Alejandro S Mechaly

Jordi Viñas

Dr Francesc Piferrer
email: piferrer@icm.csic.es

Gene structure analysis of kisspeptin-2 (Kiss2) in the Senegalese sole (Solea senegalensis): Characterization of two splice variants of Kiss2, and novel evidence for metabolic regulation of kisspeptin signaling in nonmammalian species

Keywords: Kiss2, kisspeptin-2, puberty, fasting, alternative splicing, Solea senegalensis

Kisspeptin signaling in the brain is involved in the control of the onset of puberty in vertebrates. In this study, we present novel evidence indicating that kisspeptin may link energy balance and reproduction. For that purpose, we determined the complete gene structure of kisspeptin in a teleost fish, the Senegalese sole (Ss). In contrast to the situation evident in several fish, in this species only Kiss2 was found. Yet, two Ss Kiss2 isoforms generated by alternative splicing through intronic retention were detected: Ss Kiss2_v1, producing the functional protein, and Ss Kiss2_v2, coding for a truncated, non-functional protein. Specific qPCRs showed that the expression of these two isoforms varied differently in brain and gonads throughout maturation. In addition, and in contrast to what has been observed in mammals, fasting increased hypothalamic mRNA levels of Ss Kiss2_v1, which also caused a concomitant rise in pituitary Ss LH and Ss FSH mRNA. Together, these data indicate the impact of the nutritional status on Kiss mRNA expression as a potential regulatory mechanism for the metabolic control of reproduction in non-mammalian species, albeit with some significant differences with respect to the situation described in mammals.

A c c e p t e d M a n u s c r i p t

Introduction

In vertebrates, reproduction is subject to a complex regulation within the hypothalamic-pituitary-gonadal (HPG) axis (for reviews see [START_REF] Gottsch | A role for kisspeptins in the regulation of gonadotropin secretion in the mouse[END_REF][START_REF] Aparicio | Kisspeptins and GPR54the new biology of the mammalian GnRH axis[END_REF][START_REF] Tena-Sempere | GPR54 and kisspeptin in reproduction[END_REF][START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF][START_REF] Oakley | Kisspeptin signaling in the brain[END_REF]. In the last years, it has become established that the peptide kisspeptin has an important role in the control of reproduction, particularly relating to the onset of puberty as evidenced mostly by studies in mammals [START_REF] Gottsch | A role for kisspeptins in the regulation of gonadotropin secretion in the mouse[END_REF][START_REF] Tena-Sempere | GPR54 and kisspeptin in reproduction[END_REF][START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF][START_REF] Oakley | Kisspeptin signaling in the brain[END_REF][START_REF] Roseweir | The role of kisspeptin in the control of gonadotrophin secretion[END_REF] and fishes [START_REF] Parhar | Laser-captured single digoxigenin-labeled neurons of gonadotropin-releasing hormone types reveal a novel G protein-coupled receptor (Gpr54) during maturation in cichlid fish[END_REF][START_REF] Elizur | The KiSS1/GPR54 system in fish[END_REF][START_REF] Carrillo | Hormonal and environmental control of puberty in perciform fish: the case of sea bass[END_REF]Zohar et al., 2009;[START_REF] Taranger | Control of puberty in farmed fish[END_REF]. Hence, mRNA levels of kisspeptin and its receptor are highest at the time of the onset of puberty in rats (Rattus norvegicus) and rhesus macaque (Macaca mulatta), where an increase in expression patterns for both genes were observed in juveniles entering puberty [START_REF] Navarro | Advanced vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54[END_REF][START_REF] Shahab | Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates[END_REF]. Moreover, exogenous administration of kisspeptin activates the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) in mammals [START_REF] Funes | The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system[END_REF][START_REF] Matsui | Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat[END_REF][START_REF] Messager | Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor 54[END_REF], amphibians [START_REF] Moon | Molecular cloning of the bullfrog kisspeptin receptor Gpr54 with high sensitivity to Xenopus kisspeptin[END_REF] and teleost fishes [START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF][START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF].

In mammals, there is only one kisspeptin gene (Kiss1), with the exception of the platypus, a mammalian monotreme, which has two genes (Kiss1 and Kiss2) [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF].

The presence of two kisspeptin genes is common in fishes, as observed in medaka (Oryzias latipes), zebrafish (Danio rerio), goldfish (Carassius auratus), European sea bass (Dicentrarchus labrax) and chub mackerel (Scomber japonicus) [START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF][START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF][START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF][START_REF] Selvaraj | Molecular characterization, tissue distribution, and mRNA expression profiles of two Kiss genes in the adult male and female chub mackerel (Scomber japonicus) during different gonadal stages[END_REF]. As for the receptor, two Kissr genes, A c c e p t e d M a n u s c r i p t 4 originated through one or two rounds of gene or chromosome duplication and named Kiss1r, Kiss2rA and Kiss2rB [START_REF] Mechaly | Gene structure and regulation of the Kiss-1 receptor-2 (Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes[END_REF], are also observed in medaka, zebrafish and goldfish [START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF][START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF][START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF]. However, this situation is not universal, since only one kisspeptin gene, Kiss2, and one receptor, Kiss2rA, are present in the orange-spotted grouper (Epinephelus coioides) [START_REF] Shi | Molecular identification of the Kiss2/Kiss1ra system and its potential function during 17alpha-methyltestosterone-induced sex reversal in the orangespotted grouper, Epinephelus coloides[END_REF], grass puffer (Takifugu niphobles) [START_REF] Shahjahan | Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season[END_REF], Senegalese sole (Solea senegalensis) [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] and Atlantic halibut (Hippoglossus hippoglossus) [START_REF] Mechaly | Gene structure and regulation of the Kiss-1 receptor-2 (Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes[END_REF], indicating that the paralogous gene has been lost during evolution [START_REF] Mechaly | Gene structure and regulation of the Kiss-1 receptor-2 (Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes[END_REF]. Furthermore, in the Senegalese sole, a splice variant isoform of the Kiss2rA gene was observed in the transcript [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF], and in amphibian clawed frog (Xenopus tropicalis), a third paralogous for both the ligand (Kiss1b) and the receptor (Kiss1rb) [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF] have been reported. Thus, the evolution of kisspeptin and its receptor in vertebrates involves gene duplications, appearance of alternative splicing variants as well as gene loss, although the course of events and their relationships are still not completely clear. Therefore, a more exhaustive analysis is necessary to understand the phylogenetic relationships among these gene duplications and isoforms in the kisspeptin family.

