The role of salinity on genome-wide DNA methylation dynamics in European sea bass gills.

Epigenetic modifications, like DNA methylation, generate phenotypic diversity in fish and ultimately lead to adaptive evolutionary processes. Euryhaline marine species that migrate between salinity-contrasted habitats have received little attention regarding the role of salinity on whole-genome DNA methylation. Investigation of salinity-induced DNA methylation in fish will help to better understand the potential role of this process in salinity acclimation. Using whole-genome bisulfite sequencing, we compared DNA methylation patterns in European sea bass (Dicentrarchus labrax) juveniles in seawater and after freshwater transfer. We targeted the gill as a crucial organ involved in plastic responses to environmental changes. To investigate the function of DNA methylation in gills, we performed RNAseq and assessed DNA methylome-transcriptome correlations. We showed a negative correlation between gene expression levels and DNA methylation levels in promoters, first introns and first exons. A significant effect of salinity on DNA methylation dynamics with an overall DNA hypomethylation in freshwater-transferred fish compared to seawater controls was demonstrated. This suggests a role of DNA methylation changes in salinity acclimation. Genes involved in key functions as metabolism, ion transport and transepithelial permeability (junctional complexes) were differentially methylated and expressed between salinity conditions. Expression of genes involved in mitochondrial metabolism (tricarboxylic acid cycle) was increased, whereas the expression of DNA methyltransferases 3a was repressed. This study reveals novel links between DNA methylation, mainly in promoters and first exons/introns, and gene expression patterns following salinity change.

Hypo-Osmoregulatory Roles of Vasotocinergic and Isotocinergic Systems in the Intestines of Two European Sea Bass Lineages.

European sea bass (Dicentrarchus labrax) are a major aquaculture species that live in habitats with fluctuating salinities that are sometimes higher than in seawater (SW). Atlantic and West-Mediterranean genetic lineages were compared regarding intestinal neuropeptide receptor expression in SW (36%) and following a two-week transfer to hypersalinity (HW, 55%). Phylogenetic analysis revealed seven neuropeptide receptors belonging to the arginine vasotocine (AVTR) family and two isotocin receptors (ITR). Among AVTR paralogs, the highest mRNA levels were recorded for v1a2, with a two- to fourfold upregulation in the European sea bass intestinal sections after transfer of fish to HW. Principal component analysis in posterior intestines showed that v1a2 expression grouped together with the expression and activity of main ion transporters and channels involved in solute-coupled water uptake, indicating a possible role of this receptor in triggering water absorption. v1a1 expression, however, was decreased or did not change after transfer to hypersaline water. Among ITR paralogs, itr1 was the most expressed paralog in the intestine and opposite expression patterns were observed following salinity transfer, comparing intestinal sections. Overall, different expression profiles were observed between genetic lineages for several analyzed genes which could contribute to different osmotic stress-related responses in D. labrax lineages.

Effects of temperature and salinity on antioxidant responses in livers of temperate (Dicentrarchus labrax) and tropical (Chanos Chanos) marine euryhaline fish.

Temperature and salinity are abiotic factors that affect physiological responses in aquaculture species. The European sea bass (Dicentrarchus labrax) is a temperate species that is generally farmed at 18 °C in seawater (SW). In the wild, its incursions in shallow habitats such as lagoons may result in hyperthermal damage despite its high thermal tolerance. Meanwhile, the milkfish (Chanos chanos), a tropical species, is generally reared at 28 °C, and in winter, high mortality usually occurs under hypothermal stress such as cold snaps. This study compared changes in hepatic antioxidant enzymes (superoxide dismutase, SOD; and catalase, CAT) in these two important marine euryhaline aquaculture species in Europe and Southeast Asia, respectively, under temperature challenge combined with hypo-osmotic (fresh water, FW) stress. After a four-week hyper- or hypo-thermal treatment, hepatic SOD activity was upregulated in both species reared in SW and FW, indicating enhanced oxidative stress in European sea bass and milkfish. The expression profiles of sod isoforms suggested that in milkfish, the increase in reactive oxygen species (ROS) was mainly at the cytosol level, leading to increased sod1 expression. In European sea bass, however, no obvious difference was found between the expression of sod isoforms at different temperatures. A lower expression of sod2 was observed in FW compared to SW in the latter species. Moreover, no significant change was observed in the mRNA expression and activity of CAT in the livers of these two species under the different temperature treatments, with the exception of the lower CAT activity in milkfish challenged with SW at 18 °C. Taken together, our results indicated that the antioxidant responses were not changed under long-term hypoosmotic challenge but were enhanced during the four-week temperature treatments in livers of both the temperate and tropical euryhaline species.

