Wafa Achouak

Arnaud Laveilhé

Sylvain Fochesato

Marie Bertrand-Huleux

Joris Tulumello

Mohamed Barakat

Thierry Heulin

Phytobeneficial Traits under the Control of Multiple Crosstalks' Belowground

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

Antifungal activity of P. brassicacearum NFM421 Wt, ∆phlD and ∆gacA towards Fusarium culmorum (Fc), Fusarium graminearum (Fg), Microdochium nivale (Mn).

Expression of phlD and hcnA in P. brassicacearum NFM421 grown alone, or in competition with K. sacchari NO9 (NF/NO9), R. alamii YAS34 (NF/YAS), or both competitors (Triple) in the absence (green bars) or in the presence (red bars) of 300 µM FeCl 3 .

In planta analysis of the expression (q-RT-PCR) of phlD and hcnA in P. brassicacearum In planta colocalization of rfp-tagged P. brassicacearum NFM421 with: gfp-tagged R. alamii YAS34 (A), and gfptagged K. sacchari NO9 (B).

NFM421
A B

Conclusion

Microbial cultures and growing conditions Fungal-bacteria interaction experiments were performed using potato dextrose agar (PDA) medium.

Bacterial competitions: pure culture and co-culture in CAA medium +/-300 µM FeCl 3.

phlD and hcnA gene expression RNA extracted from bacteria, retro-transcribed using phlD or hcnA primers. Real-time PCR was performed in triplicate, and mRNA relative expression was normalized to the 16S reference gene.

In planta phlD and hcnA gene expression Surface sterilized seeds of Brassica napus were inoculated with one or a mixture of the three bacterial species. Correlations with a p-value >0.05 (Spearman correlation test) were selected to build networks (package igraph 1.2.2 using R 3.4.3). The determination of the betweeness and degree for every nodes and their size made it possible to identify the hubs.

Colocalization of competing bacteria

Relative gene expression

Relative gene expression

  Expression of the P. brassicacearum NFM421 genes phlD and hcnA grown alone (NF), or in the presence of Fusarium culmorum (NF vs. Fc), Fusarium graminearum (NF vs. Fg), or Microdochium nivale (NF vs. Mn).

  grown alone, or in competition with K. sacchari NO9 (NF/NO9), R. alamii YAS34 (NF/YAS), or both competitors (Triple) in the absence (green bars) or in the presence (red bars) of 300 µM FeCl 3 . (The data represent the relative gene expression with 16S as reference gene using the ΔC t method with efficiency correction.) Phytobeneficial Traits under the Control of Multiple Crosstalks' Belowground Arnaud LAVEILHE, Sylvain FOCHESATO, Marie HULEUX, Joris TULUMELLO, Mohamed BARAKAT, Philippe ORTET, Thierry HEULIN and Wafa ACHOUAK, Lab of Microbial Ecology of the rhizosphere (LEMiRE). BIAM UMR 7265 CNRS-CEA-Aix Marseille University. F-13108 Saint Paul Lez Durance France Pseudomonads play crucial roles in plant growth promotion and the control of plant diseases. Specifically, many fluorescent pseudomonads with biocontrol abilities protect plants from soil-borne diseases by producing antimicrobial secondary metabolite(s) such as hydrogen cyanide (HCN) and 2,4-diacetylphloroglucinol (DAPG) (Haas & Défago, 2005). Among them, Pseudomonas brassicacearum described as the major rootassociated bacterium in the rhizosphere of Arabidopsis thaliana and Brassica napus (Achouak et al., 2000; 2004). Various living organisms (i.e. other rhizobacteria, fungi and plants) might interfere with the efficiency of biocontrol agents. In this work, we assessed the expression of phytobeneficial genes, phlD and hcnA, in P. brassicacearum co-cultivated with three phytopathogenic fungi, Fusarium culmorum, Fusarium graminearum and Microdochium nivale, and two other rhizobacteria, Kosakonia sacchari NO9 and Rhizobium alamii YAS34 in the presence or absence of plant, as well as in relation to iron availability. We also investigated the impact of the introduction of these rhizobacteria on the assembly and network of the plant root-associated microbiota in a natural soil. • Antifungal activity of NFM421 strain depends only on DAPG. HCN production is suppressed by fungi. • In the presence of NO9 and YAS34 the antifungal activity of NFM421 is modulated in relation to iron availability. • In planta, NFM421 increases the expression of phlD in the presence of NO9 strains with which it shares the same microniche on the root surface. • Inoculation of the plant with these bacteria led to a shift of microbiota assemblage, hubs and ecological network. Schnider-Keel et al., 2000 Autoinduction of 2,4-diacetylphloroglucinol biosynthesis in the bio-control agent Pseudomonas fluorescens CHA0 and repression by the bacterial 194. metabolites salicylate and pyoluteorin. J. Bacteriol. 182:1215-1225. Santaella C., Sch0ue M., Berge O., Heulin T., & W. Achouak . (2008) Role of exopolysaccharide produced by Rhizobium sp. YAS34 in the colonization of Arabidopsis thaliana and Brassica napus and biofilm formation on roots. Environ. Microbiol 10(8):2150-63 Achouak W., Conrod S., Cohen V., and Heulin. (2004) Phenotypic variation of Pseudomonas brassicacearum as a plant root colonization strategy. Mol Plant Microbe Interact. 17:872-9. Achouak W., L. Sutra, T. Heulin, J-M. Meyer, N. Fromin, S. Degraeve, R. Christen, and L. Gardan. (2000). Description of Pseudomonas brassicacearum sp. nov. and Pseudomonas thivervalensis sp. nov., two root-associated bacteria isolated from Arabidopsis thaliana and Brassica napus. Int. J. Syst. Evol. Microbiol 50:9-18. Achouak W, Abrouk D, Guyonnet J, Barakat M, Ortet P, Simon L, Lerondelle C, Heulin T, Haichar Fez (2018) Root exudates mediated host plant selection of microbiota. FEMS Microbiol Ecol Principal coordinates analysis (PCoA) of unweighted UniFrac distances and rarefaction curve for the root-adhering soil and the roots of B. napus microbiota Correlation network of the roots and RAS : (control) Correlation network of the roots and RAS : (inoculated)Metacommunity-scale network of the control and the inoculated rhizosphere. The size of circles roughly represents relative scores of betweenness centrality.

