Developmental and Comparative Immunology 41 (2013) 631–643

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Developmental and Comparative Immunology

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Characterization of the CCR3 and CCR9 genes in miiuy croaker anddifferent selection pressures imposed on different domains betweenmammals and teleosts

0145-305X/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.dci.2013.06.015

⇑ Corresponding authors. Tel.: +86 580 2550826.E-mail addresses: wangrixin1123@126.com (R. Wang), tianjunxu@163.com

(T. Xu).

Zhihuang Zhu, Rixin Wang ⇑, Liping Ren, Tianjun Xu ⇑Laboratory for Marine Living Resources and Molecular Engineering, College of Marine Science, Zhejiang Ocean University, Zhoushan, PR China

a r t i c l e i n f o

Article history:Received 28 April 2013Revised 22 June 2013Accepted 24 June 2013Available online 29 June 2013

Keywords:Miichthys miiuy (miiuy croaker)Chemokine receptorCCR3CCR9Expression patternMolecular evolution

a b s t r a c t

The innate immune system can recognize non-self through pattern recognition receptors and provides afirst line of antimicrobial host defense. Thus innate immunity plays a very important role in resistanceagainst major bacterial disease in vertebrates. In the innate immune responses, the chemokine receptorsact as the main receptors of chemokines which are released at the sites of infection, inflammation andinjury. In this study, the Miichthys miiuy CCR3 and CCR9 genes were cloned and characterized, showingthat MIMI-CCR3 possesses a highly conserved DRYLA motif similar to that of other fishes. MIMI-CCR3 andCCR9 were ubiquitously expressed in all tested tissues and the expressions were significantly up-regulated after infection with Vibrio anguillarum except that of CCR9 in spleen. Evolutionary analysisshowed that all the ancestral lineages of CCR3 and CCR9 in mammals and teleosts underwent positiveselection, indicating that the ancestor of terrestrial animals further evolved to adapt to terrestrialenvironments and the continuous intrusion of microbes stimulated the evolution of CCR genes in the ances-tor of teleost. Multiple ML methods were used to detect the robust candidates for sites under positive selec-tion. In total, 11 and 8 positively selected sites were found in the subsets of current mammal and teleostCCR3 genes, and 3 and 2 sites were detected in the subsets of current mammals and teleosts in CCR9. Inter-estingly, for mammal CCR3 and CCR9 genes, the robust candidates of positively selected sites were mainlylocated in the extracellular domains which were the ligand binding and pathogen interaction regions. Forteleost CCR3 and CCR9 genes, the positively selected sites were not only located in the extracellulardomains but also in the C-terminal and intracellular domains, indicating mammals and teleosts experi-enced different selection pressures upon their N-terminus, C-terminus and intracellular loops of CCRs.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

The chemokines are a family of structurally related cytokines oflow molecular weight, which can regulate immune cell migrationunder inflammation, immune surveillance, organogenesis andnormal physiological conditions (Moser et al., 2004; Zlotnik andYoshie, 2000). They are not only functionally involved in leukocyteactivation and migration, but also regulate the immune responsesand differentiation of the recruited cells (Esche et al., 2005; Kunkelet al., 1995). Therefore, they have been classified as key regulatorsin the immune response, acting as a bridge between innate andadaptive responses. The importance of the chemokines has grownin recent years, as it has become recognized that they are key play-ers in many disease processes (Muller et al., 2001).

Chemokines can be divided into 5 subfamilies (CXC, CC, XC,CX3C, and CX) based on the arrangement of four conservedcysteine residues involved in the formation of disulfide bonds(Nomiyama et al., 2010). Upon their binding to the chemokinereceptors (ChemRs) on the cell surface, the complexes initiate aseries of intracellular signalling pathways, resulting in variousphysiological and pathological processes. In many cases, ChemRsare promiscuous, capable of binding multiple ligands, just as cer-tain ligands can bind multiple receptors (Laing and Secombes,2004). ChemRs have been characterized and classified into fourfamilies according to the subfamily of chemokine ligands that theybind or the arrangement of their first two conserved cysteine resi-dues in chemokines (Zlotnik and Yoshie, 2000). The receptors thatbind to CXC chemokines are designated as CXC chemokine receptors(CXCRs), those to CC chemokines as CCRs, those to C chemokines asCRs, and those to CX3C chemokines as CX3CRs, analogically.

All the known ChemRs are seven transmembrane domain(7-TM) G protein-coupled receptors (GPCRs). So far, 18 genes

632 Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643

encoding ChemRs with standard chemotactic functions have beenidentified in the human genome: 6 CXCR genes, 10 CCR genes, 1 CRgene, and 1 CX3CR gene (Nomiyama et al., 2011). Although CCRshave been broadly investigated during the past few years and alarge number of mamml CCRs have been discovered, informationon the existence of CCRs in teleost fish is still rather limited withonly a few reports from rainbow trout (Daniels et al., 1999; Dixonet al., 2013), zebrafish and pufferfish (DeVries et al., 2006; Liu et al.,2009). Eleven CCRs named CCR1 to CCR11 have been identified inmost mammal species (Zlotnik et al., 1999) and teleost CCRs showrelative higher similarity to mammalian CCRs at the amino acidlevels (Nomiyama et al., 2011). The teleosts lack CCR4, but containCCR4 and 8 group receptors, which resemble CCR4 and CCR8 ofother species. And the fish-specific CCR11 and CCR12 group recep-tors may substitute for these missing receptors (Nomiyama et al.,2011).

