Use of benthic vs planktonic organic matter by sandy-beach organisms: A food tracing experiment with 13C labelled diatoms

Journal of Experimental Marine Biology and Ecology 407 (2011) 309–314

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Journal of Experimental Marine Biology and Ecology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j e m b e

Use of benthic vs planktonic organic matter by sandy-beach organisms: A food tracing experiment with 13C labelled diatoms Tatiana F. Maria a, b,⁎, Marleen De Troch a, Jan Vanaverbeke a, André M. Esteves b, Ann Vanreusel a a b

Ghent University, Biology Department, Marine Biology, Krijgslaan 281-S8, B-9000 Ghent, Belgium Universidade Federal de Pernambuco, Av. Prof. Moraes Rêgo, S/N, Departamento de Zoologia, Cidade Universitária, Recife, Pernambuco 50670-901, Brazil

a r t i c l e

i n f o

Article history: Received 13 April 2011 Received in revised form 22 June 2011 Accepted 29 June 2011 Available online 23 July 2011 Keywords: Benthic food webs Ecosystem functioning Macrofauna Meiofauna Stable isotopes

a b s t r a c t Benthic diatoms are assumed to be of minor importance as food sources in sandy-beach food webs, since they are typically scarce in sandy-beach sediments; whereas organic matter derived from land and sea is known to be more important in sandy-beach food webs. In order to test if benthic and planktonic diatoms play a minor or major role, respectively, in sandy-beach food webs, a laboratory experiment was conducted. Labelled planktonic and benthic diatoms, enriched in the stable carbon 13C isotope, were offered as food sources to sandy-beach macrofauna and meiofauna communities. Uptake of both types of diatoms occurred with most of the species, but benthic diatoms were preferentially consumed by two macrofaunal species and all meiofaunal species. This result reveals the importance of benthic carbon sources in both macrofaunal and interstitial food webs, and suggests a link between both food webs through the common use of benthic diatoms. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Food webs in sandy-beach sediments are believed to be constituted of two apparently unconnected parts, a macrofaunal and an interstitial food web (McLachlan and Brown, 2006). The former is known to depend largely on direct input of phytoplankton, macrophyte detritus, carrion and stranded algae (Bergamino et al., 2011) whereas the latter is based on dissolved and particulate organic matter (McLachlan and Brown, 2006). This organic matter may be derived from both terrestrial and marine ecosystems, due to the position of sandy beaches between land and sea (Scapini, 2003). This dual input compensates for the virtual absence of primary producers and the microbial loop in sandy-beach sediments (McLachlan and Brown, 2006). Indeed, sandy beaches have very little in situ primary production compared with rocky shores and estuarine mudflats (Brown & McLachlan, 1990; Inglis, 1989). The resident primary producers are mainly represented by epipsammic organisms, which form a biofilm on the sediment, and a few epipelic diatoms, the free-living forms (Speybroeck et al., 2008a). On sheltered beaches, these may contribute somewhat to primary productivity, although never in large amounts (Hartwig, 1978; Leach, 1970). A recent stable isotope field study, however, demonstrated that the diet of sandy-beach organisms – independently of the type of food web that they belong to – depends on benthic and planktonic organic matter (Maria et al., submitted for

⁎ Correspondent author. Tel.: +32 92648523; fax: +32 92648598. E-mail address: [email protected] (T.F. Maria). 0022-0981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2011.06.028

