Dietary administration of a postbiotic, heat-killed Pediococcus pentosaceus PP4012 enhances growth performance, immune response and modulates intestinal microbiota of white shrimp, Penaeus vannamei.

The efficacy of postbiotics on the immune-related gene expression and gut microbiota of white shrimp, Penaeus vannamei remains unexplored. A commercial heat-killed postbiotic Pediococcus pentosaceus PP4012 was used to evaluate the growth performance, intestinal morphology, immunological status, and microbial community of white shrimp after dietary administration in this study. White shrimp (0.040 ± 0.003 g) were divided into three treatments; a control, inanimate P. pentosaceus (105 CFU g feed-1) at low concentration (IPL) and inanimate P. pentosaceus (106 CFU g feed-1) at high concentrations (IPH). The diets of IPL and IPH significantly increased final weight, specific growth rate and production compared to the control group. Shrimp fed with IPL and IPH significantly utilized feed more efficiently than those fed the control diet. The IPH treatment significantly lowered the cumulative mortality rate compared to the control and IPL diet following Vibrio parahaemolyticus infection. No significant difference was observed for Vibrio-like and lactic acid bacteria in intestine of shrimp fed with the control diet and the experimental diets. Adding inanimate P. pentosaceus significantly improved immune responses such as lysozyme and phagocytic activity compared to the control group. However, the total hemocyte count, phenoloxidase activity, respiratory burst, and superoxide dismutase activity were not significantly different among treatments. The immune-related genes alf, pen3a, and pen4 expression were significantly higher in shrimp fed IPL diet compared with control and IPH. Taxonomic identification of bacterial genera in all dietary groups belonged to two predominant phyla, Proteobacteria and Bacteroidota. An abundance of Photobacterium, Motilimonas, Litorilituus, and Firmicutes bacterium ZOR0006 were identified in the intestine of shrimp fed postbiotic diets. Unique microbes such as Cohaesibacter was discovered in the shrimp fed IPL while Candidatus Campbellbacteria, uncultured Verrucomicrobium DEV114 and Paenalcaligenes were discovered in the intestines of shrimp fed IPH diet. Collectively, these data suggest that including heat-killed P. pentosaceus, particularly IPH, can enhance growth performance, promote microbial diversity, elevate immune responses, and increase shrimp's resistance to V. parahaemolyticus.

Effects of Panax notoginseng Water Extract on Immune Responses and Digestive Enzymes in White Shrimp Litopenaeus vannamei.

Panax notoginseng (Burk) F. H. Chen is a traditional Chinese herbal medicine commonly used in clinical applications. This study examined the effects of the Panax notoginseng water extract (PNWE) on the immune responses and digestive enzyme activity of Litopenaeus vannamei (L. vannamei). The PNWE (50, 100, and 200 µg (g shrimp)-1) was injected into L. vannamei to analyze the immune response parameters, including the total haemocyte count (THC), granular haemocytes (GC), semi-granular haemocytes (SGC), hialin haemocyte (HC), the respiratory burst (RB), the phagocytic ratio (PR), the phagocytic index (PI), and phenoloxidase (PO). We evaluated the activity of the intestinal digestive enzymes (trypsin, chymotrypsin, amylase, and lipase), the histopathology, and the intestine Vibrio numbers. The results showed that different concentrations of the PNWE significantly increased THC, GC, SGC, PO and RB activity, the PR, and the PI of L. vannamei while reducing the HC. In addition, the PNWE also significantly increased the chymotrypsin, trypsin, and amylase activity of L. vannamei. Furthermore, 50 µg (g shrimp)-1 of PNWE regulated the lipase activity. Additionally, different concentrations of the PNWE significantly reduced the Vibrio numbers in the intestine without damaging the hepatopancreas and intestine tissues. These results indicate that the PNWE improves the immune responses of L. vannamei by increasing the haemocyte count and regulating intestinal digestive enzymes.

Sarcodia suae modulates the immunity and disease resistance of white shrimp Litopenaeus vannamei against Vibrio alginolyticus via the purine metabolism and phenylalanine metabolism.

