SW-acclimated Fish Research Articles

Myo-inositol supplement helps the performance of seawater-acclimated Nile tilapia, Oreochromis niloticus

Seawater (SW)-acclimated Nile tilapia, Oreochromis niloticus, reared under a salinity 30 environment had lower growth and survival than the freshwater (FW)-acclimated fish. It was hypothesized that cells of the SW-acclimated fish had not been able to synthesize an adequate level of a compatible osmolyte, myo-inositol (MI), in adjusting to the salinity 30 environment. In this study, MI supplements, at 250, 500, and 750 mg/kg pellets, were provided to the fish through top-dressing. After the 30-day feeding trial, the following parameters were determined: final body weights; survival; biomass increase; feed conversion ratio (FCR); plasma osmolality and ions; and two transcripts in the gills mips250 and mipa1 encoding enzymes responsible for MI biosynthesis. The SW-acclimated O. niloticus receiving 500-mg MI supplement had significantly higher survival, biomass increase, and lower FCR than those of the SW-acclimated fish receiving no supplement. At 500-mg MI supplemental level, the increasing values of plasma osmolality and Na+ observed in SW-acclimated fish were significantly attenuated. The transcript mipa1, but not mips250, was markedly up-regulated in the SW-acclimated O. niloticus, compared with that of the FW-acclimated fish. Again, MI at 500-mg supplement attenuated the up-regulation significantly. This study suggests that MI supplement at the optimum level enhanced the performance of SW-acclimated O. niloticus, and through yet unknown mechanisms, attenuated some of their physiological responses to the osmotic stress.

Mechanisms of Ca2+ uptake in freshwater and seawater-acclimated killifish, Fundulus heteroclitus, and their response to acute salinity transfer.

Killifish (Fundulus heteroclitus) has been extensively used as a model for ion regulation by euryhaline fishes. Na+ and Cl- dynamics have been well studied in killifish, but few studies have addressed that of Ca2+. Therefore, this study aimed to characterize Ca2+ fluxes in freshwater (FW) and seawater (SW)-acclimated killifish, their response to salinity transfer, and to elucidate the mechanisms of Ca2+ influx in FW and SW. SW killifish displayed a significantly higher Ca2+ influx rate than that of FW fish, while Ca2+ efflux rates were comparable in both salinities. Ca2+ influx was saturable in FW (Km = 78 ± 19µmol/L; Jmax = 53 ± 3nmol/g/h) and influx by SW killifish was linear up to 7mmol/L Ca2+. In SW-acclimated fish, 36% of Ca2+ influx was attributed to "intestinal Ca2+ intake", likely caused by drinking, whereas intestinal Ca2+ intake in FW contributed to < 2% of total. Throughout the study, results suggested that "cation competition" in SW modulates Ca2+ influx. Therefore, we hypothesized that SW-acclimated fish actually have a higher affinity Ca2+ influx system than FW-acclimated fish but that it is competitively inhibited by competing SW cations. In agreement with this cation competition hypothesis, we demonstrated for the first time that "extra-intestinal" Ca2+ influx was inhibited by Mg2+ in both FW and SW-acclimated killifish. Following acute salinity transfer, extra-intestinal Ca2+ influx was rapidly regulated within 12-24h, similar to Na+ and Cl-. Ca2+ influx in FW was inhibited by La3+, an epithelial Ca2+ channel blocker, whereas La3+ had no significant effect in SW.

