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The traditional thesis of the osmorespiratory compromise is that low branchial water and ion permeability would be traded off for increased O 2 permeability at times of elevated O 2 demand. However, there is growing evidence of independent regulation of these permeabilities in hypoxia-tolerant fish. Using 0.5-g zebrafish previously maintained under normoxia at 25°C, we investigated responses to acute temperature challenges (15°C or 35°C), acute hypoxia (15 min at 10% or 5% air saturation), as well as longer-term exposures to 10% hypoxia, on O 2 consumption (MO 2 ), diffusive water flux, and net sodium loss rates. Exposure to 35°C increased, and 15°C decreased all three rates, with diffusive water flux showing the lowest temperature sensitivity, and Na + loss the greatest. Acute 10% and 5% hypoxia increased diffusive water flux and net Na + loss, and it reduced MO 2 . All these responses reflected the traditional osmorespiratory compromise. However, during prolonged 10% hypoxia, MO 2 recovered, diffusive water flux decreased below control levels, and Na + loss rate remained elevated, even during posthypoxia recovery. Overall, zebrafish do not fit standard patterns previously seen in either hypoxia-tolerant or -intolerant fish but are clearly able to adjust the effective permeabilities of their gills to O 2 , water, and ions independently during acute temperature and hypoxia exposures.

• Common carp exposed to environmentally relevant concentrations of copper (Cu), zinc (Zn) and cadmium (Cd). • Significant Cu and Cd accumulation in the gills, whereas Zn levels remained stable. • General Na decrease in all tissues counteracted by induction of H+-ATPase gene expression. • Strong induction of metallothionein gene expression. • Antagonistic mutual inhibition of Cd and Zn uptake. The aquatic environment is continuously under threat because it is the final receptor and sink of waste streams. The development of industry, mining activities and agriculture gave rise to an increase in metal pollution in the aquatic system. Thus a wide occurrence of metal mixtures exists in the aquatic environment. The assessment of mixture stress remains a challenge considering that we can not predict the toxicity of a mixture on the basis of single compounds. Therefore the analysis of the effects of environmentally relevant waterborne mixtures is needed to improve our understanding of the impact of metal pollution in aquatic ecosystems. Our aim was to assess whether 10 % of the concentration of the 96 h LC 50 (the concentration that is lethal to 50 % of the population in 96 h) of individual metal exposures can be considered as a "safe" concentration when applied in a trinomial mixture. Therefore, common carp were exposed to a sublethal mixture of Cu 0.07 ± 0.001 μM (4.3 ± 0.6 μg/L), Zn 2.71 ± 0.81 μM (176.9 ± 52.8 μg/L) and Cd 0.03 ± 0.0004 μM (3.0 ± 0.4 μg/L) at 20 °C for a period of one week. Parameters assessed included survival rate, bioaccumulation and physiological biomarkers related to ionoregulation and defensive mechanisms such as MT induction. Our results showed a sharp increase in Cu and Cd concentration in gills within the first day of exposure while Zn levels remained stable. The accumulation of these metals led to a Na drop in gills, liver and muscle as well as a decreased K content in the liver. Biomarkers related to Na uptake were also affected: on the first day gene expression for H+-ATPase was transiently increased while a concomitant decreased gene expression of the Na+/H+ exchanger occurred. A fivefold induction of metallothionein gene expression was reported during the entire duration of the experiment. Despite the adverse effects on ionoregulation all fish survived, indicating that common carp are able to cope with these low metal concentrations, at least during a one week exposure. [ABSTRACT FROM AUTHOR]

The mosquito Aedes aegypti is largely confined to tropical and subtropical regions, but its range has recently been spreading to colder climates. As insect biogeography is tied to environmental temperature, understanding the limits of A. aegypti thermal tolerance and their capacity for phenotypic plasticity is important in predicting the spread of this species. In this study, we report on the chill coma onset (CCO) and recovery time (CCRT), as well as low-temperature survival phenotypes of larvae and adults of A. aegypti that developed or were acclimated to 15°C (cold) or 25°C (warm). Cold acclimation did not affect CCO temperatures of larvae but substantially reduced CCO in adults. Temperature and the duration of exposure both affected CCRT, and cold acclimation strongly mitigated these effects and increased rates of survival following prolonged chilling. Female adults were far less likely to take a blood meal when cold acclimated, and exposing females to blood (without feeding) attenuated some of the beneficial effects of cold acclimation on CCRT. Lastly, larvae suffered from haemolymph hyperkalaemia when chilled, but cold acclimation attenuated the imbalance. Our results demonstrate that A. aegypti larvae and adults have the capacity to acclimate to low temperatures, and do so at least in part by better maintaining ion balance in the cold. This ability for cold acclimation may facilitate the spread of this species to higher latitudes, particularly in an era of climate change., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2019. Published by The Company of Biologists Ltd.)

The purpose of this study was to investigate Na+, Cl-, K+, Ca2+, and Mg2+ levels in the plasma of freshwater and marine/estuarine teleosts collected at different salinities (0 to 34) from the estuarine and freshwater portions of the São Gonçalo channel in Southern Brazil. Any relationship between plasma ion levels and salinity and the capacity of ionic regulation of teleosts found at three or more different salinities (Genidens barbus and Micropogonias furnieri) was also investigated. Results showed no relationship between plasma ion levels and salinity when considering all species together, but the two species collected from three or more different salinities showed a significant positive relationship between plasma ion levels and salinity, indicating that G. barbus and M. furnieri have a high capacity to regulate plasma ion levels at both low and high salinities.