Red seaweeds have several biofunctional properties, including immunomodulatory, antitumor, antioxidant, and antibacterial activities. In this study, we examined the effects of diets containing Sarcodia suae on the immune response, immune-related gene expressions, and disease resistance against Vibrio alginolyticus in white shrimp Litopenaeus vannamei. In addition, 1H NMR metabolomics was applied to analyze the metabolites extracted from shrimp fed with S. suae and their functions in regulating immunity. A diet containing only fish meal was used as the control diet (S0), and three diets containing different concentrations of S. suae powder, 2.5% (S2.5), 5% (S5), and 7.5% (S7.5) were used as experimental diets. Shrimp were fed diets for 20 days. Compared to the control group (S0), results showed that (1) shrimp fed diets supplemented with 5-7.5% of S. suae powder significantly increased anti-V. alginolyticus activity; (2) phagocytic activity (PA) increased in all shrimp fed with S. suae, but total haemocyte count (THC) only increased in S7.5 group; and (3) the expression of glutathione peroxidase (GPx) in haemocyte were significantly higher in S7.5 groups. Results from the 1H NMR analysis revealed that 19 heapatopancreatic metabolites were matched and identified among groups. Based on the KEGG enrichment analysis, the up-regulated metabolites in the shrimp fed S5 and S7.5 diets were primarily due to the metabolism of purine and phenylalanine and their respective pathways. Results from these trials reveal that diets containing S. suae can increase immune response, thereby increasing shrimp resistance to V. alginolyticus. The purine and phenylalanine metabolic pathways may be considered as the relevant pathways for optimizing immunomodulatory responses.

Improvement in the probiotic efficacy of Bacillus subtilis E20-stimulates growth and health status of white shrimp, Litopenaeus vannamei via encapsulation in alginate and coated with chitosan.

The aim of this study was to increase the efficacy of probiotic Bacillus subtilis E20 by encapsulating the probiotic in alginate and coating it with chitosan. The protective effect was evaluated by firstly ensuring the viability of encapsulated probiotics in simulated gastrointestinal fluid (SGF) and simulated intestinal fluid (SIF) conditions and then at different storage temperatures. In addition, the encapsulated probiotic was incorporated into the diet to improve the growth performance and health status of white shrimp, Litopenaeus vannamei. B. subtilis E20 has the ability to survive in SGF when encapsulated in 1.5-2% alginate and coated with 0.4% chitosan. Furthermore, viability increased significantly in SIF compared to the probiotic encapsulated in 1% alginate and coated with 0.4% chitosan and the non-encapsulated probiotic. Longer storage time and adverse conditions affected probiotics' survival, which was improved by the encapsulation with significantly higher viability than the non-encapsulated probiotic at different temperatures and storage duration. Encapsulation of B. subtilis E20 and dietary administration at 107 CFU kg-1 decreased shrimp mortality after a Vibrio infection, thereby improving shrimp's disease resistance, while the non-encapsulated probiotic required 109 CFU kg-1 to achieve better resistance. Although the best results of growth performance, immune response, and disease resistance against Vibrio alginolyticus were found in the shrimp fed with the diets supplemented with encapsulated probiotic at >108 CFU kg-1, shrimp's growth performance and health status improved after being fed 107 CFU kg-1 encapsulated probiotic for 56 days. Together, the results of this study prove that encapsulation could improve the viability of probiotic in different gastrointestinal conditions and adverse storage temperatures. Overall, lower concentrations of encapsulated probiotic B. subtilis E20 (107 CFU kg-1) was able to increase the growth performance and health status of shrimp.

Probiotic, Bacillus subtilis E20 alters the immunity of white shrimp, Litopenaeus vannamei via glutamine metabolism and hexosamine biosynthetic pathway.

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 109 cfu kg diet-1. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

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.

Overcoming sorafenib evasion in hepatocellular carcinoma using CXCR4-targeted nanoparticles to co-deliver MEK-inhibitors.

