Advertisement

Apidologie

, Volume 43, Issue 5, pp 549–560 | Cite as

Effects of multiple Bt proteins and GNA lectin on in vitro-reared honey bee larvae

  • Harmen P. HendriksmaEmail author
  • Stephan Härtel
  • Dirk Babendreier
  • Werner von der Ohe
  • Ingolf Steffan-Dewenter
Original article

Abstract

The honey bee is a key non-target arthropod in environmental risk assessments of genetically modified crops. We analyzed for the first time combined effects of three Bt proteins conferring insect resistances, and a CP4-protein conferring an herbicide resistance as simultaneously expressed in one GM maize. Furthermore, the biosafety of Galanthus nivalis agglutinin (GNA lectin), a candidate protein for pest control was tested. Under worst-case exposure scenario, by using controlled in vitro larvae rearing, the combination of Bt proteins showed no adverse effects on bee larvae. In contrast, the GNA lectin was toxic at a 144 h LD50 of 16.3 μg/larva. The prepupal weight was found to differ between the larvae collection days and between the colonies used for the experiment, explaining up to five times more data variance than the protein treatments (N = 709 prepupae). In conclusion, neither single nor a mix of different Bt proteins were found harmful to honey bee larvae.

Keywords

Apis mellifera Bacillus thuringiensis environmental risk assessment genetically modified crops Cry protein 

Notes

Acknowledgements

This is publication No. 1 produced within the project Assessing and Monitoring the Impacts of Genetically Modified Plants on Agro-ecosystems (AMIGA), funded by the European Commission in the Framework programme 7. THEME [KBBE.2011.3.5-01]. This study was also funded by the German Federal Ministry of Education and Research (Bundesministerium für Bildung und Forschung BMBF, project 0315215E). The experiments were performed at the Universities of Bayreuth and Würzburg (Bavaria).

Supplementary material

13592_2012_123_MOESM1_ESM.pdf (2.6 mb)
Supplement P Photographs of the in vitro rearing experiments (PDF 2709 kb)
13592_2012_123_MOESM2_ESM.doc (44 kb)
Supplement S Post hoc survival statistics between treatment differences (DOC 44 kb)
13592_2012_123_MOESM3_ESM.doc (32 kb)
Supplement W Post hoc statistics on prepupal weight (DOC 32 kb)

References

  1. Andersen, M.E., Dennison, J.E. (2004) Mechanistic approaches for mixture risk assessments—present capabilities with simple mixtures and future directions. Environ. Toxicol. Pharmacol. 16, 1–11PubMedCrossRefGoogle Scholar
  2. Arpaia, S. (1996) Ecological impact of Bt-transgenic plants: 1 Assessing possible effects of CryIIIB toxin on honey bee (Apis mellifera L) colonies. J. Genet. Breed. 50, 315–319Google Scholar
  3. Aupinel, P., Fortini, D., Michaud, B., Marolleau, F., Tasei, J.N., Odoux, J.F. (2007) Toxicity of dimethoate and fenoxycarb to honey bee brood (Apis mellifera), using a new in vitro standardized feeding method. Pest Manage Sci. 63, 1090–1094CrossRefGoogle Scholar
  4. Babendreier, D., Kalberer, N., Romeis, J., Fluri, P., Bigler, F. (2004) Pollen consumption in honey bee larvae: a step forward in the risk assessment of transgenic plants. Apidologie 35, 293–300CrossRefGoogle Scholar
  5. Babendreier, D., Kalberer, N.M., Romeis, J., Fluri, P., Mulligan, E., Bigler, F. (2005) Influence of Bt-transgenic pollen, Bt-toxin and protease inhibitor (SBTI) ingestion on development of the hypopharyngeal glands in honeybees. Apidologie 36, 585–594CrossRefGoogle Scholar
  6. Babendreier, D., Reichhart, B., Romeis, J., Bigler, F. (2008) Impact of insecticidal proteins expressed in transgenic plants on bumblebee microcolonies. Entomol. Exp. Appl. 126, 148–157CrossRefGoogle Scholar
  7. Belzunces, L.P., Lenfant, C., Di Pasquale, S., Colin, M.E. (1994) In vivo and in vitro effects of wheat germ agglutinin and Bowman-Birk soybean trypsin inhibitor, two potential transgene products, on midgut esterase and protease activities from Apis mellifera. Comp. Biochem. Physiol. 109, 63–69CrossRefGoogle Scholar
  8. Chambers J.M. (1992) Linear models. Chapter 4 of Statistical Models in S eds. J.M. Chambers, T.J. Hastie, Wadsworth & Brooks/Cole.Google Scholar
  9. Cox D.R., Oakes D. (1990) Analysis of survival data London: Chapman & Hall 201 p.Google Scholar
  10. Crailsheim, K., Schneider, L.H.W., Hrasnigg, N., Bühlmann, G., Brosch, U., Gmeinbauer, R., Schöffmann, B. (1992) Pollen consumption and utilization in worker honeybees (Apis mellifera carnica): dependence on individual age and function. J. Insect Physiol. 38, 409–419CrossRefGoogle Scholar
  11. De Maagd, R.A., Bravo, A., Crickmore, N. (2001) How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends Genet. 17, 193–199PubMedCrossRefGoogle Scholar
  12. Duan, J.J., Marvier, M., Huesing, J., Dively, G., Huang, Z.Y. (2008) A Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae). PLoS One 3, e1415PubMedCrossRefGoogle Scholar
  13. Duan, J.J., Lundgren, J.G., Naranjo, S., Marvier, M. (2010) Extrapolating non-target risk of Bt crops from laboratory to field. Biol. Lett. 6, 74–77PubMedCrossRefGoogle Scholar
  14. European Food Safety Authority (2010) Scientific opinion on application (EFSA-GMO-NL-2007-39) for the placing on the market of insect resistant and herbicide tolerant genetically modified maize MON89034 × MON88017 for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto, EFSA Journal, 8, Article 1564.Google Scholar
  15. Finney, D.J. (1971) Probit Analysis. Cambridge University Press Cambridge, UKGoogle Scholar
  16. Fox, J. (2002) An R and S-PLUS companion to applied regression. Sage, Thousand Oaks, CA. 312 pGoogle Scholar
  17. Glare, T.R., O'Callaghan, M. (2000) Bacillus thuringiensis: Biology, Ecology and Safety. John Wiley and Sons Ltd, Chichester, UKGoogle Scholar
  18. Hanley, A.V., Huang, Z.Y., Pett, W.L. (2003) Effects of dietary transgenic Bt corn pollen on larvae of Apis mellifera and Galleria mellonella. J. Apic. Res. 42, 77–81Google Scholar
  19. Hendriksma, H.P., Härtel, S., Steffan-Dewenter, I. (2011a) Honey bee risk assessment: New approaches for in vitro larvae rearing and data analyses. Meth. Ecol. Evol. 2, 509–517CrossRefGoogle Scholar
  20. Hendriksma, H.P., Härtel, S., Steffan-Dewenter, I. (2011b) Testing pollen of single and stacked insect-resistant Bt-maize on in vitro reared honey bee larvae. PLoS One 6, e28174. doi: 10.1371/journal.pone.0028174 PubMedCrossRefGoogle Scholar
