Characterization of six IL-17 family genes in miiuy croaker and evolution analysis of vertebrate IL-17 family

Fish & Shellfish Immunology 49 (2016) 243e251

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Short communication

Characterization of six IL-17 family genes in miiuy croaker and evolution analysis of vertebrate IL-17 family Qiong Yang a, 1, Yuena Sun a, b, 1, Xiurong Su b, Taiwu Li b, Tianjun Xu a, * a b

Laboratory of Fish Biogenetics & Immune Evolution, College of Marine Science, Zhejiang Ocean University, Zhoushan, 316022, China School of Marine Sciences, Ningbo University, Ningbo 315211, Zhejiang Province, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 25 October 2015 Received in revised form 15 December 2015 Accepted 18 December 2015 Available online 22 December 2015

Interleukin-17 (IL-17) family is a cytokine family which is one of the major signaling molecules family involved in immunity. Six member of IL-17 family cytokines (IL-17A-F) were found in mammals. In fish, all IL-17 family genes except IL-17B and IL-17E have been isolated and identified. Besides, IL-17N is uniquely found from teleosts. IL-17 family genes are widely studied in mammals, but have not been widely reported in lower vertebrates. In this study, we identify six IL-17 family genes (IL-17A/F1-3, IL17C, IL-17D, IL-17N) from miiuy croaker, using LPS and poly (I:C) to infect miiuy croaker in order to analyze the expression response to bacteria and virus and expression in normal tissues. Challenge experiment showed that miiuy croaker IL-17 family genes exhibited more sensitive response to the poly (I:C) than the LPS. The expression of IL-17 in un-stimulated tissues showed that different gene has expressed in different tissues. Through the analysis of IL-17 family members exist in various representative species to study the evolution of the IL-17 family, and the result showed IL-17A/F, IL-17B, IL-17C, and IL-17D should be present in early gnathostomes species. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Interleukin Cytokine Miiuy croaker Molecular evolution Expression

1. Introduction Cytokines play a significant role in immune-regulation which are broad and loose category of small proteins. The interleukin-17 (IL-17) family is one of the most ancient cytokine families comprise of six cytokines, IL-17A to IL-17F. IL-17A was first identified and named CTLA8 [1] that is produced by activated T cells [2]. And then the other five members of IL-17 family, namely IL-17B, IL17C, IL-17D, IL-17E (IL-25) have been reported [3]. In this family, the degree of homology among IL-17A and the other members was only 16%e50% [4]. IL-17F shared the highest amino acid sequence similarity with IL-17A and acts as an proinflammatory cytokine as well as IL-17A that can promote the release of immune-related molecules (proinflammatory cytokines, chemokines and antimicrobial peptides) [5,6]. And these two genes are known produced by Th17 cells, CD8þcells, gdT cells and NK cells [7e9] and have been considered can be share the similar biological functions. IL-17A also can up-regulated expression many target genes like IL-6 by synergizing with TNF-a [10]. IL-17B and IL-17C have been reported

* Corresponding author. E-mail address: [email protected] (T. Xu). 1 Contributed equally. http://dx.doi.org/10.1016/j.fsi.2015.12.031 1050-4648/© 2015 Elsevier Ltd. All rights reserved.

have the similar function in inducing the mRNA expression of inflammatory cytokines such as IL-1b, IL-6, and IL-23 in the 3T3 cell line and peritoneal exudate cells with IL-17A [11]. Compared with IL-17A and IL-17F, these two IL-17 family members just expression promote monocytic cell line THP-1 to release TNF-a and IL-1b which IL-17A has a small effect [12]. IL-17D was deemed to be prior and variously expressed in tissues and also be reported as the most evolutionary conserved one in the IL-17 family [13,14]. And IL-17D was found can be adjust the production of cytokines in endothelial cells and performed an hold-up effect on hemopoiesis in vitro [15]. Although it can not stimulate the prolif-eration of immune cells all by itself, it is capable to stimulate the production of other cytokines from target tissues. IL-17E which is also named as IL-25 had close homology to the rest of IL-17 family members is principally expressed by Th2 cells. IL-17E stimulate Th2 cytokines (IL-4, IL-5 and IL-13) to generate their production and augmenting the expression of eotaxin and tissue eosinophilia [16,17]. As an important significant cytokine which activates downstream pathways to induce the expression of anti-microbial peptides, cytokines and chemokines through their correspondent receptors, many studies have investigated IL-17 family members in mammals, but still have not been widely reported in lower vertebrates. In 2006, five IL-17 members (IL-17A/F1-3, IL-17C and IL-17D)

