Elisa C P Catao

Nicolas Gallois

Fabyenne Fay

Benjamin Misson

Jean-François Briand
email: briand@univ-tln.fr

Metal resistance genes enrichment in marine biofilm communities selected by biocidecontaining surfaces in temperate and tropical coastal environments

Keywords: Complex communities, antifouling surface, copper tolerance, anthropogenic effect

Microorganisms able to form biofilms in marine ecosystems are selected depending on immersed surfaces and environmental conditions. Cell attachment directly on toxic surfaces like antifouling coatings suggests a selection of tolerant (or resistant) organisms with characteristics conferring adaptive advantages. We investigated if environment would drive metal resistance gene abundance in biofilms on artificial surfaces. Biofilms were sampled from three surfaces (a PVC reference and two antifouling coatings) deployed in three coastal waters with dissimilar characteristics: The Mediterranean Sea (Toulon) and Atlantic (Lorient) and Indian (Reunion) Oceans. The two coatings differed in metals composition, either Cu thiocyanate and Zn pyrithione (A3) or Cu2O (Hy). Metal resistance genes (MRG) specific to copper (cusA, copA, cueO) or other metals (czcA and pbrT)

were monitored with qPCR in parallel to the microbial community using 16S rRNA gene metabarcoding. A lower α-diversity on A3 or Hy than on PVC was observed independent on the site. Weighted Unifrac suggested segregation of communities primarily by surface, with lower site effect. Metacoder log2 fold change ratio and LeFSe discrimination suggested Marinobacter to be specific of Hy and Altererythrobacter, Erythrobacter and Sphingorhabdus of A3. Likewise, the relative abundance of MRG (MRG/bacterial 16S rRNA) varied between surfaces and sites. A3 presented the greatest relative abundances for cusA, cueO and czcA. The latter could only be amplified from A3 communities, except at Toulon. Hy surface presented the highest relative abundance for copA, specifically at Lorient. These relative abundances were correlated with LeFSe discriminant taxa. Dasania correlated positively with all MRG except cueO. Marinobacter found in greater abundance in Hy biofilm communities correlated with the highest abundances of copA and Roseovarius with czcA. These results prove the selection of specific communities with abilities to tolerate metallic biocides forming biofilms over antifouling surfaces, and the secondary but significant influence of local environmental factors.

The capsule of no more than two lines that summarizes the main finding:

Marine biofilm communities were selected by metal-releasing surfaces before environmental drivers, and enriched in metal resistance genes in temperate and tropical coastal environments

Introduction

Submersion of a surface in aquatic ecosystems leads to microbial colonization within minutes. The microbial biofilm community formed is driven by environmental features as salinity and temperature [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] Lau | Roles of bacterial community composition in biofilms as a mediator for larval settlement of three marine invertebrates[END_REF][START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF][START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF], nutrients [START_REF] Lawes | Elevated nutrients change bacterial community composition and connectivity: high throughput sequencing of young marine biofilms[END_REF], but also substrata (Catao et al., 2019;[START_REF] Celikkol-Aydin | 16S rRNA gene profiling of planktonic and biofilm microbial populations in the Gulf of Guinea using Illumina NGS[END_REF][START_REF] Faÿ | Effect of biocidal coatings on microfouling: In vitro and in situ results[END_REF][START_REF] Kirstein | Mature biofilm communities on synthetic polymers in seawater -Specific or general?[END_REF][START_REF] Oberbeckmann | Microbes on a bottle: substrate, season and geography influence community composition of microbes colonizing marine plastic debris[END_REF][START_REF] Pinto | The composition of bacterial communities associated with plastic biofilms differs between different polymers and stages of biofilm succession[END_REF].

Biocides-containing surfaces are used in maritime industry as well as in medicine to prevent the adhesion of microorganisms. To limit marine biofouling on submerged structures, the efficacy of antifouling coatings remains often related to high concentrations of metallic biocides, even though alternative strategies are being increasingly used [START_REF] Lejars | Fouling release coatings: A nontoxic alternative to biocidal antifouling coatings[END_REF]. Since 2012, in Europe, those biocides must be controlled by the EU Biocidal Products Regulation (BRP, Regulation EU 528/2012) to reduce environmental contamination. Copper-based compounds became the main biocides, and zinc-based co-biocides are also often present in the coating formulation. These metals were reported to exert a selection on the naturally occurring organisms [START_REF] Ancion | Three common metal contaminants of urban runoff (Zn, Cu & Pb) accumulate in freshwater biofilm and modify embedded bacterial communities[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] Corcoll | Copper affects composition and functioning of microbial communities in marine biofilms at environmentally relevant concentrations[END_REF] and can co-select for resistant strains [START_REF] Flach | Does antifouling paint select for antibiotic resistance?[END_REF].

In fact, coatings are still colonized by a reduced number of tolerant or resistant organisms [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] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF][START_REF] Corcoll | Copper affects composition and functioning of microbial communities in marine biofilms at environmentally relevant concentrations[END_REF][START_REF] Muthukrishnan | Long-term microfouling on commercial biocidal fouling control coatings[END_REF].

Biofilms are often referred as an ecological advantage that provide protection to the cells from a toxic environment [START_REF] Hall-Stoodley | Bacterial biofilms: from the Natural environment to infectious diseases[END_REF][START_REF] Hoyle | Bacterial resistance to antibiotics: the role of biofilms[END_REF]. Their ability to develop over toxic surfaces is considered dependent on tolerance mechanisms including exopolymeric production and/or resistance genes [START_REF] Flemming | Bacteria and archaea on Earth and their abundance in biofilms[END_REF]. Regarding metallic biocides, bacteria can react to elevated concentrations by expressing metal resistance systems as: P-type ATPases, metallothioneins, resistance-nodulation-cell-division (RND) efflux pumps and/or CDF transporters [START_REF] Bruins | Microbial resistance to metals in the environment[END_REF][START_REF] Nies | Efflux-mediated heavy metal resistance in prokaryotes[END_REF].

