Mosquitoes stand out as the most important group of vector insects due to the number of different disease agents they transmit and the magnitude of health problems these diseases cause. Owing to the resurgence of mosquito-borne disease worldwide, new methods for mosquito control based upon sound ecological understanding of mosquito biology is needed. Larval mosquitoes present a tantalizing target for such methods. The nature of microbial communities in larval mosquito habitats is a prime element in understanding how mosquito larvae exploit natural food, grow, and develop into adults. A long term objective of our proposed research is to understand the role that microorganisms play as food items and potential control agents for mosquito larvae. An implicit aim of our proposed research is to expand characterization of the bacterial community in larval mosquito habitats. Successes in cloning larvicidal toxin genes in cyanobacteria and other hosts suggest that this method may provide a method of sustained larval control. We have documented that gram negative bacteria of diverse general are found in larval habitats. Broad host- range plasmids are now available for DNA transfer into gram-negative bacteria. Thus, another research objective is to select bacteria from larval habitats as recombinant candidates for DNA transfer and gene expression of the cryIVD gene of Bacillus thuringiensis serovar. Israelensis (coding for a 72kDa larvicidal toxin). We will initially insert the plasmid-borne gene into a lab-strain pseudomonas putida, and then expand this effort to include selected environmental gram-negative bacterial stains which we have already isolated and characterized from mosquito habitats. Finally, we aim to introduce recombinants into laboratory microcosm habitats and examine the persistence of these organisms by utilizing drug resistance markers and PCR-based detection of the cryD gene fragment.
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