Discovery of an aquaporin (CrAQP2) in the freshwater larval midge, Chironomus riparius and its role in response to road de-icers

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.

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.

Cadmium transport by the gut and Malpighian tubules of Chironomus riparius

Aquaporins (AQPs) accelerate the movement of water and other solutes across biological membranes, yet the molecular mechanisms of each AQP's transport function and the diverse physiological roles played by AQP family members are still being defined. We therefore have characterized an AQP in a model organism, Drosophila melanogaster, which is amenable to genetic manipulation and developmental analysis. To study the mechanism of Drosophila Malpighian tubule (MT)-facilitated water transport, we identified seven putative AQPs in the Drosophila genome and found that one of these, previously named DRIP, has the greatest sequence similarity to those vertebrate AQPs that exhibit the highest rates of water transport. In situ mRNA analyses showed that DRIP is expressed in both embryonic and adult MTs, as well as in other tissues in which fluid transport is essential. In addition, the pattern of DRIP expression was dynamic. To define DRIP-mediated water transport, the protein was expressed in Xenopus oocytes and in yeast secretory vesicles, and we found that significantly elevated rates of water transport correlated with DRIP expression. Moreover, the activation energy required for water transport in DRIP-expressing secretory vesicles was 4.9 kcal/mol. This low value is characteristic of AQP-mediated water transport, whereas the value in control vesicles was 16.4 kcal/mol. In contrast, glycerol, urea, ammonia, and proton transport were unaffected by DRIP expression, suggesting that DRIP is a highly selective water-specific channel. This result is consistent with the homology between DRIP and mammalian water-specific AQPs. Together, these data establish Drosophila as a new model system with which to investigate AQP function.

The juvenile stages of the yellow fever mosquito, Aedes aegypti, are found in semi‐permanent water storage containers, septic tanks, and natural coastal rock pools. Changing climates and anthropogenic pollutants affect the ionic (salt) composition of these mosquito breeding sites which can influence the spread and distribution of vector populations. These habitats impose different challenges to the salt and water balance of the mosquitoes which is accomplished by regulation of salt and water transport across epithelial barriers. Aquaporins (AQPs) are transmembrane proteins first discovered as selective water channels and can be classified by their transport capabilities to other small solutes. Six aquaporin isoforms have been identified in A. aegypti mosquitoes, AaAQP1‐6, and here we investigated the protein localization and abundance levels of AaAQP2 and AaAQP6 in the osmoregulatory organs of the aquatic larvae reared in either ion‐poor water (IPW; reverse‐osmosis water), artificial freshwater (FW), or brackish water (BW; 30% seawater). Using custom antibodies with immunohistochemistry and western blotting it was found that AaAQP2‐like and AaAQP6‐like immunofluorescence was primarily intracellular in the Malpighian tubules (MTs) and gastric caeca (GC). Rearing salinity did not affect protein abundance of AaAQP2 in MTs, GC and hindgut which appeared as a putative dimer of ~ 45 to 50 kDa; however, in the hindgut of BW reared larvae an additional band of ~ 30 kDa was apparent which may represent the monomer. Western blots for AaAQP6 are currently being optimized. Results suggest that AaAQP2 and AaAQP6 are required in these osmoregulatory organs regardless of the salinity of the external environment during development. Furthermore, AaAQP2 may have additional functions in the hindgut of larvae that develop in higher salt environments.Support or Funding InformationNSERC ‐ DISCOVERY RGPIN‐2018‐05841

Amphibian aquaporins and adaptation to terrestrial environments: A review

The mosquito, Aedes aegypti, is the primary vector for the arboviral diseases Zika, dengue fever, chikungunya, and yellow fever that affect millions of people worldwide. The larvae of A. aegypti reside in hypo‐osmotic freshwater habitats, where they face dilution of bodily fluids due to the influx of water. The Malpighian tubules (MTs) are one of the major contributors to osmoregulation, whereby, they function in concert with the rectum, to remove excess water from the hemolymph, thus maintaining ionic homeostasis. The epithelium of the Malpighian tubules actively transports ions from the hemolymph into the lumen which creates an osmotic gradient that drives paracellular and transcellular water movement in the same direction. Our previous study has shown that this transcellular water transport occurs through at least one of the six aquaporins (AQP) that A. aegypti possesses, AQP5. In the present study, the function of other AQPs with high relative mRNA abundance in the MTs were studied with the aid of double stranded RNA knockdown. The MT fluid secretion rate and ionic composition of secreted fluid were assessed using Ramsay assay and ion selective microelectrode (ISME), respectively. Hemolymph ion concentrations and larval survival were measured to gauge effects of AQP knockdown on osmoregulation in the larvae. Water transport across the Malpighian tubules of the larval mosquito is a critical physiological process because it is the first essential step in eliminating the excess water, a consequence of living in hypo‐osmotic habitats. Our study sheds light on the relative contribution of AQPs in this vital process.Support or Funding InformationNatural Sciences and Engineering Research Council of Canada (NSERC) discovery grant awarded to Andrew Donini

