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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.

Recently, a “Na + /NH 4 + exchange complex” model has been proposed for ammonia excretion in freshwater fish. The model suggests that ammonia transport occurs via Rhesus (Rh) glycoproteins and is facilitated by gill boundary layer acidification attributable to the hydration of CO 2 and H + efflux by Na + /H + exchanger (NHE-2) and H + -ATPase. The latter two mechanisms of boundary layer acidification would occur in conjunction with Na + influx (through a Na + channel energized by H + -ATPase and directly via NHE-2). Here, we show that natural ammonia loading via feeding increases branchial mRNA expression of Rh genes, NHE-2, and H + -ATPase, as well as H + -ATPase activity in juvenile trout, similar to previous findings with ammonium salt infusions and high environmental ammonia (HEA) exposure. The associated increase in ammonia excretion occurs in conjunction with a fourfold increase in Na + influx after a meal. When exposed to HEA (1.5 mmol/l NH 4 HCO 3 at pH 8.0), both unfed and fed trout showed differential increases in mRNA expression of Rhcg2, NHE-2, and H + -ATPase, but H + -ATPase activity remained at control levels. Unfed fish exposed to HEA displayed a characteristic reversal of ammonia excretion, initially uptaking ammonia, whereas fed fish (4 h after the meal) did not show this reversal, being able to immediately excrete ammonia against the gradient imposed by HEA. Exposure to HEA also led to a depression of Na + influx, demonstrating that ammonia excretion can be uncoupled from Na + influx. We suggest that the efflux of H + , rather than Na + influx itself, is critical to the facilitation of ammonia excretion.

Recent molecular evidence points towards a capacity for ammonia transport across the skin of adult rainbow trout. A series of in vivo and in vitro experiments were conducted to understand the role of cutaneous ammonia excretion ( J amm ) under control conditions and after 12-h pre-exposure to high environmental ammonia (HEA; 2 mmol/l NH 4 HCO 3 ). Divided chamber experiments with bladder-catheterized, rectally ligated fish under light anesthesia were performed to separate cutaneous J amm from branchial, renal, and intestinal J amm . Under control conditions, cutaneous J amm accounted for 4.5 % of total J amm in vivo. In fish pre-exposed to HEA, plasma total ammonia concentration increased 20-fold to approximately 1,000 μmol/l, branchial J amm increased 1.5- to 2.7-fold, and urinary J amm increased about 7-fold. Urinary J amm still accounted for less than 2 % of total J amm . Cutaneous J amm increased 4-fold yet amounted to only 5.7 % of total J amm in these fish. Genes (Rhcg1, Rhcg2, Rhbg, NHE-2, v-type H + -ATPase) known to be involved in ammonia excretion at the gills of trout were all expressed at the mRNA level in the skin, but their expression did not increase with HEA pre-exposure. In vitro analyses using [ 14 C] methylamine (MA), an ammonia analog which is transported by Rh proteins, demonstrated that MA permeability in isolated skin sections was higher in HEA pre-exposed fish than in control fish. The addition of basolateral ammonia (1,000 μmol/l) to this system abolished this increase in permeability, suggesting ammonia competition with MA for Rh-mediated transport across the skin of HEA pre-exposed trout; this did not occur in skin sections from control trout. Moreover, in vitro J amm by the skin of fish which had been pre-exposed to HEA was also higher than in control fish in the absence of basolateral ammonia, pointing towards a possible cutaneous ammonia loading in response to HEA. In vitro MA permeability was reduced upon the addition of amiloride (10 −4  mol/l), but not phenamil (10 −5  mol/l) suggesting a role for a Na/H-exchanger (NHE) in cutaneous ammonia transport, as has been previously described in the skin of larval fish. Overall, it appears that under control conditions and in response to HEA pre-exposure, the skin makes only a very minor contribution to total J amm , but the observed increases in cutaneous J amm in vivo and in cutaneous J amm and MA permeability in vitro demonstrate the capacity for ammonia transport in the skin of adult trout. It remains unclear if this capacity may become significant under certain environmental challenges or if it is merely a remnant of cutaneous transport capacity from early life stages in these fish.