This study analyzed the chemical composition and anesthetic potential of essential oil (EO) of Nectandra megapotamica in fat snook (Centropomus parallelus). For the extraction of EO by hydrodistillation, leaves were separated in young (EO-Y) or old (EO-O), and the chemical composition of the EOs was determined by CG-MS. The anesthetic potential was assessed by the evaluation of induction and recovery time of anesthesia and stress response from anesthesia and transport. Three experiments were carried out: i) four different concentrations of each EO were tested to evaluate anesthesia induction and recovery time; ii) two concentrations of EO-O were tested for the evaluation of its effects on stress parameters (glucose, lactate, and Na+ and K+ plasma levels) caused by anesthesia; and iii) fish were transported in plastic bags, supplied with two concentrations of EO-O for the evaluation of water quality and mortality. All experiments were performed on fish acclimated to 0 and 33 ppt salinity. The main constituents of the Y and O-EOs were bicyclogermacrene (46.5/34.6%), α-pinene (26.8/26.2%), β-pinene (7.9/12.3%), and germacrene D (9.6/9.1%). Mild sedation was achieved at 30 °L L-1(1.3-3.2 min) and deep anesthesia at 150 °L L-1(5.6-8.0 min) with both EOs. The recovery time ranged from 1-10 min. The EO-O was not able to avoid the stress of anesthesia evidenced by elevated glucose and lactate plasma levels observed in all groups. Plasma levels of Na+ and K+ were not significantly affected by treatments. During transport, the use of EO-O did not prevent deterioration in water quality and the post-transport mortality. In conclusion, the EO of N. megapotamica has anesthetic activity in fat snook, but it was not able to prevent the stress of anesthesia and transport.

Amphibious fishes have evolved a variety of physiological modifications allowing them to survive in water and air. In air, the amphibious mangrove rivulus, Kryptolebias marmoratus, uses its skin as a site of ionoregulation. Skin ionocytes actively transport ions into/out of the body; however, it is unclear if there are specific morphological or functional changes occurring in skin ionocytes during air exposure. We used two microscopy techniques to describe skin ionocyte morphology and to investigate their plasticity after salinity challenges and air exposure. Immunohistochemical staining in air-exposed fish revealed ionocytes with Na + /K + ATPase (NKA), Na + /H + exchanger (NHE3b) and cystic fibrosis transmembrane conductance regulator (CFTR) immunoreactivity, whereas ionocytes from aquatic fish had only NKA (freshwater) or NKA and CFTR (brackish and hypersaline water). Following salinity challenges, we noted increases in the number and area of ionocyte apical surfaces, indicating that skin ionocyte activity increased in high salinity environments compared with control conditions. Furthermore, we show increased ionocyte area during air exposure suggesting increased ionocyte activity in all salinity conditions. Using energy dispersive X-ray spectroscopy to analyze the skin surface, we report decreases in magnesium, phosphorous, and sulfur after 7 days in air compared with fish in water, suggesting ionic movement in the skin surface during air exposure. Our study highlights morphological and functional features of skin ionocytes that are involved in ionoregulation in an air-exposed amphibious fish., (© 2018 Wiley Periodicals, Inc.)

Two major strategies are used by most fish to maintain energy homeostasis under hypoxia. One is to utilize alternative metabolic pathways to increase energy production, and the other is to limit energy expenditure by suppressing energy-consuming processes, especially ionoregulation. Some anabantoid fishes live in tropical rivers, where hypoxic environments occur frequently. We previously found that under ambient hypoxia, anabantoid fishes do not downregulate Na + /K + -ATPase (NKA) activity to conserve energy in gills but instead increase the frequency of air-breathing respiration (ABR). In addition to the hypoxic condition, another factor that may cause cellular hypoxia in fish is abnormally high environmental temperatures. The frequency of such extreme thermal events has increased due to global climate change. In the present study, we examined whether the anabantoid fish, Macropodus opercularis employs the two strategies mentioned above to resist both ambient hypoxic and elevated thermal (cellular hypoxic) conditions. Results indicate that neither glucose metabolism nor gill NKA activity were altered by hypoxia (DO = 1.5 ± 1 mg/L), but glucose metabolism was increased by thermal stress (34 ± 1°C). NH 4 + excretion and ABR frequency were both increased under hypoxia, thermal or hypoxic-and-thermal treatments. In fish that were restricted from breathing air, increased mortality and glucose metabolism were observed under hypoxic or thermal treatments. These results suggest that for M. opercularis , increasing ABR is an important strategy for coping with unmet oxygen demand under hypoxic or thermal stress. This behavioral compensation allows anabantoid fish to physiologically withstand hypoxic and thermal stresses, and constitutes a mechanism of stress resistance that is unavailable to water-breathing fishes.

