Fanny Louis
email: fannylouis@univ-reims.fr

Béatrice Rocher

Iris Barjhoux

Florence Bultelle

Odile Dedourge-Geffard

Véronique Gaillet

Isabelle Bonnard

Laurence Delahaut

Sandrine Pain-Devin

Alain Geffard

Odile Dedourge- Geffard

Séverine Paris-Palacios

Elise David

Seasonal monitoring of cellular energy metabolism in a sentinel species, Dreissena polymorpha (bivalve): Effect of global change?

Keywords: energy, ATP, Dreissena polymorpha, biomarkers

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

Introduction

Depending on scenarios, a 0.3 to 0.7°C increase is predicted in the mean Earth surface temperature by 2019 to 2035 (IPCC, 2014). The global climate change also includes the appearance of "exceptional" climatic events. These occurrences also bring important floods or droughts and may have an impact on aquatic populations. Since cellular energy production is dependent on multiple factors such as temperature, food availability or oxygen content [START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF], these events could significantly affect the metabolism management of organisms. Indeed, all biochemical and physiological processes are closely related to the amount of energy available within organisms [START_REF] Frieder | Metabolic cost of calcification in bivalve larvae under experimental ocean acidification[END_REF][START_REF] Somero | The physiology of climate change: how potentials for acclimatization and genetic adaptation will determine "winners" and "losers[END_REF]. Energy metabolism management is also closely related to natural environmental fluctuations. Seasonal temperature variations may lead to the establishment of adaptation and acclimation processes such as metabolic restructuring. Indeed, the aerobic metabolism can be favoured in order to improve the energy available for fitness-related functions including growth and reproduction [START_REF] Hochaka | Biochemical adaptation: Mechanism and process in physiological evolution[END_REF][START_REF] Salin | Variation in the link between oxygen consumption and ATP production, and its relevance for animal performance[END_REF][START_REF] Sokolova | Physiological adaptations to high intertidal life involve improved water conservation abilities and metabolic rate depression in Littorina saxatilis[END_REF][START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. Environmental factors directly modulate cellular and mitochondrial energy management and the energy allocation to vital functions such as maintenance, growth, reproduction and reserves storage (Amiard and Amiard-Triquet, 2008;[START_REF] Hong | Seasonal variations in the hemocyte parameters, gonad development, energy storage and utilization of the giant honeycomb oyster Hyotissa hyotis (Linnaeus 1758) in Jeju Island off the south coast of Korea[END_REF][START_REF] Verslycke | Cellular energy allocation and scope for growth in the estuarine mysid Neomysis integer (Crustacea: Mysidacea) following chlorpyrifos exposure: a method comparison[END_REF]. In invasive species, reproduction represents a large part of energy allocation [START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF].

Seasonal modulations of water physicochemical parameters influence the gonadal maturation and spawning regulation. Fluctuations in energy investment priorities between vital functions have already been observed during the spawning in marine bivalves [START_REF] Cheung | Population dynamics and energy budgets of green-lipped mussel Perna viridis (Linnaeus) in a polluted harbour[END_REF][START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF][START_REF] Petes | Intertidal mussels exhibit energetic trade-offs between reproduction and stress resistance[END_REF]. In this way, natural fluctuations monitoring could be a potential lead to understand energy metabolism management and ATP production in the model species, Dreissena polymorpha, along one reproduction cycle.

Mitochondria are the key organelles involved in multiple essential cellular processes [START_REF] Lane | Power, sex, suicide: mitochondria and the meaning of life[END_REF][START_REF] Sokolov | Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas[END_REF]. They provide more than 90% of cellular ATP and serve as a cellular centre connecting energy metabolism, stress response, signalling and cell survival [START_REF] Lane | Mitochondria: Key to Complexity[END_REF][START_REF] Monlun | Mitochondria as Molecular Platforms Integrating Multiple Innate Immune Signalings[END_REF][START_REF] Naquet | Metabolic adaptation of tissues to stress releases metabolites influencing innate immunity[END_REF]. Studies on different model species have demonstrated that mitochondria are particularly sensitive to environmental stressors [START_REF] Kurochkin | Top-down control analysis of the cadmium effects on molluscan mitochondria and the mechanisms of cadmium-induced mitochondrial dysfunction[END_REF][START_REF] Sokolov | Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas[END_REF][START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. In such context, mitochondria are considered as a stress target and/or the coordinating centre of the adaptive cellular response [START_REF] Eisner | Mitochondrial dynamics in adaptive and maladaptive cellular stress responses[END_REF][START_REF] Sokolova | Interactive effects of metal pollution and temperature on metabolism in aquatic ectotherms: implications of global climate change[END_REF]. Positive thermal stress is known to reduce ATP production leading thus to an increase of oxidative phosphorylation (OXPHOS) activity and of reactive oxygen species (ROS) production associated with oxidative damage in bivalve species [START_REF] Cherkasov | Seasonal variation in mitochondrial responses to cadmium and temperature in eastern oysters Crassostrea virginica (Gmelin) from different latitudes[END_REF][START_REF] Hraoui | Mitochondrial thermosensitivity in invasive and native freshwater mussels[END_REF][START_REF] Sokolova | Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)[END_REF]. The environmental temperature increase has been associated with a higher respiration rate, leading to a strong proton leak and a gradual mitochondria dissociation in Crassostrea virginica [START_REF] Sokolova | Physiological adaptations to high intertidal life involve improved water conservation abilities and metabolic rate depression in Littorina saxatilis[END_REF][START_REF] Sokolova | Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)[END_REF]. An increase of mitochondrial damage could also engage the pathway of apoptosis in order to eliminate damage and ensure survival [START_REF] Vakifahmetoglu-Norberg | The role of mitochondria in metabolism and cell death[END_REF].

