Stephane Desjardins

Emilie Belkai

Dominique Crete

Laurie Cordonnier

Jean-Michel Scherrmann

Florence Noble
email: <florence.noble@parisdescartes.fr>

Cynthia Marie-Claire

Desjardins Stephane

Belkai Emilie

Crete Dominique

Cordonnier Laurie

Noble Florence

NAVVEC, Neuropsychopharmacologie des addictions. Vuln rabilit Marie-Claire Cynthia

Effects of chronic morphine and morphine withdrawal on gene expression in rat

Keywords: Analysis of Variance, Animals, Behavior, Animal, drug effects, Gene Expression Profiling, Gene Expression Regulation, drug effects, Leukocytes, Mononuclear, drug effects, metabolism, Male, Morphine, administration & dosage, pharmacology, Narcotics, administration & dosage, pharmacology, Oligonucleotide Array Sequence Analysis, RNA, Messenger, genetics, metabolism, Rats, Rats, Sprague-Dawley, Substance Withdrawal Syndrome, genetics, metabolism, Time Factors '

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Opiates are among the most largely used addictive drugs. Abused opiates primarily interact with opioid receptors in the central μ nervous system and induce long-term behavioral and cellular alterations. A current challenge in opiate addiction is to understand the molecular alterations underlying the adaptative changes observed. The structure of different classes of opioid receptors, the gene encoding opioid receptors, and the signal transduction pathways triggered by opioids have been investigated extensively on neuronal cells in the last fourth decades. Chronic morphine is associated with differential gene expression in several brain structures ( ; 2004 [START_REF] Albertson | Distinctive profiles of gene expression in the human nucleus accumbens associated with cocaine and heroin abuse[END_REF] difficult to extrapolate all the results obtained from the animal brain to the patients.

Interestingly, in the case of several psychological and neurologic troubles including bipolar disorder, schizophrenia and Parkinson disease, a correlation in gene expression modulation between blood cells and brain has been recently demonstrated ( ; Bowden , 2006 et al.

). These results suggest that studies of the transcription level of certain genes in blood cells may give insights into their [START_REF] Du | Genomic profiles for human peripheral blood T cells, B cells, natural killer cells, monocytes, and polymorphonuclear cells: comparisons to ischemic stroke, migraine, and Tourette syndrome[END_REF] modulation in the brain. Moreover, in the case of major depressive disorder studies of gene expression modulations showed that peripheral leucocytes exhibit unique changes in the mRNA levels of several genes that are receptive to antidepressant treatment (Iga et al 2008).

Therefore, study of gene expression in peripheral blood mononuclear cells (PBMC) may be useful for a better understanding of brain and neurological disease related pathways, and monitoring of therapeutic responses.

On immune cells, opioid receptors with similar pharmacological and biochemical characteristics to neuronal cells have been reported ( ; ; ; ; ; ; ; [START_REF] Bidlack | Detection and function of opioid receptors on cells from the immune system[END_REF][START_REF] Gaveriaux | Identification of kappa-and delta-opioid receptor transcripts in immune cells[END_REF][START_REF] Mccarthy | Opioids, opioid receptors, and the immune response[END_REF][START_REF] Sharp | Evidence for opioid receptors on cells involved in host defense and the immune system[END_REF][START_REF] Sharp | Multiple opioid receptors on immune cells modulate intracellular signaling[END_REF]Suzuki et al., 2001aSuzuki et al., 2001b ; ). Moreover, morphine has been shown to interfere with lymphocytic cells functions following [START_REF] Suzuki | Morphine upregulates mu opioid receptors of human and monkey lymphocytes[END_REF][START_REF] Wick | Differential expression of opioid receptor genes in human lymphoid cell lines and peripheral blood lymphocytes[END_REF] binding to these receptors ( ; ). Chronic morphine treatment has been shown to alter a number of immune al showed morphine immunosuppressive effects during an escalating dose protocol in mice and these effects were abolished in mu-receptor knockout animals (

). [START_REF] Gaveriaux-Ruff | Abolition of morphine-immunosuppression in mice lacking the mu-opioid receptor gene[END_REF] In order to facilitate the interplay between morphine-treated rat models and opiate-dependent patients we chose to work on an easily accessible tissue: PBMCs. To evaluate whether transcriptional profiling of PBMC could serve to identify genes regulated by morphine rats were chronically treated with morphine during five days, and the behavioral signs of spontaneous withdrawal were observed at different times after the last injection to select the time point at which the animals displayed the minimum withdrawal signs for subsequent analyses.

High-density oligonucleotide arrays were used to assess global differential gene expression in the PBMCs. The changes in gene expression were also investigated for several transcripts by quantitative PCR 12h and 36h after the last injection when spontaneous withdrawal signs were minimum and maximum respectively. The regulations of the selected genes were also studied 4h before the last injection in order to estimate the influence of the withdrawal on the observed effects. The results showed that chronic morphine regulates gene expression in PBMCs and interestingly among these genes several have previously been found modulated by morphine treatment in rat brain.

Materials and Methods

Animals and drugs

Male Sprague-Dawley rats (Janvier, Le Genest-Saint-Isle, France) weighing 180 250 g at the beginning of the experiments were housed four per cage. Animals were maintained under standard laboratory conditions (21 1 C, 55 60 relative humidity, 12h light/dark ± °-% cycle, food and water ) for 3 to 5 days before the beginning of the experiments. Care and treatments of animals conformed to the ad libitum ethical standards and guidelines promulgated by the European Communities Council Directive (86/609/EEC). All efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. Morphine chlorhydrate (Francopia, France) was dissolved in saline (0.9 NaCl). Morphine and saline solutions (0.1mL per 100 g of body weight) were injected % intraperitoneally.

Animal Treatments

Dependence was induced in rats by repeated intraperitoneal injections of morphine twice daily at 08.00 AM and 19.00 PM. Morphine doses were progressively increased from 10 to 40 mg kg injection: first day 2 10 mg kg , second day 2 20 mg kg , third day 2 30 mg

1 - × 1 - × 1 - ×
kg , fourth and fifth days 2 40 mg kg . Control group was treated with saline following the same procedure.

1

- × 1 -

Behavioral studies

Spontaneous withdrawal syndromes were observed for 15 min in circular plexiglass observation boxes (30 cm wide; 40 cm high) 12h, 18h and 36h after the last morphine injection. Two classes of withdrawal signs were measured: counted signs and observed signs. The number of bouts of teeth chattering, wet dog shakes, mastication and tremor were counted and the sum is a score for each counted signs.

Ptosis, defecation, abnormal posture were observed over periods of 3 min, with one point being given for the presence of each sign during each period. The number of periods showing the sign was then counted (maximum score 5). Global withdrawal score was calculated as the sum of scores for all signs. Body weight was measured during the treatment and before observation period.

PBMC isolation and RNA extraction

PBMC isolation was carried out at 3 time-points: 4h before the last injection, 12h and 36h after the last injection on three independent animal groups. Animals were anaesthetized using pentobarbital (100 mg kg ) at the mentioned time points and blood was collected by 1 intracardiac ponction (8 10 mL per animal). Rat PBMCs were isolated from whole blood by density gradient centrifugation on -Ficoll-Paque Plus lymphocyte separation medium (GE Healthcare Life Science, France). The layer with PBMCs was removed and ® washed twice with saline 0.9 and the recovered cells were used directly for total RNA extraction. Total RNA was isolated with Rneasy % Micro Kits (Qiagen, Courtaboeuf, France), following the manufacturer s protocol. Each RNA sample was prepared from PBMCs ' individually isolated from one animal whole blood. Quantification of total RNA was assessed by spectrophotometry using a Nanodrop ® instrument (Nyxor, France).

Microarray target preparation and Affymetrix hybridization

The quality of the RNA samples was determined by electrophoresis using RNA labs chips (Agilent ).

Microarray analysis

DNA-Chip analyzer (DChip) version 2006 ( ) was used to analyze Affymetrix data. A comparison between the five Li and Wong, 2001

hybridizations with morphine treatment and the five controls generated a change of P-value. The average percentage of probe sets that were called as present was 60.7 among the different hybridizations except for one of the morphine array (53.5 ), this array was then % %

excluded from the subsequent study. Multiple comparisons of four morphine-treated against five control chips were therefore performed.

To identify potentially regulated transcripts we chose a cut-off of 50 fold change (treated vs.Control Assay Signal Ratio) and p<0.05.

%

Results are displayed as mean of fold induction and/or reduction. The selected transcripts after the Dchip analysis were characterized using NetAffix Analysis center ( ) and FatiGO ( ) ( ; ™ https://www.affymetrix.com + http://www.fatigo.org [START_REF] Al-Shahrour | Babelomics: a systems biology perspective in the functional annotation of genome-scale experiments[END_REF]. We used online tools that are specifically designed to assist investigators to classify the functional roles of the identified al., 2005 differentially expressed genes. The FatiGO utilizes the Gene Ontology (GO) database provided by the GO consortium ( +) and other databases as KEGG pathway. We compared overexpressed or underexpressed gene lists with rat http://www.geneontology.org/ RAE 230.2 array list. Then a Fisher s exact test was used to check for significant over representation of GO terms in one of the sets.

'

Multiple test correction to account for the multiple hypothesis tested (one for each GO term) was applied (FDR adjusted test).

Reverse transcription and Real-Time PCR

Independent batches of RNA were obtained from PBMCs of morphine and saline-treated rats. Total RNAs were reverse transcribed in a final volume of 20 L containing 1X RT-PCR buffer (Invitrogen , France), 500 M each deoxynucleotide triphosphate, 20 U of RNasin

μ ® μ
RNase inhibitor (Promega, France), 10 mM DTT, 100 U of Superscript II RNase H reverse transcriptase (Invitrogen, France), 1.5 M μ random hexamers (Amersham-Pharmacia , France), and 1 g of total RNA. Samples were incubated at 20 C for 10 min and 42 C for 30

® μ °°min,
and reverse transcriptase was inactivated by heating at 99 C for 5 min. PCR primers were chosen with the assistance of Oligo 6.42 °software (MedProbe, Norway). The primer nucleotide sequences used in this study are listed in . Fluorescent PCR analysis was Table I performed using a LightCycler instrument (Roche Diagnostics, France). The cDNAs were diluted 40-fold and 5 L were added to the o generate an external standard curve for each gene. We also quantified transcripts of the gene coding for the Ribosomal Protein Large, P0 (Rplp0), a component of the 60S subunit of ribosomes, as the endogenous RNA control and normalized each sample on the basis of its RPLP0 content.

