Jennifer Roche

Sébastien Hergalant

Amandine Depp

Imen Ben Ammar

Anne-Gaëlle Lafont

Tomas Policar

Pascal Fontaine

Sylvain Milla
email: sylvain.milla@univ-lorraine.fr

First identification of dopamine receptors in pikeperch, Sander lucioperca, during the pre-ovulatory period

Keywords: dopamine, RNA-sequencing, de novo transcriptome assembly, pikeperch

Dopamine (DA) is a ubiquitous neurotransmitter exerting a range of pleiotropic actions through two DA receptor families, the D1 and the D2. To date in vertebrates, a maximum of four receptor subtypes have been identified within the D1 family, D 1 (former D 1A ), D 5 (former D 1B ), D 6 (former D 1C ) and D 7 (former D 1E ), while the D2 family encloses five subtypes, D 2 , D 3 , D 4 , D 8 (former D 2like or D 2l ) and D 9 (former D 4-related sequence or D 4-rs ). In teleosts, no study has investigated in parallel all the DA receptors to identify and localize the whole receptor repertoire from both families. In pikeperch, Sander lucioperca, a species of interest for aquaculture development, the existence, number and location of the DA receptors are totally unknown. To address these questions, RNA-seq with de novo transcriptome reconstruction, functional annotation and phylogenetic analysis were performed to characterize the transcript repertoire of DA receptors in the brain of female pikeperch at the pre-ovulatory period. Ten different cDNA were identified and showed to belong to the D1 family: two D 1 , one D 5a , one D 6a and one D 6b and to the D2 family: two spliced variants of D 2 , one D 3 , one D 8 and one D 9. Unlike zebrafish, the subtypes D 4 and D 7 have not yet been isolated in pikeperch. As expected D 1 , D 3 , D 8 and D 9 are mostly expressed in brain parts except for the cerebellum (D 1 and D 3 ).

The inter-species differences in the number of DA receptors and the inter-organ differences in the gene expression of all receptors support the complexity of the dopaminergic actions in vertebrate.

Introduction

Dopamine (DA) is an ubiquitous neurotransmitter found in both central and peripheral nervous systems in many vertebrate species. In the central nervous system, it exerts a range of pleiotropic actions ranging from the control of locomotion, learning, emotion and sexual behaviour to the regulation of pituitary hormone release. This neuro-hormone also plays multiple roles in the peripheral tissues like regulation of the gastrointestinal motility, respiration, blood pressure and insulin secretion [START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Ben-Jonathan | Dopamine as a prolactin (PRL) inhibitor[END_REF][START_REF] Rubí | Minireview: New roles for peripheral dopamine on metabolic control and tumor growth: let's seek the balance[END_REF].

DA actions are mediated through seven transmembrane domain receptors (or G-protein coupled receptors GPCR) divided into two receptor families, namely D1 and D2 families.

These receptor families are distinguished according to their intron-exon gene organization, their primary structure, their pharmacological properties and the stimulatory (D1 family) or inhibitory (D2 family) effects on adenylate cyclase throughout their signalling pathways.

Notably, the D1 genes of the D1 family are intronless, their deduced protein structure exhibit a short third cytoplasmic loop and a long C-terminal tail while the genes of the D2 family contains introns, and the corresponding receptors a long third cytoplasmic loop and a short Cterminal tail [START_REF] Missale | Dopamine receptors: from structure to function[END_REF]. According to the authors and databases, different nomenclatures of genes and receptor subtypes exist, often leading to confusion when it comes to the designation of DA receptors in vertebrates [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. Also, the set of genes and receptors existing into each family is dependent of vertebrate groups. During vertebrate evolution, many DA receptor genes and subtypes have been lost in mammals in comparison with non-mammalian species. Mammals kept only two subtypes in the D1 family (D 1A /D 1 and D 1B /D 5 ) and three subtypes in the D2 family (D 2 , D 3 and D 4 ) each encoded by one gene [START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. In addition, in this vertebrate subgroup, the presence of introns in the D2 family allowed the generation of two receptor variants, D 2long (D 2L ) and D 2short (D 2S ) by an alternative splicing of the D 2 gene [START_REF] Dal Toso | The dopamine D2 receptor: two molecular forms generated by alternative splicing[END_REF]. This phenomenon has already been described in an amphibian, the bullfrog, Rana catesbeiana, but has not yet been found in teleosts, except for the goldfish, Carassius auratus [START_REF] Nakano | Molecular cloning of bullfrog D2 dopamine receptor cDNA: tissue distribution of three isoforms of D2 dopamine receptor mRNA[END_REF][START_REF] Popesku | Evidence for alternative splicing of a dopamine D2 receptor in a teleost[END_REF]. To date, a maximum of nine DA receptor subtypes have been reported in the clade of Osteichthyes [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. Four (former D 2l ) and D 9 (former D 4rs ). In zebrafish, Danio rerio, these nine DA receptors subtypes have been isolated [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. In this species, except for D 7 , D 8 (D 2b in [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF], D 3 and D 9 (D 4b in [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF] encoded by a unique gene, the D 1 , D 5 , D 6 , D 2 and D 4 receptor subtypes are encoded by two paralogous genes giving two distinct receptors per subtype, D 1a and D 1b , D 5a and D 5b , D 6a and D 6b , D 2a and D 2b (called D 2a and D 2c, respectively in [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF], and D 4a and D 4b (called D 4a and D 4c , respectively in [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. As suggested by Yamamoto et al. (2015), these copies likely result from the 3R teleost-specific genome duplication.

However, the duplicated copies resulting from 3R may have not been conserved in all teleost species. For example, in common carp, Cyprinus carpio, and in Nile tilapia, Oreochromis niloticus, only one copy of the D 2 receptor gene has been identified [START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF][START_REF] Levavi-Sivan | Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary[END_REF] whereas in European eel, Anguilla anguilla, rainbow trout, Oncorhynchus mykiss, and goldfish, at least two D 2 receptor genes have been found [START_REF] Vacher | Distribution of dopamine D 2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF][START_REF] Popesku | Evidence for alternative splicing of a dopamine D2 receptor in a teleost[END_REF]. In rainbow trout, these duplicated copies may result directly from the salmonid-specific genome duplication 4R [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF]. As observed in vertebrate species, there is a high diversity and complexity in the transcript profile of DA receptors, which should be deciphered for each species separately.

This high complexity raises the question about the specificities of each receptor in their respective biological functions. The literature provides some indications that the DA receptors roles depend on the receptor subtype and its location. For instance, receptors of the D2 family seems to play an inhibitory or stimulatory role in mammal locomotion whereas receptors of the D1 family would have little or no effect [START_REF] Jackson | Dopamine receptors: molecular biology, biochemistry and behavioural aspects[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF]. However, a synergistic effect between D2 and D1 receptor activation was suggested to increase forward locomotion [START_REF] Missale | Dopamine receptors: from structure to function[END_REF]. In pikeperch, Sander lucioperca, in vivo experiments suggested that the D1 receptor family, but not D2 family, is involved in the regulation of sex-steroid production during the final oocyte meiotic maturation [START_REF] Roche | D1, but not D2, dopamine receptor regulates steroid levels during the final stages of pikeperch gametogenesis[END_REF]. In zebrafish, [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF] suggested that D 2 (D 2a and D 2c ), D 3 and D 8 (D 2b ) receptor subtypes may be involved in visual function. Also, they showed that receptors D 2a and D 8 (or D 2b ) are both expressed in the pineal gland but not at the same developmental stage, leading to the possibility of two different roles within this tissue [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF]. The study of DA receptor functions is often managed through the investigation of their central and peripheral distributions. However, unlike mammals, data regarding the location of DA receptors are very scarce in teleosts. Using in situ hybridization or q-PCR analyses, some studies showed that D 2 receptors are differentially expressed in the brain of species such as European eel, rainbow trout, Nile tilapia, and zebrafish [START_REF] Vacher | Distribution of dopamine D 2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study[END_REF][START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF][START_REF] Levavi-Sivan | Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF]. D 2A and D 2B

