L ' Honoré

Lorin-Nebel Catherine

Blondeau-Bidet Eva

Julie Perez

Veyrunes Frédéric

Farcy Emilie

Thibaut L'honoré
email: thibautlhonore@gmail.com

Intraspecific variation in freshwater tolerance has consequences for telomere dynamics in the euryhaline teleost Dicentrarchus labrax

Keywords: telomere length, telomerase reverse transcriptase mRNA expression, freshwater tolerance, cell dynamics, energy metabolism, oxidative stress

Abstract

Stressful events can alter organism physiology at several levels triggering allostatic responses.

Telomeres are well-conserved repetitive DNA sequences mainly localised at chromosome's ends, playing a crucial role in DNA stability. Analyses of telomere dynamics are new tools to assess consequences of environmental stress in non-model organisms like fish. In this study, the relationship between freshwater tolerance and telomere dynamics was investigated in the gills of the European sea bass Dicentrarchus labrax. Fluorescent in situ hybridisation of telomeric sequences revealed distal telomeres as well as intrachromosomal telomeres known as interstitial telomere sequences. In order to better understand telomere dynamics in the gills of D. labrax, we used quantitative PCR to measure telomere length and mRNA expression of the catalytic subunit of telomerase reverse transcriptase tert. For the calculation of the relative telomere length, two reference genes were tested: the single copy gene mc2r, encoding melanocortin 2 receptor and the multicopy gene 18S, encoding the 18S ribosomal RNA. We proposed a novel normalisation method to calculate the relative telomere length using both, single and multiple copy genes as references. Cell dynamics was also investigated by measuring mRNA expression of genes involved in apoptosis (caspase 8 and 9), cell proliferation (proliferation cell nuclear antigen), aerobic mitochondrial metabolism (ATP citrate-synthase), anaerobic metabolism (lactate dehydrogenase a) and antioxidant enzymatic defences (superoxide dismutase 1 and 2, catalase). Following a 15-days fresh water exposure, telomere dynamics was not significantly modified in the gills of freshwater tolerant fish. But freshwater intolerant fish exhibited telomere attrition relative to saltwater controls, and lower expression of tert in gills relative to freshwater tolerant fish. This modification of telomere dynamics in intolerant individuals was found to be correlated with lower antioxidant enzymatic defences, a higher aerobic metabolic marker and a lower cellular turnover. These data bring new perspectives for the use of telomere dynamics as an integrative marker to

Introduction

Marine organisms living in fluctuating environments such as lagoons and estuaries have to constantly deal with abiotic stressors (salinity, temperature, oxygen). A strong physiological plasticity is required to be able to face salinity, temperature and oxygen level fluctuations [START_REF] Claireaux | Influence of temperature, oxygen and salinity on the metabolism of the European sea bass[END_REF]. The European sea bass Dicentrarchus labrax (Linnaeus, 1758) is a demersal fish of high commercial interest which inhabits coastal waters. D. labrax enters estuaries, lagoons and sometimes ascending rivers most likely to feed [START_REF] Rogdakis | Feeding habits and trophic level of sea bass (Dicentrarchus labrax) in the Messolonghi-Etoliko lagoons complex (Western Greece)[END_REF]). In D. labrax, a strong intra-specific variability was highlighted regarding its capacity to tolerate hyperthermia [START_REF] Ozolina | Intraspecific individual variation of temperature tolerance associated with oxygen demand in the European sea bass (Dicentrarchus labrax)[END_REF], hypoxia (Claireaux et al., 2013;[START_REF] Joyce | Individual variation in whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance in a marine teleost[END_REF] and freshwater exposure [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF], 2020), suggesting an inter-individual difference in the capacity to tolerate harsh environmental conditions. [START_REF] Burton | Can environmental conditions experienced in early life influence future generations?[END_REF] highlighted that exposure to stressful conditions in early life stages can have long-term and inter-generational effects on physiology and fitness in several taxa, including fish. It is questionable whether repeated stress encountered throughout life by fish such as D. labrax migrating seasonally in transitional waters, has a negative impact on fitness.

Few studies have examined the potential use of telomere length as an integrative marker of stress exposure in fish [START_REF] Anchelin | Premature aging in telomerasedeficient zebrafish[END_REF][START_REF] Henriques | Telomerase is required for zebrafish lifespan[END_REF][START_REF] Naslund | Telomere dynamics in wild brown trout: effects of compensatory growth and early growth investment[END_REF][START_REF] Debes | Is telomere length a molecular marker of past thermal stress in wild fish?[END_REF]. Telomeres are well conserved terminal regions of eukaryotic chromosomes, composed of repetitive sequences of TTAGGG in vertebrates [START_REF] Blackburn | A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena[END_REF]. Telomeres ensure multiple functions in preserving chromosome stability, including protecting the ends of chromosomes from degradation and preventing chromosomal end fusion [START_REF] Blackburn | Structure and function of telomeres[END_REF]. Telomere length (TL) and telomerase activity are commonly used to study ageing in higher vertebrates [START_REF] Aubert | Telomeres and aging[END_REF][START_REF] Saretzki | Telomeres, Telomerase and Ageing BT -Biochemistry and Cell Biology of Ageing: Part I Biomedical Science[END_REF].

Telomerase plays a crucial role in chromosome stability and cell viability by extending the distal 3' end of eukaryotic linear chromosome over replications [START_REF] Blackburn | Telomeres and telomerase: their mechanisms of action and the effects of altering their functions[END_REF]. This enzymatic complex consists of the telomerase reverse transcriptase (TERT) catalytic subunit, the telomerase RNA component (TERC) involved in the replication of the telomere sequence, and other associated proteins contributing to elongate telomeres localised at the end of the chromosomes [START_REF] Blackburn | Telomeres and telomerase: their mechanisms of action and the effects of altering their functions[END_REF][START_REF] Smith | Structural biology of telomeres and telomerase[END_REF]. In most non-mammal species such as birds and fish, telomere dynamics relies on two opposite forces: telomere attrition and telomere restoration, supported by telomerase. In human, chronic oxidative stress and life stressors can accelerate telomere attrition by decreasing telomerase activity or tert expression levels [START_REF] Epel | Accelerated telomere shortening in response to life stress[END_REF][START_REF] Houben | Telomere length assessment: biomarker of chronic oxidative stress?[END_REF]Starkweather et al. 2014). In ecological studies, telomere length provides a mechanistic link between environmental condition, life history traits and fitness [START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF][START_REF] Haussmann | Telomeres: Linking stress and survival, ecology and evolution[END_REF][START_REF] Monaghan | Organismal stress, telomeres and life histories[END_REF][START_REF] Mathur | Perceived stress and telomere length: A systematic review, meta-analysis, and methodologic considerations for advancing the field[END_REF]. According to recent meta-analyses focused on ecological studies in nonmodel vertebrates [START_REF] Angelier | Do glucocorticoids mediate the link between environmental conditions and telomere dynamics in wild vertebrates? A review[END_REF][START_REF] Mclennan | Telomere elongation during early development is independent of environmental temperatures in Atlantic salmon[END_REF][START_REF] Wilbourn | The relationship between telomere length and mortality risk in non-model vertebrate systems: A meta-analysis[END_REF], we still lack crucial basic data to fully understand: (i) the influence of abiotic factors, such as salinity or temperature, on telomere length, (ii) the intra-specific variation in telomere dynamics and the drivers of this intra-specific variation and (iii) the potential link between telomere attrition, lifespan and mortality risk, especially in bony fish species.

