Effects of chitin from Daphnia similis and its derivative, chitosan on the immune response and disease resistance of white shrimp, Litopenaeus vannamei.

Daphnia similis chitin and its derivative chitosan were prepared as immunostimulants to boost the immune response and determine the ability to control infectious disease caused by Vibrio alginolyticus in white shrimp, Litopenaeus vannamei. Three experimental diets supplemented with 0% chitin or chitosan (control) and 0.4% chitin or 0.4% chitosan were fed to shrimp for 56 days. Dietary inclusion of 0.4% chitosan accelerated shrimp growth compared to chitin and control. The survival and disease resistance of shrimp increased significantly when fed chitin and chitosan diets, after pathogenic injection, as indicated by the up-regulated immune responses in respiratory burst (RB), superoxide dismutase (SOD), and phagocytic activity (PA). There were no significant differences in the total haemocyte count (THC), phenoloxidase (PO)activity, and lysozyme (LYZ) activity among the groups. No significant differences were observed for prophenoloxidase system-related gene expressions among groups. However, shrimp fed chitin, and chitosan expressed significantly higher levels of antimicrobial proteins (penaeidin 3a, crustin, and anti-lipopolysaccharide factor 2) in the haemocytes than in control. The gene expressions of catalase and heat shock protein 70 increased in the hepatopancreas of shrimp fed chitosan diet compared to the chitin and control diet. The O-linked N-acetylglucosamine transferase (ogt) was significantly higher in the haemocytes of shrimp fed chitosan and chitin than the control, but ogt was only significantly higher in the hepatopancreas of shrimp fed chitosan. Dietary chitin and chitosan also showed positive effects on the transcription of peritrophin-like protein. These findings suggest that both chitin and chitosan from D. similis are efficacious at boosting the immunity of shrimp by preventing and controlling infectious diseases caused by Vibrio and have great potential to be used as a feasible immunostimulant that significantly contributes to the circular economy.

Isolation and characterization of antimicrobial peptides derived from Bacillus subtilis E20-fermented soybean meal and its use for preventing Vibrio infection in shrimp aquaculture.

Bacillus subtilis E20-fermented soybean meal (FSBM) was found to produce antimicrobial peptides (AMPs) with great antimicrobial activity against Vibrio alginolyticus (VA) and V. parahaemolyticus (VP). Three AMPs were purified with a 5 kDa ultrafiltration, Sephadex G-15 column and reverse-phase high-performance liquid chromatography (RP-HPLC). The FSB-AMP, HTSKALLDMLKRLGK, identified by an RP-nano-ultrapure liquid chromatography (UPLC) electrospray ionization (ESI)-tandem mass spectroscopic (MS/MS) analysis exhibited the highest bactericidal activity against VA and VP compared to the others. The antimicrobial activity assessment indicated that FSB-AMP inhibited the growth of VA and VP with minimal inhibitory concentrations of 72.5 and 72.5 µM. Alterations in the morphology of VA were observed by scanning electronic microscopy, and membrane disruption of VA and VP was confirmed by fluorescent microscopy with propidium iodide staining. The FSB-AMP was then incorporated into the diet of white shrimp, Litopenaeus vannamei, and a protective effect in shrimp against VP infection was recorded as well as for shrimp fed a diet containing 15% fish meal replaced by B. subtilis E20-FSBM. Results demonstrated that B. subtilis E20-FSBM could be a biofunctional ingredient to prevent vibriosis in shrimp aquaculture.

Plasma immune protein analysis in the orange-spotted grouper Epinephelus coioides: Evidence for altered expressions of immune factors associated with a choline-supplemented diet.

