Climatic fluctuations may significantly alter the taxonomic and biochemical composition of phytoplankton blooms and subsequently phytodetritus, the food source for the majority of deep-sea communities. To examine the response of abyssal benthic communities to different food resources we simulated a food sedimentation event containing diatoms and coccolithophorids at Station M in the NE Pacific. In one set of experiments we measured incorporation of diatomC and coccoN into the macrofauna using isotopically enriched 13C-diatoms and 15N-coccolithophores. In a second experiment we measured incorporation of C and N from dual-labelled (13C and 15N) diatoms. The second experiment was repeated 2 months later to assess the effect of seasonality. The simulated food pulses represented additions of 650 – 800 mg C m−2 and 120 mg N m−2 to the seafloor. In all cases rapid incorporation of tracer was observed within 4 days, with between 20% and 52% of the macrofauna displaying evidence of enrichment. However, incorporation levels of both diatomC and coccoN were low (<0.05% and 0.005% of the added C and N). Incorporation of labelled diatoms was similar during both June and September suggesting that the community was not food limited during either period. We found no evidence for selective ingestion of the different food types in the metazoan fauna suggesting that macrofauna do not have strong preferences for diatom vs. coccolithophore dominated phytodetrital pulses. C∶N ratios from both experiments suggest that the metazoan macrofauna community appear to have higher C demands and/or assimilation efficiencies compared to N. Concomitantly, the foraminifera preferentially selected for diatomN over coccoN, and we suggest that this may be related to foraminiferal requirements for intracellular nitrate. These experiments provide evidence that abyssal faunal feeding strategies are in part driven by an organism's internal stoichiometric budgets and biochemical requirements.
Citation: Jeffreys RM, Burke C, Jamieson AJ, Narayanaswamy BE, Ruhl HA, Smith KL Jr, et al. (2013) Feeding Preferences of Abyssal Macrofauna Inferred from In Situ Pulse Chase Experiments. PLoS ONE 8(11): e80510. https://doi.org/10.1371/journal.pone.0080510
Editor: Martin Solan, University of Southampton, United Kingdom
Received: June 6, 2013; Accepted: October 3, 2013; Published: November 26, 2013
Copyright: © 2013 Jeffreys et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by Natural Environment Research Council Grant (UK) awarded to UW and the National Science Foundation (US) and the David and Lucile Packard Foundation awarded to KLS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The abyssal seafloor is a vast ecosystem covering ∼54% of the Earth's surface . Abyssal communities are energy limited and rely on the input of particulate organic matter (POM) produced through photosynthesis in the surface waters for food , . A portion of the flux of POM is deposited at the seafloor as large aggregates of intact phytoplankton cells known as phytodetritus , , and a tight coupling between this external food source and abyssal benthic community structure and functioning has been documented –. Recent time-series data from two abyssal observatories have revealed that climate-driven variations in food supply in terms of its timing and magnitude, has resulted in broad changes in both community structure and ecosystem functioning –.
Energy limitation at abyssal depths (>4000 m) has led to a reduced standing stock of macrofaunal organisms when compared to smaller size classes i.e. bacteria and meiofauna . In spite of this the macrofauna are an important facet of abyssal communities. Macrofauna can influence organic matter processing, burial and nutrient cycles through feeding processes , , bioturbation ,  and remineralization . More recently macrofaunal activity (e.g. grazing and microhabitat destruction) has been linked to lower incorporation of labelled carbon by bacteria , .
Given the connectedness of climate to deep sea systems and that macrofaunal communities are known to be sensitive to changes in food supply , , , the importance of understanding feeding preferences/resource selection and the consequences for biogeochemical processes in the deep sea is increasing. Macrofauna respond within 36 hours to phytodetritus deposition by ingestion and bioturbation –. Pulse chase experiments have demonstrated that macrofauna can dominate the incorporation of labelled carbon –. These experiments also show differential utilization of phytodetritus and nutrients between macrofaunal taxa , –. Similarly, lipid distributions of deep-sea polychaetes show selective ingestion of fatty acids and sterols indicative of microalgae , . In shallower waters, pulse-chase experiments have shown that macrofauna are capable of selecting for a particular type of microalgae, i.e. ice algae over phytoplankton , . In the deep sea little is known about macrofaunal resource selection. While macrofauna have been shown to have long-term variations in functional group abundances in polychaetes, for example, the links to food supply remain unclear .
The long-term abyssal monitoring site ‘Station M’ in the NE Pacific provides a good setting to study macrofaunal resource selection. Benthopelagic coupling has been studied in detail at Station M since 1989 , . The macrofauna are known to respond to pulses of phytodetritus and climate-driven changes in food supply , , . This study aims to test the hypotheses: (1) that macrofauna will preferentially select for a particular type of phytodetritus (2) uptake of simulated phytodetrital pulses varies seasonally and, (3) that processing of phytodetritus by macrofauna will be dictated by internal biochemical (i.e. C and N) demands. The results are discussed in the context of macrofaunal community and food web structure.
Materials and Methods
Station M (34°50′ N, 123°00′ W) is located at a water depth of ca. 4100 m in the Northeast Pacific. This site is located at the base of the Monterey deep-sea fan, ∼220 km west of Point Conception, California. An overview of the biology, chemistry and oceanography of Station M are given in Smith and Druffel . Fluxes of particulate organic carbon (POC) and particulate total nitrogen (PTN) are generally highest during the spring and autumn months, reaching up to 25 mg C m−2 d−1 and 3.4 mg N m−2 d−1, respectively (50 metres above bottom, m.a.b.) and display high inter-annual variability , , . Detrital aggregates are present at the seafloor from June to December, with highest occurrences observed during the months of September and October . Detrital aggregate organic carbon (OC) at the seafloor can reach peaks of 161 mg C m−2 with aggregates having OC and TN values ranging from 5 to 110 mg OC g−1 and 2 to 13 mg N g−1, respectively , . This study took place within the US EEZ on the US RV Western Flyer cruise PULSE 52 (5th–10th June 2007) and PULSE 53 (17th–24th September 2007). The experiments and sampling were conducted as part of a routine sampling event and no specific permission was needed for this sampling. The experiments and sampling did not involve vertebrates or any endangered/protected species.
Cultivation of labelled food sources
The centric diatoms Thalassiosira weissflogii, Chaetoceros mullerri and Skeletonema costatum (Coscinodiscophyceae) and the coccolithophore Emiliania huxleyi (Prymnesiophyceae) were chosen as food sources as they have been observed both in the California current spring bloom and within detrital aggregates sampled at the seafloor at Station M , . Algae were cultured in artificial seawater using L1 medium  at 16°C (light∶dark = 16∶8; salinity = 35; pH = 7.8 to 8.2; duration = 21 days; . The artificial seawater medium inoculating the T. weissflogii, C. mullerri and S. costatum was amended with 13C-bicarbonate (99% atom % enriched NaH13CO3, Cambridge Isotope Laboratories). Similarly, the medium inoculating E. huxleyi and S. costatum was amended with 15N-sodium nitrate (98% atom enriched Na15NO3). Algae were harvested by centrifugation (1500 rpm at 16°C for 15 mins) and washed 3 times in an isotonic solution to remove excess label. Algae were flash frozen using liquid nitrogen, N2(l) to reduce cell damage and then lypholized. Cell sizes and the biochemical composition of the amended food sources are given in Table 1. Three different diatom species were used in these experiments, which may present difficulty in comparing experimental data. However, the biochemical composition of these three genera, are known to be similar  and we feel comparisons between diatoms (including the three species used) and coccolithophorids are justified.
