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.     

Unidirectional Na(+) and Ca (2+) fluxes in two euryhaline teleost fishes, Fundulus heteroclitus and Oncorhynchus mykiss, acutely submitted to a progressive salinity increase

Na⁺ and Ca²⁺ regulation were compared in two euryhaline species, killifish (normally estuarine-resident) and rainbow trout (normally freshwater-resident) during an incremental salinity increase. Whole-body unidirectional fluxes of Na⁺ and Ca²⁺, whole body Na⁺ and Ca²⁺, and plasma concentrations (trout only), were measured over 1-h periods throughout a total 6-h protocol of increasing salinity meant to simulate a natural tidal flow. Killifish exhibited significant increases in both Na⁺ influx and efflux rates, with efflux slightly lagging behind efflux up to 60% SW, but net Na⁺ balance was restored by the time killifish reached 100% SW. Whole body Na⁺ did not change, in agreement with the capacity of this species to tolerate daily salinity fluctuations in its natural habitat. In contrast, rainbow trout experienced a dramatic increase in Na⁺ influx (50-fold relative to FW values), but not Na⁺ efflux between 40 and 60% SW, resulting in a large net loading of Na⁺ at higher salinities (60–100% SW), and increases in plasma Na⁺ and whole body Na⁺ at 100% SW. Killifish were in negative Ca²⁺ balance at all salinities, whereas trout were in positive Ca²⁺ balance throughout. Ca²⁺ influx rate increased two- to threefold in killifish at 80 and 100% SW, but there were no concomitant changes in Ca²⁺ efflux. Ca²⁺ flux rates were affected to a larger degree in trout, with twofold increases in Ca²⁺ influx at 40% SW and sevenfold increases at 100% SW. Again, there was no change in Ca²⁺ efflux with salinity, so plasma Ca²⁺ concentration increased in 100% SW. As the killifish is regularly submitted to increased salinity in its natural environment, it is able to rapidly activate changes in unidirectional fluxes in order to ensure ionic homeostasis, in contrast to the trout.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

Effects of NaCl salinity on growth, morphology, photosynthesis and proline accumulation of Salvinia natans

The effects of NaCl salinity on growth, morphology and photosynthesis of Salvinia natans (L.) All. were investigated by growing plants in a growth chamber at NaCl concentrations of 0, 50, 100 and 150 mM. The relative growth rates were high (ca. 0.3 d⁻¹) at salinities up to 50 mM and decreased to less than 0.2 d⁻¹ at higher salinities, but plants produced smaller and thicker leaves and had shorter stems and roots, probably imposed by the osmotic stress and lowered turgor pressure restricting cell expansion. Na⁺ concentrations in the plant tissue only increased three-fold, but uptake of K⁺ was reduced, resulting in very high Na⁺/K⁺ ratios at high salinities, indicating that S. natans lacks mechanisms to maintain ionic homeostasis in the cells. The contents of proline in the plant tissue increased at high salinity, but concentrations were very low (<0.1 μmol g⁻¹ FW), indicating a limited capacity of S. natans to synthesize proline as a compatible compound. The potential photochemical efficiency of PSII (F_v/F_m) of S. natans remained unchanged at 50 mM NaCl but was reduced at higher salinities, and the photosynthetic capacity (ETR_max) was significantly reduced at 50 mM NaCl and higher. It is concluded that S. natans is a salt-sensitive species lacking physiological measures to cope with exposure to high NaCl salinity. At low salinities salts are taken up and accumulate in old leaves, and high growth rates are maintained because new leaves are produced at a higher rate than for plants not exposed to salt.     

Age/size effects on juvenile green sturgeon, Acipenser medirostris, oxygen consumption, growth, and osmoregulation in saline environments

