Re-identification and characterization of grass carp Ctenopharyngodon idella TLR20

A specific CpG oligodeoxynucleotide induces protective antiviral responses against grass carp reovirus in grass carp Ctenopharyngodon idella

The polymorphism and haplotype of TLR3 gene in grass carp ( Ctenopharyngodon idella) and their associations with susceptibility/resistance to grass carp reovirus

Vitamin E deficiency depressed fish growth, disease resistance, and the immunity and structural integrity of immune organs in grass carp (Ctenopharyngodon idella): Referring to NF-κB, TOR and Nrf2 signaling

Molecular cloning, characterization and expression analysis of QM gene from grass carp ( Ctenopharyngodon idellus) homologous to Wilms' tumor suppressor

Pattern recognition receptors in grass carp Ctenopharyngodon idella: I. Organization and expression analysis of TLRs and RLRs

Identification and functional characterization of the CSF1R gene from grass carp Ctenopharyngodon idellus and its use as a marker of monocytes/macrophages

Both T-bet and GATA-3, Th1/Th2 lineage-specific transcription factors, play important roles in the development of T cells and Th1/Th2 differentiation. In this study, T-bet and GATA-3 genes were cloned from grass carp (Ctenopharyngodon idellus). The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp T-bet contained 608 aa, which possessed a T-box DNA binding domain. The putative primary structure of the polypeptide deduced from the cDNA sequence of grass carp GATA-3 contained 396 aa, which possessed two consensus zinc finger domains (C-X(2)-C-X(17)-C-X(2)-C). The YxKxHxxxRP motif, KRRLSA and LMEKs/n sequences were also conserved in this GATA-3. Phylogenetic analysis indicated that grass carp T-bet and GATA-3 group with their known counterparts with zebrafish T-bet and GATA-3 as the closest neighbor, respectively. RT-qPCR results showed that grass carp T-bet gene was highly expressed in head kidney, followed by spleen, and low expressed in gill, liver, kidney, and intestine, while GATA-3 gene was highly expressed in intestine, followed by spleen, and low expressed in gill, liver, kidney, and head kidney. Grass carp is one of the "four important domestic fish" in China and often infected by grass carp reovirus (GCRV). As yet, there is no evidence that T-bet and GATA-3 (Th1/Th2 subsets) are involved in anti-virus immune of teleost fish. In this study, by RT-qPCR, we analyzed the expression dynamics of grass carp T-bet and GATA-3 genes with IFN-γ gene in response to GCRV infection for the first time. The expression dynamics showed that three genes might be crucially modulated by in vivo GCRV infection: (1) GCRV mainly induced a T-bet expression profile comparing to the GATA-3 expression, while the higher expression profiles of IFN-γ correlated with the up-regulation of T-bet; (2) T-bet/IFN-γ and GATA-3 expression changes suggest that in GCRV-infected grass carp, the common immune state of head kidney further heightens, whereas the common physiological state of intestine transforms to an anti-virus immune state. From this finding, we realize that GCRV mainly induces a Th1 response, and Th1 cell-mediated recognition mechanisms play very important roles in anti-virus cellular immune of grass carp.

Functional characterization of an interleukin 20 like homologue in grass carp Ctenopharyngodon idella

Submerged macrophytes play a pivotal role in the restoration of shallow lakes. Compared to meadow-forming Vallisneria, canopy-forming Myriophyllum spicatum exhibits characteristics that may render it the dominant species. However, M. spicatum may hamper recreational and commercial activities. Herbivorous fish may potentially regulate the biomass and interspecific competition between the two plant species. We conducted an enclosure experiment to elucidate the effects of grass carp (Ctenopharyngodon idella) and Wuchang bream (Megalobrama amblycephala) on the biomass ratio and morphological traits of M. spicatum and V. denseserrulata. Grass carp significantly reduced the biomass, density, and relative growth rate of both plant species, while Wuchang bream had no significant effect on any of these variables. Accordingly, the biomass ratio of M. spicatum to V. denseserrulata was significantly lower in the grass carp treatment than in both the fish-free controls and the Wuchang bream treatment. Wuchang bream significantly decreased the individual height of V. denseserrulata, whereas grass carp substantially reduced the height of both plant species. Our findings suggest that Wuchang bream may be more appropriate for maintaining meadow-forming species such as Vallisneria than grass carp, though it faces challenges in controlling both the biomass and height of canopy-forming species like M. spicatum.

Mitogen-activated protein kinase kinase kinase 3 (MEKK3) is an evolutionarily conserved Ser/Thr protein kinase of the MEKK family that is essential for the host immune response to pathogen challenges in mammals. However, the immune function of MEKK3s in lower vertebrate species, especially in bony fish, remains largely unknown. In this study, a fish MEKK3 (designated CiMEKK3) gene was cloned and identified from grass carp (Ctenopharyngodon idella). The present CiMEKK3 cDNA encoded a 620 amino acid polypeptide containing a conserved S-TKc domain and a typical PB1 domain. Several potential immune-related transcription factor-binding sites, including activating protein 1 (AP-1), nuclear factor kappa B (NF-κB) and signal transducer and activator of downstream transcription 3 (STAT3), were observed in the 5’ upstream DNA sequence of CiMEKK3. A phylogenetic tree showed that CiMEKK3 exhibits a close evolutionary relationship with MEKK3s from Cyprinus carpio and Carassius auratus. Quantitative real-time PCR analysis revealed that CiMEKK3 transcripts were widely distributed in all selected tissues of healthy grass carp, with a relatively high levels observed in the gill, head kidney and intestine. Upon in vitro challenge with bacterial pathogens (Aeromonas hydrophila and Aeromonas veronii) and pathogen-associated molecular patterns (PAMPs) (lipopolysaccharide (LPS), peptidoglycan (PGN), L-Ala-γ-D-Glu-mDAP (Tri-DAP) and muramyl dipeptide (MDP)), the expression levels of CiMEKK3 in the intestinal cells of grass carp were shown to be significantly upregulated in a time-dependent manner. In vivo injection experiments revealed that CiMEKK3 transcripts were significantly induced by MDP challenge in the intestine; however, these effects could be inhibited by the nutritional dipeptides carnosine and Ala-Gln. Moreover, subcellular localization analysis and luciferase reporter assays indicated that CiMEKK3 could act as a cytoplasmic signal-transducing activator involved in the regulation of NF-κB and MAPK/AP-1 signaling cascades in HEK293T cells. Taken together, these findings strongly suggest that CiMEKK3 plays vital roles in the intestinal immune response to bacterial challenges, which will aid in understanding the pathogenesis of inflammatory bowel disease in bony fish.