in European eel and the D 2 receptors in rainbow trout display high expression levels in most of the brain areas such as the olfactory bulbs, the telencephalon, the optic tectum, and also in the pituitary [START_REF] Vacher | Distribution of dopamine D 2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF]. However, in European eel, only D 2A , but not D 2B , is expressed in the retina, the olfactory epithelium, the spinal cord and the adipose tissue [START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF]. Similarly, receptors from D1 family are differentially expressed according to the brain areas but only D 1,1 and D 1,2 (D 1A1 and D 1A2) are expressed in the pituitary [START_REF] Kapsimali | Distribution of the mRNA encoding the four dopamine D1 receptor subtypes in the brain of the European eel (Anguilla anguilla): comparative approach to the function of D1 receptors in vertebrates[END_REF]. Regarding the location in the peripheral tissues, there are also inter-species peculiarities. Indeed, D 2 receptors are not detected in European eel ovaries, liver, kidney, muscle and gills whereas in Nile tilapia, D 2 receptor gene expression was measured in these tissues, except for kidney and muscle [START_REF] Levavi-Sivan | Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF]. More information about the presence and abundance level of all DA receptors would provide indications about their respective roles in teleost physiology. To date, experimental studies about DA receptors are very scarce, making genomic and protein databases the main informative sources. No investigation has aimed to identify and locate the full DA receptors repertoire for one given teleost species in a single study.

Pikeperch is a percid fish from the perciform order and a species of interest for aquaculture diversification [START_REF] Kestemont | Biology and culture of percid fishes: Principles and practices, Biology and Culture of Percid Fishes: Principles and Practices[END_REF]. Contrary to the situation in cyprinids, salmonids, or anguillids, no data have been evidenced so far on the existence, number and location of DA receptors in percid fishes. Furthermore, few information exists on the expression profile of DA receptors during the final stages of reproduction, a period of huge behavioural and endocrine balance changes. To address these questions, we gathered the repertoire of DA receptors in the brain of maturing pikeperch using whole transcriptome reconstruction from RNA sequencing, analysed their cDNA, inferred protein sequences, compared them with other species and investigated a multi-tissue gene expression pattern of these receptors.

Material & Methods

Animal and sampling procedures

The experiment was performed according to the European and French legislation for fish spleen and liver were also collected and also stored at -80°C for further DA receptor gene expression analysis.

Evaluation of oocyte maturation stages

Before tissue samplings, oocyte maturation stages were evaluated for each female according to the classification by Żarski et al. (2012). Briefly, oocytes were sampled using a catheter (CH06; 1.2 mm internal and 2 mm external diameter) and placed in Serra's solution (ethanol/formalin/glacial acetic acid, 6:3:1 v/v/v). After slowly mixing oocytes in Serra's solution and waiting (about 5 min) until the cytoplasm of the oocyte has become clarified, the oocyte maturation stage was evaluated under the binocular microscope, magnification × 4 (Motic® SFC-11 Series, Motic Asia, Hong Kong, China). In pikeperch, the final stages of maturation were divided into seven morphological stages, from stage I to stage VII (ovulation; Żarski et al., 2012). This allowed the determination of the advancement of the oocyte meiotic maturation at the sampling time. Females were evaluated between the stages II and V. Whole transcriptome-Seq was then performed using a HiSeq 2500 instrument, generating 125-nucleotide-long paired-end reads which were compiled in FASTQ files complying the format standard. Quality control of sequence reads was done using FastQC v0.11.5 software (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). Illumina universal adapters were removed with cutadapt v1.11 [START_REF] Martin | Cutadapt removes adapter sequences from high-throughput sequencing reads[END_REF] discarding trimmed reads shorter than 30 nucleotides, with parameters " -a AGATCGGAAGAGC -A AGATCGGAAGAGC -m 30 -no-indels -O 5 ". Low quality reads were also filtered, discarding bases with a Phred quality score < 20.

De novo transcriptome assembly

The de novo transcriptome assembly was carried out using Trinity v2.2.0 [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF][START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] et al., 2016). Transcripts were then quantified using Kallisto v0.43 [START_REF] Bray | Near-optimal probabilistic RNA-seq quantification[END_REF]. Identification of likely coding regions within each transcript was performed with Transdecoder v3.0.1 (http://github.com/Transdecoder), which makes use of blastp on SwissProt. Each putative peptide was aligned with hmmer3.1 [START_REF] Eddy | Accelerated Profile HMM Searches[END_REF] on the PFAM database [START_REF] Finn | The Pfam protein families database: towards a more sustainable future[END_REF] to match with a protein domain. Signal peptide and transmembrane domain predictions were checked out with signalP v4.1. [START_REF] Petersen | SignalP 4.0: discriminating signal peptides from transmembrane regions[END_REF] and TMHMM v2.0c [START_REF] Krogh | Predicting transmembrane protein topology with a hidden markov model: application to complete genomes[END_REF], respectively. Rnammer-1.2 [START_REF] Lagesen | RNAmmer: consistent and rapid annotation of ribosomal RNA genes[END_REF]) was used to mark potential rRNA remaining transcripts. All the data produced was finally compiled and submitted to Trinotate v3.0.1 (https://github.com/Trinotate) to populate an SQLite database representing the functionally annotated transcriptome, along with blast homologies, as well as gene ontology (GO), orthology (eggNOG) and pathway (KEGG) informations [START_REF] Ashburner | Gene Ontology: tool for the unification of biology[END_REF][START_REF] Huerta-Cepas | eggNOG 4.5: a hierarchical orthology framework with improved functional annotations for eukaryotic, prokaryotic and viral sequences[END_REF][START_REF] Kanehisa | KEGG: new perspectives on genomes, pathways, diseases and drugs[END_REF] The Gene Ontology Consortium, 2017). Annotations were further enriched with custom blasts on a subset of the trEMBL database (http://www.uniprot.org) containing all known and predicted DA receptor sequences.

These populated whole transcriptome databases (one for each sample) were extensively used as a dedicated tool for searching and identifying the DA receptors in pikeperch brain. Among the three females, a total of 30 cDNA sequences were obtained. Whenever possible, each identified DA receptor was computationally confirmed using three ways: 1) multiple alignments with orthologous receptors of closely related species, 2) secondary structures alignments (RaptorX, [START_REF] Källberg | Template-based protein structure modeling using the RaptorX web server[END_REF] with well-characterized DA receptors of the same type were performed to validate an localize functional domains, and 3) the receptor should be found in at least two samples. The degree of identity (%), e-values and bit scores of DA receptor protein sequences between pikeperch and other species were given by blastp [START_REF] Camacho | BLAST+: architecture and applications[END_REF].

Phylogenetic analysis

Amino acid sequences of 74 osteichthyan receptors from the D1 family (D 1 , D 5 , D 6 , D 7 ), and 75 osteichthyan receptors from the D2 family (D 2 , D 3 , D 4 , D 8, D 9 ) were retrieved from NCBI (https://www.ncbi.nlm.nih.gov), Ensembl (http://www.ensembl.org/index.html) and Uniprot databases (Supplementary Table 3). These osteichthyan species included sarcopterygians (tetrapods and a basal sarcopterygian, the coelacanth, Latimeria chalumnae), and actinopterygians (teleosts and a non-teleost actinopterygian, the spotted gar, Lepisosteus oculatus). Human, Homo sapiens, adrenergic receptor sequences were used as outgroup.

