chemokine receptors in atlantic salmon

Chemokine receptors in Atlantic salmonUnni Grimholt a Helena Hauge b Anna Germundsson Hauge b Jong Leong c Ben F Koop c

a Soeren Jaabaeksgate 10B 0460 Oslo Norwayb Norwegian Veterinary Institute PO Box 750 Sentrum 0106 Oslo Norwayc Centre for Biomedical Research Department of Biology University of Victoria PO Box 3020 STN CSC Victoria Canada

A R T I C L E I N F O

Article historyReceived 8 July 2014Revised 9 November 2014Accepted 10 November 2014Available online

KeywordsAtlantic salmonChemokine receptorsWhole genome duplicationInflammation

A B S T R A C T

Teleost sequence data have revealed that many immune genes have evolved differently when com-pared to other vertebrates Thus each gene family needs functional studies to define the biological roleof individual members within major species groups Chemokine receptors being excellent markers forvarious leukocyte subpopulations are one such example where studies are needed to decipher individ-ual gene function The unique salmonid whole genome duplication that occurred approximately 95 millionyears ago has provided salmonids with many additional duplicates further adding to the complexity anddiversity Here we have performed a systematic study of these receptors in Atlantic salmon with par-ticular focus on potential inflammatory receptors

Using the preliminary salmon genome data we identified 48 chemokine or chemokine-like recep-tors including orthologues to the ten receptors previously published in trout We found expressed supportfor 40 of the bona fide salmon receptors Eighteen of the chemokine receptors are duplicated and whentested against a diploid sister group the majority were shown to be remnants of the 4R whole genomeduplication with subsequent high sequence identity The salmon chemokine receptor repertoire of 40expressed bona fide genes is comparably larger than that found in humans with 23 receptors Diversi-fication has been a major driving force for these duplicate genes with the main variability residing inligand binding and signalling domains

copy 2014 Elsevier Ltd All rights reserved

1 Introduction

Chemokine receptors (CRs) and their ligands play an impor-tant role in coordination of cell trafficking in many biologicalprocesses They are predominantly expressed on the surface of leu-kocytes and because they dictate migration of these cells betweentissues they are crucial for an effective immune responseChemokines and their receptors have traditionally been divided intotwo main functional categories homeostatic chemokines and theirreceptors are involved in basal cell trafficking and homing while in-ducible chemokines and their receptors are involved in inflammatoryresponses There are also a few receptors with dual function in ad-dition to some atypical chemokine receptors (Bonecchi et al 2010Cancellieri et al 2013)

CRs belong to the large family of G-protein-coupled seven trans-membrane receptors with four extracellular and four intracellulardomains The extracellular N-terminal part of the receptor is re-sponsible for ligand binding while the intracellular domains includingthe C-terminus are involved in intracellular signalling (Neel et al

2005 Szpakowska et al 2012) CRs are named according to thechemokine class they bind CCRs bind to CC-chemokines CXCRs bindto CXC chemokines XCR binds to XC chemokines and CX3CR bindsto CX3C chemokines where X is any amino acid and C is cysteine(Allen et al 2007 Charo and Ransohoff 2006)

In general there are fewer receptors than chemokines with ap-proximately 20 receptors versus 50 ligands identified in mammalsso most receptors bind more than one ligand With the exceptionof atypical CRs ligand binding causes conformational changes inthe receptors that in turn trigger intracellular signals causing cel-lular events such as directional cellular migration

Homeostatic chemokines are constitutively expressed and areimportant for normal cell trafficking and for the development andmaintenance of the immune system (Moser and Loetscher 2001Proudfoot 2002) The human homeostatic receptors are CCR7 CCR9CCR10 CXCR4 and CXCR5 where for instance CCR7 is expressed oncells destined for lymph nodes CCR9 directs leukocytes to the in-testine CCR10 directs T-cells to skin and intestine and CXCR5 directsB-cells to lymph node follicles (Charo and Ransohoff 2006) CXCR4is widely expressed with multiple functions including a role in thecentral nervous system (Bonecchi et al 2010 Tran and Miller 2003)

Expressions of inflammatory chemokines are induced by me-diators such as tumour necrosis factor interferon-γ or microbialproducts associated with infection or trauma (Charo and Ransohoff

Corresponding author Soeren Jaabaeksgate 10B 0460 Oslo Norway Tel +4792661039

E-mail address unnigrimholtgmailcom (U Grimholt)

httpdxdoiorg101016jdci2014110090145-305Xcopy 2014 Elsevier Ltd All rights reserved

Developmental and Comparative Immunology (2014) ndash

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Please cite this article in press as Unni Grimholt Helena Hauge Anna Germundsson Hauge Jong Leong Ben F Koop Chemokine receptors in Atlantic salmon Developmental andComparative Immunology (2014) doi 101016jdci201411009

Contents lists available at ScienceDirect

Developmental and Comparative Immunology

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2006) A classic example would be a pathogen recognised by a toll-like receptor which then induces expression of secreted chemokine(Kaisho 2012) In humans the inflammatory chemokine recep-tors are CCR1-3 CCR5 CXCR1 CXCR2 and CX3CR1 The receptorsCCR4 CCR6 CCR8 CXCR3 CXCR6 and XCR1 have dual functions par-ticipating in both inflammatory as well as homeostatic processes

Most of the atypical or silent CRs also bind chemokines but thisbinding does not induce a signalling cascade with subsequent cellmigration Instead several of these receptors have regulatory rolesHuman atypical receptors are CCBP2 (D6) CCRL1 (CCX-CKR) CCRL2DARC and CXCR7 (RDC1) CCBP2 has been shown to act as a scav-enger for CC-chemokines and can drastically reduce the amount ofligand available for other CRs (Graham et al 2012) The chemokine-like receptor CMKLR1 does not bind to a chemokine but may havemultiple functions as it can regulate CCRL2 activity through com-petitive binding to the ligand chimerin (Yoshimura and Oppenheim2011) Some atypical receptors have recently been renamed to ACKRswhere CCBP2 is now ACKR2 CXCR7 is ACKR3 and CCRL1 is ACKR4(Bachelerie et al 2014)

CRs have been identified in many teleost species with the primaryfocus on teleosts with sequenced genomes ie fugu tetraodonmedaka stickleback and zebrafish (Bajoghli et al 2009 Chang et al2007 DeVries et al 2006 Diotel et al 2010 Huising et al 2003bLiu et al 2009 Nomiyama et al 2011 Oehlers et al 2010 Sasadoet al 2008 Verburg-van Kemenade et al 2013 Xu et al 2010)Results from these studies show clear-cut teleost orthologues tomammalian homeostatic receptors but orthology to mammalianinflammatory CRs is less obvious A few publications also exist onCRs in salmonids ie CCR6 CCR7 CCR99b CCR13 (CCR3) IL8R(CXCR1) CXCR2 CXCR3a CXCR3b and CXCR4 (Daniels et al 1999Dixon et al 2013 Ordas et al 2012 Xu et al 2014 Zhang et al2002) There are also a few accepted teleost chemokine receptorligand pairs such as CCR6 with CCL20-like ligands and CCR7 withCCL1921-like ligands (Laing and Secombes 2004) However thefunctional role of most fish CRs remains unresolved Understand-ing the specific function of individual receptors and identifying theirligands is essential for understanding teleost homeostasis andinflammation

To broaden our understanding of CRs in salmonids we made useof the salmon genome (Davidson et al 2010) to identify receptorsand study their evolution and potential function From a disease pre-vention point of view we paid particular attention to receptorspotentially involved in inflammation Of the 48 receptors identi-fied several have potential roles in inflammation being expressedin immunologically important tissues Most importantly thesalmonid-specific whole genome duplication event approx 95million years ago (Macqueen et al 2013) has had a significant impacton the receptor repertoire

2 Material and methods

21 Bioinformatics

Using available CR sequences from published articled andorretrieved from GenBank BLASTN (Altschul et al 1997) and TBLASTN(Schaffer et al 2001) searches were initially performed against bothexpressed and genomic Atlantic salmon resources at NCBI GenBankcGRASP [cGRASP Internet 2009 (Rondeau et al 2014)] andor theSalmonDB in Chile (Di Genova et al 2011) Identified genomic se-quences from the latest Atlantic salmon genome assembly (GenBankAGKD000000003) or from the Northern pike genome assembly(AZJR000000001) were subjected to gene prediction analysis usingGenScan (Burge and Karlin 1997) FGENESH (Solovyev et al 2006)andor Augustus (Stanke et al 2006) Predicted ORFs were testedthrough alignment with similar sequences from other speciesand sometimes changed using expressed match in Spidey (Spidey

Internet 2013) Spidey was also used to define exon intronboundaries

To assess evolutionary relationships and orthology all identi-fied amino acid sequences were aligned using ClustalX2011 (Larkinet al 2007) ClustalX was also used to calculate percentage iden-tity Phylogenetic analyses were performed using the neighbor-joining method (Saitou and Nei 1987) The percentages of replicatetrees in which the sequences clustered together in the bootstraptest (1000 replicates) are shown next to the branches (Felsenstein1985) The tree is drawn to scale with branch lengths in the sameunits as those of the evolutionary distances used to infer the phy-logenetic tree The evolutionary distances were computed using thep-distance method (Nei and Kumar 2000) and are in the units ofthe number of amino acid differences per site All ambiguous po-sitions were removed for each sequence pair Evolutionary analyseswere conducted in MEGA5 (Tamura et al 2011)

Secondary structure including transmembrane domains were pre-dicted and visualised using Rhythm (Rose et al 2009) Predictionof N-linked glycosylation sites was performed using NetNGlyc 10(Gupta and Brunak 2002) while tyrosine sulfation sites were pre-dicted using Sulfinator (Monigatti et al 2002) O-linked glycosylationsites were predicted using NetOGlyc 31 (Julenius et al 2005)

22 Northern pike cDNA and genomic DNA

Pike (Esox lucius) genome transcriptome and genetic map dataare described fully in Rondeau et al (2014) In brief DNA from asingle pike individual (Leong et al 2010) was submitted directlyto BGI (httpwwwgenomicscnenindex) for Illumina sequenc-ing DNA libraries of 180 bp were constructed for paired-endsequencing and libraries of 2 kb and 6 kb fragments were con-structed for mate-pair sequencing and assembly Fragment assemblyused ALLPATHS-LG (Gnerre et al 2011) The resulting contigs ge200 bpwere screened and trimmed for vector and contamination whichproduced 94267 contigs (N50 = 16909 bioproject PRJNA221548accession GenBankAZJR00000000) and 5688 scaffolds ge1000 bp witha total genome size of 877777613 bp

23 Tissue transcriptomes and analysis

For transcriptome data tissues were extracted from a single 1year old presmolt juvenile male Atlantic salmon RNA from 11 tissuesndash brain eye gill hind gut head kidney heart kidney liver musclestomach spleen ndash were extracted and submitted to BGI for Illuminasequencing Contig assembly used Trinity (Haas et al 2013) Theresulting set of transcripts was reduced by retaining those with asignificant BLASTX (Altschul et al 1997) match to the SwissProt orGene Ontology protein databases (le10minus5) or had a predicted openreading frame ge300 bp Further only those transcripts that mappedto our genome assembly using BLAT (Kent 2002) were retained Toremove possible alleles from our assembly we retained a singlelongest representative of transcripts that were ge98 similar over aminimum length of 300 bp as determined by BLASTN (Altschul et al1997) This curated set represents our RNA-seq referencetranscriptome FPKM values were then determined for each tran-script for each of the 11 different tissues

Pike chemokine receptors were identified using known salmongenes as queries that were BLASTed against the pike genome andtranscriptome (Rondeau et al 2014) Identified contigs were furtherexamined as earlier

24 RNA extraction

Three healthy Atlantic salmon weighing 70ndash80 g (AquaGen breed)kept in a freshwater flow system at 12 degC with regular feeding weresacrificed by overexposure to Finquelreg (ScanAqua AS) and tissues

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were collected in RNAlaterreg (Invitrogen) and stored at minus80 degC untilfurther processing Total RNA was extracted using RNeasyreg Mini Kit(Qiagen) followed by DNase-treatment with RQ1 RNase free DNase(Promega) to remove genomic DNA contamination All protocols wereaccording to the manufacturerrsquos instructions The concentration oftotal RNA was measured using a NanodropTM 2000 spectrophotom-eter (Thermo Scientific) and the samples were stored in RNase-free water at minus80 degC

25 Real-time PCR

To evaluate the levels of CR expression in different tissues 6 μgof total RNA from three individuals (2 μg each) were pooled andreverse transcribed in 60 μl reaction volume using RevertAidtrade HMinus First Strand cDNA Synthesis Kit (Fermentas) with oligo d(T)18primers according to the manufacturerrsquos instructions Pooling of RNAfrom three individuals was performed to reduce the effect of indi-vidual variation Correct amplification of each CR gene was confirmedby examining the melting curve analysing product size and frag-ment sequence SYBR Greenreg Real-time PCR assays (QuantiTect SYBRGreen PCR Kit Qiagen) were optimised on 10-fold serial dilutionsof cDNA and in 25 μl reaction volume Two microlitres cDNA and03 μM of each primer (listed in SF5c) were used for all assays exceptfor 18S where a 11000 dilution of cDNA was used PCR was per-formed in triplicate in 96-well optical plates on an Mx3005 real-time thermal cycler (Stratagene) The PCR cycling conditions were95 degC for 15 min 42 cycles of 94 degC for 50 s 60 degC for 15 s and 72 degCfor 1 min and finally 95 degC for 1 min 55 degC for 30 s and 95 degC for30 s Validation of assays and data handling were according to theMxPro Manual and baseline and cycle threshold (CT) were set man-ually Each assay was tested on different samples in the same plateto ensure optimal reproducibility and the 18S reference gene assaywas included in all plates Real-time PCR efficiencies were calcu-lated from the given slopes in MxPro software The correspondingreal-time PCR efficiency (E) of one cycle in the exponentialphase of each gene was calculated according to the equationE = 10[minus1slope] on 10-fold serial dilutions of cDNA for each assay (Pfaffl2001) The relative expression of CCR transcripts was normalisedto the geometric mean of the CT value of 18S and then presentedas relative expression compared to the corresponding control groupsusing the Pfaffl method

26 Sequencing

CR PCR-products were sequenced using the ABI Prism Big DyeTerminator Cycle sequencing kit on an ABI 3130xl Genetic Analyseraccording to the manufacturerrsquos instructions

3 Results and discussion

To identify salmon CRs we performed various blast searchesagainst GenBank nucleotides ESTs and ultimately preliminary andfinal salmon genome scaffolds (GenBank version AGKD01-AGKD03contigs) using salmonid zebrafish and human CR sequences asqueries A total of 48 salmon chemokine and chemokine-like re-ceptors were identified (Fig 1 Appendix S1) with amino acidsequence identity ranging from 19 to 98 (Appendix S2) Forty fiveof these sequences appear to be bona-fide CRs while three are mostlikely pseudogenes (ssCXCR72b ssCCRL12b and ssCMKLR2b)

Nomenclature for human CRs relies on ligand binding Al-though many teleost CC and CXC ligands have been identified (Alejoand Tafalla 2011 Laing and Secombes 2004 Nomiyama et al 2008Peatman and Liu 2007) only a few receptorndashligand pairs have beenestablished (Bajoghli 2013) Thus current nomenclature for teleostCRs relies on homology to mammalian receptors and where

homology is questionable different names have been suggested bydifferent authors (DeVries et al 2006 Dixon et al 2013 Liu et al2009 Nomiyama et al 2011) Based on our phylogenetic analysiswe have adopted some of the nomenclature suggested by othersbut also propose some changes to distinguish between evolution-ary and most likely functionally distinct sequences (see Table 1)

31 Phylogenetic classification

By performing a phylogenetic analysis we identified the CCR6CCR7 CCR9 CCR9B CCR13 IL8R (CXCR12) CXCR2 CXCR3a CXCR3band CXCR4 receptor sequences previously identified in salmonids(Daniels et al 1999 Dixon et al 2013 Huising et al 2003a Ordaset al 2012 Xu et al 2014) (Appendices S1 and S3) in addition to38 new sequences We and Xu et al redefined the Zhang et al (2002)CXCR12 sequence to CXCR1 while the sequence defined as CXCR3aby Xu et al (2014) clusters with the receptor we have defined asCXCR8 being quite distinct from the other sequences defined asCXCR3 Some Salmo salar (ss) sequences are clear-cut orthologuesto human CCR6 CCR7 CCR9 CXR4 CXCR7 XCR CCRL1 CCBP2 andCMKLR1 while other sequences here defined as ssCCR1 ssCCR2ssCCR3 ssCCR4 ssCCR5 ssCXCR1 ssCXCR2 ssCXCR3 ssCXCR5ssCXCR6 and ssCXCR8 show less clear-cut orthology to human CRsequences (Fig 1)

