More than 10 years after commercialization, Vip3A-expressing MIR162 remains highly efficacious in controlling major Lepidopteran maize pests: laboratory resistance selection versus field reality.

MIR162, a maize event that expresses Vip3Aa20 (Vip3A) approved for commercial cultivation around 2010, has been excellent for control of major Lepidopteran pests. However, development of fall armyworm (FAW) resistance to Vip3A is a serious concern. Resistant colonies selected in the laboratory can serve as valuable tools not only for better understanding of Vip3A's mode of action (MOA) and mechanism of resistance (MOR) but also for screening novel leads of new MOA that will help control FAW in case resistance to Vip3A in the field becomes a reality. We selected a Vip3A-resistant FAW strain, FAWVip3AR, by subjecting a FAW founder population containing field genetics to Vip3A exposure. FAWVip3AR had >9800-fold resistance to Vip3A by diet surface overlay bioassays and resistance was stable. Feeding bioassays using detached leaf tissues or whole plants indicated that FAWVip3AR larvae readily fed and completed the full life cycle on Vip3A-expressing MIR162 maize plants and leaf tissues that killed 100% of susceptible larvae. Yet, FAWVip3AR faced at least two challenges. First, FAWVip3AR suffered an apparent disadvantage (incomplete resistance) when feeding on MIR162 in comparison to FAWVip3AR feeding on Vip3A-free isoline AX5707 maize; and second, FAWVip3AR showed a fitness costs in comparison to a Vip3A-susceptible strain when both fed on AX5707. We also demonstrated that, >10 years after commercialization, MIR162 and Vip3A remain highly efficacious against field populations of three major Lepidopteran pests from different geographic locations and FAW strains resistant to other Bacillus thuringiensis (Bt) toxins that are currently on the market.

Resistance Allele Frequency to Cry1Ab and Vip3Aa20 in Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) in Louisiana and Three Other Southeastern U.S. States.

The corn earworm/bollworm, Helicoverpa zea (Boddie), is a pest species that is targeted by both Bacillus thuringiensis (Bt) maize and cotton in the United States. Cry1Ab and Vip3Aa20 are two common Bt toxins that are expressed in transgenic maize. The objective of this study was to determine the resistance allele frequency (RAF) to Cry1Ab and Vip3Aa20 in H. zea populations that were collected during 2018 and 2019 from four southeastern U.S. states: Louisiana, Mississippi, Georgia, and South Carolina. By using a group-mating approach, 104 F2 iso-lines of H. zea were established from field collections with most iso-lines (85) from Louisiana. These F2 iso-lines were screened for resistance alleles to Cry1Ab and Vip3Aa20, respectively. There was no correlation in larval survivorship between Cry1Ab and Vip3Aa20 when the iso-lines were exposed to these two toxins. RAF to Cry1Ab maize was high (0.256) and the RAFs were similar between Louisiana and the other three states and between the two sampling years. In contrast, no functional major resistance allele (RA) that allowed resistant insects to survive on Vip3Aa20 maize was detected and the expected RAF of major RAs with 95% probability was estimated to 0 to 0.0073. However, functional minor RAs to Vip3Aa20 maize were not uncommon; the estimated RAF for minor alleles was 0.028. The results provide further evidence that field resistance to Cry1Ab maize in H. zea has widely occurred, while major RAs to Vip3Aa20 maize are uncommon in the southeastern U.S. region. Information that was generated from this study should be useful in resistance monitoring and refinement of resistance management strategies to preserve Vip3A susceptibility in H. zea.

Populations of Helicoverpa zea (Boddie) in the Southeastern United States are Commonly Resistant to Cry1Ab, but Still Susceptible to Vip3Aa20 Expressed in MIR 162 Corn.

The corn earworm, Helicoverpa zea (Boddie), is a major pest targeted by pyramided Bacillus thuringiensis (Bt) corn and cotton in the U.S. Cry1Ab is one of the first insecticidal toxins used in Bt crops, while Vip3A is a relatively new toxin that has recently been incorporated into Cry corn with event MIR 162 and Cry cotton varieties to generate pyramided Bt traits targeting lepidopteran pests including H. zea. The objectives of this study were to determine the current status and distribution of the Cry1Ab resistance, and evaluate the susceptibility to Vip3Aa20 expressed in MIR 162 corn in H. zea in the southeastern U.S. During 2018 and 2019, 32 H. zea populations were collected from non-Bt corn (19 populations), Cry corn (12), and Cry/Vip3A cotton (1) across major corn areas in seven southeastern states of the U.S. Susceptibility of these populations to Cry1Ab and Vip3Aa20 was determined using diet-overlay bioassays. Compared to a known susceptible insect strain, 80% of the field populations were 13- to >150-fold resistant to Cry1Ab, while their response to Vip3Aa20 ranged from >11-fold more susceptible to 9-fold more tolerant. Mean susceptibility to each Bt toxin was not significantly different between the two groups of the populations collected from non-Bt and Bt crops, as well as between the two groups of the populations collected during 2018 and 2019. The results show that resistance to Cry1Ab in H. zea is widely distributed across the region. However, the Cry1Ab-resistant populations are not cross-resistant to Vip3Aa20, and H. zea in the region is still susceptible to the Vip3Aa20 toxin. Vip3Aa20 concentrations between 5 and 10 µg/cm2 may be used as diagnostic concentrations for susceptibility monitoring in future. Additional studies are necessary to elucidate the impact of the selection with Bt corn on resistance evolution in H. zea to Vip3A cotton in the U.S.