Ionoregulation in the euryhaline killifish Fundulus heteroclitus has been intensively studied over the last two decades using a variety of techniques. However, there has been limited use of pharmacological inhibitors to identify proteins involved in ion transport for this species. In this study, we used a range of pharmacological inhibitors (EIPA, DAPI, ethoxzolamide, bumetanide, bafilomycin, phenamil, hydrochlorothiazide) to investigate the proteins involved in Na + transport in freshwater (1 mM Na + ) acclimated F. heteroclitus. Our results indicate that Na + uptake under these conditions is sensitive to both EIPA (NHE-specific inhibitor) and DAPI (putative ASIC-specific inhibitor), but not to any of the other inhibitors. Results for EIPA are consistent with previous studies indicating F. heteroclitus relies solely on NHE2 for Na + transport across the apical membrane of ionocytes. In contrast, results for DAPI are surprising given previous studies that have indicated the H + -ATPase is basolaterally located in F. heteroclitus and so cannot contribute to Na + uptake via ASIC. The lack of bafilomycin sensitivity in the current study is consistent with a basolaterally located H + -ATPase. This suggests that DAPI is not an ASIC-specific inhibitor as has been previously assumed, and that it may also inhibit NHE2. Finally, we did not observe Na + uptake to be sensitive to ethoxzolamide, suggesting that carbonic anhydrase may not be involved in generating the H + needed to maintain NHE activity in freshwater as has been previously proposed., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Adult coho salmon (Oncorhynchus kisutch) prematurely die when they return from the ocean to spawn in urban watersheds throughout northwestern North America. The available evidence suggests the annual mortality events are caused by toxic stormwater runoff. The underlying pathophysiology of the urban spawner mortality syndrome is not known, and it is unclear whether closely related species of Pacific salmon are similarly at risk. The present study co-exposed adult coho and chum (O. keta) salmon to runoff from a high traffic volume urban arterial roadway. The spawners were monitored for the familiar symptoms of the mortality syndrome, including surface swimming, loss of orientation, and loss of equilibrium. Moreover, the hematology of both species was profiled by measuring arterial pH, blood gases, lactate, plasma electrolytes, hematocrit, and glucose. Adult coho developed behavioral symptoms within a few hours of exposure to stormwater. Various measured hematological parameters were significantly altered compared to coho controls, indicating a blood acidosis and ionoregulatory disturbance. By contrast, runoff-exposed chum spawners showed essentially no indications of the mortality syndrome, and measured blood hematological parameters were similar to unexposed chum controls. We conclude that contaminant(s) in urban runoff are the likely cause of the disruption of ion balance and pH in coho but not chum salmon. Among the thousands of chemicals in stormwater, future forensic analyses should focus on the gill or cardiovascular system of coho salmon. Because of their distinctive sensitivity to urban runoff, adult coho remain an important vertebrate indicator species for degraded water quality in freshwater habitats under pressure from human population growth and urbanization., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The Tambaqui is a model neotropical teleost which is of great economic and cultural importance in artisanal fisheries and commercial aquaculture. It thrives in ion-poor, often acidic Amazonian waters and exhibits excellent regulation of physiology down to water pH 4.0. Curiously, however, it is reported to perform poorly in aquaculture at pH 8.0, an only slightly alkaline pH which would be benign for most freshwater fish. In initial experiments with Tambaqui of intermediate size (30-50 g), we found that ammonia excretion rate was unchanged at pH 4, 5, 6, and 7, but elevated after 20-24 h at pH 8, exactly opposite the pattern seen in most teleosts. Subsequent experiments with large Tambaqui (150-300 g) demonstrated that only ammonia, and not urea excretion was increased at pH 8.0, and that the elevation was proportional to a general increase in MO 2 . There was an accompanying elevation in net acidic equivalent excretion and/or basic equivalent uptake which occurred mainly at the gills. Net Na + balance was little affected while Cl - balance became negative, implicating a disturbance of Cl - versus base exchange rather than Na + versus acid exchange. Arterial blood pH increased by 0.2 units at pH 8.0, reflecting combined metabolic and respiratory alkaloses. Most parameters recovered to control levels by 18-24 h after return to pH 6.0. With respect to large Tambaqui, we conclude that a physiology adapted to acidic pH performs inappropriately at moderately alkaline pH. In small Tambaqui (4-15 g), the responses were very different, with an initial inhibition of ammonia excretion rate at pH 8.0 followed by a subsequent restoration of control levels. Elevated ammonia excretion rate occurred only after return to pH 6.0. Furthermore, MO 2 , plasma cortisol, and branchial vH + ATPase activities all declined during pH 8.0 exposure in small Tambaqui, in contrast to the responses in larger fish. Overall, small Tambaqui appear to cope better at pH 8.0, a difference that may correlate with their natural history in the wild.

