Responses of growth performance, immunity, disease resistance of shrimp and microbiota in Penaeus vannamei culture system to Bacillus subtilis BSXE-1601 administration: Dietary supplementation versus water addition.

This study evaluated the effects of Bacillus subtilis BSXE-1601, applied either as dietary supplementation or water addition, on growth performance, immune responses, disease resistance of Penaeus vannamei, and microbiota in shrimp gut and rearing water. During the 42-day feeding experiment, shrimp were fed with basal diet (CO and BW group), basal diet supplemented with live strain BSXE-1601 at the dose of 1 × 109 CFU kg-1 feed (BD group) and 15 mg kg-1 florfenicol (FL group), and basal diet with strain BSXE-1601 added to water at the concentration of 1 × 107 CFU L-1 every five days (BW group). Results showed that dietary supplementation of strain BSXE-1601 significantly promoted growth performance of shrimp, both in the diet and water, enhanced disease resistance against Vibrio parahaemolyticus (P < 0.05). The BD and BW groups exhibited significant increases in acid phosphatase, alkaline phosphatase, lysozyme, peroxidase, superoxide dismutase activities, phenonoloxidase content in the serum of shrimp compared to the control (P < 0.05). Meanwhile, the expression of immune-related genes proPO, LZM, SOD, LGBP, HSP70, Imd, Toll, Relish, TOR, 4E-BP, eIF4E1α, eIF4E2 were significantly up-regulated compared to the control (P < 0.05). When added in rearing water, strain BSXE-1601 induced greater immune responses in shrimp than the dietary supplement (P < 0.05). Chao1 and Shannon indices of microbiota in rearing water were significantly lower in BD group than in the control. The microbiota in rearing water were significantly altered in BD, BW and FL groups compared to the control, while no significant impacts were observed on the microbiota of shrimp gut. When supplemented into the feed, strain BSXE-1601 obviously reduced the number of nodes, edges, modules in the ecological network of rearing water. The results suggested that dietary supplementation of BSXE-1601 could be more suitable than water addition in the practice of shrimp rearing when growth performance, non-specific immunity, disease resistance against V. parahaemolyticus in shrimp were collectively considered.

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.

Effects of Enterococcus faecium (R8a) on nonspecific immune gene expression, immunity and intestinal flora of giant tiger shrimp (Penaeus monodon).

In this study, Penaeus monodon were gave basic feed supplemented with three levels of Enterococcus faecium. Then, the expression of non-specific immunity-related genes, and the activities of total antioxidant capacity (T-AOC), superoxide dismutase (SOD), malondialdehyde (MDA), acid phosphatase (ACP), alkaline phosphatase (AKP), phenol oxidase (PO) were evaluated. Meanwhile, the disease resistance test and intestinal flora determination were conducted. The results showed that the MDA levels of 2% and 5% E. faecium groups were significantly lower than that of the control group (P < 0.05). While the SOD and T-AOC and ACP and AKP of experimental groups were significantly higher (P < 0.05), the PO of experimental groups were significantly lower than that of the control group (P < 0.05). In addition, the expressions of immunity-related genes (tlr22, dorsal, lysozyme, crustin, imd, and relish) in the 2% and 5% E. faecalis groups were significantly greater than those in the control group (P < 0.05). After P. monodon was challenged with Vibrio parahaemolyticus for 7 days, the average cumulative mortality of P. monodon in the 2% and 5% groups were significantly lower than that in the 0% group (P < 0.05). With the increase of feeding time, the number of effective OTUs in each group showed a downward trend. At the 14th d, Proteobacteria, Bacteroidetes and Firmicutes, the dominant flora in the intestinal tract of P. monodon. In summary, supplied with E. faecium could increase the expression of non-specific immunity-related genes, enhance the immune capacity of P. monodon.

Application of nZnO supported with nanoclay for improving shrimp immunity.