The existence of paralogous genes and isoforms in fish poses the interesting question as to what is the precise function of each gene. In the European sea bass, Kiss2 was found to be the most potent elicitor of gonadotropin (LH and FSH) secretion [START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF]. In zebrafish, habenular Kiss1 and hypothalamic Kiss2 regulate reproduction but Kiss2 is more potent in stimulating gonadotropin synthesis and secretion [START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF]. In goldfish, Kiss1 acts directly at the pituitary, inducing the secretion of LH, prolactin and growth hormone [START_REF] Lu | Alternative splicing in teleost fish genomes: same-species and crossspecies analysis and comparisons[END_REF]. However, administration of the Kp10 peptide failed to A c c e p t e d M a n u s c r i p t elicit differences in LH mRNA levels [START_REF] Li | Structural and functional multiplicity of the kisspeptin/GPR54 system in goldfish (Carassius auratus)[END_REF]. In medaka, only hypothalamic Kiss1 neurons of the nucleus ventralis tuberis (NVT) were involved in estrogen feedback [START_REF] Kanda | Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes)[END_REF], and it was suggested that Kiss2 neurons play a role in non-reproductive functions [START_REF] Mitani | Hypothalamic Kiss1 but not Kiss2 neurons are involved in estrogen feedback in medaka (Oryzias latipes)[END_REF]. Intraperitoneal injection of Kiss2-10 in mature orange-spotted grouper resulted in increased GnRH1 mRNA levels in the hypothalamus and FSHb in the pituitary [START_REF] Shahjahan | Elevation of Kiss2 and its receptor gene expression in the brain and pituitary of grass puffer during the spawning season[END_REF]. Together, these results provide ample evidence of the participation of kisspeptin in fish reproduction although the roles of Kiss1 and Kiss2 vary across species.

In addition to its role in the central control of reproduction, there is also increasing evidence showing that kisspeptin signaling is involved in other processes [START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF][START_REF] Roseweir | The role of kisspeptin in the control of gonadotrophin secretion[END_REF]. These include a potentially important autocrine and/or paracrine effects in the ovary [START_REF] Gaytan | Expression of KiSS-1 in rat oviduct: possible involvement in prevention of ectopic implantation?[END_REF], placenta [START_REF] Hiden | Kisspeptins and the placenta: regulation of trophoblast invasion[END_REF], and immune system [START_REF] Cardon | Dysregulation of kisspeptin and neurogenesis at adolescence link inborn immune deficits to the late onset of abnormal sensorimotor gating in congenital psychological disorders[END_REF], as well as the participation of kisspeptin in metastasis (Makri et al., 2008), vasoconstriction [START_REF] Mead | Kisspeptins are novel potent vasoconstrictors in humans, with a discrete localization of their receptor, G proteincoupled receptor 54, to atherosclerosis-prone vessels[END_REF] and stimulation of insulin secretion [START_REF] Bowe | Kisspeptin stimulation of insulin secretion: mechanisms of action in mouse islets and rats[END_REF]. Of particular interest is the possible role of kisspeptin linking energy balance and reproduction [START_REF] Popa | The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction[END_REF], since recent observations indicate that the impacts of negative energy balance towards reproduction can be modulated by kisspeptins [START_REF] Tena-Sempere | GPR54 and kisspeptin in reproduction[END_REF]. Hence, in rats, Kiss1 is sensitive to nutritional conditions, with a decrease in expression during negative energy balance status, thus contributing to reproduction suppression [START_REF] Fernández-Fernández | Novel signals for the integration of energy balance and reproduction[END_REF]. Furthermore, Kiss1 neurons are direct targets of leptin, suggesting that reproductive deficits associated with leptin-deficient states may be attributable, in part, to diminished expression of Kiss1 [START_REF] Smith | KiSS-1 neurons are direct targets for leptin in the ob/ob mouse[END_REF]. However, the regulation of the metabolic condition and its subsequent impact on reproductive function through kisspeptin and its receptor is far from being well understood. Nothing is known about The aim of this study was to evaluate the possible role of kisspeptin signaling in situations of negative energy balance in a non-mammalian model. For this purpose, we first cloned, characterized and analyzed the gene structure of Kiss in the Senegalese sole and the levels of expression in the brain and gonads of pubertal and mature fish. This species has only one Kissr gene, Kiss2r, and thus we also wanted to search for the possible existence of paralogous Kiss genes in this species.

Experimental Procedures

Animals and tissue sample collection

Senegalese sole were obtained from the IFAPA research center in El Puerto de Santa María (Cádiz, SW Spain). Fish stocks were maintained at the Experimental Aquarium Facilities of the Institute of Marine Sciences, under natural conditions of temperature and photoperiod and fed two times a day with a commercial diet (Skretting, Spain). Animals were treated according to the approved institutional guidelines on the use of animals for research purposes and in agreement with the European regulations of animals' welfare (ETS No. 123,01/01/91). Fish were anesthetized with an overdose of St. Louis, MO, USA) and sacrificed by decapitation. Tissues were quickly removed under RNase-free conditions, frozen in liquid nitrogen and stored at -80ºC until used. Biometric

RNA isolation and cDNA synthesis

Total RNA was isolated with TRIZOL Reagent (Invitrogen, Carlsbad USA), its quality was checked in a 1.5% agarose gel and quantity measured in a Nanodrop® ND-1000 spectrophotometer (Nanodrop® Technologies Inc, Wilmington, DE, USA). All RNAs were treated with Dnase I (Invitrogen, Carlsbad, CA, USA) to remove any possible genomic DNA contamination. cDNA synthesis was performed following the manufacturer's instructions using 500 ng of RNA with Superscript III Reverse Transcriptase and random hexamer primers (Invitrogen, CA, USA) in a 20 μl reaction volume.

Molecular cloning of kisspeptin in the Senegalese sole

Degenerated PCR primers based on the polypeptide sequences YNLNSFGLRY for Kiss1 and FNL/Y/FNPFGLRF for Kiss2 were designed to discriminate between the two genes. Degenerated PCR primers were designed based on the sequence of the conserved region of Kp10 of zebrafish-1, medaka-1, sea bass-1 and goldfish-1 for the Kiss1, and fugu-2, pufferfish-2, sea bass-2, goldfish-2, stickleback-2 for the Kiss2 (Supplementary Table 1). PCR amplifications were carried out from four Senegalese sole genomic DNA libraries constructed with the Universal Genome Walker Kit (Clontech Laboratories Inc, CA, USA.).

