Aude Lucasson

Xing Luo

Shogofa Mortaza

Julien De Lorgeril

Eve Toulza

Bruno Petton

Jean-Michel Escoubas

Camille Clerissi

Lionel Dégremont

Yannick Gueguen

Delphine Destoumieux-Garzόn

Annick Jacq
email: annick.jacq@universite-paris-saclay.fr

Guillaume Mitta
email: mitta@univ-perp.fr

A core of functionally complementary bacteria colonizes oysters in Pacific Oyster Mortality Syndrome

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

INTRODUCTION

Introduced from Asia to a broad range of countries, Crassostrea gigas has become one of the world's main cultivated species. Since 2008, juvenile stages of C. gigas have suffered massive mortality events, especially in France [START_REF] Efsa | Oyster mortality[END_REF]. In subsequent years, this so-called Pacific Oyster Mortality Syndrome (POMS) [START_REF] Paul-Pont | Influence of husbandry practices on OsHV-1 associated mortality of Pacific oysters Crassostrea gigas[END_REF] has become panzootic. POMS has been observed in all coastal regions of France [START_REF] Martenot | Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010[END_REF][START_REF] Renault | Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas[END_REF][START_REF] Segarra | Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008[END_REF] and numerous other countries worldwide [START_REF] Peeler | Investigation of mortality in Pacific oysters associated with Ostreid herpesvirus-1 muVar in the Republic of Ireland in 2009[END_REF][START_REF] Lynch | A previously undescribed ostreid herpes virus 1 (OsHV-1) genotype detected in the pacific oyster, Crassostrea gigas, in Ireland[END_REF][START_REF] Efsa Poahw | Oyster mortality[END_REF][START_REF] Abbadi | Identification of a newly described OsHV-1 microvar from the North Adriatic Sea (Italy)[END_REF][START_REF] Burioli | Complete genome sequence of Ostreid herpesvirus type 1 microVar isolated during mortality events in the Pacific oyster Crassostrea gigas in France and Ireland[END_REF][START_REF] Burioli | Ostreid herpesvirus type 1 genomic diversity in wild populations of Pacific oyster Crassostrea gigas from Italian coasts[END_REF].

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

The copyright holder for this preprint this version posted November 16, 2020. ; https://doi.org/10.1101/2020.11. [START_REF] Pernet | Deciphering the effect of food availability, growth and host condition on disease susceptibility in a marine invertebrate[END_REF].384644 doi: bioRxiv preprint 3 This situation has promoted a research effort that has revealed a series of factors contributing to the disease, including infectious agents that interact with seawater temperature and oyster genetics [START_REF] Azéma | Genetic parameters of resistance to Vibrio aestuarianus, and OsHV-1 infections in the Pacific oyster, Crassostrea gigas, at three different life stages[END_REF][START_REF] Roux | Oysters and Vibrios as a Model for Disease Dynamics in Wild Animals[END_REF][START_REF] Pernet | Mass mortalities of Pacific oysters Crassostrea gigas reflect infectious diseases and vary with farming practices in the Mediterranean Thau lagoon, France[END_REF][START_REF] Pernet | Environmental anomalies, energetic reserves and fatty acid modifications in oysters coincide with an exceptional mortality event[END_REF][START_REF] Pernet | Deciphering the effect of food availability, growth and host condition on disease susceptibility in a marine invertebrate[END_REF][START_REF] Petton | Temperature influence on pathogen transmission and subsequent mortalities in juvenile Pacific oysters Crassostrea gigas[END_REF]. Dramatic POMS mortality events have coincided with the recurrent detection of Ostreid herpesvirus variants in moribund oysters [START_REF] Martenot | Detection of different variants of Ostreid Herpesvirus 1 in the Pacific oyster, Crassostrea gigas between 2008 and 2010[END_REF][START_REF] Renault | Analysis of clinical ostreid herpesvirus 1 (Malacoherpesviridae) specimens by sequencing amplified fragments from three virus genome areas[END_REF][START_REF] Segarra | Detection and description of a particular Ostreid herpesvirus 1 genotype associated with massive mortality outbreaks of Pacific oysters, Crassostrea gigas, in France in 2008[END_REF]. The involvement of other etiological agents has also been suspected. In particular, bacterial strains of the genus Vibrio have been shown to be associated with the disease [START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF][START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF]. Recently, integrative molecular approaches performed on susceptible and resistant families of oysters have revealed the complex etiology of POMS [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. These studies demonstrated that infection by Ostreid herpesvirus type 1 µVar (OsHV-1) is a critical step in POMS pathogenesis. OsHV-1 leads to an immunocompromised state by altering hemocyte physiology. This results in dysbiosis and bacteremia, which ultimately results in oyster death [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. Several bacterial genera are involved in this secondary infection [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]; one of them (Vibrio) was found to include opportunistic pathogens that cause hemocyte lysis [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF]. This observation occurred in a specific infectious environment in French Brittany [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. Whether the observed mechanisms of POMS pathogenesis are conserved in other infectious environments remains unknown. Also of importance, Vibrio species are not the only bacteria that colonize oyster tissues during the secondary bacterial infection. Several bacterial genera, including Arcobacter, Marinobacterium, Marinomonas, and Psychrobium are also found to massively colonize oysters [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. The mechanisms underlying the colonizing capacity of these species remain to be elucidated.

In the present study, we investigated whether POMS pathogenesis is conserved across environments, and which biological functions are expressed by the bacterial consortia that causes oyster death. We compared pathogenesis in oyster biparental families that display contrasting phenotypes (resistant or susceptible to POMS) in two infectious environments (the Atlantic Bay of Brest environment, and the Mediterranean Thau Lagoon environment). We found that the sequence of events is conserved in both infectious environments: intense viral replication in susceptible oysters, followed by a secondary bacteremia caused by a conserved bacterial consortium that results in oyster death. Using metabarcoding and metatranscriptomics, we identified members of the core colonizing bacterial consortium, and characterized their highly active metabolism and key adaptive responses to the within-host environment. We also identified a series of genus-specific functions required for colonization, which reveal specific strategies that the core consortium uses to adapt and colonize oysters. Finally, we identified cooperative processes used by the core consortium to meet their metabolic requirements; these processes may dictate the conservation of the core consortium across environments.

METHODS

Production of biparental oyster families

C. gigas families were produced as described in [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] De Lorgeril | Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome[END_REF]. Briefly, oysters were produced at the Ifremer hatchery in Argenton in March 2015. Three susceptible families (F 11 , F 14 , and F 15 ) and three resistant families (F 21 , F 23, and F 48 ) were used as recipients, and 15 families were used as donors. All families were maintained under controlled bio-secured conditions to ensure their specific pathogen-free status.

