Morinda citrifolia fruit extract enhances the resistance of Penaeus vannamei to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a gram-negative facultative anaerobic bacterium implicated as the causative agent of several shrimp diseases. As part of the effort to provide biocontrol and cost-effective treatments, this research was designed to elucidate the effect of Morinda citrifolia fruit extract on the immunity of Penaeus vannamei postlarvae (PL) to V. parahaemolyticus. The methanol extract of M. citrifolia was vacuum evaporated, and the bioactive compounds were detected using gas chromatography‒mass spectrometry (GC‒MS). Thereafter, P. vannamei PL diets were supplemented with M. citrifolia at different concentrations (0, 10, 20, 30, 40, and 50 mg/g) and administered for 30 days before 24 h of exposure to the bacterium V. parahaemolyticus. A total of 45 bioactive compounds were detected in the methanol extract of M. citrifolia, with cyclononasiloxane and octadecamethyl being the most abundant. The survival of P. vannamei PLs fed the extract supplement was better than that of the control group (7.1-26.7% survival greater than that of the control group) following V. parahaemolyticus infection. Shrimp fed 50 mg/g M. citrifolia had the highest recorded survival. The activities of digestive and antioxidant enzymes as well as hepatopancreatic cells were significantly reduced, except for those of lipase and hepatopancreatic E-cells, which increased following challenge with V. parahaemolyticus. Histological assessment of the hepatopancreas cells revealed reduced cell degeneration following the administration of the plant extracts (expecially those fed 50 mg/g M. citrifolia) compared to that in the control group. Therefore, the enhanced immunity against V. parahaemolyticus infection in P. vannamei could be associated with the improved hepatopancreas health associated with M. citrifolia fruit extract supplementation.

Complete genome sequence and characterization of a novel Enterococcus faecium with probiotic potential isolated from the gut of Litopenaeus vannamei.

Litopenaeus vannamei, the Pacific whiteleg shrimp, is one of the most marketable species in aquaculture worldwide. However, it is susceptible to different infections causing considerable losses in production each year. Consequently, using prebiotics that promotes the proliferation of beneficial bacteria and strengthen the immune system is a current strategy for disease control. In this study, we isolated two strains of E. faecium from the gut of L. vannamei fed with agavin-supplemented diets. These isolates showed antibacterial activity against Vibrio parahaemolyticus, Vibrio harveyi and Vibrio alginolyticus, most likely due to peptidoglycan hydrolase (PGH) activity. Furthermore, we sequenced the genome of one isolate. As a result, we observed three proteins related to the production of bacteriocins, a relevant trait for selecting probiotic strains since they can inhibit the invasion of potential pathogens. Additionally, the genome annotation showed genes related to the production of essential nutrients for the host. It lacked two of the most common factors associated with virulence in Enterococcus pathogenic strains (esp and hyl). Thus, this host-probiotic-derived strain has potential application not only in shrimp health but also in alternative aquatic environments, as it is adapted to coexist within the gut shrimp microbiota, independently of the diet.

Comparative effects of different bacterial lipopolysaccharides on modulation of immune levels to improve survival of the black tiger shrimp.

