Pierre-Yves Pascal
email: pypascal@univ-lr.fr

Christine Dupuy

Pierre Richard

Anne-Gaelle Haubois

Nathalie Niquil

Influence of environment factors on bacterial ingestion rate of the deposit-feeder Hydrobia ulvae and comparison with meiofauna

Keywords: Deposit-feeding, Hydrobia ulvae, gastropod, bacteria, grazing, environmental factors, Ammonia tepida, nematodes, mudflat

Deposit feeders are able to process a considerable volume of sediment, containing large quantities of associated bacteria. However, conclusions concerning the trophic role played by benthic bacteria in marine sediments are still not fully elucidated. This study deals with bacterivory by the gastropod Hydrobia ulvae, one of the most abundant deposit-feeding species in intertidal mudflats in Western Europe. Ingestion rates of bacteria were determined during grazing experiments using 15 N pre-enriched bacteria. Grazing experiments were performed in order to measure effects of abiotic (temperature, salinity and luminosity) and biotic (bacterial and algal abundances) factors on ingestion rates of bacteria by H. ulvae of an intertidal mudflat (Brouage, Marennes-Oléron, France). The mean ingestion rate of bacteria by H. ulvae was 1149 ngC ind -1 h -1 . The general trend showed a temperature effect with an optimum around 30°C, and the assimilation rate was significantly lower at 5°C. Bacterial assimilation did not significantly differ between salinity 18 and salinity 31. Ingestion was the same in light and in dark conditions. Results were compared with those of other grazing experiments conducted simultaneously in similar conditions with two other grazers with different size and feeding modes: the foraminifera Ammonia tepida and a nematode community from the superficial sediment of the Brouage mudflat. Hydrobia ulvae and nematodes presented a feeding behavior less influenced by environmental changes than A.

Introduction

In pelagic environments, bacteria are heavily grazed and consequently play a major role in food webs [START_REF] Azam | The ecological role of water-column microbes in the sea[END_REF]. In the benthic environment, bacteria are generally 1000 times more abundant than in pelagic systems, reaching abundances of about 10 9 cells cm 3 [START_REF] Schmidt | Constancy of bacterial abundance in surficial marine sediments[END_REF]. However, microbial food web research in sediment is in its infancy and the trophic significance of benthic bacteria remains elusive (Review in [START_REF] Kemp | Direct uptake of detrital carbon by the deposit-feeding polychaete Euzonus mucronata (Treadwell)[END_REF].

The deposit-feeder Hydrobia ulvae is one of the most abundant species of macrofauna inhabiting intertidal mudflats in Western Europe [START_REF] Bachelet | Post-recruitement phase population dynamics of the intertidal gastropod Hydrobia ulvae (Pennant)[END_REF][START_REF] Barnes | Reproductive strategies in contrasting populations of the coastal gastropod Hydrobia ulvae: longevity and life-time egg production[END_REF][START_REF] Sola | Population dynamics, reproduction, growth, and secondary production of the mud-snails Hydrobia ulvae (Pennant)[END_REF]. Deposit feeders typically process at least one body weight of sediment daily [START_REF] Lopez | Ecology of deposit-feeding animals in marine sediments[END_REF]. This sediment includes highly digestible and nutritious microphytobenthos and bacteria, less digestible plant debris and completely indigestible refractory detritus [START_REF] Rice | Early diagenesis of organic matter and the nutritional value of sediment[END_REF]. Diatoms have been found to be a major source of nutrition for H. ulvae [START_REF] Fenchel | Particle size-selection of two deposit feeders: the Amphipod Corophium volutator and the Prosobranch Hydrobia ulvae[END_REF][START_REF] Jensen | The importance of diatoms and bacteria in the diet of Hydrobia species[END_REF]Lopez and Cheng, 1983a;[START_REF] Bianchi | The importance of the microalgae, bacteria and particulate organic matter in the somatic growth of Hydrobia totteni[END_REF]Haubois et al., 2005a). However bacteria have also been found as food for Hydrobia species [START_REF] Cammen | Ingestion rate: an empirical model for aquatic deposit feeders and detritivores[END_REF][START_REF] Jensen | The importance of diatoms and bacteria in the diet of Hydrobia species[END_REF][START_REF] Bianchi | Nutrition and food limitation of deposit-feeders. II. Differential effects of Hydrobia totteni and Ilyanassa obsoleta on the microbial community[END_REF][START_REF] Levinton | Nutrition and food limitation of deposit-feeders. I. The role of microbes in the growth of mud snails (Hydrobiidae)[END_REF]. Due to high abundances of H. ulvae in intertidal mudflats, carbon flow from bacteria to snails may be a significant pathway in this type of environment.

The objective of the present study was to quantify the bacterial ingestion rate of H. ulvae and to investigate how this rate varies with abiotic (temperature, salinity and luminosity) and biotic (bacterial and algal abundance) factors. Intertidal mudflats are subject to large and quick changes in many environmental factors at short time scales (circadian and tidal cycles) [START_REF] Guarini | Modeling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF] and these variations may significantly influence snail feeding behaviour.

Bacterivory of H. ulvae was then compared to that of other grazers of different sizes and feeding modes, the foraminifera Ammonia tepida and a nematode community from surficial sediment of an intertidal mudflat (Brouage-Marennes Oléron-France). Bacterial ingestion of both grazers have been previously described (Pascal et al., 2008b;Pascal et al., In press). All grazing experiments were performed simultaneously in similar conditions using stable isotope enriched prey ( 13 C enriched algae and 15 N enriched bacteria).

Experimental procedure

Study site

The Brouage intertidal mudflat is located in the eastern part of the Marennes-Oléron Bay (Atlantic coast of France). Meteorological conditions exhibit a strong seasonality typical of a temperate climate. Range of temperature and salinity in emerged sediments are more extreme during summer tidal cycles [START_REF] Guarini | Modeling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF]. Minimum and maximum mud temperatures are 5°C and 34°C respectively. The maximum daily range of mud temperature due to emersion and immersion cycle reaches 18°C [START_REF] Guarini | Modeling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF]. Salinity of overlaying water is controlled by the Charente River freshwater input, ranging from 25 to 35 over the year [START_REF] Héral | Caractéristiques saisonnières de l'hydrologies du complexe estuarien de Marennes-Oléron (France)[END_REF]. Salinity of the upper layers of sediment may also decrease with rainfall. The sediment surface irradiance shifts from dark during submersion and night emersions to high levels of incident light during daytime emersions. This irradiance can reach 2000 M of photons m -2 s -1 [START_REF] Underwood | Primary production by phytoplancton and microphytoplancton in estuaries[END_REF]. Details of numerous benthic organisms and processes are available concerning this intertidal zone (gathered in [START_REF] Leguerrier | Numerical analysis of the food web of an intertidal mudflat ecosystem on the Atlantic coast of France[END_REF][START_REF] Leguerrier | Modeling the impact of oyster culture on a mudflat food web in Marennes-Oléron Bay (France)[END_REF][START_REF] Degré | Comparative analysis of the food webs of two intertidal mudflats during two seasons using inverse modelling: Aiguillon Cove and Brouage Mudflat, France[END_REF].

Preparation of 15

N enriched bacteria Superficial sediment (1 cm depth) was collected on the Brouage mudflat (45,55,074 N;

1,06,086 W). One cm 3 of the collected sediment was added to 20 cm 3 of bacterial liquid culture medium and kept in darkness for 24 hours at 13°C. The composition of this culture medium was previously described in Pascal et al. (2008a). This primary culture was then subcultured for 24 hours under the same conditions to get approximately 2 10 9 cells cm 3 .

