Eva Blondeau-Bidet

Junya Hiroi

Catherine Lorin-Nebel
email: catherine.lorin@umontpellier.fr

Ion uptake pathways in European sea bass Dicentrarchus labrax

Keywords: Teleost, Osmoregulation, Ion transporters, Na + uptake, NHE3, NCC2

Introduction

The gill is a key organ involved in Na + and Cl -absorption in freshwater-acclimated fish contributing to the maintenance of body fluid ionic homeostasis. As multifunctional organ, the fish gill is also a crucial organ responsible for acid-base balance and nitrogen excretion [START_REF] Dymowska | Structure and function of ionocytes in the freshwater fish gill[END_REF][START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Hwang | Gill ionic transport, acid-base regulation, and nitrogen excretion[END_REF]. Among teleost species, a diversity of ionocyte subtypes have been identified in fresh water (FW) notably in zebrafish Danio rerio, rainbow trout Oncorhynchus mykiss, medaka Oryzias latipes and Mozambique tilapia Oreochromis mossambicus [START_REF] Dymowska | Structure and function of ionocytes in the freshwater fish gill[END_REF][START_REF] Hsu | A new model for fish ion regulation: identification of ionocytes in freshwater-and seawater-acclimated medaka (Oryzias latipes)[END_REF][START_REF] Hwang | Ion uptake and acid secretion in zebrafish (Danio rerio)[END_REF]. Apical Na + uptake mechanisms have been thoroughly discussed in teleosts in the last decade and implicate Na + /H + -exchangers (NHEs), Na + channels (acid-sensing ion channels, ASICs) energized by an electrical gradient generated by apical Vtype H + ATPase (VHA) and/or Na + /Cl -cotransporters (fish-specific NCC2/NCC-like (slc12a10)) coupled to basolateral NKA and chloride channels [START_REF] Hwang | Ion regulation in fish gills: recent progress in the cellular and molecular mechanisms[END_REF][START_REF] Kumai | Mechanisms and regulation of Na + uptake by freshwater fish[END_REF].

Na + /H + exchangers (SLC9A family) are considered as main actors in apical Na + uptake, acid excretion, and can be functionally coupled to ammonia excretion in fish gill ionocytes. In freshwater-type ionocyte models of several species, NHE2 and/or NHE3 have been shown to localize in apical membranes to lead to an acid-trapping mechanism facilitating NH4 + excretion via basolateral RHBG and apical RHCG1. Extracellular NH3 then combines with H + to form NH4 + covering the gill boundary layer, which leads to an increase in pH and favors the functioning of NHE (2/3) to take up Na + in exchange for H + [START_REF] Cooper | Marine, freshwater and aerially acclimated mangrove rivulus (Kryptolebias marmoratus) use different strategies for cutaneous ammonia excretion[END_REF][START_REF] Shih | Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water[END_REF][START_REF] Weihrauch | Ammonia and urea transporters in gills of fish and aquatic crustaceans[END_REF]. In the zebrafish cell model (HR cell), NHE3b, VHA and RHCG1 seem to colocalize in apical membranes and basolateral Na + /K + -ATPase is weakly expressed [START_REF] Shih | Ammonia excretion by the skin of zebrafish (Danio rerio) larvae[END_REF][START_REF] Yan | Gene expression of Na + /H + exchanger in zebrafish H + -ATPase-rich cells during acclimation to low-Na + and acidic environments[END_REF]. NHE (2/3) has also been identified in PNA+ ionocytes of rainbow trout (for a review, see [START_REF] Dymowska | Structure and function of ionocytes in the freshwater fish gill[END_REF]. NHE2a has been shown in cuboidal cells of killifish Fundulus heteroclitus, the cell type responsible for Na + uptake [START_REF] Edwards | Molecular identification of Na + -H + exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus[END_REF][START_REF] Laurent | Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na + but not Cluptake in freshwater[END_REF]. As VHA is expressed in basolateral membranes of killifish cuboidal cells, Na + uptake seems not to be coupled to active proton excretion in this species [START_REF] Katoh | Vacuolar-type proton pump in the basolateral plasma membrane energizes ion uptake in branchial mitochondria-rich cells of killifish Fundulus heteroclitus, adapted to a low ion environment[END_REF]. There is also evidence that NHE2 and NHE3 are expressed in gill ionocytes of teleosts maintained in seawater (SW), playing critical roles in acid secretion [START_REF] Chen | Na + /H + exchanger 3 is expressed in two distinct types of ionocyte, and probably augments ammonia excretion in one of them, in the gills of the climbing perch exposed to seawater[END_REF][START_REF] Claiborne | A mechanism for branchial acid excretion in marine fish: identification of multiple Na + /H + antiporter (NHE) isoforms in gills of two seawater teleosts[END_REF][START_REF] Liu | Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism[END_REF][START_REF] Weihrauch | Ammonia and urea transporters in gills of fish and aquatic crustaceans[END_REF]. In many teleost species, nhe3 mRNA expression is high in FW compared to SW [START_REF] Bollinger | Does Japanese medaka (Oryzias latipes) exhibit a gill Na + /K + -ATPase isoform switch during salinity change ?[END_REF][START_REF] Hiroi | Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish[END_REF][START_REF] Inokuchi | Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range of salinities[END_REF][START_REF] Watanabe | Na + /H + exchanger isoform 3 expressed in apical membrane of gill mitochondrion-rich cells in Mozambique tilapia Oreochromis mossambicus[END_REF] except in the climbing perch Anabas testudineus, where the opposite pattern has been shown with increased nhe3 mRNA and NHE3 protein expression in SW [START_REF] Chen | Na + /H + exchanger 3 is expressed in two distinct types of ionocyte, and probably augments ammonia excretion in one of them, in the gills of the climbing perch exposed to seawater[END_REF]. In this latter species, NKA α1c-immunoreactive ionocytes express NHE3 and RHCG2 in basolateral membranes and lack apical RHCG1, the exact function of these cells remains to be determined. Several paralogs have been identified in teleosts for nhe (1, 2, 3) probably due to putative genome duplication events [START_REF] Glasauer | Whole-genome duplication in teleost fishes and its evolutionary consequences[END_REF][START_REF] Meyer | From 2R to 3R: evidence for a fish-specific genome duplication (FSGD)[END_REF]. Two or three nhe2 paralogous genes have been identified in F. heteroclitus, Takifugu rubripes, Gasterosteus aculeatus, and the longhorn sculpin Myoxocephalus octodecemspinosus but functional differences remain to be analyzed [START_REF] Edwards | Molecular identification of Na + -H + exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus[END_REF]. Contrary to NHE2 and 3, NHE1 has been shown to be expressed in basolateral membranes and is mainly involved in pH homeostasis and cell volume regulation of epithelial and non-epithelial cells [START_REF] Tse | Structure/function studies of the epithelial isoforms of the mammalian Na + /H + exchanger gene family[END_REF]. Its expression increases following exposure to environmental hypercapnia in SW of several species and has been considered as protein involved in H + excretion to compensate for intracellular acidosis [START_REF] Edwards | The effect of environmental hypercapnia and salinity on the expression of NHE-like isoforms in the gills of a euryhaline fish (Fundulus heteroclitus)[END_REF].

