Research Project: Sustainable Pest Management for Arid-Land Agroecosystems

Location: Pest Management and Biocontrol Research

2022 Annual Report

Objectives
Objective 1: Investigate the behavior, biology, demography and ecology of the major pests, and their natural enemies, of cotton and other western U.S. crops, with emphasis on pest movement, feeding, ecology, and conservation of natural enemies. Sub-objective 1A: Develop biological control-informed thresholds for L. hesperus in cotton (Naranjo, Vacant Entomologist) Sub-objective 1B: Characterize the demographics and dispersal patterns of B. tabaci and L. hesperus, natural enemies, and pollinators in a cotton field embedded with push and pull companion plants (Fabrick, Hagler, Vacant Entomologist) Sub-objective 1C: Identify arthropod demography and life stage-specific predation on L. hesperus inhabiting desert-adapted cotton breeding lines (Hagler, Vacant Entomologist) Sub-objective 1D: Test the efficacy on CSB of insecticides typically used in cotton pest management systems. (Brent, Vacant Entomologist) [NP304, C3, PS3A, 3B, and 3C] Objective 2: Examine non-target effects of new GE crops and determine efficacy and non-target effects of insecticidal seed treatments. Sub-objective 2A: Assess effects of Lygus-active Bt cotton on the pests L. hesperus and B. tabaci, and on the natural enemy community and its biological control function (Naranjo, Vacant Entomologist) Sub-objective 2B: Determine the contribution of F. occidentalis on B. tabaci control and the impact of insecticidal seed treatments on the natural enemy community associated with B. tabaci and L. hesperus in cotton (Naranjo, Vacant Entomologist) Objective 3: Investigate the physiology, biochemistry, and molecular biology of major pests of cotton and other arid land crops to develop new and improve existing management approaches such as those based on gene silencing or editing. Sub-objective 3A: Evaluate oral RNAi in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3B: Identify and functionally characterize sex determination genes in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3C: Develop and use CRISPR/Cas gene editing to create gene knockouts in L. hesperus (Brent, Fabrick, Hull) Sub-objective 3D: Identify Bt resistance mechanisms and fitness costs in the lepidopteran cotton pests, Pectinophora gossypiella and Helicoverpa zea (Fabrick, Hull, Naranjo) Sub-objective 3E: Develop tools for the genetic-based manipulation of CSB development for future use in precision-guided biorational pest management. [NP304, C3, PS3A, 3B, and 3C]

Approach
Objective 1: Biological control-informed thresholds, which determine pesticide treatment using the density of pests and their predators, will be developed for L. hesperus in cotton using experimental field research and data mining. Densities of L. hesperus and natural enemy communities will be manipulated and monitored to identify key predators of L. hesperus. Predictions of ratios that enable biological control will be tested and compared to conventional threshold models. Companion plantings of vernonia and marigold will be tested, with lab and field approaches, for their efficacy in protecting cotton by drawing pests away from the crop and towards areas with high predator density. Protein marking will be used to track movement and predator feeding patterns on all life stages, and to determine whether the impact of drought-tolerant cotton isolines on pest colonization and predator success. Objective 2: Cotton engineered to express the Bacillus thuringiensis (Bt) toxin selective for L. hesperus will be tested for non-target effects on natural enemies. Field studies will compare Bt and non-Bt cottons with and without additional insecticides. Sweep net sampling and sticky cards will measure the abundance of common predators of L. hesperus and B. tabaci. Biological control function will be assessed using established thresholds for B. tabaci and direct measures of predation. The impact of insecticidal seed treatments on the natural enemies of B. tabaci and L. hesperus in cotton will be assessed using field-based inclusion cage studies with young cotton plants containing whitefly eggs exposed to adult and immature thrips. To assess early-season and season-long efficacy and non-target impacts of cotton seed-treatments, field studies will compare population densities of B. tabaci, thrips, and other arthropods exposed to cotton with and with seed treatment. Objective 3: The efficacy of oral RNAi will be assessed in L. hesperus by feeding or injecting dsRNA for genes involved in ovary function. To determine if digestive tract nucleases destroy dsRNA before it can be effective, luminal contents and gut homogenates will be assessed for enzymatic activity. To identify genes involved in dsRNA uptake from the gut, homologs of endocytotic pathway genes will be identified then silenced by RNAi to determine function. The role of parental RNAi will be tested by injecting adult L. hesperus females with dsRNA targeting the eye pigmentation genes and examining embryo eye color. Sex determination gene homologs in L. hesperus will be identified, their expression measured, and function determined by RNAi. CRISPR/Cas gene driver methods will be optimized for L. hesperus, using injections and electroporation to modify embryos. Bt toxin resistance mechanisms in pink bollworm and corn earworm relying on mutations in the ABC transporter and midgut cadherin genes will be examined by toxicity screening and cellular localization. Determination of whether a fitness tradeoff occurs in the corn earworm with Bt toxin resistance will be made in susceptible and resistant strains fed toxic and non-toxic diets by comparing life history traits and flight performance.