All salmon sequences cluster with zebrafish orthologues al-though some subsets of genes have expanded or contracteddifferently in the two species Based on convincing bootstrap valuesthe salmon CCR2 CCR4 and CCR5 sequences are interesting inflam-matory receptor candidates clustering with the human inflammatoryreceptors CCR1-5 and CX3CR1 We have thus defined this clade asinflammatory-like receptors group A (Fig 1) Zebrafish orthologuesto the ssCCR4 sequence are called either CCR4Lab (Nomiyama et al2011) or CCR8 (Liu et al 2009) where we propose use of CCR4 todistinguish these sequences from the highly divergent zebrafish se-quence called CCRL4c which is an orthologue to the gene we defineas ssCCR2 The salmon sequences defined as ssCCR2 and ssCCR4 forma stable clade together with the human inflammatory receptorsCCR1-5 and a similar clustering was seen when including zebrafishCR sequences (Liu et al 2009) The salmon ssCCR5 sequences forma clade with zebrafish sequences defined as either drCCR25 (Liuet al 2009) drCCR11 (Nomiyama et al 2011) or drCX3CR1 (DeVrieset al 2006) These sequences may be functional analogues to thehuman inflammatory CX3CR1 receptor but for now we have chosento use the CCR5 nomenclature Support for these genes having animportant role in inflammation also comes from zebrafish wheretwo CCR5 orthologues (zfCCR2-2 and zfCCR5 Fig 1) were shownto be uniquely expressed in immunologically important tissues (Liuet al 2009) Teleost ligands for these receptors are not defined butseveral candidates have been suggested (Montero et al 2009)

The remaining two human inflammatory receptors CXCR1 andCXCR2 cluster with the salmon ssCXCR1 and ssCXCR2 sequencesBased on convincing bootstrap values we thus chose to define thiscluster as inflammatory-like receptors group B (Fig 1) A CXCR1orthologue has previously been published in trout (Zhang et al 2002)and both ssCXCR1 and ssCXCR2 have orthologues in trout andzebrafish (Nomiyama et al 2011 Xu et al 2014) The functionaldistinction between human CXCR1 and CXCR2 sequences has beendebated (Stillie et al 2009) but recent data suggest they couple withdistinct G protein-coupled receptor kinases (GRKs) to mediate andregulate leukocyte function (Raghuwanshi et al 2012) The IL8ligand alias CXCL8 in mammals recruits polymorphonuclear leu-kocytes (neutrophils basophiles and eosinophils) to the site ofinfection and these cells express both CXCR1 and CXCR2 on thesurface In carp two lineages of CXCL8 have been describedand they both have a crucial role in recruitment of neutrophilic

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ssCCR91a ssCCR91b drCCR9a

drCCR9b ssCCR92a

ssCCR92b CR92b hsCCR9 hsCC

hsCCR7 drCCR7

ssCCR7a ssCCR7b ssCCR

hsCCR6 ssCCR62

drCCR6b drCCR6a ssCCR61a ssCCR6 1b hsCXCR6

ssCXCR6 drCCR10

hsCCRL1 ACKR4 ssCCRL11a ssCCRL11b

drCCRL1a drCCRL1b

ssCCRL12a ssCCRL12b

ssCXCR21 ssCXCR22 drCXCR1c drCdrCd CXCXCCC

hsCXCR1 hsCXCR2

ssCXCR11 ssCXCR12

dreCXCR1 ssCXCR31a ssCXCR31b

ssCXCR32 drCXCR3b

drCXCR3a ssCXCR8a ssCXCR8b drCXCR32 drCXCR32hsCCR10

hsCXCR3 hsCXCR5

ssCXCR5 drCXCR5b ssCXCR42a ssCXCR42b ssCXC

hsCXCR4 ssCXCR41a

ssCXCR41b drCXCR4a drCXCR4b

drXCR1 drXCR1b

drXCR1c ssXCR1a ssXCR1b

drXCR1Lc drXdrXC hsXCR1

ssXCR2 drCCR121 (CCR3-1) drCCR122 (CCR3-2)

drCCR123 (CCR3-3) ssCCR3a ssCCR3b

ssCCR1 drXCR1Ld

ssCCBP2 drCCBP2

hsCCBP2 (ACKR2) drCCR112 (CCR2-2) drCCR11d

drCCR11c (CCR5) drCCR111 (CCR2-1 CX3CR1)

ssCCR5a ssCCR5b ssCCR5b hsCX3CR1 ssCCR4a

ssCCR4b drCCR4La

drCCR4Lb drCCR4Lc

ssCCR2a ssCCR2b ss C

hsCCR4

hsCCRL2 (ACKR5) hsCCR1

hsCCR3 hsCCR2

hsCCR5 hsCXCR7 (ACKR3) ssCXCR71a

ssCXCR71b drCXCR7a

drCXCR7b ssCXCR72a ssCXCR72b

hsGPER1 (CMKRL2) ssCMKRL2a

ssCMKRL2b hsCMKLR1 ssCMKRL1

drCMKRL1 hsDARC (ACKR1)

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Inflammatory-like group A

XCR-like

Inflammatory-like group B

Fig 1 Phylogeny of Atlantic salmon zebrafish and human CRs Phylogenetic tree of salmon zebrafish and human chemokine receptor sequences Red font indicates salmonCCRs while some alternative zebrafish names are given in parentheses Success in percentage per 1000 bootstrap trials is shown on each node Human CCRs are shadedaccording to function ie green are inflammatory receptors pink are dual function receptors yellow are homeostatic receptors and those without background colour areatypical receptors Three clusters of salmon inflammatory-like and XCR1-like receptor sequences of particular interest are shown Sequences and references are gathered insupplementary file Appendix S1

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granulocytes during the early phase of inflammation (van der Aaet al 2010) At least one of these lineages is present in salmonidsand represents a potential ligand for the CXCR1 and CXCR2 recep-tors (Chen et al 2013) In trout this CXCL8 variant was shown tospecifically attract a monocyte-like sub-population while the un-related CC chemokine CK6 specifically attracted a macrophage-like cell sub-population (Montero et al 2008)

The receptor sequences here defined as ssCCR3 but defined asCCR13 by Dixon et al (2013) cluster with zebrafish sequencesdefined as CCR3 (Liu et al 2009) or CCR12 (Nomiyama et al 2011)(Fig 1) The ssCCR1 sequence has an orthologue defined as XCR1Ldin zebrafish that together with the CCR3 sequences mentioned earlierform a separate clade alongside salmon zebrafish and human se-quences defined as XCR1 Based on convincing bootstrap values wedefine this clade as XCR-like receptors (Fig 1) Evidence support-ing these genes as interesting candidates for further studies comesfrom zebrafish where an orthologue to the salmon CCR3 se-quences (zfCCR3-2) was found to be uniquely expressed in spleen

kidney and gills The ssXCR2 gene may be an expressed pseudogenewith a deleted N-terminal region thus disrupting the7-transmembrane structure but this needs to be verified by morethorough studies Although XCR sequences have been identified inteleosts previously (Crozat et al 2010 Nomiyama et al 2011) theXCL ligand has not been agreed upon although some candidate se-quences have been suggested (Gilligan et al 2002 Nomiyama et al2008) A salmon orthologue to the human atypical CCBP2 se-quence is phylogenetically related to the ssCCR1-5 and XCRsequences

The remaining salmon receptors cluster with dual function ho-meostatic or atypical human receptors (Fig 1) As noted previouslyin zebrafish there is clear-cut orthology between teleost CCR6 CCR7CCR9 and CCRL1 sequences (Liu et al 2009) Potential salmon ligandsfor the CCR6 CCR7 and CCR9 receptors have been suggested as CK8CK10CK12 and CK9 respectively as these ligands cluster with mam-malian CCR6 CCR7 and CCR9 ligands (Laing and Secombes 2004)However such assumptions may be misleading as sequence iden-

Table 1Atlantic salmon CR references with trout zebrafish and human orthologues

Gene name Genbank mRNATSA match

Genomic accession Zebrafish orthologue Humanorthologue

Trout ref

CCR1 DW581300 TSA AGKD030597051 5994-11468 XCR1Ld nmCCR2a DW540320 TSA AGKD030265061 9208-10254 CCR4Lc nmCCR2b nm TSA AGKD030068871 85547-86575 CCR4Lc nmCCR3a DY717613 TSA AGKD030083391 1504-2592 CCR123 (CCR3-3) nm Dixon et al 2013CCR3b ACN11153 TSA AGKD030161071 299769-300881 nm nmCCR4a EG840655 TSA AGKD030068871 (-L) 82144-83187 CCR4La+b (CCR8-12) nmCCR4b EG775179 nt AGKD030265061 4575-5630 nm nmCCR5a CX353926 TSA AGKD030068871 95781-96845 CCR11 (CCR25) nmCCR5b nm nt AGKD030265061 18558-19616 nm nmCCR61a NM001139972 TSA AGKD030625381 18246-19421 CCR6a CCR6 Dixon et al 2013CCR61b nm TSA AGKD030093511 25921-27111 nm nmCCR62 nm TSA AGKD030018471 205631-206966 CCR6b nmCCR7a DY719066 TSA AGKD030107251 286009ndash287822 CCR7 CCR7 Ordas et al 2012CCR7b DY730093 nt AGKD030250831 110755-112501 nm nmCCR91a NP001133990 TSA AGKD030187951 65371-66767 CCR9a CCR9 Daniels et al 1999CCR91b ACI34134 nt AGKD03032792119966-23112 nm nmCCR92a nm TSA AGKD030066971 9923-12219 CCR9b nmCCR92b nm TSA AGKD030040781 128853-129911 nm nm Dixon et al 2013XCR1a nm TSA AGKD030108591 38153-39160 XCR1+b + c and XCR1Lc XCR1XCR1b nm nt AGKD030076071 36321-37340 nm nmXCR2 nm TSA AGKD030043711 4805-5718 nm nmCXCR11 DY725174 TSA AGKD030045131 488-1558 nm CXCR12 Zhang et al 2002CXCR12 nm TSA AGKD030091181 8235-9317 CXCR1 nmCXCR21 DW566408 nt AGKD030375221 6239-7318 CXCR1c nm Xu et al 2014CXCR22 nm TSA AGKD030021101 138592-139671 nmCXCR31a NP001133965 TSA AGKD030391321 5838-7407 CXCR3a+b nmCXCR31b nm nt AGKD030424941 23419-24848 nm nmCXCR32 DY730916 TSA AGKD030391341 22614-23927 nm nm Xu et al 2014CXCR41a NP001158765 nt AGKD030132151 347401-348477 CXCR4a+b CXCR4CXCR41b EG7564891 TSA AGKD030050531 292622-293620 CXCR4 Daniels et al 1999CXCR42a CK898894 TSA AGKD030322161 4028ndash5086 nm nmCXCR42b nm TSA AGKD030035791 94798-95853 nm nmCXCR5 nm TSA AGKD030594121 57-1929 CXCR5b CXCR5CXCR6 nm TSA AGKD030144711 118094ndash119377 CCR10 CXCR6CXCR71a nm nt AGKD030006751 40646-41776 CXCR7a CXCR7CXCR71b nm TSA AGKD030233091 66937-68070 nm nmCXCR72a nm TSA AGKD030047171 23331- 24786 CXCR7b nmCXCR72bᴪ nm nt pseudogene AGKD030744181 66ndash842 nmCXCR8a nm TSA AGKD030391341 41583-42698 CXCR32 nm Xu et al 2014CXCR8b GE781327 nt AGKD030424941 6656-7762 nm nmCCRL11a nm TSA AGKD030189281 5621-6703 CCRL1a CCRL1CCRL11b EG877626 TSA AGKD030145861 46887-47990 nm nmCCRL12a nm nt AGKD030040351 162432-163631 CCRL1b nmCCRL12bᴪ nm nt pseudogene AGKD030375671 309437-310606 nmCCBP2 DW566026 TSA AGKD030273891 155215-156330 CCBP2 CCBP2CMKLR1 GE7728933 TSA AGKD030141151 214415-215431 CMKLR1 CMKLR1CMKLR2a DY7319832 TSA AGKD030041791 202945-204021 nm nmCMKLR2bᴪ nm TSA pseudogene AGKD030304771 28926-29958 nm nm

Identical coloured genes are linked within contigs Abbreviation nm means no matching EST or orthologue while nt defines no matching shotgun transcript Ref is ref-erence and ᴪ defines likely pseudogenes Zebrafish orthologues in parentheses derive from Liu et al 2009 The trout CCR7 and CXCR8 sequences may be either a or b orthologues

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tity between mammalian and teleost chemokines is low This isexemplified by trout CK12 that shows a weak phylogenetic clus-tering with the human CCR7 ligands CCL1921 But when studiedin further detail CK12 was in fact shown to be a chemokine pro-duced by epithelial cells of mucosal tissues through which theseperipheral tissues recruit immature B- and T-like lymphocytes(Montero et al 2011) Thus although weakly similar to humanCCL1921 in phylogenetic studies in functional studies CK12 behavesmore like the human CCL6 CCL14 CCL15 chemokines

Salmon sequences defined as CXCR3 CXCR5 and CXCR8 clusterweakly with human CXCR3 CXCR5 and CCR10 (Fig 1) but there areno clear-cut orthology between these teleost and human se-quences The salmon CXCR6 sequence variably clusters with eitherthe human CXCR6 or the human CCR10 sequences The salmonCXCR4 sequence forms a clade with other CXCR4 sequences clus-tering either with CXCR7CMKLR or CXCR3-5 sequences dependingon which sequences are included The CXCR4 ligand CXCL12 is fairlyconserved between mammals and teleosts suggesting potentiallysimilar functional roles in for instance organogenesis or in brain func-tion (Diotel et al 2010 Sasado et al 2008 Verburg-van Kemenadeet al 2013) Interestingly in zebrafish the CXCL12 gene is dupli-cated and the two a and b variants were shown to have acquireddifferent functions primarily due to one amino acid difference(Boldajipour et al 2011) Additional CXC ligands have been iden-tified in teleosts but the individual pairing between receptors andligands remains unclear (Chen et al 2013) Salmon orthologues tothe atypical or silent chemokine receptors CMKRL CMKLR CCRL1CXCR7 and DARC were also found (Fig 1) Sequences with convinc-ing identity to human CCRL1 have previously been described in manyteleosts (Liu et al 2009 Nomiyama et al 2011)

32 Gene organisation and regional syntenies

With a few exceptions mammalian CRs have a typical one exonopen reading frame (ORF) gene organisation This is also true forthe majority of salmon genes ie ssCCR2 ssCCR3 ssCCR5 ssCCR6CXCR1 CXCR2 CXCR6 CXCR7 ssXCR1 CCRL1 CMKLR1 and CCBP2all share this one exon gene structure (Appendix S2 and data notshown) The salmon genes CCR4ab CCR9 CXCR4 CXCR8 share anexon intron structure with many other zebrafish and human CRsie one or two smaller exons followed by a larger exon The tworemaining salmon receptors ie CCR7 and CXCR3 both have aquite unusual gene structure where both genes have an introndividing the larger ORF which in ssCCR7ab is preceded by onesmaller exon The zebrafish CXCR3 orthologue (drCXCR3bENSDARG00000007358) also has this additional intron separatingthe larger exon while the zebrafish CCR7 orthologue does not Thesalmon CCR1 gene has a more unusual gene structure with fourmedium sized exons

Some salmon genes are closely linked such as the duplicatessCXCR3ndashssCXCR8 genes and the duplicate ssCCR4ndashssCCR2ndashssCCR5genes (Table 1) Although denoted drCXCR3ab(ENSDARG00000070699 ENSDARG00000007358) and drCXCR32(ENSDARG00000041041) by Nomiyama et al (2011) these zebrafishsequences are related to the salmon ssCXCR3 and ssCXCR8 se-quences respectively (Fig 1) and are also closely linked within a 19 kbregion on zebrafish chromosome 16 Thus this linkage existed priorto the split between salmonids and cyprinids more than 250 MYA(Near et al 2012) In zebrafish the CCR4Lb (CCR82) gene is du-plicated on chromosome 16 (ENSDARG00000095789 andENSDARG00000086616) with an un-annotated orthologue to thesalmon ssCCR2 gene located 18 kb downstream (drCCR4LcXP_0026648441) The remaining salmon contigs contain one geneonly but once the salmon genome scaffolds are published more re-ceptors may be linked

33 Assessing secondary structure

There are many structural features conserved between salmonand other CR sequences including the G-coupled protein seven trans-membrane signatures CR sequences typically bear one cysteineresidue in each extracellular domain where the N-terminal and ex-tracellular loop (ECL3) cysteines (C1 and C6 Figs 2 and 3) form adisulphide bridge in all known CRs except human CXCR5 and CXCR6(Wu et al 2010) In salmon ssCXCR31a and the atypical ssCMKLRreceptors form the exceptions that lack this bridging potential Thecysteines connecting ECL1 and ECL2 are also present in all salmonCRs (C2and C3 Figs 2 and 3) except for ssCMKLR2b that is most likelya pseudogene having a 6-transmembrane domain structure Addi-tional cysteines are also found in other domains but their functionalrelevance is unclear

