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Broad Goals: To use an integrative approach involving genomics, biochemistry, physiology, toxicology and chemical ecology to identify and define target sites for innovative, environmentally-friendly termiticides. Target sites that receive major focus will be tied to caste differentiation and lignocellulose digestion. <ol> <LI>Caste differentiation target sites: a.Identify and characterize novel hormones and semiochemicals involved in termite caste differentiation. b.Identify suites of developmental genes that respond to active hormones and semiochemicals. c.Functionally characterize key developmental genes using RNA interference. d.Design and test novel control agents targeted to protein products encoded by highly influential caste regulatory genes. <LI> Target sites in the gut: a. Use genomic approaches to sequence from the termite gut transcriptome, and identify a large number of lignocellulose digestion/assimilation-related genes as candidate termiticide target sites. b. Use metagenomic approaches to sequence from the termite gut symbiont metagenome (prokaryotic and eukaryotic) and identify a large number of potential termiticide target sites. c. Use RNA interference and protein/enzyme biochemistry approaches to functionally characterize relevant genes from the termite gut and gut symbionts. d . Design and test prototype termiticides that are specifically targeted to protein products encoded by key lignocellulose digestion/ assimilation genes. </ol> Expected Outputs: This research is expected to reveal important genes involved in termite caste differentiation, lignocellulose digestion, and nutrition, as well as new strategies for termite pest management that involve disruption of chemical communication, caste homoestasis, and expression of developmental and nutrition-related genes. Pest management technologies resulting from this work will include environmentally-friendly, termite-specific chemicals and interfering RNAs that specifically silence termite genes.
Non-Technical Summary: This research will integrate bioassay, genomic, biochemistry, physiology, toxicology and chemical ecology approaches to define termite molecular physiology, and subsequently apply this information in designing novel pest management tools. The rationale that underlies this research is that, once the nature of highly specific aspects of termite caste differentiation and gut function are known, this information will significantly improve our understanding of termite development, endocrinology, symbiosis, and lignocellulose digestion/ assimilation. This information, in turn, is expected to lead to (i) a greater ability to target termites with highly specific control agents, (ii) an improved ability to protect structures and urban landscapes from damage by termites, and (iii) enhanced protection of natural and urban landscapes from damage caused by overuse of non-specific, highly toxic soil termiticides. For example, hundreds of thousands of gallons of liquid soil termiticides are applied pre-construction across the Southeastern US each year. The majority of these applications may be made un-necessarily, and could possibly be causing irreparable environmental damage. In this respect, this research has the potential to lead to more environmentally-benign alternatives for termite IPM. <P> Approach: OBJECTIVE 1a. The working hypothesis in this objective is that termite soldier head extracts work synergistically with JH to induce phenotypic caste differentiation, and that only a subset of extract components will have impacts on caste differentiation. This hypothesis will be tested by using a combination of model caste-differentiation bioassays, thin layer chromatography and gas chromatography - mass spectrometry analysis to fractionate and identify active soldier head extract components. OBJECTIVE 1b. This objective will be met using model bioassays to expose worker termites to active semiochemicals, followed by determination of gene expression levels in workers at precise time intervals during the bioassays. OBJECTIVE 1c. This hypothesis will be met by injecting double-stranded RNAs (dsRNAs) corresponding to specific gene sequences into worker termites. After injection, workers will be subjected to model caste differentiation bioassays for observation of developmental impacts. OBJECTIVE 1d. This objective will met using approaches that include model bioassays, gene expression characterization, and other biochemistry and physiological approaches to examine hormone titers, hormone binding, and relevant enzyme biochemistry. -------------------- OBJECTIVE 2a. This objective is based upon the working hypothesis that a R. flavipes gut-specific cDNA library will contain a robust sampling of termite-genome-derived genes that collaborate to accomplish lignocellulose digestion and assimilation. This objective will be accomplished using a high quality normalized cDNA library (already made) representing symbiont-free termite gut tissues, then by sequencing several thousand expressed sequence tags (ESTs) from this library. OBJECTIVE 2b. Justification for the approach taken in this objective is that (i) the termite hindgut fermentation chamber contains numerous species of unculturable bacteria and protozoa that interact in lignocellulose processing, and (ii) decades of one-dimensional biochemistry research has yielded few leads on collaborative lignocellulose processing in termites. New pyrosequencing technology available at the UF-ICBR will be used. OBJECTIVE 2c. The goal of this sub-objective is to functionally characterize relevant genes identified under Objectives 2a and 2b that encode potentially vulnerable target sites for novel termiticides. The hypothesis to be tested under this objective is that relevant target genes will play critical and quantifiable roles in termite digestion and nutrient assimilation. OBJECTIVE 2d. Using information from Objective 2c, the goal of this objective is to design and test candidate materials as novel termiticides. Our hypothesis here is that prototype toxins that are targeted to the most vulnerable target sites will cause pronounced disruptions in termite feeding and/or survival.