This study discloses the nanoscale silicate platelet-supported nZnO (ZnONSP) applied as novel feed additives in aquaculture. The preparation of the nanohybrid (ZnO/NSP = 15/85, w/w) was characterized by UV-visible spectroscopy, powder X-ray diffraction and transmission electron microscope. The effects of ZnONSP on growth, zinc accumulation, stress response, immunity and resistance to Vibrio alginolyticus in white shrimp (Penaeus vannamei) were \demonstrated. To evaluate the safety of ZnONSP, shrimps (2.0 ± 0.3 g) were fed with ZnONSP containing diets (200, 400 and 800 mg/kg) for 56 days. Dietary ZnONSP did not affect the weight gain, specific growth rate, feed conversion ratio, survival rate, zinc accumulation, and the expression of heat shock protein 70 in tested shrimps. To examine the immunomodulatory effect of ZnONSP, shrimps (16.6 ± 2.4 g) were fed with the same experimental diets for 28 days. Dietary ZnONSP improved the immune responses of haemocyte in tested shrimps, including phagocytic rate, phagocytic index, respiratory burst, and phenoloxidase activity, and upregulated the expression of several genes, including lipopolysaccharide, ß-1,3-glucan binding protein, peroxinectin, penaeidin 2/3/4, lysozyme, crustin, anti-lipopolysaccharide factor, superoxide dismutase, glutathione peroxidase, clotting protein and α-2-macroglobulin. In the challenge experiment, shrimps (17.2 ± 1.8 g) were fed with ZnONSP containing diets (400 and 800 mg/kg) for 7 days and then infected with Vibrio alginolyticus. Notably, white shrimps that received ZnONSP (800 mg/kg) showed significantly improved Vibrio resistance, with a survival rate of 71.4 % at the end of 7-day observation. In conclusion, this study discovers that ZnONSP is a new type of immunomodulatory supplement that are effective on enhancing innate cellular and humoral immunities, and disease resistance in white shrimp.

A native strain of Bacillus subtilis increases lipid accumulation and modulates expression of genes related to digestion and amino acid metabolism in Litopenaeus vannamei.

In the field of shrimp aquaculture, the utilization of probiotics represents a promising avenue, due to the well-documented benefits conferred by these microorganisms. In the current study, a Bacillus subtilis strain, referred to as strain E, was isolated from the gastrointestinal tract of the shrimp Litopenaeus vannamei and subsequently identified via molecular methods and phylogeny. The probiotic potential of strain E was characterized, and its application as a feed shrimp additive was evaluated in a 45-day experiment. Several parameters were assessed, including zootechnical performance, muscle tissue proximate composition, hepatopancreas lipid concentration, and the expression of genes associated with digestion, amino acid metabolism, and antioxidant defense mechanisms in various shrimp tissues. Although no significant impact on zootechnical performance was observed, supplementation with strain E led to an increase in lipid concentration within both muscle and hepatopancreas tissues. Furthermore, a marked decrease in the expression of genes linked to digestion and amino acid metabolism was noted. These findings suggest that the addition of the B. subtilis strain E to shrimp feed may enhance nutrient absorption and modulate the expression of genes related to digestion and amino acid metabolism.

The Yin Yang 1 of Penaeus vannamei regulates transcription of the small subunit hemocyanin gene during Vibrio parahaemolyticus infection.

Hemocyanin is a respiratory protein, it is also a multifunctional immune molecule that plays a vital role against pathogen invasion in shrimp. However, the regulation of hemocyanin gene expression in shrimp hemocytes and the mechanisms involved during pathogen infection remains unclear. Here, we used DNA pull-down followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the Yin Yang 1 transcription factor homolog in Penaeus vannamei (PvYY1) as a key factor that modulates transcription of the small subunit hemocyanin gene of P. vannamei (PvHMCs) in hemocytes during Vibrio parahaemolyticus AHPND (VPAHPND) infection. Bioinformatics analysis revealed that the core promoter region of PvHMCs contains two YY1 motifs. Mutational and oligoprecipitation analyses confirmed that PvYY1 could bind to the YY1 motifs in the PvHMCs core promoter region, while truncation of PvYY1 revealed that the N-terminal domain of PvYY1 is essential for the transactivation of PvHMCs core promoter. Besides, the REPO domain of PvYY1 could repress the activity of the PvHMCs core promoter. Overexpression of PvYY1 significantly activates the promoter activity of PvHMCs core promoter, while PvYY1 knockdown significantly decreases the expression level of PvHMCs in shrimp hemocytes and survival rate of shrimp upon infection with VPAHPND. Our present study provides new insights into the transcriptional regulation of PvHMCs by PvYY1 in shrimp hemocytes during bacteria (VPAHPND) infection.

