Diverse genetic basis of Vip3Aa resistance in five independent field-derived strains of Helicoverpa zea in the US.

BACKGROUND: Practical resistance of Helicoverpa zea to Cry proteins has become widespread in the US, making Vip3Aa the only effective Bacillus thuringiensis (Bt) protein for controlling this pest. Understanding the genetic basis of Vip3Aa resistance in H. zea is essential in sustaining the long-term efficacy of Vip3Aa. The objectives of this study were to characterize the inheritance of Vip3Aa resistance in four distinct field-derived H. zea strains (M1-RR, AC4-RR, R2-RR and R15-RR), and to test for shared genetic basis among these strains and a previously characterized Texas resistant strain (LT#70-RR). RESULTS: Maternal effects and sex linkage were absent, and the effective dominance level (DML) was 0.0 across Vip3Aa39 concentrations ranging from 1.0 to 31.6 µg cm-2, in all H. zea resistant strains. Mendelian monogenic model tests indicated that Vip3Aa resistance in each of the four strains was controlled by a single gene. However, interstrain complementation tests indicated that three distinct genetic loci are involved in Vip3Aa resistance in the five resistant H. zea strains: one shared by M1-RR and LT#70-RR; another shared by R2-RR and R15-RR; and a distinct one for AC4-RR. CONCLUSION: Results of this study indicate that Vip3Aa resistance in all H. zea strains was controlled by a single, recessive and autosomal gene. However, there were three distinct genetic loci associated with Vip3Aa resistance in the five resistant H. zea strains. The information generated from this study is valuable for exploring mechanisms of Vip3Aa resistance, monitoring the evolution of Vip3Aa resistance, and devising effective strategies for managing Vip3Aa resistance in H. zea. © 2024 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Resistance Allele Frequency of Helicoverpa zea to Vip3Aa Bacillus thuringiensis Protein in the Southeastern U.S.

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.

A Modified F2 Screen for Estimating Cry1Ac and Cry2Ab Resistance Allele Frequencies in Helicoverpa zea (Lepidoptera: Noctuidae).

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.

Effective dominance and redundant killing of single- and dual-gene resistant populations of Helicoverpa zea on pyramided Bt corn and cotton.

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.

Effects of cross-pollination among non-Bt and pyramided Bt corn expressing cry proteins in seed mixtures on resistance development of dual-gene resistant Helicoverpa zea.

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 F1 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. © 2022 Society of Chemical Industry.

Status of Cry1Ac and Cry2Ab2 resistance in field populations of Helicoverpa zea in Texas, USA.

Helicoverpa zea is a major target pest of Bt corn and Bt cotton. Field-evolved resistance of H. zea to Cry1 and Cry2 proteins has been widely reported in the United States. Understanding the frequency of resistance alleles in a target insect is critical for Bt resistance management. Despite multiple cases of practical resistance to Cry proteins having been documented in H. zea, there are no data on the current status of alleles conferring resistance to Cry1Ac and Cry2Ab2 in field populations of this pest. During 2018-2019, a total of 106 F2 families for Cry1Ac and 120 F2 families for Cry2Ab2 were established using mass mating and light trap strategy. We screened 13,568 and 15,360 neonates using a discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The results showed that 93.4% and 35.0% of the F2 families could survive on the discriminatory dose of Cry1Ac and Cry2Ab2, respectively. The estimated resistance allele frequency for Cry1Ac in H. zea ranged from 0.4150 to 0.4975 and for Cry2Ab2 ranged from 0.1097 and 0.1228. These data indicate that the frequency of alleles conferring resistance to Cry1 and Cry2 proteins in H. zea in Texas are high. In addition, our data suggest the resistance to Cry1Ac and Cry2Ab2 in the screened families of H. zea varies from recessive to dominant. The information in this study provides precise estimates of Cry resistance allele frequencies in H. zea and increases our understanding of the risks to the sustainability of Bt crops.

Early Warning of Resistance to Bt Toxin Vip3Aa in Helicoverpa zea.

