Honey bees (Apis mellifera L.) are the most important pollinators of many agricultural crops worldwide and are a key test species used in the tiered safety assessment of genetically engineered insect-resistant crops. There is concern that widespread planting of these transgenic crops could harm honey bee populations.
We conducted a meta-analysis of 25 studies that independently assessed potential effects of Bt Cry proteins on honey bee survival (or mortality). Our results show that Bt Cry proteins used in genetically modified crops commercialized for control of lepidopteran and coleopteran pests do not negatively affect the survival of either honey bee larvae or adults in laboratory settings.
Although the additional stresses that honey bees face in the field could, in principle, modify their susceptibility to Cry proteins or lead to indirect effects, our findings support safety assessments that have not detected any direct negative effects of Bt crops for this vital insect pollinator.
Citation: Duan JJ, Marvier M, Huesing J, Dively G, Huang ZY (2008) A Meta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae). PLoS ONE 3(1): e1415. doi:10.1371/journal.pone.0001415
Academic Editor: Andy Hector, University of Zurich, Switzerland
Received: November 2, 2007; Accepted: December 14, 2007; Published: January 9, 2008
Copyright: © 2008 Duan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: M. Marvier was supported by EPA grant CR-832147-01.
Competing interests: Two of the authors, Jian Duan and Joseph Huesing, are employed by Monsanto Company, which produces and markets Bt crops.
Currently, all commercialized genetically engineered insect resistant crops are based on crystalline (Cry) proteins encoded by genes derived from the soil dwelling bacterium Bacillus thuringiensis (Bt). Studies on the mode of action and toxicology of Bt Cry proteins have established that these proteins are toxic to select groups of insects –. Cry proteins currently produced in commercialized Bt crops target insects in the orders Lepidoptera (moths) and Coleoptera (beetles). Because of this specificity, most experts feel it is unlikely that these Bt crops would impact honey bee (Hymenoptera: Apis mellifera L.) populations [e.g., 5], . Nevertheless, because of their importance to agriculture – the economic value of honey bee pollination for U.S. agriculture has been estimated to be worth $0.15–19 billion per year  – honey bees have been a key test species used in environmental safety assessments of Bt crops , . These assessments have involved comparisons of honey bee larval and adult survival on purified Cry proteins or pollen collected from Bt crops versus survival on non-Bt control material.
To date, no individual tests involving Bt crops or Cry proteins that target Lepidoptera or Coleoptera have shown significant impacts on honeybees , . Despite this, there have been suggestions in the popular press that Bt proteins produced in insect resistant crops might be contributing to recent declines in honeybee abundance , . Given this speculation about potential adverse impacts of Bt crops on honeybees and the possibility that small sample sizes may have undermined the power of prior risk assessment experiments (Table 1: studies to date have rarely employed more than 2–6 replicates per treatment), a formal meta-analysis, combining results from existing experiments, may provide more definitive answers. Meta-analysis increases statistical power and can reveal effects even when each of the individual studies failed to do so due to low replication , . A recent meta-analysis, synthesizing results from 42 field studies involving Bt cotton and maize , did not examine effects on honey bees because very few studies have reported field data for this group [but see 15]. Here we report a meta-analysis of 25 laboratory studies (Table 1) that focused on the chronic and/or acute toxicity of Bt Cry proteins or Bt plant tissues (pollen) on honey bee larvae and adults.
To locate studies of the nontarget effects of Bt crops for honey bees, we used multiple search criteria (e.g. Apis mellifera/honey bees and Bt/Bacillus thuringiensis) in the online databases Agricola, BioAbstracts, PubMed, and ISI Web of Science. Additional studies were found by searching the reference lists of empirical and review papers, performing general internet searches, and sending a list of references accompanied by a request for additional suggestions to over 100 researchers who are knowledgeable about studies of nontarget effects of Bt crops. Requests were also made under the US Freedom of Information Act to obtain relevant studies submitted by industry scientists to the US Environmental Protection Agency.