To prevent loss from disease, immunostimulants have been used as dietary supplements to improve immunity and survival of shrimps. Among the various types of immunostimulants, there is increasing evidence that a diet enriched with bacterial lipopolysaccharide can reduce the mortality rate of shrimp under exposure to pathogens. Here, the immunostimulatory effects of bacterial lipopolysaccharide (LPS) from various bacterial sources were explored. Bacterial LPS was extracted from a shrimp pathogen, Vibrio harveyi and its effects were compared with the commercially available LPS from the non-shrimp pathogen, Escherichia coli. Our results revealed that the LPS from V. harveyi was different in molecular size but contained similar functional groups to that from E. coli. To understand their molecular mechanisms, bacterial LPS from the two sources were applied as a supplementary diet and fed to juvenile shrimp for 4-week feeding period before tissue samples were collected for transcriptomic analysis by next generation sequencing. Gene expression profiling revealed that major immune-related genes such as pattern recognition proteins (PRPs), proteinases and proteinase inhibitors, prophenoloxidase systems (proPO system), antimicrobial peptides (AMPs), signaling transduction pathways, heat shock proteins (HSPs), oxidative stress responses, and other immune-related molecules such as mucins and peritrophins were modulated in the groups of shrimp fed with bacterial LPS from both sources, but at different levels. The results suggest that bacterial LPS could modulate shrimp immune system, and different LPS sources led to different activation of immune pathways. Additionally, metabolic-related genes were affected by LPS, suggesting that energy was required for immune stimulation. In the V. harveyi pathogen challenge trial, all shrimp groups fed with diets containing LPS from both bacterial sources showed better survival than the control group without LPS. When comparing groups fed with LPS supplemented diets, the higher concentration of LPS (8 µg/body weight) from E. coli resulted in a better survival rate than a lower concentration (4 µg/body weight). Conversely, shrimp fed with a diet containing LPS from V. harveyi showed a lower survival rate when a higher dose of LPS (8 µg/body weight) was administered than the group fed with a lower concentration of LPS (4 µg/body weight). This could be due to overstimulation of shrimp immune responses, especially by LPS derived from shrimp pathogens, resulting in a reverse effect. These results confirm that immunity in shrimp upon administration of bacterial LPS depends on the origin and dose of the LPS administered.

Shrimp production, the most important diseases that threaten it, and the role of probiotics in confronting these diseases: A review.

Probiotics are increasingly used in aquaculture to protect cultivated organisms from pathogens and improve water quality and feed efficiency. Shrimp are the most commonly cultivated aquaculture species worldwide. In this current review, we will explore the current challenges that impact shrimp aquaculture and summarize how probiotics are utilized to improve production. Probiotics are live microorganisms that are described as friendly bacteria or healthy bacteria supplemented to the shrimp gut through feeds. Probiotics have been demonstrated to increase gastrointestinal stability, secrete antibacterial compounds, compete with pathogens to prevent intestinal adhesion, compete for the nutrients required for pathogen survival and produce antitoxin effects. Probiotics can also modulate the immune system and control the allergic response of the body. Therefore, this review sheds light on the challenges faced in shrimp production as well as the different types of probiotics, their methods of application, their modes of action, their role in improving shrimp production, and their influence on water quality.

Process optimization for fermented siwu decoction by multi-index-response surface method and exploration of the effects of fermented siwu decoction on the growth, immune response and resistance to Vibrio harveyi of Pacific white shrimp (Litopenaeus vannamei).

The purpose of this study was to explore the optimal fermentation technology of Chinese herbal medicine formula-Siwu Decoction and the effects of fermented Siwu Decoction (FSW) on the growth performance, immune response, intestinal microflora and anti microbial ability of Litopenaeus vannamei. Response to surface methodology (RSM) was used to optimize the fermentation process of Siwu Decoction. The optimal fermentation conditions were obtained as follows: inoculation amount of mixed strains was 4.5%, fermentation time was 36 h, and the ratio of material to liquid was 20%. A total of 1260 shrimps were selected and divided into seven groups, three in parallel in each group. The dietary level of each group was as follows: Control （No additions）, USW1 (0.2% unfermented herbal medicine), USW2 (0.5% unfermented herbal medicine), USW3 (0.8% unfermented herbal medicine), FSW1 (0.2% fermented herbal medicine), FSW2 (0.5% fermented herbal medicine), FSW3 (0.8% fermented herbal medicine). The immune response and antioxidant defense ability of hemocytes and intestine were measured at 21 and 42 days of feeding and the intestinal flora and growth performance were measured at 42 days of feeding, after that, a 7-day challenge test against Vibrio harveyi was conducted. The results showed that fermented Siwu Decoction significantly improved the growth performance and body composition of Litopenaeus vannamei; significantly increased the total number of hemocytes, phagocytic activity, antibacterial activity and bacteriolytic activity of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei; the addition of fermented Siwu Decoction significantly increased the gene expression level of hemocytes and intestinal tract of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei. The abundance of Bacillus increased, while the abundance of Vibrio decreased. After Vibrio harveyi challenge, the cumulative mortality of FSW group was significantly lower than that of control group. Fermented Siwu Decoction may be a potential physiological enhancer in aquaculture, and can be widely used in aquaculture.

