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.     

Dynamic gene expression of GH/PRL-family hormone receptors in gill and kidney during freshwater-acclimation of Mozambique tilapia

In teleosts, prolactin (PRL) and growth hormone (GH) act at key osmoregulatory tissues to regulate hydromineral balance. This study was aimed at characterizing patterns of expression for genes encoding receptors for the GH/PRL-family of hormones in the gill and kidney of Mozambique tilapia (Oreochromis mossambicus) during freshwater (FW)-acclimation. Transfer of seawater (SW)-acclimated tilapia to FW elicited rapid and sustained increases in plasma levels and pituitary gene expression of PRL₁₇₇ and PRL₁₈₈; plasma hormone and pituitary mRNA levels of GH were unchanged. In the gill, PRL receptor 1 (PRLR1) mRNA increased markedly after transfer to FW by 6 h, while increases in GH receptor (GHR) mRNA were observed 48 h and 14 d after the transfer. By contrast, neither PRLR2 nor the somatolactin receptor (SLR) was responsive to FW transfer. Paralleling these endocrine responses were marked increases in branchial gene expression of a Na⁺/Cl^? cotransporter and a Na⁺/H⁺ exchanger, indicators of FW-type mitochondrion-rich cells (MRCs), at 24 and 48 h after FW transfer, respectively. Expression of Na⁺/K⁺/2Cl^? cotransporter, an indicator of SW-type MRCs, was sharply down-regulated by 6 h after transfer to FW. In kidney, PRLR1, PRLR2 and SLR mRNA levels were unchanged, while GHR mRNA was up-regulated from 6 h after FW transfer to all points thereafter. Collectively, these results suggest that the modulation of the gene expression for PRL and GH receptors in osmoregulatory tissues represents an important aspect of FW-acclimation of tilapia.     

Differential responses in gills of euryhaline tilapia, Oreochromis mossambicus, to various hyperosmotic shocks

Euryhaline tilapia (Oreochromis mossambicus) survived in brackish water (BW; 20‰) but died in seawater (SW; 35‰) within 6 h when transferred directly from fresh water (FW). The purpose of this study was to clarify responses in gills of FW tilapia to various hyperosmotic shocks induced by BW or SW. In FW-acclimated tilapia, scanning electron micrographs of gills revealed three subtypes of MR cell apical surfaces: wavy-convex (subtype I), shallow-basin (subtype II), and deep-hole (subtype III). Density of apical surfaces of mitochondrion-rich (MR) cell in gills of the BW-transfer tilapia decreased significantly within 3 h post-transfer due to disappearance of subtype I cells, but increased from 48 h post-transfer because of increasing density of subtype III cells. SW-transfer individuals, however, showed decreased density of MR cell openings after 1 h post-transfer because subtype I MR cell disappeared. On the other hand, relative branchial Na⁺/K⁺-ATPase (NKA) α1-subunit mRNA levels, protein abundance, and NKA activity of the BW-transfer group increased significantly at 6, 12, and 12 h post-transfer, respectively. In the SW-transfer group, relative mRNA and protein abundance of gill NKA α1-subunit did not change while NKA activity declined before dying in 5 h. Upon SW transfer, dramatic increases (nearly 2-fold) of plasma osmolality, [Na⁺], and [Cl⁻] were found prior to death. For the BW-transfer group, plasma osmolality was eventually controlled by 96 h post-transfer by enhancement of NKA expression and subtype III MR cell. The success or failure of NKA activation from gene to functional protein as well as the development of specific SW subtype in gills were crucial for the survival of euryhaline tilapia to various hyperosmotic shocks.     