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 (CT min ), 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., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

Hemolymph calcium homeostasis in insects is achieved by the Malpighian tubules, primarily by sequestering excess Ca 2+ within internal calcium stores (Ca-rich granules) most often located within type I (principal) tubule cells. Using both the scanning ion-selective electrode technique and the Ramsay secretion assay this study provides the first measurements of basolateral and transepithelial Ca 2+ fluxes across the Malpighian tubules of an Orthopteran insect, the house cricket Acheta domesticus. Ca 2+ transport was specific to midtubule segments, where 97% of the Ca 2+ entering the tubule is sequestered within intracellular calcium stores and the remaining 3% is secreted into the lumen. Antagonists of voltage-gated (L-type) calcium channels decreased Ca 2+ influx ≥fivefold in adenosine 3',5'-cyclic monophosphate (cAMP)-stimulated tubules, suggesting basolateral Ca 2+ influx is facilitated by voltage-gated Ca 2+ channels. Increasing fluid secretion through manipulation of intracellular levels of cAMP or Ca 2+ had opposite effects on tubule Ca 2+ transport. The adenylyl cyclase-cAMP-PKA pathway promotes Ca 2+ sequestration whereas both 5-hydroxytryptamine and thapsigargin inhibited sequestration. Our results suggest that the midtubules of Acheta domesticus are dynamic calcium stores, which maintain hemolymph calcium concentration by manipulating rates of Ca 2+ sequestration through stimulatory (cAMP) and inhibitory (Ca 2+ ) regulatory pathways., (© 2017 Wiley Periodicals, Inc.)

Larvae of Aedes aegypti , the yellow fever vector, inhabit a variety of aquatic habitats ranging from freshwater to brackish water. This study focuses on the gastric caecum of the larvae, an organ that has not been widely studied. We provide the first measurements of H + , K + and Na + fluxes at the distal and proximal gastric caecum, and have shown that they differ in the two regions, consistent with previously reported regionalization of ion transporters. Moreover, we have shown that the regionalization of vacuolar H + -ATPase and Na + /K + -ATPase is altered when larvae are reared in brackish water (30% seawater) relative to freshwater. Measurements of luminal Na + and K + concentrations also show a 5-fold increase in Na + /K + ratio in the caecal lumen in larvae reared in brackish water relative to freshwater, whereas transepithelial potential and luminal pH were unchanged. Calculated electrochemical potentials reveal changes in the active accumulation of Na + and K + in the lumen of the gastric caecum of freshwater versus brackish water larvae. Together with the results of previous studies of the larval midgut, our results show that the caecum is functionally distinct from the adjacent anterior midgut, and may play an important role in osmoregulation as well as uptake of nutrients., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

The goal of this study was to identify whether Pacific hagfish (Eptatretus stoutii) possess glucocorticoid and mineralocorticoid responses and to examine the potential role(s) of four key steroids in these responses. Pacific hagfish were injected with varying amounts of cortisol, corticosterone or 11-deoxycorticosterone (DOC) using coconut oil implants and plasma glucose and gill total-ATPase activity were monitored as indices of glucocorticoid and mineralocorticoid responses. Furthermore, we also monitored plasma glucose and 11-deoxycortisol (11-DOC) levels following exhaustive stress (30 min of agitation) or following repeated infusion with SO 4 2- . There were no changes in gill total-ATPase following implantation with any steroid, with only very small statistical increases in plasma glucose noted in hagfish implanted with either DOC (at 20 and 200mgkg -1 at 7 and 4days post-injection, respectively) or corticosterone (at 100mgkg -1 at 7days post-injection). Following exhaustive stress, hagfish displayed a large and sustained increase in plasma glucose. Repeated infusion of SO 4 2- into hagfish caused increases in both plasma glucose levels and SO 4 2- excretion rate suggesting a regulated glucocorticoid and mineralocorticoid response. However, animals under either condition did not show any significant increases in plasma 11-DOC concentrations. Our results suggest that while there are active glucocorticoid and mineralocorticoid responses in hagfish, 11-DOC does not appear to be involved and the identity and primary function of the steroid in hagfish remains to be elucidated., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Anthropogenic sources increase freshwater salinity and produce differences in constituent ions compared with natural waters. Moreover, ions differ in physiological roles and concentrations in intracellular and extracellular fluids. Four freshwater taxa groups are compared, to investigate similarities and differences in ion transport processes and what ion transport mechanisms suggest about the toxicity of these or other ions in freshwater. Although differences exist, many ion transporters are functionally similar and may belong to evolutionarily conserved protein families. For example, the Na + /H + -exchanger in teleost fish differs from the H + /2Na + (or Ca 2+ )-exchanger in crustaceans. In osmoregulation, Na + and Cl - predominate. Stenohaline freshwater animals hyperregulate until they are no longer able to maintain hypertonic extracellular Na + and Cl - concentrations with increasing salinity and become isotonic. Toxic effects of K + are related to ionoregulation and volume regulation. The ionic balance between intracellular and extracellular fluids is maintained by Na + /K + -adenosine triphosphatase (ATPase), but details are lacking on apical K + transporters. Elevated H + affects the maintenance of internal Na + by Na + /H + exchange; elevated HCO 3 - inhibits Cl - uptake. The uptake of Mg 2+ occurs by the gills or intestine, but details are lacking on Mg 2+ transporters. In unionid gills, SO 4 2- is actively transported, but most epithelia are generally impermeant to SO 4 2- . Transporters of Ca 2+ maintain homeostasis of dissolved Ca 2+ . More integration of physiology with toxicology is needed to fully understand freshwater ion effects. Environ Toxicol Chem 2017;36:576-600. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.)

