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.     

Effects of endothelin receptor type-A and type-B antagonists on prostaglandin F2alpha-induced luteolysis of the sheep corpus luteum

Three experiments were designed to examine the mechanisms that govern prostaglandin (PGF2alpha)-induced regression of the sheep corpus luteum. Evidence is presented supporting the involvement of endothelin 1 (EDN1) in PGF2alpha-induced luteolysis. Experiment 1 measured effects of PGF2alpha when actions of EDN1 were blocked by sustained administration of a type-A endothelin (EDNRA) or type-B endothelin (EDNRB) antagonist in vivo. Experiment 2 examined antisteroidogenic actions of PGF2alpha and EDN1 in the presence of an EDNRA or EDNRB antagonist in Day-8 luteal minces. In experiment 3, luteal cellular expression of EDNRA and EDNRB was determined immunohistochemically. Relative gene expression of EDNRA and EDNRB receptors was examined by real-time RT-PCR in Day-8 sheep corpora lutea. EDNRA, but not EDNRB, participated in antisteroidogenic actions of EDN1. During the first 12 h after PGF2alpha-induced luteolysis, EDNRA antagonist did not prevent a decline in serum progesterone concentrations. Early actions of PGF2alpha are either direct or mediated by something other than EDN1. However, beyond 12 h after PGF2alpha, serum progesterone concentrations increased in EDNRA antagonist-treated animals until they were the same as saline-treated controls, whereas an EDNRB antagonist had no effect in vivo or in vitro. The EDNRA antagonist negated the antisteroidogenic actions of EDN1 but only partially abolished the actions of PGF2alpha in vitro. In contrast, the EDNRB antagonist was ineffective in abolishing antisteroidogenic actions of EDN1 and PGF2alpha. Whereas real-time RT-PCR demonstrated high expression of EDNRA and low expression of EDNRB, immunohistochemically, only EDNRA was located in small steroidogenic, endothelial, and smooth muscle cells. In summary, studies in ovine corpora lutea provided strong evidence that: 1) EDNRA, but not EDNRB, mediates antisteroidogenic actions of EDN1, 2) actions of PGF2alpha are both independent of and dependent upon mediation by EDN1, and 3) small steroidogenic cells are targets for antisteroidogenic effects of EDN1. Furthermore, the results from experiment 1 suggest that the intermediary role of EDN1 may be more important in later stages of luteal regression.     

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.     

A divergent CFTR homologue: highly regulated salt transport in the euryhaline teleost F.heteroclitus

The killifish,Fundulus heteroclitus, is a euryhalineteleost fish capable of adapting rapidly to transfer from freshwater (FW) to four times seawater (SW). To investigate osmoregulation at amolecular level, a 5.7-kilobase cDNA homologous to human cysticfibrosis transmembrane conductance regulator (hCFTR) was isolated froma gill cDNA library from SW-adapted killifish. This cDNA encodes aprotein product (kfCFTR) that is 59% identical to hCFTR,the most divergent form of CFTR characterized to date. Expression ofkfCFTR in Xenopus oocytes generatedadenosine 3',5'-cyclic monophosphate-activated,Cl-selective currentssimilar to those generated by hCFTR. In SW-adapted killifish,kfCFTR was expressed at high levels in the gill, opercular epithelium, and intestine. After abrupt exposure of FW-adapted killifish to SW, kfCFTR expression in the gill increasedseveralfold, suggesting a role for kfCFTR in salinity adaptation. Undersimilar conditions, plasma Na⁺levels rose significantly after 8 h and then fell, although it is notknown whether these changes are directly responsible for the changes inkfCFTR expression. The killifish provides a unique opportunity to understand teleost osmoregulation and the role of CFTR.

     

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.     

IGF-I and branchial IGF receptor expression and localization during salinity acclimation in striped bass

