Javier Angel Tesán-Onrubia
email: javier.tesan@mio.osupytheas.fr

Marc Tedetti

François Carlotti

Melissa Tenaille

Loic Guilloux

Marc Pagano

Benoit Lebreton

Gaël Guillou

Pamela Fierro-González

Catherine Guigue

Loïc Guilloux

Sandrine Chifflet

Théo Garcia

Ismail Boudriga

Malika Belhassen

Amel Bellaaj Zouari

Daniela Bănaru
email: daniela.banaru@mio.osupytheas.fr

Spatial variations of biochemical content and stable isotope ratios of size-fractionated plankton in the Mediterranean Sea (MERITE-HIPPOCAMPE campaign)

Keywords: Phytoplankton, zooplankton, δ 13 C, δ 15 N, plankton trophic structure, contaminants

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

Introduction

Phyto-and zooplankton play a central role in the functioning of marine ecosystems by producing, transforming and transferring the organic matter up to planktivorous species [START_REF] Bănaru | Implementation of an end-to-end model of the Gulf of Lions ecosystem (NW Mediterranean Sea). I. Parameterization, calibration and evaluation[END_REF][START_REF] Chen | Diet and trophic interactions of Mediterranean planktivorous fishes[END_REF]. Plankton thus represents the main source of carbon fueling the marine food webs [START_REF] Bănaru | Trophic structure in the Gulf of Lions marine ecosystem (northwestern Mediterranean Sea) and fishing impacts[END_REF][START_REF] Cresson | Primary production and depth drive different trophic structure and functioning of fish assemblages in French marine ecosystems[END_REF]. Phyto-and zooplankton are composed of a myriad of organisms of great diversity in terms of size, metabolism, physiology, and diet that are governed by different trophic pathways, and complex ecological interactions. As taxonomic composition strongly differs between planktonic size-fractions, a size-based approach has become widely used to study the structure and functioning of the planktonic compartment [START_REF] Rau | C-13/C-12 and N-15/N-14 variations among size-fractionated marine particles -implications for their origin and trophic relationships[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Carlotti | Zooplankton community structure, biomass and role in carbon fluxes during the second half of a phytoplankton bloom in the eastern sector of the Kerguelen Shelf (January-February 2005)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. In pelagic food webs, body size determines rates of production [START_REF] Banse | Adult Body Mass and Annual Production/Biomass Relationships of Field Populations[END_REF], energy requirements [START_REF] Brown | Toward a Metabolic Theory of Ecology[END_REF], mortality rates [START_REF] Hirst | Mortality of marine planktonic copepods: global rates and patterns[END_REF] and predator-prey interactions [START_REF] Cohen | Body Sizes of Animal Predators and Animal Prey in Food Webs[END_REF][START_REF] Ljungström | Body size adaptions under climate change: zooplankton community more important than temperature or food abundance in model of a zooplanktivorous fish[END_REF]. The biochemical composition of the different plankton size-fractions provides information on their energetic content, which may influence prey selection [START_REF] Carlotti | Zooplankton community structure, biomass and role in carbon fluxes during the second half of a phytoplankton bloom in the eastern sector of the Kerguelen Shelf (January-February 2005)[END_REF], Harmelin-Vivien et al., 2019;[START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF][START_REF] Chen | Temporal variation in prey selection by adult European sardine (Sardina pilchardus) in the NW Mediterranean Sea[END_REF][START_REF] Chen | Diet and trophic interactions of Mediterranean planktivorous fishes[END_REF], while the carbon and nitrogen isotopic ratios (δ 13 C and δ 15 N values) of the plankton size-fractions provide information on the fluxes of organic matter within the planktonic food webs [START_REF] Peterson | Multiple stable isotopes used to trace the flow of organic matter in estuarine food webs[END_REF][START_REF] Cabana | Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes[END_REF][START_REF] Vander Zanden | Variation in δ15N and δ13C trophic fractionation: Implications for aquatic food web studies[END_REF].

Besides its central role in the functioning of marine ecosystems, plankton is now recognized as a key gateway of inorganic and organic contaminants into the marine food web [START_REF] Tao | Factors affecting annual occurrence, bioaccumulation, and biomagnification of polycyclic aromatic hydrocarbons in plankton food webs of subtropical eutrophic lakes[END_REF][START_REF] Chouvelon | Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-zooplankton-small pelagic fish marine food web[END_REF][START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF]. Phytoplankton is exposed to contaminants via water. Bioconcentration of contaminants in phytoplankton is driven mainly by partition equilibrium processes between the cells and the surround ing water [START_REF] Frouin | Partitioning and bioaccumulation of PCBs and PBDEs in marine plankton from the Strait of Georgia, British Columbia, Canada[END_REF]. For a given species or cell size of phytoplankton, and a given physico-chemical habitat (temperature, organic carbon content, etc.), the bioconcentration factors of organic contaminants may be directly correlated with their octanol-water partitioning coefficients (log Kow), i.e., their degree of lipophilicity/hydrophobicity [START_REF] Frouin | Partitioning and bioaccumulation of PCBs and PBDEs in marine plankton from the Strait of Georgia, British Columbia, Canada[END_REF]. Correspondingly, the bioconcentration of a given contaminant has been shown to increase with decreasing size of phytoplankton [START_REF] Fan | Phenanthrene Accumulation Kinetics in Marine Diatoms[END_REF]. Bioaccumulation processes in zooplankton are highly complex due to the entry of contaminants by both the water aqueous phase (bioconcentration) and diet, the trophic interactions and/or transfers between phytoplankton and various zooplankton, and the contaminant removal processes used by these organisms [START_REF] Tiano | PCB concentrations in plankton size classes, a temporal study in Marseille Bay, Western Mediterranean Sea[END_REF]Alekseenko et al., 2018;[START_REF] Tao | Factors affecting annual occurrence, bioaccumulation, and biomagnification of polycyclic aromatic hydrocarbons in plankton food webs of subtropical eutrophic lakes[END_REF]. Biomagnification may also occur between the low and high trophic level organisms in the marine food webs [START_REF] Chouvelon | Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-zooplankton-small pelagic fish marine food web[END_REF].

Therefore, the uptake, accumulation, and transfer of contaminants within the planktonic food webs may be strongly influenced by its characteristics such as the size-fraction distribution, the biochemical/energetic content (Hennig, 1986;[START_REF] Mason | Bioaccumulation of mercury and methylmercury[END_REF][START_REF] Wu | Accumulation, subcellular distribution and toxicity of inorganic mercury and methylmercury in marine phytoplankton[END_REF], the trophic interactions and the fluxes of organic matter [START_REF] Peterson | Multiple stable isotopes used to trace the flow of organic matter in estuarine food webs[END_REF][START_REF] Cabana | Modelling food chain structure and contaminant bioaccumulation using stable nitrogen isotopes[END_REF][START_REF] Vander Zanden | Variation in δ15N and δ13C trophic fractionation: Implications for aquatic food web studies[END_REF]. Characterizing the content of phyto-, and zooplankton in terms of size-fraction, biochemical/energetic content and stable isotope ratios (δ 13 C and δ 15 N) may thus provide key information on the structure, functioning, and trophic interactions of this planktonic food webs, but also on the capacity of accumulation and transfer of organic and inorganic contaminants within this planktonic network and potentially their transfer to higher trophic levels.

The Mediterranean Sea has a high diversity of planktonic and exploited resources, and contrasted biogeographical regions impacted by climate change and human activities (Durrieu de Madron et al., 2011;[START_REF] Mayot | Influence of the Phytoplankton Community Structure on the Spring and Annual Primary Production in the Northwestern Mediterranean Sea[END_REF]. As the Mediterranean Sea is mainly oligotrophic, its autotrophic biomass is dominated by small-size pico-and nanophytoplankton groups [START_REF] Leblanc | Nanoplanktonic diatoms are globally overlooked but play a role in spring blooms and carbon export[END_REF][START_REF] Boudriga | Ultraphytoplankton community structure in subsurface waters along a North-South Mediterranean transect[END_REF]. These groups play a key role in the biomass and energy transfer to zooplankton [START_REF] Bănaru | Seasonal variation of stable isotope ratios of size-fractionated zooplankton in the Bay of Marseille (NW Mediterranean Sea)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF][START_REF] Leblanc | Nanoplanktonic diatoms are globally overlooked but play a role in spring blooms and carbon export[END_REF].

However, coastal areas impacted by riverine and urban inputs may locally be more enriched and sometimes can even be characterized by higher phytoplankton biomass dominated by larger cells such as dinobionts and diatoms [START_REF] Harmelin-Vivien | Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean)[END_REF]. [START_REF] Champalbert | Characteristics of zooplankton standing stock and communities in the western Mediterranean Sea: relations to hydrology[END_REF] highlighted spatial differences in the zooplankton diversity and biomass, with a lower diversity and a higher biomass in coastal waters relative to offshore areas. The Mediterranean Sea is also strongly exposed to chemical contamination due to the intensive human activities in the bordering countries, its semi-closed status that limits the dilution of contaminants, and the relatively short ventilation and residence times its waters (Durrieu de Madron et al., 2011;[START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF].

