were placed, alone or with 3 mg  of one of the different fractions of semolina or debris. the amount of debris added was  proportional to the quantity of dust produced by 40 adults in 10000 durum wheat kernels,  in a week. then, a first instar larva was added.  different groups of 30 replicates were controlled after 1, 2, 3, 4, 10 days to verify  the time needed by larvae to drill and enter into the kernels. a further group of 30 replicates for each substrate were observed until adults  emergence. adults emergence was controlled every three days. data were submitted to anova and duncan’s multiple range test (spss.17). development on different particle size of semolina or on debris in each cylinder 0.5 or 6 mm of semolina (30, 70 mesh) or debris were placed. first  instar larvae were placed over 0.5 mm or in the middle of 6 mm substrate. thirty replicates, for each substrate and for each thickness, were controlled after  10 days, 30 replicates were observed until adult emergence. adults emergence was  controlled every three days.  data were submitted to anova and duncan’s multiple range test (spss.17). results development on durum wheat kernels with semolina or debris larvae drilled durum wheat kernels within two days when different particle size of  semolina were added; the number of larvae was the same with the different particle size  (tab. 1). a lower number of larvae was observed when debris were added to kernels.  the lowest number of larvae, 21, and a longer period, from 1 to 10 days, was observed  on durum wheat kernels without the addition of dust.  the number of individuals that became adult was constant in all the tests, with a 30%  mortality (tab. 2). adults emerged between the 32nd and the 35th day from egg hatching,  unless on clean kernels where the last emerged adult was observed on the 62nd day. development on different particle size of semolina or on debris first instar larvae placed in the cylinder with 0.5 mm of semolina or debris leaved  the substrate and the few left larvae were not able to complete the development; when  placed in the middle of a 6 mm substrate larvae remained in the substrate and survival  was higher (tab. 3). an equal number of individuals completed the development to adult in 6 mm of  semolina as in the tests with kernels. when development was on debris, a lower number  of emerged adults was observed.  emerging of adults was observed on semolina from the 23rd day to the 35th day; on  debris emerging of adults started on the 35th day and finished on the 47th day (tab. 4). journal of entomological and acarological research, ser. ii, 43 (1), 201136 tab. 1 number of first instar larvae (30 replicates) of rhyzopertha dominica (f.) entering durum wheat kernels, alone or with 3 mg of semolina or debris, after 1, 2, 3, 4 and 10 days and mean period (days) (s.d.). durum wheat kernels number of larvae in the kernels day mean period (days) (s.d.) 1st 2nd 3rd 4th 10th total + semolina 30*  25 4 29 1.1(0.35)b + semolina 40* 25 4 29 1.1(0.35)b + semolina 50*  27 2 29 1.1(0.26)b + semolina 70*  25 4 29 1.1(0.35)b + debris 24 0 0 2 26 1.2(0.81)b only kernels 10 2 1 4 4 21 3.4(3.44)a anova: f=11.818 p< 0.01 duncan’s multiple range test. * semolina sieved with 30 or 40, 50, 70 mesh sieves. tab. 2 number of adults of rhyzopertha dominica (f.) and mean period (s.d.) of adult emerging on durum wheat kernels, alone or with 3 mg of semolina or debris. durum wheat kernels number of adults emerging daily n mean period (days) (s.d.) 32 35 62 total + semolina 30*  17 2 19 32.3(0.94) n.s. + semolina 40* 20 20 32.0(0.00) n.s. + semolina 50*  14 4 18 32.2(0.77) n.s. + semolina 70*  19 2 21  32.3(0.90) n.s. + debris  17 1 18  32.2(0.70) n.s. only kernels 18 0 1 19 33.6(6.88) n.s. anova: f=0.739 p=0.596 duncan’s multiple range test. * semolina sieved with 30 or 40, 50, 70 mesh sieves. tab. 3 number of first instar larvae of rhyzopertha dominica (f.) alive or escaped after 10 days on different particle size of semolina or debris (thick 0.5 and 6 mm). substrate thickness (mm) 0.5 6 alive escaped alive escaped semolina 30* 12 10 22 0 semolina 70* 13 9 22 0 debris 17 7 20 0 * semolina sieved with 30 and 70 mesh sieves. 37l. limonta, m.c. morosini, d.p. locatelli: r. dominica on durum wheat kernels and semolina conclusions in this research, at 28 °c and 70% r.h., first instar larvae of rhyzopertha dominica  were able to drill sound durum wheat kernels, differently from the observation of howe  (1950) on wheat. development and the number of emerging adults were not significantly  influenced by the addition of semolina to the kernels. the number of first instar larvae,  that succesfully drilled sound kernels within 10 days, was higher adding semolina or  debris. almost all first instar larvae penetrated within the second day when semolina  was added; the time increased when only kernels were provided.  in the tests with 0.5 mm semolina, first instar larvae could not develop or fled.  in tests with 6 mm semolina, the number of emerging adults was the same as the one  observed on kernels and the development period was shorter, while with 6 mm debris  a longer development period and a lower number of emerging adults were observed. in  tests carried out with semolina, due to the small granulation, klys (2006) observed no  population development as adults showed a scarce mobility, turned over and starved. the mean period of development was shorter on semolina than on kernels. first  instar larvae inside the kernels became adults in 32 days, while larvae reared on semolina  developed to adults in 27 days.  first instar larvae placed in the middle of 6 mm of debris took 40 days to emerge as  adults. howe (1950), observed development faster on wheat kernels than the one on wheat  flour, but during the experiment the substrates were repeatedly sieved, disturbing larvae,  while in these tests the emerging of adults was verified without sieving the substrate.  references birch l.c., 1945 - a contribution to the ecology of calandra oryzae l. and rhyzopertha dominica fab. in stored wheat. - trans. r. soc. south australia 69 (1): 140-149. crombie a.c., 1944 - on intraspecific and interspecific competition in larvae of graminivorous  insects. - j. exp. biol. 20 (2): 135-151. elek j. a., 1994 - methods for collecting eggs and monitoring egg-hatch and immature development  of rhyzopertha dominica (f.) (coleoptera: bostrichidae). - j. stored prod. res. 30 (4): 261265. tab. 4 number of adults of rhyzopertha dominica (f.) and mean period (s.d.) of adult emerging on semolina of durum wheat kernels (thick 6 mm). substrate number of adults emerging daily n mean period (days) (s.d.)23 26 29 32 35 38 41 44 47 semolina 30* 1 9 8 1 1 20 27.8(2.65)b semolina 70* 4 9 7 20 26.4(2.23)b debris  0 0 0 0 4 3 3 3 2 15 40.2(4.31)a anova: f= 100.221 p<0.01 duncan’s multiple range test. * semolina sieved with 30 and 70 mesh sieves. journal of entomological and acarological research, ser. ii, 43 (1), 201138 golebiowska z., 1969 - the feeding and fecundity of sitophilus granarius (l.), sitophilus oryzae  (l.) and rhyzopertha dominica (f.) in wheat grain. - j. stored prod. res. 5: 143-155. howe r.w., 1950 - the development of rhyzopertha dominica (f.) (col., bostrichidae) under  constant conditions. - ent. monthly mag. 86: 1614-1618. kłys m., 2006 - nutritional preferences of the lesser grain borer rhizopertha dominica (f.)  (coleoptera, bostrichidae) under conditions of free choiche of food. - j. plant prot. res. 46  (4): 359-368. kumawat k.c., (2007) - effect of abiotic factors on biology of rhyzopertha dominica (fab.) on  wheat. - ann. plant prot. sci. 15 (1): 111-115. osuji f.n.c., 1982 - development of the lesser grain borer, rhyzopertha dominica, in maize  kernels as affected by site of larval entry. - ent. exp. & appl., 31: 391-394. özkaya h., özkaya b., colakoglu a.s., 2009 - technological properties of a variety of soft and  hard bread wheat infested by rhyzopertha dominica (f.) and tribolium confusum du val. - j.  food, agric. & env. 7 (3-4): 166-172. lidia limonta, matteo carlo morosini, daria patrizia locatelli - dipartimento di protezione  dei sistemi agroalimentare e urbano e valorizzazione delle biodiversità- dipsa, università  degli studi di milano,via celoria 2, 20133 milano - italy. e-mail: lidia.limonta@unimi.it accepted 21 april 2011 j. ent. acar. res..indd b. habibpour, a. cheraghi, m.s. mossadegh evaluation of cellulose substrates treated with metarhizium anisopliae (metschnikoff) sorokin as a biological control agent against the termite microcerotermes diversus silvestri (isoptera: termitidae) abstract this article is the first report on the promising effect of an entomopathogenic fungus, metarhizium anisopliae (metschnikoff) sorokin to control populations of microcerotermes diversus silvestri. biological control is an alternative to the long-term usage of chemical pesticides. m. anisopliae, the causal agent of green muscardine disease of insects, is an important fungus in biological control of insect pests. bait systems can eliminate entire colonies of subterranean termites. baiting reduces adverse environmental impacts caused by organochlorine and organophosphate pesticides in the control of termites and creates sustainable protection of buildings against their invasion. treated-sawdust bait was applied by two methods: a) combination of treated sawdust and untreated filter paper, and b) combination of treated sawdust and untreated sawdust. when combinations of treated sawdust and untreated sawdust were used, lc50 and lc90 were 8.4×10 6 and 3.9×107 (spore/ml), respectively. with the use of improved bait formula and more virulent strains, we hope to achieve better control of termite colonies and enable pathogens to become a useful element in the integrated pest management system. key words: termites, entomopathogenic fungus, biological control, sawdust, feeding. introduction microcerotermes diversus silvestri (isoptera: termitidae) is an extremely destructive pest of wood and is considered to be the major termite species in iran, iraq and oman. methods for the control of termites, including chemical control, baiting system and wood protection, have hardly been investigated scientifically in iran. current management of subterranean termites in iran mainly involves the application of a soil insecticide to reduce or isolate their foraging populations (habibpour, 2006). in other parts of the world insecticidal baits have been shown to be an effective alternative to conventional soil insecticides for remedial termite control (su, 1991). bait systems can eliminate entire colonies of subterranean termites (su & scheffrahn, 1996). biological control is recognized as a realistic alternative to chemical pesticides (bayon et al., 2000). the study of pathogens for termite control started as early as 1965 (wang & j. ent. acarol. res. ser. ii, 43 (2): 269-275 30 september 2011 powell, 2004). metarhizium anisopliae (metschnikoff) sorokin, the causal agent of green muscardine disease of insects, is an important fungus in biological control of insect pests (tajik ghanbalani et al., 2009). fungi exhibit qualities which can make them ideal for this application, including a slow-acting nature similar to that of successful chemicals, the ability to self replicate, and the ability of fungal spores to be spread by termite social behavior (grace & zoberi, 1992). baiting reduces adverse environmental impacts caused by organochlorine and organophosphate pesticides in the control of termites and creates sustainable protection of buildings against their invasion (su, 1991). bait systems are composed of two parts: a) a slow-acting toxicant, and b) a nutritive substrate, such as sawdust, with absorbent additive materials (including sugars, nitrogenous compounds and pheromones). foraging termites acquire slow-acting toxicants by feeding on the bait and then transfer it to other individuals in the colony through trophallaxis (guadalupe & morales-ramos, 2001). this study was conducted to examine the effect of the fungus m. anisopliae on the termite m. diversus. materials and methods collection of termites: termites were collected from wooden blocks of beech (fagus orientalis lipsky) (3×6×20 cm3) placed in infected soil in ahvaz (iran). worker termites were used for this experiment. fungal isolate: in this research m. anisopliae (demi 001) was used. the strain was originally isolated from rhynchophorus ferrugineus olivier (coleoptera: curculionidae) and stored at the iranian research institute of plant protection. preparation of media: fungi were cultured on sabouraud dextrose agar with 1% yeast extract medium. petri dishes were placed in an incubator (28 ± 1°c with 85 ± 5% r.h.) for two weeks. preparation of fungal suspension: to prepare the fungal suspension, 1 ml of polysorbate monoleate (tween 80®) was added to 100 ml sterile distilled water and spores were harvested from the media surface with a shallow scalpel cut and placed into the solution. spore suspension concentration was determined using a haemocytometer. experiment: after preliminary trials, concentrations of 1.1×105, 2.7×106, 3.7×107 and 3.5×108 (spore/ml) were selected for testing. treated sawdust bait was applied by two methods: a) combination of treated sawdust and untreated filter paper, and b) combination of treated sawdust and untreated sawdust. a) in this test 2 g of sugarcane molasses and 2 g of agar mixed in 100 ml of fungus spore suspension were used. this combination was placed in shaker for 30 minutes. then 25 g of beech sawdust had been added and mixed well. at this stage the bait was ready for testing. also pieces of filter paper (whatman® cat no 1001 42) were used. the filter paper with a diameter of 42 mm cut into two halves and these pieces were used in this experiment. four grams of journal of entomological and acarological research, ser. ii, 43 (2), 2011270 bait were placed inside a plastic petri dish together with a piece of filter paper in other side of the dish. the filter paper was moistened with sterile distilled water. b) in this method the bait was prepared as in the previous test and the same sawdust was prepared and sterile distilled water had been used instead of spore suspension. four grams of bait were placed inside a petri dish (9 cm diameter, 1 cm height) after weighing and 4 g of untreated sawdust was placed in the other side. one hundred worker termites were added into every petri dish. sterile distillated water was applied instead of spore suspension in control treatment. four replications were carried out for every treatment. petri dishes were incubated in 28 ± 1°c with 85 ± 5% r.h. in the dark. the rate of mortality had been registered for 14 days. analysis of variance performed by sas (9.1). graphs were drawn by software excel 2007. comparison of means calculated by lsd test at the 0.05 level. results the rate of lc50 and lc90 in both methods is summarized in tab. 1. when combinations of treated sawdust and untreated sawdust was used in one petri dish, lc50 and lc90 were 8.4×106 and 3.9×107 (spore/ml), respectively (df=14, f=23 and p<0.0001). also when combination of treated sawdust and untreated filter paper was used in one petri dish, lc50 and lc90 were 4.8×10 6 and 3.3×107 (spore/ml), respectively (df=14, f=13 and p<0.0001) (tab. 1). the rate of lt50 and lt90 in both methods is summarized in tab. 2. in both techniques the rate of lt50 and lt90 were decreased with increasing concentration of spore suspension in bait. the lowest level of lt50 lt90 related to the concentration of 3.5×10 8 (spore/ml) in both methods that over 90% of the population were killed in less than a week. when filter paper was applied instead of sawdust in untreated section, the rate of lt50 and lt90 were low (tab. 2). the mean comparisons of termites mortality with the two methods is reported in fig. 1. the highest mortality was observed with concentrations of 3.7×107 and 3.5×108 b. habibpour et al.: evaluation of cellulose substrates treated with metarhizium anisopliae tab. 1 the rate of lc50 and lc90 in both methods. * treated sawdust and untreated sawdust. ** treated sawdust and untreated filter paper. 271 (spore/ml). the rate of mortality was enhanced by increasing the concentration of spore suspension in the bait. there was no significant difference between concentrations of 3.7×107 and 3.5×108 (spore/ml). the lowest level of mortality (less than 10%) was observed with concentration of 1.1×105 (spore/ml). the level of mortality with concentration of 2.7×106 (spore/ml) was less than 10% in combination of treated sawdust and untreated sawdust and was less than 30% in combination of treated sawdust and untreated filter paper (fig. 1). the mean comparisons of feeding in combination of treated sawdust and untreated filter paper method is reported in fig. 2. the highest level of feeding was achieved from fig. 1 comparison of mean mortality in both methods. the same letter above bars indicates absence of signifi cant differences (lsd test, p = 0.05). * sus: treated sawdust and untreated sawdust. ** suf: treated sawdust and untreated fi lter paper. tab. 2 the rate of lt50 and lt90 in both methods. * treated sawdust and untreated sawdust . ** treated sawdust and untreated filter paper. journal of entomological and acarological research, ser. ii, 43 (2), 2011272 combination treated sawdust in concentration of 2.7×106 (spore/ml) that didn’t exhibit significant different with control treatment and concentrations of 1.1×105 and 3.7×107 (spore/ml). but there was significant different with concentration of 3.5×108 (spore/ml) and were placed on a higher level. the highest feeding activity occurred in concentration of 1.1×105 (spore/ml) from filter paper (fig. 2). the mean comparisons of feeding for combination of treated sawdust and untreated sawdust method is reported in fig. 3. no significant different was detected from treated part. furthermore, there were no significant different between rate of feeding from fig. 2 comparison of mean feeding in combination of treated sawdust and untreated fi lter paper method. the same letter above bars indicates absence of signifi cant differences (lsd test, p = 0.05). * s: treated sawdust. ** uf: untreated fi lter paper. fig. 3 comparison of mean feeding for combination of treated sawdust and untreated sawdust method. the same letter above bars indicates absence of signifi cant differences (lsd test, p = 0.05). * s: treated sawdust. ** us: untreated sawdust b. habibpour et al.: evaluation of cellulose substrates treated with metarhizium anisopliae 273 untreated sawdust in different concentrations except concentration of 3.5×108 (spore/ ml) which was lower than the other. overall rate of feeding was less in treated part than in the untreated part in all concentrations (fig. 3) discussion and conclusions when combination of untreated sawdust was applied as untreated part, lc50 and lc90 exhibited higher level in comparison of untreated filter paper. probably this is because of the higher attractiveness of a sawdust bait for termite than the filter paper. comparison between lc50 and lc90 prepared acceptable information about performance of bait in different concentrations. the rate of lc50 and lc90 decreased with increasing concentration and due to lower level of these two parameter when untreated filter paper used instead of untreated sawdust. mean comparisons of mortality revealed the importance of choosing the appropriate concentration in both methods. collectively no significant different observed between rates of mortality in every concentration in two methods. figs. 2 and 3 offer considerable information regarding the amount of eaten untreated and treated parts. according to fig. 3 it can be expressed that termites feed less from treated baits. however, it should not be assumed that fungi are repellent for termites in high concentration and that the termites avoided the bait containing fungi. it should be noted that population of termites did decreas regarding to the rate of lc50 and lc90 in both methods in less than a week. probably the reason of lower feeding rate in high concentrations with comparison of control and lower concentrations is related to this case. wang & powell (2004) declared that m. anisopliae was not repellent for reticulitermes flavipes kollar (iso.: rhinotermitidae) and coptotermes formosanus shiraki (iso.: rhinotermitidae) in cellulose powder bait with effective concentrations. they suggested use of attractive baits as a suitable alternative for increasing performance of fungi against these two implanted termites. habibpour et al. (2006) in research about laboratory evaluation of chemical additives as feeding stimulants for m. diversus represented that additive nitrogenous compounds such as lesitin may increase efficacy of toxicant baits against termites in field condition. using of such cases may increase efficacy of entomopathogenic fungi against of termites. habibipour et al. (2008) applied borax-treated bait against of m. diversus and exploded that level of mortality and rapid of mortality of m. diversus depend on toxin concentration that was according to the present study. collectively this research investigated possibility of using entomopathogenic fungus m. anisopliae in form of bait against m. diversus and trying to raise awareness of performance of this fungus. according to this findings fungus in combination of sawdust bait revealed efficient alternative in vitro and therefore it can be a candidate for optimizing performance and field trials. it may take a long treatment period and many treatment sites to eliminate field colonies using m. anisopliae. most field studies failed to eliminate termite colonies by using fungal pathogens. these failed experiences had prevented the fungus from becoming a stand-alone termite treatment measure. developing a palatable formulation with appropriate concentration is the key to improve its efficacy. with the study of improved bait formula and virulent journal of entomological and acarological research, ser. ii, 43 (2), 2011274 strains, we hope to achieve better control of termite colonies and enable pathogens to become a useful element in the integrated pest management system. acknowledgements this research was supported by shahid-chamran university of ahvaz, iran. references bayon i.l., ansard d., brunet c., girardi s., paulmier i., 2000 biocontrol of reticulitermes santonensis by entomopathogenic fungi improvement of the contamination process. the in irg/wp/doc 00-10359, 14-19 may. grace j.k., zoberi m.h., 1992 experimental evidence for transmission of beauveria bassiana by reticulitermes flavipes workers (isoptera: rhinotermitidae). sociobiology, 20: 23–28. guadalupe r.m., morales-ramos j.a. 2001 bait matrix for delivery of chitin synthesis in hibitors to the formosan subterranean termites (isoptera: rhinotermitidae). journal of economic entomology, 94 (2): 506-510. habibpour b., 2006 laboratory and field evaluation of bait-toxicants for suppression subterranean termite populations in ahvaz (iran). phd dissertation, department of plant protection, college of agriculture, shahid-chamran university, ahvaz, iran. habibpour b., mossadegh m.s., moharramipour s., 2006 laboratory evaluation of chemical additives as feeding stimulants for microcerotermes diversus silvestri (isoptera: termitidae). the scientific journal of agriculture, 29 (2): 33-42. habibpour b., mossadegh m.s., moharramipour s., fathi m., 2008 toxicity of borax bait to microcerotermes diversus (silvestri) (isoptera: termitidae) under laboratory conditions. journal of knowledge of agriculture, 18 (4): 171-185. su n.y., 1991 evaluation of bait–toxicants for suppression of subterranean termite populations. sociobiology, 19: 211-220. su n.y., scheffrahn r.h., 1996 fate of subterranean termite colonies (isoptera) after bait applications – an update and review. sociobiology, 23: 253-275. tajick ghanbalani m.a., asgharzadeh a., hadizadeh a.r., mohammadi sharif m., 2009 a quick method for metarhizium anisopliae isolation from cultural soils. american journal of agricultural and biological science, 4 (2): 152-155. wang c., powell j.e., 2004 cellulose bait improves the effectiveness of metarhizium anisopliae as a microbial control of termites (isoptera: rhinotermitidae). biological control, 30: 523-529. behzad habibpour, department of plant protection, college of agriculture, shahid-chamran university of ahvaz, iran. e-mail: habibpour_b@scu.ac.ir amir cheraghi, department of plant protection, college of agriculture, shahid-chamran university of ahvaz, iran. mohammad saeed mossadegh, department of plant protection, college of agriculture, shahid chamran university of ahvaz, iran. b. habibpour et al.: evaluation of cellulose substrates treated with metarhizium anisopliae 275 jear2012 [journal of entomological and acarological research 2015; 47:4955] [page 79] malaria vector species composition and relative abundance in mutare and mutasa districts, zimbabwe s. sande,1 m. zimba,1 p. chinwada,1 h.t. masendu,2 a. makuwaza3 1department of biological science, university of zimbabwe, harare; 2abt associates inc., mount pleasant, harare; 3national institute of health research, harare, zimbabwe abstract regular entomological monitoring is important to determine changes in mosquito species composition and relative densities of malaria vectors in relation to vector control interventions. a study to gain insights into malaria vector species composition and relative abundance was undertaken in mutare and mutasa districts, zimbabwe. two methods; indoor resting catches and larval sampling were used to collect indoor resting adults and larvae from may 2013 to april 2014. mosquitoes collected as adults and reared from larvae that were identified morphologically as potential malaria vectors were further processed to sibling species by polymerase chain reaction (pcr). morphological identification of anopheline mosquitoes showed presence of two complexes: an. funestus and an. gambiae. the total number of female members of the an. funestus group and an. gambiae complex collected by both methods from the two sites was 840 and 31 respectively. malaria vector species of both complexes were more abundant in mutare than in mutasa. the pcr-based assays showed the presence of four sibling species: an. funestus sensu stricto (90.8%, 267/294) and an. leesoni (5.1%, 15/294), of an. funestus group; an. arabiensis (41.9%, 13/31) and an. quadriannulatus (48.4%, 15/31) of the an. gambiae complex. about 4% and 5% of specimens of an. gambiae complex and an. funestus group respectively did not amplify. of the two identified malaria vector sibling species, an. funestus sensu stricto was more abundant (95.4%, 267/280) than an. arabiensis (4.6%, 13/280), suggesting the replacement to secondary vector of an. arabiensis, which was previously the predominant vector species. an. funestus sensu stricto and an. arabiensis, the most important vectors of human malaria were identified in this study, but their resting and biting habits as well as insecticide susceptibility are unclear. further studies on vector behaviour are therefore recommended. introduction malaria in zimbabwe is a serious public health problem, causing morbidity, mortality and poverty although control efforts aimed at the vector mosquito are set up annually (lukwa et al., 2014). the most important vector species of human malaria in africa belong to an. gambiae complex and an. funestus group. in zimbabwe, malaria is transmitted by vector species belonging to an. gambiae and an. funestus (masendu et al., 2005). members of the an. gambiae complex include eight sibling species: an. gambiae sensu stricto giles, an. arabiensis patton, an. bwambae white, an. melas theobald, an. merus dönitz, an. quadriannulatus theobald, an. amharicus hunt, coetzee and fettene and an. comorensis brunhes, le goff and geoffroy. within the complex, an. gambiae sensu stricto (hereafter referred to as an. gambiae) and an. arabiensis are the major human malaria vectors in sub-saharan africa (gregory & yoosook, 2013). previous studies on an. gambiae complex in zimbabwe documented four members; an. gambiae, an. arabiensis, an. merus and an. quadriannulatus in various combination of sympatry (masendu et al., 2005). the wide distribution of an. arabiensis, often in association with the nonvector an. quadriannulatus, confirmed its status as the principal human correspondence: shadreck sande, 2557 glaudina park, harare, zimbabwe. tel.: +2634.304.702 mobile: +263.773.031.291 fax: +2634.798.566. e-mail: shadrecksande@rocketmail.com key words: abundance; anopheline; mosquito; species. contributions: ss, conception of the problem, design, collection, pcr assays, analysis, interpretation and drafting of final article; mz, pc, and htm, responsible for all the stages of the research, including analysis and interpretation as well as critically reviewing the final draft for academic worth; am, data collection, pcr assays, analysis, interpretation and drafting of final article. acknowledgements: the authors would like to thank the university of zimbabwe for providing learning facilities, mr. nzira lukwa, medical entomologist for the national institute of health research for providing equipment for mosquito collection and dr. j. mberikunashe, national malaria control programme manager for providing vehicle for field work as well as the communities in mutare and mutasa districts for allowing the collection of mosquitoes in their houses. funding: the national malaria control programme funded this research project. conflict of interest: the authors declare no potential conflict of interest. conference presentation: this paper was presented at zimbabwe national malaria conference, 2014 september 1-4, kadoma, zimbabwe. received for publication: 13 janaury 2015. revision received: 20 june 2015. accepted for publication: 20 june 2015. ©copyright s. sande et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:4955 doi:10.4081/jear.2015.4955 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:4955 no nco mm er cia l u se on ly [page 80] [journal of entomological and acarological research 2015; 47:4955] malaria vector in zimbabwe (masendu et al., 2005; munhenga, 2010). historically, the an. funestus group consists of at least nine african species: an. funestus sensu stricto (hereafter referred to as an. funestus), an. rivulorum, an. vaneedeni, an. leesoni, an. confusus, an. fuscivenosus, an. brucei, an. parensis and an. aruni (gillies & de meillon, 1968; gillies & coetzee, 1987). recently, new sibling species including an. rivulorumlike (from west africa), an. funestus-like (from malawi) and an. funestuslike-like (from zambia) (spillings et al., 2009) have been isolated. in the an. funestus group, an. funestus is the only member that is implicated as an important vector of malaria in sub-saharan africa (coetzee et al., 2000). the sympatric occurrence of an. funestus with minor vectors an. rivulorum and an. leesoni has been found in several countries in africa (wilkes et al., 1996; cohuet et al., 2003) but has not been reported in zimbabwe. the importance of the above vectors in malaria transmission differs depending on their feeding and resting preference behavior, seasonal abundance and vectorial capacity (coluzzi, 1984). these differences therefore, contribute to the varied malaria epidemiological patterns observed in africa and, subsequently, different vector behaviour may require different strategies for optimal vector control. currently, the two most common vector control strategies are indoor residual spraying (irs) and long lasting insecticidal nets (llins), accounting for almost 60% of global investments in malaria control (who, 2013). molineaux & gramiccia (1980) concluded that differences in vector behavior and insecticide resistance cause the failure of irs and/or mosquito nets in suppressing malaria transmission in any setting. as such, different vector species may have different insecticide susceptibility status at any given locality and time, thus, insecticides for vector control need to be selected carefully to achieve maximum impact on malaria transmission. current vector control studies in zimbabwe, especially on species composition and relative abundance in relation to malaria control focus mainly on members of the an. gambiae complex. this focus on only an. gambiae complex and marginalizing the an. funestus group may be due to fairly easy larval collection and adaptability of the an. gambiae complex to field insectary and laboratory conditions as well as the scarcity of members of the an. funestus group. the scarcity of an. funestus in zimbabwe is believed to be associated with consistent implementation of irs which commenced in the early 1950s and expanded in 1980s following national independence in 1980 and scaled up in the mid-1990s (munhenga, 2010). an. funestus was last reported about ten years ago by masendu et al. (2005) only at buffalo ranch in chiredzi district of masvingo province in the southern region of zimbabwe. more recently, it was reported in honde valley, zimbabwe (choi et al., 2014). despite continued irs programmes in place, there is a real possibility of an. funestus resurgence as the focus of entomological studies has been almost exclusively on the an. gambiae complex. consequently, the current malaria species composition and densities, especially of an. funestus remain unknown in the study areas. up-to-date information on species composition and densities of both primary and secondary vectors is crucially needed to properly devise and implement vector control activities to prevent malaria transmission and to assess their effectiveness. therefore, this study aimed to determine malaria vector species composition and relative abundance in burma valley and zindi in mutare and mutasa districts, respectively. materials and methods study areas the study was undertaken in burma valley administrative ward (19° 11’ s, 32° 48’ e), mutare district, and zindi administrative ward (18° 22’ s, 32° 56’ e), mutasa district of manicaland province, zimbabwe (figure 1). studies were done from may 2013 to april 2014. the sites are situated in rural areas with similar ecological settings. the ecological features of the sites, however, show that they consist predominantly of tropical savanna, grassland and woodland ecosystem characterized by grass, trees and bushes, widely spaced and typically associated with the tropical wet and dry climate type. the climate is tropical with annual temperature, relative humidity and rainfall ranges of 18-30°c, 65-85% and 900-1200 mm, respectively (taylor & mutambu, 1986). additionally, the climate presents three clearly different seasons: a cold-dry season between april and august, with little rains in april and at times extremely cold in july, hot and dry season in august through to october, completely without rains, and a hot-wet season stretching from november to march with a potential of high rainfall during the months of december and february (taylor & mutambu, 1986). both sites are valleys (679 and 766 meters above sea level for burma valley and zindi, respectively) with several streams and rivers that flow into mozambique. majority of the community members are subsistence farmers who grow mainly maize, bananas and yams along the river banks. income is derived from the sale of their agricultural produce in mutare, harare and bulawayo urban markets. the farmers use mainly pyrethroid and organophosphate classes of insecticides to protect their crops against several agricultural pests. selection of study sites was based on the ecological conditions which are potential breeding sites for malaria vectors. malaria transmission in the two study areas is a major public health problem and occurs seasonally, especially in november through to may with a peak in march/april (lukwa et al., 2014). in june and july, the two sites experience the lowest malaria transmission (lukwa et al., 2014). several strategies which included irs, llins, larval source management, intermittent preventive treatment in pregnancy, diagnosis and case management as well as social behaviour change communication were put into action to control both the malaria vectors and parasites, to reduce malaria transmission in the study sites. among these interventions, irs, llins and malaria case management have been scaledup to cover most villages of the study areas (lukwa et al., 2014). mosquito larval sampling and rearing weekly physical examinations of natural and human-made mosquito larval habitats were conducted to determine the availability of potential breeding sites for both an. gambiae sensu lato (s.l.) and an. funestus group for two months for five days per month from september to october 2013. types of breeding sites were categorised into humanmade and natural in origin and recorded. larval collection was performed once a month from november 2013 to april 2014. where mosquito larvae were present, 5-10 dips, depending on the size of the habitat, were taken using standard dippers of 350 ml capacity. samples were classified into: i) group of 1st and 2nd instars; ii) group of 3rd and 4th of instars; and iii) pupae. the instars were identified morphologically using taxonomic keys of gillies & de meillon (1968). the number of 1st to 4th instar larvae of each anopheline species collected per dip represented the larval density in each breeding site. immediately after morphological determination of species and larval density assessment, the anopheles larvae were placed in plastic jugs and taken to entomological field laboratories/insectaries for rearing according to who (2003) guidelines. all larvae were kept at room temperature and fed with ground fish food. the adults that emerged were transferred to the laboratory at national institute of health research (nihr) in harare. the specimens were killed by anaesthetizing with drops of acetyl acetate placed on a large filter paper that was held above the adults container. emerged adults were identified morphologically into species complexes (an. gambiae and an. funestus) using taxonomic keys of gillies & coetzee (1987). afterwards, they were individually preserved on silica gel in well-labelled eppendorf tubes prior to polymerase chain reaction (pcr) assays. other anophelines were morphologically identified (gillies & coetzee, 1987), recorded and discarded. article no nco mm er cia l u se on ly indoor collections indoor resting adult mosquitoes were collected by the pyrethrum spray catch (psc) method (who, 2003) in twenty conveniently selected bedrooms in burma valley and zindi, ten bedrooms apiece for one day per month from may 2013 to april 2014. the selected bedrooms were visited between 6:30 and 10:00 a.m. every study day to collect adult mosquitoes. anophelines were sorted and identified morphologically into an. gambiae s.l. and an. funestus group as well as other anophelines (gillies & coetzee, 1987). female specimens were preserved in labelled eppendorf tubes containing silica gel as drying agent awaiting further processing at nihr. males were recorded and discarded. polymerase chain reaction species identification polymerase chain reaction was carried out using deoxyribonucleic acid (dna) extracted from two legs of each morphologically identified specimen. an. gambiae s.l. specimens were pcr-assayed using a protocol described by scott et al. (1993) and amplified using specific diagnostic primers for an. gambiae, an. arabiensis and an. quadriannulatus. as for an. funestus group, samples were assayed following the methods of koekemoer et al. (2002) with minor modifications as detailed by spillings et al. (2009) using primers for an. funestus, an. leesoni, an. vaneedeni, an. parensis, an. rivulorum, an. rivulorumlike and an. funestus-like. the results of pcr amplification were visualized on 1% agarose gel by ethidium bromide staining. data analysis data were analysed using analysis of variance (anova) at 95% confidence limit. the relative abundance of the species was expressed as the percentage of the total number of anopheles collected. ethical considerations confidentiality and voluntary participation was assured to the households members who were included for psc. signed informed consent form was obtained from each participant before collecting mosquitoes from the bedrooms. results larval habitat census a total of 42 habitats positive for breeding of aquatic stages of mosquitoes were identified in the study areas (figure 2). rain pools were temporary, shallow wells, yam plantations and river banks were semipermanent, while irrigation channels as well as marshes were permanent mosquito habitats. of these breeding sites, 23 were in burma valley and 19 in zindi. for both study sites, the overwhelming majority of the anopheline-positive habitats were human-made (88.1%, 37/42) with the remainder natural in origin (11.9%, 5/42). chances of sampling anopheline larvae were higher in irrigation channels in zindi, but highly heterogeneous in yam plantations in burma valley area. all larval habitats were located within 2 km of the homesteads of both study sites. larval habitat support and relative abundance habitat support for larval development differed at the two study sites. in burma valley, 34.8% (8/23) of the habitats had only anopheline, while 17.4% (4/23) had only culicine, and were visited 12 times. in zindi, 15.8% (3/19) of the habitats had anopheline only and 36.8% (7/19) had only culicine, and were visited 10 times. this gave a com [journal of entomological and acarological research 2015; 47:4955] [page 81] article figure 1. map of manicaland province, zimbabwe, showing the study sites. no nco mm er cia l u se on ly [page 82] [journal of entomological and acarological research 2015; 47:4955] bined total of 22 longitudinal samples in 12 months for the two study areas. sympatry in anopheline and culicine larvae was found in 47.8% (11/23) of the habitats in burma valley and 47.4% (9/19) in zindi, suggesting that the mosquito larvae from the subfamilies anophelinae and culicinae coexist in almost half of the habitats. a total of 3227 immature stages of anopheline larvae (2438 early instars, 789 late instar) were collected in burma valley and 1621 (872 early instars, 749 late instars) in zindi. pupae were observed in 28.6% (12/42) of larvae-positive sites. anopheles larvae were more abundant in burma valley than in zindi (anova: df=6; f=12.11; p=0.00). the mean density of anopheles larvae over the entire sampling efforts was 4.2 and 1.8 larvae per dip in burma valley and zindi, respectively. the majority of an. funestus larvae were collected in irrigation channels, marshes and shallow wells, whilst an. gambiae s.l. larvae were found in rain pools, yam plantations and river banks. species composition of anopheline larvae of the approximately 4848 anopheles larvae collected, a total of 4690 adult mosquitoes emerged. from these, two malaria species complexes were morphologically identified, namely, an. funestus group and an. gambiae s.l. the an. funestus group accounted for (1.9%, 87/4690) of the adults, while an. gambiae s.l. constituted (0.2%, 10/4690), with an. pretoriensis, a non-malaria vector species contributing the majority (97.9%, 4593/4690) in both study sites. overall, the densities of adults of the an. funestus group and an. gambiae s.l. that emerged from the larval collections were low, but the an. funestus group was about 8 times more abundant than an. gambiae s.l. species composition and abundance of adult anopheles mosquitoes a total of 5625 anopheles adults, including males and females were collected from larval sampling and indoors through psc methods (table 1). of the total collections, an. pretoriensis was the most abundant while the remainder was shared between the an. funestus group and an. gambiae s.l. overall, more anopheline mosquitoes were collected from aquatic stages (i.e., reared from larvae) than indoors (i.e., as adults), but indoor collections by psc method had more malaria vector mosquitoes than larval collection. of note, the results showed that the combined female an. funestus group and an. gambiae s.l. were approximately 5 times more abundant than males (table 1). species composition and abundance of female adult malaria vectors the species composition and the relative abundance of female malaria vector mosquitoes are presented in table 2. altogether, more than 800 female an. funestus group and an. gambiae s.l. mosquitoes were collected. as observed from table 2, there was no difference in species composition in the two sites (anova: df=1; f=3.25; p=0.17), though the relative abundance of an. funestus group and an. gambiae article figure 2. types of mosquito breeding habitats in burma valley and zindi, mutare and mutasa districts respectively, zimbabwe: shallow well (a), yam plantation (b), irrigation channel (c), rain pool (d), marsh (e) and river bank (f). no nco mm er cia l u se on ly s.l. was more in burma valley than in zindi (anova: df=9; f=3.65; p=0.00). all in all, members of the an. funestus group were 27 times more abundant than an. gambiae s.l. in both sites (table 2). polymerase chain reaction analysis of an. funestus group sibling species from a total of 840 females that had been morphologically identified as members of the an. funestus group, 294 specimens were pcr assayed. two sibling species were identified: an. funestus (90.8%, 267/294) at a position of 505 base pairs (bp) and an. leesoni (5.1%, 15/294) at 146 bp (figure 3). twelve specimens (4.1%, 12/294) failed to amplify after two replicates, though the positive control amplified successfully. polymerase chain reaction based assays to identify sibling species of an. gambiae s.l. out of a total of 31 females positively identified to be an. gambiae s.l., 48.4% (15/31) were identified as an. quadriannulatus while 41.9% (13/31) were an. arabiensis (figure 4). about 9.7% (3/31) of the an. gambiae s.l. tested could not be identified to specific species by pcr using the then available primers specific for an. gambiae, an. arabiensis and an. quadriannulatus sibling species. percentage species composition and abundance of an. funestus and an. arabiensis in burma valley and zindi an. funestus and an. arabiensis sibling species were present in both study sites, though both species were less abundant in zindi than in burma valley (table 3). the relative abundance of an. arabiensis was generally low in both sites. overall, an. funestus was more abundant than an. arabiensis in both sites (anova: df=2; f=14.20; p=0.02) (table 3). discussion and conclusions the study showed the presence of two anopheles complexes: an. gambiae and an. funestus in burma valley and zindi. both complexes contain important malaria vectors in sub-saharan africa (coetzee et al., 2000). morphologically identified adults reared from larval collections revealed the presence of predominantly an. pretoriensis (a nonmalaria vector) and relatively low numbers of adults of the an. funestus group and an. gambiae s.l. the members of the an. funestus group were, however, relatively more abundant than members of an. gambiae [journal of entomological and acarological research 2015; 47:4955] [page 83] article table 1. percentage mosquito species composition sampled in burma valley and zindi as identified by morphological means (actual numbers in parentheses). sampling method study area n an. funestus group an. gambiae s.l. an. pretoriensis male female male female psc burma valley 795 16.0 80.6 0.5 2.8 0.1 zindi 140 9.3 87.1 0.7 2.9 0 rfl burma valley 3141 0.3 1.1 0.1 0.1 98.4 zindi 1549 0.1 2.8 0.1 0.1 96.9 total 5625 2.6 14.9 0.2 0.6 81.7 psc, pyrethrum spray catch; rfl, reared from larvae. table 2. species composition (%) and abundance of an. funestus group and an. gambiae s.l. adult females by sampling method in burma valley and zindi areas (count data in parentheses). sampling method study area total number an. funestus group an. gambiae s.l. psc burma valley 663 96.7 (641) 3.3 (22) zindi 126 96.8 (122) 3.2 (4) rfl burma valley 37 91.9 (34) 8.1 (3) zindi 45 95.6 (43) 4.4 (2) total 871 96.4 (840) 3.6 (31) psc, pyrethrum spray catch; rfl, reared from larvae. table 3. percentage of species composition and abundance of an. funestus and an. arabiensis in burma valley and zindi areas (count data in parentheses). study area total number an. funestus an. arabiensis burma valley 169 95.3 (161) 4.7 (8) zindi 111 95.5 (106) 4.5 (5) total 280 95.4 (267) 4.6 (13) no nco mm er cia l u se on ly [page 84] [journal of entomological and acarological research 2015; 47:4955] s.l. (anova: df=4; f=4.44; p=0.04). these findings are consistent with previous studies in zimbabwe that demonstrated dominance of an. pretoriensis reared from larvae over other anopheline species (masendu et al., 2005). this study clearly showed that malaria vectors belonging to the an. funestus group and an. gambiae s.l. breed in sympatry in the study sites. most of the mosquito larval habitats in the study areas are humanmade; suggesting that malaria transmission in the study areas is derived mainly from human modification and manipulation of the ecosystem. while the irrigation facilities are important to sustain food security, mitigating measures need to be put in place to minimize breeding of vector mosquitoes. in the present study, members of the an. funestus group were more abundant than members of an. gambiae s.l. from both adult and larval collections. previous work by mpofu (1985), taylor and mutambu (1986) and masendu et al. (2005) had shown members of an. gambiae s.l. to be the more abundant malaria vector in zimbabwe. using pcr, the present study identified two species: an. funestus and an. arabiensis that transmit malaria in the study sites. pcr identification of sibling species of the an. funestus group and an. gambiae s.l. is of great importance to the malaria control programme in zimbabwe. molecular analysis of the an. funestus group established the presence of an. funestus and an. leesoni. this is in agreement with studies by oyewole et al. (2005) but in contrast with other previous studies in africa which recorded an. funestus in sympatry with minor vectors an. rivulorum and an. leesoni (wilkes et al., 1996). the failure to amplify 5.1% of the an. funestus group could be due to morphological misidentification of some specimens and/or emergence of other sibling species. meanwhile, pcr assay of an. gambiae s.l. also revealed two common members: an, quadriannulatus and an. arabiensis, but could not exclude the possibility that other members of the complex might be article figure 3. identification of members of an. funestus group from burma valley and zindi: negative control (lane a), positive control (lane b), 100 base pair molecular ladder (lane i), an. funestus (505 base pair) (lanes c, d, f and r), an. leesoni (146 base pair) (lanes g, h, j, m, n and o), an. funestus and an. leesoni mixed dna (lane k), no amplification (lanes e, l, p, q, s and t). figure 4. species identification of members of an. gambiae s.l. from burma valley and zindi: an. quadriannulatus (150 base pair) (lanes 1, 3-8, 10, 12-14 and 16), an. arabiensis (315 base pair) (lanes 2, 11 and 15), 100 base pair molecular marker (lane 9). no nco mm er cia l u se on ly amongst the specimens (9.7%) that failed to amplify. it is likely that the unidentified an. gambiae s.l. could be an. merus as the protocol by scott et al. (1993) used in this study did not include primers to identify an. merus which masendu et al. (2005) reported as being common in some parts of zimbabwe. polymerase chain reaction assays revealed an. funestus to be more abundant than an. arabiensis, confirming the status of the former as a major malaria vector mosquito in both sites. this is in sharp contrast with results from work by mpofu (1985), taylor & mutambu (1986) and masendu et al. (2005) which had shown an. arabiensis to be a primary vector while an. gambiae and an. funestus were secondary vectors in zimbabwe. the sharp increase in an. funestus population in the presence of irs implementation in this study sites cannot be explained in this study. it is more likely that pyrethroid-resistant an. funestus survived undetected while an. arabiensis and to a larger extent, an. gambiae, remained susceptible to pyrethroids which have been used for irs and treatment of llins over the years in the study areas. in general, species replacement for various reasons, especially as a result of irs and llins is not a new phenomenon (kitau et al., 2012). recent data from east africa showed changes in sibling species following the scaling-up of itns/llins, with an. arabiensis becoming the dominant species in habitats that previously supported sympatric an. gambiae and an. arabiensis populations (kitau et al., 2012). a similar change in species composition and abundance was reported during the implementation of irs in zimbabwe; example being the observation of an. funestus in most parts of zimbabwe by mpofu (1985), which was isolated in later studies by masendu et al. (2005) only at buffalo ranch in chiredzi district. as revealed by the findings of this study, heterogeneity of vector species composition and dominance of an. funestus is of major significance in malaria control. the results have important implications for malaria epidemiology and control given that an. funestus is a more efficient vector than an. arabiensis. low densities of an. funestus have the potential to sharply increase levels of malaria transmission (morgan et al., 2010). this study also revealed that there is information gap on the resting, biting and insecticide susceptibility status of malaria vectors in the study sites. the information is crucial for planning, implementation and evaluating malaria vector control strategies. additionally, the emergence/upsurge of an. funestus in areas under irs and llins use requires urgent attention to prevent possible malaria outbreaks. mitigation measures for irrigation projects or farming should be put in place to minimize malaria vector breeding. references choi k.s., christian r., nardini l., wood o.r., agubuzo e., muleba m., munyati s., makuwaza a., koekemoer l.l., brooke b.d., hunt r.h., coetzee m., 2014 insecticide resistance and role in malaria transmission of anopheles funestus populations from zambia and zimbabwe. parasit vectors. 7: 464. coetzee m., craig m., le sueur d., 2000 distribution of african malaria mosquitoes belonging to the anopheles gambiae complex. parasitol. today. 16: 74-77. cohuet a., simard f., toto j.c., kengne p., coetzee m., fontenille d., 2003 species identification within the anopheles funestus group of malaria vectors in cameroon and evidence for a new species. am. j. trop. med. hyg. 69: 200-205. coluzzi m., 1984 heterogeneities of the malaria vectorial system in tropical africa and their significance in malaria epidemiology and control. bull. world health organization 62: 107-113. gillies m.t., coetzee m., 1987 a supplement to the anophelinae of africa south of the sahara (afro tropical region). south africa institute for medical research, johannesburg. publications of the south africa institute for medical research: 55. gillies m.t., de meillon b., 1968 the anophelinae of africa south of the sahara. south africa institute for medical research, johannesburg. publications of the south africa institute for medical research: 54. gregory c.l., yoosook l., 2013 speciation in anopheles gambiaethe distribution of genetic polymorphism and patterns of reproductive isolation among natural populations. intech. available from: http://dx.doi.org/10.5772/56232 kitau j., oxborough r.m., tungu p.k., matowo j., malima r.c., magesa s.m., bruce j., mosha f.w., rowland m.w., 2012 species shifts in the anopheles gambiae complex: do llins successfully control anopheles arabiensis? plos one 7: e31481. koekemoer l.l., kamau l., hunt r.h., coetzee m., 2002 a cocktail polymerase reaction assay to identify members of the anopheles funestus (diptera: culicidae) group. am. j. trop. med. hyg. 66: 804-811. lukwa n., sande s., makuwaza a., chiwade t., netsa m., asamoa k., vazquez-prokopec g., reithinger r., williams j., 2014 nationwide assessment of insecticide susceptibility in anopheles gambiae populations from zimbabwe. malaria j. 13: 408. masendu h.t., hunt r.n., koekemoer l.l., brooke b.d., 2005 spatial and temporal distributions and insecticide susceptibility of malaria vectors in zimbabwe. afr. entomol. 13: 25-34. molineaux l., gramiccia g., 1980 the garki project: research on epidemiology and control malaria in the sudan savanna of west africa. world health organization. geneva, switzerland. available from: http://garkiproject.nd.edu/static/documents/garkiproject.pdf morgan j.c., irving h., okediv l.m., steven a., wondji c.s., 2010 pyrethroid resistance in an anopheles funestus population from uganda. plos one 29: e11872. mpofu s.m., 1985 seasonal vector density and disease incidence pattern in an area of zimbabwe. trans. r. soc. trop. med. hyg. 79: 169-175. munhenga g., 2010 characterization of resistance mechanisms in the major malaria vector anopheles arabiensis from southern africa. phd thesis university of witwatersrand, south africa: 5-12. oyewole i. o., ibidapo a.c., oduola a.o., obansa j.b., awolola s.t., 2005 -molecular identification and population dynamics of the major malaria vectors in a rainforest zone of nigeria. nigerian soc. exp. biol. 17: 171-178. scott j.a., brogdon w.g., collins f.h., 1993 identification of single specimens of the anopheles gambiae complex by the polymerase chain reaction. am. j. trop. med. hyg. 49: 520-529. spillings b.l., brooke b.d., koekemoer l.l., chiphwanya j., coetzee m., hunt r.h., 2009 a new species concealed by anopheles funestus giles, the major malaria vector in africa. am. j. trop. med. hyg. 81: 510-515. taylor p., mutambu s.l., 1986 a review of the malaria situation in zimbabwe with special reference to the period 1972-1981. trans. r. soc. trop. med. hyg. 80: 12-19. who, 2003 malaria entomology and vector control. learners’ guide. who/cds/cpe/smt/2202.18 rev.1 part 1. world health organization, geneva. who, 2013 an operational manual for indoor residual spraying for malaria transmission control and elimination. world health organization, geneva. available from: http://www.int/malaria/publications/atoz/9789241505123/en wilkes t.j., matola y.g., charlwood j.d., 1996 anopheles rivulorum, a vector of human malaria in africa. med. vet. entomol. 10: 108-110. [journal of entomological and acarological research 2015; 47:4955] [page 85] article no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2016; 48:4911] [page 1] abstract this study was conducted in order to describe and measure the morphological characters of adults and larval stages of the cicada cicadatra persica distributed in erneh, syria. eggs, newly hatched nymphs, fully developed nymphs, exuviae and adults were described and measured. the mean lengths of eggs (1.9±0.1 mm) were the same as newly hatched nymphs (1.8±0.174 mm), whereas the mean length of the fully developed nymphs (3.1±0.185 cm) was longer than the adults. several parts of adults’ body (males and females) were measured. the results show that the mean of females bodies (2.60±0.118 cm) were larger than males ones (2.45±0.192 cm), nevertheless, there were no significant differences between them. introduction little is known about the species cicadatra persica kirkaldy 1909, which is distributed in middle east countries and surrounding areas. c. persica was reported for the first time in macedonia and its song was recorded and analyzed (gogala & trilar, 1998, 2003). kartal & zeybekoğlu (1996) described the internal and external structure of the reproductive system of its adults. researches on c. persica distribution in syria are limited. the only researches conducted on this species were by dardar et al. (2012, 2013a, 2013b), dardar and belal (2013) emphasizing the morphological differences among egg nests and adult individuals of c. persica. in this study, pictures are given for adults, larval stages and eggs of c. persica and some morphological characters are measured. materials and methods collecting individuals adults and larval stages of c. persica were collected from apple fruit orchards in the village erneh that is located in al-sheikh mountain in the south west of syria. the eggs were collected by cutting the twigs that hold egg nests from apple fruit orchards. the newly hatched nymphs were collected by using the traps described by dardar et al. (2012) and fixed in an ethanol and glycerin solution to maintain body soft to aid the study with a stereomicroscope. the fully developed nymphs were collected by digging in the soil during adults’ emergence period. adults were collected by hand and with emergence traps described by dardar et al. (2012) from the orchards at sunset and sunrise times, when the insects are collected in larger numbers. morphometric study morphological characters of adults and larval stages identified by moulds (2005) were studied by using a stereomicroscope (olympus sz61, optical lens: 10x, body lens: 2x). the length of body of adults (n=11 males, n=11 females) and fully development nymphs (n=8) was measured in cm by a ruler; 34 and 30 morphological characters of males (n=11) and females (n=11) of c. persica were measured. the length of different parts of body of adults was measured with the stereomicroscope with a magnification of 0.67. the length of the body and different parts of the body of the newly hatched nymphs (n=6) was also measured with a magnification of 4.5. the real length of different part of the body was measured in mm. correspondence: marah a. derdar, department of insects research, administration of plant protection research, general commission for scientific agricultural research, damascus, syria. e-mail: marah.derdar@hotmail.com hamzeh m.r. belal, department plant protection, faculty of agriculture, university of damascus, damascus, syria. e-mail: cecehamz@scs-net.org key words: cicadidae; morphological characters; egg nest; apple orchards; erneh. acknowledgements: the general commission for scientific agricultural research supported this research. we thank the biological control studies and research center, which is dependent to the faculty of agriculture, university of damascus, syria. we also thank the farmer ismael masoud in village of erneh for permitting us to collect samples from his orchards. received for publication: 16 december 2014. revision received: 22 march 2015. accepted for publication: 13 october 2015. ©copyright m.a. derdar and h.m.r. belal, 2016 licensee pagepress, italy journal of entomological and acarological research 2016; 48:4911 doi:10.4081/jear.2016.4911 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2016; volume 48:4911 morphometry of adults and larval stages of cicadatra persica (cicadidae: hemiptera) distributed in erneh, syria m.a. derdar,1 h.m.r. belal2 1department of insects research, administration of plant protection research, general commission for scientific agricultural research, damascus; 2department plant protection, faculty of agriculture, university of damascus, damascus, syria no nco mm er cia l u se on ly [page 2] [journal of entomological and acarological research 2016; 48:4911] statistical analysis means and standard deviations were calculated for the length of different parts of adults’ body and larval stages using spss software (ibm corp., armonk, ny, usa). two independent samples t-test were carried out for the length of some morphological characters in order to compare males and females of c. persica. results and discussion morphometric study the eggs the color of the newly laid eggs is white (figure 1) when newly laid and then it changes gradually into black light yellow (figure 2), dark yellow, light rosy, and completely rosy (figure 3) before hatching. the mean length of the egg of c. persica was 0.1±1.9 mm (n=6). the newly hatched nymph the body of the nymph is rosy. the head is conical and ends with a long rostrum. ocelli are absent; there are two pairs of eyes and antennas on the both lateral sides of the head (figure 4). it has sucking mouthparts. the pronotum is well developed and longer than mesonotum and metanotum in order to support the fore legs to dig in the soil. the wing pads are absent. the sixth pairs of legs are transparent. the fore femur is large and has a vertical spine on the lateral side of the leg. in this age, we cannot distinguish between the genital organs of males and females. each antenna consists of 7 transparent segments (figure 5). the lengths of 24 morphological characters for newly hatched nymphs (n=6) of c. persica were measured (table 1). the fully development nymph the color of the body is brown (figure 6). the head is triangular and held two pairs of eyes and antennas. each antenna consists of 7 segments. the ocelli are absent. it has sucking mouthparts. mesonotum is larger than pronotum and hold fore wing pads. the metanotum is small and hold the hind wing pads (figure 7). there are six pairs of legs, and the fore legs hold 3 spines that are different in size on the lateral side of the leg (figure 8). the abdomen consists of 8 segments end with the genital organ. the mean length of the body’s nymph was 3.1±0.185 cm (n=8). article figure 1. white eggs. figure 2. yellowish eggs. figure 3. rosy eggs. figure 4. the newly hatched nymph. no nco mm er cia l u se on ly [journal of entomological and acarological research 2016; 48:4911] [page 3] article figure 5. antenna of the newly hatched nymph. figure 6. the full development nymph. figure 7. full development nymph (ventral view). figure 8. fore legs of the full development nymph. table 1. the measurements (in mm) of the morphological characters for newly hatched nymphs of cicadatra persica (n=6). the character mean±std. the character mean±std. body length 1.8±0.174 tibia 3 0.34±0.014 width of head capsulate 0.30±0.025 tarsi 1 0.11±0.00 pronotum width 0.375±0.0748 tarsi 2 0.12±0.026 coxa 1 0.22±0.031 tarsi 3 0.15±0.018 coxa 2 0.16±0.035 claw 1 0.04±0.011 coxa 3 0.22±0.031 claw 2 0.05±0.006 trochanter 1 0.16±0.027 claw 3 0.05±0.011 trochanter 2 0.13±0.025 scape 0.11±0.008 trochanter 3 0.12±0.030 pedicel 0.11±0.010 femur 1 0.25±0.033 1 0.10±0.005 femur 2 0.21±0.070 2 0.08±0.009 femur 3 0.27±0.034 segments of flagellum of antenna 3 0.07±0.010 tibia 1 0.20±0.034 4 0.05±0.005 tibia 2 0.25±0.021 5 0.05±0.005 std., standard deviation. no nco mm er cia l u se on ly [page 4] [journal of entomological and acarological research 2016; 48:4911] the adult the adult emerges from the final stage of nymph by molting that takes about 30-40 min. the newly emerged adult is soft and dull. it remains quit at rest without any movement on her exuvia, and takes her natural color and her wings begin to harden gradually (figure 9). then it flies and leaves its exuvia (figure 10) on the soil surface, trunks, or weeds. the body of adult is black; the head is triangular and hold two pair of compound eyes, 3 ocelli and one pair of antennae. each antenna consists of 5 segments (figure 11). it has sucking mouthparts. the wings are membranous with black veins and orange color at the base. the abdomen consists of 8 segments end with the genital organ. the male thirty-four morphological characters for males (n=11) of c. persica were measured (table 2). the female thirty morphological characters for females (n=11) of c. persica were measured (table 3). article figure 9. molting. figure 10. exuviae of cicadatra persica. figure 11. the antenna of cicadatra persica. table 2. measures (in cm) of the morphological characters for males (n=11) of cicadatra persica. the character mean±std. the character mean±std. body length 2.45±0.192 coxa 2 3.61±0.305 width of head capsulate 9.04±1.162 coxa 3 3.45±0.317 pronotum width 11.34±1.248 trochanter 1 3.10±0.349 wings span 7.02±0.199 trochanter 2 2.40±0.366 fore wing 3.20±0.109 trochanter 3 2.04±0.258 hind wing 1.809±0.083 femur 1 4.78±0.606 post clypeus 4.36±0.445 femur 2 5.18±0.611 operculum 6.96±1.000 femur 3 5.30±0.761 aedeagus 5.76±0.998 tibia 1 5.18±0.663 pygofer 6.454±0.656 tibia 2 6.68±0.791 upper lap 0.61±0.255 tibia 3 7.78±0.885 basal lap 0.57±0.127 tarsi 1 2.61±0.353 dorsal peak of pygofer 1.26±0.262 tarsi 2 2.61±0.321 claspers 1.80±0.475 tarsi 3 2.64±0.443 uncus 0.46±0.112 claw 1 1.2±1.924 sternite viii 6.91±0.702 claw 2 0.72±0.075 coxa 1 5.49±0.737 claw 3 0.70±0.077 std., standard deviation. table 3. measures (in cm) of the morphological characters for females (n=11) of cicadatra persica. the character mean±std. the character mean±std. body length 2.60±0.118 trochanter 1 2.88±0.252 width of head capsulate 8.20±0.340 trochanter 2 2.17±0.210 pronotum width 10.91±0.712 trochanter 3 1.82±0.236 wings span 7.04±0.311 femur 1 4.34±0.238 fore wing 3.19±0.158 femur 2 4.60±0.190 hind wing 1.84±0.067 femur 3 4.77±0.253 post clypeus 3.94±0.298 tibia 1 4.55±0.225 ovipositor 9.68±0.531 tibia 2 5.78±0.646 abdominal segment 9.30±0.703 tibia 3 6.97±0.320 ovipositor sheath 3.84±0.361 tarsi 1 2.34±0.192 valvifer 2 4.97±0.450 tarsi 2 2.34±0.163 dorsal peak 0.94±0.143 tarsi 3 2.34±0.197 coxa 1 4.71±0.359 claw 1 0.61±0.070 coxa 2 3.45±0.163 claw 2 0.64±0.052 coxa 3 3.33±0.168 claw 3 0.62±0.075 std., standard deviation. no nco mm er cia l u se on ly conclusions there was no significant differences in the body length, wingspan, or pronotum between males and females of c. cicadatra (p=0.279, p=0.148, p=0.251). however, the results show that there were significant differences in the width of head’s capsulate and length of the third tibia (p=0.001, p=0.001); the mean width of head’s capsulate of the male was bigger than female’s one, while the mean length of the third tibia of the male was bigger than female’s one. references dardar m.a., belal h.m.r., 2013 morphological differences among egg nests and adult individuals of cicadatra persica (hemiptera: cicadidae), distributed in erneh, syria. zookeys. 319: 11-25. dardar m.a., belal h.m.r., basheer a.m., 2012 observation on some biological aspects of cicadatra persica (cicadidae: hemiptera) in apple fruit orchards in erneh, syria. j. entomol. acarol. res. 44: 56-59. dardar m.a., belal h.m.r., basheer a.m., 2013a a study on egg nests of cicadatra persica (hemiptera: cicadidae) distributed in erneh, syria. ann. soc. entomol fr. 49: 273-276. dardar m.a., belal h.m.r., basheer a.m., 2013b occurance of cicadatra persica on apple trees malus domestica in erneh, syria. j. insect sci. 13: 1-5. gogala m., trilar t., 1998 first record of the cicadatra persica kırkaldy. 1909 from macedonia, with description of its song. acta entomol. sloven. 6: 5-15. gogala m., trilar t., 2003 video analysis of wing clicking in cicadas of the genera cicadatra and pagiphora (homoptera: auchenorrhyncha: cicadodea). acta entomol. sloven. 11: 5-15. kartal v., zeybekoğlu ü., 1996 an investigation on the morphology of genital organs and oviposition capacity of cicadatra persica kirkaldy, 1909 (cicadidae, hemiptera). zoology. 1: 59-62. moulds m.s., 2005 an appraisal of the higher classification of cicadas (hemiptera: cicadoidea) with special reference to the australian fauna. records austr. museum. 57: 375-446. [journal of entomological and acarological research 2016; 48:4911] [page 5] article no nco mm er cia l u se on ly jear2012 abstract asir region in the southwest of saudi arabia has been a subject for expansion of agricultural projects, urbanization, which presumably have impact on distribution of phlebotomine sandflies. few reports are available on sandflies in this region which is an important focus of cutaneous leishmaniasis. therefore, this study aimed at updating the species composition, distribution and periodical fluctuation of sandflies in this region. specimens were monthly collected by the center for disease control light traps for one year in four localities representing different altitudes. in five other, collections were twice during the year period. ten species (six phlebotomus and four sergentomyia) were identified, of which p. arabicus (32%) was the most common followed by p. bergeroti (29%) and p. sergenti (15%). of the reported species, s. palestinensis is considered a new record from asir. sandflies were more common and maximum biodiversity was observed in lowlands and not in high altitudes. at different altitudes, the two commonest species were more active during spring. sandfly density (sandfly/trap) was directly related to temperature and inversely related to altitude, relative humidity (rh) and wind velocity (p<0.05). to sum up, the distribution and abundance of sandflies in asir are influenced by a combination of different factors: temperature, rh, wind velocity and altitude. introduction phlebotomine sandflies (diptera: psychodidae) are widespread in the tropics and subtropics (lane, 1993). they include many potential vectors for both cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). cl is an endemic disease in many areas of saudi arabia including the western provinces (al-qurashi et al., 2000; el-badry et al., 2008). the disease is more common on the foothills and high plateau of asir region in the southwest of the kingdom (al-zahrani et al., 1989; abdelwahab & abdoon, 2005). vl infections also recovered from the south-west of the kingdom (al-zahrani et al., 1988a; ibrahim et al., 1995; al-jaser, 2006). several reports are available on the distribution of the sandfly fauna in several regions of saudi arabia (lewis & büttiker, 1980, 1982; killick-kendrick et al., 1985; morsy & al seghayer, 1992; abu-zinada, 1999; aldawood et al., 2004; al barrak, 2005; el-badry et al., 2009; doha & samy, 2010) revealing the presence of 25 species with the dominance of p. papatasi (mustafa et al., 1994). in only three occasions (lewis & büttiker, 1982; abdelwahab & abdoon, 2005; alahmed et al., 2010), the sandflies were surveyed in asir with 22 species identified. during the past few decades, saudi arabia has viewed tremendous efforts in social development and urbanization in all provinces, and insect fauna, particularly sandflies was affected. the expansion of agricultural projects, urbanization and development of water resources, lead to creation of new breeding sites for sandflies (alahmed et al., 2010). the asir area has also been a subject for various development and tourist projects as well as resettlement programs which presumably have impact on distribution of sandflies. however, few reports (lewis & büttiker, 1982; abdelwahab & abdoon, 2005; alahmed et al., 2010) described sandflies in asir region although cl has long been recognized as an important public health problem in this area (al-zahrani et al., 1988b). therefore, this study aimed at identifying and updating the sandfly species composition, their geographical distribution, periodical abundance and species diversity in some localities representing different altitudes in asir region as one of cl-endemic areas in saudi arabia. the study could be important for implementing any large-scale control project. materials and methods study area asir region (19°00′n, 42°00′e to 19°00′n, 43°00′′e) in the southcorrespondence: mohamed a. kenawy, department of entomology, faculty of science, ain shams university, abbassia, cairo 11566, egypt. tel.: +202.24821633 / 24821096 / 24821031, ext: 711 fax: +202.26839622. mobile: +2.01223540005 / 01111151554. e-mail: mohamedkenawy85@yahoo.com key words: sandflies; species composition; species diversity; seasonal abundance; asir region; saudi arabia. acknowledgements: the authors would like to thank dr. bahira el sawaf, entomology department, faculty of science, ain shams university cairo, egypt for helpful comments and suggestions that really improved the manuscript. received for publication: 21 february 2015. revision received: 23 march 2015. accepted for publication: 23 march 2015. ©copyright m.a. kenawy et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:5016 doi:10.4081/jear.2015.5016 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. distribution and periodicity of sandflies (diptera: phlebotominae) along different altitudes in asir region, southwest of saudi arabia m.a. kenawy,1 h.a. al ashry,2 m. shobrak3 1department of entomology, ain shams university, abbassia, cairo, egypt; 2trap pest control and garden maintenance co. ltd., jeddah, kingdom of saudi arabia; 3biology department, science college, taif university, taif, kingdom of saudi arabia [page 56] [journal of entomological and acarological research 2015; 47:5016] journal of entomological and acarological research 2015; volume 47:5016 no nco mm er cia l maximum biodiversity was observed in lowlands and not in high altino nco mm er cia l maximum biodiversity was observed in lowlands and not in high altitudes. at different altitudes, the two commonest species were more no nco mm er cia l tudes. at different altitudes, the two commonest species were more active during spring. sandfly density (sandfly/trap) was directly related to no nco mm er cia l active during spring. sandfly density (sandfly/trap) was directly related to temperature and inversely related to altitude, relative humidity (rh) and no nco mm er cia l temperature and inversely related to altitude, relative humidity (rh) and wind velocity (p<0.05). to sum up, the distribution and abundance of no nco mm er cia l wind velocity (p<0.05). to sum up, the distribution and abundance of ease is more common on the foothills and high plateau of asir region in no nco mm er cia l ease is more common on the foothills and high plateau of asir region inthe southwest of the kingdom (al-zahrani no nco mm er cia l the southwest of the kingdom (al-zahrani abdoon, 2005). vl infections also recovered from the south-west of the no nco mm er cia l abdoon, 2005). vl infections also recovered from the south-west of the kingdom (al-zahrani no nco mm er cia l kingdom (al-zahrani several reports are available on the distribution of the sandfly fauna no nco mm er cia l several reports are available on the distribution of the sandfly fauna no nco mm er cia l in several regions of saudi arabia (lewis & büttiker, 1980, 1982; no nco mm er cia l in several regions of saudi arabia (lewis & büttiker, 1980, 1982; no nco mm er cia l correspondence: mohamed a. kenawy, department of entomology, faculty no nco mm er cia l correspondence: mohamed a. kenawy, department of entomology, faculty of science, ain shams university, abbassia, cairo 11566, egypt. no nco mm er cia l of science, ain shams university, abbassia, cairo 11566, egypt. tel.: +202.24821633 / 24821096 / 24821031, ext: 711 fax: +202.26839622. no nco mm er cia l tel.: +202.24821633 / 24821096 / 24821031, ext: 711 fax: +202.26839622. mobile: +2.01223540005 / 01111151554. no nco mm er cia l mobile: +2.01223540005 / 01111151554. e-mail: mohamedkenawy85@yahoo.comno nco mm er cia l e-mail: mohamedkenawy85@yahoo.com key words: sandflies; species composition; species diversity; seasonal abun-no nco mm er cia l key words: sandflies; species composition; species diversity; seasonal abunus e tropics and subtropics (lane, 1993). they include many potential vectors us e tropics and subtropics (lane, 1993). they include many potential vectors for both cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). us e for both cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl).cl is an endemic disease in many areas of saudi arabia including the us e cl is an endemic disease in many areas of saudi arabia including the western provinces (al-qurashi us e western provinces (al-qurashi ease is more common on the foothills and high plateau of asir region inus e ease is more common on the foothills and high plateau of asir region in on ly on ly phlebotomine sandflies (diptera: psychodidae) are widespread in theon ly phlebotomine sandflies (diptera: psychodidae) are widespread in the tropics and subtropics (lane, 1993). they include many potential vectorson ly tropics and subtropics (lane, 1993). they include many potential vectors for both cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). on ly for both cutaneous leishmaniasis (cl) and visceral leishmaniasis (vl). west of saudi arabia has an area of 81,000 km² and a population of 1,913,392 (2010 census). it is a mountainous area divided into 3 distinct topographical zones: i) sarawat asir which is a mountain range extending north-south along the coastal plains of the red sea and that rise to almost 3000 m at jebel sawdah near abha the capital; ii) asir plateau and iii) tehamah plain (tehama) which is a narrow sandy coastal strip of lowlands at sea level. the region receives more rainfall than the rest of the country and falling in two seasons: spring (march and april) and summer (june to august). temperature in asir highlands is generally lower than in the other part of the region and the rest of the kingdom as well. the coastal plain zone is generally characterized by lower rainfall, high temperature and relative humidity (rh). the study included nine localities representing different altitudes. collection of sandflies sandfly collections were carried out monthly (june 2009 to may 2010) in abha, bishah, muhayil and tanomah (al namas), and twice during the year period in the sarat abidah, rejal almaa, balqarn, al bark and tathleth. in each locality, 2-3 collection sites were selected to represent different habitats: wadies (valleys), farms, dumpsites and sheep raising farms. sandflies were collected by the center for disease control (cdc) miniature light traps (john w hock co, gainesville, fl, usa). the traps (3 traps/site/night) were set before sunset and collected after sunrise next morning. collected phlebotomines were preserved in 70% alcohol, cleaned in chloral hydrate: phenol (1:1 vol/vol) and then mounted in puri’s medium for identification using different keys (lewis, 1982; lane, 1986; kakarsulemankhel, 2009, 2010). along with fly collections, the elevation of the collection site above sea level, the weather temperature, rh and wind velocity (km/h) were recorded. periodical abundance the periodic abundance of the common phlebotomus spp. was examined in localities representing lowlands, moderately altitude areas and highlands. statistical analysis the relations of sandfly density (no/trap) to the locality altitude and weather conditions (temperature, rh and wind velocity) were examined respectively by simple and multiple regression analysis. the slopes of the regression equations were tested for deviation from 0 by t-test. for the four localities that were monthly surveyed during the year period, the diversity of the sandfly based on the simpson (1d) and shannon (h) indices was examined. the paleontological statistics (past) software ver. 2.08 was used for data analysis (hammer et al., 2001). results species composition and relative abundance a total of 972 sandflies (454 males and 518 females) of two genera were collected: phlebotomus which was more common (621 fly: 63.89%) and sergentomyia (351 fly: 36.11%). six hundred forty flies (246 males and 394 female) were identified into 10 species (table 1): 6 phlebotomus and 4 sergentomyia. phlebotomus arabicus was the most common species (32.19%) followed by p. bergeroti (29.38%), p. sergenti (15.00%), p. orientalis (11.09%), p. papatasi (5.94%), s. clydei (3.44%), and p. alexandri (2.03%). s. africana, s. palestinensis and s. christophersi were rare (0.31% each). based on the present and previous surveys a complete list of sandfly species (8 phlebotomus spp. and 15 sergentomyia spp.) reported in asir is presented in table 2. geographical distribution the species composition varied among the surveyed localities (figure 1). the reported species were encountered in all altitudes, except s. christophersi and s. africana which were reported only in low lands (<500 m) and p. alexandri and s. palestinensis which were collected only from highlands (>2000 m). sandflies were more common (8 species) and more abundant in lowlands (48.10% of collected flies) than in moderately altitude areas (27.99%) or in high lands (23.9%) (table 3). regression analysis indicated that fly density (fly/trap) was inversely related to the altitude (b=–0.003, p<0.05). species diversity the diversity for the sandfly sampled monthly in the four localities was examined. the results revealed maximum diversity in muhayil (low altitude) with the highest simpson index (1-d=0.69) and shannon index (h=1.48). on the other hand, abha (high altitude) represented the site with the minimum diversity indices (1-d=0.59 and h=1.10). the rest of the localities exhibited medium biodiversity indices (figure 2). effect of weather conditions regression analysis indicated that the fly density (fly/trap) was directly related to temperature (b=0.413, p<0.01) and inversely related to rh (b=–0.002, p<0.05) and wind velocity (b=–0.170, p<0.05). periodic abundance the seasonal activity patterns of the two common species (p. arabicus and p. bergeroti) across the different altitudes were examined (figures 3 [journal of entomological and acarological research 2015; 47:5016] [page 57] article table 1. relative abundance of reported sandfly species in asir region. genus species no. % phlebotomus p. (phlebotomus) bergeroti parrot 188 29.38 p. (phlebotomus) papatasi (scopoli) 38 5.94 p. (paraphlebotomus) alexandri sinton 13 2.03 p. (paraphlebotomus) sergenti parrot 96 15.00 p. (adlerius) arabicus theodor 206 32.19 p. (larroussius) orientalis parrot 71 11.09 sergentomyia s. (parrotomyia) africana (newstead) 2 0.31 s. (parrotomyia) palestinensis (adler & theodor) 2 0.31 s. (sintonius) christophersi (sinton) 2 0.31 s. (sintonius) clydei (sinton) 22 3.44 total no. (male/female) 640 (246/394) sex ratio m:f 1:1.60 no nco mm er cia l spp. was examno nco mm er cia l spp. was examined in localities representing lowlands, moderately altitude areas and no nco mm er cia l ined in localities representing lowlands, moderately altitude areas and the relations of sandfly density (no/trap) to the locality altitude no nco mm er cia l the relations of sandfly density (no/trap) to the locality altitude and weather conditions (temperature, rh and wind velocity) were no nco mm er cia l and weather conditions (temperature, rh and wind velocity) were examined respectively by simple and multiple regression analysis. no nco mm er cia l examined respectively by simple and multiple regression analysis. the slopes of the regression equations were tested for deviation from no nco mm er cia l the slopes of the regression equations were tested for deviation from 0 by t-test. for the four localities that were monthly surveyed during no nco mm er cia l 0 by t-test. for the four localities that were monthly surveyed during the year period, the diversity of the sandfly based on the simpson (1no nco mm er cia l the year period, the diversity of the sandfly based on the simpson (1d) and shannon (h) indices was examined. the paleontological stano nco mm er cia l d) and shannon (h) indices was examined. the paleontological statistics (past) software ver. 2.08 was used for data analysis (hammerno nco mm er cia l tistics (past) software ver. 2.08 was used for data analysis (hammer species diversity no nco mm er cia l species diversitythe diversity for the sandfly sampled monthly in the four localities no nco mm er cia l the diversity for the sandfly sampled monthly in the four localities was examined. the results revealed maximum diversity in muhayil no nco mm er cia l was examined. the results revealed maximum diversity in muhayil (low altitude) with the highest simpson index (1-d=0.69) and no nco mm er cia l (low altitude) with the highest simpson index (1-d=0.69) and us e than in moderately altitude areas (27.99%) or in high lands (23.9%) us e than in moderately altitude areas (27.99%) or in high lands (23.9%)(table 3). regression analysis indicated that fly density (fly/trap) was us e (table 3). regression analysis indicated that fly density (fly/trap) wasinversely related to the altitude (b=–0.003, p<0.05). us e inversely related to the altitude (b=–0.003, p<0.05). species diversityus e species diversity on ly (figure 1). the reported species were encountered in all altitudes, on ly (figure 1). the reported species were encountered in all altitudes, s. africana on lys. africana which were reported only in low on ly which were reported only in lowp. alexandri on lyp. alexandri and on lyand on ly s. palestinensis on ly s. palestinensis lected only from highlands (>2000 m). sandflies were more common (8on ly lected only from highlands (>2000 m). sandflies were more common (8 species) and more abundant in lowlands (48.10% of collected flies)on ly species) and more abundant in lowlands (48.10% of collected flies) than in moderately altitude areas (27.99%) or in high lands (23.9%)on ly than in moderately altitude areas (27.99%) or in high lands (23.9%) [page 58] [journal of entomological and acarological research 2015; 47:5016] and 4). generally, sandflies were more active during spring months (march to may; mean temperature=30°c, rh=24%, wind=6 km/h) with moderate activity during summer (june to august; mean temperature=36°c, rh=35%, wind=7 km/h) and autumn months (september to november; mean temperature=29°c, rh=33%, wind=9 km/h) and very low density or absent during winter months (december to february; mean temperature=20°c, rh=47%, wind=14 km/h). discussion and conclusions the present study is a report of the results of an entomological survey of sandflies in asir region. during the study, 10 sandfly species were identified: 6 belong to genus phlebotomus and 4 to genus sergentomyia. phlebotomus spp. were dominating (ca 96%), which is a palaearctic feature (lewis & büttiker, 1980), its distribution is in agreement with the previous observation (alahmed et al., 2010). all the encountered species in this study were reported earlier from asir region (lewis & büttiker, 1982; abdelwahab & abdoon 2005; alahmed et al., 2010) except s. palestinensis, which is considered a new record from this region, and it occurs only at high altitude (tanomah). the species was previously reported from al baha, makkah, al madinah and jizan (lewis & büttiker, 1982). of the identified phlebotomus species (612 fly), p. arabicus (ca 34%) and p. bergeroti (ca 31%) were the commonest species followed by p. sergenti, p. orientalis, p. papatasi and p. alexandri. p. bergeroti was reported as a rare species in saudi arabia (nadim et al., 1979; büttiker et al., 1982), however, abdelwahab & abdoon (2005) and alahmed et al. (2010) indicated that in asir, p bergeroti is the most abundant species while p. sergenti is second in abundance. in al baha, p. bergeroti is also the most abundant constituting 41.7% of the collected flies (doha & samy, 2010). phlebotomus alexandri revealed also very low density (abdelwahab & abdoon, 2005; alahmed et al., 2010). phlebotomus ara article table 2. a list of sandfly species reported in asir region during the present (1) and previous surveys (2. lewis & büttiker, 1982; 3. abdelwahab & abdoon, 2005; 4. alahmed et al., 2010). species source 1 2 3 4 phlebotomus (phlebotomus) papatasi (scopoli) x x x x p. (phlebotomus) bergeroti parrot x x x x p. (paraphlebotomus) alexandri sinton x x x x p. (paraphlebotomus) kazeruni theodor & mesghali x p. (paraphlebotomus) saevus parrot & martin x p. (paraphlebotomus) sergenti parrot x x x p. (larroussius) orientalis parrot x x x x p. (adlerius) arabicus theodor x x x x sergentomyia (sergentomyia) antennata (newstead) x x s. (sergentomyia) fallax (parrot) x x s. (sergentomyia) taizi lewis x s. (sergentomyia) schwetzi (adler, theodor & parrot) x s. (sintonius) tiberiadis (adler, theodor & lourie) x x s. (sintonius) adleri (theodor) x x s. (sintonius) calcarata (parrot) x x s. (sintonius) christophersi (sinton) x x x s. (sintonius) clydei (sinton) x x x s. (parrotomyia) palestinensis (adler & theodor) x s. (parrotomyia) africana (newstead) x x x s. (parrotomyia) magna (sinton) x s. (grassomyia) dreyfussi parrot x x s. (grassomyia) squamipleuris (newstead) x s. (neophlebotomus) sonyae lewis x table 3. distribution of sandfly species in different altitudes of the monthly surveyed localities. lowlands moderate altitude highlands <500 m >1000 m >2000 m locality muhayil bishah tanomah abha no. of species 8 6 6 4 collected flies: no. (%) 165 (48.10) 96 (27.99) 34 (9.91) 48 (13.99) no nco mm er cia l x x x x no nco mm er cia l x x x x no nco mm er cia l no nco mm er cia l x no nco mm er cia l x x no nco mm er cia l x no nco mm er cia l no nco mm er cia l x x x no nco mm er cia l x x x x x x x no nco mm er cia l x x x x no nco mm er cia l no nco mm er cia l x x x x no nco mm er cia l x x x x x x no nco mm er cia l x x no nco mm er cia l no nco mm er cia l x x no nco mm er cia l x x x no nco mm er cia l x no nco mm er cia l no nco mm er cia l (adler, theodor & parrot) no nco mm er cia l (adler, theodor & parrot) x no nco mm er cia l x (adler, theodor & lourie) no nco mm er cia l (adler, theodor & lourie) x x no nco mm er cia l x x no nco mm er cia l no nco mm er cia l no nco mm er cia l x x no nco mm er cia l x x x x no nco mm er cia l x x no nco mm er cia l no nco mm er cia l (sinton) no nco mm er cia l (sinton) x x xno nco mm er cia l x x xno nco mm er cia l x x xno nco mm er cia l x x x us e us e 1 2 3 4 us e 1 2 3 4 x x x x us e x x x x us e us e x x x xus e x x x x x x x xus e x x x x on lytable 2. a list of sandfly species reported in asir region during the present (1) and previous surveys (2. lewis & büttiker, 1982; 3. on lytable 2. a list of sandfly species reported in asir region during the present (1) and previous surveys (2. lewis & büttiker, 1982; 3. on ly on ly 1 2 3 4on ly 1 2 3 4 bicus is rare (abdelwahab & abdoon, 2005), in contrast to our findings and that of alahmed et al. (2010). phlebotomus orientalis was considered rare (abdelwahab & abdoon, 2005), but represented 5.85% of the collected flies in abha (alahmed et al., 2010) and 12% in the present study. among the sergentomyia species (28 sandflies), s. clydei was dominating (ca 79%), while s. christophersi, s. africana and s. palestinensis each represented about 7%, numbers similar to previous findings. s. christophersi, s. clydei, s. africana numbered 37, 31 and 24, respectively out of 558 collected sandflies (alahmed et al., 2010). several investigators (büttiker et al., 1982; lewis & büttiker, 1982; aldawood et al., 2004; al barrak, 2005; el-badry et al., 2008) reported p. papatasi as a dominant species in saudi arabia. however in this study, p. papatasi was not common and represented only about 6% of the collected sandflies. only 13 specimens (2.38%) (alahmed et al., 2010) and 19 specimens (0.95%) (lewis & büttiker, 1980) of p. papatasi were previously collected in asir. this confirms the marked affinity of sandfly fauna of asir (abdelwahab & abdoon, 2005) as in southern sinai, egypt (el sawaf et al., 1987) where p. bergeroti and p. sergenti constituted the highest percentage of collected sandflies whereas p. papatasi was a poorly represented species. sandflies were more common and abundant in lowlands respect to higher altitudes. the lowest abundance was observed in highlands in agreement with the previous observation (büttiker et al., 1982). in asir region (abdelwahab & abdoon, 2005), the highest abundance of phlebotomus sandflies was found in tehamah foothills in lowland and in the coastal plain while the lowest fly densities were reported at higher altitudes in sarawat mountains and asir plateau. this was confirmed in the present study by regression analysis, which indicated that the density of flies inversely related to the altitude (p<0.05). meanwhile, the results revealed maximum diversity indices in muhayil (lowland) and minimum ones in abha (highland) due to the low richness of the species in this locality (n=4 spp). this may indicate that the lowlands are the most favourable sites for the breeding and activity of sandflies. lower fly abundance at higher altitudes may be due to low temperature (annual mean=21.6°c), fog and strong winds (annual mean=11.92 km/h) in comparison with lowlands (annual mean of temp=32.03°c and of wind velocity=6.43 km/h) it was reported (abdelwahab & abdoon, 2005) that the species composition of the sandflies is not affected by altitude, although it was found (büttiker et al., 1982) that the species spectrum shows a tendency for more species to occur at higher altitudes than in the tehamah districts (lowlands). moreover, in sinai, egypt (el sawaf et al., 1987), a remarkable difference in sandfly species composition at different altitudes was observed. the present study indicated that although most sandfly species were encountered in all altitudes, some species were found restricted to certain altitudes: s. christophersi and s. africana in lowlands and p. alexandri and s. palestinensis in highlands. this agrees with previous research (büttiker et al., 1982) that is p. papatasi and p. bergeroti prefer lower altitudes whereas p. orientalis and p. arabicus are species of higher altitudes. the knowledge of the seasonal activity of sandflies is of importance in predicting the period of maximum risk of leishmania transmission and for carrying out an effective control program. results indicated that in different altitudes, phlebotomus flies were more active during spring, with moderate activity during summer and autumn and very low density or absent during winter seasons. almost similar observations [journal of entomological and acarological research 2015; 47:5016] [page 59] article figure 1. the surveyed localities and sandfly species distribution in asir region. no nco mm er cia l no nco mm er cia l u se and for carrying out an effective control program. results indicated that us e and for carrying out an effective control program. results indicated that in different altitudes, us e in different altitudes, spring, with moderate activity during summer and autumn and very low us e spring, with moderate activity during summer and autumn and very low density or absent during winter seasons. almost similar observationsus e density or absent during winter seasons. almost similar observations on ly with previous research (büttiker on ly with previous research (büttiker prefer lower altitudes whereas on ly prefer lower altitudes whereas species of higher altitudes. on lyspecies of higher altitudes. the knowledge of the seasonal activity of sandflies is of importance on lythe knowledge of the seasonal activity of sandflies is of importance in predicting the period of maximum risk of on ly in predicting the period of maximum risk of and for carrying out an effective control program. results indicated thaton ly and for carrying out an effective control program. results indicated that phlebotomus on ly phlebotomus [page 60] [journal of entomological and acarological research 2015; 47:5016] were previously reported (abdelwahab & abdoon, 2005) where the highest abundance of sandflies was recorded during the spring and summer (march to september) and the lowest fly abundance was throughout the period from november to february. morsy et al. (1995) found that the greatest number of p. papatasi in riyadh occurred most commonly during the summer season with two peaks in june and september. during the winter season no insects were found and then the population density started to appear again from march. regression analysis indicated that fly density was directly related to the weather temperature (p<0.01) and inversely related to rh and wind velocity (p<0.05). previous analysis (abdelwahab & abdoon, 2005) showed a significantly positive correlation between fly density and temperature and negative correlation with rh at asir foothills. cutaneous leishmaniasis is an endemic disease in many areas of saudi arabia (al-qurashi, 2000; el hassan, 2013) and is widespread in villagers of the asir plateau (al-zahrani et al., 1988b). the disease is more common on the foothills and high plateau of asir region (alzahrani et al., 1989). visceral leishamniasis infections occur in jizan (al-zahrani et al., 1988a; ibrahim et al., 1995; al-jaser, 2006). of the reported sandfly species, several are implicated as vectors of leishmaniasis in asir and other regions of the kingdom. p. bergeroti is assumed as a secondary vector of leishmaniasis in asir mountain plateau (büttiker et al., 1982) and as a suspected vector of l. tropica (anthroponotic cutaneous leishmaniasis) in makka (lewis, 1982) and in some african countries as well as in egypt, iran, oman and yemen (maroli et al, 2013). based on dominance of this species and the recorded increase in the number of leishmaniasis cases after few months of its peak of abundance (abdelwahab & abdoon, 2005) may highlight its role as a probable vector of leishmania in asir area. recently, p. bergeroti was found positive for leishmania-like flagellate infection in al baha (doha & samy, 2010). however, studies are needed to clarify the possible role of this highly abundant species in leishmania transmission in asir where this disease is widely spread; p. papatasi is the main vector of cl in many parts of saudi arabia (lewis & büttiker, 1982; mondragon-shem et al., 2015), may be the major vector in al qassim region (lewis & büttiker, 1980; al barrak, 2005) and is a proven vector of l. tropica in many countries including yemen, iraq, egypt, algeria, iran, jordan, morocco and tunisia (maroli et al., 2013). moreover, p. papatasi, was found to be a proven vector of l. major (zoonotic cutaneous leishmaniasis) in the sinai peninsula, egypt (samy et al., 2014). although, p. papatasi is the most widespread and dominant species in all investigated areas of saudi arabia (mustafa et al., 1994; alahmed et al., 2010), however, in the present study this species was not common (6% of collected sandflies). this may indicate that this species has little or no role in leishmania transmission in asir region. this assumption agrees with what has been found in kuwait where p. papatasi did not appear as an urban species and was not regarded as a possible vector of leishmania in that country (abdelwahab & abdoon, 2005); p. sergenti is a proven vector of l. tropica in the southern region of saudi arabia (al-zahrani et al., 1988b; doha & samy, 2010), and in several other countries (maroli et al., 2013); p. alexandri and p. orientalis are suspected to be the main vectors of l. donovani (vl) in the southwestern part of saudi arabia (alzahrani et al., 1997). the role of the two species in transmitting l. donovani is fully documented in other countries. p. alexandri is the main vector from north africa to western china (lane, 1993) while p. orientalis is a proven vector in sudan (maroli et al., 2013); p. arabicus was found naturally infected with leishmania-like flagellates in al baha (doha & samy, 2010). in conclusion, the present findings indicate that the distribution and abundance of sandflies in asir region are influenced by a combination of ecological and topographical factors (temperature, rh, wind velocity and altitude). the obtained results could be important for the successful implementation of leishmaniasis control programs. among the reported species, s. palestinensis is considered a new record from asir. article figure 2. sandfly diversity indices for monthly sampled localities. figure 3. periodical abundance of phebotomus bergeroti at different altitudes (low: muhayil; moderate: bishah; high: tanomah and abha localities). figure 4. periodical abundance of phlebotomus arabicus at different altitudes (low: muhayil; moderate: bishah; high: tanomah and abha localities). no nco mm er cia l transmission in asir where this disno nco mm er cia l transmission in asir where this disis the main vector of cl in many parts of saudi arabia no nco mm er cia l is the main vector of cl in many parts of saudi arabia ., 2015), may be the no nco mm er cia l ., 2015), may be the major vector in al qassim region (lewis & büttiker, 1980; al barrak, no nco mm er cia l major vector in al qassim region (lewis & büttiker, 1980; al barrak, in many countries includno nco mm er cia l in many countries including yemen, iraq, egypt, algeria, iran, jordan, morocco and tunisia no nco mm er cia l ing yemen, iraq, egypt, algeria, iran, jordan, morocco and tunisia , was found to be a proven no nco mm er cia l , was found to be a proven (zoonotic cutaneous leishmaniasis) in the sinai no nco mm er cia l (zoonotic cutaneous leishmaniasis) in the sinai ., 2014). although, no nco mm er cia l ., 2014). although, p. papatasi no nco mm er cia l p. papatasi most widespread and dominant species in all investigated areas of no nco mm er cia l most widespread and dominant species in all investigated areas of ., 1994; alahmed no nco mm er cia l ., 1994; alahmed et al no nco mm er cia l et al the present study this species was not common (6% of collectedno nco mm er cia l the present study this species was not common (6% of collected sandflies). this may indicate that this species has little or no role inno nco mm er cia l sandflies). this may indicate that this species has little or no role inno nco mm er cia l u se us e us e o nlyon ly on ly on ly figure 2. sandfly diversity indices for monthly sampled localities. on ly figure 2. sandfly diversity indices for monthly sampled localities. on ly on ly references abdelwahab a.i., abdoon m.a.a., 2005 distribution and population dynamics of phlebotomus sandflies (diptera: psychodidae) in an endemic area of cutaneous leishmaniasis in asir region, southwestern of saudi arabia. j. entomol. 2: 102-108. abu-zinada n.y., 1999 a spotlight survey of sandflies of the genus phlebotomus in jeddah, saudi arabia. j. egypt. soc. parasitol. 29: 85-89. alahmed a.m., kheir s.m., al khereji m.a., 2010 distribution of sandflies (diptera: psychodidae) in saudi arabia. res. bult food sci. agric. res. center, king saud univ. 171: 5-23. al barrak a.s., 2005 a study on abundance and control of sandflies (diptera: psychodidae) in al qassim region, saudi arabia. p. j. bio. sci. 8: 326-329. aldawood a.s., alahmed a.m., kheir s.m., hussein s.m., 2004 population dynamics of sandflies (diptera: psychodidae) in hanifa valley, riyadh, saudi arabia. p. j. bio. sci. 7: 464-467. al-jaser m.h., 2006 studies on the epidemiology of malaria and visceral leishmaniasis in jizan area, saudi arabia. j. king saud univ. sci. 19: 9-19. al-qurashi a.r., ghandour a.m., osman m., al-juma m., 2000 dissemination in cutaneous leishmaniasis due to leishmania major in different ethnic groups in saudi arabia. intl. j. dermatol. 39: 832-836. al-zahrani m.a., lane r.p., ching c.i., asiry m.a., peters w., 1997 biology of phlebotomus sandflies (diptera: psychodidae) in two contrasting leishmaniasis foci in south-west saudi arabia. bull. entomol. res. 87: 3. al-zahrani m.a., peters w., evans d.a., 1988a visceral leishmaniasis in man and dogs in south-west saudi arabia. trans. r. soc. trop. med. hyg. 82: 857. al-zahrani m.a., peters w., evans d.a., chin c., smith v., lane r.p., 1988b phlebotomus sergenti, a proven vector of leishmania tropica in saudi arabia. trans. r. soc. trop. med. hyg. 82: 416. al-zahrani m.a., peters w., evans a., smith v., ching chin i., 1989 leishmania infecting man and wild animals in saudi arabia. 6. cutaneous leishmaniasis of man in the south-west. trans. r. soc. trop. med. hyg. 83: 621-628. büttiker w., al-ayed l.h., al-wabila.h., assalhy h.s., rashed a.m., shareefi d.m., 1982 medical and applied zoology in saudi arabia. a preliminary study on leishmaniasis in two areas of asir region. fauna of saudi arabia 4: 509-519. doha s.a., samy a.m., 2010 bionomics of phlebotomine sandflies (diptera: psychodidae) in the province of al-baha, saudi arabia. mem. inst. oswaldo cruz, rio de janeiro 105: 850-856. el-badry a.a., al juhani a., el kheir i., al zubainy s., 2009 sandflies distribution and bionomics in al madinah al munawwarah region, western of saudi arabia. res. j. parasitol. 4: 1-11. el-badry a.a. al-juhani a., ibrahim el-k.d., al zubiany s., 2008 distribution of sandflies in el nekheil province in al madinah al munawwarah region, western of saudi arabia. parasitol. res. 103: 151-156. el hassan a.m., 2013 cutaneous leishmaniasis in al-ahsa oasis in saudi arabia and in sudan: a comparative study. saudi j. med. sci. 1: 64-71. el sawaf b.m., shoukry a., el said s., lane r.p., kenawy m.a., beier j.c., abdel sattar s., 1987 sandfly species composition along an altitudinal transect in southern sinai, egypt. ann. parasitol. hum. comp. 26: 467-473. hammer ø., harper d.a.t., ryan p.d., 2001 past: paleontological statistics software package for education and data analysis. available from: http://www.nhm2.uio.no/norlex/past/past.exe ibrahim e.a., al-zahrani m.a., nawarani o.a., 1995 visceral leishmaniasis in gizan. ann. saudi med. 15: 671. kakarsulemankhel j.k., 2009 taxonomic review of sandflies of the subgenus phlebotomus rondani and berte (diptera: psychodidae). pak. entomol. 31: 71-92. kakarsulemankhel j.k., 2010 taxonomic review of sandflies of the subgenus paraphlebotomus theodor (diptera: psychodidae). pak. entomol. 32: 125-147. killick-kendrick r., leaney a. j., peters w., rioux j.a., bray r.s. 1985 zoonotic cutaneous leishmaniasis in saudi arabia: incrimination of phlebotomus papatasi as a vector in al hassa. trans. r. soc. trop. med. hyg. 79: 252-255. lane r.p., 1986 the sandflies of egypt (diptera: phlebotominae). bull. br. mus. nat. hist. (ent.) 52: 1-35. lane r.p., 1993 sandflies (phlebotomine). in: lane r.p., crosskey r.w. (eds.), medical insects and arachnids. chapman and hall, london: 78-119. lewis d. j., 1982 a taxonomic review of the genus phlebotomus (diptera: psychodidae). bull. br. mus. nat. hist. (ent.) 45: 121-209. lewis d. j., büttiker w., 1980 insects of saudi arabia, diptera: fam. psychodidae, subfamily: phlebotominae. fauna of saudi arabia 2: 252-282. lewis d. j., büttiker w., 1982 insects of saudi arabia. the taxonomy and distribution of saudi arabian phlebotomine sandflies (diptera: psychodidae). fauna of saudi arabia 4: 353-397. maroli m., feliciangeli m.d., bichaud l., charre, r..n., gradoni l., 2013 phlebotomine sandflies and the spreading of leishmaniases and other diseases of public health concern. med. vet. entomol. 27: 23-47. mondragon-shem k., al-salem w.s., kelly-hope l., abdeladhim m., al-zahrani m.h., valenzuela j.g., acosta-serrano a., 2015 severity of old world cutaneous leishmaniasis is influenced by previous exposure to sandfly bites in saudi arabia. plos negl. trop. dis. 9: e0003449. morsy t.a., abou el-ela r.g., rifaat m.m., al dakhil m.a., 1995 the seasonal and daily activities of phlebotomus papatasi in riyadh, saudi arabia. j. egypt. soc. parasitol. 25: 699-711. morsy t.a., al seghayer s.m., 1992 a brief note on phlebotomine sandflies in riyadh, saudi arabia. j. egypt. soc. parasitol. 22: 437440. mustafa m.b., hussein s.m., ibrahim e.a., al seghayer s.m., al amri s.a., gradoni l., 1994 phlebotomus papatasi (scopoli), vector of zoonotic cutaneous leishmaniasis in riyadh province, saudi arabia. trans. r. soc. trop. med. hyg. 88: 40. nadim a., seyedi-rashti m.a., ashi j., 1979 cutaneous leishmaniasis in saudi arabia: an overview.bull. soc. pathol. exot. filialis. 72: 237-344. samy a.m., campbell l.p., peterson a.t., 2014 leishmaniasis transmission: distribution and coarse-resolution ecology of two vectors and two parasites in egypt. rev. soc. bras. med. trop. 47: 57-62. who, 2010 control of the leishmaniasis. who technical report series 949: 186 pp. [journal of entomological and acarological research 2015; 47:5016] [page 61] article no nco mm er cia l asis in man and dogs in south-west saudi arabia. trans. r. soc. no nco mm er cia l asis in man and dogs in south-west saudi arabia. trans. r. soc. al-zahrani m.a., peters w., evans d.a., chin c., smith v., lane no nco mm er cia l al-zahrani m.a., peters w., evans d.a., chin c., smith v., lane , a proven vector of no nco mm er cia l , a proven vector of leishmania no nco mm er cia l leishmania in saudi arabia. trans. r. soc. trop. med. hyg. 82: 416. no nco mm er cia l in saudi arabia. trans. r. soc. trop. med. hyg. 82: 416. al-zahrani m.a., peters w., evans a., smith v., ching chin i., no nco mm er cia l al-zahrani m.a., peters w., evans a., smith v., ching chin i., infecting man and wild animals in saudi arabia. no nco mm er cia l infecting man and wild animals in saudi arabia. 