Christine Dupuy
email: cdupuy@ifremer.fr

André Vaquer

Thong Lam-Höai

Claude Rougier

Nabila Mazouni

Jacques Lautier

Yves Collos

Solange Le Gall

Virginie Pons

Feeding rate of the oyster Crassostrea gigas in a natural planktonic community of the Mediterranean Thau Lagoon

Keywords: Bivalve, oyster, food source, Thau Lagoon, microbial food web, heterotrophic protist, picophytoplankton, trophic link

The Mediterranean Thau Lagoon is an important European oyster farming area. Oyster growth levels are among the highest in France, although chlorophyll a concentration is low. Previous studies have demonstrated that picophytoplankton, nano-microphytoplankton, dinoflagellates and loricate ciliates such as tintinnids are abundant in the Thau Lagoon. Moreover, heterotrophic flagellates and aloricate ciliates have not been investigated. The aim of this study was to assess picophytoplankton, protist and zooplankton abundances in water columns of the Thau Lagoon and to understand the particular structure of the Thau microbial food web, which may explain such a paradoxical oyster growth. In oligotrophic waters in the Thau Lagoon, the picoeukaryote Ostreococcus tauri is the dominant autotrophic picoplankter with a maximum Summer abundance. On 17 August 1998, the picophytoplankton and nanophytoplankton abundances were not as high as expected and we observed the development of large diatoms. At this time, available carbon resources arose from microphytoplankton (84.5 %) and picoplanktonic cells represented only 1.27 % in terms of carbon. The heterotrophic cells were few in abundance and constitued only < 14 % of carbon resources. In order to evaluate the importance of the "protozoan trophic link" for energy transfer from "microbial food web" to large benthic suspension feeders, the oyster Crassostrea gigas was offered a planktonic community as potential prey. In the grazing experiment, all > 5 µm flagellates, microphytoplankton, dinoflagellates, ciliates and large zooplankton were retained by the oyster gills. Only < 5 µm flagellates and picoeukaryotic cells, Ostreococcus tauri, were not very well retained (45 % and 2 %). The high clearance rates of Crassostrea gigas found in this experiment can be explained by a low suspended particulate matter (0.65 mg l -1 ). Oysters adapted their retention mechanism when they lived in oligotrophic waters. These results indicate that, under the given experimental conditions, picophytoplankton did not represent a valuable trophic resource for farmed oysters because (1) Crassostrea can not retain picoparticles and (2) the picoplankton represented a poor available carbon resource to be transferred via a weak heterotrophic protist community. In the oyster pends of the Thau Lagoon during this study, which followed a rainfall event, microphytoplanktonic primary producers, in particular diatoms, could be considered as the main food sources for bivalve suspension feeders.

INTRODUCTION

Oysters obtain energy resources by filtering particles from sea water and their growth depends upon the nutritive value of the retained seston [START_REF] Berg | Temporal and spatial variations in the composition of seston available to the suspension feeder Crassostrea virginica[END_REF], related to the trophic capacity of coastal waters [START_REF] Héral | Shellfish Culture Development and Management -Aquaculture International Seminar[END_REF]. Marine lagoons appear as original entities, compared to adjoining ecosystems. The Mediterranean Thau Lagoon is an important European oyster farming area, with standing stocks of Crassostrea gigas estimated at 40, 000 tons. Oyster growth levels are among the highest in France [START_REF] Goyard | REMORA, résultats nationaux, année[END_REF], although chlorophyll a concentration is low (< 2 µg l -1 , [START_REF] Frisoni | Contribution à l'étude du phytoplancton dans le domaine paralique[END_REF] relative to other coastal ecosystems. The particular structure of the Thau microbial food web may explain such a paradoxical oyster growth. In the oceans, more than 50 % of the primary production is due to unicellular organisms less than 3 µm in size [START_REF] Platt | Photosynthesis of picoplankton in the oligotrophic ocean[END_REF][START_REF] Li | Autotrophic picoplankton in the tropical ocean[END_REF][START_REF] Glover | Contribution of Synechococcus spp. to size-fractioned primary productivity in three water masses in the Northwest Atlantic Ocean[END_REF]), which constitute a nutrient source of particulate and dissolved organic matter for heterotrophic organisms. In the Thau Lagoon, Ostreococcus tauri, a picoeukaryote, whose size is less than 1 µm, is responsable for most of the primary production in the summer [START_REF] Courties | Smallest eukaryotic organism[END_REF]. Furthermore, high levels of dissolved organic matter (DOM) in the Lagoon provide a potential for high bacterial production. However, such small-sized picoparticles are not efficiently retained by gills of bivalves, particularly oysters (< 20 % for picoparticles, [START_REF] Shumway | Particle selection, ingestion, and absorption in filter-feeding bivalves[END_REF][START_REF] Héral | Shellfish Culture Development and Management -Aquaculture International Seminar[END_REF][START_REF] Riisgård | Efficiency of particle retention and filtration rate in 6 species of North east American bivalves[END_REF][START_REF] Barillé | No influence of food quality, but ration-dependent retention efficiencies in the japanese oyster Crassostrea gigas[END_REF].

Protists, which consume bacteria and phytoplankton, are abundant in coastal ecosystems [START_REF] Revelante | Microzooplankton distribution in the Northern Adriatic Sea with emphasis on the relative abundance of ciliated protozoans[END_REF]Sherr et al. 1986 a;[START_REF] Fenchel | Marine plankton food chains[END_REF][START_REF] Leakey | Planktonic ciliates in Southampton water: abundance, biomass, production, and role of pelagic carbon flow[END_REF]. They are preyed upon by numerous organisms of zooplankton, particulary copepods [START_REF] Berk | Ciliates as a food source for marine planktonic copepods[END_REF][START_REF] Pr | Feeding behaviour, prey detection and capture efficiency of the copepod Acartia tonsa feeding on planktonic ciliates[END_REF][START_REF] Gifford | The microzooplankton-mesozooplankton link: consumption of planktonic protozoa by the calanoid copepods Acartia clausi Dana and Neocalanus plumchrus Murukawa[END_REF][START_REF] Hartmann | Predation on ciliates by the suspension-feeding calanoid copepod Acanthodiaptomus denticornis[END_REF] and thus have been suggested as a major trophic link between picoplankton and micro or macroplankton [START_REF] Porter | Ciliate protozoans as links in freshwater planktonic food chains[END_REF][START_REF] Conover | Interrelations between microzooplankton and other plankton organisms[END_REF]Sherr et al. 1986 b;[START_REF] Stoecker | Predation on protozoa: its importance to zooplankton[END_REF]. Likewise, protists may represent a trophic link between picoplankton and filter-feeding bivalves. Some data support this assumption. Tintinnids were observed in oyster's stomachs [START_REF] Paulmier | La nutrition des huîtres en relation avec les sources trophiques[END_REF]). Moreover, filter-feeding benthic molluscs retain protists, as shown by contaminations of bivalves by toxic flagellates [START_REF] Sournia | Le Phytoplancton nuisible des Côtes de France. De la biologie à la prévention[END_REF]. In a mixed cell suspension of phytoplankton and dinoflagellates, six different species of bivalves were able to selectively clear and digest dinoflagellates [START_REF] Shumway | Particle selection, ingestion, and absorption in filter-feeding bivalves[END_REF]. The mussel Geukensia demissa removed microbiota from a salt-marsh water column with different effectiveness according to the cell type [START_REF] Kemp | Effects of filter-feeding by the ribbed mussel Geukensia demissa on the water-columm microbiota of a Spartina alterniflora salt-marsh[END_REF]. Crassostrea gigas easily consume a non-toxic microdinoflagellate [START_REF] Bardouil | Feeding responses of Crassostrea gigas (Thunberg) to inclusion of different proportions of toxic dinoflagellates in their diet[END_REF]. Ingestion and assimilation of bacterial carbon via heterotrophic flagellates were demonstrated in mussels [START_REF] Kreeger | Ingestion and assimilation of carbon from cellulolytic bacteria and heterotrophic flagellates by the mussels Geukensia demissa and Mytilus edulis (Bivalvia, Mollusca)[END_REF]. Similarly, the oyster Cr. gigas retained and ingested a cultured bacterivorous ciliate, Uronema sp. (Le [START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF]. In an Atlantic coastal oyster pond, the field community of hetero/mixotrophic protists was 90 % retained and represented the main energy resource for oysters [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. Heterotrophic protists may thus constitute an alternative or complementary food resource for oysters, allowing indirect recovery of DOM and picoplanktonic production, otherwise not accessible to them.

