Research Project: Biologically-Based Products for Insect Pest Control and Emerging Needs in Agriculture

Location: Biological Control of Insects Research

2021 Annual Report

Objectives
Objective 1: Identify new molecular components of insect immune signaling. Objective 2: Determine the biology of insect cell line establishment and associated cryopreservation technologies to establish next-generation insect cell lines to meet specific needs of academic and indus¬trial partners, such as the need for honey bee cell lines. Objective 3: Identify genomic structural variants and metabolites contributing to corn rootworm toxin resistance phenotypes and develop genetic markers to assess rootworm resistance. Objective 4: Determine the influence of microbiomes on the performance of selected agricultural pests, including the spotted wing Drosophila.

Approach
1A: Existing A. tristis genetic databases will be used to identify candidate PGF2a synthase genes based on sequence similarities to known PGF2a synthase genes from other organisms. 1B: Utilizing standard molecular biology techniques, candidate PGF2a synthase genes will be cloned and their biological function will be verified experimentally. 1C: Advanced molecular biology techniques and chemical agents will be used to impair PGF2a synthase function and any effects on immune system function and reproductive health will be measured experimentally. 2A: Variations in DNA, RNA and protein expression patterns will be compared between primary cultures and replicating cell lines using next-generation bioinformatic tools and analyses. 2B: Cell lines will be genetically engineered through the introduction of plasmid DNA coding for proteins of interest, and the expression and proper function of the introduced proteins will be verified in cell-based fluorescent reporter assays. 2C: Various techniques and proprietary technologies will be tested for their ability to improve the survival rate of cells as they are frozen and stored for long time periods. 3A: Structural variants of ABC transporters will be identified from genetic data produced from Bt toxin-resistant and toxin-sensitive lab colonies of corn rootworm. Changes in ABC transporter expression levels will be evaluated following exposure of corn rootworms to Bt toxins. The functional role of these variants in Bt toxin resistance will be evaluated by gene knockdown experiments. 3B: Genetic markers for variants of ABC transporters, as well as other genes, associated with Bt toxin resistance will be identified from genetic databases, and their presence in field populations and other corn rootworm strains will be evaluated using standard molecular biology techniques. 3C: Metabolites that are up- or down-regulated in Bt-resistant and Bt-sensitive lab colonies of corn rootworm will be measured using standard analytical chemistry techniques. Compounds with significantly different expression patterns will be linked to metabolic pathways and their functional role in Bt toxin resistance or sensitivity will be evaluated experimentally. 4A: The gut microbiome of wild Drosophila suzukii will be compared to a microbial database to characterize its community structure. Statistical modeling will be used to further determine whether certain microbes tend to co-occur. Further models will test whether individual and co-associated groups of microbes seem particularly suited to colonizing the wild fly gut. 4B: Lipid content of the fly guts will be measured. Statistical analyses will then be used to determine whether specific microbes are associated with increased or diminished fat content in the fly host. Germ-free flies will be generated and a portion of them will be exposed to select microbes to verify their influence on the host. 4C: The genomes of microbes of interest will be sequenced and genes and genetic pathways will be associated with host lipid content. Finally, germ-free flies will be exposed to bacteria modified to lose or gain these genes to verify their influence on the host.

Progress Report
Objective 1. We identified a new molecular element of insect eicosanoid signaling systems, a thromboxane A2, which has not been identified in any insect species. Similarly, for goal 1.2, a thromboxane A2 synthetase has been cloned and the enzyme amino acid sequence analyzed. Objective 2. Goal 2.1: We purified mRNA from eight insect cell lines and generated transcriptomes for each. Goal 2.5. We generated cell lines and froze them at different temperatures using various methods to facilitate an evaluation of freezing times on cell survival in 2022 and 2023. Objective 3, Goal 3.1. Larval and adult western and northern corn rootworms were collected from Corn Belt sites to initiate studies of resistance genetic variants in these two species. A larval northern corn rootworm transcriptome was generated to enable identification of the variants in this insect species. Goal 3.3. Larval Western and northern corn rootworms were collected to initiate multiomic analyses of responses to insect toxins, alterations in diet nutrient composition, and treatment with double stranded RNA knockdowns of selected targets. Initial results indicate that tryptophan and asparagine levels are limiting in artificial diets, while Lysine and phenylalanine are in excess. Objective 4, Goal 4.1, Collection and dissection of Spotted wing Drosophila midguts, DNA extraction and culture of the fly-associated microbes was initiated. Goal 4.2, Collaborations to assist with field collection of the specimens and to assist with data analyses were established with scientists at the Universities of Missouri and Oregon. Four laboratory, and two field-derived colonies of Spotted wing Drosophila were established during Summer 2021. Protocols for the removal and replacement of bacteria in the fly were developed.

