Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay of Biscay (France)

Estuarine, Coastal and Shelf Science xxx (2016) 1e12

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Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay of Biscay (France)
cile Masse
a, b, *, Guillaume Meisterhans a, b, Bruno Deflandre a, b, Guy Bachelet a, b, Ce
lie Ciutat a, b, Florence Jude-Lemeilleur a, b, Line Bourasseau a, b, Sabrina Bichon a, b, Aure a, b a, b
mare a, b, Fre
de
ric Garabetian a, b , Natalie Raymond , Antoine Gre Nicolas Lavesque a b

Universit
e de Bordeaux, EPOC, UMR 5805, F-33615 Pessac, France CNRS, EPOC, UMR 5805, F-33615 Pessac, France

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 January 2015 Received in revised form 10 December 2015 Accepted 5 January 2016 Available online xxx

Changes in (1) main sediment characteristics, and (2) benthic bacterial and macrofaunal communities were simultaneously addressed along an innereouter transect within the West Gironde Mud Patch (Bay of Biscay, NE Atlantic), through the sampling of three stations (E: inner part, C: central part and W: outer part) during July 2010. Except in the top centimetre where a sandy layer was found at station E, all sediments were muddy and tended to be coarser and richer in photosynthetic pigments and particulate organic carbon and nitrogen at stations C and W than at station E. Maximum oxygen penetration depth was also lower at station E than at stations C and W. These results are consistent with: (1) the occurrence of strong hydrodynamics precluding current sedimentation in the inner part of the WGMP, and (2) conversely, inputs of particles originating from the Gironde Estuary in its central and outer part. Prokaryotic cell abundances were lower at station E than at stations C and W. Bacterial community composition also differed more clearly at station E as compared to stations C and W. Conversely, macrofaunal abundances and species richness decreased monotonously from station E to station W (i.e., along the innereouter gradient). Macrofaunal composition strongly differed at station E on one side, and stations C and W on the other side. These results are consistent with the current paradigm regarding the long term effect of major rivers on benthic macrofauna given that strong hydrodynamics at station E precludes the sedimentation of fine particles originating from the Gironde Estuary. At last, we found no overall correlation between benthic bacterial and macrofaunal compositions, which may however clearly result from limitations in our sampling design. © 2016 Elsevier Ltd. All rights reserved.

Keywords: Atlantic Ocean West Gironde Mud Patch Macrobenthos Bacterial community

1. Introduction In soft bottom coastal sediments, bioturbation-mediated interactions between benthic macrofaunal and microbial communities are critical for key biogeochemical processes including organic matter remineralisation (Kristensen and Kostka, 2005; Solan and Wigham, 2005; Aller, 2014). Bioturbation by engineer macrobenthic species induces the differentiation of biogenic structures from the bulk sediment (Jones et al., 1994; Cardinale et al., 2004; Mermillod-Blondin and Rosenberg, 2006). This affects microbial community density or biomass (Aller and Yingst,


um National d’Histoire Naturelle, * Corresponding author. Present address: Muse CRESCO, F-35800 Dinard, France.
). E-mail address: [email protected] (C. Masse

1985; Reichardt, 1989; Aller, 1994), composition (Marinelli et al., 2002; Bertics and Ziebis, 2009; Laverock et al., 2010) or functions such as potential exoenzyme activity (Stief et al., 2004), nitrification and/or denitrification (Svensson and Leonardson, 1996; Gilbert et al., 2003; Bertics et al., 2012), nitrogen fixation (Bertics et al., 2010, 2012) or sulphate reduction (Bertics and Ziebis, 2010). In the field, community bioturbation potential can be assessed from the inventories of macrofaunal species through the functional classification of organism traits associated with sediment mixing (Queiros et al., 2013). Besides, the analysis of benthic macrofaunal community composition has proved to be a useful indicator of the environmental ecological status (Dauer, 1993; Borja et al., 2000). Along a gradient of organic matter enrichment, oxygen depletion and/or physical disturbances, structural changes in benthic macrofaunal communities follow a generalized successional model

http://dx.doi.org/10.1016/j.ecss.2016.01.011 0272-7714/© 2016 Elsevier Ltd. All rights reserved.


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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characterized by typical spatio-temporal gradients in species richness, biomass and abundances (e.g. Pearson and Rosenberg, 1978; Rosenberg, 2001). Changes in the spatial patterns of macrofaunal community composition relate to the abiotic filter that structures communities at the regional and/or local scale and, therefore, reflect changes in the benthic habitat (Pearson and Rosenberg,
mare et al., 2009). 1978; Dauer, 1993; Borja et al., 2000; Gre Up to now, factors influencing the kilometre-scale distribution of bacterial communities inhabiting marine sediments have seldom been addressed. Changes in macro- and meiofaunal community composition have been related to changes in bacterial biomass along a seaward transect in a Mediterranean beach (Papageorgiou et al., 2007). Density and biomass of bacteria, meio- and macrofauna have also been shown to change relatedly along a transect of quality and quantity of organic matter in the NW Mediterranean Sea (Albertelli et al., 1999) and in the deep Eurasian Arctic Ocean € ncke et al., 2000). Water depth, sediment edaphic conditions (Kro and distance between sites explained the spatial patterns of bacterial community composition in Mediterranean (Polymenakou et al., 2005; Semprucci et al., 2010) and Pacific sediments (Hewson et al., 2007). Surprisingly, no study however simultaneously addressed macrofaunal and bacterial community composition. As mentioned above, various studies have shown that bioturbation can result in micro-niche differentiation and subsequent structural and compositional changes in sediment bacterial communities at the centimetre scale of macrofaunal burrows (Marinelli et al., 2002; Bertics and Ziebis, 2009; Laverock et al., 2010). These studies were however based on laboratory experiments and in situ data is still clearly lacking. The West Gironde Mud Patch (WGMP) is a 420 km2 clay-silt sedimentary patch of ca. 4 m in thickness, located on the French Atlantic coast, 25 km off the mouth of the Gironde Estuary in the Bay of Biscay (Jouanneau et al., 1989). About 50% of the terrigenous particle discharge from the Gironde Estuary (Lesueur et al., 1989), i.e. 1.5
106 tons of particles per year (Castaing and Jouanneau, 1987) are deposited in the WGMP. Sedimentary processes are different between the inner and outer parts (Weber et al., 1991). In the outer part, surficial sediments are supplied with fine particles originating from the Gironde Estuary while in the inner part, surficial sediments are supplied with inorganic sandy shelf deposits. This induces a gradient in the surficial sediment grain size in the WGMP with smaller particles in its outer part (Jouanneau et al., 1989). Moreover, the differentiated sedimentary processes result in an innereouter increasing gradient in organic carbon content and lability, which is reflected in micro-biomass respiration, as measured by ETS (Electron Transfer System) activities (Relexans et al., 1992). While sedimentary processes have been extensively studied in the WGMP (Jouanneau et al., 1989; Lesueur et al., 1989, 2002; Weber et al., 1991; Gadel et al., 1997; Parra et al., 1999), the associated biota was only poorly investigated (Relexans et al., 1992) and the composition of its benthic macrofaunal communities has never been assessed despite bioturbation was evidenced by core radiography in the outer part of the WGMP (Jouanneau et al., 1989). The present study therefore aimed at documenting changes in both benthic macrofaunal and bacterial communities along the innereouter gradient already described for the WGMP and to assess how these changes support the current paradigms regarding: (1) sedimentary processes in the WGMP and (2) the long term effect of large rivers on benthic macrofauna within their prodeltas. In addition, we hypothesized that both (1) bioturbationmediated interactions between benthic macrofauna and bacteria and (2) the prominent action of the innereouter gradient on these two biological compartments would result in linked distribution patterns of benthic macrofaunal and bacterial communities in the WGMP. A secondary objective therefore consisted in assessing the

