Effect of fed dietary yeast (Rhodotorula paludigena CM33) on shrimp growth, gene expression, intestinal microbial, disease resistance, and meat composition of Litopenaeus vannamei.

Yeast is a health-promoting and bio-therapeutic probiotic that is commonly used in aquaculture. Rhodotorula paludigena CM33 can accumulate amounts of intracellular carotenoids and lipid, which are regarded as nutritionally beneficial compounds in various aspects. The aim of this study was to evaluate the impact of different levels of R. paludigena CM33 (RD) incorporated in a dietary composition at 0% (control), 1% (1% RD), 2% (2% RD), and 5% (5% RD) on the growth of shrimp (Litopenaeus vannamei), their immune-related gene expression, intestinal health, resistance to Vibrio parahaemolyticus (VPAHPND) infection, and meat composition. The results showed significant improvements in the specific growth rate, weight gain, and survival of shrimp fed with 1% RD, 2% RD, and 5% RD, which were higher than the control group after 4 weeks of administration. The administration of 5% RD group resulted in a decrease in cumulative mortality upon VPAHPND challenge when compared to the control group. Furthermore, the expression levels of immune-responsive genes, including proPO system (prophenoloxidase-2: PO2), antioxidant enzyme (superoxide dismutase: SOD, glutathione peroxidase: GPX, and catalase: CAT), JAK/STAT pathway (signal transducer and activator of transcription: STAT, gamma interferon inducible lysosomal thiol reductase: GILT), IMD pathway (inhibitor of nuclear factor kappa-B kinase subunit beta and epsilon: IKKb and IKKe), and Toll pathway (Lysozyme) genes, were up-regulated in the 5% RD group. In the context of microbiota, microbiome analysis revealed that the main phyla in shrimp intestines were Proteobacteria, Firmicutes, Bacteroidota, Campilobacterota, Actinobacteriota, and Verrucomicrobiota. At the genus level, Vibrio was found to be reduced in the 5% RD group, whereas the abundance of potentially beneficial bacteria Bifidobacterium was increased. The 5% RD group showed a significant increase in the levels of crude protein and crude lipid, both of which are essential nutritious components. Our results show the capability of R. paludigena CM33 as a probiotic supplement in shrimp feed in improving growth, antimicrobial responses against VPAHPND, and meat quality by increasing protein and lipid content in shrimp.

Peroxiredoxin-4 supplementation modulates the immune response, shapes the intestinal microbiome, and enhances AHPND resistance in Penaeus vannamei.

Peroxiredoxin-4 from Penaeus vannamei (LvPrx4) is considered a damage-associated molecular pattern (DAMP) that can activate the expression of immune-related genes through the Toll pathway. We previously demonstrated that the recombinant LvPrx4 (rLvPrx4) can enhance shrimp resistance against Vibrio parahaemolyticus, causing acute hepatopancreatic necrosis disease (VPAHPND), which causes great production losses in shrimp farming. Herein, we showed that the rLvPrx4 had a thermal tolerance of around 60 °C and that the ionic strength had no noticeable effect on its activity. We discovered that feeding a diet containing rLvPrx4 to shrimp for three weeks increased the expression of the immune-related genes LvPEN4 and LvVago5. Furthermore, pre-treatment with rLvPrx4 feeding could significantly prolong shrimp survival following the VPAHPND challenge. The shrimp intestinal microbiome was then characterized using PCR amplification of the 16S rRNA gene and Illumina sequencing. Three weeks of rLvPrx4 supplementation altered the bacterial community structure (beta diversity) and revealed the induction of differentially abundant families, including Cryomorphaceae, Flavobacteriaceae, Pirellulaceae, Rhodobacteraceae, and Verrucomicrobiaceae, in the rLvPrx4 group. Metagenomic predictions indicated that some amino acid metabolism pathways, such as arginine and proline metabolism, and genetic information processing were significantly elevated in the rLvPrx4 group compared to the control group. This study is the first to describe the potential use of rLvPrx4 supplementation to enhance shrimp resistance to VPAHPND and alter the composition of a beneficial bacterial community in shrimp, making rLvPrx4 a promising feed supplement as an alternative to antibiotics for controlling VPAHPND infection in shrimp aquaculture.

A comparative study on effects of dietary three strains of lactic acid bacteria on the growth performance, immune responses, disease resistance and intestinal microbiota of Pacific white shrimp, Penaeus vannamei.