The initial response of the IGF-I system and the expression and cellular localization of IGF type-I receptor (IGF-IR) were studied in the gill of a euryhaline teleost during salinity acclimation. Exposure of striped bass (Morone saxatilis) to hyperosmotic and hypoosmotic challenges induced small, transitory (<24 h) deflections in hydromineral balance. Transfer from freshwater (FW) to seawater (SW) induced an initial decrease in plasma IGF-I levels after 24 h in both fed and fasted fish. There was an overall decrease in liver IGF-I mRNA levels after SW transfer, suggesting that decreased plasma levels may be due to a decline in hepatic IGF-I synthesis. No changes were observed in gill IGF-I mRNA, but SW transfer induced an increase in gill IGF-IR mRNA after 24 h. Transfer from SW to FW induced an increase in plasma IGF-I levels in fasted fish. In fed fish, no significant changes were observed in either plasma IGF-I, liver, or gill IGF-I mRNA, or gill IGF-IR mRNA levels. In a separate experiment, FW-acclimated fish were injected with saline or IGF-I prior to a 24-h SW challenge. Rapid regain of osmotic balance following SW transfer was hindered by IGF-I. Immunohistochemistry revealed for the first time in teleosts that IGF-IR and Na(+)-K(+)-ATPase are localized in putative chloride cells at the base of the lamellae, identifying these cells in the gill as a target for IGF-I and IGF-II. Overall the data suggest a hyperosmoregulatory role of IGF-I in this species.     

A critical analysis of transepithelial potential in intact killifish (Fundulus heteroclitus) subjected to acute and chronic changes in salinity

We investigated the in vivo salinity-dependent behavior of transepithelial potential (TEP) in Fundulus heteroclitus (3-9 g) using indwelling coelomic catheters, a technique which was validated against blood catheter measurements in a larger species (Opsanus beta; 35-70 g). In seawater (SW)-acclimated killifish, TEP was +23 mV (inside positive), but changed to -39 mV immediately after transfer to freshwater (FW). Acute transfer to dilute salinities produced a TEP profile, which rapidly attenuated as salinity increased (0, 2.5, 5 and 10% SW), with cross-over to positive values between 20 and 40% SW, and a linear increase thereafter (60, 80 and 100% SW). TEP response profiles were also recorded after acute transfer to comparable dilutions of 500 mmol L(-1) NaCl, NaNO3, Na gluconate, choline chloride, N-methyl-D-glutamate (NMDG) chloride, or 1,100 mosmol kg(-1) mannitol. These indicated high non-specific cation permeability and low non-specific anion permeability without influence of osmolality in SW-acclimated killifish. While there was a small electrogenic component in high salinity, a Na+ diffusion potential predominated at all salinities due to the low P Cl/P Na (0.23) of the gills. The very negative TEP in FW was attenuated in a linear fashion by log elevations in [Ca2+] such that P Cl/P Na increased to 0.73 at 10 mmol L(-1). SW levels of [K+] or [Mg2+] also increased the TEP, but none of these cations alone restored the positive TEP of SW-acclimated killifish. The very negative TEP in FW attenuated over the first 12 h of exposure and by 24-30 h reached +3 mV, representative of long-term FW-acclimated animals; this reflected a progressive increase in P Cl/P Na from 0.23 to 1.30, probably associated with closing of the paracellular shunt pathway. Thereafter, the TEP in FW-acclimated killifish was unresponsive to [Ca2+] (also to [K+], [Mg2+], or chloride salts of choline and NMDG), but became more positive at SW levels of [Na+]. Killifish live in a variable salinity environment and are incapable of gill Cl(-) uptake in FW. We conclude that the adaptive significance of the TEP patterns is that changeover to a very negative TEP in FW will immediately limit Na+ loss while not interfering with active Cl(-) uptake because there is none. Keeping the shunt permeability high for a few hours means that killifish can return to SW and instantaneously re-activate their NaCl excretion mechanism.     

Effect of salinity on expression of branchial ion transporters in striped bass (Morone saxatilis)

The time course of osmoregulatory adjustments and expressional changes of three key ion transporters in the gill were investigated in the striped bass during salinity acclimations. In three experiments, fish were transferred from fresh water (FW) to seawater (SW), from SW to FW, and from 15-ppt brackish water (BW) to either FW or SW, respectively. Each transfer induced minor deflections in serum [Na+] and muscle water content, both being corrected rapidly (24 hr). Transfer from FW to SW increased gill Na+,K+-ATPase activity and Na+,K+,2Cl- co-transporter expression after 3 days. Abundance of Na+,K+-ATPase alpha-subunit mRNA and protein was unchanged. Changes in Na+,K+,2Cl- co-transporter protein were preceded by increased mRNA expression after 24 hr. Expression of V-type H+-ATPase mRNA decreased after 3 days. Transfer from SW to FW induced no change in expression of gill Na+,K+-ATPase. However, Na+,K+,2Cl- co-transporter mRNA and protein levels decreased after 24 hr and 7 days, respectively. Expression of H+-ATPase mRNA increased in response to FW after 7 days. In BW fish transferred to FW and SW, gill Na+,K+-ATPase activity was stimulated by both challenges, suggesting both a hyper- and a hypo-osmoregulatory response of the enzyme. Acclimation of striped bass to SW occurs on a rapid time scale. This seems partly to rely on the relative high abundance of gill Na+,K+-ATPase and Na+,K+,2Cl- co-transporter in FW fish. In a separate study, we found a smaller response to SW in expression of these ion transport proteins in striped bass when compared with the less euryhaline brown trout. In both FW and SW, NEM-sensitive gill H+-ATPase activity was negligible in striped bass and approximately 10-fold higher in brown trout. This suggests that in striped bass Na+-uptake in FW may rely more on a relatively high abundance/activity of Na+,K+-ATPase compared to trout, where H+-ATPase is critical for establishing a thermodynamically favorable gradient for Na+-uptake.     

