SUN Zhenlong,GAO Qinfeng,,DONG Shuanglin,Paul K.S.Shin,and WANG Fang

1) Key Laboratory of Mariculture,Ministry of Education,Ocean University of China, Qingdao 266003, P.R.China

2) Department of Biology and Chemistry and State Key Laboratory in Marine Pollution, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P.R.China

1 Introduction

The sea cucumberApostichopus japonicus(Skelenka)is a commercially important marine species for aquaculture in China.In the last two decades,the farming ofA.japonicushas been rapidly developed (Chen,2004).The total production of this species in China has reached 93000 tons in 2008 with a 19.4% annual increase compared with that in 2007 (Ministry of Agriculture of China,2009).In order to meet the requirement for rapid extension of sea cucumber farming,extensive research has been conducted onA.japonicusregarding genetics (Liet al.,2009),energetics (Liuet al.,2009; Yuanet al.,2009),thermo-tolerance (Donget al.,2010),immunology(Guet al.,2011),and nutrology (Seo and Lee,2011).However,few studies have investigated the feeding ecology and physiology ofA.japonicusto date (Zhouet al.,2006; Renet al.,2010).Previous research has shown thatA.japonicusshows the state of dormancy,e.g.,aestivation and hibernation during winter due to the extreme temperature conditions,with the feeding and metabolic activities considerably depressed and even ceased.In contrast,A.japonicusexhibits active feeding and high growth rate in warm seasons such as spring and autumn(Liao,1980; Yanget al.,2005; Yuanet al.,2007; Baoet al.,2010).

Understanding the feeding habit of sea cucumbers is of importance to the improvement of relevant aquaculture techniques.As an obligate deposit-feeding species,A.japonicusis fed by several food sources,mainly including benthic organisms such as microalgae,nematode,and copepod autochthonously produced in sediment,as well as the allochthonous items settled from water column such as planktonic microalgae and detritus of micro- and macroalgae (Hua,1989; Zhanget al.,1995).The feeding behavior of sea cucumbers as well as their digestion and absorption of food nutrients are generally related to environmental factors of their habitat,particularly the quality and quantity of available food sources (Slater and Carton,2010; Zamora and Jeffs,2011).Paltzatet al.(2008) found thatParastichopus californicusStimpson actively selects organic material from the sediment,thus significantly decreasing the content of sedimentary organic matter in a sea-cucumber-oyster polyculture system in British Columbia,Canada.Slater and Carton (2009) have reported similar results on the feeding activity ofAustralostichopus mollisin sea-cucumber-mussel poly-culture ecosystems in temperate regions.A recent field investigation by Slater and Carton (2010) have revealed the obvious differentiation in carbon (C) and nitrogen (N) stable isotope ratios ofA.mollisin two habitats in Kenndey Bay,New Zealand.These authors indicated that different food sources were ingested and utilized by the sea cucumber in response to the changes in environmental conditions.More recently,a laboratory experiment by Gaoet al.(2011) has demonstrated that the mixture of sedimentary substance containing microalgae and the macroalgaeSargassum thunbergiiorGracilaria lemaneiformisenhanced the growth ofA.japonicuscompared with the pure food ingredient of macroalgae.

Utilization of various food sources by aquatic animals has traditionally been evaluated by direct gut content analysis,i.e.,morphological identification of the food items taken up by the consumers (Lehane and Davenport,2002; Akin and Winemiller,2006).Ruizet al.(2007),for example,investigated the feeding habit of the sea cucumberAthyonidium chilensisin a subtidal ecosystem and quantified various ingested food itemsviagut morphemetryin situ.These authors found that the macroalgae and micro-invertebrates were the main prey items ofA.chilensis.Presently,quantitative evaluation on the uptake of potential food items by sea cucumbers are lacking,mainly because of the technical difficulties in taxonomical identification of the sea cucumber food items caused by the small-size and digestion damages to gut contents (Gaoet al.,2006; Winget al.,2008).Furthermore,application of direct gut content analysis only represents an instant snapshot of the food ingested by animals.This has led to an inevitable bias in quantifying the contribution of various potential items to the consumer’s food assimilation due to different digestibility of the food items.As a result,the food items with low digestibility tend to be retained in the gut,thus can be identified as dominant food sources.The stable isotope approach offers distinct advantages over conventional dietary techniques because (1) evaluation of food sources by the stable isotope method is based on the assimilated instead of ingested constituents; and (2)assimilated matter represents the time-integrated utilization of food (Hobson and Welch,1992).Accordingly,stable isotopes have increasingly been applied as tracers to follow the flux of organic matter or pollutants along food chains or food webs in terrestrial and aquatic environments (e.g.,Fry and Sherr,1984; Riéraet al.,1996;Riéra and Hubas,2003; Shimodaet al.,2007; Kharlamenkoet al.,2008).

The objectives of the present study were (1) to quantify the seasonal changes in the relative contribution of potential food sources to the feeding requirements ofA.japonicusin a farm pond; and (2) to provide valuable data on the feeding habit ofA.japonicusfor improvement of sea cucumber farming techniques,particularly for optimization of the food environment in farm waters.

