Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand.

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Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand

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João C.R. Cardoso ⇑, Rute C. Félix, Marlene Trindade, Deborah M. Power Comparative Endocrinology and Integrative Biology, Centre of Marine Sciences, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal

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Article history: Available online xxxx Keywords: Comparative genomics Secretin receptor Evolution Ray-finned fishes Sarcopterygii Structure

a b s t r a c t The secretin receptor (SCTR) is a member of Class 2 subfamily B1 GPCRs and part of the PAC1/VPAC receptor subfamily. This receptor has long been known in mammals but has only recently been identified in other vertebrates including teleosts, from which it was previously considered to be absent. The ligand for SCTR in mammals is secretin (SCT), an important gastrointestinal peptide, which in teleosts has not yet been isolated, or the gene identified. This study revises the evolutionary model previously proposed for the secretin-GPCRs in metazoan by analysing in detail the fishes, the most successful of the extant vertebrates. All the Actinopterygii genomes analysed and the Chondrichthyan and Sarcopterygii fish possess a SCTR gene that shares conserved sequence, structure and gene environment synteny with the tetrapod homologue. Phylogenetic clustering and gene environment comparisons revealed that fish and tetrapod SCTR shared a common origin and diverged early from the PAC1/VPAC subfamily group. In teleosts SCTR duplicated as a result of the fish specific whole genome duplication but in all the teleost genomes analysed, with the exception of tilapia (Oreochromis niloticus), one of the duplicates was lost. The function of SCTR in teleosts is unknown but quantitative PCR revealed that in both sea bass (Dicentrarchus labrax) and tilapia (Oreochromis mossambicus) transcript abundance is high in the gastrointestinal tract suggesting it may intervene in similar processes to those in mammals. In contrast, no gene encoding the ligand SCT was identified in the ray-finned fishes (Actinopterygii) although it was present in the coelacanth (lobe finned fish, Sarcopterygii) and in the elephant shark (holocephalian). The genes in linkage with SCT in tetrapods and coelacanth were also identified in ray-finned fishes supporting the idea that it was lost from their genome. At present SCTR remains an orphan receptor in ray-finned fishes and it will be of interest in the future to establish why SCT was lost and which ligand substitutes for it so that full characterization of the receptor can occur. Ó 2014 Elsevier Inc. All rights reserved.

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1. Introduction

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Secretin (SCT) was identified over 100 years ago in the groundbreaking experiments of Bayliss and Starling that revealed it was a regulator of exocrine pancreatic secretion (1902) (Bayliss and Starling, 1902). This heralded a new era in the study of regulatory systems and the term ‘‘hormone’’ was adopted for chemical messengers produced in one organ, released into the circulation and acting on a distant target organ (Starling, 1905). In 1981, Jensen and Gardner identified a receptor for SCT (SCTR) in the human pancreas and in 1991 it was cloned from the rat NG108-15 cell line (Ishihara et al., 1991). SCTR is a member of the Class 2 B1 subfamily

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⇑ Corresponding author. E-mail addresses: [email protected] (J.C.R. Cardoso), [email protected] (R.C. Félix), [email protected] (M. Trindade), [email protected] (D.M. Power).

of G-Protein Coupled Receptors (GPCRs), which is a group of seven helical cell transmembrane receptors. In mammals, SCT is a 27 amino acid peptide and is involved in many functions (Lam et al., 2008). In the brain it is a potent neuropeptide and controls the secretion of several neuropeptides (Yamagata et al., 2008; Chu et al., 2009; Velmurugan et al., 2010) and at the periphery it regulates body water homeostasis (Chu et al., 2007, 2009), relaxes vascular smooth muscle (Fara and Madden, 1975; Bell and McDermott, 1994) and enhances secretion of reproductive hormones (Kasson et al., 1986; Kimura et al., 1987). It also inhibits feeding behaviour and regulates fat and protein metabolism (Sekar and Chow, 2013). Tetrapod SCTR belongs to the PAC1/VPAC subfamily group (a.k.a. secretin-GPCRs or E-group) that shared the same ancestral gene as the vertebrate glucagon and parathyroid receptors and emerged after the protostome-deuterostome divergence (Harmar, 2001; Cardoso et al., 2004, 2005, 2006; Schioth and Fredriksson, 2005; Hwang et al., 2013; Mirabeau and

http://dx.doi.org/10.1016/j.ygcen.2014.05.025 0016-6480/Ó 2014 Elsevier Inc. All rights reserved.

Please cite this article in press as: Cardoso, J.C.R., et al. Fish genomes provide novel insights into the evolution of vertebrate secretin receptors and their ligand. Gen. Comp. Endocrinol. (2014), http://dx.doi.org/10.1016/j.ygcen.2014.05.025

