Jeff G. Richards

Na+/K+-ATPase alpha-isoform switching in gills of rainbow trout (Oncorhynchus mykiss) during salinity transfer.

SUMMARY We identified five Na+/K+-ATPase α-isoforms in rainbow trout and characterized their expression pattern in gills following seawater transfer. Three of these isoforms were closely related to other vertebrate α1 isoforms (designated α1a, α1b and α1c), one isoform was closely related to α2 isoforms (designated α2) and the fifth was closely related to α3 isoforms (designated α3). Na+/K+-ATPase α1c- and α3-isoforms were present in all tissues examined, while all others had tissue specific distributions. Four Na+/K+-ATPase α-isoforms were expressed in trout gills (α1a, α1b, α1c and α3). Na+/K+-ATPase α1c- and α3-isoforms were expressed at low levels in freshwater trout gills and their expression pattern did not change following transfer to 40% or 80% seawater. Na+/K+-ATPase α1a and α1b were differentially expressed following seawater transfer. Transfer from freshwater to 40% and 80% seawater decreased gill Na+/K+-ATPaseα 1a mRNA, while transfer from freshwater to 80% seawater caused a transient increase in Na+/K+-ATPase α1b mRNA. These changes in isoform distribution were accompanied by an increase in gill Na+/K+-ATPase enzyme activity by 10 days after transfer to 80% seawater, though no significant change occurred following transfer to 40% seawater. Isoform switching in trout gills following salinity transfer suggests that the Na+/K+-ATPase α1a- andα 1b-isoforms play different roles in freshwater and seawater acclimation, and that assays of Na+/K+-ATPase enzyme activity may not provide a complete picture of the role of this protein in seawater transfer.

Reciprocal expression of gill Na+/K+-ATPase alpha-subunit isoforms alpha1a and alpha1b during seawater acclimation of three salmonid fishes that vary in their salinity tolerance.

SUMMARY The upregulation of gill Na+/K+-ATPase activity is considered critical for the successful acclimation of salmonid fishes to seawater. The present study examines the mRNA expression of two recently discovered α-subunit isoforms of Na+/K+-ATPase (α1a and α1b) in gill during the seawater acclimation of three species of anadromous salmonids, which vary in their salinity tolerance. Levels of these Na+/K+-ATPase isoforms were compared with Na+/K+-ATPase activity and protein abundance and related to the seawater tolerance of each species. Atlantic salmon (Salmo salar) quickly regulated plasma Na+, Cl– and osmolality levels within 10 days of seawater exposure, whereas rainbow trout (Oncorhynchus mykiss) and Arctic char (Salvelinus alpinus) struggled to ionoregulate, and experienced greater perturbations in plasma ion levels for a longer period of time. In all three species, mRNA levels for theα 1a isoform quickly decreased following seawater exposure whereasα 1b levels increased significantly. All three species displayed similar increases in gill Na+/K+-ATPase activity during seawater acclimation, with levels rising after 10 and 30 days. Freshwater Atlantic salmon gill Na+/K+-ATPase activity and protein content was threefold higher than those of Arctic char and rainbow trout, which may explain their superior seawater tolerance. The role of the α1b isoform may be of particular importance during seawater acclimation of salmonid fishes. The reciprocal expression of Na+/K+-ATPase isoforms α1a and α1b during seawater acclimation suggests they may have different roles in the gills of freshwater and marine fishes; ion uptake in freshwater fish and ion secretion in marine fishes.

Ionoregulatory disruption as the acute toxic mechanism for lead in the rainbow trout (Oncorhynchus mykiss)

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) CaCO(3)). 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 [Ca(2+)], [Na(+)] and [Cl(-)] occurred after 48 h of exposure through to 120 h, with increases in plasma [Mg(2+)], ammonia, and cortisol. No marked changes in PaO(2), PaCO(2), 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 Ca(2+) 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.

Acute waterborne nickel toxicity in the rainbow trout (Oncorhynchus mykiss) occurs by a respiratory rather than ionoregulatory mechanism

The acute mechanism of toxicity of waterborne nickel (Ni) was investigated in the rainbow trout (Oncorhynchus mykiss) in moderately hard ( approximately 140 mg l(-1) as CaCO(3)) Lake Ontario water, where the 96-h LC(50) 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 Ca(2+) was observed in juvenile trout exposed for 48-60 h to 15.6 mg Ni l(-1) as NiSO(4). 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 NiSO(4), plasma ions (Na(+), Cl(-), Ca(2+), and Mg(2+)) were all well conserved. However, mean arterial oxygen tension dropped gradually to approximately 35% of control values. This drop in P(aO(2)) was accompanied by an acidosis primarily of respiratory origin. P(aCO(2)) 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 LC(50). 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.