Briefly, two rounds of PCR were performed: first, PCR amplification was carried out using a primer (AP1) provided in the kit and degenerate for Kiss2 (1PCR_S_Kiss2 and 1PCR_AS_Kiss2). Second, a nested PCR was performed by the primer combination AP2 and A c c e p t e d M a n u s c r i p t degenerate and nested Kiss2 PCR primers, 2PCR_S_Kiss2 and 2PCR_AS_Kiss2, using a 1/50 dilution of the product of the primary PCR. In all cases, PCR conditions were as follows: initial denaturation at 94°C for 25 s, followed by 7 cycles at 94°C for 25 s and 26°C for 3 min, and 32 cycles at 94°C for 25 s and 67°C for 3 min, and 67°C for 7 min after the final cycle. The full-length cDNAs of Kiss2 was obtained from a Senegalese sole brain cDNA library constructed in the UNI ZAP-XR vector. PCR reactions using gene-specific primers in combination with primers annealing in the UNI Zap-XR vector were performed to obtain the cDNA ends.

Gene structure, sequence alignment and phylogenetic analysis

To assess the genomic gene structure, the coding of the gene was amplified by the primer combination Kiss2_Ex1_F1 and Kiss2_Ex2_R2 using genomic DNA as template.

Genomic DNA was extracted using a biopsy of muscle (~50 mg of tissue) incubated overnight at 55°C in 1 ml of lysis solution (10 mM Tris-HCl, pH 7.5; 25 mM EDTA, pH 8; 0.5% SDS; 100 mM NaCl), and 10 µl of proteinase K (10 mg/ml). DNA was cleaned by a wash of equal volume of phenol/chloroform and a wash of chloroform/ isoamyl alcohol.

Then, 5 µl of RNase A (10 mg/ml) were added and incubated at 55°C for 30 min. Finally, DNA was precipitated with 95% ethanol, washed with 70% ethanol and eluted in 50 µl of sterile water.

The gene structure was constructed using the nucleotide and predicted peptide sequence of Ss Kiss2 compared with known Kiss2 sequences available through GenBank.

The presence and location of the putative signal peptide cleavage site of the Kiss2 of fishes A c c e p t e d M a n u s c r i p t were assessed using the prediction protein servers of the Center for Biological Sequence Analysis (http://www.cbs.dtu.dk/services/). The 5'UTR and 3´UTR regions were localized upstream and downstream of the start codon (ATG) and stop codon (TGA), respectively. Phylogenetic analysis was performed using multiple alignments of the DNA sequence corresponding to the 30 nucleotides that codified the 10 aa of Kp10. A total of 38 sequences were aligned using Clustal W [START_REF] Thompson | CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positionspecific gap penalties and weight matrix choice[END_REF] and edited in Bioedit [START_REF] Hall | BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT[END_REF]. A phylogenetic tree was constructed by the neighbor-joining method [START_REF] Saitou | The Neighbor-Joining method -a new method for reconstructing phylogenetic trees[END_REF] using the Kimura two parameters distance [START_REF] Kimura | Simple model for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences[END_REF]. All phylogenetic analyses were conducted on MEGA version 4 [START_REF] Tamura | MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) Software Version 4.0[END_REF].

Tissue expression pattern of Ss Kiss2

The tissue distribution of Ss Kiss2 in adult animals was analyzed by RT-PCR in different tissues. Total RNA and cDNA synthesis were carried out following the protocol described in section 2.2. The resulting cDNA was used to amplify Ss Kiss2 by RT-PCR using the Platinum Taq DNA Polymerase (Invitrogen). The PCR cycling conditions for Ss Kiss2 were: 5 min at 94°C; 35 cycles of 30 s at 94°C, 30 s at 60°C, 30 s at 72°C and a final extension of 5 min at 72°C. Genomic DNA contamination in cDNA was checked with a primer combination encompassing the Intron I (Kiss2_Ex1_F1 and Kiss2_Ex2_R2) (supplementary Table 1). Amplification of the beta-actin gene was used as RNA quality control. No template control (NTC) was included to ensure that no contamination had occurred. PCR products were run on 1.5% agarose gel and subsequently sequenced to verify their authenticity. The expression pattern of Ss Kiss2_v1 and Ss Kiss2_v2 was analyzed in whole brain of pubertal and mature fish (as classified in [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF] by real-time quantitative PCR (qPCR) (n = 4 -6). RNA and cDNA were obtained following the protocol described above. The Ss Kiss2 primers used for qPCR were designed to amplify each Ss Kiss2 isoform (see results and Supplementary Table 2). The qPCR amplification reaction mixture contained 2 µl of diluted cDNA (1:3) (freshly synthesized from 500 ng of RNA), 4 µM of each primer, and 10 µl of Power SYBRs Green PCR Master Mix (Applied Biosystems, Foster City, CA) in a final volume of 20 µl. Thermal cycling conditions comprised 95°C for 10 min, 40 cycles at 95°C for 10 s, and 60°C for 30 s. At the end of the PCR cycles, the qPCR products were analyzed using a dissociation curve step to confirm that only a single PCR product was amplified. NTC reactions for every primer pair were also included on each reaction plate to check for external DNA contamination. The amplification efficiency (E) of each primer set/ target gene was assessed as E = 10 (-1/slope) as determined by linear regression of a series of dilutions of the input RNA. The qPCR reactions were performed with an ABI Prism 7900 Sequence Detection Systems (Applied Biosystems).

