DOI: 10.13102/sociobiology.v65i2.2345Sociobiology 65(2): 280-284 (June, 2018) Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 Estimating colonies of Plebeia droryana (Friese, 1900) (Hymenoptera: Apidae: Meliponini): adults, brood and nest structure Introduction Stingless bees are found in tropical and subtropical regions all around the world (Sakagami, 1982) and they have populated tropical regions for over 65 million years – longer than Apis, the honeybees (Camargo & Pedro, 1992; Michener, 2000). However, stingless bees have 50 times more species and, as emphasized here, differ from Apis in many biologically significant ways (Roubik, 2006). The colonies have individuals active every day that perform their activities and, therefore, have supported climate impact of the environment (Roubik, 1989; Hansell, 1993). The colonies of stingless bees present structural, behavioral and physiological adaptations, which protect bees from the external environment (Roubik & Peralta, 1983). Their nests are perennial, and they are active throughout the year (Michener, 1974; Sakagami, 1982). Abstract Estimate of stingless bee colonies including nest structures and quantitative brood and adult individuals are scarce. Here, we describe a new approach to estimate colonial parameters from nest structure, adults and brood. We used five colonies of Plebeia droryana (Friese, 1900) to evaluate colony size and weight of adult and brood. Nest architecture in P. droryana is similar to the species of the same genus but differ to the other stingless bees. In this species, we counted a total of 9 to 12 brood combs and a total of 19 to 25 food pots in the nests. The number of individuals in the colonies is considered small and our estimate was based on individual and group weight. Our study approach may contribute to further detailed studies of the species nest and considering the stingless bees to the pollination of agricultural crops and native flora of tropical regions, it is important to add information about the biology of P. droryana. Sociobiology An international journal on social insects YS Roldão-Sbordoni, FS Nascimento, S Mateus Article History Edited by Denise Alves, USP, Brazil Received 19 October 2017 Initial acceptance 04 December 2017 Final acceptance 13 March 2018 Publication date 09 July 2018 Keywords Stingless bees; nest estimative; population size. Corresponding author Yara Sbrolin Roldão-Sbordoni Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto São Paulo (USP) Departamento de Biologia, Laboratório de Comportamento e Ecologia de Insetos Sociais Avenida Bandeirantes, 3900 - CEP 14040-901 Ribeirão Preto-SP, Brasil. E-Mail: yara.roldao@hotmail.com Stingless bees usu ally build their nests in holes in trees, cavity in the soil, and even in cavities in human constructions, but some also construct aerial nests (Nogueira-Neto, 1997), and some species that regularly establish their nests within nests of Nasutitermes or ants (Michener, 2000). In contrast to the Apini, new stingless bee nests are started by workers going back and forward from an existing old colony. They transfer building materials and food to the new site. Ultimately, a young queen flies to the new site, workers stay there, and independence from the old colony is gradually attained over a period of weeks or months (Michener, 1946; Inoue et al., 1984; Van-Veen & Sommeijer, 2000). Nests are made of wax secreted from the metasomal terga mixed with resins and gums collected by the foragers. Some species add mud, feces, or other materials in several parts of the nest. In all species, the composition and texture differ in different parts of the nest (Michener, 2000). In some species, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto São Paulo - USP, Departamento de Biologia, Laboratório de Comportamento e Ecologia de Insetos Sociais, Ribeirão Preto-SP, Brazil RESEARCH ARTICLE - BEES Sociobiology 65(2): 280-284 (June, 2018) 281 the brood combs are covered by layers of wax involucrum and the storage pots are made with cerumen. Several bee species build a strong structure