Journal of Insect Conservation

, Volume 17, Issue 5, pp 1071–1079 | Cite as

Non-lethal method of DNA sampling in euglossine bees supported by mark–recapture experiments and microsatellite genotyping

  • Cíntia Akemi OiEmail author
  • Margarita M. López-Uribe
  • Marcelo Cervini
  • Marco Antonio Del Lama


Non-lethal sampling methods are of great interest for conservation genetic studies to prevent the death of individuals in populations that are threatened or in decline. With this aim, we tested a non-lethal method of partial antennae removal for DNA sampling in two euglossine bee species: Euglossa cordata and Eulaema nigrita. We validated the survival of the individuals through mark–recapture experiments during 16 months. The quality and quantity of the tissue for DNA analysis was verified through amplification and genotyping of nine and eleven microsatellite loci, respectively. Our results from the mark–recapture experiments showed equal recapture rates of individuals with intact and removed antennae (E. cordata χ2 = 2.492, df = 1, p = 0.114; E. nigrita χ2 = 1.683, df = 1, p = 0.194). Microsatellite loci were successfully genotyped in 97.1 and 97.6 % of the E. cordata and E. nigrita individuals, respectively. Our results validate the feasibility of using antennae tissue for DNA genetic analysis without compromising the survival of individual bees.


Tissue sampling Antennae Orchid bees Euglossa cordata Eulaema nigrita 



We acknowledge support from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior) for the master degree fellowship to C. A. Oi and from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico) and FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo, Processo 2004/15801-0) for the financial research support. We are thankful to N. Mortari for laboratory setting courtesy, and C. H. da Silva, J. C. Almeida and O. Lino e Silva for help in the field. We also would like to thank F. T. T. Hanashiro, K. Nagamati Jr., L. Packer, J. Gibbs and three anonymous reviewers for commentaries and suggestions that improved this manuscript.