The present study evaluated the growth performance, immune responses, disease resistance and intestinal microbiota in Penaeus vannamei fed diets supplemented with three strains of lactic acid bacteria (LAB). The basal diet (control, CO) supplemented with Lactobacillus plantarum W2 (LA), Pediococcus acidilactici Nj (PE), Enterococcus faecium LYB (EN) and florfenicol (FL), respectively, formed three LAB diets (1 × 1010 cfu kg-1) and a florfenicol diet (15 mg kg-1, positive control), were fed to shrimp for 42 days. Results indicated that specific growth rate, feed efficiency rate, and disease resistance of shrimp against Vibrio parahaemolyticus in the treatment groups were significantly improved versus the control (P < 0.05). Compared with the control, acid phosphatase, alkaline phosphatase, phenonoloxidase, total nitric oxide synthase, peroxidase, superoxide dismutase activities, total antioxidant capacity, and lysozyme content in the serum and the relative expression levels of SOD, LZM, proPO, LGBP, HSP70, Imd, Toll, Relish, TOR, 4E-BP, eIF4E1α and eIF4E2 genes in the hepatopancreas of LAB groups were enhanced to various extents. Intestinal microbiota analysis showed that the LA and EN groups significantly improved microbial diversity and richness, and LAB groups significantly altered intestinal microbial structure of shrimp. At the phylum level, the Verrucomicrobiota in the LA and PE groups, the Firmicutes in the EN group, and the Actinobacteriota in the PE and EN groups were enriched. Moreover, the CO group increased the proportion of potential pathogens (Vibrionaceae and Flavobacteriaceae). The potential pathogen (Vibrio) was reduced, and potential beneficial bacteria (Tenacibaculum, Ruegeria and Bdellovibrio) were enriched in response to dietary three strains of LAB. When the intestinal microbiota homeostasis of shrimp is considered, L. plantarum and E. faecium showed better effects than P. acidilactici. However, due to the concerns on the possible potential risks of E. faecium strains to human health, L. plantarum W2 is more suitable for application in aquaculture than E. faecium LYB. Considering collectively the above, Lactobacillus plantarum W2 could be applied as better probiotic to improve the growth performance, non-specific immunity, disease resistance and promote intestinal health of P. vannamei.

Improving red-color performance, immune response and resistance to Vibrio parahaemolyticus on white shrimp Penaeus vannamei by an engineered astaxanthin yeast.

Astaxanthin (AST), a super antioxidant with coloring and medical properties, renders it a beneficial feed additive for shrimp. This study conducted a white shrimp feeding trial of 3S, 3'S isoform AST, which was derived from metabolic-engineered Kluyveromyces marxianus fermented broth (TB) and its extract (TE) compared to sources from two chemically synthetic ASTs (Carophyll Pink [CP] and Lucantin Pink [LP]), which contain 3S, 3'S, 3R, 3'S (3S, 3'R) and 3R, 3'R isoforms ratio of 1:2:1. The effects on red coloration, immune parameters and resistance to Vibrio infection were evaluated. Four AST sources were incorporated into the diets at concentrations of 0 (control), 100 mg kg-1 (TB100, TE100, CP100, and LP100), and 200 mg kg-1 (TB200, TE200, CP200, and LP200). Results revealed that in week 4, shrimps that received AST-supplemented feeds, especially TB100, TB200, and TE200, significantly increased redness (a*) values. Immune responses including phagocytosis activity, superoxide-anion production, phenoloxidase activity, and immune-related genes were examined on days 0, 1, 2, 4, 7, 14, 21, and 28. Generally, shrimps that received AST-supplemented feeds exhibited higher immune responses on days 7 and 14 than the control feed. Gene expression levels of superoxide dismutase and glutathione peroxidase were significantly upregulated on days 7 and 14 in shrimps that received AST-supplemented feeds, while genes of penaeidins, antilipopolysaccharide factor, and lysozyme were upregulated on days 4, 7, and 14, especially received TB200 and TE200. Furthermore, shrimps that received TB100, TE100, CP100, and LP100 7 days were then challenged with Vibrio parahaemolyticus and the result demonstrated higher survival rates especially TB100 at 168 h than the control feed. In conclusion, incorporating AST into the diets enhanced shrimp red coloration, immune parameters, and resistance against V. parahaemolyticus infection. The K. marxianus-derived AST exhibited higher performance than did chemical AST to be a potential feed additive in shrimp aquaculture.

Dietary supplementation of Salvinia cucullata in white shrimp Litopenaeus vannamei to enhance the growth, nonspecific immune responses, and disease resistance to Vibrio parahaemolyticus.

The current study investigates the effect of ethanolic extract of Salvinia cucullata (EESC) on growth, non-specific immune parameters, and disease resistance to Vibrio parahaemolyticus in Litopenaeus vannamei. The in-vitro cytotoxicity investigation was performed on shrimp hemolymph hemocytes to assess the toxicity and immunological responses with various concentrations of EESC, and no significant difference in cell viability was seen across dosages, but substantial changes in Phenol Oxidase (PO) and phagocytosis were reported. The in-vivo investigation was conducted on white shrimp for 56 days using varied amounts of 0 (control), 5 (EESC5), 10 (EESC10), and 20 (EESC20) g kg-1 containing feeds and challenged against Vibrio parahaemolyticus. The shrimp fed the EESC10 diet gained the most weight, had the highest specific growth rate (SGR) and had a better feed conversion ratio (FCR). The highest cumulative survival percentage was noted on the EESC10 diet-fed shrimps followed by EESC20 and EESC5 groups after the bacterial challenge with V. parahaemolyticus. The results of immune parameters such as total protein, total carbohydrate, coagulation time, total hemocytes count (THC), superoxide dismutase (SOD), ProPO, and phagocytosis levels were better in the EESC10 group. EESC5 and EESC20 groups were also shown better immunomodulatory effects than the control group. In conclusion, the oral administration of EESC was found to be an effective functional feed additive to improve the growth, immune parameters, and disease resistance against V. parahaemolyticus in L.vannamei.

