Transgenic crops producing Bacillus thuringiensis (Bt) toxins kill some key insect pests, but evolution of resistance by pests can reduce their efficacy. The predominant strategy for delaying pest resistance to Bt crops requires refuges of non-Bt host plants to promote survival of susceptible pests. To delay pest resistance to transgenic cotton producing Bt toxin Cry1Ac, farmers in the United States and Australia planted refuges of non-Bt cotton, while farmers in China have relied on “natural” refuges of non-Bt host plants other than cotton. Here we report data from a 2010 survey showing field-evolved resistance to Cry1Ac of the major target pest, cotton bollworm (Helicoverpa armigera), in northern China. Laboratory bioassay results show that susceptibility to Cry1Ac was significantly lower in 13 field populations from northern China, where Bt cotton has been planted intensively, than in two populations from sites in northwestern China where exposure to Bt cotton has been limited. Susceptibility to Bt toxin Cry2Ab did not differ between northern and northwestern China, demonstrating that resistance to Cry1Ac did not cause cross-resistance to Cry2Ab, and implying that resistance to Cry1Ac in northern China is a specific adaptation caused by exposure to this toxin in Bt cotton. Despite the resistance detected in laboratory bioassays, control failures of Bt cotton have not been reported in China. This early warning may spur proactive countermeasures, including a switch to transgenic cotton producing two or more toxins distinct from Cry1A toxins.

Evolution of pest resistance reduces the efficacy of insecticidal proteins from Bacillus thuringiensis (Bt) used in sprays or in transgenic crops. Although several pests have evolved resistance to Bt crops in the field, information about the genetic basis of field-evolved resistance to Bt crops has been limited. In particular, laboratory-selected resistance to Bt toxin Cry1Ac based on recessive mutations in a gene encoding a toxin-binding cadherin protein has been identified in three major cotton pests, but previous work has not determined if such mutations are associated with field-selected resistance to Bt cotton. Here we show that the most common resistance alleles in field populations of cotton bollworm, Helicoverpa armigera , selected with Bt cotton in northern China, had recessive cadherin mutations, including the deletion mutation identified via laboratory selection. However, unlike all previously studied cadherin resistance alleles, one field-selected cadherin resistance allele conferred nonrecessive resistance. We also detected nonrecessive resistance that was not genetically linked with the cadherin locus. In field-selected populations, recessive cadherin alleles accounted for 75–84% of resistance alleles detected. However, most resistance alleles occurred in heterozygotes and 59–94% of resistant individuals carried at least one nonrecessive resistance allele. The results suggest that resistance management strategies must account for diverse resistance alleles in field-selected populations, including nonrecessive alleles.

Extensive planting of crops genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) has suppressed some major pests, reduced insecticide sprays, enhanced pest control by natural enemies, and increased grower profits. However, rapid evolution of resistance in pests is reducing these benefits. Better understanding of the genetic basis of resistance to Bt crops is urgently needed to monitor, delay, and counter pest resistance. We discovered that a point mutation in a previously unknown tetraspanin gene in the cotton bollworm (Helicoverpa armigera), a devastating global pest, confers dominant resistance to Cry1Ac, the sole Bt protein produced by transgenic cotton planted in China. We found the mutation using a genome-wide association study, followed by fine-scale genetic mapping and DNA sequence comparisons between resistant and susceptible strains. CRISPR/Cas9 knockout of the tetraspanin gene restored susceptibility to a resistant strain, whereas inserting the mutation conferred 125-fold resistance in a susceptible strain. DNA screening of moths captured from 23 field sites in six provinces of northern China revealed a 100-fold increase in the frequency of this mutation, from 0.001 in 2006 to 0.10 in 2016. The correspondence between the observed trajectory of the mutation and the trajectory predicted from simulation modeling shows that the dominance of the mutation accelerated adaptation. Proactive identification and tracking of the tetraspanin mutation demonstrate the potential for genomic analysis, gene editing, and molecular monitoring to improve management of resistance.

The 'natural refuge strategy" for delaying insect resistance to transgenic cotton that produces insecticidal proteins from Bacillus thuringiensis (Bt) relies on refuges of host plants other than cotton that do not make Bt toxins. We tested this widely adopted strategy by comparing predictions from modeling with data from a four-year field study of cotton bollworm (Helicoverpa armigera) resistance to transgenic cotton producing Bt toxin Cry1Ac in six provinces of northern China. Bioassay data revealed that the percentage of resistant insects increased from 0.93% in 2010 to 5.5% in 2013. Modeling predicted that the percentage of resistant insects would exceed 98% in 2013 without natural refuges, but would increase to only 1.1% if natural refuges were as effective as non-Bt cotton refuges. Therefore, the results imply that natural refuges delayed resistance, but were not as effective as an equivalent area of non-Bt cotton refuges. The percentage of resistant insects with nonrecessive inheritance of resistance increased from 37% in 2010 to 84% in 2013. Switching to Bt cotton producing two or more toxins and integrating other control tactics could slow further increases in resistance.

The cotton bollworm Helicoverpa armigera, is one of the world’s major pest of agriculture, feeding on over 300 hosts in 68 plant families. Resistance cases to most insecticide classes have been reported for this insect. Management of this pest in agroecosystems relies on a better understanding of how it copes with phytochemical or synthetic toxins. We have used genome editing to knock out a cluster of nine P450 genes and show that this significantly reduces the survival rate of the insect when exposed to two classes of host plant chemicals and two classes of insecticides. Functional expression of all members of this gene cluster identified the P450 enzymes capable of metabolism of these xenobiotics. The CRISPR-Cas9-based reverse genetics approach in conjunction with in vitro metabolism can rapidly identify the contributions of insect P450s in xenobiotic detoxification and serve to identify candidate genes for insecticide resistance.