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Q. Yang et al. / Fish & Shellfish Immunology 49 (2016) 243e251

have been found in zebrafish and that is the first time IL-17 family has been reported in fish [18]. In previously reports, we found that the fish IL-17 family members are different form mammals, IL-17N was only found in fish as a novel gene member of IL-17 family while IL-17B and IL-17E have not been isolated and identified [19e21]. And IL-17A/Fs are several genes have been found in fish which have close location on the same chromosome in addition to the high homology of both isoforms and difficult to identify direct homologues [19]. IL-17A/F has high similarity with both IL-17A and IL-17F and it is difficult to distinguish IL-17A and IL-17F in fish, thus, IL-17A and IL-17F homologs nominated as IL-17A/F1-3 have been revealed in fish. IL-17 family genes in fish have not been thoroughly researched. The miiuy croaker (Miichthys miiuy) as one of the most important species in the marine industry facing many diseases, researches on immune-related genes and immune response of miiuy croaker have been studied and reported [22,23]. In this study, we identified six IL-17 family genes (IL-17A/F1-3, IL-17C, IL-17D and IL-17N) in miiuy croaker to study their characterization, evolution, expression patterns and immune responses to lipopolysaccharide (LPS) and polyinosinic: polycytidylic acid (usually abbreviated poly (I:C)) stimulation to get better undstanding of the specific immunity and immune response of miiuy croaker immune genes.

245

the presence of them in miiuy croaker IL-17 family members genomic regions. And MEGA5 and DNAman were used to do the multiple alignment. We constructed a phylogenetic tree (Table S1 of Supporting information) by MEGA5 program according to the genetic distance analysis conducted using the neighbor-joining method with 1000 bootstrapping replications [28]. Moreover, aligned of sequences under codon model using MUSCLE software for its high accuracy and speed [29,30]. 2.4. Analysis of six miiuy croaker IL-17 family genes expression

We temporarily cultured miiuy croakers which collected from Zhoushan Fisheries Research Institute in the seawater tank and divided them into two groups (control group and injection group). We collected ten tissues (liver, spleen, kidney, intestines, stomach, blood, eye, gill, skin, and brain) from three fishes of the control group. And from LPS and ploy (I:C) injected miiuy croaker (at 12 h, 24 h, 36 h, fishes were killed), we severally collected head kidney. All the samples were immediately frozen in liquid nitrogen and then stored at 80  C [24].

Total RNA was extracted from those fish samples using Trizol reagent (Qiagen) according to the manufacturer's instructions and the cDNA was synthesized utilizing a QuantScript RT Kit (TIANGEN) following the manufacturer's instructions and stored at 20  C until use. We used ten healthy tissues to test the expression level in different tissues. Each cDNA sample was taken from three different miiuy croaker individuals. We conducted which was used to PCR reaction by Veriti Thermal Cycler (Applied Biosystems, USA) and the reactions were carried out as followed: 5 min at 95  C, then followed by 35 cycles of 30 s at 94  C, 30 s at 58  C and 40 s at 72  C, and then 10 min at 72  C, at last 4  C forever. And the PCR products were visualized on a 2% agarose gel using gene specific primers (IL17A/F1-3-F/R,IL-17CeF/R, IL-17D-F/R and IL-17NeF/R) and internal reference b-actin specific primers and we designed six primers for IL-17 family genes and the primers of b-actin (b-actin-F/ R) used as internal control (Table S2 of Supporting information). We also used real-time quantitative PCR (qRT-PCR) to determine the mRNA expression patterns of six miiuy croaker IL-17 family genes in ten different tissues of healthy individuals and in head kidney of LPS injected and poly (I:C) injected miiuy croakers (at 12 h, 24 h, 36 h). Using the 2DDCT method, we determined the relative gene expression data [31]. To analyze the results we used a one-way analysis of variance (ANOVA) statistical test followed by Duncan's multiple comparison tests. As the mean ± SE (standard error), we considered expression differences significant at the 95% confidence level (p < 0.05).

