Expression Profiles of Branchial FXYD Proteins in the Brackish Medaka Oryzias dancena: A Potential Saltwater Fish Model for Studies of Osmoregulation

FXYD proteins are novel regulators of Na⁺-K⁺-ATPase (NKA). In fish subjected to salinity challenges, NKA activity in osmoregulatory organs (e.g., gills) is a primary driving force for the many ion transport systems that act in concert to maintain a stable internal environment. Although teleostean FXYD proteins have been identified and investigated, previous studies focused on only a limited group of species. The purposes of the present study were to establish the brackish medaka (Oryzias dancena) as a potential saltwater fish model for osmoregulatory studies and to investigate the diversity of teleostean FXYD expression profiles by comparing two closely related euryhaline model teleosts, brackish medaka and Japanese medaka (O. latipes), upon exposure to salinity changes. Seven members of the FXYD protein family were identified in each medaka species, and the expression of most branchial fxyd genes was salinity-dependent. Among the cloned genes, fxyd11 was expressed specifically in the gills and at a significantly higher level than the other fxyd genes. In the brackish medaka, branchial fxyd11 expression was localized to the NKA-immunoreactive cells in gill epithelia. Furthermore, the FXYD11 protein interacted with the NKA α-subunit and was expressed at a higher level in freshwater-acclimated individuals relative to fish in other salinity groups. The protein sequences and tissue distributions of the FXYD proteins were very similar between the two medaka species, but different expression profiles were observed upon salinity challenge for most branchial fxyd genes. Salinity changes produced different effects on the FXYD11 and NKA α-subunit expression patterns in the gills of the brackish medaka. To our knowledge, this report is the first to focus on FXYD expression in the gills of closely related euryhaline teleosts. Given the advantages conferred by the well-developed Japanese medaka system, we propose the brackish medaka as a saltwater fish model for osmoregulatory studies.     

The expression of gill Na,K-ATPase in milkfish,Chanos chanos,acclimated to seawater,brackish water and fresh water

Juvenile milkfish Chanos chanos (Forssk?l, 1775) were transferred from a local fish farm to fresh water (FW; 0 per thousand ), brackish water (BW; 10 per thousand, 20 per thousand ) and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. The blood and gill of the fish adapted to various salinities were analyzed to determine the osmoregulatory ability of this euryhaline species. No significant difference was found in plasma osmolality, sodium or chloride concentrations of milkfish adapted to various salinities. In FW, the fish exhibited the highest specific activity of Na, K-ATPase (NKA) in gills, while the SW group was found to have the lowest. Relative abundance of branchial NKA alpha-subunit revealed similar profiles. However, in contrary to other euryhaline teleosts, i.e. tilapia, salmon and eel, the naturally SW-dwelling milkfish expresses higher activity of NKA in BW and FW. Immunocytochemical staining has shown that most Na, K-ATPase immunoreactive (NKIR) cells in fish adapted to BW and SW were localized to the filaments with very few on the lamellae. Moreover, in FW-adapted milkfish, the number of NKIR cells found on the lamellae increased significantly. Such responses as elevated NKIR cell number and NKA activity are thought to improve the osmoregulatory capacity of the milkfish in hyposaline environments.     

Time-course changes in the expression of Na, K-ATPase and the morphometry of mitochondrion-rich cells in gills of euryhaline tilapia (Oreochromis mossambicus) during freshwater acclimation

Changes in expression of Na, K-ATPase (NKA) and morphometry of mitochondrion-rich (MR) cells in gills of tilapia were investigated on a 96-hr time course following transfer from seawater (SW) to fresh water (FW). A transient decline in plasma osmolality and Na+, Cl- concentrations occurred from 3 hrs onward. Gills responded to FW transfer by decreasing NKA activity as early as 3 hrs from transfer. This response was followed by a significant decrease in the NKA isoform alpha1-mRNA abundance, which was detected by real-time PCR at 6 hrs post transfer. Next, a decrease of alpha1-protein amounts were observed from 6 hrs until 24 hrs post transfer. Additionally, during the time course of FW transfer, modifications in number and size of subtypes of gill MR cells were observed although no significant difference was found in densities of all subtypes of MR cells. These modifications were found as early as 3 hrs, evident at 6 hrs (exhibition of 3 subtypes of MR cells), and mostly completed by 24 hrs post transfer. Such rapid responses (in 3 hrs) as concurrent changes in branchial NKA expression and modifications of MR cell subtypes are thought to improve the osmoregulatory capacity of tilapia in acclimation from hypertonic SW to hypotonic FW.     