In this context, the aims of this work, carried out in the frame of the MERITE-HIPPOCAMPE campaign, were: 1) to investigate the structure and functioning (in terms of trophic interactions and organic matter fluxes) of the planktonic food webs of the Mediterranean Sea through the characterization of the biomass, biochemical/energetic content and δ 13 C/δ 15 N ratios of several size-fractions of phyto-and zooplankton ranging from 0.7 to > 2000 µm, 2) to explore variations in size-spectra and spatial variations of these characteristics (biochemical/energetic content and δ 13 C/δ 15 N ratios), and relate these variations to their composition (via cytometry and imagery analyses) and also to the specificities of the studied areas, and 3) to discuss the implications of these findings regarding the accumulation and transfer of contaminants in the planktonic food webs.

To our knowledge, this work represents the first study to use a wide variety of approaches, and methodologies, such as biochemical analyses (proteins, carbohydrates, lipids and energy), analyses of isotopic ratios (δ 13 C, δ 15 N), cytometry analyses and mixing models (MixSiar) on fine size-fractions of phyto-and zooplankton ranging from 0.7 to > 2000 µm for the purpose of studying plankton food webs. The use of innovative sampling techniques, such as the deployment of in situ pumps equipped with a sequential filtration system, or the towing of a MultiNet device in chlorophyll maximum layer (CML), enabled us to obtain large amounts of size fractionated phyto-and zooplankton, material rarely acquired together.

Material and methods

Study area and sampling

The MERITE-HIPPOCAMPE cruise was conducted in the Mediterranean Sea in spring 2019, from April 13 to May 14, onboard the R/V Antea [START_REF] Tedetti | HIPPOCAMPE cruise, RV Antea[END_REF]Tedetti et al., 2023). Ten stations were sampled from the French coast (La Seyne-sur-Mer, Northwestern Mediterranean) to the Gulf of Gabès in Tunisia (Southeastern Mediterranean) in coastal and offshore locations, including the Gulf of Lion, the Ligurian and Algerian consensus regions, the North Balearic Front and Sicilian Channel areas, and the gulfs of Hammamet and Gabès, which presented different hydrological, biogeochemical and bloom conditions, and different levels of anthropogenic pressures (Fig. 1, Table S1). Phytoplankton and zooplankton were sampled at each station at the CML, during spring bloom (April to May), when maximum primary and secondary production occurs [START_REF] Liénart | Dynamics of particulate organic matter composition in coastal systems: Forcing of spatio-temporal variability at multi-systems scale[END_REF][START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF].

Suspended particulate matter (SPM) was collected and filtered using McLane Large Volume

Water Transfer System Samplers (WTS6-142LV, 4-8 L min -1 ), hereinafter referred to as

McLane in situ pumps. One pump was mounted with a regular 142-mm filter-holder holding one 142-mm-diameter filter. This pump was equipped with a ~ 0.7-µm-pore-size precombusted pre-weighed GF/F filter (Whatman) and covered with a 60-µm-pore-size homemade sock-type pre-filter so that the filtered particle size-fraction was 0.7-60 µm. A second McLane in situ pump was mounted with a mini-Multiple Unit Large Volume in situ Filtration System (MULVFS) filter holder composed of baffle tubes on the top followed by successive baffle and filter support plates, for sequential filtration with three different filters (142-mm diameter) [START_REF] Bishop | Getting good particles: Accurate sampling of particles by large volume in-situ filtration: Getting good particles[END_REF]. The filter series used were one ~ 0.7-µm-pore-size pre-combusted (450 °C, 6 h), pre-weighed GF/F filter, one 2.7-µm-pore-size pre-combusted, pre-weighed GF/D filter (Whatman), and one 20-µm-pore-size pre-cleaned (HCl 0.05% v/v) pre-weighed home-made Nylon filter so that the filtered particle size fractions were 0.7- 2.7, 2.7-20, and > 20 µm (hereafter labeled 20-60 µm). The pumps were deployed in the CML between 40 and 60 min. The volumes of filtered seawater ranged between 169 and 300 L depending on the stations. After pump deployment, filters were 'dried' by connecting the filter holder to a vacuum pump, stored in pre-combusted (450 °C, 6 h) aluminum sheets, and conserved at -20 °C and then freeze-dried. Then, punches with a known surface area were made into filters to obtain subsamples for the different analyses.

Zooplankton was sampled using a Multinet Plankton Sampler (Midi type with 0.25-m 2 aperture, Hydro-Bios), referred to hereafter as 'MultiNet', towed horizontally in the CML. The MultiNet position was maintained stable at the defined layer by means of a V-fin deflector and controlled vessel speed (2.5 knots), and real-time control of its position from the onboard desk-unit. The MultiNet was mounted with five individual (exchangeable) 2.5-m-long nylon nets with a mesh size of 60-µm and cod ends of the same mesh size. The MultiNet frame was equipped with various sensors: two Hydro-Bios flowmeters (one at the mouth and the other on the side) to assess the volume of water filtered by the nets, a CTD sensor and a total chlorophyll a (TChla) fluorometer. Connected to the onboard desk unit via the electromechanical cable, these captors inform on in situ depth, TChla concentration, filtered volume and flow rate, allowing the operator to decide to open and close the nets. Nets were closed when the flow rate reached a threshold value to limit clogging. Filtered volumes for each net reached up to 185 m 3 . Once the five nets were filled, the MultiNet was hauled back on board, and the five cod ends linked by a helicoidal bucket connector were carefully recovered. The fifth net remained open until the end of the operation, filtering the layer between the CML and the surface. The cod ends were rinsed out with local seawater, and their content transferred to pre-cleaned 10-L PFA bottles. The MultiNet was then returned to the water. This operation was repeated several times to get sufficient amounts of plankton for all possible further contaminant content analyses [START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF]. In the clean onboard container lab, plankton collected in PFA bottles was then size-fractionated through a column of five stainless steel sieves (60, 200, 500, 1000 and 2000 µm mesh-size) by wet-sieving with seawater previously filtered onto GF/F filters and stored in stainless steel jerrycans to obtain the following size-fractions: 60-200, 200-500, 500-1000, 1000-2000 and > 2000 µm. Samples were stored in pre-combusted (450 °C, 6 h) Pyrex bottles, conserved at -20 °C and freeze-dried. Large fractions (> 2000 µm) were most likely under-sampled compared to smaller fractions. These fractions should be carefully treated because they represent a relatively lowand difficult to estimatebiomass, form colonies, and may actively evade capture by swimming. The detailed methods to determine zooplankton group composition analysis presented in this paper and related to our results are detailed in Fierro-González et al.

(2023).

Analyses

Particulate organic carbon and nitrogen

Samples (i.e., 22-mm diameter filter or 1 mg dry weight DW zooplankton) were leached with 100 μL of sulfuric acid (H2SO4 0.5 mol L -1 ) to remove any inorganic carbon. Samples were then stored in 25 mL Schott ® glass bottles for subsequent analyses. Filter blanks were conditioned with 1 mL of ethanol and with 600 mL of 0.2-μm filtered seawater.

Determination of particulate organic carbon (POC) and nitrogen (PON) concentrations were carried out simultaneously on the same sample using the persulfate wet-oxidation procedure according to [START_REF] Raimbault | Simultaneous determination of particulate organic carbon, nitrogen and phosphorus collected on filters, using a semiautomatic wet-oxidation method[END_REF].

Suspended particulate matter and plankton dry weight

Suspended particulate matter dry weight (SPMDW) was measured in the 0.7-60 and 20-60-µm filters gravimetrically by weighing the tare, wet and dry filters with a precision balance (d = 0.01 mg). Briefly, we subtracted from the dry filter the tare and the salts associated with the evaporated water, considering a salinity of 38. Tests conducted with filtered seawater evidenced a higher mass after drying than the salt present in water, and most likely related to the highly hygroscopic characteristics of salts. A ratio of 1.2 between the observed mass and the theoretical mass associated with salt was applied to correct the water bonded to salt. When biomasses were low, as for 0.7-2.7 and 2.7-20-µm filters, high uncertainty in weighing increased the margin of error. SPMDW in the 0.7-2.7 and 2.7-20-µm filters were thus estimated by multiplying their relative fraction of POC by the biomass measured gravimetrically in the 0.7-60-µm size-fraction. POC can be employed as a proxy of SPMDW, based on the correlations assessed by [START_REF] Trimble | The role of suspended particulate matter in 234Th scavenging and 234Th-derived export fluxes of POC in the Canada Basin of the Arctic Ocean[END_REF]. SPMDW at the CML is mainly composed of POC (around 50%) but also contains biogenic silica, calcium carbonate and lithogenic matter [START_REF] Bishop | The chemistry, biology, and vertical flux of particulate matter from the upper 400 m of the equatorial Atlantic Ocean[END_REF][START_REF] Krasakopoulou | Spatial and temporal distribution patterns of suspended particulate matter and particulate organic carbon in the Saronikos Gulf (eastern Mediterranean, Greece)[END_REF]. On the basis of these observations, relative POC was used to assess the contribution of the different size-fractions to the total biomass. Plankton dry weight (Plankton DW) of size fractions 60-200, 200-500, 500-1000, 1000-2000, and > 2000 µm were determined after drying samples on pre-weighed GF/F filters (60 °C, 24 h) and then re-weighed with a microbalance, taking into consideration the volumes of water filtered by the MultiNet [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF].