correlation between the composition of benthic bacterial and macrofaunal communities. 2. Material and methods 2.1. Sampling Three stations located on a NEeSW (innereouter) transect within the WGMP were sampled in July 2010 on board of the RV ^tes de la Manche during the BIOMIN-1 cruise: station E Co (45 450 648N; 1310458W), station C (45 400 962N; 1410508W) and station W (45 350 808N; 1510918W) (Fig. 1). Five replicate cores were collected at each station using a multiple corer (Oktopus GmbH, Germany) fitted with 8 polycarbonate tubes (inner diameter 95 mm), which allowed the sampling of the top 20 cm of sediment and the overlying bottom water without disturbing the sedimentewater interface. One single core was used to assess sediment characteristics (i.e. median grain size (D50), Particulate Organic Carbon (POC), Particulate Organic Nitrogen (PON)). Three cores were used to characterize chloropigments (i.e. chlorophyll a and phaeophytin a) and bacterial abundance and community composition. All these 4 cores were sliced every 0.5 cm from 0 to 2 cm depth, every cm from 2 to 5 cm depth, and every 2 cm from 5 to 11 cm depth. The 1e1.5 and 1.5e2 cm slices were pooled, as well as the 3e4 and 4e5 cm, and the 7e9 and 9e11 cm, for prokaryotes and bacterial assessments for the sake of homogeneity with a previous cruise. For bacterial community composition, sediment was collected in a sterile container and homogenized with a sterile spatula. Within 2 h after collection, 1 gWW of sediment of each slice per core was preserved in 2 mL of 3.7% formalin for later determination of prokaryotic abundances. Another gWW of sediment was preserved in 1 mL of preservative buffer (100 mM TriseHCl [pH 8.0], 100 mM EDTA [pH 8.0], 1.5 NaCl and 1% [wt/vol] cetyltrimethylammonium bromide) (Zhou et al., 1996) for later analysis. All sediment samples were stored at 80  C until treatment. Oxygen vertical microprofiles were assessed on a fifth sediment core. Benthic macrofauna was sampled with a Hamon grab (0.25 m2, 3 replicates). Sediment was sieved through a 1-mm mesh and the remaining fraction was fixed in 4% formalin. 2.2. Sediment analyses D50 was assessed using a Malvern® Master Sizer laser microgranulometer. POC and PON were measured using a ThermoFinnigan® Flash Elemental Analyser Series 1112. Chlorophyll a and phaeophytin a were measured according to Neveux and Lantoine (1993) using a Perkin Elmer® spectrofluorometer. Chloropigments were not assessed in the 9e11 cm slices. Oxygen microprofiles were assessed at in situ temperature in a temperature-controlled incubator kept in darkness at 100 mm depth increments with Unisense® OX100 Clark-type sensors (Revsbech, 1989). Microelectrodes were connected to a highsensitivity 4-ways picoammeter. Linear calibrations were achieved between the bottom-water oxygen content, precisely determined by a Winkler titration and the zero oxygen in the anoxic part of the sediment. Steady-state profiles (n ¼ 11 at stations E and W and n ¼ 7 at station C) were randomly performed in the collected sediment core from each site to account for spatial microheterogeneity. The position of the sedimentewater interface was determined from a break in the oxygen-concentration gradient. Maximum oxygen penetration depths were assessed using the ^ne, 2010). software PRO2FLUX (Deflandre and Duche


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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Fig. 1. Map of the West Gironde Mud Patch showing the locations of the 3 sampled stations (E, C, W) along a NEeSW (innereouter) geographical transect. Sedimentary map modified from Allen and Castaing (1977).

2.3. Prokaryotic abundances Prokaryotic cells were desorbed from sediment particles using a protocol modified from Duhamel and Jacquet (2006). Tween 80 (0.5% final) and sodium pyrophosphate (0.1% final) were gently mixed with each sediment sample (30 min, 720 rpm) and the resulting suspension was sonicated for 20 min. The cellular fraction containing prokaryotes was purified using a migration on Gentodenz density gradient (1.310 g mL1) according to Amalfitano and Fazi (2008). Cellular fractions were preserved in formalin (3.7% final) at 80  C until flow cytometry analysis at the Plateforme
anologique de Banyuls-surCytom
etrie-Imagerie (Observatoire Oce Mer). Extemporaneously, cellular fractions were diluted (1:100 v:v) in artificial filtered (0.22 mm) seawater and stained during 10 min with SYBR®Green I (50 mL mL1; 25 X). A standard 0.97 mm fluorescent bead suspension was then added as an internal reference to each sample before running, under a low flow rate (10 mL min1), allowing a single cell analysis (Amalfitano and Fazi, 2008). Data were acquired using the CellQuest® software. Cytogram analysis provided event numbers, identified as prokaryotic cell numbers, during the counting time (1 min). Counts were subsequently normalized to flow rate and to the mass of extracted sediment to calculate a density number in cells gDW of sediment1. Stained prokaryotic cells were excited at 488 nm and discriminated

according to their right angle light scatter (SSC, related to cell size) and green fluorescence (FL1) emission measured at 530 ± 30 nm. Photosynthetic prokaryotes were eliminated by discrimination on a plot of FL1 versus red fluorescence (FL3) (Lebaron et al., 2002). 2.4. Bacterial community composition Bacterial community composition was characterized by ARISA (Automated Ribosomal Intergenic Spacer Analysis), a PCR-based whole-community fingerprinting method (Fisher and Triplett, 1999). DNA extraction was achieved on 700 mL of homogenized thawed sediment sample. DNA was extracted and purified by coupling a bead beating method (Lysing matrix E tubes and Fast Prep; MP Biomedicals®: two runs at 5.5 m s1 during 30 s) to the UltraClean® Soil DNA Isolation Kit (MO BIO Laboratories). The amount of extracted and purified DNA was quantified by spectrofluorimetry (Perkin Elmer® LS 55) using SYBR®Green and a standard DNA of calf thymus solution at 1 mg mL1. PCR amplification of the 16Se23S rDNA intergenic spacer was carried out using 50 FAM labelled S-D-Bact-1522-B-S-20 (50 -TGC GGC TGG ATC CCC TCC TT-30 ) and L-D-Bact-132-a-A-18 primers (50 CCG GGT TTC CCC ATT CGG-30 ) (Normand et al., 1996). The final reaction mix (25 ml) consisted of 1X PCR buffer (Promega), 1.5 mM


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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MgCl2, 0.3 mg mL1 bovine serum albumine, 5% DMSO, 200 mM of each deoxynucleoside triphosphate (Invitrogen), 0.5 mM of each primer (Invitrogen), 0.25 U of Taq polymerase (Promega) and 10 ng of template DNA at about 1 ng mL1. Amplification was performed in a thermocycler (Eppendorf AG), consisting in an initial denaturation at 94  C for 5 min, followed by 35 cycles of denaturation (94  C, 1 min), annealing (55  C, 1 min) and extension (72  C, 1 min) and a final extension (72  C, 10 min). For each extracted DNA sample, triplicate PCR assays were performed using 3
10 ng of template DNA. Amplification products of the 3 assays were pooled and purified using QIAquick PCR Purification Kit (QIAgen). The purified amplification products were then quantified using the spectrofluorimetric method previously described for extracted DNA quantification. Finally, 2 mL of amplification product adjusted by dilution to about 10 ng mL1 were mixed with 0.5 mL GeneScan 1200 LIZ internal size standard (Applied Biosystems) and 9 mL Hi-Di formamide (Applied Biosystems). The mixture was denatured at 95  C for 3 min and fragments were discriminated using an ABI 3730XL automated sequencer (Applied Biosystems) operated by the Plateforme Genome-Transcriptome Pierroton (INRA, Bordeaux, France). The resulting electrophoregrams were analysed using the Applied Biosystems Peak Scanner software. Peak sizes <200 bp and >1200 bp were considered as background noise and eliminated. An “optimal divisor” (Od) was then determined to remove fluorescence background within remaining peaks (Osborne et al., 2006). Peaks contributing less than 0.1% (i.e. Od value) of the total amplified DNA (as determined by relative fluorescence intensity) were eliminated from profiles as being indistinguishable from baseline noise. Binning was carried out according to Ramette (2009) under the R software (http://cran.r-project.org) using the algorithm “Interactive binner” (http://www.ecology-research. com). Finally, a matrix of relative abundances (fluorescence) of the different Intergenic Transcribed Spacers (ITS) representing the Operational Taxonomic Units (OTU) was generated. 2.5. Benthic macrofauna Benthic macrobionts were sorted, counted and identified to the lowest possible taxonomic level. Biomass was determined at the phylum level as ash-free dry weight (AFDW) after desiccation (60  C, 48 h) and calcination (450  C, 4 h). This allowed assessing benthic macrofaunal abundance, biomass and species richness. The bioturbation potential of each taxa was assessed by considering their scores of mobility in the sediment (Mi) and sediment reworking (Ri) as provided by Queiros et al. (2013). Mi scores were 1 for organisms that lived in fixed tubes, 2 for organisms with limited movement, 3 for organisms with slow but free movement through the sediment matrix, 4 for organisms with free movement via burrow system. Ri scores were 1 for epifauna, 2 for surficial modifiers, 3 for upward and downward conveyors, 4 for biodiffusors and 5 for regenerators. The higher its score of Mi and Ri, the more the taxa can be considered as a strong bioturbator. The analysis of the Ri and Mi scores listed in Queiros et al. (2013) for 1030 taxa shows that: (1) the Ri*Mi distribution is clearly bimodal with two maxima at 6 and 12, and (2) only ca. 25% of the considered taxa have a Ri*Mi  12. This led us to consider that taxa with Ri*Mi  12 were potentially strong bioturbators. The cumulated relative abundance of those taxa was considered as an index of the bioturbation potential at each of the three sampled stations. 2.6. Statistical analyses The significance of differences in D50, POC and PON relative to depth and between stations could not be tested due to the lack of

