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The fish gill serves many physiological functions, among which is the excretion of ammonia, the primary nitrogenous waste in most fishes. Although it is the end-product of nitrogen metabolism, ammonia serves many physiological functions including acting as an acid equivalent and as a counter-ion in mechanisms of ion regulation. Our current understanding of the mechanisms of ammonia excretion have been influenced by classic experimental work, clever mechanistic approaches, and modern molecular and genetic techniques. In this review, I will overview the history of the study of ammonia excretion by the gills of fishes, highlighting the important advancements that have shaped this field with a nearly 100-year history. The developmental and evolutionary implications of an ammonia and gill-dominated nitrogen regulation strategy in most fishes will also be discussed. Throughout the review, I point to areas in which more work is needed to push forward this field of research that continues to produce novel insights and discoveries that will undoubtedly shape our overall understanding of fish physiology. [ABSTRACT FROM AUTHOR]

The mechanism(s) of sodium, chloride and pH regulation in teleost fishes has been the subject of intense interest for researchers over the past 100 years. The primary organ responsible for ionoregulatory homeostasis is the gill, and more specifically, gill ionocytes. Building on the theoretical and experimental research of the past, recent advances in molecular and cellular techniques in the past two decades have allowed for substantial advances in our understanding of mechanisms involved. With an increased diversity of teleost species and environmental conditions being investigated, it has become apparent that there are multiple strategies and mechanisms employed to achieve ion and acid-base homeostasis. This review will cover the historical developments in our understanding of the teleost fish gill, highlight some of the recent advances and conflicting information in our understanding of ionocyte function, and serve to identify areas that require further investigation to improve our understanding of complex cellular and molecular machineries involved in iono- and acid-base regulation. [ABSTRACT FROM AUTHOR]

In teleost fish, branchial ionocytes are important sites for osmoregulation and acid-base regulation by maintaining ionic balance in the body fluid. During the early developmental stages before the formation of the gills, teleost ionocytes are localized in the yolk-sac membrane and body skin. By comparing with teleost fish, much less is known about ionocytes in developing embryos of elasmobranch fish. The present study investigated the development of ionocytes in the embryo and larva of cloudy catshark, Scyliorhinus torazame. We first observed ionocyte distribution by immunohistochemical staining with anti-Na+/K+-ATPase (NKA) and anti-vacuolar-type H+-ATPase (V-ATPase) antibodies. The NKA- and V-ATPase-rich ionocytes appeared as single cells in the gill filaments from stage 31, the stage of pre-hatching, while the ionocytes on the body skin and yolk-sac membrane were also observed. From stage 32, in addition to single ionocytes on the gill filaments, some outstanding follicular structures of NKA-immunoreactive cells were developed to fill the inter-filament region of the gill septa. The follicular ionocytes possess NKA in the basolateral membrane and Na+/H+ exchanger 3 in the apical membrane, indicating that they are involved in acid-base regulation like single NKA-rich ionocytes. Three-dimensional analysis and whole-mount immunohistochemistry revealed that the distribution of follicular ionocytes was limited to the rostral side of gill septum. The rostral sides of gill septum might be exposed to faster water flow than caudal side because the gills of sharks gently curved backward. This dissymmetric distribution of follicular ionocytes is considered to facilitate efficient body-fluid homeostasis of catshark embryo. [ABSTRACT FROM AUTHOR]

An important goal of environmental and comparative physiology research is to identify species or populations that may be susceptible to environmental change such as heat wave events that are predicted to become more frequent and intense in the future. This study tested the hypothesis that fishes inhabiting alkaline lakes face significant physiological challenges, which results in reduced thermal tolerance. Brook stickleback (Culaea inconstans) were collected from an alkaline lake (pH 9.3) in Alberta, Canada and held under neutral conditions in the laboratory. Subsequently, fish were acutely exposed (4 d) to neutral (pH 7) or alkaline (pH 9.5) waters at 10 or 25°C. Exposure to alkaline water reduced critical thermal maximum (CTmax) in stickleback by approximately 1°C, but thermal acclimation capacity ("thermal plasticity") was unaffected by alkaline exposure. Alkaline conditions resulted in physiological disturbances characteristic of exposure to high pH including elevated whole-body ammonia and lactate concentrations. Acute warming to CTmax in alkaline-exposed fish resulted in reductions in whole-body sodium and chloride concentrations. In addition, alkaline exposure compromised recovery from exercise at elevated temperatures. Overall, these results suggest that the physiological disturbances observed in response to alkaline exposure may render fish more susceptible to acute warming, reducing thermal tolerance. [ABSTRACT FROM AUTHOR]

The insect ion transport peptide (ITP) and its alternatively spliced variant, ITP-like peptide (ITP-L), belong to the crustacean hyperglycemic hormone family of peptides and are widely conserved among insect species. While limited, studies have characterized the ITP/ITP-L signaling system within insects, and putative functions including regulation of ion and fluid transport, ovarian maturation, and thirst/excretion have been proposed. Herein, we aimed to molecularly investigate Itp and Itp-l expression profiles in the mosquito, Aedes aegypti, examine peptide immunolocalization and distribution within the adult central nervous system, and elucidate physiological roles for these neuropeptides. Transcript expression profiles of both AedaeItp and AedaeItp-l revealed distinct enrichment patterns in adults, with AedaeItp expressed in the brain and AedaeItp-l expression predominantly within the abdominal ganglia. Immunohistochemical analysis within the central nervous system revealed expression of AedaeITP peptide in a number of cells in the brain and in the terminal ganglion. Comparatively, AedaeITP-L peptide was localized solely within the pre-terminal abdominal ganglia of the central nervous system. Interestingly, prolonged desiccation stress caused upregulation of AedaeItp and AedaeItp-l levels in adult mosquitoes, suggesting possible functional roles in water conservation and feeding-related activities. RNAi-mediated knockdown of AedaeItp caused an increase in urine excretion, while knockdown of both AedaeItp and AedaeItp-l reduced blood feeding and egg-laying in females as well as hindered egg viability, suggesting roles in reproductive physiology and behavior. Altogether, this study identifies AedaeITP and AedaeITP-L as key pleiotropic hormones, regulating various critical physiological processes in the disease vector, A. aegypti. [ABSTRACT FROM AUTHOR]