Finally, bacteria were collected in 0.2 µm filtered seawater after 3 centrifugations (3500 g, 10 mn, 20°C), frozen in liquid nitrogen and kept frozen at -80°C until grazing experiments.

Preparation of 13 C enriched algae

An axenic clone of the diatom Navicula phyllepta (CCY 9804, Netherlands Institute of Ecology NIOO-KNAW, Yerseke, The Netherlands), the most abundant diatom species in the study area (Haubois et al., 2005b), was cultured in medium described by [START_REF] Antia | The survival of axenic cultures of marine phytoplankton algae from prolonged exposure to darkness at 20°C[END_REF] and containing NaH 13 CO 3 (4 mM). Diatoms were concentrated by centrifugation (1500 g, 10 mn, 20 °C), washed three times to remove the 13 C-bicarbonate, and freeze-dried.

Quantification of bacteria and algae abundance

In order to determine the ratio between enriched and non-enriched preys in microcosms, abundances of bacteria and algae were assessed. To separate bacteria from sediment particles, incubation in pyrophosphate (0.01M for at least 30 min) and sonication (60 W) were performed. Bacteria from both sediment and culture were labelled using 4.6-diamidino-2phenylindole dihydrochloride (DAPI) (2500 µg l -1 ), filtered onto 0.2 µm Nucleopore black filter [START_REF] Porter | The use of DAPI for identifying and counting aquatic microflora[END_REF] and then counted by microscopy. We verified the absence of ciliates and flagellates in the bacterial culture during this microscope observation step. The abundance of diatoms in the sediment was assessed using Chl a as a proxy, measured using fluorometry [START_REF] Lorenzen | A method for the continuous measurement of in vivo chlorophyll concentration[END_REF].

Grazing experiments

The top centimeter of sediment was collected during ebb tide from the same study area at midday on March 13, 2006. It was sieved on 500 µm, 200 µm and 50 µm in order to concentrate respectively H. ulvae, A. tepida and nematodes. Before sieving, snails were placed on natural sediment and kept for 24 h at 20°C in the dark. Time between sieving and the start of the grazing experiment never exceeded two hours in order to avoid starvation bias in feeding behavior [START_REF] Calow | The feeding strategies of two freshwater gastropods. Ancylus fluviatilis Mull. and Planorbis contortus Linn. (Pulmonata) in terms of ingestions rates and absorption efficiencies[END_REF]. Seventeen handpicked specimens of H. ulvae were placed in polypropylene Petri dishes (ø = 9 cm). This density was chosen in order to avoid a densitydependence effect on the individual ingestion due to space limitation [START_REF] Blanchard | Measurement of ingestion rate of Hydrobia ulvae (Pennant) on intertidal epipelic microalgae: the effect of mud snail density[END_REF].

A fraction of the sediment passing through the 50 µm mesh was mixed with the 15 N enriched bacteria. This slurry contained 10.5 10 8 bacteria cm 3 , 15 N enriched bacteria being twice as abundant as non-enriched ones. Four cm 3 of this slurry were put into each microcosm. Each experiment was carried out in triplicate, along with at least one control. Control samples were frozen (-80°C) in order to kill any grazers.

The calculation of bacterial ingestion rate relies on the assumption that enriched preys accumulate in snail's gut at a constant rate, and that no egestion of labelled materials occurs during incubation time. A kinetic study was run for 1 to 12 hours including the 2 hour run that was used for all other experiments. Incubations were made under the following standardized conditions that were close to the mean values recorded on the study site: temperature (20°C), salinity (31), luminosity (darkness), bacterial abundance (10.5 10 8 bacterial cells cm 3 ) and algal abundance (15 µgChla g dry sediment -1 ). For each experiment to determine the effects of environmental factors, only one incubation factor was modified so as to determine its effect on H. ulvae's grazing activity. After the sieving step, snails were transferred without acclimation into different microcosms to simulate short-term changes of environmental factors. To test the effect of temperature, the snails were placed at 5°C, 15°C, 30°C and 40°C: these temperatures are in the range of those measured in the study area [START_REF] Guarini | Modeling the mud surface temperature on intertidal flats to investigate the spatio-temporal dynamics of the benthic microalgal photosynthetic capacity[END_REF].

The effect of salinity was investigated by placing Hydrobia in microcosms with a salinity of 18. To decrease salinity, cultured bacteria were rinsed with 0.2 µm filtered-sea-water diluted with 0.2 µm filtered distilled water. Such decrease in salinity can occur in field conditions when sediment is exposed to heavy rainfall. The light effect was tested with a light intensity of 83 M of photons m -2 s -1 . Bacterial abundance was modified adding various quantities of bacteria enriched in 15 N. Bacterial abundances (total enriched and non-enriched) tested were 4, 7 and 17 cells cm 3 wt sed -1 with respectively the following ratio between abundance of total and enriched bacteria: 6.1, 2.0 and 1.3. Algal abundance was modified by adding various quantities of cultured N. phyllepta enriched in 13 C while bacterial abundances (total enriched and non-enriched) were kept constant at 10 10 8 cells cm 3 . Algal abundance (total enriched and non-enriched) were 26, 64 and 114 µgChla g dry sed -1 with respectively the following ratio between abundance of total and enriched algae: 2.4, 1.3 and 1.2.

Incubations were stopped by freezing microcosms at -80°C. Samples were thawed and H.

ulvae were separated by hand from their shell and the 17 specimens of each microcosm were pooled and homogenized using a Potter-Eveljhem.

Isotope analysis and calculations

15 N and 13 C of prey (bacteria and algae) and grazers were measured using an EA-IRMS (Isoprime, Micromass, UK). Nitrogen isotope composition is expressed in the delta notation ( 15 N) relative to air N 2 : 15 N = (( 15 N/ 14 N) sample / ( 15 N/ 14 N) reference )-1 1000. Carbon isotope composition is expressed in the delta notation ( 13 C) relative to Vienna Pee Dee Belemnite (VPDB): 13 C = (( 13 C/ 12 C) sample / ( 13 C/ 12 C) reference )-1 1000.

Incorporation of 15 N is defined as excess above background 15 N (control experiment) and is expressed in terms of specific uptake (I). I was calculated as the product of excess 15 N (E)

and biomass of N per grazer. I was converted to bacterial carbon grazed using the C/N ratio of bacteria. E is the difference between the background (F background ) and the sample (F sample ) 15 N fraction: E = F sample -F background , with F = 15 N / ( 15 N + 14 N) = R / (R + 2) and R = the nitrogen isotope ratio. For the F background , we used control values measured with killed grazers (frozen).

For H. ulvae we used the highest value measured in control ( 15 N = 12.42 and 13 C = -13.72).

R was derived from the measured 15 N values as: R = (( 15 N/1000)+1) RairN 2 where RairN 2 = 7.35293 10 -3 [START_REF] Mariotti | Apports de la géochimie isotopique à la connaissance du cycle de l'azote[END_REF]. The uptake of bacteria was calculated as Uptake = (I (% C enriched bacteria / % N enriched bacteria )) / (F enriched bacteria incubation time). This uptake was multiplied by the ratio between the abundance of total and enriched bacteria determined by DAPI counts.

Incorporation of 13 C was calculated analogously, with

F = 13 C / ( 13 C + 12 C) = R / (R + 1),
RairN 2 is replaced by R VPDB = 0.0112372 and Uptake = I / (F enriched bacteria incubation time).

