Sophie M Prud'homme
email: prudhomme.sophie@gmail.com

David Renault
email: david.renault@univ-rennes1.fr

Jean-Philippe David
email: jean-philippe.david@univ-grenoble-alpes.fr

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

A multiscale approach to decipher the molecular mechanisms involved in the direct and intergenerational effect of ibuprofen on the mosquito Aedes aegypti

ABSTRACT:

The anti-inflammatory ibuprofen is a ubiquitous surface water contaminant. However, the chronic impact of this pharmaceutical on aquatic invertebrate population remains poorly understood. In the model insect Aedes aegypti, we investigated the intergenerational consequences of parental chronic exposure to an environmentally relevant concentration of ibuprofen. While exposed individuals did not show any phenotypic changes, their progeny showed accelerated development and an increased tolerance to starvation. In order to understand the mechanistic processes underpinning the direct and intergenerational impacts of ibuprofen, we combined transcriptomic, metabolomics and hormone kinetics studies at several life-stages in exposed individuals and their progeny. This integrative approach revealed moderate transcriptional changes in exposed larvae consistent with the pharmacological mode of action of ibuprofen. Parental exposure led to lower levels of several polar metabolites in progeny eggs, and to major transcriptional changes at the following larval stage. These transcriptional changes, most likely driven by changes in expression of numerous transcription factors and epigenetic regulators, led to ecdysone signalling and stress response potentiation. Overall, the present study illustrates the complexity of the molecular basis of the intergenerational pollutant response in insects and the importance of considering the entire life cycle of exposed organisms and of their progeny, in order to fully understand the mode of action of pollutants and their impact of ecosystems.

INTRODUCTION:

Since the early 2000s, the worldwide surface water contamination by pharmaceutical products and the associated ecotoxicological consequences on aquatic organisms has been an issue of increasing concern. [START_REF] Beek | Pharmaceuticals in the Environment-Global Occurrences and Perspectives[END_REF][START_REF] Fent | Ecotoxicology of Human Pharmaceuticals[END_REF] Ibuprofen, a non-steroidal anti-inflammatory drug used for general pain relief, is one of the most commonly detected pharmaceutical products. [START_REF] Ashton | Investigating the Environmental Transport of Human Pharmaceuticals to Streams in the United Kingdom[END_REF][START_REF] Loos | EU-Wide Survey of Polar Organic Persistent Pollutants in European River Waters[END_REF] Due to its widespread use, ibuprofen is chronically released into the aquatic environment, via urinary excretion and the direct disposal of unused or outdated pills in urban wastewater. [START_REF] Fent | Ecotoxicology of Human Pharmaceuticals[END_REF] Despite its relatively short life-time in water, [START_REF] Buser | Occurrence and Environmental Behavior of the Chiral Pharmaceutical Drug Ibuprofen in Surface Waters and in Wastewater[END_REF] the continuous release of ibuprofen causes a phenomenon of pseudo-persistence in aquatic ecosystems. [START_REF] Hernando | Environmental Risk Assessment of Pharmaceutical Residues in Wastewater Effluents, Surface Waters and Sediments[END_REF] This results in measured concentrations usually ranging from 0.005 to 5 µg/L in European surface water, up to tens of µg/L at several sites worldwide. [START_REF] Ashton | Investigating the Environmental Transport of Human Pharmaceuticals to Streams in the United Kingdom[END_REF][START_REF] Loos | EU-Wide Survey of Polar Organic Persistent Pollutants in European River Waters[END_REF][START_REF] Marsik | Non-Steroidal Anti-Inflammatory Drugs in the Watercourses of Elbe Basin in Czech Republic[END_REF][START_REF] Nannou | Occurrence of Pharmaceuticals in Surface Waters: Analytical Method Development and Environmental Risk Assessment[END_REF][START_REF] Matongo | Pharmaceutical Residues in Water and Sediment of Msunduzi River, KwaZulu-Natal, South Africa[END_REF][START_REF] Rivera-Jaimes | Study of Pharmaceuticals in Surface and Wastewater from Cuernavaca, Morelos, Mexico: Occurrence and Environmental Risk Assessment[END_REF] Several studies have illustrated the impact of chronic exposure to ibuprofen at individual or sub-individual level on several freshwater invertebrates such as mollusks, cnidarians and crustaceans. [START_REF] Heckmann | Chronic Toxicity of Ibuprofen to Daphnia Magna: Effects on Life History Traits and Population Dynamics[END_REF][START_REF] Hayashi | Reproduction Recovery of the Crustacean Daphnia Magna after Chronic Exposure to Ibuprofen[END_REF][START_REF] Han | Endocrine Disruption and Consequences of Chronic Exposure to Ibuprofen in Japanese Medaka (Oryzias Latipes) and Freshwater Cladocerans Daphnia Magna and Moina Macrocopa[END_REF][START_REF] Aguirre-Martínez | Caffeine, Ibuprofen, Carbamazepine, Novobiocin and Tamoxifen Have an Effect on Corbicula Fluminea (Müller, 1774)[END_REF][START_REF] Contardo-Jara | Exposure to Human Pharmaceuticals Carbamazepine, Ibuprofen and Bezafibrate Causes Molecular Effects in Dreissena Polymorpha[END_REF][START_REF] Pounds | Acute and Chronic Effects of Ibuprofen in the Mollusc Planorbis Carinatus (Gastropoda: Planorbidae)[END_REF][START_REF] Pascoe | Do Pharmaceuticals Affect Freshwater Invertebrates? A Study with the Cnidarian Hydra Vulgaris[END_REF] Hence, the occurrence, persistence and ecotoxicological data currently available for this pharmaceutical product have led several authors to classify ibuprofen as a priority pollutant for ecotoxicological risk assessment. [START_REF] Zuccato | Identification of the Pharmaceuticals for Human Use Contaminating the Italian Aquatic Environment[END_REF][START_REF] Besse | Human Pharmaceuticals in Surface Waters[END_REF] In mammals, the anti-inflammatory effect of ibuprofen is due to an inhibition of prostaglandin synthesis through competitive fixation on a cyclooxygenase (COX) active site. [START_REF] Charlier | Dual Inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) as a New Strategy to Provide Safer Non-Steroidal Anti-Inflammatory Drugs[END_REF] Prostaglandins belong to the eicosanoids family and are messengers of the inflammatory reaction. These lipid compounds are synthetized from arachidonic acid oxidation by COX1 and COX2. [START_REF] Charlier | Dual Inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) as a New Strategy to Provide Safer Non-Steroidal Anti-Inflammatory Drugs[END_REF] This mode of action (MOA) has also been demonstrated in freshwater fish exposed to ibuprofen. [START_REF] Jeffries | Chronic Exposures to Low and High Concentrations of Ibuprofen Elicit Different Gene Response Patterns in a Euryhaline Fish[END_REF][START_REF] Patel | Testing the "read-across Hypothesis" by Investigating the Effects of Ibuprofen on Fish[END_REF] Surprisingly, the molecular and physiological alterations induced in ibuprofen-exposed invertebrates remain poorly documented, despite the fact that invertebrates are usually remarkably abundant in water ecosystems and are an important part of the food web. Acute exposure of Daphnia magna led to the modification of expression of several enzymes involved in eicosanoids synthesis, and was associated with impaired reproductive function. [START_REF] Heckmann | Systems Biology Meets Stress Ecology: Linking Molecular and Organismal Stress Responses in Daphnia Magna[END_REF] Similarly, Milan et al. demonstrated that exposure of the manila clam Ruditapes philippinarum to concentrations ranging from 0.1 to 1 mg/L over 7 days modifies the transcription rate of several genes involved in cell respiration, arachidonic acid metabolism, eicosanoid metabolism, immunity and inflammatory response. [START_REF] Milan | Gene Transcription and Biomarker Responses in the Clam Ruditapes Philippinarum after Exposure to Ibuprofen[END_REF] It is important to note that these transcriptomic-based studies were performed at unrealistic exposure concentrations. As the transcriptomic responses elicited by a pollutant largely depend on the exposure concentration, mechanistic studies performed at high concentrations may not be representative of the transcriptional modifications elicited in organisms at environmental concentrations. [START_REF] Jeffries | Chronic Exposures to Low and High Concentrations of Ibuprofen Elicit Different Gene Response Patterns in a Euryhaline Fish[END_REF] A recent study on the freshwater mussel Dreissena polymorpha using more environmentally realistic concentrations demonstrated that acute exposure induced decreased COX activity at concentrations ranging from 10 to 100 µg/L, though the molecular mechanisms involved in the resulting physiological changes were not assessed in this study. [START_REF] André | Cumulative Effects of Ibuprofen and Air Emersion in Zebra Mussels Dreissena Polymorpha[END_REF] Understanding the inter-, trans-and multigenerational impact of pollutants is an area of increasing interest and constitutes a major challenge in ecotoxicology, as it allows a better grasp of the long-term consequences of the chronic exposure of populations to pollutants. [START_REF] Bachère | Parental Diuron-Exposure Alters Offspring Transcriptome and Fitness in Pacific Oyster Crassostrea Gigas[END_REF][START_REF] Kimberly | Multigenerational Contaminant Exposures Produce Non-Monotonic, Transgenerational Responses in Daphnia Magna[END_REF][START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF][START_REF] Reátegui-Zirena | Transgenerational Endpoints Provide Increased Sensitivity and Insight into Multigenerational Responses of Lymnaea Stagnalis Exposed to Cadmium[END_REF][START_REF] Windsor | Endocrine Disruption in Aquatic Systems: Up-Scaling Research to Address Ecological Consequences[END_REF] In a recent study, we evaluated the short and long-term impact of chronic exposure to an environmentally-relevant concentration of ibuprofen (1µg/L) on the life-history traits of the A. aegypti mosquito. [START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF] Although no changes were observed in exposed F0 individuals, this work demonstrated transgenerational consequences of ibuprofen exposure, leading to an increased developmental rate in the F1 progeny grown in an unpolluted environment. As a vector of several arboviruses responsible for major human diseases such as dengue fever, yellow fever, chikungunya or zika, a huge amount of ecological, physiological and genomic data are available for this mosquito species. In particular, the hormonal and metabolic regulation of key developmental and reproductive events have been extensively characterized. [START_REF] Briegel | Metabolic Relationship between Female Body Size, Reserves, and Fecundity of Aedes Aegypti[END_REF][START_REF] Margam | Ecdysteroid Titers and Developmental Expression of Ecdysteroid-Regulated Genes during Metamorphosis of the Yellow Fever Mosquito, Aedes Aegypti (Diptera: Culicidae)[END_REF][START_REF] Timmermann | Larval Growth and Biosynthesis of Reserves in Mosquitoes[END_REF] Its genome has been sequenced and continuously annotated since 2007, leading to an improved understanding of the molecular basis of its physiology over the past 10 years. [START_REF] Nene | Genome Sequence of Aedes Aegypti, a Major Arbovirus Vector[END_REF] This insect species thus represents a valuable biological model for delineating the interactions between genotypic and phenotypic responses in aquatic insects, and thus improving our understanding of the impacts of pollutants on individuals.

