Xiaobo Wen

The intestinal microbial diversity in mud crab (Scylla paramamosain) as determined by PCR‐DGGE and clone library analysis

To identify the intestinal microbial diversity in mud crab and to investigate the bacterial difference in the intestinal microbiology between wild crabs (WC), pond‐raised healthy and diseased crabs (DC).

Enhancement of the immune response and protection against Vibrio parahaemolyticus by indigenous probiotic Bacillus strains in mud crab (Scylla paramamosain)

In a previous study, bacterial communities of the intestine in three populations of crabs (wild crabs, pond-raised healthy crabs and diseased crabs) were probed by culture-independent methods. In this study, we examined the intestinal communities of the crabs by bacterial cultivation with a variety of media. A total of 135 bacterial strains were isolated from three populations of mud crabs. The strains were screened for antagonistic activity against Vibrio parahaemolyticus using an agar spot assay. Antagonistic strains were then identified by 16S rRNA gene sequence analysis. Three strains (Bacillus subtilis DCU, Bacillus pumilus BP, Bacillus cereus HL7) with the strongest antagonistic activity were further evaluated for their probiotic characteristics. The results showed that two (BP and DCU) of them were able to survive low pH and high bile concentrations, showed good adherence characteristics and a broad spectrum of antibiotic resistance. The probiotic effects were then tested by feeding juvenile mud crabs (Scylla paramamosain) with foods supplemented with 10(5) CFU/g of BP or DCU for 30 days before being subjected to an immersion challenge with V. parahaemolyticus for 48 h. The treated crabs showed significantly higher expression levels of immune related genes (CAT, proPO and SOD) and activities of respiratory burst than that in controlled groups. Crabs treated with BP and DCU supplemented diets exhibited survival rates of 76.67% and 78.33%, respectively, whereas survival rate was 54.88% in crabs not treated with the probiotics. The data showed that indigenous mud-associated microbiota, such as DCU and BP, have potential application in controlling pathogenic Vibriosis in mud crab aquaculture.

Characterization of a novel anti-lipopolysaccharide factor isoform (SpALF5) in mud crab, Scylla paramamosain.

Anti-lipopolysaccharide factors (ALFs), the potential antimicrobial peptides that bind and neutralize lipopolysaccharide (LPS), are common effectors of innate immunity in crustaceans. In this study, a novel isoform of ALFs (SpALF5) was isolated from the hemocytes of mud crab Scylla paramamosain. The full-length 975bp SpALF5 contains a 375bp open reading frame (ORF) encoding 125 amino acids. Although SpALF5 exhibits a low degree of nucleotide homology with other reported ALFs, it contains the conserved amino acid sequence with a signal peptide and a LPS-binding domain including two conservative cysteine residues. The genomic organization of SpALF5 consists of four exons and three introns, with each intron containing one or more tandem repeats. Unlike most of ALFs mainly distributed in crab hemocytes, SpALF5 transcript was predominantly observed in the brain, muscle and skin, while barely detected in the hemocytes in our study. In situ hybridization assay also showed that SpALF5 mRNA was localized in brain, muscle and skin tissues of mud crab. Further, SpALF5 transcript was significantly up-regulated after challenge with LPS, polyinosinic polycytidylic acid (PolyI:C) (with the except of that in brain), Vibrio parahemolyticus or white spot syndrome virus (WSSV). The recombinant SpALF5 protein showed a varying degree of binding activity towards bacteria and fungus. Moreover, in vitro, the recombinant SpALF5 revealed a strong antimicrobial activity against Gram-negative bacteria (V. parahemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) and fungus (Sacchromyces cerevisiae), but could only inhibited the growth of some Gram-positive bacteria like Staphylococcus aureus. The results suggest that SpALF5 is a potent immune protector and plays an important role in immune defense against invading pathogens in S. paramamosain.