replication. Changes in these parameters were thus only qualitatively assessed. Univariate One-Way PERMANOVAs (Anderson, 2001; McArdle and Anderson, 2001) were used to assess differences between stations in maximum oxygen penetration depth and macrofaunal abundance, total biomass, species richness and Simpson's diversity index. Univariate Two-Ways PERMANOVAs were used to assess differences between stations and sediment depths in chloropigments, prokaryotic abundance, and bacterial Simpson's diversity index. In all cases we used untransformed data and Euclidean distance as (dis)similarity measure. Multivariate One-Way PERMANOVA was used to assess differences in benthic macrofaunal composition between stations. A SIMilarity PERcentage analysis (SIMPER) was performed to identify the taxa contributing most to intra-station similarity and to interstation dissimilarity (Clarke and Warwick, 2001). A multivariate Two-Ways PERMANOVA was used to assess differences in bacterial composition between stations and sediment depths. One-Way designs always consisted of a factor ‘Station’ fixed with three levels: E, C and W. Two-Ways designs consisted of two crossed factors: ‘Station’ fixed with the same three levels, and ‘Sediment depth’, fixed with 10 or 7 levels depending on the considered dependent variable(s). Both bacterial and macrofaunal community compositions were also assessed using non-metric Multi Dimensional Scaling nMDS (Clarke and Warwick, 2001). For both bacterial and macrofaunal community composition analyses, we always used untransformed data and the BrayeCurtis index as a measure of similarity (Clarke et al., 2006). The relationship between macrofaunal and bacterial communities' composition was addressed by assessing the significance of the correlation between bacterial and macrofaunal similarity (untransformed data, BrayeCurtis similarity) matrices using a Mantel test (Mantel, 1967). As there were no direct associations between collected sediment cores for bacterial community analysis and benthic grabs for macrofaunal community analysis, a total of 216 ¼ 3!
3!
3! different combinations of core and grab pairs (i.e. 3! combinations of core and grab pairs per station) was possible. These 216 combinations were exhaustively analysed using Mantel tests and the HolmeBonferroni procedure (Holm, 1979) to correct p-values for multiple comparisons. All statistics were performed using the PRIMER 6 package (Clarke and Warwick, 2001) and its PERMANOVA add on (Anderson, 2001). 3. Results 3.1. Sediment characteristics Vertical profiles of D50 tended to be different at station E as compared to stations C and W (Fig. 2). At station E, D50 tended to be coarser in the top 10 mm (19e46 mm) than in the 10e100 mm deep sediments (8e10 mm; Fig. 2A). Conversely, POC and PON tended to be lower in the top 10 mm (0.44e0.65% DW and 0.05e0.07% DW, respectively) than in the 10e100 mm sediment (0.59e0.95% DW and 0.09e0.12% DW, respectively; Fig. 2B and C). At stations C and W, D50 (20e22 mm and 18e28 mm, respectively), POC (0.93e1.07% DW and 1.01e1.29% DW) and PON (0.12e0.13% DW and 0.12e0.16% DW, respectively) conversely tended to be more homogeneous within the whole sediment column. Overall and irrespective of the above-mentioned strong changes observed in the top mm at station E, both POC and PON tended to be lowest at station E, intermediate at station C and highest at station W (Fig. 2). Both ‘Station’ and ‘Sediment depth’ had a significant effect on chlorophyll a and phaeophytin a concentrations (p < 0.003 in all cases, Fig. 2D and E), with a significant interaction between these


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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D50 (μm)

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Fig. 2. Vertical sediment profiles of (A) median grain-size (D50), (B) particulate organic carbon (POC), (C) particulate organic nitrogen (PON), (D) chlorophyll a and (E) phaeophytin a concentrations at the 3 sampled stations. Horizontal bars are standard deviations.

two factors for chlorophyll a (p < 0.001) but not for phaeophytin a (p ¼ 0.408). For chlorophyll a, this mostly resulted from the occurrence of subsurface peaks at stations C and W but not at station E where chlorophyll a tended to decrease uniformly with sediment depth. Phaeophytin a concentrations tended to decrease

with sediment depth, and to be lower at station E than at stations W and C. Maximum oxygen penetration depth was significantly lower at station E (2.9 ± 0.1 mm) than at stations C (3.9 ± 0.8 mm) and W (3.7 ± 0.1 mm) (p < 0.001, Fig. 3).


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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Maximum oxygen penetration depth (mm)

E

3.2. Prokaryotic abundances

Stations C

W Both ‘Station’ and ‘Sediment depth’ had a significant effect on prokaryotic abundances (p ¼ 0.002 and p ¼ 0.008, respectively) with no interaction between these two factors (p ¼ 0.824). Prokaryotic abundances decreased with sediment depth from 3.3, 4.0 and 2.9
107 cells gDW1 in the 0e10 mm layer to 0.3, 1 and 1.5
107 cells gDW1 in the 70e100 mm layer at stations E and C and W, respectively (Fig. 4). The overall (i.e., integrated over the whole sampled sediment column) average prokaryotic cell abundance was lower at station E (1.1
107 cells gDW1) than at stations W (2.0
107 cells gDW1) and C (2.3
107 cells gDW1).

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4 3.3. Bacterial community compositions

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Fig. 3. Maximum oxygen penetration depth in the sediments of the 3 sampled stations. Vertical bars are standard deviations.

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Overall, 306 OTU ranging from 200 to 1120 bp were detected at the 3 studied stations (n ¼ 60), with 274 OTU at station E (n ¼ 20), 191 OTU at station C (n ¼ 20) and 227 OTU at station W (n ¼ 20). Fifty-two percent of the retrieved OTU were present at the 3 sampled stations. ‘Station’ and ‘Sediment depth’ did not significantly affect bacterial Simpson's diversity index (p ¼ 0.216 and p ¼ 0.622, respectively), which ranged from 0.031 ± 0.001 (station E, 30e50 mm) to 0.114 ± 0.057 (station E, 20e30 mm) (data not shown). Bacterial communities compositions was much more heterogeneous at station E (38% intra-group Bray Curtis similarity) than at stations C and W (59 and 61% intra-group Bray Curtis similarities, respectively) (Fig. 5). There was no significant effect of ‘Sediment depth’ on bacterial community composition (p ¼ 0.270). Conversely, bacterial community composition significantly differed between stations (p < 0.001) with a BrayeCurtis similarity of 36% between stations E and C, 34% between stations E and W and 57% between stations C and W. 3.4. Benthic macrofauna

100 Fig. 4. Vertical profiles of prokaryotic abundances along the sediment column at the 3 sampled stations. Horizontal bars are standard deviations.

