Location: Biological Control of Insects Research2017 Annual Report
1a. Objectives (from AD-416):
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.
1b. Approach (from AD-416):
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.
3. Progress Report:
Subobjective 1A. We conducted the work with another pest, beet army worms. We discovered the first known insect intracellular PLA2, expressed the recombinant enzyme and characterized the enzyme. We determined the gene structure and silenced the gene with a dsRNA construct. Silencing the gene effectively disabled an immune response to bacterial infection. The findings were reported in a publication in invited seminars. Subobjective 2A. We surveyed the western corn rootworm microbiome in eggs, larvae and adults. The collection of bacteria was found to be soil and growth-stage dependent. Differences in larval microbiome were found to decline with the age of the insect and converged to a similar microbiome in adults. Findings were presented at the 26th International Working Group on Ostrinia and Other Maize Pests, Beijing, China (April, 2017). Objective 3. We have tested colonies of eight different entomopathogenic nematode species, including two Heterorhabditis species and six Steinernema species, for infectivity against the western corn rootworm. All colonies were able to infect the rootworm and cause mortality. Colonies differed in rate of infection, percent mortality and production of nematode progeny. The results suggest better performance by species in the Heterorhabditis family for the control of western corn rootworm larvae in laboratory experiments. We will present our findings at the annual meeting of the Society of Invertebrate Pathology (August, 2017). 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 in a published the paper. We used the cell line in research to document prostaglandin actions in the cell line and reported these findings in another peer-reviewed publication.
1. The diversity of bacteria western corn rootworm larvae encounter in the soil is reflected on their surfaces and in their digestive tracts (microbiome). Microbiomes influence many aspects of insect growth, nutrition, reproduction, Bacillus thuringiensis resistance, and pathogen resistance. The microbiome role in the western corn rootworm lifecycle is unknown. ARS researchers in Columbia, Missouri conducted the first survey of the rootworm microbiome from egg to adult life stages. Over 1100 bacterial species were putatively identified in soil and insect samples from two different geographic regions. The results showed that insects reared in differing soils contained significantly different compositions of bacterial species. However, over the life cycle, from egg to adults, differences between the soil types and insects declined, and converged on a very similar microbiome that contained approximately 14 core bacterial species. These findings will be used by researchers and industry to advance their understanding of rootworm nutrition and toxin resistance.
2. Artificial diet for western corn rootworm toxin bioassays. A major difficulty in performing the Environmental Protection Agency-mandated monitoring for resistance to Bacillus thuringiensis (Bt) toxins in field populations is having an artificial diet that can be used in the assay of several different toxins. ARS researchers in Columbia, Missouri formulated a new diet and demonstrated that diet supports rootworm development and is compatible with bioassays involving Bt toxins found in current maize varieties and newly discovered toxins. The new formulation will be used by researchers, seed industry, monitoring laboratories, and government regulatory agencies to standardize methods used to monitor for rootworm resistance.
Büyükgüzel, E., Erdem, M., Tunaz, H., Küçük, C., Atilgan, U., Stanley, D.W., Büyükgüzel, K. 2016. Inhibition of eicosanoid signaling leads to increased lipid peroxidation in a host/parasitoid system. Comparative Biochemistry and Physiology - Part A: Molecular & Integrative Physiology. 204 (2017) 121-128.