Genome-wide characterization of Toll-like receptor gene family in common carp (Cyprinus carpio) and their involvement in host immune response to Aeromonas hydrophila infection

Accepted Manuscript Genome-wide characterization of toll-like receptor gene family in common carp (Cyprinus carpio) and their involvement in host immune response to Aeromonas hydrophila infection

Yiwen Gong, Shuaisheng Feng, Shangqi Li, Yan Zhang, Zixia Zhao, Mou Hu, Peng Xu, Yanliang Jiang PII: DOI: Reference:

S1744-117X(17)30062-X doi: 10.1016/j.cbd.2017.08.003 CBD 470

To appear in: Received date: Revised date: Accepted date:

30 March 2017 25 August 2017 26 August 2017

Please cite this article as: Yiwen Gong, Shuaisheng Feng, Shangqi Li, Yan Zhang, Zixia Zhao, Mou Hu, Peng Xu, Yanliang Jiang , Genome-wide characterization of toll-like receptor gene family in common carp (Cyprinus carpio) and their involvement in host immune response to Aeromonas hydrophila infection, (2017), doi: 10.1016/ j.cbd.2017.08.003

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ACCEPTED MANUSCRIPT Genome-wide characterization of Toll-like receptor gene family in common carp (Cyprinus carpio) and their involvement in host immune response to Aeromonas hydrophila infection

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Peng Xu4, and Yanliang Jiang*,1,3

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Yiwen Gong†,1,2, Shuaisheng Feng†,1, Shangqi Li1, Yan Zhang1, Zixia Zhao1, Mou Hu3,

Key Laboratory of Aquatic Genomics, Ministry of Agriculture; CAFS Key

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Laboratory of Aquatic Genomics and Beijing Key Laboratory of Fishery

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Biotechnology, Chinese Academy of Fishery Sciences, Beijing, China College of Fisheries and Life Science, Shanghai Ocean University, Shanghai, China

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Hangzhou Qiandaohu Xunlong Sci-Tech Development Company Limited, Quzhou,

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Fujian Collaborative Innovation Center for Exploitation and Utilization of Marine

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Biological Resources, Xiamen University, Xiamen 361102, China

Equal contribution

Corresponding author; E-mail address: [email protected]

ACCEPTED MANUSCRIPT Abstract The Toll-like receptor (TLR) gene family is a class of conserved pattern recognition receptors, which play an essential role in innate immuity providing efficient defense against invading microbial pathogens. Although TLRs have been extensively

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characterized in both invertebrates and vertebrates, a comprehensive analysis of TLRs

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in common carp is lacking. In the present study, we have conducted the first

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genome-wide systematic analysis of common carp (Cyprinus carpio) TLR genes. A set of 27 common carp TLR genes were identified and characterized. Sequence

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similarity analysis, functional domain prediction and phylogenetic analysis supported

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their annotation and orthologies. By examining the gene copy number of TLR genes across several vertebrates, gene duplications and losses were observed. The

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expression patterns of TLR genes were examined during early developmental stages

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and in various healthy tissues, and the results showed that TLR genes were ubiquitously expressed, indicating a likely role in maintaining homeostasis. Moreover,

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the differential expression of TLRs was examined after Aeromons hydrophila

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infection, and showed that most TLR genes were induced, with diverse patterns. TLR1, TLR4-2, TLR4-3, TLR22-2, TLR22-3 were significantly up-regulated at minimum one timepoint, whereas TLR2-1, TLR4-1, TLR7-1 and TLR7-2 were significantly down-regulated. Our results suggested that TLR genes play critical roles in the common carp immune response. Collectively, our findings provide fundamental genomic resources for future studies on fish disease management and disease-resistance selective breeding strategy development.

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Keywords: Toll-like receptor, TLR, Common carp, Immune response, Bacterial

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infection

ACCEPTED MANUSCRIPT 1. Introduction The Toll-like receptor (TLR) family is a class of conserved pattern recognition receptors (PRRs), which can recognize pathogen-associated molecular patterns (PAMPs). PAMPs are highly conserved small molecules associated with pathogens,

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including a variety of different molecular structures such as lipopolysaccharide (LPS),

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lipoprotein, lipoteichoic acid, peptidoglycan, mannose and nucleic acid. The

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recognition of PAMPs by TLRs triggers the innate immune system, and regulates the adaptive immune response to protect the host against pathogen invasion (Rebl et al.,

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2010). TLRs are type I transmembrane proteins that contain an extracellular

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N-terminus with a leucine-rich repeat region (LRR) and an intracellular C-terminus with a Toll–interleukin (IL)-1 receptor (TIR) domain (Tong et al., 2015). Using the

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extracellular LRR containing domain, TLRs recognize the PAMPs’ ligands to trigger

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the activation of signaling cascades (Palti, 2011), to activate downstream molecules and initiate the expression of target genes such as interleukin (IL), tumor necrosis

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factor (TNF), and type I interferon (IFN) (Kawai and Akira, 2010).