Overall, 1556 individuals belonging to 45 taxa/species were collected during the present study. Macrofaunal abundance, species richness, and species composition all significantly differed between

Fig. 5. nMDS plot based on the BrayeCurtis similarities of bacterial composition in each sediment sample (i.e., Stations x Sediment depth) collected at the 3 sampled stations.


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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1.6 Molluscs Polychaetes Crustaceans Echinoderms Cnidaria Nemerta Phoronida

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W

Fig. 6. Benthic macrofaunal abundance (A), biomass (B), species richness (C) and Simpson's diversity index (D) at the 3 sampled stations. Vertical bars are standard errors.

stations (p ¼ 0.004, p ¼ 0.033 and p ¼ 0.006, respectively), but not total biomass and the Simpson's diversity index (p ¼ 0.285 and p ¼ 0.106, respectively) (Fig. 6). A total of 1221 individuals (407 ± 69 ind. 0.25 m2), belonging to 32 taxa/species (23.3 ± 2.0 species grab1), with a total biomass of 1.2 ± 0.2 gAFDW 0.25 m2, were collected at station E (Fig. 6 and Table 1). The average Simpson's index was 0.457 ± 0.098. Four species (Kurtiella bidentata, Amphiura filiformis, Owenia fusiformis and Malmgrenia andreapolis) contributed for 90% to the 72.4% intra-station BrayeCurtis similarity. Among those, the bivalve K. bidentata contributed to 66.8% of intra-station similarity. Seven species out of 32 (22%), representing 21% of the total abundance, were classified as potentially strong bioturbators. In station C, 246 individuals (82 ± 26 ind. 0.25 m2) were collected, belonging to 22 taxa (13.7 ± 2.0 species grab1), for a total biomass of 0.9 ± 0.4 gAFDW 0.25 m2 (Fig. 6 and Table 1). The average Simpson's index was 0.243 ± 0.011. Six taxa/species (Rissoidae, Scalibregma inflatum, Terebellides stroemii, A. filiformis, Hilbigneris gracilis and Nephtys incisa) contributed for 90% to the 40.1% intra-station similarity. Among those, the Rissoidae contributed to 39.2% to the intra-station similarity. Nine species out of 22 (41%), representing 41% of the total abundance were classified as potentially strong bioturbators. A total of 89 individuals (29.7 ± 3.8 ind. 0.25 m2) belonging to 21 taxa (12.0 ± 1.2 species grab1) and with a total biomass of 0.5 ± 0.1 gAFDW 0.25 m2 (Fig. 6 and Table 1) were collected at station W. The average Simpson's index was 0.220 ± 0.066. Six taxa/

species (Rissoidae, Oestergrenia digitata, T. stroemii, Callianassa subterranea, H. gracilis and M. andreapolis) contributed for 90% of the 54.8% intra-station similarity. The Rissoidae accounted for 49.4% of this intra-station similarity. Nine species out of 21 (43%), representing 33% of the total abundance, were classified as potentially strong bioturbators. Benthic macrofaunal composition clearly differed at station E as compared to stations C and W (Fig. 7). Nine species contributed for 90% of the average dissimilarity between station E and either station C and station W. Among them, K. bidentata characterizing station E clearly contributed most (i.e., >60%). In terms of abundances, station E was dominated by bivalves versus polychaetes at stations C and W. In terms of biomass, all three stations were dominated by echinoderms: ophiurids (i.e., mainly A. filiformis) at station E and holothurids (i.e., Leptopenctata elongata and O. digitata) at stations C and W (Fig. 6, Table 1).

3.5. Correlation between bacterial and macrofaunal community compositions Based on the analysis of the 216 possible combinations of cores and grabs originating from the same station and using both Mantel tests and the HolmeBonferroni method to correct p-value for multiple comparisons, there was no significant correlation between the bacterial and macrofaunal correlation matrices (Supplementary Table 1).


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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Table 1 Abundance (mean per 0.25 m2 ± standard error), mobility (Mi) and sediment reworking scores (Ri) of benthic macrofaunal taxa in stations E, C and W in the West Gironde Mud Patch. A: amphipods; An: anthozoans; B: bivalves; C: cumaceans; D: decapods; G: gastropods; H: holothurians; O: ophiuroids; P: polychaetes; Ph: phoronids. Ri and Mi are from Queiros et al. (2013). Taxa in bold are those classified as potentially strong bioturbators. (score of mobility in the sediment): 1 for organisms that live in fixed tubes, 2 for organisms with limited movement, 3 for organisms with slow and free movement through the sediment matrix, and 4 for organisms with free movement via burrow system. Ri (score of sediment reworking): 1 for epifauna, 2 for surficial modifiers, 3 for conveyors and 4 for biodiffusors (from Queiros et al., 2013). Species/Taxa

E

Abra alba (B) Abra nitida (B) Ampelisca spinipes (A) Ampharete acutifrons (P) Amphiura filiformis (O) Callianassa subterranea (D) Chaetozone sp. (P) Chamelea striatula (B) Corbula gibba (B) Cylichna cylindracea (G) Diastylis bradyi (C) Diastylis laevis (C) Edwardsia claparedii (An) Euclymene oerstedi (P) Euspira nitida (G) Glycera tridactyla (P) Heteromastus filiformis (P) Hilbigneris gracilis (P) Kurtiella bidentata (B) Labioleanira yhleni (P) Lagis koreni (P) Leptopentacta elongata (H) Malmgrenia andreapolis (P) Melinna palmata (P) Nemertea Nephtys incisa (P) Nucula nucleus (B) Oestergrenia digitata (H) Ophiura ophiura (O) Owenia fusiformis (P) Paraonidae (P) Phaxas pellucidus (B) Philocheras bispinosus (D) Pholoe baltica (P) Phoronis muelleri (Ph) Phyllodoce lineata (P) Prionospio malmgreni (P) Processa nouveli holthuisi (D) Rissoidae (G) Scalibregma inflatum (P) Spiophanes kroyeri (P) Sternaspis scutata (P) Terebellides stroemii (P) Upogebia deltaura (D) Venus casina (B)

0.7 0.3 10.7 e 61.3 0.3 e 0.3 2.0 2.3 e 4.0 e 1.7 0.3 2.0 1.0 6.3 270.7 e 2.0 e 9.0 4.3 e e 0.7 e 2.7 7.0 0.3 0.3 1.0 1.7 1.3 e 0.7 e 4.7 4.0 0.3 e e 1.0 2.0

± 0.3 ± 0.3 ± 10.2 ± 8.7 ± 0.3 ± 0.3 ± 1.0 ± 0.3 ± 1.5 ± 0.9 ± 0.3 ± 0.6 ± 0.6 ± 0.7 ± 58.1 ± 1.1 ± 2.3 ± 1.7

± 0.7 ± ± ± ± ± ± ±

1.4 0.6 0.3 0.3 1.0 0.3 0.3

± 0.3 ± 1.8 ± 2.1 ± 0.3

± 0.0 ± 1.0

C

W

e e 1.0 1.3 2.3 0.7 0.3 e 0.7 e e e 4.7 e e e e 3.3 1.0 0.3 e 0.3 0.3 0.7 0.3 7.0 e 1.0 e e e e e e e e e 0.3 25.7 22.3 0.7 1.7 6.0 e e

e 0.3 0.3 e e 1.3 e e e e 0.3 e e e e 0.7 0.7 3.3 e 0.3 e e 1.3 e e 0.7 e 3.0 e e 0.3 e 0.7 e e 0.3 e 0.3 12.0 1.0 0.3 0.7 1.3 e 0.3

± 0.6 ± 0.9 ± 0.3 ± 0.3 ± 0.3 ± 0.3

± 3.7

± 2.0 ± 0.6 ± 0.3 ± 0.3 ± 0.3 ± 0.7 ± 0.3 ± 3.2 ± 0.6

± 0.3 ± 10.7 ± 11.8 ± 0.7 ± 1.2 ± 2.5

± 0.3 ± 0.3

± 0.3

± 0.3

± 0.3 ± 0.7 ± 2.0 ± 0.3

± 0.3

± 0.3 ± 0.0

± 0.3 ± 0.7

± 0.3 ± 0.3 ± 3.6 ± 0.6 ± 0.3 ± 0.3 ± 0.3 ± 0.3

Mi

Ri

2 2 1 2 3 4 2 2 2 3 3 3 2 1 3 3 2 3 2 3 1 3 3 1 3 3 3 3 2 1 3 2 4 2 1 3 2 4 3 4 1 3 1 4 2

