Morpholino gene knockdown in adult Fundulus heteroclitus: role of SGK1 in seawater acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism.     

Salinity regulates claudin mRNA and protein expression in the teleost gill

The teleost gill carries out NaCl uptake in freshwater (FW) and NaCl excretion in seawater (SW). This transformation with salinity requires close regulation of ion transporter capacity and epithelial permeability. This study investigates the regulation of tight-junctional claudins during salinity acclimation in fish. We identified claudin 3- and claudin 4-like immunoreactive proteins and examined their expression and that of select ion transporters by performing Western blot in tilapia (Oreochromis mossambicus) gill during FW and SW acclimation. Transfer of FW tilapia to SW increased plasma osmolality, which was corrected after 4 days, coinciding with increased gill Na+-K+-ATPase and Na+-K+-2Cl(-) cotransporter expression. Gill claudin 3- and claudin 4-like proteins were reduced with exposure to SW. Transfer to FW increased both claudin-like proteins. Immunohistochemistry shows that claudin 3-like protein was localized deep in the FW gill filament, whereas staining was found apically in SW gill. Claudin 4-like proteins are localized predominantly in the filament outer epithelial layer, and staining appears more intense in the gill of FW versus SW fish. In addition, tilapia claudin 28a and 30 genes were characterized, and mRNA expression was found to increase during FW acclimation. These studies are the first to detect putative claudin proteins in teleosts and show their localization and regulation with salinity in gill epithelium. The data indicate that claudins may be important in permeability changes associated with salinity acclimation and possibly the formation of deeper tight junctions in FW gill. This may reduce ion permeability, which is a critical facet of FW osmoregulation.     

Mitogen activated protein kinase 14-1 regulates serum glucocorticoid kinase 1 during seawater acclimation in Atlantic killifish, Fundulus heteroclitus

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies of environmental toxicants and osmoregulation. Previous research in our laboratory has shown that acute acclimation to seawater is mediated by an increase in SGK1. SGK1 promotes the trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane of the gill within the first hour in seawater resulting in increased chloride secretion. Although we have shown that the increase in gill SGK1 does not require activation of the glucocorticoid receptor, the mechanisms that mediate the rise SGK1 during acute acclimation is unknown. To test the hypothesis that mitogen activated protein kinase (MAPK14) is responsible for the rise in SGK1 we identified the coding sequence of killifish MAPK14-1 and designed a translational blocking vivo-morpholino targeting MAPK14-1. Injection of the MAPK14-1 vivo-morpholino resulted in a 30% reduction of MAPK14-1 and a 45% reduction in phosphorylated-MAPK14-1 protein in the gill of killifish transitioned from freshwater to seawater. Knock down of phosphorlyated-MAPK14-1 completely blocked the rise in SGK1 mRNA and protein in the killifish gill, providing the first direct and in vivo evidence that MAPK14-1 is necessary for acute seawater acclimation.     

Tissue Distribution, Effects of Salinity Acclimation, and Ontogeny of Aquaporin 3 in the Marine Teleost, Silver Sea Bream (Sparus sarba)

The purpose of the present study was to ascertain the tissue-specific expression of the water channel protein, aquaporin 3 (AQP3), during salinity acclimation and larval development of silver sea bream (Sparus sarba). A cDNA fragment encoding aquaporin 3 (aqp3) from silver sea bream gill was cloned and from the deduced amino acid sequence a polyclonal antibody was prepared. AQP3 was found to be present in gill, kidney, liver, brain, heart, and spleen but not in whole blood. The abundance of AQP3 was significantly highest in gills of hypoosmotic (6 ppt) and isoosmotic (12 ppt) acclimated sea bream when compared to seawater (33 ppt) and hypersaline (50 ppt)- acclimated sea bream. Spleen tissue also displayed significantly high levels of AQP3 protein in hypoosmotic and isoosmotic salinities whereas the AQP3 abundance in brain, liver, heart, and kidney remained unchanged across the range of salinities tested. The ontogenetic profile of AQP3 was also investigated from developing sea bream larvae and AQP3 was first detected at 14 days posthatch (dph) and increased steadily up to 28–46 dph. In conclusion, this study has demonstrated that AQP3 expression is modulated in gill and spleen tissue of salinity acclimated sea bream and that it can be detected relatively early during larval development.     

