Gonzalez-Araya Ricardo

Robert René
email: rene.robert@ifremer.fr

René Robert

Larval development and fatty acid composition of Ostrea edulis (L.) fed four different single diets from conditioning to pre-settlement

Keywords: broodstock conditioning, diet, larval development, Ostrea edulis

Survival, growth and fatty acid composition of Ostrea edulis larvae (L.) fed four different single species, microalgal diets, Tisochrysis lutea (T), Chaetoceros neogracile (Cg), Skeletonema marinoi (Sm) or Tetraselmis suecica (Ts) from broodstock to pre-settlement, were studied. Lower larval growth (5.5 μm to 6.5 μm/d) was recorded in progeny continuously fed single S. marinoi or T. suecica, whereas good growth was achieved with single T. lutea (7.8 μm/d). Larvae, originated from broodstock receiving Sm or Ts, exhibited growth compensation when fed a bispecific balanced diet (TCg). This did not occur when broodstock and larvae were fed Cg or T, for which single or mixed diets led to similar larval growth. Furthermore, survival was high (>90%) regardless of microalgal diet, except for larvae fed from broodstock to pre-settlement T (53%) or Ts (2%). There were significant differences in 20:5 (n-3) and 22: 6 (n-3) contents in polar and neutral fractions of O. edulis expelled larvae dependent on broodstock diet, as well as throughout larval development, but no clear trend was apparent when comparing fatty acid (FA) relative composition of both fractions of O. edulis larvae fed different diets at release or prior to settlement. In contrast, such correlation occurred when FA was expressed in absolute content but exclusively for larvae-fed single diets and was particularly noticeable between 22: 6 (n-3) and growth and survival. In the present work, broodstock nutritional deficiencies have been revealed in O. edulis progeny, compensated thereafter by feeding the larvae a mixed diet, and in this balanced condition, no obvious relation with larval development indicators was found with main fatty acid contents.

Introduction

Broodstock conditioning is an important step in the molluscan hatchery process. Indeed, conditioning allows the extension of the reproductive period [START_REF] Helm | Hatchery culture of bivalves, a practical manual[END_REF] and leads generally to better gamete quality [START_REF] Utting | Techniques for the hatchery conditioning of bivalve broodstocks and the subsequent effect on egg quality and larval viability[END_REF].

The importance of temperature in molluscan reproduction has been thoroughly described (e.g. [START_REF] Mann | Some biochemical and physiological aspects of growth and gametogenesis in Crassostrea gigas and Ostrea edulis grown at sustained elevated temperatures[END_REF][START_REF] Fabioux | Temperature and photoperiod drive Crassostrea gigas reproductive internal clock[END_REF]Maneiro, Silva, Pazos, Sánchez & Pérez-Parallé, 2017a), but the role of nutrition in quantitative aspects of reproduction is being recognized increasingly (e.g. [START_REF] Enriquez-Diaz | Gametogenesis, reproductive investment, and spawning behavior of the Pacific giant oyster Crassostrea gigas: evidence of an environment-dependent strategy[END_REF]. In O. edulis, a 6% daily ration has been shown to be highly effective for broodstock conditioning [START_REF] Utting | Procedures for maintenance and hatchery-conditioning of bivalve broodstocks[END_REF], and the importance of food quality based on different microalgal diets was pointed out by Millican and Helm (1994). Since that pioneering work, only a few dedicated studies have been carried out (e.g. [START_REF] Berntsson | Effects of broodstock diets on fatty acid composition, survival and growth rates in larvae of the European flat oyster, Ostrea edulis[END_REF]Maneiro, Pérez-Parallé, Silva, Sánchez & Pazos, 2017b). Indeed research on O. edulis reproduction was shelved following Marteilia refringens and Bonamia ostreae disease outbreaks from 1970-1980 that caused a rapid decline in the flat oyster industry in France [START_REF] Buestel | History, status, and future of oyster culture in France[END_REF] and throughout Europe [START_REF] Laing | A feasibility study of native oyster (Ostrea edulis) stock regeneration in the United Kingdom. CARD Project FC1016, Native oyster Stock Regeneration -A review of Biological[END_REF][START_REF] Bromley | Bad moves: Pros and cons of moving oysters -A case study of global translocations of Ostrea edulis Linnaeus, 1758 (Mollusca: Bivalvia)[END_REF]. However, O.

edulis remains an emblematic species in Brittany (North West of France), with an annual production of 1000 -1500 tons [START_REF] Robert | A glimpse of the mollusc industry in Europe[END_REF]. This interest increased quite recently because of high oyster C. gigas juvenile mortalities (e.g. Petton, Pernet, Robert & Boudry, 2013;[START_REF] Azema | Genetic parameters of resistance to Vibrio aestuarianus, and OsHV-1 infections in the Pacific oyster, Crassostrea gigas, at three different life stages[END_REF]. Accordingly, we revisited flat oyster feeding requirements during conditioning and showed that, based upon ecophysiological and biochemical studies, some microalgae more effectively supported gametogenesis [START_REF] González-Araya | A physiological and biochemical approach to selecting the ideal diet for Ostrea edulis (L.) broodstock conditioning (Part A)[END_REF]González-Araya, Lebrun, Quéré & Robert, 2012a). Specificall, Chaetoceros neogracile and Skeletonema marinoi were ingested and assimilated well; whereas, Tetraselmis suecica was poorly absorbed with no significant enrichment in the main fatty acids and sterols in any tissue at the end of a 6 week feeding experiment. Tisochrysis lutea was found to be moderately effective as a diet (assimilation efficiency = 20%) and a highly efficient source of essential lipids (e.g 22:5 (n-6)) (González- [START_REF] González-Araya | A physiological and biochemical approach to selecting the ideal diet for Ostrea edulis (L.) broodstock conditioning (Part A)[END_REF]. On the other hand, larval development has shown to be related to the type of food delivered during conditioning (Berntsson et al., 1997), with apparent but inconsistent compensation when larvae are fed a mixed diet (González- Araya et al., 2012b).

Moreover, several studies [START_REF] Helm | Fatty acid composition of early non-feeding larvae of the European flat oyster, Ostrea edulis L[END_REF][START_REF] Jonsson | Larval growth and settlement of the European oyster (Ostrea edulis) as a function of food quality measured as fatty acid composition[END_REF] have suggested that some PUFAs are essential for survival and development of O. edulis larvae as demonstrated for C. gigas larvae (da Costa, [START_REF] Delaunay | The effect of monospecific algal diets on growth and fatty acid composition of Pecten maximus (L.) larvae[END_REF].

Such interactions between broodstock diets and subsequent larval development were addressed in the present work by feeding O. edulis larvae the same single diets as broodstock and comparing larval performance to larvae fed a bispecific diet used as reference for oyster development in our laboratory [START_REF] Villa | Influence of food density and temperature on ingestion, growth and settlement of Pacific oyster larvae Crassostrea gigas[END_REF]Petton, Le Souchu, Mingant & Robert, 2009). We hypothesised that broodstock nutritional deficiencies could be revealed and compensated at larval development by feeding the larvae a mixed, balanced diet.

Materials and methods

Broodstock conditioning and larval rearing

A total of 360 18-month-old flat oysters ( ≈ 5 cm length and 0.5 g flesh dry weight) originating from Brittany (Bay of Cancale, France) were distributed homogeneously in translucent, 50-L tanks (30 oysters per tank for an equivalent biomass: 1kg total weight and 16 g dry flesh weight). Triplicate tanks were set up for each of the four single species diets tested. Seawater was maintained at 19 °C in a flow-through system at a flow rate of 12 L h -1 , and the oysters were fed continuously by means of a peristaltic pump, at 900 µm 3 µL -1 per feeding condition to limit pseudofaeces deposition [START_REF] González-Araya | A physiological and biochemical approach to selecting the ideal diet for Ostrea edulis (L.) broodstock conditioning (Part A)[END_REF]. Seawater at ambient salinity (34 g L -1 ) was filtered on 1µm polypropylene filter media following UV treatment. Four microalgal diets were delivered during broodstock conditioning (40 days): Tisochrysis lutea (formely named Isochrysis affinis galbana or T-Iso: CCAP 927/14 = T), Chaetoceros neogracile (UTEX LB2658 = Cg), Skeletonema marinoi (CCAP 66/4 = Sm), or Tetraselmis suecica (CCAP 1077/3= Ts). Microalgal size, growth, biochemical profiles, and other characteristics have been already detailed in exponential and stationary phases [START_REF] Robert | Amélioration des productions phytoplanctoniques en écloserie de mollusques: caractérisation des microalgues fourrage[END_REF][START_REF] González-Araya | A physiological and biochemical approach to selecting the ideal diet for Ostrea edulis (L.) broodstock conditioning (Part A)[END_REF]. Accordingly, only a brief outline will be given here with total fatty acid compositions, expressed as relative and absolute contents (Table 1).

From each treatment, released larvae were distributed in 5-L, translucent, methacrylate cylinders and reared in flow-through conditions with UV treatment (González- [START_REF] Araya | Influence of diet assemblage on Ostrea edulis broodstock conditioning and subsequent larval development[END_REF]. Larval expulsion was asynchronous, but broodstock fed different diets released first batches of larvae within 10 days meaning that they were not reared simultaneously but in a closed window-time (April). Larvae at a density of 5 mL -1 were distributed, in triplicate for each larval diet, in seawater at 22 °C and 34 g L -1 salinity.

