Jiasong Zhang

Effect of dietary poly-β-hydroxybutyrate (PHB) on growth performance, intestinal health status and body composition of Pacific white shrimp Litopenaeus vannamei (Boone, 1931).

ABSTRACT In the present study, the effect of dietary supplementation of poly‐&bgr;‐hydroxybutyrate (PHB) on the growth performance, intestinal digestive and immune function, intestinal short‐chain fatty acids (SCFA) content and body composition of Pacific white shrimp Litopenaeus vannamei (Boone, 1931) was evaluated. The shrimp was fed for 35 days with four different diets: 0%, 1%, 3% and 5% PHB supplemented feed. The results indicated that supplementation of PHB significantly increased the growth performance of the shrimp, and the feed conversion rate (FCR) in 3%PHB treatment group was significantly lower than the control (P < 0.05). The intestinal amylase, lipase and trypsin activity in the three PHB treatment groups were all significantly higher than that of the control (P < 0.05), but the pepsin activity were only significantly affected by 3%PHB treatment (P > 0.05). The activities of intestinal immune enzymes such as total antioxidant capacity (T‐AOC) and inducible nitric oxide synthase (iNOS) was significantly induced by 3%PHB treatment (P < 0.05), while lysozyme (LSZ) activity was significantly affected by 5%PHB treatment and nitric oxide (NO) content was significantly induced in three PHB treatments. Meanwhile, PHB induced significantly the expression level of intestinal heat shock protein 70 (HSP70), Toll and immune deficiency (Imd) gene. HE staining showed that PHB induced the intestinal health status of L. vannamei. Intestinal SCFA content analysis revealed that the content of propionic and butyric acid of 3%PHB treatment were significantly higher than that of the control (P < 0.05). Body composition analysis showed that the crude protein in 3% and 5%PHB treatments, and the crude lipid in 1% and 5%PHB treatments were all significantly higher than the control (P < 0.05). These results revealed that PHB could improve the growth performance, modulated intestinal digestive and immune function, increased intestinal SCFA content and body composition in L. vannamei, and the optimum dietary PHB requirement by L. vannamei was estimated at 3% (w/w) diet. HIGHLIGHTSThe effect of dietary poly‐&bgr;‐hydroxybutyrate (PHB) supplementation in Litopenaeus vannamei was studied.PHB improved the growth performance and feed utilization of L. vannamei.PHB increased the intestinal digestive and immune function of L. vannamei.PHB influenced the intestinal structure and SCFA content of L. vannamei.PHB changed the body composition of L. vannamei.

Effects of dietary Lactobacillus plantarum in different treatments on growth performance and immune gene expression of white shrimp Litopenaeus vannamei under normal condition and stress of acute low salinity

ABSTRACT A 45‐day feeding trial followed by an acute stress test of low salinity was done to evaluate effects of Lactobacillus plantarum on growth performance and anti‐stress capability of white shrimp (Litopenaeus vannamei). Shrimp were randomly allocated in 15 tanks (100 shrimp per tank) and divided into 5 treatments with 3 replicates. Triplicate tanks were fed with a control diet or diets containing different treatments of L. plantarum (fermentation supernatant (FS), live bacteria (LB), dead bacteria (DB) and cell‐free extract (CE) of L. plantarum) as treatment groups. Growth performance including weight gain (WGR), feed conversion ratio (FCR) and specific growth rate (SGR) were determined after feeding 45 days. Anti‐stress capacity was evaluated by determining the gene expression of ProPO, SOD and Lys in gut of shrimp at the end of feeding trial and again at 96 h post‐stress test. Results indicated that supplementation of L. plantarum into diet had significantly improved growth performance of L. vannamei. On the other hand, L. plantarum supplementation had no significant effects on the gene expression of SOD and Lys in gut of shrimp cultured under normal condition for 45 days. Supplementation of L. plantarum had increased survivability of L. vannamei having higher survival rates compared to the control group. However, statistical analysis showed no significant difference between the control group and treatments. Compared with the control group, supplementation of L. plantarum significantly improved the resistance of L. vannamei against the stress of acute low salinity, as indicated by higher survival rate as well as higher transcript levels of ProPo, SOD and Lys gene. Our findings suggested that L. plantarum, especially cell‐free extract of L. plantarum has improved the anti‐stress capacity of L. vannamei and could serve as a potential feed additive that helps shrimp to overcome environmental stresses. HighlightsDietary L. plantarum in four different treatments were used in the experiment.L. plantarum has significantly improved growth parameters (FBW, WGR and SGR) of L. vannamei.Cell‐free extract of L. plantarum showed the highest growth parameters.L. plantarum improved the resistance of L. vannamei against the stress of acute low salinity.

