Mathilde Bertrand
email: mathildebertrand@outlook.fr|christophe.lebigre@ifremer.fr

Pablo Brosset

Philippe Soudant

Christophe Lebigre

Spatial and ontogenetic variations in sardine feeding conditions in the Bay of Biscay through fatty acid composition

Keywords: fatty acids, lipids, Sardina pilchardus, small pelagic fish, NE Atlantic, size-at-age

Food characteristics are amongst the most influential factors determining the fish life history traits as quantitative and qualitative changes in individuals' diet can lead to a decline in the energy allocated to their growth, and hence influence natural populations' characteristics. The size-at-age and weight of European sardines (Sardina pilchardus) in the Bay of Biscay (BoB) have decreased substantially over the last decade, especially for the youngest age classes, and the factors underlying such changes have not yet been identified. We therefore analysed the fatty acid (FA) composition in the neutral (NL) and polar (PL) lipids in samples collected across the BoB to determine whether the diet of sardines changes with their ages. We found that the

Introduction

Food characteristics are one of the most important biotic factors influencing individuals' fitness as it allows animals to extract energy from their environment. This energy is then allocated to life history traits and any alteration in its quantity or quality leads to a decrease in survival, growth, and reproductive success [START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF][START_REF] Stearns | The evolution of life histories[END_REF].

Furthermore, the environment governs communities' composition and dynamic by controlling the abundance and assemblage of primary producers and their consumers [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Hauss | Changes in N:P stoichiometry influence taxonomic composition and nutritional quality of phytoplankton in the Peruvian upwelling[END_REF]. For instance, [START_REF] Hixson | Climate warming is predicted to reduce omega-3, long-chain, polyunsaturated fatty acid production in phytoplankton[END_REF] and [START_REF] Pethybridge | Spatial patterns and temperature predictions of tuna fatty acids: tracing essential nutrients and changes in primary producers[END_REF] predicted a decrease in the nutritional quality of phytoplankton along increasing sea surface temperature, potentially (i) affecting the recruitment and dynamics of forage fish (and their predators), and (ii) having consequences for human food safety [START_REF] Budge | Estimating concentrations of essential omega-3 fatty acids in the ocean: supply and demand[END_REF] since fish may become less abundant and/or less nutritious. In other words, changes at the base of trophic chains can strongly influence the fitness of all species in the upper trophic levels, making it crucial to understand as such changes may have important consequences for stock productivity, ecosystem dynamics and fisheries yields [START_REF] Carozza | Metabolic impacts of climate change on marine ecosystems: implications for fish communities and fisheries[END_REF][START_REF] Martino | Using otolith chronologies to understand long-term trends and extrinsic drivers of growth in fisheries[END_REF].

The European sardine (Sardina pilchardus) plays a central role in the transfer of energy between planktonic compartments and higher trophic levels in pelagic food webs [START_REF] Certain | Investigating the coupling between small pelagic fish and marine top predators using data collected from ecosystem-based surveys[END_REF][START_REF] Cury | Small pelagics in upwelling systems: patterns of interaction and structural changes in "wasp-waist" ecosystems[END_REF]. In the Bay of Biscay (BoB), there has been a marked decrease in size-at-age, weight-at-age and body condition of sardine (Doray et al., 2018b;Véron et al., 2020aVéron et al., , 2020b)), a pattern similar in another small pelagic fish at the same trophic level, the European anchovy (Engraulis encrasicolus, Doray et al., 2018b). More specifically between 2000 and 2015, mean length of sardine decreased particularly at age 1 fish (ca. from 18.5 to 15 cm, i.e. 20%) and body condition declined by 15% on average across all ages (Doray et al., 2018b;Véron et al., 2020a). However, their size-at-maturity (ca. 14 cm) did not change and their age-at-maturity remains at 1 year in the BoB (Véron et al., 2020b). These declines have started while the stock had a relatively low exploitation rate and the increase in harvest was unrelated to the decline in fish growth and selective mortality [START_REF] Boëns | Determinants of growth and selective mortality in anchovy and sardine in the Bay of Biscay[END_REF], making it unlikely that the recent decline in morphometry of sardines in this area can be attributed to fisheries-induced evolution. Therefore, such a decline in size-at-age and body condition is more likely to be driven by environmental changes in the BoB, though the actual mechanism remains elusive (Véron et al., 2020a(Véron et al., , 2020b)). Moreover, these phenotypic trends are similar to those observed in the Gulf of Lions since 2008, in which food quantity and quality have been identified as the main drivers leading to the decline of sardines size and body condition and the subsequent collapse of its fishery [START_REF] Brosset | Spatio-temporal patterns and environmental controls of small pelagic fish body condition from contrasted Mediterranean areas[END_REF](Brosset et al., , 2015b;;[START_REF] Saraux | Small pelagic fish dynamics: a review of mechanisms in the Gulf of Lions[END_REF]. As sardine's stock of the BoB sustains major French and Spanish fisheries (totalling 32,299 tons landed in 2018; [START_REF] Ices | Sardine (Sardina pilchardus) in divisions 8.a-b and 8.d (Bay of Biscay)[END_REF], it is important to determine whether bottom-up processes also explain the decline in size-at-age and body condition in this species.

A bottom-up control of the phenotypic characteristics of sardines may result from changes in their diet: a decrease in the quantity of food available per individual due to densitydependent competition (the survival rate of juveniles increases in this stock; Doray et al., 2018b;[START_REF] Van Beveren | Rapid changes in growth, condition, size and age of small pelagic fish in the Mediterranean[END_REF], and/or a decrease in the quality of food (as it seems to be the case with sardines of the Gulf of Lions; [START_REF] Bachiller | A trophic latitudinal gradient revealed in anchovy and sardine from the Western Mediterranean Sea using a multi-proxy approach[END_REF][START_REF] Brosset | Linking small pelagic dietary shifts with ecosystem changes in the Gulf of Lions[END_REF][START_REF] Saraux | Small pelagic fish dynamics: a review of mechanisms in the Gulf of Lions[END_REF]. Sardines primarily feed on small species of zooplankton (copepods, decapods, cirripedes, fish eggs and cladocerans) and phytoplankton (diatoms and dinoflagellates) but whose contribution to individuals' diet varies depending on fish length, season and region considered [START_REF] Costalago | Comparison of the feeding apparatus and diet of European sardines Sardina pilchardus of Atlantic and Mediterranean waters: ecological implications[END_REF]Garrido et al., 2008a;[START_REF] Van Der Lingen | Trophic dynamics of small pelagic fish[END_REF]. Indeed, during sardine spawning periods (October to May, with peaks in November and April; [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], their broad distribution appears fragmented by the presence of cold bottom water [START_REF] Bellier | Historical fluctuations in spawning location of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) in the Bay of Biscay during 1967-73 and 2000[END_REF]. Thus, in Spring the biomass of sardines is higher along the coast in the Southern BoB, near the Loire estuary, and in the waters South-West of Brittany (Doray et al., 2018a). In general, this population structure coincides with the distribution of sardine eggs [START_REF] Bellier | Historical fluctuations in spawning location of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) in the Bay of Biscay during 1967-73 and 2000[END_REF][START_REF] Petitgas | Hydro-plankton characteristics and their relationship with sardine and anchovy distributions on the French shelf of the Bay of Biscay[END_REF] and younger sardines are usually located in the Southern BoB [START_REF] Silva | Geographic variability of sardine dynamics in the Iberian Biscay region[END_REF]. In the BoB, the abundance of primary producers and zooplankton is also strongly structured spatially as the amount of chlorophyll-a is particularly high near the coasts and estuaries (Adour, Gironde, Loire; [START_REF] Huret | A multi-decadal hindcast of a physical-biogeochemical model and derived oceanographic indices in the Bay of Biscay[END_REF] and large copepods are particularly abundant near the shelf-break of the BoB [START_REF] Dessier | The spring mesozooplankton variability and its relationship with hydrobiological structure over year-to-year changes (2003-2013) in the southern Bay of Biscay (Northeast Atlantic)[END_REF].