with cerumen, mud, and resin called scutellum or batumen (Zucchi & Sakagami, 1972). Some of the supports and batumen plates are of tough material, and the external sheet of batumen around some exposed nests is harder and brittle (Michener, 2000). Nest characteristics such as the nesting site, architectural complexity, and building materials may be taxon-specific and provide an excellent opportunity to assess information about underlying behavioral evolution of the respective taxa and higher groups (Rasmussen & Camargo, 2008). The nest architecture of stingless bees consists of an entrance, access tunnel, food pots (nectar and pollen storage) and brood area, with horizontal combs or in cluster (Zucchi & Sakagami, 1972; Wille & Michener, 1973; Michener, 1974). Michener (1974) described the colony size of various social bees, especially the genus Trigona. In addition, Lindauer and Kerr (1960) presented an estimate of adult workers in colonies of stingless bee colonies. They evaluated 10 species and among them Plebeia droryana (formerly known as Trigona droryana) had 2,000 to 3,000 adults. Wille and Michener (1973) showed another estimate in which measured nest structures as food pots and brood cells diameter and length. Using Lindauer and Kerr (1960) estimative references to P. droryana and other bees, Duarte et al. (2016) studied nest architecture of Tetragona clavipes and presented structures and individuals estimative. The genus Plebeia Schwarz, 1938 consists a very diverse group and is distributed in the Neotropical region. Plebeia is morphologically the most primitive taxa within stingless bees and is mainly found in the southeast region in Brazil (Wittmann, 1989; Michener, 1990; Witter et al., 2007). The stingless bee P. droryana has a wide geographic distribution, which includes Argentina, Bolivia, Paraguay and some Brazilian states as Bahia, Espírito Santo, Minas Gerais, Paraná, Pernambuco, Rio Grande do Sul, Rio de Janeiro, Santa Catarina and São Paulo (Moure et al., 2007; Camargo & Pedro, 2013). These bees are small (4 mm) and not aggressive, build royal cells for queen production (Nogueira-Neto, 1970). Although some estimative recordings for P. droryana nests and other stingless bee species have been made, studies on basic biology aspects are still scarce. The stingless bees are important for pollination of agricultural crops and native flora of tropical regions and income source as the meliponiculture. Therefore, new studies that provide information about the biology of Neotropical stingless bees and can collaborate with the conservation of the species by measuring the nest structures and to allow better handling conditions are necessary (Cortopassi-Laurino et al., 2016; Jaffé et al., 2015). Here, we present a description of the nest structure and an evaluation of adults and brood of P. droryana. Moreover, we provide a new method to estimate stingless bee colonies. Material and methods Study site The study was carried out in the Campus of the University of São Paulo at Ribeirão Preto, Brazil (21°10’39’’S, 47°48’24’’W). Five colonies of the stingless bee P. droryana were kept in hives made with wood (14.0 ́ 14.0 ́ 12 cm; walls: 2 cm; volume of 2.352 cm3) in the Laboratório de Comportamento e Ecologia de Insetos Sociais. The hives were connected to the outside via plastic tubes through which the bees could freely exit and enter in the nest. The bees collected pollen and nectar freely because they no received artificial feeding. Colony size We evaluated colony size by weighting P. droryana colonies (hereafter PD1, PD2, PD3, PD4 and PD5) between 17th September and 27th September 2013. We used carbon dioxide to anesthetize individuals and to preserve the structure of the nest. We weighted the combs, the involucrum wax and the adult individuals. We estimated the weight of food pots before removing the combs. Workers and males were weighted together, but males were found only in one nest. We counted the number of the food pots (honey and pollen), brood combs with egg, larvae (small and medium) and pupae, resin piles and