Adrian Chmielewski

Jaysen Nelayah

Hakim Amara

Jérôme Creuze

Damien Alloyeau

Guillaume Wang

Christian Ricolleau

Direct measurement of the surface energy of bimetallic nanoparticles: Evidence of Vegard's rule-like dependence

Keywords: numbers: 65.80.-g, 68.35.Md, 68.37.Lp

We use in situ transmission electron microscopy to monitor in real-time the evaporation of gold, copper and bimetallic copper-gold nanoparticles at high temperature. Besides, we extend the Kelvin equation to two-component systems to predict the evaporation rates of spherical liquid mono-and bimetallic nanoparticles. By linking this macroscopic model to experimental TEM data, we determine the surface energies of pure gold, pure copper, Cu50Au50 and Cu25Au75 nanoparticles in the liquid state. Our model suggests that the surface energy varies linearly with the composition in liquid Cu-Au nanoalloy, i.e. it follows a Vegard's rule-like dependence. To get atomic-scale insights into the thermodynamic properties of Cu-Au alloys on the whole composition range, we perform Monte Carlo simulations employing N -body interatomic potentials. These simulations at microscopic level confirm the Vegard's rule-like behavior of the surface energy obtained from experiments combined with macroscopic modeling.

The surface energy of materials is a very important quantity for physics and chemistry since it drives a wide range of phenomena such as phase transition, crystal melting, evaporation and growth. For nanostructures, it is even more important as it controls not only their 3D morphology and their reactivity with respect to different environments, but it also plays an essential role in Ostwald ripening as well as in the growth rate of facets during nanoparticle (NP) coarsening. Thanks to the Wulff [1] and Wulff-Kaishew [2] relations giving the equilibrium shape of free and supported crystallites, respectively, surface energy ratios for low index facets of some metals were determined [3,4]. However, absolute surface energy determination is still rather difficult. For bulk and micron-size particles, several approaches were developed based on the temperature dependence of the 3D equilibrium shape, [5][6][7][8] on the detailed study of the 2D equilibrium morphology and its thermal fluctuations [9][10][11][12][13][14] or on a combination of the two previous approaches [15].

In the case of nanosized particles, Vermaak et al. showed that surface energy and the surface stress are equal for spherical NPs as long as their properties are similar to those of liquid droplets [16,17]. This approximation is also valid if the strain rates can be considered as quasi-static at high temperatures. Several studies discussed the values of surface stress and surface energies and the origin of the discrepancies when they are different [18][19][20][21][22]. Another approach, based on the Kelvin equation [23] that relates equilibrium vapor pressure to surface curvature, was developed by Blackman et al. and Sambles et al. [24][25][26]. It derives a thermodynamic model of the evaporation of spherical liquid droplets and solid particles maintained at constant temperature. From the nonlinear relation between the time of complete evaporation and particle size, the mean surface energies of solid silver, [25], liquid lead [25] and liquid and solid gold [26] were successfully measured. Again, in NPs verifying Kelvin effect, a close approach that studies the linear relationship between the evaporation temperature and the inverse of the particle size was used to estimate the surface energies of silver [27] and PbS [28].

An alternative for determining surface energies of nanostructures is the direct computer simulation by using ab initio calculations [29][30][31][32] or semi-empirical potentials [33,34]. In order to improve the accuracy of theoretical predictions, Medasani et al. have used both techniques to calculate the surface energy of pure NPs over a wide range of particle size [22]. However, the case of bimetallic NPs at given temperatures, which is much more complex, was not addressed.

In this letter, we extend Sambles approach [25] to determine experimentally the surface energies of monometallic copper and gold NPs as well as bimetallic Cu-Au NPs by monitoring their evaporation at high temperatures by transmission electron microscopy (TEM) imaging. Adjustment of experimental TEM data by a macroscopic model based on an extended Kelvin equation suggests that the surface energy varies linearly with particle composition in liquid Cu-Au nanoalloys. This Vegard's rule-like tendency is confirmed by simulations performed at atomic-scale on the whole composition range for Cu-Au NPs in the liquid state.

TEM experiments were carried out using a JEOL JEM-ARM200F spherical aberration corrected electron microscope equipped with a cold field emission gun operated here at 80 kV and an ultra-fast OneView camera (Gatan) for TEM imaging. In situ heating was performed using a slightly modified version of the Atmosphere TEM environmental gas cell from Protochips Inc. with heating capabilities up to 1000 • C. Gold (Au), copper (Cu) and bimetallic Cu 50 Au 50 and Cu 25 Au 75 nanoparticles were synthesized by alternated pulsed laser deposition (PLD) technique in a high vacuum chamber at 10 -5 Pa. Details on the experimental procedure for particle synthesis are published elsewhere [35]. Here, the nominal thickness of metal deposited for all three specimens was fixed at 2 nm and the temperature of the SiN substrate was held at 450 • C in order to obtain spherical shaped NPs with diameter below 10 nm at high temperatures. The composition of the bimetallic alloyed NPs were determined from lattice fringe spacing measurements in HRTEM images of single NPs at room temperature prior to their heating and evaporation at high temperatures. NP deposition was carried out directly on the 50-nm thick electron transparent silicon nitride membrane of the small heating E-chip that composes the gas cell. To enhance image contrast during TEM imaging of NPs at high temperatures, the SiN membrane of the larger E-chip of the gas cell was removed. The evaporation of copper, gold, Cu 50 Au 50 and Cu 25 Au 75 nanoalloys in vacuum were studied at 725 • C, 800 • C, 700 • C and 750 • C respectively. The three samples were heated up from room temperature to these temperatures at a rate of 1 • C/s. During the heat ramps and the evaporation process, the electron beam was turned off except for TEM imaging at regular intervals. In this way, we minimised unwanted electron beam effects like local increases in the temperature of the irradiated areas [36] and/or structural modifications of the NPs that could adversely accelerate particle evaporation. In general, NPs size and shape evolve during the annealing as a consequence of Ostwald ripening and/or coalescence. The crystalline state evolves as well with the temperature from solid to liquid to gas (melting / evaporation) or directly from solid to gas (sublimation). It is well known that the melting temperature of small NPs decreases as the particle size decreases [37,38]. Here, we focus on the evaporation kinetics of NPs with radius φ < 3 nm which are all in the liquid state at high temperature (> 700 • C) as shown by their uniform image contrast and the absence of lattice fringes in their TEM images (see discussion in Sec. I of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]). We followed the variation of the NPs size as a function of time at a given temperature. For the three systems, there is a clear decrease of the particle radius with time as predicted by the Kelvin equation. [25]. When the NPs are imaged under continuous illumination, TEM observations show substantial particle sintering via dynamic diffusion and Ostwald ripening (see Fig. 2s of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]). These transformations are characteristic of electron beam-induced effects as they are observed even in the absence of any heating. Under intermittent electron illumination, particle mobility on the heated SiN support is suppressed which is a strong indication that the effect of the electron beam is largely minimized and that the evaporation of NPs observed experimentally is activated primarily by the thermal heating of the SiN support. Figures 1(a)-(d) compare the TEM images of a set of Cu NPs at 725 • C at different times over a 115.5 minutes period. These TEM images show that the NPs are completely evaporated over this period. The full followup of particle evaporation is shown in Fig. 3S of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]. The evaporation curves of a pure Au NP (•) and a pure Cu NP (•) is presented in Fig. 1(e). The uncertainties in the determination of particle radius were estimated by measuring the latter along two perpendicular directions in the TEM images. The evaporation time t of a monometallic liquid NP of radius r at a temperature T is given by (see Sec. IV and V of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]):

t(r) = B A E 0 B r -E 1 B r (1)
with:

A = P ∞ ρ l M l 2πRT 1 2 ; B = 2γM l RT ρ l and E 0 (x) = exp(-x) x ; E 1(x) = ∞ x exp(-y) y dy
where γ is the surface energy of the metal, M l the molar mass of the liquid phase, ρ l its density, P ∞ the equilibrium vapor pressure over a flat surface of the liquid and R the ideal gas constant. By fitting the experimental data in Fig. 1(c) to the Kelvin model according to Eq. 1, we extract the surface energy for liquid gold and copper NPs. We obtain a mean surface energy of 0.93 ± 0.23 J.m -2 and 2.03 ± 0.16 J.m -2 . These surface energies are averaged over four Au and three Cu NPs respectively.