In microbial communities, the specific resistance to copper was studied on marine sediments [START_REF] Besaury | Expression of copper-resistance genes in microbial communities under copper stress and oxic/anoxic conditions[END_REF][START_REF] Besaury | Abundance, activity, and diversity of Archaeal and Bacterial communities in both uncontaminated and highly copper-contaminated marine sediments[END_REF][START_REF] Besaury | Abundance and diversity of copper resistance genes cusA and copA in microbial communities in relation to the impact of copper on Chilean marine sediments[END_REF][START_REF] Roosa | Bacterial metal resistance genes and metal bioavailability in contaminated sediments[END_REF], tailing dam [START_REF] Chen | Bacterial heavy-metal and antibiotic resistance genes in a copper tailing dam area in Northern China[END_REF] and in terrestrial samples as manure compost [START_REF] Guo | Responses of antibiotic and heavy metal resistance genes to bamboo charcoal and bamboo vinegar during aerobic composting[END_REF]. This resistance seems to be mediated by 1) the efflux ATP-ase pump copA [START_REF] Rensing | CopA: An Escherichia coli Cu(I)-translocating P-type ATPase[END_REF]; 2) the cusA protein related to chemo-osmotic copper extrusion and part of the RND family [START_REF] Outten | The Independent cue and cus Systems Confer Copper Tolerance during Aerobic and Anaerobic Growth in Escherichia coli[END_REF][START_REF] Piddock | Multidrug-resistance efflux pumps ? not just for resistance[END_REF] and the 3) cueO encoding for a multicopper oxidase [START_REF] Silveira | Co-transfer of resistance to high concentrations of copper and first-line antibiotics among Enterococcus from different origins (humans, animals, the environment and foods) and clonal lineages[END_REF]. The genes above have been described from Escherichia coli and Enterococcus faecium and seem to be present in certain Bacteria and few Archaea. Other genes can be monitored to detect resistance to other metals or co-and cross-resistance. As for example, the czcA gene which codes for the antiporter cation-anion for efflux of Co +2 /Zn +2 /Cd +2 is also part of the RND family [START_REF] Nies | Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34[END_REF].

Together with this gene, the complex of proteins encoded by pbr have been identified from the indigenous plasmid pMOL30 of the bacteria Ralstonia metallidurans with the functions of uptake and efflux of Pb [START_REF] Borremans | Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34[END_REF].

Only one study on the Swedish west coast have reported the effect of metal released coating on the co-selection of antibiotic and metal resistance genes (MRG) using a metagenomics approach [START_REF] Flach | Does antifouling paint select for antibiotic resistance?[END_REF]. Furthermore, MRG have been linked to sediment and biofilm microbial communities in freshwater lakes [START_REF] Song | Bacterial community rather than metals shaping metal resistance genes in water, sediment and biofilm in lakes from arid northwestern China[END_REF]. However, to our knowledge, no work has yet linked the specific MRG occurrence, using quantitative PCR, with the selection of specific taxa within marine biofilm communities, and most specifically related to human-made surfaces commonly deployed in the sea. The selection of specific communities on metal-containing surfaces in comparison to inert surfaces lead us to explore the hypothesis that the community selection was driven more strongly by metal containing surfaces than environmental conditions in three dissimilar coastal sites in both temperate and tropical ecosystems. Then, we wonder if correlation might be established more particularly between selected microbial taxa and the abundance of some of metal resistance genes in the different ecosystems.

Material and methods

Experimental design and environmental parameters

Immersions were performed in French harbors at the Toulon Bay (43°06'25" N; 5°55'41" E) in the North-Western Mediterranean Sea, Lorient (Kernevel harbour, 47°43'8.0178" N; 3°22'7.2048" W) in the south coast of Brittany in the Atlantic Ocean, and Le Port, Reunion Island (20°56'03" S; 55°17'0.6" E) in the Indian Ocean.

Biofilm was studied over panels (Figure S1) immersed in the euphotic zone (from 0.5 to 2 m deep) for 30 days in the summer season, from 15 th June to 15 th July 2016 at Lorient and Toulon, and 1 st to 29 th December 2016 at Reunion.

Water chemistry was analysed in the three sites during the immersion period according to physical, nutritional and trace metals (Catão et al.,submitted,Table S1). Briefly, water quality in the three coastal sites differed significantly. At Reunion, water presented high signals of contamination in terms of phosphate and Pb concentrations. Lorient was also considered eutrophic with the highest levels of nitrate between the three sites. Toulon was characterized by the highest salinity but lowest phosphate and silicate concentrations. Toulon also presented the highest contamination of Cu and Zn. Due to sample loss, Lorient could not be evaluated regarding trace metal contamination.

Surface parameters

PVC panels of 5 × 5 cm were either sandblasted or painted with one of the two commercially available antifouling paints (in triplicates): a solvent-based Nautix A3 white selfpolishing paint (hereafter named A3) and a water-based Boero Hydrocoat blue paint (hereafter abbreviated as Hy). A3 and Hy are chemically-active paints as they contain metallic biocides that are continuously released during immersion to hamper formation of biofouling on human-made surfaces deployed in the sea. Furthermore, A3 and Hy differed in biocides composition and formulation, as the former contained 219 g.L -1 (15%) copper thiocyanate (CuSCN; 114.5 g Cu) and 58.4 g.L -1 of Zn (4% of Zn pyrithione -ZPT, and 3% ZnO) and Hy had 844.8 g.L -1 (749.3 g Cu) of copper oxide (Cu2O) (40.23%), but no Zn. Information on the polymer and metals used were obtained directly from the providers or in technical sheets.

Biofilm sampling

After scraping the biofilms on the 5 x 5 cm panels with a sterile scalpel, samples were frozen in liquid nitrogen and kept at -80 °C until DNA extraction. For flow cytometry analysis, biofilms were also scraped but fixed in 10 mL solution of artificial seawater (ASW; Sigma-Aldrich) containing 0.25% glutaraldehyde (Sigma-Aldrich). Cells were enumerated as previously described [START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF].