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Since first discovered in the mid-1980s (Anderson et al., 1985), more than 21 TLRs have been identified (Kawai and Akira, 2010). In humans, 10 functional TLRs have been described, named TLR1-TLR10. With a few exceptions, equivalents of these 10 TLRs have been described in fish. During the past a few decades, the emergence of genomic research and draft whole genome sequences of several fish species have showed that at least 16 TLRs existed in teleost (Temperley et al., 2008; Zhang et al., 2013a; Tong et al., 2015; Solbakken et al., 2016; Liao et al., 2017). Fish

ACCEPTED MANUSCRIPT TLRs are structurally highly similar to the mammalian TLRs, but exhibit distinct features and large diversity, which may be correlated to the aquatic habitat and the diverse evolutionary history. For instance, there are TLRs unique to fish, which form a so-called “fish-specific” family of TLRs including TLR19, 20, 21, 22 and 23 (Rebl

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et al., 2010). In addition, several TLR members are limited to only one or several fish

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species, such as TLR26 only reported in channel catfish (Zhang et al., 2013a), TLR27

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in coelacanth (Latimeria chalumnae), spotted gar (Lepisosteus oculatus) and elephant shark (Callorhinchus milii) (Wang et al., 2015), a novel gene TLR28 in miiuy croaker

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(Miichthys miiuy) (Wang et al., 2016b). All TLRs are classified into six major

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subfamilies, designated as TLR1, TLR3, TLR4, TLR5, TLR7, and TLR11 (Meijer et al., 2004; Zhang et al., 2014; Wang et al., 2016a). TLR1-subfamily comprising TLR1,

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TLR2, TLR6, TLR10, TLR14, TLR18 and TLR25, can recognize lipopeptides.

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TLR3-subfamily contains only TLR3 gene, and activates immune response via double-stranded RNA intermediates (Huang et al., 2011). TLR4-subfamily,

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containing only TLR4 gene, is responsible for detecting LPS. TLR5-subfamily

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including TLR5M and TLR5S mainly recognize monomeric flagellin, the structural component of bacterial flagella (Hwang et al., 2010). TLR7-subfamily comprises TLR7, TLR8 and TLR9, of which TLR7 and TLR8 binds to single-stranded viral RNA while TLR9 binds to viral or bacterial DNA containing unmethylated CpG motifs (Palti et al., 2010a; Xinxian et al., 2016). TLR11-subfamily consists of “fish-specific” TLRs, and TLR 11, TLR 12, TLR13 from mice which may be considered as orthologue to those fish-specific TLRs, and can recognize protozoan

ACCEPTED MANUSCRIPT parasite, dsRNA, unmethylated CpG-DNA (Pietretti et al., 2014; Rauta et al., 2014; Sundaram et al., 2012). Teleost occupy a pivotal position in the evolution of innate and adaptive immunity, therefore, it draws growing attention for comparative studies of immune

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defense mechanisms between lower vertebrates and mammals (Rebl et al., 2010). The

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common carp, Cyprinus carpio, not only is an important aquaculture fish species

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mainly cultured in Asia and Europe (Jiang et al., 2014a), but also serve as a good research model for teleost immune studies (Feng et al., 2016; Jiang et al., 2016).

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Several TLRs have been identified in common carp, including TLR1 (Fink et al.,

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2016), TLR2 (Fink et al., 2016), TLR3 (Falco et al., 2014; Yang and Su, 2010), TLR7 (Tanekhy et al., 2010), TLR9 (Kongchum et al., 2011a), TLR20 (Pietretti et al., 2014).

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However, these studies mainly focused on a single TLR gene. The comprehensive

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information of TLR family in common carp is limited. In the past decade, due to the well-developed sequencing technologies with drastically reduced cost, common carp

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draft whole genome sequences plus numerous other genomic resources have been

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developed (Christoffels et al., 2006; Ji et al., 2012; Jiang et al., 2014b; Xu et al., 2011; Xu et al., 2014), which allow us to systematically examine the TLR gene family at genome scale. In this study, by utilizing all available genomic resources, we identified a set of 27 TLR genes in common carp with analysis of their sequence structures and functional domains. The common carp is a known allotetraploid and has experienced an additional round of whole genome duplication (WGD) compared to other teleosts, which may cause difficulties with assembly and misannotation of genes. To further

ACCEPTED MANUSCRIPT confirm the annotation, we conducted phylogenetic analysis. In addition, the expression patterns of TLR genes during common carp early developmental stages and in various healthy tissues were established. Furthermore, we examined the regulated expression of TLR genes in spleen after Aeromonas hydrophila infection.

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Our systematic study of TLR genes in common carp provided fundamental genomic

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resources for better understanding the activation of the innate immune system in

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response to invading pathogens in teleost.

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2. Materials and Methods

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2.1. Ethics Statement

All sampling procedures involving the handling and treatment of animals during

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this study were approved by the Animal Care and Use committee of the Centre for

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Applied Aquatic Genomics at the Chinese Academy of Fishery Sciences prior to initiation. The fish were euthanized by using MS-222 (80 mg/L immersion) before

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sampling, and all efforts were made to minimize suffering.