Status was verified by i) the absence of OsHV-1 DNA using qPCR, (see below) and ii) a low Vibrio load (~10 cfu/g tissue) on selective culture medium (thiosulfate-citrate-bile salts-sucrose agar) [START_REF] Petton | Factors influencing disease-induced mortality of Pacific oysters Crassostrea gigas[END_REF].

Oysters remained free of any abnormal mortality throughout larval development, at which time experimental infections were started.

Mesocosm experimental infections

The experimental infection protocol consisted of a cohabitation assay between donors (exposed to pathogens naturally present in the environment) and recipient specific pathogen-free oysters [START_REF] Petton | Temperature influence on pathogen transmission and subsequent mortalities in juvenile Pacific oysters Crassostrea gigas[END_REF][START_REF] Bruto | Vibrio crassostreae, a benign oyster colonizer turned into a pathogen after plasmid acquisition[END_REF]. Details of the experimental infection protocol (e.g. biomass, oyster weight, experimental duration, and tank volume) were as described in [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF][START_REF] De Lorgeril | Differential basal expression of immune genes confers Crassostrea gigas resistance to Pacific oyster mortality syndrome[END_REF]. Briefly, donor oysters were deployed at Logonna Daoulas (lat 48.335263, long 4.317922) in French Brittany (Atlantic environment) and at Thau Lagoon (lat 43.418736, long 3.622620) (Mediterranean environment). Oysters were deployed in farming areas during the infectious period, and remained in place until the onset of mortality (< 1%). Donors were then brought back to the laboratory (in Argenton) and placed in tanks, each containing recipient oysters from the three resistant and the three susceptible families. Experimental infections took place in July 2015 and September 2015 for the Atlantic and Mediterranean exposures, respectively. For each (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. experimental infection, mortality rate was monitored, and 10 oysters were sampled in triplicate from each oyster family shucking at 7 time points (0, 6, 12, 24, 48, 60, and 72 hours post-infection). The shell was removed and the whole oyster was flash frozen in liquid nitrogen. Oyster pools were ground in liquid nitrogen in 50 ml stainless steel bowls with 20mm diameter grinding balls (Retsch MM400 mill). The powders obtained were stored at -80°C prior to RNA and DNA extraction.

DNA extraction and quantification of OsHV-1 and total bacteria.

DNA extraction was performed as described in [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF] using the Nucleospin tissue kit (Macherey-Nagel).

DNA concentration and purity were checked with a NanoDrop One (Thermo Scientific).

Quantification of OsHV-1 and total bacteria were performed using quantitative PCR (qPCR) with a Roche LightCycler 480 Real-Time thermocycler (qPHD-Montpellier GenomiX platform, Montpellier University, France). Absolute quantity of OsHV-1 was calculated by comparing the observed Cq values to a standard curve of the OsHV-1 DNA polymerase catalytic subunit (AY509253) amplification product cloned into the pCR4-TOPO vector (Invitrogen). Relative quantification of total bacteria 16S rDNA gene was calculated by the 2 -ΔΔCq [START_REF] Pfaffl | A new mathematical model for relative quantification in real-time RT-PCR[END_REF] method with the mean of the measured threshold cycle values of two reference genes (Cg-BPI, GenBank: AY165040 and Cg-actin, GenBank: AF026063) (de Lorgeril, Lucasson et al. 2018).

Analyses of bacterial microbiota

Bacterial metabarcoding was performed using 16S rRNA gene amplicon sequencing. Libraries were generated using the Illumina two-step PCR protocol targeting the V3-V4 region [START_REF] Klindworth | Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencingbased diversity studies[END_REF]. A total of 252 libraries (six families × seven sampling time points × three replicates × two infectious environments) were paired-end sequenced with a 2 × 250 bp read length at the Genome Quebec platform on a MiSeq system (Illumina) according to the manufacturer's protocol. A total of 41,012,155 pairs of sequences were obtained. Metabarcoding data was processed using the FROGS pipeline [START_REF] Escudie | FROGS: Find, Rapidly, OTUs with Galaxy Solution[END_REF]. Briefly, paired reads were merged using FLASH [START_REF] Magoc | FLASH: fast length adjustment of short reads to improve genome assemblies[END_REF]. After cleaning steps and singleton filtering, 26,442,455 sequences were retained for further analyses. After denoising and primer/adapter removal with cutadapt, clustering was performed using SWARM, which uses a two-step clustering algorithm with a threshold corresponding to the maximum number of differences between two Operational Taxonomic Units (OTU) (denoising step d = 1; aggregation distance = 3) [START_REF] Mahe | Swarm: robust and fast clustering method for amplicon-based studies[END_REF]. Chimeras were removed using VSEARCH [START_REF] Rognes | VSEARCH: a versatile open source tool for metagenomics[END_REF]. Resulting OTUs were annotated using Blast+ against the Silva database (release 128).

Bacterial metatranscriptomic analysis

Powder obtained from the frozen oysters was resuspended in Trizol, and total RNA was extracted using a Direct-zol TM RNA Miniprep kit. Polyadenylated mRNAs (i.e. oyster mRNAs) were removed using a MICROBEnrich TM Kit (Ambion). cDNA oriented sequencing libraries were prepared as described in [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF] using the Ovation Universal RNA-Seq system (Nugen). Library preparation included steps to remove oyster nuclear, mitochondrial, and ribosomal RNAs, as well as bacterial rRNAs [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF].

A total of 36 libraries (three families × two sampling timepoints × three replicates × two infectious environments) were sequenced by the Fasteris company (Switzerland, https://www.fasteris.com) in paired-end mode (2 × 150 bp) on an Illumina HiSeq 3000/4000 to obtain 200-300 million clusters per sample (Supplementary Table 1).

Figure S1 presents a schematic of data processing. Raw Illumina sequencing reads from 72 fastq files (R1 and R2) were trimmed using Trimmomatic v0.38, and rRNA reads (both eukaryotic and bacterial) were removed using Sortme RNA v2.1b with the rRNA Silva database (release 128). At this stage, about 9% of reads were removed, underscoring the efficiency of experimental rRNA removal during library preparation (Supplementary Figure 1).