This study aimed to unravel the regulatory roles of choline in activating immune responses and disease resistance of the orange-spotted grouper Epinephelus coioides. Fish were fed a choline-supplemented diet at 1 g kg-1 of feed for 30 days. Fish fed a fish meal basal diet without choline-supplement served as controls. At the end of the feeding trial, fish were challenged with Vibrio alginolyticus. Meanwhile, plasma proteomics of fish in each group were also evaluated by two-dimensional gel electrophoresis (2-DE), and differentially expressed proteins were identified by tandem mass spectrophotometry (MS/MS), then a Western blot analysis or real-time polymerase chain reaction was used to confirm differential expressions of immune-enhancing proteins. Results showed that choline significantly increased survival of E. coioides 48 days after being injected with V. alginolyticus. From maps of plasma proteins, a comparative analysis between the control and choline groups revealed that 111 spots matched, with 26 altered expression spots in the choline group. Of these 26 spots, 16 were upregulated and 10 downregulated. After protein identification by reverse-phase nano-high-performance liquid chromatography-electrospray ionization MS/MS analysis, eight of 26 proteins were found to be immune-related proteins, all of which were upregulated, including complement 3 (C3), alpha-2-macroglobulin-P-like isoform (A2M), fibrinogen beta chain precursor (FBG), and immunoglobulin heavy constant mu (Ighm) proteins. Expression of the A2M protein and A2M enzyme activity in plasma of fish fed choline significantly increased compared to the control group. Additionally, A2M messenger (m)RNA transcripts were also upregulated in the liver and kidneys. Significantly higher C3 expressions at both the mRNA and protein levels were detected in the liver of fish in the choline group. Moreover, FBG gene expressions in the liver and kidneys significantly increased, while Ighm increased in the kidneys and spleen of fish in the choline group. Our results suggest that dietary administration of choline can protect grouper against bacterial infections through activating the complement system, thereby inducing antiprotease activity and natural antibodies that play important roles in the innate immune system of fish.

Current applications, selection, and possible mechanisms of actions of synbiotics in improving the growth and health status in aquaculture: A review.

Synbiotics, a conjunction between prebiotics and probiotics, have been used in aquaculture for over 10 years. However, the mechanisms of how synbiotics work as growth and immunity promoters are far from being unraveled. Here, we show that a prebiotic as part of a synbiotic is hydrolyzed to mono- or disaccharides as the sole carbon source with diverse mechanisms, thereby increasing biomass and colonization that is established by specific crosstalk between probiotic bacteria and the surface of intestinal epithelial cells of the host. Synbiotics may indirectly and directly promote the growth of aquatic animals through releasing extracellular bacterial enzymes and bioactive products from synbiotic metabolic processes. These compounds may activate precursors of digestive enzymes of the host and augment the nutritional absorptive ability that contributes to the efficacy of food utilization. In fish immune systems, synbiotics cause intestinal epithelial cells to secrete cytokines which modulate immune functional cells as of dendritic cells, T cells, and B cells, and induce the ability of lipopolysaccharides to trigger tumor necrosis factor-α and Toll-like receptor 2 gene transcription leading to increased respiratory burst activity, phagocytosis, and nitric oxide production. In shellfish, synbiotics stimulate the proliferation and degranulation of hemocytes of shrimp due to the presence of bacterial cell walls. Pathogen-associated molecular patterns are subsequently recognized and bound by specific pattern-recognition proteins, triggering melanization and phagocytosis processes.

Screening probiotic candidates for a mixture of probiotics to enhance the growth performance, immunity, and disease resistance of Asian seabass, Lates calcarifer (Bloch), against Aeromonas hydrophila.

Six bacteria, including, Lactobacillus casei M15, Lac. plantarum D8, Lac. pentosus BD6, Lac. fermentum LW2, Enterococcus faecium 10-10, and Bacillus subtilis E20, and one yeast, Saccharomyces cerevisiae P13 were selected as probiotics for Asian seabass, Lates calcarifer, by tracking the growth performance and disease resistance of fish against Aeromonas hydrophila in the first trial. The probiotic efficiency screening results showed that B. subtilis E20 and Lac. pentosus BD6, and S. cerevisiae P13 and Lac. fermentum LW2 respectively improved either the growth performance or disease resistance. Therefore, these four probiotics were then selected to prepare a probiotics mixture, and this was incorporated in equal proportions into diets for Asian seabass at levels of 0 (control), and 106 (MD6), 107 (MD7), 108 (MD8), and 109 (MD9) colony-forming units (cfu) (kg diet)-1. A synergistic effect of the combined probiotics was investigated in this study, and the probiotics mixture was able to improve both the growth performance and health status of fish. After 56 days of feeding, fish fed the MD9 diet had a higher final weight and percentage of weight gain. In addition, protein contents in the dorsal muscle of fish fed the MD8 and MD9 diets were significantly higher compared to the control. For the pathogen challenge test, fish fed the MD7, MD8, and MD9 diets had significantly lower cumulative mortalities after A. hydrophila infection compared to those of fish fed the control and MD6 diets, which might have been due to increased respiratory bursts, decreased superoxide dismutase activity in leucocytes, and increased phagocytic activity. Therefore, we considered that the probiotics mixture could adequately provide probiotic efficiency for Asian seabass, and the diet containing 109 cfu (kg diet)-1 probiotic mixture is recommended to improve the growth and health status of Asian seabass.

Dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity against Aeromonas hydrophila in Nile tilapia (Oreochromis niloticus).

Bacillus amyloliquefaciens has attracted attention as a probiotic in aquaculture due to its immunostimulatory activity against pathogenic infection. Xylanases are extensively used in animal feed to degrade plant ingredients, enhancing nutrient utilization and increasing the growth rate of various animals. In the present study, the effects of dietary supplementation with B. amyloliquefaciens and xylanase-expressing B. amyloliquefaciens R8 on the growth of Nile tilapia (Oreochromis niloticus) and immunity against Aeromonas hydrophila were evaluated. The results showed that the xylanase activity in the intestine, weight gain (WG), feed efficiency (FE) and condition factor (CF) of Nile tilapia fed B. amyloliquefaciens R8 for 2 months were significantly increased compared with those of the fish fed the control diet and B. amyloliquefaciens. Moreover, the mRNA expression of growth- and metabolism-related genes, such as insulin-like growth factor-1 (igf-1), glucokinase (GK), glucose-6-phosphate 1-dehydrogenase (G6PD), and glucose-6-phosphatase (G6Pase), was significantly induced in Nile tilapia fed administered B. amyloliquefaciens R8, and this group also exhibited a higher survival rate than the control fish following a challenge with A. hydrophila. The phagocytic activity and respiratory burst activity of head kidney leukocytes as well as the serum lysozyme activity of B. amyloliquefaciens R8-fed Nile tilapia were significantly higher than those of fish fed the control diet for 2 months. Superoxide dismutase (SOD) levels in the head kidney leukocytes of Nile tilapia fed B. amyloliquefaciens R8 differed from those of fish fed the control diet, but this was not significant. These results indicate that dietary supplementation with xylanase-expressing B. amyloliquefaciens R8 improves growth performance and enhances immunity and disease resistance against A. hydrophila in Nile tilapia.

Effects of hirami lemon, Citrus depressa Hayata, leaf meal in diets on the immune response and disease resistance of juvenile barramundi, Lates calcarifer (bloch), against Aeromonas hydrophila.

The present study was conducted to evaluate the dietary supplementation of leaf meal from Citrus depressa Hayata on the growth, innate immune response, and disease resistance of juvenile barramundi, Lates calcarifer. Four diets were formulated to contain 0% (control), 1% (C1), 3% (C3), and 5% (C5) leaf meal, respectively. During a 56 d feeding trial, fish survival, growth performance, and feed efficiency were not significantly different among all groups. For immune response, respiratory burst, superoxide dismutase and lysozyme activities were not significantly different among all groups. However, fish fed the C5 diet for 56 d had significantly higher phagocytic activity. Also, fish fed C3 and C5 diets had significantly higher Mx gene expressions in spleens and head kidneys with nerve necrosis virus injections after 24 h. Disease resistance against Aeromonas hydrophila was increased by the C5 diet. In this study, barramundi fed on a diet containing 5% C. depressa Hayata leaf meal had significantly better innate immune response and disease resistance against A. hydrophila.

Improvement in non-specific immunity and disease resistance of barramundi, Lates calcarifer (Bloch), by diets containing Daphnia similis meal.