  Bacteria were tagged by plasmids encoding fluorescent proteins. YAS34 strain pHC60::gfp (Santaella et al., 2008); NO9 strain pBBR1MCS-2::mkate; NFM421::rfp or NFM421::gfp (Achouak et al., 2004). Root colonization was imaged by CLSM. Microbiota profiling & microbial network Comparison of microbiota from the rhizosphere of inoculated plants vs control plants by DNA extraction from the root-adhering soil and the roots and bulk soil; 16S rrs amplicon sequencing on a MiSeq instrument in paired-end 2 x 300 bp mode (Illumina) (Achouak et al., 2019).


Marie Usal
email: marie.usal@hotmail.fr

Christophe Regnault
email: christophe.regnault@gmail.com

Sylvie Veyrenc
email: sylvie.veyrenc@univ-grenoble-alpes.fr

Karine Couturier
email: karine.couturier@univ-grenoble-alpes.fr

Cécile Batandier
email: cecile.batandier@univ-grenoble-alpes.fr

Anne-Laure Bulteau
email: anne-laure.bulteau@ens-lyon.fr

David Lejon
email: dlejon@rovaltainresearch.com

Bruno Combourieu
email: bruno.combourieu@rovaltainresearch.com

Thomas Lafond
email: thomas.lafond@univ-rennes1.fr

Muriel Raveton
email: muriel.raveton@univ-grenoble-alpes.fr

Stéphane Reynaud
email: stephane.reynaud@univ-grenoble-alpes.fr

Concomitant exposure to benzo[a]pyrene and triclosan at environmentally relevant concentrations induces metabolic syndrome with multigenerational consequences in Silurana (Xenopus) tropicalis

Keywords: endocrine disruptors, metabolic syndrome, amphibian population decline, transgenerational, Silurana (Xenopus) tropicalis

Numerous studies suggest that amphibians are highly sensitive to endocrine disruptors (ED) but their precise role in population decline remains unknown. This study shows that frogs exposed to a mixture of ED throughout their life cycle, at environmentally relevant concentrations, developed an unexpected metabolic syndrome. Female Silurana (Xenopus) tropicalis exposed to a mixture of benzo[a]pyrene and triclosan (50 ng.L -1 each) from the tadpole stage developed liver steatosis and transcriptomic signature associated with glucose intolerance syndrome, and pancreatic insulin hyper secretion typical of pre-diabetes. These metabolic disorders were associated with delayed metamorphosis and developmental mortality in their progeny, both of which have been linked to reduced adult recruitment and reproductive success. Indeed, F1 females were smaller and lighter and presented reduced reproductive capacities, demonstrating a reduced fitness of ED-exposed Xenopus. Our results confirm that amphibians are highly sensitive to ED even at concentrations considered to be safe for other animals. This study demonstrates that ED might be considered as direct contributing factors to amphibian population decline, due to their disruption of energetic metabolism.

Introduction

In recent decades, amphibian wetland populations have declined dramatically worldwide, with an extinction rate 211 times faster than for other species [START_REF] Roelants | Global patterns of diversification in the history of modern amphibians[END_REF].

Fragmentation and loss of habitat has been described as one of the main causes, along with other threats such as the introduction of exotic species, emerging infectious diseases, and global climate change [START_REF] Blaustein | Amphibian Population Declines: Evolutionary Considerations[END_REF][START_REF] Hayes | The cause of global amphibian declines: a developmental endocrinologist's perspective[END_REF]. In addition to these welldescribed factors, several studies suggest that wetland pollution by endocrine disruptors (ED) is also a key contributing factor to this decline by altering amphibian fitness (Fedorenkova et al., 2012). Although several authors claim that the vulnerability of amphibians to ED is relatively similar to that of other species [START_REF] Kaplan | Effects of Endocrine Disrupting Chemicals on Amphibian Metamorphosis and Mitochondrial Membrane Permeability Transition[END_REF], there is growing evidence that once the symptoms of ED toxicity are detected in amphibian populations, population decline and community destructuring are unavoidable [START_REF] Hayes | The cause of global amphibian declines: a developmental endocrinologist's perspective[END_REF]. ED may act on crosstalk between the thyroid and reproductive axes (Duarte-Guterman et al., 2014) thus disrupting organogenesis and gonadal differentiation in larvae (Crump et al., 2002;Veldhoen et al., 2006;[START_REF] Kaplan | Effects of Endocrine Disrupting Chemicals on Amphibian Metamorphosis and Mitochondrial Membrane Permeability Transition[END_REF][START_REF] Kloas | Endocrine Disruption in Aquatic Vertebrates[END_REF][START_REF] Langlois | Fadrozole and finasteride exposures modulate sex steroid-and thyroid hormone-related gene expression in Silurana (Xenopus) tropicalis early larval development[END_REF]Lorenz et al., 2011;Shi et al., 2014). ED may also interfere with the gonadotrop axis in adults during the breeding season, causing disruptions of egg and sperm maturation [START_REF] Hayes | Herbicides: Feminization of male frogs in the wild[END_REF][START_REF] Hayes | Demasculinization and feminization of male gonads by atrazine: Consistent effects across vertebrate classes[END_REF][START_REF] Mosconi | Environmental Estrogens and Reproductive Biology in Amphibians[END_REF]Urbatzka et al., 2006Urbatzka et al., , 2007)). In addition, there are growing concerns that ED might also be involved in metabolic disorders (Casals-Casas and Desvergne, 2011). However, despite experimental evidence from mammals and epidemiological correlations in humans suggesting that ED could induce metabolic diseases [START_REF] Alonso-Magdalena | Bisphenol A exposure during pregnancy disrupts glucose homeostasis in mothers and adult male offspring[END_REF][START_REF] Jiang | Mitochondrial dysfunction in early life resulted from perinatal bisphenol A exposure contributes to hepatic steatosis in rat offspring[END_REF]Chevalier and Fénichel, 2015a;Chevalier and Fénichel, 2015b;Susiarjo et al., 2015;Chevalier andFénichel, 2016, Medic-Stojanoska, 2019), their potential involvement in metabolic disorders associated with amphibian population decline has been largely neglected. The first evidence that ED could alter frog metabolism was shown in S. tropicalis after acute exposure to the suspected ED triclosan

A C C E P T E D M

A N U S C R I P T 4 (TCS) [START_REF] Regnault | Metabolic and immune impairments induced by the endocrine disruptors benzo[a]pyrene and triclosan in Xenopus tropicalis[END_REF] and the well-known benzo[a]pyrene (BaP) [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF][START_REF] Regnault | Metabolic and immune impairments induced by the endocrine disruptors benzo[a]pyrene and triclosan in Xenopus tropicalis[END_REF]. More recently, two independent controlled exposures of S. tropicalis throughout their life cycle to these two ED at environmentally relevant concentrations induced a metabolic syndrome featuring a pre-diabetes state. The exposed animals produced progeny that metamorphosed later, were smaller and lighter at metamorphosis, and had reduced reproductive success, thus linking metabolic disorders to putative population decline [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF].

