BACKGROUND Pyramided Bacillus thuringiensis (Bt) crops producing multiple Bt proteins with different modes of action are widely planted in the United States. Helicoverpa zea is a major target pest of pyramided Bt crops and has evolved practical resistance to both Cry1 and Cry2 proteins in some regions of U.S. However, little information is available regarding redundant killing and the dominance of resistance for insects possessing multiple resistance on pyramided Bt crops. In this study, we evaluated redundant killing and the dominance of resistance for H. zea strains resistant to Cry1 or Cry1 + Cry2 on pyramided Bt corn and cotton. RESULTS We found that the Cry1‐resistant H. zea was incompletely dominant on Cry1Ac + Cry1F cotton. Pyramided crops producing Cry2 and/or Vip3Aa proteins showed a complete redundant killing against the Cry1‐resistant H. zea. The Cry1 + Cry2‐resistant H. zea displayed incompletely recessive to completely dominant resistance on pyramided Bt crops containing Cry1 and/or Cry2 proteins. The redundant killing was complete for the Cry1 + Cry2‐resistant H. zea on pyramided Bt crops producing Vip3Aa protein. CONCLUSION The dominant resistance of Cry1 and Cry2 in H. zea on pyramided Bt crops deviates from the assumption of functionally recessive resistance underlying the high‐dose refuge strategy. However, the assumptions of complete redundant killing are achieved for both Cry1‐ and Cry1 + Cry2‐resistant H. zea on pyramided Bt crops. These results suggest that the pyramided strategy could be valuable for increasing the durability of Bt technology for managing H. zea, a pest with inherently low susceptibility against Cry proteins. © 2022 Society of Chemical Industry.

Evaluating the frequency of resistance alleles is important for resistance management and sustainable use of transgenic crops that produce insecticidal proteins from Bacillus thuringiensis. Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) is a major crop pest in the United States that has evolved practical resistance to the crystalline (Cry) proteins in Bt corn and cotton. The standard F2 screen for estimating resistance allele frequency does not work well for H. zea because successful single-pair matings are rare. In this study, we developed and implemented a modified F2 screen for H. zea that generates F1 progeny by crossing three laboratory susceptible female moths with one feral male moth instead of single-pair crosses. During 2019–2020, we used this modified method to establish 192 F2 families from 623 matings between susceptible females and feral males from Arkansas, Louisiana, Mississippi, and Tennessee. From each F2 family, we screened 128 neonates against discriminating concentrations of Cry1Ac and Cry2Ab in diet overlay bioassays. Based on these discriminating concentration bioassays, families were considered positive for resistance if at least five larvae survived to second instar, including at least one to third instar. The percentage of positive families was 92.7% for Cry1Ac and 38.5% for Cry2Ab, which yields an estimated resistance allele frequency (with 95% confidence interval) of 0.722 (0.688–0.764) for Cry1Ac and 0.217 (0.179–0.261) for Cry2Ab. The modified F2 screen developed and implemented here may be useful for future resistance monitoring studies of H. zea and other pests.

Helicoverpa zea is a major target pest of Bt crops expressing Cry and/or Vip3Aa proteins in the U.S.A. Widespread practical resistance of H. zea to the Cry1 and Cry2 proteins makes Vip3Aa the only effective Bt protein against this pest. Understanding the frequency of resistance alleles against Vip3Aa in field populations of H. zea is crucial for resistance management and the sustainability of Vip3Aa technology. Using a modified F2 screen method by crossing susceptible laboratory female moth with feral male moth of H. zea, we successfully screened a total of 24,576 neonates from 192 F2 families of H. zea collected from Arkansas, Louisiana, Mississippi, and Tennessee during 2019–2020. We found five F2 families containing ≥3rd instar survivors on the diagnostic concentration of 3.0 µg/cm2 Vip3Aa39. Dose-response bioassays confirmed the high levels of Vip3Aa resistance in these F2 families, with an estimated resistance ratio of >909.1-fold relative to the susceptible strain. The estimated resistance allele frequency against Vip3Aa in H. zea for these four southern states is 0.0155 with a 95% CI of 0.0057-0.0297. These data should provide critical information for understanding the risks of Vip3Aa resistance in H. zea and help design appropriate resistance management strategies for the sustainability of the Vip3Aa technology.

BACKGROUND: Seed mixture strategy can guarantee the compliance of planting non-Bt crops to host the susceptible insects for resistance management. However, pollen movement between Bt and non-Bt corn in the mixed plantings could reduce the efficacy of this strategy for ear-feeding insects. Few studies have evaluated the effects of cross-pollination among non-Bt and pyramided Bt corn in seed mixtures on the resistance development of insects possessing multiple resistances. Here, we provided the first study to investigate whether cross-pollination in mixed plantings of pyramided Bt corn producing Cry1A.105 and Cry2Ab2 would increase the dominance of resistance of dual-gene resistant populations of Helicoverpa zea, a target of pyramided Bt corn and cotton in the USA. RESULTS: We compared the survival and development of susceptible, dual-gene resistant (resistance to both Cry1 and Cry2 proteins) and heterozygous genotypes of H. zea in the laboratory on non-Bt and pyramided Bt corn ears collected from mixed plantings and structured plantings in the field. We found higher fitness for F 1 heterozygous insects than for the susceptible insects of H. zea on both pyramided Bt corn and non-Bt corn in the mixed plantings.CONCLUSION: These results suggest that cross-pollination in mixed plantings will significantly increase the dominance of resistance by supporting survival of heterozygous insects for dual-gene resistant populations of H. zea, and therefore accelerate evolution of resistance to pyramided Bt crops.