Dr François Lecoquierre
email: francois.lecoquierre@chu-rouen.fr

Kévin Cassinari

Pascal Chambon

Gaël Nicolas

Sarah Malsa

Régine Marlin

Yvon Assouline

Jean-François Fléjou

Thierry Frebourg

Claude Houdayer

Odile Bera

Stéphanie Baert-Desurmont

Patients with 10q22.3q23.1 recurrent deletion syndrome are at risk for juvenile polyposis AUTHORS

Juvenile polyposis syndrome (JPS) is a rare autosomal dominant predisposition to hamartomatous polyps within the gastrointestinal tract, at high risk for malignant transformation. BMPR1A and SMAD4 loss-of-function variants account for 50% of the cases.

More specifically, point mutations and structural abnormalities in BMPR1A lead to a highly

INTRODUCTION

Juvenile polyps are a rare histologic type of digestive hamartoma. They can occur anywhere in the gastro-intestinal tract mucosa from the stomach to the rectum and are isolated in most of the affected individuals [START_REF] Jelsig | Juvenile Polyps in Denmark From 1995 to 2014[END_REF]. The association of multiple juvenile polyps (typically more than five) defines juvenile polyposis syndrome (JPS). Juvenile polyps can cause bleeding and anemia, and are at risk for malignant transformation, particularly in JPS, positioning JPS as a digestive cancer predisposition syndrome. Unlike isolated juvenile polyps, JPS often results from germline heterozygous genetic variants leading to autosomal dominant transmissions of the disease in families. Two genes have been associated with JPS: SMAD4 and BMPR1A. Both genes combined account for about half of the JPS cases [START_REF] Aretz | High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome[END_REF]. Similar to several other predispositions to digestive polyps and cancer, pathogenic variants act via a loss-of-function (LOF) mechanism, and a second hit in the wild-type allele can occasionally be found within the polyps [START_REF] Blatter | Somatic alterations in juvenile polyps from BMPR1A and SMAD4 mutation carriers[END_REF]. Both sequence variants and copy number variations leading to loss-of-function alleles have been reported. It is well established that the expression of the disease is highly heterogeneous in affected individuals, in terms of age at diagnosis, symptoms and number of polyps. Despite BMPR1A haploinsufficiency being known as a cause of JPS, the penetrance of juvenile polyps in patients with large deletions including BMPR1A has been questioned. Notably, in the 7.3Mb 10q22.3q23.1 recurrent deletion syndrome, reported in 15 families within the literature and associated with a syndromic developmental disorder of variable severity, JPS hasn't been described in any patient. Here, we present a patient harboring this recurrent deletion, who developed a severe juvenile polyposis syndrome at the age of 25. We then discuss the seemingly low penetrance of JPS in pediatric patients with 10q22.3q23.1 deletion in the literature.

CLINICAL REPORT

Clinical description

We report a 32 year-old female patient presenting with the association of polyposis coli and a developmental disorder. No known history of either developmental abnormalities or digestive tumors was identified in her family. She was born from healthy nonconsanguineous parents, after a full-term uneventful pregnancy. Clinical examination at birth revealed a systolic heart murmur due to a ventricular septal defect. Pediatric follow-up showed delayed psychomotor acquisitions, as well as facial dysmorphic features and convergent strabismus. At the age of 25, diffuse digestive polyposis was diagnosed in a context of iron deficiency anemia associated with digestive pain and diarrhea. Gastroscopy revealed more than 30 polyps ranging from 5 to 20 mm. Colonoscopy revealed 25 sessile or pedunculated polyps ranging from 3 to 15 mm in the rectum and the sigmoid colon, and one 30 mm pedunculated polyp in the left colonic angle. A few polyps were collected and their anatomopathological examination at this point was in favor of adenomas. Therefore, coloproctectomy was performed in order to control the polyposis. Anatomopathological analysis of the excised colic tissues showed no adenomas but refined the diagnosis to juvenile polyps. Follow-up of gastric lesions also identified hamartomas compatible with juvenile polyps. Patient was lost to follow-up for 13 months. She developed a gastric cancer, with peritoneal carcinomatosis at the time of diagnosis, from which she died at the age of 32.

Molecular analysis

After obtaining informed written consent from the patient's legal guardian, we included this patient in our in-house workflow for the molecular analysis of mendelian predisposition to digestive polyposis and colorectal cancer, as recently described [START_REF] Baert-Desurmont | Optimization of the diagnosis of inherited colorectal cancer using NGS and capture of exonic and intronic sequences of panel genes[END_REF]. Briefly, sequencing of BMPR1A as well as 9 other genes was performed by targeted NGS on DNA extracted from whole blood sample. Simultaneously to single nucleotide variants (SNVs) detection, we performed copy number variants (CNVs) detection with the CANOES software, which is based on read depth profile comparison to a set of control samples [START_REF] Backenroth | CANOES: detecting rare copy number variants from whole exome sequencing data[END_REF]. This approach showed a complete heterozygous deletion of the BMPR1A gene. PTEN, which is also located in chr10q23.2, was not involved in this deletion according to CANOES. The deletion was validated by Multiplex Ligation Probe Analysis (MLPA) in the proband. Segregation analysis showed that it was not inherited from the asymptomatic mother, but no sample was available from the father. We hypothesized that a contiguous gene syndrome could explain both the juvenile polyposis and the developmental disorder presented by this patient. We therefore refined the CNV boundaries by array-CGH. Agilent SurePrint 4x180k microarray kit was used with standard protocol.

Deletion of the whole BMPR1A gene was confirmed by the identification of a deletion encompassing chr10(hg19):81651077-88906961 (Fig. 1). The deleterious nature of the deletion regarding the digestive phenotype was clear since heterozygous loss-of-function mutations in BMPR1A, either by point mutations or whole gene deletions, are known to cause juvenile polyposis syndrome [START_REF] Aretz | High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome[END_REF][START_REF] Howe | Germline mutations of the gene encoding bone morphogenetic protein receptor 1A in juvenile polyposis[END_REF]. This deletion was not present in general population databases such as the Database of Genomic Variants (DGV) or the Exome Aggregation Consortium (ExAC). On the other hand, twenty similar deletions were reported in the Decipher database (https://decipher.sanger.ac.uk/, lastly assessed 2019-04-23), which gathers CNV data from patients with presumed genetic affections and mostly developmental disorders. Most of the Decipher cases in which parental information was available occurred de novo or from an affected parent (Supplementary Table 1). The clinical phenotype of these patients, as well as other patients from the literature, is presented in the discussion. The large size of the deletion (7.3Mb), its absence in control population databases and its apparent enrichment in patients with developmental disorders allowed us to consider this deletion as pathogenic with full contribution to the phenotype, both regarding the digestive and the extra digestive presentation of the proband.

DISCUSSION

We identified a deletion of BMPR1A in our proband, in a context of severe gastric and colonic juvenile polyposis, which led to the diagnosis of 10q22q23 recurrent deletion. Of note, it has been observed that gastric polyps were rare in juvenile polyposis caused by alterations in BMPR1A [START_REF] Aretz | High proportion of large genomic deletions and a genotype phenotype update in 80 unrelated families with juvenile polyposis syndrome[END_REF].