Larvae were fed continuously the same single-species, microalgal diets as delivered to broodstock at 1,500 µm 3 µL -1 equivalent T. lutea volume (≈ 40 cells) or the mixed diet T. lutea plus C. neogracile (TCg: v/v), which has been shown to be an effective bispecific diet for Crassostrea gigas larvae (Rico- Villa, Le Coz, Mingant & Robert, 2006;[START_REF] Kheder | Effect of nutrition on C. gigas larval development and the evolution of physiological indices. Part A: quantitative and qualitative diet effects[END_REF]. Each broodstock diet was split into treatments with two larval diets, i.e., 8 different larval conditions were assessed: BTLT, BTLTCg, BCgLCg, BCgLTCg, BSmLSm, BSmLTCg, BTsLTs, and BTsLTCg. Thus, the designation BTLT means that broodstock (B) was fed T. lutea (T) and larvae (L) expelled from them were also fed T. lutea; whereas, BSmLTCg means that broodstock was fed S. marinoi and larvae expelled from them were fed T. lutea plus C. neogracile (TCg).

Larval length and survival were quantified on days 3, 6, 9 and 11 under the light microscope by use of an image analysis technique (WinImager 2.0 and Imaq Vision Builder 6.0 software for image capture and treatment, respectively) and direct counting of empty shells for dead larvae estimation. The number of pediveligers ready to set (competent larvae possessing an eyespot) was estimated at the end of the experiment, on day 11.

Biochemical procedures

About 10 000 larvae were sampled, on each larval treatment, at 0 (released larvae) and 11 days (end of larval rearing); the samples were collected by filtration on pre-ignited (at 450° C) GF/D filter pads (3 µm porosity). For lipid extraction, the filter pads were placed separately in tubes containing a mixture of chloroform-methanol (2:1, v/v) with 0.01 wt% butylated hydroxytoluene (BHT) as antioxidant, closed under nitrogen, shaken and frozen at -20°C.

An aliquot of the sample (chloroform-methanol mixture) was evaporated to dryness, with a known amount of 23:0 fatty acid (as internal standard) and lipids were recovered with three washings of 500 µl each of CHCl 3 -MeOH (98:2, v/v). This was placed on top of silica gel microcolum (30 x 5 mm ID, Kieselgel, 70-230 mesh (Merk), previously heated to 450° C and deactivated with 5% water (Marty, Delaunay, Moal & Samain, 1992). The neutral lipids were eluted with 10 ml of CHCl 3 -MeOH (98:2) and the polar lipids were recovered with 10 ml of MeOH. The neutral and polar fractions were taken for methylation and analysis by chromatography.

Statistical procedures

Analysis of variance (ANOVA) was used to test for treatment effects. Statistical analyses were performed using STATISTICA software (version 8.0). Significant differences were detected between the means at the 5% threshold and a posteriori multiple comparison test between the means (Tukeys' test), after transformation of percentage data by the function [arcsin (racine x i/100)].

Results

Larval growth

The size of expelled larvae ranged from 172 µm (± 2.1) to 179 µm (± 2.7) with no significant difference when oysters were fed either diatoms or T. lutea. In contrast, newly-released larvae were significantly smaller (p< 0.01) when broodstock were fed T. suecica (165 µm ± 3.5).

Larvae originating from broodstock fed S. marinoi and fed a similar diet had lower growth (Fig. 1A), with a daily length increment of ≈ 5.5 µm d -1 (Fig. 2A). The supply of a mixed larval diet (TCg) improved growth somewhat (Fig. 1B), with daily growth of 6.0 µm d -1 (Fig. 2A). Similarly larvae fed T. suecica as broodstock exhibited relatively good growth until day 7 (Fig. 1A), with a daily length increment of 6 µm d -1 (Fig. 2A); whereas, a noticeable improvement in larval development was recorded when these larvae were fed mixed diet TCg (Fig. 1B), leading to a daily length increment of ≈ 8.0 µm d -1 (Fig. 2A). Larvae originated from broodstock fed T. lutea or C. neogracile exhibited the highest growth when fed broodstock-based diets (Fig. 1A), with 7.5 and 7.1 µm d -1 daily length increments, respectively (Fig. 2A), but no significant differences (p>0.05) were found when larvae were fed mixed diets (Fig 1B ), with daily length increments of 8.3 and 7.8µm d -1 respectively (Fig. 2A).

Larval survival

Under most experimental conditions, larval survival was high, > 90% on day 11, except when larvae and broodstock were fed single T. lutea (53%) or T. suecica (2% : Fig. 2B).

For this last condition, mortalities took place progressively with moderate larval survival on day 3 (40%) increasing to (100%) on day 7; whereas, larvae fed T. lutea exhibited low mortalities until day 11 when mortality was sudden.

Larval competence

No clear relationship between diet and competence to metamorphosis wa found (Fig. 2C). At the end of the larval rearing period (day 11), 25% of pediveligers were eyed when larvae originating from broodstock fed T or Ts, were fed TCg. However, eyed larvae accounted for 46-50% of the total in larvae originating from broodstock fed C. neogracile or S. marinoi, respectively (Fig. 2C).

Biochemical composition

The biochemical contents of the four microalgae used here as food f230or O. edulis have been described in a previous work reporting on broodstock conditioning (González- Araya et al., 2010), so only a brief outline will be given here. With 10.2% of total fatty acids, T. lutea was rich in 22:6 (n-3) (DHA) but very poor (0.33%) in 20:5(n-3) (EPA). With ≈ 22-23% of total fatty acids, C. neogracile and S. marinoï were rich in 20:5 (n-3) but relatively poor (1.1%) to moderate (4.4%) in 22:6 (n-3). With 30.8% T. suecica was rich in 16:0 (vs 6.7-11.1% for the other species). In contrast, with only 0.23% T. suecica almost lacked 22:6 (n-3); whereas, it contained moderate amount (4.4%) of 20:5 (n-3). 16:0, 20:5 (n-3) and 22:6 (n-3) were the dominant fatty acids in O. edulis larvae with values varying from 6.7% to 21.6% in polar fraction (Table 2) and 3.3% to 23.3% in neutral fraction (Table 4) regardless larval stage or diet.

At release, a similar relative amount of 16:0 in the polar fraction was recorded in larvae regardless broodstock diet, with values fluctuating from 12.3% to 13.7% (Table 2). In contrast, when expressed in absolute content, the larvae originating from broodstock fed S. marinoi exhibited a significantly lower concentration (p<0.05) with 0.7 ng larva -1 vs 1.1-1.6 ng larva -1 for the other diets (Table 3). Similarly, significant differences (p<0.05) in 16:0 relative content were found in neutral fraction of expelled larvae, with values varying from 14% to 23.3%; accordingly, broodstock diet interfered with initial biochemical composition of larval storage (Table 4). Such effects were also found when fatty acid composition was expressed in absolute content with, however, no differences (p<0.05) in initial larval biochemical content when broodstock were fed T. lutea or T. suecica (Table 5). Despite initial stores, no apparent enrichment in 16:0 occurred during O. edulis larval development because 16:0 relative content remained quite similar (p> 0.05), with values varying from 10.6 to 15.9% in larval polar fraction (Table 2). In contrast, there was an overall decrease in this FA in the larval neutral fraction throughout larval development regardless of diet (Table 4).

There were significant differences (p<0.05) in 20:5 (n-3) relative and absolute contents within O. edulis expelled larvae dependent upon broodstock diet, with values fluctuating from 6.7% to 19.1% and from 0.7 to 2.4 ng larva -1 in the polar fraction (Tables 2,3) and from 3.3% to 15.8% and 3.5 to 25.1 ng larva -1 in the neutral fraction (Tables 4,5). Thus, larvae originating from broodstock fed C. neogracile or S. marinoi exhibited the highest EPA concentration in both fractions (polar: 19.1% and 17.1% (Table 2); neutral: 15.8% and 14.7% (Table 4) respectively). When expressed as absolute FA content, this was the case only for C. neogracile, with 2.4 ng larva -1 and 25.1 ng larva -1 in the polar and neutral fractions, respectively (Tables 3,5). In contrast, progeny originating from broodstock fed T. lutea showed the lowest relative contents (6.7% and 3.3%, respectively, in polar and neutral fractions: Tables 2,4); a finding confirmed when FA composition was expressed as absolute content for the neutral fraction (Table 5) but not for the polar fraction (Table 3). Surprisingly the initial content of expelled larvae originated form broodstock fed T. suecica exhibited relatively high percentage content of 20:5 (n-3) in both fractions (15.2 and 11.9% respectively in polar and neutral fractions: Tables 2,4), which was confirmed for the absolute content (1.2 ng larva -1 and 9.3 ng larva -1 respectively in polar and neutral fractions: Tables 3,5). In both fractions, 20:5 (n-3) concentration increased significantly (p<0.05) in larvae fed T. lutea that had originated from broodstock also fed T. lutea (15.9% vs 6.7% and 18.1% vs 3.3%, respectively in polar and neutral fractions: Tables 2,4). This enhancement was more acute when FA composition was expressed in absolute content (13.3 vs 0.7 ng larva -1 and 333.5 vs 3.5 ng larva -1 respectively in polar and neutral fractions: Tables 3,5) A similar finding was obtained with broodstock and larvae solely fed C. neogracile, with EPA larval relative content increasing to 25.7% vs 19.1% and 24.2% vs 15.8%, respectively, in polar and neutral fractions: Tables 2,4); an acute enhancement in the neutral fraction was also found when expressed as absolute FA content (235.8 ng larva -1 vs 25.1 ng larva -1 (Table 5).