To further enrich bacterial sequences, reads were successively mapped by Bowtie2 [START_REF] Langmead | Fast gapped-read alignment with Bowtie 2[END_REF] on a multifasta file containing Crassostrea gigas genome sequence v9, complemented by C. gigas EST (available from NCBI), and a multifasta file containing the sequences of OsHV-1 (present in diseased oysters) and other viral sequences previously associated with bivalves [START_REF] Rosani | A Needle in A Haystack: Tracing Bivalve-Associated Viruses in High-Throughput Transcriptomic Data[END_REF]. Unmapped reads, which represented 4-10% of the starting reads (depending on conditions) were retained for further analysis (Supplementary Table 1). After a new filtering step using Trimmomatic, all remaining reads corresponding to the 36 samples were pooled (516,786,580 reads, 36-150 nt) and assembled using Trinity v2.3.2 in paired-end mode to build a reference metatranscriptome (1,091,409 contigs, 201 -15,917 nt). The resulting metatranscriptome was further annotated using Diamond BlastX against the NCBI nr protein database [START_REF] Buchfink | Fast and sensitive protein alignment using DIAMOND[END_REF]. 48.4 % of the contigs aligned with a protein in the database, and were further assigned to a taxon using Megan 6-LR Community Edition [START_REF] Huson | MEGAN-LR: new algorithms allow accurate binning and easy interactive exploration of metagenomic long reads and contigs[END_REF]. Sequences were annotated at different taxonomic levels from species to phylum. Out of the 1,091,409 contigs, 352,473 contigs aligned with bacterial proteins by BlastX with an E-value ≤ 01 e -06.

Each protein was assigned to 31 functional categories (Supplementary Table 2). Out of the 352,473 best hits, 74,015 were annotated as "hypothetical", "unknown", or "unnamed", and were assigned to the category "Unknown function". The remaining hits corresponded to 15,504 different protein names that were manually assigned to one of the 30 remaining functional categories.

Quantification of gene expression and data normalization

For each of the 36 samples used for the assembly of the metatranscriptome, reads were mapped back onto the metatranscriptome by Bowtie2 in paired-end mode. Raw counts per features (i.e. per contig) were computed using HTseq-Count [START_REF] Anders | HTSeq--a Python framework to work with high-throughput sequencing data[END_REF]. For each contig, and for each sample, raw counts were normalized to TPM (Transcripts per Kilobase / Million = Mapped reads / Length of contig (kb) / Scaling factor with Scaling factor = total reads in a sample / 1,000,000), which corrects for contig length and differences in read number in the different samples. Supplementary Table 3 presents a complete list of contigs, their length, the encoded feature annotation, and the TPM value of the feature in each sample. In many cases, one protein could be encoded by several contigs, either because gene assembly into a contig was incomplete, or because of the existence of contig isoforms. Altogether, the 352,473 contigs encoded 225,965 unique proteins.

When analyzing the functional enrichment in the seven genera that predominated in the microbiota of diseased oysters (see Results section), contigs that encoded the same protein were merged into a single feature/protein, and their expression levels were summed. The expression level of a category was calculated by summing up the expression level of all the proteins assigned to the category. In addition, expression values were normalized from TPM to a value corrected for the relative proportion of the genus activity in the sample such that the ratio of the average normalized expression level of the three replicates for a given functional category in two conditions (i.e. T60 or 72 vs T0) reflected a specific enrichment (Enrichment Factor or EF) of the function, rather than a general increase of the genus in the population. First, a pseudo count of one read was added to each gene in each condition/replicate, and the resulting counts were normalized by dividing by the total number of counts of the genus in a given condition/replicate, then multiplying by 10,000. For a given function/protein or functional category, an enrichment factor (EF) between the two conditions (i.e. T60 or T72 vs T0) was further defined as the ratio of the average normalized expression level of the three replicates at T60 or 72 over the average expression level at T0.

Statistical analyses

Survival curves were used to determine differential mortality kinetics between oyster families with the non-parametric Kaplan-Meier test (Mantel-Cox log-rank test, p < 0.05, GraphPad_Prism 6.01). For OsHV-1 and total bacteria quantifications, significant differences between resistant and susceptible oyster families were determined using the non-parametric Mann Whitney test (p < 0.05, GraphPad_Prism 6.01). For bacterial metabarcoding, statistical analyses were performed using R v3.3.1 (http://www.R-project.org, [START_REF]R: a language and environment for statistical computing[END_REF]). Principal coordinate analysis (PCoA, "phyloseq") on a Bray-Curtis distance matrix (ordinate, "phyloseq") was performed to determine dissimilarities between samples. Multivariate homogeneity of group dispersions was tested between bacterial assemblages of the six oyster families using 999 permutations (permutest, betadisper, "vegan"). DESeq2 ("DESeq", [START_REF] Love | Moderated estimation of fold change and dispersion for RNAseq data with DESeq2[END_REF]) from the OTUs to the higher taxonomic ranks was used to identify candidate taxa whose abundance changed between the initial and final time points of the experiment. The abundance of a bacterial taxon was considered significantly modified when the adjusted p-value for multiple testing with the Benjamini-Hochberg procedure, which controls the false discovery rate (FDR), was < 0.05. Heatmaps of significant genera were computed using relative abundances and the heatmap.2 function "ggplots" [START_REF] Mcmurdie | phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data[END_REF]. For bacterial metatranscriptomics, significant differences in expression between two conditions (i.e. T60 or T72 vs T0) were assessed by a paired Student's t-test (p < 0.05).

Metabarcoding and RNAseq sequence data will be made available through the SRA database (BioProject accession number PRJNA423079 with SRA accession SUB7738644 for bacterial metabarcoding and SUB8110348 for Bacterial metatranscriptomic). Other data generated from this study are included in the published version of this article and its supplementary files.

RESULTS

Primary OsHV-1 infection and secondary bacteremia are conserved in POMS, independent of the infectious environment

Six C. gigas families were subjected to two experimental infections that mimicked disease transmission in the wild. We previously reported high variability in the dynamics of mortality and percentage survival of oyster families confronted with an Atlantic infectious environment. Specifically, the F11, F14, and F15 families were highly susceptible (survival rate < 4% after 330h) to POMS, whereas the F21, F23, and F48 families were highly resistant (survival rate > 82% after 330h) [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF]. Similar results were obtained in the present study when the same oyster families were confronted with a Mediterranean infectious environment: families F11, F14, and F15 were susceptible (survival rates < 9%), whereas families F21, F23, and F48 were resistant (survival rates > 88%) (Figure 1). Thus, these oyster families displayed similar phenotypes when confronted with two different infectious environments (Mantel-Cox log-rank test, p < 0.0001 for each comparison of resistant vs. susceptible oyster families). Susceptible and resistant oyster families are hereafter referred to as S (S F11 , S F14, and S F15 ) and R (R F21 , R F23 , and R F48 ), respectively. green, and susceptible oyster families (S F11 , S F14 , and S F15 ) are presented in red. At each time point (indicated by asterisks on the arrow), 10 oysters were sampled 3 triplicates from each family in each tank for barcoding, qPCR, and metatranscriptomic analysis. Data for the Atlantic infectious environment are extracted from [START_REF] De Lorgeril | Immune-suppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF] and shown for comparison.

