Clément Coclet

Cédric Garnier

Gaël Durrieu

Sébastien D'onofrio

Nicolas Layglon

Jean-François Briand

Benjamin Misson
email: misson@univ-tln.fr

Impacts of copper and lead exposure on prokaryotic communities from contaminated contrasted coastal seawaters: the influence of previous metal exposure

Keywords: prokaryotic communities, copper contamination, metal exposure history, coastal seawater, mesocosm experiment, high throughput sequencing

Our understanding of environmental factors controlling prokaryotic community is largely hampered by the large environmental variability across spatial scales (e.g. trace metal contamination, nutrient enrichment, physico-chemical variations) and the broad diversity of bacterial pre-exposure to environmental factors. In this article, we investigated the specific influence of copper (Cu) and lead (Pb) on prokaryotic communities from the uncontaminated site, using mesocosm experiments. In addition, we studied how pre-exposure (i.e. life history) affects communities, with reference to previous metal exposure on the response of three prokaryotic communities to similar Cu exposition. This study showed a stronger influence of Cu contamination than Pb contamination on prokaryotic diversity and structure. We identified 12, and 34 bacterial families and genera, respectively, contributing to the significant differences observed in community structure between control and spiked conditions. Taken altogether, our results point towards a combination of direct negative responses to Cu contamination and indirect responses mediated by interaction with phytoplankton. These identified responses were largely conditioned by the previous exposure of community to contaminants.

INTRODUCTION

Marine coastal environments are especially vulnerable to anthropogenic pressures such as chemical contamination [START_REF] Halpern | A Global Map of Human Impact on Marine Ecosystems[END_REF][START_REF] Mermex Group | Marine ecosystems ' responses to climatic and anthropogenic forcings in the Mediterranean[END_REF]. For example, copper (Cu) contamination in coastal environments is mainly caused by urban and industrial wastes [START_REF] Levin | The function of marine critical transition zones and the importance of sediment biodiversity[END_REF][START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF], or antifouling coatings close to harbors [START_REF] Turner | Marine pollution from antifouling paint particles[END_REF]. Lead (Pb) contamination is mainly associated with historical short-term events [START_REF] Xu | The source of natural and anthropogenic heavy metals in the sediments of the Minjiang River Estuary (SE China): Implications for historical pollution[END_REF]Dang et al. 2015aDang et al. , 2015b) ) or historical fuel consumption for nautical traffic [START_REF] Callender | Heavy Metals in the Environment -Historical Trends[END_REF]. Cu contamination is mainly encountered in seawaters of harbor areas while Pb contamination are found in sediments, probably due to past events (Dang et al. 2015b). In Toulon Bay, Cu and Pb concentrations can be enriched up to more than 100-fold above geochemical background levels depending on the compartment (e.g. sediment or water column) [START_REF] Casas | Relation between metal concentration in water and metal content of marine mussels (Mytilus galloprovincialis): impact of physiology[END_REF][START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF][START_REF] Jean | Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem[END_REF][START_REF] Cossa | Origin and accumulation of trace elements in sediments of the northwestern Mediterranean margin[END_REF]Dang et al. 2015b;[START_REF] Twining | Metal contents of phytoplankton and labile particulate material in the North Atlantic Ocean[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF].

Thus, the unique feature of Toulon Bay relates to a gradient of multiple trace metal elements (TME) with very low concentrations in open parts comparable to levels measured in open Mediterranean Sea [START_REF] Morley | Distribution and behaviour of some dissolved trace metals in the western Mediterranean Sea[END_REF]. By comparison, most enclosed and anthropized areas exhibit very high TME concentrations (Dang et al. 2015a;[START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]. Therefore, Toulon bay is a model ecosystem for the study of ecological consequences of TME contamination in the marine coastal environment.

In Toulon Bay where TME gradients are wider than classical physico-chemical gradients, TME contamination have been proposed to influence directly the bacterioplankton diversity and structure [START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]. Several field studies clearly suggested TMEs as structuring factors of abundance, diversity and composition of bacterioplanktonic communities by selecting metal resistant taxa [START_REF] Sheeba | Role of heavy metals in structuring the microbial community associated with particulate matter in a tropical estuary[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF][START_REF] Goni-Urriza | Consequences of contamination on the interactions between phytoplankton and bacterioplankton[END_REF]. A microcosmbased study revealed that artificial mixture of contaminants (nutrients, metals, and herbicides) induced a strong inhibition of growth, production, respiration and significant modifications of the functional patterns of bacterioplankton community [START_REF] Pringault | Consequences of contaminant mixture on the dynamics and functional diversity of bacterioplankton in a southwestern Mediterranean coastal ecosystem[END_REF]. Furthermore, both industrial effluent discharge and the resuspension of metal-contaminated marine sediments have been shown to affect microbial diversity in a resuspension experiment [START_REF] Zouch | Effect of Acidic Industrial Effluent Release on Microbial Diversity and Trace Metal Dynamics During Resuspension of Coastal Sediment[END_REF]. TME contamination typically found in coastal environments have also been observed to affect phytoplanktonic communities [START_REF] Mackey | Phytoplankton responses to atmospheric metal deposition in the coastal and open-ocean Sargasso Sea[END_REF][START_REF] Lafabrie | Impact of contaminated-sediment resuspension on phytoplankton in the Biguglia lagoon (Corsica, Mediterranean sea)[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF], which can have indirect consequences for bacterioplankton community [START_REF] Goni-Urriza | Consequences of contamination on the interactions between phytoplankton and bacterioplankton[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF].

Life history of a microbial community can be defined as its past exposure to a multitude of environmental changes, like top-down (e.g. grazing pressure, exposition to toxic substances) as well as bottom-up (e.g. phytoplanktonic production) control.

