Lea Joly

Christophe Loots

Cédric L Meunier

Maarten Boersma

Sophie Collet

Valerie Lefebvre

Jose-Luis Zambonino-Infante

Carolina Giraldo
email: carolina.giraldo@ifremer.fr

Joly Lea

Loots Christophe

Boersma Maarten

Collet Sophie

Giraldo Carolina

Maturation of the digestive system of Downs herring larvae (Clupea harengus, Linnaeus, 1758): identification of critical periods through ontogeny

published or not. The documents may come

Introduction

North Sea herring is a commercially and ecologically important stock of North Atlantic herring (Clupea harengus). This stock has experienced high biomass fluctuations, with an almost complete fisheries induced collapse in the past, resulting in the closure of the fishery in the 1980's [START_REF] Dickey-Collas | Lessons learned from stock collapse and recovery of North Sea herring: A review[END_REF]. Since then, the stock has recovered and, as a result of the very tight regulations, is currently sustainably exploited. Nevertheless, years of poor recruitment, i. e. the quantity of juvenile fish entering the adult population, are regularly reported despite high adult biomass levels. This suggests that other poorly understood factors are responsible for variability in larval survival [START_REF] Payne | Recruitment in a changing environment: The 2000s North Sea herring recruitment failure[END_REF]).

The economic implications of fish stock inter-annual variability have motivated research on population dynamics and fish biology for more than a century [START_REF] Hart | [END_REF]. [START_REF] Hjort | Fluctuations in the great fisheries of northern Europe viewed in the light of biological research[END_REF] was the first to point out larval survival, and not only spawning stock biomass, as a key factor determining annual recruitment and size of fish populations. He defined the transition from endogenous to exogenous nutrition as the critical period ("Critical period hypothesis"), i.e. the period when the larvae start feeding, and are most sensitive to starvation. This first-feeding stage has been shown to be a bottleneck period for Western Baltic Sea herring, where young larvae reaching this critical period before the plankton bloom contribute little to the surviving year-class [START_REF] Polte | Survival bottlenecks in the early ontogenesis of Atlantic herring (Clupea harengus, L.) in coastal lagoon spawning areas of the western Baltic Sea[END_REF]. Different recruitment hypotheses based on hydrodynamic and trophodynamic factors have been put forward since, and it is now well accepted that biological and physical processes act in concert to determine the survival of fish at early life stages [START_REF] Houde | Emerging from Hjort's shadow[END_REF], from fertilization to metamorphosis and beyond. For instance, feeding success and subsequent growth rate are advocated as two of the most important factors influencing larval survival and recruitment [START_REF] Anderson | A Review of Size Dependant Survival During Pre-recruit Stages of Fishes in Relation to Recruitment[END_REF]Bergenius et al. 2002;Jenkins and King 2006;Robert et al. 2007).

Thus far, condition and physiological state of field-caught larvae have been used as indicators of suitable environmental condition and potential recruitment success. These are typically measured by indices, including morphometric, histological and biochemical measurements [START_REF] Ferron | An appraisal of condition measures for marine fish larvae[END_REF]. These indices are used to assess directly or indirectly the nutritional status of organisms, or to infer their ability to bear starvation [START_REF] Ehrlich | Morphological and histological changes during the growth and starvation of herring and plaice larvae[END_REF]. Some of them are widely used in larval fish surveys, such as the relative content of RNA to DNA in tissue, or the size corrected weight of individuals. Recently, [START_REF] Denis | Ontogenetic changes in the larval condition of Downs herring: use of a multi-index approach at an individual scale[END_REF] observed that Downs herring larvae, one of the most important spawning components of North Sea herring, undergo a change in their condition at around 13 mm. The authors observed a reduction in instantaneous growth rate (by RNA: DNA) along with an increase in ingestion rate and suggested this to be a possible critical period resulting from a change in the energy-allocation strategy (i.e., from growth to energy reserves). Because the response of RNA:DNA ratios differs with larval size [START_REF] Foley | A review and assessment of the potential use of RNA:DNA ratios to assess the condition of entrained fish larvae[END_REF] the former hypothesis of a second critical period needs to be validated with additional independent and well-controlled approaches. It is known that ontogeny and organogenesis of the digestive system can influence the physiological status of larvae [START_REF] Suthers | Significance of larval condition: comment on laboratory experiments[END_REF]. Hence, knowledge of the development and maturation of the digestive tract, as well as the dynamics of energy storage by accessory glands, would be very helpful for a precise interpretation of field data. Furthermore, the study of ontogenic transitions could be of importance to determine critical periods during the development at which suboptimal feeding conditions could directly affect survival and recruitment.

Marine fish larvae are not fully developed at hatch and undergo simultaneous major ontogenic changes in morphology, anatomy and physiology. Growth and development are linked to the nutritional status of individuals which relies on internal storage, and after first feeding, on food quality, availability and functionality of digestive organs [START_REF] Ronnestad | Feeding behaviour and digestive physiology in larval fish : current knowledge , and gaps and bottlenecks in research[END_REF]. Maturation of the larval digestive tract is defined as the acquisition of adult digestive features; this process has been widely studied for digestive enzymes in teleosts, especially in the context of aquaculture (Zambonino [START_REF] Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]. Most fish species lack a morphologically distinct and functional stomach (characterized by the appearance of gastric glands, (Zambonino Infante and Cahu 2001)) before metamorphosis, and digestion processes in larvae mainly depend on other digestive organs, especially the pancreas [START_REF] Ronnestad | Feeding behaviour and digestive physiology in larval fish : current knowledge , and gaps and bottlenecks in research[END_REF]). Among pancreatic enzymes, trypsin activity has been related to food ingestion [START_REF] Pedersen | Variations in the content of trypsin and trypsinogen in larval herring (Clupea harengus) digesting copepod nauplii[END_REF]) and has been used to experimentally identify starving from well-fed herring larvae. Trypsin measurements reflect changes in food availability and nutritional condition over shorter timescales than RNA/DNA ratios which has been estimated > 5 days [START_REF] Ueberschär | A comparison of the nutritional condition of herring larvae as determined by two biochemical methods -tryptic enzyme activity and RNA/DNA ratio measurements[END_REF][START_REF] Foley | A review and assessment of the potential use of RNA:DNA ratios to assess the condition of entrained fish larvae[END_REF]. Intestine maturation is characterized by changes in enterocytes enzymatic activities, i.e. a decrease in cytosolic activities concomitant with a sharp increase in brush border membrane enzyme activities (Zambonino [START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF].

Consequently, digestive enzymes activities are used as indicators of larval development and potential of survival. Although these aspects are essential to understand larval recruitment, which is necessary to assess stock fluctuations, there is a striking lack of information on the ecologically and economically important North Sea C. harengus. Teleosts have similar developmental patterns, but large species-specific variations exist regarding organ differentiation timing and metabolic efficiency [START_REF] Gisbert | Nutritional cellular biomarkers in early life stages of fish[END_REF]. For a more complete assessment of potential recruitment success it is hence essential to acquire in-depth knowledge on the processes during larval development, both structural changes as well as changes in physiology.

In the present study, we focused on North Sea herring (Downs component) and aimed to (1) describe the development and maturation of the digestive tract and associated organs (oesophagus, stomach, intestine, liver and pancreas), and (2) identify physiological changes that could potentially be linked to critical periods during ontogeny. To do so, herring larvae were reared in the laboratory from hatch to the last larval stage, and digestive enzyme activities (trypsin, alpha amylase, leucine-alanine peptidase, aminopeptidase N and alkaline phosphatase) as well as histological structure of digestive organs were assessed at different times through development. Hence, the present study provides important basic information on the structural and biochemical changes in the digestive system throughout the larval stages of herring that can be useful for future work assessing the condition of field-caught larvae in relation to environmental conditions.

Materials and methods

Artificial fertilization

Mature herring from the Downs component were obtained in November 2018 from a local organization of fishermen (Coopérative Maritime Etaploise, CME) in Boulogne-Sur-Mer (France).

Artificial fertilization was carried out using ready-to-spawn fish at the Ifremer Manche-Mer du Nord laboratory. Eggs of 27 females (mean standard length: 27.2  (SD) 1.92 cm; mean weight: 166.4  32.54 g) were stripped-spawned in seawater and placed on four plastic plates (PVC) of 490 cm 2 each before being fertilized with the sperm of 9 males (mean standard length: 28.0  3.84 cm; mean weight: 179.2  32.0 g). The eggs and sperm were incubated for 10 minutes at 13.5°C and 32.7 psu, before rinsing. To ensure genetic diversity, on each plate eggs from at least six females were spread and the mixed sperm of at least two males was added for the fertilization. Along with the four PVC plates a smaller slide of 21 cm 2 was used to monitor closely the fertilization and eggs development without having to take out of water the eggs intended for the experiment. Eggs were strippedspawned on the slide and fertilized with the same protocol in order to give us an approximation on fertilization success. The fertilization success of the slide was of 42%. Around 200 eggs were laid on 21 cm 2 , thus we can estimate that approximately 4600 eggs were present on each plate of the experiment.

The plates were transported to Ifremer-Centre de Bretagne laboratory in boxes with oxygenated and thermoregulated sea water (variation range: 13.5 to 15°C). Eggs were then incubated in a 200 L tank in the dark. For the entire experiment, natural seawater was pumped from the Bay of Brest (32.5 psu, pH 8.0), passed through a sand filter, heated to avoid natural temperature fluctuations, filtered (2 µm) and UV sterilized (PZ50, 75W, Ocene, France).