We next compared pathogenesis between the two infectious environments by monitoring OsHV-1 load, microbiota dynamics, and bacterial abundance in the three resistant and three susceptible oyster families (Figure 2). OsHV-1 DNA was detected in all families, regardless of whether they were confronted with the Atlantic or Mediterranean infectious environment (Figure 2a). However, very intense viral replication occurred only in the susceptible oyster families: viral DNA loads were 2 to 3 logs higher than in resistant oysters at 24 h (Figure 2a). 4). Sufficient sequencing depth was confirmed by species richness rarefaction curves (Supplementary Figure 2). Changes in microbiota composition were greater in susceptible oysters than in resistant oysters at all taxonomic ranks (Supplementary Figure 3). Indeed, for the Atlantic infectious environment, 52, 43, and 54 OTUs significantly differed (in terms of relative proportion between the start and end of the experiment) in susceptible oysters S F11 , S F14 and S F15 , respectively; only 1, 11, and 9 OTUs significantly differed in resistant oysters R F21 , R F23 and R F48 , respectively (Supplementary Table 5). The same trend was observed in the Mediterranean infectious environment. 11, 47, and 43 OTUs significantly differed in S F11 , S F14 and S F15 , respectively, as opposed to 2, 8, and 6 OTUs in R F21 , R F23 and R F48 , respectively.

PCoA on a Bray-Curtis dissimilarity matrix (beta diversity) revealed higher microbiota dispersion in susceptible oyster families than in resistant families in both infectious environments (multivariate homogeneity of groups dispersion, d.f. = 1; p = 0.016 and p = 0.020 for Atlantic and Mediterranean environments, respectively) (Figure 2b and2c). This disruption of the bacterial community structure occurred in susceptible oysters between 24 h and 48 h, concomitantly with the active replication of OsHV-1. In addition, susceptible oyster families displayed a significantly greater bacterial load than resistant oysters when confronted with either the Atlantic or the Mediterranean infectious environment (Mann Whitney test, p < 0.05; Figure 2d). This increase started at 60 h and continued to until the end of the experiment (72 h). Total bacterial abundance in susceptible oysters was more than 5-fold higher at 72 h than at T0, which indicated bacterial proliferation. In contrast, total bacterial load remained stable in resistant oysters.

A core of bacterial genera colonizes oysters during secondary bacterial infection in POMS

All bacterial genera that changed significantly in abundance during the two experimental infections (Atlantic and Mediterranean) in susceptible oyster families are reported in Supplementary Table 5 (DESeq2, adjusted p < 0.05). We focused on genera representing > 2% of the bacteria in at least one sample for each susceptible oyster family confronted with each infectious environment (Figure 3). In the Atlantic infectious environment, the corresponding OTUs represented a total of 4%, 0.8%, and 46% of total bacteria at the beginning of the experiment (T0), as opposed to 73%, 75%, and 72% at 72 h for S F11 , S F14, and S F15 , respectively (Supplementary Table 5). In the Mediterranean infectious environment, these OTUs increased from 2%, 6%, and 7% at T0 to 47%, 56%, and 56% at 72 h for S F11 , S F14 , and S F15 , respectively. Nine to twenty genera increased significantly in abundance between T0 and 72 h. Ten genera (Arcobacter, Cryomorphaceae, Marinobacterium, Marinomonas, Proxilibacter, Pseudoalteromonas, Psychrilyobacter, Psychrobium, Psychromonas, and Vibrio) were common to almost all (5 of 6) susceptible oyster families and both infectious environments (Figure 3).

Most of the remaining genera (Aquibacter, Aureivirga, Fusibacter, Neptunibacter, Peredibacter, Pseudofulbibacter) were shared by at least two families in one infectious environment. One genera (Salinirepens) increased significantly in all susceptible oysters in the Atlantic infectious environment only. These results show that a core of bacterial genera colonizes oysters during the POMS secondary bacterial infection, independent of the infectious environment. In resistant oyster families, several taxa also vary significantly in abundance over time. Most of these taxa are the same taxa identified in susceptible oyster families (Supplementary Figure 4), but they are present in lesser quantities. These taxa represent between 4% to 23% of the reads sequenced at 72h in resistant oysters, whereas they represent 47% to 75% of the reads sequenced in susceptible oysters (Supplementary Table 5).

Seven genera are responsible for most bacterial gene expression in diseased oysters

To understand the colonization success of certain genera, we analyzed the gene expression of the colonizing microbiota. As described in the Methods section, we used metatranscriptomics to determine the functional mechanisms underlying colonization for different bacterial genera in the three different oyster genetic backgrounds and two different infectious environments. RNAseq was performed after removal of rRNA, oyster mRNA, and mitochondrial RNA using custom probes. We sequenced the remaining RNA of the susceptible families S F11 , S F14 , and S F15 , in both the Atlantic and Mediterranean infectious environments, at T0 and just before oyster mortality occurred (i.e. at 60 h and 72 h for the Atlantic and the Mediterranean infectious environments, respectively). Three biological replicates were analyzed for each condition, corresponding to a total of 36 biological samples. After removal of the remaining non-bacterial and ribosomal reads, we assembled and annotated a reference metatranscriptome. We aligned the translated contigs with the NCBInr protein database, and used MEGAN for taxonomic assignments. We obtained 352,473 contigs that aligned with bacterial proteins with an e-value ≤ 10 -6 . In many cases, a single protein could be encoded by several contigs, either because the gene was not fully assembled or because of contig isoforms. In total, our assembled metatranscriptome encoded 225,965 unique proteins. These protein were functionally and taxonomically annotated, and their expression level was determined for each condition. Further analyses were restricted to proteins assigned at the genus or species levels.

To analyze the contribution of each genus to the overall bacterial gene expression, we added the level of expression of all proteins assigned to a given genus (measured in Transcripts Per Million, or TPM).

We focused on genera that contributed to more than 2% of total gene expression in at least one oyster sample (Figure 4). increased relative activity of the genus. Genera shown contributed at least 2% of the total transcriptional activity in at least one sample of diseased oysters. Bacterial genera that were overrepresented according to metatranscriptomics alone for all conditions in the diseased oysters are in red, while genera that were overrepresented according to both metabarcoding and metatranscriptomics are underscored. (Atl: Atlantic, Med: Mediterranean, T0: before infection, T60/72: diseased) Seven genera were consistently found to contribute the majority of transcriptomic activity in diseased oysters, displaying a strong increase in the number of transcripts compared to healthy oysters.