Nowadays, many studies showed that mutations in some CCRscould be beneficial for lowering disease impact and providing somedegree of resistance to pathogens (Alkhatib et al., 1997). In addi-tion, there is increasing evidence that CCRs were involved in thepathogenesis of many diseases, such as acquired immunodefi-ciency syndrome (AIDS), multiple sclerosis and Alzheimer’s disease(Bajetto et al., 2001). Thereinto, CCR3 plays a vital role in allergicinflammation. Studies on the guinea pig showed that CCR3 block-ade could inhibit antigen-induced eosinophil accumulation(Sabroe et al., 1998). According to its pivotal role in eosinophilrecruitment and allergic responses, CCR3 showed a potential ther-apeutic target for the treatment of allergic inflammatory disease(Weston et al., 2006). And, the receptor of Thymus-expressed che-mokine (TECK), CCR9, was predominantly expressed on gut-hom-ing thymus dependent (T) and bursa (B) dependent lymphocytes,especially the immunoglobulin A (IgA)-secreting plasma cells(Papadakis et al., 2000; Kunkel et al., 2003; Hieshima et al., 2004;Pabst et al., 2004), suggesting that the TECK-CCR9 interactionplayed an important role in the mucosal immunity of the intestine.

Miiuy croaker, Miichthys miiuy, is an important marine fish thatis mainly distributed from the western Japan Sea to the East ChinaSea. In China, it is commercially important food fish specie with aworldwide market demand, due to its good taste, abundant nutri-ents and medicinal value, and has been widely cultured since thelate 1990s. Currently, the issue of environment pollution is moreand more serious, so the fish culture presents even greater chal-lenges than ever. In order to improve fish health and increase theprofits, we need to better understand the teleost immune system(Sommerset et al., 2005). Further understanding the immune sig-nificance in fish is needed to protect the fish against such infectiousdisease. In order to elucidate the immune mechanisms, a series ofthe immune-related genes have been carried out and reported inthis species (Cheng et al., 2011; Meng et al., 2012; Sun et al.,2012; Xu et al., 2011a, 2012).

CCRs genes were involved in the pathogenesis of many diseases,the immune response of CCRs of miiuy croaker against Gram-negative bacteria (e.g. Vibrio anguillarum) is largely unknown,despite the fact that is one of the most menacing bacteria inaquaculture (Toranzo and Barja, 1990). The aim of the presentstudy was to clone CCR3 and CCR9 genes in miiuy croaker andidentify tissues that expressed these receptors and understandtheir expression pattern during an infection by pathogenic bacte-ria. Because of the critical role of CCRs in signaling immuneresponses, they are expected to experience purifying selection tomaintain conformation and functionality of ligand binding andsignaling (Kunstman et al., 2003). But they are also expected toexperience positive selection pressure in response to pathogenhijacking due to their role as targets of pathogen entry (Shields,2000). Thus, our particular goals are also to explore the molecularevolution on these two subfamilies of CCRs.

2. Materials and methods

2.1. Fish sampling and challenge experiments

Healthy miiuy croakers (meanweight 810 g) were obtainedfrom Zhoushan Fisheries Research Institute (Zhejiang, China). Allfish were maintained in aerated water tanks at ambient tempera-ture (25 �C) and under a natural photoperiod. After one week ofacclimatizing, the challenge experiments of miiuy croaker wereperformed as previously described (Xu et al., 2011c). Fish were ran-domly divided into two groups, injection and control groups. In theinjection group, fish were injected intraperitoneally with 1 ml bac-teria suspension, which was made after V. anguillarum (AquaticPathogen Collection Centre of Ministry of Agriculture, China) cen-trifuge to approximately 3.0 � 107 colony forming units (CFU)/mlin phosphate-buffered saline while the control fish were chal-lenged with 1 ml phosphate-buffered saline and were maintainedin separate tanks. The infected and control fish samples were killedat 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h after injection, respectively.Three tissues (liver, spleen and kidney) of three individuals wereremoved and then kept at �80 �C until use. Immediately followingtissue excision, samples were placed into 1 ml of Trizol reagent andhomogenized.

For full-length cDNA cloning and expression analysis, ten tissuesamples (liver, spleen, kidney, intestines, heart, muscle, gill, brain,eye, and fin) of healthy miiuy croaker were also collected fromthree adult miiuy croaker and stored at �80 �C until RNAextraction.

2.2. RNA isolation and cDNA synthesis

Total RNA was extracted from the various tissues of adult indi-viduals by Trizol reagent (Qiagen) in light of the manufacturer’sinstructions. cDNA was synthesized utilizing a QuantScript RT Kit(TIANGEN) according to the manufacturer’s protocol, and thenwas stored at �20 �C for later.

2.3. Cloning the full-length cDNAs of miiuy croaker CCR3 and CCR9

In a preliminary study, we have identified the immune genes ofmiiuy croaker from the spleen cDNA library (Xu et al., 2010). OneEST sequence (GW670745) similar to other fish CCR3 gene And an-other EST sequence (GW669493) similar to other fish CCR9 genewere obtained. In order to isolate the full length cDNAs of CCR3and CCR9 genes, each clone was separately sequenced from bothforward and reverse directions with vector primers, then acquiringsequence repeat three times. Finally, the full-sequence of miiuycroaker cDNAs of CCR3 and CCR9 were acquired by forward and re-verse overlapping sequence. Meanwhile, these two genes were ob-tained through high-throughput transcriptome sequencing,primers (Mimi-CCR3-F/R and Mimi-CCR9-F/R, Table S1 of Support-ing information) designed to verify the correctness of two genessequence. The PCR conditions were: a pre-denaturalization at94 �C for 4 min; 35 cycles of denaturation at 94 �C for 45 s, anneal-ing at 56 �C for 45 s, extension at 72 �C for 2 min; and a finalextension at 72 �C for 10 min. Joint verification of the above twomethods allows us to obtain the final two CCRs cDNA sequence.