The pre-exposure of communities to harmful contaminants like TME can affect the microbial ecology of the marine system and therefore is of major concern (Hughes- [START_REF] Hughes-Martiny | Microbial biogeography : putting microorganisms on the map[END_REF][START_REF] Bissett | Life history determines biogeographical patterns of soil bacterial communities over multiple spatial scales[END_REF]Bell et al. 2013;[START_REF] Sjöstedt | Repeated disturbances affect functional but not compositional resistance and resilience in an aquatic bacterioplankton community[END_REF][START_REF] Ward | Conserved Microbial Toxicity Responses for Acute and Chronic Silver Nanoparticle Treatments in Wetland Mesocosms[END_REF]. However, most studies exploring patterns in microbial community diversity and structure in response to environmental change have focused on direct effects, without consideration of life history. Previous TME exposure can lead to an increase of metal tolerance in microbial communities by the elimination of the most sensitive individuals and the replacement of sensitive species by tolerant ones [START_REF] Gustavson | Pollution-induced community tolerance (PICT) in coastal phytoplankton communities exposure to copper[END_REF]. [START_REF] Serra | Copper accumulation and toxicity in fluvial periphyton: The influence of exposure history[END_REF] found that copper pulse exposure may be toxic to periphyton communities while continuous copper exposure may lead to community adaptation, which is often related to changes in species composition. All of these studies are in agreement with the pollution-induced community tolerance (PICT) model [START_REF] Blanck | Functional Testing of Aquatic Biota for Estimating Hazards of Chemicals[END_REF][START_REF] Blanck | A Critical Review of Procedures and Approaches Used for Assessing Pollution-Induced Community Tolerance (PICT) in Biotic Communities[END_REF]). However, while numerous studies developed the PICT approach based on photosynthetic efficiency, extracellular enzyme activities or respiration activity [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination ?[END_REF][START_REF] Soldo | Tolerance of Oocystis nephrocytioides to copper: Intracellular distribution and extracellular complexation of copper[END_REF][START_REF] Serra | Copper accumulation and toxicity in fluvial periphyton: The influence of exposure history[END_REF][START_REF] Tlili | Signification écologique de la tolérance acquise des communautés microbiennes des biofilms de rivières à une contamination d'origine anthropique[END_REF], little is known about how metal exposure impacts diversity and composition of marine prokaryotic communities previously exposed to similar contamination.

Assuming that the life history of its planktonic communities in Toulon Bay would mainly reflect TME exposure history due to the strong and chronic Cu and Pb contamination gradients of the area (Layglon et al., under revision), in this study we aimed to answer two main questions: (1) to what extent do Cu and Pb contamination affect prokaryotic communities from uncontaminated site at relevant concentrations; and (2) does prokaryotic TME exposure history play a role in shaping prokaryotic diversity after a Cu exposure?

MATERIALS AND METHODS

Study area, seawater collection, processing, and storage

The study was conducted in Toulon Bay, located in the northwestern part of the Mediterranean Sea (Figure 1 and Supplementary Material). Seawater used for the first mesocosm experiment was collected at 1-m depth from site 41p (low contaminated site) (Figure 1) on May 10 th 2016 and seawater used for the second mesocosm experiment was collected at 1-m depth from both sites LAZ (intermediate contaminated site) and 6ext (highly contaminated site) on May 2 nd 2017 (Figure 1).

The 3 sampling seawaters used for mesocosm experiments were geochemically contrasted. They were representative of the North-West to South-East decreasing gradient of trace metal contamination (mainly Cu and Pb) previously observed in Toulon Bay (Figure 1). Minimal concentrations were recorded in the open-sea sampling site (41p), while maximal concentrations were recorded in the northern and enclosed part of the little bay (6ext) where the TME contamination is chronic [START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF][START_REF] Coclet | Metals contamination as a toxic and structuring factor for ultraphytoplankton in a multicontaminated Mediterranean coastal area[END_REF]Layglon et al., under revision). Details on sample's information are given in the supplementary Table 1. Seawater was collected using an aircompressed Teflon pump (AstiPure TM II, Saint-Gobin) connected with Teflon tubing. It was filtered on site through a 5-10 µm cellulose Polycap filter (Whatman) connected to the Teflon tubing after the pump in order to remove the largest grazers. Filtered seawater was collected in 50-L low-density polyethylene (LDPE) tanks (Nalgene  ).

Back to the lab, each mesocosm was filled with 20-L of filtered seawater. The remaining seawater was filtered through 0.2 µm Teflon Polycap filters (Whatman) to eliminate all organisms, then stored at 4°C and subsequently used to re-fill mesocosms during the experiment. All the sampling material, bottles, tanks, and filters were pre-cleaned following rigorous protocols, detailed in supplementary information and in [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF], in order to keep metal concentrations as close as possible to their initial levels.

Mesocosm setup, and experimental design

For both mesocosm studies, twelve 20-L LDPE tanks (Nalgene  ) were enclosed in a large box covered of plastics in order to avoid contamination in TMEs from ambient particles. Fresh air was continuously provided inside the box by a filtered-equipped fan in order to avoid humidity accumulation and to equilibrate temperature with the surrounding room. The tanks were filled with 5-10 µm filtered water, and thus included a natural marine microbial community mainly composed of free-living organisms. Seawater in each tank was continuously mixed with a Teflon magnetic stir bar used at low speed and oxygenized with 0.2 µm-filtered and humidified ambient air. The 12 tanks were submitted to a daily light/dark cycle of 14/10h, using full solar spectrum aquariophilly neon tubes and a surface light irradiance of 50 µmol.m 2 .s 1 of white light. In the first year, the temperature of the room and of the mesocosms' box were kept constant at 19 ± 1.6°C, and 23 ± 1.5°C, respectively. In the second year, the room temperature and the mesocosm interior were kept constant at 19 ± 2.2°C and, 22 ± 2.0°C, respectively. To maintain resource availability during the experiments, each tank had a continuous opened water circulation system, using a peristaltic pump (Minipuls Evolution  , Gilson), to renew 50% of the total water volume in a week by 0.2 µm filtered seawater sampled at the same site.