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2.2. Identification of TLR gene family in common carp genome and analysis TLRs genes from zebrafish and human were obtained from Ensembl (release 85; http://asia.ensembl.org/) and NCBI (http://www.ncbi.nlm.nih.gov/) databases (Supplementary file 1), and used as the query sequences to search against all available common carp genomic resource by stand-alone BLAST tools, with an E-value cutoff of 1e-10. Then reciprocal BLAST searches were conducted by using the candidate common carp TLR gene sequences as queries to verify the veracity of candidate genes.

ACCEPTED MANUSCRIPT Additionally, the coding sequences were confirmed by BLAST searches against NCBI non-redundant protein sequence database (nr). For TLR protein analysis, the web-based simple modular architecture research tool (SMART; Letunic et al., 2006) was used for conserved domains identification

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N-glycosylation were analyzed by using ExPASy server

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and annotation, with default parameters. The molecular weight, theoretical PI, and

2.3. Phylogenetic analysis of TLR gene family

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(http://web.expasy.org/protparam/ ).

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Phylogenetic analysis was conducted with reference TLR proteins from zebrafish

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(Danio rerio), human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), lizard (Anolis carolinensis), frog (Xenopus tropicalis), medaka (Oryzias

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latipes), fugu (Takifugu rubripes), stickleback (Gasterosteus aculeatus), salmon

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(Salmo salar) and catfish (Ictalurus punctatus). All reference protein identifiers are shown in Supplementary file 1. Multiple protein sequences were aligned by ClustalW

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with default parameters. We performed maximum likelihood analysis in MEGA7

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(Kumar et al., 2016) with bootstrap test of 1000 replicates. The best-fit model was the JTT+I+G model which uses a Jones-Taylor-Thornton (JTT) matrix and incorporates a proportion of invariant sites (+I) and the gamma distribution for modeling rate heterogeneity (+G). The maximum likelihood trees were constructed using MEGA7 with Subtree-Pruning-Regrafting – Extensive (SPR level 5) as the LM Heuristic Methods. 2.4. Bacterial challenge and sample collection

ACCEPTED MANUSCRIPT Six-month old healthy common carp (180 ± 25 g in weight and 15 ± 3 cm in length) were obtained from common carp farms located at Zhengzhou, Henan, China. The fish were randomly divided into control group and treatment group. Treated group was intraperitoneally injected with 0.1 ml of A. hydrophila cultured in LB

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medium (1×108 CFU/ml), while control group was injected with same volume of

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sterilized LB broth. The bacteria were isolated from diseased fish in Dongxi Lake

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(Wuhan, China), followed by a series of biochemical test to confirm it to be A. hydrophila by the Key Lab of Freshwater Animal Breeding, Huazhong Agricultural

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University (Jiang et al., 2016). The spleen from the two groups of common carp was

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collected at 4 h, 12 h, and 24 h post challenge. 5 replicate fish were selected from both treated and control for each of the three time points - 30 fish in total.

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Ten tissues were collected from 3 healthy adult common carps including brain,

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heart, spleen, liver, kidney, intestine, gill, muscle, skin and blood. Pooled samples of 10 egg mass/fries were collected from eight developmental stages post fertilization (0

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hour post fertilization (hpf), 12 hpf, 24 hpf, 36 hpf, 48 hpf, 3 day post fertilization

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(dpf), 5 dpf, and 7 dpf). All sample collected were immediately submerged into 10 ml RNAlaterTM (Ambion, USA), following the manufacturer’s protocol. Tissues were stored at -80ºC until RNA extraction. 2.5. RNA isolation and quantitative real-time PCR analysis QIAGEN RNeasy Mini kit was used for extracting total RNA and the synthesis of cDNA was conducted by using SuperScriptIII Synthesis System (Life Technologies). qRT-PCR was performed on an ABI PRISM 7500 Real-Time Detection System (Life

ACCEPTED MANUSCRIPT Technologies) using SYBR Green Master Mix reagent (Transgen Biotech, Beijing, China). PCR programs was conducted with initial polymerase activation at 94°C for 30 s; 40 cycles at 94°C for 5 s, 56°C for 15 s, 72°C for 34 s. The 20 uL reaction mixture contains 0.4 uL forward primer, 0.4 uL reverse primer, 10 uL TransStart Top

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Green qPCR Super Mix, 1 uL cDNA template (100 ng/mL), and 8.2 uL ddH2O.

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Common carp β-actin gene was used as the internal standard. To determine the gene

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expression patterns in healthy tissues, Ct values from the tissue with the lowest expression values were used as control group. For samples in different early

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developmental stages, 0 hpf was used as control group. Expression level of treated

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group transcript was determined as a relative expression ratio to control group and all samples were normalized to the expression levels of β-actin in the same sample. All

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statistical analysis were done using REST software (Pfaffl et al., 2002), with ANOVA

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3. Results

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analysis. P values less than 0.05 were considered to be significant.

