Cloning, immunolocalization and functional analysis of calcitonin receptor–like receptor 1 (AaegGPRCAL1; Diuretic Hormone 31 (DH31) receptor) in females of mosquito Aedes aegypti (Diptera: Culicidae)

In mosquitoes, the calcitonin-like diuretic hormone 31 (DH31), known as mosquito natriuretic peptide (MNP), increases sodium and fluid secretion. Although there are three predicted calcitonin receptor-like receptors (GPRCAL1-GPCRCAL3) in the Aedes aegypti genome, the specific contribution of each receptor to rapid fluid excretion in response to DH31 is unknown in female mosquitoes. Thus, we cloned the full length cDNA encoding AaegGPRCAL1, analyzed receptor expression in the M. tubules of non-blood fed females and investigated the role of AaegGPRCAL1 in fluid excretion by RNA interference. Analyses of RNAi effectiveness were performed by qPCR and immunohistochemistry. The RNAi phenotype was evaluated by secretion Ramsay assays with M. tubules in vitro, and by in vivo measurements of postprandial fluid excretion from females enclosed in a humidity chamber. The receptor was immunolocalized in principal cells of M. tubules. The M. tubules from females that underwent knock-down of AaegGPRCAL1 exhibited lower rate of fluid secretion in vitro, and in vivo, silenced females exhibited reduction in the fluid excreted as compared to those of the controls. These results are in agreement with the observed reduction of receptor signal in the principal cells of dsRNA injected females. To our knowledge, this is first evidence of an RNAi-induced phenotype allowing quantification of the contribution of single family B GPCR to water homeostasis in any invertebrate.