1 INTRODUCTION

The sea cucumber Apostichopus japonicus is the most sought-after species among the edible aspidochirotid holothurians in China and is used extensively in Chinese traditional medicine (Zhang, 1958). Ineffective fishery management in the past century, however, has allowed overfishing which has led to declining stocks and the erosion of the fisheries (Purcell et al., 2013). To restore the natural resources, and meet the increasing world market demand, there has been a recent focus on sea cucumber seed production, farming, ranching, and wild-stock enhancement in China (Chen, 2004). The annual production of A. japonicus in China reached 10000 metric tons dry weight by 2010(Hu et al., 2010) with the retail price of dry sea cucumber ranging from USD 300 to 500/kg (Purcell et al., 2010). At present, sea cucumber aquaculture is one of the most profitable marine-culture industries in China. However, the traditional Chinese technique of growing A. japonicus(monoculture with prepared diets) results in low water-use efficiency (i. e. the entire water column remains unutilized). Additionally, being a relatively new and rapidlydeveloping sector, there has been a lag between commercial production and scientific study. As a result, there has been little research to address the potential ecological effects of large-scale sea cucumber aquaculture in China and elsewhere.

Apostichopus japonicus is a typical deposit-feeding sea cucumber that consumes relatively large quantities of sediment to assimilate a fraction of the organic matter present including bacteria, benthic microalgae, protozoa, and detritus from plants and animals (Zhang et al., 1995). Deposit feeders such as A. japonicus are likely candidates for polyculture or integrated multitrophic aquaculture (IMTA) as they are able to consume the feces and excess supplied feed of the cocultured species, potentially reducing the amount of organic matter accumulating in the benthos (Yingst, 1976 ; Uthicke, 1999). Indeed, inclusion of sea cucumbers in IMTA systems has recently been examined in China and other countries. For example, in China, A. japonicus was able to utilize biodeposits (i.e. feces and pseudofeces) from cultured scallops (Zhou et al., 2006) and co-culturing A. japonicus with jellyfish (Rhopilema esculenta) was shown to alleviate benthic nutrient loading and provide a secondary cash crop (Ren et al., 2014). Additionally, the co-culture of A. japonicus with juvenile charm abalone (Haliotis discus h a nnai) in an indoor seawater system was shown to decrease inorganic nitrogen levels and provide for better growth of abalone compared with monoculture (Kang et al., 2003). In New Zealand, Slater and Carton (2009) showed that grazing of the Australasian sea cucumber (Australostichopus mollis) underneath green-lipped mussel (Perna canalicul us) farms resulted in a reduction of total organic carbon, chlorophyll a, and phaeopigments associated with biodeposition from these farms. Finally, in Canada, Paltzat et al.(2008) showed that California sea cucumbers (Parastichopus californicus) grown in trays underneath Pacific oysters (Crassostrea gigas) were capable of using the biodeposits generated by the shellfish.

Shrimp culture is one of the most profitable aquaculture activities, playing an important role in the overall world marine-culture production, with the global production of farmed shrimp reaching 2.5 million metric tons in 2011(FAO, 2012). In China, mariculture of the Chinese white shrimp (Fenneropenaeus chinensis) in coastal ponds is currently occurring at a relatively small scale, due primarily to the presence of white spot syndrome virus (WSSV) and to a depressed market. Additionally, research has demonstrated that largescale shrimp culture can cause environmental degradation (particularly due to farm effluents including residual diets and fertilizers)(Funge-Smith and Briggs, 1994), and increase levels of pathogenic microorganisms (Hunter et al., 1987 ; Chen et al., 1990). A thorough understanding of the various physical, chemical, and biological processes that may affect water quality and shrimp production is required to successfully minimize negative environmental impacts and maximize aquaculture yield (Boyd, 1986). Belton and Little (2008) suggested that integrated aquaculture, such as IMTA, may be a viable option for reducing various negative environmental impacts of shrimp culture. Unfortunately, up to the present, polyculture of shrimp with other species has not been a common practice as there has been a lack of knowledge regarding appropriate methodologies (Martínez-Porchas et al., 2010). Wang et al.(1998) assessed the ecological roles and optimum stocking densities in intensive polyculture of F. chinensis and Taiwanese red tilapia hybrids (Oreochromis mossambicus× O. niloticus, random mating). Cruz-Suárez et al.(2010) suggested that, when co-cultured with juvenile whiteleg shrimp (Litopenaeus vannamei), the green seaweed Ulva clathrata had the potential to diminish the need for artificial feed as well as improve shrimp quality. To date, however, the potential ecological or biological effects of co-culturing Chinese white shrimp with sea cucumbers remain poorly understood.