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3.1 Identification of TLR genes in common carp Utilizing all available genomic resources, a total of 27 TLR genes in common carp genome were identified, including 19 newly identified and 8 published elsewhere (Table 1). The newly identified TLRs were TLR1, TLR2-1, TLR2-2, TLR4-3, TLR5, TLR7-2, TLR8-1, TLR8-2, TLR8-3, TLR18-1, TLR18-2, TLR19, TLR21-1, TLR21-2, TLR22-1, TLR22-2, TLR22-3, TLR25-1, TLR25-2. All complete coding sequences of the newly identified TLR genes were deposited to DDBJ database with

ACCEPTED MANUSCRIPT accession number of LC150761-LC150779. Full length coding sequences were obtained of all TLR family members except TLR4-4 and TLR7-2. Detailed information of their corresponding genomic sequences, coding sequences, number of exons are summarized in Table 1. The number of amino acid of TLR genes ranges

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from 733 to 1149. As shown in Table 1, of all TLR genes, the genomic sequences of

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14 TLRs are intronless. There are 6 TLRs comprising 2 exons, 5 TLRs comprising 3

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exons, 1 TLR comprising 5 exons and 1 TLR comprising 6 exons.

To better understand the biological function of TLR genes, we looked into the

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characteristics of proteins, including the molecular mass, theoretical PI, potential

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N-glycosylation sites, and the functional domains. The molecular weight of TLR proteins ranged from 56 to 130 kDa (Table 2). The theoretical PI of most TLRs, 21

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out of all 27 TLR proteins, were within 7.0~9.0. Glycosylation in TLR is likely to

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influence receptor surface representation, trafficking and pattern recognition (Weber et al., 2004). The number of potential N-glycosylation sites varied in common carp

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TLRs, ranging from 6 to 21 (Table 2). The functional domains of TLR genes were

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predicted based on their protein sequences. As shown in Fig. 1, all TLRs harbor a Toll/Interleukin-1 receptor (TIR) domain, and various number of Leucine-rich repeat (LRR) domains, ranging from 5 to 17 LRRs. The classic transmembrane domain was detected in vast majority of TLRs by SMART. LRR-carboxyl domain exists in most TLR genes except in TLR18-1, TLR18-2, TLR19, TLR20, TLR21-1, and TLR21-2. The similar domain organization may not indicate their relatedness, thus the phylogenetic analysis is needed for identification of the relationship.

ACCEPTED MANUSCRIPT 3.2. Phylogenetic analysis In order to properly annotate the common carp TLR genes, as well as identify their phylogenetic relationship, phylogenetic analysis was conducted. A total of 155 full-length amino acid sequences of TLRs from 12 species were used for constructing

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phylogenetic tree. As shown in Fig. 2, six major clades are resolved, corresponding to

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6 TLR subfamilies: TLR1-subfamily which consist of TLR1, TLR2, TLR18, and

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TLR25, TLR7-subfamily which consist of TLR7, TLR8 and TLR9, TLR3-subfamily, TLR5-subfamily, TLR4-subfamily, TLR11-subfamily which consist of TLR19,

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TLR20, TLR21 and TLR22. Overall, the phylogenetic analysis supported the

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annotation of common carp TLR genes. Except for TLR25 which was not identified in zebrafish, each common carp TLR gene was first clustered with its respective

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counterparts of zebrafish, then grouped with other orthologous genes of other teleost

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and other vertebrates, with strong bootstrap support (Fig. 2). TLR25 appeared to be a quite unique TLR gene, which was only found in catfish and common carp so far. The

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phylogenetic tree showed that the common carp TLR25 was clustered with catfish

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TLR25, and formed a subclade with TLR18 gene of teleost, indicating the close evolutionary-relationship between TLR18 and TLR25. 3.3 Gene copy number analysis of TLRs The phylogenetic tree revealed the copy number of several TLR genes was double or more in common carp than in other species. To further determine the variation of TLR gene copy number in different vertebrates, we searched literatures as well as public databases. Up to present, a total of 28 TLR genes were annotated across

ACCEPTED MANUSCRIPT a broad spectrum of species, including 26 TLRs reported in early literatures (Temperley et al., 2008), in addition with two newly identified TLRs, TLR27 (Wang et al., 2015) and TLR28 (Wang et al., 2016b). Thirteen TLRs named TLR1~TLR13 were identified in human and mouse, with one single copy for each gene. In chicken,

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TLR1 and TLR2 were found duplicated. In frog, TLR14 was found duplicated. While

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in fish, multiple copies were reported for several TLRs including TLR2, TLR3, TLR4,

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TLR5, TLR6, TLR18, TLR20, TLR21, TLR22 and TLR25. In the present study, 14 distinct TLR genes of all 28 TLRs were identified in common carp: TLR1, TLR2,

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TLR3, TLR4, TLR5, TLR7, TLR8, TLR9, TLR18, TLR19, TLR20, TLR21, TLR22

