Rongquan Wang

Isolation and characterization of 25 novel EST-SNP markers in grass carp (Ctenopharyngodon idella)

The grass carp (Ctenopharyngodon idella) is one of the most important freshwater fish species in China. In this study, we reported the isolation and characterization of 25 novel single nucleotide polymorphism (SNP) markers from 47 wild individuals (collected from Yangtze River). The ranges of observed heterozygosity (Ho), expected heterozygosity (He) and polymorphic information content were 0.0213–0.5532, 0.0213–0.5059 and 0.0208–0.3749, respectively. Six of the characterized loci were significantly deviated from Hardy–Weinberg equilibrium (Pxa0<xa00.05). Except for two loci, which were in the same unigene, the rest had no linkage disequilibrium between loci pairs. Those novel EST-SNP markers may be useful for conservation strategies and further genetic improvement of grass carp.

Molecular cloning and functional characterization of the NFIL3/E4BP4 transcription factor of grass carp, Ctenopharyngodon idella.

NFIL3 (nuclear factor interleukin 3-regulated) is an important bZIP transcription factor in the immune response and immune cells development. Here, we identified the NFIL3 gene from grass carp (Ctenopharyngodon idella; gcNFIL3). The deduced amino acid sequence of gcNFIL3 is 468 residues with a typical bZIP domain. Phylogenetics demonstrated that gcNFIL3 clustered closely with NFIL3 of zebrafish. Real-time PCR revealed gcNFIL3 is constitutively expressed in all tissues examined. Its expression was significantly upregulated in head kidney and trunk kidney after stimulation by bacteria. Immunofluorescence microscopy revealed that gcNFIL3 is mainly expressed in the nucleus. Overexpression of gcNFIL3 reduces Aeromonas hydrophila invasion and proliferation. In CIK cells, gcNFIL3 could induce the activation of NF-kappa B and upregulates the expression of IL10 and IFN. These results indicated that gcNFIL3 has immunoregulatory properties and might play a role in the immune response of fish.

Mannan-binding lectin-associated serine Protease-1 (MASP-1) mediates immune responses against Aeromonas hydrophila in vitro and in vivo in grass carp

Abstract The mannan‐binding lectin‐associated serine protease‐1 (MASP‐1) gene is a crucial component of the lectin pathway in the complement and coagulation cascade. Although MASP‐1 has been found in the immune system of teleosts, its immune functions in response to bacterial infection are unclear. In this study, we identified a MASP‐1 homolog (gcMASP‐1) in the grass carp (Ctenopharyngodon idella). The full‐length 3308‐bp gcMASP‐1 cDNA includes a 2160‐bp open reading frame encoding a protein composed of 719 amino acids with epidermal growth factor‐like, complement control protein, and trypsin‐like domains. gcMASP‐1 shares a high similarity with MASP‐1 counterparts in other species, and it is most closely related to Cyprinus carpio MASP‐1 and Sinocyclocheilus anshuiensis MASP‐1. Transcription of gcMASP‐1 was widely distributed in different tissues and induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMASP‐1 was also affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over‐expressing gcMASP‐1, transcript levels of almost all components, except gcMBL and gcC5, were significantly enhanced, and gcIL1&bgr;, gcTNF‐&agr;, gcIFN, gcCD59, gcC5aR1, and gcITG&bgr;‐2 were significantly upregulated after exposure to A. hydrophila; gcMASP‐1 interference downregulated the transcript levels after A. hydrophila challenge. In addition, gcMASP‐1 activated NF‐&kgr;B signaling. These findings indicate the vital role of gcMASP‐1 in innate immunity in C. idella. HighlightsWe identified gcMASP‐1, a MASP‐1 homolog, in grass carp.gcMASP‐1 transcription was induced by A. hydrophila in vivo and in vitro.gcMASP‐1 downregulation suppressed the immune response to A. hydrophila.gcMASP‐1 over‐expression activated NF‐&kgr;B signaling.Our findings confirm gcMASP‐1 involvement in innate immunity in grass carp.

