Effects of potential probiotic Bacillus subtilis T13 on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumber Apostichopus japonicus.

A feeding experiment was conducted to determine influences of potential probiotic Bacillus subtilis T13 (isolated from intestine of healthy sea cucumbers) on growth, immunity and disease resistance against Vibrio splendidus infection in juvenile sea cucumbers Apostichopus japonicus. Animals were fed with diets containing B. subtilis T13 at 0, 10(5), 10(7) and 10(9) CFU/g for 30 days, respectively. At the end of the growth trial, fifteen sea cucumbers from each aquarium were sampled for immune indices measurement. Then twenty sea cucumbers from each replicate were challenged with V. splendidus. Results showed that administration of B. subtilis T13 had significant effect on the specific growth rates (SGR) of sea cucumbers (P < 0.05). Phagocytosis, respiratory burst activity and total nitric oxide synthase (T-NOS) activity were significantly improved in coelomocytes of sea cucumbers fed with T13 at 10(9) CFU/g diet (P < 0.05). The highest values of the total coelomocytes counts (TCC) and superoxide dismutase (SOD) activity were found in sea cucumbers fed diet containing T13 at 10(9) CFU/g. The cumulative mortality after V. splendidus challenge decreased significantly in sea cucumbers fed with T13 at dose of 10(9) CFU/g (P < 0.05). The present study confirmed the potential beneficial effects of B. subtilis T13 as dietary probiotic in juvenile A. japonicus.

Effects of dietary n-3 highly unsaturated fatty acids on growth, nonspecific immunity, expression of some immune related genes and disease resistance of large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans).

The study was conducted to investigate the effects of dietary n-3 highly unsaturated fatty acid (n-3 HUFA) on growth, nonspecific immunity, expression of some immune related genes and disease resistance of juvenile large yellow croaker (Larmichthys crocea) following natural infestation of parasites (Cryptocaryon irritans). Six isoproteic and isolipidic diets were formulated with graded levels of n-3 HUFA ranging from 0.15% to 2.25% of the dry weight and the DHA/EPA was approximately fixed at 2.0. Each diet was randomly allocated to triplicate groups of fish in floating sea cages (1.0 × 1.0 × 1.5 m), and each cage was stocked with 60 fish (initial average weight 9.79 ± 0.6 g). Fish were fed twice daily (05:00 and 17:00) to apparent satiation for 58 days. Results showed that moderate n-3 HUFA level (0.98%) significantly enhanced growth compared with the control group (0.15% HUFA) (P < 0.05), while higher n-3 HUFA levels (1.37%, 1.79% and 2.25%) had detrimental effects on the growth though no significance was found (P > 0.05). Nitro blue tetrazolium (NBT) positive leucocytes percentage of head kidney and serum superoxide dismutase (SOD) activity increased with increasing n-3 HUFA from 0.15% to 0.60%, and decreased with further increase of n-3 HUFA from 0.60% to 2.25% (P < 0.05). Serum lysozyme activity increased significantly as n-3 HUFA increased from 0.15% to 1.37%, and then decreased with n-3 HUFA from 1.37% to 2.25% (P > 0.05). There were no significant differences in phagocytosis index (PI) of head kidney leucocytes among dietary treatments (P > 0.05). The hepatic mRNA expression of Toll-like receptor 22 (TLR22) and Myeloid differentiation factor 88 (MyD88) was significantly up-regulated in fish fed the diets with low or moderate levels, while in kidney this increment was only found at specific sampling time during the natural infestation of parasites. The 13 d cumulative mortality rate following natural infestation of parasites decreased with n-3 HUFA increased from 0.15% to 0.60% (P < 0.05), and significantly increased with n-3 HUFA from 0.60% to 2.25% (P < 0.05). Results of this study suggested that fish fed low or moderate dietary n-3 HUFA had higher growth, nonspecific immune responses, expression levels of some immune related genes and disease resistance of large yellow croaker following natural infestation of parasites and dietary n-3 HUFA may regulate fish immunity and disease resistance by altering the mRNA expression levels of TLR22 and MyD88.

Transcriptional up-regulation of a novel ferritin homolog in abalone Haliotis discus hannai Ino by dietary iron.