Intraspecific divergence of ionoregulatory physiology in the euryhaline teleost Fundulus heteroclitus: possible mechanisms of freshwater adaptation

SUMMARY We examined intraspecific variation in ionoregulatory physiology within euryhaline killifish, Fundulus heteroclitus, to understand possible mechanisms of freshwater adaptation in fish. Pronounced differences in freshwater tolerance existed between northern (2% mortality) and southern (19% mortality) killifish populations after transfer from brackish water (10 g l-1) to freshwater. Differences in Na+ regulation between each population might partially account for this difference in tolerance, because plasma Na+ was decreased for a longer period in southern survivors than in northerns. Furthermore, northern fish increased Na+/K+-ATPase mRNA expression and activity in their gills to a greater extent 1-14 days after transfer than did southerns, which preceded higher whole-body net flux and unidirectional influx of Na+ at 14 days. All observed differences in Na+ regulation were small, however, and probably cannot account for the large differences in mortality. Differences in Cl- regulation also existed between populations. Plasma Cl- was maintained in northern fish, but in southerns, plasma Cl- decreased rapidly and remained low for the duration of the experiment. Correspondingly, net Cl- loss from southern fish remained high after transfer, while northerns eliminated Cl- loss altogether. Elevated Cl- loss from southern fish in freshwater was possibly due to a persistence of seawater gill morphology, as paracellular permeability (indicated by extrarenal clearance rate of PEG-4000) and apical crypt density in the gills (detected using scanning electron microscopy) were both higher than in northern fish. These large differences in the regulation of Cl- balance probably contributed to the marked differences in mortality after freshwater transfer. Glomerular filtration rate and urination frequency were also lower in southerns. Taken together, these data suggest that northern killifish are better adapted to freshwater environments and that minimizing Cl- imbalance appears to be the key physiological difference accounting for their greater freshwater tolerance.

Ionoregulatory changes in different populations of maturing sockeye salmon Oncorhynchus nerka during ocean and river migration.

SUMMARY 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 (α1b). 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 α1b 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 in plasma chloride concentration and osmolality.

Limited extracellular but complete intracellular acid-base regulation during short-term environmental hypercapnia in the armoured catfish, Liposarcus pardalis.

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.

Wild Arctic Char (Salvelinus alpinus) Upregulate Gill Na+,K+‐ATPase during Freshwater Migration

The successful acclimation of eurhyhaline fishes from seawater to freshwater requires the gills to stop actively secreting ions and start actively absorbing ions. Gill Na+,K+‐ATPase is known to be an integral part of the active ion secretion model of marine fishes, but its importance in the active ion uptake model of freshwater fishes is less clear. This study, conducted in the high Arctic, examines gill Na+,K+‐ATPase regulation in wild anadromous arctic char returning to freshwater from the ocean. Gill Na+,K+‐ATPase activity, protein expression, and mRNA expression of Na+,K+‐ATPase isoforms α1a and α1b were monitored in arctic char at three points along their migration route to and from Somerset Island, Nunavut, Canada: out at sea (Whaler’s Point), in seawater near the river mouth (Nat’s Camp), and after entering the Union River. Arctic char collected from the Union River had more than twofold greater gill Na+,K+‐ATPase activity. This was associated with a significant increase (threefold) in Na+,K+‐ATPase isoform α1a mRNA expression and a significant increase in plasma sodium and osmolality levels compared with seawater char. Compared with char sampled from Whaler’s Point, Na+,K+‐ATPase isoform α1b mRNA expression was decreased by ∼50% in char sampled at Nat’s Camp and the Union River. These results suggest that the upregulation of gill Na+,K+‐ATPase activity is involved in freshwater acclimation of arctic char and implicate a role for Na+,K+‐ATPase isoform α1a in this process. In addition, we discuss evidence that arctic char go through a preparatory phase, or “reverse smoltification,” before entering freshwater.

Impact of Ontogenetic Changes in Branchial Morphology on Gill Function in Arapaima gigas

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 (∼67.5 g), which still have lamellae, larger fish (∼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 (26.5 ± 1.9 vs. 16.5 ± 1.5 mmHg) and \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland,xspace} \usepackage{amsmath,amsxtra} \usepackage[OT2,OT1]{fontenc} \newcommand\cyr{ \renewcommand\rmdefault{wncyr} \renewcommand\sfdefault{wncyss} \renewcommand\encodingdefault{OT2} \normalfont \selectfont} \DeclareTextFontCommand{\textcyr}{\cyr} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \landscape