The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs.

Serine protease cascades regulate important insect immune responses namely melanization and Toll pathway activation. An important component of these cascades are clip-domain serine protease homologs (cSPHs), which are non-catalytic, but essential for activating the enzyme prophenoloxidase (PPO) in the melanization response during septic infections. The activation of cSPHs requires their proteolytic cleavage, yet factors that control their activation and the complexity of their interactions within these cascades remain unclear. Here, we report the identification of CLIPA28 as a novel immune-related cSPH in the malaria vector Anopheles gambiae. Functional genetic analysis using RNA interference (RNAi) revealed that CLIPA28 is essential for the melanization of Plasmodium berghei parasites in refractory mosquitoes, and for mosquito resistance to fungal infections. We further show, using combined biochemical and genetic approaches, that CLIPA28 is member of a network of at least four cSPHs, whereby members are activated in a hierarchical manner following septic infections. Depletion of the complement-like protein TEP1 abolished the activation of this network after septic infections, whereas, depletion of the serine protease inhibitor 2 (SRPN2) triggered enhanced network activation, even in naïve mosquitoes, culminating in a dramatic reduction in cSPHs hemolymph levels, which paralleled that of PPO. Our data suggest that cSPHs are engaged in complex and multilayered interactions within serine protease cascades that regulate melanization, and identify TEP1 and SRPN2 as two master regulators of the cSPH network

The environment and species affect gut bacteria composition in laboratory co-cultured Anopheles gambiae and Aedes albopictus mosquitoes

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 mosquito-species 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 intra- and 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

The environment and species affect gut bacteria composition in laboratory co-cultured Anopheles gambiae and Aedes albopictus mosquitoes

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 mosquito-species 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 intra- and 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