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Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): Evidence from carbon stable isotope
Aquaculture 319 (2011) 272–276
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): Evidence from carbon stable isotope Qin-Feng Gao ⁎, Yansu Wang, Shuanglin Dong, Zhenlong Sun, Fang Wang Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, China
a r t i c l e
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Article history: Received 28 December 2010 Received in revised form 26 June 2011 Accepted 30 June 2011 Available online 8 July 2011 Keywords: Sea cucumber Apostichopus japonicus (Selenka) Feeding Growth Carbon stable isotope
a b s t r a c t The present experiment compared the effects of 6 different diet types, including benthic matter collected from natural sea water (diet A), brown algae Sargassum thunbergii (diet B), red algae Gracilaria lemaneiformis (diet C), mixture of benthic matter and G. lemaneiformis (diet D), mixture of benthic matter and S. thunbergii (diet E) and mixture of G. lemaneiformis and S. thunbergii (diet F) on the growth of sea cucumber Apostichopus japonicus. The sea cucumber fed mixture diet containing benthic matter showed the higher specific growth rate (SGR) relative to the single feed ingredient and sea cucumbers fed diet D showed the maximum SGR. Carbon stable isotope evidence revealed the different feeding preference of sea cucumbers among the various feed ingredients. The results of the present study suggested that G. lemaneiformis might be more preferable to the nutritional requirements of sea cucumber relative to the traditional sea cucumber feed S. thunbergii. G. lemaneiformis which has been widely cultured with higher availability and lower feed cost is suitable for the manufacture of sea cucumber feed in aquaculture practice. Addition of benthic matter collected from the natural sea waters containing microalgae, bacterial and muddy materials is helpful to improve the feeding and absorption of macroalgae by sea cucumber and subsequently to enhance the growth and production of sea cucumber. © 2011 Elsevier B.V. All rights reserved.
1. Introduction As a commercially important marine species for aquaculture in China, the farming of sea cucumber Apostichopus japonicus (Selenka) has been developing rapidly in the last decades (Liao, 1980). The total production of sea cucumber in China has reached 93,000 tons in 2008 with19.4% annual increase compared to that in 2007 (MOAC, 2009). However, studies on the biological and ecological characteristics of this species, including its feeding habit, are scarce so far relative to its booming extension of farming scale. The natural distribution of A. japonicus in Asia covers the subtidal zone from 35°N to 44°N along the coast of Russia, China, Japan and Korea (Sloan, 1984; Yuan et al., 2009). As feeding habit is one of the most important foundations for advancing aquaculture techniques, of the previous studies on the biology and ecology of sea cucumber, considerable research efforts have been made to the feeding ecology and physiology of various sea cucumber species worldwide. Asha and Muthiah (2006) compared the effects of 5 single microalgae species and their mixture on the larval growth, development and survival of the Indian commercial sea cucumber Holothuria spinifera at laboratory conditions so as to explore the feeding preference of this species. Singh et al. (1998) studied the feeding response of the dendrochirote
⁎ Corresponding author. Tel./fax: + 86 532 82032435. E-mail address: [email protected] (Q.-F. Gao). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.06.051
sea cucumber Cucumaria frondosa to changing food concentrations and found the significant positive relationship between the stomach content of the experimental animals and the seston chloropigment concentration. Another 3-yr in situ field observation by Singh et al. (1999) revealed that day length and the seston quality were the principal factors influencing the feeding seasonality of C. frondosa. More recently, Slater et al. (2009) and Zhou et al. (2006) examined the food availability of farming wastes from bivalve cultivation by their local sea cucumber species, Australostichopus mollis and A. japonicus, respectively. Slater and Jeffs (2010) explored the limitation of benthic sediment characteristics, including the food conditions, on the localized distribution of A. mollis. Other reports on the studies regarding various aspects of sea cucumbers, especially for the local species A. japonicus in China, include its genetics (Li et al., 2009), energetics (Yuan et al., 2009), thermology (Dong et al., 2010), larval development (Li et al., 2010) etc. Previous studies showed that sea cucumber, as typical depositfeeding species, might take up organic matter in sediment in the forms of bacteria, prozotoa, benthic microalgae and detritus of macroalgae such as S. thunbergii and sea grass such as Zosterophyllum spp. as food sources. Previous studies on the utilizations of food sources by A. japonicus were conducted with traditional means of direct gut content observations (Gao, 2008). However, trophic models based on dietary observations can only represent an instant snapshot of food ingested by the animals. The application of stable isotopes may overcome such limitations. Stable isotopes change in a predictable
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way when they are transferred along trophic levels (Fry, 2006; Peterson and Fry, 1987). Moreover, stable isotope approach offers distinct advantages over conventional dietary techniques since (1) evaluation of food sources is based on assimilated instead of ingested food, and (2) assimilated matter represents time-integrated utilization of food (Hobson and Welch, 1992). This method may be particularly useful in revealing the food sources of sea cucumber because the recognition of gut content is difficult, even impossible sometimes, due to the small size and the digestive damage of the food particles. There has been increasing application of stable isotopes as tracers to follow the flux of organic matter or pollutants along food chains or food webs in both terrestrial and aquatic environments (e.g., Bearhop et al., 2000; Collier et al., 2002; Gao et al., 2006; Vizzini et al., 2002). Benthic matter is one of the most important food sources of sea cucumber under natural condition (Slater and Jeffs, 2010). In aquaculture practice, to meet the food requirement of cultured sea cucumber with higher stocking density relative to that under natural conditions, artificial feed is generally supplemented to the sea cucumber and powder of macroalgae such as Thallus laminariae, S. thunbergii and S. fusiforme is the main ingredient of the formulated diet used in the intensive sea cucumber cultivation (Slater et al., 2009; Yuan et al., 2006). Brown algae are the most favorite macroalgae for the manufacture of sea cucumber feed (Asha and Muthiah, 2007). However, large-scale culture of Sargassum spp. has not been developed so far and the source of Sargassum spp. for sea cucumber feed predominantly relies upon natural collection. As a result, the cost of Sargassum spp. is high and the inadequate supply of Sargassum spp. limits the development of sea cucumber cultivation. Accordingly, it is the urgent need to find substitute algae materials for sea cucumber feed. The red algae (Rhodophyta) G. lemaneiformis is a species for cultivation in northern China newly introduced from southern China in recent years and has been successfully grown in large scale (Mao et al., 2006). The high production and easy procurability of G. lemaneiformis as well as its subsequent low price make it potentially feasible to use this seaweed species as new material of sea cucumber feed. The availability of G. lemaneiformis by sea