Characterization analysis of TLR5a and TLR5b immune response after different bacterial infection in grass carp (Ctenopharyngodon idella)

Grass carp is the most commonly consumed fish species in Hong Kong. The allergenicity of grass carp and its allergen content are yet to be reported. This study characterized the major allergen in grass carp and investigated its allergenicity. Sixty-nine subjects with history of IgE-mediated allergic reaction to grass carp were recruited. The protein content in steamed grass carp extract was resolved by SDS-PAGE, and the major allergen was identified by immunoblotting with serum from subjects allergic to grass carp. The identity of allergen was elucidated by mass spectrometry and amino acid sequence obtained by amplifying the specific gene from cDNA library of grass carp. The cross-reactivity between parvalbumins from grass carp and other phylogenetically close (common carp) or commercially important (cod and salmon) species was investigated by competitive inhibition ELISA. A major IgE-binding protein was found at approximately 9kDa and identified as parvalbumin by immunoblotting and mass spectrometry. Grass carp parvalbumin was more allergenic than common carp, salmon, and cod parvalbumins despite sharing high sequence homology. This newly identified major allergenic parvalbumin isoform from grass carp was registered as Cten i 1 in the World Health Organization and International Union of Immunological Societies allergen database. Grass carp parvalbumin is identified as the major fish allergen in Hong Kong. The strong allergenicity of Cten i 1 contributes to the high IgE reactivity of grass carp. Grass carp, among other fish species, should be considered when managing fish-allergic patients.

Cloning and characterization of the grass carp (Ctenopharyngodon idella) Toll-like receptor 22 gene, a fish-specific gene

The gene and virus-induced expression of IRF-5 in grass carp Ctenopharyngodon idella

Grass carp (Ctenopharyngodon idellus) has been one of the most important traditional domestic freshwater aquaculture fish in China. Its habitat includes major rivers and lakes in China, but it is most abundant in Yangtze River and its adjacent lakes. In the present study, the genetic diversity and population structure of grass carp from Yangtze River system were investigated using microsatellite DNA markers. In total 109 individuals were collected from four sites of the Yangtze River System including Yibin, Sichuan; Jiayu, Hubei; Dongting Lake, Hunan and Poyang Lake, Jiangxi. A full-sib family was prepared using artifical fertilization. Genomic DNA was extracted from ethanol-preserved fin tissues by using phenol-chloroform method. Using a set of microsatellite primers originally reported in common carp (Cyprinus carpio), we carried out many cross-species PCR amplifications. PCR products were separated on 7.5% nondenaturing polyacrylamide gels, stained with ethidium bromide in water and visualized with ultraviolet. A successfully amplified locus with clear banding pattern (i.e. one or two bands) was subject to test for genetic segregation in a full-sib grass carp family before it was considered to be used in population analysis. Purified PCR products were cloned into E. coli DH5α strain using pMD-18T vector, and positive clones were selected and sequenced by Shanghai BioAsia Biotech Company for confirmation of repeat motifs. The size of alleles was scored by comparison with pBR322 DNA/Msp I markers. Five microsatellite primers successfully amplified six loci with reasonable polymorphism in grass carp. The number of alleles in these loci ranged from 3 to 7. These six microsatellite loci were inherited with Mendelian mode in a test full-sib grass carp family. Sequencing analysis for the PCR products in grass carp confirmed that all these six loci contained core repeat motifs, and some di-nucleotide motifs were similar to those in common carp (e.g. CA repeat) while others were new in grass carp, especially those tri-nucleotide repeats (i.e. TTA and GGA). Six microsatellite loci in grass carp, i.e. MFW1-1, MFW1-2, MFW5, MFW15, Koi3 and Koi20, were applied to subsequent studies on genetic diversity and possible population structure of grass carp samples from the Yangtze River System including river and its major adjacent lakes. The results showed that the number of alleles of a population ranged from 3.8 to 4.8 on average, and mean observed heterozygosity (Ho) from 0.40 to 0.57, while mean expected heterozygosity (H E) from 0.48 to 0.65. After applying Hardy-Weinberg Equilibrium (HWE) test, several loci were found to be significantly deviated from HWE in some populations in which deficiency of heterozygotes was apparent. Genetic distance analysis showed that the largest distance was between Sichuan and Dongting Lake population and the smallest distance was between Jiayu and Poyang Lake population. However, the analysis of molecular variance (AMOVA) indicated that almost majority of the variance in the grass carp was within populations (95.60%), and only small proportion was among populations (4.40%). The lower level of overall genetic diversity and particularly lower Fixation Index (Fst=0.044) indicated that the grass carp in Yangtze River system had a relatively lower genetic variation and its genetic structure between populations was weak.