Data were analyzed using the comparative cycle threshold (Ct) method [START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF] (DDCt method) to calculate relative changes in gene expression. Fold change (the relative quantification, RQ) was calculated from the DDCt and normalized to the reference gene betaactin. Determinations were carried out in technical triplicates for the Ss Kiss2_v1 Ss 2.7. Effect of fasting and feeding conditions on the expression of kisspeptin and its receptor in adult Senegalese sole Adult male and female Senegalese sole were randomly distributed into two 600-liter tanks. The nutritional study was conducted for a period of 15 days after a 5-day period of acclimation, with seawater temperature at 20 ± 1ºC. One group of fish (n = 8) was fed ad libitum (5 times a day) and used as the control group ("Fed"). Another group (n = 7) was maintained under food deprivation conditions ("Fasting"). The initial mean BW (g) and SL (cm) were similar in both groups: Fed; 885,71 ± 140,53 g and 33,14 ± 1,99 cm; Fasting; 871,43 ± 121,85 g 32, 57 ± 1,97 cm with non-significant differences (BW, P = 0.68; SL, P = 0.59). After 15 days of differential feeding all fish were sacrificed and the hypothalamus, stomach and pituitary were quickly removed and keep at -80 o C under. Biometric information, SL (cm), BW (g), gonad and liver weight (g) were obtained for all the fishes. The gonadosomatic (GSI) and hepatosomatic (HSI) indices were calculated as the percent of the corresponding organ weight with respect to the total body weight. Total RNA isolation and DNA treatment and cDNA synthesis were carried out as described in section 2.2. qPCR amplification reaction was performed for each cDNA sample in triplicate in 10 µl of the reaction mixture containing 5 µl of 10x master mix (Express SyBr green, Invitrogen), 0.2 µl of each primer (10 µM), and 2 µl of a 1/10 diluted sample from the cDNA synthesis. Reaction mixtures without a template were run as controls. The cycling conditions were as: 95°C for 10 A c c e p t e d M a n u s c r i p t min, followed by 40 cycles at 95°C for 10 s, 60°C for 30 s, and 72°C for 20 s. The expression pattern was analyzed by qPCR using the Ss Kiss2_v1 and Ss Kiss2_v2 and Ss Kiss2r_v1 and Ss Kiss2r_v2 in hypothalamus and stomach, whereas the Ss LH and Ss FSH (taken from [START_REF] Cerdà | Molecular cloning of Senegalese sole (Solea senegalensis) folliclestimulating hormone and luteinizing hormone subunits and expression pattern during spermatogenesis[END_REF] were assessed in pituitary for both experimental conditions (for full list of primers see Supplementary Table 2).

8. Statistical analyses

Data are shown as mean ± standard error of the mean (S.E.M.). All statistical analyses were performed using the SPSS v16 software. The significance of pairwise comparisons of the mRNA levels of pubertal and mature fishes in brain and gonads of Ss Kiss2_v1 and Ss Kiss2_v2 were determined using a one-way ANOVA followed by Fisher's least significant difference (LSD) test for both isoforms. Statistical differences between fed and fasting fish were analyzed by a Student t-test for the biometric data as well as the mRNA levels of Ss Kiss2_v1 and Ss Kiss2_v2, Ss Kiss2r_v1 and Ss Kiss2r_v2 in the hypothalamus and stomach and Ss FSH and Ss LH in the pituitary. Differences were accepted as significant when P<0.05.

Results

Cloning and sequence analysis of Ss Kiss2 gene

Although PCRs were carried out to specifically find the two Kiss genes (Kiss1 and Kiss2) in the Senegalese sole (Ss), only Ss Kiss2 was detected. The complete coding region of fully spliced Ss Kiss2 comprised 390 bp that predicts a 129 amino acid (aa) protein (Fig. 1A and B). The sequence of Ss Kiss2 has been deposited into GenBank with accession number HM116743. Sequence analysis identified a N-terminal with 19 aa residue of the signal peptide and the ten conserved aa corresponding to the Kp10 region (Fig. 1B). A high level of aa sequence identity was observed only in the decapeptide region Kp10 between Ss Kiss2 and zebrafish Kiss2 (90%), zebrafish Kiss1 (60%) and human Kiss1 (70%), while the degree of nucleotide conservation was reduced to 20% in adjacent regions of the gene (Table 1).

Genomic structure of Ss Kiss2 gene

In addition to the coding region, the genomic sequence of the Ss Kiss2 was also obtained. Ss Kiss2 is organized into 2 exons, exon 1 with 109 bp and exon 2 with 281 bp, and one intron of 36 bp (Fig. 2). The sequence obtained comprises a prepro Ss Kiss2 cDNA that spans 432 bp and includes a 36 bp 5′-untranslated region (5′-UTR), a 390 bp open reading frame (ORF) encoding a putative preprohormone of 129 a, and a 6 bp 3′ UTR (Fig. 2). The consensus ATTAAA polyadenylation signal and the poly (A) tail were not found. The molecular architecture of prepro Ss Kiss2 includes a 19 aa peptide, the Kiss2 peptide, a putative processing site, and a kisspeptin-associated peptide. The deduced sequence of Ss Kiss2 peptide Kp10 consists of 10 aa. The proteolytic cleavage site (GKR) is conserved in all Kiss2 genes analyzed so far (Fig. 1B and Fig. 2).

Detection of two transcript variants in the mRNA of the Kiss2 of Senegalese sole

Specific RT-PCR with a primer set that amplifies a region that encompasses the intron generates two different products of 390 and 426 bp (Fig. 3A) named hereafter Ss Kiss2_v1 A c c e p t e d M a n u s c r i p t 14 and Ss Kiss2_v2, respectively. The presence of genomic contamination in the RNA extraction was discarded with DNAse treatment of all RNAs. Furthermore, A PCR with primers flanking intron 1 were designed to amplify both Ss Kiss2 isoforms and used to evaluate the expression of Ss Kiss2 in several tissues. The Ss Kiss2_v2 transcript was detected in all tissues analyzed, whereas the Ss Kiss2_v1 was only detected in the ovaries, testis, stomach and brain, with a higher expression in this latter tissue (Fig. 3A). Negative controls with nonreverse transcribed RNA samples (no-template control, NTC) did not yield positive signals.

Sequence analysis revealed that Ss Kiss2_v2 retained the entire intron 1 (Fig. 3B) and revealed the presence of all the features characteristic of an alternative splicing mechanism in the intron 1, including a 5´ss donor (5´) splice site, consensus sequence (5´) splice site (CS 5´ss), branch point (BP), polypirimidine track (PT), 3´ss, acceptor (3´) splice site, and consensus sequence (3´) splice site (CS 3´ss) (Fig. 3C). The putative translation to aa of the Ss Kiss2_v2 isoform revealed the presence of two premature termination codons (PTCs) in the intron.

Sequence comparison and phylogenetic analysis of Ss Kiss2 in vertebrates

Amino acid sequence comparison of the peptide Kp10 among several vertebrate groups showed a complete fixation of aas 2 (Asn), 6 (Phe), 7 (Gly), 8 (Leu) and 9 (Arg).

