We measured unidirectional ion fluxes of fish collected directly from the Rio Negro, an extremely dilute, acidic blackwater tributary of the Amazon. Kinetic analysis of Na(+) uptake revealed that most species had fairly similar J(max) values, ranging from 1,150 to 1,750 nmol g(-1) h(-1), while K(m) values varied to a greater extent. Three species had K(m) values <33 micromol L(-1), while the rest had K(m) values >or=110 micromol L(-1). Because of the extremely low Na(+) concentration of Rio Negro water, the differences in K(m) values yield very different rates of Na(+) uptake. However, regardless of the rate of Na(+) uptake, measurements of Na(+) efflux show that Na(+) balance was maintained at very low Na(+) levels (<50 micromol L(-1)) by most species. Unlike other species with high K(m) values, the catfish Corydoras julii maintained high rates of Na(+) uptake in dilute waters by having a J(max) value at least 100% higher than the other species. Corydoras julii also demonstrated the ability to modulate kinetic parameters in response to changes in water chemistry. After 2 wk in 2 mmol L(-1) NaCl, J(max) fell >50%, and K(m) dropped about 70%. The unusual acclimatory drop in K(m) may represent a mechanism to ensure high rates of Na(+) uptake on return to dilute water. As well as being tolerant of extremely dilute waters, Rio Negro fish generally were fairly tolerant of low pH. Still, there were significant differences in sensitivity to pH among the species on the basis of degree of stimulation of Na(+) efflux at low pH. There were also differences in sensitivity to low pH of Na(+) uptake, and two species maintained significant rates of uptake even at pH 3.5. When fish were exposed to low pH in Rio Negro water instead of deionized water (with the same concentrations of major ions), the effects of low pH were reduced. This suggests that high concentrations of dissolved organic molecules in the water, which give it its dark tea color, may interact with the branchial epithelium in some protective manner.

SUMMARYThe salinity tolerance of the `California' Mozambique tilapia(Oreochromis mossambicus × O. urolepis hornorum), a current inhabitant of the hypersaline Salton Sea in California, USA, was investigated to identify osmoregulatory stress indicators for possible use in developing a model of salinity tolerance. Seawater-acclimated (35 g l–1) tilapia hybrids were exposed to salinities from 35–95 g l–1, using gradual and direct transfer protocols, and physiological (plasma osmolality, [Na+],[Cl–], oxygen consumption, drinking rate, hematocrit, mean cell hemoglobin concentration, and muscle water content), biochemical(Na+, K+-ATPase) and morphological (number of mature,accessory, immature and apoptotic chloride cells) indicators of osmoregulatory stress were measured. Tilapia tolerated salinities ranging from 35 g l–1 to 65 g l–1 with little or no change in osmoregulatory status; however, in fish exposed to 75–95 g l–1 salinity, plasma osmolality, [Na+],[Cl–], Na+, K+-ATPase, and the number of apoptotic chloride cells, all showed increases. The increase in apoptotic chloride cells at salinities greater than 55 g l–1, prior to changes in physiological and biochemical parameters, indicates that it may be the most sensitive indicator of osmoregulatory stress. Oxygen consumption decreased with salinity, indicating a reduction in activity level at high salinity. Finally, `California' Mozambique tilapia have a salinity tolerance similar to that of pure Mozambique tilapia; however, cellular necrosis at 95 g l–1 indicates they may be unable to withstand extreme salinities for extended periods of time.

We examined the metabolic and ionoregulatory responses of the Amazonian cichlid, Astronotus ocellatus, to 20 h exposure to severe hypoxia (0.37 +/- 0.19 mg O(2)/l; 4.6% air saturation) or 8 h severe hypoxia followed by 12 h recovery in normoxic water. During 20 h exposure to hypoxia, white muscle [ATP] was maintained at normoxic levels primarily through a 20% decrease in [creatine phosphate] (CrP) and an activation of glycolysis yielding lactate accumulation. Muscle lactate accumulation maintained cytoplasmic redox state ([NAD(+)]/[NADH]) and was associated with an inactivation of the mitochondrial enzyme pyruvate dehydrogenase (PDH). The inactivation of PDH was not associated with significant changes in cytoplasmic allosteric modulators ([ADP(free)], redox state, or [pyruvate]). Hypoxia exposure caused an approximately 65% decrease in gill Na(+)/K(+) ATPase activity, which was not matched by changes in Na(+)/K(+) ATPase alpha-subunit protein abundance indicating post-translational modification of Na(+)/K(+) ATPase was responsible for the decrease in activity. Despite decreases in gill Na(+)/K(+) ATPase activity, plasma [Na(+)] increased, but this increase was possibly due to a significant hemoconcentration and fluid shift out of the extracellular space. Hypoxia caused an increase in Na(+)/K(+) ATPase alpha-subunit mRNA abundance pointing to either reduced mRNA degradation during exposure to hypoxia or enhanced expression of Na(+)/K(+) ATPase alpha-subunit relative to other genes.