The role of aquaporin 3 in teleost fish

The aquaporin isoform, AQP3 has now been identified in a number of different teleost fish species, with additional DNA sequence information on AQP3 genes in further fish species available in genome databases. In zebrafish (Danio rerio), the AQP3 gene is present as two duplicate isoforms resulting from a teleostean fish genome-wide duplication. A further splicoform/isoform has also been identified in rainbow trout (Oncorhynchus mykiss). The identification of these AQP3 isoforms in other fish species is consequently explored. The role of AQP3 in physiological/osmoregulatory processes, in various teleost organs is then described. In teleost gill, AQP3 is expressed in 'chloride' cells, and in some species, in other epithelial cell types, where it may have a number of different functions including the prevention of dehydration. In eel esophagus, immunohistochemistry shows that AQP3 is expressed in surface epithelial cells in the anterior esophagus, but in mucus cells within the epithelium of the posterior esophagus. In eel intestine, AQP3 is found in macrophage-like cells and probably plays no part in osmoregulatory processes. In the rectum, as in the posterior esophagus AQP3 is expressed in mucus cells. In eel kidney, AQP3 is expressed in a subset of renal tubules, and localizes to the apical pole of tubule cells. There is no apparent change in the location or protein abundance of renal AQP3 following the acclimation of eels from freshwater to seawater.     

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

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

     

Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus

Previous research has suggested that northern populations of the euryhaline killifish (Fundulus heteroclitus) are better adapted to freshwater environments than their southern counterparts. In this study, we examined whether this adaptation has come at an ionoregulatory cost in seawater, by comparing published data for northern killifish to newly acquired data on the molecular responses of southern killifish to seawater transfer. After abrupt transfer from brackish water (10 per thousand) to seawater, Na,K-ATPase activity, Na,K-ATPase alpha(1a) mRNA expression, and NKCC1 mRNA expression increased 1 and 4 days after transfer in the gills of southern fish (by 2-3-fold), but increased at 1 day and not 4 days after transfer in northern fish. Small increases in mRNA expression were observed in both populations at 14 days. CFTR expression also increased in southern and northern fish at 1 and 4 days into seawater, and was also elevated at 14 days in northern fish. Because fish from both southern and northern populations maintained plasma Na(+) and Cl(-) balance after seawater transfer, the differences in activity and expression could not have been caused by differences in plasma ion levels. Instead, some other regulatory factor may account for the differences in expression between populations. This study shows that freshwater adaptation in northern populations of killifish has not necessarily come at a significant ionoregulatory cost in seawater, but has altered the molecular responses of their gills to seawater transfer compared to southern killifish.     

Effects of environmental salinity on gill endothelin receptor expression in the killifish, Fundulus heteroclitus

We recently determined that rapid changes in environmental salinity alter endothelin-1 (EDN1) mRNA levels in the euryhaline killifish, Fundulus heteroclitus, so we hypothesized that EDN1 may be a local regulator of gill ion transport in teleost fishes. The purpose of the present study was to examine the effects of changes in environmental salinity on the gill endothelin receptors: EDNRA, EDNRB, and EDNRC. Using quantitative real-time PCR, we determined that after a fresh water (FW) to seawater (SW) transfer, there is a two to threefold increase in gill EDNRA and EDNRB mRNA levels. Likewise, we found a two to three fold increase in gill EDNRA and EDNRB protein concentration. In addition, killifish that have acclimated to FW for 30 days had significantly lower EDNRA mRNA and protein levels than SW killifish. ENDRA were immunolocalized to the mitochondrion-rich cells of the killifish gill, suggesting that EDN1 signaling cascades may affect MRC function. EDNRB were found throughout the gill vasculature and on lamellar pillar cells. We previously immunolocalized EDN1 to the pillar cell suggesting that EDN1 acts as an autocrine signaling molecule and potentially regulates pillar cell tone and lamellar perfusion. We conclude that EDN1 is physiologically active in the teleost gill, and regulated by environmental salinity. Future functional studies examining the physiological role of this system are necessary to completely understand EDN1 in the fish gill.     