Effect of salinity and temperature on the expression of genes involved in branchial ion transport processes in European sea bass.

The responses of European sea bass to temperature increase and salinity decrease were investigated measuring mRNA expression levels of main genes involved in ion transport. Juvenile fish were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred for two weeks to either fresh water (FW) or SW at the respective temperature. Unlike temperate conditions, there is no change in Na+/K+-ATPase α1a (nka α1a) and Na+/H+ exchanger 3 (nhe3) mRNA expression following FW transfer in warm conditions. This is linked to the high expression of these genes in warm SW compared to temperate SW. Na+/Cl--cotransporter (ncc2a) expression however is increased following FW transfer in temperate and warm conditions. Main transporters involved in ion excretion (Na+/K+/2Cl--1 cotransporter, nkcc1 and cystic fibrosis transmembrane conductance regulator, cftr) as well as nitrogen excretion (Rh-glycoproteins, rhcg1 and rhbg) and acid-base regulation (V-H+-ATPase, vha-a and b) are highly expressed in SW warm conditions vs FW warm. Overall, our results suggest a higher activation of ion transport processes in warm conditions and more strikingly in SW. This is linked to a strong interplay between diverse ion transporters in order to coordinate physiological responses at the gill level.

Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes.

European sea bass Dicentrarchus labrax undertake seasonal migrations to estuaries and lagoons that are characterized by fluctuations in environmental conditions. Their ability to cope with these unstable habitats is undeniable, but it is still not clear how and to what extent salinity acclimation mechanisms are affected at temperatures higher than in the sea. In this study, juvenile sea bass were pre-acclimated to seawater (SW) at 18°C (temperate) or 24°C (warm) for 2weeks and then transferred to fresh water (FW) or SW at the respective temperature. Transfer to FW for two weeks resulted in decreased blood osmolalities and plasma Cl- at both temperatures. In FW warm conditions, plasma Na+ was ~15% lower and Cl- was ~32% higher than in the temperate-water group. Branchial Na+/K+-ATPase (NKA) activity measured at the acclimation temperature (Vapparent) did not change according to the conditions. Branchial Na+/K+-ATPase activity measured at 37°C (Vmax) was lower in warm conditions and increased in FW compared to SW conditions whatever the considered temperature. Mitochondrion-rich cell (MRC) density increased in FW, notably due to the appearance of lamellar MRCs, but this increase was less pronounced in warm conditions where MRC's size was lower. In SW warm conditions, pavement cell apical microridges are less developed than in other conditions. Overall gill morphometrical parameters (filament thickness, lamellar length and width) differ between fish that have been pre-acclimated to different temperatures. This study shows that a thermal change affects gill plasticity affecting whole-organism ion balance two weeks after salinity transfer.

Immunolocalization and expression of Na(+)/K(+) -ATPase in embryos, early larval stages and adults of the freshwater shrimp Palaemonetes argentinus (Decapoda, Caridea, Palaemonidae).

The euryhaline shrimp Palaemonetes argentinus exemplifies an evolutionary transition from brackish to freshwater habitats that requires adequate osmoregulatory capacities. Hyperosmoregulation is functional at hatching and it likely begins during the embryonic phase allowing this species to develop entirely in fresh water. Here, we investigated the Na(+)/K(+)-ATPase α-subunit gene (nka-α) expression using quantitative real-time PCR and localized Na(+)/K(+)-ATPase (NKA) in ion-transporting epithelia through immunofluorescence microscopy. We reared shrimps from spawning to juvenile stages at two salinities (1, 15 ‰) and maintained adults for 3 weeks at three salinity treatments (1, 15, 25 ‰). nka-α gene expression was measured in: (1) embryos at an early (SI), intermediate (SII) and late (SIII) stage of embryonic development; (2) newly hatched larvae (Zoea I, ZI); and (3) isolated gill tissue of adults. The nka-α expression was low in SI and SII embryos and reached maximum levels prior to hatching (SIII), which were similar to expression levels detected in the ZI. The nka-α expression in SIII and ZI was highest at 15 ‰, whereas salinity did not affect expression in earlier embryos. In SIII, in ZI and in a later zoeal stage ZIV, NKA was localized in epithelial cells of pleurae, in the inner-side epithelium of branchiostegite and in the antennal glands. Gills appeared in the ZIV but NKA immunolabeling of the cells of the gill shaft occurred in a subsequent developmental larval stage, the decapodid. Extrabranchial organs constitute the main site of osmoregulation in early ontogenetic stages of this freshwater shrimp.