To describe the evaporation of bimetallic Cu-Au NPs, we extend Sambles et al. [25] approach to spherical liquid bimetallic A-B particle under the following hypotheses. We assume that the composition of the liquid phase does not vary during the evaporation process. As a result, the compositions of liquid and vapor phases are always similar and equal to the initial composition of the liquid NP. Moreover, the bimetallic metal vapor in equilibrium with the liquid NP is considered as a mixture of ideal gases A and B. Finally, we consider that the bimetallic liquid phase behaves like an ideal liquid mixture with the partial pressure of each component following Raoult's law. As we will see later, the hypothese of an ideal liquid mixture is validated by atomic scale modeling. Under these conditions, the Kelvin equation of a bimetallic A-B alloy is given by (see Sec. V of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]):

P r P ∞ = exp 2γ(αM A + M B ) (α + 1)RT ρ A,B r (2)
where P ∞ is the total vapor pressure of the twocomponent gas system in equilibrium with a semi-infinite bimetallic A-B planar surface and P r , that of the gas in equilibrium with the curved surface of a spherical bimetallic liquid droplet of radius r at temperature T . α = n A /n B is the molar ratio, M A and M B are the molecular weights of the two metals and ρ A,B is the liquid density of the mixed compound. From the extended Kelvin equation, the evaporation time t of a bimetallic alloyed NP with radius r is also defined through Eq.( 1) but with (see Sec. V of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]):

A = P ∞ (1 + α) α ρ A M A 2πRT 1 2 + 1 ρ B M b 2πRT 1 2
(3)

B = 2γ(αM A + M B ) RT (1 + α)ρ A,B (4)
Figure 2 shows the evaporation curves at 700 • C of five Cu 50 Au 50 NPs with initial radii less than 3 nm. Adjustment of experimental evaporation curves of seven bimetallic NPs by the model based on the extended Kelvin equation gives a mean surface energy for liquid Cu 50 Au 50 nanoalloy equal to 1.530 ± 0.076 J.m -2 . This value is nearly the average between the experimentallydetermined surface energies of liquid copper and liquid gold. For Cu 25 Au 75 NPs, a mean surface energy of 1.230 ± 0.047 J/m 2 is obtained by analyzing six NPs with radii below 3 nm (Sec. VI of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF] for the corresponding evaporation curves). Thus, determination of the absolute values of the surface energy of the monometallic and bimetallic NPs suggests that the surface energy for Cu-Au nanoalloy follows a Vegard's rule-like behavior. The exact shape of the NPs are not known as TEM images only give their two-dimensional projection perpendicular to the substrate. However, the NPs are most probably greater than a hemisphere such that the experimentally-measured radii projected in the plane of the substrate are equal to their radius of curvature which appears in the Kelvin equation. Indeed, Au, Cu and Cu-Au NPs are loosely bound to the SiN support due to the hydrophobic nature of the latter. This hydrophobicity increases when the SiN film upon heating as residual moisture are taken off. As metal adhesion is reduced, the contact angles of Au, Cu and Cu-Au NPs are expected to be close to 180 • above 700 • C. On Si 3 N 4 film, Cu shows low wettability with a contact angle of about 130 • above 1000 • C [54]. When the truncation is greater or equal to a hemisphere as in our experiments, the average change in the rate of evaporation is of the order of 6 % with are within the uncertainties of the surface energies determined by our model.

To get insight at atomic-scale and investigate the thermodynamic properties of Cu-Au alloys, we perform Monte Carlo (MC) simulations in the canonical ensemble (with fixed concentrations). This approach, unlike others found in the literature [55][56][57], enables thermodynamic study over a broad-spectrum of temperatures (0 < T < 2000 K) on the whole composition range at the atomic scale. In this context, we develop a specific N -body interatomic potential derived from the second moment approximation of the tight-binding scheme(TB-SMA) [42,43,58] perfectly adapted to deal with large systems (∼1000-10000 atoms) similar to those observed experimentally. The accuracy of TB-SMA models depends critically on the validity of the database to which the parameters of the potentials are fitted. Here, we sum- marize only the crucial points of the TB-SMA potential and refer the reader to Sec. VII of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF] for more details. For pure elements, they are obtained by fitting on several bulk physical quantities (lattice parameters, cohesive energies and elastic constants). Note that fitting the TB-SMA parameters to experimental cohesive energies leads to an underestimation (∼ a factor 2) of the surface energies since it is well known that such potentials are not always adapted to describe physical properties from bulk to surface [59]. Nevertheless, these deviations do not prevent us to describe qualitatively structural properties of Cu-Au nanoalloys as we will see in the following. Improving the accuracy usually implies increasing the number of parameters, which can blur the physical transparency of the model [60][61][62]. Besides, we can notice that the Au surface energy calculated is lower than the one of Cu, in agreement with DFT calculations, which is in favor of the Au surface segregation, as already discussed in [55]. For the Cu-Au system, the potential has been fitted both to DFT calculations [63] and to the experimental bulk phase diagram [47], in particular to experimental order/disorder transition temperatures. This TB-SMA potential is then implemented in a MC code to relax the structures at finite temperatures [START_REF] Frenkel | Understanding Molecular Simulation[END_REF] (see Sec. VIII of the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]).

As a first step, we focus on liquid NPs to investigate their structural properties (physical and chemical states) by using canonical MC simulations at different temperatures. A typical example is presented in Fig. 3 where the starting configuration is a cuboctahedron containing 2507 atoms and has size of 4 nm comparable to those studied experimentally. Interestingly, a visual inspection shows no segregation effect in the liquid state. This is confirmed in a more quantitative way with the analysis of the density profiles along the radius of the NPs depicted in Fig. 3 and whatever the concentration and the size of the NPs (see Sec. VIII in the Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]). The calculation of the surface energy with the composition is therefore relevant since a disordered alloy is formed at the surface of the NP. Actually, the same phenomena can be observed for a slab (see Figure 7S(a) in Supplemental Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]). As a result, a slab will be considered in the following since the definition of γ for alloys is straightforward in this case. Indeed, surfaces can be created by dividing an infinite crystal into two parts. The energy needed to cut the bonds and bring the two resulting parts to infinity determines the surface energy :

γ = E slab -E bulk 2S , (5)
where S is the surface area. E slab and E bulk represent the total energies of the slab and the bulk containing both N atoms at different concentrations, respectively. Here, we use Monte Carlo simulations to calculate these quantities at temperatures where the system is liquid. In the present calculations, the surface energy converges properly for large enough slabs. More precisely, we consider a slab size equal to 36.14 × 36.51 × 36.90 Å3 containing 2520 atoms. As shown in Fig. 4 where two temperatures are considered, surface energies vary linearly with the composition of the alloy following the theoretical Vegard's rule-like behavior. Unexpectedly, this is not the case for E slab and E bulk (see Figure 7S(b) in Suppplementary Material [START_REF]for additional TEM data, the generalization of Kelvin equation to a twocomponent sytem and derivation of the evaporation ki[END_REF]). Moreover, by taking into account the factor 2 discussed previously, experimental and theoretical data are in very good agreement. Therefore, it can be concluded that our model based on the Kelvin equation is robust to determine the surface energy of an alloy for all concentrations and not only for the equiatomic composition. In summary, by combining in situ TEM observations of the thermally-activated evaporation of Au, Cu and Cu 50 Au 50 NPs and an extended Kelvin equation for twocomponent systems, we have determined the surface energy of these metals in the liquid phase. Our results show that the surface energy of liquid bimetallic Cu-Au nanoalloys follows a Vegard's rule-like dependence. This dependence was confirmed by Monte Carlo simulations using an optimized tight binding TB-SMA potential that described very well the thermodynamic properties of the Cu-Au nanoalloy. This work constitutes a major step in the determination of surface energy of alloyed nanoparticles experimentally which is still a challenge.

The authors acknowledge the Région Ile-de-France for convention SESAME E1845 for the support of the JEOL JEM-ARM200F installed at Paris Diderot University. J.C and H.A. thank F. Ducastelle and B. Legrand for fruitful discussions.

FIG. 1 .

 1 FIG. 1. (a)-(d) In situ TEM monitoring of the evaporation of a set of Cu NPs at 725 • C (e) Evaporation curves of a Au NP at 800 • C and a Cu NP (indicated by a white arrow in (a)-(d)) at 725 • C. Circles correspond to the experimental data adjusted by the evaporation of single-component system obtained from Kelvin equation (full line). Values of the surface energy extracted from the fitting of experimental data are given in insert.

FIG. 2 .

 2 FIG. 2. Evaporation curves of liquid Cu0.5Au0.5 NPs at 700 • C. Experimental data (circles) are modeled by the extended Kelvin equation for two-component system (full line).

  FIG. 3. Properties of Cu50Au50 NPs calculated for MC runs at (a) 300 K and (b) 1500 K. (left) Equilibrium configurations (right) Density and composition profiles along the radius of the NP (orange: Au, blue: Cu).

2 FIG. 4 .

 24 FIG. 4. Variation of the surface energy of Au1-xCux systems in the liquid state as a function of the Cu composition. The full black line is a linear fit of experimental data.