Nevertheless, the first and last positions (1 and 10) are considered the most important in the Kp10 classification [START_REF] Roa | New frontiers in kisspeptin/GPR54 physiology as fundamental gatekeepers of reproductive function[END_REF]. The Kiss1 form presented either a tyrosine (Y) in the position one and either a Phenylalanine (F) or Tyrosine in the last position corresponding to the Y-Y and Y-F forms, respectively. On the contrary, Kiss2 corresponded only to the F-F form (Fig. 4A). Since the Kiss gene presents very low levels of conservation among A c c e p t e d M a n u s c r i p t 15 vertebrates, which make it difficult for correct alignment of the gene, the phylogenetic analysis of a total of 27 species (38 sequences in total) was restricted to 30 nucleotides of the Kp10 region. The phylogenetic analysis (Fig. 4B) clustered two different groups corresponding to Kiss1 and Kiss2.

Comparison of Ss Kiss2_v1 and Ss Kiss2_v2 expression levels during development

The mRNA levels of the Ss Kiss2_v1 and Ss Kiss2_v2 isoforms were analyzed separately by a specific qPCR in Senegalese sole male and female in brain and gonads (see supplementary table 1 for specific primers used in the qPCRs). In the brain, both isoforms were detected in both pubertal and mature fish of both sexes, with an overall higher and significant (P < 0.05) difference in expression in the mature females with respect to males for each of the isoforms (Fig. 5A). In the gonads, the two isoforms were also detected in both sexes and stages of development. However, and also contrary to the situation in the brain, the differences were stage-related, with a significantly (P < 0.001) high expression in pubertal females (Fig. 5B).

Fasting effects on key genes of the reproductive axis

Fifteen days of fasting resulted in a significant reduction in BW (-5.63%) while control fish maintained their BW (P < 0.001) (Fig. 6A). The HSI was reduced in the fasted fish although did not show statistical differences (P = 0.278) (Fig. 6B). Similarly, the GSI did not show statistical differences between the two different nutritional conditions, although a slight increase in GSI could be detected for males and females, respectively (Figs. 6C andD).

Changes in these indices indicate the effects of fasting in somatic vs. gonadal tissues. In the hypothalamus, induced fasting increased mRNA levels of Ss Kiss2_v1 (P = 0.016), Ss Kiss2_v2 (P = 0.018), Ss Kiss2r_v1 (P = 0.03) and Ss Kiss2r_v2 (P = 0.044) (Fig. 7A). Similarly, expression levels of Ss LH and Ss FSH in the pituitary increased up to ~ 4 fold (P = 0.035 and P = 0.007, respectively) (Fig. 7B). Although fasting increased the levels of Ss Kiss2_v1 and Ss Kiss2_v2, no statistically significant changes were detected for the Ss Kiss2_v1 (P = 0.362), Ss Kiss2_v2 (P = 0.245), Ss Kiss2r_v1 (P = 0.324) and Ss Kiss2r_v2 (P = 0.790) in the stomach (Fig. 7C).

Discussion

The presence of both Kiss paralogous genes (Kiss1 and Kiss2) was thoroughly examined in the Senegalese sole, although only Ss Kiss2 was found indicating that Kiss1 most probably was lost in this species. Additional studies including synteny analysis of KISS1 and cognate neighboring genes such as golt1a identified in other species (van Aerle et al., 2008;[START_REF] Kanda | Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes)[END_REF][START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF][START_REF] Um | Molecular coevolution of kisspeptins and their receptors from fish to mammals[END_REF], or searching for possible pseudogenes for Kiss1, would ultimately confirm the loss of Kiss1 in this species. The absence of some genes of the kisspeptin system has been in fact observed in other species: stickleback (Gasterosteus aculeatus), sockeye salmon (Oncorhynchus nerka), orange-spotted grouper and grass puffer. Regarding the receptor, it should be noted that in the Senegalese sole only one paralogous gene, Ss Kiss2r, was found [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]. The presence of only one gene for the ligand and the receptor was also recently reported in an another teleost fish, the orange-spotted grouper [START_REF] Shi | Molecular identification of the Kiss2/Kiss1ra system and its potential function during 17alpha-methyltestosterone-induced sex reversal in the orangespotted grouper, Epinephelus coloides[END_REF]. However, it is still premature to conclude that the loss of a variant of the ligand implies a loss of a variant of the receptor or vice versa; we simply point out these two situations. In any case, the loss of the second paralogous gene in these species is probably a consequence of the genome reduction that occurred during evolution [START_REF] Smith | Patterns of genome size diversity in the ray-finned fishes[END_REF] in the absence of stable adaptive advantages due to subfunctionalization of the two genes.

The results of our phylogenetic analyses (Fig. 4B andC) supports the overall classification of kisspeptin into Kiss1 and Kiss2 as previously proposed by [START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF], and are also in accordance with the classification of kisspeptins into the YY, YF and FF forms, with a total correspondence of the FF form with Kiss2. Our analysis also reveals two Kiss gene duplication events, the first occurring early in vertebrate evolution, giving rise to Kiss1 and Kiss2, and before the appearance of jawed fish since both genes are already found in sea lampreys. However, our interpretation of the second duplication is contrary to that of [START_REF] Um | Molecular coevolution of kisspeptins and their receptors from fish to mammals[END_REF], which involved only Kiss1 and gave rise to the Kiss1A and Kiss1B ortholog genes. [START_REF] Um | Molecular coevolution of kisspeptins and their receptors from fish to mammals[END_REF] considered that this duplication occurred prior to the appearance of teleosts. Our analysis, in contrast, indicates that these paralogous genes are never found in teleosts. Thus, the second duplication most likely occurred coinciding with the colonization of land, appearing either already in the Sarcopterygians (lungfishes and relatives) or later with the rise of the amphibians, as is evidenced in the clawed frog. Since Kiss1b has never been found in reptiles, birds or mammals, we opted to place this duplication in the amphibians as the most plausible possibility (Fig. 4C). However, future studies with more Sarcopterygian and amphibian species sequences should clarify the precise timeline of the appearance of the second gene duplication. Thereafter, Kiss2 invariably was lost in mammals [START_REF] Um | Molecular coevolution of kisspeptins and their receptors from fish to mammals[END_REF], with only one exception: a monotreme, Platypus (Ornithorhynchus anatinus) [START_REF] Lee | Molecular evolution of multiple forms of kisspeptins and GPR54 receptors in vertebrates[END_REF]. In fish, the opposite occurred, since while A c c e p t e d M a n u s c r i p t 18 some species retained both genes [START_REF] Kanda | Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes)[END_REF][START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF][START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF][START_REF] Um | Molecular coevolution of kisspeptins and their receptors from fish to mammals[END_REF]Akasome et al., 2010), others apparently lost Kiss1, but never Kiss2, as observed in the present study.