Effect of dietary Clostridium butyricum on growth, intestine health status and resistance to ammonia stress in Pacific white shrimp Litopenaeus vannamei

&NA; The present study evaluated the effect of dietary Clostridium butyricum (CB) on growth, intestine microstructure, intestine digestive and immune function, intestine short‐chain fatty acids (SCFA) content and body composition of Pacific white shrimp Litopenaeus vannamei. The shrimp was fed for 56 d with diets containing different levels of C. butyricum (1 × 109 cfu/g): 0% (Control), 0.25% (CB1), 0.5% (CB2) and 1.0% (CB3) as treatment groups, followed by an acute ammonia stress test for 72 h. The results indicated that dietary supplementation of C. butyricum decreased the feed conversion rate (FCR) and increased the growth performance of shrimp. Compared with the control group, after shrimp fed with C. butyricum 56 d, intestine amylase and protease activity in the three C. butyricum group increased, while lipase activity was only affected in the CB1 and CB2 group. Total antioxidant capacity (T‐AOC) content, lysozyme (LSZ) activity, and the relative expression level of Toll and immune deficiency (Imd) gene all increased in three C. butyricum groups. Inducible nitric oxide synthase (iNOS) activity increased in the CB2 and CB3 group, heat shock protein 70 (HSP70) gene expression level increased in the CB3 group, while nitric oxide (NO) content was not affected by C. butyricum. After shrimp exposed to ammonia stress, intestine immune biochemical parameters (T‐AOC, LSZ, iNOS and NO) and genes (HSP70, Toll and Imd) expression level of C. butyricum group was higher than that of the control. HE stain showed that C. butyricum increased the intestine epithelium height of L. vannamei. These results revealed that C. butyricum could improve the growth performance, increased intestine SCFA content and body crude protein content, modulated intestine digestive capacity, and enhanced intestine immune function of L. vannamei against ammonia stress. HighlightsEffect of dietary Clostridium butyricum supplementation in Litopenaeus vannamei was studied.C. butyricum improved the growth performance and feed utilization of L. vannamei.C. butyricum enhanced the intestine structure, digestive enzyme and SCFA content of L. vannamei.C. butyricum increased the intestine immune function of L. vannamei resistance to ammonia stress.C. butyricum increased body crude protein content of L. vannamei.

Intestine oxidative stress and immune response to sulfide stress in Pacific white shrimp Litopenaeus vannamei.

