A comparison of aquaporin expression in mosquito larvae (Aedes aegypti) that develop in hypo-osmotic freshwater and iso-osmotic brackish water.

The mosquito Aedes aegypti vectors the arboviral diseases yellow fever, dengue, Zika and chikungunya. Larvae are usually found developing in freshwater; however, more recently they have been increasingly found in brackish water, potential habitats which are traditionally ignored by mosquito control programs. Aedes aegypti larvae are osmo-regulators maintaining their hemolymph osmolarity in a range of ~ 250 to 300 mOsmol l-1. In freshwater, the larvae must excrete excess water while conserving ions while in brackish water, they must alleviate an accumulation of salts. The compensatory physiological mechanisms must involve the transport of ions and water but little is known about the water transport mechanisms in the osmoregulatory organs of these larvae. Water traverses cellular membranes predominantly through transmembrane proteins named aquaporins (AQPs) and Aedes aegypti possesses 6 AQP homologues (AaAQP1 to 6). The objective of this study was to determine if larvae that develop in freshwater or brackish water have differential aquaporin expression in osmoregulatory organs, which could inform us about the relative importance and function of aquaporins to mosquito survival under these different osmotic conditions. We found that AaAQP transcript abundance was similar in organs of freshwater and brackish water mosquito larvae. Furthermore, in the Malpighian tubules and hindgut AaAQP protein abundance was unaffected by the rearing conditions, but in the gastric caeca the protein level of one aquaporin, AaAQP1 was elevated in brackish water. We found that AaAQP1 was expressed apically while AaAQP4 and AaAQP5 were found to be apical and/or basal in the epithelia of osmoregulatory organs. Overall, the results suggest that aquaporin expression in the osmoregulatory organs is mostly consistent between larvae that are developing in freshwater and brackish water. This suggests that aquaporins may not have major roles in adapting to longterm survival in brackish water or that aquaporin function may be regulated by other mechanisms like post-translational modifications.

Anti-diuretic activity of a CAPA neuropeptide can compromise Drosophila chill tolerance.

For insects, chilling injuries that occur in the absence of freezing are often related to a systemic loss of ion and water balance that leads to extracellular hyperkalemia, cell depolarization and the triggering of apoptotic signalling cascades. The ability of insect ionoregulatory organs (e.g. the Malpighian tubules) to maintain ion balance in the cold has been linked to improved chill tolerance, and many neuroendocrine factors are known to influence ion transport rates of these organs. Injection of micromolar doses of CAPA (an insect neuropeptide) have been previously demonstrated to improve Drosophila cold tolerance, but the mechanisms through which it impacts chill tolerance are unclear, and low doses of CAPA have been previously demonstrated to cause anti-diuresis in insects, including dipterans. Here, we provide evidence that low (femtomolar) and high (micromolar) doses of CAPA impair and improve chill tolerance, respectively, via two different effects on Malpighian tubule ion and water transport. While low doses of CAPA are anti-diuretic, reduce tubule K+ clearance rates and reduce chill tolerance, high doses facilitate K+ clearance from the haemolymph and increase chill tolerance. By quantifying CAPA peptide levels in the central nervous system, we estimated the maximum achievable hormonal titres of CAPA and found further evidence that CAPA may function as an anti-diuretic hormone in Drosophila melanogaster We provide the first evidence of a neuropeptide that can negatively affect cold tolerance in an insect and further evidence of CAPA functioning as an anti-diuretic peptide in this ubiquitous insect model.

Functional plasticity of the gut and the Malpighian tubules underlies cold acclimation and mitigates cold-induced hyperkalemia in Drosophila melanogaster.

At low temperatures, Drosophila, like most insects, lose the ability to regulate ion and water balance across the gut epithelia, which can lead to a lethal increase of [K+] in the hemolymph (hyperkalemia). Cold acclimation, the physiological response to a prior low temperature exposure, can mitigate or entirely prevent these ion imbalances, but the physiological mechanisms that facilitate this process are not well understood. Here, we test whether plasticity in the ionoregulatory physiology of the gut and Malpighian tubules of Drosophila may aid in preserving ion homeostasis in the cold. Upon adult emergence, D. melanogaster females were subjected to 7 days at warm (25°C) or cold (10°C) acclimation conditions. The cold-acclimated flies had a lower critical thermal minimum (CTmin), recovered from chill coma more quickly, and better maintained hemolymph K+ balance in the cold. The improvements in chill tolerance coincided with increased Malpighian tubule fluid secretion and better maintenance of K+ secretion rates in the cold, as well as reduced rectal K+ reabsorption in cold-acclimated flies. To test whether modulation of ion-motive ATPases, the main drivers of epithelial transport in the alimentary canal, mediate these changes, we measured the activities of Na+/K+-ATPase and V-type H+-ATPase at the Malpighian tubules, midgut, and hindgut. Na+/K+-ATPase and V-type H+-ATPase activities were lower in the midgut and the Malpighian tubules of cold-acclimated flies, but unchanged in the hindgut of cold-acclimated flies, and were not predictive of the observed alterations in K+ transport. Our results suggest that modification of Malpighian tubule and gut ion and water transport probably prevents cold-induced hyperkalemia in cold-acclimated flies, and that this process is not directly related to the activities of the main drivers of ion transport in these organs, Na+/K+- and V-type H+-ATPases.

A mosquito entomoglyceroporin, Aedes aegypti AQP5, participates in water transport across the Malpighian tubules of larvae.