Elevated Na+/K+-ATPase responses and its potential role in triggering ion reabsorption in kidneys for homeostasis of marine euryhaline milkfish (Chanos chanos) when acclimated to hypotonic fresh water

The milkfish (Chanos chanos) is an economic species in Southeast Asia. In Taiwan, the milkfish are commercially cultured in environments of various salinities. Na⁺/K⁺-ATPase (NKA) is a key enzyme for fish iono- and osmoregulation. When compared with gills, NKA and its potential role were less examined by different approaches in the other osmoregulatory organs (e.g., kidney) of euryhaline teleosts. The objective of this study was to investigate the correlation between osmoregulatory plasticity and renal NKA in this euryhaline species. Muscle water contents (MWC), plasma, and urine osmolality, kidney histology, as well as distribution, expression (mRNA and protein), and specific activity of renal NKA were examined in juvenile milkfish acclimated to fresh water (FW), seawater (SW 35‰), and hypersaline water (HSW 60‰) for at least two weeks before experiments. MWC showed no significant difference among all groups. Plasma osmolality was maintained within the range of physiological homeostasis in milkfish acclimated to different salinities, while, urine osmolality of FW-acclimated fish was evidently lower than SW- and HSW-acclimated individuals. The renal tubules were identified by staining with periodic acid Schiff’s reagent and hematoxylin. Moreover, immunohistochemical staining showed that NKA was distributed in the epithelial cells of proximal tubules, distal tubules, and collecting tubules, but not in glomeruli, of milkfish exposed to different ambient salinities. The highest abundance of relative NKA α subunit mRNA was found in FW-acclimated milkfish rather than SW- and HSW-acclimated individuals. Furthermore, relative protein amounts of renal NKA α and β subunits as well as NKA-specific activity were also found to be higher in the FW group than SW and the HSW groups. This study integrated diverse levels (i.e., histological distribution, gene, protein, and specific activity) of renal NKA expression and illustrated the potential role of NKA in triggering ion reabsorption in kidneys of the marine euryhaline milkfish when acclimated to a hypotonic FW environment.     

Two isoforms of the Na+/K+/2Cl- cotransporter are expressed in the European eel (Anguilla anguilla)

Two cDNA isoforms of the NKCC1 secretory cotransporter have been isolated from the European eel. The NKCC1a isoform exhibited mRNA expression in a wide range of tissues in a similar fashion to mammals, whereas NKCC1b was expressed primarily in the brain. The effect of freshwater (FW) to seawater (SW) transfer on NKCC1a expression was dependent on the developmental stage. In non-migratory yellow eels, NKCC1a mRNA expression in the gill was transiently up-regulated 4.3-fold after 2 days but also subsequently by 2.5-6-fold 3 weeks after SW transfer. Gill NKCC1a expression was localised mainly in branchial chloride cells of SW acclimated yellow eels. In contrast to yellow eels, NKCC1a mRNA abundance was not significantly different following SW acclimation in silver eel gill. NKCC1a mRNA abundance decreased in the kidney following SW acclimation and this may correlate with lower tubular ion/fluid secretion and urine flow rates in SW teleosts. Kidney NKCC1a mRNA expression in silver eels was also significantly lower than in yellow eels, suggesting some pre-acclimation of mRNA levels. NKCC1a mRNA was expressed at similar low levels in the middle intestine of FW- and SW-acclimated yellow or silver eels, suggesting the presence of an ion secretory mechanism in this gut segment.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

The ClC-3 chloride channel and osmoregulation in the European Sea Bass, Dicentrarchus labrax

