Qi Zhang

Mohammad Wahiduzzaman

Sujing Wang

Stefan Henfling

Narjes Ayoub

Effrosyni Gkaniatsou

Farid Nouar

Clémence Sicard

Charlotte Martineau

Yuanjing Cui

Guillaume Maurin
email: guillaume.maurin@univ-montp2.fr

Guodong Qian
email: gdqian@zju.edu.cn

Christian Serre
email: christian.serre@espci.fr

Multivariable Sieving and Hierarchical Recognition for Organic Toxics in Nonhomogeneous Channel of MOFs

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

Separation and purification are distinctly important in analytical chemistry and in diverse industrial processes. [START_REF] Karan | Sub-10 nm polyamide nanofilms with ultrafast solvent transport for molecular separation[END_REF] Large quantities of artificial organic compounds such as persistent organic pollutants (POPs), pesticides, medicines, and dyes are produced annually, which are significant threats to global water resources. Therefore both green chemistry and environmental protection call for isolation and/or reuse of toxic organic compounds in liquid phase based on separation processes. [START_REF] Ali | New Generation Adsorbents for Water Treatment[END_REF] The most commonly used separation technologies at the industrial level such as liquid-liquid extraction and distillation lead to increased cost and vast energy consumption, raising toxicity issues due to the employment of organic solvents in large volumes. [START_REF] Van De Voorde | Adsorptive separation on metal-organic frameworks in the liquid phase[END_REF] Porous adsorbents are promising for the isolation of various toxics via adsorbent/membrane-based sieving and purification. [START_REF] Zhao | Metal-Organic Frameworks for Separation[END_REF] Active charcoal, zeolites and resins are commercial benchmark materials currently used for molecular sieving. However, these adsorbents are far from recognizing different organic molecules efficiently due to their lack of contrivable opening pore size, the existence of pore size gaps and shortage in well-designed pore chemistry.

Tremendous varieties of organics lead to multiple physical and chemical variates such as charge, size, shape, polarity and aromaticity, and this complex scenario increases the difficulty to separate specific substrates. Designing porous materials with refined tunable porosity, [START_REF] Huang | An in situ self-assembly template strategy for the preparation of hierarchical-pore metal-organic frameworks[END_REF][START_REF] He | Polarized three-photon-pumped laser in a single MOF microcrystal[END_REF][START_REF] Sakaida | Crystalline coordination framework endowed with dynamic gate-opening behaviour by being downsized to a thin film[END_REF][START_REF] Wang | Reversed thermo-switchable molecular sieving membranes composed of two-dimensional metal-organic nanosheets for gas separation[END_REF][START_REF] Cai | A Modulator-Induced Defect-Formation Strategy to Hierarchically Porous Metal-Organic Frameworks with High Stability[END_REF][START_REF] Chung | Computational Screening of Nanoporous Materials for Hexane and Heptane Isomer Separation[END_REF][START_REF] Yu | Confinement of pyridinium hemicyanine dye within an anionic metal-organic framework for two-photonpumped lasing[END_REF] appropriate surface charge properties, 12; 13 specific functionalization [START_REF] Cui | Photonic functional metal-organic frameworks[END_REF][START_REF] Desai | A Water-Stable Cationic Metal-Organic Framework as a Dual Adsorbent of Oxoanion Pollutants[END_REF][START_REF] Peng | A versatile MOF-based trap for heavy metal ion capture and dispersion[END_REF][START_REF] Dong | Confinement of Aggregation-Induced Emission Molecular Rotors in Ultrathin Two-Dimensional Porous Organic Nanosheets for Enhanced Molecular Recognition[END_REF][START_REF] Xiao | Integration of Plasmonic Effects and Schottky Junctions into Metal-Organic Framework Composites: Steering Charge Flow for Enhanced Visible-Light Photocatalysis[END_REF][START_REF] Yang | Encapsulating highly catalytically active metal nanoclusters inside porous organic cages[END_REF][START_REF] Lustig | Metalorganic frameworks: functional luminescent and photonic materials for sensing applications[END_REF] and excellent water stability [START_REF] Howarth | Chemical, thermal and mechanical stabilities of metal-organic frameworks[END_REF][START_REF] Cadiau | Design of Hydrophilic Metal Organic Framework Water Adsorbents for Heat Reallocation[END_REF][START_REF] Li | Capture of organic iodides from nuclear waste by metal-organic frameworkbased molecular traps[END_REF] is envisioned to realize multivariate sieving of a wide variety of organics. New advanced porous materials such as Metal-Organic Frameworks (MOFs), covalent organic polymers (COPs), porous organic cages and porous polymers have been considered recently as affordable alternatives in this direction. Zhao et al. and Li et al. reported size-and/or charge-selective capture of dyes by the use of MOFs with distinct pore sizes. 24; 25 However, the corresponding experiments were performed in organic solvent, which hampers the practical use of these MOFs in aqueous solution. Very recently, Ghosh et al. reported a base-resistant ionic MOF as an ion-exchange sorbent for dyes in water. [START_REF] Desai | Base-Resistant Ionic Metal-Organic Framework as a Porous Ion-Exchange Sorbent[END_REF] Porous polymers such as P-CDP and PFCMP-0 have been shown to only remove contaminants rather than their isolation owing to the large size gaps of pores. 27; 28 These cases are limited in terms of ultrafine tuning of sieving "gates" at the Angström level, which lowers the precision of size separation. Nevertheless, COP-99, a porous network polymer, has been shown recently to realize charge-and size-based aqueous separation of water-soluble organics. [START_REF] Byun | Charge-specific size-dependent separation of water-soluble organic molecules by fluorinated nanoporous networks[END_REF] Moreover, several studies reported the separation of organics by different MOFs involving a thermodynamic conformational or kinetics-driven separation and/or size exclusion. [START_REF] Zhang | Goal-directed design of metal-organic frameworks for liquid-phase adsorption and separation[END_REF] However, these adsorptive separations can only recognize organics based on two variates (charge and size) in aqueous phase and other recognition is rarely realized as the weak interaction can be affected by water in channel. [START_REF] Adil | Gas/vapour separation using ultra-microporous metal-organic frameworks: insights into the structure/separation relationship[END_REF][START_REF] Bozbiyik | Shape selective properties of the Al-fumarate metal-organic framework in the adsorption and separation of n-alkanes, iso-alkanes, cyclo-alkanes and aromatic hydrocarbons[END_REF][START_REF] Bobbitt | Metal-organic frameworks for the removal of toxic industrial chemicals and chemical warfare agents[END_REF][START_REF] Hartlieb | CD-MOF: A Versatile Separation Medium[END_REF] Therefore there is a need to expand the list of adsorbents to achieve multivariable sieving of organics besides the charge and size-like mechanisms achieved so far. Furthermore, most of the previous studies have been devoted to explore charged dyes while toxic molecules encountered in real conditions such as high carcinogenic multivariable toxics, e.g. persistent organic pollutants (POPs) and pesticides, have not been investigated so far.

To target in the multivariate sieving of a broader variety of toxic molecules, the molecular sieves should combine different recognition mechanisms and specific sites are needed to interact with toxic molecules. Inspired by multivariable recognition of ligands in biology through multiple NCIs, involving dispersed variations of electromagnetic interactions, we hypothesized similar artificial channels within MOFs might interact with toxic molecules and separate different mixtures. [START_REF] Neel | Exploiting non-covalent π interactions for catalyst design[END_REF] However, in the field of porous molecular sieves, only charge interactions are widely used for charge-based separation in charged frameworks, [START_REF] Desai | Base-Resistant Ionic Metal-Organic Framework as a Porous Ion-Exchange Sorbent[END_REF] and no one applied other NCIs such as various π-effects in the field of aqueous toxics sieving. [START_REF] Pavlakos | Noncovalent Lone Pair⋅⋅⋅(No-π!)-Heteroarene Interactions: The Janus-Faced Hydroxy Group[END_REF] In this regard, we synthesized a series of isostructural MIL-140s and examined their sieving performance for both dye stuff and various practical toxics. We found that these MOFs can not only accomplish charge-and size-based separation of charged dye mixtures, but also realize isolation of a wider range of practical toxics such as POPs and pesticides with very high separation effect. To reveal the separation mechanisms, an outside-in their theoretical model, hierarchical recognition, is presented: (i) the surface charge state, which acts as a first barrier for charged molecules through the external surface of the porous materials, (ii) the sieving "gate", which works through the discrimination of organics of different dimensions via a size exclusion effect (Scheme 1a) and (iii) NCIs (NCIs stands for Non-Covalent Interactions) with attractive sites present at the pore wall, which is similar to ligand recognition in transporters and based on nonregular electronic distribution created by either a non-regular shape of the porosity or by the incorporation of functional groups (see Scheme 1b andc). Both the separation experiments and theoretical calculation demonstrate the key role of hierarchical recognition for multivariable sieving.

RESULTS AND DISCUSSION

Structural Design and Characterization

In order to design continuous water-stable triangular channels as well as control the potential energy surface of pores by regulating the length and chemistry of ligands, an isoreticular series of water stable microporous Zr dicarboxylate MOFs with triangular pores, namely MIL-140s or [ZrO(O2C-R-CO2)] (MIL stands for Materials from Institut Lavoisier; R=organic spacer) has been selected for the systematic sieving performances of complex toxics molecules through the combination of advanced experimental and computational approaches. [START_REF] Pearson | Hard and Soft Acids and Bases[END_REF] MIL-140s differ from the family of most versatile Zr-based UiO series MOFs (UiO stands for University of Olso) in water stability, it is admitted that the extended analog of UiO-66, such as UiO-67 or [Zr6O4(OH)4(O2CC12H8CO2)12], was degradable under humidity. [START_REF] Decoste | Stability and degradation mechanisms of metal-organic frameworks containing the Zr6O4(OH)4 secondary building unit[END_REF] The current set of MIL-140s solids was completed through the design of either first examples of functionalized analogues of MIL-140C (MIL-140C-2NO2, -2CO2H) or new larger pores candidates bearing longer organic linkers (MIL-140E and MIL-140F). The synthesis of MIL-140s is based on the solvothermal reaction of ZrCl4 and dicarboxylic linkers. This involves here 1,4-H2BDC (1,4- A computational assisted strategy based on the interplay between density functional theory (DFT) geometry optimizations and X-Ray Powder Diffraction data (XRPD) were considered to propose structure models for the newly synthesized MOFs with the corresponding cell parameters reported in Table S1. As shown in Figure 1, all MIL-140s crystallize in the monoclinic space group C2/c (n°15) (see Table S1, Figure S8-S10 and Data S1-S4), and they are built from the infinite edge sharing Zr-oxide chains bridged by dicarboxylic ligands. The Zr atoms are seven-coordinated-three µ3-O oxygen atoms, and four oxygen atoms from the dicarboxylic ligands-all Zr 4+ ions have the same coordinated environment (Figure 1C). This delimits one-dimensional triangular channels of free apertures, from 3.2 to 7.2 Å when standing from MIL-140A to MIL-140F along the c axis (see Figure S1 and Table S2). Thus, these MOFs possess tunable pore sizes to sieve organics with a wide variety of dimensions. And on the other hand, the arrangement mode of ligand on pore wall indicates that these MOFs allow flipping of the benzene rings (see Figure 1A andB). The experimental BET areas for MIL-140 (A, B, C, E, F) range from 390 (20) to 1320 (20) m 2 .g -1 and these values are in good agreement with the theoretical N2accessible area calculated from the crystal structures as summarized in Table S2 and Figure S15. In the case of partially substituted MIL-140C-2CO2H (with BPDC-2CO2H of 16.7 % replacing BPDC in the framework) and MIL-140C-2NO2 (with BPDC-2NO2 of 22.2 % replacing BPDC in the framework, see Figure S2 andS3), the lower experimental BET areas and pore volumes compared to MIL-140C are attributed to the steric hindrance created by the presence of the functional groups.

