Insectes Sociaux

, Volume 60, Issue 1, pp 111–118 | Cite as

A technique to artificially inseminate leafcutter ants

  • S. P. A. den BoerEmail author
  • J. J. Boomsma
  • B. Baer
Technical Article


Artificial insemination (AI), the instrumental transfer of semen from the male to female reproductive organs, offers excellent opportunities to study mating system adaptations as it allows paternity to be experimentally manipulated. AI techniques have been developed for many animals, but rarely for ants, where they would be particularly useful as most species do not mate under controlled lab conditions. Here, we describe an AI technique for Atta leafcutter ants involving (1) the collection of ejaculates via induction of natural ejaculation, (2) storage in glass capillaries, and (3) transfer to queens using a modified AI equipment as used for honeybees. Queens were fixed and anesthetized in a queen holder, after which the sting chamber was opened with two steel hooks, the tip of the semen-containing glass capillary was inserted into the bursa copulatrix and the semen slowly expelled. Sperm was successfully stored in the spermatheca of queens, and some queens produced a small colony as a result. We could furthermore confirm the earlier observations that Atta semen is directly transferred to the spermatheca rather than to the bursa copulatrix as in most other eusocial insects. The technique that we present here can offer novel opportunities to study mating events such as sperm transfer, sperm competition, and cryptic female choice in ants. At present, the number of queens that produce colonies after AI remains low. However, this number will likely increase, as our results indicate that rearing conditions after AI influence colony founding success of artificially inseminated Atta queens.


Ejaculate Mating biology Social insects Spermatheca Sperm Seminal fluid 



We thank the Smithsonian Tropical Research Institute (STRI) in Panama for facilities and logistic support and the Autoridad Nacional de Ambiente (ANAM) for issuing collection permits. This work was supported by a Marie Curie International Outgoing Fellowship to SPAdB, a grant from the Danish National Research Foundation to JJB and several grants from the Australian Research Council to BB.


