Inheritance and fitness cost of laboratory-selected resistance to Vip3Aa in Helicoverpa zea (Lepidoptera: Noctuidae).

The polyphagous pest Helicoverpa zea (Lepidoptera: Noctuidae) has evolved practical resistance to transgenic corn and cotton producing Cry1 and Cry2 crystal proteins from Bacillus thuringiensis (Bt) in several regions of the United States. However, the Bt vegetative insecticidal protein Vip3Aa produced by Bt corn and cotton remains effective against this pest. To advance knowledge of resistance to Vip3Aa, we selected a strain of H. zea for resistance to Vip3Aa in the laboratory. After 28 generations of continuous selection, the resistance ratio was 267 for the selected strain (GA-R3) relative to a strain not selected with Vip3Aa (GA). Resistance was autosomal and almost completely recessive at a concentration killing all individuals from GA. Declines in resistance in heterogeneous strains containing a mixture of susceptible and resistant individuals reared in the absence of Vip3Aa indicate a fitness cost was associated with resistance. Previously reported cases of laboratory-selected resistance to Vip3Aa in lepidopteran pests often show partially or completely recessive resistance at high concentrations and fitness costs. Abundant refuges of non-Bt host plants can maximize the benefits of such costs for sustaining the efficacy of Vip3Aa against target pests.

Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea.

Evolution of pest resistance reduces the benefits of widely cultivated genetically engineered crops that produce insecticidal proteins derived from Bacillus thuringiensis (Bt). Better understanding of the genetic basis of pest resistance to Bt crops is needed to monitor, manage, and counter resistance. Previous work shows that in several lepidopterans, resistance to Bt toxin Cry2Ab is associated with mutations in the gene encoding the ATP-binding cassette protein ABCA2. The results here show that mutations introduced by CRISPR/Cas9 gene editing in the Helicoverpa zea (corn earworm or bollworm) gene encoding ABCA2 (HzABCA2) can cause resistance to Cry2Ab. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains. A multiple concentration bioassay with one of these strains revealed it had > 200-fold resistance to Cry2Ab relative to its parental susceptible strain. All Cry2Ab-resistant individuals tested had disruptive mutations in HzABCA2. We identified five disruptive mutations in HzABCA2 gDNA. The most common mutation was a 4-bp deletion in the expected Cas9 guide RNA target site. The results here indicate that HzABCA2 is a leading candidate for monitoring Cry2Ab resistance in field populations of H. zea.

Effects of gene flow between Bt and non-Bt plants in a seed mixture of Cry1A.105 + Cry2Ab corn on performance of corn earworm in Arizona.

BACKGROUND: Using natural populations of Helicoverpa zea from Arizona, we tested the hypotheses that gene flow between Bacillus thuringiensis (Bt) plants and non-Bt plants in a seed mixture of 10% non-Bt corn and 90% Bt corn producing Cry1A.105 and Cry2Ab reduces larval performance on ears from non-Bt plants, or increases performance on ears from Bt plants. RESULTS: Gene flow was not detected in blocks of non-Bt or Bt corn but was extensive in seed mixtures. Analyses of larval weight and abundance over a period of 3 to 4 weeks did not indicate consistent effects of gene flow on development rate and survival. However for non-Bt plants, the ear area damaged and percentage of ears with exit holes were significantly lower in the seed mixtures than blocks. By contrast, the percentage of ears with exit holes and ear damage did not differ significantly between the seed mixtures and blocks for Bt plants. Nearly 100% of the ears were damaged and the damaged area was substantial, showing that H. zea is a major ear-feeding pest in Arizona. Relative to non-Bt corn, the pyramided Bt corn did not significantly reduce the percentage of damaged ears and only reduced the ear area damaged by 21 to 39%, indicating that H. zea may have evolved resistance to Cry1A.105, Cry2Ab, or both. CONCLUSIONS: Our results indicate that gene flow between Bt and non-Bt plants in seed mixtures reduced effective refuge size, and that block refuges may be needed to manage the evolution of H. zea resistance to Bt corn in Arizona. © 2020 Society of Chemical Industry.

Gene Flow Between Bt and Non-Bt Plants in a Seed Mixture Increases Dominance of Resistance to Pyramided Bt Corn in Helicoverpa zea (Lepidoptera: Noctuidae).