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Joly, 2013; Cardoso et al., 2014). In vertebrates’ six functional PAC1/ VPAC members exist and they are named after the neuropeptides that activate them. The pituitary adenylate cyclase-activating polypeptide (PACAP) receptor (PAC1) and vasoactive intestinal peptide (VIP) receptors (VPAC1 and VPAC2) are stimulated by PACAP and VIP with the exception of fish VPAC2 that is activated by peptide histidine isoleucine (PHI) (Wu et al., 2008). Growth hormone releasing hormone (GHRH) receptor (GHRHR) is activated by GHRH and PACAP-related peptide (PRP) stimulates the PRP receptor (PRPR) and has not so far been identified in mammals (Tam et al., 2013). Subsequent to isolation of rat SCTR (Ishihara et al., 1991) homologues were isolated in human (Chow, 1995; Jiang and Ulrich, 1995; Patel et al., 1995), mouse (Vassilatis et al., 2003), cattle (Meuth-Metzinger et al., 2005) and rabbit (Svoboda et al., 1998). Recently, SCTR was isolated in chicken, amphibian (Xenopus laevis and Rana rugulosa) and African lungfish (Protopterus dolloi) (Tam et al., 2011; Wang et al., 2012). Characterization of the nonmammalian receptors revealed that in common with mammals, SCT stimulates a rise in intracellular cAMP and increases mobilization of intracellular calcium (iCa2+) (Tam et al., 2011; Wang et al., 2012) and the chicken and amphibian peptide homologues share a conserved role and stimulate pancreatic secretion (Dockray, 1975a, 1975b; Tam et al., 2011). SCTR is the most recently identified member of the vertebrate neuropeptide Class 2 B1 (GPCR) family in teleosts (Wang et al., 2012; Hwang et al., 2013) from which both receptor and SCT genes were thought to be absent (Cardoso et al., 2005, 2006, 2007a; Roch et al., 2009; Cardoso et al., 2010; Tam et al., 2011). Recently, using different strategies and combining gene linkage synteny analysis allied to DNA amplification techniques the first teleost SCTR cDNA was isolated from the zebrafish brain (Wang et al., 2012). Intriguingly so far no gene encoding the isolated receptor has been identified in the zebrafish genome assembly (Wang et al., 2012) however SCTR genes have been identified in other teleost genomes (Hwang et al., 2013). Teleost fishes comprise the largest and most speciose group of vertebrates on earth. Their evolution was affected by a specific whole genome duplication (teleost specific gene duplication, TSGD or 3R) that occurred early in the Teleostei radiation (Volff, 2005; Ravi and Venkatesh, 2008). Fish genomes have a higher rate of chromosomal rearrangements, gene-linkage disruptions and contain faster evolving protein-coding sequences compared with mammals (Ravi and Venkatesh, 2008; Lu et al., 2012). This rapid evolution has been shown to affect the existence and persistence of other Class 2 B1 GPCRs in the genome and the evolutionary context of the fish SCTR remains to be established (Cardoso et al., 2004, 2005; Fradinger et al., 2005; Cardoso et al., 2007b; Roch et al., 2009; Irwin and Prentice, 2011; Hwang et al., 2013). The present study will integrate and extend the model previously developed for PAC1/VPAC subfamily group evolution by integrating and extending knowledge about SCTR and SCT in the fishes in relation to the chordates and terrestrial vertebrates.

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2. Methods

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2.1. Sequence database searches and data retrieval

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SCTR genes were procured in the lamprey (jawless fish), cartilaginous fish (holocephalan), ray-finned fishes and in lobe-finned fish genomes publicly available. Nine genomes from ray-finned fishes were explored which included two pufferfish (Tetraodon nigroviridis; Takifugu rubripes); stickleback (Gasterosteus aculeatus); nile tilapia (Oreochromis niloticus); medaka (Oryzias latipes); platyfish (Xiphophorus maculatus); Atlantic cod (Gadus morhua); cavefish

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(Astyanax mexicanus) and the primitive freshwater ray-finned fish the spotted gar (Lepisosteus oculatus); the genomes were available from the ENSEMBL (http://www.ensembl.org, accessed October 2013) database. The lobe-finned fish coelacanth (Latimeria chalumnae) genome assembly was accessed from the ENSEMBL (http://www.ensembl.org, accessed October 2013) database. The bait was the deduced amino acid sequence of the zebrafish (Danio rerio) SCTR homologue (ACC86056.1, (Wang et al., 2012)) of the human receptor. Searches for SCTR transcripts were also performed in teleost fish (taxid:32443) EST databases available from NCBI (http://www.ncbi.nlm.nih.gov/, accessed October 2013). Searches in the genomes of the marine lamprey (Petromyzon marinus, http://www.ensembl.org, accessed October 2013) and the Japanese lamprey (Lethenteron japonicum, http://jlampreygenome. imcb.a-star.edu.sg, November 2013) and in the cartilaginous fish, the elephant shark (Callorhinchus milii, http://esharkgenome.imcb. a-star.edu.sg, November 2013) were also carried out using the zebrafish SCTR, the hagfish (Eptatretus burger) and lamprey VPACs (Ng et al., 2012). The genomes of terrestrial vertebrates, the amphibian (Xenopus tropicalis), Anole lizard (Anolis carolinensis) and chicken (Gallus gallus) were also mined for the receptor gene homologues. The existence of SCTR in early chordate genomes, the acorn worm (Saccoglossus kowalevskii, https://www.hgsc.bcm.edu/), sea urchin (Strongylocentrotus purpuratus, http://metazoa.ensembl.org/), amphioxus (Branchiostoma floridae, http://genome.jgi-psf.org/ Brafl1/Brafl1.home.html) and Ciona (Ciona intestinalis, http:// www.ensembl.org/) were also explored. Searches for other members of the PAC1/VPAC subfamily (PAC1, VPAC1, VPAC2, GHRHR and PRPR) were also performed in the above-mentioned genomes. The existence of a putative SCT gene in genome assemblies was determined using the databases described above and also teleost EST libraries that were searched with the X. laevis (NP_001267 540.1), chicken (NP_001020004.1) and human (NP_068739.1) mature peptide transcripts.