Dietary supplementation of curcumin-loaded chitosan nanoparticles stimulates immune response in the white leg shrimp Litopenaeus vannamei challenged with Vibrio harveyi.

Pathogen infections in shrimps trigger the release of reactive oxygen species (ROS) as a part of immune response. The excessive accumulation of ROS causes the production of oxidative stress, which leads to oxidative damage of the biomolecules in the host cells. The inclusion of dietary antioxidants is known to mitigate oxidative stress and stimulate immunity. Curcumin, a potential antioxidant was encapsulated in chitosan nanoparticles to surge its bioavailability and was administered orally to Vibrio harveyi challenged and non-challenged Litopenaeus vannamei. The non-challenged shrimps fed with curcumin-loaded chitosan nanoparticles (Cur-CSNPs) showed a significant increase (p ≤ 0.05) in the specific growth rate, daily growth coefficient and survival rate. A significant increase (p ≤ 0.05) in the phenoloxidase activity, total hemocyte count and superoxide dismutase activity was observed in both the challenged and non-challenged shrimps fed with Cur-CSNPs. Additionally, a significant increase (p ≤ 0.05) in the relative mRNA expression of lysozyme, cMnSOD and lectin was observed in the Cur-CSNPs fed shrimps. The findings of this research suggest that Cur-CSNPs reinforce the immune system of L. vannamei against V. harveyi infection. Moreover, the non-challenged shrimps showed improvement in the growth parameters in addition to immunostimulation. Thereby a routine inclusion of dietary Cur-CSNPs could mitigate the oxidative damage caused by the incidence of environmental or pathogen-mediated oxidative stress.

Curcumin-mediated sono/photodynamic treatment preserved the quality of shrimp surimi and influenced its microbial community changes during refrigerated storage.

Shrimp surimi is widely acknowledged as a value-added shrimp product due to its delicious taste, rich flavor, and nutrition. However, the refrigerated shrimp surimi is prone to deterioration due to rapid microbial growth during storage. The present study sought to assess the effects of curcumin-mediated sono/photodynamic treatment on bacterial spoilage and shrimp surimi quality stored at 4 °C. The total viable count (TVC), microbiota composition, and quality parameters, including the total volatile basic nitrogen (TVB-N), thiobarbituric acid reactive substance (TBARs), and pH were investigated. The results showed that the spoilage bacteria in shrimp surimi rapidly increased with a surge on day 2 during refrigeration storage. The Psychrobacter and Brochothrix were identified as the Specific Spoilage Organisms (SSOs), which were also positively correlated with TVB-N and TBARs. The results further elucidated that the sono/photodynamic treatment could significantly inhibit the growth of SSOs on the surface and interior of shrimp surimi and delay shrimp surimi quality deterioration. In conclusion, the sono/photodynamic treatment as a non-thermal sterilization method could be a reliable and potential method for inactivating spoilage microorganisms and preserving shrimp surimi quality.

Guava (Psidium guajava) leaf extract enhances immunity, growth, and resistance against Vibrio parahaemolyticus in white shrimp Penaeus vannamei.