The effect of temperature on telomere attrition was the main environmental abiotic parameter analysed in fish. In mosquitofish Gambusia holbrooki, a decrease from 25°C to 20°C for 24 h was associated with a decrease in telomere length [START_REF] Rollings | Telomeric attrition with age and temperature in Eastern mosquitofish (Gambusia holbrooki)[END_REF]. Conversely, an increase in temperature from 20°C to 30°C for 1 month triggered telomere attrition in the Siberian sturgeon Acipenser baerii [START_REF] Simide | Age and heat stress as determinants of telomere length in a long-lived fish, the siberian sturgeon[END_REF]. Regarding the relationship between telomere attrition and ageing in fish, studies are controversial. In the zebrafish, telomere length has been observed to increase from larvae to adult stages and to shorten significantly in older individuals (Anchelin et al., 2011). Additionally, Hatakeyama et al. (2016) showed in the medaka that telomeres do not shorten linearly with age, but shortening dynamics depends on growth rate and level of telomerase activity at each life stage. Therefore, it appears that telomere dynamics is particularly variable and nonlinear in fish.

Previous experimental studies performed in juvenile D. labrax at different ages have shown that about 25 to 30% of individuals are unable to acclimate successfully to experimental transfer from seawater to fresh water [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF][START_REF] Honoré | Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass[END_REF]. The freshwater intolerant phenotype exhibits several characteristics: failure in hydromineral balance regulation, decrease in swimming capacities, downregulation of gluco-and mineralocorticoid receptors involved in both stress response and osmoregulation and, ultimately, death [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF], 2020). Recently, [START_REF] Angelier | Do glucocorticoids mediate the link between environmental conditions and telomere dynamics in wild vertebrates? A review[END_REF] raised new hypotheses suggesting a trade-off between immediate survival and telomere maintenance/protection, which would transitionally lead to shortened telomeres during an "emergency state". In this study, we compare extreme phenotypes regarding freshwater tolerance (tolerant vs intolerant) in order to determine if D. labrax exhibiting contrasted freshwater tolerance differ in telomere dynamics.

The gill was considered as a somatic tissue of interest to study the relationship between hypoosmotic stress and telomere dynamics as the branchial epithelium exhibits a rapid cell turnover and a strong morphological plasticity [START_REF] Nilsson | Gill remodeling in fish -a new fashion or an ancient secret?[END_REF][START_REF] Kang | The acute and regulatory phases of time-course changes in gill mitochondrion-rich cells of seawater-acclimated medaka (Oryzias dancena) when exposed to hypoosmotic environments[END_REF]). In D. labrax , gills are able to remodel within 1 to 2 weeks in response to fluctuations of environmental factors like salinity, oxygen availability and temperature (Sollid and Nilsson 2006;[START_REF] Lorin-Nebel | The Na + /K + /2Clcotransporter in the sea bass Dicentrarchus labrax during ontogeny: involvement in osmoregulation[END_REF][START_REF] Nilsson | New insights into the plasticity of gill structure[END_REF][START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF]. Such plasticity in the response to environmental change has been demonstrated to be associated to elevated cellular dynamics, such as cell renewal and apoptosis [START_REF] Sollid | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]Tzaneva et al. 2014;[START_REF] Sales | Proliferation, survival and cell death in fish gills remodeling: From injury to recovery[END_REF][START_REF] Mierzwa | Regeneration of the gill filaments and replacement of serotonergic neuroepithelial cells in adult zebrafish (Danio rerio)[END_REF]. In addition, an increased number of gill mitochondrion-rich cells (MRCs) has been shown in hypo-osmotic environments in numerous species including D. labrax [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF], suggesting a raise of energetic demand to fuel active ion transport [START_REF] Evans | The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste[END_REF]. Interestingly, freshwater intolerant D. labrax were previously characterised by a higher density of branchial MRCs compared to freshwater tolerant fish [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF], suggesting metabolic disorders in freshwater intolerant D. labrax .

Since mitochondria are known to be the main source of ROS production in cells [START_REF] Lambert | Reactive oxygen species production by mitochondria[END_REF], an increase in mitochondria may also trigger an increased production of metabolic ROS, as a by-product of cellular respiration [START_REF] Quijano | Interplay between oxidant species and energy metabolism[END_REF]. In vitro, oxidative stress was shown to be a major factor triggering DNA damage and accelerated telomere shortening in human endothelial cells, through the reduction of telomerase activity [START_REF] Kurz | Chronic oxidative stress compromises telomere integrity and accelerates the onset of senescence in human endothelial cells[END_REF][START_REF] Ahmed | Impact of oxidative stress on telomere biology[END_REF]. In vivo studies showing a direct link between oxidative stress and telomere dynamics are more scarce [START_REF] Boonekamp | Does oxidative stress shorten telomeres?[END_REF]. Recent reviews by Reichert & Stier (2017) and Chatelain et al (2019) concluded that there is strong evidence from both experimental and correlative in vivo studies in vertebrates that oxidative stress induces effects on telomere dynamics, with tissue-dependent, life stage-dependant and sex-dependant variations. Nevertheless, more experimental studies are required to further understand the influence of oxidative stress on telomere dynamics in vivo.