Osmoregulation in larvae and juveniles of two recently separated Macrobrachium species: Expression patterns of ion transporter genes.

In this comparative study, osmoregulatory mechanisms were analyzed in two closely related species of palaemonid shrimp from Brazil, Macrobrachium pantanalense and Macrobrachium amazonicum. A previous investigation showed that all postembryonic stages of M. pantanalense from inland waters of the Pantanal are able to hyper-osmoregulate in fresh water, while this species was not able to hypo-osmoregulate at high salinities. In M. amazonicum originating from the Amazon estuary, in contrast, all stages are able to hypo-osmoregulate, but only first-stage larvae, late juveniles and adults are able to hyper-osmoregulate in fresh water. The underlying molecular mechanisms of these physiological differences have not been known. We therefore investigated the expression patterns of three ion transporters (NKA α-subunit, VHA B-subunit and NHE3) following differential salinity acclimation in different ontogenetic stages (stage-V larvae, juveniles) of both species. Larval NKAα expression was at both salinities significantly higher in M. pantanalense than in M. amazonicum, whereas no difference was noted in juveniles. VHA was also more expressed in larvae of M. pantanalense than in those of M. amazonicum. When NHE3 expression is compared between the larvae of the two species, further salinity-related differences were observed, with generally higher expression in the inland species. Overall, a high expression of ion pumps in M. pantanalense suggests an evolutionary key role of these transporters in freshwater invasion.

Molecular characterization and expression of Na+/K+-ATPase α1 isoforms in the European sea bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer.

The Na+/K+-ATPase (NKA) is considered as the main pump involved in active ion transport. In the European sea bass, Dicentrarchus labrax, we found two genes encoding for the alpha 1 subunit isoforms (NKA α1a and NKA α1b). NKA α1a and NKA α1b isoform amino acid (aa) sequences were compared through phylogeny and regarding key functional motifs between salmonids and other acanthomorph species. Analysis of aa sequences of both isoforms revealed a high degree of conservation across teleosts. The expression pattern of both nka α1a and nka α1b was measured in the gill, kidney and posterior intestine of fish in seawater (SW) and transferred to fresh water (FW) at different exposure times. Nka α1a was more expressed than nka α1b whatever the condition and the tissue analyzed. After long-term salinity acclimation (2.5 years) either in FW or SW, transcript levels of nka α1a were higher in the kidney followed by the posterior intestine and the gill. Compared to SW conditions, expression of nka α1a in FW was significantly increased or decreased, respectively, in gill and posterior intestine. In contrast, branchial nka α1b was significantly decreased in FW-acclimated fish. Short-term FW acclimation seems to rapidly increase nka α1a transcript levels in the kidney unlike in gill tissues where different gene expression levels are detected only after long-term acclimation.

Salinity effects on osmoregulation and gill morphology in juvenile Persian sturgeon (Acipenser persicus).

