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.     

Large‐scale mortality and limited expression of heat shock proteins of aestivating sea cucumbers Apostichopus japonicus after acute salinity decrease

This study deals with the mortality and related physiological responses of aestivating sea cucumber Apostichopus japonicus to acute salinity decrease. Aestivating and active sea cucumbers were exposed to a decrease in salinity (from 30 to 20 psu) at a rate of 2.5 psu every 6 h, and then maintained at 20 psu for 96 h. The mortality of aestivating sea cucumbers was ~30%, which was significantly higher than that of active sea cucumbers (~10%). This result indicated that sea cucumbers in aestivation were more susceptible to hypo‐salinity stress. To elucidate the underlying physiological mechanisms, the osmotic pressure in coelomic fluid and the levels of hsp70 and hsp90 mRNA in aestivating and active sea cucumbers were measured. No significant difference in osmoregulation was observed between the two groups. The osmotic pressure of coelomic fluid in both groups changed with decrease in ambient salinity. There were significant differences in the time course and magnitude of hsp70 and hsp90 expression between the two groups. After exposure to decreased salinity, aestivating sea cucumbers showed a delayed up‐regulation of hsp70 and hsp90 expression compared with animals in active state, and these levels decreased rapidly to control values. The expression of hsp70 and hsp90 in aestivating sea cucumbers were significantly lower than those in active sea cucumbers after salinity change. The differences in hsp70 and hsp90 expression between the states may partly explain the higher mortality of sea cucumbers in aestivation when exposed to low salinity.     

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.     

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.     

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.     

Feasibility of Offshore Co‐culture of Abalone,Haliotis discus hannai Ino,and Sea Cucumber,Apostichopus japonicus,in a Temperate Zone

Feasibility of offshore co‐culture of abalone and sea cucumber was investigated in Northern China. Survival and growth of abalone, Haliotis discus hannai Ino, and sea cucumber, Apostichopus japonicus, co‐cultured in abalone cages from suspended longlines, in the offshore area, were examined. Abalone and sea cucumbers were co‐cultured at density ratios of both 3:1 and 6:1 for 1 yr. Abalone were fed with fresh kelp and no additional feed was given to sea cucumbers. Survival of abalone and sea cucumber was 100% for all treatments. Abalone and sea cucumber grew well; the body weight (BW) of abalone and sea cucumber was nearly doubled and had reached a commercial size. There were no significant differences in the growth rates for both abalone and sea cucumber between the two density treatments. The specific growth rate of BW of abalone (SGRbw) was highest in June, with a value of 0.536%/d. Growth rate of sea cucumber (SGRsc) was highest in December, reached 1.84%/d, with an annual average SGRsc of 0.182%/d. Results suggested that the offshore co‐culture of abalone and sea cucumber was feasible offshore. The co‐culture of abalone with sea cucumbers may provide an additional valuable crop without additional financial input.     

Effect of dietary carbohydrate levels on growth performance,non‐specific immune enzymes and acute response to low salinity and high temperature of juvenile sea cucumber Apostichopus japonicus

This study was performed to determine the optimum dietary carbohydrate (CHO) levels of sea cucumber, based on the parameters of growth, digestive enzymes, digestibility, non‐specific immune enzymes and acute low‐salinity (20 g/L) stress and high‐temperature (30°C) stress tolerance. Diets with eight different CHO (dextrin) levels (32.9, 107.6, 192.5, 257.2, 316.8, 428.0, 482.4 and 572.8 g/kg) were fed to sea cucumber juveniles (0.49 ± 0.01 g) for 60 days. Significant higher amylase activity was observed in sea cucumbers fed diet with CHO ranging between 32.9 and 192.5 g/kg than that of other treatments (p < .05). The sea cucumbers fed with 192.5 g/kg CHO showed significantly higher acid phosphatase activity than the treatments of 482.4 and 572.8 g/kg CHO (p < .05), and significantly higher alkaline phosphatase activity than other treatments (p < .05, except 257.2 g/kg). The treatments of 428.0–572.8 g/kg were found significantly lower values than other treatments in apparent digestibility coefficients for dry matter and crude protein (p < .05). The sea cucumbers fed with 192.5, 257.2 and 316.8 g/kg CHO showed better tolerance to high‐temperature (30°C) and low‐salinity (20 g/L) stress than other treatments. In brief, the optimal dietary CHO level for the growth of juvenile sea cucumber is 177.96 g/kg. However, excessive CHO will inhibit amylase enzyme activity and decrease digestibility, resulting in low growth of sea cucumber.     

