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Here, we describe a laboratory experiment as part of an upper-level vertebrate physiology course for biology majors to investigate the physiological response of vertebrates to osmoregulatory challenges. The experiment involves measuring plasma osmolality and Na+-K+-ATPase activity in gill tissue of teleost fish acclimated to water of differing salinity. We describe results obtained using the widely available goldfish (Carassius auratus) and a common baitfish, the Gulf killifish (Fundulus grandis). The procedures described are generally applicable to other fish species, and they provide an alternative to the experimental use of humans or other mammalian species to investigate osmoregulation mechanisms. In addition to reenforcing the conceptual material covered in lecture, this laboratory exercise trains students in a wide range of laboratory and analytical skills, such as calculating and performing dilutions, pipetting, tissue sampling and homogenizing, preparing standard curves, conducting enzymatic assays, and analyzing and interpreting results. Typical student results are presented and discussed, as are common experimental and conceptual mistakes made by students. [ABSTRACT FROM AUTHOR]

Abstract: For most of the last century, the need to obtain sufficient oxygen to meet the respiratory requirements of the tissues was viewed as the primary selective pressure driving gill development in teleost fish. Recently, however, it has been suggested that ionoregulatory pressures may actually be more important. This manuscript reviews the theoretical and empirical evidence dealing with the functional ontogeny of the gill in the context of the oxygen and ionoregulatory hypotheses. Gas and ion exchange are subject to similar geometric constraints in developing fish. Both initially are exclusively cutaneous but shift to the gill with tissue growth because of declining surface-to-volume ratios. Based on the appearance of mitochondria-rich cells (MRCs), ionoregulatory activity shifts to the gill in advance of gas exchange. In every species examined to date, MRCs appear on the developing gill in advance of secondary lamellae, the definitive gas exchange structure of the adult gill. Biochemical and histochemical studies indicate that these early branchial MRCs are actively involved in ion exchange. In some cases, the specific activity is many times greater than in the adult gill. In contrast, O2 microelectrode and hemoglobin ablation experiments suggest that the early gill contributes little O2 to the general systemic circulation. Any oxygen taken up appears to be consumed locally. Functional ablation experiments with zebrafish indicated that the larval gill became essential for ion balance well before it was needed for O2 uptake. Similar experiments with rainbow trout, however, found that the gill became essential in terms of gas and ion exchange at about the same time. On balance, the evidence appears to favour the ionoregulatory hypothesis but the oxygen hypothesis cannot be absolutely rejected without more information. Some of the major deficiencies in our knowledge regarding the transition from cutaneous to branchial gas and ion exchange are highlighted and potential implications of the ionoregulatory hypothesis are discussed. [Copyright &y& Elsevier]

Abstract: The physiological effects of waterborne and waterborne plus dietborne copper exposure were determined in the euryhaline copepod Acartia tonsa at different salinities (5, 15 and 30ppt). Copepods were exposed (48h) to a reported 48-h LC50 for copper (CuCl2), which had been previously determined under the same experimental conditions. Whole body copper accumulation, ion concentrations (Na+, Cl−, Mg2+), and Na+, K+-ATPase activity were the endpoints measured in all experimental groups. Feeding rate was also measured in fed experimental groups. In copper-exposed copepods, whole body copper accumulation was dependent on salinity, decreasing as salinity increased. However, it was similar in copepods exposed to waterborne and waterborne plus dietborne copper, irrespective the salinity tested. Waterborne copper exposure induced a disturbance of the whole body Na+ concentration in all salinities tested. This effect was characterized by an increased whole body Na+ concentration in seawater (salinity 30ppt) and a decreased whole body Na+ concentration at lower salinities (5 and 15ppt). The ionoregulatory imbalance in low salinity (5ppt) was associated with an inhibition of the whole body Na+, K+-ATPase activity, as observed in freshwater fish and crustaceans. When copepods were exposed to waterborne plus dietborne copper, the physiological effects described were only observed at a low salinity (5ppt) and were associated with a marked inhibition of the feeding rate. Taken altogether, the data suggest that the physiological effects induced by waterborne copper exposure in A. tonsa acclimated to higher salinities (15 and 30ppt) are due to a combined effect of food restriction and copper exposure. Differential physiological responses to waterborne and waterborne plus dietborne copper cannot be ascribed to differences in whole body copper burden. [Copyright &y& Elsevier]

The Amazonian oscar is extremely resistant to hypoxia, and tolerance scales with size. Overall, ionoregulatory responses of small (~15 g) and large oscars (~200 g) to hypoxia were qualitatively similar, but the latter were more effective. Large oscars exhibited a rapid reduction in unidirectional Na+ uptake rate at the gills during acute hypoxia (Po2 ~10 mmHg), which intensified with time (7 or 8 h); Na+ efflux rates were also reduced, so net balance was little affected. The inhibitions were virtually immediate (1st h) and preceded a later 60% reduction (at 3 h) in gill Na+-K+-ATPase activity, reflected in a 60% reduction in maximum Na+ uptake capacity without change in affinity (Km) for Nat Upon acute restoration of normoxia, recovery of Na+ uptake was delayed for 1 h. These data suggest that dual mechanisms may be involved (e.g., immediate effects of O2 availability on transporters, channels, or permeability, slower effects of Na+-K+-ATPase regulation). Ammonia excretion appeared to be linked indirectly to Na+ uptake, exhibiting a Michaelis-Menten relationship with external [Na+], but the Km was less than for Na+ uptake. During hypoxia, ammonia excretion fell in a similar manner to Na+ fluxes, with a delayed recovery upon normoxia restoration, but the relationship with [Na+] was blocked. Reductions in ammonia excretion were greater than in urea excretion. Plasma ammonia rose moderately over 3 h hypoxia, suggesting that inhibition of excretion was greater than inhibition of ammonia production. Overall, the oscar maintains excellent homeostasis of ionoregulation and N-balance during severe hypoxia. [ABSTRACT FROM AUTHOR]

A diet containing an inert marker (ballotini beads, quantified by X-radiography) was used to quantify the transport of two essential minerals, Ca2+ and Mg2+ from the diet during the digestion and absorption of a single meal of commercial trout food (3% ration). Initially, net uptake of Ca2+ was observed in the stomach followed by subsequent Ca2+ fluxes along the intestine which were variable, but for the most part secretory. This indicated a net secretion of Ca2+ along the intestinal tract resulting in a net assimilation of dietary Ca2+ of 28%. Similar handling of Ca2+ and Mg2+ was observed along the gastrointestinal tract (GI), although net assimilation differed substantially between the cations, with Mg2+ assimilation being close to 60%, mostly a result of greater uptake by the stomach. The stomach displayed the highest net uptake rates for both cations (1.5 and 1.3 mmol kg−1 fish body mass for Ca2+ and Mg2+, respectively), occurring within 2 h following ingestion of the meal. Substantial secretions of both Ca2+ and Mg2+ were observed in the anterior intestine, which were attributed to bile and other intestinal secretions, while fluxes in the mid and posterior intestine were small and variable. The overall patterns of Ca2+ and Mg2+ handling in the GI tract were similar to those observed for Na+ and K+ (but not Cl−) in a previous study. Overall, these results emphasize the importance of dietary electrolytes in ionoregulatory homeostasis. [ABSTRACT FROM AUTHOR]