The effect of abrupt and 5-day gradual salinity transfers from freshwater (FW) to 11 ‰ Caspian Sea brackish water (BW) was investigated in juvenile Persian sturgeon Acipenser persicus with three different weight groups: 1-2 g (1.62 ± 0.27 g), 2-3 g (2.55 ± 0.41 g) and 3-5 g (4.28 ± 0.76 g). Mortality rates, blood osmotic pressure, gill morphology and branchial Na+, K+-ATPase (NKA) activity were measured 4 and 10 days after abrupt transfer and 9 and 15 days after the initial gradual transfer (i.e. 4 and 10 days after reaching Caspian Sea salinity). Fish under 3 g could not survive increased salinity, and the blood osmotic pressure of the remaining surviving fish increased and remained elevated. However, heavier fish were able to survive and successfully acclimate, even to rapid salinity change with osmotic pressure reduced to Caspian Sea osmolality levels. At the gill level, the developmental increase in chloride cell volume and a higher NKA content most probably allow juveniles weighing more than 2 g to sharply increase NKA activity if the fish are transferred to BW. The rapid chloride cell proliferation occurring with increased salinity should strengthen this acclimation response. Therefore, a drastic physiological change occurs when fish weigh more than 2 g that allows migration to higher salinities. The triggering signal on chloride cells must be further investigated in order to optimize this functional step.

Differential distribution of V-type H(+)-ATPase and Na (+)/K (+)-ATPase in the branchial chamber of the palaemonid shrimp Macrobrachium amazonicum.

V-H(+)-ATPase and Na(+)/K(+)-ATPase were localized in the gills and branchiostegites of M. amazonicum and the effects of salinity on the branchial chamber ultrastructure and on the localization of transporters were investigated. Gills present septal and pillar cells. In freshwater (FW), the apical surface of pillar cells is amplified by extensive evaginations associated with mitochondria. V-H(+)-ATPase immunofluorescence was localized in the membranes of the apical evaginations and in clustered subapical areas of pillar cells, suggesting labeling of intracellular vesicle membranes. Na(+)/K(+)-ATPase labeling was restricted to the septal cells. No difference in immunostaining was recorded for both proteins according to salinity (FW vs. 25 PSU). In the branchiostegite, both V-H(+)-ATPase and Na(+)/K(+)-ATPase immunofluorescence were localized in the same cells of the internal epithelium. Immunogold revealed that V-H(+)-ATPase was localized in apical evaginations and in electron-dense areas throughout the inner epithelium, while Na(+)/K(+)-ATPase occurred densely along the basal infoldings of the cytoplasmic membrane. Our results suggest that morphologically different cell types within the gill lamellae may also be functionally specialized. We propose that, in FW, pillar cells expressing V-H(+)-ATPase absorb ions (Cl(-), Na(+)) that are transported either directly to the hemolymph space or through a junctional complex to the septal cells, which may be responsible for active Na(+) delivery to the hemolymph through Na(+)/K(+)-ATPase. This suggests a functional link between septal and pillar cells in osmoregulation. When shrimps are transferred to FW, gill and branchiostegite epithelia undergo ultrastructural changes, most probably resulting from their involvement in osmoregulatory processes.

Coping with salinity extremes: Gill transcriptome profiling in the black-chinned tilapia (Sarotherodon melanotheron).

Steeper and sometimes extreme salinity gradients increasingly affect aquatic organisms because of climate change. Hypersalinity habitats demand powerful physiological adaptive strategies. Few teleost species have the capacity to spend their whole life cycle in salinities way over seawater levels. Focusing on the multifunctional gill, we unraveled the tilapia S. melanotheron key strategies to cope with different environmental conditions, ranging from freshwater up to hypersaline habitats. De novo transcriptome assembly based on RNAseq allowed for the analysis of 40,967 annotated transcripts among samples collected in three wild populations at 0, 40 and 80 ‰. A trend analysis of the expression patterns revealed responses across the salinity gradient with different gene pathways involved. Genes linked to ion transport, pH regulation and cell surface receptor signaling were mainly upregulated in the high salinity habitat. We identified tight junction proteins that were critical in high salinity habitats and that were different from the well-known tightening junctional proteins identified and expressed in fresh water. Expression profiles also suggest a change in the vascular tone that could be linked to an osmorespiratory compromise not only in fresh water, but also in high salinity environments. A striking downregulation of genes linked to the immune system and to the heat shock response was observed suggesting an energetic trade-off between immunity and acclimation/adaptation in the hypersaline habitat. The high expression of transcripts coding for immune and heat shock response in the freshwater habitat suggests the establishment of powerful mechanisms to protect gills from environmental threats and to maintain protein integrity. Non-directional expression trends were also detected with an upregulation of genes only in the hypersaline habitat (80 ‰) or only in the marine habitat (40 ‰). Unravel physiological strategies in S. melanotheron populations will help to better understand the molecular basis of fish euryhalinity in salinity-contrasted environments.

