Histochemical localization and characterization of AKP,ACP, NSE,and POD from cultured <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

We investigated the distribution of four enzymes involved in the immune response of Apostichopus japonicus. We collected samples of the tentacles, papillate podium, integument, respiratory tree, and digestive tract and stained them for acid phosphatase (ACP), alkaline phosphatase (AKP), non-specific esterase (NSE) and peroxidase (POD) activity. The distribution and content of ACP, AKP, NSE, and POD differed among the tissues. The coelomic epithelium of the tentacle, papillate podium, and integument and the mucous layer of respiratory tree were positive for ACP activity. The coelomic epithelium and cuticular layer of the tentacle, papillate podium, and integument and the mucous layer and tunica externa of the respiratory tree and digestive tract stained positive or weakly positive for AKP activity. Almost all the epithelial tissues stained positive, strongly positive, or very strongly positive for NSE activity. The cuticular layer of the tentacle and integument and the mucous layer, tunica submucosa, and tunica externa of the respiratory tree and digestive tract stained positive for POD activity. We hypothesize that these enzymes play a role in the immune response in A. japonicus.     

Screen and effect analysis of immunostimulants for sea cucumber,Apostichopus japonicus

Immunostimulants may improve disease resistance of aquaculture animals by promoting the nonspecific immunity response of the organisms. Five types of saccharides, including chitosan, yeast polysaccharide, burdock oligosaccharide, seaweed polysaccharide and lentinus edodes polysaccharide, were screened for potential use as immunostimulants by using spectrophotometry. The saccharides were injected into Apostichopus japonicus, a sea cucumber, and the lysozyme and superoxide dismutase (SOD) activities of the coelomic fluid and epidermal slime were monitored in six consecutive days. The results show that the lysozyme activity of the animal's coelomic fluid was significantly stimulated on day 2, day 4 and day 6 after the injection of the saccharides (P<0.05). The effects of chitosan and yeast polysaccharide were the most notable. The lysozyme activity of the epidermal slime was significantly increased by chitosana, yeast polysaccharide, seaweed polysaccharide, and burdock oligosaccharide on day 1 and day 2 after the injection (P<0.05). The SOD activity of the coelomic fluid was significantly promoted by the saccharides on day 2 and day 4 post-injection (P<0.05), while the SOD activity of the epidermal slime increased on day 2. These findings indicate that chitosan and yeast polysaccharide are the most effective immunostimulants and potential healthy anti-disease feedstuff for A. japonicus.     

Seasonal biochemical changes in composition of body wall tissues of sea cucumber Apostichopus japonicus

Seasonal variation in proximate,amino acid and fatty acid composition of the body wall of sea cucumber Apostichopus japonicus was evaluated.The proximate composition,except for ash content,changed significantly among seasons(P,0.05).Alanine,glycine,glutamic acid and asparagic acid were the most abundant amino acids.Total amino acid and essential amino acid contents both varied clearly with seasons(P,0.05).16:0 and 16:1n7 were the primary saturated fatty acid(SFA) and monounsaturated fatty acid(MUFA) respect...     

Identification and characterization of 43 microsatellite markers derived from expressed sequence tags of the sea cucumber (Apostichopus japonicus)

The sea cucumber Apostichopus japonicus is a commercially and ecologically important species in China. A total of 3056 potential unigenes were generated after assembling 7597 A. japonicus expressed sequence tags (ESTs) downloaded from Gen-Bank. Two hundred and fifty microsatellite-containing ESTs (8.18%) and 299 simple sequence repeats (SSRs) were detected. The average density of SSRs was 1 per 7.403 kb of EST after redundancy elimination. Di-nucleotide repeat motifs appeared to be the most abundant type with a percentage of 69.90%. Of the 126 primer pairs designed, 90 amplified the expected products and 43 showed polymorphism in 30 individuals tested. The number of alleles per locus ranged from 2 to 26 with an average of 7.0 alleles, and the observed and expected heterozygosities varied from 0.067 to 1.000 and from 0.066 to 0.959, respectively. These new EST-derived microsatellite markers would provide sufficient polymorphism for population genetic studies and genome mapping of this sea cucumber species.     