Freshwater fish actively take up ions via specific transporters to counter diffusive losses to their hypotonic environment. While much is known about the specific mechanisms employed by teleosts, almost nothing is known about the basal fishes, such as white sturgeon (Acipenser transmontanus) which may offer insight into the evolution of osmo- and ionoregulation in fishes. We investigated Na + uptake in juvenile white sturgeon in the presence and absence of transporter inhibitors. We found that sturgeon acclimated to 100μmoll -1 Na + have Na + uptake kinetics typical of teleosts and that a Na + /H + exchanger (NHE) is the predominant transporter for Na + uptake. White sturgeon are tolerant to hypercarbia-induced respiratory acidoses and recover blood pH (pH e ) at 1.5kPa PCO 2 but not at higher PCO 2 (6kPa PCO 2 ) where they preferentially regulate intracellular pH (pH i ). It was hypothesized that during exposure to hypercarbia Na + uptake would increase at CO 2 tensions at which fish were capable of pH e regulation but decrease at higher tensions when they were preferentially regulating pH i . We found that Na + uptake did not increase at 1.5kPa PCO 2 , but at 6kPa PCO 2 Na + uptake was reduced by 95% while low water pH equivalent to 6kPa PCO 2 reduced Na + uptake by 71%. Lastly, we measured net acid flux during hypercarbia, which indicates that net acid flux is not associated with Na + uptake. These findings indicate Na + uptake in sturgeon is not different from freshwater teleosts but is sensitive to hypercarbia and is not associated with pH e compensation during hypercarbia., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Although iono-regulatory processes are critical for survival of crustaceans during the molt cycle, the mechanisms involved are still not clear. The Na + /K + /2Cl - cotransporter (NKCC), a SLC12A family protein that transports Na + , K + and 2Cl - into cells, is essential for cell ionic and osmotic regulation. To better understand the role of NKCC in the molt osmoregulation, we cloned and characterized a NKCC gene from the mud crab, Scylla paramamosain (designated as SpNKCC). The predicted SpNKCC protein is well conserved, and phylogenetic analysis revealed that this protein was clustered with crustacean NKCC. Expression of SpNKCC was detected in all the tissues examined but was highest in the posterior gills. Transmission electron microscopy revealed that posterior gills had a thick type of epithelium for ion regulation while the anterior gills possessed a thin phenotype related to gas exchange. During the molting cycle, hemolymph osmolality and ion concentrations (Na + and Cl - ) increased significantly over the postmolt period, remained stable in the intermolt and premolt stages and then decreased at ecdysis. Meanwhile, the expression of SpNKCC mRNA was significantly elevated (26.7 to 338.8-fold) at the ion re-establishing stages (postmolt) as compared with baseline molt level. This pattern was consistent with the coordinated regulation of Na + /K + -ATPase α-subunit (NKA α), carbonic anhydrase cytoplasmic (CAc) isoform and Na + /H + exchanger (NHE) genes in the posterior gills. These data suggest that SpNKCC may be important in mediating branchial ion uptake during the molt cycle, especially at the postmolt stages., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Milkfish, a species within the primitive teleost lineage Otocephala, can survive in water conditions ranging from hypo- to hyper-saline. This study explored the effects of environmental salinity on apical morphologies of ionocytes and the expression of villin homologs in the gills of milkfish acclimated to either seawater (SW) or fresh water (FW). Scanning electron microscopy revealed that the ionocytes in the gill filaments of SW and FW milkfish, respectively, cellular apical morphologies were hole-type and squint-type. The flat-type ionocytes were observed in the gill lamellae of FW milkfish. Furthermore, apical surfaces of some lamellar ionocytes exhibited microvilli. Villin 1 is a microvilli marker expressed in the epithelial cells of various vertebrates. In the phylogenetic tree of villin 1 homologs, primitive teleosts exhibit villin 1-like (VILL) and villin 1 proteins. Two mRNA sequences, villin 1 and VILL, were identified from the milkfish transcriptome by next generation sequencing. Low but constant expression of villin 1 (gene and protein) was observed in the gills for both SW and FW fish. VILL gene and protein expression levels in the gills were higher in FW fish, compared to SW fish. Double immunofluorescence staining demonstrated that VILL protein was present in some lamellar ionocytes of FW milkfish, but not in the filament ionocytes of either FW or SW milkfish. Taken together, these findings indicated that the VILL expression of ionocytes is hypoosmotic-dependent. The VILL might be involved in the formation of microvilli in the lamellar ionocytes for hyperosmoregulation of the milkfish., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In the present study, the effect of copper was examined in the common goldfish (Carassius auratus auratus). Fish were fasted and exposed to either a high (0.84μM), a low (0.34μM) or a control copper concentration (0.05μM) for 1 and 7days. Swimming performance was not affected by either fasting or copper exposure. Food deprivation alone had no effect on ionoregulation, but low plasma osmolality levels and plasma Na + were noticed in fasted fish exposed to Cu for 7days. Both gill Na + /K + -ATPase and H + -ATPase activities were undisturbed, while both kidney ATPase activities were up-regulated when challenged with the high Cu levels. Up-regulated kidney ATPase activities likely acted as compensatory strategy to enhance Na + reabsorption. However, this up-regulation was not sufficient to restore Na + to control levels in the highest exposure group., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Developing freshwater fish must compensate for the loss of ions, including sodium (Na(+)), to the environment. In this study, we used a radiotracer flux approach and pharmacological inhibitors to investigate the role of sodium/hydrogen exchange proteins (Nhe) in Na(+) uptake in rainbow trout (Oncorhynchus mykiss) reared from fertilization in soft water (0.1mM Na(+)). For comparison, a second group of embryos/larvae reared in hard water (2.2mM Na(+), higher pH and [Ca(2+)]) were also included in the experiment but were fluxed in soft water, only. Unidirectional rates of Na(+) uptake increased throughout development and were significantly higher in embryos/larvae reared in soft water. However, the mechanisms of Na(+) uptake in both groups of larvae were not significantly different, either in larvae immediately post-hatch or later in development: the broad spectrum Na(+) channel blocker amiloride inhibited 85-90% of uptake and the Nhe-inhibitor EIPA also caused near maximal inhibitions of Na(+) uptake. These data indicated Na(+) uptake was Nhe-mediated in soft water. A role of Nhe3b (but not Nhe2 or Nhe3a) in Na(+) uptake in soft water was also supported through gene expression analyses: expression of nhe3b increased throughout development in whole embryos/larvae in both groups and was significantly higher in those reared in soft water. This pattern of expression correlated well with measurements of Na(+) uptake. Together these data indicate that in part, rainbow trout embryos/larvae reared in low Na(+) soft water maintained Na(+) homeostasis by an EIPA sensitive component of Na(+) uptake, and support a primary role for Nhe3b., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In teleost fishes, it is well-established that the gill serves as an important ionoregulatory organ in addition to its primary function of respiratory gas exchange. In elasmobranchs, however, the ionoregulatory function of the gills is still incompletely understood. Although two types of ionocytes, Na(+)/K(+)-ATPase (NKA)-rich (type-A) cell and vacuolar-type H(+)-ATPase (V-ATPase)-rich (type-B) cell, have been found in elasmobranch fishes, these cells were considered to function primarily in acid-base regulation. In the present study, we examined ion-transporting proteins expressed in ionocytes of Japanese-banded houndshark, Triakis scyllium, reared in full-strength seawater (SW) and transferred to diluted (30%) SW. In addition to the upregulation of NKA and Na(+)/H(+) exchanger type 3 (NHE3) mRNAs in the type-A ionocytes, we found that Na(+), Cl(-) cotransporter (NCC, Slc12a3) is expressed in a subpopulation of the type-B ionocytes, and that the expression level of NCC mRNA was enhanced in houndsharks transferred to a low-salinity environment. These results suggest that elasmobranch gill ionocytes contribute to NaCl uptake in addition to the already described function of acid-base regulation, and that NCC is most probably one of the key molecules for hyper-osmoregulatory function of elasmobranch gills. The existence of two types of ionocytes (NHE3- and NCC-expressing cells) that are responsible for NaCl absorption seems to be a common feature in both teleosts and elasmobranchs for adaptation to a low salinity environment. A possible driving mechanism for NCC in type-B ionocytes is discussed., (Copyright © 2016 Elsevier Inc. All rights reserved.)