The first aim of this study was to determine the occurrence and the localisation of telomeres in D. labrax genome using fluorescence in situ hybridisation (FISH) in order to test if interstitial telomeric sites are detected. The head kidney was used for karyotyping because of its high cell renewal [START_REF] Bertollo | Direct chromosome preparation from freshwater teleost fishes[END_REF]. Then, an acute 2 weeks freshwater stress was used to test whether osmotic stress affects telomere dynamics in the gills of 5-month-old D. labrax exhibiting contrasted freshwater tolerance capacities, as previously described in L 'Honoré et al. (2019). Telomere attrition was evaluated using relative TL measurement using q-PCR and the mRNA expression of tert was measured as a proxy of telomere maintenance. To better understand cell dynamics and the potential influence of oxidative stress and energy metabolism on telomere dynamics, mRNA expression of genes involved in apoptosis (caspase 8 and 9), cell proliferation (proliferation cell nuclear antigen), aerobic mitochondrial metabolism (ATP citrate-synthase), anaerobic metabolism (lactate dehydrogenase a) and antioxidant enzymatic defences (superoxide dismutase 1 and 2, catalase) were measured.

Osmotic stress and individual tolerance to fresh water may differentially influence telomere dynamics where telomere attrition would reflect the harshness of the environment an individual has experienced. We hypothesised that non-tolerant fish to fresh water will exhibit shorter telomeres than tolerant fish, as a consequence of oxidative and physiological stress. If telomeres shorten to critical levels in the gill tissue, this may trigger organ dysfunction, as previously shown in gut and muscle zebrafish [START_REF] Carneiro | Short telomeres in key tissues initiate local and systemic aging in zebrafish[END_REF]. This could have consequence on general physiology and survival since the fish gill is a multifunctional organ involved in gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste [START_REF] Evans | The multifunctional fish gill: dominant site of gas exchange, osmoregulation, acid-base regulation, and excretion of nitrogenous waste[END_REF]. As short telomeres induce senescence in cells and hence reduce the regenerative capacity of the corresponding tissues, it has been suggested that TL might affect various fitness parameters [START_REF] Monaghan | Do telomere dynamics link lifestyle and lifespan?[END_REF]. In fact, TL has been linked to survival and reproductive success in some bird species [START_REF] Haussmann | Longer telomeres associated with higher survival in birds[END_REF][START_REF] Pauliny | Age-independent telomere length predicts fitness in two bird species[END_REF]. From an evolutionary ecology point of view, telomere-induced selection could occur if telomere attrition differently affects relative fitness among individuals [START_REF] Olsson | Evolutionary ecology of telomeres: a review[END_REF].

Evidence for causal effects of telomere traits on life history and fitness-related parameters is still limited. In this study, we investigate the consequence of an acute osmotic stress in the non-model euryhaline species D. labrax to test whether intraspecific differences in osmoregulatory capacities have consequences on TL maintenance.

Materials and methods

Origin of animals

Fish were issued from in vitro fertilisation of unrelated wild native West Mediterranean breeders (40 males and 23 females) in order to obtain a large genetic diversity. Sea bass were grown at the Ifremer Station at Palavas-les-flots (Hérault, France) under a 16/8 hours light/dark photoperiod in seawater (SW) at 20°C. Food was proposed ad libidum.

2. Fluorescence in situ hybridisation on telomere sequence DNA and microscope analysis Karyotype analysis and fluorescent in situ hybridisation were performed at the CytoEvol facilities of UMR ISEM of the LabEx CeMEB (Montpellier, France). Cephalic kidney of two males and 2 females (10 month-old) were sampled and processed as described in [START_REF] Ozouf-Costaz | First insights into karyotype evolution within the family Mormyridae[END_REF]. Fluorescence in situ hybridisation (FISH) was performed following the same procedure as described in [START_REF] Ozouf-Costaz | First insights into karyotype evolution within the family Mormyridae[END_REF], using an oligonucleotide telomeric probe (TTAGGG)7 labelled with Cy3 at its 5' end (biomers.net, Ulm, Germany) and counterstaining the chromosomes with DAPI (4',6-diamidino-2-phenylindole)-antifade mounting medium solution (Vectashield, Vector Laboratories, Peterborough, UK). Three slides were prepared per individual and preparations were analysed using a Zeiss Axioplan 2 Imaging epifluorescence microscope equipped with a cooled charge couple devise camera and Cytovision 7.4 software (Applied Imaging, San Jose, CA).

Experimental exposure to freshwater

Five month-old D. labrax juveniles (N=1525, 4.20 ± 0.09 cm, 0.87 ± 0.06 g) were experimentally exposed to fresh water according to L 'Honoré et al. (2019). Briefly, fish were transferred from SW to brackish water (BW) at 15 ppt for 24h before being transferred to fresh water (FW) for 2 weeks. A no replication experimental setup, where intolerant fish and tolerant fish are maintained in the same tank and exact same conditions, was chosen because we expected from previous studies that the FW intolerant phenotype represents about one third of the experimental cohort [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF][START_REF] Honoré | Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass[END_REF], thus requiring an elevated number of animals (N=1525). In addition, the detection of FW intolerant phenotype also requires an elevated number of individuals swimming in shoals in order to be able to observe abnormal individual behaviour within the shoal as described in L 'Honoré et al. (2019). After 2 weeks of freshwater challenge, tolerant and intolerant phenotypes were sorted, measured and weighted. More precisely, fish exhibiting erratic swimming, isolation from the shoal associated with low reflexes and stronger pigmentation were identified as the freshwater-intolerant phenotype (FW-I). These animals were characterised by an incapacity to maintain hydromineral balance in FW [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF][START_REF] Honoré | Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass[END_REF]. The three experimental groups analysed were: seawater controls (SW, 6.30 ± 0.12 cm, 2.84 ± 0.14 g), freshwater tolerant fish (FW-T, 5.28 ± 0.10 cm, 1.47 ± 0.10 g) and freshwater intolerant fish (FW-I, 5.20 ± 0.10 cm, 1.25 ± 0.08 g). At the end of the exposure, fish were euthanised in 100 ppm of benzocaine and the first left gill arc was dissected, flash frozen in liquid nitrogen and stored respectively dry or in RNAlater (Quiagen, Valencia, CA) at -80°C until gDNA and mRNA extraction.

gDNA extraction

Genomic DNA (gDNA) extraction was performed using the Maxwell ® 16 Buccal Swab LEV DNA Purification Kit (Promega, Charbonières, France). Samples were eluted in 50 µL of ultrapure water. Quantity was measured fluorometrically using a Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific), concentrations ranged from 60 to 200 µg mL -1 . Purity was verified using the NanoDrop TM One/One C Spectrophotometer (Thermo Scientific, Waltham, MA, USA) through A260/A280 and A260/A230 ratios. DNA quality was checked using Bioanalyzer 2100 (Santa Clara, CA, United States).