DNA extracted with PowerBiofilm DNA isolation kit (Qiagen, Courtaboeuf, France) was used for 16S rRNA gene amplification with the primers 515F-Y/926R [START_REF] Parada | Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples: Primers for marine microbiome studies[END_REF] as previously described [START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF]. Picogreen (Quant-iT™ PicoGreen® dsDNA Assay kit, ThermoFischer Scientific) quantified PCR products were pooled in equimolar mixes before Illumina MiSeq 2 × 250 pb sequencing or diluted to 1 ng/µL for qPCR quantification. Raw sequences can be found under the project PRJNA608293 in NCBI database, and under the samples number SAMN14169847, SAMN14169848, and SAMN14169852 to 58.

Bioinfomatic and statistical analyses

A total of 429,545 paired-end raw sequences were treated in R v3.5.3 with DADA2 default pipeline for 16S rRNA [START_REF] Callahan | Exact sequence variants should replace operational taxonomic units in marker-gene data analysis[END_REF] (Supp informations). ASV taxonomy assignment was performed against SILVA database 132. α-and β-diversity, as well as significance tests (PERMANOVA) were calculated in QIIME2 after matrix rarefaction to smallest sample (2399 sequences). All statistical analysis or graphical representation were performed in R (version 3.5).

Metacoder was used to display Log2 fold change of relative abundance of each taxon in pairwise comparisons between the surfaces.For each taxa level, metacoder calculates the abundance pertaxon.

Furthermore, biomarkers were detected for each site between the three surfaces with LEfSe [START_REF] Segata | Metagenomic biomarker discovery and explanation[END_REF]. The discriminant taxa were filtered from the biom table and their relative abundance was correlated (Spearman) with metal resistance genes abundance (calculated as the ratio between targeted gene over 16S rRNA copies, for each gene).

Quantitative PCR (qPCR)

Ribosomal (archaeal and bacterial 16S rRNA genes) and metal resistance genes (cusA, copA, cueO, czcA, pbrT) were quantified with BioRad CFX Connect System. Each reaction (20 µL) contained 1X SSO Advanced Universal Sybr Green Supermix, 0.3 µM of each primer and 4 µL of diluted DNA (1ng/ µL). Primers, gene size and amplification cycles are presented in Table S2. Fluorescence was read after each cycle at 72 o C. A melting curve (95 o C for 10s and incremental increase of 0.5 o C from 65 to 95 o C) was performed at end of each run to ensure specificity. The standard curves (details on Table S3) were obtained by a 10-fold dilution of linearized plasmids containing the reference gene. Copy number was estimated from the concentration of linearized plasmid (ng) multiplied by the Avogadro number and divided by the size of the vector summed to the partial gene times 660 (bp weight g.mol -1 ) times 10 9 ng.g -1 .

The origin of each gene fragment is described in Table S3. Both 16S rRNA genes and czcA were amplified from environmental samples and cloned in PGEM T easy. copA was synthetized by Eurofins Genomics and delivered within a pEX-A128 vector. The remaining genes (cusA, cueO and pbrT) were synthesized by ThermoFischer Scientific. All vectors contain ampicillin gene resistance for clone selection.

All qPCR were performed in technical replicates to ensure reproducibility, especially in low-gene abundance quantification. We accepted only technical replicates with less than 15% of relative standard deviation and only samples that had number of copies above the detection limit.

Diluted DNA was estimated to have no significant PCR inhibition as indicated by assays on serially diluted samples for bacterial 16S rRNA amplification (data not shown).

Results

Density and diversity within prokaryotic biofilm communities

Cell density for heterotrophic prokaryotes and phototrophic microorganisms varied from 2.6 × 10 5 to 2.6 × 10 7 and 6.4 × 10 3 to 1.1 × 10 8 cells cm -2 , respectively (Figure S2). In all three sites, PVC presented significantly higher abundances of both cellular communities. However, at Reunion, biofilms grown on Hy surfaces presented similar heterotrophic cell density as found for PVC.

Across the 27 samples, 175,747 paired-end Illumina reads remained after treatment according to DADA2 pipeline. Archaea were present in only one sample (one of the replicates of PVC immerged in Toulon) and corresponded to an ASV of the family of Nitrosopumilaceae.

Archaeal sequences were consequently removed for further analyses. Similar trends were found for α-diversity indexes (Chao1, PD or Pielou) as for densities. PVC exhibited the highest richnness and diversity and no significant difference could be noticed between the two coatings in all the three sites (ANOVA; p < 0.05) (Figure S3). However, we detected a significant difference between sites only for Hy, with Reunion exhibiting the highest phylogenetic diversity and Toulon the lowest.

Concerning their taxonomic structure, bacterial communities present within the biofilms were firstly segregated by surface and then by site (Figures 1 andS3). The first axis indicated segregation of A3 and Hy and the differences between sites within each surface. With smaller contribution, the second axis pointed at the greater distinction between the communities over the control (PVC) and the metals-containing surfaces (A3 and Hy). Surface presented greater pseudo-F values, indicating a greater effect-size of the differences between communities in each surface (Table S4). In all four metrics used to detect the similarity between communities, Hy biofilms were the most distinct (Figure S4). However, the phylogenetic signal incorporated in Unifrac (weighted and unweighted) distances indicated greater proximity of the biofilms developed over the two biocidal surfaces. Likewise, Bray-Curtis and Jaccard separated the communities present at Reunion, while Lorient and Toulon seemed more similar. Nevertheless, Unifrac suggested lower influence of site on biofilm communities, as PERMANOVA indicated no significant difference between sites with Weighted Unifrac distances (Table S4). Site differentiation within each surface seemed to be more due to presence/absence (Jaccard) of certain ASVs, or even their relative abundance (Bray-Curtis), rather than communities phylogenetically closeness.

β-diversity differences between surfaces and sites were also apparent in the relative abundance of certain families (Figure S5). Flavobacteriaceae (27.2% ± 13.3) and Rhodobacteriaceae (13.8% ± 5.1) were the most abundant families except on A3 from both Reunion and Toulon, respectively. Sphingomonadaceae and Marinobacteriaceae completed the top three most abundant families on A3 (19.9% ± 2.5) and Hy (13.2% ± 2.7) respectively.

Saprospiraceae was abundant on biocidal in Lorient (8.9% ± 1.3) and Toulon (7.1% ± 1.9).