Microtubule‐dependent changes in morphology and localization of chloride transport proteins in gill mitochondria‐rich cells of the tilapia,Oreochromis mossambicus

The tilapia (Oreochromis mossambicus) is a euryhaline fish exhibiting adaptive changes in cell size, phenotype, and ionoregulatory functions upon salinity challenge. Na⁺/Cl^? cotransporter (NCC) and Na⁺/K⁺/2Cl^? cotransporter (NKCC) are localized in the apical and basolateral membranes of mitochondria‐rich (MR) cells of the gills. These cells are responsible for chloride absorption (NCC) and secretion (NKCC), respectively, thus, the switch of gill NCC and NKCC expression is a crucial regulatory mechanism for salinity adaptation in tilapia. However, little is known about the interaction of cytoskeleton and these adaptive changes. In this study, we examined the time‐course of changes in the localization of NKCC/NCC in the gills of tilapia transferred from fresh water (FW) to brackish water (20‰) and from seawater (SW; 35‰) to FW. The results showed that basolateral NKCC disappeared and NCC was expressed in the apical membrane of MR cells. To further clarify the process of these adaptive changes, colchicine, a specific inhibitor of microtubule‐dependent cellular regulating processes was used. SW‐acclimated tilapia were transferred to SW, FW, and FW with colchicine (colchicine‐FW) for 96 h. Compared with the FW‐treatment group, in the MR cells of colchicine‐FW‐treatment group, (1) the average size was significantly larger, (2) only wavy‐convex‐subtype apical surfaces were found, and (3) the basolateral (cytoplasmic) NKCC signals were still exhibited. Taken together, our results suggest that changes in size, phenotype, as well as the expression of NCC and NKCC cotransporters of MR cells in the tilapia are microtubule‐dependent. J. Morphol. 277:1113–1122, 2016. © 2016 Wiley Periodicals, Inc.     

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.     

Ionoregulatory and endocrine responses to disturbed salt and water balance in Mozambique tilapia exposed to confinement and handling stress

This study assessed the endocrine and ionoregulatory responses by tilapia (Oreochromis mossambicus) to disturbances of hydromineral balance during confinement and handling. In fresh water (FW), confinement and handling for 0.5, 1, 2 and 6 h produced elevations in plasma cortisol and glucose; a reduction in plasma osmolality was observed at 6 h. Elevations in plasma prolactins (PRL₁₇₇ and PRL₁₈₈) accompanied this fall in osmolality while no effect upon growth hormone (GH) was evident; an increase in insulin-like growth-factor I (IGF-I) occurred at 0.5 h. In seawater (SW), confinement and handling increased plasma osmolality and glucose between 0.5 and 6 h; no effect on plasma cortisol was seen due to variable control levels. Concurrently, both PRLs were reduced in stressed fish with only transient changes in the GH/IGF-I axis. Next, the branchial expression of Na⁺/K⁺/2Cl^? cotransporter (NKCC) and Na⁺/Cl^? cotransporter (NCC) was characterized following confinement and handling for 6 h. In SW, NKCC mRNA levels increased in stressed fish concurrently with elevated plasma osmolality and diminished gill Na⁺, K⁺-ATPase activity; NCC was unchanged in stressed fish irrespective of salinity. Taken together, PRL and NKCC participate in restoring osmotic balance during acute stress while the GH/IGF-I axis displays only modest responses.     

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.     

Early life stage salinity tolerance of wild and hatchery-reared juvenile pink salmon Oncorhynchus gorbuscha

Salinity tolerance in wild (Glendale) and hatchery (Quinsam) pink salmon Oncorhynchus gorbuscha (average mass 0·2 g) was assessed by measuring whole body [Na⁺] and [Cl^?] after 24 or 72 h exposures to fresh water (FW) and 33, 66 or 100% sea water (SW). Gill Na⁺, K⁺‐ATPase activity was measured following exposure to FW and 100% SW and increased significantly in both populations after a 24 h exposure to 100% SW. Whole body [Na⁺] and whole body [Cl^?] increased significantly in both populations after 24 h in 33, 66 and 100% SW, where whole body [Cl^?] differed significantly between Quinsam and Glendale populations. Extending the seawater exposure to 72 h resulted in no further increases in whole body [Na⁺] and whole body [Cl^?] at any salinity, but there was more variability among the responses of the two populations. Per cent whole body water (c. 81%) was maintained in all groups of fish regardless of salinity exposure or population, indicating that the increase in whole body ion levels may have been related to maintaining water balance as no mortality was observed in this study. Thus, both wild and hatchery juvenile O. gorbuscha tolerated abrupt salinity changes, which triggered an increase in gill Na⁺, K⁺‐ATPase within 24 h. These results are discussed in terms of the preparedness of emerging O. gorbuscha for the marine phase of their life cycle.     