In this context, the present study aims at understanding the molecular basis of the direct and intergenerational disturbances caused by ibuprofen exposure in A. aegypti, and how these link to phenotype modifications. We developed an integrative approach combining lifespan and stress tolerance measurement with metabolomics, transcriptomics, and hormone kinetics to assess the general physiological state of directly-exposed individuals and their progeny. As well as providing new insights into the direct consequences of ibuprofen exposure, this approach allowed us to understand the molecular basis of the disturbed phenotype in the progeny of exposed individuals.

METHODS:

Animal rearing

A laboratory strain of A. aegypti (Liverpool strain) was provided by the Pasteur institute (Paris, France) and used for all experiments. The rearing conditions used for aquatic stages and imagoes (i.e. mature life stage in insects) were the same as in Prud'homme et al, [START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF] and are detailed in SI-1.

Chronic exposure of mosquitoes to ibuprofen

Ibuprofen (purity 98%, CAS no. 15687-27-1), was purchased from Sigma-Aldrich (St. Louis, MO). Ten mg/L stock solutions were prepared in absolute ethanol (HPLC grade) at the beginning of the experiment and aliquots were stored at -20°C. The contaminated media was freshly prepared with tap water and aliquots of ibuprofen stock solution to reach concentrations of 1 µg/L. The final solvent (ethanol) concentration was 0.002% for the contaminated and control media. Chronic exposure to ibuprofen or solvent-control, lasting from larvae hatching to imagoes emergence, was conducted with static renewal of the media, replaced every 48h. F1 spawn obtained from F0 exposed and control individuals were raised in unpolluted media.

In order to ensure effective concentrations of ibuprofen in the culture media, the contaminated media were freshly prepared in two separate glass dishes and kept in the same light and temperature conditions as in our experiment. Larvae were added to one of the dishes at a density of 1 larva/mL to compare the change in ibuprofen concentration in the culture media, with or without the presence of larvae. 500 mL aliquots were sampled at 0, 24, and 48h (without larvae) and at 48h (with larvae) and stored at -20 °C until ibuprofen concentration measurement. Ibuprofen was extracted by solid phase extraction and measured by LC-HRMS/MS (SI-2). Dosage accuracy was determined by measuring concentration of analytical standard in five independent extraction replicates of a reference media with 1µg/L of analytical standard. Initial concentrations of ibuprofen were close to the nominal concentration (1 µg/L) and remained relatively stable over 48h, with or without larvae (Figure S1).

Experimental design

All F0 and F1 populations were raised at a density of 1 larvae/mL, in the same light, temperature and food conditions as described in SI-1. In these conditions, imago emergence occurred from the 7 th (males) and 9 th (females) days after hatching. In Aedes aegypti, mating occurs immediately after female emergence, and females are mature to spawn 72h after emergence. Egg maturation is initiated by blood feeding, and eggs are spawned on the 3rd day following the blood-meal. In our experiment, a blood-meal was provided to F0 females 15 days after the start of our experiment (1 st stage larvae hatching), to ensure their mating status and sexual maturity. Egg fertilization and embryonic development are concomitant to spawning, and dried eggs enter into a quiescent phase at the end of embryonic development. The hatching of quiescent eggs occurs in the 1 st hour following egg immersion.

In a first experiment, populations of 2,000 individuals were either exposed to ibuprofen and solvent or solvent only (controls). Experiments were performed in quadruplicate concomitantly for both ibuprofen exposure and controls, and all replicates were raised in parallel. Individuals were collected at different time points, corresponding to different developmental stages in exposed populations (F0) and their progeny subsequently grown in unpolluted medium (F1) (Figure S2). These individuals were further used to perform individual phenotypic characterization (Figure S2: ③, ⑪), to monitor hormone levels and the transcription level of key associated genes during egg maturation (Figure S2: ⑦,⑧) and to perform a transcriptome-wide gene expression study by RNA-seq (Figure S2: ①,⑥,⑩). In a second experiment, two independent sets of 2,000 individuals were exposed to ibuprofen or solvent alone (control) in the same conditions detailed above, and individuals were collected at several developmental stages to perform a comparative directed metabolomic analysis by GC-MS (Figure S2: ②,④,⑤,⑨).

Phenotypic responses to chronic exposure

Imago lifespan

F0 imagoes from each replicate population were collected in a 10-hour window after emergence. Females and males were separated before mating and placed in distinct cages in order to assess the lifespan of virgin individuals (3 replicate cages per condition). A total of at least 59 virgin males and 32 virgin females were isolated from ibuprofen exposed and control populations respectively. At least 52 primiparous females from each F0 population replicate were collected one day after their first egg-laying and placed in separate cages. Primiparous females emerged in a time laps of 4 days. The lifespan of these females is thus considered ± 2 days.

Imagoes had access to water and sucrose ad libitum. The number of dead individuals in each cage was assessed daily until the death of the last surviving imago.

Progeny tolerance to starvation

The emerging imagoes remaining after sampling for lifespan measurement, hormonal and transcriptional analysis were collected twice a day and males and females were placed in net cages for mating, as previously described. [START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF] F1 eggs spawned after the first blood-meal were collected and air-dried for one week. F1 egg batches (≈ 1000 eggs) were then simultaneously immersed to elicit hatching as described. [START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF] For each replicate population, 3 groups of 20 newly hatched larvae (< 1h) were isolated, resulting in 12 groups per parental exposure condition. These larvae were raised in unpolluted media with no food resources in order to assess starvation-survival in the F1 progeny. Surviving larvae were counted daily until the death of the last surviving larvae. Dead individuals were regularly removed to avoid necrophagy. Larvae that did not respond to contact stimulation were considered dead.