The uptake measured was multiplied by the ratio between the abundance of total and enriched diatom, determined from fluorometrical measurements.

Enriched N. phyllepta carbon consisted of 22.95 ± 0.54% 13 C. The C/N ratio of enriched bacteria was 3.49 and bacterial nitrogen consisted of 2.88 ± 0.03% 15 N. The individual average weight of H. ulvae was 0.54 ± 0.08 mg and each specimen was composed on average of 184 ± 19 µgC and 43 ± 4 µgN (N = 72 samples of at least 17 specimens each). Ingestion rate as gC bacteria gC H. ulvae -1 h -1 was obtained by dividing ingestion rate of bacteria (gC ind -1 h -1 ) by H. ulvae mean weight (gC ind -1 ).

Variations of uptake rates according to salinity and irradiance were tested using bilateral independent-samples two-tailed tests. One-way analyses of variance (ANOVA) were used in order to test the impact of temperature and algal and bacterial abundance on uptake rates of bacteria and algae. The Tukey test was used for post-hoc comparisons.

Results

The kinetic experiment showed that accumulation of bacteria in H. ulvae increased linearly during the first two hours of incubation and then levelled off (Fig. 1). The linear regression slope for the first two hours indicated an uptake rate of 1149 ngC ind -1 h -1 equivalent to 6.43 10 -3 gC bacteria gC H. ulvae -1 h -1 (r 2 = 0.98). The linear regression slope between five and twelve hours was more than seven times lower than for the two first hours and indicated an uptake rate of 145 µgC ind -1 h -1 equivalent to 0.81 10 -3 gC bacteria gC H. ulvae -1 h -1 (r 2 = 0.98).

Ingestion of bacteria increased from 462 to 1277 ngC ind -1 h -1 when temperature increased from 5°C to 30°C, and then decreased reaching 1059 µgC ind -1 h -1 at 40°C (Fig. 2). Ingestion rate of bacteria by H. ulvae was significantly decreased at 5°C (F = 10; p<0.01), but ingestion rates observed at 10, 20, 30 and 40°C were not significantly different.

The ingestion rate measured for a salinity of 31 (1149 ± 285 ngC ind -1 h -1 ) was similar to the one measured for a salinity of 18 (1085 ± 58 ngC ind -1 h -1 ) (two-tailed test, p = 0.20).

The ingestion rate observed under light conditions (1478 ± 246 ngC ind -1 h -1 ) was similar to the one observed in darkness (1149 ± 285 ngC ind -1 h -1 ) (two-tailed test, p = 0.72).

Ingestion rates of bacteria were significantly linked to bacterial abundance in microcosms (F = 38; p<0.001) (Fig. 3). Ingestion rate increased linearly from 38 ± 13 to 1117 ± 93 ngC ind -1 h -1 ) when bacterial concentrations increased from 4 to 10 10 8 cells cm 3 and increased, though not significantly to 1604 ± 366 ngC ind -1 h -1 for a bacterial concentration of 17 10 8 cells cm 3 .

Dual labeling of prey allowed simultaneous assessment of the ingestion of bacteria and algae. When algal concentration increased from 15 to 114 gChla g -1 with constant bacterial abundance (10.5 10 8 cells cm 3 ), the ingestion rate of algae remained constant (F = 3.3; p = 0.11) (Fig. 4). However, ingestion rate of bacteria remained constant for algal concentration in the sediment between 15 and 64 µgChla g dry wt sed -1 but significantly decreased at the highest algal concentration of 114 µgChla g dry wt sed -1 (F = 4.4; p < 0.05). As a result, when algal abundance increased, the fraction of algae in the diet of H. ulvae increased.

Grazing experiments were simultaneously performed with two other grazers: the foraminifera A. tepida and a nematode community. Feeding behaviour of A. tepida (Pascal et al., 2008b) and nematodes (Pascal et al., In press) has been previously described. All grazing incubations were conducted at the same time and in similar conditions, making comparisons between grazers possible. The effects of environmental factors (temperature, salinity and luminosity) on ingestion rates of bacteria are summarized in Table 1. Among the different grazers tested, A. tepida was the most affected by salinity and temperature. Light only affected nematodes and increased their feeding activity. Classification of grazers according to their maximal ingestion rates of bacteria and algae reported per grazer weight gave the following list arranged in ascending order: A. tepida, nematode community and H. ulvae (Table 1). For the dual labelling experiment, ratios between algae and bacteria ingested were measured for each grazer, at each algal concentration. Comparison of those ratios between grazers reflected their respective ability to discriminate between food sources (i.e. algae and bacteria). At the three algal concentrations tested, A. tepida and nematodes showed a higher ratio of algae:bacteria ingested than H. ulvae, suggesting a better ability to preferentially ingest algae over bacteria compared to the gastropod (Fig. 5).

Discussion

Kinetic experiment

During the 12 hour incubation, H. ulvae first accumulated the enriched bacteria linearly over 2 hours (Fig. 1). This suggests that ingestion rate of bacteria was constant and that no egestion of recently ingested labelled material occurred during this period. After 4 hours of incubation, the accumulation rate of bacteria by H. ulvae decreased. This may have two nonexclusive origins: egestion of labelled bacteria and a decrease in feeding activity over time. As all other grazing experiments were performed during two hours, they consequently reflected the ingestion rates of H. ulvae.

Linear accumulation of labelled diatom or bacteria by H. ulvae had been previously recorded from 45 min [START_REF] Fenchel | Particle size-selection of two deposit feeders: the Amphipod Corophium volutator and the Prosobranch Hydrobia ulvae[END_REF] to 2 hours [START_REF] Blanchard | Measurement of ingestion rate of Hydrobia ulvae (Pennant) on intertidal epipelic microalgae: the effect of mud snail density[END_REF]Haubois et al., 2005a). Molluscs have complex digestive tracts allowing partitioning of food particles within the gut. Relatively indigestible material passes quickly to the intestine and is subjected to extracellular digestion. More nutritious material like bacteria and algae is diverted to the digestive gland where it undergoes intracellular digestion. For Hydrobia totteni, gut residence time is 30-40 min (Lopez and Cheng, 1983b) whereas digestive gland residence time is 5 hours [START_REF] Kofoed | Time-dependent absorption in deposit feeders[END_REF]. Assuming a similar situation for H. ulvae, the absence of egestion during the two hour long incubations would mean that all bacteria are diverted to the digestive gland. In their grazing experiment with enriched diatoms, [START_REF] Sokolowski | Cd transfer in the deposit-feeder prosobranch Hydrobia ulvae (Pennant) from the benthic diatoms: the kinetics of rapid Cd assimilation and efflux[END_REF] observed that during the first 4 h phase of experiment, accumulation rate was 3 times higher than during the last 12 h phase. We observed exactly the same ratio between accumulation rates found during the grazing periods 0-2 h and 0-12 h. Those similar results suggest that digestive processes for bacteria and algae may be similar.

Range of ingestion rates

To our knowledge, there is no data dealing with the ingestion rate of bacteria by Hydrobia to compare with our values. However, concerning algal ingestion our data are consistent with those previously measured. In the present study, ingestion rates fluctuated between 1.2-1.8 µgC ind -1 h -1 . In the literature, ingestion rates of snails fed with diatoms are 0.5-2.9 µgC ind -1 h -1 for Hydrobia truncata [START_REF] Forbes | The role of sediment particle size in nutritional energetics of a surface deposit-feeder. I. Ingestion and absorption of sedimentary microalgae by Hydrobia truncata (Vanatta)[END_REF]) and 1.2 [START_REF] Sokolowski | Cd transfer in the deposit-feeder prosobranch Hydrobia ulvae (Pennant) from the benthic diatoms: the kinetics of rapid Cd assimilation and efflux[END_REF], 1.12-1.33 [START_REF] Blanchard | Measurement of ingestion rate of Hydrobia ulvae (Pennant) on intertidal epipelic microalgae: the effect of mud snail density[END_REF] and 0.04-2.08 (Haubois et al., 2005a) for H. ulvae.

