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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Abstract Ion uptake mechanisms are diverse in fish species, certainly linked to duplication events that have led to the presence of a multitude of paralogous genes. In fish, Na+ uptake involves several ion transporters expressed in different ionocyte subtypes. In the European sea bass Dicentrarchus labrax , several key transporters potentially involved in Na+ uptake have been investigated in seawater (SW) and following a 2 weeks freshwater (FW) acclimation. Using gel electrophoresis, we have shown that the Na+/H+-exchanger 3 (nhe3, slc9a3) is expressed in gills and kidney at both salinities. Quantitative realtime PCR analysis showed a significantly higher nhe3 expression in fresh water (FW) compared to SW. Its apical localization in a subset of gill ionocytes in freshwater-acclimated fish supports the role of NHE3 in Na+ uptake. Interestingly, NHE3-immunopositive cells also express basolateral Na+/K+/2Cl− cotransporter 1 (NKCC1) and are mainly localized in gill lamella. Among the three nhe2 (slc9a2) paralogs, only nhe2c shows differential branchial expression levels with higher mRNA levels in SW than in FW. The increased branchial expression of the ammonia transporter rhcg1 (Rhesus protein), nhe3 and cytoplasmic carbonic anhydrase (cac) in FW could indicate the presence of a functional coupling between ion transporters to form a Na+/NH4+ exchange complex. Acid-sensing ion channel 4 (asic4) seems not to be expressed in sea bass gills. Na+/Cl- cotransporter (ncc2a or ncc-like) is about three times more expressed in FW compared to SW suggesting coupled Na+ and Cl− uptake in a subset of gill ionocytes. Besides the main pump Na+/K+-ATPase, branchial NCC2a and NHE3 may be key players in ion uptake in sea bass following a long-term freshwater challenge. Highlights • Transcript levels of ion transporters were investigated in Dicentrarchus labrax gills. • Key branchial transporters involved in freshwater acclimation were identified. • A focus was made on the expression of Na+/H+-exchanger (NHE) paralogs. • The Na+/Cl- - cotransporter (NCC2a) was identified for the first time in sea bass. • Different ionocytes subtypes were compared in filaments vs lamella [ABSTRACT FROM AUTHOR]

Abstract: 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 10min to 30days. TRPV4 mRNA expression was detected in gills, kidney and brain. In gills, the expression increased significantly in FW from 24h to 30d. In contrast, in the kidney, the TRPV4 expression decreased from 10min to 7d of exposure to FW and then it increased at 30d. 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. [Copyright &y& Elsevier]

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 2 weeks 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 (V apparent ) did not change according to the conditions. Branchial Na + /K + -ATPase activity measured at 37 °C (V max ) 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. [ABSTRACT FROM AUTHOR]

Abstract: Coastal marine ecosystems are submitted to variations of several abiotic and biotic parameters, some of them related to global change. Among them the ultraviolet-B (UV-B) radiation (UVBR: 280–320nm) may strongly impact planktonic fish larvae. The consequences of an increase of UVBR on the osmoregulatory function of Dicentrarchus labrax larvae have been investigated in this study. In young larvae of D. labrax, as in other teleosts, osmoregulation depends on tegumentary ion transporting cells, or ionocytes, mainly located on the skin of the trunk and of the yolk sac. As early D. labrax larvae passively drift in the top water column, ionocytes are exposed to solar radiation. The effect of UVBR on larval osmoregulation in seawater was evaluated through nanoosmometric measurements of the blood osmolality after exposure to different UV-B treatments. A loss of osmoregulatory capability occured in larvae after 2 days of low (50μWcm−2: 4h L/20h D) and medium (80μWcm−2: 4h L/20h D) UVBR exposure. Compared to control larvae kept in the darkness, a significant increase in blood osmolality, abnormal behavior and high mortalities were detected in larvae exposed to UVBR from 2 days on. At the cellular level, an important decrease in abundance of tegumentary ionocytes and mucous cells was observed after 2 days of exposure to UVBR. In the ionocytes, two major osmoeffectors were immunolocalized, the Na+/K+-ATPase and the Na+/K+/2Cl− cotransporter. Compared to controls, the fluorescent immunostaining was lower in UVBR-exposed larvae. We hypothesize that the impaired osmoregulation in UVBR-exposed larvae originates from the lower number of tegumentary ionocytes and mucous cells. This alteration of the osmoregulatory function could negatively impact the survival of young larvae at the surface water exposed to UVBR. [Copyright &y& Elsevier]