Studies had to meet a series of criteria in order to be included in this analysis. Specifically, studies had to: (i) involve Bt Cry proteins that are either lepidopteran-active (Cry1, Cry2, or Cry9 class) or coleopteran-active (Cry3 class) and that were either expressed in Bt plant tissues or produced by genetically modified B. thuringiensis, Escherichia coli, or Pseudomonas fluorescens strains (i.e. we excluded studies testing formulations of whole or lysed B. thuringiensis bacterial cells or spores, which might contain a mixture of different toxins, surfactants, and inert carrier ingredients) (ii) measure the effects of ingestion of the cry protein for honey bees of the species Apis mellifera; (iii) have occurred in a laboratory setting; (iv) report survival (or conversely mortality) as a response variable; (v) include a comparison to a non-transgenic control (typically sugar water or, for tissue studies, pollen from a non-transgenic plant variety); (vi) present treatment means, accompanied by standard deviations (s) and sample sizes (n) (or the author directly provided these values to us) necessary to calculate the metric of effect size, Hedges' d  (i.e., we required n1>0, n2>0, n1+n2>2, and s1(n1−1)+s2(n2−1)>0); and (vii) have been written in English. Measures of standard error, , were transformed to standard deviations () as needed. Available studies reported a range of response variables including survival, growth, development, and abundance. We focused only on survival (or mortality) data to maximize consistency among studies and reduce issues of non-independence when studies reported multiple metrics for the same sets of bees. Application of these criteria yielded data from a suite of 25 suitable publications or reports (Table 1). The Cry proteins used in these studies include those intended for use primarily in Bt corn, cotton, and potato. For those studies reporting data for multiple concentrations of a particular Cry protein, we included data for only the highest reported dosage. If data were reported as repeated measures over time for a particular life history stage (e.g. the number of adult bees alive on each of 14 days following dosage), we included data for only the final time point. Applying these criteria, in combination with the fact that several studies reported multiple independent experiments or measures of survival for multiple stages, yielded a total of 39 independent assessments of the effects of Bt proteins on honeybee survival (Table 1).
For each study, we recorded details about the Cry protein and its origin, the dose and duration of exposure, and the control treatment. When necessary, we scanned data figures and used Adobe Photoshop software to extract means and measures of within treatment variance. Authors provided raw data in several instances (noted in Table 1).
Quantitative data synthesis
Hedges' d was calculated for each study as the difference between the means of the Bt Cry protein and control treatments divided by the pooled standard deviation and weighted by the reciprocal of the sampling variance . The sign of Hedges' d was reversed for studies that reported mortality rather than survival. Negative values therefore indicate lower survival (whereas positive values indicate higher survival) in Bt Cry protein treatments compared to non-Bt control treatments. Bias-corrected bootstrapped 95% confidence intervals (CIs) were used to determine if specific effect sizes differed significantly from zero. Within group and between group heterogeneities were calculated using fixed effects models in MetaWin v.2 . Fixed effects models are generally considered to be inferior due to their bias toward finding effects (Type I bias) . However, Type I error is not an issue for any of our findings (see below), and we used this model deliberately to make the analysis less conservative in case Bt has weak effects. Moreover, mixed models collapse to fixed models when no variation remains after accounting for differences among groups and sampling error , and this was the case for all of the analyses presented here.
When all studies were combined, no statistically significant effect of Bt Cry protein treatments on survival of honey bees was detected (N = 39, d = 0.025, 95% CI = −0.128 to 0.171). When data for lepidopteran-active and coleopteran-active Bt Cry proteins were compared using a fixed categorical meta-analysis model, the above pattern of no significant effects held true for each class of protein (Fig. 1). No significant difference in effect sizes was detected between lepidopteran-active and coleopteran-active proteins (Q = 0.668, df = 1, P = 0.25); nor was any significant within-group heterogeneity detected for effect sizes calculated for either lepidopteran-active (Qw = 12.828, df = 29, P>0.99) or coleopteran-active proteins (Qw = 5.893, df = 8, P = 0.66). Mean effect sizes also did not differ (Q = 0.012, df = 1, P = 0.90) between studies that were peer-reviewed (N = 20, d = 0.015, 95% CI = −0.153 to 0.245) versus not peer-reviewed (N = 19, d = 0.039, 95% CI = −0.190 to 0.293).
