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.

Characterization of the lipidomic profile of clam Meretrix petechialis in response to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus is a devastating pathogen of clam Meretrix petechialis, which brings about huge economic losses in aquaculture breeding industry. In our previous study, we have found that Vibrio infection is closely associated with lipid metabolism of clams. In this study, an untargeted lipidomics approach was used to explore the lipid profiling changes upon Vibrio infection. The results demonstrated that the hepatopancreas of clams was composed of five lipid categories including fatty acyls, glycerolipids, glycerophospholipids, sphingolipids and sterol lipids. And the content of lipid classes altered during Vibrio infection, implying that Vibrio infection altered intracellular lipid homeostasis in clams. Meanwhile, a total of 200 lipid species including 82 up-regulated and 118 down-regulated significantly were identified in response to Vibrio infection, of which ceramide (Cer), phosphatidylcholine (PC) and triglyceride (TG) accounted for the largest proportion. Notably, all Cers showed a significantly decreased trend while nearly all TG species were increased significantly during Vibrio infection, which suggested that Cer and TG could be determined as effective biomarkers. Furthermore, these differentially expressed lipid species were enriched in 20 metabolic pathways and sphingolipid metabolism was one of the most enriched pathways. These results evidenced how the lipid metabolism altered in the process of Vibrio infection and opened a new perspective on the response of marine bivalves to pathogen infection.

TLR4 involved in immune response against Vibrio Parahaemolyticus by MyD88-dependent pathway in Crassostrea hongkongensis.

Vibrio parahaemolyticus (V. parahaemolyticus) is a salt-loving gram-negative bacterium, and is the leading cause of mortality in cultured shellfish in recent years. Toll-like Receptor 4 (TLR4) is a classical pattern recognition receptor (PRRs) that recognizes pathogen-associated molecular patterns (PAMPs) of pathogenic microorganism and activates the immune response. However, the function and signal pathway of TLR4 in oyster are still unknown. In this study, a new TLR4 gene was identified from the Crassostrea hongkongensis (C. hongkongensis). The ChTLR4 contained an open reading frame of 2643 bp, encoding 880 amino acids with seven leucine-rich repeat (LRR) domains and a Toll/IL-1R (TIR) domain. The ChTLR4 shared the highest sequence identity (83.0%) with TLR4 of Crassostrea gigas. Tissue expression analysis revealed that ChTLR4 showed the highest constitutive expression in the gill and hepatopancreas, and was significantly upregulated in immune tissues post V. parahaemolyticus infection, especially in gill and hemocytes. Moreover, TLR4 silencing significantly inhibited the immune-enzyme activities, including SOD, CAT, ACP, AKP in gill and LZM in hemolymph supernatant, and increased MDA content in hemolymph supernatant. Meanwhile, the antimicrobial activities of the hemolymph supernatant were also significantly inhibited by TLR4 silencing. These data demonstrated that the ChTLR4 involved in innate immune response of C. hongkongensis against V. parahaemolyticus challenge. Finally, qRT-PCR analysis showed that ChTLR4 silencing clearly inhibited the expression of genes in TLR4-MyD88 pathway, indicating that MyD88-dependent pathway played a crucial role in ChTLR4-mediated immune response against V. parahaemolyticus.

Transcriptome analysis of Macrobrachium rosenbergii hemocytes reveals in-depth insights into the immune response to Vibrio parahaemolyticus infection.

Macrobrachium rosenbergii as one of the common freshwater prawn species in Southeast Asia, which breeding industry is seriously threatened by vibriosis and causes high mortality. In this study, the RNA-seq was employed for assessing the M. rosenbergii hemocytes transcriptomes following Vibrio parahaemolyticus challenge. After challenge for 6 h (h), there were overall 1849 DEGs or differentially expressed genes, including 1542 up-regulated and 307 down-regulated genes, and there was a total of 1048 DEGs, including 510 up-regulated genes and 538 down-regulated genes, after challenge for 12 h. Mitogen-activated protein kinase (MAPK) immune-related pathways, Toll, immune deficiency (IMD), and Janus kinase (JAK)/signal transducer and activator of transcription (STAT) were among the immune pathways where a lot of the DEGs were connected. The expression patterns of 18 chosen immune-related genes were examined utilizing qRT-PCR or quantitative real-time polymerase chain reaction, which revealed that the V. parahaemolyticus infection activated the M. rosenbergii's immune response. Permutational multivariate analysis of variance (PERMANOVA) showed that V. parahaemolyticus infection modulated immune regulation and apoptosis pathways. The gathered information provided new insight into M. rosenbergii's immunity and suggested a novel approach to fight against bacterial infection.