RNA extraction and reverse transcription

RNA extraction was performed using the total RNA extraction kit that includes a DNase step (Nucleospin ® RNA, Macherey-Nagel, Germany). Quantity and purity of extraction products were verified using a UV spectrophotometer (NanoDrop™ One/OneC Spectrophotometer, Thermo Scientific, Waltham, MA, USA). RNA quality was checked using Bioanalyzer 2100 and RIN levels were comprised between 6 and 9 (mean RIN = 8.15). Reverse transcription was performed using one microgram of RNA using the qScript™ cDNA SuperMix (Quanta Biosciences™) providing all necessary components for first-strand synthesis: buffer, oligo(dT) primers, random primers and qScript reverse transcriptase.

Target genes selection

Key genes were selected to better understand cell dynamics and the potential influence of oxidative stress and energy metabolism on telomere dynamics. Proliferating cell nuclear antigen (pcna) was used as a cell proliferation marker [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF]) whereas caspase 8 and 9 (casp 8, casp 9) were used as extrinsic and intrinsic cell apoptosis markers respectively [START_REF] Olsson | Caspases and cancer[END_REF][START_REF] Paiola | Oestrogen, an evolutionary conserved regulator of T cell differentiation and immune tolerance in jawed vertebrates?[END_REF]. Mitochondrial superoxide dismutase 1 (sod1), cytosolic superoxide dismutase 2 (sod2) and catalase (cat) were selected as antioxidant enzymes because superoxide anion (O2 .-) and hydrogen peroxide (H2O2) are the main ROS formed by mitochondria. Lactate dehydrogenase is a key enzyme in the control of energy metabolism composed of four polypeptide subunits encoded by two genes: ldh-a and ldh-b [START_REF] Driedzic | Lactate -The preferred aerobic fuel of metabolism of the fish heart[END_REF]. In this study, ldh-a was investigated as a marker for anaerobic glycolysis [START_REF] Almeida-Val | Anoxia-and hypoxiainduced expression of LDH-A* in the Amazon Oscar, Astronotus crassipinis[END_REF]Valvona et al. 2016). Gene encoding citrate synthase (cs) was selected as a marker of aerobic metabolism [START_REF] Roche | Monovalent cation requirement for ADP inhibition of pyruvate dehydrogenase kinase[END_REF][START_REF] Elcock | Evidence for electrostatic channeling in a fusion protein of malate dehydrogenase and citrate synthase[END_REF][START_REF] Goldenthal | Cloning and molecular analysis of the human citrate synthase gene[END_REF].

Quantitative real-time polymerase chain reaction

Telomere length measurement and relative mRNA gene expression quantification was realised using 384-wells plates filled with an Echo®525 liquid handling system (Labcyte Inc., San Jose, CA, USA). Each well contained a mix composed by 1.5 µL of LightCycler-FastStart DNA Master SYBR-Green I™ Mix (Roche, Manheim, Germany), 0.27 µL of each primer (forward and reverse primers at 0.9 µM final concentration), 0.23 µL of ultrapure water and 1 µL of cDNA or gDNA. For pcna, tert, casp8, casp9, sod1, sod2, cat, cs, ldh-a, 18S and l13 cDNA amplification, efficiency (E) of each primer pair was tested using standard curves performed on all-samples pools of cDNA (Table 1).

For mRNA expression analyses, the q-PCR conditions 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 reference genes 18S and l13 were chosen according to previous studies performed in sea bass [START_REF] Mitter | Evaluation of candidate reference genes for QPCR during ontogenesis and of immune-relevant tissues of European sea bass (Dicentrarchus labrax)[END_REF]. Relative mRNA expressions were normalised against two reference genes, l13 and 18S, according to the method of Vandesompele et al. (2002) and expressed using the comparative ∆∆Ct method (Ct, threshold cycle number) described by [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF], with SW fish as a reference. For all samples, measurements were run in triplicates, and no-template control (water) Ct was above 40.

For TL measurement, the q-PCR conditions were adapted from [START_REF] Cawthon | Telomere length measurement by a novel monochrome multiplex quantitative PCR method[END_REF] with some modifications as follows: 15 min denaturation at 95 °C followed by 2 cycles (94° C for 15 s, 49° C for 15 s) followed by 35 cycles (95 °C for 15 s, 62 °C for 10 s and 74 °C for 15 sec).

Telomere primers used to amplify telomeric hexamer repeats were TEL G and TEL C as described in [START_REF] Cawthon | Telomere length measurement by a novel monochrome multiplex quantitative PCR method[END_REF]. Efficiency of each primer pairs reported on Table 1 were obtained by standard curves performed on all-sample pools of gDNA (tel, mc2r and 18S).

Relative TL calculation was performed using the ratio between telomere repeat copy number and reference gene copy number known as T/R ratio. T/R ratio was calculated with the ∆∆Ct method described in [START_REF] Cawthon | Telomere measurement by quantitative PCR[END_REF] and normalised against two reference genes, a single copy gene mc2r and a multicopy gene 18S [START_REF] Wang | Robust measurement of telomere length in single cells[END_REF]).

Formulas used "E" as primers efficiency as indicated in Table 1. The condition SW was used as the control condition for the ∆∆Ct calculation. An inter-plate assay was performed to investigate the potential variability between two different q-PCR runs. Inter-assay validation was performed in duplicates with mc2r and 18S on gDNA of 16 samples. labrax [START_REF] Sola | A karyotype analysis of the genus Dicentrarchus by different staining techniques[END_REF]. Fluorescent in situ hybridisation of telomeric sites revealed that telomere sequences were localised distally, as expected. Interstitial telomeric sequences (ITS) localised proximally were also observed. FISH does not allow a precise detection so we cannot conclude about any inter-individual differences in signal intensity or localisation of telomere sequences.

Relative telomere length measurements

Method validation

Primer efficiency of the single copy gene mc2r and the multicopy gene 18S were at 2.0 (Table 1). The primers specificity was checked using the melting point (Tm) of the product for each primer pair and displayed a unique pike at the expected temperature. The inter-plate Pearson correlation r² were respectively above 0.92 and 0.99 for the T/R ratio with mc2r and 18S as reference genes (Pearson test, P < 0.0001 for each gene, Figs 1Sa-b). Coefficients of variation (CV) did not exhibited values > 3% for both intra-assay CV and inter-assay CV as resumed in Table 1S.