publication). Dense mats of microphytobenthos are not typically found on sandy-beach sediments (McLachlan and Brown, 2006) because of the high hydrodynamic wave action and tidal regime. Hence, the importance of microphytobenthos in sandy-beach food webs is often neglected. However, the contribution of a food source to the diet of beach organisms may be more related to its quality than its quantity (Tenore, 1983). Furthermore, the overlap of the natural isotopic signatures of benthic and planktonic primary producers in a sandy beach did not allow elucidation of the carbon sources of all beach-inhabiting heterotrophic organisms (Maria et al., submitted for publication). This overlap largely occurs in coastal food-web studies, and hinders the interpretation of food web interactions (Mutchler et al., 2004). Tracer stable isotope experiments with pre-labelled food sources are an alternative and efficient way to overcome situations of redundant isotope signatures of potential food sources (Evrard et al., 2010; Galván et al., 2008; Herman et al., 2000). Another aspect in the complexity of the food web on sandy beaches is the co-occurrence of two heterogenic size-class groups (McLachlan, 1983), the macrofauna and meiofauna, and the role of different carbon sources in their diet. Macrofaunal organisms prefer detritus and phytoplankton as a direct food source, and therefore the majority (90%) of the sandy-beach macrofauna is represented by filter-feeders and/or detritivores (Ansell et al., 1978; Gianuca, 1983; McLachlan and Bate, 1984; McLachlan and Brown, 2006; McLachlan and Romer, 1990; Steele, 1976). In contrast, several feeding modes are known for meiofauna, with a preference for detritus and benthic diatoms as food sources (Giere, 2009). In this study, we investigated the response of sandy-beach organisms to the availability of epipsammic benthic and pelagic

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diatoms. The null hypothesis tested here was: there is no difference in the uptake of benthic vs pelagic diatoms as a food source. This hypothesis was tested for meiofaunal and macrofaunal organisms and discussed in relation to their feeding types. 2. Material and methods 2.1. Culture and labelling technique of diatoms Two monoclonal strains of marine epipsammic benthic (Seminavis robusta) and planktonic diatoms (Phaeodactylum tricornutum) were obtained from the diatom culture collection of the research group Protistology and Aquatic Ecology (Biology Department, UGent). The clones from which the lineage of S. robusta was started were isolated from a sample collected in November 2000 from the ‘Veerse Meer’, a brackish-water lake in Zeeland, The Netherlands. P. tricornutum was isolated from Blackpool, UK in 1956 by the Culture Collection of Algae and Protozoa, UK. Epipsammic were preferred over epipelic benthic diatoms because of their dominance in sandy-beach sediments (Speybroeck et al., 2008a) and ease of cultivation (DeTroch, personal communication). Seminavis spp. are abundantly found in the Westerschelde estuary and nearby beaches (Sabbe K., pers. comm.) and P. tricornutum is also reported from sites along the shores of the North Sea (Hendey, 1964). Diatoms were grown in f/2 medium (Guillard, 1975) based on filtered and sterilised artificial seawater (salinity of 35, Instant Ocean® salt, Aquarium Systems, France) in sterile tissueculture vials (S. robusta) and Erlenmeyer flasks (P. tricornutum) in a climate room at 16 ± 1 °C with a 12:12 h light-dark period and 25– 50 μmol photons m −2 s −1. For labelling of the diatom cells with 13C, 5 ml of a solution with NaH 13CO3 (sodium bicarbonate, 13C, 99%, Cambridge Isotope Laboratories, Inc.; 336 mg in 100 ml milliQ H2O) was added per 100 ml culture medium. S. robusta was harvested after 23 d and P. tricornutum after 14 d of incubation. Both species were harvested one day before the experimental set-up. To harvest, the medium was rinsed away and benthic-diatom cells were extracted by washing 3× with artificial sea water; planktonic diatoms were triple centrifuged (3000 rpm, 10 min) to remove non-incorporated 13C. After rinsing, the concentrated diatom suspensions were kept at 16 °C under dark conditions to avoid further growth of the diatoms and potential loss of 13C prior to the start of the experiment. To estimate the density of diatom cells in cultures, the cells were homogeneously suspended by shaking and then 50 μl of the cell suspension was transferred into a well of a multidish (96 wells). In a few minutes, after all the cells settled to the bottom of the well, they were counted under a Zeiss Axiovert 135 inverted microscope (Zeiss Gruppe, Jena, Germany). The densities obtained were used to estimate the densities that were applied in the experimental microcosms (see further). To evaluate the 13C incorporation in the diatom cells, an aliquot of concentrated diatom culture was pipetted into 2.5 × 6 mm pre-treated (4 h at 550 °C to remove any contaminating organic C) Al cups and dried overnight at 60 °C for further analysis. This labelling technique yielded an increase in δ 13C from − 15 ± 3.7‰ to 2680 ± 147‰ (S. robusta) and from − 7 ± 0.6‰ to 2961 ± 179‰ (P. tricornutum). 2.2. Collection of natural beach communities One day after the diatoms were harvested, sediment from the upper 5 cm was collected from the middle intertidal level of the ultradissipative sandy beach at De Panne (51°05′30″N, 02°34′01″E) in front of the nature reserve “Westhoek reservaat” on 24 August 2010 during low tide. The intertidal area is approximately 440 m wide and has four runnels parallel to the water line. The sampling area was restricted to a sandbar in order to ensure a homogeneous nematode community composition, as runnels and sandbars are known to

