The role of the V‐type H + ‐ATPase in CAPA‐treated Malpighian tubules in the yellow fever mosquito, Aedes aegypti

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 utilize their highly adapted excretory system that includes the Malpighian ‘renal’ tubules (MTs), which are under rigorous control by several hormones to regulate transepithelial movement of ions/water. Originating in the nervous system, the mosquito anti-diuretic hormone is a member of the CAPA peptide family and is a homolog of 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. Active ion transport in A. aegypti MTs is driven by the principal cells, whose brush-border membrane is densely packed by the V-type H+-ATPase (VA), serving as the primary energizer for transepithelial secretion of electrolytes and water. Due to its predominant role in fluid secretion, the VA is a likely target for diuretic and anti-diuretic factors. The effect of endocrine factors and the VA inhibitor, bafilomycin, on fluid secretion rate by MTs was measured using an in vitro bioassay. Bafilomycin was found to inhibit fluid secretion stimulated by serotonin (5HT) and the calcitonin-related diuretic hormone (DH31), while having no inhibitory action on MTs stimulated with the corticotropin-releasing factor (CRF)-related diuretic hormone. CAPA and bafilomycin treatment led to alkalization of the secreted fluid suggesting inhibition of the apical VA, which may lead to constrained entry of cations (Na+ and K+) across the apical membrane of MTs. VA activity was increased in adult female MTs treated with DH31, whereas CAPA treatment reduced VA activity, comparable to saline levels. Furthermore, a VA antibody was used to determine protein localization and expression in DH31 and CAPA-treated MTs. In DH31-incubated MTs, the VA was exclusively localized to the apical memrane of the MTs, whereas VA immunoreactivity was observed in both the apical membrane and cytosol of CAPA-treated tubules. These results suggest a novel mechanism for CAPA inhibition, blocking the VA to hinder fluid secretion. A. aegypti mosquitoes are vectors of a variety of pathogens leading to illnesses, such as Dengue fever, Yellow fever, and the Zika virus. The current results advance our knowledge of the anti-diuretic control of the A. aegypti MTs, suggesting that CAPA targets the VA to inhibit diuresis. Investigating the pathway of CAPA inhibition and its role in countering diuresis will help provide a deeper understanding of this critical physiological process. Studying the excretory system and discerning the underlying mechanisms behind ion and fluid transport may aid in developing novel pest management strategies to better control Aedes population and lessen its prominence as a disease vector.