Abstract: The purpose of this review is to collate physiological knowledge on salmonid eggs and larvae in relation to water quality criteria. Salmonid genera reviewed include Coregonus, Thymallus, Salvelinus, Salmo, and Oncorhynchus spp. When physiological data for salmonids are lacking, the zebrafish and medaka models are included. The primary focus is on the underlying mechanisms involved in the hydro-mineral, thermal, and respiratory biology with an extended section on the xenobiotic toxicology of the early stages. Past and present data reveal that the eggs of salmonids are among the largest shed by any broadcast spawning teleost. Once ovulated, the physicochemical properties of the ovarian fluid provide temporary protection from external perturbations and maintain the eggs in good physiological condition until spawning. Following fertilisation and during early development the major structures protecting the embryo from poor water quality are the vitelline membrane, the enveloping layer and the chorion. The vitelline membrane is one of the least permeable membranes known, while the semi-permeable chorion provides both physical and chemical defense against metals, pathogens, and xenobiotic chemicals. In part these structures explain the lower sensitivity of the eggs to chemical imbalance compared to the larvae, however the lower metabolic rate and the chronology of gene expression and translational control suggest that developmental competence also plays a decisive role. In addition, maternal effect genes provide a defense potential until the mid-blastula transition. The transition between maternal effect genes and zygotic genes is a critical period for the embryo. The perivitelline fluids are an important trap for cations, but are also the major barrier to diffusion of gases and solutes. Acidic environmental pH interferes with acid–base and hydromineral balance but also increases the risk of aluminium and heavy metal intoxication. These risks are ameliorated somewhat by the presence of ambient humic acid. High temperatures during development may be teratogenic, cause sexual bias, or long-term effects on muscle cellularity. Xenobiotics cause inhibition of neural acetylcholine esterase and carboxylases and disrupt the normal signalling pathways of hormones by binding to relevant receptors and mimicking their actions. A complex suite of genes is activated in response to environmental or parentally transmitted xenobiotics. The primary defense mechanism in embryos involves resistance to uptake but later biotransformation via the aryl hydrocarbon receptor (AHR)-mediated activation of members of the cytochrome mixed-function mono-oxygenase superfamily (CYP1A, CYP2B, and CYP3A) and subsequent glucuronidation or glutathionation. Due to the number of duplicate or triplicate genes coding for intermediates in the signalling pathways, and cross-talk between nuclear orphan receptors and steroid hormone receptors, a large number of complications arise in response to xenobiotic intoxicaton. One such syndrome, known as blue-sac disease causes an anaphylactoid response in hatched larvae due to increased permeability in the vascular endothelium that coincides with AHR-mediated CYP induction. Early embryos also respond to such xenobiotic insults, but apparently have an immature translational control for expression of CYP proteins, which coincides with a lack of excretory organs necessary for the end-point of biotransformation. Other syndromes (M74 and Cayuga) are now associated with thiamine deficiency. Where possible guidelines for water quality criteria are suggested. [Copyright &y& Elsevier]

Mosquito larvae are found in diverse aquatic habitats ranging from freshwater to hypersaline water and must often deal with rapid changes in habitat salinity. We transferred larvae of Aedes aegypti from freshwater to 30% seawater, or vice versa, and measured the time course of changes in their hemolymph ion concentrations, using ion-selective microelectrodes. We also reported the Michaelis-Menten kinetics of Na+ and Cl- transport by the anal papillae for the first time using the scanning ion-selective electrode technique (SIET). Hemolymph concentrations of Na+, Cl- and H+ increased within 6 h, when larvae were transferred from freshwater to seawater and decreased within 6 h, when transferred from seawater to freshwater. Kinetic parameters for Na+ and Cl- transport by the anal papillae were altered after only 5 h following transfer between freshwater (FW) and 30% seawater (30%SW). The Jmax (maximum transport rate) for both ions decreased when larvae were transferred to 30%SW, whereas the Kt (a measure of transporter affinity) increased for Na+ transport but was unaltered for Cl- transport, suggesting that Na+ and Cl- uptake are independent. Data reveal significant changes in ion transport by the anal papillae of mosquito larvae when they are faced with changes in external salinity such that Na+ and Cl- uptake decrease in higher salinity. The alterations in Na+ and Cl- uptake may be a consequence of changes in hemolymph ion levels when larvae encounter altered salinity. The rapid changes in ion transport described here compliment the previously observed long term alterations in the morphology and ultrastructure of the anal papillae. [ABSTRACT FROM AUTHOR]

Abstract: Rainbow trout, common carp, and gibel carp were exposed to sublethal Cu levels (1.0 or 1.7 μM) for 1 week. In rainbow trout, arterial oxygen tension (P aO2 ) remained normal and there was no indication of anaerobic metabolism. P aO2 was considerably lower in common and gibel carp and Cu exposure decreased this further. The decrease was transient for common carp but persistent in gibel carp and coincided with an elevation in arterial carbon dioxide tension (P aCO2 ) indicating that all gas exchange was compromised in both cyprinid species. The disturbed gas exchange resulted in acidosis, which was respiratory and metabolic for common carp but mainly respiratory for gibel carp. Gibel carp produced ethanol as end product of their alternative anaerobic pathway. The hypothesis that hypertrophy and hyperplasia, resulting in increased diffusion distances, are reducing P aO2 appeared invalid. Hypoventilation seems a more likely cause. Ionoregulatory parameters responded more uniform among species. Fast and pronounced decreases in plasma sodium and chloride developed for all three species, independent of the observed gill damage. Rainbow trout lost 20% of their plasma Na in the first 3 days, while common and gibel carp had only lost 13 and 16% respectively at that time. This difference might be crucial when challenged with Cu exposure and allow a fish to survive the first shock phase and supports it the hypothesis that sodium turnover is a key factor in predicting Cu toxicity. [Copyright &y& Elsevier]

Abstract: The possible nephrotoxic effects of waterborne lead exposure (as Pb(NO3)2) were investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Kidney lead accumulation was time-dependent, increasing upon exposure to 0.57±0.01mgdissolvedPbL−1 for up to 96h with a significantly higher burden occurring in the posterior kidney compared to the anterior segment. Urine analyses in trout exposed to 1.20±0.09mgdissolvedPbL−1 revealed a significant increase in urinary lead excretion rate throughout 96h of exposure. Urine flow rate and glomerular filtration rate (GFR) were not impacted with the exception of a significant decrease in GFR from 84 to 96h in lead-exposed trout. Urine pH decreased significantly over time in lead-exposed fish. Correspondingly, urine ammonia excretion rate showed a marked increase from 48h onwards. In experimental fish, urine glucose excretion was significantly greater by 96h while urine lactate, urea and protein excretion were not significantly altered by lead exposure. The urine excretion rate of Ca2+ increased significantly by approximately 43% after only 24h of lead exposure, and was maintained at a higher rate than controls for up to 96h. Magnesium excretion increased in a time-dependent fashion, reaching a two- to three-fold rise by 96h. In contrast, rates of Na+ and Cl− excretion were decreased in experimental fish by approximately 30% by 48h, this trend continuing for the duration of lead-exposure. There were no changes in any of these parameters in similarly treated control fish. Clearance ratio analyses indicated progressive decreases in the net reabsorption efficiencies of the renal system for Ca2+, Mg2+, Pb, and glucose, suggesting that the active tubular transport mechanisms for these substances were inhibited by lead exposure, while Na+, K+, Cl−, lactate, and protein reabsorptions were unaffected. Net ammonia secretion increased. We conclude that changes in renal function both reflect and help to minimize some of the associated disturbances in systemic physiology. Lead-induced ionoregulatory toxicity in rainbow trout, particularly the disturbance of Ca2+ homeostasis, is not exclusively a branchial phenomenon, but is in part a result of disruption of ionoregulatory mechanisms at the kidney. This action of lead outside the gills is critical to consider when developing guidelines for water quality. [Copyright &y& Elsevier]

Abstract: Two trials were conducted to determine the effect of sudden decrease in salinity of raw and potassium-fortified inland saline water on western king prawn Penaeus latisulcatus osmoregulation, ionoregulation and condition. Prawns were subjected to salinity decrease over 1 h from 32 to 25 ppt in the first trial and from 27 to 20 ppt in the second trial in three water types: inland saline water with potassium fortified to 100% and 80% of the marine water concentration (IS100, IS80), and raw inland saline water (ISW). In the first trial condition and ingestion rate were monitored over 19 days following salinity change. In the second trial condition, haemolymph osmo- and iono-regulation were recorded over 48 h following salinity change. In the first trial, 100% mortality was observed in ISW by day 13, with final survival 94% in IS80 and 100% in IS100. Tail muscle moisture content increased significantly (P <0.05) over time in both trials and in all water types, suggesting loss of energy reserves. In the second trial, serum osmolality, sodium concentration and osmoregulatory capacity decreased following salinity change, stabilising by 24 h in IS100 and IS80 but continuing to decrease till 48 h in ISW, suggesting partial breakdown of osmoregulatory function in the potassium-deficient medium. Prawns were stronger regulators of divalent than monovalent cations. These trials demonstrate that potassium-deficient inland saline water requires fortification with potassium to allow prawn survival and efficient osmoregulation. [Copyright &y& Elsevier]

