Clemence Gourtay
email: clemence.gourtay@uqar.ca

Denis Chabot

Celine Audet

Lauriane Madec

Christine Huelvan

Loick Ducros

Guy Claireaux

David Mazurais

Jose-Luis Zambonino-Infante

Clémence Gourtay

Céline Audet

Loïck Ducros

José-Luis Zambonino-Infante

Dr Clémence Gourtay

Céline Audet

Ducros

Effect of thermal and nutritional conditions on fatty acid metabolism and oxidative stress response in juvenile European sea bass (Dicentrarchus labrax)

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Many fish species have life history strategies in which successive ontogenetic stages occupy different habitats. In those characterized by the production of large amounts of small pelagic eggs and the lack of parental care, the survival of eggs, larvae, and juveniles strongly depends on abiotic and biotic environmental factors acting on their habitats [START_REF] Cushing | Population production and regulation in the sea: A fishery perspective[END_REF][START_REF] Sogard | Size-selective mortality in the juvenile stage of teleost fishes: a review[END_REF][START_REF] Juanes | Role of habitat in mediating mortality during the post-settlement transition phase of temperate marine fishes[END_REF]Houde, 2008). Coastal zones and estuaries are productive nursery grounds for a large number of marine species because they provide refuge from predators together with high food availability and enhanced survival, development, and growth of early stages [START_REF] Beck | The role of nearshore ecosystems as fish and shellfish nurseries[END_REF][START_REF] Able | Do surf zones in New Environ[END_REF]. However, coastal and estuarine nursery areas are subjected to a wide range of anthropogenic and natural stressors. Coastal habitats in particular face anthropogenic activities, including global warming, which indirectly influence trophic food webs [START_REF] Harley | The impacts of climate change in coastal marine systems[END_REF][START_REF] Lehodey | Climate variability, fish, and fisheries[END_REF][START_REF] Brander | Global fish production and climate change[END_REF][START_REF] Rijnsdorp | Resolving the effect of climate change on fish populations[END_REF][START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO2 emissions scenarios[END_REF][START_REF] Hixson | Climate warming is predicted to reduce omega-3, long-chain, polyunsaturated fatty acid production in phytoplankton[END_REF].

Lipids represent an important energy supply and play a crucial role as major components of cell membranes [START_REF] Sargent | The lipids[END_REF]. In marine trophic networks, polyunsaturated fatty acids (PUFA) are mainly supplied by the dominant phytoplankton species, such as diatoms and dinoflagellates [START_REF] Hu | Microalgal triacylglycerols as feedstocks for biofuel production: Perspectives and advances[END_REF][START_REF] Lang | Fatty acid profiles and their distribution patterns in microalgae: A comprehensive analysis of more than 2000 strains from the SAG culture collection[END_REF]. The n-3 PUFA (also known as omega 3), particularly eicosapentenoic acid (EPA) and docosahexaenoic acid (DHA), are naturally abundant for primary consumers. However, it has been shown that phytoplankton growth and biochemical composition, including fatty acids (FA), are affected by temperature (e.g., [START_REF] Arts | Lipids in aquatic ecosystems[END_REF]. An increase in water temperature results in a decrease in the omega 3 content of phytoplankton species [START_REF] Ackman | Marine phytoplankter fatty acids[END_REF][START_REF] Thompson | Effects of variation in temperature. II. On the fatty acid composition of eight species of marine phytoplankton[END_REF][START_REF] Renaud | Effect of temperature on growth, chemical composition and fatty acid composition of tropical Australian microalgae grown in batch cultures[END_REF][START_REF] Guschina | Mechanisms of temperature adaptation in poikilotherms[END_REF]. At least two recent studies have shown a global decline of n-3 PUFA in trophic networks while n-6 tends to be more abundant [START_REF] Colombo | A fundamental dichotomy in long-chain polyunsaturated fatty acid abundance between and within marine and terrestrial ecosystems[END_REF][START_REF] Hixson | Climate warming is predicted to reduce omega-3, long-chain, polyunsaturated fatty acid production in phytoplankton[END_REF]. The high availability of n-3 PUFA in phytoplankton may explain the lack of selection pressure on its biosynthesis pathway in several marine fish species and thus why piscivorous fish have lost the capacity for de novo synthesis of n-3 PUFA [START_REF] Tocher | Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional Gadus morhua L.)[END_REF].

In most other fish species, it has been shown that marine species have low capacities to convert 18-carbon fatty acids into n-3 PUFA [START_REF] Owen | Elongation and desaturation of dietary fatty acids in turbot (Scophthalmus maximus) and rainbow trout (Salmo gairdneri)[END_REF][START_REF] Mourente | In vivo metabolism of [1-14C] linolenic acid (18:3n-3) and [-14C] eicosapentaenoic acid (20:5n-3) in marine fish: time-course of the desaturation / elongation pathway[END_REF][START_REF] Sargent | The lipids[END_REF][START_REF] Geay | Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet[END_REF][START_REF] Geay | Characteristics of fads2 gene expression and putative promoter in European sea bass (Dicentrarchus labrax): Comparison with salmonid species and analysis of CpG methylation[END_REF] due to apparent deficiencies in one or more steps of the biosynthesis pathway [START_REF] Ghioni | Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4n-3, to eicosapentaenoic acid, 20:5n-3, in a cell line from the turbot, Scophthalmus maximus[END_REF][START_REF] Tocher | Fatty acid metabolism in marine fish: low activity of 5 desaturation in gilthead sea bream (Sparus aurata) cells[END_REF][START_REF] Santigosa | Effect of diets containing a purified soybean trypsin inhibitor on growth performance, digestive proteases and intestinal histology in juvenile sea bream (Sparus aurata L.)[END_REF].