In situ, TCS is found in wastewater effluent due to its use as an antimicrobial agent in household and personal-care products such as cosmetics, soap, toothpaste, shampoo and textiles [START_REF] Levy | Molecular basis of triclosan activity[END_REF][START_REF] Piccoli | Determination of triclosan in personal health care products by liquid chromatography (HPLC)[END_REF][START_REF] Mcavoy | Measurement of triclosan in wastewater treatment systems[END_REF][START_REF] Møretrø | Assessment of the antibacterial activity of a triclosan-containing cutting board[END_REF]McArthur et al., 2012). It is released into natural aquatic environments and, in 2000, it was among the top seven organic wastewater pollutants found in streams by the United States Geological Survey [START_REF] Mcavoy | Measurement of triclosan in wastewater treatment systems[END_REF][START_REF] Barber | Endocrine disrupting alkylphenolic chemicals and other contaminants in wastewater treatment plant effluents, urban streams, and fish in the Great Lakes and Upper Mississippi River Regions[END_REF]. In wetlands, this emerging contaminant is therefore mixed with other ED; including the well-known BaP, classified as a reproductive ED by several countries, including the United States and all the European Union member states [START_REF] Petersen | Characterization of circannual patterns of metabolic recovery from activity in Rana catesbeiana at 15°C[END_REF]United States Government, 2010). BaP enters surface water mainly via atmospheric fallout, urban run-off, municipal and industrial effluent, and oil spillages or leakages (Srogi, 2007). TCS and BaP are both found in effluent from wastewater treatment plants [START_REF] Weir | Concentrations of Hydrophobic Organic Pollutants in U.S. Wastewater Treatment Plants and in Receiving Surface Waters Modeled from EPA Biosolids Monitoring Data[END_REF][START_REF] Gardner | The significance of hazardous chemicals in wastewater treatment works effluents[END_REF], in water streams [START_REF] Galloway | Occurrence of pharmaceuticals and other organic wastewater constituents in selected streams in northern Arkansas[END_REF] and in lakes (Whitacre, 2010). Although it is common practice to assess the possible environmental risk of chemicals individually, this approach may over or underestimate the real environmental impact of mixtures of xenobiotics [START_REF] Relyea | A cocktail of contaminants: how mixtures of pesticides at low concentrations affect aquatic communities[END_REF][START_REF] Godoy | What do we know about the ecotoxicology of pharmaceutical and personal care product mixtures? A critical review[END_REF].

BaP and TCS concentrations in surface water vary from 0.1 to 237 ng.L -1 (Trapido and Veldre, 1996;[START_REF] Olivares-Rubio | Relationship between biomarkers and endocrine-disrupting compounds in wild Girardnichthys viviparus from two lakes with different degrees of pollution[END_REF] and 0.8 to 74 ng.L -1 [START_REF] Lindström | Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater[END_REF][START_REF] Blair | Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern[END_REF] respectively. Thus the impact of these chemicals in mixture should be studied within this range of concentrations.

A C C E P T E D M A N U S C R I P T 5

In this context, this study aims at understanding the precise impact of a mixture of BaP and TCS on S. tropicalis at environmental concentrations. The effects of BaP and TCS were studied over two generations of S. tropicalis. The parental F0 generation of Xenopus was exposed to the mixture (50 ng.L -1 of each ED) for 12 months from the tadpole stage to the mature adult stage. The impacts of these contaminants on the Xenopus' metabolism were studied by examining both the transcriptome and overall liver physiology. Multigenerational impacts were studied by monitoring the development and reproductive capacity of their progeny not exposed to the ED (F1 generation).

Methods

Animals

Seven-day old Silurana (Xenopus) tropicalis were acclimatized for one week in Marc's Modified Ringer's (MMR) 0.1X medium under controlled conditions (25°C, photoperiod 12h:12h).

The frogs were fed daily ad libitum with trout food (pellets Aquatic 3, Special Diets Services).

The density of Xenopus was 50 tadpoles per 2 L of 0.1X MMR (two tanks per exposure condition). Between 30 (prior to metamorphosis) to 150 days (complete metamorphosis), the density of frogs was adjusted to 30 animals per 4 L. After 150 days, the density of animals was adjusted to 10 animals per 4 L (four tanks per conditions). All experiments were performed in an accredited animal house (Rovaltain research company, n° A 26 004 1401) and approved by the ethics committee ComEth Grenoble -C2EA -12 (animal welfare agreement n° 02545.03).

Exposure

The Xenopus were collectively exposed to the ED-mixture under the controlled conditions, in identical tanks, with identical animal densities as described above. One-quarter of the medium was renewed daily using an MMR solution containing BaP (Sigma-Aldrich) and TCS

A C C E P T E D M

A N U S C R I P T 6 (Sigma-Aldrich) at an equimolar concentration of 200 ng.L -1 and the medium in its entirety was changed weekly. The control frogs were exposed to dimethyl sulfoxide (DMSO, vehicle) at a concentration of 4/1000 (v:v). As described in [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF], pollutant concentrations in the medium were continually monitored using GC-MS/MS, and confirmed that the average weekly BaP/TCS concentrations were both equal to 50 ng.L -1 . These concentrations match the BaP and TCS concentrations found in polluted ponds (Trapido and Veldre, 1996;[START_REF] Lindström | Occurrence and environmental behavior of the bactericide triclosan and its methyl derivative in surface waters and in wastewater[END_REF][START_REF] Blair | Pharmaceuticals and personal care products found in the Great Lakes above concentrations of environmental concern[END_REF][START_REF] Olivares-Rubio | Relationship between biomarkers and endocrine-disrupting compounds in wild Girardnichthys viviparus from two lakes with different degrees of pollution[END_REF]. Xenopus mortality and development were monitored weekly and the body mass and snout-vent length of each animal was measured at the metamorphic stage. After 12 months' exposure, four females from each exposure condition were used for glucose tolerance test. Eight others females were rapidly killed by concussion and immediately dissected to sample the organs. Blood was collected through an intra-cardiac puncture.