cucumber, however, has not been evaluated to the present. In the present study, the effects of benthic matter collected from natural waters, brown algae S. thunbergii and red algae G. lemaneiformis on the growth of sea cucumber A. japonicus were compared. Especially, we quantified the absorption efficiency of sea cucumber among above three food ingredients using carbon stable isotope. The objectives of the present study are to compare the suitability of different food sources for feeding cucumber, including natural benthic matter, traditional sea cucumber feed S. thunbergii and the newly developed red algae G. lemaneiformis so as to provide scientific evidence for optimizing ingredients of sea cucumber feed. 2. Materials and methods 2.1. Diet preparation Six diet formula, either single form or mixture of benthic matter, brown algae S. thunbergii and red algae G. lemaneiformis were used in the experiment. Diets A, B and C are the single form of benthic matter, S. thunbergii and G. lemaneiformis, respectively, and diets D, E and F were the mixture (1:1) of benthic matter–G. lemaneiformis, benthic matter–S. thunbergii and S. thunbergii–G. lemaneiformis, respectively. To reduce the disturbance of substantial muddy substance in the sediment directly collected from the seabed on the experiment, benthic matter was collected by means of deposition. The wave-shape (S-shape) plastic plates (40 cm × 40 cm) which were used in sea cucumber breeding were installed 15 cm below the water surface in an aquaculture pond. One week after installation, the plastic plates were retrieved for experimental use. Microscopic observation found
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that the surface of plastic plates were basically covered by the substances settled from surrounding water body, including detritus, fine clay particles, and benthic algae, especially diatom species such as Navicula spp. Skeletonema spp. and Nitzschia spp. Dried macroalgae S. thunbergii and G.lemaneiformis were ground and sieved with 0.08 mm mesh size. The algae powder was pelletized with a feed processing machine for experimental use. 2.2. Experiment The experiment was carried out at the laboratory of Qingdao National Ocean Scientific Research Center, Ocean University of China and lasted for 3 months. Sea cucumbers were collected from an adjacent commercial farm where the sea cucumbers were cultured without supplement of artificial feed. The sea cucumbers were acclimated for 10 days at the experimental temperature of 17 °C, which is same as the temperature of the farm where the sea cucumbers were collected. One of our previous experiments has shown that 10 days acclimation was long enough for sea cucumbers to adapt the laboratory conditions (Liu et al., 2009). After acclimation, the sea cucumbers were allocated into 6 triplicate groups contained in glass aquaria fed with diet A, B, C, D, E and F as described above, respectively. The difference in the body weight of the sea cucumbers was not significant between the 6 diet groups (ANOVA, p N 0.05, Table 1). The pelletized algae powder was directly fed to the sea cucumbers once every day. The amount of food supplied to each aquarium is equal (5% of the sea cucumber weight, the ration requirement which was determined in our previous study [Liu et al., 2009]) and the ratios of 2 food ingredients in diets D, E and F were 1:1. For diets A, D and E containing the benthic matter, the plastic plates which were used to collect the benthic matter were retrieved from the pond every day just before feeding the sea cucumbers and placed on the bottom of the aquaria immediately. The food density on unit area of the plastic plates was estimated and the plate area supplied to the sea cucumbers was adjusted so as to keep the food amount supplied to sea cucumbers consistent throughout the experiment. To estimate the food quantity of the benthic matter on the plastic plates, samples of benthic matter were removed from unit area of the plates with filtered seawater and a brush. The dry weight of the benthic matter was measured after drying in an oven at 80 °C for 24 h and organic content was determined after combustion in a Muffle furnace at 450 °C for 6 h (Gao et al., 2008). The area of plates supplied to the experimental aquaria was adjusted according to the food density on unit area of the plate surface so as to keep the consistency of the food amount supplied to the sea cucumbers throughout the study. Prior to the start of the experiment, the initial wet weights of the sea cucumbers were measured individually as described in Dong et al. (2006). Initial individual wet weights of the sea cucumbers were not statistically different among the 6 diet groups (ANOVA, p N 0.05). Wet and dry weights of other 10 sea cucumber individuals were measured to calculate the water content of the experimental sea cucumbers. Table 1 The Initial and final wet weight (WW: g) and dry weight (DW: g) of the experimental sea cucumbers for the six diet groups (mean ± SD, n = 3). Values with different superscript marks in the same row represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description. Diet A Initial WW 9.68 ± 0.07 DW 0.53 ± 0.00 Final WW 12.36 ± 2.33ab DW 0.70 ± 0.06ab
Diet B
Diet C
Diet D
Diet E
Diet F
9.49 ± 0.07 0.52 ± 0.01 15.50 ± 0.56b 0.75 ± 0.06b
9.49 ± 0.03 0.52 ± 0.01 12.90 ± 1.80ab 0.71 ± 0.03ab
9.50 ± 0.08 0.52 ± 0.01 18.17 ± 1.03c 1.00 ± 0.11c
9.61 ± 0.14 0.53 ± 0.00 12.06 ± 2.41b 0.78 ± 0.03b
9.56 ± 0.15 0.53 ± 0.01 11.50 ± 1.47a 0.64 ± 0.05a
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Accordingly, the initial dry weights of the experimental sea cucumbers were estimated based on their wet weights and the water content for calculating their specific growth rate (SGR) after the experiment. During the experiment, aeration was provided continuously and one-third volume of the water of each aquarium was changed every day with sand-filtered seawater. The experimental temperature was 17 ± 0.5 °C, salinity 30–32 psu, DO N 6.5 mg l − 1 and photoperiod 13:11 (L/D). 2.3. Sample determination Samples of benthic matter on the plastic plates were rinsed with filter seawater and a brush as described above. To determine the carbon stable isotope ratio of the benthic matter, the samples were treated following Gao et al. (2006). Benthic matter removed from the plates was filtered with Whatman GF/F glass fiber filters under vacuum suction of less than one-third atmospheric pressure. The residue on the filters was treated with 1.2 N isotonic HCl to remove carbonates prior to stable isotope measurements. After the experiment, all the experimental sea cucumbers were collected, dried and dry weights were measured. The specific growth rate (SGR: % d − 1) of each individual was calculated as: SGR = ðlnDWt −lnDW0 Þ = t × 100 where DWt and DW0 are the final and initial dry weights of the sea cucumber individuals, respectively, and t the culture duration in days. The experimental sea cucumbers, together with those collected before experiment, were dissected and the body tissue was dried at 80 °C for 24 h and ground and sieved for stable isotope measurements. After pretreatment, carbon isotope ratios of macroalgae powder, benthic matter and sea cucumbers were determined using an elemental analyzer coupled with an isotope ratio mass spectrometer (EA-IRMS, ThermoFinnigan MAT Delta-plus). Results of isotope ratios were expressed in standard δ-unit notation, which is defined as follows: 13
δ C=
h i Rsample = Rstandard –1 × 1000
where R is the 13 C: 12 C ratio. The values were reported relative to the Vienna Pee Dee Belemnite standard (PDB). A laboratory working standard (glycine) was run for every 10 samples. Analytical precision was ±0.1‰. Carbon concentrations of all samples were determined using a CHNS/O Analyzer (PE2400 Series II, PerkinElmer).