Dicentrarchus labrax migrates between sea (SW), brackish and fresh water (FW) where chloride concentrations and requirements for chloride handling change: in FW, fish absorb chloride and restrict renal losses; in SW, they excrete chloride. In this study, the expression and localization of ClC-3 and Na⁺/K⁺-ATPase (NKA) were studied in fish adapted to SW, or exposed to FW from 10 min to 30 days. In gills, NKA-α1 subunit expression transiently increased from 10 min and reached a stabilized intermediate expression level after 24 h in FW. ClC-3 co-localized with NKA in the basolateral membrane of mitochondria-rich cells (MRCs) at all conditions. The intensity of MRC ClC-3 immunostaining was significantly higher (by 50 %) 1 h after the transfer to FW, whereas the branchial ClC-3 protein expression was 30 % higher 7 days after the transfer as compared to SW. This is consistent with the increased number of immunopositive MRCs (immunostained for NKA and ClC-3). However, the ClC-3 mRNA expression was significantly lower in FW gills. In the kidney, after FW transfer, a transient decrease in NKA-α1 subunit expression was followed by significantly higher stable levels from 24 h. The low ClC-3 protein expression detected at both salinities was not observed by immunocytochemistry in the SW kidney; ClC-3 was localized in the basal membrane of the collecting ducts and tubules 7 and 30 days after transfer to FW. Renal ClC-3 mRNA expression, however, seemed higher in SW than in FW. The potential role of this chloride channel ClC-3 in osmoregulatory and osmosensing mechanisms is discussed.     

Osmoregulation and expression of ion transport proteins and putative claudins in the gill of southern flounder (Paralichthys lethostigma)

The southern flounder is a euryhaline teleost that inhabits ocean, estuarine, and riverine environments. We investigated the osmoregulatory strategy of juvenile flounder by examining the time-course of homeostatic responses, hormone levels, and gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein expression after salinity challenge. Transfer of freshwater (FW)-acclimated flounder to sea water (SW) induced an increase in plasma osmolality and cortisol and a decrease in muscle water content, plasma insulin-like growth factor I (IGF-I) and hepatic IGF-I mRNA, all returning to control levels after 4 days. Gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein levels were elevated in response to SW after 4 days. Transfer of SW-acclimated flounder to FW reduced gill Na(+),K(+)-ATPase and Na(+),K(+),2Cl(-) cotransporter protein, increased plasma IGF-I, but did not alter hepatic IGF-I mRNA or plasma cortisol levels. Gill claudin-3 and claudin-4 immunoreactive proteins were elevated in FW versus SW acclimated flounder. The study demonstrates that successful acclimation of southern flounder to SW or FW occurs after an initial crisis period and that the salinity adaptation process is associated with changes in branchial expression of ion transport and putative tight junction claudin proteins known to regulate epithelial permeability in mammalian vertebrates.     

Physiological preparedness and performance of Atlantic salmon Salmo salar smolts in relation to behavioural salinity preferences and thresholds

This study investigated the relationships between behavioural responses of Atlantic salmon Salmo salar smolts to saltwater (SW) exposure and physiological characteristics of smolts in laboratory experiments. It concurrently described the behaviour of acoustically tagged smolts with respect to SW and tidal cycles during estuary migration. Salmo salar smolts increased their use of SW relative to fresh water (FW) from April to June in laboratory experiments. Mean preference for SW never exceeded 50% of time in any group. Preference for SW increased throughout the course of smolt development. Maximum continuous time spent in SW was positively related to gill Na⁺, K⁺‐ATPase (NKA) activity and osmoregulatory performance in full‐strength SW (measured as change in gill NKA activity and plasma osmolality). Smolts decreased depth upon reaching areas of the Penobscot Estuary where SW was present, and all fish became more surface oriented during passage from head of tide to the ocean. Acoustically tagged, migrating smolts with low gill NKA activity moved faster in FW reaches of the estuary than those with higher gill NKA activity. There was no difference in movement rate through SW reaches of the estuary based on gill NKA activity. Migrating fish moved with tidal flow during the passage of the lower estuary based on the observed patterns in both vertical and horizontal movements. The results indicate that smolts select low‐salinity water during estuary migration and use tidal currents to minimize energetic investment in seaward migration. Seasonal changes in osmoregulatory ability highlight the importance of the timing of stocking and estuary arrival.     