Transcriptome analysis of Pacific white shrimp (Liptopenaeus vannamei) after exposure to recombinant Vibrio parahaemolyticus PirA and PirB proteins.

Vibrio parahaemolyticus is a Gram-negative bacterium commonly found in marine and estuarine environments and is endemic among the global shrimp aquaculture industry. V. parahaemolyticus proteins PirA and PirB have been determined to be major virulence factors that contribute significantly to the development of acute hepatopancreatic necrosis disease. Our previous work had demonstrated the lethality of recombinant PirA and PirB proteins to Pacific white shrimp (Liptopenaeus vannamei). To understand the host response to these proteins, recombinant PirA and PirB proteins were administered using a reverse gavage method and individual shrimp were then sampled over time. Shrimp hepatopancreas libraries were generated and RNA sequencing was performed on the control and recombinant PirA/B-treated samples. Differentially expressed genes were identified among the assayed time points. Differentially expressed genes that were co-expressed at the later time points (2-, 4- and 6-h) were also identified and gene associations were established to predict functional physiological networks. Our analysis reveals that the recombinant PirA and PirB proteins have likely initiated an early host response involving several cell survival signaling and innate immune processes.

Dietary supplementation of synbiotic Leuconostoc mesenteroide B4 and dextran improves immune regulation and disease resistance of Penaeus vannamei against Vibrio parahaemolyticus.

White shrimp (Penaeus vannamei) is an important culture species in Taiwan but often encounters disease infection by Vibrio parahaemolyticus that cause acute hepatopancreatic necrosis disease (AHPND). This study investigates the effects of dietary supplementation of Leuconostoc mesenteroide B4 and its fermentate (dextran) on the immune response, intestinal morphology, disease resistance, and immune-related gene expression in white shrimp. In comparison to the control group, the shrimp fed with a diet containing B4+dextran (107 CFU B4/g feed and 0.05% dextran) for 14, 28, 42 and 56 days had a significantly higher feed efficiency, weight gain and specific growth rate. A significantly higher villus height in the intestine and higher survival rate after challenging with V. parahaemolyticus was recorded for the B4+dextran group. Flow cytometry analysis demonstrated that the group that had ingested B4+dextran had a higher total hemocyte count and a higher proportion of semi-granulocytes, but a lower percentage of granulocytes compared to the control group. The shotgun metagenomic results in the midgut revealed that Leuco. mesenteroides was barely found in the midgut of the shrimp, suggesting that this microbe and its transient presence in the midgut is not the direct mechanism underlying the improved shrimp growth in the treated sample. Instead, dextran, a key ingredient in the B4 fermentate, on the dynamic of the microbial populations in shrimp, possibly promoting the diversity of gut microbes, especially the beneficial microbes, and thereby rendering protection against AHPND. In terms of comparing the gene expression between the control and synbiotic groups, pre- and post-bacterial challenge, a higher expression level of immune genes was mostly found in the B4+dextran group after challenging it with V. parahaemolyticus (group B4+dextran-VP) in the hepatopancreas and hemocyte. In contrast, the transcript level of immune-related genes was found to be higher in the B4+dextran group than other combinations in the midgut. Taken together, this study found that dietary addition of synbiotic Leuco. mesenteroides B4 and dextran can improve the growth performance, intestinal morphology and microbiome, regulation of immune genes and disease resistance against V. parahaemolyticus infection in white shrimp.

Antibacterial activity and immunomodulatory role of a proline-rich antimicrobial peptide SpPR-AMP1 against Vibrio campbellii infection in shrimp Litopenaeus vannamei.

