Aedes aegypti Rhesus glycoproteins contribute to ammonia excretion by larval anal papillae

Abstract

In larval Aedes aegypti, transcripts of the Rhesus-like glycoproteins AeRh50-1 and AeRh50-2 have been detected in the anal papillae, sites of ammonia (NH 3 /NH 4 + ) excretion; however, these putative ammonia transporters have not been previously localized or functionally characterized. In this study, we show that the AeRh50s co-immunolocalize with apical V-type H + -ATPase as well as with basal Na + /K + -ATPase in the epithelium of anal papillae. The doublestranded RNA-mediated knockdown of AeRh50-1 and AeRh50-2 resulted in a significant reduction in AeRh50 protein abundance in the anal papillae, and this was coupled to decreased ammonia excretion. The knockdown of AeRh50-1 resulted in decreased hemolymph [NH 4 + ] and pH whereas knockdown of AeRh50-2 had no effect on these parameters. We conclude that the AeRh50s are important contributors to ammonia excretion at the anal papillae of larval A. aegypti, which may be the basis for their ability to inhabit areas with high ammonia levels.

Ammonia excretion in aquatic invertebrates: new insights and questions

“…* Significant difference compared with controls (dotted line, control). A and B are fromChasiotis et al (2016); C and D have been composed by the authors utilizing data fromChasiotis et al (2016) andDurant et al (2017).…”

Evidence that Rh proteins in the anal papillae of the freshwater mosquitoAedes aegyptiare involved in the regulation of acid base balance in elevated salt and ammonia environments

“…The inhibition of basolateral Na + /K + -ATPase (NKA), which functions to maintain a negative cell potential and low [Na + ] in the cytosol, causes significant reductions in NH 4 + efflux at the anal papillae (Chasiotis et al, 2016;Patrick et al, 2006). The efflux of H + driven by apical V-type H + -ATPase (VA) contributes to the negative intracellular voltage generated by NKA, facilitating NH 3 efflux likely through an ammonia-trapping mechanism at the apical membrane whereby NH 3 combines with H + on the external side, which maintains an NH 3 gradient favoring excretion (Chasiotis et al, 2016;Durant et al, 2017;Weihrauch et al, 2009). Inhibition of VA also results in a significant reduction in ammonia excretion at the anal papillae epithelium, implicating VA and NKA in the process of ammonia excretion, which is likely to stem from establishing the negative electrical potential of the cytosol and ammonia trapping.…”

“…Interestingly, inhibition of an apical Na + /H + exchanger 3 (NHE3, SLC 9 family) decreases NH 4 + excretion at the anal papillae in A. aegypti larvae, suggesting that this transporter may either contribute to ammonia trapping or perhaps directly conduct NH 4 + across the apical membrane (Chasiotis et al, 2016). Aedes aegypti possess two homologs of the vertebrate Rhesus glycoproteins (Rh proteins), AeRh50-1 and AeRh50-2, which have been localized to the apical and basolateral membranes of the anal papillae epithelium (Durant et al, 2017). It has been suggested that Rh proteins may transport NH 3 as well as CO 2 ; however, the specific transport substrate(s) of Rh proteins remains to be determined (Kustu and Inwood, 2006;Weihrauch et al, 2004;Weiner and Verlander, 2017).…”

“…It has been suggested that Rh proteins may transport NH 3 as well as CO 2 ; however, the specific transport substrate(s) of Rh proteins remains to be determined (Kustu and Inwood, 2006;Weihrauch et al, 2004;Weiner and Verlander, 2017). In A. aegypti larvae, knockdown studies of both AeRh50-1 and AeRh50-2 proteins results in a significant reduction in ammonia efflux (Durant et al, 2017). A decrease in [NH 4 + ] in the hemolymph coupled with an acidification of hemolymph was also observed in response to AeRh50-1 knockdown and it was suggested that this may occur if AeRh50-1 was capable of CO 2 transport.…”

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

“…Double-stranded RNA (dsRNA) was synthesized and column-purified using the MEGAscript® RNAi Kit (Invitrogen, Carlsbad, CA) following the recommended protocol using primers for dsCAPAr synthesis (see Table S3) and primers as reported previously for dsARG 64 , which is an ampicillin resistance gene cloned from standard sequencing plasmid (pGEM T-Easy) that served as a negative control. A Nanoject Nanoliter Injector (Drummond Scientific, Broomall, PA) was used to inject one-day old female mosquitoes with 1μg (in ∼140nL) of either dsCAPAR or dsARG.…”