Sorafenib is a RAF inhibitor approved for several cancers, including hepatocellular carcinoma (HCC). Inhibition of RAF kinases can induce a dose-dependent "paradoxical" upregulation of the downstream mitogen-activated protein kinase (MAPK) pathway in cancer cells. It is unknown whether "paradoxical" ERK activation occurs after sorafenib therapy in HCC, and if so, if it impacts the therapeutic efficacy. Here, we demonstrate that RAF inhibition by sorafenib rapidly leads to RAF dimerization and ERK activation in HCCs, which contributes to treatment evasion. The transactivation of RAF dimers and ERK signaling promotes HCC cell survival, prevents apoptosis via downregulation of BIM and achieves immunosuppression by MAPK/NF-kB-dependent activation of PD-L1 gene expression. To overcome treatment evasion and reduce systemic effects, we developed CXCR4-targeted nanoparticles to co-deliver sorafenib with the MEK inhibitor AZD6244 in HCC. Using this approach, we preferentially and efficiently inactivated RAF/ERK, upregulated BIM and down-regulated PD-L1 expression in HCC, and facilitated intra-tumoral infiltration of cytotoxic CD8+ T cells. These effects resulted in a profound delay in tumor growth. Thus, this nano-delivery strategy to selectively target tumors and prevent the paradoxical ERK activation could increase the feasibility of dual RAF/MEK inhibition to overcome sorafenib treatment escape in HCC.

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.

Dietary administration of Gynura bicolor (Roxb. Willd.) DC water extract enhances immune response and survival rate against Vibrio alginolyticus and white spot syndrome virus in white shrimp Litopeneaus vannamei.

Gynura bicolor (Roxb. & Willd.) DC., a perennial plant belonging to the Asteraceae family, is originated from the tropical area of Asia. The total hemocyte count (THC), phenoloxidase (PO) activity, respiratory bursts (RBs), superoxide dismutase (SOD) activity, and lysozyme activity were examined after white shrimp Litopenaeus vannamei had been fed diets containing the water extract of G. bicolor at 0 (control), 0.5, 1.0, and 2.0 g (kg diet)(-1) for 7-28 days. The results indicated that these parameters increased accordingly with the amount of extract and time. THCs of the shrimp fed the G. bicolor diets at 1.0 and 2.0 g (kg diet)(-1) were significantly higher than that fed the control diet for 14-28 days. For the shrimp fed the G. bicolor diets at 0.5, 1.0, and 2.0 g (kg diet)(-1), the PO, RBs, and lysozyme activities reached the highest levels after 7 days, whereas SOD activity reached the highest levels after 14 days. In a separate experiment, white shrimp L. vannamei fed the diets containing the G. bicolor extract for 28 days were challenged with Vibrio alginolyticus at 3 × 10(6) cfu shrimp(-1) and white spot syndrome virus (WSSV) at 1 × 10(3) copies shrimp(-1). The survival rate of the shrimp fed the G. bicolor diets was significantly higher than that of the shrimp fed the control diet at 48-144 h post challenge V. alginolyticus and WSSV. For the shrimp fed the G. bicolor diets at 0.5, 1 and 2 g (kg diet)(-1) under challenges of V. alginolyticus and WSSV, their LPS- and ß-1,3-glucan-binding protein (LGBP) and peroxinectin (PE) mRNA expressions were significantly higher than those of the challenged control shrimp at 12-96 and 24-144 h post-challenge, respectively. We concluded that dietary administration of a G. bicolor extract could enhance the innate immunity within 28 days as evidenced by the increases in immune parameters (PO, RBs, and lysozyme) and antioxidant enzyme (SOD) activities of shrimp to against V. alginolyticus and WSSV infections.

Effects of the water extract of Gynura bicolor (Roxb. & Willd.) DC on physiological and immune responses to Vibrio alginolyticus infection in white shrimp (Litopenaeus vannamei).

Gynura bicolor (Roxb. & Willd.) DC is widely distributed in certain areas of Asia and is very popular in vegetarian cuisine in Taiwan. To investigate the regulatory roles of G. bicolor in various functions in crustaceans, we examined innate non-specific immune responses (including total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs), and superoxide dismutase (SOD) activity), physiological responses (including haemolymph glucose, lactate, and lipids), and gene expressions (including prophenoloxidase (proPO), lipopolysaccharide- and b-1,3-glucan-binding protein (LGBP), and peroxinectin (PE) mRNA transcripts) to the pathogen Vibrio alginolyticus in white shrimp (Litopenaeus vannamei) that were individually injected with the water extract from G. bicolor at 2, 4, and 8 µg g(-1). Results indicated that PO, RBs, SOD activity, proPO, LGBP, and PE mRNA transcripts of shrimps receiving the water extract of G. bicolor at 2, 4, and 8 µg g(-1) significantly increased after challenge with V. alginolyticus for 96 h. However, no significant difference in the THC was seen at any dose. L. vannamei injected with the water extract of G. bicolor at all doses respectively maintained lower glucose, lactate, and lipid levels in response to V. alginolyticus challenge at 12-36, 24-36, and 24-48 h. Survival rates at 24-72 h of L. vannamei that received G. bicolor at any dose was significantly higher than those of shrimp that received saline. It was concluded that the water extract of G. bicolor can maintain physiological homeostasis and enhance immunity against V. alginolyticus infection in L. vannamei.