This makes these fishes highly dependent upon dietary sources of n-3 PUFA [START_REF] Ghioni | Low C18 to C20 fatty acid elongase activity and limited conversion of stearidonic acid, 18:4n-3, to eicosapentaenoic acid, 20:5n-3, in a cell line from the turbot, Scophthalmus maximus[END_REF][START_REF] Tocher | Fatty acid metabolism in marine fish: low activity of 5 desaturation in gilthead sea bream (Sparus aurata) cells[END_REF]. To what extent the n-3 PUFA decline induced by global climate change may impact species that depend on phytoplankton to fulfill their needs whether as primary or secondary consumers is not yet known. However, it has also been reported that most of the genes coding for the main components of this biosynthesis pathway are present and that their transcription could be up/down regulated by some nutritional or environmental factors [START_REF] Dias | Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax)[END_REF][START_REF] Gomez-Requeni | Effects of dietary amino acid profile on growth performance, key metabolic enzymes and somatotropic axis responsiveness of gilthead sea bream (Sparus aurata)[END_REF][START_REF] Figueiredo-Silva | Blackspot seabream (Pagellus bogaraveo) lipogenic and glycolytic pathways appear to be more related to dietary protein level than dietary starch type[END_REF]. Thus it is possible to study the regulation of lipid metabolism and the FA synthesis pathway that directly or indirectly result from climate change.

Environmental and nutritional factors may alter the amount of functional protein involved in lipid metabolism through a range of transcriptional (e.g., activity of transcription factors), post-transcriptional (e.g., mRNA stability), translational, and post-translational (e.g., phosphorylation) mechanisms. The transcriptional regulation of genes involved in lipogenesis by PUFA is well documented. PUFA are known to bind and directly influence the activity of a variety of transcription factors including peroxisome proliferator activated receptors (PPAR), which in turn have been shown to regulate the transcription of many genes involved in lipid homeostatic processes [START_REF] Jump | The biochemistry of n-3 polyunsaturated fatty acids[END_REF]. There appears to be several ppar variants expressed in fish tissues: is mainly expressed in the liver, is mainly expressed in adipose tissue, and has been found in most tissues [START_REF] Boukouvala | Molecular characterisation of three peroxisome proliferator-activated receptors from the sea bass (Dicentrarchus labrax)[END_REF]. Sterol regulatory element binding protein-1 (srebp-1) is another key transcription factor known to be involved in fatty acid synthesis assembly [START_REF] Horton | SREBPs: activators of the complete program of cholesterol and fatty acid synthesis in the liver[END_REF]. It is well documented that expression of the ppar and srebp-1 genes are regulated at the transcriptional level by dietary FA in mammals [START_REF] Lindi | Impact of the Pro12Ala polymorphism of the PPAR-c2 gene on serum triacylglycerol response to n23 fatty acid supplementation[END_REF][START_REF] Juliano | Fatty acids, cholesterol, and sphingolipids: interactive regulators and targets for SREBP processing[END_REF][START_REF] Maclaren | Peroxisome proliferator-activated receptor (PPAR) expression in cultured bovine endometrial cells and response to omega-3 fatty acid, growth hormone and agonist stimulation in relation to series 2 prostaglandin production[END_REF]. Similar results have been reported in fish [START_REF] Leaver | Three peroxisome proliferator-activated receptor isotypes from each of two species of marine fish[END_REF][START_REF] Leaver | Multiple peroxisome proliferator-activated receptor beta subtypes from Atlantic salmon (Salmo salar)[END_REF][START_REF] Jordal | Complete replacement of dietary fish oil with a vegetable oil blend affects liver lipid and plasma lipoprotein levels in Atlantic salmon (Salmo salar L.)[END_REF][START_REF] Dong | Cloning and characterization of SREBP-1 and PPAR-Lateolabrax japonicus, and their gene expressions in response to different dietary fatty acid profiles[END_REF], including the European sea bass [START_REF] Geay | Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet[END_REF][START_REF] Geay | Characteristics of fads2 gene expression and putative promoter in European sea bass (Dicentrarchus labrax): Comparison with salmonid species and analysis of CpG methylation[END_REF].

In mammals, ppar and srebp-1 are the main transcription factors regulating the mRNA expression of elongase and desaturase (Nakamura andNara, 2002, 2003), which are key enzymes involved in PUFA biosynthesis. The first step of the bioconversion pathway requires the delta 6-desaturase gene (fads2; [START_REF] Sprecher | Metabolism of highly unsaturated n-3 and n-6 fatty acids[END_REF]. Another important enzyme system is the fatty acid synthetase complex (FAS), which catalyzes the key lipogenesis pathway in fishes [START_REF] Sargent | The lipids[END_REF]. The main products are saturated 16:0 (palmitic acid) and 18:0 (stearic acid) FA. In the carnivorous fishes blackspot seabream (Pagellus bogaraveo; [START_REF] Figueiredo-Silva | Blackspot seabream (Pagellus bogaraveo) lipogenic and glycolytic pathways appear to be more related to dietary protein level than dietary starch type[END_REF], European sea bass (Dicentrarchus labrax; [START_REF] Dias | Regulation of hepatic lipogenesis by dietary protein/energy in juvenile European seabass (Dicentrarchus labrax)[END_REF], and gilthead sea bream (Sparus aurata; [START_REF] Gomez-Requeni | Effects of dietary amino acid profile on growth performance, key metabolic enzymes and somatotropic axis responsiveness of gilthead sea bream (Sparus aurata)[END_REF], dietary fat intake has been shown to inhibit FAS activity.

