Prescilla Perrichon
email: prescilla.perrichon@gmail.com

Farida Akcha

Karyn Le Menach

Manon Goubeau

Hélène Budzinski

Xavier Cousin

Paco Bustamante

Parental trophic exposure to three aromatic fractions of polycyclic aromatic hydrocarbons in the zebrafish: consequences for the offspring

Keywords: Polycyclic Aromatic Hydrocarbons, parental transmission, fish early-life stages, PhotoMotor Response, cardiac performance, PAH metabolites, genotoxicity Brut Arabian Light, EROD: EthoxyResorufin-O-Deethylase, dpf: days postfertilization, HFO: Heavy Fuel Oil, HMW: High Molecular Weight, hpf Low Molecular Weight, PAHs: Polycyclic Aromatic Hydrocarbons, PY: Pyrolytic, PMR: PhotoMotor Response, RT-PCR: Real-Time Polymerase Chain Reaction

In recent decades, PAH emissions due to extensive anthropogenic activities have risen sharply causing considerable pollution of aquatic ecosystems. This pollution represents a threat for organisms, among them fish. Consequently, prenatal stress can have important repercussions, and may impact survival and population recruitment. To investigate this point, eggs were collected from zebrafish exposed during 6 months by trophic route to three aromatic fractions from two different origins, pyrolytic (PY) and petrogenic (light (BAL) and heavy (HFO) fractions) sources.

Chronic dietary exposure of the parents were performed at environmentally relevant concentrations (0.3X, 1X and 3X; 1X represents an environmental concentration measured in French estuary). In order to explore the consequences of parental exposure for the next first generation, toxic responses were studied in both embryos and larvae using a multiscale approach. Toxic effects were assessed by looking at hatching success, developmental abnormalities, photomotor response and heartbeat.

The level of PAH metabolites and EROD activity in fish larvae were measured to assess exposure to PAHs. Egg production of parents was significantly reduced compared to the Control; hence little information was available for BAL and HFO offspring. The size of larvae from PY parents was found to increase despite a reduced yolk sac compared to Control larvae. Furthermore, a high level of behavioral stress was observed in larvae originating from parents exposed to three-fold the environmental concentration. The cardiac activity was reduced in a concentration-dependent manner for the PY exposure group. No effect was however observed on biotransformation markers (cyp1a, EROD), nor on the level of DNA damage for all PY, BAL and HFO offspring. The absence of significant differences in metabolite levels may indicate a potential early depuration of transferred compounds or no PAH-transmission. The disruptions observed at the individual level in the next generation could impact on the longer-term, surviving population.

Introduction

In recent decades, increasing industrialization and urbanization has resulted in significant chemical pollution of the aquatic environment [START_REF] Shen | Global atmospheric emissions of polycyclic aromatic hydrocarbons from 1960 to 2008 and future predictions[END_REF]. Among organic pollutants, polycyclic aromatic hydrocarbons (PAHs) are ubiquitous and semi-persistent pollutants in the environment [START_REF] Laflamme | The global distribution of polycyclic aromatic hydrocarbons in recent sediments[END_REF]. They are lipophilic compounds with two or more fused benzene rings. PAHs can be divided into two groups: the Low-Molecular-Weight (LMW) PAHs (PAHs with 2 or 3-benzenic rings) and the High-Molecular-Weight (HMW) PAHs (PAHs presenting more than 3-benzenic rings). PAHs are produced from three pathways: i) pyrolytic processes such as incomplete combustion of organic matter from natural or anthropogenic sources (pyrogenic origin), ii) diagenesis of sedimentary organic matter giving form to fossil fuel (petrogenic origin), iii) natural precursor transformations during early diagenesis processes (diagenetic origin) (Neff, 1979). Although they may be produced naturally, their predominant emission is from anthropogenic sources, including burning of fossil fuel, coal tar, wood, waste incineration, urban runoff, maritime transport discharges and petroleum spills (Neff, 1979).

Depending on their origin, PAHs are generally present in complex mixtures that can be characterized by their molecular fingerprint. Indeed, a predominance of HMW compounds associated with the presence of less or non-alkylated PAHs is representative of a pyrolytic mixture.

Conversely, a petrogenic mixture is characterized by a majority of LMW PAHs and the presence of methylated compounds [START_REF] Abrajano | High molecular weight petrogenic and pyrogenic hydrocarbons in aquatic environments[END_REF]Sakari, 2012).

Due to their physico-chemical properties, most PAHs exhibit hydrophobic and lipophilic properties making them relatively insoluble in water. They have a high capacity to adsorb onto suspended particles or non-polar matrices which respectively favors their accumulation in sediments and in the lipid tissue of organisms. Fish have the ability to biotransform PAHs to more water-soluble metabolites, hence reducing bioaccumulation and transfer efficiency in higher levels of marine food chains [START_REF] Meador | Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms[END_REF]. Nonetheless, benthic invertebrate preys represent important pathways of PAH exposure for demersal fish. Indeed, many invertebrate species such as mollusks can bioaccumulate high PAH concentrations in their fatty tissues [START_REF] Peterson | Long-term ecosystem response to the Exxon Valdez oil spill[END_REF][START_REF] Varanasi | Bioavailability and biotransformation of aromatic hydrocarbons in benthic organisms exposed to sediment from an urban estuary[END_REF] due to the lower biotransformation capability [START_REF] Bustamante | Bioaccumulation and metabolisation of (14)C-pyrene by the Pacific oyster Crassostrea gigas exposed via seawater[END_REF][START_REF] Lee | Uptake, metabolism and discharge of polycyclic aromatic hydrocarbons by marine fish[END_REF][START_REF] Palmork | Distribution and elimination of [9-14C]Phenanthrene in the Horse mussel (Modiola modiolus)[END_REF]. PAH toxicity in fish can be directly linked to biotransformation by enzymatic systems such as those from cytochrome P450, which metabolize PAHs into reactive metabolites [START_REF] Meador | Bioaccumulation of polycyclic aromatic hydrocarbons by marine organisms[END_REF][START_REF] Van Der Oost | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. Some of the metabolites produced are more toxic than the parent compounds, presenting, for example, a high reactivity for the DNA leading to genotoxic effects [START_REF] Dû-Lacoste | Comparative study of different exposure routes on the biotransformation and genotoxicity of PAHs in the flatfish species, Scophthalmus maximus[END_REF][START_REF] Wessel | Genotoxic and enzymatic effects of fluoranthene in microsomes and freshly isolated hepatocytes from sole (Solea solea)[END_REF][START_REF] Wessel | Relationship between PAH biotransformation as measured by biliary metabolites and EROD activity, and genotoxicity in juveniles of sole (Solea solea)[END_REF]. However, DNA damage is not necessary linked to activation of enzymatic systems such as ethoxyresorufin-O-deethylase (EROD) activity. A case in point is fluoranthene which is not a potential inducer of EROD activity, while its produced-metabolites exhibit genotoxic effects [START_REF] Wessel | Genotoxic and enzymatic effects of fluoranthene in microsomes and freshly isolated hepatocytes from sole (Solea solea)[END_REF]. Furthermore, PAH metabolization can also lead to the production of reactive oxygen species (ROS) which may induce an oxidative stress [START_REF] Frenzilli | The Comet assay for the evaluation of genotoxic impact in aquatic environments[END_REF].

In aquatic organisms, exposure to PAHs can thus lead to detrimental defects from a developmental, physiological and behavioral point of view [START_REF] Bihanic | Environmental concentrations of benz[a]anthracene induce developmental defects and DNA damage and impair photomotor response in Japanese medaka larvae[END_REF][START_REF] Incardona | Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development[END_REF]2004;[START_REF] Perrichon | Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays[END_REF]. PAHs' impact on reproductive success is particularly studied due to its consequence on population recruitment. Indeed, PAHs can act as endocrine disruptors that interfere and thus lead to adverse reproductive and developmental effects [START_REF] Cooper | Endocrine disruptors and reproductive development : a weight-ofevidence overview[END_REF][START_REF] Hawliczek | Developmental toxicity and endocrine disrupting potency of 4-azapyrene, benzo[b]fluorene and retene in the zebrafish Danio rerio[END_REF]Monteiro et al. 2000a;Monteiro et al. 2000b). Previous studies showed a reduction of the plasma level of gonad hormones in fish after PAH exposure (Monteiro et al., 2000a;[START_REF] Thomas | Reproductive life history stages sensitive to oil and naphthalene in Atlantic croaker[END_REF]. [START_REF] Thomas | Reproductive life history stages sensitive to oil and naphthalene in Atlantic croaker[END_REF] highlighted a blockage of sexual maturation, impairments of ovarian recrudescence and induction of oocyte atresia in female Atlantic croaker (Micropogonias undulatus) following exposure to water-soluble fractions of diesel fuel and naphthalene. Similarly, a delay in gonad maturation has been reported in female English sole from sites with high concentrations of PAHs [START_REF] Casillas | Inducibility of spawning and reproductive success of female english sole (Parophrys vetulus) from urban and nonurban areas of puget sound, Washington[END_REF][START_REF] Johnson | An analysis in support of sediment quality thresholds for polycyclic aromatic hydrocarbons (PAHs) to protect estuarine fish[END_REF][START_REF] Johnson | Contaminant effects on ovarian development in English sole (Parophrys vetulus) from Puget Sound, Washington[END_REF] as well as in female Atlantic croaker exposed in the laboratory to benzo[a]pyrene [START_REF] Thomas | Teleost model for studying the effects of chemicals on female reproductive endocrine function[END_REF]). An exposure to a PAH-contaminated sediment has been shown to reduce spawning in sole [START_REF] Johnson | An analysis in support of sediment quality thresholds for polycyclic aromatic hydrocarbons (PAHs) to protect estuarine fish[END_REF]. PAHs can hence have an effect on the parents themselves but also on their offspring. The physiological condition of a female at the time of vitellogenesis was reported as a modulation source for the offspring generation. Prenatal stress (i.e. chemical contamination) could thus affect the optimal development of the early life stage in the future generation [START_REF] Eriksen | Prespawning stress in farmed Atlantic salmon Salmo salar: maternal cortisol exposure and hyperthermia during embryonic development affect offspring survival, growth and incidence of malformations[END_REF][START_REF] Gagliano | Hormonally mediated maternal effects shape offspring survival potential in stressful environments[END_REF][START_REF] Mccormick | Experimental test of the effect of maternal hormones on larval quality of a coral reef fish[END_REF]. Uptake of pollutants can occur during ovogenesis and consequently accumulate in the vitellin reserve of the embryos. Relatively few studies reported potential parental transmission of PAHs in offspring generation. Following maternal exposure, the transfer of anthracene in the vitellus of fathead minnows eggs has been shown by Tilghman [START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF]. More recently, [START_REF] Pollino | Physiological changes in reproductively active rainbowfish (Melanotaenia fluviatilis) following exposure to naphthalene[END_REF] demonstrated a positive correlation between the occurrences of abnormal larvae in offspring with naphthalene-exposed female rainbowfish (Melanotaenia fluviatilis). The transfer of PAHs to the embryos was associated with mortality, hatching delay and growth alterations [START_REF] Hose | Uptake of benzo[α]pyrene by gonadal tissue of flatfish (family pleuronectidae) and its effects on subsequent egg development[END_REF][START_REF] Nye | The effect of maternal exposure to contaminated sediment on the growth and condition of larval Fundulus heteroclitus[END_REF][START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF].

