Identification and functional analysis of <i>CLIPA</i> and <i>CLIPE</i> in the innate immune response of <i>Aedes aegypti</i>

Serine protease cascades play a pivotal role in the innate defense of mosquitoes. These cascades consist of catalytic serine proteases and their non-catalytic homologs, collectively termed CLIPs, which are classified into five subfamilies. While the catalytically active CLIPB/Cs have been extensively studied, the biochemical functions of the CLIPA and CLIPE remain less explored. In vivo studies have demonstrated that CLIPAs in Anopheles gambiae are crucial for defense against a range of pathogens. At the functional level, however, some Anopheles CLIPA homologs exhibit properties that notably diverge from those of their counterparts in Aedes aegypti. Consequently, the functional landscape of CLIPA and CLIPE in Ae. aegypti remains largely uncharted. Our systematic investigation yielded several key findings. We first identified the CLIPA and CLIPE subfamily members in Ae. aegypti and characterized their distinct domain architectures. Subsequent screening revealed specific CLIPA/E members that are transcriptionally responsive to infection by S. aureus and E. coli. Functional RNAi assays demonstrated that CLIPA4, CLIPA5, CLIPA11, CLIPA14, CLIPE8, and CLIPE11 are critical for antibacterial defense, significantly impacting mosquito survival upon bacterial challenge. Mechanistic studies further showed that CLIPA4 and CLIPE8 regulate the expression of transcription factors Rel1 (Toll pathway) and Rel2 (IMD pathway), consequently altering the expression profiles of key antimicrobial peptides. Additionally, these two genes were found to be essential for maintaining normal phenoloxidase activity in the hemolymph, indicating their role in modulating the melanization response. Collectively, our RNAi-based approach establishes that specific CLIPA/E subfamily members are crucial components of the antibacterial innate immune response in Ae. aegypti, functioning through the regulation of humoral immune pathways.