Site specific distinct taxa on biocide-containing surfaces

Over 1.5 thousand taxa have been parsed by metacoder from which only 9 were singletons (remaining 1551 taxa). Due to the taxon redundancy in higher levels of taxonomic hierarchy, 889 taxon_id remained for relative abundance calculation. Metacoder graphical representation of the log2 fold change of taxon proportions between surfaces provided symmetrical results to compare the surfaces two by two and to display the differences over the taxonomic hierarchy.

The family Marinobacteriaceae (Gamma-proteobacteria), and most specifically the genus Marinobacter, were significantly more present on Hy than on PVC or A3 surfaces.

Sphingomonadaceae (Alpha-proteobacteria) were also more abundant on Hy than on PVC, but less abundant than on A3, especially the genus Altererythrobacter (Figure 2 Most of the taxa described above were also detected as biomarkers by LeFSe (LDA > 3)

when comparing the three surfaces regardless of site. For example, Aquimarina was detected as biomarker for Hy, and Alteromonas was considered a Hy biomarker but did not present significant log2 fold change against either of the two other surfaces. Lastly, families detected as PVC biomarkers were Rhodobacteriaceae, Saprospiraceae, Pirellulaceae, Rubritaleaceae and Hyphomonadaceae However, many of those surface biomarkers were detected specifically in one or two sites (Figure S7). Only Micavibrionaceae was distinctly found on A3 in all three sites, and

Metal resistance genes (MRG) abundance

Bacterial abundance, estimated by the abundance of 16S rRNA gene, was not different between sites, except for A3 at Reunion. The bacterial density ranged between 3.0 × 10 6 and 1.1 × 10 8 copies.cm -2 (Figure S8). Archaeal 16S rRNA gene quantification indicated between 5.5 × 10 -5 and 8.1 × 10 -4 less Archaea than Bacteria, but with no significant variation of archaeal density between surfaces or sites. In addition, it is noteworthy that Archaea were not amplified from all PVC at Reunion or from two of three PVC at Lorient. Considering MRG, cusA gene presented an average density (3.8 × 10 7 ± 5.3 × 10 7 copies.cm -2 ) similar to bacterial 16S rRNA gene. The other metal resistance genes (copA, cueO, czcA, pbrT) ranged from 2.1 × 10 2 to 4.9 × 10 5 copies.cm -2 .

Due to slight differences in 16S rRNA densities among samples, we have analysed functional genes abundance as a ratio of each MRG by the prokaryotic ribosomal gene abundance (Archaea+ Bacteria abundances of 16S rRNA). The relative abundance of MRG (MRG/16S rRNA gene) varied between sites and surfaces (Figure 3). A3 presented the greatest relative abundances (irrespective of the site) for cusA, cueO and czcA. Otherwise, A3 immersed at Reunion presented a community with significantly higher relative abundance of cusA and czcA, when compared to A3 at Lorient and Toulon. In fact, czcA could only be amplified from A3 communities, except in Toulon.

The A3 ratio of cueO/16S rRNA did not differ from Hy at Reunion, was similar to Hy and PVC at Toulon, but was the highest at Lorient. Also, in this site, the Hy surface presented the highest ratio for copA. Finally, pbrT relative abundance showed no difference between the communities developed on the two antifouling surfaces and was only amplified in one PVC sample from Toulon.

Concerning the sites, we could detect all MRG at Toulon only. Besides, regarding the abundance of MRG, Toulon was the only site where there was no difference in MRG relative densities between the biofilms grown on the antifouling surfaces and on PVC. Lastly, in Toulon, cusA was significantly higher on PVC compared to the other sites.

Specific taxa positively correlated to resistance genes

Relative abundance for each MRG was used to calculate their correlation in each site and/or surface against taxa found discriminant for each surface in each site by LeFSe (LDA > 2) (Figure 4). Dasania (Spearman's ρ = 0.73, p-value < 0.001, n=27) and Alteromonas (ρ = 0.75, p-value < 0.001, n=27) showed the strongest correlation at the genus level with cusA whatever the site and coatings.

Marinobacter, a discriminant taxon for Hy, had the greatest positive correlation with the abundance of copA (ρ = 0.64, p-value < 0.001, n=27). Other genera found on Hy also correlated

Discussion

Our study of antifouling surfaces immersed in temperate and tropical harbor waters aimed to provide providing new ecological insights to the selection of biofilm communities according to their potential resistance to metals on those surfaces. These findings might also contribute to further grasp the mechanisms that allow the colonization of such coatings depending on the local water characteristics.

Communities were selected by surface, and by site to a lesser degree AF coatings contain metallic biocide embedded in an organic matrix, which both differ for the two coatings here. Hy contained greater concentration of Cu2O within an aqueous-based resin, whereas A3 was conceived as an organic solvent matrix containing CuSCN and ZnO. These differences could probably explain the variability in community composition as well as in MRG relative abundance. We have chosen three sites with contrasting environmental conditions but with a common factor: the anthropogenic pressure. Human effect in coastal waters is often regarded as contamination either by nutrients or pollutants and can potentially exert selective pressure over microbial communities [START_REF] Nogales | Anthropogenic perturbations in marine microbial communities[END_REF]. Microbial communities are influenced by trace metal contamination, whether in freshwater [START_REF] Ancion | Three common metal contaminants of urban runoff (Zn, Cu & Pb) accumulate in freshwater biofilm and modify embedded bacterial communities[END_REF], seawater [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] Webster | Site-specific variation in Antarctic marine biofilms established on artificial surfaces[END_REF] or sediments [START_REF] Gillan | Structure of sediment-associated microbial communities along a heavy-metal contamination gradient in the marine environment[END_REF][START_REF] Misson | Chemical multi-contamination drives benthic prokaryotic diversity in the anthropized Toulon Bay[END_REF]. Even though planktonic communities in our sites might thus present already a tolerance to metals as shown in the Toulon Bay [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]2020), the contribution of harbour relevant trace metals concentrations to microbial biofilm structuration, among other covarying factors, is less evident (Briand J.-F., pers. com, Catao et al submitted). Furthermore, as one explanation to the relatively low impact of seawater metals concentrations on biofilm community structure could be the ability of EPS to trap metals, hence preventing the contact with biofilm organisms and consequently their selection, the actual level of tolerance to metals in biofilm community on inert surface, like plastics, remains to be compared to biofilm community from non-contaminated ecosystems.