6. cutaneous leishmaniasis of man in the south-west. trans. r. no nco mm er cia l 6. cutaneous leishmaniasis of man in the south-west. trans. r. büttiker w., al-ayed l.h., al-wabila.h., assalhy h.s., rashed no nco mm er cia l büttiker w., al-ayed l.h., al-wabila.h., assalhy h.s., rashed a.m., shareefi d.m., 1982 medical and applied zoology in saudi no nco mm er cia l a.m., shareefi d.m., 1982 medical and applied zoology in saudi arabia. a preliminary study on leishmaniasis in two areas of asir no nco mm er cia l arabia. a preliminary study on leishmaniasis in two areas of asir region. fauna of saudi arabia 4: 509-519.no nco mm er cia l region. fauna of saudi arabia 4: 509-519. doha s.a., samy a.m., 2010 bionomics of phlebotomine sandfliesno nco mm er cia l doha s.a., samy a.m., 2010 bionomics of phlebotomine sandflies (diptera: psychodidae) in the province of al-baha, saudi arabia. -no nco mm er cia l (diptera: psychodidae) in the province of al-baha, saudi arabia. lewis d. j., büttiker w., 1982 insects of saudi arabia. the taxonono nco mm er cia l lewis d. j., büttiker w., 1982 insects of saudi arabia. the taxono-my and distribution of saudi arabian phlebotomine sandflies no nco mm er cia l my and distribution of saudi arabian phlebotomine sandflies(diptera: psychodidae). fauna of saudi arabia 4: 353-397. no nco mm er cia l (diptera: psychodidae). fauna of saudi arabia 4: 353-397. maroli m., feliciangeli m.d., bichaud l., charre, r..n., no nco mm er cia l maroli m., feliciangeli m.d., bichaud l., charre, r..n., us e (diptera: psychodidae). bull. br. mus. nat. hist. (ent.) 45: 121-209. us e (diptera: psychodidae). bull. br. mus. nat. hist. (ent.) 45: 121-209. lewis d. j., büttiker w., 1980 insects of saudi arabia, diptera: us e lewis d. j., büttiker w., 1980 insects of saudi arabia, diptera:fam. psychodidae, subfamily: phlebotominae. fauna of saudi us e fam. psychodidae, subfamily: phlebotominae. fauna of saudi arabia 2: 252-282.us e arabia 2: 252-282. lewis d. j., büttiker w., 1982 insects of saudi arabia. the taxono-us e lewis d. j., büttiker w., 1982 insects of saudi arabia. the taxonomy and distribution of saudi arabian phlebotomine sandfliesus e my and distribution of saudi arabian phlebotomine sandflies on ly bull. br. mus. nat. hist. (ent.) 52: 1-35. on ly bull. br. mus. nat. hist. (ent.) 52: 1-35. on ly lane r.p., 1993 sandflies (phlebotomine). in: lane r.p., crosskey on ly lane r.p., 1993 sandflies (phlebotomine). in: lane r.p., crosskey r.w. (eds.), medical insects and arachnids. chapman and hall, on lyr.w. (eds.), medical insects and arachnids. chapman and hall, lewis d. j., 1982 a taxonomic review of the genus on ly lewis d. j., 1982 a taxonomic review of the genus (diptera: psychodidae). bull. br. mus. nat. hist. (ent.) 45: 121-209.on ly (diptera: psychodidae). bull. br. mus. nat. hist. (ent.) 45: 121-209. lewis d. j., büttiker w., 1980 insects of saudi arabia, diptera: on ly lewis d. j., büttiker w., 1980 insects of saudi arabia, diptera: 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 [journal of entomological and acarological research 2012; 44] [page 1] editorial g.c. lozzia, university of milano, italy the journal of entomological and acarological research (jear), formerly the bollettino di zoologia agraria e di bachicoltura of the institute of entomology of the università degli studi, milano, was founded in 1928 by remo grandori. minos martelli and luciano süss were in charge of the journal until december 2011. in january 2012, the editor came to a big decision and began to explore the possibility of publishing a new online open access version of the jear. the aim was to reach out to researchers in the field all over the world, and i was nominated editor in chief. the journal is structured in four major parts according to subject area: entomology, stored product pests, insect ecology and acarology. these are run by highly qualified internationally recognized section editors who are supported by a new editorial board made up of scientists from all over the world. the jear publishes papers on systematics of geographical speciation and evolution, ecological system dynamics, phenology and supervised pest control, stored product pests, medical, urban and veterinary entomology, plant insect interaction and ecosystems, biological control and agroecology, and all other subjects related to the four subject areas. i hope you like the new edition of the journal, and in particular those articles submitted by our younger colleagues; the future of modern entomology and acarology is in their hands. i would also like to thank the former editorial board and director, and all the colleagues who will work with me on this interesting and stimulating new project. the editor in chief prof. giuseppe carlo lozzia journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2012; volume 44 no nco mm er cia l u se on ly jear2012 abstract indoor residual house spraying using lambda-cyhalothrin, deltamethrin and dichlorodiphenyltrichloroethane (ddt) was conducted in zhombe resettlement area, zimbabwe. a total of 204/219 (93.1%), 224/260 (86.2%) and 257/325 (79.1%) rooms were sprayed with lambda-cyhalothrin, deltamethrin and ddt wettable powders respectively. bioassays were conducted on sprayed walls and roofs using 3-5 day old laboratory reared susceptible anopheles gambiae sensu lato mosquitoes placed in world health organization cones. bioassays conducted on sprayed walls (1 month), showed that efficacy of lambda-cyhalothrin was the same with ddt but different with deltamethrin and this trend continued in the 2nd month. during the 3rd month, lambda-cyhalothrin killed more mosquitoes than deltamethrin (p=1.931¥10-14), ddt killed more mosquitoes than deltamethrin (p=0.0001) and lambda-cyhalothrin killed more mosquitoes than ddt (walls). efficacy of lambda-cyhalothrin and ddt was the same 4 months post spray (p=0.487), notable differences were seen in lambdacyhalothrin and deltamethrin (p=2.57¥10-6), ddt and deltamethrin (p=2.17¥10-8). efficacy of lambda-cyhalothrin and ddt was the same 5 months post spray (p=0.244), major differences were found in lambdacyhalothrin and deltamethrin (p=0.000), ddt and deltamethrin (p=5.18¥10-5) and this trend continued in the 6th month. one month after spraying roofs, mortality of mosquitoes due to lambdacyhalothrin/deltamethrin (p=2.56¥10-5), lambda-cyhalothrin/ddt (p=1.2¥10-7) and deltamethrin/ddt (p=0.013) were significantly different and this continued in the 2nd month. however, 3 months after spraying, mortality due to lambda-cyhalothrin/deltamethrin (p=1.46¥10-6), lambda-cyhalothrin/ddt (p=0.048), and deltamethrin/ddt (p=0.004) were significantly different and this continued in the 4th month. five months after spraying roofs, mortality due to lambda-cyhalothrin/ deltamethrin (p=0.000) and deltamethrin/ddt (p=6.6¥10-7) were significantly different. six months after spraying, lambdacyhalothrin/deltamethrin (p=0.34), lambda-cyhalothrin/ddt (p=0.982), and deltamethrin/ddt (p=0.64) were not significantly different. when using exit window traps, no mosquitoes were collected from rooms sprayed with each of the insecticides over a 6-month period. however, 17, 6, 14, 7, 2 and 3 fed an. gambiae sl mosquitoes were collected in the 1st, 2nd, 3rd, 4th, 5th and 6th month respectively from unsprayed rooms and none of them died after 24 h. introduction the use of residual insecticides for indoor residual house spraying (irs) remains an essential component of malaria control in many parts of the world, including zimbabwe (eilsele, et al., 2010). residual insecticides have life spans of 3-6 months for pyrethroids (raghavendra et al., 2011) and 8 months to 2 years for organo-chlorines (taylor et al., 1981). irs targets mosquitoes that are endophillic (indoor resting) that will eventually pick up a lethal dose before they die. in a study conducted in kenya, irs reduced mosquito vector density and disease incidence for a period of 6 months (zhou et al., 2010). molineaux & gramiccia (1980) attributed vector exophily to the failure to interrupt malaria transmission following irs with the insecticide propoxur in nigeria. sharp et al. (2007) observed that the number of anopheles gambiae sensu stricto mosquitoes was reduced from 25.5 to 1.9 per trap per 100 nights after spraying with a pyrethroid. world health organization (who, 2006) set the criteria for knock down as ≥95% and 24 h mortality as ≥80% and a spraying coverage of >80% is required in order to interrupt transmission. the epidemiology and history of malaria control in zimbabwe is well correspondence: nzira lukwa, national institute of health research, p.o. box cy573, causeway, harare, zimbabwe. tel: +263.4.797052 fax: +263.4.253979. e-mail: nziraa33@yahoo.co.uk key words: spraying, lambdacyhalothrin, deltamethrin, ddt, anopheles gambiae sensu lato. contributions: nl, slm, research concept and design, data collection tools, data analysis and interpretation, manuscript first draft, revision and final approval; am, pm, improving methodology, data collection tools, data analysis, manuscript revision and final approval. acknowledgements: the authors would like to thank mrs m. gidi, mr f. chinyanya, mr mupindu and mrs rwizi for their support. this study could not have been possible without authority from dr chimusoro and mrs vimbayi chikwavaire. several people assisted in many ways and are duly acknowledged. received for publication: 5 june 2012. revision received: 25 august 2012. accepted for publication: 28 august 2012. ©copyright n. lukwa et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e10 doi:10.4081/jear.2012.e10 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. the residual effect of lambda-cyhalothrin, deltamethrin and dichlorodiphenyltrichloroethane in zhombe, kwekwe district, zimbabwe n. lukwa,1 a. makuwaza,1 s.l. mutambu,1 p. munosiyei2 1national institute of health research, causeway, harare; 2bindura university of science education, zimbabwe [page 46] [journal of entomological and acarological research 2012; 44:e10] journal of entomological and acarological research 2012; volume 44:e10 no nco mm er cia l u se on ly documented by taylor & mutambu (1986). malaria vector control started in the 1940s and has witnessed the coming and going of such insecticides as dieldrin and benzene hexachloride and the introduction of synthetic pyrethroids such as deltamethrin, alpha-cypermethrin, and lambda-cyhalothrin. irs remains an essential component of the national malaria control programme (nmcp) in zimbabwe. information from previous work indicates that the malaria vectors in zimbabwe are still susceptible to dichlorodiphenyltrichloroethane (ddt) and pyrethroids (manokore et al., 2000) although munhenga et al. (2008) documented resistance to permethrin and ddt of mosquitoes collected from one locality in gwave, zimbabwe. the residual effect of ddt, lambda-cyhalothrin and deltamethrin was monitored in zhombe resettlement area, zimbabwe. material and methods study area zhombe is one of the malarious areas in kwekwe district that is not sprayed by the nmcp during its annual spraying cycle. malaria spraying by the nmcp focuses on the highly burdened districts. insecticides ddt 75wp is a wettable powder containing 75% dichloro-diphenyltrichloro-ethylene, insectokill 10wp contains 10% lambda-cyhalothrin wettable powder and deltaguard 5wp contains 5% deltamethrin wettable powder. deltamethrin (moderately toxic) has an acute toxicity of lethal dose for 50% effect (ld50) oral, rat. >5000 mg/kg and ld50 dermal, rat. >2000 mg/kg. lambda-cyhalothrin (moderately toxic) has an acute toxicity of ld50 oral, rat. 144 mg/kg and ld50 dermal, rat. 696 mg/kg. ddt (moderately to slightly toxic) has an acute toxicity of ld50 oral, rat. 113-800 mg/kg and ld50 dermal, rat. 2500-3000 mg/kg spraying was conducted using an xpert 8l hudson sprayer (h.d. hudson manufacturing company, chicago, il, usa), pressurised to 55 psi (pounds per square inch). one hut in zimbabwe constitutes one room only. a total of 204/219 (93.1%) rooms were sprayed with lambdacyhalothrin at 30 mg/m2, 224/260 (86.2%) rooms were sprayed with deltamethrin at 25 mg/m2, 257/325 (79.1%) rooms were sprayed with ddt at 2 mg/m2 and 205 rooms served as the control. all insecticides were sprayed in march 2010. mosquitoes anopheles gambiae sensu lato mosquito larvae were collected from the study sites through larval scooping. the larvae were placed in larval bowls (rearing dishes) in a simulated field insectary and reared to adult stage by the provision of fish food. the resulting female adults were given 10% sugar water. the mosquito colony is susceptible to 4% ddt, 0.05% deltamethrin, 0.05% lambda-cyhalothrin, 0.5% etofenprox, 0.15% cyfluthrin and 0.75% permethrin. residual effect bioassays were conducted on randomly selected rooms (table 1) that were sprayed with ddt, lambda-cyhalothrin or deltamethrin in accordance with who guidelines (2006). three who cones (8.5 cm in diameter at the base and 5.5 cm high) were fastened on different positions on the wall and other 3 on the roof. ten to fifteen an. gambiae sl mosquitoes were aspirated and placed in each who cone and exposed for 30 min. mosquitoes were retrieved using an aspirator, placed in a holding cup before recording knock down rate. these mosquitoes were provided with 10% sugar solution in a 50 ml bottle that contained a wick and this was done over a period of 6 months. mortality was scored after 24 h. by adding up all mosquitoes that died in each cone for each surface and dividing by the number of cones, the mean mortality per month for each insecticide was calculated. effect of insecticides on mosquito behaviour this was done monthly using exit window traps (ewt) mounted at 1 window opening at each homestead at 5 p.m. five rooms for each insecticide were used every month for 6 months and this included 5 unsprayed rooms that served as controls. all the other windows and openings were then closed. mosquito collection was done using an aspirator and placing mosquitoes in a holding cup by 10 a.m. the following day. data analysis the data analysis was performed with the anova test (analysis of variance) using the 95% confidence limit. mortality of mosquitoes at each time pont was analyzed for the 3 insectides. results results for bioassays conducted on sprayed walls are shown in table 2. no mortality occurred in the 2 control rooms that were used per month, with a total of 12 control rooms over the 6-month period. the mortality for mosquitoes exposed to walls sprayed with lambda-cyhalothrin (1 month) was the same with ddt but significantly different with deltamethrin. two months after spraying, mortality for mosquitoes due to lambdacyhalothrin was significantly different with deltamethrin (p=0.003) and ddt (p=0.03). during the same period, ddt and deltamethrin were significantly different (p=1.31¥10-5). during the 3rd month, lambda[journal of entomological and acarological research 2012; 44:e10] [page 47] article table 1. number of rooms used for bioassays. months post lambda-cyhalothrin deltamethrin ddt control spraying rooms where rooms where rooms where rooms where rooms where rooms where rooms walls were used roofs were used walls were used roofs were used walls were used roofs were used used 1 4 4 6 5 6 5 2 2 7 8 6 4 6 5 2 3 7 7 8 6 4 4 2 4 4 6 4 4 4 4 2 5 5 5 4 4 6 9 2 6 7 9 6 6 4 1 2 ddt, dichlorodiphenyltrichloroethane. no nco mm er cia l u se on ly [page 48] [journal of entomological and acarological research 2012; 44:e10] cyhalothrin killed more mosquitoes than deltamethrin (p=1.931¥10-14), ddt killed more mosquitoes than deltamethrin (p=0.0001) and lambdacyhalothrin killed more mosquitoes than ddt (all results were significantly different). efficacy of lambda-cyhalothrin and ddt was the same 4 months post spray (p=0.487), notable differences were seen in lambdacyhalothrin and deltamethrin (p=2.57¥10-6), ddt and deltamethrin (p=2.17¥10-8). efficacy of lambda-cyhalothrin and ddt was the same 5 months post spray (p=0.244), major differences were found in lambdacyhalothrin and deltamethrin (p=0.000), ddt and deltamethrin (p=5.18¥10-5). efficacy of lambda-cyhalothrin and ddt was the same 6 months post spray (p=0.427), major differences were found in lambdacyhalothrin and deltamethrin (p=3.7¥10-7), ddt and deltamethrin (p=1.58¥10-9). no mortality was observed with the control mosquitoes over the 6-month period. results for bioassays conducted on sprayed roofs are shown in table 3. one month after spraying, the mortality of mosquitoes due to lambda-cyhalothrin/deltamethrin (p=2.56¥10-5), lambda-cyhalothrin/ddt (p=1.2¥10-7) and deltamethrin/ddt (p=0.013) were significantly different. results for 2 months after spraying, mortality due to lambdacyhalothrin/deltamethrin (p=3.65¥10-11), lambda-cyhalothrin/ddt (p=9.18¥10-12), and deltamethrin/ddt (p=2.26¥10-5) were significantly different. three months after spraying, mortality due to lambdacyhalothrin/deltamethrin (p=1.46¥10-6), lambda-cyhalothrin/ddt (p=0.048), and deltamethrin/ddt (p=0.004) were significantly different. four months after spraying, mortality due to lambda-cyhalothrin/ deltamethrin (p=1.46¥10-6), lambda-cyhalothrin/ddt (p=0.048), and deltamethrin/ddt (p=0.004) were significantly different. five months after spraying, mortality due to lambda-cyhalothrin/deltamethrin (p=0.000) and deltamethrin/ddt (p=6.6¥10-7) were significantly different apart from lambda-cyhalothrin/ddt (p=0.811). six months after spraying, lambda-cyhalothrin/deltamethrin (p=0.34), lambdacyhalothrin/ ddt (p=0.982), and deltamethrin/ddt (p=0.64) were not significantly different. no mortality was observed with the control mosquitoes over the 6-month period. effect of insecticides on mosquito behaviour after installing one exit window trap per room for a total of 5 rooms per treatment per month, no mosquitoes were collected from rooms sprayed with insecticides over a 6-month period. however, 17, 6, 14, 7, 2 and 3 fed an. gambiae sl mosquitoes were collected in the 1st, 2nd, 3rd, 4th, 5th and 6th month respectively from unsprayed rooms and none of them died after 24 h. discussion and conclusions spraying coverage was above 80% as required by who (2006) in lambda-cyhalothrin and deltamethrin sprayed villages although slightly low in ddt sprayed villages. we did not carry out a study on level of product acceptability in the study villages and therefore cannot explain these observed differences. the number of rooms used for bioassays sometimes differed in some months due to either availability of mosquitoes or presence of the head of a household to give consent. during the first month on sprayed walls, deltamethrin was not as effective as either lambda-cyhalothrin of ddt but this trend was totally different on sprayed roofs over the same period. in the second and third month, the efficacy of these 3 insecticides was totally different on the walls and roofs. during the 4th month, efficacy of lambda-cyhalothrin and ddt on walls was the same but different with deltamethrin but all roofs were totally different over the same period. in the 5th month, efficacy of lambda-cyhalothrin was the same both on the walls and roofs but different with deltamethrin and this trend in the sixth month on walls, however, mortality on the roof was the same. these results were obtained when a susceptible colony of an. gambiae sl mosquitoes was used. our observations on ddt did not go beyond 6 months and therefore cannot be compared with observations by taylor et al. (1981). insecticide manufacturers claim longer residual periods on their products, making it necessary for control programmes to verify such claims. the residual effect of each insecticide formulation largely depends on the quality of the product (for the manufacturer) and the quality of spraying (for control programmes). studies on mosquito behaviour have shown that no mosquitoes were collected in huts sprayed with any of the insecticides. these results imply that mosquitoes died after getting in contact with sprayed surfaces and therefore could not exit sprayed houses. even if the mosquito population in the study villages had exophilic tendencies, they died before leaving the sprayed houses. in comparison with mosquitoes that were continuously caught from article table 2. bioassays conducted on walls. months lambdadeltamethrin ddt post cyhalothrin spraying 1 107/107 (100%)a 151/161 (93.8%)b 161/161 (100%)a range (100%) range (88-100%) range (100%) 2 214/218 (98.2%)c 161/172 (93.6%)d 171/171 (100%)e range (90-100%) range (87-100%) range (100%) 3 199/200 (99.5%)f 203/225 (90.2%)g 120/126 (95.2%)h range (90-100%) range (87-100%) range (93-100%) 4 117/120 (97.5%)i 105/117 (89.7%)k 118/120 (98.3%)i range (90-100%) range (86-97%) range (93-100%) 5 148/153 (96.7%)m 97/107 (90.7%)n 166/174 (95.4%)m range (87-100%) range (88-97%) range (92-97%) 6 186/211 (88.2%)p 125/163 (76.7%)q 96/107 (89.7 %)p range (81-95%) range (72-88%) range (80-94%) ddt, dichlorodiphenyltrichloroethane. same letter in the same row denotes no significant difference and different letter in the same row denotes significance difference. table 3. bioassays conducted on roofs. months lambdadeltamethrin ddt post cyhalothrin spraying 1 99/99 (100%)a 130/138 (94.2%)b 132/136 (97%)c range (100%) range (90-100%) range (96-100%) 2 223/223 (100%)d 106/112 (94.6%)e 1291/131 (98.5%)f range (100%) range (92-100%) range (98-100%) 3 200/203 (98.5%)g 166/179 (92.7%)h 110/114 (96.5%)i range (90-100%) range (88-100%) range (90-100%) 4 170/178 (95.5%)j 112/124 (90.3%)k 103/111 (92.8%)l range (90-100%) range (88-100%) range (90-100%) 5 140/151 (92.7%)m 112/128 (87.5%)n 238/256 (92.9%)m range (88-100%) range (85-90%) range (90-100%) 6 224/256 (87.5%)p 138/160 (86.3%)p 24/28 (85.7 %)p range (81-94%) range (74-90%) range (84-90%) ddt, dichlorodiphenyltrichloroethane. same letter in the same row denotes no significant difference and different letter in the same row denotes significance difference. no nco mm er cia l u se on ly ewt installed in unsprayed houses, there are indications that these mosquitoes did not die after being in contact with unsprayed houses and there had to leave these houses under natural conditions. it can be assumed that mosquitoes that were in contact with sprayed structures died and therefore could not be collected from ewt and these results are in agreement with observations of bruce-chawett (1964). mosquitoes collected from unsprayed houses were blood fed and did not die, as observed by sharp et al. (2007). in conclusion, ddt and lambda-cyhalothrin had residual effects of 6 months as compared with deltamethrin that had a residual effect of 5 months. references bruce-chwatt l.j., 1964 changing tides of chemotherapy of malaria. br. med. j. 1: 581-586. eilsele t.p., larsen d., steketee r.w., 2010 protective efficacy of interventions for preventing malaria mortality in children in plasmodium falciparum endemic areas. int. j. epidemiol. 39: 88-101. manokore v., murahwa f.c., chirebvu e., 2000 absence of insecticide resistance in anopheles gambiae s.l. after four decades of residual house spraying in gokwe district, zimbabwe. j. med. entomol. 37: 286-288. molineaux l., gramiccia g., 1980 the garki project. world health organization, geneva. munhenga g., masendu h.t., brooke b.d., hunt r.h., koekemoer l.k., 2008 pyrethroid resistance in the major malaria vector anopheles arabiensis from gwave, a malaria-endemic area in zimbabwe. malaria j. 7: 247. raghavendra k., barik t.k., reddy b.p., sharma p., dash a.p., 2011 malaria vector control: from past to future. parasitol. res. 108: 757-79. sharp b.l., ridl f.c., govender d., kuklinski j., klienschmidt i., 2007 malaria vector control by indoor residual insecticide spraying on the tropical island of bioko, equatorial guinea. malaria j. 6: 52. taylor p., crees m.j., hargreaves k., 1981 duration of anopheles arabiensis control in experimental huts sprayed with ddt and decamethrin. t. zimbabwe sci. assoc. 61: 1-13. taylor p., mutambu s.l., 1986 a review of the malaria situation in zimbabwe with special reference to the period 1972–1981. t. roy. soc. trop. med. h. 80: 12-19. world health organization, 2006 guidelines for testing mosquito aldulticides for indoor residual spraying and treatment of mosquito nets. who/cds/whopes/gcdpp/2006.3. available from: http://whqlibdoc.who.int/hq/2006/who_cds_ntd_whopes_gcd pp_2006.3_eng.pdf zhou g., githeko a.k., minakawa n., yan g., 2010 communitywide benefits of targeted indoor residual spray for malaria control in the western kenya highland. malaria j. 9: 67. [journal of entomological and acarological research 2012; 44:e10] [page 49] article no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2015; 47:5090] [page 69] the first record of dendrothrips aspersus (thysanoptera: thripidae) from iran k. minaei department of plant protection, college of agriculture, shiraz university, shiraz, iran abstract the species dendrothrips aspersus bhatti, 1971 is reported for the first time from iran, based on the materials collected on grasses. this species was endemic to their originated region and is recorded for the first time outside their native range. the host records of d. aspersus in both india and iran are discussed. moreover, the number of thrips species that have been recently recorded from iran are tabulated. introduction in the most recent treatment of the insect order thysanoptera, 9 families have been recognised (mound, 2011b). however, most of the species belongs to these two families: phlaeothripidae and thripidae. although thripidae is the second largest family of thysanoptera, it is much more abundant than phlaeothripidae in temperate regions (thripswiki, 2015), and this situation is also true for iran, where 125 species (including one species group) of thripidae versus 45 species of phlaeothripidae (minaei, 2013) were collected. a large proportion of thripids are flower and leaf-feeders. in iran, three genera (dendrothrips usel, iranodendrothrips alavi, minaei & fekrat, pseudodendrothrips schmutz) with seven species are known in dendrothripinae. an identification key for those genera and species including four species in dendrothrips are also available (alavi et al., 2014). the purpose of this paper is to report dendrothrips aspersus as the fifth species in this genus in iran. this is also the first record of this species outside india. materials and methods thrips specimens were collected into ethanol (70%) and then mounted on to the glass slides in canada balsam. the photomicrographs and measurements were taken using a motic ba310 microscope with motic image plus 2.0 ml software. most of the specimens are deposited in department of plant protection, shiraz university, iran (ppsu) with two females in australian national insect collection, canberra (csiro). results dendrothrips aspersus bhatti, 1971 dendrothrips aspersus bhatti, 1971: 349. female macroptera: body generally yellow (figure 1), antennal segments v-viii brown; pronotum yellow with brown spots medially and anterolaterally; tergites with brown markings on lateral thirds, two pairs of longitudinal markings joined by two transverse markings on iii-vii, but the posterior transverse marking usually absent on ii and v; forewing white with three brown spots; major body setae pale. antennae 8-segmented (figure 2a), segments iii and iv each with simple sense-cones, iii to vi each with two to five rows of microtrichia on dorsal and ventral surface; segments v cylindrical; vi not constricted basally, its inner sense-cone exceeding apex of segment viii. head transverse (figure 2b), wider than long; ocellar setae pair i absent, pairs ii and iii minute, ocellar setae iii located just outside the triangle (in front of posterior ocelli). pronotum wider than long (figure 2b), granulate, without distinct transverse lines, with no elongate setae, shallowly concave at each side near posterior margin, with about 20 discal setae; posterior margin with about 10 setae; ferna divided; prospinasterum well developed. mesonotum sculptured with transverse anastomosing striae (figure 2c), without campaniform sensilla; pair of median setae sitcorrespondence: kambiz minaei, department of plant protection, college of agriculture, shiraz university, shiraz, iran. e-mail: kminaei@shirazu.ac.ir acknowledgements: i am grateful to l. a. mound (csiro, canberra, australia) as well as k. tyagi (molecular systematics division, zoological survey of india, kolkata, india) for help me in identification of the species discussed in this paper. j. s. bhatti kindly sent me a copy of his valuable paper on indian dendrothrips. two anonymous reviewers provided me with detailed criticisms and corrections to an earlier draft. key words: dendrothripinae; iran, grass; new record; thrips. received for publication: 9 february 2015. revision received: 14 june 2015. accepted for publication: 14 june 2015. ©copyright k. minaei, 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:5090 doi:10.4081/jear.2015.5090 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:5090 no nco mm er cia l u se on ly [page 70] [journal of entomological and acarological research 2015; 47:5090] uated medially. metascutum reticulate distinctively with no campaniform sensilla, pair of minute median setae situated far from anterior margin. forewing with 3 grayish spots along anterior margin and one next to scale along posterior margin, not covered with microtrichia, with costal margin downturned, scale with three to four veinal seate and one discal seta; remaining wing setae small and finely acute. tarsi one-segmented. lateral sides of abdominal tergites ii-viii with reticulations; tergites iii to vii with microtrichia along posterior margin behind s1 and s2 setae; tergite viii with posteromarginal comb complete; tergites ix and x with some rows of microtrichia; tergite ix longer than tergite x, s1 slightly longer than s2; tergite x not divided (figure 2d). remarks: the species is distinguished from other recorded species of dendrothrips in iran (d. phyllireae, d. saltatrix, d. karnyi, d. deggeri) by the absence of prominent posteroangular setae as well as eight segmented antennae. the posteroangular setae are not developed in both deggeri and phyllireae as do in aspersus. however, the number of antennal segments in deggeri and phyllireae are 9 and 7 respectively while this is 8 for aspersus. measurements (one female, in micrometers): body length 910. head, length (width across cheeks) 78 (157). pronotum median length (width) 92 (183). fore wing length 6665. tergite ix median length s1 setae length 40, s2 setae length 43. ovipositor length 160. antennal segments i-viii length: 22, 26, 30, 28, 26, 22, 9, 11. material studied: iran, fars province, shiraz, 1 female on cynodon dactylon, 15.viii.2014 (km 1241) (in anic); same locality and plant, 2 females, 16. viii.2014 (km 1243); same locality and plant, 3 females, 29. viii. 2014 (km 1250); same locality and plant, 6 females (1 in anic), 5. ix. 2014 (km 1255, 1256). discussion and conclusions dendrothrips aspersus was described by bhatti in 1971 from india based on specimens collected on zizyphus flowers (family rhamanaceae) and leaves of acacia (family fabaceae) with an identification key to 13 species of dendrothrips. recently, bhagat (2011) collected this species on the same plants from jammu and kashmir state of india. there is no report of this species outside india, so this is the first record outside this sub-continent. the fauna of iran shares many species with the european mediterranean area (minaei, 2013), but oriental region has also a considerable influence on the iranian fauna. the presence of d. aspersus in iran confirms that statement. the species of the genus dendrothrips are mainly associated with two plant families, oleaceae and flacourtiaceae (marullo, 2003), however, two species have been collected on vitex sp (fam. lamiaceae). these include d. minutus (ananthakrishnan, 1961) as well as d. karnyi (zur strassen, 2003). furthermore, d. aspersus has just apparently collected on ziziphus sp. and acacia sp. despite the foregoing, all specimens of d. aspersus in this study were collected from leaves of grasses (family poaceae), which were grown up in an olive garden, but there are no documents, which demonstrate that olive (family oleaceae) may be a host for the species. the host plant recognition in thysanoptera is difficult (mound, 2013). mound (1999, 2011a) demonstrated that among dendrothripinae, none of dendrothrips species is associated with grasses. from the last two decades the number of thrips species described or recorded in iran increased. minaei (2013) reported 202 species (including one species group) from iran. later on, 19 species including one new genus and 10 new species have been recorded from iran (table 1). some of the species were synonymised (table 2), considering the reports of thrips viminalis (rahemi et al., 2010), of two haplothrips species, h. cahirensis (trybom 1911) and h. knechteli priesner (fallahzadeh & saghaei, 2012) not in minaei checklist (minaei, 2013), the number of species of thysanoptera known from iran grows to 223. article figure 1. dendrothrips aspersus, female. figure 2. dendrothrips aspersus, female. a) antenna; b) head and pronotum; c) mesonotum and metanotum; d) abdominal tergites vii-x. no nco mm er cia l u se on ly references alavi j., minaei k., fekrat l., 2014 the iranian dendrothripinae (thysanoptera: thripidae) with description of a new genus and species. zootaxa 3860: 479-486. alavi j., modarres awal m., fekrat l., minaei k., 2013 the genus mycterothrips (thysanoptera: thripidae) in iran, with three new species. zootaxa 3718: 345-356. ananthakrishnan t.n., 1961 studies on some indian thysanoptera vi. zool. anzieger 167: 259-271. bhagat r.c., 2011 species richness and host-plant diversity of thrips (thripidae: thysanoptera) in jammu & kashmir. indian j. appl. pure biol. 26: 85-89. bhatti j.s., 1971 five new species of dendrothrips uzel, with a key to the indian species (thysanoptera: thripidae). orient. insects 5: 345-359. fallahzadeh m., saghaei n., 2012 new data on the fauna of thysanoptera in fars province-iran. afr. j. agric. res. 7: 5548-5552. fekrat l., manzari s., 2014 first report of scolothrips dilongicornis (thysanoptera, thripidae) from iran. j. entomol. soc. iran 34:43-44. gholami n., fekrat l., manzari s., 2014 first record of thrips juniperinus (thys.: thripidae) from iran. j. entomol. soc. iran 34: 65-66. jahangiri sisakht n., habibpour b., ramezani l., 2014 the first report of the species thrips italicus (thys.: thripidae) from iran. j. entomol. soc. iran 34: 21-22 [in persian]. marullo r., 2003 host relationships at plant family level uzel in dendrothrips (thysanoptera: thripidae: dendrothripinae) with a new australian species. aust. j. entomol. 42: 46-50. minaei k., 2013 thrips (insecta, thysanoptera) of iran: a revised and updated checklist. zookeys 330: 53-74. minaei k., 2014a a new species of eremiothrips from iran (thysanoptera: thripidae). acta entomol. mus. nat. pragae 54: 29-34. minaei k., 2014b new record of predatory thrips, aeolothrips melaleucus (thysanoptera, aeolothripidae) from iran. linzer biol. beitr. 46: 637-642. minaei k., abdollahi m., 2015 predators of leaf-feeding mites, scolothrips (thysanoptera: thripidae), in iran with first description of the female of scolothrips tenuipennis. zool. ecol25: 36-66. minaei k., alavi j., fekrat l., aleosfoor m., 2014 first record of the genus eryngyothrips from iran with description of a new species (thysanoptera: thripidae). acta entomol. mus. nat. pragae 54: 455-460. minaei k., mound la., 2014a new synonymy in the wheat thrips, haplothrips tritici (thysanoptera: phlaeothripidae). zootaxa 3802: 596-599. minaei k., mound la., 2014b the genus sitothrips (thysanoptera: thripidae) with a new grass-living species from southern iran. zootaxa 3884: 594-596. minaei k., mound la., 2014c the liothrips-lineage of thrips (thysanoptera: phlaeothripidae) from iran with the first record of micropterous morph of a liothrips species. zootaxa 3889: 107-117. minaei k., mound la., 2015 thysanoptera disjunct distribution between western america and the mediterranean with a new psilothrips species (thripidae) from iran. dtsch. entomol. z. 62: 1-7. mirab-balou m., 2013 a newly recorded species of the genus thrips (insecta: thysanoptera) from iran. nat. monten. 12: 251-254. mirab-balou m., 2014a a newly recorded species of the genus haplothrips (insecta: thysanoptera) from iran. j. crop prot. 3: 557-561. mirab-balou m., 2014b first report of the genus and species of nesothrips brevicollis (bagnall) (thysanoptera: phlaeothripidae) from iran. j. crop prot. 3: 99-103. mirab-balou m., jamali j., tong x.l., 2014a neohydatothrips ilamensis n. sp. (insecta: thysanoptera: a new species from iran. arch. biol. sci. 66: 969-973. mirab-balou m., nourollahi kh., jamali j., 2014b a new species of the genus anaphothrips (thysanoptera: thripidae) from ilam province, iran. far east. entomol. 273: 21-24. miramirkhani n., fekrat l. manzari, s. sadeghi namghi h., 2014 a newyly recorded genus and species of phlaeothripinae, karnyothrips flavipes (jones, 1912) (thysanoptera: phlaeothripidae) from iran. proceeding of the 21th iranian plant protection congress. p. 430. mound l.a., 1999 saltatorial leaf-feeding thysanoptera (thripidae, dendrothripinae) in australia and new caledonia, with newly recorded pests of ferns, figs and mulberries. aust. j. entomol. 38: 257-273. mound l.a., 2011a grass-dependent thysanoptera of the family thripidae from australia. zootaxa 3064: 1-40. mound l.a., 2011b order thysanoptera haliday, 1836. pp 201-202 in: zhang z.-q. (ed.), animal biodiversity: an outline of higherlevel classification and survey of taxonomic richness. zootaxa 3148:1-237. [journal of entomological and acarological research 2015; 47:5090] [page 71] article table 1. the species recorded in iran after the checklist by minaei (2013). species* family reference aeolothrips melaleucus aeolothripidae minaei, 2014b anaphothrips microptera thripidae mirab-balou et al., 2014b eremiothrips eshghii thripidae minaei, 2014a eryngyothrips banihashemii thripidae minaei et al., 2014 haplothrips verbasci phlaeothripidae mirab-balou, 2014a iranodendrothrips kamalii thripidae alavi et al., 2014 karnyothrips flavipes phlaeothripidae miramirkhani et al., 2014 neohydatothrips ilamensis thripidae mirab-balou et al., 2014a nesothrips brevicollis phlaeothripidae mirab-balou, 2014b mycterothrips mahvelatensis thripidae alavi et al., 2013 mycterothrips nastarani thripidae alavi et al., 2013 mycterothrips sanubari thripidae alavi et al., 2013 psilothrips zygophylli thripidae minaei & mound, 2015 scolothrips dilongicornis thripidae fekrat & manzari, 2014 scolothrips tenuipennis thripidae minaei & abdollahi, 2015 sitothrips izadpanahi thripidae minaei & mound, 2014b thrips juniperinus thripidae gholami et al., 2014 thrips italicus thripidae jahangiri et al., 2014 thrips longiceps (bagnall) thripidae mirab-balou, 2013 *full details of scientific names are provided by thripswiki (2015). table 2. nomenclature changes in thrips species recorded in iran after the checklist by minaei (2013). species change reference haplothrips cerealis synonymised with h. tritici minaei & mound, 2014a ataliothrips reuteri moved to liothrips minaei & mound, 2014c no nco mm er cia l u se on ly [page 72] [journal of entomological and acarological research 2015; 47:5090] mound l.a., 2013 homologies and host-plant specificity: recurrent problems in the study of thrips. fla. entomol. 96: 318-322. rahemi s., hashemi khabir z., sadeghi se., moharramipour s., shojai m., zeinali s., 2010 report of willow thrips thrips viminalis uzel (thy.: thripidae) from iran. j. forest range protect. res. 8: 89-91. thripswiki, 2015 thripswiki-providing information on the world’s thrips. available from: http://thrips.info/wiki/ accessed: 10 jan, 2015. zur strassen r., 2003 die terebranten thysanopteren europas und des mittelmeer-gebietes. die tierwelt deutschlands und der angrenzenden meeresteile 74: 1-277 [in german]. article no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2012; 44:e1] [page 3] notes on some lepidoptera tortricidae from central asia p. trematerra università degli studi del molise, dipartimento di scienze animali, vegetali e dell’ambiente, campobasso, italy abstract faunistic data of some lepidoptera tortricidae collected in mountainous localities of kazakhstan, kyrgyzstan and turkmenija, are reported. the total number of species recorded is 69; some of them are of special biogeographical interest. introduction in the present paper, lepidoptera tortricidae are reported to have been found among material collected in the mountainous localities of kazakistan, kirgizstan and turkmenija. the specimens were taken in the period from 1974 to 2000 by colleagues sergei v. churkin (moscow, russia), vidmandas karalius, povilas ivinskis, jan miatleuski, and aidas saldaitis (vilnius, lithuania). specimens were collected applying electrical lamps of different types. part of the material was trapped during the daytime, and a few species were raised from larvae. microlepidoptera fauna of central asia is less known. some parts of the region were not completely inventoried and studied. the present paper provides new data on the distribution of some tortricidae (total number of species 69). among other tortricids of particular interest were: phtheochroa kenneli (obraztsov), cochylimorpha halophilana (christoph), c. alternana (stephens), c. emiliana (kennel), c. pirizanica (razowski), and c. woliniana (schleich), phalonidia contractana (zeller), agapeta zoegana (linnaeus), eugnosta lathoniana (hübner), cochylidia rupicola (curtis) and c. moguntiana (rössler), eana samarcandae (razowski), cnephasia cupressivorana (staudinger) and c. longana (haworth), clepsis moeschleriana (wocke) and c. praeclarana (kennel), endothenia hebesana (walker), celypha ermolenkoi kostyuk, pelochrista griseolana (zeller) and p. medullana (staudinger), pammene fasciana (linnaeus) (razowski, 1979, 2002, 2003 and 2009; kuznetsov, 1989; brown, 2005; alipanah, 2009; trematerra, 2010a and 2010b). list of taxa tortricidae subfamily tortricinae tribe tortricini acleris scabrana ([denis & schiffermüller], 1775) material examined. 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, leg. miatleuski j. and karalius v. distribution. w europe to russian far east, asia minor and north america. tribe cochylini phtheochroa inopiana (haworth, [1811]) material examined. 3 males, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg.; 1 male and 1 female, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m, kyzyl-oi, 26.07.00, s. churkin leg. distribution. entire palaearctic, from iberian peninsula and british islands to japan. phtheochroa kenneli (obraztsov, 1944) material examined. 1 male, kazakistan s.w. mangishlak pen. w. 45 km s.e. from aktan, karagije loc., 10.30.04.99, miatleuski leg. distribution. from s europe, s ukraine, crimea, lover volga area, se urals and caucasus (dasghestan), asia. cochylimorpha halophilana (christoph, 1872) material examined. 1 male, turkmenija bachardeno r. kov ata, 1981.ix.17, p. ivinskis. distribution. from central se europe (lower volga, s ural mts.), caucasus to iran and afghanistan. cochylimorpha asiana (kennel, 1899) material examined. 1 male, kirgizstan, s. issyk-kul l., kadzhisai v., 1.07.00, 1800 m, s. churkin leg. distribution. se europe (ukraine and russia), libya, caucasus, turkmenia and from kazakhstan to c asia and mongolia, iran, afghanistan, china. cochylimorpha cultana (lederer, 1855) material examined. 1 male, turkménija, kara-kala r. ai-dere, 1985.v.8, p. ivinskis. distribution. in europe known from spain, s france and s ural mts; widely distributed in asia (w turkestan to alai, shansi, hoengshan in china) and n africa (algeria, tunisia). correspondence: pasquale trematerra, università degli studi del molise, dipartimento di scienze animali, vegetali e dell’ambiente, via de sanctis, 86100 campobasso, italy. e-mail: trema@unimol.it key words: lepidoptera tortricidae, faunistc records, central asia. received for publication: 23 february 2012. revision received: 14 march 2012. accepted for publication: 23 march 2012. ©copyright p. trematerra, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e1 doi:10.4081/jear.2012.e1 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2012; volume 44:e1 no nco mm er cia l u se on ly [page 4] [journal of entomological and acarological research 2012; 44:e1] cochylimorpha alternana (stephens, 1834) material examined. 1 male and 1 female, european p. of kazakistan, ryn-kum sandy steppe nw. kandagash loc., 0517.06.1999, miatleuski j. leg. and karalius v. leg. distribution. w palaearctic, from france and british islands eastward to ural mts. and asia minor, also libya. cochylimorpha emiliana (kennel, 1919) material examined. 1 female, turkménija, ai-dere, 1984.v.15, p. ivinskis. distribution. known from east tannuola, mongolia. cochylimorpha pirizanica (razowski, 1963) material examined. 1 female, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg.; 2 females, kirgizstan (fergana valley), 27 km s.osh, 19.07.2000, 1200 m, s. churkin leg. distribution. iran: fars province, pirezan and muk road. cochylimorpha woliniana (schleich, 1868) material examined. 1 female, kirgizstan, s. issyk-kul l., kadzhisai v., 1.07.00, 1800 m, s. churkin leg. distribution. known from europe, mainly from its southern and median parts; c. woliniana luteola (kuznetsov, 1975) in w siberia and mongolia. phalonidia latifasciana (razowsky, 1970) material examined. 1 female, turkménija, ai-dere, 1984.v.14, p. ivinskis. distribution. europe: s urals; c asia: kazakhstan. phalonidia contractana (zeller, 1847) material examined. 1 female, turkmenija bachardeno r. kov ata, 1981.ix.17, p. ivinskis; 1 male, kazakistan, s.e. balcanas vill. ili riv. env., 15-20.08.94, saldaitis leg.; 1 male, kirgizstan, s. toktogul l., 15 km ne karakul v., 1300-1400 m, 17.06.2000, s. churkin leg.; 2 males, kirgizstan, south chatkal, 5 km. e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg. distribution. widely distributed from iberian peninsula to ural mts. and greece. also found in asia minor, israel, afghanistan and kashmir, china (xinjiang). phalonidia affinitana (douglas, 1846) material examined. 1 female, european p. of kazakistan, rynkum sandy steppe nw. kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. known from w europe to ural mts. and caucasus. agapeta hamana (linnaeus, 1758) material examined. 1 male and 2 females, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 0517.06.1999, miatleuski j. and karalius v. leg.; 2 females, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. and karalius v. leg.; 1 female, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg.; 1 male and 1 female, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m, kyzyl-oi, 26.07.00, s. churkin leg. distribution. w palaearctic, excluding the southern territories, north to 66° (kazakhstan, transcaucasia, c asia, w & s siberia, asia minor, iran, afghanistan, mongolia, w china and n india). agapeta zoegana (linnaeus, 1767) material examined. 6 males, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. w palaearctic species known from w europe to ural mts. and asia minor. eugnosta lathoniana (hübner, 1800) material examined. 1 male, kazakistan, s.w. mangishlak pen. w. 45 km s.e. from aktan, karagije loc., 10.30.04.99, miatleuski leg. distribution. s europe from iberian peninsula to peloponnese, from germany to ural mts. and caucasus, also algeria, asia minor, armenia. eupoecilia sanguisorbana (herrich-schäffer, 1860) material examined. 1 male, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. distribution. europe excluding southern areas, ural mts., w kazakhstan, china (heilongjiang). aethes xanthina (falkovitsh, 1963) material examined. 1 female, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg.; 1 female, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m., kyzyl-oi, 26.07.00, s. churkin leg; 1 female, turkménija, ai-dere, 1984.v.13, p. ivinskis. distribution. se europe, (ural mts.); also known from turkmenia and iran. aethes dilucidana (stephens, 1852) material examined. 1 male and 1 female, kirgizstan, south chatkal, 5 km. e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg.; 1 female, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg. distribution. w palaearctic species, from europe to algeria; s siberia, kazakhstan, turkmenia (the literature data concerning asia require re-examination). aethes bilbaensis (rössler, 1877) material examined. 1 male, kirgizstan, s chatkal, 5 km e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg.; 1 female, kirgizstan, se toktogul l, sargata v., 900-, 10.07.1999, 1100 m, s. churkin leg. distribution. w palaearctic species, its area extends from nw africa and iberian peninsula to greece and s ural mts., also known from asia minor, n lebanon, iran, afghanistan and turkmenia; crimea, caucasus, transcaucasia and w kazakhstan. aethes spirana (kennel, 1899) material examined. 1 female, turkménija, kara-kala r. ai-dere, 1985.v.8, p. ivinskis. distribution. kazakhstan, uzbekistan, tadzhikistan, c asia, alexander mts., iran. cochylidia rupicola (curtis, 1834) material examined. 1 female, turkménija, ai-dere, 1984.v.14, p. ivinskis. distribution. west palaearctic species, recorded from europe and asia minor. cochylidia moguntiana (rössler, 1864) material examined. 3 males and 3 females, kirgizstan, s. issykkul l., kadzhi-sai v., 1800 m, 1.07.00, s. churkin leg. distribution. europe from spain to balkan peninsula, c europe to ural mts.; siberia, buryatia, chita, afghanistan, china. n pakistan. cochylis posterana (zeller, 1847) material examined. 1 female, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg.; 1 male, kirgizstan, s issykarticle no nco mm er cia l u se on ly kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg; 2 females, turkménija, ai-dere, 1984.v.15, p. ivinskis; 1 male and 3 females, turkménija, kara-kala r. ai-dere, 1985.v.7-8, p. ivinskis. distribution. europe, ukraine, european russia east as far as transkaspia. cochylis piana (kennel, 1919) material examined. 1 male, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. distribution. kazakhstan, tadzhikistan, uzbekistan, mongolia, iran, afghanistan, c asia, djarkent, china (shaanxi, sinjiang). cochylis amoenana (kennel, 1899) material examined: 1 male and 2 females, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg. distribution. asia minor, n caucasus, transcaucasia, tadzhikistan, afghanistan, pakistan, iran. tribe cnephasiini eana samarcandae (razowski, 1958) material examined: 1 male and 1 female, kirgizstan, s. toktogul l., 15 km ne karakul v., 1300-1400 m, 17.06.2000, s. churkin leg.; 4 males and 8 females, kirgizstan, south chatkal, 5 km e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg. distribution. recorded from samarkand (uzbekistan). cnephasia asiatica (kuznetsov, 1956) material examined: 2 males, turkménija, ai-dere, 1974.v.11, p. ivinskis; 2 females, turkménija, ai-dere, 1984.v.8-15, p. ivinskis; 1 female, turkménija, kara-kala r. ai-dere, 1985.v.8, p. ivinskis; turkménija, ai-dere, 1984.v.8, p. ivinskis. distribution. turkmenistan. cnephasia communana (herrich-schäffer, 1851) material examined. 3 males and 4 females, turkménija, ai-dere, 1984.v.7-13, p. ivinskis. distribution. w palaearctic species, from iberian peninsula to karelia and caucasus; transcaucasia, trans-ural, s. siberia, kazakhstan and mountains of turkmenia, also libya and asia minor. cnephasia cupressivorana (staudinger, 1871) material examined. 1 female, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg.; 1 female, kirgizstan, inn. tianshan, naryn r. vall, kazarman vic., 1400 m., 21.07.2000, s. churkin leg. distribution. southern europe from balkan peninsula to rumania, austria and asia minor. cnephasia longana (haworth, [1811]) material examined. 1 female, kirgizstan, se toktogul l, sargata v., 900 10.07.1999, 1100 m, leg. s. churkin. distribution. known from europe to mediterranean and east to w european russia. tribe archipini epagoge grotiana (fabricius, 1781) material examined. 3 males, european p. of kazakistan dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg. and karalius v. leg. distribution. from w and n europe to ukraine and s ural mts., trans-ural and asia minor. periclepsis cinctana (denis & schiffermüller, 1775) material examined. 2 males, european p. of kazakistan dzhanibek env. kandagash loc. 21-27.06.1999. leg. miatleuski j., karalius v. distribution. known all over europe; the data from asia require confirmation. archips betulanus (hübner, [1787]) material examined. 1 female, kirgizsky, mts s slopes, karakol r., balyktyr., 2300 m, 13.07.1999, s. churkin leg. distribution. palaearctic species, probably excluding the south. archips rosanus (linnaeus, 1758) material examined. 2 males, kirgizstan, tash-kumyr v., 1000 m, 18.06.2000, s. churkin leg.; 1 female, kirgizstan, s chatkal, 5 km e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg. distribution. palaearctic species; introduced in north america. pandemis cinnamomeana (treitschke, 1830) material examined. 2 females, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg. distribution. w europe to ural mts., siberia, mongolia, russian far east, nw china, korea, japan. pandemis chondrillana (herrich-schäffer, 1860) material examined. 8 males and 1 female, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 0517.06.1999, miatleuski j. leg., karalius v. leg.; 1 male, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg., karalius v. leg.; 1 female, kirgizstan, s. toktogul l., 15 km ne karakul v., 1300-1400 m, 17.06.2000, s. churkin leg. distribution. europe, ukraine and southern areas of european russia; also recorded in asia minor, iran, afghanistan and in kazakhstan through siberia to mongolia, c asia and nw china. clepsis moeschleriana (wocke, 1862) material examined: 1 male, kirgizstan, s issyk-kul l., ottuk v., 1650, 29.06.2000, s. churkin leg. distribution. a boreo-alpine species. polar ural, altai mts.; in nearctics spread from labrador to alaska, known from other parts of canada and usa. clepsis praeclarana (kennel, 1899) material examined: 1 female, kirgizstan, s. issyk-kul l., kadzhisai v., 1.07.00, 1800 m, s. churkin leg. distribution. se europe, georgia, sw siberia, saisan and mongolia. clepsis neglectana (herrich-schäffer, 1851) material examined. 5 males and 1 female, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg.; 1 male, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg. distribution. widely distributed in europe; also known from n africa, cyprus and lebanon; in c asia reaching kirgizstan and tuva. clepsis trifasciata (trematerra, 2010) material examined. 1 male, holotype, labelled as follows: kirgizstan, s issik-kul l., kadzhi-sai v., 1.07.2000, 1800 m, s. churkin leg.; paratype, 1 male with same label (trematerra, 2010). distribution. known from kirgizstan. subfamily olethreutinae tribe bactrini bactra lacteana caradja, 1916 material examined. 1 male, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg. and karalius v. leg. distribution. europe except for northern areas, caucasus, kazakhstan, w mongolia, russia (from s siberia to primorsk), china, japan. bactra bactrana (kennel, 1901) material examined. 1 male, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. [journal of entomological and acarological research 2012; 44:e1] [page 5] article no nco mm er cia l u se on ly [page 6] [journal of entomological and acarological research 2012; 44:e1] distribution. canary islands, s spain, italy, marocco, algeria, malta, egypt, asia minor, arabia, mesopotamia, iran, iraq, caucasus, afghanistan, caspian area, tadzhikistan, uzbekistan, pakistan, india, and ethiopia. tribe olethreutini endothenia hebesana (walker, 1863) material examined. 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg. and karalius v. leg. distribution. from scandinavia, finland, european nw russia, w siberia and s primorsk, nw china, nearctic region. celypha rufana (scopoli, 1763) material examined. 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg., karalius v. leg. distribution. n c and e europe, transcaucasia, s siberia, tuva, cisbaikal, and korea. celypha ermolenkoi kostyuk, 1980 material examined. 1 female, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m, kyzyl-oi, 26.07.00, s. churkin leg. distribution. ukraine (crimea). celipha cespitana (hübner, [1814-1817]) material examined. 2 males, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg.; 1 male and 3 females, kirgizstan, south chatkal, 5 km. e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg.; 10 males, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg.; 1 male, inn. tian-shan naryn r. vall, kazarman vic., 1400 m., 21.07.2000, s. churkin leg. distribution. w europe, to ural mts., transcaucasia, asia minor, near east, iran, siberia, c asia, cisbaikal, tuva, primorsk, manciuria, korea, japan, north america. tribe eucosmini thiodia torridana (lederer, 1859) material examined. 2 females, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg. distribution. europe excluded n and sw parts, ural mts., transcaucasia, asia minor, kazakhstan, turkmenia, s siberia, amurzeya-area, priamure, s primorsk, s sakhalin, kunashir islands. thiodia lerneana (treitschke, 1835) material examined. 1 female, kazakistan, s.e. balcanas vill. ili riv. env., 15-20.08.94, leg. saldaitis; 3 females, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. distribution. spain, italy, austria to hungary and balkan peninsula, s e europe, transural, kazakhstan, turkmenia. thiodia citrana (hübner, [1796-99]) material examined. 1 female, european p. of kazakistan, rynkum sandy steppe nw kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. europe from spain to karelia, lebanon, caucasus, transcaucasia, asia minor, iran, kazakhstan, turkmenia, s siberia, s. primorsk, china, japan. epinotia thapsiana (zeller, 1847) material examined. 1 male, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. distribution. canary islands, southern and central areas of europe to e european russia, transcaucasia, asia minor, iran, s siberia, kazakhstan, tadzhikistan, turkmenia, korea. pelochrista griseolana (zeller, 1847) material examined. 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg. and karalius v. leg. distribution. spain and sicily. pelochrista medullana (staudinger, 1879) material examined. 1 male, european p. of kazakistan, ryn-kum sandy steppe nw kandagash, loc. 05-17.06.1999, miatleuski j. leg. and karalius v. leg.; 2 males, kirgizstan, toktogul area, karasù l., 2100 m, 16.06.2000, s. churkin leg.; 1 male, kirgizstan, suusamyr mts., kokemeren r, 5 km n kyzyl-oi, 1800 m, 24-25.06.2000, s. churkin leg. distribution. balkan peninsula, europe, asia minor, iran. pelochrista modicana (zeller, 1847) material examined. 1 male, turkmenija, morgunovka, 1974.v.3, p. ivinskis. distribution. s e europe, asia minor, transcaucasia, turkmenia, kazakhstan. pelochrista arabescana (eversmann, 1844) material examined. 11 males, kazakistan, sw mangishlak pen.w. 45 km se from aktan, karagije loc., 10.30.04.99, miatleuski leg. distribution. central, southern and eastern europe, caucasus, transcaucasia, iran, s siberia, kazakhstan, turkmenia, mongolia, china. eucosma lugubrana (treitschke, 1830) material examined. 1 female, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m, kyzyl-oi, 26.07.00, s. churkin leg. distribution. europe, kazakhstan. eucosma conterminana (guenée, 1845) material examined. 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. and karalius v. leg.; 2 males and 12 females, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg.; 2 females, (kirgizstan) fergana valley, 27 km s.osh, 1200 m., 19.07.2000, s. churkin leg. distribution. europe, caucasus, ural mts, transcaucasia, asia minor, iran, kazakhstan, c asia, s siberia, mongolia, priamure, s primorsk, china. eucosma catoptrana (rebel, 1903) material examined. 1 male, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. europe, kazakhstan. eucosma metzneriana (treitschke, 1830) material examined. 1 male, kirgizstan, s issyk-kul l., ottuk v, 1650 m., 29.06.2000, s. churkin leg.; 1 male, kirgizstan, inn. tianshan naryn r. vall, kazarman vic., 1400 m, 21.07.2000, s. churkin leg. distribution. europe, s russia, ukraine, karelia, caucasus, transcaucasia, asia minor, iran, kazakhstan, c asia, mongolia, siberia, amur-zeya area, priamure, s primorsk, kuanhsien, korea, japan. eucosma luciana (kennel, 1919) material examined. 1 male, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 05-17.06.1999, leg. miatleuski j. and karalius v. distribution. e europe, transcaucasia, s siberia, mongolia russian far east, nw china, japan. eucosma pupillana (clerck, 1759) material examined. 10 males and 9 females, european p. of kazakistan, ryn-kum sandy steppe nw kandagash loc., 0517.06.1999, miatleuski j. leg. and karalius v. leg.; 2 males and 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., article no nco mm er cia l u se on ly 21-27.06.1999, miatleuski j. leg. and karalius v. leg.; 1 male, kirgizstan, s. toktogul l., 15 km ne karakul v., 1300-1400 m, 17.06.2000, s. churkin leg.; 2 males, kirgizstan, south chatkal, 5 km. e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg.; 1 female, kirgizstan, s issyk-kul l., ottuk v, 1650 m, 29.06.2000, s. churkin leg. distribution. europe, ural mts., asia minor, iran, kazakhstan, tadzhikistan. eucosma ephedrana (christoph, 1877) material examined. 1 male, kirgizstan, s. issyk-kul l., kadzhi-sai v., 1.07.00, 1800 m, s. churkin leg. distribution. transcaucasia, c asia, iran, afghanistan. eucosma clarescens (kuznetsov, 1964) material examined. 1 female, european p. of kazakistan, rynkum sandy steppe nw kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. europe, kazakhstan, mongolia. gypsonoma oppressana (treitschke, 1835) material examined. 1 male, kirgizstan, e suusamyr mts., kovjuksu r., 1650 m, kyzyl-oi, 26.07.00, s. churkin leg. distribution. europe, trascaucasia, kazakhstan, tadzhikistan. epiblema foenellum (linnaeus, 1758) material examined. 1 female, kirgizstan, south chatkal, 5km. e aflatun v., karasu r., 1350 m, 19.06.2000, s. churkin leg. distribution. known from palaearctic and oriental regions. epiblema junctanum (herrich-schäffer, 1856) material examined. 1 female, european p. of kazakistan, rynkum sandy steppe nw kandagash loc., 05-17.06.1999, miatleuski j. leg. and karalius v. leg. distribution. europe, asia minor, iran, c asia, kazakhstan, siberia, priamure, s primorsk. notocelia cynosbatella (linnaeus, 1758) material examined. 3 males, turkménija, kara-kala r. ai-dere, 1985.v.8, p. ivinskis; turkménija, ai-dere, 1984.v.14-15, p. ivinskis. distribution. europe, ural mts., caucasus, asia minor, near east, iran, trascaucasia, kazakhstan, siberia, amur-zeya area priamure, s primorsk, s sakhalin. tribe grapholitini cydia pomonella (linnaeus, 1758) material examined. 1 male and 1 female, european p. of kazakistan, dzhanibek env. kandagash loc., 21-27.06.1999, miatleuski j. leg. and karalius v. leg. distribution. w palaearctic, worldwide (introduced). pammene fasciana (linnaeus, 1761) material examined. 1 female, azerbaidän, lenkoran, aurora, 1987.viii.4, p. ivinskis. distribution. europe, karelia, ukraine. references alipanah h., 2009 – synopsis of the cochylini (tortricidae: tortricinae: cochylini) of iran, with the description of a new species. zootaxa, 2245: 1-31. brown j.w., 2005 – tortricidae (lepidoptera). in: world catalogue of insects 5. apollo book aps, stenstrup, denmark: 1-741. kuznetsov v.i., 1989 – family tortricidae (olethreutidae, cochylidae) – tortricid moths. in: medvedev g.s., keys to the insects of the european part of the ussr, volume iv, lepidoptera, part i. – e.j. brill, leiden, the netherlands: 279-991. razowski j., 1979 – revision of the genus clepsis guenée (lepidoptera, tortricidae). part i. acta zool. cracov. xxiii (8): 101198. razowski j., 2002 – tortricidae (lepidoptera) of europe, volume 1, tortricinae and chlidanotinae. frantisek slamka, bratislava: 1247. razowski j., 2003 tortricidae of europe. volume 2. olethreutinae. frantisek slamka, bratislava: 1-301. razowski j., 2009 – tortricidae of the palaearctic region, volume 2, cochylini. frantisek slamka, bratislava: 1-195. trematerra p., 2010a – clepsis trifasciata sp. n. with notes on some lepidoptera tortricidae from kirgizstan. j. entomol. acarol. res. ser. ii 42 (1): 1-10. trematerra p., 2010b lepidoptera tortricidae from se european russia with description of ceratoxanthis saratovica sp. n. j. entomol. acarol. res. ser. ii 42 (1): 19-26. [journal of entomological and acarological research 2012; 44:e1] [page 7] article no nco mm er cia l u se on ly jear2012 abstract oilseeds such as flax, canola, safflower, soybean and sunflower, which are annual plants, provide the world’s major source of vegetable oils, although the highest oil yield comes from oil-bearing tree fruits. one of the most popular oil seeds is safflower (carthamus tinctorius l.), which belongs to the asteraceae family. due to the ability of this plant to grow in dry and semi-dry conditions, safflower oil has the potential to be a commercially profitable product in iran. seasonal populations of safflower capsule flies were studied in kohgiluyeh safflower farms, iran, from march to may in 2008 and 2009. four yellow sticky traps were used to monitor populations of fruit flies in the safflower farms. traps were checked once a week during the sampling period. the traps were emptied weekly into insect collection vials containing 70% ethanol. data were analysed with a two-way anova. the relation between abiotic factors and species abundance was analysed with multiple linear regression. the results emphasized that acanthiophilus helianthi was the most serious pest of safflower under the ecological conditions found in gachsaran, being present in the field throughout three months of the year (march to may). chaetorellia carthami was present in safflower fields from march to may, but in significant numbers only during april and may. terellia luteola was present in safflower fields from march to may and in significant numbers only in late april, it does not seem to be a serious pest in safflower farms under gachsaran’s ecological conditions. introduction safflower (carthamus tinctorius l.) is a member of the compositae or asteraceae family (emongor, 2010). it is a multi-purpose oilseed crop grown mainly for its high quality edible oil, as well as for bird seed (karimi, 2000). initially, safflower oil was used as a source of oil in the manufacture of paint, but today it is widely used as edible oil for cooking, and in the production of margarine and salad oil (tinay, 2001). safflower is also grown for its flowers, which are used as; cut flowers, in colouring and food flavouring, for the manufacture of dyes for the textile industry, livestock forage, as vegetable, herbal teas and for medicinal purposes (karegar et al., 2004). in china, safflower is grown as a medicinal plant for the treatment of cardiovascular diseases, male and female fertility, lowering blood cholesterol, as well as various types of rheumatism and respiratory diseases (rennie et al., 2003). safflower, is now cultivated on approximately one million hectares of land and annually about 700,000 tons of seed are produced (zeynali, 2001). iran, once known as persia, used to be a centre for the commercial cultivation of safflower in the ancient world and it continues to cultivate this oil seed to this day (golkar et al., 2010). at present, approximately 1000 hectares of land are under safflower cultivation in iran, which produces approximately 700 tons of seed annually (karegar et al., 2004). due to the ability of the plant to thrive in arid and semi-arid lands, safflower has the potential to become a commercially profitable product in iran (karimi, 2000). like other crops, safflower is susceptible to various diseases and insect attacks (majidi et al., 2011). due to water restrictions and the amount of arable land, one of the methods used to increase production is to reduce the damage caused by pests and plant diseases. in nature, insect populations fluctuate depending on environmental factors. broadly speaking, these environmental factors can be divided into biotic factors, such as natural enemies and plants, and abiotic factors, such as temperature, relative humidity and precipitation. from ecological studies, vital information can be obtained by monitoring changes in insect population numbers that result from changes in environmental factors. studies of potential pests are necessary in order to meet the challenges of providing protection for both crops and livestock (den & walton, 1997). fruit flies of the family tephritidae (order: diptera) are one of the most serious pests of fruits and vegetables. they cause enormous economic losses in the production of fruits and vegetables throughout the world (korneyev & konovalov, 2010). in the iranian province of correspondence: karim saeidi, department of plant protection, agricultural and natural resources research and education center, kohgiluyeh va boyerahmad province, p.o. box 351, yasouj, iran. tel.: +98.741.3334821/+98.741.3334821 fax: +98.741.3334011. e-mail: saeidi391@yahoo.com key words: fruit flies; acanthiophilus helianthi; chaetorellia carthami; terellia luteola; population fluctuation; kohgiluyeh va boyerahmad province. received for publication: 1 september 2014. revision received: 27 march 2015. accepted for publication: 30 april 2015. ©copyright k. saeidi et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:4684 doi:10.4081/jear.2015.4684 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. population dynamics of safflower capsule flies (diptera: tephritidae) in kohgiluyeh safflower farms of iran k. saeidi,1 s. mirfakhraei,2 f. mehrkhou2 1department of plant protection, agricultural and natural resources research and education center, kohgiluyeh va boyerahmad province, yasouj; 2department of plant protection, college of agriculture, urmia university, urmia, iran [page 50] [journal of entomological and acarological research 2015; 47:4684] journal of entomological and acarological research 2015; volume 47:4684 no nco mm er cia l relation between abiotic factors and species abundance was analysed no nco mm er cia l relation between abiotic factors and species abundance was analysed with multiple linear regression. the results emphasized that no nco mm er cia l with multiple linear regression. the results emphasized that was the most serious pest of safflower under no nco mm er cia l was the most serious pest of safflower under the ecological conditions found in gachsaran, being present in the no nco mm er cia l the ecological conditions found in gachsaran, being present in the field throughout three months of the year (march to may). no nco mm er cia l field throughout three months of the year (march to may). was present in safflower fields from march to no nco mm er cia l was present in safflower fields from march to may, but in significant numbers only during april and may. no nco mm er cia l may, but in significant numbers only during april and may. manufacture of paint, but today it is widely used as edible oil for cookno nco mm er cia l manufacture of paint, but today it is widely used as edible oil for cook-ing, and in the production of margarine and salad oil (tinay, 2001). no nco mm er cia l ing, and in the production of margarine and salad oil (tinay, 2001). safflower is also grown for its flowers, which are used as; cut flowers, no nco mm er cia l safflower is also grown for its flowers, which are used as; cut flowers, in colouring and food flavouring, for the manufacture of dyes for the no nco mm er cia l in colouring and food flavouring, for the manufacture of dyes for the no nco mm er cia l textile industry, livestock forage, as vegetable, herbal teas and for no nco mm er cia l textile industry, livestock forage, as vegetable, herbal teas and for medicinal purposes (karegar no nco mm er cia l medicinal purposes (karegar no nco mm er cia l no nco mm er cia l correspondence: karim saeidi, department of plant protection, agricultural no nco mm er cia l correspondence: karim saeidi, department of plant protection, agricultural and natural resources research and education center, kohgiluyeh va no nco mm er cia l and natural resources research and education center, kohgiluyeh va boyerahmad province, p.o. box 351, yasouj, iran.no nco mm er cia l boyerahmad province, p.o. box 351, yasouj, iran. tel.: +98.741.3334821/+98.741.3334821 fax: +98.741.3334011.no nco mm er cia l tel.: +98.741.3334821/+98.741.3334821 fax: +98.741.3334011. us e carthamus tinctorius us e carthamus tinctorius or asteraceae family (emongor, 2010). it is a multi-purpose oilseed us e or asteraceae family (emongor, 2010). it is a multi-purpose oilseedcrop grown mainly for its high quality edible oil, as well as for bird seed us e crop grown mainly for its high quality edible oil, as well as for bird seed (karimi, 2000). initially, safflower oil was used as a source of oil in theus e (karimi, 2000). initially, safflower oil was used as a source of oil in the manufacture of paint, but today it is widely used as edible oil for cook-us e manufacture of paint, but today it is widely used as edible oil for cookon ly on ly carthamus tinctoriuson ly carthamus tinctorius or asteraceae family (emongor, 2010). it is a multi-purpose oilseed on ly or asteraceae family (emongor, 2010). it is a multi-purpose oilseed kohgiluyeh va boyerahmad, there are 18 known species of fruit flies, however, the species considered to be the most serious pests of fruits and vegetables number less than ten (gilasian & merz, 2008). the majority of these species are polyphagous, with high fecundity and the ability to spread quickly over a wide area, thus making them serious pests for the growers of fruits and vegetables. effective management of this fly on safflower crops requires a better understanding of the species’ seasonal dynamics in a particular locality. to achieve effective pest control measures, actions need to be targeted at periods of maximum population build-up and at the most vulnerable stage of the crop (saeidi, 2006). the present study was carried out to monitor population fluctuations of fly species associated with safflower damage in the gachsaran region of iran. despite the fact that the threat of pest infestation is a serious problem that hinders the cultivation of safflower on a commercial scale, no comprehensive or useful information about safflower pests in kohgiluyeh va boyerahmad province and other parts of the country could be found. materials and methods study site kohgiluyeh-va-boyerahmad is a mountainous province situated in south west of iran. about 3/4 of the area is rugged and plains comprise only 1/4 of the province area (figure 1). the study was performed from march to may in 2008 and 2009 at the safflower farm in the agricultural research station, gachsaran. this site has a warm climate (mean minimum temperature of 15°c and a mean maximum temperature of 46°c), and it is characterized by annual precipitation of about 250-300 mm. the total land area of the safflower farm surveyed covered an area of 0.5 ha. the site was selected because it represented a large area of cultivated safflower that is commonly infested by fruit flies. fly trapping four yellow sticky traps were used to monitor the populations of fruit flies in the safflower farm. the traps were composed of polyethylene plates with dimensions 20×20 cm produced by the agro science british company. the traps were installed at a height of 80 cm from the ground, 200 m away from the field edge and 800 m apart from each other. traps were checked once a week during the sampling period. the sticky traps were completely cleaned after each survey and re-glued if necessary. collection and identification of traps catches the traps were emptied weekly into insect collection vials containing 70% ethanol. the insects collected were sent to a laboratory for identification and counting. identification was based on the morphological characteristics of the collected specimens using a taxonomic key developed by the iranian fruit fly initiative (mohamadzade namin et al., 2010). samples of the identified insects have been deposited at the department of plant protection, agricultural and natural resources research center of yasouj, iran. incubation of flower heads each week 50-safflower flower heads were collected and placed into plastic vessels. the mean room temperature during the incubation period was 23°c, while the relative humidity for the same period varied between 55% and 60%. the flower heads were inspected every other day to remove fruit flies pupae, until there no were pupae present in the flower heads. the pupae were then placed in plastic bottles (diameter 8 cm and height 15 cm) lined at the bottom with moist tissue paper for emergence. emerged flies were collected by aspirator and then counted. climatic data data on temperature, precipitation and relative humidity of the study area were obtained from the local weather station at the gachsaran agricultural research station. data analysis data was subjected to two-way anova. the correlation between abiotic factors and species abundance was analyzed with spss multiple linear regression. results and discussion relationship of safflower capsule flies and abiotic factors a total of 7633 fruit flies were captured in the yellow sticky traps during both years, and consisted of 88.8% (6780) a. helianthi, 7.6% (585) c. carthami and 3.5% (268) t. luteola. in addition, 854 fruit flies emerged from the incubated flower heads of which 70.8% (605) were a. helianthi, 20.0% (171) c. carthami and 9.1% (78) t. luteola. a total of 3446 fruit flies were captured in the yellow sticky traps from march to may 2008. there was a significant difference among months in 2008 [degree of freedom (df)=2; f=3.59; p=0.041]. the highest [journal of entomological and acarological research 2015; 47:4684] [page 51] article figure 1. the geographical position of kohgiluyeh va boyerahmad province on map of iran (saeidi et al., 2015). no nco mm er cia l 46°c), and it is characterized by annual precipitation of about 250-300 no nco mm er cia l 46°c), and it is characterized by annual precipitation of about 250-300 mm. the total land area of the safflower farm surveyed covered an area no nco mm er cia l mm. the total land area of the safflower farm surveyed covered an area of 0.5 ha. the site was selected because it represented a large area of no nco mm er cia l of 0.5 ha. the site was selected because it represented a large area of four yellow sticky traps were used to monitor the populations of fruit no nco mm er cia l four yellow sticky traps were used to monitor the populations of fruit flies in the safflower farm. the traps were composed of polyethylene no nco mm er cia l flies in the safflower farm. the traps were composed of polyethylene plates with dimensions 20×20 cm produced by the agro science british no nco mm er cia l plates with dimensions 20×20 cm produced by the agro science british company. the traps were installed at a height of 80 cm from the no nco mm er cia l company. the traps were installed at a height of 80 cm from the ground, 200 m away from the field edge and 800 m apart from each no nco mm er cia l ground, 200 m away from the field edge and 800 m apart from each other. traps were checked once a week during the sampling period. the no nco mm er cia l other. traps were checked once a week during the sampling period. the sticky traps were completely cleaned after each survey and re-glued if no nco mm er cia l sticky traps were completely cleaned after each survey and re-glued if collection and identification of traps catchesn on -co mm er cia l collection and identification of traps catchesn on -co mm er cia l no nco mm er cia l u se 20.0% (171) us e 20.0% (171) a total of 3446 fruit flies were captured in the yellow sticky traps from us e a total of 3446 fruit flies were captured in the yellow sticky traps frommarch to may 2008. there was a significant difference among months us e march to may 2008. there was a significant difference among months in 2008 [degree of freedom (df)=2; f=3.59; p=0.041]. the highestus e in 2008 [degree of freedom (df)=2; f=3.59; p=0.041]. the highest on lya total of 7633 fruit flies were captured in the yellow sticky traps duron lya total of 7633 fruit flies were captured in the yellow sticky traps dur-ing both years, and consisted of 88.8% (6780) on lying both years, and consisted of 88.8% (6780) and 3.5% (268) on lyand 3.5% (268) t. luteola on lyt. luteola emerged from the incubated flower heads of which 70.8% (605) were on ly emerged from the incubated flower heads of which 70.8% (605) were on ly 20.0% (171) on ly 20.0% (171) c. carthamion ly c. carthami a total of 3446 fruit flies were captured in the yellow sticky traps from on ly a total of 3446 fruit flies were captured in the yellow sticky traps from [page 52] [journal of entomological and acarological research 2015; 47:4684] number of individuals was obtained in may (figure 2), and the number of flies captured of each species was also significantly different (df=2; f=133.72; p=0.000). the highest number of specimens came from a. helianthi (3780), followed by c. carthami (296) and t. luteola (111) (figure 3). acanthiophilus helianthi was the species with the highest number of individuals (p<0.05) emerging from incubated flower heads with 225 (63.2%) individuals, followed by c. carthami and t. luteola with 101 (28.3%) and 30 (8%) specimens, respectively (table 1). similar results were obtained in 2009. a total of 4187 fruit flies were captured in the yellow sticky traps from march to may 2009. there was a significant difference found in the various months in 2009 (df=2; f=6.46; p=0.005). the highest number of specimens was obtained in may (figure 4), and the number of flies captured was also significantly different between species (df=2; f=133.72; p=0.000). the highest number of individuals belonged to a. helianthi (3000), followed by c. carthami (289) and t. luteola (157) (figure 5). acanthiophilus helianthi was still the most significant species (p<0.05) with the highest number of individuals emerging from incubated flower heads at 380 (76.3%) individuals, followed by c. carthami and t. luteola with 70 (14%) and 48 (9.6%) specimens, respectively (table 2). examining damaged safflower’s flower heads across the semi-arid areas in kohgiluyeh va boyerahmad province, in the south-west of iran, it was observed that the safflower capsule fly and two other flies, namely terellia luteola and chaetorellia carthami, were infecting the flower heads of this crop. overall, a. helianthi was the most abundant insect species. figure 6 gives population dynamics of the different fruit fly species during the study period (2008). the number of flies per trap per week for a. helianthi was significantly higher (p<0.05) in the three months from march to may than those for c. carthami and t. luteola. actually, there was no significant difference found between c. carthami and t. luteola numbers. the highest number of flies per trap per week for a. helianthi was recorded in mid-may, while the highest catch for c. carthami was recorded in early may. whereas, the highest trap catch for t. luteola was recorded at the end of march and april. table 2 shows the number of fruit fly species coming from incubated flower heads during the study period (2009). the trapped numbers for the three fruit fly species were not significantly different (p<0.05). the mean number of a. helianthi captured during the study was significantly higher than those of c. carthami and t. luteola. a. helianthi was the most dominant (p<0.05) fruit fly species that emerged from the incubated fruits during the peak safflower months from march to may. table 3 shows the correlations matrix for the three fruit fly species. the occurrence of a. helianthi was negatively correlated with that of c. carthami. the occurrence of a. helianthi was also negatively correlated with temperature, but positively correlated with relative humidity. however, populations of c. carthami were positively correlated with temperature, but negatively correlated with both relative humidity and rainfall. populations of t. luteola did not show any significant correlations in these measures. relationship of safflower capsule flies and abiotic factors (2009) a total of 4187 fruit flies were captured in the yellow sticky traps from march to may 2009. out of these 90.2% (3780) were a. helianthi, 7.1% (296) c. carthami and 2.6% (111) t. luteola. in addition, 498 fruit flies emerged from the incubated flower heads of which 76.3% (380) were a. helianthi, 14% (70) c. carthami and 9.6% (48) t. luteola (table 2). figure 7 shows population dynamics of the different fruit fly species during the study period (2009). the number of flies per trap per week for a. helianthi was significantly higher (p�0.05) in the three months from march to may, than those for c. carthami and t. luteola. in fact, article figure 2. number of safflower capsule flies found in different months 2008. figure 3. number of safflower capsule flies for each species in 2008. figure 4. number of safflower capsule flies in the months of 2009. figure 5. number of safflower capsule flies for each species in 2009. no nco mm er cia l was recorded in early may. whereas, the highest trap catch no nco mm er cia l was recorded in early may. whereas, the highest trap catch table 2 shows the number of fruit fly species coming from incubatno nco mm er cia l table 2 shows the number of fruit fly species coming from incubated flower heads during the study period (2009). the trapped numbers no nco mm er cia l ed flower heads during the study period (2009). the trapped numbers for the three fruit fly species were not significantly different no nco mm er cia l for the three fruit fly species were not significantly different captured during the study no nco mm er cia l captured during the study c. carthami no nco mm er cia l c. carthami and no nco mm er cia l and t. luteola no nco mm er cia l t. luteola was the most dominant (p<0.05) fruit fly species that no nco mm er cia l was the most dominant (p<0.05) fruit fly species that emerged from the incubated fruits during the peak safflower months no nco mm er cia l emerged from the incubated fruits during the peak safflower months table 3 shows the correlations matrix for the three fruit fly species. no nco mm er cia l table 3 shows the correlations matrix for the three fruit fly species. was negatively correlated with that of no nco mm er cia l was negatively correlated with that of helianthino nco mm er cia l helianthi was also negatively correlatedno nco mm er cia l was also negatively correlatedno nco mm er cia l no nco mm er cia l figure 3. number of safflower capsule flies for each species in no nco mm er cia l figure 3. number of safflower capsule flies for each species in no nco mm er cia l 2008. no nco mm er cia l 2008. us e o nly there was no significant difference found between c. carthami and t. luteola. the highest number of flies per trap per week for a. helianthi was recorded in late may, while the highest catch for c. carthami was recorded in early may. the highest trap number for t. luteola was recorded in the first week of april. figure 8 shows the percentage of fruit fly species extracted from the incubated flower heads during the study period (2009). the trap catch numbers for the three fruit fly species were not significantly different (p<0.05). however, the mean number of a. helianthi captured during the study was significantly higher than those of c. carthami and t. luteola. a. helianthi was the most dominant (p<0.05) fruit fly species that emerged from the incubated fruits in the peak safflower season months from march to may. table 4 shows the correlations matrix for the three fruit fly species. the occurrence of a. helianthi was negatively correlated with that of c. carthami. furthermore, the occurrence of a. helianthi was also negatively correlated with temperature, but positively correlated with relative humidity. in addition, populations of c. carthami were positively correlated with temperature, but negatively correlated with both relative humidity and rainfall. populations of t. luteola did not show any significant correlations with abiotic factors. the production of safflower in asia is threatened by three major insect pests, namely; aphids (homoptera: aphididae), stem borers (lepidoptera: noctuidae) and fruit flies (diptera: tephritidae). however, only the latter cause large-scale economic damage to the safflower flower heads (kutuk and ozgur, 2003). for example, yield losses due to fruit flies of more than 45% have been reported in west asia (khouzama et al., 2002) and between 28% to 85% in iran (keyhanian, 2008; hasanshahi and askarianzadeh, 2012). studies on the species of fruit flies associated with safflower in the gachsaran agricultural research station showed that a. helianthi, c. carthami and t. luteola were the most important fruit fly species. the results from the present study demonstrated that a. helianthi was the dominant species from march to may. it was also the dominant fruit fly species that emerged from incubated safflower flower heads. the dominance of this fruit fly species coincided with the production of flower heads in both early and late maturing safflower varieties. this could be due to the absence of flower heads on the alternative host plants such as weeds. in addition, the study period represents the arid period at the gachsaran station, which is conducive to the population growth of a. helianthi (jakhmola and yadav, 1980). as a consequence, a. helianthi causes an enormous amount of damage to safflower flower heads, resulting in complete seed loss if appropriate control measures are not taken (merz, 2008; gharajerdaghi et al., 2012). the results showed that the patterns of fruit fly population fluctuations in the safflower farm and during the study were similar (figures 2 and 4). the population appeared in march, started increasing in april and reached its maximum in may of both years (2008 and 2009). hasanshahi and askarianzade (2012) reported similar results from the tehran province (iran). they stated that the peak population of a. helianthi was observed in may. this peak period of safflower capsule fly population coincides with the ripening of the safflowers. keyhanian (2008) reported different results from ghom province in iran. he carried out an experiment to determine the seasonal abundance and loss assessment of the safflower capsule fly on safflowers. the results showed that the adults of a. helianthi appeared on the safflower crop between the 1st week of april up to 4th week of june, and an infestation of capsules by a. helianthi larvae was observed from the 1st of april to the end of june. thereafter it declined, which was attributed to the maturity of the crop. its maximum population in the 1st and 2nd [journal of entomological and acarological research 2015; 47:4684] [page 53] article table 1. fruit fly species recovered from incubated safflower flower heads in 2008. month number of flower heads incubated number of fruit fly species emerged acanthiophilus helianthi chaetorellia carthami terellia luteola march 200 25 11 5 april 200 85 40 9 may 200 115 50 16 table. 2 fruit fly species recovered from incubated safflower flower heads in 2009. month number of flower heads incubated number of fruit fly species emerged acanthiophilus helianthi chaetorellia carthami terellia luteola march 200 90 10 5 april 200 120 20 12 may 200 170 40 31 figure 6. mean number of fruit flies per trap per week of the three fruit fly species from march to may 2008. no nco mm er cia l were the most important fruit fly species. the no nco mm er cia l were the most important fruit fly species. the was the no nco mm er cia l was the dominant species from march to may. it was also the dominant fruit fly no nco mm er cia l dominant species from march to may. it was also the dominant fruit fly species that emerged from incubated safflower flower heads. no nco mm er cia l species that emerged from incubated safflower flower heads. the dominance of this fruit fly species coincided with the production no nco mm er cia l the dominance of this fruit fly species coincided with the production of flower heads in both early and late maturing safflower varieties. this no nco mm er cia l of flower heads in both early and late maturing safflower varieties. this could be due to the absence of flower heads on the alternative host no nco mm er cia l could be due to the absence of flower heads on the alternative host plants such as weeds. in addition, the study period represents the arid no nco mm er cia l plants such as weeds. in addition, the study period represents the arid no nco mm er cia l period at the gachsaran station, which is conducive to the population no nco mm er cia l period at the gachsaran station, which is conducive to the population (jakhmola and yadav, 1980). as a consequence, no nco mm er cia l (jakhmola and yadav, 1980). as a consequence, causes an enormous amount of damage to safflower flower no nco mm er cia l causes an enormous amount of damage to safflower flower no nco mm er cia l no nco mm er cia l u se on ly on ly on ly [page 54] [journal of entomological and acarological research 2015; 47:4684] generation was seen at the last week of may and the 1st week of june, respectively. one of the most important reasons for the different results obtained in gachsaran and ghom are due to the different climatic conditions and vegetation types present in these two areas. references den, d.r., walton m.p., 1997 methods in ecological and agricultural entomology. cab international. wallingford, oxon. emongor v., 2010 safflower (carthamus tinctorius l.) the underutilized and neglected crop: a review. asian j. plant sci. 9: 299-306. gharajedaghi y.k., khaghaninia s., farshbaf pour abad r., 2012 an investigation of the fruit flies (diptera: tephritidae) fauna in ajabshir region with the new record from iran. mun. ent. zool. 7: 1. gilasian e., merz b., 2008 the first report of three genera and fifteen species of tephritidae (diptera) from iran. j. entomol. soc. iran 27: 11-14. golkar p., arzani a., rezaei m., 2010 inheritance of flower colour and spinelessness in safflower (carthamus tinctorius l.). j. genetics. 89: 124-127. hasanshahi g.h., askarianzadeh a., 2012 effect of drought stress on the damage safflower fly, acanthophilus helianthi rossi (dip.: tephritidae) on three cultivars of safflower, carthamus tinctorius l. in tehran region. iranian j. entomol. res. 4: 28-39. jakhmola s.s., yadav h.s., 1980 incidence of and losses caused by capsule fly, acanthiophilus helianthi rossi in different varieties of safflower. indian j. entomol. 42: 48-53. karegar a., abdulahi r., arslan b., 2004 assessing of heritability and variance components of yield and some agronomic traits of different safflower (carthamus tinctorius l.), cultivars. asian j. plant sci. 6: 554-557. article table 3. correlation coefficients (r) between the occurrence of fruit flies and climatic parameters in 2008. a b c d e f acanthiophilus helianthi (a) 1 chaetorellia carthami (b) �0.442* 1 terellia luteola (c) �0.365 �0.210 1 precipitation (d) 0.292 0.345 0.128 1 relative humidity (e) 0.678* �0.535* 0.008 0.731* 1 temperature (f) �0.410* 0.176* 0.205 �0.556 0.660* 1 numbers with asterisks are significant (p<0.05). figure 7. mean number of fruit flies per trap per week of the three fruit fly species from march to may 2009. table 4. correlation coefficients (r) between the occurrence of fruit flies and climatic parameters in 2009. a b c d e f acanthiophilus helianthi (a) 1 chaetorellia carthami (b) �0.453* 1 terellia luteola (c) �0.389 �0.230 1 precipitation (d) 0.299 0.369 0.136 1 relative humidity (e) 0.692* �0.565* 0.009 0.767* 1 temperature (f) �0.450* 0.182* 0.215 �0.585 0.689* 1 numbers with asterisks are significant (p<0.05). figure 8. percentage of fruit fly species from incubated flower heads from march to may 2009. means with different letters are significantly different at p<0.05. no nco mm er cia l jakhmola s.s., yadav h.s., 1980 incidence of and losses caused by no nco mm er cia l jakhmola s.s., yadav h.s., 1980 incidence of and losses caused bycapsule fly, no nco mm er cia l capsule fly, safflower. indian j. entomol. 42: 48-53. no nco mm er cia l safflower. indian j. entomol. 42: 48-53. karegar a., abdulahi r., arslan b., 2004 assessing of heritability no nco mm er cia l karegar a., abdulahi r., arslan b., 2004 assessing of heritability and variance components of yield and some agronomic traits of difno nco mm er cia l and variance components of yield and some agronomic traits of dif) between the occurrence of fruit flies and climatic parameters in 2008. no nco mm er cia l ) between the occurrence of fruit flies and climatic parameters in 2008. no nco mm er cia l no nco mm er cia l a b c d e f no nco mm er cia l a b c d e f 1 no nco mm er cia l 1 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l heads from march to may 2009. means with different letters are no nco mm er cia l heads from march to may 2009. means with different letters are no nco mm er cia l u se hasanshahi g.h., askarianzadeh a., 2012 effect of drought stress us e hasanshahi g.h., askarianzadeh a., 2012 effect of drought stress on the damage safflower fly, us e on the damage safflower fly, tephritidae) on three cultivars of safflower, us e tephritidae) on three cultivars of safflower, l. in tehran region. iranian j. entomol. res. 4: 28-39.us e l. in tehran region. iranian j. entomol. res. 4: 28-39. jakhmola s.s., yadav h.s., 1980 incidence of and losses caused byus e jakhmola s.s., yadav h.s., 1980 incidence of and losses caused by acanthiophilus helianthius e acanthiophilus helianthi on ly teen species of tephritidae (diptera) from iran. j. entomol. soc. on ly teen species of tephritidae (diptera) from iran. j. entomol. soc. golkar p., arzani a., rezaei m., 2010 inheritance of flower colour on lygolkar p., arzani a., rezaei m., 2010 inheritance of flower colourand spinelessness in safflower ( on lyand spinelessness in safflower (carthamus tinctorius on lycarthamus tinctorius genetics. 89: 124-127. on ly genetics. 89: 124-127. hasanshahi g.h., askarianzadeh a., 2012 effect of drought stresson ly hasanshahi g.h., askarianzadeh a., 2012 effect of drought stress on the damage safflower fly, on ly on the damage safflower fly, karimi a., 2000 genetic variation in a safflower germplasm grown in rain fed cold dry lands. j. agron. 5: 50-52. keyhanian a.k., 2008 seasonal abundance of the safflower fly, acanthiophilus helianthi rossi (diptera: tephritidae), an infestation on safflower, carthamus tinctorius l. in ghom province, iran. pajouhesh-va-sazandegi 78: 57-62. khouzama m.k., kalash s.m., white i.m., 2002 flower head infesting fruit flies (diptera: tephritidae) on thistles (asteraceae). lebanon j. nat. his. 36: 617-626. korneyev s.v., konovalov s.v., 2010 review of the fruit flies (diptera: tephritidae) of lugansk region (ukraine). summery. 1: 35-38. kutuk m., ozgur a.f., 2003 faunistical and systematical studies on the genus tephritis latreille, 1804 (diptera: tephritidae) in the south west of turkey along with new records. turk. entomol. derg 27: 243-252. majidi m.m., tavakoli v., mirlohi a., sabzalian, m.r., 2011 wild safflower species (carthamus oxyacanthus bieb.): a possible source of drought tolerance for arid environments. austr. j. crop sci. 5: 1055-1063. merz b., 2008 order diptera, family tephritidae. arthropod fauna of the uae1: 643-661. mohamadzade namin s., nozari j., najarpoor a., 2010 the fruit flies (diptera: tephritidae) in the fauna of ardabil province, with new records for iran. ukrainska entomofaunistyka 1: 35-41. rennie k.l., hughes j., lang r., 2003 nutritional management of rheumatoid arthritis: a review of the evidence. j. hum. nutr. dietetics. 16: 97-109. saeidi k., 2006 biology safflower fly, acanthiophilus helianthi (diptera: tephritidae) in gachsaran region. annual report office of yasooj agricultural research center: 10-19. saeidi k., mirfakhraei s., mehrkhou f., valizadegan o., 2015 biodiversity of insects associated with safflower (carthamus tinctorius) crop in gachsaran, iran. j. entomol. acarol. res. 47: 26-30. tinay a.h., 2001 changes in fatty acids composition during seed growth and physiochemical characteristics of oil extracted from four safflower cultivars. plant foods hum. nutr. 56: 385-395. zeynali e., 2001 simulating gfdl predicted climate change impact on rice cropping in iran. j. agric. sci. technol. 3: 81-90. [journal of entomological and acarological research 2015; 47:4684] [page 55] article no nco mm er cia l u se on ly on rice cropping in iran. j. agric. sci. technol. 3: 81-90. on ly on rice cropping in iran. j. agric. sci. technol. 3: 81-90. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 [journal of entomological and acarological research 2015; 47:5256] [page 109] efficacy of insect-proof nets used in tunisian tomato greenhouses against tuta absoluta (meyrick) (lepidoptera: gelechiidae) and potential impact on plant growth and fruit quality a. harbi,1 k. abbes,1 b. dridi-almohandes,2 b. chermiti1 1laboratory of entomology and biological control, department of plant protection; 2conventional and organic vegetable crops unit, high agronomic institute of chott-mériem, university of sousse, tunisia abstract insect-proof screens constitute efficient physical means of protecting horticultural crops against insect pests and their use has become widespread. however, they may have a negative impact on plant growth and fruit quality by modifying climatic parameters of greenhouses. in case of tomato crops, they are used mainly against white flies and the tomato leaf miner tuta absoluta (meyrick). in tunisia, tomato plastic tunnels are often netted following two modalities: i) complete netting of the greenhouse under the plastic screen (total netting); or ii) netting only doors and lateral aeration windows (partial netting). weekly monitoring of t. absoluta in two tomato greenhouses with different netting setups using pheromone traps and sampling of leaves and fruits showed no differences in the levels of infestation by the pest with a maximum average values of 6.66 eggs/leaf, 4.16 larvae/leaf and 4.16 mines/leaf. the maximum infestation rate of leaves was 86.66% and that of fruits was 10.83%. no effects of the netting setup used on plant growth parameters were detected. however, the study of fruit quality parameters revealed significant decrease in sugar contents in tomato fruits when using total netting setup (4.26°brix versus 3.68°brix). recommendations regarding the combined use of pheromones traps and insect-proof nets are given and possibilities to enhance the efficiency of nets as physical barrier against t. absoluta are explored. introduction the tomato leafminer tuta absoluta (meyrick) (lepidoptera: gelechiidae) is ranked among the most devastating insect pests of mediterranean open field and greenhouse tomato cultivations (desneux et al., 2010, 2011; lebdi et al., 2011; abbes et al., 2012b). originating from south america, this moth invaded the mediterranean region since 2006 through spain (urbaneja et al., 2007; desneux et al., 2010). currently, the pest continues its spread through asia and subsaharan africa (desneux et al., 2011; brévault et al., 2014). t. absoluta has a high reproductive fitness as it can complete up to 12 generations per year depending on climatic conditions (tropea garzia et al., 2012). damage is caused by the mining larvae attacking leaves, stems and fruits and losses can reach up to 100% (guenaoui, 2008; chermiti et al., 2009; tropea garzia et al., 2012). besides, this pest has been reported to develop resistance to many commonly sprayed insecticides (siqueira et al., 2001; guedes & picanço, 2012; haddi et al., 2012; roditakis et al., 2015). furthermore, it has been lately demonstrated that females of some populations of t. absoluta from france and tunisia are able to reproduce parthenogenetically in the laboratory which may make its management difficult task (megido et al., 2012, 2013a; abbes & chermiti, 2014). several control tactics have been developed and integrated in pest management schemes targeting this economic pest including male annihilation using sex pheromone traps, mating disruption and biological control mainly through the use of mirid bugs (mollá et al., 2011; lebdi et al., 2011; abbes et al., 2012a, 2012b; cocco et al., 2013; megido et al., 2013b). in tunisian unheated tomato plastic tunnels, the control strategy based principally on the use of the male monitoring of this pest has been supported by the use of insect-proof nets to avoid the migration of gravid females from the outside and the re-infestations of the pheromone-treated crops (abbes et al., 2012b). physical barriers have been used in horticulture as reliable and efficient tools to protect tomato cultivations and nurseries from several key insect pests such as thrips, white flies and t. absoluta (castellano et al., 2008; harbi et al., 2012). nevertheless, they may have a negative effect correspondence: brahim chermiti, laboratory of entomology and biological control, department of plant protection, high agronomic institute of chottmériem, chott-mériem 4042, university of sousse, tunisia. tel.: +216.28156148. e-mail: chermiti54@yahoo.fr key words: climatic parameters; leafminer; physical barrier; sugar contents. acknowledgements: the authors wish to thank rabeb fersi for her technical assistance during the experiments. we also wish to acknowledge the valuable comments from anonymous reviewers that substantially improved the quality of the paper. received for publication: 27 april 2015. revision received: 4 august 2015. accepted for publication: 4 agust 2015. ©copyright a. harbi et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:5256 doi:10.4081/jear.2015.5256 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:5256 no nco mm er cia l u se on ly [page 110] [journal of entomological and acarological research 2015; 47:5256] on plant growth and fruit quality by modifying climatic parameters of greenhouses (teitel, 2006; valera et al., 2006). in the mediterranean region, unheated plastic tomato tunnels protected with insect proof nets have been frequently infested by t. absoluta especially near the openings leading to extensive insecticide applications (abbes & chermiti, 2011; harbi et al., 2012; cocco et al., 2013; biondi et al., 2015). this can eventually increase the possibilities of insecticide resistance in t. absoluta populations. furthermore, many insecticides used against t. absoluta have been reported as not compatible with the natural enemies used for the biological control of this pest owing their possible acute and long term toxicological side effects on non target arthropods (carvalho et al., 2003; biondi et al., 2012a, 2012b, 2013). in tunisia, two setups of insect proof nets are commonly used in tomato tunnels: i) some growers prefer to completely cover the plastic tunnels with insect-proof net and to overlay the plastic film to ensure hermetic isolation of the greenhouse from the external environment and to reduce the entrance of t. absoluta gravid females; or ii) some others prefer to put the insect proof net only in the aeration windows and doors which is a more economical option. this study was carried out aiming to: i) evaluate the efficacy of these two insect-proof net setups to control t. absoluta in greenhouses when combined with male monitoring; ii) assess possible impacts of both netting setups on plant growth and quality parameters of tomato fruits. the efficiency of netting strategies is discussed and recommendations for a better control of t. absoluta using insect-proof nets are given. materials and methods experimental greenhouses the study was conducted in two tomato greenhouses located in the region of bekalta (tunisian central coast). tomato plants of the cultivar sahel were grown from seeds in a pest free nursery and transplanted on 10th november 2009 with a density of 3 plants/m². experimental greenhouses were 64 m long, 8.5 m wide and 3 m high. they were protected with white-coloured insect-proof net of 6×8 threads/cm² and covered with plastic screens. the first greenhouse (hereafter called t) was completely covered with insect-proof screen and with polyethylene plastic with three aeration strips 1.5 m large each spaced out by 19.8 m. conversely the second greenhouse (hereafter named p) was covered with polyethylene plastic film and insect-proof screens were installed only in the three aeration strips of the same size of greenhouse t. in both greenhouses, the resulting aeration rate was of 13.7%. male monitoring in addition to insect-proof nets, the adult population dynamics were monitored by placing in each greenhouse one sex pheromone water trap (tutasun®, koppert biological systems, the netherlands). traps were installed on 11th december 2009 at 40 cm height from the ground level in the centre of greenhouses. sex pheromone lures (pherodis®, koppert biological systems) loaded with 0.5 mg of synthetic sex pheromone of t. absoluta and they were renewed every five to six weeks. trapped males were monitored every week and the level of water in the traps was adjusted as needed. insecticide treatments as a part of the integrated pest management scheme against t. absoluta, the experimental crops were sprayed with a bacillus thuriengiensis var. kurstaki based insecticide (bactospeine 16000, valent biosciences corp., usa). sprays were based on the level of male weekly catches of the moth in the pheromone traps with a threshold of 30 males/trap/week (megido et al., 2013b). sprays were performed on 9th february, 3rd march 2010, 7th and 30th april and 10th, 15thand 17th may 2010. sampling the assessment of the preimaginal populations of t. absoluta in both greenhouses was carried out from 20th december 2009 to 2nd june 2010 through random sampling of leaves as described by gravena (1991) for leafminers and leaf borers. the sampling protocol consists on the examination of the first fully developed leaf from the upper third of tomato plants; since young leaves are the preferential oviposition site for t. absoluta (silva et al., 1998; leite et al., 1999). for each greenhouse, 30 fully developed leaves were collected weekly from the upper part of the tomato plant and the number of all present instars of the tomato leafminer was counted in laboratory using a leica ms5 stereomicroscope (leica, germany). the infestation rate of fruits per plant was assessed weekly by counting infested and total number of fruits directly 20 tomato plants randomly chosen in each studied greenhouse. air temperature and air relative humidity were monitored in both greenhouses using data loggers type watch dog® (spectrum technologies, usa). assessment of plant growth and quality parameters of tomato fruits to compare the impact of the netting setup on tomato plant growth, 12 plants were randomly chosen in each greenhouse. the growth parameters were measured weekly starting with day 100 and continuing to day 142 after transplanting. the investigated parameters were: plant height, stem diameter at 30 cm from the apex and height of the first fruit cluster. the study of quality parameters in the tomato crops was based on fruit sampling in four ripening stages (s1: turning, s2: pink, s3: lightred, and s4: red-ripe). thirty fruits for each ripening stage were randomly chosen in three different dates for each greenhouse. the identification of the ripening stages of tomatoes was based on external fruit colour (viskelis et al., 2008). the considered fruit quality parameters were weight, firmness, ph, total soluble solid (tss), acidity and dry matter. the weight of sampled fruits was determined with a precision electronic scale type sartorius basic® model b 120s (sartorius, germany). to assess the firmness of fruits, a flat headed stainless steel cylindrical probe of 2 mm in diameter was used. penetration test started when the probe got in contact with tomato surface, and finished when the probe penetrated the tissue to a depth of 8 mm. probe speed during penetration test was 1 mm/s. thirty tomatoes of each ripening stage were tested in two opposite locations in the middle of the fruit. on the other hand, during the cropping cycle, three measures of ph were performed for each ripening stage of tomato fruits at 128, 135 and 142 days after transplanting. ten homogeneous fruits per ripening stage per date were homogenised and ph was measured with a jenway® digital ph-metre model 3505 (jenway, uk). three replicates per ripening stage and per date were performed. regarding tss, three measures were taken at 128, 135 and 142 days after transplanting on ten homogeneous fruits for every ripening stage using an atago® digital refractometer model dr-m2 (atago, france). for each date, measurements were replicated three times. the titrable acidity (ta) was determined by titration of the compost powder sample homogenised with 0,01n naoh using phenolphthaleinindicator (expressed as g/l of citric acid) (caliman et al., 2010). the titration was replicated three times per ripening stage and per date. finally, the dry matter was calculated for each sampled tomato fruit from the weight difference before and after oven drying of ten fruits at 70°c during 72 h (wuzhong, 2002). article no nco mm er cia l u se on ly statistical analysis the mean density of eggs and larvae on leave and the infestation rates of leaves and fruits as well as mean values of the plant growth parameters (plant height, stem diameter at 30 cm from the apex and the height of the first fruit cluster) and fruit quality parameters (weight, firmness, ph, tss, acidity and dry matter) were compared using student’s t test at the significance level of 5% using the statistical software spss (statistical package for the social sciences, version 17.0). results climatic parameters in the greenhouse t, the average temperature ranged from 11.6°c recorded on 17th february 2010 to 24.1°c on 2nd june 2010 (figure 1a). the lowest minimum temperature was registered on 13th january 2010 (1.4°c) whereas the highest was 43.5°c registered on 2nd june 2010. with regards to relative humidity (rh) (figure 1b), average values were between 59% recorded on 6th january 2010 and 87.4% on 10th march 2010. in the greenhouse p, the average temperature ranged between 12.2°c and 23.6°c recorded on 17th february 2010 and 2nd june 2010, respectively. the lowest minimum temperature was 2.7°c recorded on 9th february 2010 while the highest was 39.3°c on 2nd june 2010 (figure 2a). the average rh was between 48.6% recorded on 6th january 2010 and raised to 84.6% n 102010 with a minimum of 20.7% observed in january and a maximum of 100% during february, march and april (figure 2b). male monitoring the monitoring of the imaginal population of t. absoluta in greenhouse t showed a low population density from the beginning of the study towards 7th april 2010 during which the number of captured adults per trap and per week was relatively low and peaked at 75 males in on 3rd march 2010. starting from 7th april 2010, the number of caught males increased progressively to reach a maximum of 207 males trapped on 21st april 2010. after that, capture decreased to values ranging from 97 to 138 individuals per trap per week towards the end of the cropping cycle. similar flight pattern was observed in greenhouse p starting with low trap catches from the beginning of the trial until 4th april 2010 when 58 individuals/trap/week were recorded (figure 3b). the number of captured males increased gradually to reach a maximum of 296 males per trap per week on 12th may 2010 and thereby decreasing at the end of the cultivation owing the end of the cropping cycle and the absence of young leaves, preferential oviposition site for t. absoluta. [journal of entomological and acarological research 2015; 47:5256] [page 111] article figure 1. maximum, minimum and average daily temperature (a) and relative humidity (b) in the greenhouse t during the study. figure 2. maximum, minimum and average daily temperature (a) and relative humidity (b) registered in the greenhouse p during the study. no nco mm er cia l u se on ly [page 112] [journal of entomological and acarological research 2015; 47:5256] population dynamics and infestation parameters on leaves the assessment of the population dynamics of t. absoluta in greenhouses t and p revealed the absence of all biological instars of the insect during the beginning of the trial until 14th april 2010 in greenhouse t and 21st april 2010in greenhouse p (figure 4). the low population density until april is confirmed by the absence of mines and a low infestation rate on leaves not exceeding 3.3% in greenhouse t and 10% in greenhouse p (figure 5). the population density increased article figure 3. tuta absoluta male capture in pheromone traps in greenhouses t (a) and p (b). (♦) indicates lure renewal and (↓) indicates bt sprays. figure 4. dynamics of juvenile instars of tuta absoluta in greenhouses t (a) and p (b). (↓) indicates bt sprays. figure 5. mean number of larval galleries of tuta absoluta per leaf (a) and infestation rate of leaves (b) in greenhouses t and p. (↓) indicates bt sprays. no nco mm er cia l u se on ly gradually to reach a maximum of 9.7 individuals per leaf on 26th may 2010 in greenhouse t and 8.3 individuals per leaf on 19th may 2010 in greenhouse p (figure 4a and b). this high population density caused infestation rates on leaves on 2nd june 2010 in both greenhouses (figure 5b). similarly, the number of mines per leaf remained low until april, then increased progressively to reach a maximum of 4.2 mines per leaf on 2nd june 2010 in greenhouse t and 2.6 mines per leaf on 19th may 2010 in greenhouse p (figure 5b). estimation of tomato fruit loss the assessment of fruit losses started on 28 april 2010 until the end of the trial. the fruit infestation rate remained low in the beginning with a maximum of 2% in both greenhouses. the final infestation rates were 4.3% and 11% in greenhouses t and p, respectively without a significant difference (t-test, p>0.05) (figure 6). impact of insect-proof net on plant growth and quality of tomato fruits impact on plant growth measurement of tomato plants height from day 100 to day 142 after transplantation showed that plants have the same growth speed without significant differences (t-test, all p>0.05) (figure 7a). the mean growth rate ± standard error (se) was 2.5±0.21 cm/day in greenhouse t and 2.7±0.14 cm/day in greenhouse p. similarly, stems’ diameters were not significantly different (t-test, all p>0.05). the average diameter ±se of the stems increased from 1.6±0.19 to 1.7±0.25cm after 42 days in greenhouse t and from 1.6±0.22 to 1.8±0.25 cm in greenhouse p during the same period (figure 7b). on the other hand, the average height of the first bunch showed no significant differences between studied greenhouses (t-test, p=0.11>0.05). indeed, for 100-day aged plants, the average height±se of the first bunch was about 26.3±2.46 cm in greenhouse t and 28.8± 4.64 cm in greenhouse p (figure 7c). impact on tomato fruit quality the pattern of tomato fruit firmness during ripening was similar in both studied greenhouses was similar. it decreased from the first ripening stage (turning) to the fourth ripening stage (red-ripe) from an average of 3.46 kg/cm² to 1.15 kg/cm² respectively without significant differences between greenhouses t and p within each ripening stage (t-test, all p>0.05) (figure 8a). the ph of tomato juice does not reflect its concentration in acids and is generally between 4 and 4.5 (grasselly et al., 2000). in this study, the ph was between 4.01 and 4.16 in the greenhouse t and between 4.02 and 4.14 in greenhouse p (figure 8b), without significant difference between greenhouses t and p within each ripening stage (t-test, all p>0.05). tss as measured by the refractometric index increased in greenhouse p from 4.08°brix in the turning stage to 4.27°brix in the red-ripe stage (figure 8c). in greenhouse t, it decreased from 4.11°brix in the turning stage to 3.69°brix in the red-ripe stage while marking a slight increase at pink stage reaching 4.13° brix. tss at red-ripening stage was significantly higher in greenhouse p than on greenhouse t (t test, p=0.03) (figure 8c). [journal of entomological and acarological research 2015; 47:5256] [page 113] article figure 6. percentage of fruits infested by tuta absoluta in the experimental greenhouses. figure 7. tomato growth parameters in experimental greenhouses: plant height (a); plant diameter (b) and height of the first bunch (c). no nco mm er cia l u se on ly [page 114] [journal of entomological and acarological research 2015; 47:5256] ta represented by the citric acid content, showed the same pattern as the tss with a maximum of 4.54 g/l and 4.76 g/l and a minimum of 3.52 g/l and 4.14 g/l in greenhouses t and p, respectively without a significant differences in all ripening stage (t-test, all p>0.05) (figure 8d). the dry matter (dm) in tomato fruits in greenhouse t ranged from 5.67% in the turning stage to 5.15% in the red-ripe stage (figure 8e). on the other hand, in greenhouse p it decreased from 5.73% in the turning stage to 4.93% in the red-ripe stage (figure 8e) without significant differences (t-test, all p>0.05). considering that tomato fruits in greenhouse p had in the red-ripe stage higher tss than those of the greenhouse t, the average fruit weight in the first greenhouse (89.17 g) was higher than that recorded in greenhouse t (63.94 g), even though no significant difference was observed (t-test, p=0.284) (figure 8f). discussion and conclusions measurements of air temperature and relative humidity in tomato greenhouses with different netting modalities revealed similar climatic article figure 8. mean (±se) tomato quality parameters values in the greenhouses t and p. a) firmness; b) ph (b); c) total soluble solid; d) titrable acidity; e) dry matter; f) weight. ripening stages: turning (s1); pink (s2); light-red (s3); red-ripe (s4). means within each ripening stage marked with different letters are significantly different by t-test (p<0.05). no nco mm er cia l u se on ly conditions. this can be explained by the same percentage of aeration. however, it may also depend on the physical properties of the insectproof nets used such as transmissivity (the ability to transmit radiation) and reflectivity (the ability to reflect radiation). as a general rule, nets are non�uniform materials and the effect of their radiometric properties on the crops should be taken into consideration. on the other hand, despite the netting of the experimental greenhouses, t. absoluta infested the studied crops even though the number of leaf mines and fruit damage were relatively low. furthermore, bt-based insecticides specifically targeting t. absoluta were applied 7 times during the cropping season which reduced the density of larvae on leaves and the infestations on leaves and fruits. however, repeated sprayings of bt without alternating insecticides with different active ingredients and modes of action can result in emergence of bt-resistant populations of t. absoluta (braham et al., 2012). this was the case for many other lepidopteran pests such as spodoptera frugiperda (j. e. smith) and helicoverpa armigera (hübner) (wu, 2007; zhu et al., 2015). the flight patterns of the males of t. absoluta captured in sex pheromone traps were comparable in studied greenhouses as well as the population dynamics of the pest and the infestation rates on leaves and fruits. these indicate that both tested insect-proof net setups can provide the same levels of protection against t. absoluta. in both cases, the integration of sex pheromone traps with insect-proof nets and bt sprays resulted in a relatively low level of crop infestation. the early detection of the presence of adults of t. absoluta with pheromone traps may allow targeting first instar larvae known to be the most susceptible to bt. this can be very effective in tackling early the pest as the following larval instars are endophytic and hard to be targeted with insecticides acting through contact or ingestion. when combining sex pheromone trapping and insect proof nets, it is important to ensure that nets are well installed and holes-free and traps are well maintained (adjustment of the level of water for water traps or renewal of sticky boards in case of delta traps). abbes and chermiti (2011) reported that the use of pheromone traps without insect-proof net combined with bad trap maintenance can result in higher infestation rate by raising the density of males in the greenhouse and consequently the mating chances. however, in tunisia, we noticed that during the achievement of some agricultural practices such as pruning and especially harvest, some growers open the greenhouses’ doors allowing the increase of the imaginal populations of t. absoluta and by consequence leaf and fruit infestation rates especially towards the end of the cropping cycle. recent studies on the efficiency of a-cypermethrin-treated nets reported their potential to prevent the entrance of adults of t. absoluta inside the greenhouses through their repellence and long term side effects on pest life history parameters and reproductive fitness (biondi et al., 2015). this can ameliorate the efficiency of insect-proof nets considering the combined effects of the physical barrier and the insecticide. furthermore, some tunisian growers prefer to maintain their netted tomato greenhouses closed even after the end of the cropping cycle in order to avoid the dissemination of the pest. this good agricultural practice can reduce the level of inoculum of t. absoluta in the next tomato cropping seasons (in greenhouses and open field) (abbes et al., 2012b). thus, the use of a-cypermethrin-treated nets can make this strategy more efficient in reducing the imaginal population of the pest. in this study, similar plant growth parameters were registered in both greenhouses suggesting that the type of netting setups as described has no direct impact on the vegetative growth of tomato plant. in addition, most fruit quality parameters (firmness, ph, ta, dm and weight) were comparable under both tested cropping conditions. however, tss was higher in the greenhouse equipped with insectproof nets in the aeration strips p at the end of the study. this can be explained by the fact that netting the entire greenhouse can decrease the solar radiation which may result in the decrease of the tss in the fruits especially near maturation (red-ripe ripening stage). this phenomenon is known as the shading factor which can vary according to the net material and colour (castellano et al., 2008). these parameters should be also taken in consideration when using insect-poof nets. based on our findings, it can be concluded that the total netting of the greenhouses is not economically rewarding since the netting of openings can give the same level of protection against t. absoluta. this netting setup can be ameliorated by using insecticide-treated nets to enhance the role of the physical barrier. references abbes k., chermiti b., 2011 comparison of two marks of sex pheromone dispenserscommercialized in tunisia 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légumes, paris: 222 pp. gravena s., 1991 manejo integrado de pragas do tomateiro. in: anais do 2° encontro nacional de produção e abastecimento de tomate, 8-11 october, jaboticabal, brazil: 105-157. guedes r.n.c., picanço m.c., 2012 the tomato borer tuta absoluta in south america: pest status, management and insecticide resistance. eppo bull. 42: 211-216. guenaoui y., 2008 nouveau ravageur de la tomate en algérie: première observation de tuta absoluta, mineuse de la tomate invasive, dans la region de mostaganem, au printemps 2008. phytoma. 