Mating

At the adult stage, five F0 females and ten F0 males were selected from the ED-mixture batch and from control batch to mate naturally. Two injections of human chorionic gonadotropin (hCG) were performed in the dorsal lymph sac of animals to induce the mating behavior (Showell and Conlon, 2009). After hatching, the tadpoles were bred in the same control conditions as described above and the F1 development was monitored weekly. The following criteria were used to characterize the development of F1 Xenopus: time from hatching to metamorphosis, snout-vent length and weight at the metamorphic and adult stages. Afterwards, the mature adults of the F1 generation were mated, as described above, to assess their reproductive capacity.

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A C C E P T E D M

A N U S C R I P T 7

Glucose tolerance test

Glycaemia was measured by digital sampling at 0, 1, 2, and 4 hours after intraperitoneal glucose injection (1 mg.g -1 fresh mass) using a glucometer (Accu-Chek performa, Roche Diagnostics, Meylan, France). The statistical comparison was performed on the area under the curve obtained after glucose injection.

Hepatosomatic index and hepatotoxicity

The hepatosomatic index (HSI) was calculated by dividing the weight of the liver by the body weight and hepatoxicity was evaluated by measuring alanine aminotransferase activity in serum as previously described in [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF].

Histological observations

The liver (same lobe/frog) and the pancreas were collected, embedded in Tissue OCT (Labonord -VWR, Templemars, France), rapidly frozen in liquid nitrogen, and conserved at -80°C. Using a cryostat (CM3050 S, Leica, Nussloch, Germany), histological transverse sections 7 μm thick were produced and mounted on Super-Frost plus microscope slides (Labonord). The frozen sections of liver were stained with Hematoxylin and Erythrosin (HE) and Gomori's trichrome to study tissue organization, with Oil Red O (ORO) to assess total lipid hepatic content and with Periodic Acid Schiff (PAS) staining to assess glycogen content. All staining and procedures have been previously described in [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF]. PAS scoring was performed by two people independently. The PAS staining score (0-4) was assigned according to the percentage of glycogen in the cells, with a score of 0 corresponding to no glycogen, and a score of 4 corresponding to a cell full of glycogen. For ORO, lipid quantifications were performed as previously described [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF]. In short, an image segmentation was processed using R, G and B layer similarity, with the default sharpness and compactness parameters. The object size parameter was set to 10. Then, using a 2-class typology (lipid or not), several training Insulin production was quantified by immunohistochemistry on frozen pancreas sections as previously described [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF].

Hepatic mitochondrial respiration and mitochondrial oxidative stress evaluation

The mitochondria were prepared as described in [START_REF] Garait | Fat intake reverses the beneficial effects of low caloric intake on skeletal muscle mitochondrial H(2)O(2) production[END_REF]. Complex I and complex II-mediated mitochondrial respiration (State 2) was evaluated with 5 mM glutamate, 2.5 mM malate, and 5 mM succinate. State 3 respiration was measured after the addition of adenosine diphosphate (ADP, 500 mM). The respiratory control index (RCI) was defined as the ratio: state 3/state 2.

To evaluate the mitochondrial oxidative stress, aconitase activity was measured spectrophotometrically at 340 nm as previously described [START_REF] Bulteau | Redox-Dependent Modulation of Aconitase Activity in Intact Mitochondria[END_REF]. Citrate synthase activity was measured as control of mitochondria density by following the rate of appearance of thionitrobenzoic acid at 412 nm in the presence of 10 μg protein, 100 mM Tris-HCl pH 8.0, 0.1%Triton X100, 300 mM acetylCoA, 100μM DTNB, and 500 μM oxaloacetate (Srere, 1969).

Liver proteasome activity

As previously described in Bulteau et al. (2002), the peptidase activity of the proteasome was assayed using a fluorogenic peptide, succinyl-Leu-Leu-Val-Tyr-7-Amido-4-Methylcoumarin (LLVYAMC) (Sigma-Aldrich, Saint Quentin Falavier, France).

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A C C E P T E D M A N U S C R I P T 9 2.

Liver RNA extraction and sequencing

The complete method is largely detailed in [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF]. Briefly, for each biological replicate, the RNAqueous®-4PCR Kit (Ambion, USA) was used to extract the total RNA from 15 mg of liver and according to the manufacturer's instructions. RNA-seq libraries were prepared using the TruSeq Stranded mRNA sample Prep kit (Illumina) and sequenced on an Illumina GAIIx sequencer as 75 bp reads by Hybrigenics-Helixio (Clermont-Ferrand, France).

Read mapping and gene expression quantification and differential analysis were performed using Trimmomatic (v0.32.1) [START_REF] Bolger | Trimmomatic: a flexible trimmer for Illumina sequence data[END_REF], Bowtie-0.12.7 / TopHat 2 (v0.6) software (Trapnell et al., 2009), Cufflinks (v0.0.7), Cuffmerge (v0.0.6) and Cuffdiff (v0.0.7) (Trapnell et al., 2012) tools implemented in a Galaxy pipeline( http://galaxyproject.org).Differentially expressed genes were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and General Ontology terms (GO-term) enrichment analysis, using the online functional annotation tool DAVID (Database for Annotation, Visualisation and Integrated Discovery, http://david.abcc.ncifcrf.gov) [START_REF] Huang | Bioinformatics enrichment tools: paths toward the comprehensive functional analysis of large gene lists[END_REF] as previously described [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF].

The significance was calculated using a modified Fisher's Exact test (p < 0.05). Heat maps reflecting gene expression profiles sharing enriched annotation pathways or other gene expression associated with these pathways, were generated using TM4 Multi experiment Viewer (MeV) software [START_REF] Saeed | hormone-associated gene expression and disrupts postembryonic anuran development[END_REF].

Muscle glycogen content

Forty mg of thigh muscle were lysed in 40% KOH (100°C, 30 minutes) and cooled, before glycogen precipitating with 100% ethanol (-20°C, overnight). After centrifugation at 10,000 x g for 15 minutes at 4°C, the glycogen pellet was hydrolyzed into glucose by 2M HCl at 100°C for 3 hours. After 2M NaOH addition, the concentration of glucose was measured using the GAGO-20 kit (Sigma Aldrich, France), in accordance with the manufacturer's instructions.