Biochemical and energy content

Biochemical compounds (proteins, carbohydrates, and lipids) were extracted in triplicate from freeze-dried filters and zooplankton samples and expressed in µg mg -1 DW. Absorbance of the extracts was then measured at different wavelengths using a spectrophotometer (Shimadzu, UV-1280). Briefly, proteins were extracted using a Folin phenol reagent following the [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF] method and measured at 700 nm. Carbohydrates were extracted using the phenol-sulfuric acid reaction described in [START_REF] Dubois | Colorimetric Method for Determination of Sugars and Related Substances[END_REF] and measured at 490 nm. Finally, lipids were extracted with a monophasic methanoldichloromethane solution [START_REF] Bligh | A rapid method of total lipid extraction and purification[END_REF] and measured at 360 nm. Ashes and residual organic compounds not recovered by these standard biochemical assays (chitin for example) may be estimated by subtracting the weight of the biochemical compounds from the total dry weight, but were not presented in this study. The energy content (Ei) was estimated by summing the three biochemical compounds after converting them into energetic units (21.4 kJ g -1 for proteins, 17.2 kJ g -1 for carbohydrates and 35.6 kJ g -1 for lipids) [START_REF] Postel | 4 -Biomass and abundance[END_REF]. The plankton energy amount (ET) provided by plankton per cubic meter per station at CML (kJ m -3 ) was calculated for all the plankton size-fractions by multiplying the SPMDW or the biomass (Bi in mg DW m -3 ) of each size-fraction at a given station by its energy content (Ei in kJ mg -1 DW) and the sum of ET of all size-fractions represents the plankton total energy amount, ETS: ET = Bi × Ei.

Stable isotope analyses (δ 13 C and δ 15 N)

The SPMDW collected on filters was scraped off with a scalpel. Zooplankton samples were ground to a fine powder with an agate mortar and pestle. Presence of carbonates on samples can bias the measurement of δ 13 C values as they are enriched in 13 C compared to organic matter [START_REF] Pinnegar | Differential fractionation of δ13C and δ15N among fish tissues: implications for the study of trophic interactions[END_REF]. Therefore, samples were acidified prior to the measurement of δ 13 C values of the particulate organic matter (POM). Samples were acidified using HCl 1% and immediately rinsed with MilliQ-water. Measurements of δ 15 N values of the POM were carried out on raw samples. For zooplankton, approximately 0.5 mg of powder was weighed into a tin cup (8 × 5 mm) using a precision balance (d = 0.01 mg). Carbon and nitrogen stable isotope analyses were performed using a continuous-flow isotope-ratio mass spectrometer (Delta V Plus, Thermo Scientific) with a Conflo IV interface coupled to an elemental analyzer (EA Isolink, Thermo Scientific). Analyses were conducted at the Littoral, Environment and Societies Joint Research Unit stable isotope facility (CNRS -University of La Rochelle, France). The δ 13 C and δ 15 N values are expressed in δ notation as deviations from standards (Vienna Pee Dee Belemnite for δ 13 C and N2 in air for δ 15 N), in ‰, according to the formula: where X is 13 C or 15 N, Rsample is the isotopic ratio of the sample and Rstandard is the isotopic ratio of the standard. Calibration was carried out using reference materials (USGS-61, -62, -63 for both carbon and nitrogen). The analytical precision of the measurements was < 0.10‰ for carbon and nitrogen based on analyses of USGS-61 and USGS-63 used as laboratory internal standards.

Cytometry and imagery analyses

For flow cytometry analyses, seawater was sampled with Niskin and Go-Flo bottles at the CML. Seawater was filtered through a silk mesh filter with 100-µm pore size and then immediately fixed with a 2% paraformaldehyde solution. Samples were incubated for 15 min at 4 °C in the dark and then stored in liquid nitrogen onboard and at -80 °C in the laboratory until analysis. The autotrophic pico-and nanoplankton were analyzed using a CyFlow ® Space flow cytometer (SysmexPartec) equipped with a blue diode pumped solid -state laser (20 mW; 488 nm) and a red diode laser (25 mW; 638 nm). Seawater samples were thawed in the dark, filtered through a nylon mesh filter with a porosity of 30 μm (CellTrics ® , SysmexPartec), and mixed with flow check high-intensity beads of 2-μm diameter (Polysciences, USA). Cells were characterized based on their scatter and fluorescence signals (Khammari et al., 2018) and data were analyzed with the FloMax software (Sysmex Partec), which directly calculates the cell concentration (cells cm -3 ) of the resolved cell groups. Carbon biomass of each cell group was estimated according to Khammari et al., (2020). The contributions of zooplankton, phytoplankton (microalgae), and detritus components to the total Plankton DW were estimated using imagery methods (FlowCAM and ZOOSCAN) according to Fierro-González et al.

(2023).

Bayesian mixing model

The contributions of the different size-fractions of phytoplankton (0. 7-2.7, 2.7-20 and20-60 µm) as food resources to zooplankton size-fractions and their confidence intervals were estimated using a Bayesian mixing model using the R package MIXSIAR [START_REF] Parnell | Bayesian stable isotope mixing models[END_REF]; https://github.com/brianstock/MixSIAR). This modeling approach incorporates variability of potential food source isotopic compositions for consumers and of trophic fractionation factors (TFF) and generates probability distributions of food source contributions. The consumers (i.e., zooplankton) were grouped in the 60-500, 500-2000, and > 2000-µm size-fractions based on statistical differences in their isotopic compositions.

Zooplankton size-fractions with similar isotopic compositions were thus considered to consume the same food resources. Contributions were computed using δ 13 C and δ 15 N values.

As there is no well-established set of TFFs for plankton, we computed mixing model estimates using the mean TFF values of 1.70 ± 0.38 ‰ for δ 13 C and 2.40 ± 0.26 ‰ for δ 15 N (mean ± standard error (SE)), as established by [START_REF] Tiselius | Daily changes in δ15N and δ13C stable isotopes in copepods: equilibrium dynamics and variations of trophic level in the field[END_REF].

Trophic level

The trophic level (TL) of consumers was calculated following the equation proposed by [START_REF] Post | Using Stable Isotopes to Estimate Trophic Position: Models, Methods, and Assumptions[END_REF]:

where δ 15 N consumer is the isotopic composition of the consumer, δ 15 Nbaseline is the isotopic composition of the size-fraction 0.7-2.7 µm considered as baseline with a trophic level of 1 and TFF δ 15 N was fixed to 2.40 ± 0.26 ‰ [START_REF] Tiselius | Daily changes in δ15N and δ13C stable isotopes in copepods: equilibrium dynamics and variations of trophic level in the field[END_REF]. TL of each size fraction was weighted to its respective biomass per station (TLB):

where I is represented by each size fraction from 60-200 to > 2000 µm and n is the number of size-fractions.

Data treatment

The effect of size-fractions and geographical area on the SPMDW, PlanktonDW, POC, PON, C/N, carbon biomass of phytoplankton, biochemical composition, energy content, isotopic composition and trophic levels were tested by means of one-way ANOVA or non-parametric Kruskal-Wallis tests after testing for normality and homogeneity of variances, followed by appropriate paired comparison tests, using the software Statistica 12. Spearman's rank order correlation tests were used to assess the significance of the correlations between our results and some environmental variables [START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF].

Results

Plankton dry weight, TChla, POC, PON and C:N

Significant differences in SPMDW were observed between the different size-fractions (H = 23.2, p < 0.0001) (Table 1). When considering all stations, the highest mean SPMDW was measured in the 2.7-20 µm size-fraction (0.18 ± 0.04 mg DW L -1 , n = 10) and the lowest in the 20-60 µm fraction (0.04 ± 0.02 mg DW L -1 , n = 10). Generally, these concentrations decreased from coastal stations to offshore stations, except for St9, which exhibited the highest SPMDW in the 2.7-20 µm size-fraction (Table 1). The size-fractions 0.7-2.7 and 2.7-20 µm presented the lowest values at St15. The highest SPMDW in the 20-60-µm size-fraction were observed at St17, St19 and St4. TChla concentrations in the > 0.7-µm fraction ranged from 0.21 (St17) to 1.54 µg L -1 (St9) with a mean value of 0.76 ± 0.14 µg L -1 (n = 10) (Table 1). The 60-200 and 200-500 µm zooplankton size-fractions showed the highest PlanktonDW (Table 1). POC and PON concentrations increased with phytoplankton size with the lowest values in the 0.7-2.7 µm size-fraction (11.4 and respectively 2.1 µg C L -1 ) and the highest ones in the 0.7-60 µm size-fraction (42.8 and respectively 6.9 µg C L -1 ) (Table S2). The lowest POC and PON values were observed at St11 for the 0.7-2.7 µm size-fraction and at St2 for the 2.7-20 µm size-fraction (Table S2). The C:N ratios in the 0.7 to 60 µm sizefractions ranged from 5.7 at St19 to 8.5 at St9. The 0.7-2.7 µm size-fraction presented significantly lower C:N ratios than the 2.7-20-µm and the 20-60 µm size-fractions (Table S2).