To investigate the chemical properties of the channels, we further analysed the electronic distribution of MIL-140s. We found that the channels of MIL-140s possess particular electronic distribution on its pore wall. As revealed in Figure 1E and Figure S4, in the sectional view, yellow-to-cyan colors indicate high-to-low electron densities, MIL-140s present two distinct electronic regions in the edges of the triangular channels: i.e. an electron-poor zone A and electron-rich zone B surrounding the ligands arranged perpendicular and parallel to the channel respectively. The introduction of -CO2H and -NO2 groups in MIL-140C-2CO2H and MIL-140C-2NO2 act as a subtle modifier of the electronic distribution in the pore wall and afford extra electron rich region (see Figure 1E). The electronic distribution calculation reveal that MIL-140s have heterogeneous potential energy surface owing to either the non-regular shape of the porosity and or the incorporation of functional groups.

Charge and Size-based Separation of Dye Mixtures

It is known that hydrothermally stable MIL-101(Cr) or [Cr3(μ3-O)(OH)(H2O)2(O2CC6H4CO2)3]•nH2O was intensively studied for various organic adsorption due to its large surface areas and mesoporous cages accessible through large microporous windows (see Figure S15 andS16). MIL-101(Cr)s were thus investigated here for the sake of comparison and understanding the importance of pore architecture for multivariable molecular sieving. [START_REF] Férey | A Chromium Terephthalate-Based Solid with Unusually Large Pore Volumes and Surface Area[END_REF] Experimental BET areas of MIL-101(Cr)s solids vary from 1520 to 3250 m 2 .g -1 (see Table S4) and their pore sizes are over 2.5 nm, accessible through large microporous windows. In order to reveal the nature of the external surface chemistry, zeta potentials of MIL-140s and MIL-101(Cr)s were first examined as a function of pH. As shown in Figure S6, under neutral conditions, pH≈6.8, which corresponds typically to the value of the solutions to be purified, MIL-140s solids all exhibit a negative zeta potential a priori in favor of repulsion of negatively charged organics out of the channel. MIL-101(Cr) exhibits on the contrary a positively charged zeta potential. Whereas, the functionalized MIL-101(Cr)s exhibit negatively charged surface at around pH 6.8, in the order of MIL-101(Cr)-NO2 (only -10 mV)>MIL-101(Cr)-CO2H>MIL-101(Cr)-SO3H, which is consistent with decreasing pka values and thus increasing acidic character (see table S3 and Figure S5). These results suggest that a fine-tuning of the nature of the functional groups allows a control of the surface charge properties.

Following the surface charge characterization, we investigated the relatively easy chargeand size-based separation in water. Four model organics were first selected including an anionic Methyl Orange (MO, 5.3 Å) and three cationic organics: methylene blue (MB, 4.7 Å), Nile blue (NB, 6.1 Å) and Rhodamine 6G (R6G, 10.1 Å) (see Figure 2C). We selected only the positively charged MIL-101(Cr) and the negatively charged MIL-101(Cr)-SO3H as representative candidates to study the separation performances. As shown in Figure 2A, MIL-101(Cr)s only realize charge-based separation. The positively charged MIL-101(Cr) adsorbs negative MO almost exclusively, as previously reported, [START_REF] Haque | Adsorptive removal of methyl orange from aqueous solution with metal-organic frameworks, porous chromium-benzenedicarboxylates[END_REF] while the negatively charged MIL-101(Cr)-SO3H shows an opposite trend extracting almost only the MB from the solution. As shown in Figure 2B, size exclusion-based separation for NB and R6G was however not successful, the mesoporous MIL-101(Cr)-SO3H allows both small and large organics to pass through the pores, while MIL-101(Cr) shows weak adsorption for both NB and R6G. These results undoubtedly demonstrate the impact of chemical groups such as -SO3H acidic groups attributed to the strong NCIs that occur between the ionized -SO3 - moieties and the positively charged molecules, and size sieving calls for ultrafine regulation of channel size. MIL-140s were then investigated for both charge-and size-based separation (see Figure S17 andS18). As shown in Figure 2A, MIL-140s show a preferred adsorption for the positive MB over the negative MO as a result of their negative surface charge (see Figure S6). The separation results show that channel size affects the separation performance (see Figure 1D and Figure S7). MIL-140A (3.2 Å) precludes from the penetration of MB (4.7 Å) through its channels. When one considers MIL-140F with the largest pore size, the adsorption capacity of organics increased, with however a limited selectivity toward MO. Noteworthy, other MIL-140s can realize a selective adsorption of MB due to the charge barrier. Specifically, MIL-140C partially substituted with BPDC linker bearing -CO2H and -NO2 groups allow a complete selective adsorption of MB due to the much higher electron density of the pore walls, which is evidenced in Figure 1E. These results indicate that the electronic distribution in pores markedly influences the molecular separation of charged dyes. To further reveal the mechanism of MO/MB separation in MIL-140s, we conducted DFT calculations to evaluate the adsorption induced structural deformation of MOF ligands/guest molecules upon adsorption in the confined pores. DFT calculations showed that MB is arranged along the channel tightly and parallel to the confined pore wall with a lower degree of freedom and at the vicinity of the functional groups. The DFT calculations further evidence that the presence of guest molecules induce a significant reorientation of the phenyl rings, which emphasizes that the MOF adapts its geometry to accommodate the organics (see Figure 2D andE

Multivariable Sieving of Pesticides and POPs

The above mentioned dyes are model molecules. On the other hand, charge and size are the two most common variates, which cannot satisfy the molecular sieving of a broader range of organics. The more frequent representative toxics such as pesticides and polycyclic aromatic hydrocarbons (PAHs), which have potential carcinogenicity and mutagenicity, call for a careful separation and recovery. The excellent separation results for charge and size encouraged us to further evaluate the sieving performance of these practical toxics in water. We therefore studied the isolation performance for pesticides in water of MIL-140s, and compared their performances with representative water-stable MIL-101(Cr) and MIL-101(Cr)-SO3H (3-D pores), as well as benchmark porous solids such as MCM-41 and activated charcoals. As shown in Figure 3A, three representative pesticides: paraquat (5.5 Å), difenzoquat (6.6 Å) and clopyralid (7.4 Å) were selected. As shown in their molecular structure and electrostatic potentials, these organics have similar sizes but differ in chemical properties, thus allowing to explore the influence of the NCIs-based recognition besides size sieving.

Separation experiments of paraquat/difenzoquat are firstly performed. As shown in Figure 3B, the separation of paraquat and difenzoquat is hardly achieved using MCM-41 and porous activated charcoal. These results indicates that MCM-41 and porous activated charcoal have equivalent affinities toward both paraquat and difenzoquat. Positively charged MIL-101(Cr) rejecting cationic paraquat and difenzoquat, while negatively charged MIL-101(Cr)-SO3H adsorbing both paraquat and difenzoquat. All abovementioned porous materials thus fail in multivariable sieving toward new variables. MIL-140s on the contrary exhibit multivariable recognition based on the preferential NCIs. As the two positive pesticides exhibit almost similar dimensions (5.5 Å and 6.6 Å respectively), the former two levels of recognition, namely surface charge barrier and "sieving gate", are not applicable. We even found that difenzoquat was preferentially extracted although it possesses 1 Å larger size than paraquat (Figure 3B). At equilibrium, separation factor between a preferred toxic A and a lesser adsorbed toxic B is defined as (yA/yB)/(xA/xB), in which y and x are the mole fractions in the adsorbed phase and the solution respectively. A former study revealed that MIL-140C exhibited only a low separation factor around 2.5 for isolation of hydroquinone and catechol from water. [START_REF] Van De Voorde | Isolation of Renewable Phenolics by Adsorption on Ultrastable Hydrophobic MIL-140 Metal-Organic Frameworks[END_REF] In the present study, MIL-140C-2CO2H and MIL-140C-2NO2 exhibit on the contrary extremely high separation factors for paraquat and difenzoquat (Figure 3C).

We further selected MIL-140C-2CO2H as a representative adsorbent to investigate the origin of the NCIs-based recognition by molecular simulations. Typically, the DFToptimized geometries for both the empty and the guest loaded MIL-140C-2CO2H were then considered to evaluate the electronic density fluctuations (∆ ) upon adsorption using the formula: ∆ = @ -( + ). From the analysis of such a 3-D electron density map of MIL-140C-2CO2H loaded with difenzoquat, we noticed a significant charge transfer between the MOF and the guest that occurs mainly in the vicinity of the cations (see Figure 3D). These calculations reveal that the channel undergoes a higher deformation upon adsorption of difenzoquat resulting from a reorientation of the phenyl rings that tends to optimize the interactions between difenzoquat and the pore wall. The DFT simulation also reveal that the aromatic rings of the confined difenzoquat molecule freely rotates with respect to the central pyrazol node (see Figure S21). As shown in Figure 3E, the phenyl rings of the difenzoquat and the MOF pore wall interact in a displaced-face-to-face or edge-to-face configurations, which is an optimal configuration for π-π interaction. DFT simulation also reveals that electrondeficient -CH3 of difenzoquat points to electron-rich -CO2H of the pore wall after interaction. The difenzoquat can also interact with phenyl rings via the cation-π interaction when there is no -CO2H in the pores according to previous research in chemical recognitions. [START_REF] Dougherty | The Cation-π Interaction[END_REF] On the other hand, the simulation results did not show obvious configuration change for MIL-140C-2CO2H enclosed with linear paraquat, which indicates the interaction between heterocyclic and pore wall is weak.