Statistical analysis

All series of data were analyzed with GraphPad Prism 4.0 software. For behavioral experiment, three independent experiments were

Results

Behavioral experiment

Physical dependence was induced in male Sprague-Dawley rats by repeated i.p injections of morphine twice daily and the withdrawal signs were monitored in order to determine the time points for the transcriptional studies. The weight gain was monitored every 12h throughout morphine treatment and withdrawal, as shown on . A significant increase of 18 was observed in control rats and the figure 1a % weight of rats treated with morphine increased by 13 during the five days of morphine treatment. This difference was not statistically % significant. The weight of the control group continued to increase during 4 days after the last injection of saline; while morphine withdrawal induced a significant reduction on weight gain in rats treated with the alkaloid. A maximum difference was observed in body weights of control versus morphine-treated rats 36h after the last injection (p<0.001) (

). Figure 1a Morphine-withdrawn animals showed behavioral signs of opiate withdrawal, including wet dog shakes, teeth chattering, diarrhea, mastication, abnormal posture and ptosis. The calculated global withdrawal scores are presented in . Withdrawal signs were figure 1b observed at 12h, 18h and 36h after the last injection and global withdrawal scores of morphine-treated animals were significant (p<0.001) at each time-point. Twelve hours after the last morphine injection, all the withdrawal signs were already present but the global withdrawal score was less important than those calculated at 18h and 36h after the last injection. The 12h and 36h time points displaying the minimum and maximum withdrawal scores were therefore selected for the subsequent studies.

Regulation of gene expression in the PBMCs

The Affymetrix GeneChip Rat Genome RAE 230 2.0 was used to identify genes differentially regulated in the PBMCs of rats at transcripted loci with various levels of matches to known genes. A selective list of the genes modulated in this study is presented in Table . We used NetAffix Analysis Center and FatiGO to identify the signalling pathways and genes families regulated by chronic II ™ +

morphine (

). The results revealed several genes families that have been previously described as modulated by chronic morphine in Figure 2 the brain. We found that 7 of the 276 annoted genes regulated belong to the G-protein coupled receptor protein signalling pathway, these % include G-protein coupled receptors and G-protein coupled protein. Signal transduction pathways were also represented such as mitogen activated protein kinase pathway and cAMP pathway. Other regulated genes have a role in immunity and defense process interleukin 1 receptor, type I (Illr1), interferon beta 1 (Ifnb1), and a role in pro-apoptotic Tissue inhibitor of metalloproteinase 3 (Timp3), Bcl2 modifying factor (Bmf), guanine nucleotide binding protein, alpha q (Gnaq), NCK-associated protein 1 (Nckap1), and anti-apoptotic pathways Sphingosine kinase 1 (Sphk1), Chemokine (C-C motif) ligand 2 (Ccl2), Fibroblast growth factor 8 (Fgf8) . Three [ ] 5-hydroxytryptamine receptors 6, 7 and 2a (Htr6, Htr7 and Htr2a), and calcium channels were also identified.

Validation of differentially regulated genes by quantitative RT-PCR at 12h

In order to validate the regulation of 24 selected genes we performed quantitative real-time RT-PCR on samples collected at the same time-point (12h) as for the array analysis (minimum withdrawal). We chose genes of the G protein pathway (Gprk5, Gng11), genes of the Mapk pathway (similar to mitogen-activated protein kinase kinase kinase kinase 5 isoform 2), mitogen-activated protein kinase 7 (Mapk7)

and genes known to be modulated in the brain by morphine treatment (Adcy5, Adenosine kinase, Htr2a). Since morphine promotes cells apoptosis, we also chose to confirm the regulation of genes implicated in this process such as Bmf, Nckap1, Timp3, and also genes implicated in cell adhesion such as Integrin beta 1 (Itgb1) and integrin alpha 6 (Itga6). Out of the 18 tested genes 12 were validated by Q-RT-PCR ( ). Furthermore, the fold-changes obtained were similar to those calculated in the array experiments ( and Table III Table II ). We also tested 6 genes that belong to the tested families and almost fulfill the criteria detailed in the material and methods table III section: regulator of G-protein signalling 10 (Rgs10) (1.46), membrane interacting protein of Rgs16 (Mir16) (1.39), mitogen-activated protein kinase 8 interacting protein 3 (Mapk8ip3) (1,35), adaptor-related protein complex 1, sigma 1 subunit (Ap1s1) (1.36), calpain 2 (Capn2) (1.4), RAS, guanyl releasing protein 2 (Rasgrp2) (1.22). We found that the regulations of Rgs10, Mir16, Capn2, Rasgrp2, were verified by quantitative real-time RT-PCR at 12h ( ).

Table III

Regulation of differentially regulated genes by quantitative RT-PCR at 36h

In order to compare the expression levels at the minimum and maximum withdrawal scores in our model, we carried out an additional independent experiment at 36h to analyze the differential expression levels between morphine-treated and control rats of 18 genes confirmed or not at 12h. Four groups of genes could be distinguished according to their pattern of regulation at 12h and 36h. Four genes + + (Adcy5, Mir 16, Camk4, and Anxa3) were significantly up-regulated at 12h but significantly down-regulated at 36h. A group with Itga6 and Rasgrp2 showed a sustained up-regulation at 12h and 36h. A group with Adk, Nckap1, Gng11, Gprk5, Rgs10, Capn2, Fgl2, Sod2, Timp3, Ap1s1, and Itgb1 showed a significant up-regulation at 12h but no modulation at 36h. And a fourth group composed of Mapk8ip3 and Htr2A was not significantly modulated at 12h but significantly regulated at 36h.

Regulation of the validated genes during chronic morphine treatment

In order to know how the validated genes were being expressed before the onset of the withdrawal process, quantitative real time PCR was performed on PBMCs of rat sacrificed on day 5. For all the tested genes the regulations observed 4h before the last morphine injection confirmed those obtained 12 hours after the last morphine injection ( ). Furthermore, the fold changes observed at 4h were very Table III similar to those obtained at 12h. In addition no significant regulation was found at 4h for the 7 genes that were not modulated at 12H.

+ -+

Discussion

The aim of this study was to identify differentially expressed genes in blood cells of morphine-dependent rats in spontaneous withdrawal situation. The large majority of studies carried out to date have used opioid receptor antagonist-precipitated withdrawal syndrome. However, this experimental approach greatly differs from the clinical setting where the opiate withdrawal syndrome occurs as a result of drug intake cessation. To better mimic these conditions, spontaneous withdrawal was evaluated. We observed classical signs used in behavioral studies and found low levels of withdrawal scores. Indeed spontaneous withdrawal induces milder behavioral modulation than the naloxone-precipitated one. As expected, the severity of opiate withdrawal varied as a function of the time interval between the last morphine administration and testing. In order to study the modifications in gene expressions in morphine-dependent rats and to minimize the transcriptional effects of withdrawal, the 12h time point was selected for microarray analysis. At this time point, withdrawal + symptoms and loss of weight were at their minimum. At 36h, the withdrawal signs were maximum, and this time point was therefore selected for comparison with the 12h results. An additional time point (4h before the last injection) was chosen in order to assess the modulation of the selected candidate genes before the beginning of the withdrawal process.

Several studies have demonstrated characteristic blood genomic profiles for different brain disorders and neurologic diseases (Du et al.

; ;

). A study of gene expression in PBMCs from schizophrenic patients suggested a correlation , [START_REF] Sharp | Multiple opioid receptors on immune cells modulate intracellular signaling[END_REF][START_REF] Tang | Blood gene expression profiling of neurologic diseases: a pilot microarray study[END_REF]et al. Tang , 2004 et al. between the gene expression modulations patterns in PBMCs and the severity of the schizophrenic syndrome (

). In the [START_REF] Bowden | Preliminary investigation of gene expression profiles in peripheral blood lymphocytes in schizophrenia[END_REF] case of migraine suffering patients differential gene expression modulations were found in platelets as compared to control patients ( ). Tang and coworkers also found a specific gene expression pattern in PBMCs from Tourette syndrome patients ( Hershey , 2004 et al. Tang ). Moreover in the case of depression the low level of expression of alpha2a adrenergic receptors observed in the platelets of , 2005 et al.

depressive patients could be increase after a treatment with an antidepressive compound (

). These results suggest [START_REF] Garcia-Sevilla | Regulation of platelet alpha 2A-adrenoceptors, Gi proteins and receptor kinases in major depression: effects of mirtazapine treatment[END_REF] a correlation between the gene expression regulations in the brain and the blood cells in these pathologies. The identification of an easily accessible tissue (blood) where modifications of gene expression following chronic morphine treatment could give insight to those occurring in the CNS would facilitate clinical research on the mechanisms of addiction in humans (

). In this study, we [START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF] used PBMCs as a model to study blood expression profile following morphine treatment.

Our results describe the regulation of various genes and pathways in PBMCs of rats treated with morphine. 17 out the 24 tested genes were validated by quantitative real-time PCR at 12h. Interestingly the modulations observed on the last day of the morphine treatment + were similar to those obtained at the 12h time point of the spontaneous withdrawal but evolved as the withdrawal state worsens. Among + the regulated pathways in PBMCs some have been previously identified in vitro in transcription studies. Stefano et al have showed a regulation of apoptosis and cell adhesion genes in human peripheral blood leukocytes after acute morphine treatment ( ).

Stefano

, 2005 et al.

In addition morphine has been shown to modulate immune responses (

). [START_REF] Eisenstein | Opioid modulation of immune responses: effects on phagocyte and lymphoid cell populations[END_REF] Interestingly, among the genes found regulated in PBMCs several have been previously reported to be involved in responses to morphine in different brain regions. The two major transcription pathways (Mapk and G protein coupled receptor) reported to be regulated by morphine treatment in brain ( ; ; ) were also found regulated in PBMCs. [START_REF] Asensio | Stimulation of mitogen-activated protein kinase kinases (MEK1/2) by mu-, delta-and kappa-opioid receptor agonists in the rat brain: regulation by chronic morphine and opioid withdrawal[END_REF][START_REF] Fan | Acute and chronic morphine treatments and morphine withdrawal differentially regulate GRK2 and GRK5 gene expression in rat brain[END_REF][START_REF] Traynor | Regulators of G prtein signaling and drugs of abuse[END_REF] Opioid receptor activations lead to physiological responses via coupling to inhibitory guanine nucleotide-binding regulatory protein (Gold ), modulating several effectors, such as adenylyl cyclases, and voltage-gated Ca channels. In the present study, modulations , 2003 et al. ). In the present study [START_REF] Nestler | Molecular basis of long-term plasticity underlying addiction[END_REF] we observed an increase in the expression of several mitogen-activated protein kinases (Mapk7, similar to Mapkkkk5). Mapk pathway has been shown to be activated in various regions of rat brain by opioid receptors coupled to G-proteins ( ).

