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Septicemia is a systemic inflammatory response to bacterial infection that results in a hyper-inflammatory state, which could lead to septic shock and death in grass carp (Ctenopharyngodon idella). The aim of this study was to determine the underlying mechanism of microRNA (miR-130a) in bacteria-infected grass carp. Expression levels of miR-130a against Aeromonas hydrophila (A. hydrophila) infection in Ctenopharyngodon idella kidney cells (CIK) were analyzed. Luciferase reporter assay, quantitative reverse transcription-polymerase chain reaction were performed to explore whether Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 (CiGadd45bb) was a target of miR-130a. MiR-130a mimic, inhibitor and miR-control were transfected to CIK respectively. After transfection, the expression levels of proinflammatory genes were determined. Here we show that CiGadd45bb as a target of miR-130a. We also confirmed that miR-130a levels were significantly higher after being stimulated for 4 h and lower after 12 h (P < 0.01). Overexpressing miR-130a strikingly inhibited p38, JNK, ERK and TNF-a genes (P < 0.01) and silencing miR-130a activated p38, JNK, ERK, TNF-a, IFN and IL-8 (P < 0.01). Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-130a in grass carp., (Copyright © 2023. Published by Elsevier Ltd.)

The intestinal microbiome plays a pivotal role in the nutritional digestion and metabolism of the grass carp (Ctenopharyngodon idella). Here, we characterized the digesta and mucosal microbiome of the anterior, middle, and posterior intestine of the grass carp, using 16S rRNA next-generation sequencing. Based on 16S rRNA amplicon data, Proteobacteria, Firmicutes and Bacteroides were the dominant phyla in the intestine of grass carp. Our results also showed that microbial communities of the middle intestine exhibited higher alpha diversity indices compared with the anterior and posterior intestine. The clustering of microbial communities that had either colonized in the digesta or were attached to the mucosa, were significantly tighter in the posterior intestine, based on average unweighted Unifrac distances (P < 0.05). The digesta or mucosa of the anterior and middle intestines were similar in microbial composition, but were significantly different to the posterior intestine (P < 0.05). In digesta and mucosa samples from the posterior intestine, we observed a significantly increased abundance of cellulose-degrading microbiomes, such as Bacteroides, Clostridiales and Spirochaetia (P < 0.05). Our results suggested that the microbiomes of the posterior intestine, either attached to the mucosa or colonized in the digesta, were distinct from the microbiomes of the anterior and middle intestine in grass carp., (Copyright © 2021 Elsevier Inc. All rights reserved.)

There appears to be a close correlation between intestinal microbiotas and obesity. Still, our understanding of the relationship between the intestinal microbiota and body-mass in grass carp (Ctenopharyngodon idella) remains limited. Herein, we explored this association in the anterior, middle, and posterior intestine of cohabitating grass carp by using next-generation sequencing of the 16S rRNA gene. The results showed that alpha diversity indices of the low-weight-gain (LWG) groups were higher than that of the high-weight-gain (HWG) groups. HWG groups possessed the decreased ratio of Bacteroidetes to Firmicutes compared with that in the LWG groups. Principal coordinate analysis (PCoA) and analysis of similarities (ANOSIM) revealed that there were significant differences between the HWG and LWG groups. Furthermore, linear discriminant analysis (LDA) coupled with effect size (LEfSe) showed that the order Clostridiales were significantly abundant in the HWG groups. Phylogenetic molecular ecology networks (pMENs) showed a lower average path distance (GD), higher average clustering coefficient (avgCC), and higher average degree (avgK) in the HWG group. Our results suggested that there appeared to be a tight correlation between the intestinal microbiota and body-mass in grass carp. The study provides a referable resource for establishing the relationship between intestinal microbiotas and economic traits, which also lays a foundation for the progress of new fish probiotic in the future., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Complement component 4 (C4) has critical immunological functions in vertebrates. In the current study, a C4 homolog (gcC4) was identified in grass carp (Ctenopharyngodon idella). The full-length 5458 bp gcC4 cDNA contained a 5148 bp open reading frame (ORF) encoding a protein of 1715 amino acids with a signal peptide and eight conservative domains. The gcC4 protein has a high level of identity with other fish C4 counterparts and is phylogenetically clustered with cyprinid fish C4. The gcC4 transcript shows wide tissue distribution and is inducible by Aeromonas hydrophila in vivo and in vitro. Furthermore, its expression also fluctuates upon lipopolysaccharide or flagellin stimulation in vitro. During infection, the gcC4 protein level decreases or increases to varying degrees, and the intrahepatic C4 expression location changes. With gcC4 overexpression, interleukin 1 beta, tumor necrosis factor alpha, and interferon transcripts are all upregulated by A. hydrophila infection. Meanwhile, overexpression of gcC4 reduces bacterial invasion or proliferation. Moreover, gcC4 may activate the NF-κB signaling pathway. These findings demonstrate the vital role of gcC4 in the innate immunity of grass carp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