Figueiredo

Biological membranes rich in PUFA are highly sensitive to oxidation due to their high level of unsaturation [START_REF] Pamplona | Double bond content of phospholipids and lipid peroxidation negatively correlate with maximum longevity in the heart of mammals[END_REF][START_REF] Hulbert | On the importance of fatty acid composition of membranes for aging[END_REF]; this oxidation can lead to the production of reactive oxygen species (ROS). ROS are generated as by-products of cellular metabolism, and mitochondria are a primary site for ROS production [START_REF] Barja | Mitochondrial oxygen consumption and reactive oxygen species production are independently modulated: implications for aging studies[END_REF]. Higher temperatures may promote ROS production, especially in organisms rich in membrane PUFA, and this could lead to various cellular and tissular dysfunctions [START_REF] Kawatsu | Studies on the anemia of fish -III. An example of macrocytic anemia found in brook trout, Salvelinus fontinalis[END_REF]Watanabe et al., 1970;Murai and Andrews, 1974;[START_REF] Sakai | Severe oxidative stress is thought to be a principal cause of jaundice of yellowtail Seriola quinqueradiata[END_REF]. The relative rates of peroxidation of different acyl chains have been used to convert the fatty acid composition of a particular membrane into the peroxidation index (PI; see Hulbert et al., 2007, for details). The PI represents the theoretical susceptibility of membrane lipid composition to lipid peroxidation, and it appears to be homeostatically regulated with respect to dietary PI. In practice, malondialdehyde (MDA) is one lipid peroxidation product that can be measured [START_REF] Frankel | Lipid oxidation[END_REF]. Under most physiological states, ROS production is closely matched by the antioxidant response. Antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), and peroxidases, form an important part of the antioxidant response [START_REF] Lesser | Oxidative stress in marine environments: biochemistry and physiological ecology[END_REF]. In fish species, COX and CS activity have been monitored because they are known to be adjusted to the maximum aerobic metabolism capacity in fish [START_REF] Childress | Depth-related enzymatic activities in muscle, brain and heart of deep-living pelagic marine teleosts[END_REF]Thuesen andChildress, 1993, 1994).

European seabass migrate offshore to spawn, and the developing larvae drift to sheltered coastal or estuarine nurseries where they spend their first few years of life [START_REF] Holden | The biology, movements and populations dynamics of bass, Dicentrarchus labrax, in English waters[END_REF][START_REF] Kelley | The importance of estuaries for sea bass Dicentrarchus labrax (L.)[END_REF]Vinagre et al., 2009). During that period, juvenile sea bass exhibit rapid growth and are faced with a broad spectrum of environmental constraints and feeding conditions, making it an attractive model organism to examine the regulatory response of the PUFA biosynthesis pathway. The aim of this study was to test the effects of two levels of n-3 PUFA dietary contents on different molecular (transcript expression) and biochemical (MDA, enzymatic activity) variables related to FA metabolism and antioxidant response, respectively, in European sea bass juveniles raised at two different temperatures. To obtain realistic responses in terms of a global change scenario, we exposed European sea bass to temperatures of 15°C (current average temperature to which the Atlantic sea bass population is exposed) and 20°C (to mimic the temperature rise that is expected over the next 80 years; [START_REF] Gattuso | Contrasting futures for ocean and society from different anthropogenic CO2 emissions scenarios[END_REF]. We hypothesized that a diet poor in n-3 PUFA would activate lipid biosynthesis while a diet richer in n-3 PUFA would lead to oxidative stress and would worsen under higher temperature conditions.

Materials and methods

Feeding experiment

The protocols for fish maintenance, diet composition, and nutritional conditioning are detailed in [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF] [18:3n-3]; EPA+DHA = 0.73% DM), while the reference diet (RD) contained 50% colza oil and 50% fish oil, the latter being richer in EPA and DHA (20:5n-3, 22:6n-3; EPA+DHA = 1.65% DM). Diets were isoenergetic and contained the same percentages of proteins and lipids (Table 1).

At d-93, juveniles (0.75 ± 0.02 g; 3.57 ± 0.02 cm; mean ± SD) were divided among 12 indoor 500 L tanks supplied with filtered and aerated natural seawater, six of which were maintained at 15°C and the other six at 20°C. Each tank contained 300 fish, representing a mean biomass of 263.93 ± 0.28 g. During the following 150 days, fish were fed one of the two experimental diets. Feeding took place for 7 h during daytime (08:00 to 15:00) using an automatic distributor (treadmill, 2 cm h -1 ); daily visual control was done to ensure that the food distributed had been consumed and that fish were fed ad libitum. Each diet × temperature combination was replicated in three tanks.

Tissue sampling

To account for the impact of temperature alone on fish growth, sampling dates were calculated in degree-days Peroxidation index, enzyme activity, and malondialdehyde assay

The peroxidation index (PI) was calculated for total dietary lipids and the polar fraction of muscle lipids (see [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF] for details of fatty acid content) following the equation provided by [START_REF] Hulbert | Life and death: metabolic rate, membrane composition, and life span of animals[END_REF]:

PI = (0.025 × % monoenoics) + (1 × % dienoics) + (2 × % trienoics) + (4 × % tetraenoics) + (6 × % pentaenoics) + (8 × % hexaenoics).
Liver samples were reduced to powder using a mixer mill (MM 400, Retsch, 30 Hz, 30 s) with liquid nitrogen. To extract total proteins, 100 mg of liver powder was diluted in 1 mL lysis buffer (150 mM NaCl, 10 mM Tris-HCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X100, 0.5% Igepal, 1% phosphatase inhibitor cocktail II, 2% NaPPi, and 1 tablet EDTA-free protease inhibitor cocktail, pH adjusted to 7). Samples were then homogenized on ice using a pro polytron® (BioBlock Scientific; Illkirch, Bas-Rhin, France). For protein extraction, samples were left on ice for 40 min and then centrifuged (3-30K Sigma, 6000 g, 1 h, 4°C). The middle phase containing the protein extract was recovered with a Pasteur pipette and centrifuged (3-30K Sigma, 10000 g, 45 min, 4°C). The resulting middle phase containing the protein extract was recovered with a Pasteur pipette, separated into aliquots, and stored at 80°C for the enzyme activity assay.

