Trophic relationship of benthic invertebrate fauna from the continental slope of the Sea of Japan

Deep-Sea Research II 86–87 (2013) 34–42

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Trophic relationship of benthic invertebrate fauna from the continental slope of the Sea of Japan b ¨ Vladimir I. Kharlamenko a,n, Angelika Brandt b, Serguei I. Kiyashko a, Laura Wurzberg a b

A.V. Zhirmunsky Institute of Marine Biology, Far East Branch, Russian Academy of Sciences, Palchevskogo 17, 690059 Vladivostok, Russia Biozentrum Grindel und Zoologisches Museum, Universit¨ at Hamburg, Martin-Luther-King-Platz 3, 20146 Hamburg, Germany

a r t i c l e i n f o

a b s t r a c t

Available online 7 August 2012

The Sea of Japan continental slope food web was examined by analysis of stable C and N isotopes and fatty acid compositions in ten species of common benthic organisms and in sediment and particulate organic matter. A considerable range of d13C and d15N values was found for benthic species, with d13C values of  22.3% in crinoids (Heliometra glacialis) to  16.1% in asteroids (Ctenodiscus crispatus) and with d15N values of 5.3% in foraminifera (Elphidium sp.) to 15.5% in C. crispatus. Polyunsaturated fatty acids were the most abundant of the fatty acids in the total lipids of all investigated species. The organisms’ individual fatty acid compositions show the importance of a variety of food sources, including phytoplankton, detritus, foraminiferans and zooplankton, for megabenthic species. Additionally, the presence of considerable amounts of the 20:4(n-6) and 20:1(n-13) fatty acids indicates the importance of the benthic microbial loop in the nutrition of some of the studied animals. & 2012 Elsevier Ltd. All rights reserved.

Keywords: Sea of Japan Continental slope Bathyal Invertebrate trophic position Stable isotopes Fatty acids

1. Introduction The causes of high species diversity in deep-sea ecosystems have been subject to increasing scientific attention. One of the major foci of such research is the investigation of trophic relationships within these ecosystems. Generally, the basis of the deep-water benthic food web is food particles, including phytodetritus and zooplankton remains (faecal pellets, carcasses and exuviae), sinking from the upper water layers and forming macroaggregates (‘‘marine snow’’), but the remains of coastal macrophytes and larger vertebrates and invertebrates also contribute to the food fluxes (Gooday et al., 1990; Sokolova, 1986; Vetter and Dayton, 1999). Thus, different types of detritus are available to deep-sea organisms, making the specialisation of detritus feeders likely (Carney, 2005). The Sea of Japan (SJ) is distinct from the other marginal North Pacific seas because of the shallowness of the straits connecting it with the Pacific Ocean. The SJ deep waters, which are cold and well-aerated, are formed by the sinking of cooled surface water (Zenkevich, 1963). Primary production in the SJ is much lower than in the other North Pacific Ocean regions; consequently, export fluxes to the seafloor in the SJ are lower than in other regions (Shuntov, 2001). Carnivorous zooplankton do not inhabit the bathypelagic zone of the SJ (Vinogradov, 1973). The combination of the cold waters and the lack of carnivorous zooplankton results in large quantities of zooplankton carcasses sinking to the seafloor, where they become food for benthic animals (Vinogradov and

n

Corresponding author. Tel.: þ7 4232 310937. E-mail address: [email protected] (V.I. Kharlamenko).

0967-0645/$ - see front matter & 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.dsr2.2012.08.007

Sazhin, 1978). Plant residues are also typically present on the SJ seafloor (Mokievskii, 1954). The continental slope of the SJ is large, covering approximately 40% of the total area of the seafloor (Zenkevich, 1963). Except for some commercially exploited species, the composition and biomass of the fauna inhabiting the northwestern continental slope of the SJ have only been studied in a limited area (Deryugin, 1939; Levenstein and Pasternak, 1973; Mokievskii, 1954). Moreover, food-web studies based on microscopic stomach content analyses have only been conducted for two species (Chuchukalo et al., 2011; Sokolova, 1982). Traditional methods for studying trophic relationships in marine ecosystems include direct observation of feeding animals and analysis of gut contents. These methods are often unsuitable for deep-sea organisms because of the small size of the organisms in these taxa and the poor state of most food material when it reaches the seafloor. Direct observation is generally only possible by camera, and feeding processes have only been captured in a limited number of cases. Gut contents depend on the periodicity of the food supply and may be regurgitated upon sample retrieval. Therefore, alternative methods such as analysing the natural distribution of stable carbon and nitrogen isotopes in food-web compartments have been used to study trophic relationships in deep-sea ecosystems. The isotope method provides time-integrated averages of ingested food items (e.g., Hobson and Welch, 1992) and was successfully used in studies of trophodynamics in deep-sea ecosystems (Fanelli et al., 2011; Iken et al., 2001; Polunin et al., 2001). Additionally, analysing fatty acid (FA) compositions can enhance the quality of such ecological studies by capturing complex feeding interactions. Many potential food sources have specific FA compositions that can be used to trace

V.I. Kharlamenko et al. / Deep-Sea Research II 86–87 (2013) 34–42

the utilisation of the food source by consumers. For this reason, FA analyses have been used extensively for the study of trophic relationships in marine food webs (Dalsgaard et al., 2003; Kelly and Scheibling, 2012). The aim of this study was to identify the main food sources and analyse the trophic interactions of the common macrobenthic and megabenthic species in the SJ bathyal zone (500–2000 m) using stable isotopes and FA analysis. In this study we put more emphasis to the assessment of the trophic role of phytoplankton, zooplankton and bacteria on the continental slope of SJ. The trophic role of phytoplankton, zooplankton and bacteria in SJ can differ from their role in the other Far Eastern Seas due to its special characteristics.