With R 2 = 0.10, a non-significant (p>0.05) linear relationship between mean daily growth and EPA relative content was found, regardless of larval diet. In contrast, when expressed in absolute FA content, a relationship with growth occurred for larvae fed single diets (R 2 = 0.51 : Fig. 3A) but did not exist for larvae fed mixed diets (R 2 = 0.03 : Table 6 and Fig. 3C). Similar, general trends were confirmed for the other larval development descriptors (e.g., competence: Table 6 and Fig. 3 B,D). There were significant differences (p<0.05) in 22:6 (n-3) content of O. edulis expelled larvae between broodstock diet treatments, with values fluctuating from 12.5% to 21.6% in the polar fraction (Table 2) and from 8.0% to 15.5% in the neutral fraction (Table 4), which was confirmed when FA composition was expressed as absolute content for the neutral fraction (Table 5) but not for the polar fraction (Table 3). Larvae originating from broodstock fed T. lutea or T. suecica exhibited the highest DHA concentrations in both fractions (polar: 21.6% and 20.2% (Table 2); neutral: 15.5% and 13.6% (Table 4) respectively). However such a relationship was only confirmed for T. lutea when biochemical composition was expressed in absolute content in the neutral fraction (Table 5). With the exception of larvae expelled from broodstock fed S. marinoi, 22:6 (n-3) concentration decreased significantly (p<0.05) in both fractions throughout larval development, regardless of larval diet (Tables 2,4); whereas, no clear trend could be established when FA composition of larvae was expressed as absolute content (Tables 3,5).

With R 2 = 0.09, a non-significant (p>0.05) linear relation between mean daily growth and DHA relative content was found. In contrast, when expressed in absolute FA content, a significant relationship with growth was found for larvae fed single diets (R 2 = 0.82 : Fig. 3A), but not for larvae fed mixed diets (R 2 = 0.03: Table 6 and Fig. 3C). A similar, general trend was confirmed for the other larval development descriptors and specifically for survival (R 2 = 0.97 vs 0.19 : Table 6).

Diets had lower influence on 20:4(n-6) (ARA) content of expelled larvae, in both fractions (polar: 3.7% to 4.7% (Table 2); neutral: 1.0% to 2.4% (Table 4),) as well as throughout larval development during which significant (p<0.05) decreases occurred for larvae fed C. neogracile or T. suecica (Tables 2,4). This trend was not confirmed when FA composition was expressed in absolute content (Tables 3,5).

With R 2 = 0.02, no linear relationship between mean daily growth and DHA relative content was found. In contrast, when expressed as absolute FA content, a relationship with growth was found for larvae fed single diets (R 2 = 0.74 : Fig. 3A), but did not exist for larvae fed mixed diets (R 2 = 0.00: Table 6 and Fig. 3C). Similar, general trends were confirmed for the other larval development descriptors, and for survival (R 2 = 0.82 vs 0.15 : Table 6).

Discussion

Live microalgae are essential for successful larval performance, especially bivalve metamorphosis that relies on energy reserves stored during two developmental stages [START_REF] Labarta | Energy, biochemical substrates and growth in the larval development, metamorphosis and postlarvae of Ostrea edulis[END_REF]. The first stage corresponds to embryonic development wherein endogenous reserves are provided by the egg [START_REF] Bayne | Aspects of the metabolism of Mytilus edulis during starvation[END_REF], and the second stage prior to metamorphosis, which depends on reserves stored during all of larval life. The latest depends on the food value of algal diets supplied to growing larvae combined with endogenous reserves [START_REF] Whyte | Biochemical and energy changes during embryogenesis in the rock scallop Crassadoma gigantea[END_REF].

The nutritional value of different live microalgae is determined by ingestibility (cell size/shape), digestibility, and nutrient content [START_REF] Brown | Nutritional properties of microalgae for mariculture[END_REF]. The nutritional role in bivalve nutrition has been emphasized for essential polyunsaturated fatty acids (PUFAs), especially the n-3 fatty acids eicosapentaenoic (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) necessary for successful growth and development [START_REF] Langdon | The effect of algal and artificial diets on the growth and fatty acid composition of Crassostrea gigas spat[END_REF][START_REF] Thompson | Nutritional value of diets that vary in fatty acid composition for larval pacific oysters (Crassostrea gigas)[END_REF]Martinez-Fernandez, Acosta-Salmon & Southgate, 2006). These lipids play important structural roles in regulating cell-membrane fluidity and other cellular functions [START_REF] Hazel | Thermal adaption in biological membranes: functional significance of changes in phospholipid molecular species composition[END_REF][START_REF] Hall | Eicosapentaeonic acid regulates scallop (Placopecten magellanicus) membrane fluidity in response to cold[END_REF]. In general, bivalves have limited or no ability to synthesize PUFAs or to elongate shorter-chained compounds (da Costa et al., 2016) but require these for growth and development [START_REF] Waldock | Fatty acid metabolism in young oysters, Crassostrea gigas: polyunsaturated fatty acids[END_REF][START_REF] Whyte | Influence of algal diets on biochemical composition and energy reserves in Patinopecten yessoensis (Jay) larvae[END_REF][START_REF] Chu | Metabolism of palmitic linoleic and linolenic acids in adult oysters, Crassostrea virginica[END_REF]Marty, Delaunay, Moal & Samain, 1992;[START_REF] Delaunay | The effect of monospecific algal diets on growth and fatty acid composition of Pecten maximus (L.) larvae[END_REF]. Although the effects of nutrition on larval growth and survival have been studied extensively in a wide range of molluscs (see review of Marshall, McKinley & Pearce, 2012), the effects of food on broodstock and subsequent larval development performances have received less attention.

In the present work, initial larval size was related to diet quality expressed as the type of microalgae delivered during broodstock conditioning. Thus, when larval growth relies on energetic reserves stored during embryogenesis, larvae released from broodstock fed single T. suecica were probably poorly enriched compared to larvae from broodstock fed other microalgal diets. Such larval size variability upon release (from 178 to 186 µm), related to broodstock diets, have been already reported (Berntsson et al., 1997) with, however, no statistical differences between treatments. This result may have been attributable to the richness of natural seston in incoming seawater. Indeed in the reported experiment, unfed oysters released larvae of similar size to those expelled by fed oysters. In contrast, in our experiment seawater was 1-µm filtered and UV-treated, meaning that there was no apparent bias in our feeding conditions. However, no clear trend could be detectable in fatty acid composition of newly released larvae originated from broodstock fed T. suecica, when analysing specific fatty acids such as DHA, with initial content as high as that recorded in progeny originated for broodstock fed T. lutea (20 vs 22% and 1.6 vs 2.2 ng larva -1 for relative and absolute contents respectively). A transfer from original oyster reserve (stored before conditioning) might explain that compensation because T. suecica lacked DHA (0.23%) and has been shown to be poorly assimilated in gonad and other tissues during O. edulis broodstock conditioning [START_REF] González-Araya | A physiological and biochemical approach to selecting the ideal diet for Ostrea edulis (L.) broodstock conditioning (Part A)[END_REF]. The situation was similar when considering FA ratios as (n-3)/(n-6), 22:6/20:5, 22:5/20:4 that did not reveal any outliers nor explanatory values.

