Seawater Adaptation Research Articles

Identification and apical membrane localization of an electrogenic Na+/Ca2+exchanger NCX2a likely to be involved in renal Ca2+excretion by seawater fish

Seawater (SW) contains ∼10 mM Ca(2+), yet marine fish must drink seawater as their major water source. Thus marine teleosts fish need to excrete Ca(2+) to maintain whole body Ca(2+) homeostasis. In the intestine, seawater Ca(2+) interreacts with epithelial-secreted HCO(3)(-) by the intestinal epithelium, and the resulting CaCO(3) precipitates, which is rectally excreted. Recently the transporters involved in intestinal HCO(3)(-) secretion were identified. Ca(2+) is also excreted by the kidney, but the protein(s) involved in renal Ca(2+) excretion have not been identified. Here we identified a candidate transporter by using SW pufferfish torafugu (Takifugu rubripes) and its closely related euryhaline species mefugu (Takifugu obscurus), which are becoming useful animal models for studying molecular mechanisms of seawater adaptation. RT-PCR analyses of Na(+)/Ca(2+) exchanger (NCX) family members in various torafugu tissues demonstrated that only NCX2a is highly expressed in the kidney. Renal expression of NCX2a was markedly elevated when mefugu were transferred from freshwater to seawater. In situ hybridization and immunohistochemical analyses indicated that NCX2a is expressed in the proximal tubule at the apical membrane. NCX2a, expressed in Xenopus oocytes, conferred [Ca(2+)](out)- and Na(+)-dependent currents. These results suggest that NCX2a mediates renal Ca(2+) secretion at the apical membrane of renal proximal tubules and has an important role in whole body Ca(2+) homeostasis of marine teleosts.

Growth, osmoregulation and endocrine changes in wild Atlantic salmon smolts and post-smolts during marine migration

We have examined physiological parameters associated with seawater adaptability, growth and energetics, as well as major endocrine regulators of these processes in wild migrating Atlantic salmon smolts and post-smolts from the river through the fjord, coastal areas and the open ocean. Muscle RNA/DNA ratio suggests that growth rate increases soon after entry into seawater and continues to increase after the post-smolts reach the offshore banks and the feeding grounds in the Norwegian Sea. Post-smolts prioritize rapid growth and protein deposition in spring and summer, and their energy intake during this period is so high that deposition of energy is possible in addition to muscle growth. An increase in thyroxine (T4) and triiodothyronine (T3) levels was observed, suggesting a major activation of hepatic conversion of T4 to T3 in post-smolts in seawater, probably related to the high metabolic activity and rapid growth and development of the post-smolts. Decreased plasma growth hormone (GH) levels were observed from the river through the fjord, with levels around 2ngml−1 in rapidly growing post-smolts, concurrent with an increase in circulating insulin-like growth factor I (IGF-I). An increase in pituitary GH expression levels and hepatic GH receptor (GH-R) and local IGF-I mRNA levels suggest a physiological basis for the changes in circulating GH and IGF-I levels. Receptor expression in brain and pituitary suggests that both hormones are actively involved in the growth and differentiation of these tissues during the critical early marine phase. Gill Na+,K+-ATPase (NKA) activity increased to post-smolt levels above 20μmol ADP mgprot.÷1h÷1, probably representing long-term NKA activity levels of Atlantic salmon in seawater. Concurrent with the changes in NKA activity the expression of the NKA α1b isoform remained high in post-smolts, while the expression of the NKA α1a decreased from smolts to post-smolts. Both cystic fibrosis transmembrane conductance regulator (CFTR) I and II showed a reduction in mRNA levels from smolts to post-smolts, and remained stable at low expression levels in seawater.

Detection of QTL with effects on osmoregulation capacities in the rainbow trout (Oncorhynchus mykiss)