2. Materials and methods All materials analysed were obtained in August 2010 during the SoJaBio (Sea of Japan Biodiversity Studies) expedition aboard the R/V Akademik Lavrentyev. Sampling stations were positioned on the northwest continental slope of the Sea of Japan in water depths of 500–1600 m (Fig. 1). Megabenthic species were collected with an Agassiz trawl (AGT) and an epibenthic sledge (EBS, Brenke, 2005). Bottom sediments were collected with a multiple corer (MUC), and the upper three centimetres of sediment were subsampled for analysis. Samples of particulate organic matter (POM) were obtained by filtering approximately 300 cm3 of the bottom water collected in the MUC over precombusted (at 450 1C for 4 h) GF/F filters. These samples contained mainly resuspended POM from the flocculent layer of the bottom sediments, rather than suspended organic matter from the water column. Megabenthic organisms were dissected onboard after collection, and tissue subsamples were either stored in vials with a chloroform–methanol mixture at  20 1C for FA analysis or immediately oven-dried at 60 1C and stored in a desiccator for isotope analysis. The individuals of all megabenthic species sampled were adults of the same size class. Foraminiferans were immediately separated from the total meiobenthos onboard and processed in the same way as the megabenthic samples. Lipids were extracted from invertebrate tissues, filters and sediment organic matter (SOM) using the Bligh and Dyer extraction method (Bligh and Dyer, 1959). Fatty acid methyl esters (FAMEs) were prepared from the total lipids according to the generally accepted Carreau and Dubacq procedure (Carreau and Dubacq, 1978) and purified with a preparative thin layer chromatography in benzene. The 4,4-dimethyloxazoline derivatives of the FAs were prepared according to Svetashev (2011). The FAMEs

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were analysed on a Shimadzu GC 2010 chromatograph with the use of a fused quartz capillary (30 m  0.25 mm) column coated with SUPELCOWAX 10 (Supelco). FAs were identified based on the gas liquid mass spectrometry data from the FAMEs and on the 4,4-dimethyloxazoline derivatives of the FAs. Mass spectrometry was performed on a Shimadzu GCMS QP5050A spectrometer using MDN 5S columns (temperature gradient from 160 to 260 1C at 2 1C/min). All spectra were obtained by using the electron impact method at 70 eV. For the isotopic analysis, muscle tissues were sampled from molluscs (foot and mantle muscles) and crustaceans (leg and abdominal muscles), tube feet were sampled from asteroids, and arm fragments were sampled from ophiuroids and crinoids. Ovendried samples were ground to a fine powder using an agate mortar and pestle, and the 0.5 mg subsamples were packaged into tin caps. Because of the low lipid content of all the isotopic samples analysed, neither lipid extraction nor lipid correction was performed. Ophiuroid and crinoids subsamples, which contained the skeletal mineral carbonate, and samples of bottom sediments and POM were treated with 1 M HCl and re-analysed for carbon isotopic compositions. A composite sample of foraminiferans was analysed after acidification in a silver cup according to Jaschinski et al. (2008). Isotopic analysis was performed at the Laboratory of Stable Isotopes (Far Eastern Geological Institute, DVO RAN) using a system of devices: a FlashEA 1112 elemental analyser, a ConFlo IV interface, and a MAT 253 isotope mass spectrometer (ThermoQuest, Germany). Sample isotopic ratios were expressed in the conventional d notation as parts per thousand (%) according to the following equation: 
 d13 C or d15 N ¼ Rsample Rstandard =Rstandard  1000, where R¼ 13C/12C or 15N/14N. The d values were expressed relative to the international reference standards of Pee Dee Belemnite (PDB) for carbon and atmospheric N2 for nitrogen. The internal laboratory standard was measured after every sixth sample during analysis to control the data quality. Based on the standard deviation (SD) of the replicates of the laboratory standard, internal precision was 70.1% for both d13C and d15N. The relative trophic level (TL) of the megabenthic consumers was calculated by using the following equation (Post, 2002):   TL ¼ d15 Nconsumer d15 Nbase =Dd15 N þTLbase , where d15Nconsumer is the d15N value of the species in question, and d15Nbase and TLbase are the d15N and TL values of the baseline species, respectively. Dd15N is the 15N-enrichment factor per trophic level; we used a Dd15N value of 3.4% (Minagawa and Wada, 1984; Post, 2002). We chose the selective deposit feeder Megayoldia sp. as the baseline species and assumed that it had a TL value of 2. Statistical analysis was performed using Statistica 6.0 and PRIMER 6 software. Data were analysed with one-way ANOVA and Tukey’s test at a significance level of p o0.05. The FA profiles between species were compared using principal component analysis (PCA). The FAs that contributed to less than 1.0% of the total FA composition were omitted from statistical analysis. 3. Results 3.1. Stable isotope composition of invertebrates, SOM, POM and foraminifera

Fig. 1. Map of SojaBio study area. The stars indicate point where samples on continental slope were collected.

The isotopic analysis revealed a considerable range of d13C and d15N values for the 9 megabenthic species (Table 1). No correlation between the carbon and nitrogen isotopic values was found for any of the megabenthic samples analysed (r2 ¼0.02; p¼0.36; N ¼42).

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V.I. Kharlamenko et al. / Deep-Sea Research II 86–87 (2013) 34–42

Table 1 d 13C and d15N values of benthic invertebrates, bottom sediments and particulate organic matter (mean values 7 standard deviation). Mean values marked with the same letters are not significantly different (p 40.05, ANOVA Tukey HSD test). N ¼number of samples. Group/source

Species

POM

SOM

Foraminifera Mollusca: Bivalvia

Elphidium sp. (ca. 60 ind. pooled) Robaia robai

Megayoldia sp.