harbour dissimilar nematode communities (Gingold et al., 2010). An aquarium with the bottom previously removed was used as a core. The 20 aquaria, each with a surface area of 156.25 cm 2, were filled in the field with 500 ml of undisturbed sediment. Sea water was also collected. In the laboratory, the sediment was covered by 500 ml of ambient seawater previously filtered on a pre-combusted glass-fibre filter (Whatman GF/F, 25 mm diameter) to remove any debris or organisms. Aquaria were kept aerated for 1 day in a dark, controlledtemperature room (15 °C) until the start of the experiment, in order to stabilise. 2.3. Experimental design To test whether sandy-beach organisms have a preference for benthic or planktonic diatoms, aquaria were randomly allocated to four treatments: (1) B*: a total of 15 × 10 6 cells of S. robusta (labelled) (2) P*: a total of 30 × 10 6 cells of P. tricornutum (labelled) (3) B*P: a total of 7.5 × 10 6 cells of S. robusta (labelled) and 15 × 10 6 cells of P. tricornutum (unlabelled) (4) BP*: total of 7.5 × 10 6 cells of S. robusta (unlabelled) and 15 × 10 6 cells of P. tricornutum (labelled) A control containing only sediment and sterile seawater with no addition of diatoms was also included in the experiment in order to calculate excess 13C (above background) (see further). The number of cells in each treatment was standardised for total biomass added per experimental unit. This equal-biomass standard was based on the overall cell size in our diatom cultures, where S. robusta was almost double the size of P. tricornutum. The level of food supply provided was considered as very saturated (De Troch et al., 2005; 2007). The diatoms were spread evenly over the surface of each aquarium with a Pasteur pipette and left to settle for 1 h. Next, the microcosms were covered with an opaque lid and the aeration restarted. All experimental units were aerated and placed randomly on shelves at 15 ± 1 °C in a dark, temperature-controlled room. The uptake of labelled diatoms by macrofauna and meiofauna was analysed after 5 d of grazing. Previous experiments showed that meiofauna uptake was constant after 4 d of grazing (e.g. De Troch et al., 2005). 2.4. Sample processing After 5 d the experiment was terminated by removing the seawater, filtering it over a 38 μm sieve and adding 2% glutaraldehyde to each aquarium. The fauna was extracted from the sediment by 10× decantation over a sieve of 1 mm for macrofauna and 38 μm for meiofauna. After decantation, the extracted macrofauna and meiofauna were sorted and identified under a binocular stereomicroscope. Meiofauna was sorted into Turbellaria (60 individuals), Copepoda (30–50 individuals) and Nematoda. Nematodes were split into Sigmophoranema rufum (115 individuals), Enoplolaimus litoralis (60 individuals), “1B” (150 individuals), and “2A” (170 individuals) and were picked separately. 1B and 2A represented non-selective deposit feeders and epistrate feeders, respectively, according to the classification of Wieser (1953). All organisms were hand-picked with a fine sterile needle under a binocular microscope, rinsed twice in MQ water to remove adhering particles, and transferred to a drop of MQ water in 2.5 × 6 mm pre-treated (4 h at 550 °C to remove any exogenous organic carbon) Al cups. Subsequently, these cups were oven-dried overnight at 60 °C, pinched closed and stored under dry atmospheric conditions until further analysis. For macrofauna, Scolelepis squamata, Eteone sp., Bathyporeia pilosa, Bathyporeia sarsi and Eurydice pulchra were ground and homogenised, and sub-samples of 1.0 mg of each species were transferred to Ag cups stored in multi-well microtitre plates under dry atmospheric

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conditions until analysis. Crustaceans were decalcified by addition of 0.25 M HCl for 15 min.

