A new system for the culture and stock enhancement of sea cucumber,Apostichopus japonicus (Selenka),in cofferdams

A new multilayer, plate‐type system for the culture and stock enhancement of sea cucumbers in cofferdam was developed. To optimize and evaluate the system, four experimental designs were implemented using polyethylene (PE)‐corrugated sheets of various colours, interval spacing and shapes/styles. Results showed that a system equipped with black PE‐corrugated sheets attracted more animals than either blue, green, transparent or a selection of mixed sheets (six transparent sheets in the upper layer and five black sheets in the lower layer) (P<0.05). Also, more animals gathered in the system with oblique‐angled sheets (30° to the base plate) than either a wavy (the bottom and every second sheet was at an angle of 10° to the base plate) or parallel arrangement (P<0.05), and more animals assembled in the system with 2 cm between sheets than spacings of 3, 4 or 5 cm (P<0.05). As expected, the upper layers of the systems attracted more animals than lower layers in most cases except for those with transparent and mixed oblique‐angled sheets with a 3 cm spacing (P<0.05). Thus, a system with black, oblique‐angled‐corrugated sheets and 2 cm spacing is recommended for Apostichopus japonicus (Selenka) culture and stock enhancement in cofferdams or ponds.     

Suspended culture of the sea cucumber Apostichopus japonicus below a Pacific oyster raft – potential for integrated multi‐trophic aquaculture

To discuss the possibility of co‐culturing Pacific oyster with the sea cucumber Apostichopus japonicus, a field experiment was conducted in an oyster farm. Apostichopus japonicus juveniles (mean wet weight, 0.08 g) were cultured below an oyster raft and at a control station for 216 days, and the wet weight and stable isotope ratios (δ¹³C, δ¹⁵N) were analysed together with settling organic matter (OM) collected using sediment traps. All sea cucumbers cultured below the raft survived (survival rate, 100%), while at the control station one individual disappeared (96%). During 216 days, the juveniles at the oyster and control stations grew to a mean weight of 5.5 and 2.6 g, attaining respective specific growth rates of 2.0% and 1.6% (paired t‐test, P < 0.001). Settlement rates of carbon and nitrogen at the oyster station were ~5 times larger than those at the control station. The stable isotope analysis showed that settling OM at both stations originated from coastal phytoplankton and that phytoplankton represented the primary food source for A. japonicus. The rapid growth of A. japonicus at the oyster station was concluded to be due to the abundant supply of oyster biodeposits, which could be ingested by this species.     

Microbial communities in sea cucumber (Apostichopus japonicus) culture pond and the effects of environmental factors

This study investigated microbial community composition as well as their correlation with environmental factors of Apostichopus japonicus culture ponds in northern China by 16S rRNA gene amplicon sequencing. The results showed that microbiota richness varied consistently with diversity in the pond ecosystem. Microbiota richness and diversity were highest in sediment, followed by gut of A. japonicus and water. The dominant bacterial phylum in the pond ecosystem is Proteobacteria. Gammaproteobacteriaeria and Flavobacteria are two dominant bacterial classes in the ecosystem. There is significant difference (p < 0.05) between dominant bacterial communities at the levels of order, family and genus. There is also remarkable regional difference (p < 0.05) between microbial community composition in the pond ecosystems. Specifically, microbial community composition in Changhai and Yingkou show a high similarity, so do those of Laoting and Rushan. According to the redundancy analysis of the microbial community composition and pond environmental factors, chemical oxygen demand is the dominant environmental factor determining microbial community composition in pond water; sulphide has the greatest influence on the microbial community composition in pond sediment; the rest of environmental factors have varied influence on microbial community composition in pond ecosystems.     