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and TLR25 (Table 3). Among those common carp TLR genes, nine members appeared to have been duplicated, including TLR2, TLR3, TLR4, TLR7, TLR8,

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TLR18, TLR21, TLR22 and TLR25. Comparing to several representative vertebrates

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and teleost (human, mouse, chicken, lizard, frog, zebrafish, catfish, stickleback, medaka, fugu, tetraodon), common carp harbored the most number of TLR genes

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(Table 3). It is interesting that only one single copy of TLR5 exist in common carp

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while two or more copies exist in other fish species. Teleost fish appeared not to have TLR6, TLR10-13, TLR15-TLR17, TLR24. Up to present, TLR28 were only found in miiuy croaker (Wang et al., 2016b). 3.4 Tissue expression of TLR genes in common carp In order to determine the expression patterns of TLR genes, quantitative real-time PCR was performed using gene-specific primers in ten healthy tissues, including blood, brain, gill, heart, intestine, kidney, liver, muscle, skin, and spleen. Tissue

ACCEPTED MANUSCRIPT expression of all TLR genes were detected, expect TLR20 because of difficulties in primer design for specific amplification. As shown in Fig. 3, TLR genes were widely expressed in all tested tissues, but exhibited tissue preference, indicating that different TLR genes might be involved in different biological process under healthy conditions.

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For instance, TLR4-1, TLR7-1 and TLR19 were expressed at high level in the brain;

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TLR22-2 were highly expressed in the heart; TLR2-1, TLR2-2, TLR3-1, TLR4-2,

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TLR5, TLR8-3, TLR22-1 and TLR22-2 were highly expressed in the spleen; TLR8-3, TLR9, TLR18-1, TLR18-2 and TLR22-2 were highly expressed in the kidney;

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TLR2-1, TLR4-1 and TLR8-3 were highly expressed in the intestine; TLR1, TLR3-2,

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TLR7-1 and TLR7-2 were highly expressed in the gill; TLR1, TLR5, TLR25-1 and TLR25-2 were highly expressed in the muscle; TLR4-2 were highly expressed in the

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skin. Most TLR genes were expressed at relatively low levels in kidney, muscle and

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blood (Fig. 3). Several TLR genes were extremely highly expressed in the spleen, such as TLR2-2 (Fig. 3).

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3.5 Expression of TLR genes during early developmental stages

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To determine the expression of TLR genes during common carp early developmental stages, fertilized eggs and newly hatched fries were collected at eight developmental timepoints, including 0 hpf, 12 hpf, 24 hpf, 36hpf, 48 hpf, 3 dpf, 5 dpf, and 7 dpf. Quantitative real-time PCR was performed to analyze the expression level of each TLR gene. As shown in Fig. 4, most of the tested TLR genes can be detected throughout all tested timepoints, but were expressed at extremely low levels within 24 hpf. Overall, the expression level of TLRs were increased at 36 hpf, decreased at 48

ACCEPTED MANUSCRIPT hpf, and increased again at 7 dpf. TLR4-4 and TLR8-1 were hardly detected during all tested developmental stages. 3.6 Expression of TLR genes in the common carp spleen following bacterial infection Transcriptional regulation of TLRs after A. hydrophila infection were examined

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in the spleen. As shown in Fig. 5, the expression of all tested TLR genes appeared to

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be affected by bacterial challenge, but differed in the timing and extent/direction of

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regulation. At the 4 h after infection, TLR4-2, TLR4-3,TLR22-2, TLR22-3 were significantly up-regulated (P < 0.05), while the expression level of

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TLR7-2,TLR7-2,TLR21-2 were significantly down-regulated at the same time (P <

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0.05). At the second detective timepoint (12 h), TLR1 and TLR22-2 were significantly up-regulated (P < 0.05), while at the 24 h after infection, the expression

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of most TLR genes were decreased, with TLR2-1 and TLR4-2 significantly

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down-regulated (P < 0.05). Of all, TLR1 at 12 h and TLR2-2 at 24 h were observed as the most up-regulated genes with 15-fold and 9-fold increment, respectively. In

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contrast, the most down-regulated genes were observed at 24 h, TLR4-2 and TLR2-1

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with 50-fold and 11-fold reduction, respectively.

4. Discussion Toll-like receptors (TLRs) perform a vital role in innate immue system through the recognition of pathogen associated molecular patterns. Although TLRs have been extensively characterized in both invertebrates and vertebrates, a systematic analysis

ACCEPTED MANUSCRIPT of TLRs phylogenetics and expression in common carp is lacking. Here, we presented a comprehensive analysis of all common carp TLRs, that a set of 27 TLR genes were identified and further investigated by using genomic, transcriptomic and phylogenetic tools.