Total protein concentrations of the liver homogenates were assayed in triplicate according to the Bio-Rad DC Protein Assay (ref: 5000116, Bio-Rad), which is based on the Lowry assay [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF].

Specific CAT activity was measured in triplicate according to [START_REF] Aebi | Catalase in vitro[END_REF] by reading absorbance at 550 nm every 0.25 s for 1 min.

The specific enzyme activity of citrate synthase (CS) was measured in triplicate according to [START_REF] Srere | Citrate synthase[END_REF] and [START_REF] Bergmeyer | Enzymes as biochemical reagents[END_REF]. The assay medium contained 100 mM Tris-HCl (pH 8), 10 mM DTNB, and 2 mM acetyl-CoA. Liver homogenates and assay media were protected from light and incubated for 5 min in NUNC F-bottom 96-well plates (Thermo Scientific); 5 mM oxaloacetate solution was added to initiate the reaction. The kinetics of colouring solutions were monitored at 25°C by reading absorbance at 412 nm every 10 s for 4 min.

The lipid peroxide assay was adapted from the thiobarbituric acid reactive substances (TBARS) protocol (Uchiyama and Mihara, 1978). Liver samples were ground on ice using a POLYTRON PT-MR 2100 with distilled water containing 1% BHT in methanol, with 50 mg of liver for 1 mL of grinding solution. A total of 500 µL of 1% TBA and 1.5 mL of 1% phosphoric acid were added to 500 µL of liver homogenate. This mixture was heated for 30 min at 100°C and cooled for 30 min on ice, at which point 2 mL of butanol were added and the mixture vortexed. Protein precipitates were removed by centrifugation at 800 g. The absorbance of the supernatant was read in NUNC F-bottom 96-well plates (Thermo Scientific) with a

Multiskan GO spectrophotometer (Thermo Scientific) at 532 nm. The results were calculated using an MDA standard curve with butanol as the solvent.

mRNA expression analysis RNA extraction

Liver samples were reduced to powder using a mixer mill (MM 400, Retsch, 30 Hz, containing oligo (dT), random primers, and RNaseA inhibitor; reverse transcriptase. Reverse transcription (RT) negative controls were also performed on each sample using the same reaction mix except for the reverse transcriptase (substituted by water).The cDNA synthesis reaction was incubated for 5 min at 25°C followed by 30 min at 42°C and terminated by incubation for 5 min at 85°C to inactivate the enzyme. RT was performed using a T 100 Thermal-cycler (Bio-Rad Laboratories Inc.). cDNA samples were use.

Quantitative real-time RT-PCR analysis

The analysis of mRNA expression in liver tissue of European sea bass was carried out by qPCR using the primers listed in Table 2. Primers were designed using Primer3plus

(http://primer3plus.com/) based on sequences available from the Genbank database (https://www.ncbi.nlm.nih.gov/genbank/). mRNA expression was quantified using a C1000 touch thermal cycler (CFX96 system, Bio-Rad Laboratories Inc.). For each primer pair, qPCR efficiencies were estimated by standard curves resulting from serial dilutions (from 1/10 to

Results

Liver mass and protein content

Results showing the effect of diet, temperature, and time on liver mass and hepatic protein content are shown in Table 3A. After 720 dd of feeding, liver mass was significantly higher (+48%) in juveniles reared at 20°C than at 15°C, and diet had no significant effect (Table 3A). At 1660 dd, the increase in liver mass was higher for juveniles reared at 20°C (+67%) than

for those reared at 15°C (+59%), while liver mass was still higher (+58%) in juveniles reared at 20°C than at 15°C. Again, no diet effect was detected. Liver protein concentration decreased significantly over time (0.12 ± 0.01 mg protein mg liver -1 at 720 dd vs. 0.10 ± 0.00 mg protein mg liver -1 at 1660 dd), and no significant effect of diet or of temperature was observed.

Peroxidation index and MDA

The PI of the RD diet was 34% higher than that of the LD diet (15.86 ± 1.78 vs. 10.52 ± 0.50). Accordingly, the PI in the polar lipids of juveniles fed RD was significantly higher (+18%) compared to fish fed LD (45.36 ± 6.96 vs. 38.50 ± 5.45; Table 3A). The temperature × diet interaction was significant although no difference was detected with the post-hoc test (Table 3A). Surprisingly, MDA content was not affected by diet or temperature; however, MDA content increased with time (2.06 ± 0.48 vs. 2.77 ± 0.45 nmol mg protein -1 ; Table 3A).

