Genetically engineered crops that produce insecticidal toxins from Bacillus thuringiensis (Bt) are grown widely for pest control. However, insect adaptation can reduce the toxins' efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to provide susceptible insects to mate with resistant insects. Variable farmer compliance is one of the limitations of this approach. Here we report the benefits of an alternative strategy where sterile insects are released to mate with resistant insects and refuges are scarce or absent. Computer simulations show that this approach works in principle against pests with recessive or dominant inheritance of resistance. During a large-scale, four-year field deployment of this strategy in Arizona, resistance of pink bollworm (Pectinophora gossypiella) to Bt cotton did not increase. A multitactic eradication program that included the release of sterile moths reduced pink bollworm abundance by >99%, while eliminating insecticide sprays against this key invasive pest.

Evolution of resistance by insect pests can reduce the benefits of insecticidal proteins from Bacillus thuringiensis (Bt) that are used extensively in sprays and transgenic crops. Despite considerable knowledge of the genes conferring insect resistance to Bt toxins in laboratory-selected strains and in field populations exposed to Bt sprays, understanding of the genetic basis of field-evolved resistance to Bt crops remains limited. In particular, previous work has not identified the genes conferring resistance in any cases where field-evolved resistance has reduced the efficacy of a Bt crop. Here we report that mutations in a gene encoding a cadherin protein that binds Bt toxin Cry1Ac are associated with field-evolved resistance of pink bollworm (Pectinophora gossypiella) in India to Cry1Ac produced by transgenic cotton. We conducted laboratory bioassays that confirmed previously reported resistance to Cry1Ac in pink bollworm from the state of Gujarat, where Bt cotton producing Cry1Ac has been grown extensively. Analysis of DNA from 436 pink bollworm from seven populations in India detected none of the four cadherin resistance alleles previously reported to be linked with resistance to Cry1Ac in laboratory-selected strains of pink bollworm from Arizona. However, DNA sequencing of pink bollworm derived from resistant and susceptible field populations in India revealed eight novel, severely disrupted cadherin alleles associated with resistance to Cry1Ac. For these eight alleles, analysis of complementary DNA (cDNA) revealed a total of 19 transcript isoforms, each containing a premature stop codon, a deletion of at least 99 base pairs, or both. Seven of the eight disrupted alleles each produced two or more different transcript isoforms, which implicates alternative splicing of messenger RNA (mRNA). This represents the first example of alternative splicing associated with field-evolved resistance that reduced the efficacy of a Bt crop.

Evolution of resistance in pests threatens the long-term efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays and transgenic crops. Previous work showed that genetically modified Bt toxins Cry1AbMod and Cry1AcMod effectively countered resistance to native Bt toxins Cry1Ab and Cry1Ac in some pests, including pink bollworm (Pectinophora gossypiella). Here we report that Cry1AbMod and Cry1AcMod were also effective against a laboratory-selected strain of pink bollworm resistant to Cry2Ab as well as to Cry1Ab and Cry1Ac. Resistance ratios based on the concentration of toxin killing 50% of larvae for the resistant strain relative to a susceptible strain were 210 for Cry2Ab, 270 for Cry1Ab, and 310 for Cry1Ac, but only 1.6 for Cry1AbMod and 2.1 for Cry1AcMod. To evaluate the interactions among toxins, we tested combinations of Cry1AbMod, Cry1Ac, and Cry2Ab. For both the resistant and susceptible strains, the net results across all concentrations tested showed slight but significant synergism between Cry1AbMod and Cry2Ab, whereas the other combinations of toxins did not show consistent synergism or antagonism. The results suggest that the modified toxins might be useful for controlling populations of pink bollworm resistant to Cry1Ac, Cry2Ab, or both.

Transgenic crops producing Bacillus thuringiensis (Bt) proteins kill key insect pests, providing economic and environmental benefits. However, the evolution of pest resistance threatens the continued success of such Bt crops. To delay or counter resistance, transgenic plant “pyramids” producing two or more Bt proteins that kill the same pest have been adopted extensively. Field populations of the pink bollworm (Pectinophora gossypiella) in the United States have remained susceptible to Bt toxins Cry1Ac and Cry2Ab, but field-evolved practical resistance to Bt cotton producing Cry1Ac has occurred widely in India. Here we used two rounds of laboratory selection to achieve 18,000- to 150,000-fold resistance to Cry2Ab in pink bollworm. Inheritance of resistance to Cry2Ab was recessive, autosomal, conferred primarily by one locus, and independent of Cry1Ac resistance. We created a strain with high resistance to both toxins by crossing the Cry2Ab-resistant strain with a Cry1Ac-resistant strain, followed by one selection with Cry2Ab. This multi-toxin resistant strain survived on field-collected Bt cotton bolls producing both toxins. The results here demonstrate the risk of evolution of resistance to pyramided Bt plants, particularly when toxins are deployed sequentially and refuges are scarce, as seen with Bt cotton and pink bollworm in India.