Effect size is Hedge's d, and error bars represent bias-corrected bootstrap 95% confidence intervals. Positive mean effect sizes indicate improved survival when exposed to Cry proteins compared to water or sugar-water control treatments. N = number of lines of independent data summarized by each bar.
No significant effects on survival occurred with either larval or adult stages. This pattern was consistent when data from studies using lepidopteran-active and coleopteran-active Bt Cry proteins were analyzed either together (Fig. 2a) or separately (Fig. 2b&2c). No significant differences in effect sizes were detected between larvae and adults in any of the above analyses (Fig. 2a: Q = 0.093, df = 1, P = 0.69; Fig. 2b: Q = 0.298, df = 1, P = 0.47; Fig. 2c: Q = 0.064, df = 1, P = 0.80), nor were any significant within-group heterogeneities detected for the effect sizes calculated for either larvae (Fig. 2a: Qw = 9.523, df = 23, P>0.99; Fig. 2b: Qw = 3.875, df = 17, P>0.99; Fig. 2c: Qw = 4.746, df = 5, P = 0.45) or adults (Fig. 2a: Qw = 9.772, df = 14, P = 0.78; Fig. 2b: Qw = 8.656, df = 11, P = 0.65; Fig. 2c: Qw = 1.084, df = 2, P = 0.58).
The lack of adverse effects of Bt Cry proteins on both larval and adult honey bees is consistent with prior studies on the activity-spectrum and mode of action of different classes of Bt Cry proteins. To date, with the exception of a possible ant-specific Cry22 toxin patent application, no class of Bt Cry protein has been found to be directly toxic to hymenopteran insects . Although studies of acute toxicity performed in a laboratory setting may overlook sub-lethal or indirect effects that could potentially reduce the abundance of honeybees in a field setting, our findings strongly support the conclusion that the Cry proteins expressed in the current generation of Bt crops are unlikely to have adverse direct effects on the survival of honey bees. Additional analyses that included all available performance variables (survival, growth and development) similarly showed no adverse effect of Bt treatments. We do not report these results in depth here because they are potentially compromised by issues of non-independence – it is inappropriate to simultaneously include multiple measures taken on the same groups of bees. Unfortunately, few studies reported performance measures other than survival, and this prevented us from conducting separate analyses on these aspects of performance.
Although only laboratory data are synthesized here, the overall finding of no effect is consistent with the data available from a recent, well-replicated field study . Additionally, the fact that laboratory studies typically expose honey bees to doses of Cry proteins that are ten or more times those encountered in the field provides additional reassurance that toxicity in the field is unlikely. However, the need for additional studies in the field may be warranted if stressors such as heat, pesticides, pathogens, and so on are suspected to alter the susceptibility of honey bees to Cry protein toxicity.
Assessment of the potential risks of Bt crops for honey bees has become increasingly refined over time. However, these studies continue to be characterized by the use of very low replication with potentially limited statistical power. Based on retrospective power analyses of their data, Rose et al.  recommend that “laboratory studies to measure adult bee survival should test at least six cohorts of 50 bees per treatment to detect a 50% reduction with 80% statistical power.” However, this level of replication is 1.5–3 times greater than that used in many of the similar studies performed to date (Table 1). Modest increases in the replication of these and similar studies examining potential adverse effects of transgenic crops would likely help to improve public confidence in findings of no effect . In addition, meta-analysis of data from available studies testing similar hypothesis is an effective tool for quantitatively synthesizing the collective evidence regarding the safety of genetically modified crops.
We thank Chanel McCreedy, Christina Mogren, and Kathleen Powers (all from Santa Clara University) and James Regetz (National Center for Ecological Analysis and Synthesis) for assistance with the database. Thanks also to Roy Fuchs (Monsanto Company) for helpful comments.
Conceived and designed the experiments: MM JD. Analyzed the data: MM JD. Wrote the paper: MM JD ZH JH GD.
- q. Maggi VL (1993) Evaluation of the dietary effect(s) of B.t.t. protein on honey bee larvae: California Agricultural Research Inc. U.S. EPA MRID number: 429322-09.
