Regulation of dietary astragalus polysaccharide (APS) supplementation on the non-specific immune response and intestinal microbiota of sea cucumber Apostichopus japonicus.

Astragalus polysaccharide (APS) plays important roles in antibacterial, antiviral and antiparasitic activities in mammals, birds and aquatic animals. However, the relationship between non-specific immune responses and intestinal microbiota in sea cucumber (Apostichopus japonicus) after dietary APS supplementation has not been reported to date. Here, the effect of dietary APS supplementation on the non-specific immune response and intestinal microbial composition and species distribution of sea cucumber was explored. We found that although there was no significant effect on sea cucumber growth, the enzymatic activity and expression level of immune- and antioxidant-related genes changed after dietary APS supplementation. Furthermore, the intestinal microbial composition and species distribution of sea cucumber were different at the phylum and genus levels after dietary APS supplementation. The phyla Proteobacteria and Bacteroidetes were significantly different between the APS2 group and CK group. The results of PCA and PCoA analysis also showed that the APS2 group was significantly different compared to the other groups. Finally, analysis of the relationship between non-specific immune responses and the intestinal microbiota showed that the expression level of NF-κB was significantly correlated with intestinal microbiota at the genus level. This finding suggests that dietary APS supplementation might affect the non-specific immune response and intestinal microbiota of sea cucumber through the NF-κB signalling pathway; the appropriate added level was 800 mg/kg. Taken together, our results lay a foundation for further understanding the relationship between non-specific immune responses and intestinal microbial of sea cucumber.

The influence of three antibiotics on the growth, intestinal enzyme activities, and immune response of the juvenile sea cucumber Apostichopus japonicus selenka.

The global abuse and misuse of antibiotics in the treatment and prevention of bacterial infections has resulted in the ubiquitous existence of these drugs in aquatic environments, which causes frequent antimicrobial resistance and pollution in ecosystems. However, the chronic effects of antimicrobial agents on aquatic animal growth and health have not been fully evaluated. In the present study, three typical antibiotics (tetracycline, erythromycin, and norfloxacin) were administered orally to juvenile sea cucumbers Apostichopus japonicus for 45 days, to mimic the long-term use of antibiotics. As a result, tetracycline and erythromycin promoted the growth and digestive activity of lipase, pepsin, and trypsin, but norfloxacin did not show significant prompting effect on digestive activity and even retarded the weight gain of the sea cucumbers. The mortality was higher in antibiotic treated groups between the 2nd and 4th days after challenge with Vibrio splendidus. At the same time, lower immune-related parameters were found in antibiotic feeding juveniles, suggesting that the use of antibiotics might weaken the immune defense system of sea cucumbers. This study revealed that antibiotic administration could facilitate the growth of sea cucumbers to varying degrees yet coupled with high risks of impaired immune function and compromised disease resistance.

Immune-related proteins detected through iTRAQ-based proteomics analysis of intestines from Apostichopus japonicus in response to tussah immunoreactive substances.

Apostichopus japonicus is a species of sea cucumber that is extensively bred as a marine delicacy because of its high nutritive and medicinal value. Immunostimulants are usually used to enhance the immunity of sea cucumber against diseases, but the physiological function of immunostimulants is poorly understood. In this study, we fed A. japonicus individuals with a diet supplemented with different concentrations of tussah immunoreactive substances (TIS), and then subjected their intestines to iTRAQ-based proteomic analysis. A total of 51 differentially expressed proteins were detected in response to TIS, 13 proteins were upregulated, while 38 proteins were reduced. These proteins are involved in phagocytosis, tissue protection, cell apoptosis and energy metabolism. Among these 51 proteins, 7 proteins (GLO2, ACOX, CTTN, MARK, FADD, CSTA and CASP6) related to immunity with functional annotation in sea cucumber were further analyzed. In addition, the upregulated expression of 4 immune-related proteins (GLO2, ACOX, CTTN and MARK) was validated by qRT-PCR. The findings of this study gave further insight into the mechanism by which TIS might enhance the immunity of A. japonicus.

Effects of l-tryptophan on the growth, intestinal enzyme activities and non-specific immune response of sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress.