Isotopically labelled food choice experiments were conducted in situ using a prototype of the Oceanlab spreader mesocosms , . Each spreader consisted of a transparent acrylic tube (diameter: 29 cm, height 50 cm) with a centrally fixed cartridge of isotopically labelled food sources, which is released by depressing an elastically tensioned plunger. Spreaders were deployed by the ROV Tiburon, releasing known doses of isotopically labelled phytodetritus onto replicate 0.066 m−2 areas of the seafloor.
During June 2007 three spreaders were deployed containing a slurry of C. mulleri and E. huxleyi representing an addition of 0.8 g C m−2 and 0.12 g N m−2, equivalent to ∼40% and 58% of the annual POC and PTN flux, at the seafloor (Experiment 1). The fourth spreader was deployed containing the dual labelled diatom S. costatum slurry, here an addition of 0.7 g C m−2 and 0.12 g N m−2, equivalent to ∼35% and 58% of the annual POC and PTN flux, respectively was applied to the seafloor (Experiment 2). Following this during September 2007 four replicate spreaders were deployed containing a suspension of the diatom T. weissflogii, representing an addition of 0.65 g C m−2, equivalent to 34% of the annual POC flux at the seafloor.
Spreaders were deployed on undisturbed areas of the seafloor and after any resuspended sediment had settled the experiments commenced. Experiments were terminated after four days and sub-sampled using 70 mm diameter push cores recovered by the ROV. Three pushcores from each spreader were retrieved in June and a single core from each spreader in September. Cores for macrofaunal analyses were sectioned 1 cm intervals to 5 cm in June and at 0–2 cm and 2–5 cm in September. In order to compare data between seasons we combined data obtained (abundance, biomass, label incorporation) from the 1 cm intervals in June to 0–2 cm and 2–5 cm for comparison with September. Background/control push cores were taken close to the spreaders at the start of the experiments to provide natural stable isotope values of the macrofauna and sediments (n = 8 in June and n = 3 for September).
Sampling procedures and isotope analyses
Sectioned cores were wet-sieved through a 250 mm mesh, using filtered seawater and fixed in buffered 4% formaldehyde solution. Sections were sorted under x12 and x20 magnification. Macrofauna were identified to phylum/sub-phylum and polychaetes to the lowest taxonomic level i.e. genus or species and abundances were recorded. Macrofauna were rinsed in Milli Q water placed in tin cups and dried at 60°C. Organisms containing calcareous parts were decalcified in double boated silver cups with 2 M HCl and dried as above. Cores for sediment isotopes were lyophilised prior to analysis. Lyophilised sediments were decarbonated by addition of excess 1 M HCl, incubated for 24 h at 30°C in an acid-fumed environment and dried to constant weight.
Macrofaunal and sediment total C and N contents and isotopic ratios were determined using a Flash EA 1112 Series Elemental Analyser connected via a Conflo III to a DeltaPlus XP isotope ratio mass spectrometer (Thermo Finnigan). Isotope ratios were calculated with respect to CO2 and N2 reference gases injected with each sample. Isotopic values of gases were directly referenced against IAEA reference materials USGS40 and USGS41 (both L-glutamic acid), both certified for δ13C (%VPDB) and δ15N (%air N2). The C and N content of the samples were calculated from the area output of the mass spectrometer calibrated against the National Institute of Standards and Technology (NIST) standard reference material 1547 peach leaves, which was analysed with every batch of ten samples. Long-term isotope measurement precisions relative to a quality control standard (milled flour) were: total C = 40.3±0.42%, δ13C = −25.5±0.29‰, total N = 1.7±0.04% and δ15N = 0.367±0.0002‰ (mean ± SD, n = 200). Isotope ratio data were expressed in δ units (‰) and used to estimate faunal 13C and 15N uptake and incorporation.
Owing to natural variation observed in the natural abundance stable isotopic data in both this study and that of Sweetman & Witte , fauna from the experiments were considered to be enriched in 13C and 15N when their isotopic composition was >−14‰ and >20‰, respectively. Nematodes and foraminifera had high natural δ15N values and were considered to be enriched in 15N if their isotopic composition was >25‰. Foraminifera also had isotopically heavy natural δ13C values and were therefore considered to be enriched in 13C when their signatures were >−5‰.
Enrichment of 13C or 15N in macrofauna was calculated as excess above natural abundance levels and is expressed as specific uptake: Δδ = (δsample – δbackground). Specific uptake is a qualitative measure of label uptake based on isotope ratio data. Incorporation of 13C and 15N into faunal biomass is a quantitative measure  and was calculated as the product of the excess atom % of 13C or 15N (difference in atom %13C or 15N between sample and background) and C or N content (expresses as unit weight): 13C or 15N incorporation (unit wt13C or 15N) = (atom% 13Csample or 15Nsample – atom % 13Cbackground or 15Nbackground) × (unit wt C or N of organism). Incorporation of 13C or 15N was then adjusted to account for algal labeling, yielding total C or N uptake: uptake (unit wt C or N) = 13C or 15N incorporation/15 atom % (for 13C labelled diatoms) or 7 atom % (for 15N labelled coccolithophores). The data used in the incorporation and biomass specific calculations are given in Tables S1 to S3.
Unfortunately, δ15N analysis was not conducted on the polychaetes from Exp. 2. Only the elemental C and δ13C values were determined on fauna from September.
The multivariate community data on major groups (standardized fourth root transformed, Bray-Curtis similarity was used to calculate resemblance) was analysed by means of non-parametric permutational (9999 permutation) ANOVA (PERMANOVA), ,  to assess differences between controls and experimental cores, between seasons and between sediment layers. The data set was analysed using a 3-factor mixed model design factors: Treatment (experiment or control) - fixed Season - fixed and Sediment Depth - fixed in PERMANOVA+ for PRIMER. For the experimental data each organism analysed represented a data point in the matrices.
Isotope data from the June experiments were analysed in PERMANOVA. Models were run on normalized fourth root transformed, Euclidean distance similarity matrices. The δ13C and δ15N tracer incorporation data from the two experiments (Exp. 1 diatomC and coccoN; Exp.2 diatomC and diatomN) was analysed by using mixed model designs to assess (1) if there were differences in incorporation of C and N between Exps.1 and 2, (2) if there were differences in the incorporation of C and N between taxa and (3) if there were differences in incorporation between sediment layers. Model factors include: Experiment - fixed, Taxonomic group - fixed and Sediment Depth - fixed. The taxonomic groups analysed in the model included: Foraminifera, Nematoda and Crustacea. The data were classified into two sediment layers: 0 to 2 cm and 2 to 5 cm.
The response of the polychaetes to the food choice experiments were examined by means of a 2 factor mixed model to assess (1) if there were differences in the incorporation of C and N at the family level (2) if there were differences in incorporation between sediment layers. Model factors include: Polychaete family - fixed and Sediment Depth - fixed. Differences between polychaete feeding type - fixed were analysed separately in a 1-factor model with unrestricted permutation of the raw data. Differences in polychaete δ13C tracer incorporation between the two experiments were analysed by means of a 1-factor model, experiment - fixed with unrestricted permutation of the raw data.
The same data analysis procedures were carried out in PERMANOVA on both the biomass specific incorporation results and on the natural abundance C∶N ratios, δ13C and δ15N values of the fauna from the control cores.
Univariate data, e.g. sediment 13C values and incorporation of diatom carbon during the September experiments were tested for normality and eveness (Shapiro-Wilk's and Levene's tests, respectively). If the data met these assumptions differences between factors e.g. taxon or sediment depth were tested for using a one-way analysis of variance was used (ANOVA) and if the data were not normally or evenly distributed then a Kruskal-Wallis or a Mann-Whitney U-test was applied.
