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ARS Home » Midwest Area » Columbia, Missouri » Biological Control of Insects Research » Research » Research Project #429499

Research Project: Insect Biotechnology Products for Pest Control and Emerging Needs in Agriculture

Location: Biological Control of Insects Research

2018 Annual Report

Objective 1. Develop dsRNA (i.e., RNAi-based gene silencing) and, potentially, other genetic constructs to silence eicosanoid signaling and other immune-related genes in pest insect species, including the squash bug. Subobjective 1A: Clone, express and characterize a recombinant PLA2 from the squash bug, Anasa tristis and other pest insect species and test the influence of silencing PLA2 on standard immune parameters. Subobjective 1B: Identify, clone and silence POX genes in squash bugs, then determine the influence of gene silencing on selected cellular immune parameters. Subobjective 1C: Determine the influence of suppressing insect immune signaling on pest insect life history. Objective 2. Use conventional and molecular methods to develop and optimize western corn rootworm artificial diets. Subobjective 2A. Improve and standardize an artificial diet for rearing the western corn rootworm. Subobjective 2B. Determine molecular and cellular components contributing to WCR survival of Bt intoxication. Objective 3. Improve control of western corn rootworm with the entomopathogenic nematode Heterorhabdidtis bacteriophora by determining the influence of local soil and climate conditions on the survival of the nematode, and the attractiveness of the nematode to the corn root alarm signal (E)-ß-caryophyllene. Objective 4. Establish research-ready cell lines from midgut and other tissues of corn rootworm, fall armyworm, and other pest insect species in support of biotechnology (e.g. RNAi research) products for pest control.

1A. Squash bug PLA2 will be cloned, expressed and characterized with respect to temperature, pH, and substrate specificity. Gene expression in selected tissues, and the influence of microbial infections on gene expression, will be determined. Gene silencing of PLA2(s) will be conducted to determine its influence on one or more cellular immune reactions. 1B. Squash bug peroxinectin genes will be identified and tissue- and life-stage specificity determined. Quantitative methods, such as microaggregation and nodulation assays, will be developed to test for suppression of specific immune parameters resulting from silencing POX genes. 1C. Survivorship time will be measured to determine changes in susceptibility to infection in insects that are immunosuppressed by injection with pharmaceutical eicosanoid biosynthesis inhibitors, or dsRNA gene-silencing constructs. Treated and control insects will be artificially infected with known doses of selected microbes. 2A. Improvement of an artificial diet for rearing and bioassays of the western corn rootworm (WCR) will be developed by optimizing diet texture, presentation, feeding stimulants, pH, nutrients, anti-microbial compounds, and WCR development time. RNA-seq analysis will be used to direct optimization of performance traits. 2B. Expression- or sequence-variant based differences associated with survival of Bt intoxication by resistant larvae will be identified and confirmed by documenting gene expression differences between diet-reared Bt-resistant and susceptible larvae. 3. Entomopathogenic nematodes most infectious for WCR will be determined using mortality bioassays. Entomopathogenic nematode strains most responsive to maize root attractants will be selected using olfactometer choice assay methods. WCR infesting entomopathogenic nematodes will be selected for enhanced overwintering capability and improved desiccation survival. 4. Various standard tissue dissociation techniques will be used, alone or in combination, to generate new insect cell lines. A variety of media and/or media supplements will be assessed for cell attachment and proliferation. Specialized cell culture flasks or plates coated with attachment factors and/or containing gels for 3D support will be tested for their ability to facilitate cell attachment and proliferation. Plate inserts to co-culture tissue explants with previously established cell lines or selected tissues will be evaluated.

Progress Report
Subobjective 1A. Reported on a novel calcium-independent cellular enzyme that acts in insect immunity in 2015. Advanced that finding with new work showing the mechanism of activatingthe enzyme during the onset of microbial infection. Specifically, infection leads to formation of a small signal molecule which activates the enzyme. The activation leads to formation of biochemical signal molecules that mediate insect immune functions. This work identified new genes necessary for insect immune reactions, and revealed new targets for suppressing insect immune responses to bacteria and other microbial biological control agents. Findings were published in a peer-reviewed journal. Subobjective 1B. Many aspects of mammalian immunity are mediated by signal molecules. Discovery that these signal molecules also mediate insect immunity raised the question of their biosynthesis. It was determined that they are biosynthesized through a mechanism distinctly different from the mechanism known in mammals. This work points to still another target gene in the insect immune signaling system that may be the basis of advanced pest management technologies based on suppressing specific target genes. The finding was published in a peer-reviewed journal. Subobjective 1C. Documented virtually all insects collected in field sites had experienced and recovered from microbial infections. In Sub-objective 1C, ddocumented biological costs associated with successful immune reactions to infection, registered as life history changes. Specifically, recovery from infections led to developmental delays and reduced survivorship to adulthood. This was reported in a peer-reviewed journal. Objective 2A. Reported a new publically available corn rootworm diet formulation that supports improved weight gain, larval development and survival. The diet is compatible with the four insecticidal Bt proteins marketed for corn rootworm control. Our new diet facilitates discrimination between resistant and non-resistant rootworm colonies. Findings were presented at the November, 2017 International Organization for Biological Control Workshop (Mexico) and the November, 2017 Annual Meeting of the Entomological Society of America (Denver, CO). Objective 2B. Rootworm larvae were tested for differences in gene expression between larvae that were susceptible or resistant to an insect-specific Bt toxin. Candidate genes were suppressed in artificial diet bioassays to determine their roles in digestive tract responses to Bt intoxication. This enabled identification of specific gene expression issues caused by Bt intoxication. Findings were presented at the November 2017 Entomological Society of America, and the June 2018 Arthropod Genomics Symposium. Objective 3. Tested the viability of several entomopathogenic nematode species of the Steinernema genus for biocontrol of western corn rootworm larvae using three protocols. Laboratory experiments determined nematode movement patterns in the presence and absence of rootworm. Greenhouse assays in small pots determined the ability of nematodes to reduce rootworm numbers. Field studies determined nematode ability to protect corn roots from heavy rootworm infestation. Inconsistencies between the performance of specific nematode species between lab, greenhouse, and field demonstrated that some nematode species performed better in the field than in lab or greenhouse assays. These have greater potential for biological control of rootworm infestation. Findings were presented at the November 2017 International Organization for Biological Control Workshop in Merida, Mexico, the November 2017 meeting of the Entomological Society of America. Objective 4. Many insect cell lines have been established, although most of them were made from ovaries of juvenile insects. Advanced the field of insect cell lines by establishing what is called “next generation” cell lines, lines derived from specific tissues or from insects that are intractable for cell line generation. Established 16 cell lines from specific tissues. Many of these cell lines have been transferred to other research programs, documented by MTAs.