Analysis of fatty acid composition of sea cucumber Apostichopus japonicus using multivariate statistics

Fatty acids （FAs） provide energy and also can be used to trace trophic relationships among organisms. Sea cucumber Apostichopus japonicus goes into a state of aestivation during warm summer months. We examined fatty acid profiles in aestivated and non-aestivated A. japonicus using multivariate analyses （PERMANOVA, MDS, ANOSIM, and SIMPER）. The results indicate that the fatty acid profiles of aestivated and non-aestivated sea cucumbers differed significantly. The FAs that were produced by bacteria and brown kelp contributed the most to the differences in the fatty acid composition of aestivated and non- aestivated sea cucumbers. Aestivated sea cucumbers may synthesize FAs from heterotrophic bacteria during early aestivation, and long chain FAs such as eicosapentaenoic （EPA） and docosahexaenoic acid （DHA） that produced from intestinal degradation, are digested during deep aestivation. Specific changes in the fatty acid composition of A. japonicus during aestivation needs more detailed study in the future.     

Isolation,Identification, and Quantitative Determination of Saponin in Apostichopus japonicus by HPLC-DAD

Apostichopus japonicus is an important invertebrate that is widely used as a tonic food in Asian countries.The purpose of this study is to purify and identify a class of compound,the saponins,from the body wall of A.japonicus,and to establish a new me-thod to determine the quantity of saponins in the sea cucumber.In this study,the saponins of A.japonicus,cladoloside A(CA),were ob-tained from 80%ethanol extract by column chromatography for the first time and were characterized using the spectral method.The resulting purified saponins were then profiled using ¹HNMR,¹³CNMR,and ESI-MS,which revealed the CA molecular formula of C₅₃H₈₂O₂ and contained a triterpenoid backbone,a methylglucopyranosyl moiety,a quinovopyranosyl,and two xylopyranosyls.A me-thod for the quantitative determination of CA,comprising microwave-assisted extraction,high-performance liquid chromatography,and diode array detector method,was established.Extraction efficiency was optimized by changing microwave power,extraction sol-vent,volume,time,and temperature.Results showed that under the optimum conditions(extraction time of 10 min,temperature of 45℃,and solvent of 25 mL 70%ethanol under 400 W),the detection limit of CA was 0.0015 mg mL^?1,and the recoveries of CA from samples at spiking levels of 10,20,and 50μg g^?1 ranged from 90.1%-104%.The proposed method was successfully applied to ana-lyze the saponins in different tissues of A.japonicus collected in different seasons.The method developed in this study can provide quantitative technical support for the quality control of A.japonicus.     

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopus japonicus. In this study, four monoclonal antibodies (MAbs) (3C1, 3D9, 2F2, 2A8) against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria. However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens ...     

Effects of photoperiod on daily activity rhythm of juvenile sea cucumber, Apostichopus japonicus (Selenka)

In nature, Apostichopus japonicus exhibits a behavioral characteristic of emerging at night and sheltering during the day. Hence, it was commonly believed that longer and darker light conditions are better for a population of A. japonicus. In aquaculture situations therefore, animals have commonly been cultured without lighting, especially during juvenile-hatching culture. However, how the length of darkness each day affects A. japonicus and what the mechanism is of behavioral response of A. japonicus to different photoperiods remain unclear, and are specifically addressed in this study. We applied nine photoperiod treatments (light:dark (LD) cycles at 0:24, 3:21, 6:18, 9:15, 12:12, 15:9, 18:6, 21:3, and 24:0 under 500 lx) to 60 individuals (29.73 ± 0.23 g) per treatment (with 3 replicates). The environmental conditions were set as: water temperature = 16.9 ± 0.3°C, dissolved oxygen>6.0 mg/L, ammonia<0.3 mg/L, pH = 7.8-8.2, and salinity = 30-32. Under experimental conditions of continuous darkness as well as continuous light, cyclic nocturnal activity patterns of A. japonicus (viz. the animals emerged and fed at night and sheltered during the day) were observed. However, they spent more and less time, respectively, moving and feeding under continuous darkness and continuous light, than those under a natural light cycle. Under photoperiods with 6-12 h of light, the animals showed one sheltering behavior transition and two emerging behavior transitions. These behavior transitions appeared to be governed by some internal physiological factors and induced by daily light variation. The behavior of the animals was significantly affected by different photoperiods, and the distribution rate (DR) of the animals emerging at "daytime" increased with lengthening "light time" (except for the LD 3:21 cycle). We also found that there was no significant difference in mean DR per day under LD cycles of 6:18, 9:15, 15:9, and 12:12. Mean DRs per day were significantly lower for these photoperiods, than for those under LD cycles of 0:24 and 3:21, and significantly higher than for those under LD cycles of 18:6, 21:3 and 24:0. These results imply that 6-15 h light per day were the similar photoperiods to natural sunlight. From the viewpoint of activity and feeding behaviors, these might be the optimal photoperiods for rearing A. japonicus.     