Differential expression of branchial Na+/K(+)-ATPase of two medaka species, Oryzias latipes and Oryzias dancena, with different salinity tolerances acclimated to fresh water, brackish water and seawater

Previous studies on non-diadromous euryhaline teleosts introduced a hypothesis that the lowest level of gill Na⁺/K⁺-ATPase (NKA) activity occurs in the environments with salinity close to the primary natural habitats of the studied species. To provide more evidence of the hypothesis, two medaka species, Oryzias latipes and O. dancena, whose primary natural habitats are fresh water (FW) and brackish water (BW) environments, respectively, were compared from levels of mRNA to cells in this study. The plasma osmolalities of O. latipes and O. dancena were lowest in the FW individuals. The muscle water contents of O. latipes decreased with elevated external salinities, but were constant among FW-, BW-, and seawater (SW)-acclimated O. dancena. Expression of NKA, the primary driving force of ion transporters in gill ionocytes, revealed different patterns in the two Oryzias species. The highest NKA α-subunit mRNA abundances were found in the gills of the SW O. latipes and the FW O. dancena, respectively. The pattern of NKA activity and α-subunit protein abundance in the gills of O. latipes revealed that the FW group was the lowest, while the pattern in O. dancena revealed that the BW group was the lowest. Immunohistochemical staining showed similar profiles of NKA immunoreactive (NKIR) cell activities (NKIR cell number × cell size) in the gills of these two species among FW, BW, and SW groups. Taken together, O. latipes exhibited better hyposmoregulatory ability, while O. dancena exhibited better hyperosmoregulatory ability. Our results corresponding to the hypothesis indicated that the lowest branchial NKA activities of these two medaka species were found in the environments with salinities similar to their natural habitats.     

Expression and distribution of Na, K-ATPase in gill and kidney of the spotted green pufferfish, Tetraodon nigroviridis, in response to salinity challenge

Freshwater (FW) spotted green pufferfish (Tetraodon nigroviridis) were transferred directly from a local aquarium to fresh water (FW; 0 per thousand ), brackish water (BW; 15 per thousand ), and seawater (SW; 35 per thousand ) conditions in the laboratory and reared for at least two weeks. No mortality was found. To investigate the efficient mechanisms of osmoregulation in the euryhaline teleost, distribution and expression of Na,K-ATPase (NKA) in gill and kidney of the pufferfish were examined and the osmolality, [Na+] and [Cl-] of the blood were assayed. The lowest levels of both relative protein abundance and activity were found to be exhibited in the BW group, and higher levels in the SW group than FW group. In all salinities, branchial NKA immunoreactivity was found in epithelial cells of the interlamellar region of the filament and not on the lamellae. Relative abundance of kidney NKA alpha-subunit, as well as the NKA activity, was found to be higher in the FW pufferfish than fish in BW or SW. Renal NKA appeared in the epithelial cells of distal tubules, proximal tubules, and collecting tubules, but not in glomeruli, in fish groups of various salinities. Plasma osmolality and chloride levels were significantly lower in FW pufferfish than those in BW and SW, whereas plasma sodium did not differ among the groups. Although identical distributions of NKA were found in either gill or kidney of FW-, BW- or SW-acclimated spotted green pufferfish, differential NKA expression in fish of various salinity groups was associated with physiological homeostasis (stable blood osmolality), and illustrated the impressive osmoregulatory ability of this freshwater and estuarine species in response to salinity challenge.     

Identification of FXYD protein genes in a teleost: tissue-specific expression and response to salinity change

It is increasingly clear that alterations in Na+-K+-ATPase kinetics to fit the demands in specialized cell types is vital for the enzyme to execute its different physiological roles in diverse tissues. In addition to tissue-dependent expression of isoforms of the conventional subunits, alpha and beta, auxiliary FXYD proteins appear to be essential regulatory components. The present study identified genes belonging to this family in Atlantic salmon by analysis of expressed sequence tags. Based on the conserved domain of these small membrane proteins, eight expressed FXYD isoforms were identified. Phylogenetic analysis suggests that six isoforms are homologues to the previously identified FXYD2, FXYD5, FXYD6, FXYD7, FXYD8, and FXYD9, while two additional isoforms were found (FXYD11 and FXYD12). Using quantitative PCR, tissue-dependent expression of the different isoforms was analyzed in gill, kidney, intestine, heart, muscle, brain, and liver. Two isoforms were expressed in several tissues (FXYD5 and FXYD9), while six isoforms were distributed in a discrete manner. In excitable tissues, two isoforms were highly expressed in brain (FXYD6 and FXYD7) and one in skeletal muscle (FXYD8). In osmoregulatory tissues, one isoform was expressed predominantly in gill (FXYD11), one in kidney (FXYD2), and one equally in kidney and intestine (FXYD12). Expression of several FXYD genes in kidney and gill differed between fresh water and seawater salmon, suggesting significance during osmoregulatory adaptations. In addition to identifying novel FXYD isoforms, these studies are the first to show the tissue dependence in their expression and modulation by salinity in any teleosts.     