Studies based on stable isotopes and stomach contents showed that sardines' diet changed as they aged (especially in spring and summer; [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF][START_REF] Costalago | Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species[END_REF][START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF] and that there was no significant spatial pattern in fish diet within the BoB [START_REF] Chouvelon | Trophic ecology of European sardine Sardina pilchardus and European anchovy Engraulis encrasicolus in the Bay of Biscay (north-east Atlantic) inferred from δ 13 C and δ 15 N values of fish and identified mesozooplanktonic organisms[END_REF]. However, the characterisation of lipids and their variation may allow us to learn more about the sardines' diet and the constraints exerted by the modification of planktonic communities on their biology. Indeed, lipids are a source of energy (neutral lipids, hereafter NL) and underpin the properties of cell membranes (polar lipids, hereafter PL; [START_REF] Hulbert | Polyunsaturated fats, membrane lipids and animal longevity[END_REF][START_REF] Tocher | Metabolism and functions of lipids and fatty acids in teleost fish[END_REF]. Lipids comprise saturated and unsaturated carbon chains called fatty acids (FA) that are particularly useful biomarkers of organisms' diet [START_REF] Cartes | Temporal changes in lipid biomarkers, especially fatty acids, of the deep-sea crustaceans Boreomysis arctica and Nematoscelis megalops: implications of their biological cycle and habitat near the seabed[END_REF][START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Meyer | Abiotic and biotic drivers of fatty acid tracers in ecology: a global analysis of chondrichthyan profiles[END_REF][START_REF] Riquelme-Bugueño | Fatty acid composition in the endemic Humboldt Current krill, Euphausia mucronata (Crustacea, Euphausiacea) in relation to the phytoplankton community and oceanographic variability off Dichato coast in central Chile[END_REF]. Indeed, the FA synthesis chains differ between zoo-and phytoplankton meaning that the presence or absence of some FA can reflect changes in sardines' diet [START_REF] Graeve | The selection and analysis of fatty acid ratios: a new approach for the univariate and multivariate analysis of fatty acid trophic markers in marine pelagic organisms[END_REF]. Thus, FA have been used as qualitative and semi-quantitative food web biomarkers and have proven to be a valuable method to define food web relationships, trophic positioning, and the dietary behaviours of marine species [START_REF] Turchini | Fish oil replacement in finfish nutrition[END_REF]Xu et al., 2020). FA can also inform us about the reproductive status of fish as NL are strongly solicited during reproduction [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] Rosa | Bioenergetics of small pelagic fishes in upwelling systems: relationship between fish condition, coastal ecosystem dynamics and fisheries[END_REF] and largest sardines may have a longer laying period due to their larger quantities of NL [START_REF] Nunes | Integrating fish size, condition, and population demography in the estimation of Atlantic sardine annual fecundity[END_REF]Zwolinski et al., 2001). Furthermore, marine fish must find essential fatty acids (EFA; e.g. n-3 and n-6 polyunsaturated FA at 20 and 22 carbons) in their food as they have little or no ability to synthesise them de novo [START_REF] Ahlgren | Fatty acid ratios in freshwater fish, zooplankton and zoobenthos -are there specific optima?[END_REF][START_REF] Hulbert | Polyunsaturated fats, membrane lipids and animal longevity[END_REF][START_REF] Sargent | Lipid nutrition of marine fish during early development: current status and future directions[END_REF]. It has been shown that EFA deficiencies can affect many vital functions such as growth, survival, stress resistance, and immune system [START_REF] Benítez-Santana | Dietary n-3 HUFA deficiency induces a reduced visual response in gilthead seabream Sparus aurata larvae[END_REF]Izquierdo, 1996;[START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF]. Therefore, EFA composition and quantity can enable us to test if fish nutritional needs are fully satisfied, both qualitatively and quantitatively [START_REF] Sargent | Requirements, presentation and sources of polyunsaturated fatty acids in marine fish larval feeds[END_REF]. Consequently, the variation in sardines' FA composition and concentration reflects their food characteristics [START_REF] Bandarra | Seasonal changes in lipid composition of sardine (Sardina pilchardus)[END_REF] and their phenology (growth or reproduction; [START_REF] Pacetti | Effect of spawning on furan fatty acid profile of edible muscle and organ tissues from sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus)[END_REF], making these markers invaluable to examine whether bottom-up processes are acting on the phenotype of this species.

The aim of this article is therefore (i) to characterise the FA composition of sardine muscles in both NL and PL fractions, (ii) to determine whether it is associated with sardine endogenous characteristics (sex, age, weight, sexual maturity) and/or spatial distribution in the BoB, and (iii) to understand the variations in sardine feeding conditions according to the different FA useful to identify prey groups. Considering the spatial structure in food resources (i.e. primary production and zooplankton) and sardines' age across the BoB, we hypothesised that S. pilchardus FA composition changes according with the sampling location and individuals' age and that these changes are more visible in the NL than in the PL, due to a different FA turnover rate between these two lipid fractions.

Materials and methods

Acquisition of sardine samples: PELGAS 2018

The PELGAS survey, led by Ifremer, takes place in the BoB every May since 2000. Its main purpose is to estimate the biomass of small pelagic fish by acoustic detection, to inform the European Union on the status of the pelagic stocks in this area (Doray et al., 2018b). At each sampling location, called station, the fish are sorted by species and size (three size classes are defined in sardines: < 15 cm; 15-20 cm; > 20 cm). Muscle samples were collected in five sardines per size classes at 16 stations in May 2018 (PELGAS 2018, https://doi.org/10.17600/18000419). As only one or two different size classes were observed per station, our sampling is less homogeneous than we would have desired (Figure 1, Table 1). Muscle is an interesting tissue in clupeid species as it stores most of the lipid reserves (Brosset et al., 2015a;[START_REF] Lloret | Condition and health indicators of exploited marine fishes[END_REF] at fairly constant levels year round except at the beginning of the spawning season (May is the end of sardine spawning, Garrido et al., 2008a), and is recommended for human nutrition (omega 3 dietary supply; [START_REF] Pacetti | Effect of spawning on furan fatty acid profile of edible muscle and organ tissues from sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus)[END_REF]. For sardines, the biological parameters routinely collected were: body length, total fresh weight, age, sex and sexual maturity. Age is determined by microscopic reading of otoliths' growth rings (calcified parts of the inner ear of fish; ICES, 2011). Sex and sexual maturity stages are determined by macroscopic analyses of the gonads and assigned such as: (1) immature, (2) developing, (3) pre-spawning, (4) spawning, (5) partial post-spawning, and (6) post-spawning (Véron et al., 2020b). The average sardine size-at-age and their standard deviations were: 15.0 ± 1.0 cm for age 1, 17.8 ± 1.1 cm for age 2, 19.4 ± 0.6 cm for age 3, 20.7 ± 0.7 cm for age 4, 21.0 ± 1.1 cm for age 5, 21.7 ± 1.3 cm for age 6 and older (one individual for ages 7, 8 and 10).