Deviation of habitat salinity during seasonal gonad recrudescence affects plasma sex steroid levels and suppresses gonadal maturation in an euryhaline fishEtroplus suratensis

Impact of osmoregulation on plasma sex steroid levels and gonadal histo-architecture was monitored to elucidate the effects of deviation from habitat salinity on gonadal recrudescence in an active reproductive season of an euryhaline fish Etroplus suratensis (pearlspot). Fish were maintained in three different salinities of 0 ppt Fresh Water (FW), 15 ppt Brackish Water (BW) and 30 ppt Sea Water (SW) for a period of 60 days. Plasma osmolality values were found to be significantly highest in SW-acclimated fish accompanied by highest levels of plasma K⁺ and Cl¯ ions. The progress of gonadal recrudescence was higher in BW followed by FW and SW as evident from the cellular features of gonads and increased level of plasma sex steroids, such as, in case of female and 11-keto Testosterone and Testosterone in case of males. Plasma cortisol levels were comparatively higher in fish of both sexes in SW group. Significantly high levels of cortisol in SW suggest its role in hypo-osmoregulation and associated stress. This study clearly reveals that salinity changes during the active reproductive phase can suppress the steroid-mediated gonad recrudescence maximally under hypo-osmoregulation in an euryhaline fish.

A Stenohaline Medaka, Oryzias woworae, Increases Expression of Gill Na(+), K(+)-ATPase and Na(+), K(+), 2Cl(-) Cotransporter 1 to Tolerate Osmotic Stress.

The present study aimed to evaluate the osmoregulatory mechanism of Daisy's medaka, O. woworae,as well as demonstrate the major factors affecting the hypo-osmoregulatory characteristics of euryhaline and stenohaline medaka. The medaka phylogenetic tree indicates that Daisy's medaka belongs to the celebensis species group. The salinity tolerance of Daisy's medaka was assessed. Our findings revealed that 20‰ (hypertonic) saltwater (SW) was lethal to Daisy's medaka. However, 62.5% of individuals survived 10‰ (isotonic) SW with pre-acclimation to 5‰ SW for one week. This transfer regime, "Experimental (Exp.) 10‰ SW", was used in the following experiments. After 10‰ SW-transfer, the plasma osmolality of Daisy's medaka significantly increased. The protein abundance and distribution of branchial Na(+), K(+)-ATPase (NKA) and Na(+), K(+), 2Cl(-) cotransporter 1 (NKCC1) were also examined after transfer to 10‰ SW for one week. Gill NKA activity increased significantly after transfer to 10‰ SW. Meanwhile, elevation of gill NKA αα-subunit protein-abundance was found in the 10‰ SW-acclimated fish. In gill cross-sections, more and larger NKA-immunoreactive (NKA-IR) cells were observed in the Exp. 10‰ SW medaka. The relative abundance of branchial NKCC1 protein increased significantly after transfer to 10‰ SW. NKCC1 was distributed in the basolateral membrane of NKA-IR cells of the Exp. 10‰ SW group. Furthermore, a higher abundance of NKCC1 protein was found in the gill homogenates of the euryhaline medaka, O. dancena, than in that of the stenohaline medaka, O. woworae.

Past seawater experience enhances seawater adaptability in medaka, Oryzias latipes.

BackgroundDuring the course of evolution, fishes have acquired adaptability to various salinity environments, and acquirement of seawater (SW) adaptability has played important roles in fish evolution and diversity. However, little is known about how saline environments influence the acquirement of SW adaptability. The Japanese medaka Oryzias latipes is a euryhaline species that usually inhabits freshwater (FW), but is also adaptable to full-strength SW when transferred through diluted SW. In the present study, we examined how past SW experience affects hyposmoregulatory ability in Japanese medaka.ResultsFor the preparation of SW-experienced fish, FW medaka were acclimated to SW after pre-acclimation to 1/2 SW, and the SW-acclimated fish were transferred back to FW. The SW-experienced fish and control FW fish (SW-inexperienced fish) were transferred directly to SW. Whereas control FW fish did not survive direct transfer to SW, 1/4 of SW-experienced fish adapted successfully to SW. Although there were no significant differences in blood osmolality and plasma Na+ and Cl− concentrations between SW-experienced and control FW medaka in FW, increments in these parameters following SW transfer were lower in SW-experienced fish than in control FW fish. The gene expression of SW-type Na+, K+-ATPase (NKA) in the gills of SW-experienced medaka increased more quickly after direct SW transfer compared with the expression in control FW fish. Prior to SW transfer, the density of NKA-immunoreactive ionocytes in the gills was higher in SW-experienced fish than in control FW fish. Ionocytes expressing CFTR Cl− channel at the apical membrane and those forming multicellular complexes, both of which were characteristic of SW-type ionocytes, were also increased in SW-experienced fish.ConclusionThese results indicate that past SW experience enhances the capacity of Na+ and Cl− secretion in ionocytes and thus hypoosmoregulatory ability of Japanese medaka, suggesting the presence of epigenetic mechanisms involved in seawater adaptation.Electronic supplementary materialThe online version of this article (doi:10.1186/s40851-016-0047-2) contains supplementary material, which is available to authorized users.