Sequence comparison of the Ss Kiss2 with other kisspeptin orthologs indicated that this prepropeptide contains a 19 aa signal peptide, followed by a 78 aa (Kp), which is longer than the corresponding 54 aa peptide described in humans [START_REF] Lee | KiSS-1, a novel human malignant melanoma metastasis-suppressor gene[END_REF]. Proteolytic processing of the full-length 145 aa of the kisspeptin protein in humans results in shorter peptides, with 54 (Kp54; previously known as metastin), 14 (Kp14), 13 (Kp13) or 10 (Kp10) aas [START_REF] Kotani | The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54[END_REF][START_REF] Ohtaki | Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor[END_REF]. Thus, a similar structure of kisspeptins appears to be conserved from fish to mammals. For instance, in goldfish, a Kp56, Kp27, Kp16, Kp13 and Kp10 were also observed [START_REF] Lu | Alternative splicing in teleost fish genomes: same-species and crossspecies analysis and comparisons[END_REF]. However, and similar to was observed in mammals, a sequence analysis of the Ss Kiss2 showed a lack of the cleavage sites that would yield shorter peptides. Nonetheless, the possibility of producing shorter peptides cannot be completely disregarded since they can be generated by degradation [START_REF] Popa | The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction[END_REF]. The last 10 aa of Kp78 corresponding to Kp10 are followed by three aa (GKR) that correspond to a putative site of terminal cleave and amidation, which is well conserved in all mammalian [START_REF] Kotani | The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54[END_REF][START_REF] Ohtaki | Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor[END_REF] and fish Kiss genes [START_REF] Biran | Molecular identification and functional characterization of the kisspeptin/kisspeptin receptor system in lower vertebrates[END_REF] (Fig. 1B).

Interestingly, the last 29 aa resulting from the cleavage of Ss Kiss2 indicate a kisspeptinassociated peptide. Although a kisspeptin-associated peptide has never been explicitly described for kisspeptin and its function would have to be confirmed, it is important to recall a similar situation with GnRH, where the existence of a GnRH-associated peptide (GAP) is required for proper processing of the precursor and production of the mature GnRH peptide [START_REF] De Roux | GnRH receptor and GPR54 inactivation in isolated gonadotropic deficiency[END_REF].

A c c e p t e d M a n u s c r i p t

Two splice variants of the Kiss2 ligand were observed in this study and named Ss Kiss2_v1 and Ss Kiss2_v2 for the shorter and longer isoform, respectively. Ss Kiss2_v2 retained the 36 bp of the intron. Sequence analysis of Ss Kiss2_v2 revealed the presence of several features characteristic of an alternative splicing mechanism (Fig. 3C). The deduced aa sequence of the intron shows 10 residues with two PTC. Thus, Kiss2_v2 most likely leads to the generation of a putative truncated product that lacks the Kp10 region, which would result in a completely non-functional peptide. The generation of mRNA variants containing PTCs is viewed as a characteristic result of alternative splicing [START_REF] Ben-Dov | Genome-wide analysis of alternative Pre-mRNA splicing[END_REF]. Furthermore, the occurrence of alternative splicing is expected to be higher in species with compact genomes, as in the Senegalese sole [START_REF] Smith | Patterns of genome size diversity in the ray-finned fishes[END_REF] as compared to the lowest frequency observed in species (e.g., zebrafish) in which duplication of the genomes occurred [START_REF] Lu | Alternative splicing in teleost fish genomes: same-species and crossspecies analysis and comparisons[END_REF]. At present, the biological significance of Ss Kiss2_v2 is not known, but based on the fact that the active peptide Kp10 is localized in exon 2 in the Senegalese sole as well as in the other species examined so far (Oakley et al., 2010), the retention of intron 1 could serve as a way to control the relative expression amounts of the functional Ss Kiss2 isoform. A similar situation was found regarding the receptor in the Senegalese sole. In this case, intron 3 could be retained due to an alternative splicing mechanism to form truncated and non-truncated isoforms coded by the same gene [START_REF] Mechaly | Identification of two isoforms of the kisspeptin-1 receptor (kiss1r) generated by alternative splicing in a modern teleost, the Senegalese Sole (Solea senegalensis)[END_REF]. However, in the receptor, a hypothetical non-functional protein could be generated due the presence of two PTCs in intron 3, located in the middle of transmembrane domain 4. Future studies at the protein level are required to analyze the physiological significance of the two Kiss2 isoforms. Alternative splicing is emerging as an important mechanism to control gene expression [START_REF] Izquierdo | Simple principle to explain the evolution of pre-mRNA splicing[END_REF]. Thus, it is surprising that splice variants of the Kiss genes have been A c c e p t e d M a n u s c r i p t 20 detected so far only in Pleuronectiformes [START_REF] Mechaly | Gene structure and regulation of the Kiss-1 receptor-2 (Kiss1r-2) in the Atlantic halibut: Insights into the evolution and regulation of Kiss1r genes[END_REF], but not in any other vertebrate species. Therefore, and to the best of our knowledge, the Senegalese sole is the only species in which the machinery necessary for the occurrence of two splice variants in both the Kiss ligand and Kiss1 receptor genes has been identified. This observation, together with the fact that in this species we detected only one paralogous gene for both the ligand and the receptor provides an interesting opportunity for the study of gene regulation in the kisspeptin system.

