Huaiping Zheng

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.

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.

Effects of phenanthrene on hepatic enzymatic activities in tilapia (Oreochromis niloticus ♀ × O. aureus ♂).

The effects of phenanthrene (Phe) on hepatosomatic index (HSI) and hepatic enzymatic activities in hybrid tilapia (Oreochromis niloticus female x O. aureus male) were investigated via the static freshwater exposure at dosage of 50, 100, and 400 microg/L for 4-14 d. Compared with the control group, HSI was significantly decreased (P < 0.05) at 400 microg/L at day 14. Increased enzymatic activities (P < 0.05) were observed for catalase (CAT), glutathione peroxidase (GPx) and superoxide dismutase (SOD) at either 100 or 400 microg/L at day 8 and 14, as well as for CAT at 50 microg/L at day 14, except for GPx at 400 microg/L at day 8. Ethoxyresorufin O-deethylase (EROD) activity was significantly increased (P < 0.05) at all dosage at day 4 as well as at 50 microg/L at day 8, but significantly decreased at either 100 or 400 microg/L at day 14 (P < 0.05). Glutathione-S-transferase (GST) activity was not affected. The results suggest that CAT, GPx, SOD and EROD, as well as HSI in tilapia may be used as the biomarkers or indexes for evaluating or monitoring the pollution of polycyclic aromatic hydrocarbons (PAHs) such as Phe.

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.

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.

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.

Differential responses of a thioredoxin-like protein gene to Vibrio parahaemolyticus challenge in the noble scallop Chlamys nobilis with different total carotenoids content

ABSTRACT Being lack of specific immune system, both enzymes and non‐enzymatic antioxidants play crucial roles in immune of invertebrates. In the present study, in order to investigate immune roles of enzyme (thioredoxin, TRX) and antioxidants (carotenoids), Golden scallops with golden shell and golden muscle rich in carotenoids content and Brown scallops with brown shell and white muscle less carotenoids content of the noble scallop Chlamys nobilis were challenged by Vibrio parahaemolyticus for 48 h. Firstly, a cDNA of TRX protein gene from the scallop (named as CnTRX) was cloned and characterized. The cDNA contains 1280 bp, consisting of a 5′ ‐UTR of 99 bp, a long 3′ ‐UTR of 860 bp and a 321 bp open reading frame (ORF) encoding 106 amino acids. Phylogenetic analysis showed that CnTRX had a closer evolution relationship with TRX from Chlamys farreri. CnTRX was ubiquitously expressed in all examined tissues including intestine, adductor, mantle, gonad, gill, kidney, hepatopancreas and hemolymph, and the highest expression level was detected in the hemolymph. Next, CnTRX transcripts were significantly up‐regulated in V. parahaemolyticus group in comparison with PBS control group. Moreover, CnTRX transcripts were significantly higher in Golden scallops than that of Brown ones at 6 h, 12 h and 24 h with bacteria challenge (P < 0.05). The present result indicates that both CnTRX and carotenoids are important factors involved in the immune defense against bacteria challenge in the noble scallop. HighlightsA novel thioredoxin gene called CnTRX was cloned in the Chlamys nobilis.Golden scallops and Brown scallops with different carotenoids content were acutely challenged by Vibrio parahaemolyticus.CnTRX transcripts of the scallop were up‐regulated under bacterial stress.Transcripts were significantly higher in Golden scallops than that of Brown ones.Both CnTRX and carotenoids can play immune roles under bacterial stress in the noble scallop.

Two novel serine proteases from Scylla paramamosain involved in the synthesis of anti-lipopolysaccharide factors and activation of prophenoloxidase system

Serine proteases (SPs) are important in various immune responses, including prophenoloxidase (proPO) activation, antimicrobial peptides (AMPs) synthesis, and hemolymph coagulation in invertebrates. In this study, SP3 and SP5 of mud crab (Scylla paramamosain) were studied. SP3 and SP5 were expressed in all examined tissues (mainly in hemocytes), and are associated with the immune responses of mud crab to Vibrio parahemolyticus and Staphylococcus aureus, as well as interacted with TRAF6, and are involved in the activation of anti-lipopolysaccharide factors (ALFs) probably through the TLR/NF-κB pathway. Depletion of SP3 inhibited the expression of ALF1, ALF2, ALF3, and ALF6, while knockdown of SP5 significantly decreased ALF5, and ALF6. Furthermore, both SP5 and TRAF6 regulated the PO activity in the hemolymph of mud crab. Overexpression assay showed that both SP3 and SP5 could enhance the promoter activities of ALFs in mud crab. Taken together, the results of this study indicate that SP3 and SP5 might play important roles in the immune system of mud crab against pathogen invasion.

Transcriptome-seq provides insights into sex-preference pattern of gene expression between testis and ovary of the crucifix crab (Charybdis feriatus)

The crucifix crab, Charybdis feriatus, which mainly inhabits Indo-Pacific region, is regarded as one of the most high-potential species for domestication and incorporation into the aquaculture sector. However, the regulatory mechanisms of sex determination and differentiation of this species remain unclear. To identify candidate genes involved in sex determination and differentiation, high throughput sequencing of transcriptome from the testis and ovary of C. feriatus was performed by the Illumina platform. After removing adaptor primers, low-quality sequences and very short (<50 nt) reads, we obtained 80.9 million and 66.2 million clean reads from testis and ovary, respectively. A total of 86,433 unigenes were assembled, and ~43% (37,500 unigenes) were successfully annotated to the NR, NT, Swiss-Prot, KEGG, COG, GO databases. By comparing the testis and ovary libraries, we obtained 27,636 differentially expressed genes. Some candidate genes involved in the sex determination and differentiation of C. feriatus were identified, such as vasa, pgds, vgr, hsp90, dsx-f, fem-1, and gpr. In addition, 88,608 simple sequence repeats were obtained, and 61,929 and 77,473 single nucleotide polymorphisms from testis and ovary were detected, respectively. The transcriptome profiling was validated by quantitative real-time PCR in 30 selected genes, which showed a good consistency. The present study is the first high-throughput transcriptome sequencing of C. feriatus. These findings will be useful for future functional analysis of sex-associated genes and molecular marker-assisted selections in C. feriatus.