This study aimed to explore the consequences of parental exposure (F0) to PAHs for the next first offspring generation (F1) under chronic dietary exposure conditions. Parents were chronically exposed during their whole lifecycle, through diet, to three aromatic fractions of pyrolytic and petrogenic origin, at concentrations which were relevant from an environmental point of view. This study focused on the early life stages of offspring. Multiscale approach was undertaken in order to assess a wide range of fish toxic response, using phenotypic markers (hatching success, developmental abnormalities), behavioral (PhotoMotor Responses), physiological (e.g. heartbeat) and cellular/subcellular markers (DNA damage, apoptosis, oxidative stress). In addition to these effect markers, the degree of exposure to PAHs was evaluated with the level of EROD activity, cyp1a induction and PAH-metabolites in offspring larvae.

Materials and Methods

Chemicals

The Arabian light crude oil (BAL 100) and the heavy fuel oil from the Erika fuel oils (No.

2) used for this study was provided by Cedre (Centre of Documentation, Research and Experimentation on Accidental Water Pollution, France). PAH spiking of food and chemical analysis were performed with dichloromethane solvent from Acros Organics (Thermo Fisher Scientific, Geel, Belgium). The ethyl-4-aminobenzoate (benzocaïne) was purchased from Sigma-Aldrich (St Quentin Fallavier, France).

Preparation of PAH-contaminated food

Four sizes of food were prepared (i.e. ≤ 125 µm, 125-315 µm, 315-500 µm, ≥ 500 µm) from ground commercial food INICIO Plus 0.5 mm (Biomar, France), in order to adapt the food to the fish morphology throughout the lifecycle (from larvae to adult stages). Each food lot was contaminated by the PAH aromatic fractions as described hereafter. The aromatic fractions were a pyrolytic fraction (PY) extracted from sediments collected in the highly polluted site of Oissel (Seine estuary, France) and two petrogenic fractions extracted from Brut Arabian Light oil (BAL)

and heavy Erika fuel oil (HFO). Extraction and quantification of these aromatic fractions based on the 16 priority PAHs listed by US-EPA were performed according to procedures described in [START_REF] Budzinski | Evaluation of sediment contamination by polycyclic aromatic hydrocarbons in the Gironde estuary[END_REF] and [START_REF] Cachot | Characterization of toxic effects of sediment-associated organic pollutants using the lambda transgenic medaka[END_REF]. PAH and alkylated PAH concentrations were reported in [START_REF] Vignet | Chronic dietary exposure to pyrolytic and petrogenic mixtures of PAHs causes physiological disruption in zebrafish -Part I: Survival and growth[END_REF] and given in Fig. 1 Supplementary data. Dry food was hence spiked with the three PAH fractions according to [START_REF] Vicquelin | A new spiked sediment assay using embryos of the Japanese medaka specifically designed for a reliable toxicity assessment of hydrophobic chemicals[END_REF]. Briefly, the amount of food required for the experiment was mixed with dichloromethane (Solvent Control) and each tested aromatic extract in a round-bottom glass flask. Then, solvent was completely evaporated with a rotary evaporator (RV10 Basic VWR International) equipped with a heating water bath (HB10 Basic, VWR International) at 45 °C and until total evaporation. Finally, spiked-food was stored overnight in darkness at room temperature under a fume hood to ensure complete residual solvent elimination. For each aromatic fraction, three concentrations were prepared: 0.3X, 1X and 3X. The nominal environmental concentration 1X represents the total PAH concentration measured in mollusks from the Seine estuary corresponding to 5 µg PAHs g -1 of dry food. PAH and alkylated PAH concentrations measured in contaminated-food were also reported in [START_REF] Vignet | Chronic dietary exposure to pyrolytic and petrogenic mixtures of PAHs causes physiological disruption in zebrafish -Part I: Survival and growth[END_REF] and

given in Table 1 Supplementary data.

Fish exposure

Parental dietary exposure (F0 Generation)

Wild-type zebrafish (TU strain, Tübingen, Germany) were used for the experiment. The parental dietary exposure was performed during the whole 9 months of the zebrafish lifecycle (larvae, juvenile, and adult). The parental generation was produced from fifty viable eggs per condition, provided by healthy spawning of zebrafish. Then, the eggs were transferred to Petri dishes (100 mm) filled with 30 mL of artificial water (0.33 mM CaCl2, 0.33 mM MgSO4, 5 mM NaCl and 0.17 mM KCl) and incubated at 28 ± 0.5 °C until exposure started. Maintenance of the standard conditions of zebrafish was already described in [START_REF] Perrichon | Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays[END_REF]. The trophic exposure was then performed from 5 days post-fertilization (dpf) larvae which correspond to the mouth opening stage. The procedure was already described in [START_REF] Vignet | Chronic dietary exposure to pyrolytic and petrogenic mixtures of PAHs causes physiological disruption in zebrafish -Part I: Survival and growth[END_REF]. Briefly, larvae were transferred in 1 L tanks in the breeding room and fed ad libitum with aromatic fraction-spiked food twice a day. In order to improve growth, larvae were then transferred to 3 and 10 L tanks from 12 dpf and 3 months post-fertilization (mpf) respectively. From this latter stage, diet rations distributed were set at 2% of the total biomass. During the 9 months of dietary exposure, a distribution of non-contaminated food with living Artemias sp. nauplii (INVE, Belgium) was given in supplement once a day. Breeding exposure was performed in a constant aeration and flow water systems with daily water renewal set at 40% in order to maintain optimal water quality. Water and room temperature were kept at 27 °C with a 14 h light/10 h dark photoperiod.

Offspring production (F1 Generation)

Eggs were obtained according to two distinct methods. Three spawning boxes (1 L, AquaSchwarz, Germany) were placed directly into the breeding tank the evening before spawning day. In addition, random pairwise mating was performed. One male and one female were placed together in a spawning box on the evening preceding egg production. With both methods, spawning and fertilization took place within 30 min after the onset of light in the morning. They were alternatively used in order to collect eggs twice a week. Fertilized eggs were selected by microscopic exam and viable eggs were distributed in batches of 30 into plastic Petri dishes (100 mm diameter) filled with 30 mL of artificial water and incubated at 28 ± 0.5 °C until analysis.

Biological analysis

Hatching and developmental abnormalities

Sublethal endpoints were recorded as described in [START_REF] Lammer | Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Physiol Part C[END_REF] scoring matrix (score of 0-3) was applied following occurrence and severity of these abnormalities:

(0) healthy larvae, (1) one abnormality or mildly-affected larvae, (2) two abnormalities or moderately affected larvae, (3) three abnormalities or more or severely affected larvae. Results

were expressed in percentage.

Impact on behavioral: swimming activity

PhotoMotor Response on 120 hpf larvae were monitored, following the procedure described by [START_REF] Péan | Long-term dietary-exposure to non-coplanar PCBs induces behavioral disruptions in adult zebrafish and their offspring[END_REF] with modifications by [START_REF] Perrichon | Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays[END_REF]. Briefly, the locomotor activity of larvae was recorded during a light/dark change challenge in a temperature-controlled box (28 ± 0.5 °C). Recording included three periods of 5 min: 5 min Light On (1), 5 min Dark (D) and 5 min Light On (2). Video analyses were performed with Ethovision XT 8.5 software (Noldus, The Netherlands). The behavioral performances of larvae were assessed by measuring the swimming distance (cm) during these three periods. This distance was calculated considering the center point of the larvae between two consecutive X-Y coordinates summed over a 5-min periods.

Effects on cardiac activity

Cardiac activity was recorded at 28 ± 0.5 °C on 120 hpf larvae. Larvae were introduced in 1 mm wide lines molded in agarose (2%), immobilized with 3% methylcellulose and positioned in a lateral view. An acclimation of 2 hours, in a temperature-controlled environment (28 ±0.5 °C), was performed before analysis in order to stabilize heartbeat. The heart area of each fish was recorded under an inverted microscope (Olympus SZX9, 40x) coupled to a camera (DMK 31AU03, The Imaging Sources, Germany) and to IC Capture 2.2 software (The Imaging Sources, Germany).

Larvae were placed under the microscope for two minutes before recording their heartbeat in order to acclimatize them to the light microscope. For each larvae, three successive videos of 30 seconds were acquired. Video analysis was performed in three successive processing steps: i) A video conversion was made in images stack to greyscale using VirtualDub 1.9.11 software (http://www.virtualdub.org/). A total of 450 images were extracted per 30 seconds of video; ii) An images analysis was performed using ImageJ software [START_REF] Schneider C A | NIH Image to ImageJ: 25 years of image analysis[END_REF], focusing on two ROIs (Regions Of Interest) manually delimited and corresponding to the atrium and ventricle, both cardiac chambers. The integrated analysis of grey intensity was performed for each image in both ROI previously converted in order to calculate the alternate circulation between both chambers;

and iii) Calculation of cardiac frequency for both chambers from a Single Spectrum (Fourier)

analysis was made on integrated data of grey intensity with Statistica 9.0 software (StatSoft, USA).

Cardiac frequencies were then corrected to obtain data expressed in beat.min -1 .

EROD activity

EROD activity as an indicator of phase I biotransformation activity was determined following the procedure described by [START_REF] Perrichon | Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays[END_REF]

DNA integrity of the offspring

DNA strand break measurements by the alkaline comet assay were performed on 24 hpf embryos. Pools of five dechorionated embryos for four samples per concentration were digested with 1 mg.mL -1 of phosphate-buffered saline 1X/collagenase IV from Clostridium histolyticum (PBS 1X, 137 mM NaCl • KCl 2.7 mM • Na2HPO4 10 mM • KH2PO4 1.8 mM, pH 7.4, Sigma-Aldrich, Germany) solution during 45 minutes at room temperature. Cell suspension was filtered through 48 µm gauze into a 1.5 mL reaction tube in order to separate individual cells from tissue debris. Following a centrifugation for 10 minutes at 2300 rpm at room temperature, the cells pellet was resuspended with 30 µl of PBS. The viability of the dissociated embryo cells was evaluated by a Trypan blue exclusion test. Then, the comet assay was performed as described by [START_REF] Akcha | Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds[END_REF]. Two slides were prepared per sample. DNA was stained with 70 µL of GelRed™ solution

(1/10000) for one hour at 4 °C in the dark. Slides were analyzed using a fluorescence microscope (Olympus BX60, 400x) coupled to a Luca-S EMCCD camera (Andor™ technology, Northern Ireland) and imaging analysis software (Komet 6.0, Andor™ Technology, Northern Ireland).

Genotoxic assessment was assessed by measuring the DNA percentage in the comet tail (50 cells per slide).

Gene expressions

Larvae were sampled at 96 hpf and stored in RNA Later (Sigma-Aldrich) at -20 °C until used.

Total RNA were extracted on 20 pooled larvae using Trizol® Reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions with chloroform/ethanol purifications. Total RNA concentration extracted was quantified by spectrophotometry at 260 nm. Furthermore, purity of RNAs was verified by measuring the A260/A230 nm and A260/A280 nm ratios and by electrophoresis on a 1.8% agarose gel with ethidium bromide staining. To avoid the possibility of genomic DNA contamination, the RNA sample was digested by RNase-free DNase I (Promega Madison, USA) and then purified.

Then, a reverse transcription of RNA was performed. First-strand cDNA was synthetized from total 1 µg RNA using a reaction mix including 500 ng of oligo(dT)15, 250 ng of random hexamer primers (Promega, Madison, USA), 10 mM of deoxyribonucleotide triphosphates

PAH-metabolites analysis in larvae

Larvae were sampled at 120 hpf and stored at -80 °C until used. Metabolites were extracted on 50 pooled larvae and three replicates per concentration. Larvae were manually homogenized in 3-4 mL of sodium acetate buffer (pH 5.0). An enzymatic deconjugation was performed for 16 hours at 37 °C after addition to 20 µL of beta-glucuronidase enzyme from Helix pomatia-2 (≥100 000 units.mL -1 , Sigma-Aldrich, Germany) using a method adapted by [START_REF] Mazéas | Solid-phase extraction and purification for the quantification of polycyclic aromatic hydrocarbon metabolites in fish bile[END_REF].