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Common carp TLR genes appear to have duplicated gene copies. Comparing to

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human, mouse, chicken, frog, tetraodon, fugu, medaka, stickleback, catfish and

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zebrafish, the total number of TLRs identified in common carp was the most (Table 3). TLR2, TLR3, TLR4, TLR7, TLR8, TLR18, TLR21, TLR22, and TLR25 appeared to

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be duplicated, which may be resulted from whole genome duplication or tandom

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(localized) duplication. It has been widely accepted that, two rounds of large-scale gene duplication took place early in vertebrate evolution, and an additional round of

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duplication named teleost-specific whole genome duplication occurred in the common

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ancestor of all teleost (Glasauer et al., 2014; Van de Peer et al., 2009). Further analysis using molecular markers (David et al., 2003), linkage map (Zhang et al.,

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2013b), and whole genome sequencing analysis (Xu et al., 2014) indicated that

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common carp had experienced the 4th round whole genome duplication (4R WGD), which resulted in more copies for many genes in common carp than most other teleost. Duplicated gene copies located close to each other are more likely the result of tandom (localized) duplication caused by unequal crossing over. Tandom (localized) duplication is believed to be one of the dominant mechanisms contributing to genome evolution (Pan and Zhang, 2008). In our data, for instance, TLR7-1, TLR7-2, TLR8-1, TLR8-3 were closely located on common carp linkage group 40, which could be

ACCEPTED MANUSCRIPT resulted from tandom duplication. Besides gene duplications, gene losses were observed as well. Comparing to tetrapods, TLR6, TLR10, TLR11, TLR12, TLR13, TLR15 were not found in teleost fish, which was likely resulted from the gene losses after the split of teleost fish from tetrapods during evolution. It was noteworthy that

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multiple copies of TLR5 were identified in other teleost while only one single copy

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was found in common carp, which appeared as a result of lineage-specific gene losses.

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TLR5 is responsible for the recognition of bacterial flagllin in vertebrate. Two types of TLR5 have been identified in some fish species such as rainbow trout (Tsujita et al.,

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2004), seabream (Munoz et al., 2013), Japanese flounder (Hwang et al., 2010), catfish

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(Baoprasertkul et al., 2007; Zhang et al., 2013a), grass carp (Jiang et al., 2015). While in some other fish species such as Atlantic cod, the loss of TLR5 were reported

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(Solbakken et al., 2016). It is likely that the flagellin detection is implemented by the

ligand profiles.

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retained copy of TLR5, or covered by other PRR families due to the overlapping

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Interestingly, most of the common carp TLR genes are intronless. The

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exon-intron structure of TLRs genes varied between species in our data exemplified by the intronless TLR1 in common carp, pufferfish, chicken and mammals in contrast to the intron-containing TLR1 in zebrafish (Wu et al., 2008). Furthermore, it also varied within duplications such as one and two introns for TLR2-1 and TLR2-2 in common carp, respectively, while the genomic TLR2 gene in zebrafish and channel catfish were intronless (Rebl et al., 2010). Its orthologue from pufferfish comprised ten introns, and even eleven introns in Japanese flounder. Longer or more introns

ACCEPTED MANUSCRIPT could delay regulatory response due to the cost of splicing which would take more time (Jeffares et al., 2008). We found that in common carp that TLRs tend to have few or no introns. Thus, we speculated that this could provide the common carp immune system with a highly efficient TLR response.

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Without in vivo experiment, we conducted bioinformatic and molecular analysis

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including protein characterics and functional domain prediction, phylogenetic analysis

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and expression detection to investigate the biological function of common carp TLR genes and to verify if they are behaving as other vertebrate TLRs. The functional

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domain prediction of common carp TLRs showed that the TIR domain, which was

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crucial for ligand binding and signal transduction in innate immunity, was highly conserved across all TLR gene members, whereas the LRR domain, which was

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involved in pathogen recogniciton (Bell et al., 2003), varied in the number and

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position. Moreover, the phylogenetic analysis apparently suggested its high conservation, with each common carp TLR gene fell into their respective clades with

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orthologous genes from other species (Fig. 2), which also well supported the

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annotation of common carp TLRs. In addition, six major clusters were apparently formed by phylogenetic analysis with 155 TLR genes from 12 species, which were consistent with the classification of TLRs suggested by Roach et al. (Roach et al., 2005), including six subfamilies TLR1, TLR3, TLR4, TLR5, TLR7 and TLR11. To further assess the biological roles, the expression patterns of TLR genes were examined in various common carp healthy tissues, during different developmental stages, and after bacteria A. hydrophila infection. During the common carp early

ACCEPTED MANUSCRIPT developmental stages, TLR genes except TLR2-1 were expressed at extremely low levels within 24 hpf, implying that most of TLR genes play important roles in the early development after 24 hpf. Although exhibiting unique tissue-specific expression patterns, the common carp TLR genes were widely expressed in all tested healthy

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tissues, implying a likely role in maintaining homeostasis. The highest expression

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level for some TLR genes were detected in the spleen, a major immune organ in fish,

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suggesting a critical role of TLRs in host immune response activities. Therefore, we further determined the expression patterns of TLR gene family in common carp

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spleen, following bacteria A. hydrophila infection. Most TLRs were induced