Another Na + uptake model involves an apical Na + channel electrochemically coupled to the apical VHA. As epithelial Na + channels (ENaC) present in mammals are not present in teleost genomes, including the European sea bass, another Na + channel might be present in apical membranes of ionocytes to take up Na + in Na + -poor environments. The presence of apical ASIC4 (acid-sensing ion channel 4) has been shown in gill ionocytes of two teleost species, the rainbow trout (in PNA+ ionocytes) [START_REF] Dymowska | Acid-sensing ion channels are involved in epithelial Na + uptake in the rainbow trout Oncorhynchus mykiss[END_REF] and zebrafish (in HR cells) [START_REF] Dymowska | The role of acid-sensing ion channels in epithelial Na + uptake in adult zebrafish (Danio rerio)[END_REF]. Zebrafish HR cells express NHE3b in apical membranes suggesting colocalization of VHA, NHE3b and ASIC4 in this cell type [START_REF] Guh | Insights into molecular and cellular mechanisms of hormonal actions on fish ion regulation derived from the zebrafish model[END_REF]. ASIC4 could thus, beside NHE3b, be part of a secondary Na + uptake mechanism in these cells.

Coupled Na + /Cl -uptake has been suggested to occur through Na + /Cl -cotransporters from the SLC12 family. Na + /Cl -cotransporters are called ncc1 (slc12a3) or ncc2 (or ncc-like) (slc12a10). The latter have been analyzed in different teleost species [START_REF] Breves | Pituitary control of branchial NCC, NKCC and Na + , K + -ATPase alpha-subunit gene expression in Nile tilapia, Oreochromis niloticus[END_REF][START_REF] Hiroi | Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish[END_REF][START_REF] Hsu | A new model for fish ion regulation: identification of ionocytes in freshwater-and seawater-acclimated medaka (Oryzias latipes)[END_REF][START_REF] Inokuchi | Functional classification of gill ionocytes and spatiotemporal changes in their distribution after transfer from seawater to freshwater in Japanese seabass[END_REF][START_REF] Takei | Diverse mechanisms for body fluid regulation in teleost fishes[END_REF][START_REF] Wang | Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)[END_REF]. Among different paralogs belonging to the ncc2 family, zebrafish slc12a10.2 was specifically expressed in the gills and its expression is stimulated in a low-Cl -environment and not in a low-Na + environment [START_REF] Wang | Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)[END_REF]). Hwang's group also showed that slc12a10.1 seems to be predominantly expressed in the kidney and not in the gills of this species. In Mozambique tilapia O. mossambicus however, branchial ncc2 (slc12a10) was highly expressed in FW compared to SW [START_REF] Inokuchi | Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range of salinities[END_REF]. In another tilapia species, Oreochromis niloticus, ncc2 (slc12a10.2) has been shown to be activated by prolactin [START_REF] Breves | Pituitary control of branchial NCC, NKCC and Na + , K + -ATPase alpha-subunit gene expression in Nile tilapia, Oreochromis niloticus[END_REF].

In the European sea bass Dicentrarchus labrax, Na + uptake mechanisms as well as ionocyte subtypes have not been clearly identified in FW. Sea bass are euryhaline species that undertake seasonal migrations to low-salinity habitats like estuaries and lagoons and thus have to be able to rapidly activate efficient ion uptake and/or ion secretion mechanisms according to salinity. Several studies have shown that sea bass are able to tolerate FW for several weeks [START_REF] Bodinier | 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[END_REF][START_REF] Bossus | The ClC-3 chloride channel and osmoregulation in the European Sea Bass, Dicentrarchus labrax[END_REF][START_REF] Lorin-Nebel | The Na + /K + /2 Clcotransporter in the sea-bass Dicentrarchus labrax during ontogeny: Involvement in osmoregulation[END_REF]. In late juveniles maintained in FW for two weeks, plasma Na + , contrary to plasma Cl -, was maintained constant without a significant decrease, suggesting efficient Na + uptake and reabsorption at the gill and kidney levels [START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF]. There is evidence of increased branchial nka α1a (atp1a1a) expression [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea-bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF], the predominant NKA α1 isoform in sea bass gills, contributing to increased NKA activity in FW [START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF][START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax : Gill and urinary system involvement[END_REF]. To our knowledge, no data are available on the presence and induction of diverse ion transporters favoring Na + uptake in sea bass gills in FW. This raises the question if sea bass gills present an ionocyte subtype that would be involved in proton-facilitated ammonia excretion and Na + uptake as shown in other species that experience low-salinity environments during their life cycle.

The objectives of the present study are to i) characterize main Na + /H + -exchangers (NHEs) in sea bass osmoregulatory organs ; ii) identify transporters potentially involved in branchial Na + uptake and iii) identify a new ionocyte subtype in European sea bass gills, the NHE3-type ionocyte.

Material and Methods

Experimental conditions

European sea bass Dicentrarchus labrax were obtained from the Ifremer Station at Palavas-les-Flots (Hérault, France). Two batches of fish were used in these experiments. For quantitative real-time PCR and immunocytochemistry (batch 1), the sampled fish average fork length was 20.77±1.32 cm (mean ± SD) and average weight was 86.87±20.23 g. For identification of paralogous genes in the main osmoregulatory tissues (gills, kidney and posterior intestine) (batch 2), the sampled fish average fork length was 12.32±1.70 cm (mean ± SD) and weight was 21.62±9.92 g. Fish were brought to the Montpellier University and maintained for one week in 3,500 L tanks containing natural seawater (SW) from the Mediterranean sea at 38 ‰ and 18 °C, pH=7.78 under a 12 h light/12 h dark photoperiod. Fish were then transferred to smaller 200 L tanks either containing dechlorinated tap water (FW) or SW and were maintained in this salinity until sampling. Fish were respectively maintained in SW and FW for 2 weeks (medium term, batch 1) or 4 months (long term, batch 2) before sampling. Ionic composition (in mEq.L -1 ) of the FW was Na + (0.12), K + (0.04), Ca 2+ (5.70), Mg 2+ (0.29), Cl -(0.98), NO3 -(0.06) and SO4 2-(0.61) and pH=8.58. Water was aerated and mechanically/biologically filtered (Eheim System, Lens, Pas-de-Calais, France). Temperature, salinity, oxygen and nitrogen levels were checked regularly. 10 % of the water in each tank was changed regularly using a siphon tube. The tank was then immediately refilled with water at the same salinity (either SW or dechlorinated FW) and temperature. Fish were fed with fish granules (Aphymar feed, Mèze, Hérault, France) until 2 days before sampling. At the end of the experiment, fish were anesthetized in a solution of phenoxy-2-ethanol (0.24 ml.L -1 ) (batch 1) or benzocaine (1 ml.L -1 ) (batch 2) prior to any manipulation. The experiments were conducted according to the guidelines of the European Union (directive 86/609) and of the French law (decree 87/848) regulating animal experimentation.