The effects of acute moderate (20% air O2saturation; 6-h exposure) and severe (5% air O2saturation; 4-h exposure) hypoxia on N-waste, acid-base, and ion balance in dogfish sharks (Squalus acanthias suckleyi) were evaluated. We predicted that the synthesis and/or retention of urea, which are active processes, would be inhibited by hypoxia. Exposure to moderate hypoxia had negligible effects on N-waste fluxes or systemic physiology, except for a modest rise in plasma lactate. Exposure to severe hypoxia led to a significant increase in urea excretion (J urea), while plasma, liver, and muscle urea concentrations were unchanged, suggesting a loss of urea retention. Ammonia excretion (J amm) was elevated during normoxic recovery. Moreover, severe hypoxia led to disruptions in acid-base balance, indicated by a large increase in plasma [lactate] and substantial decreases in arterial pHa and plasma , as well as loss of ionic homeostasis, indicated by increases in plasma [Mg2+], [Ca2+], and [Na+]. We suggest that severe hypoxia in dogfish sharks leads to a reduction in active gill homeostatic processes, such as urea retention, acid-base regulation and ionoregulation, and/or an osmoregulatory compromise due to increased functional gill surface area. Overall, the results provide a comprehensive picture of the physiological responses to a severe degree of hypoxia in an ancient fish species.

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 (CT max ) 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 CT max 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.

Killifish (Fundulus heteroclitus) has been extensively used as a model for ion regulation by euryhaline fishes. Na+ and Cl− dynamics have been well studied in killifish, but few studies have addressed that of Ca2+. Therefore, this study aimed to characterize Ca2+ fluxes in freshwater (FW) and seawater (SW)-acclimated killifish, their response to salinity transfer, and to elucidate the mechanisms of Ca2+ influx in FW and SW. SW killifish displayed a significantly higher Ca2+ influx rate than that of FW fish, while Ca2+ efflux rates were comparable in both salinities. Ca2+ influx was saturable in FW (Km = 78 ± 19 µmol/L; Jmax = 53 ± 3 nmol/g/h) and influx by SW killifish was linear up to 7 mmol/L Ca2+. In SW-acclimated fish, 36% of Ca2+ influx was attributed to “intestinal Ca2+ intake”, likely caused by drinking, whereas intestinal Ca2+ intake in FW contributed to < 2% of total. Throughout the study, results suggested that “cation competition” in SW modulates Ca2+ influx. Therefore, we hypothesized that SW-acclimated fish actually have a higher affinity Ca2+ influx system than FW-acclimated fish but that it is competitively inhibited by competing SW cations. In agreement with this cation competition hypothesis, we demonstrated for the first time that “extra-intestinal” Ca2+ influx was inhibited by Mg2+ in both FW and SW-acclimated killifish. Following acute salinity transfer, extra-intestinal Ca2+ influx was rapidly regulated within 12–24 h, similar to Na+ and Cl−. Ca2+ influx in FW was inhibited by La3+, an epithelial Ca2+ channel blocker, whereas La3+ had no significant effect in SW.

Freshwater fish must maintain the concentration of salts in their blood within a narrow range, but this status is challenged by the prevailing water chemistry and its fluctuations. This paper provides a framework for understanding how fish in nature will respond to forecasted future changes in water chemistry. Abstract Alterations in water chemistry can challenge resident fish species. More specifically, chemical changes that disrupt ion balance will negatively affect fish health and impact physiological and ecological performance. However, our understanding of which species and populations are at risk from ionoregulatory disturbances in response to changing freshwater environments is currently unclear. Therefore, we propose a novel framework for incorporating ionoregulatory physiology into conservation management of inland fishes. This framework introduces the concepts of fundamental chemical niche, which is the tolerable range of chemical conditions for a given species based on laboratory experiments, and realized chemical niche, which is the range of chemical conditions in which a species resides based on distribution surveys. By comparing these two niches, populations that may be at risk from ionoregulatory disturbances and thus require additional conservation considerations can be identified. We highlight the potential for commonly measured ionoregulatory traits to predict fundamental and realized chemical niches but caution that some traits may not serve as accurate predictors despite being important for understanding ionoregulatory mechanisms. As a sample application of our framework, the minimum pH distribution (realized niche) and survival limit pH (fundamental niche) of several North American fishes were determined by systematic review and were compared. We demonstrate that ionoregulatory capacity is significantly correlated with a realized niche for many species, highlighting the influence of ionoregulatory physiology on fish distribution patterns along chemical gradients. Our aim is that this framework will stimulate further research in this field and result in a broader integration of physiological data into conservation management decisions for inland waters.