The maximal ingestion rates of algae and bacteria by Hydrobia ulvae were higher than meiofaunal rates. Nevertheless, individual weight of H. ulvae is more than one hundred and one thousand times higher than A. tepida and nematodes respectively (Table 1). Body size is an important determinant of many physiological processes and maximal ingestion rate is generally inversely correlated to body size (e. g. [START_REF] Moloney | General allometric equations for rates of nutrient uptake, ingestion, and respiration in plankton organisms[END_REF]. The present study focuses only on two potential prey, bacteria and algae, although other food sources are available in sediment. For instance Dissolved Organic Material may constitute an important food supply for nematodes [START_REF] Lopez | Feeding biology of the brackish-water Oncholaimid nematod Adoncholaimus thalassophygas[END_REF][START_REF] Meyer-Reil | Uptake of organic matter by meiofauna organisms and interrelationships with bacteria[END_REF][START_REF] Montagna | Competition for dissolved glucose between meiobenthos and sediment microbes[END_REF][START_REF] Jensen | Feeding ecology of free-living aquatic nematodes[END_REF] and foraminifera [START_REF] Schwab | Metabolism in the protozoan Allogromia laticollaris Arnold[END_REF]. Nematodes and foraminifera may be principally dependent on those other resources and present consequently low ingestion rates of bacteria and algae.

Effect of abiotic factors on bacterial ingestion rate

The general trend showed a temperature effect with an optimum around 30°C. However, except at the lowest temperature (5°C), differences between feeding rates of H. ulvae observed in the present study were not significant, indicating a limited influence of temperature. In a similar manner, [START_REF] Barnes | Variation in feeding rate of the intertidal mudsnail Hydrobia ulvae in relation to the tidal cycle[END_REF] did not detect changes in feeding activity of H. ulvae during in situ experiments with the same range of temperature. Ingestion rates observed at salinities of 18 and 31 were not significantly different in the present study.

Grudeno & André (2001) also observed that shell growth of juvenile. H. ulvae was unaffected by salinity in the range of 15-30. Light did not affect the ingestion of bacteria by H. ulvae.

The literature gives conflicting results concerning the effect of light. [START_REF] Barnes | Daily activity rythms in the intertidal Gastropod Hydrobia ulvae (Pennant)[END_REF] found that crawling activity of snails was higher in darkness whereas [START_REF] Orvain | Environmental and behavioural factors affecting activity in the intertidal gastropod Hydrobia ulvae[END_REF] observed an increase of H. ulvae crawling activity with light. However in Orvain & Sauriau's experiment, light may have induced formation of algal biofilm, affecting microphytobenthic distribution and thus indirectly affecting snail activity levels.

In intertidal mudflats, the surficial centimeter of sediment is subjected to fast and large environmental variations. The ability of a grazer to sustain feeding activity when environmental conditions fluctuate can be interpreted as an adaptation to this habitat. All compared grazers in the present study came from the top centimeter of sediment of the same study area and grazing incubations were performed in similar conditions. Compared to other grazers, the feeding response of the foraminifera A. tepida presented the largest ranges of variation indicating that they may present a low adaptation to rapid environmental changes (Table 1). Nevertheless, A. tepida is considered as one of the most tolerant species of foraminifera to temperature and salinity variations [START_REF] Bradshaw | Laboratory experiment on the ecology of foraminifera[END_REF][START_REF] Walton | The genus Ammonia Brünnich, 1772: its geographic distribution and morphologic variability[END_REF] and more generally to environmental changes [START_REF] Samir | The response of benthic foraminifera and ostracods to various pollution sources: a study from two lagoons in Egypt[END_REF][START_REF] Armynot Du | Foraminiferal proxies for pollution monitoring in moderately polluted harbors[END_REF][START_REF] Bouchet | Effects of short-term environmental disturbances on living benthic foraminifera during the Pacific oyster summer mortality in the Marennes-Oléron Bay (France)[END_REF]. A. tepida may be able to survive starvation when the environment is unfavourable and may await optimal conditions to feed and develop. An alternate explanation could be that when conditions are hostile, foraminifera move from the top layers of sediment to deeper layers [START_REF] Severin | Laboratory experiments on the vertical movement of Quinqueloculina impressa Reuss through sand[END_REF][START_REF] Severin | Laboratory observations of the rate of subsurface movement of a small miliolid foraminifer[END_REF][START_REF] Groß | Sediment interactions of foraminifera: implications for food degradation and bioturbation processes[END_REF]. Feeding activity of nematodes and H. ulvae appeared to be more independent of environmental variables. The nematode community was mainly composed of three species, Chromadora macrolaima, Daptonema oxycerca and Ptycholaimellus jacobi (Pascal et al., In press), known to dwell and feed in surface sediment. Moreover, H. ulvae is considered the most environmentally tolerant of Northwest European Hydrobia [START_REF] Hylleberg | The effect of salinity and temperature on egestion in mud snails (Gastropoda: Hydrobiidae). A study in niche overlap[END_REF][START_REF] Lassen | Tolerance to abiotic factors in mudsnails (Hydrobiidae)[END_REF]. However caution must be taken in interpretation of our results because the effects of each environmental factor on feeding behaviour of snails were studied independently whereas in natural environment, all these factors covary. Moreover, seasonal acclimatising capacity was not taken into account [START_REF] Barnes | Variation in feeding rate of the intertidal mudsnail Hydrobia ulvae in relation to the tidal cycle[END_REF].

Feeding response to bacterial and algal abundances

In fine grained environments, Hydrobia ingests mouthfuls of sediment containing organic food source, including bacteria and microalgae [START_REF] Kofoed | The feeding biology of Hydrobia ventrosa (Montague). I. The assimilation of different components of food[END_REF]Lopez and Cheng, 1983b;[START_REF] Levinton | What is the role of particulate organic mater in benthic invertebrate nutrition?[END_REF]. Due to high size and feeding mode of H. ulvae, the snail probably has a very low ability to ingest selectively very small preys such as bacterial cells. In opposition, larger preys such as diatom cells can be selectively ingested from sediment by Hydrobia [START_REF] Fenchel | Character displacement and coexistance in mud snails (Hydrobiidae)[END_REF][START_REF] Lopez | The availability of microorganisms attached to sediment particles as food for Hydrobia ventrosa (Montague) Gastropoda, Prosobranchia[END_REF][START_REF] Lopez | Epipsammic browsing and deposit-feeding in mud snails (Hydrobiidae)[END_REF].

Indiscriminant ingestion of bacteria by H. ulvae implies that bacterial ingestion is exclusively dependent on ingestion rate of sediment and the concentration of bacteria in sediment. This type of feeding indicates that relationships between bacterial ingestion and bacterial concentration in sediment can be described by (i) a power law relation, (ii) a linear increase or (iii) null or decreasing relation. Those relations mean that when prey concentration increases, ingestion rate of sediment respectively (i) increases, (ii) remains constant or (iii)

decreases. In the present study, the ingestion rate of bacteria first increased linearly with bacterial concentration and then levelled off at the highest concentration (Fig. 3). This relation may involve a constant ingestion rate of sediment at the lowest concentration of bacteria and a decrease of ingested sediment at the highest bacterial concentration.