Phylogenetic analysis and protein structure

Phylogenetic analyses of nhe2 (slc9a2) (Fig. 1), ncc (slc12a3) (Fig. 2) and nhe3 (slc9a3) (Fig. 1S) have been performed. Nucleotide sequences of different teleosts and mammalians were obtained at Ensembl or NCBI (Table 1, 2, 1S). Sea bass sequences were obtained from the sea bass genome project [START_REF] Tine | European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation[END_REF]. Multiple nucleotide alignments were performed with MUSCLE V3.8.31 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] and ambiguous regions were removed with Gblocks V0.91b [START_REF] Talavera | Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments[END_REF]. Best model of evolution was selected using Modelgenerator V.85 [START_REF] Keane | Assessment of methods for amino acid matrix selection and their use on empirical data shows that ad hoc assumptions for choice of matrix are not justified[END_REF] following the corrected Akaike Information Criterion (with four discrete gamma categories) and used to construct phylogenetic trees. Phylogenetic trees were inferred using the maximum likelihood method implemented in the PhyML program (v3.1/3.0 aLRT) [START_REF] Guindon | A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood[END_REF] with the General Time Reversible (GTR) model of substitution [START_REF] Tavaré | Some probabilistic and statistical problems in the analysis of DNA sequences[END_REF] with discrete-gamma distributed rate variation and with the transversional model (TVM) [START_REF] Posada | Using MODELTEST and PAUP* to select a model of nucleotide substitution[END_REF] with discrete-gamma distributed rate variation and estimated proportion of invariable sites, combined to the best "Nearest Neighbor Interchange" (NNI). The trees were generated and robustness of the phylogeny assumption was evaluated by the approximate likelihood test (aLRT) SH-like branch support and by bootstrapping procedure from 1000 data set replicates. As outgroup species, we used sequences of chicken (Gallus gallus), rabbit (Oryctolagus cuniculus) and human (Homo sapiens) for phylogenetic analyses on nhe3.

Multiple protein sequence alignment and amino acid identities (in %) were performed with MUSCLE V3.8.31 [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF] implemented in Geneious® 9.1.8 software (Biomatters). The prediction of membrane topology was performed with PolyPhobius [START_REF] Käll | An HMM posterior decoder for sequence feature prediction that includes homology information[END_REF]. The prediction of potential N-glycosylation was done using ScanProsite (http://www.expasy.ch/tools/scanprosite/).

RNA extraction and complementary DNA (cDNA) synthesis

Total RNA was extracted from gills, kidney and posterior intestine using Trizol ® reagent and processed according to the manufacturer's instructions. RNA quantity was assessed by measuring the A260/A280 ratio using the NanoDrop® ND-1000 V3300 Spectrophotometer (Nanodrop Technology Inc., Wilmongton, DE, USA) and RNA integrity was checked using an Agilent Bioanalyzer 2100 equipped with an RNA Nano Chip (Agilent Technologies, CA, USA). RNA Integrity Numbers (RIN) were greater than 7. One microgram of total RNA was treated with DNase I amplification grade (Invitrogen™, Life Technologies) and the first strand of complementary DNA (cDNA) was generated using Random Primers oligonucleotides (Invitrogen™) and the M-MLV reverse transcriptase (Invitrogen™), following the manufacturer's guidelines. PCR products were sequenced by Sanger sequencing in order to validate the identity of the amplified sequences in comparison with the sea bass genome.

Tissue distribution analysis using RT-PCR

Tissue distribution of nhe1 (slc9a1), nhe1β-like (slc9a1β-like), nhe2a (slc9a2.1), nhe2b (slc9a2.2), nhe2c (slc9a2.3) and nhe3 (scl9a3) were examined in gills (first right gill arch), posterior intestine and posterior kidney of sea bass. cDNA of 4 animals per salinity (SW and FW) were pooled in equal amounts. For PCR amplification, 1µL of diluted cDNA (at 1/10 for nhe1 (slc9a1) , nhe1β-like (slc9a1β-like), nhe2a (slc9a2.1), nhe2b (slc9a2.2), nhe2c (slc9a2.3) and at 1/20 for nhe3 (slc9a3) was used as template in a final reaction volume of 25 µL containing 5 µL (1X) of 5X Green GoTaq ® Flexi Buffer, 0.5 µL (0.2 mM) of dNTP mix (10 mM each), 0.13 µL (1.25 units) of GoTaq ® G2 Flexi DNA Polymerase (Promega, Madison, USA), 2 µL (2 mM) of MgCl2 0.5 µL (0.2 µM) of each primer (Table 3) and 15.87 µL of nuclease-free water. Conditions used in the PCR were as follows : 2 min denaturation at 95 °C followed by 35 cycles (95 °C for 30 s, 61 °C for 45 s and 72 °C for 1 min) followed by a final elongation step at 72 °C for 4 min. The resulting PCR products were visualized by electrophoresis on a 2 % agarose gel containing 0.5 μg/mL GelRed ® (Biotium, USA) and viewed under UV light on a Gel Doc 1000 system (Bio-Rad, UK).

Quantitative real-time RT-PCR (qRT-PCR)

Eight fish per salinity treatment were analyzed for gene expression studies. An Echo ® 525 liquid handling system (Labcyte Inc., San Jose, CA, USA) was used to dispense 0.75 μL of LightCycler-FastStart DNA Master SYBR-Green I™ Mix (Roche, Mannheim, Germany), 0.037 μL of each primer (at 0.5 μM), 0.21 μL of ultra-pure water and 0.5 μL of diluted cDNA into a 384-well reaction plate.

Each sample was run in triplicate. The qRT-PCR conditions were as follows: denaturation at 95 °C for 10 min, followed by 45 cycles of amplification (95 °C, 15 s), hybridization (60 °C, 5 s) and elongation (72 °C, 10 s), and a final step at 40 °C for 30 s. A melting curve program was performed to control the amplification specificity. Ultra-pure water was used as a no-template control in the qRT-PCR.

Efficiencies were between 1.9 and 2.1 according to the considered primer pair.

The expression level was normalized using the expression of the elongation factor ef1α and ubiquitin like and ribosomal protein S30 fusion gene (fau). ef1α has been already successfully used in this species [START_REF] Blondeau-Bidet | Molecular characterization and expression of Na + /K + -ATPase α1 isoforms in the European sea-bass Dicentrarchus labrax osmoregulatory tissues following salinity transfer[END_REF] and fau was recommended for sea bass qRT-PCR normalization by [START_REF] Mitter | Evaluation of candidate reference genes for QPCR during ontogenesis and of immunerelevant tissues of European sea bass (Dicentrarchus labrax)[END_REF]. Relative expression levels were calculated using the comparative Ct method (threshold cycle number) by averaging the results normalized against both fau and ef1α [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]).

Ct of both reference genes did not vary according to salinity conditions.

