Genome-wide identification and characterization of TRAF genes in the Yesso scallop (Patinopecten yessoensis) and their distinct expression patterns in response to bacterial challenge

Fish & Shellfish Immunology 47 (2015) 545e555

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Genome-wide identification and characterization of TRAF genes in the Yesso scallop (Patinopecten yessoensis) and their distinct expression patterns in response to bacterial challenge Jing Wang, Ruijia Wang*, Shuyue Wang, Mengran Zhang, Xiaoli Ma, Pingping Liu, Meiwei Zhang, Xiaoli Hu, Lingling Zhang, Shi Wang**, Zhenmin Bao Ministry of Education Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, 5 Yushan Road, Qingdao 266003, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 August 2015 Received in revised form 21 September 2015 Accepted 29 September 2015 Available online 3 October 2015

The tumor necrosis factor (TNF) receptor associated factors (TRAFs) are the major signal transducers for the TNF receptor superfamily and the interleukin-1 receptor/Toll-like receptor (IL-1R/TLR) superfamily, which regulate a variety of cellular activities and innate immune responses. TRAF genes have been extensively studied in various species, including vertebrates and invertebrates. However, as one of the key component of NF-kB pathway, TRAF genes have not been systematically characterized in marine invertebrates. In this study, we identified and characterized five TRAF genes, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, in the Yesso scallop (Patinopecten yessoensis). Phylogenetic and protein structural analyses were conducted to determine their identities and evolutionary relationships. In comparison with the TRAF genes from vertebrate species, the structural features were all relatively conserved in the PyTRAF genes. To gain insights into the roles of TRAF genes during scallop innate immune responses, quantitative real-time PCR was used to investigate the expression profiles in the different stages of scallop development, in the healthy adult tissues, and in the hemocytes after bacterial infection with Micrococcus luteus and Vibrio anguillarum. Based on the qRT-PCR analysis, the expression of most of the PyTRAFs was significantly induced in the acute phases (3e6 h) after infection with Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria, and many more dramatic changes in PyTRAFs expression were observed after V. anguillarum challenge. Notably, the strong response in the upregulation of PyTRAF6 post-bacterial challenge was distinct from that previously reported in scallops and crabs but was similar to that of other shellfish, Echinodermata and even teleost fish. The high level expressions of PyTRAFs in the hemocytes and the gill, and their specific expression patterns after challenges provide insights into the versatile roles and responses of TRAFs in the innate immune system against Gram-negative bacterial pathogens in bivalves. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Patinopecten yessoensis Gram-positive and Gram-negative infection Innate immune PyTRAF

1. Introduction The Toll-like receptor (TLR) family, the interleukin-1 receptor (IL-1R) family the RIG-I-like receptor (RLR) family, and the Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are a group of intracellular signaling molecules with crucial functions initiating the signal transduction pathways. As important regulators, these genes are involved in a wide spectrum of cellular

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (R. Wang), [email protected] (S. Wang). http://dx.doi.org/10.1016/j.fsi.2015.09.050 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

responses, including cell proliferation, apoptosis, differentiation and death, that mediate a wide variety of biological functions, including adaptive and innate immunity, embryonic development, stress response and bone metabolism [1e5]. Binding of TNFRassociated factors (TRAFs) to TNFRs typically induces signaling cascades that lead to the activation of nuclear factor kappa B (NFkB) and mitogen-activated protein kinases (MAPKs) and, ultimately, to the regulation of cell survival [4,6e8]. TRAF genes are ubiquitous and conserved in animal kingdom [5]. The genes were originally identified as signaling transducers of the TNFR superfamily using biochemical purification techniques and yeast two-hybrid screening aimed at the identification of signal