Significant effect on periphyton biofilm diversity was only reported on higher copper concentration (>0.32 µM), rarely measured in natural seawater [START_REF] Corcoll | Copper affects composition and functioning of microbial communities in marine biofilms at environmentally relevant concentrations[END_REF] and at least ten times concentrations measured in our sites (20 to 50 nM for dissolved copper, (Catão et al., submitted)). However, we assume that the amount of copper and zinc directly in contact with microorganisms trying to colonize a biocidal substrate should be much more considerable as we could see significant differences between biofilm communities over the inert PVC and the antifouling coatings -A3 and Hy. That means that especially copper and zinc released by the coatings applied a strong selection pressure on the potentially already tolerant microbial community that will colonise substrates in each harbor.

Toxicity and consequently community selection by metal-enriched surfaces depend on the gradual release of toxic metals. Our observations were made in communities developed after onemonth immersion, which can be long in terms of biofilm succession, but is coherent with the highest release rates observed from metals-containing coatings [START_REF] Valkirs | Measurement of copper release rates from antifouling paint under laboratory and in situ conditions: implications for loading estimation to marine water bodies[END_REF]. Leaching of these compounds varies with as well hydrodynamics [START_REF] Valkirs | Measurement of copper release rates from antifouling paint under laboratory and in situ conditions: implications for loading estimation to marine water bodies[END_REF] and environmental factors as the increased Cu leaching and lower Zn release in sites with increased salinity [START_REF] Singh | Leaching of copper and zinc from spent antifouling paint particles[END_REF]. Despite significant differences in β-diversity between all three surfaces, A3

and Hy did not differ from each other in terms of cell density (phototrophs and heterotrophic prokaryotes) nor regarding α-diversity, but both selected for lower cell number and diversity than PVC. However, within each biocidal surface, we could detect sites with higher or less cell density, richness and diversity. The variation in metals composition between these two coatings did not seem to affect significantly their diversity or cell number, contrary to other trends observed before of greater effect of Cu than Zn in microfouling density inhibition [START_REF] Agostini | Surface coatings select their micro and macrofouling communities differently on steel[END_REF].

As seen before, the selection of certain organisms by lifestyle, ie planktonic vs biofilm (Catão et al., submitted.;[START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF][START_REF] Song | Bacterial community rather than metals shaping metal resistance genes in water, sediment and biofilm in lakes from arid northwestern China[END_REF] or metal releasing surface [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] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF][START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF][START_REF] Tagg | Paint particles are a distinct and variable substrate for marine bacteria[END_REF] prevails over environmental factors. Slight variations observed within communities from the same surface between the three sites might be related to the different ranges of nutrients, metal contamination, physical characteristics or even exposure of the immersed surfaces to the hydrodynamics found in each one of the three ports (Catão et al., submitted.). Toulon and Reunion sites are semi-enclosed and more protected from marine currents with also higher temperatures, salinity and lead concentrations. On the other hand, Lorient site exhibits fluctuant salinity and dissolved organic carbon as influenced by the mixing of freshwater in the sea, which might lead as well to dilution of metallic contamination [START_REF] Briand | Spatio-temporal variations of marine biofilm communities colonizing artificial substrata including antifouling coatings in contrasted french coastal environments[END_REF]. In this site there was no difference of chao1 richness between PVC and A3, which can be a sign of lower diffusion of copper from A3 surface due to water characteristics, as lower temperature and salinity.

The comparison of communities that colonized a same type of surface in three different sites indicated the presence of deterministic factors (according to niche-based theory) as judged by the greater site-to-site similarity (low β-diversity) [START_REF] Chase | Predators temper the relative importance of stochastic processes in the assembly of prey metacommunities[END_REF]. These factors can be interspecies tradeoffs or competition, and/or the environmental conditions. We showed here that surface characteristics imply selection of certain taxa independently from the site.

Taxonomic indication of competitive fitness over antifouling surfaces

Copper contamination affected microbial community structure in marine sediments [START_REF] Gillan | Structure of sediment-associated microbial communities along a heavy-metal contamination gradient in the marine environment[END_REF], and promoted changes in bacterial community composition and modification of the EPS in in vitro groundwater treatment for Cu removal [START_REF] Vilchez | Dominance of sphingomonads in a copper-exposed biofilm community for groundwater treatment[END_REF].

Flavobacteriaceae seems to be widespread and core species in marine biofilms [START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF][START_REF] Salta | Marine biofilms on artificial surfaces: structure and dynamics[END_REF], with few specific taxa found on antifouling surfaces. Similarly, Planctomycetes, Verrucomicrobia and Saprospiraceae, groups often found in marine biofilms [START_REF] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF][START_REF] Dang | Microbial surface colonization and biofilm development in marine environments[END_REF][START_REF] Oberbeckmann | Microbes on a bottle: substrate, season and geography influence community composition of microbes colonizing marine plastic debris[END_REF][START_REF] Pinto | The composition of bacterial communities associated with plastic biofilms differs between different polymers and stages of biofilm succession[END_REF], were only detected on PVC, which reflects the apparent lack of organisms resistant to metals in these phyla and family. However, counter-examples could be noticed in each of these groups with the genera Aquamarina and Maritimimonas (Flavobacteriaceae), identified as biomarkers on A3, and previously detected on paint particles [START_REF] Tagg | Paint particles are a distinct and variable substrate for marine bacteria[END_REF], Roseovarius (Rhodobacteraceae) or BD7-11 (Planctomycetes), underlining that bioindication remains difficult at high taxonomic levels.