We further conducted the separation experiments of heterocyclic toxics such as paraquat and clopyralid. Clopyralid possesses indeed an electron-rich -CO2H group, and a "σ-hole" near the Cl moieties (Figure 3A). [START_REF] Brown | Halogen bonding anion recognition[END_REF] Thus these two molecules not only differ in terms of dimensions (paraquat; 5.5 Å and clopyralid; 7.4 Å) but also in electronic density distribution due to the presence of different functional groups. As shown in Figure 4A, the blue and red blocks represent paraquat and clopyralid are preferentially adsorbed respectively. Clopyralid is 2 Å larger than paraquat, which leads to its lower uptake when the pore size is small. The sieving results show that MIL-140A (3.2 Å) repulses both clopyralid and paraquat. MIL-140B and MIL-140C capture more paraquat, with a separation factor of 17.6 and 11.4 respectively (Figure 4B). These results show very high correlation with the molecule sizes, small paraquat is preferentially extracted. When the channel size increases, more clopyralid can be extracted in MIL-140E and MIL-140F, with a separation factor of 2.2 and 6.9 respectively (Figure 4B). We also observed that -CO2H and -NO2 decorated MOFs show opposite separation results with MIL-140C. To evaluate the interaction mechanism, DFT calculation is conducted with MIL-140C-2CO2H containing clopyralid. DFT calculation reveals that the adsorption of clopyralid in the MIL-140C-2CO2H induces a shift of electron density distributions near -CO2H and -Cl, and we can also observe a rotation of the phenyl ring and a slight deformation of the framework (Figure 4C). The formation of hydrogen-bonds between -CO2H groups from the pore wall and the -CO2H function of the clopyralid illustrates the strong host/guest interactions resulting into a significant charge transfer as depicted in Figure 4D. All above-mentioned separation results show that MIL-140s can accomplish the multivariable sieving of two heterocyclic organics based on the competition between size effect and NCIs, which means that we can selectively extract one organic from the mixture according to our requirements. As a contrast, porous activated charcoal shows a high but non selective uptake for these heterocyclic organics while MCM-41 can only extract paraquat from the mixture. Positively surface charged MIL-101(Cr) extracts very few of these two heterocyclic molecules, while MIL-101(Cr)-SO3H adsorbed both the paraquat and clopyralid.

We then extended the study to the sieving of nonpolar PAH, such as pyrene. Their separation from charged organics in water is topical, however none of the commercial sieves such as porous activated charcoal are able to make it. According to the former results and dimensions of the toxics, MIL-140B is selected to conduct the separation of pyrene/MO and pyrene/R6G. As shown in Figure S22, the separation results show that MIL-140B can extract pyrene from the aqueous solution of both MO and R6G with high separation factor (147 and 231 respectively). To intuitively observe the adsorption of pyrene with MIL-140B, powder of MIL-140B was directly added in the cloudy mixture of pyrene and water. As shown in Figure S23, the mixture becomes clear and few pyrene can be detected in water. These experiments show that hydrophobic MIL-140B congenitally adsorb pyrene owing to the π-π interactions as we discussed in former examples. In the case of dye molecules, the reason MIL-140B excludes MO and R6G is the same as we verified in former dye experiments, which is surface-charge repulsion for MO and size exclusion for R6G.

Recycling Performance and Interference Experiment

MIL-140C-2CO2H and MIL-101(Cr)-SO3H were finally investigated to assess the sustainability and reusability of MOF sieves through multiple cycled sieving-desorption tests over MB/MO. Remarkably, these MOFs were shown to be still efficient for the separation of MB/MO after five cycles (see Figure S24). Moreover, the integrity of their frameworks was kept as demonstrated by PXRD (see Figure S25 and S26) carried out after sieving, which is consistent with the excellent water stability of MIL-140s and MIL-101(Cr)s. These results are of great interest in a view of potential applications. To highlight the superiority of these solids, various functionalized UiO-67s bearing a same initial ratio of functional groups as for the preparation of MIL-140C-2CO2H and MIL-140C-2NO2 were also synthesised. However, the PXRD patterns of UiO-67s after sieving demonstrated their poor water stability (see Figure S27). This result highlights that MIL-140s can not only serve as multivariable sieves but also possess adequate water-stability, comparable here to the MIL-101(Cr)s, mainly due to the more hydrophobic character of MIL-140s as well as their infinite metal oxide building units. Interference experiment is then conducted to evaluate the influence of disturbing ions for separation. Sodium chloride is selected as the interfering ions. As shown in Figure S32, the separation performance of MIL-140C-2CO2H is not affected with the existence of tenfold moles of sodium chloride in water.

DISCUSSION

In summary, we discovered that a series of distinctive MOF (MIL-140s) with nonhomogeneous electronic distribution in channels can induce various NCIs with guest molecules, leading to the high performance separation of both dyes and practical organic toxics. The fundamental sieving mechanism of MIL-140s for the sieving of organics has been demonstrated through separation experiments and DFT theoretical calculations, indicating that the separation results match well with a presented outside-in theoretical model (hierarchical recognition). The sieving mechanisms follow several chemical and/or physical patterns: (i) surface charge-based barrier at the external surface of MIL-140s as primary recognition, (ii) a sieving "gate" at the Angström level within the pores as a secondary recognition and finally (iii) NCIs-based mechanism within the channels to obtain a third-level recognition. The strategy we presented in this work is expected to expand the applications of aqueous molecular sieving by targeting more variables in the way similar to translocator in organisms. This further discerns the design of new generations of host-guest systems particularly in the area of porous sieves, and promisingly this strategy could be extended to other related areas such as functional membranes, chemical sensors and biomimetic molecular devices.

  benzenedicarboxylic acid), 2,6-H2NDC (2,6-naphthalenedicarboxylic acid), 4,4'-H2BPDC (4,4'-biphenyldicarboxylic acid), previously used to produce MIL-140A, B and C, as well as new linkers such as H2BPDC4'-dicarboxylic acid), 4,4'-H2SDC (4,4'-Stilbenedicarboxylic acid) and 4,4'-H2EDC (4,4'-Ethyne-1,2-diyldibenzoic acid) that were employed for the first time to produce analogues of the MIL-140 structure type. The so-obtained MIL-140s with a formula [ZrO(O2C-R-CO2)] are denoted as MIL-140A (R=C6H4), B (R=C10H6), C (R=C12H8), C-2CO2H (R=(C14O4H8)x(C12H8)1-x), C-2NO2 (R=(C12N2O4H6)x(C12H8)1-x), E (R=C14H10) and F (R=C14H8) (see Figure 1D and Figure S11-S14).

  ). The major structural changes of the MOF ligands we have observed can be characterized by either a twist of aromatic rings around the central C-C bond or by a ripple effects resulting locally to skewed ligands. As depicted in Figure S19, we can see that in the presence of MB: i) aromatic rings of the biphenyl ligands in MIL-140-C-2CO2H are mutually rotated leading to an ~55° dihedral angle change compared to their pristine state, and ii) the phenyl rings are skewed into convex or concave shapes to best accommodate the MB molecules within the pores. For size exclusion-based sieving, we investigated the sieving performance of NB/R6G in MIL-140s. As shown in Figure 2B, NB (6.1 Å) is easier to be adsorbed by MIL-140s than R6G (10.1 Å) due to its lower dimension and the limited channel size. Typically, MIL-140s, except for MIL-140A (smallest pore diameter) and MIL-140F (largest pore diameter), allow an efficient separation of the organics. The preferential DFT-optimized geometry of NB in MIL-140E is illustrated in Figure 2F and it emphasizes a significant guest-induced distortion of the MIL-140E framework caused by the π-π interactions between NB and the MOF framework. The adsorption capacities MOFs for dye molecules are highly related to the channel size and pore environment, as analysed above (see Figure S28-S31 and Table S4-S5).To further understand the importance of NCIs in these separation experiments, we conducted similar separation of MB/MO with MIL-140C and MIL-140-C-2NO2 in ethanol. As shown in Figure S20, all of these MOFs become invalid for separation of MB/MO mixtures. This results indicate that organic solvents obstruct NCIs between guest molecules and pore wall.

ACKNOWLEDGMENTS

The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (Nos. 51432001, 516320008, U1609219 and 61721005), Chinese national scholarship committee (CSC), and Zhejiang Provincial Natural Science Foundation of China (No. LD18E020001). This work was also partially supported by Centre National de la Recherche Scientifique and University of Versailles St Quentin en Yvelines. E. G. thanks the ANR-11-LABEX-0039 for funding. We also thank Dr. Saad Sene, Dr. Monica Gimenez Marques, Kevin Dedecker, Dr Marvin Benzaqui, Xinhang Jiang and Jia Wang for their contribution to the tests and revision. Guillaume Maurin thanks Institut Universitaire de France for its support. Qi Zhang and Mohammad Wahiduzzaman contributed equally to this work.

EXPERIMENTAL PROCEDURES

Synthesis of [ZrO(O2C-R-CO2)](MIL-140s)

MIL-140C was obtained by reflux for 24 h with a mixture of ZrCl4 (116.5 mg, 0.5 mmol) in 2 mL DMF, 4,4'-H2BPDC (4,4'-biphenyldicarboxylic acid) (121 mg, 0.5 mmol) in DMF (20 mL) and acidic acid (2.29 mL, 40 equivalent of ZrCl4). The white powder was collected by filtration and washed with DMF and methanol. MIL-140C-2NO2 was achieved with similar method, but using the mixture of H2BPDC-2NO2/H2BPDC (H2BPDC-2NO2:H2BPDC=1:3, 1:2, 1:1) and acidic acid (1.65 mL) in 9 mL DMF. MIL-140C-2CO2H was achieved with similar method, but using the mixture of the H2BPDC-2CO2H/H2BPDC (H2BPDC-2CO2H:H2BPDC=1:3, 1:2, 1:1) and acidic acid (1.4 mL) in 10 mL DEF. MIL-140E was achieved with similar method, but using acidic acid (1.145 mL) in 16 mL DMF. MIL-140F was achieved with similar method, but using acidic acid (1.65 mL) in 11 mL DMF. MIL-140A and MIL-140B were synthesized as the reported method. [START_REF] Guillerm | A series of isoreticular, highly stable, porous zirconium oxide based metal-organic frameworks[END_REF] Synthesis of MIL-101(Cr)s MIL-101(Cr)s were synthesized with the published methods, 39; 45 except for MIL-101(Cr)-SO3H for which a new protocol was used. For MIL-101(Cr), Cr(NO3)3•9H2O (400 mg, 1 mmol (Aldrich, 99%)), fluorhydric acid (1mmol), 1,4-benzene dicarboxylic acid (164 mg, 1 mmol (Aldrich 99%)) were added in 4.8 mL H2O, the mixture was put in a hydrothermal bomb which was heated for 8h at 220°C; For MIL-101(Cr)-CO2H, the starting materials CrCl3 (198 mg, 1.25 mmol) and 1.25 mmol of ligand (equimolar mixture of the two derivatives) were introduced into a 20 mL Teflon reactor, after the addition of 5 mL of deionized water, the reactor was heated to 180 °C within 1 h and held for 96 h; MIL-101(Cr)-NO2 was achieved by a hydrothermal reaction of CrCl3 and H2BDC-NO2 (molar ratio CrCl3 : H2BDC-NO2 : H2O = 1.6 : 1 : 278) at 180 °C for 96 hours. MIL-101(Cr)-SO3H was achieved with a new method without HF and Cr(VI), adapted from a reported synthesis of MIL-101(Cr) using tetramethylammonium (TMAOH) solutions, [START_REF] Yang | Synthesis of metal-organic framework MIL-101 in TMAOH-Cr(NO3)3-H2BDC-H2O and its hydrogen-storage behavior[END_REF] Cr(NO3)3•9H2O (400 mg, 1 mmol), aqueous solution of TMAOH (5 mL, 0.025 M), H2BDC-SO3Na (536 mg, 2 mmol) were added into a 23 mL Teflon reactor, the bomb was put in the oven for 24 h at 190°C. The powder of MIL-101(Cr)s was washed with water and ethanol for three times and dried at 150°C overnight before the test.

