Yubang Shen

Transcriptome Analysis of the Innate Immunity-Related Complement System in Spleen Tissue of Ctenopharyngodon idella Infected with Aeromonas hydrophila.

The grass carp (Ctenopharyngodon idella) is an important commercial farmed herbivorous fish species in China, but is susceptible to Aeromonas hydrophila infections. In the present study, we performed de novo RNA-Seq sequencing of spleen tissue from specimens of a disease-resistant family, which were given intra-peritoneal injections containing PBS with or without a dose of A. hydrophila. The fish were sampled from the control group at 0 h, and from the experimental group at 4, 8, 12, 24, 48 and 72 h. 122.18 million clean reads were obtained from the normalized cDNA libraries; these were assembled into 425,260 contigs and then 191,795 transcripts. Of those, 52,668 transcripts were annotated with the NCBI Nr database, and 41,347 of the annotated transcripts were assigned into 90 functional groups. 20,569 unigenes were classified into six main categories, including 38 secondary KEGG pathways. 2,992 unigenes were used in the analysis of differentially expressed genes (DEGs). 89 of the putative DEGs were related to the immune system and 41 of them were involved in the complement and coagulation cascades pathway. This study provides insights into the complement and complement-related pathways involved in innate immunity, through expression profile analysis of the genomic resources in C. idella. We conclude that complement and complement-related genes play important roles during defense against A. hydrophila infection. The immune response is activated at 4 h after the bacterial injections, indicating that the complement pathways are activated at the early stage of bacterial infection. The study has improved our understanding of the immune response mechanisms in C. idella to bacterial pathogens.

Next-generation sequencing identified microRNAs that associate with motile aeromonad septicemia in grass carp.

MicroRNAs (miRNAs) modulate many biological processes and can be up-or down-regulated in a variety of diseases states, including in bacterial infection. In this study, we characterized miRNAs associated with susceptibility or resistance to motile Aeromonas hydrophila in grass carp (Ctenopharyngodon idella), a commercially important farmed fish species in China. Using Illumina next-generation sequencing, we detected 185 miRNAs in A. hydrophila-susceptible (SGC) and -resistant grass carp (RGC) during immune activation. Twenty-one miRNAs were found to be differentially expressed between SGC and RGC, with 23.8% (5 out 21) exhibiting elevated expression in SGC. Further investigation of a selected five miRNAs indicated differences in the timing and tissue sources of expression. Target genes likely regulated by these miRNAs were identified using computational prediction combined with transcriptome expression data. MiRNA targets of differentially expressed mRNAs included those with functions important for bacterial resistance, such as tlr4 and nfil3-6. In addition, we demonstrated that nfil3-6 and tlr4 genes are direct targets of let-7i and cid-miRn-118. The present study suggested that the multiple miRNAs, displaying diverse kinetics and tissue sources on antibacterial immune processes.

Molecular cloning, characterization and immunological response analysis of Toll-like receptor 21 (TLR21) gene in grass carp, Ctenopharyngodon idella

TLR21, a non-mammalian Toll-like receptor, has been recently identified in fishes, frogs and birds. In the present study, the full-length cDNA sequence of TLR21 (CiTLR21) from Ctenopharyngodon idella has been isolated and characterized. The CiTLR21 full-length cDNA sequence consists of 3578bp, with an open reading frame (ORF) of 2958bp encoding 985 amino acid residues. The putative CiTLR21 protein contains a signal peptide sequence, 17 leucine-rich (LRR) motifs, a transmembrane region and a Toll/interleukin-1 receptor (TIR) domain. The CiTLR21 gene is expressed in a wide range of tissues with the highest expression in skin. Upon induction by Aquareovirus, CiTLR21 expression is significantly down-regulated in liver and spleen, whereas is significantly up-regulated in liver and spleen after Aeromonas hydrophila infection. These results suggest that CiTLR21 plays an important role in Aquareovirus and A. hydrophila-related diseases. This work may provide the basis for further investigations into the immune system of grass carp and other teleost fishes.

