In the present study, 21 forkhead box (Fox) genes were identified in Andrias davidianus, including 13 full-length genes and eight partial sequences. Phylogenetic analysis showed that most were conserved in other investigated amphibians, whereas the Foxk1 gene was found exclusively in A. davidianus. Molecular evolution analysis indicated that most Fox genes underwent purifying selection, whereas two sites of the adFoxp4 gene showed positive selection and were located on the adFoxp4 protein surface. Expression profiles of all Fox genes identified were analysed in the hypothalamic-pituitary-gonad axis by reverse transcription-quantitative polymerase chain reaction. Eighteen genes exhibited sexually dimorphic expression (15 ovary-biased and three testis-biased genes), whereas two genes showed no difference between ovary and testis. Further investigation of 12 selected sexually dimorphic Fox genes showed changes in the expression profile of 11 genes in the ovary of larvae reared at high temperatures (28°C). The results of the present study provide information on Fox genes in an amphibian and suggest that they play key roles in sexual development and reproduction in A. davidianus.

Half-smooth tongue sole (Cynoglossus semilaevis) is one of the most valuable marine aquatic species in Northern China. Given to the rapid development of aquaculture industry, the C. semilaevis was subjected to disease-causing bacteria Vibrio anguillarum. It therefore is indispensable and urgent to understand the mechanism of C. semilaevis host defense against V. anguillarum infection. In the present study, the extensively analysis at the transcriptome level for V. Anguillarum disease in tongue sole was carried out. In total, 94,716 high quality contigs were generated from 75,884,572 clean reads in three libraries (HOSG, NOSG, and CG). 22,746 unigenes were identified when compared with SwissProt, an NR protein database and NT nucleotide database. 954 genes exhibiting the differentially expression at least one pair of comparison in all three libraries were identified. GO enrichment for these genes revealed gene response to biotic stimulus, immune system regulation, and immune response and cytokine production. Further, the pathways such as complement and coagulation cascades and Vibrio cholerae infection pathways were enriched in defensing of pathogen. Besides, 13,428 SSRs and 118,239 SNPs were detected in tongue sole, providing further support for genetic variation and marker-assisted selection in future. In summary, this study identifies several putative immune pathways and candidate genes deserving further investigation in the context of development of therapeutic regimens and lays the foundation for selecting resistant lines of C. semilaevis against V. anguillarum.

MicroRNAs (miRNA) play key regulatory roles in diverse biological processes. Cynoglossus semilaevis is an important commercial mariculture fish species in China. To identify miRNAs and investigate immunerelated miRNAs of C. semilaevis, we performed high-throughput sequencing on three small RNA libraries prepared from C semilaevis immune tissues (liver, head kidney, spleen, and intestine). One library was prepared under normal conditions (control, CG); two were prepared during Vibrio anguillarum infection, where vibriosis symptoms were obvious and non-obvious (HOSG and NOSG, respectively). We obtained 11,216,875, 12,313,404, and 11,398,695 clean reads per library, respectively. Bioinformatic analysis identified 452 miRNAs, including 24 putative novel miRNAs. We analyzed differentially expressed miRNAs between two libraries using pairwise comparison. For NOSG-CG, there was significant differential expression of 175 (38.72%) miRNAs. There was significant differential expression of 215 (47.57%) miRNAs between HOSG and CG. Compared with CG, The HOSG-NOSG comparison revealed significantly different expression of 122 (26.99%),miRNAs respectively. Real-time quantitative PCR (RT-qPCR) experiments were performed for 10 miRNAs of the three samples, and agreement was found between the sequencing and RT-qPCR data. For miRNAs that were significantly differentially expressed, functional annotation of target genes by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analysis indicated that a set of miRNAs that were expressed highly abundantly and significantly differentially were might involved in immune system development and immune response. To our understanding, this is the first report of comprehensive identification of C semilaevis miRNAs being differentially regulated in immune tissues (liver, head kidney, spleen, and intestine) in normal conditions relating to V. anguillarum infection. Many miRNAs were differentially regulated upon pathogen exposure. This work provides an opportunity for further understanding of the molecular mechanisms of miRNA regulation in C semilaevis host-pathogen interactions. (C) 2013 Elsevier Ltd. All rights reserved.