Effects of dietary melatonin on antioxidant and immune function of the Pacific white shrimp (Litopenaeus vannamei), as determined by transcriptomic analysis.

Melatonin (MT) is regarded as an antioxidant and immunostimulant that can efficiently scavenge free radicals and activate antioxidant enzymes. The aim of this study was to investigate the effects of dietary MT on the growth performance and immune function of the Pacific white shrimp (Litopenaeus vannamei). Six groups of L. vannamei were supplemented with dietary MT at 0, 22.5, 41.2, 82.7, 165.1, and 329.2 mg/kg levels for 2 months. RNA-Seq analysis was performed to obtain transcriptome data of the control group and the group supplemented with dietary MT at 82.7 mg/kg BW. In total, 1220 DEGs (799 up-regulated and 421 down-regulated) were identified. Pathways and genes related to growth performance and immune function were verified by real-time quantitative polymerase chain reaction. The total hemocyte count, phagocytosis rate, and respiratory burst were significantly increased in the MT (82.7 mg/kg BW) group as compared to the control group. Analysis of antioxidant-related enzymes in the hepatopancreas showed that dietary MT (82.7 mg/kg BW) significantly increased activities of superoxide dismutase (SOD), glutathione peroxidase (GPx), and catalase, while dietary MT at 41.2 mg/kg BW significantly increased activities of glutathione S-transferase, lysozyme (LZM), and phenoloxidase (PO). At the transcriptional level, dietary MT up-regulated expression levels of genes associated with antioxidant immunity and growth, which included PO, SOD, LZM, GPx, chitin synthase, ecdysone receptor, calcium-calmodulin dependent protein kinase I, and retinoid X receptor. In conclusion, dietary MT may improve the growth performance and immune function of L. vannamei to some extent.

Effects of dietary dihydromyricetin on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei).

A 56-day culture trial was conducted to evaluate the effects of dietary dihydromyricetin (DMY) on growth performance, antioxidant capacity, immune response and intestinal microbiota of shrimp (Litopenaeus vannamei). 840 healthy shrimp (1.60 ± 0.21 g) in total were fed with four different levels of DMY diets at 0 (Control), 100 (D1), 200 (D2), and 300 (D3) mg/kg, respectively. Samples were collected after the culture trial, and then, a 7-day challenge experiment against Vibrio parahaemolyticus was conducted. The results demonstrated that DMY significantly enhanced the activity of protease, amylase and lipase as well as the expression of lipid and protein transport-related genes (P < 0.05). The results of plasma lipid parameters indicated that DMY reduced lipid deposition, manifested by significantly (P < 0.05) decreased plasma total cholesterol (T-CHO), triglyceride (TG) and low-density lipoprotein cholesterol (LDL-C). The expression of genes involved in fatty acid ß-oxidation and triglyceride catabolism was significantly up-regulated (P < 0.05), and genes involved in triglyceride synthesis were significantly down-regulated in DMY groups when compared to control group (P < 0.05). Moreover, dietary DMY also significantly (P < 0.05) increased the total antioxidant capacity (T-AOC), antioxidant enzymes activity and glutathione (GSH) content of shrimp, and a significant increase of total hemocytes count (THC), phagocytic rate (PR), antibacterial activity (AA) and bacteriolytic activity (BA) was observed in DMY groups (P < 0.05). The addition of DMY to the diet significantly augmented immune response by up-regulating the expression of genes related to toll-like receptors (Toll) signaling pathway, immune deficiency (IMD) signaling pathway and intestinal mucin. Furthermore, dietary DMY could modulate the composition and abundance of intestinal microbiota. In conclusion, DMY showed promising potential as a functional feed additive for shrimp to improve the growth performance and physiological health.