Effects of diatom concentration in prepared feeds on growth and energy budget of the sea cucumber Apostichopus japonicus (Selenka)

Eight test diets were used in the experiment: seven fresh diatom (Cylindrotheca fusiformis) diets in which diatom occupied 100%, 83.3%, 50%, 33.3%, 20%, 14.3% and 11.1% in dry matter of the feeds (marked as D100, D83, D50, D33, D20, D14 and D11), and a formulated diet (70% Sargassum thunbergii powder, 20% sea mud and 10% fish meal, based on dry weight, marked as ST). The results showed that the specific growth rates (SGRs) of the animals fed diets D33, D20, D14 and D11 were not significantly different from the SGR of those fed diet ST. However, the energy allocated to growth for the animals fed with diet D14 was twice that of diet ST. For sea cucumbers fed diet D14 the largest expenditure part was allocated to respiration (55.4%), but for those fed diet ST, the largest part was allocated to faeces (62.7%). The organic content of diet D14 was also much lower than that of diet ST. Frozen diatom diets containing 14% (dry matter weight) fresh diatom could be an environmentally friendly feed as an alternative option for macroalgae powder.     

Requirement of vitamin E of growing sea cucumber Apostichopus japonicus Selenka

This feeding trial was conducted to determine the vitamin E requirement of growing sea cucumber Apostichopus japonicus Selenka. Six isonitrogenous and isoenergetic experimental diets were formulated to contain graded levels of vitamin E (6.7, 81.2, 159.3, 237.8, 314.6, 395.9 mg/kg diet). Each diet was assigned randomly to 30 growing sea cucumber with initial body weight 15.43 g in triplicates for 8 weeks. Survival rate was not affected by dietary vitamin E; meanwhile, both the weight gain (WG) and specific growth rate were presented the trend of increasing first and then kept stable. With the increasing of dietary vitamin E, crude lipid content of body wall was evaluated firstly and dropped afterwards. When dietary vitamin E contents were lower than 159.3 mg/kg, vitamin E contents of body wall were increased by dietary vitamin E levels, but there were no more differences when dietary vitamin E higher than 159.3 mg/kg. There were minor effects on digestive enzymes of intestine by dietary vitamin E. Contents of malondialdehyde were decreased, while the total superoxide dismutase activity was increased first and then decreased with the increasing of dietary vitamin E. Activities of intestinal alkaline phosphatase were increased, and glutamic oxaloacetic transaminase were decreased first and then increased with the increasing of dietary vitamin E. In conclusion, analysis by a linear regression equation of WG or vitamin E contents in body wall indicated that the optimum requirement of vitamin E for growing sea cucumber (initial body weight 15.43 g) was 165.2–187.2 mg/kg diet.     

Fatty acid composition in sea cucumber Apostichopus japonicus fed with microbially degraded dietary sources

Fatty acids (FAs) in the body walls of sea cucumber, Apostichopus japonicus, reared under diet‐administered conditions were analysed to determine the accumulative status of dietary FAs. Four test diets different in diet source and microbial degradation, that is, a diet composed of powdered pennate diatoms (diet PD), one microbially degraded of diet PD (diet PDM), one composed of brown algae [tangle weed, Saccharina (Laminaria) longissima (Miyabe); diet BA], and one microbially degraded of diet BA (diet BAM), were fed to juvenile A. japonicus, respectively, for 2 months and the FA composition of the juvenile body wall was determined. FAs in body wall were influenced and changed by diet source and microbial degradation of diet. Degradation significantly promoted juvenile's growth and increased iso‐15:0 in the body wall; however, iso‐15:0 was not a growth factor. FA 20:4n6 and 20:5n3 were dominant in all groups despite being a minor dietary constituent, suggesting that these FAs can be readily accumulated or synthesized in the animal.     