Supplementing artemisinin positively influences growth, antioxidant capacity, immune response, gut health and disease resistance against Vibrio parahaemolyticus in Litopenaeus vannamei fed cottonseed protein concentrate meal diets.

Artemisinin (ART) is a kind of Chinese herbal medicine worth exploring, which obtains various physiological activities. In order to study the prebiotic effect of ART on Litopenaeus vannamei fed cottonseed protein concentrate meal diets, six groups of isonitrogenous and isolipid diets were prepared (including the fish meal control group, FM; cottonseed protein concentrate replacing 30% fishmeal protein and supplementing ART groups: ART0, ART0.3, ART0.6, ART0.9, and ART1.2). The feeding trials was lasted for 56 days. The results showed that the final body weight, weight gain and specific growth rate of the ART0.6 group were the highest, yet the feed coefficient rate of the ART0.6 group was the lowest significantly (P < 0.05). There was no significant difference in survival rate among treatments (P > 0.05). In serum, the content of malondialdehyde in ART0 group was the highest (P < 0.05); the activities of superoxide dismutase, catalase, phenol oxidase and lysozyme increased firstly and then decreased among the ARTs groups (P < 0.05). The activities of intestinal digestive enzymes (including the trypsin, lipase and amylase) showed an upward trend among the ARTs groups (P < 0.05). The histological sections showed that the intestinal muscle thickness, fold height and fold width in the FM group were significantly better than those in the ART0 group; while the mentioned above morphological indexes in the ART0 group were significantly lowest among the ARTs groups (P < 0.05). Sequencing of intestinal microbiota suggested that the microbial richness indexes firstly increased and then decreased (P < 0.05); the bacterial community structure of each treatment group was almost close; the relative abundance of pathogenic bacteria decreased significantly (P < 0.05), such as the Proteobacteria and Cyanobacteria at phylum level, besides the Vibrio and Candidatus Bacilloplasma at genus level. In intestinal tissue, the relative expression levels of TOLL1, TRAF6 and Pehaeidih3 showed up-regulated trends, while the expression of Crustin and LZM firstly up-regulated and then down-regulated (P < 0.05). The challenge experiment suggested that the cumulative mortality of FM group was significantly lower than that of ART0 group; besides the cumulative mortality firstly increased and then decreased between the ARTs groups (P < 0.05). In conclusion, the dietary supplementation of ART can improve the growth, antioxidant capacity, immune response, gut health and disease resistance of the shrimp. To be considered as a dietary immune enhancer, the recommended supplementation level of ART in shrimp's cottonseed protein concentrate meal diets is 0.43%.

Dietary of Lactobacillus paracasei and Bifidobacterium longum improve nonspecific immune responses, growth performance, and resistance against Vibrio parahaemolyticus in Penaeus vannamei.

This study investigated the effects of two probiotics, namely Lactobacillus paracasei and Bifidobacterium longum, as feed additives on growth performance, nonspecific immunity, immune-related gene expression, and disease resistance against Vibrio parahaemolyticus in Penaeus vannamei. The experimental diets were prepared using L. paracasei and B. longum at concentrations of 105 and 107 CFU/g; these diets were referred to as P5, P7, B5, and B7. After 8 weeks of the diets, regarding growth performance, the B7 group showed the highest weight gain rate (890.34 ± 103.65%), special growth rate (4.08 ± 0.19%), and feed conversion rate (1.52 ± 0.19%) compared with the other groups. Moreover, the total hemocyte counts were significantly increased (p < 0.05) in the P7 groups on day 14 during the 28-day feeding trial. The phagocytosis rate in all experimental groups was increased on day 14 and was persistently significantly activated to day 21, especially in the P7 and B5 group. The phagocytic index of the P7 group showed a significant increase on day 14 and persistent activation to day 21. In the analysis of respiratory burst activity and phenoloxidase activity, the P7 and B5 groups showed a significant increase on day 7 and persistent activation to day 21. The expression level of the immune-related genes of superoxide dismutase, clotting protein, Penaeidin2, Penaeidin3, Penaeidin4, anti-LPS factor, crustin, and lysozyme was significantly increased in the experimental groups, especially in the P7 group. Furthermore, the optimum conditions of feed additives were determined in challenge trials conducted using P7 and B5. Shrimps fed P7 and B5 showed an increased survival rate (72.73% and 66.67%) after the V. parahaemolyticus challenge. In sum, the results revealed that B. longum, as a feed additive at 107 CFU/g, enhanced growth performance. L. paracasei at 107 CFU/g and B. longum at 105 CFU/g can enhance nonspecific immune responses and immune-related gene expression, and 107 CFU/g L. paracasei has the highest resistance ability for V. parahaemolyticus. Thus, dietary supplementation with L. paracasei and B. longum may be a valuable approach in white shrimp aquaculture.

Identification of antibacterial metabolites produced by a marine bacterium Halobacillus marinus HMALI004.

