Location: Biological Control of Insects Research
Project Number: 5070-22000-038-000-D
Project Type: In-House Appropriated
Start Date: Aug 19, 2020
End Date: Aug 18, 2025
Objective:
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.
