Regulation of the gut-specific carboxypeptidase: A study using the binary Gal4/UAS system in the mosquito Aedes aegypti

• We generated CP-Gal4 Aedes aegypti mosquitoes with a midgut-specific expression.
• The CP-Gal4 drives expression in the female mosquito midgut after blood feeding.
• Insulin and Target-of-Rapamycin regulate the carboxypeptidase CP gene.
• RNA interference silencing of the insulin receptor has confirmed this finding.

Pathogen transmission by mosquitoes is tightly linked to blood feeding which, in turn, is required for egg development. Studies of these processes would greatly benefit from genetic methods, such as the binary Gal4/UAS system. The latter has been well established for model organisms, but its availability is limited for mosquitoes. The objective of this study was to develop the blood-meal-activated, gut-specific Gal4/UAS system for the yellow-fever mosquito Aedes aegypti and utilize it to investigate the regulation of gut-specific gene expression. A 1.1-kb, 5′ upstream region of the carboxypeptidase A (CP) gene was used to genetically engineer the CP-Gal4 driver mosquito line. The CP-Gal4 specifically activated the Enhanced Green Fluorescent Protein (EGFP) reporter only after blood feeding in the gut of the CP-Gal4 > UAS-EGFP female Ae. aegypti. We used this system to study the regulation of CP gene expression. In vitro treatments with either amino acids (AAs) or insulin stimulated expression of the CP-Gal4 > UAS-EGFP transgene; no effect was observed with 20-hydroxyecdysone (20E) treatments. The transgene activation by AAs and insulin was blocked by rapamycin, the inhibitor of the Target-of-Rapamycin (TOR) kinase. RNA interference (RNAi) silence of the insulin receptor (IR) reduced the expression of the CP-Gal4 > UAS-EGFP transgene. Thus, in vitro and in vivo experiments have revealed that insulin and TOR pathways control expression of the digestive enzyme CP. In contrast, 20E, the major regulator of post-blood-meal vitellogenic events in female mosquitoes, has no role in regulating the expression of this gene. This novel CP-Gal4/UAS system permits functional testing of midgut-specific genes that are involved in blood digestion and interaction with pathogens in Ae. aegypti mosquitoes.

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