Progress 10/01/23 to 09/30/24
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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. Artificial Intelligence (AI)/Machine Learning (ML) Neither artificial intelligence (AI) nor Machine Learning (ML) methods were used for this project during FY2024. ACCOMPLISHMENTS 01 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. 02 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. 03 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. 04 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. 05 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.
Impacts
(N/A)
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. ÿ-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.
- 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.
- 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.
- 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.
|
Progress 10/01/22 to 09/30/23
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. This report documents fiscal year (FY) 2023 progress for project 2020- 22620-023-000D, ⿿Sustainable Pest Management for Arid-Land Agroecosystems, which began in July 2020. Under Sub-objective 1Bi, ARS researchers in Maricopa, Arizona, conducted Y-tube olfactometer bioassays to measure lygus bug responses to the odor of various plants to identify which species attract or repel major cotton pests. The goal is to find plants that can be placed adjacent to cotton (companion plants) that attract (lure) the pests away from cotton. Ultimately, companion planting practices could reduce pesticide use in cotton. An additional host plant test was initiated to examine lygus response to stinknet, an aggressive invasive weed. There is anecdotal evidence that this weed might serve as an overwintering host for lygus and its natural enemies. For Sub-objective 1Bii, ARS researchers developed a large-scale feeding choice arena to measure whitefly host plant preference and dispersal behavior in a greenhouse containing cotton (the target agronomic crop), vernonia (a preferred host plant), and basil (a non-preferred host plant). The three plant types were strategically placed within the greenhouse to simulate a real-world cropping system. The study showed that marked whiteflies released in the greenhouse's center preferentially dispersed to vernonia, suggesting that the plant has potential as a cotton companion crop. A similar study has been initiated testing the host plant preferences of lygus. If found to lure both species away from cotton, such a system could reduce pesticide usage. Under Sub-objective 1D, the invasive cotton seed bug (CSB) has been tested for its 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 parts per million), five petri dishes, each containing five adult CSB, were tested for the effect on insect mortality. Several insecticides were identified that are very effective even at low dosages. The most promising five candidate insecticides will be tested now under lab conditions with open cotton bolls that have been sprayed. This will simulate more natural conditions prior to actual in situ testing. In support of Objective 2, the examination of potential effects of new genetically engineered crops and insecticidal seed treatments on non- target insect species, has now been fully completed, with all collected data published. Under Sub-objective 3A, the efficacy of oral RNA interference (RNAi) in Lygus hesperus was found to be limited by two main 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 has consistently failed to identify a quality candidate for SID-1, a channel protein critical in cellular uptake of dsRNAs. Delivery of dsRNAs in conjunction with various nanoparticles that enhanced dsRNA stability and uptake in other systems also failed to promote consistent uptake or target transcript knockdown. Topical application of dsRNA-linked nanoparticles mixed with various surfactants likewise was ineffective at producing an RNAi effect. 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 of gene function rather than control applications. For Sub-objective 3B, Lygus hesperus transcriptomic datasets were searched for potential homologs of sex determination genes structurally and functionally conserved in model insects (e.g., fruitfly, red flour beetle, silkmoth). Searches have identified 20 of the 24 genes that comprised the initial search set and include early sex determination genes (sex lethal, transformer 2, doublesex, and fruitless) as well as important downstream genes such as intersex. These five key genes have been cloned, sequence validated, sex-specific expression assessed. Studies to determine the developmental effects of gene knockdown have been initiated. Under Sub-objective 3C, ARS researchers validated the viability of applying clustered regularly interspaced short palindromic repeats CRISPR/ Cas9-mediated gene editing to study gene function in Lygus. Cardinal and cinnabar, two genes previously shown to play a role in Lygus eye pigmentation, were targeted by designing single guide RNA (sgRNA) for each. This was injected, along with one of two Cas9 enzymes, into newly laid Lygus eggs. Clear phenotypic effects were observed in developing embryos with experimental eggs exhibiting pink eyes, whereas control eggs were characterized by brown eyes. Atypical coloration in the experimental