Location: Biological Control of Insects Research
2016 Annual Report
Objectives
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.
Approach
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. We identified and cloned a gene encoding the enzyme responsible for the first step in biosynthesis of eicosanoids (PLA2) from the squash bug. Using an alternative pest, we identified, cloned and characterized a PLA2 that is completely new to insect science. We expressed the gene in bacterial cells and documented its role in insect immunology. Work on the squash bug PLA2 is progressing.
Subobjective 2A. We improved the ratio of the ingredients in the only publicly available artificial diet for rearing the western corn rootworm. The performance of corn rootworm reared on the new formulation was comparable to, or exceeded, that of rootworm reared on all known private diet formulations. Two manuscripts reporting the findings are in progress, and we will present our findings at the 2016 International Congress of Entomology (September, 2016).
Objective 3. We have established colonies of six entomopathogenic nematodes, including two Heterorhabditis species and four Steinernema species. These colonies have been used to initiate insect infectivity and behavioral observation and selection studies. We have been invited to present our findings at the 2016 International Congress of Entomology (September, 2016).
Objective 4. We selected beginning cell culture protocols and established the first squash bug cell line. We reported the new cell line at the 2016 World Congress of In Vitro Biology (June, 2016) and drafted the manuscript for publication in an appropriate professional journal, which will be submitted in 2016.
Accomplishments
1. Discovery of biochemical signaling to control internalization of infecting bacteria by insect blood cells. Circulating insect blood cells clear microbial pathogens from circulation by internalizing them. The process is tightly regulated, although the regulating mechanism is not clearly understood, which hinders progress in suppressing the process. ARS researchers in Columbia, Missouri, in an international collaboration, identified a signal system that regulates the internalization. This discovery will be used by scientists globally because it identifies a new specific molecular target for suppressing insect immune reactions to microbial biological control agents.
2. Discovery of a mechanism certain insect pests use to response to agricultural pesticides with enhanced reproduction. Most pest insect species respond to continued exposure to agricultural pesticides by developing resistance to the pesticides. A few species, however, have gone beyond classical resistance to using pesticides to advantage the pest. ARS researchers at Columbia, Missouri, in an international collaboration, identified the mechanism of enhancing reproduction. Pesticides stimulate production and storage of fats that are used by the pest to enhance egg development. We found that silencing, separately, two key genes in fat production eliminates the enhanced reproduction. Scientists will use this new information to improve insect pest management technologies.
3. Improved diet formulation for rearing western corn rootworm. A major limitation in developing control measures for one of the most costly agricultural pests, the western corn rootworm, is the lack of an effective and standardized diet for rearing the insect. ARS researchers in Columbia, Missouri, refined the formulation of a published diet resulting in improved survival, weight gain and development of western corn rootworm. The new formulation will be used by researchers, industry and government regulatory agencies to standardize methods used to create new control methods and to monitor for resistance.
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Review Publications
Chang, C.L., Goodman, C.L., Ringbauer Jr., J.A., Geib, S.M., Stanley, D.W. 2016. Larval x-ray irradiation influences protein expression in pupae of the Oriental fruit fly, Bactrocera Dorsalis. Archives of Insect Biochemistry and Physiology. 92(3):192-209.
Chang, C.L., Villalun, M.V., Geib, S.M., Goodman, C.L., Ringbauer Jr., J.A., Stanley, D.W. 2015. Pupal x-ray irradiation influences protein expression in adults of the oriental fruit fly, Bactrocera dorsalis. Journal of Insect Physiology. 76:7-16.
Dong, S., Zhang, H., Chen, X., Stanley, D.W., Yu, X., Song, Q. 2015. The neuropeptide bursicon acts in cuticle metabolism. Archives of Insect Biochemistry and Physiology. 89(2):87-97 doi: 10.1002/arch.21227.
Ge, L., Jiang, Y., Xia, T., Song, Q., Stanley, D.W., Kuai, P., Lu, X., Yang, G., Wu, J. 2015. Silencing a sugar transporter gene reduces fecundity, growth and development in the brown planthopper, Nilaparvata lugens (Stal) (Hemiptera: Delphacidae). Scientific Reports. doi: 10.1038/SREP.2015-12194.
Morales Ramos, J.A., Rojas, M.G., Shelby, K., Coudron, T.A. 2015. Nutritional value of pupae versus larvae of Tenebrio molitor (Coleoptera: Tenebrionidae) as food for rearing Podisus maculiventris (Heteroptera: Pentatomidae). Journal of Economic Entomology. 109(2):564-571.
Park, Y., Stanley, D.W. 2015. Physiological trade-off between cellular immunity and flight capability in the wing-dimorphic cricket, Gryllus firmus. Journal of Asia Pacific Entomology. 18(3):553–559.
Shirk, P.D., Perera, O.P., Shelby, K., Furlong, R.B., LoVullo, E.D., Popham, H.J. 2015. Unique synteny and alternate splicing of the chitin synthases in closely related heliothine moths. Gene. 574(1):121-139.
Wu, S., Xu, G., Stanley, D.W., Huang, J., Ye, G. 2015. Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis. Scientific Reports. doi: 10.1038/SREP-12247.