BackgroundThere is increasing evidence that the ability to adapt to seawater in teleost fish is modulated by genetic factors. Most studies have involved the comparison of species or strains and little is known about the genetic architecture of the trait. To address this question, we searched for QTL affecting osmoregulation capacities after transfer to saline water in a nonmigratory captive-bred population of rainbow trout.ResultsA QTL design (5 full-sib families, about 200 F2 progeny each) was produced from a cross between F0 grand-parents previously selected during two generations for a high or a low cortisol response after a standardized confinement stress. When fish were about 18 months old (near 204 g body weight), individual progeny were submitted to two successive hyper-osmotic challenges (30 ppt salinity) 14 days apart. Plasma chloride and sodium concentrations were recorded 24 h after each transfer. After the second challenge, fish were sacrificed and a gill index (weight of total gill arches corrected for body weight) was recorded. The genome scan was performed with 196 microsatellites and 85 SNP markers. Unitrait and multiple-trait QTL analyses were carried out on the whole dataset (5 families) through interval mapping methods with the QTLMap software. For post-challenge plasma ion concentrations, significant QTL (P < 0.05) were found on six different linkage groups and highly suggestive ones (P < 0.10) on two additional linkage groups. Most QTL affected concentrations of both chloride and sodium during both challenges, but some were specific to either chloride (2 QTL) or sodium (1 QTL) concentrations. Six QTL (4 significant, 2 suggestive) affecting gill index were discovered. Two were specific to the trait, while the others were also identified as QTL for post-challenge ion concentrations. Altogether, allelic effects were consistent for QTL affecting chloride and sodium concentrations but inconsistent for QTL affecting ion concentrations and gill morphology. There was no systematic lineage effect (grand-parental origin of QTL alleles) on the recorded traits.ConclusionsFor the first time, genomic loci associated with effects on major physiological components of osmotic adaptation to seawater in a nonmigratory fish were revealed. The results pave the way for further deciphering of the complex regulatory mechanisms underlying seawater adaptation and genes involved in osmoregulatory physiology in rainbow trout and other euryhaline fishes.

Effects of 17β-estradiol, 4-nonylphenol, and β-sitosterol on the growth hormone-insulin-like growth factor system and seawater adaptation of rainbow trout (Oncorhynchus mykiss)

Previous studies show that successful adaptation of euryhaline teleost fish to seawater (SW) involves the GH-IGF system. The increasing occurrence, distribution, and concentration of environmental contaminants, including environmental estrogens (EE), in aquatic habit over recent time may compromise the hypoosmoreegulatory ability of fish. In this study, we used rainbow trout (Oncorhynchus mykiss) to assess the effects of EE on the GH-IGF system and adaptation to increased salinity. Juvenile trout (ca. 30g) were exposed to either low (10μg/l) or high (100μg/l) concentrations of β-sitosterol, 4-n-nonylphenol (NP), or 17β-estradiol (E2) for 28days in fresh water (FW); after which, fish were exposed to 20‰ SW. Plasma chloride levels in control fish rose initially, and then declined to initial levels after 48h. By contrast, plasma chloride levels in all EE-treated groups except β-sitosterol low increased and remained elevated over initial levels after 48h. Levels GH receptor 1 (GHR 1), GHR 2, insulin-like growth factor-1 (IGF-1), and IGF-2mRNAs in liver of control fish increased 6–12h after SW exposure. In gill, levels of GHR 1, GHR 2, IGF-1, IGF-2, IGF receptor 1A (IGFR1A), and IGFR1B mRNAs increased in control fish 6–12h after 20‰ SW exposure. Levels of IGFR1A and IGFR1B mRNAs in white muscle and of IGFR1A mRNA in red muscle increased in control fish 6–12h after 20‰ SW exposure. Expression of all mRNAs in liver, gill, and red and white muscle declined from peak levels in control fish by 48h after transfer. Exposure of fish to β-sitosterol, NP, and E2 abolished or attenuated normal salinity-induced changes in the expression of GHR, IGF, and IGFR1 mRNAs in all tissues. These results indicate that EE reduces salinity adaptation by inhibiting components of the GH-IGF system.

Expression of aquaporins 3, 8 and 10 in the intestines of freshwater- and seawater-acclimated Japanese eels Anguilla japonica