In the Thau Lagoon, loricate ciliates such as tintinnids are abundant [START_REF] Lam-Hoai | Tintinnids and rotifers in a northern Mediterranean coastal lagoon. Structural diversity and function through biomass estimations[END_REF]; but, heterotrophic flagellates and aloricate ciliates had not yet been studied. The aim of this study is to assess protist and zooplankton abundances in the water column of the Thau Lagoon and investigate their potential use by oysters as trophic resource. Specific questions to be addressed were: 1) do the different microbiota from the planktonic community represent a substantial energy resource in the Thau Lagoon water columns? 2) is oyster filtration effective to account for the removal of a sufficient trophic resource from cleared microbiota? Our experimental design to study the effect of oyster filtration on a field planktonic community was to monitor changes in the abundance of the different microbiota in a 1300 ml tray, on a time scale of 30 minutes.

MATERIALS AND METHODS

The Thau Lagoon (France, 3° 36' E, 43° 24' N) is spread over 75 km 2 , with a mean depth of 5 m and is connected to the sea by 3 narrow channels. Shellfish breeding is carried out in 3 areas off the northernwestern shore (fig. 1).

Oyster collection and acclimation

Oysters were collected in the middle of August 1998 from the NW farming area (station Z, fig. 1) of the Thau Lagoon. Fifty adult Crassostrea gigas (1 year old, shell length about 5 cm) were transported to the laboratory, removed of epibionts and acclimatized overnight to the ambiant field temperature of 25°C, in GF/C (1.2 µm, Whatman) filtered water. Just before the experiment, 10 active filtering oysters were selected and only 3 oysters were used to perform the retention experiments.

Planktonic community collection

The planktonic community devoted to assess protist abundance in the Thau Lagoon and provided as potential food to the experimental oysters, came from station Z (fig. 1), in the middle of the northern farming area, near the NW bank of the Lagoon (5 m in depth). Water samples were collected on 17 August 98, from the subsurface (50 cm), using a sampling 5 l "Niskin" bottle whose central latex cord had been replaced with silicone tubing and held at field temperature (25°C) in an opaque carboy until use for enumeration of protist and for oyster retention experiment.

Pigments determination

Sea water aliquots (from 30 to 50 ml) were filtered under a 10 mm Hg vacuum through a 2.5 cm GF/F filter, which was kept in Corning glass tubes at -20°C until extracted. Triplicate filters were ground in 4 ml of 90 % acetone. Samples were stored in the dark at 4 °C for 24 h; at the end of the extraction period, the tubes were centrifugated for 10 min at 1 200 x g. Concentrations of chlorophyll a and pheopigment were measured using a Perkin-Elmer LS50 b spectrofluorometer and pigment amounts calculated according to [START_REF] Neveux | Spectrofluorometric assay of chlorophylls and phaeopigments using the least squares approximation technique[END_REF].

Phytoplankton: taxonomy, enumeration and gross growth rates

Sea water triplicates were fixed by formaldehyde (4% final concentration) for microscopic determination of nano-microphytoplankton and enumerated in Utermöhl settling chambers. For flow cytometry analysis of picophytoplankton (< 2 µm), samples were formaldehyde-fixed (0.5% final concentration) and stored in liquid nitrogen, according to [START_REF] Troussellier | Flow cytometric analysis of coastal lagoon bacterioplankton and picoplankton: fixation and storage effects[END_REF]. Subsamples (200-500 µl) were analyzed with an ACR-1400-SP flow cytometer (Brucker Spectrospin, Wissembourg, France). Picoplanktonic cell sizes were defined by using fluorescent beads (2 µm diameter PolySciences) and controlled by filtration through Nuclepore polycarbonate membranes (2 µm porosity). Cells > 2 µm group together nano and microphytoplankton up to 50 µm diameter (upper boundary set by the cytometer). Numeration variability was evaluated from triplicate sample measurements. Standard deviations (SD) were between 1 and 7 % for picoplanktonic numerations and between 5 and 41 % for the largest phytoplanktonic cells, a high value related to their low abundance in the sample volume. This variability is similar or slightly inferior to that obtained from 5 samples during each experiment: either 4 to 11 % for picoplankton in control and grazing experiments and 20 to 35 % for cells greater than 2 µm in control experiments. Phytoplankton gross growth rates and mortality by grazing were determinated in situ at station Z and simultaneously over 24 h using the dilution technique of [START_REF] Landry | Estimating the grazing impact of marine micro-zooplankton[END_REF] as modified by [START_REF] Landry | A refined dilution technique for measuring the community grazing impact of microzooplankton with experimental tests in the central equatorial Pacific[END_REF]. No prefiltration was performed, as large diatoms (size greater than 200 µm) were present.

Flow cytometry was used to distinguish size classes. The variability was estimated from duplicate samples by the software of Statview 4, i.e. 3 % for the gross growth rate and 21 % for the grazing mortality.

Ciliates and flagellates: taxonomy and enumeration

Ciliates, dinoflagellates and flagellates were fixed, stained and enumerated according to methods modified from [START_REF] Haas | Improved epifluorescence microscopy for observing planktonic microorganisms[END_REF], [START_REF] Caron | Technique for enumeration of heterotrophic and phototrophic nanoplankton, using epifluorescence microscopy, and comparison with other procedures[END_REF] and [START_REF] Sherr | Staining of heterotrophic protists for visualisation via epifluorescence microscopy[END_REF] modified by [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF].

Samples were made in triplicate. Sizes of all cells (length and width) were measured through a calibrated ocular micrometer. The mean cell volume of each taxon was calculated by equating the shape to standard geometric configurations. The cell volume was converted into carbon units, using a theoretical carbon/volume ratio of 0.17 pg Carbon (C) µm -3 [START_REF] Putt | An experimentally determined carbon:volume ratio for marine "oligotrichous" ciliates from estuarine and coastal waters[END_REF], corrected for glutaraldehyde fixative [START_REF] Leakey | A comparison of fixatives for the estimation of abundance and biovolume of marine planktonic ciliate populations[END_REF]. For Tintinnina protists, besides enumerations according to Utermöhl, counts of individuals retained by a 40 µm mesh-size collector were proceeded by image analysis [START_REF] Lam-Hoai | Tintinnids and rotifers in a northern Mediterranean coastal lagoon. Structural diversity and function through biomass estimations[END_REF].

Zooplankton

The large zooplankton was sampled from triplicate mixed vertical tows from the 2 m deep water column were performed, using a 40 µm mesh-sized net (input volume: 356 l). Zooplankter counts, size measures and biovolume estimations were made using an image analysis technique [START_REF] Lam-Hoai | Tintinnids and rotifers in a northern Mediterranean coastal lagoon. Structural diversity and function through biomass estimations[END_REF]. Cell volume was converted into carbon units as reported above. Zooplankton abundances at station Z were evaluated by means of two sampling methods: mixed vertical tows from the 2 m deep water column with a 40 µm mesh-sized net and sampling with 5 l "Niskin" bottle filtered through the same mesh size. Similar results were obtained: 22 identical taxa out of 30 were identified in both sample types.

Experimental protocol for the study of protist retention by oysters

The possible influence of oyster filtration upon the natural planktonic community was studied by comparing the changes in plankton abundances in triplicate suspensions with or without filtering oysters during 30 min at ambient temperature (25°C). At the start of the feeding period, 3 replicate incubations , each containing one oyster (mean dry tissue weight 1.86 ± 0.15 g) were assessed: each oyster was transferred into an individual 1500 ml pyrex round tray containing 1300 ml of natural unfiltered Lagoon water from station Z, gently homogeneized with a magnetic rod to prevent sedimentation. Two experimental treatments were performed, each in triplicate: suspensions were (1) allowed to evolve as controls, or (2) delivered to actively filtering oysters. Water samples were collected every 5 min to assess picophytoplankton, nano-microphytoplankton, flagellate, dinoflagellate and ciliate abundances. Diatoms were enumerated at t0 and t15 min and large zooplankton abundances at t0 and t30 min. It is to be noticed that at the very low natural food concentration used in this study (seston load = 0.65 mg l -1 ), there was no visible production of pseufaeces. Dry tissue weight of each oyster was recorded at the end of the experiment to express the carbon resource from retained particles per g of oyster dry tissue.