Clinical phenotype associated to 10q22q23 deletion

We systematically reviewed the phenotype of patients harboring a similar 10q22.3q23.1 deletion in both Decipher database (20 patients, Supplementary table 1) and in the literature (15 families, Table 1). Various degree of phenotypic information was available for 12/20 patients within the Decipher database. The most recurrent consistent features included global developmental delay or intellectual disability, delayed language, behavioral abnormalities and facial dysmorphic features. Some of the less commonly reported features seen in more than one proband include talipes equinovarus, generalized hypotonia and teeth abnormalities. Among the twelve patients with available clinical information, digestive polyposis was mentioned in one single patient. The literature has been recently reviewed [START_REF] Coelho Molck | A New Case of the Rare 10q22.3q23.2 Microdeletion Flanked by Low-Copy Repeats 3/4[END_REF] and comprises 25 patients from 15 families described in 7 studies [START_REF] Alliman | Clinical and molecular characterization of individuals with recurrent genomic disorder at 10q22.3q23.2[END_REF][START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF][START_REF] Coelho Molck | A New Case of the Rare 10q22.3q23.2 Microdeletion Flanked by Low-Copy Repeats 3/4[END_REF][START_REF] Petrova | A Boy with an LCR3/4-Flanked 10q22.3q23.2 Microdeletion and Uncommon Phenotypic Features[END_REF][START_REF] Reddy | Oligoarray (105K) CGH analysis of chromosome microdeletions within 10q22.1q24.32[END_REF][START_REF] Singh | Interstitial deletion of 10q23.1 and confirmation of three 10qdel syndromes[END_REF][START_REF] Van Bon | The phenotype of recurrent 10q22q23 deletions and duplications[END_REF], including one family with 11 individuals [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF]. Strikingly, none of these patients had had a diagnosis of JPS or even juvenile polyps (Table 1).

Mechanism and candidate genes for the non-digestive abnormalities

Recurrence of deletions with the same boundaries is explained by the mechanism of nonallelic homologous recombination (NAHR), mediated by low copy repeats (LCRs), as seen on the "Segmental Dups." track in Figure 1B. Intriguingly, the amount of reported deletions with boundaries within these LCRs is quite low compared to other NAHR mediated recurrent syndromes. Indeed, Van Bon et al. evaluated its prevalence to 0.016% within their cohort of intellectual disability/multiple congenital abnormality [START_REF] Van Bon | The phenotype of recurrent 10q22q23 deletions and duplications[END_REF], which seems to be much less frequent than other recurrent deletions. It has been proposed that the large genomic distance between the two LCRs was responsible for this quite infrequent rearrangement event. The deletion encompasses 47 genes, including 7 genes flagged by OMIM as involved in Mendelian diseases: ANXA11, MAT1A, CDHR1, RGR, LDB3, BMPR1A and GLUD1. However, besides BMPR1A, none of these genes is known to cause a disease by a heterozygous loss-of-function mechanism. The gene or the minimal region involved in the neurodevelopmental abnormalities in this contiguous gene syndrome is still unknown. Two genes appear to be good candidates since they reach the statistical significance of loss-offunction intolerance as defined by ExAC pLI (probability of loss-of-function intolerance) [START_REF] Lek | Analysis of protein-coding genetic variation in 60,706 humans[END_REF]. The first one, GRID1, is mainly expressed in the brain and encodes an ionotropic glutamate receptor subunit that has been associated to schizophrenia by GWAS analysis [START_REF] Treutlein | Dissection of phenotype reveals possible association between schizophrenia and Glutamate Receptor Delta 1 (GRID1) gene promoter[END_REF]. A small number of truncating variants and deletions are present in population databases (ExAC and DGV, respectively). The second one, WAPAL, is ubiquitously expressed, with a central role in chromatin regulation via cohesin positioning on the genome [START_REF] Busslinger | Cohesin is positioned in mammalian genomes by transcription, CTCF and Wapl[END_REF]. WAPAL is drastically depleted in truncating variants in ExAC despites its high predicted mutability and no deletion of this gene are reported in DGV, suggesting that individuals with loss of function alleles are not included in these control populations, potentially due to a severe pediatric disorder. In addition to our patient, several patients from the literature had heart malformations, notably ventricular and atrioventricular septal defect, patent ductus arteriosus and valvular defects. The association of cardiac abnormalities and 10q22q23 deletions had already been observed, and 2 candidate genes have been proposed. Breckpot et al. proposed the implication of BMPR1A since they identified a de novo intragenic deletion of BMPR1A in a patient with atrioventricular septal defect, short stature, delayed puberty and facial dysmorphic features [START_REF] Breckpot | BMPR1A is a candidate gene for congenital heart defects associated with the recurrent 10q22q23 deletion syndrome[END_REF].

However, this clinical presentation does not match JPS caused by loss-of-function variants of BMPR1A, questioning the implication of BMPR1A heterozygous loss-of-function variants in the cardiac phenotype. GRID1 has been pointed out as another candidate, as Van Bon et al.

identified an intragenic GRID1 deletion in a patient with atrioventricular septal defect [START_REF] Van Bon | The phenotype of recurrent 10q22q23 deletions and duplications[END_REF]). Yet the inheritance of this CNV was unknown, and the deletion was predicted to potentially result in an in-frame exon skipping. Also, the expression of GRID1 is very low in the heart according to the GTEx database. Thus, there is no certainty about the mechanism underlying heart defects in 10q22q23 deletions.

The risk of JPS in 10q22.3q23.1 deletion

The risk of juvenile polyposis in 10q22.3q23.1 deletion has been debated. The implication of BMPR1A has made several authors anticipate the risk of JPS in their patients [START_REF] Alliman | Clinical and molecular characterization of individuals with recurrent genomic disorder at 10q22.3q23.2[END_REF][START_REF] Petrova | A Boy with an LCR3/4-Flanked 10q22.3q23.2 Microdeletion and Uncommon Phenotypic Features[END_REF][START_REF] Reddy | Oligoarray (105K) CGH analysis of chromosome microdeletions within 10q22.1q24.32[END_REF][START_REF] Singh | Interstitial deletion of 10q23.1 and confirmation of three 10qdel syndromes[END_REF], but the observation of juvenile polyps in carriers had still never been described in the literature [START_REF] Petrova | A Boy with an LCR3/4-Flanked 10q22.3q23.2 Microdeletion and Uncommon Phenotypic Features[END_REF]. We can hypothesize that most patients are currently diagnosed at pediatric age in the context of genetics investigations of a developmental disorder, and that they might be too young to be symptomatic regarding the digestive presentation. Indeed, most patients in the literature have been evaluated at less than 6 years (Table 1) while the median age at diagnostic in BMPR1A related juvenile polyposis has been shown to be 18 year-old [START_REF] Aytac | Genotype-defined cancer risk in juvenile polyposis syndrome[END_REF], but some polyps are sometimes diagnosed within the first decade [START_REF] Lieberman | Variable Features of Juvenile Polyposis Syndrome With Gastric Involvement Among Patients With a Large Genomic Deletion of BMPR1A[END_REF]. However, Balciuniene et al. described a three-generation family with numerous individuals carrying the deletion, none of whom being described as with digestive symptoms [START_REF] Balciuniene | Recurrent 10q22-q23 deletions: a genomic disorder on 10q associated with cognitive and behavioral abnormalities[END_REF], suggesting an incomplete penetrance for polyposis. Alliman et al. described a patient carrying a similar deletion, in whom colonoscopy was performed at the age of 17 years 10 month, revealing no polyps. Another patient from the same study underwent esophagogastroduodenoscopy at age 4 months, which was also normal (Table 1).