Harmful algal blooms kill fish populations worldwide, as exemplified by the haptophyte microalga Prymnesium parvum. The suspected causative agents are prymnesins, categorized as A-, B-, and C-types based on backbone carbon atoms. Impacts of P. parvum extracts and purified prymnesins were tested on the epithelial rainbow trout fish gill cell line RTgill-W1 and on the human colon epithelial cells HCEC-1CT. Cytotoxic potencies ranked A > C > B-type with concentrations spanning from low (A- and C-type) to middle (B-type) nM ranges. Although RTgill-W1 cells were about twofold more sensitive than HCEC-1CT, the cytotoxicity of prymnesins is not limited to fish gills. Both cell lines responded rapidly to prymnesins; with EC50 values for B-types in RTgill-W1 cells of 110 ± 11 nM and 41.5 ± 0.6 nM after incubations times of 3 and 24 h. Results of fluorescence imaging and measured lytic effects suggest plasma membrane interactions. Postulating an osmotic imbalance as mechanisms of toxicity, incubations with prymnesins in media lacking either Cl−, Na+, or Ca2+ were performed. Cl− removal reduced morphometric rearrangements observed in RTgill-W1 and cytotoxicity in HCEC-1CT cells. Ca2+-free medium in RTgill-W1 cells exacerbated effects on the cell nuclei. Prymnesin composition of different P. parvum strains showed that analog composition within one type scarcely influenced the cytotoxic potential, while analog type potentially dictate potency. Overall, A-type prymnesins were the most potent ones in both cell lines followed by the C-types, and lastly B-types. Disturbance of Ca2+ and Cl− ionoregulation may be integral to prymnesin toxicity. [ABSTRACT FROM AUTHOR]

Fish mortality is associated with harmful algal blooms, although whether toxicity is related directly to the presence of cyanotoxins or the prevailing water chemistry remains unclear. Similarly, while planktivorous fish may be exposed to toxin through the diet, the hazard posed by waterborne extracellular toxin to carnivorous fish is less well understood. In this study rainbow trout (Oncorhynchus mykiss) were exposed for up to 28 d to waterborne microcystin-LR at nominal concentrations of 1.5 and 50 µg L−1 (measured values 2 and 49 µg L−1, respectively). The former represents the Canadian drinking water guideline, and the latter an elevated environmental level. This study hypothesised that waterborne toxin exposure would specifically impact gill function, and given the importance of this tissue in freshwater fish ion regulation, effects on plasma ions and branchial ion transporter activity would be observed. Microcystin-LR exposure resulted in a significant and persistent hypocalcaemia at the higher exposure concentration, but plasma sodium and branchial activities of the sodium/potassium ATPase, proton ATPase and calcium ATPase enzymes remained unaffected. An in vitro assessment failed to show any effect of microcystin-LR on branchial calcium ATPase activity even at exposure concentrations as high as 1000 µg L−1. A transient increase in hepatic alkaline phosphatase activity was also observed at 49 µg L−1, but there were no effects of toxin exposure on branchial or hepatic lactate dehydrogenase activity. These results suggest that microcystin-LR exposure does not have a general effect on ion regulation, but instead produces a novel and specific impact on calcium metabolism in rainbow trout, although the mechanism underlying this effect remains unknown.

Freshwater habitats of the Neotropics exhibit a gradient from relatively neutral, ion-rich whitewater to acidic, ion-poor blackwater. Closely related species often show complementary distributions among regions with divergent water quality, suggesting that distinct osmoregulatory environments may be a major constraint to fish distribution and an important driver of Neotropical fish diversity. To provide a foundation for further investigation, we reconstructed the gill filament transcriptome of Cichla ocellaris monoculus, a highly exploited Neotropical cichlid that inhabits a broad range of habitats, in experimental conditions meant to mimic opposing endpoints of the whitewater-blackwater gradient. The optimal combined transcriptome contained 185,480 transcripts (> 200 bp) and contained 281.9 Mbp (N50 2,648 bp), from which 17,379 putatively orthologous protein-coding loci were annotated. We observed evidence of utilization of peptide hormone signaling that may have activated signaling cascades to moderate the permeability of the gills in response to these physicochemical challenges. Expression of genes related to paracellular tight junctions and transcellular ion transport indicated responses broadly similar to euryhaline fishes in fresh versus seawater. These results provide a useful foundation for investigating the axes of acclimation or constraint for Neotropical fishes facing increasing habitat modification due to anthropogenic forces, and more broadly the proximate and ultimate modes of diversification in the hyperdiverse Amazon fish fauna. [ABSTRACT FROM AUTHOR]