Ingestion of algae by snails was not influenced by algal concentration in the range of concentration tested (Fig. 4). Contrarily, Hydrobia had been found previously to have increasing algal ingestion rate when algal concentration increased in a similar range of values [START_REF] Forbes | Changes in feeding and crawling rates of Hydrobia truncata (Prosobranchia: Hydrobiidae) in response to sedimentary chlorophyll a and recently egested sediment[END_REF][START_REF] Forbes | The role of sediment particle size in nutritional energetics of a surface deposit-feeder. I. Ingestion and absorption of sedimentary microalgae by Hydrobia truncata (Vanatta)[END_REF]Haubois et al., 2005a). The ability of a deposit feeder to alter its ingestion rate depends on its sensory capacity to recognize food quality [START_REF] Taghon | Optimal foraging by deposit-feeding invertebrates: roles of particle size and organic coating[END_REF]. This perception may differ between the freeze-dried diatoms in the present study and live diatoms in other studies, which could explain the different responses observed.

The ratio between algae and bacteria ingested denotes grazer ability to select diatoms from the sediment/bacteria aggregate. At each algal concentration, this ratio was always higher for A. tepida and nematodes, indicating higher algal selection efficiency. The nematode community was composed mainly of epigrowth feeders (75%) and non selective deposit feeders (21%). Epigrowth feeders puncture diatom cells with their teeth to ingest cell contents. Consequently, they are mainly dependant on algal resources [START_REF] Jensen | Feeding ecology of free-living aquatic nematodes[END_REF]. A.

tepida uses a network of pseudopodia to gather and ingest food particles. This feeding mode allows foraminifera to be highly selective in ingested food [START_REF] Lee | Tracer experiments in feeding littoral foraminifera[END_REF][START_REF] Lee | Trophic dynamics and niches of salt marsh foraminifera[END_REF]. Ammonia may also greatly depend on algal resources, as this foraminifera was found to ingest rapidly and with high efficiency fresh algal deposits [START_REF] Moodley | Ecological significance of benthic foraminifera: 13 C labelling experiments[END_REF]. [START_REF] Montagna | The effect of freshwater inflow on meiofaunal consumption of sediment bacteria and microphytobenthos consumption of sediment bacteria and microphytobenthos in San Antonio Bay, Texas[END_REF] ulvae, but this hypothesis can reasonably be rejected as Hydrobia is not able to select diatoms according to the cell size [START_REF] Levinton | The body size-prey-size hypothesis and Hydrobia[END_REF]Haubois et al., 2005a). In sandy sediment, Hydrobia presents epipsammic browsing activity by taking particles into the buccal cavity, scraping off attached microorganisms and then spitting out the particles [START_REF] Lopez | Epipsammic browsing and deposit-feeding in mud snails (Hydrobiidae)[END_REF]. As a result, gut contents and even fecal pellets of Hydrobia can contain more diatoms than the offered sandy sediment [START_REF] Fenchel | Particle size-selection of two deposit feeders: the Amphipod Corophium volutator and the Prosobranch Hydrobia ulvae[END_REF][START_REF] Lopez | The availability of microorganisms attached to sediment particles as food for Hydrobia ventrosa (Montague) Gastropoda, Prosobranchia[END_REF].

Conversely, in fine grained sediment, Hydrobia ingests mouthfuls of sediment containing organic food, including microalgae [START_REF] Kofoed | The feeding biology of Hydrobia ventrosa (Montague). I. The assimilation of different components of food[END_REF]Lopez and Cheng, 1983b;[START_REF] Levinton | What is the role of particulate organic mater in benthic invertebrate nutrition?[END_REF]. Results of the present study also suggest that H. ulvae feeding on muddy sediment present a limited ability to discriminate between algae and the sediment/bacteria aggregate. [START_REF] Taghon | Variable ingestion rate and its role in optimal foraging behavior of marine deposit feeders[END_REF] pointed out that for animals having limited particle selection ability, foraging strategies are mainly a function of ingestion and digestion processes. Indeed, in the present study H. ulvae appeared to decrease the rate of ingested sediment at high algal and bacterial concentrations. In the present study, labelled prey were distributed homogeneously in sediment. Feeding rates and feeding behaviour of H. ulvae may be different when algae are condensed in biofilm.

In the Brouage mudflat, H. ulvae and meiofauna are present, on average, in similar biomasses throughout the year [START_REF] Degré | Comparative analysis of the food webs of two intertidal mudflats during two seasons using inverse modelling: Aiguillon Cove and Brouage Mudflat, France[END_REF]. The present study suggests that H. ulvae ingests bacteria at a higher rate than meiofaunal do. In the study area, benthic bacteria would therefore be grazed to a higher extent by macrofauna than by meiofauna.

Figure captions

Figure 1 .

 1 Figure 1. Bacteria uptake by H. ulvae (mean SD, N =3) as function of incubation time

Figure 2 .

 2 Figure 2. Bacterial ingestion rate by H. ulvae (mean SD, N =3) as function of

Figure 3 .

 3 Figure 3. Bacterial ingestion rate by H. ulvae (mean SD, N =3) as function of bacterial abundance (10 8 cells cm 3 ). Different letters above bars indicate significant differences

Figure 4 .

 4 Figure 4. Ingestion rates of algal carbon(mean SD) and bacterial carbon (mean SD) by H. ulvae (ngC ind -1 h -1 ) as a function of algal abundance ( gChla g dry wt sed -1 ).Bacterial abundance was kept constant (1.05 10 9 cells cm 3 ). * above bars indicate

Figure 5 .

 5 Figure 5. Ratio between algae and bacteria taken up by three different grazers (the

Fig. 1

 1 Fig. 1

  Fig. 2

  Fig. 4

  also observed that nematodes demonstrate a high efficiency in selective ingestion of algae in comparison with other meiofaunal groups. H. ulvae appeared less proficient in algal selection than meiofaunal grazers. N. phyllepta, the algal species used in the present study may have been too small (<30 µm) to allow selective ingestion by H.

Table captions Table 1 .

 captions1 Comparison of the feeding activity of three different grazers (the foraminifera A. tepida, the Brouage mudflat nematode community and the gastropod H. ulvae).

			Meiofauna	Macrofauna
		Ammonia tepida	Nematode community	Hydrobia ulvae
	Individual weight (gC organic ind -1 )	1.0 10 -6	1.3 10 -7	1.8 10 -4
		Effect of environmental factors on ingestion rate of bacteria
	Ratio between ingestion rates of bacteria at 30 and 10°C	3.6	1.6	1.5
	Effect of salinity (18 against 31 ‰)	Negative	None	None
	Effect of luminosity (Darkness against 83 µM of photons m -2 s -1 )	None	Positive	None
			Maximal ingestion rates of bacteria and algae
	Maximal ingestion rate of bacteria (10 -3 gC bacteria gC grazer -1 h -1 )	0.06	0.92	7.45
	Maximal ingestion rate of algae (10 -3 gC algae gC grazer -1 h -1 )	0.94	5.08	9.56

Table 1

 1

Table ( s)

 (

Acknowledgements

We thank Amélie Sallon for her help in sample preparation, Gaël Guillou for isotopic analyses and Utte Wollenzien from NIOO-CEMO for giving us a Navicula phyllepta strain.

We are grateful to Johnson Galen for English corrections. The "Conseil Général de Charente Maritime", the programme ECCO, the programme PNEC -Chantier Littoral Atlantique and the ANR-VASIREMI financially supported this work.