Planktonic group composition

Different plankton groups contributed to the biomass of the different size-fractions. The sizefraction 0.7-2.7 µm was on average dominated in biomass by Synechococcus spp. (4.6 µg C L -1 or ~ 52%) and picoeukaryotes (4.0 µg C L -1 or ~ 45%) (Table S3). The size-fraction 2.7-20 µm was dominated in biomass by the nanoeukaryotes (20.0 µg C L -1 ~ 88%) and represented the highest biomass among size-fractions. The highest cumulated biomass in the range 0.7-20 µm was found at St1, St9 and St19 (Table S3). POC concentration and the carbon biomass of phytoplankton size-fractions between 0.7 and 20 µm showed a significant linear correlation (R 2 = 0.66; p = 0.018) (Fig. S1). Carbon biomass estimated by flow cytometry represented around 86% of the POC measured on filters (y = 0.86x + 11.86). Total biomass was dominated by detritus and phytoplankton in the 60-200 and 200-500 µm sizefractions (68.8 and 59.2%, respectively), which decreased with the increasing size-fractions (10.5% in the > 2000-µm size-fraction) (Table S4). Copepods dominated in the zooplankton biomass in the 60 to 2000 µm size-fractions (between 54.3% and 91.9%) while crustaceans and gelatinous dominated the > 2000 µm size-fraction (Table S4).

Biochemical and energy content

The Mediterranean plankton sampled had for all size-fractions and stations combined, except the 0.7-60-µm fraction, mean dry weight concentrations (± SE) of 222.6 ± 6.9 µg mg -1 DW (n = 204) for proteins, 96.9 ± 6.5 µg mg -1 DW (n = 203) for carbohydrates, 35.1 ± 1.5 µg mg -1 DW (n = 197) for lipids and an Ei of 7.7 ± 0.2 kJ g -1 DW (n = 196).

Differences between plankton size-fractions

Considering all stations combined, the mean concentration of proteins increased with size and was significantly higher in large fractions of zooplankton (500-1000 and 1000-2000 µm) than in small fractions of phytoplankton (0.7-2.7 and 2.7-20 µm). The highest mean protein concentration was recorded in the 1000-2000 µm size-fraction and the lowest in the largest (> 2000 µm) size-fraction (Fig. 2A). The mean concentration in carbohydrates decreased with size. The concentrations measured in phytoplankton (0.7-60 and 20-60 µm) were significantly higher than those of the large zooplankton (1000-2000 and > 2000 µm) (Fig. 2B). The mean concentration in lipids was quite homogeneous between size-fractions, with the highest values measured in the size-fractions 20-60 and 1000-2000 µm, and lowest in the size-fractions 0.7-2.7 and > 2000 µm (Fig. 2C). Energy content showed an increasing trend with size for both phyto-and zooplankton size-fractions. The highest values were measured in the 20-60 µm and 1000-2000 µm size-fractions, respectively, and the lowest in the sizefractions 0.7-2.7 and > 2000 µm. (Fig. 2D).

Differences between stations

To investigate the differences between stations, the size-fractions were grouped in two fractions: the phytoplankton fraction (fractions between 0.7 and 60 µm) and the zooplankton fraction (fractions between 60 and 500 µm that were sampled at all the stations). The highest mean protein concentrations in phyto-and zooplankton fractions were recorded at St9, St11 and St15, and the lowest at St17 (Fig. 3A,B). The highest mean carbohydrate concentrations were observed at St9 for the phytoplankton (Fig. 3C). The highest mean lipid concentrations and energy values for phyto-and zooplankton were measured at St1, St2, St9 and St11, and the lowest at St3, St4 and St17 (Fig. 3E-H). Overall, greater differences appeared between stations than between size-fractions for most of these biochemical compounds (Table S5).

ETS ranged from 1.17 kJ m -3 at St3 to 6.30 kJ m -3 at St9 with a mean value (± SE) of 2.37 ± 0.49 kJ m -3 (n = 10) (Fig. 4). High ETS were also observed at coastal stations St1, St4, St17 and St19. The 0.7-2.7 and 2.7-20 µm size-fractions represented the largest energetic reservoirs with mean values of 0.65 ± 0.09 kJ m -3 (n = 10) and 1.16 ± 0.37 kJ m -3 (n = 10), respectively (Fig. 4). The 200-500-µm fraction represented the largest energy reservoir for the zooplankton with a mean of 0.164 kJ m -3 . Although the 0.7-2.7 and 2.7-20 µm fractions had lower Ei (6.2 and 6.3 kJ g -1 DW, respectively) than those measured in the > 60 µm sizefractions (between 7.0 and 9.5 kJ g -1 DW) (Fig. 2), their dry weight was about one order of magnitude higher (Tables 1 andS2), thus enhancing their major contribution of phytoplankton to the total plankton energy amount available in the system (Fig. 4).

Stable isotope compositions (δ 13 C and δ 15 N)

For all size fractions and stations combined, except the 0.7-60 µm fraction, the plankton δ 13 C values ranged from -26.6 to -17.4 ‰, with a mean of -23.0 ± 0.1‰ (n = 136), whereas the δ 15 N values ranged from -0.6 to 5.7‰, with a mean of 2.6 ± 0.1‰ (n = 136).

Differences between plankton size-fractions

Considering all stations combined, the mean δ 13 C and δ 15 N values increased with size, especially in the fractions ranging from 0.7 to 60 µm (Fig. 5, Table S6). Significantly higher mean δ 13 C values were reported in the 20-60 µm fraction than in the 0.7-2.7 µm fraction. The mean δ 15 N values of the size-fractions from 0.7 to 60 µm were all significantly different. Size-fractions between 60 and 2000 µm showed slightly higher δ 13 C values than those of smaller fractions. The highest mean δ 13 C and δ 15 N values were measured in the 1000 to > 2000 µm size-fractions (Fig. 5, Table S6).

Differences between stations

As for the biochemical composition, the size-fractions were grouped into two fractions: the phytoplankton fraction (fractions between 0.7 and 60 µm) and the zooplankton fraction (fractions between 60 and 500 µm that were sampled at all the stations). The mean δ 13 C and δ 15 N values followed the same spatial variations between phyto-and zooplankton fractions (Fig. 6A-D). For both phyto-and zooplankton, the highest mean δ 13 C values were measured at St4 and St17. The highest mean δ 15 N values were detected in phytoplankton at St4, while the lowest ones were observed at St9. In zooplankton, the highest mean δ 15 N values were measured at St10, and the lowest ones at St9, St17, and St19 (Fig. 6A-D). Overall, in contrast to the biochemical composition, greater differences were highlighted between size-fractions than between stations for both δ 13 C and δ 15 N values (Table S5).

Trophic levels

For all zooplankton size-fractions (60 to > 2000 µm) and stations combined, the TL ranged from 1.5 to 3.6 with a mean of 2.2 ± 0.1 (n = 76). When considering all stations combined, mean TL significatively increased with size (F = 5.2, p = 0.001) (Fig. 7). The highest mean TL (2.6 ± 0.2, n = 10) was measured for the 1000-2000 µm size-fraction and the lowest TL (1.9 ± 0.1, n = 20) in the 60-200 µm fraction. For the 60-200, 200-500, and 500-1000 µm size-fractions, differences in mean TLs were overall significant between stations (H = 18.7, p = 0.027; H = 18.9, p = 0.026 and H = 16.3, p = 0.037, respectively) with the highest mean trophic levels by size-fraction at St10. Mean TL weighted by the plankton biomass (TLB) had values larger than 2 at St2, St3 in northern coastal waters, and at St9, St10 and St11 in offshore waters (Fig. 8). The cumulated biomass of the zooplankton groups (60 to > 2000 µm) of the sampled stations was significantly and negatively correlated with TLB (R 2 = 0.61, p = 0.020) (Fig. S2).

Trophic flows in the plankton food web

The Mediterranean plankton food web analysis highlighted size predation (Fig. 9, Fig. S3).

For all stations combined, the 0.7-2.7 µm fraction was mainly consumed by the 60-500-µm fraction, while the consumption of the 2.7-20 µm fraction was similar between the 60-500 and 500-2000-µm fractions. The > 2000 µm size-fraction mainly consumed the 20-60 µm size fraction (Fig. 9, Fig. S3).