In addition, a piece of muscle was taken from each selected sardine and stored in the freezer at -80°C until lipid analysis in the laboratory. Overall, we collected 100 individual muscle samples during the 2018 survey. Before any manipulation, the glassware was heated to 450°C for 6 hours and the metal 171 or Teflon materials were rinsed with acetone to avoid contamination of the samples. We first 172 solidified muscle samples in liquid nitrogen and passed them through a ball mill (1 min at 30 173 oscillations/sec). We retrieved between 200 and 250 mg of shred for each sample and added 174 6 mL of a CHCl3/MeOH mixture (2/1, v/v) to extract lipids [START_REF] Mathieu-Resuge | Trophic ecology of suspension-feeding bivalves inhabiting a north-eastern Pacific coastal lagoon: comparison of different biomarkers[END_REF]. We To ensure that samples were not degraded, we performed High-Performance Thin-Layer Chromatography (HPTLC) on total lipids (TL) based on [START_REF] Olsen | The rapid analysis of neutral and polar marine lipids using doubledevelopment HPTLC and scanning densitometry[END_REF]. This TL plate allowed us to quickly visualise the different classes of PL and NL, including the free FA potentially appearing during the degradation of the samples. The samples analysed had no significant concentrations of free FA (<1% of TL) indicating that samples' degradation was absent or limited.

Fatty acid composition

To determine the composition of FA, lipid extracts stored at -20°C were first shaken for 20 min, centrifuged for 15 min at 3,000 rpm and 1 mL of each sample was transferred to new vials and evaporated to dryness. Dry lipid extracts were then re-suspended three times with 500 µL of CHCl3/MeOH (98/2; v/v) and gently deposited on top of a silica micro-column [START_REF] Marty | Changes in the fatty acid composition of Pecten maximus (L.) during larval development[END_REF]. We eluted NL with 10 mL CHCl3/MeOH (98/2; v/v) in a 22 mL vial and PL with 20 mL MeOH in another 22 mL vial (Le [START_REF] Grand | Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum[END_REF]. Upstream, an internal standard composed of 20 µL of C23:0 (0.115 µg/µL) has been added to each 22 mL vial. After the elution, the NL and PL fractions were evaporated to dryness using a Genevac centrifugal evaporator (program: Low Boiling Point; temperature: 30°C).

After evaporation, the dry NL and PL fractions were re-suspended three times with 500 µL CHCl3/MeOH (2/1; v/v) and transferred into 7 mL vials. These were evaporated dry under N2 flow and we then added 800 µL of H2SO4/MeOH (3.4%) and incubated the samples for 10 min at 100°C after vortexing [START_REF] Budge | Studying trophic ecology in marine ecosystems using fatty acids: a primer on analysis and interpretation[END_REF][START_REF] Grand | Membrane phospholipid composition of hemocytes in the Pacific oyster Crassostrea gigas and the Manila clam Ruditapes philippinarum[END_REF]. Heat and sulphuric acid catalyse the cleavage of ester bonds and methanol provided the CH3 groups for the formation of FA methyl esters (FAME). We extracted FAME by adding 800 µL of hexane and 1.5 mL of hexane-saturated distilled water, and by shaking and centrifuging the NL and PL fractions 1 min at 1,000 rpm. FAME solubilise in hexane while the catalyst and glycerol mix with water, resulting in two phases. We discarded the denser aqueous phase and repeated this step twice by adding only 1.5 mL of hexane-saturated distilled water. Finally, the samples were delicately placed in the freezer at -20°C without removing the aqueous phase. After several hours, we quickly transferred the unfrozen upper organic phase into 2 mL vials, which are flushed with N2 and stored in a refrigerator prior analysis.

We analysed our FAME one by one in gas chromatography coupled with a flame ionisation detector (GC-FID; [START_REF] Couturier | State of art and best practices for fatty acid analysis in aquatic sciences[END_REF] to quantify their concentration. Our samples were simultaneously analysed on two capillary columns (polar and apolar) to confirm the identity of FAME. The elution order of FAME is not the same on these two columns which produces two different chromatograms (one per capillary column).

The area of C23:0 (ca. 1,000 µV/min) has been checked using the software Galaxie Chromatography Data System (version 1.9.3.2) on all chromatograms to ensure proper reading of the samples by the GC-FID. Then, we assigned a FA to each peak by comparing the retention times of the two chromatograms of a sample (polar and apolar columns) with reference chromatograms [START_REF] Couturier | State of art and best practices for fatty acid analysis in aquatic sciences[END_REF]. The "NL FA" and "PL FA" data of the individuals were then processed by a predefined R script which calculates the mass (1) and mass percentage (2) of a given FA within NL and PL, based on the formulas below: M , = , :

:

(1)

P , = , ∑ , (2)
with MFA the mass of a given FA, AFA its area on the chromatogram, AC23:0 the area of the reference FA (C23:0), MC23:0 the mass of the reference FA (known as equal to 2.3 µg), and PFA the mass percentage of a given FA. The mass values obtained were finally related to concentrations (µg FA/mg wet weight).

Statistical analyses

Statistical analyses were performed using R (version 3.5.1; R Core Team, 2018) and all significance thresholds were set to α = 0.05. First, we calculated the total amount of FA in both each FA. Among the 35 FA, we selected those >0.30% in TL (14:0, 15:0, 16:0, 17:0, 18:0, 16:1n- 7, 16:1n-9, 18:1n-7, 18:1n-9, 20:1n-9, 20:1n-11, 22:1n-9, 22:1n-11, 24:1n-9, 16:2n-4, 18:2n-6, 18:3n-3, 18:4n-3, 20:2n-6, 20:4n-3, 20:4n-6, 20:5n-3, 21:5n-3, 22:5n-3, 22:5n-6, 22:6n-3, iso17:0) and some <0.30% in TL with ecological significance as biomarkers (20:0, 20:1n-7, 16:2n-7, 16:3n-4, 16:3n-6, 16:4n-1, 18:3n-4, iso15:0). Indeed, 16:2n-7, 16:3n-4, and 16:4n-1 belong to diatoms 16-carbon PUFA [START_REF] Cañavate | Advancing assessment of marine phytoplankton community structure and nutritional value from fatty acid profiles of cultured microalgae[END_REF], 16:3n-6 is specific to some Chlorodendrophyceae class (chlorophyta phylum; Jónasdóttir, 2019), iso15:0 belongs to bacteria FA [START_REF] Remize | Study of synthesis pathways of the essential polyunsaturated fatty acid 20:5n-3 in the diatom Chaetoceros muelleri using 13 C-isotope labeling[END_REF], and 20:0, 20:1n-7 and 18:3n-4 are potential indicators of elongation activity (from 18:0, 18:1n-7 and 16:3n-4, respectively; Soudant, personal communication).