groups largely persisted throughout the nymphal stages and into early adult development, which is clear evidence of a persistent loss of gene function. Under Sub-objective 3Di, experiments using CRISPR/Cas9 gene editing to modify the ATP-binding cassette gene ATP-binding cassette sub-family A member 2 (ABCA2) in a susceptible strains of pink bollworm and corn earworm have been completed and the work has been published. The work demonstrated the power of using CRISPR/Cas9 gene editing for validating the in vivo function of genes involved in resistance to Bacillus thuringiensis (Bt) transgenic crops and showed that ABCA2 is a functional receptor of Cry2Ab in two important agricultural insect pests. For Sub-objective 3Dii, cellular localization of the wild-type pink bollworm cadherin (PgCad1_s) transmembrane protein and four mutant cadherins (PgCad1_r1, r2, r3, and r4) was determined and immunofluorescence methods were optimized and used to screen cell lines for cross-reactivity to PgCad1 antibodies. Plasmids corresponding to each of the five PgCad1-Venus fusion proteins and mCherry-labeled cellular organelle marker proteins were constructed and sequence-verified. Whereas wild-type PgCad1_s-Venus localized primarily within the plasma membrane of stably transformed Tni cells, the four mutant cadherins were retained intracellularly within the endoplasmic reticulum. These results indicate that the cadherin mutations alter the cellular trafficking of the known Cry1Ac midgut receptor, thereby contributing to functional resistance. To confirm this within actual pink bollworm midgut tissue, fixation and embedding methods that are compatible with immunostaining with custom anti-PgCad1 antibodies is required. Hence, cell lines expressing each of the five untagged cadherin proteins were provided to a University of Arizona collaborator, who tested numerous methods of cell fixation and embedding. Five different custom-made, anti-PgCad1 antibodies were tested against cultured cells expressing wild-type PgCad1_s under various fixation/embedding conditions. At least one PgCad1 antibody and a fixation/embedding method has been identified for future immunohistochemistry experiments with Cry1Ac-resistant pink bollworm strains harboring mutant PgCad1 alleles. In support of Sub-objective 3Diii, the studies to determine if toxin resistance alters the flight capability or propensity of the corn earworm were delayed primarily to allow for the rebuild of a new, improved flight mill apparatus. Specifically, research published by ARS scientists highlight the efficiency and ease of use for this newly built flight monitoring system. Diet bioassay experiments and flight mill tests to compare GA with GA-R and GZ with GZ-R corn earworm strains will be initiated in FY24. Artificial Intelligence (AI)/Machine Learning (ML) Neither artificial intelligence (AI) nor Machine Learning (ML) methods were used for this project during FY2023. ACCOMPLISHMENTS 01 Increase in global resistance to genetically engineered Bacillus thuringiensis (Bt) crops. Transgenic plants producing insecticidal proteins from Bacillus thuringiensis (Bt) are grown widely to control pests, but evolution of insect resistance has reduced the efficacy of Bt crops. An ARS scientist at Maricopa, Arizona, and collaborators at the University of Arizona, analyzed global resistance monitoring data for the first 25 years of cultivation of Bt crops including corn, cotton, soybean, and sugarcane. A total of 73 cases were examined including 24 pest species from 12 countries with cases of practical resistance arising from three in 2005 to 26 in 2020. Practical resistance was documented in some populations of 11 pest species, collectively affecting nine widely used crystalline (Cry) Bt toxins in seven countries. Conversely, 30 cases reflect no decrease in susceptibility to Bt crops in populations of 15 pest species in nine countries. The remaining 17 cases provide early warnings of resistance, which entail genetically-based decreases in susceptibility without evidence of reduced efficacy. Numerous factors that can lead to sustained susceptibility to Bt crops were reviewed, providing improvements for the sustainability of current and future transgenic insecticidal crops. 02 CRISPR/Cas9 gene editing feasible in Lygus. CRISPR/Cas9-mediated gene editing is the foremost tool available for functional genomics approaches and targeted genomic engineering applications; however, the technique is species-specific and often requires significant optimization, especially in non-model organisms such as Lygus hesperus. The applicability of the CRISPR/Cas9 system for this species was successfully demonstrated by a team of ARS researchers in Maricopa, Arizona. Eggs injected with the Cas9 enzyme and sgRNAs targeting either cardinal or cinnabar, two eye pigmentation genes, developed red eyes instead of brown eyes typical of wildtypes. Although atypical coloration in both experimental groups largely persisted throughout the nymphal stages, adult manifestation of the phenotype was limited to the cinnabar group, and atypical eye coloration was associated with disruptions in the sequence of the respective target genes. The heritability and persistence of the red eye phenotype across multiple generations demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can be applied to L. hesperus and that eye pigmentation genes are useful for tracking genetic manipulation. 