Larval rearing

Most of the eggs hatched on the 9 th day of incubation (starting point of the experiment: 1 dph) and larvae were kept until 69 days post hatch (dph). Three days after hatching (3 dph) about 4500 larvae were counted and distributed equally in three 38 litres conical black tanks (1500 larvae by tank), constituting the replicates of the experiment. A continuous flow-through system of 20 L h-1 was used and the temperature was set to 13°C (13.3 ± 0.25°C). Oxygen saturation was measured daily with an oximeter (WTW Oxi 340, Bioblock scientific) and was always higher than 98%. In order to prevent any food limitation, the daily food quantity was distributed four times during the day to maintain an ad libitum level, ensuring that there were always prey in the tank during the day. To be sure to allow proper feeding through time we used an increasing range of living prey sizes from phytoplankton to 24 h old nauplii, before weaning with feed granules.

Rhodomonas salina (Strain 2002) and Oxyrrhis marina (Strain 21.89) were obtained from the Culture Collection of algae (University of Göttingen, Germany). R. salina were grown in 10 L bottles under constant light in enriched Conway medium to provide quick growth (N and P concentrations doubled). O. marina were cultivated in a 5 L bottle under constant lighting and fed R. salina. Nauplii of the commercially available Artemia salina (VNBS, Viepearl) were hatched in 60 L tanks after 24 h at 28°C, and directly used to feed larvae early in the development. Thereafter, artemia nauplii were enriched 24 h (A1) in a mixture of fish oil (FO) and baker yeast (BY) (For 1 Million artemii = 4.3 g FO and 11.1 g BY).

The feeding protocol of [START_REF] Moyano | Thermal impacts on the growth, development and ontogeny of critical swimming speed in Atlantic herring larvae[END_REF] was followed for early feeding. Yolk sac larvae were fed the microalgae R. salina (mean: 30 000 cells mL -1 ) and the heterotrophic protist Oxyrrhis marina (mean: 250 cells mL -1 ). Two days after mouth opening, Artemia salina nauplii (A0) were supplied in the tanks (mean: 70 A0 larvae -1 d -1 ). From 22 to 39 dph a mix of A. salina nauplii (mean: 140 A0 larvae -1 d -1 ) and 24 h old enriched artemii (mean: 140 A1 larvae -1 d -1 ) were added. After that, A1 were supplied in decreasing quantities (mean: 200 A1 larvae -1 d -1 ) while introducing feed granules (Neo Supra, Le Gouessant, 58% proteins, 13% lipids) until 49 dph (from 0.2 to 0.5 g tank -1 d -1 ). From 49 to 69 dph, larvae were fed only with the granules (1 g tank -1 d -1 ). The lighting sequence of the tanks was modeled on aquaculture protocols. The lighting period was of 24 h a day, progressively increased from 1 to 59 lux along the development, in order to maximize larval feeding, in particular for young herring larvae which possess only cone cells prior to metamorphosis [START_REF] Bell | The appearance of rods in the eyes of herring and increased didocosahexaenoyl molecular species of phospholipids[END_REF].

Larval sampling

A total of 761 larvae of C. harengus were sampled and measured (Total Length, TL in mm) throughout the experiment to estimate growth rates. A subsample of those individuals were used for enzyme analysis. Larvae were collected with a sieve in the morning, before feeding, and euthanized in ice water. As larvae are fragile organisms, homogeneous sampling through the tanks inevitably leads to a slight increase in mortality. Thus, for histological individual analysis we decided to limit the number of larvae sampled at the beginning of development and to increase it progressively according to the progress and success of the rearing. The samplings were adjusted to not go below a minimum, non-stressful, density of 25-30 larvae.L -1 per tank. Four vitelline larvae were directly sampled from the hatching tank at 3 dph. After that, from 2 to 13 larvae per tank were sampled at 12,19,21,25,28,35,49 and 69 dph. The larvae were preserved for 48 h in Bouin's solution, then rinsed and preserved in 70% ethanol in the dark until analysis. To describe the morphological development, larval stage and size were defined under binocular observations for 4 larvae (at 3 dph) + ((2-10 larvae) * 7 (sampling dates) * 3 (replicates)) + (13 larvae * 3 (replicates)) = 169 larvae. To have a reference value on the level of shrinkage of the conservation technique, larvae sampled at 69 dph were measure before and after conditioning (13 larvae per tank). A total of 35 larvae were used to histologically describe the digestive system development through time.

The developmental stage (Fig. 1) for each larva was determined according to [START_REF] Doyle | A Morphological Staging System for the Larval Development of the Herring[END_REF]: 1) yolk sac larval stage, 2) pre-flexion stage; yolk sac absent and straight notochord, 3) flexion of the notochord stage and 4) post flexion stage with visible pelvic fins. We sampled larvae for enzyme activity analyses from 14 dph onwards and pooled their tissue for spectrophotometric assays. Between 30 and 50 larvae were sampled in each tank for enzymes analysis at 14, 21, 28, 35 and 49 dph. The larvae were put in vials and stored at -80°C, yielding a total of 5 (sampling days) * 3 (replicates) = 15 samples.

Organogenesis of the digestive system

Organogenesis of the digestive system was studied using histology. Individuals were dehydrated and embedded in paraffin blocks, as described by Di [START_REF] Pane | Ontogenetic shift in the energy allocation strategy and physiological condition of larval plaice (Pleuronectes platessa)[END_REF], substituting the xylene by diasolv (non-Carcinogenic, Mutagenic or Reprotoxic substitute of xylene/toluene). Sagittal section of 7 µm were made with an automated-microtome (Leica, RM2255) and mounted on slides. Larval sections were dewaxed and rehydrated in successive baths of diasolv and alcohol progressively less concentrated (100 %, 95%, 70%) and tap water. Two histochemical colorations were used on different larvae. Half of them were stained using alcian blue (AB) pH 2.5 alone. The other half was double-stained with Periodic acid-Schiff (PAS) to stain glycogen and neutral mucosubstances, and alcian blue (AB) pH 2.5 for acid mucosubstances. Then all the slides were stained with Groat's hematoxylin and picro-indigo carmine for topographic coloration. Between 3 to 6 larvae per sampling date were observed under a microscope (Leica, DM6B) to describe the development of the larval digestive system. We focused on the histological organization of the digestive tract (oesophagus, stomach, intestine) and accessory digestive glands (liver and pancreas) (Table 1). Presence of zymogen, inactive enzymatic precursor, in the pancreas and glycogen, indicative of energy reserve, in the liver were examined. The liver condition and reserve amount were scored based on hepatocytes vacuoles characteristics following the protocol developed by Di [START_REF] Pane | Ontogenetic shift in the energy allocation strategy and physiological condition of larval plaice (Pleuronectes platessa)[END_REF]. Image acquisition was done with a software (LAS X) and a camera (Leica, DMC4500) connected to the microscope. [START_REF] Cahu | Early weaning of sea bass (Dicentrartrus labrax) larvae with a compound diet: effect on digestive enzymes[END_REF]) in order to obtain pancreatic and intestinal segments. Samples were homogenized in five volumes (w/v) of ice-cold distilled water.

Brush borders were extracted and purified from the intestinal segment [START_REF] Crane | Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small digfish (Scyliorhinus canicula )[END_REF]).

Homogenates of younger larvae and pancreatic segments from older larvae were used to determine activities of pancreatic enzymes, i.e. trypsin and alpha amylase, according to [START_REF] Holm | Metabolism and Hormonal Regulation High and Low Inhibitor Soybean Meals Affect Human Duodenal Proteinase Activity Differently : In Vivo Comparison with Bovine Serum Albumin[END_REF] and Métais and Bieth (1968) respectively. Cytosolic leucine-alanine peptidase activity was assessed from intestinal homogenates [START_REF] Nicholson | A One-Step for Intestinal Peptide Acid Oxidase Assay Hydrolase Activity A number of methods have been described for the assay of intestinal peptide hydrolase activity . The spectrophotometric assay described by Josefsson and Lindberg ( 1 ) is not suitable[END_REF]. Assays of the brush borders enzymes were performed for alkaline phosphatase and aminopeptidase N following [START_REF] Bessey | Rapid coloric method for determination of alcaline phosphatase in five cubic millimeters of serum[END_REF] and [START_REF] Maroux | The aminopeptidase from hog-intestinal brush border. Métais P, Bieth J (1968) Détermination de l'α-amylase par une microtechnique[END_REF], respectively. All measurements were carried out using a spectrophotometer (Thermo Scientific, Evolution 201) at 37°C.

For each analysis, protein content was determined in the sample with Bradford's method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]). All the activity measurements were done three times for each sample and averaged to give a value per tank and time. Enzymatic activities were expressed as specific activities in mUnit mg protein -1 and segmental activities, i.e. the total activity of one enzyme per larva in the intestinal segment. The level of intestinal maturation was calculated as the ratio of brush borders enzymes related to cytosolic enzymes based on the segmental activities (Zambonino [START_REF] Infante | Partial Substitution of Di-and Tripeptides for Native Proteins in Sea Bass Diet Improves Dicentrarchus labrax Larval Development 1[END_REF].

Statistical analyses

Data analyses were performed using the R software (R core Team, 2019) with an alpha level of 0.05. Differences in growth rates among tanks were investigated with a linear mixed-effects model (LMEM) using the package "lme4" and the function lmer [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. "Length" was the outcome variable with "dph" used as a fixed effect and "tank" as a random effect. Following recommendation by [START_REF] Barr | Random effects structure for confirmatory hypothesis testing: Keep it maximal[END_REF] we fit a maximal random effects structure which allows for random slopes (for the different tanks) and random intercepts. The function ranova() from the "lmerTest" package [START_REF] Kuznetsova | lmerTest Package: Tests in Linear Mixed Effects Models[END_REF]) was then used to test the effect of the random effect on the model (likelihood ratio tests), and normality of residuals was checked. The random effect "tank"

was not significant (P > 0.05) and so replicates of the different tanks were pooled for subsequent analysis. The data are expressed as mean  SD. Enzymes data normality was checked with Shapiro's test and homoscedasticity with Bartlett's test. As normality and homoscedasticity were respected, specific activities and enzymatic ratio data were compared by one-way ANOVA, with time as a factor, followed by post hoc HSD Tukey tests when significant differences were found.

