An anti-diuretic hormone receptor in the human disease vector,Aedes aegypti: identification, expression analysis and functional deorphanization

Abstract

Insect CAPA neuropeptides, which are homologs of mammalian neuromedin U, have been described in various insect species and are known to influence ion and water balance by regulating the activity of the Malpighian ‘renal’ tubules (MTs). A number of diuretic hormones have been shown to increase primary fluid and ion secretion by the insect MTs and, in the adult female mosquito, a calcitonin-related peptide (DH31) also known as mosquito natriuretic peptide, increases sodium secretion at the expense of potassium to remove the excess salt load acquired upon blood-feeding. An endogenous mosquito anti-diuretic hormone was recently described, having inhibitory activity against select diuretic factors and being particularly potent against DH31-stimulated diuresis. In the present study, we have functionally deorphanized, both in vitro and in vivo, a mosquito anti-diuretic hormone receptor (AedaeADHr). Expression analysis by quantitative PCR indicates the receptor is highly enriched in the MTs, and fluorescent in situ hybridization confirms expression within principal cells. Characterization using a heterologous system demonstrated the receptor was highly sensitive to mosquito CAPA peptides. In adult females, AedaeADHr transcript knockdown using RNAi led to the abolishment of CAPA-peptide induced anti-diuretic control of DH31-stimulated MTs. The neuropeptidergic ligand is produced within a pair of neurosecretory cells in each of the six abdominal ganglia, whose axonal projections innervate the abdominal neurohaemal organs (known as the perivisceral organs), where these neurohormones are released into the open circulatory system of the insect. Furthermore, pharmacological inhibition of PKG/NOS signalling abolished the anti-diuretic activity of AedaeCAPA-1, which collectively confirms the role of cGMP/PKG/NOS in this anti-diuretic signalling pathway.SignificanceInsects are by far the most successful and abundant group of organisms on earth. As a result of their small size, insects have a relatively large surface area to volume ratio, raising the potential for rapid gain or loss of water, ions and other molecules including toxins – a phenomenon that applies to insects living in both aquatic and terrestrial environments. In common with many other organisms, hormones are key regulators of the excretory system in insects, and numerous factors control the clearance of excess water and ions (i.e. diuretics) or retention of these elements (i.e. anti-diuretics). Here we characterized an endogenous anti-diuretic hormone receptor in the human disease vector, Aedes aegypti, demonstrating its expression is highly enriched in the Malpighian ‘renal’ tubules and is necessary for eliciting anti-diuretic control of this key component of the mosquito excretory system.

Another fly diuretic hormone: tachykinins increase fluid and ion transport by adultDrosophila melanogasterMalpighian ‘renal’ tubules

“…Standard curves were obtai ned using serial dilutions of the MTs cDNA templ ate from female w 1118 yieldi ng primer efficiencies ranging from 90-103%. For each replicate, the ∆∆Ct method was used to determine the relative quantification (RQ) value, which was normalized to the geometri c mean of RpL32 and alpha-tubulin reference genes [67].…”