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3. Results 3.1. Diatom uptake by meiofauna

2.5. Isotope analysis All isotope measurements were performed on a PDZ Europa ANCAGSL elemental analyser interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK) by the UC Davis Stable Isotope Facility (University of California-Davis, USA). Minimal He dilution was applied because of the low biomass of meiofauna samples. δ 13C isotope data were corrected from the measurement of empty Al or Ag control cups. The values of δ 13C were standardised per unit carbon of organism as recommended by Middelburg et al. (2000). Incorporation of 13C is reflected as excess 13C (above background) and expressed as total C uptake (I) in μg of 13C per individual and calculated as the product of excess 13C (E) and individual biomass (organic carbon). Excess 13C is the difference between the 13C fraction of the control (Fcontrol, i.e., based on the natural signature of organisms that did not feed on labelled diatoms) and the sample (Fsample), where F = 13C /( 13C + 12C) = R / (R + 1). The carbon isotope ratio (R) was derived from the measured δ 13C values as R = (δ 13C / 1000 + 1)*RVPDB, with RVPDB = 0.0112372 as the carbon isotope ratio of the reference material (Vienna Pee Dee Belemnite - VPDB) (Fry 2006). Since the labelled diatoms had different initial δ 13C signatures (see before), the uptake per unit carbon of organism was further standardised, taking into account the proportion of 13C in each diatom food source. The amount of diatom carbon that was taken up by organisms and expressed per unit carbon of organism was therefore corrected with a factor derived from the atomic 13C percentage of the diatom food source. 2.6. Data analysis Differences in uptake among the treatments were tested by means of one-way analysis of variance (ANOVA). When significant differences were detected, Tukey's HSD test was applied to test for pairwise differences, using 95% confidence limits. Prior to ANOVA, Cochran's Ctest was used to check the assumption of homoscedasticity; if the data did not fulfil this requirement, data were log(x + 1) transformed. When the requirements for parametric statistics were not met even after transformation, the data were compared using the non-parametric Kruskal–Wallis ANOVA by ranks with subsequent pairwise comparison, and 95% confidence limits. All tests were performed using the Statistica 7.0 software package.

All meiofauna organisms were enriched in all treatments (Fig. 1A). In the treatments with only labelled benthic or planktonic food sources, copepods showed the highest Δδ 13C values, followed by turbellarians and epistrate feeders (2A nematodes) in the benthic and planktonic treatments, respectively (Fig. 1A). In the treatments with mixed benthic and pelagic diatoms, turbellarians and copepods showed the highest Δδ 13C when benthic diatoms were labelled, but when planktonic diatoms were labelled, copepods had the highest Δδ 13C followed by epistrate-feeding nematodes (Fig. 1A). Comparing the four diatom treatments in terms of uptake per unit of organism carbon, there was a significant difference among treatments for all meiofauna taxa (Fig. 1B, Table 1). Higher uptake per unit of organism carbon occurred in the treatment with labelled benthic diatoms (B*) for turbellarians, non-selective deposit-feeding nematodes (1B), epistrate-feeding nematodes (2A), S. rufum and E. litoralis; while harpacticoid copepods showed a higher uptake of benthic diatoms in the mixed treatment B*P (Fig. 1B). The post-hoc analyses (Tukey test or Post-hoc paired comparison for non-homoscedastic data, Table 2) showed that the benthic-diatom treatment (B*) differed significantly from the planktonic-diatom treatment (P*) (p b 0.01). All species/groups showed a lower uptake in the planktonic-diatom treatment (P*) compared to the benthicdiatom treatment (B*). In the mixed treatment with labelled planktonic diatoms (BP*), the uptake per unit of organism carbon was lower for almost all organisms. Both single treatments differed from the mixed-diatom treatments, except in the case of epistratefeeding nematodes and S. rufum, where there was no difference between the treatments with benthic diatoms (B*) and the mixed diet of labelled benthic diatoms and unlabelled planktonic diatoms (B*P). In addition, turbellarians did not show significant difference between the planktonic treatment (P*) and both mixed diets (B*P and BP*) (Table 2). 3.2. Diatom uptake by macrofauna There was little enrichment in the polychaetes S. squamata and Eteone sp. and the isopod E. pulchra, whereas high enrichment was observed for the amphipods B. sarsi and B. pilosa (Fig. 2A). There was no significant difference between the latter species in terms of Δδ 13C within each treatment (p N 0.05) (Fig. 2A). Comparing uptake per unit of organism carbon, there was no significant difference among treatments for S. squamata and E. pulchra, whereas a significant