Calculation of specific retention percentages, clearance rates and retained resource

To investigate the occurence of a differential grazing by the oyster among the various taxons of picophytoplankton, nano-microphytoplankton, flagellate and ciliate protists and large zooplankton, the percentage of cells retained between 0 and 15 min was calculated. Clearance rates were estimated for each planktonic type, as previously reported [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]) between 0 and 15 min. During the first five minutes of the experiment, individual variations in the settling of a regular oyster filtration prevented any reliable study of the protist abundance evolution in the triplicate suspensions: therefore, we selected the subsequent sampling time (15 min) as the most appropriate "standard" time in our clearance experiment. Defined as the theoretical water volume entirely cleared from particles per unit time (l h -1 ) (Bayne & Widdows 1978), the clearance rate was calculated according to [START_REF] Coughlan | The estimation of filtering rate from the clearance of suspensions[END_REF], for the main taxonomic groups from planktonic suspension. Taking into account that weight specific filtration decreases with increasing body size, standardized clearance rates were calculated according to [START_REF] Riisgård | Efficiency of particle retention and filtration rate in 6 species of North east American bivalves[END_REF]. Specific contribution of the various planktonic taxa to the particulate resource retained by oysters was expressed as particulate organic carbon (POC) retained per unit time and per g of oyster dry tissue (µg C h -1 g -1 ). It was calculated by multiplying the field carbon resource of each taxon (µg C l -1 ) by the corresponding standardized clearance rate (l h -1 g -1 ). Initial picophytoplankton, nanomicrophytoplankton, diatom, flagellate, dinoflagellate, ciliate and large zooplankton abundances from the triplicate experiments with or without a filtering oyster were compared using a t-Student test (data were previously tested for normality using the Kolmogorov-Smirnov test). Changes in these microbial abundances were followed in triplicate controls during the 30 min experiment by comparing with regression test aliquots sampled at 5 time points (0, 5, 10, 15 and 30 min).

RESULTS

Planktonic resource available in the Thau Lagoon in August 1998

Taxonomic composition and standing stocks of field microbiota were evaluated in order to estimate the relative contribution of micro-organisms to the particulate organic carbon (POC) resource.

Picophytoplankton

Picophytoplankton was the most abundant autotrophic plankter at the time of our investigation in the oyster breeding area. It was mainly constituted of picocells exhibiting the same flow cytometric characteristics as the eukaryotic Ostreococcus tauri, which is the dominant autotrophic taxon in this < 2 µm size-class of the Thau Lagoon community during the summer period [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF]. Mean abundance of this eukaryotic picophytoplankter was 2.5  0.3 x 10 7 cells l -1 , which represents more than 97 % of the total autotrophic community density. However, these tiny cells accounted for a very low biovolume (0.52 µm 3 ) and a subsequent low carbon cell content (0.089 pg C cell -1 ).

Diatoms

The microphytoplankton was mostly constituted in August 1998 by large diatoms, such as Rhizosolenia setigera, Lioloma pacificum, Nitzschia longissima and colonial diatoms, such as Pseudo-Nitzschia seriata, Thalasionema nitzschioides, Rhizosolenia striata, Skeletonema costatum, Leptocylindricus sp. and several species of Chaetoceros. The average abundance of these algae was 4  0.8 x 10 5 cells l -1 , mostly represented by Skeletonema costatum and Chaetoceros sp. Other taxons (Hemiaulus and Pleurosigma) were scarce. Diatom sizes (table 1) ranged from 15 µm for Skeletonema costatum up to a maximum value of 541 µm for Lioloma pacificum. Biovolumes were high and carbon cell contents fluctuated between  25 pg C cell -1 for Leptocylindrus and Nitszchia longissima and  5 000 pg C cell -1 for a large diatom, Guinardia striata.

Flagellates, dinoflagellates

Flagellate abundance in the oyster farming area was 2.9 ± 1.2 x 10 5 cells l -1 , among which 20 % were heterotrophic. Cell lengths ranged from 3 to 19 µm. Dinoflagellate abundance was 1.1 ± 0.3 x 10 4 cells l - 1 , with cell sizes ranging from 35.5 µm for an unidentified dinoflagellate up to 146 µm for Ceratium sp.

(table 2).

Ciliates

Aloricate ciliates (total abundance 7.2 ± 2.1 x 10 3 cells l -1

) were dominated by the subclass Choreotrichia, mainly represented by the order Haptorida (5.1 ± 2.2 cells l -1 ) and dominant type was Mesodinium sp. Common taxa from the order Oligotrichida (Strombidium sp., 1.8 ± 1 x 10 3 cells l -1 ) were also observed. Ciliate sizes ranged from a minimal 12.5 µm length for a Mesodinium sp., up to a maximum value of 209 µm for Favella sp. (table 3). Loricate ciliates were mostly represented by Tintinnina with abundance of 137 ± 66 cells l -1 among which 13 large cells l -1 were retained by the 40 µm mesh of the zooplancton collector. Tintinnina biovolumes, as well as carbon cell contents, were much higher than for other assemblages (table 3).

Large zooplankton

In the farming area (station Z), 68 zooplanktonic organisms from 27 identified taxa could be grouped into 5 major groups, arranged in a decreasing abundance order (table 4). The total biovolume, estimated from image analysis was 47 x 10 6 µm 3 l -1 . Meroplankton, such as Polychaeta larvae, Cirripeda nauplii and nectobenthic taxa were almost absent.

Potential planktonic carbon resource

In order to estimate the potential planktonic resource available for oysters in the farming area, the particulate organic carbon (POC) relative to each taxonomic group was estimated. By multiplying for each microbiota the specific carbon cell content by the field abundance, we estimated the relative contribution of each taxon to the total carbon resource of the planktonic community (table 5).

Microplanktonic diatoms, which have large biovolumes, constituted the first trophic resource (161.5 µg C l -1 ), in spite of their low abundance in the Thau Lagoon on 17 August 1998. Dinoflagellates, represented the second carbon resource (16.6 µg C l -1 ). On the contrary, picoeukaryotic cells, with a very small biovolume (0.52 µm 3 ), accounted for a minimal carbon biomass (2.4 µg C l -1 ), despite their high field density. Ciliates, flagellates and large zooplankton were not important in terms of biomass in Thau waters at the time of our experiment.

Gross growth rates and grazing mortality of autotrophic plankton was estimated in situ using incubations after dilution manipulations: the subsequent reduction of grazing pressure thereby uncoupling growth and mortality. The small nanophytoplankton exhibited the highest gross growth rate: 3.27 day -1 (table 6). The gross growth rate of other phytoplankters averaged 2.6 day -1 . The grazing mortality of picoplankton in a size range between 0.7 and 0.9 µm was the highest, with 3.32 day -1 .

Thus, the whole daily picoplanktonic production was consumed by heterotrophic planktonic grazers (G/Ke = 1.29, table 6).

Retention by oysters of trophic resource cleared from the planktonic microbiota of Thau

Lagoon in August 1998

Grazing experiments

The planktonic suspension used for oyster grazing experiments, which was collected in subsurface at station Z, exhibited a complex taxonomic composition: 2.5 x 10 7 picophytoplankters l -1 , 4 x 10 5 diatoms l -1 from nano-microplankton, 2.2 x 10 5 ± 2 x 10 4 flagellates l -1 , 1.2 x 10 4 ± 3.2 x 10 3 dinoflagellates l -1 , 7.4 x 10 3 ± 2 7 x 10 3 ciliates l -1 , 68 large zooplankters l -1 .

Since all experimental suspensions originated from the same water sample, initial microbial abundances showed no significant difference between suspensions without (control) or with filtering oysters (t Student test, n= 6, p >> 0.05). In the same way, the chlorophyll a concentration was similar at to in control suspensions (1.86 µg l -1 ) and oyster treatments (1.82 µg l -1 ), and pheophytin initial concentrations were identical in control and oyster trays.

In control suspensions gently homogeneized, chlorophyll a concentrations remained constant

(1.82 µg l -1 ) until the end of the experiment; similarly, pheophytin concentrations were steady in controls. Abundances of picoeukaryotes, >2 µm phytoplankton, flagellates, dinoflagellates and ciliates remained virtually constant for 30 min (fig. 2). Results of regression tests were r 2 = 0.204, p >> 0.05 for picoeukaryotes, r 2 = 0.18, p >> 0.05 for flagellates, r 2 = 0.12, p >> 0.05 for dinoflagellates and r 2 = 0.0019, p >> 0.05 for ciliates.