2.4. qRT-PCR analysis of CCR3 and CCR9 expression

Three pairs of primers (CCR3-RT-F/R, CCR9-RT-F/R and b-actin-RT-F/R) were designed and subsequently used in the study of theMIMI-CCR genes expression (Table S1 of Supporting information).The mRNA expression patterns of CCR3 and CCR9 genes in differenttissues (heart, muscle, kidney, eye, gill, intestine, brain, spleen, fin

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and liver) of healthy miiuy croakers and in three tissues (liver,spleen and kidney) of infected and health miiuy croakers weredetermined using real-time quantitative PCR (qRT-PCR). qRT-PCRwas conducted on a 7500 RT-PCR system (Applied Biosystemss,USA) using a SYBRsR premix ExTaq™ kit (TaKaRa). The reactioncarried out without the template was used as blank control. PCRamplification was performed in triplicate for each sample andcycling conditions were as follows: 10 s at 95 �C, followed by 40cycles consisting of 5 s at 95 �C and 34 s at 60 �C. Dissociation curveanalysis was performed after each assay to determine target spec-ificity. Expression of b-actin was used as the internal control forMIMI-CCR genes expression analysis. The primers b-actin-RT-F/R(Table S1 of Supporting information) were used for RT-PCR ofb-actin expression. Comparisons between groups were made byone-way analysis of variance followed by a Duncan test for identifi-cation of the statistically distinct groups. Differences betweenmeans were considered significant at the 95% confidence level(P < 0.05). All data were expressed as the mean ± SE (standard error).

2.5. Taxonomic coverage, sequence alignment and phylogenetic treeconstruction

All the CCRs sequences used in this study were retrieved fromGenBank (http://www.ncbi.nlm.nih.gov/Genbank/) and Ensemble(http://www.ensemble.org/) database (Table S2 and S3 of Support-ing information). The SignalP 4.0 Server was adopted for signalpeptide prediction (Nielsen et al., 1997). MUSCLE software was uti-lized for the alignment of sequences under codon model for itshigh accuracy and speed (Edgar, 2004). To reduce the error infor-mation, the final multisequencing alignment of sequences wasmodified manually. The potential protein domains of amino acidsequences were forecasted via SMART program (http://smart.rem-bi-heidelberg.de/; Letunic et al., 2006). Structural predictions werealso carried out using TMHMM server version 2.0 (http://www.cbs.dtu.dk/services/TMHMM). To study the evolutionaryrelationship between MIMI-CCR3 and CCR9 with other vertebrateCCRs, a phylogenetic tree including some vertebrate CCR sequences(Table S2 of Supporting information) was constructed based on thegenetic distance of the deduced amino acid sequences by theneighbour-joining (NJ) method using the MEGA5 program (Tamuraet al., 2011). The phylogenetic tree was bootstrapped 1000 timesusing the Poisson model (Jukes, 1969).

Then all the CCR3 and CCR9 gene sequences (Table S3 of Sup-porting information) were selected to construct phylogenetic treefor following evolutionary analysis, respectively. The divergenceand percent identity values of each sequence pair in the alignmentwere calculated using the MEGALIGN program of DNASTAR(Clewley and Arnold, 1997). jModeltest software was used to selectthe optimal substitution model for the data (Author, 2008), andBayesian information criterion (BIC) was used to choose theoptimal model. Phylogenetic trees were constructed with theBayesian approach by MrBayes v3.2 (Ronquist and Huelsenbeck,2003), and the program was run with 5,000,000 generations withburn-in of the first 25% trees.

2.6. Evolution analyses

The subset of all CCR3 sequences was used to determinewhether the diverse environments had affected selective pres-sures in ancestral lineages of mammals and teleosts (the repre-sentative of typical aquatic or terrestrial creatures) in CCR3genes. The selective pressures in ancestral lineages of mammalswere analyzed using the maximum likelihood (ML) methods inCODEML program of PAML v 4 (Yang et al., 2000). Firstly, theone-ratio model, which assumes all branches have only one ra-tio, was used to identify the selective pressures in all CCR3

genes. And then the free ratio model, which allows varied xratios in each branch, was implemented to check whether thismodel suitably fitted the data compared with the one-ratio mod-el by the likelihood ratio test (LRT). Thirdly, several two-ratiomodels which allow different x values between the ancestorsof mammals or teleosts and all other branches were tested tocheck whether they fitted the data significantly better than theone-ratio. Finally, we used the branch-site model to detect theinterested foreground lineages which included the ancestrallineages of mammals and teleosts.

The subsets of mammalian and teleost CCR3 sequences, bothof which used the sequence of Xenopus tropicalis as the outgroup, were used to account for the different functional andstructural constraints experienced by individual site-domains inthe site model of CODEML program, respectively (Yang et al.,2000). The Hyphy package in the Data Monkey Web Server(http://www.datamonkey.org) was also implemented to detectthe candidates for positive selection (Pond and Frost, 2005). InCODEML, six site models were used on the teleost and mamma-lian CCR3 sequences subsets to investigate the possible posi-tively selected sites in current genes. In all cases, twice thedifferences of log-likelihood values (2DlnL) between each twonested models were calculated following a chi-squared distribu-tion with degrees of freedom equaling the differences in thenumber of parameters between the nested models (Yang,1997). The Bayes empirical Bayes (BEB) in the case of modelsM2a and M8 was used to calculate the Bayesian posterior prob-ability (BPP) of the codon sites under a positive selection (Yanget al., 2005). In Hyphy, the best fitting nucleotide substitutionmodels were searched for through the automatic model selectiontool available on the server. All sequences of each group ofmammals and teleosts were analyzed under five distinct models:single likelihood ancestor counting (SLAC), fixed-effect likelihood(FEL), random effect likelihood (REL), fast unconstrained bayesianappRoximation (FUBAR) and mixed effects model of evolution(MEME) (Pond and Frost, 2005; Murrell et al., 2012, 2013). Weidentified codons and accepted them with P values <0.1 for SLAC,FEL and MEME, Bayes Factor >50 for REL and posterior probabil-ity >0.9 for FUBAR as candidates for positive selection.

The subset of all CCR9 sequences was used to detect the selec-tive pressures on the ancestors of ancestral lineages of mammals,teleosts and the evolutionary common ancestry of birds and mam-mals in CCR9 genes. The subsets of mammalian and teleost CCR9sequences, both of which used the sequence of Latimeria chalumnaeas the out group, were used to calculate the selected pressuresimposed in current CCR9 genes. Detailed method was the same asthat above described.

