The yellow fever mosquito Aedes aegypti is the major vector of arboviruses, causing numerous devastating human diseases, such as dengue and yellow fevers, Chikungunya and Zika. Female mosquitoes need vertebrate blood for egg development, and repeated cycles of blood feeding are tightly linked to pathogen transmission. The mosquito’s posterior midgut (gut) is involved in blood digestion and also serves as an entry point for pathogens. Thus, the mosquito gut is an important tissue to investigate. The miRNA aae-miR-275 (miR-275) has been shown to be required for normal blood digestion in the female mosquito; however, the mechanism of its action has remained unknown. Here, we demonstrate that miR-275 directly targets and positively regulates sarco/endoplasmic reticulum Ca 2+ adenosine triphosphatase , which is implicated in active transport of Ca 2+ from the cytosol to the sarco/endoplasmic reticulum. We utilized a combination of the gut-specific yeast transcription activator protein Gal4/upstream activating sequence (Gal4/UAS) system and miRNA Tough Decoy technology to deplete the endogenous level of miR-275 in guts of transgenic mosquitoes. This gut-specific reduction of miR-275 post blood meal decreased SERCA mRNA and protein levels of the digestive enzyme late trypsin. It also resulted in a significant reduction of gut microbiota. Moreover, the decrease of miR-275 and SERCA correlated with defects in the Notch signaling pathway and assembly of the gut actin cytoskeleton. The adverse phenotypes caused by miR-275 silencing were rescued by injections of miR-275 mimic. Thus, we have discovered that miR-275 directly targets SERCA , and the maintenance of its level is critical for multiple gut functions in mosquitoes. Author summary Female mosquitoes transmit numerous devastating human diseases. The mosquito gut, in addition to its primary function as a site of blood digestion, represents the entry point for pathogen colonization in mosquito vectors. The conserved microRNA, miR-275, was shown to be required for blood digestion and egg development. In this study, we investigated the target of miR-275 contributing to the regulation of mosquito gut functions. We achieved spatiotemporal suppression of miR-275 using a transgenic Tough Decoy RNA approach in the A . aegypti female mosquito gut. Furthermore, we have uncovered that miR-275 targets sarco/endoplasmic reticulum Ca 2+ – adenosine triphosphatase (SERCA) , affecting numerous gut functions including blood digestion, production of digestive proteases, and assembly of the gut actin cytoskeleton. SERCA is essential for maintenance of Ca 2+ homeostasis, and its disturbance, in humans, leads to cardiac hypertrophy, heart failure and cancers. Therefore, the finding that the miRNA miR-275 targets SERCA not only contributes to the knowledge of mosquito gut regulation but also significantly adds to the general understanding of mechanisms governing this critical molecule.
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