Schulz

, 2004 et al.

Electrophysiological studies have suggested a functional relationship between the opioid and serotoninergic systems in the rat prefrontal cortex involving the and Htr2a receptors (

). Moreover, an implication of Htr2a receptors in the dopamine release, ). They are involved in several cellular functions [START_REF] Uings | Cell receptors and cell signalling[END_REF] including the modulation of G-protein-coupled receptors signalling (

). In this study we found that morphine treatment [START_REF] Berg | Integrins regulate opioid receptor signaling in trigeminal ganglion neurons[END_REF] upregulated two integrins genes (Itgb1 and Itga6) in rat PBMCs. Moreover, the severity of the withdrawal symptoms did not affect the expression of these two genes. Interestingly, Itgb1 has been recently shown to regulate opioid receptor signalling in sensory neurons (Berg ). , 2007 et al. In conclusion, our data show that chronic morphine treatment induces transcriptional modifications in PBMCs that evolve with the withdrawal state of the animals. This suggests that PBMC gene expression patterns could be indicative of the disease stage. For some of the modulated genes the PBMC regulations confirmed previously reported brain modulations. In order to better characterize the correlations suggested here, a study of the modulation of the differentially regulated genes obtained in PBMCs, in brain structures involved in addiction, is currently carried out. This approach could help to identify important diagnostic/prognostic markers as well as potential therapeutic targets and mechanistic pathways.

Table I

Primer sequences used for SYBR Green-based real-time quantitative polymerase chain reaction.

Gene gene forward primer (sense) gene reverse primer (antisense)

Adcy5 5 ATGGGTCTGCTTTTCCACT 3 ′ ′ 5 GGAGGTGTGAGGAGAGAATG 3 ′ ′ Adk 5 TGAGCGTTCCTGCCTTGA 3 ′ ′ 5 CCCTCTACGGTAATAATG 3 ′ ′ Anxa3 5 CTGTGGAGGAGATGATTGAAGA 3 ′ ′ 5 TCGTACAGCACTCCAGCA 3 ′ ′ Ap1s1 5 CTCCAGCCCTACATACAAAC 3 ′ ′ 5 CCCTAAAGCCTTCAGTGG 3 ′ ′ Bmf 5 GAGCCAAGTAGTCAGTTACCAC 3 ′ ′ 5 GTCCAATCCAGTCCAGTCAT 3 ′ ′ Camk4 5 CATCCTACACCCTCCACAAGTCT 3 ′ ′ 5 ACAAGGCTGGTCCCCCTC 3 ′ ′ Capn2 5 CAGCAAGCCCTCTTAGACT 3 ′ ′ 5 TCTGAGCCCTGTTCTCC 3 ′ ′ Fgl2 5 TATCGCTTTTTGGCTGAACA 3 ′ ′ 5 AGAGGCTGAAGACATTTGCT 3 ′ ′ Gng11 5 TCCAGAAGACAAGAATCCC 3 ′ ′ 5 ATTTCCCTCCAGAGTTCCT 3 ′ ′ Gprk5 5 ACTTGGAAGCAGAACTTGTAAC 3 ′ ′ 5 TGGGGGAACAGTATCCTATC 3 ′ ′ Htr2a 5 CAGGCGAGTCACAGGATAG 3 ′ ′ 5 CACAGCAGTAAAAGACAGCA 3 ′ ′ Itga6 5 GAAGTGAGGACCCTTGCTGATG 3 ′ ′ 5 CGAACCTGTGCCTTAGTGACAA 3 ′ ′ Itgb1 5 AAAGCAAATGCCAAATCTTGTG 3 ′ ′ 5 TTCCTTCTTGTAAAAATGTCGTA 3 ′ ′ Mapk7 5 CCCGCTGACATTGAGTCT 3 ′ ′ 5 GTGGGTCTCAGGGTTCTTG 3 ′ ′ Mapk8ip3 5 TGGAGAGCAACTGACAAGA 3 ′ ′ 5 AGTGCCCCTGAAGAGTAAG 3 ′ ′ Mir16 5 AAAGGAATCCAGGTTGTTAGTT 3 ′ ′ 5 CAAGGTGGGACTCGTAGTAAT 3 ′ ′ Nckap1 5 GGAAGCAGACTCAGTTTCTC 3 ′ ′ 5 CAGCAGGGTCCAAGGTAG 3 ′ ′ Ptpn22_predicted 5 CAAGCAAGCCTACAGAATG 3 ′ ′ 5 TGGTCCTTTGGGTTTTG 3 ′ ′ Ralgps2 5 GAAGGCAAAAAACCCACAGT 3 ′ ′ 5 CCACCACAGACACATTCTTA 3 ′ ′ Rasgrp2 predicted 5 GAAGCCTTTGATGACTCTGG 3 ′ ′ 5 GGGATTGCTGATAGAAGTGG 3 ′ ′ similar to Mapkkkk5 isoform 2 5 CTCACAGCAATCTCTACATCC 3 ′ ′ 5 CATTCGTCGTCTTGTCACT 3 ′ ′ Rgs10 5 GGCGGACTGAGGAAGAG 3 ′ ′ 5 CACAACAAAGCAATGACATC 3 ′ ′ RPLP0 5 GGGGCCACCTGGAGAACAAC 3 ′ ′ 5 GCA GCA GCT GGC ACC TTA TTG 3 ′ ′ SOD2 5 GGCTTGGCTTCAATAAGGAG 3 ′ ′ 5 ATCCCCAGCAGTGGAATA 3 ′ ′ Timp3 5 GAAGCCTCTGAAAGTCTCTG 3 ′ ′ 5 CCCACCTCTCCACAAAGT 3 ′ ′

Table II

Affymetrix data at 12h of selective genes. Fold changes compare PBMC s treated rats to PBMC s saline rats. In bracket, fold change of different probe sets on the array

Table III

Validation and evolution of differentially regulated genes by quantitative RT-PCR at 4h, 12h and 36h of the last morphine injection. Data represent fold change in PBMCs of morphine treated rat (n

- + + = 12

  to yield a total volume of 10 L. The PCR reagents were obtained from the FastStart DNA Master SYBR Green I kit μ (Roche Diagnostics, France). The reaction buffer contained 4 mM MgCl and 0.5 M of each primer. Amplification protocols consisted of 2 μ 35 40 cycles of denaturation at 94 C (5 s), annealing at 64 C (5 s), and extension at 72 C (10 s). cDNAs from a naive rat blood were used -°°°t

®

  performed, 15 morphine-treated rats and 15 control rats were compared at each time-point. Data were analyzed by two-way ANOVA with Bonferonni post-test. For body weight study three independent experiments were performed, 28 treated and placebo rats were compared at each time-point. Data were analyzed by two-way repeated measures ANOVA with Bonferonni post-test. For real-time quantitative RT-PCR results, three additional experiments at 4h, 12h and 36h were carried out and the RNAs from 12 14 morphine-treated rats and rats were compared. Data were analyzed using Student s test between groups at each time point. Differences with <0.05 -' p between experimental groups at each point were considered statistically significant.

  last morphine injection. To obtain a set of differentially expressed genes and global gene expression profiling, we used a combinaison of value (P<0.05) and fold change value (>1.5 or <0.66). Comparison of chronic morphine with control condition revealed P-812 (596 up-regulated and 215 down-regulated) differentially expressed (50 fold or more) gene transcripts in PBMCs of treated rats. Of % the 812 significantly modulated candidates, only 394 were annotated and represented 276 distinct genes, whereas the others (51.5 ) were %

  2+of genes belonging to two major components of the development of opiate addiction were found in the PBMCs: the G-protein coupled receptor signalling (Gprk5, gng11, Rgs 10, Mir 16) and the cAMP signalling (Adcy5) pathways. Adcy5 has been shown to play an important role for the development of morphine behavioral effects ( ). Nestler et al have shown that decreased activation of Kim , 2006 et al. ERK1/2 might be relevant in modulating long lasting forms of synaptic plasticity in opiate addiction (

  serotonin receptor Htr2a by morphine treatment are consistent with these findings. Integrins are transmembrane receptors allowing the interactions of cells with the extracellular matrix (

  Figure 1 a) Evolution of weight variation as compared to weight before the first injection, n 15 rats per treatment. Body weight of each rat was = measured before the injection (twice daily from 5 days) or the observation (from 12h to 96h after the last injection). Three independent + experiments were achieved. Results are expressed as mean .SEM. p<0.05, p<0.01, p < 0.001, NS: non significant Two-way repeated ± * ** *** measure ANOVA. b) Evolution of global withdrawal score. Mean SEM of opiate withdrawal score (n 15 rats per treatment) at 12h, 18h ± = + + and 36h after the last injection. Two Way ANOVA p<0.05; p<0.01; p<0.001. + * ** ***

  14) as compared to saline treated rats (n 10 12).

  was done by Student test * p<0.05, ** p<0.01,

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

[START_REF] Sullivan | Evaluating the comparability of gene expression in blood and brain[END_REF]

Ackowledgements:

This work was supported by the Consortium national de recherche en genomique (CNRG) and the Mission Interministerielle de Lutte contre la Drogue et la Toxicomanie (MILDT).