The difference in SGRs of sea cucumbers and carbon stable ratios between the 6 feed types were compared with one way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons with significance level (p value) of 0.05 (Zar, 2009). For diet D, E and F in which 2 feed ingredients were used, the contribution of each ingredient to the total food absorption of sea cucumbers were compared with Student's t test to determine the food preference of sea cucumbers between the 2 food ingredients. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of variance with Kolmogorov–Smirnov test and Levene's test, respectively. The statistical analyses were performed using the software SPSS for Windows, Release 16.0 (Norušis, 2008; SPSS Inc., 2008). 3. Results 3.1. Growth Wet and dry weights of the sea cucumber individuals before (initial) and after (final) the experiment was listed in Table 1. At the beginning of the experiment, there was no significant difference in the initial body weights between the sea cucumbers allocated to the 6 feed groups (ANOVA, p N 0.05). Final body weights, both wet and dry weights, of the sea cucumbers showed significant differences after experiment (ANOVA, p b 0.05). Minimum and maximum body weights of the sea cucumbers occurred in diet F (mixture of S. thunbergii and G. lemaneiformis) and diet D (mixture of G. lemaneiformis and benthic matter), respectively. The body weights of the sea cucumbers in other 4 diet groups showed the intermediary values. The specific growth rates (SGR) showed the same trend as the final body weights for the 6 diet groups (Fig. 1). 3.2. Carbon stable isotope and food contribution Carbon stable isotope ratios (δ 13C) and carbon content of the sea cucumbers and feeds used in the experiment were listed in Table 2. The δ 13C values of the 3 food sources used in the experiment, i.e., benthic matter, S. thunbergii and G. lemaneiformis, were significantly different (ANOVA, p b 0.05). Except for diet A, sea cucumbers fed diets B, C, D, E and F showed obvious 13 C depletion due to the uptake of isotopically light S. thunbergii or G. lemaneiformis. The δ 13C values (−14.9‰) of the sea cucumbers fed benthic matter (diet A) showed most 13C-enriched compared to those from other 5 diet groups and did not change remarkably relative to the initial δ 13C value (−14.7‰) of the sea cucumbers before the start of the experiment. Moreover, the average δ 13C value of −14.9‰ for the sea cucumbers from diet A was very near to that of the benthic matter (−16.0) corrected with 1‰ of
2.4. Isotope mixing model and statistical analysis
13 0 13 13 0 13 δ CX −δ CM ½CX fX;B + δ CY –δ CM ½CY fY;B = 0 fX;B + fY;B = 1 where fX,B and fY,B represent the fractions of assimilated biomass (B) of sources X, Y, respectively, in the mixture M. [C]X and [C]Y represent the C concentrations in food sources X, Y. Isotopic signatures for the sources were corrected for trophic fractionation as designated by the prime (') symbol. Average fractionation effects of 1‰ for carbon isotope were used to correct stable isotope shifts for each trophic level (McClelland and Valiela, 1998; Peterson & Fry, 1987).
0.9
c
0.8 0.7
SGR (% d-1)
For diets D, E and F where the mixture of 2 feed materials was involved, 2-source concentration-weighted isotope mixing model was used to evaluate the respective contributions of the feed ingredients to the food of the sea cucumbers as follows (Phillips, 2001; Phillips & Koch, 2002):
0.6 0.5
b b ab
ab
0.4
a
0.3 0.2 0.1 0
Diet A
Diet B
Diet C
Diet D
Diet E
Diet F
Diet treatments Fig. 1. Specific growth rates (SGR: % d− 1) of the experimental sea cucumbers for the 6 diet groups. Values with different marks represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description.
Q.-F. Gao et al. / Aquaculture 319 (2011) 272–276 Table 2 Carbon stable isotope ratios (δ13C: ‰) and carbon content (%) of the experimental sea cucumbers and feeds (mean ± SD, n = 3). Values with different superscript marks in the same column, within the table block of feed or sea cucumber, represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description.