Antimicrobial peptides (AMPs) constitute one of the most promising sources of natural molecules used for the design of effective antimicrobial agents alternative to antibiotics. Previously, we have showed that a crab proline-rich AMP designated as SpPR-AMP1 is a potent AMP that exhibited antimicrobial activity against both Gram-positive and Gram-negative bacteria. Here, we demonstrated the importance of SpPR-AMP1 peptide in treating a virulent acute hepatopancreatic necrosis disease (AHPND) Vibrio campbellii VH-639 isolate and eliciting the innate immune response to counter the AHPND infection in shrimp Litopenaeus vannamei. SpPR-AMP1 exhibited a strong antimicrobial activity against V. campbellii VH-639 at MIC value of 0.195-0.39 µM. Scanning electron microscopy (SEM) revealed the membrane disruption potential of SpPR-AMP1 against the V. campbellii VH-639 cells. The in vivo effect of SpPR-AMP1 in shrimp L.vannamei was investigated and the results showed that SpPR-AMP1 was capable of modulating the innate immune response by stimulating the expression levels of AMP transcripts in shrimp hemocytes. Moreover, treatments with SpPR-AMP1 could promote the resistance of shrimp against V. campbellii VH-639 infection as demonstrated by a significant increase in shrimp survival rate and decrease in both the bacterial load and the expression levels of bacterial PirA and PirB toxin gene transcripts in the infected shrimp. These results suggest the potential of SpPR-AMP1 peptide with the combined antimicrobial and immunoenhancing capabilities as promising antimicrobial agent to treat V. campbellii VH-639 causing AHPND infection in shrimp aquaculture.

RNA-seq analysis revealing the immune response of Neocaridina denticulata sinensis gill to Vibrio parahaemolyticus infection.

Vibrio parahaemolyticus causes serious economic losses to the shrimp farming industry. There is still a lack of adequate understanding of the changes in the overall response of N. denticulata sinensis caused by V. parahaemolyticus, particularly with respect to gill tissue, which is severely damaged by the pathogen. In this study, a total of 1358 differentially expressed genes were identified between the PBS control and Vibrio stimulation groups using transcriptome sequencing techniques. After further screening and analysis, many immune-related genes were obtained, involving lysosome pathway, metabolic process, chitin-binding protein, and serine protease family members. In addition, we randomly selected six DEGs in the lysosome pathway for qRT-PCR verification, and the results showed that their expression patterns were consistent with the RNA-seq. The results demonstrate the molecular regulation of the gill tissue response to V. parahaemolyticus infection in N. denticulata sinensis, contributing to the understand of the complex and efficient innate immune system and defense mechanisms in crustaceans.

Shrimp Lipid Droplet Protein Perilipin Involves in the Pathogenesis of AHPND-Causing Vibrio parahaemolyticus.

Acute hepatopancreatic necrosis disease (AHPND), caused by a unique strain of Vibrio parahaemolyticus (Vp (AHPND)), has become the world's most severe debilitating disease in cultured shrimp. Thus far, the pathogenesis of AHPND remains largely unknow. Herein, in Litopenaeus vannamei, we found that a Vp (AHPND) infection significantly increased the expression of lipid droplets (LDs) protein LvPerilipin, as well as promoted the formation of LDs. In addition, the knockdown of LvPerilipin increased the shrimp survival rate in response to the Vp (AHPND) infection, and inhibited the proliferation of Vp (AHPND). Furthermore, we demonstrated that LvPerilipin depletion could increase the production of reactive oxygen species (ROS), which may be responsible for the decreased Vp (AHPND) proliferation. Taken together, our current data for the first time reveal that the shrimp lipid droplets protein Perilipin is involved in the pathogenesis of Vp (AHPND) via promoting LDs accumulation and decreasing ROS production.

Integrative analyses of mRNA and microRNA expression profiles reveal the innate immune mechanism for the resistance to Vibrio parahaemolyticus infection in Epinephelus coioides.