De novo Assembly of the Grass Carp Ctenopharyngodon idella Transcriptome to Identify miRNA Targets Associated with Motile Aeromonad Septicemia

Background De novo transcriptome sequencing is a robust method of predicting miRNA target genes, especially for organisms without reference genomes. Differentially expressed miRNAs had been identified previously in kidney samples collected from susceptible and resistant grass carp (Ctenopharyngodon idella) affected by Aeromonas hydrophila. Target identification for these differentially expressed miRNAs poses a major challenge in this non-model organism. Results Two cDNA libraries constructed from mRNAs of susceptible and resistant C. idella were sequenced by Illumina Hiseq 2000 technology. A total of more than 100 million reads were generated and de novo assembled into 199,593 transcripts which were further extensively annotated by comparing their sequences to different protein databases. Biochemical pathways were predicted from these transcript sequences. A BLASTx analysis against a non-redundant protein database revealed that 61,373 unigenes coded for 28,311 annotated proteins. Two cDNA libraries from susceptible and resistant samples showed that 721 unigenes were expressed at significantly different levels; 475 were significantly up-regulated and 246 were significantly down-regulated in the SG samples compared to the RG samples. The computational prediction of miRNA targets from these differentially expressed genes identified 188 unigenes as the targets of 5 conserved and 4 putative novel miRNA families. Conclusion This study demonstrates the feasibility of identifying miRNA targets by transcriptome analysis. The transcriptome assembly data represent a substantial increase in the genomic resources available for C. idella and will provide insights into the gene expression profile analysis and the miRNA function annotations in further studies.

Expression of complement component C7 and involvement in innate immune responses to bacteria in grass carp

Activation of the complement system, which forms a major part of the innate immune system, results in the formation of the terminal complement complex. The complement component, C7, plays an integral role in the assembly of this complex within target cell membranes. In this study, C7 was isolated and characterized from grass carp, an important cultured fish in China. The predicted amino acid sequence of C7 cDNA (2644 bp) exhibited 55.4 and 48.3% homology with trout C7-1 and zebrafish C7, respectively. The grass carp C7 gene was consisted of 18 exons and 17 introns. C7 gene expression was detected in the trunk kidney, liver, head kidney, skin, spleen, heart and intestine. Significant changes in C7 transcript expression (>20-fold) were detected following Aeromonas hydrophila infection, indicating C7 involvement in innate immune responses to bacteria in teleost fish.

Molecular cloning, characterization and expression patterns of HSP60 in the grass carp (Ctenopharyngodon idella)

HSP60 is a highly immunogenic molecule, which is able to activate a large number of T cell types and is implicated in a variety of autoimmune diseases. The grass carp (Ctenopharyngodon idella), a freshwater fish species of the family Cyprinidae, accounts for the third biggest value (USD 4.8 billion) at single species level of major cultured fish species in the world. Here, we isolated and characterized the HSP60 cDNA from grass carp (designated as CiHSP60). Its cDNA was 2434 bp in length and encoded a putative protein of 575 amino acids. BLAST analysis revealed that the CiHSP60 gene shared a high similarity with other known HSP60 sequences. CiHSP60 contained all three classical HSP60 family signatures. The mRNA of CiHSP60 was constitutively expressed in all tested tissues of untreated grass carp, including brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine, and fin, with the highest expression level in the blood. CiHSP60 transcript was present in unfertilized eggs, which suggests that CiHSP60 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiHSP60 gene in grass carp after the challenge with the bacterium Aeromonas hydrophila. A clear time-dependent expression pattern of CiHSP60 was found after the bacterial challenge, and the mRNA expression reached a maximum level at three days post challenge, and returned to control levels after seven days. The upregulated mRNA expression of CiHSP60 in grass carp after bacterial challenge indicates that the HSP60 gene is inducible and involved in immune responses. These results suggest that CiHSP60 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in grass carp.