In the present study, field research was undertaken to assess the feasibility of growing A. japonicus with F. chinensis in a large earthen pond. Co-culture of the two species was compared with the monoculture of each species to examine the effects of growing sea cucumbers with shrimp on both the environment and species production (growth and survival). Ecological effects were assessed by examining various water and sediment characteristics including suspended particulate matter, total organic matter, total organic carbon, total nitrogen, C/N ratio, and chlorophyll a.

2 MATERIAL AND METHOD 2.1 Study site and experimental enclosures

A 6-month trial was carried out in an earthen pond located in Jinghai Bay, Shandong Province, China (36°55′N, 122°10′E) from May to November 2008. The quadrate pond was approximately 2 ha (length×width×depth: 200 m×100 m×2 m) and had routine water exchange (40%–60% per day) because of tidal activity. The bottom of the pond was a mixture of mud and sand. Three culture treatments with A. japonicus and F. chinensis were established: monoculture of sea cucumbers (C), monoculture of shrimp (S), and co-culture of the two species (CS). Four replicate enclosures (length×width×height: 8 m×8 m×2.5 m) for each treatment were set up in the center of the pond in a 4×4-array using polyethylene netting of 0.5-cm mesh size and bamboo stakes (Fig. 1a, b). The treatment enclosures were set up in a fully randomized pattern. Each enclosure was situated 8 m apart from other enclosures to avoid potential interactions among replicates due to sedimentation and bioturbation. Water flow in the pond was from the inlet to the outlet, but only for approximately 1 h at high tide. After high tide, the gates both at the inlet and outlet were shut and the pond became essentially a closed system where the water flow was determined by the wind force and direction (there may have been little to no flow if there was no wind). Our former study on monoculture of sea cucumbers revealed that there were no significant differences in sedimentation rate throughout the experimental area of the same culture pond (Ren et al., 2010), therefore, there should be no significant differences in natural sediment load among the enclosures within the 50–60 m distance of the experimental area in the present study. Artificial reefs made of plastic tubes were placed in each C and CS enclosure to supply shelters for the sea cucumbers, four plastic tubes (length×diameter: 40 cm×15 cm) being tied together and laid uniformly on the bottom of each enclosure with one group of tubes per square meter (Fig. 1c). Once the shrimp were released (June 15, 2008), the enclosures were covered with netting to stop the shrimp from jumping out. The nets of all replicate enclosures were cleaned every 15 d to remove fouling organisms and to ensure efficient water exchange and good water quality in the enclosures. The cultured animals depended solely on natural food, with no additional feed or fertilizers being supplied during the experiment.

2.2 Experimental animals

Hatchery-reared sea cucumber juveniles (mean±SE wet weight: 5.0±0.2 g) were stocked on April 2, 2008 at a density of 10 juveniles/m² in each C and CS enclosure. Hatchery-reared shrimp juveniles (mean±SE length: 1.1±0.5 cm, mean±SE wet weight: 1.2±0.1 g) were released into the S and CS enclosures on June 15, 2008 at a density of 3 juveniles/m². Shrimp were cultured during the summer months when A. japonicus typically undergoes aestivation. The shrimp were harvested after 3 months of culture by net capture on September 20, 2008. The sea cucumbers were harvested by SCUBA diving on November 15, 2008. At the end of the experiment, the whole wet weights of 50 shrimp and 100 sea cucumbers per enclosure were measured. Before measurement, sea cucumbers were put on a cotton towel to drain excess water until a constant weight was achieved. Yields of the two species were expressed on a gram per square meter basis. Survival rates of cultured animals at the end of the experiment were calculated by counting the remaining surviving individuals and expressing this number as a percentage of the initial stocking number in each enclosure. Specific growth rates (SGR) at the end of the experiment were calculated as a percentage of the initial wet weights and expressed on a per-day basis.

2.3 Experimental sampling

Cylindrical sedimentation traps (length×diameter: 550 mm×110 mm) were used for monthly collection of settling particles in each replicate enclosure. One trap was set up in each of the four corners of each enclosure and deployed for 10 d every month. The opening of each trap was covered with netting (mesh size: 0.5 cm) to prevent large nekton and small fish entering. Once the traps were retrieved, they were left to stand for ~6 h, after which the overlying seawater in each trap was gently removed by siphoning. The sediment collected in each trap was then rinsed with distilled water and dried at 60℃ for 48 h (to constant weight) to determine the quantity of suspended particulate matter (SPM), which was expressed on a dry weight per unit area per day basis (g/(m² ·d)).