Fig. 1. Meiofauna uptake: (A) expressed as Δδ13C among the treatments; (B) expressed as total uptake per unit of organism carbon. B*: benthic diatoms labelled, B*P: benthic diatoms labelled and planktonic diatoms unlabelled, P*: planktonic diatoms labelled and BP*: benthic diatoms unlabelled and planktonic diatoms labelled.

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Table 1 Variance analyses on total uptake (μg13C.μgC−1) of meiofauna and macrofauna based on ANOVA (parametric data) and Kruskal–Wallis (non-parametric data) analyses. df: degree of freedom, MS(treat): mean square of the treatment, MS(res): mean square of the residual, p: probability. Significant results (p b 0.05) are indicated in bold. df Meiofauna Turbellaria Copepoda 1B 2A Sigmophoranema rufum Enoplolaimus litoralis Macrofauna Scolelepis squamata Eurydice pulchra Bathyporeia sarsi Bathyporeia pilosa

MS(treat)

MS(res)

F-ratio

p-value

3, 2, 3, 3, 3, 3,

8 6 8 8 8 8

0.04 0.17 – 6.95 – –

0.002 0.001 – 0.015 – –

18.82 118.05 – 462.92 – –

b 0.001 b 0.001 0.016 b 0.001 0.021 0.016

2, 2, 3, 3,

3 3 8 8

0.08 0.00 1925.87 3.68

0.032 0.017 393.851 0.127

1.13 4.21 5.27 29.03

0.432 0.134 0.032 b 0.001

–: Values not given by Kruskal–Wallis analysis.

difference among treatments was found in both Bathyporeia species (Table 1). The lowest uptake occurred in the treatment of unlabelled benthic diatoms and labelled planktonic diatoms — BP* (Fig. 2B). The Tukey test indicated that there was no difference among the treatments B*, P* and B*P, and these three treatments differed from the mixed treatment BP* (Table 2). 4. Discussion Overall, the highest uptake by meiofauna was noted in the benthic-diatom treatment (B*), followed by the treatment with labelled benthic diatoms and unlabelled planktonic diatoms (B*P), and then followed by the planktonic-diatom treatment (P*). The lowest uptake was found in the treatment with unlabelled benthic diatoms and labelled planktonic diatoms (BP*). The exceptions to this pattern were S. rufum and epistrate feeders, which showed no difference between B* and B*P. Therefore, the higher significant uptake in the treatment with solely benthic diatoms and/or in the mixed treatment where benthic diatoms were labelled shows the preference for benthic diatoms over pelagic ones by all meiofauna organisms. Diatoms were reported previously as part of the turbellarian diet (Straarup, 1970). For macrofauna organisms, only amphipods (Bathyporeia spp.) showed no significant differences in the total uptake per unit of carbon between the treatment with only benthic cells (B*) and the single planktonic (P*) treatment. This suggests that these Bathyporeia species did not discriminate between both food sources. However, in the mixed treatments a clear preference for benthic diatoms was

Table 2 Paired comparisons based on the results of table 1 following ANOVA (parametric) and Kruskal–Wallis analyses (non-parametric).

