Research Project: Sustainable Pest Management for Arid-Land Agroecosystems

Location: Pest Management and Biocontrol Research

2025 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 is the final report which documents the five-year progress of project 2020-22620-023-000D, titled, “Sustainable Pest Management for Arid-Land Agroecosystems”, which expired in June 2025 and is being replaced by new project titled, “Sustainable Insect Pest Management for Arid-Land Crops”, which is currently undergoing OSQR review. Under Sub-objective 1A, intended studies were delayed due to critical scientific vacancies during much of the project. The first experimental studies are now underway. Once cotton fields are sufficiently developed, abundance measures of predatory arthropods and Lygus hesperus will be collected and analyzed to identify predator species associated with reductions in L. hesperus abundance. Experiment will be replicated in FY26. In FY27 the predator to prey ratios (thresholds) will be tested in comparison with the conventional pest-only thresholds. Under Sub-Objective 1B, three open field studies conducted in FY24 are currently being replicated. The first study is to characterize the demographics of pests, natural enemies, and pollinators in deficit-irrigated cotton. Deficit irrigation was found to produce higher populations of L. hesperus, but did not change predator populations. The second study is to characterize the dispersal and recolonization behavior of cotton pests, L. hesperus and Bemisia tabaci, and their natural enemies in cotton fields following localized pesticide applications. The applications led to rapid recolonization of cotton by B. tabaci, while natural enemy populations were drastically reduced and did not recover adequately to limit the reproduction of B. tabaci. The third study is to characterize the pest, natural enemy, and pollinator assemblages associated with cotton embedded with drought-resistant potential trap crops. Plots of a common commercial cultivar of Bt cotton (NextGen 3195) were established with and without embedded rows planted with sesame. Data suggests that sesame is preferred by B. tabaci over cotton, but can also host substantial populations of natural enemies, especially big-eyed bugs, lacewings, and assassin bugs, in addition to phytophagous species such as leafhoppers and cotton fleahoppers. Sesame may not be a preferred host for L. hesperus, but its abundant flowers and extrafloral nectaries make it a promising nursery plant for several natural enemies and a valuable nectar resource for pollinators. Under Sub-objective 1C, the intended studies were not initiated due to multiple critical scientific vacancies during most of the project. Under Sub-objective 1D, the invasive cotton seed bug (CSB) was tested for susceptibility to twelve contact insecticides typically used in a cotton cropping systems, many of which are effective against other hemipterans. The standard petri dish assay technique was adapted for the test, and three experimental replicates have been completed. For each insecticide and dosage (0, 1, 10, 100, and 1000 ppm), five dishes, each containing five adult CSB, were tested for the effect on insect mortality. Several insecticides were identified as very effective even at low dosages. The most promising five candidate insecticides are being tested under lab conditions with treated open cotton bolls. This simulates more natural conditions prior to actual in situ testing. CSB was also tested for susceptibility to double-stranded RNA (dsRNA)-mediated RNA interference (RNAi). Oral RNAi (RNAi), which introduces dsRNAs via food and/or water uptake, was ineffective, preventing the use of oral RNAi for control applications. In contrast, target transcript levels were reduced following the injection of the corresponding dsRNAs, suggesting studies of gene function are possible. Under Sub-objective 2A, replicated field plot studies were conducted to assess the non-target impacts of a new transgenic cotton with efficacy against L. hesperus and thrips and to assess impacts on biological control services. Treatments included: transgenic Bacillus thuringiensis (Bt) cotton (MON88702), that produces an endogenous toxin, and its 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 insecticide as an alternative control 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 also allowed the evaluation of potential risks of this new Bt cotton 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 is complete, analyses have been completed and are currently being readied for publication. Results suggest that a large community of non-target arthropods are not affected by the Bt cotton and that biological