2.2. Identification of IL-17s in miiuy croaker

2.5. Evolution analyses

To identify the IL-17 family genes from miiuy croaker, utilizing the BLASTP and TBLASTN programs with a query set of other fish's IL-17s to search the miiuy croaker transcriptome [25] and a private database of miiuy croaker whole-genome sequences (unpublished). Using MAFFT, six IL-17 family gene cDNA sequences were found in cDNA database aligned with the scaffolds.

We found that IL-17C and IL-17D were widely present both in mammals and teleosts, thus, another two phylogenetic trees (Fig. S1) were constructed to test the positive slection in mammals and teleosts via the Bayesian approach using MrBayes v3.1.2 which is running 5,000,000 generation with 25% of trees burned [32]. The sequences we used to construct these two trees were retrieved from GenBank (Table S3 of Supporting information). To explore whether the diverse environments had affected selective pressures in ancestral lineages of mammals and teleosts in IL-17C and IL-17D, we used the maximum likelihood (ML) methods in CODEML program of PAML v4 [33]. A free-ratio model, site-specific model, a branch-specific model and a branch-site model are included in the PAML [34]. The one-ratio modelwas used to identify the selective pressures and the free ratio model was used to check whether this model suitably fitted the data compared with the one-ratio model by the likelihood ratio test (LRT). The two-ratio models were applied to check if they fitted the one-ratio model. The branch-site model was used to detect the foreground lineages that included the ancestral lineages of mammals and teleosts [35]. We used the

2. Materials and methods 2.1. Sample preparation

2.3. Sequence analysis and phylogenetic tree construction Open Reading Frame Finder (http://www.ncbi.nlm.nih.gov/gorf/ gorf.html) was used to predict the open reading frame (ORF) of miiuy croaker IL-17 family genes and the SMART was used to characterize the potential protein domains of amino acid sequences [26]. All other species IL-17 family genes were gained from the GenBank (http://www.ncbi.nlm.nih.gov/Genbank) and the Ensemble Genome Browser (http://www.ensembl.org) database. IL-17 family genes synteny map locations of neighboring genes in other species were obtained from Genomicus [27]. Using BLAST programs and those genes in other species as queries, we searched