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transducers of TNFR2 [9]. To date, up to seven TRAFs have been found in mammals, including two vertebrate specific members, TRAF1 and TRAF5 [5,10e13]. With respect to invertebrates, fewer TRAFs have been identified. Only three were reported in insects [12], and one in nematodes [11]. Most TRAF proteins are characterized by a C-terminal TRAF domain, which is composed of an Nterminal coiled-coil region (TRAF-N) and a conserved C-terminal bsandwich (TRAF-C). This domain is responsible for proteineprotein interactions, including TRAF oligomerization as well as interactions with upstream regulators and downstream effectors [8,14]. Instead of the classical TRAF-C domain, the C-terminus of the TRAF7 contains seven WD40 repeats, which play a role similar to that of the TRAF domain in the interaction with protein kinases [15]. Although the N-terminal regions of TRAF proteins are less conserved, most of the TRAFs, with the sole exception of TRAF1, have a distinctive Nterminal RING finger domain followed by a variable number of zincfinger domains [16]. The RING finger domain of TRAF proteins is critical for downstream effector functions, and the zinc-finger domain mediates DNA binding and/or proteineprotein interactions [17,18]. The RING finger domain is also found in many E3 ubiquitin ligases. Indeed, increasing evidence indicates that in addition to their role as adaptor proteins, the TRAFs (including TRAF2, TRAF3, TRAF6, and TRAF7) may play important roles in the promotion of ubiquitination events [19e22]. Although the TRAF genes share similar structures and signal pathways, each member of the gene family has an overlapping yet distinct physiological role in biological processes, which was confirmed in the comparative analysis of TRAF-deficient murine strains [23]. Numerous studies of TRAF genes were conducted to investigate their roles in innate immune systems [8,23,24]. For example, the inhibition of TRAF2 leads to lymphadenopathy and an increased number of B cells, which indicates that TRAF2 is involved in the regulation of lymphocytes, and TRAF3 is an important regulator of specific innate immune receptors and plays a role in the negative regulation of the NF-kB signaling pathway. Additionally, TRAF6 is an important participant in the signaling of the innate immune Toll-like receptors (TLRs), and the simple overexpression of TRAF7 results in cell death [15,25e28]. TRAF7 is also associated with MAPK
ERK kinase kinase 3 (MEKK3), which is a key signaling molecule in the TNF-induced NF-kB activation pathway [29]. To our knowledge, the innate immune regulation by TRAFs in mollusks has been rarely reported. Two TRAF3 homologues, PfTRAF3 and CgTRAF3, were cloned and characterized from Pinctada fucata and Crassostrea gigas, respectively. Moreover, two TRAF6 homology genes, CfTRAF6 and MyTRAF6, were identified and characterized from Chlamys farreri and Mizuhopecten yessoensis, respectively. Based on an investigation of the expression profiles of hemocytes under the stress of Gram-negative bacteria, the genes were suggested to participate in the immune response [30e33]. The first homolog of TRAF7 in mollusk was isolated from Crassostrea hongkongensis through the screening of a suppression subtractive library, and the temporal expression of ChTRAF7 in the hemocytes following bacterial infection indicated that the ChTRAF7 may play an important role in the signal transduction of the immune response of oysters [34]. The Yesso scallop (Patinopecten yessoensis, Jay, 1857) is one of the most important aquaculture species in China [35]. It has been reported that a large-scale outbreak of bacterial disease led to severe mortality of scallops [36,37]. To better understand the mechanisms of the immune responses and defenses of scallops and for the healthy development of the scallop industry, we have identified and functionally characterized some important genes acting on innate immunity in the past [38e40]. However, our understanding of the TRAF gene family in scallops remains limited. In this paper, we identified five TRAF genes, TRAF2, TRAF3, TRAF4, TRAF6 and

TRAF7, in the Yesso scallop and analyzed the expression profiles at the different stages of development, in healthy scallop tissues, and in the hemocytes after infection with Micrococcus luteus and Vibrio anguillarum, thereby providing insights into the regulated function of this gene family in the scallop innate immune mechanisms of scallops. 2. Materials and methods 2.1. Database mining, gene identification and sequence analysis To identify TRAF genes, the transcriptome [35] and the whole genome sequence databases of the Yesso scallop (unpublished data) were searched using all available TRAF protein sequences of invertebrates, including Zhikong scallop (C. farreri), limpet (Lottia gigantea), Jinjiang oyster (C. hongkongensis), Pacific oyster (C. gigas), mussel (Mytilus galloprovincialis), pearl oyster (Pinctada martensii), Hawaiian squid (Euprymna scolopes), fruit fly (Drosophila melanogaster) and worm (Caenorhabditis elegans), and some vertebrate sequences, including human (Homo sapiens), mouse (Mus musculus), chicken (Gallus gallus), xenopus (Xenopus tropicalis), and zebrafish (Danio rerio) from NCBI (http://www.ncbi.nlm.nih.gov) and Ensembl (http://useast.ensembl.org). TBLASTN was used to obtain the initial pool of TRAFs transcriptome sequences from the Yesso scallop, and then, BLASTN was performed to verify the cDNA sequences by comparing the transcriptome sequences with the whole genome sequences [41]. The genomic structures of PyTRAFs were determined by mapping cDNA sequences to genomic DNA using BLASTN. ORF (open reading frame) finder (http://www.ncbi. nlm.nih.gov/gorf/gorf.html) and DNAstar (version 7.1) were employed to predict the amino acid sequences. Furthermore, the predicted amino acid sequences were confirmed by BLASTP against the NCBI non-redundant protein sequence database. The conserved domains were identified using the simple, modular architecture research tool (SMART) (http://smart.embl.de/). The putative isoelectric (PI) points and molecular weights were computed using the Compute pl/Mw tool (http://web.expasy.org/compute_pi/). The secondary structures of the Yesso scallop TRAF proteins were predicted using Geneious 7.1.7 (http://www.geneious.com/), and the tertiary structures were predicted using Phyer2 (http://www.sbg. bio.ic.ac.uk/phyre2/). 2.2. Phylogenetic analysis The TRAF proteins from other vertebrates and invertebrates listed in the previous section were used for phylogenetic analysis with the Yesso scallop TRAFs. The amino acid sequences of TRAF proteins from these species were retrieved from the NCBI and Ensembl Genome Browser. Phylogenetic trees were constructed using MEGA 6 with the Neighbor-Joining method [42]. Bootstrapping with 5000 replications was conducted to evaluate the phylogenetic tree. 2.3. Sample collection and bacteria treatment Two-year-old healthy Yesso scallops were collected from natural populations in Dalian zhangzidao Fishery Group Co. (Liaoning Province, China) in January 2014. All the procedures involved in the handling and the treatment of scallops during this study were approved by the Ocean University of China Institutional Animal Care and Use Committee (OUC-IACUC) prior to the initiation of the study. The scallops were acclimated in the laboratory for one week before the start of the experiment. The filtered and aerated seawater was maintained at a constant 8  C, which is within the optimum temperature range for the growth of the scallops.