Interferon regulatory factor 1 (IRF1) was known to play key roles in antiviral defense in several species, and some other important biological processes. In this report, full length cDNA of IRF1 from Cynoglossus semilaevis (CsIRF1) was identified. It was of 1,455 bp, containing a 5' UTR of 104 bp, a 3' UTR of 541 bp with a poly (A) tail and an ORF of 810 bp encoding a putative protein of 269 amino acids. The putative CsIRF1protein contained one conserved IRF domain (1-113aa), and two low complexity regions (140-158aa and 230-242aa, respectively). Phylogenetic analysis showed that CsIRF1 was conserved in the teleost evolutionary branch, which was independent of mammalian, birds and amphibians. Additionally, CsIRF1 had the 96% homology with marine fishes, while 66% with freshwater fishes. The expression profiles of CsIRF1was analyzed by quantitative real-time PCR in healthy tissues and in immune tissues challenged with different pathogens [Vibrio anguillarum and Lymphocystis disease virus (LCDV)], respectively. CsIRF1 was widely expressed in healthy tissues of Cynoglossus semilaevis and with the highest expression in blood, as much as 19 times of that in liver. V. anguillarum and LCDV both induced the CsIRF1 gene expression distinctly in liver, with the peak value reached to 98-fold at 6 h and 25-fold at 24 h, respectively. The bacteria induced CsIRF1 suddenly up-expression in each detected tissues. However, at the initial stage of the challenge of virus LCDV, the CsIRF1 expression in blood and spleen were up regulated; on the contrary, its expression in liver and head kidney were down regulated, 0.3 and 0.4-fold 6 h post virus injection, respectively. These results suggested that CsIRF1 gene might involve in not only antiviral activity but also antibacterial procedure, indicating its vital role in Cynoglossus semilaevis innate defense system.

Ceruloplasmin is a ferroxidase combining more than 95% copper atoms in plasma that plays roles in copper transport and regulates iron homeostasis.In this study,a fulllength cDNA of ceruloplasmin was cloned from channel catfish Ictalurus punctatus.The cDNA of ceruloplasmin has a length of 4 110bp,composed of a 25bp 5′UTR,an 860bp 3′UTR and a 3 225bp ORF,encoding apolypeptide of 1,074amino acids.Alignments showed that catfish ceruloplasmin was conserved among others.Quantitative real-time PCR was conducted to detect expression profiles of ceruloplasmin in normal tissues and four tissues(liver,headkidney,intestine and spleen)after infection by different pathogens:Edwardsiella tarda,Streptococcus iniae,Aeromonas hydrophila,and channel catfish Hemorrhage Reovirus(CCRV).The expression profile in liver was the highest in normal tissues.S.iniaeinduced the expression of ceruloplasmin to the highest extent,and CCRV virus to the lowest extent.These results provide a basis for further research aimed at exploring the precise immune-related molecular mechanism of ceruloplasmin in channel catfish.

采用RACE方法克隆了半滑舌鳎（Cynoglossus semilaevis）PKR基因（CsPKR）,获得了CsPKR全长cDNA序列为2907bp,其中包含1959bp的开放阅读框,100bp的5′非编码区和848bp的3′非编码区。保守结构域分析显示推导的CsPKR氨基酸在N端存在两个特异的dsRBD（dsRNA binding domain dsRBD）结构域,在C端具有多个保守的激酶活性位点,包括ATP结合位点和底物配体结合位点,以及活性环结构A-loop。系统进化树分析显示鱼类、两栖类、鸟类及哺乳类的PKR具有共同的进化起源,CsPKR与牙鲆PKR的亲缘关系最为密切。荧光定量PCR结果显示,CsPKR基因在健康鱼多个组织中广泛表达,在脑中的表达最高,头肾中表达最低。经鳗弧菌和淋巴囊肿病毒分别感染后,CsPKR基因在免疫相关组织中呈现上调表达趋势,其中感染鳗弧菌12h后在血液中表达量较对照组上升了9.28倍,在感染淋巴囊肿病毒24h后,在肝脏中的表达达到最大,为对照组的9.97倍。以上结果暗示CsPKR基因在半滑舌鳎响应细菌和病毒免疫应答中起重要作用。