Transcriptome and Expression Profiling Analysis of the Hemocytes Reveals a Large Number of Immune-Related Genes in Mud Crab Scylla paramamosain during Vibrio parahaemolyticus Infection

Background Mud crab Scylla paramamosain is an economically important marine species in China. However, frequent outbreaks of infectious diseases caused by marine bacteria, such as Vibrio parahaemolyticus, result in great economic losses. Methodology/Principal Findings Comparative de novo transcriptome analysis of S. paramamosain infected with V. parahaemolyticus was carried out to investigate the molecular mechanisms underlying the immune response to pathogenic bacteria by using the Illumina paired-end sequencing platform. A total of 52,934,042 clean reads from the hemocytes of V. parahaemolyticus-infected mud crabs and controls were obtained and assembled into 186,193 contigs. 59,120 unigenes were identified from 81,709 consensus sequences of mud crabs and 48,934 unigenes were matched proteins in the Nr or Swissprot databases. Among these, 10,566 unigenes belong to 3 categories of Gene Ontology, 25,349 to 30 categories of KEGG, and 15,191 to 25 categories of COG database, covering almost all functional categories. By using the Solexa/Illuminas DGE platform, 1213 differentially expressed genes (P<0.05), including 538 significantly up-regulated and 675 down-regulated, were detected in V. parahaemolyticus-infected crabs as compared to that in the controls. Transcript levels of randomly-chosen genes were further measured by quantitative real-time PCR to confirm the expression profiles. Many differentially expressed genes are involved in various immune processes, including stimulation of the Toll pathway, Immune Deficiency (IMD) pathway, Ras-regulated endocytosis, and proPO-activating system. Conclusions/Significance Analysis of the expression profile of crabs under infection provides invaluable new data for biological research in S. paramamosain, such as the identification of novel genes in the hemocytes during V. parahaemolyticus infection. These results will facilitate our comprehensive understanding of the mechanisms involved in the immune response to bacterial infection and will be helpful for diseases prevention in crab aquaculture.

Characterization of microRNAs in Mud Crab Scylla paramamosain under Vibrio parahaemolyticus Infection

Background Infection of bacterial Vibrio parahaemolyticus is common in mud crab farms. However, the mechanisms of the crab’s response to pathogenic V. parahaemolyticus infection are not fully understood. MicroRNAs (miRNAs) are a class of small noncoding RNAs that function as regulators of gene expression and play essential roles in various biological processes. To understand the underlying mechanisms of the molecular immune response of the crab to the pathogens, high-throughput Illumina/Solexa deep sequencing technology was used to investigate the expression profiles of miRNAs in S . paramamosain under V. parahaemolyticus infection. Methodology/Principal Findings Two mixed RNA pools of 7 tissues (intestine, heart, liver, gill, brain, muscle and blood) were obtained from V. parahaemolyticus infected crabs and the control groups, respectively. By aligning the sequencing data with known miRNAs, we characterized 421 miRNA families, and 133 conserved miRNA families in mud crab S . paramamosain were either identical or very similar to existing miRNAs in miRBase. Stem-loop qRT-PCRs were used to scan the expression levels of four randomly chosen differentially expressed miRNAs and tissue distribution. Eight novel potential miRNAs were confirmed by qRT-PCR analysis and the precursors of these novel miRNAs were verified by PCR amplification, cloning and sequencing in S . paramamosain . 161 miRNAs (106 of which up-regulated and 55 down-regulated) were significantly differentially expressed during the challenge and the potential targets of these differentially expressed miRNAs were predicted. Furthermore, we demonstrated evolutionary conservation of mud crab miRNAs in the animal evolution process. Conclusions/Significance In this study, a large number of miRNAs were identified in S . paramamosain when challenged with V. parahaemolyticus, some of which were differentially expressed. The results show that miRNAs might play some important roles in regulating gene expression in mud crab under V. parahaemolyticus infection, providing a basis for further investigation of miRNA-modulating networks in innate immunity of mud crab.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) participates in anti-lipopolysaccharide factors (ALFs) gene expression in mud crab