The first mesocosm experiment was performed from May 10 th to June 17 th 2016 over a 6-week period. Twelve pre-cleaned 20-L LDPE (Nalgene  ) tanks were filled with 5-10 µm filtered water from site 41p (Figure 1). A 2-week acclimation period was carried out to establish relatively stable conditions in each tank. After the acclimation period, seawater from the 12 tanks was pooled, homogenized and then tanks were re-filled. Following the acclimation period, seawater in tanks from the first mesocosm study were assigned to four treatments: (1) site control (41p CTRL, i.e. no TME added to 41p seawater), (2) Cu addition (150 nM) (41p Cu), Pb addition (7 nM) (41p Pb), and (4) Cu and Pb addition (150 nM + 7 nM, respectively) (41p Cu+Pb) (Supplementary Figure 1). The Cu treatments were spiked with a stock solution of Cu nitrate (10 -3 M) and the Pb treatments were spiked with a stock solution of Pb nitrate (10 -3 M). According to the initial Cu and Pb concentrations measured in the 41p seawater used to fill the tanks, final concentrations of 150 nM and 7 nM for Cu and Pb respectively, were targeted. The water supplies were simultaneously spiked with similar concentrations of Cu, Pb, or both depending of treatment to maintain metal concentrations in tanks.

The second mesocosm was performed from May 2 nd to June 12 th 2017 over a 6-week period. Six 20-L LDPE (Nalgene  ) tanks were filled with 5-10 µm filtered water from each sampling site (LAZ and 6ext sites) (Figure 1). The treatments in the second mesocosm experiment were (1) site control (LAZ CTRL, i.e. no TME added to LAZ seawater), (2) Cu addition to LAZ seawater (150 nM) (LAZ Cu), (3) site control (6ext CTRL, i.e. no TME added to 6ext seawater), and (4) Cu addition to 6ext seawater (150 nM) (6ext Cu) (Supplementary Figure 2). The Cu treatments were spiked with a stock solution of Cu nitrate (10 -3 M). According to the initial Cu concentrations measured in LAZ and 6ext seawaters used to fill the tanks, final concentration of 150 nM for Cu were targeted. The water supplies were simultaneous spiked with Cu depending of treatment to maintain metal concentrations in tanks.

Experimental time plan and sampling

After the acclimation period, seawater was sampled from the 12 tanks using a peristaltic pump connected with Teflon tubing at different times over the course of the first mesocosm experiment (0, 3, 7, 10, 14, 17, 21, and 25 days) after the Cu/Pb spikes, and the second mesocosm experiment (0, 3, 7, 10, 14, 17, 21, 25 and 28 days) after the Cu spikes. At each sampling time, 333 mL of seawater was sampled in each replicate of a given experimental condition and pooled in 1L FEP bottles (Nalgene  ) to obtain composite samples for each condition. It was then immediately resampled for future trace metals (TM), dissolved organic carbon (DOC), and total nitrogen (TN) quantifications as previously described [START_REF] Omanović | Trace metal detection in Šibenik Bay, Croatia: Cadmium, lead and copper with anodic stripping voltammetry and manganese via sonoelectrochemistry. A case study[END_REF][START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary : The Krka River estuary[END_REF], and fully explained in Supplementary Material. Additionally, seawater (10-mL) for flow cytometry analysis were directly collected in 15-mL centrifuge tubes (Falcon) from each of 12 tanks, fixed with 0.25% (final concentration) glutaraldehyde, and stored at -80°C until analysis. Finally, at 0, 7, 14, 21, and 25 days, 1-L of seawater from each of 12 tanks was collected in 1-L HDPE bottles and filtered through 0.2 µm polycarbonate membranes (Millipore). Filters were stored at -80°C until DNA extraction.

Seawater physico-chemical measurements and analyses

Temperature (°C), pH, dissolved oxygen concentration (mg.L -1 ) and saturation (%) were directly monitored daily into tanks, over the course of both mesocosm experiments, using LDO10105 (Hach) multiparameter probe. Differential pulse anodic stripping voltammetry (DPASV) was used to measure Cd, Cu, Pb, and Zn dissolved concentrations. Details about instruments, analysis conditions, and data treatment are provided in Supplementary material and elsewhere [START_REF] Oursel | Dynamics and fates of trace metals chronically input in a Mediterranean coastal zone impacted by a large urban area[END_REF][START_REF] Cindrić | Evidencing the natural and anthropogenic processes controlling trace metals dynamic in a highly stratified estuary : The Krka River estuary[END_REF]. DOC and TN concentrations were determined using a TOC-V CSH analyzer (Shimadzu) (further details in [START_REF] Dang | Sedimentary dynamics of coastal organic matter: An assessment of the porewater size/reactivity model by spectroscopic techniques[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF].

Flow cytometry analysis

Autotrophic prokaryotes (Synechococcus-like, SYN), photosynthetic picoeukaryotes (PPE) and photosynthetic nanoeukaryotes (PNE) populations were characterized and enumerated using a BD Accury TM C6 (BD Biosciences) flow cytometer, as previously described [START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]. Heterotrophic prokaryotes were enumerated after staining with SYBR Green as previously described [START_REF] Cabrol | Inhibitory effects of sodium azide on microbial growth in experimental resuspension of marine sediment[END_REF].