Biological parameters, immune enzymes, and histological alterations in the livers of grass carp infected with Aeromonas hydrophila

ABSTRACT Aeromonas hydrophila is the causative agent of bacterial septicemia that is frequently observed in grass carp, Ctenopharyngodon idellus. In this study, we evaluated the biological parameters and immune enzymes in the liver of grass carp following A. hydrophila infection and quantified the alterations in liver histology using a semi‐quantitative system. For the biological parameters, we found that the liver somatic index (LSI) was more sensitive than Fultons condition factor (CF) and was significantly decreased at three days post‐injection (DPI). At the immune enzyme level, the level of peroxidase (POD) in the liver significantly increased at 1 and 3 DPI. The activity of alkaline phosphatase (ALP) significantly increased at 3 DPI. Similarly, acid phosphatase (ACP) activity significantly increased at 1, 3, and 5 DPI. Histologically, the results indicated that the liver index at 3, 5, and 7 DPI was significantly higher than that of control groups. The regressive alterations as the highly variable reactions patterns and its index at 5 DPI was significantly higher than that of 1, 21 DPI, and the control groups. Based on our results, we suggest that grass carp resist A. hydrophila infection via an innate immune mechanism in the liver. The findings of this study will help elucidate the underlying mechanisms of resistance to A. hydrophila infection. HighlightsWe attempted a novel perspective to quantified alterations observed on histology using a semi‐quantitative system.We evaluated biological parameters and immune enzymes in grass carp following A. hydrophila infection.Our finding indicated the vital role of liver in innate immunity against A. hydrophila in grass carp.

Identification and functional analysis of the toll-like receptor 20.2 gene in grass carp, Ctenopharyngodon idella.

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197xa0bp, with an open reading frame of 2835xa0bp that encoded a 944xa0amino acid polypeptide. Relatively, high expression levels of gctlr20.2 were observed in the spleen, head kidney, liver and brain tissues, with lower expression levels in the trunk kidney, intestine and heart tissues. Inxa0vivo and inxa0vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C.xa0idella kidney cells stimulated with lipopolysaccharide, flagellin or polyinosinic-polycytidylic acid. Overexpression of gctlr20.2 increased the expression of il1β, il8 and tnf-α, but not ifn, and also increased the activity of the nf-κB signal pathway. Silencing, via siRNA-tlr20.2, inhibited gctlr20.2 transcription by 65.7% and down-regulated the expression of inflammatory cytokine genes, but not tnf-α. This study increases understanding of the immune system in C.xa0idella.

Genetic variation analysis of wild populations of grass carp (Cteno-pharyngodon idella) using microsatellite markers: Genetic variation analysis of wild populations of grass carp (Cteno-pharyngodon idella) using microsatellite markers

Twelve microsatellites were used to analyze the genetic diversity and genetic structure of eight wild populations of grass carp, among which six populations from Yangtze River (Hanjiang, Wujian, Jiujiang, Shishou, Mudong, and Wanzhou), one population from Pearl River and Heilongjiang River for each, Zhaoqing, and Nenjiang, respectively. Twelve markers showed highly polymorphic and all the eight populations contained high genetic variations. The variations of six populations of Yangtze River and Zhaoqing population of Pearl River were higher than Nenjiang population of Heilongjiang River. Bottleneck analysis revealed that four populations (Zhaoqing, Nenjiang, Mudong, and Wangzhou) had experienced a recent genetic bottleneck, and the effective population size was reduced. Pairwise FST and AMOVA analysis detected significant genetic difference among populations. The pairwise population genetic distances and the UPGMA tree demonstrated that the genetic distances between six populations of Yangtze River and Zhaoqing population were closer and clustered together earlier, as compared to those populations with Nenjiang population. The genetic structure simulation analysis suggested that there were five logic populations of all individuals. The genetic structures of Zhaoqing and Nenjiang populations were shown with independent separation, but the genetic structures of populations from Yangtze River were shown with fuzzy distribution. The high diversity was found in the wild grass carp from three major watersheds in China, which would supply a basis for future genetic improvement. However, the bottleneck effect of some populations should be taken into account in the practical breeding programs.