For freshwater risk assessment, integrative biomarkers are required in an active biomonitoring perspective. As the energy metabolism is crucial for all physiological reactions, several studies were realised to develop new energy markers in different model species. The energy cost of metabolism management against environmental disturbances have been discussed in several reviews [START_REF] Calow | Physiological costs of combating chemical toxicants: Ecological implications[END_REF][START_REF] Calow | How do physiological responses to stress translate into ecological and evolutionary processes?[END_REF][START_REF] Sokolova | Energy homeostasis as an integrative tool for assessing limits of environmental stress tolerance in aquatic invertebrates[END_REF]. The scope for growth and/or the cellular energy charge was investigated in Dreissena polymorpha along a pollution gradient [START_REF] Smolders | Cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization[END_REF] or after a metal stress [START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF]. These studies pointed out a decrease of the energy allocation to growth and reproduction in presence of stress. Furthermore, the measurement of energy reserves is a useful tool to provide information about the global organism's energy available for maintenance, growth and reproduction [START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF][START_REF] Sprung | Physiological energetics of the zebra mussel Dreissena polymorpha in lakes III. Metabolism and net growth efficiency[END_REF]. The adenylate energy charge (AEC) is used as a biomarker of the global health status of the organisms (Le Gal et al., 1997[START_REF] Smolders | Cellular energy allocation in zebra mussels exposed along a pollution gradient: linking cellular effects to higher levels of biological organization[END_REF]. After a chemical contamination, [START_REF] Sokolova | Resistance to freshwater exposure in White Sea Littorina spp. I: Anaerobic metabolism and energetics[END_REF][START_REF] Sokolova | Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)[END_REF] highlighted the decrease of the AEC in the oyster (Crassostrea virginica) and in the White Sea periwinkles (Littorina spp).

The zebra mussel, Dreissena polymorpha is an invasive filter feeder bivalve widespread in European freshwaters. Due to several biological characteristics such as its abundance, wide geographical distribution and high filtration activity, D. polymorpha has been used as a model species for freshwater biomonitoring for several years [START_REF] De Lafontaine | Biomarkers in zebra mussels (Dreissena polymorpha) for the assessment and monitoring of water quality of the St Lawrence River (Canada)[END_REF][START_REF] Faria | Characterization of the multixenobiotic resistance (MXR) mechanism in embryos and larvae of the zebra mussel (Dreissena polymorpha) and studies on its role in tolerance to single and mixture combinations of toxicants[END_REF][START_REF] Faria | Multi-biomarker responses in the freshwater mussel Dreissena polymorpha exposed to polychlorobiphenyls and metals[END_REF][START_REF] Kerambrun | Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study[END_REF][START_REF] Pain | Identification of multixenobiotic defence mechanism (MXR) background activities in the freshwater bivalve Dreissena polymorpha as reference values for its use as biomarker in contaminated ecosystems[END_REF][START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF]. At the cellular level, mitochondria activities are usually reflected by the electron transport system (ETS). [START_REF] Potet | Integrated multi-biomarker responses in two dreissenid species following metal and thermal cross-stress[END_REF] have observed an increase of ETS activity under a metal stress and a decrease after a thermal or dietary stress in D. polymorpha. At the molecular level, some units of the OXPHOS were followed (ATP synthase and cytochrome c oxidase). It was shown that the relative gene expression level of ATP synthase increased in summer [START_REF] Kerambrun | Variations in gene expression levels in four European zebra mussel, Dreissena polymorpha, populations in relation to metal bioaccumulation: A field study[END_REF].

In this study, cellular energy production (ATP) and management were monitored during one reproduction cycle in situ. The whole energy metabolism was targeted through different functions such as energy balance regulation, mitochondrial density and aerobic/anaerobic metabolism. Digestive gland was principally targeted for gene expression and enzymatic activities. Indeed, this organ, similarly to vertebrate liver, play a key role in detoxification mechanisms and has already been noticed as a target for bioaccumulation and biomarker analysis in mollusks (Faggio et al., 2018;Rodrigo and Costa, 2017). Furthermore, most of literature focused on digestive glands concerning biomarker analyses analysis (Faria et al. 2014;[START_REF] Potet | Integrated multi-biomarker responses in two dreissenid species following metal and thermal cross-stress[END_REF][START_REF] Potet | Differential tolerance to nickel between Dreissena polymorpha and Dreissena rostriformis bugensis populations[END_REF][START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF]. In parallel, energy reserves, condition index and the gonadal stage were analysed. As the respiratory chain may be a major source of ROS production, lipid peroxidation content was also measured. Our approach allows a better understanding of the physiology of Dreissena polymorpha, under changing natural fluctuations.

Energy metabolism studies in Dreissena polymorpha are still too few and are only taking in account one or 2 parameters concerning energy management [START_REF] Louis | Energy allocation in two dreissenid species under metal stress[END_REF]Weber et al., 2020). A previous study of our laboratory aimed to characterise the reproduction cycle of the same population of dreissenids. Indeed, [START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF] studied D. polymorpha gonadal development stage as well as reserves storage for one year. Since this sentinel species is commonly used for in situ monitoring, it is essential [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] to characterise the response pattern of monitored markers along the years and (2) to develop new and complemental markers of energy metabolism.

Material and Methods

Organisms collection

Specimens were hand-collected in the Meuse channel (Euville, France, E05°37'28'', N48°44'59'') for one reproduction cycle from May 2018 to June 2019 with a monthly or bimonthly frequency. This site was chosen since data about physiology responses were available on this population of D. polymorpha as well as the reproduction cycle characterisation along one year [START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF]. Organisms with a length between 15 and 25mm were selected in a depth of 1.5m maximum. Organisms were measured and weighted in order to determine the condition index using the following formula: CI= fresh weight / volume (length x height x width). For gene expression and enzymatic activity analysis (n=12 for each sampling time), digestive glands were dissected and snap frozen. For reserve contents measurement (n=12), the whole organism was dissected and snap frozen.

Whole body mussels were frozen for energy nucleotide contents analysis (n=10). All of these procedures were done in the field, except for organisms intended to the gonadal development analysis that were quickly transported to the laboratory in field water (n=12). All marker measurements were realized without taking into account organisms' gender. Dissections and freezing were performed at the sampling site in order to avoid mRNA and protein degradation and to limit marker measurements bias.

Environmental parameters

At each sampling time, physicochemical parameters were recorded. Water samples (10 L) were carried to the laboratory and processed for analysis in black container. Ammonium (NH4), nitrates (NO2), nitrites (NO3) and chlorophyll a (chl a) contents were measured by spectrophotometric analyses. Chemical oxygen demand and suspended matter were measured by electrochemical and gravimetric methods, respectively. The temperature was recorded every 10min from June 2018 to June 2019 using two probes (TidbiT v2 Temp) installed on the site. The average temperature was calculated for each month.