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2.2. Phylogenetic analysis

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Phylogenetic analysis of SCTR was performed using the deduced amino acid sequence of the seven TM domains and intra and extracellular loops (Supplementary File 1). Eighty-three sequences, from 19 different vertebrates including 14 fish were used and contained representatives of the SCT, PACAP, VIP, GHRH and PRP receptors and of the GCG and PTH receptor families. The receptor sequence alignment was submitted to ProtTest (2.4) to select the best model to study protein evolution according to the Akaike Information Criterion (AIC) statistical model (Abascal et al., 2005). Bootstrapping analysis was used to assign measures of accuracy of the phylogenetic clades (Felsenstein, 1985). ML analysis was constructed in the PhyML program (v3.0 aLRT) with receptor sequences that were aligned using ClustalW (2.0.3) (Thompson et al., 1997). Data was also analysed using the NJ method (Saitou and Nei, 1987) implemented in the Mega 5.2 program (Tamura et al., 2011). The phylogenetic trees were built using the JTT substitution model (Jones et al., 1992). ML analysis included fixed proportion of invariant sites (0.02), 4 gamma-distributed rate categories to account for rate heterogeneity across sites and gamma shape parameter was fixed (1.03). Reliability for internal branching was assessed using 100 bootstrap replicates. NJ analysis was performed with 4 gamma-distributed rate categories, fixed gamma parameter (1.03) and reliability of internal branches assessed using 1000 bootstrap replicates. ML and NJ trees were rooted with the vertebrate PTHR subfamily cluster that contained the receptors from human (ENSP00000321999), chicken (ENSG ALP00000 008782), zebrafish (ENSDARP00000091807 and ENS-

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DARP00000027674) and spotted-gar (ENSLOCP00000011589 and ENSLOCP00000014456).

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2.3. Sequence comparisons

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Multiple amino acid sequence alignments were generated using ClustalW (v2) tool (http://www.genome.jp/tools/clustalw/) and GeneDoc (http://iubio.bio.indiana.edu/) was used to annotate the receptor alignments and to calculate percentage of sequence identity/similarity. The N-terminal (N-ted) region of the fish and terrestrial vertebrates SCTRs were compared by aligning the N-terminal coding region up to conserved TM1. The conserved cysteine (C) residues were identified and used to anchor the alignment. The signal peptide was identified and N-glycosylation consensus sites (N-x-T/ S) were predicted. Residues known to be functionally important in rat (Miller et al., 2007) were annotated. Gaps were introduced to maximize sequence homology.

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2.4. Gene structure and gene linkage analysis

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The structure and gene environment neighbouring the rayfinned fish and coelacanth SCTR genes were characterized and compared to tetrapods. The zebrafish SCTR gene is not predicted in the zebrafish genome assembly and its structure was deduced. Its gene organization (exons/introns) was deduced using the coding region of the full-length zebrafish SCTR cDNA. The gene environments of the vertebrate SCTR and SCT genes were characterized and neighbouring genes identified based upon the ENSEMBL database gene annotation and complemented with sequence homology searches. Short-range gene linkage comparisons included human, chicken, lizard, Xenopus, the coelacanth and 8 ray-finned fish (tetraodon, stickleback, tilapia, medaka, cod, zebrafish, cavefish and gar).

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2.5. RNA extractions and cDNA synthesis

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Gills, liver, heart, stomach, spleen, kidney, testis, ovary, brain and several gut sections (duodenum, mid-gut and hind-gut) were collected from tilapia (Oreochromis mossambicus, n = 3) and sea bass (Dicentrarchus labrax, n = 3) and RNA extracted using TriReagent (Sigma Aldrich, Spain). Total RNA was treated with 1 U DNase (DNA-free Kit, Ambion, UK) for 30 min at 37 °C according to the manufacturer’s instructions. The DNase treated total RNA (500 ng) was denatured at 65 °C for 5 min, quenched on ice for 5 min and used for cDNA synthesis in a 20 ll reaction volume containing 10 ng of pd(N)6 random hexamers (GE Healthcare, UK), 2 mM dNTPs, 100 U of MMLVRT and 20 U RNasinÒ Plus RNase inhibitor (Promega, Spain). The cDNA was synthesized for 10 min at 20 °C followed by 60 min at 42 °C and 72 °C for 5 min and used to characterize receptor expression in tilapia and sea bass.

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2.6. Quantitative-PCR (q-PCR)

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Expression of the duplicate tilapia and single sea bass SCTR was carried out using quantitative real-time PCR (q-PCR). The tilapia and sea bass SCTRs were amplified using transcript specific primers in a 15 ll reaction volume that contained 300 nM of forward and reverse primer, SsoFast EvaGreen supermix (Bio-Rad, Portugal) and 2 ll of template cDNA (diluted 1:5). Two reference genes (EF1a and 18S) were used to calculate transcript relative expression units (Supplementary Table 1). The q-PCR analysis was performed in duplicate reactions (70% aa similarity). The predicted mature coding regions of the duplicate tilapia SCTRs are 82% similar (59% identical). In contrast, Oni_17436 shares high identity with cod SCTR (70% identity) and also with the fresh water fish representatives, the zebrafish, (63%) and cavefish (62%). The tilapia paralogue SCTR (Oni_12054) is more related to the marine teleost homologues and is 71%, 84% and 88% identical to the takifugu, stickleback and sea

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Fig. 1. Dendogram showing members of the PAC1/VPAC gene family in species representing some of the major vertebrate lineages, especially teleost fishes. The number of predicted genes for members of the SCTR family is indicated. Accession numbers are available in Supplementary Table 2. Searches in teleost genomes were complemented with EST mining. ⁄Predicted based upon sequence similarity searches; ⁄⁄retrieved from EST data; ni not identified in the genome; ? Sequenced and assembled genome is not publicly available. The teleost specific genome duplication (TSGD or 3R) is indicated. The two hagfish VPACs were obtained from (Ng et al., 2012) and the lamprey homologues from (Ng et al., 2012) and from the database (KE993774.1). A putative PAC1 (KE998314.1/KE993672.1) receptor gene was also retrieved from the Japanese lamprey genome assembly.

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bass, respectively. Sequence comparisons of the teleost SCTRs revealed that they share 40–50% aa identity with the other PAC1/ VPAC members and the zebrafish SCTR is 43% and 44% identical in aa sequence with the PAC1 and VPAC1A/B, respectively and 42% and 47% with the GHRHR and PRPR.