Effects of dietary peptide intake on growth,energy budget,body composition and non‐specific immunity of the sea cucumber Apostichopus japonicus (Selenka)

In this study, a 60‐day feeding trial was conducted to determine the effects of feed supplementating the feed of Apostichopus japonicus with peptides on its growth, energy budget, body composition and immune responses. Sea cucumbers were fed with five experimental diets supplemented with different proportions of peptides: 0 (D1), 12.5 (D2), 25 (D3), 37.5 (D4) and 50 (D5) g/kg in basal diet. Our results suggested that specific growth rate (SGR) and ratio of viscera to body wall (RVBW) of sea cucumbers fed with D4 were significantly improved. Relative to D1, ingestion rate (IR) and faeces production rate (FPR) for D3 and D4 were reduced considerably. Notably, the energy intake increased when peptide level increased from 0 to 50 g/kg. Meanwhile, the energy deposited for growth increased, and the energy loss decreased when peptide level increased from 0 to 37.5 g/kg. The sea cucumbers in D4 had the highest level of crude fat and lowest crude ash. The activities of immunoenzyme, such as SOD, CAT, T‐AOC, ACP and AKP, increased with peptide increase. Results suggested that supplementation of the feed with 25–37.5 g/kg peptides could significantly improve the growth performance, body composition and immune capacity of Apostichopus japonicus.     

Effects of differently processed diets on growth,immunity and water quality of the sea cucumber,Apostichopus japonicus (Selenka, 1867)

The type of formulated diet used in aquaculture systems affects the feed efficiency and water quality. In this study, the water quality, growth performance and immunity of the sea cucumber, Apostichopus japonicus (Selenka, 1867), fed diets produced using four different processing methods (extruded pellet diet, cold‐bonded pellet diet, flake diet and mash diet) were quantified for 60 days to identify the optimal feed type for culturing of this species. Sea cucumbers grew faster, the feed conversion ratio was much lower, and immunity indicators were higher when fed the extruded pellet diet compared to the other diets. Sea cucumbers fed the extruded pellet diet had the highest specific growth rate (0.96), whereas A. japonicus fed the cold‐bonded pellet diet had the lowest specific growth rate (0.51). The lysozyme activity of sea cucumbers fed the extruded pellet diet was higher (57.52) than that of A. japonicus fed the cold‐bonded pellet diet (39.22), flake diet (37.32) and mash diet (32.68). When animals were fed the mash diet, the ammonia–nitrogen, nitrite–nitrogen and phosphate productions were higher than in animals fed the other diets. These results indicate that the extruded pellet diet is the optimum feed type for use in the culture 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.     

Effects of l‐tryptophan on the performance,energy partitioning and endocrine response of Japanese sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress

The effects of dietary tryptophan on growth performance, energy budget and endocrine response of sea cucumber Apostichopus japonicus (Selenka) were studied, to test whether the tryptophan could mitigate the crowding stress response of the sea cucumber. Four density treatments of the sea cucumber (i.e. 4, 8, 16 and 32 individuals per 40 L water, represented as L, ML, MH and H) were fed with diets containing 0 (control), 1%, 3% and 5% l ‐tryptophan, respectively, for 75 days. The results showed that the specific growth rate (SGR) and feed conversion ratio (FCE) of A. japonicus decreased with increasing in the stocking densities but increased after supplementation of 1% and 3% tryptophan. The energy allocation of the A. japonicus was affected significantly by stocking density and dietary tryptophan treatments (p < .05). In general, the energy consumed (C) and energy used for growth (G) decreased with the increasing in the stocking densities. The highest proportion of energy deposited to growth occurred in each 3% treatment (L, 6.38%; ML, 6.12%; MH, 4.56% and H, 4.14%), which were significantly higher than the controls (p < .05) respectively. Higher cortisol, glucose and lactate levels of A. japonicus were recorded in the control treatments of MH and H, but l ‐tryptophan supplementation could reduce the levels of cortisol, glucose and lactate of the test animals. The present results proved that the growth performance of the sea cucumber was inhibited by high stocking densities, while dietary supplementation of tryptophan could significantly mitigate the crowding stress, and improve the growth of sea cucumber.     