An annual faecal 16S amplicon sequencing of individual sea cucumber (Apostichopus japonicus) demonstrates the feeding behaviours against eukaryotes in natural environments

To fill in the gaps in knowledge as to how individual sea cucumber Apostichopus japonicus behave consuming eukaryotic food sources in natural environments, eukaryotic communities in the faeces of sea cucumbers and sediments were analysed through one whole year based on 16S rRNA gene sequencing of the organelle genomes. A total of 390 eukaryotic features were obtained, and 99.7% of the features were assigned to chloroplasts. The eukaryotic communities in faeces and sediments showed seasonal fluctuations through one whole year based on Bray‐Curtis distance and community composition. Comparison of eukaryotic communities between faeces and sediments showed that 12 families including Chaetocerotaceae and Laminariaceae were more abundant in faeces than in sediments, suggesting that sea cucumbers may choose sediment containing these algal taxa more often compared with others in natural environments. All features of Laminariaceae were assigned to Saccharina japonica, which is consistent with the fact that this alga is one of the most suitable diets in the aquaculture of A. japonicus. Assessments of individual 16S amplicon sequences of both faecal and sediment samples could be an alternative tool to help us understand dynamic feeding behaviours of sea cucumber populations in contributing to bioresource conservation and development of a superior approach to aquaculture.     

Effects of kelp residues fermented with probiotics on the culture of sea cucumber,Apostichopus japonicus

The kelp aquaculture production in China is the largest in the world, and a large amount of kelp residue is produced by kelp processing. Kelp residues contain substantial quantities of crude fibre, protein, and residual alginic acid, and may be used as feedstuff for aquaculture animals. In this study, we used probiotics to ferment kelp residues to improve kelp nutrient content and then fed the fermented kelp to the sea cucumber, Apostichopus japonicus. To study the effect of fermented feed on sea cucumber, its growth performance, digestive enzyme activity, diversity of intestinal microbiota and water quality of the sea cucumber culture water were determined. Growth performance of sea cucumber fed with fermented feed significantly (p < .01) increased when compared with sea cucumber fed with formulated feed. Amylase, cellulose and alginase activities were significantly (p < .01) higher in the fermented feed group when compared with the formulated feed group. The total number and diversity of intestinal microbiota showed a significant increase in sea cucumbers fed with the fermented feed. The water quality of the fermented feed group showed much lower ammonia and nitrite (<0.050 mg/L) levels when compared with the formulated feed group. These results suggest that kelp residues fermented with probiotics enhance the growth, digestive enzyme activities and intestinal microbiota of sea cucumbers and improve the culture water quality. Fermented kelp residues are a new supplementary nutrient source for sea cucumbers and may be applicable to other animal aquacultures.     

Immune responses of the sea cucumber Apostichopus japonicus to stress in two different transport systems

We report the effect of transport stress on the immune responses of the sea cucumber Apostichopus japonicus. The total coelomocytes counts, phagocytosis activity, reactive oxygen species production, acid phosphatase activity, phenoloxidase activity and catecholamine levels were measured in A. japonicus following both damp and water transportation methods. With both transport methods, stress resulted in significant elevation of catecholamine levels. Total coelomocytes counts increased following transportation, whereas phagocytic activity and reactive oxygen species production decreased. Damp transport significantly depressed acid phosphatase and phenoloxidase activity in A. japonicus. However, there were no significant differences in acid phosphatase and phenoloxidase activity after water transportation. These results show that transport stress induces modifications in the immune system of A. japonicus and that damp transport elicited more severe stress than water transport. This study increased understanding of the mechanism of transport stress inducing neuroendocrine responses and immune modification in A. japonicus. The information obtained should be helpful to optimize transport process in sea cucumber culture.     

A preliminary identification of genomic loci for body colour variation in the sea cucumber Apostichopus japonicus

The sea cucumber Apostichopus japonicus is one of the most economically important aquaculture species in China, which shows a distinct characteristic of body colour variation. A number of purple variants were obtained in the coast of China, which shows great potential to develop a niche market. In the present study, to understand the genetic basis of green‐purple colour variation, a genome‐wide association study based on the 2b‐RAD sequencing was conducted. As a result, 8,795 genome‐wide single nucleotide polymorphism (SNP) markers were obtained and then applied to the association analysis. Ten most associated SNP loci with p‐value <1.0 × 10^–8 were obtained, and they were assigned to 4 chromosomes (chr17, chr21, chr15 and chr05). In genomic regions neighbouring to those SNPs, 33 candidate genes with annotation were obtained and the endothelin‐converting enzyme‐1, which was reported to be involved in pigmentation variation, was identified. To our knowledge, this is the first report of the genetic locus for body colour variation in this species. These findings will be useful for the fine mapping of the determining gene for body colour variation of A. japonicus.     