Meiofauna Turbellaria Copepoda 1B 2A Sigmophoranema rufum Enoplolaimus litoralis Macrofauna Scolelepis squamata Eurydice pulchra Bathyporeia sarsi Bathyporeia pilosa

B* x B*P

B* x P*

B* x BP*

B*P x P*

B*P x BP*

P* x BP*

≠ – ≠ = = ≠

≠ – ≠ ≠ ≠ ≠

≠ – ≠ ≠ ≠ ≠

= ≠ ≠ ≠ ≠ ≠

= ≠ ≠ ≠ ≠ ≠

= ≠ ≠ ≠ ≠ ≠

– – = =

= = = =

= = ≠ ≠

– – = =

– – ≠ ≠

= = ≠ ≠

-: No comparison due to the absence of replication in one of the treatments.

observed as a higher uptake in the B*P treatment was found in comparison to the BP* treatment. The use of benthic and recently settled pelagic diatoms by Bathyporeia species was reported previously by Herman et al. (2000). Although microphytobenthos is an important food source for micro-, meio- and macrobenthos in intertidal and shallow subtidal areas (Evrard et al., 2010; Granéli and Turner, 2002; Middelburg et al., 2000; Moens et al., 2002; Sundbäck and Persson, 1981), their role remains poorly quantified for food webs on sandy beaches (Speybroeck et al., 2008a; Spilmont et al., 2005; Urban-Malinga and Wiktor, 2003). Our experiment showed for the first time that macrofaunal and meiofaunal sandy-beach organisms prefer locally produced benthic organic matter over marine pelagic food sources. The preference for diatoms – one of the components of the microphytobenthos – by members of the meiofauna and macrofauna contradicts the expected pattern of the use mainly of direct input from planktonic origin in sandy-beach food webs (Bergamino et al., 2011; McLachlan and Brown, 2006), but supports the findings of Maria et al. (submitted for publication) who demonstrated that both meiofaunal and macrofaunal food webs depend partly on benthic primary production. The broad term “sandy beach” is applied to a wide range of environments, from exposed ocean beaches to extremely sheltered sand flats (McLachlan, 1983). Within this range, local primary production is potentially more important in sheltered situations because of the low hydrodynamic forces and the very fine sediment; the combination of both variables concentrates benthic diatoms in the first centimetres of the sediment (Hartwig, 1978; Leach, 1970). Previous knowledge of sandy-beach macrofaunal food webs is based on research in high-energy systems, where pelagic diatoms have proved to be an important food source (Ansell et al., 1978; Bergamino et al., 2011; Heymans and McLachlan, 1996; Lercari et al., 2010; Steele, 1976). Meiofauna food webs in sandy beaches are poorly documented. Phytoplankton has been shown to be highly significant in the diet of meiofauna in salt marshes (Buffan-Dubau and Carman, 2000; Carman and Fry, 2002; Maddi et al., 2006), whereas microphytobenthos is important for meiofauna in intertidal mudflats (Moens et al., 2002; Rzeznik-Orignac et al., 2008) and subtidal sandy sediments (Evrard et al., 2010). Therefore the utilisation of benthic diatoms by sandy-beach organisms suggests that ultradissipative sandy beaches are more similar to tidal flats, especially in the meiofauna context, since the diet of nematodes in tidal flats is based on microphytobenthos and settled phytoplankton (Moens et al., 2002). Our experiment showed that benthic diatoms are indeed a carbon source in the sediment of ultradissipative sandy beaches, and must not be ignored in tracing and/or modelling sandy-beach food webs in dissipative extremes. However, diatom uptake by itself does not seem to be sufficient to provide the amount of carbon required to sustain the meiobenthic community (Hicks and Coull, 1983; Warwick and Price, 1979) and Bathyporeia species (Speybroeck et al., 2008b). The refutation of our null hypothesis suggested that some, but not all sandy-beach organisms feed selectively on benthic diatoms. For example, S. squamata, Eteone sp. and E. pulchra did not distinguish between the two food sources offered. The low diatom uptake by these organisms may be related to their feeding types; the two latter organisms are known to be predators (Fauchald and Jumars, 1979; Speybroeck et al., 2008a), although E. pulchra did not show the natural 15 N isotopic signature of predators at the same beach (Maria et al., submitted for publication) and S. squamata is a suspension/deposit feeder (Dauer, 1983). Among the meiofaunal organisms studied, epistrate-feeding nematodes (2A) showed the highest selectivity for benthic diatoms. This nematode feeding group is well adapted to feed on diatoms because of its buccal cavity morphology and a muscular pharynx bulb (Jensen, 1987; Moens and Vincx, 1997), and it is found

