Extracellular ionic and acid-base adjustments of Atlantic salmon presmolts and smolts in fresh water and after transfer to sea water: the effects of ovine growth hormone on the acquisition of euryhalinity

In order to better understand the basis for the acquisition of euryhalinity by juvenile salmon and the role of endogeneous stimuli, experiments have been carried out to examine the dynamics of ionic and acid-base adjustments in fresh water (FW) and after direct transfer to full salinity (32 g l⁻¹) sea water (SW) (1) on Atlantic salmon smolt during the natural period of smoltification in spring, (2) on presmolt salmon in autumn, after intraperitoneal implantation of pellets containing ovine growth hormone (oGH). During parr-smolt transformation in FW, gill Na⁺/K⁺ ATPase activity gradually rises, the plasma osmolality (Posm) is unaffected and the total CO₂ of the plasma decreases significantly while whole blood pH fluctuates slightly. Direct transfer of smolt from FW to SW provokes only a slight increase in Posm and emphasizes the acid-base balance disruptions shown in FW. An oGHimplant in a presmolt stimulates gill Na⁺/K⁺ ATPase activity in FW, and affects the acid-base balance. After SW transfer (12 days after implantation), oGH treatment prevents the increase of osmotic pressure and the restoration of the acid-base, ionic equilibrium was faster for oGH-implanted fish than for sham-operated fish. These observations show that in FW smelting salmon develop most of the systems they need for migration and growth in SW and that oGH implants induce the development of physiological characteristics of smolts in a non-natural period of smolting.     

Salinity effects on oxygen consumption, gill Na+, K+-ATPase and ion regulation in juvenile coho salmon

The metabolic response of juvenile coho salmon Oncorhynchus kisutch to different salinities was examined, using whole-animal oxygen consumption rates and gill Na⁺, K⁺-ATPase activities as indicators of osmoregulatory energetics. Coho salmon smolts were acclimated to fresh water (FW), isosmotic salinity (ISO, 10‰) and sea water (SW, 28‰) and were sampled for up to 6 weeks for plasma levels of cortisol, glucose and ions (Na⁺, K⁺, Cl⁻), gill Na⁺, K⁺-ATPase activity and oxygen consumption rates. Following an initial adjustment period, plasma constituents in SW fish returned to near-FW values, indicating that the fish were acclimated to SW by day 21. Gill Na⁺, K⁺-ATPase activities on days 21 and 42 were lowest in ISO, higher in FW and highest in SW. This result is consistent with the idea that less energy would be required to maintain ion balance in an isosmotic environment, where the ionic gradients between extracellular fluid and water would be minimal. Oxygen consumption rates of swimming fish (1 body length s⁻¹), however, did not differ significantly between the three test salinities after 6 weeks. The results of this study suggest that the metabolic response of juvenile salmonids to changes in salinity is dependent on life-history stage (e.g. fry v . smolt), and that oxygen consumption rates do not necessarily reflect osmoregulatory costs.     

Pre‐migratory differentiation of wild brown trout into migrant and resident individuals

In February to March, wild brown trout Salmo trutta were captured by electrofishing in a natural watercourse (tributaries of the River Lille Aa, Denmark), individually tagged (Passive Integrated Transponders), and released. Representatives of the tagged brown trout were recaptured on the release sites in April by electrofishing and eventually caught in downstream smolt traps ('migrants') placed in the main river or by electrofishing ('residents') on the initial sites in June. Upon each capture, smolt appearance and body size were evaluated, and a non‐lethal gill biopsy was taken and used for Na⁺,K⁺‐ATPase analysis. Based on repetitive gill enzyme analysis in individual fish, a retrospective analysis of the rate of development in individual brown trout ultimately classified as migrants or residents was performed. Two months prior to migration, a bimodal morphological and physiological (gill Na⁺,K⁺‐ATPase) development concurred and was related to the subsequent differentiation into resident and migratory fractions of each population. This differentiation was unrelated to growth rate and body size of individual fish but skewed in favour of migratory females. Individuals destined to become migrants developed a smolt‐like appearance before the onset of migration and had higher rate of change of gill Na⁺,K⁺‐ATPase activity than fish remaining residents. The rate of change of gill Na⁺,K⁺‐ATPase activity was independent of the distance migrated to the trap (3–28 km). Thus in bimodal wild brown trout populations a major increase in enzyme activity takes place before migration is initiated and is a characteristic of migratory individuals only.     