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Gram-positive (M. luteus) and Gram-negative (V. anguillarum) bacteria that were used to infect the scallops were cultured in liquid 2216E broth (5 g/L of Tryptone, 1 g/L of yeast extract, and 0.1 g/L of C6H5Fe$5H2O, pH ¼ 7.6) at 28  C and harvested by centrifugation at 2000 g for 5 min, as described by Cong et al. [43]. The pellet was suspended in filtered seawater and was adjusted to 2  107 and 1  107 CFU/mL in seawater, respectively [44,45]. A total of 240 individuals were randomly and equally divided into three groups (groups C, N and P). At 0 h, 3 h, 6 h, 12 h and 24 h post infection, 10 individuals were randomly collected from each group. Group C was employed as the control, and group P and group N received the immersion infections of M. luteus and V. anguillarum, respectively. The mantle, gill, gonad, kidney, hepatopancreas, smooth muscle, adductor muscle, foot and eye were dissected. The hemolymph samples were collected from adductor muscles using a syringe and were immediately centrifuged at 800 g, 4  C for 10 min to harvest the hemocytes [46]. All tissues were immediately frozen in liquid nitrogen and then subsequently frozen at
80  C before processing. To obtain samples representing different developmental stages, spawning, fertilization and larval culture were conducted following previously detailed protocols [47]. Briefly, to induce spawning, sexually mature scallops were exposed to the air in darkness for 1 h and then were thermally stimulated by increasing the seawater temperature from 9  C to 12  C. After fertilization, the embryos were incubated at 12e13  C until the embryos developed into juvenile scallops. The oocytes, fertilized eggs, morulae, blastulae, gastrulae, trochophore larvae, D-shaped larvae, umbo larvae, eyespots larvae and juvenile scallops were preserved in RNAlater® (SigmaeAldrich, St. Louis, MO, USA) and stored at
80  C for further analysis. 2.4. RNA extraction and quantitative real-time PCR analysis Total RNA was isolated following the method described by Hu et al. [48], and then was digested with DNase I (TaKaRa, Shiga, Japan). RNA concentration and purity were determined using a Nanovue Plus spectrophotometer (GE Healthcare, NJ, USA). The RNA integrity was assessed by agarose gel electrophoresis. First strand cDNA was synthesized using Moloney murine leukemia virus (MMLV) reverse transcriptase (Thermo, USA) following the manufacturer's protocol. All of the cDNA products were diluted to 5 ng/ml for use as the template in real-time PCR. Real-time PCR was conducted using the SsoFast™ EvaGreen® Supermix on a LightCycler 480 Real-time PCR System (Roche Diagnostics, Mannheim, Germany). The running program was as follows: 50  C for 2 min, 94  C for 10 min, and 40 cycles at 94  C for 15 s and at 62  C for 1 min. Cytochrome B (CB), DEAD-box RNA helicase (HELI) and b-actin were designated as internal reference genes for the normalization of gene expression in embryos, healthy adults and test subjects during the real-time PCR experiment, respectively [49,50]. The specificity of the primers was assessed by alignment with the P. yessoensis draft genome assembly (unpublished data) using BLASTN with an e-value of 1e
10. Melting curve analysis was also performed to verify that each primer set amplified a single product. All the primers used in the real-time PCR were designed using Primer Premier 5.0 and are listed in Table 1. Data from the real-time PCR were analyzed using the Relative Expression Software Tool (REST) version 2009 [51]. In the analysis of gene expression in healthy tissues, the tissue with the highest Ct values in general was selected as the control group for REST, and the relative expression levels of the other tissues were then calculated based on the control group. Statistical analyses of the data were performed with the SPSS (version 16.0) statistical software package using independent t-tests.

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Table 1 Summary of primers used in the study. Gene name

Primers for real-time PCR

PyTRAF2-F PyTRAF2-R PyTRAF3-F PyTRAF3-R PyTRAF4-F PyTRAF4-R PyTRAF6-F PyTRAF6-R PyTRAF7-F PyTRAF7-R Cytochrome BeF Cytochrome BeR DEAD-box RNA helicase-F DEAD-box RNA helicase-R b-actin-F b-actin-R