Gene expression

Total RNA was extracted from digestive gland using TriReagent (Euromedex) following manufacturer's protocol. RNA integrity was estimated by UV revelation after electrophoresis on 1% agarose gel in TBE (Tris Borate EDTA 0.5%) buffer. RNA purity was estimated by the measurement of the ratio of absorbance at 260 and 280nm. Reverse transcription was performed on 400ng of total RNA using Verso cDNA Synthesis Kit (Thermo Scientific) according to manufacturer's instructions.

The reaction was conducted at 42°C for 30min using a PCR Mastercycle (Eppendorf).

Gene expression levels were carried out by real-time quantitative PCR using Absolute Blue qPCR SYBR Green ROX Mix (Thermo Scientific) according to the manufacturer's protocol. Specific primers are given in Table 1. Energy balance regulation was studied through ampk (AMP-activated protein kinase) and ampd (AMP deaminase) gene expressions. Concerning the OXPHOS, three genes were selected: beta subunit ATP synthase (atp), cytochrome b (cytb), cytochrome c oxidase (cox) and succinate dehydrogenase (sdh). Another unit of the respiratory chain, the alternative oxidase (aox) was recorded. Gene expression levels were also measured for citrate synthase (cs) and lactate dehydrogenase (ldh), an anaerobic metabolism enzyme. The optimal primer quantity was evaluated across a range of concentrations between 150 and 600nM. Dilutions of the cDNA mixture between 1:10 and 1:1000 were tested to calculate the PCR efficiency. The PCR steps consisted in 15 min initial denaturation at 95°C followed by 40 cycles of heat denaturation at 95°C for 10sec, annealing at 60°C for 60sec and were carried out in a CFX Manager (BioRad) on 1/10 e of ADNc. The relative gene expression level was calculated using the Pfaffl method [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] taking into account the PCR efficiency and normalized by the geometric mean of the two housekeeping genes (actine and ribosomal protein S3) according to [START_REF] Navarro | Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults[END_REF].

Enzymatic activities

The set of enzymatic activities was individually measured in each mussel. The digestive glands were weighted and crushed in 50mM phosphate buffer containing protease inhibitors (pH 7.6, supplemented with 1mM phenylmethylsulfonyl fluoride and 1mM L-serineborate) at 8:1 (v/w) ratio.

Samples were then centrifuged (3000g, 10min, 4°C). The supernatant was collected for the enzymatic activity measurement. Assays were performed with an automated spectrophotometer (Galery, ThermoScientific) and with a spectrofluorimeter (SAFAS, Xenius). ETS, lactate dehydrogenase (LDH) as well as proteins content and lipid peroxidation were measured according to [START_REF] Garaud | Integrated assessment of ceria nanoparticle impacts on the freshwater bivalve Dreissena polymorpha[END_REF] and [START_REF] Potet | Integrated multi-biomarker responses in two dreissenid species following metal and thermal cross-stress[END_REF]. Caspase-3 activity was measured according to [START_REF] Garaud | Integrated assessment of ceria nanoparticle impacts on the freshwater bivalve Dreissena polymorpha[END_REF] in cytosolic extract.

For cytochrome c oxidase (COX) activity, supernatant was diluted to 1:45 in 100mM Tris buffer (pH 8.1). Reduced cytochrome c at 1mM (Merck) supplemented with 16% of ascorbate (Merck) was added to the previous dilution. Cytochrome c oxidation was then monitored for 3min at 550nm.

For citrate synthase (CS) activity, supernatant was diluted to 1:55 in 0.1M Tris buffer (pH 8.1) supplemented with 0.15mM DTNB (Merck) and 0.2mM acetyl co enzyme A (Merck). The activity was first monitored 3min at 420nm. A second measure was performed after the addition of 4mM oxaloacetate (Merck). Enzymatic activity was expressed in U.g -1 proteins (molar extinction coefficient (ε) reduced cytochrome c = 7040M -1 .cm -1 , ε oxaloacetate = 14140M -1 .cm -1 ).

Energy reserve contents

Organisms were weighted and crushed in 50mM phosphate buffer (pH 8) at 5:1 (v/w) ratio.

Protein, lipid and glycogen contents were measured on the homogenate. Protein content was determined by the Bradford's method. Lipid and glycogen contents were measured according to the method of [START_REF] Van Handel | Rapid determination of total lipids in mosquitoes[END_REF] and adapted by [START_REF] Plaistow | The effect of the acanthocephalan parasite Pomphorhynchus laevis on the lipid and glycogen content of its intermediate host Gammarus pulex[END_REF]. Results were expressed in mg.g -1 of fresh weight (FW).

Energy nucleotide contents

Nucleotides extraction was realised as described by [START_REF] Sokolova | Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)[END_REF]. Briefly, frozen mussels were quickly dissected and lyophilized for 48h. Samples were then crushed in 0.6M perchloric acid (Merck) solution, centrifuged (3000g, 10min, 4°C) and the supernatant was collected.

The pH of each extract was adjusted to 5 -6.5 with 5mM potassium hydroxide and 1M bicine solution.

Precipitated potassium perchlorate was removed by a second centrifugation. Extracts were then filtered (0.45µM) and stored at -80°C. Concentrations of ATP, ADP, AMP and IMP were measured with an HPLC system and Nucleosic C18 column (Nucleosil) according [START_REF] Sokolova | Cadmium exposure affects mitochondrial bioenergetics and gene expression of key mitochondrial proteins in the eastern oyster Crassostrea virginica Gmelin (Bivalvia: Ostreidae)[END_REF]. The separation was performed at a flow rate of 0.8ml.min -1 using a mobile phase of 1M phosphate buffer with 5% methanol (pH 6) for 15min. Nucleotide contents were monitored at 254nm and nucleotide quantifications were calculated using ATP, ADP, AMP and IMP standards (Sigma) of 20, 10, 2 and 10µM, respectively.

Adenylate energy charge (AEC) was calculated from the tissue concentrations of adenylates as follows:

AEC= [ATP] + 0.5 × [ADP] [ATP] + [ADP] + [AMP]

Gonadal development analysis

Dissected organisms (without byssus and shell) were fixed in Bouin's aqueous solution for 24h followed by successive alcohol dehydration baths from 50° to 100°. After deshydratation, sampled were placed in a butanol solution in order to permeate all tissues. Samples were then paraffinembedded and 7µM sections were prepared from the foot to gonads of organisms. Sections were stained with picro indigo carmin and solid nuclear red and observed with a microscope to sex the gonadsand to assign them to a development stage [START_REF] Gist | Annual reproductive cycle of the zebra mussel in the Ohio River: a comparison with Lake Erie[END_REF][START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF]. This method was based on the size and the number of present oocytes as well as spermatogenesis steps.