Fig. 1. (A) Genomic structure and genome location of miiuy croaker IL-17 family genes. Black boxes represent exons; white boxes represent untranslated regions (UTRs); full lines represent introns. (B) Sequence alignment of amino acid of miiuy croaker IL-17 family genes. The predicted signal peptide and low complexity region are marked by the pink and blue words. Cysteine residues in forming disulfide bonds are boxed and highlighted and connected with line. The domain of IL-17 is framed. (C) Schematic representation gene synteny at the IL-17 family genes loci in different fishes. The arrows indicated the transcriptional direction. Same color was shown in same genes. IL-17 family genes were in boxes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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sequence of coelacanth as the out group of all the subsets of mammals and teleosts IL-17C and IL-17D sequences and respectively utilized to analyze the positive selection. The Hyphy package in the Data Monkey Web Server (http://www.datamonkey.org) was also used to test the positive selection [36]. In CODEML, six site models were used to analyze the possible positively selected sites on the teleost and mammalian IL-17C and IL-17D sequences subsets, the Bayes empirical Bayes (BEB) in the models M2a and M8 were used to calculate the Bayesian posterior probability (BPP) of the codon sites under a positive selection [37]. We compared twice the differences of log-likelihood values (2DlnL) between each two nested models and the positive selection may contribute to the evolution if the LRT was statistically significant (p < 0.01) with the u > 1. Codons identified and accepted with P values < 0.1 for SLAC, FEL and MEME, Bayes Factor >50 for REL and posterior probability >0.9 for FUBAR as candidates for positive selection. 3. Results and discussion 3.1. Characteristics of miiuy croakerIL-17 family genes We obtained the full length of six IL-17 family genes from five scaffolds in miiuy croaker genome (Scaffold_91, 93, 396, 968 and 1737) and putatively named miiuy croaker IL-17A/F1-3, IL-17C, IL17D and IL-17N. Though miiuy croaker IL-17A/F1 and IL-17A/F2 are harbored on the scaffold_396, other miiuy croaker IL-17 family genes were found to be independently located on otherscaffolds. The cDNA sequence of miiuy croaker IL-17A/F1 has three exons and two introns (Fig. 1A) and deduced ORF of 501 bp which enconded a predicted protein of 166 amino acids. The miiuy croakerIL-17A/F2 and cDNA sequences deduced ORF of 432 bp encoded a putative protein of 143 and amino acids and has three exons and two introns. The ORF of miiuy croaker IL-17A/F3 cDNA sequence has been deduced of 495 bp that enconded a predicted protein of 164 amino acids. And miiuy croaker IL-17A/F3 sequence cDNA has four exons and three introns. The miiuy croaker IL-17C cDNA sequence also has three exons and two introns and an ORF of 528 bp which enconded a predicted protein of 175 amino acids. The miiuy croaker IL-17D cDNA consists of a sequence of two exons and one intron, an ORF of 636 bp encoding a 211 amino acid peptides. The IL-17-N cDNA sequence has three exons and two introns, and deduced ORF of 420 bp encoded a putative protein of 139 amino acid residues. The analysis of the amino acid sequences indicated that in the miiuy croaker IL-17 family all the genes have the same IL-17 domain, and miiuy croaker IL-17A/F1-3, miiuy croaker IL-17C, miiuy croaker IL-17D and miiuy croaker IL-17N proteins severally had a putative signal peptide which was determined to putatively comprise 29, 22, 22, 18, 19, 26 and 22 amino acids at the 5’-end. The miiuy croakerIL-17A/F2 also had a low complexity region (42e50 aa). Multiple alignment of miiuy croakerIL-17 family genes with each other and found in the tertiary structure of IL-17 family genes, six cysteine residues that formed disulphide bridges were conserved in the miiuy croaker IL-17 family genes (Fig. 1B). We also found several important features via alignment analysis of the miiuy croaker IL-17 family genes. In miiuy croaker IL-17 family genes, the C-terminal region showed high similarity with each other while little conservation was shown in the N-terminal regions. And the same result also had been found in human IL-17 family genes [38]. And the IL-17 domain was located in all IL-17 family members C-terminal region and showed IL-17 domain was conserved. And we have done multiple alignment with other known IL-17 family genes in vertebrates as well and showed conservation between the mammalian and teleost amino acids, especially the IL-17D and IL-17N (Fig. 1B). All IL-17 family members both in mammalian and teleost had signal peptide and the similar