Immunocytochemistry

The first left gill arch of three animals per condition was fixed for 48 h by immersion in Bouin's fixative. After rinsing in 70 % ethanol, samples were dehydrated in a graded ethanol series and embedded in Paraplast (Sigma). Transverse sections (4 µm) were cut on a Leitz Wetzlar microtome, collected on poly-L-lysine-coated glass slides and stored at room temperature until the immunostaining procedure. For co-immunolabelling, sections were dewaxed (LMR and butanol), hydrated through a descending series of ethanol baths (from 100 % to 50 %) and rinsed in phosphatebuffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM phosphate buffer, pH 7.4, Sigma). Slides were then immersed in a sodium citrate buffer (Tri-sodium citrate dihydrate in 0.05 % Tween 20, pH 6) and placed in a microwave oven (at 80 % power 2 times 1 min) to reveal antigenic sites. The slides were then incubated for 10 min in 0.02 % Tween 20 in PBS supplemented with NaCl to 286.5 mM, pH 7.3. After saturation in 5 % bovine serum albumin (BSA) in PBS at room temperature for 20 min, the slides were rinsed twice with PBS. Primary labelling was performed overnight at 4 °C (NHE3) followed by three washes in PBS, then 2h at room temperature (NKA and T4) in a wet chamber. The following primary antibodies diluted in 0.5 % BSA-PBS were used : mouse monoclonal antibody (α5) anti-Na + /K + -ATPase deposited to the DSHB by Fambrough, D.M. (DSHB Hybridoma Product α5) (Hybridoma Bank, University of Iowa) at 10 µg.mL -1 , mouse T4 (SLC12A1/A2) antibody deposited to the DSHB by Lytle, C. / Forbush III, B. (DSHB Hybridoma Product T4) at 7 µg.mL -1 and rabbit polyclonal antibody raised against rainbow trout (O. mykiss) Na + /H + -exchanger 3b (NHE3b) at 20 µg.mL -1 . NHE3 antibody was raised against a cocktail of two peptides from rainbow trout NHE3b.

The sequences of these peptides are 100 % identical to sea-bass NHE3 (SLC9A3). NHE3 antibody was already used successfully in other teleost species [START_REF] Christensen | Branchial ionocyte organization and ion-transport protein expression in juvenile alewives acclimated to freshwater or seawater[END_REF][START_REF] Seo | Expression of ion transporters in gill mitochondrion-rich cells in Japanese eel acclimated to a wide range of environmental salinity[END_REF][START_REF] Wright | Uncommon) mechanisms of branchial ammonia excretion in the common carp (Cyprinus carpio) in response to environmentally induced metabolic acidosis[END_REF]. Negative control slides without the primary antibody were also prepared. After three washes in PBS to remove unbound antibody, the sections were incubated for 1 h with the secondary antibodies at room temperature (anti-mouse Alexa Fluor ® 488 at 10 µg.mL -1 and anti-rabbit Rhodamine at 4 µg.mL -1 ). Following 3 washes, nuclei were counterstained with 4',6-diamidino-2phenylindole (DAPI) during 2 min, then washed 3 times and sections were mounted in an antibleaching mounting medium (Immunohistomount, Santa Cruz Biotechnology). Observations were done with a Leica TCS SP2 confocal microscope of the MRI platform, at Montpellier University.

Statistical analysis

Statistical analyses were performed using Graphpad Prism (version 6, GraphPad Software Incorporated, La Jolla, CA 268, USA). Due to the small sample size (N=8), a non-parametric Mann-Whitney U-test and a Kruskal-Wallis test were used for statistical comparisons. To determine differences between conditions, a post-hoc test was performed. Data are presented as means ± SEM unless noted otherwise, and the level of statistical significance was set at p < 0.05

Results

Identification of sea bass nhe paralogous genes and phylogeny

In the sea bass genome, we found several paralogous genes for Na + /H + -exchangers (slc9a) among them two nhe1 (slc9a1) (annotated slc9a1 and slc9a1β-like), three nhe2 (slc9a2) that we will call in this study (nhe2a (slc9a2.1), nhe2b (slc9a2.2) and nhe2c (slc9a2.3) according to their phylogenetic position (Fig. 1) and only one nhe3 (slc9a3) (Fig. 1S). Sea bass NHE3 is highly homologous to other NHE3 of the longhorn sculpin M. octodecemspinosus, the stickleback G. aculeatus, and the climbing perch (A. testudineus) (81 %, 78 %and 76 % respectively).

Maximum likelihood analysis generated a tree of the three D. labrax nhe2 subtypes against sequences of selected acanthomorph and salmonid species. Analysis of teleost genomes revealed the presence of one, two (nhe2a and b in F. heteroclitus and T. rubripes, nhe2b and nhe2c in M. octodecemspinosus) or three (nhe2a, b and c in G. aculeatus) nhe2 subtypes [START_REF] Edwards | Molecular identification of Na + -H + exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus[END_REF]. D. labrax nhe2a, nhe2b and nhe2c group with other nhe2a, nhe2b and nhe2c sequences (Fig. 1). The human (H. sapiens), chicken (G. gallus) and rabbit (O. cuniculus) branched at the basis of the teleost slc9a2 paralog clade with respect to their phylogenetic position. NHE2b and NHE2c amino acid sequences of D. labrax exhibited a percentage identity of 79 %, whereas NHE2a showed a lower percentage identity with NHE2b and NHE2c (48 % and 46 %, respectively).

Characterization of sea bass ncc1/ncc2 paralogous genes and phylogeny

Three ncc1/ncc2 genes have been identified in the sea bass genome called slc12a3, slc12a3-like and slc12a10.2. Slc12a3 (ncc1, 3081 bp) could not be detected in sea bass gills in this study (not shown).

A sequence of 486 bp was identified for slc12a10.2 (ncc2b), that is phylogenetically very close to slc12a3-like (2802 bp) and other teleost slc12a10 (ncc2) sequences (Fig. 2). We thus propose that sea bass slc12a3-like can be considered as ncc2 compared to our phylogenetic tree conducted on slc12a10/slc12a3 sequences [START_REF] Takei | Diverse mechanisms for body fluid regulation in teleost fishes[END_REF][START_REF] Wang | Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)[END_REF]. The tree shows two clades, the slc12a3/ncc1 clade and the fish-specific slc12a10/ncc2 clade. All teleost species examined have only one slc12a3/ncc1 gene, grouped in the same clade (blue frame, Fig. 2). The slc12a10 and slc12a3-like sequences of sea bass were very close and belonged to the ncc2 clade (green frame in Fig. 2).

The identity of the sea bass NCC1 and NCC2 (a, b) amino acid sequences were confirmed by comparison with sequences of teleost species. Interestingly, sea bass NCC1 showed a low percentage identity with sea bass NCC2a and NCC2b (48 % and 30 %, respectively). Sea bass NCC1 was highly homologous to NCC1 of killifish F. heteroclitus and stickleback G. aculeatus (81 % and 79 % respectively). Sea bass NCC2 a and b showed an amino acid sequence identity of 72 % and 82 %, respectively, with the killifish ortholog, NCC2.

Prediction of the transmembrane topology revealed the existence of 12 potential transmembranespanning segments (Fig. S2) as previously reported in zebrafish and winter flounder (Pseudopleuronectes americanus) NCCs [START_REF] Wang | Role of SLC12A10.2, a Na-Cl cotransporter-like protein, in a Cl uptake mechanism in zebrafish (Danio rerio)[END_REF][START_REF] Gamba | Primary structure and functional expression of a cDNA encoding the thiazide-sensitive, electroneutral sodium-chloride cotransporter[END_REF]). The search for potential N-glycosylation sites showed 2 highly conserved sites (Asn 364 and Asn 551) among all the analysed NCCs (see yellow highlighting, Fig. S2). The N-glycosylation site at position 411 in fish NCC1 and mammalian NCC seems to be absent in fish NCC2 sequences. In fish NCC2, two other Nglycosylation sites are present (Asn 409 and Asn 418) close to this region. At position 433, the Nglycosylation site was only conserved in mammalian NCC and teleost NCC2 sequences whereas Asn 789 was conserved in mammal NCC only. We have identified several amino acids conserved only among mammal NCC and fish NCC1 sequences (Fig. S2, red boxes). Several amino acids are conserved only among the teleost NCC1 and NCC2 sequences (Fig. S2, blue boxes). We also identified conserved amino acids between mammal and fish NCC2 sequences (Fig S2, green boxes).

mRNA expression patterns of nhe paralogs in osmoregulatory tissues

Tissue distribution of nhe paralogs were compared in gills, posterior kidney and posterior intestine/rectum in SW and FW after 4 months of salinity acclimation with fau as the internal control (Fig. 3). Nhe1 (slc9a1) was expressed in the all osmoregulatory tissues analysed, notably in the posterior intestine/rectum and FW kidney. Nhe1β-like (slc9a1β-like) was expressed in gills and kidney in SW and FW. Nhe2b (slc9a2.2) and nhe2c (slc9a2.3) were specifically expressed in the gill tissue and present at both salinities. Nhe3 (slc9a3) was highly expressed in the gills and posterior kidney at both salinities.