Oxidative metabolism and antioxidant response

Results showing the effect of diet, temperature, and time on oxidative stress indicators are also shown in Table 3A. CS activity remained stable regardless of the experimental treatment, while COX activity varied depending on diet × temperature and degree-days × temperature interactions. There was no temperature effect on COX activity at 720 dd, but fish raised at 20°C exhibited a higher COX activity at 1660 dd than fish reared at 15°C. COX activity was significantly lower in fish fed RD at 720 dd, although this diet effect was no longer present at 1660 dd. The COX/CS ratio was higher (+15%) in juveniles fed LD than in those fed RD (6.01 ± 1.21 vs. 5.09 ± 1.80). In addition, the COX/CS ratio remained stable over time for fish raised at 15°C but increased in fish raised at 20°C. CAT activity increased slightly but significantly over time at both 15°C and 20°C (70 ± 7.93 vs. 105 ± 27.01). At 20°C, however, juveniles fed the RD diet displayed CAT activity that was more than twice the activity measured in LD-fed fish. At 1660 dd, the diet effect was no longer present. SOD activity was significantly lower ( 8%) in juveniles fed RD compared to those fed LD (3.94 ± 0.36 U mg protein -1 vs. 4.25 ± 0.43 U mg protein -1 ). SOD activity significantly decreased ( 8%) with time (4.25 ± 0.31 U mg protein -1 at 1660 dd vs. 3.95 ± 0.47 U mg protein -1 at 720 dd). There was also a significant increase in SOD activity (+10%) at the higher temperature (3.91 ± 0.43 U mg protein -1 at 15 °C vs. 4.29 ± 0.33 U mg protein -1 at 20°C).

Relative mRNA expressions

The relative mRNA expression of fads2 increased with time. At 720 dd, the relative level of fads2 mRNA was highest at 20°C in juveniles fed LD and lowest at 15°C in juveniles fed RD (Table 3, Fig. 1A). After 1660 dd of feeding, there was no significant increase of the fads2 mRNA level in juveniles fed LD at 15°C or RD at 20°C; however, the level was higher in juveniles fed RD at 15°C and LD at 20°C (Table 3B, Fig. 1A). The expressions of other mRNA showed no differences according to diet: expressions of fas, , and srebp-1 mRNA were significantly lower (158%, 29%, and 24%, respectively) for fish raised at 15°C than at 20°C (Table 3B, Fig. 1B). These expressions were respectively 630%, 91%, and 118% lower at 720 dd than at 1660 dd (Table 3B, Fig. 1B). The highest relative mRNA expression for was observed at 1600 dd for fish raised at 20°C (Table 3B, Fig. 1B); no treatment effect was observed for the expression of mRNA (Table 3B).

Discussion

In a previous study, [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF] showed that the depletion of n-3 PUFA and a lower temperature (15°C) contributed to a decreased growth rate in sea bass: the depleted n-3

PUFA diet resulted in lower contents of essential fatty acids (EFA, ARA, DHA, EPA) in muscle even though a higher retention of those FA was noted in fish fed this diet [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF].

The aim of the present work was to examine to what extent these diets and temperatures (15°C and 20°C) can affect the expression of mRNA coding for enzymes/transcription factors involved in lipid metabolism and biochemical variables related to antioxidant response in liver.

We hypothesized that a diet poor in n-3 PUFA would activate lipid biosynthesis while a diet richer in n-3 PUFA would lead to oxidative stress.

We are aware that our quantitative analyses of transcripts coding for actors involved in lipid metabolism do not necessarily reflect the activities of the proteins and thus cannot be used to infer metabolic activities. However, the relative levels of transcripts reflect the transcriptional regulation of metabolic constituents, which may reflect the sensitivity of the associated metabolic pathway to environmental or dietary factors.

mRNA expression, lipid metabolism, and antioxidant enzymes

Our results based on fads2 mRNA level quantification only partly support the hypothesis of an activation of the pathway involved in lipid biosynthesis. Indeed, the relative expression of fads2 mRNA was affected by diet, but even though the highest mRNA level was observed in LD-fed fish at 20°C on the first sampling date, the situation was completely reversed at the end of the study, making the results difficult to interpret. We observed no effect of diet on fas, , , , or srebp-1 mRNA expression. Regulation at translational and post-translational levels certainly cannot be excluded, but data on mRNA expression suggest no diet effects on these indicators at the dietary PUFA levels tested here.

This indicates that LD was probably not low enough in PUFA to exert negative effects at the transcriptional level on these constituents, even though it did result in a lower growth rate in both length and mass [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF]. Comparable conclusions were drawn by Yilmaz et al. ( 2016) for juvenile sea bass and by [START_REF] Tocher | Highly unsaturated fatty acid synthesis in marine fish: Cloning, functional characterization, and nutritional Gadus morhua L.)[END_REF] for cod. Our results contrast with those obtained for sea bass by Vagner et al. (2007aVagner et al. ( , b, 2009)), who showed that the fads2 mRNA level was higher in larvae fed a low PUFA diet (0.5% or 0.7% EPA+DHA, % DM) compared to those fed a high PUFA diet (1.7% or 3.7% EPA+ DHA, % DM). González-Rovira et al.

(2009) also showed a significantly higher fads2 mRNA level in the liver of sea bass fed linseed and rapeseed oils compared to those fed fish oil, while it remained stable in individuals fed a diet based on olive oil. Because all these sea bass studies were based on different dietary formulations and different developmental stages, it is difficult to determine why our results would differ from theirs. As mentioned above, previous studies reporting modulation of the elongation and desaturation pathways used diets that were highly deficient in EPA + DHA (Vagner et al. 2007a(Vagner et al. , b, 2009)). We could have used a diet that would have been more deficient.