Accomplishments
1. Thermal sterilization of Artificial Diets for Western Corn Rootworm Larvae. The western corn rootworm is the most serious pest of corn in the U.S. Corn Belt. This highly adaptive pest has evaded nearly all management tactics developed to date. Diet assays, whereby insects are exposed to toxins on artificial diet, are a critical component of resistance monitoring programs and evaluating new toxins. Published rootworm diets contain antibiotics that reduce bacterial contamination. But antibiotic ingestion necessarily alters the rootworm gut microbiota, and this changes the outcome of toxin bioassays. Rapid heating, or Pasteurization, is one of the most widely applied techniques to reduce microbial contamination, and this could elimination of antibiotics from the mix. Using a custom-built flash sterilization unit we characterized the effects of timed intervals of thermal exposure on the quality of rootworm diet by measuring larval weight, molting and survival. Results indicated that temperatures had non-linear effects on performance of diet, but no impacts were observed on the exposure intervals evaluated. The optimum temperature of diet processing was 60°C for a duration less than 30 min. Significant losses in nutrients needed for rootworm growth and development in diet were observed when the diet was heated above 75°C even for brief durations caused a two-fold reduction in larval weight gain, and delays in larval molting. These now published findings will guide the continued development of commercially available, sterilized rootworm diets, and will also provide invaluable insights into the design and manufacture of diets for other insects. The work was funded by a SBIR Phase I grant.

Review Publications
Kim, Y., Stanley, D.W. 2021. Eicosanoid signaling in insect immunology: New genes and unresolved issues. Genes. 12(2). Article 211. https://doi.org/10.3390/genes12020211.
Zhao, I., Elsik, C.G., Hibbard, B.E., Shelby, K. 2021. Detection of alternative splicing in western corn rootworm (diabrotica virgifera virgifera LeConte) in association with eCry3.1Ab resistance using RNA-seq and PacBio iso-seq. Insect Molecular Biology. 30(4):436-445. https://doi.org/10.1111/imb.12709.
Al Baki, M., Roy, M., Lee, D., Stanley, D.W., Kim, Y. 2021. The prostanoids, thromboxanes, mediate hemocytic immunity to bacterial infection in the lepidopteran Spodoptera exigua. Developmental and Comparative Immunology. 120(7). Article 104069. https://doi.org/10.1016/j.dci.2021.104069.
Kang, D.S., Kim, S., Cotten, M.A., Sim, C. 2020. Transcript assembly and quantification by RNA-seq reveals significant differences in gene expression and genetic variants in mosquitoes of the Culex pipiens (Diptera: Culicidae) complex. Journal of Medical Entomology. 58(1):139–145. https://doi.org/10.1093/jme/tjaa167.
Paddock, K., Hibbard, B.E., Barry, J.M., Sethi, A., Mueller, A.L., Shelby, K., Pereira, A. 2021. Restoration of susceptibility following removal of selection for Cry34/35Ab1 resistance documents fitness costs in resistant population of western corn rootworm, diabrotica virgifera virgifera. Pest Management Science. 77(5):2385-2394. https://doi.org/10.1002/ps.6266.
Perera, O.P., Shelby, K., Pierce Iii, C.A., Snodgrass, G.L. 2021. Digestive gene expression profiles in the salivary glands and gut of tarnished plant bug, Lygus lineolaris (Hemiptera: Miridae). Journal of Insect Science. 21:3. https://doi.org/10.1093/jisesa/ieab028.
Ahn, S., McDonnell, R., Corcoran, J., Martin, R.C., Choi, M.Y. 2020. Identification and functional characterization of the first molluscan neuromedin U receptor in the slug, Deroceras reticulatum. Scientific Reports. 10. Article 22308. https://doi.org/10.1038/s41598-020-79047-x.
Ahn, S., Corcoran, J., Vander Meer, R.K., Choi, M.Y. 2020. Identification and characterization of GPCRs for Pyrokinin and CAPA peptides in the brown marmorated stink bug, Halyomorpha halys (Hemiptera: Pentatomidae). Frontiers in Physiology. 11:559. https://doi.org/10.3389/fphys.2020.00559.
Hamiaux, C., Carraher, C., Löfstedt, C., Corcoran, J. 2020. Crystal structure of Epiphyas postvittana pheromone binding protein 3. Scientific Reports. 10, 16366. https://doi.org/10.1038/s41598-020-73294-8.
Ahn, S., Oh, H., Corcoran, J., Kim, J., Park, K., Park, C.G., Choi, M.Y. 2020. Sex-biased gene expression in antennae of Drosophila suzukii. Archives of Insect Biochemistry and Physiology. 104(1):e21660. https://doi.org/10.1002/arch.21660.
Zhang, N., Jiang, H., Meng, X., Qian, K., Liu, Y., Song, Q., Stanley, D.W., Wu, J., Park, Y., Wang, J. 2020. Broad-complex transcription factor mediates opposing hormonal regulation of two phylogenetically distant arginine kinase genes in Tribolium castaneum. Communications Biology. 3. Article 631. https://doi.org/10.1038/s42003-020-01354-w.
Stanley, D.W., Goodman, C.L., Ringbauer Jr, J.A., Song, Q. 2020. Prostaglandins influence protein phosphorylation in established insect cell lines. Archives of Insect Biochemistry and Physiology. 105(1). Article 21725. https://doi.org/10.1002/arch.21725.
Kim, Y., Ahmen, S., Al Baki, M., Kumar, S., Kim, K., Park, Y., Stanley, D.W. 2020. Deletion mutant of PGE2 receptor using CRISPR-Cas9 exhibits larval imunosuppression and adult infertility in a lepidopteran insect, Spodoptera exigua. Developmental and Comparative Immunology. 111(10). Article 103743. https://doi.org/10.1016/j.dci.2020.103743.