2 Materials and Methods

2.1 Study Area and Sample Treatment

This study was conducted in the Homey Group International,Rongcheng City,Shandong Province,eastern China (36?54′N,122?09′E).The rectangular farm pond ofA.japonicuswas 1.3 km in length and 1.0 km in width,with water depth ranging from 2.0 to 3.0 m.There are no riverine inputs of freshwater to the pond and at least 1/3 water is exchanged weekly through tidal flushing.The farm pond is directly exposed to the sunlight without any shadow coverage.Juvenile sea cucumbers averaging 5 g in wet weight were released from the hatchery into the farm pond in spring every year.

The seabed of the pond was flattened artificially and covered with polypropylene membranes 2-yr prior to the experiment.The seabed sediment had spatially constant muddy characteristics and was composed of silt and clay mainly settled from the water column.Three sites were selected from the central part of the pond as the sampling stations and marked with floating buoys to ensure the exact positioning during each sampling cruise.

A preliminary investigation was conducted to confirm the potential food sources ofA.japonicusat each sampling station.Sea cucumbers were collected by SCUBA diving and 10 middle-size individuals (～ 70 g in weight)were selected for further analysis.The sea cucumbers were transported to laboratory immediately after collection.To confirm the potential food items ofA.japonicus,the sea cucumbers were dissected with the content collected from the entire digestive tract for microscopic examination.Direct microscopy demonstrated that the dominant food items included various microalgae species,microbenthic animals such as nematode and copepod and fragments of macroalgae.

Samples of sea cucumbers and potential food sources confirmed by microscopic observation were collected in four seasons,i.e.,September 2008 (summer),November 2008 (autumn),February 2009 (winter) and May 2009(spring).During each sampling cruise,36 adult sea cucumbers (71.85 g ± 17.44 g) were collected from the 3 sampling stations.In addition,approximately 10 kg of sediment was collected with a sediment sampler from the top 3 cm of the seabed for extraction of benthic animals and microalgae,as well as determination of typical pigment chlorophyll (Chl)alevel.

The sea cucumbers were dissected in the laboratory,with the entire digestive tract rinsed with distilled double deionized water (DDDW).Then,the sea cucumbers were dried at 55℃ for > 72 h to constant weight and ground to fine,homogeneous powder using a sterile micro-grinder and a 0.1 mm sieve.

The potential food sources including benthic microalgae,nematodes and copepods were immediately separated from surface sediment upon collection.Benthic microalgae (n= 3) were extracted following Couch (1989)and Riéraet al.(1996),Nematodes (n= 1 – 3) extracted according to Couch (1988),and copepods (n= 1) collected following Couch (1989).Thereafter,the nematodes and copepods were starved overnight in filtered seawater.Finally,all benthic microalgae,nematodes and copepods samples were collected on pre-combusted (450℃ for 6 h),ashless,glass fiber filters (Whatman GF/F) and weighed using a gentle vacuum suction (1/3 – 1/2 atmospheric pressure).The filters retaining microalgae,nematodes and copepods were dried at 55℃ for > 72 h to constant weight.Two aliquots of the samples were prepared for C and N stable isotope analysis,respectively.For C stable isotope analysis,the samples were decarbonated using HCl (35%)fume for at least 24 h until no CO2bubbles were produced,as confirmed by a dissecting microscope.According to Lorrainet al.(2003),decarbonation via HCl-acidification removes inorganic carbonate without significantly affecting the stable isotope ratios of organic C.For N stable isotope analysis,the sample was directly measured without acid treatment.All samples were stored at ?80℃ prior to stable isotope analyses.

Triplicate samples of suspended particulate organic matter (POM) were obtained from seawater by filtration and HCl treatment.Twenty-five liters of seawater was collected about 0.5 m above the seabed at each sediment sampling site with an acid-treated,pre-rinsed plexiglass water sampler (HL-CS1000mL,Shanghai).Water samples were sieved through a 120 μm mesh to remove large-size zooplankton and fragments of macroalgal debris,and then filtered through glass fiber filters.Thereafter,the filter papers were quickly rinsed with DDDW water to remove salts adsorbed on the particle surface.Here,DDDW instead of isotonic salt solution was used to avoid potential salt contamination by seawater or the isotonic solution.No obvious cell rupture occurred under the short flushing time (< 1 min) (Currinet al.,1995; Gaoet al.,2006).Direct microscopy showed that the dominant constituents retained on the filters included various phytoplankton species and fine silty particles.Few pieces of debris retained on the filters were removed with forceps under microscope.For C stable isotope analysis,the filters containing suspended POM were decarbonated with HCl- treatment as that for benthic microalgae treatment.