Mélanie Blanc

Philipp Antczak

Xavier Cousin

Christoph Grunau

Nikolai Scherbak

Joëlle Rüegg

Steffen H Keiter

The insecticide permethrin induces transgenerational behavioral changes linked to transcriptomic and epigenetic alterations in zebrafish (Danio rerio)

Keywords: Transgenerational epigenetic inheritance, developmental neurotoxicity, glutamatergic signaling, DNA methylation, pesticide

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

Introduction

Permethrin is one of the most common domestic and agricultural pyrethroid insecticides worldwide [START_REF] Demars | Assessment of Pyrethroid Contamination and Potential Mitigation Strategies in California Central Coast Surface Waters[END_REF][START_REF] Banks | Insecticide-Treated Clothes for the Control of Vector-Borne Diseases: A Review on Effectiveness and Safety[END_REF]. It is considered a safer alternative compared to many other pesticides since permethrin does not accumulate in the environment and is quickly metabolized and excreted by organisms [START_REF] Holmstead | Pyrethroid photodecomposition: permethrin[END_REF][START_REF] Imgrund | Environmental fate of permethrin[END_REF][START_REF] Zhu | Photodegradation Kinetics, Mechanism and Aquatic Toxicity of Deltamethrin, Permethrin and Dihaloacetylated Heterocyclic Pyrethroids[END_REF]. Nonetheless, a recent study showed that photodegradation products of permethrin can potentially be more harmful to aquatic organisms than the parent compound [START_REF] Zhu | Photodegradation Kinetics, Mechanism and Aquatic Toxicity of Deltamethrin, Permethrin and Dihaloacetylated Heterocyclic Pyrethroids[END_REF]. Due to increasing usage, pyrethroids are detected in human fluids in the low µg/L range [START_REF] Barr | Urinary Concentrations of Metabolites of Pyrethroid Insecticides in the General U.S. Population: National Health and Nutrition Examination Survey 1999-2002[END_REF][START_REF] Saillenfait | Pyrethroids: Exposure and health effects -An update[END_REF], and in aquatic compartments in the ng/L to high µg/L range [START_REF] Hladik | Assessing the occurence and distribution of pyrethroids in water and suspended sediments[END_REF][START_REF] Shahsavari | Groundwater pesticides residue in the southwest of Iran-Shushtar plain[END_REF][START_REF] Budd | An evaluation of temporal and spatial trends of pyrethroid concentrations in California surface waters[END_REF].

Exposure to permethrin can elicit a variety of adverse effects in various species, including neurotoxic effects and behavioral deficits [START_REF] Carloni | The impact of early life permethrin exposure on development of neurodegeneration in adulthood[END_REF][START_REF] Demicco | Developmental Neurotoxicity of Pyrethroid Insecticides in Zebrafish Embryos[END_REF][START_REF] Fedeli | In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats[END_REF][START_REF] Nunes | Acute Exposure to Permethrin Modulates Behavioral Functions, Redox, and Bioenergetics Parameters and Induces DNA Damage and Cell Death in Larval Zebrafish[END_REF][START_REF] Saito | Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice[END_REF][START_REF] Yang | Joint toxicity of permethrin and cypermethrin at sublethal concentrations to the embryo-larval zebrafish[END_REF][START_REF] Furlong | Prenatal exposure to pyrethroid pesticides and childhood behavior and executive functioning[END_REF][START_REF] Shelton | Neurodevelopmental Disorders and Prenatal Residential Proximity to Agricultural Pesticides: The CHARGE Study[END_REF], as well as oxidative stress [START_REF] Carloni | The impact of early life permethrin exposure on development of neurodegeneration in adulthood[END_REF][START_REF] Vadhana | Early life permethrin treatment leads to long-term cardiotoxicity[END_REF][START_REF] Nunes | Acute Exposure to Permethrin Modulates Behavioral Functions, Redox, and Bioenergetics Parameters and Induces DNA Damage and Cell Death in Larval Zebrafish[END_REF]. Permethrin has also been shown to act as an endocrine disrupter in fish [START_REF] Tu | Permethrin is a potential thyroid-disrupting chemical: In vivo and in silico envidence[END_REF][START_REF] Zhang | Environmentally relevant levels of λ-cyhalothrin, fenvalerate, and permethrin cause developmental toxicity and disrupt endocrine system in zebrafish (Danio rerio) embryo[END_REF].

In a previous study, we showed that early-life exposure of zebrafish (F0) to permethrin induces transgenerational disruption of larvae behavior in the F1 and F2 generations [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF].

Permethrin is not known to be mutagenic [START_REF] Pluijmen | Lack of Mutagenicity of Synthetic Pyrethroids in Salmonella Typhimurium Strains and in V79 Chinese Hamster Cells[END_REF][START_REF] Pednekar | Evaluation of Mutagenic Activities of Endosulfan, Phosalone, Malathion, and Permethrin, before and after Metabolic Activation, in the AmesSalmonella Test[END_REF], thus, transgenerational effects are more likely to be a consequence of chemically-induced, inherited changes in epigenetic patterns [START_REF] Nilsson | Chapter 2 -Definition of epigenetic transgenerational inheritance and biological impacts[END_REF]. Epigenetic mechanisms are mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence, and which can further lead to alterations of cell function and impair the physiology of an organism [START_REF] Holliday | Epigenetics: A Historical Overview[END_REF]. During fetal development and early life, exposure to epigenetic modifiers is of concern due to high epigenetic plasticity that can affect normal and pathological development later on [START_REF] Baccarelli | Epigenetics and environmental chemicals[END_REF][START_REF] Guerrero-Preston | Global DNA hypomethylation is associated with in utero exposure to cotinine and perfluorinated alkyl compounds[END_REF][START_REF] Stel | The Role of Epigenetics in the Latent Effects of Early Life Exposure to Obesogenic Endocrine Disrupting Chemicals[END_REF][START_REF] Vaiserman | Early-life Exposure to Endocrine Disrupting Chemicals and Later-life Health Outcomes: An Epigenetic Bridge?[END_REF]. Additionally, cancer, neurological disorders, and several other conditions have been associated with changes in the epigenetic landscape and were linked to chemical exposure [START_REF] Grandjean | Only One Chance: How Environmental Pollution Impairs Brain Development and How to Protect the Brains of Next Generation[END_REF][START_REF] Prusinski | Developmental exposure to endocrine disrupting chemicals alters the epigenome: Identification of reprogrammed targets[END_REF][START_REF] Zoghbi | Epigenetics and Human Disease[END_REF]. Some of these epigenetic changes may be inherited, and thereby induce adversities in the unexposed offspring, a phenomenon known as transgenerational epigenetic inheritance [START_REF] Liberman | Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms[END_REF][START_REF] Skinner | Epigenetic transgenerational actions of environmental factors in disease etiology[END_REF][START_REF] Skinner | What is an epigenetic transgenerational phenotype? F3 or F2[END_REF]. For now, most evidence is based on persistent DNA methylation changes in germ cells of exposed individuals, that can escape the epigenetic reprogramming events occurring at fertilization and sex differentiation.

They are further transferred to somatic and germ cells of the developing offspring, and remain «imprinted» in the epigenome over generations [START_REF] Liberman | Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms[END_REF][START_REF] Skinner | Environmental epigenetic transgenerational inheritance and somatic epigenetic mitotic stability[END_REF].

However, there are studies showing that other epigenetic marks, such as histone posttranslational modifications and non-coding RNA regulation, can also be inherited and likely work in concert to support inheritance phenomena [START_REF] Blake | Unravelling the complex mechanisms of transgenerational epigenetic inheritance[END_REF].

A recent work found significant associations between pyrethroid exposure and DNA methylation changes in human blood [START_REF] Furlong | An epigenome-wide association study of ambient pyrethroid pesticide exposures in California's central valley[END_REF]. In addition, previous studies have reported that permethrin affects expression of DNA methyltransferases, the enzymes in charge of DNA methylation, in the striatum of exposed rats [START_REF] Fedeli | In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats[END_REF] and in zebrafish embryos, along with other epigenetic factors [START_REF] Blanc | Environmental chemicals differentially affect epigeneticrelated mechanisms in the zebrafish liver (ZF-L) cell line and in zebrafish embryos[END_REF]. Furthermore, global DNA methylation changes have been observed in the F1 offspring upon permethrin exposure [START_REF] Bordoni | Intergenerational Effect of Early Life Exposure to Permethrin: Changes in Global DNA Methylation and in Nurr1 Gene Expression[END_REF]. Finally, specific changes in DNA methylation patterns in sperm were linked to reproductive abnormalities in the F3 offspring of rats exposed to a mixture of permethrin and the insect repellent N,N-diethyl-meta-toluamide (DEET) (Manikkam et al., 2012a).