  21. Jaber K., Haubruge E., Francis F. (2010) Development of entomotoxic molecules as control agents: illustration of some protein potential uses and limits of lectins (Review), Biotech. Agron. Soc. Environ.14, 225-241.Google Scholar
  22. James C. (2010) Global Status of Commercialized Biotech/GM Crops: 2009 ISAAA Brief No 42 ISAAA: Ithaca, NY.Google Scholar
  23. Jung-Hoffmann, I. (1966) Die Determination von Königin und Arbeiterin der Honigbiene. Z. Bienenforsch. 8, 296–322Google Scholar
  24. Khasdan, V., Sapojnik, M., Zaritsky, A., Horowitz, A.R., Boussiba, S., Rippa, M., Manasherob, R., Ben-Dov, E. (2007) Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis. Arch. Microbiol. 188, 643–653PubMedCrossRefGoogle Scholar
  25. Klein, A.M., Vaissiere, B.E., Cane, J.H., Steffan-Dewenter, I., Cunningham, S.A., Kremen, C., Tscharntke, T. (2007) Importance of pollinators in changing landscapes for world crops. Proc. R. Soc. London 274, 303–313CrossRefGoogle Scholar
  26. Konrad, R., Ferry, N., Gatehouse, A.M.R., Babendreier, D. (2008) Potential Effects of Oilseed Rape Expressing Oryzacystatin-1 (OC-1) and of Purified Insecticidal Proteins on Larvae of the Solitary Bee Osmia bicornis. PLoS One 3, e2664PubMedCrossRefGoogle Scholar
  27. Lee, M.K., Curtiss, A., Alcantara, E., Dean, D.H. (1996) Synergistic Effect of the Bacillus thuringiensis Toxins CryIAa and CryIAc on the Gypsy Moth, Lymantria dispar. Appl. Environ. Microbiol. 62, 583–586PubMedGoogle Scholar
  28. Lehrman, A. (2007) Does pea lectin expressed transgenically in oilseed rape (Brassica napus) influence honey bee (Apis mellifera) larvae? Environ. Biosaf. Res. 6, 271–278CrossRefGoogle Scholar
  29. Lima, M.A.P., Pires, C.S.S., Guedes, R.N.C., Nakasu, E.Y.T., Lara, M.S., Fontes, E.M.G., Sujii, E.R., Dias, S.C., Campos, L.A.O. (2011) Does Cry1Ac Bt-toxin impair development of worker larvae of Africanized honey bee? J. Appl. Entomol. 135, 415–422CrossRefGoogle Scholar
  30. Malone L.A., Burgess E.P.J. (2009) Impact of Genetically Modified Crops on Pollinators. pp 199–224 in: Environmental Impact of Genetically Modified Crops Eds. N. Ferry, A.M.R. Gatehouse, CAB International, Oxfordshire, UK.Google Scholar
  31. Monsanto Company (2009) Application for authorization of MON89034 × MON88017 maize in the European Union, according to Regulation (EC) No 1829/2003 on genetically modified food and feed, Accessed 11 Mar 2011: www.gmo-compassorg/pdf/regulation/maize/MON89034xMON88017_applicationpdf.
  32. Niu, G.D., Johnson, R.M., Berenbaum, M.R. (2011) Toxicity of mycotoxins to honeybees and its amelioration by propolis. Apidologie 42, 79–87CrossRefGoogle Scholar
  33. Padgette, S.R., Kolacz, K.H., Delannay, X., Re, D.B., La Vallee, B.J., Tinius, C.N., Rhodes, W.K., Otero, Y.I., Barry, G.F., Eichholz, D.A., Peschke, V.M., Nida, D.L., Taylor, N.B., Kishore, G.M. (1995) Development, identification and characterization of a glyphosate-tolerant soybean line. Crop Sci. 35, 1451–1461CrossRefGoogle Scholar
  34. Pardo-López, L., Muñoz-Garay, C., Porta, H., Rodríguez-Almazán, C., Soberón, M., Bravo, A. (2009) Strategies to improve the insecticidal activity of Cry toxins from Bacillus thuringiensis. Pept. 30, 589–595Google Scholar
  35. Peterson, R.K., Shama, L.M. (2005) A comparative risk assessment of genetically engineered, mutagenic, and conventional wheat production systems. Transgenic Res. 14, 859–875PubMedCrossRefGoogle Scholar
  36. Peumans, W.J., Smeets, K., Van Nerum, K., Van Leuven, F., Van Damme, E.J.M. (1997) Lectin and alliinase are the predominant proteins in nectar from leek (Allium porrum L) flowers. Planta 201, 298–302PubMedCrossRefGoogle Scholar
  37. Pigott, C.R., King, S.M., Ellar, D.J. (2008) Investigating the properties of Bacillus thuringiensis Cry-proteins with novel loop replacements created using combinatorial molecular biology. Appl. Environ. Microbiol. 74, 3497–3511PubMedCrossRefGoogle Scholar