Computational exploration of the MIL-140s structure and their interactions with the organics DFT-based geometry optimizations of the empty and organics-loaded MOFs were performed at the general gradient approximation (GGA) level with the functional and basis sets as specified in the SI. The organic molecule was inserted in each pore of a 1x1x3 supercell model of the respective MOFs. This supercell approach ensures translational movements of the organic molecules within the pores. MB loaded in MIL-140C-2CO2H and MIL-140C-2NO2, NB loaded in MIL-140E and toxics loaded in MIL-140C-2CO2H were considered as typical cases to investigate the preferential location of these confined molecules and the guest-induced deformation of the MOFs.

SUPPLEMENTAL INFORMATION

Supplemental

DECLARATION OF INTERESTS

The authors declare no competing interests.

Scheme 1. Theoretical model of outside in hierarchical recognition of ideal molecular sieves (A) Charge-specific recognition (the left part) and size recognition (the right part). (B)

The electronic distribution at the pore wall, the protrusions represent functional groups. (C) NCIs between parts with inverse electronic distributions on guest molecules and pore wall.


Sébastien Alfonso
email: sebastien.alfonso1@gmail.com

Bastien Sadoul

Manuel Gesto

Lucette Joassard

Béatrice Chatain

Benjamin Geffroy

Marie-Laure Bégout

Coping styles in European sea bass: The link between boldness, stress response and neurogenesis

Keywords: Personality, HPI axis, Gene transcription, Neurotransmitters

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

Introduction

Coping styles have been defined as "a coherent set of individual physiological and behavioral differences in stress responses consistent across time and context" by Koolhaas and colleagues [START_REF] Koolhaas | Coping styles in animals: current status in behavior and stress-physiology[END_REF]. They have been described in various animal species, including fish, as a continuum between two extreme phenotypes, called proactive and reactive [START_REF] Øverli | Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF][START_REF] Tudorache | Covariation between behaviour and physiology indicators of coping style in zebrafish (Danio rerio)[END_REF][START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF]. In terms of behavior, proactive fish are bolder, more aggressive, explore their environment faster, and display less flexible behavior than reactive fish [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Millot | Exploration behaviour and flight response toward a stimulus in three sea bass strains (Dicentrarchus labrax L.)[END_REF][START_REF] Øverli | Behavioral and neuroendocrine correlates of displaced aggression in trout[END_REF][START_REF] Huntingford | Coping strategies in a strongly schooling fish, the common carp Cyprinus carpio[END_REF]. Moreover, correlations between these behavioral responses across time and context have been established in several fish species [START_REF] Øverli | Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Geffroy | Behavioural syndrome in juvenile eels and its ecological implications[END_REF], as well as between behavior and physiology [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF][START_REF] Øverli | Behavioral indicators of stress-coping style in rainbow trout: Do males and females react differently to novelty?[END_REF][START_REF] Killen | fasting, fear: routine metabolic rate and food deprivation exert synergistic effects on risk-taking in individual juvenile European sea bass[END_REF][START_REF] Rey | Combining animal personalities with transcriptomics resolves individual variation within a wild-type zebrafish population and identifies underpinning molecular differences in brain function[END_REF][START_REF] Mackenzie | Screening for coping style increases the power of gene expression studies[END_REF][START_REF] Johansen | Neural plasticity is affected by stress and heritable variation in stress coping style[END_REF][START_REF] Silva | Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: Evidence for coping styles[END_REF], generally used for defining coping styles since.

Stress can be defined as the cascade of biological events that occur when an organism faces a challenge out of the normal range and attempts to reestablish physiological equilibrium [START_REF] Barton | Stress in finfish: past, present and future -a historical perspective[END_REF].

Typical stress responses of fish toward adverse events involve two main neuroendocrine axes, the brain-chromaffin axis and the hypothalamo-pituitary-interrenal (HPI) axis [START_REF] Gesto | The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time course study[END_REF][START_REF] Sumpter | The endocrinology of stress. Fish Stress and Health in Aquaculture[END_REF][START_REF] Barton | Stress in fishes: a diversity of responses with particular reference to changes in circulating corticosteroids[END_REF]. Both axes are important in the primary response to stress [START_REF] Wendelaar Bonga | The stress response in fish[END_REF][START_REF] Reid | The adrenergic stress response in fish: control of catecholamine storage and release[END_REF][START_REF] Winberg | Serotonin as a regulator of hypothalamic-pituitary-interrenal activity in teleost fish[END_REF], which is activated in various stressful contexts [START_REF] Reid | The adrenergic stress response in fish: control of catecholamine storage and release[END_REF][START_REF] Gesto | Effects of acute and prolonged naphthalene exposure on brain monoaminergic neurotransmitters in rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Gesto | beta-Naphthoflavone and benzo(a)pyrene alter dopaminergic, noradrenergic, and serotonergic systems in brain and pituitary of rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Gesto | Acute and prolonged stress responses of brain monoaminergic activity and plasma cortisol levels in rainbow trout are modified by PAHs (naphthalene, beta-naphthoflavone and benzo(a)pyrene) treatment[END_REF][START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF] and leads to the release of stress hormones, catecholamines (mostly epinephrine and norepinephrine), and corticosteroids (cortisol in teleost fish) into the systemic circulation. At the central level, several monoaminergic neurotransmitters, such as serotonin (5-HT), dopamine (DA), and norepinephrine (NE), are also known to be involved in the organization of stress responses in vertebrates [START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF]. Stress induces changes in brain monoaminergic systems, both acutely and chronically, which is especially true for the serotonergic system. For example, serotonergic activity is consistently activated upon both chronic or acute stress exposure in fish [START_REF] Gesto | The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time course study[END_REF][START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Winberg | The Effect of Stress and Starvation on Brain-Serotonin Utilization in Arctic Charr (Salvelinus-Alpinus)[END_REF]. Acute net chasing was for example demonstrated to induce a rapid increase in forebrain serotonergic activity in rainbow trout [START_REF] Gesto | The response of brain serotonergic and dopaminergic systems to an acute stressor in rainbow trout: a time course study[END_REF]. Similarly, chronic confinement stress (three times a day during four weeks) also induced elevated concentration of 5HIAA and serotonergic activity in the telencephalon of Artic Charr [START_REF] Winberg | The Effect of Stress and Starvation on Brain-Serotonin Utilization in Arctic Charr (Salvelinus-Alpinus)[END_REF]. The concentration of the brain monoamines DA, NE, and 5HT and the concentration of their associated main metabolites, 3,4-dihydroxyphenylacetic acid (DOPAC), 3-methoxy-4hydroxyphenylglycol (MHPG), and hydroxyindoleacetic acid (5HIAA), respectively, are generally used to quantify monoaminergic system activity using the turnover ratio between the monoamine and associated metabolite [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF][START_REF] Shannon | A Comparison of Biochemical Indexes of 5-Hydroxytryptaminergic Neuronal-Activity Following Electrical-Stimulation of the Dorsal Raphe Nucleus[END_REF]. This activity has been suggested to vary between individuals with different behavioral responses [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF][START_REF] Hoglund | Avoidance behavior and brain monoamines in fish[END_REF][START_REF] Brelin | Divergent stress coping styles in juvenile brown trout (Salmo trutta)[END_REF][START_REF] Winberg | Role of brain serotonin in modulating fish behavior[END_REF]. Both the stress axis (HPI) and monoaminergic systems (brain-chromaffin) have been shown to be reliable physiological markers correlated with differences in coping styles [START_REF] Øverli | Behavioral and neuroendocrine correlates of displaced aggression in trout[END_REF][START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Øverli | Stress coping style predicts aggression and social dominance in rainbow trout[END_REF][START_REF] Sørensen | Neural plasticity and stress coping in teleost fishes[END_REF][START_REF] Vindas | How do individuals cope with stress? Behavioural, physiological and neuronal differences between proactive and reactive coping styles in fish[END_REF] even if, in some species, physiological markers were not directly linked to behavioral traits related to coping styles [START_REF] Qu | Independence between coping style and stress reactivity in plateau pika[END_REF].

The HPI axis, which produces cortisol, its major end-product hormone, acts as an activator of physiological and behavioral responses [START_REF] Sumpter | The endocrinology of stress. Fish Stress and Health in Aquaculture[END_REF][START_REF] Sadoul | Stress and growth[END_REF][START_REF] Schreck | The concept of stress in fish[END_REF]. Glucocorticoid receptors (Grs) and mineralocorticoid receptor (Mr) bind cortisol [START_REF] Kiilerich | Endogenous regulation of 11deoxycorticosterone (DOC) and corticosteroid receptors (CRs) during rainbow trout early development and the effects of corticosteroids on hatching[END_REF] and are detected in various fish tissues.

They are involved in different physiological functions linked for instance to growth, reproduction, immunity and they participate in the regulation of control cortisol release [START_REF] Johansen | Cortisol receptor expression differs in the brains of rainbow trout selected for divergent cortisol responses[END_REF][START_REF] Alderman | Central and peripheral glucocorticoid receptors are involved cortisol response to an acute stressor in rainbow trout[END_REF][START_REF] Bernier | Chapter 6 regulation and contribution of the corticotropic, melanotropic and thyrotropic axes to the stress response in fishes[END_REF][START_REF] Faught | The molecular stress reponse[END_REF]. The production and release of cortisol occurs in interrenal cells, located in the teleost head kidney, and is mainly controlled by adrenocortidotropic hormone (Acth), which is released by the pituitary gland into the blood [START_REF] Sumpter | The endocrinology of stress. Fish Stress and Health in Aquaculture[END_REF]. In turn, the release of Acth is mainly controlled by corticotropin releasing-factor (Crf), a neuropeptide produced in the hypothalamus [START_REF] Wendelaar Bonga | The stress response in fish[END_REF][START_REF] Doyon | Corticotropin-releasing factor and neuropeptide Y mRNA levels are elevated in the preoptic area of socially subordinate rainbow trout[END_REF]. In addition, recent studies suggest that the regulation of stress axis and neurogenesis are closely related [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF][START_REF] Sørensen | Cortisol reduces cell proliferation in the telencephalon of rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Best | Maternal cortisol stimulates neurogenesis and affects larval behaviour in zebrafish[END_REF] and we previously proposed that fish displaying high sensitivity to stress also exhibit high levels of neurogenesis in a non-stressful context [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF].

