The objective of the proposed research is to develop more effective and environmentally safe bacteria for controlling the mosquito vectors of major human diseases including malaria, filariasis, dengue, and the viral encephalitides. These bacteria will be much more cost-effective than Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs), the two species currently used in vector control, and will be much less prone to induce mosquito resistance. To support sustainable use of bacterial larvicides in vector control, recombinant DNA technology will be used to create novel combinations of insecticidal proteins in individual strains. These will be evaluated for efficacy and resistance management properties aimed at controlling species belonging to the most important vector genera, namely, Anopheles, Aedes, and Culex. Development and use of these new bacteria will be enhanced by completion of studies focusing on improving knowledge of mechanisms underlying the synergism responsible for the high toxicity and capacity of the Cyt1A protein to delay resistance to bacterial endotoxins in vector populations. These objectives will be achieved through a comprehensive research program consisting of the following three specific aims: (1) Complete construction of recombinant bacteria with emphasis on Bacillus sphaericus as a host cell and increase knowledge of Bti and Bs parasporal assembly, (2) Assess the resistance management properties of the best Bti and Bs recombinants, and (3) Complete studies of the general mechanism by which Cyt1A synergizes endotoxins and delays resistance. New expression and chromosome-integration strategies will be used to construct Bs recombinants, and non-endotoxin genes involved in parasporal body assembly will be identified primarily by making bacterial mutants and gene knockouts. Resistant management properties and their underlying Mendelian basis will be evaluated through laboratory selections and genetic crosses. Studies of Cyt1A's mechanism of action will employ a variety of genetic and histological techniques. Bacterial larvicides developed through our studies should improve vector control and disease reduction, with concomitant health benefits accruing from reduced use of synthetic chemical insecticides. Moreover, insecticidal protein combinations identified to optimize resistance management will provide models for possibly engineering field populations of bacteria for vector control, and may also prove useful for resistance management in Bt-transgenic crops.
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