Regarding TL, CV were the highest using 18S as the reference gene (32.0% in SW, 34.7% in FW-T and 60.0% in FW-I, Fig. 1Sc), whereas they were the lowest using mc2r as the reference gene (26.6% in SW, 35.5% in FW-T and 32.0% in FW-I, Figure 2). Thus, we will consider mc2r as the best reference gene for TL since it was 3-times less variable within phenotypes.

2.2 Telomere dynamics in response to freshwater exposure: telomere length and mRNA expression of tert No significant difference in TL could be measured between SW and FW-T (P = 0.8107, Fig. 2). A significantly lower relative TL was measured in FW-I compared to FW-T and SW (P < 0.0001, Fig. 2). Regarding tert expression, no significant difference was measured between SW and FW-T (P = 0.2115, Fig. 3a). In FW-I, tert expression was significantly lower than in FW-T but not compared to SW (P = 0.0011 and P = 0.072 respectively).

mRNA expression of genes involved in cell dynamics, metabolism and antioxidant defences

Transcript levels of pcna did not differ between SW and FW-T (P = 0.9144), whereas they were significantly lower in FW-I than in SW and in FW-T (P = 0.0001 and P = 0.0003, Fig. 3b). Although we did not measure any significant difference in casp8 expression levels between SW and FW-T (P = 0.1936, Fig. 3c), we measured significant lower expression of casp8 in FW-I compared to FW-T but not compared to SW (P = 0.0137 and P = 0.0367 respectively). We did not measure any significant difference in casp9 expression levels between the three groups (Fig. 4d).

Superoxide dismutase sod1 and sod2 mRNA gene expression did not exhibit any significant differences between the three phenotypes ( Figs 4a-b). Regarding cat, no significant differences were inferred between SW and FW-T (P = 0.0455). However, FW-I displayed significantly lower expression compared to both SW and FW-T (P = 0.0005 and P = 0.0052 respectively, Fig. 4c).

Concerning cs mRNA expression levels, they were significantly lower in SW than in FW-T and FW-I (P = 0.0133 and P = 0.0101, Fig. 4d). However, no significant differences could be inferred between FW-T and FW-I (P = 0.6932), or between each group regarding ldh-a mRNA expression levels (P = 0.0469 for SW vs FW-T, P = 0.0219 for SW vs FW-I, and P = 0.3669 for FW-T vs FW-I).

Correlation between variables

Testing the Spearman coefficient correlation between all quantitative variables (Table 2), it appeared that telomere dynamics markers (TL and tert mRNA expression) were significantly and positively correlated (r = 0.48, P = 0.013). Body mass and body length were not correlated with telomere dynamics markers (P > 0.05 for both), and no significant differences could be inferred between the two phenotypes in FW (Mann-Whitney test, P = 0.3110 and unpaired t-test, P = 0.5893, for body mass and body length respectively). These correlations were all positives regarding cellular turnover markers (r = 0.48 between pcna and TL, r = 0.38 between casp8 and TL, r = 0.64 between casp8 and tert, r = 0.47 between casp9 and tert) and antioxidant enzymatic defences (r = 0.66 between sod2 and tert, r = 0.59 between TL and cat). However, the correlation between TL and metabolic marker was negative (r = -0.58 between cs and TL, r = -0.39 between ldh-a and TL).

Discussion

Method validation

As reviewed in [START_REF] Lai | Comparison of telomere length measurement methods[END_REF], the estimation of relative TL using the q-PCR method may be biased by inter-assays variations. By reproducing the same q-PCR amplification using two different plates as described in [START_REF] Appleby | Telomere length as a putative biomarker of health and disease[END_REF], we showed that the operational variability was very limited in this study. Karyotype analysis of D. labrax revealed 24 pairs of chromosomes different in size as already demonstrated in the literature [START_REF] Sola | A karyotype analysis of the genus Dicentrarchus by different staining techniques[END_REF]. In this study, we demonstrate the presence of interstitial telomeric sequences (ITS) in D. labrax.

According to Ocalewicz et al. (2013), most of the pericentromeric and ITS in fish are possible relicts of chromosome fusion events. The occurrence of ITS may potentially reduce the sensitivity of the q-PCR method for TL measurement by adding a background noise, especially if small TL changes are expected. Due to their intrachromosomal position, several authors suggested that ITS do not shorten during DNA replication or in response to ageing or stress [START_REF] Foote | Extent and variability of interstitial telomeric sequences and their effects on estimates of telomere length[END_REF]. According to a recent meta-analysis of [START_REF] Chatelain | The association between stressors and telomeres in non-human vertebrates: a meta-analysis[END_REF], the noise in telomere length resulting from interstitial repeats may not mask the differences in the length of end-cap telomeres between individuals, using the qPCR method or the TRF method.

It would be interesting to use a quantitative technique such as Q-FISH [START_REF] Lai | Comparison of telomere length measurement methods[END_REF] to further explore the proportion of ITS vs terminal telomeric sequences and to determine whether the inter-individual variability of ITS is elevated in sea bass.

For relative quantification, the choice of the reference gene may be crucial to improve the reliability of the T/R ratio calculation. While most studies used single copy genes as reference for relative TL calculation [START_REF] Lai | Comparison of telomere length measurement methods[END_REF], [START_REF] Wang | Robust measurement of telomere length in single cells[END_REF] demonstrated in single cells that a multicopy gene like 18S was more robust for this calculation. However, given that the variability among each group seemed to depend on the reference gene, we propose to use a single copy gene like mc2r to reduce bias due to the variations of a single specific reference gene.

Telomere and cell dynamics in gills following FW exposure

The use of TL as a biomarker for environmental stress exposure requires a tissue with active telomere dynamics. Previous study working on erythrocytes reported no difference in telomere length in D. labrax with age [START_REF] Horn | Telomere length change in European sea bass (Dicentrarchus labrax)[END_REF]. Gill tissue has several interesting properties: a strong plasticity associated with high cell renewal and active cell division [START_REF] Nilsson | Gill remodeling in fish -a new fashion or an ancient secret?[END_REF]Tzaneva et al. 2014), the presence of MRCs suggesting an active cellular respiration and potential increased production of metabolic ROS by-products [START_REF] Hwang | New insights into fish ion regulation and mitochondrion-rich cells[END_REF]. To our knowledge, the effect of salinity change on telomere dynamics has never been studied in euryhaline teleost. In this study, we were able to detect a significant TL reduction of about 50% in the gill of FW-I after only two weeks of freshwater stress. A strong interindividual variability in TL was observed, as expected in vertebrates [START_REF] Dugdale | Heritability of telomere variation: it is all about the environment![END_REF]Toupance et al. 2019). This highlights that telomere dynamics in gill is quickly modified. In accordance to the hypothesis of Angelier et al (2019), TL was not maintained in fish whose survival is threatened, suggesting a trade-off between immediate survival and telomere protection.