To evaluate the physiological ability to adjust to environmental variations of salinity, Carcinus maenas were maintained in 10, 20, 32 (control), 40, and 50 ppt (13.8 ± 0.6 °C) for 7 days. Closed respirometry systems were used to evaluate oxygen consumption ( M ˙ O 2 ), ammonia excretion (Jamm), urea-N excretion (Jurea-N) and diffusive water fluxes (with 3H2O). Ions, osmolality, metabolites, and acid–base status were determined in the hemolymph and seawater, and transepithelial potential (TEP) was measured. At the lowest salinity, there were marked increases in M ˙ O 2 and Jamm, greater reliance on N-containing fuels to support aerobic metabolism, and a state of internal metabolic alkalosis (increased [HCO3−]) despite lower seawater pH. At higher salinities, an activation of anaerobic metabolism and a state of metabolic acidosis (decreased [HCO3−] and increased [lactate]), in combination with respiratory compensation (decreased PCO2), were detected. TEP became more negative with decreasing salinity. Osmoregulation and osmoconformation occurred at low and high salinities, respectively, with complex patterns in individual ions; hemolymph [Mg2+] was particularly well regulated at levels well below the external seawater at all salinities. Diffusive water flux rates increased at higher salinities. Our results show that C. maenas exhibits wide plasticity of physiological responses when acclimated to different salinities and tolerates substantial disturbances of physiological parameters, illustrating that this species is well adapted to invade and survive in diverse habitats. [ABSTRACT FROM AUTHOR]

The gills are the primary site of exchange in fishes. However, during early life-stages or in amphibious fishes, ionoregulation and gas-exchange may be primarily cutaneous. Given the similarities between larval and amphibious fishes, we hypothesized that cutaneous larval traits are continuously expressed in amphibious fishes across all life-stages to enable the skin to be a major site of exchange on land. Alternatively, we hypothesized that cutaneous larval traits disappear in juvenile stages and are re-expressed in amphibious species in later life-stages. We surveyed six species spanning a range of amphibiousness and characterized cutaneous ionocytes and neuroepithelial cells (NECs) as representative larval skin traits at up to five stages of development. We found that skin ionocyte density remained lower and constant in exclusively water-breathing, relative to amphibious species across development, whereas in amphibious species ionocyte density generally increased. Additionally, adults of the most amphibious species had the highest cutaneous ionocyte densities. Surprisingly, cutaneous NECs were only identified in the skin of one amphibious species (Kryptolebias marmoratus), suggesting that cutaneous NECs are not a ubiquitous larval or amphibious skin trait, at least among the species we studied. Our data broadly supports the continuous-expression hypothesis, as three of four amphibious experimental species expressed cutaneous ionocytes in all examined life-stages. Further, the increasing density of cutaneous ionocytes across development in amphibious species probably facilitates the prolonged occupation of terrestrial habitats. [ABSTRACT FROM AUTHOR]

Extensive usage of silver in several applications may contaminate the freshwater compartment and affects the health status of aquatic organisms. In the present investigation, fingerlings of Indian major carp Labeo rohita were exposed (35 days) to sublethal concentrations of silver nitrate (AgNO3) and bioaccumulation pattern, biochemical and histological alterations were evaluated. Initially, the median lethal concentration (LC 50) of silver nitrate to the fish Labeo rohita for 96 h was calculated and it was found to be 0.035 mg L−1. To assess the toxicity of ionic silver two sublethal concentrations (0.0035 mg L−1: 1/10th of LC 50 and 0.007 mg L−1; 1/5th of LC 50) were selected and the experiment was conducted for a period of 35 days. A considerable quantity of AgNO3 was accumulated in the gill, liver and kidney tissues of Labeo rohita. Accumulation of AgNO3 was high in liver tissue compared to other organs/tissue. Membrane-bound enzyme (Na+/K+/-ATPase) activity was inhibited in both the concentrations when compared with the control group. Likewise, plasma electrolytes (Na+, K+ and Cl−) level were significantly decreased when compared with untreated groups. On the other hand, a noticeably increased level of glucose was observed in fish exposed to 0.0035 and 0.007 mg L−1 of AgNO3. Moreover, declining protein level was assessed during the initial days (7 and 14 days), and the level was increased in the rest of the exposure period. Extensive histopathological abnormalities were noticed in gill (hyperplasia, epithelial lifting, curling of lamellae) and liver (degeneration of hepatic nucleus, vacuolization, hepatic necrosis) of fish treated with silver nitrate when compared to control groups. The results obtained in the present study revealed that the bioaccumulation pattern, alterations in membrane-bound gill-ATPase activity, plasma electrolytes, biochemical and histological parameters of fish can be useful for biomonitoring the changes in the environment and health condition of fish in freshwater ecosystem contaminated with silver.

Aim: To determine whether Na+ uptake in adult zebrafish (Danio rerio) exposed to acidic water adheres to traditional models reliant on Na+/H+ Exchangers (NHEs), Na+ channels and Na+/Cl− Cotransporters (NCCs) or if it occurs through a novel mechanism. Methods: Zebrafish were exposed to control (pH 8.0) or acidic (pH 4.0) water for 0‐12 hours during which 22Na+ uptake (JNain), ammonia excretion, net acidic equivalent flux and net K+ flux (JHnet) were measured. The involvement of NHEs, Na+ channels, NCCs, K+‐channels and K+‐dependent Na+/Ca2+ exchangers (NCKXs) was evaluated by exposure to Cl−‐free or elevated [K+] water, or to pharmacological inhibitors. The presence of NCKXs in gill was examined using RT‐PCR. Results: JNain was strongly attenuated by acid exposure, but gradually recovered to control rates. The systematic elimination of each of the traditional models led us to consider K+ as a counter substrate for Na+ uptake during acid exposure. Indeed, elevated environmental [K+] inhibited JNain during acid exposure in a concentration‐dependent manner, with near‐complete inhibition at 10 mM. Moreover, JHnet loss increased approximately fourfold at 8‐10 hours of acid exposure which correlated with recovered JNain in 1:1 fashion, and both JNain and JHnet were sensitive to tetraethylammonium (TEA) during acid exposure. Zebrafish gills expressed mRNA coding for six NCKX isoforms. Conclusions: During acid exposure, zebrafish engage a novel Na+ uptake mechanism that utilizes the outwardly directed K+ gradient as a counter‐substrate for Na+ and is sensitive to TEA. NKCXs are promising candidates to mediate this K+‐dependent Na+ uptake, opening new research avenues about Na+ uptake in zebrafish and other acid‐tolerant aquatic species. [ABSTRACT FROM AUTHOR]