Apart from these two cases, the other reports do not mention any invasive screening for digestive polyps. It is possible that some patients may not have been actively investigated, while some others might have been followed after the initial publication. The identification of another closely related contiguous gene deletion syndrome including BMPR1A but also PTEN in individuals with severe forms of juvenile polyposis occurring in young infancy [START_REF] Delnatte | Contiguous gene deletion within chromosome arm 10q is associated with juvenile polyposis of infancy, reflecting cooperation between the BMPR1A and PTEN tumor-suppressor genes[END_REF], together with the apparent absence of digestive symptoms in children with the recurrent deletion of BMPR1A but not PTEN, have led some to suggest the requirement of a contiguous deletion of both genes for polyposis to take place [START_REF] Dahdaleh | Juvenile polyposis and other intestinal polyposis syndromes with microdeletions of chromosome 10q22-23[END_REF]. However, both the present case report (and the additional patient within the Decipher database) and the clear implication of BMPR1A null variants as a major cause of JPS argues that patients with 10q22.3q23.1 recurrent deletion that do not include PTEN are at substantial risk for JPS. We propose that the risk of digestive symptoms of these two entities should be stratified. On the one hand, deletions encompassing both genes might lead to a severe pediatric JPS; on the other hand, individuals harboring 10q22.3q23.1 recurrent deletion encompassing BMPR1A but not PTEN are at risk of developing JPS in young adulthood, similar to patients with pathogenic point mutations and structural variants restricted to BMPR1A. A recent study provides evidence for a high penetrance of JPS in deletions of the BMPR1A gene [START_REF] Lieberman | Variable Features of Juvenile Polyposis Syndrome With Gastric Involvement Among Patients With a Large Genomic Deletion of BMPR1A[END_REF]. In this study, the authors investigated the clinical consequences of a founder 429 kb deletion encompassing the whole coding sequence of BMPR1A in more than 50 individuals from 7 families from of a Bukharin Jewish origin. This analysis showed a full penetrance of polyps in adults, with a high degree of intra-and inter-familial nonallelic variability.

The risk of developing a colorectal cancer in patients with JPS has been estimated between 38% and 68% [START_REF] Brosens | Risk of colorectal cancer in juvenile polyposis[END_REF][START_REF] Campos | Colorectal cancer risk in hamartomatous polyposis syndromes[END_REF], and the risk of gastric and duodenal tumors between 10% and 20% [START_REF] Dunlop | Guidance on gastrointestinal surveillance for hereditary non-polyposis colorectal cancer, familial adenomatous polypolis, juvenile polyposis, and Peutz-Jeghers syndrome[END_REF]. We therefore strongly recommend the implementation of an active digestive surveillance in patients harboring 10q22.3q23.1 deletion. The surveillance protocol may be based on the existing recommendation established in JPS [START_REF] Achatz | Cancer Screening Recommendations and Clinical Management of Inherited Gastrointestinal Cancer Syndromes in Childhood[END_REF][START_REF] Syngal | ACG clinical guideline: Genetic testing and management of hereditary gastrointestinal cancer syndromes[END_REF] including, starting from 15 yearold: (i) a colonoscopy performed annually and deferred up to 3 years if no colonic polyps are found, (ii) upper gastrointestinal endoscopy and (iii) capsule endoscopy for the surveillance of the small bowel.

CONCLUSIONS

We present the first diagnosis of juvenile polyposis syndrome in a patient harboring recurrent 10q22.3q23.1 deletion. Although the penetrance of JPS in 10q22.3q23.1 deletion in still unclear, the greatest caution is required in patients harboring this recurrent CNV, which should trigger an active digestive surveillance.

FIGURES


Alyssa Azaroff

Marisol Goñi

Claire Gassie

Mathilde Monperrus

Remy Guyoneaud
email: remy.guyoneaud@univ-pau.fr

Marisol Goñi Urriza

Rémy Guyoneaud

methylating microbial communities from coastal to Capbreton Canyon sediments (North Atlantic Ocean)

Keywords: Mercury-methylation, Hg-methylating prokaryotes, 16S rDNA diversity, HgcA diversity, marine sediments

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

Introduction

Methylmercury (MeHg) is a neurotoxin that is biomagnified in aquatic food webs, with fish consumption as a primary route for human MeHg exposure [START_REF] Mason | Mercury biogeochemical cycling in the ocean and[END_REF][START_REF] Sunderland | Mercury Exposure from Domestic and Imported Estuarine and Marine Fish in the U.S. Seafood Market[END_REF]. In aquatic ecosystems, methylation of mercury (Hg) to MeHg is carried out by some anaerobic microorganisms. Studies have identified the sulfate-reducing bacteria (SRB) as the main contributors to Hg methylation [START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment[END_REF]. MeHg production has also been associated with the activity of sulfur-reducing bacteria, iron-reducing bacteria (FeRB) and methanogenic archaea [START_REF] Fleming | Mercury Methylation from Unexpected Sources: Molybdate-Inhibited Freshwater Sediments and an Iron-Reducing Bacterium[END_REF][START_REF] Hamelin | Methanogens: principal methylators of mercury in lake periphyton[END_REF][START_REF] Ranchou-Peyruse | Overview of Mercury Methylation Capacities among Anaerobic Bacteria Including Representatives of the Sulphate-Reducers: Implications for Environmental Studies[END_REF]. The recent identification of some genes required for Hg methylation, hgcA and hgcB [START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF], expanded our knowledge on the diversity of potential Hg methylators [START_REF] Gilmour | Mercury Methylation by Novel Microorganisms from New Environments[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. hgcAB genes are present in the genome of diverse sulfate, sulfur and iron reducing Deltaproteobacteria, in methanogens, but also in Clostridia (firmicutes), fermentative and acetogenic microorganisms [START_REF] Gilmour | Robust Mercury Methylation across Diverse Methanogenic Archaea[END_REF][START_REF] Gilmour | Mercury Methylation by Novel Microorganisms from New Environments[END_REF][START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF][START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. Metagenomic data suggested the presence of hgcAB in members of others microbial phyla including the Chloroflexi, Chrysiogenetes, Nitrospina, the PVC superphylum (Planctomycetes, Verrucomicrobia, Chlamydia) and Spirochaetes [START_REF] Gionfriddo | Microbial mercury methylation in Antarctic sea ice[END_REF][START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF][START_REF] Mcdaniel | Expanded Phylogenetic Diversity and Metabolic Flexibility of Microbial Mercury Methylation[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. Microbial Hg-methylating communities that have recently been described in wetlands, paddy soils, lakes, dam reservoir and sewage treatment plant effluent included methanogens, SRB, FeRB, syntrophs and also unaffiliated microorganisms [START_REF] Bravo | Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases[END_REF][START_REF] Du | Mercurymethylating genes dsrB and hgcA in soils/sediments of the Three Gorges Reservoir[END_REF][START_REF] Liu | Analysis of the Microbial Community Structure by Monitoring an Hg Methylation Gene (hgcA) in Paddy Soils along an Hg Gradient[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF][START_REF] Schaefer | Detection of a key Hg methylation gene, hgcA, in wetland soils[END_REF][START_REF] Xu | Mercury methylating microbial communities of boreal forest soils[END_REF]. Hg-methylating microbial communities in marine environment remain largely un-investigated with regards to their importance for methylation and the specific types of microorganisms involved.