Plasma ionic regulation and gill Na+/K+-ATPase changes during rapid transfer to sea water of yearling rainbow trout, Salmo gairdneri: time course and seasonal variation

Danish rainbow trout, Salmo gairdneri Richardson, (40–65 g) were transferred to 28%o sea water at intervals throughout the early spring and summer. Gill Na⁺/K⁺-ATPase of fish kept in fresh water surged distinctly during May. Simultaneously, a body silvering occurred and plasma concentrations of Cl⁻, Na⁺ and total thyroxine (T4) decreased. The seawater transfer-induced adaptive response in plasma electrolytes comprised a biphasic change, i.e., an adjustive peak phase and a regulatory phase lasting for 2 days and 1 week after transfer, respectively. Further, gill Na⁺/K⁺-ATPase activity increased to a new level after an initial lag phase of 2–3 days, but electrolyte regulation was mostly initiated prior to the adaptive change in ATPase activity. In spite of increasing pre-transfer freshwater Na⁺/K⁺-ATPase activity during the spring, the electrolyte peak level, the degree of muscle dehydration and the mortality of fish transferred to sea water increased from April to July. The apparent uncoupling of freshwater Na⁺/K⁺-ATPase activity and plasma electrolyte regulation in sea water is discussed in relation to smelting and prediction of hypo-osmoregulatory performance.     

Effects of dietary soybean meal and photoperiod cycle on osmoregulation following seawater exposure in Atlantic salmon smolts

Atlantic salmon Salmo salar juveniles were fed either fishmeal-based diets (FM) or diets in which soybean meal (SBM) partly replaced the FM from first feeding on. The fish were kept at continuous daylight during the juvenile stage. During the last 3 weeks before reaching 100 g body mass, all fish were subjected to 12L:12D. Starting at 100 g body mass, groups of 60 fish from each feeding background were subjected to continuous light for 12 weeks (short winter), or a square-wave photoperiod cycle to stimulate parr to smolt transformation with 8L:16D during the first 6 weeks, and then continuous light during the last 6 weeks (long winter). After the 12 weeks, 20 fish from each treatment were subjected to 0, 24 or 96 h seawater exposure at a water salinity of 34. Hypo-osmoregulatory ability at seawater exposure was assessed by mortality, intestinal pathology, plasma ion concentrations and osmolality, gill Na⁺/K⁺-ATPase activity and element concentrations in the cytoplasm of distal intestinal enterocytes using X-ray microanalysis. The hypo-osmoregulatory capacity was higher in fish kept at short winter than at long winter, apparently due to more rapid development of gill Na⁺/K⁺-ATPase activity. Fish fed SBM suffered typical soybean meal-induced histological alterations of the distal intestine and apparent reductions in digestive function in the more proximal gastrointestinal regions. The net osmoregulatory capacity of these fish was maintained, as indicated by higher gill Na⁺/K⁺-ATPase activity and lower plasma Na⁺, Ca²⁺ and osmolality compared to the FM-fed fish. Thus, feeding SBM did not impair the hypo-osmoregulatory ability of the Atlantic salmon following seawater exposure.     

Effects of salinity on the ionic balance and growth of juvenile turbot

The effects of salinity changes (27, 19 and 10‰) on seawater-adapted juvenile turbot were studied on their plasma osmolarity and ion concentrations, on oxygen consumption, on gill Na⁺,K⁺-ATPase activity after 3 months and on growth parameters. All plasma concentrations (except chloride) were unchanged, suggesting that fish were well adapted to their environment. Oxygen consumption was significantly decreased in the 19 and 10‰ groups, where fish weighed significantly more 105 days after transfer than fish maintained in sea water. These results, and the fact that apparent food conversion rates were lower in a diluted environment, suggest that on a long term schedule growth conditions could be improved by adaptation to brackish waters (salinities between 10 and 19‰). The effects of transfer from sea water to 27, 19, 10 and 5‰ were also followed during the first 3 weeks. With salinity 10‰ a steady state was reached on day 21 with all plasma values within the same range. The significant differences observed in osmolarity, plasma ion concentrations and Na⁺,K⁺-ATPase activity 3 weeks after transfer of juveniles to 5‰ salinity, compared with transfers in higher salinities, suggest that there is a threshold of acclimation of turbot to a hypotonic environment.     

