Expression Pattern and RNAi Inhibition of Putative Sodium‐coupled Cation‐chloride Cotransporters in the Mosquito Aedes aegypti

Abstract

The mosquito Aedes aegypti possesses three genes with sequence similarity to sodium-coupled cation-chloride cotransporters (CCCs). We began to characterize these genes through phylogenetic analysis, assessment of tissue distribution, and preliminary RNAi experiments. The protein predicted by VectorBase AAEL006180 (tentatively named aeCCC1) groups with Drosophila melanogaster Na-K-Cl cotransporter dmNCC69 (CG4357) and mammalian Na-K-Cl and Na-Cl cotransporters, whereas proteins predicted by AAEL009888 (aeCCC2) and AAEL009886 (aeCCC3) group with Manduca sexta msBSC and Drosophila CG31457. We evaluated expression of aeCCC1, aeCCC2, and aeCCC3 in tissues of Ae. aegypti larvae and adults. For aeCCC1 and aeCCC2, expression was similar between adults and larvae. In contrast, aeCCC3 was ~500-fold more abundant in larvae compared to adults. In adult female mosquitoes, aeCCC1 was most highly expressed in head and Malpighian tubules whereas aeCCC2 was most highly expressed in hindgut. In larvae, aeCCC3 was 6000-fold more abundant in anal papillae compared to Malpighian tubules. To begin assessing aeCCC1 function, we used RNAi to knock down its expression. We generated two ~500 bp dsRNAs, injected them into adult female mosquitoes, and assessed aeCCC mRNA expression 4 days after injection. Both dsRNAs reduced aeCCC1 expression compared to control mosquitoes injected with PBS. Changes in aeCCC2 and aeCCC3 expression were also detected, suggesting either direct or indirect off-target effects. Our findings suggest that the Aedes aegypti CCCs may play distinct roles in mosquito osmoregulation.