2 2 2 3 4 4 2 2 2 2 2 2 2 3 2 4 3 4 2 4 3 2 4 3 4 4 2 2 2 2 2 2 2 2 2 4 3 1 2 4 3 4 3 3 2

4. Discussion 4.1. The West Gironde Mud Patch sediments The WGMP is a mud patch contrasting with the sandy soft bottom on most of the south-eastern shelf of the Bay of Biscay on re and Dorel, 1970). Within this the Atlantic French coast (Longe mud bank, previous studies have reported substantial differences in sediment composition from the inner to the outer part (Jouanneau et al., 1989; Weber et al., 1991; Relexans et al., 1992). According to Weber et al. (1991), the WGMP surficial sediments in the inner part are characterized by organic carbon poor and sandysilty sediments while those in the outer part are temporarily enriched in chlorophyll a supplied by a constant fine sedimentation

of particles originating from the Gironde Estuary. Overall, our own results support the conclusions of these former studies. The occurrence of coarse sediment in the uppermost layer of station E might for example be linked to an episode of strong wind and heavy sea the day just before sampling (B.D. personal observation). This suggests that the inner station, which is the shallowest and the closest to the estuarine mouth, is likely to be markedly disturbed by hydrodynamics (tide, waves) as hypothesized by Jouanneau et al. (1989). Besides, our data regarding sedimentary organics characteristics (POC and PON, phaeophytin a and chlorophyll a) were consistent with the assumption that sedimentary processes provided the outer WGMP with a (fresh) organic matter supply (Relexans et al., 1992). 4.2. Bacterial community The ARISA fingerprinting method has successfully been used at different spatial scales, from a few millimetres to kilometres, to assess the dynamics of bacterial community composition in sediment (e.g. Hewson and Fuhrman, 2006; Hewson et al., 2007; Fuhrman and Steele, 2008; Bertics and Ziebis, 2009). During the present study, we observed marked differences in bacterial community fingerprints from 20-km distant stations suggesting spatial changes in benthic bacterial community composition. These changes were consistent with those of sediment POC and PON, chlorophyll a and phaeophytin a concentrations and maximum oxygen penetration depth, which all relate to the availability of organic matter for bacteria in the sediments. As previously observed in the WGMP (Relexans et al., 1992), the prokaryotic abundance tended to increase from the inner to the outer part. The highest values of prokaryotic abundance were thus observed at stations C and W where the highest values of POC, PON chlorophyll a and phaeophytin a contents were recorded. This was in agreement with a previous study reporting positive correlation between bacterial abundance and chlorophyll a sediment content (Albertelli et al., 1999). The availability of organic matter in the sediments likely controls the benthic bacterial community in the WGMP. Moreover, in agreement with a deterministic community assembly mechanism (Nemergut et al., 2013), such distance-dependent changes at the kilometre scale (i.e. geographical patterns) have been previously reported and interpreted as a local selection of dispersed OTU by environmental conditions (Hewson et al., 2007). The central and outer parts of the WGMP are the receptacle of particles coming from the Gironde Estuary (Jouanneau et al., 1989; Weber et al., 1991; Relexans et al., 1992). Free and particle-attached bacteria originating from the Gironde Estuary could settle in their sediments emphasizing the role of dispersal (sensu Nemergut et al., 2013: “movement of organisms across space […] including the rate and the order in which taxa are added to communities”) for the WGMP bacterial community. The observed patterns in bacterial community composition in the WGMP sediments could thus also result from the gradual renewal of estuarine bacterial populations by marine populations from the inner to the outer part. Additional information on the bacterial composition of the Gironde Estuary sedimentary particles and further experiments dedicated to assess the temporal dynamics of the sediment bacterial community composition would allow to further test this hypothesis. During the present study, the Simpson's diversity index, ranging from 0.04 to 0.05, was close to values reported for so-called diversified and balanced communities in mats (Nübel et al., 1999), Mediterranean plankton (Schauer et al., 2000) and phototrophic river biofilms (Lyautey et al., 2003). In marine sediments, values up to 0.01 have however been reported (Hewson et al., 2003; Zinger et al., 2011). The use of peak relative intensity to calculate diversity indices is controversial due to possible biases in PCR


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Fig. 7. nMDS plot based on the BrayeCurtis similarities of macrofaunal composition in each grab collected at the 3 sampled stations.

amplification (e.g. Wintzingerode et al., 1997) but the main limitation to an in-deeper analysis of our data is the lack of temporal replication in sampling preventing from considering e.g. seasonal variations. 4.3. Benthic macrofauna The present study is the first one considering benthic macrofauna in the WGMP. We recorded a clear decrease in benthic macrofaunal abundance and species richness from the inner to the outer part of the WGMP. Relexans et al. (1992) reported a similar general pattern (although not statistically significant due to strong temporal variability) for meiofaunal abundances. These results apparently contradict the current paradigm on the (long-term) impact of riverine inputs in controlling benthic macrofaunal characteristics and composition (Rhoads et al., 1985; Aller and Aller, 1986; Aller and Stupakoff, 1996). According to this paradigm, spatial changes in benthic macrofaunal composition off (major) rivers indeed result from 2 opposite effects, namely: (1) a reduction of benthic fauna at the immediate vicinity of the river mouth due to the inputs of large quantities of sediments resulting in high sedimentation rates and instability, and (2) a positive effect further offshore resulting from moderate organic enrichment. Such a ^ ne River prodelta in spatial pattern was recently found in the Rho periods of high river flow (Bonifacio et al., 2014). Conversely these authors also reported a monotonous inshore/offshore decrease in both benthic macrofaunal and sedimentary organics quantitative characteristics following a dry period. They attributed this last ^ne River, which resulted in the pattern to lower inputs from the Rho lack of negative consequences of sediment deposition even in the very inner part of the prodelta. In agreement with several previous studies (including Jouanneau et al., 1989), our own granulometry results support that sedimentation of fine particles currently does not occur at station E (see the section above). Moreover, our results also show that benthic macrofaunal composition at this station clearly differs from those at stations C and W, as it is dominated by suspension-feeders and more specifically by a species (e.g. K. bidentata) slightly favoured by strong hydrodynamical conditions. Our overall interpretation is thus that the source of disturbance present at station E is not an excess of sedimentation and the associated consequences in

terms of organic contents and sediment instability, but is conversely linked to strong hydrodynamical conditions, which prevent such a deposition. Since the sediments present at station E are either coarser (surface) or much older (deep) than at stations C and W, this pattern also account for the occurrence of lower POC and PON contents at station E conversely to what is observed in the ^ ne River Prodelta following dry periods (Bonifacio et al., 2014). Rho We did not observe major differences (except for abundances and biomass) either in sedimentary organics characteristics and benthic macrofaunal composition between stations C and W, as could be expected from the above-mentioned paradigm (Rhoads et al., 1985; Aller and Aller, 1986; Aller and Stupakoff, 1996). Bonifacio et al. (2014) were also unable to detect similar changes in ^ne River prodelta in spite of a sedimentary organics within the Rho larger number of sampled stations and sampling depths. Conversely, they showed that this gradient was apparent for benthic macrofaunal composition except following dry periods. Our interpretation is thus that the lack of difference between stations C and W recorded during the present study may result from different causes: (1) the fact that these stations were located too close on the sampled transect, and (2) the fact that the magnitude of such differences is likely affected by the flow regime of the considered River (Bonifacio et al., 2014). Since the present study was achieved during a low flow period, it would certainly prove interesting to sample the same transect at different periods of the year and to include at least a supplementary outer station during future studies in the WGMP. According to the Queiros et al. (2013) classification, potentially strong bioturbators (i.e. species with Ri
Mi > 12) were more numerous at station E than at stations C and W. However, at station E, 73.9% of potentially strong bioturbator individuals were ophiurids (A. filiformis) which only rework the top centimetre of the sediment column while 61% and 51% of potentially strong bioturbator individuals were deeply burrowing polychaetes at station C (Scalibregma inflatum) and station W (Hilbigneris gracilis, Malmgrenia andreapolis). Moreover, according to our own observations and based on its Ri
Mi score of 6, the bioturbation potential of the large burrowing echinoderm, Oestergrenia digitata, which was only recorded at station C and W, seems clearly underestimated. The functional classification in Queiros et al. (2013) therefore likely underestimated actual sediment reworking at stations C and W