Proliferating cell nuclear antigen (Pcna), neurogenic differentiation factors 1 and 2 (Neurod1 and Neurod2), and the early growth response protein (Egr1) are important markers of neurogenesis and neuronal development [START_REF] Sørensen | Neural plasticity and stress coping in teleost fishes[END_REF][START_REF] Burmeister | Evolutionary conservation of the egr-1 immediate-early gene response in a teleost[END_REF]. While the regulation of stress axes might be an important feature to characterize coping styles in fish, there is still a paucity of information regarding the differences in neurogenesis between coping styles. Additional data are therefore needed in order to better understand the mechanisms associated to the behavioral responses.

Differences in behavioral and physiological responses to stress may have direct implications on growth, foraging activities, and the resulting fitness of wild fish species [START_REF] Sadoul | Stress and growth[END_REF][START_REF] Réale | Personality and the emergence of the pace-of-life syndrome concept at the population level[END_REF][START_REF] Geffroy | Differential effects of behaviour, propensity to migrate and recruitment season on glass eels and elvers' growing performance[END_REF]. In the context of aquaculture, better knowledge of individual diversity can help to improve the welfare and management of the fish [START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF], as proactive fish sometimes show better feeding motivation and feed efficiency than reactive fish [START_REF] Øverli | Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Martins | The consistency of individual differences in growth, feed efficiency and feeding behaviour African catfish Clarias gariepinus (Burchell 1822) housed individually[END_REF][START_REF] Martins | Comparison of feed intake behaviour and stress response in isolated and non-isolated African catfish[END_REF].

The European sea bass Dicentrarchus labrax is a predatory species, widely distributed around Europe and West Africa [START_REF]The state of world fisheries and aquaculture[END_REF], that has a key ecological role in the marine food chain [START_REF] Whitehead | Fish of the North-eastern Atlantic and Mediterranean[END_REF]. It is also one of the most highly farmed fish species in Europe [START_REF] Tveteras | Estimates & trends 2011-2012[END_REF]. Therefore, additional knowledge of coping styles in European sea bass is required to improve both its management and welfare under conditions of aquaculture [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Ferrari | Heritability of boldness and hypoxia avoidance in European seabass, Dicentrarchus labrax[END_REF] and to better understand and predict population dynamics [START_REF] Sih | Ecological implications of behavioural syndromes[END_REF][START_REF] Conrad | Behavioural syndromes in fishes: a review with implications for ecology and fisheries management[END_REF].

Here, we had two main objectives. First, we monitored the boldness of the fish, by assessing the behavior of European sea bass over time and context testing for behavioral consistency between shy and bold fish. A group risk taking test (GRT) and open field test (OFT) were performed at 227 and 749 days post fertilization (dpf), respectively. Second, we aimed to describe the underlying physiological mechanisms associated with boldness. The blood and brains of the fish were sampled after the OFT to measure cortisol concentration in the plasma, the transcription of genes involved in stress regulation (mr, gr1, gr2, crf), neurogenesis (pcna, neurod1, neurod2), and neuronal development (egr1) and, finally, the concentration of monoamines (DA, 5HT, NE) and associated metabolites (DOPAC, 5HIAA) in whole brain.

Material and methods

Experiments were authorized by ethics committee agreement APAFIS#7098 and all procedures involving animals were in accordance with the ethical standards of the institution and followed the recommendations of Directive 2010/63/EU.

Fish rearing and pit-tagging

The reproduction and larval rearing of European sea bass, were performed at Ifremer, Palavas-les-flots (France, 34250). Fish were produced from third-generation domesticated West Mediterranean brood stock on February 27, 2016, applying a full factorial mating design combining 10 dams and 50 sires by in vitro fertilization. At 223 dpf, ~3000 fish were anesthetized using 200 ppm of benzocaine (benzocaine E1501, Sigma, Saint Louis, MO, USA), measured for body mass (g) and standard length (cm), and pit-tagged in the muscle, just in front of the dorsal fin, with an ISO PIT tag (8 x 1.4 mm) to ensure individual identification.

Fish were maintained until 457 dpf following standard procedures [START_REF] Chatain | Estimation et amélioration des performances zootechniques de l'élevage larvaire de Dicentrarchus labrax et de Sparus auratus[END_REF]. Thereafter, they were reared in six different 5-m 3 tanks within a recirculating bio-filtered system at a temperature of 20.9 ± 1.3°C, pH = 7.4, and a salinity of 37 PSU under a 10/14 light/dark photoperiod. The fish were fed a standard marine fish aquaculture diet (Neo-grower, Le Gouessant, France) ad libitum using a self-feeder.

Group risk-taking test

The group risk-taking test (GRT) is a standardized test to measure boldness in fish and was adapted from Ferrari and colleagues [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF]. The GRT was performed at 227 dpf with 1,468 fish and the following day, it was repeated with the other 1,468 naive fish (mass = 18.8 ± 1.2 g; standard length = 10.3 ± 1 cm).

The GRT consists of placing all the fish into one half of a 1.5 -m 3 tank, separated in two by an opaque PVC divider (Figure 1.A). Fish were gently transferred from their home tank to one half of the experimental tank under light anesthesia (100 ppm Benzocaine, same stock solution as before) between 10:00 and 11:00 am. The side containing the fish was covered with a black opaque curtain to create a sheltered area. Both halves of the tank received water of the same quality, oxygenation, and exchange rate. After an habituation period of 2 h, the door (a circular opening of 12 cm Ø in the centre or the divider) was opened and the fish were left with the possibility to exit the sheltered area, therefore exhibiting a risk-prone behavior reflecting boldness level. The door was equipped with a PIT-tag detection antenna (DORSET, The Netherlands) connected to a control device. The PIT-tag detection antenna allowed the monitoring of fish behavior during the GRT by obtaining the exact exit time (latency to exit (s)) of each fish leaving the sheltered area for the first time. As the test lasted for 24 hours, a latency of 1,440 min was assigned to fish that did not exit the sheltered area before the end of the test. Once the outcome of the GRT was analyzed, the individuals were categorized as bold (fish that exited the shelter) or shy (fish that stayed in the shelter during the entire experiment). They were then transferred back to their home tanks and reared together until the open field test (OFT) was performed, 16 months later.

Open field test

The open field test (OFT) was adapted from that of Benhaim and colleagues [START_REF] Benhaim | Self-feeding behaviour and personality traits in tilapia: A comparative study between Oreochromis niloticus and Sarotherodon melanotheron[END_REF] and performed at 749 dpf (341 ± 4 g) using a sub-sample of fish (n = 57, half of them bold and the other half shy, based on their GRT categorization). The OFT consists of placing an individual fish in an open and novel arena (75 x 75 cm with a water height of 23 cm, Figure 1.B) and tracking its swimming activity. The fish was placed in the closed sheltered area for a habituation period of 5 min. The door was then opened, allowing the fish to explore its novel environment while being video-tracked. Videos were taken with a DMK 31AU03 camera (The Imaging Source, Germany) and the recorded behavior of each fish then analyzed using EthoVision XT 13.1 software (Noldus, The Netherlands) by the same operator (SA). The latency to exit the shelter (s) was recorded for each individual. A latency of 1,200 s was assigned to fish that did not exit the shelter at the end of the test. Fish that exited the shelter were categorized as bold, whereas those that did not were categorized as shy. The total distance travelled (cm), number of transitions between shelter and arena, and the time spent in the shelter (s) were also recorded.

Blood and brain sampling

Following the OFT, fish were gently removed from the arena and euthanized using 1,500 ppm benzocaine. Blood and brain were sampled without knowing the boldness level of the fish.

Blood was sampled from the caudal vessels using lithium heparinized syringes [START_REF] Sadoul | Measuring cortisol, the major stress hormone in fishes[END_REF]. Then, blood was centrifuged (4 min at 13,000 x g) to obtain plasma samples, which were stored at -20°C until analysis. The plasma cortisol concentration was determined by ELISA (RE52061, IBL International, Hamburg, Germany) using a Synergy-HT microplate reader (BioTek Instruments, Winooski, VT, USA) following manufacturer's instructions.

The brain was extracted, divided along the sagittal plane, and placed directly in liquid nitrogen. The left half was used for monoamine analyses and the right half for gene transcription analyses.

Brain monoamine analyses

The left half of the fish brain was used for HPLC analysis of brain monoamines. Tissues were weighed and immediately homogenized in 1 mL perchloric acid (PCA) solution (0.4 M PCA and 0.1 mM EDTA) with a Sonopuls ultrasonic homogenizer (Bandelin, Germany).

Homogenates were then centrifuged for 10 min at 14 000 x g (at 4°C). Supernatants were diluted 20 times in the PCA solution and 20 µL of the dilution injected into the high performance liquid chromatography (HPLC) system.

The diluted supernatants were analyzed high performance liquid chromatography with electrochemical detection (HPLC-EC). The HPLC mobile phase consisted of 73.9 mM NaH2PO4, 0.1 mM Na2EDTA, and 0.58 mM sodium 1-octanesulfonate in deionized water with 15.3% (v/v) methanol. The pH of the mobile phase was adjusted to 3.0 with orthophosphoric acid. The chromatographic separation was performed at 0.9 mL/min with a Reprosil-Pur 120 C18-AQ column (150 x 4 mm, 5 µm particles) (Dr. Maisch, Germany) and electrochemical detection of the separated compounds was performed using an ESA Coulochem II detector. The detection system included a double analytical cell with oxidation potentials set at 0 mV (first electrode) and +340 mV (second electrode). A conditioning cell at + 400 mV was used before the analytical cell to oxidize potential interfering compounds. The rest of the HPLC system consisted of a Shimadzu LC-10AD pump and a Midas autosampler (Spark Holland, The Netherlands). Acquisition and integration of the chromatograms were performed using ClarityTM HPLC software (DataApex Ltd, Czech Republic).