Cardiomya beringensis

Mollusca: Scaphopoda Crustacea: Decapoda Echinodermata: Crinoidea Echinodermata: Ophiuroidea Echinodermata: Asteroidea

Fustiaria nipponica Chionoecetes japonicus Eualus biungus Heliometra glacialis Ophiura leptoctenia Ctenodiscus crispatus

Station

Depth (m)

Gear

N

d15N

d13C

B7 B6 Mean B6 A3 Mean B6 A2 B6 Mean B7 A2 B6 Mean B7 B6 Mean B7 A3 B6 B7 B7 A2 B6 A3 Mean

532 1075

MUC MUC

1075 1530

MUC MUC

1075 582 1075

MUC AGT AGT

532 582 1075

AGT AGT AGT

532 1075

AGT AGT

532 1623 1011 532 532 582 1075 1623

AGT AGT EBS AGT AGT AGT AGT AGT

1 1 2 1 2 3 1 3 3 6 3 3 1 7 1 3 4 3 3 5 3 4 3 1 3 7

0.6 1.8 1.27 0.8 5.4 6.3c 7 0.1 6.0c 7 0.5 5.3 8.2b 70.4 8.0b 7 0.1 8.1b 70.3 6.0c 7 0.4 6.6c 7 0.4 6.5 6.3c 7 0.4 12.8 12.9a 7 0.3 12.9a 7 0.2 9.3b 71.0 13.5a 7 0.3 11.5 70.2 9.6b 70.4 9.1b 70.1 12.6a 7 1.1 13.6 15.5 70.3 14.0a 7 1.6

 25.0  24.3  24.77 0.5  22.6  23.0a 7 0.2  22.9a 7 0.3  20.0  18.2c 7 0.4  18.9c 7 0.5  18.6c 7 0.6  18.1c 7 0.2  17.6c 7 0.3  18.0  17.9c 7 0.3  20.0  19.8b 70.1  19.8b 70.1  18.5c 7 0.8  19.9b 70.1  19.7b 70.3  22.3a 7 0.5  21.1a 7 0.4  16.7d 70.9  17.4  16.1d 70.5  16.5d 70.7

The most depleted d13C values were observed for POM (  24.7%) and SOM (  23.0%). The mean d13C values of the megabenthic species ranged from  22.3% (Heliometra glacialis, a crinoid) to  16.5% (Ctenodiscus crispatus, an asteroid). The d13C values of H. glacialis and the ophiuroid Ophiura leptoctenia did not differ significantly from the d13C values measured for SOM. Significant differences in d13C were found between the group of H. glacialis and O. leptoctenia and the group of Eualus biunguis (a shrimp), Chionoecetes japonicus (a crab) and Cardiomya behringensis (a septibranch bivalve). A third group consisted of the infaunal species R. robai and Megayoldia sp. (protobranch bivalves) and Fustiaria nipponica (a scaphopod mollusc), which had mean d13C values significantly different from those of the other two groups. The POM samples also had the most depleted d15N values. The d15N values of SOM were enriched by 4.8% relative to POM. The d15N values were low for Elphidium sp. (foraminifera) and Megayoldia sp., and they did not differ significantly from the d15N values of SOM. Four zoobenthic species (R. robai, O. leptoctenia, F. nipponica and H. glacialis) compose a separate group with d15N values ranging from 8.1% to 9.8%. The species with the highest d15N values were the predators C. japonicus, C. behringensis and E. biunguis and the deposit feeder C. crispatus. Four megabenthic species (bivalves Megayoldia sp., R. robai, and C. behringensis and asteroid C. crispatus) were sampled at more than one station (Table 1). We found no significant intraspecies differences in either d13C or d15N values for bivalve molluscs sampled at different water depths. The samples of the asteroid C. crispatus showed a significant enrichment of d15N values by ca. 3% with increasing water depth from 500 to 1600 m. We suggest that these intraspecies variations in C. crispatus were not the result of ontogenetic differences because all individuals studied were from the same size class. No significant spatial variations were found for the d13C values of C. crispatus.

3.2. Fatty acid composition of invertebrates, SOM, POM and foraminifera The POM and SOM samples collected on the continental slope contained large amounts of the FAs 16:0, 18:1(n-9) and 18:2(n-6) (Table 2). The ratio of 16:1(n-7) to 16:0 was 0.13 in POM and 0.5 in SOM. In addition to the high 18:2(n-6) values, another notable result was the presence of an elevated level of 16:1(n-10), especially in POM. Low levels of polyunsaturated FAs (PUFAs) and FAs typical of bacteria were found in both SOM and POM. High levels of docosahexaenoic acid (22:6(n-3) or DHA) and eicosapentaenoic acid (20:5(n-3) or EPA) found in foraminiferan FAs distinguished them from SOM and POM, whereas the levels of arachidonic acid (20:4(n-6) or AA) were the same in both groups. The FA compositions of most species studied were dominated by PUFAs (Table 3). The PUFA content ranged from 33.8% in R. robai to 58.4% in C. japonicus. The dominant PUFAs in most species were EPA and DHA. However, AA played a major role in C. crispatus and F. nipponica. The monounsaturated FA (MUFA) content of the samples ranged from 24.5% to 40.5%. In three of the megabenthic species, the MUFA content was higher than the PUFA content. In all of the analysed species, 18:1(n-9) and 18:1 (n-7) were the major MUFAs, with the exception of C. crispatus, for which 20:1(n-13) was the predominant MUFA. The highest levels of 20:1(n-9) and 22:1(n-9) were observed in C. behringensis. The saturated FA (SFA) content ranged from 12.1% to 26.0%, with the highest values measured in E. biunguis. The non-methyleneinterrupted dienoic fatty acid (NMID) content ranged from 0.07% to 4.23%. The maximum level of NMIDs was found in the scaphopods and the minimum level of these FAs was found in the decapods. The highest bacterial FA percentages were found in O. leptoctenia and C. crispatus (3.16–3.81%). Only FAs composing more than 1% of the total FA content were used to conduct the principal component analysis (PCA). The relative contents of these FAs differed significantly among the

V.I. Kharlamenko et al. / Deep-Sea Research II 86–87 (2013) 34–42

Table 2 Fatty acid composition (in % of total fatty acids) of particulate organic matter, bottom sediment and foraminifera total lipids (mean values7 standard deviation). N ¼number of samples. Fatty acids