To determine whether there was a spatial structure in sardines' diet at the scale of the BoB, we performed dendrograms based on the dissimilarities in the values of the extracted principal component axes between stations for each lipid fraction. These dissimilarities were established based on Euclidean distances since our variables were quantitative and clusters were identified using the "complete" method. To interpret RDA, PCA and dendrograms, we focused on fatty acids trophic markers (FATM), which are specific to various groups of prey and accumulate when consumed [START_REF] Sargent | Marine wax esters[END_REF][START_REF] Sargent | Ecological investigations on the zooplankton community in Balsfjorden, northern Norway: lipids and fatty acids in Meganyctiphanes norvegica, Thysanoessa raschi and T. inermis during mid-winter[END_REF].

Fatty acids trophic markers used

In this study, the contribution of macrozooplankton to the diet of sardines was not included due to the lack of specific enough FATM for this prey range (e.g. krill, decapods).

Therefore, we retained the FATM for three prey groups of sardines: copepods, diatoms, and non-diatom phytoplankton. The FATM of herbivorous copepods such as Calanus spp. and other calanoids (e.g. Temora spp.) are 20:1n-9 and 22:1n-11 [START_REF] Kattner | Polar herbivorous copepods -different pathways in lipid biosynthesis[END_REF]; those of diatoms are 20:5n-3 and 16-carbon FA such as 16:1n-7, 16:2n-4, 16:2n-7, 16:3n-4, 16:4n-1 [START_REF] Cañavate | Advancing assessment of marine phytoplankton community structure and nutritional value from fatty acid profiles of cultured microalgae[END_REF]; while others FATM such as 18:2n-6, 18:3n-3, 18:4n-3, 18:5n-3 and 22:6n-3

represent FATM from non-diatom primary producers [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Napolitano | Fatty acids as trophic markers of phytoplankton blooms in the Bahia Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)[END_REF][START_REF] Pethybridge | Spatial patterns and temperature predictions of tuna fatty acids: tracing essential nutrients and changes in primary producers[END_REF]. We also focussed on the EPA/DHA ratio (i.e.

eicosapentaenoic acid to docosahexaenoic acid, 20:5n-3/22:6n-3) commonly used as an indicator of trophic relationships. This ratio decreases with increasing carnivory since DHA is highly conserved in food webs [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Scott | Species differences, origins and functions of fatty alcohols and fatty acids in the wax esters and phospholipids of Calanus hyperboreus, C. glacialis and C. finmarchicus from Arctic waters[END_REF].

Results

Sardine fatty acids profiles in the Bay of Biscay

Overall, we identified 56 and 57 FA in NL and PL fractions, respectively (4 FA were specific to NL and 5 FA were specific to PL, Table S1). We found in NL 30% SFA, 27% MUFA, and 40% PUFA (including 35% n-3 and 4.4% n-6, Table S1) whereas PL consisted in 32% SFA, 8% MUFA, and 59% PUFA (including 55% n-3 and 3.5% n-6, Table S1). The remaining percentages are associated with dimethyl acetal FA (exclusively PL), branched FA, and unknown FA (Table S1). The EPA/DHA ratio was 0.68 for NL and 0.23 for PL (Table S1). The FA contributing most to the quantitative differences between NL and PL are: 14:0, 16:0, 16:1n-7, 18:1n-9, 20:1n-9, 22:1n-11, 18:4n-3 and 22:6n-3 (Figure 2). In general, there was a greater inter-individual variability in NL than in PL for the 27 FA, since the average coefficients of variation were 34% (ranging from 11% to 97%) and 29% (ranging from 9% to 80%), respectively (Figure 2).

The relationship between endogenous variables and fatty acids profiles

The redundancy analyses highlighted that the contribution of the spatial and endogenous variables was similar in NL (21 and 19% respectively, Table 2) while spatial variables explained more variance than endogenous variables in PL (18 and 14% respectively, Table 2). While sardines' weight, sex and maturity stage explained a very limited amount of variance in FA, individuals' age explained nearly 20% of the total variation in FA (Table 2, Figure 3). More specifically, the increase in individuals' age is associated with major changes in the content of some FA (Figure 4 and Figure 5). In NL, we observed an increase in FA specific of copepods (20:1n-9 and 22:1n-11, Figure 3A and Figures 4B&C) and a decline in FA specific of phytoplankton (20:5n-3, i.e. diatoms, Figure 3A and Figure 4D). For other FA non-associated with specific prey groups, there were increases (e.g. 20:1n-11, Figure 4A) and declines with sardines' age (e.g. 15:0, 18:4n-3, Figures 4E&F). Some of these FA also changed similarly in PL (Figure 3B), which was particularly evident for the increase in 20:1n-9 (i.e. copepods, Figure 4H) and the decrease in 20:5n-3 (i.e. diatoms, Figure 4I) with sardines' age. As for NL, an increase (20:4n-3, Figure 4G) and a decline (15:0, iso15:0, Figure 4J&K) with sardines' age were observed for other FA non-associated with specific prey groups. We found a decrease in the EPA/DHA ratio with age in NL and PL fractions, although the ratio was three times greater in NL (Figure 5). Noteworthy, changes seemed to occur primarily between the sardines aged 1 and 2 and those older than 2 years old, both in NL and PL fractions (Figure 4 and Figure 5).

Spatial variability of fatty acids profiles

To determine whether there was a spatial structure in the 35 main FA, we first carried out PCA to produce integrative values of FA profiles for each individual. For NL, the first axis of the PCA (PC1_NL) was positively related to 20:1n-9 and 22:1n-11 (copepods FATM) and negatively related to 22:6n-3 and iso17:0 (non-diatom phytoplankton and bacterial FATM, respectively; Figure S1A and Table S2). The second axis (PC2_NL) was positively related to a mix of 20:5n-3, 18:3n-3 and 18:4n-3 (diatoms and non-diatom phytoplankton FATM) and negatively related to 22:5n-6 (non-diatom phytoplankton FATM mainly present in Prymnesiophyceae, Pavlovophyceae, Pelagophyceae and Raphydophyceae; Figure S1A and Table S2). The third axis (PC3_NL) was positively related to 16:4n-1, 16:1n-7 and 16:2n-4

(FATM typical of diatoms, Figure S1B and Table S2). For PL, the first axis of the PCA (PC1_PL)

was negatively related to 16:1n-7, 16:3n-4 and 20:5n-3 (diatoms FATM) and positively related to 22:6n-3 (abundant in haptophytes and dinophytes, Figure S1C and Table S2), while the second axis (PC2_PL) was negatively related to 20:1n-9 and 22:1n-11 (FATM of copepods) and 18:2n-6, 18:3n-3 and 18:4n-3 (non-diatom phytoplankton FATM, Figure S1C and Table S2).