617: 18-19 haddi k., berger m., bielza p., cifuentes d., field l.m., gorman k., rapisarda c., williamson m.s., bass c., 2012 identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (tuta absoluta). insect biochem. mol. biol. 42: 506-513. harbi a., abbes k., chermiti b., 2012 evaluation of two methods for the protection of tomato crops against the tomato leafminer tuta absoluta (meyrick) under greenhouses in tunisia. eppo bull. 42: 317-321. lebdi g. k., skander m., mhafdhi m., belhadj r., 2011lutte intégrée contre la mineuse de la tomate, tuta absoluta meyrick (lepidoptera: gelechiidae) en tunisie. faunistic entomol. 2011: 125-132. leite g.l.d., picanço m., lucia t.m.c., della moreira m.d.,1999 role of canopy height in the resistance of lycopersicon hirsutum f. glabratum to tuta absoluta (lep., gelechiidae). j. appl. entomol. 123: 459-463. megido r.c., brostaux y., haubruge e., verheggen f., 2013a propensity of the tomato leafminer, tuta absoluta (lepidoptera: gelechiidae), to develop on four potato plant varieties. am. j. potato res. 90: 255-260. megido r.c., haubruge e., verheggen f.j., 2012 first evidence of deuterotokous parthenogenesis in the tomato leafminer, tuta absoluta (meyrick) (lepidoptera: gelechiidae). j. pest sci. 85: 409-412. megido r.c., haubruge e., verheggen f.o., 2013b pheromonebased management strategies to control the tomato leafminer, tuta absoluta (lepidoptera: gelechiidae). biotechnologie, agronomie, société et environnement 17: 475-482. mollá o., gonzález-cabrera j., urbaneja a., 2011 the combined use of bacillus thuringiensis and nesidiocoris tenuis against the tomato borer tuta absoluta. biocontrol 56: 883-891. roditakis e., vasakis e., grispou m., stavrakaki m., nauen r., gravouil m., bassi a., 2015 first report of tuta absoluta resistance to diamide insecticides. j. pest sci. 88: 9-16. silva c.c., jham g.n., picanço m., leite g.l.d., 1998 comparison of leaf chemical compositionand attack patterns of tuta absoluta (meyrick) (lepidoptera: gelechiidae) in three tomato species. agron. lusitana 46: 6171. siqueira h.a.a., guedes r.n.c., fragoso d.b., magalhaes l.c., 2001 -abamectin resistance and synergism in brazilian populations of tuta absoluta (meyrick) (lepidoptera : gelechiidae). int. j. pest manage. 47: 247-251. teitel m., 2006 the effect of screens on the microclimate of greenhouses and screenhouses a review. acta hortic. 719: 575-586. tropea garzia g., siscaro g., biondi a., zappalà l., 2012 tuta absoluta, a south american pest of tomato now in the eppo region: biology, distribution and damage. eppo bull. 42: 205-210. urbaneja a., vercher r., navarro v., garcía marí f., porcuna j.l., 2007 la polilla del tomate,” tuta absoluta”. phytoma espana 194: 16-23. valera d., álvarez a., molina f., 2006 aerodynamic analysis of several insect-proof screens used in greenhouses. spanish j. agric. res. 4: 273-279. viskelis p., jankauskiene j., bobinaite r., 2008 content of carotenoids and physical properties of tomatoes harvested at different ripening stages. in: foodbalt, 3rd baltic conference on food science and technology, jelgava, latvia: 166-170 wu k., 2007 monitoring and management strategy for helicoverpa armigera resistance to bt cotton in china. j. invert. pathol. 95: 220-223. wuzhong n., 2002 yield and quality of fruits of solanaceous crops as affected by potassium fertilization. better crop. int. 16: 6-8. zhu y.c., blanco c.a., portilla m., adamczyk j., luttrell r., huang f., 2015 evidence of multiple/cross resistance to bt and organophosphate insecticides in puerto rico population of the fall armyworm, spodoptera frugiperda. pest. biochem. physiol. 122: 15-21. article no nco mm er cia l u se on ly jear2012 abstract the gum tree thrips, thrips australis (bagnall) is recorded from shiraz, fars province, iran for the first time. variation in color and structure of species is discussed and illustrations are provided. introduction the genus thrips linnaeus is the second largest genus in the thysanoptera, and currently includes 286 species worldwide (mound, 2012). however the genus is absent from the neotropics, apart from introduced species (mound and marullo, 1996). most species in the genus are flower living, although a few are known to breed on leaves (mound, 1997; mound and kibby, 1998). some members of the genus are well known as pests in various parts of the world, such as t. angusticeps uzel, t. flavus schrank, t. hawaiiensis (morgan), t. meridionalis priesner, t. tabaci lindeman (moritz et al., 2004) as well as iran (minaei et al., 2007). however, for many species there is little information available on their biology, geographical distributions, host associations and structural variation. recent years have seen much study into the genus thrips. the species of thrips from the indian region were revised and 33 species were recognized in that area (bhatti, 1980). palmer (1992) has given identification keys to 91 species from oriental and pacific islands. nakahara (1994) has treated 62 species from the new world. a key is provided for 8 species from central america (mound and marullo, 1996). ten pest species of the genus have been treated and a key has been given by mound and kibby (1998). mound and masumoto (2005) provided an identification key to 41 species from australia, new zealand and new caledonia. an illustrated key is provided to 23 species of the genus thrips from peninsular malaysia (mound and azidah, 2009). thirty-four species are recorded from africa (mound, 2010) and subsequently an illustrated key is provided to distinguish the 33 species of genus thrips recorded from china (zhang et al., 2011). finally, an internet based interactive key has been prepared for 26 species of this genus, including potential invaders from california, north america (hoddle et al., 2012). in iran, 26 species of the genus are listed (table 1) (bhatti et al., 2009), although the names of two of them, t. iranicus and t. pistaciae, remain in doubt because they cannot be recognized from their original descriptions. the purpose of this paper is to report another thrips species from iran, with illustrations and observed variations within the iranian specimens. materials and methods the specimens of thrips australis discussed below were collected in shiraz, fars province, iran, by breaking up white flowers of eucalyptus camaldulensis onto a plastic tray. the specimens were removed with a fine brush into a collecting vial containing 95% ethyl alcohol. microscopic slides were mounted into canada balsam after dehydration through a series of ethanol using a form of the protocol given in world thysanoptera (http://anic.ento.csiro.au/thrips/field_lab/index. html). microphotographs were obtained using a dino-lite microscope, eyepiece camera. digital images were enhanced and plates prepared using adobe photoshop™. terminology generally follows mound et al., (1976) and mound and masumoto (2005). most specimens are deposited at the department of plant protection collection, shiraz university, shiraz, iran. results thrips australis (bagnall) isoneurothrips australis bagnall, 1915 (p. 592) thrips lacteicorpus girault, 1926 (p. 17) thrips mediolineus girault, 1926 (p. 18) anomalothrips amygdali morgan, 1929 (p. 5) isoneurothrips marisabelae ortiz, 1973 (p. 119) correspondence: kambiz minaei, department of plant protection, college of agriculture, shiraz university, shiraz, iran. e-mail: kminaei@shirazu.ac.ir key words: thrips australis, eucalyptus camaldulensis, fars province, new record. acknowledgements: during my visit to csiro ecosystem sciences, canberra, australia in 2009, dr. laurence mound, encouraged me to collect t. australis from eucalyptus trees in iran. he also kindly gave me a slide mount of the species at that time. dr. ahmad reza khosravi, depatment of biology, shiraz university kindly recognized the species of eucalyptus. received for publication: 16 july 2012. revision: not required. accepted for publication: 27 august 2012. ©copyright k. minaei, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e9 doi:10.4081/jear.2012.e9 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. first report of an endemic australian thrips, thrips australis (thysanoptera: thripidae) on eucalyptus in shiraz, iran k. minaei department of plant protection, college of agriculture, shiraz university, shiraz, iran [page 42] [journal of entomological and acarological research 2012; 44:e9] journal of entomological and acarological research 2012; volume 44:e9 no nco mm er cia l u se on ly specimens of this species were collected from the white flowers of eucalyptus camaldulensis at shiraz and this is the first report of t. australis in iran. the specimens were compared with one specimen of this species from new zealand, and also with the available published literature. although variation in color and structure was observed within the iranian specimens (tables 1 and 2), they were distinguishable from other thrips species by almost a complete row of forewing (figure 1a), six (instead of five) marginal setae on clavus (figure 1a), and a bullet shaped antennal segment vi (figure 1b). diagnosis macropterous body typically yellow usually with brown markings medially on tergites iii-viii, tergites ix-x brown; antennal segment i white, ii-iii brownish yellow (sometimes i-ii white, iii brownish yellow, remaining segments almost brown; forewing usually shaded; major setae except ocellars and postoculars dark. antennae 7-segmented (figure 1c), iiiiv with forked sensorial, vi large and bullet-shaped, vii short (figure 1b). the head is bigger in width than in length (figure 1d) with ocellar setae iii arising within ocellar triangle. pronotum with 2 pairs of short stout postero-angular setae (figure 1d); posterior margin with 6, 7 or 8 setae. mesonotum with lines of sculpture close to campaniform sensilla. metanotum reticulate medially (figure 1e), median setae arise behind anterior margin, campaniform sensilla present. forewing first vein with almost uninterrupted row of setae, clavus with 6 marginal and 1 discal setae (figure 1a). abdominal tergite ii with 4 lateral setae, tergites v-viii with paired ctenidia, marginal comb not developed medially on tergite viii (figure 1f); ix with 2 pairs of campaniform sensilla; pleurotergites commonly with more than 5 discal setae (figure 1g). sternite ii with 2 pairs of marginal setae, iii-vii with 3 pairs. sternites with a large number of discal setae (figure 1h); the number of setae increased toward sternites vii; 3 small setae (sometimes 2 or 4) on sternite ii but 14-32 developed setae on sternite vii, in irregular double rows (figure 1h). material examined. 7 females, iran, fars province, shiraz, from eucalyptus camaldulensis, 21.06.2012 (km853); 15 females, same locality, from eucalyptus camaldulensis, 24.06.2012 (km856); 17 females, shiraz, from eucalyptus camaldulensis, 24.06.2012 (km861). discussion and conclusions although the species is native to australia, it has been introduced around the world wherever eucalyptus trees are grown (mound, 2010). so it seems likely that the presence of this species in iran is not surprising. color and size both vary, and as a result the species has been described under five other names (see above). the variation in color of antennal segments and two character states reported here (table 2), as well as some other variations which have already been mentioned above (diagnosis section), support the variability of the species. in addition to australia (mound and masumoto, 2005), thrips australis has been recorded from many other parts of the world, including egypt (priesner, 1965), japan (miyazaki and kudo, 1988), the pacific regions (palmer, 1992), europe (zur strassen, 2003), united states (nakahara, 1994), central america (mound and marullo 1996), brazil (monteiro, 2002), peninsular malaysia (mound and azidah, 2009), africa (mound, 2010), britain (collins, 2010), china (zhang et al., 2011) and north america (hoddle et al., 2012). sakimura (1967) and kirk (1987) have questioned whether t. australis is native to australia on the basis that this species has been found in so many countries around the world. however, neither of these authors considered the many field observations that associate this thrips with white eucalyptus flowers, both in australia and in other countries (mound and masumoto, 2005). in kenya, t. australis is known from crops such as tomato, capsicum, french bean, sunflower and carrot (icipe, 2012). however, there are currently no records from any crops in iran. [journal of entomological and acarological research 2012; 44:e9] [page 43] article table 1. thrips species recorded in iran. no. thrips species 1 thrips albopilosus uzel 2 thrips angusticeps uzel 3 thrips atratus haliday 4 thrips coloratus schmutz 5 thrips dubius priesner 6 thrips euphorbiae knechtel 7 thrips flavus schrank 8 thrips fraudulentus (priesner) 9 thrips fuscipennis haliday 10 thrips hawaiiensis (morgan) 11 thrips iranicus yakhontov 12 thrips major uzel 13 thrips mareoticus (priesner) 14 thrips meridionalis (priesner) 15 thrips minutissimus linnaeus 16 thrips nigropilosus uzel 17 thrips pelikani schliephake 18 thrips physapus linnaeus 19 thrips pillichi priesner 20 thrips pistaciae yakhontov 21 thrips simplex (morison) 22 thrips tabaci lindeman 23 thrips trehernei priesner 24 thrips verbasci (priesner) 25 thrips vuilleti (bagnall) 26 thrips vulgatissimus haliday table 2. variation in color and structure of among 39 specimens of t. australis collected in shiraz. character characteristics number of specimens color of antennal i-ii white, iii brownish yellow 13 segments* i white, ii-iii brownish yellow 26 number of posteromarginal 6 20 setae on pronotum 7 11 8 8 number of discal setae 2 14 on sternites ii 3 18 4 7 *antennal segments iv-vii in all specimens brown. no nco mm er cia l u se on ly [page 44] [journal of entomological and acarological research 2012; 44:e9] article figure 1. t. australis (a) forewing; (b) antennal segments vi-vii; (c) left antenna; (d) head and pronotum; (e) metanotum; (f) abdominal tergites vii-ix; (g) abdominal pleurotergites iv-vi; (h) abdominal sternites v-vii. no nco mm er cia l u se on ly references bagnall r.s., 1915 brief descriptions of new thysanoptera. vi. ann. mag. nat. hist. 15: 588-597. bhatti j.s., 1980 species of the genus thrips from india. syst. entomol. 5: 109-166. bhatti j.s., alavi j., zur strassen r., telmadarraiy z., 2009 thysanoptera in iran 1938-2007. an overview. part 1. thrips. 7: 182. collins d.w., 2010 thysanoptera of great britain: a revised and updated checklist. zootaxa. 2412: 21-41. girault a.a., 1926 three new thysanoptera from australia. insecut. inscit. menstr. 14: 17-18. hoddle m.s., mound l.a., paris d., 2012 thrips of california 2012. http://keys.lucidcentral.org/keys/v3/thrips_of_california/thrips_of_ california.html accessed: 12.07.2012. icipe 2012 occurrence of thrips australis hood, 1915 in east africa. http://thrips.icipe.org/index.php?option=com_content&view=article&id=131&itemid=156 accessed: 30.06.2012. kirk, w.d.j., 1987 a key to the larvae of some common australian flower thrips (insecta: thysanoptera), with a host-plant survey. aust. j. zool. 35: 173-185. minaei k., azemayeshfard p., mound l.a., 2007 the thrips genus-group (thysanoptera: thripidae) in iran. j. entomol. soc. iran. 27: 29-36. miyazaki, m., kudo, i., 1988 bibliography and host plant catalogue of thysanoptera of japan. misc. publ. natl. inst. agro-environ. sci. 3: 1-246. morgan a.c., 1929 a new genus and five new species of thysanoptera foreign to the united states. proc. entomol. soc. wash. 31: 1-9. moritz g., mound l.a., morris d.c., goldarazena a., 2004 pest thrips of the world visual and molecular identification of pest thrips. cd-rom. brisbane: cbit. mound l.a., 1997 biological diversity. in: lewis t, (ed). thrips as crop pests. cab international, wallingford, uk: 197-215. mound l.a., 2010 species of the genus thrips (thysanoptera, thripidae) from the afro-tropical region. zootaxa. 2423: 1-24. mound l.a., 2012thysanoptera (thrips) of the world a checklist. http://www.ento.csiro.au/thysanoptera/worldthrips.html accessed: 13.07.2012. mound l.a., azidah a.a., 2009 species of the genus thrips (thysanoptera) from peninsular malaysia, with a checklist of recorded thripidae. zootaxa. 2023: 55-68. mound l.a., kibby g., 1998 thysanoptera: an identification guide. cab international institute of entomology and british museum (natural history), london, uk: 70. mound l.a., marullo r., 1996 the thrips of central and south america: an introduction. mem. entomol. int. 6:1-488. mound l.a., masumoto m., 2005 the genus thrips (thysanoptera, thripidae) in australia, new caledonia and new zealand. zootaxa. 1020: 1-64. mound l.a., morison g.d., pitkin b.r., palmer j.m., 1976 thysanoptera. handbooks for the identification of british insects, vol. 1. royal entomological society of london (res), london, uk: 1-79. nakahara s., 1994 the genus thrips linnaeus (thysanoptera, thripidae) of the new world. us depart. agr. tech. bull. 1822: 1183. ortiz m.p., 1973 una nueva species de isoneurothips bagnall (thysanoptera: thripidae) del perù. rev. peruana entomol. 16: 117-120. palmer j.m., 1992 thrips from pakistan to the pacific: a review. bull. brit. mus. nat. hist. (entomol.) 61: 1-76. priesner h., 1965 a monograph of the thysanoptera of the egyptian deserts. pub. inst. desert egypte 13: 1-549. sakimura k., 1967 a preliminary review of the genus isoneurothrips and the subgenus thrips (isothrips). pac. insects 9: 429-436. zhang h., xie y., li z., 2011 identification key to species of thrips genus from china (thysanoptera, thripidae), with seven new records. zootaxa. 2810: 37-46. zur strassen r., 2003 die terebranten thysanopteren europas und des mittelmeer-gebietes. die tierwelt deutschlands 74: 1-271. [article in german]. [journal of entomological and acarological research 2012; 44:e9] [page 45] article no nco mm er cia l u se on ly jear2012 abstract pseudosmittia fabioi boggero, zaupa & rossaro, 2014 was found to be conspecific with prosmittia verae krasheninnikov & makarchenko, 2008 after examination of original description and illustrations. accordingly, ps. fabioi is placed in junior synonymy with pr. verae, new synonymy. the transfer of ps. fabioi to the genus prosmittia allows to state that the female of ps. fabioi (boggero et al., 2014) is the first description of the female of the genus prosmittia. introduction recently eugenji makarchenko sent to the senior author a paper in cyrillic, with english diagnosis, describing a new species belonging to the genus prosmittia brundin, 1956: prosmittia verae krasheninnikov & makarchenko, 2008. from an examination of the drawings it was immediately apparent that the described species is identical with pseudosmittia fabioi, boggero, zaupa & rossaro, 2014. the apparent synonymy was confirmed after an accurate examination of the original description given in cyrillic and kindly translated by martin spies. the genus prosmittia was established by brundin (1956) for pseudosmittia jemtlandica brundin, 1947; cranston & oliver (1988) synonymized prosmittia with pseudosmittia goetghebuer, 1933, but sæther & ferrington (1993) reestablished the prosmittia genus because of a unique combination of characters, separating it from pseudosmittia, that is the lack of acrostichals, a moderate costal extension, r4+5 ending near to the wing apex, distal to m3+4. the genus includes 15 palaearctic species (ashe & o’connor, 2012), three of these, pr. jemtlandica brundin, 1947, pr. rectangularis tuiskunen, 1985, pr. valentinae, baranov, 2011, have been known from europe (sæther et al., 2000), one, pr. verae krashennikov & makarchenko, 2008 from caucasus, nine, pr. furudoseptima (sasa & arakawa, 1994), pr. hibaraundecima (sasa & suzuki, 1998), pr. itachituberculata (sasa & kawai, 1987), pr. itachinidiocura (sasa & kawai, 1987), pr. kamiquarta (sasa & hirabayashi, 1991), p. kibaprima (sasa & sumita, 2001) pr. taishodeea (sasa & tanaka, 2001), pr. togacurva (sasa & okazawa, 1992) and pr. yakitaira (sasa & suzuki, 2000) from japan, two, pr. tauiensis makarchenko & makarchenko, 2007 and pr. anyuiica makarchenko & makarchenko, 2009, are known from russian far east. females, pupae and larvae of all the known species were until now unknown. on the basis of the present synonymy it appears that the female of prosmittia is described in boggero et al. (2014). the present paper formally establishes the synonymy between pr. verae and ps. fabioi allowing to extend the distribution of the pr. verae to the mediterranean area. the terminology and abbreviations used in the description follow sæther (1980). prosmittia verae krasheninnikov & makarchenko, 2008 prosmittia verae krasheninnikov & makarchenko, 2008: 359 (original description). pseudosmittia fabioi boggero, zaupa & rossaro, 2014: 1 (original description). syn. nov. description a complete description of the species is in krasheninnikov & makarchenko (2008) and in boggero et al. (2014). comparisons of measurements on pr. verae and ps. fabioi are given in table 1 (head, body, wings) and table 2 (legs). here are summarized the characters supporting the inclusion of the species in prosmittia and the synonymy of the two species. correspondence: bruno rossaro, defens department of food, environmental and nutritional sciences, università degli studi di milano, via celoria 2, i 20133 milano, italy. e-mail: bruno.rossaro@unimi.it key words: synonymy; chironomidae; orthocladiinae; prosmittia; pseudosmittia. acknowledgments: a particular thank go to martin spies (staatliche naturwissenschaftliche sammlungen bayerns zoologische staatssammlung münchen, germany) and eugenji makarchenko (institute of biology and soil sciences, far east branch of russian academy of sciences, vladivostok), for the precious information given, essential to establish the synonymy. received for publication: 12 march 2015. revision received: 29 april 2015. accepted for publication: 10 june 2015. ©copyright b. rossaro et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:5151 doi:10.4081/jear.2015.5151 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. pseudosmittia fabioi boggero, zaupa & rossaro, 2014 (diptera: chironomidae: orthocladiinae) a new junior synonym of prosmittia verae krasheninnikov & makarchenko, 2008 b. rossaro,1 s. zaupa,2 a. boggero2 1department of food, environmental and nutritional sciences, university of milano; 2cnr-institute of ecosystem study, verbania pallanza (vb), italy [page 62] [journal of entomological and acarological research 2015; 47:5151] journal of entomological and acarological research 2015; volume 47:5151 no nco mm er cia l species (ashe & o’connor, 2012), three of these, no nco mm er cia l species (ashe & o’connor, 2012), three of these, brundin, 1947, no nco mm er cia l brundin, 1947, no nco mm er cia l cyrillic, with english diagnosis, describing a new species belonging to no nco mm er cia l cyrillic, with english diagnosis, describing a new species belonging to krasheninnikov no nco mm er cia l krasheninnikov & makarchenko, 2008. from an examination of the drawings it was no nco mm er cia l & makarchenko, 2008. from an examination of the drawings it was no nco mm er cia l baranov, 2011, have been known from europe (sæther no nco mm er cia l baranov, 2011, have been known from europe (sæther pr. verae no nco mm er cia l pr. verae krashennikov & makarchenko, 2008 from caucasus, nine, no nco mm er cia l krashennikov & makarchenko, 2008 from caucasus, nine, furudoseptima no nco mm er cia l furudoseptima suzuki, 1998), no nco mm er cia l suzuki, 1998), diocura no nco mm er cia l diocura no nco mm er cia l no nco mm er cia l correspondence: bruno rossaro, defens department of food, no nco mm er cia l correspondence: bruno rossaro, defens department of food, environmental and nutritional sciences, università degli studi di milano, no nco mm er cia l environmental and nutritional sciences, università degli studi di milano, key words: synonymy; chironomidae; orthocladiinae; no nco mm er cia l key words: synonymy; chironomidae; orthocladiinae; acknowledgments: a particular thank go to martin spies (staatlichen on -co mm er cia l acknowledgments: a particular thank go to martin spies (staatliche us e unique combination of characters, separating it from us e unique combination of characters, separating it from that is the lack of acrostichals, a moderate costal extension, r us e that is the lack of acrostichals, a moderate costal extension, r near to the wing apex, distal to m us e near to the wing apex, distal to m species (ashe & o’connor, 2012), three of these, us e species (ashe & o’connor, 2012), three of these, brundin, 1947, us e brundin, 1947, pr. rectangularisus e pr. rectangularis on ly description given in cyrillic and kindly translated by martin spies. on ly description given in cyrillic and kindly translated by martin spies. was established by brundin (1956) for on lywas established by brundin (1956) forbrundin, 1947; cranston & oliver (1988) synon lybrundin, 1947; cranston & oliver (1988) synwith on ly with pseudosmittiaon ly pseudosmittia & ferrington (1993) reestablished the on ly & ferrington (1993) reestablished the unique combination of characters, separating it from on ly unique combination of characters, separating it from wing costal vein extending beyond tip of r4+5 by less than 50 µm; all veins without setae. r4+5 distal with respect to m3+4, vein cu1 curved, sinusoidal, anal lobe reduced, alula and squama bare. hypopygium. tergite ix with 12-14 setae, with narrow, rounded-triangular and microtrichia-covered anal point 35-46 µm long. laterosternite ix with 2-3 setae. gonocoxite 165-176 µm long, dorsal part of inferior volsella bare, fingerlike, ventral part long, rounded, covered with short setae. gonostylus 57-66 µm long, covered with setae, subapically narrowed, distally on outer edge with angular projection about 7-8 µm long; macroseta 10-11 µm long. transverse sternapodeme 94-16 µm long, lateral projections reduced. virga small, forming one spine 17-24 µm in length. hypopygium ratio: ratio of length of gonocoxyte to length of gonostylus 2.67-2.96. distribution pr. verae was known only from the type locality on the agura river at the foot of little akhun (north caucasus), now its distribution is extended to the mediterranean area in sardinia (boggero et al., 2014). discussion and conclusions the inclusion of the genus prosmittia in pseudosmittia (cranston & oliver, 1988) had the effect that in the holarctic key of genera (cranston et al., 1989) prosmittia was not included, so boggero et al. (2014), ignoring sæther & ferrington (1993), sæther et al. (2000) and [journal of entomological and acarological research 2015; 47:5151] [page 63] article table 1. range of lengths (in mm or µm) and proportions of segments in prosmittia verae and pseudosmittia fabioi. pr. verae (n=3) ps. fabioi (n=10) body color brown brownish-black body length 2.3-2.4 mm 2.3-2.6 mm wing length 1.7-2.0 mm 1.5-1.7 mm body length/wing length 1.5 1.53 vertical setae 3-5 3-6 postorbital setae 2-5 4-5 clypeals 6 6 length of terminal flagellomere 346-468 µm 364-368 µm ar 0.76-0.94 0.80-0.86 palpomere lengths 22-24; 33-47; 83-110; 70-94; 94-138 22-24; 44-45; 91-93; 78-80; 92-94 head wide 380-413 µm 420-428 µm dorsocentrals 7-9 5-7 acrostichals 0 0 prealars 3 3 scutellars 4 4-8 vr 1.43 1.38 table 2. range of lengths (in µm) and proportions of segments in prosmittia verae and pseudosmittia fabioi (male, n=3). p. verae fe ti ta1 ta2 ta3 p1 517-605 605-748 292-363 204-264 116-127 p2 545-671 578-677 259-325 171-209 110-127 p3 589-660 616-776 314-418 176-242 160-198 ta4 ta5 lr bv sv p1 55-66 50-61 0.45-0.49 3.26-3.39 3.73-3.98 p2 50-55 49-55 0.45-0.48 3.64-3.75 4.15-4.35 p3 61-72 55-61 0.51-0.54 3.24-3.36 3.43-3.84 p. fabioi fe ti ta1 ta2 ta3 p1 530-564 636-664 294-318 212-226 122-128 p2 572-598 577-603 265-277 175-179 109-121 p3 586-604 649-683 335-361 204-210 167-179 ta4 ta5 lr bv sv p1 59-63 60-62 0.46-0.48 3.22-3.23 3.97-3.86 p2 43-49 56-58 0.46-0.46 3.69-3.63 4.34-4.34 p3 54-64 52-54 0.52-0.53 3.29-3.25 3.69-3.57 fe, femur; ti, tibia; ta1-5, tarsomeres 1-5; lr, leg ratio, ratio of metatarsus to tibia; bv, beinverhältnisse, combined length of femur, tibia, and basitarsus (ta1) divided by combined length of tarsomeres 2-5; sv, schenkelscheine-verhältnis, ratio of femur plus tibia to metatarsus (ta1). no nco mm er cia l no nco mm er cia l no nco mm er cia l m no nco mm er cia l m 0.76-0.94 no nco mm er cia l 0.76-0.94 no nco mm er cia l no nco mm er cia l no nco mm er cia l 22-24; 33-47; 83-110; 70-94; 94-138 no nco mm er cia l 22-24; 33-47; 83-110; 70-94; 94-138 380-413 no nco mm er cia l 380-413 µ no nco mm er cia l µm no nco mm er cia l m no nco mm er cia l no nco mm er cia l no nco mm er cia l 7-9 no nco mm er cia l 7-9 no nco mm er cia l 0 0 no nco mm er cia l 0 0 no nco mm er cia l no nco mm er cia l 3 3 no nco mm er cia l 3 3 4 no nco mm er cia l 4 no nco mm er cia l no nco mm er cia l no nco mm er cia l table 2. range of lengths (in no nco mm er cia l table 2. range of lengths (in µ no nco mm er cia l µ no nco mm er cia l µ no nco mm er cia l µm) and proportions of segments in no nco mm er cia l m) and proportions of segments in no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l us e us e us e us e us e us e 6 6us e 6 6us e m us e m on ly on ly brownish-black on ly brownish-black on ly on ly on ly 2.3-2.6 mm on ly2.3-2.6 mm on ly on ly on ly on ly on ly [page 64] [journal of entomological and acarological research 2015; 47:5151] overlooking the presence of a wing costal extension, were erroneously induced to include ps. fabioi in the genus pseudosmittia. the discovering of pr. verae description (krasheninnikov & makarchenko, 2008) and a careful subsequent reexamination of ps. fabioi allowed to conclude that ps. fabioi, with a moderate costal extension, r4+5 ending distal to m3+4 and absence of acrostichals, can be included beyond any doubt in the genus prosmittia; the shape of hypopygium emphasized the strict similarity with the one of pr. verae. the small differences observed in morphometric characters can be reasonably interpreted as differences between populations of the same species in different areas. the lack of acrostichals resolves also the question about the presence or absence of acrostichals in pseudosmittia (boggero et al., 2014): the present results allow confirming that acrostichals are present in all pseudosmittia (ferrington & sæther, 2000 or 2011) and absent in all prosmittia. another consequence of the transfer of ps. fabioi from pseudosmittia to prosmittia is that the description of female of ps. fabioi is the first description of female belonging to the genus prosmittia; in prosmittia the female antenna is characterized by six antennal flagellomeres, adding an additional character in a generic separation of prosmittia from pseudosmittia, because all the species of pseudosmittia of which females are described possess only five antennal flagellomeres. references ashe p., o’connor j.p., 2012 a world catalogue of chironomidae (diptera). part 2. orthocladiinae. irish biogeographical society and national museum of ireland, dublin. xvi+968 pp. boggero a., zaupa s., rossaro b., 2014 pseudosmittia fabioi sp. n., a new species from sardinia (diptera, chironomidae, orthocladiinae). j. entomol. acarol. res. 46: 1-5. brundin l., 1947 zur kenntnis der schwedischen chironomiden arkiv zool. bd. 39a. hf. 3: 1-95. brundin l., 1956 zur systematik der orthocladiinae (diptera, chironomidae) rep. inst. freshwater res. drottningholm. 37: 5-185. cranston p.s., oliver d.r., 1988 additions and corrections to the nearctic orthocladiinae (diptera, chironomidae). can entomol. 120: 425-462. cranston p.s., oliver d.r., sæther o.a., 1989 the adult males of orthocladiinae (diptera: chironomidae) of the holarctic region. keys and diagnoses. in wiederholm t. (ed.), chironomidae of the holarctic region. part 3. adult males. entomol. scand. suppl. 34: 165-352. ferrington l.c., sæther o.a., 2011 a revision of the genera pseudosmittia edwards, 1932, allocladius kieffer, 1913, and hydrosmittia gen. n. (diptera: chironomidae, orthocladiinae). zootaxa 2849: 1-314. krasheninnikov a.b., makarchenko e. a., 2008 prosmittia verae sp. n., a new chironomid species (diptera:chironomidae: orthocladiinae) from the environs of sochi town (the northern caucasus). caucasian entomol. bull. 4: 359-361. sæther o.a., 1980 glossary of chironomid morphology terminology (diptera: chironomidae). entomol. scand. suppl. 14: 1-51. sæther o.a., ashe p., murray d.a., 2000 family chironomidae. papp l. and darvas b. (eds), contributions to a manual of palaearctic diptera (with special reference to the flies of economic importance). science herald, budapest. 4. a.6.: 113-334. sæther o.a., ferrington l., 1993 redescription of prosmittia jemtlandica (brundin, 1947), with a review of the genus. j. kans. ent. soc. 66: 257-262. article no nco mm er cia l sæther o.a., ferrington l., 1993 redescription of no nco mm er cia l sæther o.a., ferrington l., 1993 redescription of jemtlandica no nco mm er cia l jemtlandicaent. soc. 66: 257-262. no nco mm er cia l ent. soc. 66: 257-262.u se sæther o.a., ashe p., murray d.a., 2000 family chironomidae. us e sæther o.a., ashe p., murray d.a., 2000 family chironomidae. papp l. and darvas b. (eds), contributions to a manual of us e papp l. and darvas b. (eds), contributions to a manual ofpalaearctic diptera (with special reference to the flies of economic us e palaearctic diptera (with special reference to the flies of economic us e importance). science herald, budapest. 4. a.6.: 113-334.us e importance). science herald, budapest. 4. a.6.: 113-334. sæther o.a., ferrington l., 1993 redescription of us e sæther o.a., ferrington l., 1993 redescription of on ly sp. n., a new chironomid species (diptera:chironomidae: on ly sp. n., a new chironomid species (diptera:chironomidae: orthocladiinae) from the environs of sochi town (the northern on ly orthocladiinae) from the environs of sochi town (the northern caucasus). caucasian entomol. bull. 4: 359-361. on lycaucasus). caucasian entomol. bull. 4: 359-361.sæther o.a., 1980 glossary of chironomid morphology terminology on lysæther o.a., 1980 glossary of chironomid morphology terminology (diptera: chironomidae). entomol. scand. suppl. 14: 1-51.on ly (diptera: chironomidae). entomol. scand. suppl. 14: 1-51. sæther o.a., ashe p., murray d.a., 2000 family chironomidae.on ly sæther o.a., ashe p., murray d.a., 2000 family chironomidae. papp l. and darvas b. (eds), contributions to a manual of on ly papp l. and darvas b. (eds), contributions to a manual of 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract the presence of entomopathogenic beauveria bassiana on pleasing fungus beetle episcapha quadrimacula has been reported on fruiting bodies of ganoderma boninense for the first time in malaysia. short paper erotylidae, also known as pleasing fungus or spore-feeding beetles, is a family of beetles comprising mainly of detritusand fungus-associated beetle species. furthermore, erotyliids were proposed to have little to no economic importance in most parts of the world (mishra & meyerrochow, 2006). in a few studies conducted in oil palm plantations with palms attacked by ganoderma boninense or basal stem rot (bsr) disease, episcapha quadrimacula beetles from the family of erotylidae were reported to be one of the most common insects found to propagate in the fruiting bodies of g. boninense. more than 80% of the e. quadrimacula larvae were reported with g. boninense basidiospores (chung, 2011; seman, 2013). dispersal of the basidiospores was proposed not only by mean of winds and could also be assisted by insects, namely e. quadrimacula (chung, 2011; seman, 2013). however, the information related to the aids of e. quadrimacula in moving or transferring basidiospores from palm to palm and spread of ganoderma disease is limited. in 2014, ganoderma fruiting bodies were collected in paloh oil palm estate, paloh, johor (2°13’n, 103°12’e). approximately 2-3% of the fruiting bodies collected (n=60 fruiting bodies collected) had fungalinfested e. quadrimacula (figure 1a-b). all the infested e. quadrimacula beetles collected were sent to pathology laboratory for isolation. beauveria bassiana was isolated from these infested beetles (figure 1c-d). to the best of our knowledge, this is the first record of b. bassiana reported from e. quadrimacula beetles in malaysian oil palm plantation (farr & rossman, 2015). dna of the pure culture was extracted (fastdna spin kit, mp biomedicals, usa) from 2-week-old b. bassiana isolate grown on mea. the internal transcribed spacers (its) of the rdna and b-tubulin gene were amplified separately and sequenced (macrogen, korea). similarity search and analyses were conducted using the blast search algorithm in ncbi genbank. the sequences with accession number of kt183365 (b-tubulin) and kt183365 (its) showed 100% similarity with b. bassiana (jn713134) and 99% similarity with b. bassiana (ay334537) using b-tubulin gene and its regions, respectively. sequences from this study were combined with other existing sequences from genbank were analysed using neighbour joining approach and b. bassiana isolate from current study clustered with other existing b. bassiana isolates (figure 2a-b). furthermore, based on both morphological characteristics and phylogenetic analysis, this current beauveria isolate is identified as b. bassiana. pathogenicity study was conducted using the current b. bassiana isolate in triplicate (5 beetles per replicate). beetles were sprayed with approximately 100 ml of conidial suspension (5×106 conidia/ml). a separate set of beetles were sprayed with sterile water and acted as control. all the beetles were placed in a moist chamber and kept at 24±2°c for 2 weeks. treated dead beetles were transferred to sterile petri dishes with moistened filter papers at 24°c and observed for signs of conidial formation. control beetles showed no external mycelia (figure 1e-f). beauveria bassiana was re-isolated only from the infested beetles to satisfy koch’s postulates (figure 1e-f). in conclusion, e. quadrimacula beetles are susceptible to the infestation by b. bassiana, demonstrating mortality and with external mycelia after exposed to conidia from b. bassiana. in china, b. bassiana was isolated from a wide-range of insects from 16 different families and two separate orders coleoptera and lepidoptera (teng, 1996; farr & rossman, 2015). in malaysia, b. bassiana was found to be pathogenic toward metisa plana (oil palm bagworms) and proposed to use as biocontrol agent for m. plana (ramla ali et al., 1993). thus far, this is the first observation of b. bassiana recorded to be pathogenic toward e. quadrimacula or erotyliids. correspondence: yit kheng goh, advanced agriecological research sdn bhd, no. 11 jalan teknologi 3/6, taman sains selangor 1, kota damansara, 47810 petaling jaya, selangor darul ehsan, malaysia. tel: +603.61517924 fax: +603.61517081. e-mail: gohykheng@aarsb.com.my key words: basidiomata; b-tubulin; ganoderma boninense; its; oil palm. acknowledgements: we would like to thank boustead plantations holdings and kuala lumpur kepong berhad for their permission and financial support to the project. received for publication: 20 august 2015. revision received: 4 april 2016. accepted for publication: 7 april 2016. ©copyright y.k. goh et al., 2016 licensee pagepress, italy journal of entomological and acarological research 2016; 48:5492 doi:10.4081/jear.2016.5492 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. first record of entomopathogenic beauveria bassiana (ascomycota: hypocreales) on pleasing fungus beetle episcapha quadrimacula (coleoptera: erotylidae) in malaysia y.k. goh,1 t.m. teo,1 n.f. marzuki,1 s.s. tan,1 r. subramanian,2 i. hasim,1 y.k. goh,1 k.j. goh1 1advanced agriecological research sdn bhd, petaling jaya, selangor darul ehsan, malaysia; 2advanced agriecological research sdn bhd, paloh, johor, malaysia [page 314] [journal of entomological and acarological research 2016; 48:5492] journal of entomological and acarological research 2016; volume 48:5492 no nco mm er cia l u se on ly [journal of entomological and acarological research 2016; 48:5492] [page 315] short paper figure 1. beauveria bassiana and episcapha quadrimacula: a) e. quadrimacula beetle infested with b. bassiana on oil palm trunk infected with ganoderma boninense; b) e. quadrimacula with b. bassiana conidia and mycelia (ventral view of the beetle); c) morphology of b. bassiana colony on mea (front); d) reverse view of b. bassiana colony morphology on mea; e and f) control and treated e. quadrimacula (e: dorsal view, and f: ventral view). scale bars: a, e&f (1 cm) and b (1 mm). no nco mm er cia l u se on ly [page 316] [journal of entomological and acarological research 2016; 48:5492] references chung g.f., 2011 management of ganoderma diseases in oil palm plantations. the planter 87: 325-339. farr d.f., rossman a.y., 2015 fungal databases. syst. mycol. microbiol. lab., ars, usda. available from: http://nt.arsgrin.gov/fungaldatabases/ accessed: june 09, 2015. mishra m., meyer-rochow v.b., 2006 fine structure of the compound eye of the fungus beetle neotriplax lewisi (coleoptera, cucujiformia, erotylidae). invert. biol. 125: 265-278. ramlah ali a.s., basri m.w., ramle m., 1993 pathogenicity test on beauveria bassiana (balsamo) against oil palm bagworm (metisa plana wlk). elaeis 5: 92-101. seman i.a., 2013 estado actual de la investigación y desarrollo (i+d) sobre ganoderma en malasia. palmas (columbia) 34 (special volume 1): 100-118. teng s.c., 1996 fungi of china. mycotaxon, ltd., ithaca, ny: 586 pp. short paper figure 2. phylogenetic analyses of beauveria bassiana: neighbour joining tree illustrating the position of b. bassiana isolate from current study (in bold) compared with other beauveria species and other entomopathogenic fungal species using b-tubulin (a) and its (b) primer sets, respectively. only bootstrap values of 50% or greater from 1000 bootstrap replications are indicated on the respective branches. branch lengths illustrated are with the scale bar of 0.02 and 0.005 substitutions per nucleotide position, respectively. no nco mm er cia l u se on ly jear2012 journal of entomological and acarological research 2012; volume 44:e4 abstract the sweetpotato ipomoea batatas l. (convolvulaceae) has been one of the most important foods for pacific islanders for centuries. however, the yield levels have been declining in the recent past due to the presence of sweetpotato weevils cylas formicarius (fabricius) (coleoptera, brentidae), euscepes postfasciatus (fairmaire) and daealus tuberosus (zimmer man) (coleoptera, curculionidae). therefore, urgent management or eradication methods are sought in the mariana islands (guam, rota, saipan, and tinian). however, the management or eradication of these weevil pests requires accurate assessments of the target pest density. currently, no advice is provided to growers on the best method for sampling sweetpotato for weevil pests, although pheromone-based traps or chemicals are being used. this study defines the results of field counts designed to adjust relative sampling techniques for three sweetpotato weevil pests by inspecting plants visually and at random in the field with an absolute measure of population density. significant relationships were detected between the relative four sampling sites between the three weevil pests. in the dry and wet season, 90% and 35.5%, respectively, of population density of c. formicarius was noticed in rota. this density of the population levels of this species is significantly lower in saipan, guam and tinian. no incidence of e. postfasciatus and d. tuberosus was observed on guam. however, e. postfasciatus is identified as the second most destructive pest in rota, tinian and saipan in both the dry and wet seasons. likewise, d. tuberosus is the third major pest as the recorded population density ranged from 12.5% to 2.5%. also, it is evident from the sampling study that the population densities of all three weevils are significantly higher in the dry season than the wet season. introduction sweetpotato, ipomoea batatas l. (convolvulaceae) has been an important crop and has been used as food for several centuries (o’brien, 1972). it is believed to have originated in tropical america and spread extensively to other climatically suitable areas in the tropics, subtropics and even in warmer areas of the temperate zone (yen, 1982). this crop is also considered to be the sixth most important crop in the world, after wheat, rice, maize, white potato and barley (vietmeyer, 1986). nearly 92% of the sweetpotato crop is produced in asia and the pacific islands (chalfont et al., 1990). sweetpotatoes also contain significant levels of protein in addition to carbohydrates (loebenstein and thottappilly, 2009). however, recent years have seen a gradual decrease in the areas under sweetpotato cultivation. sweetpotatoes are in the same family as morning glories (ipomoea spp.) and its leaves resemble the leaves of the sweetpotato vines (austin et al., 1991). although the amount of sweetpotato produced per capita declined in usa up till 2006, the total area of cultivation and amount per capita produced has been increasing (economic research service, 2011). the sweetpotato has been a staple food for pacific islanders for centuries and is the most widely produced crop (nandawani and tudela, 2010). sweetpotato is considered an important food crop in the mariana islands. this crop is grown continuously throughout the year. however, its total harvested area and productivity is declining due to high infestation of the sweetpotato weevils, cylas formicarius (fabricius) (coleoptera, brentidae), euscepes postfasciatus (fairmaire) (west indian sweetpotato weevil) and daealus tuberosus (zimmerman) (coleoptera: curculionidae) (zimmerman, 1948) (figure 1). although c. formicarius is a major pest of cultivated sweetpotatoes, it is also a pest of stored sweetpotatoes. however, e. postfasciatus and d. tuberosus are also well-known as post-harvest pests of sweetpotatoes. many farmers and homeowners spray toxic pesticides to control these weevils. since the grubs bore inside the tubers and the vines, and the adults are nocturnally active, the chemicals have been shown to reduce the weevil population with variable degrees of success (jansson et al., 1987; chalfont correspondence: gadi v.p. reddy, western pacific tropical research center, university of guam, mangilao, guam 96923, usa. e-mail: reddy@uguam.uog.edu key words: population estimation, quadrat, cylas formicarius, euscepes po stfasciatus, daealus tuberosus. acknowledgements: this project was supported by fy 2011 pacific islands area conservation innovation grants (pia-cig) program, grant agreement no. 69-9251-11-902, the natural resources conservation service (nrcs)usda;western integrated pest management center (wipmc) award # 200751120-03885 / university of california, davis sub award # 07 -001492guam3, and usda hatch funds (project# gua0561). the usda is an equal opportunity provider and employer. a rule of reason will be applied as to the need for the statement in specific situations. we also thank r. gumataotao for his help during the field survey work. received for publication: 3 january 2012. revision received: 16 march 2012. accepted for publication: 2 april 2012. ©copyright g.v.p. reddy et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e4 doi:10.4081/jear.2012.e4 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. estimation of the population density of the sweetpotato weevils on the mariana islands g.v.p. reddy,1 j. mcconnel,1 a.e. badilles2 1western pacific tropical research center, university of guam, mangilao, guam; 2northern marianas college, cooperative research extension/education service (crees), rota, mp, usa [page 20] [journal of entomological and acarological research 2012; 44:e4] no nco mm er cia l u se on ly et al., 1990). although pheromone-based traps would be useful in estimating the population levels, monitoring and controlling these weevils, the pheromone compounds have only been identified and used for c. formicarius (jackson and bohac, 2006). however, trap catches are known to be affected by weather, topographical conditions and/or flora. cultural controls, such as the use of resistant cultivars of i. batatas, noninfested planting material and crop rotation, along with various management systems have been shown to reduce pest numbers (jansson et al., 1987; chalfont et al., 1990). approximately 30 alternative wild hosts have been recorded as sweetpotato weevil hosts, particularly for c. formicarius and multiple species of ipomoea (hill, 1983). reports of wild hosts of e. postfasciatus are very limited, probably due to its restricted distribution. in the mariana islands, littlebell and aiea morning glory, ipomoea triloba l. (convolvulaceae) are widespread and serve as alternative hosts for c. formicarius. this vine is reported to grow around sweetpotato fields and has been recorded as an alternate host of e. postfasciatus (pemberton, 1943). interestingly, both species of c. formicarius and e. postfasciatus are reported to feed on the leaves and roots of carrots daucus carota l. (apiaceae) and radish raphanus sativus l. (brassi caceae) in hawaii (muruvanda et al., 1986). the management and eradication of these weevils are vital for sweetpotato production in the mariana islands and also other parts of the world. estimating pest densities is one of the best basic themes for management and eradication programs the accomplishment of which depends significantly on how precisely the densities are estimated (ito and yamamura, 2008). the general aim of the study to acquire reliable estimates in of weevil density was determined by densities estimated by the quadrat method in the mariana islands. materials and methods study sites to obtain reliable estimates of the weevil’s density, three to four sweetpotato fields were selected on the islands of guam, rota, saipan and tinian (table 1). the sampling plans were developed according to the methodology described in southwood (1978) and pedigo and buntin (1993). two sets of samples, one in the dry and one in the wet season, were taken once the sweetpotato plants in each field were 8090 days old. the field sites of the 14 sampling sites, determined with a 12-channel global positioning system (garmin corp., taiwan), are listed in table 1. within each field site, three to four 0.4 acre plots were randomly chosen for estimation of the sweetpotato weevils (table 1). within each plot, one quadrat sample with the physical unit size of 1x1 m was taken. a wooden frame was placed horizontally above each quadrat, and all c. formicarius, e. postfasciatus and d. tuberosus adults inhabiting the vegetation underneath the frame or falling to the ground were identified and counted. because of the uncertainties regarding the behavior of the beetles, the inspection of the canopy and the ground lasted 10 min. data analyses to analyze field population densities per quadrat, a nested anova model was applied to test for significant differences between fields within islands and between islands. this was done by using the glimmix procedure sas version 9.13 (sas institute inc., 2009). tukey hsd test was used to make multiple comparisons for significant differences between island population levels for the wet and the dry seasons. to compare the population levels on the islands, the percentage of c. formicarius, e. postfasciatus and d. tuberosus adults inhabiting each island in the dry and wet season was calculated. this was done by disregarding the weevils outside the sweetpotato fields and taking into account the size of sweetpotato fields on each island. to obtain island populations, the average density per quadrat was multiplied by the sweetpotato area on each island and translated into a percentage of occupation, also referred to as percentage of incidence, by means of dividing the island population by the combined number of weevils living on all islands. results the results from the estimation of the population density indicated the population variation among four islands. during the dry season, 90% of population density of c. formicarius was noticed in rota, which was followed by saipan (75.5%), tinian (58.5%) and guam (54.5%) (figure 2). a significant difference was observed (p<0.05) between population levels from guam to saipan, tinian and guam. however, there was no significant difference between population levels in guam and tinian. in the wet season, the population levels were recorded as 35.5% in rota which was significantly higher than guam (22.5%), article [journal of entomological and acarological research 2012; 44:e4] [page 21] table 1. summary of study sites with geographical locations. island locality geographical locations no. of quadrat of fields samples per field (n) guam latte heights 13.26°n, 144.48°e, 79.9 m, a.s.l. 3 dededo 13.30°n, 144.51°e, 96.9 m, a.s.l. 4 mangilao 13.43°n, 144.80°e, 54.3 m, a.s.l. 3 aes, yigo 13.31°n, 144.52°e, 138.0 m, a.s.l. 4 rota sinapalo 14.17°n, 145.24°e, 179.8 m, a.s.l. 4 gagani farm 14.12°n, 145.17°e, 27.4 m, a.s.l. 3 mangloña farm14.13°n, 145.17°e, 175.0 m, a.s.l. 4 sinapalo-ii 14.17°n, 145.24°e, 179.8 m, a.s.l. 4 tinian tago wells 14.97°n, 145.62°e, 9.4 m, a.s.l. 3 marpo heights 14.98°n, 145.65°e, 17.7 m, a.s.l. 3 virginia lizama 14.97°n, 145.63°e, 21.9 m, a.s.l. 4 saipan garapan area 15.20°n, 145.72°e, 14.3 m, a.s.l. 3 tanapag zone 15.24°n, 145.76°e, 16.2 m, a.s.l. 3 capitol hill 15.21°n, 145.75°e, 233.8 m, a.s.l. 4 a.s.l., above sea level. figure 1. sweetpotato weevils recorded in the marianas islands. no nco mm er cia l u se on ly saipan (26.0%) and tinian (13.5%). no incidence of e. postfasciatus was observed on guam (figure 3). in the dry season, 45.0% of e. postfasciatus was recorded in rota which was significantly (p<0.05) higher than in saipan (26.5%) and tinian (2.5%). in the wet season, 22.0% of the population was observed in rota which is significantly higher than in saipan (12.0%) and tinian (5.0%). no prevalence of d. tuberosus was noted in guam (figure 4). in the dry season, a significantly higher (p<0.01) population (25.0%) of d. tuberosus was observed in rota than in saipan (12.5%) and tinian (7.5%). similar population trends were observed in the wet season. significantly higher population was observed in rota (12.5%), which was followed by saipan (5.5%) and tinian (2.5%). overall, there was a significant difference (p< 0.001) between population levels in the dry season and the wet season among the four islands. discussion according to chalfont et al. (1990), no sampling methods have been developed. also, a reliable sampling method for c. formicarius is not available. there are several methods for sampling insect populations in agricultural crops, including visual assessment, beat cloth, sweep net and d-vac sampling that have been used around the world (kogan and pitre, 1980). in the present study, visual assessment was used for sampling because of the uncertainties regarding the nature of the pest species. weevil populations are thickly clumped in sweetpotato fields (jansson et al., 1990). also, between 82% and 91% of the total population is below the surface of the soil, and between 78% and 89% of the total population is found in the area extending from 10 cm above the ground to 15 cm below the ground (jansson et al., 1990). therefore, reliable estimates of weevil population may be obtained by sampling the plant (chalfont et al., 1990) as carried out in the present study. nevertheless, all these techniques can be viewed as being relative sampling techniques. this will help to plan their use in decision making against thresholds based on absolute population densities in order to calibrate the relative sampling technique to the absolute population in the field (duffield et al., 2005). likewise, hanson (1967) suggested that the population density of animals from total counts on sample plots requires the variance of the counts to be kept fairly low. as the plot size is increased, it reduces the variance due to clumping, but at the same time, the increase in the size of individual plots often leads to a reduction in the number of plots. based on this theory, the number of counts taken from the present study is appropriate to count the population from the field. although nearly 30 alternative wild hosts have been recorded as sweetpotato weevil hosts (hill, 1983), the weevil population prefers and attacks primarily i. batatas. moreover, the islands do not have all the alternative hosts. therefore, insect densities outside sweetpotato fields are not required for calculating a weevil incidence for each island. the present results indicate a higher incidence of c. formicarius in all the four islands, particularly in rota where it reached 90% population level in the dry season. this indicates the severity of the pest and is a warning to growers. in fact, if the necessary control methods are not adopted, the whole crop could be lost. there have been previous reports of the damage caused by this weevil. according to sutherland (1986) and chiranjeevi et al. (2003), c. formicaries can cause considerable damage, with reports of losses ranging from 5% to 100%. this weevil infestation ranges from 20% to 50% on many growing farms and can even reach 100% in some seasons and varieties (ames et al., 1996). in louisiana, losses from c. formicarius range from 5 to 97% in areas where the weevil can be found (cockerham et al., 1954). according to messenger (1954), e. postfasciatus is confined to the tropical areas of the western hemisphere, mainly around the article [page 22] [journal of entomological and acarological research 2012; 44:e4] figure 2. mean percentage of incidence (±sem, standard error mean) of cylas formicarius on sweetpotato fields in the mariana islands. bars with different letters are significantly different between population levels (nested anova, tukey hsd, p<0.05). figure 3. mean percentage of incidence (±sem, standard error mean) of euscepes postfasciatus on sweetpotato fields in the mariana islands. bars with different letters are significantly different between population levels (nested anova using poisson’s model, tukey hsd, p<0.05). figure 4. mean percentage of incidence (± sem, standard error mean) of daealus tuberosus on sweetpotato fields in the mariana islands. bars with different letters are significantly different between population levels (nested anova using poisson’s model, tukey hsd, p<0.05). no nco mm er cia l u se on ly article caribbean. it is also found in hawaii, fiji, tonga and on okinawa island in japan. the results from the present study indicated that e. postfasciatus is the second most destructive pest of sweetpotatoes in the mariana islands. this weevil was recorded for the first time in rota during 1946 (zimmerman, 1948). it is believed that this weevil was accidentally introduced into okinawa from hawaii or saipan (kohama, 1990). in hawaii, a conservative estimate by sherman and tamashiro (1954) of the loss due to this pest was reported to be from 10% to 20% of the crop. this weevil is distributed throughout the okinawa islands in japan, where it heavily infests sweetpotatoes (yasiuda and kohama, 1990). although d. tuberosus was first recorded on sweetpotatoes in 1946 on guam (zimmerman, 1948), this species was not found or did not cause damage to sweetpotatos in guam in the present study. however, the present sampling studies have shown d. tuberosus to be the third most destructive pest in rota, saipan and tinian. this species was also first recorded in 1946 on rota (zimmerman, 1948) and, since there was no quarantine regulation within these islands, spread to saipan and tinian. in conclusion, the present sampling study revealed that c. formicarius is the most destructive pest on sweetpotato in all the islands. this was followed by e. postfasciatus and d. tuberosus whichcaused damage to sweetpotatoes in the commonwealth of northern mariana islands (cnmi). therefore, the management or eradication of these weevil pests is urgently required to save sweetpotatoes in the mariana islands. references austin d.f., jansson r.k., wolfe g.w., 1991 – convolvulaceae and cylas: a proposed hypothesis on the origins of this plant/insect relationship. trop. agr. 68: 162-170. ames t., smit n.e.j.m., braun a.r., o’sullivan j.n., skoglund l.g., 1996 sweetpotato: major pests diseases, and nutritional disorders. international potato center (cip), lima, perú: 152 pp. chalfont r.b., jansson r.k., seal d.r., schalk j.m., 1990 ecology and management of sweet potato insects. annu. rev. entomol. 35: 157-180. chiranjeevi c., reddy d.d.r., gour t.b., reddy y.n., sultana a., 2003 comparative biology of sweet potato weevil cylas formicarius fabricius on vines and tubers of sweet potato. j. res. angrau 31: 17-21. cockerham k.l., deen o.t., christian m.b., newsom l.d., 1954 the biology of the sweet potato weevil. la aes. bull. 483: 30. duffield s.j., winder l., chapple, d.g. 2005 calibration of sampling techniques and determination of sample size for the estimation of egg and larval populations of helicoverpa spp.(lepidoptera: noctuidae) on irrigated soybean. aust. j. entomol. 44: 293-298. economic research service (ers), 2011 u.s. department of agriculture (usda), food availability (per capita) data system. available from: http://www.ers.usda.gov/data/foodconsumption hanson h.r., 1967 estimating the density of an animal population. j. res. lepidoptera 6: 203-247. hill d., 1983 agricultural insect pests of the tropics & their control. university of cambridge university press, cambridge: 516 pp. ito y., yamamura k., 2008 estimation of insects for sterile insect release method and models for the determination of released insects. in: sterile insect release method: techniques for the eradication of invasive pest). kaiyusha, tokyo: 19-40. jackson d.m., bohac j.r., 2006 evaluation of pheromone traps for monitoring sweetpotato weevils. j. agric. urb. entomol. 23: 141158. jansson r.k., bryan h.h., sorensen, k.a., 1987 within-vine distribution and damage of sweetpotato weevil, cylas formicarius elegantulus (coleoptera: curculionidae), on four cultivars of sweetpotato in southern florida. fla entomol. 70: 523-526. jansson r.k., mcsorley r., 1990 -sampling plans for the sweetpotato weevil (coleoptera: curculionidae) on sweet potato in southern florida. j. econ. entomol. 83: 1901-1906. kogan m., pitre h.n., 1980 general sampling methods for aboveground populations of soybean arthropods. in: kogan m., herzog d.c., (eds.) sampling methods in soybean entomology. springerverlag, new york, usa: 30-60. kohama t., 1990 invasion and colonization of the sweetpotato weevils in okinawa and current problems for their control. shokubutu boueki (plant protection) 44: 115-117. loebenstein g., thottappilly g. 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[journal of entomological and acarological research 2012; 44:e4] [page 23] no nco mm er cia l u se on ly jear2012 abstract the tomato russet mite, aculops lycopersici (tryon, 1917) (acari: eriophyidae) is reported for the first time on pepino (solanum muricatum aiton) in ordu and samsun provinces in turkey. short paper aculops lycopersici (tryon, 1917) (acari: eriophyidae) is known as tomato russet mite or tomato rust mite. it was described as phyllocoptes lycopersici tryon in 1917 from samples collected in queensland, australia. the same mite was later described as a new species by massee (1937) with the same name, and later by keifer (1940) as phyllocoptes destructor (synonymized by keifer, 1952). geographical distribution of a. lycopersici, first described from queensland, australia, has been expanded to include the afro-tropical region, australian region, east palaearctic (east of the border line here defined), near east (asian turkey, caucasian russian republics, georgia, armenia, azerbaijan, lebanon, syria, israel, jordan, sinai peninsula (egypt), arabian peninsula, iran, iraq), nearctic region, neotropical north african region (not including the sinai peninsula), oriental regions (de lillo, 2004; anonymous, 2012a). aculops lycopersici occurs on some solanaceae (jeppson et al., 1975; özman-sullivan & öcal, 2005), few convolvulaceae and rosaceae. it is mostly known as a pest of tomatoes, but also damages potato (solanum tuberosum l.), eggplant (brinjal) (solanum melongena l.), tobacco (nicotiana tabacum l.), bell pepper (capsicum annuum l.), jerusalem cherry (solanum pseudocapsicum l.), petunia (petunia hybrida l.), tomatillo (physalis philadelphica lam.), cherry pepper (capsicum annuum var. annuum l.), hairy nightshade (solanum sarrachoides (sendtner)), black nightshade (nightshade) (solanum nigrum l.), small flowered nightshade (solanum nodiflorum jacq.), popolo (solanum nelsonii dunal.), horse nettle (solanum carolinense l.), jimson weed (datura stramonium l.), tolguacha (datura meteloides dunal.), chinese thorn apple (datura quercifolia kunth), amethyst (browallia speciosa hook.), poha (cape gooseberry) (physalis peruviana l.) (perring, 1996; duso et al., 2010), hot pepper (capsicum frutescens l.), sweet potato (ipomoea batatas (l.)), long-spined thornapple (datura ferox l.), thornapple (datura innoxia p.mill.), wild tomato (lycopersicon peruvianum l.), red currant tomato (lycopersicon pimpinellifolium (l.)), native gooseberry (physalis minima linn.), jamaican forget me not (browallia americana l.) (solanales: solanaceae) (craemer, 2002), field bindweed (convolvulus arvensis l.), morning glory (pharbitis nil l.) (perring, 1996; duso et al., 2010; anonymous, 2012b) and tall morning glory (ipomoea purpurea l.) (solanales: convolvulaceae) (hoy, 2011), wild blackcurrant (ribes americanum mill.), wild gooseberry (ribes hirtellum michx.) and blackberry (rubus caesius l.) (rosales: rosaceae) (perring, 1996; duso et al., 2010). larrain (2002) recently found this mite on pepino (solanum muricatum aiton) in chile. in august 2010 and september 2011, aculops lycopersici and its typical symptoms such as bronzing of leaves, withering and change of stem color from green to brown were found on s. muricatum plants in kayabaşı village in ordu and terme province in samsun, both in the black sea region of turkey. the leaves expected to be contaminated were collected in individual polyethylene bags in august 2010 and september 2011 and brought to the laboratory. the mites on the leaves were collected by brush under a microscope. eriophyids were preserved in vials containing 70% ethanol. the mites were moved separately to watch glasses containing lactophenol as a clearing medium. each mite was mounted in a drop of hoyer’s medium and the microscope slides were dried at 60°c. the slide mounts were studied under different magnifications (walter & krantz, 2009). the mites were identified according to keifer et al. (1982). the species was confirmed by mariusz lewandowski. these were the first records of a. lycopersici infection on pepino in turkey. since there is no literature on a lycopersici of pepino fruit grown in turkey, this plant was recorded as a new host for turkey. in turkey, the species was initially recorded on open-air tomato crops in adana and içel provinces (şekeroğlu & özgür, 1984), and later [journal of entomological and acarological research 2012; 44:e20] [page 115] journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2012; volume 44:e20 first report of aculops lycopersici (tryon, 1917) (acari: eriophyidae) on pepino in turkey r. akyazi university of ordu, faculty of agriculture, department of plant protection, ordu, turkey correspondence: rana akyazi, university of ordu, faculty of agriculture, department of plant protection, ordu, turkey. tel. +9.452.234.50.10 fax: +9.452.234.66.32. e-mail: ranainak@hotmail.com key words: aculops lycopersici, pepino, solanum muricatum, turkey. acknowledgments: we thank mariusz lewandowski (department of applied entomology, faculty of horticulture and landscape architecture, warsaw university of life sciences, warsaw, poland) for confirmation of the eriophyid species. received for publication: 16 august 2012. revision received: 30 october 2012. accepted for publication: 14 november 2012. ©copyright r. akyazi, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e20 doi:10.4081/jear.2012.e20 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly on tomato plants in field and glasshouses at adana, içel, antalya, manisa, i̇zmir, samsun, tokat provinces of turkey (öncüer et al., 1992; can & çobanoğlu, 2004; madanlar & öncüer, 1994; yaşarakıncı & hıncal, 1997; yoldaş et al., 1999; hıncal et al., 2002; i̇nal, 2005; yanar et al., 2008; güncan et al., 2010). tomato russet mite was also found on s. nigrum in the tomato fields in tokat province (yanar et al., 2008). so it seems likely that the presence of this species on s. muricatum in ordu and samsun provinces, where plants of the solanaceae family can be grown, should not be surprising. however, there are currently no records from any plants in ordu and from s. muricatum in turkey. references anonymous, 2012a aculops lycopersici. available from: http://www. faunaeur.org/distribution_table.php anonymous, 2012b tomato russet mite (aculops lycopersici) host plants/species affected. available from: http://www.plantwise.org/ ?dsid=56111&loadmodule=plantwisedatasheet&page=4270&site =234 can m., çobanoğlu s., 2004 antalya i̇li kumluca yöresinde sebze üretimi yapılan plastik ve cam seralarda bulunan akar (acarina) türlerinin tanımı, konukçuları ve yoğunluklarının belirlenmesi üzerine araştırmalar [studies on the determination of mite (acarina) species their hosts and population densities on greenhouse vegetable in kumluca antalya]. a. ü. fen bilimleri enstitüsü, yüksek lisans tezi, ankara. craemer c., 2002 aculops lycopersici, host notes. available from: http://ecoport.org/ep?arthropod=18684&entitytype=ar****&entit ydisplaycategory=ar****3000 de lillo e., 2004 fauna europaea: eriophyoidea. in: magowski̇ w. fauna europaea: acariformes. fauna europaea version 1.1. available from: http://www.faunaeur.org/full_results.php?id=93787 duso c., castagnoli m., simoni s., angeli g., 2010 the impact of eriophyoids on crops: recent issues on aculus schlechtendali, calepitrimerus vitis and aculops lycopersici. exp. appl. acarol. 51: 151-168. i̇nal b., 2005. bafra ve çarşamba ovalarında çeşitli kültür bitkisi alanlarında bulunan acarina türleri üzerinde faunistik çalışmalar (faunistic studies on the acarina species found on various crops in bafra and çarşamba plains). ondokuz mayıs üniversitesi fen bilimleri enstitüsü, yüksek lisans tezi, samsun. güncan a., madanlar n., yoldaş z., ersi̇n f., tüzel y., 2010 i̇zmir ilinde örtüaltı organik sebze üretiminde topraküstü zararlılarının durumu [status of above-ground pests in organic vegetable production under greenhouse conditions in i̇zmir province]. türk. entomol. derg. 34: 503-513. hincal p., yaşarakinci n., çinarli i̇., 2002 i̇zmir ilinde domates pas akarı (aculops lycopersici massee) (acarina: eriophyidae)‘nın popülasyon seyri, doğal düşmanları ve kimyasal mücadelesi üzerinde araştırmalar [the researches on the population developments of aculops lycopersici (massee) (acarina:eriophyidae) and its natural enemies, the chemical control of the pest in i̇zmir]. bit. kor. bült. 42: 9-22. hoy m.a., 2011 agricultural acarology: introduction to integrated mite management. -crc press, taylor and francis group, boca raton, london, new york. jeppson l.t., keifer h.h., baker e.w., 1975 mites injurious to economic plants.university of california press, berkley, ca. keifer h.h., 1940 eriophyid studies. x. bull. calif. dept. agric. 29: 160-179. keifer h.h., 1952 the eriophyid mites of california (acarina, eriophyidae). bull. calif. insect surv. 2: 1-123. keifer h.h., baker e.w., kono t., delfinado m., styer w.e., 1982 an illustrated guide to plant abnormalities caused by eriophyid mites in north america. u.s. department of agriculture, agricultural research service, agriculture hand book, washington, dc. larrain s.p., 2002 insect and mite pest incidence on sweet pepinos solanum muricatum (ait.) cultivated in the iv region, chile. agr. tec. 62: 15-26. madanlar n., öncüer c., 1994 i̇zmir ilinde sera domatesi zararlısı olarak aculops lycopersici (massee) (acarina, eriophyidae) [aculops lycopersici (massee) (acarina, eriophyidae) a pest of greenhouse tomatoes in i̇zmir province]. türk. entomol. derg. 18: 237-240. massee a.m., 1937 an eriophyid mite injurious to tomato. bull. ent. res. 28: 403. öncüer c., karsavuran y., yoldaş z., durmuşoğlu e., 1992 sanayi domateslerinde görülen zararlılar, yayılış ve bulaşma oranları üzerinde araştırmalar [investigation on process tomato pests and their distribution and infestation rates]. in: türkiye ii. entomoloji kongresi bildirileri, adana: 705. özman-sulli̇van s.k., öcal h., 2005 sebzelerde bulunan eriophiyoid akarlar [eriophyoid mites on vegetables]. gap iv. tarım kongresi bildirileri, cilt 1, şanlıurfa: 334-341. perring t.m., 1996 vegetables. in: lindquist e.e., sabelis m.w., bruin j., (eds.). eriophyid mites their biology, natural enemies and control. world crop pests. elsevier, amsterdam: 593-610. şekeroğlu e., özgür a.f., 1984 a new tomato pest in çukurova, aculops lycopersici (massee) (acarina: eriophyidae). turk j. plant protect. 8: 211-213. tryon h., 1917 report of the entomologist and vegetable pathologist. queensland dept. agric. and stock rept., queensland: 49-63. walter d.e., krantz g.w., 2009 a manual of acarology. in: krantz g.w., walter d.e., (eds.). collection, rearing and preparing specimens. texas tech university press, lubbock, tx: 83-97. yanar d., ecevi̇t o., kadioğlu i̇., 2008 tokat yöresinde domates ekim alanlarında zarar oluşturan domates pas akarı aculops lycopersici (massee) (acari: eriophyidae) [tomato rust mite aculops lycopersici (massee) (acari: eriophyidae) causing damage in tomato production areas of tokat]. g.o.p üniv. zir. fak. derg. 25: 1-5. yaşarakinci n., hincal p., 1997 i̇zmir’de örtüaltında yetiştirilen domates, hıyar, biber ve marulda bulunan zararlı ve yararlı türler ile bunların popülasyon yoğunlukları üzerinde araştırmalar [the research on determining the pests and beneficial species and their population densities on the tomato, cucumber, pepper and lettuce glasshouses in i̇zmir]. bit. kor. bült. 37: 79-89. yoldaş, z., madanlar n., gül a., onoğur e., 1999 investigations on integrated control practices in vegetable glasshouses in izmir. acta hort. 491: 453-460. short paper [page 116] [journal of entomological and acarological research 2012; 44:e20] no nco mm er cia l u se on ly jear2012 abstract we report the results of recording lepidoptera in the lower sangro valley during a period of 22 years. the investigations were devoted to macroheterocera and were carried out in the two regional nature reserves oasi di serranella and lecceta di torino di sangro. the listing also includes some microlepidoptera as non-target species, as well as occasionally observed butterflies. the 401 recorded species are presented in a table indicating both the locality of the records and the observed flight times and periods of activity. fifteen species are published for the abruzzo region for the first time; 2 species are new for the italian peninsula. introduction we investigated the fauna of the lower sangro valley from 1988, with a particular focus on macroheterocera. we also aimed to verify occurrence and distribution of the lepidoptera of central italy. the butterflies observed and the few microlepidoptera, collected together with the macroheterocera are also listed. materials and methods the study was carried out in two regional protected areas: the oasi di serranella (42°07’35” n, 14°22’40” e) and the lecceta di torino di sangro (42°13’50” n, 14°32’20” e). in addition, light catches were carried out twice (july 13th and 15th 2000) on the beach of the sangro valley (42°14’31” n, 14°31’30” e, at sea level), but with little success. the oasi di serranella was investigated at irregular intervals a total of 49 times from 1988 to 2010. the main focus was on observations at night. rhopalocera were registered only during occasional walks in the protected area. pheromones, provided by e. priesner, were used once on june 21st 1991 to look for sesiidae. the lecceta di torino di sangro was intensively studied with respect to night active macrolepidoptera for two years (2006-2007), excluding the slope towards the sea. different investigation times were fixed to facilitate observation of the broadest possible spectrum of species. in 2006, investigations were carried out during the periods from may 16th to june 16th and from september 12th to october 12th. in 2007, they were carried out from july 20th to august 3rd. in total, there were 26 night catches. occasionally, during daytime selection of light catching sites, some species of butterfly were observed and registered. active observation was necessary due to the lighting methods employed. these consisted of a gaze tower with a 250 watt compound light bulb and a black light tube connected to a super actinic tube. a generator served as energy source. easily recognizable species were recorded at the illuminated site and samples were collected. species not determinable with certainty were also sampled. recordings have been archived in the collection of the author. some of the microlepidoptera were given to a. werno, who compiled a list of identified species that has been integrated into the list of results (table 1) and marked in the column footnotes. the collected specimens were determined by the author. identification literature (e.g., fibiger, 1990-2010; hausmann, 2001, 2004; mironov, 2003; weigt, 2009) and comparable collections were consulted where necessary. dissection was performed on species that were difficult to identify and individual specimens that were anomalous. all recorded data are stored in an oracle databank. specific individual data on certain species are available from the author on request. informations about the distribution of the species have been found in the literatures (prola et al., 1978a, 1978b; prola & racheli, 1979, 1980; teobaldelli, 1976; sciarretta & zahm, 2002; parenzan & porcelli 20052006, 2006-2007). journal of entomological and acarological research 2012; volume 44:e14 [page 64] [journal of entomological and acarological research 2012; 44:e14] contribution to the knowledge of the lepidoptera fauna of the lower sangro valley in the abruzzo region of central italy n. zahm entomologist correspondence: norbert zahm, ludwig-uhland-str. 34, d-66839 schmelz, germany. tel. +49.6887.4357. e-mail: nzahm@t-online.de key words: lower sangro valley, macroheterocera. acknowledgments: the author would like to thank dr. a. natale and m. pellegrini for their permission to carry out the research and their active support with its realization. hi would also like to thank dr. e. priesner (†), who supplied the pheromones. thanks go to a. werno for the determination of some microlepidoptera, to dr. h. schreiber for the english translation and s. and c. gaia and h. gay for the carful proofreading, and to r. hinsberger, e. müller and m. pellegrini for the photographs. received for publication: 26 april 2012. revision received: 14 november 2012. accepted for publication: 14 november 2012. ©copyright n. zahm, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e14 doi:10.4081/jear.2012.e14 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly t ab le 1 . l is t o f re su lt s. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty ti ne id ae 1 n em ap og on a ge nj oi g .p et er se n , 1 95 9 x 27 -v ii -2 00 7 n ig ht w ) ps yc hi da e 2 o ik et ic oi de s lu te a (s ta u d in g er , 1 87 0) x 20 -v ii -2 00 7 n ig ht g ra ci lla ri id ae 3 eu ca ly bi te s au ro gu tt el la (s te ph en s, 1 83 5) x 24 -v ii -2 00 7 n ig ht w ) 4 ca ly bi te s ph as ia ni pe nn el la (h ü bn er , 1 81 3) x 20 -v ii -2 00 7 n ig ht w ) o ec op ho ri da e 5 ep ic al lim a fo rm os el la (d en is & s ch if fe rm ü ll er , 1 77 5) x 20 -v ii -2 00 7 n ig ht w ) 6 ce ph al is ph ei ra fe rr ug el la (d en is & s ch if fe rm ü ll er , 1 77 5) x 20 -v ii -2 00 7 n ig ht w ) co le op ho ri da e 7 co le op ho ra o no br yc hi el la ze ll er , 1 84 9 x 24 -v ii -2 00 7 n ig ht w ) bl as to ba si da e 8 bl as to ba si s ph yc id el la (z el le r, 1 83 9) x 20 -v ii -2 00 7 n ig ht w ) au to st ic hi da e 9 o eg oc on ia d ea ur at el la (h er ri ch -s ch äf fe r, 1 85 4) x 3vi ii -2 00 7 n ig ht w ) g el ec hi id ae 10 br yo tr op ha s en ec te lla (z el le r, 1 83 9) x 31 -v ii -2 00 7 n ig ht w ) 11 ps eu do te lp hu sa s ca le lla (s co po li , 1 76 3) x 27 -v ii -2 00 7 n ig ht w ) 12 sc ro bi pa lp a er ga si m a (m ey ri ck , 1 91 6) x 27 -v ii -2 00 7 n ig ht w ) li m ac od id ae 13 ap od a lim ac od es (h u fn ag el , 1 76 6) x 19 -v 16 -v i n ig ht se si id ae 14 pa ra nt hr en e ta ba ni fo rm is (r o tt em bu rg , 1 77 5) x 21 -v i19 91 d ay 1) co ss id ae 15 co ss us c os su s (l in n ae u s, 1 75 8) x x 5vi 3vi ii n ig ht 16 pa ra hy po pt a ca es tr um (h ü bn er , 1 80 8) x 8vi i 22 -v ii n ig ht 17 ze uz er a py ri na (l in n ae u s, 1 76 1) x x 19 -v 3vi ii n ig ht to rt ri ci da e 18 ph th eo ch ro a in gr id ae h u em er , 1 99 0 x 16 -v -2 00 6 n ig ht 4) 8 ) 19 co ch yl is h yb ri de lla (h ü bn er , 1 81 3) x 15 -v ii -2 00 0 n ig ht 20 co ch yl is m ol lic ul an a ze ll er , 1 84 7 x 27 -v ii i19 92 n ig ht 21 ac le ri s sp ar sa na (d en is & s ch if fe rm ü ll er , 1 77 5) x 24 -v ii -2 00 7 n ig ht w ) 22 cl ep si s co ns im ila na (h ü bn er , 1 81 7) x 20 -v ii -2 00 7 n ig ht w ) 23 en do th en ia o bl on ga na (h aw o rt h , 1 81 1) x 20 -v ii -2 00 7 n ig ht w ) 24 eu de m is p ro fu nd an a (d en is & s ch if fe rm ü ll er , 1 77 5) x 20 -v ii 3vi ii n ig ht w ) 25 ce ly ph a ru fa na (s co po li , 1 76 3) x 20 -v ii -2 00 7 n ig ht w ) 26 lo be si a bo tr an a (d en is & s ch if fe rm ü ll er , 1 77 5) x 27 -v ii -2 00 7 n ig ht w ) 27 ep in ot ia fe st iv an a (h ü bn er , 1 79 9) x 7vi -2 00 6 n ig ht 28 cy di a fa gi gl an da na (z el le r, 1 84 1) x 27 -v ii -2 00 7 n ig ht w ) pt er op ho ri da e 29 ag di st is h ey de ni ( ze ll er , 1 85 2) x 15 -v ii -2 00 0 n ig ht 30 ag di st is ta m ar ic is (z el le r, 1 84 7) x 30 -v ii -2 00 1 n ig ht 31 am bl yp ti lia a ca nt ha da ct yl a (h ü bn er , 1 81 3) x x 16 -v 15 -v ii n ig ht 32 cn ae m id op ho ru s rh od od ac ty la (d en is & s ch if fe rm ü ll er , 1 77 5) x 12 -v i20 06 n ig ht 33 o xy pt ilu s di st an s (z el le r, 1 84 7) x 13 -i x20 06 n ig ht 34 st an ge ia s ic el io ta (z el le r, 1 84 7) x 12 -i x20 06 n ig ht 35 pt er op ho ru s pe nt ad ac ty la (l in n ae u s, 1 75 8) x 29 -v -2 00 5 n ig ht 36 m er ri fi el di a m al ac od ac ty lu s (z el le r, 1 84 7) x 15 -v ii -2 00 0 n ig ht 37 em m el in a m on od ac ty la (l in n ae u s, 1 75 8) x x x 15 -v 30 -v ii n ig ht py ra lid ae 38 la m or ia a ne lla (d en is & s ch if fe rm ü ll er , 1 77 5) x 20 -v ii -2 00 7 n ig ht w ) 39 g al le ri a m el lo ne lla (l in n ae u s, 1 75 8) x 27 -i x20 06 n ig ht 40 ac te ni a br un ne al is ( tr ei ts ch ke , 1 82 9) x 27 -v ii -2 00 7 n ig ht w ) 41 h er cu lia fu lv oc ili al is (d u po n ch el , 1 83 4) x 31 -v ii -2 00 7 n ig ht 9) t o b e co n ti n u ed o n n ex t p ag e. article [journal of entomological and acarological research 2012; 44:e14] [page 65] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 42 en do tr ic ha fl am m ea lis (d en is & s ch if fe rm ü ll er , 1 77 5) x 12 -i x20 06 n ig ht 43 et ie lla z in ck en el la (t re it sc h ke , 1 83 2) x 7vi 20 -v ii n ig ht w ) 44 o nc oc er a se m ir ub el la (s co po li , 1 76 3) x x 8vi i 24 -v ii n ig ht 45 ph yc it a ro bo re lla ( d en is & s ch if fe rm ü ll er , 1 77 5) x 20 -v ii 27 -v ii n ig ht w ) 46 ac ro ba si s ob liq ua (z el le r, 1 84 7) x 27 -v ii 3vi ii n ig ht w ) 47 ap om ye lo is c er at on ia e (z el le r, 1 83 9) x 20 -v ii -2 00 7 n ig ht w ) 48 h om oe os om a si nu el la (f ab ri ci u s, 1 79 3) x 27 -v ii 31 -v ii n ig ht w ) 49 ph yc it od es a lb at el la (r ag o n o t, 1 88 7) x 24 -v ii 3vi ii n ig ht w ) 50 ep he st ia w el se ri el la (z el le r, 1 84 8) x 20 -v ii -2 00 7 n ig ht w ) 51 ep he st ia e lu te lla ( h ü bn er , 1 79 6) x 27 -v ii -2 00 7 n ig ht w ) 52 ep he st ia p ar as it el la st au d in g er , 1 85 9 x 20 -v ii -2 00 7 n ig ht w ) 53 em at he ud es p un ct el la (t re it sc h ke , 1 83 3) x 8vi i19 94 n ig ht 54 ca la m ot ro ph a pa lu de lla (h ü bn er , 1 82 4) x 24 -v ii 6vi ii n ig ht 55 ag ri ph ila la ti st ri a (h aw o rt h , 1 81 1) x 27 -i x20 06 n ig ht 56 ag ri ph ila g en ic ul ea (h aw o rt h , 1 81 1) x 12 -i x20 06 n ig ht 57 ca to pt ri a fa ls el la (d en is & s ch if fe rm ü ll er , 1 77 5) x 24 -v ii -2 00 7 n ig ht w ) 58 el op hi la n ym ph ae at a (l in n ae u s, 1 75 8) x 22 -v ii -2 01 0 n ig ht 59 el op hi la ri vu la lis (d u po n ch el , 1 83 4) x x 8vi i 12 -i x n ig ht 60 ap or od es fl or al is (h ü bn er , 1 80 9) x x 6vi i 3vi ii n ig ht 61 cy na ed a de nt al is (d en is & s ch if fe rm ü ll er , 1 77 5) x 27 -i x20 06 n ig ht 62 h yd ri ri s or na ta lis (d u po n ch el , 1 83 2) x x 22 -v ii 27 -i x n ig ht 63 py ra us ta d es pi ca ta (s co po li , 1 76 3) x 28 -v -1 98 9 n ig ht 64 si to ch ro a ve rt ic al is (l in n ae u s, 1 75 8) x 15 -v -2 00 6 n ig ht 65 o st ri ni a nu bi la lis (h ü bn er , 1 79 6) x 28 -v -1 98 9 n ig ht 66 d ia se m io ps is ra m bu ri al is (d u po n ch el , 1 83 4) x 27 -i x20 06 n ig ht 67 pa lp it a un io na lis (h ü bn er , 1 79 6) x 8x20 06 n ig ht 68 d ol ic ha rt hr ia p un ct al is (d en is & s ch if fe rm ü ll er , 1 77 5) x 28 -v -1 98 9 n ig ht la si oc am pi da e 69 la si oc am pa tr if ol ii ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 15 -i x 22 -i x n ig ht 70 m ac ro th yl ac ia ru bi (l in n ae u s, 1 75 8) x 29 -v ii -1 99 0 d ay n ig ht 71 o do ne st is p ru ni ( li n n ae u s, 1 75 8) x 12 -i x20 06 n ig ht sa tu rn iid ae 72 sa tu rn ia p yr i ( d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v -2 00 6 n ig ht sp hi ng id ae 73 m im as ti lia e (l in n ae u s, 1 75 8) x 6vi 30 -v ii n ig ht 74 sm er in th us o ce lla ta (l in n ae u s, 1 75 8) x x 18 -v 6vi ii n ig ht 75 la ot ho e po pu li (l in n ae u s, 1 75 8) x x 18 -v 14 -i x n ig ht 76 ag ri us c on vo lv ul i( li n n ae u s, 1 75 8) x 19 -i x20 06 n ig ht 77 m ac ro gl os su m s te lla ta ru m (l in n ae u s, 1 75 8) x 28 -v -1 98 9 d ay 2) 78 h yl es li vo rn ic a (e sp er , 1 77 9) x x 19 -v 25 -v n ig ht 79 d ei le ph ila e lp en or (l in n ae u s, 1 75 8) x x 15 -v 6vi ii n ig ht 80 d ei le ph ila p or ce llu s (l in n ae u s, 1 75 8) x 24 -v ii -1 98 9 n ig ht h es pe ri id ae 81 th ym el ic us a ct eo n (r o tt em bu rg ,1 77 5) x 28 -v 21 -v i d ay 82 o ch lo de s ve na ta (b re m er & g re y, 1 85 3) x 12 -i x20 06 d ay 83 g eg en es n os tr od am us ( fa br ic iu s, 1 79 3) x 31 -v ii -2 00 9 n ig ht 3) 1 0) pa pi lio ni da e 84 ip hi cl id es p od al ir iu s (l in n ae u s, 1 75 8) x 29 -v ii -1 99 0 d ay 85 pa pi lio m ac ha on l in n ae u s, 1 75 8 x 21 -v i 28 -v ii d ay t o b e co n ti n u ed o n n ex t p ag e. article [page 66] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty pi er id ae 86 eu ch lo e au so ni a (h ü bn er , 1 80 4) x 24 -v 28 -v d ay 87 ap or ia c ra ta eg i( li n n ae u s, 1 75 8) x 28 -v -1 98 9 d ay 88 pi er is b ra ss ic ae (l in n ae u s, 1 75 8) x 24 -v 16 -i x d ay 89 pi er is m an ni i( m ay er , 1 85 1) x 16 -i x20 06 d ay 90 pi er is ra pa e (l in n ae u s, 1 75 8) x 28 -v 5vi d ay 91 pi er is n ap i ( li n n ae u s, 1 75 8) x 28 -v 12 -i x by d ay 92 po nt ia e du sa ( fa br ic iu s, 1 77 7) x 5vi -2 00 6 d ay 93 co lia s cr oc eu s (g eo ff ro y in f o u rc ro y, 1 78 5) x 28 -v 12 -i x d ay 94 g on ep te ry x rh am ni (l in n ae u s, 1 75 8) x 16 -v -2 00 6 d ay ly ca en id ae 95 ly ca en a ph la ea s (l in n ae u s, 1 76 1) x 28 -v -1 98 9 d ay 96 sa ty ri um il ic is (e sp er , 1 77 9) x 5vi -2 00 6 d ay 97 cu pi do a rg ia de s (p al la s, 1 77 1) x 28 -v ii -1 98 8 d ay 98 cu pi do a lc et as (h o ff m an n se g g , 1 80 4) x 29 -v ii -1 99 0 d ay 99 ce la st ri na a rg io lu s (l in n ae u s, 1 75 8) x 5vi -2 00 6 d ay 10 0 g la uc op sy ch e al ex is (p o d a, 1 76 1) x 28 -v -1 98 9 d ay 10 1 pl eb ei us id as (l in n ae u s, 1 76 1) x 28 -v -1 98 9 d ay 10 2 ar ic ia a ge st is (d en is & s ch if fe rm ü ll er , 1 77 5) x 12 -i x20 06 d ay 10 3 po ly om m at us th er si te s (c an te n er , 1 83 5) x 5vi 28 -v ii d ay 10 4 po ly om m at us ic ar us (r o tt em bu rg , 1 77 5) x 24 -v 12 -i x d ay n ym ph al id ae 10 5 br en th is d ap hn e (d en is & s ch if fe rm ü ll er , 1 77 5) x 5vi -2 00 6 d ay 10 6 va ne ss a at al an ta (l in n ae u s, 1 75 8) x 5vi -2 00 6 d ay 10 7 va ne ss a ca rd ui ( li n n ae u s, 1 75 8) x 7vi i 12 -i x d ay 10 8 in ac hi s io ( li n n ae u s, 1 75 8) x 7vi i20 09 d ay 10 9 po ly go ni a cal bu m ( li n n ae u s, 1 75 8) x 24 -v 21 -v i d ay 11 0 po ly go ni a eg ea (c ra m er , 1 77 5) x 7vi i20 09 d ay 11 1 m el it ae a ci nx ia ( li n n ae u s, 1 75 8) x 24 -v -2 00 6 d ay 11 2 m el it ae a ph oe be ( d en is & s ch if fe rm ü ll er , 1 77 5) x 5vi 28 -v ii d ay 11 3 m el it ae a at ha lia (r o tt em bu rg , 1 77 5) x 5vi -2 00 6 d ay 11 4 li m en it is re du ct a st au d in g er , 1 90 1 x 12 -i x20 06 d ay 11 5 pa ra rg e ae ge ri a (l in n ae u s, 1 75 8) x x 16 -v 12 -i x d ay 11 6 la si om m at a m eg er a (l in n ae u s, 1 76 7) x x 16 -v 16 -i x d ay 11 7 co en on ym ph a ar ca ni a (l in n ae u s, 1 76 1) x 28 -v 5vi d ay 11 8 co en on ym ph a pa m ph ilu s (l in n ae u s, 1 75 8) x 24 -v 29 -v ii d ay 11 9 py ro ni a ce ci lia (v al la n ti n , 1 89 4) x 7vi i20 09 d ay 12 0 m an io la ju rt in a (l in n ae u s, 1 75 8) x 24 -v 12 -i x d ay 12 1 m el an ar gi a ga la th ea ( li n n ae u s, 1 75 8) x 5vi 7vi i d ay 12 2 h ip pa rc hi a fa gi (s co po li , 1 76 3) x 27 -i x20 06 d ay 12 3 h ip pa rc hi a st at ili nu s (h u fn ag el , 1 76 6) x 28 -v ii 16 -i x d ay 12 4 br in te si a ci rc e (f ab ri ci u s, 1 77 5) x 5vi 12 -i x d ay d re pa ni da e 12 5 th ya ti ra b at is ( li n n ae u s, 1 75 8) x x 18 -v 3x n ig ht 12 6 h ab ro sy ne p yr it oi de s (h u fn ag el , 1 76 6) x x 16 -v 3x n ig ht 12 7 te th ea o cu la ri s (l in n ae u s, 1 76 7) x x 18 -v 31 -v ii n ig ht 12 8 cy m at op ho ri na d ilu ta d en is & s ch if fe rm ü ll er , 1 77 5) x 12 -i x 12 -x n ig ht 12 9 w at so na lla b in ar ia (h u fn ag el , 1 76 7) x x 6vi i 15 -i x n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [journal of entomological and acarological research 2012; 44:e14] [page 67] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 13 0 w at so na lla u nc in ul a (b o rk h au se n , 1 79 0) x 16 -v 8x n ig ht 13 1 ci lix h is pa ni ca pe re z d eg re g o ri o & a l., 2 00 2 x 4vi ii 15 -x n ig ht 4) g eo m et ri da e 13 2 lo m as pi lis m ar gi na ta (l in n ae u s, 1 75 8) x 18 -v 3vi ii n ig ht 13 3 li gd ia a du st at a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 3vi ii n ig ht 13 4 st eg an ia tr im ac ul at a (v il le rs , 1 78 9) x x 15 -v 27 -v ii i n ig ht 13 5 h el io m at a gl ar ea ri a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 15 -v 27 -v ii i d ay n ig ht 13 6 m ac ar ia a lte rn at a (d en is & s ch if fe rm ü ll er , 1 77 5) x 18 -v 27 -v ii i n ig ht 13 7 m ac ar ia a rt es ia ri a (d en is & s ch if fe rm ü ll er , 1 77 5) x 18 -v 27 -v ii i n ig ht 13 8 g od on el la a es ti m ar ia ( h ü bn er , 1 80 9) x 8vi i 3vi ii n ig ht 13 9 te ph ri na a re na ce ar ia (d en is & s ch if fe rm ü ll er , 1 77 5) x 24 -v ii 6vi ii n ig ht 14 0 o pi st ho gr ap ti s lu te ol at a (l in n ae u s, 1 75 8) x x 16 -v 3x n ig ht 14 1 ep io ne re pa nd ar ia (h u fn ag el , 1 76 7) x 28 -v 3vi ii n ig ht 14 2 ap ei ra s yr in ga ri a (l in n ae u s, 1 75 8) x x 12 -v i 22 -v ii n ig ht 14 3 en no m os q ue rc ar ia (h ü bn er , 1 81 3) x 5vi 27 -i x n ig ht 14 4 se le ni a de nt ar ia (f ab ri ci u s, 1 77 5) x x 27 -v ii i 22 -i x n ig ht 14 5 se le ni a lu nu la ri a (h ü bn er , 1 78 8) x 22 -i x20 06 n ig ht 14 6 cr oc al lis e lin gu ar ia (l in n ae u s, 1 75 8) x x 22 -v ii 1x n ig ht 14 7 m en op hr a ab ru pt ar ia (t h u n be rg , 1 79 2) x x 16 -v 6x n ig ht 14 8 sy no ps ia s oc ia ri a (h ü bn er , 1 79 9) x x 15 -v 17 -i x n ig ht 14 9 pe ri ba to de s rh om bo id ar ia (d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 15 -x n ig ht 15 0 pe ri ba to de s um br ar ia (h ü bn er , 1 80 9) x x 28 -v 15 -x n ig ht 15 1 h yp om ec is p un ct in al is ( sc o po li , 1 76 3) x x 18 -v 4vi ii n ig ht 15 2 as co ti s se le na ri a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 25 -v 3vi ii n ig ht 15 3 em at ur ga a to m ar ia (l in n ae u s, 1 75 8) x 28 -v 6vi ii d ay n ig ht 15 4 eu m an ni a le pr ar ia (r eb el , 1 90 9) x 22 -v ii 4vi ii n ig ht 4) 15 5 ca be ra e xa nt he m at a (s co po li , 1 76 3) x x 18 -v 3x n ig ht 15 6 ca m pa ea h on or ar ia (d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 8x n ig ht 15 7 se m ia sp ila te s oc hr ea ri a (r o ss i, 17 94 ) x 14 -i x20 06 n ig ht 15 8 co m ib ae na b aj ul ar ia (d en is & s ch if fe rm ü ll er , 1 77 5) x 24 -v 6vi n ig ht 15 9 h em is to la s ic ili an a pr o u t, 1 93 5 x x 29 -v 16 -v i n ig ht 16 0 th al er a fi m br ia lis (s co po li , 1 76 3) x 22 -v ii 4vi ii n ig ht 16 1 h em it he a ae st iv ar ia (h ü bn er , 1 78 9) x x 5vi 21 -v i n ig ht 16 2 ch lo ri ss a vi ri da ta ( li n n ae u s, 1 75 8) x 21 -v i 24 -v ii n ig ht 16 3 ph ai og ra m m a et ru sc ar ia (z el le r, 1 84 9) x 15 -v 27 -v ii i d ay n ig ht 16 4 id ae a co ns an gu in ar ia (l ed er er , 1 85 3) x 6vi i20 08 n ig ht 16 5 id ae a ru st ic at a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 6vi i 6vi ii n ig ht 16 6 id ae a fi lic at a (h ü bn er , 1 79 9) x x 18 -v 6x n ig ht 16 7 id ae a el on ga ri a (r am bu r, 1 83 3) x x 18 -v 5vi n ig ht 16 8 id ae a ob so le ta ri a (r am bu r, 1 83 3) x x 27 -v ii 4vi ii n ig ht 16 9 id ae a fu sc ov en os a (g o ez e, 1 78 1) x 6vi i20 08 n ig ht 17 0 id ae a se ri at a (s ch ra n k, 1 80 2) x x 15 -v 15 -x n ig ht 17 1 id ae a su bs er ic ea ta (h aw o rt h , 1 80 9) x x 16 -v 6x n ig ht 17 2 id ae a di m id ia ta (h u fn ag el , 1 76 7) x 13 -i x20 06 n ig ht 17 3 id ae a tr ig em in at a (h aw o rt h , 1 80 9) x 19 -v 22 -i x n ig ht 17 4 id ae a in fi rm ar ia (r am bu r, 1 83 3) x x 20 -v ii 30 -v ii n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [page 68] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 17 5 id ae a os tr in ar ia (h ü bn er , 1 81 3) x 7vi 16 -v i n ig ht 10 ) 17 6 id ae a di st in ct ar ia (b o is d u va l, 1 84 0) x 7vi 24 -v ii n ig ht 4) 17 7 id ae a ru br ar ia (s ta u d in g er , 1 90 1) x x 3vi ii 6vi ii n ig ht 17 8 id ae a av er sa ta (l in n ae u s, 1 75 8) x x 18 -v 27 -i x n ig ht 17 9 id ae a de ge ne ra ri a (h ü bn er , 1 79 9) x x 16 -v 12 -x n ig ht 18 0 id ae a st ra m in at a (b o rk h au se n , 1 79 4) x 28 -v 27 -v ii i n ig ht 18 1 sc op ul a ni gr op un ct at a (h u fn ag el , 1 76 7) x x 5vi 27 -i x n ig ht 10 ) 18 2 sc op ul a or na ta (s co po li , 1 76 3) x 18 -v 3x n ig ht 18 3 sc op ul a ru bi gi na ta ( h u fn ag el , 1 76 7) x x 4vi ii 15 -x n ig ht 18 4 sc op ul a m ar gi ne pu nc ta ta (g o ez e, 1 78 1) x x 19 -v 15 -x n ig ht 18 5 sc op ul a im it ar ia ( h ü bn er , 1 79 9) x x 18 -v 15 -x n ig ht 18 6 sc op ul a em ut ar ia (h ü bn er , 1 80 9) x 8vi i 24 -v ii n ig ht 10 ) 18 7 sc op ul a m in or at a (b o is d u va l, 1 83 3) x x 20 -v ii 15 -x n ig ht 18 8 rh od os tr op hi a ca la br a (p et ag n a, 1 78 7) x x 18 -v 12 -v i d ay n ig ht 18 9 ti m an dr a co m ae a. sc h m id t, 1 93 1 x x 18 -v 1x n ig ht 19 0 cy cl op ho ra p up pi lla ri a (h ü bn er , 1 79 9) x 19 -v 27 -i x n ig ht 19 1 cy cl op ho ra ru fi ci lia ri a (h er ri ch -s ch äf fe r, 1 85 5) x 19 -v 24 -v ii n ig ht 19 2 cy cl op ho ra p or at a (l in n ae u s, 1 76 7) x 12 -i x 14 -i x n ig ht 19 3 cy cl op ho ra s up pu nc ta ri a (z el le r, 1 84 7) x 20 -v ii 14 -i x n ig ht 19 4 rh od om et ra s ac ra ri a (l in n ae u s, 1 76 7) x x 30 -v ii 15 -x d ay n ig ht 19 5 ly th ri a pu rp ur ar ia (l in n ae u s, 1 75 8) x 8vi i 30 -v ii n ig ht 19 6 o rt ho na m a ob st ip at a (f ab ri ci u s, 1 79 4) x x 31 -v ii 12 -x n ig ht 19 7 ca ta rh oe ru bi da ta (d en is & s ch if fe rm ü ll er , 1 77 5) x 18 -v 27 -v ii i n ig ht 19 8 ep ir rh oe a lte rn at a (o .f .m ü ll er , 1 76 4) x x 19 -v 15 -x n ig ht 19 9 ep ir rh oe g al ia ta (d en is & s ch if fe rm ü ll er , 1 77 5) x 5vi -2 00 6 n ig ht 20 0 co sm or ho e oc el la ta (l in n ae u s, 1 75 8) x 5vi -2 00 6 n ig ht 20 1 n eb ul a ab lu ta ri a (b o is d u va l, 1 84 0) x 8x 12 -x n ig ht 20 2 ec lip to pe ra s ila ce at a (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v ii -2 00 9 n ig ht 20 3 h or is m e vi ta lb at a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 22 -v ii n ig ht 20 4 h or is m e te rs at a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 29 -v 7vi n ig ht 4) 20 5 eu ph yi a fr us ta ta (t re it sc h ke , 1 82 8) x 8vi ii -2 00 3 n ig ht 20 6 pe ri zo m a bi fa ci at a (h aw o rt h , 1 80 9) x 13 -i x20 06 n ig ht 20 7 g ym no sc el is ru fi fa sc ia ta (h aw o rt h , 1 80 9) x x 25 -v 1x n ig ht 20 8 ch lo ro cl ys ti s vat a (h aw o rt h , 1 80 9) x 5vi 16 -v i n ig ht 20 9 eu pi th ec ia h aw or th ia ta d o u bl ed ay , 1 85 6 x x 18 -v 21 -v i n ig ht 21 0 eu pi th ec ia in tu rb at a (h ü bn er , 1 81 7) x 12 -i x20 06 n ig ht 21 1 eu pi th ec ia d od on ea ta g u en èe , 1 85 8 x x 16 -v 24 -v n ig ht 4) 21 2 eu pi th ec ia c en ta ur ea ta (d en is & s ch if fe rm ü ll er , 1 77 5) x x 20 -v ii 14 -i x n ig ht 21 3 eu pi th ec ia p au xi lla ri a bo is d u va l, 1 84 0 x 17 -i x20 06 n ig ht n ot od on tid ae 21 4 th au m et op oe a pi ty oc am pa (d en is & s ch if fe rm ü ll er , 1 77 5) x x 31 -v ii 6vi ii n ig ht 21 5 cl os te ra c ur tu la ( li n n ae u s, 1 75 8) x 20 -v ii -2 00 7 n ig ht 21 6 cl os te ra p ig ra (h u fn ag el , 1 76 6) x 30 -v ii -2 00 1 n ig ht 21 7 ce ru ra v in ul a (l in n ae u s, 1 75 8) x 18 -v -2 00 6 n ig ht 21 8 fu rc ul a fu rc ul a (c le rc k, 1 75 9) x x 6vi ii 13 -i x n ig ht 21 9 n ot od on ta tr it op hu s (d en is & s ch if fe rm ü ll er ), 1 77 5) x 20 -v ii -2 00 7 n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [journal of entomological and acarological research 2012; 44:e14] [page 69] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 22 0 n ot od on ta z ic za c (l in n ae u s, 1 75 8) x 31 -v ii -2 00 9 n ig ht 22 1 d ry m on ia d od on ae a (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v 24 -v n ig ht 22 2 d ry m on ia v el it ar is (h u fn ag el , 1 76 6) x 16 -v -2 00 6 n ig ht 22 3 ph eo si a tr em ul a (c le rc k, 1 75 9) x x 15 -v 27 -i x n ig ht 22 4 pt er os to m a pa lp in a (c le rc k, 1 75 9) x 18 -v 27 -v ii i n ig ht 22 5 pt ilo do n cu cu lli na (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v 12 -i x n ig ht 22 6 pe ri de a an ce ps (g o ez e, 1 78 1) x 16 -v -2 00 6 n ig ht 22 7 st au ro pu s fa gi (l in n ae u s, 1 75 8) x x 19 -v 31 -v ii n ig ht 22 8 h ar py ia m ilh au se ri (f ab ri ci u s, 1 77 5) x 16 -v i 3vi ii n ig ht 22 9 sp at al ia a rg en ti na ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 4vi ii n ig ht n oc tu id ae 23 0 ac ro ni ct a ps i( li n n ae u s, 1 75 8) x 22 -v ii -2 01 0 n ig ht 4) 23 1 ac ro ni ct a m eg ac ep ha la (d en is & s ch if fe rm ü ll er , 1 77 5) x x 18 -v 21 -v i n ig ht 23 2 ac ro ni ct a au ri co m a (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v ii 31 -v ii n ig ht 23 3 ac ro ni ct a ru m ic is (l in n ae u s, 1 75 8) x x 6vi i 8x n ig ht 23 4 cr an io ph or a lig us tr i ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 22 -i x n ig ht 23 5 cr yp hi a al ga e (f ab ri ci u s, 1 77 5) x x 24 -v ii 17 -i x n ig ht 4) 23 6 cr yp hi a oc hs i b o u rs in , 1 94 0 x x 22 -v ii 4vi ii n ig ht 4) 23 7 cr yp hi a ra pt ri cu la ( d en is & s ch if fe rm ü ll er , 1 77 5) x 4vi ii -2 01 0 n ig ht 23 8 cr yp hi a m ur al is ( fo rs te r, 1 77 1) x 22 -v ii 27 -v ii i n ig ht 23 9 id ia c al va ri a (d en is & s ch if fe rm ü ll er , 1 77 5) x 29 -v -2 00 5 n ig ht 24 0 pa ra co la x tr is ta lis (f ab ri ci u s, 1 79 4) x 16 -v i 14 -i x n ig ht 24 1 m ac ro ch ilo c ri br um al is ( h ü bn er , 1 79 3) x 28 -v 27 -v ii i n ig ht 24 2 h er m in ia g ri se al is (d en is & s ch if fe