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.     

The osmotic response of salmon louse,Lepeophtheirus salmonis (Copepoda: Caligidae), during the transition from sea water to fresh water

Summary The osmotic changes in haemolymph and body tissues of the ectoparasitic salmon louse,Lepeophtheirus salmonis, have been studied upon transfer from sea water (SW) to dilute sea water (37% SW), and then to fresh water (FW). The parasite shows osmoconformity in SW but hyperosmotic regulation in 37% SW regardless of whether it is attached to the salmon host or free swimming in the water. The same conclusion is reached by haemolymph Cl^– measurements. In FW, the osmotic tolerance and response of attached and free swimming parasites differ: Attached animals maintain steady haemolymph osmolality and Cl^– concentration and survive for at least 1 week, while free swimming parasites quickly become diluted and start to die within 8 h.Acclimation to 37% SW is accompanied by changes in body tissue water content and in the content of ninhydrin positive substances and specific amino acids which suggest the presence of cell volume regulation. Glycine is the dominating free amino acid in the cephalothorax tissues but alanine, proline and taurine also occur in high amounts. Lysine is found to increase significantly during FW acclimation of attached parasites. A breakdown of cell volume regulation is suggested to limit the survival of attached salmon louse in fresh water.Abbreviations FW fresh water - NPS ninhydrin positive substances - SW sea water     

Prolactin regulation in the coho salmon,Oncorhynchus kisutch

Summary Parr and smolt sea water acclimated coho salmon,Oncorhynchus kisutch were subjected to gradual and direct transfers to fresh water. Plasma osmotic pressure, Na⁺, K⁺, Ca⁺⁺ and Mg⁺⁺ were similar in freshwater (FW) fish and seawater (SW) transferred controls for the 24 h following transfer. In spite of the similarity in osmotic pressure and ion levels, plasma cortisol concentrations were significantly increased immediately following salinity change while both pituitary and plasma prolactin decreased indicating enhanced secretion by the pituitary and clearance from the blood. In vitro experiments showed greater incorporation of tritiated leucine into prolactin (PRL) cells immediately after transfer to FW while prolactin injections into intact fish lowered activity in rostral pars distalis (RPD) cells as measured by the same technique, providing evidence of hormonal feedback. These experiments show that the increased synthesis and release of PRL that occurs in coho following movement into FW is not obviously correlated with plasma osmotic pressure, Na⁺ or Ca⁺⁺ concentrations as has been observed in other species of teleosts.Abbreviations FW freshwater - SW seawater - PRL prolactin - RPD rostral pars distalis     

Ion fluxes and diffusion potentials in the Dungeness crab,Cancer magister

Summary Transepithelial potentials (TEP's) were measured in Dungeness crabs exposed to a variety of experimental media. The TEP's can be accounted for as diffusion potentials. In sea water (SW) theP _Na/P _Cl ratio (calculated by substitution in the Goldman equation) was 0.68, but in dilutions of SW the value increased, reaching a maximum of 3.33 in freshwater (FW). When the calcium and magnesium concentrations in the diluted media were maintained at SW levels theP _Na/P _Cl remained close to that in SW.The effluxes of Na and Cl were monitored in crabs exposed to the experimental media and the TEP's were measured simultaneously. After transfer from SW to FW the decrease in Na efflux was considerably less than expected from the change in potential alone, indicating an increased permeability to sodium, while transfer from SW to 500 mM NaCl caused a 3.4-fold increase in Na efflux without any associated change of potential. These results indicate an increase in the permeability of the gill epithelium to Na as the ambient concentrations of Ca and Mg decline. The Cl effluxes are not dependent on the external concentration of divalent ions, but about 30% of the Cl efflux may be due to exchange diffusion.Abbreviations FW freshwater - SW sea water - TEP transepithelial potential This project was in part supported by faculty research grants from Fordham University, Bronx, NY and Towson State University, Towson, MD to GDR     

Ionic and osmotic regulation capabilities of juvenile Gulf of Mexico sturgeon, Acipenser oxyrinchusde sotoi