Gene expression

Nhe paralogous genes were quantified in SW and FW (Fig. 4A). Among them, nhe2a (slc9a2.1) showed a nearly not detectable band (Fig. 3) and has therefore not been quantified in sea bass gills. Significant expression differences between salinities were observed for nhe1β-like (slc9a1β-like), nhe2c (slc9a2.3) and nhe3 (slc9a3) transcripts (Fig. 4A). Nhe1β-like (slc9a1β-like) and nhe2c (slc9a2.3) expression were significantly higher in SW than in FW, respectively by 1.7-fold (p < 0.01) and by 28-fold (p < 0.001). Conversely, mRNA levels of nhe3 (slc9a3) were significantly higher in FW than in SW, by 2.8-fold (p < 0.01). The expression of nhe3 (slc9a3) was high compared to nhe2c (slc9a2.3) mRNA levels, by 17-fold in SW (p = 0.024) and by 1351-fold in FW (p < 0.0001).

The relative expression of nkcc1 (slc12a2) and ncc2a (slc12a3-like) (Fig. 4B) were examined in SW and FW. mRNA levels of nkcc1 (slc12a2) was significantly higher in SW than in FW, by 1.7-fold (p = 0.001). The expression of ncc2a (slc12a3-like) showed the opposite trend, with a 3-fold overexpression in FW compared to SW (p = 0.0003).

In the sea bass genome, two rhcg sequences have been identified, named rhcg1 and rhcg. We renamed rhcg to rhcg2 according to its phylogenetic position (not shown). Among the 17 different carbonic anhydrase genes detected in the sea bass genome, we quantified the cahz gene that grouped with the teleost CA2-like-a clade, as defined by [START_REF] Lin | Carbonic anhydrase 2-like a and 15a are involved in acid-base regulation and Na + uptake in zebrafish H + -ATPase-rich cells[END_REF] including medaka CA2-like a and trout cytoplasmic CAc [START_REF] Esbaugh | Cytoplasmic carbonic anhydrase isozymes in rainbow trout Oncorhynchus mykiss : comparative physiology and molecular evolution[END_REF][START_REF] Gilmour | New insights into the many functions of carbonic anhydrase in fish gills[END_REF]. CAC amino-acid sequence of D. labrax exhibited a percentage identity of 84 % and 81 % respectively with CA2-like a of O. latipes and CAc of O. mykiss, respectively. The CAC sequence of D. labrax showed a lower homology with CA2-like b of O. latipes and CAb of O. mykiss (70 % and 78 %, respectively). We considered this gene as the cytoplasmic carbonic anhydrase, and therefore we called it cac in this paper.

Relative expression of branchial vha-a (atp6v1a), vha-b (atp6v1b2), nka α1a (atp1a1a), nka α1b (atp1a1b), cytoplasmic carbonic anhydrase cac, sodium bicarbonate cotransporter nbc1 (slc4a4), ammonia transporters (rhbg, rhcg1 and rhcg2) are shown in Fig. 4C. Acid-sensing ion channel 4 gene (asic4) has also been tested as a potential Na + uptake channel, but results fell outside the range of quantifiable values (Ct>31) and could not be interpreted. FW challenge induced nka α1a (atp1a1a) expression by 2-fold (p = 0.0006) as well as the presumably apical ammonia transporter rhcg1, by 4fold (p = 0.0001). Expression of both vha subunit-a and b (atp6v1) were low and did not change according to salinity. Rhcg2 was well expressed at both salinities and cac showed a slightly but not significantly higher expression in FW (p = 0.06). Transcript levels of reference genes ef1α and fau did not change between salinities (p > 0.57 and p > 0.87, respectively).

Immunolocalization

Na + /K + -ATPase was coimmunostained with NHE3 to identify NHE3-immunopositive ionocytes in FW (Fig. 5). NHE3 was localized in apical membranes of ionocytes expressing NKA (Fig. 5A, B1, B2). NHE3-expressing cells were generally localized on lamella and showed a large apical surface (Fig. 5A). Few NHE3-immunopositive cells have been detected on filaments. Higher magnification images (Fig. 5B1,B2) showed that apical NHE3 is in contact with the surrounding water whereas basolateral surfaces of NHE3-expressing cells are in contact with blood.

We also used NHE3/T4 (NKCC1,2/NCC) coimmunostaining in SW (Fig. 6A1,A2) and FW gills (Fig. 6B1, B2, C1, C2). In SW, T4 immunostaining was detected in all ionocytes that are located on filaments and showed a strong basolateral staining (Fig. 6A1). NHE3 could not be detected at this salinity (Fig. 6A2). In FW, NHE3-immunopositive cells were also immunopositive for T4, but on basolateral membranes (Fig. 6B1,B2,C1,C2). Most ionocytes present on filaments showed a low basolateral T4 staining and no apical NHE3 (Fig. 6C2,arrow).

NKA/T4 (NKCC1,2/NCC) coimmunostained gills from freshwater-exposed sea bass showed intracellular colocalization of basolateral NKCC1 (T4) and NKA in some lamellar cells indicated in Fig. 7 (indicated by '*'). These cells did not exhibit apical T4 immunolabeling. Another ionocyte subtypes was localized on the filament and at the base of the lamella with an apical T4 immunostaining, presumably NCC2. NCC2 cells, contrary to the previous described ionocyte subtype, did no show strong basolateral T4 (NKCC1). Two image from a z-stack were shown (Fig. 7A1,A2)

showing several ionocytes with faint apical T4 staining identified by a number. Cells without number did not show clear apical T4 throughout the z-stack.

Discussion

In the last decade, different sodium uptake pathways have been investigated in several teleost species (zebrafish, medaka, trout and tilapia notably) [START_REF] Dymowska | Structure and function of ionocytes in the freshwater fish gill[END_REF][START_REF] Hwang | Mechanisms of ion transport in freshwater fishes[END_REF] but it is important to complete ion uptake models in other ecologically relevant euryhaline species that encounter strong salinity fluctuations in their natural habitat as the European sea bass. The sea bass is an important species for aquaculture and fisheries and its genome has been fully sequenced, assembled and annotated [START_REF] Tine | European sea bass genome and its variation provide insights into adaptation to euryhalinity and speciation[END_REF]. The sea bass is therefore an excellent model for analyzing osmoregulatory mechanisms at different salinities.