The DRY motif known to be important for intracellular signal-ling in classical CRs is located right after the third transmembranedomain in most salmon CRs (Figs 2 and 3) The exceptions aressCCR1 ssXCR1ab and ssCMKLR12 which should then be classi-fied as atypical CRs according to this definition However the twosalmon XCR sequences share a HRY motif with their human andmouse XCR orthologues and the salmon ssCKLMRs share a DRC motifwith their human orthologues suggesting unique intracellular sig-nalling and potentially also different functions for these orthologuesThe atypical nature of the ssCCR1 receptor remains to be estab-lished The salmon ssCCBP2 sequence on the other hand does containa DRY motif unlike its atypical human ssCCBP2 counterpart thusquestioning the atypical nature of this salmon molecule

All full-length sequences contain the typical CR seven trans-membrane domains with four potential extracellular and fourintracellular domains including the N- and C-terminal sequences(Figs 2 and 3) Most N-terminal salmon CR sequences contain fromone to three N-glycosylation sites in addition to several potentialtyrosine O-sulfation sites equivalent to that found in human CRs(Bannert et al 2001 Liu et al 2008) The exceptions are ssCCR7bssCCR91ab and ssCCR92b that completely lack N-linkedglycosylation sites but they may use O-linked glycosylation insteadSeveral salmon CRs also have predicted N-linked glycosylation sitesin intracellular and extracellular domains where for instance theECL2 domain displays fifteen such sites suggesting a functional rel-evance Dixon et al (2013) noted that the CCR6 ECL2 domain is muchlarger in teleost than in mouse and human sequences further point-ing to a potentially more complex function for this domain in teleosts

The C-terminus of salmon CR sequences contain several motifsknown to be important for intracellular signalling such as thedileucine motif known to interact with adaptor proteins found inssCCR7 ssCCR9 and ssCXCR6 sequences [DE-XXXL-(IL) (Matteraet al 2011)] Some sequences also have predicted C-terminal N-or O-linked glycosylation sites that may participate in intracellu-lar transport or regulation

34 Three R or 4R duplications

Many teleost CR genes have duplicates where some are seem-ingly unique to salmon such as CCR2 CCR3 and CCR4 (Fig 1) Othergenes show simple orthology between salmon and zebrafish suchas ssCCR1drXCR1Ld ssCXCR6drCCR10 and ssdrCXCR5 sequences(Fig 1 Table 1) Some subsets of genes have expanded andor con-tracted differently such as the four CCR11 and XCR genes in zebrafishThere are also examples of old gene duplications that occurred priorto the split between zebrafish and salmonids such as ssCCR6162 ssCCR9192 CCRL1112 and CXCR7172 However apronounced difference between the two species is the multipleyounger gene duplications observed in Atlantic salmon There are18 genes that occur in duplicate with sequence identities rangingfrom 82 to 95 (CCR2 CCR3 CCR4 CCR5 CCR61 CCR7 CCR91

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20 40 60 80 100ssCCR1 ------------------------------MMNLSESWKTMVNETSSVNDSDYTDEGYDDEKHVKLC----------DEVGGLEEVTAGCFLVIFLLSVTGNGLL 65ssCCR2a -----------------------------------------MNTTEATST-DDYSGDNYYGNMISPC-------STGTSLTQGSNYQPILFYLVFTLGLTGNSLV 56ssCCR2b -----------------------------------------MNTTEATSTDDYYGYD-------SPC-------STGTSLTQGSNYQPILFYLVFTLGMTGNSLV 50ssCCR3a -------------------------------MADYEDFLAFFNEDNFTDYNNSVDTSYVVDEMVNLC-------AKTEVNRFGAKFIPTFYTINFLLSVVGNGLV 67ssCCR3b -------------------------------MAEYKDFLDLFSDENDMDYN-YTDPIYVVDKVVNFC-------VTADVNRFGAKFTPILYTINFLLSFFGNGLV 66ssCCR4a MNTTGYPVHTTEGGNTTTIPFSSVSVENGNSSSYAYENSYSYAYGTHFADAFEVTTYDYSDYDDGIC----------EYKPHGASFLPVLYSLFFILGFLGNVLV 95ssCCR4b --------------------------------MNITGYPVHTTASTHFADAFEVTTYDYNNYDDGVC----------KYNAHGASFLPVLYSLFFILGFLGNVLV 63ssCCR5a -----------------------------------MPDKDMEPTTEYNYSSYYDDTEG-LYRSE-PC-------NTANVKEFGRVFLPTLYSLVFIVGFIGNGLV 61ssCCR5b ----------------------------------------MEPTTDYNYSAYYDGIEGLDTSEGQPC-------NNANVKEFGRVFLPTLYSLVFIVGFIGNGLV 58ssCCR61a -----------------------------------------MNHTDNGEETVNNSVAY-DYDLVEPC-------NMEDNNSVERVVRLYIHSVICILGLLGNILV 56ssCCR61b -----------------------------------------MDGTGYSESTNGITEDYGEMDYVEPC-------QMTKNNSVERVVRLYIHSVICILGLLGNILV 69ssCCR62 -------------------------------------MNEMCTDAYDYDNTENYTKDY-PDDNEYIC-------NLNPNRDMEIVIQTYFHSFICAFGFCGNALV 60ssCCR7a ----------MTAVKDIQILVPALLIWTYFETCFSQNENMTTEFTTDYTDYPTDKTDLDYDHWTQQC-------QKESNRHFRSWFMPTFYSLICFLGLVGNILV 88ssCCR7b ----------MTAVKDIRILVPALLIWTYFETCFSQNEKMTTEFITDYT---MDKTDLDYEYWTQQC-------QKESNRYFRSWFMPTFYSFICFLGLVGNILV 85ssCCR91a ------------------------------MPIIGDLVTSPMVSEVYDYDSSFTPTAGEDDLEDFMC-------DKSPVRAFRGQYEPPLYWTIIILGGLGNLTV 68ssCCR91b ------------------------------MPVIGDMVTSPMDSEVYDYDSSFTPTVGEDGLDDFMC-------DKSAVRAFRGQYEPPLYWSIVILGGLGNLTV 68ssCCR92a -------------------------------ME-WPLFTALPTDETLSGDYTDDY-GTFTETPGGLC-------DKSWGREFRALYEPPLFWLIFVLGAVGNLMV 96ssCCR92b -------------------------------MESPSSFTIFPTFETGSGDYTEDYEGGFTETPGGMC-------DKSWVREFRGLYEPPLFWLIFALGAVGNLMV 67ssXCR1a ---------------------------------------------MEYNETN-ITYDYDYDYKDEVC-------NKEGVVKFGSIATPAFFSVVTILSLAGNILV 52ssXCR1b ---------------------------------------------MGDIETNGTDYGYDDYYTDEVC-------NKAGVVKFGSIATPAFFSVVTILSLAGNILV 53ssXCR2 ---------------------------------------------------------------------------------------------------------- ssCXCR11 ------------------------------MTELEQPYVLDYDYNSTNDSYNFNITSFDLDSNTLSC-------AAQPLGPSAVIFLCVLHIAIFLLAVPGNLLV 68ssCXCR12 ------------------------------MADPNISYLLTLEDFGEYFNYTDFNTTYELDENTLIC-------DTSPISSGVTVVLCALYVLILLLAIPGNLVV 68ssCXCR21 -------------------------------------MQDMDYADSPYSDIFNCTYPPIDELKAAPC--------SVSILGLSSVGLMVTYIIVFVLSVLGNGVV 60ssCXCR22 -------------------------------------MPEMDVDLSLFVEFLNFTYPPIDELMGVPC--------NVSILGLSSVGLMITYITVFILSVMGNSVV 60ssCXCR31a ----------------------------------------MDLDLGGIFLENSTYNYDEDYVYKEEC-----SPEDGVGVRFGTVFLPMLYSLTLVLGLVGNVLV 60ssCXCR31b ----------------------------------MANVTDMDLDLGGIFLENSTYNYDEDYVYKEEC-----SPEDGVGVRFGTVFLPMLYSLTLVLGLVGNGLV 66ssCXCR32 ------------------------------MDSLTANGEKFTITISGGDLDNYYDEYNNYTDTSDTCCSTGEVCSLEEGMSFDAVFLPVFYSLTLVLGLLGNGLV 75ssCXCR41a ----------------------------MSSFYEVEHIFLDNTSYEE------SGDFDLDLGFEEPC------N-RVGGDYFQRIFLPTVYGIIFLLGIVGNGLV 64ssCXCR41b ----------------------------MSTYYETI-IFYNDNSSEE------SGDYDLG--YEEPC------N-RVSGDDFQRIFLPTVYGIIFLLGIVGNGLV 61ssCXCR42a -----------------------------MSYYEHFVIPESDYDYNDTSSGFGSGLGDFGTGFEEPC------D-QLLSPSVQRIFLPVVYGIIFTLGITGNGLV 69ssCXCR42b -----------------------------MSYYEHFVIQESDYDYNDTISGFGSGLGDFGAGFEEPC------DRELLSPSVQSIFIPVVYGFIFTLGITGNGLV 70ssCXCR5 ------------------------------MTYDKGSFEDGDLFFGFDNYSDLESPNNSSGDTEYTC------NDGAGLQLFHTVFQPLVYSLVFFLGLTGNGLM 69ssCXCR6 ------------------------------MDLTSFFDMDYDHSLATGDYFDYNDTSTRGYMLIERC-------EASEQQLTIKVFQTCVFLLVFLLGLLGNSLV 68ssCXCR71a ----------------------------------MNSFDLDELFDTWEDLNLTGLLENGTRVEMGGC-------PTAFDRSALLHSMCILYVFIFVVGLAANGLV 64ssCXCR71b ----------------------------------MSSFDLVELLDTWEDLNLTGLLENGTRVEMVGC-------PTAFDRSALLHSMCILYIFIFVVGLAANGLV 64ssCXCR72 --------------------------------MSLSVNELTELMEMWAELNFTGDNMSSHHVEALLC-------PAGFSHAAVLYTLSVLYIFIFLVGLAANTLV 66ssCXCR8a ---------------------------------MDHVKATTDYYIYEDSYN-YSPETGSSQSSGVPC-------NQDGIMDFTRSYSPVVYSLVFVLALLGNILV 64ssCXCR8b ---------------------------------MDHVNATTDYYIYEDIYNSSSSETGSSQSS-VPC-------YKDGIMDFTQSYSPVVYSLVFVLALVGNILV 64ssCCRL11a ---------------------------------------MDLVE--DYDYYDNLTLNYSYEDYHTVC-------EKADVRSFAGLFLPVVYSVCVAVGLAGNSLV 57ssCCRL11b ---------------------------------------MDLTEEDDYDYHNNLTLNYSYEDYHTVC-------EKADVRSFAGLFLPVVYGACVVVGLAGNSLV 59ssCCRL12 -----------------------MSLYSLTSQRTERMEMDEE-DYNYDFGNTSSNDSDDYDDYHSVC-------DKAEVRSFGRLFLPVVYALALVVGVAGNALV 74ssCCBP2 ------------------------------------MDLNIPELTDDYNYSHYYDYGDEPLDGFGLC-------EKAHVKVFGRIFLPISYIIICTLSIIVNILF 62ssCMKLR1 -----------------------------------------------MEDFDYKEYGEDYTADNETYENTSVSGSVTFNHPRSFSVETGINILISLLGLSGNAIV 67ssCMKRL2a -----------------------------------------MIFIAIENRMEMENSTMVYSDVTTGM-------DSVLDTRHLDIISLVVYCVAFVLGPIGNGLV 57ssCMKRL2b -------------------------------------------------------------------------MVYSDVTTGMDIISLVVYCVAFVLGPTGNGLV 32

1MT1Clanimret-N

Fig 2 Amino acid sequence alignment of Atlantic salmon CRs Amino acid alignment of all identified Atlantic salmon chemokine receptor sequences (see Appendix S1 for references) Residues in red font define transmem-brane regions while blue font residues define the DRY motif known to be involved in CR signalling (Allen et al 2007) The lacking DRY motif in CCR1 XCR and CMKLR sequences are boxed Purple shaded residues are N-linkedglycosylation sites yellow shading shows Y-linked sulfation sites green shading shows dileucine motifs important for binding to AP2 while grey shaded residues represent potential O-linked glycosylation (N-terminal) orphosphorylation sites (C-terminal) (Blom et al 2004 Borroni et al 2010) Regions and conservedsemi-conserved cysteine residues are numbered and shown below the alignment Abbreviations used are ECL = extra-cellularloop ICL = intracellular loop TM = transmembrane domain CCRL12b and CXCR72b are likely pseudogenes with no transcript support and thus not included

