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

Location: Biological Control of Insects Research

2020 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
This is a brief summary of the project life (2015-2020), which will be replaced by a new project entitled “Biologically-based products for insect pest control and emerging needs in agriculture”. In Subobjective 1A we researched a protein (PLA2) that releases fatty acids from certain classes of fats. We cloned PLA2s from cell lines representing two insect pests, corn earworm and tobacco budworm. We expressed and characterized the proteins and a manuscript for publication in a peer reviewed journal is in preparation. We also discovered and characterized a new PLA2 that acts in immunity and development and a second PLA2 that acts in digestion. We characterized digestive PLA2s in two mosquito species. Findings were published in peer-reviewed journals. In Subobjective 1B, we identified a protein responsible for generating biochemical signals, although the work is not complete. In Subobjective 1C, we reported in a peer-reviewed journal that recovery from microbial infections delays or inhibits larval development. In Subobjective 2B, we identified gene expression and genetic differences between Bacillus thuringiensis (Bt)-resistant and susceptible rootworms. Blocking expression of a specific gene target on rootworm midgut tissues reversed resistance of Bt-resistant larvae, exposing a unique and novel resistance mechanism. We identified metabolic responses that differed between feeding Bt-susceptible and resistant rootworms, and corn root responses to rootworm feeding. We conducted studies of the role that gut bacteria play in the rootworm from egg to adult. Our results showed that although WCR may migrate long distances, or be collected from diverse regions, their microbiomes converged upon a similar gut bacterial profile during maturation to adults. In Objective 3, we screened multiple species of entomopathogenic nematodes collected from several Missouri locations and other states to determine which were the most effective controls for corn rootworm infestations in greenhouse and field. The most effective species of nematode was bred for several generations to increase attraction to an alarm chemical released from rootworm damaged corn roots. We then conducted field trials of these nematodes with combinations of two other rootworm biocontrol agents (bacteria and fungi) which demonstrated that mixtures of the nematodes with bacteria were most effective in protecting corn roots from rootworm damage. In Subobjective 4A, we established next-generation cell lines from squash bugs (two lines), southern armyworms (seven lines, three from nervous tissues, two from testes and two from fat body, a liver-like organ), fall armyworms (16; two from midgut, three from aorta, nine from central nervous system, and four from testes) and western corn rootworms (three from larvae). We characterized all the cell lines and reported on them in a series of papers in a peer reviewed journal. We used the squash bug cell lines in research to determine the influence of selected biochemical signals on cell biology, also published in a peer-reviewed paper. We are working to establish cell lines from honey bees and collaborated on a peer reviewed paper on the topic.

Accomplishments
1. Diet-toxicity bioassays of western and northern corn rootworms. Western and northern corn rootworms are major pests of corn production in the U.S. Corn Belt, but they are rapidly evolving resistance to all commercially available genetically modified corn varieties. The Environmental Protection Agency has mandated that registrants of Bt crops develop resistant colonies to facilitate understanding mechanisms that pests may use in surviving Bt crops and this is a major concern for the industry. Currently, diet-toxicity evaluations of each major maize seed company’s Bt toxins are conducted on proprietary artificial diets, which prevents direct comparisons of toxicity data from different seed companies. ARS researchers at Columbia, Missouri, developed advanced high throughput design and screening methods to formulate high-performing diets for use in diet-toxicity bioassays of western and northern corn rootworms. Several Industry and academic researcher groups were trained on-site to prepare and use the diets developed by ARS researchers in their own research. A Phase I Small Business Innovation Research grant was awarded to the collaborative group to further improve the packaging and shipping stability of both diets. These diets benefit researchers, industry and extension workers who must rapidly, inexpensively, easily and reproducibly assess rootworm resistance against genetically modified corn in the field, and evaluate new toxins using diet-toxicity assays.