A C C E P T E D

M A N U S C R I P T 10 2.

Transcription profile of muscle glucose transporter

Total RNA was isolated from 40 mg muscle using TRIzol RNA isolation reagent (Invitrogen, Life Technologies, Carlsbad, USA) according to the manufacturer's instructions.

RNA was purified with an RNA Clean & Concentrator kit to remove sugars present in large quantities (R1015, Ozyme zymo Research). After DNAse I treatment of 1.5 µg total RNA, cDNA synthesis was performed with SuperScript III and oligo-dT 20 primer (Invitrogen) according to the manufacturer's instructions. Real-time quantitative PCR reactions were performed on the iQ5 system (Bio-Rad, Hercules, USA) with 0.45 μM of each primer, 7.5 μL of iQ SYBR Green supermix (Bio-Rad) and 3 μL of cDNA diluted (1:100). The primers used (Table S1) were designed and validated in silico with NCBI primer blast. For each gene analyzed, a melt curve analysis was performed to check for the unique presence of the targeted PCR product and the absence of significant primer dimers. The selected housekeeping genes (RPS7 and RPL27) were validated with several RNAseq experiments [START_REF] Regnault | Impaired liver function in Xenopus tropicalis exposed to benzo[a]pyrene: transcriptomic and metabolic evidence[END_REF][START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF]. The ΔΔCT method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] taking Into account PCR efficiency was used for data analysis was used for data analysis.

Oocyte lipid content

Five to ten mg of oocytes were crushed in 500 µl of 1:1 chloroform/methanol then kept at 4°C for one hour. The samples were centrifuged at 18 000g for 30 minutes then 100 µl of supernatant was placed in a glass tube. The samples were placed in a dry bath at 95°C for 10 minutes to obtain lipid deposition. Once the solvent evaporated, 200 µl of 95° sulfuric acid was added and the samples are kept at 95°C for 10 minutes. Once the samples had cooled, 4.8 mL of phospho-vanillin reagent (Sigma, V1104) was added. After 10 minutes, lipids were measured spectrophotometrically at 535 nm.

A C C E P T E D

M A N U S C R I P T 11 2.

Statistics

For F0 females, phenotypic data are expressed as the mean ± SEM and derived from 4 individual experiments. Since the statistical distribution of the data was unknown (Gaussian or not) for four replicates, Wilcoxon's test was used to compare the ED-mixture exposure population to the control population.

The distributions of animal development times (mortality, age to metamorphosis) and female maturity time for F0 and F1 generations were compared between the control populations and the ED-exposed population using a Kaplan-Meyer log-rank test.

Results and discussion

Effect of the ED-mixture on the development of F0 animals and their progeny (F1)

The ED-mixture at environmental concentrations did not significantly impact the mortality of F0 exposed animals compared to the F0 control (Fig. 1A). Xenopus exposed to the EDmixture showed no delay in metamorphosis (Fig. 1B) nor any significant modification in size or weight at the time of metamorphosis (Fig. 1C andD). Furthermore, the ED-mixture did not impact the time to maturity of F0 females (Fig. 1E). Taken together, these results confirmed those obtained for exposure to a single ED, with the exception of the time to metamorphosis, which has been shown to be significantly delayed for Xenopus exposed to BaP and TCS individually [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF].

In order to test the effect of parental exposure on their progeny, five pairs of F0 Xenopus were mated. Despite their sexual maturity, one of the five exposed females rejected the male leading to 80% successful amplexus at 3 hours post-stimulation compared to 100% for the control (Fig. 1F). In addition, a lower number of hatched eggs per amplexus was observed in ED-mixture-exposed animals (Fig. 1G andS1A).

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A C C E P T E D M A N U S C R I P T 12

The F1 progeny, only exposed to the ED-mixture at the gamete stage, showed significant mortality during the first 150 days of development (Fig. 1H) and significantly delayed metamorphosis (the 100% population metamorphosis level was reached 20 days later) (Fig. 1I).

Moreover, F1 metamorphic Xenopus were lighter but there was no significant decrease in size (Fig. 1J and1K). In comparison to single exposure to BaP or TCS, the effect of exposure to the ED-mixture on progeny age at metamorphosis appeared to be equivalent to BaP exposure, whereas the effect on progeny weight was equivalent to TCS exposure [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF].

These disturbances could play a major role in the ongoing decline of the amphibian population.

Delayed metamorphosis could affect progeny survival in cases of early desiccation of temporary ponds [START_REF] Rumrill | Delayed effects and complex life cycles: How the larval aquatic environment influences terrestrial performance and survival[END_REF]. Along with lower body weight at metamorphosis, it could also reduce adult recruitment and the chances of successful reproduction (Smith, 1987, Todd et al., 2011). At the adult stage, F1 females ancestrally exposed to the ED-mixture were smaller and lighter (Fig. S2A andB), factors which have been shown to impact on overwintering survival rates in several amphibian species in temperate environments [START_REF] Hayes | Pesticide Mixtures, Endocrine Disruption, and Amphibian Declines: Are We Underestimating the Impact?[END_REF]. Moreover, reduced post-metamorphic growth has previously been showed to impact reproductive success since female weight correlates with clutch weight in several amphibian species [START_REF] Kuramoto | Correlations of quantitative parameters of fecundity in amphibians[END_REF][START_REF] Browne | Reproduction and larval rearing of amphibians[END_REF]. F1 females from ED-mixture-exposed progenitors also showed delayed sexual maturity, taking more than 80 days to reach 80% of mature Xenopus in the population compared to the controls (Fig. 1L). These effects were not found in genitors suggesting a multigenerational worsening of the phenotype. In addition, a reduction in F1 reproductive success and consequent decrease in the fitness of F0 progenitors was observed. Indeed, whereas amplexus was successful for 100% of controls at 3 hours post-stimulation, this was the case for only 40% of F1 progeny from exposed progenitors (Fig. 1M). F1 mating produced eggs with highly variable hatching rates, although the overall trend showed a dramatic decrease in the number of hatched eggs from the F1 offspring of ED-mixture-exposed parents (Fig. 1N andS1B). Variable hatching rates have been described in natural mating in S. tropicalis (Showell and Conlon, 2009) and

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13 these results should be interpreted with caution. However, since the same transgenerational effects have previously been observed in S. tropicalis exposed to each ED separately [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF] reaching a total of 15 mating pairs for exposed animals, these results still suggest a decrease in reproduction success that will need to be confirmed in future studies.