Description of the macrofaunal community structure at Station M was based upon specimens recovered from both background and experimental cores (n = 20 for June and n = 7 for September) and each core was treated as a single replicate. There was no significant difference in the macrofaunal density, biomass dry weight or biomass C normalised to m2 between background cores and the experimental cores from each spreader (Table 2). There was no significant difference in density (ind. m−2) between seasons (Fig. 1a & 1b, Table 2). There were significant differences in density between sediment depths (Fig. 1a & 1b, Table 2). Simper analyses revealed, foraminifera and crustaceans were responsible for ∼38% and 19% of the variation in density between sediment depths, respectively. The majority (∼80%) of foraminifera were located at 2–5 cm, whilst 58% of crustaceans were located in the upper 2 cm of the sediment (Fig. 1a & 1b). Macrofaunal biomass (dry weight mg m−2) did vary significantly between both seasons and horizons (Fig. 1c &1d, Table 2). Foraminifera and molluscs accounted for 32% and 26% of the seasonal variation and 34% and 25% of the variation between sediment depths. There were no significant differences in biomass C between seasons or sediment depths (Fig. 1e & 1f, Table 2).
Bars represent means ±1 standard deviation, n = 20 for June and 7 for September.
The foraminifera were numerically dominant representing >70% of the community. The majority of foraminifera in the samples were agglutinated and calcareous species e.g. Globobulimina and Cyclammina. The nematodes represented <10% of the total macrofaunal community. Crustaceans were the dominant metazoan macrofaunal group followed by the polychaetes accounting for >70% and ∼15%, of the metazoan density respectively. Conversely, the polychaetes were the dominant taxon in terms of biomass C and N (Fig. 1e, &1g).
A total of 23 polychaete species were identified from the June experiments. Polychaete families were numerically dominated by the Cirratulidae and Paraonidae (Fig. 2a & 2b). Dominant species within the cirratulids were Aphelochaeta spp. and Monticellina siblina (Fig. 2e). The dominant genus within the paraonids was Aricidea spp.. In terms of biomass cirratulids and paraonids were still important but a few families with large-sized individuals became significant contributors to total biomass C and N e.g. Trichobranchidae and Hesionidae (Fig. 2a).
Relative proportions of abundance, biomass (dry weight), biomass C and biomass N of polychaete feeding types in (c) June and (d) September. Relative proportions of abundance, biomass C and biomass N of polychaete species in (e) June.
Polychaetes were classified into feeding types according to Fauchald and Jumars , and were dominated by surface deposit feeders (SDF; Fig. 2c & 2d) and subsurface deposit feeders (SSDF). In terms of biomass C and N SDF were the most important group followed by SSDF. Surface deposit feeding polychaetes were represented by the cirratulids, spionids and trichobranchids. The subsurface deposit feeders contained paraonids and capitellids. Predator/scavengers included hesionids, dorvilleids, lumbrinerids and syllids.
Background isotope levels and food web structure
The natural abundance δ13C, δ15N values and C∶N ratios of sediments and fauna are summarised in Fig. 3. No significant difference in the isotopic composition of the sediments was observed between depths (Figs. 3a, 3b; δ13C Mann-Whitney U test: p = 0.245; δ15N F1,6 = 0.801, p = 0.405). There was no significant difference in faunal δ13C composition between June and September (Mann-Whitney U-test: p = 0.981) and so data in Fig. 3a include δ13C values of fauna from both June and September. Faunal carbon and nitrogen isotopes exhibited wide ranges in values, particularly within the foraminifera. The majority of consumers were enriched in 13C and 15N compared to surficial sediments (Fig. 3a, 3b).
Data are means (±1 standard deviation) from a series of 8 pushcores. For the sediments and foraminifera black symbols represent surface samples (0–2 cm) and grey symbols represent sub-surface samples (2–5 cm).
Values of δ13C were significantly different between taxonomic group and sediment depth and these factors interacted (Fig. 3a, Table 3). Pairwise comparisons (Table S4) revealed that polychaetes were significantly enriched in 13C compared to crustaceans and nematodes in both sediment depths i.e. 0–2 cm and 2–5 cm. Foraminiferans were significantly enriched in 13C compared to all taxa in the 2–5 cm sediment layer. Foraminifera in surface sediments were significantly depleted in 13C compared to deeper sediments (PERMANOVA p = 0.002, t = 9.39, unique perms = 700). Deeper living crustaceans were significantly depleted in 13C compared to surface dwellers (p = 0.001, t = 9.3932, unique perms = 1287).
There were significant differences in δ15N values between taxonomic groups and sediment depth (Fig. 3b, Table 3.) Pairwise comparisons revealed that polychaetes were significantly enriched in 15N compared to crustaceans and foraminifera. Fauna found deeper in the sediment were significantly lighter compared to those in the upper 2 cm (mean δ15N = 13.4±3.9‰ for 0–2 cm and 11.4±3.0‰ for 2–5 cm). There were significant differences in C∶N ratio between taxonomic groups and sediment depth (Fig. 3c, Table 3). Pairwise comparisons revealed that foraminifera had significantly higher C∶N ratios than all other taxa (Table S5). Fauna in the upper 2 cm of the sediment had lower C∶N ratios than those living deeper (mean C∶N = 4.7±1.5 for 0–2 cm and 7.9±5.2 for 2–5 cm).
Macrofaunal response to different food sources
Incorporation of phytodetrital C and N.
Rapid ingestion of label occurred in all experiments. During June in Exp. 1∼25% and 20% of the animals analysed had incorporated 13C and 15N, respectively. In Exp. 2∼52% and 42% of the animals analysed had incorporated 13C and 15N, respectively. During September ∼49% of the animals analysed had incorporated 13C. No significant difference in label incorporation between taxonomic groups (foraminifera, nematoda and crustacea) was evident (Fig. 4, for June see Table 4; for September including all groups, Kruskal-Wallis: p = 0.218). Significant differences were noted between sediment depths in June (Table 4), >75% of algal carbon and nitrogen was incorporated in the upper 2 cm of the sediment (Fig. 4f–g). No significant difference in algal carbon incorporation was noted in September (Mann-Whitney U-test: p = 0.067, Fig. 4f–g). There was no significant difference in incorporation of diatom carbon between June (Exp. 2) and September (Mann-Whitney U-test: p = 0.413; Fig. 4b & 4c).
Vertical distribution of (f) phytoC and (g) phytoN. In plots f and g, dark grey bars represent incorporation from Exp. 1, light grey bars represent incorporation from Exp. 2 and white bars represent mean incorporation of tracer from September. Note the data presented from Exp. 2 in June represents total incorporation µg C m−2 or µg N m−2. Number of replicates in Exp. 1 = 3 and Exp. 2 = 1 and September = 3.
The PERMANOVA model results (based on label incorporation in crustaceans, nematodes and foraminiferans) indicated no significant difference between the two experiments in June (Fig. 4a, b, d&e Table 4). There was no correlation between the incorporation of diatomC and coccoN in Exp. 1 (rs = 0.957, p = 0.05) or between diatomC and diatomN in Exp. 2 (rs = 0.393, p = 0.05). Consequently, in Exp. 1, there was no significant difference between the proportion of diatomC and coccoN incorporated by the macrofaunal community as a whole (C = 0.012±0.004% and N = 0.008±0.001%; F1,4 = 2.581, p = 0.183).