Loma salmonae infections of salmonids culminate in the development of branchial xenomas and subsequent focal hyperplasia of the lamellar or filament epithelium following xenoma rupture and spore release. The effects of this acute branchial disruption upon net ionic flux rates and plasma electrolyte concentrations were determined in juvenile rainbow trout given an experimental oral exposure to L. salmonae. Mean numbers of branchial xenomas peaked at week 5 post-exposure (PE), which coincided with a reduction in the specific growth rate, although there were no significant differences in mass, length or condition of Loma-exposed fish compared with unexposed controls. Following exposure, negative net whole body Na+ and K+ fluxes decreased, whereas net Cl− fluxes remained unchanged compared with non-exposed control fish. At week 3 PE during the initial branchial xenoma formation stage, there was a significant negative whole body net K+ flux in Loma-exposed trout compared with other points during the exposure and subsequent infection. Additionally, Loma-exposed fish had marginally elevated plasma Na+ and Cl− concentrations, whilst K+ levels remained unchanged, compared with control fish. Although there was a progressive decrease in leucocrit, haematocrit remained unchanged over the course of the Loma exposure and subsequent infection. These results suggest that ionic compensation can occur at the gills during the development of xenomas during exposure to L. salmonae and the resultant infection, therefore allowing defence of plasma electrolyte concentrations, unlike the acute ionic disturbances seen with some other parasitic diseases. [ABSTRACT FROM AUTHOR]

Abstract: Two waterborne Cu exposures were performed to investigate if Cu is an ionoregulatory toxicant at all salinities in the killifish, Fundulus heteroclitus. A 30-day flow through exposure in 0 (FW), 5, 11, 22, and 28ppt (SW) and three [Cu]''s (nominal 0, 30, and 150μgCuL−1) revealed no apparent Cu induced mortality at the intermediate salinities and high mortality in FW and SW. Fish were sampled at 4, 12, and 30 days after the start of the exposure and both Na+/K+ adenosine triphosphatase (Na+/K+ ATPase) and carbonic anhydrase (CA) activity in the gill and intestine as well as whole body [Na+], and [Cl−] were measured. At the high [Cu] a reduction of whole body [Na+] after 4 days of exposure in FW was the only physiological parameter influenced. A second static 24h Cu exposure was performed in FW, 5, 13, and 29ppt (SW) and two [Cu]''s (nominal 0 and 110μgCuL−1). In addition to the parameters listed above, ammonia flux was measured at all salinities and Na+ flux was measured in FW fish. Cu affected ionoregulation in FW where decreased Na+ uptake associated with inhibition of Na+/K+ ATPase led to decreased whole body [Na+] after 24h. The only affected parameter in SW was net ammonia excretion suggesting that Cu is not an ionoregulatory toxicant in SW at the concentrations employed. We propose that physiology rather than chemistry explain much of the variation in Cu toxicity seen across salinities. [Copyright &y& Elsevier]

Abstract: Western king prawns, Penaeus latisulcatus are a candidate species for culture in inland saline water. Western king prawn survival, growth, condition, osmo- and iono-regulation was studied when reared in potassium-fortified inland saline water for 202 days. PL40 prawns were stocked into three media in 250 L tanks: inland saline water with potassium fortified to 80% (IS80) of the marine water concentration, 100% (IS100) of the marine water concentration and marine water (MW). By the conclusion of the trial, survival was 53% in IS80, 64% in IS100 and 68% in MW. Mean prawn weight, total length, carapace length, condition factor and moult interval were significantly higher (P <0.05) in MW than in IS100 and IS80. Specific growth rate of prawns in MW was significantly higher (P <0.05) than in IS80. There were no significant differences (P >0.05) in osmoregulatory capacity of prawns between various media. Serum Na+, K+, Ca2+and S concentrations were influenced by their concentration in the medium. Ca2+ was the only major cation hyper-regulated and tended to be accumulated, while Mg2+ was maintained at a much lower concentration in the serum than in the medium. Moisture contents in hepatopancreas, tail muscle and exoskeleton and organosomatic indices improved from IS80 to IS100 to MW. The lower growth rates and condition factors of prawns reared in potassium-fortified inland saline water suggests the presence of limiting factors other than potassium concentration. Western king prawns are stronger regulators of divalent cations than monovalent cations and the extra energy required to regulate ions in inland saline water may have been the major cause of lower growth rates. [Copyright &y& Elsevier]

Abstract: This study examined the impact of elevated dietary Ca2+ on the responses to chronic dietary Pb exposure in juvenile rainbow trout. Trout were fed reference (0.3μgPb/g, ∼20mgCa2+/g) and Pb-enriched diets (∼50 or 500μgPb/g) in the presence of background Ca2+ (∼20mgCa2+/g) or (∼60mgCa2+/g) of added Ca2+ (as CaCO3) for 42 days. The quantitative order of Pb accumulation in tissues reflected the exposure pathway of Pb via the diet (per tissue wet weight): gut>bone>kidney>liver>spleen>gill>carcass>brain>white muscle. The anterior intestine accumulated the most Pb per tissue wet weight, while the bone accumulated the most Pb per fish weight. Pb concentrations were much higher in the posterior kidney than the anterior kidney. Simultaneous addition of Ca2+ to the diet had an overall protective effect in all the tissues analysed in reducing Pb accumulation. The RBCs accumulated 100 times more Pb when compared to the plasma, while the whole blood δ-aminolevulinic acid dehydratase was inhibited in the high treatment group without added Ca2+, by the end of the exposure. Neither plasma Cl−, K+, Mg2+ nor Na+, K+-ATPase activities in the gills, mid- and posterior intestine were affected. However, there were mild disruptions in plasma Na+ and Ca2+ levels in the elevated Pb and Ca2+ treatment groups, and a significant up-regulation in Na+, K+-ATPase activity at the anterior intestine in fish fed the high Pb diets with background or added Ca2+. By day 42, Pb levels in most tissues had either stabilized or started to decrease, indicating some capacity for regulation of accumulated loads. We conclude that elevated dietary Ca2+ levels will be protective in reducing Pb burdens in freshwater juvenile rainbow trout exposed to environments contaminated with waterborne Pb. [Copyright &y& Elsevier]

Abstract: The gulf toadfish, Opsanus beta, is a marine teleost fish with an aglomerular kidney that is highly specialized to conserve water. Despite this adaptation, toadfish have the ability to survive when in dilute hypoosmotic seawater environments. The objectives of this study were to determine the joint role of the kidney and intestine in maintaining osmotic and ionic balance and to investigate whether toadfish take advantage of their urea production ability and use urea as an osmolyte. Toadfish were gradually acclimated to different salinities (0.5, 2.5, 5, 10, 15, 22, 33, 50 and 70 ppt (1.5%, 7.5%, 15%, 30%, 45%, 67%, 100%, 151% and 212% seawater)) and muscle tissue, urine, blood and intestinal fluids were analyzed for ion and in some cases urea concentration. The renal and intestinal ionoregulatory processes of toadfish responded to changes in salinity and when gradually acclimated, toadfish maintain a relatively constant plasma osmolality at environmental salinities of 5 to 50 ppt. However, at salinities lower (2.5 ppt) or higher (70 ppt) than this range, a significant deviation from resting plasma and urine osmolality as well as changes in muscle water content was measured, suggesting osmoregulatory difficulties at these salinities. The renal system compensates for dilute seawater by reducing Na+ reabsorption by the bladder, which allowed excess water to be excreted. In the case of hypersalinity, Na+ reabsorption was increased, which resulted in a conservation of water and the concentration of Mg2+, Cl−, SO4 2− and urea. A similar pattern was observed within the gastrointestinal system. Notably, Mg2+, HCO3 − and SO4 2− were the dominant ions in the intestinal fluid under control and hypersaline conditions due to the absorption of Na+, Cl− and water. When exposed to dilute seawater conditions, the absorption of Na+ was greatly reduced which likely increased water elimination. As a result of decreased environmental levels and a reduction in drinking rate, Mg2+ and SO4 2− in intestinal fluids under hypoosmotic conditions were greatly reduced. While urea did play a minor role in renal osmoregulation, toadfish appear to preferentially regulate Na+ and to some extend Cl− in urine and intestinal fluids. [Copyright &y& Elsevier]