To evaluate the relative contribution of macroalgae to food uptake by the sea cucumber,triplicate samples of dominant macroalgae species were collected by SCUBA diving during each sampling cruise,handpicked from the pond shore,or removed from farming facilities such as rafts and floating buoys connected with anchor.Preliminary analysis indicated that there were no obvious differences in the isotopic values among macroalgae species and their decayed detritus in the farm pond.Hence,fresh macroalgae instead of debris were sampled for stable isotope analysis.These macroalgae species commonly prosper in spring and depress in summer,with an obvious seasonal succession typical in temperate regions (Fong and Zedler,1993; Shimodaet al.,2007).The collected samples were examined under a dissecting microscope,and sediments and epibiota on the leaf surface were cleaned.The samples were rinsed with DDDW,dried at 55℃,and ground into powder (< 0.1 mm) with a sterile mortar and pestle.The dried samples were stored at?80℃ prior to further analysis.

Our major objective was to quantify the relative contributions of potential food sources separated from surface sediment to the food taken up byA.japonicususing stable isotope techniques.Therefore,the isotopic ratios of potential food items were specifically measured in replacement of the bulk stable isotope analysis for the sediment.

2.2 Measurements of Environmental Conditions

During each sampling cruise,the temperature and salinity of seawater were measuredin situ～ 0.5 m above the seabed.One liter of sea water (n= 3) was collected from the same water depth for typical pigment Chlaanalysis.The Chlawas extracted from seawater and surface sediment with 90% acetone and measured with a spectrophotometer following Strickland and Parsons (1977).

2.3 Measurement of Stable Isotope and Elemental Concentrations

The C and M isotope ratios were determined using an elemental analyzer coupled with an isotope ratio mass spectrometer (EA-IRMS,ThermoFinnigan MAT Deltaplus).Results of the isotope ratios were expressed in standard δ-unit notation as follows:

whereXis13C or15N,andRis13C :12C ratio for C or15N :14N ratio for N.The values were reported relative to the Vienna Pee Dee Belemnite standard (PDB) for C and to air N2for N.A laboratory working standard (glycine) was run for every 10 samples.Analytical precision was ±0.1‰ and ± 0.2‰ for C and N,respectively.The C and N levels were determined using a CHNS/O Analyzer(PE2400 Series Ⅱ,PerkinElmer).

2.4 Statistical Analysis and Isotope Mixing Model

One-way analysis of variance (ANOVA) with Turkey’s test for multiple comparisons was used to compare the differences of environmental parameters,i.e.,water temperature,salinity and Chlalevels of water and surface sediment among the four cruises and to examine the seasonal variability of isotopic values and potential food sources ofA.japonicus.Prior to ANOVA analysis,raw data were tested for normality of distribution and homogeneity of variance with Kolmogorov-Smirnov test and Levene’s test,respectively (Zar,2009).All statistical analyses were performed with SPSS 16.0 (SPSS Inc.,2008).

The relative contribution of each potential food source to sea cucumber assimilation was evaluated with the software ‘Isosource’ (Phillips and Gregg,2003).For C and N isotopes,average fractionation effects of 1‰ (Peterson and Fry,1987; McClelland and Valiela,1998; McCutchanet al.,2003) and 1.6‰ (Vanderklift and Ponsard,2003)were respectively used to correct stable isotope shifts for each trophic level.The N isotopic fractionation of 3‰ –4‰ for each trophic level is widely used in the trophic relationship studies using stable isotope analysis.A recent review by Vanderklift and Ponsard (2003),however,concluded that the extent of isotopic discrimination of ammonotelic invertebrates,includingA.japonicus,is lower than those of ureotelic or uricotelic organisms.On average,ammonotelic invertebrates show isotopic fractionation of 1.6‰ instead of 3‰ – 4‰ for each trophic level as used in this study.

3 Results

3.1 Environmental Conditions

The environmental conditions of the sea cucumber pond exhibited obvious seasonality (Table 1).The temperature significantly fluctuated in different seasons,with the highest value observed in summer and lowest value in winter (F3,11= 33953.00,P< 0.05).The salinity was below 30 in summer and autumn,significantly lower than that in spring and winter (F3,11= 261.33,P< 0.05).The seawater Chlalevel was significantly higher in summer and autumn compared to those in spring and winter (F3,11=52.57,P< 0.05).However,the surface sediment showed no significant differences in Chlalevel amongst four seasons,despite that the pigment level was higher in spring and autumn than in summer and winter (F3,11= 1.09,P<0.05).

3.2 Isotopic Composition and Food Contributions

The δ13C and δ15N values ofA.japonicusranged from?18.38‰ to ?16.09‰ and 12.02‰ to 12.68‰,respectively (Table 2).Significant differences were observed in the δ13C values of the sea cucumber among the four seasons (F3,11= 61.53,P< 0.01),with13C enriched in summer and depleted in autumn.As for N stable isotope,the δ15N values ofA.japonicusshowed no obvious seasonal fluctuations,except that the highest δ15N value observed in autumn was significantly different from those in spring and summer (F3,11= 12.21,P< 0.05).