Statistical analysis

The survival curves for F0 imagoes exposed to ibuprofen or solvent-control and of their F1 larvae progeny were compared using a Kaplan-Meyer survival test performed with R software. [START_REF]R: A Language and Environment for Statistical Computing[END_REF]

Ecdysteroid dynamics during vitellogenesis

Four females were collected from each F0 replicate population 30 min before and 4, 8, 16, 20, 24, 36 and 48h after blood feeding in order to assess ecdysteroid titer dynamics during vitellogenesis (Figure S2,⑧). The collected females were isolated in 250 µL methanol (MeOH) pro-analysis (Merk, Germany) and stored at -20°C.

Total ecdysteroids from whole animals were extracted with MeOH and re-dissolved in enzymatic immunoassay (EIA) buffer (SI-3). Ecdysteroids were quantified using an EIA adapted from the method described in Porcheron et al. [START_REF] Porcheron | Development of an Enzyme Immunoassay for Ecdysteroids Using Acetylcholinesterase as Label[END_REF] (SI-3). Ecdysteroids titers were estimated using a 20-hydroxyecdysone (20E) calibration curve (8 to 4,000 fmol; Sigma-Aldrich, USA) diluted in EIA reaction buffer, and titers were expressed as 20E equivalents. All measurements were performed in duplicate and the results were expressed as mean values ± SEM. Eight to 16 females were assessed at each time-point for each exposure condition. Mean ecdysteroid titer at each time-point was compared between exposed and control females using a Student's t-test, after data checking for normality (Shapiro-Wilk test) and homoscedasticity (F-test). The resulting p-values were corrected for multiple comparison (8 time-points) using the FDR method.

Gene transcription dynamics during vitellogenesis

The transcription levels of 5 ecdysone related genes (74EF, VtgA, VtgR, Lp, LpRov, Table S1) were assessed at several stages in the vitellogenic process. Four pools of 5 to 10 blood-fed females were collected from each F0 replicate 30 min before and 4h, 8h, 16h, 20h, 24h, 36h and 48h after blood feeding and stored in TRIzol (Invitrogen Life Technologies, USA) at -80°C. Total RNAs were extracted from each sample (SI-4) and realtime quantitative PCR (RT-qPCR) reactions were performed in triplicate on the iQ5 system (Bio-Rad, USA) (SI-4). The primer sequences are listed in Table S1. The primers used for RT-qPCR were designed using Beacon Designer™ 5.10 software, except for the rpl32, VtgR and LpRov genes, for which the primers were already published. [START_REF] Bonizzoni | RNA-Seq Analyses of Blood-Induced Changes in Gene Expression in the Mosquito Vector Species, Aedes Aegypti[END_REF][START_REF] Clifton | The Fate of Follicles after a Blood Meal Is Dependent on Previtellogenic Nutrition and Juvenile Hormone in Aedes Aegypti[END_REF][START_REF] Perez | Aedes Aegypti Pharate 1st Instar Quiescence Affects Larval Fitness and Metal Tolerance[END_REF] Data analysis was performed according to the ∆∆Ct method, [START_REF] Pfaffl | A New Mathematical Model for Relative Quantification in Real-Time RT-PCR[END_REF] taking into account PCR efficiency and using the housekeeping genes encoding the ribosomal protein RPL32 (AAEL00339) and actin (AAEL001673) for dual-gene normalization. The results were expressed as the mean transcription ratio between ibuprofen exposed and control females at each time point. Wilcoxon tests were performed to estimate the significant changes at each gene transcription level between ibuprofen-exposed and control females at each time point. The resulting p-values were corrected for multiple comparison (8 time-points) using the FDR method.

Metabolomic GC-MS analysis

The metabolomic experiments were performed on F0 4 th stage larvae (L4F0), F0 male and female imagoes (respectively MalF0 and FemF0), and F1 eggs 4h after laying (EggF1), obtained from the two population replicates exposed to ibuprofen or the two control populations. Three batches of 10 4 th stage F0 larvae (34-40 hours after moult) were collected from each replicate of both exposure conditions. Four batches of 10 virgin F0 females and 4 batches of 10 virgin F0 males were collected 72h after emergence from each replicate of both exposure conditions. Batches of individuals were also sampled for cytosolic protein content analysis (SI-5).

Following the F0 population breeding, three batches of 100 F1 eggs were collected in the 4 hours following egglaying from each replicate of both exposure conditions. Samples were snap frozen in liquid N 2 before being stored at -80°C. The samples were then lyophilized, weighed, and stored at -20°C until metabolite extraction using methanol/chloroform/water (2/1/2) solid/liquid extraction. The detailed extraction and derivatization procedure is provided in the supplementary information (SI-6.1/2). The metabolomics approach was directed toward 53 polar metabolites representatives of the insect metabolome (Table S2). Metabolite quantification was performed using Gas Chromatography-Mass Spectrometry (GC-MS) on a system consisting of a CTC CombiPal autosampler (PAL System, CTC Analytics AG, Zwingen, Switzerland), a Trace GC Ultra chromatograph and a Trace DSQII quadrupole mass spectrometer (Thermo Fischer Scientific Inc, Waltham, MA, USA). The detailed quantification procedure is provided in the supplementary information (SI-6.3). Metabolite concentrations were expressed as nmol/mg of dry mass. Concentration data were imported in Metaboanalyst 3.0 for the statistical analyses. [START_REF] Xia | MetaboAnalyst 3.0--Making Metabolomics More Meaningful[END_REF] Data were paretoscaled before the statistical analyses and individual metabolite concentrations were compared according to the exposure group using a multivariate approach by PCA and supervised orthogonal partial least square discriminant analyses (OPLS-DA) (SI-6.4). Individual concentrations of the metabolites were compared using Wilcoxon rank-sum test. The resulting p-values were adjusted using Benjamini & Hochberg's method (FDR).

Transcriptomic analysis by RNAseq

The transcriptomic analysis was performed on F0 4 th stage larvae (L4F0), F0 imago females (FemF0) and 1 st stage larvae from F1 progeny (L1F1). In each of the four F0 biological replicate populations, batches of 35 exposed or control 4 th stage larvae were collected 34h to 40h following moult to perform the transcriptomic analysis (Figure S2,①). Batches of 30 mature virgin females, raised with ad libitum access to water and 10% sucrose, were collected 72 hours after emergence in each F0 independent replicate, after being instantly killed by brief immersion in 70% ethanol (Figure S2,⑥). Batches of 300 newly-hatched F1 larvae (< 30 min) spawned by each F0 replicate were reared in unpolluted medium with food. These larvae were collected 10h after hatching for the transcriptome analysis of the unexposed progeny (F1) (Figure S2,⑩). Collected L4F0 samples were immediately stored in TRIzol reagent (Invitrogen Life Technologies, USA) at -80°C. FemF0 and L1F1 samples were immediately immerged in RNAlater (Ambion, USA) and stored at 4°C for 24h, and then at -80°C before RNA extraction. The procedure for total RNA extraction is detailed in the supporting information (SI-7.1). Out of the four independent RNA samples collected for each life-stage and each exposure condition, the three showing the best quality profile on BioAnalyseur 2100 (Agilent Technologies, USA) were used for the high throughput RNA sequencing (RNAseq) transcriptomic analysis. c-DNA libraries were prepared from 400ng total RNA using Illumina TruSeq Stranded mRNA Sample Preparation kits (Illumina Inc., USA) following the manufacturer's instructions. Libraries were sequenced as 75 bp single reads (L4F0) or 150 bp single reads (FemF0 and L1F1) on an Illumina platform by Helixio-Hybrigenics, France. Raw-data processing was performed on the galaxy.prabi.fr web service and the computing facilities of the LBBE/PRABI (Lyon, France). Data were first filtered according to size and quality parameters using Trimmomatic [START_REF] Bolger | Trimmomatic: A Flexible Trimmer for Illumina Sequence Data[END_REF] (SI-7.2). Tophat2 43 (v0.6) was used to map filtered reads to the A. aegypti reference genome (Aaeg L3.3 assembly and gene set, http://vectorbase.org). The following data processing was based on the Cufflink RNA-seq workflow (v2.2.0; http://cole-trapnell-lab.github.io/ cufflinks/), [START_REF] Trapnell | Differential Analysis of Gene Regulation at Transcript Resolution with RNA-Seq[END_REF][START_REF] Trapnell | Differential Gene and Transcript Expression Analysis of RNA-Seq Experiments with TopHat and Cufflinks[END_REF] using the Cufflink, Cuffmerge and Cuffdiff suite of tools to identify differentially transcribed genes (DTG) between ibuprofen and control individuals (L4F0, FemF0) or progeny (L1F1) (SI-7.2). Genes were considered as differentially transcribed when the transcription ratio TR (ibuprofen vs. control) was >1.2 in either direction, with a corrected p-value (q-value; Benjamini-Hochberg correction) below 0.05. Only genes for which transcription level was superior to 0.5 FPKM in at least one of the two considered conditions were considered, in order to avoid expression level inaccuracy. The median transcriptional fold-changes for each transcriptomic analysis were compared using a Kruskal-Wallis test followed by a Dunn's test, using FDR to adjust the p-values.