structure showed that every IL-17 family gene in different species had the similar function. 3.2. Comparison of gene synteny with other species We analyzed six IL-17 family genes (IL-17A/F1-3,IL-17C, IL-17D and IL-17N) on the scaffolds inmiiuy croaker to make sure the gene order and orientation and compared with other fish homologues (Fig. 1C). The miiuy croaker IL-17A/F1 and IL-17A/F2 were found has synteny relationship, and these two genes were adjacent tightly as the same situation in tilapia, platyfish and zebrafish. But the surrounding genes of IL-17A/F1 and IL-17A/F2 in miiuy croaker were different from these three species while the surrounding genes of the other three fishes were almost the same. The partial genomes region respectively containing IL-17A/F3, IL-17C and IL17D loci in fish were aligned, and the results indicated that in fish, the region of these three IL-17 family members were conserved suggesting the evolutionary conservation. Comparing fugu and miiuy croaker IL-17N surrounding genes, only one gene is same in these two fishes. The locations in tandem on the chromosome of IL-17A/F1 and IL-17A/F2 in teleost were similar to chromosome locations of IL-17A and IL-17F in mammals [19,21]. And IL-17A/F1 has been reported in grass carp that could recruit immune cells by production of CXCL-8 just like mammalian IL-17 A and F [21] and IL-17A/F2 has been examined in rainbow trout and finding that is capable of inducing the gene expression of proinflammatory cytokine IL-6, chemokine CXCL-8 and antimicrobial peptide b-defensin in splenocytes [39]. The similarity of chromosome locations may showed similarity of gene function. 3.3. Phylogenetic analysis Using MEGA5, we aligned miiuy croaker IL-17 family genes with other known IL-17 family genes and constructed a phylogenic tree (Fig. 2A). The result showed that miiuy croaker IL-17 gene members were independently grouped closely with their counterparts. We found an interesting thing that IL-17A/F were grouped together andmiiuy croaker IL-17N formed its own sub-cluster. Moreover, Ciona intestinalis IL-17 genes were isolated from other sub-clusters. And we found an interesting thing that in IL-17A/F group most IL17A/F2 and IL-17A were grouped together and so were IL-17A/F3 and IL-17F. This result showed that the same ancestral gene evolved to IL-17 family gene in fish and other vertebrates [20]. To better know about the evolution of IL-17 family genes, we counted human, frog, coelacanth, spotted gar, cave fish, zebrafish, fugu, elephant shark, lamprey and miiuy croaker IL-17 family genes (Fig. 2B) by using Ensemble Genome Browser and blasting their genomes. We found that lamprey has only one IL-17 gene and according to the phylogenic tree constructed above this gene should be IL-17D. IL-17E was only found in mammals and IL-17N was only found in teleosts. Frog, coelacanth and spotted gar have IL-17B, IL17C, IL-17D and IL-17F. IL-17A/F1-3 and IL-17C, IL-17D and IL-17N was found in teleosts expect cave fish. IL-17B is only presented in a small number of fish, such as channel catfish. In cave fish, we found IL-17A, IL-17B, IL-17C, IL-17D, IL-17F and IL-17N. IL-17 family genes of cave fish both had teleosts and tetrapods characteristics. All these species had IL-17D, thus we speculated about IL-17D was the most ancient gene in IL-17 family. And along with the evolution of species, IL-17C was then showed out. And IL-17A/F and IL-17B and IL-17C were also present in early gnathostomes species. And then, IL-17A/F may expanded to several other genes, as IL-17A/F1-3 in teleosts and IL-17A and IL-17F in mammals. IL-17N may also be one of IL-17A/Fs. IL-17E maybe appeared after the divergence of birds and mammals and was possible diverged by IL-17C.

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Fig. 2. (A) Phylogenetic relationship of miiuy croakerIL-17 family members and other representative known IL-17 family members. MEGA5 used to generate a maximum likelihood tree, and alignments of all the amino acid sequences were aligned by MUSCLE. Orange word on the tree represent miiuy croaker IL-17 family genes. (B) Diagram to show evolution of IL-17 family in 1R, 2R and 3R species. The black boxes indicated the existence of genes. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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Table 1 Likelihood ratio tests of branch-models and branch-site models on IL-17C and IL-17D genes. Gene IL-17C

IL-17D

IL-17C

IL-17D

a b c

Model Branch-model A: Omega ¼ 1 B: One-ratio C: Free-ratio A: Omega ¼ 1 B: One-ratio C: Free-ratio Branch-site model 1:Null-mammals 2:mammals 3:Null-fishes 4:fishes 1:Null-mammals 2:mammals 3:Null-fishes 4:fishes