Prior to PAH-metabolites extraction, internal standards solution was added to samples. Metabolites were extracted by a Solid-Phase Extraction (SPE) procedure using octadecyl cartridge (500 mg, 3 cc, Bakerbond C18-SPE) and eluted in 100% methanol solvent. After re-concentration of organic extracts with gas nitrogen, samples were purified by SPE using amino-column (500 mg, 3 cc, Supelco NH2-SPE) and eluted with dicholoromethane/methanol solution (80/20, v/v). Behavioral data for PY treatment were analyzed using a one-way repeated measure ANOVA (R-ANOVA) with exposure concentrations as a dependent factor and light (Light On(1), Light Off and Light On(2)) as a within factor. A post-hoc Newman Keuls test was performed each time a difference was found to be significant. For behavioral data of BAL and HFO treatments, normality and homoscedasticity were not validated; therefore non-parametric Mann-Whitney tests were used.

In order to test significant differences between three challenge periods, Kruskal-Wallis tests were conducted with multiple pairwise comparisons.

Gene expressions were analyzed with a Relative Expression Software Tool REST-2009©

(Qiagen, http://www.REST.de.com). The gene expression level was given in a relative expression ratio compared to the control condition.

The significance of the results was ascertained at α=0.05.

Results and Discussion

Following 6 months of parents' exposure to the three aromatic fractions (PY, BAL and HFO), PAHs-metabolites were detected in whole larvae of the F0 generation showing that a parental contamination had occurred [START_REF] Vignet | Chronic dietary exposure to pyrolytic and petrogenic mixtures of PAHs causes physiological disruption in zebrafish -Part I: Survival and growth[END_REF]. The mean number of spawning events and batch fecundity obtained from these exposed parents were also reported by these authors (Vignet, 2014).

Parents' reproduction of 3X concentrations for the three treatments was severely impaired. Similar results were recorded in 1X concentrations for fish exposed to BAL and HFO aromatic fractions.

These complex mixtures have direct consequences on egg production with no eggs or few eggs obtained for these concentrations. Consequently, analyses of the offspring were not possible in the case of any egg production. For samples with an insufficient number of eggs, statistical analyses have not been performed and thus the results were given on an indicative basis.

Morphological impairments in offspring larvae (F1 generation)

No hatching delay was observed in offspring larvae whatever the nature and the concentration of the PAH extract tested. Hatching rates in offspring larvae from PY and BAL exposed parents were not significantly different whatever the tested concentrations (Fig. 1A, 1B) (P>0.05).

Statistical analyses revealed a decrease in the hatching rate (2.0-fold) in larvae from HFO-0.3X parents compared to the Solvent Control group (P<0.001) at the first hatching day (48 hpf) (Fig. 1C). However, no significant difference was recorded for the following days, with hatching rates averaging 100%. Regarding these latter results, it should be noted that this decrease observed at 48 hpf, probably did not lead to the PAH parental transmission but only to the hatching process unsynchronization in zebrafish [START_REF] Kimmel | Stages of embryonic development of the zebrafish[END_REF]. Only one spawning was obtained from HFO-1X exposed parents, consequently statistical analysis was not possible. Data are thus represented on an indicative basis. Therefore, our results did not show any alteration of hatching success in the first generation offspring from parents exposed to three PAH complex mixtures. In spite of the fact that relatively few studies reported results about PAHs transmission to offspring generation, [START_REF] Hose | Uptake of benzo[α]pyrene by gonadal tissue of flatfish (family pleuronectidae) and its effects on subsequent egg development[END_REF] have however demonstrated a reduction of hatching success in offspring eggs after BaP injection in the gonads of sexually mature flatfish. Similarly, [START_REF] Hall | Anthracene reduces reproductive potential and is maternally transferred during long-term exposure in fathead minnows[END_REF] have reported that maternally transferred anthracene reduced hatching success in the offspring eggs of fathead minnows (Pimephales promelas). In addition, [START_REF] Nagler | Exposure of male american plaice (Hippoglossoides platessoides) to contaminated marine sediments decreases the hatching success of their progeny[END_REF] have proved a significant decline in the number of hatched larvae because of sperm quality alteration in male American plaice (Hippoglossoides platessoides) exposed to contaminated sediment.

Biometric measurements in offspring larvae originating from PY, BAL and HFO fractionsexposed parents are presented in Table 3. Offspring larvae from PY-0.3X treatment were slightly longer than Solvent Control and PY-1X larvae (P<0.001 both). Yolk sac surface and ratio yolk sac/whole body surfaces of larvae from 0.3X and 1X concentrations were slightly reduced compared to Solvent Control larvae (P<0.001 both). Larvae originating from HFO-0.3X parents were slightly longer than Solvent Control larvae (P<0.001). No significant difference in yolk sac surface, in ratio yolk sac/whole body surfaces were detected in these offspring larvae (P>0.05). In addition, Table 3 gives a summary of abnormal larvae proportions and details of abnormalities and their associated severity in larvae. No significant morphological abnormalities were recorded between tested concentrations compared to Solvent Control, whatever the parental treatment (P>0.05). Abnormal individuals (and mainly mild-affected) ranged from 3.34 to 11.1% for PY offspring larvae, from 21.9 to 25.9% for BAL offspring larvae and from 18.0 to 19.8% for HFO offspring larvae. Taken together, these results showed that PAHs transmitted from the parents induced some biometric impairment. However, the toxic action of PAHs transmitted following these treatments will be not so severe as to induce edemas, skeletal, craniofacial deformations in offspring larvae. These results are in accordance with those of [START_REF] Nye | The effect of maternal exposure to contaminated sediment on the growth and condition of larval Fundulus heteroclitus[END_REF] who demonstrated that offspring larvae from contaminant-exposed mothers were longer with smaller yolk sacs than larvae produced by unexposed mothers. From those results, two hypothesis were proposed to explain these differences in size : i) the spawning event from contaminant-exposed mothers took place earlier or ii) a faster development in offspring embryos which will consume more yolk reserves compared to reference embryos. In the present study, development of offspring embryos was assessed during 96 hpf, from 30 min after spawning and no growth delay were recorded whatever the parental treatment considered. In addition, a greater number of hatched larvae would be then observed earlier. Therefore, the first hypothesis is unlikely. Thus, a greater consumption of nutritional reserves in offspring larvae from exposed parents is possible to offset potential physiological failure before hatching, leading to faster development, which would explain longer larvae at 96 hpf. Survival time in starved offspring larvae during 15 dpf was not assessed in the present study, but this parameter will be determinant to assess this potential consumption of reserves and therefore the resistance to starvation. A reduced ability by larvae to resist starvation could lead to a decline in the fish population.

Behavioral disruptions in offspring larvae (F1 generation)

Behavioral responses were analyzed in order to assess the potential developmental neurotoxic effects of parental transmission. These responses are currently used to assess the toxicity of various compounds through the analysis of swimming capacity, stress-regulating systems and motor responses (perception and reaction) to stimuli [START_REF] Champagne | Translating rodent behavioral repertoire to zebrafish (Danio rerio): relevance for stress research[END_REF][START_REF] Drapeau | Development of the locomotor network in zebrafish[END_REF][START_REF] Esch | Locomotor activity assay in zebrafish larvae: influence of age, strain and ethanol[END_REF][START_REF] Linney | Zebrafish as a neurotoxicological model[END_REF]. Figure 2 presents the measurements of distance moved per 5-min intervals in offspring larvae from PY, BAL and HFO-exposed parents following a light/dark challenge. Whatever the tested-aromatic fraction, response patterns of offspring larvae were similar, dissociating three swimming activity phases as reported in the literature: i) basal swimming activity phase (Light On(1)), ii) challenge phase (Light Off) leading to an increasing swimming activity and iii) the recovery phase (Light On(2)) when the light is turned on again, where swimming activity was comparable to that observed in the basal phase [START_REF] Kokel | Rapid behavior-based identification of neuroactive small molecules in the zebrafish[END_REF][START_REF] Macphail | Locomotion in larval zebrafish: Influence of time of day, lighting and ethanol[END_REF][START_REF] Padilla | Assessing locomotor activity in larval zebrafish: Influence of extrinsic and intrinsic variables[END_REF]. The swimming activity of larvae from PY-3X parents was significantly higher than the other concentrations during both light periods (P<0.05) (Fig. 2A).

Our results clearly demonstrated hyperactivity for 3X offspring larvae from PY-exposed parents during all behavioral challenges. The ability of these larvae to swim a greater distance could be due to the greater length and/or a possible neurotoxic stimulation of transferred-pyrolytic mixture.

The significant lack of morphological data for this condition did not support the first hypothesis.

Furthermore, results obtained in PY-0.3X offspring larvae did not reflect these findings. While these larvae had a high swimming activity, these results primarily demonstrate a high level of basic stress, a lower tolerance during the stress period and lower stress recovery. Regarding offspring larvae from BAL-0.3X parents (Fig. 2B), while no significant difference was observed in the basal swimming activity phase, their PMR pattern tended to be similar to the PMR pattern in PY-3X larvae (P>0.05). Indeed, these larvae showed a significant increase in the distance moved during the recovery phase (Light On(2), P=0.01). Regarding HFO treatment (Fig. 2C), PMR of 0.3X offspring larvae was significantly reduced during the all light/dark challenge compared to Solvent Control larvae (Light On(1): P<0.001, Light Off: P=0.002, Light On(2): P<0.001). Their behavioral responses were in total opposition with those of PY and BAL larvae, where 0.3X offspring larvae displayed a significant early lethargic activity whatever the swimming activity phase. No data were available for 1X and 3X concentrations in either BAL or HFO offspring larvae. According to current understandings, this is the first study concerning behavioral assessment of complex PAHs mixtures in the offspring of contaminated fish and relatively few studies have dealt with the impact of organic pollutant on the fish PMR. Therefore, we cannot directly compare effects with those reported in the literature. However, using a similar approach, Péan et al. ( 2013) have reported stimulation of this PMR in offspring zebrafish larvae originating from fish contaminated with polychlorinated biphenyls. Their results proved that a potential transmission of organic pollutants may alter the neurotoxic development of offspring. Recently, our previous studies have reported a reduction of basal swimming activity and response to light stress in ELS of zebrafish and an increase in Japanese medaka following sediment contact exposures with two unique PAH, fluoranthene (3-benzenic rings PAH) and benzo[a]pyrene (5-benzenic rings PAH) respectively [START_REF] Perrichon | Influence of sediment composition on PAH toxicity using zebrafish (Danio rerio) and Japanese medaka (Oryzias latipes) embryo-larval assays[END_REF]. A reduction in the swimming activity behavioral pattern was also recorded in Japanese medaka after exposure to benzo[a]anthracene (4-benzenic rings) [START_REF] Bihanic | Environmental concentrations of benz[a]anthracene induce developmental defects and DNA damage and impair photomotor response in Japanese medaka larvae[END_REF].

The present study demonstrated a parental influence in the neurotoxic behavior of their offspring.

These modifications in swimming activity could be critical for vital functions like their visual acuity, their hunting behavior or their escape response to predation or for the spawning migrations, which could be significantly reduced [START_REF] Gonçalves | The joint effect of polycyclic aromatic hydrocarbons on fish behavior[END_REF]. However, it is unknown whether any of the above observations translate to physiological defects and to fitness in surviving fish over the longer-term.

