CAPA Neuropeptides Inhibit VA Activity in Malpighian Tubules of the Mosquito, Aedes Aegypti

Abstract

Abstract Haematophagus insects, such as the female Aedes aegypti mosquito, face the challenge of excess ion and water intake after engorgement on a blood meal. To cope with this, adult female A. aegypti have a specialized excretory system that includes the Malpighian tubules (MTs), which are under rigorous control by several neuroendocrine factors to regulate transepithelial movement of ions/water. Produced in the CNS, the mosquito anti-diuretic hormone is a member of the CAPA peptide family, which share homology to the vertebrate neuromedin U peptide. CAPA peptides inhibit fluid secretion of MTs stimulated by select diuretic factors, 5HT and DH31 through the NOS/cGMP/PKG pathway. However, the anti-diuretic signalling mechanism and downstream cellular targets, such as ion channels and transporters, remain unclear. To study whether the V-type H+-ATPase (VA) plays a role in CAPA inhibition, we performed fluid secretion assays in MTs treated with diuretics and bafilomycin, a known VA inhibitor. Bafilomycin significantly inhibited DH31-stimulated fluid secretion 30 min post treatment compared to diuretic controls (p<0.05, n=22). Similar, however, delayed responses were seen in 5HT-stimulated MTs, while no affect was observed in DH44-stimulated secretion. An indirect way to measure whether CAPA inhibits VA activity was to measure the pH of the secreted fluid from diuretic-stimulated MTs treated with CAPA. In DH31-stimulated MTs supplemented with CAPA, there was an immediate significantly higher pH at 40 min, increasing up to 7.73±0.038 compared to control, 7.56±0.038 (p=0.0007, n=20). The pH of 5HT-stimulated MTs treated with CAPA was seen to significantly increase up to 7.75±0.061 (p=0.03, n=10) at the 60 min mark, in agreement with the delayed response previously seen. Unlike the effects observed with DH31 and 5HT, CAPA did not alter the pH of the secreted fluid in DH44-stimulated MTs. Alkalization of the secreted fluid in response to CAPA suggests inhibition of the proton pump, which may lead to constrained cation entry across the apical membrane of the MTs. To understand Na+/K+-ATPase (NKA) and VA activity in response to CAPA, we performed activity assays in diuretic-stimulated MTs. Adult female MTs treated with DH31 resulted in an increase of both NKA and VA activity compared saline controls. As expected, MTs incubated with both DH31 and AedaeCAPA-1 had a lower NKA and VA activity (p<0.05) resulting in activity levels comparable to saline control levels. The results thus far could suggest a novel mechanism for CAPA inhibition, blocking the VA to hinder fluid secretion. Investigating the pathway of CAPA inhibition and its role in countering diuresis will help provide a deeper understanding of the critical process of diuresis and its signaling mechanism.