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.     

Transcriptional changes in epigenetic modifiers associated with gene silencing in the intestine of the sea cucumber, Apostichopus japonicus (Selenka), during aestivation

The sea cucumber, Apostichopus japonicus, undergoes aestivation to improve survival during periods of high-temperature. During aestivation, the metabolic rate is depressed to reduce the consumption of reserved energy. We evaluated the role of epigenetic modification on global gene silencing during metabolic rate depression in the sea cucumber. We compared the expression of epigenetic modifiers in active and aestivating sea cucumbers. The expression of three genes involved in DNA methylation and chromatin remodeling (DNA (cytosine-5)-methyltransferase 1, Methyl-CpG-binding domain protein 2), and Chromodomain-helicase-DNA-binding protein 5) was significantly higher during aestivation (Days 20 and 40). Similarly, we observed an increase in the expression of genes involved in histone acetylation (Histone deacetylase 3) and Histone-binding protein RBBP4) during the early (Days 5 and 10) and late phases (Days 20 and 40) of aestivation. There was no change in the expression of KAT2B, a histone acetyltransferase. However, the expression of histone methylation associated modifiers (Histone-arginine methyltransferase CARMER and Histone-lysine N-methyltransferase MLL5) was significantly higher after 5 d in the aestivating group. The results suggest that the expression of epigenetic modifiers involved in DNA methylation, chromatin remodeling, histone acetylation, and histone methylation is upregulated during aestivation. We hypothesize that these changes regulate global gene silencing during aestivation in 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.     

Metabolomics analysis for skin ulceration syndrome of Apostichopus japonicus based on UPLC/Q-TOF MS

Skin ulceration syndrome(SUS) is the main diseases affected the development of sea cucumber(Apostichopus japonicus) culture industries.To better observe the changes in the sea cucumber A.japonicus with SUS and understand the pathogenesis of the disease,activities of superoxide dismutase(SOD),catalase(CAT),and level of malondialdehyde(MDA) in coelomic fluid were detected using the Assay Kit and metabolite s in the body wall were assessed using ultra-performance liquid chromatography and quadrupoletime of flight mass spectrometry.The re sults indicated that level of MDA was increased during SUS compared with healthy individuals(P0.01),but activities of SOD and CAT were reduced(P0.05).In metabolomics analysis,metabolites,such as adenosine,choline,betaine aldehyde,palmitic acid,and taurine,were found to be upregulated and 2-oxoadipic acid,anthranilic acid(vitamin L1),thioetheramide-PC,cholesterol-3-sulfate,and pentadecanoic acid were downregulated(VIP1 and P0.1).Pathway enrichment analysis indicated most enrichment of KEGG pathways were mainly related to energy metabolism,immunity,and osmoregulation such as ABC transporters,glycine,serine and threonine metabolism,tryptophan metabolism and neuroactive ligand-receptor interaction.Our study reflected the difference in enzyme activity and metabolites between A.japonicus with SUS and those without,which will provide reference data for investigating SUS.     

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.     

Microsatellite DNA polymorphisms and the relation with body weight in sea cucumber Apostichopus japonicus

The relationship between microsatellite polymorphism and body weight of captive bred Chinese sea cucumber Apostichopus japonicus was investigated in two local populations in Dalian.Among ten loci discovered, nine show changes except for A J07 loci. Seven loci were found highly polymorphic in both populations. For each locus in two populations, the average number of alleles is 6.428 6 and 6.285 7, the average observed heterozygosity at 0.225 7 and 0.245 9, the expected heterozygosity at 0.776 8 and 0.748 8, the polymorphism information content (PIC) at 0.709 2 and 0.674 6, respectively. Further analysis show significant correlation between A. japonicus body weight and occurrence markers AJ02 and AJ04. The findings of the relation may be helpful for molecular breeding,as well as the marker-assisted selection of sea cucumbers.     

Effect of water temperature on digestive enzyme activity and gut mass in sea cucumber Apostichopus japonicus （Selenka）, with special reference to aestivation