It is now admitted that Na + uptake is strongly linked to acid-base regulation and nitrogen excretion in specific ionocyte subtypes [START_REF] Weihrauch | Ammonia and urea transporters in gills of fish and aquatic crustaceans[END_REF][START_REF] Wright | A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins[END_REF]. Na + uptake can also be coupled to Cl -uptake in species that express Na + /Cl -cotransporters from the NCC2 (SLC12A10) clades [START_REF] Hiroi | New insights into gill ionocyte and ion transporter function in euryhaline and diadromous fish[END_REF][START_REF] Hsu | A new model for fish ion regulation: identification of ionocytes in freshwater-and seawater-acclimated medaka (Oryzias latipes)[END_REF] or mediated by a Na + channel coupled to apical VHA [START_REF] Dymowska | Structure and function of ionocytes in the freshwater fish gill[END_REF]. The predominance of one mechanism of ion uptake over the other is highly dependent on the environmental stimuli (ion strength, salinity, pH) and the considered species.

Unlike zebrafish and trout, sea bass asic4 seems not to be expressed in FW gills. Other ASICs should be analyzed in future to explore if other members of the ENaC/DEG (degenerin) family might be involved in Na + uptake in sea bass gills.

NHE3 as a key protein in gills of sea bass maintained in fresh water

We have shown for the first time in European sea bass that branchial nhe3 is significantly more expressed following a FW transfer and its expression is much higher than all other nhe paralogous genes analyzed in this study, clearly showing its major role in sea bass gills, notably in FW. It has to be noted that nhe3 is also highly expressed in the posterior kidney and could thus also have a role in renal Na + re-uptake and acid secretion. Only one NHE3 isoform is present in the sea bass genome and it is highly homologous to NHE3 of species showing only one NHE3 paralogous protein and potentially sharing a common NHE3 function. Immunolocalization of NHE3 shows an apical localization in a subset of ionocytes that also express Na + /K + -ATPase as previously shown in rainbow trout PNA+ MRC, zebrafish HR cells and Mozambique tilapia type-III ionocytes [START_REF] Hiroi | Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish[END_REF][START_REF] Hwang | Mechanisms of ion transport in freshwater fishes[END_REF][START_REF] Inokuchi | Gene expression and morphological localization of NHE3, NCC and NKCC1a in branchial mitochondria-rich cells of Mozambique tilapia (Oreochromis mossambicus) acclimated to a wide range of salinities[END_REF]. We have not detected NHE3 in apical membranes of gill ionocytes from SW fish unlike previous data obtained in other species like Japanese sea bass [START_REF] Inokuchi | Functional classification of gill ionocytes and spatiotemporal changes in their distribution after transfer from seawater to freshwater in Japanese seabass[END_REF]. NH4 + -dependent Na + uptake has been identified in medaka, trout and zebrafish [START_REF] Shih | Rhcg1 and NHE3b are involved in ammonium-dependent sodium uptake by zebrafish larvae acclimated to low-sodium water[END_REF][START_REF] Wright | A new paradigm for ammonia excretion in aquatic animals: role of Rhesus (Rh) glycoproteins[END_REF][START_REF] Wu | Ammoniumdependent sodium uptake in mitochondrion-rich cells of medaka (Oryzias latipes) larvae[END_REF]. In zebrafish HR cells, low-sodium water is a trigger for the differentiation of an apical protein complex involving NHE3b, RHCG1 and V-type H + -ATPase [START_REF] Shih | Ammonia excretion by the skin of zebrafish (Danio rerio) larvae[END_REF]. Our study shows that vha (a and b subunits) is not highly expressed and expression is not induced following FW challenge in sea bass gills. [START_REF] Sinha | Nutritional Status as the Key Modulator of Antioxidant Responses Induced by High Environmental Ammonia and Salinity Stress in European Sea Bass (Dicentrarchus labrax)[END_REF] however have shown that VHA activity is increased at low salinity (2.5 ‰) compared to SW. It is thus possible that posttranscriptional modifications activate VHA in sea bass FW gills. The involvement or not of VHA in the above-mentioned functional complex as well as its localization in ionocytes remain to be elucidated in sea bass.

Interestingly, expression of sea bass rhcg1 and nka α1a increase significantly following FW challenge, however we do not know if RHCG1 is localized in NHE3-expressing cells as shown in other species [START_REF] Nakada | Localization of ammonia transporter Rhcg1 in mitochondrion-rich cells of yolk sac, gill, and kidney of zebrafish and its ionic strength-dependent expression[END_REF]. Sea bass gill ionocyte density has previously been shown to increase significantly in FW compared to SW, notably due to an increase in lamellar ionocytes [START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF]. We have shown in this study that NHE3-immunopositive cells are essentially located on lamella. The slightly higher cytoplasmic carbonic anhydrase (cac) expression in FW gills could indicate an increased proton and bicarbonate supply for different transporters expressed in apical and basolateral membranes. Interestingly, NHE3-expressing cells also show basolateral T4 immunostaining which suggests the presence of NKCC1 in these cells as shown recently in the Japanese sea bass Lateolabrax japonicus [START_REF] Inokuchi | Functional classification of gill ionocytes and spatiotemporal changes in their distribution after transfer from seawater to freshwater in Japanese seabass[END_REF]. In NHE3-expressing cells from the FW condition, T4 staining intensity is clearly lower than in ionocytes from fish maintained in SW condition. This may be linked to the lower branchial mRNA levels of nkcc1 (slc12a2) in FW compared to SW. SW-type ionocytes may change their molecular setup and differentiate into FW-type cells expressing apical NHE3 and basal NKA and NKCC1. This hypothesis has been raised recently by Kaneko's group who reported in Japanese sea bass gills a transformation of SW-type cells to NHE3-type cells concomitant with a shift in their distribution from filaments to lamellae (Inokushi et al., 2017). In this earlier study, 10 days acclimated fish were used, similarly to our study were 2 weeks-transferred fish were analyzed. In Japanese sea bass, NHE3-NKCC1 expressing cells were called intermediate cells whereas a small percentage of NHE3 cells without basolateral NKCC1 were considered as fully FW-type cells. We have previously reported in European sea bass the presence of intermediate cells in FW lamella expressing basal NKCC1 using the T4 antibody in a time-series study following FW transfer [START_REF] Lorin-Nebel | The Na + /K + /2 Clcotransporter in the sea-bass Dicentrarchus labrax during ontogeny: Involvement in osmoregulation[END_REF]. If European sea bass gills transform SW-type ionocytes into NHE3-NKCC1 cells upon FW transfer, this would certainly contribute to the high capacity of sea bass to maintain constant Na + levels in highly fluctuating salinities like Mediterranean lagoons.