Euryhaline teleosts possess excellent adaptability to a wide range of environmental salinities. Although intestinal water absorption is important for seawater (SW) adaptation, little is known about the molecular mechanisms of the water-transporting pathway in the intestine. We have cloned three homologs of the mammalian aquaporins (AQPs) 3, 8 and 10 from the intestine of the SW-acclimated Japanese eel. The deduced amino-acid sequences shared 47–98% homology with other known respective AQP isoforms. Topology prediction of the identified sequences showed six membrane-spanning domains, intracellular N- and C-terminal tails, and two NPA (asparagine–proline–alanine) motifs in the second and fifth connecting loops, all of which are highly conserved among known AQPs. Reverse transcription PCR analysis revealed that AQP3 was expressed in various tissues, whereas the expression of AQP 8 and 10 mRNAs was detected predominantly in the intestine. The expression levels of AQPs 1, 3, 8 and 10 in the anterior and posterior intestines and the rectum were determined by real-time quantitative PCR and compared for FW (freshwater)- and SW-acclimated eels. AQP1 expression levels in the posterior intestine and rectum were significantly higher in the SW-acclimated eel than in the FW-acclimated fish. AQP3 expression in the SW-acclimated eel was only higher in the rectum than that in the FW-acclimated eel. Expression levels of AQPs 8 and 10 in the intestinal segments tended to be higher in the SW-acclimated eel than in the FW-acclimated eel. These results showed that intestinal AQP expression is closely related to SW adaptation, suggesting the presence of a water-absorbing mechanism associated with multiple AQP isoforms in the intestinal tract.

Acute salinity challenges in Mozambique and Nile tilapia: Differential responses of plasma prolactin, growth hormone and branchial expression of ion transporters

The responses of Mozambique and Nile tilapia acclimated to fresh water (FW) and brackish water (BW; 17‰) were compared following acute salinity challenges. In both species, plasma osmolality increased to above 450 mOsm by 2 h after transfer from FW to seawater (SW); these increases in osmolality were accompanied by unexpected increases in plasma prolactin (PRL). Likewise, PRL receptor gene expression in the gill also increased in both species. In Nile tilapia, hyperosmotic transfers (FW to BW and SW) resulted in increased plasma growth hormone (GH) and in branchial GH receptor gene expression, responses that were absent in Mozambique tilapia. Branchial gene expression of osmotic stress transcription factor 1 (OSTF1) increased in both species following transfer from FW to SW, whereas transfer from BW to SW induced OSTF1 expression only in the Nile tilapia. Branchial expression of Na +/Cl − cotransporter was higher in FW in both species than in BW. Branchial gene expression of Na +/K +/2Cl − cotransporter (NKCC) increased after transfer from BW to SW in Mozambique tilapia, whereas expression was reduced in the Nile tilapia following the same transfer. The difference in the SW adaptability of these species may be related to a limited capacity of Nile tilapia to up-regulate NKCC gene expression, which is likely to be an essential component in the recruitment of SW-type chloride cells. The differential responses of GH and OSTF1 may also be associated with the disparate SW adaptability of these two tilapiine species.

Changes in gene expression for GH/PRL/SL family hormones in the pituitaries of homing chum salmon during ocean migration through upstream migration

Gene expression for growth hormone (GH)/prolactin (PRL)/somatolactin (SL) family hormones in the pituitaries of homing chum salmon were examined, because gene expression for these hormones during ocean-migrating phases remains unclear. Fish were collected in the winter Gulf of Alaska, the summer Bering Sea and along homing pathway in the Ishikari River–Ishikari Bay water system in Hokkaido, Japan in autumn. The oceanic fish included maturing adults, which had developing gonads and left the Bering Sea for the natal river by the end of summer. The absolute amounts of GH, PRL and SL mRNAs in the pituitaries of the maturing adults in the summer Bering Sea were 5- to 20-fold those in the winter Gulf of Alaska. The amount of GH mRNA in the homing adults at the coastal seawater (SW) areas was smaller than that in the Bering fish, while the amount of PRL mRNA remained at the higher level until fish arrived at the Ishikari River. The gill Na +,K +-ATPase activity in the coastal SW fish and the plasma Na + levels in the brackish water fish at the estuary were lowered to the levels that were comparable to those in the fresh water (FW) fish. In conclusion, gene expression for GH, PRL and SL was elevated in the pituitaries of chum salmon before initiation of homing behavior from the summer Bering Sea. Gene expression for GH is thereafter lowered coincidently with malfunction of SW adaptability in the breeding season, while gene expression for PRL is maintained high until forthcoming FW adaptation.