Progress Report
This report documents progress for project 2020-22620-023-000D, titled “Sustainable Pest Management for Arid-Land Agroecosystems” which was started in July 2020 and continues research from project 2020-22620-022-000D titled, “Ecologically Based Pest Management in Western Crops Such as Cotton.” The following documents the research progress made in fiscal year (FY) 2022. Under Sub-objective 1B, tests are underway to measure lygus bug and whitefly olfactory response to various plants. The methods used for testing include a Y-tube olfactometer and behavior monitoring software. The tests indicate that video and computer automated systems can effectively track the insect's response to various plant odors. However, optimization of the software required more time and effort than anticipated. Tests are now being conducted to determine which plants might attract or repel major cotton pests. The goal is to find plants that can be planted adjacent to cotton (companion plants) that attract (lure) the pests away from cotton. Ultimately, companion planting practices could reduce pesticide use in cotton. For Sub-objective 1B, Hypothesis 1Bii, an insect feeding choice arena assay was developed to measure lygus bug and whitefly host plant preference for feeding activity. In pilot tests, the arena proved very effective. Currently, a wide variety of plant species are being tested under greenhouse conditions for their attractiveness to lygus and whitefly. In addition, a novel method for marking lygus and whitefly was developed. This method is being used to monitor lygus and whitefly plant preference under more realistic conditions than feeding choice arena tests. Lygus and whitefly mark-release-recapture greenhouse studies are underway to monitor their dispersal characteristics within large greenhouses that contain a wide variety of potential host plants (a mini agroecosystem). Ultimately, the data obtained from the feeding choice and greenhouse studies will be used to develop an effective trap cropping system for cotton growers. If successful, such a system could reduce pesticide usage. Under Sub-objective 2A, replicated large field plot studies were conducted for a final year to assess the non-target impacts of a new transgenic cotton with efficacy against Lygus hesperus and thrips, and to assess off-target impacts on biological control services. Treatments included: transgenic Bacillus thuringiensis (Bt) cotton (MON88702), that produces an endogenous toxin, and its non-toxic near isoline (DP393) without additional insecticides; both cultivars with the addition of a material that selectively controls thrips; and a positive control of DP393 sprayed with a broad-spectrum material that represents an alternative control agent for Lygus. Bt and non-Bt seeds did not have added insecticidal seed treatments, as is common for the industry. These treatment combinations enabled comparison of the Bt trait alone on non-targets, a comparison of the Bt trait to a conventional control alternative, and comparison of the thrips trait and a conventional control alternative on the abundance of natural enemies and potential biological control function on key pests. Because thrips are also predators in the system, this will also allow us to evaluate if the planting of this new Bt cotton poses any risks to current levels of biological control on whitefly and mites. Extensive sampling quantified the abundance of pests and natural enemies in the system, data collection has been completed for the final year