Will the Widespread Use of Bt Crops Lead to the Development of Insect Resistance to Bt?

Bacillus thuringiensis (Bt), a widespread soil bacterium, produces insecticidal proteins called Bt toxins (1). There are many Bt strains that produce characteristic sets of toxins, each with its own activity spectrum that targets larvae of specific insect species. For example, some Bt toxins kill larvae of particular species of moths and butterflies; others kill larvae of certain species of beetles or mosquitoes. Bt sprays have been used to control insects since the 1920s (1), but use of specific Bt toxins has increased dramatically since 1996 with the introduction of GE crops.

Bt toxins are also called Cry toxins because they exist as crystals inside the bacterium. Full length Cry toxins are inactive until cleaved to generate their active form in the insect midgut (2, 3). Binding of activated forms of Cry toxins to receptors in the midgut is generally believed to be essential for toxicity. According to one model (4, 5), after binding to midgut receptors, activated toxins form oligomers that create pores in midgut membranes, causing contents to leak, ultimately killing the larvae. The precision of Bt proteins for certain insects and their lack of effects in mammals are due to the specificity of receptor binding (6).

Bt corn producing Cry1Ab and Bt cotton producing Cry1Ac account for the majority of the 494 million acres (200 million hectares) (hectare = ha = 2.47 acres) of Bt crops grown worldwide during the ten-year period from 1996 to 2007 (8). These two Bt crops kill some key lepidopteran pests, including European corn borer (Ostrinia nubilalis) on maize and pink and cotton bollworm (Pectinophora gossypiella and Helicoverpa armigera) and tobacco budworm (Heliothis virescens) on cotton. As of November 2011, total of 2041 applications have been filed with the Animal and Plant Health Inspection Service (APHIS) of the US Department of Agriculture (USDA) to conduct small-scale field tests, with six denied, 35 pending and 378 withdrawn (9). Of these small scale field tests, APHIS has issued permits for 131 field tests of GE Bt varieties, although the actual number conducted is not known. These field tests represent 17 different plant species engineered with Bt genes; these include alfalfa, apple, corn, cotton, cranberry, eggplant, grape, peanut, persimmon, poplar, potato, rapeseed, rice,  tobacco, tomato, walnut and white spruce.

Evolution of insect resistance to Bt toxins can reduce the long-term effectiveness of Bt crops (10, 11, 12, 13). Strains of many pests have been selected for resistance to Bt toxins in the laboratory, and two lepidopteran insects, Plutella xylostella and Trichoplusia ni, have evolved resistance to Bt sprays in the field and in greenhouses, respectively (14, 15). The primary strategy in the field for delaying insect resistance to Bt crops is planting refuges of non-Bt crops near Bt crops (16, 10, 12). This strategy is based on the idea that insects feeding on plants in the refuge are not selected for resistance, because those plants do not make Bt toxins. Under ideal conditions, insect resistance to Bt toxins is recessive. Thus, heterozygous offspring, produced when homozgygous resistant insects mate with susceptible insects, are killed by the Bt crop. Models predict that resistance can be postponed substantially if the rare homozygous resistant insects surviving on a Bt crop mate with the more abundant susceptible insects from refuges (10, 11). The strategy is called the high-dose/refuge strategy because the created plants produce Bt toxin concentrations high enough to kill heterozygous insects, thereby making resistance functionally recessive (12).

In the United States and some other countries, refuges of non-Bt crops are required (16). A 2005 survey showed that U.S. farmers believe refuges are effective in managing resistance (17); 91% of farmers were found to meet the regulatory requirements for refuges associated with Bt corn (18). A study of Bt cotton revealed compliance with the refuge strategy was higher than 88% in five of six years from 1998 to 2003 (19). In addition to mandating non-Bt crop refuges, the U.S. Environmental Protection Agency (EPA) requires monitoring for field resistance to provide early warning of resistance development (16). In Arizona, where Bt cotton producing Cry1Ac has been used widely since 1997 and pink bollworm has been under intense selection for resistance, a statewide surveillance system for resistance exists. From 1997 to 2004, results of laboratory bioassays of insects derived annually from 10 to 17 cotton fields statewide showed no net increase in mean frequency of pink bollworm resistance to Bt toxin (15). DNA screening from 2001 to 2005 also showed that resistancelinked mutations remained rare in pink bollworm field populations (20). Sustained efficacy of Bt cotton has contributed to long-term regional suppression of pink bollworm (21).