  1. Ackerman JD, Montalvo AM (1985) Longevity of Euglossine bees. Biotropica 17(1):79–81CrossRefGoogle Scholar
  2. Allendorf FW, Holenlohe PA, Luikart G (2010) Genomics and the future of conservation genetics. Nat Rev Genet 11(10):697–709. doi: 10.1038/nrg2844 PubMedCrossRefGoogle Scholar
  3. Augusto S, Garófalo CA (2011) Task allocation and interactions among females in Euglossa carolina nests (Hymenoptera, Apidae, Euglossini). Apidologie 42(2):162–173. doi: 10.1051/apido/2010040 CrossRefGoogle Scholar
  4. Beja-Pereira A, Oliveira R, Alves PC, Schwartz MK, Luikart G (2009) Advancing ecological understandings through technological transformations in noninvasive genetics. Mol Ecol Resour 9:1279–1301. doi: 10.1111/j.1755-0998.2009.02699.x PubMedCrossRefGoogle Scholar
  5. Bischoff I (2003) Population dynamics of the solitary digger bee Andrena vaga Panzer (Hymenoptera, Andrenidae) studied using mark–recapture and nest counts. Popul Ecol 45(3):197–204. doi: 10.1007/s10144-003-0156-6 CrossRefGoogle Scholar
  6. Bouga M, Klossa-Kilias E, Alahiotis S, Killias G (2005) Non-lethal DNA sampling of wing tips discriminates subspecies of Apis mellifera occurring in Greece. J Apic Res 44(4):195–196. doi: 10.3896/IBRA. Google Scholar
  7. Brosi BJ (2009) The effects of forest fragmentation on euglossine bee communities (Hymenoptera: Apidae: Euglossini). Biol Conserv 142(2):414–423. doi: 10.1016/j.biocon.2008.11.003 CrossRefGoogle Scholar
  8. Campanella JJ, Smalley JV (2006) A minimally invasive method of piscine tissue collection and an analysis of long-term field-storage conditions for samples. BMC Genet 7:32. doi: 10.1186/1471-2156-7-32 PubMedCrossRefGoogle Scholar
  9. Casquet J, Thebaud C, Gillespie RG (2012) Chelex without boiling, a rapid and easy technique to obtain stable amplifiable DNA from small amounts of ethanol-stored spiders. Mol Ecol Resour 12:136–141. doi: 10.1111/j.1755-0998.2011.03073.x PubMedCrossRefGoogle Scholar
  10. Cerântola NCM, Oi CA, Cervini M, Del Lama MA (2010) Genetic differentiation of urban populations of Euglossa cordata Linnaeus 1758 from the state of São Paulo, Brazil. Apidologie 42(2):214–222. doi: 10.1051/apido/2010055 CrossRefGoogle Scholar
  11. Châline N, Ratnieks FLW, Raine NE, Badcock NS, Burke T (2004) Non-lethal sampling of honey bee, Apis mellifera, DNA using wing tips. Apidologie 35(3):311–318. doi: 10.1051/apido:2004015 CrossRefGoogle Scholar
  12. Chaves PB, Paes MF, Mendes SL, Strier KB, Louro ID, Fagundes V (2006) Noninvasive genetic sampling of endangered muriqui (Primates, Atelidae): efficiency of fecal DNA extraction. Genet Mol Biol 29(4):750–754. doi: 10.1590/S1415-47572006000400028 CrossRefGoogle Scholar
  13. Cohen J (1969) Statistical power analysis for the behavioral sciences (1st ed). New York, Academic Press. doi: 10.1126/science.169.3941.167 Google Scholar
  14. Desjardins P, Conklin D (2010) NanoDrop microvolume quantitation of nucleic acids. J Vis Exp 45. doi: 10.3791/2565
  15. Donald HM, Wood CW, Benowitz KM, Johnson RA, Brodie ED, Formica VA (2012) Nondestructive sampling of insect DNA from defensive secretion. Mol Ecol Resour 12(5):856–860. doi: 10.1111/j.1755-0998.2012.03154.x PubMedCrossRefGoogle Scholar
  16. EMBRAPA. Embrapa dados meteorológicos [Internet]. Embrapa Pecuária Sudeste. Accessed 06 May 2012
  17. Faul F, Erdfelder E, Lang A-G, Buchner A (2007) G*Power 3: a flexible statistical power analysis program for the social, behavioural, and biomedical sciences. Behav Res Methods 39:175–191. doi: 10.3758/BF03193146 PubMedCrossRefGoogle Scholar
  18. Fincke O, Hadrys H (2001) Unpredictable offspring survivorship in the damselfly. Megaloprepus coerulatus, shapes parental behavior, constrains sexual selection, and challenges traditional fitness estimates. Evolution 55(4):762–772. doi: 10.1111/j.0014-3820.2001.tb00812.x PubMedCrossRefGoogle Scholar
  19. Freiria GA, Ruim JB, Souza RF, Sofia SH (2012) Population structure and genetic diversity of the orchid bee Eufriesea violacea (Hymenoptera, Apidae, Euglossini) from Atlantic forest remnants in southern and southeastern Brazil. Apidologie 43(4):392–402. doi: 10.1007/s13592-011-0104-y CrossRefGoogle Scholar
  20. Gould EM, Taylor MA, Holmes SJ (2011) A more consistent method for extracting and amplifying DNA from bee wings. Apidologie 42:721–727. doi: 10.1007/s13592-011-0077-x CrossRefGoogle Scholar