  1. Aiken D.E., Waddy S.L., Moreland K. and Polar S.M. 1984. Electrically induced ejaculation and artificial insemination of the American lobster Homarus americanus. J. Crust. Biol. 4: 519-527Google Scholar
  2. Axner E. 2008. Updates on reproductive physiology, genital diseases and artificial insemination in the domestic cat. Reprod. Domest. Anim. 43: 144-149Google Scholar
  3. Baer B., Armitage S.A.O. and Boomsma J.J. 2006. Sperm storage induces an immunity cost in ants. Nature 441: 872-875Google Scholar
  4. Baer B. and Boomsma J. 2004. Male reproductive investment and queen mating-frequency in fungus-growing ants. Behav. Ecol. 15: 426 - 432Google Scholar
  5. Baer B. and Boomsma J.J. 2006. Mating biology of the leaf-cutting ants Atta colombica and A. cephalotes. J. Morphol. 267: 1165-1171Google Scholar
  6. Baer B., Morgan E.D. and Schmid-Hempel P. 2001. A nonspecific fatty acid within the bumblebee mating plug prevents females from remating. Proc. Natl Acad. Sci. U.S.A. 98: 3926-3928Google Scholar
  7. Baer B. and Schmid-Hempel P. 1999. Experimental variation in polyandry affects parasite loads and fitness in a bumble-bee. Nature 397: 151-154Google Scholar
  8. Baer B. and Schmid-Hempel P. 2000. The artificial insemination of bumblebee queens. Insect. Soc. 47: 183-187Google Scholar
  9. Ball D.E., Mirenda J.T., Sorensen A.A. and Vinson S.B. 1983. Instrumental insemination of the fire ant, Solenopsis invicta. Entomol. Exp. Appl. 33: 195-202Google Scholar
  10. Benzie J.A.H., Kenway M., Ballment E., Frusher S. and Trott L. 1995. Interspecific hybridization of the tiger prawns Penaeus monodon and Penaeus esculentus. Aquaculture 133: 103-111Google Scholar
  11. Boomsma J.J., Keller L. and Nielsen M.G. 1995. A comparative analysis of sex-ratio investment parameters in ants. Funct. Ecol. 9: 743-753Google Scholar
  12. Chen M., Zhang G. and Mainka S.A. 1994. Semen evaluation of giant pandas (Ailuropoda melanoleuca) at the Wolong Reserve. Zoo Biol. 13: 83-86Google Scholar
  13. Chen S.H. and Wallach E.E. 1994. Five decades of progress in management of the infertile couple. Fertil. Steril. 62: 665-685Google Scholar
  14. Cobey S.W. 2007. Comparison studies of instrumentally inseminated and naturally mated honey bee queens and factors affecting their performance. Apidologie 38: 390-410Google Scholar
  15. Collins A.M. 2004. Functional longevity of honey bee, Apis mellifera, queens inseminated with low viability semen. J. Apic. Res. 43: 167-171Google Scholar
  16. Davis N.T. 1964. Studies of the reproductive physiology of Cimicidae (Hemiptera) II. Artificial insemination and the function of the seminal fluid. J. Insect Physiol. 11: 355-366Google Scholar
  17. de Miranda J.R. and Fries I. 2008. Venereal and vertical transmission of deformed wing virus in honeybees (Apis mellifera L.). J. Invertebr. Pathol. 98: 184-189Google Scholar
  18. den Boer S.P.A., Baer B. and Boomsma J.J. 2010. Seminal fluid mediates ejaculate competition in social insects. Science 327: 1506-1509Google Scholar
  19. den Boer S.P.A., Baer B., Dreier S., Aron S., Nash D.R. and Boomsma J.J. 2009a. Prudent sperm use by leafcutter ant queens. Proc. R. Soc. Lond., B, Biol. Sci 276: 3945-3953Google Scholar
  20. den Boer S.P.A., Boomsma J.J. and Baer B. 2008. Seminal fluid enhances sperm viability in the leafcutter ant Atta colombica. Behav. Ecol. Sociobiol. 62: 1843-1849Google Scholar
  21. den Boer S.P.A., Boomsma J.J. and Baer B. 2009b. Honey bee males and queens use glandular secretions to enhance sperm viability before and after storage. J. Insect Physiol. 55: 538-543Google Scholar
  22. Duvoisin N., Baer B. and Schmid-Hempel P. 1999. Sperm transfer and male competition in a bumblebee. Anim. Behav. 58: 743-749Google Scholar
  23. Evans G. and Maxwell W.M.C. 1987. Salamons Artificial Insemination of Sheep and Goats. Butterworths, Sydney.Google Scholar
  24. Evison S. and Hughes W. 2011. Genetic caste polymorphism and the evolution of polyandry in Atta leaf-cutting ants. Naturwissenschaften 98: 643-649Google Scholar
  25. Fjerdingstad E.J. and Boomsma J.J. 1997. Variation in size and sperm content of sexuals in the leafcutter ant Atta colombica. Insect. Soc. 44: 209-218Google Scholar
  26. Fjerdingstad E.J. and Boomsma J.J. 1998. Multiple mating increases the sperm stores of Atta colombica leafcutter ant queens. Behav. Ecol. Sociobiol. 42: 257-261Google Scholar