On the other hand, the organisms enriched on A3 and Hy compared to PVC were mainly from α-and γ-Proteobacteria. The genera Altererythrobacter and Erythrobacter were already discriminant for biocidal coatings, including under shear stress in Toulon Bay [START_REF] Dobretsov | Living on the edge: biofilms developing in oscillating environmental conditions[END_REF][START_REF] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF]. However, when comparing the three different coastal waters, we could observe variations with lower values for these genera at Reunion. Furthermore, another Sphingomonadaceae genus was found specific of biocidal surfaces at Lorient -Sphingorhabdus.

These findings are important to highlight the variability of lower rank taxa depending on site, even if site was less relevant than surface type, as shown before between two biocidal coatings immerged in different locations in the Mediterranean Sea [START_REF] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF]. Among γ-Proteobacteria, Alteromonas or Dasania were already reported on AF coatings in dissimilar environments as the Swedish coast [START_REF] Flach | Does antifouling paint select for antibiotic resistance?[END_REF] or the Sea of Oman [START_REF] Dobretsov | Living on the edge: biofilms developing in oscillating environmental conditions[END_REF].

Lastly, in terms of Archaea, sequencing with prokaryotic universal primers [START_REF] Parada | Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples: Primers for marine microbiome studies[END_REF] detected Archaea (Thaumarchaeota) only in one biofilm (0.06 %), and archaeal-specific primers for qPCR [START_REF] Einen | Enumeration of Archaea and Bacteria in seafloor basalt using real-time quantitative PCR and fluorescence microscopy: Enumeration of Archaea and Bacteria in seafloor basalt[END_REF] confirmed little abundances in our marine biofilms (10 to 100 thousands less than Bacteria), without detectable enrichment on metal-releasing coatings whatever the site.

MRG were enriched on A3 and Hy, and varied from site to site

The lack of metals on PVC surface lead to greater cell number (using flow cytometry) and bacterial diversity in the biofilms formed as already noticed at two of the three sites (Toulon and Lorient) with copper releasing coatings (e.g. [START_REF] Camps | Antifouling coatings influence both abundance and community structure of colonizing biofilms: a case study in the Northwestern Mediterranean Sea[END_REF][START_REF] Briand | Spatio-temporal variations of marine biofilm communities colonizing artificial substrata including antifouling coatings in contrasted french coastal environments[END_REF]. However, no significant difference was observed for the copy number of the bacterial 16S rRNA gene on PVC compared to AF coatings, except A3 at Reunion. Three hypotheses can be drawn and contribute together to these results : (i) a lower coverage of the qPCR primers for the communities developed on the PVC, leading to underestimation of the community, (ii) a greater copy number of the 16S rRNA gene within the organisms that were found on A3 and Hy, which would explain the difference found between cytometry and qPCR, or, (iii) the much greater number of eukaryotes on PVC panels (Figure S1) which lead to less efficient prokaryotic DNA amplification by the qPCR primers. However, a higher actual copy number of the bacterial 16S rRNA gene on PVC would induce a lower relative MRG abundance on this coating, amplifying the difference between metalreleasing coatings and PVC.

Specific copper resistance within the marine biofilms was detected for the first time by the relative abundance of three genes, copA, cusA and cueO. They were identified on all the coatings except copA on PVC at Lorient and Reunion, with an expected global increase on copper-releasing surfaces, except at Toulon where PVC showed surprising high abundances of MRG as well.

Certain organisms possess multiple genes to cope with environments rich in Cu. Acidithiobacillus ferrooxidans is an example, portraying cop and cus, and Cu-specific periplasmic chaperones [START_REF] Almárcegui | New copper resistance determinants in the extremophile Acidithiobacillus ferrooxidans : a quantitative proteomic analysis[END_REF]. This differential distribution of MRG according to environment was expected, as found in copper contaminated Chilean marine sediments, with a greater abundance of copA with respect to cusA [START_REF] Besaury | Abundance and diversity of copper resistance genes cusA and copA in microbial communities in relation to the impact of copper on Chilean marine sediments[END_REF] or along an urban stream [START_REF] Roberto | Distribution and cooccurrence of antibiotic and metal resistance genes in biofilms of an anthropogenically impacted stream[END_REF].

Despite the fact that MRG (copA) can be found as well in Crenarchaeota [START_REF] Orell | Molecular characterization of copper and cadmium resistance determinants in the biomining thermoacidophilic archaeon Sulfolobus metallicus[END_REF], archaeal abundance in biofilm community remained low, as already reported [START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF], Catao et al., 2019), limiting the possibility to discuss their role in metal resistance in the biofilm community.

Even though copA gene is the most used to monitor copper resistance, czcA gene was also well correlated with Cu availability in sediments [START_REF] Roosa | Bacterial metal resistance genes and metal bioavailability in contaminated sediments[END_REF]. In addition, even if we focused on copper due to its massive utilization as a biocide for antifouling coatings, it was important in this study to try to cover mechanisms of resistance for additional metals, especially in waters containing high concentrations of other trace metals as Zn or Pb. Interestingly, A3, that included Zn unlike Hy, showed a higher czcA relative ratio than Hy at Lorient and Reunion, consistent with its definition of RND antiporter cation-anion for efflux of Co +2 /Zn +2 /Cd +2 . MRG have been described regarding one or more specific metals. However, these genes can also correlate with non-target metals [START_REF] Roosa | Bacterial metal resistance genes and metal bioavailability in contaminated sediments[END_REF], as the proteins by them encoded can have a broad range of action [START_REF] Conroy | Differing ability to transport nonmetal substrates by two RND-type metal exporters: Substrate specificity of metal RND exporters[END_REF]. Moreover the tolerance may vary depending on the organisms [START_REF] Nishino | Virulence and drug resistance roles of multidrug efflux systems of Salmonella enterica serovar Typhimurium[END_REF] and not only on the metal concentration. pbrT was not specifically enriched on biocidal coatings at Lorient and Reunion, despite dissimilar environmental Pb concentrations in these two sites. This could indicate either a co-selection or a non-specific action for this gene. Hence, quantification of multiple MRG might be used to detect cross-resistance in the selected communities over antifouling surfaces despite the presence of only one or two metals.