Results

Size distribution during developmental stages

LMEM fixed effect estimate of the variation of length over time was of 0.22 mm.d -1 (Table 2). All larvae presented a yolk-sac and hatched without a functional mouth but with fully pigmented eyes. The mouth opened at 3 dph (8-9 mm) and the yolk sac was fully resorbed at 8 dph (9-10 mm).

Phytoplankton was observed in the intestine from 5 dph (8-10 mm), and artemia nauplii from day 7, before total resorption of the vitellus. Additional to size measurements, the developmental stage was determined for 169 larvae sampled for histological analysis. Size ranges between each stage are well separated (Fig. 2). Stage 1 larvae were found at 3 dph, the larvae were thin and long with a straight notochord, had a visible intestine and vitelline reserves. In stage 2 larvae between 12 and 35 dph, the vitellus was totally resorbed and the caudal and dorsal fins started to differentiate. For stage 3 larvae between 21 and 35 dph, body height was higher, the intestine bigger and the caudal, dorsal and anal fins fully differentiated.

Melanophores were also present along the superior part of the intestine and some above the head and on the caudal fin. For stage 4 larvae from 35 to 69 dph, all the body features were bigger, the whole body was thicker and the pelvic fins started to differentiate. Melanophores were present all over the body and concentrated above the intestine, on the dorsal part of the body, above the head, around the mouth and on the dorsal fin. The measurements came from individuals stored successively in Bouin's solution and 70% ethanol for histology. The conditioning method can result in shrinkage of the larvae. Individuals measured before and after conservation shrank by an average of 11.2 % ( 7.4 %, n = 39).

Digestive system development

Yolk-sac stage (stage 1)

At 3 dph, larvae were around 8 mm and the mouth was open with the yolk sac still present. The oesophagus was relatively short, narrow and folded, with an important amount of goblet cells (Fig. 3c). Only acid mucosubstances (AB positive, Fig. 4a) were detected. The stomach was not yet morphologically differentiated and appeared as a simple cavity with short and mostly flattened epithelial cells (Fig. 3d). In the intestine, the enterocytes formed a straight thick layer with some goblet cells in between, producing acid mucosubstances (AB positive). Microvilli forming the brush border at the top of the cells were visible (Fig. 3e). Pancreas and liver had an elongated shape going from the back of the pectoral fins to the beginning of the intestine. Pancreatic cells were organized into weakly distinct clusters with a moderate amount of zymogen (Fig. 3g). Only exocrine pancreas was visible. The liver had large cells with large vacuoles (Fig. 3f) and a pronounced pink (PAS-positive)

tint highlighting an important quantity of glycogen in the cells. At 12 dph, larvae were around 10 mm, the tissular organization was quite similar to the one described for stage 1 except for the yolk sac which was completely resorbed. The goblet cells in the upper part of the oesophagus were also tinted in blue, revealing acid mucosubstances production (Fig. 4b). Some stage 2 larvae showed few violet goblet cells in the lower part of the oesophagus, revealing both acid and neutral mucosubstances production (violet tint: AB and PAS positive).

Post-flexion stage (Stage 4)

Stage 4 is the last stage before metamorphosis into a juvenile. Internal and external morphology of the larvae become increasingly closer to that of adults (Fig. 5a). The oesophagus and its longitudinal folds were longer and the interstice wider than in stage 3 (Fig. 5b). As for the stage 3, two distinct parts were identified by the goblet cells mucus production, the coloration was stronger than before suggesting an increase in the quantity of acid and neutral mucosubstances produced respectively in the superior and inferior part of the organ (Fig. 4d). Epithelial cells of the stomach were higher at 49 dph (Fig. 5c). At 69 dph a coloration appeared in the stomach, the stomacal glands were not observed. Most of the larvae presented a strong blue coloration (AB positive: acid mucosubstances)

for two third of the stomach length and a strong pink coloration (PAS positive: neutral mucosubstances) in the last part of the stomach directly connected to the pylorus. At 69 dph, the intestine was larger, the villi were prominent and the ceca pyloric were visible after the pylorus (Fig. 5e,d). At 49 dph the acini were still distinct but at 69 dph the structure of the exocrine pancreas was more diffuse. At the end of the stage 4, one large endocrine islet was still present in the middle of the pancreas and small islets were observed on either side of the larger one (Fig. 5f). The pancreas expanded further along the intestine. The liver of stage 4 larvae, like for stage 3, was highly variable with some individuals presenting numerous and large vacuoles (Fig. 5g,6a) while for others the vacuoles and amount of glycogen were reduced or absent (Fig. 6b,c).

Activity of digestive enzymes

The level of maturation of the digestive system was determined based on the variations in the intestinal enzymes activities (Cahu and Zambonino Infante, 1995). Trypsin specific activity remained constant from 14 to 49 dph (31.0  5.11 mU mg protein -1 ; ANOVA, F (4,10) = 1.67, P = 0.233) (Table 3). Amylase specific activity was different through time (ANOVA, F (4,10) = 12.14, P < 0.01), a post hoc Tukey test showed that the activity was higher at 14 dph (P < 0.05) and decreased later from 5079  470.6 to a mean of 818.1  324.7 mU mg protein -1 at 49 dph. Intestinal enzymes specific activities were tested for larvae at 28, 35 and 49 dph. No statistical significant differences were found for the intestinal enzymes specific activities Aminopeptidase N (ANOVA, F (2, 6) = 2.79, P = 0.139),

Alkaline phosphatase (ANOVA, F (2, 6) = 4.80, P = 0.057) and Leucine-alanine peptidase (ANOVA, F (2, 6) = 0.637, P = 0.561), at the three different ages. The evolution of the ratio between segmental activities of the brush border and cytosolic enzymes differed through time (AP/Leu-Ala: ANOVA, F

(2,6) = 9.55, P = 0.014; AminP/Leu-Ala: ANOVA, F (2,6) = 8.37, P = 0.018), a post hoc Tukey test highlighted a shift between 28 and 35-49 dph (Fig. 7, P < 0.05).

Table 3 Summary of the specific activities (mU mg protein -1 ) of pancreatic (trypsin and amylase) and

Discussion

Here, we report for the first time a detailed description of the development and maturation of the digestive system of Down's herring larvae, an important component of the North Sea herring stock.

The larval growth rate obtained during our experiment is similar to other experiments on Atlantic herring [START_REF] Ehrlich | Morphological and histological changes during the growth and starvation of herring and plaice larvae[END_REF][START_REF] Moyano | Thermal impacts on the growth, development and ontogeny of critical swimming speed in Atlantic herring larvae[END_REF]) and field data estimates on Downs [START_REF] Denis | Ontogenetic changes in the larval condition of Downs herring: use of a multi-index approach at an individual scale[END_REF]. The combination of histological and enzymatic approaches allowed us to identify and discuss developmental periods at which herring larvae may be more vulnerable to fluctuating environmental conditions and suboptimal feeding conditions.

Maturation and functionality of the digestive system

Anatomical and cellular changes of the oesophagus, stomach and intestine as well as accessory glands drive the digestion processes, the efficiency of nutrients transport and absorption. Functional timing of development is the major difference between fish species and appears to be genetically programmed [START_REF] Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF].

Oesophagus

In teleosts, the alimentary canal generally develops from an undifferentiated straight tube at the yolk-sac stage to a segmented one where the oesophagus, the stomach and the intestine are differentiated. For Down's herring larvae, at 3 dph, all digestive organs, including the liver and pancreas were already distinguishable. With the opening of the mouth at around 3 dph, presence of * * microplankton in the gut was observed before yolk sac resorption in agreement with previous observations [START_REF] Checkley | Selective feeding by Atlantic herring (Clupea harengus) larvae on zooplankton in natural assemblages[END_REF][START_REF] Munk | Foraging behaviour and prey size spectra of larval herring Clupea harengus[END_REF]. [START_REF] Busch | Transition from endogenous to exogenous nutrition: Larval size parameters determining the start of external feeding and size of prey ingested by Ruegen spring herring Clupea harengus[END_REF] showed that the first feeding, on prey larger than phytoplankton cells, is allowed by the decreasing of the yolk sac diameter. He argued that the oesophagus diameter increases by mechanical action in response to a decrease in the pressure exerted by the yolk sac when it is consumed. Consumption of larger preys is also facilitated by the presence of goblet cells that play different roles but are mostly involved in the lubrication of the digestive system (Zambonino [START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF]. Goblet cells can be present before the onset of exogenous feeding or appear later in the development depending of the species [START_REF] Lazo | Ontogeny of the Digestive Tract[END_REF]). In addition, they also differ in their histochemical features. For example, in dover sole, senegal sole and yellowtail flounder, oesophagus goblet cells only secrete acid mucosubstances (Zambonino [START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF], whereas a mix of neutral and acid glycoconjugates were observed for the california halibut and white seabream. The nature of this secretion varies along the ontogeny of herring larvae, there is an initial production of acid mucus during stage 1 and 2 and an increased production of neutral mucosubstances for stages 3 and 4. The changes in histochemical properties of the glycoconjugates is species-specific, for the siberian sturgeon goblet cells in the larval oesophagus first started to produce neutral glycoproteins only, and later a mix of neutral and acid mucins [START_REF] Gisbert | Histochemistry of the development of the digestive system of Siberian sturgeon during early ontogeny[END_REF].