The salinity tolerance, and hydromineral regulation capabilities of three size groups (small 110–170 g; medium 230–290 g, large 460–700 g; n=48 for each group) of 13-month-old juvenile Gulf of Mexico sturgeon were investigated. Fish (n=6 for each salinity) were transferred directly from freshwater (FW) to a series of experimental salinity treatments (0, 5, 10, 15, 20, 25, 30, and 35 parts per thousand (ppt)). Fish were also acclimated in brackish water (20 ppt) for 2 weeks and transferred to a salinity of 34 ppt. In this condition juvenile Gulf of Mexico sturgeon adapted to saltwater (SW) and maintained their hydromineral balance. FW adapted sturgeon (n=6) had an average blood hemotocrit of 28.2±0.8%, plasma osmolality of 260.7±1.6 mOsm kg⁻¹ H₂O, and plasma ion concentrations of 135.7±1.2 mM l⁻¹ Na⁺, 106.9±1.9 mEq l⁻¹ Cl⁻, and 2.9±0.1 mM l⁻¹ K⁺. In SW adapted sturgeon (n=8) blood parameters averaged 26.9±0.7% for hematocrit, 294.2±2.3 mOsm kg⁻¹ H₂O for osmolality, 152.0±1.7 mM l⁻¹ Na⁺, 149.2±1.4 mEq l⁻¹ for Cl⁻, and 3.1±0.1 mM l⁻¹ K⁺. The method of transfer (abrupt or slow acclimation) directly affected fish survival and the time they took to achieve ionic and osmotic regulation. This SW adaptation appears to be related to body size, the larger the fish the easier the adaptation process. A threshold size of about 170 g was apparent for the fish to adapt to saltwater after 2 weeks of acclimation. Chloride cells were present in both FW and SW adapted sturgeon with SW and brackish water fish having chloride cells significantly (P<0.05) more numerous (561±53 and 598±45 cells mm⁻²) and larger in size (41.0±3.85 and 34.2±4.49 μm²) than FW adapted sturgeon (10±1.0 cells mm⁻² and 22±2.53 μm²). Few chloride cells were observed in the opercular membrane, however, none were found in the pseudobranch and spiracle.     

Changes of glycogen metabolism in the gills and hepatic tissue of tilapia (Oreochromis mossambicus) during short-term Cd exposure

The aim of the study was to test the hypothesis that the mechanism of glycogen metabolism has taken place in gills rather than in liver during Cd exposure. Male tilapia were exposed to 44.45 μM ambient Cd for 12 h, and we found blood glucose significantly increased, however, lactate levels showed no significant changes. The glycogen phosphorylase (GP) activity increased immediately after 0.75 to 3 h of Cd exposure in the gills, and after 1 to 6 h in the liver, respectively. In addition, the glycogen level depleted faster in the gills than in the liver. Plasma cortisol level increased from 0.25 to 1 h and recovered after 3 h, while the glucagon did not significantly change during Cd exposure. Glucocorticoid receptor (GR) mRNA expression decreased after 0.75 h in the gills, while it significantly increased after 6 h in the liver. Ca²⁺, Na⁺, Cl⁻, and K⁺ significantly decreased upon Cd exposure within 6 h following Cd-induced toxic stress. We suggested that the cortisol is the spontaneous stimulation of glycogen metabolism in the gills, and it triggers a subsequent energy supply later in the liver. Taken together, the profile of glycogen metabolism between gills and liver during Cd-exposure stress provide good support to our hypothesis.     