However, LD was already sufficiently low in n-3 PUFA to significantly slow the growth rate, and an effect on fads2 mRNA level in the liver was expected. Obviously, the level of depletion we used does not seem to dramatically affect regulation of the lipid metabolism pathway because the requirements are still met in this species [START_REF] Skalli | Impact of essential fatty acid sea bass (Dicentrarchus labrax)[END_REF], even though this was not tested at all the possible steps from mRNA to the number of proteins.

There is growing evidence that inclusion of high levels of PUFA in fish diet, because of their high degree of unsaturation, can induce oxidative lipid damage [START_REF] Álvarez | Dietary fish oil and digestible protein modify susceptibility to lipid peroxidation in the muscle of rainbow trout (Oncorhynchus mykiss) and sea bass (Dicentrarchus labrax)[END_REF][START_REF] Luo | Effect of dietary arachidonic acid levels on growth performance, hepatic fatty acid profile, intermediary metabolism and antioxidant responses for juvenile Synechogobius hasta[END_REF]. Thus, we were expecting lower response indicators of oxidative stress in fish fed The COX/CS ratio was higher in LD-fed fish, suggesting higher aerobic metabolism and possibly indicating a functional adaptation of mitochondria to adjust energy demand and metabolic fuel availability (i.e., lipids biosynthesis). PI is a theoretical index that is calculated with in vitro oxidation rate values [START_REF] Holman | Autoxidation of fats and related substances[END_REF][START_REF] Cosgrove | The kinetics of the autoxidation of polyunsaturated fatty acids[END_REF][START_REF] Hulbert | Life and death: metabolic rate, membrane composition, and life span of animals[END_REF], and it does not account for all the in vivo processes that occur to deal with ROS production. CAT activity (degradation of hydrogen peroxide into water and oxygen) was twice as high in RD-fed juveniles compared to those fed LD, and they showed the highest PI which is consistent with our expectations but only at 20°C. On the other hand, MDA content remained stable regardless of diet, but we are aware that the method we used was not the most reliable one, even though it is commonly used in fish studies (Mourente et al., 2002;[START_REF] Simonato | Biomarkers of waterborne copper exposure in the neotropical fish Prochilodus lineatus[END_REF][START_REF] Sobjak | Assessment of the oxidative and neurotoxic effects of glyphosate pesticide on the larvae of Rhamdia quelen fish[END_REF]. However, the activity of SOD (catalyzing the dismutation of superoxide radicals into oxygen or hydrogen peroxide) was higher in juveniles fed LD than in those fed RD, suggesting the presence of more substrate to be catalyzed.

Temperature and diet effects

Higher temperatures in combination with poor n-3 PUFA could affect the lipid metabolism pathway. The relative mRNA expressions of fas, , and srebp-1 increased over time and with acclimation temperature, but the temperature effect was independent of the diet effect, indicating that a positive transcriptional regulation of lipid metabolism may occur at high temperatures. The fads2 mRNA expression tended to increase over time, but not significantly due to the high inter-individual variability within experimental treatments at 1660 dd. Such a variability in relative mRNA expression has been observed in this species [START_REF] Geay | Regulation of FADS2 expression and activity in European sea bass (Dicentrarchus labrax, L.) fed a vegetable diet[END_REF], and more investigation is required to determine the reasons. [START_REF] Gourtay | Will global warming affect the functional need for essential fatty acids in juvenile sea bass (Dicentrarchus labrax)? A first overview of the consequences of lower availability of nutritional fatty acids on growth performance[END_REF] showed that higher growth was observed in juveniles fed the same experimental diets at 20°C compared to 15°C, accompanied with higher polar DHA and ARA contents and neutral DHA content (only at 1660 dd for this parameter). Again, it must be remembered that the quantity of transcripts measured by qPCR does not necessarily reflect the amount of proteins, especially since temperature may affect the transcription translation relationship.

Nevertheless, our results support the hypothesis that acclimation temperature enhanced the expression of mRNA associated with regulation of the lipid metabolism.

In several organisms including fish, it has been shown that COX and CS activities reflect maximum aerobic metabolic capacity [START_REF] Childress | Depth-related enzymatic activities in muscle, brain and heart of deep-living pelagic marine teleosts[END_REF]Thuesen andChildress, 1993, 1994). Elevated temperature stimulates metabolic processes in ectotherms, increasing ATP demand and, therefore, electron transfers along the mitochondrial respiratory chain, production as a side product, which can imply higher likelihood of oxidative stress, but only if the rates of ROS production exceed the system's capacity to eliminate those ROS that result in oxidative stress. Increased environmental temperature has been associated with oxidative stress in fish [START_REF] Parihar | Lipid peroxidation and ascorbic acid status in respiratory organs of male and female freshwater catfish Heteropneustes fossilis exposed to temperature increase[END_REF]Heise et al., 2006a, b;[START_REF] Lushchak | Effects of different environmental oxygen levels on free radical processes in fish[END_REF][START_REF] Bagnyukova | Oxidative stress and antioxidant defenses in goldfish liver in response to short-term exposure to arsenite[END_REF]. Vinagre et al. ( 2012) studied the impact of temperature on oxidative stress in sea bass juveniles and found that lipid peroxidation and catalase activity were very sensitive to environmental temperature. Accordingly, SOD activity was higher at 20°C than at 15°C, and the highest CAT activity was observed at 720 dd in fish fed LD at 20°C.

Liver mass increased with time and temperature, but proteins remained relatively stable.

This means that total enzymatic activity (per mg of tissue) was related to liver mass only. At 20°C, total enzymatic activity should be higher and thus enable fish to cope with oxidative stress. The absence of change with temperature in the MDA content, which is one of the products of lipid peroxidation [START_REF] Frankel | Lipid oxidation[END_REF], again did not match the original hypothesis.