Table 1 Environmental conditions in the sea cucumber farm pond of Apostichopus japonicus in four seasons during September 2008 to May 2009

The stable isotopic ratios of POM showed significant seasonal fluctuations in both δ13C and δ15N values (F3,11=587.20,P< 0.01 for δ13C; andF3,11= 47.74,P< 0.01 for δ15N).For C stable isotope of POM,the δ13C values were higher in summer and autumn than those in winter and spring.As for N stable isotope of POM,15N was significantly depleted in summer as compared to the other three seasons.Similarly,the13C values of benthic microalgae extracted from surface sediment was most enriched in summer,with no significant differences from the other three seasons.The δ15N values of benthic microalgae significantly varied in different seasons,with the lowest value observed in autumn (F3,11= 661.48,P< 0.01 for δ13C; andF3,11= 9.68,P< 0.01 for δ15N).For benthic animals extracted from surface sediment,harpacticoid copepod had higher C and N isotopic ratios than nematode.

As for the macroalgae,a total of 8 dominant species were collected during the four sampling cruises.Due to different growth seasons,the sampling of several species was not satisfactory in the depression seasons.Most macroalgae species were collected in autumn or spring only.Dominant macroalgae species was unavailable in autumn due to the degradation of most macroalgae species in summer.In winter,only the PhaeophytaEctocarpussp.was found in the pond.In general,the δ13C values stabilized between ?16‰ and ?15‰,except the highest value (?11.54‰) in the ChlorophytaUlva pertusaand lowest value (?20.06‰) in the RhodophytaGracilaria bursa-pastoriin summer.The δ15N values of most macroalgae species had no significant differences between autumn and spring,except that the15N value ofU.pertusawas markedly depleted in summer compared to winter.

The dual C and N isotope plots for 3 sampling seasons illustrated the incorporation of food sources into the tissue ofA.japonicus(Fig.1).Computation with “Isosource” (Phillips and Gregg,2003) quantified the relative contributions of various food sources to the total food assimilation ofA.japonicusin winter,spring and summer(Table 3).The calculation of food contributions in autumn was unavailable because the sea cucumbers stopped feeding in summer.In general,macroalgae was the dominant food source of sea cucumbers during the 1-yr investigation period,with the relative contribution averaging 54.4% – 63.2% in winter and spring and 28.1% in autumn.Benthic microbenthos,i.e.,copepod and nematode,were also important food sources of sea cucumber,which respectively contributed 22.6% – 39.1% and 6.3% –22.2% on average to the sea cucumber feeding.The relative contributions of other food sources,i.e.,POM and benthic microalgae were lower compared to those of macroalgae and benthic animals.The contributions of different food sources showed substantial seasonal changes,despite that macroalgae or copepod was the dominant food source ofA.japonicus.Generally,the contributions of tested food sources (except for macroalgae) peaked in winter,while that of macroalgae increased in winter and spring.

Fig.1 Dual stable isotope plots of δ13C and δ15N (mean ± SD)for the sea cucumber Apostichopus japonicus and its potential food sources in a farm pond in three sampling seasons during November 2008 to May 2009.POM,suspended particulate organic matter.The stable isotope ratios of food sources were corrected with the average fractionation effects of 1‰ and 1.6‰ for C and N,respectively.A,November 2008; B,February 2009; and C,May 2009.

Table 3 Contribution of potential food sources (%) to sea cucumber dietary consumption in a farm pond in three sampling seasons during November 2008 to May 2009

4 Discussion

Results from the present study showed that substantial seasonal shifts occurred in the C and N stable isotope ratios of the sea cucumberA.japonicusand its potential food sources,i.e.,POM,macroalgae,benthic microalgae and benthic animals such as nematode and harpacticoid copepod.Such seasonal variations were mainly due to the changes in the abiotic environmental conditions and/or the biotic physiological processes (Canuelet al.,1995;Ganneset al.,1998; Schmidtet al.,1999; Gaoet al.,2006;Kharlamenkoet al.,2008).

The δ13C values ofA.japonicusshowed significant differences among four seasons while the δ15N values exhibited no obvious seasonal fluctuations.Both δ13C and δ15N values were highest in summer compared with those in other seasons.Such seasonal pattern of C and N stable isotopes could be attributed to the excessive expenditure of nutritional reserve in summer.Previous studies have shown that adultA.japonicusexhibits the state of dormant aestivation under extremely high water temperature in summer.During the dormancy,the alimentary tract of the sea cucumber degenerates,thus considerably depressing and even ceasing the feeding.As a result,the nutritional budget ofA.japonicusshows a net consumption in the summer aestivation state,resulting in notable biomass losses (Liao,1980; Yanget al.,2005; Yuanet al.,2007; Baoet al.,2010).During dormancy,the prior consumption of isotopically light12C or14N in the body wall or gut tissues leads to the enrichment of13C and15N in summer (Peterson and Fry,1987; Peterson,1999).On the other hand,biological components,e.g.,proteins,lipids and carbohydrates in various tissues potentially result in different isotopic fractionations.For instance,the tissues containing higher proportions of lipids usually exhibit higher negative C isotopic ratios than those with lower lipid contents,owing to the discrimination against13C during lipid synthesis (Tieszenet al.1983; Sunet al.2012).During the aestivation of sea cucumber,net consumption of energy substrates including lipids and proteins substantially reduces the contents of biological components in summer than in the other seasons,particularly spring and autumn when the sea cucumber grows rapidly with substantial accumulation of biological constituents (Baoet al.,2010).Hence,seasonal fluctuations in the biological composition of sea cucumber might be another factor contributing to the isotopic differences among the four seasons.