The mature pattern of stage 3 and stage 4 herring larvae in terms of histochemical properties is similar to the one of sea bream [START_REF] Sarasquete | Glyconjugates in epidermal, branchial and digestive mucous cells and gastric glands of gilthead sea bream, Sparus aurata, Senegal sole, Solea senegalensis and Siberian sturgeon, Acipenser baeri development[END_REF], with a large amount of neutral mucosubstances secreted in the distal oesophageal zone and acid mucosubstances secreted in the proximal oesophageal zone.

Stomach

The presence of pyloric ceca and gastric glands in the stomach are commonly used to histologically define the transition from larvae to juvenile stage, and to an adult mode of digestion (Zambonino [START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF]. No gastric glands were observed or found during the experiment, suggesting that metamorphosis from a digestive point of view is not totally completed. However, the height of the stomacal cells starts to increase during the stage 3 and until the end of the stage 4 (~30 mm at 69 dph). The stomach was the latest organ to start its development, and by 69 dph it was not fully developed into the Y-shaped form typical of adult clupeids [START_REF] Whitehead | The West African Pygmy Herring Sierrathrissa leonensis: General features, Visceral Anatomy, and Osteology[END_REF], allowing for the ingestion of large preys.

Pancreas

In the exocrine pancreas, enzyme precursors are stored in the form of zymogen granules that were present throughout the development of herring larvae. The most studied pancreatic enzymes are alpha amylase, involved in the hydrolysis of glycogen and starch [START_REF] Lazo | Ontogeny of the Digestive Tract[END_REF], and trypsin, considered as the most important proteolytic enzyme for fish early-life stages [START_REF] Ronnestad | Feeding behaviour and digestive physiology in larval fish : current knowledge , and gaps and bottlenecks in research[END_REF]. The expression pattern of these enzymes has been well described during larval development, with a lowering amylase activity, while there is an increase in trypsin activity [START_REF] Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]). In the present study, we observed the decrease in amylase activity but trypsin activity remained unchanged during the two months of herring larval development. As trypsin intestinal activity is stimulated by food ingestion [START_REF] Pedersen | Fate of trypsin and assimilation efficiency in larval herring (Clupea harengus) following digestion of copepods[END_REF], and given that we sampled in the morning before the first-feeding of the day, the stable value along the experiment might represent the basal trypsin content of larval herring. This suggests that herring larvae have an early functional exocrine pancreas, and that its maturation continues beyond 49 dph (18.5  2.4 mm TL), as indicated by the decrease in amylase activity until 49 dph and the progressive change from a compact organ to a more elongate and diffuse one, like in herring adult.

Intestine

The intestine development generally corresponds to the maturation of enterocytes which is characterized by a sharp decrease in cytosolic enzymes activities in parallel to an increase in enzymes activities in brush border membranes [START_REF] Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]. These changes mark the beginning of the adult mode of digestion in fish larvae. In the present study, we assayed a set of intestinal enzymes belonging to these different enterocyte compartments. Leucine-alanine peptidase located in the cytosol, [START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF]) and aminopeptidase N located in the brush border [START_REF] Govoni | The physiology of digestion in fish The physiology of digestion in fish larvae[END_REF] ensure proteins digestion, while alkaline phosphatase is involved in the hydrolysis of phosphorylated proteins and may also be involved in amino-acids transport [START_REF] Cara | Assessment of digestive enzyme activities during larval development of white bream[END_REF]. In addition to playing a role in nutrient transport and absorption, intestinal alkaline phosphatase is a key protective enzyme in fish involved in gastrointestinal health, limiting inflammation [START_REF] Lallès | Intestinal alkaline phosphatase in the gastrointestinal tract of fish: biology, ontogeny, and environmental and nutritional modulation[END_REF]. Ratios of brush border enzymes to cytosolic enzymes activities are largely used as indicators of intestinal maturation. In herring, the change from a larval to an adult mode of digestion in enterocytes occurred between 28 (15.0  2.2 mm TL) and 35 dph (16.9  2.0 mm TL), the size class and dph correspond to stage 3 larvae. The brush border structure was visible since the start of the development but was not mature until the end of the stage 3, at 35 dph when larvae were around 16 mm. In the meantime, the size of the intestine increased with more prominent villi. Stage 4 larvae were observed close to 35 dph, suggesting a synchronism between the physiological and the morphological developmental changes. As the larvae were pooled to assess enzyme activity our results reflect the general pattern of digestive maturation in herring larvae, and help to identify the moment when most of the individuals have reached this particular developmental sequence. Further analysis using other techniques (e.g. radioimmunoassay or fluorescence technique) might be useful to assess the degree of individual variability on the maturation of the digestive system linked to ontogeny.

Critical periods and implications for Downs herring recruitment

The critical period was defined by [START_REF] Hjort | Fluctuations in the great fisheries of northern Europe viewed in the light of biological research[END_REF] as the major mortality event occurring during larval development if suitable environmental conditions are not met and was hypothesized to happen at transition from endogenous to exogenous feeding [START_REF] Houde | Emerging from Hjort's shadow[END_REF]). In the present study, we observed that Downs herring larvae started to feed early and before the exhaustion of vitelline reserves.

Moreover, liver of stages 1 and 2 larvae depicted large and numerous glycogen vacuoles showing that young herring larvae tend to develop energy storage when food is available. Glycogen plays an important role as cellular source of energy to be used later on in the development [START_REF] Furukawa | Changes in glycogen concentration and gene expression levels of glycogen-metabolizing enzymes in muscle and liver of developing masu salmon[END_REF]) and to cope with environmental stress like changes in food availability [START_REF] Gisbert | Nutritional cellular biomarkers in early life stages of fish[END_REF]. Later on in the ontogeny, significant changes in liver reserves were observed. Heterogeneity in liver vacuoles size along with a decrease in glycogen was observed during stage 3 and 4, and some larvae were found with small or no vacuole. Glycogen was still present quite homogeneously but in lower quantity compared to smaller larvae and the general appearance of hepatocytes was less inflated.

Vacuoles depletion in the liver can be explained by the transition from stage 2 to stage 3 which entails a high energetic cost. Transition to stage 3 implies important morphological changes such as the development and growth of caudal and dorsal fins, as well as an increase in body height.

Anatomically the organs of the digestive system also increased in size, all these changes likely being high energy demanding. These results are consistent with the decrease in growth rate observed for wild collected Downs herring larvae between 13 and 14 mm [START_REF] Denis | Ontogenetic changes in the larval condition of Downs herring: use of a multi-index approach at an individual scale[END_REF]. Growth rate continues to decrease with the increase of larval length which may be indicative of a shift after 14 mm to a storage-oriented development strategy. Our results do not directly support this hypothesis since vacuoles in the liver were not more numerous in stage 4 larvae. These larvae are at the onset of metamorphosis, a challenging period where major physiological changes (e.g. increase in muscle development [START_REF] Batty | Development of swimming movements and musculature of larval herring (Clupea harengus)[END_REF], muscle fiber recruitment and surface increased sharply after 25 mm in Clyde herring [START_REF] Johnston | Embryonic temperature modulates muscle growth characteristics in larval and juvenile herring[END_REF]) also occur and require a huge amount of energy. While this could explain the depletion of liver vacuoles, change in the type of storage linked to a shift from a glycogen-based metabolism to a lipid-based one can be another hypothesis. Further analyses of larval lipid content and lipid class composition would be useful and complementary to disentangle and confirm energetic trade-offs between glycogen and lipids dynamics linked to larval ontogeny and nutritional condition.

For North Sea herring, the critical period in the wild was assessed to occur before 19 mm [START_REF] Nash | The influence of life history dynamics and environment on the determination of year class strength in North Sea herring (Clupea harengus L.)[END_REF] since abundance of larger larvae are used to estimate the recruitment index at one year and are well correlated with abundance of age 1 herring the year after (ICES 2020). Then, rather that the transition from stage 1 to stage 2, the transition from stage 2 to 3 appears more as the critical period for North Sea herring. According to our results on the variation pattern of liver vacuoles and reserves during larval ontogeny, variability in the mortality rate experienced during this critical period may rely on the ability of larvae to make reserves before the transition, i.e. during stage 2. Stage 2 larvae in a favorable feeding environment could be able to store reserves while maintaining efficient growth, which would lead to a better potential of survival when passing to the next development stage. The use of liver reserve in stage 2 larvae as an indicator of potential of survival/mortality rate should then be investigated. Downs herring larvae, caught each year during the IBTS could be analysed using histology to estimate year to year differences in proportion of individuals with liver reserves. These results should be then correlated to annual variations in recruitment and environmental parameters. The use of the RNA/DNA ratio may easiest for long term monitoring of larval herring condition, but since it does not directly measure reserve storage, and can differ from the histological index (Di [START_REF] Pane | Ontogenetic shift in the energy allocation strategy and physiological condition of larval plaice (Pleuronectes platessa)[END_REF], it first require calibration.

Conclusion

The combination of histological and histochemical techniques gave us significant insights on the ontogeny of the digestive system for Downs herring larvae. All the digestive organs were present at hatching, except for the stomach. Digestive accessory glands were even well developed before mouth opening and containing an important amount of reserves (zymogen, glycogen). The use of biochemical measurements combined with histology was complementary and indicated not only the ontogeny of cell structures but also their functionality (i.e. brush borders of the enterocytes). The fluctuations in liver reserves quantities suggest that the transition from stage 2 to 3 and the end of the stage 4, at the onset of metamorphosis into juveniles, are highly demanding in energy. These results could serve as a reference for nutritional histological condition of herring larvae from field studies. These transitions can be perceived as critical or challenging periods for larvae that have to cope with a changing environment in the wild. Our results suggest that only larvae that were able to store sufficient energy during the first-feeding stage could pass these critical periods, develop and survive. Our findings are relevant to help explain inter-annual fluctuations in Downs herring larvae survival and recruitment. Therefore, inter-annual variations in the level of energy storage (e.g. based on the evaluation of the amount of vacuoles in the liver) in first-feeding stages should be examined and considered as potential predictors of recruitment variability.