Samuel P Caro
email: samuel.caro@cefe.cnrs.fr

Léo Pierre

Matthieu Bergès

Raldi Bakker

Claire Doutrelant

Francesco Bonadonna

Mutual mate preferences and assortative mating in relation to a carotenoid-based color trait in blue tits

Keywords: bird, carotenoids, color, mate-choice, sexual selection

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Choosing mates is one of the most important decisions that many sexually reproducing animals have to take. Mate choice indeed impacts an individual's current reproductive success and fitness [START_REF] Andersson | Sexual selection[END_REF]. Both direct and indirect benefits, such as access to a high-quality territory, parental care, reduced likelihood of getting sexually transmitted disease, or genetic quality for the offspring, can be derived from selecting an appropriate partner [START_REF] Fisher | The evolution of sexual preference[END_REF][START_REF] Trivers | Parental investment and sexual selection[END_REF][START_REF] Smith | Theories of sexual selection[END_REF]. In conventional sexrole species, females are typically considered the choosing sex, while males spend their time advertising [START_REF] Jones | Mate choice and sexual selection: What have we learned since Darwin?[END_REF][START_REF] Davies | An introduction to behavioural ecology[END_REF]. As a consequence, mate choice has been mostly studied in terms of male traits and behaviours that affect female preference. However, conspicuous traits are present both in males and females of many species [START_REF] Amundsen | Why are female birds ornamented?[END_REF][START_REF] Dale | The effects of life history and sexual selection on male and female plumage colouration[END_REF][START_REF] Hare | Sexual selection and its evolutionary consequences in female animals[END_REF][START_REF] Doutrelant | Evolution of female coloration: What have we learned from birds in general and blue tits in particular[END_REF]. While these traits in females have long been considered non-functional byproducts of sexual selection on male traits [START_REF] Lande | Sexual dimorphism, sexual selection, and adaptation in polygenic characters[END_REF][START_REF] Price | Sexual selection, selection load and quantitative genetics of zebra finch bill colour[END_REF][START_REF] Kraaijeveld | The evolution of mutual ornamentation[END_REF], the possibility that males could also choose their female partners has received comparatively less attention [START_REF] Amundsen | Why are female birds ornamented?[END_REF][START_REF] Clutton-Brock | Sexual selection in females[END_REF][START_REF] Courtiol | The evolution of mutual mate choice under direct benefits[END_REF][START_REF] Doutrelant | Evolution of female coloration: What have we learned from birds in general and blue tits in particular[END_REF]. Like male ornaments, female ornaments could signal reproductive or survival qualities and be involved in male mate choice [START_REF] Hare | Sexual selection and its evolutionary consequences in female animals[END_REF]. Mutual choosiness is predicted to be particularly important when benefits of choices are evident for both sexes, in particular in monogamous species where reproductive rates are similar for males and females, and in species with biparental care [START_REF] Johnstone | Mutual mate choice and sex differences in choosiness[END_REF][START_REF] Kokko | Why is mutual mate choice not the norm? Operational sex ratios, sex roles and the evolution of sexually dimorphic and monomorphic signalling[END_REF][START_REF] Kraaijeveld | The evolution of mutual ornamentation[END_REF]. Male mate preferences have been documented in various taxa, including fishes [START_REF] Sargent | Male mate choice in fishes[END_REF][START_REF] Amundsen | Male mate choice selects for female coloration in a fish[END_REF][START_REF] Roberts | Male mate choice contributes to behavioural isolation in sexually dimorphic fish with traditional sex roles[END_REF][START_REF] Schlupp | Male mate choice in livebearing fishes: an overview[END_REF], insects [START_REF] Bonduriansky | The evolution of male mate choice in insects: a synthesis of ideas and evidence[END_REF][START_REF] Byrne | Evidence for adaptive male mate choice in the fruit fly Drosophila melanogaster[END_REF], reptiles [START_REF] Olsson | Male-preference for large females and assortative mating for body size in the sand lizard (Lacerta agilis)[END_REF][START_REF] Lebas | The role of colour in signalling and male choice in the agamid lizard Ctenophorus ornatus[END_REF], mammals [START_REF] Domb | Sexual swellings advertise female quality in wild baboons[END_REF][START_REF] Fitzpatrick | Exaggerated sexual swellings and male mate choice in primates: testing the reliable indicator hypothesis in the Amboseli baboons[END_REF], and birds [START_REF] Amundsen | On the function of female ornaments: male bluethroats prefer colourful females[END_REF][START_REF] Griggio | Males prefer ornamented females: a field experiment of male choice in the rock sparrow[END_REF][START_REF] Pryke | The relative role of male vs. female mate choice in maintaining assortative pairing among discrete colour morphs[END_REF][START_REF] Kimmitt | Male courtship preference during seasonal sympatry may maintain population divergence[END_REF]). In birds for example, male bluethroats (Luscinia s. svecica) have been reported to spend more time close to colorful than drab females in a binary choice experiment [START_REF] Amundsen | On the function of female ornaments: male bluethroats prefer colourful females[END_REF]. Female coloration positively correlated with tarsus length and body mass, suggesting that coloration can also signal phenotypic quality in females [START_REF] Amundsen | On the function of female ornaments: male bluethroats prefer colourful females[END_REF][START_REF] Roulin | Female barn owls (Tyto alba) advertise good genes[END_REF][START_REF] Griggio | Males prefer ornamented females: a field experiment of male choice in the rock sparrow[END_REF][START_REF] Weiss | Female-specific color is a signal of quality in the striped plateau lizard (Sceloporus virgatus)[END_REF][START_REF] Doutrelant | Female coloration indicates female reproductive capacity in blue tits[END_REF][START_REF] Williams | Physiological adaptation for breeding in birds[END_REF]. In crested auklets (Aethia cristatella), both males and females were attracted by opposite-sex models having experimentally increased crests on the front-head [START_REF] Jones | Mutual sexual selection in a monogamous seabird[END_REF]. On the other hand, evidence for male mate preference has failed to be shown in other studies [START_REF] Muma | Male traits expressed in females: direct or indirect sexual selection?[END_REF][START_REF] Dale | Male pied flycatchers do not choose mates[END_REF]. Since reproduction is often less costly for males than females [START_REF] Williams | Physiological adaptation for breeding in birds[END_REF], and additional paternities can be gained from fertilizing multiple females, males might not always need to be choosy (but see [START_REF] Pizzari | Sophisticated sperm allocation in male fowl[END_REF]. Male choosiness might even be counterselected if choosy males face a competitive disadvantage [START_REF] Fitzpatrick | Exaggerated sexual swellings and male mate choice in primates: testing the reliable indicator hypothesis in the Amboseli baboons[END_REF]. These contrasted hypotheses highlight the need for additional work on male sexual preferences to understand how often males contribute to sexual selection.

For a long time, it has also been considered that individuals always try to mate with the highest quality partners. In practice, this has assumed that regardless their own condition or phenotype, females (historically considered the choosing sex, see above) should always choose the best males, and therefore that there is little inter-individual variation in preferences [START_REF] Cotton | Sexual selection and condition-dependent mate preferences[END_REF]. Several studies have however challenged this view and shown that individuals may vary substantially in their mate preferences, with preferences that can sometimes be predicted from the subject's own phenotype.

Female black field crickets (Teleogryllus commodus) raised on a protein-rich diet were more responsive and choosy to male call quality than females reared on poorer diets [START_REF] Hunt | Female mate choice as a condition-dependent life-history trait[END_REF]. Females of the smooth toadlet (Uperoleia laevigata), an Australian frog, selected mates that were about 70% of their own body weight [START_REF] Robertson | Female choice increases fertilization sucess in the Australian frog, Uperoleia laevigata[END_REF].

In zebra finches (Taeniopygia guttata), females that were reared in enlarged broods, and that were concomitantly considered as being of poorer quality than females raised in small broods, were shown to prefer males that were themselves of lower quality [START_REF] Holveck | Low-quality females prefer low-quality males when choosing a mate[END_REF]; see Griggio and Hoi, 2010 for a similar example in sparrows; but see [START_REF] Wang | No mutual mate choice for quality in zebra finches: Time to question a widely-held assumption[END_REF]. Similarly, male and female great tits (Parus major) have been shown to prefer opposite-sex individuals that were of similar heterozygosity levels as themselves, even if in the case of relatively homozygous individuals it meant having offspring of low heterozygosity level [START_REF] Zandberg | Direct fitness benefits explain mate preference, but not choice, for similarity in heterozygosity levels[END_REF]. In the frog and the great tit examples, the authors also showed that these preferences are adaptive because pairing with the preferred phenotypes led to higher fertilization rates and fitter offspring, respectively [START_REF] Robertson | Female choice increases fertilization sucess in the Australian frog, Uperoleia laevigata[END_REF][START_REF] Zandberg | Direct fitness benefits explain mate preference, but not choice, for similarity in heterozygosity levels[END_REF]. Such kind of selfreferent phenotype matching, leading to assortative mating, could be quite widespread. It has however been relatively little studied experimentally so far.

Here we study experimentally the mate preferences of captive blue tits. Blue tits are socially monogamous, both sexes care for the offspring, and both sexes are ornamented [START_REF] Andersson | Ultraviolet sexual dimorphism and assortative mating in blue tits[END_REF], characteristics that, according to what we have reported above, are ideal for testing mutual mate preference [START_REF] Hunt | Preferences for ultraviolet partners in the blue tit[END_REF] and assortative mating [START_REF] Fargevieille | Assortative mating by colored ornaments in blue tits: space and time matter[END_REF]. In blue tits both the UV-blue crest and the yellow chest have been suggested to function as secondary sexual signals (but see [START_REF] Parker | What do we really know about the signalling role of plumage colour in blue tits? A case study of impediments to progress in evolutionary biology[END_REF] for a meta-analysis on male UV coloration). Most studies focused on the role that blue crest color variation plays on intra-sexual competition and on intersexual selection [START_REF] Hunt | Blue tits are ultraviolet tits[END_REF]1999;e.g. Delhey et al., 2003;[START_REF] Kurvers | No consistent female preference for higher crown UV reflectance in Blue Tits Cyanistes caeruleus: a mate choice experiment[END_REF][START_REF] Remy | Mediating male-male interactions: the role of the UV blue crest coloration in blue tits[END_REF][START_REF] Parker | What do we really know about the signalling role of plumage colour in blue tits? A case study of impediments to progress in evolutionary biology[END_REF]. The role of the yellow-colored chest has received less attention, but there is correlational and experimental evidence that the intensity of yellow chroma or brightness may reflect an individual's condition and parental ability [START_REF] Senar | Brighter yellow blue tits make better parents[END_REF][START_REF] Saks | Carotenoid-based plumage coloration of male greenfinches reflects health and immunocompetence[END_REF][START_REF] Peters | The condition-dependent development of carotenoid-based and structural plumage in nestling blue tits: Males and females differ[END_REF][START_REF] Doutrelant | Female coloration indicates female reproductive capacity in blue tits[END_REF][START_REF] Ferns | Carotenoid plumage hue and chroma signal different aspects of individual and habitat quality in tits[END_REF][START_REF] Reudink | Plumage brightness predicts non-breeding season territory quality in a long-distance migratory songbird, the American redstart Setophaga ruticilla[END_REF][START_REF] Williams | Physiological adaptation for breeding in birds[END_REF][START_REF] Garcia-Navas | Plumage yellowness predicts foraging ability in the blue tit Cyanistes caeruleus[END_REF][START_REF] Midamegbe | Female blue tits with brighter yellow chests transfer more carotenoids to their eggs after an immune challenge[END_REF]. The yellow color could thus potentially be used as a reliable signal of quality in a mate-choice context. Contrary to the blue color that is structurally based, the yellow color is conferred by carotenoids exclusively acquired through food [START_REF] Goodwin | The biochemistry of the carotenoids[END_REF][START_REF] Hill | Bird coloration: function and evolution[END_REF][START_REF] Isaksson | Sex and age differences in reflectance and biochemistry of carotenoid-based colour variation in the great tit Parus major[END_REF]. In captivity, where the right balance of carotenoids is difficult to provide, blue and great tits rapidly loose their yellow-based coloration that becomes light-grey (see Fig. S1A), showing how plastic this trait is. Carotenoids are also thought to contribute to prevention of oxidative stress and regulation of immune function [START_REF] Blount | Carotenoid modulation of immune function and sexual attractiveness in zebra finches[END_REF][START_REF] Biard | Carotenoid availability in diet and phenotype of blue and great tit nestlings[END_REF][START_REF] Simons | What does carotenoid-dependent coloration tell? Plasma carotenoid level signals immunocompetence and oxidative stress state in birds-A meta-analysis[END_REF][START_REF] Koch | Do carotenoid-based ornaments entail resource trade-offs? An evaluation of theory and data[END_REF].