We present the first data on changes in ionoregulatory physiology of maturing, migratory adult sockeye salmon Oncorhynchus nerka. Fraser River sockeye were intercepted in the ocean as far away as the Queen Charlotte Islands (∼850 km from the Fraser River) and during freshwater migration to the spawning grounds; for some populations this was a distance of over 700 km. Sockeye migrating in seawater toward the mouth of the Fraser River and upriver to spawning grounds showed a decline in gill Na+,K+-ATPase activity. As a result, gill Na+,K+-ATPase activity of fish arriving at the spawning grounds was significantly lower than values obtained from fish captured before entry into freshwater. Plasma osmolality and chloride levels also showed significant decreases from seawater values during the freshwater migration to spawning areas. Movement from seawater to freshwater increased mRNA expression of a freshwater-specific Na+,K+-ATPase isoform (α1a) while having no effect on the seawater-specific isoform (crib). In addition, gill Na+,K+-ATPase activity generally increased in active spawners compared with unspawned fish on the spawning grounds and this was associated with a marked increase in Na+,K+-ATPase alb mRNA. Increases in gill Na+,K+-ATPase activities observed in spawners suggests that the fish may be attempting to compensate for the osmotic perturbation associated with the decline ill plasma chloride concentration and osmolality. [ABSTRACT FROM AUTHOR]

Abstract: The estuarine crab Chasmagnathus granulatus (Crustacea, Decapoda, Brachyura) inhabits salt marshes along the South Atlantic coast from Rio de Janeiro (Brazil) to Patagonia (Argentina). In the present study, salinity tolerance (0–45‰; 16–1325 mOsm/kg H2O) and hemolymph osmotic and ionic (Na+, Cl−, and K+) regulation in both female and male C. granulatus were analyzed in summer and winter. Results showed that both female and male C. granulatus are euryhaline. Mortality was only observed in extremely low salinity (0‰; 16 mOsm/kg H2O) for both sexes. For females, the LT50 at 0‰ salinity was similar in summer (20.1 h) and winter (17.4 h). Males were more tolerant to salinity than females in both seasons, and mortality was observed only in summer (LT50 = 50.9 h). Results from freshly collected crabs or long-term (16-day) osmotic and ionic regulation experiments in the laboratory showed that male C. granulatus is a better hyper-osmoregulator than female in summer and winter. However, a hypo-osmoregulatory ability was only observed in females experimentally subjected to salinity 40‰ (1176±11 mOsm/kg H2O) in both seasons. In both sexes, hyper-osmotic regulation was achieved by hyper-regulating hemolymph Na+, Cl−, and K+ concentration. In females, hypo-osmotic regulation was achieved by hypo-regulating hemolymph Na+ and Cl− concentration. Long-term (16-day) osmotic and ionic regulations in different salinities were similar in males or females collected and tested in summer and winter. Despite this lack of a seasonal effect on hemolymph osmoregulatory and ionoregulatory patterns in males or females, a marked seasonal difference in the dynamics of these processes was observed for both sexes. In the first 2 days after hypo-osmotic shock (20‰→5‰; 636→185 mOsm/kg H2O), variations in female osmolality and ion (Na+ and Cl−) concentration were larger and faster in winter than in summer, while in males the opposite was observed. Furthermore, a seasonal effect on the crab response to hyper-osmotic shock (20‰→40‰; 636→1176 mOsm/kg H2O) was only observed in males. A new osmolality and ion (Na+ and Cl−) concentration steady state was faster achieved in winter than in summer. Regarding sexual differences, females showed a better capacity to hypo-regulate the hemolymph osmolality and Na+ concentration than males, even after a sudden increase in salinity (hyper-osmotic shock) in both seasons. On the other hand, males showed a better capacity to hyper-regulate the hemolymph osmolality and Na+ concentration than females, even after a sudden decrease in salinity (hypo-osmotic shock), especially in winter. Taken together, results reported in the present study suggest the need to consider both sex and collection season as important factors in future osmotic and ionic regulation studies in estuarine crabs. [Copyright &y& Elsevier]

The mechanism of Pb-induced disruption of Na+ and Cl- balance was investigated in the freshwater rainbow trout (Oncorhynchus mykiss). Na+ and Cl- influx rates were reduced immediately in the presence of 2.40 ± 0.24 and 1.25 ± 0.14 µM Pb, with a small increase in efflux rates occurring after 24-h exposure. Waterborne Pb caused a significant decrease in the maximal rate of Na+ influx without a change in transporter affinity, suggesting a noncompetitive disruption of Na+ uptake by Pb. Phenamil and bafilomycin markedly reduced Na+ influx rate but did not affect Pb accumulation at the gill. Time-course analysis in rainbow trout exposed to 0, 0.48, 2.4, and 4.8 µM Pb revealed time- and concentration-dependent bronchial Pb accumulation. Na+-K+-ATPase activity was significantly reduced, with 4.8 µM exposure resulting in immediate enzyme inhibition and 0.48 and 2.4 µM exposures inhibiting activity by 24 h. Reduced activity was weakly correlated with gill Pb accumulation after 3- and 8-h exposures; this relationship strengthened by 24 h. Reduced Na+ uptake was correlated with gill Pb burden after exposures of 3, 8, and 24 h. Immediate inhibition of branchial carbonic anhydrase activity occurred after 3-h exposure to 0.82 ± 0.05 or 4.30 ± 0.05 µM Pb and continued for up to 24 h. We conclude that Pb-induced disruption of Na+ and Cl- homeostasis is in part a result of rapid inhibition of carbonic anhydrase activity and of binding of Pb with Na+-K+-ATPase, causing noncompetitive inhibition of Na+ and Cl- influx. [ABSTRACT FROM AUTHOR]

Of the fish species tested in chronic Ni exposures, rainbow trout (Oncorhynchus mykiss) is the most sensitive. To develop additional Ni toxicity data and to investigate the toxic mode of action for Ni, we conducted acute (96-h) and chronic (85-d early life-stage) flow-through studies using rainbow trout. In addition to standard toxicological endpoints, we investigated the effects of Ni on ionoregulatory physiology (Na, Ca, and Mg). The acute median lethal concentration for Ni was 20.8 mg/L, and the 24-h gill median lethal accumulation was 666 nmol/g wet weight. No effects on plasma Ca, Mg, or Na were observed during acute exposure. In the chronic study, no significant effects on embryo survival, swim-up, hatching, or fingerling survival or growth were observed at dissolved Ni concentrations up to 466 µg/L, the highest concentration tested. This concentration is considerably higher than the only other reported chronic no-observed-effect concentration (<33 µg/L) for rainbow trout. Accumulation of Ni in trout eggs indicates the chorion is only a partial barrier with 36%, 63%, and 1% of total accumulated Ni associated with the chorion, yolk, and embryo, respectively. Whole-egg ion concentrations were reduced by Ni exposure. However, most of this reduction occurred in the chorion rather than in the embryos, and no effects on hatching success or larval survival were observed as a result. Plasma ion concentrations measured in swim-up fingerlings at the end of the chronic-exposure period were not significantly reduced by exposure to Ni. Nickel accumulated on the gill in an exponential manner but plateaued in trout plasma at waterborne Ni concentrations of 118 µg/L or greater. Consistent with previous studies, Ni did not appear to disrupt ionoregulation in acute exposures of rainbow trout. Our results also suggest that Ni is not an ionoregulatory toxicant in long-term exposures, but the lack of effects in the highest Ni treatment precludes a... [ABSTRACT FROM AUTHOR]

The main objective of this study was to compare the development of seawater adaptation and changes in body composition of smolt produced using three different strategies, and to investigate growth during the first month in seawater. Smolt released at different times of the year experience different environmental conditions. In northern Norway there are large annual variations in seawater temperature and photoperiod, and both are known to influence growth rates of salmonids. Three groups of smolts were raised and released 15 April (Group 1, large 1+ smolt), 15 June (Group 2, medium sized 1+ smolt) and 15 September (Group 3, medium sized 0+ smolt). The 0+ out-of-season smolt (group 3) transferred to seawater in September had the best hypo-osmoregulatory ability of the three groups after one month in seawater. These fish also had the highest growth rate (assessed using TGC), and deposited body fat during their first month in seawater. By contrast large 1+ salmon released in April had poorer hypo-osmoregulatory ability, failed to grow during their first month in the sea, and appeared to mobilise body fat stores to support metabolism during this critical phase. The medium-sized 1+ smolt, transferred to seawater in June, occupied an intermediate position. Although they appeared to experience some problems with hypo-osmotic regulation, the fish grew during their first month in seawater, and they were able to maintain their concentration of body fat. Our findings conclude that the 0+ out-of-season smolt were not disadvantaged during the early phase of seawater rearing compared to the smolt that were produced by a ‘traditional’ strategy and transferred to seawater in spring. Indications were that the 0+ smolt fared better following transfer to seawater than did the fish making up both the large 1+ (group 1) and the medium-sized 1+ (group 2) groups. [Copyright &y& Elsevier]