Interestingly, the quick telomere attrition measured in FW-intolerant fish was correlated to a significant lower tert expression compared to FW-tolerant fish, suggesting an altered capacity to maintain TL in intolerant fish facing freshwater stress. Conversely, transfer from seawater to fresh water did not trigger any significant change in tert expression and TL in FWT. In European hake Merluccius merluccius and in Atlantic cod Gadus morhua, tert expression was found higher in early developmental stages suggesting an higher telomerase demand possibly linked with elevated tissue renewal and long-term cell proliferation capacity maintenance (López de [START_REF] De Abechuco | Molecular cloning and measurement of telomerase reverse transcriptase (TERT) transcription patterns in tissues of European hake (Merluccius merluccius) and Atlantic cod (Gadus morhua) during aging[END_REF]). However, there is no clear trend concerning the relationship between ageing and telomerase activity [START_REF] Hatakeyama | The teleost Oryzias latipes shows telomere shortening with age despite considerable telomerase activity throughout life[END_REF][START_REF] Henriques | Telomerase is required for zebrafish lifespan[END_REF][START_REF] Saretzki | Telomeres, Telomerase and Ageing BT -Biochemistry and Cell Biology of Ageing: Part I Biomedical Science[END_REF]). Regarding cell dynamics, freshwater transfer is expected to increase cell population renewal associated with branchial epithelium remodelling occurring during hypoosmotic acclimation [START_REF] Nilsson | Gill remodeling in fish -a new fashion or an ancient secret?[END_REF][START_REF] Masroor | Effect of combined stress (salinity and temperature) in European sea bass Dicentrarchus labrax osmoregulatory processes[END_REF]. We observed no significant changes in mRNA expression of cell dynamics markers of apoptosis casp8, casp9 or proliferation pcna in FW-T after 2 weeks of exposure. Most of the cellular changes have probably been completed in successfully acclimated D. labrax within 2 weeks of freshwater exposure [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF]). In the gill of FW-I, casp8 and pcna levels were significantly down-regulated compared to the freshwater tolerant condition, suggesting a slowdown of cell dynamics in the gills of intolerant fish. This is consistent with the results of [START_REF] Carneiro | Short telomeres in key tissues initiate local and systemic aging in zebrafish[END_REF], which observed a decreased cell proliferation in the gut and testis of tert-/-mutants zebrafish using PCNA immunostaining. Conversely, in cellular in vitro models, a link between tert overexpression, cell survival and increased cell proliferation has been shown [START_REF] Dagarag | Genetic manipulation of telomerase in HIV-specific CD8+ T cells: enhanced antiviral functions accompany the increased proliferative potential and telomere length stabilization[END_REF][START_REF] Aubert | Telomeres and aging[END_REF]. Given that we highlighted a correlation between cellular dynamics and telomere dynamics in D. labrax, we can hypothesise that the reduction of cellular dynamics observed in FW-I may be associated to a reduction of tert expression or telomerase activity. The reduction of cell dynamics in intolerant fish may be possibly due to an exhaustion of energetic reserves allocated to osmoregulatory processes. Due to technical and ethical limitations regarding the number of individuals used for this study, no replication of freshwater and seawater treatment was performed. Therefore, we cannot exclude a batch effect between SW and FW fish or other confounding factor that may influence within salinity treatment response.

Metabolism and antioxidant defences following freshwater exposure

Freshwater exposure differentially affected energy metabolism and antioxidant enzymatic defences in D. labrax according to their individual freshwater tolerance capacity. Freshwater exposure significantly increased mRNA gene expression of the citrate synthase gene, a marker of aerobic metabolism. Acclimation of teleosts to different environmental salinities causes depletion of energy which is used to regulate the functioning of various highly energyconsuming pumps and ion transporters in gill MRCs [START_REF] Chang | Regulation of glycogen metabolism in gills and liver of the euryhaline tilapia (Oreochromis mossambicus) during acclimation to seawater[END_REF][START_REF] Hwang | New insights into fish ion regulation and mitochondrion-rich cells[END_REF]Tseng and Hwang 2008). In tilapia gill epithelial cells, Tseng et al. (2008) have shown that citrate synthase and LDH proteins were induced after transfer from FW to SW, confirming the active role of these enzymes to fuel active ion-pumping and fish osmoregulation. During salinity challenges either from SW to FW or from FW to SW, an increase in lactate contents and LDH activities has been reported in the gills of several euryhaline teleost fish [START_REF] Vijayan | Food-deprivation affects seawater acclimation in tilapia: hormonal and metabolic changes[END_REF][START_REF] Polakof | Food deprivation alters osmoregulatory and metabolic responses to salinity acclimation in gilthead sea bream Sparus auratus[END_REF]Tseng et al. 2008) indicating the involvement of monocarboxylate metabolites in gill energy consumption during osmoregulation. In this study, no significant changes in mRNA expression of ldh-a could be inferred according to the Bonferonni adjusted p-value of 0.0169. Present mRNA gene expression data should be taken with caution since they do not reflect the concentration and/or activity of the related protein. Therefore, additional biochemical analyses (e.g. activity of key enzymes of aerobic and anaerobic metabolic pathways such as LDH, citrate synthase or citrate oxidase) would be necessary to confirm the hypothesis of a metabolic distress in FW-intolerant fish. But data from this and previous studies [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF][START_REF] Honoré | Are European sea bass as euryhaline as expected? Intraspecific variation in freshwater tolerance[END_REF][START_REF] Honoré | Inter-individual variability in freshwater tolerance is related to transcript level differences in gill and posterior kidney of European sea bass[END_REF] converge to this hypothesis. After two weeks in FW, the cost of acclimation is maintained elevated in FW-I compared to FW-T and SW. This is consistent with results of previous studies in sea bass showing that (i) intolerant fish over-absorbed ions in the gills to compensate a renal failure (L 'Honoré et al., 2020), (ii) intolerant fish exhibit and overabundance of MRCs in gills [START_REF] Nebel | Differential freshwater adaptation in juvenile sea-bass Dicentrarchus labrax: involvement of gills and urinary system[END_REF]) and (iii) intolerant fish exhibit a change in gluco-and mineralocorticoids regulatory pathways, underlying impairment of hydromineral balance and stress response regulation (L 'Honoré et al., 2020). Thus, in species exhibiting intraspecific variability in abiotic stress tolerance such as salinity in killifish Fundulus heteroclitus [START_REF] Scott | Intraspecific variation in gene expression after seawater transfer in gills of the euryhaline killifish Fundulus heteroclitus[END_REF], temperature [START_REF] Ozolina | Intraspecific individual variation of temperature tolerance associated with oxygen demand in the European sea bass (Dicentrarchus labrax)[END_REF] or hypoxia [START_REF] Joyce | Individual variation in whole-animal hypoxia tolerance is associated with cardiac hypoxia tolerance in a marine teleost[END_REF] in D. labrax, differences in gill TL should be further investigated to test whether differential patterns of tolerance to physiological stress have consequence on telomere attrition, and possibly on tissue functioning as suggested by [START_REF] Carneiro | Short telomeres in key tissues initiate local and systemic aging in zebrafish[END_REF].