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3.2. Phylogeny of the fish secretin receptors

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Phylogenetic analysis revealed that the fish SCTRs clustered and that the vertebrate SCTRs were on a branch independent of other Class 2 subfamily B1 GPCRs (Fig. 2, Supplementary Fig. 1). The topology of ML and NJ trees suggests that SCTR diverged early from the other PAC1/VPAC subfamily members and that strong evolutionary pressure presumably, limited gene duplication and gene number expansion in the SCTR branch. In contrast, the PAC1/VPAC ancestral genes underwent multiple rounds of gene duplication that gave rise to present day sub-family members. The recently identified VPACs from the Japanese lamprey and hagfish (Ng et al., 2012) cluster with vertebrate PAC1, VPACs, PRPR and GHRHR receptors (Fig. 2, Supplementary Fig. 1) and not with vertebrate SCTR. In teleosts, two SCTR clusters were resolved that had strong bootstrap support and, the duplicate tilapia receptors each clustered with one of the branches. Oni_12054 clustered with the Percomorpha members and Oni_17436 clustered with the cod and representatives of the Otomorpha (zebrafish, catfish and cavefish) lineage suggesting that during the teleost radiation duplication of the SCTR gene occurred but the duplicates were selectively eliminated from their genomes. The available sequence for salmoniformes SCTR is very incomplete and lacks the region from TM4 to TM7 and in phylogenetic analysis tends to group with Percomorpha fish homologues within the tilapia Oni_12054 cluster (Fig. 2, Supplementary Fig. 1).

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3.3. Sequence conservation of the fish SCTR with tetrapods

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The fish SCTRs contain the seven TM domains characteristic of GPCRs and the N-terminal ectodomain (N-ted) is large with the conserved cysteine residues and N-glycosylation (N-x-T/S) motifs (Fig. 3). Comparison of the N-terminus of the teleost fish (zebrafish, tilapia, stickleback), lobe-finned fish (coelacanth, lungfish) and the tetrapod Xenopus, chicken and human SCTRs reveals conservation of the seven conserved cysteine’s (C) of which six are likely to form three disulphide bridges (C2-C5; C3-C6 and C4-C7). Three N-glycosylation consensus sites are conserved in human, chicken and lobe-finned fish but only two are preserved in teleosts (Fig. 3, Supplementary Fig. 2). The seven cysteines identified in the vertebrate SCTR are also conserved in the fish and tetrapod PAC1 and VPAC members. The N-glycosylation motif between C3-C4 in SCTR is shared by the vertebrate PAC1s and VPAC1s. The N-glycosylation motif at C6 in SCTR is also conserved within the vertebrate PAC1 and VPAC2/PHIR N-ted sequences. Despite the overall low sequence identity of the teleost and tetrapod SCTR, the N-terminal amino acids involved in the activation of the mammalian receptor are conserved in the teleost and lobefinned fish receptors and include, aspartic acid (D71) between C3 and C4, tryptophan (W76) next to C4, proline (P90) next to C5, glycine and tryptophan (G111-W112) located between C6 and C7 and asparagine (N128) after C7 (Furness et al., 2012). The same amino acids are also conserved in the N-ted of vertebrate PAC1, VPAC1 and VPAC2 (Fig. 3). Motifs localized within SCTR intracellular loops and carboxyl-terminal tail that affect mammalian receptor signalling and G protein coupling (Garcia et al., 2012) are also conserved in the fish receptor. Conserved amino acids include R164 in the first intracellular loop, H168 at the beginning of TM2 and K314, and L315, within the third intracellular loop and TM6. The highly

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Fig. 2. Evolutionary relationship of the vertebrate secretin receptors with the other Class 2 B1 GPCRs. Phylogenetic analysis was performed using the maximum likelihood (ML) method using 83 sequences retrieved from 19 different vertebrates. Reliability of internal branches was assessed using 100 bootstrap replicates. Only the bootstrap support higher than 50% is represented. To facilitate observation of the vertebrate secretin receptor (SCTR) branch, the pituitary adenylate cyclaseactivating polypeptide (PAC1), vasoactive intestinal peptide (VPAC1 and 2), growth hormone releasing hormone (GHRHR), PACAP-related peptide (PRPR), glucagon (GCGR) and parathyroid hormone 1 (PTH1R) receptor clusters are condensed. The GCGR branch included sequences from human (ENSP00000383558), chicken (ENSGALP00000041802), zebrafish (ENSDARP00000095410 and ENSDARP0000001 3211) and spotted gar (ENSLOCP00000017159). PTH1R receptor subfamily cluster contained sequences from human (ENSP00000321999), chicken (ENSGALP000 00008782), zebrafish (ENSDARP00000091807 and ENSDARP00000027674) and spotted gar (ENSLOCP00000011589 and ENSLOCP00 000014456). The sea bass (D. labrax, Dla) SCTR EST sequence was kindly provided by Adelino Canário. Tree was rooted with the vertebrate PTH1R cluster. A similar tree was obtained with the Neighbour joining method (Supplementary Fig. 1). VPAC sequences from hagfish (Ebu, E. burger) and lamprey (Lja, L. japonicum) were obtained from (Ng et al., 2012) and a new lamprey VPAC gene (Lja 1) was deduced from genome assembly (KE993764.1). The putative elephant shark SCTR in scaffold_45 and other Class 2 B1 GPCRs and the lamprey PAC1 receptor were not used in the analysis, as the retrieved sequences were very incomplete. Species names and accession numbers of the receptor genes are available in Supplementary Table 2.

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conserved FQGbbVxxbYCFxNxExQ (where x represent any amino acid residue and b any hydrophobic amino acid residue) of TM7 in tetrapod Class 2 B1 members is also conserved in fish but in the teleost the first F is mutated to a T. The IRIL motif characteristic of VPAC (within TM5) and L171 in TMD2, crucial for mammalian SCTR activation (Di Paolo et al., 1999; Solano et al., 2001), are also present in the teleost and lobe-finned fish SCTRs (Supplementary Fig. 2).