control function, measured on sentinel whitefly prey, is not altered by the Bt crop. Further, the technology is moderately effective in controlling Lygus and thrips and may allow growers to forego one or more application of insecticides for pest management. For Sub-objective 2B, the intended studies were not initiated due to multiple critical scientific vacancies during the project. Under Sub-objective 3A, the efficacy of oral RNAi in L. hesperus was found to be limited by two factors, extra-oral enzymatic degradation of dsRNAs and limited uptake of dsRNAs by the midgut. Although the degradative effects of salivary gland nucleases can be bypassed via administering dsRNAs in sugar water, those dsRNAs fail to undergo uptake by midgut cells. Midgut nuclease activity, however, had limited effects on dsRNA stability and searches of Lygus transcriptomic data have consistently failed to identify a quality candidate for SID-1, a protein critical for dsRNA uptake. Delivery of dsRNAs in conjunction with various nanoparticles that enhanced dsRNA stability and uptake in other systems also failed to promote uptake or target transcript knockdown. Topical application of dsRNA-linked nanoparticles mixed with various surfactants was also ineffective at knockdown. Similarly, injection of dsRNAs into gravid females as a means of inducing RNAi in oocytes was ineffective. The only viable approach appears to be injection, limiting the utility of RNAi to studies gene function studies rather than control applications. In support of Sub-objective 3B, L. hesperus transcriptomic datasets were mined for potential homologs of sex determination genes structurally and functionally conserved in model insects (e.g., fruitfly, red flour beetle, silkmoth). Most of the genes sought were identified, including sex lethal, transformer 2, doublesex (dsx), fruitless, and intersex. In most species studied to date, the development of female- and male-specific traits is regulated by sex-specific splicing of dsx. Despite sequence similarity with other dsx genes, disruption of dsx function via RNAi and CRISPR-based gene editing revealed that L. hesperus dsx is atypical in that it does not undergo sex-specific splicing and only functions in male development. The development of female traits is thus driven by either an unknown gene or by another gene in the sex determination cascade that also has a highly atypical function. Under Sub-objective 3C, the viability of applying CRISPR/Cas9-mediated gene editing to study gene function in L. hesperus was validated. Genes regulating eye pigmentation (cardinal and cinnabar), cuticular coloration (aaNAT), sex differentiation (dsx), and spermatogenesis (beta-tubulin2) were functionally knocked-out by injecting Lygus eggs with target-specific guide RNAs and the Cas9 enzyme. Strong phenotypic effects were observed for all targets. Discernable changes to eye and cuticle color persisted throughout the life of the insects. dsx knockout resulted in female dominant sex ratios. beta-tubulin2 knockout yielded sterile males. Except for dsx, the edited genes were heritable over multiple generations, indicating that CRIPSR-based editing offers a viable approach for developing new control strategies. For Sub-objective 3D, the mechanisms and costs of resistance to Bt toxins in engineered crops were studied. Under Sub-objective 3Di, CRISPR/Cas9 gene editing showed that the ATP-binding cassette transporter 2 protein ABCA2 functions as a receptor of the Cry2Ab Bt toxin in both Helicoverpa zea and Pectinophora gossypiella. This supports ABCA2 as a leading candidate for resistance to Cry2Ab expressing crops. Under Sub-objective 3Dii, recombinant PgCad1 proteins corresponding to four known mutant alleles were produced in cultured insect cells to study how they confer resistance to Cry1Ac Bt toxin. None of the mutant PgCad1 proteins translocated to the cell surface, indicating cellular trafficking issues. These cells also lost susceptibility to Cry1Ac. Those producing unmodified PgCad1 had expression on the cell surface and remained susceptible. These data indicate that an underlying mechanism of Cry1Ac resistance involves the lack of a functional cadherin receptor on the cell surface, rather than direct interference of toxin-receptor binding. To determine if resistance to Bt toxins results in fitness costs, Under Sub-objective 3Diii, strains of H. zea moths resistant to Cry1Ac or Vip3Aa were flown on rotary flight mills and compared with moths from susceptible strains. While Cry1Ac resistance did not influence flight performance, Vip3Aa resistance reduced survival of males engaging in long-distance flight, having implications for how H. zea populations are managed.