For delaying evolution of pest resistance to transgenic corn producing Bacillus thuringiensis (Bt) toxins, limited data are available to compare the effectiveness of refuges of non-Bt corn planted in seed mixtures versus blocks. Here we addressed this issue in the ear-feeding pest Helicoverpa zea Boddie by measuring its survival and development in the laboratory on ears from field plots with 90% Cry1A.105 + Cry2Ab corn and 10% non-Bt corn planted in a seed mixture or blocks. We compared a strain of H. zea selected for resistance to Cry1Ac in the laboratory, its parent strain not selected in the laboratory, and their F1 progeny. The relative survival of the F1 progeny and dominance of resistance were higher on ears from Bt plants in the seed mixture than the block. Half of the kernels in ears from non-Bt plants in the seed mixture produced both Cry1A.105 and Cry2Ab. However, survival on ears from non-Bt plants did not differ between the block and seed mixture. In simulations based on the observed survival, resistance to Cry1A.105 + Cry2Ab corn evolved faster with the seed mixture than the blocks, because of the higher dominance of resistance in the seed mixture. Increasing the refuge percentage improved durability of Cry1A.105 + Cry2Ab corn more for the blocks than the seed mixture. These findings imply that, for a given percentage of non-Bt corn, resistance of H. zea and other ear-feeding pests to multi-toxin Bt corn is likely to evolve faster for seed mixtures than blocks.

Seasonal Declines in Cry1Ac and Cry2Ab Concentration in Maturing Cotton Favor Faster Evolution of Resistance to Pyramided Bt Cotton in Helicoverpa zea (Lepidoptera: Noctuidae).

Under ideal conditions, widely adopted transgenic crop pyramids producing two or more distinct insecticidal proteins from Bacillus thuringiensis (Bt) that kill the same pest can substantially delay evolution of resistance by pests. However, deviations from ideal conditions diminish the advantages of such pyramids. Here, we tested the hypothesis that changes in maturing cotton producing Cry1Ac and Cry2Ab affect evolution of resistance in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), a pest with low inherent susceptibility to both toxins. In terminal leaves of field-grown Bt cotton, the concentration of both toxins was significantly higher for young, squaring plants than for old, fruiting plants. We used laboratory bioassays with plant material from field-grown cotton to test H. zea larvae from a strain selected for resistance to Cry1Ac in the laboratory, its more susceptible parent strain, and their F1 progeny. On young Bt cotton, no individuals survived to pupation. On old Bt cotton, survival to pupation was significantly higher for the lab-selected strain and the F1 progeny relative to the unselected parent strain, indicating dominant inheritance of resistance. Redundant killing, the extent to which insects resistant to one toxin are killed by another toxin in a pyramid, was complete on young Bt cotton, but not on old Bt cotton. No significant fitness costs associated with resistance were detected on young or old non-Bt cotton. Incorporation of empirical data into simulations indicates the observed increased selection for resistance on old Bt cotton could accelerate evolution of resistance to cotton producing Cry1Ac and Cry2Ab in H. zea.

Effects of seasonal changes in cotton plants on the evolution of resistance to pyramided cotton producing the Bt toxins Cry1Ac and Cry1F in Helicoverpa zea.

BACKGROUND: In pests with inherently low susceptibility to Bacillus thuringiensis (Bt) toxins, seasonal declines in the concentration of Bt toxins in transgenic crops could accelerate evolution of resistance by increasing the dominance of resistance. Here, we evaluated Helicoverpa zea survival on young and old cotton plants that produced the Bt toxins Cry1Ac and Cry1F or did not produce Bt toxins. RESULTS: Using a strain selected for resistance to Cry1Ac in the laboratory, its parent strain that was not selected in the laboratory, and their F1 progeny, we showed that resistance to Cry1Ac + Cry1F cotton was partially dominant on young and old plants. On Cry1Ac + Cry1F cotton, redundant killing was incomplete on young plants but nearly complete on old plants. No significant fitness costs on non-Bt cotton occurred on young plants, but large recessive costs affected survival on old plants. Simulation models incorporating the empirical data showed that the seasonal changes in fitness could delay resistance to Cry1Ac + Cry1F cotton by inducing low equilibrium frequencies of resistance alleles when refuges are sufficiently large. CONCLUSION: Our results suggest that including effects of seasonal changes in fitness of pests on Bt crops and refuge plants can enhance resistance risk assessment and resistance management. © 2017 Society of Chemical Industry.

Validation of a Landscape-Based Model for Whitefly Spread of the Cucurbit Yellow Stunting Disorder Virus to Fall Melons.

The cucurbit yellow stunting disorder virus (CYSDV) transmitted by Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) has caused significant reductions in fall melon (Cucumis melo L.) yields in Yuma County, Arizona. In a recent landscape-based study, we found evidence that cotton and spring melon fields increased abundance of B. tabaci and spread of CYSDV infection in fall melon fields. Here, we show that a statistical model derived from data collected in 2011-2012 and based on areas of cotton and spring melon fields located within 1,500 m from edges of fall melon fields was sufficient to retrospectively predict incidence of CYSDV infection in fall melon fields during 2007-2010. Nevertheless, the slope of the association between areas of spring melon fields and incidence of CYSDV infection was three times smaller in 2007-2010 than in 2011-2012, whereas the slope of the association between areas of cotton fields and incidence of CYSDV infection was consistent between study periods. Accordingly, predictions were more accurate when data on areas of cotton alone were used as a basis for prediction than when data on areas of cotton and spring melons were used. Validation of this statistical model confirms that crop isolation has potential for reducing incidence of CYSDV infection in fall melon fields in Yuma County, although isolation from cotton may provide more consistent benefits than isolation from spring melon.