Some of the variables that we measured showed differences during the experiment without interaction with either diet or temperature. Thus, liver protein concentration and liver SOD activity decreased over time in juveniles, while the MDA content and relative mRNA expression of fads2 increased with time. Whether these modifications related to development or with acclimation to physical experimental conditions is hard to say, but obviously they were independent of diet or temperature conditions.

Conclusion

Our objective was to use long-term experimental conditions with treatment levels that were realistic in terms of a global change scenario to test the hypothesis that a diet poor in n-3

PUFA would activate lipid biosynthesis while a diet richer in n-3 PUFA would lead to oxidative stress and would worsen under higher temperature conditions. We were unable to confirm this working hypothesis. We conclude that both levels of n-3 PUFA tested in this study were high enough so as not to impair lipid metabolism in sea bass juveniles. The combination of a decrease in dietary n-3 PUFA along with an increase in temperature conditions will modify the oxidative stress response, but at a level that juveniles can cope with.

The authors declare that they have no conflicts of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. Experiments were performed under French national regulations and registration code C2EA 74) (Authorization APAFIS 3056# 20151207173873100). Each diet (LD and RD) was manufactured in a single batch of 50 kg to supply the entire experiment. Since diets were only produced once, there was no variation during the experiment. Srebp-1 0.77 ± 0.32 0.67 ± 0.18 1.12 ± 0.28 0.98 ± 0.27 2.13 ± 0.89 1.49 ± 0.74 2.12 ± 0.66 1.99 ± 0.71 P < 0.05

List of symbols and abbreviations

  (dd) relative to the first day of the experiment (D0) at dph-93 and 0 dd. At 720 dd after the beginning of the experiment (dph-129, D36, and dph-141, D48, at 20°C and 15°C, respectively) and at 1660 dd (dph-176, D83, and dph-204, D111, at 20°C and 15°C, respectively), 32 fish were randomly sampled from each tank (three tanks per treatment, N = 96 per treatment), anaesthetized (lethal concentration of MS-222, 200 mg L -1), and their livers excised. Because livers were small, the livers of eight juveniles were pooled for a total of four pools per tank and 12 per experimental condition. Pools were stored at 80°C. Two pools per tank were used for molecular biology analysis (N = 6 per experimental treatment for a total of 24), one pool was used for enzyme activity, and the fourth was used for malondialdehyde (MDA) analysis (N = 3 per experimental condition).

  by observing the decomposition of H2O2 into oxygen and water. The reaction mixture consisted of 20 mM phosphate (pH 7) and 10 mM H2O2. We used a Multiskan GO (Thermo Scientific) plate reader with GREINER UV-Star 96 (ref: M3812, Sigma). The kinetics of the colouring solutions were monitored at 22°C by reading absorbance at 240 nm every 10 s for 4 min. The specific enzyme activity of superoxide dismutase (SOD, combined Cu, Zn-SOD, and Mn-SOD) was determined in triplicate using a commercial kit (ref: 19160, Sigma), with a NUNC F-bottom 96-well (Thermo Scientific) and Multiskan GO (Thermo Scientific) plate reader. Final absorbances were read at 450 nm after 20 min incubations at 37°C. The specific enzyme activity of cytochrome c oxidase (COX) was measured in triplicate using a commercial kit (ref: CYTOCOX1, Sigma) combined with an Evolution 21 UV-Visible Spectrometer (Thermo Scientific). The kinetics of colouring solutions were monitored at 25°C

  30 s) with liquid nitrogen. Samples (50 100 mg) were placed in 1 mL of Extractall reagent (Eurobio; Courtaboeuf, Essone, France) and homogenized using a pro polytron® (BioBlock Scientific; Illkirch, Bas-Rhin, France) tissue disruptor for 30 s. Potential contaminating DNA ., Carlsbad, CA, USA) according to the Total RNA was extracted following the tions. The quantity and purity of RNA were assessed using an ND-1000 NanoDrop® spectrophotometer (Thermo Scientific Inc.; Waltham, MA, USA). RNA integrity was determined by electrophoresis using an Agilent Bioanalyzer 2100 (Agilent Technologies Inc.; Santa Clara, CA, USA). All samples had an RNA integrity number (RIN) higher than seven and could thus be used for real-time quantitative polymerase chain reaction (qPCR) analysis. RNA samples cDNA synthesis After extraction, cDNA Synthesis kit (Bio-Rad Laboratories Inc., Hercules, CA, USA) following the 500 ng total RNA was reverse transcribed into cDNA in a volume of 20 µL that was composed of 15 µL of sample;