Multiple sequence alignments of D1 and D2 protein families were created using Clustal Omega [START_REF] Sievers | Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega[END_REF] included in SeaView version 4.6.3 [START_REF] Gouy | SeaView Version 4: A Multiplatform Graphical User Interface for Sequence Alignment and Phylogenetic Tree Building[END_REF] and manually adjusted. Calculation of the best amino acid substitution matrix was determined using the ProtTest software version 3.4.2 [START_REF] Darriba | ProtTest 3: fast selection of best-fit models of protein evolution[END_REF]. The JTT (Jones, Taylor and Thornton) protein substitution matrix was selected for both alignments. Phylogenetic analyses were performed with the resulting protein alignments using the Maximum Likelihood method with 1000 bootstrap replicates (RaxML software [START_REF] Stamatakis | RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies[END_REF], http://www.phylo.org).

Expression profile analysis of DA receptors

After total RNA extraction using Isol-RNA reagent (VWR International SAS), a Dnase treatment was performed according to the manufacturer's procedure (Fisher Scientific, Illkirch, France). Reverse transcription (RT) was then achieved using 1 µg/µL of total RNA following a previously described procedure [START_REF] Milla | Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis[END_REF]. To determine the multi-tissues mRNA profile of some DA representatives (D 1a , D 1b , D 3, D 8 and D 9 ), Real-time polymerase chain reaction (q-PCR) was carried out using a StepOne Plus system (Applied Biosystems, Fisher Scientific) as previously described [START_REF] Milla | Spleen immune status is affected after acute handling stress but not regulated by cortisol in Eurasian perch, Perca fluviatilis[END_REF]. To validate the specificity of the amplification and the absence of amplified genomic DNA, different controls were performed:

1) sample without Dnase treatment, 2) sample without reverse transcriptase and 3) negative controls with RNase-free water. Gene expression was normalized using the geometric mean of two housekeeping genes: Ribosomal Protein L8 (RPL8) and Adenosine Kinase like (AK), whose expressions were stable under these conditions (data not shown). Primers used for DA receptors and housekeeping genes were described in Table 1. To ensure that primers were specific of each DA receptor they were designed in unconservative regions such as intra and extracellular domains, specific to each receptor. All primer pairs were further checked for uniqueness and concordance within each transcriptome using blastn with 'word size 7' as parameter for small sequence matches.

Statistical analysis

Statistical analyses were performed using R version 3.4.0. For all dependent variables, homogeneity of variances was tested using Levene test (leveneTest, package 'car', [START_REF] Fox | An R companion to applied Regression[END_REF]. For relative DA receptors gene expression, data were analysed by a linear mixed model (lmer, package 'lme4', [START_REF] Bates | Fitting Linear Mixed-Effects Models using lme4[END_REF] with tissues as fixed effects, and either the fish (for repeated measures) and/or the maturation stage at the sampling time as [START_REF] Benjamini | Controlling the false discovery rate: a practical and powerful approach to multiple testing[END_REF]. Data are expressed as mean ± SEM. The level of significance used in all tests was P < 0.05.

RESULTS

Evaluation of the transcriptome assembly

After filtering and trimming low quality reads, sequencing generated an average of 79.9 million read pairs per sample. This led to an average of 272,211 contigs per fish after the Trinity [START_REF] Grabherr | Full-length transcriptome assembly from RNA-Seq data without a reference genome[END_REF][START_REF] Haas | De novo transcript sequence reconstruction from RNA-seq using the Trinity platform for reference generation and analysis[END_REF] assembly step. Re-mapping of the respective reads on each transcriptome reached 99.1% match and 97.4% complete, concordant pair realignment. N50 calculation, based on the longest transcript per "gene", was evaluated at 1000, with an average of 659 nt length (median was at 375 nt). GC content was 44.67%. After estimating their relative abundance, transcripts were flagged for sufficient expression with a minimum of 20 reads per contig required. An average of 91,052 transcripts per sample passed this filter. For our purposes, all transcripts were retained anyway until the identification step.

Putative proteomes were finally computed from each transcriptome with Transdecoder (http://github.com/Transdecoder), which produced an average of 76,976 putative proteins per sample, 40,856 being complete (from the first methionine to the stop codon) and fully annotated. A comprehensive database compiling transcriptomic results was generated at this point (see Material and Methods) and was used throughout the rest of the study.

Identification and sequence analyses of pikeperch DA receptors

Database mining and pairwise comparisons led to the identification of ten distinct cDNAs corresponding to DA receptors. Their deduced amino acid sequences were aligned with human and some teleost and DA receptors from both D1 and D2 families.

Five DA receptors, namely D1_1, D1_2, D1_3, D1_4 and D1_5 (Table 2 and Supplementary

Phylogenetic analysis

Phylogenetic analyses of actinopterygian and sarcopterygian DA receptors amino acid sequences were performed to confirm the identity of the predicted pikeperch sequences.

Regarding the D1 family (Figure 3

Gene expression profile of D 1a and D 1b

The same profile of expression was observed for both genes with a high expression in the brain (Figures 5A andB), predominantly in the olfactory bulbs, optic tectum, telencephalon and rostral ORR, medulla/rostral spinal cord and hypothalamus/IL (Figure 6). Both gene expressions were lower in the cerebellum and in the pituitary, and undetectable in muscle, adipose tissue, gills and spleen. D 1b expression was also undetectable in liver and heart.

Gene expression of D 3 , D 8 and D 9

The three genes were mainly expressed in the brain region (Figures 5C, D andE). The D 3 was mostly expressed in the telencephalon and rostral ORR, olfactory bulbs, hypothalamus/IL and pituitary. The D 8 was predominantly expressed in the optic tectum, medulla/rostral spinal cord and hypothalamus/IL while the D 9 was mostly expressed in olfactory bulbs, telencephalon and rostral ORR, and optic tectum. In the ovaries, D 3 and D 8 gene expressions were above the detection limit while the D 9 mRNA was undetectable. In the heart, D 3 had an intermediate expression level. Finally, the gene expressions were above the detection limit in the liver for D 3 and D 8 , in the heart for D 8 and D 9 , in the muscles for D 8 , in the gills for D 3 and undetectable in the remaining tissues.

Discussion

In this study, we identified ten DA receptor mRNAs isolated from the brain of pikeperch at the pre-ovulatory period.

Five DA receptor sequences were attributed to the D1 family using amino acid sequence comparisons and phylogenetic analyses. These sequences share between 52% and 93% of sequence identity with other vertebrate D 1 receptors and only 21-41% with vertebrate D 2 receptors. Besides the seven TMDs found in complete sequences and the DRY sequence specific of GPCR, these receptors share common features with D 1 receptors characterized in other vertebrate species, including mammals and teleosts. Among these characteristics, we observed the short third intracellular cytoplasmic loop and a long unvariable C terminal cytoplasmic tail enclosing a conserved cysteine residue allowing to anchor the receptor to the membrane [START_REF] Civelli | Molecular diversity of the dopamine receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Ben-Jonathan | Dopamine as a prolactin (PRL) inhibitor[END_REF]. Some conserved amino acid residues allowing the receptor functionality and regulation were also observed. For instance, we found an aspartate in TMD 2 and 3 or two serines in TMD 5 that form the narrow pocket for the ligand binding, two cysteines in the first and second extracellular loop that allow the stabilization of the receptor conformation with a disulfide bond, or also some phosphorylation/glycosylation sites in the different cytoplasmic and extracellular loops [START_REF] Civelli | Molecular diversity of the dopamine receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF]. Phylogenetic analysis assigned these five pikeperch sequences to specific DA receptor clades, D 1 , D 5 and D 6 [START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. Thus, we assume that pikeperch possesses at least five D1

family DA receptor genes that are orthologous to D 1a , D 1b , D 5a , D 6a and D 6b .