The levels of serotonin (5HT), dopamine (DA), and their main oxidative metabolites 5hydroxyindoleacetic acid (5HIAA) and 3, 4-dihydroxyphenylacetic acid (DOPAC) were quantified, along with the levels of norepinephrine (NE). The main NE metabolite, 3methoxy-4-hydroxyphenylglycol (MHPG), was below the detection limit. Quantification was carried out by comparing peak areas with those of the corresponding standards. The ratios between 5HIAA and 5HT and between DOPAC and DA were then calculated as indirect measures of the activity of the serotonergic and dopaminergic neurons, respectively [START_REF] Winberg | Roles of brain monoamine neurotransmitters in agonistic behaviour and stress reactions, with particular reference to fish[END_REF].

Gene transcription analyses

The right half of the fish brain was used for gene transcription analyses.

Extraction and reverse transcription of total RNA from the brain

Each half-brain (± 200 mg) was mechanically disrupted using a ball mill in 900 µL QIAzol lysis Reagent (Quiagen) in 2 ml RNAse-free tubes. They were then diluted two-fold before starting RNA extraction, following the manufacturer's instructions (RNeasy Plus Universal Mini Kit protocol, Qiagen, recommended initial sample size: 100 mg). The reverse transcription of total RNA to cDNA was performed as described in Geffroy and colleagues [START_REF] Geffroy | Sexually dimorphic gene expressions in eels: useful markers for early sex assessment in a conservation context[END_REF]. RNA quantity was assessed by measuring the A260/A280 ratio using the NanoDrop® ND-1000 V3300 Spectrophotometer (Nanodrop Technology Inc., Wilmington, DE, USA).

Briefly, an aliquot of each tube was diluted in diethylpyrocarbonate-treated water. Then, 1.5 μg RNA was reverse transcribed using 200 U Moloney murine leukemia virus (MMLV) reverse transcriptase (PROMEGA) and 2 μg random hexamers (Promega) in a master-mix buffer and supplemented with 25 U RNase inhibitor (RNasin; Promega) in a final volume of 15 μl. All cDNA were diluted 50 fold in nuclease-free water prior to quantitative real-time PCR (qPCR).

Quantitative real-time PCR

The transcription of genes involved in stress regulation, namely glucocorticoid receptor 1 and 2 (gr1 and gr2), the mineralocorticoid receptor (mr), and corticotrophin releasing factor (crf) was monitored. We also investigated the expression of genes involved in neurogenesis: neurogenic differentiation factors (neurod1 and neurod2) and proliferating cell nuclear antigen (pcna), as well as an early growth response gene (egr1), involved in brain development.

Primer sequences were retrieved from the literature [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF][START_REF] Pavlidis | Onset of the primary stress in European sea bass Dicentrarhus labrax, as indicated by whole body cortisol in relation to glucocorticoid receptor during early development[END_REF][START_REF] Crespo | Isolation and characterization of Ff1 and Gsdf family genes in European sea bass and identification of early gonadal markers of precocious puberty in males[END_REF] or specifically-designed using the web version of PRIMER 3 (http://primer3.ut.ee; Table 1). The qPCR experiments were conducted in a final volume of 1.5 μl, using 1 μl fish brain cDNA and 0.5 μl of an optimized concentration of primers and Fast SYBR Green PCR Master Mix (Applied Biosystems, Life technologies). Distribution of the volume into the plate was performed using a robot (Labcyte Echo525) able to distribute as low as 25 nL by acoustic transfer ensuring reliable repeatability even with small qPCR volumes. The hot start enzyme was activated for 2 min at 95°C and amplification carried out using the following program: 95°C for 3 s and 60°C for 30 s for 40 cycles. After amplification, a melting curve was obtained using the following protocol: 10 s holding at 55°C followed by sequential 0.05°C increases repeated 80 times. Technical duplicates were run for each sample. A standard curve was generated by mixing all samples and the efficiency (E) for all genes was calculated (ef1: 1.98; l13: 1.96; gr1: 1.90; gr2: 2.14; mr: 2; crf: 1.83; egr1: 2; neurod1: 1.88; neurod2: 1.88; and pcna: 1.80).

Housekeeping genes

both been recommended as housekeeping genes [START_REF] Radonic | Guideline to reference gene selection for quantitative real-time PCR[END_REF][START_REF] Bland | Development and validation of a range of endogenous controls to support the implementation of practical Taqman real-time PCR-based surveillance for fish diseases within aquaculture[END_REF] and were used in this study. The coefficients of variation were below 5% for both l13 and eef1-alpha. The amplification efficiency was 98 and 96 % for l13 and eef1-alpha respectively.

Relative levels of gene transcription were obtained using the following equation with the efficiency (E) of the target gene normalized by the efficiency and the geometric mean of the two housekeeping genes as reference (ref; l13and eef1-alpha), according to Roche diagnostic [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]: a*: from Pavlidis and colleagues [START_REF] Pavlidis | Onset of the primary stress in European sea bass Dicentrarhus labrax, as indicated by whole body cortisol in relation to glucocorticoid receptor during early development[END_REF]; b*: from Sadoul and colleagues [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF]; c*: from Crespo and colleagues [START_REF] Crespo | Isolation and characterization of Ff1 and Gsdf family genes in European sea bass and identification of early gonadal markers of precocious puberty in males[END_REF]; and d*: designed from the European sea bass genome from UCSC.

= ( ) ( ) !"#

Statistical analyses

Statistical analyses were performed using R 3.1.0 software [START_REF]R: A language and environment for statistical computing[END_REF]. All statistical analyses were Finally, a principal component analysis (PCA) was performed among fish that exited the shelter with individual continuous variables recorded in OFT (Latency to exit shelter, distance travelled, time spent in shelter and number of transitions) using ade4 package [START_REF] Dray | The ade4 package: implementing the duality diagram for ecologists[END_REF]. Individual PC scores of the two first axes of the PCA were then downloaded. PC scores from axis 2, reflecting coping styles were analyzed using linear regression as in relation to either monoamine concentration or relative level of gene transcription in the brain. Homogeneity of the model residuals was a posteriori checked using Shapiro-Wilk.

Results

Behavioral consistency across context and time between the GRT and OFT

During the GRT, 63% of the fish exited the sheltered area within the 24 hours of the test and were designated as bold. Consequently, 37% of the fish were designated as shy in the GRT. In the OFT, 58% of the fish exhibited bold behavior and exited the sheltered compartment vs.

42% which exhibited shy behavior and stayed in the sheltered compartment during the 20 min of the test. The proportion of fish with bold/shy behavior was similar between the GRT and OFT (χ² = 0.51; p = 0.48, Figure 2A). However, there was no behavioral consistency across context between the GRT and OFT for either bold or shy fish designated as such during the GRT (p = 0.28 and 0.85, respectively). Indeed, 46% of fish designated as shy during the GRT stayed shy in the OFT, whereas the other 54% became bold one year later in the OFT. For fish designated as bold during the GRT, 61% stayed bold, whereas the other 39% became shy during the OFT (Figure 2B).

3.2.Open field test

Of the 57 fish screened in the OFT, 58% were categorized as bold. These bold fish actively swam in the tank during the 20 minutes of the test (912.

Plasma cortisol concentration

Plasma cortisol concentrations were similar between bold and shy fish screened in the OFT (t = -0.10, p = 0.92, data not shown). The mean plasma cortisol concentration in bold fish was 904.3 ± 51.8 ng/mL (n = 33) and 912.7 ± 60.2 ng/mL in shy fish (n = 24).

Monoamine concentration in the brain

There was no difference in the concentration of DA or DOPAC in the brain between bold and shy fish screened in the OFT, nor the DOPAC/DA ratio (t = -0.86, p = 0.40; t = -0.58, p = 0.57; and t = -0.61, p = 0.54, respectively). For the serotonergic system, there was no difference in the concentration of 5HT or 5HIAA in the brain between bold and shy fish (t = 0.45, p=0.66 and t = -1.15, p = 0.25, respectively). However, shy fish had a significantly lower 5HIAA/5HT ratio in the brain than bold fish (t = -2.17, p = 0.03). The concentration of NE in the brain was also significantly higher in the shy than bold fish (t = 2.22, p = 0.03, Figure 3). Note that when considering bold and shy fish categories as assigned after the GRT, no difference in monoamine concentration or turnover ratio was detected (Supplementary material).

Gene transcription in the brain

The transcription of gr1 in the brain did not differ between bold and shy fish screened in the OFT (W = 312, p = 0.32), whereas gr2, mr and crf were significantly upregulated in the brains of shy fish relative to those of bold fish (W = 171, p = 0.01, W = 255, p = 0.047 and W = 145, p < 0.001, respectively).

The transcription of neurod1 and pcna in the brain did not differ between bold and shy fish (W = 358, p = 0.82 and W = 311, p = 0.31, respectively), whereas there was a trend towards the upregulation of neurod2 in shy fish relative to bold fish (W = 269, p = 0.08). The transcription of egr1 was significantly upregulated in the brains of shy fish relative to those of bold fish (t = -2.70, p < 0.01, Figure 4). Note that when considering bold and shy fish categories as assigned after the GRT, no difference in gene transcription was detected (Supplementary material).

3.6.Principal component analysis (PCA)

On one hand, PCA axis 1, which explained 52 % of the data variability observed, was mainly driven by the time spent in shelter, the distance travelled and the number of transitions between shelter and open field arena during the OFT (Table 3). On the other hand, PCA axis Regarding monoamines concentration in the brain, concentration of DA was negatively correlated with PCA axis 2 (F = 5.25, p = 0.03, R² = 0.12), i.e., bolder fish displayed higher level of DA in the brain but there was no correlation for DOPAC or DOPAC/DA ratio with PCA axis 2 (F = 0.98, p = 0.33 and F = 0.002, p = 0.96 respectively). Moreover, 5HT and 5HIAA also were negatively correlated with PCA axis 2 scores (F = 6.10, p = 0.02, R² = 0.14 and F = 7.96, p = 0.008, R² = 0.18 respectively) but there was no correlations between 5HIAA/5HT ratio and PCA axis 2 (F = 0.39, p = 0.54). Finally there was no correlation between concentration of NE in the brain and PCA axis 2 scores (F = 1.4 p = 0.25, Figure 5). Concerning gene transcription in the brain, there was no correlation between the investigated genes and PCA axis 2 scores except a negative trend between relative level of pcna and PCA axis 2 scores (F = 3.99, p = 0.07, R² = 0.08). All statistical details concerning correlations between the relative levels of other genes are given in the text of the Supplementary Material and illustrated in Figure S1.

Discussion

Here, we evaluated the boldness of European sea bass using two different challenges, which were group risk taking test (GRT) and open field test (OFT) at 227 and 749 dpf, respectively.

