No abstract

Yellow fever mosquitoes, Aedes aegypti, face diverse salt and water challenges. Larvae lose salt to the freshwater environment and gain water osmotically, whereas adults tend to lose water by evaporation. Moreover, adult females take in large quantities of salt and water during a blood meal. We hypothesize that sodium‐dependent cation‐chloride cotransporters (CCCs) contribute to the salt and water balance of Aedes aegypti by participating in epithelial salt secretion and absorption. Sequence analysis revealed three genes (AAEL006180, AAEL009888, and AAEL009886) in Aedes aegypti with similarity to vertebrate Na‐K‐Cl cotransporters. We performed multiple sequence alignment and phylogenetic analysis of the proteins encoded by the Aedes aegypti genes and orthologs in Culex and Anopheles mosquitoes, Drosophila, and other arthropods. The protein encoded by AAEL006180 groups in a clade with the human secretory Na‐K‐Cl cotransporter and a Drosophila cotransporter known to be involved in salt secretion. In contrast, AAEL009888 and AAEL009886 fall into a clade that includes sequences from other insects but not vertebrates. Moreover, both AAEL009888 and AAEL009886 have orthologs in other mosquito genera but other insects only have one ortholog, indicating that AAEL009888 and AAEL009886 probably diverged early in the evolution of mosquitoes. Amino acids that differ among the paralogs include residues known to influence ion and inhibitor binding affinities. Using qPCR, we quantified the relative gene expression of the Aedes aegypti paralogs with at least two isoform‐specific primer sets for each gene. AAEL009886 transcript is expressed at significantly greater levels in larvae compared to adults and at significantly greater levels in anal papillae compared to Malpighian tubules, suggesting that AAEL009886 may be specialized for ion absorption in larvae. In contrast, AAEL006180 and AAEL009888 transcripts are expressed at approximately the same levels in adults and larvae. To evaluate the physiological role of the proteins encoded by AAEL006180 and AAEL009888, we attempted to reduce expression of each transporter using dsRNA. Larvae exposed to dsRNA corresponding to either transporter had up to five‐fold increases in hemolymph ammonium levels compared to negative controls as well as smaller changes in other hemolymph cations. Our results point towards physiological roles for AAEL006180 and AAEL009888 in osmoregulation and ammonia balance of larval mosquitoes.Support or Funding InformationNSF‐IOS‐1557230