No Bt Resistance?

Prof. Joe Cummins questions the recent report that there has been no Bt resistance outbreaks

Worldwide, over 62 million hectares have been planted with Bt crops – GM crops engineered with Bt toxins from soil bacterium Bacillus thuringiensis - and proponents have expressed pleased surprise that Bt resistant insects do not seem to have evolved [1,2]. But there’s more than meets the eye.

Bt toxins do not represent a single gene product but are products of different genes, variants of which are present in different strains of the bacterium. Bacillus thuringiensis is fairly common in soil and creek beds, but the varieties capable of strong insect control are rare and valuable. Strains containing multiple unique toxins are designated israelensis (Bti), kurstaki (Btk), azaiwai (Bta), tenbrionis (Btt) sotto (Bts), and entomocidus (Bte), etc. The strains are differently specific for insects of the Order Lepidoptera, Diptera or Coleoptera. The individual isolated toxin proteins are designated CryI, CryII , CryIII or CryIV, but each of these may require further identification related to small sequence differences. For example, .a toxin may be designated CryIA(b), CryIIIA, CryIVD, etc. [3]. Genes for the toxins introduced into a crop plant are usually altered to enhance their activity. Some codons are modified to those preferred by plants in contrast to bacteria. Usually, an intron is introduced into the bacterial gene to enhance rapid translocation from the plant nucleus to the cytoplasm. Examples of alterations of Cry genes to enhance activity are included in patents [4,5].

Insects evolve resistant to individual Cry toxins and cross-resistance appears to be limited. Resistance is most frequently due to nuclear genes, rather than cytoplasmic genes encoded by chloroplasts and mitochondria. Resistance can be recessive, requiring two copies of the resistance allele (variant of a gene) to give protection against Bt toxin; dominant, requiring only one copy of the resistance allele to give full protection against Bt toxin; or incompletely recessive, where one copy of the allele gives partial protection. Incompletely recessive alleles are recessive at high toxin levels but become dominant as the toxin level decreases [6,7].

The specificity of resistance to Bt toxin is demonstrated in laboratory experiments with the cotton bollworm. Resistant bollworm thrives on a diet containing Cry as well as on cotton modified with a gene for Cry1Ac. But this resistant bollworm was susceptible to commercial Bt spore formulations Dipel and XenTari, which contains multiple toxins. The bollworm was resistant to Cry1Ab but not to Cry2Aa or Cry 2Ab [8].

Thus, although millions of hectares have been planted with Bt crops, the target for developing Bt resistance is much smaller because there are many different Bt toxins to which the insects must develop resistance independently. In theory, a devastating resistance to all Bt toxins could evolve, but this has not yet been observed in laboratory or field experiments. A non-recessive Cry1Ac-resistant mutant of tobacco budworm showed cross-resistance to a wide array of Bt toxins [9], but such mutations are infrequent.

As mentioned above [1,2], there have been no outbreaks of resistant pests in Bt crops, although such outbreaks have been observed in sprayed populations of diamond back moth, and many laboratory experiments produced Bt-resistant insects [10].

Bt toxins kill by binding to target sites in cell membranes of the mid-gut and disrupt the membranes. One prominent mutation in resistant bollworm involves cadherin, an adhesion protein that binds together cells in solid tissue, thereby preventing disruption of the gut cells [10]. Recently, incomplete recessive alleles of Cry1Ac and Cry2Aa have been identified in bollworm during screening of Bt-cotton crops [11]. Apparently, the finding was not considered an "outbreak", even though it could be the start of one.

To stave off the impending threat of resistance outbreaks, regulators have introduced the ‘refuge’ strategy, the planting of non-Bt crops to prevent or slow the evolution of resistance. The refuge strategy is based on the assumption that resistance will be recessive, so sensitive heterozygotes will die from consuming Bt crop. If the mutation is dominant or or incompletely recessive, resistance will spread despite the refuge.

Greenhouse tests showed that the refuge could prevent the spread of resistant mutants if it was maintained as a block of non-Bt crop, rather than as a mixed crop of Bt and non Bt plants [12]. Regulators in North America have set a minimum of 20% non-Bt crop in block-planting.

The introduction of the refuge has meant that farmers would have to deal with the potential of 20% of their crops becoming infested, so regulators allowed the refuge to be sprayed with pesticide. In a position paper produced by the Environment Protection Agency and the United States Department of Agriculture, it states that [13], "In corn growing areas (no cotton), growers should plant a minimum of 20% non-Bt corn to serve as a refuge. In areas where European corn borer (ECB), southwestern corn borer (SWCB), corn earworm (CEW), or other target lepidopteran pests have historically been high, insecticide treatment of the refuge is anticipated."

