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.     

Elver Invasion, Population Structure and Growth of Marbled eels Anguilla marmorata in a Tropical River on Réunion Island in the Indian Ocean

Winter skates, Leucoraja ocellata, exposed to 80% and 50% seawater (SW) exhibited rapid and significant weight gains followed by a slight recovery to new steady state levels within 8 days. Skates were acclimated at each salinity (100% SW [N = 16], 80% SW [N = 8], 50% SW [N = 8]), anesthetized (MS222) and bled from the caudal vein. In 100% SW, skate plasma (930mOsm/kg) was slightly hyperosmotic to the external medium (922mOsm/kg). Plasma osmolality decreased with seawater dilution, but became increasingly hyperosmotic to the bathing media. The environmental dilutions resulted in significant, but disproportionate changes in plasma Cl^–, P^–, Na⁺, Ca⁺, Mg⁺, trimethylamine oxide (TMAO) and urea concentrations. Mean corpuscular [Hb] and milliliter RBC water measurements suggest that skate red cells swelled less at each dilution than predicted for a passive erythrocyte osmometer. Concentrations of the major RBC solutes K⁺, urea, TMAO and Cl^– decreased by 8, 25, 5 and 21%, respectively in 80% SW. In 50% SW, K⁺, urea, TMAO and Cl^– concentrations decreased by 9, 47, 36 and 15%, respectively. Quantitatively, the other measured intracellular electrolytes (Mg⁺, Na⁺, P^– and Ca⁺) also exhibited disproportionate changes in concentration. Our results indicate that L. ocellata is a euryhaline elasmobranch that can tolerate significant reduction in the external salinity through the release of both ions and urea from the extracellular compartments while retaining electrolytes at the expense of urea in the intracellular compartment.     

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.     

Temporal changes in liver carbohydrate metabolism associated with seawater transfer in Oreochromis mossambicus

The metabolic aspects of ionic and osmotic regulation in fish are not well understood. The objective of this study was to examine changes in carbohydrate metabolism during seawater (SW) acclimation in the euryhaline tilapia (Oreochromis mossambicus). Hepatic activities of three key enzymes of the intermediary metabolism, phosphofructokinase, glycogen phosphorylase and glucose 6-phosphate dehydrogenase, together with glycogen content and plasma glucose concentration were measured at 0, 0.5, 1, 2, 3, 6, 12, 24, 48 and 96 h after the direct transfer of tilapia from fresh water (FW) to 70% SW. Plasma growth hormone, prolactin₁₇₇ and prolactin₁₈₈, Na⁺ and Cl⁻ concentrations were also measured. Plasma Na⁺ and Cl⁻ levels were highest at 12 h, but returned to FW levels at 24 h after transfer, suggesting the tilapia were able to osmoregulate within 24 h after transfer. Plasma glucose levels were significantly higher in 70% SW than in FW during the course of acclimation, especially in the early stages. Hepatic enzyme activities and glycogen content did not change significantly during the acclimation period. Our results suggest the possibility that glucose is an important energy source for osmoregulation during the acclimation to hyperosmotic environments in O. mossambicus.     

Effects of exogenous cortisol and seawater adaptation on thyroid hormone receptors in the smolt stage of the sockeye salmon,Oncorhynchus nerka

The objective of this investigation was to quantify how thyroid hormone receptors of the sockeye salmon, Oncorhynchus nerka, respond to salinity changes from freshwater (FW) to seawater (SW) conditions. Thyroid hormone receptors (TRs) mRNA and protein expressions levels significantly increased when the fish were transferred from FW to SW, and the plasma T₃ and T₄ levels were significantly highest at 50 % SW and then maintained as control. Moreover, these parameters were significantly lower in the cortisol-injected groups than in the control. Hence, TRs, T₃, and T₄ may play a role in SW adaptation, when the fish migrate from FW to SW environments. We showed a negative correlation between cortisol and thyroid hormone levels, and a significant increase in plasma K⁺ levels in the kidney when the fish were transferred to SW, with levels being significantly lower in the cortisol-injected group. Hence, cortisol appears to be a stress hormone, and the plasma Na⁺ and Cl^- levels significantly increased when the fish were transferred to SW, with levels being significantly lower in the cortisol-injected group. These results indicate that cortisol modulates ion transportation in the plasma.     

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.     

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.     

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     

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.     

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

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

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+ uptake through the brush border membranes of intestine from fresh water and sea-water adapted trout (Salmo gairdneri,R.)

Summary A comparative study of the mechanisms of Na⁺ absorption through brush border membranes of enterocytes from freshwater (FW) and seawater (SW) adapted trout were carried out using purified vesicle preparations. In contrast to FW trout, SW trout were found to possess a Na⁺–K⁺–Cl^– cotransport process. This finding is regarded as a major adaptation to SW since this cotransport allows an increase of ions and water absorption. Both FW and SW trout were equipped with a Na⁺–H⁺ exchange. In FW, the intestine of the trout had both a Na⁺–Na⁺ exchange and a Na⁺ conductance which may be responsible for enterocyte Na⁺ uptake along the potential gradient.     