14:0 16:0 16:1(n-10) 16:1(n-7) 7-Me-16:1n-10 16:4(n-1) 18:0 18:1(n-11) 18:1(n-9) 18:1(n-7) 18:1(n-5) 18:2(n-6) 20:1(n-13) 20:1(n-11) 20:1(n-9) 20:1(n-7) 20:2(n-9) 20:4(n-6) 20:5(n-3) 22:1(n-15) 22:1(n-13) 22:1(n-9) 21:5(n-3) 22:5(n-3) 22:6(n-3) Bacterial FAn Non-methyleneinterrupted FA (NMID)nn

POM

SOM

N¼2

N¼2

Elphidium sp. (ca. 60 ind.) N ¼1

4.77 0.1 3.2 7 1.2 4.4 21.9 7 0.7 19.2 7 1.7 22.6 3.87 4.07 1.4 3.8 2.87 0.1 8.1 7 2.1 12.4 0.47 0.0 0.97 0.4 1.1 0.17 0.0 0.37 0.1 0.7 7.97 0.4 6.8 7 1.5 7.7 0.17 0.1 0.17 0.1 0.0 17.3 7 1.6 13.2 7 1.7 9.8 1.37 1.3 4.2 7 0.1 5.0 0.57 0.1 0.27 0.1 0.0 11.7 7 2.3 11.3 7 6.5 4.5 1.77 0.0 0.57 0.0 1.0 0.77 0.0 0.47 0.1 0.0 0.37 0.0 0.27 0.0 0.4 0.17 0.0 0.0 7 0.0 0.2 0.17 0.0 0.0 7 0.0 0.0 0.47 0.0 1.6 7 0.5 2.3 2.67 0.3 3.4 7 0.5 9.7 0.97 0.1 0.67 0.0 0.0 0.57 0.0 0.27 0.0 0.4 0.0 7 0.0 0.0 7 0.0 0.0 0.77 0.3 0.0 7 0.0 0.6 0.7 7 0.0 1.3 7 0.2 0.4 1.87 0.2 1.2 7 0.5 0.6 3.97 0.3 6.5 7 1.8 1.1 0.57 0.2 0.17 0.0 0.0

Nonionella sp. (ca. 45 ind.) N¼ 1 2.1 11.3 2.1 14.9 1.2 0.9 3.9 0.0 3.9 3.1 0.0 2.9 0.3 2.4 0.0 0.0 0.0 3.0 26.1 0.0 0.4 0.0 0.0 0.0 0.8 2.9 0.0

n Bacterial fatty acids were iso-15:0, anteiso-15:0,15:0, iso-16:0, iso-17:0, anteiso-17:0,17:0, 17:1n-8, iso-18:0, 19:0. nn Non-methylene-interrupted FA were 20:2(5,11), 20:2(5,13), 22:2(7,13), 22:2(7,15).

species studied. Only loadings of the first two components of FAs that were higher than 0.3 were taken into account (Table 4). The PCA outcome shows that 18:0, 20:1(n-13), 16:0, bacterial FA, AA, EPA and DHA were the most important for distinguishing between samples along PC 1, whereas 16:1(n-7), 14:0, 16:4(n-1) and DHA were most important along PC 2 (Table 4). The PCA divided the analysed organisms into three groups according to their FA composition (Fig. 3). The first group comprises C. crispatus and F. nipponica, which had high levels of 18:1(n-9) and AA, the latter being the dominant FA in C. crispatus. The second group consists of Megayoldia sp. and R. robai, which were rich in 14:0, 16:1(n-7) and 16:4(n-1), and O. leptoctenia, which had a higher 18:1(n-11), 18:1(n-9) and 20:1(n-13) content than the bivalves. The third group comprises C. japonicus, E. biunguis, H. glacialis and C. behringensis, which are characterised by a high DHA and 18:1(n-9) content. C. behringensis and H. glacialis have lower DHA contents than the decapods.

4. Discussion 4.1. Stable isotope composition and trophic position of invertebrates Strong positive correlations between d15N and d13C values measured in animals point towards a single primary food source supporting marine communities (Fanelli et al., 2009; Polunin et al., 2001). In this study, the absence of a strong correlation between the d15N and d13C values of the animal samples may