Discussion

Plankton biochemical content

The present description of the plankton biochemical content along a north-south transect in the Mediterranean Sea appears to be unique to our knowledge. The biochemistry of phytoand zooplankton has been extensively documented in many marine environments but remains little observed in the Mediterranean Sea [START_REF] Morris | Biochemical composition of crustacean zooplankton from the eastern Gulf of Mexico[END_REF][START_REF] Danovaro | Biochemical composition of pico-, nano-and micro-particulate organic matter and bacterioplankton biomass in the oligotrophic Cretan Sea (NE Mediterranean)[END_REF]Yilmaz and Besiktepe, 2010;[START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF]. [START_REF] Jónasdóttir | Fatty Acid Profiles and Production in Marine Phytoplankton[END_REF] summarizes the biochemistry of phytoplankton by a protein / carbohydrate / lipid average ratio of 5 / 3 / 2 (with a range of 40-60, 17-40, and 16-26%), similar to [START_REF] Ríos | Chemical composition of phytoplankton and Particulate Organic Matter in the Ría de Vigo (NW Spain)[END_REF] who found a ratio of 4.9 / 3.2 / 1.9. In our study, the mean ratio of the size-fractions between 0.7 and 60 µm was on average 5.6 / 3.6 / 0.9, therefore with relatively higher proteins and lower lipids compared to previous ratios. The biochemical ratio in the > 60 µm sizefractions was 6.9 / 2.0 / 1.1, reflecting higher protein and lower carbohydrate contents compared to phytoplankton. The higher proportion of protein to carbohydrate is a good indicator of the nitrogen availability in the environment whereas the inverse pattern may reflect nitrogen-limited environments for both phyto- [START_REF] Fabiano | Vertical Distribution and Biochemical Composition of Pico-and Microparticulate Organic Matter in the Ross Sea (Antarctica)[END_REF][START_REF] Danovaro | Biochemical composition of pico-, nano-and micro-particulate organic matter and bacterioplankton biomass in the oligotrophic Cretan Sea (NE Mediterranean)[END_REF]Yilmaz and Besiktepe, 2010;[START_REF] Kim | River discharge effects on the contribution of small-sized phytoplankton to the total biochemical composition of POM in the Gwangyang Bay, Korea[END_REF] and zooplankton [START_REF] Bhat | Biochemical composition of zooplankton from the northern Arabian Sea[END_REF]. [START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF] found in spring time in the Bay of Marseille (in the same location as our station St4), for the size-fraction 200-500 µm, carbohydrate concentrations (62.4 to 66.7 mg g -1 ) similar to ours, while their concentrations in lipids (98.3 to 102.8 mg g -1 ) and proteins (292.7 to 335.7 mg g -1 ) were higher.

The protein and lipid concentrations were higher in zooplankton than in phytoplankton.

Within the zooplankton, an increase in proteins with size was observed, in agreement with [START_REF] Guisande | Biochemical fingerprints in zooplankton[END_REF]. Most of the organisms in our samples between 60 and 2000 µm were composed of copepods, and, consequently, the different fractions corresponding to different life-stages of the same group [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF] may explain the size influence on their biochemical content. The highest concentrations of carbohydrates and lipids reported in the 20-60 µm size-fraction can be related to a mixed composition of microphyto-and microzooplanktonic organisms, such as diatoms and metazoan eggs [START_REF] Danovaro | Biochemical composition of pico-, nano-and micro-particulate organic matter and bacterioplankton biomass in the oligotrophic Cretan Sea (NE Mediterranean)[END_REF][START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF]. However, an underestimation of the SPMDW in the 20-60 µm fraction with respect to that in the size-fractions from 0.7 to 60 µmdue to different estimation methods (see section 2.2.2)may explain the higher biochemical concentrations measured in the 20-60 µm fractions. Finally, the lowest protein, carbohydrate, and lipid concentrations observed in the > 2000-µm fraction were probably related to the dominance in this fraction of filter feeder organisms, such as salps and siphonophores [START_REF] Bănaru | Seasonal variation of stable isotope ratios of size-fractionated zooplankton in the Bay of Marseille (NW Mediterranean Sea)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF][START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF][START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF].

Organic contaminants have been shown to have a strong chemical affinity for lipids or proteins [START_REF] Mason | Bioaccumulation of mercury and methylmercury[END_REF][START_REF] Wu | Accumulation, subcellular distribution and toxicity of inorganic mercury and methylmercury in marine phytoplankton[END_REF]Wang, 2011, Frouin et al., 2013). Therefore, without considering the size of the organisms (which is also an important factor in the accumulation of contaminants in biota, in particular, phytoplankton), we can assume that the concentrations of organic contaminants in zooplankton of stations St1, St2, St9 and St11 (which contains more lipids and proteins) may be higher than those in the zooplankton of stations St3, St4 and St17 (which have less lipids and proteins). The biochemical content of the plankton size fractions can change due to variations of the composition of the plankton community and the nutrient inputs linked to the physical and chemical environment, thus affecting the entire trophodynamics of the ecosystem [START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF][START_REF] Chen | Temporal variation in prey selection by adult European sardine (Sardina pilchardus) in the NW Mediterranean Sea[END_REF][START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF].

Plankton total energy amount (E TS )

The ET decreased with size due to plankton biomass reduction with size. In our study, the ET in SPMDW were lower than previously measured in other areas [START_REF] Mayzaud | Seasonal changes of the biochemical composition of marine particulate matter with special reference to fatty acids and sterols[END_REF][START_REF] Fabiano | Vertical Distribution and Biochemical Composition of Pico-and Microparticulate Organic Matter in the Ross Sea (Antarctica)[END_REF][START_REF] Kim | River discharge effects on the contribution of small-sized phytoplankton to the total biochemical composition of POM in the Gwangyang Bay, Korea[END_REF]. This is most likely due to the lower phytoplankton biomass reported in the oligotrophic Mediterranean Sea, highlighting their low qualitative values even if spatial and seasonal variations may occur. In the eastern Mediterranean basin, the Ei measured in zooplankton by [START_REF] Danovaro | Biochemical composition of pico-, nano-and micro-particulate organic matter and bacterioplankton biomass in the oligotrophic Cretan Sea (NE Mediterranean)[END_REF] were even lower than in our study, probably related to higher oligotrophy in their study area. In the Bay of Marseille, the ET measured at St4 were higher for us than those recorded in similar zooplankton size-fractions during the same season in 2017 by [START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF]. Plankton showed higher ETS at St1, St4, St9 and St19. At these stations, where bloom conditions prevailed, trophic regimes were probably responsible for enhanced biomass [START_REF] Mayot | Influence of the Phytoplankton Community Structure on the Spring and Annual Primary Production in the Northwestern Mediterranean Sea[END_REF][START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF]Tedetti et al., 2023) (Table S1), which may have impacted their ETS. The 0.7-2.7, 2.7-20, and 200-500µm size-fractions represented the largest energetic reservoir in the Mediterranean phyto-, and zooplankton compartments. Our study highlights their importance as major potential sources of contaminants for their consumers.

Plankton stable isotopes ratios and composition

The fractions comprised between 0.7 and 60 µm corresponded to SPMDW made up of a complex mixture of living, detrital and lithogenic material, difficult to separate [START_REF] Lam | Size-fractionated major particle composition and concentrations from the US GEOTRACES North Atlantic Zonal Transect[END_REF][START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF]. The main limitation of food web studies relying of stable isotope ratios is to establish an acceptable baseline, using samples mainly composed of primary producers [START_REF] Harmelin-Vivien | Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean)[END_REF][START_REF] Tamelander | Base-line variations in stable isotope values in an Arctic marine ecosystem: effects of carbon and nitrogen uptake by phytoplankton[END_REF]. In our study, pico-and nanoplankton dominated the SPMDW biomass which was composed of around 86% of photosynthetic organisms, as confirmed by cytometry results [START_REF] Boudriga | Ultraphytoplankton community structure in subsurface waters along a North-South Mediterranean transect[END_REF].

Moreover, their low C:N ratios suggest a composition dominated by living photosynthetic organisms.

Few studies have measured isotopic compositions of the smallest size-fractions of plankton [START_REF] Wainright | Seasonal variation of the stable isotopic compositions of coastal marine plankton from Woods Hole, Massachusetts and Georges Bank[END_REF][START_REF] Tamelander | Base-line variations in stable isotope values in an Arctic marine ecosystem: effects of carbon and nitrogen uptake by phytoplankton[END_REF], and even fewer have separated them by size [START_REF] Rau | C-13/C-12 and N-15/N-14 variations among size-fractionated marine particles -implications for their origin and trophic relationships[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Im | Evidence for ontogenetic feeding strategies in four calanoid copepods in the East Sea (Japan Sea) in summer, revealed by stable isotope analysis[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF][START_REF] Décima | Zooplankton trophic structure and ecosystem productivity[END_REF]. The size-fractions from 0.7 to 60 µm (without considering the 0.7-60 µm fraction) have similar δ 13 C values but their δ 15 N values are rather low (-23.9 ± 0.2 ‰ and 1.4 ± 0.16 ‰, respectively) compared to those measured in other oceanic basins (from -28 to -17 ‰ and from 1 to 12 ‰, respectively) [START_REF] Wainright | Seasonal variation of the stable isotopic compositions of coastal marine plankton from Woods Hole, Massachusetts and Georges Bank[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Tamelander | Base-line variations in stable isotope values in an Arctic marine ecosystem: effects of carbon and nitrogen uptake by phytoplankton[END_REF] and in the Mediterranean Sea (from -25 to -22 ‰ and from 1 to 6 ‰, respectively) [START_REF] Rau | C-13/C-12 and N-15/N-14 variations among size-fractionated marine particles -implications for their origin and trophic relationships[END_REF][START_REF] Harmelin-Vivien | Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF][START_REF] Liénart | Dynamics of particulate organic matter composition in coastal systems: A spatio-temporal study at multi-systems scale[END_REF].