The effect of alginate oligosaccharides on intestine barrier function and Vibrio parahaemolyticus infections in the white shrimp Litopenaeus vannamei.

The intestine is a host-pathogen interaction site and improved intestinal barrier function help to prevent disease in shrimp. Alginate oligosaccharides (AOS) are derived from resourceful brown algae. The intestine protection properties of AOS were widely recognized, and their benefits in fish have been reported. Nevertheless, there are no reports on AOS in shrimp and other crustaceans. In the present work, we measured the effects of AOS on growth performance and disease resistance in the white shrimp Litopenaeus vannamei and investigated their effects on intestinal health. Shrimps with an initial weight of about 2 g were fed with diets supplemented with 0 (control), 0.07%, 0.2%, 0.6%, or 1.2% of AOS for 56 days and were sampled and challenged with Vibrio parahaemolyticus. Dietary AOS did not significantly influence weight gain or feed utilization (P > 0.05). However, AOS considerably decreased the seven-day cumulative mortality after the challenge at any dose (P < 0.05). Dietary AOS improved the intestinal structure, significantly boosted the intestinal villus height at 0.6% and 1.2% levels, and increased intestinal wall thickness by 0.2%, 0.6%, and 1.2%. The alkaline phosphatase and maltase activities were also increased, suggesting that AOS improved the intestinal condition. Redox homeostasis in intestinal was improved by AOS, as expressed by the enhanced total antioxidant capacity and decreased malonaldehyde content, partly due to the increased superoxide dismutase and catalase activities. Compared with the antioxidant system, AOS's stimulating effects on immunity were more significant. At any level, AOS significantly activated lysozyme activity, the expression of propo and two antimicrobial peptide genes (pen-3 and crusin). However, the lowest concentration of AOS did not stimulate the gene expression of all three assayed pattern recognition receptors (LGBP, Toll, and IMD), and only the highest concentration of AOS increased the expression of imd. These findings suggest that AOS are highly efficient immunostimulants, and various immune pathways in shrimp are differentially sensitive to AOS. Finally, our findings suggest that AOS significantly alter the gut microbiota and their relative abundance at the phylum, family, and genus levels. In conclusion, AOS significantly enhances disease resistance in L. vannamei, possibly attributed to improved intestinal development, increased intestinal immunity and altered microbiota. These findings could provide a basis for future studies on the practical use of AOS and its mechanisms of action.

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.

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.

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.

Effect of Clostridium butyricum on intestinal microbiota and resistance to Vibrio alginolyticus of Penaeus vannamei.

In order to evaluate the effect of Clostridium butyricum (C. butyricum) feeding on intestinal microorganisms and protection against infection by Vibrio alginolyticus (V. alginolyticus) in Penaeus vannamei (P. vannamei). We set up two groups, CG30 (fed normal feed) and CB30 (fed feed supplemented with C. butyricum), for the 30d C. butyricum feeding test, and four groups, CG (CG30 group injected with PBS), CB (CB30 group injected with PBS), VACG (CG30 group injected with V. alginolyticus), and VACB (CB30 group injected with V. alginolyticus), for the 24 h infection test. The protective effect of C. butyricum against acute V. alginolyticus infection in P. vannamei was explained in terms of survival, histopathology, changes in enzyme activity, transcriptome analysis, and immune-related genes. We found that feeding C. butyricum significantly altered intestinal microbial populations' abundance and significantly reduced Vibrio spp. In the V. alginolyticus stress test, C. butyricum improved the survival rate and alleviated pathological changes in hepatopancreatic tissues, alleviated the reduction of superoxide dismutase (SOD) and phenoloxidase (PO) activity caused by infection, and increased the lysozyme content in P. vannamei. VACB group compared with the VACG group, 1730 up-regulated differentially expressed genes (DEGs) and 2029 down-regulated DEGs were screened. Quantitative real-time PCR (qRT-PCR) showed that dietary supplementation with C. butyricum suppressed the upregulation of alkaline phosphatase (AKP) transcription factors and the downregulation of prophenoloxidase (proPO), alpha-2-macroglobulin (A2M), and anti-lipopolysaccharide factor (ALF) induced by V. alginolyticus infection. In conclusion, feed supplementation with C. butyricum changed P. vannamei's population ratio of intestinal microorganisms. Moreover, C. butyricum has the potential to act as an inhibitor of V. alginolyticus infection and enhance the resistance of P. vannamei to V. alginolyticus infection.