A novel cDNA encoding ferritin (HdhNFT) was cloned from the hepatopancreas of abalone, Haliotis discus hannai Ino. The deduced protein contains 171 amino acid residues with a predicted molecular mass (MW) about 19.8 kDa and theoretical isoelectric point (pI) of 4.792. Amino acid alignment revealed that HdhNFT shared high similarity with other known ferritins. The HdhNFT contained a highly conserved motif for the ferroxidase center, which consists of seven residues of a typical vertebrate heavy-chain ferritin with a typical stem-loop structure. HdhNFT mRNA contains a 27 bp iron-responsive element (IRE) in the 5'-untranslated region. This IRE exhibited 82.14% similarity with abalone H. discus discus and 78.57% similarity with Pacific oyster Crassostrea gigas IREs. By real-time PCR assays, the mRNA transcripts of HdhNFT were found to be higher expressed in kidney, hepatopancreas, gill, mantle and muscle than in haemocytes and gonad. Moreover, mRNA expression levels of HdhNFT in the hepatopancreas and haemocytes were measured by real-time PCR in abalone fed with graded levels of dietary iron (29.2, 65.7, 1267.2 and 6264.7 mg/kg). Results showed that the expression of the HdhNFT mRNA increased with dietary iron contents. Furthermore, the maximum value of the HdhNFT mRNA was found in the treatment with 6264.7 mg/kg of dietary iron. These data indicated that dietary iron can up-regulate HdhNFT at transcriptional level in abalone.

Interaction of dietary Bacillus subtilis and fructooligosaccharide on the growth performance, non-specific immunity of sea cucumber, Apostichopus japonicus.

A feeding experiment was conducted to investigate the interaction of probiotic Bacillus subtilis and prebiotic fructooligosaccharide (FOS) on the growth performance, immunity, intestinal microflora and disease resistance of sea cucumber (Apostichopus japonicus). Five hundred and forty individuals (initial body weight: 5.06 +/- 0.10 g, mean +/- S.E) were fed nine practical diets according to a 3 x 3 factorial design: the basal diet as the control diet supplemented with three levels of B. subtilis (0, 1.82 x 10(7) or 4.95 x 10(7) CFU g(-1) diet), crossed with 0, 0.25% or 0.50% FOS. After 8 weeks, three sea cucumbers per tank were sampled for bacterial quantification and immunity determination. Then all the sea cucumbers left were challenged by Vibrio splendidus. The results showed that dietary B. subtilis significantly increased the specific growth rate (SGR), total coelomocytes counts (TCC), phagocytosis of sea cucumbers, the counts of total viable bacteria and disease resistance to V. splendidus (P < 0.05), whereas the counts of Vibrio decreased. However, dietary B. subtilis had no significant effect on phenoloxidase (PO) activity in coelomocyte lysate supernatant (CLS) (P > 0.05). The SGR, PO activity, total viable bacterial counts (TBC) and Vibrio counts (VBC) were significantly affected by dietary FOS. In the group with 0.50% FOS, TCC, phagocytosis and PO activity significantly increased compared to the group fed without FOS in diet (P < 0.05). In the groups with 1.82 x 10(7) CFU B. subtilis/g diet, FOS supplementation remarkably decreased VBC. And higher level of FOS (0.50%) resulted in significantly higher TCC and PO activity compared with 0.25% FOS (P < 0.05). Moreover, the animals fed with diets supplemented with 0.25% and 0.50% FOS at each B. subtilis level had notably lower cumulative mortality after 14 days following V. splendidus exposure (P < 0.05). Under the experimental conditions, dietary B. subtilis and FOS had a synergistic effect on enhancing immunity and disease resistance of sea cucumber (P < 0.05).

Molecular cloning, characterization and mRNA expression of selenium-dependent glutathione peroxidase from abalone Haliotis discus hannai Ino in response to dietary selenium, zinc and iron.

A novel selenium-dependent glutathione peroxidase (Se-GPX) was cloned from abalone Haliotis discus hannai Ino (HdhGPx) by homology cloning with degenerate primers and RACE techniques. The full length of HdhGPx cDNA was 963bp with a 669bp open reading frame (ORF) encoding 222 amino acids and a 101bp eukaryotic selenocysteine insertion sequence (SECIS) in 3' untranslated region (UTR). It was showed that HdhGPx has a characteristic codon at (235)TGA(237) that corresponds to selenocysteine (SeC) as U(72). Sequence characterization revealed that HdhGPx contains a characteristic GPx signature motif 2 ((96)LGLPCNQF(103)), an active site motif ((179)WNFEKF(184)). In addition, two potential N-glycosylation sites ((112)NGTE(115) and (132)NLTQ(135)) were identified in HdhGPx. 3D modeling analysis showed that the overall structure of HdhGPx monomer had more similarity to human GPx3 than human GPx1. Relatively higher-level mRNA expression was detected in hepatopancreas, mantle and gonad by real-time PCR assays. The relative expression levels of HdhGPx mRNA in hepatopancreas and haemocytes were detected by real-time PCR in abalone fed with nine different diets containing graded levels of selenium (0.15, 1.32 and 48.7mgkg(-1)), zinc (6.69, 33.85 and 710.63mgkg(-1)) and iron (29.17, 65.7 and 1267.2mgkg(-1)) for 20weeks, respectively. The results showed that the expressions of HdhGPx mRNA were statistically higher at adequate dietary selenium (1.32mgkg(-1)), zinc (33.85mgkg(-1)) and iron (65.7mgkg(-1)) than those in low dietary minerals, respectively. But HdhGPx mRNA expression levels were down-regulated by high contents of dietary selenium (48.7mgkg(-1)), zinc (710.63mgkg(-1)) and iron (1267.2mgkg(-1)), respectively. These results indicated that adequate dietary minerals could increase the mRNA expression of HdhGPx, and then to increase the total antioxidant capacities in abalone.