Salinity tolerance of Kosteletzkya virginica. I. Shoot growth, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicotyledonous halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m^-3 NaCl, and the effects of external salinity on shoot growth and ion content of individual leaves were studied in successive harvests. Growth was stimulated by 85 mol m^-3 NaCl and was progressively reduced at the two higher salinities. Growth suppression at high salinity resulted principally from decreased leaf production and area, not from accelerated leaf death. As is characteristic of halophytic dicots. K. virginica accumulated inorganic ions in its leaves, particularly Na⁺ and K⁺. However, the Na⁺ concentration of individual leaves did not increase with time, but remained constant or even declined, seeming to be well-coordinated with changes in water content. A striking feature of the ion composition of salinized plants was the development of a dramatic gradient in sodium content, with Na⁺ partitioned away from the most actively growing leaves. Salt-treated plants exhibited a strong potassium affinity, with foliar K⁺ levels higher in salinized plants than unsalinized plants after an initial decrease. These results suggest that selective uptake and transport, foliar compartmentation of Na⁺ and K⁺ in opposite directions along the shoot axis, and the regulation of leaf salt loads over time to prevent build-up of toxic concentrations are whole-plant features which enable K. virginica to establish favourable K⁺-Na⁺ relations under saline conditions.     

Seasonal changes in androgen levels in stream- and hatchery-reared Atlantic salmon parr and their relationship to smolting

In stream-reared Atlantic salmon Salmo salar , plasma androgens were significantly greater in mature male parr than immature males and females in October, but had declined by January and did not differ significantly from immature fish throughout the spring. Immature fish in March were significantly larger and had greater gill Na⁺, K⁺-ATPase activity than their previously mature counterparts. Bimodal growth distribution was seen in hatchery-reared Atlantic salmon and a proportion of the male fish in the lower mode matured. Plasma testosterone (T) and 11-ketotestosterone (11-KT) were significantly elevated from September to December in mature male (1+ year) parr. In January, plasma androgens had declined in mature males and did not differ significantly from immature fish. By May all the hatchery fish were large enough to smolt and a proportion of the previously mature males had increased gill Na⁺, K⁺-ATPase activity. Therefore elevated androgens in the previous autumn do not prevent smolting. Parr with higher plasma T and 11-KT in April and May, that are presumably beginning to mature, had lower gill Na⁺, K⁺-ATPase activity, indicating that future maturation and associated increases in androgens may inhibit smolting.     

Salinity tolerance of Kosteletzkya virginica. II. Root growth, lipid content, ion and water relations

Abstract. Kosteletzkya virginica (L.) Presl., a dicot halophyte native to brackish tidal marshes, was grown on nutrient solution containing 0. 85, 170 or 255 mol m ³ NaCl, and the effects of external salinity on root growth, ion and water levels, and lipid content were examined in successive harvests. Root growth paralleled shoot growth trends, with some enhancement observed at 85 mol m ³ NaCl and a reduction noted at the higher salinities. Root Na⁺ content increased with increasing external NaCl, but remained constant with time for each treatment. K⁺ content, although lower in salt-grown plants after 14 d salinization, subsequently increased to levels comparable to unsalinized plants. A strong K⁺ affinity was reflected in the increased K⁺/Na⁺ selectivity of salt-grown plants and by their low Na⁺/K⁺ ratios. Cl levels rose in salinized plants and values were double or more those for Na⁺, indicating the possibility of a sodium-excluding mechanism in roots. Root phospholipids and sterols, principal membrane constituents, were maintained or elevated and the free sterol/phospholipids ratio increased in salinized K. virginica plants, suggesting retention of overall membrane structure and decreased permeability. This response, considered in light of root calcium maintenance and high potassium levels, suggests that salinity-induced changes in membrane lipid composition may be important in preventing K⁺ leakage from cells.     

Genetic differences in physiology, growth hormone levels and migratory behaviour of Atlantic salmon smolts

Out of five strains of Atlantic salmon Salmo salar of 1+ years released upstream of a fyke net in the River Gudenaa in 1996, three, Lagan, Ätran and Corrib, migrated immediately, 50% of the recaptured fish reaching the net in 3–6 days. Burrishoole and Conon fish migrated with a 15–19 day delay. Smolt development in 1997 at the hatchery showed a spring surge in gill Na⁺, K⁺-ATPase activity in all strains which was correlated with increased seawater tolerance. Differences in the timing of gill enzyme development matched the observed migration pattern well. Lagan, Ätran and Corrib strains reached high enzyme activity earlier than the Burrishoole and Conon strains, and strains with delayed enzyme development and migration showed a delayed regression of seawater tolerance compared with the early strains. Inter-strain differences in plasma growth hormone profiles could not be related to the observed patterns of Na⁺, K⁺-ATPase and seawater tolerance development. The study gives evidence of genetic influence on the timing and intensity of smolting and subsequent migration in Atlantic salmon.     