rm ü ll er , 1 77 5) x 8x20 06 n ig ht 24 3 pe ch ip og o pl um ig er al is ( h ü bn er , 1 82 5) x x 24 -v 27 -i x n ig ht 24 4 za nc lo gn at ha z el le ra lis (w o ck e, 1 85 0) x 6vi ii -1 98 8 n ig ht 24 5 za nc lo gn at ha ta rs ip en na lis tr ei ts ch ke , 1 83 5 x 13 -v i20 06 n ig ht 24 6 ca to ca la n up ta (l in n ae u s, 1 76 7) x x 17 -i x 6x n ig ht 24 7 ca to ca la e lo ca ta ( es pe r, 1 78 7) x x 13 -i x 14 -i x n ig ht 24 8 ca to ca la p ue rp er a (g io rn a, 1 79 1) x 22 -v ii 27 -v ii i n ig ht 5) 24 9 ca to ca la p ro m is sa (d en is & s ch if fe rm ü ll er , 1 77 5) x 4vi ii -2 01 0 n ig ht 25 0 ca to ca la e le ct a (v ie w eg , 1 79 0) x 16 -v ii 22 -v ii n ig ht 25 1 ca to ca la c on ju nc ta (e sp er , 1 78 7) x 21 -i x20 06 n ig ht 25 2 ca to ca la lu pi na h er ri ch -s ch äf fe r, 1 85 1 x 22 -v ii -2 01 0 n ig ht 25 3 ca to ca la n ym ph ag og a (e sp er , 1 78 7) x 8vi i19 94 n ig ht 25 4 ca to ca la n ym ph ae a (e sp er , 1 78 7) x 16 -v ii -2 00 9 n ig ht 25 5 m in uc ia lu na ri s (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v 25 -v n ig ht 25 6 cl yt ie il lu na ri s (h ü bn er , 1 81 3) x 22 -v ii 8vi ii n ig ht 25 7 o ph iu sa ti rh ac a (c ra m er , 1 77 3) x 31 -v ii -2 00 9 n ig ht 25 8 d ys go ni a al gi ra (l in n ae u s, 1 76 7) x x x 18 -v 13 -i x n ig ht 25 9 g ra m m od es b if as ci at a (p et ag n a, 1 78 7) x x x 13 -v ii 22 -i x n ig ht 10 ) 26 0 d ra st er ia c ai lin o (l ef èb vr e, 1 82 7) x 18 -v 3vi ii n ig ht 26 1 ly ge ph ila c ra cc ae (d en is & s ch if fe rm ü le r, 1 77 5) x 5vi 12 -x n ig ht 26 2 ly ge ph ila p ro ca x (h ü bn er , 1 81 3) x 16 -v 12 -x n ig ht 26 3 ca te ph ia a lc hy m is ta (d en is & s ch if fe rm ü ll er , 1 77 5) x x 5vi 4vi ii n ig ht 26 4 ae di a le uc om el as (l in n ae u s, 1 75 8) x x 19 -v 27 -i x n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [page 70] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 26 5 ty ta lu ct uo sa (d en is & s ch if fe rm ü ll er , 1 77 5) x x x 29 -v 17 -i x n ig ht 26 6 eu cl id ia g ly ph ic a (l in n ae u s, 1 75 8) x 28 -v -1 98 9 d ay 26 7 la sp ey ri a fle xu la (d en is & s ch if fe rm ü ll er , 1 77 5) x x 18 -v 27 -i x n ig ht 26 8 h yp en a pr ob os ci da lis (l in n ae u s, 1 75 8) x 22 -v ii 15 -x n ig ht 26 9 h yp en a ro st ra lis ( li n n ae u s, 1 75 8) x 20 06 6) 27 0 ri vu la s er ic ea lis (s co po li , 1 76 3) x 18 -v 6vi ii n ig ht 27 1 co lo bo ch yl a sa lic al is (d en is & s ch if fe rm ü ll er , 1 77 5) x x 18 -v 3vi ii n ig ht 27 2 ze be eb a fa ls al is (h er ri ch -s ch äf fe r, 1 83 9) x 19 -v 1x n ig ht 27 3 eu te lia a du la tr ix ( h ü bn er , 1 81 3) x 7vi -2 00 5 n ig ht 27 4 d ia ch ry si a st en oc hr ys is (w ar re n , 1 91 3) x 5vi 24 -v ii d ay n ig ht 4) 1 0) 27 5 m ac du nn ou gh ia c on fu sa (s te ph en s, 1 85 0) x 8vi i 15 -x n ig ht 27 6 au to gr ap ha g am m a (l in n ae u s, 1 75 8) x x 19 -v 15 -x n ig ht 27 7 tr ic ho pl us ia n i( h ü bn er , 1 80 3) x x 25 -v 6vi ii n ig ht 27 8 ch ry so de ix is c ha lc it es (e sp er , 1 78 9) x x 30 -v ii 22 -i x n ig ht 27 9 ab ro st ol a tr ip la si a (l in n ae u s, 1 75 8) x 16 -v -2 00 6 n ig ht 28 0 ab ro st ol a ag no ri st a d u fa y, 1 95 6 x 14 -i x20 06 n ig ht 4) 28 1 em m el ia tr ab ea lis (s co po li , 1 76 3) x x x 6vi i 17 -i x n ig ht 28 2 ac on ti a lu ci da (h u fn ag el , 1 76 6) x x 7vi 13 -v ii n ig ht 28 3 ph yl lo ph ila o bl it er at a (r am bu r, 1 83 3) x 3vi ii -2 00 8 n ig ht 10 ) 28 4 pr ot od el to te p yg ar ga ( h u fn ag el , 1 76 6) x x 15 -v 27 -i x n ig ht 28 5 o di ce s ua va (h ü bn er , 1 81 3) x 6vi i20 08 n ig ht 28 6 eu bl em m a vi ri du la ( g u en ée ) x 22 -v ii 15 -i x n ig ht 28 7 xa nt ho de s al ba go (f ab ri ci u s, 1 79 4) x 21 -i x20 06 n ig ht 28 8 am ph ip yr a py ra m id ea (l in n ae u s, 1 75 8) x x 22 -v ii 12 -i x n ig ht 4) 28 9 am ph ip yr a be rb er a ru n g s, 1 94 9 x 16 -v ii -2 00 9 n ig ht 4) 1 0) 29 0 am ph ip yr a tr ag op og in is (c le rc k, 1 75 9) x 12 -i x20 06 n ig ht 29 1 sy nt hy m ia fi xa ( fa br ic iu s, 1 78 7) x 16 -v 24 -v n ig ht 29 2 h el io th is v ir ip la ca ( h u fn ag el , 1 76 6) x 29 -v -2 00 5 n ig ht 29 3 h el io th is p el ti ge ra (d en is & s ch if fe rm ü ll er , 1 77 5) x 25 -v 8x n ig ht 29 4 h el io th is n ub ig er a h er ri ch -s ch äf fe r, 1 85 1 x 24 -v 27 -i x n ig ht 10 ) 29 5 h el ic ov er pa a rm ig er a (h ü bn er , 1 80 8) x x 5vi 6x n ig ht 29 6 el ap hr ia v en us tu la (h ü bn er , 1 79 0) x x 18 -v 27 -v ii i n ig ht 29 7 st ilb ia fa ill ae pü n g el er , 1 91 8 x 8x 12 -x n ig ht 29 8 ca ra dr in a se lin ib o is d u va l, 1 84 0 x x 29 -v 27 -i x n ig ht 4) 29 9 ca ra dr in a cl av ip al pi s (s co po li , 1 76 3) x x 8vi i 17 -i x n ig ht 4) 30 0 ca ra dr in a fla vi re na (g u en ée , 1 85 2) x x 19 -v 17 -i x n ig ht 4) 30 1 ca ra dr in a vi ci na st au d in g er , 1 87 0 x 22 -v ii 8vi ii n ig ht 10 ) 30 2 h op lo dr in a re sp er sa ( d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v ii -2 00 9 n ig ht 30 3 h op lo dr in a am bi gu a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 28 -v 8x n ig ht 7) 30 4 ch ar an yc a tr ig ra m m ic a (h u fn ag el , 1 76 6) x x 15 -v 24 -v n ig ht 30 5 sp od op te ra e xi gu a (h ü bn er , 1 80 8) x x 22 -v ii 12 -x n ig ht 30 6 se sa m ia n on ag ri oi de s (l ef èb vr e, 1 82 7) x x x 18 -v 15 -x n ig ht 4) 30 7 se sa m ia c re ti ca l ed er er , 1 85 7 x 30 -v ii 6vi ii n ig ht 30 8 pr ox en us h os pe s (f re ye r, 1 83 1) x x 15 -v 17 -i x n ig ht 30 9 d yp te ry gi a sc ab ri us cu la (l in n ae u s, 1 75 8) x x 5vi 24 -v ii n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [journal of entomological and acarological research 2012; 44:e14] [page 71] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 31 0 po ly ph ae ni s se ri ca ta (e sp er , 1 78 7) x 6vi i20 08 n ig ht 31 1 th al po ph ila m at ur a (h u fn ag el , 1 76 6) x 17 -i x 3x n ig ht 31 2 tr ac he a at ri pl ic is ( li n n ae u s, 1 75 8) x 12 -v i20 06 n ig ht 31 3 eu pl ex ia lu ci pa ra ( li n n ae u s, 1 75 8) x 24 -v 13 -v i n ig ht 31 4 ph lo go ph or a m et ic ul os a (l in n ae u s, 1 75 8) x 27 -v ii i19 92 n ig ht 31 5 ca llo pi st ri a ju ve nt in a (s to ll , 1 78 2) x 12 -v i 27 -i x n ig ht 31 6 m et ho ra sa la tr ei lle i( d u po n ch el , 1 82 7) x 31 -v ii -2 00 7 n ig ht 31 7 ip im or ph a re tu sa (l in n ae u s, 1 76 1) x 8vi i 3vi ii n ig ht 31 8 pa ra st ic ht is y ps ill on (d en is & s ch if fe rm ü ll er , 1 77 5) x 5vi 12 -v i n ig ht 31 9 m es og on a ox al in a (h ü bn er , 1 80 3) x 3x20 06 n ig ht 32 0 co sm ia d if fi ni s (l in n ae u s, 1 76 7) x 22 -v ii -2 01 0 n ig ht 32 1 co sm ia a ff in is (l in n ae u s, 1 76 7) x 8vi i19 94 n ig ht 32 2 co sm ia p yr al in a (d en is & s ch if fe rm ü ll er , 1 77 5) x 16 -v ii -2 00 9 n ig ht 32 3 xa nt hi a oc el la ri s (b o rk h au se n , 1 79 2) x x 16 -v ii 12 -i x n ig ht 10 ) 32 4 co ni st ra ru bi gi ne a (d en is & s ch if fe rm ü ll er , 1 77 5) x 12 -i x20 06 n ig ht 32 5 ep is em a gl au ci na (e sp er , 1 78 9) x 15 -x -1 98 9 n ig ht 32 6 m eg an ep hr ia b im ac ul os a (l in n ae u s, 1 76 7) x 8x20 06 n ig ht 32 7 d ry ob ot od es m on oc hr om a (e sp er , 1 79 0) x 22 -i x20 06 n ig ht 4) 32 8 d ry ob ot od es c ar bo ni s (f .w ag n er , 1 93 1) x 6x20 06 n ig ht 4) 32 9 tr ig on op ho ra fl am m ea (e sp er , 1 78 5) x 15 -x -1 98 9 n ig ht 33 0 m ni ot yp e so lie ri ( bo is d u va l, 1 84 0) x 21 -i x 8x n ig ht 33 1 o lig ia la tr un cu la (d en is & s ch if fe rm ü ll er , 1 77 5) x x 15 -v 7vi n ig ht 4) 33 2 m es ol ig ia fu ru nc ul a (d en is & s ch if fe rm ü ll er , 1 77 5) x 30 -v ii 27 -v ii i n ig ht 33 3 m es ap am ea s ec al is ( li n n ae u s, 1 75 8) x 5vi 22 -i x n ig ht 4) 33 4 m es ap am ea d id ym a (e sp er , 1 78 8) x x 8vi i 13 -i x n ig ht 4) 33 5 lu pe ri na d um er ili i( d u po n ch el , 1 82 6) x x 17 -i x 27 -i x n ig ht 33 6 n on ag ri a ty ph ae (t h u n be rg , 1 78 4) x 6vi ii -1 98 8 n ig ht 33 7 ar ch an ar a di ss ol ut a (t re it sc h ke , 1 82 5) x 6vi i 30 -v ii n ig ht 10 ) 33 8 ar ch an ar a sp ar ga ni i( es pe r, 1 79 0) x x 20 -v ii 6vi ii n ig ht 33 9 la ca no bi a w -la ti nu m ( h u fn ag el , 1 76 6) x 15 -v 29 -v n ig ht 34 0 la ca no bi a ol er ac ea ( li n n ae u s, 1 75 8) x 22 -v ii 27 -v ii i n ig ht 34 1 ae th er ia b ic ol or at a (h u fn ag el , 1 76 6) x x 6vi 31 -v ii n ig ht 34 2 h ad en a lu te ag o (d en is & s ch if fe rm ü ll er , 1 77 5) x 29 -v -2 00 5 n ig ht 34 3 h ad en a pe rp le xa (d en is & s ch if fe rm ü ll er , 1 77 5) x 13 -i x20 06 n ig ht 34 4 m yt hi m na fe rr ag o (f ab ri ci u s, 1 78 7) x x 16 -v ii 6x n ig ht 34 5 m yt hi m na a lb ip un ct a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 18 -v 15 -x n ig ht 34 6 m yt hi m na v it el lin a (h ü bn er , 1 80 8) x x 18 -v 6vi ii n ig ht 34 7 m yt hi m na s tr am in ea ( tr ei ts ch ke , 1 82 5) x x 28 -v 27 -v ii i n ig ht 34 8 m yt hi m na o bs ol et a (h ü bn er , 1 80 3) x x 15 -v 27 -v ii i d ay n ig ht 34 9 m yt hi m na z ea e (d u po n ch el , 1 82 7) x 28 -v 26 -v ii n ig ht 35 0 m yt hi m na jo an ni si bo u rs in & r u n g s, 1 95 2 x 22 -i x 27 -i x n ig ht 10 ) 35 1 m yt hi m na c on gr ua (h ü bn er , 1 81 7) x x 15 -v 15 -x n ig ht 35 2 m yt hi m na lal bu m (l in n ae u s, 1 76 7) x x 15 -v 15 -x n ig ht 35 3 m yt hi m na s ic ul a (t re it sc h ke , 1 83 5) x 15 -v 15 -x n ig ht 35 4 m yt hi m na ri pa ri a (r am bu r, 1 82 9) x x 15 -v 15 -x n ig ht 35 5 m yt hi m na lo re yi (d u po n ch el , 1 82 7) x 6vi ii -1 98 8 n ig ht 35 6 m yt hi m na u ni pu nc ta (h aw o rt h , 1 80 9) x 6vi i 15 -x n ig ht t o b e co n ti n u ed o n n ex t p ag e. article [page 72] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly t ab le 1 . c o n ti n u ed f ro m p re vi o u s p ag e. fa m il y s er ia l s pe ci es lo ca li ty d at es o f fl ig ht t im e ti m e of o bs er ve d fo ot no te no . i ii ii i fr om to ac ti vi ty 35 7 la si on yc ta c al be rla i( st au d in g er , 1 88 3) x x 16 -v 22 -i x n ig ht 35 8 ax yl ia p ut ri s (l in n ae u s, 1 76 1) x x 15 -v 27 -v ii i n ig ht 35 9 o ch ro pl eu ra p le ct a (l in n ae u s, 1 76 1) x x 15 -v 27 -v ii i n ig ht 36 0 n oc tu a pr on ub a li n n ae u s, 1 75 8 x x 19 -v 12 -x n ig ht 36 1 n oc tu a co m es h ü bn er , 1 81 3 x x 21 -i x 15 -x n ig ht 36 2 n oc tu a fi m br ia ta ( sc h re be r, 1 75 9) x x 6vi ii 1x n ig ht 36 3 n oc tu a ti rr en ic a bi eb in g er , s pe id el & h an ig k, 1 98 3 x x 6vi i 3x n ig ht 36 4 n oc tu a ja nt hi na ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 12 -v i 22 -i x n ig ht 4) 36 5 n oc tu a ja nt he (b o rk h au se n , 1 79 2) x x 13 -i x 12 -x n ig ht 4) 36 6 n oc tu a in te rje ct a h ü bn er , 1 80 3 x 6vi i20 08 n ig ht 36 7 ep ile ct a lin og ri se a (d en is & s ch if fe rm ü ll er , 1 77 5) x 24 -v ii 14 -i x n ig ht 36 8 xe st ia c -n ig ru m (l in n ae u s, 1 75 8) x x 16 -v 15 -x n ig ht 36 9 xe st ia c as ta ne a (e sp er , 1 79 8) x 22 -i x 12 -x n ig ht 37 0 xe st ia x an th og ra ph a (d en is & s ch if fe rm ü ll er , 1 77 5) x x 22 -i x 12 -x n ig ht 37 1 pe ri dr om a sa uc ia (h ü bn er , 1 80 8) x 15 -x -1 98 9 n ig ht 37 2 ag ro ti s pu ta (h ü bn er , 1 80 3) x x 11 -i x 27 -i x n ig ht 37 3 ag ro ti s ip si lo n (h u fn ag el , 1 76 6) x x 5vi 15 -x n ig ht 37 4 ag ro ti s ex cl am at io ni s (l in n ae u s, 1 75 8) x x 16 -v 27 -v ii i n ig ht 37 5 ag ro ti s cl av is (h u fn ag el , 1 76 6) x 30 -v ii -2 00 1 n ig ht 37 6 ag ro ti s se ge tu m (d en is & s ch if fe rm ü ll er , 1 77 5) x x 14 -i x 15 -x n ig ht ly m an tr iid ae 37 7 ly m an tr ia d is pa r( li n n ae u s, 1 75 8) x x 8vi i 6vi ii n ig ht 37 8 o cn er ia le de re ri (m il li èr e, 1 86 8) x 6vi i20 08 n ig ht 10 ) n ol id ae 37 9 m eg an ol a st ri gu la ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 16 -v 1x n ig ht 38 0 m eg an ol a al bu la ( d en is & s ch if fe rm ü ll er , 1 77 5) x x 29 -v 27 -v ii i n ig ht 38 1 n ol a cu cu la te lla ( li n n ae u s, 1 75 8) x 6vi ii -1 98 8 n ig ht 38 2 n ol a ae ru gu la (h ü bn er , 1 79 3) x 6vi ii -1 98 8 n ig ht 38 3 n yc te ol a re va ya na (s co po li , 1 77 2) x x 13 -v i 16 -v ii n ig ht 38 4 n yc te ol a si cu la na (f u ch s, 1 89 9) x 3vi ii -2 00 8 n ig ht 10 ) 38 5 ps eu do ip s pr as in an a (l in n ae u s, 1 75 8) x 7vi -2 00 5 n ig ht 38 6 be na b ic ol or an a (f u es sl y, 1 77 5) x 19 -v 14 -i x n ig ht 38 7 ea ri as c lo ra na (l in n ae u s, 1 76 1) x 29 -v 27 -v ii i n ig ht 38 8 ea ri as v er na na (f ab ri ci u s, 1 78 7) x x 18 -v 3vi ii n ig ht ar ct iid ae 38 9 pe lo si a m us ce rd a (h u fn ag el , 1 76 6) x x 12 -v i 27 -v ii i n ig ht 39 0 li th os ia q ua dr a (l in n ae u s, 1 75 8) x x 5vi 22 -i x n ig ht 39 1 ei le m a co m pl an a (l in n ae u s, 1 75 8) x 24 -v ii -1 98 9 n ig ht 39 2 ei le m a ca ni ol a (h ü bn er , 1 80 8) x x x 16 -v 15 -x n ig ht 39 3 d ys au xe s fa m ul a (f re ye r, 1 83 6) x x 24 -v 12 -x n ig ht 39 4 sp ir is s tr ia ta (l in n ae u s, 1 75 8) x 24 -v 28 -v d ay 39 5 ph ra gm at ob ia fu lig in os a (l in n ae u s, 1 75 8) x x 15 -v 12 -x n ig ht 39 6 cy m ba lo ph or a pu di ca (e sp er , 1 78 4) x 27 -i x20 06 n ig ht 39 7 sp ilo so m a lu te a (h u fn ag el , 1 76 6) x 29 -v -2 00 5 n ig ht 39 8 sp ilo so m a lu br ic ip ed a (l in n ae u s, 1 75 8) x x 24 -v 27 -v ii i n ig ht 39 9 rh yp ar ia p ur pu ra ta (l in n ae u s, 1 75 8) x 5vi -2 00 6 d ay 40 0 ar ct ia v ill ic a (l in n ae u s, 1 75 8) x x 15 -v 5vi n ig ht 40 1 eu pl ag ia q ua dr ip un ct ar ia (p o d a, 1 76 1) x x 27 -v ii i 22 -i x n ig ht 1) p he ro m on e ca tc h; 2 ) an ot he r sp ec im en w as fo un d de ad in si de th e ad m in is tr at io n bu ild in g in 2 00 4; 3 ) at tr ac te d by li gh t; 4) g en ita lia d is se ct ed ; 5 ) th e sp ec im en o f 2 7t h au gu st w as fo un d du ri ng d ay ti m e in 1 99 2 po se d on th e ou ts id e w al l o f t he a dm in is tr at io n bu ild in g; 6 ) th e re co rd w as a s pe ci m en fo un d de ad in si de th e ad m in is tr at io n bu ild in g; 7 ) sp ec im en s pr ev io us ly r ep or te d as h . b la nd a in 1 98 9 ha ve n ow b ee n de te rm in ed a s h . a m bi gu a; 8 ) fi rs t r ec or d fo r th e ap en ni ne p en in su la ; 9 ) fi rs t r ec or d fo r th e it al ia n m ai nl an d; 1 0) n ew fo r th e ab ru zz o re gi on ; w ) sp ec ie s pa ss ed to a . w er no fo r id en tif ic at io n. article [journal of entomological and acarological research 2012; 44:e14] [page 73] no nco mm er cia l u se on ly the investigation area the lower sangro valley is located in the chieti province of abruzzo in central italy (figure 1). it consists of a 20 km long by 2.5 km wide plain, stretching between the estuary of the river aventino into the river sangro and the estuary of the river sangro into the adriatic sea. today, the area is mainly used for agriculture. extensive industrial and commercial areas have also been developed. housing developments are only located along the perimeter. natural areas are only to be found in the immediate vicinity of the river and in the two nature reserves. the oasi di serranella has been described by zahm (1989) (figure 2). it is an area of 180 ha where the estuary of aventino river enters the sangro river. approximately 90 ha of water surface are dammed up to 90 m above sea level. the protected area essentially consists of extensive wetlands, within which a number of microhabitats with their respective vegetation can be distinguished. potamogeton species are typically present in the areas of flowing water. phragmites communis and several typha species grow in large stands of reeds. two wide strips of floodplain forests continue along the perimeter of the marshland with alnus glutinosa, a number of salix and populus species, as well as quercus robur in the drier areas. mediterranean plants grow on a xeric plateau e.g. spartium junceum, helichrysum italicum and cistus incanus. the lecceta di torino di sangro has been described by di fabrizio (2004) (figure 3). the 170 ha of the natural reserve extend from the banks of the sangro river near its estuary up and across the slopes of the costal hills (2-60 m above sea level) and down the slopes facing the coast. it is one of the last remains of the original adriatic coast forests. although called lecceta (forest of quercus ilex) it is actually a mixed deciduous forest with a dominating number of quercus ilex. co-occurring species include quercus pubescens, q. cerris, carpinus orientalis, coronilla emerus, acer neapolitanum, crataegus monogyna and ligustrum vulgare. the undergrowth is for the most part very dense, almost impenetrable. hedera helix, smilax aspera and ruscus aculeatus are well represented in the forest and brushwood area. coastal steppes and mediterranean macchia spread in open areas with cistus creticus and pistacia lentiscus. typical riverbank tree species, e.g. salix and populus species, grow along the river sangro. results the first results of the oasi di serranella (1988-1989) were published in 1989. they have been integrated in this final study in order to provide a comprehensive overview. it is unknown to the author whether other entomologists have made any recordings or published any reports on the area of investigation and, therefore, no additional data have been included. a total of 1878 single observations were recorded over many years of investigation. the study recorded 401 species, 15 of which were first recorded for the abruzzo region, one was new for the italian mainland and one for the apennine peninsula. table 2 shows the distribution of records and species of each of the three groups microlepidoptera, rhopalocera and macroheterocera. the investigation of the individual areas resulted in the following number of species: oasi di serranella 287, lecceta di torino di sangro 253, coastal area 12. the individual recorded species are shown in table 1. localities, observed flight times and observed times of activity are reported as well as the associated families. presentation of results systematics and nomenclature follow karsholt & razowski (1996) and with regards to geometridae (hausmann, 2001, 2004; mironov, 2003) as published so far. results from three localities are presented: i oasi di serranella; ii lecceta di torino di sangro; iii coastal area of the sangro valley (table 1). only the earliest and latest observations are shown. no year is given since these periods of flight are independent of year of observation. one-off observations are also listed. for reasons of clarity, author names and publication years are not presented in the text. the following 17 species are also shown in the appendix. phtheochroa ingridae (figures a1 and a5) was only recognized as an independent species and distinguished from p. rugosana in 1990 by huemer. the species is endemic for italy. the holotype come from the kalterer see (trentino-alto adige region), the paratypes come from the same region and from the veneto region. trematerra (2003) only mentions these two regions as distribution area. the 3 females from the lecceta di torino di sangro match the description and figures of these types in terms of habitus as well as genitalia. therefore, their discovtable 2. average number of records per species in each of the groups. groups records species average microlepidoptera 89 63 1.41 rhopalocera 100 44 2.27 macroheterocera 1689 294 5.74 total 1878 401 4.68 figure 1. geographical map of italy with indication of the lower sangro valley. article [page 74] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly ery on 7th may 2006 has been the first and only record of the species on the apennine peninsula to date. within italy, herculia fulvocilialis (figure a2) has only been mentioned in sicily by bassi et al. (1995), speidel (1996) and by slamka (2006). therefore, the species can be considered new for the italian mainland. gegenes nostrodamus (figures a4, a6) is very similar to g. pumilio. length of forewings and underside of wings, in particular of hindwings, provide enough evidence for a clear determination of the collected specimen. according to parenzan and porcelli (2005-2006), the species is also present in all remaining regions of the peninsula. idaea ostrinaria (figure a3), a pretty thermophilic sterrhinae, was also found by the author on the southeast slope of the majella mountain, in the orfento valley, and on the gran sasso mountain. this species was also mentioned by parenzan and porcelli (2005-2006) for all remaining regions of the peninsula. scopula nigropunctata (figure a3) appears to be more common and more widely distributed in the abruzzo region than was previously thought considering the fact that to date it has not been mentioned in literature for this region. it has also been found by the author at several locations on the majella mountain and in francavilla, and by dell’agata (grassi et al., 2003-2005, unpublished data) on the gran sasso mountain. scopula emutaria (figure a3) has also been found in francavilla together with s. nigropunctata. grammodes bifasciata (figure a3) is a tropical and subtropical species whose distribution area also includes southern europe. diachrysia stenochrysis (syn. tutti) (figures a3 and a7) and d. chrysitis often show similarities in forewing drawing, as well as in the genital structure in males, so that a clear determination is not always possible. however, in the case of the 2 available examples the features were unambiguous. apart from the single find of phyllophila obliterata (figure a3) the author could not obtain any further recordings in the abruzzo region. recordings of amphipyra berbera, heliothis nubigera, caradrina vicina and nycteola siculana (figure a3, a. berbera also figure a8) have also been made for the majella mountain. the latter has also been located on the gran sasso mountain by grassi et al. (2003-2005, unpublished data). nomenclature of c. vicina differs from nowacki & fibiger (1996) who used the genus eremodrina that has subsequently been down-graded to subgenus level by hacker (2004). xanthia ocellaris (figure a3), a very variable species, is one of the less frequent autumn species of the genus xanthia. apart from the 2 examples from the investigation area, the author could not obtain any further recordings from the abruzzo region. archanara dissoluta (figure a3) lives in wet habitats with stands of reeds where its caterpillars develop in the stems of phragmites species. the author obtained one further recording for the abruzzo region on the lower slopes of the majella mountain on the banks of the river aventino near pianimarini. mythimna joannisi(figure a3) is a species of pan-african distribution that is also found on the major western mediterranean islands, reaching continental europe on the southern and eastern coast of iberia and in southern italy. while it was discovered in lazio on the tyrrhenian side of the apennine peninsula, the species has not previously been detected on the adriatic side beyond apulia. its habitat is the reed areas near the coast where its feeding plant phragmites species grows. ocneria ledereri (figure a3) appears to be a rare species in the abruzzo region. apart from this single specimen, the author could not obtain any further recordings. conclusions this study contributes valuable information to widen our knowledge of the occurrence and distribution of the lepidoptera of central italy. fifteen species are published for the abruzzo region for the first time; 2 species are new for the italian peninsula. references bassi g., passerin d’entrèves p., speidel w., zangheri s., 1995 lepidoptera pyraloidea. in: minelli a., ruffo s., la posta s. checklist delle specie della fauna italiana. band 87 calderini, bologna. di fabrizio f., 2004 aree protette d’abruzzo. cogecstre, penne. fibiger m., (ed.), 1990 2010 noctuidae europaeae, vol. 1-12. entomological press, soro. hacker h., 2004 revision of the genus caradrina ochsenheimer, 1816, with notes on other genera of the tribus caradrina (lepidoptera, noctuidae). esperiana 10. pemberley natural history books ba, iver, bucks. figure 2. detail of riserva naturale oasi di serranella (photo m. pellegrini). figure 3. detail of riserva naturale lecceta di torino di sangro (photo m. pellegrini). article [journal of entomological and acarological research 2012; 44:e14] [page 75] no nco mm er cia l u se on ly article hausmann a., 2001 introduction, archiearinae, orthostixinae, desmobathrinae, alsophilinae, geometrinae. in: hausmann a., (ed.). the geometrid moths of europe, vol. 1. apollo books, stenstrup: 1-282. hausmann a., 2004 sterrhinae in: hausmann a., (ed.). the geometrid moths of europe, vol. 2. apollo books, stenstrup: 1-600. huemer p., 1990 eine neue phtheochroa-art aus norditalien (lepidoptera: tortricidae). nachr. bl. bayer. ent. 39: 82-87. karsholt o., razowski j., (eds.) 1996 – the lepidoptera of europe. a distributional checklist. – apollo books, stenstrup. mironov v., 2003 larentiinae ii (perizomini and eupitheciini) in: hausmann a., (ed.). the geometrid moths of europe, vol. 4. apollo books, stenstrup: 1-463. nowacki j., fibiger m., 1996 noctuidae. in: karsholt o., razowski j., (eds.). the lepidoptera of europe. apollo books, stenstrup: 251-293. parenzan p., porcelli f., 2005-2006 i macrolepidotteri italiane, fauna lepidopterorum italiae (macrolepidoptera). phytophaga xv: 5-393. parenzan p., porcelli f., 2006-2007 i macrolepidotteri italiane, fauna lepidopterorum italiae (macrolepidoptera). addenda et corrigenda i. entomologica 40: 153-221. prola c., provera p., racheli t., sbordoni v. 1978a i macrolepidotteri dell’appennino centrale. parte i. fragm. entomol. 14: 1-217. prola c., provera p., racheli t., sbordoni v. 1978b i macrolepidotteri dell’appennino centrale. parte ii. boll. ass. romana entomol. 32: 1-238. prola c., racheli t., 1979 i geometridi dell’italia centrale. parte i, oenochrominae, hemitheinae, sterrhinae, larentiinae (pars). boll. ist. entomol. univ. bologna 34: 191-246. prola c., racheli t., 1980 i geometridi dell’italia centrale. parte ii, larentiinae. boll. ist. entomol. univ. bologna 35: 29-108. sciarretta a., zahm n., 2002 i macrolepidotteri dell’ ‘abetina di rosello’ (abruzzo) con note faunistiche, biogeografiche ed ecologiche. phytophaga xii: 25-42. slamka f., 2006 pyraloidea europas, band i. bratislava (slowakei). speidel w. 1996. – pyralidae – in: karshholt o., razowski j. (eds.), the lepidoptera of europe. apollo books, stenstrup: 166-196. teobaldelli a., 1976 i macrolepidotteri del maceratese e dei monti sibillini (appennino umbro-marchigiano). note app. sperim. entomol. agr. 16: 81-346. trematerra p., 2003 catalogo dei lepidoptera tortricidae della fauna italiana: geonemia, distribuzione in italia, note biologiche, identificazione. boll. zool. agr. bachic. ser. ii 35(supp 1): 3-270. weigt h.j., 2009 eupithecia, monografie der blütenspanner mitteleuropas. – pdf-manuskript hans-joachim weigt. zahm n., 1989 lepidopteren aus der oasi di serranella (mittelitalien, abruzzen) (lepidoptera). boll. ass. romana entomol. 43: 1-9. [page 76] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly figure a1. phtheochroa ingridae (photo hinsberger). figure a3. 1st row from left: idaea ostrinaria, scopula nigropunctata, scopula emutaria. 2nd row from left: grammodes bifasciata, diachrysia stenochrysis, phyllophila obliterata. 3rd row from left: amphipyra berbera, heliothis nubigera, caradrina vicina. 4th row from left: xanthia ocellaris, archanara dissoluta, mythimna joannisi. 5th row from left: ocneria ledereri, nycteola siculana (photo hinsberger). figure a2. herculia fulvocilialis (photo hinsberger). figure a4. gegenes nostrodamus (photo hinsberger). appendix contribution to the knowledge of the lepidoptera fauna of the lower sangro valley in the abruzzo region of central italy appendix [journal of entomological and acarological research 2012; 44:e14] [page 77] no nco mm er cia l u se on ly figure a7. diachrysia stenochrysis male genitalia (photo müller). figure a8. amphipyra berbera male genitalia (photo müller). figure a5. phtheochroa ingridae female genitalia (photo müller). figure a6. gegenes nostrodamus male genitalia (photo müller). appendix [page 78] [journal of entomological and acarological research 2012; 44:e14] no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2015; 47:4838] [page 73] preliminary checklist of iranian mymarids (hymenoptera: chalcidoidea, mymaridae) h. lotfalizadeh department of plant protection, east-azarbaijan agricultural and natural resources research center, agricultural research, education and extension organization (areeo), tabriz, iran abstract twenty-seven species of mymaridae (hymenoptera: chalcidoidea) belonging to eight genera are recorded from iran: anagrus haliday (4 species), anaphes haliday (2 species), camoptoptera foerster (1 species), erythmelus enock (4 species), gonatocerus nees (10 species), mymar curtis (1 species), polynema haliday (3 species), and stephanodes enock (2 species). brief information on their known biology and hosts is provided. two genera, gonatocerus and anagrus, include about 80% of specimen composition. introduction the family mymaridae includes some of the smallest known insects: the combined lengths of three or even four adult individuals may not even equal 1 mm (annecke & doutt, 1961). more than 1400 species and 100 genera are known (noyes, 2013). this family is one of the most distinctive of the superfamily chalcidoidea, in having the antennal toruli quite far apart (3-5 times their own diameter), usually a reduced wing venation, and distinctive fringed wings (nikol’skaya, 1978). its members are abundant and easily collected using a variety of trapping methods. the hosts of mymaridae include eggs of hemiptera, psocoptera, coleoptera, orthoptera and some other insect orders (huber, 1986). but only about one quarter of the genera have hosts reported for them. as in the trichogrammatidae, several mymarids attack eggs of the aquatic insects. huber (1986) published a comprehensive review of the known hosts of the mymarids known to that date. a few species of mymarids have been responsible for biological control successes (lin et al., 2007). unfortunately, the fauna of this family has not been studied comprehensively in iran, so that only 11 species, namely anagrus atomus (l.), a. nigriceps (smits van burgst), erythmelus flavovarius (walker), e. israeliensis viggiani & jesu, e. panis (enock), e. rex (girault), gonatocerus litoralis (haliday), g. oxypygus foerster (as g. ovicenatus leonard & crosby), mymar taprobanicum ward, stephanodes reduvioli (perkins) and s. similis (foerster) were previously reported from iran (fallahzadeh & huber, 2011). fallahzadeh & huber (2011) listed 10 species from iran. later, triapitsyn (2013), haghayeghi-nosrati et al. (2013), bayegan et al. (2014) and haghayeghi-nosrati et al. (2014) added some new records and increased the number of known species from country. so, prior to present study, the total numbers of mymarid species recorded from iran were 20. the present contribution to their knowledge in iran is based mainly on new data and material accumulated in recent years by the author. also some biological and ecological studies were made by hesami et al. (2004, 2009), latifian & soleyman-nejadian (2009), and akbarzadehshoukat (1998). materials and methods the specimens were collected with a malaise trap during 2011-2013. samples were collected and labelled every 7-10 days, replacing alcohol in the collecting vessel. captures were made in different areas of iran including eastazarbaijan province: khosroshahr (n 37º 58’ 28” and e 46º 02’ 55”, elevation 1346 m). then specimens were sorted into vials with 70-80% ethyl alcohol. because of their small size, slide preparations of the entire or dissected specimens are needed, preferably using a permanent mounting medium as canada balsam, to see some of the distinguishing features. representatives from each morphospecies, both females and males, were slide-mounted into canada balsam using the technique described by noyes (1982) and modified for the mymaridae by dr. j. t. huber (pers. communication). approximately 350 slidecorrespondence: hossein lotfalizadeh, department of plant protection, eastazarbaijan agricultural and natural resources research center, agricultural research, education and extension organization (areeo), tabriz, iran. e-mail: hlotfalizadeh@gmail.com key words: hymenoptera; mymaridae; chalcidoidea; fauna; distribution; iran. acknowledgements: i would like to thank dr. triapitsyn and dr. huber for confirmation of identifications and sharing their experiences on preparation of slid. i am grateful to dr. triapitsyn for critical comments on the manuscript. received for publication: 9 january 2015. revision received: 14 june 2015. accepted for publication: 18 june 2015. ©copyright h. lotfalizadeh, 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:4838 doi:10.4081/jear.2015.4838 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:4838 [page 74] [journal of entomological and acarological research 2015; 47:4838] mounted and pinned specimens were examined during this study. identifications of genera were made from card-mounted specimens after drying from ethanol using hmds. identifications were made using the following references: huber (1986, 1988); huber & fidalgo (1997); baquero & jordana (2003); beardsley & huber (2000); matthews (1986); noyes & valentine (1989); peck et al. (1964); pricop (2010); soyka (1955, 1961); schauff (1984); triapitsyn (1978, 2002, 2006) and viggiani & jesu (1988). results list of species i-genus anagrus haliday, 1833 anagrus species parasitize mainly cicadellidae and delphacidae (huber et al., 2009). four species occur in iran while two species in the uae (huber et al., 2009), and seven species are recorded for yemen (huber et al., 2009). anagrus atomus (linnaeus, 1767) material examined: 4♂♂, east-azarbaijan, khosroshahr, 6.v.2012, h. lotfalizadeh. hesami et al. (2001) studied the morphology of a. atomus that was reared from the grape leafhopper, arboridia kermanshah dlabola (cicadellidae), in isfahan. this species has been also reported from eggs of other insects (chiappini, 1987; noyes, 2013). it is cosmopolitan and has been recorded from europe, north africa, middle east, central asia, north and south america (trjapitzin, 1978; triapitsyn & berezovskiy, 2004). anagrus atomus was reported from iran by hesami et al. (2001) and triapitsyn & berezovskiy (2004). anagrus nigriceps (smits van burgst, 1914) material examined: 4♀♀ and 1♂, east-azarbaijan, khosroshahr, 15.iii.2012, h. lotfalizadeh. this species was reported from europe, middle east, central asia, china and iran (triapitsyn & berezovskiy, 2004). anagrus avalae soyka, 1956 material examined: 20♀♀ and 17♂♂, east-azarbaijan, khosroshahr, 20.v.2012, h. lotfalizadeh. it is widely distributed in europe, north america, australia, new zealand and iran (triapitsyn & berezovskiy, 2004). triapitsyn & berezovskiy (2004) gave a list of known leafhopper hosts of this species. anagrus sp. material examined: 2♀♀ and 1♂, east-azarbaijan, khosroshahr, vii.2012, h. lotfalizadeh. ii-genus anaphes haliday, 1833 in the neighbouring countries to iran, one specimen of an unidentified species was collected in oman and one in the uae (huber et al., 2009), and also one species from syria and turkey (aeschlimann, 1986). this genus includes parasitoids of curculionidae or chrysomelidae and miridae (huber et al., 2009). some species are used as biological control agents (huber, 1992). species identification in anaphes is very difficult, and no good keys are available. anaphes sp. 1 material examined: 6♀♀ and 1♂♂, east-azarbaijan, khosroshahr, 1.ix.2012, h. lotfalizadeh. anaphes sp. 2 material examined: 1♂, east-azarbaijan, khosroshahr, 17.ix.2013, h. lotfalizadeh. iii-genus camptoptera foerster, 1856 only two specimens of one species were collected in iran; huber et al. (2009) reported a few specimens from yemen and oman. the genus was reviewed by huber (1999). camptoptera sp. material examined: 1♀, east-azarbaijan, khosroshahr, 1.xi.2010, h. lotfalizadeh. 1♀, same data, 12.x.2011. iv-genus erythmelus enock, 1909 this genus includes four species from two subgenera (erythmelus s. str. and parallelaptera enock) in iran while three species in the uae (huber et al., 2009), one species in iraq (triapitsyn, 2003), and two species in yemen (huber et al., 2009). erythmelus species parasitize miridae and tingidae. erythmelus flavovarius (walker, 1846) material examined: 1♀ and 5♂♂, east-azarbaijan, khosroshahr, 15.v.2013, h. lotfalizadeh. erythmelus israeliensis viggiani & jesu, 1985 this species was reported from karaj, alborz province, iran by triapitsyn (2003). erythmelus rex (girault, 1911) material examined: 1♀ and 3♂♂, east-azarbaijan, khosroshahr, 3.vi.2013, h. lotfalizadeh. erythmelus panis (enock, 1909) material examined: 1♀ and 4♂♂, east-azarbaijan, khosroshahr, 6.vi.2013, h. lotfalizadeh. this species belongs to the subgenus parallelaptera and is distributed in australia, europe, china, india (noyes, 2013) and iran: alborz (triapitsyn, 2003), west-azarbaijan (akbarzadeh-shoukat, 1998) and east-azarbaijan provinces. v-genus gonatocerus nees, 1834 gonatocerus, with seven known species in iran, is one of the most common genera in the country (haghayeghi-nosrati et al., 2013). this genus is divided into three subgenera: cosmocomoidea howard, lymaenon walker and gonatocerus nees ab esenbeck (triapitsyn et al., 2010; triapitsyn, 2013). among the collected species from iran two belong to cosmocomoidea (ater, oxypygus), one to gonatocerus (pictus) and four to lymaenon (aureus, litoralis, novickyi, thyrides). gonatocerus (cosmocomoidea) ater förster, 1841 material examined: 1♀, east-azarbaijan, khosroshahr, 19.iv.2013, h. lotfalizadeh. it was previously reported from iran: east-azarbaijan province by haghayeghi-nosrati et al. (2013). article gonatocerus (lymaenon) aureus girault, 1911 material examined: 5♀♀, east-azarbaijan, khosroshahr, 2.vii.2012, h. lotfalizadeh. this species widely distributed in the palaearctic, neotropcal and nearctic regions (noyes, 2013) and iran: east-azarbaijan province (haghayeghi-nosrati et al., 2013). gonatocerus (lymaenon) litoralis (haliday, 1833) material examined: 100♀♀ and 81♂♂, east-azarbaijan, khosroshahr, 8.vii.2012, h. lotfalizadeh. this species is widely distributed in the palaearctic, neotropical and nearctic regions (noyes, 2013) and in iran: east-azarbaijan and khorasan-razavi provinces (haghayeghi-nosrati et al., 2013; triapitsyn, 2013). gonatocerus (lymaenon) novickyi soyka, 1946 material examined: 2♀♀, east-azarbaijan, khosroshahr, 1.vi.2013, h. lotfalizadeh. this species was recorded from iran: east-azarbaijan province (haghayeghi-nosrati et al., 2013). gonatocerus (lymaenon) thyrides (debauche, 1948) material examined: 7♀♀, east-azarbaijan, khosroshahr, 23.xi.2013, leg. h. lotfalizadeh. this species is widely distributed in the palaearctic region (noyes, 2013) and in iran: east-azarbaijan province (haghayeghi-nosrati et al., 2013). gonatocerus (gonatocerus) longicornis nees ab esenbeck, 1834 material examined: 3♀♀, east-azarbaijan, khosroshahr, 2.ix.2011, h. lotfalizadeh. 4♀♀, gilan province, roudabr, 13.xi.2012, h. lotfalizadeh. this species was reared from zyginidia sohrab zachvatkin, 1947 (hem.: cicadellidae) on triticum aestivum l. in iran (fallahzadeh & huber, 2011). it is distributed in the palaearctic and oriental regions (noyes, 2013) and in iran: east-azarbaijan province (fallahzadeh & huber, 2011; haghayeghi-nosrati et al., 2013). gonatocerus (cosmocomoidea) oxypygus foerster, 1856 material examined: 15♀♀, east-azarbaijan, khosroshahr, 12.vii.2013, h. lotfalizadeh. this species widely distributed in the palaearctic and oriental regions (noyes, 2013) and in iran: fars (fallahzade & huber, 2011) and zanjan provinces (triapitsyn, 2013). gonatocerus (gonatocerus) pictus (haliday, 1833) material examined: 12♀♀, east-azarbaijan, khosroshahr, 12.vii.2013, h. lotfalizadeh. this species is distributed in europe (trjapitzin, 1978; noyes, 2013) and iran: east-azarbaijan province (haghayeghi-nosrati et al., 2013). gonatocerus pictus is a rare species in romania (pricop, 2010). gonatocerus (lymaenon) sp. 1 this undescribed species has been reported from iran: fars province (triapitsyn, 2013). gonatocerus (lymaenon) sp. 2 this undescribed species has been reported from iran: khorasanrazavi province as an egg parasitoid of n. tenellus on artemisia sp. and salsola sp. leaves (triapitsyn, 2013). vi-genus mymar curtis, 1829 only one species of the genus mymar have been reported from iran (bayegan et al., 2014) and two species from yemen (huber et al., 2009). annecke & doutt (1961) provided a key to females of the world species and triapitsyn & berezovskiy (2001) gave a key to females and males of the eight recognized species of mymar in the world. bayegan et al. (2014) collected this genus from a rice field. huber et al. (2009) reported cicadellidae and delphacidae as hosts of this genus. mymar taprobanicum ward, 1875 material examined: 4♀♀ gilan province, 13.xi.2012, z.-s. bayegan. 1♀, east-azarbaijan, arasbaran, vii.2013. this species is almost cosmopolitan and was reported from iran: gilan province by bayegan et al. (2014). vii-genus polynema haliday, 1833 three undetermined species of this genus were found in the northwest of iran. these species represent the three subgenera recognized by triapitsyn & fidalgo (2006): dorypolynema hayat & anis, 1999, polynema haliday, 1833 and doriclytus forester, 1847. four species of polynema have been reported from the uae and 10 species from yemen (huber et al., 2009). members of this genus are reported as parasitoids of hemiptera (cicadellidae, membracidae, miridae, nabidae, and anthocoridae) and odonata (lestidae) (huber et al., 2009). polynema (dorypolynema) sp. 1 material examined: 2♀♀ and 10♂♂, east-azarbaijan, khosroshahr, 5.v.2012, h. lotfalizadeh. polynema (polynema) sp. 2 material examined: 3♀♀ and 7♂♂, east-azarbaijan, khosroshahr, 20.xi.2012, h. lotfalizadeh. polynema (doriclytus) sp. 3 material examined: 6♀♀ and 3♂♂, east-azarbaijan, khosroshahr, 26.xi.2012, h. lotfalizadeh. viii-genus stephanodes enock, 1909 only one species was reported from iran (haghayegh-nosrati et al., 2014). stephanodes species were reviewed by huber & fidalgo (1997). stephanodes species parasitize nabidae and cicadellidae (huber et al., 2009). stephanodes reduvioli (perkins, 1905) triapitsyn & berezovskiy (2002) illustrated the diagnostic characters of the two palaearctic species. the male genitalia of s. reduvioli have a relatively longer phallobase and the aedeagus relatively narrower in the middle. this species has been reported from iran: alborz province, karaj, and shahdasht (huber & fidalgo, 1997). stephanodes similis (foerster, 1847) material examined: 2♀♀ and 1♂, east-azarbaijan, khosroshahr, 5.v.2013, h. lotfalizadeh. stephanodes similis is an egg parasitoid of nabidae and cicadellidae (huber & fidalgo, 1997). this species is widely distributed in europe (pricop, 2009), north america and argentina (triapitsyn & berezovskiy, 2002) and was recently reported from iran: east-azarbaijan province (haghayeghinosrati et al., 2014). [journal of entomological and acarological research 2015; 47:4838] [page 75] article [page 76] [journal of entomological and acarological research 2015; 47:4838] discussion and conclusions the list of mymaridae in iran includes 27 species belonging to 8 genera (table 1), 37% of them are in the genus gonatocerus with 10 species. the genera anagrus and erythmelus include 4 species, polynema 3 species, anaphes and stephanodes 2 species, and camoptoptera and mymar only 1 species. based on the present study, the list of iranian mymarids has increased from 10 reported species by fallahzadeh & huber (2011) to 27 species. a list of the mymaridae known from iran is given in table 1, which is based on the material examined and the previously recorded species. about 80% of the studied specimens belong to the genera gonatocerus and anagrus (figure 1). article table 1. list of mymaridae known from iran. genus species distribution in iranian provinces references anagrus a. atomus (l.) east-azarbaijan present study fars fallahzadeh & huber (2011) khorasan triapitsyn (1998) alborz walker et al. (1997) isfahan hesami et al. (2001) a. nigriceps (smits van burgst) east-azarbaijan present study alborz triapitsyn & berezovskiy (2004) a. avalae soyka east-azarbaijan present study alborz triapitsyn & berezovskiy (2004) anagrus sp. east-azarbaijan present study anaphes anaphes sp. 1 east-azarbaijan present study anaphes sp. 2 east-azarbaijan present study camptoptera camptoptera sp. east-azarbaijan present study erythmelus e. flavovarius (walker) alborz fallahzadeh & huber (2011) alborz fallahzadeh & huber (2011) e. israeliensis viggiani & jesu alborz triapitsyn (2003) e. rex (girault) east-azarbaijan present study alborz triapitsyn (2003) e. panis (enock) east-azarbaijan present study alborz triapitsyn (2003) gonatocerus g. ater förster east-azarbaijan haghayeghi-nosrati et al. (2013) g. aureus girault east-azarbaijan haghayeghi-nosrati et al. (2013) g. litoralis (haliday) east-azarbaijan haghayeghi-nosrati et al. (2013) khorasan-razavi triapitsyn (2013) g. novickyi soyka east-azarbaijan haghayeghi-nosrati et al. (2013) g. thyrides (debauche) east-azarbaijan haghayeghi-nosrati et al. (2013) g. longicornis nees ab esenbeck east-azarbaijan haghayeghi-nosrati et al. (2013) zanjan fallahzadeh & huber (2011) g. oxypygus foerster east-azarbaijan haghayeghi-nosrati et al. (2013) gilan present study zanjan triapitsyn (2013) g. pictus (haliday) east-azarbaijan haghayeghi-nosrati et al. (2013) gonatocerus sp. 1 fars triapitsyn (2013) gonatocerus sp. 2 khorasan razavi triapitsyn (2013) mymar m. taprobanicum ward east-azarbaijan present study gilan bayegan et al. (2014) polynema polynema sp. 1 east-azarbaijan present study polynema sp. 2 east-azarbaijan present study polynema sp. 3 east-azarbaijan present study stephanodes s. reduvioli (perkins) alborz huber & fidalgo (1997) s. similis (forester) east-azarbaijan haghayeghi-nosrati et al. (2014) figure 1. composition of mymaridae based on number of collected specimens in present study. references aeschlimann j.p., 1986 distribution and effectiveness of anaphes diana [=patasson lameerei][hym.: mymaridae], a parasitoid of sitona spp. eggs [col.: curculionidae] in the mediterranean region. entomophaga 31: 163-172. akbarzadeh-shoukat g., 1998 the first report on the occurrence of the egg parasitoid of pear lace bug in iran. appl. entomol. phytopath. 66: 44-45. annecke d.p., doutt r.l., 1961 the genera of the mymaridae. hymenoptera: chalcidoidea. entomol. mem. dept. agr. un. s. africa 5: 1-71. baquero e., jordana r., 2003 the genus gonatocerus nees (hymenoptera chalcidoidea mymaridae) in corn fields of navarra, north spain. redia 85:1-19. bayegan z.s., lotfalizadeh h., zargaran m.r., pooraiiouby r., 2014 new record of a genus and species of mymaridae (hymenoptera: chalcidoidea) from iran. turk. j. zool. 38: 655-656. beardsle, j.w., huber j.t., 2000 key to genera of mymaridae in the hawaiian islands, with notes on some of the species (hymenoptera: chalcidoidea). proc. haw. entomol. soc. 34: 1-22. chiappini e., 1987 ricerche sulla variabilità di anagrus atomus (l.) 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(doriclytus) vitripenne (hymenoptera: mymaridae). zootaxa 1362: 55-68. triapitsyn s.v., huber j.t., logarzo g.a., berezovskiy v.v., aquino d.a., 2010 review of gonatocerus (hymenoptera: mymaridae) in the neotropical region, with description of eleven new species. zootaxa 2456: 1-243. viggiani g., jesu r., 1988 considerazioni sui mimaridi italiani ed i loro ospiti. atti [xv] con. nazion. ital. entomol. [l’aquila] 15: 1019-1029. walker g.p., zareh n., bayoun i.m., triapitsyn s.v., 1997 introduction of western asian egg parasitoids into california for biological control of beet leafhopper, circulifer tenellus. pan-pac. entomol. 73: 236-242. article jear2012 [journal of entomological and acarological research 2012; 44:e7] [page 33] molecular typing of bacteria of the genus asaia in malaria vector anopheles arabiensis patton, 1905 s. epis,1,2 m. montagna,2 f. comandatore,2 c. damiani,1 a. diabaté,4 d. daffonchio,3 b. chouaia,3 g. favia1 1scuola di bioscienze e biotecnologie, università degli studi di camerino; 2divet, università degli studi di milano; 3defens, università degli studi di milano, italy; 4institut de recherche en science de santé/centre muraz, bobo-dioulasso, burkina faso abstract the acetic acid bacterium asaia spp. was successfully detected in anopheles arabiensis patton, 1905, one of the major vector of human malaria in sub-saharan africa. a collection of 45 asaia isolates in cellfree media was established from 20 individuals collected from the field in burkina faso. 16s rrna universal polymerase chain reaction (pcr) and specific qpcr, for the detection of asaia spp. were performed in order to reveal the presence of different bacterial taxa associated with this insect. the isolates were typed by internal transcribed spacer-pcr, box-pcr, and randomly amplified polymorphic dna-pcr, proved the presence of different asaia in a. arabiensis. introduction species of the genus anopheles meigen, 1818 are the most important vectors of malaria in sub-saharan africa. within the anopheles gambiae complex, composed of seven morphologically indistinguishable species of mosquitoes, anopheles arabiensis patton, 1905 and anopheles gambiae giles, 1902 sensu strictu show the wider distribution and are the most efficient malaria vectors (onyabe & conn, 2001a and 2001b). a. arabiensis prefers tropical weather and its typical habitat is the dry savannah, reproducing in natural and artificial puddles of water, but can fly for more than two kilometers from its place of birth. a. arabiensis population density varies depending on climate conditions during the year; the adults show the minimum density between march and june, while after the first rains in july reach the highest levels (i.e. august and september) then decrease in october (robert et al., 1998). the presence of a. arabiensis is favored by temperatures of 28-30°c, clean water, high concentrations of hco3and co32-, low concentrations of no3and nacl, absence of predatory invertebrates and fish (as gambusia and tilapia), presence of plants of the genus pistia (water lettuce) and absence of those of the genus lemna (duckweed) (robert et al., 1998). a. arabiensis is the only vector of malaria in dakar, senegal, and is predominant in ethiopia, sudan and in the guinean savannah. a. gambiae is vicariant of a. arabiensis in the sub-saharan region not colonized by the latter. in the last 20 years, a. arabiensis has expanded its distribution, spreading even in forests where a. gambiae was prevalent, probably because of climate change (onyabe & conn, 2001a; donnelly et al., 2001). hargreaves and colleagues (2003) demonstrated that a. arabiensis is resistant to ddt, one of the major insecticides used in malaria control program. as a result of these data, in order to control the vector, it is important to develop new integrated strategies. similar to a recently described work (chouaia et al., 2010), here we present the first description of the symbiont asaia in a. arabiensis mosquitoes and its genetic diversity. asaia is a gram-negative acetic acid bacterium associated with different genera of insect (favia et al., 2007; crotti et al., 2009; damiani et al., 2010). it has been found in the gut, salivary glands, and reproductive organs; it is transmitted vertically, horizontally and venereally from males to females. recently, it has been demonstrated that asaia is not transmitted to humans (epis et al., 2011) suggesting that asaia could be regarded only as an opportunistic pathogens and a good candidate to potentially vector antiplasmodial factors (favia et al., 2008; ricci et al., 2010). uncovering the level of diversity of the symbiont asaia of a. arabiensis would be useful for potential applications by allowing the selection of the dominant genotypes, that in this case represents the most widespread genotype among the insect populations, in order to develop paratransgenic approaches. material and methods mosquito strains origin a. arabiensis was collected from soumousso and vallée du kou (burkina faso), immediately stored in enrichment medium, and sent correspondence: bessem chouaia, defens, università degli studi di milano, via celoria 2, 20133 milano, italy. tel: +39.02.5031.9068 fax: +39.02.5031.6694. e-mail: bessem.chouaia@unimi.it key words: anopheles arabiensis, mosquito-associated bacteria, asaia spp., molecular typing. acknowledgements: this work was supported by firb-ideas (grant rbid082mlz). received for publication: 31 may 2012. revision received: 18 july 2012. accepted for publication: 19 july 2012. ©copyright s. epis, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e7 doi:10.4081/jear.2012.e7 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2012; volume 44:e7 no nco mm er cia l u se on ly [page 34] [journal of entomological and acarological research 2012; 44:e7] to the university of camerino (italy). furthermore, 20 females collected from the same localities were reared in burkina faso insectary; the laid eggs were sent and maintained in the insectary of the laboratory of parasitology at the university of camerino in order to start the new colony. isolation of asaia from a. arabiensis asaia strains were isolated from adult mosquitoes using the enrichment medium i as described by lisdiyanti et al, 2001. a. arabiensis samples were washed in 1% sodium hypochlorite for 1 min, subsequently washed three times with 0.9% nacl, and homogenized by grinding in 200 μl 0.9% nacl. the homogenate was inoculated into the enrichment medium and allowed to grow at 30°c for 3 days, with shaking. when microbial growth occurred, the microorganisms were streaked on caco3 agar plates (ph 6.8) containing 1.0% (wt/vol) d-glucose, 1.0% (wt/vol) glycerol, 1.0% (vol/vol) ethanol, 1.0% (wt/vol) bactopeptone, 0.5% (wt/vol) yeast extract, 0.7% (wt/vol) caco3, and 1.5% (wt/vol) agar as reported in chouaia et al., 2010. after 3 days growth, the colonies were collected. after the incubation period, 500 μl of enriched broth containing the asaia have been preserved in a solution of 16% glycerol. the glycerine obtained were placed at -80°c for storage. dna extraction and amplification from selected colonies dna was extracted from asaia strains and mosquitoes using commercial kit wizard genomic dna purification (promega corp., fitchburg, wi, usa) and eluted in 100 μl of elution buffer. the quantitative polymerase chain reaction (qpcr) screening of mosquitoes, for the specific detection of asaia spp., were performed with iq thermal cycler (bio-rad, hercules, ca, usa) using the previously described primers asafor (5�-gcgcgtaggcggtttacac) and asarev (5�agcgtcagtaatgagccaggtt) (favia et al., 2007). the 16s rrna gene was pcr amplified with universal bacterial 16s rrna gene primers 27f (5�-tcgacatcgtttacggcgtg) and 1492r (5�-ctacggctaccttgttacga). internal transcribed spacer (its)-pcr was performed, with primer itsf (5�-gccaaggcatccaac) and itsr (5�-gtcgtaa caaggtagccgta) (daffonchio et al., 1998). the box-pcr was performed, with box-a1 primer (5�-ctacggcaaggcgacgctgac) according to urzì et al., 2001; randomly amplified polymorphic dna (rapd)-pcrs were performed, using the rapd opa4 (5�-aatcgggctg) and opa10 (5�gtgatcgcag) primers (daffonchio et al., 1998). sequence analysis nucleotide identity searches were performed in the genbank database using the basic local alignment search tool (blast; http://blast. ncbi.nlm.nih.gov/blast.cgi) program to identify the bacterial strains most closely related to isolates and the best matching dna sequences of the sequenced amplicons. a phylogenetic tree based on the analysis of the 16s rrna sequences was inferred using the neighbor-joining method (saitou et al., 1987) with 1000 replicates as a bootstrap test and visualized using mega 4 (tamura et al., 2007), the 16s rrna sequence of acetobacter tropicalis ba 1.3 was used as an outgroup. the 16s rrna gene sequences were deposited under accession numbers from he814602 to he814621 in the european molecular biology laboratory nucleotide sequence database (http://www.ebi.ac.uk/). cluster analysis pcr fingerprintings were obtained by visualization, under uv light, of 1.2% of agarose gel electrophoresis. the profiles were analyzed using the quantity one® software (version 4.6.5, bio-rad). for each strain profile, a matrix, reporting the presence (1) or absence (0) of bands at a specific distance, was generated. the analysis of profiles of its-pcr, box-pcr and rapd-pcrs allowed obtaining a genotypic profile for each strain. to estimate the level of similarity between the strains based on the merged profiles obtained from its-pcr, box-pcr, and rapd-pcr, and to create the unweighted pair group method with arithmetic mean (upgma) tree, mvsp software (version 3.13n, kovach computing services, pentraeth, isle of anglesey, uk) was used (dunn and everitt, 1982). results forty-five isolates of asaia spp. were obtained from 20 mosquitoes. the sequences obtained by universal bacterial 16s rrna pcr confirmed the presence of asaia spp. in a. arabiensis mosquitoes. moreover, specific qpcr carried on a. arabiensis showed the presence of this bacterium associated to the studied population; asaia 16s rrna gene copies was abundant in all dnas extracted from a. arabiensis mosquitoes, with a mean of 7.8×105 asaia 16s rrna gene copies for single individual. the 45 asaia isolates from the mosquitoes exhibited the same itspcr fingerprinting profiles, presenting an amplification pattern consisting of 3 bands between 500 and 3700 bp (data not shown). the rapd-pcr using the primer set opa4 and opa10 revealed, respectively, four and three genotypes characterized by complex band patterns between 450 and 7300 bp. the analysis obtained from box-pcr showed several profiles among the strains examined. box-pcr discriminated from 4 to 6 band pattern types, with most of the bands in the range between 350 and 4500 bp. by using the software quantity one, for each strain, a band profile was obtained from the analysis of the agarose gels of the amplifications carried out on the genome of asaia from a. arabiensis. the merging of the results obtained from all the four pcrs (its-pcr, box-pcr and rapd-pcrs) allowed us to distinguish 21 genotype patterns. the tree generated with the upgma methods using the jaccard coefficient allowed clustering the different profiles in a tree (figure 1). from the analysis of this tree it was possible to identify 9 groups (from i to ix). article table 1. different groups and relative profile numbers and asaia strains. groups profile numbers asaia strains i 1 1 2 2, 3, 4, 5 3 12 ii 4 7, 8, 9, 11 5 10 6 6 iii 7 15, 16 8 14, 17, 18 9 19, 20, 21, 22 iv 10 13 v 11 36, 37 vi 12 27, 28 13 29, 30, 31 14 32, 33, 34 vii 15 35 16 23, 24, 25 viii 17 39 18 40, 41, 43 19 42 ix 20 45 21 38, 44 no nco mm er cia l u se on ly the analysis of the data allowed also to identify two more abundant strains (groups iii and vi) each of them grouping 20% of the total strains (table 1). those stains were present in all studied populations of mosquito and not restricted to only one individual. other strains were also present in more than one mosquito like the groups i, ii and vii. except for the group iv all the other groups were represented by more than one isolate present in more than one mosquito. the phylogenetic tree based on the partial 16s rrna sequence of representative strains of asaia isolated for a. arabiensis showed that the strains clustered with other strains of asaia krungthepensis (figure 2). the phylogenetic analysis of the 16s rrna did not show a high diversity of the strains as compared to the results of the molecular typing based on dna markers (figure 1). discussion and conclusions previous studies have shown that a. stephensi (favia et al., 2007), aedes aegypti, a. gambiae, ae. albopictus mosquitoes (chouaia et al., 2010) and scaphoideus titanus (crotti et al., 2009) host the bacterium asaia. here we demonstrate the presence of acetic acid bacteria asaia both in males and in females mosquitoes of a. arabiensis. the fact that asaia is also able to be transmitted horizontally and cross colonize different species (damiani et al., 2008; crotti et al., 2009) may suggest the capacity of asaia to be environmentally transmitted between a. arabiensis and a. stephensi. in order to obtain a genetic profile of each strain of asaia, the dna was amplified using box-pcr, rapd-pcrs and its-pcr. based on these profiles, a similarity tree was build. this analysis showed that, in a. arabiensis, asaia strains clustered in 21 different genotypes that can be grouped into 9 clusters. this study showed that the same a. arabiensis mosquito may harbor simultaneously different strains of the symbiont and that each strain of asaia can colonize more than a single specimen of a. arabiensis. a similar multiple infection phenomena have been reported in other mosquito species harboring asaia (chouaia et al., 2010) and insects associated with the symbiont wolbachia (werren et al., 1995). the strains isolated in this work display a higher diversity (21 genotypes from 45 strains) than those studied in the work of chouaia et al., 2010 using boxpcr, rapd-pcrs, trna-pcr and its-pcr (29 genotypes from 284 strains). the detected higher diversity can be explained by the fact that, unlike the mosquito species studied by chouaia et al. (2010), the population of a. arabiensis studied in this work is a wild population. the mosquito associated asaia is a secondary symbiont that may be acquired from the environment (damiani et al., 2008; chouaia et al., 2012). in spite of the stable lab rearing concentrations, the natural environment present complex interactions both at the biotic and abiotic levels, thus bacterial populations displaying high diversity have higher chance to adapt and survive in new environmental conditions (have higher chance of adaptations and survival in a changing environment). the presence of asaia in a. arabiensis, the most important malaria vector in sub-saharan africa, can lead to the possibility to use this symbiont in a paratransgenesis approach as support for the prevention of malaria transmission. in devising a paratransgenic approach it is essential to consider the genetic heterogeneity of the bacterium asaia and the lack of a predominant strain in a. arabiensis. references chouaia b., rossi p., epis s., mosca m., ricci i., damiani c., et al., 2012 delayed larval development in anopheles mosquitoes deprived of asaia bacterial symbionts. bmc microbiol. 12 (suppl 1): s2. [journal of entomological and acarological research 2012; 44:e7] [page 35] article figure 1. similarity tree between the profiles of asaia spp. isolated from the anopheles arabiensis mosquitoes. the tree was generated with the unweighted pair group method with arithmetic mean methods using the jaccard coefficient. we can distinguish 9 groups (from i to ix). figure 2. phylogenetic positions of the strains of asaia isolated from an arabiensis, based on 16s rrna gene sequences (neighbor joining method; kimura correction; all positions containing gaps and missing data were eliminated from the data set). values of bootstrap superior to 60% have been reported in the tree. the scale bar represents sequence divergence. the 16s rrna sequence of acetobacter tropicalis strain ba 1.3 was used as outgroup. no nco mm er cia l u se on ly [page 36] [journal of entomological and acarological research 2012; 44:e7] chouaia b., rossi p., montagna m., ricci i., crotti e., damiani c., et al., 2010 molecular evidence for multiple infections as revealed by typing of asaia bacterial symbionts of four mosquito species. appl. environ. microbiol. 76: 7444-7450. crotti e., damiani c., pajoro m., gonella e., rizzi a., ricci i., et al., 2009 asaia, a versatile acetic acid bacterial symbiont, capable of cross-colonizing insects of phylogenetically distant genera and orders. environ. microbiol. 11: 3252-3264. daffonchio d., borin s., frova g., manachini p.l., sorlini c., 1998 pcr fingerprinting of whole genomes: the spacers between the 16s and 23s rrna genes and of intergenic trna gene regions reveal a different intraspecific genomic variability of bacillus cereus and bacillus licheniformis [corrected]. int. j. syst. bacteriol. 48: 107-116. damiani c., ricci i., crotti e., rossi p., rizzi a., scuppa p., et al., 2008 paternal transmission of symbiotic bacteria in malaria vectors. curr. biol. 18: r1087-1088. damiani c., ricci i., crotti e., rossi p., rizzi a., scuppa p., et al., 2010 mosquito-bacteria symbiosis: the case of anopheles gambie and asaia. microb. ecol. 60: 644-654. donnelly m.j., licht m.c., lehmann t., 2001 evidence for recent population expansion in the evolutionary history of the malaria vectors anopheles arabiensis and anopheles gambie. mol. biol. evol. 18: 1353-1364. dunn g., everitt b.s., 1982 an introduction to mathematical taxonomy. cambridge university press, cambridge, uk. favia g., ricci i., damiani c., raddadi n., crotti e., marzorati m., et al., 2007 bacteria of the genus asaia stably associate with anopheles stephensi, an asian malarial mosquito vector. proc. natl. acad. sci. u s a. 104: 9047-9051. favia g., ricci i., marzorati m., negri i., alma a., sacchi l., et al., 2008 bacteria of the genus asaia: a potential paratransgenic weapon against malaria. adv. exp. med. biol. 627: 49-59. hargreaves k., hunt r.h., brooke b.d., mthembu j., weeto m.m., awolola t.s., et al., 2003 anopheles arabiensis and a. quadriannulatus resistance to ddt in south africa. med. vet. entomol. 17: 417-422. lisdiyanti p., kawasaki h., seki t., yamada y., uchimura t., komagata k., 2001 identification of acetobacter strains isolated from indonesian sources, and proposals of acetobacter syzygii sp. nov., acetobacter cibinongensis sp. nov., and acetobacter orientalis sp. nov. j. gen. appl. microbiol. 47: 119-131. onyabe d.y., conn j.e., 2001a population genetic structure of the malaria mosquito anopheles arabiensis across nigeria suggests range expansion. mol. ecol. 10: 2577-2591. onyabe d.y., conn j.e., 2001b the distribution of two major malaria vectors, anopheles gambie and anopheles arabiensis, in nigeria. mem. inst. oswaldo cruz. 96: 1081-1084. ricci i., damiani c., rossi p., capone a., scuppa p., cappelli a., et al., 2010 mosquito symbioses: from basic research to the paratransgenic control of mosquito-borne diseases. j. appl. entomol. 135: 487-493. robert v., awono-ambene h.p., thioulouse j., 1998 ecology of larval mosquitoes, with special reference to anopheles arabiensis (diptera: culicidae) in market-garden wells in urban dakar, senegal. j. med. entomol. 35: 948-955. saitou n., nei m., 1987 the neighbor-joining method: a new method for reconstructing phylogenetic trees. mol. biol. evol. 4: 406-425. tamura k., dudley j., nei m., kumar s., 2007 mega4: molecular evolutionary genetics analysis (mega) software version 4.0. mol. biol. evol. 24: 1596-1599. urzì c., brusetti l., salamone p., sorlini c., stackebrandt e., daffonchio d., 2001 biodiversity of geodermatophilaceae isolated from altered stones and monuments in the mediterranean basin. environ. microbiol. 3: 471-479. werren j.h., zhang w., guo l.r., 1995 evolution and phylogeny of wolbachia: reproductive parasites of arthropods. proc. biol. sci. 261: 55-63. article no nco mm er cia l u se on ly jear2012 abstract the effect of permethrin-treated africa university (au) mosquito blankets on susceptible female anopheles gambiae sensu lato mosquitoes was studied under laboratory conditions at africa university campus in mutare, zimbabwe. wash resistance (ability to retain an effective dose that kills ≥80% of mosquitoes after a number of washes) and repellence (ability to prevent ≥80% of mosquito bites) properties were studied. the au blankets were wash resistant when 100% mortality was recorded up to 20 washes, declining to 90% after 25 washes. untreated au blankets did not cause any mortality on mosquitoes. however, mosquito repellence was 96%, 94%, 97.9%, 87%, 85% and 80.7% for treated au blankets washed 0, 5, 10, 15, 20 and 25 times, respectively. mosquito repellence was consistently above 80% from 0-25 washes. in conclusion, au blankets washed 25 times were effective in repelling and killing an. gambiae sl mosquitoes under laboratory conditions. introduction malaria is one of the killer diseases affecting zimbabwe, mostly in the low veldt due to the conducive environment prevailing in these areas. the ministry of health and child welfare (mohcw) has a mandate to control malaria through the national malaria control program (nmcp) that has defined structures from head office, provincial, down to district levels. the nmcp co-ordinates all malaria control activities in the country using the integrated vector management approach that includes indoor residual spraying, insecticide treated materials (itms), larviciding and use of personal protection methods (lukwa, 1994; taylor & mutambu, 1986). personal protection is one of the malaria control methods advocated for at community level. itms have been used for some time although the use of treated blankets is relatively uncommon. long-lasting permethrin-treated nets used as blankets inhibited 35-60% of mosquitoes from entering the huts and eventually 88-98% of them died (hougard et al., 2007). the use of treated-blankets and sheets reduced malaria infection in the intervention group by 70% (kimani et al., 2006). in another study, insecticide-treated bedding protected people against malaria and leishmaniasis vectors, since the mean mortality rates were 16-30.3% (graham et al., 2002). the results on acceptability clearly showed that providing treated bedding resulted in protection from mosquito bites (graham et al., 2002). chaddars (head dress) treated with permethrin were evaluated in several villages in afghanistan with a 7.2% incidence rate of leishmaniasis for the control group and 2.5% for people using permethrintreated chaddars (reyburn et al., 2000). up to 8% of the respondents using treated chaddars for the control of leishmaniasis were convinced that chaddars prevent bites from sand flies, as compared to 28% who did not feel any change in sand fly biting (control group) (reyburn et al., 2000). permethrin-treated chaddars killed 36-70% of the mosquitoes exposed to the treatment as compared to 20-39% of the mosquitoes that were killed in the controls (rowland et al., 1999). the world health organization (who) (who, 2006) set the criteria for 24-h mortality at 80% or over. although the use of long-lasting insecticide-treated mosquito nets (llins) is an important part of malaria control in zimbabwe, treated blankets have never been used for this purpose. studies were carried out at africa university, mutare, on the possibility of using africa university (au) mosquito blanket for controling mosquitoes. materials and methods study area mosquitoes were collected from selected breeding sites from correspondence: nzira lukwa, national institute of health research, p.o. box cy573, causeway, harare, zimbabwe. tel.: +263.4.797052; +263.274664 fax: +263.4.253979. e-mail: nziraa33@yahoo.co.uk key words: an. gambiae sl, wash resistance, repellence, bioassays, permethrin. acknowledgements: the authors would like to acknowledge the support provided by mr chinyanya and ms gidi. permission to carry out the research was granted by the then provincial medical director for manicaland province (dr banda). all participants are fully acknowledged. funding was provided by africa university. ethical approval for this study was granted by the medical research council of zimbabwe. received for publication: 28 august 2012. revision received: 9 january 2013. accepted for publication: 1 february 2013. ©copyright n. lukwa et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e5 doi:10.4081/jear.2013.e5 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. wash resistance and repellent properties of africa university mosquito blankets against mosquitoes n. lukwa,1 a. makuwaza,1 t. chiwade,1 s.l. mutambu,1 m. zimba,2 p. munosiyei3 1national institute of health research, causeway, harare; 2university of zimbabwe, biological sciences, mount pleasant, harare; 3bindura university of science education, bindura, zimbabwe [page 18] [journal of entomological and acarological research 2013; 45:e5] journal of entomological and acarological research 2013; volume 45:e5 jear_2013_1:hrev_master 23/04/13 09.55 pagina 18 no n c om me rci al us e o nly chakohwa village (19°51’ s, 32°55’ e) where malaria transmission is moderate. the inhabitants are mostly subsistence farmers who specialize in growing maize. some irrigation is practised for vegetables. laboratory tests were carried out using facilities at africa university (18°52’ s, 32°42’ e) located near mutare city, zimbabwe. mosquito collection and rearing the anopheles gambiae sensu lato mosquito larvae were collected from the fringes of rivers (figure 1) using soup ladles in breeding sites in chakohwa village. the larvae were placed in rearing bowls (figure 2) and later covered using mosquito netting. all the larvae were fed on fish food until they pupated. the pupa were picked up using a 2 ml plastic pipette, placed in a small dish before being placed in 5 l plastic mosquito cages to hatch. the upper part of the mosquito cage is open and has netting, and a sleeve is provided for introducing and retrieving mosquitoes. the adults were fed on 10% sugar solution that had been daubed in cotton wool and placed on top of the cage. the adult an. gambiae sl mosquitoes from this study area have not been identified to species level. a sample of the resultant adults was used to determine levels of insecticide susceptibility and the rest were used for bioassays. the adult mosquitoes were susceptible to the 4 classes of insecticides, namely pyrethroids (lambda-cyhalothrin), organophosphates (malathion), organochlorines (dichloro-diphenyl-trichloroethane) and cabarmates (bendiocarb). repellent blanket the au mosquito blanket (also known as the chicopee insect shield) measures 187.96¥121.92 cm and weighs 567 g. it is manufactured by buzzoff™ (s.c. johnson & son, inc., st racine, wi, usa) and is treated with 0.52% permethrin at the factory. the blanket can be used as a wrapper or top sheet (i.e. placed on top of blankets). the control blanket was made by the same manufacturer but it did not contain insecticide. world health organization’s washing procedure the blanket was cut into 25¥25 cm pieces, with 6 pieces for each wash (0, 5, 10, 15, 20 and 25). each piece was individually introduced into a 1 l beaker containing 500 ml of distilled water (with 1 ml/l soap) added. the beakers were introduced into a water bath that contained water at a temperature of 30°c. the water bath was set at 155 movements per min for 10 min. after 10 min, the beakers were removed from the water bath and the pieces of blankets removed. each piece of blanket was rinsed twice for 10 min in clean distilled water in the same shaking conditions and dried under cover. all pieces of blanket were kept uncontaminated before use. bioassays (described under wash resistance properties) were only performed on dry blanket samples (who, 2005). wash resistance properties wash resistance properties were evaluated on either permethrintreated or untreated samples of au blankets that were washed using the standard who washing procedure (who, 2005). each who cone measured 8.5 cm in diameter at the base and was 5.5 cm high. each piece of was fastened to the who cone using a rubber band (6 replicates for each wash) (figure 3). a total of 5 non-blood fed (2-5 days old) an. gambiae s.l adult female mosquitoes were aspirated using a sucking tube (carried out through the small hole in the cone) and exposed to each blanket sample for 30 min. mosquitoes from each who cone were retrieved and placed in holding paper cups after being provided with 10% sugar solution in the form of a cotton swab used as a wick. mortality was recorded 24 h after exposure. this procedure was repeated for all the 6 washes (0, 5, 10, 15, 20 and 25). repellence test mosquito cages were made from 5 l buckets consisting of a sleeve for introducing mosquitoes and covered with mosquito netting on top and used for each test. five cages per wash were prepared simultaneously for each of the 6 washes. fifty starved laboratory-reared female an. gambiae s.l were placed in each mosquito cage and left to acclimatize for 1 h in studies that were replicated 5 times. each piece of blanket was wrapped around each hand (figure 4) and placed in the mosquito cage (figure 5). one hand was placed in the cage containing mosquitoes for 2 min and the number of mosquitoes probing to bite recorded as described by maharaj et al. (2010). this was carried out for untreated and treated pieces of blanket. repellence tests were performed following the test method previously described by curtis et al. (1990). percentage mosquito repellence was calculated following the method previously described by mehr et al. (1985) as follows: where bc is the mean number of bites on control; and bt is the mean number of bites on treated pieces of blanket. [journal of entomological and acarological research 2013; 45:e5] [page 19] article figure 1. fringes of mosquito breeding site. figure 2. mosquito rearing bowl with mosquito netting. jear_2013_1:hrev_master 23/04/13 09.55 pagina 19 no n c om me rci al us e o nly [page 20] [journal of entomological and acarological research 2013; 45:e5] the cut-off point for repellence was 80% or over as described by maharaj et al. (2010). data analysis an analysis of variance (anova) test of variance using the 95% confidence limit was carried out to test statistical difference. results au treated mosquito blankets were wash resistant for up to 25 washes since the mortality was above 80%, as stipulated by the who (2005) (table 1). one hundred percent mortality was realized up to 20 washes before it decreased at 25 washes when treated blankets were used. no mortality occurred with the untreated blankets. in mosquito repellent studies, more mosquitoes were biting the persons using untreated samples of the blanket than the treated, and the results were significantly different for all washes (p<0.0001) (table 2). mosquito repellent properties of treated au blankets were observed for up to 25 washes since repellence was above 80% as reported by maharaj et al. (2010). discussion and conclusions studies on wash resistance properties showed that the treated au blanket killed mosquitoes for up to 25 washes, in agreement with rowland et al. (1999). thus, enough lethal insecticide was retained on treated pieces of blanket for up to 25 washes. when blankets are not article figure 3. a) bottom of world health organization’s (who) cone; b) top of who cone covered with piece of blanket. figure 4. hand wrapped in piece of blanket during repellence test. figure 5. hand introduced into mosquito cage for repellence test. table 1. wash resistance properties of africa university treated mosquito blankets three days after washing. no. of washes mortality (%) 0 30/30 (100) 5 30/30 (100) 10 28/28 (100) 15 31/31 (100) 20 27/27 (100) 25 27/30 (90) jear_2013_1:hrev_master 23/04/13 09.55 pagina 20 no n c om me rci al us e o nly properly treated during manufacture, insecticide can be lost quickly during washing, thus losing the protective efficiency. when this happens, mosquitoes will no longer be killed and people living in malarious areas will be exposed to infective mosquito bites. the more wash resistant the treated blankets are, the longer they last in protecting vulnerable populations. the quality of the fiber used in blanket manufacture has a bearing on how long the treated blankets will last. observations of graham et al. (2002) on mortality (16-30%) of mosquitoes were not as good as ours (90-100%), implying that au treated blankets worked better than treated bedding. however, our results on mortality were slightly better than (llins) treated with permethrin but used as top sheets (88-98%) (hougard et al., 2007). on the other hand, treated au blankets have proved to be good repellents for up to 25 washes and our results were better than those of hougard et al. (2007). the treated au blankets minimize the number of mosquitoes that land and, therefore, reduce malaria transmission. results from this study show that treated blankets might offer protection from mosquito bites even if they have been washed 25 times. these results point to the use of a new concept (repellent blankets) in malaria control. references curtis c.f., lines j.d., baolin l., renz a., 1990 natural and synthetic repellents. in: curtis c.f., ed., appropriate technology in vector control. crc press, boca raton, fl: 75-92. graham k., mohamad n., rehman h., farhan m., kamal m., rowland m., 2002 comparison of three pyrethroid treatments of top sheets for malaria control in emergencies: entomological user acceptance studies in an afghan refugee camp in pakistan. med. vet. entomol. 16: 199-206. hougard j.m., martin t., guillet p.f., coosemans m., itoh t., akogbeto m., et al., 2007 preliminary field testing of a long-lasting insecticide-treated hammock against anopheles gambiae and mansonia spp. (diptera: culicidae) in west africa. j. med. entomol. 44: 651-655. kimani e.w., vulule j.m., kuria i.w., mugisha f., 2006 use of insecticide-treated clothes for personal protection against malaria: a community trial. malaria j. 27: 63. lukwa n., 1994 do traditional repellent plants work as mosquito larvicides?. cent. afr. j. med. 40: 306-325. maharaj r., maharaj v., newmarch m., crouch n.r., bhagwandin n., folb p.i., et al., 2010 evaluation of selected south african ethno-medicinal plants as mosquito repellents against the anopheles arabiensis mosquito in a rodent model. malaria j. 9: 301. mehr z.a., rutledge l.c., morales e.l., meixsall v.e., korte d.w., 1985 laboratory evaluation of controlled release insect repellent formulations. j. am. mosquito control assoc. 1: 143-147. reyburn h., ashford r., mohsen m., hewitt s., rowland m., 2000 a randomised controlled trial of insecticide treated bed nets and chaddars or top sheets, and residual spraying of interior rooms for the prevention of cutaneous leishmaniasis in kabul, afghanistan. trans. r. soc. trop. med. hyg. 94: 361-366. rowland m., durrani n., hewitt s., mohammad n., bouma m., carneiro o., et al., 1999 permethrin treated chaddars and top sheets: appropriate technology for protection against malaria in afghanistan and other complex emergencies. trans. r. soc. trop. med. hyg. 93: 465-472. taylor p., mutambu s.l., 1986 a review of the malaria situation in zimbabwe with special reference to the period 1972-1981. trans. r. soc. trop. med. hyg. 80: 12-19. world health organization, 2005 guidelines for laboratory and field testing of long-lasting insecticidal mosquito nets; who/cds/whopes/gcdpp/2005.11. world health organization, geneva. available from: http://whqlibdoc.who.int/hq/2005/who_ cds_whopes_gcdpp_2005.11.pdf world health organization, 2006 guidelines for testing mosquito adulticides for indoor residual spraying and treatment of mosquito nets; who/cds/whopes/gcdpp/2006.3. world health organization, geneva. available from: http://whqlibdoc.who.int/hq/ 2006/who_cds_ntd_whopes_gcdpp_2006.3_eng.pdf [journal of entomological and acarological research 2013; 45:e5] [page 21] article table 2. number of mosquitoes probing to bite people using treated and untreated blanket samples and their repellence effect. treated au blankets untreated au blankets no. of washes no. of mosquitoes probing to bite repellence [control-treated/control]*100 no. of mosquitoes probing to bite total (range) % total (range) 0 5 (0-2) 96.0 125 (18-37) 5 7 (1-2) 94.0 118 (17-30) 10 3 (0-1) 97.9 145 (26-33) 15 16 (3-4) 87.0 123 (23-26) 20 18 (2-5) 85.0 120 (23-26) 25 26 (4-7) 80.7 135 (24-30) au, africa university. jear_2013_1:hrev_master 23/04/13 09.55 pagina 21 no n c om me rci al us e o nly jear2012 [journal of entomological and acarological research 2016; 48:4938] [page 345] abstract the ocimum plant was traditionally used for mosquitoes repellent and control in india especially in tamil nadu. in this research, deals with the larvicidal, pupicidal and adulticidal potential of three different solvent extracts of o. canum against aedes aegypti. the overall result highlights that the chloroform extracts of o. canum were shown significant larvicidal (15.027 mg/l) activity at 24 h of exposure. the pupicidal and adulticidal activity of this plant exhibits highest mortality against a. aegypti within 24 h at the dose ranges of 89.773 mg/ml, 41.912 mg/ml respectively. the chloroform extracts contain major phyto-constituents like phenol, alkaloids, protein and tannins. thin layer chromatography profiles also provide a database for the presence of active components. gc-ms analysis of bioactive chloroform extract revealed that a total of seventeen compounds, six were considered as major and the remaining as minor compounds. the spectral studies of ft-ir denoted the functional groups of bioactive components like alkenes, ketone, hydroxyl and others. based on the outcome of results show that ocimum canum have found to potent ability for controlling the mosquitoes, it can be used as an ideal eco-friendly agent for arresting dengue fever in future. introduction mosquitoes are the most important single group of insects in terms of public health importance, which transmit variety of diseases like malaria, filariasis, dengue and japanese encephalitis. it causing millions of deaths every year (brown, 1986). mosquito-borne diseases contribute to a larger proportion of health problems in developing countries. repetition of synthetic insecticides for mosquito control has disrupted natural biological control systems and led to resurgences in mosquito populations. it also resulted in the development of resistance, undesirable effects on non-target organisms, and fostered environmental and human health concern (thomas et al., 2004). the recent research focusing for herbal preparations that do not produce any adverse side effects in the non-target organisms and easily biodegradable (kant et al., 1996). in general, plant derived compounds (phytopesticides) have been recognized as an important natural resources of insecticides (gbolade et al., 2000). several phytochemicals have been reported to exhibit harmful effect against mosquito larvae and insecticides, reproduction of inhibitors, repellent potential, ovicidal and oviposition deterrent (prajapati et al., 2005; pushpanathan et al., 2006) properties. one of the method available for the control of mosquito population is over and injudicious applications of persistent synthetic insecticides, resulting undesirable effect of synthetic insecticides. previous research has been proved the effectiveness of plant derived secondary compounds, such as saphonine (chowdhury et al., 2008), steroids (ghosh et al., 2008), isoflavonoids (josep et al., 2004), essential oil (cavalcanti et al., 2004), alkaloids and tannins (khanna et al., 2007) reported as mosquitocidal agents. dengue fever is considered as a serious public health problem in the world. in tropical countries, where the favorable environmental conditions are responsible for the proliferation of vector aedes aegypti. among the arbovirus in india, distribution of all the dengue virus type is continuously expanding. remarkably the reemergence of chikungunya virus (chik) since 2005 is posing an additional concurrent diseases burden in the country. aedes aegypti (l) (diptera: culicidae) is a fresh water breeding mosquito it is very difficult to control during rainy season.approximately 2500 million people, two fifths of the world’s population, are now at risk from dengue fever (fulmali et al., 2008; kumar et al., 2008). the who currently estimates there may be 50 million cases of dengue fever worldwide every year (who, 2011). lamiaceae have been traditionally used in developing countries for their insecticidal and repellent property against several insect correspondence: devarajan natarajan, natural drug research laboratory, department of biotechnology, school of biosciences, periyar university, salem, tamilnadu, india. fax: +91.427.2345124 (office). e-mail: mdnataraj@rediffmail.com ; natarajpu@gmail.com key words: mosquitoes repellent activity; ocimum canum; gc-ms analysis; aedes aegypti. acknowledgements: the author’s express their sincere thanks to tnscst, tamilnadu for the finance assistance under the student project scheme (tnscst/sps/td/2013-2014). acknowledged to vit university (sif) and st. joseph college, for provide spectral analysis of gc-ms for ft-ir and hplc. we also acknowledge the department of biotechnology, periyar university, salem 636 011 for providing laboratory facilities for baseline work. received for publication: 6 january 2015. revision received: 7 july 2016. accepted for publication: 7 july 2016. ©copyright o. prabhavathi et al., 2016 licensee pagepress, italy journal of entomological and acarological research 2016; 48:4938 doi:10.4081/jear.2016.4938 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2016; volume 48:4938 mosquitocidal properties of ocimum canum sims (lamiaceae) leaf extracts against dengue vector aedes aegypti l. (diptera: culicidae) o. prabhavathi, r. yuvarajan, d. natarajan natural drug research laboratory, department of biotechnology, school of biosciences, periyar university, salem, tamilnadu, india no nco mm er cia l u se on ly [page 346] [journal of entomological and acarological research 2016; 48:4938] species (ngamo et al., 2007). for example, rosmarinus officinalis and lavandulaan gustifolia showed moderate larvicidal activity (conti et al., 2010). prajapati et al. (2005) reported the essential oils from selected plants as noticeable repellent and ovicidal properties, hyptissu aveolens has useful insecticidal (amusan et al., 2005; jaenson et al., 2006) and control many stored product pests (peerzada, 1997; othira et al., 2009; conti et al., 2011). moreover, its chemical composition and biological activity might be changed due to the function origin and collection time of plants (noudjou et al., 2007). ocimum canum (lamiaceae) is a pubescent erect much branched herb, 15-60 cm high with sub-quadrangular striate branches. leaves are elliptic-lanceolate, glabrous and gland dotted strongly aromatic herb, widely distributed in throughout india (especially in fields of waste lands, plains and lower hills). it contain several volatile oils include methyl cinnamate, methylheptenone, methylnonylketone, dcamphor, citral, ocimin, methylchavicol, linalool, nevadensin, salvigenin, beta-sitosterol, betulinic, ursolic, oleanolic acids, flavanoids, pectolinarigenin-7-methylether and nevadensin, respectively. polysaccharides composed of xylose, arabinose, rhamnose and galacturonic acids (rastogi and mehrotra, 1993). the main biological properties of o. canum reported as antimicrobial, antioxidant, anthelmintic and anti-diabetic agents (bhattacharjee, 2001; chopra, 1956). the present study was focused on to perform the mosquitocidal activity and identifying the potential bioactive compounds from o. canum against dengue vector aedes aegypti. materials and methods collection of plant materials the fresh leaves of o. canum were collected (during the month of november and december 2013) from pottaneri village, mettur (tk), salem district, tamil nadu. the plant specimen was authenticated by dr. d. natarajan, assistant professor, department of biotechnology, periyar university, salem and also cross-checked with available books and herbarium records. the voucher specimen was deposited in the ndrl for further reference. the collected plants leaves were washed with tap water to remove unwanted solid dust particles and shade dried at room temperature for 10 days. the dried plant material was powdered separately using commercial electrical blender. preparation of extracts the processed plant materials (500 g) were sequentially extracted by hot extraction method in a soxhlet apparatus using three organic solvents (acetone, chloroform and hexane) for 48 to 72 h until the influx solvent changed into colorless. the plant extracts were filtered through whatman filter paper no. 1. extracts were concentrated under reduced pressure at 40°c using rotary vacuum evaporator. the dried crude extracts were weighed for calculating their extractive value and stored in an air tight container at 4°c for further bioassays. mosquito source aedes aegypti (larva, pupa and adult) mosquitos were collected from ncdc, connoor, tamil nadu, india. it was maintained at natural drug research laboratory, department of biotechnology, periyar university, salem. the larvae were kept in plastic trays containing tap water, and maintained at 27±2°c with 75-85% relative humidity under 14:10 h light and dark. larvae were fed with diet of yeast, dog biscuits and sugar solution for adult mosquitos. bioassay tests larvicidal bioassay the larvicidal activities of selected plant crude extracts were assessed as per who protocol (1981). briefly, in a container 25 fourth instar larvae were kept in 249 ml of distilled water with 1ml of different concentrations (100, 200, 300, 400 and 500 mg/l) of plant extracts. the chamber containing the control larvae received 1 ml of dmso served as negative control. after 24 h exposure, the dead larvae were counted and corrected using abbott’s formula and the percentage mortality was recorded from the average of three replicates. the average mortality percentages of three replicates were used to carry out lethal concentrations (lc50 and lc90) by probit analysis. pupicidal bioassay pupicidal activities of crude extracts was evaluated as per the modified method of modify as krishnappa et al. (2012). for the bioassay in a container, 25 pupae were kept in 249 ml of distilled water with 1 ml of extract at different concentrations (100, 200, 300, 400 and 500 mg/l) in dmso. the container received 1 ml of dmso served as negative control. all containers were maintained at room temperature (28±2) with naturally prevailing photoperiod (12:12 h/l:d) in the laboratory. any pupa was considered to be dead if did not move when prodded repeatedly with a soft brush. after exposure period, the dead larvae were counted and mortality was corrected by abbott’s (1925) formula. the percentage mortality was recorded from the average of three replicates (finney, 1971). adulticidal bioassay a total of 15 adult female mosquitoes (3-5 days old) were treated with different concentrations (100, 200, 300, 400 and 500 mg/l) of plant crude extracts impregnated with filter papers (whom 1981). the mosquitoes were allowed to acclimatize in the holding tube for 1 h and then exposed to test paper for 1 h. mortality was recorded every 10 min throughout the exposure period. mortality of mosquitoes was determined at the end of 24 h recovery period. percentage of mortality was corrected by abbott’s formula. lc50 and lc90 with 95% confidence limits were determined using probit analysis (finney, 1971). statistical analysis the average larval (adult) mortality data were subjected to probit analysis for calculating lc50 and lc90 statistics at 95 % confidence limits of upper confidence limit (ucl) and lower confidence limit (lcl) values and chi square test were calculated using the spss 14.0. phytochemical analysis the qualitative phytochemicals (phenol, alkaloids, quinones, glycosides, flavanoids, amino acids, tannins, proteins, cabhohdrate and saponin) analysis of three different extracts (acetone, chloroform, and hexane) were performed as per the methods of behera et al. (2012). thin layer chromatography thin layer chromatography was performed as per the method of bishnu et al. (2011). silica powder was added to distilled water and mixed with magnetic stirring continuously. the slurry was poured into a clean and dried slide scattered all over the slide to make a thin film. the silica plates were activated by heating them in hot air oven at 120°c for 3 h. after 3 h, the silica plates were allowed to cool at room temperature and marked about 1cm from the bottom. the extracts were loaded at the bottom center of the slide.the beaker was saturated with suitable solvent system [methanol: chloroform (7:3)].the final solvent front was marked and the plate was dried. the developed tlc plates article no nco mm er cia l u se on ly were dried and visually observed for various bands. the rf (retension frequency) value was calculated as follows: rf = distance travelled by compound distance travelled by solvent spot visualization few pieces of iodine crystals were kept in the iodine vapor container. the plates were kept in iodine vapor and left for few hours. brown colored bands were visualized. the bands were photographed under uv trans-illuminator (low beam and high beam uv light). high performance liquid chromatography the hplc system was used to quantify the bioactive compounds from chloroform extracts of o. canum. the chromatographic separation was performed on a c18 column (5 mm, 250×4.6 mm i.d.) with the column temperature of 35°c. linear gradient elution was collected (with methanol and aqueous) and the procedure as follows (v/v):5 to 25% (0 to 8 min), 25 to 55% (8 to 20 min) and 55 to 100% (20 to 30 min). the uv detector is used as 280 nm (deo et al., 2011). gc-ms analysis the gc-ms analyses of chloroform extract of plant was done by perkin elmer q-700 equipment. column temperature was programmed at 35°c-180°c for 2 min and simultaneously, increased about 4°c20°c/min. helium was used as the carrier gas at 0.9 ml/min. the mass spectrum was obtained at 70 ev ionization voltage. the identification of individual compound was done using mass spectral database (the nist (version 3.0) database). furthermore, the retention time (rt) and kovats index (ki) values of reference compounds were compared with isolated compounds for identification cheng et al. (2009). fourier transform infrared spectroscopy an arid zone ft-ir equipped with dtgs detector was used to measure the spectrum developed in chloroform extracts of o. canum. ft-ir spectrum was analyzed a thin transparent oil films were made by pressing two nacl discs (25×5 mm) of the liquid derivatives. absorption spectra were acquired at 4 cm-1 resolution and signal-averaged over 32 scans. interferegrams were fourier transformed using cosine apodization for optimum linear response. spectra were baseline corrected, scaled for mass differences and normalized to the methylene peak at 2927 cm-1. results and discussion larvicidal activity mortality percentage was calculated for its toxicity effect of plant crude extracts (acetone, hexane and chloroform) against fourth instar larvae of a. aegypti. the mortality percentage of larvae was calculated 24hrs of exposure. the highest larval mortality found in choloroform extract of o. canum plant against ivth instar larvae of ades agypti with the lc50 and lc90 values of 15.027 and 353.49 mg/l. whereas the acetone extract exhibits moderate activity with lc50 and lc90 values of 58.873 and 134.47 mg/l respectively (table 1). recently, the researcher reported that ocimum plant extract shows better larvicidal activity against larval and adults of mosquito species (pratheeba et al., 2015; murugan et al., 2016). pupicidal activity the pupal mortality of ades agypti (after treatment of o. canum plant extract) was observed at various concentrations (table 1). the [journal of entomological and acarological research 2016; 48:4938] [page 347] article t ab le 1 . m o sq u it o ci d al a ct iv it y o f o . ca n u m le af e xt ra ct s ag ai n st a . ag yp ti . o . c an u m l ar vi ci da l pu pi ci da l a du lt ic id al e xt ra ct s c on . % l c 50 lc 90 s lo pe c on . % l c 50 lc 90 s lo pe c on . % l c 50 lc 90 s lo pe m g/ l m or ta li ty m g/ l m or ta li ty m g/ l m or ta li ty ac et on e 1 00 4 2 5 8. 87 3 1 34 .4 7 -6 .3 23 1 00 4 4 4 1. 91 2 2 04 .2 5 -3 .0 23 1 00 4 2 1 60 .7 1 4 19 .2 7 -6 .7 89 2 00 4 8 2 00 4 8 2 00 4 3 3 00 5 2 3 00 4 8 3 00 4 8 4 00 5 8 4 00 5 2 4 00 5 3 5 00 6 6 5 00 8 8 5 00 5 9 ch lo ro fo rm 1 00 5 6 1 5. 02 7 3 53 .4 9 -1 .1 1 00 5 5 7 2. 26 5 1 19 .3 8 -1 0. 93 1 00 7 0 2 2. 66 2 1 56 .4 3 -2 .0 70 2 00 6 8 2 00 7 2 2 00 7 6 3 00 7 8 3 00 7 6 3 00 8 8 4 00 9 1 4 00 8 0 4 00 1 00 5 00 9 7 5 00 8 0 5 00 1 00 h ex an e 1 00 1 2 1 90 .5 0 3 41 .4 7 -1 1. 58 1 00 5 3 9 2. 26 5 1 59 .2 8 7. 93 10 0 1 2 1 08 .3 8 8 42 .3 2 -1 .3 79 2 00 2 7 2 00 5 6 2 00 3 2 3 00 4 2 3 00 7 3 3 00 5 6 4 00 5 5 4 00 7 3 4 00 6 8 5 00 5 7 5 00 7 7 5 00 6 1 co n. , c on ce nt ra tio n; l c 5 0, le th al c on ce nt ra tio n 50 ; l c 9 0, le th al c on ce nt ra tio n 90 . no nco mm er cia l u se on ly [page 348] [journal of entomological and acarological research 2016; 48:4938] mortality rate was increased on the basis of concentration/dose (low to high) of the extracts. the highest pupal mortality was observed in acetone extract with low lc50 value 41.912 mg/l respectively. similarly, the above result was supported by various scientists like selvakumar et al. (2015) reported that the better pupicidal activity of different solvents extracts of annona reticulata and gokulakrishnan et al., (2013) were identified essential oils from pogostemon cablin and evaluated the pupicidal activity against various mosquitoes including a. aegypti. adulticidal activity the result of adulticidal activity of chloroform leaves extract of o. canum show maximum adulticidal property with very low lc50 values (22.662 mg/l) compared with other solvents like acetone and hexane (table 1). the results highlights the leaf and flower of o. sanctum were tested against fourth instar larvae of aedes aegypti and they determined the lc50 values at various concentrations of extract like 425.94, 150.40, 350.78, 575.26 and 175.67 mg/l (mohamed et al., 2008). similarly, govindarajan et al. (2014) reported the larvicidal, and adulticidal potential of the chloroform extract from the erythrina indica tested against a. aegypti. the highest larval mortality and adulticidal activity were noticed in methanol extract of e. indica. hexane leaves extracts of citrus sinesis against the early fourth instars and female adult of aedes aegypti. the hexane extract from c. sinensis leaves are proved to be reasonably larvicidal but remarkably irritant against dengue vector (lc50 and lc90 values of 446.84 and 1370.96 mg/l respectively) was noticed after 24 h exposure (radhika et al., 2011). methanolic leaf extract of s. campanulata have the potential to be used as an ideal ecofriendly approach the control of mosquitoes especially a. aegypti (karthika devi et al., 2013). ethanolic and petroleum ether extracts from various parts of r. nasutus, d.elliptica, t. reidioides, h. aromatica, s. tuberose and a. calamusi were tested for their larvicidal activity potential against a. aegypti mosquitoes (naruman komalamisra et al., 2005). the larval toxicity and smoke repellent potential of albizzia amara and o. basilicum at different concentrations against aedes aegypti, resultedthat the a. amara was more effective against a. aegypti than o. basilicum (murugan et al., 2007). the adulticidal and repellent activities of crude hexane, chloroform, benzene, acetone and methanol extracts of the leaf of cassia tora leaves against a. aegypti. among them, methanol extract was showed significant activity (duraisamy amerasan et al., 2012). kamaraj et al. (2008) reported high larval mortality in methanol extracts of cryptocoryne auriculata and solanum torvum against the larvae of an. subpictus (lc50 44.21, 44.69, 53.16, 41.07, 35.32, 28.90 and 44.40 ppm; lc90 187.31, 188.29, 233.18, 142.66, 151.60, 121.05, 192.11 ppm, respectively) and cx. tritaeniorhynchus (lc50 69.83, 51.29, 81.24, 71.79, 44.42, 84.47 and 65.35 ppm; lc50 335.26, 245.63, 300.45, 361.83, 185.09, 351.41 and 302.42 ppm, respectively). bioefficacy of plant extracts differ from species to species of plants. the changes in adulticidal activity of these extracts is probably due to variation in the types and levels of active ingredients that depend not only on the genetic characteristics of the plant species but also the conditions under which they were grown and harvested (tawatsin et al., 2006). phytochemical analysis phytochemical analysis of o. canum reveals that the acetone extracts indicate the presence of phenol, saponins, tannins and proteins (table 2). chloroform extract show the presence of phenol, flavonoids, saponins, protein and carbohydrates and the hexane extracts showed the presence of flavonoids, saponins, glycosedes and proteins. saponins and proteins are presented in all the tested extracts. similarly, the results were reported that the presence of volatile oils, flavonoids, carbohydrates, phytosterols, tannins and fixed oils from the leaves extracts of o. americanum (sarma et al., 2011). the results revealed that the presence of phytochemicals viz., alkaloids, saponins, tannins, steroids, phlobatannin, terpenoids, flavinoids and cardiac glycosides in the ocimumspeceis (muhammad neem abbas et al., 2013). the phytochemical in the peels of r. sativuscontain most important phyto-constituents like tannins, saponins, flavonoids, amino acids, terpenoids, cardiac glycosides and chalcones (safia janjua et al., 2014) and also the results were suggested in o. sanctum (himal paudel chetri et al., 2008). thin layer chromatography profile for chloroform extracts of o. canum the results of thin layer chromatography profile show different band formations (figure 1) based on the solvent systems (methanol and chloroform) percentage of 5%, 10%, 20% and 25%, which shows different rf value. the rf values are 0.038, 0.192, 0.615, 0.803 and 1cm. based on the rf value suggested and assumed some bioactive components using confirmation test. the qualitative and quantitative analysis of major constituents from o. sanctum includes eugenol, qurcetine and some others by tlc (rawat et al., 2011) and ursolic acid (kedar kumar rout et al.,2012). o. gratissimum, o. sanctum and o. canum leaves extracts were showed the better band formation with the rf values of o. canum (0.513, 0.40, 0.58, 0.38 and 0.82) referred to lutein pheophytin xanthophyll oil chlorophyll b and �-carotene, o. sanctum (0.445, 0.59, 0.74, 0.934) and o. gratissimum (0.431, 0.573, 0.78) (quereshi et al., 2011). eugenol and methyl eugenol from the petroleum ether extracts of o. sanctum (nasare, 2013). high performance liquid chromatography analysis of o. canum the hplc analysis of o. canum leaf crude chloroform extracts results show major peaks (more concentration of components) at the retention times (min.) of 3.482, 5.459, 9.960 and12.688 (at wavelength of 254 nm) (table 3 and figure 2). the previous studies of hplc analysis were reported that the presence ofeugenol (joshi et al., 2011) and phenolic compounds and terpenes (shanmuga sundaram et al., 2011) identified from the leaves of o. sanctum. similarly, the total content of bioactive compounds from the species of lamiaceae family plants, flavonoids derivatives were identified from the thymus species (kulevanova et al., 2001). flavonoid content from the methanolic and aqueous extracts of o. sanctum and o. kilimandsacharicum (deo et al., 2011), the flavonoid and total phenolic contents of methanolic extract was higher than aqueous extract of stachys inflata (sayyed mehdy et al., 2011), the content of rosmarinic acid was quantified from the methanol crude extract of perilla frutescens (jing liu, 2013). article table 2. phytochemical analysis of crude extract of o. canum. phytochemical testacetone hexane chloroform phenol + + quinons flavonoids + + alkaloids amino acids saponins + + + glycosides + proteins + + + tannins + carbohydrates + -, absence;+, presence. no nco mm er cia l u se on ly fourier transform infrared spectroscopy analysis of chloroform extracts of o. canum ft-ir analysis of chloroform extracts show the presence of some functional groups with corresponding intensity peaks o-h stretching or h bending for alcohols or phenols groups of active compounds,c-h stretching which was correspond to alkenes, n-h bend for 1º amines, cc stretching was denoted the aromatic ring (table 4). c-h(-ch2x) which is assumed alkyl halides groups of bioactive compounds then cn stretching, which may indicates aliphatic amines. c-cl stretching which may denoted for alkyl halides groups of component, c-h oop for aromatics groups of bioactive component, c-cl stretching for corresponds to alkyl halides, c-br stretching for alkyl halides groups and c=c-h or ch bending denoted for alkynes (figure 3). the results of ft [journal of entomological and acarological research 2016; 48:4938] [page 349] article figure 1. thin layer chromatography profile for chloroform extracts of o. canum. figure 2. high-performance liquid chromatogram of chloroform extracts of o. canum. table 3. high-performance liquid chromatography analysis of crude extracts of chloroform leaf extract of o. canum. peak ret. tim area height area% height % 1. 3.482 19975 365 8.170 8.284 2. 5.459 4502 144 1.841 3.255 3. 9.960 133262 2438 54.504 55.264 4. 12.688 86759 1465 35.485 33.198 no nco mm er cia l u se on ly [page 350] [journal of entomological and acarological research 2016; 48:4938] ir highlights the bioactive phenyl propanoid from o. sanctum (upadhyaya et al., 2014) and crude extracts of o. bacillicum (gabi baba et al., 2012) which showed active functional groups. gc-ms analysis of chloroform extracts of o. canum gc-ms analysis of crude chloroform extracts of o. canum were identified as contain seventeen bioactive compounds (figure 4 and 5). among them, six (bicyclo[2.2.1]heptan-2-one,1,7,7-trimethyl-,(is), azulene,1,2,3,3a,4,5,6,7-octahydro-1,4-dimethyl-7-ci-methylethenyz),[ir-(1.alpha.,3a.beta,4 alp., methyl8,11,14-heptadeca-trienoate,2,6, 10,14,8, 22tetracosahexaene,2,6,10,15,19,23-hexamethyl(all-e)-, eicosane, hentriacontane)were considered as major and remaining minor compounds (caryophyllene,z,z-6,28-heptatriactontadien-2-one, z,z-6,28-heptatriactontadien-2-one, phytol, cis-1-chloro-9-octadecene, 3,7,11,15-tetramethyl-2-hexadecen-1-ol, hentriacontane, 5-acetoxy methyl-2,6,10-trimrthyl-2,9-undecadien-6-ol, 2h-1-benzopyran-6-ol, 3,4-dihydro-2,5,7,8-tetramethyl-2-(4,8,12-trimethyltridecyl),acetate,[2r-[gamma.-sitosterol) based on the retention time, peaks, molecular weight and percentage of area.similar kind of work was done by several researchers with different lamiaceae plant species i.e., stachys oblique (harmandar et al., 1997), origanum dictamnus, teucrium polium and lavandula vera (proestos et al., 2006), teucrium marum subsp. marum (ricci et al., 2005), nepeta argolica (skaltsa et al., 2000) and thymus comosus (pavel et al., 2009) and supports the findings of the present study. article table 4. fourier transform infrared spectroscopy analysis of chlorofom leaf extract of o. canum. s. no peak range types and functional group bonding pattern 1 3377.12 alcohols, phenols o-h str (s),h band (b) 2 2930.77 alkanes c-h stretching 3 1572.21 1° amines n-h (bend) 4 1403.82 aromatics c-c (str) (in ring) 5 1260.71 alkyl halides c-h (-ch2x) 6 1122.19 aliphatic amines c-n (str) 7 1031.51 aliphatic amines c-n (str) 8 923.13 carboxylic acid o-h (bend) 9 831.98 alkyl halides c-cl (str) 10 757.23 aromatics c-h ‘oop’ 11 696.11 alkyl halides c-cl str 12 651.77 alkyl halides c-br str 13 618.90 alkynes -c=c-h;ch bend figure 3. fourier transform infrared spectroscopy analysis of chloroform extract of o. canum. no nco mm er cia l u se on ly [journal of entomological and acarological research 2016; 48:4938] [page 351] article figure 4. gc-ms analysis of chloroform extracts of o. canum. no nco mm er cia l u se on ly [page 352] [journal of entomological and acarological research 2016; 48:4938] conclusions the chloroform extracts of ocimum canum showed highest mortality against a. aegypti vector in all stages. presence of potential phytoconstituents was identified using preliminary screening test and the functional groups of phytoconstituents were characterized by ft-ir and hplc. the structural derivation of the identified compounds was carried out by gc-ms analysis. the overall results concluded that the chloroform extracts contain potential bioactive compounds for the mosquitoes repellent, which can be useful for development of green based mosquitocidal 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[journal of entomological and acarological research 2016; 48:4938] [page 353] article no nco mm er cia l u se on ly [page 354] [journal of entomological and acarological research 2016; 48:4938] rastogi r.p., mehrotra b.n., 1993 compendinum of indian medicinal plants. publications and information directorate, new delhi: 454-456. rawat, p., kumar m., rahuja, n., srivastava, s.d.l., srivastava, a.k., maurya, r., 2011 synthesis and antihyperglycemic activity of phenolic c-glycosides. biorgan. med. chem. lett. 21: 28-33. ricci d., fraternale d., giamperi l., bucchini a., epifano f., burini g., curini m., 2005 chemical composition, antimicrobial and antioxidant activity of the essential oil of teucrium marum (lamiaceae). j. ethnopharmacol. 98: 195-200. rout k.k., singh r.k., barik d.p., mishra s.k., 2012 thin-layer chromatographic separation and validated hptlc method for quantification of ursolic acid in various ocimum species. j. food drug analy. 