Christine Dupuy
email: cdupuy@univ-lr.fr

Linda Rossignol

Emmanuelle Geslin

Pierre-Yves Pascal

AMMONIA TEPIDA (CUSHMAN Foraminifer

) Christine Dupuy

PREDATION OF MUDFLAT MEIO-MACROFAUNAL METAZOANS BY A CALCAREOUS FORAMINIFER, AMMONIA TEPIDA (CUSHMAN, 1926)

Keywords: Mudflat, foraminifera, Ammonia tepida, grazing, nematodes, benthic copepods, Hydrobia ulvae

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

INTRODUCTION

Foraminifera are heterotrophic protists present in both pelagic and benthic marine environments. Benthic foraminifera inhabit all water depths and populate a variety of microhabitats on and in the substrate (Murray, 1991;[START_REF] Ellison | Foraminifera and meiofauna on an intertidal mudflat, Cornwall, England: populations; respiration and secondary production; and energy budget[END_REF][START_REF] Chandler | Foraminifera may structure meiobenthic communities[END_REF]Moodley and others, 1998;[START_REF] Alve | Major aspects of foraminiferal variability (standing crop and biomass) on a monthly scale in an intertidal zone[END_REF]2002;2005;Murray and Alve, 2000). They utilize a diversity of trophic mechanisms and nutritional resources [START_REF] Goldstein | Foraminifera: a biological overview[END_REF], and probably play an important role in food webs that influences the structure of the benthic community [START_REF] Altenbach | Short term processes and patterns in the foraminiferal response to organic flux rates[END_REF]Linke and others, 1995;Moodley and others;[START_REF] Alve | Major aspects of foraminiferal variability (standing crop and biomass) on a monthly scale in an intertidal zone[END_REF]Fontanier and others, 2002;Nomaki and others, 2008;Suhr and others, 2008).

Trophic behaviors exhibited by foraminifera include selective (grazing) and indiscriminate herbivory [START_REF] Jeeps | Studies on Polystomella Lamarck (Foraminifera)[END_REF][START_REF] Sliter | Laboratory experiments on the life cycle and ecologic controls of Rosalina globularis d'Orbigny[END_REF][START_REF] Jones | Morphogroups" of agglutinating foraminifera. Their life positions and feeding habitats and potential applicability in (paleo)ecological studies[END_REF]Lee and others, 1991;Moodley and others 2002;Witte and others, 2003;Nomaki and others, 2008), symbiosis (Lee and Anderson, 1991), parasitism (Cedhagen, 1994), uptake of dissolved organic matter (DeLaca and others, 1981;[START_REF] Lipps | Biotic interactions in benthic foraminifera[END_REF], passive suspension feeding [START_REF] Lipps | Biotic interactions in benthic foraminifera[END_REF][START_REF] Cedhagen | Position in the sediment and feeding biology in Astorhiza limicola Sandahl[END_REF][START_REF] Lutze | Rupertina stabilis (Wallich), a highly adapted suspensionfeeding foraminifer[END_REF]Lutze and Thiel, 1989), and deposit feeding [START_REF] Lipps | Biotic interactions in benthic foraminifera[END_REF][START_REF] Jones | Morphogroups" of agglutinating foraminifera. Their life positions and feeding habitats and potential applicability in (paleo)ecological studies[END_REF]. The majority of foraminiferal species are assumed to be omnivorous, feeding on organic detritus, bacteria, and algae [START_REF] Lee | Nutrition and physiology of the foraminifera[END_REF][START_REF] Lipps | Biotic interactions in benthic foraminifera[END_REF]. Some foraminifera are known to feed on metazoans [START_REF] Buchanan | A contribution to the biology of Astrorhiza limicola[END_REF]Bowser andothers, 1986, 1992;[START_REF] Goldstein | Foraminifera: a biological overview[END_REF]Suhr and others, 2008), but most of them are not exclusively carnivorous and utilize carnivory in addition to at least one other trophic mechanism [START_REF] Goldstein | Foraminifera: a biological overview[END_REF]. Carnivory by planktonic foraminifera is well documented (e.g. [START_REF] Boltovskoy | Recent Foraminifera: W. Junk[END_REF]Wright, 1976, Bé andothers, 1977), but little is known about this behavior among benthic foraminifera (see review in [START_REF] Goldstein | Foraminifera: a biological overview[END_REF]. The position of foraminifera in food webs remains conjectural despite direct observations on the diets of some species [START_REF] Murray | Ecological experiments on Foraminiferida[END_REF]Anderson and Lee, 1991;Lee and others, 1991;[START_REF] Bernhard | Bacterial biofilms as a trophic resource for certain benthic foraminifera[END_REF][START_REF] Goldstein | Foraminifera: a biological overview[END_REF]Heinz and others, 2005;Pascal and others, 2008a).

Foraminifera typically use their pseudopodia to gather and ingest food (Bowser and others, 1992). Planktonic foraminifera are known to prey upon copepods and other crustaceans [START_REF] Anderson | A cytochemical fine structure study of phagotrophy in a planktonic foraminifer Hastigerina pelagica (d'Orbigny)[END_REF]Bé and others, 1977;[START_REF] Caron | Predicted and observed feeding rates of the spinose planktonic foraminifer Globigerinoides sacculifer[END_REF]Snider and others, 1984;Hemleben and others, 1988). Some larger benthic foraminifera also feed on metazoans. For example, Peneroplis pertusus (Forskål) feeds on copepods by ingesting the internal soft parts after which it discards the empty carapace (Winter, 1907cited by Goldstein, 1999). The large agglutinated foraminifera Astrorhiza limnicola (Sandahl) feeds on crustaceans and echinoderms [START_REF] Nyholm | Studies on Recent Allogromiidae, I. Micrometula hyalostriata[END_REF][START_REF] Buchanan | A contribution to the biology of Astrorhiza limicola[END_REF]; however, the same species has also been observed only as a suspension feeder [START_REF] Cedhagen | Position in the sediment and feeding biology in Astorhiza limicola Sandahl[END_REF]. Several authors [START_REF] Delaca | The morphology and ecology of Astrammina rara[END_REF]Bowser and others, 1992;Suhr and others, 2008) consider Astrammina rara (Rhumbler), an other large agglutinated foraminifera, to be carnivorous. Relatively smaller benthic foraminifera such as Elphidium and Pyrgo also have been observed capturing prey, but not feeding on them (Jepps, 1942;Suhr and others, 2008). Really small studies suggest predation by small benthic foraminifera [START_REF] Christiansen | Notes of the biology of foraminifera[END_REF][START_REF] Hallock | A predatory foraminifer, Floresina amphiphaga, n. sp., from the Florida Keys[END_REF]Cehdagen, 1994).

The purpose of this study is to examine and document the trophic interactions between the smaller benthic foraminifera Ammonia tepida and several kinds of metazoans in laboratory-controlled experiments. Ammonia tepida is known as a deposit-feeder on algae [START_REF] Lee | Nutrition and physiology of the foraminifera[END_REF]Stouff and others, 1999;Moodley and others, 2000) and bacteria (Goldstein and Corliss, 1994;Langezaal and others, 2005;Pascal and others, 2008a). Species of Ammonia have been used in numerous laboratory experiments because the genus is ubiquitous in inner-shelf, estuarine, and saltmarsh environments (Murray, 1991), where they tolerate wide ranges in temperature, salinity, and other physico-chemical parameters (e.g.. [START_REF] Bradshaw | Laboratory experiment on the ecology of foraminifera: Contributions from the Cushman Foundation for Foraminiferal Research[END_REF][START_REF] Schnitker D | Ecotype variation in Ammonia beccarii (Linné)[END_REF][START_REF] Walton | The genus Ammonia Brünnich, 1772: its geographic distribution and morphologic variability[END_REF]Debenay and others, 1998). Being hardy and readily collected, Ammonia is ideal subjects for laboratory study of living specimens.