Adaptation to freshwater in the palaemonid shrimp Macrobrachium amazonicum: comparative ontogeny of osmoregulatory organs.

The ontogeny of osmoregulatory organs was studied in two geographically isolated populations of the palaemonid shrimp Macrobrachium amazonicum, one originating from the Amazon estuary (A) and the other from inland waters of the Pantanal (P) in northeastern and southwestern Brazil, respectively. A previous investigation had shown that the estuarine population is able to hypo-osmoregulate in seawater, whereas the hololimnetic inland population has lost this physiological function. In the present study, the structural development of the branchial chamber and excretory glands and the presence of Na(+)/K(+)-ATPase (NKA) were compared between populations and between larval and juvenile stages after exposure to two salinities representing hypo- and hypertonic environments. In the newly hatched zoea I stage of both populations, gills were absent and NKA was localized along the inner epithelium of the branchiostegite. In intermediate (zoea V) and late larval stages (decapodids), significant differences between the two populations were observed in gill development and NKA expression. In juveniles, NKA was detected in the gills and branchiostegite, with no differences between populations. At all developmental stages and in both populations, NKA was present in the antennal glands upon hatching. The strong hypo-osmoregulatory capacity of the early developmental stages in population A could be linked to ion transport along the inner side of the branchiostegite; this seemed to be absent or weak in population P. The presence of fully functional gills expressing NKA appears to be essential for efficient hyper-osmoregulation in late developmental stages during successful freshwater adaptation and colonization.

Intestinal osmoregulatory mechanisms differ in Mediterranean and Atlantic European sea bass: A focus on hypersalinity.

European sea bass (Dicentrarchus labrax) migrate towards habitats where salinity can reach levels over 60‰, notably in Mediterranean lagoons. D. labrax are genetically subdivided in Atlantic and Mediterranean lineages and have evolved in slightly different salinities. We compared Atlantic and West-Mediterranean populations regarding their capacity to tolerate hypersalinity with a focus on the involvement of the intestine in solute-driven water reabsorption. Fish were analyzed following a two-week transfer from seawater (SW, 36‰) to either SW or hypersaline water (HW, 55‰). Differences among lineages were observed in posterior intestines of fish maintained in SW regarding NKA activities and mRNA expressions of nkaα1a, aqp8b, aqp1a and aqp1b with systematic higher levels in Mediterranean sea bass. High salinity transfer triggered similar responses in both lineages but at different magnitudes which may indicate slight different physiological strategies between lineages. High salinity transfer did not significantly affect the phenotypic traits measured in the anterior intestine. In the posterior intestine however, the size of enterocytes and NKA activity were higher in HW compared to SW. In this tissue, nka-α1a, nkcc2, aqp8ab and aqp8aa mRNA levels were higher in HW compared to SW as well as relative protein expression of AQP8ab. For aqp1a, 1b, 8aa and 8b, an opposite trend was observed. The sub-apical localization of AQP8ab in enterocytes suggests its role in transepithelial water reabsorption. Strong apical NKCC2/NCC staining indicates an increased Na+ and Cl- reuptake by enterocytes which could contribute to solute-coupled water reuptake in cells where AQP8ab is expressed.

Mechanisms of Na+ uptake from freshwater habitats in animals.

Life in fresh water is osmotically and energetically challenging for living organisms, requiring increases in ion uptake from dilute environments. However, mechanisms of ion uptake from freshwater environments are still poorly understood and controversial, especially in arthropods, for which several hypothetical models have been proposed based on incomplete data. One compelling model involves the proton pump V-type H+ ATPase (VHA), which energizes the apical membrane, enabling the uptake of Na+ (and other cations) via an unknown Na+ transporter (referred to as the "Wieczorek Exchanger" in insects). What evidence exists for this model of ion uptake and what is this mystery exchanger or channel that cooperates with VHA? We present results from studies that explore this question in crustaceans, insects, and teleost fish. We argue that the Na+/H+ antiporter (NHA) is a likely candidate for the Wieczorek Exchanger in many crustaceans and insects; although, there is no evidence that this is the case for fish. NHA was discovered relatively recently in animals and its functions have not been well characterized. Teleost fish exhibit redundancy of Na+ uptake pathways at the gill level, performed by different ion transporter paralogs in diverse cell types, apparently enabling tolerance of low environmental salinity and various pH levels. We argue that much more research is needed on overall mechanisms of ion uptake from freshwater habitats, especially on NHA and other potential Wieczorek Exchangers. Such insights gained would contribute greatly to our general understanding of ionic regulation in diverse species across habitats.