Mitochondria are widely recognized as a source of ROS in animal cells, where it is assumed that overproduction of ROS may conduct to an overwhelmed antioxidant system and oxidative stress [START_REF] Quijano | Interplay between oxidant species and energy metabolism[END_REF]. Therefore, an elevated mitochondrial metabolism could increase the production of ROS and would therefore require an activation of anti-oxidant defences to maintain the oxidative balance. In this study, the expression of cat, sod1 or sod2 genes, encoding enzymes involved in the main mitochondrial anti-oxidant defences, were not significantly modified after 2 weeks in freshwater in the gills of the tolerant fish compared to seawater controls. These results are consistent with [START_REF] Ghanavatinasab | Changes in gill antioxidant status in Acanthopagrus sheim exposed to different environmental salinities[END_REF], where no significant difference in SOD and CAT were observed in yellowfin seabream Acanthopagrus sheim exposed for 2 weeks in 5 ppt water. However, a significant decrease in cat expression levels was measured in the gills of FW-intolerant sea bass compared to FW-tolerant and SW. This result suggests that telomere attrition in FW-intolerant fish could be due to an imbalance between increased ROS production and downregulated antioxidant defences, leading to oxidative damage on telomeres in individuals with lower capacity to induce tert. But this hypothesis needs to be further explored by investigating pro-oxidants, other enzymatic and non-enzymatic anti-oxidant defences as well as oxidative damages.

This study suggests that, in case of elevated physiological and metabolic stress, telomere repair is not prioritised and that energetic limitation has direct consequence on telomere maintenance. These results are consistent with the hypothesis of [START_REF] Angelier | Do glucocorticoids mediate the link between environmental conditions and telomere dynamics in wild vertebrates? A review[END_REF] suggesting a trade-off between immediate protection and telomere maintenance. Additional evidence concerning energy metabolism, oxidative stress and damage would be necessary to support the preliminary results of this study. Another recent in vivo study highlighted that TL and metabolism are more tightly linked than initially thought [START_REF] Casagrande | Telomere attrition: Metabolic regulation and signalling function?[END_REF].

The results obtained in this study are in agreement with the metabolic telomere attrition concept proposed by [START_REF] Casagrande | Telomere attrition: Metabolic regulation and signalling function?[END_REF], that assumes that TL attrition is strongest during times of energy limitation. Oxidative stress may also be at stake but the relationship between ROS production and mitochondrial energy production remains to be further investigated (Salin et al., 2015). In marine teleost, there is no evidence that hyposaline stress triggers oxidative stress as shown in hepatic tissue of D. labrax [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] as well as in A. sheim gills [START_REF] Ghanavatinasab | Changes in gill antioxidant status in Acanthopagrus sheim exposed to different environmental salinities[END_REF]. But again, the gill was poorly studied and a transient increase of production of metabolic ROS, as a by-product of cellular respiration cannot by excluded. According to these hypotheses, telomere dynamics can be considered as a major determinant for cell homeostasis.

Finally, our results suggested that, in the wild, freshwater environment requiring active ionic regulation would potentially not represent a stress involving telomere shortening in fish having large salinity tolerance capacity, if salinity variation is considered solely. But in transitional waters, other environmental parameters are at stake. In particular, temperature and hypoxia have been shown to upregulate TERT expression in testis and liver of medaka and decrease TL in muscle and fin in brown trout, respectively [START_REF] Yu | Hypoxia induces telomerase reverse transcriptase (TERT) gene expression in non-tumor fish tissues in vivo: The marine medaka (Oryzias melastigma) model[END_REF][START_REF] Debes | Is telomere length a molecular marker of past thermal stress in wild fish?[END_REF]).

Multi-stress experimental studies would be necessary to further understand the influence of abiotic factors on telomere length, but the results obtained from this study bring interesting information regarding the consequences of exposure to harsh low salinity conditions and the intra-specific variation in telomere dynamics on non-model fish vertebrates, which is of particular interest for ecologists in the context of global change. Therefore, an interesting perspective of this work would be to determine whether marked fluctuations of environmental parameters, such as those encountered in transitional waters, affect TL in the wild, in association with other life-history traits markers such as otolithometry in order to gain further information on age, growth rate and habitat [START_REF] Darnaude | Validation of otolith ∂18O values as effective natural tags for shelf-scale geolocation of migrating fish[END_REF][START_REF] Bouchoucha | Otolith fingerprints as natural tags to identify juvenile fish life in ports[END_REF].