3. Results

3.1. Gene characterization of MIMI-CCR3 and CCR9 cDNA

The full length MIMI-CCR3 and CCR9 genes were successfullyobtained. The full length of CCR3 cDNA is 1297 nucleotides (nt),with an open reading frame of 1086 nt encoding a protein of 362amino acids (GenBank accession No. KC914581). The 50-UTR and30-UTR of CCR3 are 48 and 160 nt, respectively. The full length ofCCR9 cDNA is 1280 nt, with an open reading frame of 1119 ntencoding for 373 amino acids (GenBank accession No. JF427581).The 50-UTR and 30-UTR of CCR9 are 62 and 96 nt, respectively(Fig. 1a, Fig. 1c). The SMART and TMHMM program predicted thatseven transmembrane regions, an extracellular amino-terminaldomain (N-terminus), three extracellular and three intracellularloops and a cytoplasmic carboxyl-terminus (C-terminus) existedin CCR3 and CCR9 of miiuy croaker (Fig. 1b, d).

Fig. 1. (A) Nucleotide sequence of the miiuy croaker CCR3 cDNA and deduced amino acid sequence. The 50- and 30-noncoding region was lowercase and italic. The translationtransmembrane domains was indicated in blue, the conserved DRYLA motif was shown in red, and the stop codon was signed with asterisk (⁄). (B) The predict proteindomains characteristic of CCR3 and the alignment of some vital domains of miiuy croaker CCR3 with other telests. The transmembrane domains (TM) were marked with lightblue. The conservation was represented by the bar graph under the alignment. Identical residues are indicated by asterisks (⁄). (C) Nucleotide sequence of the miiuy croakerCCR9 cDNA and deduced amino acid sequence. (D) The predict protein domains characteristic of CCR9 and the alignment of C-terminal domain of miiuy croaker CCR3 withother telests. (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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3.2. Distribution of MIMI-CCR3 and CCR9 transcripts

The analysis of qRT-PCR was used to study the tissue expressionprofile of MIMI-CCR3 and CCR9 transcripts. qRT-PCR showed thatCCR3 transcripts were ubiquitously expressed in ten tissues,although their expression levels were distinctly different (Fig. 2).Higher CCR3 mRNA expression levels were detected in heat andgill, and lower levels in intestine, fin, brain, eye, kidney, liver, mus-cle, and spleen. Similarly, qRT-PCR analysis showed that CCR9swere detected in all tissues examined albeit at different levels

(Fig. 3), and high levels of CCR9 expressions were detected inspleen and gill while low expressions were detected in intestine,fin, brain, eye, kidney, liver, muscle, and heat.

3.3. Expression analysis of MIMI-CCR3 and CCR9 upon induction withV. anguillarum

To understand the potential roles of CCR3 and CCR9 of miiuycroaker upon stimulation with Gram-negative bacterial infection,the qRT-PCR was implemented to assess the relative quantity

Fig. 2. Expression of CCR3 gene in 10 tissues of miiuy croaker (A) and expression analysis in liver (B), spleen (C) and kidney (D) during 6, 12, 24, 36, 48, and 72 h of inductionwith V. anguillarum. Deviation bars represented the standard errors. Values with the same superscript are not significantly different (P > 0.05). Data indicated with asterisksymbol (⁄) are significantly different (P < 0.05) between challenged group and control group.

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changes of miiuy croaker CCR3 and CCR9 transcripts in spleen,liver and kidney (Figs. 2 and 3). The expressions at differentsampling time points were compared to those of control. In liver,MIMI-CCR3 expression decreased moderately from infectionstarting time to 48 h post-induction and reached the highestpoint at 72 h. In spleen, the expression level of CCR3 fluctuatedfrom 6 h to 24 h, but significantly increased and reached its peaklevel in 36 h, subsequently, the expression decreased graduallyto the hardpan in 72 h. In kidney, the expression gradually in-creased after an initial decrease from 0 h to 12 h, and then theexpression decreased gradually to a very low level in 36 h, fol-lowed by a rapid increase at 48 h. Last, the highest expressionlevel was detected at 72 h post-infection. In both of liver andkidney, MIMI-CCR9 expression levels fluctuated from infectionstarting time to 36 h post-induction, then sharply increasedand reached the peak level at 72 h after infection. In spleen,the expression was not up-regulated but down-regulated andmaintained lower level than normal from 6 to 72 h.

3.4. Multiple alignment and phylogenetic analysis

A phylogenetic tree (Fig. 4) constructed based on the amino acidsequences of the MIMI-CCR3 and CCR9 sequences with the other

known vertebrate CCR sequences showed that MIMI-CCR3 groupedwith the common ancestor of turbot and Japanese flounder on aseparate branch from the other known CCR3s. And MIMI-CCR9was genetically closest to the common ancestor Atlantic salmonand rainbow trout CCR9. In addition, most of fish CCRs weregrouped together and their own branches did not include othervertebrate CCRs, which indicated that the majority of fish CCRshad been generated after the divergence among these species.Moreover, most CCRs clustered largely according to nomenclature.

Then, fish CCR3 and CCR9 amino acid sequences were aligned,respectively. MIMI-CCR3 shared a highly conserved DRYLA motifin the second intracellular loop adjacent to the third transmemh-rane domain as other fishes (Fig. 1). MIMI-CCR3 deduced aminoacid sequence shared a 67.2% to 93.9% identity with the se-quences of other fish CCR3s (Table S4 of Supporting informa-tion). Similar results were obtained from the alignment of thededuced amino acid sequence of MIMI-CCR9. The identities ofCCR9 among fish ranged from 47.5% to 93.3% (Table S5 of Sup-porting information). The phylogenetic tree of CCR3s showedthat miiuy croaker and Pleuronectiformes were clustered to-gether and this clade was sister to Oreochromis niloticus(Fig. 5). And the similar topology structure was found in thephylogeny tree of CCR9s (Fig. 6). In these two phylogenetic trees

Fig. 3. Expression pattern of CCR9 gene in different tissues of miiuy croaker (A) and expression analysis in liver (B), spleen (C) and kidney (D) at 6, 12, 24, 36, 48, and 72 hpost-inoculation with V. anguillarum. Deviation bars represented the standard errors. Values with the same superscript are not significantly different (P > 0.05). Data indicatedwith asterisk symbol (⁄) are significantly different (P < 0.05) between challenged group and control group.