Investigating the mechanisms of Ni uptake and sub-lethal toxicity in the Atlantic killifish Fundulus heteroclitus in relation to salinity

The Atlantic killifish (Fundulus heteroclitus) is a resilient estuarine species that may be subjected to anthropogenic contamination of its natural habitat, by toxicants such as nickel (Ni). We investigated Ni accumulation and potential modes of Ni toxicity, in killifish, as a function of environmental salinity. Killifish were acclimated to 4 different salinities [0 freshwater (FW), 10, 30 and 100% seawater (SW)] and exposed to 5 mg/L of Ni for 96 h. Tissue Ni accumulation, whole body ions, critical swim speed and oxidative stress parameters were examined. SW was protective against Ni accumulation in the gills and kidney. Addition of Mg and Ca to FW protected against gill Ni accumulation, suggesting competition with Ni for uptake. Concentration-dependent Ni accumulation in the gill exhibited saturable relationships in both FW- and SW-acclimated fish. However SW fish displayed a lower Bmax (i.e. lower number of Ni binding sites) and a lower Km (i.e. higher affinity for Ni binding). No effect of Ni exposure was observed on critical swim speed (Ucrit) or maximum rate of oxygen consumption (MO2max). Markers of oxidative stress showed either no effect (e.g. protein carbonyl formation), or variable effects that appeared to depend more on salinity than on Ni exposure. These data indicate that the killifish is very tolerant to Ni toxicity, a characteristic that may facilitate the use of this species as a site-specific biomonitor of contaminated estuaries.

Effects of salinity and prolactin on gene transcript levels of ion transporters, ion pumps and prolactin receptors in Mozambique tilapia intestine

Euryhaline teleosts are faced with significant challenges during changes in salinity. Osmoregulatory responses to salinity changes are mediated through the neuroendocrine system which directs osmoregulatory tissues to modulate ion transport. Prolactin (PRL) plays a major role in freshwater (FW) osmoregulation by promoting ion uptake in osmoregulatory tissues, including intestine. We measured mRNA expression of ion pumps, Na+/K+-ATPase α3-subunit (NKAα3) and vacuolar type H+-ATPase A-subunit (V-ATPase A-subunit); ion transporters/channels, Na+/K+/2Cl− co-transporter (NKCC2) and cystic fibrosis transmembrane conductance regulator (CFTR); and the two PRL receptors, PRLR1 and PRLR2 in eleven intestinal segments of Mozambique tilapia (Oreochromis mossambicus) acclimated to FW or seawater (SW). Gene expression levels of NKAα3, V-ATPase A-subunit, and NKCC2 were generally lower in middle segments of the intestine, whereas CFTR mRNA was most highly expressed in anterior intestine of FW-fish. In both FW- and SW-acclimated fish, PRLR1 was most highly expressed in the terminal segment of the intestine, whereas PRLR2 was generally most highly expressed in anterior intestinal segments. While NKCC2, NKAα3 and PRLR2 mRNA expression was higher in the intestinal segments of SW-acclimated fish, CFTR mRNA expression was higher in FW-fish; PRLR1 and V-ATPase A-subunit mRNA expression was similar between FW- and SW-acclimated fish. Next, we characterized the effects of hypophysectomy (Hx) and PRL replacement on the expression of intestinal transcripts. Hypophysectomy reduced both NKCC2 and CFTR expression in particular intestinal segments; however, only NKCC2 expression was restored by PRL replacement. Together, these findings describe how both acclimation salinity and PRL impact transcript levels of effectors of ion transport in tilapia intestine.