Npa

Ln likelihood

Parameter estimates

64 65 85 59 60 117

4848.48 4555.05 22173.75 4100.951 3640.68 3590.22

u ¼ 1.00 0.11671 Variableubranch u ¼ 1.00 0.07595 Variableubranch

68 69 68 69 62 63 62 63

4495.29 4495.29 4495.29 4495.29 3607.44 3609.98 3607.81 3607.30

Model compare

Positive selection sitesb

2△lnLc (p-value)

None B vs A C vs B

n/a None

568.86(p < 0.01) 35237.40(p < 0.01)

B vs A C vs B

n/a

920.54(p < 0.01) 100.92(p < 0.01)

1 vs 2

not found

0.00(p ¼ 1)

3 vs 4

not found

0.00(p ¼ 1)

1 vs 2

not found

5.07(p ¼ 0.02)

3 vs 4

not found

1.00(p ¼ 0.31)

Numbers of parameters. Only the sites with BPP > 0.95 were shown. Twice the difference of ln [likelihood] between the two models compared.

3.4. Molecular evolution analysis IL-17C and IL-17D have been found both in mammals and teleosts and we constructed two phylogenetic trees which included the mammals and teleosts IL-17C and IL-17D sequences. Under oneratio model, the value of u for all branches in IL-17C was 0.1167 and in IL-17D was 0.0760, suggesting that these two genes underwent the purifying selection (P < 0.01, Table 1, Table 2). Compared with the one-ratio model, the result of free-ratio model showed that each branch had its independent u value (P < 0.01, Table 1). In both branch-site model and site model, no positively selected site was found in IL-17C or IL-17D (Table 1 and 2). To get farther known about the molecular evolution of IL-17C and IL-17D we implemented the other four ML methods. And finally, in IL-17C, two sites were singled out respectivelyin the subsets of mammals and teleosts, and in IL-17D, one site was singled out in the subset of teleosts (Table 3). 3.5. Quantitative analysis of IL-17 family genes expression We used ten healthy tissues mentioned above to detect the tissue expression of IL-17 family genes in miiuy croaker and the results showed that different IL-17 family gene have different expression in different tissues (Fig. 3A). IL-17A/F2 was ubiquitously expressed in all the tissues tested and expressed highly in all the tissues except intestine compared with other IL-17 family genes. IL17A/F1 weakly expressed compared with other IL-17 family genes and was seen only in stomach, liver and head kidney. IL-17A/F3 was found express in gills, liver, eyes, head kidney and blood. IL-17C and IL-17D both were seen express in intestine, gills and blood. And IL17C was also seen express in intestine weakly while IL-17D was also seen express in skin, brain and head kidney. IL-17N expressed in intestine, gills, head kidney and spleen tissue. In healthy tissues, most miiuy croaker IL-17 family genes express in kidney and gill, indicating an important role in immunity as kidney and gill are important immune organs and this is as the same as in Japanese pufferfish [20]. The result of qRT-PCR was showed that although not all the miiuy croaker IL-17 family genes were found express in healthy head kidney, the expression of every IL-17 family gene was found express in LPS-stimulated and poly (I:C)-stimulated head kidney