The mosquito Aedes aegypti is the primary vector for arboviral diseases such as Zika fever, dengue fever, chikungunya and yellow fever. The larvae reside in hypo-osmotic freshwater habitats, where they face dilution of their body fluids from osmotic influx of water. The Malpighian tubules help maintain ionic and osmotic homeostasis by removing excess water from the hemolymph; however, the transcellular pathway for this movement remains unresolved. Aquaporins are transmembrane channels thought to permit transcellular transport of water from the hemolymph into the Malpighian tubule lumen. Immunolocalization of A.aegypti aquaporin 5 (AaAQP5) revealed expression by Malpighian tubule principal cells of the larvae, with localization to both the apical and basolateral membranes. Knockdown of AaAQP5 with double-stranded RNA decreased larval survival, reduced rates of fluid, K+ and Na+ secretion by the Malpighian tubules, and reduced Cl- concentrations in the hemolymph. These findings indicate that AaAQP5 participates in transcellular water transport across the Malpighian tubules of larval Aaegypti where global AaAQP5 expression is important for larval survival.

Salinity responsive aquaporins in the anal papillae of the larval mosquito, Aedes aegypti.

The larvae of the mosquito, Aedes aegypti normally inhabit freshwater (FW) where they face dilution of body fluids by osmotic influx of water. In response, the physiological actions of the anal papillae result in ion uptake while the Malpighian tubules and rectum work in concert to excrete excess water. In an apparent paradox, the anal papillae express aquaporins (AQPs) and are sites of water permeability which, if AQPs are expressed by the epithelium, apparently exaggerates the influx of water from their dilute environment. Recently, naturally breeding populations of A. aegypti were found in brackish water (BW), an environment which limits the osmotic gradient. Given that salinization of FW is an emerging environmental issue and that these larvae would presumably need to adjust to these changing conditions, this study investigates the expression of AQPs in the anal papillae and their response to rearing in hypo-osmotic and near isosmotic conditions. Transcripts of all six Aedes AQP homologs were detectable in the anal papillae and the transcript abundance of three AQP homologs in the papillae was different between rearing conditions. Using custom made antibodies, expression of two of these AQP homologs (AQP4 and AQP5) was localized to the syncytial epithelium of the anal papillae. Furthermore, the changes in transcript abundance of these two AQPs between the rearing conditions, were manifested at the protein level. Results suggest that AQP4 and AQP5 play an important physiological role in larval responses to changes in environmental salinity.

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

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 (NH3/NH4+) 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 double-stranded 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 [NH4+] 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.

Chronic dietary salt stress mitigates hyperkalemia and facilitates chill coma recovery in Drosophila melanogaster.

Chill susceptible insects like Drosophila lose the ability to regulate water and ion homeostasis at low temperatures. This loss of hemolymph ion and water balance drives a hyperkalemic state that depolarizes cells, causing cellular injury and death. The ability to maintain ion homeostasis at low temperatures and/or recover ion homeostasis upon rewarming is closely related to insect cold tolerance. We thus hypothesized that changes to organismal ion balance, which can be achieved in Drosophila through dietary salt loading, could alter whole animal cold tolerance phenotypes. We put Drosophila melanogaster in the presence of diets highly enriched in NaCl, KCl, xylitol (an osmotic control) or sucrose (a dietary supplement known to impact cold tolerance) for 24h and confirmed that they consumed the novel food. Independently of their osmotic effects, NaCl, KCl, and sucrose supplementation all improved the ability of flies to maintain K+ balance in the cold, which allowed for faster recovery from chill coma after 6h at 0°C. These supplements, however, also slightly increased the CTmin and had little impact on survival rates following chronic cold stress (24h at 0°C), suggesting that the effect of diet on cold tolerance depends on the measure of cold tolerance assessed. In contrast to prolonged salt stress, brief feeding (1.5h) on diets high in salt slowed coma recovery, suggesting that the long-term effects of NaCl and KCl on chilling tolerance result from phenotypic plasticity, induced in response to a salty diet, rather than simply the presence of the diet in the gut lumen.

An animal homolog of plant Mep/Amt transporters promotes ammonia excretion by the anal papillae of the disease vector mosquito Aedes aegypti.

The transcripts of three putative ammonia (NH3/NH4 (+)) transporters, Rhesus-like glycoproteins AeRh50-1, AeRh50-2 and Amt/Mep-like AeAmt1 were detected in the anal papillae of larval Aedes aegypti Quantitative PCR studies revealed 12-fold higher transcript levels of AeAmt1 in anal papillae relative to AeRh50-1, and levels of AeRh50-2 were even lower. Immunoblotting revealed AeAmt1 in anal papillae as a pre-protein with putative monomeric and trimeric forms. AeAmt1 was immunolocalized to the basal side of the anal papillae epithelium where it co-localized with Na(+)/K(+)-ATPase. Ammonium concentration gradients were measured adjacent to anal papillae using the scanning ion-selective electrode technique (SIET) and used to calculate ammonia efflux by the anal papillae. dsRNA-mediated reductions in AeAmt1 decreased ammonia efflux at larval anal papillae and significantly increased ammonia levels in hemolymph, indicating a principal role for AeAmt1 in ammonia excretion. Pharmacological characterization of ammonia transport mechanisms in the anal papillae suggests that, in addition to AeAmt1, the ionomotive pumps V-type H(+)-ATPase and Na(+)/K(+)-ATPase as well as NHE3 are involved in ammonia excretion at the anal papillae.