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120 140 160 180 200 ssCCR1 LVALCRYEG-------LRRVTNLFILNLLFSDLLFTLTLPFWAVYYL--SHWMFGDLACKLLTGAYFTGLYSSIMLLTSMTVYRCVIVVASR----WTAVPRRRL 157ssCCR2a LWVLLKYMK-------LKTMTDICLLNLALSDLLLALSLPLWAYHAQG-HEFE-GDSPCKIMAGVYQVGFYSSILFVTLMSVDRYLAIVHA-----VTAMRARTL 147ssCCR2b LWVLLKYMK-------LKTMTDICLLNLALSDLLLALSLPLWAYHAQG-HEFE-GDSPCKIMAGVYQVGFYSSILFVTLMSVDRYLAIVHA-----VAAMRARTL 141ssCCR3a LCIIYKYEK-------LTSVTNIFLLNLVISDLLFASSLPFLATYYS--SEWIFGPFMCKLVGSMYFIGFYSSILFLTLMTFDRYLAVVHA-----INAAKQRRK 158ssCCR3b LCIIYKYEK-------LTCVTNIFLLNLVISDLLFASSLPFWALYYF--YGWIFGPVMCKLVGSVYFIGFYSSILFLTLMTFDRYLAVVHA-----INAAKRRRK 157ssCCR4a LWVILLGVK-------LCSMTDVCLLNLALADLLLVCTLPFLAHHAT--DQWVFGDIMCKVVLSAYHIGFYSGIFFITLMSVDRYLAIVHA-----VYAMRARTR 186ssCCR4b LWVILLRVR-------LRSMTDVCLLNLALADLLLVCSLPFLAHHAR--HQWVFGDVMCKVVLSAYHVGFYSGIFFITLMSVDRYLAIVHA-----VYAMRARTR 154ssCCR5a VCVLVKFRR-------IRSITDLCLFNLALSDLFFIISLPFWSHYATA-AKWLLGDFMCRLVTGLYMLGFYGSIFFMVILTVDRYVVIVHA-----HTMARPRSV 153ssCCR5b VYVLVKCRR-------TRSMTDLCLLNLALSDLFFVISLPFWSHYATA-AEWLLGDFMCRLVTGLYMLGFYGSIFFMMILTVDRYVVIVHA-----HKMARLRSV 150ssCCR61a IVTYA-FYK------KAKSMTDVYLLNVAIADMLFVVALPLIIYNEQS-D-WAMGTVACKVLRGAYSVNLYSGMLLLACISTDRYIAIVQAR---RSFRLR--SL 147ssCCR61b IVTYA-FYK------KAKSMTDVYLLNVAIADMLFVAALPLIIYNEQS-D-WAMGTVACKILRGAYSINLYSGMLLLACISTDRYIAIVQAR---RSFMLRSFTL 162ssCCR62 IVTYA-FYK------KAKTMTDVYLLNVAVADLLFIVALPLIIYNEQH-D-WSMGSVACKAFRGAYSINLYSGMLLLACISRDRYISIVQAR---RSFGLRSQNL 153ssCCR7a IGTYV-YFN------RLKTGTDVFLLSLSIADLLFAVSLPLWATNSMT-E-WVLGLFICKVMHTIYKVSFYSGMFLLTSISVDRYFAISKAV---SAHRHRSKAV 181ssCCR7b IGTYV-YFN------RLKTGTDVFLLSLSIADLLFAVSLPLWATNSMT-E-WVLGLFICKAMHTIYKVSFYSGMFLLTSISVDRYFAISKAV---SAHRHRSMAV 178ssCCR91a VWIYL-HFRQ-----RLKTMTDVYLLNLAVADLFFLGTLPLWAVEATQ-G-WSFSSGLCKVTSALYKINFFSSMLLLTCISVDRYVVIVQTT---MAQNSKRQRL 162ssCCR91b VWIYL-HFHQ-----RLKTMTDVYLLNLAVADLFFLGTLPFWAVEGNQ-G-WSFGLGLCKITSALYKINFFSSMLLLTCISVDRYVVIVQTT---KAQNSKRQRL 162ssCCR92a VFIFT-TVRH-----RLKTMTDVYLLNLAVADLLFLGTLPFWAADATK-G-WMFGLSLCKLLSAIYKINFFSSMLLLTCISVDRYVAIVQVT---KAHNQKNKRL 190ssCCR92b VFIFT-TVRH-----RLKTMTDVYLLNLAVADLLFLGTLPFWAADATR-G-WVFGLGLCKILSAVYKINFFSSMLLLTCISVDRYVAIVQVT---KAHNLKNKRL 161ssXCR1a LVILAKYEN-------LKSLTNIFILNLALSDLLFTFGLPFWAAYHI--WGWTFGWLLCKTVTFVFYAGFYSSVLFLTIMTIHRYLAVVHP-----LSDHGSQRG 143ssXCR1b LVILAKYEN-------LKSLTNIFILNLALSDLVFTFGLPFWAAYHI--WGWTFSRILCKTVTFVFYAGFYSSVLFLTIMTIHRYLAVVHP-----LSDHGSQRG 144ssXCR2 -------------------MTNAFMMNLALSDLVFTCGLPFWVSYHL--SGWSYGDLTCKAVSFLFYAGYYSSGIFLILMTLHRYLAVLRPLSRLVSGPSRSQ-G 83ssCXCR11 GLVIG--FSQ-----QSLTPSDVFLFHLTVADGLLALTLPFWAANTLH-G-WIFGDFLCKCLSLVMEASFYTSILFLVCISVDRYLVIVRPAK-----SRKGRRR 159ssCXCR12 GLVIA--SSK-----QPLSPSDLYLLHLAVADFLLALTLPFWAASVTV-G-WVFGDVMCKLVSIFQEVSFYASILFLTCISVDRYLVIVRAMEA----SKAARRR 160ssCXCR21 IYVVC--CMA-----RGRTTTDIYLMHLAMADLLFSLTLPFWAVYVYS-H-WIFGTFLCKFLSGLQDAAFYSGVFLLACISVDRYLAIVKTTQ------ALAQRR 150ssCXCR22 IYVVC--CMA-----RDRTTTDVYLMHLAMADLLFSLTLPFWAVYVYS-H-WIFGTFLCKFLSGLQDAAFYCGVFLLACISVDRYLAIVKATR------ALAQRR 150ssCXCR31a LVVLVQKRR-------SWSVTDTFILHLGLADTLLLVTLPLWAVQATG--EWSFGTPLCKITGAIFTINFYCSIFLLACISLDRYLSVVHAVQ---MYSR--RKP 151ssCXCR31b LVVLVQKRR-------SWRVTDTFILHLGLADTLLLLTLPLWAVQATG--EWSFGTPLCKITGAMFTINFYCSIFLLACISLDRYLSVVHEVQ---MYSL--RKT 157ssCXCR32 LLVLVQRRR-------GWSVTDTFILHLCVADILLVLTLPFWAAQATG--EWSFGTPLCKITGAIFTINFYCGIFLLACISLDRYLSVVHAVQ---MYSR--RKP 166ssCXCR41a VTVMGYQKK------VKT-MTDKYRLHLSVADLLLVFTLPFWAVDAAS--SWYFGGFLCTTVHVIYTINLYSSVLILAFISVDRYLAVVHATN---SQTTRKRKL 157ssCXCR41b LIVMGYQKK------VKT--TDKYRLHLSVADLLFVLTLPFWAVDAAS--SWYFGGFLCTAVHMIYTINLYSSVLILAFISVDRYLAVVHATN---SQTTRTFLA 153ssCXCR42a VFVLGCQRK------ARLSLTDRYRLHLSAADLLFVLALPFWAVDAAL-GDWRVGAVMCVGVHVIYTVNLYGSVLILAFISLDRYLAVVKATV---TSTTHTRQL 164ssCXCR42b VFVLGCQRK------ARLSLTDRYRLHLSAADLLFVLALPFWAVDAAL-GDWRFGAVTCVGVHVIYTVNLYGSVLILAFISLDRYLAVVKATD---TSTTHIRQR 165ssCXCR5 LTVLLKRRG-------LLRITEIYLLHLGLADLMLLATFPFALAQVSF--GVVFGDVLCKLIGLLNRLNFLCGSLLLACIGFDRYLAIVHAIT---SLQS--RRP 160ssCXCR6 IATFVLYRRL-----RLRSMTDIFLFQLALADLLLLLTLPIQAGDTLL-GHWAFGNALCKATHASYAVNTYSGLLLLACISVDRYMVVARTQEVLR---LRSRML 164ssCXCR71a LWINIRAQHTTSSS-SPRHETHLYIAHLAAADLCVCVTLPVWVSSLAQHGHWPFSELACKLTHLLFSVNLFSSIFFLACMSVDRYLSVTRPAD---SEDGGRRRK 165ssCXCR71b LWVNVRSQRTTSSS-SPRHETHLYIAHLAAADLCVCVTLPVWVSSLAQHGHWPFGEVACKLTHLLFSVNLFSSIFFLACMSVDRYLSVTRPAD---SENGGRRRK 165ssCXCR72 VWVNLRSERN-------RFETHLYILNLAVADLCVVATLPVWVSSLLQRGHWPFGEAVCKITHLVFSVNLFGSIFFLTCMSVDRYLSVALFGD---GGNS-RRKK 160ssCXCR8a LCVLMRYRTSQTGGACSFSLTDTFLLHLAVSDLLLALTLPLFAVQWAH--LWVFGVTACKISGALFSLNRYSGILFLACISFDRYLAIVHAVS---TGWK--RNT 162ssCXCR8b LCVLMRYRTSQTGGACSFSLTDTFLLHLAVSDLLLALTLPLFAVQWAR--QWVFGVAACKISGALFSLNRYSGILFLACISFDRYLAIVHAVS---TSWK--RNT 162ssCCRL11a LSVYAYHKRL-----RR-TMMDAFLVHLAVADLLLLLTLPFWAADAAR-G-WELGLPLCKLVSACYTINFTCCMLLLACVSMDRYLASIRAEGRNHGRLGRVFTR 154ssCCRL11b LAVYAYHTRL-----RR-TMTEAFLAHLAVADLLLLLTLPFWAADAAL-G-WELGLPLCKLVSACYAINFTCCMLLLACVSMDRYLASVRAEGRNQGRLGRVFTR 156ssCCRL12 VVVYASPRRL-----R--TLTDVCILNLAVADLLLLFTLPFWAADAVH-G-WWIGVAACKLTSFLYTTNFSCGMLLLACVSVDRYRALAHNAGGRAGSGPR--DR 168ssCCBP2 ISTLIKSKHH----------RKTFPMSMAISDMLFALTLPFWAVYAHN--EWIFGNDSCKTVTAIYITTLYSSILFITCISVDRYLNVVWTLS-----SWNHCTP 150ssCMKLR1 IWISGFKMR -------TSVNTTWYLSLAISDFLFCVCLPFNIVYMVT-SHWPFGLVMCKLTSSTMFLNMFSSVFLLVLISVDRCVSITFPVW-----AQNNRTI 158ssCMKRL2a IYVTSCRIK--------KTVNSVWFLNLAMADFLFTSFLLLYIINIARGYDWPFGDILCKLNSMVNVLNMFASIFLLAAISLDRCVSTWVVVW-----AHNKCTP 149ssCMKRL2b IYVTSCRIK--------KTTNSVWFLNLALADFLFTSFLLLYIINMARGYDWPFGDILCKLNSMVTVLNMFASIFLLAAISLDRCLSTWVVVW-----AHNKCTP 124 ICL1 TM2 ECL1 C2 TM3 DRY ICL2

Fig 2 (continued)

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220 240 260 280 300 ssCCR1 RYALAACTASWVVSLAASLSDVIASQVQEV------------------------------ENGTRIFTCEVLPG-----TTDEELGYYLQVFLLFVLPLIIIILC 227ssCCR2a RYGTLASIIVWVASISAALPEAIFAAVVRE------------------------------NDENSGTSCQRIYPE-DTEKTWKLLRNLGENGVGLLLCLPIMVFC 221ssCCR2b RYGTLASIIVWVASISAALPEAIFVAVVRE------------------------------NDESSGTSCQRIYPE-DTEKTWKLLRNFGENGVGLLLCLPIMVFC 215ssCCR3a IYACVSSAVVWCISLLASVKELVLYNVWKD--------------------------------PQSGHLCEETGFSKDIMDKWELVGYYQQFVIFFLLPLAMVMYC 231ssCCR3b IYACVSSAVVWCISLLASVNELVLYNVWKD--------------------------------PRVGHLCEETGFSNEIMIKWQLVGYYQQFVIFFLFPLAMVMYC 230ssCCR4a KYGAIAAVVTWLAGFLASFPEALFLKVEKH---------------------------------NEKENCRPVY-DG---HAWGIFGLFKMNTLGLLIPLVIMGFC 254ssCCR4b KYGAIAAVVTWLAGFLASFPEALFLKVEKN---------------------------------NEKENCRPVY-DG---HSWGIFALFKRIIFGLLIPLIIMGFC 222ssCCR5a RVGVTLSLFMWAVSLCASLPTIIFTKVNNE---------------------------------SGLTTCKPEYPEG---SMWRQVSYLEMNILGLLLPLSIMVIC 222ssCCR5b RLGVTLSLFMWALSLCASLPTIIFTKVNNE---------------------------------SGLTTCKPEYPEG---SMWRQVSYLEMNVLGLLLPLSVMVIC 219ssCCR61a IYSRIICAAVWNLALLLSVPTFVYYERYVPAHSTFGN-DYDNYDYNNATTPFDLENTIFLE-EENYVVCDFRFPDNATARQMKILVPSTQMAVGFFLPLLVMGFC 250ssCCR61b LYSRIICATVWSLALLLSVPTFVYYERYVPAHSFYNVSEYGFYDYRNAMTPVGLKNPISSESEEDSVVCKFRFPDNATARQMKVLVPSTQMAVGFFLPLLVMGFC 267ssCCR62 IYSRLICTAIWALAIALSVPTVIYNER--------------------------VEETILLE--GTITVCQAQFQSNRTARLMKVLVPSLQVAMGFFLPLLAMVIC 230ssCCR7a FISKVTSVVIWVMALVFSVPEMSYTNIS-------------------------------------NKTCTPYTAGS---DQVRVGIQVSQMVLGFVLPLLIMAFC 246ssCCR7b FISKVTSVVIWVMALVFSVPEMSYTNIS-------------------------------------NKTCTPYTAGS---DQVRVAIQVSQMVLGFVLPLLIMAFC 243ssCCR91a SCSKLVCACVWLLAVALALPEFMFANVK---------------------------------ELEGRDYCTMVYWSN-QDNSTKILVLALQICMGFCLPLLVMVFC 233ssCCR91b SCSKLVCTCVWLLAVVLALPEFMFANVK---------------------------------ELDGRFYCTMVYWSN-QDNRTKILVLVLQICMGFCLPLLVMVFC 233ssCCR92a SVSKLTCLAVWIISGLLALPELIFAQVKP--------------------------------DHRGNSFCVLVYTNN-LFNRTKILVLVLQICVGFCLPLLVMVLC 262ssCCR92b FVSKLVCLAVWIISGLLALPEFIFAQVKP--------------------------------DRRGNSFCVLVYPNN-LFNRTKILVLALQVCVGFCLPLLVMVLC 233ssXCR1a CYGVTISLIIWAISFGSAVPALIFSSVQKN------------------------------PHEGDHLHCEYS------VPLWKKVSTYQQN-VFFLAAFAVMAFC 211ssXCR1b CYGVTVSLVIWVVSFGAAVPALIFSSVQEN-----------------------------PHEEDIHFYCEYW------DPLWKRVGSYQQN-VFFLAAFAVIGFC 213ssXCR2 TWSAVVSLVVWTVSLLAAMPALIFTKLIITD---------SNDLKDLLDHNNPDGPSDSPAPSGEQRYCEVA------DVSWRLWGVYQQN-ILFIVTLLVVCVC 172ssCXCR11 ACRWYACTFIWALGGALSLPALFN-EAFTPP-------------------------------SGGPTRCVER-FDLGSATHWRLATRGLRHILGFLLPLVIMVAC 231ssCXCR12 EVSWGTCATVWLVGGLLSLPGLFN-HVFLLP-------------------------------GTERMTCTES-YDPGSAEAWRLVIRVLGHTLGFLLPLTVMVVC 232ssCXCR21 HLVGIVCGAVWLGAGLLSLPAVLQREAIQLE------------------------------DLGDQSICYE-NLTASSSNQWRVFVRVLRHTLGFFLPLAVMVVC 224ssCXCR22 HLVGLVCGAVWLGAGLLSLPVALQREAIQPE------------------------------DLEGQIICFE-NLTAASSDRSRVGVRVIRHVLGFFLPLSVMVVC 224ssCXCR31a WMVQASCLSVWLLSILLSIPDWHFLESVRDTRR------------------------------DKQECVHNYPSLSQSGFDWRLASRLLYHTVGFLLPSVMLLFC 226ssCXCR31b WMVQASCLSVWLLSLLLSIPDWHFLESVRDARR------------------------------DKVECVHNYLSLSQSGFDWRLASRLLYHTVGFLLPSAVLLFC 232ssCXCR32 WMVQASCMSVWLLSILLSIPDWHFLESVRDTRR------------------------------DKQECVHNYPSLSQSGFDWRLASRLLYHTVGFLLPSAVLLFC 241ssCXCR41a LADRWIYVAVWLPAAVLTVPDIVFAT------ALD--------------------------SG-SRTICQR-IYPQKTSFYWMAAFRFQHILVGFVLPGLVILTC 228ssCXCR41b --DRVIYVAVWLPAVILTVPDTVFAT------AQN--------------------------RV-SRTICQR-IYPQETSFYWMAGFRFQHILVGFVLPGLVILTC 222ssCXCR42a LARRLVYAGAWLPAGLLAIPDMVFAR------TQE--------------------------AGEGEMVCTR-LYPPENAPLWVSLFHLQTVLVGLVVPGLVLLVC 236ssCXCR42b LARRYVYAGAWLPACLLAIPDMVFAR------TQE--------------------------AGEGEMVCAR-LYPPENAPLWVSLFHLQTVLVGLVVPGLVLLVC 237ssCXCR5 RNVHLTCLALWLVCLALSVPNAVFLS-VGESPI-----------------------------DPTQLSCFF-HSHGLHANNWDLTERLLTHVLCFFLPLGVMTYC 234ssCXCR6 TVGKLASLGVWLTALLLSLPEILFSGVER--------------------------------EQEGEAHCGMNVWV--AESWRVKTATRCAQIAGFCLPFLVMVAC 235ssCXCR71a LIRHSVCMGVWLLALVASLPDTYFLRALRS-------------------------------SQGEVVLCRP-VYPEEHPREWMVGVQLSFILLGFIIPFPIITLA 238ssCXCR71b LIRRSVCVGVWLLALVASLPDTYFLQAVRS-------------------------------SHGEVVLCRP-VYPEEHSREWMVGVQLSFILLGFVLPFPVIALA 238ssCXCR72 VVRRVICILVWLLALAASVPDTYFLQAVKS-------------------------------THSDATVCRP-VYPTDNPREWMVGIQLSFIVLGFAIPFPVIAVF 233ssCXCR8a CHAQIACALIWIVCFGLSGVDIAFRQVVKMEVGRS-------------------------GDHQGLLVCQT--VFPHSSLQWEVGMPLVNLVLGFGLPLLVMLYC 240ssCXCR8b CHAQIACALIWTVCLGLSGVDIAFRQ--KMEVGRS-------------------------GDHQGLLVCQT--VFTHSSVQWQVGMPLVNLVLGFGLPLLVMLYC 238ssCCRL11a AHCGKVCLGVWAVALLLGLPDLLFSTVSE---------------------------------TSRRRVCLA-VYPSSLAQEVKACLEMVEVLLGFLVPLLVMAWC 225ssCCRL11b AHCGKVCLGVWAVAFLLGLPDLLFSRVRE---------------------------------TPGRRVCMT-VYPPSLAREVKACLEVVEVLLGFLVPLLVMMWC 227ssCCRL12 RQWILVCAVVWTTAVCLGLPDMVFFTVKN---------------------------------TPHRLACTA-IYPSSMARPAKAALELLEVLLSFLLPFLVMVVC 239ssCCBP2 MENTLVCFVVWSLSILAAAPHWTFVQEQE---------------------------------FHGQKICMYPFGEENHLPLWKILMKFQLNVFGFLTPFLIMLFC 222ssCMKLR1 PRASGVVVLVWALSAALTVPSLVHRQIKTHG---------------------------------ADTLCYTD-YQSG-----HKAVALSRFVCGFVIPLLIIVFC 224ssCMKRL2a GRAEVICVGIWLASLVCSLPFTIFRQIMHY---------------------------------GNWTMCSY-S--ISHDSSTYRNLVVFRFLLGFLIPFLVIIGS 218ssCMKRL2b GRAEA---GTWLSSASC-------------------------------------------------WAFSSHSI----------------------------IGS 149 TM4 ECL2 C3 TM5 C4

Fig 2 (continued)

