Relationships between body weight and other morphological traits in young sea cucumbers Apostichopus japonicas

Correlation, multiple regression, and path analyses were used to investigate the relationships between body weight and three other morphological traits in juvenile Japanese sea cucumbers Apostichopus japonicus. We measured live body weight(BW), body length(BL), numbers of papillae(NP), and numbers of tube feet(NF) at 60, 80, 100, and 130 days post-hatching(dph). We calculated path correlation coe cients, correlation indices( R~2), and coe cients of determination with BW as the dependent variable and the other morphological traits as independent variables. The coe cient of variation for BW was high across all age groups, and all measured morphological traits were significantly correlated( P 0.01). BL had the greatest direct ef fect on BW across all age groups(60 dph, 0.526; 80 dph, 0.404; 100 dph, 0.620;and 130 dph, 0.681), while NF had the greatest indirect ef fect on BW across all age groups(60 dph, 0.528;80 dph, 0.452; 100 dph, 0.666; and 130 dph, 0.603). Regression analyses between morphological traits and BW indicated that R 2 was greater than 0.85 only in the 100-dph specimens. The indirect ef fects of the other measured morphological traits on BW were age-dependent. The optimal regression equations,as determined with stepwise regression, were, for 60-dph specimens: BW_(60)=10~((-3.04+0.092 BL+0.014 NP+0.014 NF))( R~2 =0.632); for 80-dph specimens: BW _(80)=10~((-3.035+0.056 BL+0.017 NP+0.02 NF))( R~2 =0.686); for 100-dph specimens:BW_(100) =10~((-3.742+0.069 BL+0. 633*l g( NP)+0. 464*l g( NF)))( R~2 =0.893); and for 130-dph specimens: BW_(130)=10~((-2.472+0.065 BL+0.012 NP))( R~2 =0.774). Our work clarified the correlation between various morphological traits and body weight of a commercially-important sea cucumber species( A. japonicus). Our predictive models for body weight might be useful for the aquaculture and selective breeding of A. japonicus. These models might also provide theoretical support for the indirect selection of traits that are di cult to select directly.     

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.     

Development and characterization of novel polymorphic microsatellite markers for the Korean freshwater snail Semisulcospira coreana and cross-species amplification using next-generation sequencing

Korean freshwater snails of the genus Semisulcospira are widely distributed across East Asia.It has been a very popular nutritional food in Korea,and is an ecologically important water quality indicator because it lives only in clean water.However,no microsatellite markers have been generated to study the population genetic diversity of this genus.In the present study,we developed and characterized 18 novel microsatellite loci from Semisulcospira coreana genomic DNA.The microsatellites were isolated using 454 GS-FLX titanium sequencing and 18 markers were used for genotyping in S.coreana.In addition,we also tested the cross-species transferability of the microsatellite markers in four additional Semisulcospira spp.We identified 18 polymorphic loci and the number of alleles per loci,and their polymorphism information content values ranged from 2 to 17 and 0.203 to 0.902,respectively.The observed and expected heterozygosities of the loci ranged from 0.063 to 0.924 and 0.226 to 0.924,respectively.According to the analysis of the cross-species transferability of these markers,four species,S.forticosta,S.gottschei,S.tegulata,and S.libertina,showed a very high transferability(80%–85%).These results show that this set of nuclear markers could be useful for population genetics studies of this species and closely related species.     

Development of 57 novel polymorphic microsatellite markers in half-smooth tongue sole (Cynoglossus semilaevis)

Half-smooth tongue sole (Cynoglossus semilaevis) is a promising species for aquaculture in China.The wild population of C. semilaevis is under threat from environmental factors. Microsatellite markers are very suitable for assessing genetic diversity. Four microsatellite-enriched libraries of half smooth tongue sole (Cynoglossus semilaevis) were constructed,from which 57 polymorphic microsatellites were isolated and characterized.The polymorphism of these microsatellites was assessed by genotyping in 30 ind...     