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3.4. Secretin receptor gene structure and gene environment

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The SCTR gene is localized on chromosome 9 in zebrafish and in common with human and chicken gene homologues has 13 exons (Fig. 4). In both tetrapods and teleosts TM1 is encoded by exon 5,

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TM2 by exon 6, TM3 by exon 7, TM4 by exons 7 and 8, TM5 by exons 9 and 10, TM6 by exon 11 and TM7 by exons 12 and 13 (Fig. 4). In other teleosts such as tilapia and stickleback the predicted SCTR gene structure in ENSEMBL is variable and remains to be confirmed when the full-length receptor transcript is isolated. The zebrafish SCTR gene is 24 kb in length and in the human it is 85 kb and intron 1 is 4.7 kb and 27 kb, respectively and is the largest in the gene. The chicken SCTR gene is more compact than the zebrafish gene and is only 15 kb in length as a result of its smaller introns (data not shown). The zebrafish receptor has a similar structure to Takifugu ADCYAP1R1 (A and B) but is one exon shorter than zebrafish PHIR (Fig. 4) (Cardoso et al., 2004; Wu et al., 2008). The gene environment of SCTR in fish genomes is conserved with the tetrapod homologue region and six genes are linked (Fig. 5). The gene order and synteny of the genome region containing SCTR in the spotted gar is highly conserved with teleosts, coelacanth and tetrapods. In Percomorpha the sequence homologues of tetrapod and other fish SCTR-linked genes are split between two genome regions, although only a single SCTR is found. The tilapia is the exception and retained duplicate copies of the SCTR gene and the linked gene C1qL2.

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3.5. Tissue distribution of the teleost secretin receptor

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Seven SCTR EST clones were retrieved by screening teleost EST databases from several tissues (Supplementary Table 3). The results confirm that SCTR is transcribed in fish, and has a widespread tissue distribution and is present in the gastrointestinal tract but also in adult nervous tissue and is expressed early in fish development. The tissue distribution of the duplicate tilapia and single sea bass SCTR transcripts confirm the EST data. SCTR transcripts are abundant in the gastrointestinal system and are significantly (p < 0.05) higher in the mid-gut compared to hind-gut, duodenum, brain and ovary (Fig. 6). In tilapia, Oni_12054 is the most abundant transcript and is significantly (p < 0.05) more abundant in the stomach compared to brain, liver, duodenum, hind-gut, testis and ovary. The paralogue tilapia Oni_17436 transcripts are significantly (p < 0.05) more abundant in ovary then gill > brain and duodenum > mid-gut and hind-gut (Fig. 6). In contrast to tilapia Oni_17436 no SCTR was detected in sea bass gills.

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3.6. Secretin gene environment in fish

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In ray-finned fishes the SCT gene remains unidentified and searches carried out on available genomes and ESTs failed to identify a putative gene or transcript. The exceptions are the elephant shark (C. milii) a holocephalan (scaffold_249) and the coelacanth (JH126644.1) a lobe-finned fish that contain putative genes for SCT in their genomes. The deduced mature peptides of the cartilaginous fish and the coelacanth are 35% and 46% identical, respectively to the human homologue. The gene content of the genome region flanking SCT in coelacanth and Xenopus is identical but in chicken some rearrangements occurred. The homologue of the coelacanth SCT genome region in human is shared between chromosome 11 and 19 (Fig. 7). In lizard a syntenic region on chromosome 6 is present and the absence of a putative SCT is most likely a consequence of the incomplete nature of the genome assembly. In teleost genomes the corresponding region is present in two copies as expected due to TSGD (3R), but each has undergone gene losses, including SCT (Fig. 7). The gene environment of the cartilaginous fish SCT was not characterized as its genome was incompletely assembled. In seven teleost fish genome regions and also in the spotted gar, homologues of 5 genes that are closely linked to the coelacanth SCT

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Fig. 3. N-terminal comparisons of the secretin receptor and related Class 2 B1 family members in teleost, lobe-finned fishes and tetrapods. The N-terminal region is extracted up until the conserved TM1 and aligned. The conserved cysteines (C) are annotated in grey and bold and are numbered. Predicted N-glycosylation sites (N-x-T/S) are indicated in bold and the vertebrate SCTR signal peptide is in italics. Residues indicated by an arrow and in bold are important for the mammalian receptor ligand binding. Underlined amino acids in the human receptor sequence are functionally important in the rat SCTR (Miller et al., 2007). Complete residue conservation is annotated with a ‘‘⁄’’, partial conservation with ‘‘.’’ and the position of the amino acids present in most of the receptor families with ‘‘:’’. Gaps have been introduced to maximize sequence homology.

Fig. 4. Gene organization of the zebrafish and human secretin receptors. Gene sizes are indicated next to each gene, exons are numbered and are represented by closed boxes and the solid black line joining exons denotes the introns. Numbers indicate equivalent exons. The largest intron is located between exons 1 and 2 and is 27 kb and 4.7 kb in the human and zebrafish, respectively. The Takifugu ADCYAP1R1 gene structure has previously been reported (Cardoso et al., 2004).

already present in the ancestral genome before the divergence of the fishes. All the teleost genomes analysed contained SCTR and gene duplicates were detected in the tilapia (O. niloticus). Phylogenetic analysis suggests that SCTR and the precursor of other VPAC/ PAC1 subfamily genes arose, when an ancestral gene duplicated early in the vertebrate radiation, and that subsequently the SCTR gene was under strong evolutionary pressure. In contrast, the VPAC/PAC1 subfamily branch gave rise to the genes PAC1, VPAC (1 and 2), GHRHR and PRPR. The notion that vertebrate SCTR gene was under strong evolutionary pressures is also supported by the well-conserved gene environment both in ray-finned fish and tetrapods. Although functional analysis was not performed in the present study the abundance of the transcript in the intestine of both Mozambique tilapia and sea bass suggests it probably mediates the function initially identified by Bayliss and Starling for SCT in mammals. It is interesting that in the ray-finned fishes the ligand, SCT, was lost although it is present in the holocephalan and Sarcopterygii (coelacanth and tetrapods). The loss of key amino acids important for ligand binding in the N-ted domain of SCTR and the absence of SCT suggests that an alternative ligand probably exists in ray-finned fishes.

