Project : USDA ARS

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Research Project: Ecologically Sustainable Approaches to Insect Resistance Management in Bt Cotton

Location: Southern Insect Management Research

2022 Annual Report

Objectives
1. Determine ecological characteristics and insect-plant interactions, such as susceptibility and fitness, to help identify components that can be manipulated to minimize the evolution of resistance to Bt toxins in Helicoverpa zea. 1.A. Determine the status of Bt resistance in H. zea and develop methods to measure the subsequent fitness costs associated with survival on Bt crops relative to other wild and cultivated hosts. 1.B. Evaluate the impact of Bt corn as a primary host on subsequent H. zea damage and fitness on Bt cotton. 2. Characterize genomic, transcriptomic, and population genetic components of H. zea relative to their contribution to evolution of resistance to Bt toxins and develop computational methods for identifying interactions between gene co-regulatory networks that modulate resistance loci. 2.A. Develop computational methods to identify any gene regulatory networks that interact in responding to intoxication with Bt toxins in H. zea. 2.B. Characterize genomic, transcriptomic, and population genetic components of H. zea relative to their contribution to evolution of resistance to Bt toxins. 3. Develop and optimize early detection methods for invasive polyphagous pests of cotton.

Approach
Recent failures of transgenic crops producing Bacillus thuringiensis (Bt) insecticidal toxins to control noctuid pests and reports of field-evolved resistance to Bt toxins indicate an increase in tolerance to certain Bt toxins in pest populations. To gain a better understanding of factors contributing to the evolution of resistance and to identify components that could be manipulated to minimize the development of resistance to Bt toxins, ecological characteristics of bollworm (BW), Helicoverpa zea, and its interactions with host plants will be investigated. The long-term objective of this project is to identify ecologically sustainable approaches and develop new strategies for the efficient management of BW resistance to Bt toxins. A large-scale Bt resistance survey will be conducted by collecting BW populations from a range of wild and crop hosts across the southern U.S. Progeny from these populations will be assayed using discriminating doses of Bt toxins that compare susceptibility of field insects with control insects from laboratory colonies. This survey will serve as a basis for quantifying the incidence of Bt resistant in BW in areas where Bt corn and cotton production coexists. The fitness parameters will examine the impacts of Bt crops on tolerant insects and fitness of their offspring. This information will assist in determining the status of susceptibility of BW to Bt crops in the southern US. Impacts of Bt corn as a primary host of BW on subsequent damage and fitness on Bt cotton will be assessed using correlations between Bt toxin levels in kernels and larval survival on Bt field corn. Toxin levels in kernels and larval survival on different Bt corn hybrids will facilitate the inference of selection pressure on BW by Bt corn, which will be vital for developing insect resistance management (IRM) strategies in Bt cotton. Contribution of genetic components to Bt toxin resistance evolution will be studied using empirical and computational methods. Genetic loci linked Bt resistance will be evaluated using computational methods such as weighted gene co-regulatory network analysis to predict interactions between gene co-regulatory networks that modulate resistance. Genetic loci predicted to have a high probability of participation in modulating mode of action of Bt toxins will be used in quantitative, comparative, and population genetic studies to evaluate their roles in to Bt toxin resistance. This approach is expected to identify novel genetic loci involved in the toxin mode of action and those contributing to resistance to Bt toxins. This project will also develop novel technologies or improve upon those currently available to facilitate rapid detection of invasive pests. Species-specific antibody-based lateral flow immune assays (LFIA) will be used for rapid identification of species. When LFIA is not possible due to lack of species-specific antigens (targets) in proteins to develop antibodies, isothermal recombinase polymerase amplification (RPA) technology that can amplify species-specific DNA tagged with artificial antigens will be used to detect invasive species.

Progress Report
This project made limited progress on laboratory and field research components during the COVID19 pandemic. Most of our effort focused on maintaining and preserving resources such as insect lines with unique traits critical for research projects. However, laboratory and field research has recently resumed. Substantial progress has been made regarding insect collections of bollworm and laboratory bioassays of F1 progeny. Wild populations of Helicoverpa species from New York, Pennsylvania, Texas, and Virginia were collected during the 2020 growing season, but no DNA extractions were possible to conduct genotyping assays. Identification of gene co-regulatory networks responding to Bt toxin ingestion in tobacco budworm was completed. ATP binding cassette (ABC) transporter class C gene knock-out lines of tobacco budworm were established using CRISPR/Cas9 gene editing system. Guide RNA (gRNA) designed to ABCC3 and ABCC4 transporter genes were used to knock out the genes by injecting the in vitro assembled binary RNA-Protein complexes into freshly laid eggs. Insect lines with deletions that inactivated ABCC3 or ABCC4 gene were established by screening the F2 progeny. Bioassays were conducted to evaluate the effects of deactivating ABCC3 or ABCC4 gene on the susceptibility to Vip3Aa insecticidal protein from Bacillus thuringiensis.