Cortisol is a major osmoregulatory hormone in fishes. Cortisol acts upon the gills, the primary site of ionoregulation, through modifications to specialized ion-transporting cells called ionocytes. We tested the hypothesis that cortisol also acts as a major regulator of skin ionocyte remodelling in the amphibious mangrove rivulus (Kryptolebias marmoratus) when gill function ceases during the water-to-land transition. When out of water, K. marmoratus demonstrated a robust cortisol response, which was linked with the remodelling of skin ionocytes to increase cell cross-sectional area and Na+-K+-ATPase (NKA) content, but not when cortisol synthesis was chemically inhibited by metyrapone. Additionally, we discovered a novel morphology of skin-specific ionocyte that are spikey with multiple cell processes. Spikey ionocytes increased in density, cell cross-sectional area and NKA content during air exposure, but not in metyrapone-treated fish. Our findings demonstrate that skin ionocyte remodelling during the water-to-land transition in amphibious fish is regulated by cortisol, the same hormone that regulates gill ionocyte remodelling in salinity-challenged teleosts, suggesting conserved hormonal function across diverse environmental disturbances and organs in fishes. [ABSTRACT FROM AUTHOR]

Although blackwaters, named for their rich content of dissolved organic carbon (DOC), are often very poor in ions and very acidic, they support great fish biodiversity. Indeed, about 8% of all freshwater fish species live in the blackwaters of the Rio Negro watershed in the Amazon basin. We review how native fish survive these harsh conditions that would kill most freshwater fish, with a particular focus on the role of DOC, a water quality parameter that has been relatively understudied. DOC, which is functionally defined by its ability to pass through a 0.45‐µm filter, comprises a diverse range of compounds formed by the breakdown of organic matter and is quantified by its carbon component that is approximately 50% by mass. Adaptations of fish to acidic blackwaters include minimal acid–base disturbances associated with a unique, largely unknown, high‐affinity Na+ uptake system that is resistant to inhibition by low pH in members of the Characiformes, and very tight regulation of Na+ efflux at low pH in the Cichliformes. Allochthonous (terrigenous) DOC, which predominates in blackwaters, consists of larger, more highly colored, reactive molecules than autochthonous DOC. The dissociation of protons from allochthonous components such as humic and fulvic acids is largely responsible for the acidity of these blackwaters, yet at the same time, these components may help protect organisms against the damaging effects of low water pH. DOC lowers the transepithelial potential (TEP), mitigates the inhibition of Na+ uptake and ammonia excretion, and protects against the elevation of diffusive Na+ loss in fish exposed to acidic waters. It also reduces the gill binding and toxicity of metals. At least in part, these actions reflect direct biological effects of DOC on the gills that are beneficial to ionoregulation. After chronic exposure to DOC, some of these protective effects persist even in the absence of DOC. Two characteristics of allochthonous DOC, the specific absorbance coefficient at 340 nm (determined optically) and the PBI (determined by titration), are indicative of both the biological effectiveness of DOC and the ability to protect against metal toxicity. Future research needs are highlighted, including a greater mechanistic understanding of the actions of DOCs on gill ionoregulatory function, morphology, TEP, and metal toxicity. These should be investigated in a wider range of native fish Orders that inhabit one of the world's greatest biodiversity hotspots for freshwater fishes. Research Highlights: Dissolved organic carbon (DOC) mitigates metal toxicity and provides ionoregulatory protection to freshwater fish, particularly in ion‐poor, acidic waters.The role of DOC in this protection is reviewed and future research needs are highlighted. [ABSTRACT FROM AUTHOR]

The Amur ide (Leuciscus waleckii) is a fish in the Cyprinidae family. Compared with other Amur ide living in freshwater ecosystems, the Amur ide population in Lake Dali Nor of China is famous for its high tolerance to the alkaline conditions of 54 mM (pH 9.6). Yet, surprisingly, the ionoregulatory mechanism responsible for this remarkable alkaline adaptation remains unclear. Therefore, this study sought to investigate how bicarbonate affects the acid-base balancing and ionoregulatory responses of this animal. Here, using a comparative approach, the alkali form of Amur ide and its ancestral freshwater form living in other freshwater basins were each exposed to 50 mM (pH 9.59 ± 0.09), a level close to the alkalinity of Lake Dali Nor, and their physiological (AE1) adjustment of ions and acid-base regulation were investigated. This study highlighted differences in blood pH and serum ions (e.g., Na+, K+, Cl−, and Ca2+), Na+/K+ ATPase (NKA) activity and its mRNA level, and mRNA expression of gill transporters (Na+/H+ exchanger member 2 and/or 3, Na+/HCO3- cotransporter (NBC1), Cl−/HCO3- exchanger, Na+/Cl− cotransporter (NCC), Na+/K+/2Cl− (NKCC1), SLC26A5, and SLC26A6) for alkalinity adaptation between the two forms of Amur ide differing in alkalinity tolerance. Specifically, close relationships among the serum Na+ and mRNA levels of NCC, NKCC1, and NHE, and also NKA and NBC1, in addition to serum Cl− and bicarbonate transporters (e.g., SLC26A5 and SLC26A6), characterized the alkali form of Amur ide. We propose that this ecotype can ensure its transepithelial Cl− and Na+ uptake/base secretions are highly functional, by its basolateral NKA with NBC1 and apical ionic transporters, and especially NCC incorporated with other transporters (e.g., SLC26). This suggests an evolved strong ability to maintain an ion osmotic and acid-base balance for more effectively facilitating its adaptability to the high alkaline environment. This study provides new insights into the physiological responses of the alkaline form of the Amur ide fish for adapting to extreme alkaline conditions. This information could be used as a reference to cultivating alkaline-tolerant fish species in abandoned alkaline waters.