The temporal variations in δ13C values of POM were consistent with those of POM derived from marine source reported by other studies (Peterson,1999; Shimodaet al.,2007).In the present study,the δ13C values of POM showed marked seasonal fluctuations,with the highest value observed in summer (September 2008) and the lowest in winter (February 2009).Generally,planktonic microalgae showed the highest primary productivity in summer and the discrimination of13C vs.12C during the processes of photosynthesis was reduced.As a result,δ13C of POM showed the highest value in summer season.(Fry and Wainright,1991; Canuelet al.,1995).Similarly,the δ13C values of benthic algae extracted from surface sediment peaked in summer owing to the highest productivity of benthic microalgae in the warm season.

Assimilation of various food sources by marine consumers depends on both exogenic food availability and autogenic food preference of the animal (Wong and Cheung,2001; Mangionet al.,2004; Gaoet al.,2006).As a strategy to counteract the substantial fluctuations in the quantity and quality of organic matters potentially utilizable as food sources,marine animals show substantial feeding preference to various food components to optimize the energetic and nutritional requirements,such as the deposit-feeding holothurians speciesHolothuria atra,Holothuria leucospilota(Mangionet al.,2004),andA.mollis(Zamora and Jeffs,2011).

Macroalgae are among the most productive plants in marine ecosystems.Up to 90% of the net production of macroalgae may end up as detritus ingestible by benthic deposit-feeders,including sea cucumber (Nadon and Himmelman,2006).Hence,macroalgae and its decayed debris may be an important food source of sea cucumbers,which accounts for > 50% of C utilized by benthic animals in coastal ecosystems (Nadon and Himmelman,2006).Our results indicated thatA.japonicustook up macroalgae as the principal food source throughout the investigation period.However,the relative contribution of macroalgae to the assimilation byA.japonicuswas lower in spring and autumn than in winter,despite the prosperity of macroalgae in spring and autumn in the pond.This could be attributed to the co-existence of other food items in the pond,e.g.,benthic nematode and copepod,which commonly bloom in spring and autumn.The elevated production of such food sources potentially “diluted” the ingestion and uptake of macroalgae by sea cucumber(Francoet al.,2010; Debes and Eliasen,2006; Yanget al.,2010).

Compared to macroalgae,benthic animals represented by nematode and copepod were of less importance to the food uptake byA.japonicus.However,the presence of animal-source protein including benthic animals in the pond food likely enhanced the metabolism and growth of the sea cucumber via secondary contribution.Recently,Wanget al.(2009) have found that the addition of an appropriate amount of fish meal to the food sources ofA.japonicuswas beneficial to increase its growth,indicating that the animal protein was an essential food component for sea cucumber.

In the present study,POM was the third important food source ofA.japonicuscompared to macroalgae and benthic animals.The relative contribution of POM to the food taken up byA.japonicusin autumn was higher than that in spring and winter.It was likely that the higher productivity of microalgae in autumn,as indicated by the higher Chlalevel,elevated the assimilation of POM by sea cucumber compared to that in spring and winter.The decreased settlement of POM in spring and winter was likely caused by the enhanced wave activities associated with the strong southeast and northwest monsoon,which reduced the accumulation of POM for feeding the sea cucumber in spring and summer.

Of the tested food sources,benthic microalgae were of minimum importance to the food taken up byA.japonicus,possibly owing to its low digestibility.Previous studies indicated that benthic microalgae are mainly composed of diatom species in the coastal benthic environment in Shandong Peninsula (Jianget al.,2007).Diatoms have been thought to be hardly digested by holothurians,because frustules of unicellular diatoms have evolved protection function and additional mechanical digestion is required to crush the diatom cells (Hammet al.,2003).Despite of relatively low dietary contribution,benthic microalgae including various diatoms can be an essential food component for the sea cucumber.In one of our recent laboratory studies (Gaoet al.,2011),we found that the addition of benthic microalgae to the macroalgaeGracilaria lemaneiformisandSargassum thunbergiipowder as the food sources improved the absorption of macroalgae by sea cucumber,although macroalgae was the preferential food source when the mixture of micro- and macroalgae was supplied.

In conclusion,this study suggested that despite the deposit-feeding habit,a substantial proportion of the food assimilated byA.japonicuswas from macroalgae and POM settled from the water column.Hence,to increase the frequency of water exchange between the pond and open sea may enhance the quantity and/or quality of the food supply to sea cucumbers.As this study was conducted on the sea cucumberA.japonicuscultured in one farm pond,its representativeness needs to be tested and application of the results to other systems should be cautious.

Acknowledgements

This study was funded by the National Natural Science Foundation of China (Grant Nos.31172426 and 30871931),the Ministry of Science and Technology of China (Grant Nos.2011BAD13B03 and JQ201009) and the State Oceanic Administration of China (Grant No.200905020); it was also supported by the Program for New Century Excellent Talents in University.We thank the four anonymous reviewers for their constructive comments on the manuscript.