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

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since the occurrence of enhanced bioturbation in the central and outer parts of the WGMP is further supported by: (1) the occurrence of subsurface maxima in chlorophyll a and phaeophytin a concentrations, (2) deeper oxygen penetration at stations C and W (note that Jouanneau et al. (1989) also hypothesized that sediment compaction could also contribute to decrease oxygen penetration in the inner WGMP sediments), and (3) Sediment Profiles Images collected at the three sampled stations either during the present study or during the LEVIATHAN cruise (A. Romero Ramirez, personal communication). These results fully support previous radiographic investigations showing deeper biogenic structures in sediment cores collected in the outer than in the inner WGMP (Jouanneau et al., 1989). 4.4. Linking bacterial and macrofaunal communities In this study, we simultaneously addressed benthic bacterial and macrofaunal communities in the same well-defined marine ecosystem based on the sampling of three stations located along a putative gradient. The observed patterns (i.e., opposite spatial changes in the main community characteristics) led us to first investigate the possible correlation between bacterial and macrofaunal benthic community compositions based on in situ sampling. We found no significant correlation. However, we cannot exclude that this result may be caused by discrepancies between: (1) the spatial scales (Hamon grab vs 95 mm-diameter corer) associated with the sampling of benthic bacteria and macrofauna, and (2) the taxonomic approaches (morphometric taxonomy vs single gene based phylogeny) used in determining the diversity of benthic bacteria and macrofauna. The first of these two points has already been assessed at the centimetre scale in macrofaunal burrows and under various environmental conditions (e.g., Dobbs and Guckert, 1988; Papaspyrou et al., 2005; Bertics and Ziebis, 2009; Pischedda et al., 2011). At a larger scale, a strong link, likely through the bacterial compartment, between benthic macrofaunal functional diversity and biogeochemical cycling has also been reported in the fine sandy sediments of the North Sea (Braeckman et al., 2014). To our knowledge, the second one is yet to be tackled in relation with the ongoing development of new molecular techniques. Another limitation of our sampling design to tackle the question of the correlation between bacterial and macrofaunal compositions is the lack of correspondence between individual cores and grabs used to address these two biological compartments. This last point precludes the use of statistics for paired samples and imposes the use of multiple tests together with their associated correction, which tends to reduce the power of the test. Moreover, and because of (1) a low number of stations and (2) their location along a strong putative gradient, it should be stressed that, during the present study, even a significant correlation may have resulted either from (1) similar processes structuring both communities, and/or (2) a true interdependency between the composition of the two communities (Horner-Devine et al., 2007). It would therefore certainly prove interesting to further investigate the correlation between benthic bacterial and macrofaunal compositions based on a larger set of stations. 5. Conclusions Benthic macrofaunal and bacterial communities showed opposite patterns and statistically non-significantly correlated compositions along the innereouter gradient sampled in the WGMP. These patterns are interpreted as reflecting: (1) the link between the quantitative characteristics of sedimentary organics and both the abundance of prokaryotic cells and benthic bacterial composition, and (2) the interaction between the availability of sedimentary

organics and the level of hydrodynamics in controlling benthic macrofaunal main characteristics and composition. The lack of correlation between benthic bacterial and macrofaunal compositions may result from limitations in our sampling design. Nevertheless, such community patterns might have functional implications and should control the ecosystemic services delivered by the WGMP benthic communities in the Bay of Biscay. Acknowledgements This study stems from the projects “Diagnostic de la qualit
e des milieux littoraux” funded by R
egion Aquitaine, OSQUAR and FEBBA (20101205007) funded by R
egion Aquitaine and FEDER-EUROPE (Presage n 33732), and BIOMIN funded by LEFE-CYBER/INSU and EC2CO-PNEC/INSU CNRS. Guillaume Meisterhans was supported by
cile Masse
by a a doctoral fellowship from R
egion Aquitaine and Ce doctoral fellowship from French Minist ere de l’Enseignement Sup
erieur et de la Recherche. We thank the captain and the crew of ^tes de la Manche; Pierre Anschutz, Aure
lie Garcia, Olivier the RV Co Maire, Edouard Metzger, Alicia Romero Ramirez, and Florian Cesbron for sampling assistance and cheerful exchanges during the BIOMIN-1 cruise; Franck Salin, head of Plateforme GenomeTranscriptome Pierroton (INRA, Bordeaux, France) for ARISA analyses; Claude Courties, head of Plateforme Cytom
etrie-Imagerie
anologique de Banyuls sur Mer, Universite
Pierre (Observatoire Oce et Marie Curie) for cytometry analyses; Vincent Hanquiez, Service g
eomatique - traitement de donn
ees (EPOC) for the map. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.ecss.2016.01.011. References Albertelli, G., Covazzi-Harriague, A., Danovaro, R., Fabiano, M., Fraschetti, S., Pusceddu, A., 1999. Differential responses of bacteria, meiofauna and macrofauna in a shelf area (Ligurian Sea, NW Mediterranean): role of food availability. J. Sea Res. 42, 11e26.
partition des se
diments superficiels sur le Allen, G.P., Castaing, P., 1977. Carte de re
ol. Bassin Aquitaine 21, plateau continental du Golfe de Gascogne. Bull. Inst. Ge 255e260. Aller, R.C., 1994. Bioturbation and remineralization of sedimentary organic matter: effects of redox oscillation. Chem. Geol. 114, 331e345. Aller, R.C., 2014. Sedimentary diagenesis, depositional environments, and benthic fluxes. In: Holland, H.D., Turekian, K.K. (Eds.), Treatise on Geochemistry. Elsevier, Oxford, pp. 293e334. Aller, J.Y., Aller, R.C., 1986. General characteristics of benthic faunas on the Amazon inner continental shelf with comparison to the shelf off the Changjiang River, East China Sea. Cont. Shelf Res. 6, 291e310. Aller, J.Y., Stupakoff, I., 1996. The distribution and seasonal characteristics of benthic communities on the Amazon shelf as indicators of physical processes. Cont. Shelf Res. 16, 717e751. Aller, R.C., Yingst, J.Y., 1985. Effects of the marine deposit-feeders Heteromastus filiformis (Polychaeta), Macoma balthica (Bivalvia), and Tellina texana (Bivalvia) on averaged sedimentary solute transport, reaction rates, and microbial distributions. J. Mar. Res. 43, 615e645. Amalfitano, S., Fazi, S., 2008. Recovery and quantification of bacterial cells associated with streambed sediments. J. Microbiol. Methods 75, 237e243. Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecol. 26, 32e46. Bertics, V.J., Sohm, J.A., Magnabosco, C., Ziebis, W., 2012. Denitrification and nitrogen fixation dynamics in the area surrounding an individual ghost shrimp (Neotrypaea californiensis) burrow system. Appl. Environ. Microbiol. 78, 3864e3872. Bertics, V.J., Sohm, J.A., Treude, T., Chow, C.E.T., Capone, D.G., Fuhrman, J.A., Ziebis, W., 2010. Burrowing deeper into benthic nitrogen cycling: the impact of bioturbation on nitrogen fixation coupled to sulfate reduction. Mar. Ecol. Prog. Ser. 409, 1e15. Bertics, V.J., Ziebis, W., 2009. Biodiversity of benthic microbial communities in bioturbated coastal sediments is controlled by geochemical microniches. ISME J. 3, 1269e1285. Bertics, V.J., Ziebis, W., 2010. Bioturbation and the role of microniches for sulfate reduction in coastal marine sediments. Environ. Microbiol. 12, 3022e3034.


