: Since the inception of this project, our global hypothesis has been that microbial community dynamics in larval mosquito habitats govern larval growth and consequent adult emergence, and through direct and indirect influences of larval nutrition affect mosquito population dynamics and vectorial capacity. By understanding the controlling factors imposed by the microbial community in support or suppression of larval growth, new insights into mosquito ecology and vector control methods will be elucidated, including means of manipulating the microbial community with genetic transformation methods. In this competing continuation proposal, we will build directly upon our recent research advances with newly-framed hypotheses and new directions that involve 3 inter-related Specific Aims covering the following objectives: (1.1) Development of an axenic (germ-free) experimental system for direct analysis of the effects of individual microbial components to larval growth responses, using stable isotope tracers in field experiments and radio-isotope tracers in laboratory experiments; (1.2) Analysis of the contribution of extracellular polysaccharides associated with microbial biofilms, as well as dissolved and colloidal organic compounds, to larval nutrition and growth; (1 .3) Estimation of assimilation efficiencies of natural microbial foods; (2.1) Molecular-based characterizations of the biofilm-associated microbial community using 16S rDNA sequence libraries, and sole-carbon source utilization phenotype analysis; (2.2) Characterization of the fungat and protozoan community and biomass associated with substrate biofllms; (2.3) Analysis of the role of phosphorous limitation to microbial biomass production and mosquito growth; (3.1) Isolation on selective media of dominant Flavobacterium, Comamonas, and Pseudomonas spinosa strains; (3.2) Transformation of these bacterial strains with Cryl 1A larvicidal toxin genes by chromosomal integration using mini-TN5 transposons; and (3.3) Studies of transformant efficacy against mosquito larvae, and persistence in simulated mosquito habitats.
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