Prostaglandins and related compounds, collectively know as eicosanoids, are crucial mediators of hemocytic immune responses to bacterial infections in larvae of the tobacco hornworm Manduca sexta, and likely in all insects. Virtually nothing is known about the biochemistry of eicosanoids in insect hemocytes. The overall goal of the proposed research is to investigate eicosanoid metabolism in hemocytes of M sexta as a first step toward gaining an understanding of the physiological roles of eicosanoids in insect immunology. The research in this proposal is designed to answer basic questions: 1-How do eicosanoid precursor fatty acids become free for eicosanoid biosynthesis? 2-Which eicosanoid species are biosynthesized by hemocytes? 3-Do pharmaceutical eicosanoid biosynthesis inhibitors have the same actions in mammals and in insect hemocytes? 1-In mammals, phospholipase A2 is thought to regulate eicosanoid biosynthesis by modulating the availability of free eicosanoid-precursor fatty acids. Radioactive substrates will be used to characterize the activity of this enzyme. 2-Hemocyte microsomes will be incubated with radioactive eicosanoid-precursor fatty acids, and eicosanoid biosynthesis will be monitored by radio-chromatography. 3-Eicosanoid biosynthesis inhibitors known from mammals will be characterized by adding selected inhibitors to preparations described in step 3. Actions in larvae will be monitored by injecting the inhibitors into larvae, then determining hemolymph eicosanoid titres by radioimmunoassay. The information gained in these studies in a model insect will provide compelling insights into the mechanisms of mediating cellular immune responses in hematophagous insects of medical importance. This work has implications for developing novel pest control strategies based on compromising cellular immunity.
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