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320 340 360 380 400 420ssCCR1 YSAILRTVLVTA----T----RRRHRTVLVVFCIVVAFFVCWAPYNLFMFVSSVYTP-----VD-CGVKE-RLHVVLVVCRIVAYAHCFLNPALYMLS-HSFRRH 316ssCCR2a YISILTVLQRLR----N----SKKDRAMKLIFAIVGVFVVSWVPYNVVVFLRTLQMFDIGN--S-CEAST-QVDRAMEVTETIALAHCCVNPVIYAFVGEKFRKC 314ssCCR2b YISILTVLQRLR----N----SKKDRAMKLIFAIVGVFVVSWVPYNVVVFLQTLQMFDIGN--S-CEAST-QLDAAMEVTETIALAHCCVNPVIYAFVGEKFRKC 308ssCCR3a YVRITVRVMSTR----M----REKCRAVKLIFVIVFSFFVCWTPYNIVILLRALQMSTSHSFEP-CSD---VLDYALYVTRNIAYLYCCVSPVFYTFLGKKFQSH 324ssCCR3b YVRITVRIMSTQ----M----RGKCRAVKLIFVIIFTFFVCWTPYNVVILLRALQISTSDDSDP-CFE---VLNYALYVTRNIAYLYCCVSPVFYTFVGKKFQSH 323ssCCR4a YTQIVKRLLSCP----S----SKKQ-TIRLILIVVVVFFCCWTPYNMTAFFKALELSEVYS--S-CESSK-AIRLTLQITEAMAYSHSWLNPILYVFVGQKFRRP 346ssCCR4b YTQIVRRLLSAP----S----SKKQ-AIRLILIVVVVFFCCWTPYNMTAFFKALELSEVYS--S-CESSK-AIRLTLQITEAMAYSHSCLNPILYVFVGQKFRRP 314ssCCR5a YSRIVPMLVTIK----T----TKKHKAIKLIIIIVVVFFCFWTPYNVVILLRYLETQSYFG--D-CTTHT-NIDLAMQCTEVIAFTHCCLNPIIYAFAGQKFMSL 315ssCCR5b YSRIVPMLVNIK----T----TKKHKAIKLIIIIVVVFFCFWTPYNVVIVLRYLEAQSYFG--D-CITHK-NIDLAMQWTEVIAFTHCCLNPIIYAFVGQKFTSL 312ssCCR61a YANIIVTLLRAK----N----FQRHKAVRVVLAVVVVFIICHLPYNAALLYDTINKFK--ILP--CSQVD-ATEVAKTVTETVAYLHCCLNPVLYAFIGVKFRNH 342ssCCR61b YASVIITLLRVK----N----FQRHKAVRVVLAVVVVFIACHLPYNAALLYDTVHMFK--PQL--CGEID-TTQVAKTVTETVAYLHCCLNPVLYAFIGVRFRNH 359ssCCR62 YASILWTLLRAQ----S----TQRHKAVRVVLAVVVVFIVCHLPYNVVLLYHTVALFQ--QRE--CEVEN-IILTTLTITRSLAYLHCCLNPILYAFIGVKFRSR 322ssCCR7a YGAIVKTLCQAR----S----FEKNKAIKVIFAVVAVFLLCQVPYNLVLLLTTLDTAKGGSKD--CIYDN-SLLYASDITQCLAFMRCCLNPFVYAFIGVKFRRD 340ssCCR7b YGAIVKTLCQAR----S----FEKNKAIKVIFTLVAVFLLCQVPYNLVLLLTTLDAAKGGSKD--CIYDN-SLLYASDITQCLAFLRCCLNPFVYAFIGVKFRRD 337ssCCR91a YAGIIRTLLKTR----S----FQKHKALRVILVVVAVFVLSQLPYNTVLVMEATQAANSTETD--CSAAK-RFDVVGQMLKSLAYTHACLNPFLYVFVGVRFRRD 327ssCCR91b YAGIIRTLLKTR----N----FKKHKALRVIMVVVVVFVLSQLPYNSVLVVEATKAVNSTGMD--CDAEK-RFDVVGQVLKSLAYMHASLNPFLYVFVGERFRRD 327ssCCR92a YSVIIRTLLQAK----S----FEKHKALRVIFAVVAVFVLSQLPYNGLLVVNATQAADTTITD--CAVSE-HFDVAGQIAKSLAYTHACINPFLYVFIGVRFQKD 356ssCCR92b YSVIIRTLLQAK----S----FEKHKALRVIFAVVAVFVLSQLPYNGLLVVDATQAANTTITD--CAISG-HFDVAGQIAKSLAYTHACINPFLYVFIGVRFRKD 327ssXCR1a YVRILAAIFKSR----S----HMRNRTMNLIFSIVAVFFLGWAPYNVVIFLRLLTDHSVAPFND-CEVSM-KLDYGFYVCRLIAFSHCCLNPVFYAFVGIKFRNH 306ssXCR1b YVRILRTIFKSR----S----HMRNRTVKLIFSIVAVFFLGWAPYNVVIFLRLLHDYTVAPFNT-CEVST-WLDYGFYVCRLIAFSHCCLNPVFYAFVGIKFRNH 308ssXCR2 YSQIVVRLLRPRVRVRRQRSGGDSRSQRTARLVLGLVLVFFVGWAPYNVVIFLRTLVYKSQDGGGVGQCCVILNTMGGVWHQQYVGLLLLCDQAAGVLLLLSQPT 277ssCXCR11 YSITVSRLLQ-T----SG---FQKHRAMRVIIAVVFAFLLCWTPFHMTVMADTLMRARLVRFD--CAERN-RVDLALQVTHSLALVHSFVNPVLYAFVGEKFRGN 325ssCXCR12 YGVIVARLLR-T----RGG--FQRNRAMRVIVALVLAFLLCWMPYHLAVMADTLFWAKVVGYG--CRERS-AVDTAMFATQSLGLLHSCVNPVLYAFVGEKFRRR 327ssCXCR21 YSCTAATMFRGM----RNG--DHKHKAMRVILAVVLAFVMCWLPCNVSVLVDTLMRSGSLGEET-CEFRN-SVSVALYVTKVIAFTHCAVNPVLYAFIGQKFRNQ 321ssCXCR22 YSCTAVTLFRGV----RNG--GQKHKAMRVILAVVLAFVACWLPRNISVLVDTLMRSGSLGEET-CEFQN-NVSVALYVTEVMAFTHCAVNPVLYAFIGQKFRNQ 321ssCXCR31a YSCILLRLQ-------RGSVGLQKQRAVQVILVLVLVFFLCWTPYNITLMVGTFQGRPGEPVSGSYENGRTALENSLVVTFALACLHACLNPVLHLGLCRNFRRH 324ssCXCR31b YSCILLQLQ-------RGSQGLQKQRAVRVILALVLVFFLCWTPYNITLMVDTFQGRPGEPVSVSCENGRTAVEKSLIVTFALACLHACLNPVLHLGLCRNFRRR 330ssCXCR32 YSCILLQLQ-------RGSQSLQKQRAVRVILALVLVFFLCWTPYNITLMVDTLYSN-STLVDT-CE-SRKALDISLTATSSLGYLHCSLNPVLYAFVGVKFRHH 336ssCXCR41a YCIIIAKLSQG-----AKG-QVLKRKALKTTVILILCFFSCWLPYCVGIFVDTLMLLNVISHN--CALEQ-SLQTWILITEALAYFHCCLNPILYAFLGVKFKKS 324ssCXCR41b YCIIIAKLSQG-----SKG-QVLKRKALKTTVILVLCFFSCWLPYCVGIFVDTLMLLNVISHS--CALEQ-SLQTWISITEALAYFHCCLNPILYAFLGVKFKKS 318ssCXCR42a YCVIVSRLTRG-----PLGGQRQKRRAVRTTVALVLCFFLCWLPYCIGIAVDALLRLELIPRG--CMLES-GLGVWLAVSEPMAYAHCCLNPLLYAFLGVGFKSS 333ssCXCR42b YCVIVSRLTRG-----PLGGQRQKRRAVRTTVALVLCFFLCWLPYCIGITVDALLRLELIPRG--CTLES-GLGLWLAVSEPMAYAHCCLNPLLYAFLGVGFKSS 334ssCXCR5 YAAVAITLHHSQ----RGQRSLEKEGAIRLAALVTAVFCLCWLPYNITMLVKTLVDRGLDSGLS-CQ-SRTSLDKALVVTESLGYTHCCLNPLLYAFTGVRFRQD 333ssCXCR6 YSLIGRLLCEGR----G-QGGWRRQRTLRLMVVLVAVFLLFQLPYTVVLSLKVAGPG-AARQT--CDQWA-ATLLREYVTCTLAYTRCCLNPLLYALVGVRFRSD 331ssCXCR71a YALLAKALSSS---FSSSAVEQERRVSRKVILAYIVVFLGCWGPYHGVLLADALSLLGLVPLS--CGLEN-ALYVALHLTQCLSLLHCCFNPILYNFINRNYRYD 337ssCXCR71b YALLAQALSSSS--CSSSAVEQDRRVSRRVILAYTVVFLGCWGPYHGVLLADALSLLGLVPLS--CGLEN-ALYVALHLTQCLSLLHCCFNPILYNFINRNYRYD 338ssCXCR72 YLLLAGAIGNANPPGSSANSNQERRISRNIILTYIVVFLVCWLPYHGVLLVDTLSLLNVLPFS--CRLEK-FLYVSLHLTQCFSLIHCCINPVIYNFINRNYRYD 335ssCXCR8a YIRIFRSLC--------NASRRQKRKSLHLIVSLVSMFVLCWAPYNSFQLVESLKKLGMISGG--CQFGR-TVDIGILVSESMGLSHCALNPLLYGFVGVKFRRE 334ssCXCR8b YIRIFRSLC--------NASRRQKRKSLHLIVSLVSMFVLCWAPYNSFQLAESLKKLGVISGG--CQFGR-TVDIGILVSESMGLSHCALNPLLYGFVGVKFRSE 332ssCCRL11a YFNVGRVLGRLP----V-ESRGRRLSAIRVLLVVVGVFVVTQLPYNTVKMYRAMDSAYTLVTH--CGVSK-ALDRAAQVTESLALTHCCLNPLLYAFLGSSFRKH 322ssCCRL11b YAGVGRVLRRLP----E-ESRGRRRRAIRVLLVVVGLFVVTQLPYNAVKMCRAMDSVYTLVTH--CGVSK-ALDRAAQVTESLALTHCCLNPLLYVFLGSSFRQY 324ssCCRL12 YCWVGRALVRIG----AGVRREKRWRALRVLLAVVGVFLFTQLPYNLVKLWRTLDVIYGLVTD--CDLSK-GLDQALQVTESLALTHCCINPMLYAFIGSSFRGY 337ssCCBP2 YLRVCCAVAKVK--------VGPRRKSLKLVMIVVVVFFVLWFPYNIVSFLHSLQHLHAIYN---CATSL-HLDFAIQVTEVIAYSHGFVNPIVYAFVNKRVWKG 315ssCMKLR1 YSVIFVQLRSRP---------MKSTKPVKVMTVLIVSFFVCWVPYHTFVLLEVNLGNHSLE----------MLYTWLKVGSTMAAANSFLNPILYVLMGHDFRQT 310ssCMKRL2a YIAIWIRARRLQ----R----GTTRRSLRIIVSVVLAFFICWMPFHVLQFLDIMANG--------SPGLNLVVHIVIPLSTSLAYLNSCLNPILYVFMCDEFQKK 307ssCMKRL2b YIAIWIRAKRLQ----R----GRTCRSLRTIVSVVLAFFICWMPFHVFQFMDIMEED--------NQGLELVVHIGIPLSASLAYLNSCLNPILYVFMCDEFQKK 238 ICL3 TM6 C5 ECL3 C6 TM7 C7-8

Fig 2 (continued)

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440 460 480 500 520ssCCR1 LWSLL------CCLMGEERGGQAGGGERSVGYNMHHITPRPKRTSFGVSGP----------------------------------------------- 361ssCCR2a LGTALSRY--PLCKKLSKHAMVSSRGSENETSNTPV-------------------------------------------------------------- 348ssCCR2b LGTVLSRY--PLCKKLSKHAMVSSRGSENETSNTPV-------------------------------------------------------------- 342ssCCR3a FRKLLAKH--IPCLKSYIDTNQSSQSRTTSQKSPHTMYEY---------------------------------------------------------- 362ssCCR3b FRKLLAKR--IPCLKRHIPTSQNSNSRITSQKSPHNTYEYEKGTGLQTRV------------------------------------------------ 371ssCCR4a LIRLINKAPRRMCQFMKNYLPWDFRASRTGSVYSQTTSMDERSTAV---------------------------------------------------- 392ssCCR4b LIRLINKAPCRMCQFMKNYLPRDFRVSRTGSIYSQTTSMDERSTAVGTAT------------------------------------------------ 364ssCCR5a VLKLLRKWMP-MCFARPYVCGLSERNISVYSRSSEISSTRLL-------------------------------------------------------- 356ssCCR5b VLKLLRKWMP-FCFARPNVSELPEQKSSVYSRSSEITSTRLL-------------------------------------------------------- 353ssCCR61a FRKIVEDVW---CIGKRVMNPRRFSRVTSEMYVSTVRKSMDGSSTDNASSFTM--------------------------------------------- 392ssCCR61b FKKIVEDVW---CVGKRVMNVRRFTRVKSEIYVSTARRSVDGSSTDNASSFTM--------------------------------------------- 409ssCCR62 FRKILEDLW---CMGRKYIYPSGRSSRMTSDLYIPAHKSSDGSNKNGSSFTM---------------------------------------------- 371ssCCR7a LLKLLKDLG---CMSQERFFQYTCGKRRSSAVAMETETTTTFSP------------------------------------------------------ 381ssCCR7b LLKLLKDLG---CMSQERFFQYTCGKK-SSAAAMETETTTTFSP------------------------------------------------------ 377ssCCR91a ILKLLRIYH---CWPAKGKLSKIQGGPGRSSVMSDTDTTQALSL------------------------------------------------------ 368ssCCR91b ILKLIRIYH---CWPAQGVLSKIQGGPGRSSVMSDTDTTQALSL------------------------------------------------------ 368ssCCR92a LLRLLKLCT---CGLSQGGVSKLQAIPKRPSVMSDTETTCALAL------------------------------------------------------ 397ssCCR92b LLRLLRQYT---CGLNQRGLSKMQAVPKRPSVMSDTETTPALSL------------------------------------------------------ 368ssXCR1a LKVILQEH----CRRQSTIDSQQIRAIP--SRGSMY-------------------------------------------------------------- 336ssXCR1b LKVILLKL----CRRQSTMDTQQIRLPNIYSMGSMY-------------------------------------------------------------- 340ssXCR2 VLRVRWGQVPEPPEENVEGLLSRCYRCQ---------------------------------------------------------------------- 305ssCXCR11 LGALVRKS-RGPERGSSSRFSRSTSQTSEGNGLL---------------------------------------------------------------- 358ssCXCR12 LLQMFQKAGVMEQRASLTRASRYFSQTSEATSTFM--------------------------------------------------------------- 362ssCXCR21 FLLTLHKHELISKRVLAAYRRGSAHSTVSQRSRNTSVSL----------------------------------------------------------- 360ssCXCR22 LLVVLYKHGLISKRLMVAYRSGSANSTASQRSRNTSVTL----------------------------------------------------------- 360ssCXCR31a VLDMMR------CVEGVQNDPKLSLWDSGVVEDSPDLAEEKGTLNPITTMGQVQSTQS---------------------------------------- 376ssCXCR31b VLDMVR------CVEGVQDDPKLSLWDSGVVEDSPDQAEEKGTLNPMTTMGQVVEASCSVGLSDAVH------------------------------- 391ssCXCR32 LLDMLRSLG---CKLKSGVRLQTASRRSSMWSESGDTSHTSAIY------------------------------------------------------ 377ssCXCR41a ARNALTVSSRSSHKVLTKKR-GPISSVSTESESSSVLYS----------------------------------------------------------- 362ssCXCR41b ARNALTFSSRSSHKILTKKR-GPISSVSTESESSSALSS----------------------------------------------------------- 356ssCXCR42a ARRALTLTRTSSLKIVPRRRTGAMTSTTTESESSSLHSS----------------------------------------------------------- 372ssCXCR42b ARRALTLTRMSSLKILPRRRTGATTSTTTESESSSLHSS----------------------------------------------------------- 373ssCXCR5 LLRLLAH------------------------------------------------------------------------------------------- 340ssCXCR6 VLKLLHGVG-CLCWAVSGPHLESCTSGSPSSLGLTTLSPLPPTSPLLLPPETLAHSIKYQPPTASHLSGPTKVFLFSSRPTLPSDGLLQSTVFKTKPV 428ssCXCR71a LMKAFIFKYSTRTGLARLIEQTHVSETEYSAVAVENTPQI---------------------------------------------------------- 377ssCXCR71b LMKAFIFKYSTRTGLTRLIEQPHVSETEYSAVAVENPPQI---------------------------------------------------------- 378ssCXCR72 LMKAFIFKYSTKTGLAKLIDASHVSETEYSAVAAVENNV----------------------------------------------------------- 374ssCXCR8a LTRM--------CKGLLGQRFYTGMNGWGGQSRARRTTGSFSSAESENTSHFSVMA------------------------------------------ 382ssCXCR8b LTRM--------CKGLLGQRFYPGMKGWGGQRRTRRPTGSFSSAESENTSHFSVMA------------------------------------------ 380ssCCRL11a VLKAAKAFGERTRRR-----EEQPVEMSFNNSQAASQETSAFSI------------------------------------------------------ 361ssCCRL11b VLKAAKAFGERTKRRRGEQREDEGMEMSFNSHNTASQETSTFSI------------------------------------------------------ 368ssCCRL12 VLRVAKSLGQRLGGRMRLGGRMRGGRHGNEEPAVEISLNTHNSAGHTHSHSVSEDEDTSTFTI----------------------------------- 400ssCCBP2 FAKM----CGGKCRRRTSDEYVLECSDSTKSMSVQSGVIELQAVQSYLENNTNQPTNTERR------------------------------------- 372ssCMKLR1 LKRSVLWKIENAMAEDGRTGGRNLSKSGSFESKAFTHV------------------------------------------------------------ 339ssCMKRL2a LRQSVLLVFENAFAEDHGMNFVSSTRSLSSHLSRISRKSESLAPGEGGHLRLTGDQSDSKVETEV--------------------------------- 372ssCMKRL2b LRQSVLLVFENAFAEDHGMNFVSSTRSLSSHLSRISRKSESLAPGEGGHLRLTGDQSDSKVETEV--------------------------------- 303 C9 C-terminal