Review Publications
Ludwick, D.C., Ericsson, A.C., Meihls, L.M., Gregory, M.L., Finke, D.L., Coudron, T.A., Hibbard, B.E., Shelby, K. 2019. Survey of bacteria associated with western corn rootworm life stages reveals no difference between insects reared in different soils. Scientific Reports. 9:15332. https://doi.org/10.1038/s41598-019-51870-x.
Yang, L., Wang, J., Jin, H., Fang, Q., Yan, Z., Lin, Z., Zou, Z., Song, Q., Stanley, D.W., Ye, G. 2019. Immune signaling pathways in the endoparasitoid, Pteromalus puparum. Archives of Insect Biochemistry and Physiology. 103(2):e21629. https://doi.org/10.1002/arch.21629.
Xu, G., Teng, Z., Gu, G., Guo, L., Wang, F., Xiao, S., Wang, J., Wang, B., Fang, Q., Wang, F., Song, Q., Stanley, D.W., Ye, G. 2019. Genomic and transcriptomic analyses of glutathione S-transferases in an endoparasitoid wasp, Pteromalus puparum. Archives of Insect Biochemistry and Physiology. 103(2):e21634. https://doi.org/10.1002/arch.21634.
Xiong, S., Yu, K., Xinhai, Y., Fang, Q., Deng, Y., Xiao, S., Yang, L., Wang, B., Wang, F., Zhichao, Y., Wang, F., Song, Q., Stanley, D.W., Ye, G. 2019. Genes acting in longevity-related pathways in the endoparasitoid, Pteromalus puparum. Archives of Insect Biochemistry and Physiology. 103(2):e21635. https://doi.org/10.1002/arch.21635.
Qi, Y., Wang, J., Xu, G., Song, Q., Stanley, D.W., Fang, Q., Ye, G. 2019. Biogenic amines biosynthetic and transduction genes in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Archives of Insect Biochemistry and Physiology. 103(2):e21632. https://doi.org/10.1002/arch.21632.
Wang, J., Jin, H., Yang, L., Ye, X., Xiao, S., Song, Q., Stanley, D.W., Ye, G., Fang, Q. 2019. Genome-wide identification and analysis of genes encoding cuticular proteins in the endoparasitoid wasp Pteromalus puparum (Hymenoptera: Pteromalidae). Archives of Insect Biochemistry and Physiology. 103(2):e21628. https://doi.org/10.1002/arch.21628.
Xiao, S., Wang, B., Li, K., Xiong, S., Ye, X., Wang, J., Zhang, J., Yan, Z., Wang, F., Song, Q., Stanley, D.W., Ye, G., Fang, Q. 2019. Identification and characterization of miRNAs in an endoparasitoid wasp, Pteromalus puparum. Archives of Insect Biochemistry and Physiology. 103(2):e21633. https://doi.org/10.1002/arch.21633.
Reall, T., Kraus, S., Goodman, C.L., Ringbauer Jr, J.A., Geibel, S., Stanley, D.W. 2019. Next-generation cell lines established from the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae). In Vitro Cellular and Developmental Biology. 55:686-693. https://doi.org/10.1007/s11626-019-00394-9.
Zhou, K., Goodman, C.L., Ringbauer Jr, J.A., Song, Q., Beerntsen, B., Stanley, D.W. 2019. Establishment of two midgut cell lines from the fall armyworm, Spodoptera Frugiperda (Lepiodoptera: Noctuidae). In Vitro Cellular and Developmental Biology - Animal. 56:10-14. https://doi.org/10.1007/s11626-019-00420-w.
Guo, Y., Goodman, C.L., Stanley, D.W., Bonning, B.C. 2020. Cell lines for honey bee virus research. Viruses. 12(2):236. https://doi.org/10.3390/v12020236.
Zhou, K., Goodman, C.L., Ringbauer Jr, J.A., Stanley, D.W. 2020. Cell lines derived from the western corn rootworm larvae, Diabrotica virgifera virgifera (Chrysomelidae: Coleoptera). In Vitro Cellular and Developmental Biology - Animal. 56:281-285. https://doi.org/10.1007/s11626-020-00460-7.
Stanley, D.W., Kim, Y. 2019. Why most insects have very low proportions of C20 polyunsaturated fatty acids: the oxidative stress hypothesis. Archives of Insect Biochemistry and Physiology. 103(1):e21622. https://doi.org/10.1002/arch.21622.
Huynh, M.P., Hibbard, B.E., Vella, M., Lapointe, S.L., Niedz, R.P., Shelby, K., Coudron, T.A. 2019. Development of an improved and accessible diet for western corn rootworm larvae using response surface modeling. Scientific Reports. 9:16009. https://doi.org/10.1038/s41598-019-52484-z.
Ge, L., Zhou, Z., Sun, K., Huang, B., Stanley, D.W., Song, Q. 2020. The antibiotic jinggangmycin increases brown planthopper (BPH) fecundity by enhancing rice plant sugar concentrations and BPH insulin-like signaling. Chemosphere. 249:126463. https://doi.org/10.1016/j.chemosphere.2020.126463.
Yang, L., Wang, B., Qiu, L., Wan, B., Yang, Y., Liu, M., Wang, F., Fang, Q., Stanley, D.W., Ye, G. 2019. Functional characterization of a venom protein calreticulin in the Ectoparasitoid Pachycrepoideus vindemiae. Insects. 11(1):29. https://doi.org/10.3390/insects11010029.
Xu, G., Teng, Z., Gu, G., Qi, Y., Guo, L., Xiao, S., Wang, F., Fang, Q., Song, Q., Stanley, D.W., Ye, G. 2019. Genome-wide characterization and transcriptomic analyses of neuropeptides and their receptors in an endoparasitoid wasp, Pteromalus puparum. Archives of Insect Biochemistry and Physiology. 103(2):e21625. https://doi.org/10.1002/arch.21625.
Wu, J., Ge, L., Liu, F., Song, Q., Stanley, D.W. 2019. Pesticide-induced planthopper population resurgence in rice cropping systems. Annual Review of Entomology. 65:409-429. https://doi.org/10.1146/annurev-ento-011019-025215.
Ge, L., Zhou, Y., Gu, H., Wu, Q., Zhou, Z., Zheng, S., Stanley, D.W., Song, Q. 2019. Male selenoprotein F-like (SPF-L) influences female reproduction and population growth in Nilaparvata lugens (Hemiptera: Delphacidae). Frontiers in Physiology. 10:1196. https://doi.org/10.3389/fphys.2019.01196.
Pereira, A.E., Huynh, M.P., Sethi, A., Miles, A.L., French, B.W., Ellersieck, M.R., Coudron, T.A., Shelby, K., Hibbard, B.E. 2020. Baseline susceptibility of a laboratory strain of northern corn rootworm, diabrotica barberi (coleoptera: chrysomelidae) to bacillus thuringiensis traits in seedling, single plant, and diet-toxicity assays. Journal of Economic Entomology. 113(4):1955–1962. https://doi.org/10.1093/jee/toaa107.