No differences in larval growth were found when larvae were fed single or mixed diets, originated from broodstock previously fed T. lutea or C. neogracile, with no clear trend in main relative fatty acids composition. Moreover, when fed T. lutea, O. edulis larval growth was quite similar to that reported with Isochrysis galbana, a closely-related haptophyte (9 µm d -1 : Ferreiro, Perez-Camacho, [START_REF] Ferreiro | Changes in the biochemical composition of Ostrea edulis larvae fed on different food regimes[END_REF]. In contrast, larvae originating from broodstock fed S. marinoi showed lower growth regardless of larval diet, single or mixed. In this case, relative content of 20:5 (n-3) remained constant from release to metamorphosis except when larvae were fed single S. marinoi but decreased significantly, in both polar and neutral fractions, when mixed larval diet TCg was delivered. This result contrasts with a previous work [START_REF] Enright | Evaluation of phytoplankton as diets for juvenile Ostrea edulis L[END_REF]) that classified S. costatum (named now S. marinoi) as the second highest-ranked diatom for O. edulis growth (juvenile). Such apparent contradiction can be explained by the size of this microalga and the stage of bivalve development at which this diatom was delivered. Indeed, S. marinoi is a colonial diatom formed of spherical to cylindrical cells joined in chains. Colonies can be longer than 60 µm [START_REF] Sauriau | Artificial filament breakage of the diatom Skeletonema costatum intended for mollusk aquaculture[END_REF], but in 300-L batch cultures in Argenton, colonies are usually composed of 2 to 4 cells, and the chains rarely exceed 10-15 µm in length (Robert, unpublished data). Accordingly, this phytoplankton size is more appropriate for older bivalve stages than larvae [START_REF] Robert | Bivalve hatchery technology: the current situation for the Pacific oyster Crassostrea gigas and the scallop Pecten maximus in France[END_REF]. When fed S. marinoi, O. edulis larval performance was, nevertheless, more-closely in agreement with that reported by [START_REF] Ferreiro | Changes in the biochemical composition of Ostrea edulis larvae fed on different food regimes[END_REF], who classified this diatom as the worst tested microalga, supporting O. edulis larval growth of only 0.4 µm d -1 (12-fold lower than in the present work). On the other hand, larvae fed the mixed diet and originating from broodstock fed T. suecica showed surprisingly similar growth from day 7 as larvae released by broodstock previously fed T. lutea or C. neogracile. Larvae fed solely T. suecica exhibited lower growth, reaching however 6 µm d -1 (vs 7.3 µm d -1 for the mixed larval diet). This relatively good larval growth was, however, counterbalanced by very low larval survival (2%) that did not allow biochemical analysis. These results contrast with those reported by [START_REF] Berntsson | Effects of broodstock diets on fatty acid composition, survival and growth rates in larvae of the European flat oyster, Ostrea edulis[END_REF] for broodstock fed single T. suecica that induced higher larval growth (4.5 µm d -1 ) than larvae originated from broodstock fed T. suecica plus C. calcitrans (1.7 µm d -1 ).

No relationships were found between larval and growth main fatty acids when they were expressed in relative content. In contrast, a positive correlation was found with DHA absolute content and growth for single larval diets, regardless of microalgae species; whereas such correlation was not obvious for mixed larval diets. A similar correlation with DHA relative content of released O. edulis larvae and larval growth has been already reported (Berntsson et al., 1997) and should explained 50% of the variation in growth rate among broodstock. In contrast these authors did not find any correlation between EPA and growth as pointed out in this present work.

Generally, in the present work larval survival was high (≥ 90%), except for larvae fed T. suecica and originating from broodstock also fed T. suecica (2%). Our results partially agree with those of Fereiro et al (1990) who reported large survival fluctuations between O. edulis larval batches, fed T. suecica, from 0 to 100%, with, relatively good growth though (8.7 µm d -1 ). When broodstock were fed T. suecica and released larvae were fed our mixed diet, larval survival was similar to that reported by [START_REF] Berntsson | Effects of broodstock diets on fatty acid composition, survival and growth rates in larvae of the European flat oyster, Ostrea edulis[END_REF], with survival ≥ 90%. When continuously fed single T. suecica from broodstock through the entire larval life, deficiencies might occur, leading to high larval mortality.

We noted a similar trend for T. lutea with, however, higher survival (53%) but no clear relation with main relative fatty acid content was established. Lastly, competence to metamorphose did not reveal clearly the influence of larval diet when comparing the effects of each microalgae. However positive correlations were found with EPA absolute contents and eyed larval percentage, with single larval diets regardless of the species; such a correlation was not significant for mixed larval diets. Larvae continuously fed S. marinoi, from broodstock to pre-settlement, exhibited a low level of competence, which is in agreement with corresponding data on growth. A similar trend occurred with larvae fed single T. suecica, which is in agreement with data on survival.

Moreover, except for T. lutea, mixed diets (TC g ) led to higher larval competence, which is partially in agreement with our previous work (González -Araya et al., 2012).

When analysing the overall O. edulis development performance, larvae fed single T. lutea from broodstock to pre-settlement exhibited deficiencies which were expressed in 50% mortality. Larvae fed S. marinoi showed lower growth and competence clearly related to cell size, which was unlikely to have been consumed efficiently this early stage. Lastly, when larvae were fed T. suecica, severe nutritional deficiencies occurred, leading to the death of the entire larval population. Regardless of broodstock feeding, however, these potential deficits seem to be mitigated when expelled larvae are fed TC g , which globally led to better larval performances.

Conclusion

The present work confirmed that some broodstock nutritional deficiencies, originating from broodstock conditioning, could be compensated at early stage development when larvae are fed a mixed, balanced diet. Although larvae fed T. suecica from broodstock to pre-settlement exhibited high mortalities and moderate growth, those receiving a bispecific diet displayed good larval performance. Such partial improvement also occurred with T lutea for larval survival. However, to accurately understand the relationships between biochemical transfers from diets to broodstock, from broodstock to initial larvae, and from diets to larvae in development, a more complex experimental design should be carried out. It should be based on single, efficient diets characterized by different fatty acids and should include a starved control to specify the contributions of broodstock and larval reserves. Moreover it would also include metamorphosis determination, because, in contrast with C. gigas, O edulis larval growth is insufficient to discriminate the effects of diets. Some evidence was found, as mortality data revealed severe deficiencies that could not be related to biochemical composition because insufficient live larvae remained for analysis. Such an experiment will be reported in a forthcoming paper.

FiguresFigure 1 .

 1 Figures

  Figure 1. Mean growth and standard deviation of O. edulis larvae fed different single

Figure 2 .

 2 Figure 2. Mean daily growth (A), survival (B) and larval competence (C) (± S.D.) of O.

Acknowledgments

This work could not have been completed without the technical support of the team at the Argenton Ifremer station -C. Mingant, I. Quéau and L. Lebrun-and the LPI analytical biochemical platform -C. Quéré-all of whom we wish to thank. A great thank to G. Wikfors from Milford laboratory for improving English. We are also grateful to the Universidad de Los Lagos (MECESUP-ULA 03/02), which contributed to the partial funding of a PhD grant for the second author. This work was carried out during the SETTLE project and was partially funded by FP7/2007-2013 under agreement no.

222043.

Marshall R., Mc Kinley S., Pearce M. C. (2012). Effects of nutrition on larval growtg and survival in bivalves. Review in Aquaculture 2, 33-55. doi: https://10.1111/j.1753-5131.2010.01022.x Martinez-Fernandez, E., Acosta-Salmon H., Southgate P.C. (2006). The nutritional value of seven species of tropical microalgae for black-lip pearl oyster (Pinctada margaritifera, L) larvae. Aquaculture 257, 491-503. https://doi.org/10.1016/j.aquaculture.2006.03.022 Marty Y., Delaunay F., Moal J., Samain J.F. (1992). Changes in the fatty acid composition of Pecten maximus (L.) during larval development. Journal of

Such regression was not evaluated for survival due to low differences between most of the conditions (> 90%).


Paul Gatti
email: paul.gatti@ifremer.fr

Louise Cominassi

Erwan Duhamel

Patrick Grellier

Herve Le Delliou

Sophie Le Mestre

Pierre Petitgas

Manuella Rabiller

Jérôme Spitz

Martin Huret

Gatti Paul

Cominassi Louise

Duhamel Erwan

Grellier Patrick

Le Delliou Herve

Le Mestre

Petitgas Pierre

Rabiller Manuella

Spitz Jérôme

Huret Martin

Bioenergetic condition of anchovy and sardine in the Bay of Biscay and English Channel

Keywords: Energy density, calorimetry, small pelagic fish, Engraulis encrasicolus, Sardina pilchardus, Northeast Atlantic

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

Introduction

Fish condition is increasingly assessed in the context of fisheries and stock assessments as an indicator of the state of fish stocks in addition to biomass indices (Brosset et al., 2015b;[START_REF] Hartman | Estimating Energy Density of Fish[END_REF][START_REF] Mcpherson | Inconsistencies in measurement of fish condition: a comparison of four indices of fat reserves for Atlantic herring (Clupea harengus)[END_REF][START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF][START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF][START_REF] Van Der Lingen | Estimating the lipid content of pelagic fish in the southern Benguela by visual assessment of their mesenteric fat[END_REF]. Condition indices have displayed significant relationships with total egg production (Marshall et al., 1999) or even recruitment [START_REF] Boyd | Food, transport and anchovy recruitment in the southern Benguela upwelling system of South Africa[END_REF][START_REF] Marshall | Reconstructing the stock-recruit relationship for Northeast Arctic cod using a bioenergetic index of reproductive potential[END_REF][START_REF] Marshall | The effect of interannual variation in growth and condition on haddock recruitment[END_REF].

There are numerous ways to assess the condition of fish. Morphological condition indices, such as the Fulton index, are the most widespread as they can easily be implemented from size at age data [START_REF] Mcpherson | Inconsistencies in measurement of fish condition: a comparison of four indices of fat reserves for Atlantic herring (Clupea harengus)[END_REF], but more precise indicators based on qualitative [START_REF] Van Der Lingen | Estimating the lipid content of pelagic fish in the southern Benguela by visual assessment of their mesenteric fat[END_REF] or quantitative assessment of lipid content (Brosset et al., 2015a) are also increasingly applied, especially for small pelagic fish. Condition indices are often defined as a measure of stored energy [START_REF] Mcpherson | Inconsistencies in measurement of fish condition: a comparison of four indices of fat reserves for Atlantic herring (Clupea harengus)[END_REF]. [START_REF] Mcpherson | Inconsistencies in measurement of fish condition: a comparison of four indices of fat reserves for Atlantic herring (Clupea harengus)[END_REF] argued that condition indices should be well defined and validated before using them to monitor fish condition, as some are "only surrogates of physiological status". Fish proximate composition is based mainly on water, protein, lipid and ash content [START_REF] Elliott | Body composition of brown trout (Salmo trutta L.) in relation to temperature and ration size[END_REF].