The osmorespiratory compromise in sculpins: impaired gas exchange is associated with freshwater tolerance

We acclimated two species of sculpin, the freshwater prickly sculpin (Cottus asper) and the closely related marine Pacific staghorn sculpin (Leptocottus armatus) to freshwater ( approximately 0 g/L), brackish water (15 g/L), and seawater (30 g/L) for at least 4 wk and examined the relationships between respiration, ion regulation, gill morphology, and freshwater tolerance. The prickly sculpin successfully acclimated to all three salinities and did not experience appreciable changes in plasma osmolality, [Cl-], or mortality. Gill Na+/K+-ATPase activity was lowest in prickly sculpins acclimated to freshwater, their native salinity, and increased during acclimation to seawater. Furthermore, prickly sculpins acclimated to freshwater had a 30% higher P(crit) than fish acclimated to brackish water or seawater; P(crit) is the environmental P(O2) below which an animal can no longer maintain a routine (.-)M(O2), and an increase in P(crit) represents a compromise of respiratory gas exchange. The higher P(crit) observed in prickly sculpins acclimated to freshwater is likely a consequence of their having small, relatively thick gills that increase in thickness (by approximately 1 microm) during freshwater exposure. In contrast, the marine Pacific staghorn sculpin successfully acclimated to brackish water and seawater, but high mortality (25%) was observed after 3 wk of exposure to freshwater. Pacific staghorn sculpins exposed to freshwater suffered significant, 15%-20%, reductions in plasma osmolality and [Cl-], and these losses in plasma ions resulted in a 1.4-fold increase in gill Na+/K+-ATPase activity. Pacific staghorn sculpins have large, thin gills that are not modified in response to salinity acclimation, and as a result, these animals show no respiratory compromise during freshwater acclimation, as evidenced by the lack of change in P(crit), but show significant ion regulatory disturbance. Overall, this study suggests that gill thickening and the resulting respiratory compromise are necessary for freshwater tolerance in sculpins.     

The effect of environmental salinity on the proteome of the sea bass (Dicentrarchus labrax L.)

The European sea bass, Dicentrarchus labrax L., tolerates a range of salinities from freshwater to hyper-saline. To study differences in protein expression, fish were reared in both freshwater and seawater. After 3-month acclimation, gill and intestine epithelia were collected and the soluble protein extracted. In all, 362 spots were differentially expressed in the gills and intestines of fishes reared in seawater compared to those from freshwater. Fifty differential protein spots were excised from a colloidal Coomassie-stained gel. Nine separate protein spots were identified unambiguously by mass spectrometry and database searching. Among the six proteins over-expressed in gill cells in seawater, five were cytoskeleton proteins and one was the aromatase cytochrome P450. In gill cells under freshwater conditions, the two over-expressed proteins identified were the prolactin receptor and the major histocompatibility complex class II β -antigen. In intestinal cells under freshwater conditions, the Iroquois homeobox protein Ziro5 was upregulated over ninefold. The expression of these proteins, their possible direct or indirect roles in the adaptation of D. labrax to salinity, and their correspondences with a previous study are discussed.     

Fundulus heteroclitus acutely transferred from seawater to high salinity require few adjustments to intestinal transport associated with osmoregulation

The common killifish, Fundulus heteroclitus, has historically been a favorite organism for the study of euryhalinity in teleost fish. Despite the species' large range of salinity tolerance, studies of osmoregulation in high salinity are rare, with most previous studies focused on fish transferred between freshwater and seawater. Similarly, while branchial transport properties have been studied extensively, there are relatively few studies investigating the role of the intestine in osmoregulation in killifish. This study sought to characterize the fluid and ion transport occurring in the intestinal tract of killifish adapted to seawater, and furthermore to investigate the adjustments that occur to these mechanisms following acute transfer to high salinity (70ppt). In vivo samples of blood plasma and intestinal fluids of seawater-acclimated killifish indicated absorption of Na(+), Cl(-), and water, the relative impermeability of the intestine to Mg(2+) and SO(4)(2-), and active secretion of HCO(3)(-) into the intestinal lumen. The details of these processes were investigated further using in vitro techniques of isolated intestinal sac preparations and an Ussing chamber pH-stat titration system. However, these methods were discovered to be of limited utility under physiologically relevant conditions due to tissue deterioration. Results that could be validly interpreted suggested that there are few changes to intestinal transport following transfer to high salinity, and that adjustments to epithelial permeability occur in the first 24h post-transfer.     

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

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

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.     