of field studies, final data are being entered and verified and will be analyzed during the coming year. Preliminary results suggest that biological control function, measured on sentinel whitefly prey, is not altered by the Bt crop and that the technology is moderately effective in control of Lygus and thrips. Under Sub-objective 2B, the intended studies were not initiated due to a critical scientific vacancy in the unit. These studies will be completed when the position is filled. Under Sub-objective 3A, oral RNAi in Lygus was previously shown to be ineffective with no phenotypic effects observed and no reduction in the abundance of target mRNA transcripts. The limited degradation seen after double stranded Ribonucleic acid (dsRNAs) were incubated with midgut homogenates suggests that the ineffectiveness of oral RNAi is not gut nuclease dependent. Antisense oligonucleotides and siRNAs modified to improve stability were also ineffective as were liposomes and nanoparticles complexed with fluorescently labeled dsRNAs, both of which had limited target tissue uptake. These findings suggest that persistence of the dsRNAs in the midgut may be a limiting factor for efficacy. In addition, no evidence for parental RNAi was observed in Lygus nymphs derived from mothers injected with dsRNAs targeting eye pigmentation. In support of Sub-objective 3C, multiple dsRNAs were found to affect eye pigmentation after injection into early-stage embryos. Embryonic injection of dsRNAs targeting genes linked to other developmental processes were also effective in generating expected phenotypes. These results indicate that eggs can be transcriptionally modified and as such allows for genes expressed early in Lygus development to now be evaluated as candidates for targeted disruption. Under Sub-objective 3D, Hypothesis 3Di, experiments using CRISPR/Cas9 gene editing were able to modify the ATP-binding cassette gene ABCA2 in a susceptible strain of corn earworm. The resultant insects were found to be resistant to Cry2Ab, a toxin found in transgenic Bt crops. Resistant individuals had mutations that disrupted the function of the ABCA2 gene, with five different disruptive mutations identified. These data not only represent the first identification of a gene responsible for resistance to Cry2Ab in corn earworm but illustrate the power of using CRISPR/Cas9 gene editing for validating the in vivo function of genes involved in resistance to Bt transgenic crops. In support of Sub-objective 3D, Hypothesis 3Dii, cultured insect cells were provided to collaborators at the University of Arizona and experiments have established appropriate cellular fixation and embedding techniques require for immunofluorescence experiments. Plasmids have been constructed and sequenced to confirm production of correct PgCad1 proteins with and without the Venus marker protein. Additional plasmids producing mCherry-labeled cellular organelle markers have been prepared. Progress has been slowed by impacts of pandemic on university collaborator and staff to complete immunohistochemistry objective. For Sub-objective 3D, Hypothesis 3Diii, studies to determine if toxin resistance alters the flight capability or propensity of the corn earworm have been delayed approximately one year due to the pandemic. Flight mill assays to test the flight capacity of a commercially available strain of corn earworm have been completed. Diet bioassay experiments and flight mill tests to compare GA and GA-R corn earworm strains will be initiated in FY 2023.