The biogeochemical cycling of Hg remains understudied in the open ocean, particularly on continental margins and associated slopes [START_REF] Mason | Mercury biogeochemical cycling in the ocean and[END_REF]. Submarine canyons are large incisions into the continental margin which contribute to the enrichment of the sediments with organic matter (OM) and various trace metals [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF][START_REF] Oliveira | Heavy metal distribution in surface sediments from the continental shelf adjacent to Nazaré canyon[END_REF][START_REF] Palanques | Anthropogenic trace metals in the sedimentary record of the Llobregat continental shelf and adjacent Foix Submarine Canyon (northwestern Mediterranean)[END_REF]. Indeed, a previous study on the Capbreton Canyon (North Atlantic Ocean) has shown that the MeHg formation is driven by the OM and the sulfur content [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. Comparatively to offshore canyon sediment, coastal sediment had the highest biological Hg methylation rates whereas ambient mercury compounds concentrations were lower [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. Currently, no data exist about the microbial populations carrying hgcA in submarine canyons or even in marine sediments.

Knowledge of the relationship between community composition and MeHg production may help to elucidate the origins of the large variability in MeHg/Hg ratios in oceanic ecosystem.

Building on these earlier discoveries and recognizing the implication of microbial communities on Hg methylation in Capbreton submarine canyon sediments, this study aimed to provide a mechanistic understanding of MeHg formation in sediments along the canyon transect. For this purpose, we linked sediment geochemistry and Hg speciation with parallel analysis of the global prokaryotic diversity (16S rDNA) and microorganisms carrying hgcA. This is the first study in marine sediments, from coastal to canyon samples exploring the diversity of microorganisms involved in Hg methylation.

Materials and methods

Study area and sampling strategy

The present study was undertaken in one of the longest canyons in the world, located in the North Atlantic Ocean: the Capbreton submarine canyon. It deeply incises the Aquitaine continental slope and shelf, and runs along the Spanish coast for 300 km and sinks into the abyssal plain at 3500 meters depth. Its head is at only 200 meters from the coast, where the population and human activities, are constantly increasing. Like many coastal ecosystems, it is a highly productive and ecologically rich zone due to large and continuous continental inputs of nutrients and organic matter coming from a rich network of effluents and the Adour estuary. Surface sediments were collected with a grab sampler and a core sampler along the canyon transect starting at 1.2 km from the coast to 23.5 km. Samples were located in slopes and terraces into the canyon (n=13) and in the adjacent continental shelf (n=9; Fig. 1). G03 is considered a canyon location because it is located in a gully connected to the mouth of this canyon (Mazières et al., 2014). For each sediment sample, Hg compounds analysis (Hg(II) and MeHg) geochemical parameters as well as 16S rDNA diversity were analysed. hgcA diversity has been determined along the canyon transect at locations G03, G10, G14 and G24. On board, samples for DNA were collected in triplicate with sterile cryotubes then immediately frozen at -80 °C until analysis.

Chemical analysis

Sample processing, geochemical parameters (organic carbon (POC), total sulfur (TS)) and speciation of mercury (Hg(II) and MeHg) as well as the Hg transformation potential are detailed in a previous study [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF].

DNA extraction and bacterial community composition: 16S rDNA gene

DNA was extracted from frozen sediments with the Power Soil DNA extraction kit (Mo-Bio Laboratories, Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Diversity of the 16S rDNA were determined by sequencing the V4-V5 hypervariable regions of the 16S rDNA with universal primers V4-515F (5' GTGYCAGCMGCCGCGGTA 3') and V5-928R (5′-ACTYAAAKGAATTGRCGGGG 3') [START_REF] Parada | Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples[END_REF][START_REF] Walters | Improved Bacterial 16S rRNA Gene (V4 and V4-5) and Fungal Internal Transcribed Spacer Marker Gene Primers for Microbial Community Surveys[END_REF][START_REF] Wang | Conservative Fragments in Bacterial 16S rRNA Genes and Primer Design for 16S Ribosomal DNA Amplicons in Metagenomic Studies[END_REF]. PCR was performed using ampliTaq Gold® 360 master mix (Applied Biosystems, CA, USA), 0.5 μM of each primer and 3 ng of extracted DNA. PCR cycling was as following: after 10 min of initial denaturation at 95 °C, 30 cycles of 30 s denaturation at 95 °C, 30 s annealing at 60 °C and 40 s elongation at 72 °C with 7 min final elongation at 72 °C. Amplicons were sequenced by the Get-PlaGe sequencing service (INRA, Toulouse, France) using MiSeq 250 pb paired-end technology and reagent kit V3. Data were preprocessed using the Galaxy FROGS pipeline [START_REF] Escudié | FROGS: Find, Rapidly, OTUs with Galaxy Solution[END_REF]. Chimeraic and PhiX reads were removed, Operational Taxonomic Units (OTUs) clustering, after a de-noising step allows building fine clusters with minimal differences, with an aggregation distance equal or above 3. Data were normalized to the minimum number of reads. Taxonomic assignments were performed using the Silva database v.128 [START_REF] Pruesse | SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB[END_REF]. Sequences data have been deposited in GenBank under the accession number PRJNA608532.