These different D 1 receptor paralogs have already been identified in literature or have been released in the relevant databases. A single D 1 receptor was identified in sarcopterygian species, such as mammals, birds, and amphibians, as well as in the spotted gar, a non-teleost actinopterygian. Two D 1 paralogs were found in several teleost species, including European eel, common carp, zebrafish, and Nile tilapia [START_REF] Sugamori | D1A, D1B, and D1C dopamine receptors from Xenopus laevis[END_REF][START_REF] Demchyshyn | The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus[END_REF][START_REF] Macrae | Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes[END_REF][START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]; NCBI, Ensembl and Uniprot databases), probably as a consequence of the teleost-specific genome duplication (3R). In some species such as in Atlantic herring (Figure 3), one paralog is likely to be secondarily lost. But the pikeperch would have conserved these two D 1A paralogs, as most of the teleosts.

In the same way, two D 6 receptors were identified in pikeperch, as in other teleosts, such as Atlantic herring, Clupea harengus, zebrafish, and Nile tilapia, while a single D 6 is present in sarcopterygians and in non-teleost actinopterygian [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. In European eel, Japanese medaka, Oryzias latipes, Japanese pufferfish (fugu), Takifugu rubripes, and threespined stickleback, Gasterosteus aculeatus, only one D 6 paralog was identified [START_REF] Sugamori | D1A, D1B, and D1C dopamine receptors from Xenopus laevis[END_REF][START_REF] Macrae | Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes[END_REF][START_REF] Lamers | Cloning and sequence analysis of a hypothalamic cDNA encoding a D1C dopamine receptor in tilapia[END_REF][START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF]. These results suggest that the two D 6 identified in the pikeperch are issued from 3R, and that one copy may have been lost in some species throughout teleost radiation.

Concerning D 5 , a single receptor is present in sarcopterygians, while two D 5 receptors were evidenced in some teleosts species, such as Japanese medaka, three-spined stickleback, Nile tilapia, fugu and Atlantic herring [START_REF] Sugamori | D1A, D1B, and D1C dopamine receptors from Xenopus laevis[END_REF][START_REF] Demchyshyn | The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus[END_REF][START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF]Yamamoto et al., 2015 NCBI, Ensembl and Uniprot databases). In contrast, only one D 5 receptor, othologous to the pikeperch D1_3, was evidenced in European eel, Asian bonytongue, Scleropages formosus, zebrafish and European seabass, Dicentrarchus labrax [START_REF] Sugamori | D1A, D1B, and D1C dopamine receptors from Xenopus laevis[END_REF][START_REF] Demchyshyn | The dopamine D1D receptor. Cloning and characterization of three pharmacologically distinct D1-like receptors from Gallus domesticus[END_REF][START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF]Yamamoto et al., 2015 NCBI, Ensembl and Uniprot databases). From the phylogenetic analysis, the single spotted gar D 5 branched at the base of the teleost D 5a clade, and in polytomy with the sarcopterygian and the teleost D 5b (named D 1X in [START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF] clades. This result does not suggest that the two teleost D 5 paralogs result from 3R. As this study was based on brain transcriptome analysis, and with the absence of a pikeperch genome in the available databases, we cannot ascertain the absence of a D 5b paralog in this species.

Five receptor sequences, belonging to the D2 receptor family, have herein been identified in pikeperch. These sequences share from 33% to 95% identity with other receptors of D2 family in vertebrates and only 29-46% with receptors of D1 family. The molecular structure is also consistent with the one described in other vertebrate species: the seven TMDs, the long variable third cytoplasmic loop and a short and conserved C terminal tail ending with a conserved cysteine residue. Similarly to the receptors of D1 family, some conserved amino acid residues were also observed in pikeperch sequences of the D2 family. For instance, the primary structure possesses the aspartate in TMD 2 and 3, the two serines in TMD 5, the two cysteines in the first and second extracellular loops or the phosphorylation/glycosylation sites in the different loops [START_REF] Civelli | Molecular diversity of the dopamine receptors[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Levavi-Sivan | Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary[END_REF].

Phylogenetic analysis revealed that the pikeperch receptors of the family D2 are orthologous to D 2 , D 3 , D 8 , and D 9 . From our study based on brain transcriptome analysis, we could not evidence in the pikeperch the presence of 3R-paralogs belonging to the D2 receptor family, suggesting the loss of a 3R-duplicated D 2 , D 3 , D 8 , and D 9 during evolution in pikeperch.

All these D2 family receptors have been previously identified in vertebrates including several teleost species. For instance, a D 8 sequence was cloned in zebrafish [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF], and identified in silico in other actinopterygians, such as spotted gar, Nile tilapia, Japanese medaka and a sarcopterygian, the coelacanth [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]; NCBI, Ensembl and Uniprot databases). Phylogenetic analysis clearly revealed that the actinopterygian and sarcopterygian D 8 sequences clustered in a single clade distinct to the other vertebrate D 2 . Our results are consistent with [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF] and [START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF], suggesting that this receptor could be the product of an early gene duplication during vertebrate evolution, prior to divergence of the Osteichthyes group.

In the D 8 sister clade, encompassing D 2 sequences, two paralogs have been evidenced in European eel, Asian bonytongue, Atlantic herring and zebrafish, likely resulting from 3R [START_REF] Vacher | Distribution of dopamine D 2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study[END_REF][START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF][START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF][START_REF] Popesku | Evidence for alternative splicing of a dopamine D2 receptor in a teleost[END_REF]NCBI database). In contrast, only one D 2 receptor gene could be identified in pikeperch, as in other teleost, such as Japanese medaka, tilapia and fugu, suggesting the loss of the duplicated paralog in these species. This D 2 gene encodes for two identical receptors D 2aL and D 2aS , except for a 29 amino acid sequence missing in the third cytoplasmic loop of the D 2aS . This has already been reported in some mammals including human and rat [START_REF] Dal Toso | The dopamine D2 receptor: two molecular forms generated by alternative splicing[END_REF][START_REF] Monsma | Multiple D2 dopamine receptors produced by alternative RNA splicing[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF] but also in bullfrog [START_REF] Nakano | Molecular cloning of bullfrog D2 dopamine receptor cDNA: tissue distribution of three isoforms of D2 dopamine receptor mRNA[END_REF]. Taking advantage of the phylogenetic results, regarding the mammalian model and the full identity between both sequences, we suggest an alternative splicing of the pre-messenger of the unique D 2a gene in pikeperch. This phenomenon has been described before in goldfish [START_REF] Popesku | Evidence for alternative splicing of a dopamine D2 receptor in a teleost[END_REF], but to date this has never been confirmed in another teleost.

Nevertheless, no alternative splicing has been highlighted in a wide range of vertebrate species including xenopus [START_REF] Martens | Expression of the Xenopus D2, dopamine receptor. Tissue-specific regulation and two transcriptionally active genes but no evidence for alternative splicing[END_REF], fugu [START_REF] Macrae | Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes[END_REF], common carp [START_REF] Hirano | Dopamine receptor subtypes expressed in vertebrate (carp and eel) retinae: cloning, sequencing and comparison of five D1-like and three D2-like receptors[END_REF], rainbow trout [START_REF] Vacher | Distribution of dopamine D 2 receptor mRNAs in the brain and the pituitary of female rainbow trout: an in situ hybridization study[END_REF], zebrafish [START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF], Nile tilapia [START_REF] Levavi-Sivan | Cloning, characterization and expression of the D2 dopamine receptor from the tilapia pituitary[END_REF] and in European eel [START_REF] Pasqualini | Two distinct dopamine D2 receptor genes in the European eel: molecular characterization, tissue-specific transcription, and regulation by sex steroids[END_REF].

