At least two types of insect immune responses require activation of plasma proteins by proteases in hemolymph. One is the proteolytic processing of cytokine precursors, including the Toll ligand Sptzle. A second protease-mediated response is the activation of phenoloxidase zymogens, which then oxidize circulating diphenols to quinones, precursors for deposition of melanin on the surface of invading organisms. Such Innate immune responses are likely to affect the outcome of infections of insect vectors with the pathogens and parasites they transmit. Responses modulated by proteases in blood are not simply linear pathways, but instead are often protease webs, in which each protease may cleave multiple substrates, and active proteases may be regulated by several inhibitors. Previous work from our laboratory and others has established the lepidopteran insect, Manduca sexta, as a model system well suited for biochemical investigation of hemolymph proteases. The coming availability of a M. sexta genome sequence and new developments in proteomics technology make possible the investigation of serine protease webs in M. sexta hemolymph, using biochemical systems approaches. This research will be aimed at understanding hemolymph protease pathways, their potential cross-connections, and their regulation. The proteomics systems approach may reveal previously undiscovered protease-activated immune molecules of insects. The specific aims of the project are to: 1. Use Manduca sexta genome and transcriptome sequence data to identify and annotate genes encoding putative serine proteases and protease inhibitors and to produce reagents for further experimentation. 2. Use a systems approach for proteomics experiments to identify pathway components and the substrate repertoire of selected hemolymph proteases, followed by experiments to validate and characterize interactions using purified proteins. 3. Investigate the regulation of protease pathways in hemolymph by serpins, using in vitro, ex vivo, and in vivo experimental approaches. The long term goals of the research are to gain a thorough understanding of the regulation of protease cascades that mediate innate immune responses in Manduca, to apply this knowledge to insect vectors of human diseases, and to apply advantages of the Manduca system for fundamental studies on the regulation of serine protease activity.