Optimum dietary protein level for growth and coelomic fluid non‐specific immune enzymes of sea cucumber Apostichopus japonicus juvenile

A feeding trial was conducted to determine the optimum dietary protein level of sea cucumber Apostichopus japonicus juvenile focusing on growth performance and non‐specific immune response. Diets with seven crude protein levels (42.0, 108.9, 155.2, 216.7, 258.0, 313.3 and 357.5 g kg^?1) were fed to sea cucumber juveniles (1.05 ±0.01 g) once a day for 100 days. More than 70% survival was observed, and there was no significant difference among all treatments. The sea cucumbers fed diets containing 108.9 g kg^?1 crude protein showed significantly (P < 0.05) higher body weight gain than those of the sea cucumbers fed diets containing 42.0, 216.7, 258.0, 313.3 and 357.5 g kg^?1 crude protein. No significantly differences (P > 0.05) were observed in moisture, crude protein, crude lipid, ash and carbohydrate content of the body wall among all treatments. The coelomic fluid catalase activity of the sea cucumbers generally increased with increasing dietary protein levels. Therefore, the acid phosphatase, superoxide dismutase and lysozyme activity increased with increasing dietary protein levels at first and decreased subsequently. The relationship between dietary protein levels and body weight gain was analysed by a second‐order polynomial regression analysis model. The result indicates that the optimum dietary protein level for sea cucumber juveniles is 135.4 g kg^?1.     

Time‐series response of immune enzymes and catecholamines in juvenile sea cucumber Apostichopus japonicus during long‐term live transport

Sea cucumbers are free of protective exoskeleton and capable of autolysis under stress conditions. The live transport of small‐size juveniles to grown‐out sites induces hypoxia and congesting stress. The transport process is essential to production of outdoor healthy seedlings. Here, we evaluated the effects of damp transport and water transport on Apostichopus japonicus, an economic species inhabiting along the Asian coast. The survival after transport and immune performance of small‐size juveniles (0.05–0.1 g of immersed weight) were monitored during distinct duration of transport (1, 3, 5, 8 and 12 hr). The results revealed that there were no significant differences between the two transport systems within 5 hr. Given the survival for recovery of 48 hr and responses of immune enzymes and catecholamines during transport, water transport was less detrimental to sea cucumbers than damp transport when the transport duration was less than 8 hr. Once the duration prolonged to 12 hr, these immune parameters changed irregularly and the survival declined drastically. Water transport with a notably higher survival for juveniles was the more appropriate manner for long‐time transport. These findings provide insights into long‐term transport of small‐size A.  japonicus juveniles and might be beneficial for production of ecologically healthy seedlings.     

Effects of stocking density and body physical contact on growth of sea cucumber,Apostichopus japonicus

The present study was conducted to investigate the effects of physical contact on the growth of Apostichopus japonicus through various stocking densities, group culture and physical isolation culture. The results showed that the specific growth rates (SGR), the feed conversion efficiencies and the energy used for growth of the animal decreased with the increase in stocking density, while the coefficient of variations (CV) in group culture treatments increased. In high density treatments (4, 6 ind./40 L), the SGRs in isolated culture treatments were not different to those in group culture treatments of the same density, however, the CVs in the latter (58.0% and 60.4%, respectively) were significantly higher than those in the former (37.8% and 35.8%, respectively). The CV difference between group one and isolated one of the same density (20.2% and 24.7%, respectively) was less than that in single individual culture (32.7%), indicating that the effect of physical contact on growth variation was weaker than that of genetic factor. This study proved that the individual growth variation in sea cucumber resulted mainly from genetic factor and physical contact, while some chemical mediator might also be the factor affecting the growth rate of sea cucumber.     