03 Gene editing identifies a key receptor gene of pest resistance to transgenic Bacillus thuringiensis (Bt) crops. The corn earworm, Helicoverpa zea, is a major New World pest of many crops that has evolved resistance to transgenic corn and cotton producing different Bt protein toxins. Understanding of the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. An ARS researcher in Maricopa, Arizona, and collaborators at the University of Arizona, showed that mutations introduced by clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing in the corn earworm gene encoding an ATP-binding cassette protein (HzABCA2) causes resistance to the Cry2Ab Bt toxin. Disruptive mutations in HzABCA2 facilitated the creation of two Cry2Ab-resistant strains in which all resistant individuals tested had disruptive mutations in HzABCA2. The results demonstrate that HzABCA2 mutations can cause high levels of resistance to Cry2Ab, and that this gene is a leading candidate for monitoring corn earworm for Cry2Ab resistance in the field. 04 Complex molecular basis of lab- and field-selected pink bollworm resistance to transgenic Bacillus thuringiensis (Bt) cotton. Cotton has been genetically engineered to produce insect-killing proteins from the bacterium Bacillus thuringiensis (Bt) to control major pests that include the pink bollworm, one of the most damaging pests of cotton world-wide. Genetically engineered Bt crops have many advantages, including enhanced pest suppression, improved yields, increased farmer profits, and decreased use of conventional insecticides thereby benefiting the environment and human health; however, these benefits are reduced when pests evolve resistance to Bt crops. The pink bollworm rapidly evolved resistance to Bt cotton in India, but not in China or the United States where this invasive pest was eradicated using Bt cotton, sterile moth releases, and other tactics. An ARS researcher from Maricopa, Arizona, and collaborators from the University of Arizona, compared the genetic profiles of Bt-resistant pink bollworm taken from lab-selected strains from the United States and China and field-selected populations from India. Testing showed the genetic basis of resistance was similar across countries and between resistance selected in the lab versus the field suggesting that lab selection can be useful for identifying genes likely to be important in field-evolved resistance to Bt crops. Likely, differences in management practices among countries caused the different outcomes. 05 Development of a method to track insect dispersal. Tracking dispersal patterns in agroecosystems is essential for efficiently managing arthropod pests and conserving natural enemies and pollinators. An ARS researcher at Maricopa, Arizona, developed and refined a method to tag key cotton pests (lygus and whitefly), natural enemies, and pollinators with a fluorophore that glows brightly under ultraviolet light. The key features of this novel marking procedure are that it is inexpensive, persistent, easily detected, does not affect insect behavior, and works equally well on large and small insects (e.g., whiteflies and parasitoids). The novel fluorophore marking method will expedite future dispersal research on whiteflies and various other pest and beneficial arthropod species. 06 Monitoring lygus bug and natural enemy movement in trap-cropped strawberry fields. The lygus bug is the major strawberry pest, but prefer to feed on alfalfa and many types of native weeds. As such, intercropping strips of alfalfa (trap cropping) within strawberry production fields and allowing weeds to flower adjacent to the fields can serve as a sink for both lygus and its natural enemies. An ARS scientist at Maricopa, Arizona, teamed up with University of California scientists to study the population dynamics and dispersal characteristics of lygus and its associated predator complex in organically grown strawberry fields embedded with strips of alfalfa and surrounded by weedy vegetation. The research showed that the lygus population was up to 2.5 times higher in alfalfa and that most marked lygus and predators that emigrated from senescing weeds were recovered from alfalfa rather than strawberry. Moreover, the marked predator-to- lygus ratio found in alfalfa compared to strawberries was 5:1. Trap cropping effectively reduced the infestation of lygus in strawberries demonstrating that converting weedy areas to native perennial plantings could mitigate the risk of pest migration while conserving beneficial insects.
Impacts
(N/A)
Publications
- Liu, X., Long, G., Guo, H., Ma, Y., Gong, L., Zhang, M., Hull, J.J., Dewer, Y., Liu, L., He, M., He, P. 2023. Functional characterization of five developmental signaling network genes in the white-backed planthopper: Potential application for pest management. Pest Management Science. 79(8) :2869-2881. https://doi.org/10.1002/ps.7464.
- Hull, J.J., Brent, C.S., Fu, T., Wang, G., Christie, A.E. 2022. Mining Lygus hesperus (western tarnished plant bug) transcriptomic data for transient receptor potential channels: Expression profiling and functional characterization of a painless homolog. Comparative Biochemistry and Physiology. 44. Article 101027. https://doi.org/10.1016/j.cbd.2022.101027.