Akin,S.,and Winemiller,K.O.,2006.Seasonal variation in food web composition and structure in a temperate tidal estuary.Estuaries and Coasts,29: 552-567.

Franco,M.D.A.,Vanaverbeke,J.,Van Oevelen,D.,Soetaert,K.,Costa,M.J.,Vincx,M.,and Moens,T.,2010.Respiration partitioning in contrasting subtidal sediments: seasonality and response to a spring phytoplankton deposition.Marine Ecology,31: 276-290.

Bao,J.,Dong,S.L.,Tian,X.L.,Wang,F.,Gao,Q.F.,and Dong,Y.W.,2010.Metabolic rates and biochemical compositions ofApostichopus japonicus(Selenka) tissue during periods of inactivity.Chinese Journal of Oceanology and Limnlology,28 (2): 218-223.

Canuel,E.A.,Cloern,J.E.,Ringelberg,D.B.,Guckert,J.B.,and Rau,G.H.,1995.Molecular and isotopic tracers used to examine source of organic matter and its incorporation into the food webs of San Francisco Bay.Limnology and Oceanography,40: 67-81.

Chen,J.X.,2004.Present status and prospects of sea cucumber industry in China.In:Advances in Sea Cucumber Aquaculture and Management (ASCAM),463.Lovatelli,A.,et al.,eds.,FAO,Rome,Italy,25-38.

Couch,C.A.,1988.A procedure for extracting large numbers of debris-free,living nematodes from muddy marine systems.Transactions of the American Microscopical Society,107: 96-100.

Couch,C.A.,1989.Carbon and nitrogen isotopes of meiobenthos and their food resources.Estuarine,Coastal and Shelf Science,28: 433-411.

Currin,C.A.,Newell,S.Y.,and Paerl,H.W.,1995.The role of standing deadSpartina alternifloraand benthic microalgae in sail marsh food webs: considerations based on multiple stable isotope analysis.Marine Ecology Progress Series,121: 99-116.

Debes,H.H.,and Eliasen,K.,2006.Seasonal abundance,reproduction and development of four key copepod species on the Faroe shelf.Marine Biology Research,2: 249-259.

Dong,Y.W.,Ji,T.T.,Meng,X.L.,Dong,S.L.,and Sun,W.M.,2010.Difference in thermo-tolerance between green and red color variants of the Japanese sea cucumber,Apostichopus japonicusSelenka: Hsp70 and heat-hardening effect.Biological Bulletin,218: 87-94.

Fong,P.,and Zedler,J.B.,1993.Temperature and light effects on the seasonal succession of algal communities in shallow coastal lagoons.Journal of Experimental Marine Biology and Ecology,171: 259-272.

Fry,B.,and Sherr,E.B.,1984.δ13C measurements as indicators of carbon flow in marine and freshwater ecosystems.Contributions in Marine Science,27: 13-47.

Fry,B.,and Wainright,S.C.,1991.Diatom sources of13C-rich carbon in marine food webs.Marine Ecology Progress Series,76: 149-157.

Gannes,L.Z.,Martínez del Rio,C.,and Koch,P.,1998.Natural abundance variations in stable isotopes and their potential uses in animal physiological ecology.Comparative Biochemistry and Physiology – Part A:Molecular and Integrative Physi- ology,119: 725-737.

Gao,Q.F.,Paul,K.S.S.,Lin,G.H.,Chen,S.P.,and Cheung,S.G.,2006.Stable isotope and fatty acid evidence for uptake of organic waster by green-lipped musselsPerna viridisin a polyculture fish farm system.Marine Ecology Progress Series,317: 273-283.

Gao,Q.F.,Wang,Y.S.,Dong,S.L.,and Sun,Z.L.,2011.Absorption of different food sources by sea cucumberApostichopus japonicus(Selenka) (Echinodermata: Holothuroidea):evidence from carbon stable isotope.Aquaculture,319: 272-276.

Gu,M.,Ma,H.M.,Mai,K.S.,Zhang,W.B.,Bai,N.,and Wang,X.J.,2011.Effects of dietary beta-glucan,mannan oligosaccharide and their combinations on growth performance,immunity and resistance againstVibrio splendidusof sea cucumber,Apostichopus japonicus.Aquaculture,31: 303-309.

Hamm,C.E.,Merkel,R.,Springer,O.,Jurkojc,P.,Maier,C.,Prechtel,K.,and Smetacek,V.,2003.Architecture and mate-rial properties of diatom shells provide effective mechanical protection.Nature,421: 841-843.

Hua,H.F.,1989.The ecological habit of the sea cucumberApostichopus japonicus.Aquaculture Overseas,2: 5-8 (in Chinese with English abstract).

Hobson,K.A.,and Welch,H.E.,1992.Determination of trophic relationships within a high Artic marine food web using δ13C and δ15N analysis.Marine Ecology Progress Series,84: 9-18.

Jiang,Z.H.,Chen,R.S.,and Wang,J.,2007.Species composition and biomass of microphytobenthos in intertidal zone of Hongdao Island,Jiaozhou Bay.Marine Fisheries Research,28: 74-81 (in Chinese with English abstract).