Amphitrea, Arcobacter, Marinobacterium, Marinomonas, Oceanospirillum, Pseudoalteromonas, and

Vibrio were together responsible for up to 40% of the total bacterial transcriptomic activity detected just before the onset of oyster mortality. All are Gram-negative bacteria, and 6 out of 7 are Gammaproteobacteria. Of the six Gammaproteobacteria, Amphitrea, Marinobacterium, Marinomonas and Oceanospirillum belong to the same family (Oceanospirillaceae) and order (Oceanospirillales).

Arcobacter belongs to the class Epsilonproteobacteria. The genus Fluviicola, which belongs to the phylum Bacteroidetes, presented an increase of transcriptomic activity in the Atlantic infectious environment only. Finally, the genus Oleiphilus (order Oceanospirillales), was represented at a maximum level of 1.9 % in diseased oysters.

These results indicate that a limited number of genera participate in the secondary bacteremia that occurs in POMS. These genera are remarkably conserved between the different susceptible oyster families and infectious environments. We therefore focused on these seven genera for additional study.

Secondary bacterial colonizers display genus-specific strategies

We focused on the late phase of the infection process in our examination of the functions underpinning successful colonization by the seven genera discussed above. 31 functional categories were defined, and each transcript-encoded protein was assigned to one of them (Supplementary Table 2). For each genus and each category, an enrichment factor (EF) was calculated relative to the general increase of the genus in the bacterial population (See Materials and Methods for details, Figure 5). For all genera except Arcobacter, translation and transcription appeared to be globally overrepresented in diseased oysters. Aside from these functions, we found that each colonizing genus displayed a specific pattern of functional enrichment, which was generally conserved across oyster families and environments. For instance, adhesion, cell defense, motility, natural competence, quorum sensing, stress response, and virulence gene expression were either over-or under-represented according to the genus considered. These results suggest that each genus adopts a specific strategy to ensure colonization success.

Enriched functions are potentially important for successful colonization

In a global metatranscriptomic analysis, important overrepresented transcripts/subcategories could be hidden by the under-expression of genes classified as being in the same functional categories.

Therefore, we next focused our analysis on overrepresented transcripts (listed in Supplementary Table 6), and identified several overexpressed genes that are likely to contribute to efficient colonization. Table 1 summarizes the distribution of these overexpressed genes/subcategories within the seven genera. Rhodanese like protein 2.8.1.1 @ @ @ @ Formaldehyde detoxification Formaldehyde dehydrogenase, glutathione-independent 1.2.98.1 @ @ @ @ Hydroxyacylglutathione hydrolase 3.1.2.26 @ @ S-formylglutathione hydrolase 3.1.2.12 @ Aminoglycoside phosphotransferase 2.7.1.-@ RND family efflux transporter @ @ TolC family outer membrane protein @ @ @ @ Hg resistance

MerR family DNA-binding transcriptional regulator @ Inorganic ion Calcium transport

Ca2+ transporter @

Potassium transport K+ transport @ @ Sodium transport Na+/H+ antiporter NhaC @ @ Phosphonate utilisation

Alkyl phosphonate utilization PhnA @ @ Phosphonate transporter @ @ @ Alkaline phosphatase 3.1.3.1 @ inorganic diphosphatase @ @ PhoH (Pi starvation) @ @ @ @ Pi transport @ Polyphosphate kinase 2 2.7.4.1 @ @ Selenate transport

Selenate transporter @

Metal homeostasis Fe(3+) ABC transporter substrate-binding protein @ @ @ @ Ferric iron uptake transcriptional regulator @ @ Imelysin @ @ Bacterioferritin @ @ @ Ferritin @ Siderophore production @ @ TonB-dependent siderophore receptor @ @ @ Copper homeostasis

Copper-translocating P-type ATPase @ @ Heavy metal (Co2+, Zn2+, W…) transporter @ @@ Metal transporter @ @ Nitrogen compound metabolism Nitrosative stress Hydroxylamine reductase 1.7.99.1

Precursor metabolite and energy production and conversion ATP synthase ATP synthase 7.1.2.2 @ @ @ @ @ @ @ cytochrome C @ @ @ @ @ @ @ cytochrome C oxidase 7.1.1.9 @ @ @ @ @ @ @ Na(+)-translocating NADH-quinone reductase 7.2.1.1 @ @ @ @ @ @ @ cytochrome B @ @ @ @ cytochrome o ubiquinol oxidase 7.1.1.3 @ electron transfer flavoprotein @ @ @ @ @ electron transfer flavoprotein-ubiquinone oxidoreductase 1.5.5.1 @ @ @ hybrid-cluster NAD(P)-dependent oxidoreductase @ @ NADH-quinone oxidoreductase 7.1.1.2 @ @ @ pyruvate flavodoxin oxidoreductase 1.2.7.1 @ ubiquinol oxidase subunit II 7.1.1.7 @ ubiquinol-cytochrome c reductase 1.10.2.2 @ @ @ acetate--CoA ligase 6.2.1.1 @ @ @ pyruvate dehydrogenase 1.9.6.1 @ @ @ @ @ fumarate reductase 1.3.5.4 @ hydrogenase small subunit 1.12.5.1 @ fumarate/nitrate reduction transcriptional regulator Fnr @ @ Drug resistance

Pi homeostasis

Iron homeostasis

Metal homeostasis

Electron transfer

Acetyl-CoA biosynthesis 21 402

Mixed acid fermentation formate dehydrogenase 1.17.5.3 @ @ @ @ @ phosphoenolpyruvate synthase 2.7.9.2 @ pyruvate kinase 2.7.1.40 @ @ @ @ 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase 5.4.2.11 @ @ @ fructose-bisphosphatase 3.1.3.11 @ @ fructose-bisphosphate aldolase class II 4.1.2.13 @ @ @ @ @ @ glucose-6-phosphate isomerase 5.3.1.9 @ glyceraldehyde-3-phosphate dehydrogenase 1.2.1.12 @ @ @ @ @ phosphoglycerate kinase 2.7.2.3 @ @ @ phosphopyruvate hydratase 4.2.1.11 @ @ @ @ @ triose-phosphate isomerase 5.3.1.1 @ @ @ @ isocitrate lyase 4.1.3.1 @ @ @ @ CoA ester lyase (malate synthase) 2.3.3.9 @ malate synthase 2.3.3.9 @ @ @ @ L-alanine fermentation PRPP biosynthesis ribose-phosphate pyrophosphokinase 2.7.6.1 @ @ @ @ @ @ Lactate dehydrogenase or related 2-hydroxyacid dehydrogenase 1.1.-.-@ Ldh family oxidoreductase 1.1.5.12 @ @ Aerobic respiration control protein arcA @ 2-oxoglutarate dehydrogenase 1.2.4.2 @ @ @ @ @ @ 2-oxoglutarate synthase 1.