The proportion of bold/shy fish did not differ between the two tests (approximately 60% of the fish were categorized as bold). The proportion of bold/shy fish however differed from a previous study in European sea bass in which ~80% were identified as shy using a group risktaking test [START_REF] Ferrari | Heritability of boldness and hypoxia avoidance in European seabass, Dicentrarchus labrax[END_REF]. The high proportion of bold fish identified in our study may be attributable to small differences with their protocol and/or the level of fish domestication which could affect boldness [START_REF] Millot | Exploration behaviour and flight response toward a stimulus in three sea bass strains (Dicentrarchus labrax L.)[END_REF]. We did not find any clear behavioral consistency in boldness across context between the GRT and OFT. Indeed, only 50% of fish with shy behavior in the GRT were also categorized as shy in the OFT (522 days between the two tests). The consistency of boldness over time was previously reported in European sea bass when consecutive GRTs were performed 29 days apart [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF]. Boldness, as well as other personality traits, is known to be consistent over a short period of time, but can evolve during the life of the animal [START_REF] Polverino | Emergence and development of personality over the ontogeny of fish in absence of environmental stress factors[END_REF].

Moreover, boldness is influenced by many external factors, both biotic and abiotic [START_REF] Brown | Heritable and experiential effects on boldness in a tropical poeciliid[END_REF][START_REF] Thomson | Plasticity of boldness in rainbow trout, Oncorhynchus mykiss: do hunger and predation influence risk-taking behaviour?[END_REF][START_REF] Frost | Plasticity in animal personality traits: does prior experience alter the degree of boldness?[END_REF][START_REF] Ferrari | Unpredictability in food supply during early life influences growth and boldness in European seabass[END_REF].

Abiotic factors, such as temperature, physico-chemical characteristics of the water, or food supply, were kept constant during our experiment. However, some other factors could explain the absence of a clear boldness consistency between the GRT and OFT. The difference in social context between the two tests (group vs isolation) could be one and could partly explain the discrepancy between the results, regardless of the time window that separated the two behavioral experiments [START_REF] Webster | Boldness is influenced by social context in threespine sticklebacks (Gasterosteus aculeatus)[END_REF]. Social context is indeed very important in shaping personality traits in fish [START_REF] Geffroy | The link between social network density and rank-order consistency of aggressiveness in juvenile eels[END_REF] and may have affected our capacity to evaluate boldness in single fish in a social species, such as sea bass, as already mentioned by Ferrari and colleagues [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF].

Nevertheless, the fish expressed two divergent behavioral responses during the OFT. The majority of fish (58 %) expressed bold trait, with a high level of activity in the OFT arena, whereas shy fish expressed freezing behavior in the shelter. These two different behavioral responses have been largely described in the literature as typical proactive and reactive coping style characteristics [START_REF] Øverli | Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF][START_REF] Castanheira | Coping styles in farmed fish: consequences for aquaculture[END_REF][START_REF] Øverli | Behavioral indicators of stress-coping style in rainbow trout: Do males and females react differently to novelty?[END_REF].

Differential stress regulation of the HPI axis and the brain-chromaffin axis are often associated with the opposite behavioral responses expressed by proactive and reactive individuals [START_REF] Koolhaas | Coping styles in animals: current status in behavior and stress-physiology[END_REF][START_REF] Øverli | Evolutionary background for stress-coping styles: relationships between physiological, behavioral, and cognitive traits in non-mammalian vertebrates[END_REF][START_REF] Vindas | How do individuals cope with stress? Behavioural, physiological and neuronal differences between proactive and reactive coping styles in fish[END_REF][START_REF] Vindas | Brain cortisol receptor expression differs in Arctic charr displaying opposite coping styles[END_REF]. Here, the signal from the dopaminergic system in the whole brain was similar between bold and shy fish. The concentrations of DA and DOPAC and the DA/DOPAC turnover ratio were indeed identical between our two groups, whereas dopaminergic system signaling was found to be different between bold and shy fish in different brain parts such as optic tectum [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Koolhaas | Individual variation in coping with stress: A multidimensional approach of ultimate and proximate mechanisms[END_REF]. Possible specific differences between bold and shy fish could also be masked by the fact that the analyses were performed on whole brain [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Backstrom | Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness[END_REF]. The boldest fish screened in the OFT nevertheless displayed higher DA concentration in the whole brain compared to the less bold ones. This is consistent with previous studies suggesting that dopaminergic system may trigger active behavioral responses, including bolder behavior [START_REF] Winberg | Social Rank and Brain Levels of Monoamines and Monoamine Metabolites in Arctic-Charr, Salvelinus-Alpinus (L)[END_REF][START_REF] Winberg | Induction of Social-Dominance by L-Dopa Treatment in Arctic Charr[END_REF][START_REF] Thornqvist | Bold zebrafish (Danio rerio) express higher levels of delta opioid and dopamine D2 receptors in the brain compared to shy fish[END_REF]. Nevertheless, it is important to note that our study was performed on the whole brain, therefore we cannot conclude that the differences between bold and shy fish are representative of what would be measured in region-specific areas. Concerning the serotonergic system, the 5HT/5HIAA turnover ratio was lower in shy than bold fish following the OFT but the boldest fish displayed both higher levels of 5HT and 5HIAA in the brain than the less bold ones. Similar differences in 5HIAA have been previously observed in the hypothalamus and telencephalon of rainbow trout (Oncorhynchus mykiss) following an acute stressor [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF]; fish with a more reactive coping style showed lower levels of serotonergic activity upon exposure to stress. This supports the view of the shy and bold groups established in the OFT as exhibiting different coping styles, as differences in the serotonergic system could be involved in the determination of the individual coping style [START_REF] Puglisi-Allegra | Serotonin and stress coping[END_REF]. Another interpretation is that the differential behavior observed (exploring vs freezing in the shelter) might have driven the difference in the serotonergic response. In this case, the bold fish were more active and left the shelter and thus exposed themselves out in the open area, which might be more stressful for them, leading to higher 5HT activity. This alternative interpretation has been considered by Koolhaas and colleagues [START_REF] Koolhaas | Neuroendocrinology of coping styles: Towards understanding the biology of individual variation[END_REF] who hypothesized that "it is likely that differential neuroendocrine characteristics are mainly a consequence rather that the cause of behavioral differentiation". Finally, the noradrenergic system is generally less well-documented than the serotonergic and dopaminergic systems in relation to coping styles.

However, the higher NE concentrations we measured in the whole brain of shy fish correlate well with results found in the brainstem, telencephalon and optic tectum of rainbow trout following confinement stress [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Backstrom | Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness[END_REF].

Concerning the HPI axis, plasma cortisol concentrations measured in our study were higher than those previously found in European sea bass [START_REF] Rotllant | Response to confinement in sea bass (Dicentrarchus labrax) is characterised by an increased biosynthetic capacity of interrenal tissue with no effect on ACTH sensitivity[END_REF][START_REF] Samaras | Repeatability of cortisol stress response in the European sea bass (Dicentrarchus labrax) and transcription differences between individuals with divergent responses[END_REF]. It is likely that the social isolation during the OFT is a very strong stressor for European sea bass, as was shown for rainbow trout [START_REF] Bernier | Heads or tails? Stressor-specific expression of corticotropin-eleasing factor and urotensin I in the preoptic area and caudal neurosecretory system of rainbow trout[END_REF]. Further, there was no difference in the cortisol response between bold and shy fish categorized using the OFT. This may seem surprising, as high cortisol levels have been shown

to be predictive of the reactive coping style in sea bass in another context [START_REF] Ferrari | Consistency in European seabass coping styles: A life-history approach[END_REF]. Nevertheless, it cannot be excluded that the lack of difference may be due to the fact that, once in the OFT, bold fish take more risk and are further stressed by exploring the open field arena whereas shy fish stay in the shelter resulting in different physiological status when blood sample was taken. Moreover, such an absence of a difference in the peak cortisol response was previously reported following an acute stressor in various fish species [START_REF] Tudorache | Covariation between behaviour and physiology indicators of coping style in zebrafish (Danio rerio)[END_REF][START_REF] Vindas | Brain cortisol receptor expression differs in Arctic charr displaying opposite coping styles[END_REF], but divergences appeared during the recovery phase [START_REF] Tudorache | Covariation between behaviour and physiology indicators of coping style in zebrafish (Danio rerio)[END_REF]. The transcription in the whole brain of gr2, mr and crf, which involved in the regulation of the HPI axis, was higher in shy than bold fish, whereas gr1 transcription did not differ between the two groups. Elevated gr2, mr or crf transcription in shy fish has also been reported in other fish species and contexts [START_REF] Backstrom | Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness[END_REF][START_REF] Geffroy | Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish[END_REF][START_REF] Van Den Bos | Further characterisation of differences between TL and AB zebrafish (Danio rerio): Gene expression, physiology and behaviour at day 5 of the larval stage[END_REF][START_REF] Summers | Learning and CRF-Induced Indecision during Escape and Submission in Rainbow Trout during Socially Aggressive Interactions in the Stress-Alternatives Model[END_REF]. For example, HR rainbow trout (considered as shy) displayed higher levels of crf than LR (considered as bold) in the forebrain of rainbow trout following confinement stress [START_REF] Backstrom | Stress effects on AVT and CRF systems in two strains of rainbow trout (Oncorhynchus mykiss) divergent in stress responsiveness[END_REF]. These effects were shown to be modulated by binding proteins reducing the activity of Crf but increasing its longevity [START_REF] Flik | CRF and stress in fish[END_REF][START_REF] Samaras | Allostatic Load and Stress Physiology in European Seabass (Dicentrarchus labrax L.) and Gilthead Seabream (Sparus aurata L.)[END_REF]. In addition to its effects on the regulation of the HPI axis, Crf can interact with neurotransmission and modulate behavior [START_REF] Clements | Evidence that acute serotonergic activation potentiates the locomotor-stimulating effects of corticotropin-releasing hormone in juvenile chinook salmon (Oncorhynchus tshawytscha)[END_REF][START_REF] Lowry | Regulation of behavioral responses by corticotropin-releasing factor[END_REF][START_REF] Carpenter | Corticotropin Releasing Factor Influences Aggression and Monoamines: Modulation of Attacks and Retreats[END_REF][START_REF] Backstrom | Serotonin Coordinates Responses to Social Stress-What We Can Learn from Fish[END_REF]. Overall, Crf interacts with serotonergic and noradrenergic pathways, resulting in an increase in locomotor behavior, including non-ambulatory behavior, and has been reported to cause anxiety [START_REF] Lowry | Regulation of behavioral responses by corticotropin-releasing factor[END_REF]. In the present study, shy fish (which expressed higher levels of crf than bold fish) expressed a lower turnover of serotonergic activity and higher NE concentrations. Shy fish also displayed a freezing behavior in the shelter during the OFT. This echoes a previous study showing that intraventricular Crf injections in rats increase the anxiety state of the animal, as well as the turnover ratio between NE and its metabolite DHPG in the brain-stem locus coeruleus [START_REF] Butler | Corticotropin-Releasing Factor Produces Fear-Enhancing and Behavioral Activating Effects Following Infusion into the Locus Coeruleus[END_REF].