Vibrio parahaemolyticus, as one of the main pathogens of marine vibriosis, has brought huge losses to aquaculture. However, the interaction mechanism between V. parahaemolyticus and Epinephelus coioides remains unclear. Moreover, there is a lack of comprehensive multi-omics analysis of the immune response of grouper spleen to V. parahaemolyticus. Herein, E. coioides was artificially injected with V. parahaemolyticus, and it was found that the mortality was 16.7% in the early stage of infection, and accompanied by obvious histopathological lesions in the spleen. Furthermore, 1586 differentially expressed genes were screened by mRNA-seq. KEGG analysis showed that genes were significantly enriched in immune-related pathways, Acute-phase immune response, Apoptosis, Complement system and Cytokine-cytokine receptor interaction. As for miRNA-seq analysis, a total of 55 significantly different miRNAs were identified. Further functional annotation analysis indicated that the target genes of differentially expressed miRNAs were enriched in three important pathways (Phosphatidylinositol signaling system, Lysosome and Focal adhesions). Through mRNA-miRNA integrated analysis, 1427 significant miRNA-mRNA pairs were obtained and "p53 signaling pathway", "Intestinal immune network for IgA production" were considered as two crucial pathways. Finally, miR-144-y, miR-497-x, novel-m0459-5p, miR-7133-y, miR-378-y, novel-m0440-5p and novel-m0084-3p may be as key miRNAs to regulate immune signaling pathways via the miRNA-mRNA interaction network. The above results suggest that the mRNA-miRNA integrated analysis not only sheds new light on the molecular mechanisms underlying the interaction between host and V. parahaemolyticus but also provides valuable and new insights into resistance to vibrio infection.

The production and influence of anti-Vibrio parahaemolytics IgY against experimental infection of V. parahaemolyticus in cultured Fenneropenaeus indicus.

The increasing number of antibiotic-resistant bacteria emphasizes the need to find alternatives to complement antibiotics. Immunotherapy may also be used as a complementary treatment against pathogens that are difficult to treat with traditional antibiotics. Eggs are normal dietary components and there is practically no risk of toxic side effects of IgY given orally. In the present study, pathogenic Vibrio parahaemolyticus was isolated from infected shrimp and studied their virulence factors including LD50 (by challenging with Fenneropenaeus indicus), proteolytic and hemolytic activities. The edible antibody IgY was raised by injecting the antigen of Extra Cellular Products (ECP) of V. parahaemolyticus to Gallus gallus domesticus during layoff period with and without the herbal immunoadjuvants, Asparagus racemosus and Glycine max (V.p wo: V. parahaemolyticus ECP without adjuvant; V.p A: V. parahaemolyticus ECP with A. racemosus and V.p G: V. parahaemolyticus ECP with G. max). Eggs were collected after five weeks of immunization and anti- V. parahaemolyticus IgY was extracted and purified. Physicochemical properties of the immunized Chickens' serum and anti- V. parahaemolyticus IgY's cross reactivity, growth inhibition assay, single radial immunodiffusion assay and bacterial agglutination were studied. The results revealed that, the serum protein parameters were significantly (P ≤ 0.001) increased in experimental groups from control group. The antibody raised with immunoadjuvants had significantly (P ≤ 0.001) higher cross reactivity, growth inhibition, single radial immunoassay and bacterial agglutination when compared with and without immunoadjuvant and control groups. Further the control and experimental anti-V. parahaemolytics IgY coated artificial diets were fed to F. indicus for 60 days. After 30 and 60 dpv (days of post vaccination), shrimps from each groups were challenged with virulent V. parahaemolyticus and studied the survival, haematological and immunological parameters. The IgY coated diets (V. p A and V.p G) fed shrimps had decreased cumulative mortality, significantly (P ≤ 0.001) improved coagulase activity, total haemocyte count and oxyhaemocyanin. The immunological parameters such as prophenoloxidase, intracellular anion production, lysozyme production and phagocytosis also improved significantly (P ≤ 0.001) in IgY treated shrimps.

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

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

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

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

Potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and VP(AHPND) resistance in Pacific white shrimp (Litopenaeus vannamei, Boone, 1931).