Gill Na(+)-K(+)-2Cl(-) cotransporter abundance and location in Atlantic salmon: effects of seawater and smolting

Na(+)-K(+)-2Cl(-) cotransporter abundance and location was examined in the gills of Atlantic salmon (Salmo salar) during seawater acclimation and smolting. Western blots revealed three bands centered at 285, 160, and 120 kDa. The Na(+)-K(+)-2Cl(-) cotransporter was colocalized with Na(+)-K(+)-ATPase to chloride cells on both the primary filament and secondary lamellae. Parr acclimated to 30 parts per thousand seawater had increased gill Na(+)-K(+)-2Cl(-) cotransporter abundance, large and numerous Na(+)-K(+)-2Cl(-) cotransporter immunoreactive chloride cells on the primary filament, and reduced numbers on the secondary lamellae. Gill Na(+)-K(+)-2Cl(-) cotransporter levels were low in presmolts (February) and increased 3.3-fold in smolts (May), coincident with elevated seawater tolerance. Cotransporter levels decreased below presmolt values in postsmolts in freshwater (June). The size and number of immunoreactive chloride cells on the primary filament increased threefold during smolting and decreased in postsmolts. Gill Na(+)-K(+)-ATPase activity and Na(+)-K(+)-2Cl(-) cotransporter abundance increased in parallel during both seawater acclimation and smolting. These data indicate a direct role of the Na(+)-K(+)-2Cl(-) cotransporter in salt secretion by gill chloride cells of teleost fish.     

Adaptive branchial mechanisms in the sturgeon Acipenser naccarii during acclimation to saltwater

Variations of Na(+)/K(+)-ATPase activity and fatty-acid composition in the gills of the sturgeon Acipenser naccarii subjected to progressive acclimation to full seawater (35 ppt) were determined in relation to the hypo-osmoregulatory capacity of this species in the hyperosmotic medium. Blood samples were taken and gills arches were removed at intermediate salinity levels between 0 and 35 ppt and after 20 days at constant salinity (35 ppt). Plasma osmolality and Na(+)/K(+)-ATPase activity increased significantly with growing environmental salinity. Total saturated fatty acids (SFAs) decreased, while total polyunsaturated fatty acids (PUFAs) increased significantly with increasing salinity due mainly to changes in n-3 PUFAs (20:5n-3 and 22:6n-3). The n-3/n-6 ratio increased significantly during the acclimation process. The results show a direct relationship between salinity, increased gill Na(+)/K(+)-ATPase activity and ultrastructural changes of the gill chloride cells. Changes in the fatty-acid composition in gills of A. naccarii during progressive acclimation to full seawater suggest that variations of gill fatty acids may also have a role in osmoregulatory mechanisms.     

Arsenic inhibits CFTR-mediated chloride secretion by killifish (Fundulus heteroclitus) opercular membrane.

Killifish are euryhaline teleosts that normally experience rapid changes in the salinity of the swim water. Acclimation to seawater is mediated by cortisol, which by activating glucocorticoid receptors, upregulates CFTR mediated Cl- secretion in the gill and operculum. Arsenic, a toxic metalloid that naturally occurs in the aquatic environment, has been shown to disrupt glucocorticoid hormone-mediated regulation of genes. Because little is known about the effects of environmentally relevant levels of arsenic on ion channels and salt homeostasis, studies were conducted to examine the effects of arsenic on the ability of killifish to acclimate to increased salinity. Arsenic in the swim water or administered by intraperitoneal injection prevented acclimation. To determine if arsenic blocked acclimation by inhibiting CFTR mediated Cl- secretion (Isc), opercular membranes were isolated and mounted in Ussing chambers and the effects of arsenic on Isc were measured. Arsenic (24 hr exposure) reduced Isc in opercular membranes isolated from salt water acclimated killifish. In addition, arsenic acutely (5-10 minutes) and reversibly inhibited Isc with an IC50 = 4.1 microM (305 ppb) when applied to the apical (seawater) side of the operculum, but not when added to the basolateral side of the operculum. Arsenic (4 microM for 60 minutes) also reduced mitochondrial respiration. Thus, environmentally relevant levels of arsenic block acclimation to seawater in killifish by reversibly inhibiting CFTR-mediated Cl- secretion by the opercular membrane, in part by inhibiting mitochondrial respiration.     

