This proposal addresses the immune response of mosquitoes to filarial worms, and is a competitive renewal for years 18-22. The proposal specifically addresses melanotic encapsulation as a response of mosquitoes to filarial worms, which occurs with Armigeres subalbatus to Brugia malayi, while a similar response also occurs with a selected strain of Anopheles gambiae against malaria parasites. Because melanotic encapsulation appears to have a significant influence on vector competence, the PI plans to investigate several enzymes that participate in the melanin biosynthetic pathway. The hypothesis to be tested is that the enzymes phenylalanine hydroxylase (PAH), prophenoloxidase (proPO), dopa decarboxylase (DDC) and dopachrome conversion enzyme (DCE) influence the capacity of mosquitoes to effectively resist infection with parasites. The PI proposes 1: to obtain clones for these 4 enzymes from Aedes aegypti and Ar. subalbatus (some of which are already available); 2, use these clones to assess spatial and temporal transcription and generate recombinant proteins for antibody production, which will be used to examine spatial distribution of corresponding protein. These studies will use in situ hybridization, real-time quantitative PCR, western blotting, and light and EM-antibody staining. Third, enzyme activity assays will be used to correlate activity profiles with transcriptional activity, and to evaluate the biochemical behavior of these enzymes under varying conditions. Finally, the PI will engineer double subgenomic Sindbis virus to express antisense RNA for these genes for use in knockout strategies in Ar. subalbatus. In aggregate, these experiments are expected to provide a comparative perspective on the melanization process in a non-melanizing mosquito (Ae. aegypti) and in the well-characterized, melanizing Ar. subalbatus. These experiments will provide a better understanding of the role of each enzyme in parasite melanization, contribute to an understanding of vector competence for filarial worms, and contribute to an understanding of the overall epidemiology of mosquito-borne disease. In addition, these studies will provide new information on the participation of these enzymes in mosquito immunity.
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