  1/270) of a pool of cDNA. In the present study, qPCR efficiencies for each primer pair ranged from 95 to 100%, with R 2 > 0.99. Samples were then analyzed in triplicate. The final well (0.5 of each primer [10 µM] DNase-Supermix (Bio-Rad Laboratories Inc.) containing antibody-mediated hot-started iTaq DNA polymerase, dNTPs, MgCl2, SYBR® Green I dye, fluorescein, stabilizers, and enhancers. In each plate, negative controls (RT negative controls) were systematically included to ensure the absence of residual genomic DNA contamination. Non-template controls (H2O) were also analyzed. Each plate contained an inter-run calibrator in triplicate (dilutions of a pool of all samples). The qPCR thermal cycling included an initial activation step at 95°C for 2 min followed by 39 cycles of 5 s at 95°C and 20 s at 60°C. A melting curve was performed at the end of the amplification phase to confirm the amplification of a single product in each reaction. The corresponding Cq (quantification cycle) value was determined automatically for each sample using the Gene Expression Module of the CFX Manager software (Bio-Rad Laboratories Inc.). Cq is the number of cycles required to yield a detectable fluorescence signal. The CFX Manager Software was used to normalize the relative quantity of messenger with the The expression level of elongation factor 1 (ef1a) and ribosomal (28S) mRNA were tested to correct for loading differences or other sampling variations present in each sample. The ef1a mRNA was used for normalization in the liver tissue of juvenile fish since it did not show any significant variation of expression among experimental conditions (P > 0.05). Statistical and data analysis Relative mRNA expressions and enzyme activity data were log10 transformed to obtain normal distributions. The effects of time, temperature, and diet were tested for peroxidation index, relative mRNA expression (ppar , , , fas, srebp-1, fads2), enzyme activity (CAT, SOD, CS, COX), and MDA content using three-way ANOVAs. Normality and homoscedasticity of data were tested using Shapiro-Wilk and Levene tests, respectively. When appropriate, Tukey mean comparison tests were done because homoscedasticity was respected. (ver. 3.3.3; R Development Core Team).

LD.

  The CS enzyme catalyzes the first step of the tricarboxylic acid cycle, while COX has a specific role as terminal electron acceptor in the electron transport system. They are both key enzymes involved in aerobic metabolism and can be used as indicators of aerobic metabolic capacity (Marie et al. 2006). The tricarboxylic acid cycle in liver also supports biosynthetic processes such as lipid biosynthesis or gluconeogenesis from malate (Owen et al., 2002; Windisch et al., 2011). Accordingly, the COX to CS ratio can be used to reflect preferred metabolic pathways and relative metabolic adjustments in response to warming in a tissue. Furthermore, changes in COX activity may be related to alterations in mitochondrial membrane structure (Wodtke, 1981; O'Brien and Mueller, 2010) and those in CS activity to changes in mitochondrial matrix volume (e.g., Hardewig et al., 1999; Guderley and St-Pierre, 2002; Guderley, 2004). In our study, COX activity was the lowest in fish fed RD at 720 dd; low COX activity can suggest decreased oxidative capacities, which may support lower metabolic costs with the RD diet.

  possibly explaining the higher COX activity and COX/CS ratio recorded at 20°C. These results corroborate those of Seebacher et al. (2005) in Pagothenia borchgrevinki, which responds to warming by increasing muscle COX activity. The authors suggest that high COX activity comes along with increased oxidative capacities, and these may support elevated metabolic costs at warmer temperatures. Such circumstances are also likely to enhance ROS

FiguresFigure 1 : 1 .

 11 Figures

*

  Effect of diet, temperature, and time on liver oxidative stress indicators (N=3) and B) lipid metabolism (N=6). When factor interactions 768 were significant, groups were compared with Tukey post-hoc tests ( ; significantly different groups were assigned different letters. RD: 769 reference diet, LD: low n-3 polyunsaturated fatty acid diet, dd: degree-days, Temp.: temperature. No significant effect of diet was observed; diet column has been removed from the table.

  775

F

  15 ± 0.07 a 0.25 ± 0.07 ab 0.24 ± 0.14 ab 0.29 ± 0.28 b 2.15 ± 0.93 c 0.87 ± 0.58 bc 1.29 ± 1.07 bc 1.28 ± 0.83 c n.s. n.s. Time × Temp. × Diet P < 0.05

F

Table 1 :

 1 Composition of experimental diets. For dry matter, proteins, total lipids, triglycerides, and phospholipids, data are presented as % of dry mass. Data for specific fatty acid (FA) Calculated as %proteins × 16.7 kgJ g -1 + %total lipids × 37.7 kgJ g -1 + NFE × 16.7 kgJ g-1

	categories are presented as % of total lipids. LD: low n-3 polyunsaturated fatty acid (PUFA)
	diet, RD: reference n-3 PUFA diet; SFA: saturated FA; MUFA: monounsaturated FA; ARA:
	arachidonic acid; EPA: eicosapentaenoic acid; DHA: docosahexaenoic acid
		Low n-3 PUFA diet	Reference n-3 PUFA diet
		LD	RD
		Proximal composition (%DM)
	Dry matter	94.84	95.12
	Ash	5.16	4.88
	Proteins	50.48	50.23
	Nitrogen Free Extract (NFE) a	22.38	23.26
	Total lipids	21.98	21.63
	Triglycerides	16.99	17.05
	Phospholipids	4.70	4.71
	Energy (kJ) b	2045.41	2042.73
		% of total lipids in diet
	SFA	2.18	2.97
	Monosaturated FA	7.32	6.48
	n-3	1.93	2.68
	n-6	2.65	2.28
	n-9	6.56	5.46
	EPA+DHA	0.73	1.65
	18:1n-9	5.69	4.65
	18:2n-6	2.50	2.16
	18:3n-3	0.97	0.77
	18:3n-6	0.00	0.01
	18:4n-3	0.08	0.14
	20:4n-6	0.03	0.07
	20:5n-3	0.28	0.94
	22:5n-3	0.03	0.07

b

Table 2 :

 2 Specific primers used for quantitative PCR with Genbank accession numbers

766

*Used as housekeeping gene

Acknowledgements

Mer (Ifremer; doctoral support to C. Gourtay) and the Natural Sciences and Engineering Research Council of Canada (Discovery Grant to C. Audet; financial support to C. Gourtay). The authors are grateful to the Aquastream company and to all members of the Adaptation, Reproduction and Nutrition Lab (Ifremer, Brest), past and present, who contributed to this work.

Compliance with ethical standards

Conflict of interest