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 continuous and alternate administration of β‐glucan and mannan‐oligosaccharide on the growth,immunity and resistance against Vibrio splendidus of sea cucumber Apostichopus japonicus (Selenka)

A 4‐week growth trial was conducted to compare the effects of different feeding strategies of dietary immunostimulants on the growth, immunity and resistance against Vibrio splendidus of sea cucumbers Apostichopus japonicus (Selenka). Six feeding strategies were set, including feeding immunostimulants‐free diet continuously (control), feeding dietary β‐glucan (1.25 g kg^?1 diet) continuously, feeding dietary mannan‐oligosaccharides (MOS; 2.00 g kg^?1 diet) continuously, feeding β‐glucan 2 days followed by MOS 5 days alternately, feeding β‐glucan 5 days followed by MOS 2 days alternately and feeding β‐glucan 7 days followed by MOS 7 days alternately. The sea cucumbers fed immunostimulants showed higher specific growth rate (SGR) and lower cumulative mortality than control (P < 0.05). When sea cucumbers were fed with β‐glucan continuously, total coelomocytes counts and superoxide anion were significantly higher than control on the 4th day (P < 0.05). However, these two immune parameters were not significantly higher than those in control after the 18th day (P > 0.05). While sea cucumbers continuously fed MOS, these two immune parameters were not significantly higher than control until the 15th day. All immune parameters of the sea cucumbers fed with β‐glucan and MOS alternately were significantly higher than those in control during the experiment (P < 0.05). The sea cucumbers fed with β‐glucan 7 days followed MOS 7 days alternately showed the highest SGR and second lowest cumulative mortality. It was suggested that this feeding strategy is most suitable for sea cucumbers.     

Effects of dietary protein levels on the activity of the digestive enzyme of albino and normal Apostichopus japonicus (Selenka)

We evaluated the effects of different proportions of dietary protein (5%, 10%, 15%, 20%, 25% and 30% protein) on the activity of digestive enzymes of normal and albino Apostichopus japonicus. The experimental diets were fed for 60 days, the optimal conditions for digestive enzyme activity in sea cucumbers were studied. The optimal temperature for protease was 29.3°C and the optimal pH was 1.8. The optimal temperature for amylase was 34.3°C and the optimal pH was 6.7. The optimal temperature for cellulase was 56°C and the optimal pH was 5.9. The activity of intestinal protease increased at first and then decreased as the proportion of dietary protein increased, reaching the maximum when the proportion of protein was 19.7%. The activity of protease in the intestine of normal sea cucumber was significantly lower than that of albino sea cucumber, and the activity of amylase was significantly higher than that of albino sea cucumber. This study is expected to provide a basis for further explaining the ecological difference of albino and normal A. japonicus.     

Growth performance and digestion improvement of juvenile sea cucumber Apostichopus japonicus fed by solid‐state fermentation diet

In this study, the compound probiotics including Pseudoalteromonas. Sp D11, Bacillus subtilis A142, Saccharomyces cerevisiae Y23 and Lactobacillus plantarum L54 were applied for solid‐state fermenting the artificial diet of juvenile sea cucumber Apostichopus japonicus. The quality of solid‐state fermented (SSF) feed and its potential effects on growth performance and digestive enzyme activities were investigated. The results showed that, compared to unfermented diet, SSF feed had a better palatability quality; meanwhile, a higher ratio of crude protein, free amino acids and reducing sugars and a lower ratio of crude fibre, crude ash and alginic acid were found in SSF feed. The results of the feeding trial showed that feeding SSF feed could improve growth performance of sea cucumbers more than those fed with the unfermented diet. The results of digestive enzyme assays showed that feeding SSF feed to animals could significantly improve activities of amylase, cellulase, alginic acid enzyme and protease, which implied that fermented feed could improve digestibility of A. japonicus. In summary, the SSF feed fermented by compound probiotics had better palatability and nutrition value than unfermented feed. And feeding SSF feed could significantly improve the growth performance and digestion ability for sea cucumbers in the phases of nursery and grow‐out.     