Here, we test whether both male and female blue tits choose their partners, and whether the expressed preferences are based on the color of their breast. For this, we manipulated the chest color of captive blue tits and tested the preferences of opposite-sex individuals for variation in this phenotypic trait over two successive experiments. In the first experiment, we manipulated the chest colors of the birds that were presented as stimuli. In the second experiment, we manipulated the chest colors of both the stimulus and the subject birds. We then tested whether both males and females express preferences (experiments 1 and 2), whether they show preferences for the same color phenotypes (experiments 1 and 2), and whether their own phenotype (chest color) influences the preference they express (experiment 2).

Methods

Ethical note

The experiments run in this study were approved by the Animal Experimentation Committee of the Royal Dutch Academy of Sciences (DEC-KNAW; permit number CTE.09-04 and NIOO11.09), and the Animal Care and Use Committee Languedoc-Roussillon (permit number CEEA-LR-1047). The work performed in the field was approved by the prefectural office of Corsica and the Regional Direction of Environment (DIREN) committee (permit numbers 2009-0379, 3467 and 2015615-147).

Subjects

In experiment 1, we used 81 blue tits (Cyanistes caeruleus; 44 males and 37 females) that were taken as nestlings in the long-term studied populations in Corsica (Muro and Pirio, see [START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. Whole broods of chicks (N=17 broods) were collected from their nests when they were 7 to 10-day-old, and were transferred to the laboratory for standardized hand rearing (see [START_REF] Drent | Realized heritability of personalities in the great tit (Parus major)[END_REF][START_REF] Titulaer | Personality affects learning performance in difficult tasks in a sex-dependent way[END_REF]. Briefly, birds were transported to the Netherlands Institute of Ecology (NIOO-KNAW) by car. During the travel, and later on at the institute, chicks were fed every half-hour, for 14 hours per day (7:00 am -9:00 pm), with a diet consisting of a mixture of curd cheese, ground beef heart, baby cereal, multivitamin solution and calcium carbonate, supplemented with wax moth larvae and bee larvae, until independence. After reaching independence (about 35 days after hatching) and being molecularly sexed, the birds were temporarily transferred to an individual home cage of 0.9 x 0.4 m x 0.5 m, before being released in single-sex groups in outdoor aviaries, with acoustic but no visual contacts between aviaries. Adult diet consisted of a mixture of egg, cow heart, vitamins, and minerals, supplemented with dry food containing insects (Orlux Insect Patee, Versele-Laga, Deinze, Belgium) and peanuts. Birds also had access to mealworms, grit, and sunflower seeds. Food and water were provided ad libitum.

In experiment 2, we used 72 blue tits (40 males and 32 females) that were also taken as nestlings, aged 8 to 12-day-old, in the long-term studied population of La Rouvière, near Montpellier. Hand-rearing procedures were similar to those of experiment 1, except that it was conducted at the Center for Functional and Evolutionary Ecology (CEFE-CNRS) at Montpellier, France. Diet consisted here in a solution of hand-rearing powder (Nutribird A21 and A19, Versele-Laga, Deinze, Belgium), supplemented with wax moth larvae, bee larvae, and mealworms. After they reached independence, birds were fed with mealworms, and a cake made of eggs, sunflower margarine, sugar, flour and protein-rich pellets (Country's Best Show1-2 Crumble, Versele-Laga, Deinze, Belgium).

Both mealworms and cake were supplemented with commercial powders containing mostly vitamins and minerals (Nutribird A21, Versele-Laga; and Nekton-S, Nekton GmbH, Pforzheim, Germany). Food and water were provided ad libitum. Birds were also housed in single-sex groups in outdoor aviaries. Here however, visual and physical contact were possible through the mesh of adjacent aviaries. Aviaries housing groups of females were away from aviaries housing groups of males, with no visual and physical contacts possible between males and females. For logistic reasons inherent to food distribution, birds from a given aviary belonged to the same color treatment (provided in the food, see below), and treatments alternated between aviaries, so that each bird could see and interact with same-sex birds from both treatments.

Color treatments

In experiment 1, we manipulated the chest color of male and female blue tits using color markers (a procedure used in other studies, [START_REF] Delhey | Fertilization success and UV ornamentation in blue tits Cyanistes caeruleus: correlational and experimental evidence[END_REF][START_REF] Remy | Mediating male-male interactions: the role of the UV blue crest coloration in blue tits[END_REF][START_REF] Ligon | Social costs enforce honesty of a dynamic signal of motivation[END_REF]. Only birds used as stimuli in the mate-preference apparatus (32 birds: 16 males and 16 females, see below) were artificially colored, not the ones that were used as subjects (49 birds: 28 males and 21 females). Half of the stimulus birds (8 males and 8 females) had their chest feathers colored in yellow (Yellow group, Fig. 1A), with a marker that best mimic the natural color in free-living blue tits (Prismacolor Art Marker, canary yellow PM19, Newell Rubbermaid, Atlanta, USA), while the other half of the birds (8 males and 8 females) had their chest feathers colored in grey (Prismacolor Art Marker, warm grey PM-99, Newell Rubbermaid, Atlanta, USA), the color that blue and great tits commonly acquire when held in captivity for long periods of time (Fig. 1A).

Resulting colors were measured with a spectrometer (Avaspec, Avantes, The Netherlands) and with a DH-2000 Deuterium Tungsten Halogen Light Source and a 200 µm optic-fiber probe. Spectrograms obtained from the two kinds of artificially colored birds were close to spectrograms obtained in non-manipulated birds (wild yellow birds and captive grey birds, respectively; Fig. 1A andC, table S1).

In experiment 2, we manipulated the chest color of male and female blue tits through the addition of carotenoids in their diet. In order to test for self-reference phenotype matching mechanisms, the chest plumage of both stimulus and subject birds were manipulated early in life, when birds were starting to feed independently (i.e. about 35 days old, in early June). Thirty five birds had their diet (cake and mealworms, see above) supplemented with a powder containing 10% of canthaxanthin at a concentration of 5000 ppm (Carophyll-Red 10%, produced by DSM Nutritional Products Ltd, Basel, Switzerland), while 37 birds had their diet supplemented with a placebo made of the exact same powder as in the other group, but with no pigment in it (placebo specifically made for the experiment by DSM Nutritional Products Ltd, Basel, Switzerland).

Treatments thus only differed in the presence/absence of canthaxanthin, which colors the chest plumage in dark yellow/orange. Spectrograms of placebo and canthaxanthin tits are shown at fig. 1B. We opted for canthaxanthin rather than other carotenoids present in the diet of wild tits like lutein, or zeaxanthin because those other carotenoids have not always been efficient in enhancing the chest color of blue tits (e.g. [START_REF] Biard | Carotenoid availability in diet and phenotype of blue and great tit nestlings[END_REF], and because we had experience with canthaxanthin and knew it efficiently colors the chest plumage of blue tits (P. Perret, unpubl. data). Furthermore, using canthaxanthin allowed us to oppose a pale yellow/grey to a dark yellow/orange morph, two experimental groups that consisted in color phenotypes rarely encountered in the wild, thus avoiding to oppose a natural phenotype (natural yellow) with an artificial one (pale yellow/grey). Treatments were stopped two weeks before the start of the behavioral tests. After the first series of binary preference tests, we started the diet supplementation again, but we reversed the experimental groups, i.e. birds that were first supplemented with canthaxanthin received the placebo, and vice versa for the other group, which reversed the plumage colors of the two groups. That way each bird was tested in both groups.

Mate preference testing

About two weeks before the start of the tests, birds were settled in individual cages in rooms where opposite-sex birds could not see each other. Tested and stimulus birds were housed in separate rooms. During that time, birds were exposed to a long photoperiod (≥ 15 hours of light per day). Long photoperiods indeed trigger a cascade of neuroendocrine reactions that lead to production and secretion of sexual steroids by the gonads, particularly 17β-estradiol (E2) in females, and promote the expression of reproductive behaviors [START_REF] Dawson | Photoperiodic control of seasonality in birds[END_REF]. In songbirds, increasing plasma E2 in females, either directly through injection or implants of E2, or indirectly through exposure to long photoperiods, is often used to infer behavioral sexual preferences to a relevant stimulus like male song or male presence [START_REF] Searcy | Measuring responses of female birds to male song[END_REF][START_REF] Byers | Female mate choice and songbird song repertoires[END_REF][START_REF] Caro | Female Lincoln's sparrows modulate their behavior in response to variation in male song quality[END_REF][START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. By exposing blue tits to long days prior to testing, we increased the chances that the observed choices are actual sexual preferences and not social preferences, even though it is hard to firmly distinguish the two.

Experiment 1: Among the 81 birds used, 32 (16 males and 16 females) were used as stimulus birds. These 32 birds were randomly distributed across the two color treatments (yellow and grey markers, see above). The remaining 49 birds were not artificially colored and used as subjects. Each subject was tested only once and exposed to one pair of unrelated birds of the opposite sex. Sixteen pairs (8 pairs of each sex) of stimulus birds were formed and used in two to four mate-preference trials. Tests were performed in 2013 (April 23 to May 15), with birds that were born in 2010 and 2011.

Experiment 2: Among the 72 birds used, 24 (12 males and 12 females) were used as stimulus birds. Contrary to experiment 1, both stimulus and tested birds were subjected to the color treatments (canthaxanthin and placebo supplements in the diet, see above).

In addition, the members of the 12 pairs of stimulus birds were not selected at random here, but pairs were made of brothers or sisters. Choosing genetically similar birds for a given pair reduces any genetically-related and early-environment-related variances between the members of a pair and hence enhances the importance of the artificially manipulated color of the chest as a choice criterion during the behavioural tests. Each pair of stimulus birds was used in three to five mate-preference trials. Tests were performed in 2015 (December 16 to December 23), with birds that were born in spring.