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Fig. 2. Macrofauna uptake: (A) expressed as Δδ13C among the treatments; (B) expressed as total uptake per unit of organism carbon. B*: benthic diatoms labelled, B*P: benthic diatoms labelled and planktonic diatoms unlabelled, P*: planktonic diatoms labelled and BP*: benthic diatoms unlabelled and planktonic diatoms labelled.

in high numbers in the upper sediment layers of sandy beaches (Maria et al., submitted for publication; Platt, 1977). Since benthic diatoms are more abundant in the superficial sediment layers during low tide (Mitbavkar and Anil, 2004), this feeding strategy may explain the increases and decreases in abundances of epistrate nematodes in the superficial sediment layers during low tide and high tide, respectively (Maria et al., submitted for publication). The higher carbon uptake of epistrate feeders compared to S. rufum, a possible food competitor, supports the suggestion that S. rufum does not compete with epistrate feeders for food resources, in agreement with the findings of Maria et al. (submitted for publication). As stated by the authors, competition may be partly avoided by the spatial segregation between S. rufum and epistrate nematodes in the sediment layers. Unfortunately, the design adopted here does not enable us to reach further conclusions about competition. The relatively high benthic diatom uptake observed for copepods is in disagreement with the natural stable isotope profile that was found for sandy-beach copepods, as their natural isotopic signature did not correspond to the signature of microphytobenthos neither to the one of suspended organic matter (Maria et al., submitted for publication). In the field copepods can utilise diatoms in combination with other potential food sources whereas in the experiment there was a high availability of diatoms. However, the maximum size of 40 μm for the benthic diatoms used in this feeding experiment corresponded approximately to the lower limit of the benthic diatoms extracted from the field. Therefore, grazing on the newly added food might have been easier, resulting in the discrepancy between the experimental data and field results. Our experiment showed that benthic diatoms can be the basis of both macrofaunal (with some restrictions) and interstitial food webs. So far, the connection between macrofaunal and meiofaunal food webs in sandy-beach sediments has been presumed to exist only in the surf-zone through common utilisation of dissolved and particulate organic matter as food sources (McLachlan and Brown, 2006) or by consuming different proportions of microphytobenthos and suspended particulate matter (Maria et al., submitted for publication). Our findings demonstrated that these two food webs in intertidal sediments are connected to some degree through the use of the same benthic organic matter. Peterson (1999) stated that feeding experiments using stableisotope techniques are an effective means to reach robust conclusions about feeding strategies. However, caution is due when extrapolating the results of this laboratory experiment to more complex in situ conditions. In our experiment, some physical factors, such as wave action and tidal regime, were excluded when the intertidal sediments were left under constant subtidal conditions to avoid washing away the benthic diatoms. Besides, benthic consumers may demonstrate preference or avoidance for a particular diatom size (De Troch et al. 2006) and/or diatom species (Wyckmans et al. 2007).

Taking this into account, the outcome of this experiment indicates that benthic diatoms can play an important role in sustaining, partially, meiofaunal and part of the macrofaunal food webs in intertidal sandy-beach sediments. These findings generally correspond with what we could deduce from the natural isotopic composition of food sources and organisms from an ultradissipative sandy beach (Maria et al., submitted for publication). This approach indicates the potential contribution of benthic (epipsammic) diatoms to trophic interactions at the base of food webs of sandy beaches.

Acknowledgements We thank Niels Viaene for his support during the sampling campaign, as well as Annick Van Kenhove, Tania Nara and Guy De Smet for their help during the meiofauna picking. Olga Chepurnova and Pieter Vanormelingen are acknowledged for their assistance during the diatom cultivation and harvesting. Janet W. Reid, JWR Associates is acknowledged for her critical revision of the English. The two anonymous referees are acknowledged for their constructive comments in a previous version of this manuscript. The first author is sponsored by a PhD scholarship from Vlaamse Interuniversitaire Raad — VLIR-UOS. The second author is a postdoctoral fellow of the Research Foundation — Flanders (FWO-Flanders, Belgium). This paper contributes to FWO project G.0041.08N. [ST]

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