In order to reveal the effects of l-tryptophan (Trp) on the physiology and immune response of sea cucumber (Apostichopus japonicus Selenka) exposed to crowding stress, four density groups of sea cucumbers (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, 1, 3 and 5% l-tryptophan respectively for 75 days. The results showed that the specific growth rates (SGR) of the sea cucumber fed with diet with 3% Trp (L, 2.1; ML, 1.76; MH, 1.2; H, 0.7) were significantly higher than those fed with basal diet without Trp supplementation (P < .05). Peak amylase activity occurred at H stress density at 3% dietary Trp. Trypsin activity was higher in diet 3% in ML and MH densities than the controls, which increased by 66.4% and 53.8%. However, the lipase activity first increased and then decreased from the stocking density L to H, with highest values of 3% Trp group showed the highest value than other groups. Compared to those fed with the basal diet, sea cucumber fed diets with Trp (3%) had significantly higher phagocytic activities (0.28 OD540/106 cells, H) in coelomic fluid and respiratory burst activities (0.105 OD630/106 cells, MH) (P < .05). The results suggested that Trp cannot improve superoxide dismutase (SOD) activity at L, ML and MH densities. The alkaline phosphatase activity (AKP) significantly decreased at H stress density. Under the experimental conditions, the present results confirmed that a diet supplemented with 3% Trp was able to enhance intestinal enzyme activities, non-specific immune response and higher growth performance of A. japonicus.

Effects of dietary supplementation of four strains of lactic acid bacteria on growth, immune-related response and genes expression of the juvenile sea cucumber Apostichopus japonicus Selenka.

A feeding experiment was conducted to evaluate the effects of four strains of lactic acid bacteria (LAB) [i.e. Lactobacillus plantarum LL11 (LP), Weissella confuse LS13 (WC), Lactococcus lactis LH8 (LL) and Enterococcus faecalis LC3 (ES)] isolated from marine fish on growth, immune response and expression levels of immune-related gens in body wall of juvenile sea cucumber Apostichopus japonicus. As a result, sea cucumber had better growth performance fed supplementation of LP and ES than the control group (P < .05). Survival rate in each LAB supplementation group was significantly higher than that in control group after Vibrio splendidus challenge (P < .05). In regards to the enzyme activities, LP supplementation significantly imporved the enzyme activities of alkaline phosphatase (AKP) (P < .05), acid phosphatase (ACP) and superoxide dismutase (SOD), ES supplementation significantly imporved AKP activity (P < .05), and WC supplementation significantly imporved ACP activity (P < .05). However, lysozyme (LSZ) activity was not significantly changed in the four LAB supplementation treatments (P > .05). For the gene expression levels, different expression patterns were observed among four groups, heat shock proteins (HSP60, HSP70 and HSP90) and caspase-2 showed dramatic up-regulation at 30 d while NF-kappa-B transcription factor p65 was down-regulated at 15 d and up-regulated at 30 d, and nitric oxide synthase was down-regulated at both timepoints in almost all the four groups. In conclusion, the four LAB strains screened from marine fish supplemented in diets indicated positive effects on immune response for A. japonicus, especially, the L. plantarum LL11 and E. faecalis LC3 indicated better growth performance.

Indole reduces the expression of virulence related genes in Vibrio splendidus pathogenic to sea cucumber Apostichopus japonicus.

Indole is a metabolite of tryptophan that can be synthesized by various bacteria. In the present study, production of indole by Vibrio splendidus Vs was determined using Kovac's reagent, and m/z was further determined by HPLC-MS. Extracellular indole reached a maximum concentration of 160 µM, when OD600 of V. splendidus Vs was approximately 0.9. In addition, glucose could reduce indole level, and 1% (m/v) glucose could reduce the mRNA level of tnaA, the gene encoding tryptophanase, down to 0.2%. To investigate the effects of indole on the mRNA levels of virulence related genes of V. splendidus Vs, mRNA levels of vsm, vsh and ABC respectively related to protease activity, haemolytic activity and ABC transporter ATP-binding protein were determined. Exogenous indole supplemented at a concentration of 125 µΜ could respectively down regulate the mRNA level of vsm, vsh and ABC to 16%, 13% and 11%. Meanwhile, indole could alter the expressions of immune related gene in Apostichopus japonicus. When coelomocytes were co-cultured with exogenous indole at a concentration of 125 µΜ, the mRNA level of Ajp105 and AjLBP/BPI1, were up regulated by 1.6-fold and 2.1-fold, respectively. Combined all the results in our study suggested that indole could alter the expressions of the virulence related genes in pathogenic V. splendidus Vs as well as the immune related genes in A. japonicus.

Synbiotic dietary supplement affects growth, immune responses and intestinal microbiota of Apostichopus japonicus.

