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

Location: Biological Control of Insects Research

2022 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 1.1: We identified two new insect eicosanoids previously known in mammals, but not insects. These are thromboxanes A2 and B2. These two eicosanoids mediate insect cellular immune reactions to fungal infections. Publication of this work broadens understanding of eicosanoid signaling in insect immunology. In separate work we developed and published a manually curated database for all known insect venom proteins. The database contains comprehensive information on over 4,800 proteins and over 4,500 corresponding nucleotide sequences. One paper resulted from this work. Objective 1.2 and Objective 2.1: We generated three cell lines from tissues derived from the small hive beetle, and generated transcriptomes for each. The transcriptomes were then evaluated for differences in gene expression levels as well as the expression of specific insecticidal targets between the cell lines. One publication resulted from these studies to date. Objective: Protein extraction methods and reagents were evaluated for their performance in extracting protein from cell lines. Objective 2.3: Genomic DNA extraction methods and reagents were evaluated for their performance in extracting genetic material from cell lines. Objective 2.4: Several insect cell lines were genetically engineered to express functional recombinant proteins. In these experiments cell lines were modified to express dopamine receptors and evaluated for their ability to respond to dopamine in cell-based assays. Objective 2.5: A long-term cryopreservation study continued in which cells previously frozen in various test reagents at selected freezing temperatures were thawed and evaluated for enhanced viability and growth rates. One publication reporting these results will be submitted in fiscal year 2022. Objective 3.1: Feeding on Bacillus thuringiensis entomotoxic proteins disrupts the gut of rootworm larvae. We reported on a novel finding that a gut lining protein called peritrophin exhibited alternative splicing when susceptible and resistant western corn rootworm (WCR) larvae were fed on Bt expressing maize seedlings. Microscopic examination of the gut tissue revealed that Bt-susceptible larvae develop symptoms indicative of gut disruption, whereas resistant larvae showed much milder effects. Objective 3.3: We completed multiomic studies that documented WCR and northern corn rootworm (NCR) larval responses to dietary treatments with artificial diet modifications, entomotoxic proteins, and double stranded RNAs. We demonstrated that resistant and susceptible WCR larvae had metabolomic and gene expression responses to feeding that were unique to each of three commercially available Bt-expressing maize hybrids that target rootworms. Overall, 580 metabolites found in larvae were classified into different metabolic pathways in susceptible and resistant WCR and NCR larvae that may be involved in defense against or recovery from Bt feeding. In addition to these we completed additional multiomic studies of WCR and NCR artificial diet formulations, and a study on the effects of double stranded RNA additions on both species. We completed pasteurization optimization experiments in collaboration with a commercial insect company. Two reports of these results have been accepted for publication, with others in review. Objective 4.1: Working relationships were established with local farms and vineyards to monitor Spotted Wing Drosophila (SWD). Collaborations with other ARS units and universities were initiated to assist with specimen collection and data analyses. This work was supported in part by the Missouri Grape and Wine Institute. ARS scientists and collaborators have collected, dissected, and extracted DNA from flies, and have extracted and cultured the fly- associated microbes. Flies were captured from grapes, blueberries, raspberries, blackberries, cherries, and elderberries. Preliminary testing was performed to determine the general taxonomic composition of the SWD microbiome. Objective 4.2: Four laboratory, and two field-derived colonies of SWD were established and optimized axenic and gnotobiotic techniques were developed.

Accomplishments
1. Determination of spatial and temporal distribution patterns of grape mealybugs in mid-Missouri vineyards. An ARS researcher in Columbia, Missouri, reported on an ongoing grape mealybug monitoring study that informs grape growers about how to control these agricultural pests by defining periods of activity, thus improving the efficiency and effectiveness of insecticidal tools (https://americanvineyardmagazine.com/mealybugs-in-mid-missouri-vineyards/). Based on these data, it is now known (1) how widespread mealybugs are in mid-Missouri vineyards, (2) when male flights occur during the growing season, (3) the mealybugs present are grape mealybugs (based on genetic testing), and (4) that the grape mealybugs in Missouri vineyards harbor the viruses that cause grape leafroll disease, one of the most destructive diseases of grapevines worldwide. Grape leafroll disease reduces grape quality and vineyard production, and because there is no cure, controlling mealybug populations is critical to preventing its spread. The Missouri Grape and Wine Board is supporting this work to continue the project through the 2023 growing season. 304 3 B 2020