Sucrose and ouabain effects on the kinetic properties of a membrane-bound (Na++ K++ Mg2+) ATPase in sugar beet roots

Kinetic studies of a microsomal (Na⁺+ K⁺+ Mg²⁺)ATPase from sugar beet roots ( Beta vulgaris L. cv. Monohill) show that sucrose influences the MgATPase in different ways depending on the presence of K⁺ and/or Na⁺ 1) In the presence of the substrate MgATP and Na⁺ the effect of sucrose follows simple Michaelis-Menten kinetics. 2) In the presence of substrate together with K⁺ or (K⁺+ Na⁺), sucrose has little effect on the ATPase activity. 3) In the presence of Na⁺, onabain acts as an uncompetitive inhibitor with respect to MgATP. 4) In the presence of K⁺ or (K⁺+ Na⁺), the inhibition by ouabain is somewhat depressed and shows non-linearity when 1/v is plotted versus 1/MgATP. 5) Sucrose and Na⁺ activate in a competitive way, so that a successive increase of the Na⁺ level decreases the activation by sucrose. Both K_m and V-values are thereby changed. 6) The sucrose activation in the presence of Na⁺ is also influenced by ouabain. It is, therefore, suggested that Na⁺ may regulate the interference between the Na⁺/K⁺ pump and a sucrose sensitive system.     

Smolting and seasonal variation in the smolt characteristics of one- and two-year-old Saimaa landlocked salmon under fish farm conditions

Silvering of the skin, reduced condition factor, elevated gill Na⁺, K⁺-ATPase activity and well-developed capacity to regulate the osmotic and ionic balance in sea water were observed in 1 and 2 year old hatchery-reared Saimaa landlocked salmon Salmo salar m. sebago during April-June. Loss of hypoosmoregulatory ability and gill Na⁺K⁺-ATPase activity was observed earlier in 2 year than 1 year old fish. Coincident with changes associated with smolting both age groups showed diminished osmoregulatory capacity in fresh water. Slow growth during May-June may also be attributed to osmoregulatory difficulties in fresh water. These results support the suggestion that the developmental changes at smolting are seasonal and unrelated to any salinity changes and the idea of smolting as evidence of maladaptation of the fish to fresh water.     

Genetic basis of sodium exclusion and sodium tolerance in yeast. A model for plants

A yeast strain carrying disruptions in TRK1 and ENA genes was very sensitive to Na⁺ because uptake discriminated poorly between K⁺ and Na⁺, and Na⁺ efflux was insignificant. Transformation with TRK1 and ENA1 restored discrimination, Na⁺ efflux and Na⁺ tolerance. Increasing external Ca²⁺ increased Na⁺ tolerance almost in the same proportion in TRK1 enal cells and in trkl ENAI cells, suggesting an unspecific effect of this cation. By using a vacuolar ATPase mutant, the role of the vacuole in Na⁺ tolerance was also demonstrated. The yeast model of Na⁺ exclusion and Na⁺ tolerance may be extended to plants.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Cortisol stimulates hypo-osmoregulatory ability in Atlantic salmon, Salmo salar L.

Hypo-osmoregulatory ability in juvenile Atlantic salmon, Sulrno salur L., was improved by cortisol treatment. Implantation of a vegetable shortening pellet containing cortisol (50 mg kg⁻¹) resulted in elevated plasma cortisol titres. Maximum cortisol levels (160–170ngml⁻¹) were observed at days 6 and 12 after the implantation and dropped significantly by day 55. Cortisol-implanted fish in fresh water developed a twofold increase in gill Na⁺/K⁺-ATPase activity at days 6 and 12, and a threefold increase by day 55. Intestinal mucosa Na⁺/K⁺-ATPase activity was not affected by cortisol. Cortisol-implanted fish exposed to 28 ppt sea water for 48 h tended to show an improved ability to regulate their plasma osmolarity and reduce their ionic load. The osmo-regulatory ability attained at days 12 and 55 was further evaluated by exposing fish to 37 ppt sea water for 96 h. While all the control fish died relatively early in these tests, cortisol-implanted fish showed a clear reduction in their mortality rate. These results indicate that cortisol can induce biochemical and organismal changes during winter months that typify preadaptive events normally occurring in the spring.     

Relation of source and sink during grain filling period in wheat and some aspects of its regulation

The effect of Mg²⁺, Na⁺, K⁺, ouabain and pH on ATPase activity of purified membrane fractions enriched in plasmalemma fragments from Hordeum vulgare L. (glycophyte) and Halocnemum strobilaceum L. (halophyte) was studied. Membrane ATPases from both plants were synergistically activated by K⁺ and Na⁺ in the presence of Mg²⁺. The maximum activity of the enzymes were observed at the ratio Na/K = 2–3. Ouabain (10^-4 M) almost completely eliminated the (Na⁺+ K⁺)-stimulated component of the ATPase activity. The Na, K, Mg-ATPase of Hordeum had a single pH optimum (pH 8), but that of the Halocnemum had two optima(pH 6 and 8). It appears that similar enzymes operate in the cells of both plants studied. The higher Na, K, Mg-ATPase activity of the halophyte compared to that of the glycophyte suggests the involvement of the enzyme in the extrusion of Na⁺ from the cytoplasm of cells of both plants.     

