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

Location: Biological Control of Insects Research

2019 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. 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 activating the 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, documented 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. ARS researchers at Columbia, Missouri, collaborated with an insect diet-producing company to commercialize the western corn rootworm artificial diet formulation. An artificial diet for the Northern corn rootworm was also developed using the methods for western corn rootworm diet optimization. These newly formulated diets do not require maize root powder or other ingredients not available commercially, which will ease adoption. Possible feeding stimulants extracted from maize roots were evaluated and determined to provide no additional improvement to larval performance on artificial diets. Findings were reported in peer-reviewed journals, resulted in extramural funding from stakeholders, and were presented at the May 2019 Purdue University Microbiome Symposium, the February 2019 Bayer Insect Management Academic Summit (St. Louis, MO), the March 2019 North Central Branch of the Entomological Society of America (Cincinnati, OH), and the November 2018 Annual Meeting of the Entomological Society of America (Vancouver, BC). Objective 2B. Knockdown of a specific gene target on rootworm midgut tissues reversed resistance of Bt-resistant larvae, exposing a unique and novel resistance mechanism. Gene transcript sequencing revealed genomic differences between Bt-resistant and Bacillus thuringiensis (Bt)-susceptible rootworm colonies. Identified Bt-intoxication-specific changes in larvae and in maize root responses to rootworm chewing. Findings were reported in peer-reviewed journals and were presented at the November 2018 Entomological Society of America (Vancouver, BC), and the June 2019 Arthropod Genomics Symposium (Manhattan, KS). Objective 3. Biological control of western corn rootworm larvae by nematodes was evaluated in greenhouse mortality pot assays. All nematode species tested led a significant reduction in the number of larvae recovered from test pots compared to the controls. Larval rootworm recovery was significantly lower for the smaller nematode species than for larger species. Thus, applications of the smaller species allow greater completion of the nematode species life cycle within the body of the rootworm larvae. Field trials with combinations of three rootworm biocontrol agents (nematodes, bacteria and fungi) demonstrated that mixtures of nematodes and bacteria were the most effective in protecting maize roots from rootworm damage. Findings were reported in peer-reviewed journals and were presented at the February 2019 Bayer Insect Management Academic Summit (St. Louis, MO), and the November 2018 meeting of the Entomological Society of America (Vancouver, BC). 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 seven lines from the southern armyworm, three from nervous tissues, two from testes and two from fat body, a liver-like tissue in insects. Many of these cell lines have been transferred to other research programs, documented by Material Transfer Agreements. The work on southern armyworm was reported to the Society for In Vitro Biology meeting in June 2018 and one peer-reviewed publication is accepted for publication on the southern armyworm lines.

Accomplishments
1. Commercialization of a western corn rootworm artificial diet. Initiated commercialization of a western corn rootworm artificial diet ARS researchers at Columbia, Missouri, formulated a high-performing diet for use in bioassays that will enable the industry to standardize and simplify monitoring and screening processes. Manufacturing issues related to diet quality and ingredient availability were resolved. The need for substitutions of some components, packaging, nutrient stability, sterilization, antibiotics, and heat stable components were overcome. Industrial and academic beta-testers have reported highly positive results. The product is now marketed by Frontier Agricultural Services, catalog number WCRMO-2.