Recruitment and degeneration of mitochondrion-rich cells in the gills of Mozambique tilapia Oreochromis mossambicus during adaptation to a hyperosmotic environment

Cellular recruitment and degeneration of branchial mitochondrion-rich (MR) cells were examined in Mozambique tilapia transferred from hypoosmotic to hyperosmotic water. To examine apoptosis in the gills associated with salinity change, tilapia were directly transferred from freshwater to 70% seawater. The TUNEL assay showed that apoptotic cells in the gills were significantly increased at 1 day after transfer, which was supported by an electron-microscopic observation that gill MR cells underwent morphological changes characteristic of apoptosis such as an irregularly shaped electron-dense nucleus and distension of the tubular system. To further examine MR-cell recruitment, freshwater-acclimated tilapia were transferred either to freshwater or to 70% seawater after BrdU injection. Immunohistochemical detection of BrdU-labeled nuclei and Na(+)/K(+)-ATPase-rich MR cells allowed us to classify BrdU-labeled MR cells into two subtypes: a single MR cell and an MR-cell complex. Although newly generated single MR cells were observed similarly in both freshwater and 70% seawater-transferred fish, the density of MR-cell complexes was much higher in 70% seawater than in freshwater. Our findings indicated that transfer from hypoosmotic to hyperosmotic water enhanced apoptosis of freshwater-type MR cells, resulting in reduction in hyperosmoregulatory ability for freshwater adaptation, and stimulated the recruitment of MR-cell complexes to develop hypoosmoregulatory ability for seawater adaptation.     

Effects of environmental salinity and temperature on osmoregulatory ability, organic osmolytes, and plasma hormone profiles in the Mozambique tilapia (Oreochromis mossambicus)

The Mozambique tilapia, Oreochromis mossambicus, is capable of surviving a wide range of salinities and temperatures. The present study was undertaken to investigate the influence of environmental salinity and temperature on osmoregulatory ability, organic osmolytes and plasma hormone profiles in the tilapia. Fish were acclimated to fresh water (FW), seawater (SW) or double-strength seawater (200% SW) at 20, 28 or 35 degrees C for 7 days. Plasma osmolality increased significantly as environmental salinity and temperature increased. Marked increases in gill Na(+), K(+)-ATPase activity were observed at all temperatures in the fish acclimated to 200% SW. By contrast, Na(+), K(+)-ATPase activity was not affected by temperature at any salinity. Plasma glucose levels increased significantly with the increase in salinity and temperature. Significant correlations were observed between plasma glucose and osmolality. In brain and kidney, content of myo-inositol increased in parallel with plasma osmolality. In muscle and liver, there were similar increases in glycine and taurine, respectively. Glucose content in liver decreased significantly in the fish in 200% SW. Plasma prolactin levels decreased significantly after acclimation to SW or 200% SW. Plasma levels of cortisol and growth hormone were highly variable, and no consistent effect of salinity or temperature was observed. Although there was no significant difference among fish acclimated to different salinity at 20 degrees C, plasma IGF-I levels at 28 degrees C increased significantly with the increase in salinity. Highest levels of IGF-I were observed in SW fish at 35 degrees C. These results indicate that alterations in gill Na(+), K(+)-ATPase activity and glucose metabolism, the accumulation of organic osmolytes in some organs as well as plasma profiles of osmoregulatory hormones are sensitive to salinity and temperature acclimation in tilapia.     

Environmental Regulation of Mitochondria-Rich Cells in Chalcalburnus tarichi (Pallas, 1811) During Reproductive Migration

Chalcalburnus tarichi is an anadromous cyprinid fish that has adapted to extreme conditions (salinity 22 ‰, pH 9.8 and alkalinity 153 mEq × l^?1) in Lake Van in eastern Turkey. Changes in immunoreactivity of Na⁺/K⁺-ATPase in gill tissue and osmolarity and ion levels in plasma were investigated in C. tarichi during reproductive migration. Physicochemical characteristics and ion levels in Lake Van were high compared freshwater. Plasma osmolality and plasma ion concentrations ([Na⁺], [K⁺] and [Cl^?]) increased after transfer from freshwater to Lake Van. The mitochondria-rich (MR) cells of the gill were stained in both filament and lamellar epithelia of C. tarichi by immunocytochemistry with a specific antiserum for Na⁺/K⁺-ATPase in river fish samples. Density and area of MR cells were decreased in lake-adapted fishes. These results indicated that freshwater acclimation capacity is correlated with the size and distribution of MR cells in C. tarichi, in contrast to many teleost fishes.