Bioinformatic analysis

Sequences were demultiplexed and assigned to corresponding samples using CASAVA (Illumina). Bioinformatic analysis were performed on sequences from two sequencing MiSeq runs in the same sequencing company were used for our two mesocosm experiments. The analysis of the raw sequences was done by following the standard pipeline of DADA2 [START_REF] Callahan | DADA2 : High-resolution sample inference from Illumina amplicon data[END_REF] in RStudio (R Core Team 2017) with the following parameters: maxN= 0, maxEE= c(2,2), truncQ= 2. Briefly, the package includes the following steps: filtering, dereplication, sample inference, chimera identification, and merging of paired-end reads. DADA2 infers exact amplicon sequence variants (ASVs) from sequencing data, instead of building operational taxonomic units from sequence similarity. The taxonomy assignments were done with the SILVA v.128 database [START_REF] Pruesse | SILVA: A comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[END_REF][START_REF] Quast | The SILVA ribosomal RNA gene database project: Improved data processing and web-based tools[END_REF] and the "assignTaxonomy" function in DADA2. For some ASVs, in order to obtain a finer taxonomical resolution, DADA2 package implements a method to make species level assignments based on exact matching between ASVs and sequenced reference strains in the SILVA v.128 database, using "addSpecies" function.

Sequences classified as mitochondria or chloroplast were removed from the ASV table. The datasets from the samples were normalized by random subsampling to include an equal number of reads (n = 2078 reads). Samples with lower number of sequences than the number of reads chose to the normalization were discarded (Supplementary Table 1).

Statistical analysis

All plots and statistical analyses were performed with RStudio (R Core Team 2017).

Alpha diversity and error estimates were performed using QIIME script core_diversity_analyses.py [START_REF] Caporaso | QIIME allows analysis of highthroughput community sequencing data[END_REF], including Chao1, equitability, observed richness, Shannon and Simpson's diversity. Two-way analysis of variance (ANOVA) was applied to investigate the influences of Cu and Pb addition, experimental time, and their interaction on -diversity metrics and the abundance heterotrophic prokaryotes' community. Nonmetric multidimensional scaling (NMDS) based on Bray-Curtis distance was applied to evaluate the overall differences in prokaryotic community structure, using the vegan package in R (Oksanen et al. 2016). Global and pairwise PERMANOVA tests, using vegan and RVAideMemoire packages, respectively were applied to test significant differences between treatments and sampling time. To find representative phylotypes associated with the treatments, similarity percentage (SIMPER) analyses were first applied to screen OTUs primarily responsible for the overall dissimilarity (i.e. contributing to more than 1% of total dissimilarity) in the prokaryotic communities between treatments at each time point using vegan package.

Results

Trace metal elements and additional physicochemical parameters

Initial seawater samples (Ti) used for the first (i.e. 41p) and the second (i.e. LAZ and 6ext) experiments were geochemically contrasted and presented variations in trace metal concentrations as expected (Supplementary Table 2 and Supplementary Table 3). Total Cu, Pb, and Zn concentrations for 41p were 2.44 nM, 0.13 nM, and 5.34 nM, respectively while the concentrations were 2.5-, 3-, and 3.6-fold higher than in LAZ respectively, representing 15 nM, 0.79 nM, and 40 nM. For initial 6ext seawater, total Cu, Pb, and Zn concentrations were 15-, 18-, and 26-fold higher than in 41p respectively, representing 36 nM, 2.32 nM, and 139 nM.

At the beginning of both mesocosm experiments (T0), trace metal concentrations measured in control conditions were close to the ones encountered in initial seawater samples (Ti) from 41p, LAZ, 6ext. Thus, this confirms that no adsorption or contamination in metals occurred in our experiments. In both mesocosms, metal concentrations added were close to the targeted concentrations (150 nM of Cu and 7 nM of Pb) (Supplementary Table 2 and Supplementary Table 3). Finally, both Cu and Pb concentrations at the beginning (T0) were not different to those measured at the end (TF) of each experiment, confirming no metal loss in treatments where metals were added (Supplementary Table 2 and3).

The DOC and TN concentrations were not significantly different between the 3 initial seawaters (Supplementary Table 2 andSupplementary Table 3). For both mesocosm experiments, water temperature, pH, DO, and DOC did not exhibit significant differences among conditions but all of these parameters experienced temporal significant changes during the entire course of experiments (Kruskal-Wallis, p < 0.05). Globally, for both mesocosm experiments, pH and water temperature increased with time while DO saturation and DOC concentration decreased with time (Supplementary Table 2 andSupplementary Table 3).

Ultraphytoplanktonic and heterotrophic prokaryotes' abundances

During the first mesocosm experiment, total ultraphytoplanktonic abundances were determined by flow cytometry. Abundances were comparable between sampled seawater (Ti) from 41p and both conditions 41p CTRL and 41p Pb (1.1 ± 0.1 x 10 4 cell.mL -1 on average), with low temporal variability during the entire course of experiment (Supplementary Figure 3A). Total ultraphytoplanktonic abundances in conditions 41p Cu and 41p Cu+Pb, started to decrease 3 days after metal addition, leading to significantly lower abundances than in conditions 41p CTRL and 41p Pb until the end of the experiment (ANOVA, P < 0.001). At the beginning of the experiment (T0), heterotrophic prokaryotes' abundance measured in all conditions (11 ± 0.3 x 10 4 cell.mL -1 on average) was significantly lower than in sampled seawater (Ti) from the 41p (2.5 x 10 5 cell.mL -1 ) (ANOVA, P < 0.001) (Supplementary Figure 3B). From three days after metal addition to the end of the experiment, heterotrophic prokaryotic abundance was significantly higher in conditions 41p Cu and 41p Cu+Pb than in conditions 41p CTRL and 41p Pb (ANOVA, P < 0.001).