For the functional analysis, 20 functional classes were defined to help describe the physiological processes affected by ibuprofen exposure, and all detected genes were assigned to one these classes based on their annotation (see Table S5 and SI). DTG repartition among these functional classes was compared to the distribution of all detected genes using a Fisher's exact test where p-values were corrected for multiple-testing using the FDR method. The enrichment of some functional classes among DTG was studied independently for up-regulated genes and down-regulated genes.

RESULTS AND DISCUSSION

Phenotypic consequences of direct and parental ibuprofen exposure

Direct ibuprofen exposure did not impact the lifespan of virgin and primiparous F0 females while F0 virgin male imago showed a 23% increased lifespan (Xsq=3.8, df=1, p-value=0.051) (Figure 1). Our previous study revealed that ibuprofen exposure did not affect the development of F0 individuals or imago size, suggesting that ibuprofen exposure has limited phenotypic consequences on F0 individuals. [START_REF] Prud'homme | Impact of Micropollutants on the Life-History Traits of the Mosquito Aedes Aegypti: On the Relevance of Transgenerational Studies[END_REF] In F1 progeny, the survival of 1 st stage larvae obtained from exposed parents under starving condition was doubled compared to the controls (Xsq=18.4, df=1, p-value=1.8e -05 ) (Figure 2). These results, combined with the reduction in development time previously observed in the F1 progeny of ibuprofen-exposed individuals (Figure S3), confirms the phenotypic changes caused by chronic parental exposure to ibuprofen.

Overall transcriptomic consequences of direct and parental ibuprofen exposure

The transcriptomic analyses identified 134 differentially transcribed genes (DTGs) in F0 4 th stage larvae (L4F0), 40 DTGs in F0 female imagoes (FemF0) and 1566 DTGs in F1 1 st stage larvae (L1F1) between exposed individuals and the controls respectively. This represents 1.1%, 0.3% and 14.8% of the genes detected at each of the three life-stages studied (Figure 3,A). DTG were poorly shared among the developmental stages (Figure 3, B). Interestingly, the range of differential transcription was greater in directly exposed individuals as compared to their progeny, with median fold-changes associated to DTG that were greater in L4F0 (z=-6.27, p-value≈0) and FemF0 (z=9.03, p-value≈0) than in L1F1 (Figure 3,C). As most phenotypic variations were observed in the F1 progeny of exposed individuals (increased starvation-survival and faster development), the range of differential transcription may not necessarily be associated with evident phenotypic changes at the individual scale. Conversely, numerous but moderate transcriptional changes can cause a pronounced disruption in development, as previously demonstrated in Daphnia magna. [START_REF] Barata | Do Genotype Responses Always Converge from Lethal to Nonlethal Toxicant Exposure Levels? Hypothesis Tested Using Clones of Daphnia Magna Straus[END_REF] Molecular changes associated with direct ibuprofen exposure in 4 th stage F0 larvae Direct exposure did not affect the representation of the 53 quantified polar metabolites in 4 th stage F0 larvae (Figure S4, A, Table S6), but induced the differential transcription of 134 genes. Interestingly, the transcriptomic profile analysis did not show any stress-response related markers. Among the DTGs, 51 genes were up-regulated and 83 genes were down-regulated (Table S7 and SI). The functional enrichment analysis revealed a significant overrepresentation of genes coding for cuticular proteins and kinases/phosphatases among the up-regulated genes, and of genes involved in immunity, intra-and extra-cellular trafficking or coding for kinases/phosphatases among the down-regulated genes (Table 1, Table S7). The "ccaat/enhancer binding protein" (C/EBP, AAEL002853) was the only transcription factor showing a differential transcription in larvae following direct exposure (-1.6 fold). In insects, this transcription factor has been associated with the activation of genes involved in various physiological functions, such as immune response and iron or protein homeostasis. [START_REF] Meredith | A Novel Association between Clustered NF-kappaB and C/EBP Binding Sites Is Required for Immune Regulation of Mosquito Defensin Genes[END_REF][START_REF] Pham | Identification and Mapping of the Promoter for the Gene Encoding the Ferritin Heavy-Chain Homologue of the Yellow Fever Mosquito Aedes Aegypti[END_REF][START_REF] Zakharkin | Female-Specific Expression of a Hexamerin Gene in Larvae of an Autogenous Mosquito[END_REF] According to the functional enrichment analysis, several genes related to immune response were overrepresented amongst the down-regulated genes, including five anti-microbial peptides (CECN, CECE, DEFC, DEFD, DEFA), two clip-domain serine proteases, two prophenoloxidases (PPO4, PPO6), the C-type lectin CTLGA5 and one macroglobulin/complement (Figure 4, C, D; Table S7). Interestingly, C/EBP binding domains were detected in the upstream region of defensins in A. aegypti and shown to be essential for their regulation together with other antimicrobial peptides genes. [START_REF] Meredith | A Novel Association between Clustered NF-kappaB and C/EBP Binding Sites Is Required for Immune Regulation of Mosquito Defensin Genes[END_REF] The overrepresentation of genes involved in intra/extra-cellular trafficking was mainly due to the downregulation of all nine hexamerins annotated in the A. aegypti genome from -3.5 to -45.7 fold (Figure 4, C; Table S7). Hexamerins play a crucial role as amino acid resources for imaginal metamorphosis in holometabolous insects. Hexamerins are synthetized in the mosquito's fat body mainly during the 4 th larval stage and proteolized during the non-feeding nymphal stage, thus providing the amino acid resources needed for the development of the future imago. [START_REF] Zakharkin | Female-Specific Expression of a Hexamerin Gene in Larvae of an Autogenous Mosquito[END_REF][START_REF] Haunerland | Insect Storage Proteins: Gene Families and Receptors[END_REF] In A. aegypti, protein biosynthesis during 4 th larval stage contributes to 70 to 88% of the total protein quantity disposable for the nymphal metamorphosis. [START_REF] Timmermann | Larval Growth and Biosynthesis of Reserves in Mosquitoes[END_REF] The substantial down-regulation of hexamerin transcription in F0 4 th stage larvae exposed to ibuprofen should therefore affect the performance and protein resources of exposed pupae and imagoes. However, this down-regulation did not result in an alteration in the 15 free amino acid levels measured by GC-MS (Table S6) or the total cytosolic protein content (Figure S6) in F0 imagoes. This demonstrates that the individual scale consequences of transcriptional changes should be correlated to functional or phenotypic changes. Some studies also suggested that hexamerins could be involved in innate immune response in mosquitoes and other insects species. [START_REF] Lombardo | Novel Factors of Anopheles Gambiae Haemocyte Immune Response to Plasmodium Berghei Infection[END_REF][START_REF] Poopathi | Hexamerin a Novel Protein Associated with Bacillus Sphaericus Resistance in Culex Quinquefasciatus[END_REF][START_REF] Burmester | Evolution and Function of the Insect Hexamerins[END_REF] The down-regulated genes involved in intra/extra-cellular trafficking also included two transferrin and two ferritin coding genes (Figure 4, C; Table S7). These proteins, mainly synthesized in the fat bodies and basically involved in iron transport, are also known to play a role in immune responses in insects. [START_REF] Geiser | Insect Transferrins: Multifunctional Proteins[END_REF][START_REF] Zhou | Differential Regulation of Transferrin 1 and 2 in Aedes Aegypti[END_REF] Interestingly, C/EBP binding sites have been shown to regulate hexamerins and ferritins in the Aedes species. [START_REF] Pham | Identification and Mapping of the Promoter for the Gene Encoding the Ferritin Heavy-Chain Homologue of the Yellow Fever Mosquito Aedes Aegypti[END_REF][START_REF] Zakharkin | Female-Specific Expression of a Hexamerin Gene in Larvae of an Autogenous Mosquito[END_REF] Finally, four genes encoding odorant binding proteins, a large protein family found to be involved in immune response [START_REF] Levy | Peptidomic and Proteomic Analyses of the Systemic Immune Response of Drosophila[END_REF] were also up-regulated in exposed larvae (Figure 4, D; Table S7).