Statistical analysis

Marker responses were analysed by a one-way ANOVA to assess time effect. Parametric (Tukey) or non-parametric (Kruskal-Wallis) post-hoc tests were processed on markers and obtained pvalues were adjusted for multiple comparisons by a correction method (Benjamini and Hochberg).

Normality and homoscedasticity of the data were tested with Shapiro and Bartlett tests respectively.

Correlations were performed on the total data set. A clustered map was built on a complete linkage using Euclidean distance measures between different variables. The obtained values can be seen as a robust approximation of the Pearson correlation. A global analysis of biomarker results was also performed with a Partial Least Square Discriminant Analysis (PLS-DA) to identify the overall capacity of biomarkers to discriminate the months and to define the most relevant markers among the battery. Relevance of marker responses was based on the individual role of each marker in the overall model, estimated by the VIP (Variable Importance in the Projection). Only markers with a VIP > 1 were considered significant in the model (PLS-DA). Since only four organisms could be sampled in February (used for gene expression) and 16 in March 2019 (4 for gene expression, 4 for enzymatic activity, 4 for nucleotide contents and 4 for histology), the results obtained for these months are presented in the different figures but the data were excluded for statistical analysis. A threshold of p < 0.05 was considered significant. Statistical analyses were performed using R software (R Development Core Team, 3.5.0).

Results

Environmental parameters

Water physicochemical features are reported in Table 2 andFig

Reproduction cycle

Gonadal development stages are presented in Fig. 2. Since only one male was observed in November 2018 in histological analysis, the male gonadal index could not be determined. In 2018, males spawned their gametes between July and September (Fig. 2A) and females between May and August (Fig. 2B). The spawning events were synchronous for both sexes in 2018. Females' gonadal development seemed to be more premature than males' development in 2019. A shift was observed between males and females' gonadal stage between April and June 2019. Indeed, the spawning of oocytes began in April when males were halfway between maturation and pre-spawn stage. In addition, in June 2019, all the females had spawned their gametes whereas no male had spawned theirs. It is also necessary to take into account the sampling randomness resulting in a sex ratio rarely equals to 1:1.

Global health status

Condition index (CI) (Fig. 3A), lipid and protein contents (Fig. 3C-D) showed globally the same response variations. CI values decreased just after the spawning (July and August 2018) and then gradually increased until the next reproduction period (from September 2018 to April 2019). Lipid contents also decreased in reproduction period (from August to November 2018) and increased from April 2019. Although protein content variations were less marked, it decreased during the gametes spawning and then increased again. Concerning glycogen reserves (Fig. 3B), a significant decrease was observed in August 2018 followed by a significant increase until November 2018 (5-10mg.gFW - 1 ). From April 2019, glycogen content reached the same level as in August 2018 (< 2mg.gFW -1 ).

Mitochondria efficiency

Mitochondria efficiency was assessed in our study by the OXPHOS and the citrate synthase activity and transcriptional level. CS activity increased during the pre-reproduction and reproduction periods while trend to decrease during post reproduction and rest (Fig. 4A). Concerning COX activity, a significant decrease since April 2019 was observed (Fig. 4B). The ETS activity showed a 2-fold increase in June 2018 and came back to lower level in July and August (Fig. 4C). A gradually increase was then observed from September 2018 to June 2019. Globally, relative gene expression levels exhibit two distinct patterns. OXPHOS subunits genes (cox, atp and cytb) reach their highest levels during the pre-reproduction and reproduction periods while they exhibited a marked decrease during post reproduction and rest periods (Fig. 5A-B-C). On the contrary, citrate synthase gene expression (cs) level was significantly higher during the rest period (Fig. 5D). No difference of sdh relative gene expression level could be noticed along the year (Fig. S1).

Energy balance

Energy balance regulation was studied through ampk and ampd gene expressions (Fig. 6). The transcript levels of ampk (Fig. 6A) gradually increased from May 2018 to November 2018 and decreased significantly in April 2019. Levels stayed low until June 2019. Although no significant difference could be observed, the expression of ampd gene (Fig. 6B) showed the same pattern of variation. ATP and ADP contents presented a similar progression over the year than ampk and ampd gene expression levels (Fig. 7, part I: A-B). Energy nucleotides concentration was stable from May 2018 to September 2018. A strong increase was observed in November 2018 followed by an important reduction of ATP and ADP contents in April 2019. The pattern of AMP and IMP contents were similar but they slightly differed from the previous markers (Fig. 7, part I: C-D). Indeed, the concentration of these nucleotides were stable from May 2018 to August 2018 and gradually increased until April 2019 where they reached a steady state (p < 0.05). A correlation was observed between ATP content and the expression of ampd involved in energy balance regulation but also with cs gene expression, glycogen and lipid contents (Table S1). The AEC remained stable from May 2018 to November 2018 (0.5-0.7). Nevertheless, a significant decrease was observed in April 2019 (0.13-0.32) (Fig. 7, part II).

Stress markers

Anaerobic metabolism was assessed through ldh gene expression and LDH activity, both presenting a similar pattern of responses (Fig. 8 A-B). Values were higher in pre-reproduction and reproduction periods than during post-reproduction and rest periods. A gradual increase of aox relative gene expression was observed from May 2018 to August 2018 and it decreased until June 2019 (Fig. 8C). On the contrary, lipid peroxidation (LOOH) content was stable from May 2018 to November 2018, while it displayed a significant increase from April 2019 compared to July and August 2018 (Fig. 8D). LOOH concentration was negatively correlated with condition index, energy reserves (lipids and glycogen). Positive correlations were also observed between LOOH and LDH activity and AMP/IMP contents (Table 3). Caspase-3 (CSP3) activity was measured only on 7 sampling times (May, June, July, August and November 2018 and May and June 2019). Its activity increased significantly from November 2018 to June 2019 (Fig. 8E). Negative correlation between CSP3 activity and cytb gene expression was noticed while positive correlation with stress markers like LOOH measurements (r² = 0.57, p < 0.05) and IMP/AMP contents were observed (Table 3).