Aquaporin proteins in murine trophectoderm mediate transepithelial water movements during cavitation

Hepatic stellate cells (HSCs) are effector cells of hepatic fibrosis contributing to excessive collagen deposition and scar matrix formation. Sustained HSC activation leads to hepatic cirrhosis, a leading cause of liver-related death. Reversal of hepatic fibrosis has been attributed to the induction of HSC apoptosis. Aquaporins (AQPs) are critical proteinacious channels that mediate cellular water loss during the initiation and progression of apoptosis. This study examined AQP expression in quiescent and activated HSCs and determined the responsiveness to AQP-dependent apoptosis. Aquaporin gene and protein expressions in quiescent and activated HSCs were determined by reverse transcription polymerase chain reaction and Western blot analyses. AQP function was determined by cell swelling and apoptotic assays in the absence and presence of HgCl(2) , a non-specific AQP inhibitor. In this study, we report that activated HSCs showed no detectable expression of AQP 1, 5, 8, 9 and 12 mRNAs but expression was observed in quiescent HSCs. Similarly, AQP 0, 1, 8 and 9 protein was not detected in activated HSCs but was measured in quiescent HSCs. Dual fluorescent immunohistochemistry confirmed that AQP expression is decreased in activated HSCs in a model of liver injury. Functional studies demonstrated that quiescent HSCs were highly susceptible to osmotic challenge and apoptotic stimulus, whereas activated HSCs were less responsive. Finally, apoptosis was abrogated by the inhibition of AQP-dependent water movement. These findings demonstrate that increased resistance to apoptosis in activated HSCs is due, at least in part, to the changes in AQP expression and function that occur following HSC activation.

We assessed the effects of road deicer (NaCl) on life-history traits (survival, emergence, development time, sex ratio, and size at emergence) of Chironomus riparius under laboratory conditions mimicking near-freezing overwintering temperatures and warmer temperature fluctuations during early spring. We added 1-d-old larvae to culture chambers and acclimated them for 1 wk at 22°C. We decreased the temperature over a period of 2 d to 1–2°C. After 1 wk, we added deicing salt (NaCl) to the chambers over a period of 4 d to expose the larvae (as 2nd instars) to 0, 5, or 10 g/L salt (from deicer) concentrations. We then raised the temperature over a period of 1–2 d to fluctuate between 8–12°C and monitored adult emergence. All larvae in the 10-g/L chambers died when the temperature reached 12°C. The number of individuals that survived and the number that emerged were significantly lower, and development time was significantly longer in the 5- than in the 0-g/L chambers. Males and females emerged synchronously, sex ratio was 1∶1, and females were significantly larger than males at 0 g/L. Males emerged only slightly before females, sex ratio was male skewed (2∶1), and females were the same size as males at 5 g/L. Male size did not differ between 0 and 5 g/L, but females were significantly smaller at 5 than at 0 g/L. Road deicers can have significant lethal and sublethal population-level effects on chironomids in roadside wetlands, but these effects might be reduced if chironomids are present in nearby unaffected habitats that can act as source populations in late spring, summer, and autumn.

Aquaporins (AQPs) are a large family of membrane-bound water channels that facilitate water and small solute transport and have important physiological functions in all cells. At least thirteen types of aquaporins exist that exhibit a high level of phylogenetic homology, and many are present in ocular cells. Mouse knockout studies have demonstrated that AQPs play an important function in aqueous humor (AQH) dynamics in the anterior chamber cells and also have a role in retinal function. In contrast to the rich literature on aquaporin function, very little is known about how to assess water movement mediated by AQPs in their native environment, such as across ciliary epithelial cell membranes where AQH is manufactured. In part, the reason could be the lack of appropriate procedures for studying water movement across ciliary epithelial layers. This chapter describes methods to measure AQP function <i>in vitro</i> using ciliary epithelial cells, including a high-throughput screening assay method. In addition, a procedure to determine aquaporin function in its native environment is also presented.