In insect species, prostaglandins play a role in the immune response by mediating the induction of immune genes, and eicosanoid biosynthesis inhibition in insects has been shown to disrupt the immune response. [START_REF] Büyükgüzel | The Influence of Chronic Eicosanoid Biosynthesis Inhibition on Life History of the Greater Waxmoth, Galleria Mellonella and Its Ectoparasitoid, Bracon Hebetor[END_REF][START_REF] Büyükgüzel | Eicosanoids Mediate Galleria Mellonella Cellular Immune Response to Viral Infection[END_REF][START_REF] Durmuş | Eicosanoids Mediate Melanotic Nodulation Reactions to Viral Infection in Larvae of the Parasitic Wasp, Pimpla Turionellae[END_REF][START_REF] Isao | Eicosanoids Mediate Induction of Immune Genes in the Fat Body of the Silkworm, Bombyx Mori[END_REF] The down-regulation of immune response genes and hexamerins observed in F0 larvae (Figure 4, C-D) is thus consistent with a disruption in prostaglandin synthesis, that might be induced by the inhibition of COX activity (Figure 4,A). This is supported by the fact that the COX inhibitory activity of ibuprofen has been evidenced in other invertebrate species, including the mosquito Anopheles albimanus. [START_REF] André | Cumulative Effects of Ibuprofen and Air Emersion in Zebra Mussels Dreissena Polymorpha[END_REF][START_REF] Gil De Muñoz | Prostaglandin E2 Modulates the Expression of Antimicrobial Peptides in the Fat Body and Midgut ofAnopheles Albimanus[END_REF] As detailed above, several downregulated genes in F0 larvae are regulated by the C/EBP transcription factor. Interestingly, in mammal macrophages the pro-inflammatory activity of prostaglandins has been shown to be mediated by the activation of CREB, allowing it to bind to the C/EBP-β promoter and leading to an over-transcription of C/EBP-β expression and of its target genes. [START_REF] Na | Endogenous Prostaglandin E2 Potentiates Anti-Inflammatory Phenotype of Macrophage through the CREB-C/EBP-β Cascade[END_REF] This suggests that the under-transcription of immune response genes and hexamerins may be at least partially due to the down-regulation of C/EBP, through a disruption of prostaglandin synthesis in exposed larvae (Figure 4 A,C). However, the influence of prostaglandins on C/EBP expression has never been investigated in insect species, and this hypothesis needs to be confirmed.

Molecular changes associated with direct ibuprofen exposure in F0 imagoes

Only 40 DTG were detected in ibuprofen-exposed F0 females (Table S8 and SI). Such a small number of DTG did not allow for the identification of specific changes in physiological pathways or functions. Moreover, based on the metabolite concentrations measured, the global metabolic state of F0 females and males did not seem to be affected by ibuprofen exposure (Figure S4,B,C; Table S8). Furthermore, exposure did not affect ecdysteroid levels, nor the transcription dynamic of five key genes associated with ecdysteroid response and vitellogenesis (Figure S5). Taken together these results suggest that the transcriptional changes observed in exposed F0 4 th stage larvae have very limited consequences on imagoes, both at molecular and individual scales.

Indirect impact of ibuprofen exposure on metabolic resources of in F1 progeny eggs

The metabolic resources available for F1 progeny development were measured in 1 to 4 hour-old eggs, corresponding to the very beginning of embryonic development and before the initiation of maternal-zygotic transition. [START_REF] Biedler | Identification of Early Zygotic Genes in the Yellow Fever Mosquito Aedes Aegypti and Discovery of a Motif Involved in Early Zygotic Genome Activation[END_REF] The multivariate analysis of metabolic profiles using ACP revealed a clear segregation between the metabolic profiles of progeny obtained from exposed individuals and the controls (Figure 5,A). An OPLS-DA was performed to investigate the metabolites responsible for the separation of the two exposure groups. The model fitted well (R2X= 0.44, R2Y= 0.72 -p=0.011 according to permutation test), and showed good predictability (Q2Y= 0.66 -p= 0.007 according to permutation test), confirming the divergence of metabolic profiles. The univariate approach using the Wilcoxon rank-sum test revealed that 20 metabolites were significantly underrepresented from 12% to 81% in eggs obtained from ibuprofen-exposed parents (Figure 5,B). These 20 metabolites are all characterized by correlation coefficient p-corr ≥ |0.5| in the OPLS-DA model, confirming a good consistency between the univariate and multivariate statistical approaches. The eggs obtained from ibuprofen-exposed individuals contained lower levels of amino acids (12 out of the 17 detected amino acids), carbohydrates (maltose, sucrose), polyols (glycerol, arabitol, myoinositol and ribitol), phosphoric acid and ornithine (Figure 5, B), implying that metabolic resource internalization in eggs is affected by ibuprofen exposure. As embryonic development only relies on maternal nutrients, such changes in metabolic resources may modify the physiology and phenotype of the resulting embryo and larvae. [START_REF] Vital | Germ Band Retraction as a Landmark in Glucose Metabolism during Aedes Aegypti Embryogenesis[END_REF]

Impact of parental ibuprofen exposure on the transcriptional state of young F1 larvae

Global transcriptional responses and functional analysis. The transcriptome analysis of F1 1 st stage larvae obtained from ibuprofen-exposed parents revealed a broad change in transcriptional state compared to the controls, with 1,566 DTGs (detailed in SI). The functional enrichment analysis of up-regulated genes revealed an overrepresentation of transcripts involved in epigenetic marks and transcription regulation (Table 2), which could explain the divergent transcription profiles observed between the progeny of control versus exposed parents. Genes involved in signal transduction and cell/tissue structure were also enriched among the upregulated genes (Table 2). The 450 down-regulated genes revealed an enrichment of genes involved in protein metabolism, lipid metabolism, mitochondrial metabolism, detoxification and formation of the cuticle (Table 2). Interestingly, of the 66 genes involved in protein metabolism, 53 encoded for proteases (SI). The change in metabolic resources available for embryonic development thus seems to define a distinct physiological state in the progeny, reflected by a shift in F1 larvae transcriptional state, most likely driven by the differential expression of numerous transcription factors and epigenetic mark regulators.