  27. Fjerdingstad E.J., Boomsma J.J. and Thorén P. 1998. Multipe paternity in the leafcutter ant Atta colombica - a microsatellite DNA study. Heredity 80: 118-126Google Scholar
  28. Foote R.H. 2002. The history of artificial insemination: Selected notes and notables. J. Anim. Sci. 80: 1-10.Google Scholar
  29. Hölldobler B. and Bartz S.H. 1985. Sociobiology of reproduction in ants. In: Experimental Behavioral Ecology (Hölldobler B., Ed). G. Fischer Verlag, Stuttgart. pp 237-257Google Scholar
  30. Holt W.V., Abaigar T. and Jabbour H.N. 1996. Oestrous synchronization, semen preservation and artificial insemination in the Mohor Gazelle (Gazella dama mhorr) for the establishment of a genome resource bank programme. Reprod. Fertil. Dev. 8: 1215-1222Google Scholar
  31. Katsumata E. 2010. Study on reproduction of captive marine mammals. J. Reprod. Dev. 56: 1-8Google Scholar
  32. Kocher S.D., Tarpy D.R. and Grozinger C.M. 2010. The effects of mating and instrumental insemination on queen honey bee flight behaviour and gene expression. Insect Mol. Biol. 19: 153-162Google Scholar
  33. Korner P. and Schmid-Hempel P. 2003. Effects of sperm on female longevity in the bumblebee Bombus terrestris L. Proc. R. Soc. Lond. B, Biol. Sci 270: 227-229Google Scholar
  34. Locke S. and Peng Y. 1993. The effects of drone age, semen storage and contamination on semen quality in the honey-bee (Apis mellifera). Physiol. Entomol. 18: 144-148Google Scholar
  35. Mackensen O. and Roberts W.C. 1948. A Manual for the Artificial Insemination of Queen Bees. U.S. Dept. of Agriculture, Agricultural Research Administration, Bureau of Entomology and Plant Quarantine, 33 ppGoogle Scholar
  36. Mintzer A. 1987. Primary polygyny in the ant Atta texana: Number and weight of females and colony foundation success in the laboratory. Insect. Soc. 34: 108-117Google Scholar
  37. Richard F.J., Tarpy D.R. and Grozinger C.M. 2007. Effects of insemination quantity on honey bee queen physiology. Plos One 2: 9Google Scholar
  38. Roth T.L., Armstrong D.L., Barrie M.T. and Wildt D.E. 1997. Seasonal effects on ovarian responsiveness to exogenous gonadotrophins and successful artificial insemination in the snow leopard (Uncia uncia). Reprod. Fertil. Dev. 9: 285-295Google Scholar
  39. Ruttner F. 1975. Die instrumentelle Besamung der Bienenkönigin. Apimondia Publishing House, Bucharest.Google Scholar
  40. Schley P. 1987. Einführung in die Technik der instrumentellen Besamung von Bienenköniginnen. Köhler Offset KG, Giessen.Google Scholar
  41. Stürup M., den Boer S.P.A., Nash D.R., Boomsma J.J. and Baer B. 2011. Variation in male body size and reproductive allocation in the leafcutter ant Atta colombica: estimating variance components and possible trade-offs. Insect. Soc. 58: 47-55Google Scholar
  42. Takemura Y., Kanda T., Tamura T., Shinbo H. and Horie Y. 1996. Development of new method for artificial insemination of the silkworm, Bombyx mori. J. Seric. Sci. Jpn. 65: 456-463Google Scholar
  43. Weber N.A. 1972. Gardening Ants, the Attines. The American Philosophical Society, Philadelphia.Google Scholar
  44. Wiernasz D.C. and Cole B.J. 2003. Queen size mediates queen survival and colony fitness in harvester ants. Evolution 57: 2179-2183Google Scholar
  45. Wildt D.E. and Roth T.L. 1997. Assisted reproduction for managing and conserving threatened felids. Int. Zoo Yb. 35: 164-172Google Scholar
  46. Yue C., Schroder M., Gisder S. and Genersch E. 2007. Vertical transmission routes for deformed wing virus of honeybees (Apis mellifera). J. Gen. Virol. 88: 2329-2336Google Scholar

Copyright information

© International Union for the Study of Social Insects (IUSSI) 2012

Authors and Affiliations

  • S. P. A. den Boer
    • 1
    • 2
    • 3
    Email author
  • J. J. Boomsma
    • 1
  • B. Baer
    • 2
    • 3
  1. 1.Department of Biology, Centre for Social EvolutionUniversity of CopenhagenCopenhagenDenmark
  2. 2.Centre for Integrative Bee Research, ARC CoE in Plant Energy Biology, MCS Building M316The University of Western AustraliaCrawleyAustralia
  3. 3.Centre for Evolutionary Biology, School of Animal Biology (MO92)The University of Western AustraliaCrawleyAustralia

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