Evolution of resistance by pests can reduce the benefits of crops genetically engineered to produce insecticidal proteins from Bacillus thuringiensis (Bt). Because of the widespread resistance of Helicoverpa zea to crystalline (Cry) Bt toxins in the United States, the vegetative insecticidal protein Vip3Aa is the only Bt toxin produced by Bt corn and cotton that remains effective against some populations of this polyphagous lepidopteran pest. Here we evaluated H. zea resistance to Vip3Aa using diet bioassays to test 42,218 larvae from three lab strains and 71 strains derived from the field during 2016 to 2020 in Arkansas, Louisiana, Mississippi, Tennessee, and Texas. Relative to the least susceptible of the three lab strains tested (BZ), susceptibility to Vip3Aa of the field-derived strains decreased significantly from 2016 to 2020. Relative to another lab strain (TM), 7 of 16 strains derived from the field in 2019 were significantly resistant to Vip3Aa, with up to 13-fold resistance. Susceptibility to Vip3Aa was significantly lower for strains derived from Vip3Aa plants than non-Vip3Aa plants, providing direct evidence of resistance evolving in response to selection by Vip3Aa plants in the field. Together with previously reported data, the results here convey an early warning of field-evolved resistance to Vip3Aa in H. zea that supports calls for urgent action to preserve the efficacy of this toxin.

Genetic basis of resistance to the Vip3Aa Bt protein in Helicoverpa zea.

BACKGROUND: Helicoverpa zea is a destructive pest and target of maize and cotton expressing Cry and Vip3Aa proteins in North America. The efficacy of Cry proteins against H. zea in the USA has been largely compromised by resistance. A rapid shift towards planting Bt cotton and maize producing Vip3Aa will accelerate evolution of resistance to Vip3Aa in H. zea. Research on the genetic basis of Vip3Aa resistance in H. zea is urgently needed, and can provide fundamental information for managing resistance in this pest. Here, we characterize the inheritance of Vip3Aa resistance in H. zea. RESULTS: Susceptibility of a Vip3Aa-susceptible strain (SS), a resistant strain (RR), and progeny from different crosses against Vip3Aa39 was determined. RR was established from an F2 screening of a population from Texas sampled in 2019. RR had a resistance ratio of 45194.1-fold against Vip3Aa39 relative to SS. Maternal effects and sex linkage were absent in RR. The dominance D value, calculated based on median lethal concentration (LC50 ) values, was -1.0 and the effective dominance (DML ), calculated based on a given Vip3Aa39 concentration, was ≤0.0 at concentrations of 0.1-31.6 µg cm-2 . The test using a monogenic mode of inheritance showed that resistance to Vip3Aa in H. zea was largely due to a single gene. CONCLUSION: Results of this study indicate that Vip3Aa resistance in H. zea is monogenic, autosomal, and recessive. This information is valuable for studying the mechanism of Vip3Aa resistance, monitoring of resistance development, and designing appropriate strategies for preventive management of Vip3Aa resistance. © 2020 Society of Chemical Industry.

Inheritance of Bacillus thuringiensis Cry2Ab2 protein resistance in Helicoverpa zea (Lepidoptera: Noctuidae).

BACKGROUND: The corn earworm, Helicoverpa zea (Boddie), is a major target pest of pyramided Bt corn and cotton in the United States. Field-evolved practical resistance to Cry1 and Cry2 proteins in H. zea has been documented in multiple locations in the United States. Understanding the genetic basis of Bt resistance is essential in developing insect resistance management (IRM) strategies for the sustainable use of the Bt crop technology. In this study, we characterized the genetic bases of Cry2Ab2 resistance in H. zea using diet-overlay bioassays with two different forms of Cry2Ab2 protein. RESULTS: Laboratory bioassays using a Cry2Ab2-resistant (RR) strain, a susceptible (SS) strain, as well as cross and backcross strains, revealed that resistance to Cry2Ab2 was autosomally inherited and controlled by more than one locus. In diet bioassays, the dominance of Cry2Ab2 resistance in H. zea varied from incompletely recessive to incompletely dominant across all tested Cry2Ab2 concentrations of either Bt corn leaf powder or solubilized protein. On leaf tissue of TwinLink cotton (expressing Cry1Ab and Cry2Ae), Cry2Ab2 resistance in H. zea was completely dominant. CONCLUSION: These results have significant implications for understanding the widespread field-evolved resistance of H. zea against Cry1 and Cry2 proteins in Bt corn and cotton and should be useful in developing effective IRM strategies for H. zea. © 2020 Society of Chemical Industry.