More recently, it was reported in rainbow trout that Crf-injected fish have higher 5HT and 5

HIAA levels in various brain regions (i.e., the subpallium, raphe, and preoptic hypothalamus)

and/or express head-shaking behavior, which is related to anxiety [START_REF] Backstrom | CRF and urotensin I effects on aggression and anxiety-like behavior in rainbow trout[END_REF][START_REF] Carpenter | Corticotropin releasing factor induces anxiogenic locomotion in trout and alters serotonergic and dopaminergic activity[END_REF]. Interactions between Crf and both the serotonergic and the noradrenergic system appear promising to further explain the divergent phenotypes observed in fish coping styles [START_REF] Lowry | Regulation of behavioral responses by corticotropin-releasing factor[END_REF][START_REF] Backstrom | CRF and urotensin I effects on aggression and anxiety-like behavior in rainbow trout[END_REF][START_REF] Carpenter | Corticotropin releasing factor induces anxiogenic locomotion in trout and alters serotonergic and dopaminergic activity[END_REF].

Recent studies suggest that stress axis regulation and neurogenesis are tightly linked [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF][START_REF] Sørensen | Cortisol reduces cell proliferation in the telencephalon of rainbow trout (Oncorhynchus mykiss)[END_REF][START_REF] Best | Maternal cortisol stimulates neurogenesis and affects larval behaviour in zebrafish[END_REF].

For example, Sadoul and colleagues [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF] have shown that high levels of mr and gr in the brain correlate with high levels of pcna and neurod1 in several fish species, including the European sea bass. Here, we also detected a strong and significant correlation (at the individual level) between genes involved in the stress response and those involved in neurogenesis (supplementary material), confirming the results of Sadoul and colleagues [START_REF] Sadoul | Enhanced brain expression of genes related to cell proliferation and neural differentiation is associated with cortisol receptor expression in fishes[END_REF]. In the present study, we show that the expression of egr1 and neurod2, which are important markers of neurogenesis involved in neural activation and differentiation, respectively [START_REF] Sørensen | Neural plasticity and stress coping in teleost fishes[END_REF][START_REF] Burmeister | Evolutionary conservation of the egr-1 immediate-early gene response in a teleost[END_REF], were higher in the brains of shy fish than in those of bold fish (only a trend for neurod2). This higher propensity to display strong neurogenesis in shy fish is suggested in the literature [START_REF] Sørensen | Neural plasticity and stress coping in teleost fishes[END_REF][START_REF] Geffroy | Nature-Based Tourism Elicits a Phenotypic Shift in the Coping Abilities of Fish[END_REF][START_REF] Van Den Bos | Further characterisation of differences between TL and AB zebrafish (Danio rerio): Gene expression, physiology and behaviour at day 5 of the larval stage[END_REF][START_REF] Øverli | On the Role of Neurogenesis and Neural Plasticity in the Evolution of Animal Personalities and Stress Coping Styles[END_REF] and may be important for learning [START_REF] Sørensen | Neural plasticity and stress coping in teleost fishes[END_REF]. This is in accordance with previous results showing that shy fish are more proficient in learning and triggering self-feeders [START_REF] Benhaim | Self-feeding behaviour and personality traits in tilapia: A comparative study between Oreochromis niloticus and Sarotherodon melanotheron[END_REF][START_REF] Ferrari | First links between self-feeding behaviour and personality traits in European seabass, Dicentrarchus labrax[END_REF].

Here, both monoamine concentrations and gene transcription analyses were performed on the whole brain, including multiple regions with specific functions and patterns of monoamine synthesis and gene transcription [START_REF] Øverli | Brain monoaminergic activity in rainbow trout selected for high and low stress responsiveness[END_REF][START_REF] Vindas | Brain cortisol receptor expression differs in Arctic charr displaying opposite coping styles[END_REF][START_REF] Vindas | Early life stress induces long-term changes in limbic areas of a teleost fish: the role of catecholamine systems in stress coping[END_REF]. Significant effects were observed using the whole brain, and therefore we could expect even more significant differences between groups in specific areas of the brain. Microdissections or dissection of the different areas of the brain would have probably lead to more robust conclusions. A refined analysis would be necessary to better understand the differential pattern of responses in the different brain areas.

Overall, European sea bass characterized by two divergent behavioral phenotypes on the boldness axis, bold and shy, also display two distinct physiological responses which could be reflect the proactive and reactive differentiation, respectively. Altogether, our results suggest that the two coping styles display differences in the HPI axis, the serotonergic and noradrenergic system reactivity, and in neurogenesis. Further, and according to our results, the behavioral responses of a social animal to a challenge in isolation seem to be relevant markers of its physiological state and capacity to regulate and react to a stressor. More data are needed to verify whether such responses are specific to one time point or if they are indicators of a lifelong coping style.

Figure 1 .

 1 Figure 1. Design of the apparatus for the (A) group risk-taking test (GRT) and (B) open field

  carried out at the 95% level of significance. Values are expressed in the text as the mean ± standard error of the mean (SEM) and illustrated in the figures using boxplots. The central line of the boxplot indicates the median and the boxes the quartiles, with the whiskers covering 95% of the values. Outliers are represented by points. First, the chi-square test was performed to compare the proportion of bold versus shy fish obtained between the GRT and OFT. Then, an exact binomial test was performed to evaluate behavioral consistency between the GRT and OFT by comparing the proportion of consistent behavior with random theoretical probability (p = 0.5) for each coping style. The remaining tests (concerning physiology and behavior) were performed on fish screened in the GRT and the OFT respectively.Normality was verified for other comparisons between coping styles on behavioral and physiological variables using the Shapiro test before applying either the Student's t-test or Wilcoxon rank sum test, depending on whether normality was verified or not. Comparisons between individual coping styles in the distance travelled, the number of transitions between shelter and OFT arena, and the time spent in the shelter were performed using the Wilcoxon rank sum test.Concerning physiological variables, two individuals were discarded due to an RNA extraction failure (no gene transcription) and therefore analyses were performed on a total of 55 individuals. The Student's t-test was performed to compare plasma cortisol concentrations and monoamine concentrations in the brain (5HT, 5HIAA, 5HIAA/5HT turnover ratio, DA, DOPAC, DOPAC/DA, and NE) between coping styles measured in the OFT. The Wilcoxon rank sum test was performed to compare the transcription of genes (gr1, gr2, mr, crf, neurod1, neurod2 and pcna) between coping styles (Student t-test for egr-1).

  7 ± 60.2 cm; W = 792, p < 0.001) and moved between shelter and open field area (2.4 ± 0.4 times; W = 0, p < 0.001); whereas the shy fish stayed in the shelter. Although the bold fish exited the shelter and were active in the open arena, most nonetheless spent most of the test time in the shelter (1,000 ± 64 s), but still for less than the shy fish (W = 792, p < 0.001, Figure 2).

Figure 2 .

 2 Figure 2. Distance travelled (cm), number of transitions between shelter and arena, and time

Figure 3 .

 3 Figure 3. Concentration (ng/g brain wet mass) of dopamine (DA), 3,4-dihydroxyphenylacetic

Figure 4 .

 4 Figure 4. Relative levels of gene transcription of the mineralocorticoid receptor (mr),

2

  was mainly driven by the latency to exit the shelter and the number of transitions between shelter and open field arena during the OFT and explained 28 % of the data variability observed (Table3). PCA axis 1 was thus rather representative of activity in the OFT with individuals displaying high values for PCA axis 1 being more active in the OFT than individuals displaying low values. PCA axis 2 was rather representative of coping styles and individuals displaying high values for PCA axis 2 scores were shyer and moved more between shelter and open field arena than individuals with low values which were bolder and moved less between the two compartments of the OFT. Because of PCA axis 2 being more representative of coping styles, further analyses were performed on PCA axis 2 scores.

Figure 5 .

 5 Figure 5. Concentration (ng/g brain wet mass) of dopamine (DA), 3,4-dihydroxyphenylacetic

Table 1 .

 1 GenBank accession numbers and primer sequences for each gene used for gene transcription analyses.

		GenBank accession
	Gene		Forward primer (5' → 3')	Reverse primer (3' → 5')
		numbers
	mr	JF824641.1	GTTCCACAAAGAGCCCCAAG	AGGAGGACTGGTGGTTGATG
	gr1	AY549305 a	GAGATTTGGCAAGACCTTGACC	ACCACACCAGGCGTACTGA
	gr2	AY619996 a	GACGCAGACCTCCACTACATTC	GCCGTTCATACTCTCAACCAC
	crf	JF274994	GCAACGGGGACTCTAACTCT	GTCAGGTCCAGGGATATCGG
	neurod1	b*	TTCTCCTTCAGCGTGCACTA	GGTGCGAGTGTCCATCAAAG
	neurod2	d*	TGCGTAAAGTGGTTCCATGC	GTCGTGGGTTGGGAGAGTC
	pcna	JQ755266 c	CAGAGCGGCTGGTTGCA	CACCAAAGTGGAGCGAACAA
	egr1	d*	AACTCCAGCCTCAGTTCCTC	AGTCAGGAATCATGGGCACA
	eef1-
		AJ866727.1	AGATGGGCTTGTTCAAGGGA	TACAGTTCCAATACCGCCGA
	alpha
	l13	DT044910.1	TCTGGAGGACTGTCAGGGGCATGC	AGACGCACAATCTTGAGAGCAG

Table 2 .

 2 Contingency table showing behavior displayed by the fish during the two tests (GRT and OFT).

	Open field test
	New shy	New bold	Total

Table 3 .

 3 Loading of behavioral variables (Latency to exit shelter, distance travelled, time spent in shelter and number of transitions between shelter and open field arena during the open field test) for the two PCA axes. Values in bold are the main drivers of the related axis.

	Variable contribution	PCA axis 1	PCA axis 2
	Latency to exit shelter	0.02	0.90
	Distance travelled	0.94	-0.18
	Time spent in shelter	-0.95	0.13
	Number of transitions	0.56	0.50
	Variability explained (%)	52	28

Acknowledgements

We are grateful to Marie-Odile Vidal, François Ruelle, and Alain Vergnet for hatching and rearing the fish. We would also like to thank Mako Pegart for valuable help with the gene transcription analyses. SA received a PhD grant from ERANET ANIHWA WINFISH (ANR-14-ANWA-0008) and Ifremer and BS received a postdoctoral grant from ERANET COFASP SUSHIFISH (ANR-15-COFA-0002-01). We also thank the anonymous reviewers for helpful comments on the previous version of the manuscript.