Feed
Sea cucumber
Sample
δ13C
Carbon content
Benthic matter Sargassum thunbergii Gracilaria lemaneiformis Initial Diet A Diet B Diet C Diet D Diet E Diet F
− 16.0 ± 0.2a − 18.4 ± 0.0b − 20.2 ± 0.3c − 14.7 ± 1.1a − 14.9 ± 0.3a − 16.0 ± 0.1b − 18.0 ± 0.2d − 16.7 ± 0.5c − 16.0 ± 0.2b − 18.0 ± 0.4d
23.70 ± 1.07 29.67 ± 0.84 17.42 ± 1.48 26.14 ± 1.93 28.88 ± 0.39 30.45 ± 1.71 29.36 ± 0.85 30.55 ± 0.71 31.24 ± 0.7– 29.78 ± 1.04
the carbon stable isotope fractionation for each trophic level. In contrast to diet A, the δ13C values (−18.0‰) of the sea cucumbers from diet C showed most 13C-depleted among the 6 diet group, which was consistent to the lowest δ13C value of G. lemaneiformis used in diet C. As for diets D, E and F in which the mixtures of 2 food sources were involved, δ 13C values of the sea cucumbers were obviously shifted within the δ 13C values of their respective food sources after isotope fractionation correction indicating the incorporation and subsequent isotopic effects of the food sources on the experimental sea cucumbers. Results calculated with the 2-end member concentration-weighted dual isotope mixing model revealed that the contribution of benthic matter and G. lemaneiformis in diet D to the sea cucumber dietary consumption was 51.4 ± 0.67 and 48.6 ± 0.67%, respectively. For diet E, the contribution of benthic matter and S. thunbergii were 62.6 ± 0.83 and 37.4 ± 0.83%, respectively; for diet F, the contribution of G. lemaneiformis and S. thunbergii was 43.0 ± 0.45 and 57.0 ± 0.45%, respectively. Student's t test showed that the contributions of the 2 feed ingredients within each of diet D, E and F were significantly different (for diet D, t4 = 5.12, p b 0.05; for diet E, t4 = 37.19, p b 0.05 and for diet F, t4 = − 38.10, p b 0.05). 4. Discussion Coastal waters are characterized by large fluctuations in the quantity and quality of the organic matter which might be utilized as food sources by marine organisms as a result of the tidal and wave movements, seasonality of micro- and macroalgae growth, intermittent storm events and terrestrial input activities, etc. (Navarro and Widdows, 1997; Wong and Cheung, 2001). In face of the variations in the quantity and quality of food environment, marine animals showed active feeding preference from various food components to optimize the energetic and nutritional requirements, such as bivalve (Gao et al., 2008; Wong and Cheung, 1999), gastropod (Cheung et al., 2006). In the present study, the δ 13C values of the 3 food sources, i.e., benthic matter, S. thunbergii and G. lemaneiformis, for sea cucumbers differ significantly. Such distinct isotopic signatures implied the feasibility and possibility to trace the respective contribution of each food source taken up by sea cucumbers (Peterson & Fry, 1987) and accordingly to evaluate the food preference of sea cucumber between various food sources. Results of the present experiment revealed the feeding preference of sea cucumber A. japonicus among the 3 typical food ingredients, i.e., benthic matter, phaeophyte S. thunbergii and rhodophyte G. lemaneiformis. It appeared that A. japonicus accumulated more benthic matter against macroalgae in the diets containing the mixture of benthic matter and macroalgae (diets D and E). The respective food contribution percentage of G. lemaneiformis vs S. thunbergii in diet F showed that sea cucumber absorbed more S. thunbergii relative to G.
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lemaneiformis. As G. lemaneiformis contains lower water content (~7.5%) and higher protein content (~ 26.1%) than other macroalgae species, for example, ~12.4% water content and 10.2% protein content of another brown algae species Laminari japonica (Qi et al., 2010), G. lemaneiformis showed lower digestibility than brown algae, including S. thunbergii used in the present experiment. The lower digestibility of G. lemaneiformis might be verified by the harder texture from direct observation compared with S. thunbergii. As a result, sea cucumbers took up less G. lemaneiformis compared to S. thunbergiiin in diet F (43.0% vs 57.0%). Such result is consistent with the findings of Qi et al. (2010) that the abalone Haliotis discus showed lower feeding rate for G. lemaneiformis against that for other brown algae species S. pallidum or L. japonica. The higher digestibility of unicellular microalgae and bacteria as well as the more diverse nutritional components in the benthic matter resulted in the preference of sea cucumber to benthic matter, not G. lemaneiformis or S. thunbergiiin in diet D and E, respectively. Previous studies have shown that supplement of sea mud with suitable proportion (20%) in the feed of sea cucumber enhanced the digestion and growth of sea cucumber relative to the pure algae powder (Liu et al., 2009). In the present study, the benthic matter was collected by means of installing plastic plates in the field pond. During the collection of suspended particulate matter and the growth of microalgae on the plates, considerable muddy particles from water column settled on the plate surface. As a result, sea cucumber fed mixture of macroalgae G. lemaneiformis and benthic matter (diet D) showed higher SGR than the pure G. lemaneiformis (diet B). On the other hand, more diverse nutritional components in the mixture diets optimized the growth of sea cucumbers. Another possible reason for the higher growth rate of sea cucumbers fed mixture of G. lemaneiformis and benthic matter (diet D) is that the abundant bacteria associated with the benthic matter enhanced the digestion of G. lemaneiformis (Fenchel and Blackburn, 1979; Moore et al., 1995). A field investigation by Slater and Jeffs (2010) also suggested that higher microphytobenthic activities resulted in the higher growth rate of the Australian sea cucumber A. mollis. Since the benthic matter promoted the digestion of G. lemaneiformis and G. lemaneiformis contains higher nutrition value, sea cucumbers fed the mixture of benthic matter and G. lemaneiformis (diet D) showed better growth performance than those fed with the mixture of benthic matter and S. thunbergii (diet E). Because S. thunbergii could not improve the digestibility of G. lemaneiformis and the addition of G. lemaneiformis in diet F diluted the nutrition of, S. thunbergii, the SGR of sea cucumbers fed diet F showed lower SGR relative to those fed diet B containing pure powder of S. thunbergii. In conclusion, the results of the present study suggested that Rhodophyta G. lemaneiformis might be more preferable to the nutritional requirements of sea cucumber relative to the traditional sea cucumber feed S. thunbergii. G. lemaneiformis which has been widely cultured with higher availability and lower cost is suitable for the manufacture of sea cucumber feed in aquaculture practice. Addition of benthic matter containing microalgae, bacterial and muddy materials collected from the natural sea waters is helpful to improve the feeding and absorption of sea cucumber on maroalgae and subsequently to enhance the growth and production of sea cucumber. In the practice of large-scale sea cucumber farming, benthic matter might be obtained by means of collecting sea mud from surface sea bed or collecting the muddy substance from the farming facilities such as rafts and cages. Acknowledgements The work described in this paper was funded by the grants from the National Natural Science Foundation of China (Project No. 30871931), the Ministry of Science and Technology of China (Project No. 2011BAD13B03, JQ201009) and the State Oceanic Administration of
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Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e
Absorption of different food sources by sea cucumber Apostichopus japonicus (Selenka) (Echinodermata: Holothuroidea): Evidence from carbon stable isotope Qin-Feng Gao ⁎, Yansu Wang, Shuanglin Dong, Zhenlong Sun, Fang Wang Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, Qingdao, 266003, China
a r t i c l e
i n f o
Article history: Received 28 December 2010 Received in revised form 26 June 2011 Accepted 30 June 2011 Available online 8 July 2011 Keywords: Sea cucumber Apostichopus japonicus (Selenka) Feeding Growth Carbon stable isotope
a b s t r a c t The present experiment compared the effects of 6 different diet types, including benthic matter collected from natural sea water (diet A), brown algae Sargassum thunbergii (diet B), red algae Gracilaria lemaneiformis (diet C), mixture of benthic matter and G. lemaneiformis (diet D), mixture of benthic matter and S. thunbergii (diet E) and mixture of G. lemaneiformis and S. thunbergii (diet F) on the growth of sea cucumber Apostichopus japonicus. The sea cucumber fed mixture diet containing benthic matter showed the higher specific growth rate (SGR) relative to the single feed ingredient and sea cucumbers fed diet D showed the maximum SGR. Carbon stable isotope evidence revealed the different feeding preference of sea cucumbers among the various feed ingredients. The results of the present study suggested that G. lemaneiformis might be more preferable to the nutritional requirements of sea cucumber relative to the traditional sea cucumber feed S. thunbergii. G. lemaneiformis which has been widely cultured with higher availability and lower feed cost is suitable for the manufacture of sea cucumber feed in aquaculture practice. Addition of benthic matter collected from the natural sea waters containing microalgae, bacterial and muddy materials is helpful to improve the feeding and absorption of macroalgae by sea cucumber and subsequently to enhance the growth and production of sea cucumber. © 2011 Elsevier B.V. All rights reserved.