Mannose-binding lectin and its roles in immune responses in grass carp ( Ctenopharyngodon idella ) against Aeromonas hydrophila

ABSTRACT The complement system is a crucial component of the innate immune system that links innate and adaptive immunity via four pathways. Mannose‐binding lectin (MBL), the initiating molecule of the lectin pathway, plays a significant role in the innate immune system in mammals and fish. Herein, we identified an MBL homolog (gcMBL) in grass carp (Ctenopharyngodon idella). The full‐length 948 bp gcMBL cDNA includes a 741 bp open reading frame encoding a 246 amino acid protein with a signal peptide, collagen triple helix repeat domain, and a C‐type lectin‐like/link domain. The gcMBL protein shares low similarity with MBL counterparts in other species, and is most closely related to Cyprinus carpio MBL. Transcription of gcMBL was widely distributed in different tissues, and was induced by Aeromonas hydrophila in vivo and in vitro. Expression of gcMBL was also affected by LPS and flagellin stimulation in vitro. In cells over‐expressing gcMBL, transcripts of almost all components except gcC5 were up‐regulated, and gcMBL, gcIL1&bgr;, gcTNF‐&agr;, gcIFN, gcCD59, gcC5aR and gcITG&bgr;‐2 were significantly up‐regulated following exposure to A. hydrophila or stimulation by bacterial PAMPs. Meanwhile, gcMBL deficiency achieved by RNAi down‐regulated transcript levels following A. hydrophila challenge, and gcMBL induced NF‐&kgr;B signalling. These findings indicate a vital role of gcMBL in innate immunity in grass carp. HighlightsMannose‐binding lectin initiates the lectin pathway of innate immunity in fish.gcMBL encodes a 246 amino acid C‐type lectin in grass carp.gcMBL is mainly expressed in liver.gcMBL is up‐regulated by exposure to bacterial pathogens and PAMPs.gcMBL activates NF‐&kgr;B signalling and mediates innate immunity in teleosts.

Transcriptome analysis and histopathology of black carp (Mylopharyngodon piceus) spleen infected by Aeromonas hydrophila.

Abstract Aeromonas hydrophila causes serious economic losses to the black carp (Mylopharyngodon piceus) industry. In this study, we analyzed the spleen of disease‐resistant and susceptible black carp by RNA‐seq. Overall, a total of 5243 terms were enriched in the gene ontology (GO) analysis, and 323 related pathways were found in the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. A total of 1935 differentially expressed genes were found and were primarily involved in cell adhesion, pathogen recognition, cellular immunity, cytokines, complement systems, and iron transport. Sixteen of the differently expressed genes involved in the immune response and the accuracy of the transcriptome data were further validated by quantitative real‐time PCR (qRT‐PCR). We observed Tissue sections of the spleen infected with A. hydrophila and the control group and found that the spleen of the infected group had necrosis. HighlightsWorlds first transcriptome analysis of black carp infected with Aeromonas hydrophila.Pathological analysis of black carp infected with Aeromonas hydrophila.Results show significant difference in 1935 unigenes.GO and KEGG pathway analysis of DGEs using bioinformatics tools.Discussed important immune‐related genes and pathways.

Complement component Bf/C2b gene mediates immune responses against Aeromonas hydrophila in grass carp Ctenopharyngodon idella

ABSTRACT The complement system is a significant component of innate immunity. Here, we identified a Bf/C2 homolog (gcBf/C2b) in grass carp. gcBf/C2b shares a high similarity with Bf/C2b counterparts in other teleosts. gcBf/C2b transcription was widely distributed in different tissues, induced by Aeromonas hydrophila in vivo and in vitro, and affected by lipopolysaccharide and flagellin stimulation in vitro. In cells over‐expressing gcBf/C2b, transcript levels of all components except gcC5 were significantly enhanced, and gcBf/C2b, gcIL1&bgr;, gcTNF‐&agr;, gcIFN, gcCD59, gcC5aR1, and gcITG&bgr;‐2 were significantly upregulated after A. hydrophila challenge or stimulation with bacterial pathogen‐associated molecular patterns (PAMPs). However, gcBf/C2b in interference cells down‐regulated the transcript levels after A. hydrophila challenge, and gcBf/C2b induced NF‐&kgr;B signaling. These findings indicate the vital role of gcBf/C2b in innate immunity in grass carp. HIGHLIGHTSWe identified a C2 homolog (gcC2) in grass carp.A. hydrophila induced gcC2 expression both in vivo and in vitro.Downregulation of gcC2 suppressed the immune response to A. hydrophila.Over‐expression of gcC2 activated the NF‐&kgr;B signaling pathway.Our findings indicate the vital role of gcC2 in innate immunity in grass carp.