During the second mesocosm experiment, total ultraphytoplanktonic abundance in LAZ (1.9 x 10 4 cell.mL -1 ) was significantly higher than in 6ext seawater (7.9 x 10 3 cell.mL -1 ) (ANOVA, P < 0.001). After the acclimation period (T0), total ultraphytoplanktonic abundance followed a similar pattern in all conditions, decreasing 3 days after metal addition (Supplementary Figure 4A). From 3 days after metal addition to the end of the experiment, total ultraphytoplanktonic abundance was significantly lower in Cu-treated conditions than in LAZ and 6ext control conditions (ANOVA, P < 0.001). Heterotrophic prokaryotic abundance was not significantly different between LAZ (5.8 x 10 5 cell.mL -1 ) and 6ext (5.0 x 10 5 cell.mL -1 ) initial seawaters (Supplementary Figure 4B). At the beginning of the experiment (T0), this abundance was similar in all conditions (41 ± 1.0 x 10 4 cell.mL -1 ), while from 7 days after metal addition to the day 17, it was higher in Cu-treated conditions than in control conditions (ANOVA, P < 0.001). From 21 days after metal addition to the end of the experiment, heterotrophic prokaryotic abundance was similar in 6ext CTRL and 6ext Cu. Heterotrophic prokaryotic abundance from LAZ Cu was higher than in LAZ CTRL.

Prokaryotic alpha-diversity

For the first mesocosm experiment, all the ⍺-diversity indices except equitability varied significantly over time (Table 1A and Supplementary Figure 5). Globally, prokaryotic ⍺-diversity indices significantly decreased over time in all conditions (ANOVA, P < 0.05). After the metal addition (T0), the Chao1 index was significantly lower in Cu-treated conditions (i.e., 41p Cu and 41p Cu+Pb) relative to both initial seawaters from 41p and condition 41p CTRL (ANOVA, P < 0.001). Simpson's diversity was significantly higher in metal-treated conditions (i.e., 41p Cu, 41p Pb, and 41p Cu+Pb) relatively to condition 41p CTRL (ANOVA, P < 0.001). No significant difference was observed in equitability, observed richness, and Shannon indices between conditions.

For the second mesocosm experiment, all the ⍺-diversity indices except equitability varied significantly over time (Table 1B and Supplementary Figure 6). All prokaryotic ⍺-diversity indices significantly decreased over time in all conditions, especially after the 7 th day of the experiment (ANOVA, P < 0.05). Average Chao1, observed OTUs, and Shannon indices varied across conditions, and they were lower in condition LAZ Cu relative to LAZ CTRL (ANOVA, P < 0.05). No significant difference was observed in all ⍺-diversity indices between conditions 6ext CTRL and 6ext Cu. Finally, all ⍺-diversity indices measured in the second mesocosm experiment were lower compared to those of the first mesocosm experiment.

Prokaryotic community is strongly shaped by Cu in contrast to Pb

In the first mesocosm, the most abundant dominant phyla/classes (> 1% of all sequences across all samples) were Alphaproteobacteria, Gammaproteobacteria, Flavobacteriia and Cyanobacteria, representing 96% of the overall relative abundance. Archaea represented less than 1% of the prokaryotic community of all samples and thus were grouped with all the "other" rare taxa (< 1% of all sequences across all samples) in subsequent analysis.

Among the abundant families, 10 representative families, determined by SIMPER analysis, significantly differed treatments over time (Supplementary Table 5). Their relative abundances are shown in Figure 2. Rhodobacteraceae, Family I (Cyanobacteria), Alteromonadaceae, and SAR11 Surface 1 clade decreased in Cutreated conditions compared to 41p CTRL or 41p Pb. These families contributed to 20 ± 1.3%, 16 ± 4.6%, 6.7 ± 2.5%, and 3.0 ± 0.76% of the overall dissimilarity between these two conditions, respectively. Conversely, the relative abundance of Hyphomonadaceae, Flavobacteriaceae, and Halieaceae increased in Cu-treated conditions compared to 41p CTRL or 41p Pb and contributed to 34 ± 8.1%, 17 ± 4.4%, and 4.8 ± 1.6% respectively of the overall dissimilarity between these two groups of conditions. Moreover, 19 genera showed overall significant differences between conditions at the end of the experiment (TF) (Supplementary Table 6). Among these genera, only 4 were responsible for the structural change in Pb-treated condition.

The relative abundance of Alteromonas, Glaciecola, and unknown SAR11 Surface 2 clade significantly increased while Erythrobacter nearly disappeared when only Pb was added. Synechococcus, Litorimicrobium, and to a lesser extent, CHAB XI 27 decreased in 41p Cu compared to 41p CTRL. Conversely, Parvularcula, NS5 marine group and Hyphomonas increased in 41p Cu compared to 41p CTRL (Figure 3).

Samples from the first mesocosm study were mainly clustered by conditions (PERMANOVA, R 2 = 0.41; P < 0.001) and in a lesser extent by sampling times (PERMANOVA, R 2 = 0.34; P < 0.001) or interaction (PERMANOVA, R 2 = 0.17; P < 0.001) (Figure 4 and Supplementary Table 4A). At the beginning of the experiment (T0), prokaryotic community structures were similar between all conditions, and significantly different from the initial seawater at 41p (Supplementary Table 4B). After the 7 th day, Cu-treated conditions (41p Cu and 41p Cu+Pb) shown significantly different prokaryotic community structures from the other conditions (41p, 41p CTRL, and 41p Pb) at each sampling time, especially at the end of the experiment (TF).

Finally, there was no significant difference in communities between 41p CTRL and 41p Pb, as well as between 41p Cu and 41p Cu+Pb at each sampling time (Supplementary Table 4).