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following bacterial challenge at different timepoints, indicating their involvement in immune response. The expression pattern were different among gene members, with

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TLR1, TLR2-1, TLR4-2, TLR4-3, TLR7-1, TLR7-2, TLR21-2, TLR22-2 and

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TLR22-3 significantly differently expressed, suggesting the different functions of different gene members. Of all, TLR1 at 12 h was the most significant up-regulated

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gene, while the most significant down-regulated gene was TLR4-2 at 24 h. TLR1 can

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form heterodimer with TLR2, and recognizes a variety of triacylated lipoproteins (Takeuchi et al., 2002). The characterization of TLR1 was also reported in common carp (Fink et al., 2016), but mainly focused on the ligand-binding properties and expression pattern in healthy tissues. In the present study, TLR1 was significantly up-regulated at 12 hour post infection, indicating its important role in host immune response against bacterial infection. Due to its crucial roles in immunity, TLR1 had also been described in several fish species, such as rainbow trout (Palti et al., 2010b),

ACCEPTED MANUSCRIPT pufferfish (Wu et al., 2008), grouper (Wei et al., 2011), channel catfish (Quiniou et al., 2013; Zhang et al., 2013a), miiuy croaker (Xu et al., 2016). TLR4 as an important player in the innate immunity can recognize a major component of the outer membrane of Gram-negative bacteria, lipopolysaccharide (LPS). The fish TLR4 could

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be activated by either viral/bacterial infection, or LPS stimulation, with different

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expression patterns among various fish species, suggesting the different roles of TLR4

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in LPS sensing and augmentation of innate immunity against Gram-negative bacterial infection in different fish. It is possible that multiple fish TLR4 genes were created by

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duplications rather than speciation event and were likely evolved to express different

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ligand specificities. However, the ligand for fish TLR4 have not been confirmed. Our results showed that in common carp, the four duplicated TLR4 genes were apparently

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induced after challenging with a Gram-negative bacteria, A. hydrophila (Fig. 5), but

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with different expression patterns. TLR4-2 and TLR4-3 were up-regulated immediately after bacterial infection (4 hours), whereas TLR4-1 and TLR4-4 were

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moderately expressed. Examining the expression pattern of TLR4s in healthy tissues

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showed that TLR4-4 was expressed at extremely low level in the spleen. Therefore, we inferred that the duplicated TLR4 genes likely had different roles against bacterial invasion and functioned at different sites of host. TLR genes in common carp can be induced by other pathogens or toxic agents as well. For instance, the expression of common carp TLR5 was modulated after lipopolysaccharide, concanavalin A, and flagellin stimulation (Duan et al., 2013). Carp TLR7 expression was significantly increased in head kidney after stimulated with imiquimod (Tanekhy et al., 2010).

ACCEPTED MANUSCRIPT TLR2, TLR3, TLR7 showed a quick and prolonged elevated expression following spring viraemia of carp virus infection (Wei et al., 2016). All these studies indicated that TLRs play important roles in the innate immunity of common carp in response to pathogenic invasion. Further studies are needed to conclusively prove the downstream

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molecular regulation mechanism, and kinetics of individual receptors in the event of

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pathogen incursion.

5. Conclusions

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This is the first systematic analysis of Toll-like receptors genes in common carp.

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A total of 27 TLRs were identified and characterized from common carp whole genome sequences, and gene duplication and losses were found. High conservation of

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sequences were oberved among TLR gene memebers and across different species,

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through protein domain prediction and phylogenetic analysis. The expression profiling of TLRs were examined during early developmental stages post fertilization

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and in different healthy tissues. To further explore the roles of TLR genes in host

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defense against A. hydrophila infection, the temporal expression of TLRs were detected, and the results indiated that TLR genes were played crucial roles in defense of bacterial infection. Our findings could provide fundamental information for better undertanding the immnue response in common carp as well in other fish species.

ACCEPTED MANUSCRIPT Acknowledgements This study was supported by grants from the Central Public-interest Scientific Institution Basal Research Fund of CAFS (NO.2016PT02 and NO.2016HY-ZD0302), the National Natural Science Foundation of China (No. 31422057) and National

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Science and Technology Pillar Program (2015BAD25B01).

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ACCEPTED MANUSCRIPT Figure Legends Fig. 1. Schematic representation of the domain architecture of common carp TLR genes. Fig. 2. Phylogenetic tree of TLR genes. The phylogenetic tree was constructed using

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maximum likelihood algorithm under the JTT+I+G model of amino acid substitution

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correspond to bootstrap support values in percentages.

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as described in detail in Materials and Methods section. Numbers around the nodes

Fig. 3. Quantitative real-time PCR based expression analysis of TLR genes in

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common carp healthy tissues. Expression levels were calibrated against tissue which

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had the lowest expression level. The amplification of ß-actin was used as an internal control. The x-axis represents the relative expression levels, while the y-axis

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represents TLR genes.