This study investigated the effects of guava leaf extract (GLE) on immune responses, growth performance, and resistance to Vibrio parahaemolyticus in white shrimp (Penaeus vannamei). To examine the effect of GLE on the immune response of white shrimps, they were treated with various concentrations of GLE on hemocyte (in vitro) and were orally administered (in vivo) feed containing various concentrations of 0, 1, 5, and 10 g kg-1 GLE (control, GLE1, GLE5, and GLE10, respectively) for 28 days. Furthermore, their growth performance was evaluated for 56 days. In a separate experiment, the shrimps were challenged with V. parahaemolyticus injection after 7 days of culture. In vitro experiments indicated that GLE is nontoxic and can activate immune response. In vivo experiments revealed that the GLE5 led to the highest total hemocyte count, phenoloxidase activity, phagocytic activity, and superoxide anion production and the highest upregulation of lipopolysaccharide, ß-1,3-glucan-binding protein, peroxinectin, lysozyme, crustin, penaeidin 2, penaeidin 3, clotting protein, superoxide dismutase, and glutathione peroxidase. Moreover, better growth performance was observed in the GLE groups, with GLE5 exhibiting the highest specific growth rate, weight gain, and feed conversion rate. In addition, GLE5 enhanced resistance to V. parahaemolyticus, with a survival rate of 72.27%. In conclusion, GLE was found to be effective in enhancing nonspecific immune response and growth performance and in reducing V. parahaemolyticus infection in white shrimp.

Therapeutic effect and immune mechanism of chitosan-gentamicin conjugate on Pacific white shrimp (Litopenaeus vannamei) infected with Vibrio parahaemolyticus.

To explore the disease resistance mechanism of chitosan conjugates, chitosan-gentamicin conjugate (CS-GT) was synthesized and systematically characterized, the immune mechanism of CS-GT on Litopenaeus vannamei infected with Vibrio parahaemolyticus was further explored. The results showed that imine groups in CS-GT were effectively reduced. Dietary supplementation of CS-GT can significantly increase the survival rate, total hemocyte counts, the antioxidant and immune related enzyme activity levels of shrimps (P < 0.05), which are all dose-dependent under the experimental conditions. In addition, CS-GT can protect the hepatopancreas from invading bacteria and alleviate inflammation. Particularly, CS-GT promotes the expressions of legumain (LGMN), lysosomal acid lipase (LIPA) and Niemann-Pick type C2 (NPC2) up-regulated. It is speculated that CS-GT may stimulate the lysosome to phagocytose pathogens more effectively. In conclusions, shrimps fed with CS-GT can produce immune response via lysosome and greatly improve the disease resistance to Vibrio parahaemolyticus.

Molecular docking study of bio-inhibitors extracted from marine macro-alga Ulva fasciata against hemolysin protein of luminescence disease-causing Vibrio harveyi.

Shrimp grow-out and hatchery systems are being affected by bacterial disease particularly Vibrios. The use of chemotherapeutic agents in aquaculture practices has to lead to the development of resistance among aquatic bacteria. Thus, health management becomes of major importance in aquaculture. Under this situation, progressing bio-inhibitors from marine resources are most appropriate to be considered against pathogenic bacteria. Molecular docking is an appropriate tool in structural biology and computer-assisted drug design to predict and neutralize a target protein of known diseases. In this study, marine macro-alga Ulva fasciata was aimed at developing inhibitors against luminescence disease-causing pathogenic bacteria Vibrio harveyi. U. fasciata was collected from Thoothukudi, Tamil Nadu, India. Extract of U. fasciata was tested against growth and virulence factors of V. harveyi during Penaeus monodon larviculture. Further U. fasciata extract was subjected to GC-MS analysis to identify the biomolecules. The homology modeling of virulent protein, hemolysin of V. harveyi was designed in this study. Hence, it was aimed for molecular docking against the biomolecules identified from U. fasciata extract. During shrimp larviculture, the extract of U. fasciata (200 µg mL-1) exhibited reduction on Cumulative Percentage of Mortality (32.40%) in postlarvae against challenge of V. harveyi infection. Biomolecule Methyl dehydroabietate had showed highest binding affinity among the compounds was evaluated in molecular docking study. Statistical analysis had revealed significant differences (p < 0.05) in trials. Therefore, it was proved that the bio-inhibitors from U. fasciata will be a better option for controlling luminescence disease-causing V. harveyi in shrimp grow-out practices.

Effects of isoquinoline alkaloids from Macleaya cordata on growth performance, survival, immune response, and resistance to Vibrio parahaemolyticus infection of Pacific white shrimp (Litopenaeus vannamei).