Zebrafish is a prominent model organism for ecotoxicological and biomedical studies and is used for various routine testing for the risk assessment of chemicals and complex environmental samples [START_REF] Scholz | The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing[END_REF]. Zebrafish have genetic similarities to mammals and epigenetic pathways are also mostly conserved with some mechanistic differences such as the lack of parental imprinting [START_REF] Kamstra | Zebrafish as a model to study the role of DNA methylation in environmental toxicology[END_REF]. Besides, zebrafish are used for several established behavioral tests to assess different endpoints. For example, the novel tank allows to evaluate the activity and exploratory behavior of the fish, which may reflect alterations in anxiety [START_REF] Egan | Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish[END_REF][START_REF] Sackerman | Zebrafish Behavior in Novel Environments: Effects of Acute Exposure to Anxiolytic Compounds and Choice of Danio rerio Line[END_REF]. Due to their short life cycle, zebrafish is a model for transgenerational inheritance research [START_REF] Bugel | Zebrafish: A Marvel of High-Throughput Biology for 21st Century Toxicology[END_REF]Kamstra et al., 2014), and previous data showed that transgenerational effects observed in the zebrafish were similar to that observed in mammals (Baker et al., 2014a).

So far, no link between behavior, genetic, and potential epigenetic changes has been investigated following permethrin exposure. There is also no information available on whether these changes may persist in offspring generations and lead to transgenerational effects. In this study, we combined investigations at three different levels of biological organization in F0, F1 and F2 generations of zebrafish. To this end, we investigated in the same fish (1) the presence of a transgenerational behavioral phenotype in adults, (2) whole-brain transcriptomic changes and (3) differentially methylated regions (DMRs) that may be implicated in the heritable phenotype.

Material and Methods

Fish exposure and sampling

This work has received approval for research ethics from the Swedish Board of Agriculture, Jönköping, Sweden (#5.2.18-861/15), and a certificate of approval is available upon request.

The exposure of the F0 generation and breeding F0, F1, and F2 fish were performed as described previously [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF] and additional information is available in supplementary data (Text S1). Briefly, 2 hours-post-fertilization (hpf) AB zebrafish embryos (ZFIN ID: ZDB-GENO-960809-7) were transferred to exposure solutions containing 1 µg/L (permethrin low; PL), 10 µg/L (permethrin high; PH) permethrin (Sigma-Aldrich, PESTANAL analytical standard, purity >90% cis+trans isomers), or dimethylsulfoxide 0.01% (DMSO, Sigma-Aldrich) as solvent control (SC), and kept exposed in a flow-through system until 28 days post-fertilization. 1 µg/L is in the range of the highest permethrin concentrations found in surface water as it tends to bind to sediments where concentrations up to 20 µg/kg can be found (NORMAN EMPODAT Database, 2020). We have further demonstrated that a 28 day exposure to both 1 and 10 µg/L did not induce any observable defect on growth or survival [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF]. Chemical analysis was performed on water samples at day 0, 4, 14, 21 and 28. The results, presented in [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF], excluded cross-contamination of the solvent controls and confirmed exposure, although measured concentrations were approx. 10 times lower than the nominal ones [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF].

At 28 days post-fertilization, juveniles were transferred into clean water and bred according to standard protocols [START_REF] Westerfield | A guide for the laboratory use of zebrafish (Danio rerio). The zebrafish book[END_REF]. The F1 and F2 generations were produced by mating exposed F0 males and females and were maintained under standard (unexposed) conditions.

When they reached 4 months of age, fish were euthanized in saturated ethyl 4-aminobenzoate solution (500 mg/L, Sigma-Aldrich) and individual brains (n=4 per treatment and per sex) were dissected and snap-frozen in liquid nitrogen, then kept at -80°C until processing.

Novel tank diving test

For F0 to F2 generations, locomotor and exploratory behavior in response to a novel environment was investigated in 8-14 fish per sex and condition. Fish were tested at the age of 9 months pf (mpf; F0), 7 mpf (F1) and 8 mpf (F2). The novel tank diving test is commonly used to identify any impact on activity and anxiety level of fish via analysis of travelled distance and vertical positioning [START_REF] Levin | Anxiolytic effects of nicotine in zebrafish[END_REF]. Fish were transferred to the recording room the day before testing. They were kept in individual 3 L tanks containing system water, in visual contact with each other. Testing was performed between 1 and 5 p.m. Each individual was transferred to a 1.5 L trapezoid tank and vertical positioning was immediately recorded (Basler® acA1300 camera) for 4 min. The test duration was reduced to take into account the absence of prior incubation and the quicker relief of anxiety compared to what was initially described [START_REF] Levin | Anxiolytic effects of nicotine in zebrafish[END_REF]. Water was exchanged between each run to avoid inter-individual interferences. All behavioral data were automatically analyzed using Ethovision XT 13.0 (Noldus). The tank was divided into 3 virtual zones: top, middle and bottom [START_REF] Sackerman | Zebrafish Behavior in Novel Environments: Effects of Acute Exposure to Anxiolytic Compounds and Choice of Danio rerio Line[END_REF]. A list of measured exploratory variables is presented in Table S1. Statistical analyses were performed using Principal Component Analysis (PCA) (package factoextra, R v3.6.1) on sex-specific datasets (each including 10 metrics, 3 generations, 3 treatments) as preliminary analyses revealed that the sex was a significantly contributing factor (Text S2). One-way Analysis of Variance followed by Bonferroni´s Multiple Comparison test was applied on major principal components (PC) as well as on a selection of individual variables related to exploratory behavior (time spent in top third zone; distance travelled in top third zone; average entry duration) and defensive innate behavior (freezing as the frequency of events at a speed < 1% of the running average; angular velocity as a mean to represent erratic movements via sharp changes in direction). Data were checked for the presence of significant outliers (Grubbs' test, p≤0.05). A p-value (p)<0.05 was considered as statistically significant.

RNA-Sequencing and qPCR validation

Total DNA and RNA from four brains per treatment (PH or SC), generation, and sex were consecutively extracted for Reduced Representative Bisulfite Sequencing (RRBS) and RNA-Sequencing (RNA-Seq), respectively, using the Triprep extraction kit (Macherey-Nagel) according to the manufacturer's instructions. Quality and quantity were spectrophotometrically evaluated using a Biodrop μLITE (BioDrop, UK). One µg total RNA per sample was sent to BGI Europe for RNA sequencing. Library preparation and sequencing were handled by BGI Europe on BGISeq-500 as paired-end 150 bp reads.

We performed technical validation of the RNA-Seq results using qPCR. Within each generation, a set of genes with different biological functions was selected. Both strongly (absolute fold-change ≥ 2) and moderately regulated (absolute fold-change < 2) genes in RNA-Seq analyses were included. Comparisons were performed within generation/sex specific subdatasets (see Table S3 for full results). Primers (Eurofins Genomics, Germany) were designed using Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) (Table S2). cDNA was synthetized from 400-500 ng of RNA template and each qPCR reaction included 2X SYBR® FAST MasterMix (KAPA Biosystems), 200 nM of each primer, 2 μL of 1/10 diluted cDNA completed to 12 μL with MilliQ water. Potential gDNA contamination was controlled with purified RNA (no reverse transcriptase control) and each reaction was run in technical duplicates. Results were normalized to the expression of rpl13a and b2m as they showed the highest stability among 4 investigated genes (i.e. eef1a1, b2m, actb1 and rpl13a) (data not shown). Fold-changes were calculated using the Pfaffl method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF]. The results from the validation are available in Table S3 and revealed an overall correlation of 86% between fold-changes from both techniques (Pearson correlation).

Reduced Representative Bisulfite Sequencing (RRBS)

RRBS experiments were performed at the Environmental Epigenetics facility of the IHPE research unit, to identify differentially methylated cytosines and differentially methylated regions (DMRs) on DNA [START_REF] Meissner | Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis[END_REF]. 100 ng of purified DNA was digested with MspI and libraries were prepared for each sample using the Diagenode RRBS kit Cat No C02030033 (Diagenode, Belgium), according to the manufacturer's protocol. DNA-free water served as negative control. The 48 samples were blindly divided into eight pools to create the libraries as described in the Diagenode protocol. qPCR was used to determine the optimal number of amplification cycles. Relative fluorescence was plotted versus cycle number and the cycle number that corresponds to roughly one-third of the maximum fluorescent intensity was used for PCR amplification (19 cycles for all pools of libraries). Five µl of the PCR products were used to check quality and quantity of libraries by electrophoresis on a 1.2% agarose gel and Midori green direct staining. Purification was done on an IP-Star system (Diagenode) with Ampure XP beads (Beckman Coulter, USA). Libraries were resuspended in 20 µL of elution buffer, and size distribution and concentration were profiled with an Agilent Bioanalyzer High Sensitivity DNA Assay. Libraries were sequenced on two NextSeq550 High Output flow cells (Illumina, USA) as paired-end or single-end 150 bp reads. Randomization was applied to dispatch samples from different generations/sex/treatments on each flow cell.