Previous studies have established a correlation between a decrease in maternal egg provisioning and disturbances in offspring development such as decreased size and weight at metamorphosis, a longer larval period and decreased tadpole survival rates (Dziminski and Roberts, 2006). Indeed, oocytes need amino acids, carbohydrates and lipids from the maternal liver to ensure normal development [START_REF] Brooks | Egg quality in fish: what makes a good egg?[END_REF]. In this study, a downward trend in the lipid content in the oocytes of ED-mixture-exposed F0 females was detected (Fig. S3).

Therefore, the observed impacts on the F1 progeny could be explained by changes in lipid and carbohydrate metabolism in exposed F0 females, leading to a decrease in maternal provisioning.

Effect of ED-mixture on F0 female metabolism

Endocrine disruptor-mixture exposure induced an increase in the hepatosomatic index (HSI) of F0 Xenopus (Fig. 2A) correlated with marked liver steatosis (Fig. 2B) (Teubner et al., 2015). Moreover, lipid metabolism disruption has been recently described in aquatic animals (e.g. Gobiocypris rarus, Danio rerio, Xenopus laevis, S. tropicalis) exposed to ED [START_REF] Grün | Endocrine-disrupting organotin compounds are potent inducers of adipogenesis in vertebrates[END_REF][START_REF] Guan | Effects of bisphenol A on lipid metabolism in rare minnow Gobiocypris rarus[END_REF]Santangeli et al., 2018;[START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF]. Liver steatosis is a hallmark of non-alcoholic fatty liver disease (NAFLD) and one of the major disturbances associated with dyslipidemia [START_REF] Neuschäfer-Rube | Arylhydrocarbon receptor-dependent mIndy (Slc13a5) induction as possible contributor to benzo[a]pyrene-induced lipid accumulation in hepatocytes[END_REF][START_REF] Han | Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats[END_REF][START_REF] Hua | Alternation of plasma fatty acids composition and desaturase activities in children with liver steatosis[END_REF] demonstrating the link between a hypertrophic liver and the accumulation of lipid droplets.

Hepatic lipids can be hydrolyzed by mitochondria which perform fatty acid β-oxidation to produce ATP [START_REF] Ferré | Hepatic steatosis: a role for de novo lipogenesis and the transcription factor SREBP-1c[END_REF] but excess lipid accumulation during chronic liver steatosis has been associated with mitochondrial dysfunction [START_REF] Jiang | Mitochondrial dysfunction in early life resulted from perinatal bisphenol A exposure contributes to hepatic steatosis in rat offspring[END_REF]. In this study, exposure to

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A N U S C R I P T 14 the ED-mixture led to a 1.64-fold elevated state 2 respiratory rate (Fig. 2C) along with a 1.5-fold decreased respiratory control index (Fig. 2D) and aconitase inactivation (Fig. 2E) showing mitochondrial uncoupling and oxidative stress [START_REF] Koliaki | Adaptation of Hepatic Mitochondrial Function in Humans with Non-Alcoholic Fatty Liver Is Lost in Steatohepatitis[END_REF]. These results are confirmed by the stability of citrate synthase activity (Fig. 2F) suggesting that the observed decrease in aconitase activity was not related to variations in mitochondrial density in the liver. Lipid accumulation in the liver has been previously associated with hepatotoxicity [START_REF] Yamaguchi | Inhibiting triglyceride synthesis improves hepatic steatosis but exacerbates liver damage and fibrosis in obese mice with nonalcoholic steatohepatitis[END_REF]. The 2.5-fold increase in serum alanine amino transferase activity (Fig. 2G) confirmed this phenomenon in the ED-mixture-exposed Xenopus. Liver disruption (Fig. 2H-K) is characterized by ballooning hepatocytes (Fig. 2I), increasing melanomacrophage centers (Fig. 2I, arrow), swollen blood vessels (Fig. 2K, full arrow), necrosis and leukocyte infiltrates (Fig. 2K, dotted arrows). NAFLD encompasses steatosis and the accumulation of glycogen [START_REF] Bedossa | Histological Assessment of NAFLD[END_REF] whereas ballooned hepatocytes, inflammation, and necrosis are indicative of progression from NAFLD to non-alcoholic steatohepatisis (NASH) (Day and James, 1998;Day, 2006). Therefore, the hepatic damage in the ED-mixture-exposed animals is typical of the progression between NAFLD to NASH and corresponds to a combination of the disturbances previously described for Xenopus under single exposure to BaP and to TCS [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF]. The liver damage observed in the animals exposed to the ED-mixture was accompanied by a decrease in liver proteasome activity (Fig. 2L) suggesting endoplasmic reticulum stress and the possible development of insulin resistance (IR) [START_REF] Otoda | Proteasome dysfunction mediates obesity-induced endoplasmic reticulum stress and insulin resistance in the liver[END_REF]. This metabolic syndrome is confirmed by the accumulation of glycogen in the liver which has been associated with NAFLDinduced IR (Fig. 2M) [START_REF] Han | Long-term fatty liver-induced insulin resistance in orotic acid-induced nonalcoholic fatty liver rats[END_REF]. Moreover, despite no difference in basal serum glucose levels, ED-mixture exposure led to glucose intolerance with a 1.8-fold increase in blood glucose (Fig. 2N). This glucose intolerance is confirmed by a threefold increase in insulin production by the pancreatic β-cells (Fig. 2O). In type 2 pre-diabetes the onset of hyperglycaemia is preceded by an increased metabolic demand for insulin due to IR. This leads to a period of normal blood glucose levels, during which the insulin hypersecretion by pancreatic β-cells compensates for IR

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A N U S C R I P T 15 [START_REF] Kasuga | Insulin resistance and pancreatic β cell failure[END_REF]. In addition, the decrease in muscular glycogen content (Fig. 2P) and the reduced transcription of slc2a4 (Fig. 2Q) (coding glut4 protein; the main transporter of glucose under insulin stimulation) in ED-mixture-exposed animals confirmed the metabolic syndrome and the development of peripheral IR (Petersen et al., 2007).