METHODS

COLLECTION OF FORAMINIFERS AND METAZOANS

Foraminifera and invertebrates were sampled by scratching the superficial centimetre of sediment at low tide from the upper-intertidal zone of Brouage mudflat, located on the French Atlantic coast about 20 km south of La Rochelle, 45°54'N, 1°7'W (Fig. 1). Time between collection and experiment was minimized. This environment supports a high density of living foraminifera, especially Ammonia tepida (Pascal and others, 2008b). The meiofaunal community of this mudflat is dominated by nematodes (95%), with subsidiary copepods (2%), both of which are present throughout the year (Rzeznik-Orignac and others, 2003). The deposit-feeding gastropod Hydrobia ulvae (Pennant), known as the common mudsnail (90% of macrofauna in terms of abundance), is also a common inhabitant of intertidal mudflats in Western Europe [START_REF] Bachelet | Post-recruitement phase population dynamics of the intertidal gastropod Hydrobia ulvae (Pennant)[END_REF][START_REF] Barnes | Reproductive strategies in contrasting populations of the coastal gastropod Hydrobia ulvae. II. Longevity and life-time egg production[END_REF][START_REF] Sola | Reproduction, population dynamics, growth and production of Scrobicularia plana da Costa (Pelecypoda) in the Bidasoa estuary, Spain[END_REF]Bocher and others, 2007).

Fig 1 here

LABORATORY PROCEDURES

Sediment samples were washed through a 50-µm sieve and the >50-µm fraction was distributed among several glass Petri dishes. Extraction of living calcareous Ammonia tepida was facilitated by exposing the sediment to 80 adult Hydrobia ulvae, common adult mudsnail (Haubois and others 2004), as previously demonstrated by Rossignol and others (2007). After two days, most of the sediment had been ingested by the gastropods and excreted as fecal pellets, and the living benthic foraminifera that had eluded the gastropod were clean and readily visible for efficient picking with a very fine brush. Specimens of A. tepida were transferred to Petri dishes filled with 0.2 µm of filtered seawater from the study area. Foraminifera were acclimated for a minimum of 24 hours at 18°C with a daily light-dark cycle before the feeding experiment commenced.

Living nematodes were concentrated by first seawater-washing sediment samples through a 65-µm nylon. Sediments >65 µm were distributed on a 0.5-cm-thick layer of coarse sand (grain size: 1mm) over a 20-µm nylon mesh placed above a seawater bath and exposed to light for two days.

Following this negative phototropism method described by Rzeznik-Orignac and others ( 2004), living nematodes migrated from the sand, through the nylon mesh, and into the seawater. The living nematodes were then cleaned of organic matter. Nematodes were transferred to 0.2-µm-filtered seawater from the study area and acclimated prior to the feeding experiment, as described above for A.

tepida.

Ten live A. tepida and 10 live nematodes or copepods were placed in 0.2-µm filtered seawater on the same 5-cm-diameter Petri dish. The control dish similarly received 10 live nematodes or copepods but no foraminifera. Foraminiferal vitality was verified by observing pseudopodial activity, while nematode and copepod vitality was verified in their mobility. All feeding experiments were carried out at room temperature (18°C). Nematodes were kept in the dark, whereas copepods had a 24 hours light-dark cycle. Each part of the experiment was repeated three to five times. The behaviors of foraminifera, nematodes, and copepods were observed under a stereomicroscope regularly over 24 hours. Photographs were taken with a LEICA stereomicroscope (DMIRB 400 maximum magnifications) equipped with an Olympus DP-70 digital camera and Visilog software.

An additional series of experiments was carried out where each dish had one juvenile Hydrobia ulvae gastropod and five A. tepida, under the same light-dark cycle that was applied in the copepod experiments. Observations were made with an Olympus SZX-12 stereomicroscope equipped with a Olympus E-330 numerical camera.

SPECIMEN PREPARATION FOR ELECTRONIC MICROSCOPY

The various steps of this preparation method were performed with small microcentrifuge (Eppendorf-type) tubes that were conducive to the preservation of the fine, fragile pseudopodia. When nematodes were captured by foraminifera, samples were fixed in a solution of 2.5% glutaraldehyde diluted in 0.45-µm filtered seawater for 24 hours. To avoid desiccation and consequent loss of detail, the foraminiferal specimens with prey were washed with 0.2 M cocadylate buffer (pH 7.4). Postfixation was accomplished using 2% OsO4 diluted in the cocadylate buffer, followed by three rinses with 0.45-µm-filtered seawater and dehydration in a 507595% series of ethanol baths, plus two baths of 100% ethanol. Specimens were then immersed in HMDS (hexamethyldisilazane) for 10 minutes, after which they air-dried. Dried specimens were mounted on SEM stubs covered with carbon-conductive adhesive tape and double-coated with carbon. Observations and imaging were obtained with a Jeol 6301F SEM at the SCIAM (Service Commun d'Imagerie et d'Analyses Microscopiques) of the University of Angers.

RESULTS

PREDATOR-PREY RELATIONSHIP BETWEEN FORAMINIFERA AND NEMATODES

Acclimated specimens of Ammonia tepida extruded a dense network of pseudopodia that extended onto the dorsal and ventral surfaces of the test. The foraminifera generally oriented themselves perpendicular-to-oblique relative to the bottom of the Petri dish, and used their pseudopodial network to attach at two main points on the glass bottom (Fig. 2a).

Nematodes encountering the networks were immediately entrapped (Fig. 2b, observation using a camera and stereomicroscope), adhering to the pseudopodia or to the cyst on the ventral side of the tests (Figs. 2f-i). When stuck in the foraminiferal pseudopodia, the nematodes struggled, but rarely escaped. Some nematodes were captured by two foraminifera (Figs. 2f,2g). About 18 hours after initial contact, the foraminifera began to empty the nematode of its soft tissues (Fig. 2e). All of the prey's contents suck out within six hours (Figs. 2c,2d), leaving only an empty cuticula (Fig. 2c,2d).

Each cast-off cuticula was characterized by a hole that had been created by the predatory foraminifera (Figs. 2d). The predation of nematodes by A. tepida was observed every time these two meiofaunal organisms made contact. The control nematodes that were not in placed into association with foraminifera remained alive and unscathed after 24 hours.

Fig. 2 here

PREDATOR-PREY RELATIONSHIP BETWEEN FORAMINIFERA AND OTHER METAZOAN ORGANISMS

As with the nematodes, copepods also fell prey to A. tepida whenever the two made contact.

Despite vigorous attempts to escape, copepods could not free themselves from the pseudopodial mesh.

Often, two foraminifera trapped the same copepod (Fig. 3a). After ingesting the soft tissues of the prey, the empty carapace was discarded. The predation of these copepods by A. tepida was observed each time these two organisms were put together.

One observation was made of A. tepida feeding on a juvenile gastropod. The foraminifera attached to the aperture of a living Hydrobia and, about 20 hours later, the snail shell was empty (Fig. 3b,3c).