50 -CTTTCCTTCAGGTGGTTTTCC-30 50 -GATGCTCCTGGACTGATGTTTT-30 50 -CTAGTGCCAGACGTGAGATCG-30 50 -GTGTACTCGCAGCCCAGTTTAT-30 50 -CGCACCGTAGAATGCTCCT-30 50 -TTGGGTCACAGCGGTTAGG-30 50 -CACATTCTGTCTGGTATGGCTCT-30 50 -CGATTATAGCTCGGGATTTCAGT-30 50 -CCAGATTGTCACGCTGAAAGG-30 50 -AGATCGGTCGTAGGAGGCAC-30 50 -CCTCTCCACCCTTTCTAGTCCTTG-30 50 -CTCCTGGTTCTTCGTCTTTCTCC-30 50 -CCAGGAGCAGAGGGAGTTCG-30 50 -GTCTTACCAGCCCGTCCAGTTC-30 50 -CCAAAGCCAACAGGGAAAAG-30 50 -TAGATGGGGACGGTGTGAGTG-30

3. Results 3.1. Sequence identification and analysis Following the nomenclature of invertebrate TRAF genes, five TRAFs genes, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, were identified from the Yesso scallop transcriptome database, which were further confirmed by their presence in the draft genome sequences. The cDNA and the predicted amino acid sequences of PyTRAFs were submitted to GenBank with the accession numbers of KT367521 (PyTRAF2), KT367522 (PyTRAF3), KT367523 (PyTRAF4), KT367524 (PyTRAF6), and KT367525 (PyTRAF7). The open reading frames of PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7 were 1647, 1677, 1401, 2058 and 1953 bp, encoding 549, 559, 467, 686 and 651 amino acids, respectively. The predicted molecular weights of these PyTRAFs ranged from 53.86 to 77.86 kDa, with predicted isoelectric points (pI) that ranged from 5.67 to 7.93 (Table 2). PyTRAF2 was composed of 7 exons, PyTRAF3 had 10 exons, a total of 20 exons were found in PyTRAF7, and 6 exons were found in both PyTRAF4 and PyTRAF6 (Fig. 1). The deduced secondary structures of PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7 proteins were determined by Geneious 7.0.6, which indicated that these proteins consisted of 20, 25, 23, 25 and 26 alpha helixes; 29, 24, 30, 35 and 47 beta strands; 31, 35, 23, 57 and 44 coils; and 34, 40, 48, 64 and 55 turns, respectively (Fig. 2 and Table 2). Although the genomic structures of the PyTRAFs were various, several evolutionarily conserved domains and motifs were found in the five PyTRAFs, which were consistent with those found in other species (Supplementary Fig. 1). A RING finger domain and several zinc-finger domains at the N-terminal were identified in all PyTRAFs, which are important for signaling downstream events [17,18]. PyTRAF2, PyTRAF3, PyTRAF4 and PyTRAF6 shared a TRAF domain at the C-terminus homology region, the TRAF-C domain, which binds to the cytoplasmic domain of receptors and to other TRAF proteins. Among the five PyTRAFs, only PyTRAF6 was predicted to contain low-complexity regions (LCRs). Unlike the other family members, PyTRAF7 contained seven WD40 repeats at the Cterminus instead of the classical TRAF-C domain; however, these repeats were consistent with the TRAF7 structures found in other invertebrates. Based on analysis of the multiple sequence alignment, the functional domains of PyTRAFs were highly conserved, along with their counterparts in other species (Supplementary Fig. 1). The Ring finger domain and TRAF domain at the C-terminus were more conserved than the other domains. Compared with the other

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Table 2 The sequence features of TRAFs from Yesso scallop Patinopecten yessoensis.

Total length (bp) 50 UTR length (bp) 30 UTR length (bp) ORF length (bp) Amino acids length Weight (kDa) Theoretical pI Number of exons Number of introns Number of alpha helixes Number of beta strands Number of coils Number of turns

PyTRAF2

PyTRAF3

PyTRAF4

PyTRAF6

PyTRAF7

12582 271 142 1647 549 62.21 7.93 7 7 20 29 31 34

14833 58 259 1677 559 63.62 6.45 10 10 25 24 35 40

43512 259 2822 1401 467 53.86 7.13 6 5 23 30 23 48

25020 84 183 2058 686 77.86 6.11 6 6 25 35 57 64

77617 119 252 1953 651 72.99 5.67 20 20 26 47 44 55

PyTRAFs, two additional nuclear localization signal (NLS) motifs were captured specifically in PyTRAF4 (Supplementary Fig. 1). The phosphorylated site S426 of the vertebrate TRAF4 was not found in PyTRAF4 or in the other invertebrate TRAF4 proteins. Three low-

complexity regions of TRAF6 were characteristics of C. farreri and Patinopecten yessoensis. The tertiary structure analyses demonstrated that PyTRAF2, PyTRAF3, PyTRAF4 and PyTRAF6 shared similar protein conformational structures, including a single alphahelix (“stalk”) formed by the coiled-coil domain and a novel eightstranded anti-parallel beta-sandwich (“cap”) formed by the TRAF-C domain. PyTRAF7 exhibited a b-propeller architecture composed of seven blades with the seven C-terminal WD40 domains (Fig. 3). 3.2. Phylogenetic analysis To determine the evolutionary status of the Yesso scallop PyTRAFs, a phylogenetic tree was constructed (Fig. 4). Each TRAF protein was clearly grouped into its own clade as expected, which also provided firm phylogenetic evidence for the identities of the Yesso scallop TRAFs. Consistent with previous studies, TRAF1 and TRAF5 have been found only in vertebrates to date. Referring to the clusters of TRAF2, TRAF3, TRAF4 and TRAF6, the vertebrate TRAF members formed subclusters that were distinct compared with the invertebrate TRAFs (Fig. 4).