  38. Porcar, M., Gomez, F., Gruppe, A., Gomez-Pajuelo, A., Segura, I., Schroder, R. (2008) Hymenopteran specificity of Bacillus thuringiensis strain PS86Q3. Biol Control 45, 427–432CrossRefGoogle Scholar
  39. Potts, S.G., Biesmeijer, J.C., Kremen, C., Neumann, P., Schweiger, O., Kunin, W.E. (2010) Global pollinator declines: trends, impacts and drivers. Trends Ecol. Evol. 25, 345–353PubMedCrossRefGoogle Scholar
  40. R Development Core Team (2010). R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org.
  41. Romeis, J., Babendreier, D., Wackers, F.L. (2003) Consumption of snowdrop lectin (Galanthus nivalis agglutinin) causes direct effects on adult parasitic wasps. Oecologia 134, 528–536PubMedGoogle Scholar
  42. Romeis, J., Bartsch, D., Bigler, F., Candolfi, M.P., Gielkens, M.M.C., Hartley, S.E., Hellmich, R.L., Huesing, J.E., Jepson, P.C., Layton, R., Quemada, H., Raybould, A., Rose, R.I., Schiemann, J., Sears, M.K., Shelton, A.M., Sweet, J., Vaituzis, Z., Wolt, J.D. (2008) Assessment of risk of insect-resistant transgenic crops to nontarget arthropods. Nat. Biotechnol. 26, 203–208PubMedCrossRefGoogle Scholar
  43. Romeis, J., Hellmich, R.L., Candolfi, M.P., Carstens, K., De Schrijver, A., Gatehouse, A.M., Herman, R.A., Huesing, J.E., McLean, M.A., Raybould, A., Shelton, A.M., Waggoner, A. (2011) Recommendations for the design of laboratory studies on arthropods for risk assessment of genetically engineered plants. Transgenic Res. 20, 1–22PubMedCrossRefGoogle Scholar
  44. Romeis, J., Meissle, M., Bigler, F. (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nat. Biotechnol. 24, 63–71PubMedCrossRefGoogle Scholar
  45. Seeley, T.D. (1985) Honey bee ecology. Princeton University Press, New JerseyGoogle Scholar
  46. Sharma, P., Nain, V., Lakhanpaul, S., Kumar, P.A. (2010) Synergistic activity between Bacillus thuringiensis toxins against maize stem borer (Chilo partellus Swinhoe). Lett. Appl. Microbiol. 51, 42–47PubMedGoogle Scholar
  47. Simpson, J. (1955) The significance of the presence of pollen in the food of worker larvae of the honey-bee. Quart. J. Microscop. Sci. 96, 117–120Google Scholar
  48. Steinrücken, H.C., Amrhein, N. (1980) The herbicide glyphosate is a potent inhibitor of 5-enolpyruvyl-shikimic acid-3-phosphate synthase. Biochem. Biophys. Res. Commun. 94, 1207–1212PubMedCrossRefGoogle Scholar
  49. Stewart, S.D., Adamczyk, J.J., Knighten, K.S., Davis, F.M. (2001) Impact of Bt cottons expressing one or two insecticidal proteins of Bacillus thuringiensis Berliner on growth and survival of noctuid (Lepidoptera) larvae. J. Econ. Entomol. 94, 752–760PubMedCrossRefGoogle Scholar
  50. Wolt, J.D. (2011) A mixture toxicity approach for environmental risk assessment of multiple insect resistance genes. Environ. Toxicol. Chem. 30, 1–10CrossRefGoogle Scholar
  51. Yao, H.W., Jiang, C.Y., Ye, G.Y., Hu, C., Peng, Y.F. (2008) Toxicological assessment of pollen from different Bt rice lines on Bombyx mori (Lepidoptera: Bombyxidae). Environ. Entomol. 37, 825–837PubMedCrossRefGoogle Scholar
  52. Zuur A.F., Gende L.B., Ieno E.N., Fernandez N.J., Eguares M.J., Fritz R., Walker N.J., Saveliev A.A., Smith G.M. (2009) Mixed Effects Modelling Applied on American Foulbrood Affecting Honey Bees Larvae. Mixed effects models and extensions in ecology with R (eds A.F. Zuur, E.N. Ieno, N.J. Walker, A.A. Saveliev, G.M. Smith), pp. 447–458. Springer Science + Business Media, New York.Google Scholar

Copyright information

© INRA, DIB and Springer-Verlag, France 2012

Authors and Affiliations

  • Harmen P. Hendriksma
    • 1
    Email author
  • Stephan Härtel
    • 1
  • Dirk Babendreier
    • 2
  • Werner von der Ohe
    • 3
  • Ingolf Steffan-Dewenter
    • 1
  1. 1.Department of Animal Ecology and Tropical Biology, BiocentreUniversity of WürzburgWürzburgGermany
  2. 2.CABI-Europe SwitzerlandDelèmontSwitzerland
  3. 3.Institut für Bienenkunde (Institute for Apidology)LAVES (Lower Saxony State Office for Consumer Protection and Food Safety)CelleGermany

Personalised recommendations