SUMMARYWe 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+-ATPaseα1a 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- andα1b-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.

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 theα1a isoform quickly decreased following seawater exposure whereasα1b 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.

The seawater tolerance of Atlantic salmon (Salmo salar) smolts reared under identical hatchery conditions was assessed in two Norwegian strains: AquaGen and Imsa. Plasma ion levels were disrupted in both strains following seawater exposure, but these disruptions were more profound in the AquaGen fish. To investigate the mechanisms underlying these differences, we measured gill Na+,K+-adenosine triphosphatase (ATPase) activity and mRNA levels of Na+,K+-ATPase α-subunit and two isoforms of the cystic fibrosis transmembrane conductance regulator (CFTR). Gill Na+,K+-ATPase activity rose significantly in both strains following seawater exposure. Both Na+,K+-ATPase α-subunit and CFTR I mRNA levels were significantly elevated for the entire 2-week period following seawater exposure, whereas CFTR II levels were transiently elevated during the first 24 h only. There were no differences in enzyme activity or gene expression between strains, with the exception of CFTR II, which was significantly lower in the Imsa strain 2 weeks following seawater exposure. This suggests that although changes in mRNA and protein expression for these genes are associated with seawater transfer, they are not the basis of observed physiological differences between strains.

SUMMARY The migration of Arctic char Salvelinus alpinus from freshwater to seawater requires a substantial reorganization of the osmoregulatory tissues to regulate plasma ion levels. These modifications have an inherent metabolic cost, which must be met through the upregulation of intermediary metabolism. Arctic char intermediary metabolism was monitored during the initial 96 h of seawater acclimation through measurement of key enzymes in gill, liver, red and white muscle as well as tissue and blood free amino acid (FAA) levels, and plasma glucose and non-esterified fatty acid content. In general, seawater exposure stimulated large changes in amino acid metabolism, but no change in lipid or carbohydrate metabolism. White muscle FAA content increased significantly following seawater exposure, with levels of essential FAAs doubling after 96 h. Similar increases were seen in the plasma, suggesting a rapid mobilization of FAAs to the circulation. These changes were accompanied by significant increases in the activities of enzymes involved in amino acid metabolism in the gill, liver, red and white muscle, suggesting seawater-acclimated fish have an enhanced capacity for energy production from amino acids. Increased energy requirements were evident in the gill of seawater-acclimated char, as citrate synthase activity increased significantly. The results of this study suggest a rapid upregulation of amino acid metabolism may be critical for the successful acclimation of Arctic char to seawater.

SUMMARYFew studies have examined changes in salmon gill ion transporter expression during the transition from seawater to freshwater, a pivotal moment in the salmonid life cycle. Seawater-acclimated Atlantic salmon were transferred to freshwater and blood and gill tissue were sampled over 30days of acclimation. Salmon held in seawater had stable plasma osmolality and sodium and chloride levels throughout the experiment. Following freshwater exposure, plasma sodium and chloride levels and total osmolality decreased significantly before returning towards control levels over time. Gill H

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Inanga (Galaxias maculatus) is an amphidromous fish with a well-known capacity to withstand a wide range of environmental salinities. To investigate the molecular mechanisms facilitating acclimation of inanga to seawater, several isoforms of the Na(+), K(+)-ATPase ion transporter were identified. This included three α-1 (a, b and c), an α-2 and two α-3 (a and b) isoforms. Phylogenetic analysis showed that the inanga α-1a and α-1b formed a clade with the α-1a and α-1b isoforms of rainbow trout, while another clade contained the α-1c isoforms of these species. The expression of all the α-1 isoforms was modulated after seawater exposure (28‰). In gills, the expression of the α-1a isoform was progressively down-regulated after seawater exposure, while the expression of the α-1b isoform was up-regulated. The α-1c isoform behaved similarly to the α-1a, although changes were less dramatic. Physiological indicators of salinity acclimation matched the time frame of the changes observed at the molecular level. A 24-h osmotic shock period was highlighted by small increases in plasma osmolality, plasma Na(+) and a decrease in muscle tissue water content. Thereafter, these values returned close to their pre-exposure (freshwater) values. Na(+), K(+)-ATPase activity showed a decreasing trend over the first 72 h following seawater exposure, but activity increased after 240 h. Our results indicate that inanga is an excellent osmoregulator, an ability that is conferred by the rapid activation of physiological and molecular responses to salinity change.

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