Global analysis

A PLS-DA model was built and explained 68.2% of between-month variance with the two first axis. The first three VIP markers were atp, cs and ldh gene expressions (Table S2). Caspase-3 activity was excluded of the total data set.

The PLS model built explained 70.6% of between physico-chemical parameters variance with the two first axis. PLS allowed us to identify the importance of each parameter on biological responses. A clustered map was then built. The rows and columns were reordered according to hierarchical clustering method to identify interesting patterns. According to the cluster results (Fig. 9), stress markers as LOOH and IMP content, condition index and LDH activity were strongly positively correlated with suspended matter, nitrate and oxygen contents and negatively correlated with temperature and the content of ammonium. Conversely, aox gene expression was negatively correlated with suspended matter and oxygen content and positively correlated with temperature and the content of ammonium. Furthermore, cluster results highlighted that environmental parameters such as nitrites and chl a content had no effect on biological responses along the reproductive cycle. Some biological responses as sdh, cox, cs, ampd, atp relative gene expression and ATP content were not influenced by measured environmental parameters.

Discussion

The aim of this study was to understand the effect of natural fluctuations and intrinsic variations on global energy metabolism in the model species Dreissena polymorpha in situ.

Global status

According to our observations, D. polymorpha exhibited a typical annual reproductive cycle with a gonad development phase during fall, a maturation and spawning phase in spring-summer and, if present, a resting phase at the end of the summer (males). These observations are consistent with the reproductive patterns previously described in D. polymorpha populations [START_REF] Binelli | DDT in zebra mussels from Lake Maggiore (N. Italy): level of contamination and endocrine disruptions[END_REF][START_REF] Juhel | Impacts of microcystins on the feeding behaviour and energy balance of zebra mussels, Dreissena polymorpha: A bioenergetics approach[END_REF][START_REF] Mackie | The Zebra mussel, Dreissena polymorpha: a synthesis of European experiences and a preview for North America[END_REF][START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF]. In 2019, the expected reproductive cycle was observed but the reproduction period was asynchronous between males and females. No reason was bringing out to explain this observation. Indeed, natural media are continuously submitted to several perturbations. This asynchronous spawning could be a consequence of a potential pollution or an accumulation of different exceptional events like drought or flood periods. Concerning energy reserves, lipid content followed the reproduction cycle with a significant decrease in August 2018 concurrently with gamete emission, similarly to what is reported by [START_REF] Kwan | Metal contamination in zebra mussels (Dreissena polymorpha) along the St. Lawrence River[END_REF], [START_REF] Hassan | Gametogenesis, sex ratio and energy metabolism in Ostrea angasi: implications for the reproductive strategy of spermcasting marine bivalves[END_REF] and [START_REF] Hong | Seasonal variations in the hemocyte parameters, gonad development, energy storage and utilization of the giant honeycomb oyster Hyotissa hyotis (Linnaeus 1758) in Jeju Island off the south coast of Korea[END_REF]. Since females were mostly represented in our samples for histology analysis, the high lipid content observed until April 2019 might be explained by the accumulation of lipid nutrients in the oocytes [START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF]. According to our results, glycogen content increased after spawning of the gametes and decreased in April 2019. [START_REF] Tourari | Action de la température sur le cycle de reproduction de Dreissena polymorpha (Pallas). Etude ''in situ'' et au laboratoire[END_REF] shown that glycogen was largely used for the formation of the gametes and the gonadal tissues during gametogenesis and is especially converted into lipid materials in maturing oocytes in Dreissena polymorpha. These observations were also highlighted in oysters through an increase of glycogen content in prereproduction period followed by a net decrease during the spawning one [START_REF] Hong | Seasonal variations in the hemocyte parameters, gonad development, energy storage and utilization of the giant honeycomb oyster Hyotissa hyotis (Linnaeus 1758) in Jeju Island off the south coast of Korea[END_REF]. However, in our study, glycogen storage was significantly lower from April to June in 2019 compared to the same period in 2018. Reproduction cycle and glycogen content observations did not follow the schema described for Dreissena polymorpha and others bivalves' species [START_REF] Hong | Seasonal variations in the hemocyte parameters, gonad development, energy storage and utilization of the giant honeycomb oyster Hyotissa hyotis (Linnaeus 1758) in Jeju Island off the south coast of Korea[END_REF][START_REF] Palais | One-year monitoring of reproductive and energy reserve cycles in transplanted zebra mussels (Dreissena polymorpha)[END_REF][START_REF] Ram | Physiological aspects of zebra mussel reproduction: maturation, spawning, and fertilization[END_REF][START_REF] Ram | Spawning in the zebra mussel (Dreissena polymorpha): activation by internal or external application of serotonin[END_REF]. Glycogen might have been used simultaneously for the gamete development and spawning as well as for other cellular energy demands since glycogen is a main source of energy in organisms (Anacleto et al., 2013;Andrade et al., 2017;Cordeiro et al., 2016Cordeiro et al., , 2017)).

Energy metabolism

Globally, energy metabolism appeared to be more active during the pre-reproduction and the reproduction periods than during the post-reproduction and rest periods. The respiratory chain units (i.e. cox, atp, and cytb gene expressions) presented the same pattern of responses as the anaerobic metabolism markers (ldh gene expression and LDH activity). Study of the ETS activity during two years on D. polymorpha showed similar responses with an increase of the activity from May to June of each year [START_REF] Fanslow | Seasonal changes in the respiratory electron transport system (ETS) and respiration of the zebra mussel, Dreissena polymorpha in Saginaw Bay, Lake Huron[END_REF]. According to our results of ampk relative expression level, a significant decrease was observed from April 2019 compared to November 2018. An accumulation of AMP was simultaneously measured in the organisms. As the AMPk is activated by high AMP concentrations [START_REF] Chauhan | Spatial control of AMPK signaling at subcellular compartments[END_REF]Fredrich and Balschi, 2002), the decrease of ampk relative expression might be the result of a negative feedback. This negative feedback could also be applied to the ampd relative gene expression modulation as this enzyme is activated by AMP intracellular concentration too [START_REF] O'brien | New insights on the regulation of the adenine nucleotide pool of erythrocytes in mouse models[END_REF][START_REF] Składanowski | AMP deaminase in vitro inhibition by xenobiotics[END_REF]. Higher production of ATP should be observed consequently to counterbalance AMP accumulation and maintain a positive energy balance. However, in our study, a significant decrease in ATP content was observed from April 2019 while ETS activity seemed to remain stable. The oxygen consumed in the mitochondria is directly linked to the flow of electrons and the synthesis of ATP by the ATPsynthase. However, part of this flow can be shunted by an alternative route which bypasses the respiratory chain before complex III. Hence, especially in the presence of low levels of COX activity, the electron transport system contributes less to the driving force of the protons, and from there to the production of ATP. Thus from March to April 2019, the AOX activity could maintain the ETS level in parallel with a decrease in the ATP level. All these observations suggesting thus an imbalance of the energy metabolism.