Evolution of resistance by pests can reduce the benefits of transgenic crops that produce toxins from Bacillus thuringiensis (Bt) for insect control. One of the world's most important cotton pests, pink bollworm (Pectinophora gossypiella), has been targeted for control by transgenic cotton producing Bt toxin Cry1Ac in several countries for more than a decade. In China, the frequency of resistance to Cry1Ac has increased, but control failures have not been reported. In western India, pink bollworm resistance to Cry1Ac has caused widespread control failures of Bt cotton. By contrast, in the state of Arizona in the southwestern United States, monitoring data from bioassays and DNA screening demonstrate sustained susceptibility to Cry1Ac for 16 y. From 1996-2005, the main factors that delayed resistance in Arizona appear to be abundant refuges of non-Bt cotton, recessive inheritance of resistance, fitness costs associated with resistance and incomplete resistance. From 2006-2011, refuge abundance was greatly reduced in Arizona, while mass releases of sterile pink bollworm moths were made to delay resistance as part of a multi-tactic eradication program. Sustained susceptibility of pink bollworm to Bt cotton in Arizona has provided a cornerstone for the pink bollworm eradication program and for integrated pest management in cotton. Reduced insecticide use against pink bollworm and other cotton pests has yielded economic benefits for growers, as well as broad environmental and health benefits. We encourage increased efforts to combine Bt crops with other tactics in integrated pest management programs.

Evolution of pest resistance threatens the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Field populations of the pink bollworm (Pectinophora gossypiella), a global pest of cotton, have evolved practical resistance to transgenic cotton producing Bt toxin Cry2Ab in India, but not in the United States. Previous results show that recessive mutations disrupting an autosomal ATP-binding cassette gene (PgABCA2) are associated with pink bollworm resistance to Cry2Ab in field-selected populations from India and in one lab-selected strain from the United States (Bt4-R2). Here we discovered that an independently derived, labselected Cry2Ab-resistant pink bollworm strain from the United States (BX-R) also harbors mutations that disrupt PgABCA2. Premature stop codons introduced by mis-splicing of PgABCA2 pre-mRNA were prevalent in field-selected larvae from India and in both lab-selected strains. The most common mutation in field-selected larvae from India was also detected in both lab-selected strains. Results from interstrain crosses indicate BX-R has at least one additional mechanism of resistance to Cry2Ab that does not involve PgABCA2 and is not completely recessive or autosomal. We conclude that recessive mutations disrupting PgABCA2 are the primary, but not the only, mechanism of resistance to Cry2Ab in pink bollworm. Crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) have revolutionized management of some of the world's most devastating pests 1. In 2018, millions of farmers in 15 countries grew more than 104 million hectares of transgenic Bt crops 2. Bt crops can suppress pests, increase yield and farmer profits, and decrease conventional insecticide use, which translates to health and environmental benefits 1,3-7. However, evolution of resistance to Bt crops by pests reduces such benefits 8-11. Practical resistance to Bt crops, which is field-evolved resistance that has practical consequences for pest management, has been documented in at least nine species of major pests targeted by Bt crops 8-11. Better understanding of the mechanisms and genetic basis of resistance to Bt toxins is needed to monitor, manage, and counter pest resistance. For Cry1 toxins, the first family of lepidopteran-active crystalline (Cry) Bt toxins deployed widely in transgenic crops, the molecular genetic basis of resistance has been studied extensively 12-16. However, much less is known about this issue for Cry2 toxins, the second such family. While each of the first Bt crops produced a single toxin from the Cry1 family (e.g., Cry1Ac), most Bt crops grown now produce Cry2Ab in combination with one or more Cry1 toxins 17. For lab-selected strains of the lepidopteran pests Helicoverpa armigera and Helicoverpa punctigera, resistance to Cry2Ab is associated with reduced binding to larval midgut membranes and linked with autosomal recessive mutations that disrupt the ATP-binding cassette protein ABCA2 18,19. Deleting portions o...

BACKGROUND Better understanding of the molecular basis of resistance is needed to improve management of pest resistance to transgenic crops that produce insecticidal proteins from Bacillus thuringiensis (Bt). Here we analyzed resistance of the pink bollworm (Pectinophora gossypiella) to Bt toxin Cry1Ac, which is used widely in transgenic Bt cotton. Field‐evolved practical resistance of pink bollworm to Cry1Ac is widespread in India, but not in China or the United States. Previous work with laboratory‐ and field‐selected pink bollworm indicated that resistance to Cry1Ac is caused by changes in the amino acid sequence of a midgut cadherin protein (PgCad1) that binds Cry1Ac in susceptible larvae. RESULTS Relative to a susceptible strain, the laboratory‐selected APHIS‐R strain had 530‐fold resistance to Cry1Ac with autosomal recessive inheritance. Unlike previous results, resistance in this strain was not consistently associated with insertions or deletions in the expected amino acid sequence of PgCad1. However, this resistance was associated with 79‐ to 190‐fold reduced transcription of the PgCad1 gene and markedly lower abundance of PgCad1 protein. CONCLUSION The ability of pink bollworm and other major pests to evolve resistance to Bt toxins via both qualitative and quantitative changes in receptor proteins demonstrates their remarkable adaptability and presents challenges for monitoring and managing resistance to Bt crops. © 2019 Society of Chemical Industry

Crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. Previous work shows that resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2 in lab- and field-selected populations of the pink bollworm (Pectinophora gossypiella), one of the world’s most destructive pests of cotton. Here we used CRISPR/Cas9 gene editing to test the hypothesis that mutations in the pink bollworm gene encoding ABCA2 (PgABCA2) can cause resistance to Cry2Ab. Consistent with this hypothesis, introduction of disruptive mutations in PgABCA2 in a susceptible strain of pink bollworm increased the frequency of resistance to Cry2Ab and facilitated creation of a Cry2Ab-resistant strain. All Cry2Ab-resistant individuals tested in this study had disruptive mutations in PgABCA2. Overall, we found 17 different disruptive mutations in PgABCA2 gDNA and 26 in PgABCA2 cDNA, including novel mutations corresponding precisely to single-guide (sgRNA) sites used for CRISPR/Cas9. Together with previous results, these findings provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations.