Research Project: Sustainable Pest Management for Arid-Land Agroecosystems

Location: Pest Management and Biocontrol Research

2024 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 FY 2024 progress for project 2020-22620-023-000D, “Sustainable Pest Management for Arid-Land Agroecosystems”, which began in July 2020. In support of Sub-objective 1Biv, ARS researchers in Maricopa, Arizona, initiated three separate open field studies. The first study is designed to characterize the demographics and dispersal patterns of the whitefly (Bemisia tabaci) and western tarnished plant bug (Lygus hesperus), natural enemies, and pollinators in deficit-irrigated cotton. The second study is designed to characterize these arthropods' demographics and dispersal patterns in cotton embedded with drought-resistant trap crops. The third study is designed to characterize the dispersal and recolonization of pest and natural enemy communities to spatially localized pesticide events. All data will be captured by the end of FY24 and replicate studies will be conducted in FY25. For Sub-objective 1D, ARS researchers collected the invasive cotton seed bug (Oxycarenus hyalinipennis) from ornamental plants in southern California and evaluated their survivorship against twelve registered and one experimental insecticide formulations using contact and ingestion bioassays. Of these, six formulations induced high mortality in O. hyalinipennis nymphs and adults and were used for subsequent dose-response bioassays. Acephate, dinotefuran, flupyradifurone, and imidacloprid were found to be especially effective, with lethal concentration (LC) 50 values well below maximum labeled rates, although LC99.9 values often exceeded maximum rates. The selective options can both reduce potential damage from O. hyalinipennis and preserve natural enemies in southwestern cotton production. Under Sub-objective 3A, ARS researchers in Maricopa, Arizona, showed that RNA interference-mediated knockdown of an alkaline nuclease predominantly expressed in the L. hesperus salivary gland significantly reduced their capacity for extra-oral digestion of exogenous dsRNAs. Despite knockdown of the nuclease, oral RNAi remained ineffective as ingestion of previously validated double-stranded RNAs (dsRNAs) failed to impact target transcript levels or yield phenotypes. Injection-based RNAi remains the only viable option for this species. In support of Sub-objective 3B, ARS researchers in Maricopa, Arizona, cloned from L. hesperus genes that are putatively involved in the sex determination pathway. The biological function of each was assessed via RNAi. Following knockdown of sex lethal, transformer2, and fruitless, no obvious effects were noted on the development of adult sex characteristics despite clear reductions in the target transcripts. Knockdown of doublesex had male-specific effects, limiting testes development and sperm production. RNAi of intersex negatively impacted the development of primary and secondary sexual characteristics in both sexes. For Sub-objective 3C, ARS researchers in Maricopa, Arizona, successfully implemented CRISPR/Cas9-mediated gene editing in L. hesperus, targeting multiple genes. Disruption of aralkylamine N-acetyltransferase generated adults with altered cuticular coloration (black vs. the typical green). Follow-up experiments indicated there were no obvious fitness costs associated with the mutation, making it a good external marker of gene transformation. Knockout of doublesex yielded sex ratios that are heavily female-biased. Knockout of beta-tubulin2 generated sterile males that lack sperm. Assessments of these knockouts on fitness and ability to compete with wild-type adults for mates are ongoing. Supporting Sub-objective 3Di, ARS researchers in Maricopa, Arizona, used CRISPR/Cas9 gene editing to modify the adenosine triphosphate (ATP)-binding cassette gene ABCA2 in susceptible strains of pink bollworm and corn earworm. ABCA2 was found to be a functional receptor of Cry2Ab in these two important agricultural insect pests. The results also validated the utility of CRISPR/Cas9 gene editing in testing the putative in vivo function of genes involved in resistance to Bt toxins. Additionally, the pairing of genomic DNA sequencing of corn earworm with CRISPR/Cas9 gene editing led to the identification of novel genes responsible for resistance to Bt proteins. For example, a genome wide association study in a Cry1Ac Bt resistant strain of corn earworm, found the kinesin12 gene was associated with Bt resistance. Knockout of kinesin12 in a susceptible corn earworm strain imparted resistance to Cry1Ac. In support of Sub-objective 3Dii, ARS researchers in Maricopa, Arizona, used cultured Tni cells producing the wild-type pink bollworm cadherin (PgCad1_s) transmembrane protein and four mutant cadherins (PgCad1_r1, r2, r3, and r4) to localize cellular expression of these proteins. Transient expression of each of the five PgCad1-Venus fusion proteins and mCherry-labeled cellular organelle marker proteins showed that only the wild-type PgCad1_s-Venus protein was localized primarily within the plasma membrane of Tni cells, whereas the four mutant cadherins were retained intracellularly within the endoplasmic reticulum. Furthermore, cytotoxicity (i.e., cell swelling) was observed using Tni cells expressing the five PgCad1-Venus fusion proteins in the presence of the Cry1Ac Bt toxin. Cells producing the wild-type PgCad1_s-Venus protein showed significant cell swelling indicative of Bt intoxication, whereas no swelling was observed in cells producing the mutant PgCad1s. These results indicate that the cadherin mutations disrupt cellular trafficking of the known Cry1Ac midgut receptor, thereby contributing to functional resistance. To confirm these results within actual pink bollworm midgut tissue, fixation and embedding methods that are compatible with immunostaining with custom anti-PgCad1 antibodies is required. Hence, stable cell lines expressing each of the five untagged cadherin proteins were tested by a University of Arizona collaborator, who tested numerous methods of cell fixation and embedding. Of the five different custom-made, anti-PgCad1 antibodies that were tested against cultured cells expressing wild-type PgCad1_s under various fixation/embedding conditions, one PgCad1 antibody and a fixation/embedding method was identified for immunohistochemistry experiments with pink bollworm tissues. Pink bollworm larvae were fixed and embedded in paraffin for future hematoxylin and eosin (H&E) and immunochemistry staining. For Sub-objective 3Diii, ARS researchers performed flight studies on a Cry1Ac-resistant strain and a Vip3Aa-resistant strain using a newly designed rotary flight mill apparatus to determine if resistance has an associated fitness cost. Same aged larvae from susceptible and resistant strains, as well as a lab-reared control strain were reared on untreated diet. Newly emerged adults (either males or females) were tethered to flight mills and 8-12 individuals from each stain were simultaneously flown for 18 hours (14 h dark:4 h light) at 27 °C. Flight data indicated a significant fitness cost associated with resistance to Vip3Aa in males but not females. No fitness costs associated with flight were observed for the Cry1Ac-resistant strain. In support of Sub-objective 3E, ARS researchers in Maricopa, Arizona, tested the efficacy of RNAi techniques on the invasive cotton seed bug (Oxycarenus hyalinipennis). Directly injecting double stranded RNA (dsRNA) into the abdominal hemocoel of O. hyalinipennis caused reduced expression of a gene associated with sex development, intersex, but had no obvious effects on mortality. Attempts to induce the insects to ingest dsRNA in sucrose solution have so far been unsuccessful, but modified approaches are being attempted.