The transition from aquatic to aerial respiration is associated with dramatic physiological changes in relation to gas exchange, ion regulation, acid—base balance and nitrogenous waste excretion. Arapaima gigas is one of the most obligate extant air-breathing fishes, representing a remarkable model system to investigate (1) how the transition from aquatic to aerial respiration affects gill design and (2) the relocation of physiological processes from the gills to the kidney during the evolution of air-breathing. Arapaima gigas undergoes a transition from water- to air-breathing during development, resulting in striking changes in gill morphology. In small fish (10 g), the gills are qualitatively similar in appearance to another closely related water-breathing fish (Osteogiossum bicirrhosum); however, as fish grow (100–1000 g), the inter-lamellar spaces become filled with cells, including mitochondria-rich (MR) cells, leaving only column-shaped filaments. At this stage, there is a high density of MR cells and strong immunolocalization of Na+/K+-ATPase along the outer cell layer of the gill filament. Despite the greatly reduced overall gill surface area, which is typical of obligate air-breathing fish, the gills may remain an important site for ionoregulation and acid—base regulation. The kidney is greatly enlarged in A. gigas relative to that in O. bicirrhosum and may comprise a significant pathway for nitrogenous waste excretion. Quantification of the physiological role of the gill and the kidney in A. gigas during development and in adults will yield important insights into developmental physiology and the evolution of air-breathing. [ABSTRACT FROM AUTHOR]

Two silver-contaminated diets were prepared by exposing juvenile rainbow trout for 8 d to waterborne silver thiosulfate as Ag at either 0.1 µg/L (low-Ag diet) or 80 µg/L (high-Ag diet). The level of total Ag accumulated in whole low-Ag fish was below the detection limit of analysis. Whole high-Ag fish accumulated Ag at 21.3 nmol/g. The livers of the low- and high-Ag fish accumulated Ag at 0.43 nmol/g and 1.01 µmol/g, respectively. The Ag-contaminated fish were then fed whole to adult crayfish in an 80-d dietary study to determine the effects of long-term trophic accumulation of Ag. In a second experiment, the livers of the high-Ag trout were fed to juvenile crayfish for either one or five weeks. Accumulation of Ag was demonstrated in both adult and juvenile crayfish. Silver accumulation in juvenile crayfish peaked at approximately 650 nmol/g at three weeks, after which Ag depuration occurred. In adult crayfish that consumed the high-Ag diet, the hepatopancreas accumulated more than 90% of assimilated Ag, rising 1,000-fold over control animals to approximately 740 nmol/g at 80 d. Crayfish that consumed the low-Ag diet had small, statistically insignificant elevations of Ag in some tissues. Dietary Ag had no effect on juvenile crayfish growth or adult mortality. Disturbances in osmoregulation, which are normally associated with acute waterborne Ag exposure, were not detected. Dietary Ag also had no effect on hemolymph concentrations of Na-, Cl-, Ca2+, Mg2+, or Cu; did not affect the concentration kinetics of Na+ or Cl- influx; and had no effect on the activity of gill Na+/K+-dependent adenosine triphosphatase. Hemolymph concentrations of glucose and lactate were similarly unaffected, indicating an absence of stress-related metabolic disturbance. However, a disproportionately low number of ecdysis events occurred among crayfish that consumed the high-Ag diet. [ABSTRACT FROM AUTHOR]

We describe the gut physiology of the Lake Magadi tilapia (Alcolapia grahami), specifically those aspects associated with feeding and drinking while living in water of unusually high carbonate alkalinity (titratable base=245 mequiv l−1) and pH (9.85). Drinking of this highly alkaline lake water occurs at rates comparable to or higher than those seen in marine teleosts. Eating and drinking take place throughout the day, although drinking predominates during hours of darkness. The intestine directly intersects the esophagus at the anterior end of the stomach forming a ‘T’, and the pyloric sphincter, which comprises both smooth and striated muscle, is open when the stomach is empty and closed when the stomach is full. This unique configuration (a functional trifurcation) allows imbibed alkaline water to bypass the empty stomach, thereby avoiding a reactive mixing with acidic gastric fluids, and minimizes interference with a full stomach. No titratable base was present in the stomach, where the mean pH was 3.55, but the intestine was progressively more alkaline (foregut 6.96, midgut 7.74, hindgut 8.12, rectum 8.42); base levels in the intestinal fluid were comparable to those in lake water. The gut was highly efficient at absorbing water (76.6%), which accompanied the absorption of Na+ (78.5%), titratable base (80.8%), and Cl− (71.8%). The majority of Na+, base and water absorption occurred in the foregut by an apparent Na+ plus base co-transport system. Overall, more than 70% of the intestinal flux occurred via Na+ plus base co-transport, and less than 30% by Na+ plus Cl− co-transport, a very different situation from the processes in the intestine of a typical marine teleost. [Copyright &y& Elsevier]

The mechanism for acute toxicity of lead (Pb) in rainbow trout (Oncorhynchus mykiss) was investigated at Pb concentrations close to the 96 h LC50 of 1.0 mg dissolved Pb l−1 (0.8–1.4, 95% C.I.) determined in dechlorinated Hamilton city tap water (from Lake Ontario, hardness=140 mg l−1 CaCO3). Tissue Pb accumulation associated with death was highest in the gill, followed by kidney and liver. Significant ionoregulatory impacts were observed in adult rainbow trout (200–300 g) fitted with indwelling dorsal aortic catheters and exposed to 1.1±0.04 mg dissolved Pb l−1. Decreased plasma [Ca2+], [Na+] and [Cl−] occurred after 48 h of exposure through to 120 h, with increases in plasma [Mg2+], ammonia, and cortisol. No marked changes in PaO2, PaCO2, pH, glucose, or hematological parameters were evident. Branchial Na+/K+ ATPase activity in juvenile trout exposed to concentrations close to the 96 h LC50 was inhibited by approximately 40% after 48 h of Pb exposure. Calcium ion flux measurements using 45Ca as a radiotracer showed 65% inhibition of Ca2+ influx after 0, 12, 24 or 48 h exposure to the 96 h LC50 concentration of Pb. There was also significant inhibition (40–50%) of both Na+ and Cl− uptake, measured with 22Na and 36Cl simultaneously. We conclude that the mechanism of acute toxicity for Pb in rainbow trout occurs by ionoregulatory disruption rather than respiratory or acid/base distress at Pb concentrations close to the 96 h LC50 in moderately hard water. [Copyright &y& Elsevier]

Feed intake, growth and ionoregulation of Atlantic salmon (Salmo salar L.) were studied in fish fed diets either with or without squid extract supplementation in combination with transfer from fresh water to seawater (SW) at different times during parr–smolt transformation (10 April, 3 May and 19 June). Water temperature was held at approximately 8 °C. All groups of fish showed a temporary depression in feeding and growth after SW transfer. The depression was most severe in fish transferred in April, intermediate in fish transferred in May and shortest in fish transferred in June. Nevertheless, squid extract resulted in increased feeding and growth, especially in fish transferred to SW in June, providing evidence that squid extract contains substances that serve as feeding stimulants. The results indicate that there may be a potential to improve SW performance of salmon by adding squid extract to feed. The results also indicate that Atlantic salmon may be transferred to SW over a relatively long period during spring, but to minimise the duration of appetite depression, late transfer may be preferable. Individual feeding rates following short-time SW exposure were not correlated with plasma chloride concentrations in any of the groups, indicating that appetite suppression is not a direct result of osmo-ionoregulatory failure. [Copyright &y& Elsevier]