Effects of dissolved oxygen on survival and immune responses of scallop (Chlamys farreri Jones et Preston).

This experiment investigated the effects of dissolved oxygen (DO) on the survival and immune responses of scallop Chlamys farreri. The scallops (initial mean dry weight of soft tissue 1.52+/-0.10 g) were cultivated in the seawater with different DO levels (8.5, 6.5, 4.5, and 2.5mg l(-1), respectively) for 21 d. Each treatment had triplicate groups of 35 animals. During the experimental period, the scallops were fed with Spirulina maxima, and water temperature ranged from 15.2 degrees C to 17.5 degrees C, salinity from 29.5 per thousand to 32.5 per thousand and pH from 7.5 to 8.2. Survival, specific growth rate (SGR) and total haemocyte count (THC) were examined at the end of the study, and superoxide dismutase (SOD), acid phosphatase (ACP) and alkaline phosphatase (ALP), were examined at 12 h, 24 h, Day 7, Day 14 and Day 21 after being exposed to the graded DO levels. The lower DO levels (2.5 and 4.5mg l(-1))resulted in lower survivals of scallops, and the survival (81.7%) at 2.5mg 1(-1)DO was significantly lower than those (100.0%) at 8.5 and 6.5mg l(-1) DO. Similarly, the SGR and THC of scallop gradually reduced with decreasing DO levels, and reached significant levels at 2.5mg l(-1) DO (P<0.05). At higher DO levels (8.5 and 6.5mg l(-1)), the SOD activity maintained rather stable during the entire sampling period. At lower DO levels (4.5 and 2.5mg l(-1)), however, the SOD activity significantly increased at 12 h, and then significantly decreased to the levels below the normal. At the two lower DO levels, ACP activities had no significant changes before Day 7, and then declined to the levels that were significantly lower than the normal. Significantly higher ALP activity was only observed at 12 h in the treatment of 2.5mg l(-1) DO, but in all other treatments and sampling times it fluctuated in a narrow range. In conclusion, less than 4.5mg l(-1) DO reduced the survival and depressed the immune responses of C. farreri.

Effects of dietary pyridoxine on immune responses in abalone, Haliotis discus hannai Ino.

A feeding experiment was conducted to investigate the effects of dietary pyridoxine (PN) on the immune responses of abalone, Haliotis discus hannai Ino. Purified diets supplemented with 0, 40, 800 mg PN kg(-1) or 80 mg kg(-1) of 4-deoxypyridoxine (PN antagonist) were fed to adult abalone (initial weight 45.77 +/- 0.25 g; initial shell length 68.02 +/- 0.78 mm) for 90 days. The air-dried brown kelp, Laminaria japonica, was used as a control diet. Each diet was fed to three replicate groups of abalone in a recirculation system using a completely randomised design. The results showed that weight gain ratio (WGR) of the abalone generally increased with the level of dietary PN supplementation though no significant differences were found among the treatments (P > 0.05). Phagocytic and phenoloxidase activities were significantly higher in abalone fed diets supplemented with 800 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). Agglutination titre and respiratory burst activity were significantly higher in abalone fed diets supplemented with 40 mg PN kg(-1) than those fed the PN-free diet or the one with 4-deoxypyridoxine (P < 0.05). There were no significant differences in immunological characteristics between the abalone fed the diet containing 40 mg PN kg(-1) and those fed the diet containing 800 mg PN kg(-1) (P > 0.05). L. japonica resulted in significantly lower agglutination titre, respiratory burst and phagocytic activities than the artificial diets supplemented with 40 or 800 mg PN kg(-1) (P < 0.05). Total haemocyte count (THC), serum protein concentration, and the activities of lysozyme and acid phosphatase were not significantly affected by the dietary treatments (P > 0.05). These results demonstrate that dietary deficiency of pyridoxine suppresses the immune functions in H. discus hannai, and further investigations are needed to optimise the dietary level of this vitamin for maintaining the best immune responses in abalone.