1. Introduction As a commercially important marine species for aquaculture in China, the farming of sea cucumber Apostichopus japonicus (Selenka) has been developing rapidly in the last decades (Liao, 1980). The total production of sea cucumber in China has reached 93,000 tons in 2008 with19.4% annual increase compared to that in 2007 (MOAC, 2009). However, studies on the biological and ecological characteristics of this species, including its feeding habit, are scarce so far relative to its booming extension of farming scale. The natural distribution of A. japonicus in Asia covers the subtidal zone from 35°N to 44°N along the coast of Russia, China, Japan and Korea (Sloan, 1984; Yuan et al., 2009). As feeding habit is one of the most important foundations for advancing aquaculture techniques, of the previous studies on the biology and ecology of sea cucumber, considerable research efforts have been made to the feeding ecology and physiology of various sea cucumber species worldwide. Asha and Muthiah (2006) compared the effects of 5 single microalgae species and their mixture on the larval growth, development and survival of the Indian commercial sea cucumber Holothuria spinifera at laboratory conditions so as to explore the feeding preference of this species. Singh et al. (1998) studied the feeding response of the dendrochirote
⁎ Corresponding author. Tel./fax: + 86 532 82032435. E-mail address: [email protected] (Q.-F. Gao). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2011.06.051
sea cucumber Cucumaria frondosa to changing food concentrations and found the significant positive relationship between the stomach content of the experimental animals and the seston chloropigment concentration. Another 3-yr in situ field observation by Singh et al. (1999) revealed that day length and the seston quality were the principal factors influencing the feeding seasonality of C. frondosa. More recently, Slater et al. (2009) and Zhou et al. (2006) examined the food availability of farming wastes from bivalve cultivation by their local sea cucumber species, Australostichopus mollis and A. japonicus, respectively. Slater and Jeffs (2010) explored the limitation of benthic sediment characteristics, including the food conditions, on the localized distribution of A. mollis. Other reports on the studies regarding various aspects of sea cucumbers, especially for the local species A. japonicus in China, include its genetics (Li et al., 2009), energetics (Yuan et al., 2009), thermology (Dong et al., 2010), larval development (Li et al., 2010) etc. Previous studies showed that sea cucumber, as typical depositfeeding species, might take up organic matter in sediment in the forms of bacteria, prozotoa, benthic microalgae and detritus of macroalgae such as S. thunbergii and sea grass such as Zosterophyllum spp. as food sources. Previous studies on the utilizations of food sources by A. japonicus were conducted with traditional means of direct gut content observations (Gao, 2008). However, trophic models based on dietary observations can only represent an instant snapshot of food ingested by the animals. The application of stable isotopes may overcome such limitations. Stable isotopes change in a predictable
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way when they are transferred along trophic levels (Fry, 2006; Peterson and Fry, 1987). Moreover, stable isotope approach offers distinct advantages over conventional dietary techniques since (1) evaluation of food sources is based on assimilated instead of ingested food, and (2) assimilated matter represents time-integrated utilization of food (Hobson and Welch, 1992). This method may be particularly useful in revealing the food sources of sea cucumber because the recognition of gut content is difficult, even impossible sometimes, due to the small size and the digestive damage of the food particles. There has been increasing application of stable isotopes as tracers to follow the flux of organic matter or pollutants along food chains or food webs in both terrestrial and aquatic environments (e.g., Bearhop et al., 2000; Collier et al., 2002; Gao et al., 2006; Vizzini et al., 2002). Benthic matter is one of the most important food sources of sea cucumber under natural condition (Slater and Jeffs, 2010). In aquaculture practice, to meet the food requirement of cultured sea cucumber with higher stocking density relative to that under natural conditions, artificial feed is generally supplemented to the sea cucumber and powder of macroalgae such as Thallus laminariae, S. thunbergii and S. fusiforme is the main ingredient of the formulated diet used in the intensive sea cucumber cultivation (Slater et al., 2009; Yuan et al., 2006). Brown algae are the most favorite macroalgae for the manufacture of sea cucumber feed (Asha and Muthiah, 2007). However, large-scale culture of Sargassum spp. has not been developed so far and the source of Sargassum spp. for sea cucumber feed predominantly relies upon natural collection. As a result, the cost of Sargassum spp. is high and the inadequate supply of Sargassum spp. limits the development of sea cucumber cultivation. Accordingly, it is the urgent need to find substitute algae materials for sea cucumber feed. The red algae (Rhodophyta) G. lemaneiformis is a species for cultivation in northern China newly introduced from southern China in recent years and has been successfully grown in large scale (Mao et al., 2006). The high production and easy procurability of G. lemaneiformis as well as its subsequent low price make it potentially feasible to use this seaweed species as new material of sea cucumber feed. The availability of G. lemaneiformis by sea cucumber, however, has not been evaluated to the present. In the present study, the effects of benthic matter collected from natural waters, brown algae S. thunbergii and red algae G. lemaneiformis on the growth of sea cucumber A. japonicus were compared. Especially, we quantified the absorption efficiency of sea cucumber among above three food ingredients using carbon stable isotope. The objectives of the present study are to compare the suitability of different food sources for feeding cucumber, including natural benthic matter, traditional sea cucumber feed S. thunbergii and the newly developed red algae G. lemaneiformis so as to provide scientific evidence for optimizing ingredients of sea cucumber feed. 2. Materials and methods 2.1. Diet preparation Six diet formula, either single form or mixture of benthic matter, brown algae S. thunbergii and red algae G. lemaneiformis were used in the experiment. Diets A, B and C are the single form of benthic matter, S. thunbergii and G. lemaneiformis, respectively, and diets D, E and F were the mixture (1:1) of benthic matter–G. lemaneiformis, benthic matter–S. thunbergii and S. thunbergii–G. lemaneiformis, respectively. To reduce the disturbance of substantial muddy substance in the sediment directly collected from the seabed on the experiment, benthic matter was collected by means of deposition. The wave-shape (S-shape) plastic plates (40 cm × 40 cm) which were used in sea cucumber breeding were installed 15 cm below the water surface in an aquaculture pond. One week after installation, the plastic plates were retrieved for experimental use. Microscopic observation found