20: 865-871. saikoteswarsarma d., venkatasureshbabu a., 2011 pharmacognostic and phytochemical studies of ocimum americanum. j. chem. pharm. res. 3: 337-347. selvakumar b., gokulakrishnan j., elanchezhiyan k., deepa j., 2015 mosquito larvicidal, ovicidal and pupicidal activities of annona reticulata linn (annonaceae) against aedes aegypti (linn.), anopheles stephensi liston and culex quinquefasciatus (say) (diptera : culicidae). int. j. recent sci. res. 6: 2690-2696. shanmuga sundaram r., gowtham l., ramanathan m., manikandan p., venugopal v., kamalakannan d., nayak b.s., 2011 quantification of bioactive principles in indian traditional herb ocimum sanctum linn. (holy basil) leaves by high performance liquid chromatography. asian j. biomed. pharma. sci. 1: 35-41. skaltsa h.d., lazari d.m., loukis a.e., constantinidis t., 2000 essential oil analysis of nepeta argolica bory & chaub. subsp. argolica (lamiaceae) growing wild in greece. flavour fragr. j. 15: 96-99. sohia s.m.h., eghdami a., sadeghi f., 2011 antioxidant activity and high performance liquid chromatography analyzation of methanolic and aqueous extract of stachys inflata. org. chem. j. 1: 36-41. tawatsin, a., asavadachanukorn, p., thavara, u., 2006 repellency of essential oils extracted from plants in thailand against four mosquito vectors and oviposition deterrent effects against aedes aegypti. southeast asian. j. trop. med. public health. 37: 915-931. thomas t.g., rao s., lal s., 2004 mosquito larvicidal properties of an indigenous plant ipomoea cairica, linn. japan j. infect. dis. 57: 176-177. upadhyaya s., tewari sn., behera j., 2014 isolation and characterization of a bioactive phenylpropanoid from ocimum sanctum l. leaves through chromatographic and spectroscopic methods. am. chem. sci. j. 4: 286-297. warikoo r., ray a., sandhu j.k., samal r., wahab n., kumar s., 2012 larvicidal and irritant activities of hexane leaf extracts of citrus sinensis against dengue vector aedes aegypti l. asian pacific j. trop. biomed. 152-155. world health organization, 1981 instructions for determining susceptibility or resistance of mosquito larvae to insecticides. who/vbc-81, pp 807. world health organization, 2011 dengue and dengue haemorrhaghic fever. -who factsheet: 117. article no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract insecticide susceptibility tests using world health organization papers treated with 4% dichloro-diphenyl-trichloro-ethane (ddt), 0.05% deltamethrin, 0.05% lambda-cyhalothrin, 0.5% etofenprox, 0.15% cyfluthrin and 0.75% permethrin were conducted in kamhororo, masakadza and chilonga villages, zimbabwe. three to 5-day old female anopheles gambiae sensu lato adult mosquitoes were used. deltamethrin knocked down 100% of the mosquitoes from kamhororo, masakadza and chilonga at 35 min exposure. ddt did not knock down 100% of the mosquitoes from kamhororo and masakadza but did so in chilonga. one hundred percent knockdown was achieved for cyfluthrin when exposed to mosquitoes from kamhororo (60 min), masakadza (25 min) and chilonga (25 min). etofenprox knocked down 100% of the mosquitoes collected from kamhororo (30 min), masakadza (30 min) and chilonga (55 min). knockdown of mosquitoes due to deltamethrin, ddt, cyfluthrin, permethrin; lambda-cyhalothrin and etofenprox were different at different observation times. one hundred percent mortality due to deltamethrin, ddt, etofenprox, lambdacyhalothrin and cyfluthrin was recorded for mosquitoes collected from all the 3 sites. one hundred percent mortality due to pemethrin was recorded for mosquitoes collected from kamhororo and chilonga but mortality was 98.5% for those collected from masakadza. no knockdown or mortality occurred in the controls from each locality. the kd50 (knockdown of 50% of the mosquitoes) values were 24.4-73.7 min (ddt), 8-13 min (pemethrin), 9.4-16.3 min (cyfluthrin), 9.4-14.4 min (etofenprox), 8.7-13 min (lambda-cyhalothrin) and 12.1-15.9 min (deltamethrin). the kd90 (knockdown of 90% of the mosquitoes) values were 45.6-199.5 min (ddt), 14.7-26.5 min (pemethrin), 16.5-34.9 min (cyfluthrin), 21.8-24.4 min (etofenprox), 16.3-31.6 min (lambdacyhalothrin) and 21-25.3 min (deltamethrin). no insecticide resistance was recorded from the 3 sites. introduction malaria control is largely based on the use of long-lasting insecticide-treated nets and indoor residual spraying, but the efficacy of these control methods is endangered by the appearance of insecticide resistance in vector mosquitoes. malaria in zimbabwe causes significant mortality and morbidity although control efforts aimed at the main vector, anopheles arabiensis, are instituted annually (midzi et al., 2004, unpublished data). anopheles gambiae sensu stricto, anopheles arabiensis, and anopheles funestus sensu stricto are the most important species for malaria transmission in africa (kawada et al., 2011). insecticide resistance is a reduction in sensitivity of an insect population as reflected by repeated failure of an insecticide to achieve the expected level of control when used according to recommendations (who, 1998). insecticide resistance is mediated by behavioral, metabolic or physiological factors that result from: reduction in insecticide penetration, an increased metabolism of insecticide by metabolic enzymes and/or modification of the insecticide target site (who, 1998). world health organization (who, 1998) standards state that a mortality of 98-100% indicates susceptibility (no resistance); 80-97% journal of entomological and acarological research 2012; volume 44:e19 correspondence: nzira lukwa, national institute of health research, p.o. box cy573, causeway, harare, zimbabwe. tel. +263.4.797052; +263.274664 fax: +263.4.253979. e-mail: nziraa33@yahoo.co.uk key words: resistance, permethrin, ddt, etofenprox, deltamethrin, cyfluthrin, lambda-cyhalothrin. authors’ contributions: nl and ss, research concept and design, data collection tools, data analysis and interpretation, manuscript drafting, revision and final approval; pm, improving methodology, data collection tools, data analysis, revision and final approval; mz, submitted manuscript revision, data analysis and manuscript final approval. acknowledgments: the authors would like to acknowledge the following people who participated in data collection: vimbai chikwavaire, clever matiringe, joel mbedzi, white soko, tonderai chiwade, richard mawoyo, aleck mogove tozivepi, letters nyoni, vitalis kwashira, darlington mukotsi, gumbo, chiketa, chin’ombe, johane muchenje, cosmas bvute, peter ndaima and munjodzi vhiriri. we also thank dr s.l. mutambu, director of the national institute of health research who supported this research. funding: the authors are grateful to mitsui agro, japan, for funding this data collection study through the national malaria control programme. received for publication: 7 june 2012. revision received: 8 october 2012. accepted for publication: 14 november 2012. ©copyright n. lukwa et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e19 doi:10.4081/jear.2012.e19 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. insecticide susceptibility tests conducted in kamhororo, masakadza and chilonga villages in zimbabwe during the 2011 malaria period n. lukwa,1 s. sande,2 p. munosiyei,3 m. zimba4 1national institute of health research, causeway, harare; 2national malaria control programme, causeway, harare; 3bindura university of science education, bindura; 4university of zimbabwe, biological sciences, mount pleasant, harare, zimbabwe [journal of entomological and acarological research 2012; 44:e19] [page 107] no nco mm er cia l u se on ly suggests the possibility of resistance that needs to be confirmed and less than 80% indicates resistance. however, when more than 100 mosquitoes have been used per insecticide, less than 95% mortality strongly indicates resistance. however, no standards on knockdown times are specified to indicate resistance according to the who (1998). pyrethroid insecticide resistance in an. gambiae is mainly associated with reduced target site sensitivity arising from a single point mutation in the sodium channel gene, often referred to as knock-down resistance (awola et al., 2007). the susceptibility status of an. funestus to insecticides remains largely unknown in most parts of africa because of the difficulty in rearing field collected mosquitoes; but this is not the case with an. gambiae (morgan et al., 2010). although insecticides have been used for a very long time in zimbabwe, there are very few instances when resistance has been recorded (munhenga et al., 2008). three cases of insecticide resistance have been documented in zimbabwe; one in chiredzi involving benzene hexa-chloride (green, 1982), one involving dichloro-diphenyltrichloro-ethane (ddt) in gokwe (masendu, 2004; masendu et al., 2005, unpublished data) and one involving ddt and permethrin in gokwe (munhenga et al., 2008). munhenga et al. (2008) observed insecticide resistance to permethrin from an. arabiensis mosquitoes collected from gwave, a locality 11 km from kamhororo and 16 km from masakadza. munhenga et al., (2008) also recorded ddt resistance in gwave (68.4% in 2006) but this reversed in 2008 (96% mortality). insecticide susceptibility tests did not show any significant increase in the resistance status for either permethrin or ddt but an improvement in susceptibility over a 3-year period (awola et al., 2007). chanda et al. (2011) detected insecticide resistance to ddt, deltamethrin, lambdacyhalothrin and permethrin in both an. gambiae ss and an. funestus ss collected in zambia. abilio et al., (2011) detected insecticide resistance to lambda-cyhalothrin, permethrin and bendiocarb in an. funestus collected in mozambique. an. funestus mosquitoes were resistant to 0.75% permethrin and 0.05% deltamethrin (morgan et al., 2010). there was suspected resistance to 4% ddt but these mosquitoes were fully susceptible to bendiocarb, malathion and dieldrin (morgan et al., 2010). djogbenou et al. (2011) observed full susceptibility to chlorpyrifosmethyl and very few samples displayed resistance to carbosulfan. yewhalaw et al. (2011) observed that an. arabiensis mosquitoes were resistant to ddt, permethrin, deltamethrin and malathion, but susceptible to propoxur. djogbenou et al. (2011) noted that insecticide susceptibility differs with geographical variation and this must be taken into account in the vector control strategies. for this reason, we conducted insecticide susceptibility tests in 3 different locations in zimbabwe. materials and methods study areas mosquito collection was performed in midlands province, gokwe south district, kamhororo village (17°51’s, 28°38’e), masakadza village (17°49’s, 28°36’e) and masvingo province, chiredzi district, chilonga village (21°13’s, 31°39’e). the kamhororo river runs through the village of the same name. it starts as an artesian well and flows for over 14 km. this is the major source of water for washing and domestic animals. there are no agricultural activities taking place along the river. however, cotton is grown extensively in the village and a lot of crop spraying takes place. chances of pesticides getting into the river system are high when washing clothes and spraying equipment; washing facilities have been provided but the water flows back into the river. mosquito larval collection was performed from hoof prints (a large number of cows are present and they drink this water. masakadza village, 5 km from kamhororo, is also on the kamhororo river, but mosquitoes were collected from a swamp that also started from an artesian well and flows for 1 km (this does not flow into the kamhororo river). water is used for washing (there are no designated washing facilities) and watering animals. cotton growing is also widespread in the village. no agricultural activities near the swamp are conducted. both kamhororo and masakadza are in dry areas where rainy water is limited. chilonga village is spanned by the expansive runde river that flows for over 50 km. kitchen gardening is the most common method of farming in the villages although the river passes through large sugar estates in the low veldt district of chiredzi. mosquito collection mosquito larvae were collected from breeding sites using larval scoops and placed in white plastic dishes (figure 1). the collected larvae were morphologically identified and separated for rearing; the kamhororo field insectary was used for kamhororo and masakadza mosquitoes, the chilonga field insectary was used for chilonga mosquitoes. the identified an. gambiae sl mosquitoes were reared according to awola et al. (2007) and the adults were provided with 10% sugar solution on cotton wool placed as a wick in a 50 ml glass bottle. unfed 3-5 day old an. gambiae sl adults from the same study area were pooled together as this is the time/stage at which a sizable mosquito sample was obtained. susceptibility tests who papers treated with 4% ddt, 0.05% deltamethrin, 0.05% lambda-cyhalothrin, 0.5% etofenprox, 0.15% cyfluthrin and 0.75% permethrin were used according to the who (1998). the who (1998) states that knock-down rates should be measured every 10 min up to 60 min, but we made observations every 5 min so that we could detect even small differences. the who (1998) also states that in the event that 80% knockdown is not achieved after 60 min, the samples should be held for a further 20 min. we did not do this because two-thirds of the study sites had 80% of the mosquitoes knocked down within 60 min. the who (1998) states that 20-25 mosquitoes should be placed in each exposure tube (125 mm in length and 44 mm in diameter) but we used 15-20 mosquitoes before recording mortality after 24 h. all adult mosquitoes were removed from exposure tubes, provided with sugar water and held for 24 h. a total of 360, 240 and 236 mosquitoes from masakadza, kamhororo and chilonga were posed to treated papers, respectively. the controls consisted of 50 mosquitoes in each study site. all exposure tubes were held in the vertical position. the insecticide treated papers were used once. figure 1. collection of mosquito larvae. article [page 108] [journal of entomological and acarological research 2012; 44:e19] no nco mm er cia l u se on ly determination of kd50 and kd90 kd50 (min required to 50% knockdown of the mosquitoes) and kd90 (min required to achieve 90% knockdown of the mosquitoes) were calculated using probit analysis. this uses the regression principle and correlates fixed time with knockdown response. in circumstances in which the data are not normally distributed or do not follow a regression pattern, extrapolation is made beyond the period of observation. data analysis data was analyzed using analysis of variance (anova) at 95% confidence limit. results there was no knockdown or mortality from the 150 control mosquitoes used in this study. effect of deltamethrin on knocking down mosquitoes deltamethrin knocked down 100% of the mosquitoes from kamhororo, masakadza and chilonga after 35 min exposure to deltamethrin (figure 2). one hundred percent mortality was recorded and no insecticide resistance was observed. there was no significant difference in knockdown of mosquitoes from chilonga/masakadza (p=0.13) and chilonga/kamhororo (p=0.42) after 5 min exposure to deltamethrin but a significant difference was seen in comparison with those from kamhororo/masakadza (p=0.03) (table 1). there was no significant difference in knockdown of mosquitoes from chilonga /masakadza (p=0.22) and chilonga/kamhororo (p=0.09) at the 10 min observation time-point but a significant difference was seen in comparison with those from kamhororo/masakadza (p=0.003). there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.64), chilonga/kamhororo (p=0.47) and chilonga/masakadza (p=0.33) at the 15 min observation time-point. there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.59), chilonga/kamhororo (p=0.33) and chilonga/masakadza (p=0.301) at the 20 min observation time-point. there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.69), chilonga/kamhororo (p=0.49) and chilonga/masakadza (p=0.72) at the 25 min observation time-point. there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.27), chilonga/kamhororo (p=0.59) and chilonga/masakadza (p=0.27) at the 30 min observation time-point. there was no difference in knockdown rates from 35-60 min for mosquitoes collected from either of the study sites. effect of dichloro-diphenyl-trichloro-ethane on mosquito knockdown one hundred percent knockdown was not achieved for mosquitoes collected from kamhororo and masakadza apart from those from chilonga when exposed to ddt (figure 3). one hundred percent mortality was recorded and no insecticide resistance was observed. there was no significant difference in knockdown of mosquitoes from chilonga/masakadza (p=0.42), chilonga/kamhororo (p=0.42) and masakadza/kamhororo (p=0.27) after 5 min exposure to ddt (table 2). a significant difference was found in knockdown of mosquitoes from chilonga/masakadza (p=0.012) and kamhororo/ masakadza (p=0.048) compared with those from chilonga/kamhororo (p=0.42) for which no significant difference was found at the 10 min observation time-point. there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.057), chilonga/kamhororo (p=0.52) and chilonga/masakadza (p=0.81) at the 15 min observation time-point. there was no significant difference in knockdown of mosquitoes from kamhororo/masakadza (p=0.057), chilonga/kamhororo (p=0.085) and chilonga/masakadza (p=0.078) at the 20 min observation time-point. knockdown of mosquitoes from kamhororo/masakadza (p=0.06), chilonga/kamhororo (p=0.14) and chilonga/masakadza (p=0.32) were not significantly different at 25 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.16), chilonga/kamhororo (p=0.1) and chilonga/masakadza (p=0.11) were not significantly different at 30 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.01), chilonga/kamhororo (p=0.02) and chilonga/ masakadza (p=0.03) were significantly different at 35 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.005), chilonga/kamhororo figure 2. knockdown rate of mosquitoes due to exposure to deltamethrin. table 1. knockdown of deltamethrin at each exposure time. knockdown chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 2.5ab mean 0b mean 8.5a range (0-5) range (0) range (5-10) 10 mean 25%cd mean 7.5d mean 31.5c range (20-30) range (5-10) range (30-35) 15 mean 72.5e mean 47.5e mean 58.5e range (65-80) range (20-75) range (50-65) 20 mean 87.5f mean 77.5f mean 81.5f range (85-90) range (70-85) range (75-85) 25 mean 95g mean 87.5g mean 91.5g range (90-100) range (80-95) range (80-100) 30 mean 97.5h mean 92.5h mean 100h range (95-100) range (85-100) range (100) 35 mean 100 mean 100 mean 100 range (100) range (100) range (100) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. article [journal of entomological and acarological research 2012; 44:e19] [page 109] no nco mm er cia l u se on ly (p=0.02) and chilonga/masakadza (p=0.045) were significantly different at 40 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.000) and chilonga/kamhororo (p=0.006) were significantly different at 45min observation time apart from chilonga/masakadza (p=0.07) that were not significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.001) and chilonga/kamhororo (p=0.01) were significantly different at 50min observation time apart from those from chilonga/masakadza (p=0.097) that were not significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.004), chilonga/masakadza (p=0.04) and chilonga/kamhororo (p=0.02) were significantly different at 55 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.000), chilonga/kamhororo (p=0.000) and chilonga/masakadza (p=0.03) were significantly different at 60 min observation time. effect of cyfluthrin in knocking down mosquitoes cyfluthrin knocked down 100% of the mosquitoes from kamhororo (60 min), masakadza (25 min) and chilonga (25 min) (figure 4). one hundred percent mortality was recorded and no insecticide resistance was observed. knock down of mosquitoes from chilonga/masakadza (p=0.77) and masakadza/kamhororo (p=0.31) were not significantly different at 5 min exposure to cyfluthrin apart from those from chilonga/kamhororo (p=0.037) that were significantly different (table 3). knock down of mosquitoes from chilonga/masakadza (p=0.8), kamhororo/ masakadza figure 3. knock-down rate of mosquitoes due to dichlorodiphenyl-trichloro-ethane. figure 4. knockdown of mosquitoes due to exposure to cyfluthrin. table 2. knockdown of dichloro-diphenyl-trichloro-ethane at each exposure time. knockdown chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 0a mean 2.4a mean 0a range (0) range (0-5) range (0) 10 mean 0be mean 2.4de mean 11.5c range (0) range (0-5) range (10-15) 15 mean 10.5f mean 2.4f mean 13.5f range (0-22.2) range (0-5) range (10-15) 20 mean 36.8g mean 2.4g mean 15g range (27.8-50) range (0-5) range (10-20) 25 mean 63.2h mean 2.4h mean 25h range (38.9-94.4) range (0-5) range (15-35) 30 mean 68.4i mean 7.5i mean 28.5i range (50-94.4) range (4.8-10) range (15-45) 35 mean 78.9j mean 9.8k mean 45l range (72.2-94.4) range (9.5-10) range (35-50) 40 mean 86.8m mean 17n mean 66.5p range (83.3-100) range (10-23.8) range (60-70) 45 mean 89.5st mean 24.3q mean 81.5rt range (88.9-100) range (23.8-25) range (80-85) 50 mean 89.5wx mean 29.2u mean 83.5vx range (88.9-100) range (23.8-35) range (80-85) 55 mean 92a mean 36.6b mean 86.5c range (94.4-100) range (28.6-45) range (85-90) 60 mean 100d mean 61e mean 91.5f range (100) range (60-61.9) range (90-95) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. table 3. knockdown of cyfluthrin at each exposure time. knockdown chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 15a mean 0a mean 15a range (12-18) range (0) range (5-35) 10 mean 32.5b mean 21.4b mean 33.5b range (22.5-42.5) range (14.3-27.2) range (15-65) 15 mean 92.5d mean 45.2c mean 58.5c range (90-95) range (38.1-50) range (35-75) 20 mean 97.5f mean 69e mean 86.5ef range (95-100) range (63.6-71.4) range (70-100) 25 mean 100 g mean 78.6h mean 100g range (100) range (72.7-81) range (100) 30 mean 100ij mean 78.6j mean 100i range (100) range (72.7-81) range (100) 35 mean 100km mean 92.8k mean 100lm range (100) range (90.5-100) range (100) 40 mean 100n mean 95.2p mean 100n range (100) range (90.9-95.3) range (100) 45 mean 100q mean 95.2r mean 100q range (100) range (95.3-95.5) range (100) 50 mean 100s mean 95.2s mean 100s range (100) range (95.3-100) range (100) 55 mean 100r mean 95.2r mean 100r range (100) range (95.3-100) range (100) 60 mean 100t mean 100t mean 100s range (100) range (100) range (100) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. article [page 110] [journal of entomological and acarological research 2012; 44:e19] no nco mm er cia l u se on ly (p=0.53) and chilonga/kamhororo (p=0.42) were not significantly different at 10 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.44 and chilonga/masakadza (p=0.11) were not significantly different at 15 min observation time apart from those from chilonga/kamhororo (p=0.017) that were significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.2) and chilonga/masakadza (p=0.41) were not significantly different at 20 min observation time apart from those from chilonga/kamhororo (p=0.02) that were significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.049) and chilonga/kamhororo (p=0.03) were significantly different apart from those from chilonga/masakadza at 25 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.049) and chilonga/kamhororo (p=0.03) were significantly different at 30min observation time apart from those from chilonga/masakadza. knock down of mosquitoes from kamhororo/masakadza (p=1.8¥10-5) and chilonga/kamhororo (p=0.000) were significantly different at 35 min observation time apart from those from chilonga/masakadza. knock down of mosquitoes from kamhororo/masakadza (p=0.02) were significantly different apart from those from chilonga/kamhororo (p=0.09) and chilonga/masakadza at 40 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=9.3¥10-6) and chilonga/kamhororo (p=0.000) were significantly different at 45 min observation time apart from those from chilonga/masakadza. knock down of mosquitoes from kamhororo/masakadza (p=0.27), chilonga/kamhororo (p=0.42) and figure 5. knockdown of mosquitoes due to etofenprox. figure 6. knockdown rate of mosquitoes due to permethrin. table 4. knockdown of etofenprox at each exposure time. knockdown chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 2.6a mean 0a mean 25b range (0-48) range (0) range (20-30) 10 mean 25.6c mean 12.8c mean 45c range (23.8-27.8) range (0-26.3) range (35-55) 15 mean 48.7d mean 59d mean 66.5d range (38-61) range (40-78.9) range (50-80) 20 mean 87.2e mean 79.5e mean 85e range (83.3-90.5) range (65-94.7) range (75-90) 25 mean 94.2f mean 94.9f mean 96.5f range (88.9-95.2) range (95-100) range (95-100) 30 mean 97.4g mean 100g mean 100g range (94.4-100) range (100) range (100) 35 mean 97.4h mean 100h mean 100h range (94.4-100) range (100) range (100) 40 mean 97.4i mean 100i mean 100i range (94.4-100) range (100) range (100) 45 mean 100j mean 100j mean 100j range (100) range (100) range (100) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. table 5. knockdown of permethrin at each exposure time. knockdown chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 7.5a mean 0a mean 16.5b range (5-10) range (0) range (15-20) 10 mean 62.5c mean 25.6c mean 41.5c range (60-65) range (15.8-35) range (30-50) 15 mean 87.5d mean 64d mean 68.5d range (80-95) range (47.4-80) range (55-79) 20 mean 100e mean 87.2e mean 85e range (100) range (78.9-95) range (80-94.7) 25 mean 100 g mean 94.9f mean 96.5fg range (100) range (94.7-95) range (95-100) 30 mean 100h mean 94.9i mean 100h range (100) range (94.7-95) range (100) 35 mean 100h mean 97.4i mean 100h range (100) range (94.7-95) range (100) 40 mean 100j mean 97.5j mean 100j range (100) range (95-100) range (100) 45 mean 100j mean 97.5j mean 100j range (100) range (95-100) range (100) 50 mean 100j mean 97.5j mean 100j range (100) range (95-100) range (100) 55 mean 100j mean 97.5j mean 100j range (100) range (95-100) range (100) 60 mean 100j mean 97.5j mean 100j range (100) range (95-100) range (100) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference. article [journal of entomological and acarological research 2012; 44:e19] [page 111] no nco mm er cia l u se on ly chilonga/masakadza were not significantly different at 50 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.27), chilonga/kamhororo (p=0.42) and chilonga/masakadza were not significantly different at 55 min observation time and this was the same at 60 min. effect of etofenprox in knocking down mosquitoes one hundred percent knock down was achieved for mosquitoes collected from kamhororo (30 min), masakadza (30 min) and chilonga (55 min) when exposed to etofenprox (figure 5). one hundred percent mortality was recorded and no insecticide resistance was observed. knock down of mosquitoes from chilonga/masakadza (p=0.01) and masakadza/kamhororo (p=0.006) were significantly different at 5 min exposure to etofenprox apart from those from chilonga/kamhororo (p=0.4) that were not significantly different (table 4). knock down of mosquitoes from chilonga/masakadza (p=0.09), kamhororo/masakadza (p=0.08) and chilonga/kamhororo (p=0.44) were not significantly different at 10 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.72), chilonga/masakadza (p=0.31) and chilonga/kamhororo (p=0.7) were not significantly different at 15 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.79), chilonga/masakadza (p=0.61) and chilonga/kamhororo (p=0.69) were not significantly different at 20 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.72), chilonga/kamhororo (p=0.66) and chilonga/masakadza (p=0.24) were not significantly different at 25 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/masakadza (p=0.27) and chilonga/kamhororo (p=0.42) were not significantly different at 30 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/masakadza (p=0.27) were not significantly different at 35 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/masakadza (p=0.27) were not significantly different at 40 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/masakadza (p=0.27) were not significantly different at 50 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/masakadza (p=0.27) were not significantly different at 55 min observation time. knock down of mosquitoes from kamhororo/masakadza; chilonga/kamhororo and chilonga/masakadza were not significantly different at 60 min observation time. effect of permethrin in knocking down mosquitoes one hundred percent knock down was achieved for mosquitoes collected from kamhororo (20 min), masakadza (30 min) and chilonga (20 min) when exposed to permethrin (figure 6). one hundred percent mortality was recorded for mosquitoes collected from kamhororo and chilonga apart from those from masakadza (98.5%). no insecticide resistance was recorded from the 3 sites. knock down of mosquitoes from chilonga/masakadza (p=0.04) and masakadza/kamhororo (p=0.003) were significantly different at 5 min exposure to permethrin apart from those from chilonga/kamhororo (p=0.09) that were not significantly different (table 5). knock down of mosquitoes from chilonga/masakadza (p=0.09), kamhororo/masakadza (p=0.21) and chilonga/kamhororo (p=0.06) were not significantly different at 10 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.08, chilonga/masakadza (p=0.16) and chilonga/kamhororo (p=0.31) were not significantly different at 15 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.96), chilonga/ masakadza (p=0.09) and chilonga/kamhororo (p=0.24) were not significantly different at 20 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.2) and chilonga/masakadza (p=0.49) were not significantly different at 25 min observation time apart from those from chilonga/kamhororo (p=0.000) that were significantly different. knock down of mosquitoes from kamhororo/masakadza (p=2.25¥10-5) and chilonga/kamhororo (p=0.000) were significantly different at 30 min observation time apart from chilonga/masakadza. knock down of mosquitoes from kamhororo/masakadza (p=2.25¥10-5) and chilonga/kamhororo (p=0.000) were significantly different at 35 min observation time apart from those from chilonga/masakadza. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/ masakadza were not significantly different at 40min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/ kamhororo (p=0.42) and chilonga/masakadza were not significantly diffigure 7. knockdown rate of mosquitoes due to exposure to lambda-cyhalothrin. table 6. knockdown of lambda-cyhalothrin at each exposure time. knock down chilonga kamhororo masakadza (min) (%) (%) (%) 0 mean 0 mean 0 mean 0 range 0 range 0 range (0) 5 mean 10ab mean 2.5b mean 20a range (0-20) range (0-5) range (15-30) 10 mean 65c mean 10d mean 35cd range (60-70) range (10) range (15-60) 15 mean 82.5f mean 35e mean 55ef range (80-85) range (25-45) range (15-80) 20 mean 95g mean 62.5g mean 66.7g range (90-100) range (45-80) range (25-90) 25 mean 100h mean 90h mean 86.7h range (100) range (90) range (60-100) 30 mean 100i mean 100i mean 88.3i range (100) range (100) range (65-100) 35 mean 100j mean 100j mean 100j range (100) range (100) range (100) same letter in the same row denotes no significant difference; different letter in the same row denotes significant difference article [page 112] [journal of entomological and acarological research 2012; 44:e19] no nco mm er cia l u se on ly ferent at 50 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/kamhororo (p=0.42) and chilonga/ masakadza were not significantly different at 55 min observation time. knock down of mosquitoes from kamhororo/masakadza, chilonga/ kamhororo (p=0.42) and chilonga/masakadza were not significantly different at 60min observation time. one hundred percent knock down was achieved for mosquitoes collected from kamhororo (30 min), masakadza (35 min) and chilonga (25 min) when exposed to lambda-cyhalothrin (figure 7). one hundred percent mortality was recorded for mosquitoes from all the study sites and no resistance was recorded. knock down of mosquitoes from chilonga/masakadza (p=0.67) and chilonga/ kamhororo (p=0.54) were significantly different at 5 min exposure to lambda-cyhalothrin apart from those from masakadza/kamhororo (p=0.038) that were significantly different (table 6). knock down of mosquitoes from chilonga/masakadza (p=0.18) and kamhororo/masakadza (p=0.23) were not significantly different at 10 min observation apart from those from chilonga/kamhororo (p=0.008) that were significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.51) and chilonga/masakadza (p=0.37) were not significantly different at 15 min observation time apart from those from chilonga/kamhororo (p=0.04) that were significantly different. knock down of mosquitoes from kamhororo/masakadza (p=0.89), chilonga/masakadza (p=0.37) and chilonga/kamhororo (p=0.22) were not significantly different at 20 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.85), chilonga/masakadza (p=0.49) and chilonga/kamhororo (p=0.9) were not significantly different at 25 min observation time. knock down of mosquitoes from kamhororo/masakadza (p=0.49), chilonga/kamhororo and chilonga/masakadza (p=0.49) were not significantly different at 30 min observation time. one hundred percent knockdown of mosquitoes was reported for mosquitoes collected from the 3 study areas. determination of kd50 and kd90 the kd50 values were 24.4-73.7 min (ddt), 8-13 min (pemethrin), 9.4-16.3 min (cyfluthrin), 9.4-14.4 min (etofenprox), 8.7-13 min (lambda-cyhalothrin) and 12.1-15.9 min (deltamethrin) (figure 8). the kd90 values were 45.6-199.5 min (ddt), 14.7-26.5 min (pemethrin), 16.5-34.9 min (cyfluthrin), 21.8-24.4 min (etofenprox), 16.3-31.6 min (lambda-cyhalothrin) and 21-25.3 min (deltamethrin) (figure 9). discussion the time required to knock-down 100% of the mosquitoes was compared and the results indicated that there was no difference when mosquitoes were exposed to different sources of deltamethrin. ddt only managed to knock-down 100% of the mosquitoes collected from chilonga and could not knock-down mosquites from either kamhororo or masakadza. this might be due to the great pressure being placed on the mosquitoes from kamhororo and masakadza through intensive application of pesticides for cotton growing while this is not the case figure 8. kd50 values in minutes. dichloro-diphenyl-trichloro-ethane (ddt) values were extrapolated by probit analysis because 100% knockdown was not achieved. figure 9. kd90 values. dichloro-diphenyl-trichloro-ethane (ddt) values were extrapolated by probit analysis because 100% knockdown was not achieved. article [journal of entomological and acarological research 2012; 44:e19] [page 113] no nco mm er cia l u se on ly with chilonga (approx. 600 km from these 2 sites). it is worth noting that gwave (a village not very far away from kamhororo and masakadza where insecticide resistance has been detected) serves as a potential reservoir of ddt resistant mosquitoes, as observed by masendu (2004), masendu et al. (2005, unpublished data) and munhenga et al. (2008). problems with not achieving 100% knockdown when ddt was used might be an indication of knock-down resistance, as observed by awola et al. (2007). however, djiegbe et al. (2011) demonstrated that a high frequency of resistant genes does not necessarily translate into resistance in an. gambiae sl mosquitoes. it is important to study this mechanism in the follow-up studies. there was no great difference between the times required to knockdown 100% of the mosquitoes due to lambda-cyhalothrin and permethrin from the 3 sites. this is interesting because munhenga et al. (2008) recorded insecticide resistance from mosquitoes from gwave but this has not been observed for mosquitoes from kamhororo and masakadza in terms of knock-down time. the times required for 100% knockdown of mosquitoes from chilonga and masakadza (for cyfluthrin) were very similar but were abnormally high for kamhororo; the reasons for this are not known. interestingly, the time required for 100% knockdown of mosquitoes from chilonga (etofenprox) was higher than that of kamhororo and masakadza; the reasons for this are not known. it may be linked to pest control on sugar estates since there is no sugar cane cultivation in either kamhororo or maskadza. in general, knock-down rates of mosquitoes appeared to be differ according to their sources and this was also time dependent. this trend was also observed for the different insecticides under study. this highlights the need to study all the insecticide classes in order to understand this trend as this may provide useful information on the possibility of insecticide resistance developing in some localities in zimbabwe. it is also important to cover all geographical areas since this study was only carried out in dry areas where malaria is prevalent. mortality rates are encouraging from all the study sites when considering all the insecticides used in this study. no insecticide resistance was observed at the study sites. it is important to monitor trends in pemethrin response for mosquitoes from masakadza (near gwave where pemethrin resistance has been reported by munhenga et al. 2008). interestingly, this trend was not observed in kamhororo that is nearer gwave than masakdaza. thus, the absence of ddt and permethrin resistance agrees with observations of dabire et al. (2008). the kd50 and kd90 values obtained from all the study sites (for ddt) appeared to be within the same range but these were abnormally high for mosquitoes collected from kamhororo. this may signal future problems with ddt use in kamhororo, but according to munhenga et al. (2008), reversal of resistance may occur. we are not sure whether this will happen for mosquitoes collected from kamhororo. otherwise, our results show that the tested insecticides have reasonable knock-down rates in the study areas. use of probit analysis showed the difference between knock-down rates. one major observation from this program is that it extrapollates results when 100% knock down is not achieved and at times, this goes beyond the study time. these results agree with earlier observations that cases of insecticide resistance are very rare in zimbabwe, in agreement with munhenga et al. (2008), green (1982), masendu (2004) and masendu et al. (2005, unpublished data). unfortunately, munhenga et al. (2008) did not detect any knock-down resistance or mutations from gwave but no information is available for kamhororo. knock-down rates from our study agree with findings of djogbenou et al. (2011) that insecticide susceptibility differs according to geographical variations. more studies on this should be conducted across the country. references abilio a.p., kleinschmidt i., rehman a.m., cuamba n., ramdeen v., mthembu d.s., et al., 2011 the emergence of insecticide resistance in central mozambique and potential threat to the successful indoor residual spraying malaria control programme. malaria j. 2: 110. awola t.s., oduola a.o., oyewole i.o., obansa j.b., amajoh c.n., koekemoer l.l., coetzee m., 2007 dynamics of knockdown pyrethroid insecticide resistance alleles in a field population of anopheles gambiae s.s. in south-western nigeria. j. vector-borne dis. 44: 181-188. chanda e., hemingway j., kleinschmidt i., rehman a.m., ramdeen v., phiri f.n., et al., 2011 insecticide resistance and the future of malaria control in zambia. plos one 6: e24336. dabire k.r., diabate a., agostinho f., alves f., manga l., faye o., baldet t., 2008 distribution of the members of anopheles gambiae and pyrethroid knock-down resistance gene (kdr) in guineabissau, west africa. bull. soc. pathol. exotique. 101: 119-123. djegbe i., boussari o., sidick a., marting t., ranson h., chandre f., et al., 2011 dynamics of insecticide resistance in malaria vectors in benin: first evidence of the presence of l1014s kdr mutation in anopheles gambiae from west africa. malaria j. 10: 261. djogbenou l., pasteur n., akogbeto m., weill m., chandre f., 2011 insecticide resistance in the anopheles gambiae complex in benin: a nationwide survey. med. vet. entomol. 25: 256-67 green c.a., 1982 malaria epidemiology and anopheline cytogenetics. in: pal r., kitzmiller j.b., kanda t., (eds.). cytogenetics and genetics of vectors. elsevier biomedical, amsterdam: 21-29. kawada h., dida g.o., ohashi k., komagata o., kasai s., tomita t., et al., 2011 multimodal pyrethroid resistance in malaria vectors, anopheles gambiae s.s., anopheles arabiensis, and anopheles funestus s.s. in western kenya. plos one 6: e22574. masendu h.t., 2004 vector mosquitoes and their significance in malaria epidemiology and control in zimbabwe. phd thesis university of witwatersrand, south africa: 1-10 morgan j.c., irving h., okediv l.m., steven a., wondji c.s., 2010 pyrethroid resistance in an anopheles funestus population from uganda. plos one 29: e11872. munhenga g., masendu h.t., brooke b.d., hunt r.h., koekemoer l.k., 2008 pyrethroid resistance in the major malaria vector anopheles arabiensis from gwave, a malaria-endemic area in zimbabwe. malaria j. 7: 247. yewhalaw d., wassie f., steubaut w., spanoghe p., van bortel w., denis l., et al., 2011 multiple insecticide resistance: an impediment to insecticide-based malaria vector control program. plos one 12: e16066. who (world health organization), 1998 report of the who informal consultation. test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces. world health organization, geneva. available from: http://www.who.int/malaria/publications/ atoz/who_cds_cpc_ mal_98_12/en/index.html [page 114] [journal of entomological and acarological research 2012; 44:e19] article no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract the aim of this paper was to analyze the distribution of chironomids (diptera, chironomidae), and determine their substrate preferences, from two hydrosystems located in northeastern algeria: the kebir-east and the seybouse wadis. sixty-five species were recorded in 49 sampling sites distributed along the main courses of the two hydrographic nets and their tributaries. the majority of taxa comprised cosmopolitan species widely distributed along these two hydrosystems. cricotopus (cricotopus) bicinctus showed the highest abundance and frequency of occurrence (29.52%) and was widespread in almost all the sampling sites. species richness ranged from 4 to 23, shannon diversity between 0.15 and 0.90, evenness from 0.23 to 1. a cluster analysis was carried out to represent the different groups of sites sharing similar species composition. agglomerative cluster analysis grouped the sampling sites into four clusters according to the community data. an indval analysis was then carried out to detect indicator species for each group of the sampling sites. cricotopus (isocladius) sylvestris was indicator of the first group of the sampling sites. orthocladius pedestris, rheocricotopus chalybeatus and c. bicinctus were indicators of the second group, and polypedilum cultellatum of the third group. the fourth group was not characterized by any species. indval analysis allowed also to determine species preferences for substrate size: corynoneura scutellata and dicrotendipes nervosus emphasized a preference to fine gravel, and glyptotendipes pallens to fine sand. introduction mediterranean wetlands are under tremendous pressure due to numerous factors like demography, human encroachment and climate change (hollis, 1992; hulme et al., 2001). loss of wetland biodiversity can only be mitigated through critical knowledge of threats (battisti et al., 2008; gibbs, 2000). such knowledge is compromised when local biodiversity is not well understood, as is the case of northeastern algeria which houses a wide spectrum of wetlands, many of international importance (samraoui & samraoui, 2008). despite their ecological and biogeographical interests, the aquatic communities of northeastern algeria have attracted few systematic studies (samraoui & menai, 1999; samraoui & corbet, 2000; annani et al., 2012; samraoui et al., 2012). the chironomidae (diptera) constitute a highly diversified group of aquatic insects frequently occurring in high density in different kinds of ecosystems (coffman & ferrington, 1984). the chironomidae are of great significance in the structure and function of lotic systems due to their great abundance, diversity and occurrence (cranston, 1995). the larvae of this family are fundamental components in freshwater food webs, occupying different habitats within river basins, with their distribution determined by several factors; among them, substrate size has an important role in the spatial distribution of macroinvertebrate assemblages (sanseverino & nessimian, 2001; brooks et al., 2005). the understanding of the relationship between species and environment is essential; therefore, every assessment will be more accurate if habitat preferences and indicator species are known (legendre & legendre, 1998; mcgeoch & chown, 1998; tickner et al., 2000). despite their importance, little is known of habitat preferences of chironomids, especially in the southern mediterranean, including algeria (lounaci et al., 2000a; lounaci et al., 2000b; arab et al., 2004; belaidi et al., 2004; chaib et al., 2011a; chaib et al., 2011b). sampling in several wadis (water courses with very irregular hydrocorrespondence: nadjla chaib, département de génie des procédés, faculté de technologie, université du 20 août 1955, bp 26 route el-hadaëk, skikda, algeria. e-mail: nadjla21@yahoo.fr key words: algeria, chironomidae, spatial distribution, substrate-type, indval analysis, cluster analysis. acknowledgements: the work was supported by the algerian ministère de l’enseignement supérieur et de la recherche scientifique (dgrsdt/m.e.s.r.s.) and the king saud university deanship of scientific research, research group project no: rgp-vpp-135. dsfp, king saud university, saudi arabia. received for publication: 11 october 2012. revision received: 12 november 2012. accepted for publication: 21 december 2012. ©copyright n. chaib et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e2 doi:10.4081/jear.2013.e2 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. chironomid (diptera, chironomidae) species assemblages in northeastern algerian hydrosystems n. chaib,1,2 a. fouzari,2 z. bouhala,2 b. samraoui,2,3 b. rossaro4 1département de génie des procédés, faculté de technologie, université du 20 août 1955, skikda, algeria; 2laboratoire de recherche et de conservation des zones humides, université du 8 mai 1945 guelma, algeria; 3center of excellence for research in biodiversity, king saud university, riyadh, saudi arabia; 4department of food, environmental and nutritional sciences (defens), università degli studi di milano, milano, italy [page 4] [journal of entomological and acarological research 2013; 45:e2] journal of entomological and acarological research 2013; volume 45:e2 jear_2013_1:hrev_master 23/04/13 09.55 pagina 4 no n c om me rci al us e o nly logic regime) in kabily du djurdjura, northern algeria (moubayed et al., 2007) generated a list of 87 chironomid species from this area: 8 belonged to tanypodinae, 3 to diamestinae, 57 to orthocladinae and 19 to chironomiae; 10 species were not described. a total of 53 species were new records for algeria, 25 of which being also new records for north africa. a survey of chironomids from the kebir-east wadi and its tributaries in northeastern algeria (chaib et al., 2011b) generated a list of 37 widespread chironomid species in the palearctic. they include 5 tanypodinae, 15 orthocladiinae, 4 tanytarsini and 13 chironomini. a similar study was carried out in the seybouse basin, where 45 chironomid species were collected. this study aims to analyze the composition of chironomidae assemblages in the northeastern algerian hydrosystems along the kebir-east and the seybouse wadis. the spatial distribution of the assemblages was examined and chironomids were correlated with substrate size in order to investigate substrate preferences. materials and methods study area forty-nine sampling sites were chosen along the main course of the kebir-east (23) and the seybouse wadis (26) and their tributaries on the base of land-use and anthropogenic impacts (chaib & samraoui, 2011; chaib et al., 2011a, 2011b; khelifa et al., 2011) (figure 1, table 1). according to the subdivision of hydrographic nets in the eastern region of algeria by the agence des bassins hydrographiques (abhcsm), our two river systems, the subject of this paper, belong to two different basins: i) the kebir-east belongs to the watershed of the côtiers constantinois est in the extreme north-east of algeria, and covers a catchment area of 1600 km2; and ii) the seybouse basin is the largest sub-basin in the northeastern region, covering an area of 6570 km2. these two fluvial systems are an important source of water in the northeastern algeria, since they supply water for irrigation of large agricultural areas extending from the regions of guelma to el kala. both the seybouse and kebir-east basins represent a mosaic of geomorphodynamic natural conditions, as well as diverse levels of manmade disturbances of a variety of origins (physical: bouhalloufa and mexa dams for the kebir-east and bouhamdane dam for the seybouse; chemical: presence of non-point pollutions, and municipal and industrial wastewater). the climate is typically mediterranean with a hot and dry summer from june to september, and a cold and rainy winter from october to may. the substratum of the kebir-east wadi is composed either of ancient sediments (marls and sandstone) of the algerian local marine miocene (equivalent to the continental aquitanien), degraded slightly on the surface in the east, or more recent plio-quaternary sediments corresponding to alluviums of the high and middle terraces of the kebir-east wadi valley. the recent quaternary sediments in the valley of kebireast wadi comprise silt, sand and stones (marre, 1987). the watershed of the seybouse wadi drains water very slowly over a gentle relief from its source in the highlands of sellawa and heracta. in the uplands, it flows through a very fractured and complex structured topography, where the hydrographic net is rarely adapted to the structure (ghachi, 1986). the effluents are torrential and the longitudinal contours are irregular and stretched. the seybouse river flows through some depressions containing an alluvial water table (c.g.g., 1971; djabri et al., 2003). this allows regulation of winter precipitations received by the mountain range. when the river reaches the plain of annaba, it loses its energy and leaves behind a great load of sediments. the geomorphological characteristics of the plain, gentle slope, sand dune barrier, and inundation-prone areas allow the river to flow easily into the mediterranean sea. all chironomid samples were collected with a surber net (300 �m mesh size, 50 cm width). sampling was carried out during spring (march-may) and summer (july-september) from 2008 to 2011. ten hauls were made in the opposite sense of the current along the sam[journal of entomological and acarological research 2013; 45:e2] [page 5] article figure 1. location of the 49 sampling sites along the kebir-east and the seybouse wadis and their tributaries (northeastern algeria). jear_2013_1:hrev_master 23/04/13 09.55 pagina 5 no n c om me rci al us e o nly [page 6] [journal of entomological and acarological research 2013; 45:e2] article table 1. list of the sampled sites located along the kebir-east and seybouse wadis and their tributaries. no. watercourse names of the sampled sites code latitude longitude altitude substrate size substrate (n) (e) (m) size classes 1 o. leben leb 8°30’32’’ 36°46’56’’ 77 very coarse gravel 5 2 o. mellili mel 8°30’28’’ 36°46’50’’ 80 very fine gravel 4 3 o. kebir at r’mel souk rsk 8°30’10’’ 36°46’55’’ 80 very fine gravel 4 4 o. louar amont lam 8°22’58’’ 36°36’52’’ 652 stones 6 5 o. louar aval lav 8°21’56’’ 36°39’01’’ 200 silt 1 6 o. bougous amont bam 8°21’53’’ 36°39’06’’ 203 very fine gravel 4 7 o. bougous aval bav 8°24’27’’ 36°42’36’’ 69 very coarse gravel 5 8 o. kebir at ain assel kas 8°21’57’’ 36°45’59’’ 30 very fine gravel 4 9 oubéïra 3 ob3 8°23’10’’ 36°51’47’’ 24 silt 1 10 oubéïra 2 ob2 8°25’15’’ 36°51’29’’ 24 silt 1 11 oubéïra 1 ob1 8°24’12’’ 36°49’29’’ 22 silt 1 12 o. messida aval mam 8°24’09’’ 36°49’23’’ 22 very fine sand 2 13 o. messida amont mav 8°22’30’’ 36°47’37’’ 25 very fine sand 2 kebir-east wadi 14 o. kebir ain khiar kak 8°18’51’’ 36°46’49’’ 23 very coarse sand 3 15 o. guergour grg 8°16’52’’ 36°46’32’’ 25 very coarse sand 3 16 o. kebir guergour kgr 8°16’43’’ 36°46’36’’ 25 silt 1 17 o. bourdim brd 8°14’50’’ 36°47’22’’ 20 very fine gravel 4 18 o. zitoun zit 8°13’02’’ 36°39’06’’ 193 stones 6 19 o. dardan drd 8°13’16’’ 36°46’39’’ 15 silt 1 20 o. kebir at anenes kan 8°12’48’’ 36°47’48’’ 14 very fine sand 2 21 o. kebir at righia krg 8°09’35’’ 36°48’51’’ 11 silt 1 22 o. boulathan blt 8°06’06’’ 36°49’42’’ 8 very fine sand 2 23 o. kebir at sebaa ksb 8°09’07’’ 36°48’59’’ 10 silt 1 24 barrage sedrata bsd 36°03.516’ 7°27.209’ 744 silt 1 25 cherf à sedrata cps 36°04.479’ 7°29.640’ 747 silt 1 26 oued krab okr 36°07.210’ 7°32.780’ 778 silt 1 27 cherf à ksar sbahi cks 36°03.207’ 7°19.557’ 751 silt 1 seybouse wadi 28 oued el nil onl 36°08.380’ 7°26.731’ 775 very coarse gravel 5 29 oued dbabcha odb 36°12.945’ 7°19.047’ 609 very coarse gravel 5 30 oued el maleh oml 36°08.893’ 7°8.642’ 742 very fine sand 2 31 oued beni mheni obm 36°09.207’ 7°19.557’ 668 very fine sand 2 32 barrage ain makhlouf bmk 36°13.528’ 7°17.783 643 stones 6 33 oued el aare oar 36°13.572’ 7°19.186’ 609 stones 6 34 cherf à ain makhlouf cmk 36°14.462’ 7°18.626’ 600 stones 6 35 oued cheniouraffluent och 36°14.877’ 7°20.610’ 742 stones 6 36 cherf à ain hsainia chs 36°25.415’ 7°18.788’ 270 very fine gravel 4 37 cherf à medjez amar cma 36°26.526’ 7°18.677’ 273 very fine gravel 4 38 bouhamdane à hammam debagh bhd 36°28.012’ 7°15.673’ 305 very fine gravel 4 39 bouhamdane à mermoura bmr 36°26.522’ 7°16.292’ 480 stones 6 40 bouhamdane à medjez amar bma 36°36.592’ 7°18.615’ 274 very fine gravel 4 41 seybouse à salah salahsalah sss 36°27.697’ 7°20.382’ 251 stones 6 42 seybouse à el –fedjouj sfj 36°28.893’ 7°24.926’ 222 stones 6 43 oued zimba – effluent ozm 36°26.020’ 7°18.452’ 291 very fine sand 2 44 oued bradâa obr 36°30.803’ 7°27.037’ 285 very coarse sand 3 45 oued helia – effluent ohl 36°25.415’ 7°18.788’ 144 stones 6 46 seybouse à zemzouma szm 36°24.795’ 7°36.676’ 143 very fine sand 2 47 seybouse à boudaroua sbd 36°31.667’ 7°42.307’ 100 very fine gravel 4 48 seybouse à chihani sch 36°41.002’ 7°45.527’ 12 very coarse sand 3 49 seybouse à dreân sdr 36°39.216’ 7°46.968’ 18 very fine gravel 4 jear_2013_1:hrev_master 23/04/13 09.55 pagina 6 no n c om me rci al us e o nly [journal of entomological and acarological research 2013; 45:e2] [page 7] article table 2. list of species recorded in 49 sites from two northeastern algerian watercourses (kebir-east and seybouse wadis) between 2007 and 2011. subfamilies are presented in phylogenetic order and genera in alphabetic order. taxa frequency of occurrence (%) total abundance of species tanypodinae conchapelopia pallidula (meigen, 1818)* 1.17 103 procladius choreus (meigen, 1804)° 0.75 66 rheopelopia ornata (meigen, 1838)# 1.12 99 tanypus punctipennis meigen, 1818° 1.36 120 zavrelimyia punctatissima (goetghebuer, 1934)* 0.19 17 diamesinae sympotthastia spinifera (serra-tosio, 1968)# 0.01 1 prodiamesinae prodiamesa olivacea (meigen, 1818)° 0.05 4 orthocladiinae cardiocladius fuscus kieffer, 1924# 0.82 72 corynoneura scutellata winnertz, 1846° 0.14 12 cricotopus (cricotopus) bicinctus (meigen, 1818)° 29.52 2606 cricotopus (isocladius) sylvestris (fabricius, 1974)° 9.39 829 eukiefferiella bedmari vilchez-quero & laville, 1987* 0.25 22 eukiefferiella claripennis (lundbeck, 1890)° 0.8 71 eukiefferiella gracei (edwards, 1929)* 0.01 1 eukiefferiella ilkleyensis (edwards, 1929)* 0.09 8 eukiefferiella sp.1 (thienemann a., 1926)* 0.02 2 hydrobaenus distylus (potthast, 1914)# 0.93 82 hydrobaenus sp.1 fries, 1830* 0.02 2 limnophyes minimus (meigen, 1818)* 0.01 1 metriocnemus sp.1 van der wulp, 1874* 0.03 3 orthocladius (euorthocladius) ashei soponis, a., 1990* 0.25 22 orthocladius (euorthocladius) rivicola kieffer, 1911# 3.15 278 orthocladius (orthocladius) excavatus brundin l., 1947* 1.36 120 orthocladius (orthocladius) rubicundus (meigen, 1818)* 0.61 54 orthocladius pedestris kieffer, 1909# 3.18 281 paracladius conversus (walker, 1856)# 0.01 1 parakiefferiella gracillima (kieffer, 1924)# 0.05 4 parametriocnemus stylatus (kieffer, 1924)° 0.27 24 paraphaenocladius sp.1 thienemann, 1924* 0.01 1 paratrichocladius rufiventris (meigen, 1830)* 2.38 210 paratrissocladius excerptus walker, 1846# 0.12 11 psectrocladius (psectrocladius) psilopterus (kieffer, 1906)* 0.11 10 psectrocladius sordidellus (zetterstedt, 1838)# 0.02 2 rheocricotopus chalybeatus (edwards, 1929)# 3.32 293 rheocricotopus fuscipes (kieffer, 1909)* 1.6 141 thienemanniella vittata (edwards, 1924)° 0.33 29 tanytarsini cladotanytarsus mancus (walker, 1856)# 1.08 95 cladotanytarsus sp.1 kieffer, 1921* 0.35 31 micropsectra atrofasciata (kieffer, 1911)# 0.06 5 paratanytarsus sp.1 (thienemann a. & bause, 1913)* 1.22 108 rheotanytarsus photophilus (goetghebuer, 1921)# 0.71 63 rheotanytarsus sp.1 thienemann a. & bause, 1913* 0.51 45 tanytarsus sp.1 van der wulp, 1874° 1.11 98 chironominae chironomus plumosus (linnæus, 1758)° 2.41 213 chironomus riparius meigen, 1804# 7.15 631 chironomus sp.1 meigen, 1803* 2.7 238 cryptochironomus defectus (kieffer, 1913)* 0.32 28 cryptochironomus rostratus kieffer, 1921# 0.11 10 cryptotendipes sp.1 beck & beck, 1969* 0.01 1 dicrotendipes nervosus (stäger, 1839)° 0.82 72 einfeldia sp.1 kieffer, 1924* 0.01 1 genus near tribelos* 0.23 20 glyptotendipes pallens (meigen, 1804)# 0.08 7 harnischia fuscimana (kieffer, 1921)° 0.1 9 microchironomus tener (kieffer, 1818)# 0.01 1 microtendipes pedellus (de geer, 1776)° 0.74 65 paracladopelma camptolabis (kieffer, 1913)# 0.01 1 phaenopsectra flavipes (meigen, 1818)* 0.02 2 polypedilum (tripodura) scalaenum (schrank, 1803)° 3.09 273 polypedilum cultellatum (goetghebuer, 1931)° 8.7 768 polypedilum laetum (meigen, 1818)* 0.03 3 polypedilum nubifer (skuse, 1889)* 4.73 418 robackia sp.1 sæther o.a., 1977* 0.02 2 synendotendipes dispar (meigen, 1830)# 0.19 17 synendotendipes impar (walker, 1856)* 0.01 1 *species recorded in the seybouse wadi. °species recorded in both the kebir-east and the seybouse wadis. #species recorded in the kebir-east. total abundance was calculated as the abundance from all samples (n=49) pooled together. jear_2013_1:hrev_master 23/04/13 09.55 pagina 7 no n c om me rci al us e o nly [page 8] [journal of entomological and acarological research 2013; 45:e2] pling station, in the middle of the current and near the banks. samples were preserved in 5% formaldehyde (larvae, pupae), and then examined under a dissecting microscope. the specimens were grouped by morphotypes according to external characteristics visible through the stereomicroscope in the laboratory. subsequently permanent mounts were prepared in faure or in balsam mounting medium, to enable the taxonomic determination of the different morphotypes. dataset a list of species derived from different studies carried out in northeastern algeria between 2007 and 2011 (chaib et al., 2011b) were collected in a database. the list was based on larval collections, with species identification aided by collection of prepupae and of mature pupae. italian keys for larvae determination were used (ferrarese, 1983; ferrarese & rossaro, 1981; nocentini, 1985; rossaro, 1982) along with keys for palaearctic pupae (langton & visser, 2003). substrate size was ranged into six classes (iso 14688-1) according to the particle size: i) class 1: silt, <0.063 mm; ii) class 2: fine sand, 0.063-0.200 mm; iii) class 3: medium-coarse sand, 0.200-2 mm; iv) class 4: fine-medium gravel, 2-20 mm; v) class 5: coarse gravel, 20-63 mm; vi) class 6: cobble, 63-200 mm. data analysis quantitative samples collected in the 49 sampling sites were considered in the statistical analyses; the mean abundance of each taxon per site was considered. rare species were included in the analysis (smith et al., 2001) resulting in a total of 65 taxa. relative abundances of species were transformed by log (10) to normalize counts. to avoid a problem of logarithm zeroes, the value 1 was added to each abundance. groups of samples sharing the same type of community composition were defined using a hierarchical cluster analysis (goodall, 1973) with ward’s linkage method and euclidian distance measure. multi-response permutation procedures (mrpp) (biondini et al., 1985) were used to test the reliability of the groups obtained. the species characterizing each cluster were identified with indicator species analysis (indval; dufrêne & legendre, 1997). a second indval analysis was carried out considering the sampling sites grouped based on substrate size. this method combines information on the concentration of species abundances in a particular group and the faithfulness of occurrence of a species in a particular group. indicator values were tested for statistical significance using a randomization (monte carlo) technique (mccune & grace, 2002). the significance was tested by carrying out 10,000 indval analyses. agglomerative cluster analysis, mrpp and indval analysis were performed with the r environment 2.15.1 (r development core team, 2009). species richness, diversity and evenness indices were also calculated (shannon & weaver, 1949). results sixty-five chironomidae taxa belonging to four subfamilies were identified (table 2). orthocladiinae showed the highest generic richness (29 taxa). this subfamily showed a proportional abundance of 59%, chironominae 31%. tanytarsini and tanypodinae were the less frequent and abundant with approximately 6% and 5%, respectively. c. bicinctus showed the highest abundance (2606 ind*m-2) and frequency of occurrence (29.52%) and was widespread in almost all the sampling sites (table 2), followed by c. sylvestris and polypedilum cultellatum with total abundance of 829 and 768, and a frequency of occurrence of 9.39% and 8.7%, respectively. total abundance and the frearticle figure 2. agglomerative cluster dendrogram based on chironomid communities sampled in the kebir-east and the seybouse wadis (2008-2011) (see table 1 for site codes and names of sampling sites for each group). jear_2013_1:hrev_master 23/04/13 09.55 pagina 8 no n c om me rci al us e o nly [journal of entomological and acarological research 2013; 45:e2] [page 9] article table 3. chironomids diversity, richness and evenness for the each sampling sites (see table 1 for site codes; see figure 2 for clusters). no. sampled sites cluster diversity species evenness richness 1 leb 1 0.75 14 0.66 2 mel 2 0.64 14 0.58 3 rsk 1 0.81 11 0.75 4 lam 1 0.75 15 0.66 5 lav 3 0.81 12 0.76 6 bam 3 0.63 11 0.57 7 bav 4 0.80 14 0.73 8 kas 4 0.79 22 0.72 9 ob3 3 0.75 8 0.76 10 ob2 4 0.64 9 0.64 11 ob1 4 0.72 9 0.71 12 mam 4 0.84 16 0.76 13 mav 3 0.74 15 0.64 14 kak 1 0.64 11 0.54 15 grg 1 0.62 11 0.59 16 kgr 2 0.62 12 0.54 17 brd 2 0.80 14 0.72 18 zit 4 0.15 5 0.23 19 drd 4 0.82 13 0.76 20 kan 1 0.75 9 0.73 21 krg 4 0.71 12 0.59 22 blt 4 0.73 11 0.66 23 ksb 4 0.76 12 0.70 24 bsd 4 0.25 9 0.28 25 cps 4 0.83 19 0.72 26 okr 4 0.77 9 0.77 27 cks 3 0.90 18 0.86 28 onl 4 0.78 8 0.85 29 odb 4 0.89 15 0.87 30 oml 4 0.83 9 0.89 31 obm 4 0.63 9 0.61 32 bmk 4 0.72 7 0.77 33 oar 4 0.75 4 1.00 34 cmk 4 0.89 20 0.83 35 och 4 0.77 23 0.68 36 chs 1 0.81 17 0.74 37 cma 4 0.89 20 0.85 38 bhd 4 0.73 10 0.75 39 bmr 4 0.80 11 0.81 40 bma 4 0.70 17 0.64 41 sss 4 0.85 17 0.78 42 sfj 4 0.89 17 0.86 43 ozm 4 0.84 23 0.72 44 obr 4 0.77 12 0.76 45 ohl 4 0.78 19 0,74 46 szm 1 0.85 17 0.78 47 sbd 4 0.76 15 0.74 48 sch 4 0.81 16 0.78 49 sdr 1 0.81 15 0.73 table 4. chironomid indicator species by group of sites and substrate size (indval analysis). taxa substrate assemblages size taxa max p max p class class tanypodinae conchapelopia pallidula 4 0.378 6 0.324 procladius choreus 3 0.102 1 0.235 rheopelopia ornata 3 0.144 5 0.575 tanypus punctipennis 1 0.749 4 0.519 zavrelimyia punctatissima 4 0.292 3 0.581 diamesinae sympotthastia spinifera 2 0.057 4 0.746 prodiamesinae prodiamesa olivacea 4 0.719 4 0.356 orthocladiinae cardiocladius fuscus 2 0.042 3 0.417 corynoneura scutellata 2 0.643 4 0.032 cricotopus (cricotopus) bicinctus 2 0.011 4 0.510 cricotopus (isocladius) sylvestris 1 0.029 5 0.809 eukiefferiella bedmari 4 0.691 6 0.571 eukiefferiella claripennis 2 0.112 6 0.923 eukiefferiella gracei 4 1.000 4 0.776 eukiefferiella ilkleyensis 4 1.000 6 0.270 eukiefferiella sp.1 4 1.000 6 0.898 hydrobaenus distylus 2 0.116 4 0.807 hydrobaenus sp.1 4 1.000 3 0.455 limnophyes minimus 3 0.164 1 1.000 metriocnemus sp.1 3 0.390 1 0.757 orthocladius (euorthocladius) ashei 3 0.537 6 0.745 orthocladius (euorthocladius) rivicola 3 0.934 6 0.796 orthocladius (orthocladius) excavatus 4 0.609 2 0.342 orthocladius (orthocladius) rubicundus 3 0.750 6 0.502 orthocladius pedestris 2 0.007 4 0.129 paracladius conversus 4 1.000 2 0.314 parakiefferiella gracillima 2 0.114 4 0.271 parametriocnemus stylatus 2 0.572 4 0.297 paraphaenocladius sp.1* 4 1.000 6 0.538 paratrichocladius rufiventris 4 0.265 3 0.542 paratrissocladius excerptus 3 0.941 6 0.057 psectrocladius (psectrocladius) psilopterus 4 0.724 2 0.497 psectrocladius sordidellus 1 0.569 2 0.725 rheocricotopus chalybeatus 2 0.009 3 0.971 rheocricotopus fuscipes 4 0.387 6 0.118 thienemanniella vittata 2 0.533 1 0.139 tanytarsini cladotanytarsus mancus 1 0.586 4 0.466 cladotanytarsus sp.1 4 0.609 3 0.732 micropsectra atrofasciata 4 1.000 4 0.754 paratanytarsus sp.1 1 0.945 4 0.684 rheotanytarsus photophilus 2 0.319 5 0.539 rheotanytarsus sp.1 4 0.189 6 0.248 tanytarsus sp.1 4 0.811 3 0.764 chironominae chironomus plumosus 2 0.276 1 0.536 chironomus riparius 4 0.744 2 0.156 chironomus sp.1 4 0.661 6 0.534 cryptochironomus defectus 1 0.805 6 0.428 cryptochironomus rostratus 3 0.065 4 0.632 cryptotendipes sp.1 1 0.335 2 0.309 dicrotendipes nervosus 2 0.761 4 0.033 einfeldia sp.1 1 0.349 5 0.150 genus near tribelos 4 1.000 1 1.000 glyptotendipes pallens 3 0.379 2 0.036 harnischia fuscimana 2 0.638 6 0.276 microchironomus tener 3 0.140 4 0.756 microtendipes pedellus 3 0.655 5 0.441 paracladopelma camptolabis 1 0.357 6 0.530 phaenopsectra flavipes 3 0.306 4 1.000 polypedilum (tripodura) scalaenum 3 0.091 1 0.774 polypedilum cultellatum 3 0.038 1 0.616 polypedilum laetum 1 0.776 4 0.500 polypedilum nubifer 4 1.000 6 0.211 robackia sp.1 4 0.155 1 0.937 synendotendipes dispar 2 0.052 5 0.124 synendotendipes impar 4 1 4 0.747 significant values (p<0.05) are in italics (see figure 2 for groups of sites: cluster analysis). *species recorded in the seybouse wadi. jear_2013_1:hrev_master 23/04/13 09.55 pagina 9 no n c om me rci al us e o nly [page 10] [journal of entomological and acarological research 2013; 45:e2] quency of occurrence were calculated from all samples (n=49) pooled together. species richness ranged from 4 (site 33) to 23 (sites 35 and 43), diversity between 0.15 (site 18) and 0.90 (site 27). evenness values ranged from 0.23 (site 18) to 1 (site 33) (table 3). indicator species were determined for each group of sites and then according to substrate-type. indicator values are in table 4, along with statistical significance values calculated by randomization (monte carlo) (mccune & grace, 2002). the agglomerative cluster analysis (figure 2) grouped the sampling sites into clusters according to the chironomid species. a 4-group level of the dendrogram was chosen, the mrpp results showing that the groups obtained were statistically different (a=0.302, p<0.005). corynoneura scutellata and dicrotendipes nervosus showed the lowest p values (0.032 and 0.033, respectively) and seemed to have a preference to very fine gravel, glyptotendipes pallens presented a p value of 0.036 suggesting a preference for very fine sand substrate (table 1). it should be emphasized that the number of larvae recorded of c. scutellata and g. pallens was very low; only a few larvae were captured in some stations. d. nervosus was more abundant in stations kas (20 larvae) and sdr (12 larvae). discussion and conclusions the percentage distribution of taxa within chironomid subfamilies was in accordance with previous studies (moubayed et al., 2007; chaib et al., 2011b), with orthocladiinae as the most frequent, taxon-richest and abundant subfamily. c. bicinctus was the most abundant and widely distributed taxon, confirming that the species can tolerate a wide range of substrate size (with a preference for the sandy substrate). in contrast c. scutellata was the least abundant, present only in very fine gravel substrates. the cobble and gravel substrates held the highest number of chironomids among all substrates (table 1). it was similarly observed (campbell & meadows, 1972) that cobble substrates offer the most suitable microenvironments because they supply materials used by the larvae to construct runways and to build cases about their bodies, crevices for protection, sources of attachment, they are also a source of food since rock surfaces are covered with periphyton (mosses and algae). in contrast, silty stations housed the least number of chironomids (table 1) due to the fact that they had the least favorable physical conditions, such as high current and low values of organic matter and transparency. however, p. cultellatum was found to be dominant there. references annani f., alfarhan a.h., samraoui b., 2012 aquatic hemiptera of northeastern algeria: distribution, phenology and conservation. rev. ecol. terre vie. 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[journal of entomological and acarological research 2013; 45:e2] [page 11] article jear_2013_1:hrev_master 23/04/13 09.55 pagina 11 no n c om me rci al us e o nly 429 too many requests you have sent too many requests in a given amount of time. jear2012 [journal of entomological and acarological research 2013; 45:e8] [page 33] proteolytic compartmentalization and activity in the midgut of andrallus spinidens fabricius (hemiptera: pentatomidae) s. sorkhabi-abdolmaleki,1 a. zibaee,1 h. hoda,2 r. hosseini,1 m. fazeli-dinan3 1department of plant protection, faculty of agricultural sciences, university of guilan, rasht; 2biological control department, iranian institute of plant protection, amol; 3department of plant protection, college of agriculture and natural resources, university of tehran, iran abstract digestive proteolytic activity in the alimentary canal of andrallus spinidens, a potential biocontrol agent of lepidopteran larvae, was studied by considering enzyme compartmentalization and diversity. the alimentary canal of adults consists of a foregut, a foursectioned midgut, namely v1 to v4 (ventriculus), and a hindgut. the optimal ph for general proteolytic activity was found to be at ph 8 with a small peak at ph 6. results revealed that there are several specific proteases in the midgut of a. spinidens, including trypsin-like, chymotrypsin-like, and elastase as serine proteases, and cathepsins b, l and d as cysteine proteases, in addition to two exopeptidases of carboxyand aminopetidases. compartmentalization of digestive proteolytic activity showed that v3 is the main area of proteolytic secretion for both general and specific proteases and that v4 has the lowest enzymatic role, so that four out of the eight specific proteases found showed no activity in this section. the lowest and the highest proteolytic activity was found to be in the 1st and 4th nymphal instars, respectively. using the specific inhibitors phenylmethylsulfonyl fluoride, na-p-tosyl-l-lysine chloromethyl ketone, ntosyl-l-phenylalanine chloromethyl ketone, l-trans-epoxysuccinyl-leucylamido-(4-guanidino)-butane, cystatin, phenanthroline and ethylendiamidetetraacetic acid, we verified the presence of all specific proteases noted using both biochemical assays and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. our findings demonstrated that a. spinidens could utilize several caterpillars because of the presence of various of proteases in its midgut. introduction intensive spraying of rice fields in northern iran with diazinon, chlorpyrifos, padan and fipronyl has caused acute and chronic effects on human and other non-target organisms as well as pest resistance. although an egg parasitoid, trichogramma spp., has annually been used to suppress population outbreaks of chilo suppressalis walker (lepidoptera: crambidae), economic damage still occurs. another potential biocontrol agent is andrallus spinidens f. (hemiptera: pentatomidae), a predator of larvae, which has been given less consideration. a. spinidens is widely distributed around the world (nageswara rao, 1965) and is a specialist on rice pests in india, malaysia and iran (nageswara rao, 1965; manley, 1982; mohaghegh & najafi, 2003). both nymphs and adults are noted to feed on several caterpillars like chilo suppressalis walker (lepidoptera: crambidae), naranga aenescens moore (lepidoptera: noctuidae) and helicoverpa armigera hübner (lepidoptera: noctuidae) in rice fields of northern iran (mohaghegh & najafi, 2003). a. spinidens has five generations per year, lives for 65 days and hibernates as an adult among rice debris and weeds (zibaee et al., 2012). a. spinidens, like other hemipteran predators, uses extra-oral digestion by injecting digestive enzymes into prey to obtain nutrients required for growth and reproduction (cohen, 1998; zibaee et al., 2012). these predators pump in potent hydrolytic salivary secretions and suck in the liquefied prey parts, delivering nutrients to their digestive tract (cohen, 1993, 1995). advantages of this feeding strategy include: i) the ability to circumvent the cuticle as a feeding barrier that restricts assess to nutrients; ii) the ability to bypass defensive chemicals that cause their prey to be unpalatable; and iii) the ability to allow the use of larger prey by relatively small predators (cohen, 1998). in this case, digestive proteases have critical roles in both the digestion of proteins in the bodies of prey to serve as a main source of nutrients, and detoxification of potential defensive chemicals of animal or plant origin. a previous study revealed that salivary glands of a. spinidens have two anterior, two lateral and two posterior lobes (zibaee et al., 2012). general proteolytic activity in the salivary glands demonstrated an optimal ph of 8 and an optimal temperature correspondence: arash zibaee, department of plant protection, faculty of agricultural sciences, university of guilan-rasht, 41635-1314, iran. tel.: +98.0131.6690264 fax: +98.131.6690281. e-mail: arash.zibaee@gmx.com ; arash.zibaee@guilan.ac.ir key words: digestive protease, andrallus spinidens, compartmentalization, inhibitor. acknowledgements: this study was supported by a grant from the university of guilan. we appreciate the kind assistance of the laboratory staffs of insect physiology and toxicology at the university of guilan and the insect rearing laboratory of the biological control department, iranian institute of plant protection. received for publication: 6 november 2012. revision received: 7 january 2013. accepted for publication: 1 february 2013. ©copyright s. sorkhabi-abdolmaleki et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e8 doi:10.4081/jear.2013.e8 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e8 jear_2013_1:hrev_master 23/04/13 09.55 pagina 33 no n c om me rci al us e o nly [page 34] [journal of entomological and acarological research 2013; 45:e8] of 40°c when azocasein was used as a substrate. by using specific substrates, we found that trypsin-like, chymotrypsin-like, aminopeptidase and carboxypeptidase are active proteases in the salivary glands of a. spinidens by maximal activity of trypsin-like proteases in addition to their optimal ph of 8-9 (zibaee et al., 2012). use of specific inhibitors including soybean trypsin inhibitor, phenylmethylsulfonyl fluoride (pmsf), nap-tosyl-l-lysine chloromethyl ketone (tlck), and n-tosyl-l-phenylalanine chloromethyl ketone (tpck), significantly decreased enzymatic activity in both assessment of enzymatic condition and through the use of polyacrylamide gel electrophoresis as well as assays phenanthroline, ethylene glycol tetraacetic acid and triethylenetetramine hexaacetic acid as specific inhibitors of methalloproteases (zibaee et al., 2012). endogenous trypsin inhibitors extracted from chilo suppressalis, naranga aenescens, pieris brassicae, hyphantria cunea and ephestia kuhniella had different effects on the trypsin-like protease activity of salivary glands so that the endogenous inhibitors significantly decreased the enzyme activity except for c. suppressalis (zibaee et al., 2012). taking into account the results of our previous study and regarding the importance of a. spinidens as a potential biocontrol agent, we felt it was necessary to investigate the feeding strategy and physiology as well as the ecology of the insect. therefore, the aim of the current study was to characterize the digestive proteases in the midgut of a. spinidens by using specific substrates and diagnostic inhibitors. a major objective of sustainable agriculture is to ensure the compatibility of pest control procedures with beneficial predator and parasitoid species in order to achieve enhanced control. the information obtained here could allow us to predict the outcome when this economically important beneficial insect feeds on phytophagous pests. for instance, whether it would be ecologically compatible, or else detrimental, as through the secondary ingestion of plant materials in the pest species’ body that have been modified to express protease inhibitors. materials and methods a. spinidens rearing a colony of a. spinidens was established from adults collected from harvested rice fields in amol (mazandaran, north of iran), in late september 2011. insects were reared on the fifth instars of c. suppressalis l. (lepidoptera: crambidae) as a food source and were provided with wet cotton plugs fitted into small plastic dishes (2.5 cm diameter) to serve as moisture sources, and held at 25±1°c and 80% relative humidity. sample preparation soluble fractions insects were randomly selected and the midguts (except for 1st and 2nd instar nymphs for which the whole body was used in the experiments) were removed by dissection under a stereomicroscope in an iced saline buffer (nacl, 10 mm). the bodies were cut with a scalpel to reveal the midgut, which was accessible after removal of fat bodies and other undesirable organs. the midgut was separated from the insect body, rinsed in iced distilled water, placed in a pre-cooled homogenizer and ground up before centrifugation. equal portions of midgut and distilled water were used to obtain the desired concentration of the enzymes (w/v). homogenates were transferred separately to 1.5-ml centrifuge tubes and centrifuged (kokusan enshinki co. ltd., tokyo, japan) at 13,000 rpm for 15 min at 4°c. the supernatants were pooled and stored at –20°c for subsequent analyses (zibaee et al., 2012). microvillar-bound fraction for solubilisation of microvillar-bound enzymes (exopeptidases) in triton x-100, membrane preparations (from the primary centrifuge precipitates, above) were exposed to triton x-100 for 20 h at 40°c, at a ratio of 10 mg of triton x-100 per mg of protein, before being centrifuged at 13,000 rpm for 30 min. no sediment was visible after centrifuging this supernatant at 10,000 rpm for 60 min. the activity of the enzymes remains unchanged at –20°c, for periods of at least a month, according to previous studies (ferreira & terra, 1983; zibaee, 2012). general proteolytic assay azocasein 2% (sigma aldrich co., st. louis, mo, usa) in universal buffer (see below: 2 mm, containing succinate, glycine and 2-morpholinoethanesulfonic acid; ph range 3-12) (frugoni, 1957) was used to determine general proteolytic activity in the midgut of a. spinidens adults, based on a method described by elpidina et al. (2001). the reaction mixture consisted of 50 ml of appropriate buffer solutions, 20 ml of azocasein and 20 ml of enzyme solution. after incubation at 37°c for 60 min, proteolysis was stopped by the addition of 100 ml of 30% trichloroacetic acid (tca). precipitation was achieved by cooling at 4°c for 10 min, and then centrifuging at 13,000 rpm for 10 min. an equal volume of 2 m naoh was added to the supernatant and the absorbance was recorded at 450 nm (awareness inc., burlington, ma, usa). a blank solution consisting of all the above components except for the enzyme solution served as a control. optimal ph and temperature for general proteolytic activity the reaction mixture was the same as described above, but a different ph range of universal buffer (3-12) and temperature (20-70°c) were considered, to find the optimal ph and temperature. for the optimal ph assays, 40 ml of buffer solution (at different ph levels) was incubated with azocasein as a substrate for 10 min at 30°c as a standard temperature. then 20 ml of sample were added and the experiment was repeated as above. for the optimal temperature assays, 40 ml of buffer solution (at the optimal ph determined) was incubated with azocasein as a substrate for 10 min at different temperature regimes from 20-70°c. after addition of 20 ml of the sample, the reaction mixture was incubated at each given temperature for 60 min. the remaining steps were repeated as above. specific protease assays serine proteases trypsin-like, chymotrypsin-like, and elastase-like activities (as the three subclasses of serine proteases) were assayed using a 1-mm concentration of nabenzoyl-l-arginine-p-nitroanilide (bapna), 1 mm of nsuccinyl-alanine-alanine-proline-phenylalanine-p-nitroanilide (saapppna), and 1 mm n-succinyl-alanine-alaninealanine-pnitroanilide (saaapna) (all from sigma aldrich) as substrates, respectively. the reaction mixture consisted of 35 ml of universal buffer (ph 8 as the recommended ph for serines), 5 ml of each of the substrates and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period of 0-10 min before adding 30% tca to terminate the reaction. the absorbance of the resulting mixture was then measured spectrophotometrically at 405 nm by p-nitroaniline release. to prove the specific proteolytic activity, a negative control was provided separately for each substrate that contained all above components except for the enzyme pre-boiled at 100°c for 30 min (oppert et al., 1997). cysteine proteases cathepsin b, l and d activities (as the three subclasses of cysteine proteases) were assayed using a 1-mm concentration of z-ala-arg-arg 4article jear_2013_1:hrev_master 23/04/13 09.55 pagina 34 no n c om me rci al us e o nly metjoxy-β-naphtylamide acetate, n-benzoyl-phe-val-arg-p-nitroanilide hydrochloride (both from sigma aldrich) and cathepsin d (sigmaaldrich) as substrates, respectively. the reaction mixture consisted of 35 ml of universal buffer (ph 5 as the recommended ph for cysteines), 5 ml of each substrate and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period of 0-10 min before adding 30% tca to terminate the reaction and read at 405 nm. to prove the specific proteolytic activity, a negative control was provided separately for each substrate that contained all the above components except for the enzyme preboiled at 100°c for 30 min (oppert et al., 1997). exopeptidases activities of the two exopeptidases in the midgut of a. spinidens were obtained by using hippuryl-l-arginine and hippuryl-l-phenilalanine (both from sigma aldrich) for carboxyand aminopeptidases, respectively. the reaction mixture consisted of 35 ml of universal buffer (ph 7 as the recommended ph for exopeptidases), 5 ml of each substrate and 5 ml of enzyme solution. the reaction mixture was incubated at 30°c for a period of 0-10 min before adding 30% tca to terminate the reaction and read at 340 nm. to prove the specific proteolytic activity, a negative control was provided separately for each substrate that contained all the above components except for the enzyme pre-boiled at 100°c for 30 min (oppert et al., 1997). protease compartmentalization in the four sections of midgut to determine proteolytic compartmentalization, four-sectioned midguts of a. spinidens were separated and protease activities were measured as described above. v1 to v4 (ventriculus) sections of 10 adults were dissected and pooled separately with an equal volume of distilled water to obtain the enzymatic source. proteolytic profile in different nymphal instars general and specific proteolytic activities were measured in the five nymphal instars of a. spinidens to find their differences depending on developmental stage. whole-body homogenates for total proteolytic activity were extracted for 1th and 2nd instars, and from only the midgut for the other stages. the procedure was conducted as above (oppert et al., 1997). optimal ph determination of specific proteases universal buffer was used to find the optimal ph of the serine and cysteine proteases as well as the exopeptidases by using the specific substrates described. the reaction mixture and experiment procedure were the same as above. effect of specific inhibitors on proteolytic activity the following compounds (sigma aldrich) were used to reveal any alteration of the proteolytic activity in the midgut of a. spinidens. pmsf (1, 3, 5 mm); trypsin inhibitor, tlck (1, 3, 5 mm); chymotrypsin inhibitor, tpck (1, 3, 5 mm); cysteine protease inhibitor, l-transepoxysuccinyl-leucylamido-(4-guanidino)-butane, 1, 3, 5 mm (e-64); cystatin (1, 3, 5 mm), and metalloprotease inhibitors, including phenanthroline and ethylendiamidetetraacetic acid (edta). electrophoresis zymogram electrophoretic detection (laemmli, 1970) of proteolytic enzymes was performed using resolving and stacking polyacrylamide gels of 10% and 4%, respectively according to the method described by garcia-carreno et al. (1993) (cleaver scientific swift valley, uk) with slight modifications. non-reducing polyacrylamide gel electrophoresis (page) was carried out at 4°c at a constant voltage of 75 mv when the dye reached the bottom of the glass, and the gel was carefully separated and put into universal buffer for 15 min. gels were then washed in water and put in a solution containing casein (1%) and universal buffer (ph 8). the gel was fixed and stained overnight with 0.1% coomassie brilliant blue r-250 in methanol:acetic acid:water (50:10:40). destaining was carried out in methanol:acetic acid:water (50:10:40) for at least 2 h. characterization of protease classes in sodium dodecyl sulphate-page zymograms using specific inhibitors was done according to garcia-carreno et al. (1993) with some modifications. a total of 25 ml of the enzyme extract was mixed with 15 ml of inhibitors at a 5-mm concentration (preparation of this sample was made 15 min before onset of the experiment.), including pmsf, tlck, tpck, e-64, cystatin, phenanthroline and edta for 30 min prior to loading. electrophoresis and zymogram were carried out as described above. protein determination protein concentration was measured according to the method of lowry et al. (1951). statistical analysis the experimental design was based on completely randomized statistics and tukey’s test was used to compare data. statistical differences were considered at p≤0.05 (sas, 1997). [journal of entomological and acarological research 2013; 45:e8] [page 35] article table 1. proteolytic compartmentalization in four sections of a. spinidens midgut. proteases v1 v2 v3 v4 total 0.036±0.0005c 0.05±0.012b 0.411±0.06a 0.058±0.005b trypsin-like 0.48±0.028c 20.00±1.89b 46.32±11.98a chymotrypsin-like 9.72±0.83b 43.96±9.37a elastase 2.75±0.27b 0.37±0.06c 3.55±0.55a 2.40±0.92b cathepsin b 53.79±3.53a 2.22±0.35b 0.61±0.003c cathepsin l 72.43±2.78a 64.07±11.02ab 54.78±5.47b 54.55±0.54b cathepsin d3 2.91±0.26a 1.86.86±0.072b 1.38±0.074b aminopeptidase 11.18±0.87a 5.74±0.99b 1.01±0.11c carboxypeptidase 34.67±2.56b 76.85±5.71a 81.15±12.98a 12.86±0.33c v stands for ventriculus and numbers refer to section of the midgut by ordering. a,b,c different letters show statistical differences in each row. total protease and cathspin d activities has been shown as absorbance at 410 nm. jear_2013_1:hrev_master 23/04/13 09.55 pagina 35 no n c om me rci al us e o nly [page 36] [journal of entomological and acarological research 2013; 45:e8] results dissection of adults under a stereomicroscope revealed that the alimentary canal of a. spinidens consists of a foregut, a 4-sectioned midgut (v1-v4) and a hindgut (figure 1). in the intact alimentary canal, the region from v2 to the hindgut occurs as a complex so that these sections are not observable separately (figure 1, top). soluble fractions prepared from the midgut of a. spinidens showed proteolytic activity to be dependent on ph and temperature. specifically, activity peaked at ph 6, but a statistically different optimal ph was observed at ph 8 (figure 2). an optimal temperature of 25°c was observed, above which proteolytic activity sharply decreased so that no enzymatic activity was observed at temperatures of 60 and 70°c (figure 2). compartmentalization of proteolytic activity in different parts of the midgut showed the highest enzymatic activity in v3 for both general and specific proteolytic activity (table 1). in the case of the two exopeptidases assayed, carboxypeptidase had higher activity than aminopeptidase in all four sections of the midgut (table 1). it was found that all three assayed cathepsins had the highest activity in v1, with cathepsin l being the major cathespin in all sections (table 1). tryspinand chymotrypsin-like serine proteases showed higher enzymatic activity than elastase in v3 (table 1). table 2 shows the differences in specific proteolytic activities in the various nymphal instars of a. spinidens. all assayed proteases had the lowest activity in the 1st instar and the highest was observed in the 5th instar, indicating an increase with nymphal development for the majority of enzymes (table 2). in the 1st instars, trypsin-like proteases and cathepsin l had the highest activity, but tryspin-like proteases and cathepsin b showed the highest activities in the fifth instar (table 2). in the case of exopeptidases, the 3rd and 5th instars showed the highest activities of the aminoand carboxypeptidases, respectively (table 2). figures 3-5 show the optimal ph of specific proteases using universal buffer and specific substrates. optimal ph of the three serine proarticle figure 1. alimentary tract morphology of a. spinidens. image shows a complex consisting of three sections: foregut, midgut and hindgut; midgut has been divided into v1 to v4. figure 2. optimal ph and temperature determination of general proteolytic activity in the midgut of a. spinidens adults by using soluble fraction in the presence of azocasein as substrate. all values were compared by one-way analysis of variance by using tukey’s test (p≤0.05). points marked by different letters are significantly different. jear_2013_1:hrev_master 23/04/13 09.55 pagina 36 no n c om me rci al us e o nly teases was found to be 9, 9-10 and 11 for tryspin-like, chymotrypsin-like and elsastase proteases, respectively (figure 3). optimal ph for cysteine proteases was found to be 4 for cathepsin b and 6 for both cathepsin l and d (figure 4). aminopeptidase had significant activity from ph 8-11, with a statistically optimal ph of 10, but carboxypeptidase showed the highest activity at ph 8 (figure 5). general proteolytic activity in the midgut of a. spinidens adults was further characterized using specific protease inhibitors (table 3). significant inhibition of azocaseinolytic activity by pmsf, tlck and tpck suggested that the serine proteases made the greatest contribution to protein digestion in the midgut (table 3). however, considerable inhibition of azocaseinolytic activity by tlck and tpck showed equal importance of both trypsin-like and chymotryosin-like proteases. e-64 and cystatin also decreased proteolytic activity, but the inhibitory effect of cystatin was higher than e-64 (table 3). inhibition of general proteolytic activity by phenanthroline and espectially edta revealed the presence of metalloproteases in the midgut extract of a. spinidens adults (table 3). zymogram analysis of the compartmentalization of proteolytic activity in v1 to v4 of the midgut confirmed our biochemical assays. figure 6 reveals the sharper bands of v3 in comparison with the other sections, especially v4, where proteolytic activity is represented by just a thin band. zymogram analysis of proteolytic activity in the nymphal instars also confirmed the biochemical assays (figure 7). in addition, zymogram characterization of the protease activity in the midgut of a. spinidens using inhibitors is shown in figure 8. six bands with proteolytic activity (p1-p6) were observed in the control. different classes of proteases were detected in the presence of the specific inhibitors through the disappearance or reduced intensity of the bands compared with the control. zymograms of the general serine protease inhibitor pmsf revealed a slight inhibition of band p3 and the complete disappearance of bands p4 to p6 (figure 8). trypsin-like serine proteases were determined in zymograms using tlck as an irreversible inhibitor of trypsin-like serine protease as band p4 (figure 8). tpck, as an irreversible inhibitor of chymotrypsinlike protease, had inhibitory effects on bands p5 and p6 (figure 8). cysteine proteases were inhibited by both e-64 and cystatin through the disappearance of bands p2 and p3 (figure 8). metalloproteinases were not found in the gel system using the inhibition by edta and phenanthroline through the disappearance of p2, p5 and p6 for phenanthroline and p2, p5 and p6 for edta (figure 8). discussion and conclusions results of the current study clearly indicate that digestive proteolytic activity in the midgut of a. spinidens is dynamic and dependent on the interactions among the site of secretion, ph and protease class. meanwhile, compartmentalization of enzyme secretion and digestion [journal of entomological and acarological research 2013; 45:e8] [page 37] article table 2. specific proteolytic activity (u/mg protein) in the nymphal instars of a. spinidens. larval total trypsin-like chymotryps elastase cathepsin cathepsin cathepsin aminopeptidase carboxypeptiinstar protease in-like b l d dase 1st instar 0.039±0.004d 6.98±0.04c 0c 2.34±0.01c 0c 10.92±0.085bc 0.38±0.029b 3.96±0.14c 3.29±0.85c 2nd instar 0.24±0.021cd 6.06±0.29c 12.45±1.65b 9.4±0.38bc 0.34±0.06b 20.9±0.036b 0.065±0.005c 6.63±0.098c 7.94±0.71c 3rd instar 0.33±0.017c 20.90±1.42b 11.37±3.6b 14.76±1.23b 56.13±2.19a 7.46±0.39c 0.33±0.026b 52.9±4.19a 39.76±2.12b 4th instar 0.51±0.017b 19.59±1.25b 42.12±3.61a 19.52±1.79b 48.42±1.82ab 29.12±3.25ab 0.58±0.093a 35.73±1.2b 54.31±5.36a 5th instar 0.89±0.035a 59.91±6.14a 56.34±2.25a 53.91±7.82a 100.57±7.36a 44.18±2.26a 0.57±0.025a 40.81±2.25ab 57.66±4.16a homogenates for total proteolytic activity were extracted from whole body for 1th and 2nd instars and midgut for others. the experiment was carried out at 30°c for both substrates. total protease and cathspin d activities has been shown as absorbance at 405 nm. a,b,c,d different letters in each column show significant differences among values when tukey’s test was used at p≤0.05, n=3. figure 3. optimal ph determination for serine proteases found in the midgut of a. spinidens adults. values marked by different letters are significantly different (tukey’s test (p≤0.05). jear_2013_1:hrev_master 23/04/13 09.55 pagina 37 no n c om me rci al us e o nly [page 38] [journal of entomological and acarological research 2013; 45:e8] were observed when different activities of digestive proteases were obtained by biochemical assays and page electrophoresis. also, it was found that different protease classes are active in the midgut by using specific substrates and inhibitors as well as zymogram analysis. knowledge of the alimentary canal anatomy is necessary for a complete understanding of digestive physiology in insects. a major characteristic of the hemipteran alimentary canal is a division of the midgut into four significant sections. as saxena (1963) described, the alimentary canal of hemipterans consists of a foregut as a short tube that contains the pharynx and esophagous. the midgut is sectioned into v1, v2, v3 and v4. v1 commonly resembles a large sac in which some variation can be observed, depending on species. v2 is similar to a tube and is much thinner than v1. v3 is a long section filled by a yellowish-brownish thick liquid. v4 is a small tube-like section. no developed sections can be observed such as the ileum or colon in the hindgut. our observations on the a. spinidens alimentary canal corresponds with the description of saxena (1963). briefly, the foregut is a short tube but the midgut consists of four sections, of which the first is large, and the second is thinner and smaller than the first and third. the third section is a long tube, and the fourth is a short tube similar to the hindgut. additionally, it was found in the four sectioned midgut of a. spinidens that v3 showed the highest proteolytic activity, not only for general proteases but also specific proteases, such as the trypsin-like, chymotrypsin-like, cathepsin l and carboxypeptidase. saxena (1954) and goodchiled (1966) have attributed these morphologies and functions to the hibernation physiology of bugs. a majority of hemipterans like a. spinidens hibernate as an adult, so the following conclusions could be drawn. during diapause, insects rely on food storage in the first midgut region (v1) (saxena, 1954), which would explain why v1 is large and sac-like. the low amount of proteolytic activity seen in v1 is most likearticle figure 4. ph determination for cysteine proteases found in the midgut of a. spinidens adults. values marked by different letters are significantly different (tukey’s test: p≤0.05). figure 5. optimal ph determination for two exopeptidases found in the midgut of a. spinidens adults. values marked by different letters are significantly different (tukey’s test: p≤0.05). table 3. effect of some specific inhibitors on proteolytic activity in the midgut of a. spinidens adults. compounds concentration (mm) activity (u/mg protein) control 5 0.18±0.06a pmsf 5 0.041±0.003d tlck 5 0.079±0.008c tpck 5 0.082±0.002c e-64 5 0.133±0.023ab cystatin 5 0.073±0.01cd phenanthroline 5 0.143±0.36ab edta 5 0.097±0.008b azocasein (2%) was used as substrate. a,b,c,d statistical differences have been shown by different letters following one-way analysis using tukey test (p≤0.05). pmsf, phenylmethylsulfonyl fluoride; tlck, na-ptosyl-l-lysine chloromethyl ketone; tpck, n-tosyl-l-phenylalanine chloromethyl ketone; e-64, l-transepoxysuccinyl-leucylamido-(4-guanidino)-butane; edta, ethylendiamidetetraacetic acid. jear_2013_1:hrev_master 23/04/13 09.55 pagina 38 no n c om me rci al us e o nly ly due to salivary enzymes that entered the gut along with the food consumed (saxena, 1954, 1963). the role of v2 is to connect the main storage site (v1) with the digestion and absorption site (v3) (saxena, 1954; goodchiled, 1966). v2 may also have some function in storage and digestion of food (goodchiled, 1966). the fourth region of the midgut is a place where symbionts are accommodated in nezara viridula l. (hemiptera: pentatomidae) and possibly in other hemipterans (chapman, 1998) like eurygaster integriceps puton (hemiptera: scutelleridae) (goodchiled, 1966). additionally, it was found that the digestion process in the midgut of a. spinidens takes about 12-24 h, as currently seen in starved and fed adults, so the sac shape of v1 may provide a storage chamber for the gradual digestion of food. there are two major types of proteases in insects, known as exoand endoproteases. exopeptidases attack protein molecules from the n-terminal end (aminopeptidases) and the c-terminal end (carboxypeptidases) (terra & ferriera, 2005). the enzymes liberate one amino acid residue at each catalytic step. endopeptidases break internal bonds of protein molecules, so there are different types of these proteases because amino acid residues vary along their peptide chain (terra & ferriera, 2005). there are three subclasses of endopeptidases involved in digestion according to their active site group: serine, cysteine, and aspartic proteases, and metalloproteases that are further classified as trypsin, chymotrypsin, elastase and cathepsins b, l and d (terra & ferriera, 2005). in serine proteases, the hydroxyl group in the side chain of a serine residue acts as a nucleophile in the reaction that hydrolyzes peptide bonds, whereas in cysteine proteases, the sulfhydryl group of a cysteine side chain performs this function (kanost & clem, 2011). in aspartic acid proteases and metalloproteases, a water molecule in the active site (positioned by interacting with an aspartyl group or a metal ion, respectively) attacks the peptide bond as a nucleophile factor (kanost & clem, 2011). the general proteolytic activity was found in the soluble fraction of a. spinidens adults that demonstrates the importance of site of secretion in the process of digestion. this phenomenon has received less attention by researchers. the activities of all the assayed specific proteases including serine, cysteine proteases and two exopeptidases were higher in the soluble fraction. soluble aminoand carboxylpeptidases are found in less evolved insects (e.g. orthoptera, hemiptera, and coleoptera: adephaga), whereas in more evolved insects (e.g. coleoptera: polyphaga, diptera and lepidoptera), these enzymes are membrane-bound. for example, zibaee (2011) found that carboxyand aminopeptidase of pieris brassicae l. (lepidoptera: pieridae) are mainly bound to the membranes of midgut cells. in contrast, mehrabadi & bandani (2011) found that both carboxyand aminopeptidase are active in the soluble fraction and no activity was observed in the sediment fraction of the hemipteran e. integriceps. the majority of studies on hemipteran gut proteases have reported that the primary gut enzymes responsible for protein digestion are of the cysteine mechanistic class, with lower activity in the serine class (terra & ferriera, 2005). stamopoulos et al. (1993) have reported only trace trypsin/chymotrypsin activity in the gut of podisus maculiventris say (hemiptera: pentatomidae), as have bell et al. (2005) on the same insect. against these studies, pascual-ruiz et al. (2009) showed that serine, cysteine, aminopeptidase and carboxypeptidase are the main proteases involved in protein digestion of p. maculiventris, so that variability of prey may affect their activity. zhu et al. (2003) and wright et al. (2006) have demonstrated activity of both cysteine and serine proteases in the midgut of lygus lineolaris and l. hesperus knight (hemiptera: miridae). these differences direct attention to the enzymes’ site of activity and the use of a negative control, as in our study. the current study indicates the need for standardization of methods for the characterization of proteolytic activity in hemipterans and all insects. also, serine activity in the midgut of a. spinidens could be attributed to the ingestion of salivary serines or utilization of prey serine proteases. pascual-ruiz et al. (2009) demonstrated that the relative activity of serine proteases in the midgut of p. maculiventris depends on the prey. the authors suggested that p. maculiventris may utilize prey proteolytic enzymes for digestion of their own tissues. their conclusion is based on the increase of trypsin and chymotrypsin activity observed in p. maculiventris feeding on lepidopteran larvae in comparison with those feeding on beetles or dipteran pupae (pascualruiz et al., 2009). optimal ph determination revealed an alkaline ph for total proteolytic activity, serine proteases and two exopeptidases with a another peak at ph 6. but an acidic ph was found for cysteine proteases. an alkaline ph is typical for activity of serine proteases (e.g., the trypsin-like and chymotrypsin-like proteases in our study) and exopeptidases (e.g., the amino and carboxypeptidases in our study) that are known to be active [journal of entomological and acarological research 2013; 45:e8] [page 39] article figure 6. zymogram of proteolytic activity in different sections of the midgut of a. spinidens. jear_2013_1:hrev_master 23/04/13 09.55 pagina 39 no n c om me rci al us e o nly [page 40] [journal of entomological and acarological research 2013; 45:e8] at ph between 7-12 (terra & ferriera, 2005). zhu et al. (2003) reported optimal gut proteolytic activity of l. lineolaris at ph 4.25 and 8.5. wright et al. (2006) found acidic midgut caseinolytic activity with an optimal ph of 4.5, which was attributed to cystein proteainases. by using specific inhibitors, these authors found an optimal ph activity of 7.5, confirming serine activity in the midgut. the frequency at which insects encounter a high-ph environment in the field is unknown, as is the identity and digestibility of the most commonly ingested protein substrates. in this case, a. spinidens is a specific biocontrol agent of chilo suppressalis. usually, caterpillars have an alkaline ph in their gut and hemolymph to overcome secondary metabolites of ingested plants. also, the feeding habits of a. spinidens require an alkaline ph in its gut for detoxifying secondary metabolites ingested along with larval body components. also, this may represent a co-evolutionary phenomenon, as enzymes secreted in the midgut of a. spinidens must show stability in the highly alkaline state of caterpillars. as the gut constitutes the majority of a caterpillar’s body, this conclusion seems to be more reliable. class-specific inhibitors are biochemically used to identify the types of proteases involved in biological processes. however, the effect of inhibitors can also be substrate-specific. direct measurement of classspecific proteolytic activities using combinations of inhibitors to inhibit one or all classes of proteases may aid in the accurate classification and quantification of digestive proteases (wright et al., 2006). different specific protease inhibitors including pmsf, tlck, tpck, cystatin, e-64, edta and phenanthroline were used to confirm the type of proteases present in the midgut of a. spinidens. pmsf highly inhibited enzymatic activity, demonstrating a major presence of serine proteases in the midgut. tlck (a trypsin-like inhibitor) and tpck (a chymotrypsin-like inhibitor) decreased enzymatic activity in different portions, demonstrating the equal roles of trypsin-like and chymotrypsinlike proteases. we also found cystatin and edta inhibitory effects, implying roles of cysteine and metalloproteinase in protein digestion by a. spinidens. zhu et al. (2003) studied the effect of eleven specific inhibitors on the proteolytic activity of l. lineolaris. bell et al. (2005) reported that the serine protease inhibitor pmsf caused the most marked reduction in the rate of proteolysis of p. maculiventris. proteolytic hydrolysis was inhibited by pmsf, tpck and e-64 in brachynema germari kolenati (hemiptera: pentatomidae), indicating the presence of chymotryptic and cysteine activity in the midgut of this insect (bigham & hosseini-naveh, 2010). presence of serine proteases in the digestive system of hemipterans and other insects is well documented. the midgut extract from rhodnius prolixus l. (hemiptera: reduviidae) also showed hydrolysis of a tryspin-like substrate (houseman, 1978). in gel assays, six proteolytic bands were observed, although the partial denaturing methodology of the electrophoresis does not preclude the presence of further proteolytic enzymes that were not detected. disappearance of proteolytic bands caused by specific inhibitors verified biochemical assays for the presence of specific inhibitors. bell et al. (2005) found four proteolytic bands in p. maculiventris and reported inhibitory effects of e-64, evidenced by the disappearance of bands. article figure 7. proteolytic zymogram in five nymphal instars of a. spinidens. from left to right, 1st to 5th nymphal instars. figure 8. zymogram analysis of the digestive proteolytic activity in a. spinidens to prove the presence of specific proteases by using inhibitors. jear_2013_1:hrev_master 23/04/13 09.55 pagina 40 no n c om me rci al us e o nly our findings in these studies show that a. spinidens utilizes several endoand exopeptidases for gut proteolysis of ingested food, as those are sensitive to inhibitors of a given mechanistic class. due to the inhibition of proteases in a. spinidens by tryspin and cysteine protease inhibitors, use of these toxic proteins in transgenic rice must be considered to prevent their possible negative effects on this predatory bug. in addition, these findings will help us to purify and characterize these proteases in future work, and to determine their importance in the digestion of prey proteins in different host regimes. this research is being continued to determine the responses of a. spinidens digestive enzymes after feeding on different prey, elucidation of the secretion mechanism in starved and fed insects in both in vivo and in vitro conditions, as well as the extraction of endogenous inhibitors of the known digestive enzymes. references bell h.a., down r.e., edwards j.p., gatehouse j.a., anghard m.r., gatehouse m.r., 2005 digestive proteolytic activity in the gut and salivary glands of the predatory bug podisus maculiventris (heteroptera: pentatomidae); effect of proteinase inhibitors. eur. j. entomol. 102: 139-145. bigham m., hosseini-naveh v., 2010 digestive proteolytic activity in the pistachio green stink bug, brachynema germari kolenati (hemiptera: pentatomidae). j. asia-pacific. entomol. 13: 221-227. chapman r.f., 1998 the insects: structure and function. 4th edition. cambridge university press, cambridge: 795 pp. cohen a.c., 1993 organization of digestion and preliminary characterization of salivary trypsin-like enzymes in a predaceous heteropteran, zelus renardi. j. insect. physiol. 39: 823-829. cohen a.c., 1995 extra-oral digestion in predaceous terrestrial arthropoda. ann. rev. entomol. 40: 85-103. cohen a.c., 1998 solid-to-liquid feeding: the inside(s) story of extraoral digestion in predaceous arthropoda. am. entomol. 44: 103117. elpidina e.n., vinokurov k.s., gromenko v.a., rudenskaya y.a., dunaevsky y.e., zhuzhikov d.p., 2001 compartmentalization of proteinases and amylases in nauphoeta cinerea midgut. arch. insect. biochem. physiol. 48: 206-216. ferreira c., terra w.r., 1983 physical and kinetic properties of a plasma-membrane-bound p-dglucosidase (cellobiase) from midgut cells of an insect (rhynchosciara americana larva). biochem. j. 213: 43-51. frugoni j.a.c., 1957 tampone universale di britton e robinson a forza ionica costante. gazz. chim. ital. 87: 403-407. garcia-carreno f.l., dimes l.e., haard n.f., 1993 substrate-gel electrophoresis for composition and molecular weight of proteinases or proteinaceous protease inhibitors. analyt. biochem. 214: 6169. goodchiled a.j., 1966 evolution of the alimentary canal in the hemiptera. biol. rev. 41: 97–140. houseman j., 1978 a thiol activated digestive protease from adults of rhodinus prolixus stal (hemiptera: reduviidae). can. j. zool. 56: 1140-1143. kanost m.r., clem r.j., 2011 insect proteases. in: gilbert l.i., ed. insect molecular biology and biochemistry. elsevier, amsterdam: 346-364. laemmli u.k., 1970 cleavage of structural proteins during the assembly of the head of bacteriophage t4. nature. 227: 680-685. lowry o.h., rosebrough n.j., farr a.l., randall r.j., 1951 protein measurement with the folin phenol reagent. j. biol. chem. 193: 265-75. manley g.v., 1982. biology and life history of the rice field predator andrallus spinidens f. (hemiptera: pentatomidae). entomol. news 93: 19-24. mehrabadi m., bandani a.r., 2011 secretion and formation of perimicrovillar membrane in the digestive system of the sunn pest, eurygaster integriceps (hemiptera: scutelleridae) in the response of feeding. arch. insect. biochem. physiol. 78: 190-200. mohaghegh j., najafi i., 2003 predation capacity of andrallus spinidens (f.) (hemiptera: pentatomidae) on naranga aenescens moore (lep.: noctuidae) under semi-field and field conditions. appl. entomol. phytopathol. 71: 57-68. nageswara rao v., 1965 andrallus (audinetia) spinidens fabr., as predator on rice pests. oryza. 2: 179-181. oppert b., kramer k.j., mcgaughey w.h., 1997 rapid microplate assay of proteinase mixtures. biotechnol. 23: 70-72. pascual-ruiz s., carrillo l., alvarez-alfagemeyy f., ruiz m., castanera p., ortego f., 2009. the effects of different prey regimes on the proteolytic digestion of nymphs of the spined soldier bug, podisus maculiventris (hemiptera: pentatomidae). bull. entomol. res. 99: 487-491. sas, 1997 sas/stat user’s guide for personal computers. sas institute, cary, nc, usa. saxena k.n., 1954 physiology of the alimentary canal of leptocorisa varicornis fabr. (hemiptera: coreidae). j. zool. soc. india. 6: 111122. saxena k.n., 1963 mode of ingestion in a heteropterous insect dysdercus koenigii (f.) (pyrrhocoridae). j. insect. physiol. 9: 4771. stamopoulos d.c., diamantidis g., chloridis a., 1993 activités enzymatiques du tube digestif du prédateur podisus maculiventris (hemimptera: pentatomidae). entom. 