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.

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.

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.

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.