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.     

The effect of Cu on gill and esophagus lipid metabolism in the rainbow trout Oncorhynchus mykiss

Rainbow trout Oncorhynchus mykiss were exposed to 0, 100, 300 and 800 μgl⁻¹ ambient Cu in brackish water (BW) for 4 days at 13 °C and subsequently transferred to either clean BW, clean fresh water (FW) or clean seawater (SW) at 16 °C. After incubation with ³²P-phosphate and ¹⁴C-acetate added as precursors to the water the fish showed a degradation, depending on previous [CU], of ³²P- and ¹⁴C-labelled gill membrane phospholipids if they had been transferred to SW or remained in BW. Corresponding experiments where the fish were exposed to Cu in BW for 12 days showed a similar subsequent degradation in SW and BW of both gill and esophagus membrane phospholipids, however to a much lesser degree in gill tissue than after 4 days. Plasma Na⁺ was similarly reduced by up to 8%, depending on previous ambient Cu, but in this case only after transfer to FW and only after 4 days of exposure. Both the effect on membrane lipid metabolism and plasma Na⁺ thus showed acclimation to ambient Cu but there was apparently no direct correlation between the two different types of observed changes in membrane function.     

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.     

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.     

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%     

The physiological response of larval <Emphasis Type="Italic">Chironomus riparius</Emphasis> (Meigen) to abrupt brackish water exposure

The physiological response of larval Chironomus riparius was examined following direct transfer from freshwater (FW) to brackish water (BW; 20% seawater). Endpoints of hydromineral status (hemolymph Na⁺, Cl⁻, and K⁺ levels, hemolymph pH, body water content, and whole body Na⁺/K⁺-ATPase and V-type H⁺-ATPase activity) were examined 1, 3, 5, 12 and 24 h following BW transfer. Larvae transferred from FW to FW served as a control. Hemolymph Na⁺ and Cl⁻ levels increased following BW transfer. Hemolymph pH was initially regulated, but significantly decreased after 24 h in BW. Changes in hemolymph ions were not caused by osmotic loss of water from the hemolymph, since larvae tightly regulated total body moisture content. Furthermore, salinity did not affect hemolymph K⁺. When larvae were transferred to BW, Na⁺/K⁺-ATPase (NKA) activity did not significantly alter relative to FW control animals. In contrast, V-type H⁺-ATPase (VA) activity in C. riparius significantly decreased in BW. In FW-reared C. riparius, whole body NKA and VA activities were equivalent. However, in the isolated gut with intact Malpighian tubules of FW-reared C. riparius, VA activity was significantly greater than whole body while NKA activity was equivalent. This suggested that gut and/or Malpighian tubule VA activity contributes significantly to whole body VA activity and that a decline in whole body VA activity in BW may be closely linked to alterations in the physiology of gut and Malpighian tubule tissue. Taken together, data indicate that VA is important for ion uptake in FW and that the NKA does not play a major role in regulating ion homeostasis when larvae are acutely exposed to BW.     

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 acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments

The recent model showed that seawater (SW) mitochondrion-rich (MR) cells with hole-type apical openings secrete Cl^? through the transporters including the Na⁺, K⁺-ATPase (NKA), Na⁺, K⁺, 2Cl^? cotransporter (NKCC), and cystic fibrosis transmembrane conductance regulator (CFTR). The present study focused on the dynamic elimination of the Cl^? secretory capacity and illustrated different phases (i.e., acute and regulatory phases) of branchial MR cells in response to hypoosmotic challenge. Time-course remodeling of the cell surfaces and the altered expressions of typical ion transporters were observed in the branchial MR cells of SW-acclimated brackish medaka (Oryzias dancena) when exposed to fresh water (FW). On the 1st day post-transfer, rapid changes were shown in the acute phase: the flat-type MR cells with large apical surfaces replaced the hole-type cells, the gene expression of both Odnkcc1a and Odcftr decreased, and the apical immunostaining signals of CFTR protein disappeared. The basolateral immunostaining signals of NKCC1a protein decreased throughout the regulatory phase (> 1 day post-transfer). During this period, the size and number of NKA-immunoreactive MR cells were significantly reduced and elevated, respectively. Branchial NKA expression and activity were maintained at constant levels in both phases. The results revealed that when SW-acclimated brackish medaka were transferred to hypoosmotic FW for 24 h, the Cl^? secretory capacity of MR cells was eliminated, whereas NKCC1a protein was retained to maintain the hypoosmoregulatory endurance of the gills. The time-course acute and regulatory phases of gill MR cells showed different strategies of the euryhaline medaka when subjected to hypoosmotic environments.