The potential synbiotic effects of a Bacillus mixture and chitosan on growth, immune responses and disease resistance against Vibrio parahaemolyticus, the causative agent of acute hepatopancreatic necrosis disease (AHPND) in Pacific white shrimp, were intensively investigated. Three effective strains of Bacillus amyloliquefaciens (A), Bacillus pumilus (P) and Bacillus subtilis (S) were mixed in pairs at a ratio of 5 × 108:5 × 108 CFU/kg diet and coated with the prebiotic chitosan (C) at a concentration of 20 mL/kg diet. Five different feed treatments were used to feed experimental shrimp for 5 weeks: control (control, no synbiotics), chitosan (coat, C) and the synbiotic treatments PAC, PSC and ASC. At week 5, the final length, final weight gain, weight gain, length, average daily gain, specific growth rate and feed conversion ratio, measured as growth parameters, were significantly upregulated in the PSC and ASC groups compared with the control and coat groups (P < 0.05). This result was consistent with the expression analysis of two growth-related genes (Rap-2a and GF-II) in the hepatopancreas and intestines of treated shrimp, as determined using qRT-PCR. The prebiotic chitosan and synbiotics PAC, PSC and ASC strongly induced significant differences in the expression of the Rap-2a and GF-II genes in the target organs compared with the expression in the control group at various time points (P < 0.05). Additionally, application of the synbiotic treatments also significantly enhanced the hepatopancreas characteristics and epithelial and intestinal wall thicknesses of the shrimp compared with the control. Interestingly, all the synbiotic treatments elevated phagocytic activity significantly at weeks 3 and 5 compared with that in the other groups. qRT-PCR analysis of immune-related genes also indicated that the prebiotic group and all synbiotic groups showed strong expression of anti-lipopolysaccharide (ALF) and prophenoloxidase (proPO) genes in the intestine. Finally, the synbiotic groups PAC, PSC and ASC exhibited stronger VPAHPND resistance at 120 h after exposure than the chitosan coat and control groups, with survival rates of 41.7 ± 11.55, 41.7 ± 0.00, 52.8 ± 5.77, 30.6 ± 15.28 and 22.2 ± 5.77%, respectively (P < 0.05). Based on the obtained information, all synbiotics were recommended for improved growth and immune responses, while ASC was the best for disease resistance against VPAHPND in Pacific white shrimp.

Dietary SYNSEA probiotic improves the growth of white shrimp, Litopenaeus vannamei and reduces the risk of Vibrio infection via improving immunity and intestinal microbiota of shrimp.

The growth performance, immunological status, and intestinal microbiology of white shrimp, Litopenaeus vannamei, were evaluated after dietary administration of the commercial probiotic SYNSEA. Shrimp were fed a control diet (without probiotic supplement) and two levels of SYNSEA probiotic, a low concentration of SYNSEA (LSL) containing 105 CFU (g diet)-1Bacillus subtilis and 105 CFU (g diet)-1 lactic acid bacteria (LAB), and a high concentration of SYNSEA (LSH) containing 106 CFU (g diet)-1B. subtilis and 106 CFU (g diet)-1 LAB, for 12 weeks. Shrimp fed with the LSL diet significantly increased growth performance as well as final weight and feed efficiency compared to the control, but not the LSH diet. After being orally challenged with Vibrio parahaemolyticus, shrimp fed with LSL diet prior to the challenge or fed with LSL and pathogen simultaneously showed significantly lower mortality compared to the control. SYNSEA probiotic significantly improved shrimp immune response, including lysozyme activity in LSL and LSH groups, and phagocytic activity in the LSL group in comparison to the control. In addition, the gene expressions of anti-lipopolysaccharide factor 2 in LSL and LSH groups, and penaeidin 4 in LSL were also up-regulated. Although there was no significant difference among groups for hepatopancreas and intestinal morphology, the muscular layer thickness and villi height were slightly improved in the intestines of shrimp fed SYNSEA. The 16S rDNA gene amplicon sequence analysis using next-generation sequencing revealed a significant decrease in α-diversity (Margalef's species richness) after oral administration of SYNSEA due to an increase in the relative abundance of beneficial bacteria in the gut flora of shrimp, such as Lactobacillus, Shewanella, and Bradymonadales and a decrease in harmful bacteria, such as Vibrio, Candidatus_Berkiella, and Acinetobacter baumannii. Together the data suggest that the provision of SYNSEA probiotic at 105 CFU (g diet)-1B. subtilis and 105 CFU (g diet)-1 LAB can improve shrimp growth, enhance immunity, and disease resistance status of the host. In addition, these findings conclude that SYNSEA probiotic has great preventive and therapeutic potential for Vibrio infection in shrimp aquaculture.

A kelch motif-containing protein KLHDC2 regulates immune responses against Vibrio parahaemolyticus and white spot syndrome virus in Penaeus vannamei.