Changing expression patterns: focus on “The transcription factor, glial cell missing 2, is involved in differentiation and functional regulation of H+-ATPase-rich cells in zebrafish (Danio rerio)”

euryhaline species of fish have a remarkable osmo- and ionoregulatory capacity. Some euryhaline species like the Mozambique tilapia can directly be transferred from freshwater to seawater and vice versa, i.e., osmolarity of the external fluid changes by ∼1,000 mOsm. Many others can tolerate this

Salinity effects on the expression of osmoregulatory genes in the euryhaline black porgy Acanthopagrus schlegeli

Black porgy is a marine euryhaline species with a capacity to cope with demands in a wide range of salinities and thus is a perfect model-fish to study osmoregulatory responses to salinity-acclimated processes and their hormonal control. The present study was performed to understand the regulatory changes in hormone, hormone receptors and important osmoregulatory genes in pituitary, gill, intestine and kidney in response to acute salinity stress. Transcript levels were analyzed by quantitative real-time PCR following acute salinity challenge by direct transfer of seawater (SW) acclimatized fish to fresh water (FWBP) and vice versa (SWBP). SW acclimation significantly increased plasma osmolality and intestine Na +/K +-ATPase (NKA) activity while FW acclimation increased plasma cortisol and branchial NKA activity. Plasma osmolality and chloride concentration decreased in FWBP whereas GH levels remained unchanged in both FWBP and SWBP. Comparative analysis of gene profiles between FWBP and SWBP showed that pituitary prolactin transcript increased significantly in FWBP. Prolactin receptor ( PRLR) transcripts increased in gill of FWBP while it decreased in gill and kidney of SWBP. NKA transcripts increased in gill of both FWBP and SWBP, while it decreased in intestine of FWBP and increased in intestine and kidney of SWBP. Glucocorticoid receptor ( GR) transcripts decreased in intestine and kidney of FWBP while it increased in gill and intestine of SWBP. No significant changes were observed in growth hormone receptor ( GHR) transcripts of both FWBP and SWBP in pituitary, gill, intestine and kidney. Our current data demonstrated the correlation between PRLR gene expression in relation to FW adaptation, and GR gene expression in relation to SW adaptation in euryhaline black porgy. The results indicate that black porgy has an excellent osmoregulatory capacity and is capable of withstanding large variations in salinity.

Potent osmoregulatory actions of homologous adrenomedullins administered peripherally and centrally in eels

The teleost adrenomedullin (AM) family consists of three groups, AM1/AM4, AM2/AM3, and AM5. In the present study, we examined the effects of homologous AM1, AM2, and AM5 on drinking and renal function after peripheral or central administration in conscious freshwater eels. AM2 and AM5, but not AM1, exhibited dose-dependent (0.01-1 nmol/kg) dipsogenic and antidiuretic effects after intra-arterial bolus injection. The antidiuretic effect was significantly correlated with the degree of associated hypotension. To avoid the potential indirect osmoregulatory effects of AM-induced hypotension, infusion of AMs was also performed at nondepressor doses. Drinking was enhanced dose-dependently at 0.1-3 pmol.kg(-1).min(-1) of AM2 and AM5, matching the potency and efficacy of angiotensin II (ANG II), the most potent dipsogenic hormone known thus far. AM2 and AM5 infusion also induced mild antidiuresis, while AM1 caused antinatriuresis. Additionally, AMs were injected into the third and fourth ventricles of conscious eels to assess their site of dipsogenic action. However, none of the AMs at 0.05-0.5 nmol induced drinking, while ANG II was highly dipsogenic. AM2 and ANG II injected into the third ventricle increased arterial pressure while AM5 decreased it in a dose-dependent manner, and both AM2 and AM5 decreased blood pressure when injected into the fourth ventricle. These data suggest that circulating AM2 and AM5 act on a target site in the brain that lacks the blood-brain barrier. Collectively, the present study showed that AM2 and AM5 are potent osmoregulatory hormones in the eel, and their actions imply involvement in seawater adaptation of this euryhaline species.

Physiological seawater adaptation in juvenile Atlantic salmon (Salmo salar) autumn migrants

Summary About 25 % of juvenile Atlantic salmon (Salmo salar) migrating downstream in the River Frome in southern England do so in the autumn rather than in the spring. Here, we examine the physiological status of these fish with regard to those features that adapt them to sea water during the parr–smolt transformation (i.e. gill Na+K+ ATPase activity; the number, size and type of chloride cells on the gill lamellae; salinity tolerance and relative plasma thyroid levels). Autumn migrants, and those fish which subsequently reside in the tidal reaches during the winter, are not sufficiently physiologically adapted to permit permanent or early, entry into the marine environment. It is not known what proportion of autumn migrating fish survive and return to spawn as adults. If significant numbers do return, however, the production from tidal reach habitats must be taken into account in the development of salmon stock management strategies, especially monitoring and assessment programmes, and in the evaluation of factors affecting stocks.