Total organic matter (TOM) content was determined from weight losses of known quantities of dry samples ashed at 500℃ for 6 h in a muffle furnace (Byers et al., 1978). Dry samples for every replicate and month were vacuum sealed for analysis of total organic carbon (TOC) and total nitrogen (TN) at the end of the experiment. After acidification with 0.1-N HCl, the samples were analyzed for TOC and TN content using a PerkinElmer CHN Analyzer (Model 2400, PerkinElmer, Waltham, Massachusetts, USA)(Holmer et al., 2007). TOM, TOC, and TN of settling particulates were expressed as percentages of the total dry weight of the sample for comparison with sediment data. C/N ratios of the settling particulates were also determined. Temperature, salinity, pH, and dissolved oxygen (DO) of the seawater in the pond were determined every 2 d, outside the enclosures. Three replicate measurements of each variable were taken at a depth of 1 m. Water temperature, salinity, pH, and DO were measured using a water quality analyzer (Multi 350i, Xiamen Lawlink Development Co., Ltd., Xiamen, China).

Sediment samples from the surface (depth: 0–1 cm) of the pond bottom close to each trap within each enclosure were collected monthly using a cylindrical metal corer (diameter: 8 cm). The sediment samples were processed within 3 h after arrival at the laboratory or stored at -20℃ for, at most, 2 d before analyzing. Methods for analyzing TOM, TOC, and TN were the same as for those for the sediment-trap particles mentioned above. Seawater was also sampled monthly at five fixed points established in the experimental pond using a 2.5-L depth-locating polymethylmethacrylate (PMMA) water sampler (Model WB-PM, Beijing Purity Instrument Co. Ltd., Beijing, China). Concentrations of chlorophyll a in monthly water and sediment samples were analyzed using the fluorescence method. The samples were processed and extracted using 90% acetone for 24 h (Orr and Grady, 1953) and then measured with a fluorometer (Trilogy ^® Laboratory Fluorometer, Turner Designs, Sunnyvale, California, USA). Water samples were preserved in Lugol's iodine solution for later identification of major phytoplankton groups. Cell counts of the dominant groups were conducted using a phytoplankton-counting chamber and a compound microscope.

2.4 Statistical analysis

The statistical software SPSS 13.0(IBM, Armonk, New York, USA) was used for data analysis. For the various sedimentation and sediment variables, a mean enclosure value was calculated for each sampling point based on the four replicate samples collected in each enclosure. Normality of the data was evaluated using the Kolmogorov-Smirnov test and homogeneity of variances with Levene's test. The data of sedimentation rates, sediment OM content, sediment TOC content, sediment TN content and chlorophyll a were log transformed to satisfy assumptions of normality. All other data were normal and homoscedastic without transformation. A series of one-way ANOVAs was conducted, followed by Student-Newman-Keuls (SNK) tests, to compare the differences between treatments within the same months. Temporal effects were not of interest in the present study and were not examined statistically. Differences were considered to be significant at P＜0.05. The comparisons of whole wet weight, growth rate, survival rate, and yield of the animals between monoculture and co-culture were analyzed using one-way ANOVAs (all data were normal and homoscedastic) with differences being considered significant at P＜0.05.

3 RESULT 3.1 Water quality

Mean monthly seawater temperature during the 6-month experimental period ranged from 10.6 in November to 28.5℃ in August with an overall average of 26.4℃(Fig. 2a). Mean monthly seawater salinity ranged from 28.0 in August to 31.6 in November with an overall average of 29.7(Fig. 2b) and the mean monthly pH ranged from 7.8 in May to 8.2 in August with an overall average of 8.1(Fig. 2c).Mean DO was always above 6.0 mg/L (Fig. 2d). There were no significant differences in any month in any of the above water quality parameters (i.e. temperature, salinity, pH, DO) among the three modes of culture. The phytoplankton in the pond was dominated by dinoflagellates in the summer and by diatoms in the spring and autumn. The mean monthly chlorophyll a concentration in seawater ranged from 0.76 μg/L in November to 5.77 μg/L in July (data not shown). Although the chlorophyll a concentrations in seawater taken from CS and S enclosures were slightly higher than that from C enclosures, there were no significant differences among them in any given month.