AIMS: This study examined and characterized the extract for metabolites of Halobacillus marinus HMALI004 to understand their antibacterial activities against opportunistic marine pathogens, that is, Vibrio parahaemolyticus and Vibrio cholerae. METHODS AND RESULTS: The bacterial strain HMALI004 was characterized as H. marinus, and an antibacterial spectral test revealed its inhibition against two opportunistic marine pathogens (V. parahaemolyticus and V. cholera). Fermentation broth of strain HMALI004 was subjected to column chromatography and high-performance liquid chromatography to separate antibacterial substances. Two compounds were successfully isolated and identified as 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid by mass spectrometry (MS) and nuclear magnetic resonance. The minimal inhibition concentration (MIC) values of 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid for V. parahaemolyticus were 25 µg/ml, while their MIC values for V. cholerae were 50 and 100 µg/ml, respectively. The reactive oxygen species (ROS) production of two pathogen strains treated with 1H-pyrrole-2-carboxylic acid and 4-chloro-1H-pyrrole-2-carboxylic acid were detected to investigate the antimicrobial mechanism. The results suggested that 4-chloro-1H-pyrrole-2-carboxylic acid exerted enhanced ROS production in V. parahaemolyticus, whereas 1H-pyrrole-2-carboxylic acid had a weaker effect. Both compounds caused a significant rise in ROS production in V. cholerae, causing severe damage to the cell wall and cytoplasm, leading to cell death. CONCLUSIONS: The bacterium H. marinus HMALI004 was isolated from a shrimp pond and was found to produce antimicrobial compounds, which could inhibit the growth of opportunistic marine pathogens V. parahaemolyticus and V. cholerae by increasing ROS. SIGNIFICANCE AND IMPACT OF THE STUDY: Successfully isolated antibacterial-producing strain, H. marinus HMALI004, and its antimicrobial compounds could be used as biological control agents for marine pathogens.

Dietary supplementation with probiotic Rhodobacter sphaeroides SS15 extract to control acute hepatopancreatic necrosis disease (AHPND)-causing vibrio parahaemolyticus in cultivated white shrimp.

Cultivation of Penaeus vannamei (Pacific white shrimp) is faced with the serious problem of acute hepatopancreatic necrosis disease (AHPND), caused by Vibrio parahaemolyticus that carries plasmids containing binary toxin genes. The disease is typically moderated by the use of antibiotics. To investigate the control of AHPND and maintenance of water quality without the use of antibiotics, the supplementation of shrimp feed with anti-vibrio compounds from a crude extract of probiotic Rhodobacter sphaeroides SS15 was evaluated. The experimental design comprised four treatments: two that were challenged with AHPND-causing V. parahaemolyticus SR2 at a density of 6.0 × 105 cells mL-1 and two that were not challenged. The unchallenged groups comprised a control group that received commercial feed only (CF) and a group that received CF supplemented with 0.27% (w/w) of the extract of R. sphaeroides SS15 (modified CF: MCF). The treatments challenged with V. parahaemolyticus SR2 comprised a challenge group that received CF only (challenge CF: CF-SR2) and a challenge group that received modified CF (challenge MCF: MCF-SR2). V. parahaemolyticus SR2 was inoculated at the start of cultivation and at day 48 at the same cell density. No significant difference in growth performance was found among all treatments. All water quality parameters were better in the two treatments that received modified CF but excess nitrite, due to overfeeding in low salinity (5-8 ppt), caused shrimp mortality in all treatments. Vibrio populations were much higher in the CF treatments than in the modified CF treatments. After the first challenge, the survival rate was about 67% in both the CF-SR2 and MCF-SR2 treatments, compared with approximately 83% in the unchallenged treatments. One day after the second challenge, mortality in the CF-SR2 treatment was 100%, whereas 16.67% survived in the MCF-SR2 treatment. The survival rate was roughly 27% higher in the MCF treatment than in the CF treatment. The hepatopancreas and gut of both modified CF treatments showed no sign of AHPND. Via better water quality and trained immunity, the anti-vibrio compounds in the modified CF have great potential to increase the survival of cultivated shrimp infected with AHPND-causing strain SR2.

A gelatinized lipopeptide diet effectively modulates immune response, disease resistance and gut microbiome in Penaeus vannamei challenged with Vibrio parahaemolyticus.

