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.     

Effects of Hanseniaspora opuntiae C21 on the growth and digestive enzyme activity of juvenile sea cucumber Apostichopus japonicas

The effects of a diet containing Hanseniaspora opuntiae C21 on growth and digestive enzyme activity were estimated in juvenile Apostichopus japonicus. Groups of sea cucumbers were fed diets containing H. opuntiae C21 at 0(control), 10 4, 10 5, and 10 6 CFU(colony-forming units)/g feed. Results showed that after 45 d the specific growth rate(SGR) of sea cucumbers fed a C21-supplemented diet at 10 4 CFU/g feed was significantly higher than that of the control( P 0.05). Intestinal trypsin and lipase activities were significantly enhanced by C21 administration at 10 4 and 10 5 CFU/g feed compared with the control( P 0.05). After feeding for 23–42 d, C21 was demonstrated by denaturing gradient gel electrophoresis to be present in the intestine of sea cucumbers. In addition, after feeding the C21-supplemented diets for 15 d, the sea cucumbers were switched to an unsupplemented diet and C21 was confirmed to be capable of colonizing the intestine for at least 31 d after cessation of feeding. In conclusion, C21 was shown to successfully colonize the intestine of juvenile A. japonicus via dietary supplementation, and improve growth and digestive enzyme activity.     

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.     

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.     

波吉卵囊藻胞外滤液对铜绿微囊藻转录水平的影响

【目的】探索波吉卵囊藻胞外滤液对铜绿微囊藻的抑制机理。【方法】采用Illumina平台,对使用BG11培养基(对照组)和波吉卵囊藻胞外滤液(实验组)培养的铜绿微囊藻(Microcystis aeruginosa)进行转录组测序,筛选差异表达基因并进行通路富集分析,并对部分差异表达基因进行qRT-PCR验证。【结果】与对照组相比,波吉卵囊藻胞外滤液处理后的铜绿微囊藻组中筛选1483个表达差异显著的基因,其中上调表达基因788个,下调表达基因695个。GO功能分析将差异基因划分为35个子类别,包括催化活性、结合、代谢过程、细胞过程、膜、膜部分等。KEGG通路分析中,所有差异基因聚集在108个通路,其中84个差异基因注释在代谢功能类别中,而氧化磷酸化通路富集最显著。氧化磷酸化通路中,相关基因表达以上调为主(26个DEGs,20个上调,6个下调)。qRT-PCR验证结果与转录组测序结果的表达变化趋势一致。【结论】波吉卵囊藻滤液引起铜绿微囊藻氧化磷酸化途径上基因表达上调,ATP合成效率提高。     

Effect of culture methods on individual variation in the growth of sea cucumber Apostichopus japonicus within a cohort and family

There is substantial individual variation in the growth rates of sea cucumber Apostiehopus japonicus individuals. This necessitates additional work to grade the seed stock and lengthens the production period. We evaluated the influence of three culture methods （free-mixed, isolated-mixed, isolated-alone） on individual variation in growth and assessed the relationship between feeding, energy conversion efficiency, and individual growth variation in individually cultured sea cucumbers. Of the different culture methods, animals grew best when reared in the isolated-mixed treatment （i.e., size classes were held separately）, though there was no difference in individual variation in growth between rearing treatment groups. The individual variation in growth was primarily attributed to genetic factors. The difference in food conversion efficiency caused by genetic differences among individuals was thought to be the origin of the variance. The level of individual growth variation may be altered by interactions among individuals and environmental heterogeneity. Our results suggest that, in addition to traditional seed grading, design of a new kind of substrate that changes the spatial distribution of sea cucumbers would effectively enhance growth and reduce individual variation in growth of sea cucumbers in culture.     

Ecological effects of co-culturing the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis> with the Chinese white shrimp <Emphasis Type="Italic">Fenneropenaeus chinensis</Emphasis> 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.     

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.     

Metabolic rates and biochemical compositions of Apostichopus japonicus (Selenka) tissue during periods of inactivity

Estivation, hibernation, and starvation are indispensable inactive states of sea cucumbers Apostichopus japonicus in nature and in culture ponds. Generally, temperature is the principal factor that induces estivation or hibernation in the sea cucumber. The present study provided insight into the physiological adaptations of A. japonicus during the three types of inactivity (hibernation, estivation, and starvation) by measuring the oxygen consumption rates (Vo₂) and biochemical compositions under laboratory conditions of low (3°C), normal (17°C) and high (24°C) temperature. The results show that the characteristics of A. japonicus in dormancy (hibernation and estivation) states were quite different from higher animals, such as fishes, amphibians, reptiles, and mammals, but more closely resembled a semi-dormant state. It was observed that the shift in the A. japonicus physiological state from normal to dormancy was a chronic rather than acute process, indicated by the gradual depression of metabolic rate. While metabolic rates declined 44.9% for the estivation group and 71.7% for the hibernation group, relative to initial rates, during the 36 d culture period, metabolic rates were not maintained at constant levels during these states. The metabolic depression processes for sea cucumbers in hibernation and estivation appeared to be a passive and an active metabolic suppression, respectively. In contrast, the metabolic rates (128.90±11.70 μg/g h) of estivating sea cucumbers were notably higher (107.85±6.31 μg/g h) than in starving sea cucumbers at 17°C, which indicated that the dormancy mechanism here, as a physiological inhibition, was not as efficient as in higher animals. Finally, the principle metabolic substrate or energy source of sea cucumbers in hibernation was lipid, whereas in estivation they mainly consumed protein in the early times and both protein and lipid thereafter.     

Domestication of marine microalga Nannochloropsis oceanica to freshwater medium and the physiological responses

Marine microalga Nannochloropsis oceanica LAMB0001 were domesticated(-73 0 generations,-two days each)to adapt freshwater BGll medium.A number of freshwater medium adapted colonyderived strains were obtained.The strains were verified phylogenetically to be N.oceanica LAMB0001 based on the 18 S ribosomal RNA gene.Freshwater-medium adapted strain(FA1)grew faster in the BG11 medium prepared with freshwater than wild-type N.oceanica grew in f/2 medium prepared with seawater.We assumed that(1)the expression patterns of the genes that expressed differentially between FA1 and the wild-type N.oceanica exposing to the BG11 medium(WT-F)have been reprogrammed;(2)the physiological processes in which these genes involved have been modified;and(3)a Gene Ontology(GO)term or a KEGG pathway enriched by DEGs between FA1 and WT-F has been up-or down-regulated if it was enriched simultaneously by up-or down-regulated DEGs between FA1 and WT-F,respectively.Under these assumptions,we found that FA1 reprogrammed the expression patterns of a set of genes that involved in cell adhesion,membrane and membrane integrity,material transportation,cell movement,and cellular signaling network.These changes in cellular functions and metabolic pathways indicate that the microalga modified its gene expression pattern in a wide function range and at a high regulation rank in order to adapt to the freshwater medium.It is feasible to domesticate marine microalgae to a freshwater habitat,which may aid to modify their cultivation performances.     

Succession and seasonal variation in epilithic biofilms on artificial reefs in culture waters of the sea cucumber <Emphasis Type="Italic">Apostichopus japonicus</Emphasis>

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 Asia- Pacific 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.     

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.