- Guo, H., Liu, X., Long, G., Gong, L., Zhang, M., Ma, Y., Hull, J.J., Dewer, Y., He, M., He, P. 2022. Functional characterization of developmentally critical genes in the white-backed planthopper: Efficacy of nanoparticle- based dsRNA sprays for pest control. Pest Management Science. 79(3):1048- 1061. https://doi.org/10.1002/ps.7271.
- Fabrick, J.A., Heu, C.C., LeRoy, D.M., DeGain, B.A., Yelich, A.J., Unnithan, G.C., Wu, Y., Li, X., Carriere, Y., Tabashnik, B.E. 2022. Knockout of ABC transporter gene ABCA2 confers resistance to Bt toxin Cry2Ab in Helicoverpa zea. Scientific Reports. 12. Article 16706. https:// doi.org/10.1038/s41598-022-21061-2.
- Tabashnik, B.E., Fabrick, J.A., Carriere, Y. 2023. Global patterns of insect resistance to transgenic Bt crops: The first 25 years. Journal of Economic Entomology. 116(2):297-309. https://doi.org/10.1093/jee/toac183.
- Stahlke, A.R., Chang, J., Chudalayandi, S., Heu, C.C., Geib, S.M., Scheffler, B.E., Childers, A.K., Fabrick, J.A. 2023. Chromosome-scale genome assembly of the pink bollworm, Pectinophora gossypiella, a global pest of cotton. G3, Genes/Genomes/Genetics. 13(4). Article jkad040. https:/ /doi.org/10.1093/g3journal/jkad040.
- Fabrick, J.A., Li, X., Carriere, Y., Tabashnik, B. 2023. Molecular genetic basis of lab- and field-selected Bt resistance in pink bollworm. Insects. 14(2). Article 201. https://doi.org/10.3390/insects14020201.
- Guo, H., Long, G., Liu, X., Ma, Y., Zhang, M., Gong, L., Dewer, Y., Hull, J.J., Wang, M., Wang, Q., He, M., He, P. 2023. Functional characterization of tyrosine melanin genes in the white-backed planthopper and utilization of a spray-based nanoparticle-wrapped dsRNA technique for pest control. International Journal of Biological Macromolecules. 230. Article 123123. https://doi.org/10.1016/j.ijbiomac.2022.123123.
- Tabashnik, B.E., Carriere, Y., Wu, Y., Fabrick, J.A. 2023. Global perspectives on field-evolved resistance to transgenic Bt crops: A special collection. Journal of Economic Entomology. 116(2):269-274. https://doi. org/10.1093/jee/toad054.
- Wang, M., Wang, J., Yasen, A., Fan, B., Hull, J.J., Shen, X. 2023. Determination of key components in the Bombyx mori p53 apoptosis regulation network using Y2H-Seq. Insects. 14(4). Article 362. https://doi. org/10.3390/insects14040362.
- Weaver, M.A., Callicott, K.A., Mehl, H.L., Opoku, J., Park, L.C., Fields, K., Mandel, J.R. 2022. Characterization of the Aspergillus flavus population from highly aflatoxin-contaminated corn in the United States. Toxins. 14(11). Article 755. https://doi.org/10.3390/toxins14110755.
- Ma, Y., Gong, L., Zhang, M., Liu, X., Guo, H., Hull, J.J., Long, G., Wang, H., Dewer, Y., Zhang, F., He, M., He, P. 2023. Two antennae-enriched carboxylesterases mediate olfactory responses and degradation of ester volatiles in the German cockroach Blattella germanica. Journal of Agricultural and Food Chemistry. 71(12):4789-4801. https://doi.org/10.1021/ acs.jafc.2c08488.
- Teng, H., Zuo, Y., Yuan, J., Fabrick, J.A., Wu, Y., Yang, Y. 2022. High frequency of ryanodine receptor and cytochrome P450 CYP9A186 mutations in insecticide-resistant field populations of Spodoptera exigua from China. Pesticide Biochemistry and Physiology. 186. Article 105153. https://doi. org/10.1016/j.pestbp.2022.105153.
- Tabashnik, B.E., Unnithan, G.C., Yelich, A., Fabrick, J.A., Dennehy, T.J., Carriere, Y. 2022. Responses to Bt toxin Vip3Aa by pink bollworm larvae resistant or susceptible to Cry toxins. Pest Management Science. 78(10) :3973-3979. https://doi.org/10.1002/ps.7016.
- Bordini, I., Naranjo, S.E., Fournier, A., Ellsworth, P. 2023. Spatial scale of non-target effects of cotton insecticides. PLOS ONE. 18(5). Article e0272831. https://doi.org/10.1371/journal.pone.0272831.