A Simple and Sensitive Plant-Based Western Corn Rootworm Bioassay Method for Resistance Determination and Event Selection.

We report here a simple and sensitive plant-based western corn rootworm, Diabrotica virgifera virgifera LeConte (Coleoptera: Chrysomelidae), bioassay method that allows for examination of multiple parameters for both plants and insects in a single experimental setup within a short duration. For plants, injury to roots can be visually examined, fresh root weight can be measured, and expression of trait protein in plant roots can be analyzed. For insects, in addition to survival, larval growth and development can be evaluated in several aspects including body weight gain, body length, and head capsule width. We demonstrated using the method that eCry3.1Ab-expressing 5307 corn was very effective against western corn rootworm by eliciting high mortality and significantly inhibiting larval growth and development. We also validated that the method allowed determination of resistance in an eCry3.1Ab-resistant western corn rootworm strain. While data presented in this paper demonstrate the usefulness of the method for selection of events of protein traits and for determination of resistance in laboratory populations, we envision that the method can be applied in much broader applications.

Brush border membrane binding properties of Bacillus thuringiensis Vip3A toxin to Heliothis virescens and Helicoverpa zea midguts.

The binding properties of Vip3A, a new family of Bacillus thuringiensis insecticidal toxins, have been examined in the major cotton pests, Heliothis virescens and Helicoverpa zea. Vip3A bound specifically to brush border membrane vesicles (BBMV) prepared from both insect larval midguts. In order to examine the cross-resistance potential of Vip3A to the commercially available Cry1Ac and Cry2Ab2 toxins, the membrane binding site relationship among these toxins was investigated. Competition binding assays demonstrated that Vip3A does not inhibit the binding of either Cry1Ac or Cry2Ab2 and vice versa. BBMV protein blotting experiments showed that Vip3A does not bind to the known Cry1Ac receptors. These distinct binding properties and the unique protein sequence of Vip3A support its use as a novel insecticidal agent. This study indicates a very low cross-resistance potential between Vip3A and currently deployed Cry toxins and hence supports its use in an effective resistance management strategy in cotton.

The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin.

The Vip3A protein, secreted by Bacillus spp. during the vegetative stage of growth, represents a new family of insecticidal proteins. In our investigation of the mode of action of Vip3A, the 88-kDa Vip3A full-length toxin (Vip3A-F) was proteolytically activated to an approximately 62-kDa core toxin either by trypsin (Vip3A-T) or lepidopteran gut juice extracts (Vip3A-G). Biotinylated Vip3A-G demonstrated competitive binding to lepidopteran midgut brush border membrane vesicles (BBMV). Furthermore, in ligand blotting experiments with BBMV from the tobacco hornworm, Manduca sexta (Linnaeus), activated Cry1Ab bound to 120-kDa aminopeptidase N (APN)-like and 250-kDa cadherin-like molecules, whereas Vip3A-G bound to 80-kDa and 100-kDa molecules which are distinct from the known Cry1Ab receptors. In addition, separate blotting experiments with Vip3A-G did not show binding to isolated Cry1A receptors, such as M. sexta APN protein, or a cadherin Cry1Ab ecto-binding domain. In voltage clamping assays with dissected midgut from the susceptible insect, M. sexta, Vip3A-G clearly formed pores, whereas Vip3A-F was incapable of pore formation. In the same assay, Vip3A-G was incapable of forming pores with larvae of the nonsusceptible insect, monarch butterfly, Danaus plexippus (Linnaeus). In planar lipid bilayers, both Vip3A-G and Vip3A-T formed stable ion channels in the absence of any receptors, supporting pore formation as an inherent property of Vip3A. Both Cry1Ab and Vip3A channels were voltage independent and highly cation selective; however, they differed considerably in their principal conductance state and cation specificity. The mode of action of Vip3A supports its use as a novel insecticidal agent.