Effect of ED-mixture on F0 female liver transcriptome

The liver transcriptome of ED-mixture-exposed Xenopus showed 120 genes differentially transcribed. This corresponds to 1.3% of the total number of genes detected with about 78% of genes up-regulated (Table S2). KEGG pathway enrichment indicated a link between ED-mixture exposure and "transcriptional misregulation in cancer" (5-fold) and "ABC transporters" (8-fold) (Table 1). GOterm enrichment analysis of the differentially transcribed genes indicates a link between ED mixture exposure and "nucleotide binding" (4-fold), "steroid hydroxylase activity" (18-fold), "ATPase activity coupled to transmembrane movement of substances" (10-fold), "chloride channel activity" (8-fold), "carbohydrate binding" (4-fold), "regulation of hormone secretion" (64-fold), "negative regulation of T-helper 2 cell differentiation" (80-fold), "transport" (3.2-fold), "amino acid transport" (13-fold), "cell proliferation" (3-fold), "negative regulation of apoptotic process" (3-fold), "liver regeneration" (16-fold), and "multicellular organism development" (2-fold) (Table 1). Several of the enriched KEGG pathways and GOterms have to be taken as a whole and suggest that a metabolic syndrome associated with liver steatosis and histopathological consequences occurred in the ED-mixture-exposed animals. Moreover, more than 32% of the differentially transcribed genes not necessarily highlighted by the enrichments are associated with NAFLD/NASH and their consequences including inflammation, cell death/proliferation and cancer (Fig. 3) and confirmed the global IR phenotype. This IR phenotype is characterized by a global dysregulation of genes involved in hepatic carbohydrate and lipid metabolism [START_REF] Bechmann | The interaction of hepatic lipid and glucose metabolism in liver diseases[END_REF]. One of the first symptoms of IR in the liver is the activation of neoglucogenic genes due to the absence of insulin signaling (Bechmann et al.,

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A N U S C R I P T 16 2012), and ED-mixture-exposed Xenopus showed a marked over-transcription of the three genes encoding glucose-6-phosphatase (G6PC, g6pc and g6pc2) (3.59-fold, 2.94-fold and 3.04fold respectively) (Fig. 3). IR syndrome has also been associated with a dysregulation of genes regulating lipid metabolism, leading to the accumulation of triglycerides (main form), free fatty acids or cholesterol in the liver [START_REF] Naik | Genomic aspects of NAFLD pathogenesis[END_REF]. Several genes involved in de novo fatty acid synthesis appeared over-transcribed in ED-mixture-exposed animals including ATP-citrate lyase (acly, 2.86-fold) and lipase hormone-sensitive (lipe, 3.59-fold) (Fig. 3). It has been demonstrated that acly is a key lipogenic enzyme for fatty acid biosynthesis [START_REF] Wang | Abrogation of hepatic ATP-citrate lyase protects against fatty liver and ameliorates hyperglycemia in leptin receptor-deficient mice[END_REF] and that the increased activity of hepatic lipases positively correlates with steatosis in humans [START_REF] Miksztowicz | Hepatic lipase activity is increased in nonalcoholic fatty liver disease beyond insulin resistance[END_REF]. Moreover, the scaffold attachment factor B (safb) and the SREBF chaperone (scap) genes associated with type 2 diabetes and lipid accumulation appear to be stimulated (2.61-fold and 3.60-fold respectively) (Fig. 3) [START_REF] Felder | The SREBF-1 locus is associated with type 2 diabetes and plasma adiponectin levels in a middle-aged Austrian population[END_REF][START_REF] Yuan | Advanced glycation end products (AGEs) increase human mesangial foam cell formation by increasing Golgi SCAP glycosylation in vitro[END_REF]Mastrocola et al., 2015) . This dysregulation of lipid metabolism can be explained by BaP's capacity to activate the arylhydrocarbon receptor (AhR) [START_REF] Neuschäfer-Rube | Arylhydrocarbon receptor-dependent mIndy (Slc13a5) induction as possible contributor to benzo[a]pyrene-induced lipid accumulation in hepatocytes[END_REF][START_REF] Moyer | Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFβ, and IDO1[END_REF].

In addition to the over-transcription of genes involved in lipid synthesis, fatty acid binding protein 1 (fapb1) (3.76-fold) and low-density lipoprotein receptor (ldlr, 2.41-fold) genes coding proteins involved in fatty acid and cholesterol uptake appeared over-transcribed (Fig. 3). This phenomenon is a key aggravating factor in NAFLD [START_REF] Guan | Effects of bisphenol A on lipid metabolism in rare minnow Gobiocypris rarus[END_REF][START_REF] Mukai | Silencing of FABP1 ameliorates hepatic steatosis, inflammation, and oxidative stress in mice with nonalcoholic fatty liver disease[END_REF] and has been already described in the development of type 2 diabetes (Mansego et al., 2012;[START_REF] Ramakrishnan | The association between insulin and low-density lipoprotein receptors[END_REF].

Several studies have shown that steatosis is associated with reactive oxygen species (ROS) production by mitochondria which causes cell damage [START_REF] Vial | Effects of a high-fat diet on energy metabolism and ROS production in rat liver[END_REF]Lockman et al., 2012). Nevertheless, mitochondria control ROS levels by inducing their scavenging system as the uncoupling protein 2 (ucp2) which plays a protective role by ameliorating oxidative stress [START_REF] Rousset | The biology of mitochondrial uncoupling proteins[END_REF]. Here, we found an under-transcription of ucp2 (0.19-fold) in ED-mixtureexposed animals suggesting a lack of protective effect (Fig. 3). Moreover, this phenomenon was

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accompanied by the under-transcription of glutathione peroxidase 4 (gpx4), which has also associated with ROS protection in fatty livers (0.40-fold) (Ucar et al., 2013). A breakdown in antioxidant defenses has been shown to play a significant role in oxidative stress associated with NASH (Ucar et al., 2013) and confirmed the observed inactivation of aconitase in EDmixture-exposed animals (Fig. 3).

In type 2 diabetes, lipotoxicity and oxidative stress are key pathogenic processes in NAFLD which, by inducing inflammatory processes, induce progression to NASH (Takahashi and Fukusato, 2014). Leukocyte infiltrates and the dysregulation of key genes involved in inflammation such as bcl6 (2.64-fold), nfkbiz, (2.12-fold), cd40 (4.02-fold) and fcn3 (12.24-fold) were both observed in ED-mixture-exposed Xenopus (Fig. 3). In NASH, the inflammatory processes correlate with hepatocellular proliferation (VanSaun et al., 2013). Hepatic transcriptomic data indicated the over-transcription of genes involved in the inhibition of apoptosis (araf, birc7, fabp1, fmn2), the under-transcription of genes activating apoptosis (anxa1, anxa5, ucp2, cyr61, myc), along with the over-transcription of genes involved in cell proliferation and cancer (cuzd1, eps8, glul, srrt, tacc2, cd40, etv6, bcl6) in ED-mixture-exposed animals (Fig. 3). Taken together, these results suggest a global dysregulation of genes involved in the balance between apoptosis and proliferation in ED-mixture-exposed Xenopus, with a trend towards the upregulation of proliferation processes which confirms the progression from NAFLD to NASH.