The kelch motif-containing proteins are widely present in organisms and known to be involved in various biological processes, but their roles in immunity remain unclear. In this study, a kelch motif-containing protein KLHDC2 was identified from Pacific white shrimp Penaeus vannamei and its immune function was investigated. The klhdc2 gene was widely expressed in shrimp tissues and its protein product was mainly present in the nucleus. Expression of klhdc2 was regulated by shrimp NF-κB family members Dorsal and Relish, and changed after immune stimulation. KLHDC2 could enhance the immune defense against Vibrio parahaemolyticus in shrimp but inhibit that against white spot syndrome virus (WSSV). Further analyses showed that KLHDC2 did not affect the phagocytosis of hemocytes but regulated the expression of a series of immune effector genes. KLHDC2 has a complex regulatory relationship with Dorsal and Relish, which may partly contribute to its positive role in antibacterial response by regulating humoral immunity. Moreover, the regulatory effect of KLHDC2 on WSSV ie1 gene contributed to its negative effect on antiviral response. Therefore, the current study enrichs the knowledge on the Kelch family and helps to learn more about the regulatory mechanism of shrimp immunity.

Sequence and functional features of a novel scavenger receptor homolog, SCARA5 from Yellow drum (Nibea albiflora).

As an important member in SR-As, member 5 (SCARA5) can swallow apoptotic cells and foreign bodies, and participate multiple signaling pathways to inhibit tumor occurrence, development growth and metastasis. To explore its immune function, SCARA5 was identified from the yellow drum (Nibea albiflora) according to its transcriptome data, and its full-length cDNA was 6968 bp (named as NaSCARA5, GenBank accession no: MW070211) encoding 497 amino acids with a calculated molecular weight of 55.12 kDa, which had the typical motifs of SR family, such as transmembrane helix region, coil region, Pfam collagens region and SR region. BLASTp and the phylogenetic relationship analysis illustrated that the sequences shared high similarity with known SCARA5 of teleosts. Quantitative real time RT-PCR analysis showed that NaSCARA5 was expressed in intestine, stomach, liver, kidney, gill, heart and spleen, with the highest in the spleen (24.42-fold compared with that in heart). After being infected with Polyinosinic:polycytidylic acid (PolyI:C), Vibrio alginolyticus and Vibrio parahaemolyticus, NaSCARA5 mRNA were up-regulated with time dependent mode in spleen, which suggested that NaSCARA5 might play an important role in the immune process of fish. The extracellular domain of NaSCARA5 was successfully expressed in BL21 (DE3), and yielded the target protein of the expected size with many active sites for their conferring protein-protein interaction functions. After being purified by Ni-NAT Superflow resin and renatured, it was found to bind all the tested bacteria (V.parahaemolyticus,V.alginolyticus and Vibrio harveyi). The eukaryotic expression vector of the NaSCARA5-EGFP fusion protein was constructed and transferred into epithelioma papulosum cyprini (EPC) cells, and it was mainly expressed on the cell membrane indicating that NaSCARA5 was a typical transmembrane protein. The aforementioned results indicated that NaSCARA5 played a significant role in the defense against pathogenic bacteria infection as PRRs, which may provide some further understandings of the regulatory mechanisms in the fish innate immune system for SR family.

Effect of low-frequency noise on the survival rate and immunity of infected Vibrio parahaemolyticus sea slug (Onchidium reevesii).

Anthropogenic noise in the marine environment has become a global environmental pollutant that affects the behavior, physiology and immunity of marine animals. However, the resistance of marine animals to pathogens while under the influence of noise is a topic that has received little attention. To assess the immune defense response of sea slugs against pathogens when exposed to low frequency noise, we performed 120 h exposure experiments on sea slugs after a Vibrio parahaemolyticus application in low frequency noise at 500 Hz and 1000 Hz. We found that after the infection with V. parahaemolyticus, the survival rate of the sea slugs decreased, the apoptosis rate and reactive oxygen species (ROS) production of hemocytes increased significantly (P < 0.05), the proliferation of hemocytes accelerated, the activities of enzymes such as superoxide dismutase (SOD), catalase (CAT), alkaline phosphatase (AKP), alanine transaminase (ALT) and lysozyme (LZM) in the hepatopancreas increased significantly, and the expression of TNF signaling pathway-related genes (TNF-α, FADD, Caspase 8, Caspase 3) and Hsp70 genes were generally upregulated. In addition, exposure of sea slug after infected with V. parahaemolyticus to low frequency noise resulted in a significant increase in both antioxidant and immune parameters, which were positively correlated with frequency. The results showed that noise frequency and exposure time had an interactive effect on the above indicators. In summary, low-frequency noise exposure increases the risk of pathogenic infections in sea slugs and exacerbates the negative effects on the antioxidant capacity and immune metabolism of the organism.