Accomplishments
1. Selective control options for cotton pests. Selective tools, including selective insecticides and transgenic cotton, have played a crucial role in reducing insecticide usage and conserving arthropod predator populations within the Integrated Pest Management (IPM) plan for Arizona cotton. To provide growers with informed recommendations, an ARS scientist in Maricopa, Arizona, collaborated with University of Arizona scientists to test the impact of two insecticides for control of whitefly and lygus bugs in cotton. Under field conditions and using appropriate plot sizes, multi-year experiments were conducted with spiromesifen and indoxacarb to assess their impacts on non-target arthropods. Assessments were made on over 27 arthropod taxa through community analyses, individual predator abundance, and biological control function via novel predator to prey ratios. Both insecticides demonstrated patterns of selectivity relative to the untreated control and similar to those of other selective insecticides used as positive controls. Therefore, indoxacarb and spiromesifen are fully selective insecticides, safe for the non-target arthropod community and compatible with the IPM plan in Arizona cotton, which relies heavily on biological control. These results provide growers and pest management advisors with additional tools to control cotton pests and will continue to enable conservation biological control.

2. Mismatch between lab-generated and field-evolved resistance to Bt crops in Helicoverpa zea. Crops genetically engineered to produce insect-killing crystalline (Cry) proteins from the bacterium Bacillus thuringiensis (Bt) are used to control some major pests, including Helicoverpa zea (corn ear worm) one of the most important crop pests in the United States. This moth has evolved practical resistance to several Cry proteins, including Cry1Ac. Although mutations in single genes that can confer resistance to Cry proteins have been identified in lab-generated strains of H. zea, the genetic basis of field-evolved resistance to Cry proteins in H. zea has remained elusive. An ARS scientist from Maricopa, Arizona, and collaborators, used a population genomic approach to analyze the genetic basis of field-evolved resistance to Cry1Ac in H. zea in samples from 17 sites across seven states of the southern United States. Extensive gene flow occurred among all populations studied and field-evolved resistance was not associated with mutations in 20 single candidate genes previously implicated in resistance or susceptibility to Cry proteins. Instead, resistance in field samples was associated with increased copy number of a cluster of nine trypsin genes. However, trypsin gene amplification was also observed in a susceptible sample and was not found in all resistant samples, implying that this amplification does not always confer resistance and mutations in other genes also contribute to field-evolved resistance to Cry1Ac in H. zea. The mismatch between lab-generated and field-evolved resistance in H. zea is unlike other cases of Bt resistance and creates challenges for managing this pest.

3. CRISPR-based gene editing introduces heritable mutations that can be harnessed for Lygus pest management. The emergence of CRISPR as a gene editing tool to introduce heritable mutations has unprecedented potential for destabilizing pest insect populations. This technology, however, remains to be developed and vetted for many insect pest populations. For the first time, ARS researchers at Maricopa, Arizona, used CRISPR technologies in Lygus hesperus, a major crop pest, to disrupt the function of genes involved in key molecular pathways controlling eye pigmentation, cuticle coloration, the development of male sex characteristics, and sperm formation. Further, the introduced mutations were stable across multiple generations. These results confirm that CRISPR technologies can be used with non-model insects, which opens new avenues for pest management.

4. Genetic-based sterilization provides a tool for suppressing Lygus populations. Genetic manipulation of genes associated with sperm formation has been proposed as a means of suppressing insect pest populations. ARS researchers at Maricopa, Arizona, used RNAi and CRISPR gene editing technologies to disrupt the function of a testis-specific gene (beta-tublin2) in Lygus hesperus, a major crop pest. The resulting adult males were sterile with sperm that was severely depleted. These results demonstrate that Lygus reproductive capacity can be manipulated at the gene level and highlight the potential for genetic-based control measures in suppressing troublesome insect pest populations.

5. Identification of effective insecticides for controlling cotton seed bug. Researchers at Maricopa, Arizona, screened 13 insecticides cleared for use in cotton fields for their efficacy against the invasive cotton seed bug. Four compounds were identified with rapid knockdown and high efficacy (>75% mortality). These findings present growers with much needed and immediately available control tools should the invasive insect migrate into cotton growing regions of the southwestern United States.