37

indicate the utilisation of multiple primary food sources by the SJ continental slope benthos. The range of d15N values indicates that the benthic community on the SJ continental slope had at least four relative trophic levels (Fig. 2). The primary consumers in this ecosystem (the foraminiferan Elphidium sp. and the protobranch bivalve Megayoldia sp.) are much more enriched in 13C relative to POM and SOM one can expected, suggesting that POM and SOM are main food sources for these consumers (Fig. 2). Additionally, these benthic consumers were more 13 C-enriched than the primary consumers in the planktonic food web of this region of the SJ (Park et al., 2011). The selective deposit feeder Megayoldia sp. had high amounts of diatom FA markers (Table 3), suggesting that it feeds on the fresh organic matter of settled diatoms. Diatom blooms are known to produce organic matter that is more 13C-enriched than other planktonic primary producers (Fry and Wainright, 1991). The flow of 13C-enriched planktonic diatoms from adjacent neritic waters may also contribute to the diet of this inhabitant of the continental slope. It has been reported that the bivalve Yoldia hyperborea (in the same family as Megayoldia sp.) strongly depends on the input of fresh algal material, despite the high availability of organic matter in sediment (Stead and Thompson, 2003). Furthermore, in phytoplankton settling experiments, Y. hyperborea has been shown to extend its palp proboscides over the sediment surface and maintain close contact with the area of the highest algal concentration (Stead and Thompson, 2006). Another selective deposit feeder, the protobranch bivalve R. robai was collected from the same stations as Megayoldia sp. but had higher d15N values and occupied a higher trophic position (TL 2.5) than Megayoldia sp. The FA composition analysis of R. robai reflected the consumption of refractory and recycled material, which is enriched with the heavier nitrogen isotope. Therefore, it is likely that R. robai utilises subsurface food, whereas Megayoldia sp. ingests food from the surface layers of the sediment. The third trophic level is occupied by the scaphopod mollusc F. nipponica, the ophiuroid O. leptoctenia, and the crinoid H. glacialis (Fig. 2). Scaphopod molluscs are known to be microcarnivores or microomnivores that feed mainly on foraminiferans (Langer et al., 1995; Reynolds, 2002). The isotopic composition of F. nipponica was consistent with a diet of foraminiferans, as demonstrated by the close agreement between the d15N and d13C values, assuming trophic enrichment (1% for 13C and 3.4% for 15N). The suspension feeder H. glacialis and the selective deposit feeder O. leptoctenia had similar nitrogen and carbon isotopic compositions which corresponded, assuming trophic enrichment, to feeding on SOM or on zooplankton primary consumers (Fig. 2). The analysis of the FA markers did not support the feeding of either of these consumers on resuspended refractory SOM (Fig. 3). Both species contained relatively high amounts of n-3 PUFAs, suggesting a substantial diet of fresh organic matter. The crinoid H. glacialis contained FAs, namely 20:1(n-9) and 22:1(n-11), characteristic of zooplankton, primarily copepods. High amounts of the FA 20:1(n-13) occurred in the brittle star O. leptoctenia, suggesting that the microbial food web contributes to the feeding of this species. Our FA data showed that the diets of O. leptoctenia and H. glacialis differ significantly despite the similarity of their isotopic compositions. Both echinoderms ingest zooplankton, but O. leptoctenia also utilises the microbial food web. The nitrogen isotopic compositions of the megabenthos indicate that the fourth trophic level of the SJ continental slope benthic community consists of the large crab C. japonicus, the shrimp E. biunguis, the septibranch bivalve C. behringensis, and the sea star C. crispatus. The literature on gut content analysis characterises C. japonicus as a benthic predator, ingesting shrimp, ophiuroids, polychaetes, squids, and juveniles of its own species (Chuchukalo et al., 2011). The d15N value of C. japonicus is therefore indicative of its status as a top benthic predator (TL

38 Table 3 Fatty acid composition (in % of total fatty acids) of the total lipids of benthic invertebrates (mean7 standard deviation). Mean values marked with the same letters are not significantly different (p 40.05, ANOVA Tukey HSD test). N—number of samples. Table contains only fatty acids with concentrations 41%.n Cardiomya behringensis Robaia robai N ¼3 N ¼3

14:0 16:0 16:1 (n-7) 7-Me-16:1n-10 16:4 (n-1) 18:0 18:1 (n-11) 18:1 (n-9) 18:1 (n-7) 18:2 (n-6) 18:4 (n-3) 20:1 (n-13) 20:1 (n-11) 20:1 (n-9) 20:1 (n-7) 20:2 (n-6) 20:4 (n-6) 20:5 (n-3) 22:1 (n-13) 22:1 (n-9) 22:5 (n-3) 22:6 (n-3) 24:6 (n-3) Bacterial fatty acidsnn Non-methylene-interrupted FA (NMID)nnn

1.72c 7 0.43 11.09b 7 1.00 5.06b 7 0.62 0.21 7 0.08 0.00d 7 0.00 4.79b,c 7 0.85 0.00 7 0.00 11.63a 7 2.50 8.84a,b 7 0.71 1.10 7 0.34 0.38 7 0.20 4.36c 7 0.67 0.00c 7 0.00 4.22a 7 0.54 1.89b 7 0.26 0.08 7 0.07 2.22c 7 0.34 10.14d 7 1.28 0.87b 7 0.22 1.94a 7 0.22 0.57b,c 7 0.07 11.34b 7 1.26 0.00 7 0.00 2.01a,b 7 0.06 2.90b 7 0.51

n

Megayoldia sp. Fustiaria nipponica Chionoecetes japonicus Eualus biunguis Heliometra glacialis Ophiura leptoctenia Ctenodiscus crispatus N¼3 N¼3 N¼3 N ¼5 N¼3 N ¼3 N ¼6

4.99b 7 0.79 5.32b 7 0.79 11.67b 7 1.98 11.54b 7 1.41 14.51a 7 0.73 16.45a 7 0.99 0.25 7 0.07 0.067 0.11 0.84b 7 0.04 2.28a 7 0.22 2.51d 7 0.30 2.15d 7 0.57 0.46 7 0.10 0.397 0.06 2.22c 7 0.26 2.29c 7 0.42 a 10.31 7 0.20 8.19a,b 7 0.43 0.84 7 0.06 1.087 0.05 1.24a 7 0.24 1.47a 7 0.19 2.62c 7 0.60 1.07c,d 7 0.20 0.36c 7 0.09 0.00c 7 0.00 0.36c 7 0.09 0.27c 7 0.06 0.03d 7 0.03 3.29a 7 0.35 0.00 7 0.00 0.067 0.03 3.25c 7 0.56 2.37c 7 0.42 b,c 20.27 7 4.57 26.55a,b 7 2.68 0.21b 7 0.06 0.23b 7 0.09 0.17c 7 0.01 0.30c 7 0.17 0.60b,c 7 0.21 0.30c 7 0.05 3.08c,d 7 0.56 1.227 0.24 0.00 7 0.00 0.00 7 0.00 a,b 2.89 7 0.59 1.20b 7 0.06 a,b 3.39 7 0.75 1.84b,c 7 0.46

1.08c 7 0.09 5.73c 7 1.02 4.70b 7 0.33 0.257 0.06 0.00d 7 0.00 6.25a,b 7 0.09 0.157 0.03 1.96c 7 0.10 9.40a 7 0.67 0.887 0.10 0.137 0.17 2.33c 7 0.08 0.29c 7 0.02 2.52b 7 0.15 0.13d 7 0.03 1.31a 7 0.21 13.01b 7 1.16 18.69c 7 0.16 0.15b,c 7 0.06 0.17c 7 0.00 4.51a 7 0.32 1.62d 7 0.09 0.00 7 0.00 3.01a 7 1.00 4.23a 7 0.93