Kharlamenko,V.I.,Kiyashko,S.I.,Rodkina,S.A.,and Imbs,A.B.,2008.Determination of food sources of marine invertebrates from a subtidal sand community using analyses of fatty acids and stable isotopes.Russian Journal of Marine Biology,34: 101-109.

Lehane,C.,and Davenport,J.,2002.Ingestion of mesozooplankton by three species of bivalves:Mytilus edulis,Cerastoder- ma eduleandAequipecten opercularis.Journal of the Marine Biological Association of the United Kingdom,82:615-619.

Li,Q.,Chen,L.,and Kong,L.,2009.A genetic linkage map of the sea cucumber,Apostichopus japonicus(Selenka),based on AFLP and microsatellite markers.Animal Genetics,40:678-685.

Liao,Y.,1980.TheAspidochirote holothuriansof China with erection of a new genus.In:Echinoderms:Present and Past.Proceeding of European Colloquium on Echinoderms,Brussels.Jangoux,M.ed.,Balkema,A.A.,Rotterdam,Netherlands,115-120.

Liu,Y.,Dong,S.L.,Tian,X.L.,Wang,F.,and Gao,Q.F.,2009.Effects of dietary sea mud and yellow soil on growth and energy budget of the sea cucumberApostichopus japonicus(Selenka).Aquaculture,286: 266-270.

Lorrain,A.,Savoye,N.,Chauvaud,L.,Paulet,Y.M.,and Naulet,N.,2003.Decarbonation and preservation method for the analysis of organic C and N contents and stable isotope ratios of low-carbonated suspended particulate material.Analytica Chimica Acta,491: 125-133.

Mangion,P.,Taddel,D.,Frouin,P.,and Conand,C.,2004.Feeding Rate and Impact of Sediment Reworking by Two Deposit Feeders Holothuria leucospilota and Holothuria atra on a Fringing Reef (Reunion Island,Indian Ocean),Echinoderms.Nebelsick,J.H.,and Heinzeller,T.,eds,Taylor and Francis Group,London,311-317.

McClelland,J.W.,and Valiela,I.,1998.Changes in food web structure under the influence of increased anthropogenic nitrogen inputs to estuaries.Marine Ecology Progress Series,186: 259-271.

McCutchan,J.H.,Lewis,W.M.,Kendall,C.,and McGrath,C.C.,2003.Variation in trophic shift for stable isotope ratios of carbon,nitrogen,and sulfur.Oikis,102: 378-390.

Ministry of Agriculture of China,2009.China Fisheries Yearbook,2008.China Agriculture Publisher,Beijing,China,24-28 (in Chinese).

Nadon,M.O.,and Himmelman,J.H.,2006.Stable isotopes in subtidal food webs: have enriched carbon ratios in benthic consumers been misinterpreted?Limnology and Oceanography,51: 2828-2836.

Paltzat,D.L.,Pearce,C.M.,Barnes,P.A.,and McKinley,R.S.,2008.Growth and production of California sea cucumbers(Parastichopus californicusStimpson) co-cultured with suspended Pacific oysters (Crassostrea gigasThunberg).Aqua-culture,275: 124-137.

Peterson,B.J.,1999.Stable isotopes as tracers of organic matter input and transfer in benthic food webs: a review.Acta Oecologica,20: 479-487.

Peterson,B.J.,and Fry,B.,1987.Stable isotopes in ecosystem studies.Annual Review of Ecology and Systematics,18: 293-320.

Phillips,D.L.,and Gregg,J.W.,2003.Source partitioning using stable isotopes: coping with too many sources.Oecologia,136:261-267.

Ren,Y.C.,Dong,S.L.,Wang,F.,Gao,Q.F.,Tian,X.L.,and Liu,F.,2010.Sedimentation and sediment characteristics in sea cucumberApostichopus japonicus(Selenka) culture ponds.Aquaculture Research,42: 14-21.

Riéra,P.,Richard,P.,Grémare,A.,and Blanchard,G.,1996.Food source of intertidal nematodes in the Bay of Marennes-Oléron (France),as determined by dual stable isotope analysis.Marine Ecology Progress Series,142: 303-309.

Riéra,P.,and Hubas,C.,2003.Trophic ecology of nematodes from various microhabitats of the Roscoff Aber Bay (France):Importance of stranded macroalgae evidenced through δ13C and δ15N.Marine Ecology Progress Series,260: 151-159.

Ruiz,J.F.,Ibá?ez,C.M.,and Cáceres,C.W.,2007.Gut morphometry and feeding of the sea cucumberAthyonidium chilensis(Semper,1868) (Echinodermata: Holothuroidea).Journal of Marine Biology and Oceanography,42: 269-274(in Spanish with English abstract).

Seo,J.Y.,and Lee,S.M.,2011.Optimum dietary protein and lipid levels for growth of juvenile sea cucumberApostichopus japonicus.Aquaculture Nutrition,17: e56-e61.

Schmidt,O.,Scrimgeour,C.M.,and Curry,J.P.,1999.Carbon and nitrogen stable isotope ratios in body tissue and mucus of feeding and fasting earthworms (Limbricus festivus).Oecologia,118: 9-15.

