Dissecting The Melanization Immune Response In The Malaria Vector Anopheles Gambiae

Melanization is among the first lines of defense in arthropods following injury or infection. Manipulating mosquito melanization can influence vector competence for Plasmodium, compromise antifungal defense, and reduce longevity. Phenoloxidase (PO), the rate limiting melanization enzyme, is activated downstream of hierarchical protease cascades containing CLIP-domain serine proteases (CLIP-SPs) from subfamilies B and C and serine protease homologs (CLIP-SPHs) from subfamily A. The identification and characterization of melanization cascade components in mosquitoes is difficult due to their small size and hemolymph volume. Consequently, our understanding of the An. gambiae melanization cascade is incomplete. In this thesis, I developed a screening method that can identify positive and negative regulators of melanization with fewer mosquitoes and less experimental effort than traditional biochemical and imaging-based approaches. The screen, known as the melanization-associated spot assay (MelASA), is based on the quantification of mosquito melanotic excreta, which is a proxy for PO activity. This straightforward assay facilitated the discovery that the serine protease CLIPC9 is required for microbial melanization and an enhanced dsRNA injection site melanization response in CTL4/CTLMA2 knockdown mosquitoes. CLIPC9 likely functions downstream of mosquito complement. Moreover, CLIPC9 activation and localization to bacteria is regulated by members of the CLIPA subfamily. Traditionally viewed as ‘co-factors’ for the prophenoloxidase activating CLIPBs, this work demonstrates that CLIPAs can also control the activation of the catalytic proteases driving melanization. This thesis identifies CLIPC9 as a new player in the melanization hierarchy and provides the field with an efficient approach for dissecting the complex protease cascades controlling this important immune response.