Hg methylation community composition: hgcA gene cloning and sequencing hgcA gene diversity was studied in four surface sediments along the canyon gradient from the coast to the offshore (G03, G10, G14 and G24). Primers targeting hgcAB sequences were adopted from [START_REF] Christensen | Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment[END_REF][START_REF] Christensen | Development and Validation of Broad-Range Qualitative and Clade-Specific Quantitative Molecular Probes for Assessing Mercury Methylation in the Environment[END_REF] and hgcAB genes were amplified with the primer set ORNL-HgcAB-uni 268F (5′-AAYGTCTGGTGYGCNGVCGG-3′) and ORNL-HgcAB-uni 167R (5′-CABGCNCCRCAYTCCATRCA-3′). PCR was performed using ampliTaq Gold® 360 master mix (Applied Biosystems, CA, USA) and 1 μM of each primer, with 2 min initial denaturation at 95 °C, 5 cycles of 30 s denaturation at 96 °C, 30 s annealing at 68 °C and 30 s elongation at 72 °C, then 30 cycles of 30 s denaturation at 96 °C, 30 s annealing at 63 °C and 1 min elongation at 72 °C with 7 min final elongation at 72 °C. PCR products were purified using GE Healthcare illustra™ GFX™ PCR DNA and Gel Band Purification Kit (GE Healthcare, Chicago, Illinois, US) according to the manufacturer's instructions. Purified products were ligated into a pCR TM 2.1-TOPO®, with TOPO TM TA Cloning TM Kit (Invitrogen, Carlsbad, CA, US), which was used to transform One Shot™ Mach1™ T1 Phage-Resistant Chemically Competent E. coli. Transformed E. coli were grown on lysogeny broth (LB) agar plates with 100 μg mL -1 ampicillin, 64 µg ml -1 X-gal and 160 mM IPTG. Successful transformants picked and screened by colony PCR with M13 primers (M13F; 5'GTAAAACGACGGCCAG-3' and M13R; 5'-CAGGAAACAGCTATGAC-3'). PCR was performed using ampliTaq Gold® 360 master mix (Applied Biosystems, CA, USA) and 0.2 µM of each primer, with 10 min initial denaturation at 95 °C, 30 cycles of 30 s denaturation at 95 °C, 30 s annealing at 54 °C and 40 s elongation at 72 °C with 10 min final elongation at 72 °C. Clones of the correct sized inserts were sequenced by SANGER sequencing at Eurofins Genomics (Ebersberg, Germany). Forward sequences were only used and converted to protein with ExPaSy tool (Swiss Institute of Bioinformatics, Lausanne, Switzerland) then, trimmed with Chromas Pro (Technelysium software). The obtained protein sequences were aligned with MUSCLE [START_REF] Edgar | MUSCLE: multiple sequence alignment with high accuracy and high throughput[END_REF].The alignment was trimmed to the amplicon, and a tree was generated using MEGAX [START_REF] Kumar | MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms[END_REF], with the Maximum Likelihood method based on the Poisson correction model [START_REF] Zuckerkandl | Evolutionary divergence and convergence in proteins. In : Evolving genes and proteins[END_REF] (with n replication bootstraps = 500). Phylogenetic analyses were processed using HgcA proteins homemade database including HgcA sequences from isolated strains' genomes and from metagenomes obtained from NCBI (https://www.ncbi.nlm.nih.gov/) and Integrated Microbial Genomes and Microbiomes platform (IMG/M, developed by the Joint Genome Institute, CA, USA, http://img.jgi.doe.gov/) [START_REF] Markowitz | IMG: the integrated microbial genomes database and comparative analysis system[END_REF] and previous studies (Supporting information, Table S3). HgcA sequences were also collected from Pfam database [START_REF] Finn | The Pfam protein families database: towards a more sustainable future[END_REF] using custom Pfam models (corrinoid-iron sulfur proteins (CFesP) classified under Pfam3599) according to [START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. From these references, we selected only sequences with the conservative cysteine in HgcA [START_REF] Smith | Site-Directed Mutagenesis of HgcA and HgcB Reveals Amino Acid Residues Important for Mercury Methylation[END_REF] within the CWA motif. All reference sequences are compiled in Supporting information, Table S3. Sequences are archived in GenBank under submission number .2316824

Statistical analysis

Pearson correlations were calculated between chemical parameters using the R software environment version (http://www.r-prorject.org/). To remove redundancy of the chemical parameters data in order to avoid collinearity, we used the variance inflation factor (VIF) with the mctest package (Imdadullah et al., n.d.) in R using the Farrar-Glauber test. Multivariate analysis (Principal Component Analysis (PCA) were performed with FactomineR and Vegan package in R [START_REF] Lê | FactoMineR: An R Package for Multivariate Analysis[END_REF][START_REF] Oksanen | Multivariate Analysis of Ecological Communities in R: Vegan Tutorial[END_REF]. PCA for 16S rDNA diversity analysis was performed at the phylum level because of PCA constrains, with relative abundance above > 0.05%. We used the factoextra package [START_REF] Kassambara | Package 'factoextra'. Extract and visualize the results of multivariate data analyses[END_REF] for extracting and visualizing the results. Hierarchical Clustering on Principal Components (HCPC) were performed using FactomineR package in R. Distances were calculated with the Canberra method and classification was applied to draw the trends of the dataset.

Results

Characterization of habitats and microbial community assemblies

In this study, we used previous chemical parameters in order to discriminate different habitats in the Capbreton canyon [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. The variance in the relationship between depth, TS content, POC content, Hg(II) concentrations and MeHg proportion (selected among 10 parameters with the variance inflation factor, VIF, Supporting information, Table S1) was explained by two principal components contributing to 82.1% of the total variance contribution (Fig. 2). Surface sediments were separated into four distinct clusters corresponding to different areas in the Capbreton Canyon, with sediments from the mouth of the canyon, intermediate canyon, deep canyon and continental shelf (Fig. 2).

Overall, the geochemical composition of sediments was dependent on the location (i.e.

Canyon vs Continental Shelf), with an enrichment of TS, POC and Hg(II) levels in intermediate and offshore canyon locations (Fig. 2). Continental shelf sediments were characterized by a lower fine fraction content (<63µm), highlighting that Capbreton canyon enhances the transport and the accumulation of particles in the sediments [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. In contrast, higher MeHg proportions of total Hg were found in coastal locations suggesting higher methylation activity close to the coast.

The composition of sediment microbial communities was investigated based on 16S rDNA gene sequences, normalized at 5024 reads per sample allowing assignment of 2600 OTUs for the whole set of data. Overall, the global diversity was quiet close across samples (Chao1 ranging from 1208 to 1685) and replicates (low standard deviation) and no particular trends in alpha diversity could be observed (Supporting information, Table S2). Proteobacteria was the dominant phylum in terms of richness and relative abundance; they represented 921 OTUs and an average of 49.6 ± 3.6% of the total relative abundance, followed by Bacteroidetes and Planctomycetes representing 12.6 ± 5.7% and 8.6 ± 1.9%, respectively. A PCA analysis was performed on the relative abundance of phyla, in order to discriminate assemblies of microbial communities. The variance of the relationship between the relative abundance of phyla, was explained by the two first principal components contributing with 62.5% of the total variance (Fig. 3). Although the global pattern composition was uniform accross all locations, relative abundance variations led to three separate microbial community clusters along the canyon axis, distinguishing communities from mouth, intermediate and offshore locations (Fig. 3). Here, the distance to the coast seems to drive the microbial composition. Those multivariate analyses discriminated four different habitats colonized by three distinct microbial communities, themselves allocated along the canyon axis gradient.

Microorganisms involved in the Hg methylation: 16S rDNA versus hgcA gene

A previous study showed that the methylmercury proportion of total Hg decreased along the canyon axis, associated with a decrease of Hg methylation likely of biotic origin [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. hgcA diversity was investigated in one sediment of each of previously defined habitat (three sediments into the canyon along the canyon axis (G03, G10 and G24) and one from the continental shelf (G14)) in order to observe the diversity of Hg-methylating microbial communities. In addition, known groups containing Hg-methylators were assessed using the 16S rDNA marker.