Physiological disruptions in offspring larvae (F1 generation)

Behavior impairments observed previously may be caused by physiological alterations.

Therefore, cardiac activity in 120 hpf offspring larvae from parents exposed to three aromatic fractions was measured and represented in Fig. 3 [START_REF] Barrionuevo | O2 consumption and heart rate in developing zebrafish (Danio rerio): influence of temperature and ambient O2[END_REF].

Numerous studies have documented cardiac defects following PAH exposure. The toxic action of unique predominant PAHs (from 3 to 5-benzenic rings PAHs) in the cardiovascular system of zebrafish mainly leads to pericardial edema, blockage of blood circulation and bradycardia [START_REF] Incardona | Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development[END_REF](Incardona et al., , 2009[START_REF] Incardona | Defects in cardiac function precede morphological abnormalities in fish embryos exposed to polycyclic aromatic hydrocarbons[END_REF]. Reduction in heart rate has also been reported in herring embryos and zebrafish embryos after respectively water-soluble fraction exposure of Alaska crude oil [START_REF] Middaugh | Preliminary observations on responses of embryonic and larval pacific herring, Clupea pallasi, to neutral fraction biodegradation products of weathered Alaska north slope oil[END_REF][START_REF] Middaugh | Responses of embryonic and larval inland silversides, Menidia beryllina, to a water-soluble fraction formed during biodegradation of artificially weathered Alaska North Slope crude oil[END_REF] and after sediment eluates/extracts exposure [START_REF] Strmac | Effects of sediment eluates and extracts from differently polluted small rivers on zebrafish embryos and larvae[END_REF]. Recently, [START_REF] Milinkovitch | Dispersant use as a response to oil spills: toxicological effects on fish cardiac performance[END_REF]2012) recorded cardiac impairments in terms of contraction velocity and not contraction force following a water-soluble fraction exposure of crude oil in golden grey mullets (Liza aurata). Similarly, the excitation-contraction coupling in cardiomyocytes of juvenile bluefin and yellowfin tunas was disrupted after exposure to Deepwater Horizon crude oil [START_REF] Brette | Crude oil impairs cardiac excitation-contraction coupling in fish[END_REF]. For similar oil, [START_REF] Incardona | Deepwater Horizon crude oil impacts the developing hearts of large predatory pelagic fish[END_REF] have shown serious defects in heart development with circulatory disruption and irregular atrial arrhythmia in three pelagic fish: bluefin tuna, yellowfin tuna and an amberjack. These physiological responses (bradycardia) observed in PY offspring larvae could indicate metabolic disruptions (e.g. respiratory distress) and thus alter fish fitness and survival [START_REF] Claireaux | Linking swimming performance, cardiac pumping ability and cardiac anatomy in rainbow trout[END_REF]. Our results are in agreement with those obtained by [START_REF] Lucas | Aerobic metabolism and cardiac activity in the descendants of zebrafish exposed to pyrolytic polycyclic aromatic hydrocarbons[END_REF] who assessed aerobic metabolic performance in offspring fish from PY-3X parents. These authors have recorded a decrease of active metabolic rate in 2 month-old fish originating from exposed parents, thus reflecting earlier cardio-respiratory impairments. Taken together, the present results are in agreement with those of [START_REF] Davoodi | Effects of exposure to petroleum hydrocarbons upon the metabolism of the common sole Solea solea[END_REF] who demonstrated that cardiac defects observed in sole (Solea solea) exposed to petroleum hydrocarbons induced a reduced AMR. However, we cannot attribute the changes in swimming activity to the heart disruption.

A lack of data in other biological parameters excluded any specific conclusions as to the potential PAH transmission and toxicity effects in offspring for BAL and HFO treatments.

Genotoxic effects in offspring (F1 generation)

Genotoxic effects on offspring embryos and larvae following the PAH exposure of parents were investigated through the analysis of detoxification, oxidative processes and DNA damage.

Whatever the tested fractions (PY, BAL or HFO), no significant differences in levels of EROD activity were observed in 96 hpf offspring larvae compared to respective Solvent Control (P>0.05) (Fig. 4). Similarly, comet assay did not reveal any significant differences (P>0.05) in the levels of DNA strand breaks measured earlier in 24 hpf offspring embryos from contaminated parents, whatever the tested-concentrations for PY, BAL and HFO fractions (Fig. 5). Unfortunately, no data were available for all 3X concentrations and HFO-1X. Furthermore, relative expressions of target genes compared to respective Solvent Control are presented in Table 4. Data obtained in larvae offspring were normalized to three housekeeping genes: eef1, βactin, gapdh. No data were available for BAL-3X and HFO-3X. Regarding offspring larvae from parents exposed to PY aromatic fraction, no significant expression change was revealed between all concentrations for genes involved in the detoxification (cyp1a, ahr2), antioxidant defense (gpx4a, cat, sod(Cu/Zn), sod(Mn)) and the apoptosis processes (p53, bax). Similarly, no significant differences in terms of detoxification (cyp1a, ahr2) processes were found at 0.3X and 1X concentrations for BAL and HFO compared to their respective Solvent Control (P>0.05). With regards to Sod(Mn), expression levels were 1.30-fold up-regulated in larvae offspring from BAL-1X parents (P=0.006). Expression of gene encoding for gpx4 was 0.59-fold repressed in offspring larvae from HFO-1X parents (P=0.018). The expression levels of others genes encoding for antioxidant defenses were not significantly different compared to Solvent Control. However, apoptosis processes appeared slightly impacted in offspring larvae from BAL-0.3X parents, with expression levels 1.47-fold and 1.30-fold higher than Solvent Control for p53 and bax respectively (P=0.03 for both conditions).

In contrast, no significant change in either genes was observed in 1X offspring larvae. For offspring larvae from HFO-1X parents, the p53 level was significantly up-regulated (P=0.03).

Taken together, these results showed no evidence of repairable or unrepairable DNA damage or potential activation of detoxification processes (ahr2, cyp1a, EROD) despite parental exposure to PAHs that are potential inducers of these activities. These results are in agreement with those directly found in the parental generation of our offspring larvae [START_REF] Larcher | Chronic dietary exposure of zebrafish to PAH mixtures results in carcinogenic but not genotoxic effects[END_REF]). Indeed, these authors have recorded a high incidence of tumors related to a refractory detoxification bioactivation or DNA damage (DNA strand breaks and micronuclei) via dietary exposure, whatever the aromatic fraction considered. In parallel, a kinetic of cyp1a expression related to the larval feeding condition was performed and had demonstrated that following a 24-hour starvation period, an early activation of cyp1a occurred after a new feeding event with contaminated food, but rapidly returned to background level. This might explain the absence of genotoxic effects in parental generation and consequently in the first generation of offspring in case of genetically-based response transmission.

Furthermore, fish have a high ability to biotransform PAHs, and are therefore able to eliminate a significant part of these compounds [START_REF] Aas | PAH metabolites in bile, cytochrome P4501A and DNA adducts as environmental risk parameters for chronic oil exposure: a laboratory experiment with Atlantic cod[END_REF][START_REF] Budzinski | Link between exposure of fish (Solea solea) to PAHs and metabolites : Application to the " Erika " oil spill[END_REF][START_REF] Jonsson | Bioconcentration, biotransformation, and elimination of polycyclic aromatic hydrocarbons in sheepshead minnows (Cyprinodon variegatus) exposed to contaminated seawater[END_REF]). PAH-metabolites (OH-PAH) concentrations were then measured in offspring larvae in order to identify parental contamination transmission. The hydroxynaphtalene (OH-N) and dihydroxybiphenyl (2-OH-Bi) were predominant metabolites produced in offspring larvae from PY, BAL and HFO fractions (Table 5). A relatively low level of 1-hydroxypyrene (1-OH-Pyr) metabolite was also present in larvae. However, the amount of metabolites was not statistically different between tested concentrations, from each origin fractions (P>0.05), which could explain the absence of a genotoxic effect. Furthermore, among these PAH-metabolites measured, none are potentially genotoxic (Le Dû-Lacoste et al., 2013).

Regarding, toxicity associated with the three aromatic fractions, the HFO fraction appeared to have greater toxicity with an induced-mortality, growth and reproductive disruptions, which lead to a significant reduction in egg production, followed by BAL and PY fractions (Vignet 2014).

Although HFO and BAL fractions were the most reprotoxic, only a narcotic effect appeared in offspring larvae of HFO-0.3X, which was not closely linked to cardiac activity or genotoxic impairments. Furthermore, the slight induction of p53 and bax involved in pro-apoptotic processes for both of these fractions, lead us to believe that processes of cellular reparation were induced to offset the potential detrimental effects, which might also explain the absence of genotoxic effects.

Finally, regarding biological responses, no conclusion can be drawn about contamination transmission or a stress transfer via neurotransmitters to the eggs.

Conclusion

Whereas early reproduction alteration was shown to lead to a significant reduction of produced eggs under environmentally relevant conditions, our study also demonstrated that longterm and chronic dietary exposures of zebrafish to PAH mixtures (from their first meal) led to impairments in their first offspring generation. Using a multiscale approach of toxicity endpoints, the present study reported some disruptions on the individual level. Larval offspring from parents exposed to PY fraction showed a higher degree of adverse effects than offspring from BAL and HFO parents. Indeed, mild morphometrical changes were recorded and bradycardias were significantly induced in larvae from PY parents. Behavioral disruptions were shown at the highest exposure concentration (3X), with basal hyperactive swimming and low reaction to second stress.

From acquired data, behavioral changes (lethargy and hyperactivity post-stress) were also demonstrated in offspring from BAL and HFO parents. It appears surprising that these effects were not related to bioactivation of processes involved in detoxification, oxidative stress or DNA damage which might be expected to explain a major part of these disruptions. A supplementary focus on the underlying mechanisms of lipid peroxidation should be performed in order to identify potential disruptions in the energetic mechanisms. Similarly, metabolic hormones (e.g. cortisol)

should be analyzed in order to validate these biological responses, which are potentially released into the nutritive yolk sac during ontogenesis closely associated with the female condition.

Furthermore, the lack of data and experiments in the literature compels us to be cautious in our conclusions. Multigenerational studies have proven to be an integrated approach for the toxicity assessment of chemicals and strengthen the predictive effects. These ecotoxicological studies should be widely undertaken to evaluate the potential for the exposed population to maintain its numbers in the future.

(

  dNTPs) solutions to a final volume of 10 µl. The reaction was initiated using M-MLV Reverse Transcriptase 5X (Promega, Madison, USA) following manufacturer's instructions. Reaction was incubated for 1 h at 42 °C in an Eppendorf Mastercycler and inactivated by heating for 15 min at 70°C. The cDNA mixture was stored at -20°C until a real-time PCR analysis.PCR primers were designed with Primer3 software (Rozen and Skaletsky, http://bioinfo.ut.ee/primer3-0.4.0/). The accession numbers of the 11 genes used in our study and the corresponding primers are reported in

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 1

 123451 Fig. 1. Hatching kinetic in 120 hpf offspring larvae originating from (A) PY, (B) BAL and (C)

  and Perrichon et al.

	(2014). Hatching success was monitored daily. Biometric analysis (standard length, whole body
	and yolk sac surface) and abnormalities were recorded in each Solvent Control and PAH-exposed
	group. Five categories of abnormalities were scored: 1-Edemas formation (brain, pericardia, yolk
	sac); 2-Skeletal deformities (scoliosis, lordosis, tail bud deformities); 3-Craniofacial deformities
	(jaw, development of eyes, head deformities); 4-Cardiac deformities (anemia, hemorrhage,
	atrium/ventricle size, blood circulation heart position); 5-Yolk sac malabsorption. Moreover, a

  . To summarize, 96 hpf larvae were incubated in 7-ethoxyresorufin for 5 hours. Levels of produced resorufin into gastrointestinal cavity were observed by fluorescence microscopy (excitation/emission, 560 nm/580 nm) and quantified by imaging analysis software ImageJ (Schneider et al., 2012) coupled with HeatMap Histogram plugin (Péan S., http://www.samuelpean.com/heatmap-histogram/). Fluorescence results were expressed in integrated density of pixels (arbitrary unit).