A feeding experiment was conducted to investigate the effects of dietary administration of synbiotic with Bacillus lincheniformis WS-2 (CGMCC No. 12813) and alginate oligosaccharides (AOS) on the growth, innate immune response, and intestinal microbiota of the sea cucumber Apostichopus japonicus and its resistance to Vibrio infection. Sea cucumbers were given a control diet (non-supplemented), pro diet (basal diet plus 1 × 109 cfu (g diet)-1B. lincheniformis WS-2), syn diet (basal diet plus 1 × 109 cfu (g diet)-1B. lincheniformis WS-2 and 10 g (kg diet) -1 AOS) or pre diet (basal diet plus 10 g (kg diet) -1 AOS) over a period of 60 days, and the growth performance and various innate immune parameters of the animals were evaluated after 30 and 60 days of feeding. No significant difference in growth performance was observed between the group fed with the syn and the group fed with the pro diet, but both these groups exhibited significant (P < 0.05) enhancement in growth performance compared to the control group. At the same time, both syn and pro diets also resulted in the animals having significantly higher levels of amylase, protease and alginate lyase activities compared to the con diet. Individuals fed with the syn or pro diet showed enhanced levels of various immune enzyme activities, compared to those fed with the con diet. At the end of the growth period, the sea cucumbers were challenged with Vibrio splendidus via intraperitoneal injection. The survival rates of sea cucumbers fed with the syn, pro or pre diet were significantly improved compared to that of sea cucumbers fed with the con diet, with sea cucumbers fed with synbiotic having the highest survival. In addition, increased proportions of Bacillus and Lactococcus were found in the intestinal tract of sea cucumbers fed with the syn diet (9.5% and 7.3%) compared to those of sea cucumbers fed with the pro diet (6.1% and 4.6%), con diet (4.0% and 3.4%), or pre diet (5.2% and 6.8%) after 60 days of feeding. Furthermore, the proportion of Vibrio in the intestinal tracts of sea cucumbers fed with the pro diet (2%) or syn diet (3.1%) was lower than that of sea cucumbers fed with the con diet (5.5%) or pre diet (3.8%), although no significant difference was detected between the pro diet and syn diet groups (P > 0.05). Overall, the results suggested that dietary synbiotic consisting of Bacillus lincheniformis and alginate oligosaccharides (AOS) could have positive benefit for sea cucumber aquaculture.

Effects of tussah immunoreactive substances on growth, immunity, disease resistance against Vibrio splendidus and gut microbiota profile of Apostichopus japonicus.

Tussah immunoreactive substance (TIS) comprises a number of active chemicals with various bioactivities. The current study investigated the effects of these substances on the sea cucumber Apostichopus japonicus. The specific growth rate (SGR) of TIS-fed sea cucumbers was significantly enhanced, whereas no significant difference in SGR was observed between those soaked in antibiotics and those fed with basal diet only. TIS also improved the immune response of the animals when given at a dose of 1.0% or 2.0%, as shown by increased phagocytic, lysozyme, superoxide dismutase, alkaline phosphatase, acid phosphatase, and catalase activities following injection with live Vibrio splendidus. At a dose of 1.0% or 2.0%, TIS significantly enhanced the immune ability (P < 0.05) of the sea cucumbers, but except for lysozyme activity, other immune indices were reduced one day after the animals were injected with Vibrio splendidus. However, the values of these immune indexes were still significantly higher compared to those of the control groups (P < 0.05). Intestinal micro flora counts and high-throughput sequencing showed that dietary TIS could improve the amount of probiotic bacteria, yielding a 6-fold increase in Bacillus and 10-fold increase in Lactobacillus for sea cucumbers fed with 2.0% TIS diet compared to the control. Furthermore, TIS-containing diet also greatly reduced the number of harmful bacteria, with the number of Vibrio in sea cucumbers fed with 1%TIS diet decreased by 67% compared to the control. The results thus indicated that TIS increased the growth of sea cucumbers and enhanced their resistance to V. splendidus infection by improving the immunity of the animals. TIS also improved the gut microbiota profiles of the animals by increasing the probiotics and reducing the harmful bacteria within their guts.