636 Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643

(Fig. 5, Fig. 6), aquatic and terrestrial organisms were recoveredas a monophyletic group, respectively.

3.5. Tests for selection in ancestral lineages of iconic animals

The codon-based models were utilized to test for heterogeneousselection pressures imposed on CCR3 genes between fish andmammals which stood for typical aquatic and terrestrial organ-isms. Phylogenetic tree (Fig. 5) was utilized to detect the positiveselection in ancestral lineages of mammals and teleosts. Firstly,x for all branches was calculated under one-ratio model and its va-lue was 0.1979 (P < 0.01, Table 1), showing that the entire CCR3genes underwent the purifying selection. Second, the free-ratiomodel showed a better fit to our data than the one-ratio model(P < 0.01, Table 1). Then two two-ratio models were used to testwhether the foreground lineages of mammal and teleost CCR3sunderwent different selection pressures compared with the back-ground lineages. The results showed that the ancestors of teleostand mammals experienced significantly different selective pres-sures from the background branches ratio (P < 0.01, Table 1).Lastly, the branch-site models were conducted to detect whetherpositively selected sites existed in ancestral lineages of mammals(posterior probability (PP = 1.00)) and teleosts (PP = 1.00; Fig. 5).

We found four and three positively selected sites in ancestral lin-eages of mammals and teleosts, respectively (Table 1).

Likewise, phylogenetic tree (Fig. 6) was used to analyze theselection pressures acting on the CCR9 genes. We found that entireCCR9 genes underwent the purifying selection. And five and onepositively selected sites were detected in ancestral lineages ofmammals and teleosts but not in that of the evolutionary commonancestry of birds and mammals (Table 2).

3.6. Positively selected sites in CCR3 and CCR9 genes of extant animals

Multiple ML methods were implemented to test the selectivepressures imposed on individual site-domains. For mammal CCR3s,site model detected 7 and 14 potential positively selected sites un-der M2a and M8 model and the results of the LRT test statistic(2DlnL) of M1a-M2a and M7-M8 comparisons were 16.72(P < 0.01, Table 3) and 24.28 (P < 0.01, Table 3). The other ML meth-ods also detected positively selected sites in this group (Table 4). Inorder to improve the accuracy of results, we considered that siteswith evidence of selection must be detected in at least two of theML methods. So we found total 11 sites as the robust candidatesfor sites under positive selection (Table 4). For teleost CCR3s, 9positively selected sites were checked under M8 model (P < 0.01,

Fig. 4. Phylogenetic tree of partial vertebrate CCR genes was constructed using MEGA5 with NJ method. Numbers of each node indicated the bootstrap values. MIMI-CCR3and CCR9 were shadowed with red and blue circles. (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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Table 3), and 8 sites were singled out for candidates of robustpositively selected sites (Table 4). For CCR9 genes, one and sixpositively selected sites were detected in M8 models of mammaland teleost subsets (Table 5). Then, the robust positively selectedsites were picked out by comparing for each ML method. Finally,three and two sites were singled out in the subsets of mammalsand telesots, respectively (Table 6).

4. Discussion

The modern vertebrate immune system is divided into innateand adaptive systems. Innate immunity provides a first line of anti-microbial host defense, and contributes to the establishment ofadaptive immune responses, indicating that it is not a redundantdefense mechanism (Fearon and Locksley, 1996; Medzhitov andJaneway, 1997). Innate and adaptive immunities function coopera-tively to control invading pathogens recognized as non-self (Fearonand Locksley, 1996; Fearon, 1997). Thus innate immunity plays avery important role in resistance against major bacterial diseasein vertebrates. In the innate immune responses, CCRs acts as themain receptors of chemokines which are low molecular weightcytokines that are released at the sites of infection, inflammation,and injury (DeVries et al., 2006). The interactions of various

chemokines with CCRs on leukocytes allow activation and chemo-taxis of neutrophils, eosinophils, lympgocytes, and monocytes formigration to the sites of evolving inflammation (Xu et al.,2011b). In this study, we cloned the CCR3 and CCR9 genes frommiiuy croaker. They all possessed a traditional structure with se-ven transmembrane regions, an extracellular N-terminal domain,three extracellular and three intracellular loops and a cytoplasmicC-terminus. The most highly conserved DRYLA motif was found inthe second intracellular loop adjacent to the third transmembranedomain of fish CCR3 (Fig. 1). This motif has been implicated inG-protein interaction and signalling, though other intracellular do-mains are also involved (Strosberg, 1991). MIMI-CCR3 and CCR9genes all shared higher identities with their orthologous genes inother fish (Table S3 of Supporting information). The phylogenetictrees conducted based on these two genes showed that CCR genesindependently evolved in aquatic and terrestrial organisms (Figs. 5and 6). The overall topology of the tree was consistent with tradi-tional taxonomy and phylogenetic transition, showing that miiuycroaker had high similarity with other teleosts. The result was sim-ilar with that of the amino acid identity analysis.

In order to explore the important role of CCR3 and CCR9 in in-nate resistance, we carried out experiments on gene expressionprofiles of them under normal conditions and pathogen infectionfrom expression analysis. The miiuy croaker CCR3 and CCR9

Fig. 5. Phylogenetic tree of all collected CCR3 genes was constructed using MrBayeswith Bayesian method. Bayesian posterior probabilities values were indicated. TheDarwin selection pressures were detected by the branch-site models in theancestral lineages of mammals (in red) and teleosts (in blue). The positive selectedsites with posterior probabilities larger than 0.95 were showed on the correspond-ing lineages. (For interpretation of the references to color in this figure legend, thereader is referred to the web version of this article.)