Influence of TME exposure history on copper effects

Prokaryotic community composition and structure

Concerning prokaryotic communities sampled from 3 geochemically contrasted sites (a low contaminated site (41p), an intermediately contaminated site (LAZ), and a highly contaminated site (6ext)), the most abundant phyla/classes (> 1% of all sequences across all samples) were Alphaproteobacteria, Gammaproteobacteria, Flavobacteriia and Cyanobacteria, representing 94% of the overall relative abundance. At the family taxonomic rank, Rhodobacteraceae (25 ± 1.5%), Flavobacteriaceae (16 ± 1.1%), Hyphomonadaceae (11 ± 1.3%), SAR11 Surface 1 clade (6.3 ± 0.89%), and Halieaceae (6.0 ± 0.59%) included the majority of the total sequences (Figure 5). As for the first mesocosm experiment, Archaea represented less than 1% of the prokaryotic community of all samples, and they were considered in the group "other" corresponding to the rare taxa in further analyses.

NMDS plot and PERMANOVA tests showed significant clusters corresponding to both sampling times and conditions (Figure 6 and Supplementary Table 7A). At all taxonomic rank, Cu addition (PERMANOVA, R 2 = 0.58; P < 0.001) showed a greater influence on community variations than time (PERMANOVA, R 2 = 0.14; P < 0.001) or interaction between time and experimental condition (PERMANOVA, R 2 = 0.20; P < 0.001) (Supplementary Table 7A). Prokaryotic community structures were significantly similar between initial seawaters from LAZ and 6ext and significantly different to the initial seawater from 41p (Supplementary Table 7B). At the beginning of the experiment (T0), similarly to what was observed for 41p in the first experiment, the prokaryotic community structures in each control conditions were not significantly dissimilar from related Cu-treated ones (LAZ CTRL vs LAZ Cu, and 6ext CTRL vs 6ext Cu). After the 7 th day, and until the end of the experiment, significant differences were clearly observed between Cu-treated and non-Cu-treated communities for 41p and LAZ. Conversely, pairwise comparisons showed no significant difference between 6ext CTRL and 6ext Cu, whatever the sampling time.

Representative taxa responsible for structural changes between control and Cutreated conditions

SIMPER analyses revealed that 12 representative families significantly differed initial seawaters, but also control and Cu-treated conditions throughout experiment duration (Figure 5 and Supplementary Table 8). SAR11 Surface 1 and Surface 2 clades, Hyphomonadaceae, and Family I (Cyanobacteria) were higher in 41p than in LAZ and 6ext, while Flavobacteriaceae and Rhodobacteraceae were more abundant in LAZ and 6ext than in 41p (Supplementary Table 8). Finally, major differences between LAZ CTRL and LAZ Cu were explained by the variation of relative abundances of Hyphomonadaceae, Rhodobiaceae, Cryomorphaceae and Alteromonadaceae.

At a lower taxonomic rank, among the 176 genera recorded, 34 significantly differed control and Cu-treated conditions at the end of the experiment (TF) (Figure 3 and Supplementary Table 9). In addition, the genera Salinihabitans and Rhodobium discriminating 41p CTRL from 41p Cu, decreased in LAZ Cu compared to LAZ CTRL while Jannaschia, unknown Cryomorphaceae, and Hirschia increased in LAZ Cu compared to LAZ CTRL. Ruegeria decreased in 6ext Cu compared to 6ext CTRL while Formosa, OCS116 clade, and Methylotenera increased in 6ext Cu compare to 6ext CTRL. Finally, representative genera responsible for structural changes between control conditions and Cu-treated conditions for 41p and LAZ were mainly abundant genera while for 6ext, representative genera were mainly rare genera (< 1% of all sequences across all samples).

Discussion

Copper exposure induced changes in prokaryotic community

In both mesocosm experiments, temporal variations in prokaryotic community structure were observed regardless of metal additions. However, metal addition led to stronger changes of community structure than the experimental setup or the experiment time. Copper exposure killed more than 99% of the ultraphytoplanktonic community. The selective pressure of metals on phytoplankton and more precisely on ultraphytoplankton is now well documented [START_REF] Echeveste | Toxic thresholds of cadmium and lead to oceanic phytoplankton: Cell size and ocean basin-dependent effects[END_REF][START_REF] Lafabrie | Impact of contaminated-sediment resuspension on phytoplankton in the Biguglia lagoon (Corsica, Mediterranean sea)[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF]. In parallel to the phytoplankton mortality, we observed an increase in heterotrophic prokaryotes' abundance and a decrease in their -diversity if they originated from the lower and intermediately contaminated sites. This abundance increases thus seemed to relate the development of a limited number of opportunistic r-strategists that efficiently use the organic resources released by the phytoplanktonic decay (Odum, 1969;Pianka, 1970). This behavior has already been observed with the highly reactive marine microbial community [START_REF] Buchan | Master recyclers: features and functions of bacteria associated with phytoplankton blooms[END_REF][START_REF] El-Swais | Seasonal assemblages and short-lived blooms in coastal north-west Atlantic Ocean bacterioplankton[END_REF]. Taken altogether, these observations highlight the strong interdependence between prokaryotic and phytoplanktonic communities, which could partly explain Cu-responses of microbial communities through indirect trophic relationships.