Taking into account all taxa, in Exp. 1 incorporation of diatomC and coccoN was not significantly different between taxonomic groups (Fig. 4a & d, Table 4). Significantly greater amounts of both diatomC and coccoN (>95%) were assimilated in the upper 2 cm of sediment (Fig. 4f & 4g, Table 4). No significant differences were noted between polychaete families or feeding types (PERMANOVA p = 0.227 and p = 0.8997, respectively).
It was only possible to compare incorporation of diatomC by the polychaetes between the two experiments. There were marginal differences in polychaete incorporation of diatomC between these two experiments (Fig. 4a & 4b; Table 4). In Exp. 1 Terebellides stroemii was responsible for ∼87% of the diatomC assimilated by the polychaetes, whilst in Exp. 2. Aricidea spp. and Prionospio sp. assimilated ∼49% and 48% of diatomC, respectively.
Biomass specific incorporation of phytodetrital C and N.
The biomass specific incorporation did not differ significantly between Exp. 1 and Exp. 2 in June (Fig. 5, Table 5). However, differences in biomass specific incorporation of phytodetritus between taxonomic groups and also between sediment depths were significant (Fig. 5, Table 5). There was a significant two-way interaction between experiment and taxon (Table 5). Pairwise comparisons revealed that in Exp. 2 Foraminifera had significantly higher biomass specific incorporation of diatomC and diatomN compared to nematodes (p = 0.034, t = 2.7171) and crustaceans (p = 0.016, t = 2.7236; Fig. 5b, 5e). Foraminifera had significantly higher biomass specific incorporation of diatomN (Exp. 2) compared to coccoN (Exp. 1: p = 0.039, Fig. 5d & 5e). In Exp. 1 there were no significant differences in biomass specific incorporation of diatomC and coccoN between taxonomic groups (Fig. 5a & 5d; p = 0.232, t = 1.9953). Biomass specific C incorporation of the polychaetes was not significantly different between experiments, families or feeding types (p = 0.071, p = 0.783 and p = 0.918, respectively, Fig. 5a & 5b). There was no significant difference in biomass specific incorporation of diatom C between June (Exp.2) and September (Mann-Whitney U-test: p = 0.801; Fig. 5b & 5c). No significant differences between taxonomic groups were noted in biomass specific incorporation of diatom C in September (Kruskal-Wallis: p = 0.333; Fig. 5c). There was no significant difference in biomass specific incorporation of diatom C between sediment depths in September (Mann-Whitney U-test: p = 0.059; Fig. 5f).
Vertical distribution of biomass specific uptake of (f) phytoC and (g) phytoN. In plots f and g, dark grey bars represent incorporation from Exp. 1, light grey bars represent incorporation from Exp. 2 and white bars represent mean incorporation of tracer from September. Note the data presented from Exp. 2 in June represents total biomass specific uptake µg C mg−1 or µg N mg−1. Number of replicates in Exp. 1 = 3 and Exp. 2 = 1 and September = 3.
Absolute incorporation C∶N ratios in June (Exps. 1 and 2) ranged from 2.3 to 367 (Fig. 6b & 6e). Biomass specific C∶N ratios ranged from 0.23 to 45.54 (Fig. 6c & 6f). The largest ranges in somatic C∶N ratios were seen in the foraminifera (Fig. 6d). As a result of the small number of organisms that simultaneously ingested 13C and 15N phytodetritus we were unable to test for significant differences in absolute incorporation and biomass specific C∶N ratios. Somatic C∶N ratios from both control and experimental cores were not significantly different (Table 5). Significant differences were observed in somatic C∶N ratios between taxonomic groups (Table 5). Foraminfera had higher C∶N ratios than all other taxa (Figs. 3c, 6a, 6d; Table 5).
Fluxes of POC and PTN to the seafloor were both reaching seasonal highs leading up to and during this study (Fig. 7). Concomitantly, estimates of macrofaunal densities were high but comparable to estimates made by Drazen et al. , when particulate fluxes were of a similar magnitude (12 –18 mg C m−2 d−1 at 600 m.a.b.) and exceeded estimates made by Sweetman and Witte  during a period of low particulate flux (∼8 mg C m−2 d−1 at 600 m.a.b.). The macrofaunal community at Station M during this study may not be food limited as natural food inputs via particulate organic matter flux are at or near the seasonal high. Faunal biomass (relative to C and N) was comparable to estimates for the Whittard canyon and Indian margin , ; both of the aforementioned study sites are environments sustained by an abundance of organic matter.
Filled circles and open triangles represent the carbon and nitrogen fluxes respectively. Grey-filled squares represent the C/N ratio of flux material. Solid and dashed lines represent flux material from sediment traps moored 600 m.a.b and 50 m.a.b., respectively. There is no data available from the 50 m.a.b. trap from August to November.
Food availability can influence community composition . Community composition (in terms of density) during this study resembled the community described by Drazen et al.  and Sweetman and Witte . Switches in the dominant taxonomic groups with respect to biomass were noted in the community composition between periods of high (this study) and low  food availability, as well as over longer time scales .
The biomass of the foraminifera increased between June and September and significant inputs of POC were observed at Station M from May to September (Fig. 7), which may have been responsible for this increased biomass. Foraminifera are known to respond rapidly to inputs of fresh POC through reproduction and growth , , .
The polychaete community composition is also further evidence of high food availability at Station M during June 2007. The three dominant families, Cirratulidae, Paraonidae and Spionidae are known to be opportunistic and respond quickly to organic enrichment , .
Macrofaunal feeding preferences
Macrofaunal stable carbon isotope ratios indicate that the basal food resource is organic C originating from the particulate flux. Macrofaunal δ13C values closely mirrored those of the sediments and previous measurements on POC . This can be confirmed by the ingestion of labelled phytodetritus in the experiments.
Faunal incorporation of labelled phytodetritus in all experiments was low <0.005%, and in agreement with previous pulse chase experiments at Station M . At the time of this study Station M did not appear to be food-limited and so the low incorporation of labelled phytodetritus may be a result of satiation of the macrofaunal community or provide evidence of dependence on semi-labile detritus, as demonstrated for the PAP macrofaunal community . The majority of labelled phytodetritus was incorporated in the upper 2 cm of the sediment, in agreement with previous pulse chase studies at abyssal sites and demonstrating that surface deposit feeding community is relatively more important in the processing of OM in abyssal settings than the deeper dwelling fauna , . A conspicuous facet of the macrofaunal community at Station M is the opposing zonation of the metazoans and the forminifera. This suggests that the surface dwelling metazoans may play a more substantial role in OM processing at Station M, results from the diatom only treatment support this.
In the diatom treatments incorporation of label by the metazoan macrofauna was an order of magnitude higher compared to the mixed algal treatment. This discrepancy may be a result of experimental design as we were unable to trace the amount of coccoC incorporated by the fauna in the mixed algal treatment. Foraminiferal incorporation of carbon was also comparable during both sampling periods. The response of the foraminifera in this study was muted compared to the response observed by Enge et al. , at the same site and to foraminifera in other areas e.g. the Indian and Pakistan margins, Sagami Bay , , . The study of Enge et al.  considered foraminifera larger than 250 µm and macrofaunal foraminifera are known to exhibit a retarded response to phytodetritus when compared with smaller foraminifera . Despite the retarded response the proportion of carbon processed by the foraminifera compared to metazoans was high, up to 46% and 90% of the C added in June and September, respectively and is in agreement with previous studies in the N. Atlantic , . The majority (>70%) of tracer uptake was observed in foraminifera in the top 2 cm of sediment, this suggests that: (1) surface dwelling foraminifera are important in carbon processing or (2) deeper dwelling foraminifera migrate in the sediment towards the added food source. Migration of foraminifera towards simulated food pulses has been reported previously; by Nomaki et al.  and Koho et al. . This seems likely given that the majority of foraminifera in terms of density and biomass were concentrated at 2–5 cm.