Compliance with Ethical Standards

Conflict of interest The authors declare they have no conflict of interest.

Ethic approval Fish experiments were conducted at the Ifremer-Centre de Bretagne facilities (agreement number: B29-212-05) following French national regulations and authorized by the Regional Ethics Committee (authorization number 16513-2018082709221792).

Fig. 1

 1 Fig. 1 Developmental stages of herring larvae: (1) yolk sac stage -3 dph, 8-9 mm TL , (2) pre-flexion stage -12 dph, 9-14 mm TL, (3) flexion stage -28 dph, 12-18 mm TL and (4) post-flexion stage, the larvae is cut in two parts because it was unfortunately too long to fit entirely under the binocular -69 dph, 15-30 mm TL, binocular observations

Fig. 2

 2 Fig. 2 Boxplot representation of the larval total length (mm) observed for each developmental stage

Fig. 3

 3 Fig. 3 Sagittal sections of Downs herring larvae (yolk sac stage, 8 mm, 3 days post hatch), colored with alcian blue pH2.5, Groat's hematoxylin and picro-indigo carmine. A Section of the head and yolk sac, Magnification x200, scale bar = 100 µm. B General view of the larva, Scan x200, scale bar = 300 µm. Magnification x1000; oil immersion for C, D, E, F and G. C Details of the oesophagus with goblet cells, scale bar = 20 µm. D Section of the stomach cavity, scale bar = 10 µm. E Section of the intestine with goblet cells, scale bar = 20 µm. F Details of the liver with large vacuoles and glycogen (pink), scale bar = 10 µm. G Details of the pancreas with zymogen granules, scale bar = 10 µm. Abbreviations: BB Brush Borders, Gc Goblet cells, I Intestine, Li Liver, N Notochord, St stomach, V

  to appear at 19 and 21 dph when larvae were around 11-12 mm and at the end of the stage 2. The distinction between the stomacal cavity and the intestine was clearly visible as a structured pylorus. In the intestine, two regions could be distinguished: the mid-and the hindgut, separated by a simple constriction of the organ. The epithelial structure was homogeneous for both parts, enterocytes began to form villi and still contained goblet cells. The endocrine pancreas was visible.Flexion stage (Stage 3)At 25 dph, larvae were around 14 mm and started transitioning into stage 3. The global morphology of the digestive tract remained the same but the organs became larger and thicker. The oesophagus interstice was wider and longer. A clear pattern appeared in the mucosubstances secretion by the goblet cells of the oesophagus: the proximal half produced acid mucus and the distal part neutral mucus (Fig.4c). The oesophagus opened up to a stomacal cavity extended in the upper part. The epithelial cells height of the stomach increased and the villi of the intestine became larger. The endocrine portion of the pancreas took the form of one large cluster of cells called islet, with a central position. Before 28 dph, exocrine pancreatic cells seemed to be regrouped but without a clear pattern. At 28 dph, cells were organized into distinct and circular acini. The pancreas became more elongated, protruding beyond the beginning of the intestine. The liver underwent major changes at the beginning of stage 3, between 25 and 28 dph. Cells were smaller and the vacuoles were reduced or absent for some larvae. At 35 dph (end of the stage 3), the liver showed both large cells with huge vacuoles and small cells with smaller vacuoles. The stain remained pink through the stage highlighting the presence of glycogen reserves.

Fig. 5 Fig. 6

 56 Fig. 5 Sagittal sections of Downs herring larvae (stage 4, 27 mm, 69 days post hatch), colored with alcian blue pH2.5, Groat's hematoxylin and picro-indigo carmine. A. General view of the larva, Scan x200, scale bar = 0.5 mm. B Section of the oesophagus and upper part of the stomach, Magnification x200, scale bar = 100 µm. C Details of the stomach, Magnification x1000; oil immersion, scale bar = 10 µm. D . Section of the pylorus and start of the intestine with goblet cells, prominent villi and a small part of pyloric ceca, Magnification x200, scale bar = 100 µm. E Details of de villus structure of the intestine and the Brush Border, Magnification x1000; oil immersion, scale bar = 10 µmF Details of the pancreas organization with pancreatic islets and exocrine pancreas, Magnification x200, scale bar

Fig. 7

 7 Fig. 7 Intestinal maturation of the enterocytes during Clupea harengus larval ontogeny indicated by the change in ratio in segmental activities of brush border enzymes (AP: alkaline phosphatase, aminP: aminopeptidase N) / cytosolic enzymes (Leu-Ala: leucine-alanine peptidase). Results are presented in mean  SD (n=3). * indicate significant differences between ratios (P < 0.05)

Table 1

 1 Synthesis of the histological structures observed for the digestive system development description[START_REF] Infante | Ontogeny and physiology of the digestive system of marine fish larvae[END_REF][START_REF] Ronnestad | Feeding behaviour and digestive physiology in larval fish : current knowledge , and gaps and bottlenecks in research[END_REF][START_REF] Pane | Evaluating the histological-based condition of wild collected larval fish: A synthetic approach applied to common sole (Solea solea)[END_REF]

	Organs	Tissue	Features
		Tube shape	short and	elongated	folded
	Oesophagus		narrow
		Epithelium	simple	pseudo-stratified stratified

Larvae from the first two sampling dates (14 and 21 dph) were entirely homogenized with a polytron (PT -MR2100, Polytron, NR: 324412). Older ones

(28, 35, 49 dph)

were dissected and measured on ice under a binocular microscope

Table 2

 2 Estimated model fixed effects of the variation of length over time using "tank" as a random effect for 761 observations.

	Effect	Estimate	Std. Error	df	t	P value
	Intercept	8.87	0.47	2.18	18.59	0.001
	Dph	0.22	0.01	2.15	18.10	0.002

Acknowledgment

Funding for this study was provided by the AWI -MARUM -IFREMER (AMI) Partnership Program awarded to the "CoCktAIL" (Climate ChAnge effects on fIsh Larvae) project (2018)(2019)(2020)(2021)(2022). The PhD Grant for L. Joly is supported by IFREMER and AWI. Additional support was provided by the French government and the region Hauts-de-France in the framework of the project CPER MARCO 2014-2020. The authors thank Prof. Philippe Koubbi for his valuable guidance regarding histological analysis as well as Clara Ortu, Marie-Anais Leprêtre, and IFREMER technicians and researchers (Margaux Denamiel, Fabien Lebon, Coline Lazard, Stéphane Karasiewicz, David Mazurais, Arianna Servili) for their valuable help in the laboratory for the fertilization procedure. Logistical aid was also provided by the Nausicaä Centre National de la Mer and in particular by Stéphane Hénard. Finally, this work would have not been possible without the implication of local fishermen (CME, From Nord) in Boulogne sur Mer willing to support science and research on highly important local fish species.

Data availability All data generated or analyzed during this study are included in this published article.


Vanderplancke Gwenaëlle

Claireaux Guy

Quazuguel Patrick

Huelvan Christine

Corporeau Charlotte

Mazurais David

Zambonino-Infante José-Luis

Exposure to chronic moderate hypoxia impacts physiological and developmental traits of European sea bass (Dicentrarchus labrax) larvae

Keywords: Hypoxia, Fish larvae, Energy metabolism, Maturation

Since European sea bass (Dicentrarchus labrax) larvae occurred in coastal and estuarine waters at early life stages, they are likely to be exposed to reduced dissolved oxygen waters at a sensitive developmental stage. However, the effects of hypoxia at larval stage, which depend in part on fish species, remain very poorly documented in European sea bass. In the present study, the impacts of an experimental exposure to a chronic moderate hypoxia (40 % air saturation) between 30 and 38 days post-hatching on the physiological and developmental traits of European sea bass larvae were assessed. This study was based on the investigation of survival and growth rates, parameters related to energy metabolism [Citrate Synthase (CS) and Cytochrome-c Oxidase (COX) activities], and biological indicators of the maturation of digestive function [pancreatic (trypsin, amylase) and intestinal (Alkaline Phosphatase "AP" and Aminopeptidase-N "N-LAP") enzymes activities]. While condition of hypoxia exposure did not induce any significant mortality event, lower growth rate as well as CS/COX activity ratio was observed in the Hypoxia Treatment group. In parallel, intestinal enzyme activities were also lower under hypoxia. Altogether, the present data suggest that sea bass larvae cope with moderate hypoxia by (1) reducing processes that are costly in energy and (2) regulating mitochondria functions in order to respond to energy-demand conditions. Both these effects are associated with a delay in the maturation of the digestive function.

Introduction

Since larvae of marine fish species hatch at an undeveloped stage, their development and ontogeny are liable to be greatly affected by biotic and abiotic conditions. This is especially true in coastal areas, which are nurseries for many marine fish species and where ecosystems are particularly impacted by human activities and global change. Among the environmental factors affecting the world's coastal waters, hypoxia is a major problem and is forecast to increase further under the combined effects of spreading coastal eutrophication and global warming [START_REF] Diaz | Overview of hypoxia around the world[END_REF][START_REF] Diaz | Marine benthic hypoxia: A review of its ecological effects and the behavioural responses of benthic macrofauna[END_REF]. Hypoxia episodes often coincide with periods of annual recruitment and the growth of benthic and pelagic fish species occurring in late spring and early summer [START_REF] Breitburg | Episodic hypoxia in Chesapeake Bay : Interacting effects of recruitment, behavior, and physical disturbance[END_REF][START_REF] Pihl | Effects of periodic hypoxia on distribution of demersal fish and crustaceans[END_REF][START_REF] Pihl | Hypoxia-induced structural changes in the diet of bottom-feeding fish and crustacea[END_REF].

