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

Location: Southern Insect Management Research

2023 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 substantial progress on laboratory and field research components during the past year, despite access to facilities limited until the end of March 2022. Wild populations of Helicoverpa species from New York, Pennsylvania, Texas, and Virginia were collected during the 2021 growing season, and some DNA extractions were possible to conduct genotyping assays. Genotyping was also done on bollworm samples collected from the Florida panhandle during the growing seasons and winter months from 2019 to 2021. ATP binding cassette (ABC) transporter class G (ABCG) gene knock-out lines of tobacco budworm were established using CRISPR gene editing system. Guide RNA (gRNA) designed for the ABCG transporter gene were used to delete exons 2 of the ABCG gene by injecting the in vitro assembled binary RNA-Protein complexes into freshly laid eggs. Insect lines with deletions that inactivated ABCG gene were established by screening the second filial generation (F2) progeny. Bioassays were conducted to evaluate the effects of deactivating the ABCG gene on the susceptibility to Cry1Ac insecticidal protein from Bacillus thuringiensis. Ecologically relevant thermal tolerance and mortality differences between resistant and susceptible bollworms populations were identified using bioassays. Resistant progeny of insects which continued diapause for more than three months had significantly lower mortality at lower temperatures than susceptible individuals from any parental background. Data indicate that thermal tolerance is highly correlated with toxin susceptibility and parental diapause history. This information will be helpful in developing trait resistance management strategies and IPM recommendations for land management.

Accomplishments
1. Effectiveness of crystalline and vegetatively produced Bt toxins for control of bollworm in cotton. ARS researchers in Stoneville, Mississippi, conducted insect bioassays and field efficacy trials with bollworms to evaluate the association between these two key aspects of resistance to crystalline transgenic insecticides. Higher levels of bollworm survival in laboratory bioassays were generally associated with decreased efficacy of Bt cotton plants in the field. The findings of these studies show that planting field corn hybrids that produce vegetative insecticidal toxins in cotton-growing regions of the United States likely expedite the development of bollworm resistance to this insecticide. Information gained from these studies supports recommendations from a Scientific Advisory Panel convened by the US EPA that effectively limit selection for resistance to vegetatively produced toxins in corn where bollworm is not a major economic pest, and thereby help to preserve its efficacy against bollworm in cotton where it is a major economic pest.

2. Evaluation of bollworm larval behavior on Bt cotton. Foliar insecticides and insecticidal proteins from Bacillus thuringiensis (Bt) in transgenic cotton are standard tools used for bollworm management in cotton. Bollworm larvae feed on flower buds (squares), fresh flowers, wilting flower corollas (bloom tags), and bolls in cotton. ARS researchers at Stoneville, Mississippi, evaluated bollworm survival in flowers of different transgenic cotton cultivars treated with various synthetic insecticides. Bollworm exposure to sub-lethal doses of insecticidal Bt proteins in certain transgenic cotton and foliar insecticides, which are commonly used for supplemental control, likely expedites the development of insect resistance to these toxicants. Integrated pest management strategies for bollworm management in cotton will be improved across the southern U.S. to account for insecticide timing and larval location within the plant canopy.

3. Functional genomics of Bt resistance in tobacco budworm. ARS researchers at Stoneville, Mississippi, used Clustered regularly interspersed short palindromic repeats (CRISPR) genome editing reagents to establish tobacco budworm knock-out lines of ATP binding cassette (ABC) transporter classes G (ABCG), C3 (ABCC3), and class C4 (ABCC4) to evaluate the role of these gene in the Bacillus thuringiensis (Bt) toxins Cry1Ac and Vip3a mode of action. Bioassays indicated 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. Evaluation of Bt resistance in bollworm populations. Although the survival rate of H. zea on many corn hybrids expressing Bt proteins is high, the impact of insects developing on these Bt proteins and their fitness characteristics in the subsequent generation is not understood. The fitness of the progeny of these H. zea which are deposited on Bt cotton, is of great interest due to the high management costs of these caterpillar pests on cotton. ARS researchers in Stoneville, Mississippi, evaluated the fitness of F1 progeny of H. zea collected from both Bt and non-Bt corn varieties as it relates to their susceptibility to commonly used insecticide, chlorantraniliprole, for their control. Diet-incorporated bioassays were used to examine the susceptibilities of these insect populations to a diamide insecticide. The response of populations collected from either Bt or non-Bt corn was similar for 2022 and fell within the ranges from previous years. For Bt corn, the estimated insecticide concentration that controls half of the pest population (LC50 value) ranged from 17.7ng – 43.9 ng/ml of diet, while the estimated concentration for those collected from non-Bt corn ranged from 16.9ng – 39.3 ng/ml of diet.

5. Evaluation of critical minimum temperatures on diapausing and non-diapausing Bt resistant and susceptible cotton bollworm pupae. Bioassays conducted by ARS researchers at Stoneville, Mississippi, indicated ecologically relevant thermal tolerance and mortality differences between resistant and susceptible bollworms populations. Resistant offspring produced from individuals whose diapause was continuous and unbroken for more than three months experienced significantly lower mortality at lower temperatures than susceptible individuals from any parental background. Data suggest that toxin susceptibility and parental diapause history are crucial in thermal tolerance. Results from these experiments will aid in developing trait resistance management strategies and IPM recommendations for land management, including crop residue destruction practices for growers across the southern U.S.