garbage dump. We also estimated the population size of adult and immature individuals. To estimate adult population, we weighted separately 42 workers in order to know the individual weight, and divided by the total weight of nest individuals, representing the total estimated number of the nest. We used a formula to calculate the total weighted of adult bee population and nest material: Totalcol size = (In w + C + Boxs + Q + W) + Totalap where, the total colony size (Totalcol size) was equal to the sum of the involucrum wax (Inw), combs (C), box with structures (Boxs), queen (Q), waste (W) and the total weight of the adult individuals (Totalap - estimative). The waste was the variation found in the colonies weighted because the manipulation of the colonies and lost of some materials (wax, honey and others). To estimate immature individuals, we weighted one comb and counted the number of cells. We measured the comb (length x width) and related the number of cells with the number of combs for each nest (Fig 2). The data were analyzed with Microsoft Excel. Results and discussion Nest and colony estimative Nests had from 9 to 12 brood combs and 19 to 25 food pots. Also, there were one to four resin piles and one to three garbage dumps (Table 1). In the total combs in every nest, we found a proportion of three to four combs with eggs and larvae stage, and six to eight combs with pupae stage (Table 1). We counted one pollen pot only in the PD4 nest. In the PD1 nest, we observed one royal cell and one giant male individual (Fig 1; Table 1). YS Roldão-Sbordoni, FS Nascimento, S Mateus – Estimating Plebeia droryana colonies282 The characteristics of nest architecture in P. droryana were similar to those described by Zucchi and Sakagami (1972) and Wille and Michener (1973) in other species. We found the occurrence of an involucrum and the resin pile, typical of this species. In the brood area, the involucrum layers keep the temperature by retaining the heat production from brood metabolism (Wille, 1983; Engels et al., 1995; Barbosa et al., 2013). The presence of garbage dump deposit was observed in this study such as previously reported by Nogueira-Neto (1997) in other stingless bee species. Weight Estimative Although some authors described previously the nest structures, the weight of them was not reported as in our study. The weight of the five colonies ranged from 987.7 to 1109.9g (Table 2). The brood combs weighted 31g in the PD1 colony, 44.1g in the PD2 colony, 38.3g in the PD3 colony, A B C D Fig 1. Nest architecture of the stingless bee Plebeia droryana. (A) Food pots (honey and pollen). (B) Resin pile. (C) The nest with all structures. (D) The amount of the combs (brood area). Scale bar 3 mm. PD1 PD2 PD3 PD4 PD5 Mean ± SD Total brood combs 11 10 10 9 12 10.40 ± 1.14 Combs (eggs and small larvae) 3 4 3 3 4 3.40 ± 0.55 Combs (larvae medium and pupae) 8 6 7 6 8 7.00 ± 1.00 Total adult individuals estimative 2507 1548 2414 1917 1069 1891.00 ± 601.45 Total immature estimative 4158 5915 5137 4212 4727 4829.80 ± 727.25 Food pots 25 22 19 19 22 21.40 ± 2.51 Resin piles 1 4 2 2 1 2.00 ± 1.22 Garbage dump 1 3 3 3 2 2.40 ± 0.89 Table 1. Number of the nest structures, brood combs, adult individuals estimative and immature estimative and the mean ±SD, respectively. Fig 2. Brood comb of the Plebeia droryana used to estimate immatures. Scale bar = 3 cm. Sociobiology 65(2): 280-284 (June, 2018) 283 31.4g in the PD4 and PD5 colonies (mean of 35.2 ± 5.82g). The weight of the involucrum wax varied between 3.6 to 8.9g (mean±SD = 6.62 ± 2.0g) (Table 2). Queens weighted 0.045g (PD1 colony), 0.058g (PD2 colony), 0.053g (PD3 colony), 0.048g (PD4 colony) and 0.049g (PD5 colony). The average of the weight to the total adult individuals was of 13.6g to PD1 colony, 8.4g to PD2 colony, 13.1g to PD3 colony, 10.4 g to PD4 colony and 5.8g to PD5 colony (mean: 10.26 ± 3.26) (Table 2). Finally, the weight of foragers and comb workers were obtained (Fig 1) and we used the total weight (42 individuals) for the total individual nest estimative (Fig 3). The number