Shengkang Li

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.

A de novo transcriptome of the noble scallop, Chlamys nobilis, focusing on mining transcripts for carotenoid-based coloration

BackgroundThe noble scallop Chlamys nobilis Reeve displays polymorphism in shell and muscle colors. Previous research showed that the orange scallops with orange shell and muscle had a significantly higher carotenoid content than the brown ones with brown shell and white muscle. There is currently a need to identify candidate genes associated with carotenoid-based coloration.ResultsIn the present study, 454 GS-FLX sequencing of noble scallop transcriptome yielded 1,181,060 clean sequence reads, which were assembled into 49,717 isotigs, leaving 110,158 reads as the singletons. Of the 159,875 unique sequences, 11.84% isotigs and 9.35% singletons were annotated. Moreover, 3,844 SSRs and over 120,000 high confidence variants (SNPs and INDELs) were identified. Especially, one class B scavenge receptor termed SRB-like-3 was discovered to express only in orange scallops and absent in brown ones, suggesting a significant association with high carotenoid content. Down-regulation of SRB-like-3 mRNA by RNA interference remarkably decreased blood carotenoid, providing compelling evidence that SRB-like-3 is an ideal candidate gene controlling carotenoid deposition and determining orange coloration.ConclusionTranscriptome analysis of noble scallop reveals a novel scavenger receptor significantly associated with orange scallop rich in carotenoid content. Our findings pave the way for further functional elucidation of this gene and molecular basis of carotenoid deposition in orange scallop.

Identification and characterization of the related immune-enhancing proteins in crab Scylla paramamosain stimulated with rhubarb polysaccharides.

Recently, considerable interest has been focused on immunostimulants to reduce diseases in crab aquaculture. However, information regarding to the related immune-enhancing proteins in crabs is not available yet. In this study, rhubarb polysaccharides were tested for enhancement of the immune activity in crab Scylla paramamosain. Compared with those in the control group, values of, phenoloxidase (PO), alkaline phosphatase (AKP) and alkaline phosphatasein (ACP) activity in the, experimental group were improved significantly 4 d after the treatment. Furthermore, 15 and 17 altered proteins from haemocytes and hepatopancreas, respectively, were found in rhubarb polysaccharide-treated crabs using 2-DE approach. Of these, hemocyanin, chymotrypsin, cryptocyanin, C-type lectin receptor, and ferritin protein were identified by mass spectrometry. In addition, RT-PCR, analysis showed that the mRNA levels of hemocyanin and chymotrypsin increased about 2.4- and 1.4-fold in the experiment group. Moreover, the hemocyanin gene in S. paramamosain (SpHMC) was, cloned and characterized. SpHMC contains one open reading frame of 2022 bp and encodes a polypeptide of 673 amino acids. It is clustered into one branch along with crab hemocyanin in a phylogenetic tree. The mRNA transcripts of SpHMC were detected mainly in the tissues of, hepatopancreas, hemocyte and intestines, and its levels were up-regulated significantly in hemocytes, of S. paramamosain treated with Vibrio parahemolyticus, Beta streptococcus or poly I:C for 6-48 h. Taken together, these studies found 5 related immune-enhancing proteins and a novel heomcyanin homologue with potential pathogen-resistant activities in crab.

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.

Cloning and characterization of a novel hemocyanin variant LvHMCV4 from shrimp Litopenaeus vannamei

Recently, we found 3 variants of hemocyanin subunit with higher molecular weight in shrimp Litopenaeus vannamei (Named as LvHMCV1-3). In this study, a novel L. vannamei hemocyanin variant (Named as LvHMCV4) was further cloned and characterized. Bioinformatic analysis predicted that LvHMCV4 contains one open reading frame of 2137 bp and encodes a polypeptide of 678 amino acids. It shares 84-99% cDNA sequences identity to that of the classical form of L. vannamei hemocyanin (LvHMC, AJ250830.1) and LvHMCV1-3. LvHMCV4 possesses a conserved structure characteristic of the hemocyanin family and can be clustered into one branch along with other arthropod hemocyanins in a phylogenetic tree. Further, the full-length DNA of LvHMCV4 contains 2660 bp and two introns, which are located at the 80-538 bp and 2063-2227 bp regions, respectively. In addition, the mRNA transcript of LvHMCV4 was expressed highly in the hepatopancreas, lymphoid, brain and hemocytes, and weakly in the heart, intestine and gill, while no expression was found in the muscle, stomach and gut. Infection by Escherichia coli K12, Vibrio parahaemolyticus, Vibrio alginolyticus, Vibrio fluvialis, Streptococcus pyogenes or Staphylococcus aureus up-regulated significantly LvHMCV4 mRNA expression in the hepatopancreas. Furthermore, the recombinant protein of LvHMCV4 (rLvHMCV4) was prepared, which showed agglutination activities against six pathogenic bacteria at concentrations ranging from 15.6 to 125 μg/ml. When co-injected with V. parahaemolyticus in L.vannamei, rLvHMCV4 significantly increased the survival rate after 48 h injection. Together, these studies suggested that hemocyanin variant, LvHMCV4, might be involved in shrimp resistance to pathogenic infection.

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.

Differential immune response of vitellogenin gene to Vibrio anguillarum in noble scallop Chlamys nobilis and its correlation with total carotenoid content

Vitellogenin (Vg), an egg yolk precursor protein, not only functions as a source of nutrients and a nonpolar molecular carrier that combine and transfer lipids, proteins, vitamin and carotenoids to oocytes during the oogenesis. but also links with the immune defense in many oviparous animals. To investigate whether Vg plays a immune defensive role in noble scallop Chlamys nobilis, an acute Vibrio anguillarum infection experiment was conducted in orange and brown scallops with different carotenoids content. qRT-PCR result showed that Vg transcripts were significantly up-regulated after challenge with V. anguillarum in orange and brown shell scallops compared to the control group and Vg expression reached the highest spot at 6 h, indicated that Vg possessed an immune function in the noble scallop. Interestingly, a significantly positive correlation between Vg transcript levels and total carotenoids content in the ovary was observed, indicating that Vg gene expression was up regulated by carotenoids. The results suggest that Vg is a potent immune protector and carotenoid may linked with Vg plays an important role in host immune system against pathogens in noble scallop C. nobilis.