Fig. 1. Structure of PyTRAF genes. The light blue boxes indicate the 30 UTRs and the 50 UTRs. The dark blue boxes indicate the exons. The horizontal bars indicate the introns. Additionally, the genes are shown according to the length (bp) of the gDNA and the mRNA sequences, which were obtained from the genome databases and transcriptome of P. yessoensis, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 2. The conserved domains of the Yesso scallop TRAFs. The horizontal gray bars represent amino acid sequences without predicted functional domains, whereas the colored boxes represent the regions with successfully predicted domains. Annotated domains: RING, RING domain (rose); zf, Zinc-finger (yellow); cc, coiled-coil (dark gray); TRAF-C, TRAF-C domain (blue); and W, WD40 domain (light green). Protein domains are shown relative to the length of and the position in the amino acid sequences. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 3. The predicted secondary and tertiary structures of PyTRAFs. A. The secondary structures of PyTRAFs. The pink cylinders represent alpha helixes; the orange straight arrows represent beta strands; the wavy lines indicate coils; and the curved arrows represent turns. B. The predicted tertiary structures of PyTRAFs. PyTRAF2, PyTRAF3, PyTRAF4 and PyTRAF6 shared similar protein conformational structures. The coiled-coil domain forms a single alpha-helix (“stalk”), and the TRAF-C domain forms an eight-stranded anti-parallel beta-sandwich (“cap”). PyTRAF7 has a b-propeller architecture that is composed of seven blades with the seven C-terminal WD40 domains. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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3.3. Spatiotemporal expressions of PyTRAFs With real-time PCR, the expression profiles of PyTRAF genes were first analyzed in the ten developmental stages of the Yesso scallop. The PyTRAFs had different expression patterns during developmental stages from the oocytes to the juvenile mollusks (Fig. 5A). The expression of PyTRAF3 displayed a double-peak pattern in the ten developmental stages. The 1st peak was seen in the five early developmental stages, which included the oocytes, fertilized eggs, morulae, blastulae and gastrulae, and the expression of PyTRAF3 increased 0.39e13.59-fold relative to the D-shaped larvae. The 2nd peak included the five following stages of the trochophore larvae, D-shaped larvae, umbo larvae, eyespots larvae and juveniles, during which the expression of PyTRAF3 increased 0.22e9.13-fold relative to the D-shaped larvae. The expression patterns of PyTRAF2 were distinct; PyTRAF2 was highly expressed in the early developmental stages with the highest expression in the zygote for which the expression increased 13.1-fold relative to the D-shaped larvae. Then, the expression of PyTRAF2 declined sharply to reach the lowest level in the umbo larval stage before the expression started to gradually recover. The expression pattern of PyTRAF6 did not change significantly during the first eight developmental stages. The maximum level of expression (3.13-fold increase compared with the D-shaped larvae) of PyTRAF6 was detected in the eyespots larvae, which then dropped slightly in the juvenile mollusks. Additionally, PyTRAF4 was rarely expressed in early development stages and the expression level of PyTRAF7 was relatively low during all developmental stages. The expression profiles of PyTRAFs genes in healthy Yesso scallops were also determined in ten tissues: hepatopancreas, mantle, gill, gonad, kidney, smooth muscle, adductor muscle, hemocytes, foot and eye. In general, PyTRAFs were ubiquitous in all the tissues but were primarily expressed in the hemocytes, gill, and muscles (Fig. 5B). The highest level of PyTRAF2 expression was in the hemocytes, which was followed by the levels of expression in the kidney and the striated muscle. The expression of PyTRAF2 in the hemocytes was 5.4-fold higher than that in the hepatopancreas. PyTRAF3 and PyTRAF4 were also most highly expressed in the hemocytes, and the expressions were 37.35-fold and 8.89-fold higher than that in the hepatopancreas, respectively. After the hemocytes, PyTRAF3 and PyTRAF4 were highly expressed in the gill and the muscles. PyTRAF7 was also most highly expressed in the hemocytes (2.87-fold increase relative to that in the hepatopancreas). The corresponding expression level in mantle, muscles and gill was lower than that in hemocytes but was higher than that in the other tissues. Unlike the other PyTRAFs, the highest expression of PyTRAF6 was in the gill (15.07-fold increase relative to the hepatopancreas), which was followed by the expression in the mantle. 3.4. Temporal expressions of PyTRAFs in response to bacterial infection

Fig. 4. The phylogenetic tree was constructed based on the protein sequences of PyTRAFs, in addition to those of other species. Accession numbers of TRAFs in the other species are attached in the Supplementary Table 1.