The alpha diversity in the different Capbreton canyon sediments was similar (Supporting information, table S2). Deltaproteobacteria, which includes most of the known Hgmethylating bacteria, were the most abundant and accounted for 50% of the Proteobacteria and 25% of the total reads (Fig. 4). Among phyla known to contain Hg-methylators members, Planctomycetes were observed at 8.6 ± 1.9% of total reads and Chloroflexi at 3.0 ± 1.9% of total reads. The relative abundance of the operational taxonomic units (OTUs) from 16S rDNA within the Deltaproteobacteria group, showed the dominance of Desulfobacterales over Desulfuromonadales, NB1-J, Sva0485, Desulfarculales and Syntrophobacterales (Fig. 5), with maximum total relative abundance of 20.8%, 5.4%, 4.9%, 1.8%, 2.2% and 3.3%, respectively. Genera within the Deltaproteobacteria known to methylate Hg (Desulfobacteraceae, Desulfobulbaceae and Desulfuromonodaceae) were dominated by Desulfobulbus and other uncultivated or unaffiliated genera. The relative abundance of Desulfuromonadales decreased along the canyon transect like the unknown genus of Desulfobacterales. SEEP, SRB1, Desulfobulbus follow the trend of the Desulfobacterales order trend and SVA0081 increased along this transect. Although Geobacter sp. were found as a crucial Hg-methylator in continental aquatic ecosystems [START_REF] Bravo | Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases[END_REF][START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF][START_REF] Xu | Mercury methylating microbial communities of boreal forest soils[END_REF], here, abundance of Geobacteraceae members peaked to only 0.17 ± 0.10% in the G03 location. Those results based on 16S rDNA may indicate that the bacterial sulfur-reduction, i.e sulfate and sulfur reduction, is the dominant metabolic guild in these marine sediments.

A total of 84 HgcA clones were obtained from locations G03, G10, G14 and G24 (n= 19, 22, 21, 22, respectively). They were mainly related to the Deltaproteobacteria (21% of the total HgcA clones), dominated by the SRB (10 HgcA clones) followed by ferrireducers (Desulfuromodales) and syntrophs (Syntrophobacterales) (Fig. 6 and Fig. 7). HgcA clones were also related to recent new clades like Chloroflexi, Atribacteria, Archae, PVC superphylum, Spirochaetes, Elusimicrobia and Bacteriodetes (Fig. 6). Nevertheless, 61 % of the HgcA clones were unaffiliated (unaffiliated 1 to unafilliated 7 groups). While 18 clones of these unafilliated HgcA clones were unkown, 22 and 11 HgcA clones were likely related to Deltaproteobacteria (BSR like) and PVC superfphylum / Aminicenantes, respectively (Fig. 6 and Fig. 7) All together, almost half of HgcA clones (43 out of 84) obtained from the Capbreton submarine canyon sediments are likely related to Deltaproteobacteria with dominance of sulfate reducers. While HgcA clones cannot be representative of the Hgmethylators diversity due to the low number of clones per location and the selective approach used (cloning sequencing), those results follow the trend observed with the 16S rDNA approach, suggesting that microbial methylation is dominated by sulfate reduction.

Discussion

Geochemical conditions along Submarine canyons offer a diversity of ecological niches

We identified four distinct habitats along the canyon transect (Fig. 2) where Hg species, OM and TS contents were enriched. OM composition and TS play important roles in Hg biogeochemistry like MeHg formation and accumulation, as seen elsewhere [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF][START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF][START_REF] Choi | Environmental factors affecting mercury methylation in estuarine sediments[END_REF][START_REF] Cossa | Origin and accumulation of trace elements in sediments of the northwestern Mediterranean margin[END_REF]. Higher biotic methylation in coastal samples as compared to the offshore sediments as determined in a previous work, may be explained by a higher OM lability [START_REF] Wakeham | Limits of our knowledge, part 2: Selected frontiers in marine organic biogeochemistry[END_REF]) and a differential sequestration of particulate matter along the canyon (i.e. gravitational sedimentation, hemipelagic sedimentation, remobilization). Otherwise, Cyanobacteria that might have come from the water released by the nearby Adour estuary [START_REF] Goñi-Urriza | Bacterial community structure along the Adour estuary (French Atlantic coast): influence of salinity gradient versus metal contamination[END_REF], decrease along the canyon axis. As a primary producer, Cyanobacteria may contribute to the local OM, with higher production in coastal sediments than offshore sediments, and thus might control the MeHg production [START_REF] Lázaro | Cyanobacteria as regulators of methylmercury production in periphyton[END_REF][START_REF] Müller | Bacterial interactions during sequential degradation of cyanobacterial necromass in a sulfidic arctic marine sediment[END_REF]. Moreover, Capbreton Canyon is still active, meaning it is still filled with particulate matter [START_REF] Gaudin | Past and present sedimentary activity in the Capbreton Canyon, southern Bay of Biscay[END_REF], where fine particulate matter is transported along the canyon transect (gravitational sedimentation), confirmed by the higher fine fraction measured in offshore sediment relative to the coastal sediments [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. According to [START_REF] Mestre | Spatial variability of marine bacterial and archaeal communities along the particulate matter continuum[END_REF] microbial community composition varied with particle size where, Bacteria were more diverse in larger size-fractions, whereas Archaea were more diverse in smaller particles determining diverse ecotypes with distinct size-fraction preferences. Considering colonization of the four habitats by three distinct bacterial communities (Fig. 2 and Fig. 3), different abundances of known groups containing Hgmethylators (Fig. 4) were correlated with the diverse metabolisms of Hg methylators identified along this canyon (Fig. 5), our results highlighted that geochemical conditions controlled both the global microbial communities and thus the Hg methylating microbial communities.

Hg-methylating communities in submarine canyon sediments: global and functional approaches

For the first time, in submarine canyon sediments, we identified Hg methylating communities using both 16S rDNA and hgcA gene. The organic carbon remineralization in marine sediments is dominated by bacterial sulfate reduction [START_REF] Jørgensen | A Thiosulfate Shunt in the Sulfur Cycle of Marine Sediments[END_REF][START_REF] Jørgensen | The sulfur cycle of a coastal marine sediment (Limfjorden, Denmark)1[END_REF] performed by Deltaproteobacteria who are known to be the main group of Hg-methylating microorganisms [START_REF] Bravo | Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases[END_REF][START_REF] Christensen | Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations[END_REF][START_REF] Compeau | Sulfate-Reducing Bacteria: Principal Methylators of Mercury in Anoxic Estuarine Sediment[END_REF][START_REF] Parks | The genetic basis for bacterial mercury methylation[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. Nevertheless, although 16S rDNA analysis showed the Desulfobacterales as dominant, hgcA analysis showed SRB were also related to Desulfovibrionales. That is in accordance with the results of SRB identified in the water column in this canyon [START_REF] Colin | Distribution of Sulfate-Reducing Communities from Estuarine to Marine Bay Waters[END_REF]. hgcA clones within the "unafilliated 4", likely affiliated with the Deltaproteobacteria [START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF], might be related to the uncultured or unknown bacteria from the 16S rDNA analysis (Fig. 5 and Fig. 6). We suggest putative Hg methylating bacteria of unknown genera within the Desulfubacteraceae, Desulfobulbaceae and Sva1033 within the Desulfuromonadaceae. The relative abundance of those genera followed the trend of Hg methylation potential along the canyon axis, suggesting their involvement in this process. Moreover, Sva1033 was related to Desulfuromonas palmitatis [START_REF] Ravenschlag | High Bacterial Diversity in Permanently Cold Marine Sediments[END_REF] and [START_REF] Kerin | Mercury Methylation by Dissimilatory Iron-Reducing Bacteria[END_REF][START_REF] Kerin | Mercury Methylation by Dissimilatory Iron-Reducing Bacteria[END_REF] demonstrated that the latter, in pure culture under iron reducing conditions, was able to methylate Hg. Although sulfate reducing bacteria are widely studied and accepted as the major bacteria involved in mercury methylation in aquatic environments, these sulfate but also sulfur reducing bacteria remain largely unknown in marine environments.