Abstract The effects of sulfide stress on oxidative stress and immune response in intestine of Pacific white shrimp Litopenaeus vannamei were evaluated in the present study. Oxidative stress parameters, immune enzymes activity and immune gene mRNA expression level were detected in intestine of L. vannamei after the exposure of 5.0 mg/L sulfide stress 72 h. The duration of sulfide stress influenced the shrimp survival, and the cumulative mortality rate was 30.0% and 33.3% at 48 h and 72 h respectively. HE staining showed that sulfide stress caused the intestine tissue damage symptoms. Compared with the control group, after exposed to sulfide stress, the content of lipid peroxidation (LPO), malondialdehyde (MDA) and ROS production (·O2− generation capacity) increased. Total antioxidant capacity (T‐AOC) activity increased at 6 h and decreased at 48 h. Superoxide dismutase (SOD) activity increased in the entire experiment. Inducible nitric oxide synthase (iNOS) activity and nitric oxide (NO) content increased to the highest at 6 h and 12 h respectively, and both decreased at 48 h. The relative mRNA expression level of heat shock protein 70 (HSP70) gene decreased at 6 h and increased to the highest at 48 h. The relative mRNA expression level of hypoxia inducible factor 1&agr; (HIF‐1&agr;) gene increased at 12 h and decreased to a lower level at 72 h. The relative mRNA expression level of Toll and immune deficiency (Imd) gene increased to the highest at 12 h and 24 h respectively, and both decreased at 48 h. These results revealed that sulfide stress could induce oxidative stress and immune response via confusion of immune enzymes activity and gene expression level in intestine of L. vannamei. HighlightsEffect of sulfide stress on oxidative stress and immune response in intestine of Litopenaeus vannamei was studied.Sulfide stress caused the oxidative stress and tissue damage in intestine of L. vannamei.Sulfide stress influenced the immune enzymes activity in intestine of L. vannamei.Sulfide stress induced the expression of immune genes in intestine of L. vannamei.

Dietary effects of succinic acid on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei

ABSTRACT Organic acids acts as an growth promoter and antimicrobial agent in aquaculture. The present study investigated the effects of a natural organic acid ‐ succinic acid (SA) on the growth, digestive enzymes, immune response and resistance to ammonia stress of Litopenaeus vannamei. The shrimps were firstly fed with diets containing different levels of SA: 0% (Control), 0.25% (SA1), 0.50% (SA2), and 1.0% (SA3) (w/w) for 56 days, followed by an acute ammonia stress for 48h. The results indicated that dietary of SA improved the growth of shrimp, and increased the survival rate of shrimp after ammonia stress for 48h. The amylase, lipase and pepsin activity increased in hepatopancreas in three SA group, while trypsin activity was only increased in the SA1 and SA2 groups. At 56d, T‐NOS activity, proPO and HSP70 gene expression level increased in the three SA group, PO activity increased in the SA1 and SA2 groups, T‐AOC content and Toll gene expression level increased in the SA2 and SA3 groups, Trx and SOD gene expression level increased in the SA2 group, while Imd, GS and GDH gene expression level was no changes. After exposure to ammonia stress for 48h, immune biochemical parameters (T‐AOC and PO) and genes (proPO, HSP70, Trx and GDH) expression level increased in the three SA group, T‐NOS activity, Toll, Imd and GS gene expression level increased in the SA2 and SA3 groups, while SOD gene expression level increased in the SA1 and SA2 groups. These results indicated that SA improved growth, enhanced digestive and immune capacities of L. vannamei against ammonia stress, and may be a potential feed additive for shrimp. The optimal dietary supplementation dosage is 0.50% (w/w) in diet. HIGHLIGHTSEffects of dietary succinic acid (SA) supplementation in Litopenaeus vannamei was studied.SA improved the growth performance and feed utilization of L. vannamei.SA influenced the digestive enzymes of L. vannamei.SA increased the immune capacity of L. vannamei resistance to ammonia stress.

Effects of dietary poly-β-hydroxybutyrate (PHB) on microbiota composition and the mTOR signaling pathway in the intestines of litopenaeus vannamei