CDNA ENCODING GROWTH HORMONE FROM HUMPBACK GROUPER (Cromileptes altivelis)

Growth hormone (GH) that plays an important role in growth, reproduction, seawater adaptation, and immune function was isolated and sequenced from humpback grouper, Cromileptes altivelis. The cDNA was isolated from pituitary using RT-PCR. The 618 bp open reading frame encodes a 205 amino acid (aa) protein, which represents an 18 aa signal peptide followed by a 187 aa mature GH polypeptide. The fragment contained conserved domain of somatotropin–1, somatotropin–2, casein kinase II phosphorylation, protein kinase C phosphorylation, N–myristoylation and N–glycosilation. The similarity of deduced protein of humpback grouper GH was 65.0 - 89.5% with other fishes

Plasma non-esterified fatty acids of elasmobranchs: Comparisons of temperate and tropical species and effects of environmental salinity

We investigated the influence of environments with different average temperatures and different salinities on plasma NEFA in elasmobranchs by comparing species from tropical vs. cold temperate marine waters, and tropical freshwater vs. tropical marine waters. The influence of the environment on plasma NEFA is significant, especially with regard to essential fatty acids (EFA) and the n-3/ n-6 ratio. n-3/ n-6 ratios in tropical marine elasmobranchs were lower by two-fold or more compared with temperate marine elasmobranchs, because of higher levels of arachidonic acid (AA, 20:4 n-6) and docosatetraenoic acid (22:4 n-6), and less docosahexaenoic acid (DHA, 22:6 n-3), in the tropical species. These results are similar to those in earlier studies on lipids in teleosts. n-3/ n-6 ratios and levels of EFA were similar between tropical freshwater and tropical marine elasmobranchs. This suggests that the observation in temperate waters that marine fishes have higher levels of n-3 fatty acids and n-3/ n-6 ratios than freshwater fishes may not hold true in tropical waters, at least in elasmobranchs. It also suggests that plasma NEFA are little affected by freshwater vs. seawater adaptation in elasmobranchs. Likewise, we found that plasma NEFA composition and levels were not markedly affected by salinity acclimation (2 weeks) in the euryhaline stingray Himantura signifer. However, in contrast to our comparisons of freshwater-adapted vs. marine species, the level of n-3 fatty acids and the n-3/ n-6 ratio were observed to significantly decrease, indicating a potential role of n-3 fatty acids in salinity acclimation in H. signifer.

Regulation of Ion Transport in Eel Intestine by the Homologous Guanylin Family of Peptides

Since the gene expression of guanylin peptides and their receptors, guanylyl cyclase Cs, is enhanced in the intestine of seawater (SW)-adapted eels compared with fresh water (FW)-adapted fish, the guanylin family may play an important role in SW adaptation in eels. The present study analyzed the effect of three homologous guanylin peptides, guanylin, uroguanylin and renoguanylin, on ion movement through the eel intestine, and examined the target of guanylin action using Ussing chambers. The middle and posterior parts of the intestine, where water and ion absorption occurs actively in SW eels, exhibited serosa-negative transepithelial potential, while the anterior intestine was serosa-positive. Mucosal application of each guanylin in the middle or posterior intestine reduced the short-circuit current (Isc) dose dependently and reversed it at high doses, and reduced electric tissue resistance. The effects were greater in the middle intestine than in the posterior intestine. All three guanylins showed similar potency in the middle segment, but guanylin was more potent in the posterior segment. 8-bromo cGMP mimicked the effect of guanylins. The intestinal response to guanylin was smaller in FW eels. The mucosal presence of NPPB utilized as a CFTR blocker, but not of other inhibitors of the channels/transporters localized on the luminal surface in SW fish intestine, inhibited the guanylin-induced decrease in Isc. In eels, therefore, the guanylin family may be involved in osmoregulation by the intestine by binding to the receptors and activating CFTR-like channels on the mucosal side through cGMP production, perhaps resulting in Cl(-) and HCO3(-) secretion into the lumen.