Bioinformatic analyses

RNA-Sequencing data analysis

Quality of the fastq files was checked using the FastQC software (v0.11.8) [START_REF] Andrews | FastQC: a quality control tool for high throughput sequence data[END_REF] and reads were mapped to the last version of the zebrafish genome (Ensembl GRCz11, annotation GRCz11.97) with StAR (v2.7.0) [START_REF] Dobin | STAR: ultrafast universal RNAseq aligner[END_REF]. QC statistics are available in Excel Table S1. Read count was performed with HTSeq (v0.11.2) [START_REF] Anders | HTSeq-a Python framework to work with high-throughput sequencing data[END_REF] and differential expression analysis with DESeq2 (v1.24.0) [START_REF] Love | Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2[END_REF]. A gene showing an adjusted p-value for multiple comparisons (padj)≤0.05 (Benjamini-Hochberg correction) was considered as a significantly differentially expressed gene (DEG). Gene Set Enrichment Analysis (GSEA v4.0.3) was used for functional enrichment against Gene Ontology (GO, 2019-07-31) and REACTOME (2019-10-02) databases [START_REF] Reimand | Pathway enrichment analysis and visualization of omics data using g:Profiler, GSEA, Cytoscape and EnrichmentMap[END_REF][START_REF] Subramanian | Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles[END_REF]. The ranking metric used in GSEA analyses was the Walt statistic given by DESeq2 [START_REF] Esteve-Codina | Chapter Four -RNA-Seq Data Analysis, Applications and Challenges[END_REF]. At first, a gene set showing a padj≤0.05 and abs (normalized enrichment score) (NES)>1.5 was considered as significantly enriched. The statistical threshold was further relaxed to a padj≤0.25 to include borderline yet biologically relevant findings to the analysis, as advised by GSEA documentation.

RRBS data analysis

All analyses were done on the Galaxy instance of the UMR 5244 -"Interactions Hôtes-Pathogènes-Environnements" laboratory http://bioinfo.univ-perp.fr [START_REF] Afgan | The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2018 update[END_REF].

Quality of the sequencing data was evaluated using FastQC (v0.71), and one replicate from F0 males was discarded due to poor sequencing quality. Adaptors were automatically trimmed using TrimGalore! (v0.4.3.1) (Krueger, 2012) and reads were aligned with Bismark (v0.22.1) [START_REF] Krueger | Bismark: a flexible aligner and methylation caller for Bisulfite-Seq applications[END_REF] to the GRCz11 version of the zebrafish genome, with the adjusted parameter "L,0,-0.6" for optimal alignment [START_REF] Kamstra | Differential DNA methylation at conserved non-genic elements and evidence for transgenerational inheritance following developmental exposure to mono(2ethylhexyl) phthalate and 5-azacytidine in zebrafish[END_REF]. Methylation calling was performed with Bismark extractor. QC statistics are available in the Excel Table S8. Data were normalized to median read count and cytosine positions showing a coverage below 5 and above the 99.9 percentile were discarded. At this stage, we observed a batch effect between the two flow cells that were used in the experiment (Figure S1A). The built-in methylKit batch correction (R package v1.12) [START_REF] Akalin | MethylKit: a comprehensive R package for the analysis of genome-wide DNA methylation profiles[END_REF] is based on a principal component association of available metadata. This type of batch correction has limited application, particularly with high dimensional datasets, as it focuses on the removal of a whole principal component that may contain more than just variation associated to batch. For this reason, the authors provided ways of extracting and reimporting batch-corrected values into their framework. We tested the built-in version and found that overall batch specific differences were still observed (Figure S1B). Currently, no fully appropriate batch correction methodology has been published for methylation data. For this reason, we extracted the percentage of methylation for each identified position and used an Empirical Bayes Framework previously developed to correct for batch effects in continuous datasets, ComBat [START_REF] Johnson | Adjusting batch effects in microarray expression data using empirical Bayes methods[END_REF]. We provided the methodology with the batch information as well as the model matrix relating to sex, treatment, and generation of the fish to remove as much technical information as possible while preserving biological information (Figure S1C). The resulting matrix was rescaled to 0-100 to be integrated back into a methylKit object. To test whether the correction removed any biological information, we compared the results from a within batch differentially methylated position analysis. This showed that 100% of the regions identified after correction were also identified in the dataset before correction and that there was a >97% correlation between differential methylation values from both datasets (Figure S2), showing that the batch correction was successful and did not create false-positive DMRs. However, the normalization step reduced the number of significant DMRs by a factor of approximately 5, potentially leading to underestimation of the amount of methylation changes. The output was used in methylkit to perform differential analyses on artificially segmented 300 bp tiles [START_REF] Kamstra | Differential DNA methylation at conserved non-genic elements and evidence for transgenerational inheritance following developmental exposure to mono(2ethylhexyl) phthalate and 5-azacytidine in zebrafish[END_REF]. DMRs were defined as 300 bp tiles including at least 4 cytosines showing a 5X coverage. An initial cut-off of 10% differential methylation and padj≤0.05 was used for statistical significance, which was further relaxed to a padj≤0.25 as for RNA-Seq data in order to discuss biologically meaningful findings. Significant DMRs were visualized in the Integrative Genome Viewer (IGV v. 2.8.0) [START_REF] Robinson | Integrative genomics viewer[END_REF] to compare methylation levels of specific cytosine positions between the different generations.

DMRs were associated to the closest transcriptional start site using the genomation package with default parameters (v. 1.18.0) [START_REF] Akalin | Genomation: a toolkit to summarize, annotate and visualize genomic intervals[END_REF]. Functional enrichment was performed using gprofiler2 (v.0.1.9) [START_REF] Reimand | gProfileR: Interface to the'g: Profiler'Toolkit[END_REF] against the Gene Ontology and Reactome databases. A custom background containing associated genes from the full list of 300 bp tiles was used. A gene set was considered significantly enriched at a padj≤0.05.

Both RNA-Seq and RRBS data have been deposited in NCBI's Gene Expression Omnibus [START_REF] Edgar | Gene Expression Omnibus: NCBI gene expression and hybridization array data repository[END_REF] and are accessible through GEO Series accession number GSE154020 (reviewer access token czwvcykovjajxkj) and GSE154206 (reviewer access token sxuhiycgbryxhkv), respectively.

Results

Behavioral effects

Behavioral analyses were performed in permethrin and control fish from all generations using the novel tank diving test. PCA analysis yielded one major component (PC1) representing most of the explanatory variables (i.e., global locomotor activity) and explaining 46% and 53% of the total variability in behavior in males and females, respectively (Table S4). Positive scores were assigned to high exploratory and locomotor activity. In F0 males exposed to 10 µg/L (PH), we observed a significant reduction in PC1, i.e. a lower locomotor activity compared to control fish (p=0.024, Figure 1A). This was not seen in F0 males from the lower exposure dose (PL).

Although not significant, F0 females showed the same tendency (p=0.070, Figure S3). No effect on global locomotor activity was observed in the F1 and F2 generations (Figure 1A,S3).

In males, but not in females, PC2 specifically described anxiety-like behavior (distance travelled, and time spent in top third area) and explained 19% of the total variability in behavior (Table S4). Positive PC2 scores were assigned to reduced anxiety. Significant reduction in anxiety-like behavior was shown in PH F2 males (p=0.030) and the same tendency was observed in PH F1 males (p=0.094; Figure 1B). PH F1 males specifically displayed a significant increase in the total distance travelled in Top area (p=0.035; Figure 2A) and an increase in the time spent in this zone (p=0.070; Figure 2B). Same trends were observed for PL F1 males (p=0.089 for distance in Top and p=0.082 for time in Top). PH F2 males showed a significant increase in the time spent in Top area (p=0.048) but no change in the distance travelled in this zone compared to controls (p=0.293; Figure 2B). No effect was observed in PL F2 males.

Besides, the data collected did not indicate any difference in average entry duration (Figure S4), nor in freezing episodes and erratic movement frequency as markers for defensive innate behavior measurements (Figure S5-S6). Overall, exploratory behavior endpoints suggest that both F1 and F2 PH males were less anxious than their SC congeners. No effect on behavior was observed in F1 and F2 females (Figure S4-S8).