0.30c 70.16 12.94b 71.11 1.22c 70.22 0.06 70.03 0.00d 70.00 1.67d 70.11 0.00 70.00 10.37 a,b 70.96 7.79b 70.79 0.78 70.10 0.10 70.02 0.00d 70.00 0.09c 70.16 1.82b 70.65 0.18d 70.04 0.05 70.04 3.03c 70.38 29.03a 71.26 0.00c 70.00 0.00c 70.00 0.45cd 70.02 21.14a 70.17 0.00 70.00 1.06b 70.07 0.21d 70.28

Other fatty acids were 16:1 (n-10), 16:4 (n-1), 18:1 (n-5), 18:2 (n-4), 21:5 (n-3), 22:4 (n-6), 22:5 (n-6), 24:1 (n-9). Bacterial fatty acids were iso-15:0, anteiso-15:0,15:0, iso-16:0, iso-17:0, anteiso-17:0,17:0, 17:1n-8, iso-18:0, 19:0. nnn Non-methylene-interrupted FA were 20:2(5,11), 20:2(5,13), 22:2(7,13), 22:2(7,15). nn

1.55c 7 0.46 21.29a 7 2.56 4.60b 7 1.33 0.18 7 0.11 0.00d 7 0.00 1.84d 7 0.25 0.03 7 0.04 8.48a,b 7 1.46 9.79a,b 7 1.24 1.25 7 0.21 0.21 7 0.02 0.00d 7 0.00 1.18b 7 0.21 0.82c 7 0.12 0.27d 7 0.05 0.00 7 0.00 3.49c 7 0.80 18.81c 7 1.05 0.00c 7 0.00 0.17c 7 0.12 0.35c,d 7 0.07 17.59a 7 3.16 0.00 7 0.00 1.36b 7 0.22 0.07d 7 0.11

1.70c 7 0.09 9.57b,c 7 0.57 3.98b 7 0.37 0.24 7 0.24 0.54c 7 0.06 4.46c 7 0.66 0.00 7 0.00 6.92b 7 1.15 2.04d 7 0.16 0.89 7 0.30 1.08a 7 0.16 0.00d 7 0.00 1.96a 7 0.59 1.94b 7 0.48 1.12c 7 0.06 0.00 7 0.00 3.98c 7 0.83 25.01a,b 7 3.24 1.95a 7 0.81 0.03c 7 0.06 0.72b 7 0.05 7.16b,c 7 2.15 6.20b 7 0.91 2.14a,b 7 0.93 0.55c,d 7 0.05

7.87a 7 2.39 8.40b,c 7 0.50 5.77b 7 0.63 0.16 7 0.15 0.03d 7 0.06 3.65c 7 0.96 2.13a 7 0.99 9.14a,b 7 2.94 4.64c 7 0.57 0.84 7 0.04 0.08 7 0.07 8.54b 7 0.53 0.44c 7 0.38 0.33c 7 0.38 0.42d 7 0.10 0.60 7 0.15 2.12c 7 0.47 17.32c 7 2.01 0.80b 7 0.02 0.89b 7 0.20 0.37b,c.d 7 0.09 2.85d 7 0.54 8.90a 7 1.19 3.16a 7 0.68 1.07c 7 0.15

0.75c 70.22 3.94c 70.93 1.15c 70.58 1.19a 70.18 0.00d 70.00 6.46a 70.47 0.16 70.13 2.98b,c 70.87 5.98b,c 70.93 1.14 70.84 1.23a 70.31 11.87a 71.84 0.00c 70.00 0.00c 70.00 1.24c 70.12 0.47 70.38 32.07a 73.76 9.60d 73.65 0.04c 70.06 0.90b 70.17 0.12d 70.10 1.42d 70.25 0.00 70.00 3.81a 71.00 1.22c 70.48

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Fatty acids

Fig. 2. d13C versus d15N biplot showing stable isotope composition of suspended particulate organic matter (POM), bottom sediment organic matter (SOM) and all consumers (solid symbols) collected on the Sea of Japan continental slope. Data on isotopic composition of epipelagic primary consumers (open symbols) (from Park et al., 2011) are shown for comparison. Dotted lines represent trophic levels (TL) based on d15N data. A trophic shift box illustrates the range in stable isotope values expected for fauna consuming SOM only. Cb—Cardiomya behringensis, Cc—Ctenodiscus crispatus, Cj—Chionoecetes japonicus, cop—copepods, E—Eualus biungus, Esp—Elphidium sp., euph—euphausiids, F—Fustiaria nipponica, H—Heliometra glacialis, M—Megayoldia sp., R—Robaia robai, O—Ophiura leptoctenia.

 0.06 0.22

0.21  0.24

0.11 0.01

 0.36  0.03

0.19  0.11

 0.04 0.26

 0.36  0.15

0.30 0.14

0.05 0.07

 0.08 0.01

0.31  0.33

0.01 0.10

 0.36 0.01

 0.16 0.23

39

 0.38  0.08 0.10 0.45 0.36 0.13  0.09 0.47 PC1 0.05 PC2 0.40

18:1n-11 18:1n-9 18:1n-7 20:1n-13 20:1n-11 20:1n-7 20:4n-6 20:5 n-3 22:1 n-13 22:5 n-3 22:6 n-3 24:6 n-3 Bacterial FA Non-methylene-interrupted FA 16:1n-7 16:4n-1 18:0 14:0 16:0

Table 4 Loadings of the first two components of fatty acids used for the PCA.

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Fig. 3. Biplot of the first and second PC derived from the fatty acid composition of individual specimens of invertebrate of continental slope the Sea of Japan. PC1 explained 30.0% of the variability between species. PC2 explained 18.9% of the variability. Cb—Cardiomya behringensis, Cc—Ctenodiscus crispatus, Cj—Chionoecetes japonicus, H—Heliometra glacialis, M—Megayoldia sp., O—Ophiura leptoctenia, R—Robaia robai, F—Fustiaria nipponica, E—Eualus biunguis.