The green sturgeon, Acipenser medirostris, is an anadromous species that migrates from freshwater (FW) to seawater (SW) relatively early in its life history, although the ages and sizes of juveniles at SW entry are not known. Developmental constraints of osmoregulatory organs may either prohibit (i.e., due to salinity tolerance limits) or minimize (i.e., due to substantial osmoregulatory or ionoregulatory energetic costs) SW entry in small fish. Interestingly, larger green sturgeon are often encountered in brackish water (BW) estuaries, perhaps due to an energetic advantage in occupying these near-isosmotic environments. To test hypotheses concerning fish-size effects on the energetic costs of occupying habitats of different salinities, we measured oxygen consumption rates in green sturgeon representing three age groups (100, 170, and 533 days post hatch; dph), which were acclimated for 5 weeks to one of three salinities (FW, <3‰; BW, 10‰; or SW, 33‰). Also, after 7 weeks, final wet masses were compared and blood and muscle tissue samples were taken to assess osmoregulatory abilities. There were no differences in body-mass-adjusted oxygen consumption rates between any salinities or ages, indicating that the energetic costs were not prohibitively high to occupy any of these salinities. The only mortalities occurred in the 100 dph SW group, where 23% of the fish died, from apparent starvation. Final wet masses were comparable between FW and BW for each age group and with the 533 dph SW group, but were lower in SW groups at 100 and 170 dph. Similarly, osmoregulatory abilities, in terms of plasma osmolality, Na⁺, K⁺, lactate, and protein concentrations, and muscle water content, were comparable in FW and BW groups at all ages, and with the SW group at 533 dph. These results indicated an age/body size effect in hyperosmotic adaptability, and that juvenile green sturgeon may be found in FW or BW at any age, but only have the ability to enter SW by 1.5 years (75 cm, 1.5 kg) of age.     

Plasma and erythrocyte solute properties of juvenile bull sharks, Carcharhinus leucas, acutely exposed to increasing environmental salinity

Plasma and erythrocyte solute properties were examined in freshwater (FW) acclimated juvenile Carcharhinus leucas following acute transfer to 75% seawater (SW), and 100% SW. Blood samples were taken at 0, 12 and 96 h following transfer to 75% SW and 24 and 72 h after transfer to 100% SW. A control group in FW was subjected to the same sampling regime. Upon transfer of C. leucas to 75% and 100% SW, plasma Na⁺, Cl⁻, K⁺, Mg²⁺, Ca²⁺, urea and TMAO concentrations all increased significantly but disproportionately. Plasma Na⁺ and Cl⁻ increased immediately, followed by an increase in plasma urea. Erythrocyte urea and TMAO concentrations increased significantly following transfer to 75% and 100% SW; however, changes in erythrocyte inorganic ion concentrations were insignificant. Haematocrit, haemoglobin and mean cell haematocrit did not differ significantly after transfer to seawater; however, plasma water was slightly reduced after 24 and 72 h in 100% SW. Red blood cell (RBC) water content was elevated 24 h after transfer to 100% SW but returned to FW levels after 72 h. These results demonstrate that the transfer from 75% to 100% SW presents C. leucas with a greater osmoregulatory challenge than transfer from FW to 75% SW, despite the larger concentration gradient in the latter. In summary, C. leucas tolerate rapid and significant increases in salinity by rapidly increasing plasma osmolality to be hyperosmotic to the environment whilst maintaining a tight regulation of their intracellular fluid environment.     

Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na+ but not Cl− uptake in freshwater

Fundulus heteroclitus (killifish) is a model organism for ionoregulatory studies, particularly because of its opercular epithelium, although the gills are the major sites of ion exchange. Whereas Na⁺ and Cl⁻ are excreted through the gills in seawater (SW), the killifish is unusual in taking up only Na⁺ and not Cl⁻ at the gills in freshwater (FW). We describe morphological changes in the branchial epithelium following transfer from an acclimation medium of 10% SW to 100% SW or FW. In 10% SW, mitochondria-rich cells resemble typical seawater chloride cells (SWCCs) with accessory cells. After transfer to 100% SW, no change occurs in pavement cell (PVC) morphology or mitotic rate (measured by bromo-deoxyuridine technique), although the density of SWCC apertures increases several fold because of the uncovering of buried SWCCs by PVCs, in accord with increased rates of Na⁺ and Cl⁻ efflux. After transfer to FW, PVC morphology remains unchanged, but SWCCs and accessory cells are quickly covered by PVCs, with many undergoing apoptosis or necrosis. The mitotic rate doubles by 10–14 h but typical freshwater chloride cells (FWCCs) do not appear. Instead, a wedge-shaped cell type that is moderately rich in apically oriented mitochondria, with a large ovoid nucleus, thin cytoplasmic layer, paucity of vesicular-tubular network, and variably villous surface rapidly (by 3 h) and progressively appears in the filament epithelium, by both uncovering and mitosis. This cell type is similar to that recently identified as the site of Na⁺ uptake in the FW trout gill. We propose the new term “cuboidal cell” for this cell, based on its morphology, to avoid confusion with traditional terminology (of PVC). We hypothesize that the cuboidal cells are the sites of active Na⁺ uptake in FW F. heteroclitus and suggest that the lack of Cl⁻ uptake is attributable to the absence of typical FWCCs previously described in teleosts.This work was supported by NSERC Discovery grants (to C.M.W.) and by an NSERC International Fellowship (to P.L.). C.M.W. is supported by the Canada Research Chair Program.     