Accomplishments
1. A high-quality meta-analysis shows that transgenic Bacillus thuringiensis (Bt) maize does not impact non-target organisms. Transgenic Bt crops are widely used globally for control of several key insect pests. However, on-going debates continue to question the environmental safety of these crops, particularly their impacts on non-target organisms. An ARS researcher in Maricopa, Arizona, in collaboration with two scientists from Agroscope in Switzerland, developed a high-quality database of published studies that compared abundance and ecological function of non-target organisms on Bt and non-Bt maize and conducted a systematic review using meta-analyses to assess non-target effects of Bt maize. Analyses revealed no effects of Bt maize on most invertebrate groups when no insecticides were applied. In contrast, abundance was often larger on untreated Bt maize compared with non-Bt maize treated with insecticides to control the Bt-targeted pest. This high-quality study confirms previously published work and will be of value to growers, biotechnology regulators and a public concerned about potential environmental risks of transgenic crops.

2. Genomics uncovers novel genetic basis of resistance to Bt toxin Cry1Ac in the corn earworm. Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) have many benefits and are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefits. Together with University of Arizona collaborators, an ARS researcher in Maricopa, Arizona, produced the first chromosome-level genome assembly and discovered a novel gene for resistance to Bt toxin in Helicoverpa zea (corn earworm), which is highly damaging pest of cotton and corn in the United States. The findings provide direct application for genomics and detection of novel Bt resistance genes.

3. A new insect marking technique provides a tool for studying whitefly and lygus host plant preference. Lygus bug and whitefly have a broad host plant range and exhibit feeding preferences between the plant types. An ARS researcher in Maricopa, Arizona, developed a method to mark lygus and whitefly with a fluorescent dye to monitor their distribution within a diverse agroecosystem that includes cotton as the focal crop. This dye is easily detected on the pests using an ultraviolet light. This method gives researchers a new and inexpensive tool for monitoring intercrop dispersal of pests and beneficials. Knowledge of insect dispersal patterns allows growers to implement more cost-effective and environmentally benign pest control (e.g., trap crops).

4. Gene editing of eye pigmentation genes in Lygus hesperus provides direct validation of gene function. Lygus hesperus is a key pest of cotton and other crops throughout the western United States, and new pest management strategies are needed. Powerful new molecular genetic biocontrol tools are becoming available that can cause species-specific disruption of critical biological processes in pests. However, such genetic biocontrol strategies require expansion of molecular knowledge and tool development in each target pest species. ARS scientists in Maricopa, Arizona, used CRISPR/Cas9 gene editing to knockout out eye color genes in the insect pest Lygus Hesperus. The team produced stable L. hesperus strains with eye color mutants that differed from wild-type individuals, demonstrating not only that CRISPR gene editing functions in L. hesperus, but that such eye pigmentation genes are useful for tracking the successful genetic manipulation in this pest species.

5. Mortality dynamics of whitefly in a multi-host agroecosystem. Whiteflies are a key pest of many agronomic and horticultural crops in the desert southwestern United States. Pest population dynamics are governed by complex, interacting factors involving its cultivated and wild host plants, seasonality, movement, and demography. An ARS researcher in Maricopa, Arizona, and collaborators from the University of Arizona, used life tables to measure sources and rates of mortality on multiple host plants in three regions of Arizona. They found that predation by arthropod natural enemies was the largest source of mortality on most host plants. They also found that total mortality is extremely high during winter months on several host plants but relatively low on spring crops like cantaloupes leading to an ecological release that ultimately drives regional dynamics of the pest during the summer and fall. This detailed understanding provides clues to the success of this invasive pest in our agroecosystems and facilitates opportunities for improved pest management at a broader landscape scale.

6. Determination of optimal plot size to assess non-target effects in cotton. The assessment of non-target effects from new insecticides and new genetically modified crops is essential for developing sustainable cotton pest management strategies. These assessments need to be conducted in plots of sufficient size to measure true treatment effects representative of commercial agriculture. An ARS researcher in Maricopa, Arizona, collaborated with researchers from the University of Arizona to identify the optimal plots size for non-target investigations. They compared square plots of 0.015 to 0.060 hectares in size and measured treatment effects (two insecticide and an untreated control) for natural enemy and pest abundance, arthropod community structure, arthropod diversity, and biological control function. The smallest plot examined was able to readily discern treatment effects. Results are useful for researchers in cotton and other crop, and for industry and regulatory agencies that frequently use field plots to assess environmental effects of new insecticides and other pest control technologies.