Penaeus vannamei is one of the most economically vital shrimp globally, but infectious diseases have hampered its proper production and supply. As antibiotics pose a huge threat to the environment and humankind, it is essential to seek an alternative strategy to overcome infection and ensure proper culture and production. The present study investigates the effect of an anti-infective biosurfactant derivative lipopeptide MSA31 produced by a marine bacterium on the growth performance, disease resistance, and the gut microbiome of P. vannamei when challenged with pathogenic Vibrio parahaemolyticus SF14. The shrimp were fed with a commercial and lipopeptide formulated diet for 60 days and the growth performance was analyzed. The lipopeptide fed shrimp group showed enhanced growth performance and specific growth rate with improved weight gain than the control group. The challenge experiment showed that the survival rate was significant in the lipopeptide fed group compared to the control group. The results revealed 100% mortality in the control group at the end of 12 h of challenge, while 50% of the lipopeptide diet-fed group survived 24 h, which indicates the enhanced disease resistance in shrimp fed with a lipopeptide diet. The test group also showed higher levels of digestive and immune enzymes, which suggests that the lipopeptide diet could positively modulate the digestive and immune activity of the shrimp. The gut microbiome profiling by Illumina high-throughput sequencing revealed that the most abundant genera in the lipopeptide diet-fed group were Adhaeribacter, Acidothermus, Brevibacillus, Candidatus, Mycobacterium, Rodopila, and Streptomyces, while opportunistic pathogens such as Streptococcus, Escherichia, Klebsiella, Neisseria, Rhizobium, and Salmonella were abundant in the control diet-fed shrimp. Also, lipopeptide diet-fed shrimp were found to have a high abundance of ammonia and nitrogen oxidizing bacteria, which are essential pollutant degraders. Therefore, the study reveals that the dietary supplementation of lipopeptide in shrimp aquaculture could positively modulate the gut microbiome and enhance the shrimp's overall health and immunity in an eco-friendly manner.

Effects of dietary synbiotics supplementation methods on growth, intestinal health, non-specific immunity and disease resistance of Pacific white shrimp, Litopenaeus vannamei.

The present study aims to investigate the effects of dietary synbiotics supplementation methods on growth, feed utilization, hepatopancreas and intestinal histology, non-specific immunity and microbiota community of Pacific white shrimp (Litopenaeus vannamei). A control diet was designed to contain 18% fish meal (CON), and then 3 g kg-1 synbiotics (Bioture, consisting of Bacillus subtilis, Saccharomyces cerevisiae, ß-glucan and mannan oligosaccharide, etc) was supplemented to the control diet with three methods, directly adding in diets for pelleting (DAP), spraying diets after pelleting at once (SDA), spraying diets before feeding every day (SDE). Shrimp with initial body weight of 1.5 ± 0.12 g were fed one of the four diets for 56 days. The results showed that dietary synbiotics significantly increased the weight gain (WG), apparent digestibility coefficient (ADC) of crude protein (CP) and dry matter (DM), hepatopancreatic protease activity and decreased feed conversion ratio (FCR) (P < 0.05). Among the three synbiotics-added diets, SDE group showed the best growth with significantly higher WG than DAP group (P < 0.05). Serum activities of total superoxide dismutase, catalase, acid phosphatase, lysozyme and alkaline phosphatase of synbiotics-added groups were significantly higher, and serum malondialdehyde level was significantly lower than those of the control (P < 0.05). The intestinal villus width and villus number were also increased by the supplementation of synbiotics. The cumulative mortality was reduced in the three synbiotics-added groups after challenging with Vibrio parahaemolyticus (P < 0.05), and SDE group showed a significantly lower mortality than the control and DAP groups (P < 0.05). In intestinal microbiota composition, the abundance of Lactococcus tended to increase and Vibro tended to decreased in SDA and SDE groups. In conclusion, dietary synbiotics improved the growth, feed utilization, intestine health and non-specific immunity of Pacific white shrimp, and spraying synbiotics on diet presented better performance than adding synbiotics in diet for pelleting.

Marine macroalga-associated heterotroph Bacillus velezensis as prospective therapeutic agent.

Marine macroalgae and their accompanying microbial flora were proved to be the reservoir of potential bioactive compounds with promising pharmacological applications. Heterotrophic bacteria concomitant with the marine algae were isolated and screened for their antibacterial potential against clinically recognized pathogens. The bacterial isolate with greater bioactive properties was identified as Bacillus velezensis MBTDLP1 (phylum Firmicutes), which was isolated from the marine macroalga Laurencia papillosa, by integrated morphological, biochemical and molecular characterization. B. velezensis showed promising antibacterial property against methicillin-resistant Staphylococcus aureus and Vibrio parahemolyticus with inhibition zone of 32-36 mm. Organic ethyl acetate extract of the isolate also displayed prospective antibacterial activity against the test pathogens (minimum inhibitory concentration 7.5-15 µg/mL), coupled with promising antioxidant (IC50 0.1-0.9 mg/mL against oxidants), anti-inflammatory (IC50 0.01 mg/mL against 5-lipoxygenase), and carbolytic enzyme attenuation properties (IC50 0.1-0.4 mg/mL in response to α-amylase and α-glucosidase). Significant anticancer potential against breast carcinoma (MCF-7) cells (IC50 0.03 mg/mL) coupled with lesser cytotoxicity to the normal fibroblast (3T3L) cells (IC50 0.14 mg/mL) were also recognized. The apoptosis assay could give reasonable outcome as the organic extract of B. velezensis induced apoptosis to 81% of the cancer cells while maintaining almost 60% viability in normal cells. The results put forward that B. velezensis MBTDLP1 could be used to isolate bioactive compounds with therapeutic potential and biomedical applications.

Pandanus tectorius fruit extract promotes Hsp70 accumulation, immune-related genes expression and Vibrio parahaemolyticus tolerance in the white-leg shrimp Penaeus vannamei.

Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.