Effects of low salinity media on growth, condition, and gill ion transporter expression in juvenile Gulf killifish, Fundulus grandis

The Gulf killifish, Fundulus grandis, is a euryhaline teleost which has important ecological roles in the brackish-water marshes of its native range as well as commercial value as live bait for saltwater anglers. Effects of osmoregulation on growth, survival, and body condition at 0.5, 5.0, 8.0 and 12.0‰ salinity were studied in F. grandis juveniles during a 12-week trial. Relative expression of genes encoding the ion transport proteins Na⁺/K⁺-ATPase (NKA), Na⁺/K⁺/2Cl⁻ cotransporter(NKCC1), and cystic fibrosis transmembrane conductance regulator (CFTR) Cl⁻ channel was analyzed. At 0.5‰, F. grandis showed depressed growth, body condition, and survival relative to higher salinities. NKA relative expression was elevated at 7 days post-transfer but decreased at later time points in fish held at 0.5‰ while other salinities produced no such increase. NKCC1, the isoform associated with expulsion of ions in saltwater, was downregulated from week 1 to week 3 at 0.5‰ while CFTR relative expression produced no significant results across time or salinity. Our results suggest that Gulf killifish have physiological difficulties with osmoregulation at a salinity of 0.5‰ and that this leads to reduced growth performance and survival while salinities in the 5.0-12.0‰ are adequate for normal function.     

Sodium Efflux and Potential Differences across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium extrusion (J_out^Na) was measured across the gills of rainbow trout, Salmo gairdneri, adapted to sea water (SW) using a gill-irrigation system of small volume. The potential difference (TEP) was also measured under similar conditions. J_out^Na was usually between 100–250 µeq (100 g)^–1 h^–1, about an order of magnitude faster than in fresh water (FW)-adapted trout, but slower than has been reported for any other marine teleost. The TEP was between 10–11 mV, body fluids positive to SW. When the external medium was changed from SW to FW J_out^Na was reduced to about 25 % of the initial value, and the TEP was reduced by 40–50 mV (i.e. body fluids negative by 30–40 mV). Addition of either Na⁺ or K⁺ in SW concentrations reversed the changes; J_out^Na increased and the gill repolarized. The electrical behavior and sodium efflux in irrigated trout gill is qualitatively the same as has been reported for unanaesthetized, free-swimming fish of other species. Thus, the irrigated gill provides an adequate model for studying the mechanism of sodium extrusion in marine teleosts.     

Osmo- and ionoregulatory responses of green sturgeon (<Emphasis Type="Italic">Acipenser medirostris</Emphasis>) to salinity acclimation

The green sturgeon is a long-lived, highly migratory species with populations that are currently listed as threatened. Their anadromous life history requires that they make osmo- and ionoregulatory adjustments in order to maintain a consistent internal milieu as they move between fresh-, brackish-, and seawater. We acclimated juvenile green sturgeon (121 ± 10.0 g) to 0 (freshwater; FW), 15 (estuarine; EST), and 24 g/l (SF Bay water; BAY) at 18°C for 2 weeks and measured the physiological and biochemical responses with respect to osmo- and ionoregulatory mechanisms. Plasma osmolality in EST- and BAY-acclimated sturgeon was elevated relative to FW-acclimated sturgeon (P < 0.01), but there was no difference in muscle water content or abundance of stress proteins. Branchial Na⁺, K⁺-ATPase (NKA) activity was also unchanged, but abundance within mitochondrion-rich cells (MRC) was greater in BAY-acclimated sturgeon (P < 0.01). FW-acclimated sturgeon had the greatest NKA abundance when assessed at the level of the entire tissue (P < 0.01), but there were no differences in v-type H⁺ATPase (VHA) activity or abundance between salinities. The Na⁺, K⁺, 2Cl⁻ co-transporter (NKCC) was present in FW-acclimated sturgeon gills, but the overall abundance was lower relative to sturgeon in EST or BAY water (P < 0.01) where this enzyme is crucial to hypoosmoregulation. Branchial caspase 3/7 activity was significantly affected by acclimation salinity (P < 0.05) where the overall trend was for activity to increase with salinity as has been commonly observed in teleosts. Sturgeon of this age/size class were able to survive and acclimate following a salinity transfer with minimal signs of osmotic stress. The presence of the NKCC in FW-acclimated sturgeon may indicate the development of SW-readiness at this age/size.     