Taking into account the involvement of the third cytoplasmic loop in the coupling to the protein G, it has been proposed that this splicing could be involved in the functional diversity of the D 2 receptors [START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Ben-Jonathan | Dopamine as a prolactin (PRL) inhibitor[END_REF][START_REF] Callier | Evolution and cell biology of dopamine receptors in vertebrates[END_REF]. Indeed, [START_REF] Senogles | A region of the third intracellular loop of the short form of the D2 dopamine receptor dictates Gi coupling specificity[END_REF] suggested that the splice variants could be coupled to a different Gi protein. Other authors showed that, besides their distinct expression in the brain, the D 2S receptor could be presynaptically located while the D 2L may be found in the postsynaptic region in mammals [START_REF] Khan | Prominence of the dopamine D2 short isoform in dopaminergic pathways[END_REF]. Conversely, [START_REF] Tress | Alternative splicing may not be the key to proteome complexity[END_REF] suggested that most annotated splice variants could lead to unfunctional proteins at a cellular level and a debate remains open about the physiological roles of these variants.

Relatively to D 3 , a single copy has been evidenced in some mammals, sauropsids and some teleosts such as fugu, zebrafish, Japanese medaka, European seabass, and Nile tilapia [START_REF] Macrae | Analysis of the dopamine receptor family in the compact genome of the puffer fish Fugu rubripes[END_REF][START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Boehmler | Evolution and expression of D2 and D3 dopamine receptor genes in zebrafish[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]NCBI database). This suggests that one of the 3R duplicated D 3 paralogs may have been lost shortly after the teleost emergence.

In the same way, a single copy of D 9 was cloned in zebrafish [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF] and was identified in databases for some other teleost species such as Nile tilapia, Japanese medaka, in the non-teleost actinopterygian spotted gar and the sarcopterygian coelacanth (Yamamoto et al., 2015;NCBI, Ensembl and Uniprot databases). This again suggests an early loss of one of the D 9 3R-paralogs during teleost radiation.

Concerning the D 4 subtype, 3R duplicated paralogs are present in some teleosts, such as zebrafish, Atlantic herring, Asian bonytongue and European eel, while a single copy has been evidenced in Nile tilapia, platyfish, Japanese medaka, and fugu [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF]; NCBI, Ensembl and Uniprot databases). This suggests that different loss events may have occurred during teleost radiation. In the present transcriptome, no DA receptor orthologous to D 4 has been evidenced in the pikeperch.

Few studies have investigated D 4 and D 9 subtypes in teleosts besides [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF], which reported three distinct genes encoding those receptors, two genes encoding the D 4 subtype (D 4a and D 4b (or D 4c )) and a single gene encoding the D 9 subtype. As suggested in previous studies, the existence of D 4 and D 9 subtypes would result from a duplication event that occurred occurred before the split of sarcopterygians and actinopterygians [START_REF] Boehmler | D4 dopamine receptor genes of zebrafish and effects of the antipsychotic clozapine on larval swimming behaviour[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF].

Several hypotheses might explain the absence of D 5b , D 2b , both D 4 paralogs and D 7 in all female pikeperch specimens analysed in the present study. First, some receptors like D 2b and D 7 may have been lost during vertebrate evolution in most of the teleost lineages [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Yamamoto | Classification of dopamine receptor genes in vertebrates: nine subtypes in Osteichthyes[END_REF]. Other paralogs, like D 5b , may have been lost only in some perciform species, including pikeperch. Second, we could also speculate that the genes encoding some of these receptors in fact correspond to unfunctional pseudogenes [START_REF] Prince | Splitting pairs: the diverging fates of duplicated genes[END_REF] whose transcripts remain undetectable in the brain. To address these questions, an exhaustive genome sequencing in pikeperch would be useful. Third, the present study analysed the transcripts during the prespawning period, a highly specific physiological situation. This event of final oocyte maturation is synchronous with huge alterations of the brain and gonad transcriptomes. We may preclude low and barely detectable gene expression of these receptors before ovulation in pikeperch (Aegerter et al., 2004). Fourth, we cannot rule out the fact that the lack of identification may stem from methodological limitations (e.g. assembly, detection thresholds, incomplete references/databases).

All DA receptors do not have an undetectable gene expression in pikeperch. Indeed, all the multi-tissues gene expression patterns revealed a specific and measurable expression for D 1a , D 1b , D 3 , D 8 and D 9 .

Despite the rough examination, the gene expression study showed relatively high expression of DA receptors in brain regions compared to peripheral organs. This would be in accordance with the importance of DA in neurophysiological functions [START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF]. The brain profile also showed a part-specific gene expression of these receptors.

For instance, the two D 1A are ubiquitously expressed in the brain except for the cerebellum where the expression is slightly lower. These low levels of D 1A mRNA in the cerebellum have previously been reported in mammals and European eel [START_REF] Mansour | A comparison of D1 receptor binding and mRNA in rat brain using receptor autoradiogaphy and in situ hybridization techniques[END_REF][START_REF] Mengod | Visualization of dopamine D1, D2 and D3 receptor mRNA's in human and rat brain[END_REF][START_REF] Laurier | Heterogeneous tissue-specific transcription of dopamine receptor subtype messenger RNA in rat brain[END_REF][START_REF] Cardinaud | Early emergence of three dopamine D1 receptor subtypes in vertebrates[END_REF][START_REF] Kapsimali | Distribution of the mRNA encoding the four dopamine D1 receptor subtypes in the brain of the European eel (Anguilla anguilla): comparative approach to the function of D1 receptors in vertebrates[END_REF]. On the contrary, some receptor mRNA are present in the brain parts related to sensory perception (e.g. olfactory bulb) or reproductive function (e.g. optic recess region). In situ hybridization would be useful to locate the DA receptor genes more precisely. For example, if a DA receptor gene is colabelled with GnRH transcripts, it indicates a possibility that DA directly interacts with GnRH neurons.

Pituitary showed intermediate and low transcript abundance for the two D 1A , and an increased expression level for the D2 family receptors. Using specific DA receptor antagonists, it was demonstrated that the D2 family receptors, rather than the D1 ones, might be involved in gonadotropin secretion [START_REF] Dufour | Neuroendocrine control by dopamine of teleost reproduction[END_REF][START_REF] Fontaine | The dopaminergic neurons controlling anterior pituitary functions: anatomy and ontogenesis in zebrafish[END_REF]. Nevertheless, we recently showed that in vivo treatment with an antagonist of the family D1 increased the T and E2 plasmatic levels, while no effect was observed with an antagonist of the family D2 [START_REF] Roche | D1, but not D2, dopamine receptor regulates steroid levels during the final stages of pikeperch gametogenesis[END_REF]. We thus imagine that this divergence between both DA receptor families localization in the pituitary is not linked to the activation of the gonadotropic axis in pikeperch at this pre-ovulatory period. In the gonad, the gene expression is either just above the quantification limit (D 1a and D 9 ) or simply low (D 1b , D 3 , D 8 ). Given this level of detection, we cannot rule out any direct effect of DA through its receptors on ovarian mechanisms [START_REF] Isobe | The role of the dopaminergic system in the rat ovary[END_REF][START_REF] Venegas-Meneses | Effects of ovarian dopaminergic receptors on ovulation[END_REF].

Finally, depending on the receptor type, the expression level is also just detectable in heart, liver or gills, which might be put in relation with involvements of DA in osmoregulatory mechanisms, heart beats and energetic metabolism in teleost fish [START_REF] Missale | Dopamine receptors: from structure to function[END_REF][START_REF] Rubí | Minireview: New roles for peripheral dopamine on metabolic control and tumor growth: let's seek the balance[END_REF]. In the spleen, muscle and adipose tissue, no expression was observable, which allows us to conclude on the non-involvement of these receptors in these tissues.