Two proprotein convertase subtilisin/kexin type 9 (PCSK9) paralogs from the tropical sea cucumber (Stichopus monotuberculatus): Molecular characterization and inducible expression with immune challenge.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a multifunctional protein that widely exists in eukaryotic species. In this study, two PCSK9 paralogs, named StmPCSK9-1 and StmPCSK9-2, were identified from the tropical sea cucumber (Stichopus monotuberculatus). The cDNAs of StmPCSK9-1 and StmPCSK9-2 are 1330 kb and 1508 kb in size, respectively. The open reading frames (ORF) for StmPCSK9-1 and StmPCSK9-2 cDNAs are 1128 and 1167 bp in length, encoding the proteins of 375 and 388 amino acids with the deduced molecular weights of 38.76 and 41.07 kDa, respectively. In accord with other members in PCSK9 family, the two StmPCSK9 paralogs possessed the inhibitor_I9 and peptidase_S8 functional domains, seven active sites, a catalytic triad and two calcium binding sites. For the gene structure, the splicing of the two StmPCSK9 paralogs was relatively conserved. In addition, the mRNA expression of StmPCSK9-1 and StmPCSK9-2 was only detected in the sea cucumber intestine and coelomocytes, and the expression levels of both the two StmPCSK9 paralogs were higher in intestine. Moreover, StmPCSK9-2 was found to be a cytoplasm protein without signal peptide, and show no response to the immune challenge. On the contrary, StmPCSK9-1 was a secreted protein and the transcriptional expression of StmPCSK9-1 was significantly up-regulated by lipopolysaccharides (LPS) treatment and slightly down-regulated by polyriboinosinic polyribocytidylic acid [Poly (I:C)] challenge in in vitro experiments performed in the cultural primary coelomocytes, suggesting that the StmPCSK9-1 may play critical roles in the innate immune defense of sea cucumber, S. monotuberculatus, against bacterial and/or viral infections.

Use of phages to control Vibrio splendidus infection in the juvenile sea cucumber Apostichopus japonicus.

In the present study, we isolated 3 bacteriophages with the ability to control Vibrio splendidus, a bacterium known to cause disease in the juvenile sea cucumber. These bacteriophages were designated as vB_VspS_VS-ABTNL-1 (PVS-1), vB_VspS_VS-ABTNL-2 (PVS-2) and vB_VspS_VS-ABTNL-3 (PVS-3). The ability of the 3 phages to inhibit the growth of V. splendidus VS-ABTNL was tested in vitro using each of the 3 phages individually or in the form of a cocktail of all 3 phages in the proportion of 1:1:1. All treated cultures produced a significant (P < 0.05) inhibition of growth of V. splendidus VS-ABTNL compared with untreated V. splendidus VS-ABTNL with the cocktail being superior to any of the 3 phages used individually. The lytic capability of the 3 phages was subsequently determined with a Spot Assay Technique performed with 4 isolates of V. splendidus, 3 other Vibrio species and 2 environmental isolates. Both PVS-1 and PVS-2 were lytic to all 4 isolates of V. splendidus while PVS-3 only inhibited the growth of 3 of them. V. splendidus VS-ABTNL was more susceptible to phage PVS-2 than the other 2 phages. In an in vivo performance trial, 360 sea cucumbers (23 ± 2 g) were randomly assigned to 1 of 6 treatments. Each treatment was housed in 3 PVC tanks (38 cm × 54 cm × 80 cm) with 20 sea cucumbers per tank. Six diets were prepared including an unsupplemented control diet, antibiotic treatment diet, 3 diets containing 1 of the 3 phages individually and a diet containing a cocktail of all 3 phages. After 60 days of feeding, all sea cucumber were challenged with V. splendidus VS-ABTNL by immersion in sea water containing a bacterial concentration of 6 × 10(6) CFU/mL for 2 days. The survival rate of sea cucumbers during the next 10 days was 18% for the unsupplemented diet, 82% for the antibiotic treatment, 82% for the phage cocktail, 65% for phage PVS-1, 58% for phage PVS-2 and 50% for phage PVS-3. There were no significant differences in weight gain, ingestion rate or feed conversion among sea cucumber fed the 4 phage treatments compared with those fed the unsupplemented diet (P > 0.05). The levels of nitric oxide synthase and acid phosphatase of sea cucumbers fed phage-containing diets were significantly (P < 0.05) increased compared with those fed the control diet. However, no significant differences (P > 0.05) were detected among the 4 phage-fed treatments. An additional study was conducted in which 60 healthy sea cucumbers (23 ± 2 g) were randomly assigned to a control, an untreated group and a test group to investigate the effects of injecting phages by coelomic injection on the survival rate and enzyme activities in the coelomic fluid of the sea cucumbers. The control was injected with 1 ml of sterilized seawater while the untreated group and the test group were injected with the same volume of V. splendidus-ABTNL culture (3 × 10(5) CFU/mL). Then, the test group was injected with 1 ml of the 3 phage cocktail (MOI = 10). After 48 h, the activities of lysozyme, acid phosphatase and superoxide dismutase were elevated in the untreated group while the levels of these enzymes in the test group were similar to the blank control. After 10-day observation, the survival rate of the sea cucumber was 100% for the blank control, 80% for the test group and 20% for the negative control. The overall results of this experiment indicate that phage therapy increased the survival of sea cucumber infected with V. splendidus VS-ABTNL. The above results demonstrate that using phages, especially a combination of different phages, may be a feasible way to control Vibrio infection in the sea cucumber industry.