38: 493-499. terra w.r., ferreira c., 2005 biochemistry of digestion. in: gilbert l.i., iatrou k., gill s.s., eds. comprehensive molecular insect science, vol. 3. elsevier, amsterdam: 171-224. wright m.k., brandt s.l., coudron t.a., wagner r.m., habibi j., backus e.a., huesing j.e., 2006. characterization of digestive proteolytic activity in lygus hesperus knight (hemiptera: miridae). j. insect physiol. 52: 717-728. zibaee a., 2012 digestive enzymes of large cabbage white butterfly, pieris brassicae l. (lepidoptera: pieridae) from developmental and site of activity perspectives. ital. j. zool. 79: 13-26. zibaee a., hoda h., fazeli-dinan m., 2012 role of proteases in extra-oral digestion of a predaceous bug, andrallus spinidens fabricius (hemiptera: pentatomidae). j. insect. sci. 12: 51. zhu y.c., zeng f., oppert b., 2003. molecular cloning of trypsin-like cdnas and comparison of proteinase activities in the salivary glands and gut of the tarnished plant bug lygus lineolaris (heteroptera: miridae). insect biochem. mol. biol. 33: 889-899. [journal of entomological and acarological research 2013; 45:e8] [page 41] article jear_2013_1:hrev_master 23/04/13 09.55 pagina 41 no n c om me rci al us e o nly jear2012 [journal of entomological and acarological research 2013; 45:e1] [page 1] isotrias penedana sp. n. a new species of lepidoptera (tortricidae: chlidanotinae: polyorthini) from portugal p. trematerra department of agricultural, environmental and food sciences, university of molise, campobasso, italy abstract a new species of tortricidae (lepidoptera: chlidanotinae: polyorthini), isotrias penedana sp. n., is described. the new species was collected in serra da peneda, in the peneda-gerês national park located in the north-western region of portugal. i. penedana differs from other species of the genus isotrias by shape of the forewing and the male genitalia. in the new species forewing markings are completely atrophied or indistinct. the socii are short and broad, covered with long setae, and the costa of the valva is hardly concave basally. the imago and male genitalia are illustrated. introduction a new tortricid isotrias penedana sp. n., of the subfamily chlidanotinae tribe polyorthini, collected in serra da peneda, portugal, is described. serra da peneda, is in the peneda-gerês national park located in the north-western region of portugal, in the old province of minho, now in the district of viana do castelo. the peneda-gerês national park presents remarkable biological, geological and archaeological features. the foothills of the serra do gerês along with the foothills of the serra da cabreira constitute the frontier between the euro-siberian and mediterranean regions, which confers on peneda-gerês a floral and phytogeographic importance. vegetation coverage of serra da peneda, serra do gerês, serra amarela and serra do soajo, as well as the mourela and castro laboreiro plateaus, is dominated by four distinct biomes: oak forest, scrubland, marshy meadows and riparian vegetation. climatically the area is strongly influenced by the atlantic, resulting in the highest rainfall in portugal. isotrias penedana sp. n. material examined. 1 male, holotypus, labelled as follows: serra da peneda, north-west portugal, june 2012, leg. martin corley; 3 males, paratypus, serra da peneda, north-west portugal, june 2012, leg. martin corley. adult. alar expanse, male 16-20 mm (figure 1). antenna greenish brown. head and palpi light greenish-olive brown. frons and vertex concolorous with palpus. thorax and tegula light greenish-brown. forewing ground colour whitish-creamy with yellowish-brown or amber spots. strigulation dense, more or less fine, yellow to amberochreous. fasciae almost completely atrophied or indistinct. cilia whitish-ochreous with two dark bands. hind wing pale greenish, distally greenish, cilia whitish with pale grey scales. male genitalia (figures 2-6). tegumen reported in figure 3; uncus rather slender with terminal rounded plate in apical portion; dorsal edge of uncus gradually curved downward. socius short and broad, covered with long setae. gnathos long; arm of gnathos strongly expanding terminally; spines of outer surface of gnathos arm atrophying (figure 4). the valva is subrectangular, broad and moderately elongated provided with a long sacculus terminating in one or two ventral spines; the transformed setae on inner surface of valva are numerous. costa of valva hardly concave basally (figure 5). external edge of cucullus slighly arched. juxta linked with valva; transtilla broad, tapering, medially plate provided with a pair of sublateral funnel-shaped processes directed proximally. aedeagus long and simple, pointed, weakly sclerotized in distal part; with fairly long coecum penis (figure 6). female genitalia. unknown. distribution. known only from three sites in serra da peneda and the castro laboreiro plateau, minho, north-west portugal. host. unknown. biology. males were found flying in the afternoon or early evening in mid-june in small sloping marshy meadows at altitudes from 770 m at podre to 1180 m at portos. the vegetation in these meadows was rich and varied, including a range of graminoids, carex and juncus species, together with centaurea sp., cirsium dissectum, achillea millefolium, conopodium majus, lotus uliginosus, etc. in every case correspondence: pasquale trematerra, università degli studi del molise, dipartimento di agricoltura, ambiente e alimenti, via de sanctis, 86100 campobasso, italy. e-mail: trema@unimol.it key words: isotrias penedana sp. n., lepidoptera tortricidae, chlidanotinae, polyorthini, new species, portugal. acknowledgements: the author would like to express his thanks to jozef razowski (poland), martin corley (england) and to gian mario fazzini (italy) for providing the study material. received for publication: 31 october 2012. revision received: 20 december 2012. accepted for publication: 21 december 2012. ©copyright p. trematerra, 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e1 doi:10.4081/jear.2013.e1 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e1 jear_2013_1:hrev_master 23/04/13 09.55 pagina 1 no n c om me rci al us e o nly [page 2] [journal of entomological and acarological research 2013; 45:e1] article figure 1. isotrias penedana sp. n., adult. figure 2. isotrias penedana sp. n., male genitalia. figure 3. isotrias penedana sp. n., male genitalia. tegumen laterally. figure 4. isotrias penedana sp. n., male genitalia. tegumen frontally. figure 5. isotrias penedana sp. n., male genitalia. valva. figure 6. isotrias penedana sp. n., male genitalia. aedeagus. jear_2013_1:hrev_master 23/04/13 09.55 pagina 2 no n c om me rci al us e o nly there were some shrubs of genista florida at the drier margins of the meadows. remarks. the western palaearctic genus isotrias meyrick, 1895, consists of 9 species, 8 known from europe and one from north africa (lucas, 1954; trematerra, 1993; razowski, 1984, 1987, 2002; brown, 2005). isotrias cuencana (kennel, 1899), present in spain (albarracín, cuenca, guadalaviar, las palomas, monteagudo de las salinas, torres de albarracín); i. huemeri trematerra, 1993, found in south italy (monti del pollino); i. hybridana (hübner, 1817) found in europe, excluding the northern territories: portugal, spain, france, belgium, germany, poland, austria, italy, czech republic, slovakia, slovenia, croatia, ex yugoslavia, bosnia and herzegovina, macedonia, albania, hungary, bulgaria, greece and ukraine; i. joannisana (turati, 1921) distributed in central and south italy, reported also for france and spain (?); i. martelliana trematerra, 1990, from south italy (monti del pollino, cozzi dell’anticristo); i. penedana trematerra, 2013, from serra da peneda, north-west portugal; i. rectifasciana (haworth, 1811), european species distributed from british islands and france to denmark and greece, east europe and asia minor (?); i. stramentana (guenée, 1845), present in south west europe: spain, france, switzerland, germany, italy, croatia; i. buckwelli lucas, 1954, cited from morocco. according to razowski (1984, 1987) the genus isotrias requires a thorough revision, the differences between the species are slight especially in the genitalia. the external characters are in many cases of importance, however in specimens from the same locality there are dark and pale examples. diagnosis. the new species i. penedana differs from other species of the genus isotrias by shape of forewing and the male genitalia. in the new species forewing markings are completely atrophied or indistinct. socii are short and broad, covered with long setae; costa of valva is hardly concave basally. the type specimens of i. penedana are deposited in the trematerra collection, university of molise, campobasso, italy, and one paratype in the private collection of martin corley, farington, england. etymology. the new species is named after serra da peneda, the area from which the type series comes. references brown j., 2005 world catalogue of insects. volume 5. tortricidae (lepidoptera). apollo books, stenstrup: 1-741. lucas d., 1954 nouveaux lépidoptères nord-africains. bull. soc. sci. nat. phys. maroc. xxxiv: 35-39. razowski j., 1984 palaearctic polyorthini (lepidoptera, tortricidae). acta zoolog. cracov. 27: 287-298. razowski j., 1987 the genera of tortricidae (lepidoptera). part i: palaearctic chlidanotinae and tortricinae. acta zoolog. cracov. 30: 141-355. razowski j., 2002 tortricidae of europe. volume 1. tortricinae and chlidanotinae. frantisek slamka, bratislava: 1-247. trematerra p., 1993 isotrias huemeri sp. n. (lepidoptera tortricidae) from southern appennine. boll. zool. agr. bachic. ser. ii 25: 11-17. [journal of entomological and acarological research 2013; 45:e1] [page 3] article jear_2013_1:hrev_master 23/04/13 09.55 pagina 3 no n c om me rci al us e o nly jear2012 abstract this study estimates the parasitization levels and fecundity of the ectoparasitic mite varroa destructor oudemans in drone brood of bee colonies located in northern greece. based on successive observations in spring and early summer, the study also examines whether early entrapment of mites into the drone brood cell decreases the mite population levels in the succeeding generation. varroa populations in drone brood were extremely high (approx. 40%) in early spring, although numbers dropped significantly (approx. 20%) after the entrapment and removal of mites into the drone brood (t=4.14518, p=0.0136, mann-whitney: p=0.005). in most cases, more than half of the inspected cells were occupied with two or more parental mites. no significant differences were found in the reproductive performance of the varroa mites between the two successive generations in spring and early summer (t=-0.607, p=0.554, mann-whitney: p=0.128). the reproductive performance of v. destructor ranged from 1.5-3 progeny per female individual (m1:1.673, se=0.09 and m2:2.02, se: 0.44 for the first and second generations, respectively). a positive and significant correlation was observed between the drone and the mite populations (y=0.830+1.153x, f=8.851, p=0.41, r2:0.689 and y=0.319+0.968x, f=45.276, r2: 0,938, p=0.07 for the first and second mite generations, respectively). there were no significant differences in the number of infested and non-infested cells during the first observations (m1: 105.2, se: 25.0, m2: 170.0 se: 40.0, t=-1.38, p=0.203, mann-whitney: n1:81.0, n2:142.5, p=0.0656). on the contrary, during the second observations the number of infested cells was significantly lower (m1: 27.6, se:8.1, m2:262.8, se:69.0, t=-3.39, p=0.027, mann-whitney: p=0.012, n1:20, n2:340). introduction the mite varroa destructor (jacobsoni) oudemans (anderson & trueman, 2000) is the most important ectoparasite of bee colonies that causes epizooty on its host apis melifera (hymenoptera: apididae). the specimens belong to the superorder anactinotrichida of the order mesostigmata (or gamasida) (evans, 1992) and is characterized by the fact that all of its life stages are closely related to those of its host. female individuals feed on the hemolymph of immature and adult bees, whereas nymphs and adult males feed only on immature bees (colin et al., 1997). females of the parasite enter the brood cells before cell capping and their reproductive period coincides with a capped period during which a. melifera complete their metamorphosis from the last larval stage through the pupal stage to the adult stage (crane, 1978; ifantidis, 1983; martin, 1994; fries et al., 1994; rosenkranz et al., 2010). the specimen was first described as a parasite of apis cerana in java by oudemans (1904). later it was identified in the southeast asian malay peninsula, singapore in 1944 and in 1952 in the eastern coastal region of the former ussr (crane, 1978; rosenkranz et al., 2010). in the indian peninsula, the mite was first recorded in pakistan in 1955 and a few years later, in 1958 and 1959, it was identified in japan and china, respectively. in the early 1960s, the parasite was introduced in colonies of the western bee a. melifera from the asian honeybee species apis cerana fabricius (martin, 1995). the parasite seems to have crossed the mediterranean sea in 1975 through the importation of colonies from romania to tunisia. in greece, however, the regular presence of the parasite in bee colonies was observed in the late 1970s and early 1980s (ifantidis 1983, 1984). since then, its presence has been regularly observed, although possible alterations in its parasitic potential as related to regional host resistance has not yet been evaluated in detail. despite the fact that the original host of the varroa mite is not damaged to any appreciable degree, mainly because infestation occurs in drone brood and mites become entrapped in dying drone brood (rath & drescher, 1990; fries et al., 2006), it causes significant problems to the western honeybee a. melifera. it is thought that one of the reasons for this is the absence of a long period of co-evolutionary relationship, as in the case of a. cerana (rath, 1999; rosenkranz et al., 2010) and, therefore, in europe the mite populations must be regularly controlled to avoid colony collapse. journal of entomological and acarological research 2012; volume 44:e18 [page 98] [journal of entomological and acarological research 2012; 44:e18] fecundity and trapping of varroa destructor (mesostigmata: varroidae) in greek drone brood of apis melifera (hymenoptera: apididae) petros t. damos laboratory of applied zoology and parasitology, department of plant protection, faculty of agriculture, aristotle university of thessaloniki, greece correspondence: petros t. damos, laboratory of applied zoology and parasitology, department of plant protection, faculty of agriculture, aristotle university of thessaloniki, 54124 thessaloniki, greece. e-mail: damos@agro.auth key words: mite, varoatosis, reproduction, bee colonies, apiculture. acknowledgements: the author acknowledges the fruitful discussions made with m. ifantidis on trial design and management. received for publication: 15 august 2012. revision received: 24 october 2012. accepted for publication: 29 october 2012. ©copyright p.t. damos, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e18 doi:10.4081/jear.2012.e18 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly generally, the reproductive potential and related fecundity of v. jacobsoni on a. melifera brood can be extremely high. there are instances in which a mother mite may reproduce a maximum of 7 offspring in drone cells and 6 in worker cells, although numbers are normally 5-6 and 4-5, respectively (ifantidis, 1984; martin, 1994, 1995). however, the actual parasitization levels can be lower and are actually difficult to estimate, while the region and the bee tribes observed, and breeding towards tolerance, all influence reproductive performance (de jong et al., 1984; de guzman et al., 2007; rosenkranz et al., 2010). fecundity of v. destructor is considered to be among the most important fitness parameters that affect the population dynamics and parasitization levels, especially in drone brood. furthermore, since it is known that v. desctructor infest drone cells 5 to 8 times more than the worker cells (schulz, 1984; woyke, 1987; fuchs, 1990; martin 1995), this attribute prompted a biotechnical control method referred to as the drone brood trapping method, in which mites are first trapped in drone brood and then removed from the hive (schulz et al., 1983; rosenkranz & engels, 1985). the scope of this study was to estimate the population levels and fecundity of v. destructor in drone broods of bee colonies located in northern greece, and to address the question of whether reproductive performance has changed towards tolerance compared to previous related studies. furthermore, the study aimed to re-evaluate under precise conditions whether early entrapment of varroa mites into the drone brood cell decreases the mite populations of the succeeding generation. data analysis and interpretation continue, and information is still being collected on the population dynamics and fecundity of v. destructor in an attempt to provide some evidence on region-specific host-parasite interactions that may help improve varroatosis management. materials and methods the study was carried out in the apiary of the laboratory of apiculture and sericulture of aristotle university of thessaloniki in greece during the spring and early summer of 2004. honeybee populations an established honeybee population of a. melifera was used during the experimental trials. these populations had a case record of considerable v. destructor infestations. the bee population consisted of 50 honeybee hives located in eastern thessaloniki in northern greece. the bee colonies were separated by variable distances between them and were located only a few meters above sea level. the hives used in the trials were standard beehives (frame size 366¥222 mm) each containing 20 honeycombs. throughout the trials, a distributed sample of 6 beehives was randomly chosen for experimentation. colonies were fed with sugar dough through the winter season since in several cases honey stores were considered insufficient for survival. no mite-specific control measures or any kind of chemical treatment were performed, and over recent years the colonies had been allowed to swarm freely. estimation of mite generations to estimate the reproductive performance of v. destructor (i.e., number of life varroa offspring per cell) of succeeding mite generations, the drone cells must be unsealed at a precise time point at which progeny are present and can be distinguished from mothers. to do this, a specific procedure was followed. first, a drone comb was placed in each hive for male bee offspring to be deposed in each cell by the queen. then each day the number of the new sealed drone cells was recorded and marked. this was made possible by the construction of a detailed drone cell map consisting of transparent plastic foil membranes in which each day the new sealed cells were marked with a waterproof colored marker. based on the drone cell map, the detailed age of each sealed cell could be estimated with accuracy. in addition, since ontogenesis of v. destructor coincides with the developmental stages of the bee host a. melifera, its distinctly morphological features were also used as a criterion. therefore, a number of cells were carefully opened and inspected during the period in which host pupae had pink and purple colored eyes and their dorsal shield was yellow (ifantidis, 1983). after this developmental stage, such observations are no longer useful given that the parent mites and their offspring are no longer distinguishable and counts could be biased (ifantidis, 1983). inspections were carried out over two successive periods in spring and early summer. the first observation period started on april 20th during which the first treatment was carried out while the second started on may 20th during which the second treatment was carried out. each observation period lasted approximately two weeks, depending upon the drone development. inspections were carried out of a sample that corresponded to 10-30% of the sealed drone cells. fecundity of v. destructor the drone combs were successively removed each day from the beehives and transferred to the laboratory for inspection in a stereoscope. the comb was divided into four parts and a representative number of cells was randomly chosen from each quarter and opened. individuals were carefully pulled out of their cells with forceps. the body of each individual and its evacuated cell were examined using cold light apparatus. the number of the anchored mites on the drone brood, as well as the number of those trapped in the base of the cell and free moving mites, was also recorded. after estimating the parental mite pressure and the number of mite progenies, each of the drone combs was placed inside a parallelogram shaped cage. the cages consisted of a thin steel mesh (2¥2 mm) that held each of the drone brood. each of the cages allows access from the drone brood to the mites after breaking the sealed cells when the drone has emerged, but not access to the drone host. each of the drone brood confined on the mesh tray was placed vertically on a transparent plastic foil which had commercial vaseline® around the edge to trap the free-moving mites. the mesh trap construction was placed inside environmental chambers at 35-36°c and the drone brood was left to breed until the adult emerged. each day, the plastic foil was inspected for newly trapped mites. each day, all mite individuals (e.g., adult daughter mites and nymphal stages) were counted and removed. successive observations were carried out until the end of drone emergence. in order to count the phoretic mites, the emerged drone adults were placed inside a soap-water solution (5%) for 24 h and the mixture was shaken in order to wash all mites. mites and bees were counted to establish absolute infestation rates (number of mites/number of bees ¥100). finally, for confirmation, all the unsealed drone brood that was used during each of the treatments was further examined in the laboratory to assess the absolute number of cells infested. the total number of infected cells in each of the drone comb was carefully counted. cell infection was identified according to the presence of characteristic white-colored varroa excreta. statistical analysis data were examined to verify whether they met the assumptions of normality, and all statistical comparisons were made using two-sample t-tests. the hypothesis of h0:m1-m2=d0 versus h1:m1-m 2≠d0, where m1 and m2 are the population means and d0 is the hypothesized difference between the two population means, was tested for a α=0.05 level of significance. for comparative reasons, a two-sample non-parametric mann-whitney rank test was also performed to test the hypothesis of h0:n1=n2 versus h1:n1≠n2, where n is the population median. finally, linarticle [journal of entomological and acarological research 2012; 44:e18] [page 99] no nco mm er cia l u se on ly ear regression was performed to detect covariance between drone brood and mite population variables. results parental mite load in drone brood figure 1 shows the parasitization levels (%) in a drone brood of a. melifera caused by parental v. destructor mites during the two successive treatments. in general, the parasitization level was high in all of the hives examined and during both treatment periods. the number of cells that were occupied by varroa parental mites before capping, and during the first observation period, was significantly higher than during the second (t=4.14518, p=0.0136, mann-whitney: p=0.005). in particular, during the first treatment period, parasitization ranged from 30-50% (m1:36.084, se:6.476, n1:41.6), while during the second it ranged from 18 to 23% (m2:7.81, se:2.139, n2:11.2). multiple parasitization multiple parasitization caused by v. destructor parents per cell of a. melifera drone brood is shown in figure 2. this infection load corresponds to the number of the inspected cells in which more than one parental mite was discovered with respect to the total number of cells inspected (values in brackets). during the first observation period, and in all experimental hives, 10-35% of the drone cells were infested with figure 1. parasitization levels (%) in drone brood of a. melifera caused by parental v. destructor mites during the (a) first and (b) second treatments. figure 2. multiple parasitization levels per drone brood cells caused by v. destructor mites of a. melifera during the (a) first and (b) second treatments. multiple parasitization corresponds to the percentage of cells infected with more than one parental mite. article [page 100] [journal of entomological and acarological research 2012; 44:e18] no nco mm er cia l u se on ly more than one parental mite before capping (m1:16.705, se:4.075, n1=15.5) while during the second observation period the multiple infections corresponded to 0.2-5% of the total drone cells inspected (m2:1.857, se:0.609, n2=2) and were, therefore, significantly lower (t=3.325, p=0.006, mann-whitney: p=0.0081). figure 3 shows a comparison of the new sealed drone brood infected with one, 2, 3, and more than 3 mites for each time treatment. during the first observation period, over 50% of the inspected cells were occupied by only one mite, while during the second period the number of inspected cells occupied by only one mite was significantly lower at close to 40% (t=6.18, p=0.000, mann whitney: p=0.008). furthermore, during the first observation, the number of cells occupied with 2 mites corresponded to 20% of the total parasitization levels, while during the second observation period, this dropped by 3% (t=4.06, p=0.010, mann whitney: p=0.0081). finally, a surprisingly higher number of cells infected with more than 3 mites was observed during the second observation time compared to the first (t=-3.02, p=0.029, mann whitney: p=0.0137). reproductive capacity and absolute mite infections the reproductive capacity of varroa mites is shown in figure 4 corresponding to the number of progeny mites in each cell. there were significant differences in reproduction of v. destructor between the two observation time periods (t=-3.29, p=0.009, mann-whitney: p=0.025) figure 3. v. destructor multiple parasitization levels in drone brood cells of a. melifera and during the (a) and (b) second treatments (i.e., first and second mite generations). comparison of multiparasitization corresponds to 4 different mite loads (1, 2, 3 and >3 mites per inspected cell). figure 4. fecundity of v. destructor parasitizing a. melifera drone brood during the (a) first and (b) second observation periods (i.e., first and second mite generations). fecundity corresponds to the actual number of female progeny produced by parental mites that entered the cell before capping and were discovered during inspection. article [journal of entomological and acarological research 2012; 44:e18] [page 101] no nco mm er cia l u se on ly and fecundity ranged from 1.5 to 3 progeny per female mite (m1:1.620, se=0.09, n1=1.62 and m2:2.02, se:0.24, n2=2.5 for the first and second observation periods, respectively). figure 5 presents the absolute number of mites (mothers and progeny) and drones for each hive and for the two successive treatments. the total number of mites corresponds to those that were free moving and that anchored on drone adults. the number of drones ranged from 275 (n50) to 1041 (n7) and from 140 (n18) to 1076 (n7) for the first and second observation periods, respectively; no significant differences were found (m1:631.0, se:130.6, m2:467.4, se:194, t=0.74, p=0.501, mann-whitney: p=0.522, n1:549, n2:220). in contrast, the absolute number of mites during the first period ranged from 244 (n15) to 1343 (n7) and was significantly higher compared to that observed during the second generation and which ranged from 17 (n18) to 352 (n21), respectively (m1:619.4, se:185.8, m2:118.2, se:64, t=2.86, p=0.046, mannwhitney: p=0.025, n1:435.5, n2:40). figure 6 shows the linear regressions between absolute number of mites (mothers and progeny) and drone brood adults of each experimental hive, and for the two successive observations that were carried out during the two successive treatments in spring and early summer. in both cases, positive correlations were observed between the numbers of drone brood and varroa mites. therefore, the increase in drone density results in a significant increase in the number of mites during the first (y=0.830+1.153x, f=8.851, p=0.41, r2:0.689) (figure 1) and the second (y=0.319+0.968x, f=45.276, r2:0.938, p=0.07) (figure 2) observation. figure 5. absolute counts of total parasitic load of v. jacobsoni (mite parents and offspring) and total number of emerged a. melifera brood (adult drones) during the (a) first and (b) second treatments (i.e., first and second mite generations). figure 6. linear regression between total parasitic load of v. destructor (mite parents and offspring) and total number of emerged a. melifera brood (adult drones) during (a) the first and (b) second treatments (i.e., first and second mite generations. regressions for parameter estimates were based on data shown in figure 5). article [page 102] [journal of entomological and acarological research 2012; 44:e18] no nco mm er cia l u se on ly mite excreta and cell infestation levels in drone brood figure 7 illustrates the total number of cells in each drone comb and the number that was infested by mites as estimated by mite excreta after adult emergence. no significant differences were found in the number of infested and non-infested cells during the first observation period (m1: 105.2, se:25.0, m2:170.0, se:40.0, t=-1.38, p=0.203, mannwhitney: p=0.0656, n1:81.0, n2:142.5). in contrast, during the second observation, the number of infested cell was significantly lower than non-infested cells (m1:27.6, se:8.1, m2:262.8, se:69.0, t=-3.39, p=0.027, mann-whitney: p=0.012, n1:20, n2:340). figure 8 shows the percentage of parasitization levels in drone brood cells as estimated by mite counts performed during the first and second observation periods on a representative number of cells, and by cell infections that were estimated by mite excretes present on the total number of unsealed cells observed at the end of each treatment. no significant differences were found in the parasitization levels estimated by representative mite counts performed during the drone breeding period or by observing mite excretes after drone emergence. parasitization levels were close to 40% during the first observation period (m1:0.402, se:0.029, m2:0.382, se:0.022, t=1.05, p=0.323, mann-whitney: p=0.378, n1:0.4160, n2:0.3730) and close to 10% (m1:0.109, se:0.011, m2:0.104, se:0.0073, t=0.36, p=0.731, mannwhitney: p=0.834, n1:0.112, n2:0.112) during the second observation period, regardless of the estimation method used. figure 7. total number of infested and non-infested a. melifera drone brood cells based on v. destructor excretes during (a) the first and (b) second treatments (i.e., first and second mite generations). figure 8. percentage of infested a. melifera drone brood cells by v. destructor estimated by mite counts, after drone emergence, and by excreta inspection during (a) the first and (b) second treatments (i.e., first and second mite generations). article [journal of entomological and acarological research 2012; 44:e18] [page 103] no nco mm er cia l u se on ly discussion the parasitic species v. destructor causes some of the most serious damage to bee colonies worldwide. this study evaluates parasitization levels and reproductive capacity of the drone brood of a. melifera in northern greece and during two successive observation times. the observation periods were chosen because at these times the mite completes, on average, less than two reproductive cycles (fries & rosankranz, 1996). results show that the parasitization level in the drone brood was extremely high during the first observation periods (close to 40%) although this was significantly lower during the second observation period (dropping to close to 20%). the fact that the inspected drone brood of the first treatment was finally removed and replaced with a new one was probably the reason why there were significantly lower mite population levels detected during the second treatment since the initial parasitization level of the hives was removed. these results agree with those from similar studies conducted in other regions (engels et al., 1984; maul et al., 1988; rosenkranz & renz, 2003; calderone, 2005) and show the usefulness of the trapping comb method which, along with other measures, could be used in greece as a potential biotechnical approach to remove mites from beehives and decrease the initial parasitic load. the multiple parasitization per drone brood cells of v. destructor was lower compared to cell infections caused by single mites, especially during the first observation period. therefore, during the first generation, more than the half the inspected cells were occupied with only one mite while during the second period the number of inspected cells occupied with only one mite was lower. in contrast, a surprisingly higher percentage of multiparasitization (e.g., >3 mites/cell) was observed during the second observation period. this could be related to the higher fecundity rates that were registered during the second generation, as well as to the lower number of drone brood cells that were available for occupation. as a result, multiple parasitization was extremely high during that period. although it is generally difficult to evaluate the observed multiple parasitization levels, in a behavioral context it is known that the varroa mites must invade brood cells as quickly as possible because they cannot reproduce on adult bees (boot et al., 1994). to the best of our knowledge, there is no evidence to suggest that there is a preferential mite invasion into unoccupied cells and the observed invasions of more than one mite into brood cells could be related to the distance between the mite present on the bee and the brood cells. in addition, the total parasitic load of the colonies, including also the parasitized worker brood, could exert some influence on the observed multiple parasitization levels. nevertheless, although it is difficult to interpret the observed differences in triple infections between first and second observations, some studies relate cell infections to bee behavior, the total number of mites, as well as to the number of cells available for invasion. according to boot et al.(1994), the mites invade the cells when a bee is present just in front of the cell opening and this procedure is quite fast (few minutes). therefore, differences in observed multiple invasions could be due to the ratio of the mites on bees and the available cells to be occupied as quickly as possible. furthermore, since infested cells seem to be just as attractive to mites as non-infested cells (fuchs, 1985), the distance between mite and cell may be the key factor affecting the number of cells occupied by more than one mite, while the temporary immobilization of female varroa mites at the bottom of the open brood cells (ifantidis, 1988) permits additional mites to enter without directly interfering with the mite inhabitants already present. concerning the actual reproduction rate of v. destructor, this is generally difficult to be measured since it depends, among others, on mite fertility and not only fecundity (rosenkranz et al., 2010). therefore, in the current study, we address the challenge of measuring observed fecundity that corresponds to the number of viable adult offspring per mother mite. more precisely, it was found that number of viable offspring in drone broods was close to 1.7 during the first observation period and close to 2 during the second. the results of v. destructor obtained in this study are within reasonable limits of the different values reported by earlier studies (schultz, 1984; büchler, 1994; ifantidis, 1990; martin, 1994; donze & guerin, 1994; martin, 1995). in worker cells, for instance, the actual mean number of mature daughters per parental mite is recorded to be 1.3 (schultz, 1984), 1.4 (büchler, 1994), 1.45 (martin, 1994), or even 0.86, which is much lower (ifantidis, 1990). on the other hand, although in the drone brood all offspring theoretically have the same possibility of developing fully, because the period of sealed brood is longer than in worker cells (approx. 14 days), the reproductive rate differs among drone and worker brood (dade, 1977; boot et al., 1992; donze & guerin, 1994; martin 1995). according to martin (1995), it reaches 4 individuals, while this number drops to 1.7 (ifantidis, 1984) or 2.2 (martin 1995) when the mean number of viable female offspring per mother mite is taken into account (ifantidis, 1990). therefore, although it is known that mite populations vary according to bee genotype, mite genotype, geographical location, and climatic conditions, the levels of drone brood parasitization in the current study are slightly lower than the levels reported from other countries (rosenkranz, 1999; de guzman et al., 2007; de jong et al., 1984; calderon & venn, 2008). however, it is quite difficult to judge whether the low mite reproduction observed in the current study is the result of bee colony tolerance towards infestation or whether other factors play a role. it is known, for instance, that selective breeding of a. melifera colonies is not only correlated with the amount of brood and related honey yields, but also to the colony performance towards parasites (spivak & gilliam, 1993; spivak, 1996; rosenkranz, 1999; lodesani et al., 2002; rosenkranz et al., 2010). since in the current work there is no available information concerning the total number of the worker brood, the actual strength of the colonies towards mite parasitization and the observed lower fecundity levels could also be related to other factors. however, these results suggest a high correlation between the drone brood and mite populations, and the lower reproductive capacity recorded could also be due to a lower colony population strength. it is thought that mites have a higher probability of causing infection if more brood cells are available for invasion, and this affects the populations (boot et al., 1993, 1994; martin & kepm, 1997). in addition, mites seem to spend longer periods in a phoretic phase when brood is scarce (calis et al., 1990; lodesani et al., 2002). in addition, there were no significant differences in the parasitization level as estimated by absolute measures and by examining the mite excreta; this method can also be used to calculate initial parasistization levels. furthermore, mite population in the experimental colonies decreased through the study to significant levels after drone brood removal. considering that the invasion potential of varroa mites into drone brood cell is significantly higher than in worker cells (fuchs, 1990) current results performed in greek drone broods are in agreement with prior findings (schulz et al., 1983; crane, 1984; rosenkranz & engels, 1985; boecking & drescher, 1992; boot et al., 1994) and support the view that drone brood trapping in early spring can be used as a biotechnical method to mitigate varroa infection levels in bee colonies. conclusions although varoatosis has been considered to be among the most serious problems in beekeeping for the last 30 years, research is still ongoing to provide a rational means to find a solution (conte & gregorc, 2009; le conte et al., 2010; carreck et al., 2010). the results obtained in the current study, in general, agree with related studies that were article [page 104] [journal of entomological and acarological research 2012; 44:e18] no nco mm er cia l u se on ly conducted in other regions (maul et al., 1988; bailey & ball, 1991; rosenkranz & renz, 2003; calderone, 2005; fakhimzadeh, 2001; lodesani, 2004; delaplane et al., 2005). slight differences compared to other studies (rosenkranz, 1999; de guzman et al., 2007; de jong et al., 1984; calderon & venn, 2008) could be explained by the tolerance of different honeybee colonies towards infestation in different geographical regions, and other factors could also play a role. given our results, it is feasible that the reproductive potential and the varroa infection levels are still quite high and remain an important beekeeping issue. therefore, improvement and development of suitable new diagnostic tools to recognize infestation levels are essential to estimate region specific epidiological aspects of varroa. furthermore, the development and optimization of safe and effective rational management options, including the mite trapping technique, may improve beekeeping practices towards integrated management of varroa. in conclusion, since mite reproduction is an essential specific regional characteristic, increasing our knowledge of the reproductive performance of v. destructor in bee colonies of northern greece 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and varroa jacobsoni mites on honeybees. j. apic. res. 26: 196-202. article [page 106] [journal of entomological and acarological research 2012; 44:e18] no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. 429 too many requests you have sent too many requests in a given amount of time. jear2012 [journal of entomological and acarological research 2015; 47:5135] [page 65] abstract a faunistic study of the subfamily oscinellinae was investigated in shabestar region (east azarbaijan province, iran) during 2013-2014. four species aphanotrigonum bicolor nartshuk, 1964, dicraeus sabroskyi beschovski, 1977, lasiambia albidipennis (strobl, 1893) and lasiambia coxalis (von roser, 1840) are newly recorded from iran. in addition, one genus lasiambia sabrosky, 1941 is recorded for the first time from iran. the diagnostic characters and photos of the recorded species are provided. introduction the family chloropidae (diptera; commonly called frit flies or grass flies) with 204 genera and more than 2500 described species is a moderately large family of acalyptratae (nartshuk, 2012a). the larvae of this family have a very diverse biology. most chloropid larvae develop in grass, many species such as dicraeus raptus (haliday, 1838), meromyza saltatrix (linnaeus, 1761) and oscinella frit (linnaeus, 1758) are pests of cereal crops by feeding from seed, bud and shoots, some species of the genus lipara cause galls on stems, few such as lasiosina cinctipes (meigen, 1830) are saprophagous, small numbers such as thaumatomyia notata (meigen, 1830) are predators of some insects and some of them such as liohippelates collusor (townsend, 1895) (commonly called eye gnats) are very annoying pests for humans by attracting and transmitting diseases to the eye (sabrosky, 1941; chvala et al., 1974; deeming and al-dhafer, 2012). species of the subfamily oscinellinae differ from the other closest subfamilies of chloropidae by the following combination of characters: ocellar setae long, straight or recurved and convergent, without incurved humeral setae: vein c along margin of wings reaching to vein m1+2; hypopygium usually with well-developed cerci and surstylus (nartshuk et al., 1988; nartshuk and andersson, 2013). some new taxonomic studies and catalogue on the world fauna of chloropids are as follows: wheeler (2003, 2007) described a new species and genera of this family from freshwater wetlands in eastern canada and costa rica, respectively. wheeler and forrest (2003) described seven new species from galapagos islands. kubik (2006) described two new species from zambia. nartshuk (2012a, 2012b) provided a checklist of the world genera of the family chloropidae and also described three new species from southern sardinia, respectively. nartshuk and andersson (2013) provided a book about species of this family. nartshuk and fedoseeva (2011a, 2011b) provided a key to the species of the genus meromyza meigen, 1830. like many other families of diptera, the chloropidae of iran are poorly known. only seventeen species belonging to eleven genera of the subfamily oscinellinae have been previously recorded from iran (nartshuk, 1984; modarres-awal, 2011; khaghaninia et al., 2014a, 2014b). this study adds four species of this subfamily for the iranian insect fauna. materials and methods adult specimens were collected by standard sweep netting in grassland habitats from shabestar region located in northern west of iran during 2013-2014. the samples were killed in a killing jar containing potassium cyanide. the species were identified based on beshovski (1976); nartshuk et al. (1988); nartshuk (2011); nartshuk & andersson (2013). hypopygium and ovipositors were cleared in 10% koh. images were obtained using a microscope (nikon sms 1000) equipped with a camera (olympus 10 µ). the material examined is deposited in collections of the following institutions: ichmm: insect collection of professor hasan maleki milani, university of tabriz, tabriz, iran and culs: czech university of life sciences collections. results four species and one genus marked with an asterisk are newly reported for the iranian oscinellid fauna [aphanotrigonum bicolor correspondence: samad khaghaninia, university of tabriz, department of plant protection, faculty of agriculture, 51664, tabriz, iran. e-mail: skhaghaninia@gmail.com key words: chloropidae; oscinellinae; shabestar; iran; new records. acknowledgements: the authors sincerely thank dr. stepan kubik, department of zoology and fisheries, faculty of agrobiology, food and natural resources, czech university of life sciences, who kindly assistance in identifying the materials. received for publication: 5 march 2015. revision received: 30 april 2015. accepted for publication: 12 june 2015. ©copyright r. namaki khamneh et al., 2015 licensee pagepress, italy journal of entomological and acarological research 2015; 47:5135 doi:10.4081/jear.2015.5135 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2015; volume 47:5135 new records of the subfamily oscinellinae (diptera; chloropidae) from iran r. namaki khamneh,1 s. khaghaninia,1 e. gilasian2 1department of plant protection, university of tabriz, tabriz; 2insect taxonomy research department, iranian research institute of plant protection, tehran, iran no nco mm er cia l erately large family of acalyptratae (nartshuk, 2012a). the larvae of no nco mm er cia l erately large family of acalyptratae (nartshuk, 2012a). the larvae of this family have a very diverse biology. most chloropid larvae develop no nco mm er cia l this family have a very diverse biology. most chloropid larvae develop (haliday, 1838), no nco mm er cia l (haliday, 1838), (linnaeus, no nco mm er cia l (linnaeus, 1758) are pests of cereal crops by feeding from seed, bud and shoots, no nco mm er cia l 1758) are pests of cereal crops by feeding from seed, bud and shoots, cause galls on stems, few such as no nco mm er cia l cause galls on stems, few such as (meigen, 1830) are saprophagous, small numbers no nco mm er cia l (meigen, 1830) are saprophagous, small numbers (meigen, 1830) are predators of some no nco mm er cia l (meigen, 1830) are predators of some no nco mm er cia l described two new species from zambia. nartshuk (2012a, 2012b) prono nco mm er cia l described two new species from zambia. nartshuk (2012a, 2012b) pro-vided a checklist of the world genera of the family chloropidae and also no nco mm er cia l vided a checklist of the world genera of the family chloropidae and also described three new species from southern sardinia, respectively. no nco mm er cia l described three new species from southern sardinia, respectively. nartshuk and andersson (2013) provided a book about species of this no nco mm er cia l nartshuk and andersson (2013) provided a book about species of this family. nartshuk and fedoseeva (2011a, 2011b) provided a key to the no nco mm er cia l family. nartshuk and fedoseeva (2011a, 2011b) provided a key to the no nco mm er cia l no nco mm er cia l correspondence: samad khaghaninia, university of tabriz, department of no nco mm er cia l correspondence: samad khaghaninia, university of tabriz, department of plant protection, faculty of agriculture, 51664, tabriz, iran. no nco mm er cia l plant protection, faculty of agriculture, 51664, tabriz, iran. e-mail: skhaghaninia@gmail.com no nco mm er cia l e-mail: skhaghaninia@gmail.com key words: chloropidae; oscinellinae; shabestar; iran; new records.no nco mm er cia l key words: chloropidae; oscinellinae; shabestar; iran; new records. us e chloropids are as follows: wheeler (2003, 2007) described a new us e chloropids are as follows: wheeler (2003, 2007) described a new species and genera of this family from freshwater wetlands in eastern us e species and genera of this family from freshwater wetlands in easterncanada and costa rica, respectively. wheeler and forrest (2003) us e canada and costa rica, respectively. wheeler and forrest (2003) us e described seven new species from galapagos islands. kubik (2006)us e described seven new species from galapagos islands. kubik (2006) described two new species from zambia. nartshuk (2012a, 2012b) pro-us e described two new species from zambia. nartshuk (2012a, 2012b) proon ly ocellar setae long, straight or recurved and convergent, without on ly ocellar setae long, straight or recurved and convergent, without incurved humeral setae: vein c along margin of wings reaching to vein on lyincurved humeral setae: vein c along margin of wings reaching to vein; hypopygium usually with well-developed cerci and surstylus on ly; hypopygium usually with well-developed cerci and surstylus., 1988; nartshuk and andersson, 2013). on ly., 1988; nartshuk and andersson, 2013). some new taxonomic studies and catalogue on the world fauna ofon ly some new taxonomic studies and catalogue on the world fauna of chloropids are as follows: wheeler (2003, 2007) described a newon ly chloropids are as follows: wheeler (2003, 2007) described a new species and genera of this family from freshwater wetlands in eastern on ly species and genera of this family from freshwater wetlands in eastern [page 66] [journal of entomological and acarological research 2015; 47:5135] nartshuk, 1964, dicraeus sabroskyi beschovski, 1977, lasiambia albidipennis (strobl, 1893) and lasiambia coxalis (von roser, 1840)]. locality, diagnostic characters, biology and distribution of identified species at the present study are given briefly. diagnostic characters adapted by beshovski (1976); nartshuk et al. (1988); nartshuk (2011) and nartshuk & andersson (2013). the species found genus aphanotrigonum duda, 1932 aphanotrigonum bicolor nartshuk, 1964 (figure 1a-c) material examined: (1♂): shabestar (shanejan), 38°14’12.3” n, 45°43’11.5” e, 1649 m, 4 jun. 2014; leg. r. namaki khamneh. diagnostic characters: body length 2 mm; head mainly yellow; apex of scutellum, part of pleura and shoulders yellow; vertical triangle as long as wide and black; stripes of mesonotum black (figure 1a); antennal grooves separated by narrow keel; occiput and postgena black; antenna yellow; postpedicel rounded; arista yellow; legs brownish-yellow; abdomen brownish-yellow (figure 1b); male genitalia: epandrium small, cerci not tapering, surstyli not narrow (figure 1c). biology: larvae are saprophagous, feeding from decomposing stem and spikelets of poaceae (nartshuk et al., 1988). distribution: turano-european (nartshuk, 2011). genus: dicraeus loew, 1873 dicraeus sabroskyi beschovski, 1977 (figure 1a and b) material examined: (4♂♂, 15♀♀): shabestar (haftcheshmeh), 38°12’24.1” n, 45°27’29.8” e, 1313 m, 19 jun. 2013; (6♂♂ 32♀♀): (til), 38°15’31.7” n, 45°28’50.8” e, 1489 m, 26 jun. 2014; leg. r. namaki khamneh. diagnostic characters: body length 2-2.5 mm; background colour gray to black; gena yellow; vertical triangle black, shiny; antenna black, postpedicel rounded; arista black; thorax black; legs black; palpi yellow (figure 2a); male genitalia: hypopygium large; cerci slendered; surstyli long, broad and with distinct setae (figure 2b). biology: larvae are phytophagous. distribution: bulgaria (beshovski, 1976). genus: lasiambia sabrosky, 1941 lasiambia albidipennis (strobl, 1893) (figure 3a-c) material examined: (1♀): shabestar (shanejan), 38°14’12.3” n, 45°43’11.5” e, 1649 m, 5 jul. 2014; leg. r. namaki khamneh. diagnostic characters: body length 2.5-3 mm, colour black; head mainly black; vertical triangle shiny black; thorax shiny black (figure 3a); vibrissal corner protruding beyond margin of eye; gena brownish; legs black (figure 3b); antenna brownish; postpedicel relatively rounded; arista mainly yellow, black in basal third; palpi brown (figure 3c). biology: the larvae are probably saprophytophagous (séguy, 1934). distribution: south-european (nartshuk, 2011). lasiambia coxalis (von roser, 1840) (figure 4a-c) material examined: (1♀): shabestar (shanejan), 38°13’39.3” n, 45°43’07.6” e, 1602 m, 5 jul. 2014; leg. r. namaki khamneh. diagnostic characters: body length 2.5 mm; shiny black species; vertical triangle shiny black; thorax shiny black (figure 4c); vibrissal corner protruding beyond margin of eye; gena yellow; occiput and postgena black; legs black and yellow; wing dark (figure 4b); antenna bicolour yellow and black; postpedicel rounded; arista black (figure 4a). biology: larvae are parasitic and develop in egg masses of chrysochaon dispar germar (orthoptera: acrididae) in other acrididae (nartshuk & andersson, 2013). distribution: europe (nartshuk & andersson, 2013). discussion and conclusions dicraeus sabroskyi is the most frequent species among the ones identified in the present study. furthermore, lasiambia albidipennis and lasiambia coxalis were collected from grasslands of shabestar article figure 1. aphanotrigonum bicolor nartshuk, 1964 (male); a) dorsal view; b) lateral view; c) dorsal view of hypopygium. no nco mm er cia l biology: larvae are parasitic and develop in egg masses of no nco mm er cia l biology: larvae are parasitic and develop in egg masses of chrysochaon dispar no nco mm er cia l chrysochaon dispar no nco mm er cia l ): no nco mm er cia l ): (til), 38°15’31.7” n, 45°28’50.8” e, 1489 m, 26 jun. 2014; leg. r. no nco mm er cia l (til), 38°15’31.7” n, 45°28’50.8” e, 1489 m, 26 jun. 2014; leg. r. diagnostic characters: body length 2-2.5 mm; background no nco mm er cia l diagnostic characters: body length 2-2.5 mm; background colour gray to black; gena yellow; vertical triangle black, shiny; antenna no nco mm er cia l colour gray to black; gena yellow; vertical triangle black, shiny; antenna black, postpedicel rounded; arista black; thorax black; legs black; palpi no nco mm er cia l black, postpedicel rounded; arista black; thorax black; legs black; palpi yellow (figure 2a); male genitalia: hypopygium large; cerci slendered; no nco mm er cia l yellow (figure 2a); male genitalia: hypopygium large; cerci slendered; surstyli long, broad and with distinct setae (figure 2b). no nco mm er cia l surstyli long, broad and with distinct setae (figure 2b). (nartshuk & andersson, 2013). no nco mm er cia l (nartshuk & andersson, 2013). distribution: europe (nartshuk & andersson, 2013). no nco mm er cia l distribution: europe (nartshuk & andersson, 2013). no nco mm er cia l no nco mm er cia l no nco mm er cia l u se vibrissal corner protruding beyond margin of eye; gena yellow; occiput us e vibrissal corner protruding beyond margin of eye; gena yellow; occiput and postgena black; legs black and yellow; wing dark (figure 4b); us e and postgena black; legs black and yellow; wing dark (figure 4b);antenna bicolour yellow and black; postpedicel rounded; arista black us e antenna bicolour yellow and black; postpedicel rounded; arista black biology: larvae are parasitic and develop in egg masses ofus e biology: larvae are parasitic and develop in egg masses of chrysochaon disparus e chrysochaon dispar on ly ): shabestar (shanejan), 38°13’39.3” n, on ly ): shabestar (shanejan), 38°13’39.3” n, 45°43’07.6” e, 1602 m, 5 jul. 2014; leg. r. namaki khamneh. on ly45°43’07.6” e, 1602 m, 5 jul. 2014; leg. r. namaki khamneh.diagnostic characters: body length 2.5 mm; shiny black on lydiagnostic characters: body length 2.5 mm; shiny black species; vertical triangle shiny black; thorax shiny black (figure 4c);on ly species; vertical triangle shiny black; thorax shiny black (figure 4c); vibrissal corner protruding beyond margin of eye; gena yellow; occiputon ly vibrissal corner protruding beyond margin of eye; gena yellow; occiput and postgena black; legs black and yellow; wing dark (figure 4b);on ly and postgena black; legs black and yellow; wing dark (figure 4b); [journal of entomological and acarological research 2015; 47:5135] [page 67] article figure 2. dicraeus sabroskyi beschovski, 1977 (male); a) lateral view; b) dorsal view of hypopygium. figure 3. lasiambia albidipennis (strobl, 1893) (female); a) dorsal view; b) lateral view; c) frontal view of head. figure 4. lasiambia coxalis (von roser, 1840) (female); a) frontal view of head; b) lateral view; c) dorsal view. no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l (strobl, 1893) (female); a) dorsal view; b) lateral view; c) frontal view of head. no nco mm er cia l (strobl, 1893) (female); a) dorsal view; b) lateral view; c) frontal view of head. no nco mm er cia l no nco mm er cia l u se on ly on ly [page 68] [journal of entomological and acarological research 2015; 47:5135] region. nartshuk & andersson (2013), reported l. coxalis from egg masses of acrididae and séguy (1934) observed l. albidipennis in the stems of brassica oleraceae, it explains why we collected just one female of each species in an area with few acridids populations as well as few wild brassicaeus plants. before this study, dicraeus sabroskyi was collected and described only from bulgaria (beshovski, 1976), suggesting that this species is very rare in palearctic region. however, a relatively high population has been collected in the shabestar region, emphasizing ecological similarity of this region with the ones where the species was found. before this study 18 species belonging to the subfamily oscinellinae were recorded from iran, present work adds other four species to the iranian checklist. the known iranian frit flies of oscinellinae are about one fourth of the subfamily chloropinae (nartshuk, 1984; modarresawal, 2011; khaghaninia et al., 2014a, 2014b). this contradicts common knowledge that oscinellinae exceed chloropinae around the world, suggesting that iranian checklist is still incomplete and needs further study. references beshovski v., 1976 dicraeus sabroskyi sp. n. diptera, chloropidae a new species from bulgaria. acta. zool. bulg. 6: 54-57. chvala m., doskocil j., mook j.h., pokorny v., 1974 the genus lipara meigen (diptera, chloropidae), systematics, morphology, behaviour, and ecology. tijdschr. ent. 117: 1-25. deeming j.c., al-dhafer h.m., 2012 chloropidae from the arabian peninsula (diptera: cyclorrhapha). zool. middle east. 58: 4-88. khaghaninia s., gharajedaghi y., namaki khamneh r. 2014a some of the chloropid flies (diptera: chloropidae) of wheat fields from east azerbaijan province with new pest records for iran. appl. res. plant prot. 3: 65-75. khaghaninia s., kudik s., garajedaghi y., 2014b new data on grass flies (diptera, chloropidae) from iran. dipterist dijest 21: 135-142. kubik s., 2006 two new species of pselaphia (diptera, chloropidae) from zambia biologia, bratislava 61: 159-160. modarres-awal m., 2011 list of agricultural pests and theirnatural enemies in iran ferdowsi university of mashhad publication, mashhad, third edition, 447 pp. nartshuk e.p., 1984 family chloropidae. in: soós á. & papp l. (eds.), catalogue of palaearctic diptera. hungarian natural history museum, budapest, 10: 222-298. nartshuk e.p., 2011 chloropidae from southern sardinia (diptera: cyclorrhapha, acalyptratae). conserv. habitat. vertebrati 5: 717732. nartshuk e.p., 2012a a check list of the world genera of the family chloropidae (diptera, cyclorrhapha, muscomorpha). zootaxa 3267: 1-43. nartshuk e.p., 2012b three new species of chloropidae (diptera) from southern sardinia. zootaxa 2318: 545-551. nartshuk e.p., andersson h., 2013 the frit flies (chloropidae, diptera) of fennoscandia and denmark. brill academic publishers, leiden, 277 p. nartshuk e.p., fedoseeva l.i., 2011a a review of the grass flies of the genus meromyza meigen, 1830 (diptera, chloropidae) of the palaearctic fauna, with a key to the species, analysis of synonymy, host specialization, and geographical distribution: part 1. entomol. rev. 91: 103-120. nartshuk e.p., fedoseeva l.i., 2011b a review of the grass flies of the genus meromyza meigen, 1830 (diptera, chloropidae) of the palaearctic fauna, with a key to the species, analysis of synonymy, host specialization, and geographical distribution: part 2. entomol. rev. 91: 778-795. nartshuk e.p., smirnov e.s., fedosceva l.i., 1988 family chloropidae. in: bei-bienko g.y. (ed.), keys to the insects of the european part of the ussr, volume 5, part 1: diptera (insecta): nhbs. gy bei-bienko, pensoft: 669-731. sabrosky c.w., 1941 the hippelates flies or eye gnats: preliminary notes. canad. entomol. 73: 23-7. séguy e., 1934 faune de france, 28. diptères (brachycères) (muscidae acalypterae et scatophagidae). lechavalier, paris, i–iv + 832 pp. wheeler t., 2003 a new brachypterous species of elachiptera becker (diptera: chloropidae) from freshwater wetlands in eastern canada. zootaxa 360: 1-6. wheeler t., 2007 two new genera of oscinellinae chloropidae (diptera) from costa rica. zootaxa 1413: 47-53. wheeler t., forrest j., 2003 the chloropidae (diptera) of the galápagos islands, ecuador. insect sys. evol. 34: 265-280. article no nco mm er cia l nhbs. gy bei-bienko, pensoft: 669-731. no nco mm er cia l nhbs. gy bei-bienko, pensoft: 669-731. no nco mm er cia l khaghaninia s., kudik s., garajedaghi y., 2014b new data on no nco mm er cia l khaghaninia s., kudik s., garajedaghi y., 2014b new data on grass flies (diptera, chloropidae) from iran. dipterist dijest 21: no nco mm er cia l grass flies (diptera, chloropidae) from iran. dipterist dijest 21: kubik s., 2006 two new species of pselaphia (diptera, chloropidae) no nco mm er cia l kubik s., 2006 two new species of pselaphia (diptera, chloropidae) modarres-awal m., 2011 list of agricultural pests and theirnatural no nco mm er cia l modarres-awal m., 2011 list of agricultural pests and theirnatural enemies in iran ferdowsi university of mashhad publication, no nco mm er cia l enemies in iran ferdowsi university of mashhad publication, sabrosky c.w., 1941 the no nco mm er cia l sabrosky c.w., 1941 the notes. canad. entomol. 73: 23-7. no nco mm er cia l notes. canad. entomol. 73: 23-7. séguy e., 1934 faune de france, 28. diptères (brachycères) no nco mm er cia l séguy e., 1934 faune de france, 28. diptères (brachycères) (muscidae acalypterae et scatophagidae). lechavalier, paris, i–iv no nco mm er cia l (muscidae acalypterae et scatophagidae). lechavalier, paris, i–iv wheeler t., 2003 a new brachypterous species of no nco mm er cia l wheeler t., 2003 a new brachypterous species of us e nartshuk e.p., smirnov e.s., fedosceva l.i., 1988 family us e nartshuk e.p., smirnov e.s., fedosceva l.i., 1988 familychloropidae. in: bei us e chloropidae. in: beius e -bienko g.y. (ed.), keys to the insects of us e bienko g.y. (ed.), keys to the insects ofthe european part of the ussr, volume 5, part 1: diptera (insecta): us e the european part of the ussr, volume 5, part 1: diptera (insecta): nhbs. gy bei-bienko, pensoft: 669-731.us e nhbs. gy bei-bienko, pensoft: 669-731. sabrosky c.w., 1941 the us e sabrosky c.w., 1941 the on ly nartshuk e.p., fedoseeva l.i., 2011b a review of the grass flies of on ly nartshuk e.p., fedoseeva l.i., 2011b a review of the grass flies of meigen, 1830 (diptera, chloropidae) of the on lymeigen, 1830 (diptera, chloropidae) of thepalaearctic fauna, with a key to the species, analysis of synonymy, on lypalaearctic fauna, with a key to the species, analysis of synonymy, host specialization, and geographical distribution: part 2. -on ly host specialization, and geographical distribution: part 2. entomol. rev. 91: 778-795.on ly entomol. rev. 91: 778-795. nartshuk e.p., smirnov e.s., fedosceva l.i., 1988 familyon ly nartshuk e.p., smirnov e.s., fedosceva l.i., 1988 family 429 too many requests you have sent too many requests in a given amount of time. jear2012 [journal of entomological and acarological research 2016; 48:5181] [page 11] abstract molecular identification is going to be more widespread in taxonomic studies of insects when traditional tools are problematic and time consuming. identification of bark beetles, as one of the most important pests of forests, based on morphological characteristics is difficult because of their small size and morphological similarities. in the current study, species-specific primers were designed to identify two most abundant and morphologically similar bark beetle species scolytus ensifer eichhoff 1881 and s. ecksteini butovitsch 1929, both found on ulmus minor miller in north of iran. these species-specific primers successfully produced a fragment size with 318 bp and 465 bp of mitochondrial cytochrome oxidase 1 (co1) gene in s. ensifer and s. ecksteini respectively. the results revealed that the multiplex polymerase chain reaction using the species-specific primers could amplify a unique band to distinguish these two species so confirmed this method as a convenient and quick tool to identify those two bark beetle species. introduction bark beetles of the subfamily scolytinae (coleoptera: curculionidae) contain nearly 6000 described species worldwide by small size less than <1 mm to 1 cm in length (jordal, 2007). the majority of scolytids are decomposer of plant tissues but some are aggressive pests of forests by significant economic and ecological damage in this ecosystem (furniss & carolin 1977; kuschel, 1995). the pest species of this group include those feed on phloem (true bark beetles), bore into the xylem and cultivate a fungal garden (ambrosia beetles). some species in the genus scolytus geoffroy, 1762 can transfer pathogenic ophiostomatoid fungi, which cause lethal dutch elm disease (ded) in ulmus (elm) trees (gibbs, 1978). at least eight scolytus species are known as vectors of ded including scolytus ensifer eichhoff, 1881 and s. pygmaeus f. 1787 (gibbs, 1978; faccoli et al., 1998). identification of bark beetles is problematic since there are paucity in morphological characteristics. meanwhile, identification of scolytine immature stages would be more challenging if a control strategy needed to be implemented for the pest species, such as vectors of dutch elm disease (santini and faccoli, 2015). s. ensifer and s. ecksteini butovitsch, 1929 are two abundant bark beetles species in guilan province (north of iran) that utilize ulmus species (ulmaceae) (amini et al., 2012). traditional identification of s. ensifer and s. ecksteini based on morphological characteristics is difficult due to wide interand intra-specific variability of their general size, shape, and color of adults. for most scolytus species, diagnostic characteristics are the presence and shape of spines on the abdominal sternites. in both species, location of spines on basal margin of the second abdominal sternite is identical (pfeffer, 1994). however, shape of the spine is slightly different and taxonomic training is necessary for its correct identification (amini & hosseini, 2012). during the last decade, molecular techniques have been used for identification of insects that lack diagnostic morphological characteristics (caterino et al., 2000) but a few molecular methods have been developed for identification of bark beetles. mitochondrial cytochrome oxidase i (co1) sequences has provided tools for identification of scolytines (stauffer et al., 1997; kelley et al. 1999; cognato & sperling, 2000; cognato & sun, 2007). johnson et al. (2008) used random amplified polymorphic dna-polymerase chain reaction (rapd-pcr) for identification of the two non-native bark beetles. jordal and kambestad (2013) used dna barcoding for identification of bark and ambrosia beetles. among molecular diagnostic techniques, the multiplex pcr method uses multiple primers in a single reaction for amplification of species-specific pcr products. this method amplifies specific sequences of genome by pcr in a single reaction simultaneously to identify any life stage of numbers of species (bej et al., 1991; henson & french, 1993). this method has been proven as a reliable correspondence: sudabe amini, department of plant protection, faculty of agricultural sciences, university of guilan, rasht, iran. tel: +98.9151602647 fax: +98.563242202. e-mail: sudabe.amini@ut.ac.ir. key words: bark beetle; cytochrome oxidase 1; molecular identification; multiplex polymerase chain reaction. acknowledgements: the authors wish to thank sarah smith and anthony cognato (united states of america, michigan state university) for confirming morphological identification. special thanks dr. arash zibaee (university of guilan) for theirs thoughtful comments and hadi sheikhnejad (university of guilan) for technical assistance with molecular work. received for publication: 29 march 2015. revision received: 6 december 2015. accepted for publication: 7 december 2015. ©copyright s. amini and r. hosseini, 2016 licensee pagepress, italy journal of entomological and acarological research 2016; 48:5181 doi:10.4081/jear.2016.5181 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2016; volume 48:5181 a multiplex polymerase chain reaction based method for rapid identification of two species of the genus scolytus geoffroy (col: curculionidae: scolytinae) in iran s. amini, r. hosseini department of plant protection, university of guilan, rasht, iran no nco mm er cia l u se on ly [page 12] [journal of entomological and acarological research 2016; 48:5181] identification method for economically important pests or predators (szalanski et al., 2003; hosseini et al., 2007; szalanski & mckern, 2007; hosseini & hajizadeh, 2011). so, objectives of the current study were to design species-specific primers and to develop a multiplex pcr method for rapid and accurate identification of the two common scolytus species present on ulmus minor. materials and methods bark beetle sampling bark beetle specimens were collected on the trunks and twigs of u. minor in three different parts of guilan province during may september 2011-2012. in total 160 samples were collected on ulmus minor. specimens were placed in 1.5 ml micro tubes, transferred to the laboratory and immediately stored at –20°c. all specimens were identified under a stereomicroscope (olympustm, model szx-12) by using taxonomic keys (pfeffer, 1994) and sent to sarah smith and anthony cognato (united states of america, michigan state university) to check the identifications based on morphological characters. all voucher specimens were deposited in natural history museum, university of guilan (rasht, iran). dna extraction dna was extracted from whole body of each identified species, separately. each species individually was homogenized in 1.5 ml tubes using a sterile plastic pestle in 50 µl of phosphate buffer saline ph 7.4 samples were incubated at 56°c for 4-5 h followed by adding 500 µl of chelex 5% (bio-rad laboratories, inc., hercules, ca, usa), and placed in water bath at 94°c for 15 min. after centrifugation at 13,000 g for 5 min, dna was transferred to a new tube and stored at –20°c (hosseini, 2010). polymerase chain reaction and sequencing three universal primers including c1-j-1718 (5’-ggaggatttggaaattgattagttcc-3’) as forward primer, c1-n-2191 (5’cccggtaaaattaaaatataaacttc -3’) (simon et al., 1994) and c1-j-2411 (5’-gctaatcatctaaaaactttaattccwgtwg-3’) as reverse primers (normark et al., 1999) were used to amplify a part of co1 in each species. pcr cocktails contained a total volume of 25 µl including 15.05 µl ddh2o, 2.5 µl reaction buffer, 0.25 µl dntps (15 mm, fisher scientific inc., hampton, new hampshire, usa), 1 µl mgcl2 (50 mm cinnagen co., tehran, iran), 1 µl of each primer (forward and reverse), 10 µm (bioneer co., daejeon, south korea), 0.2 µl of taq dna polymerase (5u/�µl, cinnagen co.) and 4 µl of dna template (5070 ng/�µl). the master mix was placed in a 0.2 ml pcr tube and amplified in a thermocycler (mastercycler gradient, eppendorf, germany) with the relevant temperature profile shown in table 1. the amplified fragments were visualized by 1.4% agarose gel in tae or tbe buffer stained in syber safe dna gel stain% (bio-rad laboratories, inc.). pcr products were sequenced using the sanger sequencing method by abi3730xl sequence analyzer (applied biosystems, foster city, ca, usa) from bioneer co. sequence alignment and primer design co1 fragments were sequenced from three individuals per species in forward direction. sequencing results were reviewed by the finch tv soft-pedia software (http://www.geospiza.com/ftvdlinfo.html) and edited manually for each species individually. the basic local alignment search tool (blast) was used to compare similarity of our nucleotide sequences with other sequences present in genbank database, (http://www.ncbi.nlm.nih.gov/blast). edited sequences were deposited in genbank database with specific accession numbers (tables 2 and 3). sequences were aligned using mega ver. 5.0 software (tamura et al., 2011) to reveal their nucleotide variation. forward primers were designed for s. ensifer and s. ecksteini according to their sequence differences by using universal reverse primer (c1-j-2411). guidelines proposed by innis and gelfand (1990) and saiki (1990) were followed to design efficient and specific primers. the primer-primer interactions were analyzed using integrated dna technologies (idt 2010; primerquest: http://eu.idtdna.com/scitools/applications/primer-quest/ default.aspx) software and those were synthesized by bioneer co. a gradient pcr program was performed using a gradient thermocycler as 35 cycles at 94°c for 1 min, 50°c as the lower temperature and 60°c as the higher temperature for 45 s, and 72°c for 1 min 30 s to optimize annealing temperature of primer pairs. a first cycle of denaturation was 94°c for 2 min and a final extension was performed at 72°c for 5 min. specificity of designed primers were evaluated using both singleplex and multiplex pcr compared with other bark beetle species including; s. pygmaeus (f.), s. rugulosus (müller), hypothenemus eruditus westwood, and taphrorychus lenkoranus reitter found on other hosts in guilan province (amini et al., 2012). multiplex polymerase chain reaction a multiplex pcr was conducted to amplify specific fragments from each bark beetle species. pcr cocktails contained a total volume of 25 µl including 4 µl of dna (20-50 ng/µl), 0.25 µl of dntp’s (15 mm), 1 µl of mgcl2 (50 mm), 4 µm of equal molar of each specific forward primers with one reverse universal primer (c1-j-2411) and 0.2 µl of taq dna polymerase (5 u/µl). cycling condition of pcr has been shown in table 1. pcr products were mixed with loading buffer and run on 1.4% agarose gel prior to be stained by syber safe dna gel stain solution (cinnagen co.). a 100 bp dna ladder (fermentas, vilnius, lithuania) was used for determining of fragment size. application of multiplex polymerase chain reaction to test efficiency of multiplex pcr for identification of the collected specimens, 70 samples of unknown adults and larvae (58 and 12 article table 1. temperature profile for amplification of cytochrome oxidase one fragments and multiplex polymerase chain reaction. c1-j-1718/ c1-j-2411 c1-j-1718/ c1-n-2191 multiplex pcr 1 94°c for 2 min 1 94°c for 2 min 1 94°c for 2 min 2 94°c for 1 min 2 94°c for 1 min 2 94°c for 1 min 3 52°c for 45 s 3 50°c for 1 min 3 53°c for 1 min 4 72°c for 1 min 4 72°c for 1 min 4 72°c for 1 min 30 s 5 go to 2 for 34 cycle 5 go to 2 for 34 cycle 5 go to 2 for 35 cycle 6 72°c for 5 min 6 72°c for 5 min 6 72°c for 5 min pcr, polymerase chain reaction. no nco mm er cia l u se on ly respectively) were collected on u. minor from different locations of guilan province and identified morphologically by expert entomologist (sarah m. smith ph.d. in michigan state university). four adults and two larvae were selected for the multiplex pcr test. all specimens were individually placed in 1.5 ml tubes and kept at –20°c for subsequent molecular assay. dna extractions were performed using the chelex 5% (bio-rad laboratories, inc.) method described earlier. results in this study, adults of three scolytus species including s. ecksteini, s. ensifer, and s. pygmaeus were identified morphologically and a part of their co1 was successfully amplified. the universal primers, c1-j1718/ c1-j-2411, amplified fragments of approximately 650 bp of co1 gene in s. ecksteini, s. pygmaeus and s. rugulosus, but amplification was not successful for s. ensifer. therefore primers c1-j-1718/ c1-n2191 were used to amplify a shorter fragment in s. ensifer. these universal primers amplified first 465 bp of co1 upstream region in s. ensifer but those amplified nearly 650 bp barcode region in other scolytus species (hebert et al., 2003). amplified fragments varied in size because of using different primers. size of fragments was 610, 650, 661 and 465 bp in scolytus rugulosus, s. pygmaeus, s. ecksteini and s. ensifer, respectively. blast searches on genbank show high similarity to others scolytus species. obtained sequences were submitted to genbank and were provide accession number (table 2). three equal molar primers including two specific forward primers and one universal reverse primer (c1-n-2191) were combined in a multiplex pcr assay to achieve a quick and robust molecular technique for identification two species of bark beetles on ulmus trees in guilan province. the optimal annealing temperature for multiplex pcr was determined to be 53°c. results indicated that the specific used primers developed were successfully amplified the expected specific fragment for s. ensifer and s. ecksteini. the size of pcr products for s. ensifer and s. ecksteini were 318 bp and 465 bp respectively. specificity tests for primer pairs showed amplification of fragments for target species although no amplification was observed in s. pygmaeus and s. rugulosus as non-target species (figure 1). our results also verified the application of multiplex pcr for successful identification of field-collected specimens (figure 2). [journal of entomological and acarological research 2016; 48:5181] [page 13] article table 2. species, primers and genbank accession numbers for mitochondrial cytochrome oxidase one sequences. species primers accession numbers scolytus ensifer (1718/2191) jx913804 scolytus ecksteini (1718/2411) jx416909, jx416907, jx416902 scolytus pygmaeus (1718/2411) jx089346, jx089347, jx089348 scolytus rugulosus (1718/2411) jx089345, jx089343, jx089342 scolytus pygmaeus (1718/2411) jx089346, jx089347, jx089348 table 3. species-specific forward primers for cytochrome oxidase one, length of primer and fragment size. species primer name primer sequence (5’ to 3’) primer length (bp) fragment size (bp) scolytus ecksteini ec-s19 gatttcctctattttggtgcta 22 465 scolytus ensifer en-s20 tttactctcgttgcccgtac 20 318 figure 1. multiplex polymerase chain reaction by combination of three equimolar primers for co1. 1: 100 bp dna ladder; 2 and 3: scolytus ecksteini; 4-5: s. ensifer; 6: s. pygmaeus; 7: s. rugulosus; 8: hypothenemus eruditus; 9: taphrorychus lenkoranus; 10: negative control. figure 2. multiplex polymerase chain reaction test for identification of field collected specimens. 1: 100 bp dna ladder; 2 and 3: positive control (scolytus ensifer and s. ecksteini); 4: unknown species larvae; 5-6: s. ensifer adults; 7-8: s. ecksteini adults; 9: s. ecksteini larvae; 10: negative control. no nco mm er cia l u se on ly [page 14] [journal of entomological and acarological research 2016; 48:5181] discussion and conclusions accurate identification of pests is fundamental in control strategy of them. in this study in the first step part of mitochondrial genome sequenced successfully and then designed primers and test primers to amplify specific fragments. the major problem in identification of bark beetles species is similarity of morphological characters such as similarity in shape of spine that is exactly on the same sternites of abdomen in both species (amini et al., 2012) that makes identification difficult, time consuming, and morphologic method needs good quality specimens and expertise in specialized taxonomy (leclercq & lecomte, 1978). above all scolytus species are vectors of fungi ophostomaid ulmi that lead to dutch elm disease and implementation of a specific control strategy for species would be a crucial challenge if identification should be made in their immature stages. according to recent studies pcrbased methods have shown to be a powerful tool in the identification of quarantined pests that have a similar morphology (hosseini et al., 2007), but the molecular technology using mtdna is easier to perform and saves time. this method can also identify damaged specimens with lack of morphological characters (judith & nicola, 2008). as bark beetles life cycle is under the bark of tree, in most of time they are not accessible in numerous and just their galleries or their vouchers are represented. so multiplex-pcr method could be an efficient identification tool when a specimen is one of only a handful of species (gariepy et al., 2008). results of this study indicated that different scolytus species could utilize and simultaneously live on the same branches of elm trees. those are very similar so their discrimination might be difficult mainly in immature stages. a rapid identification method develop for diagnosis of two important similar species of bark beetles among the bark beetles that attack elm trees. our experiments confirmed efficiency of multiplex pcr method for identification of field collected scolytus in both larval and adult stages in same time that might be compared with findings of hosseini et al. (2007) and hosseini and hajizadeh (2011). hosseini and hajizadeh (2011) designed species-specific primer and identified three mealybug species belong to different geographic location in a single reaction by multiplex pcr. in this study co1 in mtdna region is selected for design of primers, because studies have shown that this region is useful for identification of coleoptera species (paul et al., 2009; dirk et al., 2007; fang, 2009) as result showed high potential of this region of genome in discrimination of scolytus species. johnson et al. (2008) developed a rapd-pcr method to identify collected scolytus species as the two non-native bark beetles. although, the authors insist on using selected oligonucleotide primers in the rapd-pcr analysis but other studies showed rapd-pcr technique as a notoriously laboratory-dependent that could not be reproducible in other conditions (johnson et al., 2008). our study, the specific primers were successfully designed according differentiates in sequences and developed from barcoding region of s. ensifer and s. ecksteini species (co1, 5’ upstream region) and distinguished species from the other scolytid species accurate, in one reaction simultaneously. chen et al. (2013) designed species-specific primers according to co1 region of an invasive species dendroctonus valens leconte by using nested pcr method. the authors had chosen the specific region of genome by nested pcr method to design the high sensitive and specific primers for rapid and accurate distinguish beetles from the others that are abundant in china ports. chen et al. (2013) result is compared with our essay, but because in s. ensifer species pcr was not successful and dna could not amplified by universal primers, this led to choose a shorter region of co1 genome to amplify. combination of the two specific primers along with a reverse primer could successfully generated two identical different fragments, which is a major necessity in designing multiplex pcr primers. the species-specific associated primers could be successfully used in multiplex pcr to amplify products with two different sizes, which allows identification of the two bark beetle species in a single reaction. it is possible to identify a large number of scolytus species even in immature developmental stages, which might be a rapid and relatively low cost. this method proved to be an applicable technique for forestry studies, which is rapid and accurate particularly when the issue involves quarantine pests. multiplex pcr has ability to detect detecting pathogens in insect vectors (roy et al., 2005; ravikumar et al., 2011) like ambrosia beetles and it would be applicable in future study to detect fungi transported by ambrosia beetle by this innovative methods. references amini s., hosseini r., 2012 introduction and identification key for three elm bark beetles species in central part of guilan province. plant pests res. 2:13-20 [in persian]. amini s., hosseini r., sohani m., 2012 a faunal study of bark beetles (coleoptera: curculionidae: scolytinae) in guilan province in north of iran. entomofauna. 12: 169-176. bej a.k., mccarty s.c., atlas r.m., 1991 detection of coliform bacteria and escherichia coli by multiplex polymerase chain reaction, comparison with defined substrate and plating methods for water quality monitoring. app. environ. microbiol. 57: 2429-2432. caterino m.s., cho s., sperling f.a.h., 2000 the current state of insect molecular systematics: a thriving tower of babel. ann. rev. entomol. 45: 1-54. chen f., luo y., li j. g., zhao h., zong sh., shi j., 2013 rapid detection of red turpentine beetle (dendroctonus valens leconte) using nested pcr. entomol. am. 119: 7-13. cognato a.i., sperling f.a.h., 2000 phylogeny of ips degeer species (coleoptera: scolytidae) inferred from mitochondrial cytochrome oxidase i dna sequences. mol. phyl. evol. 14: 445-460. cognato a.i., sun j.h., 2007 dna based cladograms augment the discovery of a new ips species from china (coleoptera: curculionidae: scolytinae). cladistics. 23: 539-551. dirk a., michael t.m., alfried p.v., 2007 dna-based taxonomy for associating adults and larvae in multi-species assemblages of chafers (coleoptera: scarabaeidae). mol. phyl. evol. 44: 436-449. faccoli m., zanocco d., battisti a., masutti l., 1998 chiave semplificata per la determinazione degli scolytus geoffroy (coleoptera: scolytidae) italiani viventi sugli olmi. redia 81: 183-197. fang x.m., 2009 phylogenetic analysis of chrysomelidae in china inferred from mitochondrial co1 sequences (coleoptera: chrysomeloidea). j. suzhou. univ. 24: 113-117. furniss r.l., carolin v.m., 1977 western forest insects. united states department of agriculture, forest service; 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[journal of entomological and acarological research 2016; 48:5181] [page 15] article no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2016; 48:5403] [page 355] abstract the citrus leaf miner, phyllocnistis citrella stainton (lep., gracillariidae), is one of the most destructive pest of citrus and related rutaceae and ornamental plants in iran. larvae damage leaves by creating serpentine feeding mines, which have been lead to reduce yield. resistance and toxicity problems derived from synthetic insecticides have made it necessary to find more effective and healthier alternatives; therefore, bio-insecticides (i.e., bacillus thuringiensis) are becoming an important component in plant protection. the aim of the present study was to evaluate the efficiency of b. thuringiensis against p. citrella. eight b. thuringiensis concentrations were used against p. citrella l3 on orange and mortality was recorded at 1, 4, 7 and 10 days after spraying. the results showed that b. thuringiensis significantly affected mortality of p. citrella. after 1, 4, 7 and 10 days of spraying 108 concentration of b. thuringiensis had significantly caused the highest mortality to the pest with 59.8, 68.4, 73.6 and 77.0%, respectively. then the mortality percent decreased until it reached 6.5, 9.5, 39.3 and 46.7% at 101 concentration, respectively. in conclusion, the study indicated that b. thuringiensis is effective in controlling p. citrella under laboratory conditions. introduction citrus is one of the important fruit crops that are useful for human being as a source of nutrition and health product. it provides high levels of vitamin c and potassium and some of the daily requirements for essential nutrients such as folic acid and thiamine. citrus growing in iran has a very old history, which goes back as far as 330 bc (ebrahimi, 2010). in 2013, iran produced a total of four million tons of citrus from planted areas of about two hundred and ninthly thousand hectares with an average of 19,000 kg/ha (agricultural statistics, 2010). like many other agriculture crops, citrus production is hampered by problems, mainly of pests and diseases (french et al., 1997). one of the main insect pests that attack citrus is the citrus leaf miner, phyllocnistis citrella stainton (lep., gracillariidae), which adversely affect plant health and fruit development and enhance the development of canker disease (heppner, 1995; das et al., 1998; gill, 1999; liu et al., 1999; mafi & ohbayashi, 2000; michaud & grant, 2003; beattie & hardy, 2004; kahrarian, 2010). the first record of citrus leaf miner from southern and northern iran, with a dramatic increase and widespread dispersal, was noted in 1961 and 1994, respectively (amiri besheli, 2006a). the pest has five to nine generations/year, with peak periods in early summer and early autumn (amiri besheli, 2006b). p. citrella population increased over the years due to increasing cultivation of citrus and inappropriate agricultural practices applied by gardeners (margobandhu, 1993; jacas & pena, 2002; moreira et al., 2006). the control of this pest is based on chemical products. however, p. citrella has developed resistance to almost all pesticides used including organophosphates, pyrethroids, and carbamates, requiring higher doses or a mixture of several products for their effective control. these practices result in increased production costs and contamination of the environment (beattie et al., 1995). vigorous application of chemical insecticides has been used to repress p. citrella in the middle east (jafari, 1995; jafarzadeh, 2000; javan moghadam, 2001). in iran, seraj (2001) reported that abamectin and imidacloprid caused mortality higher than 70% to the pest under laboratory conditions (seraj, 2001). in addition, demir et al. (2012) in turkey and jafari (1997) in iran, reported that diflubenzeron proved to be effective against p. citrella. however, the use of synthetic pesticides is neither economic nor tolerable and has an undesirable effect on the environment due to their slow biodegradation in the environment, natural enemies and farmers (jafarzadeh, 2000; seraj, 2001). the intensive pest control programs using modern insecticides practiced in the pakistan and iran is not only a costly form of pest control, but also leads to resurgence of secondary pests such as mites and scale insects (jafari, 1996). the correspondence: karim saeidi, department of plant protection, agricultural and natural resources research and education center, kohgiluyeh va boyerahmad province, p.o. box 351, yasouj, iran. tel.: +98.741.3334821/+98.741.3334821 fax: +98.741.3334011. e-mail: saeidi391@yahoo.com key words: phyllocnistis citrella; bacillus thuringiensis; citrus leaf miner; control; insecticidal bacterium. received for publication: 29 june 2015. revision received: 12 july 2016. accepted for publication: 14 july 2016. ©copyright k. saeidi, e. saeidi, 2016 licensee pagepress, italy journal of entomological and acarological research 2016; 48:5403 doi:10.4081/jear.2016.5403 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 4.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:e journal of entomological and acarological research 2016; volume 48:5403 bio-control efficiency of bacillus thuringiensis (berliner) against the citrus leaf miner, phyllocnistis citrella stainton (lep., gracillariidae) under laboratory conditions k. saeidi,1 e. saeidi2 1department of plant protection, agricultural and natural resources research and education center, kohgiluyeh va boyerahmad, province, yasouj; 2department of veterinary, kazerun branch, islamic azad university, kazerun, iran no nco mm er cia l u se on ly [page 356] [journal of entomological and acarological research 2016; 48:5403] adverse effects of synthetic pesticides have amplified the need for effective and biodegradable pesticides. an alternative is the use of biological control such as the use of predators, parasitoids, and entomopathogens, including fungi, bacteria, viruses, and nematodes. within the bacterial group, the microorganism most widely used worldwide with the highest success in the control of several insect pests is the bacterium, bacillus thuringiensis berliner (bacillales: bacillaceae). b. thuringiensis has been shown to be useful for the control of different insect pests that affect plant crops, forest trees, or those are vectors of human diseases such as dengue and malaria (dubois & dean, 1950; mahapatro &, gupta, 2000). b. thuringiensis is a gram-positive and soil-dwelling bacterium commonly used as a biological pesticide. b. thuringiensis also occurs naturally in the gut of larvae of various types of lepidopteran, dipteran and coleopteran insects (demir et al., 2012). during sporulation, many b. thuringiensis strains produce crystal proteins (proteinaceous inclusions), that are toxic to the larvae of lepidoptera and other orders of invertebrates. because of there is no comprehensive studies on the use of b. thuringiensis against citrus leaf miner in iran, therefore, the main objective of this study were to the efficacy of b. thuringiensis against p. citrella. results presented here may be helpful for future planning of large-scale citrus cultivation in similar environmental conditions of tropics, especially for pest management purposes. material and methods in this study the stock cultures of the citrus leaf miner, p. citrella and its host plant were inevitably required for the various experiments. therefore, mass rearing both p. citrella and the host plant must be conducted. in principle, there are two main activities related to mass culturing of insect and planting of the host plant, citrus sinensis l. planting of the host plant, citrus sinensis citrus sinensis l, common known as orange is one of the host plants that are most preferred by the citrus leaf miner, p. citrella (beattie, 2004); therefore, this plant is used to rear the citrus leaf miner. the plant can be propagated through seedling and stem cutting; however, the use of seedling is preferable to stem cutting. germination of c. sinensis l. seeds was done on plastic trays (35 cm long × 25 cm wide at top; 31 cm long × 21 cm wide at bottom; and 10 cm high) filled with a mixture of mineral soil, manure and sand (1:1:1). these seeds were placed a row in planting holes spaced 5 by 5 cm apart. the mixture was kept damp in order to hasten seed turgidity and germination. the seeds started to germinate after 2-3 weeks. the seedlings, 10 to 15 cm tall, were then transplanted to plastic flower pots (20 cm top diameter × 15 cm bottom diameter × 18 cm high) containing the same mixture. plants reaching more than 50 cm tall and approximately 10 mm in stem diameter were used to culture p. citrella. all plants were kept in the greenhouse agriculture research center of yasouj, with a temperature of 33-40°c and relative humidity of 50-65% and natural photoperiod (figure 1). rearing of phyllocnistis citrella the rearing of p. citrella was initiated from newly emerged larvae collected from citrus orchards in gachsaran, iran, and maintained on potted orange seedlings. the infested orange seedlings were kept in cages of 60×60×100 cm under laboratory conditions of 35±5°c temperature, 60±10% relative humidity and 12:12 h (l:d) photoperiod at the research laboratory of agriculture yasouj, iran. the cages were covered with muslin cloth from their sides and tops to provide adequate ventilation (figure 2). potted orange plants were grown in an air-conditioned greenhouse in small pots. bacillus thuringiensis eight concentrations of b.thuringiensis var. kurstaki were selected to conduct the experiments. b. thuringiensis was obtained from a stock culture at the department of plant protection, faculty of agriculture, shiraz university. the bacteria were grown on nutrient broth to aid sporulation. the culture was then incubated on a rotary shaker (300 rpm) at 28°c for four days to ensure sporulation and cell lyses. spores and crystals were harvested by centrifugation at 1.0000 rpm for 15 min at 8°c. the pellet (3.5-4 g/l) was washed with distilled water and diluted to obtain 101-108 viable spores/ml for use in the experiments. however, in order to prepare the different concentrations, b. thuringiensis pellets were diluted in sterile distilled water to get 108 viable spores/ml and they were counted by haemocytomer slide. hereafter, subsequent dilution to 107 until 101 was made from the 108 viable spores/ml. article figure 1. citrus sinensis l., host plant for mass rearing phyllocnistis citrella in greenhouse. figure 2. the cages (60 cm long × 60 cm wide × 100 cm high) covered with muslin cloth that was used to rear phyllocnistis citrella. no nco mm er cia l u se on ly treatments the experiments were conducted in petri dishes, 5.5 cm in diameter and 1 cm in height, partially filled with a 0.5 cm thick layer of wetted cotton pad and the lid of each petri dish had a hole closed with organdie fabric for ventilation. citrus leaf discs of 10 cm2 area cut from uninfected citrus plants were placed in the dishes. third larval instars of p. citrella were gently transferred using a camel hairbrush into the petri dishes in groups of ten larvae/petri dish. larvae in control groups (n=10) were sprayed with a 1 ml of distilled water, while larvae in treatment groups (n=10) were sprayed with a 1 ml aqueous solution of the required concentrations of b. thuringiensis by using a calibrated small sprayer. the petri dishes were kept under the fore-mentioned laboratory conditions. for each concentration, three replicates were used and each petri dish contained ten p. citrella larvae. larval mortality was recorded at 1, 4, 7 and 10 days post spraying. larvae were considered dead if they did not move when lightly prodded with forceps. the overall control mortality was 0.00, 13.30, 23.33% and 33.30% at 1, 4, 7 and 10 days of the experiments. statistical analysis the statistical analysis was performed using mstat-c statistical software and the means were compared with duncans multiple range test (dmrt). the mortality resulting from b. thuringiensis treatment was adjusted for the control of mortality using abbott’s formula (aabbott, 1925). results the results showed that direct spraying of p. citrella larvae by all b. thuringiensis concentrations tested exhibited a range of mortality after 1, 4, 7 and 10 days after spraying (figure 3). p.citrella larvae mortality was significantly affected by b.thuringiensis concentration and time after bacterial application. one day after spraying 108 and 107 concentrations had significantly caused the highest mortality to the larvae with 59.8% and 54.5%, respectively. then, the mortality percent decreased significantly until it reached 6.5% at 101 concentration (f=15.44; 7, 24 df; p=0.000). four days after application, the mortality varied significantly among the different concentrations (f=10.33; 7, 24 df; p=0.000), in which it reached 68.4% at 108 concentration and started to decrease with decreasing b. thuringiensis concentrations until it reached 9.5% at 101 concentration. mortality levels were relatively higher at seven days than at one day and four days post spraying, in which the mortality levels reached up to 73.6% at 108 concentration, whereas the least mortality was recorded at 101 concentration with 39.3% (f=8.78; 7, 24 df; p=0.000). ten days after spraying, the percentage of mortality was the highest compared with 1, 4 and 7 days after application. however, there were significant differences in the mortality percent among the different concentrations of b. thuringiensis, where it was 77.0% at 108 concentration, while the least mortality of 46.7% was recorded at 101 concentration. [journal of entomological and acarological research 2016; 48:5403] [page 357] article figure 3. a) corrected mortality percent of third larval instars of phyllocnistis citrella after one day of spraying of bacillus thuringiensis in direct spray test. [different letters above bars indicate significant difference among the different b. thuringiensis concentrations]. b) corrected mortality percent of third larval instars of p. citrella after four days of spraying of b. thuringiensis in direct spray test. [different letters above bars indicate significant difference among the different b. thuringiensis concentrations]. c) corrected mortality percent of third larval instars of p. citrella after seven days of spraying of b. thuringiensis in direct spray test. [different letters above bars indicate significant difference among the different b. thuringiensis concentrations]. d) corrected mortality percent of third larval instars of p. citrella after ten days of spraying of b. thuringiensis in direct spray test. [different letters above bars indicate significant difference among the different b. thuringiensis concentrations]. no nco mm er cia l u se on ly [page 358] [journal of entomological and acarological research 2016; 48:5403] further statistical analysis of results among the different days within the same concentration of b. thuringiensis showed that there was a significant increase in mortality for all b. thuringiensis concentrations at higher treatment times (figure 3). the percentage mortality of p. citrella larvae was significantly higher after 10 days post spraying followed by 7 days and then by 4 days and the least mortality was recorded at 1 day after application for all concentrations (f=4.40; 24.18; 3, 12 df; p=0.000 -0.037). the overall corrected mortality percent of third larval instars of p.citrella in all days as a result of spraying with different concentrations of b. thuringiensis is shown in figure 4a. the 108 concentration resulted in the highest mortality with 75.6% and it was significantly higher than that caused by any of the other b. thuringiensis concentrations, while the lowest mortality was recorded for 101 concentration with only 23.7% (f=11.45; 7, 96 df; p=0.000). there was also a positive correlation of 0.655, significant (0.000) at 0.01 probability level. this means that with increasing the concentration of b. thuringiensis up to 108 there was an increase in the mortality of p. citrella. the overall corrected mortality percent of p. citrella in all concentrations of b. thuringiensis on the different days after spraying is shown in figure 4b. the results showed that the mortality was significantly increased with time and was 33.5%, 51.4%, 60.3% and 71.5% after 1, 4, 7 and 10 post spraying, respectively (f=12.55; 3, 96 df; p=0.000). also, there was a positive correlation of 0.567, significant (0.000) at 0.01 probability level. discussion the indiscriminate and injudicious use of insecticides has led to a number of adverse effects in the environment. the undesirable effects of these chemical insecticides used against insect pests in crops warrants the development of strategies that could eliminate or reduce the involvement of insecticides for controlling insect pests. the citrus leaf miner, p. citrella stainton (lep., gracillariidae) originated from southeast asia and established itself as a major pest of citrus throughout the middle east (moreira et al., 2006), where iran is located. the leaf miner attacks all cultivars of citrus, related species within the rutaceae family, and several ornamentals (french et al., 1997). plant damage is caused by leaf miner larvae as they bore through the leaf epidermis. leaves become chlorotic, often deformed, and susceptible to infection by fungi or bacteria. preliminary field trials with selected insecticides indicate the superiority of dimilin (diflubenzuron) over diazinon, zolone (phosalone) and ekamet (etrimfos) in controlling citrus leaf miner in northern iran, but it is not totally effective (amiri besheli, 2006a). but, it is well known that continuous use of chemical insecticides is neither economic nor sustainable and has a negative impact on the environment, natural enemies and gardeners. moreover, p. citrella has a long history of resistance to many chemical insecticides, and development of resistance against a chemical sometimes makes it difficult to obtain sufficiently high control (mafi & ohbayashi, 2000). therefore, efforts are needed to develop integrated pest management strategies for the management of this pest through the use of bio-pesticides. results of the present study indicated that p. citrella larval mortality was significantly affected by b. thuringiensis concentration and time after bacterial application. after 1, 4, 7 and 10 days of spraying, 108 concentration of b.thuringiensis had significantly caused the highest mortality to the pest with 58.6, 68.4, 73.6 and 77.0%, respectively. comparing the results of this study with another study conducted by amiri-beshli (amiri besheli, 2006b) to control the citrus leaf miner with b.thuringiensis, mineral oil, insecticidal emulsion (garlic extract, plant detergent soap and food additive) and insecticidal gel (plant oil and plant extracts) under the laboratory conditions, insecticidal emulsion (67%) caused higher mortality to p. citrella larvae than insecticidal gel (62%), b. thuringiensis (49%) and mineral oil (37%). the efficacy of b.thuringiensis was decreased with decreasing its concentration and was 5.3, 17.5, 19.6 and 47.0% at concentration after 1, 4, 7 and 10 days after spraying respectively. this might be due to the fact that b.thuringiensis product proteinase activity was lower in its effect to p. citrella. these results are in agreement with results obtained by beattie and hardy (beattie & hardy, 2004), who found that low concentration of b.thuringiensis caused low mortality to diaprepes abbreviates (coleoptera: curculionidae). references abbott w.s., 1925 a method of computing the effectiveness of an insecticide. j. econ. entomol. 18: 265-267. agricultural statistics, 2010 department of statistics, ministry of agriculture, islamic republic of iran. amiri besheli b., 2006a toxicity evaluation of avant, buprofezin and pyriproxifen pesticides against phyllocnistis citrella stainton (lepidoptera: gracillariidae). pak. j. biol. sci. 9: 2483-2487. amiri besheli b., 2006b efficacy of bacillus thuringiensis, mineral oil, insecticidal emulsion and insecticidal gel against phyllocnistis citrella stainton (lepidoptera: gracillariidae). plant prot. sci. 44:68-73. article figure 4. a) overall corrected mortality percent of third larval instars of phyllocnistis citrella on all days as a result of spraying with different concentrations of bacillus thuringiensis. [different small letters above bars indicate significant differences among the different b. thuringiensis concentrations]. b) all concentrations of b. thuringiensis on the different days after spraying. [different small letters above bars indicate significant differences among the different days (b) at p<0.05 (one -factor analysis of variance)]. no nco mm er cia l u se on ly beattie a., hardy s., 2004 citrus leaf miner. department of primary industries, industry and investment new south wales. beattie g.a.c., liu z.m., watson d.m., clift a.d., jiang l., 1995 evaluation of petroleum spray oils and polysaccharides for control phyllocnistis citrella stainton (lepidoptera: gracillariidae). j. aust. entomol. soc. 34: 349-353. das a., roy t.c.d., bhattacharyya b., 1998 biology of citrus leaf miner phllocnistis citrella stainton. j. agric. sci. soc. north east india. 11: 51-54. demir i., eryuzlu e., demirbag z., 2012 a study on the characterization and pathogenicity of bacteria from lymantria dispar l. (lepidoptera:lymantriidae). turk. j. biol. 36: 459-468. dubois n.r., dean d.h., 1950 synergism between cryl a insecticides crystal proteins and spores of bacillus thuringiensis, other bacterial spores and vegetative cells against lymantria dispar (lepidoptera: lymantriidae) larvae. environ. entomol. 24:17411747. ebrahimi y., 2010 the evolutionary development of citrus growing and nursery activities in iran. ramsar citrus experimental station, ramsar, iran. french j.v., legaspi j., villarreal s., saldana r., 1997 management of citrus leaf miner in texas: chemical options. subtopic. plant sci. 49: 65-70. gill r.j., 1999 citrus leaf miner found in california. california department food and agricultural. california plant pest and disease report. 18: 79-80. heppner j.b., 1995 citrus leaf miner, phyllocnistis citrella (lep., gracillariidae) on fruit in florida. florida entomol. 78: 183-186. jacas j.a., pena j.e., 2002 calling behavior of two different population of phyllocnistis citrella (lepidoptera: gracillariidae). effects of age and photoperiod. florida entomol. 85: 378-381. jafari m., 1995 introduction of new pest in mazandran citrus orchards with name citrus leaf miner phyllocnistis citrella stainton (lepidoptera: gracillariidae). twelfth iranian plant protection congress, karaj, iran. page 211. jafari m., 1996 report of the workshop on citrus leaf miner phyllocnistis citrella stainton (lepidoptera: gracillariidae) and its control in the near east. safita (tartous), syria. jafari m., 1997 effect of chemical insecticides against citrus leaf miner phyllocnistis citrella stainton (lepidoptera: gracillariidae) in nursery. phyllocnistis citrella stainton (lepidoptera: gracillariidae). thirteenth iranian plant protection congress, karaj, iran. page 119. jafarzadeh m., 2000 biology and comparison application methods imidacloprid against of citrus leaf miner phyllocnistis citrella stainton (lepidoptera: gracillariidae). master thesis gilan university. faculty of agriculture. 109 pp. javan moghadam h., 2001 citrus leaf miner in iran and in the world. j. agri. sci. zeyton. 121: 20. kahrarian m., 2010 investigation on the effects of bacillus thuringiensis var., carbaryl and diflubenzeron against the chickpea pod borer heliothis viriplaca hufn. (lepidoptera: noctuidae) in the field. j. entomol. res. 2: 295-305. liu z., beatie g., jiang l., watson d.m., 1999 volumes of petroleum spray oil required for control of phyllocnistis citrella stainton (lepidoptera: gracillariidae) in mature citrus orchards. j. australian entomol. soc. 38: 141-146. mafi s.a., ohbayashi n., 2000 toxicity of insecticides to the citrus leaf miner, phyllocnistis citrella and its parasitoids chrysocharis pentheus (hymenoptera: eulophidae). appl. entomol. zool. 41:33-39. mahapatro g.k., gupta p., 2000 evening suitable for spraying bt formulations. -insect environ. 5: 126-127. margobandhu v., 1993 insect pest of oranges in the northern circars. madras agri. j. 21: 60-68. michaud j.p., grant a.k., 2003 integrated pest management compatibility of foliar insecticides for citrus: indices derived from toxicity to beneficial insects from four orders. j. insect sci. 3: 1-8. moreira j.a., mcelfresh j.s., millar j.g., 2006 identification, synthesis and field testing of the sex pheromone of the citrus leaf miner, phyllocnistis citrella. j. chem. ecol. 32: 169-194. seraj aa., 2001 comparison of some citrus species as host citrus leaf miner phyllocnistis citrella stainton (lepidoptera: gracillariidae). j. plant pest dis. 67: 86-95. [journal of entomological and acarological research 2016; 48:5403] [page 359] article no nco mm er cia l u se on ly layout 1 [journal of entomological and acarological research 2013; 45:e11] [page 57] green synthesis of silver nanoparticles using cadaba indica lam leaf extract and its larvicidal and pupicidal activity against anopheles stephensi and culex quinquefasciatus k. kalimuthu,1,2 c. panneerselvam,1 k. murugan,1 j.