Optimization of Stocking Density for the Sea Cucumber,Apostichopus japonicus Selenka, Under Feed-Supplement and Non-Feed-Supplement Regimes in Pond Culture

Optimal stocking densities were investigated for the sea cucumber Apostichopus japonicus Selenka under feed-supplement and non-feed-supplement regimes in net enclosures for 333 d. Substantial weight loss occurred during the aestivation phase (AE).Decreased growth rates were also observed during the winter phase (WT). In contrast, sea cucumbers showed rapid growth during the spring (SP) and autumn (AU) phases. Feeding regimes considerably influenced the growth performance, i.e., sea cucumbers grew faster under feed-supplement regime than under non-feed-supplement regime (P< 0.05). The average survival rates of sea cucumbers under feed-supplement regime were higher than those under non-feed-supplement regime for both the autumn phase and spring phase,but the differences were only significant for the latter phase (P<0.05). The fitted B-N curves showed that the optimal stocking densities, in terms of net production, were 22.3 ind. m-2 for feed-supplement regime and 14.1 ind. m-2 for non-feed-supplement regime.     

Effects of water depth and substrate color on the growth and body color of the red sea cucumber,Apostichopus japonicus

Three color variants of the sea cucumber,Apostichopus japonicus are recognized,the red one is highly valued in the market. When the red variant is cultured in ponds in China,its body color changes from red to celadon in 3–6 months. The effects of water depth and substrate color on the growth and body color of this animal were investigated. Juveniles of red A. japonicus were cultured in cages suspended at a range of water depths(20,50,100,150 and 200 cm). The specific growth rate of red sea cucumbers was significantly higher in animals cultured at deeper water layers compared with those grown at shallowers. Body weights were greatest for sea cucumbers cultured at a depth of 150 cm and their survival rates were highest at a depth of 200 cm. A scale to evaluate the color of red sea cucumbers(R value) was developed using a Pantone standard color card. All stocked animals in the 9-month trial retained a red color,however the red body color was much more intense in sea cucumbers cultured at shallower depths,while animals suspended in deeper layers became pale. In a separate trial,A. japonicus were cultured in suspended cages with seven different colored substrates. Substrate color had a significant effect on the growth and body-color of red A. japonicus. The yield were greatest for A. japonicus cultured on a yellow substrate,followed by green white orange red black and blue. All sea cucumbers in the 7-month trial retained a red color,although the red was most intense(highest R value) in animals cultured on a blue substrate and pale(lowest R value) for animals cultured on a green substrate.     

Seasonal changes in food uptake by the sea cucumber Apostichopus japonicus in a farm pond: Evidence from C and N stable isotopes

This study investigated the seasonal changes in carbon (C) and nitrogen (N) stable isotope values of several typical food sources of Apostichopus japonicus in a farm pond, including particulate organic matter (POM), macroalgae, benthic microalgae and animals such as nematode and copepod. The stable isotope technique was used to quantify relative contributions of various sources to the food uptake by A. japonicus. The results showed that significant changes occurred in the C and N stable isotope values of sea cucumber food sources due to the seasonality of micro-or macroalgae prosperity and the fluctuation of environmental conditions. The sea cucumber A. japonicus exhibited corresponding alterations in feeding strategy in response to the changes in food conditions. Calculation with a stable isotope mixing model showed that macroalgae was the principal food source for A. japonicus throughout the 1-yr investigation, with the relative contribution averaging 28.1% - 63.2%. The relative contributions of other food sources such as copepod and nematode, POM, benthic microalgae to the total food uptake by sea cucumber averaged 22.6% - 39.1%, 6.3% - 22.2%, 2.8% - 6.5%, and 2.8% - 4.2%, respectively. Together these results indicated that the seasonal changes in food sources led to the obvious temporal differences in the relative contribution of various food sources utilized by A. japonicus. Such findings provide the basic scientific information for improving the aquaculture techniques of A. japonicus, particularly for optimizing the food environment of A. japonicus culture in farm ponds.     

Construction of cDNA library from intestine, mesentery and coelomocyte of Apostichopus japonicus Selenka infected with Vibrio sp. and a preliminary analysis of immunity-related genes