3.2 Animal growth and survival

Final wet weights, SGRs, survival rates, and total yields of the cultured animals in the three treatments are shown in Table 1. All four variables for sea cucumbers were significantly higher in co-culture (CS) than in monoculture (C) but, in contrast, there were no significant differences in any of these variables for shrimp cultured alone versus those with sea cucumbers (Table 1).

3.3 Sedimentation quality

The ranges of mean monthly SPM sedimentation rates in C, CS, and S enclosures were 14.5–19.5, 16.2–56.7, and 17.2–54.2 g/(m² ·d), respectively (Fig. 3). There were no significant differences in SPM sedimentation rates among different treatments in May, October, and November, whereas they were significantly higher in the CS and S treatments than in the C treatment from June to September (Fig. 3). Meanwhile, the SPM sedimentation rates in the CS and S treatments showed no significant differences in any month, both of them reaching the maximum peak in July (Fig. 3).

The ranges of mean monthly TOM content of settling particles in C, CS, and S enclosures were 13.1%–25.3%, 11.6%–33.3%, and 12.5%–34.2%, respectively (Fig. 4a). There was no difference in the TOM content of settling particles among the different treatments in any month except August, when TOM contents were significantly higher in the CS and S treatments than in the C treatment (Fig. 4a). The ranges of mean monthly TOC content in settling particles in C, CS, and S treatments were 2.6%–4.0%, 2.4%–5.7%, and 2.5%–5.4%, respectively (Fig. 4b). The TOC content in the settling particles was significantly higher in the CS and S enclosures than in C enclosures in both June and August, but this trend was reversed in July (Fig. 4b). The ranges of mean monthly TN content in settling particles in the C, CS, and S treatments were 0.26%–0.39%, 0.29%–0.64%, and 0.27%–0.56%, respectively (Fig. 4c). The TN contents in treatments CS and S were significantly higher than in treatment C in June, August, September, October, and November (Fig. 4c). The ranges of mean monthly C/N ratio in settling particles in the C, CS, and S treatments were 10.0–12.2, 8.1–11.9, and 7.9– 11.3, respectively, with C/N ratios being significantly greater in the C treatment than in the other two treatments in August, October, and November (Fig. 4d).

3.4 Sediment quality

The ranges of sediment TOM contents in the C, CS, and S treatments were 1.1%–2.8%, 1.2%–3.4%, and 1.3%–3.8%, respectively (Fig. 5a). TOM content was significantly higher in the CS and S enclosures than in the C enclosures in August and September, significantly higher in the S treatment than in the other two treatments in October and November, but not different among the three treatments from May through July (Fig. 5a). The ranges of mean monthly TOC content in sediments were 0.25%–0.60%, 0.27%–1.04%, and 0.25%–0.95% in the C, CS, and S enclosures, respectively (Fig. 5b). The ranges of mean monthly TN content in the sediments were 0.025%– 0.058%, 0.025%–0.097%, and 0.026%–0.096% in the C, CS, and S enclosures, respectively (Fig. 5c). Trends very similar to those of TOM were seen for both TOC and TN when comparing the three treatments during each month (Fig. 5b, c). The ranges of mean monthly C/N ratio of sediment samples in the C, CS, and S treatments were 9.6–12.8, 10.7–13.0, and 10.6–13.6, respectively (Fig. 5d). The values of the C/N ratios were significantly higher in sediment from the CS and S enclosures than from the C enclosures in July and August, but there were no significant differences among the three treatments in any other month (Fig. 5d). The ranges of mean chlorophyll a concentrations in the sediments of C, CS, and S enclosures were 1.5–5.7, 1.4–10.4, and 1.4–12.2 μg/g, respectively (Fig. 5e). The chlorophyll a concentration in the sediments showed no significant differences among the three treatments in the first two months (May and June). However, the sediment chlorophyll a contents in the CS and S treatments were significantly higher than in the C treatment from July to November (Fig. 5e). There were no significant differences between the CS and S treatments in July and August, whereas it was significantly higher in the S treatment than in the CS treatment in September, October, and November (Fig. 5e).