Increase in ecdysone signalling. 20E signalling is mediated by the heterodimer of nuclear receptor ecdysone receptor (EcR)/ultraspiracle (USP). EcR-USP activation by 20E leads to the transcriptional activation of three "early-response genes": Br, E74 and E75. These early-response genes code for the transcription factors responsible for the regulation of a large set of 20E target genes, so-called "early-late" and "late-response genes". [START_REF] Ou | What Goes up Must Come down: Transcription Factors Have Their Say in Making Ecdysone Pulses[END_REF] A more detailed analysis of the DTGs associated with the enriched functional classes revealed the upregulation of several 20E response genes (Table S9, A), although EcR was not detected in our analysis and its coreceptor USP was not affected. Three 20E signalling cofactors were significantly up-regulated in progeny from exposed individuals (Figure 6): (1) "trithorax-related" (trr, AAEL005380), a histone methyltransferase which is known to associate with the EcR/USP heterodimer in D. melanogaster and co-activates EcR signalling activity by modifying the chromatin structure of ecdysone responsive promoters [START_REF] Sedkov | Methylation at Lysine 4 of Histone H3 in Ecdysone-Dependent Development of Drosophila[END_REF] (2) AAEL017391, the unique ortholog (D. melanogaster, 37%) of nejire, a histone acetyltransferase which is necessary for the up-regulation of ecdysone early-responsive genes upon ecdysone signal 67 and ( 3) "cap-and-collar" (Cnc, AAEL015467, AAEL005077), which play a major role in D. melanogaster development and is crucial for the transcription of 20E early-response genes like Br and E75. [START_REF] Sykiotis | Keap1/Nrf2 Signaling Regulates Oxidative Stress Tolerance and Lifespan in Drosophila[END_REF][START_REF] Veraksa | N" Collar B Cooperates with a Small Maf Subunit to Specify Pharyngeal Development and Suppress Deformed Homeotic Function in the Drosophila Head[END_REF][START_REF] Deng | Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators[END_REF] The activity of these three cofactors has been shown to play a crucial role in the activation of 20E early-response genes. [START_REF] Sedkov | Methylation at Lysine 4 of Histone H3 in Ecdysone-Dependent Development of Drosophila[END_REF][START_REF] Bodai | Ecdysone Induced Gene Expression Is Associated with Acetylation of Histone H3 Lysine 23 in Drosophila Melanogaster[END_REF] The up-regulation of these genes suggests that larvae obtained from ibuprofen-exposed parents display a transcriptional state favourable to ecdysone signalling which could result in an overactivation of this pathway. This was confirmed by the over-transcription of two 20E early-response genes: Br (AAEL008426) and E75 (AAEL007397); four 20E early-late response genes: HR3 (AAEL009588, AAEL015043), HR4 (AAEL005850) and FTz-F1β (AAEL001304); and of two late-responsive genes npc1 (AAEL003325), AAEL005369, the unique ortholog of "crooked legs" (D. melanogaster 35%) [START_REF] Cranna | Impact of Steroid Hormone Signals on Drosophila Cell Cycle during Development[END_REF] (Figure 6). By associating the over-transcription of the transcription factor Br to the up-regulation of its direct target gene (npc1), [START_REF] Ou | What Goes up Must Come down: Transcription Factors Have Their Say in Making Ecdysone Pulses[END_REF] this observation tends to validate the transcriptional observations from a functional point of view. Apart from E74 which was not detected in our study, all the described early and early-late response genes were up-regulated in the progeny of exposed parents. This tends to confirm an overactivation of the ecdysone signalling pathway, as many steps in the ecdysone signalling hierarchy are up-regulated in our study.

The expression of enzymes involved in ecdysteroid biosynthesis is controlled by several transcription factors, allowing the accurate regulation of the hormone dynamics controlling the development of insects. In particular, the transcription factor "without children protein" (woc) up-regulates the expression of the gene coding for the enzyme allowing the first step of steroïdogenesis, and Drifter has been shown to participate in the expression of two other steroidogenic enzymes involved in ecdysteroid synthesis. [START_REF] Warren | Woc (without Children) Gene Control of Ecdysone Biosynthesis in Drosophila Melanogaster[END_REF][START_REF] Cheng | The POU Factor Ventral Veins lacking/Drifter Directs the Timing of Metamorphosis through Ecdysteroid and Juvenile Hormone Signaling[END_REF] Moreover, in addition to its coactivating role in ecdysone signalling, Cnc also plays a key role in the transcriptional activation of genes involved in ecdysone synthesis in D. melanogaster. [START_REF] Deng | Regulation of Drosophila Metamorphosis by Xenobiotic Response Regulators[END_REF] Genes encoding the steroidogenic transcription factors woc (AAEL004320), Drifter (AAEL003560) and Cnc were up-regulated in larvae obtained from exposed parents. This up-regulation may contribute to the greater ecdysone signalling induced by parental exposure, although this assumption is mitigated by the stable transcription level of key steroidogenic enzymes genes (Figure 6, Table S9).

Ecdysone signalling plays a crucial role in moult timing regulation, having a major influence on the duration of development of A. aegypti. [START_REF] Margam | Ecdysteroid Titers and Developmental Expression of Ecdysteroid-Regulated Genes during Metamorphosis of the Yellow Fever Mosquito, Aedes Aegypti (Diptera: Culicidae)[END_REF] This timing regulation is notably due to the influence of 20E signalling on cellcycle regulation. [START_REF] Cranna | Impact of Steroid Hormone Signals on Drosophila Cell Cycle during Development[END_REF] The transcriptional profile of larvae obtained from ibuprofen-exposed parents thus suggests that increased ecdysone signalling causes the accelerated development observed in this progeny.

More broadly, the up-regulation of genes involved in "signal transduction" and "cell/tissue structure" functional classes seems to fit with the greater developmental rate observed in the progeny of exposed individuals (Table 2). Indeed, Mensch et al. demonstrated that an increase in the developmental rate of Drosophila melanogaster was associated with an increased expression of genes involved in these functions. [START_REF] Mensch | Identifying Candidate Genes Affecting Developmental Time in Drosophila Melanogaster: Pervasive Pleiotropy and Gene-by-Environment Interaction[END_REF] Increase in stress response transcriptional potential. Up-regulated genes associated with "transcription regulation" include the heat-shock factor (hsf, AAEL010319), a transcription factor well described as being responsible for controlling stress-response gene expression. [START_REF] Anckar | Regulation of HSF1 Function in the Heat Stress Response: Implications in Aging and Disease[END_REF] In D. melanogaster, the regulation of HSF transcriptional activity under stress conditions is well described. The expression of heat shock proteins (HSP) under stress conditions requires the recruitment by HSF of the P-TEFb complex (Cdk9/cyclin T) to the upstream region of hsp genes. Activation of hsp70 transcription on stress exposure also requires chromatin re-modelling due to FACT complex and histone chaperone Spt6. [START_REF] Anckar | Regulation of HSF1 Function in the Heat Stress Response: Implications in Aging and Disease[END_REF] Two of these genes essential for heat-shock protein (HSPs) production on stress exposure such as cyclin t (AAEL004839) and the ortholog of spt6 (D. melanogaster, 55%) annotated as "suppressor of ty" (AAEL006956) on the A. aegypti genome were also up-regulated (Table S10).

Interestingly, the up-regulation of these genes specifically involved in hsp gene expression was associated with a basal over-transcription of 7 genes encoding HSPs or associated proteins (Figure 6, Table S10). Higher basal levels of HSP mRNA and the up-regulation of the genes necessary for HSPs production under stress exposure may result in a more efficient response to stress in the progeny of exposed parents. Interestingly, as well as regulating ecdysone signalling, the transcription factor CnC is also responsible for the xenobiotic-stress response in D. melanogaster, and the activation of its signalling confers improved tolerance to insecticides. [START_REF] Misra | Transcriptional Regulation of Xenobiotic Detoxification in Drosophila[END_REF] Several genes specifically involved in the nutritional stress response were also up-regulated in F1 larvae obtained from exposed parents: (1) the histone deacetylase coded by sir2 (AAEL006655), involved in the mobilisation of fat stores and the regulation of lipid metabolism in response to nutritional stress, which is crucial for starvation-survival in D. melanogaster 77 ; (2) the unique ortholog (D. melanogaster, 47%) of HDAC4 deacetylase AAEL002528 and lipin (AAEL005175) (Figure 6, Table S10) which are also involved in the regulation of lipid metabolism in response to food deprivation in D. melanogaster. [START_REF] Choi | Feeding and Fasting Signals Converge on the LKB1-SIK3 Pathway to Regulate Lipid Metabolism in Drosophila[END_REF][START_REF] Ugrankar | Lipin Is a Central Regulator of Adipose Tissue Development and Function in Drosophila Melanogaster[END_REF] Lipin notably up-regulates genes involved in fatty-acid oxidation under starvation conditions, allowing the metabolism to adapt to the decrease in energy resources. [START_REF] Ugrankar | Lipin Is a Central Regulator of Adipose Tissue Development and Function in Drosophila Melanogaster[END_REF] These three genes are up-regulated under starvation conditions in D. melanogaster, and Sir2 overexpression led to an increased tolerance to starvation. [START_REF] Slade | Extended Longevity and Survivorship during Amino-Acid Starvation in a Drosophila Sir2 Mutant Heterozygote[END_REF] Interestingly, in D. melanogaster imagoes starvation induces the up-regulation of the genes involved in growth, maintenance, and protein synthesis while peptidases, trafficking, detoxification and immunity proteins are down-regulated. [START_REF] Harbison | Quantitative Genomics of Starvation Stress Resistance in Drosophila[END_REF] This starvation-induced transcriptional profile is strongly consistent with the up-regulation of genes involved in cell/tissue structure and the specific down-regulation of genes involved in detoxification and proteases observed in our F1 larvae after parental exposure (Table 2).

Taken together these results suggest that the transcriptional state of the progeny of exposed parents could confer an improved response potential to stressful events, particularly nutritional stress, and may explain the better tolerance to starvation observed after parental exposure to ibuprofen.