The acute mechanism of toxicity of waterborne nickel (Ni) was investigated in the rainbow trout (Oncorhynchus mykiss) in moderately hard (∼140 mg l−1 as CaCO3) Lake Ontario water, where the 96-h LC50 for juvenile trout (1.5–3.5 g) was 15.3 mg (12.7–19.0, 95% C.L.) dissolved Ni l−1. No marked impact of Ni exposure on average unidirectional or net fluxes of Na+, Cl−, or Ca2+ was observed in juvenile trout exposed for 48–60 h to 15.6 mg Ni l−1 as NiSO4. Furthermore, when adult rainbow trout (200–340 g) were fitted with indwelling dorsal aortic catheters and exposed for 117 h to 11.6 mg Ni l−1 as NiSO4, plasma ions (Na+, Cl−, Ca2+, and Mg2+) were all well conserved. However, mean arterial oxygen tension dropped gradually to ∼35% of control values. This drop in PaO2 was accompanied by an acidosis primarily of respiratory origin. PaCO2 rose to more than double control values with a concomitant drop in arterial pH of 0.15 units. Acute respiratory toxicity was further evidenced by a significant increase in hematocrit (Ht), and plasma lactate, and a significant decrease in spleen hemoglobin (Hb). Following 117 h of exposure to 11.6 mg Ni l−1, the gill, intestine, plasma, kidney, stomach, and heart accumulated Ni significantly, with increases of 60, 34, 28, 11, 8, and 3-fold, respectively. Brain, white muscle, liver, and bile did not significantly accumulate Ni. Plasma Ni exhibited a remarkable linear increase with time to levels approximately 30-fold higher than controls. We conclude that in contrast to most other metals, Ni is primarily a respiratory, rather than an ionoregulatory, toxicant at exposure levels close to the 96-h LC50. The implications of a waterborne metal as an acute respiratory toxicant (as opposed to ionoregulatory toxicants such as Cu, Ag, Cd, or Zn) with respect to toxicity modeling are discussed. [Copyright &y& Elsevier]

Chemical speciation controls the bioavailability and toxicity of metals in aquatic systems and regulatory agencies are recognizing this as they develop updated water quality criteria (WQC) for metals. The factors that affect bioavailability may be quantitatively evaluated with the biotic ligand model (BLM). Within the context of the BLM framework, the ‘biotic ligand’ is the site where metal binding results in the manifestation of a toxic effect. While the BLM does account for the speciation and complexation of dissolved metal in solution, and competition among the free metal ion and other cations for binding sites at the biotic ligand, it does not explicitly consider either the physiological effects of metals on aquatic organisms, or the direct effect of water chemistry parameters such as pH, Ca2+and Na+ on the physiological state of the organism. Here, a physiologically-based model of survival time is described. In addition to incorporating the effects of water chemistry on metal availability to the organism, via the BLM, it also considers the interaction of water chemistry on the physiological condition of the organism, independent of its effect on metal availability. At the same time it explicitly considers the degree of interaction of these factors with the organism and how this affects the rate at which cumulative damage occurs. An example application of the model to toxicity data for rainbow trout exposed to silver is presented to illustrate how this framework may be used to predict survival time for alternative exposure durations. The sodium balance model (SBM) that is described herein, a specific application of a more generic ion balance model (IBM) framework, adds a new physiological dimension to the previously developed BLM. As such it also necessarily adds another layer of complexity to this already useful predictive framework. While the demonstrated capability of the SBM to predict effects in relation to exposure duration is a useful feature of this mechanistically-based framework, it is envisioned that, with suitable refinements, it may also have utility in other areas of toxicological and regulatory interest. Such areas include the analysis of time variable exposure conditions, residual after-effects of exposure to metals, acclimation, chronic toxicity and species and genus sensitivity. Each of these is of potential utility to longer-term ongoing efforts to develop and refine WQC for metals. [Copyright &y& Elsevier]

Developing rainbow trout were chronically exposed to silver (as AgNO3) from fertilization to swim-up, in moderately hard water (120 mg CaCO3.l−1) in the presence and absence of an additional 12 mg C/L of dissolved organic carbon (DOC, as humic acid, Aldrich). Nominal silver concentrations were 0, 0.1 and 10 μg l−1 total silver in a flow-through set-up maintained at 12 °C. The objectives of the study were to investigate the possible protective effects of DOC on growth, mortality, time to hatch and swim-up, and sublethal ionoregulatory disturbances during chronic exposure to ionic silver. Throughout development, there was a large increase in % daily mortality at 10 μg.l−1 total silver (in the absence of DOC), that was associated with an ionoregulatory disturbance, in particular a 35% reduction in whole body Na+ just prior to hatch. At nominal 10 μg.l−1 total silver, the presence of additional DOC (reducing dissolved silver to 4.7±0.3 μg l−1) resulted in a significant reduction in % daily mortality up to hatch, demonstrating a protective effect of DOC. Interestingly, DOC did not appear to mitigate the ionoregulatory disturbance, with the exception of whole body [Cl−] on day 44 of exposure. Exposure to 0.1 μg.l−1 total silver (in the absence of DOC) resulted in a statistically significant reduction in growth, and DOC did not prevent an ionoregulatory disturbance [based upon (Jin Na+), whole body Na+,K+ ATPase activity and whole body (Na+)] at this silver concentration relative to controls+DOC. DOC exerted a direct effect on growth and ionoregulatory development that complicates interpretation of the data, however, these data indicate that protective effects of DOC (in the form of Aldrich humic acid) during chronic silver exposure appear to be less than that observed during acute exposure. The ultimate goal of this and future studies is to develop a model that can predict chronic toxicity on a site-specific basis, taking into account protective effects of various ligands present in different waters, as is presently being employed for some metals during acute exposure. [Copyright &y& Elsevier]

Daphnia magna were exposed to a total concentration of 5.0±0.04 μg Ag/l, added as AgNO3 (dissolved concentration, as defined by 0.45 μm filtration=2.0±0.01 μg Ag/l) in moderately hard synthetic water under static conditions (total organic carbon=4.80±1.32 mg/l) with daily feeding and water renewal, for 21 days. There was no mortality in control daphnids and 20% mortality in silver-exposed animals. Silver exposure caused a small but significant reduction of reproductive performance manifested as a 13.7% decrease in the number of neonates produced per adult per reproduction day over the 21-day exposure. However, silver exposed daphnids also exhibited a much more marked ionoregulatory disturbance, which was characterized by a 65% decrease in whole body Na+ concentration, and an 81% inhibition of unidirectional whole body Na+ uptake. Previous work on the acute toxicity of Ag+ to daphnids has shown that Na+ uptake inhibition is directly related to inhibition of Na+,K+-ATPase activity. Therefore, we suggest that the Na+ uptake inhibition seen in response to chronic silver exposure was explained by an inhibition of the Na+ channels at the apical ‘gill’ membrane, since a 60% increase in whole body Na+,K+-ATPase activity was observed in the chronically silver-exposed daphnids. Our findings demonstrate that, in broad view, the key mechanism involved in chronic silver toxicity in D. magna, the most acutely sensitive freshwater organism tested up to now, resembles that described for acute toxicity—i.e. ionoregulatory disturbance associated with inhibition of active Na+ uptake, though the fine details may differ. Our results provide encouragement for future extension of the current acute version of the Biotic Ligand Model (BLM) to one that predicts chronic silver toxicity for environmental regulation and risk assessment. The results strongly suggest that Na+ uptake inhibition is the best endpoint to determine sensitivity to both acute and chronic toxicity in the scope of future versions of the BLM for silver. [Copyright &y& Elsevier]

The flounder, Paralichthys orbignyanus, is found in coastal and estuarine waters of the Western South Atlantic Ocean. It is being considered for aquaculture due to its high market price and wide tolerance to environmental factors such as salinity, pH, and nitrogenous compounds. The objective of this study was to characterize the ionic and osmotic regulation of P. orbignyanus over the range of its tolerated ambient salinities (0–40‰) and to evaluate the survival and growth in freshwater (0‰) and seawater (30‰) over 90 days. After 15 days of exposure to different salinities (0‰, 10‰, 20‰, 30‰ and 40‰), plasma osmolality and ionic (Na+, Cl−, K+ and Ca2+) concentrations slightly increased with salinity. The isosmotic point was estimated as 328.6 mOsm kg−1 H2O and corresponded to 10.9‰ salinity. After 90 days, survival was similar in freshwater and seawater, but osmo- and ionoregulation was significantly affected in freshwater and flounders reared in this medium showed a lower growth rate than those reared in seawater. Based on the results from this study, P. orbignyanus can be characterized as a marine/estuarine euryhaline teleost capable of hyper/hypo iono- and osmoregulation over the fluctuating salinity regime faced by this species in the environment. Furthermore, results suggest that the lower growth rate exhibited by P. orbignyanus in freshwater could be due, at least partially, to a higher energy expenditure associated to a higher branchial Na+, K+-ATPase activity in this environment. [Copyright &y& Elsevier]