ABSTRACT Tumor necrosis factor receptor‐associated factor 6 (TRAF6) is a key cytoplasm signal adaptor that mediates signals activated by tumor necrosis factor receptor (TNFR) superfamily and the Interleukin‐1 receptor/Toll‐like receptor (IL‐1/TLR) superfamily. The full‐length 2492 bp TRAF6 (Sp‐TRAF6) from Scylla paramamosain contains 1800 bp of open reading frame (ORF) encoding 598 amino acids, including an N‐terminal RING‐type zinc finger, two TRAF‐type zinc fingers and a conserved C‐terminal meprin and TRAF homology (MATH) domain. Multiple alignment analysis shows that the putative amino acid sequence of Sp‐TRAf6 has highest identity of 88% with Pt‐TRAF6 from Portunus trituberculatus, while the similarity of Sp‐TRAF6 with other crustacean sequences was 54–55%. RT‐PCR analysis indicated that Sp‐TRAF6 transcripts were predominantly expressed in the hepatopancreas and stomach, whereas it was barely detected in the heart and hemocytes in our study. Moreover, Sp‐TRAF6 transcripts were significantly up‐regulated after Vibrio parahemolyticus and LPS challenges. RNA interference assay was carried out used by siRNA to investigate the genes expression patterns regulated by Sp‐TRAF6. The qRT‐PCR results showed that silencing Sp‐TRAF6 gene could inhibit SpALF1, SpALF2, SpALF5 and SpALF6 expression in hemocytes, while inhibit SpALF1, SpALF3, SpALF4, SpALF5 and SpALF6 expression in hepatopancreas. Taken together, the acute‐phase response to immune challenges and the inhibition of SpALFs gene expression indicate that Sp‐TRAF6 plays an important role in host defense against pathogen invasions via regulation of ALF gene expression in S. paramamosain. HighlightsA novel signal adaptor in the Toll/TLR signaling pathways, Sp‐TRAF6, was characterized from the mud crabs.Be highly induced upon bacterial challenges.Sp‐TRAF6 plays an important role in host defense via regulation of ALFs gene expression.

Identification of a novel clip domain serine proteinase (Sp-cSP) and its roles in innate immune system of mud crab Scylla paramamosain.

Clip domain serine proteinases and their homologs are involved in the innate immunity of invertebrates. To identify the frontline defense molecules against pathogenic infection, we isolated a novel clip domain serine proteinase (Sp-cSP) from the hemocytes of mud crab Scylla paramamosain. The full-length 1362 bp Sp-cSP contains a 1155 bp open reading frame (ORF) encoding 384 amino acids. Multiple alignment analysis showed that the putative amino acid sequence of Sp-cSP has about 52% and 51% identity with Pt-cSP2 (AFA42360) and Pt-cSP3 (AFA42361) from Portunus trituberculatus, respectively, while the similarity with other cSP sequences was lower than 30%. However, all cSP sequences possess a conserved clip domain at the N-terminal and a Tryp-SPc domain at the C-terminal. The genomic organization of Sp-cSP consists of nine exons and eight introns, with some introns containing one or more tandem repeats. RT-PCR results indicated that Sp-cSP transcripts were predominantly expressed in the subcuticular epidermis, muscle and mid-intestine, but barely detectable in the brain and heart. Further, Sp-cSP transcripts were significantly up-regulated after challenge with lipopolysaccharides (LPS), Vibrio parahaemolyticus, polyinosinic polycytidylic acid (PolyI:C) or white spot syndrome virus (WSSV). Moreover, in vitro, the recombinant Sp-cSP revealed a strong antimicrobial activity against a Gram-positive (Staphylococcus aureus) and four Gram-negative (V. parahaemolyticus, Vibrio alginolyticus, Escherichia coli, Aeromonas hydrophila) bacteria in a dose-dependent manner. Taken together, the acute-phase response to immune challenges and the antimicrobial activity assay indicate that Sp-cSP is a potent immune protector and plays an important role in host defense against pathogen invasion in S. paramamosain.

Effects of dietary supplementation of Ulva pertusa and non-starch polysaccharide enzymes on gut microbiota of Siganus canaliculatus

Fishes represent the highest diversity of vertebrates; however, our understanding of the compositions and functions of their gut microbiota is limited. In this study, we provided the first insight into the gut microbiota of the herbivorous fish Siganus canaliculatus by using three molecular ecology techniques based on the 16S rRNA genes (denaturing gradient gel electrophoresis, clone library construction, and highthroughput Illumina sequencing), and the Illumina sequencing technique is suggested here due to its higher overall coverage of the total 16S rRNA genes. A core gut microbiota of 29 bacterial groups, covering >99.9% of the total bacterial community, was found to be dominated by Proteobacteria and Firmicutes in fish fed three different diets with/without the supplementation of Ulva pertusa and non-starch polysaccharide (NSP) enzymes (cellulase, xylanase, and β-glucanase). Diverse potential NSP-degrading bacteria and probiotics (e.g., Ruminococcus, Clostridium and Lachnospiraceae) were detected in the intestine of the fish fed U. pertusa, suggesting that these microorganisms likely participated in the degradation of NSPs derived from U. pertusa. This study supports our previous conclusion that U. pertusa-based diets are suitable for the production of S. canaliculatus with lower costs without compromising quality.