- Naranjo, S.E., Canas, L., Ellsworth, P.C. 2022. Mortality dynamics of a polyphagous invasive herbivore reveal clues in its agroecosystem success. Pest Management Science. 78(10):3988-4055. https://doi.org/10.1002/ps.7018.
|
Progress 10/01/21 to 09/30/22
Outputs
PROGRESS REPORT Objectives (from AD-416): 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 (from AD-416): 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. 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 01 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. 02 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. 03 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). 04 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. 05 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. 06 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. 07 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. 08 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.
Impacts
(N/A)
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.
|
Progress 10/01/20 to 09/30/21
Outputs
Progress Report Objectives (from AD-416): 1: Improve biological control of key pests by quantifying interactions between prey & generalist predators, including predators occupying different trophic levels, using molecular marking & gut content assays in the field & defining impacts of transgenic crops on non-target species through meta-analyses. Sub-objective 1A though 1B: See uploaded project plan. Sub-objective 1C: Examine temporal and spatial dynamics of whitefly, Lygus, predator and pollinator movements between cotton and Vernonia, a new industrial crop.(New, May, 2018) Sub-objective 1D: Assess the risk of disruption of biological control of whiteflies by the introduction of a new Bt cotton with activity against Lygus bugs and thrips.(New, May, 2018) 2: Refine resistance management strategies based on improved knowledge of host (species & phenology) & environmental (temperature) influences on inducible mechanisms of stress response in whitefly & lygus & of Cry- toxin binding & mechanisms of Bt (Bacillus thuringiensis) toxin resistance in pink bollworm. Sub-objective 2A through 2C: See uploaded project plan. 3: Refine knowledge of factors regulating mate-finding & the dynamics of reproduction in lygus & whitefly by optimizing lygus sex pheromone doses & component ratios, defining insect phenology-dependent roles of short- range cues of lygus mating receptivity, & quantifying impacts of host, environmental, & population density-based factors on whitefly sex ratios. Sub-objective 3A through 3D: See uploaded project plan. 4: Define key life history parameters including the development & survival strategies of lygus & key species of beneficial insects in relation to the environment by quantifying consequences of extreme thermal environments & defining insect stage- dependent & environment- dependent diapause responses & associated transcriptional- based & endocrine-based patterns in lygus. Sub-objective 4A through 4C: See uploaded project plan. 5: Describe molecular genetic responses, facilitating survival & adaptation in pest insects by identifying lygus & whitefly transcripts responsive to xenobiotics & environmental (thermal, water, oxidative) stressors, identify molecular targets for disruption by chemical or genetic agonists or antagonists, & develop methods to deliver dsRNA for functional disruption of aquaporins or other targets essential to maintain homeostasis. Sub-objective 5A through 5B: See uploaded project plan. Sub-objective 5C: Examine the potential of an ornamental plant to disrupt the osmotic water permeability of B. tabaci aquaporin water channel proteins using cage studies and in vitro heterologous insect cell expression functional assays(New, May, 2018). Approach (from AD-416): 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. Under Sub-objective 1Bi, preliminary tests were conducted to measure lygus bug and whitefly olfactory response to various types of plants. The methods used for testing included a Y-tube olfactometer and behavior monitoring software. The tests revealed that the computer automated system could effectively track the insect�s response to the various plant odors. Currently, a wide variety of plant species are being grown in the greenhouse. These plants will be tested to determine which 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. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until fiscal year (FY) 22. Under Sub-objective 1Bii, a novel feeding choice test arena was conceptualized, constructed, and tested for efficacy at measuring lygus bug and whitefly host plant preference for feeding and egg laying activity. The novel testing arena, coined a �guillotine� arena, was very effective, and should enhance research productivity. Currently, a wide variety of plant species are being grown in the greenhouse. Using the new arena, these plants will be tested for their attractiveness to these major cotton pests. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22. Under Sub-objective 1C, a newly developed feeding choice test arena is being used to measure lygus bug and whitefly host plant feeding preference for four different isolines of cotton. These various cotton isolines are being concurrently screened for desert adaptivity traits (e. g., heat and drought tolerance) by an ARS cotton geneticist in Maricopa, Arizona. Each variety also has traits (e.g., various trichome densities [hairiness], waxy leaf surfaces, rough leaf surfaces, etc.) that may make them more or less attractive to lygus bug and whitefly, the two most common cotton pests. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22. Under Sub-objective 2A, replicated large field plot studies were conducted to assess the non-target impacts of a new transgenic cotton with efficacy against Lygus 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 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, and the data have been compiled and entered and are currently being analyzed. 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 necessary personnel are in place. Under Sub-objective 3A, double stranded RNAs (dsRNA), which are used to reduce gene function, were designed to target genes known to influence Lygus hesperus ovarian development. These dsRNAs were either directly injected into or fed to young adult females. Several days after treatment, ovarian development in the injected group was significantly impaired relative to controls, whereas no changes in development were observed in Lygus fed the dsRNAs. Similarly, target gene transcripts were significantly reduced in Lygus injected with dsRNAs but were comparable to controls in Lygus fed dsRNAs. Transcriptome data mining identified two enzymes that could potentially degrade oral dsRNAs, one of which is expressed in the salivary gland and is activated during feeding. However, even after reducing the expression of these enzymes, orally administered dsRNAs targeting ovarian development in the nuclease impaired Lygus remained ineffectual, suggesting that biocontrol through transgenic plants expressing dsRNAs that target Lygus may not be possible. Under Sub-objective 3C, experiments were able to demonstrate that Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing can be used as a tool to assess gene function in the plant bug Lygus hesperus. Methods were optimized for embryonic delivery of a gene editing complex and guides to target the eye pigmentation genes, cinnabar and cardinal. Injected individuals that lived to adulthood and exhibited altered eye pigmentation relative to control insects were separated and allowed to reproduce. Molecular analysis of cinnabar and cardinal genes in the descendants revealed mutations specific to the target region, indicating successful gene editing and validating this tool for future studies of gene function. Under Sub-objective 3Di, experiments using CRISPR/Cas9 gene editing were able to modify the adenosine triphosphate (ATP)-binding cassette gene ABCA2 in a susceptible strain of pink bollworm. 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 26 different disruptive mutations identified. These data not only confirm the genetic basis of resistance to Cry2Ab in pink bollworm, but also shows that CRISPR/Cas9 gene editing is useful for validating the function of genes involved in resistance to Bt transgenic crops. Under Sub-objective 3Dii, plasmid DNA was created that could be inserted into cells to study expression and localization of cadherins, transport proteins implicated in Bt toxin resistance. These plasmids contained cadherin gene sequences from either susceptible or resistant strains and a gene sequence for Venus, a protein that fluoresces and serves as a location marker. Several stable insect cells lines have been produced and are ready for immunofluorescence experiments. Plasmids are currently being constructed to produce the PgCad1 proteins without the Venus marker protein. Progress has been slowed by maximized telework and reduced occupancy limits so that the study is not expected to be completed until FY22. Under Sub-objective 3Diii, the studies to determine if toxin resistance alters the flight capability or propensity of the corn earworm have been delayed due to the maximized telework requirement and reduced occupancy limits. They are now scheduled for completion in FY22. Record of Any Impact of Maximized Teleworking Requirement: The maximized telework and reduced occupancy requirements had a substantial negative impact on project research during fiscal year (FY) 21, particularly with studies requiring laboratory resources. Completion of several projects and initiation of others has been delayed to the extent that several milestones set for FY21 will have to be shifted to FY22. ACCOMPLISHMENTS 01 Transgenic cotton and sterile insect releases synergize eradication of pink bollworm from the United States. The pink bollworm is one of the world�s most invasive insects and has been a major pest of cotton in the United States since 1917. However, decades of effort and implementation of the Binational Pink Bollworm Eradication Program culminated in the USDA Secretary of Agriculture officially declaring the pink bollworm eradicated from the cotton-growing regions of the continental United States in 2018. ARS researchers in Maricopa, Arizona, and collaborators from USDA, Animal and Plant Health Inspection Service, the Arizona Cotton Research and Protection Council, and the University of Arizona used models to demonstrate that eradication was made possible by the synergistic interaction of Bacillus thuringiensis cotton and sterile insect releases. They determined that eradication saved cotton growers in the United States $224 million from 2014 to 2020 and was associated with an 82% reduction in insecticide use for all cotton pests in Arizona during this same period. The economic and social benefits achieved have wide sweeping impacts on agriculture and society and demonstrate the benefits of using agricultural biotechnology in concert with classical pest control tactics. 