Effects of dietary n-3 highly unsaturated fatty acids (HUFAs) on growth, fatty acid profiles, antioxidant capacity and immunity of sea cucumber Apostichopus japonicus (Selenka).

The present study was conducted to understand the effects of dietary n-3 highly unsaturated fatty acids (HUFAs) on growth, fatty acid profiles, antioxidant capacity and the immunity of sea cucumber Apostichopus japonicus (Selenka). Five experimental diets were prepared, containing graded levels of n-3 HUFAs (0.46%, 0.85%, 1.25%, 1.61% and 1.95%, respectively), and the 0.46% group was used as control group. The specific growth rates, fatty acid profiles, activities and gene expression of antioxidative enzymes and lysozyme of the sea cucumbers that were fed with the 5 experimental diets were determined. The results showed that the specific growth rate of sea cucumbers in all the treatment groups significantly increased compared to the control group (P < 0.05), indicating the positive effects of n-3 HUFAs on the growth of sea cucumbers. The contents of eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3) in the body wall of the sea cucumbers gradually increased with the increasing levels of n-3 HUFAs in the diets. The suitable supplement of n-3 HUFAs in diets improved the activities of superoxide dismutase (SOD) and catalase (CAT) of sea cucumbers by up-regulating the expression of SOD and CAT mRNA in sea cucumbers. However, excess n-3 HUFAs in diets caused lipid peroxidation, inhibited the expression of lysozyme (LSZ) mRNA and decreased the activities of LSZ in sea cucumbers. In summary, the suitable supplement levels of n-3 HUFAs in diets of sea cucumbers A. japonicus were estimated between 0.85% and 1.25% considering the growth performance, cost and the indicators of antioxidant capacity and immunity.

Identification and functional characterization of TNF receptor associated factor 3 in the sea cucumber Apostichopus japonicus.

TNF receptor associated factors (TRAFs) are a family of proteins primarily involved in both adaptive and innate immunity. In this study, we identified a novel TRAF3 gene in Apostichopus japonicus by transcriptome sequencing and RACE approaches (designated as AjTRAF3). The full-length of AjTRAF3 was of 2796 bp including a 5' untranslated region (UTR) of 83 bp, a 3' UTR of 1066 bp and a putative open reading frame of 1647 bp encoding a polypeptide of 548 amino acid residues. The representative domains such as a RING finger domain (residues 54-96), two TRAF domains with zinc finger structure (residues 141-228), a coiled coil and a meprin and TRAF homology (MATH) domain (residues 396-522) were all detected in the deduced amino acids of AjTRAF3. AjTRAF3 was ubiquitously expressed in all examined tissues with predominant expression in the body wall and slightly weaker in intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle. Time-course expression analysis in coelomocytes revealed that AjTRAF3 was significantly depressed towards Vibrio splendidus infection with a 0.20-fold decrease at 12 h, compared to control levels. AjTRAF3 silencing could elevate intracellular ROS levels by 2.08-fold and 2.09-fold compared to each control group in vitro and in vivo, respectively. Taken together, all these results suggested that AjTRAF3 may play a crucial role in the processes of anti-bacteria response in sea cucumber through regulating ROS production.

Regulation of dietary glutamine on the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus japonicus (Selenka).