Macrofaunal δ15N values differed between taxonomic groups indicating that fauna were feeding at different trophic levels and that there is a degree of niche separation between taxon at Station M. Assuming a trophic enrichment factor of 2.2‰ to 3.4‰ , , foraminifera appear to feed both on relatively depleted POM at the base of the food web and more degraded material. Nematodes and crustaceans were more enriched in 15N as has been previously observed at the Porcupine abyssal plain , indicating that they either feed at a higher trophic level or on other food sources e.g. degraded POM or bacteria. Crustaceans are known to prey on foraminifera . Whilst, some studies have shown that nematodes have been shown to preferentially feed on bacteria , , there is also evidence that nematodes do not select for bacterial food sources . Further to this, δ13C values of nematodes mirrored those of the sediments, suggesting a sedimentary food source. The enrichment of 15N in the nematodes at Station M could result from predation, which has been recently observed in Arctic nematodes . Polychaetes and molluscs were at the apex of the macrofaunal food web at Station M and the associated large ranges in δ15N values suggests a degree of omnivory in their diets .
There was no difference in incorporation of labelled phytodetritus between the metazoan taxonomic groups during the pulse chase experiments. This is a surprising result given the taxonomic differences in metazoan macrofaunal feeding strategies evident from the natural abundance δ13C and δ15N values. Closer inspection of the polychaete species in this study reveals a community of highly selective deposit feeders e.g. Terebellides stroemii, Prionospio spp., Tharyx kirkegaardi and Aricidea spp.. Evidence from the food web analysis and pulse chase experiments indicates that there is a high degree of plasticity in polychaete feeding strategies at Station M in agreement with the findings of Sweetman and Witte .
Dual labelling multiple food sources allows for direct appraisal of selectivity of the macrofaunal community for specific algae when present simultaneously e.g. Herman et al. . As a result of our labelling techniques food sources were significantly enriched in 13C and 15N compared to non-labelled food sources (Table 2), therefore the isotope mixing model approach used by Herman et al.  was not appropriate here. Station M is a food-limited environment, receiving significant food inputs once a year following a spring phytoplankton bloom. We suggest that if the suspected selectivity among algal food sources is indeed important, it may only be evident during certain times. Phytodetritus is usually present at Station M during late Summer and early Autumn. We did not find evidence of metazoan macrofauna selecting for a particular type of phytodetritus at Station M. In our experiments the proportions of diatomC and coccoN incorporated by the macrofaunal community in the mixed feeding experiments were similar. Futhermore, there was no significant difference in the incorporation of C or N between the mixed feeing experiments or the diatom only treatment and uptake of C and N was not correlated (section 3.3.1.). This suggests that macrofauna do not select for a particular type of phytoplankton when phytodetritus is readily available. Results from the diatom only treatment revealed that metazoans were not incorporating C and N simultaneously (Table S2), suggesting that organism stoichiometry must be considered when interpreting experimental isotope tracer data.
Macrofaunal C∶N Stoichiometry
Taxonomic differences in C∶N ratios were observed at Station M, foraminifera having the highest C∶N ratios indicating that foraminifera may have a higher demand for C and/or have higher C assimilation efficiencies. This is not a surprising result given that foraminifera grow and reproduce over short temporal scales in the presence of phytodetritus , , . In this study somatic C∶N ratios of metazoans ranged from 3 to 9, a single nematode had a value of 12. Faunal demands for C and N are driven by energy requirements for somatic growth and reproduction and are balanced against excretion of nitrogenous waste . Somatic tissues of marine invertebrates are dominated by protein and somatic C∶N ratios follow those of amino acids i.e. ranging from 1.5 to 9 . Most marine invertebrates exhibit C∶N ratios ranging from 3 to 8, e.g. , , . Organisms adapt their feeding strategies in order to maintain nutrient consumption at an optimum level in order to achieve stoichiometric homeostasis , . In doing so, the resulting C∶N ratios observed in organisms are often species-specific being regulated by a species physiology .
Based on incorporation of tracer, these experiments suggest that faunal demand and/or assimilation of C is higher than for N. In the dual-labelled diatom treatment incorporation of C by metazoans and foraminifera was an order of magnitude higher than for N (∼0.05% of added C was incorporated compared to 0.004% for N). Our results are in agreement with those for the Indian margin, where in an experiment tracing the fate of dual labelled (13C and 15N) diatoms, more C was processed relative to N . However, the phytoC:phytoN ratios for biomass incorporation coupled to the somatic C∶N ratios demonstrate a preference for N as has been shown by Hunter et al. . The higher phytoC:phytoN ratios for absolute assimilation compared to the lower biomass specific ratios may indicate preferential consumption/assimilation of carbon rich molecules such as carbohydrates and lipids as has been previously observed in shallow water corals, calanoid copepods and deep-sea foraminifera –. Furthermore, ammonotelic organisms may rapidly excret phytoN resulting in higher absolute assimilation ratios in the fauna .
The surface dwelling foraminifera had depleted natural abundance δ13C values indicating that they feed at the base of the food web on phytodetrital aggregates. Concomitantly, deeper dwelling foraminifera (2–5 cm) were enriched in 13C indicating either preferential utilisation of isotopically light cellular fatty acids during periods of low food availability. Preferential utilisation of isotopically light cellular fatty acids during periods of low food availability (fasting/starvation) can lead to enrichment in 13C by up to 4‰ –. Utilisation of cellular fatty acids by deeper dwelling foraminifera would not explain the large differences in δ13C values observed here. Large isotopic shifts have been observed in both autotrophic and heterotrophic bacteria ,  and ingestion of bacteria utilizing the reverse tricarboxylic acid pathway can lead to enrichment in 13C –. The large ranges in δ13C of foraminifera observed in this study are consistent with the ranges for the calcareous and agglutinated foraminifera at Station M during September 2007 .
We suggest from the results presented here and previous observations , , that foraminifera colonize phytodetrital aggregates and use nitrate in respiration at Station M. Foraminifera also had higher biomass specific incorporation of N compared to other taxonomic groups. Consumption of POM is proportional to faunal biomass . At the Indian margin foraminifera have also been shown to incorporate algalN in tracer experiments . The foraminifera preferentially selected for diatoms over coccolithophorids and were the only group to show evidence of preferential selection. Foraminifera may require higher levels of organic N from phytodetritus in order to catabolise this food source and fuel intracellular denitrification or to build up reserves of intracellular nitrate for respiration. Recently foraminifera have been shown to carry out complete intracellular denitrification de novo and in the presence of endobionts, in a wide range of environments –. Storage of intracellular nitrate within foraminiferal vacuoles is known to result in intracellular δ15NNO3 values ranging from 12‰ – 42‰ , . Foraminiferal δ15N values in this study ranged from 6.6‰ to 20.1‰, with three of these values being >12‰, which suggests that some foraminifera at Station M may be accumulating intracellular nitrate.