Effects of acclimation salinity on the expression of selenoproteins in the tilapia, Oreochromis mossambicus

Selenoproteins are ubiquitously expressed, act on a variety of physiological redox-related processes, and are mostly regulated by selenium levels in animals. To date, the expression of most selenoproteins has not been verified in euryhaline fish models. The Mozambique tilapia, Oreochromis mossambicus, a euryhaline cichlid fish, has a high tolerance for changes in salinity and survives in fresh water (FW) and seawater (SW) environments which differ greatly in selenium availability. In the present study, we searched EST databases for cichlid selenoprotein mRNAs and screened for their differential expression in FW and SW-acclimated tilapia. The expression of mRNAs encoding iodothyronine deiodinases 1, 2 and 3 (Dio1, Dio2, Dio3), Fep15, glutathione peroxidase 2, selenoproteins J, K, L, M, P, S, and W, was measured in the brain, eye, gill, kidney, liver, pituitary, muscle, and intraperitoneal white adipose tissue. Gene expression of selenophosphate synthetase 1, Secp43, and selenocysteine lyase, factors involved in selenoprotein synthesis or in selenium metabolism, were also measured. The highest variation in selenoprotein and synthesis factor mRNA expression between FW- and SW-acclimated fish was found in gill and kidney. While the branchial expression of Dio3 was increased upon transferring tilapia from SW to FW, the inverse effect was observed when fish were transferred from FW to SW. Protein content of Dio3 was higher in fish acclimated to FW than in those acclimated to SW. Together, these results outline tissue distribution of selenoproteins in FW and SW-acclimated tilapia, and indicate that at least Dio3 expression is regulated by environmental salinity.

The osmoregulatory effects of rearing Mozambique tilapia in a tidally changing salinity

The native distribution of Mozambique tilapia, Oreochromis mossambicus, is characterized by estuarine areas subject to salinity variations between fresh water (FW) and seawater (SW) with tidal frequency. Osmoregulation in the face of changing environmental salinity is largely mediated through the neuroendocrine system and involves the activation of ion uptake and extrusion mechanisms in osmoregulatory tissues. We compared plasma osmolality, plasma prolactin (PRL), pituitary PRL mRNA, and mRNA of branchial ion pumps, transporters, channels, and PRL receptors in tilapia reared in FW, SW, brackish water (BW) and in tidally-changing salinity, which varied between FW (TF) and SW (TS) every 6h. Plasma PRL was higher in FW tilapia than in SW, BW, TF, and TS tilapia. Unlike tilapia reared in FW or SW, fish in salinities that varied tidally showed no correlation between plasma osmolality and PRL. In FW fish, gene expression of PRL receptor 1 (PRLR1), Na+/Cl− cotransporter (NCC), aquaporin 3 (AQP3) and two isoforms of Na+/K+-ATPase (NKA α1a and NKA α1b) was higher than that of SW, BW or tidally-changing salinity fish. Gene expression of the Na+/K+/2Cl− cotransporter (NKCC1a), and the cystic fibrosis transmembrane conductance regulator (CFTR) were higher in fish in SW, BW or a tidally-changing salinity than in FW fish. Immunocytochemistry revealed that ionocytes of fish in tidally-changing salinities resemble ionocytes of SW fish. This study indicated that tilapia reared in a tidally-changing salinity can compensate for large changes in external osmolality while maintaining osmoregulatory parameters within a narrow range closer to that observed in SW-acclimated fish.