Understanding the impact of a low oxygen environment on the early life stages of fish is therefore of great importance, since fishery production relies on larval and juvenile recruitment.

Exposure of juveniles or adults from different fish species to hypoxia has been shown to notably affect fish routine metabolic rate (RMR), feed intake (FI), growth, condition factor and concentration of protein, e.g., European sea bass (Dicentrarchus labrax) [START_REF] Claireaux | Influence of temperature, oxygen and salinity on the metabolism of the European sea bass[END_REF][START_REF] Pichavant | Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass[END_REF][START_REF] Thetmeyer | Growth of European sea bass (Dicentrarchus labrax L.) under hypoxic and oscillating oxygen conditions[END_REF]; turbot (Scophthalmus maximus) [START_REF] Pichavant | Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass[END_REF][START_REF] Pichavant | Effects of hypoxia on growth and metabolism of juvenile turbot[END_REF]; Amazon oscar (Astronotus crassipinis) [START_REF] Almeida-Val | Anoxia-and hypoxia-induced expression of LDH-A* in the Amazon Oscar, Astronotus crassipinis[END_REF]; and mummichog (Fundulus heteroditus) [START_REF] Rees | Temporal dynamics in growth and white skeletal muscle composition of the mummichog Fundulus heteroclitus during chronic hypoxia and hyperoxia[END_REF]. It is supposed that some of these effects can be explained by the hypoxia-induced control of fundamental processes related to energy saving. Long-term exposure to hypoxia in fish has been also shown to affect egg development and hatching success, resulting in malformation, lower fitness and high mortality rates at larval and juvenile stages, e.g., brown trout (Salmo trutta) [START_REF] Massa | Early life development of brown trout (Salmo trutta) eggs under temporary or continuous hypoxial stress : Effects on the gills, yolk sac resorption and morphometric parameters[END_REF][START_REF] Roussel | Carry-over effects in brown trout (Salmo trutta): hypoxia on embryos impairs predator avoidance by alevins in experimental channels[END_REF]; black bream (Acanthopagrus butcheri) [START_REF] Hassell | Hypoxia impairs embryo development and survival in black bream (Acanthopagrus butcheri)[END_REF]; zebrafish (Danio rerio) [START_REF] Padilla | Oxygen deprivation causes suspended animation in the zebrafish embryo[END_REF][START_REF] Shang | Aquatic hypoxia is a teratogen and affects fish embryonic development[END_REF]; and dogfish (Scyliorhinus canicula) [START_REF] Diez | Energy exchange between the yolk and embryo of dogfish (Scyliorhinus canicula L.) eggs held under Normoxic, Hypoxic and Transient Anoxic conditions[END_REF]. These effects occur due to disturbances in a series of programmed, highly intricate and energy-consuming processes [START_REF] Ozernyuk | Adaptive specific features of energy metabolism in fish ontogenesis[END_REF][START_REF] Wu | Endocrine disrupting and teratogenic effects of hypoxia on fish, and their ecological implications[END_REF]. Although it is generally accepted that early embryonic developmental stages are particularly sensitive to stresses in fish [START_REF] Cameron | Malformation rates in embryos of North Sea fishes in 1991 and 1992[END_REF], post-hatching development also has windows of sensitivity to hypoxia. The effects of hypoxia exposure when applied at larval stages vary widely depending on fish species, hypoxia severity, exposure period and individual adaptive capacities [START_REF] Anjos | Hypoxic environments as refuge against predatory fish in the Amazonian floodplains[END_REF][START_REF] Bickler | Hypoxia tolerance in reptiles, amphibians, and fishes: Life with variable oxygen availability[END_REF]. While [START_REF] Barrionuevo | Aerobic and anaerobic metabolism for the zebrafish, Danio rerio, reared under normoxic and hypoxic conditions and exposed to acute hypoxia during development[END_REF] reported that zebrafish did not respond to moderate hypoxia before 30 days post-hatching (d.p.h.), [START_REF] Pelster | Developmental plasticity in the cardiovascular system of fish, with special reference to the zebrafish[END_REF] showed that exposure of the same species to 7 days of moderate hypoxia affected larval cardiac activity, cardiac output, heart rate, organ perfusion and blood vessel formation. Since hatching of marine fish species occurs at earlier stages of development compared with freshwater species, their ontogenic processes during the larval phase are likely more sensitive to hypoxic episodes. Low tolerance to hypoxia has been observed over the period of metamorphosis in sea bream (Pagrus major) [START_REF] Ishibashi | Ontogeny of tolerance to hypoxia and oxygen consumption of larval and juvenile red sea bream, Pagrus major[END_REF]; Japanese flounder (Paralichthys olivaceu) [START_REF] Ishibashi | Ontogenic changes in tolerance to hypoxia and energy metabolism of larval and juvenile Japanese flounder Paralichthys olivaceus[END_REF]; and bonefish (Albula sp.) [START_REF] Pfeiler | Changes in hypoxia tolerance during metamorphosis of bonefish leptocephali[END_REF]. It is supposed that this sensitivity is due to the increase of metabolic rates just before and just after the flexion stage, which is a period of dramatic molecular, physiological and behavioural changes for marine fish species such as European sea bass [START_REF] Mazurais | Transcriptomics for understanding marine fish larval development[END_REF]. The European sea bass is a highly valued fish that migrates from offshore areas to the coast just after flexion stage, which occurs between 22 and 25 d.p.h. [START_REF] Dufour | Identification of sea bass (Dicentrarchus labrax) nursery areas in the north-western Mediterranean Sea[END_REF][START_REF] Jennings | The origin and recruitment of bass, Dicentrarchus labrax, larvae to nursery areas[END_REF][START_REF] Pickett | Sea Bass: Biology, Exploitation and Conservation[END_REF]. This species can therefore be exposed to hypoxic events from this stage onward. However, few studies have been published on the effects of hypoxia on larval development of this marine fish species.

The purpose of the present study was to evaluate the impact of a chronic (between 30 and 38 d.p.h., a natural hypoxia exposure window) moderate (40% air saturation) hypoxia exposure on growth, metabolism and maturation in European sea bass larvae. To gain insight into the energy metabolism of larvae, Citrate Synthase (CS) and Cytochrome c Oxidase (COX) activities were assessed. CS and COX are key mitochondrial enzymes localized in matrix or membrane which fluctuations provide information on the properties and the numbers of the mitochondria present [START_REF] Guderley | Temperature and growth rates as modulators of the metabolic capacities of fish muscle[END_REF][START_REF] Lucassen | Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish[END_REF]. CS/COX activity ratio was used in order to estimate changing in mitochondrial size and shape in response to energydemand conditions as indicated in studies by [START_REF] Lucassen | Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish[END_REF] and [START_REF] Ibarz | Energy reserves and metabolic status affect the acclimation of gilthead sea bream (Sparus aurata) to cold[END_REF].

Finally, digestive enzyme activities [Trypsin, Amylase, Alkaline Phosphatase (AP) and Aminopeptidase-N (N-LAP)] were used as indicators of the developmental status of the fish larvae.

Material and methods

Larval rearing

Post-hatching, larvae of European sea bass were reared under normal oxygen conditions in 8 tanks at 19.1 ± 0.4 °C water temperature and 35 ± 0.2 ‰ salinity, and were fed with Artemia until 45 d.p.h according to [START_REF] Zambonino-Infante | Sea bass (Dicentrarchus labrax) larvae fed different Artemia rations: growth, pancreas enzymatic response and development of digestive functions[END_REF]. The tanks were then divided into two groups of four tanks. The Control group (or C group) was maintained under normal oxygen condition (96.6 ± 1.3 % air saturation, 7.10 mg O 2 . L -1 ), while larvae from the challenged group, also named Hypoxia Treatment group (or HT group), were subjected to moderate hypoxia (40.1 ± 2.6 % air saturation, 2.95 mg O 2 .L -1 ) for 8 days (from 30 to 38 d.p.h., Fig. 1). These hypoxia conditions had previously been determined to not induce mortality. During hypoxia exposure larvae of each group were fed with Artemia, except 24 h prior to samplings where larvae were left undisturbed and unfed. After the hypoxia exposure, larvae continue to be fed with Artemia until the end of experimentations. It was not possible to evaluate feed ingestion throughout this experiment; however, we did not notice any visible change in feeding activity in HT group during or after hypoxia exposure.

Survival rate and larval growth

Survival rate was estimated at 45 d.p.h. by counting the number of remaining larvae in each group and taking into account the larvae collected for sampling during the experiment.

Growth was evaluated on four pools (1 pool from each tank) of approximately 40 larvae per sample from the beginning of hypoxia exposure (30 d.p.h.) until 45 d.p.h. (i.e. at 30, 32, 35 and 38 d.p.h -during hypoxia exposure -and at 42 and 45 d.p.h. -after the return to normoxic conditions).

Larvae sampling

During hypoxia exposure, five samplings (at 30, 32, 35 and 38 d.p.h. and 42 d.p.h.) were performed in order to analyse CS and COX activities. For this purpose, in each treatment group, 4 samplings (1 per tank) of 35 or 40 pooled larvae were for protein concentration and enzymes activities assays. Larvae were first placed on a glass maintained at 0°C and dissected under microscope into four parts: head "H" (just behind the eye), pancreatic segment "PS" (just behind the gills) and tail "T" (point between the end of the digestive tract and the anal tail) were discarded, and the intestinal segment "IS" conserved (Fig. 2). For each group, pooled IS were then weighed, placed in ice-cold lysis buffer and stored at -80°C until proteins extraction.