Energy density (ED) is the amount of energy per unit of mass. As ash and water have null energetic potential, ED measured on the whole body mass is simply a weighted average of protein and lipid energy densities. Lipid is the storage compound for fish [START_REF] Black | The sequential mobilisation and restoration of energy reserves in tissues of Atlantic cod during starvation and refeeding[END_REF][START_REF] Cargnelli | Notes: Fish Energetics: Larger Individuals Emerge from Winter in Better Condition[END_REF] and the main driver of fish ED variability [START_REF] Anthony | Lipid content and energy density of forage fishes from the northern Gulf of Alaska[END_REF][START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF][START_REF] Spitz | Proximate composition and energy content of forage species from the Bay of Biscay: high-or low-quality food? Ices[END_REF], while proteins are the main compound of body structure thus defining the basal value of ED. The effects of numerous factors affecting ED as a bioenergetic index have been reported, e.g. species, season, area, food availability, spawning phenology, size or ontogeny [START_REF] Anthony | Lipid content and energy density of forage fishes from the northern Gulf of Alaska[END_REF][START_REF] Pedersen | Seasonal variations in the energy density of fishes in the North Sea[END_REF][START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF][START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF]. Thus, the whole body ED is a proper assessment of the amount of energy reserves and integrates daily to seasonal time scale processes as a balance between energetic expenses and gains from food.

Small pelagic fish, as forage fish species, play a key role in energy transfer [START_REF] Cury | Small pelagics in upwelling systems: patterns of interaction and structural changes in "wasp-waist" ecosystems[END_REF][START_REF] Spitz | Prey consumption by cetaceans reveals the importance of energy-rich food webs in the Bay of Biscay[END_REF]. Thus, monitoring small pelagic fish condition provides highly relevant information on the state of the whole pelagic ecosystem, as this reflects their own fitness in relation with prey availability or habitat quality, as well as their own quality as prey for numerous predators from larger fishes up to marine birds and mammals [START_REF] Anthony | Lipid content and energy density of forage fishes from the northern Gulf of Alaska[END_REF][START_REF] Payne | Proximate composition of some north-eastern Pacific forage fish species[END_REF][START_REF] Rand | Energy Density and Size of Pelagic Prey Fishes in Lake-Ontario, 1978-1990 -Implications for Salmonine Energetics[END_REF][START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF]. Indeed, predators are known to be sensitive to the species composition of the available preys with respect to prey quality [START_REF] Österblom | Junk-food in marine ecosystems[END_REF][START_REF] Spitz | Cost of Living Dictates what Whales, Dolphins and Porpoises Eat: The Importance of Prey Quality on Predator Foraging Strategies[END_REF][START_REF] Trites | The decline of Steller sea lions Eumetopias jubatus in Alaska: a review of the nutritional stress hypothesis[END_REF]. Similarly, anchovy and sardine prey on a variety of zooplankton organisms which "cannot be considered as a homogeneous resource in terms of quality" [START_REF] Dessier | Variability of energy density among mesozooplankton community: New insights in functional diversity to forage fish[END_REF]. The energetic content of zooplankton is also very variable among species, size class, area and season [START_REF] Barroeta | Energy density of zooplankton and fish larvae in the southern Catalan Sea (NW Mediterranean)[END_REF][START_REF] Dessier | Variability of energy density among mesozooplankton community: New insights in functional diversity to forage fish[END_REF]. Energetic condition indices such as ED are also of large interest for bioenergetic modelling [START_REF] Gatti | Comparing biological traits of anchovy and sardine in the Bay of Biscay: A modelling approach with the Dynamic Energy Budget[END_REF][START_REF] Hartman | Estimating Energy Density of Fish[END_REF][START_REF] Pecquerie | Modeling fish growth and reproduction in the context of the Dynamic Energy Budget theory to predict environmental impact on anchovy spawning duration[END_REF] and for food web modelling [START_REF] Albo-Puigserver | Ecological energetics of forage fish from the Mediterranean Sea: Seasonal dynamics and interspecific differences[END_REF]. Furthermore, small pelagic fish condition, e.g. lipid content, is routinely monitored as a parameter of particular interest for human consumption [START_REF] Albrecht-Ruiz | Chemical Composition of Light and Dark Muscle of Peruvian Anchovy (Engraulis ringens) and Its Seasonal Variation[END_REF][START_REF] Hale | Proximate chemical composition and fatty acids of three small coastal pelagic species[END_REF][START_REF] Marin | Variations in the fatty acid composition and nutritional value of Adriatic sardine (Sardina pilchardus Walb.) through the fishing season[END_REF][START_REF] Simat | Seasonal changes in proximate composition of anchovy (Engraulis encrasicolus, L.) from the central Adriatic[END_REF] and fish meal production.

In the Bay of Biscay, small schooling energy-rich fish such as anchovy and sardine are the key resources for a variety of predators [START_REF] Spitz | Prey consumption by cetaceans reveals the importance of energy-rich food webs in the Bay of Biscay[END_REF] In the Bay of Biscay, ED of small pelagic fish, and in particular anchovy and sardine, has already been partially assessed. [START_REF] Dubreuil | Energy density of anchovy Engraulis encrasicolus in the Bay of Biscay[END_REF] described variations of ED of anchovy with size class and season, and explored the relationship between ED and dry mass content. [START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF] studied variation in ED in the guild of small pelagic species observed in the Bay of Biscay and also showed effects of size class and season. As far as we know, there are no data on ED for anchovy and sardine in the English Channel. In this paper, we aim at updating previous studies and to investigate further the sources of variability in ED. Based on a large sampling effort in 2014 over the Bay of Biscay -English Channel area and subsequent ED measurements on numerous individual samples, we propose to analyse the effect of species, size, age, season and region for anchovy and sardine.

Material and methods

Sampling design

Juveniles and adults of anchovy and sardine were caught in 2014 during three fishery surveys, namely PELGAS [START_REF] Doray | PELGAS 2014 cruise, RV Thalassa[END_REF] and EVHOE [START_REF] Duhamel | EVHOE 2014 cruise, RV Thalassa[END_REF] in the Bay of Biscay and CAMANOC (Travers-Trolet and Verin, 2014) in the English Channel. PELGAS (April 24 th to June 5 th ) and EVHOE (October 17 th to November 16 th ) are designed for the assessment of small pelagic species in spring and of demersal species in autumn, respectively. CAMANOC (September 16 th to October 10 th ) was designed to assess demersal species in the English Channel ecosystem. Fish were caught using a pelagic trawl during PELGAS and bottom trawl during CAMANOC and EVHOE, but the latter surveys still provided samples opportunistically in most of the size ranges of both species. There is no survey in spring in the English Channel. The abundance and spatial distribution of anchovy and sardine in the English Channel in spring remain poorly known. Together, these three surveys provided samples from two regions and two contrasted seasons for the Bay of Biscay. Contrasted fish conditions are anticipated between spring that follows winter with low temperature and food availability, and autumn that follows a warm and productive period. A total of 164 anchovies and 209 sardine samples from 2014 were processed (Table 1). The onboard sampling was opportunistic and driven by the availability of the two targeted species, as well as the occurrence of wide ranges of fish size in each trawl haul. Sampling locations covered most of the areas and were representative of spatial distributions of these species (Figure 1). 2.2 Sample processing ED, i.e. the amount of energy per unit mass, was measured following the protocols of [START_REF] Dubreuil | Energy density of anchovy Engraulis encrasicolus in the Bay of Biscay[END_REF] and [START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF]. Fish length and wet weight were recorded to the nearest 0.5 cm and 1 g, respectively, on board or latter in laboratory. Fish were then individually frozen (-20°C) until lab processing. The time lag between sampling and freezing did not exceed half an hour. In the laboratory, fish were unfrozen and otoliths extracted for latter age reading (in years) by experienced readers, following the ICES guidelines [START_REF] Ices | Report of the Workshop on Age Reading of European Atlantic Sardine (WKARAS)[END_REF](ICES, , 2009)). In order to avoid water loss during sample processing and thus ensure accurate estimation of water content the abdominal cavity was not opened; which prevented any macroscopic determination of sex or sexual maturity. The whole fish was then grinded and freeze-dried during more than 48 hours. Dry mass was recorded. The dry mass content (DM %) was computed simply as the ratio between dry and wet masses. Fish were again grinded to obtain a fine homogeneous dry powder. Two subsamples of fish powder were placed in an adiabatic bomb calorimeter (IKA C-4000 adiabatic bomb calorimeter, IKA-WerkeGmbh & Co. KG) for energy measurements. If the coefficient of variation between the two measurements exceeded 3%, a third measurement was made and finally a mean among the several subsamples was calculated and assigned to each individual fish.