Finally, the third axis (PC3_PL) was negatively related to the diatoms FATM (i.e. 16:2n-7 and 16:4n-1) that were poorly explained by PC1_PL (Figure S1D, Table S2).

To account for the effect of ontogenetic diet changes in FA profiles, we carried out two separate clustering analyses (based on the values of the first three PCA axes) for NL and PL:

one with only ages 1 and 2 sardines (10 stations) and another with older sardines (9 stations).

For NL in 1 and 2 years old sardines, the clustering analysis identified four clusters of stations across the BoB (Figure 6A) distributed along a latitudinal gradient (Figure 6C). More and older sardines (panels B&D). The numbers indicate the different zones. The PC axes are defined from FATM as: PC1 positive: haptophytes and dinophytes, PC1 negative: diatoms, PC2 positive: not applicable, PC2 negative: copepods and non-diatom phytoplankton, PC3 positive: not applicable, PC3 negative: diatoms.

Discussion

Sardine fatty acids profiles in the Bay of Biscay

Although we measured and identified more FA, the sardine muscle FA profiles in NL and PL in the BoB were consistent with the composition and proportions of FA obtained in this species in other areas [START_REF] Bandarra | Sardine (Sardina pilchardus) lipid composition: does it change after one year in captivity?[END_REF][START_REF] Biton-Porsmoguer | Fatty acid composition and parasitism of European sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus) populations in the northern Catalan Sea in the context of changing environmental conditions[END_REF][START_REF] García-Moreno | Discarded species in the west Mediterranean sea as sources of omega-3 PUFA[END_REF][START_REF] Pacetti | Effect of spawning on furan fatty acid profile of edible muscle and organ tissues from sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus)[END_REF][START_REF] Pethybridge | Temporal and inter-specific variations in forage fish feeding conditions in the NW Mediterranean: lipid content and fatty acid compositional changes[END_REF]. Indeed, the most common FA and the percentages of the different categories of FA in these studies were broadly similar to those that we measured here (e.g. greater proportions of PUFA than SFA and MUFA). We found that the most abundant FA were the 16:0 and 22:6n-3 whatever the lipid fraction considered, but their proportions varied substantially between NL and PL. For instance, the proportions of 16:0 and 22:6n-3 are respectively 1.4 and three times higher in PL than in NL. These differences are similar to those reported by [START_REF] Bandarra | Seasonal changes in lipid composition of sardine (Sardina pilchardus)[END_REF][START_REF] Bandarra | Sardine (Sardina pilchardus) lipid composition: does it change after one year in captivity?[END_REF], confirming the importance of these two FA as major structural components of fish cell membranes [START_REF] Sargent | Lipid nutrition of marine fish during early development: current status and future directions[END_REF].

Our EPA/DHA ratios in NL and PL (on average 0.68 and 0.23, respectively) are similar to those found by García-Moreno et al., 2013 (0.60 for sardine oils in spring equivalent to NL), Pethybridge et al., 2014 (0.37 in March and 0.86 in July for NL of sardine muscle) and Bandarra et al., 2018 (0.25 for PL of wild sardine muscle). In our case, the EPA/DHA ratio is three times higher in NL than in PL. This ratio should decrease towards higher trophic levels as DHA is conserved especially in PL and EPA tends to decrease [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Scott | Species differences, origins and functions of fatty alcohols and fatty acids in the wax esters and phospholipids of Calanus hyperboreus, C. glacialis and C. finmarchicus from Arctic waters[END_REF]; here, a decline in the ratio is visible with the ageing of sardines, in both lipid fractions.

Sardines aged 1 and 2 years have the highest EPA/DHA ratio indicating a more herbivorous diet while those older than 2 years seem to orient their diet towards higher trophic levels such as macrozooplankton.

Ontogenetic and spatial differences in the diet of Bay of Biscay sardines through fatty acids profiles

In the BoB, the strong spatial distribution pattern in sardines' age reflects the ecology of this species in spring [START_REF] Bellier | Historical fluctuations in spawning location of anchovy (Engraulis encrasicolus) and sardine (Sardina pilchardus) in the Bay of Biscay during 1967-73 and 2000[END_REF]Doray et al., 2018a;[START_REF] Petitgas | Hydro-plankton characteristics and their relationship with sardine and anchovy distributions on the French shelf of the Bay of Biscay[END_REF]. Indeed, a majority of young individuals are found near the coast in the Southern part of the BoB [START_REF] Silva | Geographic variability of sardine dynamics in the Iberian Biscay region[END_REF] and near the nutrient rich plumes of the Gironde estuary (Doray, personal communication). Despite this spatially unbalanced age distribution, we found that sardine FA profiles were different depending on age in both NL and PL while accounting for the sampling location. This ontogenetic dietary change in NL is consistent with other studies of sardines' feeding habits based on stomach content [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF]Garrido et al., 2008a;[START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF] and stable isotopes [START_REF] Bode | Reconstruction of trophic pathways between plankton and the North Iberian sardine (Sardina pilchardus) using stable isotopes[END_REF][START_REF] Costalago | Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species[END_REF][START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF], which showed that sardines have a more varied diet as they age. According to these studies, smaller and younger sardines have a high proportion of diatoms and copepods (Microsetella spp., Corycaeus spp.) whereas larger sardines eat more fish eggs, crustacean eggs, and other copepod genera (Oncaea, Temora, Centropages). In summary, larger sardines eat more calanoid copepods than smaller ones. This pattern is consistent with our FA profiles that showed a substantial increase in FATM of herbivorous copepods between ages 1 and 3 followed by a plateau. In contrast, sardines aged 1 and 2 years show a higher concentration of small prey FATM (e.g. 20:5n-3 for diatoms). Three processes can explain this ontogenetic dietary difference: a morphological change in gills and/or a change in feeding behaviour with age or the overriding effect of prey availability. Indeed, sardines are capable of switching from non-selective filter-feeding to particulate-feeding behaviour [START_REF] Bachiller | A trophic latitudinal gradient revealed in anchovy and sardine from the Western Mediterranean Sea using a multi-proxy approach[END_REF][START_REF] Costalago | Comparison of the feeding apparatus and diet of European sardines Sardina pilchardus of Atlantic and Mediterranean waters: ecological implications[END_REF]Garrido et al., 2008a;[START_REF] Van Der Lingen | Trophic dynamics of small pelagic fish[END_REF]. [START_REF] Van Der Lingen | Trophic dynamics of small pelagic fish[END_REF] suggested that the filtering apparatus is fully developed when sardines have reached a length of 15 cm, corresponding to ca. 48% of the sardines aged 1 year sampled for this study. It has also been suggested that dietary differences in planktivorous pelagic fishes can also be explained by changes in fish feeding behaviour per se rather than by morphology [START_REF] Tanaka | Feeding habits and gill raker morphology of three planktivorous pelagic fish species off the coast of northern and western Kyushu in summer[END_REF]. The change in feeding behaviour is supported by the very different FA profiles of young and old sardines sampled in the same station. In addition, the decline in the EPA/DHA ratio with age (meaning more carnivory, Garrido et al., 2008b) may indicate a greater dietary contribution of macrozooplankton in older sardines diet (as DHA accumulates in macrozooplankton; [START_REF] Sargent | The lipid biochemistry of calanoid copepods[END_REF]Virtue et al., 2000). However, we do not identify prey for some FA strongly changing with sardines' age (e.g. 15:0) and we lack FA of prey ingested by the particulate-feeding of sardine (e.g. fish eggs, decapods), limiting investigation about macrozooplankton. Nevertheless, prey availability is probably the main driver of the overall change in FA profiles as sardines aged 1 and 2 years are living in the coastal areas of the Southern BoB, where the abundance of primary producers is very high [START_REF] Huret | A multi-decadal hindcast of a physical-biogeochemical model and derived oceanographic indices in the Bay of Biscay[END_REF] while older sardines were sampled in areas near the shelf-break where large copepods are found [START_REF] Dessier | The spring mesozooplankton variability and its relationship with hydrobiological structure over year-to-year changes (2003-2013) in the southern Bay of Biscay (Northeast Atlantic)[END_REF][START_REF] Petitgas | Ecosystem spatial structure revealed by integrated survey data[END_REF]. We lack historical values of sardine FA composition in BoB, but the stronger decline in size-at-age for sardines aged 1 and 2 years compared to older ones may reflects changes in primary production that is somehow lagged in secondary production.