Trial grow‐out culture of sea cucumber Holothuria scabra in sea cages and pens

Field trials were conducted to determine the effects of size, grow‐out period and site on the growth and survival of Holothuria scabra. Juveniles were reared in sea cages in two sites for 2 months during two different times of the year. Juveniles from Experiment 1 were subsequently grown in sea pens for 8 months in one site. The significantly lower survival particularly of the small‐sized group (<0.1 g) in Pilar is likely due to reduced water and sediment quality because of proximity to fish‐farming activities. The importance of grow‐out period was evident in the significant differences in weight increments in all size groups and biomass in the small‐sized group (P < 0.10) in Lucero during Experiment 2 when water temperature range and incidence of rainfall were higher. The decrease in the average weight of adults during the second half of the grow‐out period may be due to the interplay of the reduction in salinity, onset of sexual maturity and biomass exceeding carrying capacity in the pens. The estimated biomass threshold in the sea cages and pens in Lucero were 300 g m^?2 and 380 g m^?2 respectively. Implications of the results on the viability of small‐scale pen grow‐out culture are discussed.     

Utilization of dietary carbohydrates by sea cucumber Apostichopus japonicus (Selenka) as indicated by carbon stable isotope analysis

Carbon stable isotopes were used as trophic markers to investigate the utilization of dietary terrestrial‐source carbohydrates by sea cucumber Apostichopus japonicus. Sea cucumbers were fed by five different types of diets with the ingredients containing Sargassum muticum either without starch or with one of the four starches including corn starch, sweet potato starch, tapioca starch and potato starch. After the 70‐day feeding trial, the carbon isotopic compositions of A. japonicus appeared to reflect those of corresponding dietary components. The average contribution of corn starch (22.0%) to the growth of A. japonicus was slightly higher than the expected contribution (20%). While the proportional contributions of sweet potato, tapioca and potato starches (6.0%, 7.0% and 4.0%, respectively) were all considerably lower relative to the expected contributions. These results indicated that A. japonicus could utilize corn starch more efficiently than sweet potato, tapioca or potato starch. Moreover, A. japonicus fed diet containing corn starch showed the highest specific growth rates which were significantly higher than those fed diets containing potato or tapioca starch. The results of the present study suggested that the corn starch could be used as dietary carbohydrate source in the artificial feeds for A. japonicus farming.     

Effect of replacing dietary fish meal with silkworm (Bombyx mori L) caterpillar meal on growth and non‐specific immunity of sea cucumber Apostichopus japonicus (Selenka)

The effect of dietary substitution of silkworm (Bombyx mori L) meal (SM) for fishmeal (FM) on the growth performance and non‐specific immunity of sea cucumber (Apostichopus japonicus) (initial weight: 12.8 ± 0.16 g) was determined. Four isonitrogenous and isocaloric diets were formulated: Diet 1, which served as the control diet, contained 5% FM; Diet 2 contained 3.75% FM and 1.25% SM; Diet 3 contained 2.5% FM and 2.5% SM; and Diet 4 contained 5% SM. Other ingredients in each of the four diets were kept in the same proportion. After 8 weeks of feeding, the results showed that sea cucumbers fed Diet 2 had 18.7% increases in weight over those fed the control diet, but no significant difference was observed. No obvious difference in body wall composition was detected among the sea cucumbers fed the four different diets. Immunity analysis indicated that phagocytosis and serum alkaline phosphatase activity were not significantly (P > 0.05) affected when FM was partially or completely replaced with SM. Serum lysozyme activity of sea cucumbers fed Diet 4 showed a significant (P < 0.05) growth increase compared with those fed control diet. The results revealed that SM could be an effective substitute for FM in sea cucumber diet.     