Increase in cell signalling potential.

A detailed analysis of the up-regulated genes associated with the "signal transduction" functional class revealed their role in several signalling pathways, including insulin, MAPK, and TOR (Figure 6, Table S11). Insulin signal mainly regulates PI3K/AKT/PKB pathways, leading to the regulation of the TOR and ERK/MAPK pathways. [START_REF] Taniguchi | Critical Nodes in Signaling Pathways: Insights into Insulin Action[END_REF] Several genes belonging to the insulin signalling pathway were upregulated in the progeny of exposed individuals, including the three genes coding for PI3K (AAEL013596, AAEL002903 and AAEL000386), the regulatory proteins fak (AAEL004666) and pp2a (AAEL014042) and the effectors tsc2 (AAEL007712) and rac (AAEL008823) (Figure 6, Table S11). Genes associated to the TOR pathway were also up-regulated including the three regulators Rheb (AAEL008179), GPRFZ2 (AAEL006778) and the unique ortholog (D. melanogaster. 52%) of happyhour (AAEL017338), and the TOR substrate S6K (AAEL018120), albeit tor (AAEL000693) transcription level was not affected by parental exposure. Regulation of ERK/MAPK pathway also appeared affected by parental exposure to ibuprofen, as shc (AAEL008739), sos (AAEL012071), ras (AAEL012071), erk1/2 (AAEL007958, AAEL013939) and mnk (AAEL004704) were upregulated (Figure 6, Table S11).

Interestingly, multiple studies have demonstrated the role of insulin and TOR signalling pathways on 20E signalling regulation in insects. [START_REF] Hou | Small GTPase Rab4b Participates in the Gene Transcription of 20-Hydroxyecdysone and Insulin Pathways to Regulate Glycogen Level and Metamorphosis[END_REF][START_REF] Smith | Insulin Signaling Pathways in Lepidopteran Ecdysone Secretion[END_REF][START_REF] Caldwell | Ras Activity in the Drosophila Prothoracic Gland Regulates Body Size and Developmental Rate via Ecdysone Release[END_REF] Overexpression of the activated form of Ras in D. melanogaster leads to precocious pupation through the activation of the ecdysone signalling pathway, evidencing the role of insulin signalling pathway activation state in determining development time. [START_REF] Caldwell | Ras Activity in the Drosophila Prothoracic Gland Regulates Body Size and Developmental Rate via Ecdysone Release[END_REF] It was suggested that this action of Ras was due to the transcriptional activation of 20E synthesis genes by CnC, by favouring the interaction of CnC with chromatin and its transcriptional activity. [START_REF] Deng | Multiple Roles of Nrf2-Keap1 Signaling: Regulation of Development and Xenobiotic Response Using Distinct Mechanisms[END_REF] The TOR pathway regulates the expression of ecdysteroid synthesis related genes and 20E-response genes in response to amino acid levels, depending on the transmembrane receptor Frizzeled2 in A. aegypti. [START_REF] Weng | Frizzled 2 Is a Key Component in the Regulation of TOR Signaling-Mediated Egg Production in the Mosquito Aedes Aegypti[END_REF][START_REF] Roy | The Small GTPase Rheb Is a Key Component Linking Amino Acid Signaling and TOR in the Nutritional Pathway That Controls Mosquito Egg Development[END_REF] TOR pathway also regulates cell and body growth in response to the cellular nutritional and energetic state through a well-described mechanism in D. melanogaster. [START_REF] Danielsen | Chapter Two -Nutrient Signaling and Developmental Timing of Maturation[END_REF][START_REF] Koyama | Nutritional Control of Body Size through FoxO-Ultraspiracle Mediated Ecdysone Biosynthesis[END_REF] Interestingly, the genes encoding the TOR regulator Frizzeled 2 (gprfz2, AAEL006778) and several effectors of the TOR pathway specifically involved in the nutritional stress response, pp2a and 14-3-3 (AAEL011116), lipin and the unique ortholog (D. melanogaster, 33%) of reptor (AAEL009171), were upregulated in the progeny of exposed parents (Figure 6, Table S11). [START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF] Insulin and TOR pathways thus participate in insects, in the regulation of growth and development timing according to the availability of nutritional resources through the regulation of the ecdysone signalling pathway and regulates the implementation of the nutritional stress response. [START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF][START_REF] Colombani | A Nutrient Sensor Mechanism Controls Drosophila Growth[END_REF][START_REF] Layalle | The TOR Pathway Couples Nutrition and Developmental Timing in Drosophila[END_REF] Given the vital role of these pathways in development regulation and specific stress-response implementation, overrepresentation of these general pathways (Insulin, MAPK and TOR) and specific effectors in F1 larvae transcriptome suggests that their transcriptional state increases their potential for integrating environmental cues, especially nutritional ones, allowing the potentiation of appropriate responses.

Origin of the intergenerational consequences of ibuprofen exposure

Overall, our approach revealed a larval transcriptional response that could be consistent with the pharmacological mode of action of ibuprofen, with moderate molecular and phenotypic consequences at imaginal stages. However, parental exposure led to lower levels of several metabolites in eggs, including amino acids. This change in the metabolic resources available for embryonic development results in a distinct physiological state in the progeny, reflected by a shift in the F1 larvae transcriptional state and affecting their phenotype. The transcriptional state of the progeny displays an overactivation of the ecdysone signalling pathway resulting in the acceleration of larval development and seems to constitute preparation for a rapid and efficient response to a stressful environment, explaining the larvae's better tolerance of starvation. Thus, changes in progeny phenotype are likely to be initiated by a differential internalization of resources in eggs by exposed parents. In A. aegypti, resource internalization in eggs occurs in females during the vitellogenic phase, and the expression of genes necessary to the transport and internalization of metabolic resources are tightly regulated by ecdysone. [START_REF] Chen | The Early Gene Broad Is Involved in the Ecdysteroid Hierarchy Governing Vitellogenesis of the Mosquito Aedes Aegypti[END_REF] However, our approach failed to demonstrate any changes at molecular scale in exposed imagoes that could explain such changes in reproductive investment, especially concerning female hormonal state and metabolic resources. Assuming that F0 imagoes are no longer in direct contact with ibuprofen, the intergenerational impact could result from a delayed consequence of larval exposure, such as a modification in the expression potential of the specific genes involved in resource internalization in females, in response to 20E signalling, without influencing 20E synthesis nor the transcriptomic profile at the previtellogenic stage. These types of delayed impacts are notably suspected of being due to changes in the epigenetic regulation of transcription. [START_REF] Groh | Development and Application of the Adverse Outcome Pathway Framework for Understanding and Predicting Chronic Toxicity: I. Challenges and Research Needs in Ecotoxicology[END_REF] This must involve a differential expression of one or several genes involved in metabolic resource internalization during the egg maturation process. An untargeted transcriptomic approach during the vitellogenesis may have revealed such transcriptional changes on genes other than the best described vitellogenic genes we specifically focused on. This clearly illustrates the limit of targeted approaches for deciphering intergenerational mechanisms, since the factors involved in egg maturation and progeny production are highly multifactorial.

Towards a better understanding of the intergenerational impact of pollutants

Our study investigated the consequences of direct and parental chronic exposure to ibuprofen in the model insect A. aegypti. It illustrates the interest of a system approach in mechanistic studies, as it not only helps to deepen our understanding of the molecular basis of observed phenotypic variations, but also provides new insights into the consequences of pollutant exposure, including in the context of stress-on-stress. Such studies investigating the impacts of chronic exposure to environmentally relevant concentrations of pollutants were scarce until recent years. However, populations are often chronically exposed to micropollutants in the environment. Our work particularly emphasizes the importance of temporal scale in ecotoxicity studies and shows that considering the entire life cycle of organisms, including that of their progeny, is of major relevance to fully understand the impacts of chronic pollutant exposure and the associated mode of action. A precise understanding of the upstream molecular events leading to inter-and transgenerational impacts of chemicals is of particular interest as it is key to improving the prediction of transgenerational adverse outcomes, and to enhancing the ecological relevance of ecotoxicological risk assessment. [START_REF] Groh | Development and Application of the Adverse Outcome Pathway Framework for Understanding and Predicting Chronic Toxicity: I. Challenges and Research Needs in Ecotoxicology[END_REF] Interestingly, the consequences of parental ibuprofen exposure on F1 larvae may seem to be beneficial in terms of the survival potential of progeny larvae, raising the question of the specificity of the observed effects: are the changes occurring in F1 progeny due to a specific alteration induced by ibuprofen or do they reflect a modified reproductive strategy in response to a potentially changing environment? This adaptive aspect of the inter-and transgenerational impacts of pollutants should be carefully considered in multigenerational ecotoxicological studies, as both reproductive plasticity and specific disruption elicited by contaminants are susceptible to regulate the phenotype in the progeny.