Sodium ion transport in Neurospora crassa

Na⁺ influx and efflux in Neurospora crassa RL21a can be studied separately to calculate net Na⁺ movements. In the absence of external K⁺, Na⁺ influx was independent of the K⁺ content of the cells, but when K⁺ was present, the inhibition of Na⁺ influx by external K⁺ was higher the higher the K⁺ content. Efflux depended on the K⁺ and Na⁺ content, and on the history of the cells. Efflux was higher the higher the Na⁺ and K⁺ contents, and, in low-K⁺ cells, the efflux was also higher in cells grown in the presence of Na⁺ than when Na⁺ was given to cells grown in the absence of Na⁺. Addition of K⁺ to cells in steady state with external Na⁺ resulted in a net Na⁺-loss. In cells grown without Na⁺ this loss was a consequence of the inhibition of Na⁺ influx. In Na⁺-grown cells, addition of K⁺ inhibited Na⁺ influx and increased Na⁺ efflux.     

Development of hypoosmoregulatory ability in allis shad Alosa alosa

When different ages of juvenile allis shad Alosa alosa (18–74 days post-fertilization) were exposed for 48 h to elevated salinity (25 or 30), all ages could tolerate moderates salinity (25) whereas only the older stages could tolerate the higher salinity. In 88 days post-fertilization allis shad, acclimatation to full-strength sea water for 2 weeks did not lead to significant changes in total water content and gill Na⁺/K⁺-ATPase activity whereas total Na⁺ content increased within 5 days to values which remained stable thereafter. Together, these data suggest that juvenile allis shad develop a limited euryhalinity, which allows them to fully adapt to hyperosmotic environment immediately after transfer.     

Effects of phloridzin, metavanadate and oligomycin on membrane bound (Na++ K++ Mg2+)ATPase activity in sugar beet roots

To clarify the reaction mechanism of a (Na⁺+ K⁺+ Mg²⁺)ATPase activity in sugar beet roots ( Beta vulgaris L. cv. Monohill) phloridzin, oligomycin (inhibitors of animal ATPases) and metavanadate (NH₄VO₃) have been used. Kinetic studies showed that: 1) Phloridzin inhibition is uncompetitive with respect to MgATP and not influenced by Na⁺ or K⁺. 2) This inhibition is only found in preparations made in the absence of sucrose. 3) Oligomycin and vanadate inhibit the ATPase in different ways. Omission of sucrose from the preparation medium favours vanadate inhibition but suppresses oligomycin inhibition. 4) The kinetic pattern of the Na⁺ activation of the ATPase differs in preparations made in the absence and presence of sucrose, but that of K⁺ activation is the same. – These results indicate that inclusion as against omission of sucrose from the preparation medium causes a conformational change of the membrane fragments/vesicles, which then expose different surfaces to the surrounding medium.     

Seasonal changes in ionoregulatory variables of the glass eel Anguilla anguilla following estuarine entry: comparison with resident elvers

In the present study, glass eels Anguilla anguilla in the Minho River estuary (41·5° N, 8·5° W) decreased in size (standard length, L _S and mass, M ) from the beginning (autumn) to the end of the sampling season (summer). On the other hand elvers increased in L _S and M from spring to summer and were significantly larger than glass eels in paired comparisons. Branchial Na⁺/K⁺-ATPase and vacuolar (V-type) proton ATPase ( in vitro activities), two important ion transporting pumps, did not show significant seasonal changes in either glass eels or elvers although in glass eels Na⁺/K⁺-ATPase (activity) expression was significantly higher than in elvers. In a single month comparison Na⁺/K⁺-ATPase branchial mRNA expression was also higher in glass eels as was the protein level expression of both Na⁺/K⁺-ATPase and NKCC (Na⁺:K⁺:2Cl⁻ co-transporter). Immunofluorescence microscopy indicated apical CFTR Cl⁻ channel labelling in Na⁺/K⁺-ATPase positive chloride cell in glass eels which was absent in elvers. Whole body sodium concentration and percentage water did not show significant seasonal differences in either glass eels or elvers although there were significant differences between these two groups during some months.