  21. Gregory PG, Rinderer TE (2004) Non-destructive sources of DNA used to genotype honey bee (Apis mellifera) queens. Entomol Exp Appl 111(3):173–177. doi: 10.1111/j.0013-8703.2004.00164.x CrossRefGoogle Scholar
  22. Holehouse K, Hammond R, Bourke A (2003) Non-lethal sampling of DNA from bumble bees for conservation genetics. Insectes Soc 50(3):277–285. doi: 10.1007/s00040-003-0672-6 CrossRefGoogle Scholar
  23. Horváth MB, Martinez-Cruz B, Negro JJ, Kalmár J, Godoy JA (2005) An overlooked DNA source for non-invasive genetic analysis in birds. J Avian Biol 36(1):84–88. doi: 10.1111/j.0908-8857.2005.03370.x CrossRefGoogle Scholar
  24. Hoy M (2013) Insect molecular genetics: an introduction to principles and applications, 3rd edn. Academic Press, San Diego, 2003, 544 pp. ISBN: 0-12-357031-XGoogle Scholar
  25. Imms A (1939) Memoirs: on the antennal musculature in insects and other arthropods. Q J Microsc Sci 2:273–320Google Scholar
  26. Jander R (1976) Grooming and pollen manipulation in bees (Apoidea): the nature and evolution of movements involving the foreleg. Physiol Entomol 1(3):179–194. doi: 10.1111/j.1365-3032.1976.tb00960.x CrossRefGoogle Scholar
  27. Koch LM, Patullo BW, Macmillan DL (2006) Exploring with damaged antennae: do crayfish compensate for injuries? J Exp Biol 209:3226–3233. doi: 10.1242/jeb.02368 PubMedCrossRefGoogle Scholar
  28. Köppen W (1931) Grundriss der Klimakunde. Walter de Gruyter, BerlinGoogle Scholar
  29. Koscinski D, Crawford LA, Keller HA, Keyghobadi N (2011) Effects of different methods of non-lethal tissue sampling on butterflies. Ecol Entomol 36(3):301–308. doi: 10.1111/j.1365-2311.2011.01272.x CrossRefGoogle Scholar
  30. López-Uribe MM, Oi CA, Del Lama MA (2008) Nectar-foraging behavior of euglossine bees (Hymenoptera: Apidae) in urban areas. Apidologie 39(4):410–418. doi: 10.1051/apido:2008023 CrossRefGoogle Scholar
  31. Milet-Pinheiro P, Schlindwein C (2005) Do euglossine males (Apidae, Euglossini) leave tropical rainforest to collect fragrances in sugarcane monocultures? Rev Bras Zool 22(4):853–858. doi: 10.1590/S0101-81752005000400008 CrossRefGoogle Scholar
  32. Miotto RA, Cervini M, Figueiredo MG, Begotti RA, Galetti PM (2011) Genetic diversity and population structure of pumas (Puma concolor) in southeastern Brazil: implications for conservation in a human-dominated landscape. Conser Genet 12(6):1447–1455. doi: 10.1007/s10592-011-0243-8 CrossRefGoogle Scholar
  33. Monroe EM, Lynch C, Soluk DA, Britten HB (2010) Nonlethal tissue sampling techniques and microsatellite markers used for first report of genetic diversity in two populations of the endangered Somatochlora hineana (Odonata: Corduliidae). Ann Entomol Soc Am 103(6):1012–1017. doi: 10.1603/AN10088 CrossRefGoogle Scholar
  34. Peakall R, Schiestl FP (2004) A mark–recapture study of male Colletes cunicularius bees: implications for pollination by sexual deception. Behav Ecol Sociobiol 56(6):579–584. doi: 10.1007/s00265-004-0816-3 CrossRefGoogle Scholar
  35. Peakall R, Smouse PE (2006) Genalex 6: genetic analysis in Excel. Population genetics software for teaching and research. Mol Ecol Notes 6(1):288–295. doi: 10.1111/j.1471-8286.2005.01155.x CrossRefGoogle Scholar
  36. Piggott MP, Taylor AC (2003) Remote collection of animal DNA and its applications in conservation management and understanding the population biology of rare and cryptic species. Wildl Res 30(1):1–13. doi: 10.1071/WR02077 CrossRefGoogle Scholar
  37. Roubik DW (2001) Ups and downs in pollinator populations: when is there a decline? Conserv Ecol 5(1):2. Google Scholar
  38. Scriven JJ, Woodall LC, Goulson D (2013) Non-destructive DNA sampling from bumblebee faeces. Mol Ecol Resour 13(2):225–229. doi: 10.1111/1755-0998.12036 PubMedCrossRefGoogle Scholar
  39. Sheppard WS, Mcpheron BA (1991) Ribosomal DNA diversity in Apidae. In: Smith DR (ed) Diversity in the genus Apis. Westview Press, Boulder, CO, pp 89–102Google Scholar
  40. Sofia SH, Paula FM, Santos AM, Almeida FS, Sodré LMK (2005) Genetic structure analysis of Eufriesea violacea (Hymenoptera, Apidae) populations from southern Brazilian Atlantic rainforest remnants. Genet Mol Biol 28(3):479–484. doi: 10.1590/S1415-47572005000300026 CrossRefGoogle Scholar