应用实时荧光定量PCR技术,检测了TLR20和TLR21基因在斑点叉尾(鮰)Ictalurus punctatus感染迟钝爱德华氏菌Edwardsiella tarda、链球菌Streptococcus iniae、嗜水气单胞菌Aeromonas hydrophila和斑点叉尾(鮰)呼肠孤病毒(Channel Catfish Hemorrhage Reovirus,CCRV)后,在0、12、24、48、72 h、7d,肝脏、头肾、脾脏、肠中的时空表达特征.结果显示,4种病原均能引起斑点叉尾鲴TLR20、TLR21基因在所测免疫相关组织中表达量的变化,但呈现出不同的表达模式:感染嗜水气单胞菌后这两种基因的表达差异巨大,而TLR21基因的表达变化不大,在肝脏和头肾中表达量仅在3倍以内变化.感染爱德华氏菌后,TLR20、TLR21两种基因的表达模式类似,在肝脏中表达量显著上调.链球菌引起肝脏TLR20表达量发生4 360倍的变化,远远高于TLR21基因在同组织中的表达量(257.8倍).斑点叉尾(鮰)呼肠孤病毒感染引起TLR20、TLR21两种基因在肝脏、头肾的上调表达,肠、脾脏的下调表达.以上结果表明了TLR20、TLR21在斑点叉尾(鮰)天然免疫应答中起重要作用,为研究鱼类疾病防御机制提供了理论参考.

Expression of channel catfish TLR5 and TLR5S genes in the head kidney,intestine,liver and spleen were analyzed by quantitative real-time PCR method at 0 h,12 h,24 h,48 h,72 h and 7 d after infection with Edwardsiella tarda,Aeromonas hydrophila,Streptococcus iniae and channel catfish hemorrhage reovirus(CCRV),respectively.The results showed that TLR5 and TLR5S mRNAs were largely up-regulated by E.tarda and S.iniae and the most significant increase of TLR5S gene expression occurred in head kidney 12h after being challenged with S.iniae,which was 132-fold higher than the PBS control.After infection with A.hydrophila,TLR5 and TLR5S showed up-regulation at 12h and 24h and obvious down-regulation at 48h to 7d post-infection.TLR5 and TLR5S genes expression were suppressed by infection with CCRV in most tissues.In spleen,the expression of TLR5 was 0.017-fold while TLR5S was only 0.01-fold compared with PBS control.Among the four immune-related different tissues,the expression of TLR5 and TLR5S showed significant up-regulation in intestine and head kidney.For example,after being challenged with E.tarda and S.iniae,at time point of 12h,the expression of TLR5 increased 50.4-fold and 14.8-fold respectively in intestine,and the expression of TLR5S increased 52.8-fold and 132-fold respectively in head kidney.All data suggested that TLR5 and TLR5S were involved in the immune response of channel catfish against the intracellular bacterial and virus pathogen in a tissue-specific and pathogen-specific manner,and further confirmed that both of them played significant roles in the channel catfish innate immunity.

NLRs are a large family belonging to pattern recognition receptors which could recognize pathogen associated molecular patterns. Class II, major histocompatibility complex, transactivator (CIITA) is a member of NLR family. It is a critical transcription factor which could regulate the expression of MHC class II. In this study, a full-length cDNA of CIITA was cloned from channel catfish according to ten sequenced ESTs. This cDNA contains a 5′-UTR of 71 bp, a 3′-UTR of 238 bp and an ORF of 3,210 bp encoding 1,069 amino acids. Phylogenetic analysis showed that catfish CIITA was conserved with other CIITAs. Quantitative real-time PCR was conducted to detect the expression profiles of CIITA in normal tissues and responding to different pathogens (Edwardsiella tarda, Streptococcus iniae and channel catfish Hemorrhage Reovirus (CCRV)). The expression profile in blood was the highest (53.879-fold) in normal tissues. E. tarda and S. iniae could induce catfish CIITA in head kidney, liver and spleen. CCRV virus could also induce CIITA in head kidney and liver but reduce it in spleen. And S. iniae could induce the expression of CIITA to the highest extent and contrarily CCRV virus to the lowest extent. The expression data showed the tissue-specific and pathogen-specific expression patterns of CIITA responding to different pathogens. These expression data indicated the immune-related functions of CIITA. The data obtained in this study provide a basis for further research aimed at explore the precise immune-related molecular mechanism of CIITA in catfish.