@ citrate synthase 2.3.3.1/2.3.3.16 @ @ @ @ @ @ dihydrolipoamide dehydrogenase 1.8.1.4 @ @ @ @ fumarate hydratase 4.2.1.2 @ @ @ @ @ isocitrate dehydrogenase 1.1.1.42 @ NADP-dependent isocitrate dehydrogenase 1.1.1.42 @ @ @ @ @ @ succinate dehydrogenase 1.3.5.1 @ @ @ @ @ succinate--CoA ligase 6.2.1.5 @ @ @ @ @ Protein export and secretion system Enzyme secretion Type 2 SS @ @ @ EPS production-associated protein export Exosortase @ Folded protein export

Tat system @ @ Inner membrane protein translocation Sec system @ @ @ @ @ Toxin secretion Type 1 SS @ @ @ Virulence factor secretion/ competition Type 6 SS @ @ Glycolysis/ gluconeogenesis Glyoxylate shunt

Mixed acid fermentation

Nitrate respiration

Pentose phosphate pathway

Respiration

TCA cycle

Functions important for stress adaptation and resistance to host defenses varied over the course of pathogenesis according to the bacterial genera considered (Table 1). "Stress response" genes encoding cold-shock proteins and the ribosome-associated translation inhibitor RaiA were overexpressed by all genera. The gene encoding the stress response RNA polymerase Sigma factor RpoS was overexpressed in members of the Oceanospirillaceae family (Amphritea Marinobacterium, Marinomonas, and Oceanospirillum). In the "redox homeostasis and oxidative stress" category, peroxiredoxin genes were overexpressed by all genera, along with additional genus-specific functions related to fighting oxidative stress (superoxide dismutases, catalases, and peroxidases) (Table 1). In addition, all genera except Oceanospirillum overexpressed genes that included several nitrite and nitrate reductases (classified in "Precursor metabolite and energy production and conversion"), hydroxylamine reductases (classified in "Nitrogen compound metabolism"), and nitric-oxide reductase components (classified in "Stress response/ Nitrosative stress"), all of which could contribute to tolerance/resistance to reactive nitrogen species (RNS). RNS, like ROS, are important effectors of oyster immunity [START_REF] Destoumieux-Garzon | Vibrio-bivalve interactions in health and disease[END_REF].

"Cell defense", "Metal homeostasis" and "Virulence/ fitness" categories were more specifically encoded by a subset of bacterial genera. In the "Cell defense" category, multidrug resistance efflux pumps with a TolC component and detoxifying enzymes were overexpressed in some genera. For instance, an outer membrane channel protein TolC was overexpressed up to 16-fold (log2(EF) ≥ 4) in Pseudoalteromonas (Supplementary Table 6). Genes encoding Rhodanese, an enzyme that detoxifies cyanide, were overexpressed in Marinobacterium, Pseudoalteromonas, and Vibrio. Several arsenate reductase genes were also overexpressed in Marinobacterium, Marinomonas, and Pseudoalteromonas.

Genes encoding formaldehyde dehydrogenase, an aldehyde-detoxifying enzyme, were overexpressed in the Oceanospirillaceae. Notably, no cell defense genes were overexpressed in Arcobacter. In the "Metal homeostasis" category, 23 of 33 overexpressed genes were involved in iron homeostasis.

These genes are mainly expressed by Marinobacterium and Marinomonas. In Vibrio, the only upregulated gene of the category is annotated as a cobalt/magnesium transporter. In the "Virulence / fitness" category, a small number of overexpressed genes corresponded to putative toxins encoded by Arcobacter, Oceanospirillum, Pseudoalteromonas, and Vibrio. This has to be completed by genes from the "Protein export and secretion system" category, which are important for both virulence and interbacterial competitions. Genes encoding the general protein export system (i.e. the "Sec" system of Gram-negative bacteria [START_REF] Smets | Protein Transport Across the Bacterial Plasma Membrane by the Sec Pathway[END_REF][START_REF] Beckwith | The Sec-dependent pathway[END_REF]) were overexpressed by all genera, while type VI secretion systems were overexpressed specifically in Vibrio (e.g. V. crassostreae) and Marinomonas.

Finally, a global increase in the "Sulfur compound metabolism" category was observed in Marinobacterium (Figure 5). Overexpressed proteins of this category were also found in other genera

(Table 1 and Supplementary Table 6). Interestingly, these overexpressed proteins corresponded to enzymes that were (mostly non-redundantly) encoded by five different genera. Altogether, these enzymes constitute several sulfur metabolic pathways (Figure 6).

These results highlight functional complementation at the level of the bacterial community, and a metabolic adaptation to the within-host environment, both of which are likely important for successful oyster colonization. ). Members of the Arcobacter genus had also been associated with POMS-diseased oysters [START_REF] Lasa | Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment nextgeneration sequencing strategy[END_REF][START_REF] Lokmer | Hemolymph microbiome of Pacific oysters in response to temperature, temperature stress and infection[END_REF], but the role of this genus in pathogenesis was not investigated more deeply due to limitations of culture-based techniques [START_REF] Rahman | Improved culture enrichment broth for isolation of Arcobacter-like species from the marine environment[END_REF]. In the present study, we extended the core colonizing microbiota to five bacterial genera. The discovery of the contributions of these genera, which are responsible for up to 40% of the bacterial transcriptional activity observed in POMS, provides new insights into the pathogenesis of POMS. Altogether, our results strongly suggest that a colonizing core microbiota, rather than specific bacterial pathogens, operates as a functional unit of pathogenesis. Together with

OsHV-1, these bacteria form the POMS pathobiota. POMS secondary bacteremia may resemble periodontitis in humans, in which the evolution of the disease is characterized by the development of a pathogenic consortium comprising a limited number of species [START_REF] Yost | Functional signatures of oral dysbiosis during periodontitis progression revealed by microbial metatranscriptome analysis[END_REF][START_REF] Lamont | Polymicrobial synergy and dysbiosis in inflammatory disease[END_REF].