  Quantification procedure was based on fluorescence of reaction mixture which related to double-stranded DNA. This reaction mixture included Fast SYBR® Green Master Mix 5X (Applied Biosystems®, USA), 2.4 µL of primers at 600 nM (Eurofins MWG Operon, Germany), 5.6 µL of Milli-Q water and 2 µL of cDNA. Thermal cycling conditions were: enzymatic activation during 10 min at 95 °C, followed by 40 cycles including denaturation (95 °C, 15 s), annealing (60 °C, 40 s) and synthesis (72 °C, 30 s). Gene expression levels were quantified from the threshold cycle (CT) number and normalized to three

Table 1. Real-time PCR reactions were performed in sterile 96-well PCR plates with StepOnePlus™ instrument (Applied Biosystems®, Life Technologies USA) following the manufacturer's instructions. housekeeping genes: eef1, βactin, gapdh.

Table 1

 1 Specific primer sequences for the 11 target genes measured by real-time PCR.

	Target gene	Name	Accession Number	Primer (5' → 3')
	gapdh	glyceraldehyde-3-phosphate dehydrogenase	BC083506	GTGGAGTCTACTGGTGTCTTC a /GTGCAGGAGGCATTGCTTACA b
	eef1	eukaryotic elongation factor 1	NM_131263.1	CAGCTGATCGTTGGAGTCAA a /TGTATGCGCTGACTTCCTTG b
	βactin	beta-actin	NM_131031	CCCAGACATCAGGGAGTGATa/CACAATACCGTGCTCAATGGb
	cyp1A	cytochrome P4501A	BC094977	GACAGGCGCTCCTAAAACAG a /CTGAACGCCAGACTCTTTCC b
	ahr2	aryl hydrocarbon receptor 2	NM_131264	GCCTGGGATAAAGGAGGAAG a /CAGCTCCACCTGTCCAAAT b
	cat	catalase	NM_130912	CGCTTCTGTTTCCGTCTTTC a /CCCTGAGCATTGACCAGTTT b
	sod(Cu/Zn)	Cu/Zn-superoxide dismutase	BC055516	GTTTCCACGTCCATGCTTTT a /CGGTCACATTACCCAGGTCT b
	sod(Mn)	Mn-superoxide dismutase	CB923500	GGCCAAGGGTGATGTGAC a /ACGCTTTATGGCCTCCAAC b
	gpx4	glutathione peroxidase 4	NM_001007282.1	AGGATCCAAGTGTGGTGGAG a /GGGGTTTCCAAACAAACCTT b
	p53	bcl-2-binding component 3	NM_131327.1	GCTTGTCACAGGGGTCATTT a /ACAAAGGTCCCAGTGGAGTG b
	bax	bcl-2-associates X protein	AF231015	GGAGATGAGCTGGATGGAAA a /GAAAAGCGCCACAACTCTTC b
	Forward primer a /Reverse primer b

Table 1

 1 Specific primer sequences for the 11 target genes measured by real-time PCR.

	Target gene	Name	Accession Number	Primer (5' → 3')
	gapdh	glyceraldehyde-3-phosphate dehydrogenase	BC083506	GTGGAGTCTACTGGTGTCTTC a /GTGCAGGAGGCATTGCTTACA b
	eef1	eukaryotic elongation factor 1	NM_131263.1	CAGCTGATCGTTGGAGTCAA a /TGTATGCGCTGACTTCCTTG b
	βactin	beta-actin	NM_131031	CCCAGACATCAGGGAGTGATa/CACAATACCGTGCTCAATGGb
	cyp1A	cytochrome P4501A	BC094977	GACAGGCGCTCCTAAAACAG a /CTGAACGCCAGACTCTTTCC b
	ahr2	aryl hydrocarbon receptor 2	NM_131264	GCCTGGGATAAAGGAGGAAG a /CAGCTCCACCTGTCCAAAT b
	cat	catalase	NM_130912	CGCTTCTGTTTCCGTCTTTC a /CCCTGAGCATTGACCAGTTT b
	sod(Cu/Zn)	Cu/Zn-superoxide dismutase	BC055516	GTTTCCACGTCCATGCTTTT a /CGGTCACATTACCCAGGTCT b
	sod(Mn)	Mn-superoxide dismutase	CB923500	GGCCAAGGGTGATGTGAC a /ACGCTTTATGGCCTCCAAC b
	gpx4	glutathione peroxidase 4	NM_001007282.1	AGGATCCAAGTGTGGTGGAG a /GGGGTTTCCAAACAAACCTT b
	p53	bcl-2-binding component 3	NM_131327.1	GCTTGTCACAGGGGTCATTT a /ACAAAGGTCCCAGTGGAGTG b
	bax	bcl-2-associates X protein	AF231015	GGAGATGAGCTGGATGGAAA a /GAAAAGCGCCACAACTCTTC b
	Forward primer a /Reverse primer b

Table 3

 3 Developmental endpoints for assessing toxicity and teratogenicity of three aromatic fractions (PY, BAL, HFO) on 96 hpf offspring larvae from contaminated-genitors. Biometric values are means SEM.

			PY			BAL			HFO
		Solvent	0.3 X	1 X	Solvent	0.3 X	1 X	Solvent	0.3 X
		N=4 ; n=190	N=5 ; n=172	N=5 ; n=175	N=3 ; n=75	N=3 ; n=60	N=1 ; n=17	N=4 ; n=85	N=4 ; n=85
	Standard length (mm)	3.58 0.009 a	3.63 0.009 b	3.57 0.012 a	3.55 0.018 a	3.57 0.022 a	3.61	3.48 0.023 a	3.59 0.014 b
	Yolk sac surface (mm²)	0.26 0.002 a	0.24 0.002 b	0.25 0.002 c	0.27 0.0026 a 0.27 0.006 a	0.32	0.28 0.005 a	0.29 0.003 a
	Ratio Yolk sac/Whole larval surface	0.229 0.001 a 0.213 0.001 b 0.216 0.002 c	0.224 0.004 a 0.224 0.005 a 0.265	0.241 0.004 a 0.236 0.002 a
	Abnormal individuals (%)	6.34 1.58 a	11.1 2.20 a	8.48 3.03 a	21.9 2.01 a	25.9 2.26 a	0.00	19.8 5.13 a	18.0 3.50 a
	Severity of abnormality (Scoring/3) (%):
	No affected (Score 0)	93.7 1.58	88.9 2.20	91.5 3.03	78.1 2.01	74.1 2.26	100.0	80.3 5.13	81.0 4.25
	Mild (Score 1)	5.24 1.63	9.12 2.07	5.28 2.04	18.9 0.86	17.4 1.00	0.00	14.8 3.88	19.0 4.25
	Moderate (Score 2)	1.10 0.55	1.35 0.72	2.90 1.04	3.00 1.15	6.15 1.48	0.00	2.50 1.25	0.00 0.00
	Severe (Score 3 or more)	0.00 0.00	0.67 0.48	0.30 0.21	0.00 0.00	2.30 1.80	0.00	2.50 1.30	0.00 0.00
	Abnormalities among abnormal individuals (%):
	Edemas	2.19 1.09	1.10 0.59	0.94 0.67	8.67 1.47	6.87 2.65	0.00	14.4 6.41	10.3 5.14
	Axial Skeleton	1.46 1.10	8.47 1.74	4.58 1.80	7.29 0.40	12.3 6.81	0.00	1.44 0.72	7.72 2.39
	Craniofacial	2.09 0.73	0.79 0.57	2.21 0.92	2.50 0.96	1.15 0.88	0.00	1.44 0.72	0.00 0.00
	Cardiovascular	0.00 0.00	0.80 0.40	0.40 0.03	0.00 0.00	2.30 1.77	0.00	1.00 0.75	0.00 0.00
	Yolk sac Malabsorption	0.60 0.30	0.00 0.00	0.37 0.27	3.43 2.64	3.24 1.83	0.00	1.50 1.13	0.00 0.00
	Letters denote significant differences between experimental group (P<0.05).

Table 3

 3 Developmental endpoints for assessing toxicity and teratogenicity of three aromatic fractions (PY, BAL, HFO) on 96 hpf offspring larvae from contaminated-genitors. Biometric values are means SEM.

			PY			BAL			HFO
		Solvent	0.3 X	1 X	Solvent	0.3 X	1 X	Solvent	0.3 X
		N=4 ; n=190	N=5 ; n=172	N=5 ; n=175	N=3 ; n=75	N=3 ; n=60	N=1 ; n=17	N=4 ; n=85	N=4 ; n=85
	Standard length (mm)	3.58 0.009 a	3.63 0.009 b	3.57 0.012 a	3.55 0.018 a	3.57 0.022 a	3.61	3.48 0.023 a	3.59 0.014 b
	Yolk sac surface (mm²)	0.26 0.002 a	0.24 0.002 b	0.25 0.002 c	0.27 0.0026 a 0.27 0.006 a	0.32	0.28 0.005 a	0.29 0.003 a
	Ratio Yolk sac/Whole larval surface	0.229 0.001 a 0.213 0.001 b 0.216 0.002 c	0.224 0.004 a 0.224 0.005 a 0.265	0.241 0.004 a 0.236 0.002 a
	Abnormal individuals (%)	6.34 1.58 a	11.1 2.20 a	8.48 3.03 a	21.9 2.01 a	25.9 2.26 a	0.00	19.8 5.13 a	18.0 3.50 a
	Severity of abnormality (Scoring/3) (%):
	No affected (Score 0)	93.7 1.58	88.9 2.20	91.5 3.03	78.1 2.01	74.1 2.26	100.0	80.3 5.13	81.0 4.25
	Mild (Score 1)	5.24 1.63	9.12 2.07	5.28 2.04	18.9 0.86	17.4 1.00	0.00	14.8 3.88	19.0 4.25
	Moderate (Score 2)	1.10 0.55	1.35 0.72	2.90 1.04	3.00 1.15	6.15 1.48	0.00	2.50 1.25	0.00 0.00
	Severe (Score 3 or more)	0.00 0.00	0.67 0.48	0.30 0.21	0.00 0.00	2.30 1.80	0.00	2.50 1.30	0.00 0.00
	Abnormalities among abnormal individuals (%):
	Edemas	2.19 1.09	1.10 0.59	0.94 0.67	8.67 1.47	6.87 2.65	0.00	14.4 6.41	10.3 5.14
	Axial Skeleton	1.46 1.10	8.47 1.74	4.58 1.80	7.29 0.40	12.3 6.81	0.00	1.44 0.72	7.72 2.39
	Craniofacial	2.09 0.73	0.79 0.57	2.21 0.92	2.50 0.96	1.15 0.88	0.00	1.44 0.72	0.00 0.00
	Cardiovascular	0.00 0.00	0.80 0.40	0.40 0.03	0.00 0.00	2.30 1.77	0.00	1.00 0.75	0.00 0.00
	Yolk sac Malabsorption	0.60 0.30	0.00 0.00	0.37 0.27	3.43 2.64	3.24 1.83	0.00	1.50 1.13	0.00 0.00
	Letters denote significant differences between experimental group (P<0.05).