Fig. 6. The putative gene tree for CCR9 reconstructed by Bayesian approach with noconstraints on the topology. The Darwin selection pressures were detected in theancestral lineages of mammals (in red), teleosts (in blue) and the common ancestryof birds and mammals (in green). Bayesian posterior probabilities and positivelyselected sites in the ancestral lineages were showed. (For interpretation of thereferences to color in this figure legend, the reader is referred to the web version ofthis article.)

638 Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643

transcripts were constitutively expressed in all examined tissuesindicating that the ubiquitous expression pattern of CCR3 andCCR9 in miiuy croaker. Liu et al. (2009) studied the expression pat-terns of CCR3 and CCR9 in zebrafish, finding that zebrafish CCR3-1and CCR3-2 was widespread in various development samples butthe expression of CCR9-2 and CCR3-3 were not observed duringembryo development; CCR3-1 and CCR9-1 was widely expressedin various organs including spleen, brain, kidney, liver, gill andintestine, but the expression of CCR3-3 and CCR9-2 were not de-tected in all examined organs. Dixon et al. (2013) showed that rain-bow trout CCR9-2 was strongly transcribed in thymus andperipheral blood leukocytes (PBLs) but also in spleen, gills, hindgutand brain at lower levels. All the results showed that the expres-sions patterns of CCR3 and CCR9 were not complete the same indifferent fish. These differences in expression patterns of CCRs infish might, to some extent, be simply caused by the different tis-sues or organs examined in these studies and potential specificfunctions of CCRs in different species. Therefore, it is well worthfurther exploring the functional reasons behind these differencesin CCRs expression patterns among fishes.

In our study, miiuy croaker was injected with Gram-negativeV. anguillarum, which is one common pathogen in fishery industry(Larsen and Mellergaard, 1984). The expressions of CCR3 werechecked, showing that different expression patterns existed inthree main immune organs (liver, spleen and kidney). MIMI-CCR3 gene expression levels were up-regulated after infection withV. anguillarum in all tested tissues. However, the expression of

MIMI-CCR9 was restrained in spleen, although the expressions inliver and kidney were up-regulated after induction. The differencesin the regulation of the expression of MIMI-CCRs upon stimulationmight reflect their function difference in various tissues.

Because of the important status of fish immune in the evolu-tionary process of innate and adaptive immunity, we retrievedthe sequences of CCR3 and CCR9 genes from database and usedthese data to explore the similarities and differences between lowervertebrates and mammals in innate and adaptive immune defensemechanisms. Phylogenetic tree (Fig. 5) was utilized to detect thepositive selection on ancestral lineages of mammals (PP = 1.00)and teleosts (PP = 1.00) in CCR3 genes. Phylogenetic tree (Fig. 6)was used to detect the selective pressures on the ancestors ofancestral lineages of mammals (PP = 1.00), teleosts (PP = 1.00)and the evolutionary common ancestry of birds and mammals(PP = 1.00) in CCR9 genes. Interestingly, we all found the positivelyselected sites existing in the ancestral lineages of mammals andteleosts in both of these two genes.

The invasion of the body by pathogenic organisms triggers acellular response by the immune system that leads to the recruit-ment of leukocytes that seek out and destroy the foreign invaders.CCRs play a major role in the mobilization and activation ofthe cells of the immune system and are mainly expressed inimmune cells, which are their major target cells. Moreover, twoof the cloned CCRs have been reported to be expressed by virusesand might function in protecting the virus from immune surveillance

Table 1Likelihood ratio tests of branch-models and branch-site models on CCR3 genes.

Model Npa Ln likelihood Parameter estimates Model compare Positive selection sites 24lnLb (p-value)

Branch-modelA: Omega = 1 53 �11591.30 x = 1.0000 n/aB: One-ratio 54 �11097.45 x = 0.1979 A vs B n/aC: Free-ratio 105 �10958.12 variable x by branch B vs C n/a 278.66 (P < 0.01)D: Two-Mamc 55 �11087.77 B vs D n/a 19.36 (P < 0.01)E: Two-Teld 55 �11082.14 B vs E n/a 30.62 (P < 0.01)

Branch-site model1: Null-Mam 56 �10789.152: Mam 57 �10779.07 1 vs 2 90, 134, 147, 307 20.16 (P < 0.01)3: Null-Tel 56 �10784.764: Tel 57 �10782.47 3 vs 4 100, 165, 308 4.58(P < 0.05)

a Numbers of parameters.b Twice the difference of ln[likelihood] between the two models compared.c Mam = Ancestor branch of the mammals examined in present study.d Tel = Ancestor branch of the teleosts examined in present study.

Table 2Likelihood ratio tests of branch-models and branch-site models on CCR9 genes.

Model Npa Ln likelihood Parameter estimates Model compare Positive selection sites 24lnLb (p-value)

Branch-modelA: Omega = 1 87 �18972.75 x = 1.0000 n/aB: One-ratio 88 �17405.58 x = 0.1119 A vs B n/a 3134.34 (P < .0.01)C: Free-ratio 173 �17260.72 variable x by branch B vs C n/a 289.72 (P < 0.01)D: Two-Comc 89 �17404.93 B vs D n/a 1.30 (P = 0.25)E: Two-Mamd 89 �17403.58 B vs E n/a 4.0 (P < 0.05)F: Two-Tele 89 �17395.99 B vs F n/a 19.18 (P < 0.01)

Branch-site model1: Null-Com 90 �17173.122: Com 91 �17170.76 1 vs 2 not found 4.72 (P < 0.05)3: Null-Mam 90 �17169.064: Mam 91 �17164.79 3 vs 4 10, 14, 85, 178, 238 8.54 (P < 0.01)5: Null-Tel 90 �17174.804: Tel 91 �17171.18 5 vs 6 206 7.24(P < 0.01)

a Numbers of parameters.b Twice the difference of ln [likelihood] between the two models compared.c Com = Ancestor branch of the common ancestry of birds and mammals examined in present study.d Mam = Ancestor branch of the mammals examined in present study.e Tel = Ancestor branch of the teleosts examined in present study.