Acclimation of brackish water pearl spot (Etroplus suratensis) to various salinities: relative changes in abundance of branchial Na+/K+-ATPase and Na+/K+/2Cl− co-transporter in relation to osmoregulatory parameters

The present study was conducted to elucidate the osmoregulatory ability of the fish pearl spot (Etroplus suratensis) to know the scope of this species for aquaculture under various salinities. Juvenile pearl spot were divided into three groups and acclimated to freshwater (FW), brackish water (BW) or seawater (SW) for 15days. The fish exhibited effective salinity tolerance under osmotic challenges. Although the plasma osmolality and Na(+), K(+) and Cl(-) levels increased with the increasing salinities, the parameters remained within the physiological range. The muscle water contents were constant among FW-, BW- and SW-acclimated fish. Two Na+/K+-ATPase α-isoforms (NKA α) were expressed in gills during acclimation in FW, BW and SW. Abundance of one isoform was up-regulated in response to seawater acclimation, suggesting its role in ion secretion similar to NKA α1b, while expression of another isoform was simultaneously up-regulated in response to both FW and SW acclimation, suggesting the presence of isoforms switching phenomenon during acclimation to different salinities. Nevertheless, NKA enzyme activities in the gills of the SW and FW individuals were higher (p<0.05) than in BW counterparts. Immunohistochemistry revealed that Na(+)/K(+)-ATPase immunoreactive (NKA-IR) cells were mainly distributed in the interlamellar region of the gill filaments in FW groups and in the apical portion of the filaments in BW and SW groups. The number of NKA-IR cells in the gills of the FW-acclimated fish was almost similar to that of SW individuals, which exceeded that of the BW individuals. The NKA-IR cells of BW and SW were bigger in size than their FW counterparts. Besides, the relative abundance of branchial Na(+)/K(+)/2Cl(-) co-transporter showed stronger evidence in favor of involvement of this protein in hypo-osmoregulation, requiring ion secretion by the chloride cells. To the best of our knowledge, this is the first study reporting the wide salinity tolerance of E. suratensis involving differential activation of ion transporters and thereby suggesting its potential as candidate for fish farming under different external salinities.

Expression of ion transporters in gill mitochondrion-rich cells in Japanese eel acclimated to a wide range of environmental salinity

We examined morphological changes and molecular mechanisms of ion regulation in mitochondrion-rich (MR) cells of Japanese eel acclimated to different environmental salinities. Electron microscopic observations revealed that the apical membrane of MR cells appeared as a flat or slightly projecting disk with a mesh-like structure on its surface in eel acclimated to freshwater (FW). In seawater (SW)-acclimated eel, in contrast, the apical membrane of MR cells showed a slightly concave surface without a mesh-like structure. The mRNA expression of Na+/H+ exchanger-3 (NHE3) in deionized FW and normal SW was higher than that in normal FW and 30%-diluted SW. Expression of Na+/K+/2Cl− cotransporter-1a (NKCC1a) became higher with increasing environmental salinity. Immunofluorescence staining showed that the apical NHE3 immunoreaction was stronger in deionized FW and normal SW than in the other groups. Basolateral NKCC1 immunoreaction was most intense in normal SW. These results indicate that apical NHE3 is involved in ion uptake in fish acclimated to hypotonic environments, and that basolateral NKCC1 is important for acclimation to hypertonic environments. The relatively high expression of NHE3 in SW further indicates a possible role of NHE3 in acid-base regulation in the gills in SW-acclimated fish.