91

ACKNOWLEDGEMENTS:

This work was funded by the Centre National pour la Recherche Scientifique (CNRS), and all authors were funded by the French Ministry of Research. The authors want to thank Dr. Chantal Dauphin-Villemant for sharing her protocol for ecdysteroids dosage by EIA and for supplying the tracer and antibodies, Dr. Christophe Regnault for his assistance with sample collection and qPCR data analysis and Mr. Frederic Laporte for his assistance with qPCR execution.

SUPPORTING INFORMATION:

The Supporting Information is available free of charge on the ACS Publications website at DOI: Detailed Materials and Methods, Table S1

-S11, Figure S1-S6 (PDF)
Detailed transcriptomic data (Table sheet) COX2). [START_REF] Charlier | Dual Inhibition of Cyclooxygenase-2 (COX-2) and 5-Lipoxygenase (5-LOX) as a New Strategy to Provide Safer Non-Steroidal Anti-Inflammatory Drugs[END_REF] Prostaglandins regulate several cellular processes including the induction of immune genes [START_REF] Büyükgüzel | Eicosanoids Mediate Galleria Mellonella Cellular Immune Response to Viral Infection[END_REF][START_REF] Durmuş | Eicosanoids Mediate Melanotic Nodulation Reactions to Viral Infection in Larvae of the Parasitic Wasp, Pimpla Turionellae[END_REF] through the regulation of the transcription factor CCAAT/enhancer binding protein (C/EBP) expression [START_REF] Na | Endogenous Prostaglandin E2 Potentiates Anti-Inflammatory Phenotype of Macrophage through the CREB-C/EBP-β Cascade[END_REF]

FIGURES CAPTIONS

Figure 1 :

 1 Figure 1: Effect of ibuprofen exposure on Aedes aegypti imago lifespan. A: Lifespan of virgin females exposed to 1µg/L ibuprofen (pink curves, n = 32) or solvent control (black curve, n = 34). B: Lifespan of primiparous females exposed to 1µg/L ibuprofen (pink curves, n = 52) or solvent control (black curve, n = 54). C: Lifespan of males exposed to 1µg/L ibuprofen (pink curves, n = 59) or solvent control (black curve, n = 67). P-values are given according to a Kaplan-Meyer survival test.

Figure 2 :

 2 Figure 2: Effect of ibuprofen parental exposure on progeny survival under starvation conditions. First stage larvae from the progeny of populations exposed to 1µg/L ibuprofen (pink curves, n = 238) or solvent control (black curve, n = 237) were raised in media with no nutritional resources. P-value is given according to a Kaplan-Meyer survival test.

Figure 3 :

 3 Figure 3: Differential gene transcription in Aedes aegypti after direct or parental ibuprofen exposure.Three pools of 4 th stage larvae (L4F0, n=35) and female imagoes (FemF0, n=30) exposed to 1µg/L ibuprofen or solvent control were respectively collected 40h after moult and 72h after emergence. Three pools of F1 1 st stage larvae from each parental exposure condition (L1F1, n=300) were collected 10 hours after hatching. Gene transcription was characterized by RNAseq and statistically significant differential transcription following

Figure 4 :

 4 Figure 4: Transcriptomic response of Aedes aegypti 4th stage larvae (F0) chronically exposed to 1µg/L ibuprofen. A: Putative mechanism involved in transcriptomic perturbation induced by ibuprofen exposure. This mechanism is based on knowledge of the pharmacological mode of action of ibuprofen in vertebrates and the consequences of prostaglandin pathway disruption in insects. Under the control conditions, arachidonic acid (AA) is released from membrane phospholipids and diacylglycerol by phospholipase A2 (PLA 2 ) and phospholipase C (PLC). AA is converted in prostaglandin H 2 (PGH 2 ) by cyclooxygenases 1&2 (COX1,

  (1) or unknown mechanisms(2). By inhibiting COX activities, ibuprofen reduces PGH 2 synthesis thus reducing the expression of immune related genes and C/EBP target genes. B, C, D: The blue/orange colour-scale indicates transcription level variations in exposed larvae compared to control. B: Impact of ibuprofen exposure on transcription factors transcription rate. C: Impact of ibuprofen exposure on potential C/EBP target genes transcription rate. D: Impact of ibuprofen exposure on other immunity related genes transcription rate.

Figure 5 :

 5 Figure 5: Metabolome of Aedes aegypti F1 eggs spawned by control or ibuprofen exposed F0 individuals. A: Principal component analysis (PCA) performed with the quantification value of the 50 detected metabolites from 6 samples of 100 eggs for each condition. B: Percent change in the concentrations of metabolites significantly affected by parental exposure to ibuprofen according to a Wilcoxon rank-sum test. Bars are coloured according to the correlation score of the metabolites to OPLS-DA (R2X: 0.44, R2Y: 0.72, Q2: 0.66). *: FDR-adjusted p-value<0.05. **: FDR-adjusted p-value<0.01. a: amino acids. b: carbohydrates. c: polyols. d: intermediate metabolites.

Figure 6 :

 6 Figure 6: Effect of chronic parental ibuprofen exposure on F1 progeny gene transcription. The blue/orange colour-scale indicates transcription level variations in exposed larvae compared to control. Orthlog (unique) andpercent similarity with Drosophila melanogaster genes are given between brackets. A: Signalling pathway genes differential expression. Each gene found differentially transcribed has been assigned to specific signalling pathway according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) (D.mel) and bibliography.[START_REF] Taniguchi | Critical Nodes in Signaling Pathways: Insights into Insulin Action[END_REF][START_REF] Tiebe | REPTOR and REPTOR-BP Regulate Organismal Metabolism and Transcription Downstream of TORC1[END_REF]

Table 1 -Functional class enrichment study based on genes differentially transcribed between solvent control and Ibuprofen exposed 4 th stage larvae. The 134 genes significantly differentially transcribed in exposed larvae were used for functional class enrichment according to a Fisher's exact test, corrected for multiple comparison using

 1 Benjamini-Hochberg method.

	Page 27 of 36
		Up-regulated genes (51)
	Functional class	Enrichment	p-value	Implicated genes
	Kinases and phosphatases	x 4.45	3.33E-02 3
	Cuticle	x 15.83	2.30E-05 5
	Down-regulated genes (83)
	Functional class	Enrichment	p-value	Implicated genes
	Immunity	x 6.16	4.29E-06 11
	Kinases and phosphatases	x 4.56	5.83E-03 5
	Intra/extracellular trafficking -Chaperones	x 3.94	7.16E-06 17
	Others	x 1.65	1.14E-03 6

Table 2 -Functional class enrichment study based on genes differentially transcribed between solvent control and Ibuprofen exposed progeny (1 st stage larvae). The 1566 genes significantly differentially transcribed in exposed larvae were used for functional class enrichment according to a Fisher's exact test, corrected for multiple comparison using

 2 Benjamini-Hochberg method.

		Up-regulated genes (1116)
	Functional class	Enrichment	p-value	Implicated genes
	Epigenetic marks regulation	x 3.02	2.84E-04	15
	Transcription regulation	x 1.70	2.49E-06	65
	Signal transduction	x 2.20	2.20E-13	99
	Structure	x 1.68	1.11E-02	127
		Dow-regulated genes (450)
	Functional class	Enrichment	p-value	Implicated genes
	Lipid metabolism	x 1.64	2.93E-02	22
	Protein metabolism	x 1.54	8.37E-04	66
	Detoxification	x 2.95	2.73E-08	35
	Mitochondrial metabolism	x 2.03	2.14E-02	11
	Cuticle	x 4.95	3.19E-07	15
	Others	x 1.65	4.34E-02	10

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DATA AVAILABILITY STATEMENT:

The RNA-seq sequence data from this study have been deposited at the European Nucleotide Archive (ENA; http:// www.ebi.ac.uk/ena) under the accession numbers PRJEB23849 and PRJEB23850.