Identification and cloning of a transglutaminase from giant freshwater prawn, Macrobrachium rosenbergii, and its transcription during pathogen infection and moulting.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of the giant freshwater prawn, Macrobrachium rosenbergii, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus; tiger shrimp, Penaeus monodon; kuruma shrimp, Marsupenaeus japonicus; and crayfish, Pacifastacus leniusculus. The 2722-bp cDNA contained an open reading frame (ORF) of 2334 bp, a 72-bp 5'-untranslated region (UTR), and a 316-bp 3'-UTR containing a stop codon and a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (778 aa) was 86.67 kDa with an estimated pI of 5.4. The M. rosenbergii TG (abbreviated MrTG, accession no.: JF309296) contains a typical transglutaminase-like homologue, two putative integrin-binding motifs (RGD), ten glycosylation sites, and four calcium-binding sites; a catalytic triad is present as in arthropod TGs. Sequence comparison and a phylogenetic analysis revealed that shrimp TG can be separated into three subgroups, penaeid TG1, freshwater crustacean TG2 and marine crustacean TG2, and MrTG was more closely related to TG2 than to TG1. MrTG mRNA and TG activities were detected in all tested tissues of M. rosenbergii, with MrTG mainly being synthesised by haemocytes. There was a negative correlation between clotting time of haemolymph, and MrTG expression and TG activity of haemocytes in prawn injected with Lactococcus garvieae. The pattern of MrTG mRNA expression and TG activity in haemocytes exhibited a contrary tendency with clotting time of haemolymph during the moult stages. Those results indicate that cloned MrTG is involved in the defence response, and is probably the major functional TG for haemolymph coagulation in M. rosenbergii.

A smaller particle size improved the oral bioavailability of monkey head mushroom, Hericium erinaceum, powder resulting in enhancement of the immune response and disease resistance of white shrimp, Litopenaeus vannamei.

The effects of different particle sizes (100-150, 74-100, and <74 µm) of powder of the dried and ground stipe from the monkey head mushroom, Hericium erinaceum, on the immune response and disease resistance of white shrimp, Litopenaeus vannamei, against the pathogen, Vibrio alginolyticus, were examined. Mushroom powder with a particle size of <74 µm had a significantly higher effect on the disease resistance of shrimp compared to particle sizes of >74 µm. Mortality of shrimp after being injected with V. alginolyticus was particle size-dependent, increasing from 66.7% ± 3.3%-93.3% ± 3.3% with diets containing stipe particle sizes of <74 and 100-150 µm, respectively. The mortality of shrimp fed the diet containing <74-µm stipe powder for 28 days was significant lower than that of shrimp fed with the control diet and the diet containing 74-100-µm stipe powder after being challenged by V. alginolyticus. The optimal concentration of the <74-µm mushroom powder for enhancing the immune response and disease resistance of shrimp was 0.2 µg (g shrimp)(-1) day(-1). No significant change in the total hemocyte count, differential hemocyte count, glutathione reductase, or phagocytic activity was found in shrimp fed the control diet and mushroom powder-containing diet at a level of up to 0.2 µg (g shrimp)(-1) day(-1). Shrimp fed 0.2 µg (g shrimp)(-1) day(-1) of a mushroom-containing diet had a significantly higher disease resistance to V. alginolyticus via an increase in phenoloxidase activity, respiratory bursts, superoxide dismutase activity, and glutathione peroxidase activity. Therefore, a diet containing the stipe powder of monkey head mushroom with a particle size <74 µm at a level of 0.2 µg (g shrimp)(-1) day(-1) was found to enhance the immunity and disease resistance of shrimp.