Prolactin177, prolactin188 and prolactin receptor 2 in the pituitary of the euryhaline tilapia, Oreochromis mossambicus, are differentially osmosensitive

Two forms of prolactin (Prl), prolactin 177 (Prl(177)) and prolactin 188 (Prl(188)), are produced in the rostral pars distalis (RPD) of the pituitary gland of euryhaline Mozambique tilapia, Oreochromis mossambicus. Consistent with their roles in fresh water (FW) osmoregulation, release of both Prls is rapidly stimulated by hyposmotic stimuli, both in vivo and in vitro. We examined the concurrent dynamics of Prl(177) and Prl(188) hormone release and mRNA expression from Prl cells in response to changes in environmental salinity in vivo and to changes in extracellular osmolality in vitro. In addition, mRNA levels of Prl receptors 1 and 2 (prlr1 and prlr2) and osmotic stress transcription factor 1 (ostf1) were measured. Following transfer from seawater (SW) to FW, plasma osmolality decreased, while plasma levels of Prl(177) and Prl(188) and RPD mRNA levels of prl(177) and prl(188) increased. The opposite pattern was observed when fish were transferred from FW to SW. Moreover, hyposmotically induced release of Prl(188) was greater in Prl cells isolated from FW-acclimated fish after 6 h of incubation, while the hyposmotically induced increase in prl(188) mRNA levels was only observed in SW-acclimated fish. In addition, prlr2 and ostf1 mRNA levels in Prl cells from both FW- and SW-acclimated fish increased in direct proportion to increases in extracellular osmolality both in vivo and in vitro. Taken together, these results indicate that the osmosensitivity of the tilapia RPD is modulated by environmental salinity with respect to hormone release and gene expression.

Switching of Na+, K+-ATPase isoforms by salinity and prolactin in the gill of a cichlid fish

We identified and investigated the changes in expression of two gill Na(+), K(+)-ATPase α-subunit isoforms (α-1a and α-1b) in relationship with salinity acclimation in a cichlid fish, Mozambique tilapia. Transfer of freshwater (FW)-acclimated fish to seawater (SW) resulted in a marked reduction in α-1a expression within 24 h and a significant increase in α-1b expression with maximum levels attained 7 days after the transfer. In contrast, transfer of SW-acclimated fish to FW induced a marked increase in α-1a expression within 2 days, while α-1b expression decreased significantly after 14 days. Hypophysectomy resulted in a virtual shutdown of α-1a mRNA expression in both FW- and SW-acclimated fish, whereas no significant effect was observed in α-1b expression. Replacement therapy by ovine prolactin (oPrl) fully restored α-1a expression in FW-acclimated fish, while cortisol had a modest, but significant, stimulatory effect on α-1a expression. In hypophysectomized fish in SW, replacement therapy with oPrl alone or in combination with cortisol resulted in a marked increase in α-1a mRNA to levels far exceeding those observed in sham-operated fish. Expression of α-1b mRNA was unaffected by hormone treatment either in FW-acclimated fish or in SW-acclimated fish. The mRNA expression of fxyd-11, a regulatory Na(+), K(+)-ATPase subunit, was transiently enhanced during both FW and SW acclimation. In hypophysectomized fish in FW, oPrl and cortisol stimulated fxyd-11 expression in a synergistic manner. The clear Prl dependence of gill α-1a expression may partially explain the importance of this hormone to hyperosmoregulation in this species.

Differential effects of cortisol and 11-deoxycorticosterone on ion transport protein mRNA levels in gills of two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis)

The role of cortisol as the only corticosteroid in fish osmoregulation has recently been challenged with the discovery of a mineralocorticoid-like hormone, 11-deoxycorticosterone (DOC), and necessitates new studies of the endocrinology of osmoregulation in fish. Using an in vitro gill explant incubation approach, DOC-mediated regulation of selected osmoregulatory target genes in the gill was investigated and compared with that of cortisol in two euryhaline teleosts, Mozambique tilapia (Oreochromis mossambicus) and striped bass (Morone saxatilis). The effects were tested in gills from both fresh water (FW)- and seawater (SW)-acclimated fish. Both cortisol and DOC caused an up-regulation of the Na(+),K(+)-ATPase α1 subunit in SW-acclimated tilapia but had no effect in FW-acclimated fish. Cortisol conferred an increase in Na(+),K(+),2Cl(-) cotransporter (NKCC) isoform 1a transcript levels in FW- and SW-acclimated tilapia, whereas DOC had a stimulatory effect only in SW-acclimated fish. Cortisol had no effect on NKCC isoform 1b mRNA levels at both salinities, while DOC stimulated this isoform in SW-acclimated fish. In striped bass, cortisol conferred an up-regulation of Na(+),K(+)-ATPase α1 and NKCC transcript levels in FW- and SW-acclimated fish, whereas DOC resulted in down-regulation of these transcripts in FW-acclimated fish. It was also found that both corticosteroids may rapidly (30 min) alter the mitogen-activated protein kinase signalling pathway in gill, inducing phosphorylation of extracellular signal-regulated kinase 1 (ERK1) and ERK2 in a salinity-dependent manner. The study shows a disparate organisation of corticosteroid signalling mechanisms involved in ion regulation in the two species and adds new evidence to a role of DOC as a mineralocorticoid hormone in teleosts.