7. Estimation of economic injury levels for whiteflies attacking soybean. The economic injury level (EIL) is defined as the density of a pest that causes damage equal to the cost of preventing that damage and is a foundational element of integrated pest management. EILs have been developed for relatively few crops including soybeans, for which silverleaf whiteflies are a key pest in South America. An ARS researcher in Maricopa, Arizona, collaborated with researchers from the University of Sao Paulo, Brazil, and estimated that the EIL ranged from 2.5 to 25.5 and 0.17 to 1.79 for immature and adult whiteflies per leaflet, respectively, attacking soybean. EILs varied based on crop value, cost of control, and control efficacy. Results provide useful data for researchers and pest managers to develop economic thresholds for more efficiently managing this pest in soybean.

8. Development of a cuticular marker system for Lygus. New genetic-based control strategies have enormous potential for insect pest management. An initial step in that process is the development of a reporter system that allows for quick, non-destructive identification of modified insects. ARS researchers in Maricopa, Arizona, have identified multiple insect cuticle coloration genes and used RNAi to silence their activities. Unlike control insects, which are typically green, one group of RNAi impacted Lygus were completely black, providing a visual marker for differentiating wild-type insects from experimental groups. These results provide a valuable new tool facilitating research into genetic-based manipulation of Lygus.

Review Publications
Schutze, I., Yamamoto, P., Malaquias, J., Herritt, M.T., Merten, P., Thompson, A.L., Naranjo, S.E. 2022. Correlation-based network analysis of the influence of Bemisia tabaci feeding on photosynthesis and foliar sugar and starch composition in soybean. Insects. 13(1). Article 56. https://doi.org/10.3390/insects13010056.
Hagler, J.R., Hull, A.M., Casey, M.T., Machtley, S.A. 2021. Use of a fluorophore to tag arthropods for mark-release-recapture type research. Journal of Insect Science. 21(6). Article ieab099. https://doi.org/10.1093/jisesa/ieab099.
Heu, C.C., Gross, R.J., Le, K.P., LeRoy, D.M., Fan, B., Hull, J.J., Brent, C.S., Fabrick, J.A. 2022. CRISPR-mediated knockout of cardinal and cinnabar eye pigmentation genes in the western tarnished plant bug. Scientific Reports. 12. Article 4917. https://doi.org/10.1038/s41598-022-08908-4.
Oleisky, E.R., Stanhope, M.E., Hull, J.J., Dickinson, P.S. 2022. Isoforms of the neuropeptide myosuppressin differentially modulate the cardiac neuromuscular system of the American lobster, Homarus americanus. Journal of Neurophysiology. 127(3):702-713. https://doi.org/10.1152/jn.00338.2021.
Mao, C., Zhu, X., Wang, P., Huang, R., Zhoa, M., Hull, J.J., Lin, Y., Ma, W. 2021. Transgenic double-stranded RNA rice, a potential strategy for controlling striped stem borer (Chilo suppressalis). Pest Management Science. 78(2):785-792. https://doi.org/10.1002/ps.6692.
Hull, J.J., Brent, C.S., Choi, M.Y., Miko, Z., Fodor, J., Fonagy, A. 2021. Molecular and functional characterization of pyrokinin-like peptides in the western tarnished plant bug lygus hesperus (Hemiptera: Miridae). Insects. 12(10). Article 914. https://doi.org/10.3390/insects12100914.
Schutze, I., Naranjo, S.E., Yamamoto, P. 2022. Impact of Bemisia tabaci MEAM1 (Hemiptera: Aleyrodidae) on soybean yield and quality under field conditions. Journal of Economic Entomology. 115(3):757-766. https://doi.org/10.1093/jee/toac026.