estimated of adult individuals in the colonies was 2,507 (PD1), 1,548 (PD2), 2,414 (PD3), 1,917 (PD4) and 1,069 (PD5), mean of the 1,891 ± 601.45 (Table 1). The estimated number of immature individuals was 4,158 (PD1), 5,915 (PD2), 5,137 (PD3), 4,212 (PD4) and 4,727 (PD5), mean of the 4,829.8 ± 727.25 (Table 1). The weight of individuals has an important role for the estimative of the population. Duarte et al. (2016) have estimated nest architecture by measuring (in centimeters), and they found an approximate population of 50,927 individuals in T. clavipes by using a formula (Ihering, 1930). Our study PD1 PD2 PD3 PD4 PD5 Mean ± SD Total nest - hive 1075.3 987.7 1094.7 1042.6 1109.9 1062.04 ± 48.57 Involucrum wax 3.6 8.9 6.1 6.7 7.8 6.62 ± 2.00 Combs 31 44.1 38.3 31.4 31.4 35.24 ± 5.82 Empty wooden boxes 701.5 701.5 701.5 701.5 701.5 - Box with structures 878.1 916 702.3 985.1 1096.6 915.62 ± 145.34 Structures without the box 176.6 214.5 0.8 283.6 395.1 214.12 ± 145.32 Queen 0.045 0.0582 0.0536 0.0484 0.0499 0.051 ± 0.005 Total adult individuals 13.6 8.4 13.1 10.4 5.8 10.26 ± 3.26 Table 2. Weight of the structures and individuals in the five different nests (in grams) and the mean ± SD to each item. The weight of the empty wooden boxes was the average between all colonies studied. The “box with structures” refers to the weight of the wooden box with food pots, garbage dump and resin piles; and “structures without the box” refers to the weight without the mean of the empty wooden box, to calculate approximately the weight of the structures that remained inside the box. Individuals number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 W ei gh t ( g) 0,000 0,002 0,004 0,006 0,008 Foragers Comb workers showed an effective estimative by using the weight. Nests of P. droryana presented 1,069 to 2,507 individuals, observed also by Lindauer and Kerr (1960) that estimated to P. droryana between 2,000 and 3,000 individuals. Cortopassi- Laurino (1979) estimated the adults’ population of the P. droryana using another parameter. The colonies were considered “strong” when they had approximately 1,500 to 2,000 individuals and this result agreed with our estimated adult population (1,069 to 2,507). The immature estimate was of 4,727 individuals. Conclusion We provided a new method to estimate Plebeia droryana. The present study contributes to provide more details about the nests of this species and the estimate can be useful to further studies as well to the management and conservation of stingless bees. Acknowledgments The authors acknowledge to financial support by CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) and FAPESP (Proc. 2015/25301-9). Sam Morris helped with language editing. References Barbosa, F.M., Alves, R.M.O., Souza, B.A. & Carvalho, C.A.L. (2013). Nest architecture of the stingless bee Geotrigona subterranea (Friese, 1901) (Hymenoptera: Apidae: Meliponini). Biota Neotropica, 13: 147-152. Camargo, J.M.F. & Pedro, S.R.M. (1992). Systematics, phylogeny and biogeography of the Meliponinae (Hymenoptera, Apidae): a mini-review. Apidologie, 23: 509-522. Camargo, J.M.F. & Pedro, S.R.M. (2013). Meliponini Lepeletier, 1836. In: Moure JS, Urban D, Melo GAR (Orgs). Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region - Fig 3. Weight of individuals separately (21 foragers and 21 comb workers). The foragers were lighter because they have less adipose tissue for the flight (Michener, 1974). YS Roldão-Sbordoni, FS Nascimento, S Mateus – Estimating Plebeia droryana colonies284 online version. http://www.moure.cria.org.br/catalogue (Accessed date: 31 Aug, 2017). Cortopassi-Laurino, M. (1979). Observações sobre atividades de machos de Plebeia droryana Friese (Apidae, Meliponinae). Revista Brasileira de Entomologia, 23: 177-191. Cortopassi-Laurino, M., Imperatriz-Fonseca, V.L., Roubik, D.W., Dollin, A., Heard, T., Aguilar, I.B., Venturieri, G.C., Eardley, C. & Nogueira-Neto, P. (2006). Global Meliponiculture: challenges and opportunities. Apidologie, 37: 275-292. doi: 10.1051/apido:2006027. Duarte, R.S., Souza, J. & Soares, A.E.E. (2016). Nest architecture of Tetragona clavipes (Fabricius) (Hymenoptera: Apidae: Meliponini). Sociobiology, 63: 813-818. doi: 10. 