Multi-Toxin Resistance Enables Pink Bollworm Survival on Pyramided Bt Cotton.

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.

Cross-resistance to toxins used in pyramided Bt crops and resistance to Bt sprays in Helicoverpa zea.

To delay evolution of resistance by insect pests, farmers are rapidly increasing their use of transgenic crops producing two or more Bacillus thuringiensis (Bt) toxins that kill the same pest. A key condition favoring durability of these "pyramided" crops is the absence of cross-resistance between toxins. Here we evaluated cross-resistance in the major lepidopteran pest Helicoverpa zea (Boddie) to Bt toxins used in pyramids. In the laboratory, we selected a strain of this pest with Bt toxin Cry1Ac followed by selection with MVP II, a formulation containing a hybrid protoxin that is identical to Cry1Ac in the active portion of the toxin and 98.5% identical overall. We calculated the resistance ratio as the EC50 (concentration causing mortality or failure to develop beyond the first instar of 50% of larvae) for the laboratory-selected strain divided by the EC50 for its field-derived parent strain that was not selected in the laboratory. The resistance ratio was 20.0-33.9 (mean=27.0) for MVP II, 57.0 for Cry1Ac, 51.3 for Cry1A.105, 22.4 for Cry1Ab, 3.3 for Cry2Ab, 1.8 for Cry1Fa, and 1.6 for Vip3Aa. Resistance ratios were 2.9 for DiPel ES and 2.0 for Agree VG, which are commercial Bt spray formulations containing Cry1Ac, other Bt toxins, and Bt spores. By the conservative criterion of non-overlap of 95% fiducial limits, the EC50 was significantly higher for the selected strain than its parent strain for MVP II, Cry1Ac, Cry1A.105, Cry1Ab, Cry2Ab and DiPel ES. For Cry1Fa, Vip3Aa, and Agree VG, significantly lower susceptibility to a high concentration indicated low cross-resistance. The resistance ratio for toxins other than Cry1Ac was associated with their amino acid sequence similarity to Cry1Ac in domain II. Resistance to Cry1Ac and the observed cross-resistance to other Bt toxins could accelerate evolution of H. zea resistance to currently registered Bt sprays and pyramided Bt crops.

Large-scale, spatially-explicit test of the refuge strategy for delaying insecticide resistance.

The refuge strategy is used worldwide to delay the evolution of pest resistance to insecticides that are either sprayed or produced by transgenic Bacillus thuringiensis (Bt) crops. This strategy is based on the idea that refuges of host plants where pests are not exposed to an insecticide promote survival of susceptible pests. Despite widespread adoption of this approach, large-scale tests of the refuge strategy have been problematic. Here we tested the refuge strategy with 8 y of data on refuges and resistance to the insecticide pyriproxyfen in 84 populations of the sweetpotato whitefly (Bemisia tabaci) from cotton fields in central Arizona. We found that spatial variation in resistance to pyriproxyfen within each year was not affected by refuges of melons or alfalfa near cotton fields. However, resistance was negatively associated with the area of cotton refuges and positively associated with the area of cotton treated with pyriproxyfen. A statistical model based on the first 4 y of data, incorporating the spatial distribution of cotton treated and not treated with pyriproxyfen, adequately predicted the spatial variation in resistance observed in the last 4 y of the study, confirming that cotton refuges delayed resistance and treated cotton fields accelerated resistance. By providing a systematic assessment of the effectiveness of refuges and the scale of their effects, the spatially explicit approach applied here could be useful for testing and improving the refuge strategy in other crop-pest systems.

Effects of cotton cultivar on fitness costs associated with resistance of pink bollworm (Lepidoptera: Gelechiidae) to Bt cotton.

Fitness costs associated with insect resistance to transgenic crops producing toxins from Bacillus thuringiensis (Bt) reduce the fitness on non-Bt refuge plants of resistant individuals relative to susceptible individuals. Because costs may vary among host plants, choosing refuge cultivars that increase the dominance or magnitude of costs could help to delay resistance. Specifically, cultivars with high concentrations of toxic phytochemicals could magnify costs. To test this hypothesis, we compared life history traits of three independent sets of pink bollworm, Pectinophora gossypiella (Saunders), populations on two cotton cultivars that differed in antibiosis against this cotton pest. Each set had an unselected susceptible population, a resistant population derived by selection from the susceptible population, and the F1 progeny of the susceptible and resistant populations. Confirming previous findings with pink bollworm feeding on cotton, costs primarily affected survival and were recessive on both cultivars. The magnitude of the survival cost did not differ between cultivars. Although the experimental results did not reveal differences between cultivars in the magnitude or dominance of costs, modeling results suggest that differences between cultivars in pink bollworm survival could affect resistance evolution. Thus, knowledge of the interaction between host plants and fitness costs associated with resistance to Bt crops could be helpful in guiding the choice of refuge cultivars.