Isoquinoline alkaloids (IQs) from Macleaya cordata are promising natural products for enhancing the growth performance and overall health condition of farmed animals. The present study aimed to investigate the effects of two formulas of IQ extract, provided in either a powdered formula (IQ-E) or a water-soluble, granulated formula (IQ-WS) and containing the main active component sanguinarine at a concentration of 0.5% and 1%, respectively, on the growth, survival, immune response, and resistance to Vibrio parahaemolyticus infection of Pacific white shrimp (Litopenaeus vannamei). In Experiment 1, the postlarvae were divided into five groups (four replicates/group and 100 shrimp/tank) and fed four times/day for 30 days with a control feed, IQ-E at 200 or 300 mg/kg of feed, or IQ-WS at 100 or 150 mg/kg of feed. In Experiment 2, the surviving shrimp from Experiment 1 were redistributed into six groups (four treatment groups as in Experiment 1 plus the positive and negative controls with four replicates/group and 30 shrimp/tank) and challenged with V. parahaemolyticus by immersion at a concentration of 103 colony-forming units (CFU)/mL and were fed with the same diets for another 14 days. The results revealed that all IQ-fed shrimp in Experiment 1 had significantly enhanced survival rates and immune parameters (total hemocyte count and phagocytic, phenoloxidase, and superoxide dismutase activities) compared to the control group, even though the growth performances were similar across all groups. In Experiment 2, all IQ-fed groups showed better growth performance and survival rates compared to the positive control. Other than in the positive control group, no histopathological lesions in the hepatopancreas and the intestine were found. In summary, the current study demonstrated the benefits of using IQs from M. cordata as feed additives for improving the growth performance, survival rate, immune responses, and resistance to vibriosis of Pacific white shrimp.

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.

Understanding the role of the shrimp gut microbiome in health and disease.

With rapid increases in the global shrimp aquaculture sector, a focus on animal health during production becomes ever more important. Animal productivity is intimately linked to health, and the gut microbiome is becoming increasingly recognised as an important driver of cultivation success. The microbes that colonise the gut, commonly referred to as the gut microbiota or the gut microbiome, interact with their host and contribute to a number of key host processes, including digestion and immunity. Gut microbiome manipulation therefore represents an attractive proposition for aquaculture and has been suggested as a possible alternative to the use of broad-spectrum antibiotics in the management of disease, which is a major limitation of growth in this sector. Microbiota supplementation has also demonstrated positive effects on growth and survival of several different commercial species, including shrimp. Development of appropriate gut supplements, however, requires prior knowledge of the host microbiome. Little is known about the gut microbiota of the aquatic invertebrates, but penaeid shrimp are perhaps more studied than most. Here, we review current knowledge of information reported on the shrimp gut microbiota, highlighting the most frequently observed taxa and emphasizing the dominance of Proteobacteria within this community. We discuss involvement of the microbiome in the regulation of shrimp health and disease and describe how the gut microbiota changes with the introduction of several economically important shrimp pathogens. Finally, we explore evidence of microbiome supplementation and consider its role in the future of penaeid shrimp production.

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.

Effects of Phyllanthus amarus extract on nonspecific immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp Litopenaeus vannamei.

This study investigates the effects of Phyllanthus amarus extract (PAE) on immune responses, growth, and resistance to Vibrio alginolyticus in white shrimp (Litopenaeus vannamei). In vitro PAE treatment did not alter the cell viability of haemocytes and significantly enhanced immune parameters such as phenoloxidase (PO) activity, phagocytic activity, and superoxide anion (O2-) production. We conducted two feeding trials to examine the effects of PAE on the growth, disease resistance, and innate immune parameters of white shrimp. In the first in vivo trial, shrimps (4.01 ± 0.03 g) were fed a diet containing 0 g (control), 10 g (PAE10), 20 g (PAE20), or 40 g (PAE40) of PAE per kilogram of feed for 56 days. After the feeding period, the PAE20 group showed a significantly higher weight gain and specific growth rate than shrimp fed the control diet. Furthermore, after challenge with V. alginolyticus, shrimp fed a diet containing PAE showed significantly higher survival than those fed the control diet. The second in vivo trial (28 days) was performed to identify the mechanisms of enhanced immunity in PAE-fed shrimp. Shrimp fed the PAE20 diet generally had the highest total haemocyte count, PO activity, phagocytic activity, and O2- production, followed by the PAE40 and PAE10 groups. Thus, our results suggest that administration of 20 g of PAE per kilogram of feed can enhance immunity, growth, and resistance to V. alginolyticus in white shrimp.