The increase of immunity and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii by feeding with selenium enriched-diet.

The effects of inorganic selenium (Se) (sodium selenate, SSe) and organic selenium (seleno-l-methionine, MSe) supplementation on the immune response, antioxidant status, and disease resistance of the giant freshwater prawn, Macrobrachium rosenbergii, were studied. Five experimental diets, including a control diet (without Se enrichment), 0.5 mg (kg diet)(-1) of MSe, 1 mg (kg diet)(-1) of MSe, 0.5 mg (kg diet)(-1) of SSe, and 1 mg (kg diet)(-1) of SSe, were used. After 75 days of culture, prawn fed the Se-enriched diets had lower mortality compared to that of prawn fed the control diet after being challenged by the pathogen, Debaryomyces hansenii. No significant differences in the total hemocyte count, superoxide dismutase activity, or clearance efficiency of prawn were recorded among the control and treated groups. Significantly increased phenoloxidase and phagocytic activities in prawn fed the Se-enriched diets were found compared to the controls. Respiratory bursts of prawn fed both forms of 1 mg Se (kg diet)(-1) significantly increased compared to control prawns. For the antioxidant status analysis, glutathione peroxidase, glutathione reductase, and glutathione s-transferase of prawn fed the SSe-enriched diet at 1 mg (kg diet)(-1) were significantly increased. The results indicated that the cheaper selenium, SSe is recommended to be added in prawn feed at the concentration of 0.5 mg resulting in 1.5 mg SSe (kg diet)(-1) increased prawn immunity and disease resistance against the pathogen, D. hansenii.

Effects of hot-water extract of Toona sinensis on immune response and resistance to Aeromonas hydrophila in Oreochromis mossambicus.

Respiratory burst, lysozyme and phagocytic activities, and immunoglobulin levels in response to the pathogen Aeromonas hydrophila were examined in tilapia (Oreochromis mossanbicus, 10.65 +/- 2.5 g) injected individually with hot-water extract of Toona sinensis at 4 or 8 microg g(-1). Tilapia receiving the hot-water extract of T. sinensis at either dose had significantly increased respiratory burst, phagocytic activity and lysozyme activity towards A. hydrophila by 1 and 2 days post injection. No significant differences in total immunoglobulin levels were observed among the tilapia that received the two different doses of hot-water extract of T. sinensis at 4 and 8 microg g(-1). In another experiment, a Tilapia that had been injected with hot-water extract of T. sinensis was challenged with A. hydrophila at 5 x 10(7) colony-forming units (cfu) fish(-1). The survival of tilapia that received the hot-water extract of T. sinensis at 8 microg g(-1) was significantly higher than fish that received phosphate buffered saline and the control fish after 2 days, and at the termination of the experiment (7 days after the challenge). It was concluded that the hot-water extract of T. sinensis at 8 microg g(-1) or less had increased the immune response and resistance to A. hydrophila infection in tilapia.

cDNA cloning, identification, tissue localisation, and transcription profile of a transglutaminase from white shrimp, Litopenaeus vannamei, after infection by Vibrio alginolyticus.

Complementary (c)DNA encoding transglutaminase (TG) messenger (m)RNA of white shrimp, Litopenaeus vannamei, was cloned from haemocytes by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) using oligonucleotide primers based on the TG sequence of the horseshoe crab, Tachypleus tridentatus (accession no.: BAA02134); tiger shrimp, Penaeus monodon (AAL78166); and Pacifastacus leniusculus (AF336805). The 2638-bp cDNA contained an open reading frame (ORF) of 2172 bp, a 55-bp 5'-untranslated region (UTR), and a 411-bp 3'-UTR containing a poly A tail. The molecular mass of the deduced amino acid (aa) sequence (757 aa) was 84.9 kDa with an estimated pI of 5.2. The L. vannamei TG (abbreviated LvTG) contains a typical transglutaminase-like homologue, a putative integrin-binding motif (RGD), and four calcium-binding sites; a catalytic triad is present as in arthropod TG. Sequence comparison and phylogenetic analysis revealed that shrimp TG can be separated into two subgroups, STGS1 and STGS2, and LvTG is more closely related to STGS1 than to STGS2. LvTG mRNA and TG activities were detected in all tested tissues of L. vannamei, with LvTG mainly being synthesised in haemocytes. However, the pattern of LvTG mRNA expression was not directly correlated with TG activity. The haemocytes of L. vannamei injected with Vibrio alginolyticus showed a significant decrease of TG activity at 3 h and a significant increase of LvTG mRNA expression at 6 h followed by a notable decrease from 12 to 24 h, which indicated that cloned LvTG was involved in the immune response of shrimp. The results also imply that more than one type of TG may be involved in the defense response in L. vannamei.