Transcriptional changes in zebrafish brain

RNA-Seq analyses were performed in zebrafish whole brains from each generation (F0, F1, F2) to investigate transcriptomic changes that may be associated with the behavioral defects. The results showed an increasing number of significant differentially expressed genes (DEGs) and enriched gene sets in permethrin-exposed fish compared to controls from the F0 to F2 generation (Figure 3). Overall, there was little overlap in significant DEGs or pathways between generations (Figure 3B-C). In the F0 generation, a limited number of genes, 22 in males and 27 in females, were significantly dysregulated (Figure 3). Nonetheless, GSEA analysis revealed 95 and 69 gene sets significantly enriched in male and female, respectively (Figure 3); out of this, 19 were common to both sexes and related to transcription/translation processes, eye development, and glutamatergic synaptic activity (unblocking of NMDA receptors, glutamate binding and activation) (Figure S9). Further, in zebrafish F0 males specifically, transcriptional upregulation of NMDA receptors was accompanied by upregulation of genes linked to AMPA glutamate receptor activity (Table 1).

In the F1 and F2 generations, we observed larger transcriptomic changes in females compared to males, even though the measured behavioral changes were only observed in the latter. At first, no significant effect on glutamatergic neural circuits was observed in the F1 or F2 generation (padj≤0.05). However, when relaxing the statistical cut-off to an acceptable threshold for GSEA studies (p≤0.05, padj≤0.25) [START_REF] Subramanian | Gene set enrichment analysis: A knowledge-based approach for interpreting genome-wide expression profiles[END_REF], we observed transcriptomic enrichment in gene sets related to glutamatergic receptor activity in all generations of males, but not in females (Table 1). The persistence of this effect was specific to glutamatergic signaling as other enriched gene sets were different between generations (Excel Tables S15-S17). The direction of regulation was however opposite between F0/F1 (up) and F2 (down) (Table 1).

Of note, we also reported significant transcriptomic enrichment in epigenetic mechanisms: histone acetylation (F0_M, F2_M, F1_F, F2_F) and histone methylation/demethylation (F0_F, F1_F) pathways (Figure S10).

3.3.Epigenetic alterations in zebrafish brain

As a possible mechanism for inheritance of behavioral defects, we investigated DNA methylation changes induced by permethrin exposure, and their stability over generations in zebrafish brain using RRBS. We observed a generation-specific clustering (F0 vs F1-F2) of the methylation profiles that was independent of sex and treatment (Figure S1C). This may be explained by the higher genetic and epigenetic variability among the F0 fish group compared to F1-F2 groups due to the breeding set-up, since we included a higher number of contributing spawns in the F0 generation compared to F1 and F2 (18 vs 7) [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF]. This, however, did not prevent from assessing intra-generation and inter-condition changes in DNA methylation and further compare DMRs between generations.

We could identify several DMRs within each generation, including significant overlaps between generations (Figure 4). In the F0 generation, 110 and 121 DMRs (out of 17456 identified regions) were identified in males and females, respectively, out of which 28 were common to both sexes (Table S5).

When looking at DMR inheritance, a total of 13 and 8 DMRs were overlapping between all generations in males and females, respectively (Figure 4, Table S6). In males, these overlapping DMRs were consistently either hyper-or hypomethylated across generations with only one exception, whereas results in females suggest more complex interactions with half of the DMRs being hypomethylated in one generation and hypermethylated in the next (Figure 5).

Visualization of all inherited regions using the Integrative Genome Viewer confirmed that the most robust DMR in both sexes was on chr24 and associated to vim, encoding for a structural protein expressed in all body cell types, including in brain glial cells [START_REF] Schnitzer | Immunocytochemical demonstration of vimentin in astrocytes and ependymal cells of developing and adult mouse nervous system[END_REF][START_REF] Cerdà | Zebrafish vimentin: molecular characterization, assembly properties and developmental expression[END_REF] (Figure S11). This region showed inheritance of epigenetic alteration of eight cytosine positions from F0 to F2 generations. Within the list of genes associated with inherited DMRs in males only, from F0 to F2 generations, fmr1 (chr14, translational regulator within the nervous system), and pnocb (chr20, regulation of nociception via opioid-receptor binding activity) appeared as interesting candidates as they may be relevant to behavioral alterations [START_REF] Demin | Zebrafish models relevant to studying central opioid and endocannabinoid systems[END_REF][START_REF] Ng | Behavioral and Synaptic Circuit Features in a Zebrafish Model of Fragile X Syndrome[END_REF] (Figure S12-S13). Whereas fmr1 showed inheritance of DNA hypermethylation at the exact same cytosine positions, effects on pnocbassociated DMR were involving different cytosines and directions of regulation across generations (Figure S13).

We further performed pathway analysis using the list of genes associated to the DMRs (Figure S14). In F0 males, the gene set "AMPA glutamate receptor activity" was significantly enriched, as observed in RNA-Seq data (Table S7). When slightly relaxing the statistical cut-off (p≤0.05, padj≤0.25), the results indicated that DNA methylation changes were linked to additional pathways related to glutamatergic signaling in F0 and F2 males, but this could not be identified in F1 (Table S7).

Discussion

Developmental exposure to pyrethroids such as permethrin has been shown to induce neurobehavioral deficits in various species [START_REF] Nunes | Acute Exposure to Permethrin Modulates Behavioral Functions, Redox, and Bioenergetics Parameters and Induces DNA Damage and Cell Death in Larval Zebrafish[END_REF][START_REF] Saito | Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice[END_REF][START_REF] Furlong | Prenatal exposure to pyrethroid pesticides and childhood behavior and executive functioning[END_REF][START_REF] Shelton | Neurodevelopmental Disorders and Prenatal Residential Proximity to Agricultural Pesticides: The CHARGE Study[END_REF]. However, if and how these effects are inherited across generations is unknown. In this study, we show that early-life exposure of zebrafish to permethrin induces transgenerational behavioral alterations as well as persistent epigenetic and transcriptional modifications that are linked to glutamatergic signaling disruption.

Permethrin exposure induced a late onset reduction in overall swimming activity, findings that are in line with a recent study performed in zebrafish [START_REF] Nunes | Acute embryonic exposure of zebrafish to permethrin induces behavioral changes related to anxiety and aggressiveness in adulthood[END_REF]. The results further showed that males were more sensitive, as previously observed in mammals [START_REF] Imanishi | Prenatal exposure to permethrin influences vascular development of fetal brain and adult behavior in mice offspring[END_REF][START_REF] Saito | Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice[END_REF].

Acute exposure to permethrin induces overactivation of voltage-gated sodium channels in the nervous system, which in turn leads to constant depolarization of nerve cells, increase in the excitatory postsynaptic potential, tremors, and further paralysis and death (Vijverberg and van den Bercken, 1990). Sodium channels of invertebrates and fish are particularly sensitive to acute pyrethroid exposure compared to mammals [START_REF] Bradbury | Comparative toxicology of the pyrethroid insecticides[END_REF], which makes pyrethroid use a specific threat to aquatic communities. However, mechanisms underlying delayed neurotoxicity after low-dose exposure to permethrin are not well known and were poorly investigated in fish. In mammals, possible molecular changes were linked to neurodegeneration and synaptic morphology [START_REF] Carloni | The impact of early life permethrin exposure on development of neurodegeneration in adulthood[END_REF][START_REF] Carloni | Early life permethrin exposure induces long-term brain changes in Nurr1, NF-kB and Nrf-2[END_REF]. In addition, early-life exposure of rodents to permethrin was shown to impair glutamatergic signaling in vitro and in vivo [START_REF] Carloni | The impact of early life permethrin exposure on development of neurodegeneration in adulthood[END_REF][START_REF] Shafer | Complete inhibition of spontaneous activity in neuronal networks in vitro by deltamethrin and permethrin[END_REF], and another study linked overactivation of glutamatergic NMDA receptors to a depressive-like behavior (i.e. reduced mobility) in rats [START_REF] Cattani | Developmental exposure to glyphosate-based herbicide and depressive-like behavior in adult offspring: Implication of glutamate excitotoxicity and oxidative stress[END_REF]. Altogether, this indicates that upregulation of genes related to glutamatergic synaptic activity may support the behavioral effects observed in the F0 generation.