Like in recent studies, hgcA analysis showed that the Hg methylators are also represented by fermentative organisms, or ferrireducers (Fig. 6) [START_REF] Bae | Syntrophs Dominate Sequences Associated with the Mercury Methylation-Related Gene hgcA in the Water Conservation Areas of the Florida Everglades[END_REF][START_REF] Christensen | Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations[END_REF][START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF][START_REF] Podar | Global prevalence and distribution of genes and microorganisms involved in mercury methylation[END_REF]. This highlighted the complexity between geochemical parameters and microorganisms involved into the MeHg formation. As recently discovered, HgcA clones were related to new phyla containing hgcA [START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF], such as Aminicenantes, Bacteriodetes, Kiritimatiellaeota, Elusimicrobia and Spirochaetes (Fig. 6).

Hg methylation is a strain-specific trait [START_REF] Ranchou-Peyruse | Overview of Mercury Methylation Capacities among Anaerobic Bacteria Including Representatives of the Sulphate-Reducers: Implications for Environmental Studies[END_REF]Podar et al., 2013;[START_REF] Gilmour | Mercury Methylation by Novel Microorganisms from New Environments[END_REF]Yu et al., 2013;[START_REF] Gilmour | Robust Mercury Methylation across Diverse Methanogenic Archaea[END_REF] that cannot be predicted purely by 16S rDNA taxonomic classification. Although another recent study demonstrated that 16s rRNA gene pyrosequencing did not have sufficient resolution to identify hgcAB harboring species in soil samples [START_REF] Christensen | Determining the Reliability of Measuring Mercury Cycling Gene Abundance with Correlations with Mercury and Methylmercury Concentrations[END_REF], this approach in marine sediments could provide indications about the hgcA-carrying Prokaryotes. The unknown hgcA-carrying populations are from uncultivated organisms, and consequently their identification, cultivation and specific contribution for MeHg remain to be explored.

MeHg proportion has previously been used as a proxy for methylation efficiency [START_REF] Drott | Do Potential Methylation Rates Reflect Accumulated Methyl Mercury in Contaminated Sediments?[END_REF][START_REF] Skyllberg | Net methylmercury production as a basis for improved risk assessment of mercury-contaminated sediments[END_REF] and high MeHg proportion has also in a few cases been shown to correlate positively with the abundance of Hg(II) methylators [START_REF] Xu | Mercury methylating microbial communities of boreal forest soils[END_REF]. Relative abundance of some phyla showed a strong correlation with MeHg proportion such as Cyanobacteria or Gemmatimonadetes (Table S5) suggesting that they could be involved, indirectly or directly, in MeHg formation. This may suggest that not only the Hg methylators themselves, i.e. Deltaproteobacteria, but also the supporting and interacting bacterial communities residing in the sediment may influence the MeHg formation across the studied submarine canyon sediments [START_REF] Xu | Mercury methylating microbial communities of boreal forest soils[END_REF].

As previously demonstrated in these sediments, methylmercury production is under control of both methylation and demethylation processes [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. Although the methylation seems to be controlled by microbial activities (for example hgcA gene is used as a proxy of microbial methylation), demethylation can be both abiotic (e.g. photoreduction)

and biotic (e.g. reductive (performed by microorganisms carrying operon mer), oxidative (performed by methylotrophs)) processes [START_REF] Lu | Methylmercury uptake and degradation by methanotrophs[END_REF][START_REF] Marvin-Dipasquale | Methyl-Mercury Degradation Pathways: A Comparison among Three Mercury-Impacted Ecosystems[END_REF][START_REF] Oremland | Methylmercury Decomposition in Sediments and Bacterial Cultures: Involvement of Methanogens and Sulfate Reducers in Oxidative Demethylation[END_REF]. Such biotic demethylation should be investigated in further studies to better understand microbial involvement in the Hg cycle in marine environment.

The S cycling and OM in marine sediments: drivers of Hg biomethylation ?

The mercury bioavailability for methylating microorganisms in marine sediments and indirectly the MeHg production, is controlled OM mineralization, thus depending on geochemical parameters [START_REF] Bravo | Molecular composition of organic matter controls methylmercury formation in boreal lakes[END_REF][START_REF] Goñi-Urriza | Relationships between bacterial energetic metabolism, mercury methylation potential, and hgcA/hgcB gene expression in Desulfovibrio dechloroacetivorans BerOc1[END_REF] and the degree of OM lability. Only a small portion of the total inorganic mercury is likely available for cellular uptake [START_REF] Hsu-Kim | Mechanisms Regulating Mercury Bioavailability for Methylating Microorganisms in the Aquatic Environment: A Critical Review[END_REF]. Indeed, the relative partitioning of Hg(II) between various dissolved and particulate forms will govern the overall mobility of Hg in aquatic systems and the bioavailability of Hg to methylating microorganisms in anaerobic settings. OM could be more labile in coastal sediment [START_REF] Wakeham | Limits of our knowledge, part 2: Selected frontiers in marine organic biogeochemistry[END_REF], which may improve the microbial activities. This suggests that terrestrial inputs, from the high proximity of the canyon mouth to the coast, can influence Hg-methylating microbial composition and thus could explain the higher estimated methylation potential in coastal locations compared to the offshore sediments.

Putative Hg methylators were found among different anaerobic metabolic guilds, including sulfate and sulfur-reducing bacteria, dehalorespiring bacteria, syntrophs, and in some cases FeRB (Fig. 6) but also some newly identified micro-organims (Chloroflexi, Spirochaetes, Elusimicrobia, PVC superphylum). This suggests that Hg methylation potential is shared by organisms using a wide variety of carbon sources and electrons acceptors. This is in accordance with recent studies which demonstrated that the putative Hg methylators are phylogenetically more diverse that previously, suggesting several independent horizontal gene transfer events [START_REF] Jones | Molecular evidence for novel mercury methylating microorganisms in sulfateimpacted lakes[END_REF][START_REF] Mcdaniel | Expanded Phylogenetic Diversity and Metabolic Flexibility of Microbial Mercury Methylation[END_REF].