Bacteria were reported to carry more than one resistance system in their genomes, and even though most have been described from chromosomes, RND efflux systems were also detected in conjugative plasmids [START_REF] Gutiérrez-Barranquero | Recruitment and rearrangement of three different genetic determinants into a conjugative plasmid increase copper resistance in Pseudomonas syringae[END_REF][START_REF] Norberg | Complete nucleotide sequence and analysis of two conjugative broad host range plasmids from a marine microbial biofilm[END_REF]. This could explain greater numbers of cusA within the described biofilms. A greater abundance of cusA (10x higher) than copA was also observed for intertidal sediments [START_REF] Besaury | Expression of copper-resistance genes in microbial communities under copper stress and oxic/anoxic conditions[END_REF].

In this study, we have compared two antifouling coatings that varied in metal composition and concentration, as well as in solvent base. We assumed that aqueous matrix present in Hy was less prone than the organic one of A3 to select for certain organisms. Therefore, the differential taxa selection in Hy compared to A3, and their different behaviors towards MRG abundance, are most likely related to the greater concentration of Cu used in Hy. This surface had significantly higher copA abundance in Lorient, well correlated to the higher abundance of Marinobacter in Hy surfaces, and Dasania and Aquimarina in Hy at Lorient. Overall, in addition to the possible specific actions of these organisms, the biofilm structure can possibly affect the abundance of MRG, in terms of oxygen availability. As such, greater abundance of copA could be related to its ability to translocate Cu(I) in both aerobic and anaerobic conditions [START_REF] Besaury | Expression of copper-resistance genes in microbial communities under copper stress and oxic/anoxic conditions[END_REF][START_REF] Rensing | Escherichia coli mechanisms of copper homeostasis in a changing environment[END_REF], contrary to cueO, which is unable to oxidize Cu in the absence of O2 [START_REF] Outten | The Independent cue and cus Systems Confer Copper Tolerance during Aerobic and Anaerobic Growth in Escherichia coli[END_REF].

All three sites present coastal waters contaminated by anthropogenic marine activities, leading to the hypothesis that organisms undergo already from a selection for resistance systems to metal contamination. The overall absence of trends in the distribution of MRG between sites on coatings could be related to the similar copper and zinc concentrations, although a lower Pb contamination at Lorient was noticed. This also implies that seawater characteristics that discriminate microbial communities between sites, especially planktonic ones (Catão et al., submitted), did neither trigger the MRG type nor their relative abundances, as was reported for urban stream [START_REF] Roberto | Distribution and cooccurrence of antibiotic and metal resistance genes in biofilms of an anthropogenically impacted stream[END_REF]. Interestingly, the number of all the MRG detected on the communities developed over PVC at Toulon, thus also including czcA and pbrT, was as high as on biocidal coatings. Despite there was not huge phylogenetic differences between sites on this plastic surface, biofilm communities at Toulon appeared highly adapted to metal resistance, probably related to the historical contamination of the Bay and responsible of a chronicle metal exposure history [START_REF] Tessier | Study of the spatial and historical distribution of sediment inorganic contamination in the Toulon bay (France)[END_REF][START_REF] Coclet | Impacts of copper and lead exposure on prokaryotic communities from contaminated contrasted coastal seawaters: the influence of previous metal exposure[END_REF]. In addition, as the immersion site in the bay of Toulon is a semi-enclosed pond, we assume the residence time was much higher than in Lorient or Reunion, which could have also led to an increased adaptation of the whole community.

This could also be related to a differential adsorption of metals onto the PVC surface [START_REF] Brennecke | Microplastics as vector for heavy metal contamination from the marine environment[END_REF] which would vary depending on site because dissimilar environmental conditions lead to different organic matter and consequently distinct surface conditioning. Additionally, we cannot exclude that similar communities can produced dissimilar EPS, also depending on environmental parameters, which would possess higher metal binding capacity at Toulon. Finally, site specific taxa could further synthesize particular EPS maximizing metal binding at Toulon.

Sphingomonadaceae or Alteromonadales are selected in metals-rich environments

Primers targeting functional genes are prone to bias for certain taxonomic groups. In sediments, sequenced copA genes were found only in β-Proteobacteria, and czcA covered this class as well as α-, γ-and δ-Proteobacteria, and Cyanobacteria [START_REF] Roosa | Bacterial metal resistance genes and metal bioavailability in contaminated sediments[END_REF]. Here, most organisms presenting resistance to Cu contamination, measured by the abundance of copA and czcA, belonged to Proteobacteria. Others have also detected increased abundance of γ-Proteobacteria in biofilms on antifouling surfaces [START_REF] Flach | Does antifouling paint select for antibiotic resistance?[END_REF], class where we can find cultured organisms possessing metal RND efflux systems [START_REF] Conroy | Differing ability to transport nonmetal substrates by two RND-type metal exporters: Substrate specificity of metal RND exporters[END_REF].

Within the α-Proteobacteria, Sphingomonadaceae organisms have been described more than once from copper contaminated environments [START_REF] Balkwill | Sphingomonas and Related Genera[END_REF][START_REF] Vilchez | Dominance of sphingomonads in a copper-exposed biofilm community for groundwater treatment[END_REF].

Biofilms rich in Sphingomonas (78% relative abundance) were observed as dense aggregates induced by the presence of Cu(II), suggesting EPS as the main form of cellular detoxification [START_REF] Vilchez | Dominance of sphingomonads in a copper-exposed biofilm community for groundwater treatment[END_REF]. The ability of Sphingomonads to form biofilms is most likely related to their production of abundant EPS [START_REF] Balkwill | Sphingomonas and Related Genera[END_REF] as well as their mobility, hydrophobic cell surface due to glycosphingolipids and their ability to metabolize several substrates [START_REF] De Vries | Isolation and characterization of Sphingomonadaceae from fouled membranes[END_REF]. Most specifically, Erythrobacter was found colonizing several types of microplastics immersed in an environmental gradient [START_REF] Oberbeckmann | Environmental factors support the formation of specific bacterial assemblages on microplastics[END_REF] and was discriminant of other biocidal coatings under increased shear stress [START_REF] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF]. Besides Erythrobacter, the Rhodobacteraceae Roseovarius, here shown to be correlated with the czcA gene, was also detected as one of the most abundant genera in biofilms formed on zinc-coated surfaces [START_REF] Ding | Metagenomic analysis of zinc surface-associated marine biofilms[END_REF].