The Amur ide (Leuciscus waleckii) is a fish in the Cyprinidae family. Compared with other Amur ide living in freshwater ecosystems, the Amur ide population in Lake Dali Nor of China is famous for its high tolerance to the alkaline conditions of 54 mM (pH 9.6). Yet, surprisingly, the ionoregulatory mechanism responsible for this remarkable alkaline adaptation remains unclear. Therefore, this study sought to investigate how bicarbonate affects the acid-base balancing and ionoregulatory responses of this animal. Here, using a comparative approach, the alkali form of Amur ide and its ancestral freshwater form living in other freshwater basins were each exposed to 50 mM (pH 9.59 ± 0.09), a level close to the alkalinity of Lake Dali Nor, and their physiological (AE1) adjustment of ions and acid-base regulation were investigated. This study highlighted differences in blood pH and serum ions (e.g., Na+, K+, Cl−, and Ca2+), Na+/K+ ATPase (NKA) activity and its mRNA level, and mRNA expression of gill transporters (Na+/H+ exchanger member 2 and/or 3, Na+/ HCO 3 - cotransporter (NBC1), Cl−/ HCO 3 - exchanger, Na+/Cl− cotransporter (NCC), Na+/K+/2Cl− (NKCC1), SLC26A5, and SLC26A6) for alkalinity adaptation between the two forms of Amur ide differing in alkalinity tolerance. Specifically, close relationships among the serum Na+ and mRNA levels of NCC, NKCC1, and NHE, and also NKA and NBC1, in addition to serum Cl− and bicarbonate transporters (e.g., SLC26A5 and SLC26A6), characterized the alkali form of Amur ide. We propose that this ecotype can ensure its transepithelial Cl− and Na+ uptake/base secretions are highly functional, by its basolateral NKA with NBC1 and apical ionic transporters, and especially NCC incorporated with other transporters (e.g., SLC26). This suggests an evolved strong ability to maintain an ion osmotic and acid-base balance for more effectively facilitating its adaptability to the high alkaline environment. This study provides new insights into the physiological responses of the alkaline form of the Amur ide fish for adapting to extreme alkaline conditions. This information could be used as a reference to cultivating alkaline-tolerant fish species in abandoned alkaline waters. [ABSTRACT FROM AUTHOR]

To improve our understanding of underlying toxic mechanisms, it is important to evaluate differences in effects that a variety of metals exert at concentrations representing the same toxic level to the organism. Therefore, the main goal of the present study was to compare the effects of waterborne copper (Cu(II)), zinc (Zn(II)) and cadmium (Cd (II)) on a freshwater fish, the common carp (Cyprinus carpio), at concentrations being 0%, 25%, 50% and 100% of the 96 h LC50 (the concentration which is lethal to 50% of the population in 96 h). All the exposures were performed for a period of 1 week at 20°C. Our results show a rapid increase in the amount of copper and cadmium accumulated in the gills, while zinc only started to increase by the end of the experiment. All three metal ions increased metallothionein gene expression in both gills and liver. However, clear adverse effects were mainly observed for the Cu exposed group. Cu caused a decrease in Na level in gill tissue; it altered the expression of genes involved in ionoregulation such as Na+/K+‐ATPase and H+‐ATPase as well as the expression of oxidative stress‐related genes, such as catalase, glutathione reductase and glutathione S‐transferase. Zinc and cadmium exposure did not alter the ion levels in the gills. In addition, no obvious effect of oxidative stress was observed, except for a transient increase in glutathione reductase at the highest cadmium concentration. Common carp were exposed to several Cu (II), Zn(II) and Cd(II) concentrations for a period of 1 week at 20°C. Our results show that both Cu and Cd accumulate fast, whereas this was not the case for Zn. Most of the adverse effects were caused by Cu (e.g., on ion balance and ion transporters). All three metals triggered the response of defensive mechanisms such as metallothionein gene expression (MT). [ABSTRACT FROM AUTHOR]

Boron oxide nanoparticles (nB2O3) are manufactured for structural, propellant, and clinical applications and also form spontaneously through the degradation of bulk boron compounds. Bulk boron is not toxic to vertebrates but the distinctive properties of its nanostructured equivalent may alter its biocompatibility. Few studies have addressed this possibility, thus our goal was to gain an initial understanding of the potential acute toxicity of nB2O3 to freshwater fish and we used a variety of model systems to achieve this. Bioactivity was investigated in rainbow trout (Oncorhynchus mykiss) hepatocytes and at the whole animal level in three other North and South American fish species using indicators of aerobic metabolism, behavior, oxidative stress, neurotoxicity, and ionoregulation. nB2O3 reduced O. mykiss hepatocyte oxygen consumption (ṀO2) by 35% at high doses but whole animal ṀO2 was not affected in any species. Spontaneous activity was assessed using ṀO2 frequency distribution plots from live fish. nB2O3 increased the frequency of high ṀO2 events in the Amazonian fish Paracheirodon axelrodi, suggesting exposure enhanced spontaneous aerobic activity. ṀO2 frequency distributions were not affected in the other species examined. Liver lactate accumulation and significant changes in cardiac acetylcholinesterase and gill Na+/K+-ATPase activity were noted in the north-temperate Fundulus diaphanus exposed to nB2O3, but not in the Amazonian Apistogramma agassizii or P. axelrodi. nB2O3 did not induce oxidative stress in any of the species studied. Overall, nB2O3 exhibited modest, species-specific bioactivity but only at doses exceeding predicted environmental relevance. Chronic, low dose exposure studies are required for confirmation, but our data suggest that, like bulk boron, nB2O3 is relatively non-toxic to aquatic vertebrates and thus represents a promising formulation for further development.