Although information is still scarce and more research is needed to determine the distribution of kisspeptin in different areas of the fish brain, studies in medaka indicate high expression in two specific hypothalamic nuclei: the nucleus posterior periventricularis (NPPv) and the nucleus ventral tuberis (NVT) [START_REF] Kanda | Identification of KiSS-1 product kisspeptin and steroid-sensitive sexually dimorphic kisspeptin neurons in medaka (Oryzias latipes)[END_REF]. In the present study, using whole brains but focusing on changes in expression according to sex and maturity stage, overall higher expression levels for both isoforms was detected in mature females. On the other hand, in the gonads, significantly higher mRNA levels of both isoforms were observed in the ovaries of pubertal females. This is in accordance with the observation that Kiss2 is abundantly expressed in fish ovaries [START_REF] Felip | Evidence for two distinct KiSS genes in non-placental vertebrates that encode kisspeptins with different gonadotropin-releasing activities in fish and mammals[END_REF][START_REF] Kitahashi | Cloning and expression of kiss2 in the zebrafish and medaka[END_REF] and suggests a role for kisspeptin in ovarian development. Thus, in the Senegalese sole, the results conform what one would expect, i.e., higher levels of the mRNA in pubertal as compared to mature fish, but particularly in the ovaries rather than in the brain, where changes in expression levels were less evident.

The reproductive axis in animals is inhibited by negative energy balance produced, among other factors, by fasting [START_REF] Popa | The role of kisspeptins and GPR54 in the neuroendocrine regulation of reproduction[END_REF]. Studies relating energy balance and A c c e p t e d M a n u s c r i p t 21 reproduction via kisspeptin signaling are limited. In the mouse and rhesus macaque, fasting decreased mRNA levels of both kisspeptin and its receptor [START_REF] Luque | Regulation of hypothalamic expression of KiSS-1 and GPR54 genes by metabolic factors: Analysis using mouse models and a cell line[END_REF]Wahab et al., 2008), while it increased hypothalamic mRNA levels of Kissr, possibly through the apparent increase in sensibility to Kissr in starved animals [START_REF] Castellano | Changes in hypothalamic KiSS-1 system and restoration of pubertal activation of the reproductive axis by kisspeptin in undernutrition[END_REF]. There is no previous work regarding energy balance and the kisspeptin signaling in non-mammalian models. In this context, it is worth noting that key components of the system controlling energy balance [START_REF] Volkoff | 1a, A. clawed frog-1 E. shark-1 Rat S. senegalensis-2 Stickleback-2 S. salmon-2 C. Meckerel-2 O. spotted grouper-2 S. Bass-2 G. puffer-2 Chimpanzee Human R. macaque Sheep S. hamster Goat Mouse Pig Cattle 0.05 F-F form Kiss2 Kiss1 Y-F form Y-Y form Y-F form Y-F form S. lamprey-2[END_REF] may have opposite effects in fish and mammals. Thus, for example, ghrelin appears to have a stimulatory role in the regulation of non-mammalian, but not mammalian, reproduction [START_REF] Unniappan | Ghrelin: An emerging player in the regulation of reproduction in nonmammalian vertebrates[END_REF]. In recent years, an increasing number of mammalian appetite-regulating peptide homologs have been characterized in fish [START_REF] Volkoff | 1a, A. clawed frog-1 E. shark-1 Rat S. senegalensis-2 Stickleback-2 S. salmon-2 C. Meckerel-2 O. spotted grouper-2 S. Bass-2 G. puffer-2 Chimpanzee Human R. macaque Sheep S. hamster Goat Mouse Pig Cattle 0.05 F-F form Kiss2 Kiss1 Y-F form Y-Y form Y-F form Y-F form S. lamprey-2[END_REF], suggesting that, at least, the major components involved in the regulation of food intake have been relatively well conserved through vertebrate evolution.

Fish occupy a wide range of habitats, which pose different environmental challenges and may have led to different adaptive feeding strategies (Volkoff et al., 2009). Thus, there is the opportunity for variation across fish species and while it is true that fasting can suppress reproduction in a manner similar to what has been described in mammals, it is also possible that in other species the reverse may apply. The Senegalese sole alternates periods of normal feeding and fasting during the annual cycle in nature, as already observed for many fish species [START_REF] Costas | Feed deprivation in Senegalese sole (Solea senegalensis Kaup, 1858) juveniles: effects on blood plasma metabolites and free amino acid levels[END_REF]. This would support our observations, which, in contrast to what has been observed in the mouse and rhesus macaque, show that fasting enhanced the expression of Kiss2 and its receptor, along with LH and FSH expression. An effect of fasting on the reproductive axis has been found in sea bass, where a stimulatory effect of neuropeptide Y (NPY) on LH secretion was observed only in fasted animals (Cerdá-Reverter et al., 1999). Thus, in fasted Senegalese sole the increase in pituitary LH and FSH mRNA A c c e p t e d M a n u s c r i p t 22 levels most likely correlates with increased brain Kiss2 expression. Many fish exhibit periods of sustained negative energy balance due to reduced or suppressed food intake just prior to reproduction as a normal part of their physiology [START_REF] Milla | Corticosteroids: Friends or foes of teleost fish reproduction?[END_REF]. Our results indicate the possible existence of a link between nutritional status and reproduction in which hypothalamic kisspeptin and hypophysary gonadotropin participate. Therefore, it would be interesting in future studies to examine changes in kisspeptin genes together with changes in other genes known to be involved in feeding and energy balance, e.g., ghrelin, NPY, leptin, insulin, pro-opiomelanocortin (POMC), corticotropin-releasing hormone (CRH), among others. Furthermore, it is well established that in fish an increase in plasma cortisol levels was observed during the pre-spawning or spawning period [START_REF] Milla | Corticosteroids: Friends or foes of teleost fish reproduction?[END_REF]. A similar situation was observed in the Senegalese sole after a prolonged period of feed deprivation (21 days), where an increase in plasma cortisol levels was observed [START_REF] Costas | Feed deprivation in Senegalese sole (Solea senegalensis Kaup, 1858) juveniles: effects on blood plasma metabolites and free amino acid levels[END_REF]. For that reason, the metabolic stress suffered at 15-days of food deprivation in our study could stimulate the HPG axis in a similar way. However, it should be mentioned that these are preliminary results that need further confirmation regarding whether fasting could inhibit the HPG axis and indirectly decrease the levels of sex steroids, producing a secondary increase in Kiss2 mRNA expression levels, similar to the situation described in male rats after gonadectomy [START_REF] Arai | Factors that regulate KiSS1 gene expression in the hippocampus[END_REF]. Hence, it is tempting to speculate that kisspeptin signaling may participate in the integration of nutritional information within the reproductive axis, hence contributing to the control of reproduction in fish in response to changes in environmental conditions.