Accomplishments
1. Survey of bollworm survival on crystalline and vegetatively produced Bt toxins. The large-scale resistance survey of bollworms from multiple wild and cultivated hosts on crystalline and vegetatively produced toxins has been completed. Study by ARS researchers in Stoneville, Mississippi, indicates widespread practical resistance of bollworm in the United States to crystalline Bt toxins produced by transgenic cotton and corn, but not Vip3A. However, our results convey an early warning of field-evolved resistance to Vip3Aa in bollworm that supports calls for urgent action to preserve the efficacy of this toxin.

2. Evaluatoin of bollworm larval behaviour on Bt cotton. Evaluation by ARS researchers in Stoneville, Mississippi, of cotton expressing different Bt proteins on bollworm larval behavior and subsequent damage to fruiting structures have been completed and published. This research demonstrated that the fruiting form damage caused by bollworm in dual-gene cotton has increased over time. Findings from this study indicate that dual-gene transgenic cotton may no longer be economically adequate for bollworm control without the need for supplemental foliar insecticide applications when insect pressure is moderate to high.

3. Functional genomics of putative Bt toxin receptors in bollworm and tocacco budworm. CRISPR/Cas9 genome editing reagents were used to establish tobacco budworm knock-out lines of ABCC3 or ABCC4 to evaluate the role of these genes in the Vip3A mode of action. ARS researchers in Stoneville, Mississippi, completed bioassays indicating that knocking out either ABCC3 or ABCC4 altered the susceptibility of tobacco budworm to Vip3A. Therefore, it was concluded that neither ABCC3 nor ABCC4 transporter genes play a significant role in the mode of action of Vip3A Bt protein.

4. Efficacy of Bt protein producing field corn in controlling bollworm larvae. A large plot field trial was conducted by ARS researchers in Stoneville, Mississippi, to evaluate the efficacy of VT Double Pro (Vip3A) field corn compared to non-Bt corn in controlling bollworm larvae. Large numbers of corn earworm larvae were observed in both non-Bt corn and VT Double Pro corn. Larval development was slightly slower on VT Double Pro corn compared to non-Bt corn. Very few to no larvae were observed in corn hybrids that expressed Vip3A. Also, two small plot trials comparing Bt corn technologies were conducted. Similar results were observed in this trial as in the sentinel plots. Non Bt corn and Bt corn hybrids that did not express Vip3A had higher densities of corn earworm larvae infesting ears and greater damage than hybrids that expressed Vip3A. Very few larvae were observed in the Vip 3A expressing hybrids, and all were smaller than 3rd instar. No other caterpillar pests were observed, and no differences in yield among the hybrids were observed.

5. Comparative study of Vip3A protein producing Bt cotton and Bollgard II cotton. ARS researchers in Stoneville, Mississippi, conducted an experiment in 2021 to compare the efficacy of Bt cotton varieties containing the Bt Vip3A gene relative to non-Bt cotton and Bollgard II cotton (2-gene). Treatments were in a split-plot arrangement with four replications. The main-plot factor was insecticide spray and included plots sprayed with two applications of chlorantraniliprole or unsprayed plots. The sprayed plots were sprayed at a first flower, and the second application was made approximately two weeks after the first application. The sub-plot factor was Bt cotton type and included non-Bt, Bollgard II, Widestrike 3, TwinLink Plus, and Bollgard 3. The number of damaged squares and bolls, and the number of live larvae was recorded on multiple dates throughout the flowering period. For reporting purposes, the mean levels of percent square injury and boll injury were calculated across all samples. All plots were harvested at the end of the season and yields were converted to pounds of seed cotton per acre. Overall, bollworm densities were low to moderate in this experiment during 2021, but damage in the non-Bt cotton was consistent across multiple sample dates. Averaged across all sample dates, the mean square damage in the unsprayed non-Bt cotton was 26.8% compared to 7.8% in the sprayed non-Bt cotton (Table 2). Percent square damage in unsprayed Bt technologies ranged from 0.5 to 2.8%, while square damage in the sprayed Bt technologies ranged from 0.7 to 2.2%. As expected, the highest level of square damage occurred on Bollgard II cotton. Boll damage averaged 15.7% in the unsprayed non-Bt cotton and 5.3% in the sprayed non-Bt cotton. Boll damage ranged from 0.3% to 1.7% in unsprayed and sprayed Bt technologies. The highest level of boll damage occurred in unsprayed Bollgard II cotton. The results of these experiments will be necessary for developing control recommendations for growers across the southern U.S.

U.S. DEPARTMENT OF AGRICULTURE

Southern Insect Management Research: Stoneville, MS