Carbon cycling in marine sediments is dominated by sulfate-reduction that could mineralize 50% of the OM [START_REF] Jørgensen | The sulfur cycle of a coastal marine sediment (Limfjorden, Denmark)1[END_REF]. This is in accordance with the predominance of sulfur reducing bacteria (observed within 16S rDNA and HgcA) observed in these sediments.

Fermenters were also observed in all studied sediments (Fig. 7). This suggests a fermentative degradation of OM from complex carbon molecules to small carbon products (i.e. proprionate, pyruvate, acetate) and dihydrogen production. They could be used as electron donors for the SRB and methanogens [START_REF] Gilmour | Robust Mercury Methylation across Diverse Methanogenic Archaea[END_REF], improving the Hg biomethylation. Otherwise, OM degradation depends on the microbial composition and then both the methylating and non-methylating bacterial may control the MeHg formation [START_REF] Bravo | Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases[END_REF][START_REF] Xu | Mercury methylating microbial communities of boreal forest soils[END_REF]. Specific populations of non-Hg(II) methylating bacteria actively decomposing OM seem also to create a niche that promotes Hg(II) methylation. Also, it has been demonstrated that bioturbating fauna disturbed mechanically the electrogenic sulfur oxidation through long filamentous bacteria (Desulfobulbaceae), which likely mediated the electron transport between the deep and surface marine sediments (at the centimetre scale; 50), could have profound implication for the sulfur cycle. This highlights the complexity of interspecific relationship at different scales for the S and the Hg cycling in marine sediments [START_REF] Liu | Inorganic sulfur and mercury speciation in the water level fluctuation zone of the Three Gorges Reservoir, China: The role of inorganic reduced sulfur on mercury methylation[END_REF].

In oceans and seas, methylation of mercury occurs both in column water and sediment compartment [START_REF] Monperrus | The biogeochemistry of mercury at the sediment-water interface in the Thau Lagoon. 2. Evaluation of mercury methylation potential in both surface sediment and the water column[END_REF][START_REF] Rosati | Mercury in the Black Sea: New Insights From Measurements and Numerical Modeling[END_REF][START_REF] Smith | Episodic organic carbon fluxes from surface ocean to abyssal depths during long-term monitoring in NE Pacific[END_REF]. At a global scale, [START_REF] Boyd | Multi-faceted particle pumps drive carbon sequestration in the ocean[END_REF][START_REF] Boyd | Multi-faceted particle pumps drive carbon sequestration in the ocean[END_REF] showed that carbon sequestration in the open ocean is probably more influenced by solubility than biological activity. These insights into carbon sequestration can be taken into consideration to better understand the Hg biogeochemical cycle of mercury in ocean sediments. Although the OM is very reactive in the euphotic layer of the ocean, the carbon can directly sink to the bottom surface sediment [START_REF] Wakeham | Limits of our knowledge, part 2: Selected frontiers in marine organic biogeochemistry[END_REF] and could be an important carbon pump reacting into the Hg cycle, i.e. increase Hg methylation. Marine environment are the largest habitat on the earth accounting for more than 90% of total biosphere volume. The microorganisms colonizing marine habitats are responsible for more than 50% of the global primary production and nutrient cycling [START_REF] Lauro | The genomic basis of trophic strategy in marine bacteria[END_REF].

Carbon sedimentation at the bottom ocean of the ocean represents a significant fraction of global oceanic carbon sequestration [START_REF] Boyd | Multi-faceted particle pumps drive carbon sequestration in the ocean[END_REF] and could influence the bacterial composition, and consequently, can be a determinant for the formation of methylmercury [START_REF] Bravo | Geobacteraceae are important members of mercury-methylating microbial communities of sediments impacted by waste water releases[END_REF][START_REF] King | A Quantitative Relationship that Demonstrates Mercury Methylation Rates in Marine Sediments Are Based on the Community Composition and Activity of Sulfate-Reducing Bacteria[END_REF][START_REF] King | Sulfate-Reducing Bacteria Methylate Mercury at Variable Rates in Pure Culture and in Marine Sediments[END_REF][START_REF] King | Coupling mercury methylation rates to sulfate reduction rates in marine sediments[END_REF]. The high anthropogenic Hg levels found in the North Atlantic ocean [START_REF] Lamborg | An examination of the role of particles in oceanic mercury cycling[END_REF][START_REF] Lamborg | A global ocean inventory of anthropogenic mercury based on water column measurements[END_REF] associated with the high affinity of Hg for OM, could sink and trap Hg in those marine sediments. It is crucial to improve our knowledge of the Hg-methylating bacteria in marine sediments, in order to estimate their impact on MeHg production in a the global Hg cycle.

Conclusion

In this study, an assessment of marine Hg-methylating microbial communities was conducted in submarine canyon sediments by combining pluridisciplinary approaches including geochemical parameters, mercury speciation, global microbial diversity and hgcA-carrying bacteria diversity. Results highlighted that the distribution of microbial communities in submarine canyon sediments as well as methylation of mercury, were driven by the environmental parameters. Although Hg(II) and MeHg concentrations increased along the canyon axis from the coast to the offshore, the fraction of MeHg was higher in coastal locations due to the higher biomethylation potential estimated in coastal sediments [START_REF] Azaroff | Mercury and methylmercury concentrations, sources and distribution in submarine canyon sediments (Capbreton, SW France): Implications for the net methylmercury production[END_REF]. These results may indicate that methylation of Hg in marine sediments is driven by OM composition and the S cycle. Indeed, 16S rDNA and hgcA analysis suggested that Deltaproteobacteria dominated by sulfur-compounds-reducing bacteria are the main contributors for the methylation of Hg in marine sediments. Nevertheless, further studies are needed to identify and characterize/isolate the unaffiliated and unknown hgcA carrying microorganisms that could be novel Hg-methylators. Their methylation and demethylation capacities must be also determined in order to estimate their impacts on MeHg production in marine environments at the global scale. To fully understand and quantify the relationship between Hg, sulfur, OM and microorganisms, further studies are also needed to decipher the biogeochemical cycle of Hg in marine sediments.

  Fig.1

  Fig. 3

Acknowledgements

This work was supported by the European Union and the Adour Garonne Water Agency.

Europe commits the New Aquitaine with the European Regional Development Fund. We thank the crew of INSU-CNRS research vessel (Côte de la Manche). We gratefully thank Carl Lamborg (University of California, Santa Cruz) for his proofreading and anonymous reviewers for helpful criticize of the paper Compliance with ethical standards policy implications. Environ. Res., Mercury in Marine Ecosystems: Sources to Seafood Consumers 119, 101-117. https://doi.org/10.1016/j.envres.2012.03.013 Mazières, A., Gillet, H., Castelle, B., Mulder, T., Guyot, C., Garlan, T., Mallet, C., 2014. Highresolution morphobathymetric analysis and evolution of Capbreton submarine canyon head (Southeast Bay of Biscay-French Atlantic Coast) over the last decade using descriptive and numerical modeling. Mar. Geol. 351, 1-12.

Conflict of interest

The authors declare that they have no conflict of interest.