4.1). The more omnivorous shrimp E. biunguis had a lower trophic status (TL 3.5). The septibranch bivalve mollusc C. behringensis had an isotopic composition similar to that of top predator C. japonicus. The FA composition of C. behringensis is similar to that of the crinoid

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H. glacialis, suggesting a diet of zooplankton (Fig. 3). Septibranch bivalves have been shown to be capable of capturing live zooplankton with their inhalant siphon (Reid, 1977). However, the d15N values of C. behringensis indicate that its trophic position (TL 3.9) is one level higher than the crinoids and two levels higher than the pelagic zooplankton (Fig. 2). The Cardiomya species from the southern SJ shelf have also been shown to have d15N values higher than those of the coexisting fishes, crabs and shrimps (Antonio et al., 2010). We hypothesise that small benthic crustaceans that feed on the more 15N-enriched microbial food web (rather than zooplankton) are the prey for Cardiomya. The asteroid C. crispatus had the highest d15N values of the studied invertebrates. It is notable that the 15N-enrichment of this species increased with increasing water depth (Table 1). The apparently high trophic status of C. crispatus contradicted the feeding mode of this species, which is known to be an infaunal deposit feeder, unselectively ingesting fine sediment particles (Shick et al., 1981). High d15N values and accordingly high trophic levels (higher than those of most bottom fishes) have been reported in C. crispatus individuals from a high-latitude fjord ecosystem (Nilsen et al., 2008). One possible explanation is that C. crispatus feeds on fine refractory and recycled materials, as has been observed in deep-water benthic filter feeders (Iken et al., 2001; Mintenbeck et al., 2007). 4.2. Fatty acid composition and megabenthic food sources The FA composition of the SOM and POM samples showed the high SFA content typical of this type of organic matter because of its degradation processes. Furthermore, the SOM and POM samples were characterised by a high 18:2(n-6) content and the presence of 16:1(n-10), which in POM reached higher levels than the more common 16:1(n-7). The FAs 18:2(n-6) and 16:1(n-10) are not known to be typical components of the marine bottom sediment or POM lipids, but in some studies they were a notable constituent (1.9–5.2%) in the faecal pellets of copepods and euphausiids (Mayzaud et al., 2007) and in marine aggregates made up of mixed phytoplankton populations (Balzano et al., 2011). However, in this study, 16:1(n-10) was not detected in the analysed animals in concentrations exceeding 1%. The FA 18:2(n-6) is often considered a marker of terrestrial organic matter (Budge and Parrish, 1998; Napolitano et al., 1997), although the sources of this FA in marine sediments include fungi, algae, protozoa, cyanobacteria (Findlay et al., 1990) and seagrasses (Khotimchenko, 1993). It was shown that approximately 73% of the PUFAs preserved in marine sediments are composed of 18:2(n-6) and 18:3(n-3); consequently it was suggested that FAs of marine origin, rather than those of terrestrial origin, are selectively removed from the water column (Budge and Parrish, 1998). In the SOM and POM from the continental slope of the SJ, more than 60% of the PUFAs were 18:2(n-6) and 18:3(n-3). It should be noted that the 18:2 (n-6) content was approximately 10 times greater than the 18:3(n-3) content. This high 18:2 (n-6)/18:3(n-3) ratio excludes terrestrial plant detritus, algae and sea grasses as potential sources of 18:2(n-6), but it leaves pine pollen, fungi and protozoa as possibilities. All of the animals studied had a high PUFA content, comparable to the PUFA levels in related animals living on the SJ shelf. The protobranch molluscs from the continental slope (Megayoldia sp. and R. robai) had higher PUFA concentrations than the protobranch mollusc Acila insignis, a species from the upper sublittoral zone (Kharlamenko et al., 2011). The concentration of PUFAs in the crab C. japonicus was higher than those in the red king crab Paralithodes camtschaticus, which is found on the continental shelf (Latyshev et al., 2009), and only E. biunguis contained less PUFAs than Eualus gaimardi, which was collected on the continental shelf of Greenland (Graeve et al., 1997).