Rainbow trout embryos and larvae were continuously exposed, in a flow-through system, to 0, 0.1 µg/l (measured=0.098±0.002 µg/l) or 1.0 µg/l (measured=0.853±0.022 µg/l) total silver (as AgNO3) in moderately hard water (120 mg CaCO3/l, 0.70 mM Cl–, 1.3 mg/l dissolved organic matter and 13.7±0.1 °C) from fertilization to 1 week post-hatch. The objectives of the study were to investigate the effects of chronic silver exposure on mortality, time to hatch and growth, and on sublethal physiological indicators of toxicity. Exposure to 1.0 µg/l total silver resulted in a small, but statistically significant, increase in mortality (16%) relative to controls (12%) but interestingly, resulted in an increased rate of growth (as indicated by larval weight, length and extractable protein) and ionoregulatory development over the duration of this study. Whole body unidirectional Na+ uptake (JinNa+) increased with silver exposure concentration (both 0.1 µg/l and 1.0 µg/l total silver) just prior to and following hatch, with up to a three-fold elevation in JinNa+ in the 1.0 µg/l treatment relative to controls. Qualitatively similar changes in whole body Na+,K+-ATPase activity (per mg protein or per whole embryo or larvae) also occurred over this period. By 1 week post-hatch, there were no differences in JinNa+ among treatments and Na+,K+-ATPase activity levels in silver exposed groups were significantly reduced relative to controls. Within 2 days following hatch, there was an elevation in whole larval ammonia levels, while cortisol levels were elevated at 1 week post-hatch in the 1.0 µg/l treatment relative to controls. Ionoregulatory disturbance and elevations in both cortisol and ammonia have also been observed during acute silver exposure in adult rainbow trout, indicating that chronic and acute mechanisms of toxicity may be similar. [ABSTRACT FROM AUTHOR]

Freshwater salmonids exposed to low environmental pH typically suffer a net loss of ions, primarily Na+ and Cl-, across the gills, resulting in reduced plasma and tissue ion concentrations. However, in recent experiments in our laboratory, juvenile rainbow trout, Oncorhynchus mykiss, fed a ration of 1% body weight d-1 or greater showed no ionoregulatory disturbance during chronic, sublethal acidification. This raised the possibility that these fish had acclimated to low pH in that they would be better able to withstand further, more severe acidification than fish that had no prior experience of acid conditions: previous studies had concluded that such acclimation does not occur. This hypothesis was tested by measuring unidirectional ion fluxes during a 24 h acute acid challenge (pH 4.2) in juvenile rainbow trout that had previously been exposed to either ambient pH 6.2 (naive fish) or sublethal low pH 5.2 (acid pre-exposed fish) for 90 days, and fed a ration of either 1.0 or 0.25% d-1 (wet basis). No mortalities were observed during the acute acid challenge in the fish fed the higher ration and no differences between the two groups in the response of Na+ fluxes were observed. Sodium influx in both groups was significantly inhibited throughout the challenge and Na+ net flux was significantly stimulated over the first 6 h. Prior to the acute acid challenge, the fish fed the lower ration that had previously been exposed to pH 5.2 had significantly lower plasma ion concentrations than those fish previously exposed to pH 6.2. Both groups suffered mortalities; those of the naive fish (22% by 24 h) being markedly lower than those of the acid pre-exposed fish (68% by 24 h). However, there were no significant differences in either Na+ or Cl- fluxes between the two groups of fish during the acid challenge: both showed significant inhibition of ion influxes and significantly greater net ion losses, resulting in reduced plasma ion concentrations. These results indicate that rainbow trout are unable to acclimate to environmental acidification irrespective of the availability of dietary salts. [ABSTRACT FROM AUTHOR]

Amazonia is home to 15% (>2700, in 18 orders) of all the freshwater fish species of the world, many endemic to the region, has 65 million years of evolutionary history and accounts for 20% of all freshwater discharge to the oceans. These characteristics make Amazonia a unique region in the world. We review the geological history of the environment, its current biogeochemistry and the evolutionary forces that led to the present endemic fish species that are distributed amongst three very different water types: black waters [acidic, ion-poor, rich in dissolved organic carbon (DOC)], white waters (circumneutral, particle-rich) and clear waters (circumneutral, ion-poor, DOC-poor). The annual flood pulse is the major ecological driver for fish, providing feeding, breeding and migration opportunities, and profoundly affecting O2, CO2 and DOC regimes. Owing to climate change and other anthropogenic pressures such as deforestation, pollution and governmental mismanagement, Amazonia is now in crisis. The environment is becoming hotter and drier, and more intense and frequent flood pulses are now occurring, with greater variation between high and low water levels. Current projections are that Amazon waters of the near future will be even hotter, more acidic, darker (i.e. more DOC, more suspended particles), higher in ions, higher in CO2 and lower in O2, with many synergistic effects. We review current physiological information on Amazon fish, focusing on temperature tolerance and ionoregulatory strategies for dealing with acidic and ion-poor environments. We also discuss the influences of DOC and particles on gill function, the effects of high dissolved CO2 and low dissolved O2, with emphasis on water- versus air-breathing mechanisms, and strategies for pH compensation. We conclude that future elevations in water temperature will be the most critical factor, eliminating many species. Climate change will likely favour predominantly water-breathing species with low routine metabolic rates, low temperature sensitivity of routine metabolic rates, high anaerobic capacity, high hypoxia tolerance and high thermal tolerance., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

Physiological effects of exposure to silver (AgCln n−1; 250 μg Ag l−1 or 1000 μg Ag l−1) in seawater fish were investigated using adult starry flounders. While all fish survived up to 10 days in 250 μg Ag l−1, flounders started to die after day 4 in 1000 μg l−1. Dose-dependent increases in plasma and hepatic silver concentrations showed that silver was available for uptake. There were minimal negative effects on hematological parameters, acid-base status, and blood gases. Plasma ammonia showed a pronounced (three- to four-fold), but transient increase in flounders exposed to either 250 μg Ag l−1 or 1000 μg Ag l−1. Whole body ammonia and acid equivalent efflux measurements indicated that ammonia retention was due to a combination of stimulated production and inhibited excretion. In the 1000-μg Ag l−1 group there was a similar transient increase in plasma [magnesium], which was restored by day 4. In contrast, plasma chloride and sodium levels increased gradually towards the point when fish began to die. At 250 μg Ag l−1, the Na+/K+-ATPase activity of the intestine was unaffected but there was a two-fold increase in branchial Na+/K+-ATPase activity. The latter effect was interpreted as compensation for an elevated chloride and sodium load. The increases in plasma chloride and sodium concentrations were accompanied by a marked suppression of drinking, thereby indicating that acute silver toxicity was likely caused by a combination of elevated electrolyte concentrations and dehydration. [ABSTRACT FROM AUTHOR]

This study examined the extent to which lake sturgeon, Acipenser fulvescens, regulated serum ion concentrations and osmolarity in relation to ambient salinity. Twenty juvenile lake sturgeon, brood stock from the Moose River in the Hudson Bay drainage basin, were gradually transferred from freshwater to more saline waters at a rate of 2‰ day-1. Blood was sampled from fish at 0, 10, 15 and 25‰ following 48 h acclimation periods, Concentrations of Na+, K+, Cl , Ca2+ and osmolarity were measured. Experimentation was terminated at 25‰ due to poor fish health resulting in two mortalities. Within freshwater, lake sturgeon blood serum displays similar osmotic and ionic composition to other North American species of sturgeon. Juvenile lake sturgeon do not appear capable of homeostasis above 15‰ nor are they tolerant to environmenis exceeding 25‰. Some evidence of greater tolerance to elevated salinities with increased size was noted. The ecological potential for resource exploitation in regions of elevated salinities is discussed. [ABSTRACT FROM AUTHOR]