Effects of Chitosan-Gentamicin Conjugate Supplement on Non-Specific Immunity, Aquaculture Water, Intestinal Histology and Microbiota of Pacific White Shrimp (Litopenaeus vannamei).

When the aquaculture water environment deteriorates or the temperature rises, shrimp are susceptible to viral or bacterial infections, causing a large number of deaths. This study comprehensively evaluated the effects of the oral administration of a chitosan-gentamicin conjugate (CS-GT) after Litopenaeus vannamei were infected with Vibrio parahaemolyticus, through nonspecific immunity parameter detection, intestinal morphology observation, and the assessment of microbial flora diversification by 16S rRNA gene sequencing. The results showed that the oral administration of CS-GT significantly increased total hemocyte counts and reduced hemocyte apoptosis in shrimp (p < 0.05). The parameters (including superoxide dismutase, glutathione peroxidase, glutathione, lysozyme, acid phosphatase, alkaline phosphatase, and phenoloxidase) were significantly increased (p < 0.05). The integrity of the intestinal epithelial cells and basement membrane were enhanced, which correspondingly alleviated intestinal injury. In terms of the microbiome, the abundances of Vibrio (Gram-negative bacteria and food-borne pathogens) in the water and gut were significantly reduced. The canonical correspondence analysis (CCA) showed that the abundances of Vibrio both in the water and gut were negatively correlated with CS-GT dosage. In conclusion, the oral administration of CS-GT can improve the immunity of shrimp against pathogenic bacteria and significantly reduce the relative abundances of Vibrio in aquaculture water and the gut of Litopenaeus vannamei.

Dietary seaweed (Enteromorpha) polysaccharides improves growth performance involved in regulation of immune responses, intestinal morphology and microbial community in banana shrimp Fenneropenaeus merguiensis.

The present study was conducted to evaluate the effects of marine polysaccharides from seaweed Enteromorpha on growth performance, immune responses, intestinal morphology and microbial community in the banana shrimp Fenneropenaeus merguiensis. Two thousand and four hundred juvenile shrimps with an average body weight of 2.18 ± 0.06 g were fed for 42 d with diets containing different levels of Enteromorpha polysaccharides (EPS): 0 (control), 1, 2 and 3 g/kg as treatment groups, each of group was replicated three times with two hundred shrimps per replicate. Dietary supplementation of 1 g/kg EPS showed a consistent improvement in the final weight, weight gain, average daily gain rate (ADGR) and specific growth rate (SGR) (P < 0.05), while showed a decrease in the feed conversion ratio (FCR) of shrimp (P < 0.05). Besides, the total anti-oxidative capacity (T-AOC), superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), lysozyme (Lyz), alkaline phosphatase (ALP), and phenoloxidase (PO) activities in hemolymph were enhanced by dietary supplementation of 1 g/kg EPS (P < 0.05), while it reduced the hemolymph MDA content (P < 0.05). Shrimp fed 1 g/kg EPS supplemented diets up-regulated FmLyz, FmSOD5 and FmCLAP gene expression level of hepatopancreas and gill (P < 0.05), and also improved the intestinal FmLC2, FmLyz, FmSOD5 and FmCLAP gene expression levels (P < 0.05). In addition, shrimp fed diets containing 1 g/kg EPS increased the villus width (P < 0.05) and resulted in a higher villus surface area (P < 0.05). According to 16S rRNA sequencing results, dietary supplementation of 1 g/kg EPS tended to increase the relative abundance of Firmicutes at phylum level (P = 0.07) and decrease the relative abundance of Vibrio at genus level (P = 0.08). There was a significant positive correlation between the relative abundance of Firmicutes and mRNA expression of intestinal immune-related genes (P < 0.05). These findings revealed that dietary 1 g/kg EPS could improve growth performance, enhance nonspecific immunity and modulate intestinal function of banana shrimp F. merguiensis.