13102/ sociobiology.v63i2.1019. Engels, W., Rosenkranz, P. & Engels, E. (1995). Thermo- regulation in the nest of the Neotropical stingless bee Scaptotrigona postica and a hypothesis on the evolution of temperature homeostasis in highly eusocial bees. Studies on Neotropical Fauna and Environment, 30: 193-205. doi: 10.1080/01650529509360958. Hansell, M.H. (1993). The ecological impact of animal nests and burrows. Functional Ecology, 7: 5-12. Ihering, H.V. (1930). Biologia das abelhas melliferas do Brasil. Boletim de Agricultura da Secretária da Agricultura do Estado São Paulo, 31: 435-506. Inoue, T., Sakagami, S.F., Salmah, S. & Yamane, S. (1984). The process of colony multiplication in the Sumatran stingless bee Trigona (Tetragonula) laeviceps. Biotropica, 16: 100- 111. doi: 10.2307/2387841. Jaffé, R., Pope, N., Carvalho, A.T., Maia, U.M., Blochtein, B., Carvalho, C.A.L., Carvalho-Zilse, G.A., Freitas, B.M., Menezes, C., Ribeiro, M.F., Venturieri, G.C. & Imperatriz- Fonseca, V.L. (2015). Bees for development: brazilian survey reveals how to optimize stingless beekeeping. Plos One. 10: 1-21. doi: 10.1371/journal.pone.0121157. Lindauer, M. & Kerr, W.E. (1960). Communication between the workers of stingless bees. Bee World. 41:29-41. Michener, C.D. (1946) Notes on the habits of some Panamanian stingless bees (Hymenoptera, Apidae). Journal of the New York Entomological Society, 54: 179-197. Michener, C.D. (1974) The social behavior of the bees – a comparative study. Cambridge: Harvard University Press. 404 p. Michener, C.D. (1990). Classification on the Apidae (Hyme- noptera). Lawrence: University of Kansas Science Bulletin, 54: 75-164. Michener, C.D. (2000). The bees of the world. Cambridge: University Press, 913 p. Moure, J.S., Urban, D. & Melo, G.A.R. (2007). Catalogue of Bees (Hymenoptera, Apoidea) in the Neotropical Region. Curitiba: Sociedade Brasileira de Entomologia, 1058 p. Nogueira-Neto, P. (1970). A criação de abelhas indígenas sem ferrão. São Paulo: Chácaras e Quintais, 365 p. Nogueira-Neto, P. (1997). Vida e criação de abelhas indígenas sem ferrão. São Paulo: Nogueirapis, 445 p. Rasmussen, C. & Camargo, J.M.F. (2008). A molecular phylogeny and the evolution of nest architecture and behavior in Trigona s. s. (Hymenoptera: Apidae: Meliponini). Apidologie, 39: 102-118. doi: 10.1051/apido:2007051. Roubik, D.W. & Peralta, F.J.A. (1983). Thermodynamics in nests of two Melipona species in Brazil. ACTA Amazônica, 13: 453-466. Roubik, D.W. (1989). Ecology and natural history of tropical bees. Cambridge: Cambridge University Press, 514 p. Roubik, D.W. (2006). Stingless bee nesting biology. Apidologie, 37: 124-143. doi: 10.1051/apido:2006026. Sakagami, S.F. (1982) Stingless bees. In: Herman HR (1982) Social Insects (pp. 361-423). London: Academic Press. Van-Veen, J.W. & Sommeijer, M.J. (2000). Colony reproduction in Tetragonisca angustula (Apidae, Meliponini). Insectes Sociaux, 47: 70-75. doi: 10.1007/s000400050011. Wille, A. (1983) Biology of the stingless bees. Annual Review of Entomology, 28: 41-64. Wille, A. & Michener, C.D. (1973). The nest architecture of stingless bees with special reference to those of Costa Rica (Hymenoptera: Apidae). Revista de Biología Tropical, 21: 1-278. Witter, S., Blochtein, B., Andrade, F., Wolff, L.F. & Imperatriz- Fonseca, V.L. (2007). Meliponicultura no Rio Grande do Sul: contribuição sobre a biologia e conservação de Plebeia nigriceps (Friese 1901) (Apidae, Meliponini). Bioscience Journal, 23: 134-140. Wittmann, D. (1989). Nest architecture, nest site preferences and distribution of Plebeia wittmanni Moure & Camargo, 1989 in Rio Grande do Sul, Brazil (Apidae, Meliponinae). Studies on Neotropical, Fauna and Environment, 24: 17-23. Zucchi, R. & Sakagami, S.F. (1972). Capacidade termo- reguladora em Trigona spinipes e em algumas espécies de abelhas sem ferrão (Hymenoptera: Apidae: Meliponinae). In: Homenagem a Warwick E. Kerr (pp. 301-309). Rio Claro.