At that age, birds were sexually mature [START_REF] Silver | Immunocytochemical Distribution of Gnrh in the Brain of Adult and Posthatching Great Tit Parus-Major and Ring Dove Streptopelia-Roseogrisea[END_REF][START_REF] Dawson | Photoperiodic control of seasonality in birds[END_REF], and were set into a reproductive-like status by the long photoperiod (see above). After the end of the first series of tests, continuous exposure to a long photoperiod led to a state of photorefractoriness that coincides with the onset of a new molt of all feathers [START_REF] Dawson | Photoperiodic control of seasonality in birds[END_REF]. Once the first birds had started molting, all birds were moved to a shorter photoperiod for six weeks, which restored photosensitivity and allowed for a new cycle of stimulation of the reproductive system by the long days of April [START_REF] Silverin | Photoperiodism in male great tits (Parus major)[END_REF][START_REF] Dawson | Photoperiodic control of seasonality in birds[END_REF]. At the end of the first series of tests the color treatments were reversed for all birds (see above), so that birds from each group could grow breast feathers of the color that previously characterized the other group. The second series of tests took place in Spring 2016 (April 28 to May 3, 2016), keeping the same pairs of stimulus birds as in the first trials.

The test apparatus used for assessing potential partner preferences was identical in both experiments and similar to the one described in [START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. Briefly, two identical test chambers were used simultaneously. Each test chamber was made of a large neutral area (approximately 4m 2 ) and two semi-enclosed areas (approximately 1m 2 each) in front of the cages in which the stimulus birds were placed (Fig. S1). Artificial wooden "trees" were disposed in the neutral zone and in each stimulus zone. Each stimulus cage was constructed of wood and wire mesh, and affixed to a rolling base. A 'natural sun' light bulb (Arcadia Compact Bird Lamp 20W, Arcadia Products, Redhill, United Kingdom) was installed in the top of each stimulus bird cage to allow for UVcoloration visibility, and each cage was equipped with four wooden perches. A curtain was installed in front of each two cages to conceal the stimulus birds from the subject bird as needed throughout the testing. Three wide-angle cameras were affixed in each test chamber, with one in the neutral area, and one in each of the two stimulus zones, to capture both subject and stimulus bird activities throughout the trials (Fig. S1).

Tested and stimulus birds were introduced in their respective compartments, with the curtains closed to prevent visual contacts between them. They were given 10 minutes to acclimate to the chamber/cages. After the acclimation period, the curtains were raised, and the first half of the mate preference trial took place for 20 minutes. After that time, the curtains were closed, and the stimulus cages were switched, in order to account for any side-bias of the subject birds. The second half of the trial took place over 20 minutes. After testing, subjects were returned to their home cages. Each bird was tested only once in experiment 1, twice in experiment 2, i.e. once as canthaxanthin bird, and once as placebo bird, with four months in between the tests to allow birds to molt and change their chest plumage color.

Video analyses and data processing

Video footages from the mate preference tests were analyzed using the computer programs Observer XT (version 10.5, Noldus, Wageningen, The Netherlands) for experiment 1, and Solomon Coder (version beta 15.11.19, https://solomoncoder.com) for experiment 2. Footages were analyzed in terms of the time that subject birds spent in each of the three possible zones (neutral, right stimulus, left stimulus). Data from the two 20-min half trials of each test were pooled together for statistical analyses. Mate preference was inferred from the amount of time (in seconds) a tested bird spent near a particular stimulus bird. We also defined two other response variables that we called "interest" and "preference strength" (see [START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. Interest was calculated to estimate the overall motivation of the subject birds during the experiment. This variable indicates the proportion of time that the subjects spent close to stimulus birds, and was calculated as follows:

𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 1 + 𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 2 𝑇𝑜𝑡𝑎𝑙 𝑑𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑡ℎ𝑒 𝑡𝑒𝑠𝑡
The "total duration of the test" represents the cumulative time spent in the three possible zones of the test chamber. Preference strength indicated the strength of a subject's preference for any of the two stimulus birds it encountered in the test.

Which stimulus bird is chosen is not considered here, preference strength only illustrates the magnitude of the bias for one of the two stimuli, whichever it is. In the present case, it is thus a simple way to assess how affirmative a bird is in its choice,

and it has been shown to be context-dependent [START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. Preference strength is defined as the relative amount of time spent with the chosen individual (the bird with which the subject spent >50% of the stimulus-zone time) compared to the time spent with both stimuli, and calculated as follows:

𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑐ℎ𝑜𝑠𝑒𝑛 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 1 + 𝑇𝑖𝑚𝑒 𝑠𝑝𝑒𝑛𝑡 𝑤𝑖𝑡ℎ 𝑠𝑡𝑖𝑚𝑢𝑙𝑢𝑠 2

Statistical analyses

We used a step approach to analyze the data from simple, starting from easily understandable tests on counts, to more elaborated tests on the exact time birds spent in the different zones of the apparatus.

Interest

For both experiment 1 and 2, we started with a simple binomial test that compared the number of subject birds that spent the majority of their time in the zone of the stimulus birds with the number of birds that spent more time in the neutral zone. We next tested the effect of several explanatory variables on the level of interest. As interest is a proportion, it was arcsine transformed to achieve normality. In experiment 1, we tested whether the level of interest differed between males and females, and varied depending on the time of the day, by including the sex of the subject birds (female or male) and time of day (continuous and centered to produce a variable with a mean of zero) as explanatory variables in a linear model. Preliminary analyses of the datasets showed that the behaviors of the birds were sometimes influenced by the time at which the birds were tested. To account for this bias, time of day will be included as a nuisance variable in all analyses that include covariates. For experiment 2, we also added an effect of the treatment of the subject birds (canthaxanthin or placebo), since in this experiment we not only compared the preferences of males and females, but also considered their color phenotypes, we used a linear mixed-model with individual ID as a random intercept to account for the fact that each bird was tested on two occasions.

Preference strength

As preference strength is a proportion, it was also arcsine transformed to achieve normality. In experiment 1, we tested whether preference strength differed between males and females, and varied depending on the time of the day, by including the sex of the subject birds (female or male) and time of day (continuous and centered to produce a variable with a mean of zero) as explanatory variables in a linear model. In the model, preference strength was weighted by interest. In other words, subjects that spent more time with the stimulus birds in general (i.e. higher interest) were given more statistical weight in the analyses than subjects that were less interested (see [START_REF] Reparaz | Mate preference of female blue tits varies with experimental photoperiod[END_REF]. For experiment 2, we also added an effect of the treatment of the subject birds (canthaxanthin or placebo), since in this experiment we not only compared the preferences of males and females, but also considered their color phenotypes, and we used a linear mixed-model with individual ID as a random intercept to account for the fact that each bird was tested on two occasions.

Which birds are chosen?

For both experiments 1 and 2, we started with a simple binomial test that compared the number of subject birds that spent more time close to yellow (exp 1) or canthaxanthin (exp 2) birds than to control (exp 1) or placebo (exp 2) birds. We then tested the effect of several explanatory variables on these counts, using generalized linear (mixed) models with binomial distributions. In experiment 1, we tested whether the number of birds that spent more time close to yellow or control birds differed between males and females, and varied depending on the time of the day, by including the sex of the subject birds and time of day as explanatory variables on a binary response variable (control or yellow) in a generalized linear model. For experiment 2, we also added an effect of the treatment of the subject birds, and its interaction with sex (to test for a possible self-reference phenotype matching mechanism) on the binary response variable (canthaxanthin or placebo bird chosen) in a generalized linear mixed model with individual ID as a random intercept.

Finally, instead of only considering counts of birds (which gives the same value to all choices, independently of whether the subject spent 51 or 100% of its time with the chosen stimulus), we analyzed the exact time (in seconds) subject birds spent close to each stimulus using linear mixed models. In experiment 1, we included the sex of the subject bird (female or male), the treatment of each stimulus bird (control or yellow), and their interactions as fixed effect. Since in the dataset we considered two time values for each subject bird (one time value for each stimulus), we included subject identity as a random intercept. Like for the analysis of preference strength, time was weighted by interest. We performed the same analysis for experiment 2, adding the effect of the treatment of the subject bird (canthaxanthin or placebo), its 2-and 3-way interactions with sex of subject birds (female or male) and treatment of stimulus birds (canthaxanthin or placebo), and the effect of time of testing (continuous, centered). In experiment 2, subject identity was also introduced as a random intercept to account for the fact that each subject has been tested twice, and that there are two time values per subject for each test.

Analyses were performed in R (version 3.2.1)(R-Core-Team, 2015), using the functions binom.test, lm, glm, and the functions lmer and glmer of the package lme4 [START_REF] Bates | Fitting Linear Mixed-Effects Models Using lme4[END_REF]. Models were simplified using backward elimination of the non-significant terms, starting with the higher order interactions [START_REF] Crawley | The R book[END_REF]. P-values were obtained either by model comparisons between a model that includes and another that excluded the term of interest, or using the anova function of the package lmerTest [START_REF] Kuznetsova | lmerTest: Tests in Linear Mixed Effects Models[END_REF] in the cases of lmer analyses. Post-hoc tests following (nearly) significant interactions were performed using the package lsmeans [START_REF] Lenth | Least-Squares Means: The R Package lsmeans[END_REF]. Confidence intervals of model estimates plotted on the figures were calculated using the package bootpredictlme4 [START_REF] Duursma | bootpredictlme4: predict method for lme4 with bootstrap[END_REF].

Results

Experiment 1 Interest for stimulus birds

On average, subject birds spent 74% of their time close to the stimulus birds, with birds spending more time in the stimulus-bird zone than in the larger neutral zone in 39 of the 49 tests performed (binomial test, p<0.001). The level of interest for the stimulus birds did not differ between the sexes of the subject birds, and was not influenced by the time of day the birds were tested (table 1).

Preference strength

On average female tended to express clearer preferences than males (females: 0.67±0.10, males: 0.62±0.09; mean±sd). This sex effect on preference strength was however not fully significant (p=0.063, see table 1). There was no effect of the time of day the birds were tested (table 1).

Which stimulus bird do they choose?

If we count the number of subjects that spent more time with yellow or control birds, significantly more subjects selected control birds than yellow birds, with controls selected in 33 out of 49 tests (binomial test, p=0.02). This preference for Control birds occurred both in males and females as revealed by the absence of a sex effect on which stimulus is chosen (general linear model, z=0.30, p=0.76), and was not influenced by the time birds were tested (z=1. 23, p=0.22).