Statistical Analysis

We first explored the well documented relationship between ED and DM, initially introduced by [START_REF] Hartman | Estimating Energy Density of Fish[END_REF] but also more recently analysed in the Adriatic Sea for anchovy by [START_REF] Tirelli | Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea[END_REF]. Regressions were fitted with linear and log-log models. A posteriori test showed that residuals were normally distributed (not shown). Covariance analysis (ANCOVA) was used to determine whether the relationship between DM and ED differs among anchovy and sardine. In case of linear regressions, we also compared coefficients estimates for clupeiform species from [START_REF] Hartman | Estimating Energy Density of Fish[END_REF] with our estimates. For anchovy, we compared coefficient estimates between our linear model and the model published by [START_REF] Tirelli | Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea[END_REF] for the Adriatic Sea using Student tests. Distributions of linear regression coefficients were built by randomly drawing estimates 10.000 times in normal distributions parameterised following mean and standard deviation estimates (of linear regression coefficients). Resulting distributions were then compared to the values extracted from the literature using the Student test. In the present study a p-value threshold of 5% was considered to be significant to reject the null hypothesis.

ED and more generally condition is known to be influenced by a variety of factors, e.g.

species, season, area, size or ontogeny. We thus described qualitatively the growth patterns (in both size and wet weight) and the evolution of ED with age observed for anchovy and sardine during our three surveys. We then analysed the pattern of ED with size using quantitative tools by the mean of generalised additive models (GAM) using the R library mgcv [START_REF] Wood | Fast stable restricted maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models[END_REF]. Instead of age, which was not integrated in the model due to the scarcity of oldest fishes in our samples (see Figure 3) and the strong relationship between size and age, we considered the size. An analysis of model residuals did not demonstrate any age effect (not shown). The factorial design is not complete, but provides a description at two seasons in the Bay of Biscay, i.e. spring and autumn, and in early autumn in the English Channel. To account for the effects of region and season we thus considered the survey. The combined effect of size and survey was introduced in the GAM models fitted for each species.

Analysis of residual distribution demonstrated that data were normally distributed (not shown). As each survey corresponds to a specific combination of season and area, we expected a specific evolution of ED with size according to the survey. The following equation: , in R computing language, describes the structure of the model. The first effect is the average ED value ( ) for each survey on the whole size range. The second effect, i.e. , is the smoothed survey specific variation of ED with size around the previous average (i.e. the first effect in the model). The GAM model was thus built to integrate the factor survey and the effect of length smoothed by each modality of survey. This first set of models enabled to describe the scaling of ED with size for each species. To further compare this specific scaling of ED for anchovy and sardine, we considered in a second set of models a "standardised length" instead of total length to filter out the effect of the specific growth patterns. This standardised length is simply the ratio between the total length and the 95% upper quantiles of total length distributions for each species, i.e. 17.0 and 23.0 cm for anchovy and sardine, respectively.

Results

Energy density against dry mass content

Energy density (ED) increases with dry mass content (DM) for both species (Fig. 2), and more sharply in the case of sardine. Linear and log-log regressions were fitted to describe the relationship between DM and ED. According to ANCOVA analysis, considering a specific intercept for each species significantly improved (at a 5% threshold) the fit of the relationship in comparison with a common intercept for both species (df = 1, F = 22.56 and 22.81 for linear and log-log models, respectively). A specific slope also improved significantly the fit of the relationship (df = 1, F = 53.11 and 18.18). Intercepts and slopes in linear and log-log relationship between ED and DM are thus specific for each species. In case of ANCOVA tests for linear regressions, R² were 0.922, 0.927 and 0.936 for common intercept and slope, specific intercepts and common slope, and specific intercepts and slopes models, respectively.

In case of log-log regressions, R² were 0.930, 0.934 and 0.937 for common intercept and slope, specific intercepts and common slope, and specific intercepts and slopes models, respectively.

Figure 2. Energy density (kJ.g -1 wet mass) against dry mass content (the ratio of wet and dry masses in %). Data, linear ( ) and log-log ( .) model predictions are plotted with crosses, continuous and dotted lines, respectively. The linear model of [START_REF] Tirelli | Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea[END_REF]

fitted for Adriatic anchovy is represented by a continuous grey line (left plot).

All coefficients are significant (Table 2). R 2 are high, ranging from 0.89 to 0.94, and even higher for sardine. Student tests were used to compare coefficients of the linear model from [START_REF] Hartman | Estimating Energy Density of Fish[END_REF] 2) than anchovy, meaning that for the same increase of DM the gain of energy per unit mass is slightly higher for sardine (see Figure 2). To quantitatively describe the evolution of ED with size according to species and season GAM models were fitted and model predictions are displayed on Figure 4 and 5. The explained variance by generalised additive models are 0.64 and 0.71 for anchovy and sardine, respectively (Table 3). Most effects are highly significant, apart from the length effect for sardine in spring in the Bay of Biscay. Estimates confirm previous observations that ED is higher on average for sardine than anchovy for each combination of area and season, i.e. by 0.6, 1.3 and 4.4 kJ.g -1 for the Bay of Biscay in spring, in autumn and in the English Channel in autumn, respectively (see Table 3). In the Bay of Biscay the seasonal difference in ED is 1.5 and 2.1 kJ.g -1 for anchovy and sardine, respectively. Lower ED estimates for anchovy in the English Channel than in the Bay of Biscay are likely due to the length distribution of samples from the English Channel, mostly representatives of the smaller sizes. For anchovy in the Bay of Biscay, according to GAM predictions, ED increases up to approximately 15-16 cm in total length for both season and then seems to stabilise (Figure 4).

In the English Channel, with the lack of anchovy in the size range 14-17 cm, only a steep increase in ED with length can be described. For sardine in autumn ED increases with size up to 18 and 19 cm for the Bay of Biscay and the English Channel, respectively, then decreases strongly (Figure 5). For sardine in spring in the Bay of Biscay we still observe two phases, a first increase followed by a decrease, although the pattern is very flat and the effect of size is not significant at that season. In spring in the Bay of Biscay (Figure 5), both species display very similar values on a common size range, i.e. between 12 and 18 cm. In autumn, predicted values for the smallest sizes are also very close but diverge strongly as size increases.

The second set of GAM models including the "standardised length" instead of the total length displays the same pattern of significance and the same values for the categorical (i.e. survey) effects (not shown). Data and model fits are displayed on Figure 6. The standardised length being the ratio between total length and the 95% upper quantile of the total length distribution of each species, this explains the occurrence of values slightly exceeding 1 for this metric.

The standardised length translates the fraction of growth achieved by one individual. As expected, the patterns are really similar to those observed with the previous set of GAM models, e.g. the dome shaped increase of ED for sardine in autumn or the increase until a threshold for anchovy in the Bay of Biscay (in both spring and autumn). In addition, this representation highlights that for autumn surveys sardine displays higher ED values than anchovy at the same standardised length and also reaches its maximal ED potential earlier with respect to its "maximum size".

Discussion

Our results quantify how different factors affect the energetic condition of anchovy and sardine, obviously species and season but also region and body size, the latter in a surprisingly non monotonic allometric way as highlighted for sardine. A large number of samples were processed to draw these results. All samples have been caught in 2014, thus an extended dataset over more years would be required to further support observed patterns and refine analysis. However, the dataset containing fish individuals over a large range of size and age, up to 8 annual cohorts for sardine, this is unlikely that the main patterns described here would disagree with observations from an extended dataset.

Energy density against dry mass content

The linear and log-log relationships between energy density (ED) and percent dry mass content (DM) displayed strong fit, i.e. with R 2 values being consistently above 0.89. These relationships are generally strong for numerous species [START_REF] Hartman | Estimating Energy Density of Fish[END_REF]. These authors showed that even gathering numerous species in a single relationship can still lead to a strong fit in case of a log-log model. This is likely due to the effect of size which would override interspecific differences when gathering wide body size ranges across numerous species. But the coefficient estimates from our study, in case of linear models, displayed significant differences with previous works from [START_REF] Hartman | Estimating Energy Density of Fish[END_REF]. In addition, as we found distinct relationships between both related species through linear and log-log models, we argue that this relationship is very strong but should remain species specific.

In the case of anchovy we obtained a different relationship from that of [START_REF] Tirelli | Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea[END_REF],

which suggests that such model are species specific but also area specific. But this has to be confirmed with an extended dataset, i.e. with more years, and is likely to be drawn from distinct length and ED ranges in both datasets.

We explored the relationship between ED and DM with both linear and log-log models, which provided similar fits. [START_REF] Wuenschel | Estimating the energy density of fish: The importance of ontogeny[END_REF] suggested that linear relationships between ED (or lipid) and DM are strongly affected by ontogeny and should be stage specific.

Zenitani and Kono (2012) also described an exponential allometry between lipid content (both phospholipids and triglycerides) and body size for sardine larvae from Eastern Japan. Log-log shape would allow the description of an exponentially increasing pattern (of lipid content per unit of mass) and thus could better deal with an increase in ED with body size and ontogenetic effects occurring between larvae, juveniles and fully grown adults. Log-log models should thus be preferred, as highlighted by slightly better fits.