Effect in white shrimp Litopenaeus vannamei of Eleutherine bulbosa (Mill.) Urb. Powder on immune genes expression and resistance against Vibrio parahaemolyticus infection.

This study investigated the influence of Eleutherine bulbosa (Mill.) Urb. on the immune responses, bacterial population in the intestines, and resistance of white shrimp, Litopenaeus vannamei, against infection with Vibrio parahaemolyticus. Shrimp were fed with three dosages of powder, at 6.25 g kg-1 (P6.25), 12.5 g kg-1 (P12.5), and 25 g kg-1 (P25). One dosage of the crude extract was provided, 1.25 g kg-1 (E1.25), and the controls without administration of E. bulbosa consisted of a positive control (PC) and a negative control (NC). Feed supplementation was carried out for 30 days; then shrimp from all treatments were challenged by intramuscular injection with V. parahaemolyticus (106 cfu/mL), except for the NC. The results showed that supplementation with the powder and extract of E. bulbosa for 30 days resulted in significantly higher (P < 0.05) immune responses (total hemocyte count (THC), phenoloxidase activity (PO), respiratory bursts (RBs)), gene expression (prophenoloxidase (proPO), lipopolysaccharide- and ß-1,3-glucan-binding protein (LGBP)), and total bacterial count (TBC) compared to PC/NC. In post challenge testing, there were significantly higher levels for THC, PO, RBs, proPO, LGBP, and PE (peroxinetin), and the treatments were able to suppress V. parahaemolyticus in the intestines, hepatopancreas, and muscles and to reduce damage to the muscles and hepatopancreas. The survival rate with P12.5 was significantly higher compared to the other treatments. It was concluded that the shrimp receiving supplementation with the powder and extract of E. bulbosa had increased immunity and resistance against V. parahaemolyticus infection, with the best dosage being the P12.5 treatment.

Phloroglucinol Treatment Induces Transgenerational Epigenetic Inherited Resistance Against Vibrio Infections and Thermal Stress in a Brine Shrimp (Artemia franciscana) Model.

Emerging, infectious diseases in shrimp like acute hepatopancreatic necrosis disease (AHPND) caused by Vibrio parahaemolyticus and mortality caused by other Vibrio species such as Vibrio harveyi are worldwide related to huge economic losses in industrial shrimp production. As a strategy to prevent disease outbreaks, a plant-based phenolic compound could be used as a biocontrol agent. Here, using the brine shrimp (Artemia franciscana) as a model system, we showed that phloroglucinol treatment of the parental animals at early life stages resulted in transgenerational inherited increased resistance in their progeny against biotic stress, i.e., bacteria (V. parahaemolyticus AHPND strain and V. harveyi) and abiotic stress, i.e., lethal heat shock. Increased resistance was recorded in three subsequent generations. Innate immune-related gene expression profiles and potential epigenetic mechanisms were studied to discover the underlying protective mechanisms. Our results showed that phloroglucinol treatment of the brine shrimp parents significantly (P < 0.05) enhanced the expression of a core set of innate immune genes (DSCAM, proPO, PXN, HSP90, HSP70, and LGBP) in subsequent generations. We also demonstrated that epigenetic mechanisms such as DNA methylation, m6A RNA methylation, and histone acetylation and methylation (active chromatin marker i.e., H3K4Me3, H3K4me1, H3K27me1, H3 hyperacetylation, H3K14ac and repression marker, i.e., H3K27me3, H4 hypoacetylation) might play a role in regulation of gene expression leading toward the observed transgenerational inheritance of the resistant brine shrimp progenies. To our knowledge, this is the first report on transgenerational inheritance of a compound-induced robust protected phenotype in brine shrimp, particularly protected against AHPND caused by V. parahaemolyticus and vibriosis caused by V. harveyi. Results showed that epigenetic reprogramming is likely to play a role in the underlying mechanism.

Mannan oligosaccharide increases the growth performance, immunity and resistance capability against Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino.

This trial was conducted to investigate the effect of mannose oligosaccharides (MOS) on the growth performance, antioxidation, immunity and disease resistance of Vibro Parahemolyticus in juvenile abalone Haliotis discus hannai Ino. Four formulated diets were produced to contain 0.00 g/kg, 0.40 g/kg, 0.80 g/kg and 1.60 g/kg Actigen®, with functional ingredients of MOS, respectively. Accordingly, the experimental diets were named as A0, A4, A8 and A16. After 120-days feeding trial, the best growth performance was observed in A8 group (P < 0.05) and there was no significant difference in A0, A4 and A16 groups. With the increase of dietary MOS, the activity of the total antioxidant capacity in hepatopancreas is increasingly elevated (P < 0.05) while no significant difference was observed on activity of glutathione S-transferase (P > 0.05). The activities of superoxide dismutase and glutathione peroxidase were firstly increased and then decreased, with the highest values in A8 group (P < 0.05). Immune-related parameters were significantly affected by dietary MOS inclusion. Specifically, the activities of alkaline phosphatase and acid phosphatase in hepatopancreas and serum of abalone fed diets containing MOS were significantly higher than those of control A0 group (P < 0.05). Moreover, the highest values of both enzymes were observed in hepatopancreas of A8 group but in serum of A16 group, respectively. The lysozyme activities in hepatopancreas and serum of A4 group were significantly higher than those of other groups (P < 0.05) and there was no significant difference in A0, A8 and A16 groups (P > 0.05). The activities of cytophagy and respiratory burst in serum of abalone were not significantly affected by dietary MOS content (P > 0.05). The mRNA levels of focal adhesion kinase and integrin-linked kinase were gradually elevated with the increase of dietary MOS, with the highest value recorded in A16 group (P < 0.05). The gene expression of caspse-3 in A8 group was dramatically higher than those of other groups (P < 0.05) and there was no significant difference in A0, A4 and A16 groups (P > 0.05). The mRNA level of nuclear factor-κB was not significantly affected by dietary MOS (P > 0.05). During 56 h of V. Parahemolyticus challenge period, the accumulated mortality rate of abalone fed diets containing MOS were significantly lower than that of control A0 group in each time point (P < 0.05). Overall, the lowest rate was happened in A8 group (P < 0.05). In conclusion, MOS inclusion in diet has obviously positive effect on growth, immunity and disease resistance capability of abalone, with the optimal level of Actigen® at 0.80 g/kg in diet.

