MOLECULAR STUDY OF INSECT VECTOR ANTIHEMOSTATIC FACTORS

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In the course of feeding from vertebrates, blood feeding insects encounter components of their host's hemostatic responses, especially vasoconstriction and formation of a platelet plug. Consequently, hematophagous insects have salivary components with platelet antiaggregating activity (apyrase), and many produce salivary vasodilators. These components may also influence the course of parasite establishment by modifying the host immune response at the feeding site. Despite their importance, little is known of the molecular biology of these substances. I propose to compare, at the molecular level, apyrase and vasodilatory agents in two insects that have independently evolved blood feeding, and are significant disease vectors: the yellow fever mosquito Aedes aegypti and the triatomine bug Rhodnius prolixus. Specifically, the project involves: (1) isolating and sequencing cDNA clones for the preprotachykinin, which is processed to release vasodilatory tachykinins in Ae. aegypti. (2) isolating and sequencing cDNA clones for three NO.binding heme proteins, which function as vasodilators in A. prolixus. (3) isolating and sequencing cDNA clones for the Rhodnius apyrase, for comparison with the Aedes apyrase; (4) using these clones as probes to study regulation of the apyrase and vasodilators in both insects; (5) using the heme protein clones to produce recombinant wild-type and site-specific mutant proteins, to study the structure/function relationships and biophysics of reversible NO binding; and (6) isolating and sequencing genomic clones which include the Rhodnius heme protein genes, to study gene organization and regulation of this functionally unique group of proteins. Ultimately, this research will permit a more detailed knowledge of the biochemical and pharmacological events associated with the earliest stages of parasite transmission by blood-feeding insects. Comparison of two species, which have independently evolved blood feeding, will point to congruencies which may be common to a variety of vector insects.