The observed increase in IMP and AMP concentrations may activate glycogen phosphorylase and further enhance glycolysis and the tricarboxylic acid (TCA) cycle when the demand for ATP production increases [START_REF] Han | Regulation of glucose metabolism from a liver-centric perspective[END_REF]. Decreased glycogen content observed could be a consequence of the accumulation of IMP and AMP in organisms. CS catalyses condensation of the acetyl group of acetyl-CoA with oxaloacetate forming citrate. It represents the first step of the TCA cycle [START_REF] Eprintsev | Regulation of expression of the mitochondrial and peroxisomal forms of citrate synthase in maize during germination and in response to light[END_REF]. Products provided by TCA cycle are then used by the respiratory chain in order to produce ATP. In our study, CS activity increased during the pre-reproduction and reproduction periods as well as atp, cytb and cox relative gene expression levels. These results were already observed through an atp and cox relative expression increasing during spring and/or summer [START_REF] Kerambrun | Differentiation of sympatric zebra and quagga mussels in ecotoxicological studies: A comparison of morphometric data, gene expression, and body metal concentrations[END_REF][START_REF] Navarro | Transcriptional response of stress genes to metal exposure in zebra mussel larvae and adults[END_REF]. These results suggest an increase in the respiratory chain functioning during pre-reproduction and reproduction periods. Although it is difficult to compare a gene expression with the activity of the corresponding enzyme, a global suppression of the metabolic rate was observed when all biological responses are considered. Indeed, COX activity and ATP content decreased significantly while ETS activity and LOOH measurements increased from April 2019 to June 2019. Meanwhile, the caspase-3 (CSP3), a protease involved in apoptosis, was significantly more active. In addition, CSP3 activity was also correlated with the expression of OXPHOS-related genes and IMP concentrations. Caspase activation and apoptosis are associated with a decline in ATP levels and a production of reactive oxygen species (ROS) [START_REF] Ricci | Disruption of Mitochondrial Function during Apoptosis Is Mediated by Caspase Cleavage of the p75 Subunit of Complex I of the Electron Transport Chain[END_REF][START_REF] Sharma | Zinc Oxide Nanoparticle Induces Microglial Death by NADPH-Oxidase-Independent Reactive Oxygen Species as well as Energy Depletion[END_REF]. The changes in metabolic processes and CSP3 activity suggest an activation of apoptosis pathway likely resulting from a global stress.

A global response overview

The multivariate analysis allowed us to consider the modulations of the whole panel of markers. This method showed that some markers were positively or negatively correlated with environmental parameters. The contents of IMP and LOOH, and LDH activity are modulated by stressing conditions [START_REF] Falfushynska | Habitat pollution and thermal regime modify molecular stress responses to elevated temperature in freshwater mussels (Anodonta anatina: Unionidae)[END_REF][START_REF] Lushchak | Environmentally induced oxidative stress in aquatic animals[END_REF]. LDH activity is a marker of global stress whereas lipid peroxidation is used as an indicator of oxidative stress. AMP deaminase protein catalyses the hydrolysis of AMP to form IMP and ammonia. AMPd and AMPk proteins play a role in the energy balance and its regulation. An intracellular AMP content increase reflects an enhanced use of ATP. To avoid the accumulation of AMP, the AMPd converts it into IMP thus suggesting that some stress is challenging the organisms [START_REF] Lushchak | Environmentally induced oxidative stress in aquatic animals[END_REF]. In our study, these markers were correlated to all analysed environmental factors except for chl a and nitrite contents. Moreover, lipid peroxidation was positively correlated with IMP and lipid concentrations. The global health status of the organisms seemed to be affected in situ through the induction of stress markers like IMP and LOOH. According to our global analysis, suspended matter and nitrate concentrations seemed to be associated with an impact on energy metabolism of D. polymorpha.

Exceptional events: a long-term impact?

There was a significant increase in the frequency and the intensity of meteorological and hydrological events (EEA, 2007). In the Meuse department, drought periods were recorded in summer 2018 and 2019. The year 2018 was characterized by an exceptional drought that led to placing the entire department in a situation of alert that was extended from August to December. This prefectural decree was renewed in summer 2019 (Decree of the 8 th August 2019 on the water use). Drought events may have more effects than flood periods on aquatic organisms due to their duration and large spatial impacts [START_REF] Hrdinka | Possible impacts of floods and droughts on water quality[END_REF]. The decrease in the water column height leads to a more drastic increase in water temperature [START_REF] Van Vliet | Impact of summer droughts on the water quality of the Meuse river[END_REF]. According to our in situ observations in summer 2018, the temperature stayed above 25°C during three weeks in July and August. D. polymorpha is an ectotherm organism with a lethal temperature in acute stress between 35°C and 37°C [START_REF] Spidle | Limits to tolerance of temperature and salinity in the quagga mussel ( Dreissena bugensis ) and the zebra mussel ( Dreissena polymorpha )[END_REF]. Several studies reported optimal temperatures for the reproduction, the development and the survival of D. polymorpha between 10°C and 20°C (bij de [START_REF] De Vaate | Geographical patterns in range extension of Ponto-Caspian macroinvertebrate species in Europe[END_REF][START_REF] Ram | Spawning in the zebra mussel (Dreissena polymorpha): activation by internal or external application of serotonin[END_REF][START_REF] Sprung | Physiological energetics of the zebra mussel Dreissena polymorpha in lakes III. Metabolism and net growth efficiency[END_REF]. Drought periods observed may also have an impact on the physiology of organisms. Seasonal acclimation to different temperatures changes the tolerance window for invertebrates due to a switch in energy metabolism management in order to assure vital functions related to the fitness [START_REF] Jost | Activation of AMP-activated protein kinase in response to temperature elevation shows seasonal variation in the zebra mussel, Dreissena polymorpha[END_REF][START_REF] Lannig | Temperature-dependent effects of cadmium on mitochondrial and whole-organism bioenergetics of oysters (Crassostrea virginica)[END_REF][START_REF] Sokolova | Resistance to freshwater exposure in White Sea Littorina spp. I: Anaerobic metabolism and energetics[END_REF]. Based on our results, in 2018, temperature varied up to 20°C (November) and 25°C (August) in one day.