Species and season

Clupeiform are among the most energy rich forage fish species in the Bay of Biscay, among this family sardine displays the highest ED values and variability [START_REF] Spitz | Variability in energy density of forage fishes from the Bay of Biscay (north-east Atlantic Ocean): reliability of functional grouping based on prey quality: variability in energy density of forage fishes[END_REF].

Spitz and Jouma'a (2013) reported that ED is on average equal to 5.5 and 5.8 kJ.g -1 wet weight in spring and to 6.4 and 8.8 kJ.g -1 in autumn, for anchovy and sardine, respectively.

Our results are in agreement with these values. [START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF] showed that ED of sardine off Portugal displays a minimum in March/April and a peak in September/October. Spring and autumn ED thus exhibit minimum and maximum seasonal values for these species.

Despite of a potential lag in the seasonal cycle of plankton productivity and temperature between the English Channel, the Bay of Biscay and Portugal, as well as mesoscale processes affecting the local productivity, we also likely captured these extreme values, or were close to them, according to our sampling design in our temperate regions.

GAM models highlighted similar minima of energetic condition for both species. [START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF] showed that for sardine, minima in ED values in February/March were consistent across years irrespectively of previous energetic or environmental conditions. Considering that in spring anchovy and sardine display similar minima of absolute ED values, both species thus emerge from overwintering with a similar low or basal energetic condition after exhaustion of the lipid reserves.

Our results evidenced species specific effects on ED, with higher seasonal variations and steeper increase with both body size and DM in the case of sardine. This first suggests a higher storage capacity for sardine, i.e. it is able to store more energy (i.e. more lipid) per unit of mass. The second set of GAM models also suggests that ontogenetic scaling of storage capacity is faster in the case of sardine. The larger seasonal difference and variability in ED could also indicate larger energetic expenses between autumn and the following winter. The seasonal variability in bioenergetic condition is resulting from a combination of the seasonal cycle in plankton productivity (food availability) and of the spawning phenology [START_REF] Pedersen | Seasonal variations in the energy density of fishes in the North Sea[END_REF][START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF]. Anchovy is known to spawn in spring/summer in the Bay of Biscay [START_REF] Huret | Survey timing vs. ecosystem scheduling: Degree-days to underpin observed interannual variability in marine ecosystems[END_REF][START_REF] Motos | Reproductive biology and fecundity of the Bay of Biscay anchovy population (Engraulis encrasicholus L.)[END_REF] whereas sardine is expected to spawn in autumn and end of winter -spring with a limited spawning activity in winter [START_REF] Coombs | Spawning season and temperature relationships for sardine (Sardina pilchardus) in the eastern North Atlantic[END_REF][START_REF] Stratoudakis | Sardine (Sardina pilchardus) spawning seasonality in European waters of the northeast Atlantic[END_REF]. Sardine is thus likely to cumulate more energy during productive months (from spring to autumn) allowing larger spawning expenses over the autumn to spring season. Also, as a larger and heavier fish than anchovy, sardine is likely to face larger maintenance costs, thus requiring larger reserves to overwinter. In addition to specific energetic expenses, distinct assimilation efficiencies or diet could lead to distinct energetic conditions (which is further discussed in the last section hereafter).

Region

In autumn, except at age 0, fishes from the English Channel display larger length and wet weight at age than fishes from the Bay of Biscay. Smaller sizes at age 0 likely result from differences in birth dates and survey timing, collected juveniles in the English Channel being younger. This regional difference is less obvious for anchovy as the dataset contains only few old specimens given its very short lifespan.

Larger length, wet weight and ED at age could be due to a higher zooplankton productivity or more generally more favourable environmental conditions in the English Channel. [START_REF] Silva | Geographic variability of sardine growth across the northeastern Atlantic and the Mediterranean Sea[END_REF] showed a decrease in sardine growth pattern from the English Channel to Morocco following a latitudinal trend in temperature and zooplankton productivity. Populations at northern latitudes generally display larger growth performance, which can be explained by size related overwintering survival of juveniles involving a strong selection pressure toward faster growing individuals [START_REF] Conover | Seasonality and the scheduling of life history at different latitudes[END_REF][START_REF] Conover | Countergradient variation in growth rate: compensation for length of the growing season among Atlantic silversides from different latitudes[END_REF]. Larger individuals of a species are expected to better endure winter starvation as they display larger energy reserve [START_REF] Cargnelli | Notes: Fish Energetics: Larger Individuals Emerge from Winter in Better Condition[END_REF]. [START_REF] Silva | Temporal and geographic variability of sardine maturity at length in the northeastern Atlantic and the western Mediterranean[END_REF] displayed a similar pattern in length at first maturity of sardine across the Atlantic European coast with minimal length at maturity increasing with latitude starting from 11.0 cm in the South of Portugal until 14.5 cm in the north of the Bay of Biscay (France). This could be a consequence of the latitudinal growth pattern or could also highlight a delayed spawning onset. Delayed spawning could favour energetic allocation in growth to quickly reach larger sizes and sufficient energy storage capacity to overwinter. Moreover in the case of sardine, [START_REF] Coombs | Spawning season and temperature relationships for sardine (Sardina pilchardus) in the eastern North Atlantic[END_REF] and [START_REF] Stratoudakis | Sardine (Sardina pilchardus) spawning seasonality in European waters of the northeast Atlantic[END_REF] described a latitudinal gradient of spawning phenology in European waters associated with the temperature gradient. As the seasonal cycle of sardine ED is largely driven by spawning expenses [START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF], the ED differences between the Bay of Biscay and the English Channel in autumn could partly result from distinct spawning periods.

Instead of regional differences in growth patterns, a size related migration to the North was hypothesised by [START_REF] Furnestin | Contribution à l'étude biologique de la sardine atlantique (Sardina pilchardus WALBAUM)[END_REF] and [START_REF] Silva | Geographic variability of sardine growth across the northeastern Atlantic and the Mediterranean Sea[END_REF] in the case of sardine, based on the decrease in abundance of young and small individual with latitude. However we did catch and analyse juveniles of sardine and anchovy in the English Channel (age 0) below 10 cm in length, most of them in coastal western areas or in the Bay of Seine to the east. It seems unlikely that such small fishes actively migrated from the Bay of Biscay. Also, in a pluridisciplinary study, [START_REF] Petitgas | Anchovy population expansion in the North Sea[END_REF] showed that the English Channel was likely sharing a same population within the North Sea, rather than with the Bay of Biscay. We still lack reliable information to conclude on any population structure between the Bay of Biscay and the English Channel for both species, but we expect that distinct environments (both temperature and zooplankton abundance and phenology) may lead to distinct growth, spawning and bioenergetic strategies.

Size and age

Effects of age and size are strongly and naturally interlinked through growth. Our data displayed very similar patterns of ED with both size and age. Moreover, the analysis of GAM residuals did not reveal any effect of age (not shown), suggesting the absence of cohort effect in our dataset. We showed that energetic condition of sardine, in both the Bay of Biscay and the English Channel, strongly increased between age 0 and 1, then reached an optimum between age 2 or 3 and finally decreased thereafter. Results of Brosset et al. (2015b) highlighted a similar pattern for these species in the Gulf of Lion with a the relative condition index. These authors also argued that this decrease in condition at oldest ages is more dramatic in years of unfavourable environmental condition, in particular food availability or quality.

The effect of size on ED has been reported for many fish species [START_REF] Anthony | Lipid content and energy density of forage fishes from the northern Gulf of Alaska[END_REF], energy reserves being positively correlated with size [START_REF] Cargnelli | Notes: Fish Energetics: Larger Individuals Emerge from Winter in Better Condition[END_REF]. [START_REF] Pedersen | Seasonal variations in the energy density of fishes in the North Sea[END_REF] noticed that the effect of size is not always linear. [START_REF] Tirelli | Energy density of anchovy Engraulis encrasicolus L. in the Adriatic Sea[END_REF] described, for anchovy in the Adriatic Sea, a linear relationship between ED and size until 9.0 cm and a more variable pattern for larger sizes. Indeed, in our cases this effect is not a simple allometric relationship as ED first shows an increase with size until an optima for both species and then a steep decrease in the case of sardine. The increase in ED for juveniles and young adults is likely due to a relative decrease in water content and a concurrent increase of energy reserves, i.e. lipid content [START_REF] Wuenschel | Estimating the energy density of fish: The importance of ontogeny[END_REF].

The decrease in energetic condition for large and old fish seems a bit counter intuitive as one could expect an asymptotic trend similar to a Von Bertalanffy model. This pattern can be attributed to some decrease in energy acquisition or increase in energetic expenses, or a combination of both. Decrease in energetic gain could be either attributed to some change in diet or to a decrease in digestion or assimilation efficiency, while an increase in energetic expenses could be related to an increase in metabolic rate (e.g. respiration or cellular maintenance) or energy allocation to reproduction.