Poly-β-hydroxybutyrate (PHB) is a natural polymer of the short chain fatty acid β-hydroxybutyrate, which acts as a microbial control agent. The mammalian target of the rapamycin (mTOR) signaling pathway plays a crucial role in intestine inflammation and epithelial morphogenesis. In this study, we examined the composition of intestine microbiota, and mTOR signaling-related gene expression in Pacific white shrimp Litopenaeus vannamei fed diets containing different levels of PHB: 0% (Control), 1% (PHB1), 3% (PHB3), and 5% (PHB5) (w/w) for 35 days. High-throughput sequencing analysis revealed that dietary PHB altered the composition and diversity of intestine microbiota, and that the microbiota diversity decreased with the increasing doses of PHB. Specifically, dietary PHB increased the relative abundance of Proteobacteria and Tenericutes in the PHB1 and PHB5 groups, respectively, and increased that of Gammaproteobacteria in the three PHB groups. Alternatively, PHB decreased Alphaproteobacteria in the PHB3 and PHB5 groups. At the genus level, dietary PHB increased the abundance of beneficial bacteria, such as Bacillus, Lactobacillus, Lactococcus, Clostridium, and Bdellovibrio. The relative mRNA expression levels of the mTOR signaling-related genes TOR, 4E-BP, eIF4E1α, and eIF4E2 all increased in the three PHB treatment groups. These results revealed that dietary PHB supplementation had a beneficial effect on intestine health of L. vannamei by modulating the composition of intestine microbiota and activating mTOR signaling.

Impairment of the intestine barrier function in Litopenaeus vannamei exposed to ammonia and nitrite stress

ABSTRACT Intestine barrier serves as the front‐line of shrimp defense, which rely on its structural integrity, microbial composition, and mucus immune compounds. Mucins are the major organic components of the intestine mucus layer that contribute to the immunity of intestine mucus. In this study, we examined the histological structure, microbial composition, and mucin genes expression in the intestines of Litopenaeus vanmei under three different conditions: control, ammonia stress, and nitrite stress for 72h. H&E stain showed that ammonia and nitrite stress exposure both damaged the intestine mucosal tissue. High‐throughput 16S rDNA sequencing revealed that two stresses exposure decreased the bacterial diversity, and altered the composition of intestine microbial. Specifically, the dominant bacterial phyla Bacteroidetes abundance was increased, while Proteobacteria and Planctomycetes were decreased; at the genus level, Formosa abundance was increased and Photobacterium was decreased, opportunistic pathogens including Nautella and Pseudoalteromonas was also increased. Intestine mucus immune genes including mucin‐2 and mucin‐19 were up‐regulated, while mucin‐1, mucin‐5AC, and mucin‐5B were down‐regulated in two stress exposure groups. These results revealed that ammonia and nitrite stress harmed the intestine barrier function of L. vannamei by damaging the mucosal tissue, disrupting the composition of intestine microbial, and suppressing the immune function. HIGHLIGHTSImpairment of the intestine barrier function in Litopenaeus vannamei exposed to ammonia and nitrite stress was studied.Ammonia and nitrite stress damaged the intestine mucosal tissue.Ammonia and nitrite stress decreased the intestine bacterial diversity.Ammonia and nitrite stress disrupted the composition of intestine microbial.Ammonia and nitrite stress influenced the intestine mucin genes expression.

Effects of the Dietary Probiotic Clostridium butyricum on Intestine Digestive and Metabolic Capacities, SCFA Content and Body Composition in Marsupenaeus japonicus

A 56-day feeding trial was performed to evaluate the effects of dietary probiotic Clostridium butyricum (CB) on intestine digestive and metabolic capacities, intestine short-chain fatty acids (SCFA) content and body composition of kuruma shrimp Marsupenaeus japonicus. Shrimps were randomly allocated into 9 tanks, 30 each, and fed with diets containing different levels of C. butyricum (1 × 109 cfu g−1): 0 mg g−1 feed (Control), 100 mg g−1 feed (CB-100), 200 mg g−1 feed (CB-200), while each level was triplicated. The results indicated that compared with the control group, the intestine pepsin (Pep) activity and 5-hydroxytryptamine (5-HT) concentration of two C. butyricum groups were both increased. Amylase (AMY) and lipase (LPS) activities were only induced in CB-200 group. Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities of two C. butyricum groups showed no significant change. The α-amylase (AMY) gene expression was induced in CB-200 group, and trypsin gene expression of two C. butyricum treated groups were both induced. Intestine SCFA content and body composition analysis showed that the contents of propionic acid, butyric acid and the crude protein of two C. butyricum groups were all higher than those of control. These results revealed that C. butyricum can modulate intestine digestive and metabolic capacities, improve intestine SCFA content and body crude protein content in M. japonicus.