The present study examined the effects of dietary glutamine (Gln) on the growth, intestinal function, immunity and antioxidant capacity of sea cucumber Apostichopus japonicus (Selenka). The specific growth rate, intestinal morphology, activity of digestive enzymes, activity and gene expression of lysozyme and antioxidative enzymes of the sea cucumbers were determined after feeding 5 experimental diets with additions of increasing levels of Gln (at 0%, 0.4%, 0.8%,1.2% and 1.6%, respectively) for 60 days. We discovered that the specific growth rate of the sea cucumbers in 0.4%, 0.8% and 1.2% groups increased 35.3%, 27.3% and 24.1%, respectively, compared to the control (0%) group with significant differences. Dietary Gln can improve the intestinal function of the sea cucumbers by increasing the activities of trypsin and lipase in the intestine and the villus height and villus density of the intestine, eventhough significant differences were not observed in some groups. 0.4%-0.8% of dietary Gln can significantly increase the activity of lysozyme (LSZ) in the coelomic fluid of the sea cucumbers. Significant improvements were observed on the SOD activity in coelomic fluid of the sea cucumbers fed diets supplemented with 0.4%-1.6% of Gln compared to the control group. Similarly, the CAT activity in coelomic fluid of the sea cucumbers significantly increased in 0.8%, 1.2% and 1.6% groups compared to the control and 0.4% groups. Change pattern of the activity of CAT was consistent with the change pattern of the expression of CAT gene, indicating the dietary Gln can up-regulate the expression of CAT gene and consequently promote the secretion of CAT. However, the down-regulation of the expression of SOD gene by dietary Gln were observed in almost all of the treatment groups, which is in contrast with the change pattern of the activity of SOD, indicating the negative feedback regulation of the secretion of SOD on the expression of SOD gene. In summary, the suitable supplementation levels of Gln in diets of sea cucumber A. japonicus are 0.4%-0.8%, based on the effectiveness of dietary Gln on the growth, intestinal function, immunity and antioxidant capacity of the sea cucumbers.

Dietary Cordyceps militaris protects against Vibrio splendidus infection in sea cucumber Apostichopus japonicus.

Vibrio splendidus is the common pathogen that causes infectious diseases widely spread in cultured sea cucumber in China. Therefore, we investigated the ability of Cordyceps militaris to protect against infection caused by V. splendidus. In this study, sea cucumbers were fed with a diet containing 0 (control), 1%, 2% or 3% C. militaris for 28 days, and subsequently challenged with V. splendidus by injection with 1.0 × 10(9) cfu per animal. Parameters of immune response such as phagocytosis (PC), lysozyme (LSZ) activity, superoxide dismutase (SOD) activity, alkaline phosphatase (AKP) activity and acid phosphatase (ACP) activity were determined on days 0, 1, 3, 5 and 7 after injection. The results showed that dietary C. militaris at a dose of 2% or 3% significantly up-regulated (P < 0.05) all the immune parameters on day 0. One day after injection with V. splendidus, all the immune indices except ACP exhibited a tendency to decrease and then increase again, returning to the initial level on days 5 and/or 7 after injection. All the immune parameters of those fed with C. militaris were found significantly higher (P < 0.05) than those of the control group on day 1 after injection. Only LSZ activity of those fed with 1%- or 3%-C. militaris diet on day 5 showed significantly increases (P < 0.05) than the controls. As for ACP activity, the values remained steady with time, but with significant increase (P < 0.05) seen in sea cucumbers fed with 2%-C. militaris diet, and lasted for up to 7 days after V. splendidus injection. The cumulative mortality of sea cucumbers fed with the basal diet followed V. splendidus infection was significantly higher (P < 0.05) than those fed with 2% and 3% C. militaris diet. Under the experimental conditions, dietary C. militaris could enhance the immune responses of Apostichopus japonicus and improve its resistance to infection by V. splendidus.

Effects of dietary Bacillus cereus G19, B. cereus BC-01, and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression of immune-related genes in coelomocytes and intestine of the sea cucumber (Apostichopus japonicus Selenka).

Probiotics have positive effects on the nutrient digestibility and absorption, immune responses, and growth of aquatic animals, including the sea cucumber (Apostichopus japonicus Selenka). A 60-day feeding trial was conducted to evaluate the effects of Bacillus cereus G19, B. cereus BC-01 and Paracoccus marcusii DB11 supplementation on the growth, immune response, and expression level of four immune-related genes (Aj-p105, Aj-p50, Aj-rel, and Aj-lys) in coelomocytes and the intestine of juvenile sea cucumbers. One group was fed the basal diet (control group), while three other groups were fed the basal diet supplemented with B. cereus G19 (G19 group), B. cereus BC-01 (BC group), or P. marcusii DB11 (PM group). The growth rate of sea cucumbers fed diets with probiotics supplementation was significantly higher than that of the control group (P < 0.05). Sea cucumbers in the G19 and PM groups had a significantly greater phagocytic activity of coelomocytes compared to the control group (P < 0.05), while those in the G19 and BC groups had a greater respiratory burst activity (P < 0.05). The alkaline phosphatase (AKP) activity of coelomocytes in sea cucumbers fed diets with probiotics supplementation was significantly higher than the control group (P < 0.05). Comparatively, superoxide dismutase (SOD) activity of coelomocytes for sea cucumber in the PM group was significantly greater (P < 0.05). As for the immune-related genes, B. cereus G19 supplementation significantly increased the expression level of the Aj-rel gene in coelomocytes (P < 0.05), while B. cereus BC-01 supplementation significantly increased that of the Aj-p50 gene as compared to the control group (P < 0.05). In the intestine, the relative expression level of Aj-p105, Aj-p50, and Aj-lys genes in the PM group was significantly higher than that in the control group (P < 0.05). These results suggested that B. cereus G19 and B. cereus BC-01 supplementation could improve the growth performance and the immune response in coelomocytes, while P. marcusii DB11 supplementation could have a positive effect on the growth performance and immune response in coelomocytes and the intestine of sea cucumbers.