(Fig. 3B). In LPS-stimulated head kidney, IL-17A/F1-3 and IL-17N showed same pattern in expression, reached maximal expression at 12 h post-stimulation and at 24 h and 36 h post-stimulation expressed almost the same. IL-17C reached maximal expression at 36 h post-stimulation. IL-17A/F1-3 also showed same expression pattern in poly (I:C)-stimulated head kidney, reached maximal expression at 36 h post-stimulation and minimal expression at 24 h post-stimulation. IL-17C also reached maximal expression at 36 h post-stimulation, but at 12 h and 24 h post-stimulation expressed almost at the same. IL-17N expression increased conspicuously at 12 h post-stimulation. IL-17D expressed both in LPS-stimulated and poly (I:C)-stimulated head kidney at almost the same at all the time. Miiuy croaker IL-17 family genes were expressed in a variety of tissues, and their expression was up-regulated by LPS and poly (I:C). In this study, six IL-17 family genes were identified from miiuy croaker genome and analyzed sequence characters and examined immune-related tissues. The high similarity of C-terminal region in miiuy croaker IL-17 family genes howed high conservative. Moreover, we found every IL-17 family genes in miiuy croaker had four cysteine molecules which were important for a classical cysteine knot formation. Next, we analyzed the linear relationship of IL-17 family in miiuy croaker and found IL-17A/F1 and IL-17A/F2 were tightly located on one chromosome that showed the same result with other teleost and was similar to IL-17A and IL-17F in mammals chromosome locations and this may be related to their similar function. The region of IL-17C, IL-17D and IL-17A/F3 had conserved synteny and IL-17A/F3 did not share collinearity with IL-17A/F1 and IL-17A/F2 [20]. So far, no function of IL-17A/F3 and IL-17N have been reported yet. Phylogenetic and molecular analysis showed IL-17C and IL-17D were widely exist in teleost and mammals and through positivese selection, it may be because of the function of these genes can help the animal and fish adapt toterricolous or aquatic environment in the immune response. We also investigated miiuy croaker IL-17 family genes expression using and the result indicated that IL-17 molecule expression is biologically important for teleost immune responses. Though we studied the characterization of IL-17 family genes in miiuy croaker and analyzed vertebrate evolution of IL-17 family genes, there still remains some questions waiting to be futher research in teleost.

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Table 2 Site model tests on IL-17C and IL-17D genes in subsets of mammals and fishes. Gene

Model

Npa

Ln likelihood

Parameter estimates

Model compare

Positive selection sitesb

2 △lnLc(P-value)

IL-17C

Data set: mammals M0:one-ratio M3: discrete

30 34

3278.56 3207.52

M3 vs M0

Not analyzed

142.08(p ¼ 0.00)

M1a: nearly neutral

31

M2a: positive selection

33

3223.48

M2 vs M1

Not allowed

0.00 (p ¼ 1.00)

M7: b M8: b and u

u ¼ 0.15 u0 ¼ 0.00,P0 ¼ 0.41 u1 ¼ 0.18,p1 ¼ 0.44 u2 ¼ 0.77,P2 ¼ 0.14 u0 ¼ 0.08,P0 ¼ 0.82 u1 ¼ 1.00,p1 ¼ 0.18 u0 ¼ 0.08,P0 ¼ 0.82 u1 ¼ 1.00,p1 ¼ 0.12 u2 ¼ 1.00,P2 ¼ 0.05

31 33

3208.63 3208.63

M8 vs M7

M0:one-ratio M3: discrete

24 28

2099.27 2074.76

M3 vs M0

Not analyzed

49.03(p ¼ 0.00)

M1a: nearly neutral

25

M2a: positive selection

27

2089.86

M2 vs M1

Not allowed

0.00 (p ¼ 1.00)

M7: b M8: b and u

25 27

2077.01 2077.01

M8 vs M7

Data set: fishes M0:one-ratio M3: discrete

34 38

3178.74 3061.35

M3 vs M0

Not analyzed

234.76(p ¼ 0.00)

M1a: nearly neutral

35

M2a: positive selection

37

3094.89

M2 vs M1

Not allowed

0.00 (p ¼ 1.00)

M7: b M8: b and u

35 37

3061.93 3061.89

M8 vs M7

M0:one-ratio M3: discrete

34 38

2853.96 2792.15

M3 vs M0

Not analyzed

123.64(p ¼ 0.00)

M1a: nearly neutral

35

M2a: positive selection

37

2820.14

M2 vs M1

Not allowed

0.00 (p ¼ 1.00)