Effects of dietary soluble ß-1,3-glucan on the growth performance, antioxidant status, and immune response of the river prawn (Macrobrachium nipponense).

The effects of dietary ß-1,3-glucan on the growth performance, body composition, hepatopancreas tissue structure, antioxidant activities, and immune response of the river prawn (Macrobrachium nipponense) were investigated. In total, 900 juvenile prawns were fed one of five diets with different contents of ß-1,3-glucan (0%, 0.1%, 0.2%, and 1.0%) or 0.2% curdlan for 6 weeks. The growth rate, weight gain rate, specific growth rate, specific weight gain rate, condition factor, and hepatosomatic index of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those fed 0% ß-1,3-glucan and 0.2% curdlan (p < 0.05). The whole-body crude lipid content of prawns supplemented with curdlan and ß-1,3-glucan was significantly higher than that of the control group (p < 0.05). The antioxidant and immune enzyme activities of superoxide dismutase (SOD), total antioxidant capacity (T-AOC), catalase (CAT), lysozyme (LZM), phenoloxidase (PO), acid phosphatase (ACP), and alkaline phosphatase (AKP) in the hepatopancreas of juvenile prawns fed 0.2% ß-1,3-glucan were significantly higher than those of the control and 0.2% curdlan groups (p < 0.05), and tended to increase and then decrease with increasing dietary ß-1,3-glucan. The highest malondialdehyde (MDA) content was observed in juvenile prawns without ß-1,3-glucan supplementation. The results of real-time quantitative PCR indicated that dietary ß-1,3-glucan promoted expression of antioxidant and immune-related genes. Binomial fit analysis of weight gain rate and specific weight gain rate showed that the optimum ß-1,3-glucan requirement of juvenile prawns was 0.550%-0.553%. We found that suitable dietary ß-1,3-glucan improved juvenile prawns growth performance, antioxidant capacity, and non-specific immunity, which provide reference for shrimp healthy culture.

Comparative study of the impact of dietary supplementation with different types of CpG oligodeoxynucleotides (CpG ODNs) on enhancing intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles in Pacific white shrimp (Litopenaeus vannamei).

The CpG oligodeoxynucleotides (CpG ODNs) reportedly possess the capacity to strengthen immunity in mammals. This experiment was conducted to evaluate the impact of dietary supplementation with 17 types of CpG ODNs on intestinal microbiota diversity, antioxidant capacity, and immune-related gene expression profiles of the shrimp Litopenaeus vannamei. Diets including 50 mg kg-1 CpG ODNs wrapped in egg whites were prepared and divided into 17 different groups, with 2 control groups (normal feed and feed with egg whites). These CpG ODNs supplemented diets and the control diets were fed to L. vannamei (5.15 ± 0.54 g) three times daily at 5%-8% shrimp body weight for three weeks. The results of consecutive detection of intestinal microbiota by 16S rDNA sequencing indicated that 11 of the 17 types of CpG ODNs significantly enhanced intestinal microbiota diversity, increased the populations of several probiotic bacteria, and activated possible mechanisms relevant to diseases. The immune-related genes expression and antioxidant capacity in hepatopancreas further demonstrated that the 11 types of CpG ODNs effectively improved the innate immunity of shrimp. Additionally, histology results showed that the CpG ODNs in the experiment did not damage the tissue structure of hepatopancreas. The results suggest that CpG ODNs could be used as a trace supplement to improve the intestinal health and immunity of shrimp.