Nitrate respiration has been observed in phytodetrital aggregates at Station M and was attributed to bacteria . A number of mechanisms for nitrate accumulation in foraminifera have been suggested, these include: transport of nitrate into cells from surrounding porewaters, intracellular production of nitrate, or obtaining nitrate from symbiotic nitrifying bacteria . It is possible that foraminifera at Station M could produce intracellular nitrate via decomposition of phytoN to yield NH4+, which is then converted to nitrate via denitrification either de novo or in the presence of bacterial symbionts. However, we are not aware that this process has been observed in foraminifera to date. Alternatively, foraminifera may assimilate nitrate from surrounding porewaters, which could have been produced by decomposition of phytoN by bacteria or fauna to yield NH4+, which is then converted to nitrate by nitrifying bacteria. Koho et al.  have demonstrated that foraminifera collect nitrate both in the presence and absence of oxygen, and that foraminifera migrate towards favourable conditions i.e. towards free nitrate and or oxygen. If this is the case at Station M, it might account for the observed foraminiferal distributions. Nitrate storage/respiration in foraminifera has also recently been proposed as a transport mechanism for nitrate in sediments , . Our data suggest that foraminifera are important players in nitrogen cycling in abyssal environments and could facilitate the transport of nitrogen through the sediments. However, further work is required to determine the role of foraminifera in both organic and inorganic nitrogen cycling at Station M.
Isotopic composition of macrofauna recovered from Experiment 1 in June 2007 containing 13C-labeled diatoms and 15N-labeled coccolithophores.
Isotopic composition of macrofauna recovered from Experiment 2 in June 2007 containing 13C and 15N- labeled diatoms.
Isotopic composition of macrofauna recovered from the experiment in September 2007 containing 13C-labeled diatoms.
Pairwise comparisons of δ13C composition between taxonomic groups.
We would like to acknowledge one anonymous reviewer and Dr. Hidetaka Nomaki for their useful comments on the manuscript. We sincerely thank the Captain, officers and crew of the R.V. Western Flyer and the pilots of the ROV Tiburon for assistance at sea during PULSE 52. We are also grateful to Jacob Ellena for logistical support/assistance at sea, Amanda Khan for helping RJ with sieving sediment cores and Markus Moeseneder for collecting samples during PULSE 53. We would also like to thank Barry Thornton for his help with the isotope analyses.
Conceived and designed the experiments: RMJ UW AJ. Performed the experiments: RMJ KS HR CB. Analyzed the data: CB RJ. Contributed reagents/materials/analysis tools: UW KS BN. Wrote the paper: RJ. Gave critical revisions on the first draft, which improved the manuscript: HR UW. Participated in the critical revision of the manuscript and gave final approval of the article: RMJ CB AJ BN HR KS UW.
- 1. Smith CR, De Leo FC, Bernardino AF, Sweetman AK, Arbizu PM (2008) Abyssal food limitation, ecosystem structure and climate change. Trends Ecol Evol 23: 518–528.
- 2. Graf G (1989) Benthic Pelagic Coupling in a Deep-Sea Benthic Community. Nature 341: 437–439.
- 3. Gooday A (1988) A Response by Benthic Foraminifera to the Deposition of Phytodetritus in the Deep-Sea. Nature 332: 70–73.
- 4. Billett D, Lampitt R, Rice A, Mantoura R (1983) Seasonal Sedimentation of Phytoplankton to the Deep-Sea Benthos. Nature 302: 520–522.
- 5. Beaulieu S (2002) Accumulation and fate of phytodetritus on the sea floor. Oceanogr Mar Biol 40: 171–232.
- 6. Smith K, Kaufmann R, Baldwin R (1994) Coupling of near-bottom pelagic and benthic processes at abyssal depths in the eastern North Pacific Ocean. Limnol Oceanogr 39: 1101–1118.
- 7. Smith C, Berelson W, DeMaster D, Dobbs F, Hammond D, et al. (1997) Latitudinal variations in benthic processes in the abyssal equatorial Pacific: control by biogenic particle flux. Deep-Sea Res II 44: 2295–2317.
- 8. Bett B, Malzone M, Narayanaswamy B, Wigham B (2001) Temporal variability in phytodetritus and megabenthic activity at the seabed in the deep Northeast Atlantic. Prog Oceanogr 50: 349–368.
- 9. Ruhl HA, Ellena JA, Smith KL (2008) Connections between climate, food limitation, and carbon cycling in abyssal sediment communities. P Natl Acad Sci Usa 105: 17006–17011
- 10. Smith KL, Ruhl HA, Bett BJ, Billett DSM, Lampitt RS, et al. (2009) Climate, carbon cycling, and deep-ocean ecosystems. P Natl Acad Sci Usa 106: 19211–19218
- 11. Vardaro MF, Ruhl HA, Smith KL (2009) Climate variation, carbon flux, and bioturbation in the abyssal North Pacific. Limnol Oceanogr 54: 2081–2088.
- 12. Rex M, Etter R, Morris J, Crouse J, McClain C, et al. (2006) Global bathymetric patterns of standing stock and body size in the deep-sea benthos. Mar Ecol Prog Ser 317: 1.
- 13. Thomas C, Blair N (2002) Transport and digestive alteration of uniformly C-13-labeled diatoms in mudflat sediments. J Mar Res 60: 517–535.
- 14. Canuel EA, Spivak AC, Waterson EJ, Duffy JE (2007) Biodiversity and food web structure influence short-term accumulation of sediment organic matter in an experimental seagrass system. Limnol Oceanogr 52: 590–602.
- 15. Levin L, Blair N, DeMaster D, Plaia G, Fornes W, et al. (1997) Rapid subduction of organic matter by maldanid polychaetes on the North Carolina slope. J Mar Res 55: 595–611.
- 16. Fornes WL, DeMaster DJ, Levin LA, Blair NE (1999) Bioturbation and particle transport in Carolina slope sediments: A radiochemical approach. J Mar Res 57: 335–355
- 17. Sun M-Y, Aller RC, Lee C, Wakeham SG (1999) Enhanced degradation of algal lipids by benthic macrofaunal activity: Effect of Yoldia limatula. J Mar Res 57: 775–804
- 18. Hunter W, Veuger B (2012) Macrofauna regulate heterotrophic bacterial carbon and nitrogen incorporation in low-oxygen sediments. ISME J. doi:10.1038/ismej.2012.244.
- 19. Hunter WR, Jamieson A, Huvenne VAI, Witte U (2013) Sediment community responses to marine vs. terrigenous organic matter in a submarine canyon. Biogeosciences 10: 67–80
- 20. Drazen J, Baldwin R, Smith K (1998) Sediment community response to a temporally varying food supply at an abyssal station in the NE Pacific. Deep-Sea Res II 45: 893–913.
- 21. Hunter WR, Levin LA, Kitazato H, Witte U (2012) Macrobenthic assemblage structure and organismal stoichiometry control faunal processing of particulate organic carbon and nitrogen in oxygen minimum zone sediments. Biogeosciences 9: 993–1006
- 22. Gontikaki E, Mayor DJ, Narayanaswamy BE, Witte U (2011) Feeding strategies of deep-sea sub-Arctic macrofauna of the Faroe-Shetland Channel: Combining natural stable isotopes and enrichment techniques. Deep-Sea Res I 58: 160–172
- 23. Moodley L, Middelburg J, Soetaert K, Boschker H, Herman P, et al. (2005) Similar rapid response to phytodetritus deposition in shallow and deep-sea sediments. J Mar Res 63: 457–469.
- 24. Woulds C, Andersson JH, Cowie GL, Middelburg JJ, Levin LA (2009) The short-term fate of organic carbon in marine sediments Comparing the Pakistan margin to other regions. Deep-Sea Res II 56: 393–402
- 25. Blair N, Levin L, DeMaster D, Plaia G (1996) The short-term fate of fresh algal carbon in continental slope sediments. Limnol Oceanogr 41: 1208–1219.
