Phenoloxidase-catalyzed melanization and Spӓtzle-induced synthesis of antimicrobial proteins are universal insect defense responses against pathogen or parasite infection. In mosquitoes such as Anopheles gambiae, melanotic encapsulation is effective against certain malaria parasites. Phenoloxidases are produced as inactive prophenoloxidases (proPOs) and, upon recognition of the invading organism, activated by a serine protease (SP) cascade. POs generate reactive compounds to kill pathogens. The SP system may activate proSpӓtzle to induce antimicrobial proteins that destroy the infectious agents. Some of the clip-domain SPs known as CLIPs may participate in the cleavage activation of TEP1, a complement-like factor vital for the mosquito anti-Plasmodium responses. Having successfully expressed >30 Manduca and Drosophila proCLIPs in insect cells, we will use the baculovirus system to produce 12 A. gambiae proCLIPs as wild-type and mutant proteins with FDNR substituting their original activation sites. We plan to activate the twelve mutants using Manduca sexta PAP3 (a FDNR-specific CLIP with >21 substrate proteins from insects) and then determine specificity of the resulting CLIPs by multiplex substrate profiling. Highly specific and sensitive substrates will be developed to examine the catalytic CLIPs and, in the next project, explore how melanization is linked to TEP1 activation and how some catalytic CLIPs are regulated by A. gambiae serpins. We will test 30 possible reactions of SP SP/SPH/PO/Spӓtzle. Based on the results, we plan to reconstitute a part of the SP-SPH system for proPO and proSpӓtzle-1 activation. Acquired functional data are to be validated by in vivo infection tests via collaboration with mosquito parasitologists in the future.