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

C. Mass
e et al. / Estuarine, Coastal and Shelf Science xxx (2016) 1e12 Bonifacio, P., Bourgeois, S., Labrune, C., Amouroux, J.M., Escoubeyrou, K., Buscail, R.,
tion, G., Bichon, S., Desmalades, M., Romero-Ramirez, A., Lantoine, F., Ve re, B., Deflandre, B., Gre
mare, A., 2014. Spatiotemporal changes in surface Rivie ^ ne sediment characteristics and benthic macrofauna composition off the Rho River in relation to its hydrological regime. Estuar. Coast. Shelf Sci. 151, 196e209.
rez, V., 2000. A marine biotic index to establish the ecological Borja, A., Franco, J., Pe quality of soft-bottom benthos within European estuarine and coastal environments. Mar. Pollut. Bull. 40, 1100e1114. Braeckman, U., Yazdani Foshtomi, M., Van Gansbeke, D., Meysman, F., Soetaert, K., Vincx, M., Vanaverbeke, J., 2014. Variable importance of macrofaunal functional biodiversity for biogeochemical cycling in temperate coastal sediments. Ecosystems 17, 720e737. Cardinale, B.J., Gelmann, E.R., Palmer, M.A., 2004. Net spinning caddisflies as stream ecosystem engineers: the influence of Hydropsyche on benthic substrate stability. Funct. Ecol. 18, 381e387.
dimentaires actuels d'origine Castaing, P., Jouanneau, J.M., 1987. Les apports se
ans. Bull. Inst. Ge
ol. Bassin Aquitaine 41, 53e61. continentale aux oce Clarke, K.R., Warwick, R.M., 2001. Change in Marine Communities: an Approach to Statistical Analysis and Interpretation, second ed. PRIMER-E, Plymouth. Clarke, K.R., Somerfield, P.J., Chapman, M.G., 2006. On resemblance measures for ecological studies, including taxonomic dissimilarities and a zero-adjusted BrayeCurtis coefficient for denuded assemblages. J. Exp. Mar. Biol. Ecol. 330 (1), 55e80. Dauer, D.M., 1993. Biological criteria, environmental health and estuarine macrobenthic community structure. Mar. Pollut. Bull. 26, 249e257. ^ne, J.C., 2010. PRO2FLUX - a software program for profile Deflandre, B., Duche quantification and diffusive O2 flux calculations. Environ. Model. Softw. 25, 1059e1061. Dobbs, F.C., Guckert, J.B., 1988. Callianassa trilobata (Crustacea: Thalassinidea) influences abundance of meiofauna and biomass, composition, and physiologic state of microbial communities within its burrow. Mar. Ecol. Prog. Ser. 45, 69e79. Duhamel, S., Jacquet, S., 2006. Flow cytometric analysis of bacteria- and virus-like particles in lake sediments. J. Microbiol. Methods 64, 316e332. Fisher, M.M., Triplett, E.W., 1999. Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl. Environ. Microbiol. 65, 4630e4636. Fuhrman, J.A., Steele, J.A., 2008. Community structure of marine bacterioplankton: patterns, networks, and relationships to function. Aquat. Microb. Ecol. 53, 69e81. Gadel, F., Jouanneau, J.M., Weber, O., Serve, L., Comellas, L., 1997. Traceurs organ
po ^ts de la vasie re Ouest-Gironde (Golfe de Gascogne). Oceanol. iques dans les de Acta 20, 687e695. Gilbert, F., Aller, R.C., Hulth, S., 2003. The influence of macrofaunal burrow spacing and diffusive scaling on sedimentary nitrification and denitrification: an experimental simulation and model approach. J. Mar. Res. 61, 101e125.
mare, A., Labrune, C., Vanden Berghe, E., Amouroux, J.M., Bachelet, G., Gre Zettler, M.L., Vanaverbeke, J., Fleischer, D., Bigot, L., Maire, O., Deflandre, B., Craeymeersch, J., Degraer, S., Dounas, C., Duineveld, G., Heip, C., Herrmann, M., Hummel, H., Karakassis, I., Kedra, M., Kendall, M., Kingston, P., Laudien, J., Occhipinti-Ambrogi, A., Rachor, E., Sarda, R., Speybroeck, J., Van Hoey, G., Vincx, M., Whomersley, P., Willems, W., Wlodarska-Kowalczuk, M., Zenetos, A., 2009. Comparison of the performances of two biotic indices based on the MacroBen database. Mar. Ecol. Prog. Ser. 382, 297e311. Hewson, I., Fuhrman, J.A., 2006. Spatial and vertical biogeography of coral reef sediment bacterial and diazotroph communities. Mar. Ecol. Prog. Ser. 306, 79e86. Hewson, I., Jacobson-Meyers, M.E., Fuhrman, J.A., 2007. Diversity and biogeography of bacterial assemblages in surface sediments across the San Pedro Basin, Southern California borderlands. Environ. Microbiol. 9, 923e933. Hewson, I., Vargo, G.A., Fuhrman, J.A., 2003. Bacterial diversity in shallow oligotrophic marine benthos and overlying waters: effects of virus infection, containment, and nutrient enrichment. Microb. Ecol. 46, 322e336. Holm, S., 1979. A simple sequentially rejective multiple test procedure. Scand. J. Stat. 6, 65e70. Horner-Devine, M.C., Silver, J.M., Leibold, M.A., Bohannan, B.J.M., Colwell, R.K., Fuhrman, J.A., Green, J.L., Kuske, C.R., Martiny, J.B.H., Muyzer, G., Ovreas, L., Reysenbach, A.L., Smith, V.H., 2007. A comparison of taxon co-occurrence patterns for macro- and microorganisms. Ecology 88, 1345e1353. Jones, C.G., Lawton, J.H., Shachak, M., 1994. Organisms as ecosystem engineers. Oikos 69, 373e386. Jouanneau, J.M., Weber, O., Latouche, C., Vernet, J.P., Dominik, J., 1989. Erosion, nondeposition and sedimentary processes through a sedimentological and radiore” (Bay of isotopic study of surficial deposits from the “Ouest-Gironde vasie Biscay). Cont. Shelf Res. 9, 325e342. Kristensen, E., Kostka, J.E., 2005. Macrofaunal burrows and irrigation in marine sediment: microbiological and biogeochemical interactions. In interactions between macro- and microorganisms in marine sediments. In: Kristensen, E., Haese, R.R., Kostka, J.E. (Eds.), Coastal and Estuarine Studies 60American Geophysical Union, Washington DC, pp. 125e157. €ncke, I., Vanreusel, A., Vincx, M., Wollenburg, J., Mackensen, A., Liebezeit, G., Kro Behrends, B., 2000. Different benthic size-compartments and their relationship to sediment chemistry in the deep Eurasian Arctic Ocean. Mar. Ecol. Prog. Ser. 199, 31e41.