Contribution of Na/Na and Cl/Cl exchanges to sodium and chloride fluxes in the crabGoniopsis cruentata

Summary Sodium or chloride efflux and transepithelial potentials (TEP) were measured in crabs exposed to seawater concentrations ranging from 150 to 25% SW. In crabs acclimated to 150% SW the Na⁺ efflux (3.8 mmol/h·100 g) was significantly higher than the Cl^– efflux (2.1 mmol/h·100 g), but both fluxes decreased to about 0.6 mmol/h·100 g in crabs from 50 or 25% SW. The TEP varied linearly from –1 mV (blood negative) in 150% SW, to –11 mV in 25% SW. In 150 and 100% SW the calculated components of the ion fluxes (i.e., diffusive, urinary, active uptake or extrusion) added up to less than one-half of the isotopically measured values. In 50 and 25% SW the measured effluxes were fully accounted for by their calculated components. In crabs transferred from 150% SW to low-Na 150% SW (=TRIS ASW), the Na⁺ efflux decreased abruptly, from 3.7 to 0.6 mmol/h; the Cl^– efflux decreased much less, from 1.9 to 1.5 mmol/h. A large fraction of the Na⁺ (or Cl^–) fluxes in crabs from concentrated SW meets the criteria for exchange diffusion, which decreases or disappears as the external concentration of each ion is lowered. This suggests that changes of the permeability to ions, in response to alterations of environmental salinity, may not constitute an important adaptive strategy in this species.Abbreviations SW seawater - TEP transepithelial potential - TRIS ASW artificial seawater 150%     

Dietary lipid composition affects the gene expression of gill Na+/K+-ATPase α1b but not the α1a isoform in juvenile fall chinook salmon (Oncorhynchus tshawytscha)

We assessed the effects of dietary fatty acid composition on sodium–potassium ATPase (Na⁺/K⁺-ATPase) activity and isoform expression in the gills of juvenile fall chinook salmon, Oncorhynchus tshawytscha by supplementing diets with either anchovy oil (AO) or AO blended with canola oil (CO) so that CO comprised 0% (0CO), 11% (11CO), 22% (22CO), 33% (33CO), 43% (43CO), or 54% (54CO) of the measured dietary lipid content. The effects of diet were assessed in freshwater (FW) following 104 days of diet manipulation, in response to 24-h seawater (SW) transfer at this time, and following an additional 35 days of SW acclimation. Gill Na⁺/K⁺-ATPase activity was not significantly affected by diet at any sampling time, and there were no consistent effects of diet on the expression of the Na⁺/K⁺-ATPase α1a isoform. As dietary CO increased, Na⁺/K⁺-ATPase α1b mRNA decreased in fish held in FW, with the 43CO and 54CO diet groups having significantly lower levels than fish fed the 0CO and 11CO diets. Twenty-four-hour SW challenge did not affect the expression of the Na⁺/K⁺-ATPase α1a isoform in any diet group, but this isoform was down-regulated in all diet groups following 35 days of SW acclimation. Na⁺/K⁺-ATPase α1b expression levels increased in response to 24-h SW transfer and SW acclimation only in fish fed the 54CO diet. The effects of the two extreme diets (0CO and 54CO) were also assessed at various time points during 104 days of rearing in FW. Na⁺/K⁺-ATPase α1b mRNA levels were greater in fish fed diet 0CO versus those fed diet 54CO at all times during the FW culture period. These data demonstrate that dietary fatty acid composition can influence the gill Na⁺/K⁺-ATPase isoform physiology of juvenile fall-run chinook salmon prior to SW transfer.     