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.

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.

Effect of nanoclay supported nanosilver on the growth inhibition of aquatic pathogens and immunomodulatory effect in Penaeusvannamei.

Hybrid of nanosilver and nanoscale silicate platelet (AgNSP) is a safe, non-toxic nanomaterial which has been applied in medical use due to its strong antibacterial activity. The application of AgNSP in aquaculture was first proposed in the present study by evaluating the in vitro antibacterial activities against four aquatic pathogens, in vitro effects toward shrimp haemocytes as well as the immune responses and disease resistance in Penaeus vannamei fed with AgNSP for 7 days. For evaluating the antibacterial activities of AgNSP in culture medium, the minimum bactericidal concentration (MBC) values against Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus and Vibrio parahaemolyticus were 100, 15, 625 and 625 mg/L, respectively. Moreover, the inhibition of pathogen growth over a period of 48 h could be achieved by the appropriate treatment of AgNSP in culturing water. In freshwater containing bacterial size of 103 and 106 CFU/mL, the effective doses of AgNSP against A. hydrophila were 12.5 and 450 mg/L, respectively while the effective doses against E. tarda were 0.2 and 50 mg/L, respectively. In seawater with same bacterial size, the effective doses against V. alginolyticus were 150 and 2000 mg/L, respectively while the effective doses against V. parahaemolyticus were 40 and 1500 mg/L, respectively. For the in vitro immune tests, the superoxide anion production and phenoloxidase activity in haemocytes were elevated after in vitro incubation with 0.5-10 mg/L of AgNSP. In the assessment of dietary supplemental effects of AgNSP (2 g/kg), no negative effect on the survival was found at the end of 7 day feeding trail. In addition, the gene expression of superoxide dismutase, lysozyme and glutathione peroxidase were up-regulated in haemocytes taken from shrimps received AgNSP. The following challenge test against Vibrio alginolyticus showed that the survival of shrimp fed with AgNSP was higher than that of shrimp fed with control diet (p = 0.083). Dietary AgNSP improved the Vibrio resistance of shrimp by increasing 22.7% of survival rate. Therefore, AgNSP could potentially be used as a feed additive in shrimp culture.

Dietary supplementation of ß-1, 3-glucan improves the intestinal health of white shrimp (Litopenaeus vannamei) by modulating intestinal microbiota and inhibiting inflammatory response.

The phenomenon of intestinal dysfunction is widely observed in white shrimp (Litopenaeus vannamei) culture, and ß-1,3-glucan has been confirmed to be beneficial in intestinal health with a lack understanding of its underlying mechanism. Proteobacteria, Firmicutes, and Actinobacteria served as the predominant phyla inhabiting the intestine of white shrimp, whilst a significant variation in their proportion was recorded in shrimp fed with basal and ß-1,3-glucan supplementation diets in this study. Dietary supplementation of ß-1,3-glucan could dramatically increase the microbial diversity and affect microbial composition, concurrent with a notable reduction in the ratio of opportunistic pathogen Aeromonas, gram-negative microbes, from Gammaproteobacteria compared to the basal diet group. The benefits for microbial diversity and composition by ß-1,3-glucan improved the homeostasis of intestinal microbiota through the increase of specialists' number and inhibition of microbial competition caused by Aeromonas in ecological networks; afterward, the inhibition of Aeromonas by ß-1,3-glucan diet dramatically suppressed microbial metabolism related to lipopolysaccharide biosynthesis, followed by a conspicuous decrease in the intestinal inflammatory response. The improvement of intestinal health referred to the elevation in intestinal immune and antioxidant capacity, ultimately contributing to the growth of shrimp fed ß-1,3-glucan. These results suggested that ß-1,3-glucan supplementation improved the intestinal health of white shrimp through the modulation of intestinal microbiota homeostasis, the suppression of intestinal inflammatory response, and the elevation of immune and antioxidant capacity, and subsequently promoted the growth of white shrimp.

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.