The impact of a brominated flame retardant on smoltification and olfactory function in Atlantic salmon (Salmo salar L.) smolts

Despite a reduction in exploitation of salmon stocks throughout the NE Atlantic, there continues to be a decline in many populations. The factors regulating these populations remain poorly understood, although there is evidence that environmental conditions experienced in freshwater can effect survival in the marine environment. The aim of this study was to investigate the impact of a brominated flame retardant (hexabromocyclododecane, HBCD) on the parr-smolt transformation in juvenile salmon. It is during this pre-adaptive period to marine life that olfactory imprinting to the natal river is considered to occur. Fish were exposed to low levels of HBCD for 30 days over the peak smoltification period in freshwater, and then transferred to clean seawater for 20 days. Fish were sampled weekly to assess changes in some of the physiological parameters associated with smoltification, and olfactory response to conspecific smolt urine was measured using an electro-olfactogram (EOG). Exposure to HBCD did not affect seawater adaptability, although there was some disruption of plasma thyroid hormone levels, as well as a reduction in olfactory function to conspecific smolt urine. The results are discussed in relation to the marine survival and successful homing of adult salmon.

Effects of somatostatin on the growth hormone-insulin-like growth factor axis and seawater adaptation of rainbow trout (Oncorhynchus mykiss)

Growth hormone (GH) has been shown to contribute to the seawater (SW) adaptability of euryhaline fish both directly and indirectly through insulin-like growth factor-1 (IGF-1). This study examined the role of somatostatin-14 (SS-14), a potent inhibitor of GH, on the GH–IGF-1 axis and seawater adaptation. Juvenile rainbow trout (Oncorhynchus mykiss) were injected intraperitoneally with SS-14 or saline and transferred to 20 ppt seawater. A slight elevation in plasma chloride levels was accompanied by significantly reduced gill Na+, K+-ATPase activity in SS-14-treated fish compared to control fish 12 h after SW transfer. Seawater increased hepatic mRNA levels of GH receptor 1 (GHR 1; 239%), GHR 2 (48%), and IGF-1 (103%) in control fish 12 h after transfer. Levels of GHR 1 (155%), GHR 2 (121%), IGF-1 (200%), IGF-1 receptor A (IGFR1A; 62%), and IGFR1B (157%) increased in the gills of control fish 12 h after transfer. SS-14 abolished or attenuated SW-induced changes in the expression of GHR, IGF-1, and IGFR mRNAs in liver and gill. These results indicate that SS-14 reduces seawater adaptability by inhibiting the GH–IGF-1 axis.

Larval Exposure to 4-Nonylphenol and 17β-Estradiol Affects Physiological and Behavioral Development of Seawater Adaptation in Atlantic Salmon Smolts

Population declines of anadromous salmonids are attributed to anthropogenic disturbances including dams, commercial and recreational fisheries, and pollutants, such as estrogenic compounds. Nonylphenol (NP), a xenoestrogen, is widespread in the aquatic environment due to its use in agricultural, industrial, and household products. We exposed Atlantic salmon yolk-sac larvae to waterborne 10 or 100 microg L(-1) NP (NP-L or NP-H, respectively), 2 microg L(-1) 17beta-estradiol (E2), or vehicle, for 21 days to investigate their effects on smolt physiology and behavior 1 year later. NP-H caused approximately 50% mortality during exposure, 30 days after exposure, and 60 days after exposure. Mortality rates of NP-L and E2 fish were not affected until 60 days after treatment, when they were 4-fold greater than those of controls. Treatment with NP-L or E2 as yolk-sac larvae decreased gill sodium-potassium-activated adenosine triphosphatase (Na+,K(+)-ATPase) activity and seawater (SW) tolerance during smolt development, 1 year after exposure. Exposure to NP-L and E2 resulted in a latency to enter SW and reduced preference for SW approximately 2- and 5-fold, respectively. NP-L-exposed fish had 20% lower plasma insulin-like growth factor I (IGF-I) levels and 35% lower plasma triiodothyronine (T3). Plasma growth hormone and thyroxine (T4) were unaffected. Exposure to E2 did not affect plasma levels of IGF-I, GH, T3, or T4. Both treatment groups exhibited increased plasma cortisol and decreased osmoregulatory capacity in response to a handling stressor. These results suggest that early exposure to environmentally relevant concentrations of NP, and other estrogenic compounds, can cause direct and delayed mortalities and that this exposure can have long-term, "organizational" effects on life-history events in salmonids.