In grass carp (Ctenopharyngodon idella), septicemia is a systemic inflammatory response to bacterial infection. Once infected bacteria, a hyper-inflammatory state that could lead to septic shock and death. There is increasing evidence that microRNAs are involved in the regulation of the inflammatory response. Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 ba and bb (CiGadd45ba and CiGadd45bb) are two subtypes of Gadd45b. In the present study, miR-148 was confirmed to be involved in the inflammatory response after infection with Aeromonas hydrophila. Dual-luciferase reporter assays and miRNA expression profiling confirmed that miR-148 targeted both CiGadd45ba and CiGadd45bb. Transfection with miR148 mimics and inhibitors altered the expression levels of proinflammatory genes, suggesting that miRNAs regulate the immune response in grass carp. Our results provide a theoretical basis for studying the molecular mechanism underlying the regulation of inflammation by miR-148 in grass carp., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ctenopharyngodon idella growth arrest and DNA damage-inducible 45 ab (CiGadd45ab) is a subtype of the Gadd45a gene of the Gadd45 family in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory and apoptotic responses. However, little is known about the regulatory effects of miRNAs on CiGadd45ab expression. In the present study, CiGadd45ab was identified as a target gene of miR-23a-3p and miR-23a-5p, based on miRNA expression profiling and a dual-luciferase reporter assay. In addition, miR-23a-3p and miR-23a-5p were both confirmed to be involved in the inflammatory response following infection with Aeromonas hydrophila by targeting CiGadd45ab. Transfection with miR-23a-3p and miR-23a-5p mimics and inhibitor altered proinflammatory gene expression and apoptosis rate, thereby suggesting that miRNAs regulate immune response and anti-apoptosis by targeting CiGadd45ab in grass carp. Our results provide a theoretical basis for exploring the molecular mechanisms by which miR-23a-3p and miR-23a-5p target CiGadd45ab to regulate inflammation and apoptosis against bacterial infection in grass carp., Competing Interests: Declaration of competing interest All authors must disclose any financial and personal relationships with other people or organizations that could inappropriately influence (bias) their work. Examples of potential competing interests include employment, consultancies, stock ownership, honoraria, paid expert testimony, patent applications/registrations, and grants or other funding. Authors must disclose any interests in two places: 1. a summary declaration of interest statement in the title page file (if double-blind) or the manuscript file (if single-blind). If there are no interests to declare then please state this: ‘Declarations of interest: none'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Ctenopharyngodon idellagrowth arrest and DNA damage-inducible 45aa (CiGadd45aa) is a member of the Gadd45 family of immune-related proteins in grass carp. There is increasing evidence that microRNAs (miRNAs) are involved in the regulation of inflammatory responses and apoptosis. However, little is known about the regulatory effects of miRNAs on CiGadd45aa expression in grass carp. In the present study, CiGadd45aa was identified as a target gene of miR-731 based on miRNA expression profiling and dual-luciferase reporter assay. Our study revealed that miR-731 targets CiGadd45aa and regulates the expression of proinflammatory factors, thereby regulating immune response in grass carp. In addition, CiGadd45aa and miR-731 were both found induced apoptosis. Hence, our findings provide a theoretical basis for exploring the molecular mechanism by which miR-731 regulates inflammation and apoptosis in grass carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Growth arrest and DNA damage-inducible 45 gamma (Gadd45g) is a member of Gadd45 gene family of immunological proteins in mammals. Herein, we identified and characterised Gadd45g from grass carp. The cDNA spans over 1189 bp, with an open reading frame of 480 bp encoding a 159 amino acid protein. CiGadd45g mRNAs were expressed in all tissues investigated, with abundant expression in liver, kidney, heart, brain, blood and skin. Following infection with Aeromonas hydrophila, CiGadd45g expression was upregulated in these immune-related tissues (gill, liver, spleen, intestine, kidney and head kidney). Immune-related cytokines (p38 and JNK) and proinflammatory cytokines (IL-8, IFN-1 and TNF-α) were activated by CiGadd45g. CiGadd45g and downstream genes were regulated by microRNA miR-429b. These results indicate that CiGadd45g plays an important immune role in the response to A. hydrophila infection in grass carp., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Growth arrest and DNA damage inducible 45-beta (Gadd45B) is essential for mitogen-activated protein kinases (MAPK) activities, and involved in regulating growth, apoptosis, and DNA demethylation. In the present study, the cDNA of gcGadd45Ba and gcGadd45Bb in grass carp was identified. And the expression levels show that they were widely distributed in the tested tissues and showed significant immune responses both in vitro and in vivo after challenge with bacteria and pathogen-associated molecular patterns (PAMPs). Overexpression of Gadd45B significantly induced the expression of pro-inflammatory cytokines (IL-1β, IL-8, and TNF-α) and enhanced the phagocytosis activation of grass carp blood cells. These results indicate that Gadd45B plays an important role in innate immune responses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The Gadd45aa and Gadd45 ab genes are members of the Gadd45 family, which are critically involved in immunological and apoptosis functions. In this study, we isolated and characterized Gadd45aa and Gadd45 ab cDNA from grass carp (Ctenopharyngodon idella) (designated CiGadd45aa and CiGadd45 ab). The CiGadd45aa and CiGadd45 ab fragments spanned 1272 bp/1248 bp, which contained 474 bp/480 bp open reading frames encoding 157/159 amino acid proteins. BLAST analysis revealed that CiGadd45aa and CiGadd45 ab shared high similarity with known Gadd45a sequences. qRT-PCR analysis showed widespread and abundant expression of CiGadd45aa in gill, intestine, kidney, brain, blood, skin and fin, but low in liver, spleen, head kidney, heart, and muscle. CiGadd45 ab was expressed highly in liver, spleen and blood but at low levels in gill, intestine, kidney, head kidney, heart, brain, skin, muscle, and fin. Following challenge of grass carp with Aeromonas hydrophila, CiGadd45aa and CiGadd45 ab expression was upregulated. In immune-relevant tissues and MAPK family genes (p38, JNK and ERK) were upregulated by CiGadd45aa and CiGadd45 ab overexpression and partly downregulated by interfered in the CIK grass carp kidney cell line. In addition, transcription of the cytokine-encoding il-8 gene was upregulated/downregulated by CiGadd45aa and CiGadd45 ab overexpression and interference. These results suggest that CiGadd45aa and CiGadd45 ab play roles in innate immune responses against A. hydrophila in grass carp., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 Fulton's 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., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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 gcIL1β, gcTNF-α, gcIFN, gcCD59, gcC5aR1, and gcITGβ-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-κB signaling. These findings indicate the vital role of gcMASP-1 in innate immunity in C. idella., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