Ammonia transport and metabolism were investigated in the intestinal tract of freshwater rainbow trout which had been either fasted for 7 days, or fasted then fed a satiating meal of commercial trout pellets. In vivo, total ammonia concentrations ( T amm ) in the chyme were approximately 1 mmol L −1 across the entire intestine at 24 h after the meal. Highest chyme pH and P NH3 values occurred in the posterior intestine. In vitro gut sac experiments examined ammonia handling with mucosal (Jm amm ) and serosal (Js amm ) fluxes under conditions of fasting and feeding, with either background (control ≤0.013 mmol L −1 ) or high luminal ammonia concentrations (HLA = 1 mmol L −1 ), the latter mimicking those seen in chyme in vivo. Feeding status (fasted or fed) appeared to influence ammonia handling by each individual section. The anterior intestine exhibited the greatest Jm amm and Js amm values under fasted control conditions, but these differences tended to disappear under typical post-feeding conditions when total endogenous ammonia production (Jt amm  = Js amm  − Jm amm , signs considered) was greatly elevated in all intestinal sections. Under fasted conditions, glutamate dehydrogenase (GDH) and glutaminase (GLN) activities were equal across all sections, but the ammonia-trapping enzyme glutamine synthetase (GS) exhibited highest activity in the posterior intestine, in contradiction to previous literature. Feeding clearly stimulated the total rate of endogenous ammonia production (Jt amm ), even in the absence of a high luminal ammonia load. This was accompanied by an increase in GDH activity of the anterior intestine, which was also the site of the largest Jt amm . In all sections, during HLA exposure, either alone or in combination with feeding, there were much larger increases in endogenous Jt amm , most of which was effluxed to the serosal solution. This is interpreted as a response to avoid potential cytotoxicity due to overburdened detoxification mechanisms in the face of elevated mucosal ammonia. Thus T amm of the intestinal tissue remained relatively constant regardless of feeding status and exposure to HLA. Ammonia production by the gut may explain up to 18 % of whole-body ammonia excretion in vivo under fasting conditions, and 47 % after feeding, of which more than half originates from endogenous production rather than from absorption from the lumen.

Copper (Cu) is a persistent environmental contaminant that elicits several physiological disturbances in aquatic organisms, including a disruption in ammonia regulation. We hypothesized that exposure to Cu in a model crustacean (blue crab, Callinectes sapidus ) acclimated to brackish water (2 ppt) would lead to hyperammonemia by stimulating an increase in ammonia production and/or by inhibiting ammonia excretion. We further hypothesized that urea production would represent an ammonia detoxification strategy in response to Cu. In a pilot experiment, exposure to 0, 100, and 200 µg/L Cu for 6 h caused significant concentration-dependent increases in ammonia excretion ( J amm ). Based on these results, an acute 24-h 100 µg/L Cu exposure was conducted and this similarly caused an overall stimulation of J amm during the 24-h period, indicative of an increase in ammonia production. Terminal haemolymph total ammonia content ( T amm ) was unchanged, suggesting that while ammonia production was increased, there was no inhibition of the excretion mechanism. In support of our second hypothesis, urea excretion ( J urea ) increased in response to Cu exposure; haemolymph [urea] was unaffected. This suggested that urea production also was increased. To further test the hypothesis that J urea increased to prevent hyperammonemia during Cu exposure, crabs were exposed to high environmental ammonia (HEA; 2.5 mmol/L NH 4 HCO 3 ) for 12 h in a separate experiment. This led to a fourfold increase in haemolymph T amm , whereas J urea increased only transiently and haemolymph [urea] was unchanged, indicating that urea production likely does not contribute to the attenuation of hyperammonemia in blue crabs. Overall, Cu exposure in blue crabs led to increased ammonia and urea production, which were both eliminated by excretion. These results may have important implications in aquaculture systems where crabs may be exposed to elevated Cu and/or ammonia.

Post-hatch fishes lack a functional gill and use cutaneous surfaces for exchange with the surrounding environment. The ionoregulatory hypothesis posits that ionoregulation is the first physiological process to be limited by cutaneous exchange, necessitating its shift to the gills. We hypothesized that the ontogeny of branchial ammonia excretion (Jamm) is coupled to Na+ uptake () in accordance with the current model for exchange in freshwater. Using divided chambers, branchial and cutaneous Jamm, and oxygen consumption (MO2) by larval rainbow trout were assessed. Following hatch, the skin accounted for 97% and 86% of total Jamm and , respectively. Jamm and shifted to the gills simultaneously at 15 days post-hatch (dph) and were highly correlated (R2 = 0.951) at the gills, but not the skin, over development. Contrastingly, MO2 shifted significantly later at 27 dph, in agreement with the ionoregulatory hypothesis. Moreover, the mRNA expression and/or enzymatic activity of Rhesus proteins, Na+/H+-exchanger, H+-ATPase, Na+/K+-ATPase and carbonic anhydrase, all key components of the -exchange system, increased in the gills over larval development. We propose that the ontogeny of branchial occurs as exchange and provide evidence for a novel element to the ionoregulatory hypothesis, the excretion of potentially lethal metabolic ammonia.