-s. hwang2 1division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore, india; 2institute of marine biology, national taiwan ocean university, taiwan abstract green nanoparticle synthesis was achieved using environmentally acceptable plant extracts and eco-friendly reducing and capping agents. in the present study, activity of silver nanoparticles (agnps) synthesized using cadaba indica lam plant against anopheles stephensi and culex quinquefasciatus was determined. a range of concentrations of synthesized agnps (3.125, 6.25, 12.5, 25, 50 ppm) and crude extract (50, 100, 150, 200, 250 ppm) were tested against a. stephensi and c. quinquefasciatus. the synthesized agnps from c. indica lam were much more toxic than crude extract in both mosquito species. the cured extract high mortality values were 50% lethal concentration (lc50)=88.22, 90.84 ppm; 90% lethal concentration (lc90)=172.94, 178.55 ppm, and the agnps high mortality values were lc50=3.90, 4.39 ppm; lc90=19.04, 17.35 ppm against a. stephensi and c. quinquefasciatus, respectively. the results recorded from ultraviolet-visible spectrophotometer, scanning electron microscopy, energy dispersive x-ray and fourier transformed infrared support the biosynthesis and characterization of silver nanoparticles. these results suggest that the leaf cured extracts of c. indica lam and green synthesis of silver nanoparticles have the potential to be used as an ideal eco-friendly approach for the control of a. stephensi and c. quinquefasciatus. introduction vector-borne diseases transmitted by blood-feeding mosquitoes, such as dengue hemorrhagic fever, japanese encephalitis, malaria, and filariasis, are increasing in prevalence worldwide, particularly in tropical and subtropical zones. malaria now is responsible for illness in more than an estimated 300 million people, resulting in one million deaths per year (who, 2007). culex quinquefasciatus is a vector of lymphatic filariasis, which affects 120 million people worldwide, and approximately 400 million people are at risk of contracting filariasis, resulting in an annual economic loss of 1.5 billion dollars (who, 2002). lymphatic filariasis is a serious public health problem in india, constituting one third of the infected population in the world (who et al., 1997). mosquito-borne diseases are endemic to india due to favorable ecological conditions for the vectors, their close contact with humans, and their reproductive biology. in rubber plantations, the rich organic content, stagnant water, low light levels and protected conditions in the coconut shells used in rubber production favors intense breeding (sumodan, 2003). mosquito control is improving in many areas, but there are significant challenges, including increasing resistance to insecticides and a lack of alternative, cost-effective, and safe insecticides. this increase in insecticide resistance requires the development of strategies for prolonging the use of highly effective vector control compounds. the use of combinations of multiple insecticides and phytochemicals is one such strategy that may be suitable for mosquito control. attempts to develop novel materials as mosquito larvicides are still necessary. with the progress of nanotechnology research, many laboratories around the world have investigated silver nanoparticles (agnps) production. the development of green processes for the synthesis of nanoparticales is evolving into an important brach of nanotechnology. nanoparticles play an indispensable role in drug delivery, diagnostics, imaging, sensing, gene delivery, artificial implants, and tissue engineering (morones & elechigerra, 2005). the development of a reliable green process for the synthesis of silver nanoparticles is an important aspect of current nanotechnology research. biological methods for nanoparticle synthesis using microorganisms, enzymes, and plants or plant extracts have been suggested as possible eco-friendly alternatives to chemical and physical methods (mohanpuria et al., 2008). recently, green silver nanoparticles have been synthesized using various natural products such as nelumbo nucifera (santhoshkumar et correspondence: kandasamy kalimuthu, division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore 641 046, india. tel.: +91.770.875.5603. e-mail: biokalimuthu@yahoo.in key words: cadaba indica lam, silver nanoparticles, anopheles stephensn, culex quinquefasciatus. acknowledgments: the authors are grateful to dr. k. sasikala, professor and head, department of zoology, bharathiar university for the laboratory facilities providing for this experiment. received for publication: 12 december 2012. revision received: 31 may 2013. accepted for publication: 7 june 2013. ©copyright k. kalimuthu et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e11 doi:10.4081/jear.2013.e11 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e11 jear_2013_2:hrev_master 16/09/13 13.56 pagina 57 no nco mm er cia l =3.90, 4.39 no nco mm er cia l =3.90, 4.39 c. quinquefasciano nco mm er cia l c. quinquefasciarespectively. the results recorded from ultraviolet-visible specno nco mm er cia l respectively. the results recorded from ultraviolet-visible spectrophotometer, scanning electron microscopy, energy dispersive x-ray no nco mm er cia l trophotometer, scanning electron microscopy, energy dispersive x-ray and fourier transformed infrared support the biosynthesis and characno nco mm er cia l and fourier transformed infrared support the biosynthesis and characterization of silver nanoparticles. these results suggest that the leaf no nco mm er cia l terization of silver nanoparticles. these results suggest that the leaf tropical and subtropical zones. malaria now is responsible for illness in no nco mm er cia l tropical and subtropical zones. malaria now is responsible for illness inmore than an estimated 300 million people, resulting in one million no nco mm er cia l more than an estimated 300 million people, resulting in one milliondeaths per year (who, 2007). no nco mm er cia l deaths per year (who, 2007). lymphatic filariasis, which affects 120 million people worldwide, and no nco mm er cia l lymphatic filariasis, which affects 120 million people worldwide, and no nco mm er cia l approximately 400 million people are at risk of contracting filariasis, no nco mm er cia l approximately 400 million people are at risk of contracting filariasis, no nco mm er cia l correspondence: kandasamy kalimuthu, division of entomology, no nco mm er cia l correspondence: kandasamy kalimuthu, division of entomology, department of zoology, school of life sciences, bharathiar university, no nco mm er cia l department of zoology, school of life sciences, bharathiar university, coimbatore 641 046, india. tel.: +91.770.875.5603. no nco mm er cia l coimbatore 641 046, india. tel.: +91.770.875.5603. lam, silver nanoparticles, no nco mm er cia l lam, silver nanoparticles, us e vector-borne diseases transmitted by blood-feeding mosquitoes, us e vector-borne diseases transmitted by blood-feeding mosquitoes,such as dengue hemorrhagic fever, japanese encephalitis, malaria, us e such as dengue hemorrhagic fever, japanese encephalitis, malaria, and filariasis, are increasing in prevalence worldwide, particularly inus e and filariasis, are increasing in prevalence worldwide, particularly in tropical and subtropical zones. malaria now is responsible for illness inus e tropical and subtropical zones. malaria now is responsible for illness in more than an estimated 300 million people, resulting in one millionus e more than an estimated 300 million people, resulting in one million on ly and on ly and c. quinquefasciatus on ly c. quinquefasciatus on ly vector-borne diseases transmitted by blood-feeding mosquitoes, on ly vector-borne diseases transmitted by blood-feeding mosquitoes, [page 58] [journal of entomological and acarological research 2013; 45:e11] al., 2011), pongamia pinnata (rajesh et al., 2010), azadirachta indica (tripathi et al., 2009), glycine max (vivekanandhan et al., 2009), cinnamon zeylanicum (sathishkumar et al., 2009), and camellia sinensis (begum et al., 2009). in recent studies, potential mosquito larvicidal activity of synthesized agnps from plant extracts as well as physical methods is well documented (marimuthu et al., 2011; thirunavukkarasu et al., 2010; sap-iam et al., 2010). plants are rich sources of bioactive organic chemicals and offer an advantage over synthetic pesticides, as these are less toxic, less prone to development of resistance, and easily biodegradable. india can utilize its rich supply of herbs for such purposes, as plant extracts are not only potentially insecticides, but also can act as effective antimicrobial, antifungal, antiparasitic and anti-malarial agents. plant materials not only offer effective mosquito control agents, but also promise to be environmentally safer. therefore, an alternative approach for mosquito control is the use of natural products of plant origin. the botanical insecticides are generally pest-specific, readily biodegradable, and usually lack toxicity to higher animals (bowers, 1992). in traditional medicine systems, different parts of the plant have been described to be useful against a variety of diseases. the leaves of cadaba indica lam plant are rich in lactones, steroids, flavonoids, alkaloids, reducing sugars and tannins (peach & tracy 1955; rastogi & mehrotra, 1991). c. indica lam leaf extract is used on boils; its leaf juice is used as eye drops. against cattle fever, a decoction of fresh leaves, pepper and garlic is administered orally (reddy et al., 2007). however, the activity of the ethanol extract of the leaves was found to be most effective against bacteria and fungi (selvamani & latha, 2005). the leaf and flower liquid extract mixed with castor oil and turmeric is taken as a remedy for menorrhagia, syphilis, and as a purgative (alagesaboopathi, 2009). pathak et al. (2000) reported that the steam-distilled whole plant oil extract of tagetes minuta gave 100% mortality against larvae of anopheles stephensi, culex quinquefasciatus and aedes aegypti at doses lower than 100 ppm. volatile oil extracted from the peel of citrus fruits has also shown toxic effects on mosquito larvae as well as adults (ezeonu et al., 2001). in the present study, we report on the synthesis of silver nanoparticles, reducing the silver ions present in the solution of silver nitrate by c. indica lam leaf extract, and its efficacy against a. stephensi and c. quinquefasciatus. materials and methods materials the cadaba indica lam plants were collected in and around kaveri river bank, namakkal district, in tamilnadu, india, and identified by the taxonomist, department of botany, bharathiar university, coimbatore, india. the voucher specimen was numbered and kept in the authors’ research laboratory for further reference. silver nitrate (agno3) was purchased from precision scientific co., coimbatore, india. mosquito rearing the eggs of a. stephensi and c. quinquefasciatus were collected from the national centre for disease control field station of mettupalayam, tamil nadu, india. these were brought to the laboratory and transferred (in approximately the same aliquot numbers of eggs) into 18 cm l×13 cm w×4 cm d enamel trays containing 500 ml of water, where they were allowed to hatch. mosquito larvae were reared (and adult mosquitoes held) at 27±2°c and 75-85% relative humidity in a 14:10 (l/d) photoperiod. the larvae were fed 5-g ground dog biscuit and brewer’s yeast daily in a 3:1 ratio. the pupae were collected and transferred into plastic containers with 500 ml of water. the container was placed inside a screened cage (90 cm l×90 cm h×90 w) to retain emerging adults, for which 10% sucrose in water solution (v/v) was made available. on day 5 post-emergence, the mosquitoes were provided access to a rabbit host for blood feeding. the shaved dorsal side of the rabbit was positioned on the top of the mosquito cage in contact with the cage screen (using a cloth sling to hold the rabbit) and held in this position overnight. glass petri dishes lined with filter paper and containing 50 ml of water were subsequently placed inside the cage for oviposition by female mosquitoes. synthesis of silver nanoparticles leaves were washed with distilled water and dried for 2 days at room temperature. a plant leaf broth was prepared by placing 10 g of the leaves (finely cut) in a 300-ml flask with 100 ml of sterile distilled water. this mixture was boiled for 5 min, decanted, stored at −4°c, and used in our tests within 1 week. the filtrate was treated with aqueous 1 mm agno3 solution in an erlenmeyer flask and incubated at room temperature. as a result, in a brown-yellow solution indicating the formation of agnps, it was found that aqueous silver ions can be reduced by aqueous extract of the plant parts to generate extremely stable silver nanoparticles in water. characterization of silver nanoparticles the presence of synthesized silver nanoparticles was confirmed by sampling the reaction mixture at regular intervals and the absorption maxima was scanned by ultraviolet-visible (uv-vis) spectra at the wavelengths of 350-600 nm in a uv-3600 shimadzu spectrophotometer at 1 nm resolution. further, the reaction mixture was subjected to centrifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved in deionized water and filtered through a millipore filter (0.45 μm). an aliquot of this filtrate containing silver nanoparticles was used for scanning electron microscopy (sem) and energy dispersive x-ray (edx) studies. thin films of the sample were prepared on a carboncoated copper grid by dropping a small amount of the sample on the grid; extra solution was removed using a blotting paper, and the films on the sem grid were allowed to dry by placing it under a mercury lamp for 5 min. the surface groups of the nanoparticles were qualitatively confirmed by using fourier transformed infrared spectroscopy (ftir) (stuart, 2002), with spectra recorded by a perkin-elmer spectrum 2000 ftir spectrophotometer. larval/pupal toxicity test twenty-five larvae (instars i-iv) or pupae were placed in 249 ml of dechlorinated water in a 500-ml glass beaker, and 1 ml of the desired concentration of silver nanoparticles was added; 0.5 mg of larval food was provided for each test concentration. tests of each concentration against each instar and the pupae were replicated three times. in each case, the control comprised 25 larvae or pupae in 250 ml of distilled water. control mortality was corrected by using abbott’s formula (abbott, 1925), and percent mortality was calculated as follows: percent mortality= number of dead larvae/pupae ¥100 number of larvae/pupae introduced statistical analysis average larval mortality data were subjected to probit analysis for calculating 50% and 90% lethal concentration (lc50 and lc90) values, using the method of finney (1971). spss software, ver. 9.0 (statacorp., college station, tx, usa), was used. results were considered to be statistically significant at p<0.05. article jear_2013_2:hrev_master 16/09/13 13.56 pagina 58 no nco mm er cia l lower than 100 ppm. volatile oil extracted from the peel of citrus fruits no nco mm er cia l lower than 100 ppm. volatile oil extracted from the peel of citrus fruits has also shown toxic effects on mosquito larvae as well as adults no nco mm er cia l has also shown toxic effects on mosquito larvae as well as adults in the present study, we report on the synthesis of silver nanopartino nco mm er cia l in the present study, we report on the synthesis of silver nanoparticles, reducing the silver ions present in the solution of silver nitrate by no nco mm er cia l cles, reducing the silver ions present in the solution of silver nitrate by a. stephensi no nco mm er cia l a. stephensi and no nco mm er cia l and no nco mm er cia l lam plants were collected in and around kaverin on -co mm er cia l lam plants were collected in and around kaveri wavelengths of 350-600 nm in a uv-3600 shimadzu spectrophotometer no nco mm er cia l wavelengths of 350-600 nm in a uv-3600 shimadzu spectrophotometerat 1 nm resolution. further, the reaction mixture was subjected to cenno nco mm er cia l at 1 nm resolution. further, the reaction mixture was subjected to cen-trifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved no nco mm er cia l trifugation at 15,000 rpm for 20 min; the resulting pellet was dissolved in deionized water and filtered through a millipore filter (0.45 no nco mm er cia l in deionized water and filtered through a millipore filter (0.45 aliquot of this filtrate containing silver nanoparticles was used for no nco mm er cia l aliquot of this filtrate containing silver nanoparticles was used for scanning electron microscopy (sem) and energy dispersive x-ray no nco mm er cia l scanning electron microscopy (sem) and energy dispersive x-ray (edx) studies. thin films of the sample were prepared on a carbonno nco mm er cia l (edx) studies. thin films of the sample were prepared on a carbonus e characterization of silver nanoparticles us e characterization of silver nanoparticles the presence of synthesized silver nanoparticles was confirmed by us e the presence of synthesized silver nanoparticles was confirmed bysampling the reaction mixture at regular intervals and the absorption us e sampling the reaction mixture at regular intervals and the absorption us e maxima was scanned by ultraviolet-visible (uv-vis) spectra at theus e maxima was scanned by ultraviolet-visible (uv-vis) spectra at the wavelengths of 350-600 nm in a uv-3600 shimadzu spectrophotometerus e wavelengths of 350-600 nm in a uv-3600 shimadzu spectrophotometer at 1 nm resolution. further, the reaction mixture was subjected to cen-us e at 1 nm resolution. further, the reaction mixture was subjected to cenon ly temperature. as a result, in a brown-yellow solution indicating the foron ly temperature. as a result, in a brown-yellow solution indicating the formation of agnps, it was found that aqueous silver ions can be reduced on ly mation of agnps, it was found that aqueous silver ions can be reduced by aqueous extract of the plant parts to generate extremely stable silver on lyby aqueous extract of the plant parts to generate extremely stable silver characterization of silver nanoparticleson ly characterization of silver nanoparticles the presence of synthesized silver nanoparticles was confirmed by on ly the presence of synthesized silver nanoparticles was confirmed by results and discussion several approaches have been employed to obtain better biosynthesis of nanoparticles, which is preferable to chemical and physical methods, as it is a cost-effective and environmentally friendly method, and does not require the use of high pressure, energy, temperature, or toxic chemicals (sinha et al., 2009; goodsell, 2004). in the present study, the larvicidal and pupicidal effects of ethanol leaf extracts and synthesized agnps of c. indica lam were noted; the highest mortality was found in synthesized agnps against larvae and pupae of a. stephensi (lc50=3.90, 4.67, 10.20, 15.41, 25.27 ppm and lc90=19.04, 27.06, 47.72, 61.07, 78.32 mg/l) and c. quinquefasciatus (lc50=4.39, 5.07, 8.21, 15.44, 23.83 mg/l and lc90=17.35, 20, 35.76, 58.37, 75.33 ppm), respectively (table 1 and figure 1). the highest mortality was found against the larvae and pupae of a. stephensi (lc50=88.22, 107.34, 136.98, 169.04, 270.68 mg/l and lc90=172.94, 209.09, 284.08, 314.46, 474.85 ppm) and c. quinquefasciatus (lc50=90.84, 115.37, 145.18, 172.92, 288.86 mg/l and lc90=178.55, 211.37, 277.29, 311.29, 525.13 ppm), respectively (table 2 and figure 2). chi-square values were significant at the p≤0.05 level. fifty-percent hydroethanolic extracts of bonninghausenia albiflora whole plant, calotropis procera root, citrus maxima flower, acorus calamus rhizome, and weidelia chinensis whole plant showed acaricidal efficacy ranging from 4% to 35% within 24 h of application on rhipicephalus (boophilus) microplus. rhizome extract of a. calamus revealed that a 79.31% correlation with log concentration in probit mortality could be assigned to the concentration of the extract, and the regression line of the extract showed the lc85 as 11.26% (ghosh et al., 2011). chandran et al. (2006) synthesized silver nanoparticles by using aloe vera extract at 24 h of incubation. previous authors reported that the methanol extract of cassia fistula exhibited lc50 values of 17.97 and 20.57 mg/l for a. stephensi and c. quinquefasciatus, respectively (govindarajan et al., 2008). a 23% mortality was noted against firstinstar larvae of a. stephensi by treatment of a. ilicifolius extract at 20 ppm; this increased to 89% at 100 ppm (kovendan & murugan, 2011). mosquitocidal properties of calotropis gigantea leaf extract and bacterial insecticidal properties of bacillus thuringiensis against these mosquito vectors have been reported by kovendan et al. (2012). reduction of silver ions in the aqueous solution during reaction with the ingredients present in plant leaf extract was observed by uv-visible spectroscopy. the color change was noted by visual observation in the c. indica lam leaf extracts when incubated with agno3 solution. c. indica lam leaf extract without agno3 did not show any change in color (figure 3). the color of the extract changed to light brown within an hour, and later changed to dark brown during a 1 h incubation period, after which no significant change occurred. appearance of the yellowish brown color was an indication of formation of colloidal silver nanoparticles in the medium. the brown color could be due to the exci[journal of entomological and acarological research 2013; 45:e11] [page 59] article table 1. larvicidal activity of c. indica lam crude leaf ethnolic extract against larva and pupa of a. stephensi and c. quinquefasciatus. species life percentage of larval and pupal mortality slope lc50(lc90) 95% confidence limit x2 stages concentration of eec (ppm) lcl ucl (df=3) (instars) 50 100 150 200 250 lc50(lc90) lc50(lc90) a. stephensi i 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 ii 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 iii 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 iv 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 c. quinquefasciatus i 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 ii 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 iii 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 iv 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 lc50, 50% lethal concentration; lc90, 90% lethal concentration; eec, ethanolic extract of cadaba indica lam; control nil mortality; lcl, lower confidence limit; ucl, upper confidence limit; df, degrees of freedom. each value is the mean±sd of five replicates. table 2. larvicidal activity of synthesized silver nanoparticles using c. indica lam leaf extract against larvae and pupa of a. stephensi and c. quinquefasciatus. species life percentage of larval and pupal mortality slope lc50(lc90) 95% confidence limit x2 stages concentration of agnps (ppm) lcl ucl (df=3) (instars) 3.125 6.25 12.5 25 50 lc50(lc90) lc50(lc90) a. stephensi i 42.0±2.8 61.2±3.0 80.8±1.0 94.6±1.1 100±1.2 1.068 3.90 (19.04) 1.42 (16.48) 5.72 (23.00) 3.378 ii 40.8±2.4 55.6±3.8 71.4±2.4 88.4±1.4 98.8±1.0 1.128 4.67 (27.06) 1.43 (23.32) 7.13 (32.80) 3.376 iii 35.2±2.0 42.6±3.5 58.2±2.5 75.0±1.8 88.4±1.4 1.099 10.20 (47.72) 6.04 (40.95) 13.68 (58.18) 4.909 iv 29.8±3.4 40.0±2.6 51.2±2.2 66.4±2.4 80.2±1.0 1.010 15.41 (61.07) 10.97 (51.50) 19.51 (76.74) 4.895 pupa 21.8±3.6 33.4±2.8 42.6±2.6 55.8±2.6 68.8±1.3 0.918 25.27 (78.32) 13.80 (53.53) 45.48 (190.03) 6.095 c. quinquefasciatus i 44.2±3.0 53.2±2.4 85.8±1.0 96.2±1.0 100±1.0 1.099 4.39 (17.35) 2.37 (15.12) 5.93 (20.74) 5.051 ii 40.0±2.4 55.2±2.1 77.8±2.1 94.2±1.0 100±1.0 1.158 5.07 (20.00) 2.89 (17.40) 6.76 (23.96) 1.843 iii 36.8±1.6 43.2±2.0 63.4±2.7 82.4±2.0 95.4±1.4 1.227 8.21 (35.76) 4.99 (31.11) 10.92 (42.62) 4.851 iv 30.4±3.4 36.4±2.0 51.6±3.5 68.4±1.8 81.2±1.2 1.063 15.44 (58.37) 3.93 (42.23) 25.07 (110.23) 5.801 pupa 27.2±3.0 30.4±2.4 42.6±3.3 56.4±2.7 71.4±1.0 0.942 23.83 (75.33) 19.26 (62.54) 29.18 (97.33) 2.888 lc50, 50% lethal concentration; lc90, 90% lethal concentration; agnps, silver nanoparticles; control nil mortality; lcl, lower confidence limit; ucl, upper confidence limit; df, degrees of freedom. each value is the mean±sd of five replicates. jear_2013_2:hrev_master 16/09/13 13.56 pagina 59 no nco mm er cia l lam crude leaf ethnolic extract against larva and pupa of no nco mm er cia l lam crude leaf ethnolic extract against larva and pupa of no nco mm er cia l species life percentage of larval and pupal mortality slope lc no nco mm er cia l species life percentage of larval and pupal mortality slope lc no nco mm er cia l concentration of eec (ppm) no nco mm er cia l concentration of eec (ppm) no nco mm er cia l (instars) 50 100 150 200 250 no nco mm er cia l (instars) 50 100 150 200 250 i 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 no nco mm er cia l i 30.0±2.4 56.2±2.3 80.0±2.0 95.8±2.2 100±1.2 0.359 88.22 (172.94) 77.06 (160.36) 97.89 (189.33) 1.383 ii 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 no nco mm er cia l ii 25.6±3.6 42.2±2.4 71.8±2.6 88.4±3.0 96.2±1.0 0.374 107.34 (209.09) 95.61 (194.30) 117.80 (228.84) 1.051 iii 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 no nco mm er cia l iii 21.2±3.3 33.8±3.0 62.8±2.2 70.4±2.2 81.2±1.6 0.313 136.98 (284.08) 122.60 (258.08) 150.50 (321.54) 3.890 no nco mm er cia l iv 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 no nco mm er cia l iv 13.8±2.2 24.2±3.6 49.6±2.8 61.6±3.8 73.4±1.2 0.313 169.04 (314.46) 155.65 (285.28) 183.56 (356.63) 2.571 pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 no nco mm er cia l pupa 8.0±1.0 15.2±3.4 22.8±2.0 30.2±3.0 46.4±2.8 0.183 270.68 (474.85) 241.07 (402.53) 320.08 (605.86) 0.522 no nco mm er cia l i 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 no nco mm er cia l i 28.2±2.2 54.8±2.0 81.2±2.0 92.6±2.2 100±1.0 0.362 90.84 (178.55) 79.50 (165.69) 100.69 (195.26) 1.731 no nco mm er cia l ii 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 no nco mm er cia l ii 21.0±2.4 40.8±2.2 66.4±1.6 85.6±2.8 97.8±1.2 0.396 115.37 (211.37) 104.70 (197.44) 125.20 (230.68) 1.107 no nco mm er cia l iii 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 no nco mm er cia l iii 16.8±3.0 30.2±2.8 58.6±2.2 70.0±3.0 82.40±1.4 0.342 145.18 (277.29) 132.51 (254.27) 157.58 (309.39) 2.607 no nco mm er cia l iv 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 no nco mm er cia l iv 11.8±2.8 22.6±3.4 45.2±2.8 64.6±2.2 71.8±1.0 0.324 172.92 (311.29) 160.05 (283.70) 187.01 (350.82) 2.921 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 no nco mm er cia l pupa 8.8±2.1 15.4±2.2 24.6±3.2 31.4±3.2 40.6±2.6 0.159 288.86 (525.13) 251.76 (432.37) 357.53 (709.61) 0.387 , 90% lethal concentration; eec, ethanolic extract of no nco mm er cia l , 90% lethal concentration; eec, ethanolic extract of us e nanoparticles in the medium. the brown color could be due to the excius e nanoparticles in the medium. the brown color could be due to the excilam crude leaf ethnolic extract against larva and pupa of us e lam crude leaf ethnolic extract against larva and pupa of on ly leaf extract without agno on ly leaf extract without agno (figure 3). the color of the extract changed to light brown within an on ly (figure 3). the color of the extract changed to light brown within an hour, and later changed to dark brown during a 1 h incubation period, on lyhour, and later changed to dark brown during a 1 h incubation period,after which no significant change occurred. appearance of the yellowon lyafter which no significant change occurred. appearance of the yellowish brown color was an indication of formation of colloidal silveron ly ish brown color was an indication of formation of colloidal silver nanoparticles in the medium. the brown color could be due to the exci-on ly nanoparticles in the medium. the brown color could be due to the exci[page 60] [journal of entomological and acarological research 2013; 45:e11] tation of surface plasmon vibrations, typical of silver nanoparticles (ahmad et al., 2003; krishnaraj et al., 2010). the dark brown color of the silver colloid is attributable to surface plasmon resonance arising from the group of free conduction electrons induced by an interacting electromagnetic field (song & kim, 2008). the strong surface plasmon resonance band appears at the range of 420-480 nm and the broadening peak indicates that the particles are monodispersed (figure 4). these color changes arise because of the excitation of surface plasmon vibrations in the silver nanoparticles (mulvaney, 1996). sem (jeol-model 6390) image showing high density ag nanoparticles synthesized by c. indica lam plant extracts further confirmed the presence of ag nanoparticles (figure 5). it was shown that relatively spherical and uniform ag nanoparticles were formed with a diameter of 30-60 nm. the sem image of silver nanoparticles synthesized by plant extracts were assembled on the surface due to interactions such as hydrogen bonding and electrostatic interactions between the bioorganic capping molecules bound to the ag nanoparticles. it was found that relatively spherical and uniform silver nanoparticles were formed. the nanoparticles were not in direct contact, even within the aggregates, indicating stabilization of the nanoparticles by a capping agent (song & kim, 2008). silver nanoparticles have been characterized using sem by various investigators (durán et al., 2005; balaji et al., 2009). silver nanoparticles were synthesized using leaf extracts of acalypha indica; from the sem image, the size of the control silver nitrate obtained was more than 1000 nm, whereas synthesized silver nanoparticles measured 20-30 nm in size (krishnaraj et al., 2010). tian et al. (2007) reported that numerous flavonoids, including quercetin or quercetin 3-o-glycosides, were isolated from lotus leaves that were used for silver nanoparticle synthesis. the element analysis of the silver nanoparticles was performed using edx on the sem. figure 6 article figure 1. larvicidal activity of c. indica lam crude leaf ethnolic extract against larva and pupa of a. stephensi and c. quinquefasciatus. figure 2. larvicidal activity of synthesized silver nanoparticles using c. indica lam leaf extract against larvae and pupa of a. stephensi and c. quinquefasciatus. jear_2013_2:hrev_master 16/09/13 13.56 pagina 60 no nco mm er cia l no nco mm er cia l u se us e us e o nlyon ly shows the edx spectrum of agnps synthesized at 25ºc and 80ºc; strong signals from the silver atoms in the nanoparticles were observed, and signals from calcium, potassium, oxygen, sodium, magnesium, sulphur, ag and chloro were also recorded. the results indicate that the reaction product was composed of higher level ag nanoparticles. the agnps produced by c. indica lam leaf extract were distinct and scattered in distribution. the fourier transformed infrared spectra of agnps exhibited prominent peaks at 3453; 3288; 1790; 1638; 1384; 1114; 1077; 371; 360 cm−1 (figure 7). the sharp absorption peak at 1638 cm−1 was assigned to c=o stretching vibration in the carbonyl compounds, which may be characterized by the presence of a high content of terpenoids and flavonoids. the peaks at 1077 cm−1 correspond to c–n stretching vibration of aliphatic amines or alcohols/phenols, representing the presence of polyphenols. the absorption bands at 1088 cm−1 in the fingerprint region indicate several modes such as c–h deformation or c–o or c–c stretching, pertaining to carbohydrates. the bands at 1383 to 1431 cm−1 were assigned to scissoring modes of methylene tails, ch3 r. a broad intense band at 3402 cm−1 in both the spectra can be assigned to the n–h stretching frequency arising from the peptide linkages present in the proteins of the extract (mukherjee et al., 2008). conclusions the present study of green synthesis shows that the environmentally benign and renewable source of c. indica lam is used as an effective reducing agent for the synthesis of agnps. this biological reduction of silver nanoparticles would be a boon for the development of a clean, nontoxic, and environmentally acceptable green approach to production of agnps, involving organisms extending even to higher plants. the agnps did not exhibit any noticeable effects on c. indica lam exposure at their lc50 and lc90 values against larvae of a. stephensi and c. [journal of entomological and acarological research 2013; 45:e11] [page 61] article figure 4. ultraviolet-visible spectra of aqueous silver nitrate with c. indica lam leaf extract at different time intervals. figure 3. photographs showing change in color after adding silver nitrate (agno3) before reaction and after reaction time of 30 min. figure 5. image of scanning electron microscopic observation of synthesized silver nanoparticles. a) lower magnification (0.5 µm); b) higher magnification (1 µm). jear_2013_2:hrev_master 16/09/13 13.56 pagina 61 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l figure 3. photographs showing change in color after adding silver no nco mm er cia l figure 3. photographs showing change in color after adding silver no nco mm er cia l ) before reaction and after reaction time of 30 min. no nco mm er cia l ) before reaction and after reaction time of 30 min. no nco mm er cia l u se us e o nlyon ly [page 62] [journal of entomological and acarological research 2013; 45:e11] article figure 6. energy dispersive x-ray spectra recorded form a film, after formation of silver nanoparticles with different x-ray emission peaks labeled. cl, chloro; k, potassium; ca, calcium; o, oxygen; na, sodium; mg, magnesium; s, sulphur; ag, silver. figure 7. fourier transformed infrared spectroscopy spectrum of silver nanoparticle synthesized by reacting silver nitrate with c. indica lam leaf extract. jear_2013_2:hrev_master 16/09/13 13.56 pagina 62 no nco mm er cia l no nco mm er cia l no nco mm er cia l no nco mm er cia l figure 6. energy dispersive x-ray spectra recorded form a film, after formation of silver nanoparticles with different x-ray emission no nco mm er cia l figure 6. energy dispersive x-ray spectra recorded form a film, after formation of silver nanoparticles with different x-ray emission no nco mm er cia l peaks labeled. cl, chloro; k, potassium; ca, calcium; o, oxygen; na, sodium; mg, magnesium; s, sulphur; ag, silver. no nco mm er cia l peaks labeled. cl, chloro; k, potassium; ca, calcium; o, oxygen; na, sodium; mg, magnesium; s, sulphur; ag, silver. no nco mm er cia l no nco mm er cia l u se on ly quinquefasciatus. the agnps formed are highly stable and significant mosquito larvicides. the synthesized agnps in a methanol extract and the isolation and purification of crude methanol extracts of c. indica lam are in progress. we also seek to develop methods and techniques necessary for green synthesis of silver nanoparticles by using microorganisms, such as bacteria and fungi, for mosquito control. references abbott w.s., 1925 a method of computing the effectiveness of insecticides. j. econ. entomol. 18: 265-267. ahmad a., mukherjee p., senapati s., mandal d., khan m.i., kumar r., et al., 2003 extracellular biosynthesis of silver nanoparticles using the fungus fusarium oxysporum. colloids. surf. b. 28: 313-318. alagesaboopathi c., 2009 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high-performance liquid chromatography. sepu. 25: 88-92. tripathi a., chandrasekaran n., raichur a.m., mukherjee a., [journal of entomological and acarological research 2013; 45:e11] [page 63] article jear_2013_2:hrev_master 16/09/13 13.56 pagina 63 no nco mm er cia l chandran s.p., chaudhary m., pasricha r., ahmad a., sastry no nco mm er cia l chandran s.p., chaudhary m., pasricha r., ahmad a., sastry m., 2006 synthesis of gold nanotriangles and silver nanoparticles no nco mm er cia l m., 2006 synthesis of gold nanotriangles and silver nanoparticles using aloe vera plant extract. biotechnol. prog. 22: 577-583. no nco mm er cia l using aloe vera plant extract. biotechnol. prog. 22: 577-583. durán n., marcato p.d., alves o.l., souza g.i., esposito e., 2005 no nco mm er cia l durán n., marcato p.d., alves o.l., souza g.i., esposito e., 2005 mechanistic aspects of biosynthesis of silver nanoparticles by sevno nco mm er cia l mechanistic aspects of biosynthesis of silver nanoparticles by 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44:e8] [page 37] tortricidae (lepidoptera) from ethiopia, 2 j. razowski,1 p. trematerra2 1institute of systematics and experimental zoology, polish academy of sciences, kraków, poland; 2department of agriculture, environment and food science, university of molise, campobasso, italy abstract twenty species of tortricidae from ethiopia, oromia region, are recorded of which olethreutes didessae sp. n., ancylis colaccii sp. n., and gypsonoma giorgiae sp. n. are described as new; eucosma thalameuta meyrick, 1918, is transferred to the genus cosmetra diakonoff, 1977. introduction the tortricidae of ethiopia are insufficiently known despite 80 years ago meyrick (1932) published first faunistic paper on the microlepidoptera of ethiopia which included the data on several leafrollers. then razowski & trematerra (2010) recorded 36 species from this country of which 25 were newly described. they also characterised five collecting sites. based on so limited number of species there is no possibility to characterise the ethiopian fauna. razowski & trematerra (2010) mentioned only some species common to ethiopia and the republic of south africa, mozambique and uganda. razowski & wojtusiak (2012) found in nigeria three species known from ethiopia what is interesting zoogeographically. in the present collection there are further four species described or recorded from nigeria (lobesia hecista razowski, l. talyana razowski, l. lecta razowski, and endothenia gutturalis meyrick, 1934) and found in ethiopia. there are also some closely related species common to the two countries. material and methods this paper is based on the material collected during two italian expeditions in oromia region, southwestern ethiopia, by the entomologists of the university of molise and of the university of milano. the expeditions were realized in november 2010 and january 2012. during 2010 the itinerary of the expedition was wellega zone (didessa river, 1280 m, 11-16.11.2010), and ilubador zone (dabeda river, 1800 m, 14.11.2010). in 2012 the expedition visited wellega zone (didessa river, 1280 m, 24-28.01.2012), and ilubador zone (dabeda river, 1827 m, 26.01.2012; bedelle, mute forest, 2060 m, 27.01.2012). tortricid adults were collected during the day by net and at night from a white sheet placed behind a 160 watt mixed light. genitalia were prepared using standard methods, the abdomen was macerated in 10% koh and dissected under a stereoscopic microscope, the genitalia were separated and mounted in euparal on a glass slide. adults and slides are deposited in p. trematerra collection, campobasso (italy). systematic part: tortricidae subfamily tortricinae tribe tortricini apotoforma fustigera razowski, 1986 (figures 1, 11) material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 28.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. remarks. a. fustigera was described from mount cameroon; the ethiopian specimen has somewhat shorter subscaphium and the terminal process of the sacculus. another closely related species, a. uncifera razowski, 1964, is from natal (razowski, 1986). lozotaenia sciarrettae razowski & termaterra, 2010 material examined. 4 males and 3 females, ethiopia, ilubabor zone, bedelle, dabeta river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. remarks. l. sciarrettae was described from the bale mountains, ethiopia. metamesia episema diakonoff, 1960 material examined. 1 male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. remarks. this species was described from madagascar; recorded from ethiopia by razowski & trematerra (2010). choristoneura dinota (meyrick, 1918) (figures 2, 12) material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 16.11.2010, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, m. colacci; 1 female, ethiopia, ilubabor zone, correspondence: pasquale trematerra, department of agriculture, environment and food science, university of molise, via de sanctis, 86100 campobasso, italy. e-mail: trema@unimol.it key words: ethiopia, faunistics, lepidoptera, tortricidae, new species. acknowledgements: the authors would like to thank marco colacci, francesco parisi, andrea sciarretta, (university of molise, italy) and renato regalin (university of milano, italy) for providing the material for the study. received for publication: 6 july 2012. revision received: 7 august 2012. accepted for publication: 8 august 2012. ©copyright j. razowski and p. trematerra, 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e8 doi:10.4081/jear.2012.e8 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2012; volume 44:e8 no nco mm er cia l u se on ly [page 38] [journal of entomological and acarological research 2012; 44:e8] near bedelle, dabeda river, 1827 m, 26.01.2012, coord. utm 37p 201587e, 930253n, legg. a. sciarretta, f. parisi. remarks. c. dinota was described from malawi. it aso was recorded from kilimanjaro, tanzania (its synonymy tortrix ptilocnemis meyrick, 1920), gabon, kenya, and congo. procrica ophiograpta (meyrick, 1932) material examined. 1 male, ethiopia, ilubabor zone, bedelle, mute forest, 2060 m, 27.01.2012, coord. utm 37p 207285e, 938068n, legg. a. sciarretta, f. parisi. remarks. p. ophiograpta was described from ethiopia and then recorded by razowski & trematerra (2010) from bale mountaines (as p. ophiographa, sic!). orilesa caminosa razowski, 2012 material examined. 1 male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci; 1 male, ethiopia, wellega zone, didessa river, 1280 m, 25.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. remarks. this species is described from eala, congo. article figures 1-10. adults. apotoforma fustigera razowski, 1986 (1); choristoneura dinota (meyrick, 1918) (2); lobesia alyana razowski, 2012 (3); olethreutes didessae sp. n., holotype (4); cosmorryncha microcosma aarvik, 2004 (5); aterpia microchlamys diakonoff, 1988 (6); ancylis colaccii sp. n., holotype (7); cosmetra thalameuta (meyrick, 1918) (8); gypsonoma giorgiae sp. n., holotype (9); grapholita siderocosma diakonoff, 1978 (10). no nco mm er cia l u se on ly tribe bactrini endothenia ethiopica razowski & trematerra, 2010 material examined. 2 males, ethiopia, ilubabor zone, bedelle, mute forest, 2060 m, 27.01.2012, coord. utm 37p 207285e, 938068n, legg. a. sciarretta, f. parisi; 1 male, ethiopia, ilubabor zone, near bedelle, dabeda river, 1827 m, 26.01.2012, coord. utm 37p 201587e, 930253n, legg. a. sciarretta, f. parisi. remarks. this is an ethiopian species described from bale mountains. endothenia albapex razowski & trematerra, 2010 (figure 13). material examined. 3 males, ethiopia, ilubabor zone, bedelle, mute forest, 2060 m, 27.01.2012, coord. utm 37p 207285e, 938068n, legg. a. sciarretta, f. parisi; 1 female, ethiopia, ilubabor zone, near bedelle, dabeda river, 1827 m, 26.01.2012, coord. utm 37p 201587e, 930253n, legg. a. sciarretta, f. parisi. description. female genitalia (figure 13). sterigma short, rounded; antrum sclerite broad, concave posteriorly; ductus bursae with subterminal cingulum; signum typical of the genus. remarks. e. albapex was known to date only from ethiopia (bale mountains). it was described in the genus crotalaria razowski & brown, [in press]. the previous description (p. 56, figs 16, 59) is thus invalid; the above-mentioned characteristics are used in this new description in a valid genus. tribe olethreutini lobesia hecista razowski, 2012 material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 28.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. remarks. this species was described from nigeria. lobesia lecta razowski, 2012 material examined. 1 male, ethiopia, ilubabor zone, bedelle, mute forest, 2060 m, 27.01.2012, coord. utm 37p 207285e, 938068n, legg. a. sciarretta, f. parisi. remarks. in the male genitalia, l. lecta and l. hecista are similar to l. leucospilana (mabille, 1900) from madagascar but differ from it chiefly by a lack of a small lobe between end of the sacculus and ventral lobe of the cucullus. lobesia talyana razowski, 2012 (figures 3, 14) material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 24.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. remarks. l. talyana was described from congo; besides it is known from nigeria (razowski, 2012). olethreutes didessae razowski & trematerra, 2012 sp. n. (figures 4, 15) material examined. holotype male, ethiopia, wellega zone, didessa river, 1280 m, 26.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. diagnosis. o. didessae is closely related and similar to o. molybdachtha (meyrick, 1930) from the ivory coast but didessae has broad uncus, large lateral socii, long slender terminal process of the sacculus and broad terminally lobe from the posterior edge of the basal cavity of the valva. etymology. the specific epithet refers to the river of didessa. description. wing span 18 mm. head and thorax grey-black with black marks. forewng fairly broad; costa uniformly convex; termen weakly obique and convex. ground colour greyish with some whitish parts and bluish areas; suffusions blackish; costal strigulae small, white; divisions brown-black. markings brownish black with black marks, diffuse, incomplete, consisting of basal blotch, median fascia, and subterminal fascia. cilia grey-black. hindwing brown with broadly rounded anal area; cilia whitish cream. male genitalia (figure 15). uncus weakly sclerotized, helmetshaped, long hairy; socius large, lateral, densely bristled; gnathos and tuba analis rather weakly sclerotized; sacculus with large, sharp termination, short caudal edge marked by broad, spiny lobe from base of which a slender process extends; neck of valva very slender; cuculus elongate; aedeagus short. female not known. cosmorryncha microcosma aarvik, 2004 (figures 5, 16) material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 11.11.2010, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, m. colacci. remarks. c. microcosma was described from congo (zaire) (aarvik, 2004), and is now known from wellega zone, ethiopia. aterpia microchlamys diakonoff, 1983 (figures 6, 17) material examined. 1 male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. remarks. a. microchlamys is described from madagascar and is also recorded from mt. cameroon (razowski, 2004). tribe enarmoniini ancylis colaccii razowski & trematerra, 2012 sp. n. (figures 7, 18) material examined. holotype male, ethiopia, wellega zone, didessa river, 1280 m, 11.11.2010, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, m. colacci. diagnosis. in facies, a. colaccii is most similar to a. halisparta meyrick, 1910, from transvaal, south africa but colaccii has whitish vertex of the head (in halisparta it is rust); from a. oculifera (walsingham, 1891) from bathurst, gambia, colaccii differs by its strongly sinuate termen of forewing and anal edge of the hindwing, and a lack of brown oval spot at base of the apex (both compared species are known only from females). the male genitalia of this species differ from all known congeners by a ventral convexity of the base of the cucullus. etymology. the species is named after his collector dr marco colacci (university of molise, campobasso, italy). description. wing span 14 mm. head whitish cream, labial palpus whitish cream sprinkled brown; thorax cream, tegulae brownish. forewing slender; costa weakly convex; apex elongate-pointed; termen sinuate. ground colour cream sprinkled and suffused brown and brown ferruginous; costal strigulae whitish, divisions rust brown and brownish. remnants of markings brownish rust. cilia cream. hindwing brownish; cilia (rubbed) cream. male genitalia (figure 18). uncus reduced; socius broad, densely hairy; basal part of valva broad; neck slender expanding posterad; sacculus convexly rounded; cucullus convex ventrobasally, with slight submedian convexity; aedeagus long, slender, indistinctly tapering terminad, cornuti long and slender. female not known. tribe eucosmini cosmetra thalameuta (meyrick, 1918), comb. n. (figures 8, 19) material examined. 1 male, ethiopia, ilubabor zone, bedelle, mute forest, 2060 m, 27.01.2012, coord.utm 37p 207285e, 938068n, legg. a. sciarretta, f. parisi. remarks. c. thalameuta was described in the genus eucosma hübner, [1825] from pretoria, south africa. the type was illustrated by razowski & krüger (2007). gypsonoma giorgiae razowski & trematerra, 2012 sp. n. (figures 9, 20) material examined. holotype male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. 1 male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. diagnosis. in facies, g. giorgiae is similar to g. opsonoma (meyrick, 1918) from transvaal, the republic of south africa but giorgiae has brown forewing markings; male genitalia of giorgiae [journal of entomological and acarological research 2012; 44:e8] [page 39] article no nco mm er cia l u se on ly [page 40] [journal of entomological and acarological research 2012; 44:e8] article figures 11-17. genitalia. apotoforma fustigera razowski, 1986 (11); choristoneura dinota (meyrick, 1918) (12); endothenia albapex razowski & trematerra, 2010 (13); lobesia alyana razowski, 2012 (14); olethreutes didessae sp. n., holotype (15); cosmorryncha microcosma aarvik, 2004 (16); aterpia microchlamys diakonoff, 1988 (17). no nco mm er cia l u se on ly differ from other species in slender horn on distal edge of the basal cavity, distinct ventral incision of the valva, and a reduced membranous outer part of the cucullus (figure 20). etymology. this species is named after the daughter of the younger author. description. wing spam 13-14 mm. head grey-brown, labial palpus brownish; thorax brown with black-brown marks. forewing hardly expanding terminad; costa slightly convex; termen indistinctly concave medially, rather not oblique. ground colour white, tinged yellowish terminally; strigulation and suffusions brown; costal strigulae white, divisions blackish brown; apex concolorous with the latter marked rust posteriorly. markings blackish brown with blackish parts; basal blotch fused with postbasal fascia; median fascia atrophying subdorsally; subterminal blotch atrophied towards costa. cilia brownish. hindwing brownish; cilia cream. male genitalia (figure 20). uncus tapering terminad; socius broad, curved; valva fairly slender; sacculus simple; neck rudimentary; cucullus uniformly broad with ventroproximal group of spines; aedeagus short. tribe grapholitini cydia tytthaspis razowski & trematerra, 2010 material examined. 1 male, ethiopia, ilubabor zone, near bedelle, dabeda river, 1827 m, 26.01.2012, coord. utm 37p 201587e, 930253n, legg. a. sciarretta, f. parisi. remarks. c. tytthaspis was described from bale mountains where it was collected at 3100 m. thaumatotibia batrachopa (meyrick, 1908) material examined. 1 male, ethiopia, ilubabor zone, bedelle, dabeda river, 1800 m, 14.11.2010, legg. a. sciarretta, m. colacci. remarks. t. batrachopa was described from cape colony, south africa. it also was recorded from bale mountains, ethiopia (razowski & trematerra 2010). grapholita siderocosma diakonoff, 1978 (figures 10, 21) material examined. 1 male, ethiopia, wellega zone, didessa river, 1280 m, 24.01.2012, coord. utm 37p 215814e, 961832n, legg. a. sciarretta, f. parisi. remarks. g. siderocosma was described from the réunion island. references aarvik l., 2004 revision of the subtribe neopotamiae (lepidoptera: tortricidae) in africa. norw. j. entomol. 51: 71-122. meyrick e., 1932 entomological expedition to abyssinia, 1926-7. microlepidoptrera. trans. ent. soc. london 80: 107-120. razowski j., 1986 the data on tortricini (lepidoptera, tortricidae) published after 1966. acta zool. cracov. 29: 423-440. razowski j., 2004 review of the genera of afrotropical tortricidae (lepidoptera). acta zool. cracov. 47: 167-210. razowski j., 2012 tortricidae from the tervuren museum, 2: olethreutini (insecta: lepidoptera). genus 2: 163-182. razowski j., krüger m., 2007 an illustrated catalogue of the type specimens of tortricidae in the transvaal museum, pretoria (lepidoptera: tortricidae). shilap revta lepid. 35: 103-179. razowski j., trematerra p., 2010. tortricidae (lepidoptera) from ethiopia. j. entomol. acarol. res. ser. ii 42: 47-79. razowski j., wojtusiak j., 2012. tortricidae (lepidoptera) from nigeria. acta zool. cracov. [in press]. [journal of entomological and acarological research 2012; 44:e8] [page 41] article figures 18-21. genitalia. ancylis colaccii sp. n., holotype (18); cosmetra thalameuta (meyrick, 1918) (19); gypsonoma giorgiae sp. n., holotype (20); grapholita siderocosma diakonoff, 1978 (21). no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. layout 1 [journal of entomological and acarological research 2013; 45:e9] [page 43] a contribution to the knowledge of euphorinae (hymenoptera: braconidae), with six new records from iran s. farahani,1 a.a. talebi,1 e. rakhshani2 1department of entomology, faculty of agriculture, tarbiat modares university, tehran; 2department of plant protection, college of agriculture, university of zabol, iran abstract a survey was conducted for identification of euphorinae (hymenoptera: braconidae) in the northern provinces of iran. the specimens were collected using malaise traps during 2010-2011. in all, 9 species in four genera consisting of allurus förster, dinocampus förster, peristenus nees and perilitus nees were collected and identified. the genus allurus is recorded for the first time from iran. six species are newly recorded for the iranian fauna including allurus muricatus (haliday), peristenus pallipes curtis, peristenus relictus (ruthe), perilitus (townesilitus) bicolor (wesmael), perilitus foveolatus reinhard and perilitus rutilus (nees). morphological diagnostic characters and geographical distribution of the species are briefly discussed. a key is presented for identification of the genera and species in the studied area. introduction euphorinae förster, 1862 (hymenoptera: braconidae) is a rather large cosmopolitan subfamily of braconid wasps (van achterberg & quicke, 2000). this subfamily comprises 55 genera worldwide of which 30 genera were found in the palaearctic region (tobias, 1995; yu et al., 2012). van achterberg (1994) recognized four tribes in this subfamily (euphorini, cosmophorini, centistini, meteorini), while yu et al. (2012) listed 15 tribes (centistini, cosmophorini, cryptoxilonini, dinocampini, euphorini, helorimorphini, mannokeraiini, meteorini, myiocephalini, neoneurini, oncometeorini, perilitini, proclithrophorini, syntretini, and tainitermini). a wide range of morphological variability within the subfamily euphorinae, allow them to parasitize very different orders of insects including coleoptera, hemiptera, psocoptera, hymenoptera, neuroptera and orthoptera (shaw & huddleston, 1991). members of euphorinae develop as solitary or gregarious endoparasitoids on larvae and adults of other insects (shaw & huddleston, 1991; belokobylskij, 2000b). this subfamily is readily recognizable by the following characters: having two submarginal cells in the forewing, first metasomal tergite distinctly petiolate (van achterberg, 1993; shaw & huddleston, 1991), spiracles of first metasomal tergite usually located medially or behind middle of the tergite, vein culb of forewing absent or nearly so (van achterberg, 1993). the taxonomy of the subfamily euphorinae was studied by various authors (loan, 1974; shaw, 1985, 1996; belokobylskij, 1992; van achterberg, 1994; chen & van achterberg, 1997; haeselbarth, 1988, 1998, 1999; van achterberg and quicke, 2000; shaw & marsh, 2000; belokobylskij, 2000b; van achterberg & haeselbarth, 2003; boring, 2010; stigenberg & ronquist, 2011). comparatively, a considerable amount of data have been published about the biology of euphorinae (loan, 1983; chen & van achterberg, 1997; belokobylskij, 2000b; maetô, 1988, 1990; papp, 1994; zijp & blommers, 2002; waloff, 1967; phillips & baird, 2001; bilewicz-pawinska, 1990). the north of iran is characterized by two different ecological zones that are separated by the alborz mountains: hyrcanian (caspian) (figure 1) and iran-o-turanian zones. the hyrcanian zone includes alborz range forest steppe, caspian mixed forest and caspian lowland desert. the iran-o-turanian zone includes both mountains and plain areas dominated by a desert climate and a hot summer. these zones form diverse and vast regions of mountain, lush irrigated lowlands, wetland and desert with a great biodiversity, which is associated with the diverse flora, topographical irregularity and the xeric landscapes (heshmati, 2007). the subfamily euphorinae in iran is very poorly studied. seven tribes and 20 species are listed (as genus-species) from centistini (11), dinocampini (1-1), euphorini (3-5), meteorini (2-6), neoneurini (1-1), perilitini (2-4) and syntretini (1-2) (fallahzadeh & saghaei, 2010; ghahari et al., 2009a, 2009b, 2010; ghahari & fischer, 2011; correspondence: ali asghar talebi, department of entomology, faculty of agriculture, tarbiat modares university, p.o. box: 14115-336, tehran, iran. tel.: +98.21.48292371 fax: +98.21.48292200. e-mail: talebia@modares.ac.ir key words: braconidae, euphorinae, first records, iran. acknowledgements: we would like to thank the department of entomology, tarbiat modares university for providing financial support. the authors are most grateful to the editor and anonymous reviewers for their valuable comments and suggestions on an earlier version of this paper. our cordial thanks are also expressed to dr. cornelis van achterberg for his cooperation in this work and identification of some species. we thank mr. m. khayrandish, a. mohammadi and mr. a. nadimi for helping us in collecting the specimens. received for publication: 5 november 2012. revision received: 9 april 2013. accepted for publication: 30 may 2013. ©copyright s. farahani et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e9 doi:10.4081/jear.2013.e9 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2013; volume 45:e9 jear_2013_2:hrev_master 16/09/13 13.56 pagina 43 no nco mm er cia l subfamily euphorinae, allow them to parasitize very different orders of no nco mm er cia l subfamily euphorinae, allow them to parasitize very different orders ofinsects including coleoptera, hemiptera, psocoptera, hymenoptera, no nco mm er cia l insects including coleoptera, hemiptera, psocoptera, hymenoptera,neuroptera and orthoptera (shaw & huddleston, 1991). members of no nco mm er cia l neuroptera and orthoptera (shaw & huddleston, 1991). members of euphorinae develop as solitary or gregarious endoparasitoids on larvae no nco mm er cia l euphorinae develop as solitary or gregarious endoparasitoids on larvae and adults of other insects (shaw & huddleston, 1991; belokobylskij, no nco mm er cia l and adults of other insects (shaw & huddleston, 1991; belokobylskij, no nco mm er cia l no nco mm er cia l correspondence: ali asghar talebi, department of entomology, faculty of no nco mm er cia l correspondence: ali asghar talebi, department of entomology, faculty of agriculture, tarbiat modares university, p.o. box: 14115-336, tehran, iran. no nco mm er cia l agriculture, tarbiat modares university, p.o. box: 14115-336, tehran, iran. key words: braconidae, euphorinae, first records, iran. no nco mm er cia l key words: braconidae, euphorinae, first records, iran. acknowledgements: we would like to thank the department of entomology, no nco mm er cia l acknowledgements: we would like to thank the department of entomology, no nco mm er cia l tarbiat modares university for providing financial support. the authors are no nco mm er cia l tarbiat modares university for providing financial support. the authors are most grateful to the editor and anonymous reviewers for their valuable com-no nco mm er cia l most grateful to the editor and anonymous reviewers for their valuable com-no nco mm er cia l ments and suggestions on an earlier version of this paper. our cordialno nco mm er cia l ments and suggestions on an earlier version of this paper. our cordial us e listed 15 tribes (centistini, cosmophorini, cryptoxilonini, dinocampini, us e listed 15 tribes (centistini, cosmophorini, cryptoxilonini, dinocampini, euphorini, helorimorphini, mannokeraiini, meteorini, myiocephalini, us e euphorini, helorimorphini, mannokeraiini, meteorini, myiocephalini,neoneurini, oncometeorini, perilitini, proclithrophorini, syntretini, us e neoneurini, oncometeorini, perilitini, proclithrophorini, syntretini, and tainitermini). a wide range of morphological variability within theus e and tainitermini). a wide range of morphological variability within the subfamily euphorinae, allow them to parasitize very different orders ofus e subfamily euphorinae, allow them to parasitize very different orders of on ly large cosmopolitan subfamily of braconid wasps (van achterberg & on ly large cosmopolitan subfamily of braconid wasps (van achterberg & quicke, 2000). this subfamily comprises 55 genera worldwide of which on lyquicke, 2000). this subfamily comprises 55 genera worldwide of which30 genera were found in the palaearctic region (tobias, 1995; yu on ly30 genera were found in the palaearctic region (tobias, 1995; yu 2012). van achterberg (1994) recognized four tribes in this subfamily on ly2012). van achterberg (1994) recognized four tribes in this subfamily (euphorini, cosmophorini, centistini, meteorini), while yu on ly (euphorini, cosmophorini, centistini, meteorini), while yu listed 15 tribes (centistini, cosmophorini, cryptoxilonini, dinocampini,on ly listed 15 tribes (centistini, cosmophorini, cryptoxilonini, dinocampini, euphorini, helorimorphini, mannokeraiini, meteorini, myiocephalini, on ly euphorini, helorimorphini, mannokeraiini, meteorini, myiocephalini, [page 44] [journal of entomological and acarological research 2013; 45:e9] lashkari-bod et al., 2011; farahani et al., 2012). the objective of this study was to determine the species of the subfamily euphorinae, as a primary step to understand the situation of this large and diverse group of insects in northern provinces of iran and to provide a reference collection at tarbiat modares university. materials and methods the present study was carried out in four northern provinces including, guilan, mazandaran, alborz and qazvin provinces. the specimens were collected using 32 malaise traps; 16 traps were set up in northern slopes and 16 traps in southern slopes of the alborz mountains in both ecosystems, during 2010 and 2011 (figure 2). the specimens were extracted from the malaise traps and sorted weekly. they were then treated with 70% ethanol and finally placed on a paper plate for drying. the dried specimens were then mounted on triangular papers and labeled. the external morphology of specimens were studied using an olympus szx9 stereomicroscope. identifications were performed based on loan (1974), tobias (1995) and haeselbarth (1999). illustrations were taken using an olympus szx9 stereomicroscope and olympus ax70 microscope equipped with a sony ccd digital camera. morphological terminology follows van achterberg (1993). all the materials were deposited in the insect collection of the department of entomology, tarbiat modares university, tehran. article figure 1. habitats of guilan province located in the northern slopes of the alborz mountain. figure 2. iran-alborz, qazvin, guilan and mazandaran provinces, where the euphorinae species were collected by malaise trap. jear_2013_2:hrev_master 16/09/13 13.56 pagina 44 no nco mm er cia l no nco mm er cia l no nco mm er cia l u se figure 1. habitats of guilan province located in the northern us e figure 1. habitats of guilan province located in the northern us e us e slopes of the alborz mountain. us e slopes of the alborz mountain.o nlyon ly on ly figure 1. habitats of guilan province located in the northernon ly figure 1. habitats of guilan province located in the northern slopes of the alborz mountain. on ly slopes of the alborz mountain. results four genera including nine species of euphorinae (hym.: braconidae) were collected and identified from northern iran. they include three previously reported species, perilitus aethiops nees, perilitus stelleri (loan) and dinocampus coccinellae (schrank), and six newly recorded species for the iranian fauna, allurus muricatus haliday, peristenus pallipes curtis, peristenus relictus (ruthe), perilitus (townesilitus) bicolor (wesmael), perilitus foveolatus reinhard, perilitus rutilus (nees), which are marked with an asterisk in the text. *allurus muricatus (haliday, 1833) (figures 3a-3e) synonyms: ancylus muricatus haliday, 1833; leiophron (ancylus) muricatus haliday, 1835; leiophron muricatus reinhard, 1862; liophron muricatus marshall, 1872; centistes muricatus rudow, 1918; centistes (allurus) muricatus hellen, 1958; allurus muricatus forster, 1862; leiophron armatus wesmael, 1835. material examined: qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m a.s.l.), 24.5.2011, 1♀; 08.6.2011, 3♀; 21.6.2011, 1♂, leg. a. nadimi. diagnostic characters (female): length of body 2.5-3.0 mm; antennae 30-31 segmented; length of fore wing 2.3-2.9 mm; pterostigma slightly shorter than vein 1-r1 (about 0.9¥); marginal cell long; vein m+cu1 unsclerotized; 1-sr+m and 2-sr+m of fore wing present (figure 3b); first abdominal tergite sessile; third abdominal sternite with 2 denticles (figure 3c); hind coax with a large denticle (figure 3d); claws cleft (figure 3e). coloration: antennae dark brown; head, thorax and first abdominal tergite black; second and third abdominal tergites reddish brown but reminder tergites black; legs reddish brown. general distribution: europe, eastern and western palaearctic (yu et al., 2012). new record from iran. dinocampus coccinellae (schrank, 1802) (figures 4a, 4b) synonyms: dinocampus americanus (riley, 1888); dinocampus sculptus (cresson, 1872); dinocampus terminatus (nees, 1811). material examined: alborz province, shahriar (35º40’08.01”n, 50º56’56.64”e, 1168 m a.s.l.), 10.9.2010, 1♀; guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 24.10.2010, 1♀; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m a.s.l.), 09.5.2011, 1♀; leg. a. mohammadi. diagnostic characters (female): length of body 4.0 mm; antennae 22-23 segmented; length of fore wing 3.2 mm; pterostigma longer than vein 1-r1 (1.5¥); marginal cell short; vein m+cu1 sclerotized; 1-sr+m and 2-sr+m of fore wing present (figure 4b); first abdominal tergite sessile; dorsope and laterope absent; ovipositor slender, about as long as first abdominal tergite; claws simple. coloration: antennae dark brown; head and fore legs reddish brown; thorax, first abdominal tergite, middle and hind legs black. general distribution: australasian, europe, nearectic, neotropical, oceanic, oriental, eastern and western palaearctic (yu et al., 2012). remarks: dinocampus is small cosmopolitan genus with one species in the palaearctic region, e.g. d. coccinellae (chen & van achterberg, 1997). perilitus aethiops nees, 1834 (figures 5a, 6a, 7a, 8a) synonyms: perilitus aethiopoides (loan, 1975); perilitus brevispina (thomson, 1892); perilitus spurius (ruthe, 1856). material examined: alborz province, chalous road, shahrestanak (35º58’16.26”n, 51º21’25.80”e, 2225 m a.s.l.), 17.5.2010, 1♀; 13.6.2010, 1♂; 21.6.2010, 1♂; guilan province, roodsar, ghazichak (36º45’57.54”n, 50º19’35.22”e, 1803 m a.s.l.), 16.5.2010, 1♂; 23.5.2010, 2♂; 06.6.2010, 1♀; 13.6.2010, 2♀, 1♂; 21.6.2010, 1♀; 27.6.2010, 2♀, 1♂; 05.7.2010, 2♀, 1♂; 24.7.2010, 1♀; 01.8.2010, 2♀; 15.8.2010, 1♀; 21.8.2010, 2♀, 1♂; 03.10.2010, 1♀, 1♂; 10.10.2010, 3♂; 17.10.2010, 1♀, 3♂; 24.10.2010, 2♂; guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 23.5.2010, 1♀; 05.7.2010, 1♀; 24.10.2010, 2♀; 31.10.2010, 1♂; mazandaran province, noor, gaznasara (36º21’55.02”n, 52º06’10.74”e, 692 m a.s.l.), 06.6.2011, 1♀; 15.8.2011, 1♀; 04.9.2011, 1♂; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m a.s.l.), 24.5.2011, 2♀; 3♂; 08.6.2011, 1♀, 2♂; 21.6.2011, 2♂; leg. m. khayrandish. diagnostic characters (female): length of body 1.9-3.0 mm; antennae 19-24 segmented, first flagellar segment 2.5¥ as long as wide (figure 7a), second flagellar segment 2.0¥ as long as wide; face 1.3¥ wider than height (figure 6a); length of fore wing 2.0-2.5 mm; pterostigma longer than vein 1-r1 (2.0¥); marginal cell short; vein m+cu1 sclerotized; 1-sr+m and 2-sr+m of fore wing absent (figure 8a); first abdominal tergite petiolate, its length about 1.8¥ as long as apical width; ovipositor slender and equal or longer than first abdominal tergite; claws simple. coloration: antennae dark brown; head reddish brown, thorax and first abdominal tergite dark brown, occasionally reddish brown; legs reddish brown. [journal of entomological and acarological research 2013; 45:e9] [page 45] article figure 3. allurus muricatus (haliday, 1833), female: (a) lateral habitus; (b) fore wing; (c) abdomen (arrow); (d) hind coxa (arrow); (e) claw. jear_2013_2:hrev_master 16/09/13 13.56 pagina 45 no nco mm er cia l general distribution: europe, eastern and western palaearctic no nco mm er cia l general distribution: europe, eastern and western palaearctic (schrank, 1802) (figures 4a, 4b) no nco mm er cia l (schrank, 1802) (figures 4a, 4b) (riley, 1888); no nco mm er cia l (riley, 1888); dinocampus no nco mm er cia l dinocampus (nees, 1811). no nco mm er cia l (nees, 1811). material examined: alborz province, shahriar (35º40’08.01”n, no nco mm er cia l material examined: alborz province, shahriar (35º40’08.01”n, ; guilan province, roodsar, no nco mm er cia l ; guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 24.10.2010, 1 no nco mm er cia l orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 24.10.2010, 1 qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m no nco mm er cia l qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m ; leg. a. mohammadi. no nco mm er cia l ; leg. a. mohammadi. diagnostic characters (female): length of body 4.0 mm; no nco mm er cia l diagnostic characters (female): length of body 4.0 mm; antennae 22-23 segmented; length of fore wing 3.2 mm; pterostigma no nco mm er cia l antennae 22-23 segmented; length of fore wing 3.2 mm; pterostigma ¥ no nco mm er cia l ¥); marginal cell short; vein m+cu1 sclero-no nco mm er cia l ); marginal cell short; vein m+cu1 sclerotized; 1-sr+m and 2-sr+m of fore wing present (figure 4b); firstno nco mm er cia l tized; 1-sr+m and 2-sr+m of fore wing present (figure 4b); firstno nco mm er cia l u se us e o nly coloration: antennae dark brown; head reddish brown, thorax on ly coloration: antennae dark brown; head reddish brown, thorax on lyand first abdominal tergite dark brown, occasionally reddish brown; on lyand first abdominal tergite dark brown, occasionally reddish brown; [page 46] [journal of entomological and acarological research 2013; 45:e9] general distribution: ethiopian, europe, nearctic (introduced), oriental, eastern and western palaearctic (yu et al., 2012). remarks: this species seems to be nearest to p. stelleri, a form that can be distinguished by the apical width of the first abdominal tergite in the female (0.27-0.32) and width of the pterostigma (0.15-0.18), while the width of the first abdominal tergite (0.30-0.43) and the width of the pterostigma (0.18-0.24) of the female in p. stelleri are relatively longer (tobias, 1995). *perilitus (townesilitus) bicolor (wesmael 1835) (figures 5b, 6b, 7b, 8b) synonyms: perilitus (townesilitus) breviradialis (tobias, 1976). material examined: guilan province, roodsar, ghazichak (36º45’57.54”n, 50º19’35.22”e, 1803 m a.s.l.), 04.7.2010, 1♀; 10.7.2010, 1♀; guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 16.5.2010, mazandaran province, noor, gaznasara (36º21’55.02”n, 52º06’10.74”e, 692 m a.s.l.), 09.10.2011, 1♀; 1♀; leg. a. nadimi. diagnostic characters (female): length of body 1.8-2.2 mm; antennae 18-21 segmented, flagellum long and slender, first flagellar segment 4.0-5.0¥ as long as wide (figure 7b), second flagellar segment 4.0¥ as long as wide; face 1.1¥ wider than height (figure 6b); length of fore wing 1.5-2.0 mm; pterostigma longer than vein 1-r1 (2.0¥); marginal cell short; vein m+cu1 sclerotized; 1-sr+m and 2-sr+m of fore wing absent (figure 8b); first abdominal tergite petiolate, its length about 3.0¥ as long as apical width; ovipositor slender and longer than first abdominal tergite; claws simple. coloration: antennae dark brown (basal of flagellum yellowish); head, thorax and first abdominal tergite reddish brown, occasionally dorsal part of thorax black; legs reddish brown. general distribution: europe, eastern and western palaearctic (yu et al., 2012). new record from iran. remarks: this species is easily recognized by the length of the first and second flagellar segment compared to their width, and antennae 18-21-segmented in the female (tobias, 1995). *perilitus foveolatus reinhard 1862 (figures 5c, 6c, 7c, 8c) synonyms: perilitus sicheli giard, 1895. material examined: guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 03.10.2010, 1♀; 17.10.2010, 1♀; guilan province, roodsar, ziaz (36º52’27.18”n, 50º13’24.78”e, 490 m a.s.l.), 30.5.2010, 1♀; guilan province, roodsar, ghazichak (36º45’57.54”n, 50º19’35.22”e, 1803 m a.s.l.), 17.10.2010, 1♀; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m a.s.l.), 24.5.2011, 12♀, 1♂; 08.6.2011, 1♀; leg. m. khayrandish. diagnostic characters (female): length of body 2.5-3.0 mm; antennae 22-23 segmented, first flagellar segment 3.5¥ as long as wide (figure 7c), longer than second flagellar segment (1.2¥); face 1.1¥ wider than height (figure 6c); length of fore wing 2.2-2.5 mm; pterostigma longer than vein 1-r1 (2¥); marginal cell short; vein m+cu1 sclerotized; 1-sr+m of fore wing present and 2-sr+m absent (figure 8c); apical area between notaulices distinctly punctuate; first abdominal tergite petiolate, its length about 1.9¥ as long as apical width; ovipositor slender and longer than first abdominal tergite; claws simple. coloration: antennae very dark brown; head lighter than thorax and reddish brown; thorax and first abdominal tergite black; legs reddish brown. general distribution: europe, eastern and western palaearctic (yu et al., 2012). remarks: this species seems to be nearest to p. cornelii haeselearth from which it can be recognized by length of ovipositor sheath, which is shorter than the hind tibia (haeselbarth, 1999). *perilitus rutilus (nees, 1811) (figures 5d, 6d, 7d, 8d) synonyms: perilitus luteus herrich-schaffer, 1838; perilitus pyri (viereck, 1917); perilitus ruralis herrich-schaffer, 1838; perilitus strenuus marshall, 1887; perilitus tuberculus zaykov, 1981. material examined: guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 06.6.2010, 2♂; 13.6.2010, 3♂; 21.6.2010, 1♀; 05.7.2010, 1♀; guilan province, roodsar, ziaz (36º52’27.18”n, 50º13’24.78”e, 490 m a.s.l.), 23.5.2010, 1♂; 06.6.2010, 1♀; mazandaran province, noor, gaznasara (36º21’55.02”n, 52º06’10.74”e, 692 m a.s.l.), 27.6.2011, 1♂; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, 1541 m a.s.l.), 24.5.2011, 2♂; 08.6.2011, 4♀, 17♂; 21.6.2011, 4♀, 4♂; leg. m. khayrandish. diagnostic characters (female): length of body 2.5-3.0 mm; antennae 25-26 segmented, first flagellar segment 4.0¥ as long as wide (figure 7d), first flagellar segment as long as or slightly shorter than second segment; face 1.4¥ wider than height (figure 6d); length of fore wing 2.2-2.5 mm; pterostigma longer than vein 1-r1 (1.3¥); marginal cell short and pointed apically; vein m+cu1 sclerotized; 1-sr+m of fore wing present and 2-sr+m absent (figure 8d); first abdominal tergite petiolate, its length about 2.5¥ as long as apical width; ovipositor slender and longer than first abdominal tergite; posterior area of hind coxa distinctly transverse striate; claws simple. coloration: antennae dark brown, occasionally basal segments of antennae lighter; body yellowish brown; propodeum black; first abdominal tergite at the base pale; legs yellowish brown. general distribution: europe, nearctic (introduced), eastern and western palaearctic (yu et al., 2012). remarks: this species is taxonomically similar to p. longiradialis, from which it can be separated by the basal felagellar segments, which are thin and yellowish; antennae 23-27-segmented (28-segmented in p. longiradialis) (haeselbarth, 1999). article figure 4. dinocampus coccinellae (schrank 1802), female: (a) lateral habitus; (b) fore wing. jear_2013_2:hrev_master 16/09/13 13.56 pagina 46 no nco mm er cia l remarks: this species is easily recognized by the length of the first no nco mm er cia l remarks: this species is easily recognized by the length of the first and second flagellar segment compared to their width, and antennae no nco mm er cia l and second flagellar segment compared to their width, and antennae (figures 5c, 6c, 7c, 8c) no nco mm er cia l (figures 5c, 6c, 7c, 8c) material examined: guilan province, roodsar, orkom no nco mm er cia l material examined: guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 03.10.2010, 1 no nco mm er cia l (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 03.10.2010, 1 ; guilan province, roodsar, ziaz (36º52’27.18”n, no nco mm er cia l ; guilan province, roodsar, ziaz (36º52’27.18”n, 50º13’24.78”e, 490 m a.s.l.), 30.5.2010, 1 no nco mm er cia l 50º13’24.78”e, 490 m a.s.l.), 30.5.2010, 1♀ no nco mm er cia l ♀; guilan province, roodsar, no nco mm er cia l ; guilan province, roodsar, ghazichak (36º45’57.54”n, 50º19’35.22”e, 1803 m a.s.l.), 17.10.2010, no nco mm er cia l ghazichak (36º45’57.54”n, 50º19’35.22”e, 1803 m a.s.l.), 17.10.2010, ; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, no nco mm er cia l ; qazvin province, zereshk road (36°21’39.72”n, 50°03’55.26”e, , 1 no nco mm er cia l , 1♂ no nco mm er cia l ♂; 08.6.2011, 1no nco mm er cia l ; 08.6.2011, 1 diagnostic characters (female): length of body 2.5-3.0 mm;no nco mm er cia l diagnostic characters (female): length of body 2.5-3.0 mm; longiradialis no nco mm er cia l longiradialis u se and western palaearctic (yu us e and western palaearctic (yu remarks: this species is taxonomically similar to us e remarks: this species is taxonomically similar to us e from which it can be separated by the basal felagellar segments, which us e from which it can be separated by the basal felagellar segments, which are thin and yellowish; antennae 23-27-segmented (28-segmented in us e are thin and yellowish; antennae 23-27-segmented (28-segmented in ) (haeselbarth, 1999).us e ) (haeselbarth, 1999). on ly hind coxa distinctly transverse striate; claws simple. on ly hind coxa distinctly transverse striate; claws simple. on lycoloration: antennae dark brown, occasionally basal segments of on lycoloration: antennae dark brown, occasionally basal segments ofantennae lighter; body yellowish brown; propodeum black; first abdomon lyantennae lighter; body yellowish brown; propodeum black; first abdominal tergite at the base pale; legs yellowish brown. on ly inal tergite at the base pale; legs yellowish brown. general distribution: europe, nearctic (introduced), easternon ly general distribution: europe, nearctic (introduced), eastern and western palaearctic (yu on ly and western palaearctic (yu et alon ly et al., 2012). on ly ., 2012). remarks: this species is taxonomically similar to on ly remarks: this species is taxonomically similar to perilitus stelleri (loan, 1972) (figures 5e, 6e, 7e, 8e) material examined: guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 09.5.2010, 1♀; 16.5.2011, 1♂; leg. a. nadimi. diagnostic characters (female): length of body 3.4 mm; antennae 25 segmented, length of first flagellar segment 3.0¥ as long as wide (figure 7e), first segment about 1.3¥ as long as second segment; face 1.4¥ wider than height (figure 6e); length of fore wing 3.1 mm; pterostigma longer than vein 1-r1 (1.6¥); marginal cell short and pointed; vein m+cu1 sclerotized; 1-sr+m and 2-sr+m of fore wing absent (figure 8e); first abdominal tergite petiolate; ovipositor slender and longer than first abdominal tergite; claws simple. coloration: body very dark brown; antennae dark brown, legs and ovipositor reddish brown. general distribution: europe, western palaearctic and introduced into usa (yu et al., 2012). *peristenus pallipes curtis, 1833 (figures 9a, 9b) synonyms: peristenus barbiger (wesmael, 1835); peristenus pallipes (herrich-schaffer, 1838); peristenu punctata (provancher, 1883); peristenus tuberculifer (marshall, 1887). material examined: guilan province, roodsar, orkom (36º45’44.34”n, 50º18’11.88”e, 1201 m a.s.l.), 16.5.2010, 1♀, 6♂; 06.6.2010, 1♀; guilan province, roodsar, ghazichak (36º45’52.62”n, 50º20’10.80”e, 1787 m a.s.l.), 16.5.2010, 1♂; guilan province, roodsar, ziaz (36º52’27.18”n, 50º13’24.78”e, 490 m a.s.l.), 30.5.2010, 1♀; mazandaran province, noor, gaznasara (36º16’58.08”n, 52º10’55.62”e, 2013 m a.s.l.), 28.4.2011, 3♂; 09.5.2011, 9♀, 21♂; 25.5.2011, 7♀, 7♂; leg. m. khayrandish. diagnostic characters (female): length of body 2.7-3.0 mm; antennae 23-segmented; occipital carina complete dorsally; frons densely punctuate; mesoscutum mostly punctated; fore wing vein 1-r1 0.8¥ as long as width of pterostigma, pterostigma 2.2¥ as long as width; vein m+cu1 of fore wing unsclerotized, basal cell of fore wing densely setose, distinctly similar to first subdiscal cell (figure 9b); first abdominal tergite 1.7¥ as long as wide at apex; ovipositor shorter than first abdominal tergite and hardly visible. coloration: head and thorax uniformely black; antennae reddish brown; legs yellowish. general distribution: europe, nearctic (introduced), oriental, eastern and western palaearctic (yu et al., 2012). new record from iran. remarks: this species is taxonomically nearest to p. nitidus, from which it can be recognized by its punctuated frons and roughened mesepisternum (loan, 1974). [journal of entomological and acarological research 2013; 45:e9] [page 47] article figure 5. lateral habitus of adult perilitus species, females: (a) p. aethiops; (b) p. bicolor; (c) p. foveolatus; (d) p. rutilus; (e) p. stelleri. jear_2013_2:hrev_master 16/09/13 13.56 pagina 47 no nco mm er cia l u se us e o nly remarks: this species is taxonomically nearest to on ly remarks: this species is taxonomically nearest to which it can be recognized by its punctuated frons and roughened on lywhich it can be recognized by its punctuated frons and roughenedmesepisternum (loan, 1974). on lymesepisternum (loan, 1974). [page 48] [journal of entomological and acarological research 2013; 45:e9] *peristenus relictus (ruthe, 1856) (figures 9c, 9d) synonyms: perilitus stygicus loan, 1973. material examined: qazvin province, zereshk road (36º25’23.88”n, 50º06’37.68”e, 1926 m a.s.l.), 06.7.2011, 2♀; leg. a. mohammadi. diagnostic characters (female): length of body 2.7 mm; antennae 19 segmented; occipital carina complete, thin and weak dorsally; frons punctate; mesoscutum smooth, notaulices of mesonotum sharply impressed, finely foveolatus; fore wing vein 1-r1 0.6¥ as long as width of pterostigma, pterostigma 2.0¥ its width; vein m+cu1 of fore wing unsclerotized, basal cell of fore wing sparsely setose or largely glabrous, distinctly less setose than first subdiscal cell (figure 9d); first abdominal tergite 2.0¥ as long as wide at apex; ovipositor shorter than first abdominal tergite and hardly visible. coloration: body black; scape, pedicel and flagellomeres 1 and 2 usually reddish brown; fore leg reddish brown, mid and hind legs brownish black with base of tibia reddish brown. general distribution: europe, western palaearctic and introduced in to nearectic (yu et al., 2012). remarks: peristenus relictus and p. stygicus were described as two article figure 6. frontal view of head in perilitus species, females: (a) p. aethiops, (b) p. bicolor, (c) p. foveolatus, (d) p. rutilus, (e) p. stelleri. figure 8. fore wing in perilitus species: (a) p. aethiops; (b) p. bicolor; (c) p. foveolatus; (d) p. rutilus; (e) p. stelleri. figure 7. basal antennal segments in perilitus species: (a) p. aethiops; (b) p. bicolor; (c) p. foveolatus; (d) p. rutilus; (e) p. stelleri. figure 9. lateral habitus of adult female and fore wing in peristenus species: (a, b) p. pallipes; (c, d) p. relictus. jear_2013_2:hrev_master 16/09/13 13.56 pagina 48 no nco mm er cia l no nco mm er cia l no nco mm er cia l figure 6. frontal view of head in no nco mm er cia l figure 6. frontal view of head in perilitusno nco mm er cia l perilitusno nco mm er cia l p. foveolatusno nco mm er cia l p. foveolatusno nco mm er cia l u se on ly on ly valid species by loan (1974), however, varis & van achterberg (2001) have mentioned p. stygicus as a junior synonym of p. relictus. perlitus relictus is taxonomically nearest to p. picipes, from which it can be separated by its punctuated frons and strong notaulices (loan, 1974). key to the genera and species keys to the genera and species are the following: i) vein 1-r1 of fore wing longer than pterostigma (figure 3b); claws cleft (figure 3e); third abdominal sternite with 2 denticles (figure 3c); denticles on hind coxae large (figure 3d): allurus muricatus. vein 1-r1 of fore wing shorter than pterostigma (figure 4b, 8a–8e, 9b, 9d); claws simple; third abdominal sternite and hind coxae without denticles: 2. ii) vein m+cu1 of fore wing distinctly unsclerotized (figure 9b, 9d); ovipositor shorter than first abdominal tergite and hardly visible (genus peristenus nees): 3. vein m+cu1 of fore wing distinctly sclerotized (figure 4b; 8a-8e); ovipositor longer than first abdominal tergite: 4. iii) vein 1-r1 of fore wing 0.8¥ as long as width of pterostigma (figure 9b); antennae of female 23 segmented; antennae and legs yellowish brown: peristenus pallipes. vein 1-r1 of fore wing 0.5¥ as long as width of pterostigma (figure 9d); antennae of female 19 segmented; antennae, mid and hind legs brownish black: peristenus relictus. iv) scutellum largely rugose; occipital carina complete, ventrally separated from hypostomal carina: dinocampus coccinellae. scutellum largely smooth; occipital carina complete, ventrally joined hypostomal carina (genus perilitus nees): 5. v) vein 1-sr+m of fore wing absent (figure 8a, 8b, 8e): 6. vein 1-sr+m of fore wing present (figure 8c, 8d): 8. vi) basal segments of flagellum slender and 4.0-5.0¥ as long as width (figure 7b), lighter than rest of flagellum (figure 5b); antennae 1821 segmented: perilitus bicolor. basal segments of flagellum normal and almost 3.0¥ as long as width (figure 7a, 7c, 7d, 7e); antennae 19-25 segmented: 7. vii) antenna of female 25 segmented (31 segmented of ♂); length of pterostigma 1.6¥ as long as 1-r1 (figure 8e); length of first abdominal tergite 2.5¥ as long as apical width: perilitus stelleri. antenna of female 19-24 segmented (27-28 segmented in ♂); length of pterostigma 2.5¥ as long as 1-r1 (figure 8a); length of first abdominal tergite 1.8¥ as long as apical width: perilitus aethiops. viii) color of body ligth; first flagellar segment as long as second flagellar segment (figure 7d); antennal of female 25-26 segmented; marginal cell pointed and apex of marginal cell closer to wing apex than stigma or their middle (figure 8d): perilitus rutilus. color of body derk; first flagellar segment 1.2¥ as long as second flagellar segment (figure 7c); antennal of female 22-23 segmented; marginal cell rounded and apex of marginal cell much closer to stigma than wing apex (figure 8c): perilitus foveolatus. discussion and conclusions nine species of euphorinae were found in this study from northern provinces of iran; of these, six species were newly recorded, which increased the number of known euphorinae in iran from 20 to 26. the first records of perilitus from iran were made by hedwig (1957). other researchers who studied the perilitus fauna of iran are bartlett et al. (1978), arbab and mcneill (2001) and ghahari et al. (2010). arbab and mcneill (2001) reported p. aethiops as a parasitoid that attacks adult alfalfa weevil (hypera positica) from qazvin and hamadan provinces and ghahari et al. (2010) has recorded p. stelleri from isfahan province (cornfield). mirab-balou et al. (2008) has reported one undetermined species of the genus peristenus for the first time from iran (hamadan province). lashkari-bod et al. (2011) has recorded peristenus picipes (curtis) from fars province. fallahzadeh and saghaei (2010) erroneously reported p. rubricollis (thomson) with reference to khanjani (2004). the genus allurus of the tribe centistini is recorded here for the first time from iran. among the neighboring countries, allurus muricatus has already been recorded from kazakhstan (tobias, 1995), while another species, a. lituralis (haliday, 1835) has been recorded from turkey (yilmaz et al., 2010). the specimens were collected using malaise traps in this study, therefore the biology of the recorded species are unknown. however, according to the previous studies, allurus muricatus is considered as an adult parasitoid of sitona sp. (coleoptera: curculionidae) and stigmella sp. (lepidoptera: nepticulidae) species (aeschlimann, 1980; yu et al., 2012), but these records have been placed in doubtful hosts until they are confirmed by further investigations. this species is known to oviposit in adults and emerge from adult stages of the hosts (aeschlimann, 1980). it was found only in qazvin province from late may to late june 2011. dinocampus coccinellae is a solitary koinobiont endoparasitoid on coccinellidae but it has also been recorded as attacking chrysomelidae and curculionidae (yu et al., 2012). it parasitizes larvae, pupae and adults but only emerges from adults. bagheri (1998) has reported dinocampus coccinellae on coccinella septempunctata l. (coleoptera: coccinellidae) from isfahan. we also received some specimens of d. coccinellae from yazd province as an endoparasitoid of c. septempunctata in april 2010. belokobylskij (2000a; 2000b) introduced townesilitus as a subgenus of perilitus. many species of the genus perilitus are recorded as parasitoids of curculionoidea and chrysomeloidea. perilitus aethiops and p. stelleri are most commonly recorded as parasitoids of curculionoidea (yu et al., 2012). but perilitus bicolor and p. foveolatus are commonly recorded as parasitoids of chrysomeloidea. perilitus rutilus is a solitary endoparasitoid and emerges from the adult stage of various genera of coleoptera, such as hylobius, hypera, pityokteines, sitona and tytthaspis. many species of the genus perilitus, as well as some other genera like syntretus forster, are gregarious parasitoids (shaw and huddleston, 1991). perilitus foveolatus is a gregarious endoparasitoid on timarcha latreille (coleoptera: chrysomelidae) (haeselbarth, 1999). the genus peristenus is most commonly recorded as a parasitoid of miridae nymphs, but also rarely on chrysomelidae, cicadellidae, melandryidae and nitidulidae (loan, 1980; yu et al., 2012). some species of the genus peristenus have been used as a biological control agent against some species of the genus lygus and alfalfa plant bugs, adelphocoris lineolatus (hemiptera: pentatomidae) (clancy, 1968; coulson, 1994), whereas most host records of the genus perilitus involve coleopteran families (chrysomelidae, curculionidae, coccinellidae and tenebrionidae). some species of the genus peristenus have been used as biological control agents of sitona and hypera (coleoptera: curculionidae) and phyllotreta (coleoptera: chrysomelidae) (yu et al., 2012). moreover, increasing cases of their regulatory impact on important plant pests have been reported, making the members of the genus perilitus important biological control agents (coulson, 1987; haye, 2004). references aeschlimann j.p., 1980 the sitona (col.: curculionidae) species occurring on medicago and their natural enemies in the mediterranean region. entomophaga. 25: 139-153. [journal of entomological and acarological research 2013; 45:e9] [page 49] article jear_2013_2:hrev_master 16/09/13 13.56 pagina 49 no nco mm er cia l but only emerges from adults. bagheri (1998) has reported no nco mm er cia l but only emerges from adults. bagheri (1998) has reported no nco mm er cia l scutellum largely smooth; occipital carina complete, ventrally joined no nco mm er cia l scutellum largely smooth; occipital carina complete, ventrally joined basal segments of flagellum slender and 4.0-5.0 no nco mm er cia l basal segments of flagellum slender and 4.0-5.0¥ no nco mm er cia l ¥ as long as width no nco mm er cia l as long as width (figure 7b), lighter than rest of flagellum (figure 5b); antennae 18no nco mm er cia l (figure 7b), lighter than rest of flagellum (figure 5b); antennae 18basal segments of flagellum normal and almost 3.0 no nco mm er cia l basal segments of flagellum normal and almost 3.0¥ no nco mm er cia l ¥ as long as width no nco mm er cia l as long as width (figure 7a, 7c, 7d, 7e); antennae 19-25 segmented: 7. no nco mm er cia l (figure 7a, 7c, 7d, 7e); antennae 19-25 segmented: 7. antenna of female 25 segmented (31 segmented of no nco mm er cia l antenna of female 25 segmented (31 segmented of as long as 1-r1 (figure 8e); length of first abdomno nco mm er cia l as long as 1-r1 (figure 8e); length of first abdomas long as apical width: no nco mm er cia l as long as apical width: antenna of female 19-24 segmented (27-28 segmented in no nco mm er cia l antenna of female 19-24 segmented (27-28 segmented in as long as 1-r1 (figure 8a); length of firstno nco mm er cia l as long as 1-r1 (figure 8a); length of first coccinellae no nco mm er cia l coccinellaefrom isfahan. we also received some specimens of no nco mm er cia l from isfahan. we also received some specimens of yazd province as an endoparasitoid of no nco mm er cia l yazd province as an endoparasitoid of belokobylskij (2000a; 2000b) introduced no nco mm er cia l belokobylskij (2000a; 2000b) introduced perilitus no nco mm er cia l perilitus us e dinocampus coccinellae us e dinocampus coccinellaecoccinellidae but it has also been recorded as attacking chrysomelidae us e coccinellidae but it has also been recorded as attacking chrysomelidaeand curculionidae (yu us e and curculionidae (yu but only emerges from adults. bagheri (1998) has reported us e but only emerges from adults. bagheri (1998) has reported coccinellae us e coccinellae on us e on on ly sp. (lepidoptera: nepticulidae) species (aeschlimann, 1980; yu on ly sp. (lepidoptera: nepticulidae) species (aeschlimann, 1980; yu ., 2012), but these records have been placed in doubtful hosts until on ly ., 2012), but these records have been placed in doubtful hosts until on lythey are confirmed by further investigations. this species is known to on lythey are confirmed by further investigations. this species is known tooviposit in adults and emerge from adult stages of the hosts on lyoviposit in adults and emerge from adult stages of the hosts (aeschlimann, 1980). it was found only in qazvin province from late mayon ly (aeschlimann, 1980). it was found only in qazvin province from late may dinocampus coccinellaeon ly dinocampus coccinellae is a solitary koinobiont endoparasitoid onon ly is a solitary koinobiont endoparasitoid on [page 50] [journal of entomological and acarological research 2013; 45:e9] arbab a., mcneill m., 2001 new report of microctonus aethiopoides (hym.: braconidae) in iran. j. entomol. soc. iran. 21: 111-112. bagheri m.r., 1998 the first report of perilitus coccinellae (hym., braconidae) a parasitoid of coccinella septempunctata in isfahan. proc. 13th iranian plant protection congress, karaj, iran, 200 pp. bartlett b.r., clausen c.p., debach p., goeden r.d., legner e.f., mcmurtry j.a., et al., 1978 introduced parasites and predators of arthropod pests and weeds: a world review. handbook no. 480. agricultural research service. us dept. of agriculture, washington, d.c.: 545 pp. belokobylskij s.a., 1992 revision of the genus centistes haliday (hymenoptera: braconidae: euphorinae) of ussr far east and neighbouring territories. zool. meded. leiden. 66: 199-237. belokobylskij s.a., 2000a braconidae. in: ler p.a (ed.), key to the insects of russian far east. vol. iv. neoropteroidea, mecoptera, hymenoptera. part 4. opredelitel nasekomykh dalnego vostoka rossii. t. iv. setchatokryloobraznye, skorpionnitsy, pereponchatokrylye. ch.4. dalnauka, vladivostok: 651 pp. belokobylskij s.a., 2000b new species of the subfamily euphorinae (hymenoptera, braconidae) from east palaearctic. far east. entomol. 87: 1-28. bilewicz-pawinska t., 1990 response of parasitoids of the genus peristenus forster (hymenoptera, braconidae) to temperature changes during the diapause. entomol. fennica. 1: 189-790. boring c.a., 2010 morphology and systematics of braconid wasps. phd. diss. university of kentucky, lexington, kt: 123 pp. chen x., van achterbergh c., 1997 revision of the subfamily euphorinae (excluding the tribe meteorini cresson) (hymenoptera: braconidae) from china. zool. verhandel. leiden. 313:1-217. clancy d.w., 1968 distribution and parasitization of some lygus spp. in western united states and central mexico. j. econ. entomol. 61: 443-445. coulson j.r., 1987 udies on the biological control of plant bugs (heteroptera: miridae): an introduction and history, 1961-1983. in: hedlund r. and graham h.m (eds.), economic importance and biological control of lygus and adelphocoris in north america; ars64. usda-ars natl. techn. informat. serv., springfield, va, usa: 1-12. coulson j.r., 1994 releases of beneficial organisms in the united states and rerritories-1983; ars-131. agricultural research service. us dept. of agriculture, washington, d.c.: 113 pp. fallahzadeh m., saghaei n., 2010 checklist of braconidae (insecta: hymenoptera) from iran. mun. ent. zool. 5: 170-186. farahani s., talebi a.a., van achterberg c., rakhshani e., 2012 syntretus, a genus of euphorinae (hymenoptera: braconidae) new for iran, with first record of two species. j. crop prot. 1: 173-179. ghahari h., fischer m., 2011 a contribution to the braconidae (hymenoptera: ichneumonoidea) from north-western iran. calodema. 134: 1-6. ghahari h., fischer m., erdogan o.c., beyarslan a., havaskary m., 2009a a contribution to the knowledge of the braconid-fauna (hymenoptera, ichneumonoidea, braconidae) of arasbaran, northwestern iran. entomofauna. 19: 329-336. ghahari h., fischer m., erdo�an ö.ç., tabari m., ostovan h., beyarslan a., 2009b a contribution to braconidae (hymenoptera) from rice fields and surrounding grasslands of northern iran. mun. ent. zool. 4: 432-435. ghahari h., fischer m., hedqvist o.c., erdogan k., van achterberg k., beyarslan a., 2010 some new records of braconidae (hymenoptera) for iran. linzer biol. beitr. 42: 1395-1404. haeselbarth e., 1988 zur braconidengattung townesilitus haeselbarth & loan, entomofauna. 9: 429-460. haeselbarth e., 1998 zur braconiden-gattung perilitus nees, 1818, 1. beitrag: die perilitus falciger-gruppe (hymenoptera, braconidae). entomofauna. 19: 197-208. haeselbarth e., 1999 zur braconiden-gattung perilitus nees, 1818. beitrag: die arten mit ausgebildetem ersten cubitus-abschnitt (insecta, hymenoptera, braconidae). mitteil. münch. entomol. ges. 89: 11-46. haye t., 2004 studies on the ecology of european peristenus spp. (hymenoptera: braconidae) and their potential for the biological control of lygus spp. (hemiptera: miridae) in canada. phd. diss. university of kiel, kiel: 182 pp. hedwig k., 1957 ichneumoniden und braconiden aus den iran 1954 (hymenoptera). -jahresh. ver. vaterl. naturkd. 112: 103-117. heshmati g. a., 2007 vegetation characteristics of four ecological zones of iran. int. j. plant prod. 1: 215-224. khanjani m., 2004 field crop pests in iran. bu-ali sina university press, hamadan: 719 pp. lashkari-bod a., rakhshani e., talebi a.a., lozan a., zikic v., 2011 a contribution to the knowledge of braconidae (hym., ichneumonoidea) of iran. biharean biol. 5: 147-150. loan c.c., 1974 the european species of leiophron nees and peristenus foerster (hymenoptera: braconidae, euphorinae). trans. r. ent. soc. lond. 126: 207-238. loan c.c., 1980 plant bug hosts (hymenoptera: miridae) of some euphorine parasites (hymenoptera: braconidae) near belleville, ontario, canada. naturaliste canadien. 107: 87-93. loan c.c., 1983 host and generic relations of the euphorini (hymenoptera: braconidae). contrib. am. entomol. inst. 20: 388-397. maetô k., 1988 systematic studies on the tribe meteorini (hymenoptera: braconidae) from japan. iii. the hirsutipes group of the genus meteorus haliday. kontyu 56: 321-329. maetô k., 1990 phylogenetic relationship and host associations of the subfamily meteorinae cresson (hymenoptera, braconidae). japan j. entomol. 58: 383-396. mirab-balou m., rasoulian g. r., khanjani m., sabahi q., 2008 study on taxonomy of phytophagous bugs of the family miridae and introducing insects natural enemies of the alfalfa tarnished plant bug in hamedan alfalfa farms (west of iran). pak. entomol. 30: 55-60. papp j., 1994 the dispersion of braconid wasps in an oak forest of hungary (hymenoptera: braconidae). folia ent. hung. 55: 305-320. phillips c.b., baird d.b., 2001 geographic variation in egg load of microctonus hyperodae loan (hymenoptera: braconidae) and its implications for biological control success. biocontrol sci. techn. 11: 371-380. shaw s.r., 1985 a phylogenetic study of the subfamilies meteorinae and euphorinae (hymenoptera: braconidae). – entomography. 3: 277-370. shaw s.r., 1996 plynops, a peculiar new genus and ten new species in the tribe euphorini (hymenoptera: braconidae: euphorinae). j. hymenopt. res. 5: 166-183. shaw m.r., huddleston t., 1991 classification and biology of braconid wasps (hymenoptera: braconidae). british museum of natural history, london: 126. shaw s.r., marsh p.m., 2000 revision of the enigmatic genus marshiella shaw in the new world with the description of three new species (hymenoptera: braconidae: euphorinae). j. hymenopt. res. 9: 277-287. stigenberg j., ronquist f., 2011 revision of the west palaearctic meteorini (hymenoptera, braconidae), with a molecular characterization of hidden fennoscandian species diversity. – zootaxa. 3084: 1-95. tobias v.i., 1995 subfamily euphorinae. in: medvedev g.s. (ed.), keys to the insects of the european part of the ussr, vol. iii, article jear_2013_2:hrev_master 16/09/13 13.56 pagina 50 no nco mm er cia l (hymenoptera: braconidae) from japan. iii. the hirsutipes group no nco mm er cia l (hymenoptera: braconidae) from japan. iii. the hirsutipes groupof the genus no nco mm er cia l of the genus no nco mm er cia l coulson j.r., 1987 udies on the biological control of plant bugs no nco mm er cia l coulson j.r., 1987 udies on the biological control of plant bugs (heteroptera: miridae): an introduction and history, 1961-1983. in: no nco mm er cia l (heteroptera: miridae): an introduction and history, 1961-1983. in: hedlund r. and graham h.m (eds.), economic importance and no nco mm er cia l hedlund r. and graham h.m (eds.), economic importance and in north america; arsno nco mm er cia l in north america; ars64. usda-ars natl. techn. informat. serv., springfield, va, usa: no nco mm er cia l 64. usda-ars natl. techn. informat. serv., springfield, va, usa: coulson j.r., 1994 releases of beneficial organisms in the united no nco mm er cia l coulson j.r., 1994 releases of beneficial organisms in the united states and rerritories-1983; ars-131. agricultural research no nco mm er cia l states and rerritories-1983; ars-131. agricultural research service. us dept. of agriculture, washington, d.c.: 113 pp. no nco mm er cia l service. us dept. of agriculture, washington, d.c.: 113 pp. fallahzadeh m., saghaei n., 2010 checklist of braconidae no nco mm er cia l fallahzadeh m., saghaei n., 2010 checklist of braconidae (insecta: hymenoptera) from iran. mun. ent. zool. 5: 170-186. no nco mm er cia l (insecta: hymenoptera) from iran. mun. ent. zool. 5: 170-186. farahani s., talebi a.a., van achterberg c., rakhshani e., 2012no nco mm er cia l farahani s., talebi a.a., van achterberg c., rakhshani e., 2012 , a genus of euphorinae (hymenoptera: braconidae) newno nco mm er cia l , a genus of euphorinae (hymenoptera: braconidae) new maetô k., 1990 phylogenetic relationship and host associations of no nco mm er cia l maetô k., 1990 phylogenetic relationship and host associations of the subfamily meteorinae cresson (hymenoptera, braconidae). no nco mm er cia l the subfamily meteorinae cresson (hymenoptera, braconidae). japan j. entomol. 58: 383-396. no nco mm er cia l japan j. entomol. 58: 383-396. mirab-balou m., rasoulian g. r., khanjani m., sabahi q., 2008 no nco mm er cia l mirab-balou m., rasoulian g. r., khanjani m., sabahi q., 2008 us e ontario, canada. naturaliste canadien. 107: 87-93. us e ontario, canada. naturaliste canadien. 107: 87-93.loan c.c., 1983 host and generic relations of the euphorini us e loan c.c., 1983 host and generic relations of the euphorini(hymenoptera: braconidae). contrib. am. entomol. inst. 20: 388-397. us e (hymenoptera: braconidae). contrib. am. entomol. inst. 20: 388-397. maetô k., 1988 systematic studies on the tribe meteorinius e maetô k., 1988 systematic studies on the tribe meteorini (hymenoptera: braconidae) from japan. iii. the hirsutipes groupus e (hymenoptera: braconidae) from japan. iii. the hirsutipes group on ly ichneumonoidea) of iran. biharean biol. 5: 147-150. on ly ichneumonoidea) of iran. biharean biol. 5: 147-150. loan c.c., 1974 the european species of on ly loan c.c., 1974 the european species of peristenus foerster (hymenoptera: braconidae, euphorinae). on lyperistenus foerster (hymenoptera: braconidae, euphorinae). -trans. r. ent. soc. lond. 126: 207-238. on lytrans. r. ent. soc. lond. 126: 207-238. on ly loan c.c., 1980 plant bug hosts (hymenoptera: miridae) of someon ly loan c.c., 1980 plant bug hosts (hymenoptera: miridae) of some euphorine parasites (hymenoptera: braconidae) near belleville,on ly euphorine parasites (hymenoptera: braconidae) near belleville, ontario, canada. naturaliste canadien. 107: 87-93.on ly ontario, canada. naturaliste canadien. 107: 87-93. hymenoptera part iv. amerind publishing co., new delhi: 883 pp. van achterberg c., 1993 illustrated key to the subfamilies of the braconidae. 