Further, we showed for the first time that the indirectly exposed F1 and the unexposed F2 generations of male offspring were also affected, as they displayed a reduction in their anxiety-like behavior. While the data did not indicate a change in defensive innate behavior endpoints, other parameters that relate to anxiety, such as time spent and/or distance swam in the Top area of the tank, were altered [START_REF] Cachat | Measuring Behavioral and Endocrine Responses to Novelty Stress in Adult Zebrafish[END_REF]. This effect was significant in the highest tested concentration, whereas there was only a trend suggesting that similar alterations may happen in the low concentration. This result supports the presence of a transgenerational phenotype of developmental origin caused by F0 exposure to permethrin that confirms previous from zebrafish larvae [START_REF] Blanc | Multi-and transgenerational effects following early-life exposure of zebrafish to permethrin and coumarin 47: Impact on growth, fertility, behavior and lipid metabolism[END_REF]. Sex differences in transgenerational effects were observed following other exposure scenario and may suggest a different capacity for adaptation and recovery (Baker et al., 2014b;[START_REF] Crews | Transgenerational epigenetic imprints on mate preference[END_REF][START_REF] Vera-Chang | Transgenerational hypocortisolism and behavioral disruption are induced by the antidepressant fluoxetine in male zebrafish Danio rerio[END_REF]. In this regard, gene expression data showed that in F1 and in F2, the number of DEGs was higher in females compared to males, which may highlight sex-specific adaptive or compensatory mechanisms at play. These results further support the notion that direct effects of developmental exposure (i.e. effects in F0) can be partly dissimilar to the ones resulting from germ cell exposure (F1) or transgenerational inheritance (F2) as described in previous studies [START_REF] Alfonso | Examining multi-and transgenerational behavioral and molecular alterations resulting from parental exposure to an environmental PCB and PBDE mixture[END_REF][START_REF] Bell | An integrative framework for understanding the mechanisms and multigenerational consequences of transgenerational plasticity[END_REF][START_REF] Schmitt | Transgenerational effects of developmental exposure to chlorpyrifos-oxon in zebrafish (DANIO RERIO)[END_REF][START_REF] Blanc | An environmentally relevant mixture of polychlorinated biphenyls (PCBs) and polybrominated diphenylethers (PBDEs) disrupts mitochondrial function, lipid metabolism and neurotransmission in the brain of exposed zebrafish and their unexposed F2 offspring[END_REF]. In this study, F0 males were hypoactive, while more specific effects on anxiety relief were identified in F1 and F2 males. At the molecular level, gene expression and epigenetic changes were however similar between all three generations, and no specific similarity relevant to behavior could be identified between both F1 and F2 only. Thus, we hypothesize that behavioral alterations were partly inherited from F0 to F1 and F2, and that the hypoactive phenotype in F0 prevented from measuring more subtle behavioral changes, such as alterations in the anxiety level of F0 males. This is further strengthened by the identification of additional molecular effects specific to the F0 generation that could explain the aggravated phenotype.

Pathway analysis in F1 and F2 generations strengthened the hypothesis that behavioral alterations may be linked to glutamatergic synapse activity, as the related transcriptional changes were also specific to males. However, more investigation into the impact of the transcriptional regulation of this pathway is needed, in particular as glutamate-related pathways were upregulated in F1 but downregulated in F2, while observing similar behavioral phenotypes. We can only speculate on how these opposite patterns could lead to similar phenotypes. It is possible that despite opposite transcriptional changes, similar alterations in these pathways at the level of protein expression or function are induced in both generations.

Furthermore, F1 and F2 may show similar transcriptional changes in specific brain areas, which cannot be addressed in this study as the analyses were performed on whole brains, and thus represent overall changes. For these reasons, further investigations focusing on protein expression and functionality as well on specific brain regions may clarify the effects of permethrin on the glutamatergic system of zebrafish.

DNA methylation analyses also pointed to effects related to glutamatergic signaling, which persisted across all three generations. Changes in DNA methylation have been previously linked to late onset and transgenerational effects and have a functional role in disease etiology, development, and behavior in various species [START_REF] Liberman | Transgenerational epigenetic inheritance: from phenomena to molecular mechanisms[END_REF][START_REF] Mcghee | Paternal care in a fish: epigenetics and fitness enhancing effects on offspring anxiety[END_REF][START_REF] Tran | Neurodevelopmental Disorders and Environmental Toxicants: Epigenetics as an Underlying Mechanism[END_REF]. They could therefore be the mechanism underlying inheritance of the observed behavioral defects in male fish [START_REF] Andersen | Epigenetic marking of the zebrafish developmental program[END_REF][START_REF] Bordoni | Intergenerational Effect of Early Life Exposure to Permethrin: Changes in Global DNA Methylation and in Nurr1 Gene Expression[END_REF]Manikkam et al., 2012b). Observing sex-specific inheritance of effects in the domesticated zebrafish AB strain, which does not possess known genetic determination of sex, is somewhat unexpected. However, Vera-Chang and co-workers (2018) observed such effect in response to fluoxetine exposure, another compound affecting anxiety levels in fish [START_REF] Vera-Chang | Transgenerational hypocortisolism and behavioral disruption are induced by the antidepressant fluoxetine in male zebrafish Danio rerio[END_REF].

Fluoxetine exposure led to distinct behavioral changes in zebrafish males compared to females across generations, which could partly be attributed to sex-specific levels of sex steroids.

Interestingly, permethrin has also been shown to interfere with the male hormone system in rodents [START_REF] Kim | Potential estrogenic and antiandrogenic effects of permethrin in rats[END_REF], and with the endocrine system of zebrafish embryos [START_REF] Zhang | Environmentally relevant levels of λ-cyhalothrin, fenvalerate, and permethrin cause developmental toxicity and disrupt endocrine system in zebrafish (Danio rerio) embryo[END_REF]. In our study, no monitoring of steroids was performed, but transcriptomic and epigenetic alterations pointed to sex-specific changes in glutamate signaling. Interestingly, it was shown before that the methylation status of glutamate receptors was only involved in regulating the depression-like behavior of male rats, but not females, supporting that mechanisms are sexspecific [START_REF] Lin | The Impact and Mechanism of Methylated Metabotropic Glutamate Receptors 1 and 5 in the Hippocampus on Depression-Like Behavior in Prenatal Stress Offspring Rats[END_REF]. Thus, it is possible that similar epigenetic alterations induce sexspecific behavioral alterations in organisms, although mechanisms require further clarification.

In the present case, however, most of the methylation changes were also sex-specific. Several DMRs, inherited from the F0 to the F2 generation of males, were associated to genes implicated in the control of behavior and linked to glutamatergic signaling: vim, pnocb, and fmr1. Vimentin (vim) is an intermediate filament protein expressed in glial cells such as astrocytes [START_REF] Cerdà | Zebrafish vimentin: molecular characterization, assembly properties and developmental expression[END_REF], which is important for synapse reorganization and neuron regeneration after brain injury [START_REF] Krohn | Expression of vimentin increases in the hippocampus and cerebral cortex after entorhinal cortex lesioning and in response to transforming growth factor β 1[END_REF][START_REF] Zupanc | Potential role of radial glia in adult neurogenesis of teleost fish[END_REF]. In addition, vimentin misexpression in rat prefrontal cortex was linked to locomotor and anxiety-like behavior changes following adolescent social isolation [START_REF] Sun | Adolescent social isolation affects schizophrenia-like behavior and astrocyte biomarkers in the PFC of adult rats[END_REF], and interactions between glutamatergic signaling and vimentin expression were reported in other studies [START_REF] Kawakami | Glial and neuronal localization of ionotropic glutamate receptor subunitimmunoreactivities in the median eminence of female rats: GluR2/3 and GluR6/7 colocalize with vimentin, not with glial fibrillary acidic protein (GFAP)[END_REF][START_REF] Mcnearney | A peripheral neuroimmune link: glutamate agonists upregulate NMDA NR1 receptor mRNA and protein, vimentin, TNF-α, and RANTES in cultured human synoviocytes[END_REF]. Further, astrocyte dysregulation was incriminated in developmental neurotoxic effects of permethrin in rat [START_REF] Saito | Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice[END_REF], overall suggesting that transgenerational epigenetic dysregulation of vimentin may have profound effects on astrocyte function within the tripartite glutamatergic synapse. Prepronociceptin b (pnocb) is responsible for modulating nociception and locomotor behavior [START_REF] Demin | Zebrafish models relevant to studying central opioid and endocannabinoid systems[END_REF]. Nociceptin signaling can disturb the GABA/glutamatergic balance and favor the development of Parkinson disease, for which permethrin is under suspicion as a causal agent [START_REF] Mercatelli | NOP Receptor Ligands and Parkinson's Disease[END_REF]. Finally, glutamatergic signals regulate the trafficking of the FMR-protein encoded by fmr1 at dendrites and synapses in vitro [START_REF] Antar | Metabotropic Glutamate Receptor Activation Regulates Fragile X Mental Retardation Protein and Fmr1 mRNA Localization Differentially in Dendrites and at Synapses[END_REF], and knock-out of fmr1 produces an anxiolytic-like phenotype and impairment of avoidance learning in zebrafish [START_REF] Ng | Behavioral and Synaptic Circuit Features in a Zebrafish Model of Fragile X Syndrome[END_REF]. Methylation changes associated to the described genes and pathways implicate epigenetic regulation of neuronal development and glutamatergic synapse activity in the observed transgenerational and sex-specific behavioral effects. However, we could not well correlate DEGs and predicted cis-regulated genes from DMRs, which limits our conclusions. The lack of correlation between methylation and transcription changes is acknowledged within the field [START_REF] Kamstra | Ionizing radiation induces transgenerational effects of DNA methylation in zebrafish[END_REF][START_REF] Falisse | DNA methylation and gene expression alterations in zebrafish early-life stages exposed to the antibacterial agent triclosan[END_REF] and both technical and biological factors may be at cause. As observed in other studies [START_REF] Falisse | DNA methylation and gene expression alterations in zebrafish early-life stages exposed to the antibacterial agent triclosan[END_REF][START_REF] Kamstra | Differential DNA methylation at conserved non-genic elements and evidence for transgenerational inheritance following developmental exposure to mono(2ethylhexyl) phthalate and 5-azacytidine in zebrafish[END_REF], most of the identified DMRs were located at intergenic regions (≈70% in average, data not shown). Thus, the restrictive association of DMRs to potential cis-regulated genes, and the limited scope of RRBS analyses (screen of 3-5% of the methylome) likely underestimated functional associations. Besides, the relationship between genomic structure and gene activity is not fully understood yet and surely involves different levels of epigenetic remodeling which were not all measured in the present study [START_REF] Bemer | Unraveling the complex epigenetic mechanisms that regulate gene activity[END_REF].