02 Gene editing in pink bollworm provides direct validation of gene function and their involvement in resistance to transgenic cotton. Genetically engineered crops that produce insecticidal proteins from Bacillus thuringiensis (Bt) are important globally for managing insect pests. However, the evolution of pest resistance to Bt crops reduces their benefit. Understanding the genetic basis of such resistance is needed to better monitor, manage, and counter pest resistance to Bt crops. An ARS researcher in Maricopa, Arizona, and collaborators at the University of Arizona used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing to introduce mutations in the adenosine triphosphate (ATP)-binding cassette protein ABCA2 gene in a susceptible strain of pink bollworm, which resulted in resistance to Cry2Ab, a Bt protein produced in transgenic cotton. Overall, 26 different disruptive mutations were found, which, together with previous results, provide the first case of practical resistance to Cry2Ab where evidence identifies a specific gene in which disruptive mutations can cause resistance and are associated with resistance in field-selected populations. This study not only confirms the genetic basis of resistance to Cry2Ab in pink bollworm, but also shows that CRISPR/Cas9 gene editing is useful for the direct functional validation of genes involved in pest resistance to Bt transgenic crops. 03 Valuation of biological control in the Asia-Pacific region. Biological control of insect pests has immense economic value in many agricultural systems throughout the world. Still, this value is under appreciated by many involved in spurring innovation in and adoption of biological control research and technology. An ARS researcher in Maricopa, Arizona, in collaboration with researchers from the Chinese Academy of Sciences, Fuijan Agricultural/Forestry University, and Zhejiang University, in China, University of Queensland, Australia, and the Centre for Agriculture and Bioscience International, in the United Kingdom, estimated the economic impact of classical (introductory) biological control against 43 insect pests in food, feed, and fiber crops in the Asia-Pacific region of the world at $17.1-22.7 billion U.S. dollars, annually. In addition, biological control was shown to promote rural growth and prosperity even in marginal, poorly endowed, non-rice environments. This research provides lessons for future efforts to mitigate invasive species, restore ecological resilience, and sustainably increase the output of global food systems. 04 A desert-adapted insect pest trap crop for the cotton agroecosystem. Cotton is vulnerable to a wide array of insect pests. However, pest damage to cotton can be reduced by planting a more attractive host plant adjacent to cotton (a companion trap crop). An ARS researcher in Maricopa, Arizona, showed that vernonia, a desert-adapted plant, is strongly attractive to cotton pests and harbors an abundance of natural enemies. Further, very few arthropods were captured beyond the vernonia trap crop after being tracked using a protein immunomarking technique. The arthropods� strong attraction and fidelity to vernonia indicate that it could serve as a trap crop for the cotton pest complex and as a refuge for natural enemies. 05 Development of a new method to study predation on all the life stages of a major cotton pest. Biological control of insect pests has immense economic value in many agricultural systems, but knowledge is limited about which predator species are most important for a given pest. ARS researchers in Maricopa, Arizona, developed a novel method for studying predation on the various life stages of Lygus hesperus, a major pest of cotton and other crops. The method tagged Lygus eggs, immatures, and adults with unique proteins that were subsequently detectable in predator guts. They found that big-eyed bugs and spiders were not only the numerically dominant predator taxa in the cotton field but most frequently preyed on immature lygus. Results also showed that collops beetles and fire ants are adept at preying on the cryptic eggs, and that adult lygus, albeit at low frequencies, engaged in cannibalism on immatures. The methods described may have value in the study of predator-prey dynamics in many agroecosystems. 06 Use of artificial attractants to improve biological control in cotton. Biological control by naturally occurring predators and parasitoids is a key component of existing management programs in cotton. Plants can respond to herbivore damage by releasing volatile compounds that are attractive to a variety of natural enemies, but how these can be used to improve biological control is not well understood. ARS researchers in Maricopa, Arizona, showed that deployment of a commercially available compound (methyl salicylate) produced by damaged plants in large replicated plots failed to increase natural enemy populations, repel pest species, or improve predator to prey ratios indicative of enhance biological control in cotton. This finding was both consistent and inconsistent with similar studies in other cropping system and will be useful to pest management professionals making decisions on how to enhance management of natural enemies in cropping systems. 07 Diapause provides short and long-term benefits for a key insect pest. Lygus hesperus, a major cotton pest, survives winter conditions by entering diapause, a dormancy that limits reproductive development. Once environmental conditions are favorable, the bugs become reproductively active. An ARS researcher in Maricopa, Arizona, examined whether there are consequences for diverting resources towards diapause rather than normal development. Females that had gone through diapause were larger, had greater stores of fat, were able to mobilize the resources necessary for egg production faster than non-diapausers, and lived longer. Overall, the results indicate that short-term diapause does not have a negative impact. The resulting extra stored resources accrued may allow females to quickly take advantage of improved environmental conditions and may prolong life by shielding them against stressors. Disruption of diapause regulatory processes may render Lygus more susceptible to those stressors.