Scavenger receptor B promotes bacteria clearance by enhancing phagocytosis and attenuates white spot syndrome virus proliferation in Scylla paramamosian

ABSTRACT Phagocytosis and apoptosis are key cellular innate immune responses against bacteria and virus in invertebrates. Class B scavenger receptors (SRBs), which contain a CD36 domain, are critical pattern recognition receptors (PRRs) of phagocytosis for bacteria and apoptotic cells. In the present study, we identified a member of SRB subfamily in mud crab Scylla paramamosain, named Sp‐SRB. The full‐length cDNA of Sp‐SRB is 2593 bp with a 1629 bp open reading frame (ORF) encoding a putative protein of 542 amino acids, and predicted to contain a CD36 domain with two transmembrane regions at the C‐ and N‐terminals. Real‐time qPCR analysis revealed that Sp‐SRB was widely expressed in all tissues tested, and the expression of Sp‐SRB was up‐regulated upon challenge with Vibrio parahaemolyticus, white spot syndrome virus (WSSV), lipopolysaccharides (LPS) and polyinosinic polycytidylic acid (PolyI:C). Moreover, in vitro experiments indicated that recombinant Sp‐SRB protein (rSp‐SRB) could bind to fungi, Gram‐positive and Gram‐negative bacteria. RNA interference of Sp‐SRB resulted in significant reduction in the expression level of phagocytosis related genes, antimicrobial peptides (AMPs) and Toll‐like receptors (TLRs), which consequently led to impairment in both bacterial clearance and the phagocytotic activity of hemocytes. In addition, we found that Sp‐SRB had the ability to attenuate the replication of WSSV proliferation in mud crab S. paramamosain. Collectively, this study has shown that Sp‐SRB contributed to bacteria clearance by enhancing phagocytosis and up‐regulating the expression of AMPs possibly in a TRLs (SpToll 1 and SpToll 2)‐dependent manner. Besides, Sp‐SRB inhibited the replication of WSSV in S. paramamosian probably through enhancement of hemocytes phagocytosis of apoptotic cells. HIGHLIGHTSFull‐length of Sp‐SRB with 2593 bp was isolated from mud crab.Sp‐SRB promoted bacteria clearance by enhancing phagocytosis and up‐regulating the expression of AMPs.Sp‐SRB inhibited the replication of WSSV in S. paramamosian.

Cloning, tissue distribution, functional characterization and nutritional regulation of a fatty acyl Elovl5 elongase in chu's croaker Nibea coibor

Enzymes that lengthen the carbon chain of polyunsaturated fatty acids (PUFA) are key to the biosynthesis of the long-chain polyunsaturated fatty acids (LC-PUFA). Here we report on the molecular cloning, tissue distribution, functional characterization and nutritional regulation of a elovl5 gene from Nibea coibor. The full-length cDNA was 1315 bp, including a 5-untranslated region (UTR) of 134 bp, a 3-UTR of 296 bp and an open reading frame of 885 bp, which specified a peptide of 294 amino acids. Bioinformatics analysis showed that the deduced peptide sequence possessed all the characteristic features of microsomal fatty acyl elongases, including the so-called histidine box (HXXHH), the canonical C-terminal endoplasmic reticulum retention signal, several predicted transmembrane regions and other highly conserved motifs. Expression of elovl5 was strongly observed in stomach, and more weakly in kidney, spleen, intestine, brain, eye, liver, gill, muscle and heart. Functional characterization revealed that the chus croaker Elovl5 was able to elongate both C18 and C20 PUFA substrates. Nutritional study indicated that the hepatic expression of elovl5 could be up-regulated by low dietary n-3 LC-PUFA. These results may contribute to better understanding the LC-PUFA biosynthetic pathway and regulation mechanism in chus croaker.