Accomplishments
1. Complex molecular mechanisms of lab- and field resistance to toxic Bacillus thuringiensis (Bt) proteins and transgenic Bt crops. Insecticidal proteins from Bacillus thuringiensis (Bt) are used globally as bioinsecticides and in transgenic Bt crops to control key insect pests. However, the evolution of pest resistance to Bt has reduced their benefits. An ARS scientist at Maricopa, Arizona, and a collaborator from China highlighted the 9 global cases of field-evolved resistance to Bt crops where the underlying molecular mechanisms causing resistance are known and provided a comprehensive update on recent advances into the understanding of molecular mechanisms of laboratory-selected resistance to Bt proteins. Furthermore, new technological advances were described which have enabled greater understanding of the molecular complexities underlying the evolution of insect pest resistance to Bt crops, including those that permit the discovery of novel genetic mechanisms of resistance and for molecular monitoring of field evolved Bt resistance. Because knowledge of the causal basis of resistance is important for monitoring, managing, and countering pest resistance to Bt crops, these insights are vital for the sustainable management of many global insect pests.

2. Global populations of pink bollworm have distinct genetic structure with limited gene flow. The pink bollworm, Pectinophora gossypiella, is one of the world's most destructive pests of cotton, occurring in nearly all cotton-growing countries. Resistance to transgenic Bt cotton in Asia also represents a potential threat to cotton growing areas in Australia and elsewhere. An ARS researcher from Maricopa, Arizona, and collaborators used genomics to analyze population dynamics and connectivity patterns of pink bollworm individuals collected from fields in northwest Australia, India, Pakistan, and from four laboratory strains that originated in the United States. Five genetically distinct groups were discovered, with three in the United States, and one each in Australia and India-Pakistan. While low genetic diversity was observed within populations, there was high differentiation between populations. The high genetic differentiation between Australia and the other continents reduces concerns about gene flow to Australia, particularly from populations in India and Pakistan that have evolved resistance to transgenic insecticidal cotton.