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. • The synergistic effect of salinity-temperature on antioxidant mechanisms was noted. • Oxidative stress-related responses occurred under high temperature in temperate fish. • The same responses occurred under low temperature in the tropical milkfish. [ABSTRACT FROM AUTHOR]

European sea bass (Dicentrarchus labrax), a major aquaculture species, is distributed along the coasts of the North-Eastern Atlantic Ocean, Mediterranean and Black Sea. D. labrax enter lagoons and estuaries where salinity fluctuates and sometimes reaches levels over 60‰, notably in Mediterranean lagoons. Keeping in mind that European sea bass are genetically subdivided in an Atlantic and a Mediterranean lineage, we compared fish from Atlantic (A) and West Mediterranean (M) populations regarding their capacity to tolerate hypersalinity with a focus on the kidney, a key organ involved in water reabsorption at high salinity. Fish were analyzed following a two-week transfer from seawater (SW, 36‰) to either seawater (SW, 36‰) or hypersaline water (HW, 55‰). Plasma osmolality was significantly increased in the MHW group compared to the other groups. Plasma sodium levels were significantly increased in hypersaline water compared to seawater in both lineages whereas plasma chloride levels showed an opposite trend. In order to estimate water filtration at the kidney level, the size of renal glomeruli was investigated and showed a decreased glomerulus perimeter and area in hypersaline water compared to seawater. NKA was highly expressed in all kidney tubules notably collecting tubules and ducts. There was an effect of salinity on renal nka α1a mRNA expression with slightly lower transcript levels at 55‰ compared to 36‰. Relative protein amounts and activity of NKA however were significantly higher in fish exposed to hypersalinity regardless of their origin. AQP1a immunolabeling differed between proximal tubules subtypes and only faint AQP1a was detected in subapical parts of cells lining collecting ducts. The transcript levels of renal aqp 1a were lower in the HW group than the SW group whereas the expression of other aqp paralogs (aqp 1b , aqp 8b) did not change according to the analyzed conditions. This study showed an efficient acclimation of sea bass to high salinity by increasing active ion transport at the kidney and by decreasing the size of filtering glomeruli to minimize water loss through urine. Despite Mediterranean D. labrax are supposed to more often encounter high salinities in their habitat, their high blood osmolality in hypersaline water indicates that their overall response to hypersalinity seems not improved compared to the Atlantic lineage. However, at the kidney level, the traits analyzed differ slightly between genetic lineages, potentially as a response to high blood osmolalities in MHW. • Responses of two European sea bass genetic lineages to hypersalinity are compared. • Hydromineral balance is differently regulated in both lineages at high salinity. • Aquaporin 1a is expressed in proximal tubule I on basolateral membranes. • Renal Aquaporin 1a expression is lower in hypersaline- versus seawater. • Sea bass respond to hypersalinity by decreasing the size of renal glomeruli. [ABSTRACT FROM AUTHOR]

• Freshwater intolerance is not due to an altered ion uptake at the gills. • Cl− uptake-related genes are less expressed in the kidney of freshwater (FW) intolerant fish vs FW tolerant fish. • Branchial and renal prolactin receptors are differentially expressed between freshwater tolerance phenotypes. 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−/HCO 3 − 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. [ABSTRACT FROM AUTHOR]

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. • The expression of various branchial ion transporters is increased at 24 °C. • The induction of ion transport processes is less striking in fresh water. • In warm seawater, CFTR seems to be a key player in the regulation of Cl− balance. • Transporters regulating H+ and nitrogen excretion are highly expressed at 24 °C. [ABSTRACT FROM AUTHOR]