On the contrary, in suspensions with actively filtrating oysters, chlorophyll a concentrations dropped to 0.34 µg l -1 at the end of the grazing experiment, i.e. only 18.7 % of the initial concentration. The level of pheophytin increased, probably in relation with pigment damage, resulting from oyster grazing. The abundance of picoeukaryotes was never affected by the presence of oysters during our experiments (fig. 2a). Changes in the abundance of nano/microplanktonic particles through time are shown in fig. 2 b-e.

Phytoplanktonic cells > 2 µm decreased in triplicate oyster trays, in various ratios according to taxa (mean removal of 80 to 98 %): most of the largest protists in a size between 12.5 and 540 µm, diatoms, ciliates, dinoflagellates and large flagellates were highly retained.

Yield of cells cleared within 15 min in the oyster treatment suspensions was 92 % for microphytoplankton, 94% for dinoflagellates, 93 % for ciliates (fig. 3). Only 44 % flagellates were retained. At the end of the experiment (30 min), approximately 90 % of the large zooplankton, all dinoflagellates and ciliates and 55 % of the flagellates had been cleared by the bivalves. Flagellates in a size lower than 5 µm were poorly (45 %) retained (fig. 4).

Clearance rates and carbon resource cleared from planktonic particles

Particle abundances at 0 and 15 min were also used to estimate oyster clearance rates for each microbiota. The calculation was based on the assumption that abundance decreased exponentially through time in an enclosed suspension volume. Specific clearance rates, ie water volume entirely cleared from specific type of particles per unit time and per oyster dry tissue weight (l h -1 g -1 ), were compared among particle types (table 7). Maximum clearance rates were typical of the most efficiently retained microbiota (16.7 l h -1 g -1 for > 5 µm flagellates, 14.8 l h -1 g -1 for dinoflagellates). Conversely, clearance rates were low for poorly retained particles (6.8 l h -1 g -1 for < 5 µm flagellates) and close to zero for the picoeukaryote, showing that picoparticles are not retained by oysters. The mean clearance rate for all microbiota in a size > 5 µm averaged 11.8 l h -1 g -1 .

The carbon removed as living planktonic particles by oysters was evaluated from the calculated clearance rate and initial biomasses of microbiota in experimental suspensions. Carbon removal was dependent on taxon (table 7). The main resource arose from diatoms (81 %) and dinoflagellates (15 %).

Ciliates and flagellates poorly contributed to oyster resources and picoeukaryotic cell contribution was only 0.03 % of the trophic resource in this experiment. The oyster Crassostrea gigas retained on average 1 600 µg C h -1 g -1 from plankton. However, large zooplankton was not taken into account, as the lack of intermediary zooplankton sampling between 0 and 30 min in our protocol precludes a rigorous estimation of the clearance rate.

DISCUSSION

The aim of this study was to assess a possible contribution of picoeukaryotes in food resource intake of the oyster Crassostrea gigas. In Thau Lagoon, the picoeukaryote Ostreococcus tauri is the dominant autotrophic picoplankter [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF] with a maximum summer abundance of 2 x 10 8 cells l -1 (Chretiénnot-Dinet et al 1995). As not directly retained by oyster gills, picoeukaryotes might constitute an indirect food resource, transfered towards bivalves via heterotrophic protists. Such a trophic link between picoplankton and benthic suspension feeders was already evidenced in oysters (Le [START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF][START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. In order to test this assumption, we have evaluated the planktonic resource in Thau lagoon and investigated its utilization by oysters.

Trophic resource available in the Northern farming area of Thau Lagoon in August 1998

Inside the NW oyster farming area of Thau Lagoon (station Z), picophytoplankton abundance was 2.5 x 10 7 cells l -1 on 17 August 1998. Most of the autotrophic picocells exhibited the same flow cytometric characteristics as the picoeukaryotic Prasinophyceae, Ostreococcus tauri [START_REF] Courties | Smallest eukaryotic organism[END_REF]. Our current abundances are in the range previously observed in Thau Lagoon, ie 3.4 x 10 7 cells l -1 [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF], but lower than the maximum 10 8 cells l -1 densities at the same station in August 1992. Such high levels of picoeukaryotic abundances have been reported elsewhere in Mediterranean Sea (Magazzu & Decembrini 1995).

Abundance of the nano-microphytoplankton (ie. cells > 2 µm: diatoms, dinoflagellates and phytoflagellates) inside the oyster farming area was 6.5 x 10 5 cells l -1 , a value 7.5 times lower than mean abundance (5 x 10 6 cells l -1 ) previously observed in Thau Lagoon, and 3 times lower than previous summer data (2 x 10 6 cells l -1 ) at the same station [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF]. Diatoms were either isolated or chain associated, in a size range between 15 µm and 541 µm, constituting 62 % of the nanomicrophytoplankters and Skeletonema costatum (Thalassiosiraceae) was the dominant taxon.

Phytoflagellates represented 36.3 % and dinoflagellates only 1.7 % of the nano-microphytoplanktonic community. Among, them, 80 % were auto or mixotrophic and 20 % heterotrophic. Their sizes were between 3 and 19 µm and their total abundance (2.94 x 10 5 ± 1.2 x 10 5 cells l -1 ) was low compared to data reported in other environments: Saint-Lawrence estuary: 1.9 x 10 6 to 6 x 10 6 cells l -1 [START_REF] Lovejoy | Microbial gradients in a turbid estuary: application of a new method for protozoan community analysis[END_REF]); Limfjorden marine shallow-water: 2 x 10 6 cells l -1 (Andersen & Sorensen 1986); Parker Estuary, Massachusetts: > 10 x 10 6 heterotrophic flagellates l -1 [START_REF] Wright | Dynamics of planktonic bacteria and heterotrophic microflagellates in the Parker Estuary, northern Massachusetts[END_REF].

As far as we know, the naked ciliate community has not yet been studied in Thau Lagoon: therefore, we compared our data to results from distant areas. Our estimate of planktonic ciliate abundance (naked ciliates and Tintinninds) in the oyster farming area was 7 194 x 10 4 ± 2 10 4 ciliates l -1 . Ciliates were dominated by the order Choreotrichida, with the prevailing Haptorida taxon, Mesodinium spp.,

(5 089 ± 2 211 cells l -1 ). Oligotrichida, with the dominant taxon, Strombidium spp, (1 765 ± 1 054 cells l -1 ) were in low abundance, compared to high summer densities (9 x 10 4 cells l -1 ) observed in the Mediterranean Sea [START_REF] Rassoulzadegan | Evolution annuelle des ciliés pélagiques en Méditerrannée nord-occidentale. Ciliés oligotriches "non tintinnides" (Oligotrichina)[END_REF]. Tintinnid abundance was 209 ± 87 cells l -1 , a value slightly higher than reported data (75 cells l -1 ) from Thau Lagoon in 1994 [START_REF] Lam-Hoai | Tintinnids and rotifers in a northern Mediterranean coastal lagoon. Structural diversity and function through biomass estimations[END_REF]. In Mediterranean waters (Villefranche-sur-mer Bay), ciliates may be much more abundant: 10 4 cells l -1 [START_REF] Rassoulzadegan | Répartition des ciliés pélagiques dans les eaux de Villefranche-surmer. Remarques sur la dispersion du microzooplancton en mer et à l'intérieur des échantillons dénombrés par la méthode d'Utermöhl[END_REF].

Large zooplankton abundance in the oyster farming area was 68 000 cells m -3 , a value 20 times higher than mean value in 1994 [START_REF] Lam-Hoai | Tintinnids and rotifers in a northern Mediterranean coastal lagoon. Structural diversity and function through biomass estimations[END_REF]).

The amounts of potential carbon resources available in the field were estimated for every planktonic microbiota (table 5). Microphytoplankton was the most important resource on 17 August 1998 (161.5 µg C l -1 , ie 84.6% of the total organic carbon). Dinoflagellates (16.6 µg C l -1 ) were the second C resource. Flagellates ( 3 µg C l -1 ), ciliates ( 3 µg C l -1 ) and zooplankton ( 5 µg C l -1 ), were not responsible for trophic richness of Thau Lagoon waters and picophytoplanktonic cells represented only 1.3 % of the carbon resource. The ciliate resource in Thau was much lower than elsewhere: ciliates can represent up to 52 µg C l -1 (annual mean) in the Saint-Lawrence Estuary [START_REF] Sime-Ngando | Significance of planktonic ciliated protozoa in the Lower St. Lawrence Estuary: comparison with bacterial, phytoplankton, and particulate organic carbon[END_REF] or 63.5 µg C l -1 (in June 97) in French Atlantic coastal ponds [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF].