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that the surface of plastic plates were basically covered by the substances settled from surrounding water body, including detritus, fine clay particles, and benthic algae, especially diatom species such as Navicula spp. Skeletonema spp. and Nitzschia spp. Dried macroalgae S. thunbergii and G.lemaneiformis were ground and sieved with 0.08 mm mesh size. The algae powder was pelletized with a feed processing machine for experimental use. 2.2. Experiment The experiment was carried out at the laboratory of Qingdao National Ocean Scientific Research Center, Ocean University of China and lasted for 3 months. Sea cucumbers were collected from an adjacent commercial farm where the sea cucumbers were cultured without supplement of artificial feed. The sea cucumbers were acclimated for 10 days at the experimental temperature of 17 °C, which is same as the temperature of the farm where the sea cucumbers were collected. One of our previous experiments has shown that 10 days acclimation was long enough for sea cucumbers to adapt the laboratory conditions (Liu et al., 2009). After acclimation, the sea cucumbers were allocated into 6 triplicate groups contained in glass aquaria fed with diet A, B, C, D, E and F as described above, respectively. The difference in the body weight of the sea cucumbers was not significant between the 6 diet groups (ANOVA, p N 0.05, Table 1). The pelletized algae powder was directly fed to the sea cucumbers once every day. The amount of food supplied to each aquarium is equal (5% of the sea cucumber weight, the ration requirement which was determined in our previous study [Liu et al., 2009]) and the ratios of 2 food ingredients in diets D, E and F were 1:1. For diets A, D and E containing the benthic matter, the plastic plates which were used to collect the benthic matter were retrieved from the pond every day just before feeding the sea cucumbers and placed on the bottom of the aquaria immediately. The food density on unit area of the plastic plates was estimated and the plate area supplied to the sea cucumbers was adjusted so as to keep the food amount supplied to sea cucumbers consistent throughout the experiment. To estimate the food quantity of the benthic matter on the plastic plates, samples of benthic matter were removed from unit area of the plates with filtered seawater and a brush. The dry weight of the benthic matter was measured after drying in an oven at 80 °C for 24 h and organic content was determined after combustion in a Muffle furnace at 450 °C for 6 h (Gao et al., 2008). The area of plates supplied to the experimental aquaria was adjusted according to the food density on unit area of the plate surface so as to keep the consistency of the food amount supplied to the sea cucumbers throughout the study. Prior to the start of the experiment, the initial wet weights of the sea cucumbers were measured individually as described in Dong et al. (2006). Initial individual wet weights of the sea cucumbers were not statistically different among the 6 diet groups (ANOVA, p N 0.05). Wet and dry weights of other 10 sea cucumber individuals were measured to calculate the water content of the experimental sea cucumbers. Table 1 The Initial and final wet weight (WW: g) and dry weight (DW: g) of the experimental sea cucumbers for the six diet groups (mean ± SD, n = 3). Values with different superscript marks in the same row represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description. Diet A Initial WW 9.68 ± 0.07 DW 0.53 ± 0.00 Final WW 12.36 ± 2.33ab DW 0.70 ± 0.06ab
Diet B
Diet C
Diet D
Diet E
Diet F
9.49 ± 0.07 0.52 ± 0.01 15.50 ± 0.56b 0.75 ± 0.06b
9.49 ± 0.03 0.52 ± 0.01 12.90 ± 1.80ab 0.71 ± 0.03ab
9.50 ± 0.08 0.52 ± 0.01 18.17 ± 1.03c 1.00 ± 0.11c
9.61 ± 0.14 0.53 ± 0.00 12.06 ± 2.41b 0.78 ± 0.03b
9.56 ± 0.15 0.53 ± 0.01 11.50 ± 1.47a 0.64 ± 0.05a
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Accordingly, the initial dry weights of the experimental sea cucumbers were estimated based on their wet weights and the water content for calculating their specific growth rate (SGR) after the experiment. During the experiment, aeration was provided continuously and one-third volume of the water of each aquarium was changed every day with sand-filtered seawater. The experimental temperature was 17 ± 0.5 °C, salinity 30–32 psu, DO N 6.5 mg l − 1 and photoperiod 13:11 (L/D). 2.3. Sample determination Samples of benthic matter on the plastic plates were rinsed with filter seawater and a brush as described above. To determine the carbon stable isotope ratio of the benthic matter, the samples were treated following Gao et al. (2006). Benthic matter removed from the plates was filtered with Whatman GF/F glass fiber filters under vacuum suction of less than one-third atmospheric pressure. The residue on the filters was treated with 1.2 N isotonic HCl to remove carbonates prior to stable isotope measurements. After the experiment, all the experimental sea cucumbers were collected, dried and dry weights were measured. The specific growth rate (SGR: % d − 1) of each individual was calculated as: SGR = ðlnDWt −lnDW0 Þ = t × 100 where DWt and DW0 are the final and initial dry weights of the sea cucumber individuals, respectively, and t the culture duration in days. The experimental sea cucumbers, together with those collected before experiment, were dissected and the body tissue was dried at 80 °C for 24 h and ground and sieved for stable isotope measurements. After pretreatment, carbon isotope ratios of macroalgae powder, benthic matter and sea cucumbers were determined using an elemental analyzer coupled with an isotope ratio mass spectrometer (EA-IRMS, ThermoFinnigan MAT Delta-plus). Results of isotope ratios were expressed in standard δ-unit notation, which is defined as follows: 13
δ C=
h i Rsample = Rstandard –1 × 1000
where R is the 13 C: 12 C ratio. The values were reported relative to the Vienna Pee Dee Belemnite standard (PDB). A laboratory working standard (glycine) was run for every 10 samples. Analytical precision was ±0.1‰. Carbon concentrations of all samples were determined using a CHNS/O Analyzer (PE2400 Series II, PerkinElmer).