Litopenaeus vannamei peritrophin interacts with WSSV and AHPND-causing V. parahaemolyticus to regulate disease pathogenesis.

Peritrophins are peritrophic membrane (PM) proteins that can interact with chitin fibers via chitin-binding domains. Peritrophins have essential roles in providing porosity and strength to the PM that lines the shrimp midgut. Acute hepatopancreatic necrosis disease (AHPND), caused by strains of V. parahaemolyticus, is known to initially colonize the shrimp stomach and simultaneously disrupt its structural barriers (e.g., cuticle or epithelial tissues) to reach the hepatopancreas. Although stomach and hepatopancreas were identified as target tissues involved in AHPND pathogenesis, our results indicated that peritrophin in peritrophic membrane has a crucial role in determining not only colonization of AHPND-causing bacteria but also their tissue distribution. As the interaction between LvPeritrophin (LvPT) and WSSV (white spot syndrome virus) is not well understood, we noted that LvPT expression was upregulated in shrimp stomach challenged with either WSSV or AHPND. In an in vitro pathogen binding assay, there was strong binding of recombinant LvPT WSSV and AHPND-causing V. parahaemolyticus, and various bacteria. Furthermore, dsRNA-mediated LvPT silencing inhibited WSSV gene expression and viral genome replication. However, downregulation of LvPT gene expression increased copies of AHPND-causing bacteria in shrimp digestive tract, and facilitated bacterial colonization in stomach. In conclusion, we speculated that LvPT might regulate bacterial colonization during AHPND, whereas in WSSV infection, LvPT silencing favored the host. Although recombinant LvPT had strong binding with WSSV, the precise role of LvPT in WSSV infection needs further investigation. These findings increased our understanding of host-pathogen interactions in AHPND and WSSV infection that can be applied in shrimp aquaculture for developing effective antibacterial and antiviral strategies.

Transcriptomic analysis and discovery of genes involving in enhanced immune protection of Pacific abalone (Haliotis discus hannai) in response to the re-infection of Vibrio parahaemolyticus.

Traditionally, invertebrates were thought to lack immune memory owing to a lack of acquired immune-related factors such as immunoglobulin. Nonetheless, with the in-depth consideration of invertebrate immune priming, scholars have gradually realized that the immune defenses of invertebrates are more complex than previously imagined. In the current investigation, the survival rate of Vibrio parahaemolyticus re-infected Haliotis discus hannai (VV group) was significantly different from the other groups (p < 0.05), indicating that an enhanced immune response may commence after first exposure to the same strain of V. parahaemolyticus. The transcriptome profiles of hemocytes obtained 102,052 unigenes, and 27,449 of them were annotated successfully. Venn diagram analysis showed that 2832 DEGs commonly responded to the first and second immune responses. 1734 "immune response genes" and 1460 "potential immune-enhancing genes" were also identified. A comparison of both "immune response genes" and "potential immune-enhancing genes" revealed 1019 immune-enhancing regulatory genes and 281 essential immune-enhancing genes. According to the KEGG enrichment analysis results of ERGs and EEGs, classical immune-related signaling pathways, such as NF-kappa B signaling pathway, NOD-like receptor signaling pathway, IL-17 signaling pathway, and TLR signaling pathway were significantly enriched, indicating that they were all involved in the response to V. parahaemolyticus re-infection and were likely dominant in the immune enhancement process of H. discus hannai hemocytes. The intermolecular interactions generated by Cytoscape after re-infection of V. parahaemolyticus appear more intuitively to demonstrate that hemocytes regulation was not an independent process, but rather an intricate regulatory network. H. discus hannai demonstrated enhanced immunological activity after re-infection with V. parahaemolyticus, showing immune memory in hemocytes. The current study's findings have broadened the study of immune enhancement in invertebrates and laid the framework for future research into the molecular mechanism of immune enhancement in abalones.