To examine the expression patterns of PyTRAFs in response to bacterial infection, the Yesso scallops were challenged with M. luteus and V. anguillarum. Because of the important role in the innate immune responses in scallops [52], the expression of the PyTRAFs in the scallop hemocytes was examined at four time points (3, 6, 12 and 24 h) after M. luteus infection (Fig. 6A). Overall, the expression of PyTRAFs was different after infection, and most of the PyTRAFs were significantly up-regulated. In the induction process, PyTRAF2 and PyTRAF7 were significantly induced after 3 h with increases of 9.84-fold and 21.47-fold, respectively. After the most significant fluctuations, the expression levels of these two genes decreased sharply to a normal level (close to the control group) at 6 h post-infection and were even lower than that in the control

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Fig. 5. The expression profiles in different developmental stages and healthy adult tissues. A. Relative expression levels of PyTRAFs in different embryonic and larval stages. The relative expressions are shown as fold difference relative to the expression in D-shaped larvae. Cytochrome B was used as the internal control. B. Relative expression levels of PyTRAFs in adult tissues. Vertical bars represent the mean ± S.E. (N ¼ 3). The relative expressions are shown as fold difference relative to the expression in the hepatopancreas. DEAD-box RNA helicase was used as the internal control. Values marked with asterisks indicate significant differences from the expression in the hepatopancreas (*P < 0.05).

group (0.15-fold and 0.48-fold, respectively) at 24 h post-infection. The expressions of PyTRAF4 and PyTRAF6 were significantly upregulated at 6 h post-infection with increases of 2.91-fold and 13.92-fold, respectively. After the peak in expression, the expressions of PyTRAF4 and PyTRAF6 gradually decreased but remained higher than the typical level, even in the final sample (24 h). The overall fluctuation in the expression of PyTRAF3 was minimal, with a maximum induction in expression of 1.41-fold at 24 h postinfection. The expression profiles of PyTRAFs after V. anguillarum infection were also examined in the hemocytes of the Yesso scallop (Fig. 6B). The expression of PyTRAF2 was initially up-regulated at 3 h postinfection with a 24.7-fold increase and reached the maximum level of expression at 6 h (33.8-fold increase). The PyTRAF4 gene

was highly induced at 6 (34.5-fold increase) and 12 h (17.6-fold increase) after infection with the bacterium V. anguillarum. The expression of PyTRAF6 was initially up-regulated at 3 h postinfection with a 7.5-fold increase, and then, the expression rapidly increased by 40.5-fold at 6 h and remained at relatively higher levels (34.08- to 41.58-fold) at 12 and 24 h post-infection. The fluctuation in the expression of PyTRAF7 was greater than that in the expression of PyTRAF6; the gene was up-regulated at 3 h post-infection with a 59.75-fold increase and at 6 h with a 56.53fold increase, but the level of expression rapidly declined to a normal level at 12 h, which was followed by another significant increase (15.59-fold higher than the control group) in induction at 24 h post-infection. For PyTRAF3, the overall fluctuation in the expression was minimal. PyTRAFs demonstrated higher expression

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Fig. 6. Expression of PyTRAF genes after challenge with the Gram-positive (M. luteus) (A) and Gram-negative (V. anguillarum) (B) bacteria. Vertical bars represent the mean ± S.E. (N ¼ 10, the male and female could not be discriminated). The expression levels of PyTRAFs are the ratio of change against the level at 0 h, and b-actin was used as the internal control. The asterisks indicate significant differences (*P < 0.05).

and response level against the invading of V. anguillarum versus M. luteus. 4. Discussion The innate immune system is the first line of defense for most organisms against the invasion of pathogens. A key step in innate immunity is the detection of pathogen-associated molecular patterns (PAMPs) that are produced by infectious agents. In

vertebrates, the TLRs and the TNFRs are conserved and critical trans-membrane receptors for the innate immune response [53]. In mammals, the different TLRs specifically recognize the distinct PAMPs and activate the common and conserved signal transduction pathways [54]. In scallops, a series of components in the TLR signaling pathway and the TNFR family members have been identified [55]. As the primary signal transducers for the TNFRs and the TLR
IL-1R family, the TRAF family functions at a critical point for the activation of the downstream NF-kB signal integration.