Expression of aquaporin 3 in gills of the Atlantic killifish (Fundulus heteroclitus): Effects of seawater acclimation

Estuarine fish, such as the Atlantic killifish (Fundulus heteroclitus), are constantly and rapidly exposed to changes in salinity. Although ion transport in killifish gills during acclimation to increased salinity has been studied extensively, no studies have examined the role of aquaglyceroporin 3 (AQP3), a water, glycerol, urea, and ammonia transporter, during acclimation to increased salinity in this sentinel environmental model organism. The goal of this study was to test the hypothesis that transfer from freshwater to seawater decreases AQP3 gene and protein expression in the gill of killifish. Transfer from freshwater to seawater decreased AQP3 mRNA in the gill after 1 day, but had no effect on total gill AQP3 protein abundance as determined by western blot. Quantitative confocal immunocytochemistry confirmed western blot studies that transfer from freshwater to seawater did not change total AQP3 abundance in the gill; however, immunocytochemistry revealed that the amount of AQP3 in pillar cells of secondary lamellae decreased in seawater fish, whereas the amount of AQP3 in mitochondrion rich cells (MRC) in primary filaments of the gill increased in seawater fish. This response of AQP3 expression is unique to killifish compared to other teleosts. Although the role of AQP3 in the gill of killifish has not been completely elucidated, these results suggest that AQP3 may play an important role in the ability of killifish to acclimate to increased salinity.     

Differential design for hopping in two species of wallabies

We explored molecular and morphological alteration in gill mitochondria-rich (MR) cells of Mozambique tilapia, Oreochromis mossambicus, acclimated to deionized freshwater (DFW), freshwater (FW), 1/3-diluted seawater (1/3 SW) and seawater (SW). Scanning electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk in DFW and FW, being larger in DFW than in FW. In contrast, the apical membrane typically formed a pit structure in 1/3 SW and SW. The mRNA expression levels of Na(+)/H(+) exchanger-3 (NHE3) and Na(+)/Cl(-) cotransporter (NCC) in the gills were increased with decreasing environmental salinity, whereas Na(+)/K(+)/2Cl(-) cotransporter-1a (NKCC1a) expression was upregulated by increasing salinity. Immunofluorescence staining showed that the MR cell population of DFW- and FW-acclimated tilapia consisted mostly of MR cells with apical NHE3 and those with apical-NCC; MR cells with basolateral NKCC1a dominated in SW-acclimated tilapia. These results indicated that apical-NHE3 and apical-NCC MR cells were ion-absorbing cells, and that basolateral-NKCC1a MR cells were ion-secreting cells. In fish acclimated to 1/3 SW, both ion-absorbing and secreting cells existed in the gills, suggesting that fish in near-isotonic water were equipped with mechanisms of both hyper- and hypoosmoregulation to prepare for environmental salinity changes.     

TEP on the tide in killifish (<Emphasis Type="Italic">Fundulus heteroclitus</Emphasis>): effects of progressively changing salinity and prior acclimation to intermediate or cycling salinity

Transepithelial potentials (TEP) were measured in killifish, acclimated to freshwater (FW), seawater (SW), 33% SW or cycling salinities relevant to tidal cycles in an estuary, and subsequently subjected to salinity changes in progressive or random order. Random compared to progressive salinity changes in an upward or downward direction in FW- and SW-acclimated fish, respectively, did not greatly influence responses to salinity change. Fish acclimated to SW or 33% SW as well as those acclimated to cycling salinities behaved similarly (TEP more positive than +15 mV in 100% SW, decreasing to ~0 mV at 20–40% SW, and more negative than −30 mV in FW). In contrast, FW-acclimated fish displayed a less pronounced TEP response to salinity (0 mV in FW through 20% SW, increasing thereafter to values more positive than +10 mV at 100% SW). We conclude that when evaluated under estuarine tidal conditions, the killifish gill exhibits adaptive electrical characteristics, opposing Na⁺ loss at low salinity and favouring Na⁺ extrusion at high salinity, changes explained at least in part by the Cl⁻ to Na⁺ permeability ratio. Thus animals living in the estuaries can move to lower and higher salinities for short periods with little physiological disturbance, but this ability is lost after acclimation to FW.     