When considering the widely reported bacterial resistance to metals [START_REF] Nies | Microbial heavy-metal resistance[END_REF][START_REF] Baker-Austin | Co-selection of antibiotic and metal resistance[END_REF], the response of the prokaryotic community to Cu addition might also be due to the persistence or proliferation of resistant taxa, which can be higher than the extinction rate of sensitive taxa [START_REF] Wang | Response of Bacterioplankton Communities to Cadmium Exposure in Coastal Water Microcosms with High Temporal Variability[END_REF][START_REF] Qian | Alteration in successional trajectories of bacterioplankton communities in response to co-exposure of cadmium and phenanthrene in coastal water microcosms[END_REF]. 20

Significant differences in community structure between the control and the Cuadded conditions were found at all sampling time. Particularly, Hirschia (Hyphomonadaceae), NS4 and NS5 marine groups, Formosa and Tenacibaculum (Flavobacteriaceae) increased in Cu-treated conditions for both mesocosm experiments, indicating that they could be more resistant or take advantage from copper addition and phytoplankton decay [START_REF] Pinhassi | Changes in bacterioplankton composition under different phytoplankton regimens[END_REF]. Hyphomonadaceae were known to be abundant in plastic-attached biofilm communities [START_REF] Zettler | Life in the "plastisphere": microbial communities on plastic marine debris[END_REF][START_REF] Bryant | Diversity and Activity of Communities Inhabiting Plastic Debris in the North Pacific Gyre[END_REF][START_REF] Tender | Temporal dynamics of bacterial and fungal colonization on plastic debris in the North Sea[END_REF][START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF], especially in disturbed environments [START_REF] Briand | Spatio-Temporal Variations of Marine Biofilm Communities Colonizing Artificial Substrata Including Antifouling Coatings in Contrasted French Coastal Environments[END_REF]Coclet, et al., submitted). However, the results in this paper demonstrate for the first time the significant presence of this group in the prokaryotic community of metal contaminated seawater. Flavobacteriacea was also a major component of the microbial community in strongly disturbed and metal-contaminated environments [START_REF] Allen | The mercury resistance (mer) operon in a marine gliding flavobacterium, Tenacibaculum discolor 9A5[END_REF][START_REF] Wang | Response of Bacterioplankton Communities to Cadmium Exposure in Coastal Water Microcosms with High Temporal Variability[END_REF][START_REF] Zouch | Effect of Acidic Industrial Effluent Release on Microbial Diversity and Trace Metal Dynamics During Resuspension of Coastal Sediment[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]. Finally, the previous observation that Tenacibaculum and Formosa genera are positively selected in Cu-amended 6ext water as abundant and representative taxa from the most contaminated site (6ext) during a one-month-long survey, is in agreement with the idea of a similar selective pressure in situ as that observed in our previous field study experiment [START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]. Conversely, members of Rhodobacteraceae, Family I (Cyanobacteria, mainly Synechococcus), Alteromonadaceae, and SAR11 Surface 1 clade families have been proposed to be sensitive in situ to high trace metal levels [START_REF] Cassier-Chauvat | Responses to Oxidative and Heavy Metal Stresses in Cyanobacteria: Recent Advances[END_REF][START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF][START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF][START_REF] Corcoll | Copper affects composition and functions of microbial communities in marine biofilms at environmentally relevant concentrations[END_REF] or characteristic of marine zones away from anthropogenic influence [START_REF] Gilbert | The seasonal structure of microbial communities in the Western English Channel[END_REF][START_REF] Gilbert | Defining seasonal marine microbial community dynamics[END_REF][START_REF] Fuhrman | Marine microbial community dynamics and their ecological interpretation[END_REF].

Thus, our study reveals the sensitivity of a part of the prokaryotic community to Cu contamination representative from a harbor, by a combination of direct (i.e. toxicity) or indirect (i.e. related to phytoplankton decay) processes, as suggested recently in a field study [START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF].

Limited effects of Pb contamination on prokaryotic community

In our study, the effect of Pb exposure on prokaryotic communities was far less pronounced than the effect of Cu exposure. Pb addition using nitrate salts appeared to have limited impact on prokaryotic diversity and structure, with no detectable effect on ultraphytoplankton. Moreover, it did not prove to have a synergistic interaction with Cu. The weak toxicity of Pb on prokaryotic communities could be explained by its resistance and detoxification mechanisms, which are very efficient in microbial organisms [START_REF] Trevors | The role of microbial metal resistance and detoxification mechanisms in environmental bioassay research[END_REF][START_REF] Nies | Microbial heavy-metal resistance[END_REF][START_REF] Nayar | Environmental impact of heavy metals from dredged and resuspended sediments on phytoplankton and bacteria assessed in in situ mesocosms[END_REF][START_REF] Naik | Lead resistant bacteria: Lead resistance mechanisms, their applications in lead bioremediation and biomonitoring[END_REF].

Additionally, the dichotomy between Cu and Pb effects could be due to the difference in their concentrations added in our mesocosms as well as their bioavailability.

Indeed, to mimic the contamination levels observed in Toulon Bay, Pb concentrations were 20-fold lower than Cu concentrations, thus reducing the dose at which the cells were exposed. No data is available in the literature concerning tolerance threshold of marine bacterial communities against Pb contamination. Pb dissolved concentration was constant throughout the experiment, indicating no Pb adsorption on mesocosm materials or on microbial cells. Thus, the surface reactive behavior of Pb might not have induced a reduced bioavailability by reducing its dissolved concentration.

Therefore, our study does not allow us to explain precisely this difference of toxicity between Cu and Pb, but the non-essential nature of Pb and potential different ways to enter the cells than Cu represent hypotheses that should be studied in future research [START_REF] Han | Relating metal bioavailability to risk assessment for aquatic species: Daliao River watershed, China[END_REF].