Bioflocs substituted fishmeal feed stimulates immune response and protects shrimp from Vibrio parahaemolyticus infection.

Fishmeal is the main source of protein in the shrimp feed industry and is normally derived from trash fish. As such, the production of fishmeal has an adverse effect on the marine environment by taking away small and juvenile fish, leading to depletion of marine species. There is a need for alternative sources of protein which will substitute fishmeal in the aquaculture industry. This study evaluated the components and nutritional efficacy of bioflocs, which were used to substitute fishmeal protein. The effect of bioflocs diets on growth performance, survival rate, and immune response in shrimp compared to normal fishmeal feed were determined. Bioflocs were harvested from the shrimp ponds (C:N ratio >12:1) at Shrimp Village, Chaiya district, Surat Thani, Thailand. The total protein in bioflocs was about 48% and the total lipid was about 5% (dried weight) and the percentages of essential amino acids (EAA) and fatty acids (EFA) in bioflocs were similar to those of fishmeal feed. Shrimp fed with the different dietary bioflocs feed regimens [% to replace fishmeal; 0% (B0), 25% (B25), 50% (B50), 75% (B75), and 100% (B100)] for 42 days revealed that all growth parameters were almost similar to those of the control shrimp (shrimp fed with normal fishmeal, B0) including final body weight, weight gain, specific growth rate, and feed conversion ratio. Remarkably, the survival rates, the levels of immune parameters, and expression of immune genes (proPO-I, PEN-4 and dicer) were significantly higher in bioflocs fed shrimp, especially in B25 and B50 shrimp. Moreover, B25 and B50 bioflocs fed shrimp showed notably increased survival rates following Vibrio parahaemolyticus (V. parahaemolyticus) infection. In conclusion, the present study demonstrates that shrimp survival and immunity are enhanced by biofiocs substituted fishmeal. Significantly, the bioflocs diets activated the immune response to prevent V. parahaemolyticus infection.

Anti-PirA-like toxin immunoglobulin (IgY) in feeds passively immunizes shrimp against acute hepatopancreatic necrosis disease.

Acute hepatopancreatic necrosis disease (AHPND), caused by a toxin-producing Vibrio parahaemolyticus strain, has become a serious threat to shrimp aquaculture. The need to regulate antibiotic use prompted the development of alternative ways to treat infections in aquaculture including the use of chicken egg yolk immunoglobulin (IgY) for passive immunization. This study evaluated the protective effect of IgY against AHPND infection in Litopenaeus vannamei (Boone). IgY was isolated from eggs laid by hens immunized with recombinant PirA-like (rPirA) and PirB-like (rPirB) toxins. Whole-egg powders having IgY specific to rPirA (anti-PirA-IgY) and rPirB (anti-PirB-IgY) and IgY from non-immunized hen (control-IgY) were mixed with basal diets at 20% concentrations and used to prefeed shrimp 3 days before the bacterial challenge test. Survival rates of the challenged shrimp fed the anti-PirA-IgY, anti-PirB-IgY and control-IgY diets were 86%, 14% and 0%, respectively. Only the feed containing anti-PirA-IgY protected shrimp against AHPND. Increasing the concentration of rPirA antigen to immunize hens and lowering the amount of egg powder in feeds to 10% consistently showed higher survival rates in shrimp fed with anti-PirA-IgY (87%) compared with the control (12%). These results confirm that addition of anti-PirA-IgY in feeds could be an effective prophylactic method against AHPND infection in shrimp.

Evaluation of combination effects of Astragalus polysaccharides and florfenicol against acute hepatopancreatic necrosis disease-causing strain of Vibrio parahaemolyticus in Litopenaeus vannamei.

