The midgut microbiota of disease vectors plays a critical role in the successful transmission of human pathogens. The environment influences the microbiota composition; however, the relative mosquitospecies contribution has not been rigorously disentangled from the environmental contribution to the microbiota structure. Also, the extent to which the microbiota of the adult sugar food source and larval water can predict that of the adult midgut and vice versa is not fully understood. To address these relationships, larvae and adults of Anopheles gambiae and Aedes albopictus were either reared separately or in a co-rearing system, whereby aquatic and adult stages of both species shared the larval water and sugar food source, respectively. Despite being reared under identical conditions, clear intraand interspecies differences in midgut microbiota-composition were observed across seven cohorts, collected at different time points over a period of eight months. Fitting a linear model separately for each OTU in the mosquito midgut showed that two OTUs significantly differed between the midguts of the two mosquito species. We also show an effect for the sugar food source and larval water on the adult midgut microbiota. Our findings suggest that the mosquito midgut microbiota is highly dynamic and controlled by multiple factors.

Humoral immune responses in animals are often tightly controlled by regulated proteolysis. This proteolysis is exerted by extracellular protease cascades, whose activation culminates in the proteolytic cleavage of key immune proteins and enzymes. A model for such immune system regulation is the melanization reaction in insects, where the activation of prophenoxidase (proPO) leads to the rapid formation of eumelanin on the surface of foreign entities such as parasites, bacteria and fungi. ProPO activation is tightly regulated by a network of so-called clip domain serine proteases, their proteolytically inactive homologs, and their serpin inhibitors. In Anopheles gambiae, the major malaria vector in sub-Saharan Africa, manipulation of this protease network affects resistance to a wide range of microorganisms, as well as host survival. However, thus far, our understanding of the molecular make-up and regulation of the protease network in mosquitoes is limited. Here, we report the function of the clip domain serine protease CLIPB10 in this network, using a combination of genetic and biochemical assays. CLIPB10 knockdown partially reversed melanotic tumor formation induced by Serpin 2 silencing in the absence of infection. CLIPB10 was also partially required for the melanization of ookinete stages of the rodent malaria parasite Plasmodium berghei in a refractory mosquito genetic background. Recombinant serpin 2 protein, a key inhibitor of the proPO activation cascade in An. gambiae, formed a SDS-stable protein complex with activated recombinant CLIPB10, and efficiently inhibited CLIPB10 activity in vitro at a stoichiometry of 1.89:1. Recombinant activated CLIPB10 increased PO activity in Manduca sexta hemolymph ex vivo, and directly activated purified M. sexta proPO in vitro. Taken together, these data identify CLIPB10 as the second protease with prophenoloxidase-activating function in An. gambiae, in addition to the previously described CLIPB9, suggesting functional redundancy in the protease network that controls melanization. In addition, our data suggest that tissue melanization and humoral melanization of parasites are at least partially mediated by the same proteases.

Serine protease cascades regulate important physiological processes during infection, from complement activation and coagulation in mammals to melanization and Toll pathway activation in insects. Key components of these cascades in insects are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and the non-catalytic, clip domain serine protease homologs (cSPHs), both of which are essential for the proper functioning of these cascades. In the context of the melanization immune response of insects, CLIPs cascades converge on the activation cleavage of the zymogen prophenoloxidase to active phenoloxidase that initiates melanin biogenesis on microbial surfaces. While we have previously provided insight into the structural hierarchy of cSPHs in these cascades in the mosquito vector of malaria Anopheles gambiae, the hierarchy of cSPs as well as their functional interactions with cSPHs remain largely unknown. Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family that are key factors in the melanization response and characterize their relative contributions to the anti-bacterial melanization response and to mosquito susceptibility to bacterial infections. Based on in vivo and in vitro assays that gauge the activation cleavage profiles of candidate CLIPs, we also propose that bidirectional interactions between cSPs and cSPHs regulate signal amplification and propagation in these cascades.