DISCUSSION

The carnivorous behavior of Ammonia tepida in the laboratory has also been observed on meio-macrofauna. It appears that this carnivorous behaviour of A. tepida is not specific for the specimens of our study area. A similar carnivorous behaviour has been observed in A. tepida collected from the Japanese coast (H. Nomaki, personal communication, 2008) indicating that carnivory in A.

tepida is a general feeding strategy under laboratory conditions. But is this feeding strategy used by A. tepida in its natural environment?

In mudflat environments, due to the abundance of nematodes (on average, 42 times more abundant than foraminifera) and foraminifera, especially in the surface sediments of mudflats (Pascal and others, 2008b), contact between A. tepida and small metazoans is likely to occur very frequently.

In the Brouage mudflat, for example, nematode abundances range 8004,050  10 3 m  (mean of 2,100  10 3 m  ) with maximum abundances in winter and spring (Rzeznik-Orignac and others, 2003), while foraminifera range 20170  10 3 m  (mean of 50  10 3 foraminifera m  ) with their maximum in winter (Pascal and others, 2008b). By preying on meiofauna, A. tepida may be considered to be on a trophic level similar to metazoan consumers, but it is probably not alone among the benthic foraminifera. Microscope imaging with fatty-acid biomarker analysis strongly suggests that the diet of Astrammina rara (Rhumbler) includes polychaetes, crustaceans, molluscs, and echinoderms (Suhr and others, 2008). [START_REF] Chandler | Foraminifera may structure meiobenthic communities[END_REF] reported on Ammonia beccarii (Linné) thought to possibly be in an amensal relationship with the copepod Amphiascoides limicola (Brady). The presumption is that this is an indirect result of having consumed most of microflora, leaving the less nutritious detritus for the copepod. We suggest that the lower copepod densities observed by [START_REF] Chandler | Foraminifera may structure meiobenthic communities[END_REF] could also be due to foraminiferal predation or copepod avoidance of sediments supporting high densities of Ammonia.

However, our results suggest that A. tepida are able to directly feed on large and actively moving grazers.

Ammonia species have typically been considered herbivores, with Ammonia tepida feeding upon algae and bacteria (Goldstein and Corliss, 1994;Moodley and others, 2000;Langezaal and others, 2005;Pascal and others, 2008a). Moodley and others (2000) reported Ammonia sp. exhibiting rapid uptake of freshly deposited algal carbon. It is now evident that some of those foraminifera are likely to be omnivorous. Ammonia, the most common benthic foraminiferal genus, ubiquitous in inner shelf, estuarine, and saltmarsh environments, may be able to employ various feeding strategies according to the most available food sources. Thus, A. tepida's position in the benthic food web is complex, occupying both primary and secondary consumer positions. Thus, they may have a greater impact on benthic community structure than previously suspected. Because of their limited motility, active hunting for prey is probably not common among carnivorous foraminifera. Whereas A. tepida spreads its pseudopodial network more extensively in this feeding mode than when grazing, the trap appears to be intentionally set to entangle larger, mobile prey that unwarily wander into it. Additional experiments are needed to confirm if A. tepida switches feeding modes according to the most available food sources.

Three significant questions arise from our results:

1. How do prey remain attached to foraminiferal pseudopodia? [START_REF] Goldstein | Foraminifera: a biological overview[END_REF] stated that the pseudopodia of carnivorous foraminifera are specifically designed to catch prey. Foraminifera secrete an adhesive [START_REF] Buchanan | A contribution to the biology of Astrorhiza limicola[END_REF], possibly in their Golgi vesicles [START_REF] Goldstein | Foraminifera: a biological overview[END_REF], which is then transported to the peripheral cytoplasm and released by expulsion in the vicinity of prey (Anderson and Lee, 1991;Bowser and others, 1992). This substance is very sticky and thought to be non-toxic (Langer and Bell, 1995). We observed that the pseudopodia and ventral cyst of A. tepida are capable of retaining very active nematodes and copepods. Thus, A. tepida is able to produce an adhesive substance that can be used for capturing meio-macrofauna. However, we cannot confirm that this adhesive substance is produced only for capturing food. In the case of feeding on bacterial biofilm, [START_REF] Bernhard | Bacterial biofilms as a trophic resource for certain benthic foraminifera[END_REF], using time-lapse microscopy, revealing that biofilm parcels are transported extracellularly toward the foraminiferal cell body by pseudopodia, an observation which further implicates pseudopodial function in foraminiferal trophic mechanisms.

How do foraminifera immobilize prey?

It is not clear from our observations whether foraminifera sedate or kill their prey prior to ingestion. It is conceivable that foraminifera use narcotic agents or extracellular enzymes to immobilize or kill their prey, as [START_REF] Anderson | A cytochemical fine structure study of phagotrophy in a planktonic foraminifer Hastigerina pelagica (d'Orbigny)[END_REF] suggested for planktonic foraminifera. In either case, the digestion of prey by foraminifera usually occurs via extruded pseudopodia [START_REF] Buchanan | A contribution to the biology of Astrorhiza limicola[END_REF], and that process will eventually terminate any active resistance.

Fig. 3 here

3. How do foraminifera penetrate the cuticula of nematodes? Austin and others (2005) propose that foraminifera such as Haynesina Banner and Culver use their test ornamentation to mechanically break diatom frustules. Ammonia tepida also present pustules around the aperture of the test (Fig. 3d,e) and this ornamentation may be used in penetrating cuticula.

It has been suggested that secretions by pelagic foraminifera aid in digesting prey (Snider and others, 1984). Spindler and others (1984) claim that pelagic foraminiferal pseudopodia are not physically capable of boring through crustacean carapaces, and therefore there some substance must be secreted to dissolve the carapace at the point of entry. Benthic foraminifera such as A. tepida may also use chemical digestion. Bowser and others (1985) observed pseudopodial activities in Allogromia, and reported that pseudopodia tore small pieces from a gel, a behavior termed skyllocytosis. The authors suggested that skyllocytosis may also be used by carnivorous foraminifera to obtain prey tissues.

Skyllocytosis is an alternative hypothesis for how A. tepida penetrates the nematode cuticula.

CONCLUSIONS

Although previous studies reported Ammonia tepida as an herbivore that feeds on algae and bacteria, the species behaved a carnivore in our laboratory experiments, intentionally orienting itself and extending its sticky pseudopodial network to capture metazoans that wandered too close. We observed A. tepida preying upon nematodes, copepods, and a gastropod larva that were placed near them. Thus, the species is probably omnivorous and secondary consumer in its natural environment, possibly switching feeding modes to take advantage of the food that is most readily available and finally may potentially influence the structure of benthic communities. Further experiments are needed to determine whether A. tepida has a trophic preference when multiple food types are simultaneously available. We also need to quantify in situ carbon fluxes between nematodes, copepods, juvenile Hydrobia, and this foraminifera, and then integrate these fluxes in a food web as we continue unravelling the complexities of the carbon cycle in coastal mudflats.

ACKNOWLEGMENTS

The work was supported by the Charente Maritime Regional Council and the French ANR (National Research Agency) through the VASIREMI project "Trophic significance of microbial biofilms in tidal flats" (contract ANR-06-BLAN-0393-01). Special thanks to the technical assistance of the SIAM of the University of Angers. We are grateful to Ralf Schiebel and Frans Jorissen for their helpful comments on the manuscript. We are also grateful to Carolyn Engel-Gautier and anonymous foraminiferologist for English corrections. tepida; e detailed view of imaged.

FIGURE 1 :FIGURE 2 :FIGURE 3 :

 123 FIGURE 1: Location of the sampling site at Brouage mudflat. Extremity of the arrow: sampling station.