Although the strategies implemented to delay resistance have helped sustain efficacy of Bt crops longer than many scientists expected, field-evolved resistance to Bt crops was reported recently (22, 12, 23). Analysis of published monitoring data from the United States, Australia, China, and Spain for major lepidopteran pests targeted by Bt crops indicated field-evolved resistance in Helicoverpa zea, but not in pink bollworm or the four other insects examined (Helicoverpa armigera, Heliothis virescens, Ostrinia nubilalis, and Sesamia nonagrioides). Evaluation of the large data sets of two landmark studies (24, 25) revealed that resistance to Cry1Ac produced by Bt cotton occurred in 2003 to 2004 in some field populations of H. zea in Arkansas and Mississippi, but not in H. virescens from the same region. Resistance of H. zea to Cry1Ac has not resulted in widespread crop failures, in part because existing insecticide sprays and other tactics are still effective against this pest (12). Correspondence between monitoring data and results from computer modeling of resistance evolution suggests that the principles of the refuge strategy for these pests and Bt crops are relevant in the field. Also consistent with monitoring data, modeling suggests H. zea would evolve resistance faster than other pests, because its resistance to Cry1Ac is dominant, not recessive as with other Bt toxins (12). Monitoring data also suggest relatively large refuges may have delayed H. zea resistance to Cry1Ac in North Carolina (12). Field resistance of Busseola fusca was reported in 2007 to Cry1Ab and Cry1F in maize in South Africa (23), and in 2008 field resistance of Spodoptera frugiperda was reported in Puerto Rico (22).

First-generation GE crops produced only one Bt toxin in each plant. A second approach designed to delay resistance is called the pyramid or stacking strategy and entails combining two or more toxins in a single plant, each with different modes of action (26). If no cross-resistance exists between the two toxins, frequency of insect resistance to both toxins is much lower than that for one toxin. Importantly, tests of this approach with a model system using GE broccoli and the insect pest Plutella xylostella suggested that concurrent use of plants with one and two toxins selects for resistance to two-toxin plants more rapidly than the use of two-toxin plants alone (27). In the United States, Bollgard II® cotton producing Cry1Ac and Cry2Ab was introduced commercially in 2003 and has been grown alongside Bollgard cotton producing only Cry1Ac. On the basis of the results with the model broccoli system (28), this concurrent use of one-toxin and two-toxin Bt cotton may not optimize the benefits of the two-toxin cotton. In contrast, Australian cotton growers stopped planting cotton that produces only Cry1Ac soon after twotoxin Bt cotton became available; this strategy might result in delayed resistance development (28).

Other approaches to delaying resistance development have been suggested. The efficacy of mixing seeds of Bt and non-Bt varieties of the same crop has been debated (29); to date, evidence to resolve this issue has been limited to theoretical models and small-scale experiments (30, 31, 29, 32, 33). The practical advantage of seed mixtures in ensuring that non-Bt plants grow near Bt plants may outweigh possible advantages of spatially separate refuges (34). Another suggestion to shorten periods of insect exposure and slow evolution of insect resistance is the use of inducible promoters to drive Bt gene expression only during insect attack (35). Another approach uses knowledge of insect resistance mechanisms to design modified toxins to kill resistant insects (5), on the basis of the fact that the most common mechanism of Cry1A resistance in lepidopteran insects involves disruption of Bt toxin binding to midgut receptors (36). Mutations in midgut cadherins that bind Cry1Ac are linked with and probably cause resistance to Cry1A toxins in at least three lepidopteran pests of cotton (37, 38, 39). The role of cadherin in Bt toxicity was elucidated by silencing the cadherin gene in Manduca sexta, which reduced its susceptibility to Cry1Ab (5). Consistent with the role of cadherin in promoting toxin oligomerization demonstrated by removing an a-helix from Cry1A toxins, toxin-binding fragments of cadherin were required for oligomer formation of native Cry1A toxins, but not for Cry1A toxins lacking the α-helix. The modified Cry1A toxins killed cadherin-silenced M. sexta and Cry1A-resistant pink bollworm larvae, suggesting that modified Bt toxins might be effective against insects resistant to native Bt toxins (5).

In summary, just as insects have evolved resistance to synthetic insecticides and Bt toxins in sprays, they are evolving resistance to Bt toxins in GE crops. The elapsed time before the first cases of field resistance of insects to Bt crops were reported has been longer than what was predicted under worst-case scenarios, suggesting that management strategies may have delayed resistance development. Despite documented cases of resistance, Bt crops remain useful against most target pests in most regions. As insect resistance to Cry toxins currently deployed in Bt crops increases, other strategies to create GE crops resistant to insects are being developed, including vegetative insecticidal proteins (Vips) from Bt (40) and RNA interference (RNAi) (41, 42).

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Updated 2/16/12