Changes in branchial and intestinal osmoregulatory mechanisms and growth hormone levels during smolting in hatchery-reared and wild brown trout

Evidence of smolting was studied in Danish hatchery-reared brown trout Salmo trutta L. Twenty-four hour seawater (SW) challenge tests (28‰, 10°C) at regular intervals showed that maximal hypo-osmoregulatory ability developed within a 3–4-week period in March and April. The improved ability to regulate plasma osmolality, muscle water content and plasma total [Mg] developed asynchronously, indicating that developmental changes in the gill, the gastrointestinal system and the kidney may not necessarily concur during smolting. Gill Na⁺, K⁺-ATPase activity peaked in April at the time of optimal hypo-osmoregulatory ability. Na⁺, K⁺-ATPase a -subunit mRNA level in gills was unchanged from January until April, but decreased in May in parallel with a decrease in the activity of the enzyme. In the middle region of the intestine, Na⁺, K⁺-ATPase activity increased in February and remained high until April. In the posterior region of the intestine, the activity was stable from January until April after which it decreased. In vitro fluid transport capacitity, J_v, in the middle intestine fluctuated throughout the spring. In the posterior intestine, J_v was low until late March, when it increased fivefold until early May. Drinking rate in fish transferred to SW for 24 h surged during spring. Na⁺, K⁺-ATPase activity in the pyloric caeca was elevated from March until May, and increased in response to SW transfer in June, suggesting a hypo-osmoregulatory function of the pyloric caeca. Plasma GH levels surged in FW trout during spring, concurring with the increase in gill Na⁺, K⁺-ATPase activity and SW tolerance, but peaked in May when gill Na⁺, K⁺-ATPase activity and SW tolerance were regressing. GH levels were generally low in SW-challenged fish, and there was no consistent effect of 24-h SW exposure on GH levels. In wild anadromous trout, gill Na⁺, K⁺-ATPase activity varied seasonally as in hatchery-reared fish, but peaked at higher levels suggesting a more intense smolting in fish living in their natural environment.     

Measurement of blood volume in the elasmobranch fish Scyliorhinus canicula following acute and long‐term salinity transfers

A technique using ⁵¹chromium‐labelled erythrocytes was used to measure blood volume in Scyliorhinus canicula following long‐term and acute salinity transfers. Basal whole‐blood volume was 5·6 ± 0·2 ml 100 g^?1 (mean ±s .e .), this increased (6·3 ± 0·2 ml 100 g^?1) following +14 day acclimation to 80% sea water (SW) and decreased (4·6 ± 0·2 ml 100 g^?1) following acclimation to 120% SW. These changes were shown to be primarily due to changes in plasma volume, with no significant changes in extrapolated red‐cell volume being demonstrated. Blood volume was also measured in the same animals during 10 h acute transfer to 100% SW. Plasma volume in S. canicula during acclimation from 80% SW was significantly reduced (4·5 ± 0·3 ml 100 g^?1) after 6 h of transfer to 100% SW. Blood volume in animals during acclimation from 120% SW was significantly increased (4·8 ± 0·2 ml 100 g^?1) after 4 h of acute transfer. The osmoregulatory implications of these different timeframes during hyposaline and hypersaline transfer are discussed, along with the importance of this in vivo technique as context for in vitro studies with haemo‐dynamic stimuli.     

Effects of copper on branchial ionoregulation in the rainbow trout,Salmo gairdneri Richardson

Summary Hard- and softwater acclimated adult rainbow trout were statically exposed to copper (12.5, 25, 50, 100, and 200 ppb) for two, 12 h periods at neutral and pH 5.0. Unidirectional Na⁺, and Cl⁻, and net Na⁺, Cl⁻, K⁺, and ammonia fluxes were monitored as a measure of branchial ionoregulatory disturbance. Copper concentrations as low as 12.5 ppb led to measurable ion losses. Net Na⁺, Cl⁻, and K⁺ losses were concentration dependent and unaffected by prior acclimation to either hard- or softwater at both neutral pH and pH 5. From 12.5 to 50 ppb net NaCl losses arose primarily as a result of the inhibition ofJ _in, and at higher concentrations,J _out was also stimulated. In softwater,J _in was more resistant to inhibition than in hardwater. However, in hardwater,J _out recovered to normal levels during the second 12 h period, but no such recovery was found in softwater. Plasma NaCl was inversely correlated with [copper], while plasma glucose and ammonia increased with [copper]. At pH 5.0 and [copper] from 12.5 to 50 ppb, H⁺ contributed significantly to the total ion loss, while at 100 and 200 ppb, ion losses were no greater at pH 5.0 than at neutral pH. In no case were the effects of copper and H⁺ strictly additive.     

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.     

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.