The gut is a major route of entry in insects. However, our knowledge of insect gut function, most of which is primarily physiological, is inadequate. To enable us to better comprehend how insects adapt to and succeed in the environment, it is critical that we have a much better understanding of gut function, because the gut plays a critical role in maintaining insect homeostasis. The long-term objective of this research therefore is to establish the structure of proteins involved in ion regulation in insects. The specific objectives of this proposal are to deduce the primary structure of the vacuolar (V-type) ATPase subunits from the midgut and Malpighian tubules of the mosquito Aedes aegypti. The V-type ATPase(s) play an essential role in the maintenance of ion balance in insects, including mosquitoes. In addition these ATPase(s) also play an important role in excretion, and as a consequence, water balance. Once the subunits are characterized, their role in proton transport will be determined, and the spatial and temporal expression of the ATPase analyzed. Significant amino acid homology exists between V-type ATPase subunits from various sources. Utilizing this fact oligonucleotide primers will be made for each subunit, using the conserved domains as templates for primer design. The primers will then be used for the polymerase chain reaction (PCR) amplification of the putative subunit genes from cDNA prepared from midgut and Malpighian tubules of Ae. aegypti. These PCR products will be used for screening of cDNA libraries. However, if this strategy fails, alternative protocols, i.e., oligonucleotides based on the N-terminal sequence and/or antibodies raised against the subunits, will be used in screening the cDNA library for isolation of clones. Once the genes for these subunits are cloned, the spatial and temporal expression of the V-type ATPase will be determined using established methods. An understanding of mosquito gut function will facilitate our understanding of the mechanisms by which mosquitoes adapt to environmental conditions. Such knowledge may enable us to target the gut as a site for manipulating mosquito populations. The control of mosquitoes is critical for the disruption of many mosquito borne diseases of man.
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