Stenohaline freshwater stingrays (Potamotrygon spp.) are endemic to the very dilute (Na(+), Cl(-), Ca2(+) 300 micromol L(-1) in reference water (low DOC) to about 100 micromol L(-1) in blackwater (high DOC). In reference water, both JNain and JClin were inhibited >90%, both JNaout and JClout more than doubled, and J(Amm) did not change at pH 4.0. In blackwater, the inhibition of influxes was attenuated, the increases in outflux did not occur, and J(Amm) increased by 60% at pH 4.0. Addition of 100 micromol L(-1) Ca(2+) to reference water prevented the increases in JNaout and JClout and allowed J(Amm) to increase at pH 4.0, which demonstrates that the gills are sensitive to Ca(2+). However, addition of Ca(2+) to blackwater had no effect on the responses to pH 4.0. Addition of commercial humic acid to reference water did not duplicate the effects of natural Rio Negro blackwater at the same DOC level; instead, it greatly exacerbated the increases in JNaout and JClout at low pH and prevented any protective influence of added Ca(2+). Thus, blackwater DOC appears to be very different from commercial humic acid. Biogeochemical modeling indicated that blackwater DOC prevents Ca(2+) binding, but not H(+) binding, to the gills and that the protective effects of blackwater cannot be attributed to its higher buffer capacity or its elevated Al or Fe levels. Natural DOC may act directly at the gills at low pH to exert a protective effect and, when doing so, may override any protective action of Ca(2+) that might otherwise occur.

SUMMARY Environmental hypercapnia induces a respiratory acidosis that is usually compensated within 24-96 h in freshwater fish. Water ionic composition has a large influence on both the rate and degree of pH recovery during hypercapnia. Waters of the Amazon are characteristically dilute in ions, which may have consequences for acid-base regulation during environmental hypercapnia in endemic fishes. The armoured catfish Liposarcus pardalis, from the Amazon, was exposed to a water PCO2 of 7, 14 or 42 mmHg in soft water (in μmol l-1: Na+, 15,Cl-, 16, K+, 9, Ca2+, 9, Mg2+, 2). Blood pH fell within 2 h from a normocapnic value of 7.90±0.03 to 7.56±0.04, 7.34±0.05 and 6.99±0.02, respectively. Only minor extracellular pH (pHe) recovery was observed in the subsequent 24-96 h. Despite the pronounced extracellular acidosis,intracellular pH (pHi) of the heart, liver and white muscle was tightly regulated within 6 h (the earliest time at which these parameters were measured) via a rapid accumulation of intracellular HCO3-. While most fish regulate pHi during exposure to environmental hypercapnia, the time course for this is usually similar to that for pHe regulation. The degree of extracellular acidosis tolerated by L. pardalis, and the ability to regulate pHi in the face of an extracellular acidosis, are the greatest reported to date in a teleost fish. The preferential regulation of pHi in the face of a largely uncompensated extracellular acidosis in L. pardalis is rare among vertebrates, and it is not known whether this is associated with the ability to air-breathe and tolerate aerial exposure, or living in water dilute in counter ions, or with other environmental or evolutionary selective pressures. The ubiquity of this strategy among Amazonian fishes and the mechanisms employed by L. pardalis are clearly worthy of further study.

Hyper-saline habitats (waters with salinity >35 ppt) are among the harshest aquatic environments. Relatively few species of teleost fish can tolerate salinities much above 50 ppt, because of the challenges to osmoregulation, but those that do, usually estuarine, euryhaline species, show a strong ability to osmoregulate in salinities well over 100 ppt. Typically, plasma Na(+) and Cl(-) concentrations rise slowly or not at all up to about 65 ppt. At higher salinities ion levels do rise, but the increase is small relative to the magnitude of increase in concentrations of the surrounding water. A number of adjustments are responsible for such strong osmoregulation. Reduced branchial water permeability is indicated by the observation that with the exposure to hyper-salinities drinking rates rise more slowly than the branchial osmotic gradient. Lower water permeability limits osmotic water loss and greatly reduces the salt load incurred in replacing it. Still, increased gut Na(+)/K(+)-ATPase (NAK) activity is necessary to absorb the larger gut salt load and increased HCO(3) (-) secretion is required to precipitate Ca(2+) and some Mg(2+) in the imbibed water to facilitate water absorption. All Na(+) and Cl(-) taken up must be excreted and increased branchial salt excreting capacity is indicated by elevated mitochondrion-rich cell density and size, gill NAK activity and expression of chloride channels. Excretion of Na(+) and Cl(-) occurs against a larger gradient than in seawater and calculation of the equilibrium potential for Na(+) across the gill epithelium indicates that the trans-epithelial potential required for excretion of Na(+) climbs with salinity up to about 65 ppt before leveling off due to the increasing plasma Na(+) levels. During acute transition to SW or mildly hyper-saline waters, some species have shown the ability to upregulate branchial NAK activity rapidly and this may play an important role in limiting disturbances at higher salinities. It does not appear that the opercular epithelium, which in SW acts in a way that is functionally similar to the gills, continues to do so in hyper-saline waters. Little is know about the hormones involved in acclimation to hyper-salinity, but the few studies available suggest a role for cortisol, but not growth hormone and insulin-like growth factor. Despite the increased transport capacity evident in both the gill and gut in hyper-saline waters there is no clear trend toward increased metabolic rate. These studies provide a general outline of the mechanisms of osmoregulation in these species, but significant questions still remain.