Transient receptor potential vanilloid 4 in the European sea bass Dicentrarchus labrax: a candidate protein for osmosensing.

The Transient Receptor Potential Vanilloid 4 (TRPV4) protein is a member of the TRP ion channels superfamily that has been proposed as a potential fish osmosensor in previous studies. TRPV4 has been widely studied in mammals, particularly for its involvement in sensing the hypotonicity. The European sea bass, Dicentrarchus labrax, is a euryhaline teleost that is exposed to salinity changes due to its migrations between the sea and estuaries/lagoons. TRPV4 expression and localization in D. labrax was studied in seawater (SW)-adapted fish and in fish exposed to freshwater (FW) over different time-courses from 10 min to 30 days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24 h to 30 d. In contrast, in the kidney, the TRPV4 expression decreased from 10 min to 7d of exposure to FW and then it increased at 30 d. In the brain, its expression was relatively low in SW compared to other organs and a significant decrease occurred in FW. The TRPV4 protein was localized in the basement membranes in branchial lamellae, the cartilage of gills, the posterior pituitary gland and in the collecting ducts. Possible roles of TRPV4 are discussed.

Differential expression of two ATPases revealed by lipid raft isolation from gills of euryhaline teleosts with different salinity preferences.

In euryhaline teleosts, Na+, K+-ATPase (NKA) and V-type H + -ATPase A (VHA A) are important ion-transporters located in cell membrane. Lipid rafts (LR) are plasma membrane microdomains enriched in cholesterol, sphingolipids, and proteins (e.g., flotillin). Flotillin is a LR-associated protein, commonly used as the LR marker. Previous mammalian studies showed that LR may play a crucial role in ion exchanges. Meanwhile, studies on mammals and rainbow trout showed that NKA were found to be present mainly in LR. However, little is known about LR in fish. Therefore, the present study aimed to investigate the involvement of branchial LR in osmoregulation of tilapia and milkfish, two euryhaline teleosts with different salinity preferences, by (i) extracting LR from the gills of euryhaline teleosts; (ii) detecting the abundance of LR marker protein (flotillin-2) and ion-transporters (NKA and VHA A) in branchial LR and non-LR of fresh water- and seawater-acclimated milkfish and tilapia. The results indicated that the protein abundance of LR marker, flotillin-2, changed with environmental salinities in branchial LR of tilapia. In addition, flotillin-2 and NKA were only found in LR in both tilapia and milkfish gills, while VHA A were mainly present in non-LR. Relative protein abundance of NKA was found to be significantly higher in gills of freshwater milkfish and seawater tilapia, while VHA A was significantly higher in gills of freshwater tilapia and milkfish. This study illustrated differential distribution and salinity-dependent expression of NKA and VHA A in cell membrane of gill tissues of euryhaline teleosts with different salinity preferences.

Osmoregulatory performance and immunolocalization of Na+/K+-ATPase in the branchiopod Artemia salina from the Sebkha of Sidi El Hani (Tunisia).

Artemia salina is an extremophile species that tolerates a wide range of salinity, especially hypertonic media considered lethal for the majority of other aquatic species. In this study, A. salina cysts were hatched in the laboratory and nauplii were acclimated at three different salinities (60, 139 and 212 ppt). Once in the adult phase, their hemolymph osmolality was measured. The animals were strong hypo-osmoregulators in the entire range of tested salinities, with up to 10 fold lower hemolymph osmolalities than their surrounding environment. Immunostaining of Na+/K+-ATPase was done on sections and on whole body mounts of adults in order to localize the ionocytes in different organs. An intense Na+/K+-ATPase immunostaining throughout the cells was observed in the epithelium of the ten pairs of metepipodites. A positive immunoreactivity for Na+/K+-ATPase was also detected in the maxillary glands, in the epithelium of the efferent tubule and of the excretory canal, as well as in the anterior digestive tract. This study confirms the strong hypo-osmotic capacity of this species and affords an overview of the different organs involved in osmoregulation in A. salina adults.

Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass.

Acclimation to low salinities is a vital physiological challenge for euryhaline fish as the European sea bass Dicentrarchus labrax. This species undertakes seasonal migrations towards lagoons and estuaries where a wide range of salinity variations occur along the year. We have previously reported intraspecific differences in freshwater tolerance, with an average 30% mortality rate. In this study, we bring new evidence of mechanisms underlying freshwater tolerance in sea bass at gill and kidney levels. In fresh water (FW), intraspecific differences in mRNA expression levels of several ion transporters and prolactin receptors were measured. We showed that the branchial Cl-/HCO3- anion transporter (slc26a6c) was over-expressed in freshwater intolerant fish, probably as a compensatory response to low blood chloride levels and potential metabolic alkalosis. Moreover, prolactin receptor a (prlra) and Na+/Cl- cotransporter (ncc1) but not ncc-2a expression seemed to be slightly increased and highly variable between individuals in freshwater intolerant fish. In the posterior kidney, freshwater intolerant fish exhibited differential expression levels of slc26 anion transporters and Na+/K+/2Cl- cotransporter 1b (nkcc1b). Lower expression levels of prolactin receptors (prlra, prlrb) were measured in posterior kidney which probably contributes to the failure in ion reuptake at the kidney level. Freshwater intolerance seems to be a consequence of renal failure of ion reabsorption, which is not sufficiently compensated at the branchial level.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of Dicentrarchus labrax during ontogeny.

The expression and localization of the cystic fibrosis transmembrane conductance regulator (CFTR) were determined in four osmoregulatory tissues during the ontogeny of the sea-bass Dicentrarchus labrax acclimated to fresh water and sea water. At hatch in sea water, immunolocalization showed an apical CFTR in the digestive tract and integumental ionocytes. During the ontogeny, although CFTR was consistently detected in the digestive tract, it shifted from the integument to the gills. In fresh water, CFTR was not present in the integument and the gills, suggesting the absence of chloride secretion. In the kidney, the CFTR expression was brief from D4 to D35, prior to the larva-juvenile transition. CFTR was apical in the renal tubules, suggesting a chloride secretion at both salinities, and it was basolateral only in sea water in the collecting ducts, suggesting chloride absorption. In the posterior intestine, CFTR was located differently from D4 depending on salinity. In sea water, the basolateral CFTR may facilitate ionic absorption, perhaps in relation to water uptake. In fresh water, CFTR was apical in the gut, suggesting chloride secretion. Increased osmoregulatory ability was acquired just before metamorphosis, which is followed by the sea-lagoon migration.

Influence of salinity on the localization and expression of the CFTR chloride channel in the ionocytes of juvenile Dicentrarchus labrax exposed to seawater and freshwater.

The European sea-bass, Dicentrarchus labrax is a euryhaline teleost whose high osmoregulatory abilities allow sea-lagoon migrations. In order to investigate the mechanism underlying the acclimation of juvenile fish to salinity, CFTR was studied in long-term (6 months) freshwater (FW)- and seawater (SW)-exposed fish, and in short-term (from day 0 to day 30) FW-exposed fish. Cellular and molecular approaches were combined to determine the functions of CFTR in the gills, posterior intestine and kidney. In the kidney, the expression of CFTR transcripts and protein is low. After a direct transfer from SW to FW, the CFTR mRNA is down-regulated in the gills within 1 day, followed by a protein decrease over 7 days. In the posterior intestine, first there is a protein decrease within one day and secondly at the mRNA level in 14 days. While in the gills the regulation is transcriptional, in the posterior intestine, there is first a post-transcriptional regulation followed by a transcriptional regulation after 14 days in FW. Over a long-term exposure, there is a transcriptional regulation in both organs. Coupled to other ion transports, CFTR contributes to ion regulation and thus to the adaptation of the European sea-bass to sea-lagoon transitions.