The relative abundance of Glaciecola taxon significantly increased while

Erythrobacter became undetectable when only Pb was added. Glaciecola has been described to be an opportunistic member of prokaryotic communities, its abundance increasing rapidly following an increase in DOC concentration [START_REF] Sipler | Microbial Community Response to Terrestrially Derived Dissolved Organic Matter in the Coastal Arctic[END_REF][START_REF] Von Scheibner | Tight Coupling of Glaciecola spp . and Diatoms during Cold-Water Phytoplankton Spring Blooms[END_REF]. Since DOC concentrations in our experiment were similar in all conditions, the ability of Glaciecola to take advantage compared to other taxa in a Pb contaminated sea water remains unexplained. The genus Erythrobacter was found to be important taxa in surface-attached communities [START_REF] Zettler | Life in the "plastisphere": microbial communities on plastic marine debris[END_REF][START_REF] Dussud | Evidence of niche partitioning among bacteria living on plastics, organic particles and surrounding seawaters[END_REF][START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF], and notably in contaminated ecosystems (Catão et al. submitted;[START_REF] Briand | Spatio-Temporal Variations of Marine Biofilm Communities Colonizing Artificial Substrata Including Antifouling Coatings in Contrasted French Coastal Environments[END_REF][START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF]Coclet, et al., submitted). Thus, the strong decrease of Erythrobacter in the water after Pb addition could be explained by the ability of this genus to form biofilms in the mesocosms. Indeed, biofilms are one of the most widely distributed and successful modes of life in water environment [START_REF] Stoodley | Biofilms as Complex Differentiated Communities[END_REF][START_REF] Flemming | Biofilms: An emergent form of bacterial life[END_REF]) and a strategy that microorganisms might use to protect them from toxic metals [START_REF] Harrison | Multimetal resistance and tolerance in microbial biofilms[END_REF].

TME exposure history determines Cu effects on prokaryotic communities

Trace metal contamination gradients in Toulon bay have been already described by punctual mappings [START_REF] Jean | Protein expression from zooplankton communities in a metal contaminated NW mediterranean coastal ecosystem[END_REF]Dang et al. 2015b;[START_REF] Coclet | Trace metal contamination as a toxic and structuring factor impacting ultraphytoplankton communities in a multicontaminated Mediterranean coastal area[END_REF] or during short [START_REF] Coclet | Changes in bacterioplankton communities resulting from direct and indirect interactions with trace metal gradients in an urbanized marine coastal area[END_REF]) and long-term surveys (Layglon et al., under revision).

These studies demonstrated that TME contamination gradients persists at a pluri-annual scale in Toulon Bay. This led us to assume that prokaryotic communities sampled along this contamination gradient could present different metals exposure histories. Cu addition in both low (41p) and in intermediately (LAZ) contaminated seawaters induced important changes in prokaryotic community structure, while Cu addition in seawater originating from a chronically contaminated site (6ext) only weakly impacted the prokaryotic community structure. Based on the PICT theory [START_REF] Gustavson | Pollution-induced community tolerance (PICT) in coastal phytoplankton communities exposure to copper[END_REF][START_REF] Blanck | A Critical Review of Procedures and Approaches Used for Assessing Pollution-Induced Community Tolerance (PICT) in Biotic Communities[END_REF], this observation validates our assumption and demonstrates the adaptive potential of prokaryotic communities to withstand elevated Cu concentrations. This adaptation must have developed rapidly since the water residence time in Toulon Bay has been estimated to a few days [START_REF] Dufresne | Wind-forced circulation model and water exchanges through the channel in the Bay of Toulon[END_REF], even if this estimation is probably not representative to what happens in very enclosed docks such as the one where we sampled contaminated water (6ext).

This adaptive potential could be the result of the presence of a higher proportion of tolerant species in the most contaminated seawater, representative of a strong and chronic metal-exposure history of these communities [START_REF] Carman | Does historical exposure to hydrocarbon contamination alter the response of benthic communities to diesel contamination ?[END_REF]. This hypothesis is in agreement with the very small variation of structure after Cu addition in the community sampled from the most contaminated site. Indeed, for this community, we mainly observed changes in the relative abundance of rare taxa.

We hypothesize that mechanisms of toxicity and resistance to Cu co-occur in prokaryotic communities from low and intermediately contaminated sites. The observed dissimilarity in prokaryotic structures between these communities after Cu exposure could be the result of differences in initial community composition in 41p and LAZ seawaters. To the best of our knowledge, our study provides the first evidence that TME exposure history may play a critical role in marine prokaryotic community response to re-current metal contamination.

Conclusion

From our mesocosm experiences, we can conclude that harbor-relevant Cu exposure affects abundance, diversity and structure of prokaryotic communities from both low and intermediately TME contaminated seawaters. The effect of Cu addition is visible through both the disappearance of sensitive taxa and the selection of adapted ones. We also demonstrated that TME exposure history has a strong influence on the response of prokaryotic communities to newer-current contamination, as prokaryotic communities from contaminated site were less impacted by Cu addition. Conversely, we showed limited impacts of Pb exposure compared to Cu contamination, probably due to the low toxic potential against prokaryotes and the efficiency of its resistance and detoxification mechanisms.

Finally, our observations suggest both direct (toxic) and indirect (trophic) effects of TME contaminations, highlighting the need for studies of the complexity of both biotic and abiotic interactions.

  DNA extraction, PCR amplification, and sequencing of 16S rRNA gene amplicons DNA was extracted from the polycarbonate filters by a combination of enzymatic cell lysis (Ghiglione, Conan and Pujo-Pay 2009) and AllPrep DNA/RNA Mini Kit (QIAGEN) according to the manufacturer's instructions. Prokaryotic community structure was assessed by targeting the V4-V5 region of the 16S rRNA gene (Parada, Needham and Fuhrman 2016) and using Illumina Miseq 2 x 250pb pairedend sequencing (Genoscreen, France). The protocol for the DNA extraction and the library preparation is fully described in Coclet et al. (2019).

Acknowledgements

This study was supported by IMPRECI-M 2 research program (funded by EC2CO). C.

Coclet PhD was funded by the Provence-Alpes-Côte d'Azur region and supported by TPM. The authors wish to thank the French Navy and the LASEM for diving and sampling assistance, C. Martino for building assistance of mesocosm structure (Toulon University, France) for sampling and sample treatment assistance. The authors whish also to thank the Dr. Pedro Lebre to correct language and grammar.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.