In *correspond to incomplete sequences not included in the phylogenetic analyses.

  subtypes would be assigned to the D1 family, D 1 (former D 1A ), D 5 (former D 1B ), D 6 (former D 1C ) and D 7 (former D 1E ) while the D2 family would enclose five subtypes, D 2 , D 3 , D 4 , D 8

(

  D1_1), D 1b (D1_2), D 5a (D1_3), D 6a (D1_4), D 6b (D1_5), D 2 for two sequences sharing 100% of identity (D2_1 and D2_2), D 3 (D2_3), D 8 (D2_4) and D 9 (D2_5). Amino acid sequence alignments of the 10 putative pikeperch DA receptors with human and teleost DA receptors showed structure conservation. Two examples from D1 and D2 families: D 1a , D 1b and both D 2 are shown in Figures 1 and 2, respectively. The other sequence alignments are shown in Supplementary Figures1, 2, 3, 4 and 5. All sequences are complete except for the predicted pikeperch D 5a and D 6b for which two and four of the seven putative transmembrane domains (TMD) are missing, respectively. For both families, the TMD are highly conserved relatively to the TMD of other species. Also, the specific GPCR DRY motif is observed within all sequences, except for the predicted partial D 6b polypeptide (Supplementary Material). Regarding the D1 family (Figure1and Supplementary Materials), we observed a short third cytoplasmic loop and a long and variable C-terminal tail enclosing a cysteine amino acid at its beginning, except for the partial D 6b . In contrast, we noticed a long and variable third cytoplasmic loop and a short and relatively well-conserved cytoplasmic tail ending with a cysteine residue for all predicted receptors of the D2 family. Regarding one of both D 2 sequences, a gap in the protein sequence within the third intracellular loop (29 missing amino acids) was observed (Figure2). The two D 2 sequences were renamed D 2Long (D 2L ) and D 2Short (D 2S ), as in mammals. Highly conserved amino acid residues between pikeperch and other vertebrate species were also observed in TMD and extracellular and cytoplasmic loops. For instance, we observed one conserved aspartate in TMD II and III, one asparagine in TMD VII, two serines in TMD V and two cysteines in extracellular loops I and II (Figure2).

  ), the phylogeny analysis clustered D 6 , D 1 and D 5 sequences in three well-supported clades, with bootstrap values of 91, 98 and 67%, respectively. However, the few D 7 sequences retrieved from the available databases were not encompassed in a single monophyletic clade. The D 1 clade was composed of two distinct subclades: a sarcopterygian and an actinopterygian, with the non-teleost actinopterygian spotted gar branching at the base of the actinopterygian clade, in accordance with its phylogenetical position. Teleost D 1 sequences were separated into two clades, named D 1a and D 1b . Pikeperch D1_1 and D1_2 sequences branched individually within each one of these clades, confirming their identification as D 1a and D 1b . D 5 sequences were separated into three clades: a sarcopterygian clade, and two actinopterygian clades. The first actinopterygian clade encompassed the teleost D 5a sequences with the spotted gar D 5 branching at its base, and the second one encompassed the D 5b teleost sequences. The D1_3 pikeperch sequence branched together with the D 5a teleost sequences, confirming its place in the nomenclature as D 5a . Concerning D 6 , the actinopterygian clade, with the spotted gar D 6 sequence branching at its base, encompassed two well-supported teleost clades, each one including a pikeperch sequence belonging to the D1 family. Pikeperch D1_4 and D1_5 were thus named D 6a and D 6b , respectively. Concerning the D2 family, phylogenetic analysis clustered all vertebrate sequences in two strongly-supported clades, D 4 /D 9 (100%) on one side and D 3 /D 2 /D 8 (91%) on the other side. A clear demarcation was observed between D 4 (80%) and D 9 (89%) sequences within the D 4 /D 9 clade. Within the D 3 /D 2 /D 8 clade, D 3 (90%), D 2 (78%) and D 8 (68%) sequences diverged into three subclades. Into each one of the D 4 , D 9 , D 3 , D 2 and D 8 subclades, a well-supported actinopterygian branch was defined. The spotted gar D 4 sequence branched at the base of the two teleost D 4 clades, D 4a and D 4b , while the spotted gar D 9 branched with a single teleost D 9 clade, including the pikeperch D2_5 sequence. The spotted gar D 3 sequence branched at the base of a single teleost D 3 clade, including the D2_3 pikeperch sequence. Accordingly, these pikeperch sequences were respectively named D 9 and D 3 . Similarly, the spotted gar D 8 sequence branched together with the coelacanth D 8 one, within a single teleost D 8 clade, including the D2_4 pikeperch sequence, now called D 8 . The spotted gar D 2 sequence branched at the base of two wellsupported clades, named D 2a (73%) and D 2b (60%). The pikeperch D 2L sequence branched together with other teleost D 2a sequences. Thus, the two pikeperch D 2 sequences (D2_1 and D2_2), differing only by a contiguous sequence of 29 amino acids, corresponded to the long and short isoforms of a single D 2 gene, hereby named D 2a (D 2aL and D 2aS ). No pikeperch sequences orthologous to vertebrate D 4 were evidenced in this study. 3.4. Multi-tissue mRNA abundance of DA receptors from the D1 and D2 families in pikeperch Over the ten sequences of DA receptors found by RNA-seq, the multi-tissue gene expression pattern focused on five receptor subtypes as we failed to design good primers for the five remaining ones.

  summary, five D 1 DA receptors, D 1a , D 1b , D 5a , D 6a and D 6b and five D 2 DA receptors, D 2L and D 2S , D 3 , D 8 and D 9, sharing features specific to each DA receptor family, were identified in pikeperch during the pre-ovulatory period. Presence of two receptor families (D1 and D2), multiple receptor subtypes (D 1 , D 5 , D 6, D 2 , D 3 , D 8 and D 9 ), some teleost-specific paralogs for some subtypes (D1a and D1b, D6a and D6b) and even spliced variants (D 2aL and D 2aS ) highlight the complexity of DA receptors in teleost fish. These receptors are differentially expressed in different brain areas and in the pituitary, but also in some peripheral tissues (gonads, heart, liver, gills) indicating roles of DA through one of its receptors in different physiological functions.

Figure captions Figure 1 :

 captions1 Figure captions

Figure 2 :

 2 Figure 2: Alignment of the two deduced pikeperch D 2 receptors with those of other

Figure 3 :

 3 Figure 3: Consensus phylogenetic tree of vertebrate D1 receptor family. This

2.3. RNA-seq & de novo transcriptome assembly 2.3.1. RNA sequencing

	Total RNA from three brain samples was extracted using Isol-RNA reagent (VWR
	International SAS, Strasbourg, France) according to the manufacturer's protocol. The extracts
	were then sent to the McGill University and Genome Quebec Innovation Centre (Montreal,
	Québec, Canada) where RNA concentration was measured using a NanoDrop
	Spectrophotometer ND-1000 (NanoDrop Technologies, Inc.) and RNA integrity was assessed
	using a 2100 Bioanalyzer (Agilent Technologies). Libraries were generated from 250 ng of
	total RNA using the TruSeq stranded mRNA Sample Preparation Kit (Illumina), following the
	manufacturer's recommendations. Libraries were quantified using the Quant-iT™
	PicoGreen® dsDNA Assay Kit (Life Technologies) and the Kapa Illumina GA with Revised
	Primers-SYBR Fast Universal kit (Kapa Biosystems). Average fragment size was determined
	using a LabChip GX (PerkinElmer) instrument.