Two nhe2 paralogous genes are expressed in sea bass gills but their role in Na + uptake seems minor as they are not highly expressed in FW. Among the other nhe paralogs (besides nhe3) that have been analyzed in this study (nhe1, nhe1β-like, nhe2a, nhe2b, nhe2c), no induction has been detected in FW after the two-weeks salinity transfer. A time course sampling would be necessary to fully address the question on what nhe paralogs are induced following a FW challenge. The ubiquitous expression of nhe1 in all analyzed tissues suggests a minor role in transepithelial ion uptake and rather a role in acid removal from the cytoplasm and cell volume regulation as reported previously. No nhe1 expression difference between SW and FW has been shown in the rainbow trout in different tissues including gills, kidney and anterior intestine [START_REF] Genz | Intestinal transport following transfer to increased salinity in an anadromous fish (Oncorhynchus mykiss)[END_REF]. Few studies have quantified the expression of different nhe2 paralogs, notably nhe2c [START_REF] Edwards | Molecular identification of Na + -H + exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus[END_REF]. We have identified three nhe2 paralogous genes with only two of them well expressed in the gills, nhe2b and nhe2c. No expression has been detected in the posterior kidney and posterior intestine/rectum. This is also the case in the toadfish Opsanus tau, where nhe2 has not been detected in posterior intestine, neither in the rectum [START_REF] Esbaugh | Esophageal desalination is mediated by Na(+), H(+) exchanger-2 in the gulf toadfish (Opsanus beta)[END_REF]. It has to be noted that only one toadfish nhe2 has been investigated, that seems to play an important role in the desalinization process in the oesophagus [START_REF] Esbaugh | Esophageal desalination is mediated by Na(+), H(+) exchanger-2 in the gulf toadfish (Opsanus beta)[END_REF]. Nhe2b and more strikingly nhe2c expressions are low in sea bass gills and the involvement of NHE2 in apical Na + uptake seems thus unlikely. In trout, high levels of apical NHE2 and NHE3 have been shown in PNA + MRC, a cell type rich in NKA, but lacking VHA [START_REF] Ivanis | Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss)[END_REF]. Nhe2 sequence of trout is in the clade of sea bass nhe2b. Hypercapnia and increased cortisol levels seem to increase NHE2 levels in this species indicating a role in acid-base regulation [START_REF] Ivanis | Branchial expression and localization of SLC9A2 and SLC9A3 sodium/hydrogen exchangers and their possible role in acid-base regulation in freshwater rainbow trout (Oncorhynchus mykiss)[END_REF].

Despite its very low expression, sea bass nhe2c is about 28 times more expressed in SW than in FW suggesting its role in acid secretion as reported previously for several nhe2/3 paralogs in SW [START_REF] Chen | Na + /H + exchanger 3 is expressed in two distinct types of ionocyte, and probably augments ammonia excretion in one of them, in the gills of the climbing perch exposed to seawater[END_REF][START_REF] Claiborne | A mechanism for branchial acid excretion in marine fish: identification of multiple Na + /H + antiporter (NHE) isoforms in gills of two seawater teleosts[END_REF][START_REF] Liu | Salt secretion is linked to acid-base regulation of ionocytes in seawater-acclimated medaka: new insights into the salt-secreting mechanism[END_REF]. Contrary to trout nhe2, sea bass nhe2b and nhe2c are not expressed in the posterior kidney and intestine and nhe2a was not clearly detected in any osmoregulatory tissue. This is not the case in killifish, where NHE2a has been identified in cuboidal cells, the cell type expressing basolateral VHA and being involved in Na + uptake [START_REF] Edwards | Molecular identification of Na + -H + exchanger isoforms (NHE2) in the gills of the euryhaline teleost Fundulus heteroclitus[END_REF][START_REF] Laurent | Appearance of cuboidal cells in relation to salinity in gills of Fundulus heteroclitus, a species exhibiting branchial Na + but not Cluptake in freshwater[END_REF].

NCC2 in sea bass gills

We have shown here and in a previous study the presence of basolateral or apical T4 (NKCC1,2/NCC) immunostaining in gill ionocytes of sea bass transferred for two weeks to FW [START_REF] Lorin-Nebel | The Na + /K + /2 Clcotransporter in the sea-bass Dicentrarchus labrax during ontogeny: Involvement in osmoregulation[END_REF]. In that earlier study, it has already been reported that following FW transfer, several ionocytes remain basolaterally immunostained. This could indicate the presence of NKCC1. Other ionocyte subtypes present an apical staining, presumably NCC2. In this study we showed that apical NCC2 staining was present in several ionocytes located essentially on the filament. Compared to our previous study [START_REF] Lorin-Nebel | The Na + /K + /2 Clcotransporter in the sea-bass Dicentrarchus labrax during ontogeny: Involvement in osmoregulation[END_REF], apical immunostaining was less intense, which is probably due to different conditions used for colocalization studies with NKA. A specific NCC1 or NCC2 antibody is necessary to identify the specific paralogous NCC protein. NKCC1/NCC immunopositive ionocytes have been identified with the T4 antibody that recognizes a large epitope (38kDa peptide fragment) partially common to several SLC12A isoforms including NKCC1, NKCC2, NCC1 and also NCC2.

Apical staining using this antibody has further been shown in other teleost species in FW [START_REF] Hiroi | Evidence for an apical Na-Cl cotransporter involved in ion uptake in a teleost fish[END_REF]. In the sea bass genome, slc12a3 (ncc1), slc12a3-like (ncc2a) and slc12a10 (ncc2b) have been detected. Slc12a3-like was highly expressed in sea bass gills and was phylogenetically close to other ncc2 sequences. We therefore called it ncc2a and quantified its expression in our study. The small size of the sequence of slc12a10.2 (ncc2b) (486 pb) and its close phylogenetic position to slc12a3-like (ncc2a) suggests that these two genes may be splice variants or paralogs. Ncc2a was significantly higher expressed in FW than in SW, whereas nkcc1 (slc12a2) shows a decreased expression in FW which suggests the differentiation of ncc2a expressing ionocytes in FW conditions.

We have shown for the first time in this study the presence of different ionocyte subtypes in sea bass gills in FW with a NHE3-expressing ionocyte subtype, localized on lamella and potentially involved in Na + uptake. This NKA-rich cell expresses apical Na + /H + -exchanger (NHE3, SLC9A3) and basolateral T4, presumably NKCC1 (SLC12A2). This suggests a differentiation of a SW-type cell into a FW-type cell as a response to salinity changes that often occur in natural habitats. The presence in FW gills of ionocytes in the filament with high NKA immunostaining and apical T4 staining as well as the high branchial expression of ncc2a suggest the presence of another cell type expressing apical NCC2. It has not to be ruled out that other ionocyte subtypes exist in FW-acclimated sea bass as not all NKA-positive ionocytes showed apical NHE3 or NCC2. These other ionocyte subtypes remain to be characterized in European sea bass. The exact function of the two characterized ionocyte subtypes and the proteins they express in sea bass exposed to various environments will be investigated further in the future and will help to have a more comprehensive view on marine teleost salinity acclimation strategies.

Fig. 1

 1 Fig. 1 Phylogenic tree of nhe2 (scl9a2). Phylogram of maximum likelihood relationships between nhe2a, nhe2b and nhe2c nucleotides sequences of representative acanthomorph and salmonid species. Branch lengths represent the degree of divergence, with the scale bar indicating the distance representing 0.2 substitutions per position. Bootstrap values (in %) and SH-like aLRT values are indicated for each node if different (in red color if confident). The blue frame mainly comprises sequences of teleost nhe2a. The green frame mainly comprises sequences of acantomorph nhe2b and salmonid nhe2. The purple frame mainly comprises sequences of teleost nhe2c.

