Ammonia Excretion in an Osmoregulatory Syncytium Is Facilitated by AeAmt2, a Novel Ammonia Transporter in Aedes aegypti Larvae

Abstract

The larvae of the mosquito Aedes aegypti inhabit ammonia rich septic tanks in tropical regions of the world that make extensive use of these systems, explaining the prevalence of disease during dry seasons. Since ammonia (NH3/NH4+) is toxic to animals, an understanding of the physiological mechanisms of ammonia excretion permitting the survival of A. aegypti larvae in high ammonia environments is important. We have characterized a novel ammonia transporter, AeAmt2, belonging to the Amt/MEP/Rh family of ammonia transporters. Based on the amino acid sequence, the predicted topology of AeAmt2 consists of 11 transmembrane helices with an extracellular N-terminus and a cytoplasmic C-terminus region. Alignment of the predicted AeAmt2 amino acid sequence with other Amt/MEP proteins from plants, bacteria, and yeast highlights the presence of conserved residues characteristic of ammonia conducting channels in this protein. AeAmt2 is expressed in the ionoregulatory anal papillae of A. aegypti larvae where it is localized to the apical membrane of the epithelium. dsRNA-mediated knockdown of AeAmt2 results in a significant decrease in NH4+ efflux from the anal papillae, suggesting a key role in facilitating ammonia excretion. The effect of high environmental ammonia (HEA) on expression of AeAmt2, along with previously characterized AeAmt1, AeRh50-1, and AeRh50-2 in the anal papillae was investigated. We show that changes in expression of ammonia transporters occur in response to acute and chronic exposure to HEA, which reflects the importance of these transporters in the physiology of life in high ammonia habitats.

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

“…A previous study noted that RNAi-mediated knockdown of AeRh50-1 in the anal papillae resulted in hemolymph acidification (Durant et al, 2017). This is similar to the effect of HEA rearing whereby AeRh50 expression is downregulated and hemolymph acidification occurs (Durant and Donini, 2018). We previously speculated that this acidification could arise if AeRh50-1 is capable of transporting CO 2 and is located on the basal membrane of the anal papillae epithelium.…”

“…In a recent study, we examined the effects of rearing A. aegypti larvae in HEA on the expression of ammonia transporters in the anal papillae (Durant and Donini, 2018). We reported a decrease in the protein abundance of apical AeAmt2 and the AeRh50s, while the basolateral AeAmt1 protein abundance did not change compared with that of control larvae reared in deH 2 O.…”

“…Changes in the protein abundance of AeAmt1, AeAmt2 and AeRh50 s in response to rearing in HEA (5 mmol l −1 NH 4 Cl) have been previously documented (Durant and Donini, 2018). Here, the protein abundance of AeAmt1, AeAmt2 and AeRh50s in the anal papillae of fourth instar larvae in response to 5 mmol l −1 NaCl was examined using western blotting following an established protocol (Chasiotis and Kelly, 2008).…”

“…Western blot analysis of AeAmt1, AeAmt2 and AeRh50s was conducted according to an established protocol (Chasiotis and Kelly, 2008;Chasiotis et al, 2016;Durant et al, 2017). Custom-synthesized polyclonal antibodies raised in rabbit against AeAmt1, AeAmt2 and AeRh50s were used at dilutions of 1:500, 1:5000 and 1:2000, respectively, and have been previously validated (Chasiotis et al, 2016;Durant and Donini, 2018;Durant et al, 2017). The AeRh50 antisera is presumed to detect both AeRh50-1 and AeRh50-2 because of high epitope sequence similarity (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. The partial pressure gradient of CO 2 plays a significant role in the acid-base balance of body fluids of aquatic animals (Nawata and Wood, 2008), and in this respect it was speculated that AeRh50-1 may mediate CO 2 excretion through the anal papillae with dsRNA-mediated knockdown, resulting in accumulation of CO 2 in the hemolymph; however, no measurements of CO 2 have been performed in relation to these studies (Durant and Donini, 2018).…”