In conclusion, we have found in the Senegalese sole the Kiss2 gene, but not the Kiss1 gene, characterized its structure and defined the existence of a kisspeptin-associated peptide. We have also analyzed all the available kisspeptin sequences in vertebrates and updated the phylogenetic tree, with new information regarding the evolution of the different kisspeptin genes. We found the novel existence of two isoforms of Kiss2 and determined that they are more expressed in the female mature brain and in the ovaries of pubertal females. Finally, we present the first evidence of a relationship between food intake and kisspeptin signaling in a non-mammalian vertebrate, suggesting conserved involvement of kisspeptin in mediating energy balance effects on the reproductive axis during evolution. A c c e p t e d M a n u s c r i p t elephant shark (Callorhinchus milii) (elephant shark-1, AAVX01162971, 1-922; elephant shark-2; AAVX01172388, 1-2587), medaka (medaka-1, NM_001122921; medaka-2, NM_001160441); zebrafish (zebrafish-1, EF690279; zebrafish-2, AB439561); goldfish (goldfish-1, FJ465137; goldfish-2, FJ465138), sea bass (sea bass-1, FJ008914; sea bass-2, FJ008915), chub mackerel (chub mackerel-1, GU731672; chub mackerel-2 GU731673), grass puffer (Takifugu niphobles) (AB548304), stickleback (Gasterosteus aculeatus) (assembly BROAD S1), sockeye salmon (Oncorhynchus nerka) (AB435387), orange-spotted grouper (Epinephelus coioides) (GQ258777) and Solea senegalensis (HM116743). C) Kiss gene evolution in vertebrates inferred from the information available. D = gene duplication, L = gene lost, S = gene lost in some species, N = gene not searched. A c c e p t e d M a n u s c r i p t

The Kiss2 gene, but not the Kiss1 gene, was found in the Senegalese sole, a modern flatfish with only one type of kisspeptin receptor. However, two isoforms of Kiss2 are generated by alternative splicing in this species, with different levels of expression according to tissue and maturity status. We also present the first evidence of a relationship between food intake and kisspeptin signaling in a non-mammalian vertebrate, indicating that kisspeptin may link energy balance and reproduction.

  s c r i p t the possible kisspeptin-mediated connection between energy balance and reproduction in non-mammalian models.

  length (SL) (cm) and body weight (BW) (g) were obtained for all animals.

  Expression mRNA levels of two Ss Kiss2 isoforms (Ss Kiss2_v1 and Ss Kiss2_v2) in brain and gonads in two developmental stages.

  beta-actin genes. The RQ values for each sample were averaged and the standard error of the mean (S.E.M.) was calculated, yielding the average fold change of the target gene.

  A c c e p t e d M a n u s c r i p t 13

  ., Xu, M., MacDonald, E., Hoskins, L., 2009. Aspects of the hormonal regulation of appetite in fish with emphasis on goldfish, Atlantic cod and winter flounder: notes on actions and responses to nutritional, environmental and reproductive changes. Comp. Biochem. Physiol. A 153, 8-12. Wahab, F., Aziz, F., Irfan, S., Zaman, W., Shahab, M., 2008. Short-term fasting attenuates the response of the HPG axis to kisspeptin challenge in the adult male rhesus monkey (Macaca mulatta). Life Sci. 83, 633-637. Yang, B., Jiang, Q., Chan, T., Ko, W.K., Wong, A.O., 2010. Goldfish kisspeptin: Molecular cloning, tissue distribution of transcript expression, and stimulatory effects on prolactin, growth hormone and luteinizing hormone secretion and gene expression via direct actions at the pituitary level. Gen. Comp. Endocrinol. 165, 60-71.Zohar, Y., Muñoz-Cueto, J.A.,Elizur, A., Kah, O., 2009. Neuroendocrinology of reproduction in teleost fish. Gen.Comp. Endocrinol. 165,[438][439][440][441][442][443][444][445][446][447][448][449][450][451][452][453][454][455]

  Figure Legends

Figure 2 .

 2 Figure 2. Genomic structure of the Ss Kiss2 gene and the correspondence of the transcribed

Figure 3 .

 3 Figure 3. A) Tissue distribution of Ss Kiss2 of mature fish analyzed by RT-PCR. Agarose gel

Figure 4 .

 4 Figure 4. A) Kisspeptin-10 (Kp10) aa sequence comparison among several vertebrates.

Figure 5 .

 5 Figure 5. Ss Kiss2 expression of the two isoforms in brain A) and gonads B) in pubertal and

Figure 6 .

 6 Figure 6. Effects of fasting (15 day period) in adult Senegalese sole. A) Decrease of

Figure 7 .

 7 Figure 7. Gene expression during feeding and fasting conditions. A) Expression levels of Ss

  as fold change after data normalization against beta-actin levels Asterisks indicate statistically significant differences after the Student's t-test. *, P < 0,05; **, P < 0,01. N.S., not significant. Data shown are expressed as mean ± S.E.M. (n =7-8).

  Figure

Table 1 .

 1 Homology of the kisspeptin (Kp) and kisspeptin-10 (Kp10) region of Ss Kiss2 in comparison with human KISS1 and zebrafish Kiss1 and Kiss2.

		Kp nucleotide	Kp10 nucleotide	Kp aa	Kp10 aa
		Homology	Homology	Homology	Homology
	Human KISS1	< 20%	33%	< 20%	70%
	Zebrafish Kiss1	< 20%	57%	< 20%	60%
	Zebrafish Kiss2	< 20%	73%	33%	90%

Acknowledgements

Thanks are due to Dr. José Pedro Cañavate (IFAPA) for kindly donating the fish used in this study; Dr. A. Gómez (Institute of Aquaculture, Castellón) for helpful comments on the cloning; and Dr. F. Prat (Institute of Marine Science, Andalucía) for kindly supplying the brain Senegalese sole cDNA library used in this study. We thank Drs. M. Tena-Sempere and J.N. Nocillado for helpful comments on the manuscript. This study was carried out with the financial help of the Genoma España project "Pleurogene", and Ministry of Science and Innovation Consolider project "Aquagenomics" (CDS-2007-0002) to F.P.

Funding information: A.S.M. was supported by a predoctoral scholarship from the Spanish Ministry of Education and Science (MEC). This study was carried out with the financial help of projects "Pleurogene" and "Aquagenomics" (CDS-2007-0002) to F.P.