3. CRISPR/Cas9 gene editing modifies Lygus reproductive capacity. An advantage of CRISPR gene editing over other genetic modification methods, such as RNA interference, is heritability of the editing event, allowing gene functionality to be assessed in subsequent generations. As such, disruption of genes involved in sex differentiation and/or reproductive development can provide insights into the development of genetic-based approaches to suppress pest populations. ARS researchers in Maricopa, Arizona, used CRISPR/Cas9 gene editing to disrupt the function of two genes that regulate the reproductive capacity of the western tarnished plant bug (Lygus hesperus). Disruption of doublesex, a well-conserved gene in insect sex determination pathways, largely produced only female progeny. Disruption of beta-tubulin2, a gene frequently associated with spermatogenesis, yielded sterile males that lacked sperm. These results demonstrate that lygus reproductive capacity can be manipulated at the gene level and open the possibility of developing novel genetic-based control measures that focus on population suppression.

4. Development and refinement of a method to track insect dispersal. Tracking insect movement in agroecosystems is essential to efficiently manage arthropod pests and conserve natural enemies and pollinators. A new method for marking insects was recently described for tracking insects in their habitat. The taggant consisted of green-colored glow-in-the-dark liquid fluorophore. Research has shown that fluorophore is well retained in many insect taxa. ARS researchers in Maricopa, Arizona, examined the behavioral effects of the fluorophore mark on the lygus bug, a major economic pest, on various crops (e.g., cotton, alfalfa, strawberry, etc.). The results demonstrate that the fluorophore product has enormous potential for mark-release-recapture type research.

5. New 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 new insecticides (isocycloseram and afidopyropen) on arthropod natural enemy abundance and biological control function. Replicated field studies over two years showed that afidopyropen showed no significant differences from the untreated check across all metrics examined and was classified as a fully selective insecticide for the Arizona cotton system. In contrast, isocycloseram exhibited some negative impacts on certain metrics but was less detrimental to non-target arthropods compared to a known broad-spectrum insecticide and hence was classified as a partially selective. These results provide growers and pest management advisors with new tools for the control of cotton pests that will continue to enable conservation biological control.