The same study has detected an increase in KEGG protein families for transposase and MRG in those biofilms compared to seawater microbial communities.

Similarly to Sphingomonadaceae, the resistance of the γ-Proteobacteria Marinobacter to Cu toxicity has been linked to its sorption by the EPS [START_REF] Bhaskar | Bacterial extracellular polymeric substance (EPS): A carrier of heavy metals in the marine food-chain[END_REF]. Moreover, Alteromonas and Alteromonadaceae have been shown to be early colonizers of Cu-based surface [START_REF] Catão | Shear stress as a major driver of marine biofilm communities in the NW Mediterranean Sea[END_REF][START_REF] Chen | Early marine bacterial biofilm on a copper-based antifouling paint[END_REF], but also on PVC [START_REF] Pollet | Prokaryotic community successions and interactions in marine biofilms: the key role of Flavobacteriia[END_REF]. Two strains were recently reported to possess multiple megaplasmids with MRG including Cop, Cus and Cue sytems [START_REF] Cusick | Multiple megaplasmids confer extremely high levels of metal tolerance in Alteromonas strains[END_REF]. That family were finally found as indicators of tolerance as they were positively correlated with increasing Cu concentrations in biofilms formed in microscosms with sea water [START_REF] Corcoll | Copper affects composition and functioning of microbial communities in marine biofilms at environmentally relevant concentrations[END_REF].

Conclusions

Our study allowed the detection of specific taxa that are present on metal-releasing surfaces independently from the site, but depending on metallic biocide composition. Surface composition had great influence on cell density, bacterial diversity and composition as well as on relative abundance of MRG. This evaluation performed on biofilms grown in temperate and tropical contaminated waters, provided evidences that cell tolerance/resistance to metals may be based on several MRG with possible co-selection and not specific mechanisms. Furthermore, local contamination history may influence the basal level of MRG enrichment in biofilm community.

  and S6). Both the genera Erythrobacter and Sphingorhabdus had greater fold change on A3 when compared to PVC. Some other genera among Bacteroidetes showed greater fold change on Hy as Fabibacter, Psychroflexus and Owenweeksia. Despite greater proportion of Rhodobacteriaceae in PVC, the genus Roseovarius was more present on A3. Similarly, Aquimarina and Dokdonia were specific of A3 within the Flavobacteriaceae. Furthermore, Micavibrionaceae, Dasania (Spongiibacteraceae), Litorimonas (Hyphomonadaceae) were characteristic of A3 (compared to either Hy or PVC). Taken together, biocidal surfaces have selected Sphingomonadaceae, Caedibacteraceae, Peredibacter and Dasania.

  but to a lesser extent with this gene, namely Fabibacter (ρ = 0.50, p-value < 0.01, n=27) and Owenweeksia (ρ = 0.49, p-value = 0.01, n=27). czcAmainly detected on A3exhibited a correlation with Roseovarius (ρ = 0.61, p-value < 0.001, n=27), OM60_NOR5 clade (ρ = 0.60, pvalue < 0.01, n=27) and BD1-7 clade (ρ = 0.60, p-value < 0.01, n=27). Even though with a lower coefficient, czcA was the only gene to correlate with Altererythrobacter (ρ = 0.41, p-value < 0.05, n=27).pbrT correlated with genera present on A3, either exclusively or in common with Hy and PVC. Noteworthy, Sphingorhabdus presented a correlation of 0.53 (p-value < 0.01) and Dasania of 0.49 (p-value = 0.01). As a matter of fact, Dasania correlated positively with all MRG except cueO. This latter gene correlated with few genera, all discriminant of A3 or A3 and PVC, namely two from the most abundant families Flavobacteriaceae and Rhodobacteraceae -Maritimimonas and Roseobacter cladeNAC11 (both, ρ = 0.55, p-value < 0.01, n=27).

Figure 1 .

 1 Figure 1. Principal Coordinates Analysis (PCoA) analysis on weighted Unifrac distances within bacterial communities. Colors correspond to the three surfaces and shapes to the three sites.

Figure 2 .

 2 Figure 2. Metacoder heatmap plotted to genus level where each connected node represents a different taxonomic rank (Domain to genus from center to outwards, respectively). Nodes' size isproportional to the number of ASVs that constitute that taxonomic level. Nodes were colored according to Log2 ratio of mean proportion of each rang between A3 or Hy surfaces against PVC as control surface. Sites were not distinguished. Due to PVC higher richness, taxa that were present only on PVC were filtered out before graphical display.

Figure 3 .

 3 Figure 3. Quantification of metal resistance genes in the sampled biofilms over the three surfaces and in the three sites. Ratios were calculated with each MRG over the sum of archaeal and bacterial 16S rRNA genes abundance. Whiskers represent the smallest and the largest values. One-way ANOVA analysis was used to detect differences between sites or between surfaces, indicated respectively by upper and lower letters (Tuckey's HSD posthoc groupings, p-value<0.05).

Figure 4 .

 4 Figure 4. Lefse discriminants (LDA>2) Spearman correlation with genes abundance (gene/16S rRNA). Dotplot corresponding to the site & surface combination for which the genera were found discriminant. Heatmap of only positive correlations (with p-value < 0.05) between those genera and the MRG. Genera are ordered according to the surface(s) for which they were detected as discriminant.

  Figure1

  Figure2

Figure 4

 4 Figure 4

Acknowledgments

We thank Thomas POLLET and Raphaëlle BARRY-MARTINET (MAPIEM, UTLN), Dalyal COPIN (LBCM, UBS), Alina TUNIN-LEY and Jean TURQUET (CITEB / c/o CYROI, Sainte Clotilde, La Réunion) for their contributions to the immersion experiments at Toulon, Lorient and La Réunion, respectively. The authors also thank Christine BRESSY (MAPIEM, UTLN) for her contribution to the choice and application of AF coatings and helpful discussions. Finally, we thank Maxime Delbury of Nautix company for providing A3 coating for this study. This work was financed by the ANR project BIOPAINTROP.