The effect of water temperature on gut mass and digestive enzyme activity in sea cucumber Apostichopus japonicus, including relative gut mass (RGM), amylase, lipase, pepsin and trypsin activities were studied at temperatures of 7, 14, 21, and 28°C over a period of 40 days. Results show that RGM significantly decreased after 40 days at 21°C and markedly decreased over the whole experiment period at 28°C; however, no significant effect of duration was observed at 7 or 14°C. At 14°C, trypsin activity significantly decreased over 10 and 20 days, then increased; amylase and trypsin activity significantly decreased after 40 days at 28°C. However, no significant effect of duration was found on amylase, pepsin or trypsin activities in the other temperature treatment groups. At 28°C, lipase activity peaked in 20 days and then markedly decreased to a minimum at the end of the experiment. On the other hand, pepsin activity at 28°C continuously increased over the whole experimental period. Principle component analysis showed that sea cucumbers on day 40 in the 21°C group and in the previous 20 days in the 28°C group were in the prophase of aestivation. At 28°C, sea cucumbers aestivated at 30–40 days after the start of the experiment. It is concluded that the effect of temperature on the digestion of A. japonicus is comparatively weak within a specific range of water temperatures and aestivation behavior is accompanied by significant changes in RGM and digestive enzyme activities.     

High-throughput sequencing of 16S r RNA amplicons characterizes gut microbiota shift of juvenile sea cucumber Apostichopus japonicus feeding with three antibiotics

Sea cucumber Apostichopus.japonicus is an important marine economic species in Asian countries due to its profound nutritional and medicinal value.So far,with the rapid development of intensified artificial aquaculture of sea cucumbers,the use of antibiotics is still an inexpensive and dispensable way to treat pathogenic infections,especially during the nursery phase.However,there is little information on the effects of antibiotics on the intestinal microbiota of sea cucumber.Therefore an Illumina based sequencing method was used to examine the intestinal bacterial composition of juvenile A.japonicas following diets with three typical antibiotics(tetracycline,erythromycin.,and norfloxacin)under 15,30,and 45 d.The findings reveal that different antibiotics have distinct effects on the growth performance of juvenile sea cucumbers.However,the richness and diversity of microbiota were barely affected by antibiotics but the community composition alterations indicated that the three antibiotics exhibited their respective patterns of reshaping the intestinal bacteria of juvenile sea cucumbers.In common,the abundance of some sensitive genera with helpful functions,such as Thalassotalea,Shewanella,Sulfitobacter,and Halomonas decreased significantly with exposure to antibiotics and the abundance of multiple potential pathogenic-and suspected antibiotic-resistant microorganisms like Arcobacter,Leucothrix,and Clostridium_sensu_stricto_1 was found increased significantly in the antibiotic groups.These results suggest that low doses of antibiotics could affect the composition of the intestinal microbiota of sea cucumbers and might increase the risk of infection of the hosts.This study could help us to explore how antibacterial compounds modify the gut microbiota of sea cucumbers and provide theoretical guidance in hatchery management by scientific antibiotic use in sea cucumber mariculture.     

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber Apostichopus japonicus

Periphytic biofilms in aquaculture waters are thought to improve water quality, provide an additional food source, and improve the survival and growth of some reared animals. In the AsiaPacific region, particularly in China, artificial reefs are commonly used in the commercial farming of sea cucumbers. However, few studies have examined the epilithic biofilms on the artificial reefs. To gain a better understanding of the succession of epilithic biofilms and their ecological processes in sea cucumber culture waters, two experiments were conducted in culture waters of the sea cucumber Apostichopus japonicus in Rongcheng, China, using artificial test panels. On the test panels of succession experiment, more than 67 species were identified in the biofilms. On the test panels of seasonal variation experiment, more than 46 species were recorded in the biofilms. In both experiments, communities of epilithic biofilms were dominated by diatoms, green algae and the annelid Spirorbis sp. In the initial colonization, the dominant diatoms were Cocconeis sp., Amphora spp. and Nitzschia closterium in June, which were succeeded by species of Navicula, Cocconeis and Nitzschia(July to September), and then by Licmophora abbreviata, Nitzschia closterium and Synedra spp. in the following months. A diatom bloom in the autumn and filamentous green algae burst in the summer were also observed. Ecological indices well annotated the succession and seasonal changes in epilithic communities. Multidimensional scaling(MDS) analysis found significant differences in diatom community composition among months and seasons. Fast growth of biofilms was observed in the summer and autumn, whereas the biomass of summer biofilms was largely made up of filamentous green algae. Present results show that the components of epilithic biofilms are mostly optimal foods of A. japonicus, suggesting that biofilms on artificial reefs may contribute important nutritional sources for sea cucumbers during their growth seasons. Future works should include quantitative determination of the contribution of epilithic biofilms to the diet of A. japonicus, potential roles of epilithic biofilms in regulating the water quality of sea cucumber ponds, and the regulation of epilithic biofilms in sea cucumber culture ponds.     

Identification of five sea cucumber species through PCR-RFLP analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene (COI) was used to identifing five sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.     

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.     