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CCR92 XCR1 CXCR31 CXCR41 CXCR42 CXCR71 CXCR72 CXCR8CCRL11 CCRL12 and CMKLR2 Fig 1 Appendix S2) representing po-tential remnants of the unique salmonid WGD often defined as the4R WGD that occurred approximately 95 MYA (Macqueen et al2013)

The range of 82ndash95 identity between duplicates (Appendix S2)seems surprisingly broad assuming these genes all originated as aresult of the 4R WGD To test if some of these duplications had arisenprior to the 4R WGD we used cDNA and genomic resources fromNorthern pike [Esox lucius Esociformes (Rondeau et al 2014)] toidentify pike orthologues to salmon CRs As pike belongs to a diploidsister group of salmonids (Carmona-Antonanzas et al 2013) anygene that was duplicated prior to the 4R WGD should also appearin the Northern pike data as duplicates Initially we investigated pikecDNA (Leong et al 2010) and found orthologues to most salmonCRs with the exception of elCCR5 and elCXCR5 All salmon dupli-cates appeared as single sequences in Northern pike (Fig 4)suggesting that the salmon duplicates originated as a result of the4R WGD As one could argue that both duplicates may not be ex-pressed in pike we looked at genomic DNA for three pike genesWe found one variant only for elCCR5 (GenBank accession AZJR010402421) elCCR6 (AZJR010343871) and elXCR(AZJR010312231) further supporting the 4R origin of the eigh-teen salmon duplications

To investigate if some of these genes were duplicated after the4R WGD such as the CCR7 and CXCR8 with sequence identitiesbetween 93 and 95 we looked at trout ESTs As we found ex-pressed trout orthologues of both CCR7ab and CXCR8ab (data notshown) it seems that all the duplications occurred at the same timebut the genes have since evolved at different evolutionary rates Ex-amples are CCR3 and CCR6 that have sequence identities of 81ndash82 as opposed to CCR7 and CXCR8 that have 93ndash95 sequenceidentities Considering the phylogenetic clustering of these recep-

tors it makes sense that ssCCR3 and ssCCR6 clustering with humanXCR1 and dual function CCR6 receptors evolve faster due to poten-tial coevolution with pathogens than ssCCR7 and ssCXCR8 clusteringwith human homeostatic receptors CCR7 and CXCR5 To discrimi-nate between copies originating from the 4R WGD versus otherduplications we follow the previously introduced terminology of-a and -b for 4R WGD duplicates (Lukacs et al 2010 Shiina et al2005) as opposed to 1 and 2 for more divergent duplicates

35 Expression patterns

Gene duplications are often followed by silencing or diversifi-cations events leaving the question as to how many of the duplicatedgenes are still functional in Atlantic salmon To address this we firstperformed a thorough search of expressed GenBank resources Wefound expressed match for 24 salmon CR genes leaving 24 genesas potential pseudogenes (see Table 1) Subsequently we thenanalysed salmon CR expression under normal physiological condi-tions using RNAseq transcriptomes from various tissues As expecteddue to sheer number of sequences we found expression of sixteenadditional salmon CR genes providing expressed support for 40 ofthe 48 receptors ignoring the match for CMKLR2b being a tran-scribed pseudogene (Table 2) We did not find expressed signatureof the receptors ssCCR5b ssXCR1b ssCXCR31b ssCXCR71assCXCR72b and ssCCRL12ab suggesting they are either rarely ex-pressed or silenced pseudogenes

In teleosts head kidney (HK) has a role similar to mammalianbone marrow while the functions of mammalian lymph nodes areperformed by teleost spleen HK and most likely gills (Haugarvollet al 2008 Uribe et al 2011) This is consistent with the fact thatgills HKkidney and spleen contain most expressed CRs but also thehighest number of CR transcripts dominated by orthologues to thehuman homing receptors CCR7 CCR9 and CXCR4 Non-

Fig 3 Secondary structure of a chemokine receptor Predicted secondary structure of a salmon seven-transmembrane chemokine receptor using ssCCR3a as a model Ex-tracellular N-terminal transmembrane (cylinders) extracellular loop (ECL) intracellular loop (ICL) and intracellular C-terminal regions are shown Contact font colour codesare red for helix contact while green is membrane contact Numbered cysteines are boxed in pink and potential cysteine bonds are shown with double red lines The con-served DRY motif is boxed blue The enlarged ECL2 domain of teleost CCR6 sequences is shown with a green loop and membrane orientation is shown with IN and OUT

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ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71

elCXCR8 ssCXCR8a ssCXCR8b

100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100

elCMKRL3 elCMKRL2 ssCMKRL2a

ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100

elCCR32 elCCR3 ssCCR3a

ssCCR3b 80

100 100

elXCR1 ssXCR1a ssXCR1b

100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54

elCCR2 ssCCR2a ssCCR2b

100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35

elCCRL11 ssCCRL11a ssCCRL11b

100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100

elCXCR2 ssCXCR21 ssCXCR22

97 100

99

45

Fig 4 Phylogenetic tree of salmon and northern pike CR sequences The pike sequences are all cDNA sequences with the exception of elCCR5 (Appendix S1) Salmon genesare shown in red font and pike genes in black font Unique pike duplicate sequences are shown with green shading Success in percentage per 1000 bootstrap trials is shownon each node

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immunologically important tissues such as brain eye and heart alsoexpress many CR genes but at lower levels with the exception ofCXCR7 Older duplicates such as CCR61bCCR62 CXCR11CXCR12and CXCR41bCXCR42a display differences in expression patternsconsistent with the time frame they have had to acquire differentfunctional roles However some 4R duplicates have different ex-pression patterns such as ssCCR92ab and CCRL11ab suggestingdiversification also of these more recent duplicates Some poten-tial salmon ligands are also duplicated such as the CK8ab andCK12ab chemokines potentially interacting with the duplicate CCR6and CCR7 receptors (Laing and Secombes 2004)

As the tissue transcriptomes all originated from one fish wedecided to investigate expression of some CR genes using real-time RT-PCR Also in this study ssCCR7 had the overall highestexpression restricted to spleen HK and gills (Appendix S2) Fur-thermore the results from the RT-PCR showed that ssCCR1 washighly expressed in spleen and gills as opposed to the transcriptomestudy where CCR1 had very low expression in these tissues XCR1also showed a difference with only gills as a major organ for tran-scription using RT-PCR while the transcriptome study also showedhigh expression in HK and spleen The difference between the twostudies may be due to immune status andor genetic backgroundof the included animals In the RT-PCR study we also pooled mRNAfrom three Norwegian fishes while the transcriptomes originate fromone Canadian fish

When we compared expression patterns between different teleostgroups we also found major differences and some similarities Forinstance zebrafish CCR7 had the highest expression in brain andgills (Liu et al 2009) while salmon displayed low ssCCR7a expres-sion in brain A zebrafish analogue to the salmon ssCCR4 sequence(zfCCR8-2) was primarily expressed in the brain with minute ex-pression in other tissues as opposed to the salmon orthologue whichhad highest expression in HK and spleen In contrast the sug-gested zebrafish inflammatory receptors zfCCR2-2zfCCR5 andzfCCR3-2 were highly expressed in spleen HK and gills where thesalmon orthologues ssCCR5a and ssCCR3ab sequence displayedmedium expression levels Equivalents to ssCCR1 ssCCR2 and ssXCR1were not included in the study by Liu et al (2009) Without datafrom more individuals and different physiological conditions it isnot possible to evaluate if the intra- and inter-species differencesare true or just a product of small sample size

36 Functional diversification

Six of the 4R duplicates may have been silenced ssCCR5b XCR1bssCXCR31b ssCXCR71a ssCXCR72bψ and ssCCRL12abψ are notfound in GenBank or tissue transcriptomes but they may still betranscribed in specialised tissues or under specific biological con-ditions Other genes seem to be in the process of becoming silencedCMKLR2b for example is expressed but has a 6-transmembrane

Table 2Expression patterns of chemokine receptors in Atlantic salmon tissue transcriptomes

Gene Brain Eye Gills Gut HK Kidney Heart Liver Muscle P caecum Spleen Querylength

CCR1 009 0 059 009 128 097 007 0 0 010 546 361CCR2a 0 0 041 0 203 028 0 0 0 011 206 348CCR2b 004 0 031 018 362 059 0 0 0 005 348 342CCR3a 004 034 015 019 736 384 011 0 033 014 193 346CCR3b 024 027 169 030 609 163 024 011 085 011 524 371CCR4a 012 004 122 086 641 234 004 032 034 041 871 392CCR5a 009 005 150 097 2397 730 019 021 021 027 2290 356CCR61a 048 005 310 329 072 075 009 005 026 446 237 392CCR61b 155 022 139 040 123 059 038 017 031 029 083 392CCR62 0 005 309 012 006 023 0 0 006 004 004 392CCR7a 103 030 3303 1871 9374 2078 191 186 172 778 11072 381CCR91a 056 066 1992 403 6244 1718 049 027 192 075 4378 368CCR92a 0 014 305 012 025 0 087 128 025 022 0 397CCR92b 0 019 1204 1190 1305 391 050 052 050 328 576 340XCR1a 004 0 326 052 216 052 020 009 027 038 185 336XCR2 021 0 035 064 049 0 0 0 0 028 017 305CXCR11 008 0 016 085 1171 384 0 0 0 005 197 358CXCR12 0 006 063 030 569 251 0 0 0 0 006 362CXCR22 0 081 088 074 275 189 0 0 010 017 025 360CXCR31a 0 057 132 187 489 109 005 032 062 067 1016 376CXCR32 042 008 158 034 1271 206 015 026 005 030 1476 393CXCR41b 156 195 1997 415 19918 7916 164 098 367 121 11502 362CXCR42a 0 044 053 020 506 122 0 018 040 0 462 372CXCR42b 004 067 180 051 613 233 040 0 052 010 343 372CXCR5 0 0 011 005 215 036 005 0 0 0 220 179CXCR6 039 014 121 013 028 036 026 004 019 004 022 428CXCR71b 1313 159 2069 274 088 285 805 037 211 159 410 378CXCR72a 808 1011 1136 886 358 494 1767 174 650 598 947 378CXCR8a 028 013 652 073 1541 691 040 006 115 107 1238 382CCRL11a 0 0 132 033 0 0 0 0 023 020 023 361CCRL11b 0 045 239 067 028 067 008 004 056 011 012 361CCBP2 051 055 152 212 1273 714 119 081 190 109 365 372CMKLR1 0 007 080 037 449 246 019 011 025 016 669 339CMKLR2a 058 058 108 048 448 196 021 019 011 036 203 372CMKLR2bᴪ 060 019 048 058 482 317 021 015 042 022 195 372Total reads 58939250 60380888 59793962 59806348 59084708 61054936 58163180 58784272 61426586 61602874 60203316

Transcriptional values are given in RPKM (reads per kilobase per million mapped reads) Mapping reads back to our unpublished Atlantic salmon reference transcriptomewas done with CLC v 515 software Reads were mapped with high stringency ie greater than 95 identity over more than 90 of the total length of the query read Thetranscriptome was based on analysis of tissues of a single 1-year old individual and contained gt70000 non-redundant contigs RPKM values above 10 are shaded blue Thereceptors CCR4b CCR5b CCR7b CCR91b XCR1b CXCR21 CXCR31b CXCR41a CXCR71a CXCR8b and CCRL12a had no matching transcripts CXCR72b and CCRL12b arelikely pseudogenes while CMKLR2b is transcribed but has an error disrupting the open reading frame making it a transcribed pseudogene

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72737475767778798081828384858687888990919293949596

structure that most likely disrupts intracellular signalling But whatabout the 4R duplicates that have been retained as seemingly bona-fide expressed duplicates Are they functionally identical or havethey diversified To address these questions we investigated the se-quence variability distribution of the ssCCR3ab ssCCR61ab andssCCR92ab genes showing expression of duplicates in thetranscriptome analysis in addition to ssCCR4ab ssCCR7ab andssCCR91ab where both duplicates have matching GenBank ESTsCrystal structures of CRs suggest that the N-terminal and ECLdomains are involved in specificity and affinity docking of the ligand(Tan et al 2013 Veldkamp et al 2008 Wu et al 2010) Thus whenwe divide the sequences into transmembrane (TM) and non-transmembrane (non-TM) regions we found that 6 of all TM residuepositions and 15ndash25 of all non-TM positions were variable (Table 3)The diversity patterns match the classes we have defined for thesereceptors The potential inflammatory or dual function analoguesCCR3 CCR4 and CCR6 receptors have the highest variability in theN-terminal domain ranging from 45 to 68 The remaining threegene pairs defined as homeostatic receptors ie sCCR7 ssCCR91 andssCCR92 have lower variability in the N-terminal domain rangingfrom 13 to 25

4 Conclusion

Using the preliminary salmon genome we identified a total of48 chemokine receptors in Atlantic salmon including the ten re-ported previously Forty of these receptors seem functional withexpressed support The majority of receptors have orthologues inzebrafish while mainly the homeostatic and atypical receptors havemammalian orthologues We defined two clades with inflammatory-like salmon receptors and one clade with XCR-like receptors allpotentially important in immune responses towards pathogens Ex-pression patterns showed that a majority of the receptors areexpressed in the immunologically important tissues gills head kidneyand spleen Many salmon CRs also have roles in non-immune tissuessuch as brain and eye Eighteen of the genes exist in duplicate andwhen tested against a diploid sister group were shown to repre-sent remnants of the salmonid 4R WGD event that occurredapproximately 95 million years ago Sequence identity of 82ndash95between duplicates suggests that both diversifying as well as con-servative selection has acted upon these genes Six duplicates mayhave been silenced while others show evidence of functional di-versification The data significantly increase our knowledge of

chemokine receptors in salmonids and provide a solid foundationfor future studies defining their individual biological roles

Acknowledgement

This study was funded by the Norwegian Research Council grant206965S40 from the Havbruk program (UG HH) and partially byan NSERC grant (BFK)

Appendix Supplementary material

Supplementary data to this article can be found online atdoi101016jdci201411009

References

Alejo A Tafalla C 2011 Chemokines in teleost fish species Dev Comp Immunol35 1215ndash1222

Allen SJ Crown SE Handel TM 2007 Chemokine receptor structure interactionsand antagonism Annu Rev Immunol 25 787ndash820

Altschul SF Madden TL Schaffer AA Zhang J Zhang Z Miller W et al 1997Gapped BLAST and PSI-BLAST a new generation of protein database searchprograms Nucleic Acids Res 25 3389ndash3402

Bachelerie F Ben-Baruch A Burkhardt AM Combadiere C Farber JM GrahamGJ et al 2014 International union of pharmacology LXXXIX Update on theextended family of chemokine receptors and introducing a new nomenclaturefor atypical chemokine receptors Pharmacol Rev 66 1ndash79

Bajoghli B 2013 Evolution and function of chemokine receptors in the immunesystem of lower vertebrates Eur J Immunol 43 1686ndash1692

Bajoghli B Aghaallaei N Hess I Rode I Netuschil N Tay BH et al 2009Evolution of genetic networks underlying the emergence of thymopoiesis invertebrates Cell 138 186ndash197

Bannert N Craig S Farzan M Sogah D Santo NV Choe H et al 2001 SialylatedO-glycans and sulfated tyrosines in the NH2-terminal domain of CC chemokinereceptor 5 contribute to high affinity binding of chemokines J Exp Med 1941661ndash1673

Blom N Sicheritz-Ponten T Gupta R Gammeltoft S Brunak S 2004 Predictionof post-translational glycosylation and phosphorylation of proteins from theamino acid sequence Proteomics 4 1633ndash1649

Boldajipour B Doitsidou M Tarbashevich K Laguri C Yu SR Ries J et al 2011Cxcl12 evolution ndash subfunctionalization of a ligand through altered interactionwith the chemokine receptor Development 138 2909ndash2914