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Although several TRAFs involved in the defense response of bivalves were identified individually [30,32,56], the systematical identity of the mollusk TRAFs and the downstream transcription factors remain largely unclear. In this study, we scanned the genome and the transcriptome of a marine bivalve, P. yessoensis, and successfully identified five PyTRAFs genes. Then, the expression levels of the five PyTRAFs in different developmental stages, in the different body tissues and in the hemocytes after bacterial infections were assessed. Our work represents the first genome-wide identification of TRAF genes in mollusk and provides insights into how the expression profiles of these genes change in response to bacterial invasion. After extensive data mining with all the RNA-Seq assemblies [35], the full-length transcriptome database and the preliminary genome assembly (unpublished data), five Yesso scallop TRAFs, PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, were identified. The deduced amino acid sequences and the tertiary structures of PyTRAF genes shared highly identity with those from other invertebrate species [57e61], which suggested that the TRAF proteins were evolutionarily conserved. With the phylogenetic analyses, the identities and the relationships of these PyTRAFs were further verified. Apparently, all candidate PyTRAFs were in the TRAF gene family as the correct cluster assignment. In this scheme, the TRAF1 and TRAF2 proteins were closely related, as were the TRAF3 and TRAF5 proteins, and might have originally diversified from a common precursor. The PyTRAFs genes were expressed in all the developmental stages and in all the adult tissues of healthy Yesso scallops, which indicated that PyTRAFs had specific functions in all stages of growth and development of the scallop. Each TRAF gene showed variable expression across different larval developmental stages and different PyTRAFs exhibited distinct expression patterns (Fig. 5). These diverse patterns of expression indicated that the PyTRAFs had multi-task functions throughout the development of the scallop. PyTRAF2 and PyTRAF7 were highly expressed at the first two larval stages (oocytes and fertilized eggs), implying their functional roles during early stages of larval development. The double-peak expression pattern displayed by PyTRAF3 in the ten developmental stages implied PyTRAF3 had roles not only at early development stages but also at the stage after metamorphosis. PyTRAF4 and PyTRAF6 both had the highest expression level at eyespots larvae stage, implying their potential involvement in the metamorphosis of scallop. The expression patterns of the TRAFs genes in tissues were characterized for C. gigas, Portunus trituberculatus and C. farreri, and the expressions of these genes were high in multiple immune-related tissues [30,32,56], which reflected the crucial roles of the genes in the host immune response. In this study, all the PyTRAFs were ubiquitous in the healthy tissues (Fig. 5B), and the highest levels of expression were also detected in the tissues relevant to the innate immune system [62e65]. The highest levels of expression of PyTRAFs, except for PyTRAF6, were observed in the hemocytes, which are one of the primary immune tissues and are the locations in which the recognition and the elimination of bacterial pathogens occur in mollusks [30e33]. Unlike the other PyTRAFs, PyTRAF6 was highly expressed in the gill, which is the primary interface between aquatic organisms and the external environment and maintains the first line of defense against bacterial infection [66]. Based on the high levels of expression of PyTRAFs in the hemocytes and the gill, these genes might play important roles in the innate immune response. To provide insights into the functions of TRAFs in the innate immune response, the expression of PyTRAF genes was examined after infection with two major bacterial pathogens, M. luteus and V. anguillarum. The expression of most PyTRAFs (PyTRAF2, PyTRAF4, PyTRAF6, PyTRAF7) increased significantly in one to several periods

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post-infection, which confirmed the involvement of the PyTRAFs in the innate response against bacterial invasion in the Yesso scallop. In response to M. luteus, an acute and significant up-regulation was detected in the expression profiles of PyTRAF2 and PyTRAF7 at 3 h post-infection, which was followed by the up-regulation of PyTRAF4 and PyTRAF6 at 6 h post-infection. However, the change in the expression of PyTRAF3 was slight compared with the other PyTRAFs. TRAF3 is most recognized for the association with the CD40 receptor and was the first TRAF to be identified binding to the CD40 [67]. TRAF2, on the other hand, is able to directly associate with CD40. TRAF2, and TRAF3 associate with CD40 via a PVQET motif. Competition of TRAF2 and TRAF3 recruitment to CD40 may therefore contribute to modulating receptor signals across different cell types [68,69]. For example, TRAF3 inhibited TRAF2-mediated activation of the MAPK signals and downstream antibody production, and conversely, TRAF2 induced the ubiquitination and the degradation of TRAF3, which resulted in the activation of the noncanonical NF-kB signaling pathway [70]. The acute increase in expression in response to infection by M. luteus and V. anguillarum might indicate that PyTRAF2 and PyTRAF7 were more sensitive than the other PyTRAFs to bacterial invasion and the expressions of PyTRAFs infected by the Gramnegative bacteria were higher than those infected by the Grampositive bacterial infection. The diverse levels of expression might be relevant to the different levels of response of the innate immune response of shellfish in combating Gram-positive or Gram-negative bacteria. The differential expression patterns of TRAFs and other innate immune response-related genes might indicate a higher sensitivity of the bivalve innate immune system to the invasion of Gram-negative bacteria [71,72]. Although various patterns of expression were observed, a similar expression pattern was observed for PyTRAF2 and PyTRAF7 at each time point following the challenge with both the Gram-positive and the Gram-negative bacteria. The genes were both significantly induced at 3 h postinfection, and then, the expressions decreased at each successive point in time. The parallel expression patterns of these two PyTRAF genes might indicate that the genes had synergic physiological functions in the defense against bacteria. Simultaneously, the correlation between the expressions of PyTRAF2 and PyTRAF6 was notable; PyTRAF6, which shared the 99% sequence identity with previously reported Yesso scallop TRAF6 and displayed the roughly consistent expression pattern with the He's results [31], was upregulated when the expression of PyTRAF2 was not affected or was down-regulated. As a possible explanation, TRAF2 was thought to compete with TRAF6 for CD40 binding, which thereby limited the capacity of CD40 engagement to induce NF-kB activation [73]. Notably, the strong response in the up-regulation of PyTRAF6 post-bacterial challenge was distinct from that previously reported in the studies of TRAF6 of C. farreri and Portunus trituberculatus but was similar to that of Apostichopus japonicas, and Pinctada martensii, among others [32,56,74,75]. Moreover, the expression of TRAF6 increased in teleost fish after infection with protozoan pathogens [76,77]. TRAF6 acted as a molecular bridge that linking upstream TLRs and MyD88 with the downstream NF-kB and MAPK signaling pathways, and TRAF6 in D. melanogaster was also identified as a downstream adaptor for TLR [78,79]. Additionally, several low-complexity regions (LCRs) were predicted only in the TRAF6 proteins of mollusks. The LCRs are regions of biased composition, which typically consist of a regular repeat, cryptic repeat, and single amino acid repetitions. These LCRs can provide abundant material for new functions [80], and compared with proteins without LCRs, the proteins that contain LCRs tend to have more interact with other proteins [81]. We identified the TRAF6 contained the LCRs might have the ability to combine with other receptors and mediate other signal transductions.