NLRs are a large family of intracellular pathogen recognition receptors (PRRs) that recognize pathogen associated molecular patterns (PAMPs). In the previous study, the identification of NLRs subfamily A (NODs) and gene expression was carried out in channel catfish (Ictalurus punctatus). However, the gene expression profiles of channel catfish NODs (NOD1, NOD2, NLRC3, NLRC5 and NLRX1) after infection with various bacteria and virus are still unclear. In this study, expression of five NODs genes was analyzed by quantitative real-time PCR method. In healthy catfish tissues, all tested NODs genes were found to be ubiquitously expressed. After infection with Edwardsiella tarda, Aeromonas hydrophila, Streptococcus iniae, or channel catfish hemorrhage reovirus (CCRV), expression of NOD1, NOD2, NLRC3, NLRC5 showed a significant up-regulation in the intestine, liver and head kidney, whereas down-regulation was observed in the spleen after infection with A. hydrophila and CCRV. Expression of NLRX1 gene was up-regulated in the intestine, liver and head kidney, while obviously decreased in the spleen after infection with four pathogens. Among four different pathogens, S. iniae largely up-regulated NODs mRNAs, while CCRV only slightly enhanced NODs gene expression. Among four immune-related tissues, the order for NODs up-regulation was liver, head kidney, intestine, and spleen after infection with various pathogens. All data suggest NODs are involved in the immune responses of channel catfish against the intracellular bacterial and virus pathogens in tissue-specific and pathogen-specific manners, and provide the evidence for exploring the precise immune-related molecular mechanism of NODs in channel catfish.

A family member of inhibitor of apoptosis protein (IAP) termed baculoviral IAP repeat-containing 7 (BIRC7) from channel catfish (Ictalurus punctatus) was identified, the full length cDNA sequence of channel catfish BIRC7 (CcBIRC7) was 1686 bp, containing a 5'UTR of 93 bp, a 3'UTR of 399 bp with a poly (A) tail and an ORF of 1194 bp encoding a putative protein of 398 amino acids. The putative CcBIRC7 protein contains two BIR super-family conservative domains and a C-terminal RING finger motif. Phylogenetic analysis showed that catfish CcBIRC7 was moderately conserved with other BIRC7. Quantitative real-time PCR was conducted to examine the expression profiles of CcBIRC7 in healthy tissues and responding to different pathogens (Edwardsie(la tarda, Streptococcus iniae and Channel catfish Hemorrhage Reovirus (CCRV)). CcBIRC7 was widely expressed in healthy tissues of channel catfish and with the highest 37.28-fold expression in blood. E. tarda and S. iniae could induce CcBIRC7 gene expression drastically in head kidney, liver and spleen, which the peak value reached 31.6-fold, 613.9-fold and 34.4-fold increase by E. tarda infection, and 248.3-fold, 1540.3-fold and 120.4-fold increase post S. iniae challenge, respectively. While, CCRV virus could slightly induce CcBIRC7 expression in head kidney and liver but reduce it in spleen. The result suggested BIRC7 may play a potential role in channel catfish innate immune system against bacterial and virus infections, especially as the anti-bacteria immune gene. This is the first report of BIRC7 gene identification and its expression in fish. (C) 2012 Elsevier Ltd. All rights reserved.