The difference in SGRs of sea cucumbers and carbon stable ratios between the 6 feed types were compared with one way analysis of variance (ANOVA) followed by Tukey's test for multiple comparisons with significance level (p value) of 0.05 (Zar, 2009). For diet D, E and F in which 2 feed ingredients were used, the contribution of each ingredient to the total food absorption of sea cucumbers were compared with Student's t test to determine the food preference of sea cucumbers between the 2 food ingredients. Prior to analysis, raw data were diagnosed for normality of distribution and homogeneity of variance with Kolmogorov–Smirnov test and Levene's test, respectively. The statistical analyses were performed using the software SPSS for Windows, Release 16.0 (Norušis, 2008; SPSS Inc., 2008). 3. Results 3.1. Growth Wet and dry weights of the sea cucumber individuals before (initial) and after (final) the experiment was listed in Table 1. At the beginning of the experiment, there was no significant difference in the initial body weights between the sea cucumbers allocated to the 6 feed groups (ANOVA, p N 0.05). Final body weights, both wet and dry weights, of the sea cucumbers showed significant differences after experiment (ANOVA, p b 0.05). Minimum and maximum body weights of the sea cucumbers occurred in diet F (mixture of S. thunbergii and G. lemaneiformis) and diet D (mixture of G. lemaneiformis and benthic matter), respectively. The body weights of the sea cucumbers in other 4 diet groups showed the intermediary values. The specific growth rates (SGR) showed the same trend as the final body weights for the 6 diet groups (Fig. 1). 3.2. Carbon stable isotope and food contribution Carbon stable isotope ratios (δ 13C) and carbon content of the sea cucumbers and feeds used in the experiment were listed in Table 2. The δ 13C values of the 3 food sources used in the experiment, i.e., benthic matter, S. thunbergii and G. lemaneiformis, were significantly different (ANOVA, p b 0.05). Except for diet A, sea cucumbers fed diets B, C, D, E and F showed obvious 13 C depletion due to the uptake of isotopically light S. thunbergii or G. lemaneiformis. The δ 13C values (−14.9‰) of the sea cucumbers fed benthic matter (diet A) showed most 13C-enriched compared to those from other 5 diet groups and did not change remarkably relative to the initial δ 13C value (−14.7‰) of the sea cucumbers before the start of the experiment. Moreover, the average δ 13C value of −14.9‰ for the sea cucumbers from diet A was very near to that of the benthic matter (−16.0) corrected with 1‰ of
2.4. Isotope mixing model and statistical analysis
13 0 13 13 0 13 δ CX −δ CM ½CX fX;B + δ CY –δ CM ½CY fY;B = 0 fX;B + fY;B = 1 where fX,B and fY,B represent the fractions of assimilated biomass (B) of sources X, Y, respectively, in the mixture M. [C]X and [C]Y represent the C concentrations in food sources X, Y. Isotopic signatures for the sources were corrected for trophic fractionation as designated by the prime (') symbol. Average fractionation effects of 1‰ for carbon isotope were used to correct stable isotope shifts for each trophic level (McClelland and Valiela, 1998; Peterson & Fry, 1987).
0.9
c
0.8 0.7
SGR (% d-1)
For diets D, E and F where the mixture of 2 feed materials was involved, 2-source concentration-weighted isotope mixing model was used to evaluate the respective contributions of the feed ingredients to the food of the sea cucumbers as follows (Phillips, 2001; Phillips & Koch, 2002):
0.6 0.5
b b ab
ab
0.4
a
0.3 0.2 0.1 0
Diet A
Diet B
Diet C
Diet D
Diet E
Diet F
Diet treatments Fig. 1. Specific growth rates (SGR: % d− 1) of the experimental sea cucumbers for the 6 diet groups. Values with different marks represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description.
Q.-F. Gao et al. / Aquaculture 319 (2011) 272–276 Table 2 Carbon stable isotope ratios (δ13C: ‰) and carbon content (%) of the experimental sea cucumbers and feeds (mean ± SD, n = 3). Values with different superscript marks in the same column, within the table block of feed or sea cucumber, represent significant difference at the significance level of 0.05. For diets A, B, C, D, E and F, see the text for detailed description.