Plasma and urine of toadfish ( Opsanus tau) in sea water and 10% sea water were analyzed to assess responses of an aglomerular fish to hypoosmotic challenge. Following transfer to 10% sea water, plasma osmotic pressure decreased slowly from 318 to 241 mmol · kg H2O−1, over a period of 10–15 days. Urine osmotic pressure decreased in parallel from 299 to 207 mmol · kg H2O−1, leaving urine/plasma ratios of osmotic pressure essentially unchanged. In contrast, the volume and composition of urine changed rapidly following transfer to 10% sea water. Urine flow rate increased 110% from 3.0 to 6.3 μl · 100g−1 · h−1 and Na+ excretion increased 346%, while excretion of Mg2− and SO4 2− decreased 81% and 90%, respectively. Excretion rates for Cl− were low in seawater toadfish and decreased further in 10% sea water. An unknown sulfur-containing anion, present in the urine of seawater toadfish, contributed significantly to the composition and ionic balance in urine of toadfish in 10% sea water. These results suggest that the inability to produce strongly dilute urine obliges toadfish to lose salt in order to excrete water, in hypoosmotic media. The decrease in plasma osmotic pressure may be both a strategy to reduce osmotic and ionic gradients in dilute media and a consequence of the kidney's inability to excrete water without salt. [ABSTRACT FROM AUTHOR]

Rainbow trout, Oncorhynchus mykiss, acclimated to 33% sea water (12 mg·ml salinity) experienced significant (10 meq·1) increases in plasma [Na] and [Cl] within 5 h of exposure to 6.3 μmol copper·1 indicating severe impairment of branchial ionoregulatory capacity. All plasma ion levels subsequently stabilised once the transbranchial [Na] gradient was reduced to zero. The similar ionic strength of the external medium and their body fluids appeared to protect trout maintained in 33% sea water from further ionoregulatory stress and any secondary physiological disturbances during exposure to copper. Despite three- and fourfold greater transbranchial [Na] and [Cl] gradients, trout acclimated to full-strength sea water (35 mg·ml salinity) suffered no major changes in plasma Na, Cl, K, or Ca, blood gases or haematology during 24 h exposure to 6.3 μmol copper·1. This reduction in toxicity in full strength sea water cannot be explained by differences in copper speciation. We suggest that during acute exposure to waterborne copper, active NaCl extrusion is unaffected due to the basolateral location of the gill Na/K-ATPase, but that ionoregulatory disturbances can occur due to gill permeability changes secondary to the displacement of surface-bound Ca. However, in full strength sea water the three-fold higher ambient [Ca] and [Mg] appear to be sufficient to prevent any detrimental permeability changes in the presence of 6.3 μmol copper·1. Plasma [NH] and [HCO] were both significantly elevated during exposure to copper, indicating that some aspects of gill ion transport (specifically the apical Na/NHand Cl/HCOexchanges involved in acid/base regulation and nitrogenous waste excretion) are vulnerable to inhibition in the presence of waterborne copper. [ABSTRACT FROM AUTHOR]

The mangrove crab Ucides cordatus showed a significant reduction in its output of isosmotic urine (to 60% of submerged rates) in humid, emersed conditions when in apparent water balance. A similar, but lesser, reduction was observed in the intertidal temperature species Carcinus maenas. Some of the decrease is effected by increased water withdrawal of an isosmotic fluid in the antennal gland (inulin urine to haemolymph concentration ratio=1.27) in Ucides but most is due to the reduced filtration rate which decreases from 3.41 to 2.19 ml·100 g·day (in Carcinus from 7.37 to 5.88). In Carcinus, inulin urine to haemolymph concentration ratios are within the range 1.34-1.41 and show significant change on emersion. Comparisons of ion and inulin clearance ratios revealed interspecific differences in renal handling of Na and Cl. During emersion total Na efflux is markedly reduced in both species. Urinary Na losses are responsible for most of the efflux in Carcinus but represent only ≈50% of the total in Ucides. Significant Na uptake from interstitial water occurs in Ucides. REductions in ion and water loss appear to be mainly dependent on decreases in urine output (clearance rate). However, some released urine was found to accumulate in the branchial chambers in both species. Whether or not extrarenal reabsorption of ions from released urine occurs in the gill chambers in either crab species remains to be proved. [ABSTRACT FROM AUTHOR]

Acutely lethal (24 h) exposure of adult rainbow trout ( Oncorhynchus mykiss) to 4.9 μmol copper·l in fresh water (pH 7.9, [Ca]≈0.8 mEq·l) caused a rapid decline of plasma Na and Cl and arterial O tension, and initially a pronounced tachycardia. The internal hypoxia probably resulted from histopathologies observed in the gills of fish exposed to copper, such as cell swelling, thickening and curling of the lamellae, and haematomas. Copper cannot therefore be considered purely as an ionoregulatory toxicant during acutely lethal conditions. Mortality during exposure to copper could not simply be explained by the plasma ionic dilution, nor by the internal hypoxia, since arterial O content remained relatively unchanged. Secondary to the ionoregulatory and respiratory disturbances were a number of deleterious physiological responses which included a massive haemoconcentration (haematocrit values as high as 60%) and a doubling of the mean arterial blood pressure. The time-course of these changes suggest that cardiac failure was the final cause of death. In this respect copper exposure resembles low pH exposure in freshwater trout (Milligan and Wood 1982). Copper and H appear to be similar in both the primary site of their toxic action (the gills) and the secondary physiological consequences which result from acutely lethal exposures. Furthermore, the acute toxicity syndrome observed may be common to many metals which cause ionoregulatory and/or respiratory problems in freshwater fish. [ABSTRACT FROM AUTHOR]

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 22 Na + uptake ( J Na in ), ammonia excretion, net acidic equivalent flux and net K + flux ( J H net ) were measured. The involvement of NHEs, Na + channels, NCCs, K + -channels and K + -dependent Na + /Ca 2+ 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: J Na in 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 J Na in during acid exposure in a concentration-dependent manner, with near-complete inhibition at 10 mM. Moreover, J H net loss increased approximately fourfold at 8-10 hours of acid exposure which correlated with recovered J Na in in 1:1 fashion, and both J Na in and J H net 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., (© 2022 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.)

August Krogh made fundamental discoveries about both respiratory gas exchange and osmo/iono-regulation in fish gills. Dave Randall and co-workers identified a tradeoff between these two functions such that high functional surface area and low diffusion distance would favour O 2 uptake (e.g. exercise, hypoxia), whereas low functional surface area and high diffusion distance would favour osmo/iono-regulation (rest, normoxia). Today we call this concept the "osmorespiratory compromise" and realize that it is much more complex than originally envisaged. There are at least 6 mechanisms by which fish can change functional branchial area and diffusion distance. Three involve reorganizing blood flow pathways: (i) flow redistribution within the secondary (respiratory) lamellae; (ii) flow shunting between "respiratory" and "ionoregulatory" pathways in the filament; (iii) opening up more distal lamellae on the filament and closing non-respiratory pathways. Three more involve "reversible gill remodeling": (iv) proliferation of the interlamellar gill cell mass (ILCM); (v) proliferation of ionocytes up the sides of the lamellae; (vi) covering over the apical exposure of ionocytes by extension of pavement cells. In ways that remain incompletely understood, these mechanisms allow dynamic regulation of the osmorespiratory compromise, such that ion and water fluxes can be decoupled from O 2 uptake during continuous exercise. Furthermore, hypoxia-tolerant species can reduce branchial ion and water fluxes below normoxic levels despite hyperventilating during hypoxia. In marine fish, the osmorespiratory conflict is intensified by the greater ionic and osmotic gradients from seawater to blood, but underlying mechanisms remain poorly understood. Unlabelled Image • In fish gills, high functional surface area and low diffusion distance favour O 2 uptake. • The opposite conditions favour iono/osmoregulation. • Six (or more) mechanisms can change effective gill permeability. • Three mechanisms involve blood flow redistribution, three mechanisms involve reversible remodeling of structure. • Ion and water fluxes can be decoupled from O 2 uptake during hypoxia and exercise. [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.

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., (© 2021 Wiley Periodicals LLC.)