Using fluorescence in situ hybridization(FISH),5S rDNA gene was mapped on chromosomes of half-smooth tongue-sole(Cynoglossus semilaevis)and olive flounder(Paralichthys olivaceus)for the first time.The results showed that 5S rDNA has two hybridization signal loci in a pair of homologous chromosomes of male and female half-smooth tongue-sole,respectively;5S rDNA has two and three hybridization signal loci in a pair of homologous chromosomes of diploid and triploid olive flounder,respectively,referring to the clear and specific hybridization signals.FISH mapping of 5S rDNA genes on the chromosomes of diploid half-smooth tongue-sole and triploid olive flounder provides a feasible method for ploidy and chromosome identification of half-smooth tongue-sole and olive flounder.In addition,the coding region sequences of 5S rDNA gene of turbot(Scophthalmus maximus) and half-smooth tongue-sole were amplified using same primers designed for olive flounder 5 S rDNA gene,and the phylogenetic analysis of the 5S rDNA genes in four species of flatfishes,including plaice(Pleuronectes platessa),senegalese sole(Solea senegalensis),largemouth bass(Micropterus salmoides) and arctic lamprey(Lampetra japonica) showed that the 5S rDNA gene of five species of flatfishes shared high homology(98.3%) and gathered into a branch.The GC content was significantly higher than the AT content in conservative sequence of 5S rDNA coding region of these five species of flatfishes.

Protein disulfide isomerases (PDIs) are thought to aid protein folding and assembly by catalyzing formation and shuffling of cysteine disulfide bonds in the endoplasmic reticulum (ER). Currently, increasing evidence suggests PDIs play an important role in host cell invasion and they are relevant targets for the host immune response. However the roles of specific PDIs in teleosts are little known. Here, we characterized the Protein disulfide isomerase family A, member 6 (PDIA6) from channel catfish, Ictalurus punctatus (named as ccPDIA6). The catfish ccPDIA6 gene was homologous to those of other vertebrate species with 13 exons and 12 introns. The consensus full-length ccPDIA6 cDNA contained an ORF of 1320 bp encoding a putative protein of 439 amino acids. It had a 19 amino acid signal peptide and two active thioredoxin-like domains. Sequence of phylogenic analysis and multiple alignments showed that ccPDIA6 was conserved throughout vertebrate evolution. Southern blot analysis suggested the presence of one copy of the ccPDIA6 gene in the catfish genome. Tissue distribution shows that ccPDIA6 was expressed in all examined tissues at the mRNA level. When using the aquatic zoonotic pathogens such as Edwardsiella tara, Streptococcus iniae, and channel catfish reovirus （CCRV） to challenge channel catfish, ccPDIA6 expression was significant changed in immune-related tissues such as head kidney, intestine, liver and spleen. The results suggested that ccPDIA6 might play an important role in the immunity of channel catfish. This is the first report that the PDI gene may be involved in fish host defense against pathogen infection.

In mammals, Toll-IL-1 receptor (TIR) domain-containing adaptor molecule 1 (TICAM-1) is a signaling adaptor for TLR3 and TLR4 that activates the transcription factors IRF-3, NF-κB, and AP-1, leading to the induction of type I interferon and cytokines. TICAM is also identified in some fish species, however, the gene expression profiling of TICAM is largely unknown in teleosts. Because bacteria such as Aeromonas hydrophila, Streptococcus spp. and Edwardsiella tarda and viruses such as channel catfish virus cause a multisystemic disease responsible for severe losses in channel catfish aquaculture in China. In this study, gene expression profiling of TICAM in different immune tissues(liver, headkidney, spleen,and intestine) after infection with these pathogens assayed by quantitative RT-PCR was described. After infection with A. hydrophila, TICAM was up-regulated approximately 2.3-fold at 24 h in liver and 1.9-fold at 12 h in spleen, while expression of this gene was down-regulated in headkidney and intestine, with the lowest expression as 0.15-fold at 48 h in headkidney, 0.53-fold at 24 h in intestine, respectively. TICAM was up-regulated drastically in liver, spleen, headkidney and intestine after infection with Streptococcus spp. It reached the highest level with 23-fold in liver at 7 d post infection, and it increased about 10 times in headkidney and spleen after infection. The expression of TICAM increased in all tested tissues after infection with E.tarda, especially it was up-regulated to the highest (23.1-fold) at 7d in spleen. After infection with channel catfish virus, the gene TICAM expression was up-regulated in liver, headkidney and intestine moderately, with the highest expression of 3.7-fold in liver at 72 h, 2.8-fold in headkidney at 7 d, 1.5-fold at 24 h in intestine. However, it was down-regulated in spleen,and its lowest expression was 0.13-fold at 24 h. In conclusion, the results of this study suggest that the TICAM gene may play crucial roles in innate immunity in channel catfish.