Zuo, Y., Shi, Y., Zhang, F., Guan, F., Zhang, J., Feyereisen, R., Fabrick, J.A., Yang, Y., Wu, Y. 2021. Genome mapping coupled with CRISPR gene editing reveals a P450 gene confers avermectin resistance in the beet armyworm. PLoS Genetics. 17(7). Article e1009680. https://doi.org/10.1371/journal.pgen.1009680.
Jiang, F., Chang, G., Li, Z., Abouzaid, M., Du, X., Hull, J.J., Ma, W., Lin, Y. 2021. The hsp/co-chaperone network in environmental cold adaptation of chilo suppressalis. International Journal of Biological Macromolecules. 187:780-788. https://doi.org/10.1016/j.ijbiomac.2021.07.113.
Muscato, A.J., Walsh, P., Pong, S., Pupo, A., Gross, R.J., Christie, A.E., Hull, J.J., Dickinson, P.S. 2021. Does differential receptor distribution underlie variable responses to a neuropeptide in the lobster cardiac system? International Journal of Molecular Sciences. 22(16). Article 8703. https://doi.org/10.3390/ijms22168703.
Kan, C., Mendoza-Herrera, A., Levy, J., Hull, J.J., Fabrick, J.A., Tamborindeguy, C. 2021. HPE1, an effector from zebra chip pathogen interacts with tomato proteins and perturbs ubiquitinated protein accumulation. International Journal of Molecular Sciences. 22(16). Article 9003. https://doi.org/10.3390/ijms22169003.
Guillaume, T., Makowski, D., Libohova, Z., Bragazza, L., Sallaku, F., Sinaj, S. 2021. Soil organic carbon saturation in cropland-grassland systems: storage potential and soil quality. Geoderma. 406. Article 115529. https://doi.org/10.1016/j.geoderma.2021.115529.
Meissle, M., Naranjo, S.E., Romeis, J. 2022. Database of non-target invertebrates recorded in field experiments of genetically engineered Bt maize and corresponding non-Bt maize. BMC Research Notes. 15. Article 199. https://doi.org/10.1186/s13104-022-06021-3.
Oppert, B.S., Muszewska, A., Steczkiewicz, K., Šatovic-Vukšic, E., Plohl, M., Fabrick, J.A., Vinokurov, K.S., Koloniuk, I., Johnston, J., Smith, T.P., Guedes, R.C., Terra, W.R., Ferreira, C., Dias, R.O., Chaply, K.A., Elpidina, E.N., Tereshchenkova, V., Mitchell, M.F., Jenson, A.J., Mckay, R., Shan, T., Cao, X., Xiong, C., Jiang, H., Morrison III, W.R., Koren, S., Schlipalius, D., Lorenzen, M.D., Bansal, R., Wang, Y., Perkin, L.C., Poelcheau, M., Friesen, K.S., Olmstead, M.L., Scully, E.D., Campbell, J.F., et al. 2022. The genome of Rhyzopertha dominica (Fab.) (Coleoptera: Bostrichidae): Adaptation for success. Genes. 13(3). Article 446. https://doi.org/10.3390/genes13030446.
Benowitz, K.M., Allan, C.W., Degain, B.A., Li, X., Fabrick, J.A., Tabashnik, B.E., Carriere, Y., Matzkin, L.M. 2022. Novel genetic basis of resistance to Bt toxin Cry1Ac in Helicoverpa zea. Genetics. 221(1). Article iyac037. https://doi.org/10.1093/genetics/iyac037.
Pyc, M., Gidda, S.K., Seay, D., Esnay, N., Kretzschmar, F.K., Cai, Y., Doner, N.M., Greer, M.S., Hull, J.J., Coulon, D., Brehelin, C., Yurchenko, O., De Vries, J., Valerius, O., Braus, G.H., Ischebeck, T., Chapman, K.D., Dyer, J.M., Mullen, R.T. 2021. LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis. The Plant Cell. 33(9):3076-3101. https://doi.org/10.1093/plcell/koab179.
Hagler, J.R., Casey, M.T., Hull, A.M., Machtley, S.A. 2022. Liquid fluorophore taggants for mark-release-recapture research: A survey of potential arthropod targets. Entomologia Experimentalis et Applicata. 170:821-830. https://doi.org/10.1111/eea.13191.