We used metatranscriptomics to unveil the functions of the microbiota in relation to POMS. Bacterial metatranscriptomics from host tissues is technically challenging (due to the low proportion of bacterial transcripts in the host samples), but it provides functional information that is thought to more accurately portray the role of the microbiota in health and disease states [START_REF] Heintz-Buschart | Human Gut Microbiome: Function Matters[END_REF]. Accordingly, gene expression profiling has proven highly successful in advancing the understanding of the dynamics of disease-associated microbial populations [START_REF] Murray | Mechanisms of synergy in polymicrobial infections[END_REF]. In the case of POMS, by linking functional genes to the bacterial genera which encode them, we found a remarkably consistent relationship between the structure of bacterial communities (through 16S metabarcoding) and the functions expressed by bacterial genera in the communities (through metatranscriptomics), further supporting our hypothesis of a pathobiota composed of at least five key genera.

Among functions overexpressed in the diseased oyster-associated microbiota, we first identified core functions which were overrepresented in all dominant genera (Table 1). In addition to housekeeping functions with expression levels that reflect the rapid growth of the genus (e.g. translation and transcription, central metabolism), we found functional redundancy in two important categories required for adaptation to the within-host environment: stress response and redox homeostasis.

We found a systematic and important overproduction of various cold shock proteins in the "Stress response" functional category (Supplementary Table 6). In the diseased oyster microbiota, these proteins may be important to sustain the high level of transcription and translation we observed [START_REF] Phadtare | Unwinding activity of cold shock proteins and RNA metabolism[END_REF][START_REF] Phadtare | RNA remodeling and gene regulation by cold shock proteins[END_REF]. Redox homeostasis was another core function. Fighting oxidative stress in the host can be crucial to survival and multiplication within the host. Thus, bacteria have many lines of defense against oxidative damage [START_REF] Staerck | Microbial antioxidant defense enzymes[END_REF]. Key enzymes and proteins involved in redox homeostasis and oxidative stress response were overexpressed in several of the five genera, including superoxide dismutases (which catalyze the dismutation of superoxide in hydrogen peroxide) and catalases and peroxidases (which destroy hydrogen peroxide and other toxic peroxide compounds). All five genera overexpressed redoxin, a small redox protein which plays important roles in maintaining an intracellular reducing environment, and also acts as a cofactor in intracellular oxidoreductase reactions. Other proteins important for fighting oxidative stress were specific to one or more given genera. For example, NAD(P) reductase (which contributes to the homeostasis of reduced NAD(P)H, an important source of reducing potential) was enriched in Amphitrea, Marinobacterium, Marinomonas, and Oceanospirillum, four members of the Oceanospirillaceae family. Glutamate -cysteine ligase (which is responsible for the production of Glutathione, an important antioxidant compound, and is also involved in detoxifying reactions) was overexpressed in Amphitrea, Oceanospirillum, and Pseudoalteromonas. Earlier functional studies have identified the AhpCF complex detoxifying enzyme alkylhydroxyperoxydase as a key determinant of resistance to ROS and thus oyster colonization in Vibrio [START_REF] Vanhove | Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics[END_REF]. In the diseased oyster microbiota, this enzyme was over-produced in Marinomonas and Oceanospirillum. All of the successful genera, except Oceanospirillum, also expressed enzymes required for tolerance/resistance to RNS (Table 1). ROS and RNS are major players at the oyster-microbiota interface (Destoumieux-Garzon et al., 2020), thus it is not surprising that successful colonizers express ROS detoxifying enzymes during oyster colonization. In summary, each genus overexpressed several proteins involved in stress response, especially oxidative stress, although each genus had its own repertoire of proteins.

We found that the bacterial community associated with POMS also expresses a series of genusspecific (variable) functions. These functions include adhesion, cell defense, cell motility, metal homeostasis, natural competence, quorum sensing, sulfur metabolism, transport, and virulence (Table 1). The observation that functions of interest may be contributed by a single or multiple bacterial genera within a microbial community has also been made in the human gut [START_REF] Heintz-Buschart | Human Gut Microbiome: Function Matters[END_REF][START_REF] Schirmer | Dynamics of metatranscription in the inflammatory bowel disease gut microbiome[END_REF]. Interestingly, a function expressed by one genus can be useful for the community as a whole. Functional complementarity within the oyster-associated microbiota may therefore contribute to synergy between genera.

Evidence of cross-benefits at the community level in the oyster microbiota was found in the form of overexpressed genes related to iron homeostasis (Supplementary Table 6). The capacity to scavenge iron from the environment and the host is a well-known determinant of pathogenicity [START_REF] Begg | The role of metal ions in the virulence and viability of bacterial pathogens[END_REF][START_REF] Lemos | Iron uptake mechanisms as key virulence factors in bacterial fish pathogens[END_REF]. Except

for Vibrio, all genera overexpressed functions related to iron acquisition (e.g. iron siderophore production and transport, which enable the chelation and uptake of iron). However, in the consortium, only Marinomonas and Marinobacterium expressed siderophore-production enzymes, while other members of the microbiota expressed the receptors for iron-siderophore uptake. Similarly, virulence factors (such as secreted RTX family toxins) were expressed by Vibrio and Arcobacter, while the Type 6 Secretion System (T6SS) was expressed by Vibrio and Marinomonas. T6SS expressed by

Vibrio has been shown to contribute to the suppression of oyster cellular defenses [START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF][START_REF] Piel | Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells[END_REF]. Therefore, their expression by a few genera is likely beneficial to the whole pathobiota.

Figure 1 :

 1 Figure 1: Kaplan-Meier survival curves of oyster biparental families confronted with two different infectious environments. Resistant oyster families (R F21 , R F23 , and R F48 ) are presented in

Figure 2 :

 2 Figure 2: Primary OsHV-1 infection, bacterial dysbiosis, and secondary bacteremia are conserved in different infectious environments. (a) Early and intense replication of OsHV-1 µVar occurs in susceptible oysters (red), but not resistant oysters (green), confronted with either the Atlantic or the Mediterranean infectious environment. OsHV-1 load was quantified by qPCR and expressed as Viral Genomic Units per ng of oyster DNA (log scale) during experimental infections. Asterisks indicate significant differences between susceptible and resistant oyster families (Mann Whitney test, p < 0.05). (b-c) Principal coordinate analysis (PCoA) plot of the microbiota for susceptible (red) and resistant (green) oyster families confronted with each infectious environment. Dispersion of oyster families according to the Bray-Curtis dissimilarity matrix (beta diversity) in (b) Atlantic and (c)

Figure 3 :

 3 Figure 3: Heatmaps of bacterial genera that changed significantly in abundance over the course of infection in susceptible oysters (S F11 , S F14 , and S F15 ) in the Atlantic and Mediterranean infectious environments. Analyses were performed at the genus level. Only genera that changed significantly in abundance (DESeq2, adjusted p < 0.05) and had a relative proportion greater than 2% in at least one sample are shown. Increased intensity of color (blue) represents increased relative abundance. Genera that are consistently modified in 5 out of the 6 conditions (3 families and 2 infectious environments) are in red.