No high concentrations of bacteria-typical FAs were found in the studied species, except for cis-vaccenic acid (18:1(n-7). However, high concentrations of 18:1(n-7) were not accompanied by a decrease in the PUFA levels, as has been observed in animals feeding upon bacteria, e.g., symbiotrophic bivalves (Zhukova et al., 1992). Therefore, bacteria are not a major source of food for the species studied, but they may play a significant role as a food source for other parts of the microbial food web. The concentrations of NMIDs were low, with the highest levels found in the scaphopod F. nipponica, which likely feeds on foraminifera. The starfish C. crispatus had a notable FA composition, with very high levels of AA and the MUFA 20:1(n-13). One of the probable pathways of monoenoic fatty acid synthesis is the synthesis of 20:1(n-13) from 18:1(n-13) (Mansour et al., 2005). The FA 18:1 (n-13) was found along with 18:1(n-9) in animals from the deepsea vent system community (Colac- o et al., 2007). A notable amount of 18:1(n-13) and 20:1(n-13) was also found in the bryozoan Berenicea meandrina (Demidkova, 2010). The FA 18:1(n-13) can be synthesised by some methylotrophic bacteria, but in the time required to synthesise this FA, these bacteria can synthesise much larger quantities of the FA 18:1(n-8) (Bowman et al., 1993). We did not find 18:1(n-13) or 18:1 (n-8) in detectable concentrations in any of the studied animal species from the continental slope of the SJ. Additionally, animals obtaining organic matter from methylotrophic bacteria are significantly depleted in 13C compared with the coexisting fauna (Kiyashko et al., 2004). On the continental slope of the SJ, animals containing 20:1(n-13) were more 13C-enriched than animals that did not contain this acid. High concentrations of AA have been reported in echinoderms in general (Takagi et al., 1980) and also specifically in mud-ingesting starfishes (Howell et al., 2003), including C. crispatus (Bell and Sargent, 1985). The source of the AA in these animals is unknown. Several authors have suggested that microeukaryotic organisms are the source (Bell and Sargent, 1985; Fullarton et al., 1995), but foraminifera have also been reported ¨ to contain high amounts of AA (Suhr et al., 2003; Wurzberg et al., 2011). While agglutinated foraminifera have been found among different mineral particles and diatom, gastropod and bivalve shells in the C. crispatus gut (Sokolova, 1986), we were able to use a microscope to detect diatom remains and faecal pellets in the sediment derived from the gut contents of C. crispatus (Kharlamenko, unpublished data). It may be useful to compare the FA composition of C. crispatus with that of the sedimentdwelling scaphopod F. nipponica, for which foraminifera are the main food source. However, AA levels measured in F. nipponica were significantly lower than in C. crispatus. Additionally, the foraminiferan species analysed in this study (Elphidium sp. and Nonionella sp.) contained distinctly more EPA than AA, thereby not corroborating the previous findings of high AA levels in foraminiferans. Compared with C. crispatus and F. nipponica, AA concentrations were much lower in the lipids of the other two species consuming food from the bottom sediments (Megayoldia sp. and R. robai). These bivalves contained considerable amounts of 20:1(n-13). Because protobranch bivalves belonging to the family Yoldiidae have been shown to feed on phytoplankton (Stead and Thompson, 2003), we expected to find FAs characteristic of diatoms in the Megayoldia sp. Indeed, the diatom marker FAs 16:1(n-7), 16:4(n-1) and 20:5(n-3) (Sargent et al., 1995; Dalsgaard et al., 2003) were detected in high concentrations in the lipid composition of Megayoldia sp. Elevated concentrations of these acids have also been found in other representatives of this family, e.g., in Y. hyperborea (Parrish et al., 1996). In contrast, the proportion of diatom-characteristic FAs in R. robai, which is a representative of the family Nuculanidae, was notably lower (Table 2) than that of Y. hyperborea (Parrish et al.,

V.I. Kharlamenko et al. / Deep-Sea Research II 86–87 (2013) 34–42

1996). The proportion of these FAs was higher in R. robai than in the shallow water dwelling protobranch bivalve A. insignis, which receives a significant portion of its food from the microbial food web (Kharlamenko et al., 2011). However, compared with A. insignis, R. robai contained several times less 20:1(n-13) and 20:4(n-6), which have been used as markers of the microbial food web (Kharlamenko et al., 2011). We suggest that R. robai takes an intermediate trophic position, feeding on both settled phytoplankton and the microbial food web. High amounts of the FA 20:1(n-13) were found in both the brittle star O. leptoctenia and the starfish C. crispatus. Similar values have been detected in the coastal brittle star Amphiura elandiformis, and this FA was proposed as a useful biomarker for brittle stars (Mansour et al., 2005). However, our results indicate that 20:1(n-13) can be present in high proportions not only in brittle stars but also in other echinoderms. Furthermore, this FA was found in comparable concentrations in other invertebrates and has been proposed as a marker of microbial food webs in combination with other FA markers (Kharlamenko et al., 2011). A study of coastal crinoid gut contents found phytoplankton, protozoa and crustaceans (Rutman and Fishelson, 1969), but only inorganic particles and residues of chlorophyll were found in the deep sea crinoids (Kitazawa et al., 2007). Lipids of the crinoid H. glacialis contained the diatom-typical FAs 16:4(n-1) and 20:5(n-6) as well as FAs characteristic of zooplankton, namely 20:1(n-9) and 22:1(n-11). Therefore, despite the similarity of their isotopic composition, the diets of O. leptoctenia and H. glacialis differ considerably. Both echinoderms seem to feed mainly on zooplankton grown in the euphotic zone, but O. leptoctenia additionally utilises the benthic microbial food web, whereas H. glacialis ingests more plankton. The FA composition of the crab C. japonicus revealed high amounts of PUFAs, especially EPA and DHA, together making up more than 50% of the total FAs. The shrimp E. biunguis showed a similar FA composition, and the two decapods form one group within the PCA (Fig. 3). The bivalve C. behringensis is known to feed on live small crustaceans and contained FAs characteristic of copepods in the form of MUFAs 20 and 22 (e.g., Sargent and FalkPetersen, 1988). Furthermore, this mollusc had high levels of DHA and 18:1(n-9), giving it a position near the crustaceans in both the PCA and the stable isotope biplot.

5. Conclusion The community of invertebrate megabenthos on the continental slope of the SJ is characterised by a complex trophic structure with at least three trophic levels as indicated by the consumers’ d15N values. Stable isotope and FA trophic markers give evidence of clear resource partitioning among the species studied, even among species belonging to the same trophic guilds. Organisms occupying a similar trophic position can differ significantly in their isotopic compositions depending on the utilisation of dietary items derived from different food webs (planktonic, benthic and microbial). This difference because of diet may explain the relatively high d15N values of the selective deposit feeders R. robai and C. crispatus. The FA 20:1(n-13), generally considered rare in marine ecosystems, was found in half of the studied animals. We suggest that it indicates the importance of the benthic microbial loop in the animals’ diets. Generally, the benthic invertebrates analysed in this study had fatty acid compositions similar to the compositions found in related species from the shelf areas, suggesting a considerable supply of fresh organic matter to the continental slope of the Sea of Japan during the period of investigation.

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Acknowledgments We would like to thank the officers and crew of the R/V Akademik M. A. Lavrentyev and all of the SoJaBio participants for their help and support. The foraminiferans analysed in this study were collected and identified by Franck Lejzerowicz, a PhD student at the University of Geneva. The author thanks the two anonymous reviewers for improving this paper. The SoJaBio expedition was funded by a DFG Grant (Br 1121/37-1). This work was also supported by the Russian Foundation for Basic Research (Grant 11-04-01108-a) and Presidium of Far Eastern Branch of Russian Academy of Sciences (Grant 12-I-P30-07).

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