When we analysed separately sardines aged 1 and 2 years old and older sardines, we found that there was a clear spatial structuring in FA profiles along a geographical gradient. In 1 and 2 year old sardine, the proportion of copepods increased towards the North-Western part of the BoB, and the proportion of non-diatom phytoplankton increased in the South-Eastern part of the BoB. The proportion of diatoms was highest near the Gironde estuary and lowest near the Adour river, which is consistent with previous studies about phytoplankton distribution in spring [START_REF] Marquis | Inverse analysis of the planktonic food web dynamics related to phytoplankton bloom development on the continental shelf of the Bay of Biscay, French coast[END_REF]. Such spatial structuring was apparent in PL, but was particularly strong in NL which enabled us to identify two additional groups of stations off the Gironde estuary. Nevertheless, there are clear cycles of the primary production with high values some years (2000)(2001)(2007)(2008) and others with substantially lower concentrations [START_REF] Tocher | Metabolism and functions of lipids and fatty acids in teleost fish[END_REF][START_REF] Pigliucci | Evolution of phenotypic plasticity: where are we going now?[END_REF][START_REF] Cartes | Temporal changes in lipid biomarkers, especially fatty acids, of the deep-sea crustaceans Boreomysis arctica and Nematoscelis megalops: implications of their biological cycle and habitat near the seabed[END_REF], 2015;[START_REF] Boëns | Determinants of growth and selective mortality in anchovy and sardine in the Bay of Biscay[END_REF][START_REF] Huret | A multi-decadal hindcast of a physical-biogeochemical model and derived oceanographic indices in the Bay of Biscay[END_REF]. The overlap between primary producers and 1-year-old sardines near the Southern coast of the BoB [START_REF] Huret | A multi-decadal hindcast of a physical-biogeochemical model and derived oceanographic indices in the Bay of Biscay[END_REF][START_REF] Petitgas | Ecosystem spatial structure revealed by integrated survey data[END_REF] reveals the importance of phytoplankton for younger sardines, although sardines prefer to feed on zooplankton (Garrido et al., 2008a). Young sardines are probably more filter-feeding or consume smaller zooplankton, explaining why sardines aged 1 and 2 years do not exhibit such high copepods FATM proportion. For sardines older than 2 years old, we found greater proportions of the FA characteristics of copepods over the entire BoB and the spatial structure was primarily driven by differences in the concentration of diatoms FATM. The general dominance of copepods FATM in the Northern BoB is consistent with past studies that have described the spatial heterogeneity in the hydrobiological characteristics in spring: larger zooplankton are more abundant in the North-Western than in the South-Eastern BoB [START_REF] Petitgas | Ecosystem spatial structure revealed by integrated survey data[END_REF]. Moreover, there is a larger abundance of Calanus helgolandicus off the Gironde estuary with particularly high energy density [START_REF] Dessier | The spring mesozooplankton variability and its relationship with hydrobiological structure over year-to-year changes (2003-2013) in the southern Bay of Biscay (Northeast Atlantic)[END_REF]. This copepod species is one of the largest in the BoB and may be primarily caught by older sardines (based on stomach contents and trophic levels of the food, [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF][START_REF] Costalago | Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species[END_REF][START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF]. Sardines eat what they can find and suit their feeding mode, which depends on the environment and seasonal phenology of plankton [START_REF] Costalago | Comparison of the feeding apparatus and diet of European sardines Sardina pilchardus of Atlantic and Mediterranean waters: ecological implications[END_REF]Garrido et al., 2008a;[START_REF] Napolitano | Fatty acids as trophic markers of phytoplankton blooms in the Bahia Blanca estuary (Buenos Aires, Argentina) and in Trinity Bay (Newfoundland, Canada)[END_REF]. Consequently, the difference in diet of BoB sardines is probably due to food availability along a geographical gradient at a given time and sardines' feeding behaviour may change with their diet as they feed on larger copepods or more macrozooplankton when they age.

Lipid fractions and the ontogenetic variability in fatty acids profiles

Our study clearly shows that NL and PL vary differently with endogenous and spatial variables. Indeed, differences in sardine diet were more pronounced when studying NL than PL as has already been shown experimentally (e.g. [START_REF] Bandarra | Sardine (Sardina pilchardus) lipid composition: does it change after one year in captivity?[END_REF]. This reflects the more selective incorporation of some very specific FA into PL than into NL (e.g. n-3 and n-6 long-chain PUFA such as EFA), allowing membranes to adapt to changing environmental conditions [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF][START_REF] Soudant | Effect of food fatty acid and sterol quality on Pecten maximus gonad composition and reproduction process[END_REF]. Indeed, the FA profiles of PL undergo a stronger selective incorporation of EFA than that of NL following feeding, retaining

preferentially the FA necessary for the functioning of cell membranes [START_REF] Robin | Fatty acid profile of fish following a change in dietary fatty acid source: model of fatty acid composition with a dilution hypothesis[END_REF][START_REF] Szabo | Incorporation dynamics of dietary vegetable oil fatty acids into the triacylglycerols and phospholipids of tilapia (Oreochromis niloticus) tissues (fillet, liver, visceral fat and gonads)[END_REF]. Therefore, PL have a lower nutritional marking compared with NL that, conversely, reflect more tightly feeding variations. In addition, MUFA such as copepods FATM are preferentially oriented to energy storage [START_REF] Sargent | Lipid nutrition of marine fish during early development: current status and future directions[END_REF], explaining their greater proportion in NL than in PL. These aspects are important as many recent studies (e.g. [START_REF] Biton-Porsmoguer | Fatty acid composition and parasitism of European sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus) populations in the northern Catalan Sea in the context of changing environmental conditions[END_REF][START_REF] Pacetti | Effect of spawning on furan fatty acid profile of edible muscle and organ tissues from sardine (Sardina pilchardus) and anchovy (Engraulis encrasicolus)[END_REF] rely on TL, which is the sum of NL and PL fractions.