Effects of environmental salinity, biopsy, and GH and IGF-I administration on the expression of immune and osmoregulatory genes in the gills of Atlantic salmon (Salmo salar)

Immune-endocrine interactions are thought to be important for fish health, especially following injury of peripheral tissues. We investigated the influences of gill biopsy (surgical removal of several gill filaments with minimal bleeding) on mRNA levels of osmoregulatory and immune genes in peripheral tissues of Atlantic salmon (Salmo salar) smolts in freshwater (FW) and seawater (SW). The effects of growth hormone (GH) and insulin-like growth factor (IGF)-I, which are known as both osmoregulatory and immunomodulatory hormones, were also examined in FW- and SW-acclimated fish. Compared with the intact gill arch of the same fish, the residuary filaments after biopsy showed significant reduction in Na+,K+-ATPase (NKA) and cystic fibrosis transmembrane conductance regulator (CFTR) mRNA levels in FW fish. In contrast, mRNA levels of these genes in the filaments with biopsy were increased in SW fish. Increased caspase gene transcription was observed in the cut filaments of SW fish, but not in those of FW fish. Lysozyme gene transcription was stimulated in the residuary gill filaments after biopsy in both FW and SW. Administration of salmon GH or IGF-I increased mRNA levels of NKA, CFTR, caspase, and lysozyme in the gills after biopsy in FW fish. In SW-acclimated fish, there was no significant effect of GH or IGF-I on the transcription of osmoregulatory or immune genes. The results in this study showed tight relationships between immunity, osmoregulation, and endocrine system in fish gills, and that nonlethal gill biopsy can be used for investigations of fish health.

Expression and Localization of Aquaporin 1a in the Sea-Bass (Dicentrarchus labrax) during Ontogeny.

The successful establishment of a species in a given habitat depends on the ability of each of its developing stages to adapt to the environment. In order to understand this process we have studied the adaptation of a euryhaline fish, the sea-bass Dicentrarchus labrax, to various salinities during its ontogeny. The expression and localization of Aquaporin 1a (AQP1a) mRNA and protein were determined in different osmoregulatory tissues. In larvae, the sites of AQP1a expression are variable and they shift according to age, implying functional changes. In juveniles after metamorphosis (D32–D48 post-hatch, 15–25 mm) and in pre-adults, an increase in AQP1a transcript abundance was noted in the digestive tract, and the AQP1a location was observed in the intestine. In juveniles (D87–D100 post-hatch, 38–48 mm), the transcript levels of AQP1a in the digestive tract and in the kidney were higher in sea water (SW) than at lower salinity. These observations, in agreement with existing models, suggest that in SW-acclimated fish, the imbibed water is absorbed via AQP1a through the digestive tract, particularly the intestine and the rectum. In addition, AQP1a may play a role in water reabsorption in the kidney. These mechanisms compensate dehydration in SW, and they contribute to the adaptation of juveniles to salinity changes during sea-lagoon migrations. These results contribute to the interpretation of the adaptation of populations to habitats where salinity varies.