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Fig. 4. Quantitative real-time PCR based expression analysis of TLR genes during common carp early developmental stages post fertilization. Expression levels were

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calibrated against the developmental stage of 0 hpf. The amplification of ß-actin was

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used as an internal control. Fig. 5. The temporal expression analysis of common carp TLR genes after A. hydrophila infection in the spleen. Relative expression was measured as fold change over control samples taken at the same time point as normalized to change in expression in the ß-actin control. The results were presented as mean ± SE of fold change. Asterisks indicate statistical significance at the level of P < 0.05.

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Table 1. Summary of TLR gene family in common carp.

Gene name

Genomic length(bp)

CDS (bp)

CDS (aa)

CDS status

No. of exons

TLR1 TLR2-1 TLR2-2 TLR3-1 TLR3-2 TLR4-1 TLR4-2 TLR4-3 TLR4-4 TLR5 TLR7-1 TLR7-2 TLR8-1 TLR8-2 TLR8-3 TLR9 TLR18-1 TLR18-2 TLR19 TLR20 TLR21-1 TLR21-2 TLR22-1 TLR22-2

2394 3608 3114 3468 2975 3305 3576 3520 2487 2734 4173 3919 3985 4049 3608 3198 3942 5214 4819 2841 2949 2940 2838 2805

2394 2376 2367 2715 2715 2466 2325 2502 2393 2634 3427 3150 3047 3077 3020 4398 2579 2576 2876 2841 2949 2940 2838 2805

797 791 788 904 904 822 774 833 733 877 1149 1049 1014 1024 1005 1064 858 857 957 946 982 979 945 934

Complete Complete Complete Complete Complete Complete Complete Complete Partial Complete Complete Partial Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete Complete

1 1 2 3 3 3 6 5 1 1 2 2 1 3 2 1 2 3 2 1 1 1 1 1

A

C C

T P E

D E

M

I R

LC150761 LC150762 LC150763 KF387571(Falco et al., 2014) KF387572(Falco et al., 2014) KF582562 KF582561 LC150764 HQ229652(Kongchum et al., 2011b) LC150765 AB553573(Tanekhy et al., 2010) LC150766 LC150767 LC150768 LC150769 GU809229(Kongchum et al., 2011b) LC150770 LC150771 LC150772 KF482527(Pietretti et al., 2014) LC150773 LC150774 LC150775 LC150776

C S U

N A

T P

Accession No.

Genome location LG28 LG1 Scaffold Scaffold Scaffold Scaffold Scaffold Scaffold LG26 LG40 LG40 LG40 LG40 LG20 LG40 Scaffold Scaffold Scaffold LG32 LG32 LG32 LG12 LG35 LG7

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TLR22-3 TLR25-1 TLR25-2

2868 2454 2454

2868 2454 2454

955 817 817

Complete Complete Complete

1 1 1

LC150777 LC150778 LC150779

Scaffold LG7 LG7

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N-glycosylation sites

TLR1

80

7.88

7

TLR2-1

86

6.77

8

TLR2-2

74

6.14

6

TLR3-1

102

7.89

10

TLR3-2

56

7.62

8

TLR4-1

97

8.70

TLR4-2

76

7.82

TLR4-3

96

8.58

TLR4-4

66

7.24

TLR5

101

TLR7-1

86

TLR7-2

115

TLR8-1

117

TLR8-2

117

TLR8-3

116

TLR9

122

TLR18-1

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Molecular weight (kDa)

10 10 8 10

8.88

9

5.85

10

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Gene name

18

7.10

19

8.62

18

8.29

19

8.79

8

92

8.75

10

101

8.35

9

130

6.85

11

108

5.91

12

114

8.67

21

113

8.85

17

108

8.93

15

TLR22-2

92

8.44

14

TLR22-3

110

8.73

15

TLR25-1

94

6.41

10

TLR25-2

88

8.29

8

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TLR18-2 TLR19 TLR20

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TLR22-1

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TLR21-1 TLR21-2

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8.75

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Table 3. Comparative analysis of TLR genes of common carp with other species. Common carp Zebrafish Catfish Stickleback Medaka Takifugu Tetraodon Lizard Chicken Human Mouse 1 1 1 1 1 1 1 2 1 1 TLR1 2 1 1 1 1 1 1 2 1 1 TLR2 2 1 1 1 1 1 1 1 1 1 1 TLR3 4 2 2 1 1 1 1 TLR4 1 2 3 3 2 2 2 1 1 1 1 TLR5 1 1 TLR6 2 1 1 1 1 1 1 1 1 1 1 TLR7 3 2 2 1 1 1 1 1 TLR8 1 1 1 1 1 1 1 1 1 TLR9 1 TLR10 1 TLR11 1* 1 TLR12 1 TLR13 1 TLR15 2 1 1 1 1 1 1 TLR18 1 1 1 TLR19 1 2 1 TLR20 2 1 1 1 1 1 1 1 TLR21 3 1 1 1 TLR22 1 TLR23 2 1 TLR25 1 TLR26 Total 27 17 19 11 10 12 9 5 9 11 12 Note: * represents a pseudogene.

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