The aquatic environment is the final sink of various pollutants including metals, which can pose a threat for aquatic organisms. Waterborne metal mixture toxicity might be influenced by environmental parameters such as the temperature. In the present study, common carp were exposed for 27 days to a ternary metal mixture of Cu, Zn, and Cd at two different temperatures, 10 and 20°C. The exposure concentrations represent 10% of the 96 h-LC50 (concentration lethal for the 50% of the population in 96 h) for each metal (nominal metal concentrations of Cu: 0.08 μM; Cd: 0.02 μM and Zn: 3 μM). Metal bioaccumulation and toxicity as well as changes in the gene expression of enzymes responsible for ionoregulation and induction of defensive responses were investigated. Furthermore the hepatosomatic index and condition factor were measured as crude indication of overall health and energy reserves. The obtained results showed a rapid Cu and Cd increase in the gills at both temperatures. Cadmium accumulation was higher at 20°C compared to 10°C, whereas Cu and Zn accumulation was not, suggesting that at 20°C, fish had more efficient depuration processes for Cu and Zn. Electrolyte (Ca, Mg, Na, and K) levels were analyzed in different tissues (gills, liver, brain, muscle) and in the remaining carcasses. However, no major electrolyte losses were observed. The toxic effect of the trace metal ion mixture on major ion uptake mechanisms may have been compensated by ion uptake from the food. Finally, the metal exposure triggered the upregulation of the metallothionein gene in the gills as defensive response for the organism. These results, show the ability of common carp to cope with these metal levels, at least under the condition used in this experiment.

The aquatic environment is the final sink of various pollutants including metals, which can pose a threat for aquatic organisms. Waterborne metal mixture toxicity might be influenced by environmental parameters such as the temperature. In the present study, common carp were exposed for 27 days to a ternary metal mixture of Cu, Zn, and Cd at two different temperatures, 10 and 20°C. The exposure concentrations represent 10% of the 96 h-LC50 (concentration lethal for the 50% of the population in 96 h) for each metal (nominal metal concentrations of Cu: 0.08 μM; Cd: 0.02 μM and Zn: 3 μM). Metal bioaccumulation and toxicity as well as changes in the gene expression of enzymes responsible for ionoregulation and induction of defensive responses were investigated. Furthermore the hepatosomatic index and condition factor were measured as crude indication of overall health and energy reserves. The obtained results showed a rapid Cu and Cd increase in the gills at both temperatures. Cadmium accumulation was higher at 20°C compared to 10°C, whereas Cu and Zn accumulation was not, suggesting that at 20°C, fish had more efficient depuration processes for Cu and Zn. Electrolyte (Ca, Mg, Na, and K) levels were analyzed in different tissues (gills, liver, brain, muscle) and in the remaining carcasses. However, no major electrolyte losses were observed. The toxic effect of the trace metal ion mixture on major ion uptake mechanisms may have been compensated by ion uptake from the food. Finally, the metal exposure triggered the upregulation of the metallothionein gene in the gills as defensive response for the organism. These results, show the ability of common carp to cope with these metal levels, at least under the condition used in this experiment. [ABSTRACT FROM AUTHOR]

Acidic freshwater habitats disrupt ion-homeostasis in fishes, yet the often acidic waters of the Mekong host the second highest diversity of freshwater fish in the world. To investigate how five Mekong fish species tolerate water acidity, we measured: time to loss of equilibrium (LOE) at sustained (4 days) low pH (3.5) and net ion flux in acute low pH (3.5 and 3) in Chitala ornata , Pangasianodon hypophthalmus , Osphronemus goramy , Trichogaster pectoralis , and Monopterus albus. Our sustained low pH exposures revealed that C. ornata was least tolerant, P. hypophthalmus and M. albus were moderately tolerant, and O. goramy and T. pectoralis were highly tolerant to low pH. In general, net ion loss in acute low pH exposures was greatest in species with the shortest time to LOE in the sustained low pH exposure. We also explored how low water [Ca2+] (relative to current Mekong levels) affected ion flux at low water pH in the least tolerant C. ornata and highly tolerant T. pectoralis. In C. ornata , low water Ca2+ (56 ± 1 μmol L−1) increased net ion loss relative to high Ca2+ (342 ± 3 μmol L−1) water while no effect was observed in T. pectoralis. Finally, we find that T. pectoralis is among the most acid-tolerant fish species examined to date. • Low pH tolerance was measured in 5 Mekong fish species using net ion loss and time to LOE during continuous low pH exposure. • Net ion loss in acute low pH exposure was correlated with time to LOE in sustained low pH exposure. • Chitala ornata was the least tolerant and Osphronemus goramy and Trichogaster pectoralis the most tolerant to low pH exposure. • In 2 species investigated, low water calcium resulted in increased ion loss at pH 3.5 in C. ornata but not in T. pectoralis. • T. pectoralis had the lowest Na+ loss ever measured at pH 3 and thus is one of the most acid- tolerant fishes ever investigated. [ABSTRACT FROM AUTHOR]