11

Laverock, B., Smith, C.J., Tait, K., Osborn, A.M., Widdicombe, S., Gilbert, J.A., 2010. Bioturbating shrimp alter the structure and diversity of bacterial communities in coastal marine sediments. ISME J. 4, 1531e1544. Lebaron, P., Servais, P., Baudoux, A.C., Bourrain, M., Courties, C., Parthuisot, N., 2002. Variations of bacterial-specific activity with cell size and nucleic acid content assessed by flow cytometry. Aquat. Microb. Ecol. 28, 131e140. Lesueur, P., Tastet, J.P., Weber, O., 2002. Origin and morphosedimentary evolution of fine-grained modern continental shelf deposits: the Gironde mud fields (Bay of Biscay, France). Sedimentology 49, 1299e1320. Lesueur, P., Weber, O., Marambat, L., Tastet, J.P., Jouanneau, J.M., Turon, J.L., 1989. re de plate-forme atlantique au de
bouche
d'un estuaire: la Datation d'une vasie re  cle  vasie a l'Ouest de la Gironde (France) est d'^ age historique (VIe sie a nos jours). C. R. Acad. Sci. Paris II 308, 935e940. re, P., Dorel, D., 1970. Etude des se
diments meubles de la vasie re de la Gironde Longe
gions avoisinantes. Rev. Trav. Inst. Pe ^ches Marit. 34, 233e256. et des re
tian, F., 2003. Bacterial diLyautey, E., Teissier, S., Charcosset, J.Y., Rols, J.L., Garabe versity of epilithic biofilm assemblages of an anthropised river section, assessed by DGGE analysis of a 16S rDNA fragment. Aquat. Microbiol. Ecol. 33, 217e224. Mantel, N., 1967. The detection of disease clustering and a generalized regression approach. Cancer Res. 27, 209e220. Marinelli, R.L., Lovell, C.R., Wakeham, S.G., Ringelberg, D.B., White, D.C., 2002. Experimental investigation of the control of bacterial community composition in macrofaunal burrows. Mar. Ecol. Prog. Ser. 235, 1e13. McArdle, B.H., Anderson, M.J., 2001. Fitting multivariate models to community data: a comment on distance-based redundancy analysis. Ecology 82, 290e297. Mermillod-Blondin, F., Rosenberg, R., 2006. Ecosystem engineering: the impact of bioturbation on biogeochemical processes in marine and freshwater benthic habitats. Aquat. Sci. 68, 434e442. Nemergut, D.R., Schmidt, S.K., Fukami, T., O'Neill, S.P., Bilinski, T.M., Stanish, L.F., Knelman, J.E., Darcy, J.L., Lynch, R.C., Wickey, P., Ferrenberg, S., 2013. Patterns and processes of microbial community assembly. Microbiol. Mol. Biol. Rev. 77, 342e356. Neveux, J., Lantoine, F., 1993. Spectrofluorometric assay of chlorophylls and phaeopigments using the least squares approximation technique. Deep Sea Res. I 40, 1747e1765. Normand, P., Ponsonnet, C., Nesme, X., Neyra, M., Simonet, P., 1996. ITS analysis of prokaryotes. In: Akkermans, A.D.L., van Elsas, J.D., De Bruijn, F.J. (Eds.), Molecular Microbial Ecology Manual. Section 3.4.5. Kluwer, Dordrecht, pp. 1e12. Nübel, U., Garcia-Pichel, F., Kühl, M., Muyzer, G., 1999. Quantifying microbial diversity: morphotypes, 16S rRNA genes, and carotenoids of oxygenic phototrophs in microbial mats. Appl. Environ. Microbiol. 65, 422e430. Osborne, C.A., Rees, G.N., Bernstein, Y., Janssen, P.H., 2006. New threshold and confidence estimates for terminal restriction fragment length polymorphism analysis of complex bacterial communities. Appl. Environ. Microbiol. 72, 1270e1278. Papageorgiou, N., Moreno, M., Marin, V., Baiardo, S., Arvanitidis, C., Fabiano, M., Eleftheriou, A., 2007. Interrelationships of bacteria, meiofauna and macrofauna in a Mediterranean sedimentary beach (Maremma Park, NW Italy). Helgol. Mar. Res. 61, 31e42. Papaspyrou, S., Gregersen, T., Cox, R.P., Thessalou-Legaki, M., Kristensen, E., 2005. Sediment properties and bacterial community in burrows of the ghost shrimp Pestarella tyrrhena (Decapoda: Thalassinidea). Aquat. Microb. Ecol. 38, 181e190. Parra, M., Castaing, P., Jouanneau, J.M., Grousset, F., Latouche, C., 1999. Nd-Sr isotopic composition of present-day sediments from the Gironde Estuary, its draining basins and the West Gironde mud patch (SW France). Cont. Shelf Res. 19, 135e150. Pearson, T.H., Rosenberg, R., 1978. Macrobenthic succession in relation to organic enrichment and pollution of the marine environment. Oceanogr. Mar. Biol. Ann. Rev. 16, 229e311. Pischedda, L., Militon, C., Gilbert, F., Cuny, P., 2011. Characterization of specificity of bacterial community structure within the burrow environment of the marine polychaete Hediste (Nereis) diversicolor. Res. Microbiol. 162, 1033e1042. Polymenakou, P.N., Bertilsson, S., Tselepides, A., Stephanou, E.G., 2005. Links between geographic location, environmental factors, and microbial community composition in sediments of the eastern Mediterranean Sea. Microb. Ecol. 49, 367e378.
s, A.M., Birchenough, S.N.R., Bremner, J., Godbold, J.A., Parker, R.E., RomeroQueiro Ramirez, A., Reiss, H., Solan, M., Somerfield, P.J., Van Colen, C., Van Hoey, G., Widdicombe, S., 2013. A bioturbation classification of European marine infaunal invertebrates. Ecol. Evol. 3, 3958e3985. Ramette, A., 2009. Quantitative community fingerprinting methods for estimating the abundance of operational taxonomic units in natural microbial communities. Appl. Environ. Microbiol. 75, 2495e2505. Reichardt, W., 1989. Microbiological aspects of bioturbation. Sci. Mar. 53, 301e306. Relexans, J.C., Lin, R.G., Castel, J., Etcheber, H., Laborde, P., 1992. Response of biota to sedimentary organic matter quality of the West Gironde mud patch, Bay of Biscay (France). Oceanol. Acta 15, 639e649. Revsbech, N.P., 1989. An oxygen microsensor with a guard cathode. Limnol. Oceanogr. 34, 474e478. Rhoads, D.C., Boesch, D.F., Zhican, T., Fengshan, X., Liqiang, H., Nilsen, K.J., 1985. Macrobenthos and sedimentary facies on the Changjiang delta platform and adjacent continental shelf, East China Sea. Cont. Shelf Res. 4, 189e213. Rosenberg, R., 2001. Marine benthic faunal successional stages and related sedimentary activity. Sci. Mar. 65, 107e119.
s-Alio
, C., 2000. Spatial differences in Schauer, M., Massana, R., Pedro


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011

12

C. Mass
e et al. / Estuarine, Coastal and Shelf Science xxx (2016) 1e12

bacterioplankton composition along the Catalan coast (NW Mediterranean) assessed by molecular fingerprinting. FEMS Microb. Ecol. 33, 51e59. Semprucci, F., Boi, P., Manti, A., Covazzi Harriague, A., Rocchi, M., Colantoni, P., Papa, S., Balsamo, M., 2010. Benthic communities along a littoral of the Central Adriatic Sea (Italy). Helgol. Mar. Res. 64, 101e115. Solan, M., Wigham, B.D., 2005. Biogenic particle reworking and bacterialinvertebrate interactions in marine sediments. In: Kristensen, E., Haese, R.R., Kostka, J.E. (Eds.), Interactions between Macro-and Microorganisms in Marine Sediment. American Geophysical Union, Washington DC, pp. 105e124. Coastal and Estuarine Studies 60. Stief, P., Altmann, D., de Beer, D., Bieg, R., Kureck, A., 2004. Microbial activities in the burrow environment of the potamal mayfly Ephoron virgo. Freshw. Biol. 49, 1152e1163. Svensson, J., Leonardson, L., 1996. Effects of bioturbation by tube-dwelling

chironomid larvae on oxygen uptake and denitrification in eutrophic lake sediments. Freshw. Biol. 35, 289e300. Weber, O., Jouanneau, J.M., Ruch, P., Mirmand, M., 1991. Grain-size relationship between suspended matter originating in the Gironde estuary and shelf mudpatch deposits. Mar. Geol. 96, 159e165. Wintzingerode, F., Gobel, U.B., Stackebrandt, E., 1997. Determination of microbial diversity in environmental samples: pitfalls of PCR-based rRNA analysis. FEMS Microbiol. Rev. 21, 213e229. Zhou, J., Bruns, M.A., Tiedje, J.M., 1996. DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316e322. Zinger, L., Amaral-Zettler, L.A., Fuhrman, J.A., Horner-Devine, M.C., Huse, S.M., Welch, D.B.M., Martiny, J.B.H., Sogin, M., Boetius, A., Ramette, A., 2011. Global patterns of bacterial beta-diversity in seafloor and seawater ecosystems. PLoS One 6 (9), 1e11 e24570.


, C., et al., Bacterial and macrofaunal communities in the sediments of the West Gironde Mud Patch, Bay Please cite this article in press as: Masse of Biscay (France), Estuarine, Coastal and Shelf Science (2016), http://dx.doi.org/10.1016/j.ecss.2016.01.011