A 42-day study was conducted with barramundi, Lates calcarifer, to evaluate the effects of Daphnia meal derived from Daphnia similis on fish growth, immune response, and disease resistance to Aeromonas hydrophila. Three isonitrogenous (45%) and isolipid (10%) experimental diets were formulated to contain 0% (control), 5% (D5), and 10% (D10) Daphnia meal. Growth was depressed when fish were fed with the D10 diet for 42 days compared to control. However, the growth in fish fed with control and D5 diets for 42 days was not significantly different. By day 42, the leukocyte phagocytic activity and respiratory burst activity were significantly increased in D5 and D10 groups compared to control. Mx gene expression in the spleen and head kidney of fish after being injected with nerve necrosis virus was also significantly up-regulated in both groups compared to control. In an increased immune response, D5 and D10 fish had significantly higher survival rates than control after being challenged by A. hydrophila. Therefore, we suggest that a 5% Daphnia-meal diet could improve the barramundi immune response and disease resistance without a negative impact on growth.

Molecular characterization of two trypsinogens in the orange-spotted grouper, Epinephelus coioides, and their expression in tissues during early development.

In this study, we cloned two trypsinogens of the orange-spotted grouper, Epinephelus coioides, and analyzed their structure, expression, and activity. Full-length trypsinogen complementary (c)DNAs, named T1 and T2, were 900 and 875 nucleotides, and translated 242 and 244 deduced amino acid peptides, respectively. Both trypsinogens contained highly conserved residues essential for serine protease catalytic and conformational maintenance. Results from isoelectric and phylogenetic analyses suggested that both trypsinogens were grouped into trypsinogen group I. Both trypsinogens had similar expression patterns of negative relationship with body weight; expression was first detected at 1 day post-hatching (DPH) and exhibited steady-state expression during early development at 1-25 DPH. Both expression and activity levels significantly increased after 30 DPH due to metamorphosis. Grouper larval development is very slow with insignificant changes in total length and body weight before 8 DPH. The contribution of live food to an increase in the trypsin activity profile may explain their importance in food digestion and survival of larvae during early larval development.

Purification and characterization of trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides.

Trypsin from the pyloric ceca of orange-spotted grouper, Epinephelus coioides, was purified by fractionation with ammonium sulfate, ionic exchange, and affinity chromatography. The protein was purified 161.85-fold with a yield of 4%. Purified trypsin had an apparent molecular weight of 24 kDa according to an SDS-PAGE analysis. Optimal profiles of temperature and pH of the enzyme were 50°C and 8-10, respectively, using Nα-benzoyl-L: -arginine ethyl ester as the substrate. The results of thermal and pH stability assays showed that the enzyme was stable at temperatures of up to 50°C and in the pH range of 6-8. Trypsin activity decreased with an increasing NaCl concentration (0-0.6 M). The activity of purified trypsin was effectively inhibited by a soybean trypsin inhibitor and N-p-tosyl-L: -lysine chloromethyl ketone, and was slightly inhibited by iodoacetic acid, ethylenediaminetetraacetic acid, 1-(L: -trans-epoxysuccinyl-leucylamino)-4-guanidinobutane, and pepstatin A. Protein identification of the purified protease showed that the sequences of two peptides, LGEHNI and NLDNDIML, were highly homologous to other fish trypsins. The measurement of trypsin activity in different tissues showed that the highest activity was detected in pyloric ceca, followed by anterior intestine, middle intestine, hind intestine and spleen, but very low activities were found in other tissues. An inverse relationship between the trypsin activity in four tissues of pyloric ceca, anterior intestine, middle intestine and hind intestine and fish body weight as a result of increased pepsin in stomach indicated grouper growth status was increased.

Effects of the probiotic, Bacillus subtilis E20, on the survival, development, stress tolerance, and immune status of white shrimp, Litopenaeus vannamei larvae.