Impacts
(N/A)
Publications
- Tabashnik, B.E., Liesner, L.R., Ellsworth, P.C., Unnithan, G.C., Fabrick, J.A., Naranjo, S.E., Li, X., Dennehy, T.J., Antilla, L., Staten, R.T., Carriere, Y. 2021. Transgenic cotton and sterile insect releases synergize eradication of pink bollworm a century after it invaded the United States. Proceedings of the National Academy of Sciences(PNAS). 118(1). Article e2019115118. https://doi.org/10.1073/pnas.2019115118.
- Brent, C.S. 2021. Diapause termination and post-diapause in lygus hesperus (heteroptera: miridae). Journal of Insect Science. 21(1). Article 4. https://doi.org/10.1093/jisesa/ieaa142.
- Naranjo, S.E., Hagler, J.R., Byers, J. 2021. Methyl salicylate fails to enhance arthropod predator abundance or predator to pest ratios in cotton. Environmental Entomology. 50(2):293-305. https://doi.org/10.1093/ee/ nvaa175.
- Hagler, J.R., Casey, M.T., Hull, A.M., Machtley, S.A. 2020. A molecular approach for detecting stage-specific predation on Lygus hesperus. Journal of Insect Science. 20(6). Article 35. https://doi.org/10.1093/jisesa/ ieaa136.
- Hagler, J.R., Thompson, A.L., Machtley, S.A., Miles, C.T. 2021. Arthropod demography, distribution, and dispersion in a novel trap-cropped cotton agroecosystem. Journal of Insect Science. 21(1). Article 20. https://doi. org/10.1093/jisesa/ieab010.
- Bordini, I., Naranjo, S.E., Fournier, A., Ellsworth, P. 2020. Novel insecticides and generalist predators support conservation biological control in cotton. Biological Control. 154. Article 104502. https://doi. org/10.1016/j.biocontrol.2020.104502.
- Fabrick, J.A., Leroy, D.M., Mathew, L.G., Wu, Y., Unnithan, G.C., Yelich, A., Carriere, Y., Li, X., Tabashnik, B.E. 2021. CRISPR-mediated mutations in the ABC transporter gene ABCA2 confer pink bollworm resistance to Bt toxin Cry2Ab. Scientific Reports. 11. Article 10377. https://doi.org/10. 1038/s41598-021-89771-7.
- Guan, F., Hou, B., Dai, X., Liu, S., Liu, J., Gu, Y., Yang, Y., Fabrick, J. A., Wu, Y. 2021. Multiple origins of a single point mutation in the cotton bollworm tetraspanin gene confers dominant resistance to Bt cotton. Pest Management Science. 77(3):1169-1177. https://doi.org/10.1002/ps.6192.
- Wyckhuys, K., Lu, Y., Zhou, W., Cock, M., Furlong, M., Naranjo, S.E. 2020. Ecological pest control fortifies agricultural growth in Asia-Pacific economies. Nature Ecology and Evolution. 4:1552-1530. https://doi.org/10. 1038/s41559-020-01294-y.
- Jin, H., Abouzaid, M., Lin, Y., Hull, J.J., Ma, W. 2021. Cloning and RNAi- mediated three lethal genes that can be potentially used for Chilo suppressalis (Lepidoptera: Crambidae) management. Pesticide Biochemistry and Physiology. 174. Article 104828. https://doi.org/10.1016/j.pestbp.2021. 104828.
- Naranjo S.E. 2014. Effects of GM crops on non-target organisms. In: Ricroch A., Chopra S., Fleischer S., editors. Plant Biotechnology. Springer, Cham. p. 129-142. https://doi.org/10.1007/978-3-319-06892-3_11.
- Fleischer S.J., Hutchison W.D., Naranjo S.E. 2014. Sustainable management of insect-resistant crops. In: Ricroch A., Chopra S., Fleischer S., editors. Plant Biotechnology. Springer, Cham. p. 115-127. https://doi.org/ 10.1007/978-3-319-06892-3_10
- Hull, J.J., Gross, R.J., Brent, C.S., Christie, A.E. 2021. Filling in the gaps: A reevaluation of the Lygus hesperus peptidome using an expanded de novo assembled transcriptome and molecular cloning. General and Comparative Endocrinology. 303. Article 113708. https://doi.org/10.1016/j. ygcen.2020.113708.
|
|