Table 1

 1 related to zebrafish and tilapia D 5b or to zebrafish D 7 receptors at these levels

	Pikeperch D1_2 shared high percentage of sequence identity with zebrafish D 1b (84%), Nile
	tilapia D 1b (93%) and European eel D 1,1 (84%). D1_3 showed 66% of sequence identity with
	the human D 5 receptor while D1_4 displayed 60% of sequence identity with the human D 1
	receptor. Relatively to teleost receptors, D1_3 shared similar sequence identity with zebrafish
	D 1a and D 5a (71-72%) and a high percentage of sequence identity with the Nile tilapia D 5a
	(93%) and European eel D 5 (85%). D1_4 displayed the highest identity with the zebrafish and
	Nile tilapia D 6a (66 and 79%, respectively) and European eel D 6 receptor (65%). Finally,
	D1_5 sequence shared similar sequence identities with human D 1 and D 5 receptors (61-62%)

), belonged to the D1 family (only 21-41% of identity with human and teleost D2 receptors family). Among these five receptors, D1_1 and D1_2 displayed the highest sequence identity with human D 1 receptor (range between 72-73%). Relatively to teleost species, D1_1 displayed about 80% of sequence identity with zebrafish and European eel D 1 receptors while it displayed the highest identity with the Nile tilapia D 1a receptor (93%).

while it displayed the highest sequence identities with zebrafish D 6a and D 6b (76%), Nile tilapia D 6b (82%) and European eel D 6 (76%) receptors. No pikeperch sequences identified in this study were shared 58% of sequence identity with human D 3 while he displayed the highest sequence identities with zebrafish and Nile tilapia D 3 (75-90%). D2_4 shared highest sequence identity with Nile tilapia D 8 (89%) and zebrafish D 8 (75%) receptors. Finally, D2_5 shared 68% of sequence identity with human D 4 while he displayed the highest sequence identities with zebrafish and Nile tilapia D 9 (71-76%) receptors, respectively. Accordingly, we propose the following designation for those ten DA receptor sequences: D 1a

Table 1 : Primers used for real-time PCR

 1

		Sequences (5' -3')
		Forward	Reverse	Size (bp)
	D 1a	CTTCATCCTCAACTGCATGGT	CCTCCTGGATACAGTCTGTGG	204
	D 1b	CCAGAGAGAGACTCGTCCAAA TTGCAGAAAGCACCAAACGG	196
	D 3	TGGTGTGCTCCATCTCCAAC	GCCACAGGCAAACTGACAAC	306
	D 8	TGCCATCTCCTGCCCTTTAC	CCATCGCCTCCTTTACCAGG	255
	D 9	CGTCGTAGGGGTTTTTCTAGC GCAGAGCAGCTTGTGAAAGAC 199
	RPL8 GTTATCGCCTCTGCCAC	ACCGAAGGGATGCTCAAC	163
	AK	CTTCCTGACCGTCTCTTTGG	CCTTAGTCTCGAAGTCTTGC	209

Table 2 :

 2 Pairwise comparisons within the D1 receptor family between pikeperch, zebrafish (ZF) and human (H) receptor protein sequences

	Pikeperch		D1_1	D1_2	D1_3	D1_4	D1_5
	H D 1	Id % (Sc)	72.75 (513)	72.15 (621)	57.30 (397)	59.53 (439)	61.94 (188)
		e-value	0.0	0.0	1.88e-140	1.28e-154	4.58e-62
	H D 5	Id % (Sc)	67.45 (455)	56.76 (477)	66.13 (492)	51.84 (461)	61.24 (197)
		e-value	2.72e-162	1.54e-169	2.75e-177	1.79e-162	3.74e-65
	ZF D 1a	Id % (Sc)	81.03 (570)	82.37 (575)	71.43 (382)	65.63 (435)	62.81 (189)
		e-value	0.0	0.0	1.14e-135	4.06e-154	2.95e-63
	ZF D 1b	Id % (Sc)	79.95 (565)	84.13 (756)	59.07 (404)	62.97 (437)	66.46 (208)
		e-value	0.0	0.0	2.91e-143	1.88e-153	1.26e-69
	ZF D 5a	Id % (Sc)	64.71 (272)	64.35 (286)	72.19 (421)	58.58 (290)	66.88 (199)
		e-value	2.40e-93	1.20e-97	1.80e-152	8.15e-99	3.73e-68
	ZF D 5b	Id % (Sc)	61.45 (419)	62.08 (417)	59.13 (370)	57.65 (405)	59.24 (171)
		e-value	2.70e-149	1.75e-147	2.21e-130	7.51e-142	6.25e-56
	ZF D 6a	Id % (Sc)	68.31 (476)	57.52 (495)	61.30 (420)	65.77 (597)	76.19 (224)
		e-value	4.59e-171	3.74e-177	2.24e-149	0.0	9.17e-76
	ZF D 6b	Id % (Sc)	70.54 (475)	68.75 (480)	60.28 (409)	59.84 (533)	75.97 (228)
		e-value	4.77e-171	1.05e-171	2.08e-145	0.0	8.43e-78
	ZF D 7	Id % (Sc)	62.82 (431)	55.71 (455)	56.43 (377)	59.84 (446)	62.18 (185)
		e-value	2.50e-153	1.22e-161	1.49e-132	4.07e-157	8.21e-61
	Id = Identity; Sc = bit Score

Table 3 :

 3 Pairwise comparisons within the D2 receptor family between pikeperch, zebrafish (ZF) and human (H) protein sequences

	Pikeperch			D2_1		D2_2		D2_3		D2_4	D2_5
	H D 2L		Id % (Sc)	66.10 (602)	63.23 (557)	53.76 (438) 56.47 (456) 38.48 (262)
			e-value	0 .0		0.0		8.44e-155	2.94e-161	7.23e-87
	H D 2S		Id % (Sc)	62.50 (559)	66.37 (571)	53.68 (440) 54.98 (436) 41.18 (274)
			e-value	0.0		0.0		4.47e-156	8.36e-154	1.13e-91
	H D 3		Id % (Sc)	48.89 (388)	53.15 (403)	57.72 (449) 49.22 (368) 40.96 (276)
			e-value	1.32e-135	4.63e-142		9.23e-160	1.63e-127	1.47e-92
	H D 4		Id % (Sc)	46.88 (152)	36.49 (228)	38.22 (232) 53.49 (150) 68.04 (228)
			e-value	2.33e-44		6.86e-73		2.22e-74	1.76e-43	2.33e-73
	ZF D 2a		Id % (Sc)	73.35 (684)	69.44 (632)	51.45 (418) 54.96 (471) 33.63 (265)
			e-value	0.0		0.0		6.88e-147	1.92e-167	6.38e-88
	ZF D 2b		Id % (Sc)	73.25 (677)	71.15 (617)	53.60 (438) 58.02 (500) 37.09 (276)
			e-value	0.0		0.0		6.56e-155	1.15e-178	4.04e-92
	ZF D 3		Id % (Sc)	50.10 (447)	53.06 (456)	74.57 (666) 49.31 (386) 38.02 (270)
			e-value	3.87e-158	8.14e-162		0.0		5.62e-134	1.55e-89
	ZF D 4a		Id % (Sc)	47.62 (155)	36.62 (228)	48.54 (139) 36.59 (222) 51.39 (343)
			e-value	6.50e-46		5.54e-74		2.30e-40	3.04e-71	2.33e-119
	ZF D 4b		Id % (Sc)	35.28 (253)	37.53 (258)	36.96 (244) 35.85 (251) 54.10 (394)
			e-value	3.46e-83		2.12e-85		5.65e-80	1.90e-82	5.79e-139
	ZF D 8		Id % (Sc)	59.23 (490)	58.47 (457)	51.64 (385) 74.61 (610) 41.06 (271)
			e-value	2.56e-175	7.06e-163		5.39e-134	0.0		1.71e-90
	ZF D 9		Id % (Sc)	37.05 (249)	37.12 (253)	39.59 (262) 42.48 (274) 70.76 (535)
			e-value	3.94e-81		2.15e-83		1.35e-86	1.12e-90	0
	D 2S	=	D 2Short ;	D 2L	=	D 2Long ;	Id	=	Identity;	Sc	=	bit	Score