Review Publications
Zhang, H., Li, Y., Reall, T., Xu, Y., Goodman, C.L., Saathoff, S.G., Ringbauer Jr, J.A., Stanley, D.W. 2018. Characterization of cell lines derived from the southern armyworm, Spodoptera eridania. In Vitro Cellular and Developmental Biology. 54:749-755. https://doi.org/10.1007/s11626-018-0300-4.
Jaffuel, G., Imperiali, N., Shelby, K., Campos-Herrera, R., Geisert, R.W., Maurhofer, M., Loper, J.E., Keel, C., Turlings, T.C., Hibbard, B.E. 2019. Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes. Scientific Reports. 9:3127. https://doi.org/10.1038/s41598-019-39753-7.
Geisert, R.W., Ludwick, D.C., Hibbard, B.E. 2019. Effects of cold storage on nondiapausing eggs of the western corn rootworm (Coleoptera: Chrysomelidae). Journal of Economic Entomology. 112(2):708-711. https://doi.org/10.1093/jee/toy405.
Huynh, M.P., Bernklau, E.J., Coudron, T.A., Shelby, K., Bjostad, L.B., Hibbard, B.E. 2019. Characterization of corn root factors to improve artificial diet for western corn rootworm (Coleoptera: Chrysomelidae) larvae. Journal of Insect Science. 19(2):1-8. https://doi.org/10.1093/jisesa/iez030.
Zhao, Z., Miehls, L.M., Hibbard, B.E., Ji, T., Elsik, C., Shelby, K. 2019. Differential gene expression in response to eCry3.1Ab ingestion in an unselected and eCry3.1Ab-selected western corn rootworm (Diabrotica virgifera virgifera LeConte) population. Scientific Reports. 9:4896. https://doi.org/10.1038/s41598-019-41067-7.
Pereira, A.E., Coudron, T.A., Shelby, K., French, B.W., Bernkalu, E.J., Bjostad, L.B., Hibbard, B.E. 2019. Comparative susceptibility of western corn rootworm (Coleoptera: Chrysomelidae) neonates to selected insecticides and Bt proteins in the presence and absence of feeding stimulants. Journal of Economic Entomology. 112(2):842-851. https://doi.org/10.1093/jee/toy415.
Meihls, L., Huynh, M.P., Ludwick, D.C., Coudron, T.A., French, B.W., Shelby, K., Hitchon, A.J., Smith, J.L., Schaafsma, A.W., Pereira, A.E., Hibbard, B.E. 2018. Comparison of six artificial diets for western corn rootworm bioassays and rearing. Journal of Economic Entomology. 111(6):2727-2733. https://doi.org/10.1093/jee/toy268.
Han, G., Zhang, N., Xu, J., Jiang, H., Ji, C., Zhang, Z., Song, Q., Stanley, D.W., Fang, J., Wang, J. 2019. Characterization of a novel Helitron family in insect genomes: insights into classification, evolution and horizontal transfer. Mobile DNA. https://doi.org/10.1186/s13100-019-0165-4.
Abdulrahim, N., Othman, M., Sabri, M., Stanley, D.W. 2018. A midgut digestive phospholipase A2 in larval mosquitoes, Aedes albopictus and Culex quinquefasciatus. Enzyme Research. https://doi.org/10.1155/2018/9703413.
Ahmed, S., Stanley, D.W., Kim, Y. 2018. An insect prostaglandin E2 synthase acts in immunity and reproduction. Frontiers in Physiology. 9:1231. https://doi.org/10.3389/fphys.2018.01231.
Wang, B., Yang, Y., Liu, M., Yang, L., Stanley, D.W., Fang, Q., Ye, G. 2019. A digestive tract expressing a-amylase influences the adult lifespan of Pteromalus puparum revealed through RNAi and rescue analyses. Pest Management Science. https://doi.org/10.1002/ps.5462.
Sadekuzzaman, M., Stanley, D.W., Kim, Y. 2017. Nitric oxide mediates insect cellular immunity via phospholipase A2 activation. Journal of Innate Immunity. 10:70-81. https://doi.org/10.1159/000481524.
Stanley, D.W., Kim, Y. 2019. Prostaglandins and other eicosanoids in insects: biosynthesis and biological actions. Frontiers in Physiology. https://doi.org/10.3389/fphys.2018.01927.
Man, H.P., Hibbard, B.E., Lapointe, S.L., Niedz, R.P., French, B.W., Pereira, A.E., Finke, D.L., Shelby, K., Coudron, T.A. 2019. Multidimensional approach to formulating a specialized diet for northern corn rootworm larvae. Scientific Reports. 9:3709. https://doi.org/10.1038/s41598-019-39709-x.
Zou, D., Coudron, T.A., Zhang, L., Gu, X., Xu, W., Wu, H. 2018. Performance of Arma chinensis reared on an artificial diet formulated using transcriptomic methods. Bulletin of Entomological Research. 109:24-33. https://doi.org/10.1017/S0007485318000111.
Wang, Q., Li, J., Dang, C., Chang, X., Fang, Q., Stanley, D.W., Ye, G. 2018. Rice dwarf virus infection alters green rice leafhopper host preference and feeding behavior. Frontiers in Plant Science. https://doi.org/10.1371/journal.pone.0203364.
Sajjadian, S.M., Vatanparast, M., Stanley, D.W., Kim, Y. 2019. Secretion of secretory phospholipase A2 into Spodoptera exigua larval midgut lumen and its role in lipid digestion. Insect Molecular Biology. https://doi.org/10.1111/imb.12588.
Stanley, D.W., Kim, Y. 2019. Insect prostaglandins and other eicosanoids: from molecular to physiological actions. Advances in Insect Physiology. 56:283-343. https://doi.org/10.1016/bs.aiip.2019.01.003.
Ge, L., Zheng, S., Gu, H., Zhou, Y., Zhou, Z., Song, Q., Stanley, D.W. 2019. Jinggangmycin-induced UDP-glycosyltransferase 1-2-like is a positive modulator of fecundity and population growth in Nilaparvata lugens (Stål) (Hemiptera: Delphacidae). Frontiers in Physiology. 10:747. https://doi.org/10.3389/fphys.2019.00747.