Analyzing the exact time the subject birds spent close to each of the two stimulus birds, instead of simply accounting for which stimulus bird was preferred overall, led to similar conclusions, i.e. subjects spent more time close to control than to yellow birds (p=0.008, table 1). In this analysis, there was also a trend for an interaction between the sex of the subject and the treatment of the stimulus birds (p=0.096, see table 1), suggesting that the preference for control birds might be more pronounced in females than in males (Fig. 2A).

Experiment 2 Interest for stimulus birds

On average, subject birds spent 61% of their time close to the stimulus birds, with birds spending more time in the stimulus-bird zone than in the, larger, neutral zone in 58 of the 90 tests performed (binomial test, p=0.008). The level of interest for the stimulus birds did not differ between the treatments or sexes of the subject birds (table 2). There was however an effect of the time of the day at which the birds were tested, with birds slightly increasing their interest level as the day progresses (table 2).

Preference strength

Males did not differ from females in their preference strength (females: 0.66±0.13; males: 0.72±0.14, mean±sd) (table 2). Time of day and the treatment in which the subjects were did not influence the strength of preference either (table 2).

Which stimulus bird do they choose?

The result of the binomial test that compared the number of occasions subject birds spent more time close to canthaxanthin than to placebo birds, reveals that in the 86 tests where the birds made a choice (i.e. did not stay all the time in the neutral zone, which only occurred four times), Canthaxanthin and Placebo stimulus birds were selected in 39 and 47 cases, respectively. Thus based on counts there is no overall preference for a stimulus category (p=0.45). This analysis does not account for the sex of the subject birds, neither for their own treatment. Incorporating those factors, their interaction and the effect of time of day in a generalized linear mixed-model with a binary response variable (Canthaxanthin or Placebo) did not lead to any significant result (table S2).

By contrast, the result of the linear mixed-model using the exact time spent close to each stimulus bird (instead of the count) reveals a significant interaction between the sex of the subjects and the treatment of stimulus birds (p=0.002, Table 2, Fig. 2B).

Post-hoc analyses reveal that females spent more time close to placebo than to canthaxanthin birds (p=0.001), while males seem to spend similar amounts of times with each phenotype (p=0.33). There was also a trend for assortative choice, indicated by the (not fully significant) interaction between the treatment of the subject and the stimulus birds (p=0.058; Table 2 and fig. 3). If we run a post-hoc test on this interaction we find that Placebo subjects (males and females confounded) preferentially select Placebo stimuli (p=0.01), and canthaxanthin subjects tend to spend more time with canthaxanthin stimuli (p=0.08). This should however be interpreted cautiously since the interaction is not fully significant.

Discussion

Our experiments show that male and female blue tits do not differ in their overall interest for prospective mates. Similarly, males expressed comparable levels of preference than females, suggesting that mutual mate choice exists in this species. The only sexual difference found was in the second experiment, where the preference for paler congeners was more pronounced in females than in males. Finally, we did not find strong evidence for an assortative mate preference in blue tits, even though we found that pale birds tended to select birds that are themselves pale.

The results described here quite clearly show that male and female blue tits are mutually attracted by the opposite sex. They add to the growing body of literature that suggests that male mate choice is common in birds and other organisms (reviewed in [START_REF] Hare | Sexual selection and its evolutionary consequences in female animals[END_REF][START_REF] Doutrelant | Evolution of female coloration: What have we learned from birds in general and blue tits in particular[END_REF]. We did not measure mate choice per se in our apparatus since birds were not allowed to pair and reproduce together, but rather a sexual, or eventually social (see below), mate preference. Actual mate choice in the wild depends on a number of other parameters than mate preferences, including environmental conditions, availability of preferred phenotypes, time and effort allocated to prospecting mates, time of season and intra-sexual competition [START_REF] Jennions | Variation in mate choice and mating preferences: A review of causes and consequences[END_REF][START_REF] Cotton | Sexual selection and condition-dependent mate preferences[END_REF][START_REF] Botero | Fluctuating environments, sexual selection and the evolution of flexible mate choice in birds[END_REF][START_REF] Kuijper | A guide to sexual selection theory[END_REF][START_REF] Auld | Male mate choice in the Trinidadian guppy is influenced by the phenotype of audience sexual rivals[END_REF][START_REF] Zandberg | Direct fitness benefits explain mate preference, but not choice, for similarity in heterozygosity levels[END_REF]. Nevertheless, mate choice is the manifestation of a mate preference [START_REF] Cotton | Sexual selection and condition-dependent mate preferences[END_REF]. In any case, similar mutual mate preferences in this species were also described in earlier works by Hunt and colleagues (1999), who specifically tested the preferences for the UV-blue coloration of the crest, and in a recent meta analysis conducted on four populations over 10 years, which suggested a low but positive assortative mating for both the blue and the yellow ornaments [START_REF] Fargevieille | Assortative mating by colored ornaments in blue tits: space and time matter[END_REF].

Carotenoid-based colors have been found to signal capacity to raise an immune response [START_REF] Faivre | Immune activation rapidly mirrored in a secondary sexual trait[END_REF][START_REF] Saks | Carotenoid-based plumage coloration of male greenfinches reflects health and immunocompetence[END_REF]reviewed in Simons et al., 2012) and so could potentially signal high-quality over low-quality individuals [START_REF] Andersson | Sexual selection[END_REF][START_REF] Jones | Mate choice and sexual selection: What have we learned since Darwin?[END_REF][START_REF] Doutrelant | Female plumage coloration is sensitive to the cost of reproduction. An experiment in blue tits[END_REF][START_REF] Wells | Male size predicts extrapair paternity in a socially monogamous bird with extreme sexual size dimorphism[END_REF]. A pale plumage contains less carotenoids, it is therefore surprising that in both experiments, our blue tits did not prefer the most colored morph, corresponding to birds displaying more carotenoid in their plumage and, by extension, potentially higher quality mate. In experiment 1, pale birds were selected more often than colorful birds. This result was robust as indicated by the fact that it was already significant in the initial binomial test based on counts (number of birds that spent more time near to controls). It also suggested the existence of an assorted mating (see below). This preference for pale birds was apparent in both sexes (table 1), although it tended to be more pronounced in females (Fig. 2A). Experiment 2 confirmed these results. Even if the same binomial test did not reveal any striking preference this time, analyzing the exact time spent with the different stimulus birds, indeed showed again that female blue tits preferentially selected the least colorful males (Fig. 2B).

Preference for partners presenting lower values of signals has already been reported

in the literature, both from studies conducted in the field and in captivity. Female pied flycatchers (Ficedula hypoleuca) and house sparrows of some wild populations were described to prefer less-ornamented partners [START_REF] Saetre | A sexually selected character displacement in flycatchers reinforces premating isolation[END_REF][START_REF] Griffith | Female choice and annual reproductive success favour less-ornamented male house sparrows[END_REF].

If the reason for this preference was quite clearly established in the first case, with females that would show this preference to more surely avoid hybridization with the closely related collared flycatcher (Ficedula albicollis) [START_REF] Saetre | A sexually selected character displacement in flycatchers reinforces premating isolation[END_REF], the sparrow case was less clear. In fishes, captive male sticklebacks (Gasterosteus aculeatus) were shown to prefer drab over red females. The author argued that avoidance of redder females might in fact be a side-effect of a mechanism of avoidance of aggressive interactions in male-male competition, and not a preference for drab females per se [START_REF] Nordeide | Do male sticklebacks prefer females with red ornamentation?[END_REF]. As we can see, in the few cases where it has been reported, preference for less-ornamented partners generally arises from specific, contextdependent, situations [START_REF] Griffith | Female choice and annual reproductive success favour less-ornamented male house sparrows[END_REF], and not from a general preference for dull colors. In the case of blue tits, we can only speculate on why this preference for palechested birds occurred, and we think that four main hypotheses can be drawn from these results, hypotheses that will need to be tested in the future.

First, we know that mate preference can be influenced by genetic and cultural factors (i. e. [START_REF] Dugatkin | Interface between culturally based preferences and genetic preferences: Female mate choice in Poecilia reticulata[END_REF]. A genetically inherited preference for paler individuals seems highly improbable, but a mating preference mechanism based on sexual imprinting early in life, or on the most common social phenotype encountered thereafter, could explain the results found, at least in experiment 1. In this experiment we only colored the stimulus birds, not the subject birds, and only soon before the start of the experiment. All subjects and their aviary mates have had a pale chest since their first pre-basic molt, and were therefore used to encounter and interact with other pale congeners. However, while pale birds was the most common phenotype they met for most of the time, birds had a natural, yellow-colored chest during their first months of life in captivity (pers. observations), thanks to the carotenoids present in the food brought by their parents at the nest (chicks were brought to the lab when they were around 10-days old, but were raised in natural conditions by their parents before that, see methods). Birds only acquired a pale chest after their first pre-basic molt, which occurred a few months after birth (July for birds born in May). In addition, the parents that feed them at the nest for the first 10 days of life were of the natural, yellow phenotype. An early sexual imprinting on pale-chest birds could therefore not explain the observed preference for pale birds. It remains however possible that social associations later in life could have played a role, and this was taken into account in the design of experiment 2. In this experiment, bird food was indeed supplemented with canthaxanthin or a placebo before the onset of their first pre-basic moult, meaning that contrary to what happened in experiment 1, birds in experiment 2 went directly from their initial yellow plumage to either one of the two experimental colors, only half of which were pale. Furthermore, birds were housed in aviaries in a way that they could always see both color morphs (see methods). The preference for palechested birds in this second experiment could thus hardly be explained by early sexual imprinting, or by social habituation later in life.