Table 1 :

 1 Pairwise comparisons within the D1 receptor family between pikeperch, Nile tilapia and European eel receptor protein sequences

	Pikeperch		D1_1	D1_2	D1_3	D1_4	D1_5
	Tilapia D 1a	Id % (Sc)	92.74 (674)	79.30 (562)	66.91 (368)	62.19 (434)	61.05 (203)
		e-value	0.0	0.0	5.42e-130	1.46e-153	1.50e-68
	Tilapia D 1b	Id % (Sc)	79.36 (583)	92.66 (883)	57.14 (405)	62.40 (463)	64.29 (206)
		e-value	0.0	0.0	2.01e-143	1.53e-163	1.60e-68
	Tilapia D 5a	Id % (Sc)	67.89 (468)	59.02 (500)	93.47 (685)	55.39 (478)	61.21 (194)
		e-value	7.05e-168	6.48e-179	0.0	1.63e-169	2.48e-64
	Tilapia D 5b	Id % (Sc)	60.21 (437)	61.11 (443)	62.14 (368)	58.07 (426)	54.75 (174)
		e-value	1.94e-155	1.22e-156	6.11e-129	2.81e-149	1.82e-56
	Tilapia D 6a	Id % (Sc)	63.01 (461)	55.82 (481)	56.17 (395)	78.91 (758)	61.70 (207)
		e-value	1.21e-164	4.80e-171	8.39e-139	0.0	1.08e-68
	Tilapia D 6b	Id % (Sc)	68.27 (468)	66.30 (481)	58.97 (391)	59.25 (531)	82.47 (254)
		e-value	5.17e-168	1.27e-171	5.26e-138	0.0	1.02e-87
	Eel D 1,1	Id % (Sc)	80.00 (565)	83.62 (735)	62.60 (427)	58.50 (452)	66.06 (207)
		e-value	0.0	0.0	2.95e-152	1.08e-159	2.56e-69
	Eel D 1,2	Id % (Sc)	80.05 (598)	80.13 (731)	59.33 (408)	62.40 (467)	63.47 (201)
		e-value	0.0	0.0	7.38e-145	2.28e-165	5.22e-67
	Eel D 5	Id % (Sc)	68.73 (464)	59.91 (496)	85.27 (620)	55.18 (478)	63.19 (197)
		e-value	2.03e-166	1.94e-177	0.0	2.17e-169	2.02e-65
	Eel D 6	Id % (Sc)	70.21 (484)	69.60 (492)	63.23 (429)	65.37 (594)	75.80 (246)
		e-value	3.19e-174	4.87e-176	7.73e-153	0.0	2.22e-84
	Id	=	Identity;	Sc	=	bit	Score

Table 2 :

 2 Pairwise comparisons within the D2 receptor family between pikeperch, Nile tilapia and European eel receptor protein sequences

	Pikeperch		D2_1	D2_2	D2_3	D2_4	D2_5
	Tilapia D 2	Id % (Sc)	94.88 (918)	89.13 (844)	52.16 (429)	57.02 (496)	35.98 (271)
		e-value	0.0	0.0	5.51e-151	7.78e-177	7.16e-90
	TilapiaD 3	Id % (Sc)	51.16 (435)	52.69 (441)	89.50 (832)	51.13 (388)	37.34 (275)
		e-value	2.78e-153	4.53e-156	0.0	1.42 e-134	8.97e-92
	TilapiaD 4a	Id % (Sc)	34.73 (238)	37.62 (259)	43.72 (173)	36.13 (256)	53.39 (407)
		e-value	4.31e-77	1.39e-85	1.09e-52	5.51e-84	8.35e-144
	Tilapia D 4b	Id % (Sc)	33.04 (223)	34.66 (225)	36.22 (231)	45.55 (164)	51.68 (363)
		e-value	1.01e-71	9.68e-73	4.72e-75	3.74e-49	7.81e-127
	Tilapia D 8	Id % (Sc)	57.27 (489)	57.64 (460)	49.77 (387)	88.77 (842)	41.76 (283)
		e-value	3.30e-174	4.22e-163	4.96e-134	0.0	1.76e-94
	Tilapia D 9	Id % (Sc)	36.74 (250)	38.12 (253)	39.17 (255)	41.95 (263)	75.71 (599)
		e-value	9.26e-82	4.82 e-83	8.90e-84	1.52e-86	0.0
	Eel D 2A	Id % (Sc)	77.83 (729)	73.13 (666)	50.52 (427)	58.96 (497)	37.01 (273)
		e-value	0.0	0.0	2.45e-150	1.88 e-177	4.40e-91
	Eel D 2B	Id % (Sc)	75.48 (686)	71.73 (635)	51.50 (441)	59.04 (492)	36.77 (268)
		e-value	0.0	0.0	3.74e-156	1.63e-175	9.44e-89
	Id = Identity; Sc = bit Score

Table 3 :

 3 ID correspondence and nomenclature for DA receptor sequences 1 used in the phylogenetic analysis and the manuscript

						D1 family	D2 family
	Vertebrate species	Latin names	Receptor		Receptor
						Protein ID	Protein ID
					names		names
					D 1a	XP_018611872.1	D 2a	XP_018598250.1
					D 1b	XP_018607352.1	D 2b	XP_018610682.1
	Asian bonytongue		Scleropages
					D 5a	XP_018590910.1
	(Arowana)			formosus
					D 6a	KPP63084.1
					D 6b	KPP64030.1
					D 1b	XP_012693211.1	D 2b	XP_012677409.1
					D 5a	XP_012696109.1	D 3	XP_012671515.1
	Atlantic herring		Clupea harengus	D 5b	XP_012686723.1	D 9	XP_012687376.1
					D 6a	XP_012685303.1
					D 6b	XP_012672788.1
	Cattle			Bos taurus	D 1	Q95136	D 2	P20288
					D 5	G3X8D2	D 3	NP_001179824.1
							D 4	XP_024843396.1
	Chicken			Gallus gallus	D 1	B8YLW8	D 2	A9YZQ5
					D 5	XP_015141299.1	D 3	C5HV40
					D 7	XP_004947622.1	D 4	B6UVA0
	Clown	fish	(Sebae	Amphiprion			D 2a	A0A0C5LBQ6
	anemonefish)		sebae
	Coelacanth			Latimeria	D 1	XP_005992583.1	D 2	H3BGA1
				chalunae	D 5	H2ZWY6	D 8	XP_006010333.1
					D 6	H3BEK6	D 3	XP_014350627.1
					D 7	XP_005995307.1	D 4	H3B3R1

Acknowledgements:

We thank the DyNAMIC and LAE laboratories (University of Lorraine, France) for the access to the NanoDrop Spectrophotometer and the q-PCR thermocycler, respectively.

Funding: This work was supported by the Eurostars project [grant numbers E!9390 TRANSANDER]; the Region Lorraine and project NAZV QK1710310.

Bibliography

Aegerter, S., Jalabert, B., Bobe, J., 2004. Messenger RNA stockpile of cyclin B, insulin-like

Homo sapiens, adrenergic receptors. Red arrows indicate pikeperch sequences. Sequences used for this analyse are in Supplementary Table 3.