Mechanical Stress Induces Neuroendocrine and Immune Responses of Sea Cucumber(Apostichopus japonicus)

Grading procedure in routine sea cucumber hatchery production is thought to affect juvenile sea cucumber immunological response. The present study investigated the impact of a 3-min mechanical perturbation mimicking the grading procedure on neuroendocrine and immune parameters of the sea cucumber Apostichopus japonicus. During the application of stress, concentrations of noradrenaline and dopamine in coelomic fluid increased significantly, indicating that the mechanical perturbation resulted in a transient state of stress in sea cucumbers. Coelomocytes concentration in coelomic fluid increased transiently after the beginning of stressing, and reached the maximum in 1 h. Whereas, coelomocytes phagocytosis at 3 min, superoxide anion production from 3 min to 0.5 h, acid phosphatase activity at 0.5 h, and phenoloxidase activity from 3 min to 0.5 h were all significantly down-regulated. All of the immune parameters recovered to baseline levels after the experiment was conducted for 8 h, and an immunostimulation occurred after the stress considering the phagocytosis and acid phosphatase activity. The results suggested that, as in other marine invertebrates, neuroendocrine/immune connections exist in sea cucumber A. japonicus. Mechanical stress can elicit a profound influence on sea cucumber neuroendocrine system. Neuroendocrine messengers act in turn to modulate the immunity functions. Therefore, these effects should be considered for developing better husbandry procedures.     

Ecological effects of co-culturing the sea cucumber Apostichopus japonicus with the Chinese white shrimp Fenneropenaeus chinensis in an earthen pond

Using net enclosures in an earthen pond, we established three culture treatments with the sea cucumber Apostichopus japonicus and the Chinese white shrimp Fenneropenaeus chinensis : monoculture of sea cucumbers(C), monoculture of shrimp(S), and co-culture of the two species(CS). We measured levels of suspended particulate matter in the water column; total organic matter, total organic carbon, total nitrogen, and carbon/nitrogen ratios in both settling particles and the sediment; and chlorophyll a levels in the sediment. We then compared these variables between the three treatments. We also examined growth, survival, and yield of the two species in the different treatments. From June to September, the mean monthly suspended particulate matter sedimentation rates in the CS and S treatments were significantly( P 0.05) greater than those in the C treatment. From August to November, the mean monthly total organic matter, total organic carbon, total nitrogen, and chlorophyll a contents in the sediment in the CS and S treatments were significantly( P 0.05) greater than those in the C treatment. Final wet weight, specific growth rate, survival rate, and total yield of sea cucumbers in co-culture were all significantly greater than those of sea cucumbers in monoculture. There were no significant differences among any of these variables for shrimp reared in the two systems. The bioturbation of the sediment and fecal production of the shrimp likely supplied natural food for the sea cucumbers. Co-culture of the two species is a viable option for increasing yield per unit area, maximizing use of the water body, and diversifying crop production.     

An Evaluation on the Ratio of Plant to Animal Protein in the Diet of Juvenile Sea Cucumber(Apostichopus japonicus): Growth,Nutrient Digestibility and Nonspecific Immunity

This study was conducted to evaluate the effects of plant/animal(P/A) protein ratios(viz.1:4, 1:3, 1:2, 1:1,2:1, 3:1, 4:1) on growth performance, body composition, apparent digestibility of diets, and nonspecific immunity of juvenile sea cucumber(Apostichopus japonicus). Sea cucumbers were divided into 21 plastic tanks, and each tank was stocked with 15 individuals(initial weight: about 23.73 g). Each feed was allocated to three replicates of sea cucumbers. The feeding experiment lasted for 50 days. Results indicated that weight gain rate(WGR) and body wall weight(BWW) significantly increased as dietary ratio of P/A increased from 1:4 to 3:1, and then decreased significantly with further increase of this ratio(P 0.05). The body wall coefficient(BWC) showed a similar tendency to WGR and BWW, but no significance was detected among dietary treatments(P 0.05). The apparent digestibility of dry matter, protein and lipid increased with ratio of P/A increasing from 1:4 to 2:1(P 0.05), and then decreased with further increase of this ratio. Correspondingly, activities of trypsin and amylase were significantly increased as P/A increased from 1:4 to 2:1(P 0.05). The activities of SOD and CAT showed a similar trend with WGR, with the highest value observed in the ratio of 1:2 and 1:1, respectively. Results above showed that moderate or relatively higher ratio of P/A protein(1:1-3:1) significantly increased the growth performance, apparent digestibility, and nonspecific immunity of sea cucumber. This will contribute to improving the feed formulation for juvenile cucumbers.     

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.