Several processes may explain the low δ 15 N values of the plankton in our study: 1) in the convection areas subjected to algae blooms (such as St9), nutrients made of light isotopes are preferentially uptaken by phytoplankton [START_REF] Wainright | Seasonal variation of the stable isotopic compositions of coastal marine plankton from Woods Hole, Massachusetts and Georges Bank[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Tamelander | Base-line variations in stable isotope values in an Arctic marine ecosystem: effects of carbon and nitrogen uptake by phytoplankton[END_REF], 2) in water with poor nutrient content, ammonium, the main nitrogen source emitted through excretion and recycling, has lower δ 15 N values (Checkely and Miller, 1989), and 3) the high proportion of diazotrophs (atmospheric nitrogen fixer) in oligotrophic waters may lower δ 15 N values [START_REF] Pantoja | Stable isotope constraints on the nitrogen cycle of the Mediterranean Sea water column[END_REF][START_REF] Montoya | Nitrogen fixation and nitrogen isotope abundances in zooplankton of the oligotrophic North Atlantic[END_REF][START_REF] Koppelmann | Vertical distribution of mesozooplankton and its δ15N signature at a deep-sea site in the Levantine Sea (eastern Mediterranean) in April 1999[END_REF].

Within the 0.7 and 60 µm size-fractions, δ 13 C and δ 15 N values significantly increased with size from pico-, nano-to microplankton, similar to previous studies [START_REF] Im | Evidence for ontogenetic feeding strategies in four calanoid copepods in the East Sea (Japan Sea) in summer, revealed by stable isotope analysis[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. Differences between primary producers can be related to differences in the isotopic composition of inorganic nitrogen sources and/or fractionation between molecules made of heavy and light isotopes during physiological processes [START_REF] Ostrom | Sources and cycling of organic matter within modern and prehistoric food webs[END_REF]. A large contribution of cyanobacteria, mainly composed of Synechococcus spp. may lower δ 13 C and δ 15 N values of SPM in the 0.7-2.7 µm size-fraction [START_REF] Rau | C-13/C-12 and N-15/N-14 variations among size-fractionated marine particles -implications for their origin and trophic relationships[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. These differences in the composition may also suggest predation within the 0.7-60 µm fraction [START_REF] Onodera | Evaluation of trophic transfer in the microbial food web during sludge degradation based on 13C and 15N natural abundance[END_REF][START_REF] Armengol | Planktonic food web structure and trophic transfer efficiency along a productivity gradient in the tropical and subtropical Atlantic Ocean[END_REF]. However, an increase in δ 15 N values with the size has already been observed in autotrophs [START_REF] Karsh | Relationship of nitrogen isotope fractionation to phytoplankton size and iron availability during the Southern Ocean Iron RElease Experiment (SOIREE)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF].

The group composed of size-fractions larger than 60 µm and dominated by zooplankton, mainly copepods [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF], presented higher δ 13 C and δ 15 N values than phytoplankton.

The isotopic compositions reported here for the 200-500 µm size-fractions (-22.6 ± 0.4 ‰ and 3.2 ± 0.2 ‰, for δ 13 C and δ 15 N, respectively) were rather in the lower range of previous estimates in different oceanic basins (from -22 to -19‰ and from 2 to 8‰ for δ 13 C and δ 15 N values, respectively) [START_REF] Fry | Biomass spectra and stable isotope indicators of trophic level in zooplankton of the northwest Atlantic[END_REF][START_REF] Bode | Stable nitrogen isotope studies of the pelagic food web on the Atlantic shelf of the Iberian Peninsula[END_REF][START_REF] Yang | Regional patterns of δ13C and δ15N stable isotopes of size-fractionated zooplankton in the western tropical North Pacific Ocean[END_REF]. Overall, the isotopic compositions measured in the different zooplankton size-fractions were within the same range as those reported in the ultra-oligotrophic eastern Mediterranean basin [START_REF] Koppelmann | Trophic relationships of zooplankton in the eastern Mediterranean based on stable isotope measurements[END_REF][START_REF] Denda | Zooplankton at a seamount in the eastern Mediterranean: distribution and trophic interactions[END_REF] and in the more productive area of the Gulf of Lion [START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF][START_REF] Bănaru | Seasonal variation of stable isotope ratios of size-fractionated zooplankton in the Bay of Marseille (NW Mediterranean Sea)[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF].

Within the zooplankton size-fractions, δ 15 N values increased with size, up to the 1000-2000 µm fraction, underlining an enhancement of the predation with size [START_REF] Fry | Biomass spectra and stable isotope indicators of trophic level in zooplankton of the northwest Atlantic[END_REF][START_REF] Rolff | Seasonal variation in δ13C and δ15N of size-fractionated plankton at a coastal station in the northern Baltic proper[END_REF][START_REF] Bănaru | Seasonal variation of stable isotope ratios of size-fractionated zooplankton in the Bay of Marseille (NW Mediterranean Sea)[END_REF][START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. The δ 15 N values increased with the size-fractions, which may be due to the succession of the different life stages of copepods, coinciding with the pattern observed in their biochemical composition [START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF][START_REF] Chen | Seasonal variation in biochemical and energy content of size-fractionated zooplankton in the Bay of Marseille (North-Western Mediterranean Sea)[END_REF] related to metabolic changes during the lifespan [START_REF] Guisande | Biochemical fingerprints in zooplankton[END_REF], adaptative foraging [START_REF] Kozak | Seasonal variability of stable isotope values and niche size in tropical calanoid copepods and zooplankton size fractions[END_REF], ontogenetics [START_REF] Mauchline | The biology of Calanoid copepods[END_REF][START_REF] Im | Evidence for ontogenetic feeding strategies in four calanoid copepods in the East Sea (Japan Sea) in summer, revealed by stable isotope analysis[END_REF] or all these processes combined. Fierro-González et al. ( 2023) reported an increase in the proportion of carnivores with size. However, in the largest size-fraction (> 2000 µm), gelatinous organisms were generally overrepresented with respect to other fractions, leading to higher δ 13 C and lower δ 15 N values due probably to distinct food sources from smaller fractions [START_REF] Bănaru | Seasonal variation of stable isotope ratios of size-fractionated zooplankton in the Bay of Marseille (NW Mediterranean Sea)[END_REF][START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF].

Spatial variation in plankton isotopic compositions

The highest δ 15 N values in phytoplankton were recorded at St4, located in the Bay of Marseille, which is highly impacted by anthropogenic and terrestrial inputs. These inputs may induce, in case of declining nitrates related to intense bloom events, an increase of the δ 15 N values [START_REF] Raimbault | Distribution of inorganic and organic nutrients in the South Pacific Ocean; evidence for long-term accumulation of organic matter in nitrogen-depleted waters[END_REF][START_REF] Fey | Multi-trophic markers illuminate the understanding of the functioning of a remote, low coral cover Marquesan coral reef food web[END_REF]. Moreover, the effluents of the Marseille sewage treatment plant may be 15 N-enriched in the inorganic nitrogen pool due to bacterial denitrification [START_REF] Wainright | Seasonal variation of the stable isotopic compositions of coastal marine plankton from Woods Hole, Massachusetts and Georges Bank[END_REF][START_REF] Cabana | Comparison of aquatic food chains using nitrogen isotopes[END_REF]. The high δ 13 C values measured in phyto-and zooplankton in the Bay of Marseille (St3 and St4) and in the Gulf of Gabès (St17 and St19) may be due to higher growth rates and nutrient availability in these productive areas [START_REF] Laws | Dependence of phytoplankton carbon isotopic composition on growth rate and [CO2]aq: Theoretical considerations and experimental results[END_REF][START_REF] Bidigare | Consistent fractionation of 13C in nature and in the laboratory: Growth-rate effects in some haptophyte algae[END_REF], as well as to inputs of sedimentary organic matter in the water column during sediment resuspension events (Table S1). Moreover, the low δ 15 N values measured on the Tunisian coasts support this hypothesis and may be related to seagrass detritus [START_REF] Cresson | Spatio-temporal variation of suspended and sedimentary organic matter quality in the Bay of Marseilles (NW Mediterranean) assessed by biochemical and isotopic analyses[END_REF]. St9, located at the border of the convective area of the Northwestern Mediterranean Sea, was characterized by a post-bloom event during the campaign [START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF] and displayed the lowest δ 15 N values in phyto-and zooplanktonic organisms. The highest C:N measured at this station may suggest a higher detrital organic matter content and a higher microbial production of regenerated isotopically lighter ammonium compared to the other stations [START_REF] Maguer | Nitrogen Uptake by Phytoplankton in a Shallow Water Tidal Front[END_REF].

The relatively high δ 13 C (and δ 15 N) values measured in phyto-and zooplankton in the Bay of Marseille and the Gulf of Gabès may also be related to various inputs (sediment resuspension, effluents, etc.), and be the sign of higher levels of contaminants which could derive from the same sources. To support this hypothesis, higher trace metal concentrations have been reported in these coastal stations compared to other MERITE-HIPPOCAMPE stations [START_REF] Chifflet | Distribution and accumulation of trace metal in the planktonic food web of the Mediterranean Sea (MERITE-HIPPOCAMPE campaign)[END_REF].

Trophic levels and food web implications

The TLs determined in this study, ranging from 1.5 to 3.6, increased with size and spread out over 2 TLs. TLs lower than 2 may be related to a mixture of primary producers and consumers mainly found at some stations in the 60-200 µm size-fraction, related to clogging of the sampling device caused by phytoplankton. Further improvements in sampling methods by using larger mesh-size nets for zooplankton size-fractions collection may avoid potential clogging during bloom events and the presence of phytoplankton within the 60-200 µm sizefraction.