Na+ versus Cl- transport in the intact killifish after rapid salinity transfer

Much of the early research elucidating the general mechanisms of euryhalinity was performed on the common killifish. More recently, its opercular epithelium with abundant mitochondria-rich cells has proven to be a powerful model for analyzing the mechanisms of active NaCl transport under Ussing conditions in vitro (i.e., with isotonic saline on both surfaces, at short-circuit). However, it is unclear whether this preparation duplicates the gill under real world conditions-i.e., at open-circuit, with real seawater (SW) or freshwater (FW) on the mucosal surface. There have been only limited studies, mostly about 35 years ago, on ion transport in the intact killifish. Therefore, using radioisotopes (22Na, 36Cl), we developed and evaluated methods for the independent measurement of unidirectional Na(+) and Cl(-) influx and efflux rates and internal pools in intact killifish acclimated to 10% SW and abruptly transferred to either 100% SW or FW. Internal Na(+) pools were disturbed less than internal Cl(-) pools by transfer, and were corrected after 3 days in 100% SW or 7 days in FW. Influx and efflux rates in 10% SW were about 3000 micromol kg(-1) h(-1) and increased to 15,000-18,000 micromol kg(-1) h(-1) after transfer to 100% SW, remaining approximately equal and equimolar for Na(+) and Cl(-), and stable from 0.5 to 7 days post-transfer. After transfer to FW, Na(+) influx and efflux rates dropped to 1000-1500 micromol kg(-1) h(-1), with efflux slightly exceeding influx, and remained approximately stable from 0.5 to 7 days. However, while Cl(-) efflux responded similarly, Cl(-) influx rate dropped immediately to negligible values (20-50 micromol kg(-1) h(-1)) without recovery through 7 days. These results differ from early ion transport data in 100% SW, and demonstrate that fluxes stabilize quickly after salinity transfer. They also show that the intact animal responds more quickly than the epithelium, provide qualitative but not quantitative support for the opercular epithelium as a model for the gill under real world SW conditions, and no support for its use as a gill model under real world FW conditions, where branchial Cl(-) uptake is negligible.     

Expression of endothelin 1 and its receptors in the hypoxic pregnant rat

Endothelin 1 (EDN1) plays a primary role in the pathophysiology of hypoxia-induced fetal growth restriction in the rat. In this study we evaluated the effects of chronic maternal hypoxia on the expression of endothelin and its receptors and on receptor binding activity in the uterus and placenta of the rat, in order to elucidate their roles in hypoxia-induced fetal growth restriction. Timed-pregnant Sprague-Dawley rats were maintained in either a normoxic or a normobaric hypoxic (12% O(2)) atmosphere from Gestational Days 18-21. Uterine and placental tissues collected on Gestational Day 21 were assayed for Edn1, Ednra, and Ednrb (endothelin receptors) mRNA expression by real-time quantitative RT-PCR, for localization of EDN1 and its receptors by immunohistochemistry, for EDNRA and EDNRB protein expression by Western blot, and for receptor binding activity by homologous competitive binding assays. EDN1 mRNA expression was significantly increased in the hypoxic placenta, but not in the uterus, compared with normoxic controls. Immunohistochemistry revealed increased EDN1 specifically in the labyrinth of the placenta. Receptor mRNA levels were not significantly affected by hypoxia, but EDNRA protein expression was significantly decreased specifically in the uterine placental beds. Receptor binding decreased significantly in response to hypoxia in all tissues investigated, compared with controls. These results suggest that chronic maternal hypoxia results in increased expression of EDN1 in the placenta but not in the uterus, and that reduced binding activity, rather than regulation of receptor expression, is a mechanism by which these tissues regulate the local hemodynamic response to increased endogenous placental EDN1 in the setting of hypoxia.     

Effects of acclimation salinity on the expression of selenoproteins in the tilapia,Oreochromis mossambicus

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.     

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.