6. Endophytic control of Bt resistant cotton bollworm larvae in transgenic cotton. ARS researchers at Stoneville, Mississippi, screened commercially available corn hybrids for potential endophytic fungi. Cultivars expressing Bt proteins failed to produce endophytic growths from any tissues assayed using standard laboratory methods. Non-transgenic cultivars yielded three morphologically distinct species of naturally occurring endophytic fungi that have been isolated, purified, and cultured. Preliminary lab data suggest two candidate endophytes having entomopathogenic properties on cotton bollworm larvae and other key cotton insect pests, leading to markedly high larval mortality rates for BT resistant populations. Field evaluation is currently in progress.

7. International Cooperation / Collaboration. International collaborations with Maharana Pratap University of Agriculture and Technology (MPUAT) on population genetics of invasive old-world bollworm continued. A collaborating faculty member from MPUAT visited USDA ARS laboratory in Stoneville, Mississippi, to conduct research and training on population genetics and functional genomics of lepidopteran pests of cotton.

Review Publications
Yang, Q., Men, X., Zhang, K., Liu, M., Guo, W., Zhu, C., Zhao, W., Reddy, G.V., Ouyang, F., Ge, F. 2022. Flower strips can promote natural enemy biodiversity and biocontrol services, and prevent pest resurgence in cotton crops. Entomologia Generalis. 42:1-12. https://doi.org/10.1127/entomologia/2022/1545.
Wang, L., Reddy, G.V., Zhao, Z. 2023. The contrasting response of crop production and pest damage to ENSO cycles. Entomologia Generalis. 43:1-11. https://doi.org/10.1127/entomologia/2022/1688.
Kline, O., Phan, N.T., Porras, M.F., Chavana, J., Little C.Z., Stemet, L., Acharya R.S., Biddinger D.J., Reddy, G.V., Rajotte, E.G., Neelendra K.J. 2022. Biology, genetic diversity, and conservation of wild bees in tree fruit orchards. Biology. 12(1):31. https://doi.org/10.3390/biology12010031.
Godbold, R.E., Whitney, C.D., Gore, J., Musser, F., Catchot, A.L., Dodds, .M., Cook, D.R., Little, N. 2023. Efficacy of Bt toxins and foliar insecticides against bollworm, Helicoverpa zea (Boddie), in dried flower corollas of cotton. Journal of Cotton Science. 27(1):28-36. https://doi.org/10.56454/ZNAX3626.
Akpinar, B.A., Muslu, T., Ozturk-Gokce, Z.N., Reddy, G.V., Dogramaci, M., Budak, H. 2023. Wheat long noncoding RNAs from organelle and nuclear genomes carry conserved microRNA precursors which may together comprise intricate networks in insect responses. International Journal of Molecular Sciences. 24(3). Article 2226. https://doi.org/10.3390/ijms24032226.
Dos Santos, I., Paula-Moraes, S.V., Beuzelin, J., Hahn, D., Perera, O.P., Fraisse, C.W. 2023. Factors affecting population dynamics of Helicoverpa zea (Lepidoptera: Noctuidae) in a mixed landscape with Bt cotton and peanut. Insects. 14(4):395. https://doi.org/10.3390/insects14040395.
Stahlke, A.R., Chen, J., Tembrock, L.R., Sim, S.B., Chudalayandi, S., Geib, S.M., Scheffler, B.E., Perera, O.P., Gilligan, T.M., Childers, A.K., Hackett, K.J., Coates, B.S. 2022. A chromosome-scale genome assembly of a Helicoverpa zea strain resistant to Bacillus thuringiensis Cry1Ac insecticidal protein. Genome Biology and Evolution. 15(3). Article evac131. https://doi.org/10.1093/gbe/evac131.
Yang, F., Kerns, D.L., Little, N., Brown, S., Stewart, S., Catchot, A., Cook, D., Gore, J., Crow, W., Lorenz, G., Towles, T., Tabashnik, B.E. 2022. Practical resistance to cry toxins and efficacy of Vip3Aa in bt cotton against helicoverpa zea. Pest Management Science. 1:1-9. https://doi.org/10.1002/ps.7142.
Allen, K.C., Little, N., Perera, O.P. 2022. Susceptibilities of Helicoverpa zea (Lepidoptera: Noctuidae) populations from the Mississippi Delta to a diamide insecticide. Journal of Economic Entomology. 116(1):160–167. https://doi.org/10.1093/jee/toac180.
Flores-Rivera, X.L., Paula-Moraes, S.V., Johnson, J., Jack, C., Perera, O.P. 2022. Helicoverpa armigera (Hubner) and Helicovera zea (Boddie) (Lepidoptera: Noctuidae) in Puerto Rico: phenology of flight, analysis of hybrid presence, and insecticide performance in field crops. Journal of Pest Science. https://doi.org/10.3389/finsc.2022.1010310.