Fig. 2

 2 Fig. 2 Phylogenic tree of ncc (slc12a3), ncc2a (slc12a3-like) and ncc2b (slc12a10.2). Phylogram of maximum likelihood relationships between ncc1 and ncc2 nucleotides sequences of representative acanthomorph and salmonid species. Branch lengths represent the degree of divergence, with the scale bar indicating the distance representing 0.2 substitutions per position. Bootstrap values (in %) and SHlike aLRT values are indicated for each node if different (in red color if confident). The blue frame mainly comprises sequences of teleost ncc1. The green frame mainly comprises sequences of acantomorph ncc2.

Fig. 3

 3 Fig. 3 Tissue distribution analysis of nhe1 (slc9a1), nhe1β-like (slc9a1β -like), nhe2a (slc9a2.1), nhe2b (slc9a2.2), nhe2c (slc9a2.3) and nhe3 (slc9a3) in D. labrax acclimated to sea water or fresh water for 4 months. fau was used as the internal control.

Fig. 4

 4 Fig.4Relative expression of (A) nhe1 (slc9a1), nhe1β-like (slc9a1-βlike), nhe2b (slc9a2.2), nhe2c (slc9a2.3) and nhe3 (slc9a3), (B) nkcc1 (slc12a2) and ncc2a (slc12a3-like) (C) vha-a (atp6v1a), vha-b (atp6v1b2), nka α1a (atp1a1a), nka α1b (atp1a1b), cac,ncb1 (slc4a4) rhbg, rhcg1 and rhcg2 in gills of D. labrax juveniles exposed to SW and FW for two weeks. The expression has been normalized according to the expression of the elongation factor ef1α and the ubiquitin like and ribosomal protein S30 fusion gene fau. N= 8. Data are expressed as the mean ± SEM. For each gene, asterisks indicate significant differences between SW and FW (*p < 0.05; **p < 0.01; ***p < 0.001). FW: fresh water; SW: seawater.

Fig. 5 Fig. 6 Fig. 7

 567 Fig.5Immunolocalization of Na + /K + -ATPase (NKA) (green) and Na + /H + -exchanger 3 (NHE3) (red) in gills of 2-weeks acclimated D. labrax to fresh water using low (A) and high (B1, B2) magnifications of the gill epithelium. Nuclei were counterstained with DAPI (blue). Note that NHEexpressing ionocytes are located on lamellae. BF: bright field. Scale : 10 µm.

Fig. S2

 S2 Fig. S2 Alignment analysis of amino acid sequences of the mammalian and teleostean NCCs including D. labrax. The twelve predicted transmembrane regions (TM1-TM12) are indicated as blue arrows above the human NCC sequence. Potential N-glycosylation sites are highlighted in yellow. Red and green boxes respectively indicate conserved amino acids between mammalian NCC sequences and fish NCC1 or NCC2 sequences. Blue boxes indicated conserved amino acids among NCC sequences of fish.

Table 3 :

 3 Primer sequences used for RT-PCR and qPCR in this study. ND: not determined. F: forward 604 primer; R: reverse primer. Sequence ID indicate ID genome sequences from the sea bass genome or 605 Genbank identification numbers when available. 606

	Sequences ID	Target gene	Primer name	Sequence (from 5' to 3')	Amplicon Size	Efficiency
	DLAgn_00057430 slc9a1 / nhe1	NHE1 F NHE1 R	TATCCCACAAGTCCCACACC CAAGAGGAAGCCAAGGGAGA		1.98
	DLAgn_00204440 scl9a1-β / nhe1-β	NHE1-β F NHE1-β R	ACGGGGTTAATATGCGGCTT CAAGTTTCATGAGCAGGGCC		1.97
	DLAgn_00052750 slc9a2 / nhe2b	NHE2b F NHE2b R	CTGTCAGATCGAGGCGTTTG TCAAACACACTCAGCACAGC		1.95
	DLAgn_00136900 scl9a2 / nhe2c	NHE2c F NHE2c R	CGTTTCACCCACAATGTCCG GGGAATTGGCATTCTGGTGC		1.99
	DLAgn_00204050 slc9a3 / nhe3	NHE3 F NHE3 R	GGATACCTCGCCTACCTGAC AAGAGGAGGGTGAGGAGGAT		1.98
	DLAgn_00080120 slc12a2 / nkcc1	NKCC1 F NKCC1 R	TCAGCTCACAGTTCAAGGCC GCCGCTATGGACTCCACAA		2.08
	DLAgn_00038210 slc12a3-like / ncc2a	NCC2a F NCC2a R	ATGATGAGCCTCTTCGAGCC GCTGCTCTCATCACCTTCTGT		1.94
	DLAgn_00076370 atp6v1a / vha-a	VHA-A F VHA-A R	GGCAGTCACATCACAGGAGG CCAGCTCCATCACCACATCG		1.98
	DLAgn_00018050 atp6v1b2 / vha-b	VHA-B F VHA-B R	TTGCCATAGTCTTCGCAGCC CTTCTCGCACTGGTAGGCC		1.90
	KP400258	atp1a1.a / nka α1a	NKA a1a F NKA a1a R	CCTCAGATGGCAAGGAGAAG CCCTGCTGAGATCGGTTCC		1.89
	KP400259	atp1a1.b / nka α1b	NKA α1b F NKA α1b R	AGCAGGGCATGAAGAACAAG CCTGGGCTGCGTCTGAGG		1.99
	DLAgn_00078080 cac (cahz)	CAC F CAC R	CAGGGACCAAGTATCCTGC CCCTCCACCTTGCTCCCT		2.01
	DLAgn_00082210 slc4a4a / nbc1	NBC1 F NBC1 R	ACAGAGCACGGAACACACGG CGTCCACAGCCAGCAGTTCG		1.96
	DLAgn_00222650 rhbg	RHBG F1 RHBG R1	CCTCATGGTGACCCGAATCC GCCTGCACTCTGTCCACATA		1.97
	DLAgn_00166370 rhcg1	RHCG1 F1 RHCG1 R1	TCAGGGAATTGTGTGACCGC AGAATCAAGTCCACGCTGGG		2.01
	DLAgn_00157760 rhcg2	RHCG2 F1 RHCG2R1	TGGCTACCTGTTTGTCACGC GGATGCTCGGCGGCTTTATA		1.99
	FM004681	fau	FAU-F FAU-R	GACACCCAAGGTTGACAAGCAG GGCATTGAAGCACTTAGGAGTTG		2.07
	AJ866727	ef1α	EF1-F EF1-R	GGCTGGTATCTCTAAGAACG CCTCCAGCATGTTGTCTCC		2.09
	DLAgn_00049060 asic4	ASIC4F ASIC4R	CATCTTCGTCTCTGGTCGCC GCTCTGAATGAAGAGGCGAC		1.84
	DLAgn_00111870 slc9a2/nhe2a	NHE2a F NHE2a R	CTAGAGCCCAGGTCATTGCC GGCGTGTCCACAATACAGGA		ND
	607
	608
	609
	610

Acknowledgments

The authors would like to thank Thibaut L'Honoré for providing 4-months acclimated sea bass to fresh water as well as Waliullah Masroor for providing some sea bass gill slides and Emilie Farcy for providing fau primers. The authors also would like to thank Sophie Hermet for her help in the first immunostaining tests, Philippe Clair from the qPCR CEMEB platform as well as the Montpellier Ressources Imagerie (MRI) staff, Elodie Jublanc and Vicky Diakou, for their precious advice.