To assess how the quality and properties of the natural dissolved organic carbon (DOC) could drive different effects on gill physiology, we analysed the ionoregulatory responses of a native Amazonian fish species, the tambaqui Colossoma macropomum, to the presence of dissolved organic carbon (DOC; 10 mg l−1) at both pH 7.0 and pH 4.0 in ion‐poor water. The DOC was isolated from black water from São Gabriel da Cachoeira (SGC) in the upper Rio Negro of the Amazon (Brazil) that earlier been shown to protect a non‐native species, zebrafish Danio rerio against low pH under similar conditions. Transepithelial potential (TEP), net flux rates of Na+, Cl− and ammonia and their concentrations in plasma and Na+, K+ ATPase; v‐type H+ ATPase and carbonic anhydrase activities in gills were measured. The presence of DOC had negligible effects at pH 7.0 apart from lowering the TEP, but it prevented the depolarization of TEP that occurred at pH 4.0 in the absence of DOC. However, contrary to our initial hypothesis, SGC DOC was not protective against the effects of low pH. Colossoma macropomum exposed to SGC DOC at pH 4.0 experienced greater net Na+ and Cl− losses, decreases of Na+ and Cl− concentrations in plasma and elevated plasma ammonia levels and excretion rates, relative to those exposed in the absence of DOC. Species‐specific differences and changes in DOC properties during storage are discussed as possible factors influencing the effectiveness of SGC DOC in ameliorating the effects of the acid exposure. [ABSTRACT FROM AUTHOR]

Trout caeca are vermiform structures projecting from the anterior intestine of the gastrointestinal tract. Despite their simple gross morphology, these appendages are physically distinct along the anterior-posterior axis, and ultrastructural evidence suggests zonation of function within the structures. Individual caeca from three sections (anterior, middle and posterior) were removed from the intestine of freshwater rainbow trout and investigated for ion transport and enzyme activity. Ca2+ absorption appeared as a combination of active and passive movement, with Michaelis-Menten kinetics observable under symmetrical conditions, and was inhibited by several pharmacological agents (ouabain, La3+ and a calmodulin antagonist). There was a decrease in ion transport function from adjacent to the intestine (proximal) to the distal tip of each caecum, along with decreasing transport from anterior to posterior for the proximal portion alone. Feeding increased the JMax and KM for Ca2+ absorption within all sections, whereas ion-poor water (IPW) exposure further increased the JMax and KM for Ca2+ transport in the anterior and middle sections. Increased Na+/K+-ATPase (NKA) and citrate synthase (CS) activity rates paralleled trends seen in Ca2+ transport. Feeding in freshwater and IPW exposure increased the glycolytic capacity of the caeca via increased pyruvate kinase (PK) and decreased lactate dehydrogenase (LDH) activity, while amino acid metabolism increased with IPW exposure through increased glutamate dehydrogenase (GDH) activity. Overall, feeding and IPW exposure each altered ionoregulation within the caeca of freshwater rainbow trout in a zone-specific pattern, with the anterior and proximal portions of the caeca being most affected. Increased carbohydrate and protein metabolism fueled the increased ATP demand of NKA through CS. [ABSTRACT FROM AUTHOR]

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). NH4+ 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.

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. Amphibious fish can spend significant periods of their lives in air, and as such, require mechanisms that allow them to regulate internal ionic balance when emersed. The amphibious mangrove rivulus (Kryptolebias marmoratus) uses its skin as a site of ionoregulation in air; however, there is limited information on properties of skin ion transporting cells (ionocytes). Thus, we investigated skin ionocyte morphology of air‐exposed fish (3 hr, 1 day, and 7 days) at different salinities freshwater (FW; 0.3 ppt), brackish water (BW; 15 ppt) and hypersaline water (HSW; 45 ppt). We describe four ionocyte subtypes (A, B, C, and D) differentiated by immunoreactivity to anti‐NKA (Na +/K + ATPase), anti‐NHE3b (Na +/H + exchanger) and anti‐CFTR (cystic fibrosis transmembrane conductance regulator). We demonstrate that skin ionocytes experience plastic remodeling of internal ion transport proteins following air exposure, likely to facilitate ion transport in air. [ABSTRACT FROM AUTHOR]

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). NH4+ 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. [ABSTRACT FROM AUTHOR]

Abstract: The Malpighian tubules play a major role in haemolymph calcium homeostasis in insects by sequestering excess Ca2+ within the biomineralized granules that often accumulate in the tubule cells and/or lumen. Using the scanning ion‐selective microelectrode technique, measurements of basolateral Ca2+ transport are determined at several sites along the length of the Malpighian tubules isolated from the eight insects representing seven orders: Drosophila melanogaster (Diptera), Aedes aegypti (Diptera), Tenebrio molitor (Coleoptera), Acheta domesticus (Orthoptera), Trichoplusia ni (Lepidoptera), Periplaneta americana (Blattodea), Halyomorpha halys (Hemiptera) and Pogonomyrmex occidentalis (Hymenoptera). Ca2+ transport is specific to tubule segments containing Ca‐rich granules in D. melanogaster and A. aegypti, whereas Ca2+ transport is relatively uniform along the length of whole tubules in the remaining species. Generally, manipulation of second messenger pathways using cAMP and thapsigargin has little effect on rates of basolateral Ca2+ transport, suggesting that previous effects observed across midtubules of A. domesticus are unique to this species. In addition, the present study is the first to provide measurements of basolateral Ca2+ across single principal and secondary tubule cells, where Ca2+ uptake occurs only across principal cells. Estimated times for all tubules to eliminate the entire haemolymph Ca2+ content in each insect range from 6 min (D. melanogaster) to 19 h (H. halys) or more, indicating that rates of Ca2+ uptake by the Malpighian tubules are not always rapid. The results of the present study suggest that the principal cells of the Malpighian tubules contribute to haemolymph calcium homeostasis by sequestering excess Ca2+, often within specific tubule segments. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 MO2. 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, MO2, 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. [ABSTRACT FROM AUTHOR]