Academics have lent their authority to affirm that spraying the refuge with pesticide was necessary to control emerging resistance [14,15]. It seems clear, however, that regulators not only permit, but positively encourage pesticide spraying over not just the refuge but the entire crop [13]. The refuge strategy is really a "double whammy" strategy! And yet, the pesticide spray is hardly mentioned in government documents promoting refuges [16], or in numerous academic publications on resistance management or in association with the "miraculous" absence of Bt-resistance outbreaks.

That there have been no reported major outbreaks of Bt resistant insects in the millions of hectares of Bt crop planted may be due to two major factors that have been overlooked. The first is the numerous unique Bt alleles used in Bt-crops, and the second is the simultaneous deployment of chemical pesticide sprays in the non-Bt refuge as well as on Bt-crops.

References

1. Tabashnik B, Carriere T, Denneity T, Morin S, Sisterson M, Roush R, Shelton A and Zhao J. Insect resistance to transgenic BT crops : Lessons from the laboratory and field. J Econ Entomol 2003, 96, 1031-8.
2. Fox J. Resistance to Bt toxin surprisingly absent from pests. Nature Biotech 2003, 21,458-9.
3. Bauer L. Resistance: A threat to the insecticidal crystal proteins of Bacillus thuringiensis. Florida entomologist 1995, vol?78, 414-44
4. Baum J, Gilmer A and Mettus A. Lepidopteran resistant transgenic plants. United States Patent 2001, 6,313,378B1 pp1-151
5. Carrozi N, Rabe S, Miles P, Waren G, and DeHaan P. "ovel insecticidal toxins derived from Bacillus thuringiensis insecticidal crystal proteins. World Intelectual Property Organization 2002, WO02/15701 A2 pp1-130.
6. Liu Y, Tabashnik B, Meyer S, Carriere Y and Bartlett A. Genetics of Pink Bollworm resistance to Bacillus thuringiensis toxin Cry1Ac. J Econ Entomol 2001, 94, 248-52.
7. Tabashnik B, Liu Y, Dennehy T, Sims M, Sisterson M, Biggs R and Carriere Y Inheritance of resistance to Bt toxin Cry 1Ac in a field derived strain of pink bollworm (Lepidoptera:Gelechiidae). J Econ Entomol 2002, 95,1018-26.
8. Akhurst R, James W, Bird L, and Beard C. Resistance to the Cry 1 Ac delta endotoxin of Bacillus thuringiensis in the cotton bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae). J. Econ Entomol 2003, 96,1290-9.
9. Gould F, Martinez-Ramirez A, Anderson A, Fere J, Silva F and Moar W. Broad spectrum resistance to Bacillus thuringiensis toxins in Heliothis virescens. Proc. Natnl. Acad Sci USA 1992, 89,7986-90.
10. Morin S, Bigs R, Sisterson M et al. Three cadherin alleles associated with resistance to Bacillus thuringiensis in pink bollworm" PNAS 2003, 100, 5004-9.
11. Burd A, Gould F, Bradley J, VanDuyn J and Moar W. Estimated frequency of non recessive Bt resistance genes in Bollworm, Helicoverpa zea (Boddie) (Lepidoptera:Notuidae) in eastern North Carolina, J Econ Entomol 2003, 96,137-42.
12. Tang J, Collins H, Metz E, Earle E, Zhoa J, Roush R. and Shelton A. Greenhouse tests on resistance management of Bt transgenic plants using refuge strategies. J Econ Entomol 2001, 94,240-7.
13. EPA and USDA Position Paper "EPA and USDA position on resistance management" 1999 http://www.mindfully.org/GE/EPA-USDA-Position-27may99.htm
14. Ives A and Andow D. Evolution of resistance to Bt crops: directional selection in structured environments. Ecology Letters 2002, 5,792-801.
15. Onstad D, Guse C, Porter P, Ruschman L, Higgins R, Sloderbeck P, Peairs (spelling?OK) F and Cronholm G. Modeling the development of resistance by stalk boring Lepidopteran Insects (Crambidae) in areas with transgenic corn and frequent pesticide use. J Econ Entomol 2003, 95,1033-43
16. Macdonald P and Yarrow S. Regulation of Bt crops in Canada. J Invertebrate Pathology 2003, 83, 93-9.

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