In vertebrates, follicle-stimulating hormone (FSH), luteinizing hormone (LH), chorionic gonadotropin (CG) and thyroid-stimulating hormone (TSH) are glycoprotein hormones that play central roles in metabolism, reproduction and development. Recently, a novel heterodimeric glycoprotein hormone, called GPA2/GPB5, was discovered in humans; however, contrary to its vertebrate glycoprotein hormone relatives, the physiological role of GPA2/GPB5 has not yet been fully elucidated in any vertebrate or invertebrate. Moreover, it is unclear as to whether GPA2/GPB5 functions as a heterodimer or as individual GPA2 and GPB5 monomers in these organisms. GPA2- and GPB5-like subunits have been identified or predicted in a wide array of animal phyla including the nematodes, chordates, hemichordates, arthropods, molluscs, echinoderms and annelids. So far, molecular studies on transcript expression of the GPA2/GPB5 subunits and its putative receptor, the leucine-rich repeat-containing G protein-coupled receptor 1 (LGR1), suggests this glycoprotein hormone system plays a developmental role and may also function in hydromineral balance in invertebrates. This mini-review summarizes the current state of knowledge on the physiological actions and activity of this evolutionarily ancient heterodimeric glycoprotein hormone with a particular focus on its known functions in the invertebrates., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The euryhaline galaxiid fish, inanga (Galaxias maculatus) is widely spread throughout the Southern hemisphere occupying near-coastal streams that may be elevated in trace elements such as copper (Cu). Despite this, nothing is known regarding their sensitivity to Cu contamination. The mechanisms of Cu toxicity in inanga, and the ameliorating role of salinity, were investigated by acclimating fish to freshwater (FW), 50% seawater (SW), or 100% SW and exposing them to a graded series of Cu concentrations (0-200μgL(-1)) for 48h. Mortality, whole body Cu accumulation, measures of ionoregulatory disturbance (whole body ions, sodium (Na) influx, sodium/potassium ATPase activity) and ammonia excretion were monitored. Toxicity of Cu was greatest in FW, with mortality likely resulting from impaired Na influx. In both FW and 100% SW, ammonia excretion was significantly elevated, an effect opposite to that observed in previous studies, suggesting fundamental differences in the effect of Cu in this species relative to other studied fish. Salinity was protective against Cu toxicity, and physiology seemed to play a more important role than water chemistry in this protection. Inanga are sensitive to waterborne Cu through a conserved impairment of Na ion homeostasis, but some effects of Cu exposure in this species are distinct. Based on effect concentrations, current regulatory tools and limits are likely protective of this species in New Zealand waters., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Haemolymph calcium homeostasis in insects is achieved through the regulation of calcium excretion by Malpighian tubules in two ways: (1) sequestration of calcium within biomineralized granules and (2) secretion of calcium in soluble form within the primary urine. Using the scanning ion-selective electrode technique (SIET), basolateral Ca(2+) transport was measured at the distal, transitional, main and proximal tubular segments of anterior tubules isolated from both 3rd instar larvae and adults of the fruit fly Drosophila melanogaster. Basolateral Ca(2+) transport exceeded transepithelial secretion by 800-fold and 11-fold in anterior tubules of larvae and adults, respectively. The magnitude of Ca(2+) fluxes across the distal tubule of larvae and adults were larger than fluxes across the downstream segments by 10 and 40 times, respectively, indicating a dominant role for the distal segment in whole animal Ca(2+) regulation. Basolateral Ca(2+) transport across distal tubules of Drosophila varied throughout the life cycle; Ca(2+) was released by distal tubules of larvae, taken up by distal tubules of young adults and was released once again by tubules of adults ⩾ 168 h post-eclosion. In adults and larvae, SIET measurements revealed sites of both Ca(2+) uptake and Ca(2+) release across the basolateral surface of the distal segment of the same tubule, indicating that Ca(2+) transport is bidirectional. Ca(2+) uptake across the distal segment of tubules of young adults and Ca(2+) release across the distal segment of tubules of older adults was also suggestive of reversible Ca(2+) storage. Our results suggest that the distal tubules of D. melanogaster are dynamic calcium stores which allow efficient haemolymph calcium regulation through active Ca(2+) sequestration during periods of high dietary calcium intake and passive Ca(2+) release during periods of calcium deficiency., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