Salinity-dependent expression of the branchial Na+/K +/2Cl (-) cotransporter and Na+/K (+)-ATPase in the sailfin molly correlates with hypoosmoregulatory endurance

In the branchial mitochondrion-rich (MR) cells of euryhaline teleosts, the Na⁺/K⁺/2Cl⁻ cotransporter (NKCC) is an important membrane protein that maintains the internal Cl⁻ concentration, and the branchial Na⁺/K⁺-ATPase (NKA) is crucial for providing the driving force for many other ion-transporting systems. Hence this study used the sailfin molly (Poecilia latipinna), an introduced aquarium fish in Taiwan, to reveal that the potential roles of NKCC and NKA in sailfin molly were correlated to fish survival rates upon salinity challenge. Higher levels of branchial NKCC were found in seawater (SW)-acclimated sailfin molly compared to freshwater (FW)-acclimated individuals. Transfer of the sailfin molly from SW to FW revealed that the expression of the NKCC and NKA proteins in the gills was retained over 7 days in order to maintain hypoosmoregulatory endurance. Meanwhile, their survival rates after transfer to SW varied with the duration of FW-exposure and decreased significantly when the SW-acclimated individuals were acclimated to FW for 21 days. Double immunofluorescence staining showed that in SW-acclimated sailfin molly, NKCC signals were expressed on the basolateral membrane of MR cells, whereas in FW-acclimated molly, they were expressed on the apical membrane. This study illustrated the correlation between the gradual reductions in expression of branchial NKCC and NKA (i.e., the hypoosmoregulatory endurance) and decreasing survival rates after hyperosmotic challenge in sailfin molly.     

On the Mechanism of Sodium Extrusion across the Irrigated Gill of Sea Water-Adapted Rainbow Trout (Salmo gairdneri)

Sodium efflux (J_out^Na) across the irrigated trout gill was rapid in sea water (SW), but only about 25 % as large in fresh water (FW). The difference correlated with a change in the potential difference across the gill (TEP). The latter was about +10 mV (blood positive) in SW, but –40 mV in FW. Both flux and electrical data indicated that gills in this fish are permeable to a variety of cations including Na⁺, K⁺, Mg²⁺, choline, and Tris. They are less permeable to anions; P_Na:P_K:P_Cl was estimated to be 1:10:0.3, and P_Cl > P_gluconate. The TEP was shown to be a diffusion potential determined by these permeabilities and the extant ionic gradients in SW, FW as well as in other media. J_out^Na appeared to be diffusive in all of the experiments undertaken. Exchange diffusion need not be posited, and the question of whether there is an active component remains open.     

The influence of salinity on water balance in 0-group flounders, Platichthys flesus (L)

The water balance of 0-group flounders was investigated in a range of static [0–100% sea water (SW), 100%≡ 34%‰] and cycling salinities (2–98% SW, 12 h 25 min period). The permeability coefficients of these juvenile fish were found to be higher than those quoted for adults. The permeability of juveniles in fixed salinities decreased with increased salinity whereas animals in the tidally cycling regime showed permeability changes that were directly proportional to the ambient salinity. However, comparison of the two groups showed that animals in a cycling salinity regime were less permeable to water than animals acclimated to fixed salinities. Drinking and urine production rates fluctuated within a tidal salinity regime, and 0-group flounders were found to modify their water permeability, urine production and drinking rates simultaneously, so maintaining their blood osmotic concentration and total water content within narrow limits throughout the range of salinities.
The relevance of measurements made in fixed and tidally cycling salinities to water regulation under natural estuarine conditions is considered.     

Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na⁺,K⁺-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na⁺,K⁺-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.     

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.     

Na+-dependent Ca2+ uptake in isolated opercular epithelium of Fundulus heteroclitus

It is concluded that Ca²⁺ transport across the basolateral membranes of the ionocytes in killifish skin is mediated for the major part by a Na⁺/Ca²⁺-exchange mechanism that is driven by the (transmembrane) Na⁺ gradient established by Na⁺/K⁺-ATPase. The conclusion is based, firstly, on the biochemical evidence for the presence of a Na⁺/Ca²⁺-exchanger next to the Ca²⁺-ATPase in the basolateral membranes of killifish gill cells. Secondly, the transcellular Ca²⁺ uptake measured in an Ussing chamber setup was 85% and 80% reduced in freshwater (FW) and SW (SW) opercular membranes, respectively, as the Na⁺ gradient across the basolateral membrane was directly or indirectly (by ouabain) reduced. Thapsigargin or dibutyryl-cAMP/IBMX in SW opercular membranes reduced Ca²⁺ influx to 46%, comparable to the effects seen in FW membranes [reduction to 56%; Marshall et al. 1995a]. Basal Ca²⁺ influx across the opercular membrane was 48% lower in membranes from fish adapted to SW than in membranes from fish adaptated to FW. Branchial Na⁺/K⁺-ATPase activity was two times higher in SW adapted fish. Accepted: 29 October 1996     

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.