Short-term low-salinity tolerance by the longhorn sculpin, Myoxocephalus octodecimspinosus

The bottom-dwelling, longhorn sculpin, Myoxocephalus octodecimspinosus, is traditionally viewed as a stenohaline marine fish, but fishermen have described finding this sculpin in estuaries during high tide. Little is known about the salinity tolerance of the longhorn sculpin; thus, the purposes of these experiments were to explore the effects of low environmental salinity on ion transporter expression and distribution in the longhorn sculpin gill. Longhorn sculpin were acclimated to either 100% seawater (SW, sham), 20% SW, or 10% SW for 24 or 72 hr. Plasma osmolality, sodium, potassium, and chloride concentrations were not different between the 20 and 100% treatments; however, they were 20-25% lower with exposure to 10% SW at 24 and 72 hr. In the teleost gill, regulation of Na(+), K(+)-ATPase (NKA), Na(+)-K(+)-2Cl(-) cotransporter (NKCC1), and the chloride channel, cystic fibrosis transmembrane conductance regulator (CFTR) are necessary for ion homeostasis. We immunolocalized these proteins to the mitochondrion-rich cell of the gill and determined that acclimation to low salinity does not affect their localization. Also, there was not a downregulation of gill NKA, NKCC1, and CFTR mRNA or protein during acclimation to low salinities. Collectively, these results suggest that down to 20% SW longhorn sculpin are capable of completely regulating ion levels over a 72-hr period, whereas 10% SW exposure results in a significant loss of ions and no change in ion transporter density or localization in the gill. We conclude that longhorn sculpin can tolerate low-salinity environments for days but, because they cannot regulate ion transporter density, they are unable to tolerate low salinity for longer periods or enter freshwater (FW). The genus Myoxocephalus has three FW species, making this group an excellent model to test evolutionary and physiological mechanisms that allow teleosts to invade new low salinities successfully.     

Salinity-dependent changes in Na+/K+-ATPase content of mitochondria-rich cells contribute to differences in thermal tolerance of Mozambique tilapia

The Mozambique tilapia (Oreochromis mossambicus) is prone to osmoregulatory disturbances when faced with fluctuating ambient temperatures. To investigate the underlying causes of this phenomenon, freshwater (FW)- and seawater (SW)-acclimated tilapia were transferred to 15, 25, or 35°C for 2 weeks, and along with typically used indicators of osmoregulatory status [plasma osmolality and branchial and intestinal specific Na⁺, K⁺-ATPase (NKA) activity], we used tissue microarrays (TMA) and laser-scanning cytometry (LSC) to characterize the effects of temperature acclimation. Tissue microarrays were stained with fluorescently labeled anti-Na⁺, K⁺-ATPase antibodies that allowed for the quantification of NKA abundance per unit area within individual branchial mitochondria-rich cells (MRCs) as well as sections of renal tissue. Mitochondria-rich cell counts and estimates of size were carried out for each treatment by the detection of DASPMI fluorescence. The combined analyses showed that SW fish have larger but fewer MRCs that contain more NKA per unit area. After a 2-week acclimation to 15°C tilapia experienced osmotic imbalances in both FW and SW that were likely due to low NKA activity. SW-acclimated fish compensated for the low activity by increasing MRC size and subsequently the concentration of NKA within MRCs. Although there were no signs of osmotic stress in FW-acclimated tilapia at 25°C, there was an increased NKA capacity that was most likely mediated by a higher MRC count. We conclude on the basis of the different responses to temperature acclimation that salinity-induced changes in the NKA concentration of MRCs alter thermal tolerance limits of tilapia.     