Table 3

 3 Developmental endpoints for assessing toxicity and teratogenicity of three aromatic fractions (PY, BAL, HFO) on 96 hpf offspring larvae from contaminated-genitors. Biometric values are means SEM.

			PY			BAL			HFO
		Solvent	0.3 X	1 X	Solvent	0.3 X	1 X	Solvent	0.3 X
		N=4 ; n=190	N=5 ; n=172	N=5 ; n=175	N=3 ; n=75	N=3 ; n=60	N=1 ; n=17	N=4 ; n=85	N=4 ; n=85
	Standard length (mm)	3.58 0.009 a	3.63 0.009 b	3.57 0.012 a	3.55 0.018 a	3.57 0.022 a	3.61	3.48 0.023 a	3.59 0.014 b
	Yolk sac surface (mm²)	0.26 0.002 a	0.24 0.002 b	0.25 0.002 c	0.27 0.0026 a 0.27 0.006 a	0.32	0.28 0.005 a	0.29 0.003 a
	Ratio Yolk sac/Whole larval surface	0.229 0.001 a 0.213 0.001 b 0.216 0.002 c	0.224 0.004 a 0.224 0.005 a 0.265	0.241 0.004 a 0.236 0.002 a
	Abnormal individuals (%)	6.34 1.58 a	11.1 2.20 a	8.48 3.03 a	21.9 2.01 a	25.9 2.26 a	0.00	19.8 5.13 a	18.0 3.50 a
	Severity of abnormality (Scoring/3) (%):
	No affected (Score 0)	93.7 1.58	88.9 2.20	91.5 3.03	78.1 2.01	74.1 2.26	100.0	80.3 5.13	81.0 4.25
	Mild (Score 1)	5.24 1.63	9.12 2.07	5.28 2.04	18.9 0.86	17.4 1.00	0.00	14.8 3.88	19.0 4.25
	Moderate (Score 2)	1.10 0.55	1.35 0.72	2.90 1.04	3.00 1.15	6.15 1.48	0.00	2.50 1.25	0.00 0.00
	Severe (Score 3 or more)	0.00 0.00	0.67 0.48	0.30 0.21	0.00 0.00	2.30 1.80	0.00	2.50 1.30	0.00 0.00
	Abnormalities among abnormal individuals (%):
	Edemas	2.19 1.09	1.10 0.59	0.94 0.67	8.67 1.47	6.87 2.65	0.00	14.4 6.41	10.3 5.14
	Axial Skeleton	1.46 1.10	8.47 1.74	4.58 1.80	7.29 0.40	12.3 6.81	0.00	1.44 0.72	7.72 2.39
	Craniofacial	2.09 0.73	0.79 0.57	2.21 0.92	2.50 0.96	1.15 0.88	0.00	1.44 0.72	0.00 0.00
	Cardiovascular	0.00 0.00	0.80 0.40	0.40 0.03	0.00 0.00	2.30 1.77	0.00	1.00 0.75	0.00 0.00
	Yolk sac Malabsorption	0.60 0.30	0.00 0.00	0.37 0.27	3.43 2.64	3.24 1.83	0.00	1.50 1.13	0.00 0.00
	Letters denote significant differences between experimental group (P<0.05).

Table 1

 1 Supplementary dataTarget compounds (ng.g -1 ) measured in spiked-food with three aromatic fractions (PY, BAL, HFO) during parental exposures. Values are means ± standard deviation (SD) (N=4-7) and values without SD indicate the compound was identified in one sample.

	PY Aromatic Fraction	BAL Aromatic Fraction	HFO Aromatic Fraction

Acknowledgments: Financial support for the PhD grant was received by PP from both the Conseil Général de Charente Maritime and the Institut Français de Recherche pour l'Exploitation de la Mer (Ifremer). This work was supported by the French Agence Nationale pour la Recherche (ANR), "Contaminant, Ecosystème et Santé" program, in the framework of the ConPhyPoP (2009-002) research project and CPER A2E. This project is co-financed by the European Union with the European fund of regional development. The authors would like to thank the CEDRE for providing the crude oils necessary to the extraction of aromatic fractions. J. Lucas, L. Lyphout and C. Vignet are thanked for their help with zebrafish breeding maintenance, L. Joassard for performing the qPCR analyses and D. Leguay for technical support on zebrafish behavioral assays. This study was conducted with the approval of the Animal Care Committee of France under the official license of Marie-Laure Bégout (17-010).


Nicolás I Segovia

Julio A Vásquez

Sylvain Faugeron

Pilar A Haye
email: phaye@ucn.cl

On the advantage of sharing a holdfast: effects of density and 1 occurrence of kin aggregation in the kelp Lessonia berteroana 2 3

Keywords: fusion of individuals, density dependent, genetic relatedness, kelp, kin 23 aggregation, kin selection, microsatellites

Here we investigate the density-dependent and genetic relatedness that regulate the occurrence 26 of inter-individual (genet) fusion forming plurigenotypic organisms in the brown alga 27 Lessonia berteroana. Recruitment generally occurs at high densities in the intertidal, allowing 28 contact of neighbouring holdfasts as they grow and expand on the substrate. Algal density, on 29 the other hand, is regulated by the effects of herbivory and wave impact, which often lead to 30 low holdfast density. Herein, we investigated whether the occurrence of plurigenotypic 31 organisms and their genotypic composition (number of genotypes per plurigenotypic 32 organism) are density dependent and affected by kin selection in the intertidal kelp L. 33 berteroana. Four microsatellite loci were used to analyse DNA from 260 samples obtained 34 from shared and non-shared holdfasts, at two sites with high and two with low holdfast 35 density. Analyses showed that fusions forming plurigenotypic organisms are extremely 36 common. Interestingly, the frequency of fusions was higher in low-density sites, in which 37 100% of the plants had at least two genotypes while the average was 3.5. In high-density sites, 38 62% of plants where plurigenotypic, with an average of 2.8 genotypes per plant. Additionally, 39 we found that genotypes that shared a holdfast had a significantly higher genetic relatedness 40 than on average in the population, compatible with a kin structure. Density dependence and 41 kin structure suggest that the occurrence of plurigenotypic organisms is linked to 42 environmental quality, and that kin or multilevel selection may be favouring the fusion of 43 genetically related genets. 44 such as the population, the family or group of related individuals, the colony, and the 47 individual organism. Because most of these units can actually be nested one into the other 48 (e.g. individuals into colonies or families, colonies into populations; see Nachtomy et al. 49 2002), the limits of the individual, as a basic ecological and evolutionary unit, are 50 sometimes difficult to define. In fact, individuals of many species live in groups into which 51 they find both protection against environmental stresses (e.g. predation) and closeness to 52 other individuals for reproduction. The case of clumps and colonies is particularly 53 interesting because the functional limits between the genet, issued from a single fertilized 54 egg, and the individual organism that is made of a mixture of different genets, are most 55 often indistinguishable. Such findings have stimulated important debate around the concept 56 and definition of the individual and the organism (e.g. Nachtomy et al. 2002; Santelices 57 1999; Pepper & Herron 2008). Clumps have been reported in a wide variety of taxa, some 58 of which are algal species (Santelices et al.

Introduction 45

Different units of relevance for ecological and evolutionary processes can be delineated, first case, fusions are just a density-dependent process whereas selection and adaptation can 72 be invoked in the second hypothesis. Evidence based on higher survival rate of aggregates 73 compared to isolated individuals of red and brown algae suggest a selective advantage of 74 clumping [START_REF] Santelices | Group recruitment and early survival of Mazzaella 576 laminarioides[END_REF][START_REF] Santelices | Demographic consequences of coalescence in sporeling 578 populations of Mazzaella laminaroides (Gigartinales, Rhodophyta)[END_REF], Wernberg 2005). For 75 instance, holdfast aggregations of the kelp Ecklonia radiata have been observed in higher 76 frequency in exposed areas than in more protected ones, suggesting that aggregation 77 reduces mortality from dislodgement in exposed areas (Wernberg 2005). protective), increasing herbivory. Thus, both herbivory and WE have density-dependent 114 effects in both intertidal and subtidal Lessonia species (e.g. L. trabeculata, Vásquez 1992;115 Vásquez & Buschmann 1997) that lead to greater survival of plants at high holdfast density.

116

High holdfast density implies intraspecific competition for substrate and light, 117 limiting the settlement of new recruits [START_REF] Santelices | Recruitment, growth and survival of Lessonia nigrescens 587 (Phaeophyta) at various tidal levels in exposed habitats of central Chile[END_REF][START_REF] Andrew | Effects of wave exposure and intraspecific density on the 476 growth and survivorship of Sargassum muticum (Sargassaceae: Phaeophyta)[END_REF] 118 [START_REF] Steen | Intraspecific competition in Fucus serratus and F. evanescens 592 (Phaeophyceae: Fucales) germings: effects of settlement density, nutrient 593 concentration, and temperature[END_REF]. In this context, fusions between conspecifics may be seen as a 119 way to increase individual density without increasing substrate occupancy and to avoid 120 intraspecific competition. This kind of strategy has been found more frequently between 121 kin than unrelated individuals (Gerlash et al. 2007, Lizé et al. 2012). For example, plants 122 effectively experience strong social interactions and kin recognition to varying 123 physiological and morphological responses depending on the identity of the neighbour (de 124 [START_REF] De Kroon | Ecology: how do roots interact? Science, 318, 1562-1563. 502 parasitoid aggression in resource com-petition[END_REF][START_REF] Biedrzycki | Root exudates mediate kin recognition 483 in plants[END_REF], Wu et al. 2013). However, as the Hamilton's (1964) 125 rule predicts, fusions may be an adaptive trait only if the benefits overpass the costs, and 126 close contact among individuals within a holdfast is likely to induce strong competition 127 among them (Novoplansky 2009). This cost can probably be reduced if fusion occurs 128 among relatives, so that benefits are expressed in terms of inclusive fitness (i.e. the direct 129 contribution of each genotype to the next generation's gene pool plus their indirect 130 contribution through the progeny of their relatives). Therefore, if fusion between relatives 131 enhances survival rate, then positive kin selection is a likely explanation (File et al. 2013).

132

In this context fusions between relatives in L. berteroana would be more frequent in low 133 environmental qualities (i.e. low density) in were isolated individuals (unigenotypic 134 organisms) may not persist. 8 holdfasts/m 2 ). We use the term plant to refer to the organism identifiable macroscopically 148 on the shore, and which may be made of either a single or multiple genets.

149

From each of the 48 sampled plants, six tissue samples were collected each from a 150 different stipe selected at random, except for some small plants (i.e. less than six stipes). In 151 these cases, the samples were taken from all the available stipes (at least 5). Fresh tissue 152 samples of 2 cm in diameter were collected from the base of fronds avoiding reproductive Pyrolidone to discard polyphenols [START_REF] Martínez | Recovery and genetic diversity of the intertidal 535 kelp Lessonia nigrescens (Phaeophyceae) 20 years after EL NIÑO 1982/83[END_REF]. Extracted DNA was quantified in 158 a spectrophotometer (NanoDrop Technologies) and kept at -20ºC.

To account for the effect of plant size on the frequency of fusions, three plant-to-160 plant morphometric values were recorded for each sampled plant: maximum length, 161 holdfast diameter and number of stipes. In brown algae growth is highly related to 162 microhabitat conditions, thus kelp size is not necessarily related with age. In order to 163 consider genet-to-genet morphology, i.e., differences within the plant, for each sampled 164 stipe, we recorded the length, diameter, and number of dichotomies. The main criteria for considering loci as informative were the degree of 201 Finally, differences in the number of genotypes between densities were evaluated in a 202 single GLMM using density (D) as categorical variable following the same structure than 203 previous models.