Seasonal variations of immune parameters in the coelomic fluid of sea cucumber Apostichopus japonicus cultured in pond

In order to understand the effects of seasonal change on the immunity of sea cucumber Apostichopus japonicus cultured in pond, A. japonicus with body weight of 12.2 ± 4.5 g (sample A) and 32.6 ± 7.1 g (sample B), respectively, were collected monthly and randomly from a typical pond during a year cycle and employed for the evaluation of immunocompetence. Simultaneously, the environmental factors in the pond including water temperature, pH, salinity and dissolved oxygen (DO) were measured using a handheld multiparameter meter. The activities of acid phosphatase (ACP), alkaline phosphatase (ALP), lysozyme (LYZ), phenoloxidase (PO), superoxide dismutase (SOD), catalase (CAT) and myeloperoxidase (MPO) in the coelomic fluid were selected for the evaluation of A. japonicus immunocompetence and determined using biochemical methods. The results showed that in both samples, the activities of all determined enzymes had small values in winter and early spring, and LYZ, CAT and MPO activities also presented small values in summer, suggesting that pond‐cultured A. japonicus underwent immunosuppression twice during a year cycle, and the immunosuppression occurred in winter and early spring was more severe than that occurred in summer. In addition, most of the determined enzymes showed different variations between the two samples, and had significantly negative correlation with protein concentration, which was significantly and negatively correlated with water temperature, revealing that body weight and water temperature might have crucial effects on the immunity of A. japonicus cultured in pond.     

Carbohydrate and amino acids metabolic response to heat stress in the intestine of the sea cucumber Apostichopus japonicus

The sea cucumber Apostichopus japonicus is an economic species mainly distributed along the coast of northern China, south‐eastern Russia, Japan, Republic of Korea and Democratic People's Republic of Korea. Aquaculture industry of A. japonicus has been facing severe challenge of high temperature. In this study, we studied the mRNA expression profiles of eight key metabolic enzymes involved in carbohydrate and amino acids metabolism in A. japonicus under heat stress. The expression of hexokinase, pyruvate kinase and malate dehydrogenase showed downregulated response to heat stress, while the expression of glutamate dehydrogenase, alanine transaminase and branched‐chain aminotransferase showed upregulated response. In addition, the expression of lactate dehydrogenase and glutamate synthase showed no significant difference. We also applied ¹H NMR‐based metabolomics to investigate metabolic changes in the intestine tissue of A. japonicus under heat stress, the results of which revealed nine increased and 10 decreased metabolites in the heat stress group. These response genes and metabolites have potential to become markers for identifying severity of heat stress. More importantly, our findings suggest significant links between gene expression and metabolites changing, highlighting regulation networks of carbohydrate and amino acid metabolism in heat‐stressed A. japonicus.     

Effects of dietary n‐3 PUFA supplements on composition of n‐3 PUFA and expression of fatty acid elongase 5 (AJELOVL5) in sea cucumber,Apostichopus japonicus

This is the first comprehensive study on the effect of dietary polyunsaturated fatty acid (PUFA) levels on the expression of fatty acid elongase 5 (AJELOVL5), PUFA composition, and growth in juvenile sea cucumbers. The specific growth rate (SGR_w) was improved in n‐3 PUFA‐rich diets compared to low n‐3 PUFA diets. AJELOVL5 expression was apparently upregulated in juveniles fed lower PUFA diets relative to higher PUFA diets, with higher expression in the body wall and respiratory tree of juveniles fed diets without ɑ‐linolenic acid (ALA, 18:3n‐3) compared to juveniles fed higher ALA level diets; similar results were also detected in juveniles fed diets with lower eicosapentaenoic acid (EPA, 20:5n‐3), docosahexaenoic acid (DHA, 22:6n‐3), and none of ALA, EPA, or DHA respectively. The concentrations of ALA, EPA, and DHA in tissues were positively related to the content of dietary corresponding PUFA, with higher ALA content in juveniles fed diet ALA_12.71 than in the ALA_7.46 and ALA₀ groups. Similar results were also obtained in sea cucumber fed diets enriched with either EPA or DHA. Interestingly, considerable levels of EPA and DHA were found in the tissues of juveniles fed diets of CK₀ and DHA₀, with no specific input of EPA or DHA, showing that the sea cucumber was capable of biosynthesizing EPA and DHA from their corresponding precursors as ALA and linoleic acid (LA, 18:2n‐6).