For digestive enzymes assays, four samplings (one per tank) of 35 larvae per group were performed at 38 d.p.h. and stored at -20°C. Larvae were placed on an ice-chilled glass and dissected under a microscope into 4 parts: head (H), pancreatic segment (PS) intestinal segment (IS) and tail (T) (Fig. 2). PS and IS were then stored at -20°C for later assay of their respective enzymes, as previously described by [START_REF] Cahu | Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet : Effect on digestive enzymes[END_REF].

Extractions of proteins from larvae for analysis of CS and COX activities

Proteins were solubilized by adding 3-6 mL of ice-cold lysis buffer containing 150 mM NaCl, 10 mM Tris, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% Igepal, 1 tablet of complete EDTA-free protease inhibitor cocktail (Roche Pharma; Basel, Switzerland) in 25 mL buffer, 1% phosphatase inhibitor cocktail III (Sigma-Aldrich®; St. Louis, MO, USA) and 5mM NaPPi, pH 7.4. Total proteins and enzymes were then extracted as described by [START_REF] Foll | Long-chain n-3 polyunsaturated fatty acids dissociate phosphorylation of Akt from phosphatidylinositol 3'-kinase activity in rats[END_REF]. Briefly, total proteins and enzymes lysates were obtained after homogenization with a Pro Polytron® (BioBlock Scientific; Illkirch , Bas-Rhin , France) tissue disruptor. The homogenates obtained were then centrifuged at 3,000 g for 1 h at 4°C. At the end of the centrifugation, the interphase containing proteins and enzymes was collected by carefully pipetting and then centrifuged at 10,000 g for 45 min at 4°C in order to ensure total lipid removal. The new interphase was carefully collected, aliquoted and stored at -80°C.

Enzymatic activities assays (aerobic metabolism and larval maturation)

CS activity was assessed in Nunc™ 96-well microplates (Thermo Scientific Inc.; Waltham, MA, USA). Data were obtained with a microplate reader (Bio-Tek® Synergy™ HT; Colmar, Haut-Rhin, France) and then treated with KC4 v3 software. The assay was adapted from [START_REF] Guderley | Diet and performance in the scallop, Argopecten purpuratus: force production during escape responses and mitochondrial oxidative capacities[END_REF]. Briefly, CS activity was assayed for 10 min at wavelength λ = 412 nm and temperature T = 25°C. The microplate was prepared by mixing 20 µL of sample with 160 µL of a mix consisting of 0.25 mM Acetyl-coA, 0.125 mM DTNB and 86.25 mM Tris-HCl, pH = 8. The reaction was initiated by adding 20 µL 5 mM oxaloacetate to each well. The control used for the assay was a commercial enzyme of CS (Sigma-Aldrich® Inc.; St. Louis, MO, USA).

In parallel, COX activity was assessed using Cytochrome c Oxidase Assay Kit, CYTOCOX1 (Sigma-Aldrich® Inc.; St. Louis, MO, USA) following manufacturer's instructions. Briefly, COX activity was assayed for 1 min at wavelength λ = 550 nm and temperature T = 25°C.

The reaction was prepared by mixing 350 µL of assay buffer (10 mM Tris-HCl, 120 mM KCl, pH = 7.0), 80 µL of enzyme buffer (10 mM Tris-HCl, 250 mM sucrose, pH = 7.0) and 20 µL of sample. The reaction was then initiated by adding 50 µL of 0.22 mM Ferrocytochrome c substrate solution.

Larval development was evaluated as described by [START_REF] Cahu | Maturation of the pancreatic and intestinal digestive functions in sea bass (Dicentrarchus labrax): effect of weaning with different protein sources[END_REF].

Protocols for the homogenisation of the pancreatic segments and intestine, brush border purification, protein dosages and enzymatic assay procedures for Amylase, Trypsin, AP and N-LAP are described in [START_REF] Zambonino-Infante | Partial substitution of di-and tripeptides for native proteins in sea bass diet improves Dicentrarchus labrax larval development[END_REF]. Enzyme activities were expressed as specific activities, i.e., units per mg of proteins.

Statistical analyses

Data are presented as means ± s.e.m. (Standard Error of Mean). Statistical analyses were performed using R software [START_REF] Core | R: A language and environment for statistical computing[END_REF]. For all analyses, all response variables were checked for normality with the Shapiro test and for equality of variances using the Levene test. A regression was used in order to determine the influence of oxygen treatment on survival. A two-way ANOVAs were then used (except for survival and maturation analyses, for which a regression and a one-way ANOVA were used, respectively) to determine the influence of oxygen treatment, age of larvae (as main effects) and their interaction on each response variable. When the interaction between oxygen treatment and the age of larvae was significant, Student-test were used to determine significant differences among groups within each sampling time. An ANCOVA was used for CS/COX activity ratio analyses in order to determine the influence of oxygen treatment (as main effect) with age of larvae as co-factor.

In addition, a Student-test was used to determine significant differences among groups within each sampling time for CS/COX activity ratio and at 30, 38 and 45 d.p.h. for growth rate.

Finally, a one-way ANOVA was used in order to determine the influence of oxygen treatment on larval development. Differences were considered significant at the 95% confidence level (p-value < 0.05).

Results

No variable differed significantly between the Control (C) and the Hypoxia Treatment (HT) groups prior to the hypoxia exposure, i.e. at 30 d.p.h. just before the beginning of exposure.

Survival and growth of larvae

Survival

No significant mortality events were observed in the tanks during and after the period of exposure to hypoxia. This was confirmed by the survival rate estimated at 45 d.p.h., which was not significantly different between the two groups (C = 44.9 ± 7.2%, HT = 44.7 ± 5.3% ; p = 1).

Larval growth

Growth of larvae was monitored from the beginning of hypoxia exposure (30 d.p.h.) until 45 d.p.h. (after the return to normoxic conditions). Data were transformed using cubic root following recommendations of [START_REF] Bureau | Pattern and cost of growth and nutrient deposition in fish and shrimp: Potential implications and applications[END_REF] (Fig. 3). Globally, in both groups, the larval growth rate increased with the age of larvae. Statistical analyses revealed that larval growth was affected by oxygen treatment (two-way ANOVA: p < 0.001) and by the age of larvae (two-way ANOVA: p < 0.001). The interaction between oxygen treatment and the age of larvae was significant (two-way ANOVA: p < 0.01). While the mean larval masses were similar between the two groups at the beginning of hypoxia exposure (30 d.p.h.) (C = 4.66 ± 0.17 mg ; HT = 4.67 ± 0.16 mg ; Student-test: p > 0.5), they were found significantly different at the end of hypoxia exposure (38 d.p.h.) (C = 13.26 ± 2.29 mg ; HT = 9.23 ± 1.73 mg ; Student-test: p < 0.01). This data can be related to the significant impact of hypoxia exposure on the growth rate (two-way ANOVA interaction: p < 0.01). Furthermore, growth tend to be higher in HT group after hypoxia exposure resulting in similar mass between the two groups at 45 d.p.h. (C = 31.40 ± 2.97 mg ; HT = 27.79 ± 4.18 mg ; Student-test: p > 0.09).

Regulation of Citrate Synthase and Cytochrome c Oxidase activities

CS and COX activities in the IS of larvae were assessed from the beginning of hypoxia exposure (30 d.p.h.) until 42 d.p.h. (after the return to normoxic conditions) (Fig. 4). Overall, CS and COX specific activities increased during larval development in both groups (Fig. 4 A and Fig. 4 B) (ANCOVA: p < 0.001 and p = 0.001, respectively). During hypoxia exposure, i.e. from 30 to 38 d.p.h., CS specific activity was not impacted by hypoxia exposure while COX specific activity was higher in the HT group (ANCOVA: p < 0.001). Globally, the CS/COX activity ratio exhibit significant fluctuations throughout the experiment (Fig. 4 C) (ANCOVA: p < 0.001). During hypoxia exposure, i.e. from 30 to 38 d.p.h., CS/COX activity ratio was lower in the HT group (ANCOVA: p < 0.001). The effect of the oxygen treatment on CS/COX activity ratio is particularly marked at 32, 35 and 38 d.p.h. (Student-test: p < 0.001, p < 0.001 and p < 0.05, respectively). There was no more difference in CS/COX activity ratio between C and HT groups 96h (42 d.p.h.) after the return to normoxic conditions (Student-test: p > 0.05).

Maturation of larvae digestive functions

Activities of digestive enzymes were determined in both the C and HT groups at 38 d.p.h.

(Table 1). For pancreatic enzymes, larvae from the HT group exhibited 25% lower trypsin specific activity and showed a significantly 150% higher amylase specific activity than the C group (one-way ANOVA: p Trypsin < 0.01, p Amylase < 0.05). For the intestinal enzymes, larvae exposed to hypoxia exhibited AP and N-LAP specific activities significantly lower than those from the C group by 78% and 53%, respectively (one-way ANOVA: p AP = 0.01, p N-LAP < 0.01).