RING finger, B-box, and coiled-coil (RBCC) protein expression in branchial epithelial cells of Japanese eel, Anguilla japonica.

An RBCC (RING finger, B-box, and coiled-coil) protein was identified that belongs to the superfamily of zinc-binding proteins and is specifically expressed in the gill of eel, Anguilla japonica. Euryhaline fishes such as eels can migrate between freshwater and seawater, which is considered to be accomplished by efficient remodeling of the architecture and function of the gill, a major osmoregulatory organ. To identify molecules involved in such adaptive changes, we performed differential display using mRNA preparations from freshwater and seawater eel gills and obtained an RBCC clone among several differentially expressed clones. The clone encoded a protein of 514 amino acid residues with structural features characteristic of the RBCC protein; we therefore named it eRBCC (e for eel). eRBCC mRNA was specifically expressed in the gills with a greater extent in the gills of freshwater eels. Immunohistochemistry revealed that the expression of eRBCC is confined to particular epithelial cells of the gills including freshwater-specific lamellar chloride cells. The RING finger of eRBCC was found to have a ubiquitin ligase activity, suggesting an important regulatory role of eRBCC in the remodeling of branchial cells.     

Environmental salinity-modified osmoregulatory response in the sub-Antarctic notothenioid fish Eleginops maclovinus

In this study we assessed changes in the osmoregulatory system of juvenile sub-Antarctic Eleginops maclovinus submitted to different environmental salinities (5, 15, 32 and 45 psu) using two different acclimation trials: (1) an end-point experiment (exposure for 14 days) and (2) a time course experiment (specimens were sampled on days 1, 3, 7 and 14 post-transfer). Plasma osmolality, cortisol and metabolites (glucose, lactate and protein) values as well as Na⁺, K⁺-ATPase (NKA) activity were assessed in several osmoregulatory tissues (gills, kidney and intestine). In both trials, acclimation to different environmental salinities for 14 days induced changes in plasma metabolites (glucose, lactate and proteins) as well as cortisol values related to salinity challenges. Plasma osmolality and gill NKA activity presented a direct and positive relationship with respect to environmental salinity, while kidney NKA activity showed a “U-shaped” relationship. Anterior intestinal NKA activity increased in response to environmental salinity and apparently did not change in the middle portion of this organ, while it was enhanced in the posterior portion in environmental salinities different than seawater. Plasma metabolite values increased under hypo- and hypersaline conditions, indicating the importance of these energy substrates in extreme environments. The time course study revealed that specimens of E. maclovinus are able to accommodate their osmotic and metabolic system to respond to osmoregulatory challenges by allostatic changes.     

Acute changes in gill Na+-K+-ATPase and creatine kinase in response to salinity changes in the euryhaline teleost, tilapia (Oreochromis mossambicus)

Some freshwater (FW) teleosts are capable of acclimating to seawater (SW) when challenged; however, the related energetic and physiological consequences are still unclear. This study was conducted to examine the changes in expression of gill Na(+)-K(+)-ATPase and creatine kinase (CK) in tilapia (Oreochromis mossambicus) as the acute responses to transfer from FW to SW. After 24 h in 25 ppt SW, gill Na(+)-K(+)-ATPase activities were higher than those of fish in FW. Fish in 35 ppt SW did not increase gill Na(+)-K(+)-ATPase activities until 1.5 h after transfer, and then the activities were not significantly different from those of fish in 25 ppt SW. Compared to FW, the gill CK activities in 35 ppt SW declined within 1.5 h and afterward dramatically elevated at 2 h, as in 25 ppt SW, but the levels in 35 ppt SW were lower than those in 25 ppt SW. The Western blot of muscle-type CK (MM form) was in high association with the salinity change, showing a pattern of changes similar to that in CK activity; however, levels in 35 ppt SW were higher than those in 25 ppt SW. The activity of Na(+)-K(+)-ATPase highly correlated with that of CK in fish gill after transfer from FW to SW, suggesting that phosphocreatine acts as an energy source to meet the osmoregulatory demand during acute transfer.