Review Publications
Wu, Y., Ding, J., Xu, B., You, L., Ge, L., Yang, G., Liu, F., Stanley, D.W., Song, Q., Wu, J. 2018. Two fungicides alter reproduction of the small brown planthopper, Laodelphax striatellus by influencing gene and protein expression. Journal of Proteome Research. 17(3):978-986.
Geisert, R.W., Cheruiyot, D.J., Hibbard, B.E., Shapiro Ilan, D.I., Shelby, K., Coudron, T.A. 2018. Comparative assessment of four steinernematidae and three heterorhabditidae species for infectivity of larval diabrotica virgifera virgifera. Journal of Economic Entomology. 111(2):542-548.
Kim, Y., Ahmed, S., Stanley, D.W., An, C. 2017. Eicosanoid-mediated immunity in insects. Developmental and Comparative Immunology. 83:130-143.
Ge, L., Gu, H., Huang, B., Song, Q., Stanley, D.W., Liu, F., Yang, G., Wu, J. 2017. An adenylyl cyclase gene (NlAC9) influences growth and fecundity in the brown planthopper, Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). PLoS One.
Zhang, L., Ringbauer Jr, J.A., Goodman, C.L., Reall, T., Jiang, X., Stanley, D.W. 2018. Prostaglandin-mediated recovery from bacteremia delays larval development in fall armyworm, Spodoptera frugiperda. Archives of Insect Biochemistry and Physiology.
Ludwick, D.C., Meihls, L.N., Huynh, M.P., Pereira, A.E., French, B.W., Coudron, T.A., Hibbard, B.E. 2018. A new artificial diet for western corn rootworm larvae is compatible with and detects resistance to all current Bt toxins. Scientific Reports. 8:5379.
Huynh, M.P., Meihls, L.N., Hibbard, B.E., Lapointe, S.L., Niedz, R.P., Ludwick, D.C., Coudron, T.A. 2017. Diet improvement for western corn rootworm (Coleoptera: Chrysomelidae) larvae. PLoS One. 12(11):e0187997.
Shelby, K., Coudron, T.A. 2017. Bacterial elicitation of transcriptional response of female squash bug, Anasa tristis (De Geer). Southwestern Entomologist.
Shelby, K., Coudron, T.A., Morales Ramos, J.A. 2017. Uptake of dietary selenium by laboratory and field feeding Podisus maculiventris (Heteroptera: Pentatomidae). Florida Entomologist. 100(1):199-202.
Zou, D., Coudron, T.A., Xu, W., Gu, X., Wu, H. 2017. Development of immature tiger-fly Coenosia attenuata (Stein) reared on larvae of the fungus gnat Bradysia impatiens (Johannsen) in coir substrate. Phytoparasitica. 45(1):75-84.
Wang, Q., Han, N., Dang, C., Lu, Z., Wang, F., Yao, H., Peng, Y., Stanley, D.W., Ye, G. 2017. Combined influence of Bt rice and rice dwarf virus on biological parameters of a non-target herbivore, Nephotettix cincticeps (Uhler) (Hemiptera: Cicadellidae). PLoS One.
Ye, X., Yang, L., Stanley, D.W., Fang, Q., Li, F. 2017. Two Bombyx mori acetylcholinesterase genes influence motor control and development in different ways. Scientific Reports.
Teng, Z., Xiong, S., Xu, G., Gan, S., Chen, X., Stanley, D.W., Yan, Z., Ye, G., Fang, Q. 2017. Protein discovery: combined transcriptomic and proteomic analyses of venom from the endoparasitoid Cotesia chilonis (Hymenoptera: Brachonidae). Toxins. https://doi:10.3390/toxins9040135.
Zhang, H., Dong, S., Chen, X., Stanley, D.W., Beerntsen, B., Feng, Q., Song, Q. 2017. Relish2 mediates bursicon homodimer-induced prophylactic immunity in the mosquito Aedes aegypti. Scientific Reports. 7:43163.
Cong, D., Lu, Z., Wang, L., Wang, F., Peng, Y., Stanley, D.W., Ye, G. 2017. Does Bt rice pose risks to non-target arthropods? Results of a meta-analysis in China. Plant Biotechnology Journal. https://doi:10.1111/pbi.12698.
Goodman, C.L., Ringbauer Jr., J.A., Li, Y., Reall, T., Stanley, D.W. 2017. Cell lines derived from the squash bug, anasa tristis (coreidae: hemiptera). In Vitro Cellular and Developmental Biology - Animals. 53:417-420.