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.

Microecological Koch's postulates reveal that intestinal microbiota dysbiosis contributes to shrimp white feces syndrome.

BACKGROUND: Recently, increasing evidence supports that some complex diseases are not attributed to a given pathogen, but dysbiosis in the host intestinal microbiota (IM). The full intestinal ecosystem alterations, rather than a single pathogen, are associated with white feces syndrome (WFS), a globally severe non-infectious shrimp disease, while no experimental evidence to explore the causality. Herein, we conducted comprehensive metagenomic and metabolomic analysis, and intestinal microbiota transplantation (IMT) to investigate the causal relationship between IM dysbiosis and WFS. RESULTS: Compared to the Control shrimp, we found dramatically decreased microbial richness and diversity in WFS shrimp. Ten genera, such as Vibrio, Candidatus Bacilloplasma, Photobacterium, and Aeromonas, were overrepresented in WFS, whereas 11 genera, including Shewanella, Chitinibacter, and Rhodobacter were enriched in control. The divergent changes in these populations might contribute the observation that a decline of pathways conferring lipoic acid metabolism and mineral absorption in WFS. Meanwhile, some sorts of metabolites, especially lipids and organic acids, were found to be related to the IM alteration in WFS. Integrated with multiomics and IMT, we demonstrated that significant alterations in the community composition, functional potentials, and metabolites of IM were closely linked to shrimp WFS. The distinguished metabolites which were attributed to the IM dysbiosis were validated by feed-supplementary challenge. Both homogenous selection and heterogeneous selection process were less pronounced in WFS microbial community assembly. Notably, IMT shrimp from WFS donors eventually developed WFS clinical signs, while the dysbiotic IM can be recharacterized in recipient shrimp. CONCLUSIONS: Collectively, our findings offer solid evidence of the causality between IM dysbiosis and shrimp WFS, which exemplify the 'microecological Koch's postulates' (an intestinal microbiota dysbiosis, a disease) in disease etiology, and inspire our cogitation on etiology from an ecological perspective. Video abstract.

Virulence Genes Analysis of Vibrio parahaemolyticus and Anti-vibrio Activity of the Citrus Extracts.

Vibrio parahaemolyticus (VP) is a marine bacterium that opportunistically caused foodborne gastroenteritis in human and some diseases in marine animals. The isolated strain of V. parahaemolyticus WS001 from Samut Sakhon, Thailand has a presence of ldh (~ 450 bp) toxA (~ 333 bp) and toxB (~ 1269 bp) genes which showed pathogenicity in shrimp. This strain is suspected as low pathogenicity in human due to the lack of tdh and trh genes for encoding thermostable direct hemolysin (TDH) and TDH-related hemolysin (TRH), respectively. The shrimp pathogenic strain was tested and revealed the multi-antibiotic resistances but was susceptible to norfloxacin (10 µg/ml). Citrus peel extracts were examined because they are rich in bioactive compounds such as saponins, tannins, flavonoids, steroids, and alkaloids that are effective in anti-VP activities. The ethanolic peel extracts of Citrus aurantifolia (Christm.) Swingle and Citrus hystrix DC. were found to be more anti-VP effect than other solvent extracts by Agar disc diffusion method at an optimum concentration of 50 mg/ml and Broth micro-dilution method (MICs of 50-100 mg/ml and MBCs of 100-200 mg/ml). Thus, C. aurantifolia (Christm.) Swingle peel extract was a distinctive candidate for the development of alternative natural agent to control the spreading of diseases in shrimp.