- 1. Mendelhsohn M, Kough J, Vaituzis Z, Mathews K (2003) Are Bt crops safe? Nat Biotechnol 21: 1003–1009.
- 2. Pigott C, Ellar DJ (2007) Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiol. Molecular Biol Rev 71: 255–281.
- 3. OECD (2007) Consensus document on safety information on transgenic plants expressing Bacillus thuringiensis - derived insect control proteins. OECD Environment, Health and Safety Publications Series on Harmonization of Regulatory Oversight in Biotechnology, 2007 No. 42. Paris Environment Directorate, Organisation for Economic Co-operation and Development.
- 4. de Maagd RA, Bravo A, Berry C, Crickmore N, Schnepf HE (2003) Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu Rev Genet 37: 409–433.
- 5. O'Callaghan M, Glare TR, Burgess EPJ, Malone LA (2005) Effects of plants genetically modified for insect resistance on nontarget organisms. Annu Rev Entomol 50: 271.
- 6. Malone LA, Pham-Delegue MH (2001) Effects of transgene products on honey bees (Apis mellifera) and bumblebees (Bombus sp.). Apidologie 32: 287–304.
- 7. Council NR (2007) Status of Pollinators in North America. Washington, DC: National Academies Press.
- 8. OECD (1998) Guideline 213: Honey bees, acute oral toxicity test. Paris: OECD.
- 9. U. S. EPA (1996) Microbial Pesticide Test Guidelines. OPPTS 885.4380 Honey Bee Testing, Tier I. Washington, D.C.: United States Environmental Protection Agency.
- 10. Latsch G (2007) Collapsing colonies: are GM crops killing bees? Spiegel Online International.
- 11. McDonald J (2007) Could genetically modified crops be killing bees? San Francisco Chronicle: F4.
- 12. Arnqvist G, Wooster D (1995) Meta-analysis: synthesizing research findings in ecology and evolution. Trends Ecol Evol 10: 236–240.
- 13. Sutton A, Jones D, Abrams K, Sheldon T, Song F (2000) Methods for Meta-analysis in Medical Research. London: John Wiley.
- 14. Marvier M, McCreedy C, Regetz J, Kareiva P (2007) A meta-analysis of effects of Bt cotton and maize on non-target invertebrates. Science 316: 1475–1477.
- 15. Rose R, Dively GP, Pettis J (2007) Effects of Bt corn pollen on honey bees: Emphasis on protocol development. Apidologie 38: 368–377.
- 16. Gurevitch J, Hedges LV (1993) Meta-analysis: combining results of independent experiments. In: Scheiner SM, Gurevitch J, editors. Design and Analysis of Ecological Experiments. New York: Chapman and Hall. pp. 378–398.
- 17. Rosenberg MS, Adams DC, Gurevitch J (2000) MetaWin: Statistical Software for Meta-Analysis Version 2. Sunderland, MA: Sinauer.
- 18. Hunter JE, Schmidt FL (2000) Fixed effects vs. random effects meta-analysis models: Implications for cumulative research knowledge. Int J Select Assess 8: 275–292.
- 19. Gurevitch J, Hedges LV (1999) Statistical issues in ecological meta-analyses. 80: 1142–1149.
- 20. Marvier MA (2002) Improving risk assessment for nontarget safety of transgenic crops. Ecol Appl 12: 1119.
- 21. 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: 315–319.
- 22. Hanley AV, Huang ZY, Pett WL (2003) Effects of dietary transgenic Bt corn pollen on larvae of Apis mellifera and Galleria mellonella. J Apic Res 42: 77–81.
- 23. Liu B, Xu C, Yan F, Gong R (2005) The impacts of the pollen of insect-resistant transgenic cotton on honey bees. Biodiversity Conserv 14: 3487–3496.
- 24. Maggi VL (1993) Evaluation of the dietary effect(s) of B.t.k. endotoxin proteins on honey bee adults: California Agricultural Research Inc., U.S. EPA MRID number: 431452-07.