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gene loci on Scaffold JH126644.1 are identified but SCT gene has been lost and in teleost is split in 2 genome regions and duplicate RIC8A (in close linkage with SCT in tetrapods and coelacanth) and PRR12 genes are present (Fig. 7). The identification in this study of a putative SCT gene in cartilaginous fish genome suggests that SCT is of early origin and that gene deletion occurred after the Sarcopterygii and Actinopterygii divergence and was early in the ray-finned fish radiation. In lamprey, no homologues of the elephant shark, coelacanth or tetrapod SCT peptide, transcript or gene were retrieved. However, PACAP, VIP and GCG genes are suggested to exist in the Japanese lamprey genome (JL12590, JL3999 and JL5526) and in the marine lamprey (Irwin et al., 1999; Ng et al., 2012). Poor conservation exists between the coelacanth and frog SCT genome regions and that of the chicken and human. Human chromosome 19 (which does not contain the SCT gene) contains a region that is highly syntenic with the coelacanth and Xenopus SCT genome regions. In summary, the results suggest that during the vertebrate radiation the genome region harbouring the SCT gene underwent considerable rearrangements in ray-finned fishes and also in birds and mammals (Fig. 7).

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4.1. Fish SCTRs are highly related with the tetrapod homologues

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In the present study SCTR was identified in the coelacanth, rayfinned fishes and in the elephant shark. Our results extended the previous studies that described SCTR in teleost (Wang et al., 2012; Hwang et al., 2013) and revealed that a SCTR gene was

Fish SCTRs share a conserved structure and contain seven TM domains and large N-ted regions characteristic of tetrapod homologues. Despite the fact that ray-finned fishes and coelacanth SCTR remains an orphan, they may share similar mechanisms of receptor

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Fig. 5. Comparison of the homologous genome regions harbouring the secretin receptor across vertebrates. The receptor gene environment of 5 Sarcopterygii (human, chicken, lizard, Xenopus and the coelacanth) and 8 ray-finned fish (tetraodon, stickleback, tilapia, medaka, cod, zebrafish, cavefish and gar) are compared. Coloured and patterned block arrows represent genes (named according to HUGO gene annotation) and the arrowhead points in the direction of the predicted gene transcription and gene homologues are denoted in the same colour. Horizontal lines represent chromosome fragments obtained from the species included in the analysis. The relative length of the genome fragment (Mb) is determined from the position of the first and last gene and is indicated for each species. Open colour block arrows represent genes that were deduced based on sequence searches from the genome assembly. The SCTR gene in the X. tropicalis genome is not predicted and the receptor locus was identified in the amphibian genome assembly using the homologue receptor from X. laevis and the gene environment characterized contrasts with previous amphibian gene annotations carried out in earlier versions of the genome assembly (Tam et al., 2011). The short length of genome scaffolds in X. tropicalis means it is not possible to establish SCTR gene linkage. Only species in which the genome contains more than one linked gene are represented. Gene names and symbols: transmembrane protein 37 (TMEM37); diazepam binding inhibitor (DBI); chromosome 2 open reading frame 76 (C2Orf76); STEAP family member 3 (STEAP3); complement component 1q subcomponent-like 2 (C1qL2); macrophage receptor with collagenous structure (MARCO).

Fig. 6. Quantitative expression of tilapia and sea bass secretin receptors. Receptor expression levels relative to the geometric mean of two reference genes (EF1-a and 18S). Data corresponds to the mean ± SEM of tissue from 3 adult male Mozambique tilapia and sea bass. The exception is the ovary, which was collected from 3 females. The sea bass testis was not test.


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Fig. 7. Comparisons of the vertebrate secretin gene environment. The genome environment of the coelacanth secretin gene was characterized and compared to the homologue region in tetrapods (human, chicken, lizard, Xenopus) and 8 ray-finned fish (tetraodon, stickleback, tilapia, medaka, cod, zebrafish, cavefish and gar). In X. tropicalis the SCT gene locus was identified by comparison with the X. laevis SCT homologue (NP_001267540.1). Coloured and patterned block arrows represent genes (named according to HUGO annotation) and arrows indicate the deduced direction of transcription in the genome. Open colour block arrows represent genes that were deduced from the genome assembly. Horizontal lines represent chromosome fragments. The length of the chromosome fragment (Mb) is determined by the position of the first and last gene and is indicated for each species included in the analysis. Only the genome fragments that contain more than one linked gene are represented. Gene names and symbols: Interferon regulatory factor 3 (IRF3); Synembryn-A (RIC8A); Ras-related protein (RRAS); Proline Rich 12 (PRR12); Serine Arginine-Rich Pre-MRNA Splicing Factor (SCAF1).