The aquaculture of sea cucumber Apostichopus japonicus (Echinodermata, Holothuroidea) has grown rapidly during recent years and has become an important sector of the marine industry in Northern China. However, with the rapid growth of the industry and the use of non-standard culture techniques, epidemic diseases of A. japonicus now pose increasing problems to the industry. To screen the genes with stress response to bacterial infection in sea cucumber at a genome wide level, we constructed a cDNA library from A. japonicus Selenka (Aspidochirotida: Stichopodidae) after infecting them with Vibrio sp. for 48 h. Total RNA was extracted from the intestine, mesentery and coelomocyte of infected sea cucumber using Trizol and mRNA was isolated by Oligotex mRNA Kits. The ligated cDNAs were transformed into DH5α, and a library of 3.24×105 clones (3.24×105 cfu mL-1) was obtained with the sizes of inserted fragments ranging from 0.8 to 2.5 kb. Sequencing the cDNA clones resulted in a total of 1106 ESTs that passed the quality control. BlastX and BlastN searches have identified 168 (31.5%) ESTs sharing significant homology with known sequences in NCBI protein or nucleotide databases. Among a panel of 25 putative immunity-related genes, serum lectin isoform, complement component 3, complement component 3-like genes were further studied by real-time PCR and they all increased more than 5 fold in response to Vibrio sp. challenge. Our library provides a valuable molecular tool for future study of invertebrate immunity against bacterial infection and our gene expression data indicates the importance of the immune system in the evolution and development of sea cucumber.     

Growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus Selenka during periods of inactivity

The growth, metabolism and physiological response of the sea cucumber, Apostichopus japonicus, were investigated during periods of inactivity. The body weight, oxygen consumption rate (OCR), activities of acidic phosphatase (ACP), alkaline phosphatase (AKP), catalase (CAT) and superoxide dismutase (SOD), and content of heat shock protein 70 (Hsp70) in the body wall and coelomic fluid of A. japonicus were measured during starvation, experimental aestivation and aestivation. The results showed that the body weight of sea cucumber in the three treatments decreased significantly during the experimental period (P < 0.05). The OCR of sea cucumber reduced in starvation and experimental aestivation treatments, but increased gradually in natural aestivation treatment. The activities of ACP and AKP of sea cucumber decreased gradually in all treatments, whereas those of SOD and CAT as well as Hsp70 content decreased in the starvation and experimental aestivation treatments and increased in natural aestivation treatment. The sea cucumber entered a state of aestivation at 24℃. To some extent, the animals in experimental aestivation were different from those in natural aestivation in metabolism and physiological response. These findings suggested that the aestivation mechanism of A. japonicus is complex and may not be attributed to the elevated temperature only.     

Bacterial diversity of gut content in sea cucumber (Apostichopus japonicus) and its habitat surface sediment

This study investigated the bacterial diversity of gut content of sea cucumber(Apostichopus japonicus) and its habitat surface sediment in a bottom enhancement area using PCR-based denaturing gradient gel electrophoresis(DGGE) technique. Bacterial diversity evaluation showed that the value of the Shannon-Wiener index of gut content in different intestinal segments of A. japonicus varied between 2.88 and 3.00, lower than that of the surrounding sediment(3.23). Phylogenetic analysis showed that bacterial phylotypes in gut content and the surrounding sediment of A. japonicus were closely related to Proteobacteria includingγ-, α-, δ-and ε-proteobacteria, Bacteroidetes, Firmicute, and Actinobacteria, of which γ-proteobacteria were predominant. These results suggested that the sea cucumber A. japonicus was capable of feeding selectively, and PCR-DGGE was applicable for characterizing the bacterial community composition in gut content and the surrounding sediment of sea cucumber. Further investigation targeting longer 16S rDNA gene fragments and/or functional genes was recommended for obtaining more information of the diversity and function of bacterial community in the gut content of sea cucumber.     

Influence of dietary probiotic Bacillus TC22 and Prebiotic fructooligosaccharide on growth, immune responses and disease resistance against Vibrio splendidus infection in sea cucumber Apostichopus japonicus

The effects of probiotic Bacillus TC22 (isolated from intestine of infected sea cucumber) and prebiotic fructooligosaccharide (FOS) on growth, immunity and disease resistance in sea cucumber Apostichopus japonicus were studied. Six experimental diets were formulated with combinations of three levels of TC22 (0, 107 and 109 CFU g-1 diet) and two levels of FOS (0 and 0.5%) in a 3 × 2 factorial experiment. At the end of the 8-week feeding trial,animals were challenged by injecting Vibrio splendidus. The results revealed that the specific growth rates (SGR) of sea cucumbers were not affected by TC22 and FOS, or the interaction between TC22 and FOS (P > 0.05). However,there were significant interactions between TC22 and FOS for immune response and disease resistance in sea cucumbers (P < 0.05). When sea cucumbers were fed with TC22 at 109 CFU g?1 feed and 0.5% FOS alone or in combination, the phagocytosis, respiratory burst and phenoloxidase activity of sea cucumber coelomocytes were significantly enhanced;the disease resistance against V. splendidus infection was also increased significantly (P < 0.05).However, dietary combination of TC22 at 109 CFU g-1 diet and 0.5% FOS did not yield significantly higher efficiency than either TC22 at 109 CFU g-1 diet or 0.5% FOS used alone (P > 0.05). Therefore, further studies should examine the effects of combinations of other levels of FOS(> 0.5% or < 0.5%) and TC22 on the immunity and disease resistance of sea cucumbers.     