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 coldacclimated 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 200 mg kg −1 at 7 and 4 days post-injection, respectively) or corticosterone (at 100 mg kg −1 at 7 days 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. [ABSTRACT FROM AUTHOR]

How the growth hormone (GH)/insulin-like growth factor (IGF) system affects osmoregulation in basal vertebrates remains unknown. We examined changes in the expression of components of the GH/IGF axis and gill ion transporters during metamorphosis and following seawater (SW) exposure of sea lamprey. During metamorphosis, increases in gill nka and nkcc1 and salinity tolerance were accompanied by increases in pituitary gh , liver igf1 , gill ghr and igf1 , but not liver ghr. SW exposure of fully metamorphosed sea lamprey resulted in slight increases in plasma chloride concentrations after SW exposure, indicating a high level of SW tolerance, but no major changes in mRNA levels of gill ion transporters or components of the GH/IGF axis. Our results indicate that metamorphosis is a critical point in the lifecycle of sea lamprey for stimulation of the GH/IGF axis and is temporally associated with and likely promotes metamorphosis and SW tolerance. • The GH/IGF axis developed early in the vertebrate lineage. • Metamorphosis is key in the lifecycle of sea lamprey for stimulation of the GH/IGF axis. • The role of GH/IGF axis is osmoregulation evolved early in vertebrate evolution. • The GH/IGF axis in sea lamprey works similarly to that of teleost fishes. • In sea lamprey, gill ion secretion mechanisms develop prior to ocean entry. [ABSTRACT FROM AUTHOR]

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.A proposta deste estudo foi investigar os níveis de Na+, Cl-, K+, Ca2+, and Mg2+ no plasma de teleósteos de água doce e marinhos/estuarinos coletados em diferentes salinidades (0 a 34) nas porções de água doce e estuarinas do Canal São Gonçalo, sul do Brasil. Uma possível relação entre os níveis iônicos plasmáticos e a salinidade também foi investigada bem como a capacidade de regulação iônica dos teleósteos coletados em três ou mais salinidades (Genidens barbus e Micropogonias furnieri). Os resultados mostraram que não houve relação entre os níveis iônicos no plasma com a salinidade quando considerando todas as espécies juntas, mas as duas espécies coletadas em três ou mais salinidades mostraram uma relação significativamente positiva entre níveis iônicos no plasma e salinidade, mostrando que G. barbus e M. furnieri apresentaram uma grande capacidade para regular os íons do plasma em baixas e altas salinidades.

The sea lamprey, Petromyzon marinus, is an anadromous species in which the adults re-enter freshwater, and migrate upstream for terminal spawning. A reduction in salinity tolerance has been document in migrants although the underlying mechanisms have not been characterized. The aim of this study was to determine the capacity for marine osmoregulation in late, upstream migrants by characterizing the morphological and physiological effects of salinity challenge from a molecular perspective. For this two experiments were performed using early and late upstream migrants. Animals salinity limited was found to be 17.5‰ for late upstream migrants, thought some early migrants were able to perform at 24‰. A number of relevant blood and intestinal parameters were measured to assess ionoregulatory and biochemical changes as well as the expression of key ion-transport related proteins by immunoblotting (IB) [Na+/K+-ATPase (NKA), vacuolar-type H+-ATPase, carbonic anhydrase, and Na+:K+:2Cl-contransporter]. NKA activity was also measured, in addition to oxidative stress indicators. The Na+ and Cl- levels with plasma and intestinal fluid were quantified and it was found that in non performing animals these fluids approached environmental concentrations (osmoconforming and failure of drinking mechanism, respectively). A drop in hematocrit was also observed with plasma lactate indicating hemolytic anemia. Plasma [ALT], GST activity and [GSH] were used to assess oxidative damage to tissues. An increase in Na+/K+-ATPase activity in mid-intestine in late migrants and posterior intestine in early migrates showed a potentially adaptive ionoregulatory response to salinity increase. (FCT grant PTDC/MAR/98035/2008).

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 Ca2+)-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 HCO3− inhibits Cl− uptake. The uptake of Mg2+ occurs by the gills or intestine, but details are lacking on Mg2+ transporters. In unionid gills, SO42− is actively transported, but most epithelia are generally impermeant to SO42−. Transporters of Ca2+ maintain homeostasis of dissolved Ca2+. 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. [ABSTRACT FROM AUTHOR]

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 100 μmol l − 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 1.5 kPa P CO 2 but not at higher P CO 2 (6 kPa P CO 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 1.5 kPa P CO 2 , but at 6 kPa P CO 2 Na + uptake was reduced by 95% while low water pH equivalent to 6 kPa P CO 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. [ABSTRACT FROM AUTHOR]

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 Sp NKCC 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 (0.84 μM), a low (0.34 μM) or a control copper concentration (0.05 μM) for 1 and 7 days. 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 7 days. 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. [ABSTRACT FROM AUTHOR]

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 (0.1 mM Na + ). For comparison, a second group of embryos/larvae reared in hard water (2.2 mM 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]