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[journal of entomological and acarological research 2013; 45:e9] [page 51] article jear_2013_2:hrev_master 16/09/13 13.56 pagina 51 no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract ocimum sanctum was tested for its larvicidal and water sedimentation properties; the fruit ethanol and methanol extracts of phyllanthus emblica were tested for phytochemical, larvicidal, oviposition-deterrent and ovicidal activities. results emphasized that plant extracts have high toxicity against the egg and larvae of the malarial vector anopheles stephensi and also have water sedimentation properties. lc50 of phyllanthus emblica against anopheles stephensi larvae ranged from 33.08 ppm to 81.26 ppm and from 23.44 to 54.19 ppm for ethanol and methanol extracts, respectively. phyllanthus emblica also showed excellent ovipositional deterrent and ovicidal activities. the oviposition activity index value of ethanol and methanol extracts of phyllanthus emblica at 500 ppm were -0.80 and -0.92, respectively. ocimum sanctum includes both insecticidal secondary compounds, amino acids (glycine, lysine), vitamin c and other substances, that make treated water suitable for human consumption. water quality parameters such as color, turbidity and ph were analyzed in the water samples (pre-treatment and post-treatment of plant extracts) taken from the breeding sites of mosquitoes. hence, the plant product can be used as both mosquitocidal and water purifier. introduction mosquitoes are the most single group of insects in terms of public health significance and transmitting dreaded diseases like malaria, filariasis and dengue, etc. there are approximately 460 recognized species. while over 100 can transmit human malaria, only 30-40 commonly transmit parasites of the genus plasmodium that causes malaria which affects humans in endemic areas. the known vectors of anopheles species, which are common in india, include a. stephensi, a. culicifacies, a. fluviattis, a. minimmus, a. sudanicus and a. philippinensis. anopheles stephensi liston, 1901 (diptera: culicidae) is the most common human-biting malaria vector in india and many west asian countries, and is likely responsible for 40-50% of the annual malarial incidence (curtis, 1994). the problems of high cost, environmental risks and development of resistance in many vector mosquito species to several synthetic insecticides have revived interest in exploiting the pest control potential of plants (grainage & ahamed, 1988). conventional water treatment relies on the addition of chemicals such as alum (aluminum sulfate) as coagulants and the addition of chlorine as a bactericide. the availability of these chemicals, which depends on foreign exchange, is unreliable and unpredictable. because of economic and political constraints, the universal provision of piped water is not currently feasible, leaving millions of people without access to safe drinking water (who, 2005). this led us to look for plants with water purification properties. ocimum sanctum (holi basil), also called tulsi in india, is ubiquitous in indian tradition. tulsi is described as a sacred medicinal plant in ancient literature (kirtikar & basu, 1975). it has been used to treat malarial fevers, ringworms, and other cutaneous afflictions (butani, 1982). a variety of biologically active compounds have been isolated from the leaves, including ursolic acid, apigenin and luteolin. some other main chemical constituents of tulsi are oleanolic acid, rosmarinic acid, eugenol, carvacrol, linalool, and β-caryophyllene (merrily & winston, 2007). this paper, therefore, describes the mosquitocidal and water sedimentation properties of ocimum sanctum against malarial vector, anopheles stephensi. the plant genus phyllanthus l. (euphorbiaceae) is widely distributed in most tropical and subtropical countries. it is a very large genus consisting of approximately 550 to 750 species and is subdivided into ten or eleven subgenera. phyllanthus emblica l. has been used for the journal of entomological and acarological research 2012; volume 44:e17 [page 90] [journal of entomological and acarological research 2012; 44:e17] mosquitocidal and water purification properties of ocimum sanctum and phyllanthus emblica k. murugan,1 p. madhiyazhagan,1 a. nareshkumar,1 t. nataraj,1 d. dinesh,1 j.s. hwang,2 m. nicoletti3 1division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore, india; 2institute of marine biology, national taiwan ocean university, keelung, taiwan; 3department of environmental biology, università “la sapienza”, rome, italy correspondence: kadarkarai murugan, division of entomology, department of zoology, school of life sciences, bharathiar university, coimbatore, india. e-mail: kmvvkg@gmail.com key words: anopheles stephensi, ocimum sanctum, phyllanthus emblica, ovipositional deterrent, ovicidal, mosquitocidal, water purification. acknowledgements: the authors thank the council of scientific and industrial research csir (sanction letter no. 37(1386) emrii dated 02-062010), government of india, new delhi, for providing financial assistance. received for publication: 20 august 2012. revision received: 28 november 2012. accepted for publication: 28 november 2012. ©copyright k. murugan et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e17 doi:10.4081/jear.2012.e17 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly anti-inflammatory and anti-pyretic treatments by the rural population and for the treatment of several disorders such as the scurvy, cancer and heart diseases. the important constituent of plant leaves has antineutrophilic activity and anti-platelet properties in vitro. the extracts also have several pharmacological properties, such as anti-viral (hiv, aids, herpes virus, cytomegalovirus) antimutagenic, antiallergic, antibacterial activities (khopde et al., 2000). phyllanthus emblica l. contains a different class of secondary metabolites (calixto et al., 1998). the present study aimed to evaluate the effect of mosquitocidal and water sedimentation properties of ocimum sanctum on the malaria vector anopheles stephensi and screen phytochemicals from phyllanthus emblica l. for larvicidal, pupicidal, oviposition deterrent and ovicidal effect against the same malaria vector. materials and methods colonization, mosquito rearing and maintenance the eggs of anopheles stephensi liston, 1911 were collected from various water sources (e.g., overhead tanks) in coimbatore district, tamil nadu, india. the eggs were brought to the laboratory and were transferred to 18×13×4 cm size enamel trays containing 500 ml of water until they hatched. after hatching, the larvae were provided with dog biscuits and yeast at a 3:1 ratio and maintained at 27+2°c, 75-85% rh, under 14 h light (l): 10 h dark (d) photoperiod cycles. pupae were collected and transferred to plastic jars (12×12 cm) containing 500 ml of water, which were placed in 90×90×90 cm screened mosquito cage for adult emergence. the freshly emerged adults were maintained at 27±2°c, 75-85% rh, under 14 h l: 10 h d photoperiod cycles. the adults were provided a 20% sugar solution ad libitum, and provided with rabbits (each exposed on the dorsal side) for 30 min two times per week (hardstone et al., 2009). the males were provided with a 10% glucose solution on cotton wicks that was changed daily. blood-fed females were provided filter paper-lined cups containing water for oviposition. collection of plant material and preparation of extracts the leaves of ocimum sanctum and phyllanthus emblica fruits were collected from in and around bharathiar university campus, coimbatore, tamil nadu, india. a total of 250 g of fresh, mature leaves was rinsed with distilled water and dried in a shade. the dried leaves were put in a soxhlet apparatus (borosil glass workers ltd., worli, mumbai, india), and extracts were prepared using 100% ethanol (loba chemie pvt. ltd., mumbai, india; 99 % purity) for 72 h at 30-40°c. dried residues were obtained from 100 g of extract evaporated to dryness in a rotary vacuum evaporator. two grams of the residues were dissolved in 100 ml of ethanol (2% stock solution), from which the following concentrations were prepared: 0.5%, 0.1%, 2.0%, 4.0%, and 8.0%, using distilled water. amla fruits were cut into small pieces and ground into a uniform powder using a blender. the methanolic extract of amla was prepared by soaking 100 g of dried powdered samples in 250 ml of methanol for 12 h. the extracts were filtered by using whatman n. 1 filter paper. the filtrate was used for phytochemical screening. the preliminary qualitative phytochemical studies were performed for testing the different chemical groups present in fruit methanol extract of phyllanthus emblica (trease et al., 1978; kokate et al., 1990). laboratory plant extract toxicity tests f1-f2 larvae/pupae from the wild adult collection were used to evaluate larvicidal activity. twenty-five larvae (in stages i to iv and pupae) were placed into a 500-ml glass beaker containing 249 ml of dechlorinated water and 1 ml of desired concentrations of plant extract. test larvae and pupae were provided with food as previously described. at each tested concentration, 2-5 trials of 3 replicates were conducted concurrently. control groups of larvae exposed to ethanol served as control. mortality was corrected using abbott’s formula (abbott, 1925). ovicidal activity assay for ovicidal activity, the freshly laid eggs were collected by providing ovitraps in mosquito cages. ovitraps were kept in the cages two days after the female mosquitoes had been given a blood meal. the eggs were laid on the filter paper lining provided in the ovitrap. after scoring, 100 gravids were placed in a screen cage where ten oviposition cups were introduced for oviposition 30 min before the start of the dusk period. of these ten cups, eight were each filled with test solution of 150, 200, 250, 300, 350, 400, 450, 500 ppm, and one was filled with 100 ml of respective solvent containing water and polysorbate 80 that served as a control. a minimum of 100 eggs were used for each treatment, and the experiment was replicated 5 times. after treatment, the eggs were sieved through muslin cloth, thoroughly rinsed with tap water, and left in plastic cubs filled with dechlorinated water for hatching assessment after counting the eggs under the microscope (su & mulla, 1998). the percent egg mortality was calculated on the basis of non-hatchability of eggs with unopened opercula (chenniappan & kadarkarai, 2008). the hatching rate of eggs was assessed after 98 h post-treatment according to the method of rajkumar and jebanesan (2009). oviposition deterrence activity to study the ovipositional deterrence effect and the number of eggs deposited in the presence of extracts with different solvents of experimental plants, a multiple concentration test was carried out. for bioassay test, 20 males and 20 females were separated in the pupal stage (by size of the pupae) and were introduced into screen cages (45×45×40 cm) in a room at 27±2°c and 75-85% relative humidity (rh) with a photoperiod of 14:10 h light and dark cycles. the pupae were allowed to emerge to adults in the test cages. adults were provided continuously with 10% sucrose solution in a plastic cup with a cotton wick. they were blood fed (from pigeon) on day 5 after emergence. in the multiple concentration test, five cups, each containing 100 ml distilled water with a 9-cm piece of white filter paper for oviposition, as well as solvent extracts at concentrations of 100, 200, 300, 400 and 500 ppm, were placed in each cage. a sixth cup without extract served as a control. the positions of the plastic cups were alternated between the different replicates so as to nullify any effect of position on oviposition. five replicates for each concentration were run with cages placed side by side for each bioassay. after 24 h, the number of eggs laid in the treated and control cups were counted under a stereomicroscope. the percent effective repellency for each concentration was calculated using the following formula: where er is effective repellency, nc is number of eggs in control, and nt is number of eggs in treatment (rajkumar & jebanesan, 2009). the oviposition experiments were expressed as mean number of eggs and oviposition activity index, which was calculated using the following formula: article [journal of entomological and acarological research 2012; 44:e17] [page 91] no nco mm er cia l u se on ly where nt is total number of eggs in the test solution and ns is total number of eggs in the control solution. oviposition active index of +0.3 and above are considered as attractants while those with −0.3 and below are considered as repellents (kramer & mulla, 1979). positive values indicate that more eggs were deposited in the test cups than in the control cups and that the test solutions were attractive. conversely, negative values indicate that more eggs were deposited in the control cups than in the test cups and that the test solutions were a deterrent. field trials of plant extract larval toxicity plant extracts formulations (51 g*l-1) were applied to the water surface with a knapsack sprayer (ignition products ltd., india, 2008). pretreatment and post-treatment at 24, 48 and 72 h was conducted using a larval dipper. larvicidal efficacy was carried out against late third and early fourth-instar larvae. larvae were identified and counted to determine the relative species composition of each test site. six trials were conducted for each test site (standing water bodies) with similar weather conditions (27°c; 79% rh). the required quantity of plant extract was determined by calculating the total surface area and volume (0.25 m2 and 250 l). the required concentration was prepared using 10 times the observed laboratory lc50 values (murugan et al., 2003). percentage reduction of the larval density was calculated using the formula: where c is the total number of mosquitoes in control and t is the total number of mosquitoes in treatment. water quality parameters water quality parameters such as ph, color and turbidity were investigated using the methods of clescerl et al. (2005). to prepare the coagulants and treatment (schwarz, 2000), leaves of ocimum sanctum were shade-dried, powdered using an electric blender, and mixed with a small amount of clean water to form a paste. the paste was diluted to the required strength based on raw water turbidity. total suspended solids in raw water separated as over 50, between 50 and 150, and over 150 mg*l-1, and the final concentration used for treatment was 50, 30100 and over 150 mg*l1, respectively (schwarz, 2000). after filtering insoluble material with a fine-mesh screen or muslin cloth, the coagulant was added and stirred fast for 30 s. the treated water was then covered for 1 h without disturbance. statistical analysis the spss (version 9.0) software package was used to analyze data obtained from the bioassay. lethal concentrations (lc), lc50 and lc90, duncan multiple range test) and c2 tests were used. results ocimum sanctum leaf extract was used to test water purification properties including water color, total suspended solids and ph, and was effective in sedimentation and purification. before treatment, water color was 31 hu while after this was 12 hu. total suspended solids before treatment was 40.0 mg*l-1 and this was reduced 30.0 mg*l-1, after. similarly, ph level was 8 before treatment and 6.8 after. significant mortality was evident after the treatment of ocimum sanctum leaf extract (osle) at different concentrations against a. stephensi in laboratory (table 1). after the treatment of osle at different concentration levels (0.5-8%), 38% mortality was noted at i instar larvae by the treatment of osle at 0.5%, whereas this increased to 90% at 8% of osle treatment. mortality increased with increasing concentration. lethal concentrations (lc50 and lc90) were also calculated. the lc50 and lc90 values are represented as follows: lc50 value of i instar was 1.52%, ii instar was 2.22%, iii instar was 3.11%, iv instar was 5.13% and pupae was 6.45%. lc90 value of i instar was 7.39%, ii instar was 8.68%, iii instar was 10.07%, iv instar was 14.11% and pupae was 15.24%, respectively. an. stephensi larvae were collected exclusively in overhead water tanks and mean larval count was calculated. breeding sites treated with o. sanctum extracts, showed a reduction in an. stephensi larvae. the field experiment was carried out at drinking water tanks (0.5×0.5×1.0) with anopheles stephensi larvae and the percentage reduction/mortality was 82.5%, 87.9% and 92.4% after the 24 h, 48 h and 72 h, respectively (table 2). table 3 shows qualitative analyses of the fruit extract of phyllanthus emblica, emphasizing the presence of proteins, tannins and terpenoids. steroids were absent in phyllanthus emblica. the larvicidal and pupicidal activities of the ethanol extract of phyllanthus emblica against anopheles stephensi larvae under laboratotable 1. larvicidal activity of ocimum sanctum against malaria vector, anopheles stephensi. larval larval mortality (%)±sd lc50 95% confidence limit chi-square instars concentration of ocimum sanctum (%) (lc90) lcl ucl value and pupa 0.5 1.0 2.0 4.0 8.0 lc50 (lc90) lc50 (lc90) i 38±0.6a 43±0.3ab 58±1.2b 75±0.5c 90±0.8d 1.52067 0.85569 6.34024 (7.38616) (2.07067) (8.99671) 2.830* ii 32±0.9a 41±0.5b 50±1.0c 69±0.7d 85±1.1e 2.21933 1.57467 7.42155 (8.68104) (2.80353) (10.65323) 2.769* iii 26±1.0a 35±1.2b 46±1.4c 61±0.8d 79±1.5e 3.10581 2.47306 8.56234 (10.06631) (3.76193) 12.46361 3.384* iv 19±1.5a 25±0.8ab 39±0.6b 52±1.2c 61±0.4d 5.12732 2.94904 9.02274 (14.11467) (13.82370) (56.49114) 8.278* pupa 10±0.5a 21±1.0b 32±1.2c 46±0.8d 53±0.6e 6.44729 3.67191 9.00805 (15.23744) 570.79738 (2199.74679) 13.287* within a column means followed by the same letter(s) are not significantly different at 5% level by duncan multiple range test; *significant at p<0.001 (heterogeneity factor used in calculation of confidence limits). sd, standard deviation; lc50, lc90, lethal concentration; lcl, lower confidence limits; ucl, upper confidence limits. article [page 92] [journal of entomological and acarological research 2012; 44:e17] no nco mm er cia l u se on ly ry conditions are given in table 4. percentage mortality was 43% at 20 ppm concentration and increased to 82% at 100 ppm concentration against the first instar larvae. median lethal concentrations (33.08, 48.85, 68.28, 81.26 and 86.24 ppm) for larvae and pupae were low and significant. table 5 shows larvicidal and pupicidal activities of methanol extract of phyllanthus emblica against the malarial vector, anopheles stephensi. among the four larval stages, i instar larvae were more susceptible than the other instars. the fruit extracts also showed considerable pupal mortality. the lowest mortality observed was 20% at 20 ppm against pupae and the highest was 98% at 100 ppm against the i instar larvae. lc50 (23.44, 33.10, 42.13, 54.19 and 64.28 ppm for four instar larvae and pupae, respectively) observed for larvae and pupae were very low when compared to the ethanol extract. in the oviposition deterrent assay, gravid anopheles stephensi preferred to lay eggs in the distilled water control cups than in the cups treated with solvent extracts of phyllanthus emblica (table 6). there was also a marked difference in the number of eggs laid. observed results showed that the 500 ppm treated cups received a mean number of 49±1.15 and 19±1.53 eggs per cup in fruit ethanol and methanol extracts of phyllanthus emblica treatment while the control cups received a mean number of 456±1.50 and 470±1.30 eggs per cup. the present results indicated that the oviposition deterrence was concentration dependent, as 500 ppm of ethanol and methanol fruit extracts of experimental plant exhibits strong deterrent effect when compared with 100 ppm against oviposition. the solvent leaf extracts strongly deterred oviposition by gravid anopheles stephensi, with a significantly lower proportion of eggs being laid on ovitraps containing extracts in comparison with control solutions (p<0.05). the maximum percentage of effective repellency against oviposition was 96.93%, reported in 500 ppm followed by 95.95, 94.13, 87.01, 78.18 and 67.50% at 500, 400, 300, 200 and 100 ppm methanol extracts of phyllanthus emblica, respectively. the percentages of egg hatchability of anopheles stephensi with the fruit ethanol and methanol extracts of phyllanthus emblica are presented in table 7. the ethanol and methanol extracts of phyllanthus emblica exerted 100% mortality (no hatchability) at 400 ppm and above. very low hatchability (19±1.20% and 0%) was observed at a 350 ppm concentration of ethanol and methanol extracts of phyllanthus emblica, respectively against anopheles stephensi. almost 100% hatchability was obtained in the control. in the case of ovicidal activity, exposure to freshly laid eggs was more effective than to the older eggs. discussion and conclusions many approaches have been developed to control the mosquito menace. one such approach to prevent mosquito-borne disease is by killing mosquito at the larval stage. the current mosquito control approach is based on synthetic insecticides. even though they are effective, they created many problems, such as insecticide resistance (liu et al., 2005), pollution, and toxic side effects on humans (lixin, 2006). in the present study, osle showed higher larvicidal activities against mosquito probably due to the presence of active compounds such as eugenol and (e)-6-hydroxy-4,6-dimethyl-3-heptene-2-one (kelm & nair, 1998). the mosquito larvicidal property of leaf and flower extracts of ocimum sanctum l. against aedes aegypti and culex quinquefasciatus larvae has been previously reported (anees, 2008). mosquito breeding habitats vary from ponds, marshes, ditches, pools, drains, water containers and other similar water collections, and are often species-specific (rozendaal, 1997). the increase in the mosquito vector population and the incidence of mosquito-borne diseases (e.g., malaria, dengue, and chikungunya) is rising in india as a result of inadequate water supply systems and contamination. storage of water, often from untreated water sources, and polluted water systems serve breeding sites of mosquitoes that transmit mosquito-borne pathogens, in addition to waterborne pathogens (e.g., cholera, dysentery and typhoid) (vinod, 2011). in the present study, the treatment with coagulants at the breeding sites of mosquito had not only killed mosquito larvae but it had also water purifying properties. biopesticide spray operations had been performed in the past (murugan, 2006) for the control of vectors in the tsunami affected areas of india and the plant products such as neem and other herbal combinations showed biopesticidal potency killing mosquito larvae in the contaminated water. larvicidal effect of neem (azadirachta indica) oil cake was studied against mosquitoes. the oil cake showed good larvicidal activity against the mosquito species tested (shanmugasundaram et al., 2008). neem is derived from the neem tree azadirachta indica a. juss. (meliaceae), and its primary insecticidal components are the tetranortriterpenoid, azadirachtin and other limonoids. the effect of neem limonoids azadirachtin, salannin, deacetylgedunin, gedunin, 17-hydroxyazadiradione and deacetylnimbin on insects was investigated by senthil nathan et al. (2006). coagulants of ocimum sanctum were tested for the purifying properties at the laboratory by adding coagulants with waters from mosquito breeding sites and coagulants also had water purifying properties; treated water showed cleaning efficacy. plant product nutrients (vitamin a and c, calcium, iron and zinc) and allelochemicals (tannins, alkaloids, glycosides and saponins) not only removed solid contamitable 2. effect of ocimum sanctum treatments of drinkng water tanks malarial vector, anopheles stephensi. site no. larval density (%) before treatment after treatment 24 h 48 h 72 h 1 79 19 15 11 2 71 14 12 8 3 65 10 8 5 4 57 7 6 3 5 47 7 2 0 6 39 4 0 0 total 358 61 43 27 average 59.6 10.1 7.16 4.50 % reduction 82.50 87.9 92.4 -place: vadavalli; -habitat: drinking water; -size: 0.5×0.5×1.0; -depth: 1 cm; -species: anopheles stephensi; -stage: larvae stage; -calculation: 2.5×1.5 m; 3.10×1=31.00. table 3. phytochemical constituents present in phyllanthus emblica. phyto-constituents ethanol extract methanol extract flavonoidds + + tannins + + carbohydrates + + alkaloids + + proteins + + steroids – – terpenoids + + article [journal of entomological and acarological research 2012; 44:e17] [page 93] no nco mm er cia l u se on ly table 4. toxicity evaluation of ethanol extract of phyllanthus emblica against the malarial vector anopheles stephensi. larval % of larval mortality lc50 regression 95% confidence chi-square stages concentration (ppm) (lc90) equation limit value 20 40 60 80 100 lcl ucl lc50 (lc90) lc50 (lc90) i 43±1.32a 52±1.80b 68±2.59c 71±0.5cd 82±1.80d 33.08 y=-0.44449 18.26 42.76 1.125 (128.48) +0.01343x (110.01) (162.10) ii 36±0.70a 43±1.22b 59±1.14c 62±1.41cd 78±0.79d 48.85 y=-0.66503 38.55 57.18 1.793 (142.98) +0.01361x (122.19) (180.48) iii 20±0.65a 37±0.79b 48±1.58c 52±1.51cd 70±0.79d 68.28 y=-1.04788 60.85 76.82 2.670 (151.79) +0.01535x (131.41) (186.42) iv 16±0.35a 23±0.79b 33±1.11c 44±1.81d 68±1.06e 81.26 y=-1.44506 74.16 90.58 2.728 (153.33) +0.01778x (134.81) (183.07) pupa 12±1.59a 21±0.65b 31±1.19c 40±1.98d 64±1.06e 86.24 y=-1.57407 78.85 96.30 2.032 (156.45) +0.01825x (137.64) (186.67) within a column means followed by the same letter(s) are not significantly different at 5% level by duncan multiple range test. lc50, lc90, lethal concentration; lcl, lower confidence limits; ucl, upper confidence limits. table 5. toxicity evaluation of methanol extract of phyllanthus emblica against the malarial vector anopheles stephensi. larval % of larval mortality lc50 regression 95% confidence chi-square stages concentration (ppm) (lc90) equation limit value 20 40 60 80 100 lcl ucl lc50 (lc90) lc50 (lc90) i 49±0.70a 65±0.79b 75±0.35c 87±0.79d 98±1.27e 23.44 y=-0.51187 12.98 30.87 3.173 (82.15) +0.02183x (74.12) (93.77) ii 40±0.54a 58±0.79b 69±1.06c 82±1.08d 97±1.29e 33.10 y=-0.74820 25.11 39.27 4.043 (89.81) +0.02260x (81.53) (101.65) iii 38±0.35a 44±0.70ab 61±1.22b 76±1.41c 90±1.76d 42.13 y=-0.82596 34.42 48.39 2.881 (107.52) +0.01960x (96.59) (123.90) iv 29±0.74a 37±0.65b 53±1.51c 62±1.29d 88±1.38e 54.19 y=-1.06730 36.01 69.28 6.398 (119.26) +0.01969x (95.48) (187.81) pupa 20±0.35a 29±0.70b 48±0.93c 52±1.47cd 85±1.88d 64.28 y=-1.36664 46.70 85.52 9.100 (124.56) +0.02126x (97.90) (216.27) within a column means followed by the same letter(s) are not significantly different at 5% level by duncan multiple range test. lc50, lc90, lethal concentration; lcl, lower confidence limits; ucl, upper confidence limits. table 6. oviposition deterrence activity of ethanol and methanol extracts of phyllanthus emblica against the malarial vector anopheles stephensi. concentration ethanol methanol (ppm) number of eggs±s.e. number of eggs±s.e. treatment control er% oai treatment control er% oai 500 49±1.15a 456±1.50 89.25 -0.80 19±1.53a 470±1.30 95.95 -0.92 400 54±1.41b 389±1.71 86.11 -0.75 23±1.22ab 392±1.84 94.13 -0.88 300 62±1.72c 321±1.20 80.68 -0.67 37±1.84b 285±1.61 87.01 -0.77 200 86±1.01d 266±1.41 67.66 -0.51 48±1.73c 220±1.42 78.18 -0.64 100 98±1.52e 210±1.47 53.33 -0.36 52±1.32cd 160±1.32 67.50 -0.50 within a column means followed by the same letter(s) are not significantly different at 5% level by duncan multiple range test. s.e., standard error; er, effective repellency; oai, oviposition active index. table 7. ovicidal activity of ethanol and methanol extracts of phyllanthus emblica against eggs of anopheles stephensi. treatment extract percentage of egg hatchability±s.d. concentration of extract (ppm) 150 200 250 300 350 400 450 500 control phyllanthus emblica ethanol 96±1.24e 71±2.43d 54±1.40c 30±2.49b 19±1.20a nh nh nh 100±0.00 methanol 74±2.17d 47±1.71c 22±2.10b 14±1.03a nh nh nh nh 98±2.95 within a column means followed by the same letter(s) are not significantly different at 5% level by duncan multiple range test. s.d., standard deviation; nh, no hatchability (100% mortality). article [page 94] [journal of entomological and acarological research 2012; 44:e17] no nco mm er cia l u se on ly nants, but also greatly reduced amounts of harmful bacteria in the waste water. earlier studies examined the antimicrobial property present in the ocimum sanctum, and concluded that the component responsible was likely to be eugenol. this component has been demonstrated to have both antibacterial (nakaruma et al., 1999) and antihelmintic activities (pessoa et al., 2002). nareshkumar et al. (2011) reported mosquitocidal and water purifying properties of different plant extracts (cynodon dactylon, aloe vera, hemidesmus indicus and coleus amboinicus) on various mosquito vectors (anopheles stephensi, culex quinquefasciatus and aedes aegypti) at different water samples. in the present study, we also sought to determine whether methanol and ethanol extracts from phyllanthus emblica fruits could be used for mosquito control. we observed a functional response by all immature life stages of a. stephensi to ethanolic and methanolic extracts of phyllanthus emblica fruits. this biological activity is attributed to the compounds present in fruits, including flavonoids, phenols, and steroids that together or independently result in morbidity and mortality in a. stephensi. park et al. (2000) showed that the biological activity of the plant extracts might be due to the various compounds, including phenolics, terpenoids and alkaloids existing in plants. these compounds may jointly or independently contribute to produce larvicidal activity against mosquitoes. a piperidine alkaloid from piper longum fruit was found to be active against mosquito larvae of cx. pipiens (lee, 2000). it was recognized that the fourth larval stage of mosquitoes was more tolerant to toxicant then early instars (mulla, 1961; rettich, 1976; nareshkumar et al., 2012). larval mortality may be due to the effect of chemicals like flavonoids, alkaloids, and terpenoids. the higher mortality of mosquito larvae was due to the combined action of plant compounds that might be acting on the midgut epithelium cells exerting their toxic effects on mosquito. lethal lc50 observed in the present study is very low when compared to the earlier studies. the mangrove plant rhizophora mucronata bark and pith extract showed toxicity with lc50 values of 157.4 and 168.3 ppm, respectively, against ae. aegypti larvae (kabaru & gichia, 2001). exposure of a. stephensi larvae to sub-lethal doses of neem extracts in the laboratory, prolonged larval development, reduced pupal weight, caused high oviposition deterrence and high mortality (wandscheer et al., 2004). phyllanthus emblica l. was also effective in oviposition deterrence and ovicidal activities. adult female a. stephensi avoided oviposition in phyllanthus emblica-treated water, though some laid eggs, but these hatched in abnormal larvae. the ethanolic leaf extracts of cassia obtusifolia at high concentration (400 mg*l-1) were responsible for 92.5% oviposition deterrence effect, while 300, 200 and 100 mg*l-1 were responsible for 87.2%, 83.0%, and 75.5% deterrence effect, respectively (rajkumar & jebanesan, 2009). the leaf extract of solanum trilobatum reduced egg laying by gravid females of anopheles stephensi from 18% to 99% compared with ethanol-treated controls at 0.01, 0.025, 0.05, 0.075, and 0.1% (rajkumar & jebanesan, 2005). methanol extract of phyllanthus emblica showed more deterrence and egg mortality than the ethanol extract. the crude acetone extract of cuscuta hyaline was an effective oviposition deterrent against culex quinquefasciatus at a concentration of 80 ppm (mehra & hiradhar, 2002). it has been shown that the age of the embryos at the time of treatment plays a crucial role with regard to the effectiveness of the chitin synthesis inhibitor dimilin to culex quinquefasciatus (miura et al., 1976). the ovicidal effect of solenostemma argel was low; however, concentrations of 0.05 % and 0.1 % exhibited significant effects (p<0.05), producing 65-75% and 62.962.9%, respectively, on the first and second day after treatment, the 0.1% concentration reduced egg hatch by 33.7%, compared with control, and 100% mortality values were evident in concentrations as low as 0.025% at two days post-hatching against culex pipiens (al-doghairi et al., 2004). the seed extract of atriplex canescens showed complete ovicidal at 1000 ppm concentration in eggs of culex quinquefasciatus. the bioactive compound azadirachtin isolated from azadirachta indica showed complete ovicidal activity in eggs of culex tarsalis and culex quinquefasciatus exposed to 10 ppm concentration (murugan et al., 1996; ouda et al., 1998). the phytochemical analysis of phyllanthus emblica l. reveals the presence of alkaloids, tannins and saponins. these compounds are known to be biologically active. tannins have important roles such as stable and potent antioxidants (trease et al., 1978). herbs that have tannins as their main component are astringent in nature and are used for treating intestinal disorders such as diarrhea and dysentery (dharmananda, 2003). that of the largest group of chemicals produced by plants are the alkaloids and their amazing effect on humans has led to the development of powerful pain killer medications (raffauf, 1996). fruits are an important group of foodstuffs in the diet. these components of human diet are not adequately replaceable by any other products. plant tissues are naturally rich in nutritive or therapeutically active products of plant secondary metabolism. the consumption of fruits has been inversely associated with morbidity and mortality from degenerative diseases (aruoma, 1998; özen, 2010) and is associated with low incidences and mortality rates of cancer and heart disease (ames et al., 1993; dragsted et al., 1993). it is not known which dietary constituents are responsible for this association, but antioxidants appear to play a major role in the protective effects of plant foods (gey 1990; barberousse et al., 2008; patra & kumar, 2010). fruit contains considerable amounts of active components such as polyphenols, flavonoids, tannins, vitamins a, b, c and e, and carotenoids which are considered potent scavengers of free radicals and reactive oxygen species (rice-evans et al., 1995). the present results suggest that ocimum sanctum can be used for mosquitocidal and water purifying purposes for promoting water sustainability in developing countries. ocimum sanctum coagulum has an added advantage of having antimicrobial properties. considering the fact that ocimum sanctum coagulum can be locally produced, its use in water purification should be encouraged. this is likely to reduce the high cost of the current water treatment systems. experiments on phytochemical screening of phyllanthus emblica for their, larvicidal, pupicidal, ovipositiondeterrent and ovicidal activity have opened the possibility of further investigations on their efficiency, in view of the utilization of their higher biomass. however, the mechanism of action of the compounds from phyllanthus emblica is still not clear. studies on isolation, purification and the mechanism of action of individual compounds existing in the phyllanthus emblica are needed and these are in progress. 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c.a., miller n.j., bolwell p.g., bramley p.m., pridham j.b., 1995 the relative antioxidant activities of plant-derived polyphenolic flavonoids. free radical res. 22: 375-383. article [page 96] [journal of entomological and acarological research 2012; 44:e17] no nco mm er cia l u se on ly rozendaal j.a., 1997 mosquitoes and other biting diptera. in: vector control methods for use by individuals and communities. world health organization, geneva: 7-177. available from: http://www.who.int/whopes/resources/vector_rozendaal/en/ schwarz d., 2000 water clarification using moringa oleifera. technical information w1e, gate information service, eschborn, germany. available form: http://www.gtz.de/gate/gateid.afp accessed: 31st october 2007. senthil nathan s., kalaivani k., chung p.g., murugan k., 2006 effect of neem limonoids on lactate dehydrogenase (ldh) of the rice leaffolder, cnaphalocrocis medinalis (guene´e) (insecta: lepidoptera: pyralidae). chemosphere. 62: 1388-1393. shanmugasundaram r., jeyalakshmi t., dutt m.s., murthy p.b., 2008 larvicidal activity of neem and karanja oil cakes against mosquito vectors, culex quinquefasciatus (say), aedes aegypti (l.) and anopheles stephensi (l.). – j. environ. biol. 29: 43-5. su t., mulla m.s., 1998 ovicidal activity of neem products (azadirachtin) against culex tarsalis and culex quinquefasciatus (diptera: culicidae). j. am. mosq. cont. assoc. 14: 204-209. özen t., 2010 antioxidant activity of wild edible plants in the black sea region of turkey. grasas y aceites. 61, 86-94. trease g.e., evans w.c., 1978 a text book of pharmacognosy, 11th ed. bailliere tidall, london: 530. vinod s., 2011 deforestation and water pollution impact on mosquitoes related epidemic diseases in nanded region. – biosci. discov. 2: 309-316. wandscheer c.b., duque j.e., fukuyama y., wohlke j.l., fontana j.d., 2004 larvicidal action of ethanolic extracts from fruit endocarps of melia azedarach and azadirachta indica against the dengue mosquito aedes aegypti. toxicon. 44: 829-835. who (world health organization)/unicef (joint monitoring programme for water supply and sanitation), 2005 water for life: making it happen. world health organization, geneva. available from: http://www.who.int/water_sanitation_health/monitoring/jmp 2005/en article [journal of entomological and acarological research 2012; 44:e17] [page 97] no nco mm er cia l u se on ly jear2012 abstract cicadatra persica kirkaldy, 1909 (hemiptera: cicadidae) is regarded as a potential constraint to the productivity of apple fruit orchards in erneh (33°21’n, 35°52’e), near damascus, syria. however, no research has been conducted on this pest. this study examined adult emergence, egg laying, and hatching periods. adults emerged in early june, with an emergence peak in the fourth week of june 2011, and started laying eggs in mid-june. egg development was approximately 40 days, with the first eggs hatching on 1st august 2011 and the final hatch on 17th august 2011. the simple and relatively successful method of monitoring egg development reported here may be useful for studying the nymphal ecology and life cycle of this species. introduction cicadas are destructive pests that cause damage to some apple orchards in syria. the most obvious damage of cicadas is that caused by egg laying in small twigs. this damage causes twigs to split and die, causing a symptom called flagging. as juveniles, the larvae feed on the xylem fluid of woody plant on roots, using piercing and sucking mouthparts. the life cycles of most cicadas are long, usually involving many years spent underground as juveniles, followed by a brief adult life (26 weeks above ground) (boulard, 1990; young & bennet, 1995). fifth instars may occupy the feeding cell at the base of the emergence burrows for up to several weeks before an unknown stimulus triggers their final exit from the soil (beamer, 1982). after mating, females lay eggs in bark or twigs, the eggs hatch later in the season and the new nymphs burrow underground and begin feeding on roots. during their soil stages, the cicadas settle themselves anywhere from 50-600 mm below the surface (james et al., 1986; williams et al., 1993). in syria, the occurrence of cicadatra persica kirkaldy, 1909, was recorded and studied for the first time in apple fruit orchards during field work in erneh in june 2011 (dardar et al., 2012). c. persica kirkaldy 1909, was found for the first time in macedonia and its song was recorded and analyzed in 1998 (gogala & trilar, 1998). we report on biological studies conducted on c. persica, in apple fruit orchards in syria. materials and methods fieldwork was carried out in an apple orchard in erneh (33°21’n, 35°52’e), a village located in the alsheikh mountains in southwestern syria. the orchard was approximately 0.3 ha, and contained 128 apple trees (cultivar starking). trees were 2-2.5 m tall and were in the production stage. adult emergence adult emergence was monitored three times a week by visual observation from mid-may 2011. emergence traps were also used to determine the adult emergence period and its peak. each emergence trap was a circular tent of muslin (diameter 2 m) that was established around the trunk of the tree to a height of approximately 0.5 m (figure 1). historical damage from egg laying was unevenly distributed in the orchard; typical samples of apple trees were chosen to study adult emergence. the trees were divided into five categories based on the number of egg nests (table 1). one tree was chosen randomly from each category and an emergence trap was established. traps were checked and exuviae collected once a week from the beginning of june until the end of july. egg laying period the number of twigs on 45 apple trees damaged by newly laid egg nests were recorded every week from the first emergence of adults until late july. journal of entomological and acarological research 2012; volume 44:e12 [page 56] [journal of entomological and acarological research 2012; 44:e12] observations on some biological aspects of cicadatra persica (cicadidae: hemiptera) in apple fruit orchards in erneh, syria m.a. dardar,1 h.m.r. belal,2 a.m. basheer2 1department of insects research, administration of plant protection research, general commission for scientific agricultural research; 2department of plant protection, faculty of agriculture, university of damascus, syria correspondence: marah a. dardar, department of insects research, administration of plant protection, general commission for scientific agricultural research, syria. e-mail: marah.dardar@hotmail.com key words: cicadidae, emergence, egg nest, apple orchards, erneh. acknowledgements: this study was supported by the general commission for scientific agricultural research. we thank the grower ismael masoud in the village of erneh for permitting us to carry out this research in his orchard, and we also thank the local governing council of the village of erneh. received for publication: 4 october 2012. revision received: 5 november 2012. accepted for publication: 14 november 2012. ©copyright m.a. dardar et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e12 doi:10.4081/jear.2012.e12 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. no nco mm er cia l u se on ly egg development and hatching period traps (n=22) for newly hatched larvae were established on twigs with new egg nests. each trap was a sleeve of muslin with a small plastic container set below the damaged twig to catch hatching larvae (figure 2). the traps were checked twice a week for eight weeks beginning 4th-6th july results adult emergence the first adults were observed on 7th june 2011. adults were found in emergence traps from 14th june to 17th july, a period of five weeks. the peak of adult emergence was in the 4th week of june (23th-29th) (figure 3). egg laying period egg laying started on 14th june 2011, one week after adult emergence began. the peak of egg laying was from 25th june to 9th july with the last egg nest being found on 13th july. the egg laying period continued for one month from 14th june to 13th july (figure 4). egg development and hatching period egg hatch started on 1st august 2011, 40 days after oviposition, and continued for approximately three weeks, with a possible peak between 4th-7th august (figure 5). larvae (n=672) were captured in most traps (20 of 22). the two traps in which no larvae were recorded were established on twigs that were broken by the wind during the summer, and this stopped the flow of plant sap within these twigs, causing egg death. discussion studying the abundance of cicadas and their emergence period is important for determining the timing and the level of damage on apple trees in erneh in order to identify the most suitable date for carrying out pest control measures. adults emerged during a 5-week period from mid-june to latejuly. eggs of c. persica were laid from mid-june until mid-july, with a 2-week peak from the end of june to the beginning of july. this period is short compared with some other cicadas, such as kikihia ochrina, which lays eggs during a 5-month period (logan & connolly, 2005). eggs of c. persica on apple tree twigs were laid in june and july and hatched in august the same summer. in comparison, eggs of k. ochrina and other new zealand species probably overwinter and hatch the following summer, giving a period of egg development lasttable 1. distribution of egg nests per tree in the apple orchard. category no. nests/tree frequency 1 0 7 2 1-10 70 3 11-20 26 4 21-30 17 5 31-40 8 figure 1. emergence trap. figure 2. trap to check newly hatched larvae. figure 3. a) and b) emergence period of adults of cicadatra persica. article [journal of entomological and acarological research 2012; 44:e12] [page 57] a b no nco mm er cia l u se on ly ing 7-11 months (cumber 1952; harford 1958; logan, unpublished data). a long period of egg development has also been reported for cryptotympana facialis and graptopsaltria nigrofuscata, in whose eggs are laid in summer and first begin to hatch 10-12 months later in the following summer (moriyama & numata, 2008). methods for estimating the density of cicadas (cicada and magicicada spp.) including counts of larval skins (exuviae) and emergence traps (dybas & davis, 1962; wolda, 1989; dean & milton, 1991; milton & dean, 1992; white & sedcole, 1993; anderson, 1994) were used in this study in order to estimate the adult emergence phenomenon. these methods were time-consuming and labor-intensive, and in some cases have been recognized as poor or biased estimators of cicada densities (patterson et al., 1997). for these reasons, we suggest adult emergence and cicada numbers could be estimated according to sound levels (patterson et al., 1997) as a more precise measurement. the length of the life cycle of cicadas depends largely on the duration of the subterranean nymphal stage which can be determined by rearing or regular checking over many years. however, there have been relatively few attempts to rear cicada nymphs, and consequently the length of lifecycle is known for less than 20 of the estimated 1200 species worldwide (karban 1986; moulds & carver 1991). further attempts to rear c. persica on apple plants and under suitable conditions are necessary to determine the unknown details of its life cycle. references anderson d.c., 1994 are cicada (diceroprocta apacbe) both a “keystone” and a “critical-link” species in lower colorado river riparian communities? southwest nature 39: 26-33. beamer r.h., 1982 studies on the biology of kansas cicadidae. university of kansas – sci. bull. 18: 155-263. boulard m., 1990 contributions to general and applied entomology, 2. cicadidae (homoptera auchenorrhyncha) first part: cicadoidea. travaux du laboratoire biologie et evolution des insectes hemipteroidea. 3: 59-254. cumber ra., 1952 notes on the biology of melampsalta cruentata fabricius (hemiptera-homoptera: cicadidae), with special reference to the nymphal stages – trans. royal entomol. soc. london. 103: 219-237. dardar m.a., belal h.m.r., basheer a.m., 2012 occurrence of cicadatra persica on apple trees malus domestica in erneh, syria. j. insect sci. [in press]. dean w.r.j., milton s.j., 1991 emergence and oviposition of quintillia cf. conspersa karsch (homoptera: cicadidae) in the southern karoo, south africa j. entomol. soc. s. afr. 54: 111-119. dybas h.s., davis d.d., 1962 a population census of seventeen-year periodical cicadas (homoptera: cicadidae: magicicada) – ecology. 43: 444-459. gogala m., trilar t., 1998 first record of cicadatra persica kirkaldy, 1909 from macedonia, with description of its song acta entomol. sloven. 6: 5-15. harford b., 1958 observation of some habits and early development of melampsalta cingulata (fabr). order hemiptera. family cicadidae new zeal. entomol. 2: 4-11. james d.a., williams k.s., smith k.g., 1986 survey of 1985 periodical cicada (homoptera: magicicada) emergence sites in washington country arkansas with reference to ecological implications. proc. arkansas acad. sci. 40: 37-39. karban r., 1986 prolonged development in cicadas. in: taylor f., karban r., (eds.). evolution of insect life cycles. springer-verlag, new york, ny: 222-235. logan d., connolly p., 2005 cicadas from kiwifruit orchards in new zealand and identification of their final instar exuviae (cicadidae: homoptera) new zeal. entomol. 28: 37-48. milton s.j., dean w.r.j., 1992 an underground index of rangeland degradation: cicadas in arid southern africa oecologia. 91: 288-291. moriyama m., numata h., 2008 diapause and prolonged development in the embryo and their ecological significance in two cicada. cryptotympana facialis and graptopsaltria nigrofuscata –j. insect physiol. 54: 1487-1494. figure 4. the period of egg laying of cicadatra persica. figure 5. number of newly hatched larvae of cicadatra persica. article [page 58] [journal of entomological and acarological research 2012; 44:e12] a b no nco mm er cia l u se on ly article [journal of entomological and acarological research 2012; 44:e12] [page 59] moulds ms., carver m., 1991 superfamily cicadoidea. in: naumann i.d., carne p.b., lawrence j.f., nielsen e.s., spradberry j.p., taylor r.w., whitten m.j., littlejohn m.j., (eds.). the insects of australia. a textbook for students and research workers, vol. 1 melbourne university press, melbourne: 465-467. patterson j.i., massei g., genov p., 1997 the density of cicadas cicada orni in mediterranean coastal habitats ital. j. zool. 64: 141-146. williams, k.s., smith k.g., stephen f.m., 1993 emergence of 13yr periodical cicadas (cicadidae: magicicada): phenology, mortality, and predator satiation – ecology 74: 1143-1152. white e.g., sedcole j.r., 1993 a study of abundance and patchiness of cicada nymphs (homoptera: tibicinidae) in a new zealand subalpine shrub-grassland new zeal. j. zool. 20: 38-63. wolda h.s., 1989 seasonal cues in tropical organisms. rainfall? not necessarily! –oecologia. 80: 437-442. young d., bennet h.c., 1995 the role of the tymbal in cicada sound production j. exper. biol. 198: 1001-1019. no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract the effect of the entomopathogenic fungus, beauveria bassiana, on the biological characteristics of aphidius colemani, a parasitoid of the green peach aphid, myzus persicae, was studied under laboratory conditions. third-instar nymphs of green peach aphid were infected with 5/3×105 conidia/ml of b. bassiana, which was determined to be the lethal concentration 50 dose. they were then offered to mated female parasitoids for 24 h at different intervals. results showed that by prolonging the release intervals of parasitoids, the number of mummies and percent emergence of parasitoids were reduced. moreover, production of male offspring increased in the f1 generation of parasitoids. the interference of b. bassiana with parasitoid development was also studied by first exposing the aphid hosts to the parasitoids for 24 h and subsequently spraying them with b. bassiana 24, 48, 72, and 96 h after exposure. results showed that by prolonging fungal spraying intervals, the number of mummies and percent emergence of parasitoids were increased. it appeared that the best time for applying b. bassiana would be three to four days after parasitisation. introduction intra-guild interactions are not limited only to closely related species, but can exist between species from different kingdoms. despite the competition, there is evidence for mechanisms that reduce antagonistic interaction between natural enemies vying for common insect hosts (mesquita & lacey, 2001). competition between microorganisms and multicellular animals for nutrition and other requisites is common in nature. however, ecologists have barely started to explore the ecological and evolutionary implications of inter-kingdom competition (mesquita & lacey, 2001). aphidius colemani (hymenoptera: braconidae) is a common parasitoid of aphids, including myzus persicae (hemiptera: aphididae) (messing & rabasse, 1995). the wasp parasitizes all stages of nymphal as well as adult aphids and, under greenhouse conditions, effectively controls the green peach aphid on cotton plants, melons, peppers and cucumbers (gwan et al., 2001). in biological control, the use of pathogens can be integrated with other natural enemies, and the immediate use of a microbial control agent can protect the crop when parasitoids and predators are unable to maintain the pest population below a damage threshold (ren et al., 2010). to date, various strains of entomopathogenic fungi such as beauveria bassiana have been used to control aphids and other pests (hanh vu et al., 2007). for success in biological control of pests, entomopathogenic fungi are needed that show high toxicity to pests and low toxicity to non-target insects and other natural enemies (ren et al., 2010). according to rashki et al. (2009), aphidius matricariae and b. bassiana can be used in combination for successful biological control of m. persicae (rashki et al., 2009). however, there is little information on the congeneric wasp, a. colemani, for integrated pest management of m. persicae. the objective of this study was to determine the type of interaction between the entomopathogenic fungus, parasitoid wasp and its host, the green peach aphid. moreover, the impact of b. bassiana on biological parameters of a. colemani was evaluated. materials and methods fungus beauveria bassiana strain debi008 from the culture collection at the biocontrol laboratory of shiraz university was used. after passage of the fungus through m. persicae, it was cultured on potato dextrose agar correspondence: kambiz minaei, department of plant protection, college of agriculture, shiraz university, shiraz, iran. e-mail: kminaei@shirazu.ac.ir key words: aphidius colemani, beauveria bassiana, myzus persicae, interaction, biological control. acknowledgements: this manuscript was improved through the advice and critique kindly provided by dr. alice wells (australian biological resources scientific, canberra, australia) and two anonymous referees. the authors are particularly grateful to dr. maryam aleosfoor (plant protecting department, shiraz university) for valuable comments and guidance. dr. mohammad dadpasand and dr. hadi atashi (animal science department, shiraz university) are appreciated for helpful comments on the statistical analyses. received for publication: 19 october 2012. revision received: 11 january 2013. accepted for publication: 1 february 2013. ©copyright f. emami et al., 2013 licensee pagepress, italy journal of entomological and acarological research 2013; 45:e4 doi:10.4081/jear.2013.e4 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. interaction between the entomopathogenic fungus, beauveria bassiana (ascomycota: hypocreales) and the parasitoid wasp, aphidius colemani viereck (hymenoptera: braconidae) f. emami, m. alichi, k. minaei department of plant protection, college of agriculture, shiraz university, shiraz, iran [page 14] [journal of entomological and acarological research 2013; 45:e4] journal of entomological and acarological research 2013; volume 45:e4 jear_2013_1:hrev_master 23/04/13 09.55 pagina 14 no n c om me rci al us e o nly with yeast extract (pda) for 2 weeks at 25°c. the average viability of conidia used in the various tests was 94.7% as determined on pda using standard techniques (goettel & inglis, 1997). this involved inoculating an 8-cm petri plate containing pda with 100 ml of a suspension containing 106 conidia/ml and counting the percentage of germinated conidia in four separate areas on the plate after incubation at 25°c for 18-20 h (jenkins et al., 1998). germination was considered positive when the length of the germ tube was as long as the diameter of the conidia. during the test, the lethal concentration50 dose (5.3×105 conidia/ml) (1 ml per spray application) was used for third instar nymphs of the aphid by application in a potter spray tower (potter, 1952). host plant and insect colonies sweet peppers, capsicum annuum var. annuum, were grown individually in plastic pots (15 cm diameter, 12 cm height) to provide experimental host plants. myzus persicae and aphidius colemani from aphid mummies were collected from tomato fields in badjgah (15 km north of shiraz). m. persicae was reared on sweet peppers in a controlled environment room [21±1°c, 60±5% relative humidity (rh), 16l: 8d]. a. colemani was reared on m. persicae inside a plexiglas box (50×60×60 cm) in a controlled environment room (25±1°c, 70±5% rh, 16l: 8d). a. colemani females (4-5 days old) were used for all experiments except those to determine longevity. aphids first exposed to the parasitoid and then treated with the fungus fifteen replicated tests were conducted on separate dates, each replicate containing 40 third-instar aphids. each group was placed in a petri dish (80 mm diameter) containing a leaf of capsicum annuum, fitted into the petri dish with a hole (2 cm diameter) in the lid covered with a fine mesh screen for ventilation (baverstock et al., 2009). the leaves were fixed in 2% water-agar. aphids were exposed to a 4-5-days old female a. colemani. after 24 h, the parasitoids were removed. the replicates of parasitized aphids were sprayed with b. bassiana at a concentration of 5.3×105 conidia/ml in 0.02% tween 80, 24, 48, 72, or 96 h after exposure to the parasitoids. the control was also treated with 0.02% tween 80. after 24 h of fungal conidial application to the aphids, the lids of the petri dishes were replaced with new lids without holes and sealed with parafilm to maintain a saturated humidity to facilitate conidial germination. dead aphids were counted daily for 1 week and were placed on water agar (3 g of agar/l of water) to confirm infection by b. bassiana. during the experiment, the petri dishes were kept inverted and incubated at 25°c, under a 16:8 h photoperiod and 70-85% rh. aphids first treated with fungus and then exposed to the parasitoid forty third-instar aphids were used in each of 15 replicates. all were treated with 5.3×105 conidia/ml b. bassiana in 0.02% tween 80. the aphids were then exposed to a 4-5-days old female a. colemani at different time intervals following application of the fungus. the following combinations of treatments were tested: exposure to the parasitoid 0, 24, 48, or 72 h after application of the fungus, or no fungus (control). the control was also treated with 0.02% tween 80. fifteen replicates were set up. each group of 40 aphids was maintained on a leaf set up in 2% water-agar in a petri dish (80 mm diameter). as described above, during the first 24 h following treatments, the petri dishes were hermetically sealed. then the cover was replaced with one having a screened opening. the inverted petri dishes were kept at 70-75% rh, 25°c, and 16:8 h photoperiod. as described above, dead aphids were recorded daily for 1 week and were placed on water agar to confirm infection by b. bassiana. the number of mummies produced were recorded and the time of pre-imaginal parasitoid development, percentage of emergence, sex ratio and longevity of the f1 generation females were determined. for studying pre-imaginal parasitoid development (mummification until emergence as well as oviposition until emergence) upon formation of the mummies, they were maintained individually in a test tube until adult emergence. to investigate longevity of female parasitoids, they were maintained individually in a petri dish (80 mm diameter) under the above conditions and fed with a honey solution (60%). statistical methods the data were analyzed in a completely randomized design using proc glm in sas (1999). the treatments were compared using duncan’s multiple range test at a significance level of 0.05. the data were normalized using arcsin transformation when necessary. results aphids first exposed to the parasitoid and then treated with the fungus the number of mummies produced varied significantly among different intervals and between treatments and the control (table 1). the lowest percentage of f1 emergence of a. colemani was found for those which developed in aphids treated with b. bassiana 24 h after exposure to the female parasitoid. results showed that by prolonging the fungal spraying intervals, the number of mummies and percent emergence of parasitoids were increased. aphids first treated with fungus and then exposed to the parasitoid as seen in table 2, the number of mummies varied significantly among treatments. in all treatment groups, the numbers of mummies produced were significantly lower compared with the control. the least [journal of entomological and acarological research 2013; 45:e4] [page 15] article table 1. least squares means (±se), number of aphidius colemani mummies, and percentage of adult parasitoids emerging from myzus persicae exposed to the parasitoid and then sprayed with beauveria bassiana (bb) (5/3×105 conidia/ml) 24, 48, 72, and 96 h later. no. aphids exposed to no. aphids treated with fungus no. mummies produced by % emergence of f1 treatments ac after exposure to ac ac generation of ac only ac 40 32.0±0.77a 94.1±3.9a ac+bb (24 h) 40 35.1±1.1 9.5±0.99d 19.4±4.6e ac+bb (48 h) 40 34.9±1.2 18.4±1.75c 46.1±5.9d ac+bb (72 h) 40 33.5±1.1 25.1±1.66b 61.2±0.5c ac+bb (96 h) 40 33.2±1.3 26.2±1.15b 75.8±5.3b ac, aphidius colemani; bb, beauveria bassiana. a,b,c,d least squares means followed by the same letter in the same column are not significantly different at the 0.05 level. jear_2013_1:hrev_master 23/04/13 09.55 pagina 15 no n c om me rci al us e o nly [page 16] [journal of entomological and acarological research 2013; 45:e4] number of mummies were found when parasitism occurred 72 h after exposure to b. bassiana and the greatest numbers were recorded when exposure to the fungus and parasitoid occurred at the same time. there was a significant difference in the time period from oviposition to mummification, ranging from 5.0±0.1 to 3.2±0.1 days among treatments (table 3). in the presence of the fungus, the time between mummy formation and female emergence was decreased. the shortest interval occurred when the aphids were exposed to the parasitoids 72 h after fungal treatment or infestation. however, fungal infection of the aphids only reduced the time between mummification and male emergence of the parasitoids when the hosts were exposed to parasitoid attack 72 h after fungal infection (table 3). therefore, compared with the development of a. colemani in uninfected hosts, total developmental times for males and females were significantly shorter in hosts attacked 72 h after fungal infection. there was also a significant difference in adult female longevity of a. colemani ranging from 8.4±0.3 to 6.4±0.2 days among treatments (table 4). discussion and conclusions the number of mummies and percent emergence of parasitoids increased by prolongation of the fungal spraying intervals (table 1), suggesting that older parasitoid larvae have a high probability of winning the competition for the host aphid, possibly due to a low susceptibility to fungal infection (fransen & van lenteren, 1994). this supports the opinion that the presence of a fungistatic substance secreted by the parasitoid into the hemolymph of the host impedes the development of mycosis and enables normal parasitoid development and emergence (mesquita & lacey, 2001). the inhibitory effect of parasitoids on the development of mycosis in parasitized hosts is normally linked to the time interval between parasitism and contamination of the host by the fungus (powell et al., 1986). aphidius colemani developed normally when its host aphid (aphis gossypii) was treated with verticillum lecanii conidia 5 or 7 days after parasitization. moreover, numbers of article table 2. least squares means (±se), number of aphidius colemani mummies produced, percent adult emergence of f1 generation, and percentage of females in the f1 generation from myzus persicae sprayed with beauveria bassiana and then parasitized 0, 24, 48, and 72 h later. treatments no. of mummies produced % emergence of f1 generation percentage of females in f1 generation only ac 26.7±1.5a 89.2±1.9a 66.7±0.01a bb+ac (0 h) 10.2±0.6b 51.8±4.6b 61.6±0.06a bb+ac (24 h) 8.1±0.7b 35.8±3.5c 58.3±0.1a bb+ac (48 h) 4.3±0.6c 21.3±5.2d 39.6±0.1ab bb+ac (72 h) 2.3±0.5c 15.8±6.1d 26.7±0.1b ac, aphidius colemani; bb, beauveria bassiana. a,b,c,d least squares means followed by the same letter in the same column are not significantly different at the 0.05 level. table 3. developmental time (least squares means±se) of aphidius colemani when aphids were first exposed to beauveria bassiana and then parasitized 0, 24, 48, and 72 h later. treatments oviposition until mummification (day) mummification until emergence (day) ♀ ♂ only ac 5.0±0.1a 5.0±0.00001a 4.5±0.02a bb+ac (0 h) 4.8±0.04a 4.3±0.008b 4.5±0.1a bb+ac (24 h) 4.3±0.06b 4.8±0.06a 4.3±0.1a bb+ac (48 h) 3.5±0.08c 4.5±0.1b 4.1±0.1a bb+ac (72 h) 3.2±0.1d 4.3±0.3b 4.0±0.00001a ac, aphidius colemani; bb, beauveria bassiana. a,b,c,d least squares means followed by the same letter in the same column are not significantly different at the 0.05 level. table 4. developmental time and adult female longevity (least squares means±se) of aphidius colemani when aphids were first exposed to beauveria bassiana and then parasitized 0, 24, 48, and 72 h later. treatments oviposition until emergence (day) adult female longevity (day) ♀ ♂ only ac 10.0±0.17a 9.5±0.008a 9.8±0.03a bb+ac (0 h) 9.1±0.09b 9.3±0.3ac 8.4±0.3b bb+ac (24 h) 9.1±0.08b 8.6±0.3bc 7.0±0.3c bb+ac (48 h) 8.0±0.2c 7.6±0.3d 6.4±0.2c bb+ac (72 h) 7.5±0.2d 7.2±0.3d 6.5±0.1c ac, aphidius colemani; bb, beauveria bassiana. a,b,c,d least squares means followed by the same letter in the same column are not significantly different at the 0.05 level. jear_2013_1:hrev_master 23/04/13 09.55 pagina 16 no n c om me rci al us e o nly spores and mycelial fragments in aphid homogenates were much higher in those exposed to the fungus up to 3 days after parasitization compared with the aphids treated after 5 or 7 days (kim et al., 2005). as noted in table 2, with prolongation of the release intervals of parasitoids, the production of male offspring increased in the f1 generation of parasitoids (table 2). this is almost the same situation that antibiotic treatments cause some female parthenogenetic strains of trichogramma wasps to revert to production of male progeny (stouthamer et al., 1990). a high proportion of male offspring in many parasitic hymenoptera may indicate virginity or sperm depletion in parental females or a manipulated sex ratio (deposition of unfertilized eggs to produce male offspring) (godfray, 1994). if such constrained females reproduce, the population sex ratio will shift toward males and unconstrained females will be selected to produce more females (fauvergue et al., 1998). a female parasitoid may face several types of competition from females of her own species or from different species. the strategies evolved by parasitoids to cope with competition have implications both for the population dynamics of the species and for their use as biological control agents (boivin & brodeur, 2006). in this study, by prolonging the release intervals of parasitoids, the number of mummies produced and percent emergence of parasitoids were reduced (table 2). this may indicate the lack of larval survival potential in infected hosts, following by decreased oviposition of the adult parasitoids in the 72 h-infected hosts and fungus-killed groups. the presence of hyphal bodies or fungal metabolites in the hemolymph of the host detected by the parasitoid at the time of ovipositor insertion are apparently the cause of rejection of these hosts for oviposition (brobyn et al., 1998). fransen and van lenteren (1993) demonstrated that the parasitoid encarsia formosa laid fewer eggs in the greenhouse whitefly, trialeurodes vaporariorum, infected by the fungus aschersonia aleyrodis than in uninfected hosts. brobyn et al. (1988) found that aphelinus asychis and aphidius rhopalosiphi did not attempt to oviposit in fungus-killed aphids. furthermore, it is shown that by prolonging the release intervals of parasitoids, fungus-infected aphids have more opportunities for growth and increased competitiveness, which may lead to death of the wasp larvae (brodeur & rosenheim, 2000). on the other hand, the present results show a reduction in longevity of pre-imagos and adult parasitoids developing within hosts infected by b. bassiana (table 3), presumably because they provide lower quality food for the feeding immatures (nouhuys & laine, 2008). presence of the parasitoid wasps apparently reduces the risk of the host becoming infected with fungus as a pre-adult (lecuona et al., 2007). in most cases, the interactions between fungal pathogens and natural enemies are positive (roy & pell, 2000). the present study revealed that the best time for treating m. persicae with b. bassiana was 3-4 days after parasitation by a. colemani. therefore, with proper timing, this wasp could potentially be used in combination with b. bassiana for successful biological control of an economically important aphid such as m. persicae. references baverstock j., clark s.j., alderson p.g., pell j.k., 2009 intraguild interactions between the entomopathogenic fungus pandora neoaphidis and an aphid predator and parasitoid at the population scale. j. invert. path. 102: 167-172. boivin g., brodeur j., 2006 intraand interspecific interactions among parasitoids: mechanisms, outcomes and biological control. biol. control. 3: 123-144. brobyn p.j., clark s.j., wilding n., 1998 the effect of fungus infection of metopolophium dirhodum [hom.: aphididae] on the oviposition behaviour of the aphid parasitoid aphidius rhopalosiphi [hym.: aphidiidae]. entomophaga 33: 333-338. brodeur j., rosenheim j.a., 2000 intraguild interactions in aphid parasitoids. entomol. exp. app. 97: 93-108. fauvergue x., hopper k.r., antolin m.f., kazmer d., 1998 does time until mating affect progeny sex ratio? a manipulative experiment with the parasitoid wasp aphelinus asychis. j. evol. biol. 11: 611-622. fransen j.j., van lenteren j.c., 1993 host selection and survival of the parasitoid encarsia formosa on greenhouse whitefly, trialeurodes vaporariorum, in the presence of hosts infected with the fungus aschersonia aleyrodis. entomol. exp. app. 69: 239-249. fransen j.j., van lenteren j.c., 1994 survival of the parasitoid encarsia formosa after treatment of parasitized greenhouse whitefly larvae with fungal spores of aschersonia aleyrodis. entomol. exp. app. 71: 235-243. godfray h.c.j., 1994 parasitoids behavior and evolutionary ecology. princeton university press, princeton, nj, usa. goettel m.s., inglis g.d., 1997 fungi: hyphomycetes. in: lacey l.a., ed., manual of techniques in insect pathology. academic press, london: 213-249. gwan g.h., kim j.h., han m.w., 2001 application of aphidius colemani viereck for control of the aphid in greenhouse. j. asia pac. entomol. 4: 171-174. hanh vu v., hong s.i., kim k., 2007 selection of entomopathogenic fungi for aphid control. j. biosci. bioeng. 104: 498-505. jenkins n.e., heviefo g., langewald j., cherry a.j., lomer c.j., 1998 development of mass production technology for aerial conidia for use as mycopesticides. biocontrol news inf. 19: 29-39. kim j.j., kim k.c., roberts d.w., 2005 impact of the entomopathogenic fungus verticillium lecanii on development of an aphid parasitoid, aphidius colemani. j. invert. path. 88: 254-256. lecuona r., crespo d., rossa f., 2007 populational parameters of spalangia endius walker (hymenoptera: pteromalidae) on pupae of musca domestica l. (diptera: muscidae) treated with two strains of beauveria bassiana (bals.) vuil. (deuteromycetes). neot. entomol. 36: 537-541. mesquita a.l.m., lacey l.a,. 2001 interactions among the entomopathogenic fungus, paecilomyces fumosoroseus (deuteromycotina: hyphomycetes), the parasitoid, aphelinus asychis (hymenoptera: aphelinidae), and their aphid host. biol. control 22: 51-59. messing r.h., rabasse j.m., 1995 oviposition behaviour of the polyphagous aphid parasitoid aphidius colemani viereck (hymenoptera: aphidiidae). agric. ecosyst. environ. 25: 13-17. nouhuys s., laine a.l., 2008 population dynamics and sex ratio of a parasitoid altered by fungal-infected diet of host butterfly. r. soc. 275: 787-795. potter c., 1952 an improved apparatus for applying direct sprays and surface films with data on the electrostatic charge on atomized spray fluids. ann. appl. biol. 39: 1-28. powell w., wilding n., brobyn p.j., clark s.j., 1986 interference betwee parasitoids [hym.: aphidiidae] and fungi [entomophthorales] attacking cereal aphids. entomophaga 31: 293-302. rashki m., kharazmi a., pakdel a., allayhari h., van alphen j.j.m., 2009 interactions among the entomopathogenic fungus, beauveria bassiana (ascomycota: hypocreales), the parasitoid, aphidius matricariae (hymenoptera: braconidae), and its host, myzus persicae (homoptera: aphididae). biol. control 50: 324-328. ren s.x., ali s., huang z., wu j.h., 2010 lecanicillium muscarium as microbial insecticide against whitefly and its interaction with other natural enemies. microbiol. microbial. biotechnol. 27: 339-348. roy h.e., pell j.k., 2000 interactions between entomopathogenic fungi and other natural enemies: implications for biological control. biocontrol sci. technol. 10: 737-752. sas, 1999 user’s guide: statistics. version 9. sas institute inc., cary, nc, usa. stouthamer r., luck r.f., hamilton w.d., 1990 antibiotics cause parthenogenetic trichogramma (hymenoptera/trichogrammatidae) to revert to sex. proc. natl. acad. sci. u. s. a. 87: 242427. [journal of entomological and acarological research 2013; 45:e4] [page 17] article jear_2013_1:hrev_master 23/04/13 09.55 pagina 17 no n c om me rci al us e o nly 429 too many requests you have sent too many requests in a given amount of time. jear2012 abstract intervention measures to control the transmission of vector-borne diseases include control of the vector population. in mosquito control, synthetic insecticides used against both the larvae (larvicides) and adults (adulticides) create numerous problems, such as environmental pollution, insecticide resistance and toxic hazards to humans. in the present study, a bacterial pesticide, bacillus sphaericus (bs g3-iv), was used to control the dengue and filarial vectors, aedes aegypti and culex quinquefasciatus. bacillus sphaericus (bs g3-iv) was very effective against aedes aegypti and culex quinquefasciatus, showing significant larval mortality. evaluated lethal concentrations (lc50 and lc90) were age-dependent, with early instars requiring a lower concentration compared with later stages of mosquitoes. culex quinquefasciatus was more susceptible to bacillus sphaericus (bs g3-iv) than was aedes aegypti. fecundity rate was highly reduced after treatment with different concentrations of bacillus sphaericus (bs g3-iv). larval and pupal longevity both decreased after treatment with bacillus sphaericus (bs g3-iv), total number of days was lower in the b. sphaericus treatments compared with the control. our results show the bacterial pesticide bacillus sphaericus (bs g3-iv) to be an effective mosquito control agent that can be used for more integrated pest management programs. introduction mosquitoes are insect vectors responsible for the transmission of many diseases. mosquito-borne diseases include yellow fever, dengue fever and chikungunya, transmitted mostly by aedes aegypti; malaria, carried by the genus anopheles, and culex serves as a vector of important diseases such as west nile virus, filariasis, japanese encephalitis, st. louis encephalitis, and avian malaria. insect-transmitted disease remains a major source of illness and death worldwide. mosquitoes alone transmit disease to more than 700 million people annually and are responsible for several million deaths every year (who, 2012; taubes, 2000; kessler & guerin, 2008). management of these vectors is a serious concern in a developing country like india, due to development of pesticide resistance and for socio-economic reasons. every year, a large part of the population is affected by one or more vector-borne diseases. vector control, which includes both anti-larval and anti-adult measures, constitutes an important aspect of any mosquito control program. mosquito control using synthetic insecticides is an effective vector control strategy used extensively in daily life. synthetic insecticides are still at the forefront of mosquito-controlling efforts. however, the environmental threat that these chemicals pose affects on non-target organisms, and resistance of mosquitoes to insecticides have all increased during the last five decades (wattanachai & tintanon, 1999; amer & mehlhorn, 2006a, 2006b). in recognition of these facts, it is necessary to develop new insecticides for controlling mosquitoes that are environmentally safer, biodegradable, and more target-specific against the mosquitoes. recent negative consumer perceptions concerning the use of chemicals as larvicides have shifted research efforts towards the development of alternatives that the public perceives as natural products, such as bacterial pesticides, predators, and plant extracts. consequently, the present work deals with the insecticidal activities of natural products, such as bacterial pesticides. bacillus sphaericus is an aerobic, mesophilic, spore-forming bacterium with terminal swollen sporangia and spherical spores. as a consequence of the specific toxicity to mosquito larvae of binary toxin (bin) and mosquitocidal toxins (mtxs) produced during the sporulation and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs (bei et al., 2007). bacillus sphaericus is a naturally occurring soil bacterium that can journal of entomological and acarological research 2012; volume 44:e15 correspondence: kadarkarai murugan, department of zoology, bharathiar university, coimbatore, 641046, india. e-mail: kmvvkg@gmail.com key words: bacillus sphaericus (bs g3-iv), aedes aegypti, culex quinquefasciatus, fecundity, mosquito longevity. acknowledgments: we are very thankful to the authorities of the defence research and development laboratory (drdl), defence research and development organization, ministry of defence, government of india, tezpur for providing funds plus their isolate of bacillus sphaericus, for the successful completion of this project. received for publication: 20 august 2012. revision received: 20 november 2012. accepted for publication: 20 november 2012. ©copyright a. nareshkumar et al., 2012 licensee pagepress, italy journal of entomological and acarological research 2012; 44:e15 doi:10.4081/jear.2012.e15 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. larvicidal potentiality, longevity and fecundity inhibitory activities of bacillus sphaericus (bs g3-iv) on vector mosquitoes, aedes aegypti and culex quinquefasciatus a. nareshkumar,1 k. murugan,1 i. baruah,2 p. madhiyazhagan,1 t. nataraj1 1department of zoology, bharathiar university, coimbatore; 2defence research and development organization, defence research laboratory, assam, india [journal of entomological and acarological research 2012; 44:e15] [page 79] no nco mm er cia l u se on ly effectively kill mosquito larvae present in water. b. sphaericus has the unique property of being able to control mosquito larvae in water that is rich in organic matter. depending on the formulation and environmental conditions, b. sphaericus is generally effective 1-4 weeks after application (charles et al., 1996). the larvicidal binary toxin produced by b. sphaericus is composed of two chains, bina (42 kda) and binb (51 kda) that are deposited as parasporal crystals during sporulation (baumann et al., 1991). these crystals bind to a specific receptor present on midgut brush-border membranes, resulting in damage to the midgut cells, which further leads to the mosquito’s death (neilsenleroux & charles, 1992). bacillus sphaericus is specific and toxic against the genera culex and anopheles. it is harmless to humans, animals and the environment, and its use is recommended by the world health organization in public health programs worldwide (who, 1987). many strains of b. sphaericus have been used as a toxicant in vector control programs all over the world (regis et al., 2001). in a recent study in brazil, da silva pinto et al. (2012) cloned the bina and binb genes of bacillus sphaericus, produced recombinant binab protein in three strains of escherichia coli, and used these recombinant strains in toxicity assays against culex quinquefasciatus larvae. research on bacillus sphaericus in south asian countries and india includes a number of strains, such as bacillus sphaericus strain si-1 (hossain et al., 2007), bacillus sphaericus strain b-101 (serotype h5a, 5b) (yadav et al., 1997), bacillus sphaericus h5a5b (vcrc b42) (prabhakaran et al., 2007), bacillus sphaericus h-5a5b (manonmani & hoti, 1999), bacillus sphaericus (bs) 2362 sph-88 (serotype: h5a5b) (poopathi et al., 2009), bacillus sphaericus, c3-41, 2362, and iab59 (pei et al., 2002) for mosquito control. mosquito larvicidal activity of b. sphaericus was assessed by isolating it from ecologically different soil habitats in south india (surendran & vennison, 2011). the present study was conducted to test the larvicidal and pupacidal activities of the microbial insecticide bacillus sphaericus (bs g3-iv) on aedes aegypti and culexquinquefasciatus in the laboratory as well as in direct breeding sites. we also report the effects of bacillus sphaericus (bs g3-iv) on longevity and fecundity of aedes aegypti and culex quinquefasciatus. materials and methods collection of eggs and mosquitoes eggs of aedes aegypti were collected using oviposition traps placed in shaded areas at a height of less than 1.2 m. traps were filled with water plus a few dried leaves placed at the bottom of the container, with a muslin strip placed vertically inside the container and half-submerged in the water. culex quinquefasciatus egg rafts were collected from sewage water bodies in coimbatore district, tamil nadu, india, using cdc gravid traps (reiter 1983, 1987). these eggs were brought to the laboratory and transferred to 18¥13¥4 cm enamel trays (with separate trays for each species) containing 500 ml of water, and held for larval hatch. maintenance of larvae the mosquito larval culture was maintained in our laboratory at 27+2°c, 75-85% rh. the mosquito larvae were fed with dog biscuits and yeast (scottlabs pvt. ltd., hyderabad, india) at a 3:1 ratio. the feeding was continued until the larvae transformed into pupae. maintenance of pupae and adults the pupae were collected from the culture trays and transferred into plastic jar containers (12¥12 cm) containing 500 ml of water. these plastic jars were kept in a 90¥90¥90 cm mosquito cage for adult emergence. the emerged adults were maintained at 27±2°c, 75-85% rh, under 14 light (l):10 dark (d) photoperiod cycles. adults were fed with 10% sugar solution for a period of three days before they were given an animal for blood feeding. blood feeding of adult mosquitoes the female mosquitoes were allowed to feed on the blood of a rabbit (exposed on the dorsal side) for two days. the males were provided with 10% glucose solution on cotton wicks. the cotton was kept moist with the solution and changed every day. an egg trap (cup) lined with filter paper containing water was placed in a corner of the egg collection cage. collection and preparation of bacillus sphaericus bacillus sphaericus (bs g3-iv) with improved toxicity was collected from defence research laboratory, tezpur, assam, india. to assure good suspensions for selection and bioassay procedures, stock suspensions (1 ppm) of the primary powders were prepared in distilled water by vigorously shaking 1 g of the powder in 1000 ml of water in a screwcap glass vial. required concentrations (0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm, and 10 ppm) were prepared by serial dilution of the stock solution in distilled water. all stocks and dilutions were kept refrigerated at -4°c for no more than four months. larval/pupal assays laboratory colonies of mosquito larvae/pupae (f1 generation) were used for the larvicidal/pupacidal activity. twenty-five individual i-ivinstar larvae and pupae were introduced into a 500 ml glass beaker containing 250 ml of dechlorinated water with the desired concentrations of biopesticide. larval food was provided for the test larvae. at each tested concentration, 2-5 trials were run and each trial consisted of 3 replicates. the larvae/pupae exposed to dechlorinated water without biopesticide served as a control. the control mortalities were corrected using abbott’s formula (abbott, 1925) where: fecundity studies the fecundity experiments were conducted by taking an equal number of male and female mosquito larvae that had emerged from the control and treated sets. these were placed in individual 30¥30 cm cages for each concentration. three days after the blood meal, eggs were collected daily from small plastic bowls containing water kept in an ovitrap in the cages. fecundity was calculated from the number of eggs laid in ovitraps divided by the number of mated females. death of adults in these experiments was taken into account. longevity test the adult longevity of male and female mosquitoes (f1 generation) was also recorded. this was calculated as the number of days lived by the adult. total number of days from adult emergence to death was recorded and the means were calculated to give the mean longevity in days. field trial field applications of plant extracts were made uniformly with a knapsack sprayer on the surface of the water in each habitat. sampling of larvae was undertaken before treatment and 24, 48, 72 and 96 h after treatment by dipper sampling and counting. a separate sample was taken to determine the species composition of each larval habitat. six trials were conducted for each area with similar temperature and altiarticle [page 80] [journal of entomological and acarological research 2012; 44:e15] no nco mm er cia l u se on ly tude. the required quantity of biopesticide was determined by calculating the total surface area, and the required concentration was prepared by multiplying ten times the observed laboratory lc50 values. percent reduction of the larval density was calculated using the formula: where c total number of mosquitoes in control t total number of mosquitoes in treatment statistical analysis the data obtained from the bioassay were subjected to statistical analysis using spss (version 14.0) software (ibm corp., armonk, ny, usa). lethal concentrations (lc), lc50 and lc90, dmrt (duncan multiple range test) and c2 tests were used. results table 1 illustrates the larval (i-iv) and pupal mortality data of the dengue vector, aedes aegypti, after treatment with bacillus sphaericus (bs g3-iv) at different concentrations. the maximum mortality observed was 100% at 10 ppm concentration in i and ii instar larvae. the observed mortality rate was greatly reduced in late instar larvae and pupae. pupae showed high resistance to bacillus sphaericus (bs g3-iv), with low mortality rates of 2%, 2%, 3%, 12% and 30% at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively. duncan’s multiple range test proved that the observed mortality rates were significant at p<0.05. lc50 values for i, ii, iii and iv instar larvae were 0.60 ppm, 0.72 ppm, 2.45 ppm and 3.76 ppm, respectively, and, for pupae, 14.08 ppm. percentages of larval and pupal mortality of the filarial vector, culex quinquefasciatus, after treatment with bacillus sphaericus (bs g3-iv) at different concentrations (0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm) are shown in table 2. mortality values ranged from 22% to 100% for the larval stages, but were greatly reduced for the pupal stage. at the highest concentration (10 ppm), no larvae were found alive and 100% mortality was recorded. duncan’s multiple range test proved that the observed mortality rates were significant at p<0.05. the calculated lc50 and lc90 values are 0.07 ppm and 0.56 ppm, 0.15 ppm and 0.87 ppm, 0.29 ppm and 1.14 ppm, 0.41 ppm and 1.32 ppm, and 10.84 ppm and 25.14 ppm for i, ii, iii, iv instar larvae and pupae, respectively. adult longevity and fecundity of the dengue vector, aedes aegypti, after treatment with bacillus sphaericus (bs g3-iv) is shown in table 3. a significant reduction in adult longevity and fecundity was recorded in this experiment when compared with the control. longevity and fecundity after treatment with different concentrations of bacillus table 1. larvicidal and pupacidal activity of bacillus sphaericus on the dengue vector, aedes aegypti. larval and % larval and pupal mortality (mean±sd) standard lc50 95% confidence limit c2 value pupal stages concentration (ppm) error (lc90) lc50 lc90 0.001 0.01 0.1 1 10 (ppm) lcl-ucl lcl-ucl (ppm) (ppm) i 22±1.6d 26±0.7cd 38±0.4c 65±1.1b 100a 0.16 0.60 0.46-0.80 1.48-2.56 4.07* (1.86) ii 20±1.2d 23±0.8cd 34±1.5c 60±0.7b 100a 0.16 0.72 0.56-0.97 1.61-2.85 3.35* (2.04) iii 15±1.1d 17±1.6cd 26±2.1c 47±1.1b 98±0.5a 0.04 2.45 0.85-12.13 3.71-37.20 15.35* (6.69) iv 12±1.6d 15±0.4cd 22±2.2c 43±0.8b 90±0.4a 0.02 3.76 1.36-10.58 5.82-30.13 19.06* (9.64) pupae 2±1.2c 2±0.7c 3±1.1c 12±1.5b 30±1.5a 0.02 14.08 8.46-93.08 14.51-186.15 11.30* (24.52) means±standard deviation (sd) followed by same letter within rows indicate no significant difference (duncan’s multiple range test, p<0.05). lc50, lc90, lethal concentration; lcl, lower confidence limits; ucl, upper confidence limits. *significant at p<0.001 (heterogeneity factor used in calculation of confidence limits). table 2. larvicidal and pupacidal activity of bacillus sphaericus on the filarial vector culex quinquefasciatus. larval and % larval and pupal mortality (mean±sd) standard lc50 95% confidence limit c2 value pupal stages concentration (ppm) error (lc90) lc50 lc90 0.001 0.01 0.1 1 10 (ppm) lcl-ucl lcl-ucl (ppm) (ppm) i 38±0.4cd 43±1.1c 58±0.7b 99±0.4a 100a 0.42 0.07 0.02-0.13 0.43-0.79 2.00* (0.56) ii 35±0.9cd 39±1.6c 53±2.1b 93±0.5ab 100a 0.21 0.15 0.07-0.23 0.72-1.10 2.68* (0.87) iii 27±1.2de 32±0.7d 48±1.2c 85±1.2b 100a 0.18 0.29 0.08-0.54 0.79-2.13 5.49* (1.14) iv 22±0.9de 28±1.1d 42±2.2c 79±0.9b 100a 0.17 0.41 0.19-0.72 0.92-2.48 5.48* (1.32) pupae 11±1.5d 13±0.5cd 18±0.6c 28±1.6b 46±1.5a 0.02 10.84 6.01-90.25 14.15-243.62 8.72* (25.14) means±standard deviation (sd) followed by same letter within rows indicate no significant difference (duncan’s multiple range test, p<0.05). lc50, lc90, lethal concentration; lcl, lower confidence limits; ucl, upper confidence limits. *significant at p<0.001 (heterogeneity factor used in calculation of confidence limits). article [journal of entomological and acarological research 2012; 44:e15] [page 81] no nco mm er cia l u se on ly sphaericus (bs g3-iv) were 26.4 days (d) in males and 37.9 d in females at 0.001 ppm concentration, 21.8 d in males and 35.7 d in females at 0.01 ppm concentration, 18.9 d in males and 31.2 d in females at 0.1 ppm concentration, 15.8 d in males and 27.3 d in females at 1.0 ppm concentration, and 11.6 d in males and 21.1 d in females at 10 ppm concentration. fecundity was also reduced after treatment with bacillus sphaericus (bs g3-iv). a total of 178 eggs were recorded in the control, and the number of eggs recorded in the b. sphaericus treatments were 170, 155, 137, 116 and 82 at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively. adult longevity and fecundity of the filarial vector, culex quinquefasciatus, after treatment with bacillus sphaericus (bs g3-iv) is shown in table 4. significant reduction in adult longevity and fecundity was recorded compared with the control. longevity and fecundity recorded after treatment with different concentrations of bacillus sphaericus (bs g3-iv) were 30.9 d in males and 44.1 d in females at 0.001 ppm concentration, 25.8 d in males and 38.4 d in females at 0.01 ppm concentration, 18.2 d in males and 25.9 d in females at 0.1 ppm concentration, 11.8 d in males and 14.6 d in females at 1.0 ppm concentration, and 4.7 d in males and 7.1 d in females at 10 ppm concentration. the fecundity was also highly reduced after treatment with bacillus sphaericus (bs g3-iv). a total of 270 eggs were recorded in the control, compared with 249, 215, 178, 119 and 63 at 0.001 ppm, 0.01 ppm, 0.1 ppm, 1.0 ppm and 10 ppm, respectively, of b. sphaericus. table 5 shows the effect of bacillus sphaericus (bs g3-iv) on the dengue vector, aedes aegypti, in their breeding sites. the field trial was conducted in stagnant water bodies at vadavalli. the surface areas of the selected breeding sites were 0.6¥0.7¥0.5 m. a total of 481 larvae were found. after treatment with bacillus sphaericus (bs g3-iv), the percentage of reduction in larval density was 39.70%, 59.87%, 82.74% and 96.25% at 24 h, 48 h, 72 h and 96 h, respectively. field application of bacillus sphaericus (bs g3-iv) in the sewage water systems in vadavalli (breeding sites of the filarial vector, culex quinquefasciatus), is given in table 6. the surface areas of the selected breeding sites were 0.4¥1.7¥0.28 m. required quantity and concentration of biopesticide were calculated as 0.19 l and 2.88 ppm, respectively. a total of 788 larvae were found. after treatment with bacillus sphaericus (bs g3-iv), the percentage of reduction in larval density was 65.1%, 87.6%, 97.5% and 100% at 24 h, 48 h, 72 h and 96 h, respectively. discussion mosquitoes breed in varied habitats, such as ponds, marshes, ditches, pools, drains, water containers and other similar collections of water (rozendaal, 1997). mosquitoes such as anopheles, culex and aedes are vectors responsible for diseases such as malaria, filariasis, japanese encephalitis, dengue, dengue hemorrhagic fever, yellow fever and chikungunya. the increase in mosquito vectors and incidence of mosquito-borne diseases such as malaria, dengue, and chikungunya is rising in india due to climate change and water contamination. unclean water bodies act as temporary and permanent breeding sites of mosquitoes, which tend to spread mosquito-borne diseases, along with cholera, dysentery, typhoid, etc. understanding the ecology of mosquitoes and the mechanism of disease management is a prerequisite to adopting any type of control. in general, the population of vector species must be of sufficient size so as to promote the transmission of vector-borne diseases. if the vector population falls below a critical density, the transmission of these diseases will not be very effective. effective mosquito control is often a complex, expensive task, frequently requiring the cooperative efforts of communities as well as industry, agriculture, and state and local governments. we must be concerned with the harmful effects of synthetic pesticides on the environment and living organisms, and reports have emerged on the resurgence of several mosquito-borne diseases in the world as a consequence of increasing resistance of mosquitoes to commercial insecticides (becker et al., 2003). this has necessitated the need for research and development of environmentally safe, biodegradable, indigenous methods for vector control. we found bacillus sphaericus (bs g3-iv) to be significantly effective against aedes aegypti and culex quinquefasciatus. this may have been due to the presence of binary toxin (bin) and mosquitocidal toxins (mtxs). the bin toxin produced by bacillus sphaericus targets mosquito larval midgut epithelial cells, where it binds to cpm1 (culex pipiens maltase 1), a digestive enzyme, and causes severe intracellular damage, including dramatic cytoplasmic vacuolation (opota et al., 2011). culex quinquefasciatus was much more susceptible to bacillus sphaericus (bs g3-iv), showing 100% mortality at a 10 ppm concentration against i-iv instars. median lethal concentrations (lc50) observed were relatively low (0.07 ppm, 0.15 ppm, 0.29 ppm, 0.41 ppm, and 10.84 ppm for i, ii, iii, iv instar larvae and pupae, respectively) when compared with aedes aegypti. earlier, yousten and davidson (1982) and davidson (1983) reported that bacillus sphaericus, a spore-forming, entamopathogenic bacterium, possess potent larvicidal activity against several species of mosquito larvae. as a consequence of the specific toxicity to mosquito larvae of binary toxin (bin) and mosquitocidal toxins (mtxs) produced during the sporulation and vegetative stages, respectively, some toxic strains have been widely used for many years as biopesticides in the field in mosquito control programs (bei et al., 2007). table 3. effect of bacillus sphaericus on fecundity and longevity of dengue vector aedes aegypti in the laboratory. treatment adult longevity fecundity (ppm) (ins) male female control 29±0.2a 39±0.5a 178±0.4a 0.001 26.4±0.9b 37.9±0.6ab 170±1.6ab 0.01 21.8±0.9c 35.7±1.1b 155±1.1b 0.1 18.9±0.6d 31.2±0.6c 137±0.9c 1 15.8±0.2e 27.3±0.9d 116±0.6d 10 11.6±0.9f 21.1.±0.2e 82±1.1e means±standard deviation (sd) followed by same letter within rows indicate no significant difference (duncan’s multiple range test, p<0.05). table 4. effect of bacillus sphaericus on fecundity and longevity of the filarial vector culex quinquefasciatus in the laboratory. treatment adult longevity fecundity (ppm) (ins) male female control 34±0.2a 47±0.2a 270±0.6a 0.001 30.9±1.5ab 44.1±1.9ab 249±2.8b 0.01 25.8±1.5b 38.4±1.6b 215±3.2c 0.1 18.2±2.1c 25.9±1.1c 178±1.9d 1 11.8±0.9d 14.6±1.5d 119±1.6e 10 4.7±1.0e 7.1±0.6e 63±0.9f means±standard deviation (sd) followed by same letter within rows indicate no significant difference (duncan’s multiple range test, p<0.05). article [page 82] [journal of entomological and acarological research 2012; 44:e15] no nco mm er cia l u se on ly in the present study, aedes aegypti showed a slight reduction in mortality when compared with culex quinquefasciatus after treatment with bacillus sphaericus (bs g3-iv). this may be due to its breeding habitats, characterized by high organic matter and oxygen content, which promotes the growth and development of bacillus sphaericus. a previous study showed bacillus sphaericus exhibited only a low level of toxicity against fourth instar larvae of aedes aegypti (mulla et al., 1984), but the present isolate was more toxic when compared with those in earlier studies. usually b. sphaericus is minimally toxic to a. aegypti because this species either lacks a specific receptor for the binary toxin of b. sphaericus or has an extremely low concentration of such receptors (nielsen-le roux & charles, 1992), whereas the isolate b. sphaericus (bs g3-iv) used in the present study was improved with necessary qualities for targeting a. aegypti. production of enhanced mosquitocidal toxin by b. sphaericus 2362 and b. sphaericus 14n1 using whey permeate (wp) under submerged fermentation conditions has resulted in high mosquitocidal activity (el-bendary et al., 2008). no significant differences have been found in other factors that could affect the activity of b. sphaericus, such as the rate of ingestion of the toxins or differences in proteolytic activation, between c. quinquefasciatus and a. aegypti (aly et al., 1989). the present findings also agree with those from earlier studies pointing out the high susceptibility of culex spp. to b. sphaericus (singer, 1980; yousten, 1984; mulla et al., 1986). b. sphaericus has the unique property of being able to control mosquito larvae in water that is rich in organic matter. b. sphaericus is effective against culex spp., but is less effective against some other mosquito species (poopathi & abidha, 2010). in the present study, bacillus sphaericus (bs g3-iv) reduced larval and pupal longevity and inhibited adult emergence in both species. the life span of emerged adults was also very low when mosquito larvae were treated with bacillus sphaericus (bs g3-iv), the reduction being more pronounced in culex quinquefasciatus and less so in aedes aegypti. aedes aegypti was generally less susceptible to bacillus sphaericus (bs g3-iv), but effects on its longevity and fecundity were comparable with those seen in culex quinquefasciatus. the affected pupae also resulted in a great reduction in fecundity. adults emerging from treated larvae were morphologically normal but laid fewer eggs. the number of eggs laid by culex quinquefasciatus was very low compared with aedes aegypti. murugan et al. (2002) reported changes in fecundity after treatment with bti. combined treatment of bti with neem and pongamia showed 76% adult mortality and a reduction in fecundity (senthil nathan et al., 2004). poopathi & tyagi (2002) reported a reduction in adult longevity (17% in males and 27% in females) in culex quinquefasciatus after treatment with bacillus sphaericus (gr strain), which supports the present findings. this study showed that bacillus sphaericus (bs g3-iv) was also effective in a field environment. the percentage of reduction in larval density was observed every 24 h, and was seen to increase as the time after treatment increased. this supports the observation that the bacterial pesticide was not negatively affected by the external environment and exhibits a persistent effect. this pesticide has also been found to be eco-friendly and non-toxic to non-target organisms. mittal (2003) reported that the mosquitocidal toxins of certain strains of bacillus sphaericus and bacillus thuringiensis var israelensis h-14 (bti) are highly effective against mosquito larvae in their direct breeding sites, even at very low doses, and are also safe to other nontarget organisms. he also stated that the biolarvicide formulations of b. sphaericus are useful in the control of culex and certain anopheles spp., such as an. stephensi and an. subpictus, but are not very effective against an. culicifacies. because bacillus sphaericus distinctly affects the developmental stages of insects, it also has distinct advantages over synthetic pesticides. the specificity of the b. sphaericus toxin is in part due to differences in the number of target sites to which it binds (baumann et al., 1991). the binding of the protein toxin to the gastric caecum and posterior midgut has been observed in culex pipiens (a susceptible species) but not in resistant aedes aegypti (mittal, 2003). references abbott w.s., 1925 a method of computing the effectiveness of an insecticide. j. econ. entomol. 18: 265-267. aly c., mulla m.s., federici b.a., 1989 ingestion, dissolution, and proteolysis of the bacillus sphaericus toxin by mosquito larvae. j. invertebr. pathol. 53: 12-20. amer a., mehlhorn h., 2006a larvicidal effects of various essential oils against aedes, anopheles, and culex larvae (diptera, culicidae). parasitol. res. 99: 466-472. amer a., mehlhorn h., 2006b repellency effect of forty-one essential oils against aedes, anopheles, and culex mosquitoes. parasitol. res. 99: 478-490. table 5. effect of bacillus sphaericus against larval density of mosquito vectors in breeding sites (stagnant water) of aedes aegypti. site no. larval density (%) before treatment after treatment 24 h 48 h 72 h 96 h 1 66 39 26 9 0 2 90 56 38 16 4 3 69 40 28 12 1 4 78 45 30 14 4 5 96 59 38 16 3 6 82 51 33 16 6 total 481 290 193 83 18 average 80.16 48.33 32.16 13.83 3 % reduction 39.70 59.87 82.74 96.25 location, vadavalli; habitat, stagnant water bodies; species, aedes aegypti; stage, larvae; size, 0.6¥0.7 m; depth, 0.5 m; required quantity, 0.6¥0.7¥0.5=0.42¥0.5=0.21 l; required concentration, 2.450¥10=24.50 ppm. table 6. effect of bacillus sphaericus against larval density of mosquito vectors at the breeding sites (sewage water) of culex quinquefasciatus. site no. larval density (%) before treatment after treatment 24 h 48 h 72 h 96 h 1 140 56 22 6 0 2 126 42 14 2 0 3 138 51 18 5 0 4 120 35 12 1 0 5 134 48 17 4 0 6 130 43 14 1 0 total 788 275 97 19 0 average 131.33 45.83 16.16 13.16 0 % reduction 65.1 87.6 97.5 10 location, vadavalli; habitat, sewage water bodies; species, culex quinquefasciatus; stage, larvae; size, 0.4¥1.7 m; depth, 0.28 m; required quantity, 0.4¥1.7¥0.28=0.68¥0.28=0.19 l; required concentration, 0.288¥10=2.88 ppm. article [journal of entomological and acarological research 2012; 44:e15] [page 83] no nco mm er cia l u se on ly baumann p., clark m.a., bauman l., broadwell a.h., 1991. bacillus sphaericus as a mosquito pathogen: properties of the organism and its 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1984 bacterophage typing of mosquito pathogenic strains of bacillus sphaericus. j. invertebr. pathol. 43: 124-125. yousten a.a., davidson e.w., 1982 ultrastructural analysis of spores and parasporal crystals formed by bacillus sphaericus 2297. appl. environ. microbiol. 44: 1449-1455. zebitz c.p.w., 1984 effects of some crude and azadirachtin enriched neem azadirachita indica seed kernel extracts on larvae of aedes aegypti. entomol. exp. appl. 35: 11-14. zebitz c.p.w., 1986 effects of three neem seed kernel extracts and azadirachtin on larvae of different mosquito species. j. appl. entomol. 102: 455-463. article [page 84] [journal of entomological and acarological research 2012; 44:e15] no nco mm er cia l u se on ly jear2012 [journal of entomological and acarological research 2014; 46:715] [page 13] efficiency of mentha piperita l. and mentha pulegium l. essential oils on nutritional indices of plodia interpunctella hübner (lepidoptera: pyralidae) k. saeidi, b. hassanpour department of entomology, agricultural and natural resources research center, yasouj, iran abstract antifeedant activity of plant extracts from mentha piperita l. and mentha pulegium l. were tested against the indian meal moth, plodia interpunctella (hübner). the nutritional indices: relative growth rate (rgr), relative consumption rate (rcr), efficiency of conversion of ingested food (eci) and feeding deterrence index (fdi) were measured for first-instar larvae (15-d old). treatments were evaluated using a flour disk bioassay in the dark, at 25±1°c and 60±5% r.h. concentrations of 0, 0.1, 0.5, 0.75, 1, 1.5 and 2 ml/disk were prepared from each essential oil. after 72 h, nutritional indices were calculated. m. piperita oils were more effective than m. pulegium oils, by significantly decreasing the rgr, rcr and fdi. at the highest concentration tested (2 ml/disk), the eci (9%) was significantly reduced. introduction stored cereals, oilseeds, pulses, spices, dried fruits, tree nuts and their processed foods are important for food and trade purposes and suffer economic and quality losses due to insect pests (lamiri et al., 2001; passino et al., 2004; tapondjou et al., 2005; ali & rizvi, 2008). there are over 600 species of beetle pests and 70 species of moths capable of causing quantitative and qualitative losses (rajendran, 2002). in developing countries, damage is between 10 to 40% (shaaya et al., 1997). in some rural areas of iran that use traditional storages, damage caused by stored product insects can be as high as 80% (modarres-najafabadi et al., 2006). p. interpunctella (hübner) (indian meal moth) is considered to be the most troublesome of the moths infesting stored products in the world (phillips et al., 2000; mohandass et al., 2007). it attacks all cereal products, whole grains, dried fruits, pet foods, birdseed, dried milk and nuts (arthur et al., 1991). the larvae are generalists, as they can feed on grain products, seeds, dried fruit, dog food, and spices (arthur et al., 1990; mohandass et al., 2007). damage is caused by the larvae spinning silken threads as they feed and crawl, thus webbing the particles of food together (simmons & nelson, 1975). the control of this pest in storage systems mainly depends on fumigants such as methyl bromide or phosphine, and fogging with pyrethrins or dichlorvos. however, methyl bromide was banned in many countries starting in 2004, because of its ozone-depleting properties (hansen & jensen, 2002). synthetic pesticides have been considered the most effective and accessible means of controlling insect pests of stored products (huang & subramanyam, 2005). these chemicals are associated with undesirable effects on the environment due to their slow biodegradation and some toxic residues in products, affecting mammalian health (benhalima et al., 2004; isman, 2006; halder et al., 2010). the adverse effects of synthetic pesticides have amplified the need for an effective and biodegradable pesticide. natural products are an excellent alternative to synthetic pesticides as a means to reduce negative impacts on human health and the environment. among the various kinds of natural substances that have received particular attention as natural agents for insect management are essential oils from aromatic plants. essential oils are renewable, non-persistent in the environment and relatively safe to natural enemies, non-target organisms and human beings (halder et al., 2010). essential oils are defined as any volatile oil(s) that have strong aromatic components and that give a distinctive odor, flavor or scent to a plant. these are the by-products of plant metabolism and are commonly referred to as volatile plant secondary metabolites (koul et al., 2008). because of the intensity of plant-insect interactions, the plants have well-developed defense mechanisms against pests and are excellent sources of new insecticidal substances. their components and quality vary with geographical distribution, time of harvest, growing conditions and method of extraction (yang & zheng, 2005). effects of essential oils on stored-product insect pests have been reported on extensively (ogendo et al., 2008; park et al., 2008; benzi et al., 2009; ayvaz et al., 2010; nayamador et al., 2010; taghizadeh et al., 2010). the insecticidal activity of some essential oils from lamiaceae has been evaluated against a number of stored product insects. for example, mollai et al. (2010) found strong insecticidal activity of essential oil from satureja hortensis (lamiaceae) on p. interpunctella. the 24-h lc50 values against adults were 140 ml/l air. in another experiment, aliakbari et al. (2010) studied insecticidal activity of the essential oil from thymus daenensis (lamiaceae) on tribolium confusum (tenebrionidae). mortality was evaluated after 24 and 48 h. lc50 and correspondence: karim saeidi, department of entomology, agricultural and natural resources research center, p.o. box 351, yasouj, iran. tel.: +98.741.3334821 / +98.741.3334821 fax: +98.741.3334011. e-mail: saeidi391@yahoo.com key words: essential oil, nutritional indices, plodia interpunctella. received for publication: 3 december 2012. revision received: 31 july 2013. accepted for publication: 7 august 2013. ©copyright k. saeidi and b. hassanpour, 2014 licensee pagepress, italy journal of entomological and acarological research 2014; 46:715 doi:10.4081/jear.2014.715 this article is distributed under the terms of the creative commons attribution noncommercial license (by-nc 3.0) which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. journal of entomological and acarological research 2012; volume 44:ejournal of entomological and acarological research 2014; volume 46:715 no nco mm er cia l u se on ly [page 14] [journal of entomological and acarological research 2014; 46:715] lc95 values were 50 and 169 ml after 24-h and 42 and 103 ml after 48 h, respectively. ebadollahi & mahboubi (2011) studied fumigant activity of the essential oil from lavandula stoechas l. (lamiaceae) on tribolium castaneum. based on the results of this research, essential oils of l. stoechas should be considered as a potential control agent against t. castaneum. rafiei-karahroodi et al. (2009) studied the effect of essential oils from dracocephalum moldavica, lavandula officinalis, melissa officinalis and rosmarinus officinalis on 1-d and 3-d-old eggs of p. interpunctella. results indicated that m. officinalis and r. officinalis are suitable candidates for replacing synthetic pesticides in warehouses to control p. interpunctella. the main goal of the present study was to evaluate the insecticidal activities of essential oils from m. piperita and m. pulegium grown in iran for the control of p. interpunctella. materials and methods moth culture the indian meal moths, based on described by silhacek & miller (1972), were reared on artificial diet containing: cornmeal (26%), whole wheat flour (23%), glycerol (16%) honey (14%), ground dog meal (10%), brewers’ yeast (5%), rolled oats (4%) and wheat germ (2%) in a chamber set to a light:dark period of (11:13) and a temperature of 28±2°c. collected and dried plant specimens two plants known to have medicinal activity, m. piperita l. and m. pulegium l., were collected from their natural habitats, from different localities in iran. the identity of each plant species was verified by en. shahabedin mirinejad (botanical specialist from agriculture and natural resources researches center of kohgiloyeh and boyerahmad, yasouj), using live specimens and photographs. extraction of essential oils plant materials were air dried in the shade at room temperature (2628°c) for 20 d and stored in darkness until distillation. the essential oils were isolated from dried plant samples by hydro-distillation using a clevenger apparatus. conditions of extraction were: 50 g of air-dried sample, 1:10 plant material/water volume ratio, 3 h distillation. the essential oils were collected, dried over anhydrous sodium sulfate and stored at 4°c until use. flour disk bioassay according to the method of mohandass et al. (2007) a suspension of 10 g wheat flour in 50 ml distilled water was prepared. a micropipette was used to transfer 200-ml aliquots from the suspension onto a plastic sheet. after 4 h at room temperature, the wheat flour suspensions in the form of spherical disks were transferred to a petri dish. prepared disks were kept for 12 h to dry inside an oven, after which the weight of the flour disks was between 35-45 mg and their moisture content was approximately 15%. different concentrations of essential oils from m. piperita and m. pulegium (0.1, 0.5, 0.75, 1, 1.5 and 2 ml in 1 ml of acetone) were placed on each disk separately and held for 20 min at room temperature to allow for evaporation of the acetone. in each petri dish, one flour disk was placed along with 10 firstinstar indian meal moth larvae and held at 25±1°c and 60±5% r.h. for 3 d. at the beginning of the experiment, the weight of flour disks and larvae was measured. after 3 d, flour disks and larvae were weighed again and the number of dead larvae noted. there were 5 replicates. nutritional indices nutritional indices were calculated according to tripathi et al. (2002), with some modifications: relative growth rate (rgr)=(a-b)/(b×day) (1) where a=weight of live insects (mg) on the third day/number of live insects on the third day; b=original weight of insects (mg)/original number of insects. relative consumption rate (rcr)=d/(b×day) (2) where d=biomass ingested (mg)/number of live insects on the third day. percentage efficacy of conversion of ingested food (eci)=rgr/rcr ×100. the feeding deterrent activity was calculated as a feeding deterrent index (isman, 2006): (% fdi)=[(c–t)/c]×100 (3) where c is the weight consumption of food in the control and t is the weight consumption food in the treatment. data analysis each of the indices was calculated using a completely randomized factorial design, and five replicates were performed. the first factor in this design included three treatments, consisting of the essential oils of m. piperita, m. pulegium and a control, and the second factor consisted of six concentrations of plant essential oils: 0.1, 0.5, 0.75, 1, 1.5 and 2 ml/disk, and a control treatment. before statistical analysis, the eci and fdi nutritional indices data were normalized using an arcsin√x/100 transformation. the means were separated using duncan’s multiple range test at the 5% significance level. results plant source and dose significantly affected all nutritional indices (table 1). for rcr, eci and fdi there was a significant interaction article table 1. analysis of variance of essential oils of mentha piperita and mentha pulegium on nutritional indices of larvae of plodia interpunctella. source of variation degrees of freedom mean squares rgr rcr eci fdi plant 1 7.01×10–4* 0.066** 99.836** 5167.934** concentration 6 0.002** 0.032** 26.125** 1998.654** plant×concentration 6 3.086×10–5ns 0.003** 16.822** 176.234** error 42 2.620×10–5 3.736 4.354 0.023 rgr, relative growth rate; rcr, relative consumption rate; eci, efficiency of conversion of ingested food; fdi, feeding deterrence index; ns, non-significant; *, ** respectively significant differences at 5 and 1% level. no nco mm er cia l u se on ly [journal of entomological and acarological research 2014; 46:715] [page 15] article table 3. total average effect of essential oils of mentha piperita and mentha pulegium at various concentrations on nutritional indices of larvae of plodia interpunctella. concentration (ml/disk) standard error±average nutritional indices rgr (mg/mg/day) rcr (mg/mg/day) eci% fdi% 0.00 (control) 0.051±0.001a 0.3131±0.015a 16.288±0.453a 0.1 0.035±0.001b 0.253±0.014b 13.833±0.765a 13.281±2.050f 0.5 0.031±0.001bc 0.230±0.017c 13.478±0.749ab 20.098±2.710e 0.75 0.027±0.000c 0.210±0.015d 12.857±0.512abc 25.889±3.112d 1 0.019±0.002d 0.169±0.016e 11.242±0.765abc 32.705±3.256c 1.5 0.016±0.001d 0.116±0.023f 13.793±1.682cb 48.949±6.014b 2 0.010±0.003e 0.102±0.039g 9.384±1.812c 52.829±5.235a rgr, relative growth rate; rcr, relative consumption rate; eci, efficiency of conversion of ingested food; fdi, feeding deterrence index. a,b,c,d,e,f,gdissimilar letters in each column with using duncan’s test at level of 1% together have significant differences. table 2. effect of essential oils of mentha piperita and mentha pulegium on nutritional indices of larvae of plodia interpunctella. essential oil rgr (mg/mg/day) rcr (mg/mg/day) eci% fdi% mentha piperita 0.026±0.002b 0.219±0.160b 11.94±0.76a 34.9±3.8a mentha pulegium 0.028±0.001a 0.294±0.006a 9.64±0.44b 14.2±2.2b rgr, relative growth rate; rcr, relative consumption rate; eci, efficiency of conversion of ingested food; fdi, feeding deterrence index. a,bvalues in the same column followed by different letters are significantly different (p<0.01, duncan’s multiple range test). figure 1. effect of essential oil mentha piperita and mentha pulegium at various concentrations on nutritional indices larvae of plodia interpunctella. no nco mm er cia l u se on ly [page 16] [journal of entomological and acarological research 2014; 46:715] between plant source and dose, indicating that the effect of plant source varied significantly with dose (table 1). essential oils of m. piperita had a significantly greater negative impact on nutritional indices than did the essential oils of m. pulegium (tables 2 and 3). however, these differences were small (figure 1). discussion and conclusions in this study, to compare the anti-nutritional effects of essential oils of m. piperita and m. pulegium, parameters as indicators of nutrition were used by employing no-choice tests of the insects’ food, which had been impregnated with various concentrations of the essential oils. in these experiments, two primary outcomes were measured. the first was weight loss of the insects compared with the control during the duration of this experiment, expressed as the rgr index. second, the rcr index was measured and compared with control insects to measure whether test insects had taken less or avoided eating treated food. the effective weight loss could be related to the impact of essential oils on insect food (koul et al., 2008), and to clarify avoidance of insect feeding, fdi was used. in this experiment, it was observed that increasing the concentration and changing the type of essential oil reduced the rgr and rcr values, so that there was a greater effect with essential oil at high concentrations, and regarding essential oil type, m. piperita was found to be more effective. in terms of the mechanism of action in response to this decrease, it is clear that low concentrations of essential oils of m. piperita and m. pulegium did not show significant differences in terms of eci, but with very high essential oil concentrations, the value of the eci was reduced. even at lower concentrations of the essential oils of m. piperita and m. pulegium, there was significant inhibition of insect feeding. therefore, the impact on the rgr and rcr can be attributed to the effects of feeding deterrence or fdi. even at lower concentrations, these essential oils can effectively reduce insect feeding, as noted in the studies of other researchers. in this study, to examine the anti-nutritional properties of mint essential oils, indian meal moth was used as a model, and showed that sub-lethal concentrations of essential oils in warehouse use could prevent the insects from feeding on the stored product. therefore, we would conclude that a method to combine these essential oils with some storage products could be effective in controlling pests. references ali a., rizvi p.o., 2008. bio-efficacy of some plant leaf extracts against mustard aphid, lipaphis erysimikalt on indian mustard, brassica juncea. j. plant. protec. res. 50: 2. aliakbari j., fallahzadeh m., ghasemi a., abdizadeh r., 2010. insecticidal activity of essential oil from thymus daenensis celak against tribolium confusum dur. proc. 19th iranian plant protection congress, july 31-august 3, 2010. iranian research institute of plant protection, tehran, iran. arthur f.h., 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[journal of entomological and acarological research 2014; 46:715] [page 17] article no nco mm er cia l u se on ly 429 too many requests you have sent too many requests in a given amount of time.