- 26. Witte U, Wenzhöfer F, Sommer S, Boetius A, Heinz P, et al. (2003) In situ experimental evidence of the fate of a phytodetritus pulse at the abyssal sea floor. Nature 424: 763–766
- 27. Woulds C, Cowie GL, Levin LA, Andersson JH, Middelburg JJ, et al. (2007) Oxygen as a control on seafloor biological communities and their roles in sedimentary carbon cycling. Limnol Oceanogr 52: 1698–1709.
- 28. Levin L, Blair N, Martin C, DeMaster D, Plaia G, et al. (1999) Macrofaunal processing of phytodetritus at two sites on the Carolina margin: in situ experiments using C-13-labeled diatoms. Mar Ecol Prog Ser 182: 37–54.
- 29. Aberle N, Witte U (2003) Deep-sea macrofauna exposed to a simulated sedimentation event in the abyssal NE Atlantic: in situ pulse-chase experiments using C-13-labelled phytodetritus. Mar Ecol Prog Ser 251: 37–47.
- 30. Sweetman AK, Witte U (2008) Response of an abyssal macrofaunal community to a phytodetrital pulse. Mar Ecol Prog Ser 355: 73–84
- 31. Würzberg L, Peters J, Schüller M, Brandt A (2011) Diet insights of deep-sea polychaetes derived from fatty acid analyses. Deep-Sea Res II 58: 153–162
- 32. Jeffreys RM, Levin LA, Lamont PA, Woulds C, Whitcraft CR, et al. (2012) Living on the edge: single-species dominance at the Pakistan oxygen minimum zone boundary. Mar Ecol Prog Ser 470: 79–99
- 33. McMahon KW, Ambrose WG Jr, Johnson BJ, Sun MY, Lopez GR, et al. (2006) Benthic community response to ice algae and phytoplankton in Ny Ålesund, Svalbard. Mar Ecol Prog Ser 310: 1–14.
- 34. Sun MY, Clough LM, Carroll ML, Dai J, Ambrose WG Jr, et al. (2009) Different responses of two common Arctic macrobenthic species (Macoma balthica and Monoporeia affinis) to phytoplankton and ice algae: Will climate change impacts be species specific? J Exp Mar Biol Ecol 376: 110–121.
- 35. Laguionie-Marchais C, Billett DSM, Paterson GLD, Ruhl HA, Soto EH, et al. (2013) Inter-annual dynamics of abyssal polychaete communities in the North East Pacific and North East Atlantic—A family-level study. Deep-Sea Res I 75: 175–186
- 36. Smith K, Baldwin R, Ruhl H, Kahru M, Mitchell B, et al. (2006) Climate effect on food supply to depths greater than 4,000 meters in the northeast Pacific. Limnol Oceanogr 51: 166–176.
- 37. Smith K, Druffel E (1998) Long time-series monitoring of an abyssal site in the NE Pacific: an introduction. Deep-Sea Res II 45: 573–586.
- 38. Baldwin R, Glatts R, Smith K (1998) Particulate matter fluxes into the benthic boundary layer at a long time-series station in the abyssal NE Pacific composition and fluxes. Deep-Sea Res II 45: 643–665.
- 39. Smith K, Kaufmann R, Baldwin R, Carlucci A (2001) Pelagic-benthic coupling in the abyssal eastern North Pacific: An 8-year time-series study of food supply and demand. Limnol Oceanogr 46: 543–556.
- 40. Smith K Jr, Ruhl H, Kaufmann R, Kahru M (2008) Tracing abyssal food supply back to upper-ocean processes over a 17-year time series in the northeast Pacific. Limnol Oceanogr 53: 2655–2667.
- 41. Smith K, Baldwin R, Glatts R, Kaufmann R, Fisher E (1998) Detrital aggregates on the sea floor: Chemical composition and aerobic decomposition rates at a time-series station in the abyssal NE Pacific. Deep-Sea Res II 45: 843–880.
- 42. Beaulieu S, Smith K (1998) Phytodetritus entering the benthic boundary layer and aggregated on the sea floor in the abyssal NE Pacific: macro- and microscopic composition. Deep-Sea Res II 45: 781–815.
- 43. Venrick E (1998) Spring in the California current: the distribution of phytoplankton species, April 1993 and April 1995. Mar Ecol Prog Ser 167: 73–88.
- 44. Guillard RRL, Hargraves PE (1993) Stichochrysis immobilis is a diatom, not a chrysophyte. Phycologia 32: 234–236
- 45. Grasshoff K, Kremling K, Ehrhardt M (1983) Methods of Seawater Analysis. Weinheim: Verlag Chemie 419 pp.
- 46. Brown MR, Jeffrey SW, Volkman JK, Dunstan GA (1997) Nutritional properties of microalgae for mariculture. Aquaculture 151: 315–331.
- 47. Anderson MJ (2005) Permutational multivariate analysis of variance. Department of Statistics, University of Auckland, Auckland.
- 48. Anderson MJ, Gorley RN, Clarke KR (2008) Permanova+ for Primer: Guide to software and statistical methods. PRIMER-E Ltd.214 pp.
- 49. Fauchald K, Jumars P (1979) The diet of worms: a study of polychaete feeding guilds. Oceanogr Mar Biol Ann Rev 17: 193–284.
- 50. Gooday AJ, Hughes JA (2002) Foraminifera associated with phytodetritus deposits at a bathyal site in the northern Rockall Trough (NE Atlantic): seasonal contrasts and a comparison of stained and dead assemblages. Mar Micropaleontol 46: 83–110
- 51. Koho KA, Langezaal AM, van Lith YA, Duijnstee IAP, van der Zwaan GJ (2008) The influence of a simulated diatom bloom on deep-sea benthic foraminifera and the activity of bacteria: A mesocosm study. Deep-Sea Res I 55: 696–719
- 52. Smith CR, Hessler RR (1987) Colonization and succession in deep-sea ecosystems. Trends Ecol Evol 2: 359–363
- 53. Druffel E, Griffin S, Bauer J, Wolgast D, Wang X (1998) Distribution of particulate organic carbon and radiocarbon in the water column from the upper slope to the abyssal NE Pacific ocean. Deep-Sea Res II 45: 667–687.
- 54. van Oevelen D, Soetaert K, Heip C (2012) Carbon flows in the benthic food web of the Porcupine Abyssal Plain: The (un)importance of labile detritus in supporting microbial and faunal carbon demands. Limnol Oceanogr 57: 645–664
- 55. Enge A, Nomaki H, Ogawa N, Witte U, Moeseneder M, et al. (2011) Response of the benthic foraminiferal community to a simulated short-term phytodetritus pulse in the abyssal North Pacific. Mar Ecol Prog Ser 438: 129–142
- 56. Enge AJ, Witte U, Kučera M, Heinz P (2013) Uptake of phytodetritus by benthic foraminifera under oxygen depletion at the Indian Margin (Arabian Sea). Biogeosci Disc 10: 15305–15335
- 57. Nomaki H, Heinz P, Nakatsuka T (2006) Different ingestion patterns of 13C-labeled bacteria and algae by deep-sea benthic foraminifera. Mar Ecol Prog Ser 310: 95–108.
- 58. Sweetman AK, Sommer S, Pfannkuche O, Witte U (2009) Retarded response by macrofauna-size foraminifera to phytodetritus in a deep Norwegian fjord. J Foram Res 39: 15–22.
- 59. Moodley L, Middelburg JJ, Boschker H (2002) Bacteria and Foraminifera: key players in a short-term deep-sea benthic response to phytodetritus. Mar Ecol Prog Ser 236: 23–29.
- 60. McCutchan J, Lewis W, Kendall C, McGrath C (2003) Variation in trophic shift for stable isotope ratios of carbon, nitrogen, and sulfur. Oikos 102: 378–390.