As PL/NL ratios can change substantially with fish state, for instance depending on the sampling season or reproductive stage [START_REF] Bandarra | Seasonal changes in lipid composition of sardine (Sardina pilchardus)[END_REF], the use of TL to compare individuals from different species, locations or seasons could be biased and result in ambiguous results. Indeed, the distinction between NL and PL allowed us to better understand the variance in FA composition, which could be largely due to the fact that both lipid fractions reflect different physiological and metabolic processes.

The great variability in sardine FA profiles illustrated the importance of testing and accounting for ontogenetic changes in the species of interest, especially when focusing on the NL fraction. Xu et al. (2020) showed that within species Salmo salar and Sparus aurata, small fish have a faster FA turnover than large fish and hence that age can be a major factor in the rate of FA turnover. Whatever the temporal or spatial comparison of a species FA profile, one should ensure that the fish size and/or age distribution are similar in sampling design or consider appropriate statistical analyses to eliminate potential bias due to the ontogenetic variation in diet. Provided the dynamic nature of NL and PL profiles and their different sensitivity to recent changes in diets, future studies should be careful with the use of the TL extract and we recommend to consider NL and PL fractions separately to deeply investigate the ecological meaning of potential differences in FA profiles.

Limits and perspectives of the study

Only some FA reported in the PCA were interpreted due to the limited knowledge of the different FATM and the meaning of the dietary source signature. However, there are other FA that are related to the different PCA axes that we do not really interpret because they are not specifically synthesised by a group of species/trophic level and could not be attributed to particular preys of sardines. Even if copepods and phytoplankton represent the large majority of sardine preys, especially in spring during phytoplankton blooms [START_REF] Costalago | Ontogenetic and seasonal changes in the feeding habits and trophic levels of two small pelagic fish species[END_REF][START_REF] Dessier | The spring mesozooplankton variability and its relationship with hydrobiological structure over year-to-year changes (2003-2013) in the southern Bay of Biscay (Northeast Atlantic)[END_REF][START_REF] Koven | The effect of dietary (n-3) polyunsaturated fatty acids on growth, survival and swim bladder development in Sparus aurata larvae[END_REF], we may have missed a part of the food characterisation of sardines. Indeed, sardines' stomachs in the BoB do also contain appendicularians and decapods in spring [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF], for which we could not consider FATM. Thus, developing knowledge about new FATM specific to these groups will be important in a near future to move towards a more global description of sardines' diet with FA. In addition, there is some uncertainty in the proportions of phytoplankton FATM measured in NL as these can be underestimated if they are preferentially incorporated in PL or overestimated as they may be accumulated in copepods that eat phytoplankton [START_REF] Dalsgaard | Fatty acid trophic markers in the pelagic marine environment[END_REF]. Nevertheless, as the spatial structure of the FATM is consistent with that of the distribution of plankton in the BoB [START_REF] Petitgas | Ecosystem spatial structure revealed by integrated survey data[END_REF], it is very likely that our results are explained by different foraging behaviours (filter-feeding and particulate-feeding).

This study can therefore be expanded over time and seasons to determine whether such changes in diet are consistent within and between years and the degree to which the variation in plankton communities affect their FA profiles. Moreover, studying the prey's FA composition could help to better understand the transfer efficiencies of essential FA and the nutrition potential of plankton for small pelagic fish. To this end, a more homogenous sampling with respect to individuals' age over space is needed to disentangle more efficiently the effects of age and geographical location on sardines' FA profiles. However, even if our sampling cover one season and year, the structuration observed with the most abundant FA is consistent with the great diet variability already found with other technics in BoB [START_REF] Bachiller | Trophodynamics and diet overlap of small pelagic fish species in the Bay of Biscay[END_REF][START_REF] Chouvelon | Trophic ecology of European sardine Sardina pilchardus and European anchovy Engraulis encrasicolus in the Bay of Biscay (north-east Atlantic) inferred from δ 13 C and δ 15 N values of fish and identified mesozooplanktonic organisms[END_REF][START_REF] Chouvelon | Small pelagic fish feeding patterns in relation to food resource variability: an isotopic investigation for Sardina pilchardus and Engraulis encrasicolus from the Bay of Biscay (north-east Atlantic)[END_REF]. This confirms the potential of using FA including some FATM to provide additional information in multi-proxy studies (e.g. [START_REF] Bachiller | A trophic latitudinal gradient revealed in anchovy and sardine from the Western Mediterranean Sea using a multi-proxy approach[END_REF] and to better understand small pelagic fish population changes.

Conclusion

Our study is the first to fully characterise the composition and variability in the FA of sardines in the BoB through NL from PL. There were clear spatial and ontogenetic differences in sardines' FA profiles especially in the NL fraction. Higher total FA contents were observed in the Northern part of the BoB (area of older and larger sardines), with a dominance of FA characterising copepods, while non-diatom phytoplankton FATM prevailed in Southern BoB (area of younger and smaller sardines). Diatoms FATM were highest near the Gironde estuary.

This high dependence on younger sardines' diet to phytoplankton in spring suggests that changes in primary production (quantity and quality) may explain the stronger decline in sizeat-age of sardines aged 1 and 2 years during the last decade in the BoB. We also showed that it is important to consider both NL and PL fractions as the FA contents of NL are much more variable than those of PL and conversely, PL can provide key information about the longlasting effects of changes in individuals' diet and environmental conditions. The contribution of FA to trophic studies can clearly enable us to better understand the bottom-up control exerted by the plankton community on the characteristics of small pelagic fish and identify the food-web dynamics of the BoB pelagic ecosystem.

Figure 1 .

 1 Figure 1. Spatial distribution of sardines sampled in the Bay of Biscay, as part of PELGAS 2018.

  175then vigorously shook vials to re-suspend the shred and improve lipids' extraction. Prior to 176 their storage at -20°C, the samples were passed under a flow of N2, shaken, placed in an 177 ultrasonic bath for 10 min, and agitated for at least 20 min.

  NL and PL fractions to detect outliers and we characterised the FA of these two fractions by quantifying inter-individual variations of the most important FA. We also calculated the percentages of saturated fatty acids (SFA), monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA).Due to the unbalanced age distribution in our sampling, we performed a canonical redundancy analysis (RDA) to quantify the relationships between the composition of FA in NL and PL and endogenous factors (i.e. sex, age, weight, sexual maturity) considering geographical variables (i.e. stations' latitude and longitude) which constrained the FA data.These geographical variables are interesting since they implicitly represent a gradient of environmental conditions from the North to the South of the BoB and thus the effect of the different endogenous variables was represented without effect of the environmental variables. We then identified the overall structure of the data at the individual level and the correlations between different FA with principal component analyses (PCA) performed separately for NL and PL fatty acids, for which we extracted principal components (PC) with eigenvalues >1 (this resulted in the extraction of 3 axes for each PCA). RDA and PCA were based on 35 FA of our sardine muscles, where each FA was measured as µg/mg of wet weight of sardine muscle. These were processed by the Hellinger distance (ideal for concentration data;[START_REF] Legendre | Beta diversity as the variance of community data: dissimilarity coefficients and partitioning[END_REF] and were centred-reduced to give the same weight to

Figure 2 .