Morphologic, digestive enzymes and immunological responses of intestine from Litopenaeus vannamei after lipopolysaccharide injection

The shrimp intestine barrier serves as the first line of the host defense against pathogen infection. Lipopolysaccharide (LPS) is the cell wall component of gram-negative bacteria, which known as endotoxin and induce the intestine inflammation. In this study, the acute toxicity effects of LPS injection on the morphology, digestive enzymes and immunological responses of intestine from Litopenaeus vannamei was investigated. HE stain showed that LPS injection damaged the intestine connective and epithelium tissue. Specifically, a decrease in the activities of digestive enzymes including of amylase, lipase, trypsin and pepsin was observed. Moreover, LPS injection increased the content of oxidative stress parameters (O2- generation capacity, LPO, MDA and PC), and the expression of HIF-1α gene. Alternatively, the antibacterial activities (PO and T-NOS), and the expression of the antibacterial genes (proPO, ALF, Toll and Imd) and pathogen pattern recognition genes (LGBP and Lec) increased at first and then decreased. Significant variations in anitioxidant enzyme activity of T-AOC, and the expression of the anitioxidant-related genes (SOD, HSP70 and Trx). These results revealed that LPS injection harmed the intestine barrier of L. vannamei by damaging intestine mucosal structure, increasing oxidative stress, and suppressing the digestive and immune status.

Changes in the Intestine Microbial, Digestive, and Immune-Related Genes of Litopenaeus vannamei in Response to Dietary Probiotic Clostridium butyricum Supplementation

The intestine barrier serves as the front-line defense in shrimp. Clostridium butyricum (CB) can produce butyric acid that provides energy for the intestine epithelial cells of the host. However, the effects of dietary CB on the intestine microbiome and the digestion and immunity of the host is not clear. In this study, we therefore investigated the composition and metabolic activity of the intestine microbiome, and digestive and immune-related gene expression in Litopenaeus vannamei fed with diets containing different levels of CB: basal diet (control), 2.5 × 109 CFU kg−1 diet (CB1), 5.0 × 109 CFU kg−1 diet (CB2), and 1.0 × 1010 CFU kg−1 diet (CB3) for 56 days. Dietary CB altered the composition of the intestine microbiome. Specifically, the dominant bacterial phylum Proteobacteria was enriched in the CB3 group and weakened in the CB1 and CB2 groups. The Bacteroidetes was enriched in the CB1 and CB2 groups and weakened in the CB3 group. The Firmicutes was enriched in all three CB groups. At the genus level, the potential pathogen (Desulfovibrio and Desulfobulbus) were weakened, and beneficial bacteria (Bacillus, Clostridium, Lachmoclostridium, Lachnospiraceae, and Lactobacillus) were enriched in response to dietary CB; these might contribute to the expression of the host digestive genes (α-amylase, lipase, trypsin, fatty acid-binding protein, and fatty acid synthase) and immune-related genes (prophenoloxidase, lipopolysaccharide and β-1,3-glucan binding protein, lysozyme, crustin, and superoxide dismutase). Additionally, CB enhanced the bacterial metabolism, especially that of carbohydrates, polymers, amino acids, carboxylic acids, and amines. These results revealed that dietary CB had a beneficial effect on the intestine health of L. vannamei by modulating the composition of the intestine microbiome, enhancing the microbial metabolism activity, and promoting the digestion and immunity of the host. The optimal dietary supplementation dosage was found to be 5.0 × 109 CFU kg−1 in the diet.