Review Publications
Arora, A.K., Sim, C., Severson, D.W., Kang, D.S. 2022. Random forest analysis of impact of abiotic factors on Culex pipiens and Culex quinquefasciatus occurrence. Frontiers in Ecology and Evolution. 9. Article 773360. https://doi.org/10.3389/fevo.2021.773360.
Dang, C., Zhang, Y., Sun, C., Li, R., Wang, F., Fang, Q., Yao, H., Stanley, D.W., Ye, G. 2021. dsRNAs targeted to the brown planthopper Nilaparvata lugens: assessing risk to a non-target, beneficial predator, Cytorhinus lividipennis. Journal of Agricultural and Food Chemistry. 70(1):373-380. https://doi.org/10.1021/acs.jafc.1c05487.
Zhang, Y., Jiang, L., Ahmad, S., Zhang, J., Stanley, D.W., Miao, H., Ge, L. 2021. The octopamine receptor, OA2B2, modulates stress resistance and reproduction in Nilaparvata lugens Stål (Hemiptera: Delphacidae). Insect Molecular Biology. 31(1):33-48. https://doi.org/10.1111/imb.12736.
Huynh, M.P., Pereira, A.E., Geisert, R.W., Vella, M., Coudron, T.A., Shelby, K., Hibbard, B.E. 2021. Characterization of thermal and time exposure to improve artificial diet for western corn rootworm larvae. Insects. 12(9). Article 783. https://doi.org/10.3390/insects12090783.
Wang, Y., Goodman, C.L., Ringbauer Jr, J.A., Li, Y., Stanley, D.W. 2021. Prostaglandin A2 induces apoptosis in three cell lines derived from the fall armyworm, Spodoptera frugiperda. Archives of Insect Biochemistry and Physiology. 108(3). Article 21844. https://doi.org/10.1002/arch.21844.
Goodman, C.L., Kang, D.S., Stanley, D.W. 2021. Cell line platforms support research into arthropod immunity. Insects. 12(8). Article 738. https://doi.org/10.3390/insects12080738.
Ahmad, S., Jiang, L., Zheng, S., Chen, Y., Zhang, J., Stanley, D.W., Miao, H., Ge, L. 2021. Silencing of a putative alanine aminotransferase (ALT) gene influences free amino acid composition in hemolymph and fecundity of the predatory bug, Cyrtorhinus lividipennis Reuter. Archives of Insect Biochemistry and Physiology. 108(2). Article e21836. https://doi.org/10.1002/arch.21836.
Paddock, K.J., Pereira, A.E., Finke, D.L., Ericsson, A.C., Hibbard, B.E., Shelby, K. 2021. Host resistance to bacillus thuringiensis is linked to altered bacterial community within a specialist insect herbivore. Molecular Ecology. 30(21):5438-5453. https://doi.org/10.1111/mec.15875.
Pereira, A.E., Huynh, M.P., Carlson, A.R., Haase, A., Kennedy, R.M., Shelby, K., Coudron, T.A., Hibbard, B.E. 2021. Assessing the single and combined toxicity of the bioinsecticide spear and cry3Bb1 protein against susceptible and resistant western corn rootworm larvae (coleoptera: chrysomelidae). Journal of Economic Entomology. 114(5):2220–2228. https://doi.org/10.1093/jee/toab160.
Yang, L., Qiu, L., Fang, Q., Stanley, D.W., Ye, G. 2020. Cellular and humoral immune interactions between Drosophila and its parasitoids. Insect Science. 28(5):1208-1227. https://doi.org/10.1111/1744-7917.12863.
Corcoran, J., Goodman, C.L., Saathoff, S.G., Ringbauer Jr, J.A., Guo, Y., Bonning, B., Stanley, D.W. 2021. Cell lines derived from the small hive beetle, Aethina tumida, express insecticide targets. In Vitro Cellular and Developmental Biology - Animal. 57:849-855. https://doi.org/10.1007/s11626-021-00633-y.
Yuvaraj, J., Jordan, M.D., Zhang, D., Andersson, M., Löfstedt, C., Newcomb, R.D., Corcoran, J. 2021. Sex pheromone receptors of the lightbrown apple moth, Epiphyas postvittana, support a second major pheromone receptor clade within the Lepidoptera. Journal of Insect Biochemistry and Molecular Biology. 141. Article 103708. https://doi.org/10.1016/j.ibmb.2021.103708.
Shi, J., Jin, H., Wang, F., Stanley, D.W., Fang, Q., Ye, G. 2022. The larval saliva of an endoparasitic wasp Pteromalus puparum supresses its host immune responses. Journal of Insect Physiology. 141. Article 104425. https://doi.org/10.1016/j.jinsphys.2022.104425.
Huynh, M.P., Hibbard, B.E., Ho, K., Shelby, K. 2022. Toxicometabolomic profiling of resistant and susceptible western corn rootworm larvae feeding on Bt maize seedlings. Scientific Reports. 12. Article 11639. https://doi.org/10.1038/s41598-022-15895-z.
Roy, M., Nam, K., Kim, J., Stanley, D.W., Kim, Y. 2021. Thromboxane mobilizes insect blood cells to infection foci. Frontiers in Immunology. 12:791319. https://doi.org/10.3389/fimmu.2021.791319.