Project Summary Extracellular serine proteases and their non-catalytic homologs coordinate key defense mechanisms in insects. This includes responses of mosquitoes against pathogens and parasites that cause serious human diseases. Proteolytic cleavage generates active phenoloxidases (POs), thioester proteins (TEPs), Spätzle and other cytokines. PO catalyzes the production of reactive chemicals to sequester and kill the invading organisms. TEPs opsonize parasites, targeting them for destruction. Spätzle and other cytokines trigger intracellular signaling pathways to induce the expression of antimicrobial peptides and other defense proteins. In insect vectors of human diseases, the protease networks may be evaded or disrupted by proteins from the intruders. Knowledge of the system components and their interactions from biochemical model insects such as Manduca sexta is useful for gaining detailed molecular understanding of fundamental aspects of insect innate immunity, including these protease cascades and provides basic knowledge that can guide studies of similar systems in insect disease vectors. We have elucidated a part of the M. sexta protease network that activates proPO in response to bacteria and fungi. In this network, recognition proteins bind to microbes and activate the proteases in a cascade mode. We have annotated 193 genes encoding serine proteases and their homologs, identified 36 of the proteins in larval hemolymph, and acquired data indicating that some of them play critical roles in immune signal transduction. Based on the molecular probes, purified proteins and working experience, we propose to investigate two critical steps of the protease network, which are conserved in dipteran species, by combining hemolymph fractionation, recombinant protein production and processing, sequence and expression information, and state-of-the-art mass spectrometry. The specific aims of this project are to: 1) characterize the system initiation by examining interactions of peptidoglycans, two recognition proteins, and hemolymph protease-14 precursor (proHP14) at the domain level; 2) identify 1−2 proHP6-activating proteases and elucidate their activation mechanisms. New knowledge gained in this project will improve understanding of insect immunity at a biochemical level and stimulate related research in vector species.