The mechanism of copper (Cu) toxicity in marine invertebrates remains unclear. Therefore, marine clams (Mesodesma mactroides) were exposed (96h) to a concentration of dissolved Cu (1.6μmolL(-1)) inducing 10% mortality in sea water (30ppt). After in vivo exposure, tissue Cu accumulation (hemolymph, gill and digestive gland); hemolymph ionic (Na(+), K(+), Mg(2+) and Ca(2+)) and osmotic concentrations; tissue (gill and digestive gland) ionic concentration, enzyme (Na(+),K(+)-ATPase and carbonic anhydrase) activity, and oxygen consumption; and whole-body oxygen consumption were analyzed. Succinate dehydrogenase activity was evaluated in mitochondria isolated from gills and digestive gland and exposed (1h) in vitro to different concentrations of dissolved Cu (0.8, 7.7 and 78.7μmolL(-1)). In vivo exposure induced Cu accumulation in hemolymph, gills and digestive gland; increased Mg(2+) and decreased Ca(2+) concentration in hemolymph; decreased Mg(2+) concentration, increased Na(+),K(+)-ATPase activity and reduced carbonic anhydrase activity in gills; decreased Mg(2+) concentration, increased Ca(2+) concentration and increased Na(+),K(+)-ATPase activity in digestive gland; and reduced gill, digestive gland and whole-body oxygen consumption. Succinate dehydrogenase activity was inhibited after in vitro exposure to 78.7μmolL(-1) Cu. These findings indicate that Cu is an ionoregulatory toxicant in the marine clam M. mactroides. However, toxicity is related to disturbances in regulation of divalent cations (Mg(2+) and Ca(2+)) without effect on regulation of major monovalent cations (Na(+) and K(+)), as opposed to that observed in osmoregulating invertebrates exposed to Cu. However, other mechanism(s) of toxicity cannot be ruled out. Future studies must be performed to evaluate the consequence of the Cu-induced respiratory disturbances observed in M. mactroides., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The fate and effect of nanomaterials in the environment has raised concern about their environmental risk to aquatic organisms. Silica nanoparticles (SiO2-NPs) find its uses in various fields and are inevitably released into the environment. However, the ecotoxicological effects of SiO2-NPs on the freshwater fish remain poorly understood. The aim of this study was to evaluate the effect of different concentrations (1, 5 and 25mgL(-1)) of SiO2-NPs on certain hematological, ionoregulatory and enzymological profiles of a freshwater teleost fish Labeo rohita. Hematological parameters such as hemoglobin (Hb), hematocrit (Hct), red blood cells (RBC), white blood cells (WBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) values were altered in SiO2-NPs treated groups. Likewise, plasma electrolytes such as plasma sodium (Na(+)), potassium (K(+)) and chloride (Cl(-)) levels and Na(+)/K(+) ATPase activity in gill of SiO2-NPs treated groups were altered in all concentrations throughout the study period (96h). The alterations of these parameters were found to be dependent on dose and exposure period. The results of the present study indicate that the alterations of these parameters may relate to physiological stress system to SiO2-NPs toxicity and also demonstrate that manufactured metal oxide NPs in aquatic environment may affect the health condition of the aquatic organisms., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The euryhaline pupfish, Cyprinodon variegatus variegatus (Cvv), can successfully osmoregulate in ≥2 mM Na(+) and a freshwater population (Cyprinodon variegatus hubbsi; Cvh) osmoregulates at ≥0.1mM Na(+). We previously demonstrated that Cvv relies on an apical NKCC and NHE in the gill for Na(+) uptake in high (7mM) and intermediate (2 mM) Na(+) concentrations, while Cvh relies only on NHE for Na(+) uptake. This study investigated whether differential NHE isoform use explains differences in Na(+) uptake kinetics between these two populations. We further studied whether Cvh uses a NHE-Rh metabolon or carbonic anhydrase (CA) to overcome thermodynamic challenges of NHE function in dilute freshwater. Transfer to more dilute freshwater resulted in upregulation of nhe-2 (Cvv only) and nhe-3 (Cvv and Cvh). Relative expression of nhe-3 compared to nhe-2 was 2-fold higher in Cvv, but 200-fold higher in Cvh suggesting that nhe-3 expression is an important freshwater adaptation for Cvh. Simultaneous measurement of Na(+) and Tamm flux under various conditions provided no support for a NHE-Rh metabolon in either population. Carbonic anhydrase activity in Cvv was comparable in 7 and 2 mM Na(+) acclimated fish. In Cvh, CA activity increased by 75% in 0.1 mM Na(+) acclimated fish compared to 7 mM Na(+) fish. Ethoxzolamide had variable effects, stimulating and reducing Na(+) uptake in Cvv acclimated to 7 and 2 mM Na(+), while reducing Na(+) uptake in 7 and 0.1mM Na(+) acclimated Cvh. This suggests that CA plays important, but different roles in regulating Na(+) uptake in Cvv and Cvh., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Acid-sensing ion channels (ASICs) are epithelial Na(+) channels gated by external H(+). Recently, it has been demonstrated that ASICs play a role in Na(+) uptake in freshwater rainbow trout. Here, we investigate the potential involvement of ASICs in Na(+) transport in another freshwater fish species, the zebrafish (Danio rerio). Using molecular and histological techniques we found that asic genes and the ASIC4.2 protein are expressed in the gill of adult zebrafish. Immunohistochemistry revealed that mitochondrion-rich cells positive for ASIC4.2 do not co-localize with Na(+)/K(+)-ATPase-rich cells, but co-localize with cells expressing vacuolar-type H(+)-ATPase. Furthermore, pharmacological inhibitors of ASIC and Na(+)/H(+)-exchanger significantly reduced uptake of Na(+) in adult zebrafish exposed to low-Na(+) media, but did not cause the same response in individuals exposed to ultra-low-Na(+) water. Our results suggest that in adult zebrafish ASICs play a role in branchial Na(+) uptake in media with low Na(+) concentrations and that mechanisms used for Na(+) uptake by zebrafish may depend on the Na(+) concentration in the acclimation medium., (© 2015. Published by The Company of Biologists Ltd.)