Conclusion

In this study, we focused on finding links between exposure to a mixture of ED and amphibian population decline. We found that exposure to an environmentally realistic mixture of ED, can lead to severe metabolic impairments in amphibians. Understanding the impact of the mixture is very complex since exposure to each individual compound can induce very different effects. According to [START_REF] Relyea | A cocktail of contaminants: how mixtures of pesticides at low concentrations affect aquatic communities[END_REF], in some taxa such as amphibians, the impact of

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A N U S C R I P T 18 pollutant mixtures cannot be predicted from the effects observed from exposure to an individual compound. Here, we found that exposure to the ED-mixture induced a similar phenotype to exposure to the same ED individually [START_REF] Regnault | Unexpected metabolic disorders induced by endocrine disruptors in Xenopus tropicalis provide new lead for understanding amphibian decline[END_REF]. Our results also provide insight into the transgenerational effects of ED-mixtures, which might negatively affect the fecundity of the progeny only exposed at the gamete stage. Therefore, the results of this study highlighting metabolic disorders induced by exposure to the ED-mixture might confim the role of ED as a cause contributing to amphibian population decline.

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A N U S C R I P T 20 Bulteau, A.-L., Szweda, L.I., Friguet, B., 2002. Age-dependent declines in proteasome activity in the heart. Arch. Biochem. Biophys. 397, 298-304. Casals-Casas, C., Desvergne, B., 2011. Endocrine disruptors: from endocrine to metabolic disruption. Annu. Rev. Physiol. 73, 135-162. Chevalier, N., Fénichel, P., 2016. Obésité, diabète de type 2 et perturbateurs endocriniens.

Presse Médicale, Médecine et environnement 45, 88-97. Chevalier, N., Fénichel, P., 2015a. Endocrine disruptors: new players in the pathophysiology of type 2 diabetes? Diabetes Metab. 41, 107-115. Chevalier, N., Fénichel, P., 2015b. Bisphenol A: Targeting metabolic tissues. Rev. Endocr. Metab. Disord. 16, 299-309. Crump, D., Werry, K., Veldhoen, N., Van Aggelen, G., Helbing, C.C., 2002 Liu, X.L., Cao, H.X., Fan, J.G., 2016. MicroRNAs as biomarkers and regulators of nonalcoholic fatty liver disease. J. Dig. Dis. 17, 708-715.

Lockman, K.A., Baren, J.P., Pemberton, C.J., Baghdadi, H., Burgess, K.E., Plevris-Papaioannou, N., Lee, P., Howie, F., Beckett, G., Pryde, A., Jaap, A.J., Hayes, P.C., Filippi, C., Plevris, J.N., 2012. Oxidative stress rather than triglyceride accumulation is a determinant of mitochondrial dysfunction in in vitro models of hepatic cellular steatosis.

  manually for each class. An image classification was then run using the nearest neighboring algorithm. The results of the classifications were rasterized and the proportion of each class estimated.

  . Exposure to the herbicide acetochlor alters thyroid hormone-dependent gene expression and metamorphosis in Xenopus Laevis. Environ. Health Perspect. 110, 1199-1205. Day, C.P., 2006. From fat to inflammation. Gastroenterology 130, 207-210. Day, C.P., James, O.F.W., 1998. Steatohepatitis: A tale of two "hits"? Gastroenterology 114, 842-845. Duarte-Guterman, P., Navarro-Martín, L., Trudeau, V.L., 2014. Mechanisms of crosstalk between endocrine systems: Regulation of sex steroid hormone synthesis and action by thyroid hormones. Gen. Comp. Endocrinol., 2nd Meeting of The North American Society for Comparative Endocrinology 203, 69-85. Dziminski, M.A., Roberts, J.D., 2006. Fitness consequences of variable maternal provisioning in quacking frogs (Crinia georgiana). J. Evol. Biol. 19, 144-155. Fedorenkova, A., Vonk, J.A., Lenders, H.J.R., Creemers, R.C.M., Breure, A.M., Hendriks, A.J., 2012. Ranking ecological risks of multiple chemical stressors on amphibians. Environ.Toxicol. Chem. 31, 1416-1421.

Fig. 1 .

 1 FIGURE CAPTIONS

Fig. 2 .

 2 Fig. 2. Impact of the ED-mixture on F0 female metabolism. (A) Hepatosomatic index of control

Fig. 3 .

 3 Fig. 3. Circos plot representing the overlap between liver transcriptome of F0 females exposed

Figure 3

 3 Figure3

Acknowledgements

This work was funded by the ComUE Université Grenoble-Alpes IRS IDEX Grant, by the CNRS-INSU EC2CO program and by the Federative Structure Environmental and Systems Biology (BEeSy) of the Grenoble-Alpes University. Marie Usal was funded by the Région Auvergne-Rhône-Alpes. Christophe Regnault received funding from the French Ministry of Higher Education and Research. We thank Kim Barrett (Version Originale) for the English review of the manuscript.

Data access

RNA-seq sequence data have been deposited at EMBL-EBI (http://www.ebi.ac.uk/ena) under the accession number PRJEB18463.

 Notarstefano, V., Maradonna, F., Giorgini, E., Gioacchini, G., Forner-Piquer, I., Habibi, H.R., Carnevali, O., 2018.Effects od diethylene glycol dibenzoate and Bisphenol A on the lipid metabolism of Danio rerio. Sci. Total Environ. 636, 641-655. Shi, H., Zhu, P., Guo, S., 2014 1 -Functional pathway enrichment analysis based on hepatic genes differentially transcribed between ED-mixture-exposed and control F0 animals.

The 120 genes showing a significant differential transcription in ED-mixture-exposed animals were used for annotation enrichment using the Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool (modified Fisher's exact test with P < 0.05). The table indicates the number of genes involved in each enriched KEGG pathway or GOTerm, and the fold enrichment along with its associated P value. MF: Molecular Function and BP: Biological Process.