Conclusion

The q-PCR method performed in this study was efficient to detect relative telomere length changes in sea bass exposed to freshwater. Differences in telomere dynamics in the gills was linked with individual phenotypic plasticity related to freshwater tolerance. Lower telomere dynamics (telomere length and tert expression) in FW-I was correlated with a higher aerobic metabolism as well as a lower antioxidant defences. Figure 1 Fluorescent in situ hybridisation (FISH) of metaphase chromosomes isolated from head-kidneys of 10 month-old European sea bass using the telomeric probe (TTAGGG)7 labelled with Cy3 at its 5' end, indicated by red colour. 2 males and 2 females were analysed with N = 3 slides per fish. Scale bar: 10µm

Figure 2 Relative telomere length expressed as T/R ratio calculated using ∆∆Ct method normalised against the single copy gene mc2r in gills of 5 month-old sea bass maintained in seawater and after a transfer of 2 weeks in fresh water. Different letters denote significant differences between groups (Mann-Whitney test, Bonferroni-corrected P < 0.0167, means ± s.e.m, N=10-14). SW: control fish in seawater, FW-T: FW-tolerant fish, FW-I: FW-intolerant fish Figure 3 Relative mRNA expression of genes involved in telomere maintenance (a), cell proliferation (b) and apoptosis (c, d) in the gill of 5 month-old sea bass maintained in seawater (SW) or exposed for 2 weeks to fresh water (FW-T and FW-I). (a) telomerase catalytic subunit tert (b) proliferation cell nuclear antigen pcna (c) caspase 8 casp8 (d) caspase 9 casp9. The mRNA expression was calculated using the ∆∆Ct method with SW as a reference and normalised according to the expression of two reference genes l13 and 18S. Different letters denote significant differences between phenotypes (Mann-Whitney test, Bonferroni-corrected P < 0.0167, means ± s.e.m, N=10-15). SW: control fish in seawater, FW-T: FW-tolerant fish, FW-I: FW-intolerant fish Figure 4 Relative mRNA expression of genes involved in antioxidant defence (a, b, c) and metabolism (d, e) in the gill of 5 month-old sea bass maintained in seawater (SW) or exposed for 2 weeks to fresh water (FW-T and FW-I). (a) superoxide dismutase 1 sod1 (b) superoxide dismutase 2 sod2 (c) catalase cat (d) ATP citrate synthase cs (e) lactate dehydrogenase a (ldh-a). The mRNA expression was calculated using the ∆∆Ct method with SW as a reference and normalised according to the expression of two reference genes l13 and 18S. Different letters denote significant differences between groups (Mann-Whitney test, Bonferroni-corrected P < 0.0167, means ± s.e.m, N=10-15). SW: control fish in seawater, FW-T: FW-tolerant fish, FW-I: FW-intolerant fish Table 1 Primer sequences used for relative telomere length and gene expression analysis Table 2 Spearman r-correlation matrix. Asterisks denotes significant r using P < 0.05.

0,01 0,00 0,21 0,47* 0,28 0,78* 1,00 sod1 0,00 0,15 -0,28 -0,05 0,52* 0,15 0,14 1,00 sod2 0,24 0,23 0,18 0,66* 0,31 0,60* 0,57* 0,15 1,00 cat 0,49* 0,44* 0,59* 0,26 0,28 0,18 0,06 -0,17 0,24 1,00 cs -0,31 -0,22 -0,58* 0,11 -0,09 0,45* 0,52* 0,12 0,34 -0,66* 1,00 ldh-a -0,30 -0,21 -0,39* -0,12 -0,38* -0,04 -0,13 0,02 -0,15 -0,26 0,48* 1,00

  analyses were performed on GraphPad Prism (version 6, GraphPad Software Incorporated, La Jolla, CA 268, USA). First, Grubb's test was used on the 15 fish per condition to remove the potential outliers from the data set. Since data fitted normality test (D'Agostino-Pearson test) but not homoscedasticity test (Bartlett test), Mann-Whitney pairwise comparisons were performed, with Bonferroni adjustment (p < 0.0167). For interplate assay correlation analysis, Pearson correlation tests were used because data fitted with normality assumption. A non-parametric Spearman correlation test was performed to study the correlation between quantitative variables. Experimental values are reported as means ± s.e.m.. Results 1. FISH of DNA telomere sequences in D. labrax karyotypes Karyotype analyses confirmed the presence of 2n=48 chromosomes (Fig. 1) as expected in D.

  carassius and goldfish Carassius auratus. J Exp Biol 208:1109-1116. doi: 10.1242/jeb.01505 Sollid J, Nilsson GE (2006) Plasticity of respiratory structures -Adaptive remodeling of fish gills induced by ambient oxygen and temperature. Respir Physiol Neurobiol 154:241-251. doi: 10.1016/j.resp.2006.02.006 Starkweather AR, Alhaeeri AA, Montpetit A, et al (2014) An integrative review of factors associated with telomere length and implications for biobehavioral research. Nurs Res 63:36-50. doi: 10.1097/NNR.0000000000000009 Toupance S, Villemonais D, Germain D, et al (2019) The individual's signature of telomere length distribution. Sci Rep 9:685. doi: 10.1038/s41598-018-36756-8 Tseng Y-C, Lee J-R, Chia J, et al (2008) Regulation of lactate dehydrogenase in tilapia (Oreochromis mossambicus) gills during acclimation to salinity challenge. Zool Stud 47:473-480 Tseng YC, Hwang PP (2008) Some insights into energy metabolism for osmoregulation in fish. Comp Biochem Physiol -C Toxicol Pharmacol 148:419-429. doi: 10.1016/j.cbpc.2008.04.009 Tzaneva V, Vadeboncoeur C, Ting J, Perry SF (2014) Effects of hypoxia-induced gill remodelling on the innervation and distribution of ionocytes in the gill of goldfish, Carassius auratus. J Comp Neurol 522:118-130. doi: 10.1002/cne.23392 Valvona CJ, Fillmore HL, Nunn PB, Pilkington GJ (2016) The regulation and function of lactate dehydrogenase A: therapeutic potential in brain tumor. Brain Pathol 26:3-17. doi: 10.1111/bpa.12299

Acknowledgements

The authors wish to thank, the three anonymous referees for their helpful comments and R.

Simide for helpful suggestion in designing the method. We would like to thank Philippe Clair from the qPCR CeMEB platform for his help, the cytoEvol facilities of ISEM (labex CeMEB), and technicians of Ifremer Palavas-les-flots for the maintenance of the fish. This study was conducted with the support of LabEx CeMEB, an ANR "Investissements d'avenir"

program (ANR-10-LABX-04-01). The project used instruments that were financially supported by the 2015-2020 CPER CELIMER (funded by the French Ministry of Higher Education, Research and Innovation, the Occitanie Region, Montpellier Méditerranée Metropolis, Sète Agglopole Méditerranée, Ifremer, IRD).

Ethics

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. The experimental design has been approved by the French legal requirement concerning welfare of experimental animals (APAFIS permit no. 9045-201701068219555).