Discussion

Since they arrive on the coastline around one month after hatching [START_REF] Dufour | Identification of sea bass (Dicentrarchus labrax) nursery areas in the north-western Mediterranean Sea[END_REF][START_REF] Jennings | The origin and recruitment of bass, Dicentrarchus labrax, larvae to nursery areas[END_REF], European sea bass (Dicentrarchus labrax) are likely to experience hypoxia episodes at the end of the larval period. However, the impact of hypoxic events on their physiology at this developmental stage is still largely unknown. The present study was intended to assess the effects of moderate hypoxia (40% air saturation) applied for a duration of 8 days on 30 day-old sea bass larvae. The hypoxia treatment did not induce any significant mortality. Moreover no significant impact on skeletal deformity rate was observed in juveniles originated from larvae of HT group (data not shown). The absence of effect on survival and malformation rates in the preset study differs from previous data showing that moderate hypoxia induces lower survival rates and higher frequency of vertebral column deformities in first feeding yellowfin tuna (Thunnus albacares) [START_REF] Wexler | Temperature and dissolved oxygen requirements for survival of yellowfin tuna, Thunnus albacares, larvae[END_REF] and Atlantic salmon alevins (Salmo salar) [START_REF] Sanchez | Vertebral Column Deformity and Hypoxia in Salmo salar[END_REF] respectively. This apparent discrepancy of response is consistent with the well admitted species-specific as well as stagespecific response of fish to hypoxia constraint (Richards et al., 2009).

We observed a lower mass in larvae from the HT group at the end of hypoxia exposure resulting from a reduction of growth during the exposition compared to the C group.

Evaluation of food intake in larvae is not really easy and necessitates the use of 14 C-labelled feeds [START_REF] Kolkovski | Visual and chemical cues stimulate microdiet ingestion in sea bream larvae[END_REF], which was not possible to perform during this experiment.

However, it has been well described that hypoxia usually induces reduction in food ingestion [START_REF] Lakani | Effect of different oxygen levels on growth performance, stress response and oxygen consumption in two weight groups of great sturgeon Huso huso[END_REF][START_REF] Pichavant | Comparative effects of long-term hypoxia on growth, feeding and oxygen consumption in juvenile turbot and European sea bass[END_REF][START_REF] Pichavant | Effects of hypoxia on growth and metabolism of juvenile turbot[END_REF][START_REF] Thetmeyer | Growth of European sea bass (Dicentrarchus labrax L.) under hypoxic and oscillating oxygen conditions[END_REF].

Seven days after the return under normoxic conditions (i.e. at 45 d.p.h.), larvae from HT group recovered the same weight as those from the C group (Fig. 3) suggesting a potential compensatory growth. Yet, such a compensatory growth after hypoxia exposure was previously shown in Chinese shrimp (Fenneropenaeus chinensis) [START_REF] Wei | Compensatory growth of Chinese shrimp, Fenneropenaeus chinensis following hypoxic exposure[END_REF]; it is likely that a higher number of sampling points after the return under normoxic conditions would have permitted to point out compensatory growth in our study.

In the present study, the lower trypsin specific activity in larvae originated from the HT group suggested that one week moderate hypoxia exposure impaired ingestion of fish. As mentioned by Zambonino-Infante and [START_REF] Zambonino-Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF], pancreatic maturation in sea bass larvae has been completed by 25 d.p.h. and trypsin activity can be directly related to the dietary protein intake from 35 d.p.h. Therefore, a decrease in trypsin specific activity observed after 35 d.p.h. is generally related to undernutrition (Zambonino-Infante and [START_REF] Zambonino-Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]. Again, in the present study, even if we observed that larvae exposed to 40% air saturation hypoxia continued to have a certain feeding activity, we cannot exclude that they reduced their food consumption, compared to control larvae. Furthermore, in our study, the larvae which have experienced hypoxia exposure exhibited higher amylase specific activity at the same stage of development than larvae from C group. It is well documented that amylase specific activity decrease throughout the larval stage [START_REF] Péres | Amylase and trypsin responses to intake of dietary carbohydrate and protein depend on the developmental stage in sea bass (Dicentrarchus labrax) larvae[END_REF][START_REF] Ribeiro | Development of digestive enzymes in larvae of Solea senegalensis, Kaup 1858[END_REF][START_REF] Zambonino-Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]. This difference indicates that the normal decrease of amylase specific activity in larvae was delayed in the HT group. In parallel, the lower AP and N-LAP specific activities in larvae from the HT group revealed that the maturation of the enterocytes was impaired by the hypoxia episode. In marine fish species, particularly European sea bass, it is well admitted that the settlement of an efficient intestinal membrane digestion (characterised by the concomitant increase in AP and N-LAP specific activities) occurred around 30 d.p.h. at 19°C [START_REF] Cahu | Early weaning of sea bass (Dicentrarchus labrax) larvae with a compound diet : Effect on digestive enzymes[END_REF][START_REF] Zambonino-Infante | Ontogeny of the gastrointestinal tract of marine fish larvae[END_REF]. In the present study, these developmental delays could also have contributed to a growth depression as long as the sea bass larvae were exposed to hypoxia, as already observed in fish larvae [START_REF] Perez-Dominguez | Interrenal and thyroid development in red drum (Sciaenops ocellatus): Effects of nursery environment on larval growth and cortisol concentration during settlement[END_REF][START_REF] Wexler | Temperature and dissolved oxygen requirements for survival of yellowfin tuna, Thunnus albacares, larvae[END_REF].

We also found a down-regulation of CS/COX activity ratio in the HT group during hypoxia exposure. This down-regulation disappeared after the return in normoxic conditions (Fig. 4 C). This down-regulation of CS/COX activity ratio was mainly due to a significant increase in COX specific activity (Fig. 4 B) more than a decrease in CS specific activity (Fig. 4 A). The CS is a key enzyme localized in the mitochondrial matrix and is involved in the Krebs' cycle whereas COX is the terminal electron acceptor of the electron transport chain located in the mitochondrial inner membrane. These two enzymes are of great importance in aerobic metabolism and provide information on respiration metabolism capacity. The fluctuations of these two key mitochondrial enzymes provide information on the properties and the numbers of the mitochondria present [START_REF] Guderley | Temperature and growth rates as modulators of the metabolic capacities of fish muscle[END_REF][START_REF] Lucassen | Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish[END_REF]. CS/COX activity ratio could be used in order to estimate changes in mitochondrial size and shape in response to energy-demand conditions [START_REF] Ibarz | Energy reserves and metabolic status affect the acclimation of gilthead sea bream (Sparus aurata) to cold[END_REF]. In consequence, the decrease in CS/COX activity ratio observed in our study during the hypoxia period could reflect a lower citrate synthesis over respiratory chain capacities of mitochondria in the larvae from the HT group [START_REF] Lucassen | Mitochondrial proliferation in the permanent vs. temporary cold: enzyme activities and mRNA levels in Antarctic and temperate zoarcid fish[END_REF] as well as a modification of the size and the shape of mitochondria [START_REF] Ibarz | Energy reserves and metabolic status affect the acclimation of gilthead sea bream (Sparus aurata) to cold[END_REF]. The similar CS/COX activity ratio observed at 42 d.p.h. indicated that such mitochondrial changes were transient.

It is interesting to note that the decrease in CS/COX activity ratio in HT group coincided with a growth depression, but we were not able to establish a significant relationship between growth rate and CS/COX activity ratio. Such relationship has yet been reported by [START_REF] Mathers | Nucleic acid concentrations and enzyme activities as correlates of growth rate of the saithe Pollachius virens: growth-rate estimates of open-sea fish[END_REF] in the saithe (Pollachius virens).

Conclusion

Altogether, the results obtained suggest that sea bass larvae are able to implement physiological regulations in order to cope with a moderate decrease in ambient oxygen.

However, these regulations seemed to have a metabolic cost that impacted growth and development, but not survival. Such consequences could have a strong impact on fish larvae activities, especially on their capacities to escape predation, which could adversely affect the recruitment of sea bass. Statistical analyses revealed that larval growth rate was affected by the oxygen treatment (Two-way ANOVA: p < 0.001) and the age of larvae (Two-way ANOVA: p < 0.001).

Significant interaction between the oxygen treatment and the age of larvae (Two-way ANOVA: p < 0.01) indicated that the effects of time on growth (i.e. the growth rate) depend upon the oxygen treatment. Additional statistical analyses (represent by asterisks) indicated significant differences in mass between the Control and Hypoxia Treatment groups (Studenttest: significant codes : * p < 0.05, *** p<0.001, NS = No Significant differences). Refer to "Material and methods" section for more details on the statistical analyses activity ratio was affected by the oxygen treatment (ANCOVA: p < 0.001 and p < 0.001, respectively) and by the age of larvae (ANCOVA: p = 0.001 and p < 0.001, respectively). In parallel, CS specific activity was not affected by the oxygen treatment (ANCOVA: p > 0.7) but was affected by the age of larvae (ANCOVA: p < 0.001). Additional statistical analyses Tables Table 1:

Fig. 1 Fig. 2

 12 Figures captions:

Fig. 4

 4 Fig. 4 Changes in Citrate Synthase (CS) and Cytochrome c Oxidase (COX) activities for Control "C" (white bars) and Hypoxia Treatment "HT" (grey bars) groups. A) Specific enzymatic activity of CS, B) Specific enzymatic activity of COX and C) Ratio between enzymatic activity of CS and enzymatic activity of COX. Results are given ± s.e.m. (n = 4 pools of 40 larvae). Statistical analyses revealed that COX specific activity and CS/COX

  Specific activities of digestive enzymes. Results are given in milli-units per mg of protein (mU.mg protein -1 ) ± s.e.m. (n = 4 * 35 larvae). Within any one column, means with the same superscript letter are not significantly different (p-value > 0.05). C = Control group; HT = Hypoxia treatment group; AP = Alkaline Phosphatase; N-LAP = Aminopeptidase-N.

Funding

The first author was supported by a joint Ifremer-Région Bretagne PhD grant.

Author contributions