Development of fatty acid biomarkers for the identification of wild and aquacultured sea cucumber (Apostichopus japonicus)

In this study,the fatty acids(FAs) of the organs and tissues of sea cucumber(Apostichopus japonicus) were profiled in order to compare the FA composition of sea cucumber collected from natural habitat(wild) and cages(cultured).The differences in FA contents in dermomuscular tube,peripharyngeal annulus,gonad and intestine(with or without content) between the wild and the cultured were determined.The main fatty acids in all organs and tissues were 20:5n-3,16:1n-7,20:4n-6,22:6n-3,18:0,and 18:1n-7.The basically different FAs of body wall and digestive tube were 16:1n-7,18:1n-9 and 20:1n-11.The ratio of saturated to mono-and polyunsaturated FAs in digestive tube was independent on inside content while there was a redistribution of the total amount of n-3 and n-6 fatty acids.The comparison of FA composition of the wild and the cultured sea cucumber showed that 20:5n-3,16:1n-7 and 18:1n-7 predominated the wild while 20:4n-6 predominated the cultured.The content of branched-chain fatty acids in the wild was 3%–4% and about 9% in the cultured.The possible FAs for identifying the wild and the cultured sea cucumbers were selected.It was suggested that the indexes such as the ratio of either(n-3:n-6) to(n-7:n-6) or(n-3) +(n-7) to(n-6) may serve as the biomarkers distinguishing the wild and the cultured sea cucumber.     

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.     

Isolation and characterization of 45 Polymorphie microsatellite loci of turbot (Scophthalmus maximus) and cross-species amplification

Turbot(Scophthalmus maximus) is a flatfish species commercially important for aquaculture.In this study,we generated a microsatellite-enriched genomic DNA library for Scophthalmus maximus,and then isolated and characterized 45 microsatellite loci by genotyping 30 individuals.The observed number of alleles ranged from 2 to 19 with an average of 6.24,while the effective number of alleles ranged from 1.30 to 11.11 with an average of 3.66.The expected heterozygosities varied from 0.235 to 0.925 4 and polymorphic information content ranged from 0.204 4 to 0.903 3,with an average of 0.622.Twelve loci deviated significantly from Hardy-Weinberg equilibrium,and no significant linkage disequilibrium was observed between any pair of loci after Bonferroni correction.In cross-species amplification,five flatfish species(Paralichthys lethostigma,Verasper moseri,platichthys stellatus,Hippoglossoides dubius and Cynoglossus semilaevis) showed at least one polymorphic locus.These polymorphic microsatellite loci should prove useful for population analysis of turbot and other related species.     

Development of 101 novel EST-derived single nucleotide polymorphism markers for Zhikong scallop (Chlamys farreri)

Zhikong scallop(Chlamys farreri) is an important maricultured species in China.Many researches on this species,such as population genetics and QTL fine-mapping,need a large number of molecular markers.In this study,based on the expressed sequence tags(EST),a total of 300 putative single nucleotide polymorphisms(SNPs) were selected and validated using high resolution melting(HRM) technology with unlabeled probe.Of them,101(33.7%) were found to be polymorphic in 48 individuals from 4 populations.Further evaluation with 48 individuals from Qingdao population showed that all the polymorphic loci had two alleles with the minor allele frequency ranged from 0.046 to 0.500.The observed and expected heterozygosities ranged from 0.000 to 0.925 and from 0.089 to 0.505,respectively.Fifteen loci deviated significantly from Hardy-Weinberg equilibrium and significant linkage disequilibrate was detected in one pair of markers.BLASTx gave significant hits for 72 of the 101 polymorphic SNP-containing ESTs.Thirty four polymorphic SNP loci were predicted to be non-synonymous substitutions as they caused either the change of codons(33 SNPs) or pretermination of translation(1 SNP).The markers developed can be used for the population studies and genetic improvement on Zhikong scallop.     

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.     

Sustainability evaluation of different systems for sea cucumber (Apostichopus japonicus) farming based on emergy theory

Emergy analysis is effective for analyzing ecological economic systems. However, the accuracy of the approach is af-fected by the diversity of economic level, meteorological and hydrological parameters...     