亚硒酸钠对刺参免疫反应中体腔液酶活力的影响

为了合理评价硒对水产养殖刺参的作用,实验检测和观察了室内模拟亚硒酸钠处理及病菌感染条件下刺参体腔液免疫酶的变化、组织病理变化和相对保护率。结果表明,随着亚硒酸钠处理强度的增加,刺参体腔液中超氧物岐化酶(SOD)、谷胱甘肽过氧化物酶(GP-X)、溶菌酶(LYZ)、酚氧化酶(PO)及碱性磷酸酶(ALP)活性升高;在同时感染病菌的情况下,SOD、GP-X及LYZ活性表现为先升高后下降的趋势,PO和ALP活性则持续上升,但随亚硒酸钠浓度升高,ALP活性增加的幅度减少,而较高亚硒酸钠浓度时累计发病率上升,相对保护率下降。研究结果表明,适宜浓度的亚硒酸钠可增强刺参对病菌感染的免疫力,而病菌感染可以加剧亚硒酸钠胁迫对刺参造成的免疫功能损伤,所以适度利用硒可减轻刺参养殖病害的发生。     

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.     

Effects of light intensity on larval development and juvenile growth of sea cucumber Apostichopus japonicus

The growth rate, survival rate, development and setting rate of larval sea cucumber Apostichopus japonicus were measured under four light intensities (0, 50, 500 and 2,000 lx), and the growth rate and metabolism of the juvenile sea cucumbers were investigated under four light intensity treatments (0, 100, 1,000 and 2,500 lx). The light requirements (i.e. intensity) of the sea cucumber changed as they grew. Better growth performance, development and survival rate were observed in embryos and larvae under 500 lx treatment. However, reduced light intensity (50 lx) increased the settlement rate of the larvae. The highest specific growth rate occurred in juvenile sea cucumbers under 1,000 lx. The sea cucumbers in this group also had the lowest oxygen consumption rate and ammonia excretion rate, but the highest O:N ratio, indicating that the optimal light intensity for the juvenile sea cucumber culture was 1,000 lx. Therefore, appropriate light intensities were suggested to provide larval and juvenile sea cucumbers with better growth and development conditions.     

Effectiveness of l‐ascorbyl‐2‐polyphosphate as an ascorbic acid source for sea cucumber,Apostichopus japonicus

A feeding trial was conducted to determine the adequate dietary ascorbic acid (AsA) levels and the effects on growth, meat quality and antioxidant status of sea cucumber (10.04 ± 0.06 g), Apostichopus japonicus. l ‐ascorbyl‐2‐polyphosphate (35% AsA equivalent) was supplemented separately to the basal diet to obtain five AsA levels, 0, 598, 1473, 4676 and 14340 mg kg^?1 diet respectively. After 60‐day feeding trial, the sea cucumbers fed diets containing 598 and 1473 mg AsA kg^?1 showed significantly higher (P < 0.05) body weight gain and specific growth rate values than the sea cucumbers fed control diets. The sea cucumbers fed diets containing 1473 and 4676 mg AsA kg^?1 showed significantly higher (P < 0.05) hydroxyproline contents than those of the sea cucumbers fed diets containing 0 and 598 mg AsA kg^?1. Antioxidant enzymes such as total antioxidant capacity, superoxide dismutase and glutathione peroxidase showed increasing trends with the increasing dietary AsA levels, but no significant differences (P > 0.05) were observed when the sea cucumbers fed diets with high dietary AsA levels. The content of malondialdehyde had the opposite trend of antioxidant enzymes. In conclusion, the adequate dietary AsA level focusing on growth performance of sea cucumber is between 598 and 1473 mg kg^?1 diet. Furthermore, high level of dietary AsA (between 598 and 4676 mg kg^?1 diet) improved meat quality and antioxidant status.