204

Strictly, site has a random effect because all levels of that factor are selected as a 10 random sample from all possible levels (sites) that could be included in the study area 206 (spatial pseudoreplication). So additionally, as an exploratory analysis of the relative 207 importance of the factors in the models, according to the evidence weight of the AIC, the 208 site effect was evaluated as explanatory variable with fixed effects, and in interaction with 209 variables that GLMMs showed to have a significant effect on number of genotypes. (e.g., all five stipes with a different multilocus genotype) (Fig. 1).

251

The evaluation of the effects of plant-to-plant MV using GLMM's revealed that 252 neither total length nor holdfast diameter influenced on the number of genotypes per 253 holdfast (df: 41, p= 0.1130 and 0.1891, respectively), even when interacting with density 254 (df: 41, p= 0.0970 and 0.0879, respectively) (Table 1). Interestingly, the number of 255 genotypes varied significantly with the number of stipes per holdfast (df: 41, p = 0.00721) 256 with a significant effect of density in this model (df: 41, p = 0.00261), although the 257 interaction of density with the MV was not significant (df: 41, p= 0.15932) (Table 1). The 258 effect of the size, particularly with the number of stipes as response variable was significant 259 only in HD, while in LD there was no relationship (Fig. 2). Moreover, holdfast density had 260 a significant effect in the average number of genotypes per holdfast (df: 41, p = 0.0036)

261

(Table 1). In LD 100% of the holdfasts were plurigenotypic, whereas only 62.5 % at HD.

262

The average number of genotypes was also greater in LD, with 3.54 genotypes/holdfast, 263 versus 2.79 genotypes/holdfast in HD. This suggests that plant density explains the 264 observed data better than MV; a marginal association between number of stipes and 265 number of genotypes can be observed only in HD (Fig. 2).

266

Exploring the relative weights of each factor in the models considering the Site (St) as

272

In the PCA analyses, using genet-to-genet morphological variables, the first two axes 273 explained 95.67% of the total variance. The first axis was composed by the three variables 274 that had similar loadings, and the second axis was mainly of stipe diameter, suggesting that 275 the three genet-to-genet variables are associated to morphological variation between 276 densities. The PCA showed two groups with an overlap of few genets that were 277 significantly differentiated (one-way ANOVA; F 1,258 =52.59, p<10 -12 ) (Fig. 3). In this 278 analysis, results showed that genets in LD are in general smaller than in HD. The trend is 279 more evident for stipe diameter which tends to be thinner in LD than HD. The LDA 280 approach, with one canonical discriminant function (i.e. two classes HD-LD), showed that 281 the stipe diameter was the variable with the highest discriminant coefficient. This From a total of 39 POs, 15 in HD and 24 in LD, 30 (i.e. 77%) displayed an intra-plant mean 291 relatedness significantly higher than expected given the inter-plant pairwise relatedness in 292 the population (Table 3). From these 30 holdfasts, 66.6% occurred in HD and 79.16% in 293 LD. There was strong heterogeneity in intra-plant relatedness: site A in HD (SA-HD) had 294 the lowest number of plants (three out of seven) with significantly higher mean relatedness 295 than population level, whereas every PO in the site B (SB-HD) displayed significantly higher intra-plant mean R (Table 3). In LD, where relatedness is more common, most intra- This has been proved to explain the early benefits of coalescence in microscopic stages of 319 red algae [START_REF] Santelices | Sporeling coalescence and 581 intraclonal variation in Gracilaria chilensis (Gracilariales, Rhodophyta)[END_REF][START_REF] Santelices | Group recruitment and early survival of Mazzaella 576 laminarioides[END_REF], which show a positive 320 relationship between the number of coalescing spores and the probability of survivorship 321 [START_REF] Santelices | 584 Convergent biological processes in coalescing Rhodophyta[END_REF]Santelices 2001;[START_REF] Nelson | Life history and growth in culture of the endemic New Zealand kelp 544 Lessonia variegata J. Agardh in response to differing regimes of temperature, 545 photoperiod and light[END_REF]. Small plants (i.e. less than 5 stipes) of L. 322 berteroana were found at both densities, but in LD every small plant is formed of up to five 323 multilocus genotypes, giving further support for the early selection of PO's against 324 unigenotypic organisms in LD. On the other hand, high densities seem to result from the 325 survival of both uni-and pluri-genotypic organisms, at least during early stages.

326

There are three main ways of coalescing at the microscopic stages of kelps: (1) 327 aggregated settlement of spores within a few mm 2 producing a single PO when observable

354

In addition to that, our ordination analyses interestingly showed that plant density could be An important result of this study was the observation that genotypes sharing a holdfast 377 were more genetically related than on average in the population. This kin aggregation is not 378 expected under the paradigm of stochastic spore settlement (i.e. with no choice of the 379 settlement site). However, kin aggregation is increasingly being detected in coastal species 380 including sessile (Veliz et al. 2006) and mobile invertebrates (Selkoe et al. 2006), 381 evidencing that mixing of propagules in the water column is less extensive than previously 382 considered.

383

At least three non-exclusive hypotheses could explain this strong trend, each as 384 post-settlement processes. First, higher R-values can result from fusions between 385 sporophytes sharing the same mother and multiple fathers (siblings or half-siblings).

386

Vegetative ramification and multiple egg production of the female gametophyte is a 387 common process in Laminariales [START_REF] Muñoz | 538 Variability in per capita oogonia and sporophyte production from giant kelp 539 gametophytes (Macrocystis pyrifera, Phaeophyceae)[END_REF][START_REF] Nelson | Life history and growth in culture of the endemic New Zealand kelp 544 Lessonia variegata J. Agardh in response to differing regimes of temperature, 545 photoperiod and light[END_REF]). To determine whether genotypes do correspond to full or half-sibs, a higher number of loci would be required in 389 order to reduce the large sampling variance that lowers the precision of estimated 390 relatedness [START_REF] Lynch | Estimation of pairwise relatedness with molecular markers[END_REF]. Second, dispersal and settlement may be non-random.

391

Kin-structured dispersal (the joint dispersal of seeds or juveniles that come from a same 392 family or a same mother) is an example of process that leads to kin aggregation in the adult 393 stage. It explains some cases of small-scale genetic structure in plants (Torimaru et al. 394 2007) and genetic patchiness in marine invertebrates (Johnson & Black 1982). Such 395 dispersal modes have been reported in some seaweeds that bear unitary reproductive organs 396 like the cystocarp in red algae, which can release bunches of spores surrounded by their 397 maternal mucilage that keeps the spores together until settlement [START_REF] Aedo | Esporas de algas rojas coalescentes y no coalescentes: diferencias 472 funcionales y factores ambientales afectado la coalescencia[END_REF]). This 398 phenomenon seems unlikely to apply to kelps, which release motile spores individually that 399 can hardly maintain proximity with their relatives because of the turbulence of the coastal 400 waters were they are released.

401

The third hypothesis argues that dispersal and settlement are random but survival of 402 the young sporophytes is determined by the genetic relatedness of the surrounding 403 individuals. If fusions between genets are environmentally mediated, it is possible that the 404 aggregation between relatives provides higher benefits to the PO than non-kin aggregates 405 would. This scenario is possible whenever intra-plant competition is reduced by the genetic 406 relatedness of individuals sharing the holdfast. This is particularly noteworthy because it 407 opens to a potential role of selection in determining the occurrence of kin aggregations 408 within plants. It is not necessarily kin selection, as it is known for social animal species, 409 particularly because the existence of high relatedness between interacting individuals is not 410 by itself sufficient evidence that kin selection is the driving force (Griffin & West 2002). In 411 the case of L. berteroana, genotypes within a kin aggregate may have a higher survival up 412 to the adult stage than those within non-kin aggregates, as evidenced by the predominance 413 of PO in low-density areas, suggesting that kin aggregation brings fitness advantages over 414 random interaction. In this context, every life history trait that favours the fusion between 415 relatives to form PO should be selected for, including traits at the group level (File et al.

78

  Based on field observations of tagged sporophytes, Vásquez et al. (2008) and 79 Rodriguez et al. (2014) showed that fusions of individuals forming a PO might be recurrent 80 in the kelp Lessonia berteroana Montagne (formerly L. nigrescens, González et al. 2012). 81 This species dominates the low intertidal rocky shores of Chile and southern Peru 82 (Hoffmann & Santelices 1997) and its structure consists of a massive holdfast attached to 83 the rocky substrate. A variable number of stipes emerge from the holdfast. Stipes have 84 branches and each branch carries one frond distally. Holdfasts grow vertically and 85 horizontally leading to contact between neighbours that progressively grown and their 86 tissues mix forming a single unit that externally mimicks a single organism (Vásquez et al.

  simple expectation from this growth pattern is that the rate at which genets fuse 88 increases with density (Rodriguez et al. 2014). However, if fusions confer some kind of 89 advantage under stressful environmental conditions, then the occurrence of fusions should 90 6

135

  The goal of this study was to shed light onto the factors that regulate the fusion of 136 genets into a single organism, by assessing genetic diversity of holdfasts at low and high 137 plant density and the genotypic relatedness within plants in the kelp L. berteroana. L. berteroana were sampled in an extensive intertidal kelp bed in Lagunillas, 144 Chile (30º06'S, 71º22'W) on areas showing no evidence of recent kelp harvest. In order to 145 test for density dependence, twelve plants of different sizes were sampled in two sites of 146 low holdfast density (LD: 0.5 to 1 holdfasts/m 2 ) and two of high holdfast density (HD: 5 to 147

  153 tissue and epiphytes. Collected pieces were dried using paper towel, placed in sealed 154 individual bags with silica gel, and stored at room temperature until DNA extraction. DNA 155 was extracted from 30-40 mg of dried powdered tissue using a slightly modified version of 156 the Cetyltrimethyl Ammonium Bromide (CTAB) DNA extraction that adds Polyvinyl

  157

  loci were informative for the study, ten DNA samples from different 169 plants were randomly picked to explore eight microsatellite loci available for L. berteroana 170 (Faugeron et al. 2009). PCR's were performed in a final volume of 20 µl with: 1.5 ng of 171 DNA, 2 mM of 10X Buffer, 0.6 mM of dNTP mix, 1.8 mM of MgCl 2 , 0.2 mM of each 172 primer, 0.075 mg/mL of Bovine Serum Albumin, and 0.2 U of Taq DNA polymerase 173 (Fermentas). Temperature cycling consisted of an initial soak of three min at 95 °C, then 10 174 touchdown cycles at 95 °C for 45 sec, 60 °C-63 °C for 45 sec, and an extension of 45 sec 175 at 72 °C. Following, 30 cycles at 95 °C for 45 sec, 50 °C for 45 sec, 72 °C for 45 sec and a 176 final extension of 72 °C for 7 min. Amplicons were genotyped in an ABI Prism 3700 177 (Applied Biosystems) and the electrophenograms were visualized using GeneMarker v.

  179

180

  polymorphism in the local population and PCR amplification success. After checking for 181 polymorphism, genotypes were analysed in Microchecker 2.2 (Van Oosterhout et al. 2004) 182 to determine the probability of null alleles. The probability of finding twice the same 183 multilocus genotype by chance in each site of each density was calculated with Genealex v. 184 6 (Peakall & Smouse 2006). It was considered a sufficient number of informative loci when 185 each of the ten analysed DNA samples had different multilocus genotypes.

  more than one multilocus genotype was considered a plurigenotypic 190 organism (PO). The effect of size and density was analysed using Generalized Linear 191 Mixed Models (GLMMs) with Poisson error implemented in the lme4 package in the 192 software R (R Core Team 2013). GLMMs are appropriate for non-normal data influenced 193 by fixed and random effects (Bolker et al. 2008; Crawley 2005; Grueber et al. 2011). In 194 each model, effects and interaction of plant-to plant morphometric values (MV) (holdfast 195 diameter, total length, and total number of stipes) and density (high and low denoted as LD 196 and HD, respectively) in the occurrence of POs was evaluated using each of the three MV 197 as covariables with the Site (St) nested into Density. The correlation between plant length 198 and holdfast diameter is well known in Lessonia (Santelices 1982; Vásquez & Santelices 199 1984; Vásquez 1991), however, the correlation of these variables and the number of stipes 200 is not consistent and only occurs when reproductive stipes are considered (Vásquez 1991).