Table 3Site model tests on CCR3 genes in subset of mammals and teleosts.

Model Npa Parameter estimates Lnlikelihood

Modelcompare

Positive selection sitesb 24lnL (p-value)

Data set: mammalianM0:one-ratio 40 x = 0.0.313 �6366.48M3:discrete 44 x0 = 0.025, p0 = 0.483, x1 = 0.504, p1 = 0.431,

x2 = 1.953, p2 = 0.086�6175.84 M3 vs M0 Not analysed 381.28(P < 0.01)

M1a:nearlyneutral

41 x0 = 0.084, p0 = 0.665, x1 = 1.000, p1 = 0.335 �6196.63

M2a:positiveselection

43 x0 = 0.085, p0 = 0.654, x1 = 1.000, p1 = 0.322,x2 = 3.347, p2 = 0.024

�6188.27 M2 vs M1 20⁄⁄, 22, 46, 179, 180, 181, 191 16.72(P < 0.01)

M7:b 41 p = 0.249, q = 0.484 �6187.47M8:b and x 43 p0 = 0.953, p1 = 0.047, x = 2.449, p = 0.300,

q = 0.695�6175.33 M8 vs M7 20⁄⁄, 22, 25, 46, 95, 104, 177, 178, 179, 180,

181, 182, 191, 27124.28(P < 0.01)

Data set: teleostsM0:one-ratio 12 x = 0.109 �4173.14M3:discrete 16 x0 = 0.019, p0 = 0.644, x1 = 0.285, p1 = 0.311,

x2 = 1.748, p2 = 0.045�4060.20 M3 vs M0 Not analysed 225.88(P < 0.01)

M1a:nearlyneutral

13 x0 = 0.054, p0 = 0.832, x1 = 1.000, p1 = 0.168 �4076.86

M2a:positiveselection

15 x0 = 0.055, p0 = 0.830, x1 = 1.000, p1 = 0.162,x2 = 4.302, p2 = 0.008

�4076.86 M2 vs M1 not allowed 0.00(P = 1.00)

M7:b 13 p = 0.258, q = 1.378 �4064.09M8:b and x 15 p0 = 0.970, p1 = 0.030, x = 2.189, p = 0.322,

q = 2.274�4059.73 M8 vs M7 31, 34, 36, 111, 156, 187, 285, 290, 359 8.72(P < 0.05)

a Number of parameters.b Positively selected sites were indicated by the Bayes Empirical Bayes (BEB) approach. Asterisks indicate posterior probability P > 95% (*) and P > 99% (**).

Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643 639

Table 5Site model tests on CCR9 genes in subset of mammals and teleosts.

Model Npa Parameter estimates Lnlikelihood

Modelcompare

Positive selectionsitesb

24lnL (p-value)

Data set: mammalianM0:one-ratio 60 x = 0.0.112 �8582.02M3:discrete 64 x0 = 0.031, p0 = 0.642, x1 = 0.167, p1 = 0.245, x2 = 0.596, p2 = 0.113 �8388.52 M3 vs M0 Not analysed 387.00(P < 0.01)M1a:nearly neutral 61 x0 = 0.074, p0 = 0.888, x1 = 1.000, p1 = 0.112 �8434.64M2a:positive selection 63 x0 = 0.074, p0 = 0.888, x1 = 1.000, p1 = 0.103, x2 = 1.000, p2 = 0.009 �8434.64 M2 vs M1 not allowed 0.00(P = 1.00)M7:b 61 p = 0.417, q = 2.629 �8398.48M8:b and x 63 p0 = 0.996, p1 = 0.004, x = 1.780, p = 0.440, q = 2.941 �8394.51 M8 vs M7 191⁄⁄ 7.94(P < 0.05)

Data set: teleostsM0:one-ratio 24 x = 0.112 �8040.62M3:discrete 28 x0 = 0.013, p0 = 0.485, x1 = 0.155, p1 = 0.389, x2 = 0.656, p2 = 0.126 �7747.93 M3 vs M0 Not analysed 585.38(P < 0.01)M1a:nearly neutral 25 x0 = 0.072, p0 = 0.826, x1 = 1.000, p1 = 0.174 �7845.58M2a:positive selection 27 x0 = 0.072, p0 = 0.826, x1 = 1.000, p1 = 0.013, x2 = 4.302, p2 = 0.161 �7845.58 M2 vs M1 not allowed 0.00(P = 1.00)M7:b 25 p = 0.383, q = 2.156 �7751.71M8:b and x 27 p0 = 0.975, p1 = 0.025, x = 1.456, p = 0.434, q = 3.013 �7746.66 M8 vs M7 45, 192, 291, 335,

339, 34210.10(P < 0.01)

a Number of parameters.b Positively selected sites were indicated by the Bayes Empirical Bayes (BEB) approach. Asterisks indicate posterior probability P > 95% (*) and P > 99% (**).

Table 4Phylogenetic tests of positive selection in CCR3 genes.

Taxa Methoda

SLACb FELb RELc FUBARd MEMEb Paml-M8 Total

Mammals 20,

22

108, 179,

180, 182,194

20, 22, 46, 104, 108,

180, 181, 182, 271

20, 22,

180, 182

22, 85, 108, 179, 180, 182,

184, 191, 233, 262, 285,320

20, 22, 25, 46, 95, 104, 177, 178,

179, 180, 181, 182, 191, 271

20, 22, 46, 104, 108, 179,180, 181, 182, 191, 271

Teleosts no 94 31, 34, 36, 111, 187,188, 279, 280

no 94, 156, 184, 204, 288, 345,

355, 359

31, 34, 36, 111, 156, 187, 285,

290, 359

31, 34, 36, 94, 111, 156,187, 359

a Codons identified by more than one ML method are underlined.b Codons with P values <0.1.c Codons with Bayes factor >50.d Codons with posterior probability >0.9.

Table 6Phylogenetic tests of positive selection in CCR9 genes.