- 61. Post D (2002) Using stable isotopes to estimate trophic position: Models, methods, and assumptions. Ecology 83: 703–718.
- 62. Iken K, Brey T, Wand U, Voigt J, Junghans P (2001) Food web structure of the benthic community at the Porcupine Abyssal Plain (NE Atlantic): a stable isotope analysis. Prog Oceanogr 50: 383–405.
- 63. Nomaki H, Ogawa N, Ohkouchi N, Suga H, Toyofuku T, et al. (2008) Benthic foraminifera as trophic links between phytodetritus and benthic metazoans: carbon and nitrogen isotopic evidence. Mar Ecol Prog Ser 357: 153–164.
- 64. Ingels J, Van den Driessche P, De Mesel I, Vanhove S, Moens T, et al. (2010) Preferred use of bacteria over phytoplankton by deep-sea nematodes in polar regions. Mar Ecol Prog Ser 406: 121–133
- 65. Ingels J, Billett DSM, Van Gaever S, Vanreusel A (2011) An insight into the feeding ecology of deep-sea canyon nematodes — Results from field observations and the first in-situ 13C feeding experiment in the Nazaré Canyon. J Exp Mar Biol Ecol 396: 185–193
- 66. Guilini K, van Oevelen D, Soetaert K (2010) Nutritional importance of benthic bacteria for deep-sea nematodes from the Arctic ice margin: Results of an isotope tracer experiment. Limnol Oceanogr 55: 1977–1989.
- 67. Fonseca G, Gallucci F (2008) Direct evidence of predation in deep-sea nematodes: the case of Pontonema sp. Cah Biol Mar 49: 295–297.
- 68. Sweeting C, Jennings S, Polunin N (2005) Variance in isotopic signatures as a descriptor of tissue turnover and degree of omnivory. Funct Ecol 19: 777–784
- 69. Herman P, Middelburg J, Widdows J, Lucas C, Heip C (2000) Stable isotopes' as trophic tracers: combining field sampling and manipulative labelling of food resources for macrobenthos. Mar Ecol Prog Ser 204: 79–92.
- 70. Raubenheimer D, Simpson SJ (2004) Organismal stoichiometry: quantifying non-independence among food components. Ecology 85: 1203–1216.
- 71. Clarke A (2008) Ecological stoichiometry in six species of Antarctic marine benthos. Mar Ecol Prog Ser. doi:10.3354/meps07670.
- 72. Mincks SL, Smith CR, Jeffreys RM, Sumida PYG (2008) Trophic structure on the West Antarctic Peninsula shelf: Detritivory and benthic inertia revealed by delta C-13 and delta N-15 analysis. Deep-Sea Res II 55: 2502–2514
- 73. Frost P.C., Stelzer RS, Lamberti GA, Elser JJ (2002) Ecological stoichiometry of trophic interactions in the benthos: understanding the role of C: N: P ratios in lentic and lotic habitats. J North Am Benth Soc: 515–528.
- 74. Frost PC, Evans White MA, Finkel ZV, Jensen TC, Matzek V (2005) Are you what you eat? Physiological constraints on organismal stoichiometry in an elementally imbalanced world. Oikos 109: 18–28.
- 75. Suhr SB, Pond DW, Gooday AJ, Smith CR (2003) Selective feeding by benthic foraminifera on phytodetritus on the western Antarctic Peninsula shelf: evidence from fatty acid biomarker analysis. Mar Ecol Prog Ser 262: 153–162.
- 76. Tanaka Y, Miyajima T, Koike I, Hayashibara T, Ogawa H (2006) Translocation and conservation of organic nitrogen within the coral-zooxanthella symbiotic system of Acropora pulchra, as demonstrated by dual isotope-labeling techniques. J Exp Mar Biol Ecol 336: 110–119
- 77. Nomaki H, Ohkouchi N, Heinz P, Suga H, Chikaraishi Y, et al. (2009) Degradation of algal lipids by deep-sea benthic foraminifera: An in situ tracer experiment. Deep-Sea Res I 56: 1488–1503.
- 78. Gannes LZ, del Rio CM, Koch P (1998) Natural abundance variations in stable isotopes and their potential uses in animal physiological ecology. Comp Biochem Physiol-Part A: Mol & Integr Physiol 119: 725–737.
- 79. Doi H, Kikuchi E, Takagi S, Shikano S (2007) Changes in carbon and nitrogen stable isotopes of chironomid larvae during growth, starvation and metamorphosis. Rapid Commun Mass Spectrom 21: 997–1002
- 80. Graves GR, Newsome SD, Willard DE, Grosshuesch DA, Wurzel WW, et al. (2012) Nutritional stress and body condition in the Great Gray Owl (Strix nebulosa) during winter irruptive migrations. Can J Zool 90: 787–797
- 81. Macko SA, Fogel ML, Hare PE, Hoering TC (1987) Isotopic fractionation of nitrogen and carbon in the synthesis of amino acids by microorganisms. Chem Geol 65: 79–92.
- 82. McGoldrick DJ, Barton DR, Power M, Scott RW, Butler BJ (2008) Dynamics of bacteria–substrate stable isotope separation: dependence on substrate availability and implications for aquatic food web studies. Can J Fish Aquat Sci 65: 1983–1990
- 83. van der Meer MTJ, Schouten S, Sinninghe Damsté JS (1998) The effect of the reversed tricarboxylic acid cycle on the 13C contents of bacterial lipids. Org Geochem 28: 527–533.
- 84. Campbell B, Cary S (2004) Abundance of reverse tricarboxylic acid cycle genes in free-living microorganisms at deep-sea hydrothermal vents. Appl Environ Microb 70: 6282–6289
- 85. Williams TJ, Zhang CL, Scott JH, Bazylinski DA (2006) Evidence for Autotrophy via the Reverse Tricarboxylic Acid Cycle in the Marine Magnetotactic Coccus Strain MC-1. Appl Environ Microb 72: 1322–1329
- 86. Middleburg J, Barranguet C, Boschker H (2000) The fate of intertidal microphytobenthos carbon: An in situ 13C-labeling study. Limnol Oceanogr 45: 1224–1234.
- 87. Pina-Ochoa E, Hogslund S, Geslin E, Cedhagen T, Revsbech NP, et al. (2010) Widespread occurrence of nitrate storage and denitrification among Foraminifera and Gromiida. P Natl Acad Sci 107: 1148–1153
- 88. Risgaard-Petersen N (2003) Coupled nitrification-denitrification in autotrophic and heterotrophic estuarine sediments: On the influence of benthic microalgae. Limnol Oceanogr 48: 93–105.
- 89. Bernhard JM, Casciotti KL, McIlvin MR, Beaudoin DJ, Visscher PT, et al. (2012) Potential importance of physiologically diverse benthic foraminifera in sedimentary nitrate storage and respiration. J Geophys Rese 117: G03002
- 90. Wolgast DM, Carlucci AF, Bauer JE (1998) Nitrate respiration associated with detrital aggregates in aerobic bottom waters of the abyssal NE Pacific. Deep-Sea Res II 45: 881–892.
- 91. Koho KA, Pina-Ochoa E, Geslin E, Risgaard-Petersen N (2010) Vertical migration, nitrate uptake and denitrification: survival mechanisms of foraminifers (Globobulimina turgida) under low oxygen conditions. FEMS Microbiol Ecol 75: 273–283
- 92. Prokopenko MG, Sigman DM, Berelson WM, Hammond DE, Barnett B, et al. (2011) Denitrification in anoxic sediments supported by biological nitrate transport. Geochim Cosmochim Ac 75: 7180–7199