Effects of Pseudoalteromonas sp. BC228 on digestive enzyme activity and immune response of juvenile sea cucumber (Apostichopus japonicus)

A marine bacterium, Pseudoalteromonas sp. BC228 was supplemented to feed in a feeding experiment aiming to determine its ability of enhancing the digestive enzyme activity and immune response of juvenile Apostichopus japonicus. Sea cucumber individuals were fed with the diets containing 0(control), 105, 107 and 109 CFU g-1 diet of BC228 for 45 days. Results showed that intestinal trypsin and lipase activities were significantly enhanced by 107 and 109 CFU g-1 diet of BC228 in comparison with control(P 0.01). The phagocytic activity in the coelomocytes of sea cucumber fed the diet supplemented with 107 CFU g-1 diet of BC228 was significantly higher than that of those fed control diet(P 0.05). In addition, 105 and 107 CFU g-1 diet of BC228 significantly enhanced lysozyme and phenoloxidase activities in the coelomic fluid of sea cucumber, respectively, in comparison with other diets(P 0.01). Sea cucumbers, 10 each diet, were challenged with Vibrio splendidus NB13 after 45 days of feeding. It was found that the cumulative incidence and mortality of sea cucumber fed with BC228 containing diets were lower than those of animals fed control diet. Our findings evidenced that BC228 supplemented in diets improved the digestive enzyme activity of juvenile sea cucumber, stimulated its immune response and enhanced its resistance to the infection of V. splendidus.     

Effect of Bacillus baekryungensis YD13 supplemented in diets on growth performance and immune response of sea cucumber (Apostichopus japonicus)

The effect of a potential probiotic on the growth performance and immune response of sea cucumber(Apostichopus japonicus) was investigated. Bacillus baekryungensis YD13 isolated from sea cucumber culturing ponds was added to sea cucumber basal feed as a probiotic in different doses(0, the control; 1×104(YD134), 1×106(YD136) and 1×108(YD138) CFU g-1 of diet), and administered orally to A. japonicus(initial mean wet weight 5.44 g ± 0.17 g). The sea cucumbers were fed in 20 aquaria, 5 each treatment, for 60 d. At the end of growth trial, 20 sea cucumbers from each treatment were challenged with Vibrio splendidus. A. japonicus in YD134 and YD136 exhibited significantly better growth performance than control(P 0.05). Five non-specific immune parameters including lysozyme, acid phosphatase, alkaline phosphatase, superoxide dismutase and catalase in coelomic fluid were measured to evaluate the immune response of A. japonicus to the probiotics. Results showed that all parameters were significantly improved when YD11 was supplemented in the dose of 1×106 CFU g-1(P 0.05). The cumulative incidence and mortality after the Vibrio splendidus challenge decreased significantly in sea cucumbers of YD136. Accordingly, 1×106 CFU g-1 of YD13 in diet was recommended for the growth promotion and immune enhancement of A. japonicus.     

Nitrogen and Phosphorus Budget of a Polyculture System of Sea Cucumber （Apostichopus japonicus）, Jellyfish （Rhopilema esculenta） and Shrimp （Fenneropenaeus chinensis）

The nitrogen(N) and phosphorus(P) budget and the ecological efficiency of a polyculture system of sea cucumber(Apostichopus japonicus), jellyfish(Rhopilema esculenta) and shrimp(Fenneropenaeus chinensis) were studied in a cofferdam, 120.2 ha in size. The nutrients were supplied by spring tide inflow. In total, 139600 kg N yr-1 and 9730 kg P yr-1 input to the system; while 118900 kg N yr-1 and 2840 kg P yr-1 outflowed from the system concurrently, thus the outflow was 85.7%(N) and 29.2%(P) of inflow. The production of N and P was 889.5 kg yr-1 and 49.28 kg yr-1(sea cucumber) and 204 kg yr-1 and 18.03 kg yr-1(jellyfish and shrimp), respectively. The utilization rate of N and P by polycultured animals was 7.8‰ and 6.9‰, respectively, 21.9% and 38% higher than that of monocultured sea cucumber. Our results indicated that the polyculture system was an efficient culture system of animals and a remediation system of coastal environment as well; it scavenged 14.3% and 70.8% of N and P, respectively. Such an ecological efficiency may be improved further by increasing either the stocking density or the size of sea cucumber or both.