Nonetheless, this study identified several candidate DMRs that were inherited over three generations of zebrafish and that may have implications in the behavioral effects. However, it cannot be excluded that the observed transgenerational effects are not subsequent to DNA methylation changes. Other epigenetic mechanisms could be involved, such as micro-RNAs or histone modifications [START_REF] Blake | Unravelling the complex mechanisms of transgenerational epigenetic inheritance[END_REF]. Histone modifications are important epigenetic marks for proper nervous system development and are the target of neurotoxic compounds [START_REF] Lilja | Like a rolling histone: Epigenetic regulation of neural stem cells and brain development by factors controlling histone acetylation and methylation[END_REF][START_REF] Song | Environmental Neurotoxic Pesticide Increases Histone Acetylation to Promote Apoptosis in Dopaminergic Neuronal Cells: Relevance to Epigenetic Mechanisms of Neurodegeneration[END_REF]. To which extent these modifications can contribute to epigenetic inheritance in vertebrates is a very active domain of research [START_REF] Rose | Understanding the relationship between DNA methylation and histone lysine methylation[END_REF][START_REF] Lismer | Sperm histone H3 lysine trimethylation is altered in a genetic mouse model of transgenerational epigenetic inheritance[END_REF]. We reported previously that the histone demethylase Kdm5ba was dysregulated in zebrafish embryos exposed to permethrin [START_REF] Blanc | Environmental chemicals differentially affect epigeneticrelated mechanisms in the zebrafish liver (ZF-L) cell line and in zebrafish embryos[END_REF] and we also observed enrichment in histone modification pathways in the present work. Another study suggested the involvement of histone modifications on H3 and H4 rather than DNA methylation changes to support permethrin epigenetic effects [START_REF] Fedeli | In vivo and in silico studies to identify mechanisms associated with Nurr1 modulation following early life exposure to permethrin in rats[END_REF]. Besides, [START_REF] Valles | Exposure to Low Doses of Inorganic Arsenic Induces Transgenerational Changes on Behavioral and Epigenetic Markers in Zebrafish (Danio Rerio)[END_REF] recently showed sex-specific inheritance of histone modifications in zebrafish following ancestral exposure to arsenic; thus, suggesting that chemical exposure can induce sex-specific epigenetic alterations, which may turn into sex-specific behavioral changes. Therefore, identifying DNA regions enriched or depleted in specific histone marks may provide further insight into the epigenetic component of the observed alterations.

In conclusion, early-life exposure to permethrin induced late-onset behavioral defects in F0 adult zebrafish and a transgenerational decrease in anxiety-like behavior in males.

Transcriptional and epigenetic data indicated inherited molecular changes linked to dysregulation of glutamatergic signaling as a potential mechanism underlying the delayed and transgenerational neurobehavioral alterations in males. Functional analyses are now warranted to confirm the role of the identified pathways in the behavioral alterations, especially since some of the effects in our study were not strongly significant, possibly due to the limited number of replicates per condition. It would also be of interest to extend the phenotypical characterization to cognitive defectslearning and memory, as they could help to clarify the role of some molecular changes observed in both males and females, or the ones that are generation-specific [START_REF] Feng | Dnmt1 and Dnmt3a maintain DNA methylation and regulate synaptic function in adult forebrain neurons[END_REF][START_REF] Saito | Early-life exposure to low levels of permethrin exerts impairments in learning and memory with the effects on neuronal and glial population in adult male mice[END_REF][START_REF] Shelton | Neurodevelopmental Disorders and Prenatal Residential Proximity to Agricultural Pesticides: The CHARGE Study[END_REF]. In addition, future research may also focus on the identification of critical windows of exposure within the first 28 days, and on distinguishing between paternal and maternal contributions to germ-line mediated transgenerational epigenetic inheritance. In fact, the study of epigenetic changes in parental gametes would help characterizing the role of epigenetics in mediating the observed transgenerational effects, by understanding 1) how they could carry on information to the next generation, and 2) how these could translate into persistent sex-specific brain alterations. S8. S8. S6 and Excel Tables S9-S14.

A B

Table 1: Transcriptional enrichment in glutamate signaling related gene sets in brains from all generations of males (n=4). This table shows the Normalized Enrichment Score (NES) and associated adjusted p-value (padj) as given by the Gene Set Enrichment Analysis (GSEA) output. Headings refer to F0 males (F0_M), F1 males (F1_M) and F2 males (F2_M). Complete results from differential gene expression analyses and GSEA are available in the Excel Tables S2-S7 and S15-S17.

Figure 1 :

 1 Figure 1: PC1 (A) and PC2 (B) individual loadings after novel tank diving test in F0, F1, and F2 generations of adult male fish. #: p<0.1; *: p<0.05. PL: 1 µg/L Permethrin; PH: 10 µg/L Permethrin; SC: Solvent Control (DMSO 0.01%). Numeric values are available in TableS8.

Figure 2 :

 2 Figure 2: Univariate analysis related to anxiety-like behavior in males of the F1 and F2 generations. A: Distance travelled in top area; B: Time spent in Top area. #: p<0.1; *: p<0.05. PL: 1 µg/L Permethrin; PH: 10 µg/L Permethrin; SC: Solvent Control (DMSO 0.01%). Numeric values are available in TableS8.

Figure 3 :

 3 Figure 3: A. Distribution of differentially expressed genes (DEGs) between upregulation (UP) and downregulation (DOWN) in F0 males (F0_M), F0 females (F0_F), F1 males (F1_M), F1 females (F1_F), F2 males (F2_M) and F2 females (F2_F) following F0 early exposure to permethrin 10 µg/L (n=4). The graphic displays the total number of DEGs as well as the proportion of transcripts showing an absolute fold change above 2. B. Venn diagram showing the number of differentially expressed genes and overlapping between the 3 generations of males (padj≤0.05). Underlined numbers show significantly enriched gene sets according to Gene Set Enrichment Analysis (GSEA) results (padj≤0.05). C. Venn diagram showing the number of differentially expressed genes and overlapping between the 3 generations of females (padj≤0.05). Underlined numbers show significantly enriched gene sets according to Gene Set Enrichment Analysis (GSEA) results (padj≤0.05).

Figure 4 :

 4 Figure 4: Changes in DNA methylation in F0, F1 and F2 generations of zebrafish following F0 early-life exposure to permethrin 10 µg/L (n=4; except for treated F0 males where n=3). A. Distribution of the Differentially Methylated Regions (DMRs) between hypo-and hypermethylation within each dataset (padj≤0.05): F0 males (F0_M), F0 females (F0_F), F1 males (F1_M), F1 females (F1_F), F2 males (F2_M) and F2 females (F2_F). B. Venn diagram showing the number of DMRs and overlaps between the 3 generations in males (padj≤0.05). C. Venn diagram showing the number of DMRs and overlaps between the 3 generations in females (padj≤0.05).

Figure 5 :

 5 Figure 5: Heatmap showing inherited differentially methylated regions (padj≤0.05) in F0, F1, and F2 generations of males (left) or females (right) (n=4; except for treated F0 males where n=3). Row headings refer to genes associated to DMRs. If none, it refers to the DMR as chromosome name.starting position. Column headings refer to F0 males (F0_M), F0 females (F0_F), F1 males (F1_M), F1 females (F1_F), F2 males (F2_M) and F2 females (F2_F). Numeric values are available in TableS6and Excel Tables S9-S14.

Nevertheless, the present results show that neurobehavioral effects of pyrethroid exposure previously identified in other organisms may not be restricted to exposed generations and may therefore have profound long-term consequences on environmental health.

Acknowledgement

The authors wish to thank Jorke Kamstra for valuable comments on the manuscript, as well as Jean-François Allienne and the "Bio-Environment" platform at the University of Perpignan Via