Lysozymes are considered to be potent innate immune molecules against the invasion of bacterial pathogens. The goose-type lysozyme is one of the three major distinct lysozyme types identified in the animal kingdom including teleosts. In this report, we identified, sequenced, and characterized the goose-type lysozyme gene (CsGLys) from half-smooth tongue sole (Cynoglossus semilaevis). The full-length cDNA of CsGLys is 1191 bp in length from the transcription start site to polyadenylation site, including a 91 bp 5'-terminal untranslated region (UTR), a 452 bp 3'-terminal UTR and a 648 bp open reading frame (ORF) of encoding a polypeptide with 215 amino acids. The deduced amino acid sequence of CsGLys possesses a Goose Egg White Lysozyme (GEWL) domain with three conserved residues (E91, D104 and D121) essential for catalytic activity. The CsGLys gene consisting of 2535 bp, was similar to those of other teleost species such as Japanese flounder and large yellow croaker with five exons interrupted by four introns. The 5'-flanking region of CsGLys gene shows several transcriptional factor binding sites related to immune response. Tissue expression profile analysis by quantitative real-time reverse transcription PCR showed that CsGLys mRNA was constitutively expressed in all examined tissues with the predominant expression in skin and the weakest expression in heart. The expression of CsGLys after challenged with bacteria Vibrio anguillarum was up-regulated in blood, head kidney, liver and spleen at 12 h postinfection and it reached the peak level at the same time point with a 19.89-, 4.21-, 14.45- and 10.37-fold increase, respectively, while the CsGLys expression was down-regulated to lower level than the normal level in each tested tissues except in liver from the 48 h until 96 h. These results suggest that CsGLys might play an important role in half-smooth tongue sole host defense against the bacteria infection. (C) 2012 Elsevier Ltd. All rights reserved.

As an important adaptor protein in the intracellular signaling of interleukin-1 receptor/toll-like receptor (IL-1R/TLR) superfamily,myeloid differentiation factor 88( MyD88) plays a crucial role in the innate immune response. So far,no report about MyD88 in half smooth tongue sole( Cynoglossus semilaevis),one of the most promising and commercially important species for marine fish culture in China,is available. In this paper,the cloning,characterization and expression pattern of MyD88 in half smooth tongue sole was described. The full length MyD88 cDNA of half smooth tongue sole is 1 612 bp including a 132 bp 5′ untranslated region( UTR),a 590 bp 3′ UTR and a 858 bp open reading frame( ORF) encoding 285 amino acids. MyD88 contains a typical death domain at the N-terminal and a conservative Toll/IL-1R( TIR) domain at the C-terminal. The length of genomic DNA of MyD88 in this species is 2 923 bp with 5 exons and 4 introns. The phylogenetic tree based on 11 species MyD88 proteins showed that half smooth tongue sole MyD88 shared the closetest relationship with Japanese flounder( Paralichthys olivaceus). The next quantitative real-time reverse transcription PCR analysis revealed MyD88 gene was expressed in all tested tissues with higher expression in immune tissues and reproduction tissues including blood,liver,spleen, kidney,testis and ovary. Then,the stimulation with LPS( lipopolysaccharide),GPN( peptidoglycan) and poly I:C showed that these stimulators could induce up-regulation of MyD88 expression in PBL of half smooth tongue sole. Finally,the pathogen infection experiment with Gram negative bacteria Vibrio anguillarum,indicated that MyD88 expression was significantly increased in the spleen by nealy 180 times and in the liver by more than 60 times after 96 th hour and in the headkidney with near 5 times at 48 th hour after challenge,respectively. These results suggest that MyD88 play an important role in the innate immune system in half smooth tongue sole.