We investigated the interactive effect of ammonia toxicity, salinity challenge and nutritional status on the ecophysiological performance of European sea bass (Dicentrarchus labrax). Fish were progressively acclimated to normal seawater (32ppt), to brackish water (20ppt and 10ppt) and to hyposaline water (2.5ppt). Following acclimation to different salinities for two weeks, fish were exposed to high environmental ammonia (HEA, 20mg/L ∼1.18mM representing 50% of 96h LC50 value for ammonia) for 12h, 48h, 84h and 180h, and were either fed (2% body weight) or fasted (unfed for 7 days prior to HEA exposure). Biochemical responses such as ammonia (Jamm) and urea excretion rate, plasma ammonia, urea and lactate, plasma ions (Na(+), Cl(-) and K(+)) and osmolality, muscle water content (MWC) and liver and muscle energy budget (glycogen, lipid and protein), as well as branchial Na(+)/K(+)-ATPase (NKA) and H(+)-ATPase activity, and branchial mRNA expression of NKA and Na(+)/K(+)/2Cl(-) co-transporter (NKCC1) were investigated in order to understand metabolic and ion- osmoregulatory consequences of the experimental conditions. During HEA, Jamm was inhibited in fasted fish at 10ppt, while fed fish were still able to excrete efficiently. At 2.5ppt, both feeding groups subjected to HEA experienced severe reductions and eventually a reversion in Jamm. Overall, the build-up of plasma ammonia in HEA exposed fed fish was much lower than fasted ones. Unlike fasted fish, fed fish acclimated to lower salinities (10ppt-2.5ppt) could maintain plasma osmolality, [Na(+)], [Cl(-)] and MWC during HEA exposure. Thus fed fish were able to sustain ion-osmotic homeostasis which was associated with a more pronounced up-regulation in NKA expression and activity. At 2.5ppt both feeding groups activated H(+)-ATPase. The expression of NKCC1 was down-regulated at lower salinities in both fed and fasted fish, but was upregulated within each salinity after a few days of HEA exposure. Though an increment in plasma lactate content and a decline in energy stores were noted for both feeding regimes, the effect was more severe in feed deprived fish. Overall, several different physiological processes were disturbed in fasted sea bass during HEA exposure while feeding alleviated adverse effects of high ammonia and salinity challenge. This suggests that low food availability can render fish more vulnerable to external ammonia, especially at reduced seawater salinities., (Copyright © 2015 Elsevier B.V. All rights reserved.)