Second, more colored individuals might be more aggressive individuals [START_REF] Senar | Color displays as intrasexual signals of aggression and dominance[END_REF][START_REF] Tobias | The evolution of female ornaments and weaponry: social selection, sexual selection and ecological competition[END_REF] (but see [START_REF] Mcgraw | Carotenoid-based ornamentation and status signaling in the house finch[END_REF]. If pairing with an aggressive partner might confer direct and indirect benefits in terms of territory defense and genes conferring more vigor to offspring, it may also come at the cost of being more aggressed. In captivity, where space is often limited, pairing with an aggressive partner can lead to reproductive failure [START_REF] Caro | Non-photoperiodic factors and timing of breeding in blue tits: Impact of environmental and social influences in semi-natural conditions[END_REF]. Therefore, in confined environment like the one in which our birds are raised, mate preference rules might differ from what has been theorized [START_REF] Wang | No mutual mate choice for quality in zebra finches: Time to question a widely-held assumption[END_REF], and selecting a paler, potentially less aggressive, partner might be advantageous, especially for females that are slightly smaller than males [START_REF] Blondel | Selection-based biodiversity at a small spatial scale in a low-dispersing insular bird[END_REF]. We know in our populations that males and females with higher value of yellow chroma are more aggressive toward human intruders (Mercier-Gauthier, Doutrelant, Dubuc Messier, Charmantier, Réale, unpubl. data ), and that duller females are less aggressive toward other blue tit females [START_REF] Midamegbe | Female-female aggressiveness is influenced by female coloration in blue tits[END_REF]. Alternatively, even if we believe that the results reported here depict a sexual preference because birds were primed into a reproductive state (see methods), we cannot exclude that there might have been a social dimension to the preferences expressed, with males and females choosing a partner with which to spend time rather than to breed. In this case, selecting a less aggressive social partner would seem more appropriate. A different experimental setup with more than two possible choices, and which would include one or several individuals of the same sex as the focal bird, could have shed some light on this social paring possibility (e.g. [START_REF] Griggio | Female house sparrows "count on" male genes: experimental evidence for MHC-dependent mate preference in birds[END_REF]. In any case, like in the stickelback example [START_REF] Nordeide | Do male sticklebacks prefer females with red ornamentation?[END_REF], avoiding aggressiveness might be a possible mechanism explaining the preferences for paler individuals observed in our experiments.

Third, since potential competitors and mates are most probably assessed based on a variety of signals [START_REF] Bro-Jorgensen | Dynamics of multiple signalling systems: animal communication in a world in flux[END_REF][START_REF] Ligon | Social costs enforce honesty of a dynamic signal of motivation[END_REF], it might be the overall homogeneity of signals that matters, more than one signal taken independently from the others. In blue tits, individuals with a yellower plumage also tend to have more UV in the blue part of the plumage (Mercier Gauthier et al., unpub. data). Accordingly, [START_REF] Kurvers | No consistent female preference for higher crown UV reflectance in Blue Tits Cyanistes caeruleus: a mate choice experiment[END_REF] found that female blue tits expressed a preference for males with UV-enhanced crowns, but only if those males also had a yellower chest plumage, otherwise females selected the UV-reduced males. This suggests that choosing birds might pay attention to an ensemble of traits and prefer those individuals that are more homogenous in their different color signals. In our experiments, like in most other experiments manipulating color phenotypes, we only manipulated one single trait (i.e. the color of the chest). Since the diet that our birds receive in captivity is obviously less diversified than what free-roaming birds eat in the wild, it is possible that long-term housing in captivity induces a general decrease in plumage quality. Although we only manipulated the chest color of our birds, we also collected some blue crown feathers from the birds used in our second experiment.

Comparing the brightness, hue and UV chroma of those UV-blue feathers between the two groups shows that the diet supplemented with canthaxanthin did not affect any of those parameters (Brightness: p=0.7; hue: p=0.2; chroma: p=0.9; data not shown). This suggests that only enhancing the chest color might have increased the contrast between the different parts of the plumage, which in turn could have been counterselected. This could explain why less-colored birds were preferred in both experiments. Future studies of mate-preference, adopting a multi-modal approach manipulating several plumage traits simultaneously, will shed light on whether birds assess potential mates based on a variety of signals.

Finally, assortative mating could also explain the results described in this study. In experiment 1, all subjects had a pale chest, and they preferentially selected birds that were themselves pale, leading to a clear preference for individuals that were assorted to the subjects. This result in fact led to experiment 2, where one of the goals was to further test this hypothesis of assortative mating, presumably based on a mechanism of self-referent phenotype matching. In this second experiment, where the colors of both the stimulus and the subject birds were manipulated at an early stage, there was a trend for an interaction between the treatments of the subject and of the stimulus birds, and in the predicted direction: birds tended to spend more time with congeners of the same chest phenotype as their own. Self-reference phenotype matching was certainly not robust in our second experiment (not visible in the simple tests based on counts of birds, and interaction not fully significant when analyzing the exact time spent), but sufficiently to suggest that it might exist in blue tits, and sufficiently to call for additional studies on this specific question. A recent study testing for assortative mating for coloration in wild blue tits analyzed the matching of more than 1500 pairs across multiple populations and over 10 years. If authors found some evidence for positive assortative mating across populations, there were large spatio-temporal variations [START_REF] Fargevieille | Assortative mating by colored ornaments in blue tits: space and time matter[END_REF], confirming that mate preference can be very labile in the wild [START_REF] Chaine | Adaptive plasticity in female mate choice dampens sexual selection on male ornaments in the lark bunting[END_REF].

In conclusion, this study suggests that mutual mate preference exists in blue tits, which makes sense in a species where both sexes invest a lot in parental care [START_REF] Johnstone | Mutual mate choice and sex differences in choosiness[END_REF][START_REF] Kokko | Why is mutual mate choice not the norm? Operational sex ratios, sex roles and the evolution of sexually dimorphic and monomorphic signalling[END_REF]. Results also show that the color of the breast does influence mate preference. We additionally asked whether the phenotype of the subjects influences the preference they express, but did not fully resolve this question yet. Using a more integrative measure of quality like the one proposed by [START_REF] Wang | No mutual mate choice for quality in zebra finches: Time to question a widely-held assumption[END_REF], which takes more than one phenotypic trait into account, seems a promising way to further our understanding of the importance of phenotype matching in mate preferences. Asking this question both in captivity and in the wild also seems important to give a broader picture of the mate preference dynamics in this, and other species.

Figure 1 :

 1 Figure 1: Reflectance spectra of blue tit breast feathers. (A) Spectra of birds used in experiment 1. Colours obtained by painting the feathers with colour markers. (B) Spectra of birds used in experiment 2. Colours obtained by manipulation of the diet of the birds. (C) Spectra from wild blue tits at La Rouvière, France (measurements performed on breast feathers : see Fargevieille et al., 2017).

Figure 2 :

 2 Figure 2: Sexual differences in the color morphs selected by captive blue tits. (A) In experiment 1, control birds were preferred over yellow birds (p=0.008), and this preference for pale individuals tended to be slightly more pronounced in females than in males (interaction between sex of the chooser and treatment of the stimulus bird: p=0.096). (B) In experiment 2, this interaction was significant (p=0.002) and reveals that female blue tits spent significantly more time close to Placebo than Canthaxanthin stimuli (post-hoc test: p=0.001), while males spent similar amounts of time with each phenotype (post-hoc test: p=0.331). Small symbols represent individual data points, bigger symbols and error bars represent the statistical model estimates and S.E.

Figure 3 :

 3 Figure3: Assortative mating in captive blue tits? The nearly significant interaction between treatments of the subjects and of the stimulus birds (P=0.058) suggests that blue tits tend to prefer birds that are of the same color phenotype as theirs. This is particularly visible in placebo subjects (post-hoc test: p=0.01), less in canthaxanthin birds (p=0.08). Small symbols represent individual data points, bigger symbols and error bars represent the statistical model estimates and S.E. For the sake of simplicity, the estimates presented here come from a model including the treatments of stimulus and chooser birds, their interaction, and time of day. Sex and its interactions (table 2) were not included.

Table 1 : Analysis of the variables that potentially influence the interest of the subject birds, their preference strength, and the time spent close to the stimulus birds in experiment 1 (n=49).

 1 Variables in bold represent the minimal adequate models, eliminated variable (in grey) are presented in the reverse order in which they were removed from the model. Intercept includes Sex of subject = female, Treatment of stimulus = control.

	Trait analysed	Variable	Estimate	S.E.	F p-value
	Interest
		(Intercept)	1.06	0.04
		Time of day	-3.5e-04 2.5e-04 2.01 0.163
		Sex of subject	0.05	0.08	0.12 0.733
	Preference strength
		(Intercept)	0.96	0.02
		Sex of subject	-0.06	0.03	3.62 0.063
		Time of day	1.2e-04 8.6e-05 1.83 0.183
	Time
		(Intercept)	1039.49 52.57
		Treatment of stimulus	-202.62 74.34	7.43 0.008**
		Sex of subject	-43.77	74.82	0.34 0.560
		Time of day	-0.12	0.21	0.33 0.568
		Treatment stimulus x Sex subject	247.76 147.28 2.83 0.096

Table 2 : Analysis of the variables that potentially influence the interest of the subject birds, their preference strength, and the time spent close to the stimulus birds in experiment 2 (n=47).

 2 Variables in bold represent the minimal adequate models, eliminated variable (in grey) are presented in the reverse order in which they were removed from the model. Intercept includes Sex of subject = female, Treatment of subject and stimulus = Canthaxanthin.

	Trait analysed	Variable	Estimate	S.E.	F	p-value
	Interest
		(Intercept)	0.90	0.04
		Time of day	3.5e-04	1.5e-04 5.71	0.019*
		Sex of subject	-0.09	0.08	1.26	0.268
		Treatment of subject	-0.04	0.06	0.50	0.482
	Preference strength
		(Intercept)	0.97	0.03
		Sex of subject	0.05	0.04	1.78	0.190
		Time of day	1.1e-04	7.9e-05 2.12	0.150
		Treatment of subject	-0.04	0.04	1.29	0.262
	Time
		(Intercept)	721.74	91.52
		Sex of subject	161.50 123.59	0.28	0.609
		Treatment of stimulus	338.82 103.54	3.08	0.081

Time of day 0.35 0.17 4.27 0.044* Treatment stimulus x Sex subject -431.27 140.39 9.44 0.002**

	Treatment of subject	-39.26	72.94	0.29	0.591
	Treatment subject x Treatment stimulus	264.13 138.37	3.64	0.058
	Treatment subject x Treatment stimulus (placebo) x Sex subject	76.13	203.51	0.07	0.932

Acknowledgments

We thank Marylou Aaldering, Franca Kropman, Bart van Lith, and many other animal caretakers for their help with hand-feeding the chicks and taking good care of the birds. Maria Del Rey, Amélie Fargevieille and Benjamin Deletrez for their help with measuring feather coloration. Marcel Visser, Kate Lessells, Ralf Kurvers and Marc Naguib for helpful discussions prior to the experiments, and two anonymous reviewers for their very constructive comments. We also thank DSM Nutritional Products Ltd for providing the carotenoid products, especially the placebo, used in experiment 2.

Analyses reported in this article can be reproduced using the data provided by Caro et al. (2021).

Funding

This work was supported by the Fyssen Foundation (research grant to SPC); the Netherlands Organization for Scientific research (NWO-VENI grant to SPC), and by Wallonie-Bruxelles International (WBI-World grant to SPC).