Our estimation of microbiota in Thau Lagoon and the wide range of previously reported data show the natural variability of picophytoplankton, protist and zooplankton abundances in the field. In terms of numerical abundance, the phytoplanktonic community was overnumbered by picoeukarotes, but as regards C resource, microphytoplankton dominated in Thau Lagoon waters. In the last years, the highest picoeukaryote and nanophytoplankton densities were usually found in summer in Thau Lagoon, because of favourable environmental factors [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF]. Surprisingly, in August 1998 the picophytoplankton and nanophytoplankton abundances were not as high as expected, and we observed the development of very large diatoms; probably related to terrestrial nutrient inputs, resulting from several rainy days before sampling. However, the retrospective estimation of picophytoplankton C resource in past periods of maximal abundance: (10 8 cells l -1 in 1992; [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF] only results in a weak resource of 16 µg C l -1 . Even during picoeukaryotic bloom events, the main trophic resource would proceed from microphytoplankton, particularly diatoms.

Utilization by oysters of the planktonic trophic resource of Thau Lagoon in August 1998

Grazing experiments were carried out using a natural planktonic population from the Northern Thau Lagoon oyster farming area. Chlorophyll a, pheopigment concentrations and microbial abundances remained stationary in controls. Conversely, chlorophyll a decreased in the oyster grazing experiments (from 1.82 to 0.34 µg l -1 ), due to retention of phytoplanktonic cells by oysters; in the same time, pheopigment concentrations increased, perhaps in relation to a damage of pigments resulting from oyster grazing. Actually, diatoms (even large species such as Rhizosolenia setigera, Lioloma pacificum), dinoflagellates and ciliates were highly retained by oysters (respectively 92 %, 96 %, 93 %) during grazing experiments. Only 45 % of flagellates (size from 3 µm to 19 µm) were cleared. Their retention depended on their size: the smallest flagellates (< 5 µm), which were also the most abundant, were poorly retained (~ 48 %), compared to flagellates in a size > 5 µm (~ 80 % retained). The maintenance of picophytoplanktonic abundances, either in controls or in oyster grazing experiments, evidenced that those tiny particles (~1 µm size), were not retained by oysters gills. In our experiment, oysters retained efficiently particles larger than 5 µm diameter. Our data agree with previous reports of the known size spectrum of particle retention by oyster filtration: oyster retained less than 10 % of  1 µm particles, more than 50 % of > 3 µm particles [START_REF] Deslous-Paoli | Evolution saisonnière de la filtration de bivalves intertidaux dans des conditions naturelles[END_REF]) and 100 % for 7 µm particles in Crassostrea gigas [START_REF] Héral | Shellfish Culture Development and Management -Aquaculture International Seminar[END_REF] and Crassostrea virginica [START_REF] Riisgård | Efficiency of particle retention and filtration rate in 6 species of North east American bivalves[END_REF]; when sestonic load was low, 4 µm particles were 100 % retained [START_REF] Barillé | No influence of food quality, but ration-dependent retention efficiencies in the japanese oyster Crassostrea gigas[END_REF]).

The observation of ciliate retention agrees with the previously demonstrated retention by Crassostrea gigas of a cultured ciliate (Le [START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF]) and of a natural protist community from French Atlantic coastal ponds, with retention of 94 % for ciliates and 86 % for flagellates [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. It also corroborates the observations by [START_REF] Paulmier | La nutrition des huîtres en relation avec les sources trophiques[END_REF], who reported Tintinnids to be abundant in the stomachs of wild oysters from the Atlantic coast.

The mean clearance rate for all microbiota (except picoeukaryote) averaged 11.8 l h -1 g -1 , a value similar to the estimation calculated according to [START_REF] Riisgård | Efficiency of particle retention and filtration rate in 6 species of North east American bivalves[END_REF] for an oyster, whose weight was 1.86 g (10.7 l h -1 g -1 ). Clearance rates of oysters for flagellate, dinoflagellate and ciliate protists were between 12 and 16 liters h -1 g -1 and for diatoms 8 liters h

-1 g -1
. These values were higher than previously reported clearance rates in water with high sestonic load: 5.7 l h -1 g -1 for Phaeodactylum at high concentration (10 7 cells l -1 , ie 16 µg C l -1 ) (Fiala-Medioni et al. 1983), 5.5 l h -1 g -1 for estuary plankton of Marennes-Oléron (Deslous-Paoli et al. 1987), 4.8 l h -1 g -1 [START_REF] Bougrier | Preingestive selection of different microalgal mixtures in Crassostrea gigas and Mytilus edulis, analysed by flow cytometry[END_REF]) and 7.8 l h -1 g -1 [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]).

Conversely, with low sestonic loaded waters, clearance rate was 12 l h -1 g -1 for Phaeodactylum at 10 6 cells l -1 [START_REF] Fiala-Medioni | Relations trophiques entre huître et milieu; influence de la concentration et de la taille des particules. Bases biologiques de l'aquaculture[END_REF]) and 16 l h -1 g -1 for Isochrysis galbana at 1 x 10 5 cells l -1 , ie 0.9 mg C l -1 [START_REF] Walne | The influence of current speed, body size and water temperature on the filtration rate of five species of bivalves[END_REF]. These last values are in the range of our current data. Oysters may adjust their clearance rate in oligotrophic conditions. [START_REF] Fiala-Medioni | Relations trophiques entre huître et milieu; influence de la concentration et de la taille des particules. Bases biologiques de l'aquaculture[END_REF] and [START_REF] Deslous-Paoli | Evolution saisonnière de la filtration de bivalves intertidaux dans des conditions naturelles[END_REF] noticed that when seston load is low, bivalves increased their effort of filtration to satisfy their energy requirement.

Our current investigation supports the importance of phytoplankton in oyster nutrition [START_REF] Héral | Shellfish Culture Development and Management -Aquaculture International Seminar[END_REF][START_REF] Pastoureaud | Particle selection in the oyster Crassostrea gigas (Thunbert) studied by pigment HPLC analysis under natural food conditions[END_REF]. Evaluation of the retained resource from each taxonomic compartment during grazing experiments was calculated. The highest values of retained resource were from the microphytoplankton (1300 µg C h -1 g -1 ) at a concentration of 4 x 10 5 cells l -1 . [START_REF] Fiala-Medioni | Relations trophiques entre huître et milieu; influence de la concentration et de la taille des particules. Bases biologiques de l'aquaculture[END_REF] estimated that oyster filtering Phaedactylum tricornatum retained about 90 µg C h -1 g -1 for a phytoplankton concentration of 8 x 10 6 cells l -1 . This difference may result from the greater size and carbon content per cell of diatoms present in Thau Lagoon in August 1998 (Rhizosolenia setigera contains 4 310 pg C cell -1 ) compared to Phaedactylum tricornatum (1.6 pg C cell -1 ). Dinoflagellate represented a resource of 245 µg C h -1 g -1 . For ciliates, the retained resource was 39 µg C h -1 g -1 , a value much lower than the 126 µg C ciliates h -1 g -1 retained by oysters in Atlantic oyster ponds, where ciliates were abundant (2.3 x 10 4 cells l -1 ) [START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. From the sum of all planktonic partitions, we can estimate that oysters retained 1 600 µg C h -1 g -1 . [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF] previously observed over one year that picophytoplankton abundances were similar inside and outside the oyster farming area.

Conversely, they noticed that densities of nano-microphytoplankton, flagellates and zooplanktonic organisms were significantly higher outside the oyster farming area.