- 25. Maggi VL (1993) Evaluation of the dietary effect(s) of B.t.k. endotoxin proteins on honey bee larvae: California Agricultural Research Inc., U.S. EPA MRID number: 431452-06.
- 26. Maggi VL (1993) Evaluation of the dietary effect(s) of B.t.t. protein on honey bee adults: California Agricultural Research Inc. U.S. EPA MRID number: 429322-10.
- 28. Maggi VL (1996) Evaluation of the dietary effect(s) of purified B.t.t. protein on honey bee larvae: California Agricultural Research Inc. U.S. EPA MRID number: 441247-02.
- 29. Maggi VL (1999) Evaluation of the dietary effects of purified Bacillus thuringiensis 11231 on honey bee larvae: U.S. EPA MRID number: 449043-10.
- 30. Maggi VL (1999) Evaluation of the dietary effects of purified Bacillus thuringiensis 11231 on adult honey bees (Apis mellifera L.): U.S. EPA MRID number: 449043-11.
- 31. Maggi VL (1999) Evaluation of the dietary effect(s) on honey bee development using bacterially expressed Bt Cry1F delta-endotoxin and pollen from maize expressing Bt Cry1F delta-endotoxin: U.S. EPA MRID number: 450415-03.
- 32. Maggi VL (2000) Evaluation of the dietary effect(s) of insect protection protein 2 on adult honey bee (Apis mellifera L.): U.S. EPA MRID number: 450863-08.
- 33. Maggi VL (2000) Evaluation of the dietary effect(s) of insect protection protein 2 on honey bee larvae: U.S. EPA MRID number: 450863-07.
- 34. Maggi VL (2000) Evaluation of the dietary effects of purified Bacillus thuringiensis Cry2Ab2 protein on honey bee larvae: U.S. EPA MRID number: 453371-02.
- 35. Maggi VL (2002) Evaluation of the dietary effects of a Cry3Bb1 protein variant on adult honey bees (Apis mellifera L.): Monsanto Company MSL-17762.
- 36. Maggi VL (2002) Evaluation of the dietary effects of a Cry3Bb1 protein variant on honey bee larvae (Apis mellifera L.): Monsanto Company MSL-17761.
- 37. Maggi VL, Sims S (1994) Evaluation of the dietary effect(s) of B.t.k. endotoxin proteins on honey bee adults: California Agricultural Research Inc. and Monsanto Company. U.S. EPA MRID number: 434392-03.
- 38. Maggi VL, Sims S (1994) Evaluation of the dietary effect(s) of B.t.k. endotoxin proteins on honey bee larvae: California Agricultural Research Inc. and Monsanto Company. U.S. EPA MRID number: 434392-02.
- 39. Malone LA, Burgess EPJ, Stefanovic D (1999) Effects of a Bacillus thuringiensis toxin, two Bacillus thuringiensis biopesticide formulations, and a soybean trypsin inhibitor on honey bee (Apis mellifera L.) survival and food consumption. Apidologie 30: 465–473.
- 40. Malone LA, Burgess EPJ, Gatehouse HS, Voisey CR, Tregidga EL, et al. (2001) Effects of ingestion of a Bacillus thuringiensis toxin and a trypsin inhibitor on honey bee flight activity and longevity. Apidologie 32: 57–68.
- 41. Malone LA, Todd JH, Burgess EPJ, Christeller JT (2004) Development of hypopharyngeal glands in adult honey bees fed with a Bt toxin, a biotin-binding protein and a protease inhibitor. Apidologie 35: 655–664.
- 42. Palmer SJ, Krueger HO (1997) Cry9C protein in corn pollen: A dietary toxicity study with the honey bee (Apis mellifera): Plant Genetics Systems, N.V. U.S. EPA MRID number: 443843-02.
- 43. Sims SR (1995) Bacillus thuringiensis var. kurstaki [Cry1A(c)] protein expressed in transgenic cotton: Effects on beneficial and other non-target insects. Southwest Entomol 20: 493–500.
- 44. Sims SR (1997) Host activity spectrum of the CryIIA Bacillus thuringiensis subsp. kurstaki protein: Effects on lepidoptera, diptera, and non-target arthropods. Southwest Entomol 22: 395–404.