Bonecchi R Savino B Borroni EM Mantovani A Locati M 2010 Chemokinedecoy receptors structure-function and biological properties Curr Top MicrobiolImmunol 341 15ndash36

Borroni EM Mantovani A Locati M Bonecchi R 2010 Chemokine receptorsintracellular trafficking Pharmacol Ther 127 1ndash8

Burge C Karlin S 1997 Prediction of complete gene structures in human genomicDNA J Mol Biol 268 78ndash94

Cancellieri C Vacchini A Locati M Bonecchi R Borroni EM 2013 Atypicalchemokine receptors from silence to sound Biochem Soc Trans 41 231ndash236

Table 3Variability distribution of expressed 4R WGD duplicates

Gene CCR3ab CCR4ab CCR61a1b CCR7ab CCR91a1b CCR92a2b Total Vari

N-term 45 (2147) 68 (4160) 54 (2343) 15 (1175) 13 (752) 25 (1351) 35 (116328)TM1 20 (525) 4 (125) 0 (020) 5 (120) 10 (221) 5 (121) 8 (10132)ICL1 10 (110) 25 (28) 0 (09) 0 (08) 9 (111) 0 (011) 7 (457)TM2 0 (019) 5 (120) 5 (121) 0 (019) 5 (122) 0 (022) 2 (3123)ECL1 35 (617) 19 (316) 6 (118) 5 (120) 31 (516) 25 (416) 19 (20103)TM3 5 (120) 5 (120) 5 (119) 0 (019) 0 (020) 5 (120) 3 (4118)ICL2 5 (120) 0 (021) 17 (424) 10 (220) 4 (124) 8 (224) 8 (10133)TM4 4 (125) 0 (017) 10 (220) 0 (020) 10 (220) 10 (221) 6 (7123)ECL2 23 (626) 7 (227) 30 (2170) 3 (130) 12 (325) 8 (225) 17 (35203)TM5 4 (124) 17 (423) 15 (320) 0 (020) 4 (126) 8 (226) 8 (11139)ICL3 16 (319) 15 (320) 8 (112) 0 (016) 12 (217) 0 (017) 9 (9101)TM6 14 (322) 0 (023) 5 (122) 9 (222) 14 (322) 0 (022) 7 (9133)ECL3 30 (827) 0 (026) 40 (1025) 4 (126) 27 (726) 15 (427) 19 (30157)TM7 6 (117) 6 (118) 0 (019) 5 (120) 15 (320) 0 (019) 5 (6113)C-term 37 (2054) 14 (856) 18 (1055) 4 (246) 7 (346) 24 (1146) 18 (54303) variable

Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)

Percent variability calculated as number of variable residues divided by the total number of compared residues within individual domainsThe CCR4ab CCR91a1b and ssCCR7ab duplicates marked were not expressed in duplicate in the transcriptomes but ESTs for both genes were found in GenBank Thetransmembrane regions are shaded grey

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7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118

Carmona-Antonanzas G Tocher DR Taggart JB Leaver MJ 2013 An evolutionaryperspective on Elovl5 fatty acid elongase comparison of Northern pike andduplicated paralogs from Atlantic salmon BMC Evol Biol 13 85

Chang MX Sun BJ Nie P 2007 The first non-mammalian CXCR3 in a teleost fishgene and expression in blood cells and central nervous system in the grass carp(Ctenopharyngodon idella) Mol Immunol 44 1123ndash1134

Charo IF Ransohoff RM 2006 The many roles of chemokines and chemokinereceptors in inflammation N Engl J Med 354 610ndash621

Chen J Xu Q Wang T Collet B Corripio-Miyar Y Bird S et al 2013 Phylogeneticanalysis of vertebrate CXC chemokines reveals novel lineage specific groups inteleost fish Dev Comp Immunol 41 137ndash152

Crozat K Guiton R Contreras V Feuillet V Dutertre CA Ventre E et al 2010The XC chemokine receptor 1 is a conserved selective marker of mammalian cellshomologous to mouse CD8alpha+ dendritic cells J Exp Med 207 1283ndash1292

Daniels GD Zou J Charlemagne J Partula S Cunningham C Secombes CJ 1999Cloning of two chemokine receptor homologs (CXC-R4 and CC-R7) in rainbowtrout Oncorhynchus mykiss J Leukoc Biol 65 684ndash690

Davidson WS Koop BF Jones SJ Iturra P Vidal R Maass A et al 2010Sequencing the genome of the Atlantic salmon (Salmo salar) Genome Biol 11403

DeVries ME Kelvin AA Xu L Ran L Robinson J Kelvin DJ 2006 Defining theorigins and evolution of the chemokinechemokine receptor system J Immunol176 401ndash415

Di Genova A Aravena A Zapata L Gonzalez M Maass A Iturra P 2011SalmonDB a bioinformatics resource for Salmo salar and Oncorhynchus mykissDatabase (Oxford) 2011

Diotel N Vaillant C Gueguen MM Mironov S Anglade I Servili A et al 2010Cxcr4 and Cxcl12 expression in radial glial cells of the brain of adult zebrafishJ Comp Neurol 518 4855ndash4876

Dixon B Luque A Abos B Castro R Gonzalez-Torres L Tafalla C 2013 Molecularcharacterization of three novel chemokine receptors in rainbow trout(Oncorhynchus mykiss) Fish Shellfish Immunol 34 641ndash651

Felsenstein J 1985 Confidence limits on phylogenies an approach using thebootstrap Evolution 39 783ndash791

Gilligan P Brenner S Venkatesh B 2002 Fugu and human sequence comparisonidentifies novel human genes and conserved non-coding sequences Gene 29435ndash44

Gnerre S Maccallum I Przybylski D Ribeiro FJ Burton JN Walker BJ et al2011 High-quality draft assemblies of mammalian genomes from massivelyparallel sequence data Proc Natl Acad Sci USA 108 1513ndash1518

Graham GJ Locati M Mantovani A Rot A Thelen M 2012 The biochemistryand biology of the atypical chemokine receptors Immunol Lett 145 30ndash38

Gupta R Brunak S 2002 Prediction of glycosylation across the human proteomeand the correlation to protein function Pac Symp Biocomput 310ndash322

Haas BJ Papanicolaou A Yassour M Grabherr M Blood PD Bowden J et al2013 De novo transcript sequence reconstruction from RNA-seq using the Trinityplatform for reference generation and analysis Nat Protoc 8 1494ndash1512

Haugarvoll E Bjerkas I Nowak BF Hordvik I Koppang EO 2008 Identificationand characterization of a novel intraepithelial lymphoid tissue in the gills ofAtlantic salmon J Anat 213 202ndash209

Huising MO Stet RJ Kruiswijk CP Savelkoul HF Lidy Verburg-van KemenadeBM 2003a Molecular evolution of CXC chemokines extant CXC chemokinesoriginate from the CNS Trends Immunol 24 307ndash313

Huising MO Stolte E Flik G Savelkoul HF Verburg-van Kemenade BM 2003bCXC chemokines and leukocyte chemotaxis in common carp (Cyprinus carpio L)Dev Comp Immunol 27 875ndash888

Julenius K Molgaard A Gupta R Brunak S 2005 Prediction conservation analysisand structural characterization of mammalian mucin-type O-glycosylation sitesGlycobiology 15 153ndash164

Kaisho T 2012 Pathogen sensors and chemokine receptors in dendritic cell subsetsVaccine 30 7652ndash7657

Kent WJ 2002 BLAT ndash the BLAST-like alignment tool Genome Res 12 656ndash664Laing KJ Secombes CJ 2004 Trout CC chemokines comparison of their sequences

and expression patterns Mol Immunol 41 793ndash808Larkin MA Blackshields G Brown NP Chenna R McGettigan PA McWilliam

H et al 2007 Clustal W and Clustal X version 20 Bioinformatics 23 2947ndash2948Leong JS Jantzen SG von Schalburg KR Cooper GA Messmer AM Liao NY

et al 2010 Salmo salar and Esox lucius full-length cDNA sequences reveal changesin evolutionary pressures on a post-tetraploidization genome BMC Genomics11 279

Liu J Louie S Hsu W Yu KM Nicholas HB Jr Rosenquist GL 2008 Tyrosinesulfation is prevalent in human chemokine receptors important in lung diseaseAm J Respir Cell Mol Biol 38 738ndash743

Liu Y Chang MX Wu SG Nie P 2009 Characterization of C-C chemokine receptorsubfamily in teleost fish Mol Immunol 46 498ndash504

Lukacs MF Harstad H Bakke HG Beetz-Sargent M McKinnel L LubienieckiKP et al 2010 Comprehensive analysis of MHC class I genes from the U- S-and Z-lineages in Atlantic salmon BMC Genomics 11 154

Macqueen DJ Garcia de la Serrana D Johnston IA 2013 Evolution of ancientfunctions in the vertebrate insulin-like growth factor system uncovered by studyof duplicated salmonid fish genomes Mol Biol Evol 30 1060ndash1076

Mattera R Boehm M Chaudhuri R Prabhu Y Bonifacino JS 2011 Conservationand diversification of dileucine signal recognition by adaptor protein (AP) complexvariants J Biol Chem 286 2022ndash2030

Monigatti F Gasteiger E Bairoch A Jung E 2002 The Sulfinator predictingtyrosine sulfation sites in protein sequences Bioinformatics 18 769ndash770

Montero J Coll J Sevilla N Cuesta A Bols NC Tafalla C 2008 Interleukin 8and CK-6 chemokines specifically attract rainbow trout (Oncorhynchus mykiss)RTS11 monocyte-macrophage cells and have variable effects on their immunefunctions Dev Comp Immunol 32 1374ndash1384

Montero J Chaves-Pozo E Cuesta A Tafalla C 2009 Chemokine transcriptionin rainbow trout (Oncorhynchus mykiss) is differently modulated in response toviral hemorrhagic septicaemia virus (VHSV) or infectious pancreatic necrosis virus(IPNV) Fish Shellfish Immunol 27 661ndash669

Montero J Ordas MC Alejo A Gonzalez-Torres L Sevilla N Tafalla C 2011CK12 a rainbow trout chemokine with lymphocyte chemo-attractant capacityassociated to mucosal tissues Mol Immunol 48 1102ndash1113

Moser B Loetscher P 2001 Lymphocyte traffic control by chemokines NatImmunol 2 123ndash128

Near TJ Eytan RI Dornburg A Kuhn KL Moore JA Davis MP et al 2012Resolution of ray-finned fish phylogeny and timing of diversification Proc NatlAcad Sci USA 109 13698ndash13703

Neel NF Schutyser E Sai J Fan GH Richmond A 2005 Chemokine receptorinternalization and intracellular trafficking Cytokine Growth Factor Rev 16637ndash658

Nei M Kumar S 2000 Molecular Evolution and Phylogenetics Oxford UniversityPress New York

Nomiyama H Hieshima K Osada N Kato-Unoki Y Otsuka-Ono K TakegawaS et al 2008 Extensive expansion and diversification of the chemokine genefamily in zebrafish identification of a novel chemokine subfamily CX BMCGenomics 9 222

Nomiyama H Osada N Yoshie O 2011 A family tree of vertebrate chemokinereceptors for a unified nomenclature Dev Comp Immunol 35 705ndash715

Oehlers SH Flores MV Hall CJ OrsquoToole R Swift S Crosier KE et al 2010Expression of zebrafish cxcl8 (interleukin-8) and its receptors during developmentand in response to immune stimulation Dev Comp Immunol 34 352ndash359

Ordas MC Castro R Dixon B Sunyer JO Bjork S Bartholomew J et al 2012Identification of a novel CCR7 gene in rainbow trout with differential expressionin the context of mucosal or systemic infection Dev Comp Immunol 38302ndash311

Peatman E Liu Z 2007 Evolution of CC chemokines in teleost fish a case studyin gene duplication and implications for immune diversity Immunogenetics 59613ndash623

Pfaffl MW 2001 A new mathematical model for relative quantification in real-timeRT-PCR Nucleic Acids Res 29 e45

Proudfoot AE 2002 Chemokine receptors multifaceted therapeutic targets NatRev Immunol 2 106ndash115

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Rondeau EB Minkley DR Leong JS Messmer AM Jantzen JR von SchalburgKR et al 2014 The genome and linkage map of the northern pike (Esox lucius)conserved synteny revealed between the salmonid sister group and theneoteleostei PLoS ONE 9 (7) e102089

Rose A Lorenzen S Goede A Gruening B Hildebrand PW 2009 RHYTHM-aserver to predict the orientation of transmembrane helices in channels andmembrane-coils Nucleic Acids Res 37 W575ndashW580

Saitou N Nei M 1987 The neighbor-joining method a new method forreconstructing phylogenetic trees Mol Biol Evol 4 406ndash425

Sasado T Yasuoka A Abe K Mitani H Furutani-Seiki M Tanaka M et al 2008Distinct contributions of CXCR4b and CXCR7RDC1 receptor systems in regulationof PGC migration revealed by medaka mutants kazura and yanagi Dev Biol 320328ndash339

Schaffer AA Aravind L Madden TL Shavirin S Spouge JL Wolf YI et al 2001Improving the accuracy of PSI-BLAST protein database searches withcomposition-based statistics and other refinements Nucleic Acids Res 292994ndash3005

Shiina T Dijkstra JM Shimizu S Watanabe A Yanagiya K Kiryu I et al 2005Interchromosomal duplication of major histocompatibility complex class I regionsin rainbow trout (Oncorhynchus mykiss) a species with a presumably recenttetraploid ancestry Immunogenetics 56 878ndash893

Solovyev V Kosarev P Seledsov I Vorobyev D 2006 Automatic annotation ofeukaryotic genes pseudogenes and promoters Genome Biol 7 Suppl 1 (S10)11ndash12

Spidey Internet 2013 lthttpwwwncbinlmnihgovspideygtStanke M Tzvetkova A Morgenstern B 2006 AUGUSTUS at EGASP using EST

protein and genomic alignments for improved gene prediction in the humangenome Genome Biol 7 Suppl 1 (S11) 11ndash18

Stillie R Farooq SM Gordon JR Stadnyk AW 2009 The functional significancebehind expressing two IL-8 receptor types on PMN J Leukoc Biol 86 529ndash543

Szpakowska M Fievez V Arumugan K van Nuland N Schmit JC Chevigne A2012 Function diversity and therapeutic potential of the N-terminal domainof human chemokine receptors Biochem Pharmacol 84 1366ndash1380

Tamura K Peterson D Peterson N Stecher G Nei M Kumar S 2011 MEGA5molecular evolutionary genetics analysis using maximum likelihood evolutionarydistance and maximum parsimony methods Mol Biol Evol 28 2731ndash2739

Tan Q Zhu Y Li J Chen Z Han GW Kufareva I et al 2013 Structure of theCCR5 chemokine receptor-HIV entry inhibitor maraviroc complex Science 3411387ndash1390

Tran PB Miller RJ 2003 Chemokine receptors signposts to brain developmentand disease Nat Rev Neurosci 4 444ndash455

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87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172

Uribe C Folch H Enriquez R Moran G 2011 Innate and adaptive immunity inbteleost fish a review Vet Med (Praha) 56 486ndash503

van der Aa LM Chadzinska M Tijhaar E Boudinot P Verburg-van KemenadeBM 2010 CXCL8 chemokines in teleost fish two lineages with distinctexpression profiles during early phases of inflammation PLoS ONE 5 e12384

Veldkamp CT Seibert C Peterson FC De la Cruz NB Haugner JC 3rd BasnetH et al 2008 Structural basis of CXCR4 sulfotyrosine recognition by thechemokine SDF-1CXCL12 Sci Signal 1 ra4

Verburg-van Kemenade BM Van der Aa LM Chadzinska M 2013Neuroendocrine-immune interaction regulation of inflammation via G-proteincoupled receptors Gen Comp Endocrinol 188 94ndash101

Wu B Chien EY Mol CD Fenalti G Liu W Katritch V et al 2010 Structuresof the CXCR4 chemokine GPCR with small-molecule and cyclic peptideantagonists Science 330 1066ndash1071

Xu Q Li R Monte MM Jiang Y Nie P Holland JW et al 2014 Sequenceand expression analysis of rainbow trout CXCR2 CXCR3a and CXCR3baids interpretation of lineage-specific conversion loss and expansion ofthese receptors during vertebrate evolution Dev Comp Immunol 45 201ndash213

Xu QQ Chang MX Sun RH Xiao FS Nie P 2010 The first non-mammalianCXCR5 in a teleost fish molecular cloning and expression analysis in grass carp(Ctenopharyngodon idella) BMC Immunol 11 25

Yoshimura T Oppenheim JJ 2011 Chemokine-like receptor 1 (CMKLR1) andchemokine (C-C motif) receptor-like 2 (CCRL2) two multifunctional receptorswith unusual properties Exp Cell Res 317 674ndash684

Zhang H Thorgaard GH Ristow SS 2002 Molecular cloning and genomic structureof an interleukin-8 receptor-like gene from homozygous clones of rainbow trout(Oncorhynchus mykiss) Fish Shellfish Immunol 13 251ndash258