New osmoregulatory genes identified in the European eel (Anguilla anguilla) by a microarray study

The life cycle of the European eel includes two migratory periods, the second being the migration of indigenous freshwater (FW) eels back to the Sargasso Sea for reproduction. Various anatomical and physiological adaptations are induced which allow transition to sea water (SW). The aim of this study was to identify and characterise changes in gene expression within the major osmoregulatory tissues which accompany the physiological adaptations required for life in the SW environment. Various tissues were sampled from fish at selected intervals over a 5 month period following FW/SW transfer and RNA was isolated. Suppressive Subtractive Hybridization (SSH) was used for enrichment of differentially expressed genes. Microarrays comprising 6144 cDNAs from brain, gill, intestine and kidney libraries were hybridized with appropriate targets and analysed; 770 differentially expressed clones were selected and sequenced. Microarray results were validated by Northern blotting and qPCR for 30 genes. Some genes, such as the Na K-ATPase, are already known to be involved in osmoregulation but the functional roles of a number of others, such as the myeloid protein (associated with the immune response), need to be elucidated. In addition, the collection of full sequences for various unknown salinity-regulated genes is required to obtain more information about their precise function in SW adaptation.

Tissue-specific modulation of glucocorticoid receptor expression in response to salinity acclimation in rainbow trout

While studies clearly point to a role for cortisol signaling in seawater adaptation, very little is known about salinity impact on glucocorticoid receptor (GR) expression in fish. To this end, we investigated the temporal GR expression in the gill and liver of rainbow trout ( Oncorhynchus mykiss) to salinity exposure. Trout were subjected to gradual salinity increases (11 ppt for 1 d, 17 ppt for 2 d and 23 ppt for 2 d) over a five day period. Gill Na +, K +-ATPase α-subunit mRNA showed a transient elevation with salinity exposure, while gill cystic fibrosis transmembrane conductance regulator mRNA was not significantly affected by salinity. Liver PEPCK transcript levels showed a transient increase at day 1, but not at day 3 or day 5 of salinity exposure, while the activity of this enzyme was significantly depressed at all time points. Liver glycogen content was also significantly reduced by salinity exposure compared to the freshwater group. Gill GR transcript levels were 3-fold greater upon salinity exposure and this level was maintained over the 5 day period, while gill GR protein content remained unchanged except for a significant drop at day 1 of salinity exposure. Liver GR transcript levels showed no significant change with salinity exposure, while GR protein content was transiently elevated at day 3, but not at day 1 or day 5 of salinity exposure. The tissue-specific GR transcript response in the gill leads us to hypothesize a role for osmosensory signal transduction pathway in the regulation of GR expression in fish. Collectively, salinity exposure modulates GR expression and glucocorticoid signaling in rainbow trout.

Ontogenic change of gill chloride cells in leptocephalus and glass eel stages of the Japanese eel, Anguilla japonica

The development of gill chloride cells was examined in premetamorphic larvae (leptocephali) and juveniles (glass eels) of the Japanese eel, Anguilla japonica. Branchial chloride cells were detected by immunocytochemistry using an antiserum specific for Na+,K+-ATPase. The specificity and availability of the antiserum for the detection of Japanese eel chloride cells were confirmed by Western blot analysis. The chloride cells first appeared on the developing gill filaments in a mid larval stage of leptocephalus (32.2 mm). Both immunoreactivity and the number of chloride cells gradually increased as the fish grew to a late stage of leptocephalus over 54 mm. In glass eels just after metamorphosis, gill lamellae developed from the gill filaments, and a rich population of chloride cells was observed in the gill filaments. In glass eels collected at a coastal area, chloride cells were extensively distributed in the gill filaments. The chloride cell size decreased progressively in glass eels transferred from seawater (SW) to freshwater (FW), whereas there was no difference in cell number. In contrast, some Na+,K+-ATPase immunoreaction distinct from typical chloride cells was observed in the gill lamellae throughout FW-transferred fish, but disappeared in control fish maintained in SW for 14 days. These findings indicate that the gill and gill chloride cells developed slowly during the extremely long larval stage, followed by rapid differentiation during a short period of metamorphosis. The excellent euryhalinity of glass eels may be due to the presence of the filament chloride cells and lamellar Na+,K+-ATPase-immunoreaction, presumably being responsible for SW and FW adaptation, respectively.