Identification and cloning of a selenium dependent glutathione peroxidase from giant freshwater prawn, Macrobrachium rosenbergii.

A selenium dependent glutathione peroxidase (Se-GPx) cDNA was cloned from haemocyte by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE). The 913 bp cDNA contained an open reading frame (ORF) of 558 bp encoded a deduced amino acid sequence of 186 amino acids. The prawn Se-GPx sequence contains a selenocysteine (Sec) residue which is encoded by the unusual stop codon, (115)TGA(117). According to the molecular modeling analysis, the active site Sec residue, located in the loop between beta3 and alpha2 in a pocket on the protein surface, and hydrogen bonded to Gln(73) and Trp(141). A GPx signature motif 2, (63)LAFPCNQF(70) and active site motif, (151)WNFEKF(156), two arginine (R) residues, R(89) and R(167) contribute to the electrostatic architecture that directs the glutathione donor substrate, and two putative N-glycosylation site, (75)NNT(77) and (107)NGS(109) were observed in the prawn Se-GPx sequence. In addition, the eukaryotic selenocysteine insertion sequence element is conserved in the 3'-UTR. Comparison of amino acid sequences showed that prawn Se-GPx is more closely related to vertebrate GPx 1. The prawn Se-GPx was synthesized in haemocyte, hepatopancreas, muscle, stomach, gill, intestine, eyestalk, heart, epidermis, lymph organ, ventral nerve cord, testis and ovary. The increase of respiratory burst in haemocyte was observed in pathogen, Debaryomyces hansenii-injected prawn in order to kill the pathogen, and the up-regulation in SOD and GPx acitivity, and prawn Se-GPx mRNA transcription were involved with the protection against damage from oxidation.

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.

A second proPO present in white shrimp Litopenaeus vannamei and expression of the proPOs during a Vibrio alginolyticus injection, molt stage, and oral sodium alginate ingestion.

Prophenoloxidase (proPO) is a melanin-synthesising enzyme that plays important roles in immune responses by crustaceans. Previously, we cloned and characterized proPO-I from white shrimp, Litopenaeus vannamei. In the present study, a novel prophenoloxidase-II (proPO-II) cDNA was also cloned from haemocytes of L. vannamei using oligonucleotide primers and reverse-transcriptase polymerase chain reaction (RT-PCR). Both 3'- and 5'-regions were isolated by the rapid amplification of complementary (c)DNA end (RACE) method. The 2504-bp cDNA contained an open reading frame (ORF) of 2073 bp, an 84-bp 5'-untranslated region, and a 347-bp 3'-untranslated region containing the poly A tail. The molecular mass of the deduced amino acid sequence (691 amino acids) was 78.8 kDa with an estimated pI of 6.07. It contains two putative tyrosinase copper-binding motifs and a conserved C-terminal region common to all known proPOs. Comparisons of the amino acid sequences showed that white shrimp proPO-II is more closely related to the proPO of other penaeids than to that of crayfish, lobsters, crab, or a freshwater prawn, and is the ancestor type of known penaeid proPOs. proPO-I and proPO-II messenger (m)RNAs of shrimp were located on different loci, and were constitutively expressed mainly in haemocytes. The transcriptional regulation of these two proPOs in shrimp at different molt stages, those administered dietary sodium alginate, and those challenged with Vibrio alginolyticus were surveyed. The results showed that the proPOs may be directly involved in the acute-phase immune defence, and proPO-II may contribute earlier to immune defence in shrimp injected with V. alginolyticus, and it may be regulated by ecdysone. However, a similar effect was found by stimulating proPO-I and proPO-II mRNA expression in shrimp fed a sodium alginate-containing diet. Results of this study provide a basis for developing a comprehensive understanding of expression/function relationships of individual proPOs in shrimp.