The Effect of the Species and the Environment on Mosquito Gut Microbiome, and the Functional and Molecular Characterization of Clip Serine Proteases Regulating Mosquito Melanization

Abstract

Mosquitoes rely on both humoral and cellular innate immune responses to resist pathogens. The mosquito gut microbiome also plays an essential role in this regard, either directly through releasing toxic metabolites or indirectly through host immune priming. The environment is known to influence the mosquito gut microbiome composition; however, the relative contribution of the species and the environment in shaping the gut microbiome remains unclear. To address this aim, Anopheles gambiae and Aedes albopictus mosquitoes were co-reared over seven generations (or cohorts) at the larval stage to share the larval waters but remain physically separated by a porous net, and at the adult stages in a way that they share the same sugar food source. Midguts were dissected from selected adult mosquitoes in all these settings and extracted DNA was subjected to Miseq sequencing of the bacterial 16S rDNA to identify the species and environmental effect on gut microbial composition. Using both uni and multivariate analysis, the mosquito gut microbiome was shown to be influenced by species, cohort and food source (larval waters & adult sugars), and it appears to be as strongly affected by the host species as by food source. In the second aim of this thesis project, we characterized the molecular organization of candidate, catalytic CLIP domain serine proteases (CLIPs) that are major components of protease cascades that regulate key humoral immune responses including complement-like activity, Toll pathway activation and melanization. Combining RNAimediated gene silencing and western blotting, we show that the catalytic CLIPC9, CLIPB4 & CLIPB17 act upstream of CLIPB8 in the mosquito melanization response. Additionally, the thioester-containing protein 1 and the non-catalytic CLIP domain serine proteases SPCLIP1, CLIPA28 and CLIPA8, that play essential roles in the melanization response, function upstream of all catalytic CLIPs tested so far. We also show that CLIPB4 and CLIP17 regulate the activation of the prophenoloxidaseactivating protease (PAP) CLIPB10 independent of CLIPB8, indicating that downstream of CLIPB4 and CLIPB17, the cascade bifurcates into two branches; one converging on CLIPB8 and the other on CLIPB10. The functional significance of this bifurcation of the cascade is not clear yet but may suggest the activation of more than one PAP simultaneously leading to signal amplification and enhanced prophenoloxidase (PPO) activation.