| The main immune defense cells of sea cucumber, Apostichopus japonicas, coelomocytes, were cultured in vitro, and the bacterial cell membrane component lipopolysaccharide (LPS), was used as the pathogen to stimulate the coelomocytes of sea cucumber in vitro. By detecting three immune enzymes activity, phagocytosis and superoxide anion production of coelomocytes after stimulation, the immunological evaluation method of sea cucumber was established. Chitooligosaccharide (COS) is a novel immunostimulants for human and livestock, thus its immunepotentiate effect to sea cucumber was examined in vitro and in vivo. Results showed that COS not only efficiently enhance the immunity of coelomocytes of sea cucumber in vitro, but also improved the growth performance and decreased the cumulative mortality of sea cucumbers after pathogenic challenge, by oral administration. It indicates that COS might be the potential immunostimulant yet to be exploited for the commercial culture of sea cucumber, and there are positive consistency between in vitro /in vivo experiments of sea cucumber, therefore lay the foundation of highthroughput screening of immunostimulants for sea cucumbers in vitro in future. Detail results of the present study are presented as follow:1. The coelomocytes of sea cucumber, Apostichopus japonicas was cultured in vitro using three different culture mediums, namely CCM, HSM, L-15N, with serial low to high nutritional concentration. The MTT reduction method has been employed to detect the cells viability survived in these mediums. The results proved that the cell viability has decreased significantly at day 3 and day 5 after being cultured in vitro with CCM and HSM respectively. However, L-15N medium could keep coelomocytes of sea cucumber alive stably in vitro for at least one week, without significant viability loss. It indicates that it's crucial to successfully maintain the coelomocytes of sea cucumber in vitro with medium contains sufficient nutrient elements. The morphology of two type of major immune cell within six different coelomocytes of sea cucumber was observed. That's, spherule cell and phagocyte. The osmotic pressure of coelomic fluid of sea cucumber is equal to the sea water they lived; however, the PH of coelomic fluid is slightly lower than the sea water. Furthermore, the coelomic fluid is germy rather than sterile, which is threatening to the culture of cells in vitro. The results could be as the technical guideline to culture other coelomocytes of echinoderm in vitro successfully, to summarize, the first step is to investigate the characters of coelomoic fluid and the second is meet the nutritional requirements of coelomocytes of echinoderm.2. The coelomocytes of sea cucumbers, Apostichopus japonicus, was cultured in vitro primarily and the lipopolysaccharide (LPS), the main structure of bacterial cell membrane, was used as the pathogen to immunostimulate the coelomocytes of sea cucumbers in vitro. The stimulate concentrations of LPS are 0,0.5,5,20μg/ml respectively, and the coelomocytes are sampled after stimulated at 1 h,3 h,6 h,12 h,18 h,24 h,48 h . Five potential immune markers, phagocytic index, superoxide anion production, total nitric oxide synthase (T-NOS), total superoxide dismutase (T-SOD) and acid phosphatase (ACP), of the cultured coelomocytes were determined. LPS concentration significantly influenced phagocytic index, superoxide anion production, T-NOS and SOD, but not ACP. Superoxide anion production and T-NOS of sea cucumber coelomocytes were also influenced by duration of LPS stimulation. Significant interaction between LPS dose and stimulation time was observed on both of them. Hence, the immunological parameters, including phagocytic index, superoxide anion production, the activities of T-NOS and T-SOD can be used for rapid and cost-efficient in vitro immunonutrients / immunostimulants screening as well as optimization of administration efficacy of sea cucumber by growth trials and disease challenges. To establish correlations between the responses of coelomocytes tested in the present study and resistance to various pathogenic organisms is potentially critical to development of novel immunostimulants for health management of sea cucumber aquaculture, therefore, future research is warranted.3. The coelomocytes of sea cucumbers, Apostichopus japonicus, was cultured in vitro primarily and the chitosanoligosaccharide (COS), the enzymolysis product of chitosan, was used as the potential immunostimulant to stimulate the coelomocytes of sea cucumbers in vitro. The concentrations of COS are 0, 40, 60, 80, 120μg/ml respectively, and the coelomocytes are sampled after stimulated at 1 h,3 h,6 h,12 h,24 h. Five immune markers, phagocytic index, superoxide anion production, total nitric oxide synthase (T-NOS), total superoxide dismutase (T-SOD) and acid phosphatase (ACP), of the coelomocytes cultured in vitro were determined. COS significantly enhanced all the five immune markers in vitro in varying degrees. Overall, the immunological potentiating effect of COS to the coelomcytes of sea cucumbers indicates that COS might be the novel and promising immunostimulant applied extensively into the commercial culture of sea cucumber in the near future.4. The present study was conducted to investigate the effects of dietary chitooligosaccharide (COS) on the innate immune response and growth of sea cucumber, Apostichopus japonicus. A basal diet was supplemented with 0% (control), 100 mg/kg (low), 250 mg/kg (medium) and 600 mg/kg (high) COS to formulate four experimental diets. Each diet was randomly allocated to triplicate groups of sea cucumbers in 70-L uncirculated seawater tanks, and each tank was stocked with 25 sea cucumbers (initial average weight 7.9±0.02 g). The bottom trash of each tank was remove daily before sea cucumber were fed once at 17:00 to apparent satiation. The results of 8 weeks feeding trial showed that high COS supplementation (600 mg/kg) significantly enhanced sea cucumbers growth, total coelomocytes numbers, and total NO synthase enzyme activity (P < 0.05), whereas other supplementation (100 mg/kg,250 mg/kg) and control did not. The superoxide anion production was significantly increased by the dietary COS treatment and sea cucumber fed the diet with medium and high(250 mg/kg and 600 mg/kg)COS had significantly higher phagocytic index and superoxide dismutase activity compared with low COS and control (P < 0.05). There were no significant differences in acid phosphatase activity between sea cucumbers fed diets with and without supplementation of COS (P >0.05). The challenge experiment showed that sea cucmber fed the diet with high COS (600 mg/kg) had significantly lower cumulative mortality compared with the control and other COS groups (P < 0.05), but no significant difference was observed between the control and other supplementation groups, though there is a trend that the cumulative mortality of sea cucmbers decreased along with the increase dietary supplement of COS. Chitooligosaccharide could enhance growth and innate immunity of sea cucumber with an 8-week oral administration, and protect sea cucumber from pathogen infection, thus it could be used as a promising immunity enhancer and applied extensively in the commercial aquaculture of sea cucumber. In order to attain a better immunity and growth boosting effect, 600 mg/kg COS of diet should be assured. The results also confirmed that COS could effective enhance the sea cucumber or their coelomocytes immunity under both in vitro/ in vivo experimental condition.In conclusion, this research initially confirmed that it's feasible to highthroughput and large-scale screening of immunostimulants for sea cucumbers by using the primary cell culture technique of sea cucumber in vitro. The immunological evaluation method of sea cucumber was established by the immunostimulation of lipopolysaccharide, then the immunopotentiating effect of chitooligosaccharide (COS) to coelomocytes was screened in vitro. The oral administration experiment also confirmed that COS could enhance the sea cucumber immunity in vivo, indicating the positive consistency between in vitro /in vivo experiments results of sea cucumber. Take advantage of the economical and efficient immunostimulants screening in vitro, the development and industrialization of high performance immunostimulants will be accrearate therefore benefit the health management of sea cucumber commercial culture and even benefit the sustainable development of Chinese aquculture industry as well.
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