Our goal was to compare the internal physiological responses to acid challenge in an acidophilic tropical teleost endemic to dilute low-pH waters with those in nonacidophilic temperate species such as salmonids, which have been the subjects of most previous investigations. The Amazonian tambaqui (Colossoma macropomum), which migrates between circumneutral water and dilute acidic "blackwater" of the Rio Negro, was exposed to a graded low-pH and recovery regime in representative soft water (Na+ = 15, Cl- = 16, Ca2+ = 20 mumol L-1). Fish were fitted with arterial catheters for repetitive blood sampling. Water pH was altered from 6.5 (control) to 5.0, 4.0, 3.0, and back to 6.5 (recovery) on successive days. Some deaths occurred at pH 3.0. Throughout the regime, there were no disturbances of blood gases (O2 and CO2 tensions and contents) or lactate levels, and only very minor changes in acid-base status of plasma and red cells. However, erythrocytic guanylate and adenylate levels increased at pH's less than or equal to 5.0. Down to pH 4.0, plasma glucose, cortisol, and total ammonia levels remained constant, but all increased at pH 3.0, denoting a stress response. Plasma Na+ and Cl- levels declined and plasma protein concentration increased at pH 3.0, indicative of ionoregulatory and fluid volume disturbance, and neither recovered upon return to pH 6.5. Cortisol and ammonia elevations also persisted. Transepithelial potential changed progressively from highly negative values (inside) at pH 6.5 to highly positive values at pH 3.0; these alterations were fully reversible. Experimental elevations in water calcium levels drove the transepithelial potential positive at circumneutral pH, attenuated or prevented changes in transepithelial potential at low pH, and reduced Na+ and Cl- loss rates to the water during acute low-pH challenges. In general, tambaqui exhibited responses to low pH that were qualitatively similar but quantitatively more resistant than those previously documented in salmonids.

Soon after hatching, the osteoglossid fish Arapaima gigas undergoes a rapid transition from a water breather to an obligate air breather. This is followed by a gradual disappearance of gill lamellae, which leaves smooth filaments with a reduced branchial diffusion capacity due to loss of surface area, and a fourfold increase in diffusion distance. This study evaluated the effects these changes have on gill function by examining two size classes of fish that differ in gill morphology. In comparison to smaller fish (approximately 67.5 g), which still have lamellae, larger fish (approximately 724.2 g) without lamellae took up a slightly greater percentage of O2 across the gills (30.1% vs. 23.9%), which indicates that the morphological changes do not place limitations on O2 uptake in larger fish. Both size groups excreted similar percentages of CO2 across the gills (85%-90%). However, larger fish had higher blood PCO2 (26.51.9 vs. 16.51.5 mmHg) and HCO3(-) (40.2 +/- 2.9 vs. 33.6 +/- 4.5 mmol L(-1)) concentrations and lower blood pH (7.58 +/- 0.01 vs. 7.70 +/- 0.04) than did smaller fish, despite having lower mass-specific metabolisms, suggesting a possible diffusion limitation for CO2 excretion in larger fish. With regard to ion regulation, rates of diffusive Na+ loss were about 3.5 times higher in larger fish than they were in smaller fish, despite the lowered branchial diffusion capacity, and rates of Na+ uptake were higher by about the same amount despite 40% lower activity of branchial Na+/K+-ATPase. Kinetic analysis of Na uptake revealed an extremely low-affinity (K(m) = 587.9 +/- 169.5 micromol L(-1)), low-capacity (J(max) = 265.7 +/- 56.8 nmol g(-1) h(-1)) transport system. These data may reflect a general reduction in the role of the gills in ion balance. Renal Na+/K+-ATPase activity was 5-10 times higher than Na+/K+-ATPase activity in the gills, and urine: plasma ratios for Na+ and Cl(-) were very low (0.001-0.005) relative to that of other fish, which suggested an increased role for dietary salt intake and renal salt retention and which was representative of a more "terrestrial" mode of ion regulation. Such de-emphasis of branchial ion regulation confers greatly reduced sensitivity of diffusive ion loss to low water pH. Ammonia excretion also appeared to be impacted by gill changes. Rates of ammonia excretion in larger fish were one third less than that in smaller fish, despite larger fish having blood ammonia concentrations that were twice as high.