The effects of oral administration of Astragalus polysaccharides (APS) and florfenicol (FFC), singly or in combination, on the survival performance, disease resistance, and immunity of Litopenaeus vannamei were investigated. After challenge with an AHPND-causing strain of Vibrio parahaemolyticus (VPAHPND), shrimp were immediately fed a drug-free diet, diets containing only APS (200 mg·kg-1) or FFC (15 mg·kg-1), or diets containing low-dose (7.5 mg·kg-1 FFC + 100 mg·kg-1 APS), medium-dose (15 mg·kg-1 FFC + 200 mg·kg-1 APS), and high-dose (30 mg·kg-1 FFC+400 mg·kg-1 APS) drug combinations for 5 days. The cumulative shrimp mortality over 5 days after injection of VPAHPND in the APS + FFC combination groups was significantly lower than that in the APS or FFC alone groups (p < 0.05). Immune parameters, including the total hemocyte counts (THCs), hemocyanin (HEM) concentration, antibacterial activity, activity levels of lysozyme (LZM), and levels of acid phosphatase (ACP), alkaline phosphatase (AKP), and phenoloxidase (PO) in cell-free hemolymph, and the expression levels of the immune-related genes anti-lipopolysaccharide factor (ALF), cathepsin B (catB), crustin, lectin (Lec), lysozyme (LZM), and Toll-like receptor (TLR) in hemocytes and hepatopancreas were determined in the shrimp. The values for these immune parameters in the drug combination groups were higher than those in the APS or FFC group (p < 0.05). Finally, in the histological examinations, the histological structural alignment and integrity of the hepatopancreatic tubules in the drug combination groups was better than that in the APS and FFC groups. Under the experimental conditions, dietary APS and FFC had a synergistic effect on immunity and disease resistance among shrimp after VPAHPND infection.

Dietary values of Forsythia suspensa extract in Penaeus monodon under normal rearing and Vibrio parahaemolyticus 3HP (VP3HP) challenge conditions: Effect on growth, intestinal barrier function, immune response and immune related gene expression.

Two trials were conducted to determine the effects of dietary Forsythia suspensa extract (FSE) on shrimp, Penaeus monodon, first on growth performance, second on the immune response and immune related gene expression of shrimp. In trial 1, shrimp (mean initial wet weight about 3.02 g) were fed with five diets containing 0% (basal diet), 0.01%, 0.02%, 0.04% and 0.06% FSE in triplicate for 60 days. Growth performance (final body wet weight, FBW; weight gain, WG; biomass gain, BG) of shrimp fed FSE diets were higher (P < 0.05) than that of shrimp fed the basal diet. The survival among all the diets treatments were above 90% and no significant difference was revealed among them (P > 0.05). The antioxidant capacity (total antioxidant status, TAS; glutathione peroxidase, GSH-Px) appears in the trend of firstly increasing then decreasing with the increasing of dietary FSE levels. The highest value of TAS and GSH-Px were found in shrimp fed 0.02% FSE diet and were significantly higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Hepatopancreas malondialdehyde (MDA) of shrimp fed FSE diets were lower (P < 0.05) than that of shrimp fed the basal diet. Total haemocyte count of shrimp fed the basal diet was lower (P < 0.05) than that of shrimp fed FSE diets. Haemolymph clotting time of shrimp had the opposite trend with the total haemocyte count of shrimp. No significant differences were found in haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) and in molecular gene expression profiles of heat shock protein 70 (Hsp 70) mRNA and hypoxia inducible factor-1α (HIF-1α) mRNA in haemolymph of shrimp among all diet treatments (P > 0.05). In trial 2, a pathogenic strain of Vibrio parahaemolyticus 3HP (VP3HP) injection challenge test was conducted for 6-day after the rearing trial and shrimp survival were also compared among treatments. Survival of shrimp fed diets supplemented with 0.01%-0.02% FSE were higher than that of shrimp fed the basal and 0.06% FSE diets (P < 0.05). Dietary FSE supplementation produced stronger hepatopancreas antioxidant capacity (TAS, GSH-Px) (P < 0.05) and higher glutathione (GSH) level (P < 0.05), lower superoxide dismutase activity (SOD) (P < 0.05), higher total haemocyte count (P < 0.05), lower haemolymph clotting time (P < 0.05), lower MDA and carbonyl protein concentration (P < 0.05), lower haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) (P < 0.05), generated lower molecular gene expression profiles of HSP 70 mRNA and higher HIF-1α mRNA (P < 0.05) than the basal diet. The immune response were characterized by lower TAS and higher antioxidant enzyme activities (SOD, GSH-Px) and higher oxidative stress level (MDA and carbonyl protein) and higher haemolymph biomarkers of intestinal permeability (endotoxin and diamine oxidase) compared to levels found in trail 1. However, the total haemocyte counts and haemolymph clotting times were not changed in 0.01%-0.02% FSE diets treatments between trial 1 and trial 2 (P > 0.05). The molecular gene expression profile of Hsp 70 mRNA was increased while HIF-1α mRNA was decreased when compared to trial 1. In conclusion, results suggested that dietary intake containing FSE could enhance the growth performance and antioxidant capacity of P. monodon and furthermore reduce oxidative stress and immune depression challenged by a pathogenic strain of Vibrio parahaemolyticus stress. Considering the effect of FSE on both growth performance and immune response of P. monodon, the level of FSE supplemented in the diet should be between 0.01% and 0.02%.