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activation and signalling. The N-ted domain in fish SCTR contains conserved cysteine residues and N-glycosylation (N-x-T/S) motifs that in mammalian SCTR and other PAC1/VPAC receptors interact with the endogenous peptide hormones and stimulate receptor activity (Asmann et al., 2000; Dong and Miller, 2002; Laburthe et al., 2002, 2007; Dong et al., 2004; Furness et al., 2012). Other functional residues related to ligand binding and SCTR function in rat are also shared by the fish receptor and include leucine (L38) located between C1 and C2, arginine-asparagine (R105N106) located before C6 and tyrosine (Y138) before TM1 (Fig. 3) (Dong et al., 2003; Zang et al., 2003; Miller et al., 2007). In addition, motifs localized in receptor intracellular loops and in the C-terminus that affect receptor signalling and coupling to G proteins are also present in the fish SCTR (Garcia et al., 2012). Site-directed mutagenesis of residues in the intracellular loops of human SCTR

indicate that R164 in the first intracellular loop, H168 in TM2 and K314, L315, R330 and R333 within the third intracellular loop and TM6 affect both the cAMP and calcium signalling cascades (Okamoto et al., 1991; Garcia et al., 2012) and all these residues are present in fish SCTR. The tissue distribution of teleost SCTR transcripts mirrors that of tetrapods and it is abundant in the tilapia and sea bass gastrointestinal system and is also present in brain and to a lesser extent in other tissues. That the SCT system has a key role in the regulation of the mammalian gastrointestinal system was established more than 100 year ago by Bayliss and Starling and receptor localization in fish suggests this function has been conserved across vertebrates. In mammals, SCT is released in response to gastric acid delivery into the duodenal lumen and stimulates water, bicarbonate and enzyme secretion from the pancreas and inhibits gastric


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acid secretion and emptying (Dockray, 1975a; Chey and Chang, 2003). Subsequent studies identified mammalian SCTR in pancreas, stomach, liver, kidney, colon, heart, lung, ovary and in the brain and additional physiological roles for SCT include regulation of feeding behaviour and metabolism, reproduction, growth, neurofunctions and regulation of water homeostasis (Fara and Madden, 1975; Kasson et al., 1986; Jiang and Ulrich, 1995; Yamagata et al., 2008; Chu et al., 2009; Velmurugan et al., 2010; Sekar and Chow, 2013). In Xenopus and chicken, SCTR is also highly abundant in the gastrointestinal systems but in the lungfish, receptor expression is mostly found in the brain with only residual expression in the small intestine, pancreas and other tissues (Tam et al., 2011; Wang et al., 2012). In Xenopus SCTR is found in the small intestine, pancreas, stomach and lung but has a weak expression in brain and in chicken the receptor transcript is highly abundant in the duodenum and is found in several other sections of the gastrointestinal track, the pancreas, liver, testis and in the mid- and hind-brain (Tam et al., 2011; Wang et al., 2012). In tilapia, the duplicate receptors may have acquired divergent functions and although they are both present in brain and intestine, Oni_12054 is mainly expressed in the stomach and Oni_17436 is most abundant in ovary but substantial levels are also present in gill. In the sea bass a single SCTR gene is present and its tissue expression mostly resembles the distribution of Oni_12054 and indicates a potential role in the fish gastrointestinal tract and nervous systems but also in reproduction and water and electrolyte homeostasis and future studies will address this question. The expression of SCTR in tilapia gills (Oni_17436) but its absence from this tissue in sea bass merits further studies to understand its functional significance in teleosts. Taking into consideration the high abundance of SCTR in the teleost intestine and the role of this organ in osmoregulation and bicarbonate secretion it is tempting to speculate that receptor gene evolution in fish may be linked to osmoregulation (Wilson et al., 2002; Gregorio et al., 2013) or to modifications in gastrointestinal function (e.g: presence or absence of a stomach and existence of a long or short intestine) associated with the diversity of their feeding regimes (Ulloa et al., 2011). It remains to be established if the retention of duplicate SCTR genes is unique to tilapia or if duplicate genes also occur in other teleosts, and the functional implications of their retention.

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4.2. SCTR and SCT genes evolved differently in fish

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The presence of SCTR genes but absence of SCT genes in rayfinned fish genomes raises questions about the evolution of the ligand-receptor pair and suggests that in vertebrates they have suffered distinct evolutionary trajectories. While the genomic regions in the spotted gar, teleosts, coelacanth and tetrapods that harbour the SCTR gene share conserved synteny and gene order indicative of a common evolutionary origin, the same is not true for SCT. This suggests that there was conservative pressure on SCTR and the flanking genomic region during the radiation of the ray-finned fishes but not on SCT, which was lost from the genome. Even though the present study highlights that the SCT gene is absent in ray-finned fishes, immunohistochemical (IHC) studies have detected positive immunoreactive cells in the gastrointestinal tract of the flower fish (Pseudophoxinus antalyae (Cinar et al., 2006). The apparently positive SCT signal in IHC may be a consequence of cross-reaction of the human secretin antisera (raised against the 33–121 portion of the C-terminus) with other fish peptides. The presence in teleosts of two genome regions containing genes flanking SCTR in coelacanth and spotted gar and the duplicate SCTR in tilapia suggests that the receptor gene underwent duplication early in the teleost radiation as a consequence of the TSGD event. However, gene retention and rates of loss of the gene duplicates or genome regions/segments appear to be distinct in the

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Teleostei lineage. The duplicate tilapia SCTR evolved differently as revealed by their sequence similarity and phylogenetic clustering and differential tissue distribution. In salmon, a single copy of SCTR was identified that grouped with the Percomorpha representatives and Oni_12054 (Fig. 1). However, the incomplete salmon sequence used in the analysis limits the conclusions that can be drawn and it is not possible to exclude the existence of a second copy in salmonids of SCTR as a result of the tetraploidization event in the common ancestor of salmonids (Danzmann et al., 2008). To better understand if the distinct selective pressures on the duplicate copies of the SCTR gene in ray-finned fish is a unique gene specific event or affects other genes in the same genome region, the evolution of the SCTR-linked gene, C1qL2 (which was also duplicated in the tilapia genome) was characterized and the phylogenetic clustering obtained was similar to that of SCTR and suggests that other genes located in the same chromosome regions underwent similar evolutionary pressures (Supplementary Fig. 3). Differential retention and loss of duplicate gene copies is a common phenomenon during speciation after genome duplication and it has been mainly studied in yeast and plants but is also observed in fish (Jaillon et al., 2004; Volff, 2005; Brunet et al., 2006). If a SCTR gene exists in jawless fish is not yet known as searches failed to identify the receptor in lampreys but in the cartilaginous fish (holocephalian) genome putative SCTR and SCT genes were predicted. Studies of gut function in the 1970s suggest a putative SCT peptide exists in the intestine of lampreys and in the holocephalian fish Chimaera monstrosa (Barrington and Dockray, 1970; Nilsson, 1970) although the peptide remains to be isolated. In the present study, sequence database searches failed to identify a homologue of the Sarcopterygii SCT gene in lamprey (most likely because of the incompleteness of the genome assembly). However, putative SCTR and SCT genes were identified in the elephant shark (C. milii) genome suggesting that the genes for SCTR and SCT emerged early in the vertebrate radiation prior to the emergence of the ray-finned fishes. A fuller picture of the evolution of Class 2 subfamily B1 GPCRs and in particular SCTR and its ligand will be possible when the genome assemblies from jawless fish representatives and also from cartilaginous fish are more consolidated. The inclusion of data from other representatives of early diverging branches in the vertebrate tree such as bichirs, hagfishes and sturgeons will extend our knowledge on the origin and evolution of this and other protein gene families.