Populations of D. polymorpha are usually hand-sampling close to the shore. These populations are also submitted to the strong variations that may have an impact on physiology of mussels, changing thus their global status. Temperature is also known to have a synergic effect together with contaminants [START_REF] Cherkasov | Seasonal variation in mitochondrial responses to cadmium and temperature in eastern oysters Crassostrea virginica (Gmelin) from different latitudes[END_REF][START_REF] Lannig | Temperature-dependent effects of cadmium on mitochondrial and whole-organism bioenergetics of oysters (Crassostrea virginica)[END_REF][START_REF] Soon | Climate Change and Bivalve Mass Mortality in Temperate Regions[END_REF]. Energy cost caused by natural fluctuations and exceptional events repetition might make organisms more vulnerable to anthropic pollution at long term and impact population dynamics.

Water physicochemistry and biological responses: a potential pollution?

Physicochemical parameters like pH, temperature and ion concentrations (NO3 -, PO4 3-) are known to regulate the distribution of D. polymorpha populations [START_REF] Mcmahon | Evolutionary and physiological adaptations of aquatic invasive animals: r selection versus resistance[END_REF][START_REF] Ramcharan | Models to Predict Potential Occurrence and Density of the Zebra Mussel, Dreissena polymorpha[END_REF]. The values recorded for pH and temperature in the Meuse channel from May 2018 to June 2019 were consistent with the ecological requirements of the species [START_REF] Ramcharan | Models to Predict Potential Occurrence and Density of the Zebra Mussel, Dreissena polymorpha[END_REF]. However, a peak of nitrate and phosphate concentrations (31mg.L -1 and 0.126mg.L -1 respectively) was noticed in December 2018 in the Meuse channel 20km from the sampling site. These concentrations remained stable until February 2019 (31-25.96mg.L -1 NO3 -and 0.120-0.114mg.L -1 PO4 3-) (French Water Agency data and Table 2). According to the Water Framework Directive, a maximum concentration of 50mg.L -1 of nitrates is defined as a threshold for a "good" ecological status by the regulation (2000( /60/CE, 2000)). However, studies showed that effects on aquatic fishes might be observed at lower concentrations after 30 days of exposure (Camargo et al., 2005). [START_REF] Alonso | Short-Term Toxicity of Ammonia, Nitrite, and Nitrate to the Aquatic Snail Potamopyrgus antipodarum (Hydrobiidae, Mollusca)[END_REF] exposed the mollusc Potamopyrgus antipodarum to N-NO3 -to evaluate the LC10 at 24, 48, 72 and 96h.

The calculated LC10 values were divided by 2 between 24h (1128 ppm) and 96h (585 ppm) of exposure. Similar observations were highlighted after a 14-days chronic exposure with a LC50 divided by 10 between 1 and 14 days [START_REF] Benítez-Mora | Ecotoxicological assessment of the impact of nitrate (NO3¯) on the European endangered white-clawed crayfish Austropotamobius italicus (Faxon)[END_REF]. Camargo et al. (2005) concluded that LC measured values may be divided by a factor 18 to 20 between acute and chronic exposures. However, to our knowledge, no data are available on the effect of nitrates in D. polymorpha. Simultaneous presence of nitrates and orthophosphates is known to reflect a pesticides pollution [START_REF] Majumdar | Nitrate pollution of groundwater and associated human health disorders[END_REF]. Based on the DCE legislation, the limit of pesticides emission is 0.5mg.L -1 in freshwaters (2000( /60/CE, 2000)). In December 2018, total pesticides concentration was 0.495mg.L -1 (French Water Agency data). Several pesticides targeting the mitochondria, and especially the respiratory chain, are extensively found in freshwaters. Nevertheless, more information would be necessary to approve the hypothesis of a potential pesticide impact on organism's mitochondria.

Conclusion

This study bring deeper knowledge in the management of energy metabolism in a sentinel species, D. polymorpha, in order to develop new potential biomarkers in a biomonitoring perspective.

According to our results, three genes involved in ATP production could constitute potential biomarkers. Indeed, sdh, atp and cs genes expression were not influenced by natural seasonal fluctuations. Complementary studies are necessary to confirm their relevance as potential biomarkers.

Targeting a major part of energy metabolism and globally analysing their responses allowed us to suggest a putative impairment of mitochondrial respiratory chain, likely resulting from an in situ perturbation. Indeed, the monitored population of D. polymorpha seemed to be impacted by an external stress from April 2019. Natural environments are subjected to multiple stressors and it is impossible to know the origin of the perturbations. However, flood and drought periods associated

  . 1. Water temperature varied seasonally, the lowest values were recorded in fall (November) and the highest values were recorded in summer (August). Strong daily temperature variations were observed in August (5°C min -30°C max) and November (-2°C min -20°C max). In 2019, suspended matter values were higher than in 2018 as well as the mean of nitrates contents. May and June 2019 chl a levels were not detected. All physicochemical parameters measured along the study complied with the framework directive on water (2000/60/CE, 2000).

  with high temperature variations in only one day or during several days may sensitize the organisms to potential chemical stress and could affect vital functions. The asynchronous gonadal development between males and females observed in 2019 as well as the metabolic depletion could be a consequence of exceptional climatic events associated with the pollution. Beyond the characterisation of the energy metabolism management in D. polymorpha along one reproduction cycle, this study highlighted the relevance of in situ monitoring in particular to investigate the impact of global change on sentinel species.

Figure 1 .

 1 Figure 1. Temperature continuously measured on sampling site. The mean, minimum and maximum temperature are presented.

Figure 2 .

 2 Figure 2. Gonadal development stage for males (A) and females (B) according to the months. Different development stages are presented in terms of percentage of each stage (n=12).

Figure 3 .