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1011121314

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Chemokine receptors in Atlantic salmon

Introduction

Material and methods

Bioinformatics

Northern pike cDNA and genomic DNA

Tissue transcriptomes and analysis

RNA extraction

Real-time PCR

Sequencing

Results and discussion

Phylogenetic classification

Gene organisation and regional syntenies

Assessing secondary structure

Three R or 4R duplications

Expression patterns

Functional diversification

Conclusion

Acknowledgement

Supplementary material

References

2006) A classic example would be a pathogen recognised by a toll-like receptor which then induces expression of secreted chemokine(Kaisho 2012) In humans the inflammatory chemokine recep-tors are CCR1-3 CCR5 CXCR1 CXCR2 and CX3CR1 The receptorsCCR4 CCR6 CCR8 CXCR3 CXCR6 and XCR1 have dual functions par-ticipating in both inflammatory as well as homeostatic processes

Most of the atypical or silent CRs also bind chemokines but thisbinding does not induce a signalling cascade with subsequent cellmigration Instead several of these receptors have regulatory rolesHuman atypical receptors are CCBP2 (D6) CCRL1 (CCX-CKR) CCRL2DARC and CXCR7 (RDC1) CCBP2 has been shown to act as a scav-enger for CC-chemokines and can drastically reduce the amount ofligand available for other CRs (Graham et al 2012) The chemokine-like receptor CMKLR1 does not bind to a chemokine but may havemultiple functions as it can regulate CCRL2 activity through com-petitive binding to the ligand chimerin (Yoshimura and Oppenheim2011) Some atypical receptors have recently been renamed to ACKRswhere CCBP2 is now ACKR2 CXCR7 is ACKR3 and CCRL1 is ACKR4(Bachelerie et al 2014)

CRs have been identified in many teleost species with the primaryfocus on teleosts with sequenced genomes ie fugu tetraodonmedaka stickleback and zebrafish (Bajoghli et al 2009 Chang et al2007 DeVries et al 2006 Diotel et al 2010 Huising et al 2003bLiu et al 2009 Nomiyama et al 2011 Oehlers et al 2010 Sasadoet al 2008 Verburg-van Kemenade et al 2013 Xu et al 2010)Results from these studies show clear-cut teleost orthologues tomammalian homeostatic receptors but orthology to mammalianinflammatory CRs is less obvious A few publications also exist onCRs in salmonids ie CCR6 CCR7 CCR99b CCR13 (CCR3) IL8R(CXCR1) CXCR2 CXCR3a CXCR3b and CXCR4 (Daniels et al 1999Dixon et al 2013 Ordas et al 2012 Xu et al 2014 Zhang et al2002) There are also a few accepted teleost chemokine receptorligand pairs such as CCR6 with CCL20-like ligands and CCR7 withCCL1921-like ligands (Laing and Secombes 2004) However thefunctional role of most fish CRs remains unresolved Understand-ing the specific function of individual receptors and identifying theirligands is essential for understanding teleost homeostasis andinflammation

To broaden our understanding of CRs in salmonids we made useof the salmon genome (Davidson et al 2010) to identify receptorsand study their evolution and potential function From a disease pre-vention point of view we paid particular attention to receptorspotentially involved in inflammation Of the 48 receptors identi-fied several have potential roles in inflammation being expressedin immunologically important tissues Most importantly thesalmonid-specific whole genome duplication event approx 95million years ago (Macqueen et al 2013) has had a significant impacton the receptor repertoire

2 Material and methods

21 Bioinformatics

Using available CR sequences from published articled andorretrieved from GenBank BLASTN (Altschul et al 1997) and TBLASTN(Schaffer et al 2001) searches were initially performed against bothexpressed and genomic Atlantic salmon resources at NCBI GenBankcGRASP [cGRASP Internet 2009 (Rondeau et al 2014)] andor theSalmonDB in Chile (Di Genova et al 2011) Identified genomic se-quences from the latest Atlantic salmon genome assembly (GenBankAGKD000000003) or from the Northern pike genome assembly(AZJR000000001) were subjected to gene prediction analysis usingGenScan (Burge and Karlin 1997) FGENESH (Solovyev et al 2006)andor Augustus (Stanke et al 2006) Predicted ORFs were testedthrough alignment with similar sequences from other speciesand sometimes changed using expressed match in Spidey (Spidey

Internet 2013) Spidey was also used to define exon intronboundaries

To assess evolutionary relationships and orthology all identi-fied amino acid sequences were aligned using ClustalX2011 (Larkinet al 2007) ClustalX was also used to calculate percentage iden-tity Phylogenetic analyses were performed using the neighbor-joining method (Saitou and Nei 1987) The percentages of replicatetrees in which the sequences clustered together in the bootstraptest (1000 replicates) are shown next to the branches (Felsenstein1985) The tree is drawn to scale with branch lengths in the sameunits as those of the evolutionary distances used to infer the phy-logenetic tree The evolutionary distances were computed using thep-distance method (Nei and Kumar 2000) and are in the units ofthe number of amino acid differences per site All ambiguous po-sitions were removed for each sequence pair Evolutionary analyseswere conducted in MEGA5 (Tamura et al 2011)

Secondary structure including transmembrane domains were pre-dicted and visualised using Rhythm (Rose et al 2009) Predictionof N-linked glycosylation sites was performed using NetNGlyc 10(Gupta and Brunak 2002) while tyrosine sulfation sites were pre-dicted using Sulfinator (Monigatti et al 2002) O-linked glycosylationsites were predicted using NetOGlyc 31 (Julenius et al 2005)

22 Northern pike cDNA and genomic DNA

Pike (Esox lucius) genome transcriptome and genetic map dataare described fully in Rondeau et al (2014) In brief DNA from asingle pike individual (Leong et al 2010) was submitted directlyto BGI (httpwwwgenomicscnenindex) for Illumina sequenc-ing DNA libraries of 180 bp were constructed for paired-endsequencing and libraries of 2 kb and 6 kb fragments were con-structed for mate-pair sequencing and assembly Fragment assemblyused ALLPATHS-LG (Gnerre et al 2011) The resulting contigs ge200 bpwere screened and trimmed for vector and contamination whichproduced 94267 contigs (N50 = 16909 bioproject PRJNA221548accession GenBankAZJR00000000) and 5688 scaffolds ge1000 bp witha total genome size of 877777613 bp

23 Tissue transcriptomes and analysis

For transcriptome data tissues were extracted from a single 1year old presmolt juvenile male Atlantic salmon RNA from 11 tissuesndash brain eye gill hind gut head kidney heart kidney liver musclestomach spleen ndash were extracted and submitted to BGI for Illuminasequencing Contig assembly used Trinity (Haas et al 2013) Theresulting set of transcripts was reduced by retaining those with asignificant BLASTX (Altschul et al 1997) match to the SwissProt orGene Ontology protein databases (le10minus5) or had a predicted openreading frame ge300 bp Further only those transcripts that mappedto our genome assembly using BLAT (Kent 2002) were retained Toremove possible alleles from our assembly we retained a singlelongest representative of transcripts that were ge98 similar over aminimum length of 300 bp as determined by BLASTN (Altschul et al1997) This curated set represents our RNA-seq referencetranscriptome FPKM values were then determined for each tran-script for each of the 11 different tissues

Pike chemokine receptors were identified using known salmongenes as queries that were BLASTed against the pike genome andtranscriptome (Rondeau et al 2014) Identified contigs were furtherexamined as earlier

24 RNA extraction

Three healthy Atlantic salmon weighing 70ndash80 g (AquaGen breed)kept in a freshwater flow system at 12 degC with regular feeding weresacrificed by overexposure to Finquelreg (ScanAqua AS) and tissues

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101112131415161718192021222324252627282930313233343536373839404142434445464748495051525354555657585960616263646566

676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132


25 Real-time PCR


26 Sequencing










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Q4

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9

1011121314151617181920212223242526272829303132333435363738394041

42

4344454647

48

4950515253545556575859606162636465

666768697071

72

737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130


drCCR9b ssCCR92a


hsCCR7 drCCR7


hsCCR6 ssCCR62


ssCXCR6 drCCR10


drCCRL1a drCCRL1b

ssCCRL12a ssCCRL12b


hsCXCR1 hsCXCR2

ssCXCR11 ssCXCR12


ssCXCR32 drCXCR3b


hsCXCR3 hsCXCR5


hsCXCR4 ssCXCR41a


drXCR1 drXCR1b





ssCCR1 drXCR1Ld

ssCCBP2 drCCBP2




ssCCR4b drCCR4La

drCCR4Lb drCCR4Lc


hsCCR4


hsCCR3 hsCCR2


ssCXCR71b drCXCR7a





100

100

100

100

100

100

100

100

99

100

100

100

100

100

100

100

100

100

100

99

100

100

100

100

100

93

100

83

66

100

98

100

100

100

100

100

99

100

100

91

100

91

100

100

69

66

99

100

100

95

100

100

76

100

100

100

100

99

100

97

99

100

100

77

100

100

97

100

100

100

100

100

79

100

74

99

85

48 CCR4hsCCR8

86

93

34

40

37

68

21

80

80

93

59

51

59

95

42

23

29

33

47

30

35

22

41

43

25

25

28

01


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31

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ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



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4142434445

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72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



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12

3

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1011121314151617181920

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24

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6869

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7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































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Q8

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1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










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Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


25 Real-time PCR


26 Sequencing










ARTICLE IN PRESS



Q4

12345678

9

1011121314151617181920212223242526272829303132333435363738394041

42

4344454647

48

4950515253545556575859606162636465

666768697071

72

737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130


drCCR9b ssCCR92a


hsCCR7 drCCR7


hsCCR6 ssCCR62


ssCXCR6 drCCR10


drCCRL1a drCCRL1b

ssCCRL12a ssCCRL12b


hsCXCR1 hsCXCR2

ssCXCR11 ssCXCR12


ssCXCR32 drCXCR3b


hsCXCR3 hsCXCR5


hsCXCR4 ssCXCR41a


drXCR1 drXCR1b





ssCCR1 drXCR1Ld

ssCCBP2 drCCBP2




ssCCR4b drCCR4La

drCCR4Lb drCCR4Lc


hsCCR4


hsCCR3 hsCCR2


ssCXCR71b drCXCR7a





100

100

100

100

100

100

100

100

99

100

100

100

100

100

100

100

100

100

100

99

100

100

100

100

100

93

100

83

66

100

98

100

100

100

100

100

99

100

100

91

100

91

100

100

69

66

99

100

100

95

100

100

76

100

100

100

100

99

100

97

99

100

100

77

100

100

97

100

100

100

100

100

79

100

74

99

85

48 CCR4hsCCR8

86

93

34

40

37

68

21

80

80

93

59

51

59

95

42

23

29

33

47

30

35

22

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ARTICLE IN PRESS



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1MT1Clanimret-N


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augeAnna

Germ

undssonH

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parativeIm

munology

(2014)doi101016jdci201411009

7U

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holtet

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(2014)

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Fig 2 (continued)

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ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



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4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

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24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


drCCR9b ssCCR92a


hsCCR7 drCCR7


hsCCR6 ssCCR62


ssCXCR6 drCCR10


drCCRL1a drCCRL1b

ssCCRL12a ssCCRL12b


hsCXCR1 hsCXCR2

ssCXCR11 ssCXCR12


ssCXCR32 drCXCR3b


hsCXCR3 hsCXCR5


hsCXCR4 ssCXCR41a


drXCR1 drXCR1b





ssCCR1 drXCR1Ld

ssCCBP2 drCCBP2




ssCCR4b drCCR4La

drCCR4Lb drCCR4Lc


hsCCR4


hsCCR3 hsCCR2


ssCXCR71b drCXCR7a





100

100

100

100

100

100

100

100

99

100

100

100

100

100

100

100

100

100

100

99

100

100

100

100

100

93

100

83

66

100

98

100

100

100

100

100

99

100

100

91

100

91

100

100

69

66

99

100

100

95

100

100

76

100

100

100

100

99

100

97

99

100

100

77

100

100

97

100

100

100

100

100

79

100

74

99

85

48 CCR4hsCCR8

86

93

34

40

37

68

21

80

80

93

59

51

59

95

42

23

29

33

47

30

35

22

41

43

25

25

28

01


XCR-like



ARTICLE IN PRESS



12345








Trout ref



ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940414243444546474849505152

5354

55

565758596061626364656667686970717273

747576777879808182838485868788899091













ARTICLE IN PRESS



123456789

101112131415161718192021222324252627282930

31

32333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131


1MT1Clanimret-N


AR

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RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

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hemokine

receptorsin

Atlantic

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evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

7U

Grim

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entalandCom

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(2014)

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Fig 2 (continued)

AR

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Germ

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parativeIm

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(2014)doi101016jdci201411009

8U

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Fig 2 (continued)

AR

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INP

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Pleasecite

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inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

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eongBen

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hemokine

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Atlantic

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parativeIm

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(2014)doi101016jdci201411009

9U

Grim

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munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

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evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

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(2014)

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Fig 2 (continued)

AR

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INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

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Atlantic

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evelopmentaland

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munology

(2014)doi101016jdci201411009

11U

Grim

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parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References








Trout ref



ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940414243444546474849505152

5354

55

565758596061626364656667686970717273

747576777879808182838485868788899091













ARTICLE IN PRESS



123456789

101112131415161718192021222324252627282930

31

32333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131


1MT1Clanimret-N


AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

7U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

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Com

parativeIm

munology

(2014)doi101016jdci201411009

8U

Grim

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entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

9U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References













ARTICLE IN PRESS



123456789

101112131415161718192021222324252627282930

31

32333435363738394041424344454647484950515253545556575859606162636465

66676869707172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131


1MT1Clanimret-N


AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

7U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

8U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

9U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


1MT1Clanimret-N


AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

7U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

8U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

9U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

8U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

9U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

9U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

10U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


Fig 2 (continued)

AR

TIC

LE

INP

RE

SS

Pleasecite

thisarticle

inpress

asUnniG

rimholtH

elenaH

augeAnna

Germ

undssonH

augeJongL

eongBen

FKoopC

hemokine

receptorsin

Atlantic

salmonD

evelopmentaland

Com

parativeIm

munology

(2014)doi101016jdci201411009

11U

Grim

holtet

alDevelopm

entalandCom

parativeIm

munology

(2014)

ndash









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References









ARTICLE IN PRESS



Q5

1234

5

6789

1011121314151617181920212223242526272829303132333435363738

394041424344454647484950515253545556575859606162636465666768697071

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References

ssCXCR5 ssCXCR31a

ssCXCR31b 100

ssCXCR32 elCXCR32a

elCXCR32b 100

99

100

71


100 100

53

elCXCR41 ssCXCR41a

ssCXCR41b 94

100


99 100

100

elCXCR71 ssCXCR71a

ssCXCR71b 100

100

ssCXCR72a ssCXCR72b

100

100

ssCMKRL1 elCMKRL1

100

elCMKRL12 elCMKRL13

100 100


ssCMKRL2b 100

100 92

100

93

59

ssCCR1 elCCR1

100


ssCCR3b 80

100 100


100 100

ssXCR2 elXCR2

100

97

92

86

ssCCBP2 elCCBP2

100

54


100 100

elCCR4 ssCCR4a

ssCCR4b 100

100

84

elCCR5 ssCCR5a

ssCCR5b 100

100

94

89

56

ssCCR62 elCCR6

ssCCR61a ssCCR61b

100 100

100


100 100

86


100 100

elCCR92 ssCCR92a

ssCCR92b 92

100

100

33

ssCXCR6 elCXCR6

100

35


100 100


100 100

100

61

ssCXCR11 elCXCR11

100

ssCXCR12 elCXCR12

100 100


97 100

99

45


ARTICLE IN PRESS



123










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References










ARTICLE IN PRESS



12

34

56789

10111213141516171819202122232425262728293031323334353637383940

4142434445

46

47484950515253545556575859606162636465666768697071

72737475767778798081828384858687888990919293949596


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References


4 Conclusion



Acknowledgement




References

















Total sites21 (78372) 18 (67380) 20 (78397) 6 (22381) 11 (41368) 11 (42368) 15 (3282266)


ARTICLE IN PRESS



Q6

Q7

12

3

456789

1011121314151617181920

212223

24

252627282930313233343536373839404142434445

46

474849505152535455565758596061626364656667

6869

70

7172737475767778798081828384858687888990919293949596979899100101102103104105106107108109110111112113114115116117118






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References






























































ARTICLE IN PRESS



Q8

123456789

1011121314151617181920212223242526272829303132333435363738394041424344454647484950515253545556575859606162636465666768697071727374757677787980818283848586

87888990919293949596979899100101102103104105106107108109110111112113114115116117118119120121122123124125126127128129130131132133134135136137138139140141142143144145146147148149150151152153154155156157158159160161162163164165166167168169170171172










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References










ARTICLE IN PRESS



Q9123456789

1011121314

1516171819202122232425262728


Introduction


Bioinformatics



RNA extraction

Real-time PCR

Sequencing






Expression patterns


Conclusion

Acknowledgement


References

chemokine receptors in atlantic salmon

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