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4.3. Potential agonists of the fish SCTR

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In fish the function of SCTR is, at present, unknown and it remains to be established what ligands actually bind and activates. A gene for SCT is absent from ray-finned fish genomes nevertheless, in vivo pancreatic and gall bladder assays stimulated with pike (Esox lucius) intestine extracts suggested that a secretin-like molecule activity exists in teleosts (Dockray, 1974). However, preliminary functional studies revealed that the zebrafish SCTR is not activated by the tetrapod SCT peptide (Wang et al., 2012). The failure of tetrapod SCT to activate the fish SCTR may be related to, (a) the high sequence divergence of vertebrate SCT (Cardoso et al., 2010; Tam et al., 2011; Wang et al., 2012) or, (b) to the partial conservation of key amino acid residues in the N-ted domain of SCTR that are crucial for receptor activity and peptide binding in mammalian SCTR (Fig. 3). The lungfish orphan SCTR is potently activated by human and Xenopus SCT but also by tetrapod VIP and PACAP peptides (Tam et al., 2011). VIP and PACAP also activate the chicken and Xenopus SCTR but they are less potent than SCT. Conversely, chicken SCT binds to and activates PAC1 and VPAC (Wang et al., 2012). In contrast, human SCT does not activate the duplicate sea bream PAC1 (PAC1A and B) but the peptide stimulates

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intracellular signalling by goldfish VPAC (Chow et al., 1997; Cardoso et al., 2007a). This suggests that peptide cross-activation amongst the metazoan SCTR, PAC1 and VPACs can occur and is probably a consequence of the conservation of functionally important amino acids in all receptors. In fact early physiological assays have addressed this issue and extracts from teleost fish (pike and cod) intestine have a weak secretin-like action on the pancreas in mammals but a strong action in birds and the fish secretin-like peptide is proposed to resemble VIP rather than mammalian SCT (Dockray, 1974, 1975a). Orexin peptides share moderate amino acid similarity with STC, and in tetrapods orexin A was found to activate the lungfish and amphibian SCTRs, but not in human (Tam et al., 2011). Orexin peptides exist in teleosts (Matsuda et al., 2012) and it is likely that they may also be agonists of the teleost SCTRs or alternatively SCTR may function as a spontaneous or constitutive receptor that does not require a ligand for activation as described for many orphan GPCRs (Bond and Ijzerman, 2006; Nakashima et al., 2013). The characterization of fish and other non-mammalian tetrapod SCTR and the comparison with the well-characterized mammalian model will contribute to understanding the evolution of SCTR in vertebrates and more specifically the physiological function of SCTR in fish.

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4.4. Final considerations

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SCTR exists in ray-finned fish genomes and is expressed. The fish receptors share conserved sequence, gene structure and genome environment with the tetrapod homologues. The overlapping tissue distribution of SCTR in tetrapods and teleosts may indicate it has similar functions but this question remains to be explored in future experimental studies. The fish and the tetrapod SCTR share surprisingly low sequence similarity with the other PAC1/VPAC subfamily members that emerged from a common ancestral gene. The preceding observation and the fact that SCTR in vertebrate genomes is single copy while PAC1/VPAC are multi-copy suggests SCTR was under high conservative pressure during the vertebrate radiation. The exception is the tilapia that retained duplicate copies of SCTR and provides another example of differential retention of a duplicate gene in teleosts. A putative SCTR gene was identified in cartilaginous fish but was not found in jawless fishes genomes and the evolutionary significance is unclear as its absence may be a result of their incomplete genome assemblies. At present SCTR remains an orphan and an SCT gene is absent from ray-finned fish genomes and tissue distribution of the teleost receptors suggests it may be involved in a number of different functions. Conserved sequence motifs that are functionally important in mammalian SCTR are common to the teleost and coelacanth receptors but some are modified and if this is the basis of the lack of affinity of the fish SCTR for the tetrapod SCT peptide remains to be tested. Deorphanisation of the fish receptor and characterization of the function and evolution of SCTR and its ligand in vertebrates will be needed to elucidate the physiological consequences of the elimination of the SCT gene from ray-finned fishes genomes.

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Acknowledgments

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We are grateful to Professor Adelino Canário for providing the sequence of sea bass EST clones, Dr. Laurence Deloffre for the tilapia tissue panel and João Tavares for advice in the artwork. This study was supported by the European Regional Development Fund (ERDF) COMPETE – Operational Competitiveness Programme and Portuguese funds through FCT – Foundation for Science and Technology, under the project ‘‘PEst-C/MAR/LA0015/2013’’ and by FCT PTDC/BIA-BCM/114395/2009. JCRC is supported by auxiliary research contract FCT Pluriannual funds attributed to CCMAR.

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Appendix A. Supplementary data

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Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ygcen.2014.05. 025.

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