Calmodulin of the tropical sea cucumber: Gene structure, inducible expression and contribution to nitric oxide production and pathogen clearance during immune response.

Calmodulin (CaM) is an essential second messenger protein that transduces calcium signals by binding calcium ions (Ca(2+)) and modulating its interactions with various target proteins. In contrast to vertebrates, where CaM is well established as a cofactor for Ca(2+)-dependent physiological and cellular functions including host defense, there is a paucity of understanding on CaM in invertebrates (such as echinoderms) in response to immune challenge or microbial infections. In this study, we obtained and described the gene sequence of CaM from the tropical sea cucumber Stichopus monotuberculatus, a promising yet poorly characterized aquacultural species. mRNA expression of StmCaM could be detected in the intestine and coelomic fluid after Vibrio alginolyticus injection. Transcriptional and translational expression of StmCaM was inducible in nature, as evidenced by the expression patterns in primary coelomocytes following Vibrio challenge. This response could be mimicked by the Vibrio cells membrane components or lipopolysaccharides (LPS), and blocked by co-treatment of the LPS-neutralizing agent polymyxin B (PMB). Furthermore, inhibition of CaM activity by incubation with its inhibitor trifluoroperazine dihydrochloride (TFP) blunted the production of Vibrio-induced nitric oxide (NO) and augmented the survival of invading Vibrio in coelomocytes. Collectively, our study here supplied the first evidence for echinoderm CaM participation in innate immunity, and provided a functional link between CaM expression and antibacterial NO production in sea cucumber.

Effect of intestinal autochthonous probiotics isolated from the gut of sea cucumber (Apostichopus japonicus) on immune response and growth of A. japonicus.

The study isolated 224 bacteria from the intestine of Apostichopus japonicus, then selected and identified three of the bacteria (HS1, HS7, and HS10) which demonstrated amylase, lipase, and protease production capacity as candidate probiotics for sea cucumbers. The three potential probiotics showed no pathogenicity both in hemolytic assays on sheep blood agar plates and after immersing sea cucumbers in a suspension of the bacteria. To reveal the effects of these three potential probiotics on the innate immunity of sea cucumbers, total coelomocyte counts, respiratory burst activity, superoxide dismutase activity, lysozyme activity, acid phosphatase activity, and phagocytic activity by coelomocytes were examined after feeding with four different diets for up to 28 days. Also the specific growth rate and survival rate were investigated after a 60-day feeding trial. Sea cucumbers were fed with 4 diets: one control, three diets supplemented with 1 × 10(9) cell g(-1) of HS1, HS7, and HS10 for 28-60 days. Results showed that sea cucumbers fed diets containing HS1, HS7, and HS10 had led to an enhanced cellular and humoral immune response, notably higher total coelomocytes counts, respiratory burst activity, lysozyme activity, acid phosphatase activity, and phagocytic activity, as recorded during the four weeks of probiotics administration. On the other hand, the survival rate among dietary treatments ranged from 90.71 to 97.97% with significant improvement (P < 0.05) compared to that of the control; and the growth rate observed in the sea cucumbers fed HS1 and HS7 showed sharp increases after 60 days feeding. The present study confirmed the potential beneficial effects of Pseudoalteromonas elyakovii HS1, Shewanella japonica HS7, and Vibrio tasmaniensis HS10 as dietary probiotics in A. japonicus.

Cloning and gene expression of allograft inflammatory factor-1 (AIF-1) provide new insights into injury and bacteria response of the sea cucumber Apostichopus japonicus (Selenka, 1867).