We characterized and identified the cDNA sequence of Toll-like receptor 20.2 in Ctenopharyngodon idella (gctlr20.2); it consisted of 3197 bp, with an open reading frame of 2835 bp that encoded a 944 amino 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. In vivo and in vitro, after being challenged with Aeromonas hydrophila or grass carp reovirus (GCRV), gctlr20.2 expression was induced in C. idella 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. idella., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

MicroRNAs (miRNAs) are endogenous small non-coding RNAs that play crucial roles in numerous biological processes. However, the role of miRNAs in antibacterial defence in fish has not been fully determined. Here, we identified that nine miRNAs are differentially expressed in kidney between susceptible and resistant grass carp strains. Analysis of spatial and temporal miRNA expression patterns suggests that cid-miRn-115 and miR-142a-3p are potential regulators of anti-bacterial activity. Overexpressing of cid-miRn-115 and miR-142a-3p results in a visible change in Ctenopharyngodon idella kidney (CIK) cells immune effector activity. Bioinformatics analysis and overexpressing assay shows that cid-miRn-115 and miR-142a-3p directly regulate tlr5 expression. cid-miRn-115 and miR-142a-3p overexpressing leads to a significant decrease in tlr5 expression in CIK, thereby repressing its downstream genes, such as il-1β, il-8 and tnf-α. These findings provide a novel insight into the determination of anti-bacterial compounds in grass carp.

Rac1, a Rho GTPase, serves critical immunological functions in mammals. Here, a Rac1 homolog (gcRac1) was identified in grass carp (Ctenopharyngodon idella). The full-length 2023-base pair gcRac1 cDNA contained a 579-bp open reading frame encoding a 192-residue protein, including a conserved RHO domain and nuclear localization signal. The gcRac1 protein shares high identity with other Rac1 counterparts and phylogenetically clustered with Danio rerio Rac1. The gcRac1 transcript showed wide tissue distribution and was inducible by Aeromonas hydrophila in vivo and in vitro; its expression also fluctuated with LPS or flagellin stimulation in vitro. With gcRac1 over-expression, gcPAK1, gcIL1-β, gcTNF-α and gcIFN were basically up-regulated by A. hydrophila and bacterial PAMPs induction, while gcRac1 knockdown decreased these transcripts after A. hydrophila challenge. Over-expression of gcRac1 reduced, while its suppression facilitated, bacterial invasion. Moreover, gcRac1 could activate NF-κB signaling. These findings implicate the vital role of gcRac1 in grass carp innate immunity., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Toll-like receptors (TLRs) play a critical role in the innate immune system. Although TLR18 is an important member of this family of receptors in fish, the role of the tlr18 gene in responses to pathogen infection is still unclear. In this study, we identified and characterized the grass carp tlr18 gene (gctlr18) to further clarify the function of TLR18 in teleost fish. Gctlr18 spans over 3600 bp and encodes a polypeptide of 852 amino acids. Analysis of the deduced amino acid sequence showed that gctlr18 encodes structures typical of the TLR family, including a signal peptide, seven leucine-rich repeats (LRRs), a transmembrane region, and a (Toll-interleukin-1 receptor) TIR domain. Quantitative RT-PCR analysis showed that gctlr18 was constitutively expressed in all investigated tissues, with abundant expression in spleen, gill, heart, intestine, kidney and fin and low expression in skin, liver and brain. Following grass carp reovirus-challenge and Aeromonas hydrophila inoculation, gctlr18 transcripts were upregulated significantly in immune-relevant tissues. Stimulation of Ctenopharyngodon idella kidney (CIK) cells with purified flagellin from Salmo typhimurium, lipopolysaccharide and polyinosinic-polycytidylic acid stimulation in vitro resulted in significantly increased gctlr18 expression, reaching a peak followed by restoration of normal levels. Overexpression of gctlr18 reduced A. hydrophila invasion by 83.4%. In CIK cells, gctlr18 induced the expression of proinflammatory cytokines, including il-8, inf-1 and tnf-α. Our results indicate that gctlr18 plays a key role in innate immune responses in teleost fish., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Relative quantification is the strategy of choice for processing real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) data in microRNA (miRNA) expression studies. Normalization of relative quantification data is performed by comparison to reference genes. In teleost species, such as grass carp (Ctenopharyngodon idella), the determination of reference miRNAs and the optimal numbers of these that should be used has not been widely studied. In the present study, the stability of seven miRNAs (miR-126-3p, miR-101a, miR-451, miR-22a, miR-146, miR-142a-5p and miR-192) was investigated by RT-qPCR in different tissues and in different development stages of grass carp. Stability values were calculated with geNorm, NormFinder, BestKeeper and Delta CT algorithms. The results showed that tissue type is an important variability factor for miRNA expression stability. All seven miRNAs had good stability values and, therefore, could be used as reference miRNAs. When all tissues and developmental stages were considered, miR-101a was the most stable miRNA. When each tissue type was considered separately, the most stable miRNAs were 126-3p in blood and liver, 101a in the gills, 192 in the kidney, 451 in the intestine and 22a in the brain, head kidney, spleen, heart, muscle, skin and fin. 126-3p was the most stable reference miRNA gene during developmental stages 1-5, while 22a was the most stable during developmental stages 6-18. Overall, this study provides valuable information about the reference miRNAs that can be used to perform appropriate normalizations when undertaking relative quantification in RT-qPCR studies of grass carp., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Matrix metalloproteinase-9 (MMP-9) belongs to a family of zinc-dependent endopeptidases and is associated with vital inflammatory processes. Here, we isolated and characterized MMP-9 cDNA from grass carp (Ctenopharyngodon idella) (designated as CiMMP-9). The cDNA was 2880 bp long and encoded a putative protein of 675 amino acids, with a predicted molecular mass of 75.816 kDa and an isoelectric point (pI) of 5.25. CiMMP-9 contained all three classical MMP-9 family signatures. The mRNA of CiMMP-9 was constitutively expressed in all tested tissues of untreated grass carp, with the highest expression levels in the blood, trunk kidney, head kidney and spleen. CiMMP9 transcript was present in unfertilized eggs, which suggests that CiMMP9 transcription is maternally inherited. Fluorescent real-time quantitative RT-PCR was used to examine the expression of the CiMMP-9 gene in C. idella after being challenged with Aeromonas hydrophila. A clear time-dependent expression pattern of CiMMP-9 was found after the bacterial challenge, and mRNA expression reached a maximum level at 7 days post challenge. This indicates that MMP-9 is inducible and is involved in immune responses, thus suggesting that CiMMP-9 plays an important role in A. hydrophila-related diseases and in early embryonic development stages in C. idella., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