The flow of water and some other small molecules across cell membranes is important in many of the processes underlying reproduction. The fluid movement is strongly associated with the presence of aquaporins (AQPs) in the female and male reproductive systems. It has been suggested that AQPs mediate water movement into the antral follicle and play important roles in follicle development. AQPs are known to be involved in the early stage of spermatogenesis, in the secretion of tubule liquid and in the concentration and storage of spermatozoa. Fluid reabsorption in some regions of the male reproductive tract is under steroid hormone control and could be mediated by various AQPs. Also AQPs take part in the processes of fertilization, blastocyst formation (as the pathway for transtrophoectodermal water movement during cavitation) and implantation. Alterations in the expression and function or regulation of AQPs have already been demonstrated in disorders of the male reproductive system, such as abnormal sperm motility, the abnormal epididymis and infertility seen in cystic fibrosis, and varicocele. This article extensively reviews the distribution of AQPs in mammalian reproductive tissues and discusses their possible physiological and pathophysiological roles.

The disease vector mosquito, Aedes aegypti , is found in tropical and sub‐tropical regions around the globe and is responsible for the transmission of many deadly diseases such as dengue fever and chikungunya. Adult A. aegypti are sexually dimorphic terrestrial animals since both the males and females feed on nectar for basic nutrients, females mosquitoes require a blood meal from a vertebrate host to produce viable eggs. The blood meal poses an osmoregulatory challenge to the female mosquito because the meal is about 3x its body weight in water, plasma, and proteins. The Malpighian tubules (MTs) are the primary osmoregulatory organ, responsible for producing an ion‐rich primary urine through hormone activated ion transport driven by an H + ‐ATPase. Through diuretic hormone control, the MTs are able to begin secretion of primary urine almost immediately following the onset of blood feeding and this process requires intermembrane transporters called aquaporins (AQPs). The concentration of ions in the lumen of the MTs drives water from the hemolymph into the lumen of the MTs by osmosis to produce primary urine. Osmosis is facilitated by the expression of AQPs in the cells of the MTs. Six AQPs have been identified in A. aegypti, most of which are all selective for water and some solutes. It is hypothesized that a blood meal in female A. aegypti, with knowledge that mRNA levels of AQP1 are increased in the MTs ~3‐24hrs post blood meal, will have an effect on the localization and protein abundance of AQPs in the MTs. It was also hypothesized that the starvation of male A. aegypti would have an effect on the localization and abundance of AQPs in the MTs. Thus far, AQP1 & 4 have been localized throughout the body of both the male and female A. aegypti using immunohistochemistry. Immunolocalization of AQPs shows evidence that a blood meal has a significant effect on the localization in the female A. aegypti . It has also been found that, following Western blot analysis, a blood meal may significantly increase the protein abundance AQP1 and 4 in the MTs of the females. It has been shown that in the male A. aegypti , AQP4 abundance is significantly increased in the fat body.

Bioaccumulation of perfluoroalkyl compounds in midge (Chironomus riparius) larvae exposed to sediment

Salinization of freshwater by anthropogenic activities is a global issue and the use of de-icers in cold climates is turning out to be a major contributor to this problem. The most common de-icer is sodium chloride (NaCl) in the form of rock salt or brine, but because the harmful accumulation of NaCl in nearby freshwater systems is well recognized, newer organic de-icers, such as sugar beet juice, have been developed. Detrimental effects of NaCl on freshwater animals have been documented but the potential effects of organic based de-icers are virtually unknown. Insects are one of the most abundant groups of freshwater animals that perform services vital to ecosystem health. Hence, the aim of this study is to examine the effects of NaCl and a commercially available brine beet juice de-icer (BBJD) on the osmoregulatory physiology of a ubiquitous freshwater insect, the larvae of the midge Chironomus riparius. Larvae were exposed to sub-lethal doses of the de-icers for 24 h, and the osmoregulatory status of the larvae, and function of the Malpighian tubules (MTs) and anal papillae (AP) were assessed. Hemolymph [K+] was tightly regulated, while [Na+] was elevated by both de-icers. BBJD caused some dehydration. Larvae reduced ion uptake by the AP where Na+ secretion occurred with the NaCl treatment. BBJD also altered MT function such that the MTs cleared more K+. BBJD presents its own significant challenges to the osmoregulatory physiology of C. riparius larvae, and the relatively high levels of K+ from the sugar beets could pose a new issue for freshwater habitats.