 3 Figure 3. Condition index (n=60) (A), glycogen (B), lipids (C) and proteins (D) content (n=12) according to the months. Data are presented as median ± SD, min, max. Different letters indicate significant differences between months (p < 0.05).

Figure 4 .

 4 Figure 4. CS (A), COX (B) and ETS (C) activities according to the months. Data are presented as median ± SD, min, max (n=12). Different letters indicate significant differences between months (p < 0.05).

Figure 5 .

 5 Figure 5. atp (A), cox (B), cytb (C) and cs (D) relative gene expression levels according to the months. Data are presented as median ± SD, min, max (n=12). Different letters indicate significant differences between months (p < 0.05).

Figure 6 .

 6 Figure 6. ampk (A) and ampd (B) relative gene expression levels according to the months. Data are presented as median ± SD, min, max (n=12). Different letters indicate significant differences between months (p < 0.05).

Figure 7 .

 7 Figure 7. Part I: ATP (A), ADP (B), AMP (C) and IMP (D) contents. Part II: adenylate energy charge (AEC). Results are presented according to the months and data are expressed as median ± SD, min, max (n=12). Different letters indicate significant differences between months (p < 0.05).

Figure 8 .

 8 Figure 8. ldh relative gene expression level (A), LDH activity (B), aox relative gene expression level (C), lipid peroxidation (LOOH) (D) and caspase-3 activity (E) according to

Figure 9 .

 9 Figure 9. Hierarchical cluster analysis (complete linkage using Euclidean distance measures) applied on the PLS model. The values in the similarity matrix can be seen as a robust approximation of the Pearson correlation. The rows and columns were reordered according to some hierarchical clustering method to identify interesting patterns. SM: suspended matter; COD: chemical oxygen demand.

Figure 1

 1 Figure 1.

  Figure 3.

  Figure 5.

  Figure 6.

  Figure 7.

  Figure 8.

  Figure 9.

  Figure S1.

Table 1 .

 1 Summary of the primers used for gene expression measurements. Abbreviations, NCBI accession numbers of the original sequences of the target genes in D. polymorpha.

	Gene	Abbreviation	NCBI	F primers	R primers
	Actin	act	AF082863	CCTCACCCTCAAGTACCCCAT	TTGGCCTTTGGGTTGAGTG
	Alternative oxidase	aox	Q9P959	GTGAACCATACCCTCGCCTC	TGTAGTGACTGCGGATGGTC
	AMP deaminase	ampd	Q9DBT5	ACCCAGCCAGTGTTGTTTCA	TTTGCTCGTGGACTCAACGT
	AMP kinase α subunit	ampk	Q8BRK8	TGCTAAATGAAAGAGGTGACGC	CATGTGGTGGCATATACGATGT
	ATP synthase	atp	JQ781135	GACTCCACCTCCCGTATCCT	TCCTCTGAAAGCTCGTCCAT
	Citrate synthase	cs	MN579511	GTGCCCTGTCAGACCCTTAC	CTGTCCTGCCTTGAGTGTGT
	Cytochrome b	cytb	DQ072120.1 CGCCACTTTAAAGCGATTTTT	TGGGTCAGCAAATAGATCTGG
	Cytochrome c oxidase	cox	AAL55513	AGGCCCTGCGATAGATTTTT	AAAGGGACCCGGTAAAATTG
	Lactate dehydrogenase	ldh	Q7TNG8	TGCCCAAACTACATTGCCAAAT	ATTGGGATTGGCAAACGGGA
	Ribosomal protein S3	s3	AJ517687	CAGTGTGAGTCCCTGAGATACAAG AACTTCATGGACTTGGCTCTCTG
	Succinate dehydrogenase	sdh	Q9YHT2	TAGACCGTCCAGTTTAGCGC	CCTCTGCCACACATGTACGT

Table 2 .

 2 Environmental parameters recorded in situ or in water samples: water physicochemistry and contamination measurements; dO2: dissolved oxygen; SM: suspended matter; COD: chemical oxygen demand; NO2 -: nitrites; NO3 -: nitrates; NH4 + : ammonium; Chl a: chlorophyll a; ND: not detected.

	Month	Temperature (°C)	Conductivit y (µS.cm -1 )	dO2 (mg.L -1 )	SM (mg.L -1 )	COD (mg.L -1 )	NO2 -(mg.L -1 )	NO3 -(mg.L -1 )	NH4 + (mg.L -1 )	Chl a (µg.L -1 )	Hydrocarbon (mg.L -1 )
	May-18	22.7	497	9.1	ND	15.5	0.14	18	0.14	0.2	ND
	June-18	22.9	485	9.8	ND	17.4	0.08	15	0.10	0.4	ND
	July-18	24.2	505	9.4	4	18.9	0.13	12	0.10	0.6	0.002
	August-18	21.6	410	7.1	5	14.2	0.11	8	0.09	6.7	ND
	September-18	17.8	410	9	8	14.2	0.13	7	0.07	2.7	ND
	November-18	6.3	740	13	5	8.88	0.08	11	0.01	4.1	0.001
	February-19	5.8	521	13.2	20	16.2	0.09	26	0.09	9.8	ND
	March-19	9.3	491	10.7	24	16.5	0.12	19	0.19	1.9	ND
	April-19	12.5	520	10.4	20	40.5	0.09	21	0.02	1.5	ND
	May-19	15.8	547	9.7	25	11.7	0.19	14	0.04	ND	ND
	June-19	20.4	527	9.6	20	13.8	0.1	13	0.09	ND	ND

Table 3 .

 3 Pearson's correlations between the lipid peroxidation and the caspase-3 activity with the target markers for the total dataset. Only significant correlations for LOOH content and/or caspase activity are presented.

		Lipid peroxidation	Caspase-3 activity
		r²
	Lipids	0.59	n.s
	Glycogen	0.4	n.s
	[AMP]	0.71	0.91
	[IMP]	0.68	0.88
	Condition Index (CI)	0.57	n.s
	LDH activity	0.55	n.s
	cytb gene expression	n.s	0.51

Acknowledgment

We wish to thank Danièle Pauly for her valuable help for caspase-3 activity measurements as well as the LIEC in Metz for its welcome. This work was supported by the Grand-Est region and the University of Reims Champagne Ardenne through MEENDRE project.

Supplementary data:

Table S1. Correlations of temperature and ATP content according to target markers of the total data set using multiple linear regression. Only significant correlations were presented.