Most studies that have examined the nutritional importance of protist as a "trophic link" have focused on pelagic consumers, such as zooplankton [START_REF] Berk | Ciliates as a food source for marine planktonic copepods[END_REF][START_REF] Porter | Ciliate protozoans as links in freshwater planktonic food chains[END_REF]Sherr et al. 1986b, Jonsson & Tiselius 1990;[START_REF] Gifford | The microzooplankton-mesozooplankton link: consumption of planktonic protozoa by the calanoid copepods Acartia clausi Dana and Neocalanus plumchrus Murukawa[END_REF]; similar experiments on benthic filter-feeding consumers are scarce [START_REF] Kreeger | Ingestion and assimilation of carbon from cellulolytic bacteria and heterotrophic flagellates by the mussels Geukensia demissa and Mytilus edulis (Bivalvia, Mollusca)[END_REF][START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF][START_REF] Dupuy | Retention of ciliates and flagellates by the oyster Crassostrea gigas in French Atlantic coastal ponds: protists as a trophic link between bacterioplankton and benthic suspension-feeders[END_REF]. Trophic coupling between pelagic protists and benthic suspension-feeders is poorly documented in aquatic food models (e.g. see [START_REF] Legendre | Microbial food webs and the export of biogenic carbon in oceans[END_REF], although such a relationship could be of primary importance for C and N transfer, from the microbial food web to filter-feeder bivalves in benthos (Le [START_REF] Gall | Ingestion of a bacterivorous ciliate by the oyster Crassostrea gigas: protozoa as a trophic link between picoplankton and benthic suspension-feeders[END_REF]. In Thau Lagoon, the picoeukaryote community (average abundance 3.4 x 10 7 cells l -1 , [START_REF] Vaquer | Standing stock and dynamics of picophytoplankton in the Thau lagoon (northwest Mediterranean coast[END_REF], constituted the most abundant primary food resource for heterotrophic/mixotrophic flagellates, dinoflagellates and ciliates. However, because of the small biovolume of their cells, picoeukaryotes only represented 3 µg C l -1 . Even during summer bloom events (2 x 10 8 cells l -1 , Chretiénnot-Dinet et al.

1995), the picoeukaroyte would only supply the trophic system with a weak available biomass of 16 µg C l -1 . In addition to the low contribution of picophytoplanktonic carbon in Thau lagoon waters in August 1998, picoeukaryotic cells exhibit growth rates of 2.58 to 2.64 day -1 (table 6), which are lower than for nanoplankton (2.65 to 3.27 day -1 ). These results support the recent speculation of [START_REF] Raven | Why are there no picoplanktonic evolvers with volumes less than 10 -19 m 3 ?[END_REF], that for small cells (< 0.9 µm), there is no increase in maximum specific growth rate with decreasing cell size. Bacteria which represent about 20 µg C l -1 in Thau lagoon (Trousselier M., pers. com.) were also a potential primary food for heterotrophic/mixotrophic flagellates, dinoflagellates and ciliates. Though ciliates exhibit high gross growth efficiencies of 40 % (Johnson et al. 1982;[START_REF] Ohman | Growth kinetics of the omnivorous oligotrich ciliate Strombidium sp[END_REF], relatively low amounts of picoplanktonic C would be recovered by oysters via the protist trophic link.

In conclusion, Thau Lagoon picoplankton did not represent an important trophic resource for farmed oysters because 1) Crassostrea cannot retain picoparticles and 2) the picoplankton only represented a limited available carbon resource (in terms of biomass) to be transferred via a weak protist community.

However this assertion must be moderated because 1) the experiment was only made one time 2) the productivity of these cells is not still well known, except very punctually (for example in 08/98).

Measures of very raised growth rate of O. tauri (until 4.23 div d -1 ) were obtained in experimental conditions [START_REF] Courties | Phylotgnetic analysis and genome size of Ostreococcus tauri (Chlorophyta, prasinophyceae)[END_REF]. They would suggest an important potential productivity of picophytoplancton. In oyster pens of the Thau Lagoon during August 1998, microphytoplanktonic primary producers, in particular diatoms, could be considered as the main food source for oysters.

However, as environmental conditions were particular at the time of our experiments (influence of watershed), additional investigations are needed to further identify the energy fluxes towards farmed oysters in Thau Lagoon. Resource is expressed as POC (µg C l -1 ). Standardized clearance rate is the theoritical water volume entirely cleared from particles per unit time and standardized per oyster dry tissue weight (l h -1 g -1 ) [START_REF] Riisgård | Efficiency of particle retention and filtration rate in 6 species of North east American bivalves[END_REF]. Resource and Standardized clearance rate were multiplyed to estimate the POC resource retained by oyster (µg C h -1 g -1 ).

Figure 1 :Figure 2 :

 12 Figure 1: Location of the sampling station in Thau Lagoon: station is inside the northern A oyster farming area (from Thong Lam Hoai).

Figure 3 :

 3 Figure3: Percentage of cells retained by filtering oysters for ciliates, dinoflagellates, flagellates and picoeukaryotic cells. Abundance data (mean  SD, n = 3) were collected from triplicate incubations performed in 1300 ml lagoon water suspensions with a filtering oyster.

Figure 4 :

 4 Figure 4: Removal by the oyster Crassostrea gigas, of flagellates in a size > 5µm or < 5µm.Flagellate abundance data (mean  SD, n = 3) were collected from triplicate incubations performed in 1300 ml lagoon water suspensions with a filtering oyster.

Table 1 :

 1 Taxonomic composition, size, biovolume and carbon content of picoeukaryotic cells and microplanktonic diatoms at Z farming area on 17 August 1998.

Table 2 :

 2 Taxonomic composition, size, biovolume and carbon content of dinoflagellates at Z farming area on 17 August 1998.

						Cell length	Biovolume	Carbon per cell
	Order	Family	Genus	Species	(µm)	(x10 2 µm 3 )	(pg C cell -1 )
	Mamiellales	Prasinophyceae	Ostreococcus	tauri	1		0.005	0.089
	Centrales
		Chaetoceraceae
			Chaetoceros	spp.	18		27	452
		Rhizosoleniaceae
			Guinardia	striata fa.	111		290	4926
			Rhizosolenia	setigera fa.	460		253	4308
		Melosiraceae
			Leptocylindricus	sp.	24		1	25
		Thalassiosiraceae
			Skeletonema	costatum	15		3	45
	Pennales
			Lioloma	pacificum	541		21	363
		Nitzschiaceae
			Nitszchia	longissima	95		1	25
			Pseudonitszchia	sp.	415		21	356
		Fragilariaceae
			Thalassionema	nitzschioides	47		4	73
						Cell length	Biovolume	Carbon per cell
		Order	Family	Taxon	Species	(µm)	(x 10 3 µm 3 )	(pg C cell -1 )
	Dinophyceae
		Peridiniales
			Ceratiaceae
				Ceratium		sp.	146	1665	283025
			Peridiniaceae
				Protoperidinium	sp.	36	12	1982
				Scripsiella		sp.	32	5	766
		Gymnodiniales
			Gymnodiniaceae
				Cochlodinium	sp.	52	8	1402
				Gymnodinium	sp.	48	24	4114
				Gyrodinium		sp.	50	33	5628
		Dinophysiales
			Dinophysiaceae
				Dinophysis		sp.	40	6	968
		Ebriales
				Ebria	tripartita	45	12	2115
		Prorocentrales
			Prorocentraceae
				Prorocentrum	sp.	41	10	1675
		Dinoflagellates unidentified					35	26	4434
	Euglenophyceae
		Eutreptiales
			Eutreptiaceae	unidentified			30	1	234

Table 3 :

 3 Taxonomic composition, size, biovolume and carbon content of loricate and aloricate ciliates at Z farming area on 17 August 1998.

	Order	Suborder	Family	Taxon	Species	Cell length (µm)	Biovolume (x 10 3 µm 3 )	Carbon per cell (pg C cell -1 )
	Choreotrichida Tintinnina
			Codonellidae
				Tintinnopsis	sp.	37	13	2225
				Tintinnopsis corniger	130	71
			Tintinnidae
				Eutintinnus	fraknoii	157	116	19777
				Eutintinnus	fraknoii	128	56
			Ptychocyclidae
				Favella	serrata	179	219	37165
				Favella	serrata	209	319
		Strobilidiina
			Strobilidiidae
				Lohmaniella	sp.	12	1	174
	Oligotrichida
			Strombidiidae
				Strombidium	sp.	47	20	3479
	Haptorida
			Mesodiniidae
				Askenasia	sp.	15	2	348
				Mesodinium	sp.	27	3	536
				Mesodinium	pulex	12
			Didinnidae
				Didinium	sp.	45	36	6083
	Scuticociliatida
			unidentified			31	6	1080

Table 4 :

 4 Sizes, biovolumes and corresponding resource of the main zooplanktonic taxa > 40 µm in the farming area on 17 August 98. able 5: Contribution of various taxonomic groups to the planktonic carbon resource available for oysters at the Thau lagoon Z farming area from triplicate samples on 17 August 98.