Allograft inflammatory factor-1 (AIF-1) is an interferon (IFN)-γ-inducible Ca(2+)-binding cytokine that associates with the immune defense and inflammatory response. In this study, we reported AIF-1 gene in sea cucumber Apostichopus japonicus (AjAIF-1). The full-length cDNA of AjAIF-1 is 1541 bp with an open reading frame (ORF) of 477 bp encoding 158 amino acids. Two EF-hand Ca(2+)-binding motifs were found in the deduced AjAIF-1. AjAIF-1 was widely expressed in all tested tissues (body wall, intestine, respiratory tree, tube feet, coelomocytes and longitudinal muscle), with the highest expression in respiratory tree. After Vibrio splendidus challenge and physical injury, AjAIF-1 transcripts were significantly upregulated in coelomocytes. The mRNA expression level of AjAIF-1 in coelomocytes reached to the highest value at 4 h (3.38-folds vs. the PBS control, P < 0.05) post injection. After papilla injury, the mRNA level of AjAIF-1 in coelomocytes was upregulated, and its peak value was found at 4 h (3.88-folds vs. the control, P < 0.05). These results indicated that 1) AjAIF-1 sensitively responds to pathogen infection; 2) AjAIF-1 is involved in acute inflammatory response. Our findings gain general information about the role of AjAIF-1 in the innate immunity of A. japonicus.

Phenoloxidase from the sea cucumber Apostichopus japonicus: cDNA cloning, expression and substrate specificity analysis.

Phenoloxidase (PO) is a crucial component of the immune system of echinoderms. In the present study, the full-length cDNA of PO (AjPO) was cloned from coelomocytes of the sea cucumber Apostichopus japonicus using 3'- and 5'-rapid amplification of cDNA ends (RACE) PCR method, which is 2508 bp, with an open reading frame (ORF) of 2040 bp encoding 679 amino acids. AjPO contains a transmembrane domain, and three Cu-oxidase domains with copper binding centers formed by 10 histidines, one cysteine and one methionine respectively. Phylogenetic analysis revealed that AjPO was clustered with laccase-type POs of invertebrates. Using the isolated membrane proteins as crude AjPO, the enzyme could catalyze the substrates catechol, L-3,4-dihydroxyphenylalanine (L-DOPA), dopamine and hydroquinone, but failed to oxidize tyrosine. The results described above collectively proved that AjPO was a membrane-binding laccase-type PO. The quantitative real-time PCR (qRT-PCR) analysis revealed that AjPO mRNA was expressed in muscle, body wall, coelomocytes, tube feet, respiratory tree and intestine with the highest expression level in coelomocytes. AjPO could be significantly induced by lipopolysaccharide (LPS), peptidoglycan (PGN), Zymosan A and polyinosinic-polycytidylic acid (PolyI:C), suggesting AjPO is closely involved in the defense against the infection of bacteria, fungi and double-stranded RNA viruses.

Effects of dietary ß-glucan, mannan oligosaccharide and their combinations on growth performance, immunity and resistance against Vibrio splendidus of sea cucumber, Apostichopus japonicus.

A 4-week feeding trial was conducted to investigate the effects of dietary ß-glucan, mannan oligosaccharide (MOS) and their combinations on growth performance, immunity and disease resistance of sea cucumber Apostichopus japonicus. Sea cucumbers (1215 individuals with initial weight of 3.8 ± 0.2 g) were fed nine practical diets according to a 3 × 3 factorial design: the basal diet as the control supplemented with three levels of ß-glucan (0, 0.075, 0.15% w/w), crossed with 0, 0.1% (w/w) or 0.2% (w/w) MOS. Immune indices including total coelomocytes count (TCC), phagocytosis, superoxide anion production, superoxide dismutase (SOD) activity and total nitric oxide synthase activity (T-NOS) were measured at days 7, 11, 15, 18, 22, 25 and 29. At the end of the feeding trial, all the sea cucumbers left were weighted to monitor growth, and then were challenged by Vibrio splendidus. The results showed that dietary ß-glucan, MOS and their combinations significantly increased TCC, phagocytosis, superoxide anion production and SOD activity of sea cucumbers (P < 0.05). Only 0.15% ß-glucan and the combinations of ß-glucan and MOS significantly increased the T-NOS activity (P < 0.05). A synergistic effect was found between dietary ß-glucan and MOS. Moreover, combinations of ß-glucan and MOS prolonged the high levels of immune indices compared with ß-glucan or MOS supplementation alone. Except the 0.15% ß-glucan group, all the other treatments showed significantly lower cumulative mortality compared with control (P < 0.05). Furthermore, combination of 0.15% ß-glucan and 0.1% MOS had the best effects on enhancing disease resistance of sea cucumber. All treatments showed significantly higher specific growth rate (SGR) compared with control (P < 0.05), and the combination of 0.15% ß-glucan and 0.1% MOS was significantly higher than other treatments (P < 0.05). In conclusion, our results confirm the potential of ß-glucan and MOS as dietary immunostimulants and the synergistic effects of ß-glucan and MOS on A. japonicus.