Biochemical and physiological characterization of midgut chymotrypsin.

The mosquito is a vector of dengue, Zika, and chikungunya. The mosquito's reliance on blood facilitates the transmission of these viral pathogens to humans. Digestion of blood proteins depends on the biphasic expression of serine proteases, with trypsin-like activity contributing to most of the activity in the midgut. Other proteases found (serine collagenase- and chymotrypsin-like) are thought to contribute to digestion, but their roles are largely understudied. Thus, elucidating the activity and specific roles of all midgut proteases will help understand the complexity of the digestion process and help validate them as potential targets for the development of a new vector control strategy. Herein, we focused on characterizing the activity profile and role of chymotrypsin (AaCHYMO). Knockdown studies resulted in elimination and significant reduction of chymotrypsin-like activity in blood fed midgut extracts, while fluorescent and blood protein digestion assays revealed important substrate specificity differences. Interestingly, knockdown of AaCHYMO did not impact fecundity, indicating the presence of an intricate network of proteases working collectively to degrade blood proteins. Further, knockdown of the ecdysone receptor (EcR) led to a decrease in overall AaCHYMO expression and activity in the mosquito, which may play an important regulatory role.