Anchovy and sardine are planktivorous species feeding mainly on copepods [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF][START_REF] Plounevez | Feeding behaviour and trophic environment of Engraulis encrasicolus (L.) in the Bay of Biscay[END_REF]. [START_REF] Bode | The pelagic foodweb in the upwelling ecosystem of Galicia (NW Spain) during spring: natural abundance of stable carbon and nitrogen isotopes[END_REF] showed that the sardine diet in Northern Spain greater than 18 cm progressively shifts to smaller prey items, both smaller zooplankton organisms and phytoplankton cells, as a result of an increased filter feeding mode. Smaller zooplankton items are likely to be less energy rich and especially phytoplankton cells, which are generally described to poorly contribute as a carbon input in the diet of sardine [START_REF] Costalago | Seasonal comparison of the diets of juvenile European anchovy Engraulis encrasicolus and sardine Sardina pilchardus in the Gulf of Lions[END_REF][START_REF] Nikolioudakis | Diet of sardine Sardina pilchardus: an 'end-to-end' field study[END_REF]. Our results showed optimum values in ED around this size for the Bay of Biscay and at a slightly larger size for the English Channel, i.e. around 19 cm. Also, these slight differences in optimal size for energetic condition could be related to the latitudinal growth pattern described by [START_REF] Silva | Geographic variability of sardine growth across the northeastern Atlantic and the Mediterranean Sea[END_REF], who showed that sardine from the English Channel have a greater length at age of 2 cm than fish from the Portugal or Northern Spain.

The largest energetic expenses for fish are generally described as maintenance [START_REF] Kooijman | Waste to hurry: dynamic energy budgets explain the need of wasting to fully exploit blooming resources[END_REF] and spawning [START_REF] Jørgensen | Modelling and interpreting fish bioenergetics: a role for behaviour, life-history traits and survival trade-offs[END_REF][START_REF] Mcbride | Energy acquisition and allocation to egg production in relation to fish reproductive strategies[END_REF], two processes highly correlated with size. [START_REF] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF] showed that the seasonal cycle of sardine ED is largely driven by spawning expenses. Larger fish are expected to invest more in spawning as they display wider period of active spawning [START_REF] Nunes | Integrating fish size, condition, and population demography in the estimation of Atlantic sardine annual fecundity[END_REF], thus energetic investment in spawning is likely to significantly scale with length. As sardine spawns in both spring and autumn, lower autumnal ED values for larger individuals could thus be attributed to an earlier start of "autumn spawning", leading to an earlier decrease of energy reserves, or a later recovery of reserves associated to the previous "spring spawning".

We previously discussed a potential trade-off between food availability and temperature in sustaining maintenance requirements which is depending on fish size. The more favourable environmental condition in the English Channel, in comparison with the Bay of Biscay, may be able not only to support faster growth and higher cumulated energy reserves in autumn, but also result in a better trade-off in the environmental conditions allowing maximum energy reserves at bigger size. The decrease in ED at largest sizes in the English Channel could also be related to the increase in length at first maturity with latitude in the north-eastern Atlantic for sardine described by [START_REF] Silva | Temporal and geographic variability of sardine maturity at length in the northeastern Atlantic and the western Mediterranean[END_REF], that would delay the large energetic expenses associated with reproduction.

Conclusion

We first confirmed the validity of regression between energy density and dry mass content for anchovy and sardine, two small pelagic fish species of economic importance, and we argue that this relationship is species specific. Second, we explored the latitudinal and seasonal variation of ED for both species. In looking for a reliable index of fish (bioenergetic) condition, we thus encourage a preliminary assessment of its seasonal evolution. Sardine and anchovy show similar energy density values in spring reflecting a minimum energetic threshold. Maximum values of energy density, observed in autumn at the end of the productive season, are more variable across individuals and thus more likely to integrate spawning and environmental condition histories. Thus assessing fish condition should preferentially be based on autumn surveys. We encourage further work to cautiously consider the associated effects of size and season when planning experimental design. We finally highlighted the surprising, strong and dome shaped effect of size on the bioenergetic condition of sardine. We discussed the decrease pattern of energy density for larger and older fishes with reported patterns of spawning phenology, feeding behaviour or a trade-off between metabolic requirements and food availability. We are unable at this stage to conclude on any of these hypothesis or combination of them, which would indeed require additional individual information on maturity stages and stress responses.

  . In the Bay of Biscay and in the English Channel, anchovy and sardine fisheries have displayed large interannual fluctuations over the last three decades. Sardine landings in the Bay of Biscay are increasing since 2000 and reached 28216 tons in 2015 (ICES, 2016). The anchovy stock recovered in 2010, after a collapse in 2005 (ICES, 2012), and landings reached 28253 tons in 2015 (ICES, 2016). Anchovy landings in the English Channel remained under 25 tons per year between 1984 and 1996 but increased between 1997 and 2013 and reached 1754 tons in 2015 (ICES, 2016). Sardine landings in the subarea VII, are mainly achieved in the English Channel or close to it (ICES Subareas VIId, e and f) and reached 9314 tons in 2015 (ICES, 2016).

Figure 1 .

 1 Figure 1. Sampling locations in the Bay of Biscay and English Channel, for anchovy (left) and sardine (right) and for surveys PELGAS (circle), CAMANOC (cross) and EVHOE (triangle).

Figure 3 .

 3 Figure 3. Variation of length (first panel from top), wet weight (second panel) and energy density (in wet weight, third panel) at age, of anchovy (left) and sardine (right) observed in the Bay of Biscay in spring (survey PELGAS, white), in the Bay of Biscay in autumn (survey EVHOE, lightgrey) and in the English Channel in autumn (survey CAMANOC, darkgrey). The boxplots display the percentiles 5 and 95% (end of vertical lines), 25 and 75% percentiles (bottom and top side of the box) and finally the median (bold central line). The fourth and last panel displays the number of samples by combination of species, survey and age.

Figure 4 .

 4 Figure 4. Observed and predicted ED values by the GAM against total length for anchovy. The top left panel displays all data and fit per survey (combined modality of season and

Figure 5 .

 5 Figure 5. Observed and predicted ED values by the GAM against total length for sardine. The top left panel displays all data and fit per survey. Data and fitted values for the Bay of Biscay in spring (circle), the Bay of Biscay in autumn (triangle) and the English Channel in autumn (cross). Other panels display the model fit for each combination, with average predictions (continuous lines) and average +/-2 standard errors of the fit (dashed lines), as well as model predictions for anchovy (grey lines).

Figure 6 .

 6 Figure 6. Observed and predicted ED values by the GAM against standardised length for anchovy (grey crosses and grey lines) and sardine (black circles and black lines). Data and fits for the Bay of Biscay in spring, the Bay of Biscay in autumn and the English Channel in autumn are plotted on the first, second and third panel respectively. Model fits for each combination are displayed by average predictions (continuous lines) and average +/-2 standard errors of the fit (dashed lines).

Table 1 .

 1 Sampling design: survey, area, season, number of samples and sampled size ranges in brackets(cm)

	Survey	Area	Season -Months	Anchovy	Sardine
	PELGAS	Bay of Biscay	Spring April -June

Table 2 .

 2 Coefficients of regression models between energy density (kJ.g -1 wet mass) and dry mass content (i.e. the ratio between dry and wet masses %).For sardine, ED values observed in autumn in the English Channel are consistently higher than in the Bay of Biscay for both seasons, except at age 0 which is likely related to smaller sizes in the English Channel at that age. The pattern are the same for anchovy at age 0 and 1, and we lack older individuals from the Channel in the dataset to conclude on older ages.When considering sardine in the Bay of Biscay, ED values are higher in autumn than in spring from age 1. Whatever the season, sardine average ED values show an increase until ages 2 or 3 and a regular decrease later on. This latter pattern is maybe less clear in the Bay of Biscay in spring, and should be considered with caution due to the scarcity of oldest ages in the dataset.Finally, for age 1 and 2 sardine displays higher ED values in autumn than anchovy, both in the Bay of Biscay and in the English Channel, but similar values in spring in the Bay of Biscay.

		Species Nb	R 2	Coef.	Estimates Std. error
	Linear	Anchovy 164 0.894	intercept slope	-3.403 0.333	0.254 0.009
		Sardine 209 0.940	intercept slope	-5.85 0.428	0.226 0.008
	Log-log	Anchovy 164 0.907	intercept slope	-3.302 1.523	0.127 0.038
		Sardine 209 0.942	intercept slope	-3.995 1.740	0.101 0.030

3.2 Variation of length, weight and energy density with age

The evolution of size, wet weight and ED with age is presented in Fig.

3

. Sardines from the English Channel are larger than those from the Bay of Biscay from age 1. Anchovies from the English Channel are larger than those from the Bay of Biscay only at age 1. For both species in the Bay of Biscay, individuals display distinct sizes between spring and autumn only at age 1. Patterns in wet weight are very similar to patterns observed in total length.

Table 3

 3

	Anchovy	Sardine

. GAM summaries: estimates, associated standard errors and significance (p-value). BoB, EC, Sp and Au refer to Bay of Biscay, English Channel, Spring and Autumn, respectively.

Acknowledgments

The authors wish to thank both reviewers for their relevant advice and comments that helped in significantly improving this paper. The authors also wish to thank surveys' chiefs namely Michele Salaun, Mathieu Doray and Morgane Travers-Trollet, as well as technicians involved in the sampling at sea during surveys. This work was supported by a PhD grant from the project SEAMAN, founded by the ANR (French National Research Agency) and EU FP7 ERA-NET SEASERA, as well as the Captain project founded by FFP (France Filière Pêche).