Review Publications
Li, N., Xu, X., Li, J., Hull, J.J., Chen, L., Liang, G. 2024. A spray-induced gene silencing strategy for Spodoptera frugiperda oviposition inhibition using nanomaterial-encapsulated dsEcR. International Journal of Biological Macromolecules. 281. Article 36503. https://doi.org/10.1016/j.ijbiomac.2024.136503.
Feng, H., Zhao, Y., Yang, T., Zhou, Y., Gong, L., Zhang, M., Ma, Y., Hull, J.J., Dewer, Y., Zhang, F., Smagghe, G., He, M., He, P. 2024. Female contact sex pheromone recognition in the German cockroach (Blattella germanica) is mediated by two male antennae-enriched sensory neuron membrane proteins. Pest Management Science. 81(2):572-584. https://doi.org/10.1002/ps.8530.
Zheng, W., Jiao, P., Xu, X., Ma, W., Hull, J.J., Hua, H., Chen, L. 2025. Identification of a TOR signaling pathway gene as a candidate target for reproductive management of Adelphocoris suturalis. Journal of Integrative Agriculture. 24(4):1529-1541. https://doi.org/10.1016/j.jia.2024.08.005.
Bordini, I., Naranjo, S.E., Fournier, A., Ellsworth, P.C. 2024. Determining selectivity of isocycloseram and afidopyropen and their compatibility with conservation biological control in Arizona cotton. Pest Management Science. 81(2):639-653. https://doi.org/10.1002/ps.8460.
Hagler, J.R., Casey, M.T., Machtley, S.A., Schutze, I.X., Fabrick, J.A. 2024. Marking Helicoverpa zea (Lepidoptera: Noctuidae) with fluorophores for use in mark-release-recapture research. Annals of the Entomological Society of America. 117(6):332-339. https://doi.org/10.1093/aesa/saae027.
Legan, A.W., Allan, C.W., Jensen, Z.N., Degain, B.A., Yang, F., Kerns, D.L., Benowitz, K.M., Fabrick, J.A., Li, X., Carriere, Y., Matzkin, L.M., Tabashnik, B.E. 2024. Mismatch between lab-generated and field-evolved resistance to transgenic Bt crops in Helicoverpa zea. Proceedings of the National Academy of Sciences (PNAS). 121(47). Article e2416091121. https://doi.org/10.1073/pnas.2416091121.
Zilnik, G.L., Hepler, J.R., Merten, P., Schutze, I.X., Hoddle, C.D., Hoddle, M.S., Ellsworth, P.C., Brent, C.S. 2025. Screening of insecticides for management of the invasive Oxycarenus hyalinipennis Costa (Hemiptera: Oxycarenidae) population sourced from urban southern California. Journal of Economic Entomology. 118(2):692-699. https://doi.org/10.1093/jee/toaf014.
Hull, J.J., Le, K.P., Schutze, I.X., Heu, C.C., Gross, R.J., Fabrick, P.G., Rodriguez, J.A., Hull, A.M., Langhorst, D.R., Fabrick, J.A., Brent, C.S. 2025. RNAi-mediated knockdown of tektins does not affect male fertility in Lygus hesperus. Archives of Insect Biochemistry and Physiology. 118(4). Article e70053. https://doi.org/10.1002/arch.70053.
Cai, Y., Hou, B., Fabrick, J.A., Yang, Y., Wu, Y. 2024. The role of aquaporins in osmotic cell lysis induced by Bacillus thuringiensis Cry1Ac toxin in Helicoverpa armigera. Pesticide Biochemistry and Physiology. 204. Article 106068. https://doi.org/10.1016/j.pestbp.2024.106068.
Naranjo, S., Ellsworth, P.C. 2024. Landscape considerations in pest management: Case study of the Arizona cotton IPM system. In: Brewer, M.J., Hein, G.L., editors. Arthropod Management and Landscape Considerations in Large-scale Agroecosystems. Cambridge, MA: CABI. p. 44-47. https://doi.org/10.1079/9781800622777.0003.
Lv, J., Yang, Y., Fabrick, J.A., Wu, Y. 2025. Gene editing to enhance pesticide resistance in a beneficial predatory mite. Pesticide Biochemistry and Physiology. 212. Article 106466. https://doi.org/10.1016/j.pestbp.2025.106466.
Zhao, Y., He, M., Liu, X., Feng, H., Liu, X., Yang, T., Wang, J., Hull, J.J., Dewer, Y., Liu, L., Li, F., Smagghe, G., He, P. 2025. Multiple antennal sensory membrane neuron protein genes mediate rice plant host location in the white-backed planthopper. Journal of Agriculture and Food Chemistry. 73(22):13405-13414. https://doi.org/10.1021/acs.jafc.5c04680.