Table 6 :

 6 Phytoplankton gross growth rate and microzooplankton grazing rates. Data were collected from duplicate experiments.

				Biovolume	Carbon per animal	Field C resource
	Taxons	Biovolume (%) Length (µm)	(x 10 6 µm 3 )	(ng C cell -1 )	(µg C l -1 )
	mostly Bivalvia veligers	21	138	0.608	85		3
	Tintinnina: Favella serrata,
	Eutintinnus fraknoi, Tintinnopsis
	corniger, Helicostomella subulata	7.7	195	0.271	38		1
	Copepoda nauplii	9.3	172	0.346	48		0.5
	Appendicularia: Oikopleura dioica	31.2	518	0.83	116		1.4
	Rotatoria: Synchaeta vorax,
	Trichocerca marina	6.7	191	0.454	64		0.3
	Total of main taxa	100	247	0.64			6.2
	Abundance Carbon ressource Contribution to total POC
		(x 10 3 l -1 )	(µg C l -1 )		%
	Picophytoplankton	25 000	2.4		1.3
	Microphytoplankton	400	161.5		84.4
	< 5 µm flagellates	207	0.6		0.3
	> 5 µm flagellates	87	2.3		1.2
	Dinoflagellates	11	16.6		8.7
	Ciliates	7.2	3		1.6
	Zooplankton	0.07	5		2.6
	Sum	25 712	191		100
		Gross growth rate Ke	Grazing mortality
	Size class		(day -1 )	G (day -1 )	G/Ke
	Picoplankton (0.7-0.9 µm)	2.58	3.32		1.29
	Picoplankton (1-2 µm)		2.64	1.6		0.61
	Nanoplankton (2-4µm)		3.27	0.93		0.28
	Nanoplankton (> 4µm)		2.65	0.83		0.31

Table 7 :

 7 Contribution of various taxonomic groups to the particular resource retained by oysters, in the Z farming area of Thau Lagoon in triplicate samples on 17 August 1998.

Acknolegments: This work was supported by a research grant from IFREMER (N° 973581145) and a doctoral grant from the "Conseil Général de Charente-Maritime" (Région Poitou-Charentes). We thank the CNRS (UMR CNRS 5556, URM 5, UMR 10), the IFREMER (DEL of Sète), the LBEM (La Rochelle University) and the European Community (MAST-III-DOMTOX), contract MAST-III PL97129 for their financial support. We also thank Mr Knutsen for reviewing the English.


L Rossignol

C Dupuy
email: cdupuy@univ-lr.fr

P Y Pascal

J. -P Debenay

FOR CLEANING LIVING HARD-SHELLED FORAMINIFERA

Keywords: mudflat, Hydrobia ulvae, hard-shelled foraminifera, extraction

published or not. The documents may come

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INTRODUCTION

Laboratory studies using living foraminifera for biological and ecological investigation have been used for more than a half-century and provide important, complementary data to field-base studies (e.g., [START_REF] Myers | Culture methods for the marine foraminifera of the littoral zone[END_REF][START_REF] Le Calvez | Recherche sur les foraminifères I: Développement et reproduction[END_REF][START_REF] Jepps | Studies on Polystomella Lamarck (Foraminifera)[END_REF][START_REF] Arnold | Culture methods in the study of living Foraminifera[END_REF]. They have been increasingly used for ecological and environmental studies (e.g., [START_REF] Bradshaw | Laboratory experiments on the ecology of foraminifera: Contributions from the Cushman Foundation for[END_REF][START_REF] Bender | Calcitic cement secreted by agglutinated foraminifers grown in laboratory culture[END_REF]Bijma and others, 1990;Stouff and others, 1999;[START_REF] Khare | Laboratory experiment to record rate of movement of cultured benthic foraminifera[END_REF]Heinz and others, 2002). These studies require separation of live individuals from the sediment without harming them and efficient techniques to differentiate live and dead individuals. Vital staining, such as the use of fluorescent probes, associated with direct observation of cytoplasm and reticulopodial network allow distinguishing live individuals from dead ones [START_REF] Murray | Mortality, protoplasm decay rate, and reliability of staining techniques to recognize 'living' foraminifera: a review[END_REF]Bowser, 2000, Bernhard, 2000). However, before using these methods, it is first necessary to isolate individuals from the sediment. In samples containing a high proportion of mud, the tests are hard to discern, even more so because they are often hidden in small-particle agglutinated cysts, making observation quite difficult. The sediment must be sieved to concentrate the foraminifera before observation (e.g., Bowser and others, 1992;Linke and others, 1995;Moodley and others, 2000), but even after sieving, the tests are still scattered among the coarser sediment particles and incased in their cysts. The foraminifera may be separated from the sediment by using their negative geotaxis, which makes them crawl up the walls of their dishes or microscope slides put in the dishes [START_REF] Arnold | Field and laboratory techniques for the study of living foraminifera[END_REF]Anderson and others, 1991;[START_REF] Bernhard | Distinguishing live from dead foraminifera: Methods review and proper applications[END_REF]. However, not all foraminiferal species exhibit such behavior [START_REF] Bernhard | Distinguishing live from dead foraminifera: Methods review and proper applications[END_REF], and infaunal species must be cleaned and picked out from the sediment with a brush or pipette. This is highly time consuming and may harm living individuals (Anderson and others, 1991;[START_REF] Carey | Long-Term Culture of Marine Benthic Protists[END_REF].

This study proposes a new harmless biological technique for concentrating living hardshelled foraminifera, such as Haynesina germanica and Ammonia tepida, from muddy sediment using the feeding behavior of a small deposit-feeding gastropod, Hydrobia ulvae.

MATERIAL AND METHODS

The area selected for collecting foraminifera and Hydrobia ulvae was the upper part of the intertidal Brouage mudflat on the French Atlantic coast at latitude 45° 54' N and longitude 1° 7' W (Fig. 1). This area was selected owing to the high density of living foraminifera (110 individuals per cm 3 , Armynot du Chatelêt, per. comm.) and because Hydrobia ulvae, a common gastropod from European intertidal mudflats, is the most abundant species among the macrofauna (Haubois and others, 2004). This snail is a deposit-feeder that inhabits muddy sand-and mudflats (Hayward and others, 1998). It ingests sediment and egests inorganic particles compacted into small cylinders (feces).

Fig. 1 here

The sediment sample was collected at low tide by scraping off the first centimeter of sediment in an area where microphytobenthos was abundant (brown film on surface sediment). Seawater was collected in the same area. If the mud snails and foraminifera cannot be sampled at the same time, then it is necessary to maintain a ready stock of mud snails in the laboratory. This is quite easy since they can be kept living for several weeks if placed in sediment in a cold room (5°C). In the laboratory, 1 g of sediment was sieved with seawater through a nylon mesh of 50-µm openings to eliminate clay-and finer silt-size particles. To eliminate additional fine grains, the remaining material was gently stirred in filtered (0.2 µm) seawater and then allowed to stand for several seconds to let the foraminifera settle to the bottom. Supernatant seawater with fine suspended particles was then eliminated, and seawater re-added. This operation was repeated until the supernatant water was clear. Finally, the processed sediment and foraminifera were distributed in a glass Petri dish (16 cm diameter).

Owing to the fragility of foraminiferal tests, it was impossible to stir the sediment strongly.

Consequently, the tests remained incased in their fine-particle agglutinated cysts and flocs of fine sediments remained together with rare coarser grain.

To test the efficiency of the treatment with Hydrobia, triplicate Petri dishes were prepared, by addition of 40 and 80 specimens of Hydrobia ulvae, respectively. They were placed in a constant-temperature room (18°C) and kept at a light/dark cycle of 12h/12h for 2 days. The dishes were observed every day under a dissecting microscope. After 2 days, the number of hard-shelled foraminifera was counted in all dishes and the species were identified.