  41. Souza RO, Cervini M, Del Lama DA, Paxton RJ (2007) Microsatellite loci for euglossine bees (Hymenoptera: Apidae). Mol Ecol Notes 7(6):1352–1356. doi: 10.1111/j.1471-8286.2007.01878.x CrossRefGoogle Scholar
  42. Starks PT, Peters JM (2002) Semi-nondestructive genetic sampling from live eusocial wasps Polistes dominulus and Polistes fuscatus. Insectes Soc 49:20–22. doi: 10.1007/s00040-002-8272-4 CrossRefGoogle Scholar
  43. Suzuki KM, Arias MC, Giangarelli DC, Freiria GA, Sofia SH (2010) Mitochondrial DNA diversity of orchid bee Euglossa fimbriata (Hymenoptera: Apidae) populations assessed by PCR-RFLP. Biochem Genet 48(3–4):326–341. doi: 10.1007/s10528-009-9325-4 PubMedCrossRefGoogle Scholar
  44. Suzuki G, Inoda T, Kubota S (2012) Nonlethal sampling of DNA from critically endangered diving beetles (Coleoptera: Dytiscidae) using a single antenna. Entomol Sci 15(3):352–356. doi: 10.1111/j.1479-8298.2012.00523.x CrossRefGoogle Scholar
  45. Taberlet P, Waits L, Luikart G (1999) Noninvasive genetic sampling: look before you leap. Trends Ecol Evol 14(8):323–327. doi: 10.1016/S0169-5347(99)01637-7 PubMedCrossRefGoogle Scholar
  46. Thompson DJ, Watts PC, Saccheri IJ (2007) Conservation genetics for insects. In: Stewart AJA, New TR, Lewis OT (eds) Insect conservation biology CABI Publishing, Wallingford, pp 280–300. doi: 10.1079/9781845932541.0280
  47. Tonhasca AJ, Albuquerque GS, Blackmer JL (2003) Dispersal of euglossine bees between fragments of the Brazilian Atlantic forest. J Trop Ecol 19(1):99–102. doi: 10.1017/S0266467403003122 CrossRefGoogle Scholar
  48. Valière N, Bonenfant C, Toϊgo C, Luikart G, Gaillard JM, Klein F (2007) Importance of a pilot study for non-invasive sampling: genotyping errors and population size estimations in red deer. Conserv Genet 8(1):69–78. doi: 10.1007/s10592-006-9149-2 CrossRefGoogle Scholar
  49. Van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P (2004) Micro-checker: software for identifying and correcting genotyping errors in microsatellite data. Mol Ecol Notes 4(3):535–538. doi: 10.1111/j.1471-8286.2004.00684.x CrossRefGoogle Scholar
  50. Vila M, Auger-Rozenberg MA, Goussard F, Lopez-Vaamonde C (2009) Effect of non-lethal sampling on life-history traits of the protected moth Graellsia isabelae (Lepidoptera: Saturniidae). Ecol Entomol 34(3):356–362. doi: 10.1111/j.1365-2311.2008.01084.x CrossRefGoogle Scholar
  51. Walsh PS, Metzger DA, Higuchi R (1991) Chelex® 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10(4):506–513PubMedGoogle Scholar
  52. Wasko AP, Martins C, Oliveira C, Foresti F (2003) Non-destructive genetic sampling in fish. An improved method for DNA extraction from fish fins and scales. Hereditas 138(3):161–165. doi: 10.1034/j.1601-5223.2003.01503.x PubMedCrossRefGoogle Scholar
  53. Watts P, Thompson DJ, Daguet C, Kemp SJ (2005) Exuviae as a reliable source of DNA for population-genetic analysis of odonates. J Soc Int Odonatologica 34(2):183–187Google Scholar
  54. Wikelski M, Moxley J, Eaton-Mordas A, López-Uribe MM et al (2010) Large-range movements of neotropical orchid bees observed via radio telemetry. PLoS ONE 5(5):e10738. doi: 10.1371/journal.pone.0010738 PubMedCrossRefGoogle Scholar
  55. Wilkinson L, Engelman L (2010) SYSTAT. WIREs Comp. Stat 2:256–257. doi: 10.1002/wics.66 Google Scholar
  56. Zayed A (2009) Bee genetics and conservation. Apidologie 40(3):237–262. doi: 10.1051/apido/2009026 CrossRefGoogle Scholar
  57. Zimmermann Y, Schorkopf DLP, Moritz RFA, Pemberton RW, Quezada-Euan JJG, Eltz T (2011) Population genetic structure of orchid bees (Euglossini) in anthropogenically altered landscapes. Conserv Genet 12:1183–1194. doi: 10.1007/s10592-011-0221-1 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Cíntia Akemi Oi
    • 1
    • 4
    Email author
  • Margarita M. López-Uribe
    • 2
  • Marcelo Cervini
    • 3
  • Marco Antonio Del Lama
    • 1
  1. 1.Laboratório de Genética Evolutiva de Himenópteros, Departamento de Genética e EvoluçãoUniversidade Federal de São CarlosSão CarlosBrazil
  2. 2.Department of EntomologyCornell UniversityIthacaUSA
  3. 3.Departamento de Ciências BiológicasUniversidade Estadual do Sudoeste da BahiaJequiéBrazil
  4. 4.Laboratory of Socioecology and Social Evolution, Zoological InstituteUniversity of LeuvenLeuvenBelgium

Personalised recommendations