Taxa Methoda

SLACb FELb RELc FUBARd MEMEb Paml-M8 Total

Mammals 99,

189

99 39, 41, 64, 99, 113, 189, 264,291

99 41, 104, 127, 198, 199, 201, 203, 210, 252, 281, 304,339

191 41, 99,189

Teleosts 335 335,

339

no 335 67, 179, 197, 289, 311, 335, 339, 347 45, 192, 291, 335, 339,342

335, 339

a Codons identified by more than one ML method are underlined.b Codons with P values <0.1.c Codons with Bayes factor >50.d Codons with posterior probability >0.9.

640 Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643

(Horuk, 1994). The special evolution in the ancestral lineages ofmammals and teleosts of CCR3 and CCR9 genes might be due tothe invasion of different pathogens. When the ancestor of terres-trial animals left the water and boarded the land, they must facea completely different environment than ever. The ancestors of ter-restrial animals have to further evolve to adapt to terrestrial envi-ronments, because the pathogen of land significantly differed fromthose of the ocean. And the resident microbes are transient in thegastrointestinal tract of aquatic animals (Moriarty, 1990). Theintestinal microbiota of fish is peculiarly dependent on the externalenvironment due to the water flow passing through the digestivetract. The gastrointestinal microbiota of aquatic animals maychange rapidly with the intrusion of microbes coming from wateror food (Gatesoupe, 1999). The continuous intrusion of microbes

might stimulate the evolution of CCR genes in the ancestor ofteleost.

The CCRs have seven hydrophobic membrane-spanning do-mains, with an extracellular N-terminus and a cytoplasmic C-terminal tail (Fig. 7). The seven membrane-spanning domains arehighly conserved in CCRs, and the N-terminus is quite variableamong the different CCRs and is likely to play a major role in deter-mining ligand binding specificity (Pelchen-Matthews et al., 1999).Moreover, the N-terminal and extracellular domains had beenimplicated in receptor–ligand interaction, while the C-terminaland intracellular domains cooperated to bind and activate the Gproteins (Murphy, 1994). We used multiple ML methods to detectthe robust candidates for sites under positive selection. In total, 11and 8 positively selected sites were found in the subsets of current

Fig. 7. The predict protein domains characteristic and location of positively selected sites in the protein domains of mammal (A), teleost (B) CCR3, mammal (C) and (D) teleostCCR9. The positively selected sites were marked in the CCR3 and CCR9 diagrams of miiuy croaker and human. Red stars indicated the location of positively selected sites inextracellular or intracellular domain. Blue stars indicated the positively sites locating in transmembrane regions. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)

Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643 641

mammal and teleost CCR3 genes (Table 4). And for CCR9 genes, 3and 2 sites were detected under positive selection in the subsetsof current mammals and teleosts (Table 6). These results showeda proportion of codon sites that displayed evidence of positiveselection within the coding sequences of CCR3 and CCR9.

More interestingly, for mammal CCR3s, ten out of the elevenamino acid sites that were identified as the robust candidates ofpositively selected sites were located in the extracellular domainsof the receptor (Fig. 7a). For mammal CCR9s, two of three posi-tively selected sites were seated in the extracellular domains ofthe receptor (Fig. 7c). Metzger and Thomas (2010) also found thesimilar result that amino acid residues in extracellular domainsof CCRs evolved more quickly when they did some research onmammal CCRs. Previous studies about CCRs showed that the extra-cellular N-terminus and domains were important for both theendogenous ligand-binding functions (Pease et al., 1998; Hanet al., 1999; Zoffmann et al., 2002; Sabroe et al., 2005; Duchesneset al., 2006) and played a vital role in binding efficacy for patho-gens (Frade et al., 1997; Howard et al., 1999; Liu et al., 2003; Hoet al., 2004). Combined with the findings above, we concluded thatthe positively selected sites of CCR3 and CCR9 genes in mammals

often occurred in the extracellular domains which were the ligandbinding and pathogen interaction regions of the receptors.

However, among eight positively selected codon sites in teleostCCR3s, one was separately located in the C-terminal and the sec-ond intracellular domains, and five sites were located in the extra-cellular domains of the receptor (Fig. 7b). For teleost CCR9s, both oftwo identified positively selected sites were seated in the C-termi-nal of the receptor (Fig. 7d). The binding of ligand resulted in phos-phorylation of serine (S) and threonine (T) residues in theintracellular loops and C-terminus of the CCR by G protein-coupledreceptor kinases (GRKs) (Freedman and Lefkowitz, 1996; Fergusonet al., 1998; Krupnick and Benovic, 1998; Ferguson, 2001). Andb-arrestin can bind with higher affinity to the phosphorylatedreceptors to mediate endocytosis and plays important roles in CCRsinternalization. b-arrestin binding to CCRs can be mediatedthrough phosphorylated residues in the C-terminus and intracellu-lar loops. Moreover, the adornments of ubiquitin or palmitic acid inthe intracellular loops or C-terminal tail also have the potential toaffect the membrane association, trafficking, endocytosis, turn-over as well as signaling pathways of CCRs (Neel et al., 2005). Thesepositively selected sites in the intracellular loops and C-terminus of

642 Z. Zhu et al. / Developmental and Comparative Immunology 41 (2013) 631–643

teleost CCR3 and CCR9 genes were not located in these vital func-tional sites such as serine (S), threonine (T) residues or the sitesforming ubiquitin or palmitic acid, and might not directly influencethe membrane targeting, signaling properties and endocytosis ofthe receptors and so on, but might play an important role in someof unknown function. But we could confirm that mammals and tele-osts experienced different selection pressures upon their N-terminus,C-terminus and intracellular loops of CCRs. Because the field of CCRtrafficking in teleosts is not fully developed, a great deal of work isneeded for a better understanding of CCR function.

Acknowledgements

This study was supported by Nation Nature Science Foundationof China (31001120), Zhejiang Provincial Natural Science Founda-tion of China (Y3100013) and Important Science and TechnologySpecific Projects of Zhejiang Province (2011C14012).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.dci.2013.06.015.

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