The gene encoding matrix metalloproteinase 2 (MMP2) was cloned from grass carp (Ctenopharyngodon idella), and its expression levels during Aeromonas hydrophila infection and embryonic development stages were evaluated. The complete open reading frame of CiMMP2 was 1974 bp in length, encoding a 658-amino acid polypeptide. The deduced MMP2 protein contained four conserved domain structures, including an N-terminal signal sequence, a propeptide domain, three repeats of fibronectin-type II domain inserted in the catalytic domain and a C-terminal hemopexin-like domain. Phylogenetic analysis of MMP2s grouped grass carp with other teleosts. Detected in all fish tissues examined, CiMMP2 expression increased in the spleen and head kidney at 4 h and was significantly downregulated at 1 d after A. hydrophila infection. CiMMP2 transcripts were present in unfertilized eggs, suggesting its maternal origin. These findings implicate an important role for CiMMP2 in A. hydrophila-related diseases and early embryonic developmental stages of grass carp., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

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., (Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.)

The complement system, as a representative of innate immunity, plays a key role in the host defense against infections. C6 is the member of complement components creating the membrane attack complex (MAC). In this study, we cloned and characterized the grass carp complement component C6 (gcC6) gene. Our data showed that gcC6 gene contained a 2724bp open reading frame (ORF), a 237bp 5'-untranslated region (UTR) and a 219bp 3'-UTR. The deduced amino acid sequence of gcC6 showed 77.6% and 58.9% identity to zebrafish C6 and rainbow trout C6, respectively. GcC6 gene was expressed in a wide range of grass carp tissues, and the highest expression level of gcC6 was detected in the spleen and liver. Upon challenge with Aeromonas hydrophila, its expression was significantly up-regulated in muscle, trunk kidney, liver, head kidney, spleen, heart and intestine, whereas it was down-regulated in the brain and skin. The expression level of gcC6 was high at the unfertilized egg stage. It was significantly increased at 1 day post-hatching, but it was decreased at 10 days post-hatching. This result suggested that the complement C6 transcripts in early embryos were of maternal origin., (Copyright © 2011 Elsevier B.V. All rights reserved.)