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In conclusion, we identified complete TRAF gene family, including PyTRAF2, PyTRAF3, PyTRAF4, PyTRAF6 and PyTRAF7, for the first time in the scallop P. yessoensis. The critical functional domains and the corresponding conserved sites of biological significance in the PyTRAF protein sequences were detected via bioinformatics analyses. Moreover, the expression profiles of these genes were analyzed in different developmental stages, in healthy tissues, and in the hemocytes after bacterial infection. We concluded that all five PyTRAFs were involved in the scallop immune response. The results from this research provide the basic resources for further investigations into the immune pathways of scallops and may provide valuable information about the origins and the evolution of innate immunity systems. Acknowledgments This project was supported by National High-Tech R&D Program (863 Program, 2012AA10A402), National Natural Science Foundation of China (31322055), Doctoral Fund of Ministry of Education of China (20120132130002), Natural Science Foundation for Distinguished Young Scholars of Shandong Province (JQ201308) and Projects of Independent Innovation in Shandong Province (2013CXC80202). The authors also wish to thank Xiaohua Zhang for providing bacteria strains and technical assistance in bacterial challenge experiments. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.fsi.2015.09.050. References [1] Y. Wang, P. Zhang, Y. Liu, G. Cheng, TRAF-mediated regulation of immune and inflammatory responses, Sci. China Life Sci. 53 (2010) 159e168. [2] E.B. Kopp, R. Medzhitov, The toll-receptor family and control of innate immunity, Curr. Opin. Immunol. 11 (1999) 13e18. [3] L. Hotbauer, Osteoprotegerin ligand and osteoprotegerin: novel implications for osteoclast biology and bone metabolism, Eur. J. Endocrinol. 141 (1999) 195e210. [4] V. Baud, M. Karin, Signal transduction by tumor necrosis factor and its relatives, Trends Cell Biol. 11 (2001) 372e377. [5] R.H. Arch, R.W. Gedrich, C.B. Thompson, Tumor necrosis factor receptorassociated factors (TRAFs)da family of adapter proteins that regulates life and death, Genes Dev. 12 (1998) 2821e2830. [6] P. Xie, TRAF molecules in cell signaling and in human diseases, J. Mol. Signal. 8 (2013) 7. [7] J.R. Muppidi, J. Tschopp, R.M. Siegel, Life and death decisions: secondary complexes and lipid rafts in TNF receptor family signal transduction, Immunity 21 (2004) 461e465. [8] H. Ha, D. Han, Y. Choi, TRAF-mediated TNFR-family signaling, Current Protoc. Immunol. Chapter 11 (2009) 1e9. [9] M. Rothe, S.C. Wong, W.J. Henzel, D.V. Goeddel, A novel family of putative signal transducers associated with the cytoplasmic domain of the 75 kDa tumor necrosis factor receptor, Cell 78 (1994) 681e692. [10] L.-G. Xu, L.-Y. Li, H.-B. Shu, TRAF7 potentiates MEKK3-induced AP1 and CHOP activation and induces apoptosis, J. Biol. Chem. 279 (2004) 17278e17282. [11] H. Wajant, F. Mühlenbeck, P. Scheurich, Identification of a TRAF (TNF receptor-associated factor) gene in Caenorhabditis elegans, J. Mol. Evol. 47 (1998) 656e662. [12] A. Grech, R. Quinn, D. Srinivasan, X. Badoux, R. Brink, Complete structural characterisation of the mammalian and Drosophila TRAF genes: implications for TRAF evolution and the role of RING finger splice variants, Mol. Immunol. 37 (2000) 721e734. [13] G.-H. Cha, K.S. Cho, J.H. Lee, M. Kim, E. Kim, J. Park, et al., Discrete functions of TRAF1 and TRAF2 in Drosophila melanogaster mediated by c-Jun N-terminal kinase and NF-kB-dependent signaling pathways, Mol. Cell. Biol. 23 (2003) 7982e7991. [14] J.M. Zapata, V. Martínez-García, S. Lefebvre, Phylogeny of the TRAF/MATH Domain. TNF Receptor Associated Factors (TRAFs), Springer, 2007, pp. 1e24. [15] T. Zotti, P. Vito, The seventh ring: exploring TRAF7 functions, J. Cell. Physiol. 227 (2012) 1280e1284. [16] J.Y. Chung, Y.C. Park, H. Ye, H. Wu, All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction, J. Cell Sci. 115 (2002) 679e688.

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