Molecular cloning, expression analysis, and potential food intake attenuation effect of peptide YY in grass carp (Ctenopharyngodon idellus)

The peptide YY (PYY) is a 36 amino acid peptide involved in the food intake control in vertebrates. We have cloned and characterized a PYY gene from grass carp Ctenopharyngodon idellus. The full-length cDNA encodes a precursor protein of grass carp PYY (gcPYY) that consists of a putative 28-amino acid signal peptide, a 36-amino acid mature peptide, an amidation-proteolytic site, and a 30-amino acid carboxy-terminal extension. The gcPYY gene is comprised of 4 exons interspaced by 3 introns as seen in PYYs from other species. Amino acid alignment and gene structure comparison indicate that the structure of PYY is well preserved throughout vertebrate phylogeny. The tissue distribution and postprandial changes in gcPYY mRNA expression were evaluated by real-time PCR, which showed that the gcPYY is expressed abundantly in the central nervous system, with significantly increased expression following a single meal. During embryogenesis, the presence of gcPYY mRNA was detected in early developing embryos, and high expression levels were observed when most larvae completed their switch from endogenous nourishment to exogenous feeding. Reduced food intake by juveniles during a single meal after giving perpheral injection of gcPYY1-36 suggests a potentially important role of PYY in the food intake attenuation in grass carp.

Two novel homologs of simple C-type lectin in grass carp (Ctenopharyngodon idellus): Potential role in immune response to bacteria

C-type lectins play important roles in glycoprotein metabolism, multicellular integration and immunity. Based on their overall domain structure, they can be classified as different groups which possess different physiological functions. In this study, two novel simple C-type lectins were identified from grass carp (Ctenopharyngodon idellus), an important cultured fish in China. GcCL1 and gcCL2 share an essentially identical gene structure, a conserved promoter region shorter than 300 bp and an amino acid identity of 81.2%. Phylogenetic analysis indicated they may be products of gene duplication and could be classified as a new clade of group VII C-type lectins. Both of them were expressed in the eleven tissues examined, with the spleen having the highest abundance of transcript. The gcCL1 transcript was more abundant than gcCL2 in the majority of tissue samples from 2-yr-old grass carps, and was lower than those of gcCL2 before 15 days post-hatching. The expression of both genes was significantly up-regulated in spleen, muscle, skin, gills and hepatopancreas after induction by Aeromonas hydrophila. This is the first report that the expression of group VII C-type lectins could be induced by a pathogen, and indicates these lectins may be involved in the immune response to bacteria in fish.

Identification of TNF13b (BAFF) gene from grass carp (Ctenopharyngodon idella) and its immune response to bacteria and virus

The TNF superfamily B cell activating factor (BAFF) is a central cytokine in several diseases. A BAFF gene has been cloned from grass carp (Ctenopharyngodon idella), analyzed its structure, and investigated its expression pattern in various tissues after Aeromonas hydrophila and Aquareovirus infection. The open reading frame of grass carp BAFF (gcBAFF) consists of 804 bases encoding 267 amino acids. Phylogenetic analysis shows the gcBAFF is most closely related to other teleost BAFFs with the highest similarity to zebrafish. RT-PCR analysis shows the gcBAFF transcript is expressed in a wide range of tissues with the highest expression in skin and spleen. Upon induction by A. hydrophila and Aquareovirus, its expression is significantly up-regulated in gill, liver, kidney, spleen and skin as compared to PBS injected fish. The association of increased BAFF expression after bacterial and viral infections suggests that it plays a potentially important role in immune system of fish.

Cloning and characterization of TIMP-2b gene in grass carp

Tissue inhibitors of metalloproteinases (TIMPs) are the primary inhibitors of matrix metalloproteinases (MMPs) in tissues. In this study, a TIMP-2 gene was isolated from grass carp Ctenopharyngodon idella (CiTIMP-2b) and characterized. This cDNA sequence encoded a signal peptide, N- and C-terminal domains. CiTIMP-2b is highly homologous to the orthologous in zebrafish and other teleosts, suggesting TIMP-2b is highly conserved during teleost evolution. CiTIMP-2b gene is expressed in a wide range of tissues including blood, brain, muscle, trunk kidney, liver, head kidney, skin, spleen, heart, gill, intestine and fin, with the highest level of transcripts in spleen. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in all tissues. The CiTIMP-2b transcript is present at unfertilized eggs, which suggests that CiTIMP-2b transcript is maternally inherited. These results suggest that the CiTIMP-2b would play an important role in the A. hydrophila-related diseases and early embryonic development stages in grass carp.