4 DISCUSSION

The extensive culture of shrimp resulted in significantly higher sedimentation rates of SPM and organic matter content than sea cucumber monoculture, especially during the shrimp culture season. Ritvo et al.(1997) observed in a laboratory experiment that shrimp were able to generate significant levels of turbidity by working through the sediment. Re-suspension can be an important physical and biological mechanism for nutrient transfer across the water-sediment interface in aquatic systems (Avnimelech et al., 1999), accelerating organic matter mineralization and nutrient regeneration and thus controlling oxygen and nutrient dynamics in shallow pond systems (Blackburn et al., 1988). In the present study, the increased sedimentation rates in the two shrimp treatments were also undoubtedly caused by re-suspension of benthic sediments by shrimp bioturbation. The settling particles in the treatments with shrimp comprised mainly feces, dead plankton, and debris.

Fenneropenaeus chinensis is an omnivorous animal with a broad dietary range, the adults typically preying on small benthic crustaceans as well as some species of polychaetes, lamellibranchs, and small brittlestars in the sediment (Zhang et al., 2006). The shrimp metabolize their prey into feces, which enrich the sediment with organic matter. This may partly explain the increased concentrations of TOM, TOC, and TN in the sediment of shrimp treatments in the summer (August to September), despite the fact that no commercial diets or fertilizers were added to the enclosures. The Intermediate Disturbance Hypothesis states that the diversity and abundance of organisms are higher when disturbance is intermediate in frequency and intensity (Grime, 1973). Intermediate disturbance allows the co-existence of more species, since competitive exclusion is impeded, and may favor the colonization of opportunistic species (Huston, 1979). Laverock et al.(2010) indicated that the bioturbation of shrimp can result in significant structural and compositional changes in sediment bacterial communities, increasing bacterial diversity in surface sediments. In the present study, the increased biomass of living microalgae in the sediment in the CS and S treatments, reflected by the increased chlorophyll a level as well as the higher sea cucumber production in the CS treatment, was likely the result of the intermediate disturbance by the shrimp.

Previous studies have demonstrated that many aspidochirote sea cucumbers can, as deposit feeders, consume large quantities of sediment (Michio et al., 2003 ; Slater and Carton, 2009) and convert sedimentary organic waste into animal tissue (Ahlgren, 1998 ; Hannah et al., 2013). In the present investigation, the enriched organic matter—including shrimp feces, settling particles, benthic microalgae, and bacteria—in the sediment of co-culture systems would likely be an important natural food source for the sea cucumbers, supporting the higher growth and survival of sea cucumbers in a co-culture versus a monoculture system. As a result, the total production of sea cucumbers increased from 61.8 g/m² in monoculture to 77.9 g/m² in co-culture, for an overall percent increase of 26.0%. Shrimp production, however, was not significantly affected by co-culture with sea cucumbers. In the pond ecosystem used in the present study, the sea cucumbers were undoubtedly an important factor in nutritional recycling between water and sediment. Other ecological factors, such as the disturbance of microorganism activities in the sediment, may play important roles. Their ecological effects on the co-culture process require further study.

Interspecies' relationships should be well understood before considering the co-culture of any organisms (Lutz, 2003), since the culture of two or more species together could create problems such as competition for food and space, water quality degradation, as well as pathogen transmission from one species to another. For example, Purcell et al.(2006) found that it was not practicable to co-culture juvenile sandfish (Holothuria scabra) with juvenile western blue shrimp (Litopenaeus stylirostris) in tanks because high concentrations of ammonia-N might significantly reduce the growth of the sea cucumbers. Also, Bell et al.(2007) suggested that the co-culture of juvenile H. scabra with L. stylirostris in earthen ponds was not viable due to the aggressive behavior of the latter. In the present study, F. chinensis was cultured at a relatively low density (3 juveniles/m²) without addition of prepared feeds or fertilizers. Furthermore, the exchange in the upper water layer was capable of eliminating the negative effect of increased ammonia-N (Liu et al., 2009 ; Yu et al., 2013). There was no evidence of aggressive behavior of F. chinensis towards A. japonicus and the artificial reefs supplied shelters for A. japonicus during shrimp culture (Ren et al., 2013).

In conclusion, it is feasible to co-culture A. japonicus with a low density of F. chinensis in an earthen pond system. Indeed, co-culturing allowed for a significantly higher yield of sea cucumbers in comparison with sea cucumber monoculture, while maintaining similar SPM, TOM, TOC, and TN levels between co-culture and shrimp monoculture.

5 ACKNOWLEDGEMENT

WANG Wenqi is thanked for reviewing the manuscript and providing helpful suggestions.