In this study, the probiotic, Bacillus subtilis E20, isolated from the human health food, natto, was used for white shrimp, Litopenaeus vannamei, larvae breeding to improve the larval survival rate and development by adding probiotic to the rearing water at (control), 10(8), and 10(9) cfu L(-1) salt water once every 3 days during the 14 days of breeding experiment. Thereafter, stress tolerance and immune status of postlarvae were evaluated. Shrimp larval development was significantly accelerated after adding the probiotic to the larval rearing water at a level of 10(9) cfu L(-1). The survival rate of larvae was significantly higher in the treatment with 10(9) cfu L(-1) compared to the control and the treatment with 10(8) cfu L(-1) after all larvae had metamorphosed to postlarvae. Adding the probiotic to the shrimp larvae rearing water produced a weak inhibition of bacterial growth by an analysis of the total bacterial count and presumptive Vibrio count. For stress tests, no postlarvae died when they were reared in water in which the temperature was decreased from 30 to 2 degrees C at a rate of 0.1 degrees C min(-1). Postlarvae had significantly lower cumulate mortality in the treatments with 10(8) and 10(9) cfu L(-1) compared to the control when they were suddenly exposed to fresh water and 60 per thousand salt water. A significant decrease in the cumulative mortality of postlarvae treated with the probiotic at a level of 10(9) cfu L(-1) was recorded after the sudden transfer to 300 mg L(-1) nitrite-N compared to the control and treatment with 10(8) cfu L(-1). The analysis of immune-related gene expressions showed that the gene expression of prophenoloxidase I, prophenoloxidase II, and lysozyme of larvae were significantly increased after being reared in probiotic-containing water at the levels of 10(8) and 10(9) cfu L(-1). However, no significant difference in serine proteinase or glutathione peroxidase gene expressions was recorded in this study. It is therefore suggested that 10(9) cfu L(-1) of probiotic, B. subtilis E20 adding to rearing water for shrimp larva breeding.

Enhancement of immunity and disease resistance in the white shrimp, Litopenaeus vannamei, by the probiotic, Bacillus subtilis E20.

Effects of Bacillus subtilis E20 isolated from fermented soybean on immune parameters and the disease resistance of the white shrimp (Litopenaeus vannamei) after 98 days of B. subtilis E20 feeding were evaluated in this study. Shrimp fed B. subtilis E20-containing diets at concentrations of 10(6) (E206), 10(7) (E207), and 10(8) (E208)cfu kg(-1), respectively, had significantly increased survival rates of 13.3%, 16.7%, and 20%, compared to the control (fed no probiotic) after being challenged with Vibrio alginolyticus. There were no significant differences in the total hemocyte count, respiratory burst, or superoxide dismutase glutathione peroxidase among all treatments. Shrimp fed a higher concentration of the probiotic (E208) exhibited significant increases in phenoloxidase activity, phagocytic activity, and clearance efficiency compared to control shrimp. In addition, B. subtilis E20 showed a weaker inhibitory effect against the growth of Aeromona hydrophila with around a 0.3-cm inhibitory zone, but showed no inhibitory effects against other selected pathogens, such as white shrimp pathogens: V. alginolyticus and Vibrio vulnificus. These results suggest that the increased resistance of shrimp after B. subtilis E20 consumption occurs through immune modifications, such as increases in phenoloxidase activity, phagocytic activity, and clearance efficiency against V. alginolyticus.

Evaluation of the antibacterial activity of leaf and twig extracts of stout camphor tree, Cinnamomum kanehirae, and the effects on immunity and disease resistance of white shrimp, Litopenaeus vannamei.

Effects of essential oils and hot-water extracts isolated from leaf and twig of stout camphor tree, Cinnamomum kanehirae on antibacterial activity to pathogen of fish, abalone, marine fish and freshwater prawn, and the white shrimp, Litopenaeus vannamei immunity and disease resistance to Vibrio alginolyticus were carried out in this study. A better antibacterial activity against nine selected pathogen bacteria was recorded in twig essential oil, and the selected pathogens of both Gram-positive bacteria and Gram-negative bacteria were sensitive to the leaf and twig essential oils in the present study. No antibacterial activity was recorded in the hot-water extracts of leaf and twig. In challenge trial, a significant decrease of sensitivity to V. alginolyticus (1 x 10(6) cfu shrimp(-1)) was found in that of shrimp received hot-water extract from twig at the levels of 2 microg g shrimp(-1) compared to control. In addition, the how-water extract of twig in vitro showed greater enhanced effects on phenoloxidase activity, respiratory burst and phagocytosis of white shrimp compared to the hot-water extract of leaf. It is considered that the extracts of stout camphor tree could be a candidate to replace the chemo-therapeutants through the inhibitory effects against the growth of pathogens, and enhanced effects on shrimp immunity and disease resistance.