Feed
Sea cucumber
Sample
δ13C
Carbon content
Benthic matter Sargassum thunbergii Gracilaria lemaneiformis Initial Diet A Diet B Diet C Diet D Diet E Diet F
− 16.0 ± 0.2a − 18.4 ± 0.0b − 20.2 ± 0.3c − 14.7 ± 1.1a − 14.9 ± 0.3a − 16.0 ± 0.1b − 18.0 ± 0.2d − 16.7 ± 0.5c − 16.0 ± 0.2b − 18.0 ± 0.4d
23.70 ± 1.07 29.67 ± 0.84 17.42 ± 1.48 26.14 ± 1.93 28.88 ± 0.39 30.45 ± 1.71 29.36 ± 0.85 30.55 ± 0.71 31.24 ± 0.7– 29.78 ± 1.04
the carbon stable isotope fractionation for each trophic level. In contrast to diet A, the δ13C values (−18.0‰) of the sea cucumbers from diet C showed most 13C-depleted among the 6 diet group, which was consistent to the lowest δ13C value of G. lemaneiformis used in diet C. As for diets D, E and F in which the mixtures of 2 food sources were involved, δ 13C values of the sea cucumbers were obviously shifted within the δ 13C values of their respective food sources after isotope fractionation correction indicating the incorporation and subsequent isotopic effects of the food sources on the experimental sea cucumbers. Results calculated with the 2-end member concentration-weighted dual isotope mixing model revealed that the contribution of benthic matter and G. lemaneiformis in diet D to the sea cucumber dietary consumption was 51.4 ± 0.67 and 48.6 ± 0.67%, respectively. For diet E, the contribution of benthic matter and S. thunbergii were 62.6 ± 0.83 and 37.4 ± 0.83%, respectively; for diet F, the contribution of G. lemaneiformis and S. thunbergii was 43.0 ± 0.45 and 57.0 ± 0.45%, respectively. Student's t test showed that the contributions of the 2 feed ingredients within each of diet D, E and F were significantly different (for diet D, t4 = 5.12, p b 0.05; for diet E, t4 = 37.19, p b 0.05 and for diet F, t4 = − 38.10, p b 0.05). 4. Discussion Coastal waters are characterized by large fluctuations in the quantity and quality of the organic matter which might be utilized as food sources by marine organisms as a result of the tidal and wave movements, seasonality of micro- and macroalgae growth, intermittent storm events and terrestrial input activities, etc. (Navarro and Widdows, 1997; Wong and Cheung, 2001). In face of the variations in the quantity and quality of food environment, marine animals showed active feeding preference from various food components to optimize the energetic and nutritional requirements, such as bivalve (Gao et al., 2008; Wong and Cheung, 1999), gastropod (Cheung et al., 2006). In the present study, the δ 13C values of the 3 food sources, i.e., benthic matter, S. thunbergii and G. lemaneiformis, for sea cucumbers differ significantly. Such distinct isotopic signatures implied the feasibility and possibility to trace the respective contribution of each food source taken up by sea cucumbers (Peterson & Fry, 1987) and accordingly to evaluate the food preference of sea cucumber between various food sources. Results of the present experiment revealed the feeding preference of sea cucumber A. japonicus among the 3 typical food ingredients, i.e., benthic matter, phaeophyte S. thunbergii and rhodophyte G. lemaneiformis. It appeared that A. japonicus accumulated more benthic matter against macroalgae in the diets containing the mixture of benthic matter and macroalgae (diets D and E). The respective food contribution percentage of G. lemaneiformis vs S. thunbergii in diet F showed that sea cucumber absorbed more S. thunbergii relative to G.
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lemaneiformis. As G. lemaneiformis contains lower water content (~7.5%) and higher protein content (~ 26.1%) than other macroalgae species, for example, ~12.4% water content and 10.2% protein content of another brown algae species Laminari japonica (Qi et al., 2010), G. lemaneiformis showed lower digestibility than brown algae, including S. thunbergii used in the present experiment. The lower digestibility of G. lemaneiformis might be verified by the harder texture from direct observation compared with S. thunbergii. As a result, sea cucumbers took up less G. lemaneiformis compared to S. thunbergiiin in diet F (43.0% vs 57.0%). Such result is consistent with the findings of Qi et al. (2010) that the abalone Haliotis discus showed lower feeding rate for G. lemaneiformis against that for other brown algae species S. pallidum or L. japonica. The higher digestibility of unicellular microalgae and bacteria as well as the more diverse nutritional components in the benthic matter resulted in the preference of sea cucumber to benthic matter, not G. lemaneiformis or S. thunbergiiin in diet D and E, respectively. Previous studies have shown that supplement of sea mud with suitable proportion (20%) in the feed of sea cucumber enhanced the digestion and growth of sea cucumber relative to the pure algae powder (Liu et al., 2009). In the present study, the benthic matter was collected by means of installing plastic plates in the field pond. During the collection of suspended particulate matter and the growth of microalgae on the plates, considerable muddy particles from water column settled on the plate surface. As a result, sea cucumber fed mixture of macroalgae G. lemaneiformis and benthic matter (diet D) showed higher SGR than the pure G. lemaneiformis (diet B). On the other hand, more diverse nutritional components in the mixture diets optimized the growth of sea cucumbers. Another possible reason for the higher growth rate of sea cucumbers fed mixture of G. lemaneiformis and benthic matter (diet D) is that the abundant bacteria associated with the benthic matter enhanced the digestion of G. lemaneiformis (Fenchel and Blackburn, 1979; Moore et al., 1995). A field investigation by Slater and Jeffs (2010) also suggested that higher microphytobenthic activities resulted in the higher growth rate of the Australian sea cucumber A. mollis. Since the benthic matter promoted the digestion of G. lemaneiformis and G. lemaneiformis contains higher nutrition value, sea cucumbers fed the mixture of benthic matter and G. lemaneiformis (diet D) showed better growth performance than those fed with the mixture of benthic matter and S. thunbergii (diet E). Because S. thunbergii could not improve the digestibility of G. lemaneiformis and the addition of G. lemaneiformis in diet F diluted the nutrition of, S. thunbergii, the SGR of sea cucumbers fed diet F showed lower SGR relative to those fed diet B containing pure powder of S. thunbergii. In conclusion, the results of the present study suggested that Rhodophyta G. lemaneiformis might be more preferable to the nutritional requirements of sea cucumber relative to the traditional sea cucumber feed S. thunbergii. G. lemaneiformis which has been widely cultured with higher availability and lower cost is suitable for the manufacture of sea cucumber feed in aquaculture practice. Addition of benthic matter containing microalgae, bacterial and muddy materials collected from the natural sea waters is helpful to improve the feeding and absorption of sea cucumber on maroalgae and subsequently to enhance the growth and production of sea cucumber. In the practice of large-scale sea cucumber farming, benthic matter might be obtained by means of collecting sea mud from surface sea bed or collecting the muddy substance from the farming facilities such as rafts and cages. Acknowledgements The work described in this paper was funded by the grants from the National Natural Science Foundation of China (Project No. 30871931), the Ministry of Science and Technology of China (Project No. 2011BAD13B03, JQ201009) and the State Oceanic Administration of
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