210

  Lastly, Principal Component Analyses (PCA) and Lineal Discriminant Analysis 211 (LDA) were carried out with the log-transformed data of the three genet-to-genet variables 212 (length, diameter of the stipe and number of dichotomies) and the three plant-to-plant MV 213 variables (holdfast diameter, total length, number of stipes). The objective of these analyses 214 was to observe possible differences in both densities considering genet-to-genet variance as 215 a way to include the morphological differences within the plant and plant-to-plant 216 differences between densities. Both analyses were carried out using MASS package, and 217 the 95% confident ellipses where calculated with ELLIPSE package, both available in 218 CRAN R project (R Core Team 2013).

  occurrence of kin aggregations within a PO, we tested whether 222 genotypes sharing a holdfast were genetically closer than on average in the population. This 223 was achieved by comparing the average pairwise coefficient of relatedness R (Ritland 224 1996) of each plant with the average inter-plant pairwise R. The upper limit of the one-225 tailed 95% confidence interval for inter-plant R-values for each site was determined by 226 bootstrapping in R (R Core Team 2013) 10,000 values of mean R among ten randomly 227 12

  267a fixed predictor, the model of the interaction of Size and Density showed low values in 268 terms of relative weights (according to the AICs), suggesting that density and size did not 269 act together as explanatory variables on the occurrence of POs and the number of genotypes 270 per holdfast. Site-to-site density variations seem to explain the differences between the 271 number of POs and number of genotypes per PO in each model (Fig.1).

  282discriminant function was capable to correctly assign 89.7% of the samples to their density.283Wrong predictions were in almost all the cases (except for one) for HD samples assigned as 284 LD. On the other hand, using plant-to-plant MV, the PCA showed only one 285 undistinguishable group and the LDA could correctly assign the plant to the density of 286 origin in 47.9% of the cases.

297

  holdfast pairwise R-values are significant with only two and three POs from SA-LD and 298 SB-LD, respectively, with values within the expected range of inter-plant values. that the formation of plurigenotypic organisms (POs) in L. berteroana 305 is a highly frequent phenomenon. Thirty-nine of the forty-eight analysed plants bared at 306 least two different genets. As for a number of red and green coalescing algae (Santelices et 307 al. 1999; 2003), fusion of L. berteroana at the holdfast level results in a single macroscopic 308 organism in which the different genets are indistinguishable (Rodriguez et al 2014). The 309 average number of genotypes per holdfasts was surprisingly high, with 11 out of 35 POs 310 bearing at least 5 genotypes. Together with the observation that fusions were more frequent 311 in low-density (LD) than in high-density (HD) areas, the results indicate that the occurrence 312 of fusions is not a simple consequence of holdfast proximity. Therefore, fusions do not 313 occur during the plants' ontogeny, but instead as a consequence of reduced space when 314 plants grow and the increased distance between plants. Rodriguez et al. (2014), shows that 315 the coalescence in L. berteroana is a continuous process in which recruits may fuse with 316 other isolated recruits, groups or even in and between adults and senescent plants 317 (Rodriguez et al. 2014).

  318

  328 by naked eye; (2) recruitment of spores on top of established holdfasts; and (3) vegetative 329 ramification of the female gametophyte that then produces multiple eggs (Avila et al.330 1985) that could be fertilized by different males. This study was not designed to 331 discriminate among these possibilities. However, two major findings of this study allow 332 inferring on the causes of the fusions. On one hand, aggregated settlement should vary 333 among sites and be negatively correlated to population density in order to explain the 334 higher occurrence of PO in LD than in HD. On the other hand, multiple paternities in a 335 single ramified female gametophyte should create half-sibs relatedness, increasing the 336 degree of genetic relatedness among genotypes within a plant, compared to the population. 337 Alternatively, both aggregated and non-aggregated settlements occur everywhere, but only 338 closely settled gametophytes survive and reproduce. These points are discussed in the 339 following sections.

340 341

 340 Fusions of individuals as an environmentally mediated process16 343 The occurrence of intraspecific fusion in L. berteroana seems strongly correlated with 344 environmental quality, as well as in other kelps (Wernberg 2005, Malm & Kausky 345 2004). Indeed, differences in population density of L. berteroana result from environmental 346 quality, which is heterogeneous, and optimal conditions favour high densities. However, 347 the negative correlation between holdfast density and occurrence of POs strongly suggests 348 that POs have certain advantages over non-PO in sub-optimal conditions, i.e. in low-density 349 areas. Unigenotypic organisms only occur at HD accounting for 75% of the small plants 350 with less than five stipes, suggesting that the optimal environmental conditions allow 351 recruitment and survival of the different kinds of organisms (uni-and plurigenotypic), as 352 opposed to LD. Similar results were obtained by Malm & Kausky (204) in Fucus 353 vesiculosus in which the proportion of fused individuals was greater in wave exposed areas.

355

  effectively differentiated according to the morphology of the stipes. As is suggested by 356Novoplasnky (2009), plant morphology can be modified as a response to competition in 357 contrasting densities.358Our results suggest that the quality of the environment on L. berteroana influences 359 both the occurrence and composition of the POs and the stipe-to-stipe intra-plant 360 morphology. High density implies intraspecific competition for substrate and light. 361 Concordantly, a positive correlation between mortality of recruits and plant density has 362 been reported for other algae (e.g. of the genus Fucus, Steen & Scrosati 2004). As the 363 presence of adult conspecific inhibit recruitments (Santelices & Ojeda 1984), fusion poses 364 an additional challenge to each individual genotype, by potentially adding intra-plant 365 competition on top of intra-population competition. A precise evaluation of intra-specific 366 competition between genets within and between plants is necessary to further understand 367 the balance between both the environmental quality and the intraspecific competition. 368 Finally, our results add complexity to demographic studies in L. berteroana that 369 thus far have considered the whole plant as the individual entity (e.g. Santelices & Ojeda 370 1984, Ojeda & Santelices 1984). Our finding suggests than rather than the whole plant, it is 371 important to consider the stipes, which take into account possible differences between 372 genets into the clump.

  Such multilevel selection (Wilson 1997) on group-living traits in kelps is an 417 interesting and testable hypothesis that emerges from the pattern observed in this study. 418 419 Perspective: Fusion of individuals as an adaptive strategy 420 421 Traditionally, L. berteroana has been considered as a unitary, aclonal species (sensu 422 Santelices 1982), wherein each plant corresponds to one individual (e.g. Ojeda & Santelices 423 1984; Santelices & Ojeda 1984). Here, we showed that one organism, made of a single 424 holdfast and a variable number of stipes and fronds can be composed of one or more 425 individuals, and large kelps are most likely colonies of different genets. As several other 426 intertidal species of red or green algae share this fusion capacity, it is possible that the 427 phenomenon of coalescence in algae is an adaptation to highly stressful and heterogeneous 428 environments such as the marine intertidal rocky shore. Direct benefits (i.e. higher survival, 429 protection of the recruits) to the individual genet when integrated within a PO lead to the 430 question whether fusion or coalescence is an adaptive strategy. This may be difficult to 431 answer because it requires characterizing traits of the PO that influence the individual 432 genet, as well as the relative contribution of PO's traits and individual genets traits to the 433 fitness of the genet. It requires also measuring the exact costs and benefits of being part of a 20 PO. So far, we have shown that the number of stipes of the PO is correlated with the 435 number of genotypes, and in LD stipes tend to be thinner than HD. The number of stipes is 436 a good indicator of the reproductive potential in L. berteroana (Santelices & Ojeda 1984), 437 and therefore the observed morphological differences could mean fitness differences in 438 terms of reproductive success. 439 At the individual level, the higher number of stipes in PO may indicate that the trade-off 440 between sharing the holdfast and the opportunity to produce external structure (the stipes) 441 is reduced, as opposed to a situation in which the number of stipes would be fixed by 442 external factors. The predominance of POs in low density areas suggests that benefits 443 largely exceed the eventual costs of sharing a holdfast, despite that plants tends to be 444 smaller in low-than in high-density areas. Better evaluation of these trade-offs (e.g. by 445 quantifying the number of stipes that each genotype is able to produce within a shared 446 holdfast, compared to a solitary holdfast) would allow an exact assessment of these costs 447 and benefits at the individual level. 448 Santelices (1999) showed that genetic homogeneity and uniqueness as well as 449 physiological autonomy of algae, are relevant criteria to define an individual. In this 450 context, the limits of an individual, as a functional basic unit of organization, may vary 451 according to the questions and approaches that focus on the model organism (Pepper & 452 Herron 2008). For example, ramets, clones, colonies and clumps can be defined as 453 individual organisms, although none of these would fit into the more traditional unitary 454 organism concept (Santelices 1999). While an important debate still holds around universal 455 definitions of individual and organism (Nachtomy et al. 2002; Santelices 1999, Pepper & 456 Herron 2008 among others), the ecological and evolutionary implications of different ways 457 of organization have received relatively little attention. The propensity of most species to 458 form groups has been considered as an adaptation that may maximize the inclusive fitness 459 of the individual-genotype. The adaptation of these concepts traditionally used in social 460 animals to non-animal models is a most challenging perspective of the study of 461 plurigenotypic organisms.462

Fig. 1 .

 1 Figure Captions

  661

Fig. 2 .Fig. 3 .

 23 Fig. 2. Relationship between the number of multilocus genotypes per plant and the number

  Figure 2

Table captions 640 641 Table 1 .

 captions6411 Summary of the GLMM models using morphometric values of the plants. Table642shows the result of comparing high holdfast density (HD) with the other levels of the

	643
	644	factor. Ng: Number of genotypes; D: Density; St: sites; AIC: Akaike Information Criterion.
	645	* Significant values (p < 0.05).
	646

Table 2 :

 2 Summary of the GLMM model testing the effect of Density (D) on the Number of

	647
	648	Genotypes (Ng). St: sites; AIC: Akaike values; k: number of variables. * Significant values
		(p < 0.05).

Table 1 .

 1 Summary of the GLMM models using morphometric values of the plants. Table671shows the result of comparing high holdfast density (HD) with the other levels of the 672 factor. Ng: Number of genotypes; D: Density; St: sites; AIC: Akaike Information Criterion.

Table 3 .

 3 Estimates of intra-and inter-plant pairwise relatedness R-values. The name of the 700 sampled plant with more than one genotype is between brackets after each R-value. * 701 Indicates values significantly higher than expected from one-tailed 95% confidence interval 702 of mean inter-plant pairwise R-values.

	703

Acknowledgements 464

The authors thank Alonso Vega, Nicole Piaget, Alfonso Gonzalez, Horacio Bastías and

when distance between holdfasts exceeds 2 m, the WE is reduced (i.e. environment is less Cristian Jofré for fieldwork assistance; Raúl Vera and Javier Tapia for laboratory 466 assistance; Marcelo Rivadeneira, Jacqui Shykoff and Florence Tellier, for valued comments 467 that improved this manuscript. This study was partially supported by FONDECYT 468 1090742 to SF and CEAZA and INCAR (FONDAP 15110027) to PH.