When biomass is averaged to the TL of zooplankton (TLB), higher values are obtained at the coastal (St2, and St3), and offshore (St9, St10, and St11) stations. High adaptability in food habits of zooplankton is well known and omnivory seems to be the most common trophic pathway [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF]. Higher TLs are generally reached in microbial food webs, often observed in oligotrophic regimes [START_REF] Søreide | Seasonal food web structures and sympagic-pelagic coupling in the European Arctic revealed by stable isotopes and a two-source food web model[END_REF][START_REF] Kürten | Tracking seasonal changes in North Sea zooplankton trophic dynamics using stable isotopes[END_REF]. St10 and St11 contained carnivorous plankton, such as chaetognaths which may explain the higher TLs of the zooplankton community observed in these areas [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF]. It has been suggested that the TFF between phytoplankton and protozoans is equal to 0, contributing to an underestimation of the number of TLs in planktonic food webs [START_REF] Gutiérrez-Rodríguez | Isotopic invisibility of protozoan trophic steps in marine food webs[END_REF]. However, in this work, we suggest that the higher TLs of plankton were probably related to the microbial food web.

Coastal stations (St1,St4,St15,St17 and St19) were characterized by lower TLB. The lower TL of plankton in productive systems is classic in herbivore food webs, dominated by larger phytoplankton organisms readily available to zooplankton [START_REF] Sommer | Pelagic food web configurations at different levels of nutrient richness and their implications for the ratio fish production:primary production[END_REF][START_REF] Fileman | Grazing by Calanus helgolandicus and Para-Pseudocalanus spp. on phytoplankton and protozooplankton during the spring bloom in the Celtic Sea[END_REF]. Food chain length decreases in productive ecosystems due to an adaptive foraging of consumers which selects the most abundant low TL resources [START_REF] Kondoh | Food-chain length and adaptive foraging[END_REF]. Detritus and phytoplankton were dominant in these stations in the 60-200 µm size-fraction contributing to lowering TLB values [START_REF] Fierro-González | Zooplankton biomass, size structure, and associated metabolic fluxes with focus on its roles at the chlorophyll maximum layer during the plankton-contaminant MERITE-HIPPOCAMPE cruise[END_REF].

In addition, the zooplankton biomass was related to the trophic pathways. A negative relationship between the TLB and the zooplankton biomass was observed during this study.

Higher biomasses of zooplankton have been measured at coastal stations relative to offshore areas in relation to their primary production [START_REF] Champalbert | Characteristics of zooplankton standing stock and communities in the western Mediterranean Sea: relations to hydrology[END_REF]. Where TLs were high (St9, St10 and St11), large size fractions (500-1000 and 1000-2000 µm) were also well represented, accounting for 27 to 54% of the total zooplankton biomass. [START_REF] Espinasse | Patterns of variations in C and N stable isotope ratios in size-fractionated zooplankton in the Gulf of Lion, NW Mediterranean Sea[END_REF] have already highlighted the overrepresentation of large plankton organisms in moderately productive areas, while [START_REF] Décima | Zooplankton trophic structure and ecosystem productivity[END_REF] have reported an inverse relationship between food chain length and productivity in zooplankton. Although located in the offshore area, St9 was characterized by a still productive post-bloom situation [START_REF] Tedetti | Contamination of planktonic food webs in the Mediterranean Sea: Setting the frame for the MERITE-HIPPOCAMPE oceanographic cruise (spring 2019)[END_REF] with high TLB. Usually, during post-bloom, diatoms give way to inedible algae not consumed by zooplankton organisms and the source of organic matter is obtained through the cell lysis, fuelling microbial loop and enhancing food steps [START_REF] Sommer | Pelagic food web configurations at different levels of nutrient richness and their implications for the ratio fish production:primary production[END_REF]. High C:N ratios observed at St9 may indicate heterotrophy within the 0.7-60 µm fractions [START_REF] Harmelin-Vivien | Comparison of C and N stable isotope ratios between surface particulate organic matter and microphytoplankton in the Gulf of Lions (NW Mediterranean)[END_REF]. The post-bloom situation may induce a decrease of the resources and thus mesoplankton preying on microplankton [START_REF] Levinsen | On the trophic coupling between protists and copepods in arctic marine ecosystems[END_REF][START_REF] Basedow | Trophic positions of mesozooplankton across the North Atlantic: estimates derived from biovolume spectrum theories and stable isotope analyses[END_REF]. Hence, the results reported here highlight different trophodynamic scenarios in the Mediterranean plankton food webs and their control on planktonic biomasses, as hypothesized by [START_REF] Fry | Biomass spectra and stable isotope indicators of trophic level in zooplankton of the northwest Atlantic[END_REF].

Contrasting carbon fluxes can impact contaminant transfer. The ability of each contaminant to bioaccumulate can lead to different scenarios. In low-productive ecosystems, organic matter fluxes go through more trophic steps, which can favor a higher transfer of contaminants that will bioaccumulate such as methylmercury, PCBs or Cd [START_REF] Tiano | PCB concentrations in plankton size classes, a temporal study in Marseille Bay, Western Mediterranean Sea[END_REF][START_REF] Schartup | A Model for Methylmercury Uptake and Trophic Transfer by Marine Plankton[END_REF][START_REF] Chouvelon | Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-zooplankton-small pelagic fish marine food web[END_REF]. Conversely, in productive ecosystems, direct access to organic matter sources and reduced food steps favors higher transfer of contaminants with little or no capacity to bioaccumulate, such as the numerous trace elements Co, Ni, Cu, Ag, Pb, and Zn [START_REF] Chouvelon | Patterns of trace metal bioaccumulation and trophic transfer in a phytoplankton-zooplankton-small pelagic fish marine food web[END_REF].

Plankton food web flows

The difference in stable isotope composition between phytoplankton size-fractions made them relevant candidates for mixing model analysis of their relative contributions to zooplankton biomass [START_REF] Phillips | Best practices for use of stable isotope mixing models in foodweb studies[END_REF][START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. Overall, the contribution of the 0.7-2.7-µm size-fraction to the diet of the 60-500 µm size fraction underlines the role of the microzooplankton (60-200 µm) and of the small mesozooplankton (200-500 µm) in the transfer of organic matter from the baseline to higher trophic level consumers. The picoplankton size fraction was mainly composed of Synechococcus spp., the most abundant phytoplankton organism in the Mediterranean Sea that sustains primary production [START_REF] Boudriga | Ultraphytoplankton community structure in subsurface waters along a North-South Mediterranean transect[END_REF]. In fact, during the MERITE-HIPPOCAMPE campaign, picoplankton accounted for, on average, 27% of the total biomass of phytoplankton.

Classically, we consider that nanoflagellates and microzooplankton (from 2 to 200 µm sizefractions) may represent intermediate food steps [START_REF] Ryther | Photosynthesis and Fish Production in the Sea[END_REF][START_REF] Calbet | Mesozooplankton influences on the microbial food web: Direct and indirect trophic interactions in the oligotrophic open ocean[END_REF] ensuring the link between the 0.7-2.7 µm fraction and mesozooplankton (200-2000 µm) [START_REF] Sommer | Grazing by mesozooplankton from Kiel Bight, Baltic Sea, on different sized algae and natural seston size fractions[END_REF]. The 60 to 500 µm size-fractions were constituted of a large percentage of phytoplankton and detritus that would have contributed to lowering their stable isotope ratios. This may have consequences with regard to the mixing model results and may explain the high percentage of picoplankton estimated in the diet of 60-500 µm size-fraction.

However, in agreement with our results, and despite their small size, recent studies demonstrated that small copepods may feed on picoplankton [START_REF] Motwani | Mesozooplankton Grazing on Picocyanobacteria in the Baltic Sea as Inferred from Molecular Diet Analysis[END_REF][START_REF] Im | Evidence for ontogenetic feeding strategies in four calanoid copepods in the East Sea (Japan Sea) in summer, revealed by stable isotope analysis[END_REF][START_REF] Major | An isotopic analysis of the phytoplankton-zooplankton link in a highly eutrophic tropical reservoir dominated by cyanobacteria[END_REF].

Primary producers were also dominant in the 2.7-20 µm fractions, representing 69% of the phytoplanktonic biomass and about half of the total food supply to both the 60-500 and the 500-2000 µm size-fractions. Nanoplankton was mainly composed of nanoeukaryotes and its contribution to the planktonic food web seems essential, in agreement with the results of [START_REF] Hunt | Trophic pathways of phytoplankton size classes through the zooplankton food web over the spring transition period in the north-west Mediterranean Sea[END_REF]. Finally, the 20-60 µm fraction has low biomass and represents an important contribution only to the diet of the > 2000 µm size-fractions. TFFs for plankton should be experimentally determined and a higher number of replicates should be analyzed to improve model results and to reduce their variability.

Differences in contaminant accumulation have been reported in phytoplankton size-fractions.