Growth performance, osmoregulatory and metabolic modifications in red porgy fry, Pagrus pagrus, under different environmental salinities and stocking densities

The effects of two different environmental salinities [brackish water (BW), 12‰; sea water (SW), 39‰] and initial stock densities [low (LD), 1.0 g L−1; high (HD), 2.0 g L−1] on growth, osmoregulation, stress and energy metabolism of the fry Pagrus pagrus were investigated over a period of 45 days. Pagrus pagrus (n=80, 5.51 ± 0.25 g mean initial body weight) were randomly divided in eight groups. Growth, weight gain and specific growth rate increased in BW-acclimated fish compared with SW-acclimated fish. No differences were observed between the two stock densities tested at either environmental salinity. Plasma osmolality was lowest in BW-acclimated specimens, but the stock density had no effect on this parameter. Branchial Na+,K+-ATPase activity was positively correlated with environmental salinity, but unaltered at the renal level. Plasmatic parameters were enhanced by salinity and stocking conditions. At the hepatic level, triglyceride values were enhanced in BW-acclimated fish maintained at LD. Muscle metabolites (glycogen, glucose and lactate) increased in BW- compared with SW-acclimated fish; stock density had no influence. Our data suggest that changes in metabolic parameters could be correlated with the higher growth rates observed in P. pagrus acclimated to BW, while no significant effects due to the stocking density used were observed.

A role for inositol monophosphatase 1 (IMPA1) in salinity adaptation in the euryhaline eel (Anguilla anguilla)

This study investigated the expression and tissue distribution of inositol monophosphatase (IMPA1) and characterized its role in salinity adaptation in the eel. The coding sequence of eel IMPA1 was determined and confirmed to be orthologous to the mammalian gene/enzyme by phylogenetic analysis and structural modeling. Quantitative real-time PCR and Western blot techniques indicated up to 17-fold increases in mRNA expression and 2-fold increases in protein abundance in major osmoregulatory tissues following transfer of fish to seawater (SW). This was accompanied by up to 5-fold increases in enzyme activity, and 1.8- and 3-fold increases in inositol contents within the gill and kidney, respectively. Immunohistological studies revealed that IMPA1 protein expression predominated in SW-acclimated fish within basal epithelial/epidermal layers of the gill, esophagus, intestine, skin, and fins. SW transfer also induced a 10-fold increase in inositol content in the fin. IMPA1 immunoreactivity was also identified in chondrocytes within the cartilagenous matrix of the gills and fins, as well as in clusters of interstitial cells surrounding the kidney tubules. The observed increases in expression of IMPA1 highlight a protective role for inositol within various eel tissues following SW acclimation. This constitutes an adaptive mechanism in teleost fish naturally exposed to hypertonic environments.

TEP on the tide in killifish (Fundulus heteroclitus): 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% S: W 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 approximately 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.

Effects of seawater acclimation on mRNA levels of corticosteroid receptor genes in osmoregulatory and immune systems in trout

Influence of environmental salinity on expression of distinct corticosteroid receptor (CR) genes, glucocorticoid receptor (GR)-1 and -2, and mineralcorticoid receptor (MR), was examined in osmoregulatory and hemopoietic organs and leucocytes of steelhead trout (Oncorhynchus mykiss). There was no significant difference in plasma cortisol levels between freshwater (FW)- or seawater (SW)-acclimated trout, whereas Na+, K+-ATPase was activated in gill of SW fish. Plasma lysozyme levels also showed a significant increase after acclimation to SW. In SW-acclimated fish, mRNA levels of GR-1, GR-2, and MR were significantly higher in gill and body kidney than those in FW. Head kidney and spleen showed no significant change in these CR mRNA levels after SW-acclimation. On the other hand, leucocytes isolated from head kidney and peripheral blood showed significant decreases in mRNA levels of CR in SW-acclimated fish. These results showed differential regulation of gene expression of CR between osmoregulatory and immune systems.