In addition, experiments were carried out to address the question of whether or not the snails are ingesting any foraminifera. Twenty Hydrobia were placed for 48 hours in triplicate Petri dishes, together with sediment collected in an area rich in living foraminifera and sieved like above. At the same time, six other Petri dishes were prepared with the same sediment but without gastropods. Three of these dishes without Hydrobia were used to count the number of foraminifera at the beginning of the experiment, the three others were used as controls. At the end of the experiment, 48 hours later, the number of foraminifera was counted in the six remaining dishes (3 with Hydrobia and 3 controls without). Moreover, 50 Hydrobia were collected in an area rich in living foraminifera and sediment was collected at the same place in order to evaluate the density of living foraminifera. The shells of the Hydrobia collected in the field as well as those of the experiments (triplicate Petri dishes with 20 Hydrobia after 48 h) were broken and their living material was extracted. Because the guts of Hydrobia are very small and difficult to open without potentially breaking foraminiferal tests (if present), we used a process previously devised by one of us (Debenay, pers. comm.) for studying the gut contents of other gastropods: The living material of the gastropods was immersed for 4 days into a sodium hypochlorite solution with available chlorine of ~ 3% in order to remove organics. Control experiments, which consisted in the immersion of foraminifera with cytoplasm (potentially living) in the same solution, had shown that the tests, even agglutinated ones, were very well preserved after 4 days in the solution (Debenay, pers. comm.).

To complete this experiment, six foraminifera were left with 2 H. ulvae for 24 hours in a Petri dish, and then observed to determine if they were still alive (based on pseudopodial activity). During the first day, feces production began, but the tests were still covered with a layer of fine sediment and/or food and were not easy to distinguish. After two days, snails had cleaned all the foraminiferal tests, which had become very easy to locate and pick (5 seconds per specimen). It was easy to count them (Table 1). No improvement of the cleaning could be observed for experiments lasting more than two days. An additional benefit became evident.

RESULTS

EFFECT

The foraminifera were unable to reconstruct their agglutinated cysts after cleaning because all the fine particles of sediment were aggregated. It appeared that the foraminifera had been cleaned but not ingested by the mud snail. In the dishes without H. ulvae, the tests kept their cysts of organic and mineral particles and could hardly be distinguished (Fig. 2). Furthermore, the compaction of the sediment into feces was more efficient with 80 gastropods than with 40. The experiments were carried out with the same amount of sediment (1 g) in all the Petri dishes. After two days, the number of foraminifera (12  2 Ammonia tepida and 4  2 Haynesina germanica, proportions comparable to those found in the natural tidal flat at Brouage; Armynot du Chatelêt, per. comm.) was not significantly different between treatments with or without 20 Hydrobia ulvae (Student t test: p> 0.05) and not significantly different from numbers at the start of the treatments (Table 2). The fact that the number of individuals, including juveniles, was not lower in the dishes with Hydrobia suggests that the gastropods did not ingest any foraminifera. This inference is corroborated by the absence of foraminifera in the guts of the 60 Hydrobia from the dishes.

Table 2 here

The stomach contents of 50 gastropods collected from Brouage tidal flat contained in total only one small test (Ammonia) although foraminifera were abundant and available (~1200 living foraminifera in 50 cm 3 -89% Ammonia tepida, 8% Haynesina germanica, 3% other species). We assume that this lone foraminifera was attached on the shell or snared in the aperture of a snail but was not actually part of the gut contents. These observations suggest that Hydrobia does not ingest foraminifera in the natural environment.

In a further test, six foraminifera were placed together with two H. ulvae and observed at the start and after 24 hours. The foraminifera maintained pseudopodial activity and appeared unharmed by the gastropods (Fig. 3), even though the gastropods had scraped them clean of the agglutinated cysts that incased the tests.

Fig. 3 here

Several foraminifera extracted with this method were used for a bacterial grazing experiment. They fed normally on bacteria, showing that their health was not perceptibly affected by the cleaning process. They recovered, returned to their normal behaviors, and, when placed in sediment, they reconstruct their agglutinated cyst.

DISCUSSION

Several of methods for extraction and culture of foraminifera have been published (review in Anderson and others, 1991) but all are highly time-consuming. In this study, we describe a new method for extracting quickly and inexpensively a large number of living benthic shelled foraminifera for culture in the laboratory. This method requires only two hours of actual work to extract 1000 tests (30 minutes for preparation of Petri dishes with sediment containing foraminifera and H. ulvae and 1 h 30 min for picking 1000 tests after the sediment treatment by H. ulvae). The rest of the work is carried out by H. ulvae. In comparison, the picking of 1000 tests from untreated sediment required 20 hours. The grazing activity of Hydrobia ulvae results in the formation of small cylindrical feces of compacted sediment and cleaning of agglutinated cysts from tests of living benthic foraminifera. Most of the organic and inorganic particles, even those in the cysts around foraminiferal tests, are grazed by the mud snails and digested or packed into feces. Small isolated particles are no longer available to the foraminifera for construction of its cyst. The cleaning is harmless to the foraminifera since the gastropod does not have any feeding activity towards the foraminifera. This behavior is different from other gastropods, such as Olivella, that may selectively ingest living foraminifera, as reported by [START_REF] Hickman | Foraminiferivory: selective ingestion of Foraminifera and test alterations produced by the neogastropod Olivella[END_REF]. However, Olivella is much bigger than Hydrobia (2 cm instead of 5 mm). Moreover, unpublished studies have been carried out in the île d'Yeu laboratory on Littorina littorea, Littorina saxatilis, Gibbula umbilicalis and Monodonta lineata, temperate gastropods much bigger than Hydrobia. These studies have shown that very few foraminifera are ingested accidentally by these gastropods when they feed on algae, but that there is no selective ingestion.

After testing the abundance of cleaned tests and the incubation times on a sediment sample of 1 g, we concluded that the process is completed after 2 days with 40 H. ulvae.

About the same number of tests was cleaned in the same amount of time by either 40 or 80 H. ulvae. The only benefit of using 80 snails rather than 40 was production of feces that were more compacted, which facilitated the picking of living foraminifera. After this treatment, recovery of living specimens with a fine brush was much easier and faster. Moreover, even smaller specimens were clearly discernable, which is not the case when they are hidden in cysts of sediment particles. For the same treatment time (2 days), it is possible to increase the quantity of sediment treated by increasing the abundance of H. ulvae in bigger Petri dishes.

The active feeding of H. ulvae in Petri dishes suggest that this method could be applied to muddy samples in which the mud snail does not occur, such as fine sediments from deeper subtidal habitats or those of the shelf or slope. We suggest too that this method might be adaptable in other coastal and brackish environments where H. ulvae does not live, by using other small deposit feeders, such as Hydrobia salsa and Hydrobia totteni in USA, Hydrobia knysnaensis in South Africa, and Hydrobia buccinoides in Australia. However, these applications require testing.

  OF HYDROBIA ULVAE GRAZING ON THE SEDIMENT During all the experiments with 40 and 80 gastropods, H. ulvae fed on the bottom of the dishes and on the food aggregates surrounding living foraminifera. This activity led to the sorting of the sediment and foraminiferal tests into three components: (1) feces of H. ulvae made up of small cylinders of compacted sediment; (2) clean foraminiferal tests; (3) a few remaining mineral sediment particles.

FIGURE 1 .FIGURE 2 .

 12 FIGURE 1. Location of the sampling site: Brouage mudflat. White: subtidal area, Dark gray: tidal area.

FIGURE 3 .

 3 FIGURE 3. Pseudopodia emission of Ammonia tepida (x 200).

Table 1 here Fig. 2 here EFFECT

 1 OF HYDROBIA ULVAE ON THE FORAMINIFERA

TABLE 1 .

 1 Abundance of foraminifera (Fora g -1 dw) (dw: dry weight) in the sediment after treatment with different densities of Hydrobia (Hyd).

		Abundance	Abundance
		(Fora g -1 dw)	(Fora g -1 dw)
		40 Hyd	80 Hyd
	Ammonia
	tepida	121	129
	Haynesina
	germanica	626	655
	Sum	747	784

TABLE 2 .

 2 Abundance of foraminifera (Fora g -1 dw) (dw: dry weight) with standard deviation (SD) in the sediment before (T0) and after 48 h treatment without and with Hydrobia (Hyd).

		Abundance		Abundance		Abundance
		(Fora g -1 dw)	SD	(Fora g -1 dw)	SD	(Fora g -1 dw) SD
		T0	T0	without Hyd		20 Hyd
	Ammonia
	tepida	13	3	12	2	13	3
	Haynesina
	germanica	3	1	4	2	4	2
	Sum	16	4	17	4	17	5

ACKNOWLEDGMENTS

This work was supported by the CNRS, contract PEVS and supported by research national program ECCO. We thank Jennifer Guarini for reviewing the English. We are grateful to C.A. Brunner and the Associated Editor of JFR for their help in improving the last version of the manuscript.