Effects of environment factors on initiation of sperm motility in sea cucumber Apostichopus japonicus (Selenka)

Sperm of sea cucumber Apostichopus japonicus (Selenka) were quiescent in electrolyte NaCl solution and artificial seawater (ASW) and nonelectrolyte glucose and mannitol solutions when the osmolality was less than 200 mOsm kg-1. The sperm started to be motile as a result of increased osmolality, indicating an osmolality-dependent initiation of sperm motility in sea cucumber. After a brief incubation in hypotonic NaCl and glucose solutions with osmolalities of 200 and 400 mOsm kg-1, sperm lost partial motile ability. Sperm became immobilized when pH was 6.0 in NaCl, glucose and mannitol solutions, suggesting that an H+ release is involved in sperm activation. The decreased pH had no effect on the percentage of motile sperm in ASW, whereas it delayed the time period to reach the maximum motility (motilitymax). Extracellular Ca2+ in electrolyte solutions was not essential for motility stimulation but shortened the time of reaching motilitymax. When Ca2+ was mixed in nonelectrolyte solutions the sperm motility was completely suppressed. The K+ channel blocker, quinine, suppressed the sperm motility in electrolyte solution, showing a possible involvement of K+ transport in the process. High K+ concentration did not affect the sperm motility in NaCl solution, but decreased it in ASW and almost entirely suppressed it in nonelectrolyte solutions. The different effects of pH and K+ in ASW and NaCl solution indicate that external ions may also regulate sperm motility.     

Ecological effects of co-culturing sea cucumber Apostichopus japonicus (Selenka) with scallop Chlamys farreri in earthen ponds

Monthly changes in sedimentation and sediment properties were studied for three different culture treatments: sea cucumber monoculture (Mc), sea cucumber and scallop polyculture (Ps-c) and scallop monoculture (Ms). Results indicated that the survival rate of sea cucumber was significantly higher in Ps-c cultures than in Mc cultures. Sea cucumber yield was 69.6% higher in Ps-c culture than in Mc culture. No significant differences in body weight and scallop shell length were found between Ps-c and Ms cultures. The mean sedimentation rate of total particulate matter (TPM) was 72.2 g/(m2 d) in Ps-c cultures, with a maximum of 119.7 g/(m2 d), which was markedly higher than that of Mc (mean value). Sedimentation rates of organic matter (OM), total organic carbon (TOC), total nitrogen (TN) and total phosphorus (TP) in Ps-c cultures were also significantly higher than those in Mc cultures. TOC and TN contents of sediment increased rapidly in the first 5 months in Ms cultures and remained at a high level. TOC and TN contents in Mc and Ps-c cultures decreased during sea cucumber feeding seasons and increased during sea cucumber dormancy periods (summer and winter). The study demonstrates that co-culture of sea cucumber and scallop in earthen ponds is an alternative way to alleviate nutrient loads and improve water quality in coastal aquaculture systems. Moreover, it provides the additional benefit of an increased sea cucumber yield.     

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.     

Genetic variation in two sea cucumber （Apostichopus japonicus） stocks revealed by ISSR markers

Sea cucumber Apostichopus japonicus samples were collected in Changdao, Penglai (PL), 27 individuals, and Lingshandao, Qingdao (QD), 30 individuals, in the Shandong Peninsula, China. Ten SSR primers were used to assess the genetic variation and relationship between and within the two stocks. Respectively, for each stock, the percentage of polymorphic bands was 85.2% and 86.9%; the gene diver- sity was 0.360 5 and 0.342 8; and the Shannon’s information index was 0.515 0 and 0.499 0. At species level, the percentage of polymorphic bands was 92.2%, the total gene diversity was 0.378 9 and the Shannon’s information index was 0.550 4. The coefficient of overall genetic differentiation and the ge- netic distances between the stocks were also calculated to be 0.073 0 and 0.079 6 using the POPGENE program. Results show that the genetic diversity of the two stocks is still large but the genetic distance between the two stocks is close. A dendrogram was constructed for the 57 individuals from the two stocks, showing that the genetic structure was unitary for PL stock but complex for QD stock.     

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.