 2 Figure 2. Percentages and standard deviations of 27 fatty acids in neutral lipids (red) and polar

Figure 3 .

 3 Figure 3. Redundancy analysis (RDA) ordination diagram for neutral lipids (panel A) and polar

Figure 4 .

 4 Figure 4. Changes in the main age-related fatty acids identified by the redundancy analyses

Figure 5 .

 5 Figure 5. Changes in the EPA/DHA ratio according to the age of sardines for neutral lipids

  specifically, the Southern group was characterised by primarily negative values of the PC1_NL (i.e. more non-diatom phytoplankton and bacterial FATM) and negative values of PC3_NL (i.e. less diatoms FATM; Figure6C, area 1). The Northern group was characterised by positive values of PC1_NL indicating more FATM of copepods (Figure6C, area 4). The third and fourth groups, located in between Southern and Northern groups, showed slightly negative and positive values for PC1_NL and PC3_NL, respectively, indicating both low concentrations of copepods FATM and more FATM of diatoms (Figure6C, areas 2 and 3). The group located near the coast had particularly high PC3_NL (i.e. more diatoms 16-carbon FATM; Figure6C, area 2) compared with the group further from the coast which was also characterised by positive values of PC2_NL (i.e. a mix of diatoms and non-diatom phytoplankton FATM; Figure6C, area3). We found a very similar spatial structure in sardines older than 2 years (Figures 6B&D) with

  the notable difference that PC1_NL values were all positive (depicting a large part of copepods FATM, Figure6D). Stations located near the Gironde estuary were characterised by strong positive values of PC1_NL and PC2_NL (i.e. more copepods and a mix of diatoms and nondiatom phytoplankton FATM; Figure6D, area 2). A second group included four stations in the Northern part of the BoB in which sardines had also FA characteristic of copepods but low values of PC2_NL and PC3_NL (i.e. more non-diatom phytoplankton FATM; Figure6D, area 3).A third group was identified near the coast and characterised by a more equal content of FATM of copepods, diatoms and non-diatom phytoplankton (Figure6D, area 1).For PL, the hierarchical dendrogram for 1 and 2 years old sardines based on PCA axes values distinguished two clusters (Figures 7A&C). Southern stations had negative and positive values of PC1_PL and PC2_PL, respectively, indicating FA characteristic of diatoms (Figure7C, area 1). Northern stations had higher PC1_PL and lower PC2_PL indicating higher proportions of non-diatom phytoplankton (PC1_PL and PC2_PL) and copepods FATM (PC2_PL; Figure7C, area 2). For sardines older than 2 years, we did not find any clear spatial structure in PL (Figures 7B&D).

Figure 6 .Figure 7 .

 67 Figure 6. Hierarchical dendrogram showing the clustering of stations (panels A&B) according to the fatty acid profile on the first three principal

Table 1 .

 1 Geographical coordinates and biological parameters of sardines at the 16 sampling stations. S.D. is the standard deviation.

		Station IDs	Latitude Longitude	Number of individuals	Length (cm) Mean S.D.	Weight (g) Mean S.D.	Sex Male Female	Mean age (year)	Mean maturity stage
		W0161	43.867	-1.505	5	17.0 0.0	37.8 4.6	5	2.0	5.0
		W0162	44.065	-1.474	5	16.0 0.0	30.2 1.9	0	1.0	2.8
		W0167	44.267	-1.465	10	15.0 0.5	25.6 2.1	7	1.0	3.1
		W0176	44.868	-1.821	5	19.7 0.3	60.0 6.6	2	4.4	4.6
		W0183	45.059	-1.368	5	14.0 0.6	20.6 3.6	2	1.0	2.6
		W0184	45.036	-1.801	5	17.6 0.2	40.0 1.6	2	2.0	5.0
		W0190	45.253	-1.356	5	14.0 0.0	19.8 2.0	4	1.2	2.2
		W0207	45.127	-2.698	5	21.6 0.2	77.4 3.8	2	5.8	4.2
		W0217	45.843	-2.623	5	20.3 0.3	62.6 3.4	2	5.2	5.0
		W0247	46.685	-2.83	5	16.7 0.3	37.8 4.8	5	1.4	5.0
		W0248	46.769	-3.195	5	22.0 1.4	82.8 12.5	1	5.8	5.0
		W0254	46.887	-3.486	10	19.7 1.6	60.8 12.4	6	3.2	4.8
		W0261	47.102	-3.973	5	21.0 0.4	76.4 10.9	2	5.4	5.0
		W0272	47.345	-4.71	10	20.2 1.4	65.0 13.6	4	3.6	5.0
		W0278	47.531	-5.251	10	19.9 2.3	67.1 23.9	6	3.1	5.0
		W0290	47.207	-5.283	5	19.7 0.3	61.0 2.5	3	3.2	4.6
	168
	169	2.2. Grinding, lipid extraction and storage
	170

Table 2 .

 2 Summary of the redundancy analyses quantifying the relationship between endogenous variables and fatty acid composition of sardines (i.e. constrained effect) after accounting for the contribution of geographic effects (i.e. conditioned effect). The percentage of variance explained by each effect and variable is reported in parentheses.

		Neutral lipids	Polar lipids
	Conditioned	6.98 (20.5)	6.08 (17.9)
	Constrained (λ1)	6.44 (18.9)	4.71 (13.9)
	Unconstrained (λ2)	20.63 (60.6)	23.21 (68.3)
	Variable	λ1/ λ2	λ1/ λ2
	Age	0.26 (20.1)	0.16 (13.1)
	Weight	0.01 (1.4)	0.01 (1.1)
	Sex	0.03 (3.1)	0.03 (2.2)
	Maturity stage	0.02 (2.3)	0.02 (1.9)

Acknowledgements

We thank Mathieu Doray and Erwan Duhamel, chief survey scientists of PELGAS, for enabling us to collect these data. We are also grateful the crew of R/V 'Thalassa', particularly Hervé Barone and Laurence Pennors for their precious help during the onboard sampling. We are also grateful to two anonymous referees for providing thoughtful comments on the manuscript. PELGAS surveys are co-funded by the European Union through the Data Collection

Framework. P.B. was supported by ISblue project, Interdisciplinary graduate school for the blue planet (ANR-17-EURE-0015) which is co-funded by the French government under the program "Investissements d'Avenir" and by the Regional Council of Brittany (SAD programme).

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Xu, H., Turchini, G., Francis, D., Liang, M., Mock, T., Rombenso, A., Ai, Q., 2020. Are fish what they eat?

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