Shimoda,K.,Aramaki,Y.,Nasuda,J.,Yokoyama,H.,Ishihi,Y.,and Tamaki,A.,2007.Food sources for three species ofNihonotrypaea(Decapoda: Thalassinidea: Callianassidae) from western Kyushu,Japan,as determined by carbon and nitrogen stable isotope analysis.Journal of Experimental Marine Biology and Ecology,342: 292-312.

Slater,M.J.,and Carton,A.G.,2009.Effect of sea cucumber(Australostichopus mollis) grazing on coastal sediments impacted by mussel farm deposition.Marine Pollution Bulletin,58: 1123-1129.

Slater,M.J.,and Carton,A.G.,2010.Sea cucumber habitat differentiation and site retention as determined by intraspecific stable isotope variation.Aquaculture Research,41: e695-e702.

SPSS Inc.,2008.SPSS16.0Student Version for Windows.SPSS,Prentice Hall,Upper Saddle River,New Jersey.

Strickland,J.D.H.,and Parsons,T.R.,1977.A Practical Handbook of Seawater Analysis.Canadian Government Publishing Center,Ottawa,Canada,330pp.

Sun,Z.L.,Gao,Q.F.,Dong,S.L.,Shin,P.K.S.,and Wang,F.,2012.Estimates of carbon turnover rates in the sea cucumberApostichopus japonicus(Selenka) using stable isotope analysis: the role of metabolism and growth.Marine Ecology Progress Series,457: 101-112.

Tieszen,L.L.,Boutton,T.W.,Tesdahl,K.G.,and Slade,N.A.,1983.Fractionation and turnover of stable carbon isotopes in animal tissues: implications for δ13C analysis of diet.Oecologia,57: 32-37.

Vanderklift,M.A.,and Ponsard,S.,2003.Sources of variation in consumer-diet δ15N enrichment: a meta-analysis.Oecolo-gia,136: 169-182.

Wang,J.Y.,Song,Z.D.,Wang,S.X.,Huang.B.S.,Li.P.Y.,and Zhang,L.M.,2009.The study on the protein requirement of the sea cucumberApostichopus japonicus(Selenka) under different growth stages.Aquaculture Science and Technology Information,36: 229-233 (in Chinese with English abstract).

Wing,S.R.,McLeod,R.J.,Clark,K.L.,and Frew,R.,D.,2008.Plasticity in the diet of two echinoderm species across an ecotone: microbial recycling of forest litter and bottom-up forcing of population structure.Inter-ResearchMarine Ecology Progress Series,360: 115-123.

Wong,W.H.,and Cheung,S.G.,2001.Feeding rates and scope for growth of green mussels,Perna viridis(L.) and their relationship with food availability in Kat O,Hong Kong.Aquaculture,193: 123-137.

Yang,E.J.,Ju,S.J.,and Choi,J.K.,2010.Feeding activity of the copepodAcartia hongion phytoplankton and micro-zooplankton in Gyeonggi Bay,Yellow Sea.Estuarine,Coastal and Shelf Science,88: 292-301.

Yang,H.S.,Yuan,X.T.,and Zhou,Y.,2005.Effects of body weight and water temperature on food consumption and growth in the sea cucumberApostichopus japonicus(Selenka)with special reference to aestivation.Aquaculture Research,36: 1085-1092.

Yuan,X.T.,Yang,H.S.,Wang,L.L.,Zhou,Y.,Zhang,T.,and Liu,Y.,2007.Effects of aestivation on the energy budget of sea cucumberApostichopus japonicus(Selenka) (Echinodermata: Holothuroidea).Acta Ecologica Sinica,27: 3155-3161.

Yuan,X.T.,Yang,H.S.,Wang,L.L.,Zhou,Y.,and Gabr,H.R.,2009.Bioenergetic responses of sub-adult sea cucumberApostichopus japonicus(Selenka) (Echinodermata: Holothuroidea) to temperature with special discussion regarding its southern most distribution limit in China.Journal of Thermal Biology,34: 315-319.

Zamora,L.N.,and Jeffs,A.G.,2011.Feeding,selection,digestion and absorption of the organic matter from mussel waste by juveniles of the deposit-feeding sea cucumber,Australostichopus mollis.Aquaculture,317: 223-228.

Zar,J.H.,2009.Biostatistical analysis.5th edition,Prentice Hall,Upper Saddle River,New Jersey,USA,66-284.

Zhang,B.L.,Sun,D.Y.,and Wu,Y.Q.,1995.Preliminary analysis on the feeding habit ofApostichopus janonicusin the rocky coast waters of Lingshan Island.Marine Science,3: 11-13 (in Chinese with English abstract).

Zhou,Y.,Yang,H.S.,Liu,S.L.,Yuan,X.T.,Mao,Y.Z.,Liu,Y.,Xu,X.L.,and Zhang,F.S.,2006.Feeding and growth on bivalve biodeposits by the deposit feederStichopus japonicusSelenka (Echinodennata: Holothuroudea) co-cultured in lantern nets.Aquaculture,256: 510-520.