Trace metals reflected, for each element, contrasted accumulation patterns between small and large phytoplankton fractions [START_REF] Chifflet | Distribution and accumulation of trace metal in the planktonic food web of the Mediterranean Sea (MERITE-HIPPOCAMPE campaign)[END_REF]. Higher methylmercury concentrations were reported during a picoplanktonic bloom, probably due to a higher surface to volume ratio, favoring uptake [START_REF] Heimbürger | Methyl mercury distributions in relation to the presence of nano-and picophytoplankton in an oceanic water column (Ligurian Sea, North-western Mediterranean)[END_REF]. In addition, for PCBs, a higher accumulation was observed in smaller cells [START_REF] Axelman | Field Measurements of PCB Partitioning between Water and Planktonic Organisms: Influence of Growth, Particle Size, and Solute-Solvent Interactions[END_REF]. The different carbon size-fractions of food sources can thus influence the transfer of contaminants and concentrations in zooplankton consumers.

Conclusions

During the MERITE-HIPPOCAMPE campaign, plankton size-fractions revealed contrasted biochemical and isotopic compositions related to their size, composition and location.

Carbohydrate concentrations were the highest in phytoplankton (between 0.7 and 60 µm sizefractions) with respect to zooplankton (> 60 µm size-fractions), which displayed high protein and lipid contents increasing with size. The affinity of proteins and lipids for contaminants makes zooplankton more sensitive to their accumulation (regardless of the size of organisms).

Due to the high amount of energy contained in their biomass, the pico-and nanoplankton represents a major food resource in Mediterranean ecosystems fueling the zooplankton food webs and an essential pathway for contaminants due to the combination of small size and high energetic content. In the zooplankton community, the 200-500 µm size-fraction, dominated by copepods, showed the highest energy amount, which explains their role as essential food resource for many planktivorous fishes. Spatial variations of isotopic compositions of plankton size-fractions were also observed, revealing different carbon sources between the coast and the offshore areas, and between the Bay of Gabès (southern coast) and the Bay of Toulon (northern coast), and resulting in a different exposure to contaminants. Trophic levels revealed increasing predation with size, which can result in the biomagnification of contaminants. Trophic pathways in planktonic food webs displayed spatial variations influenced by the availability of phytoplankton resources. Higher predation occurred in low productive areas where the zooplankton community reached the highest mean trophic levels, increasing the potential exposure of planktonic food webs to contaminants. Our findings on the spatial variations of the biomass, biochemical composition, and the role of the different plankton size-fractions in the food web, as well as the different flows of organic matter in the pelagic food webs, are an essential step for the comprehension of the transfer of contaminants in the Mediterranean pelagic food webs.

Figure captions Figure 1 .

 captions1 Figure captions

Figure 2 .

 2 Figure 2. Boxplot of the concentrations of A) proteins, B) carbohydrates, C) lipids (µg mg -1 DW) and D) energy content (Ei) (kJ g -1 DW) in the different plankton size-fractions (fractions between 0.7 and 60 µm in green and > 60 µm in orange) for all stations combined. H = Kruskal-Wallis non-parametric test and the associated p-value for the respective biochemical compounds: H = 43.5, p < 0.00 01; H = 110.6, p < 0.001; H = 41.8, p < 0.0001 and H = 51.7, p < 0.0001. Mean values with different letters are significantly different (p < 0.05). The mean and median values are represented by a cross and a horizontal line, respectively, and the box lengt h is defined as the interquartile range. The minimum and maximum values are represented by whiskers. Mean values with different post-hoc letters are significantly different (p < 0.05).

Figure 3 .

 3 Figure 3. Boxplot of the concentrations of A, B) proteins, C, D) carb ohydrates, E, F) lipids (µg mg -1 DW) and G, H) energy content (Ei) (kJ g -1 DW) in the phyto-(fractions between 0.7 and 60 µmin green) and zooplankton (fractions between 60 and 500 µmin orange) fractions for each station. H = Kruskal-Wallis nonparametric test and the associated p-value for the respective biochemical compounds for phytoplankton (H = 43.4, p < 0.0001; H = 30.6, p < 0.0001; H = 34.9, p < 0.0001 and H = 42.9, p < 0.0001) and for zooplankton (H = 43.8, p < 0.0001; H = 22.9, p < 0.006; H = 38.8, p < 0.0001 and H = 38.8, p < 0.0001). Stations are grouped by geographical area: St1, St2, St3 and St4 for the Northern coast, St9, St10 and St11 for the offshore area, and St15, St17 and St19 for the Southern coast. The mean and median values were represented by a cross and a horizontal line, respectively, and the box length is defined as the interquartile range. The minimum and maximum values are represented by whiskers. Mean values with different post-hoc letters are significantly different (p < 0.05).

Figure 4 .

 4 Figure 4. Total amount of energy provided by plankton (ET in kJ m -3 ) for each of the size-fractions by station. Stations are grouped by geographical area: St1, St2, St3 and St4 for the Northern coast, St9, St10 and St 11 for the offshore area, and St15, St17 and St19 for the Southern coast.

Figure 5 .

 5 Figure 5. Mean (± standard error, SE) stable isotope compositions (δ 13 C and δ 15 N values, ‰) in the different plankton size-fractions for all stations combined. Green dots correspond to the phytoplankton size-fractions (from 0.7 to 60 µm), while the orange dots correspond to zooplankton size -fractions (> 60 µm).

Figures 6 .

 6 Figures 6. Boxplot of the A, B) δ 13 C and C, D) δ 15 N values (‰) in the phyto-(fractions between 0.7 and 60 µm in green) and zooplankton (fractions between 60 and 500 µmin orange) fractions for each station. Stations are grouped by geographical area: St1, St2, St3 and St4 for the Northern coast, St9, St10 and St11 for the offshore area, and St15, St17 and St19 for the Southern coast. Kruskall-Wallis non-parametric test were performed for A) (H = 25.5, p < 0.05), C) (H = 24.1, p < 0.05) and D) (H = 33.5, p < 0.0001), and one -way

Figure 7 .

 7 Figure 7. Boxplot of the trophic levels (TL) of the zooplankton size-fractions (60 to > 2000 µm). The median value is represented by a horizontal line and the box length is defined as the interquartile range. The minimum and maximum values are represented by whiskers. One-way ANOVA test (F = 5.17, p = 0.001). Superscript letters represent Newman-Keuls post-hoc groups and values with the different letters are significantly different (p < 0.05).

Figure 8 .

 8 Figure 8. Trophic levels (TL) of the zooplankton size-fractions (60 to > 2000 µm) and mean TL weighted by their biomass (TLB) for each station. Stations are gathered by geographical area: St1, St2, St3 and St4 for the Northern coast, St9, St10 and St11 for the offshore area, and St15, St17 and St19 for the Southern coast.

Figure 9 .

 9 Figure 9. Relative contributions of 0.7-2.7 µm (pico-), 2.7-20 µm (nano-), and 20-60 µm (microplankton) as food resources for three zooplankton size-fractions. Arrows indicate the trophic transfer between pico-POM nano-POM, and micro-POM fractions and zooplankton size fractions: 60-500 µm (in green), 500-2000 µm (in blue) and > 2000 µm (in orange). Their thickness is proportional to relative contribution in percentage, indicated close to the corresponding arrow.

Table 1 .

 1 Suspended particulate matter and plankton dry weight (SPM DW and PlanktonDW, in mg DW L -1 ) for the different plankton size-fractions and stations, as well as total chlorophyll a concentration (TChla, µg L -1 ) for the > 0.7-µm fraction. Kruskal-Wallis test and the associated p-value for SPMDW (H = 23.15, p < 0.0001) and PlanktonDW (H = 28.51, p < 0.0001). Superscript letters represent rank comparison groups and values with different letters are significantly different (p < 0.05). St = station, SE = standard -error, n = number of samples.

  Figures

  Figure 2

Table 1 .

 1

	1278

Acknowledgements

The authors wish to thank the crew of the R/V Antea and the various platforms of the Mediterranean Institute of Oceanography having contributed to the data acquisition: Plateforme Analytique de Chimie des Environnements Marins (PACEM) for the POC/PON measurements, Plateforme Régionale de Cytométrie pour la Microbiologie (PRECYM) for cytometric analyses, Microscopie et Imagerie numérique (MIM) for identification expertise and the Service Atmosphère-Mer (SAM) for the technical and operational tasks. The authors thank the LIENSs joint research unit (CNRS -La Rochelle University) for the measurement of δ 13 C and δ 15 N values. The MERITE-HIPPOCAMPE project has been funded by the crossdisciplinary Pollution & Contaminants axis of the CNRS-INSU MISTRALS program (joint action of the MERMEX-MERITE and CHARMEX subprograms) and received support from the IRD French-Tunisian International Joint Laboratory (LMI) COSYS-Med. We are grateful for the additional funding received from IFREMER, the MIO Action Sud and Transverse Axis programs (CONTAM Transverse Axis), and from the IRD Ocean Department. This study received funding by the CONTAMPUMP project (ANR JCJC #19-CE34-0001-01).

Finally, we warmly thank three anonymous Reviewers for their very relevant and useful comments and corrections on the manuscript, as well as Michael Paul for the English corrections.

Author contribution statement

Supplementary information

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Alekseenko, E., Thouvenin, B., Tronczyński, J., Carlotti, F., Garreau, P., Tixier, C., Baklouti, M., 2018. Modeling of PCB trophic transfer in the Gulf of Lions; 3D coupled model