M7: b M8: b and u

35 37

2793.09 2792.83

M8 vs M7

IL-17D

IL-17C

IL-17D

a b c

3223.48

p ¼ 0.31,q ¼ 1.33 p0 ¼ 1.00,p1 ¼ 0.00 u1 ¼ 11.91 p ¼ 0.31,q ¼ 1.33 u ¼ 0.08 u0 ¼ 0.00,P0 ¼ 0.33 u1 ¼ 0.11,p1 ¼ 0.62 u2 ¼ 0.58,P2 ¼ 0.05 u0 ¼ 0.06,P0 ¼ 0.94 u1 ¼ 1.00,p1 ¼ 0.06 u0 ¼ 0.06,P0 ¼ 0.94 u1 ¼ 1.00,p1 ¼ 0.04 u2 ¼ 1.00,P2 ¼ 0.02 p ¼ 0.61,q ¼ 1.77 p0 ¼ 1.00,p1 ¼ 0.00 u1 ¼ 33.84 p ¼ 0.61,q ¼ 5.77

3.7E-4(p ¼ 0.10)

2089.86

u ¼ 0.26 u0 ¼ 0.00,P0 ¼ 0.21 u1 ¼ 0.20,p1 ¼ 0.42 u2 ¼ 0.68,P2 ¼ 0.37 u0 ¼ 0.12,P0 ¼ 0.48 u1 ¼ 1.00,p1 ¼ 0.52 u0 ¼ 0.12,P0 ¼ 0.48 u1 ¼ 1.00,p1 ¼ 0.10 u2 ¼ 1.00,P2 ¼ 0.42

3.09E-4(p ¼ 0.10)

3094.89

p ¼ 0.50,q ¼ 0.95 p0 ¼ 0.98,p1 ¼ 0.02 u1 ¼ 1.36 p ¼ 0.51,q ¼ 1.02 u ¼ 0.09 u0 ¼ 0.01,P0 ¼ 0.37 u1 ¼ 0.09,p1 ¼ 0.45 u2 ¼ 0.39,P2 ¼ 0.02 u0 ¼ 0.07,P0 ¼ 0.87 u1 ¼ 1.00,p1 ¼ 0.13 u0 ¼ 0.07,P0 ¼ 0.87, u1 ¼ 1.00,p1 ¼ 0.09 u2 ¼ 1.00,P2 ¼ 0.04 p ¼ 0.55,q ¼ 4.34 p0 ¼ 0.99,p1 ¼ 0.01 u1 ¼ 1.00 p ¼ 0.58,q ¼ 4.94

0.09(p ¼ 0.96)

2820.14

0.52(p ¼ 0.77)

Numbers of parameters. Only the sites with BPP > 0.95 were shown. Twice the difference of ln [likelihood] between the two models compared.

Table 3 Phylogenetic tests of positive selection in IL-17C genes. Gene

IL-17C IL-17D a b c

Taxa

Mammals Teleosts Mammals Teleosts

Methoda SLACb

FELb

RELc

MEMEb

Paml-M8

Total

no no no 149

7,27 15,74,142 no 164

5,54,55,56,111 no no no

1,7,25,27,56 1,2,15,18,43,47,80,103,107,142,143,189 157,182,199 7,127,164

no no no no

7,27 15,142 no 164

Codons identified by more than one ML method are underlined. Codons with P values < 0.1. Codons with Bayes factor > 50.

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Q. Yang et al. / Fish & Shellfish Immunology 49 (2016) 243e251

Fig. 3. (A) Expression of miiuy croaker IL-17 family genes in normal tissues. Sk, skin; I, intestine; G, gill; St, stomach; L, liver; Br, brain; E, eye; K, head kidney; Sp, spleen; Bl, blood. (B) Miiuy croaker IL-17 family genes expression in head kidney at 12 h, 24 h and 36 h after injection of LPS and poly (I:C). “C” represents the control group injected with physiological saline * represents significantly different (p < 0.05) between challenged group and control group.

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