Review Publications
Ma, Y., Zhang, M., Gong, L., Liu, X., Long, G., Guo, H., Hull, J.J., Dewer, Y., He, M., He, P. 2023. Efficient nanoparticle-based CRISPR-Cas13d induced mRNA disruption of an eye pigmentation gene in the white-backed planthopper, Sogatella furcifera. Insect Science. 30(6):1552-1564. https://doi.org/10.1111/1744-7917.13203.
Hagler, J.R. 2023. Effects of fluorescent dust and protein markers on the foraging behaviour of a whitefly parasitoid, Eretmocerus emiratus (Hymenoptera: Aphelinidae). Journal of Applied Entomology. 147(9):878-882. https://doi.org/10.1111/jen.13164.
Hagler, J.R., Casey, M.T., Machtley, S.A., Merten, P. 2024. No effect of a fluorophore taggant on western tarnished plant bug, Lygus hesperus roaming and flight behavior. Entomologia Generalis. 44(1):115-119. https://doi.org/10.1127/entomologia/2023/2349.
Gan, C., Zhang, Z., Jin, Z., Wang, F., Fabrick, J.A., Wu,, Y. 2023. Helicoverpa armigera ATP-binding cassette transporter ABCA2 is a functional receptor of Bacillus thuringiensis Cry2Ab toxin. Pesticide Biochemistry and Physiology. 197. Article 105658. https://doi.org/10.1016/j.pestbp.2023.105658.
Ma, Y., Liu, T., Zhao, Y., Luo, J., Feng, H., Zhou, Y., Gong, L., Zhang, M., He, Y., Hull, J.J., Dewer, Y., He, M., He, P. 2024. RNA interference-screening of potentially lethal gene targets in white-backed planthopper Sogatella furcifera via a spray-induced and nanocarrier-delivered gene silencing system. Journal of Agricultural and Food Chemistry. 72(2):1007-1016. https://doi.org/10.1021/acs.jafc.3c05659.
Heu, C.C., Le, K.P., Gross, R.J., Schutze, I.X., Leroy, D.M., Langhorst, D.R., Brent, C.S., Fabrick, J.A., Hull, J.J. 2023. ß-tubulin functions in spermatogenesis in Lygus hesperus Knight. Journal of Insect Physiology. 152. Article 104598. https://doi.org/10.1016/j.jinsphys.2023.104598.
Wu, Y., Weng, Z., Yan, H., Yao, Z., Li, Z., Sun, Y., Ma, K., Hull, J.J., Zhang, D., Ma, W., Hua, H., Lin, Y. 2023. The microRNA-7322-5p/p38/Hsp19 axis modulates Chilo suppressalis cell-defences against Cry1Ca: An effective target for a stacked transgenic rice approach. Nature Microbiology . 21(9):1827-1838. https://doi.org/10.1111/pbi.14095.
Matheson, P., Parvizi, E., Fabrick, J.A., Siddiqui, H.A., Tabashnik, B.E., Walsh, T.K., McGaughran, A. 2023. Genome-wide analysis reveals distinct global populations of pink bollworm (Pectinophora gossypiella). Scientific Reports. 13. Article 11762. https://doi.org/10.1038/s41598-023-38504-z.
Zhou, Z., Yao, Z., Abouzaid, M., Hull, J.J., Ma, W., Hua, H., Lin, Y. 2023. Co-expression network analysis: A future approach for pest control target discovery. Journal of Agriculture and Food Chemistry. 71(19):7201-7209. https://doi.org/10.1021/acs.jafc.3c00113.
Zheng, W., Xu, X., Huang, X., Peng, J., Ma, W., Hull, J.J., Hua, H., Chen, L. 2024. Spray-induced and nanocarrier-delivered gene silencing system targeting juvenile hormone receptor components: Potential application as fertility inhibitors for Adelphocoris suturalis management. Pest Management Science. 80(8):3743-3751. https://doi.org/10.1002/ps.8077.
Nieto, D.J., Hagler, J.R., Swezey, S.L., Machtley, S.A., Bryer, J.A. 2023. Immigration of Lygus spp. (Hemiptera: Miridae) and predaceous natural enemies to trap-cropped organic strawberry. Environmental Entomology. 52(5):824-831. https://doi.org/10.1093/ee/nvad085.
Fabrick, J.A., Wu, Y. 2023. Mechanisms and molecular genetics of insect resistance to insecticidal proteins from Bacillus thuringiensis. In: Jurat-Fuentes, J.L., editor. Advances in Insect Physiology. Volume 65. Cambridge, MA: Elsevier. p. 123-183. https://doi.org/10.1016/bs.aiip.2023.09.005.
Hagler, J.R., Casey, M.T., Hull, J.J., Machtley, S.A. 2022. A labor-saving marking and sampling technique for mark-release-recapture research. Entomologia Experimentalis et Applicata. 171(2):138-145. https://doi.org/10.1111/eea.13259.
Gong, L., Ma, Y., Zhang, M., Feng, H., Zhou, Y., Zhao, Y., Hull, J.J., Dewer, Y., He, M., He, P. 2024. The melanin pigment gene black mediates body pigmentation and courtship behavior in the German cockroach Blattella germanica. Bulletin of Entomological Research. 114(2):271-280. https://doi.org/10.1017/S0007485324000166.
Niu, X., Jiang, J., Sun, Y., Hull, J.J., Ma, W., Hua, H., Lin, Y. 2024. Knockdown of MAPK p38-linked genes increases the susceptibility of Chilo suppressalis larvae to various transgenic Bt rice lines. International Journal of Biological Macromolecules. 266. Article 130815. https://doi.org/10.1016/j.ijbiomac.2024.130815.
Hull, J.J., Heu, C.C., Gross, R.J., LeRoy, D.M., Schutze, I.X., Langhorst, D.R., Fabrick, J.A., Brent, C.S. 2024. Doublesex is essential for masculinization but not feminization in Lygus hesperus. Insect Biochemistry and Molecular Biology. 166. Article 104085. https://doi.org/10.1016/j.ibmb.2024.104085.
Paul, R.L., Hagler, J.R., Janasov, E.G., McDonald, N.S., Voyvot, S., Lee, J.C. 2024. An effective fluorescent marker for tracking the dispersal of small insects with field evidence of mark-release-recapture of Trissolcus japonicus. Insects. 15(7). Article 487. https://doi.org/10.3390/insects15070487.
Ma, Y., Zhao, Y., Zhou, Y., Feng, H., Gong, L., Zhang, M., Hull, J.J., Dewer, Y., Roy, A., Smagghe, G., He, M., He, P. 2024. Nanoparticle-delivered RNAi-based pesticide target screening for the rice pest white-backed planthopper and risk assessment for a natural predator. Science of the Total Environment. 926. Article 171286. https://doi.org/10.1016/j.scitotenv.2024.171286.
Casey, M.T., Machtley, S.A., Merten, P., Hagler, J.R. 2023. A simple computerized Arduino-based control system for insect rotary flight mills. Journal of Insect Science. 23(4). Article 5. https://doi.org/10.1093/jisesa/iead053.