Figure 4 :

 4 Figure 4: Heatmap of transcriptional activity of bacterial genera in susceptible oyster families (S F11 , S F14 , and S F15 ) in the two infectious environments before infection and in diseased oysters. For each condition, results of the three replicates are shown. Increased color intensity (blue) indicates

Figure 5 .

 5 Figure 5. Functional enrichment between T0 and the onset of oyster mortality. The enrichment factor (EF) was calculated for each functional category for the seven most active genera present in all families and both environments. Each overrepresented (red) or underrepresented (blue) functional category is indicated by a bar with a width proportional to log2(EF). Red boxes around bars indicate p ≤ 0.05 (Student's t-test). Green-filled cells indicate functional categories with no gene expression (genes absent from the genome in that genus or not expressed in any conditions). Light blue-filled cells indicate values lower than the 20th percentile, while yellow-filled cells indicate values higher than the 20th percentile.

Figure 6 :

 6 Figure 6: Integrated sulfur cycle in the diseased oyster microbiota. Overexpressed sulfur metabolism pathways are shown. Enzymatic reactions are represented by arrows that are colored according to the pathway. The Enzyme Commission (E.C.) number, corresponding gene name, and genera overexpressing the gene for the enzyme catalyzing each reaction is provided in the grey rectangle beside the relevant arrow.

20 401 Table 1 .

 4011 Specific enrichment of selected functions in the seven genera. Overexpres sion of function/gene in a giv en genus is indicated by a filled out green cell. E.C. numbers are indicated whenev er applicable. Functions that are enriched in all sev en genera are in boldface. The relevant information was extracted from supplementary table S6.

	Category Role/pathways	Enriched functions/genes	E.C.. Number	A m p h i t r e a A r c o b a c t e r M a r i n o b a c t e r i u m M a r i n o m o n a s O c e a n o s p i r i l l u m P s e u d o a l t e r o m o n a s V i b r i o
	Cell defense
	Arsenic detoxification	Arsenate reductase	1.6.5.2	@ @	@
	Cyanide detoxification

DISCUSSION

  In this study, we have shown that the dysbiosis associated with POMS is conserved across infectious environments. Using metabarcoding, we demonstrated that diseased oysters affected by POMS are colonized by a common consortium of bacteria comprising ten major genera (Arcobacter, The bacterial consortium associated with POMS was remarkably similar in three different susceptible oyster families and two different environments. All diseased oyster families harbored similar microbial communities, with only a few exceptions. Remarkably, the oyster-associated microbial communities were conserved across infectious environments, although the Mediterranean and Atlantic ecosystems are likely to host diverse bacterial communities.Until recently, only members of the Vibrio genus had been repeatedly associated with POMS. These studies used culture-based approaches to investigate oyster-associated bacterial communities[START_REF] Lemire | Populations, not clones, are the unit of vibrio pathogenesis in naturally infected oysters[END_REF] Bruto et al., 2018). Vibrio species associated with POMS were characterized by key virulence factors that are required to weaken oyster cellular defenses[START_REF] Rubio | Species-specific mechanisms of cytotoxicity toward immune cells determine the successful outcome of Vibrio infections[END_REF][START_REF] Piel | Selection of Vibrio crassostreae relies on a plasmid expressing a type 6 secretion system cytotoxic for host immune cells[END_REF]

	Cryomorphaceae,	Marinobacterium,	Marinomonas,	Proxilibacter,	Pseudoalteromonas,
	Psychrilyobacter, Psychrobium, Psychromonas, and Vibrio). Using metatranscriptomics, we showed
	that five of these genera (Arcobacter, Marinobacterium, Marinomonas, Vibrio, and
	Pseudoalteromonas) were particularly transcriptionally active.

Acknowledgements

. We warmly thank the staff of the Ifremer stations of Argenton (LPI, PFOM) and Sète (LER), and the Comité Régional de Conchyliculture de Méditerranée (CRCM) for technical support in the collection of the oyster genitors and reproduction of the oysters. The authors are grateful to Philippe Clair from the qPHD platform/Montpellier genomix for useful advice. This work benefited from the support of the National Research Agency under the "Investissements d'avenir" program (reference ANR-10-LABX-04-01) through use of the GENSEQ platform (http://www.labexcemeb.org/fr/genomique-environnementale-2) from the labEx CeMEB. The present study was supported by the ANR projects DECIPHER (ANR-14-CE19-0023) and DECICOMP (ANR-19-CE20-0004), and by Ifremer, CNRS, Université de Montpellier and Université de Perpignan via Domitia. This study is set within the framework of the "Laboratoires d'Excellence (LABEX)" TULIP (ANR 10 LABX 41).

Data availability

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

High metabolic activity, revealed by a series of functional categories, was observed in all genera. In the "Translation" category, genes encoding ribosomal proteins, elongation and initiation factors, and tRNA ligases were overexpressed for all seven genera. In the "Precursor metabolite and energy production and conversion (Energy)" category, genes encoding ATP synthase and enzymes involved in glycolysis/neoglucogenesis and the TCA cycle were systematically present, as were genes encoding proteins of the respiratory chains (e.g. cytochrome-c and cytochrome oxidases). In the "Transcription and RNA metabolism (RNA)" category, genes encoding the various subunits of the RNA polymerase were overexpressed, as was the transcription housekeeping sigma factor RpoD (Supplementary

Table 6).

Redox homeostasis and oxidative stress Peroxiredoxin @ @ @ @ @ @ @ Alkyl hydroperoxide reductase 1.11.1.26 @ @ Catalase 1.11.1.6 @ @ Disulfide reductase @ @ @ Glutamate--cysteine ligase 6.3.2.2 @ @@ Peroxidase 1.11.1.7 @ @ @ Superoxide dismutase 1.15.1.1 @ @ @ @ Glutaredoxin @ @ @ @ @ @ NAD(P) reductase @ @ @ @ Redox homeostasis and oxidative stress Thioredoxin @ @ @ Stress response Cold stress Cold-shock protein @ @ @ @ @ @ @ Starvation/stationary phase Ribosome-associated translation inhibitor RaiA @ @ @ @ @ @ @ Acid stress

BolA family transcriptional regulator @ Phage shock protein PspA @ @ RIP metalloprotease RseP @ @ RNA polymerase sigma factor RpoE @ Fe-S cluster repair SufS family cysteine desulfurase @ @ General stress response Universal stress protein @ @ @ @ Heat-shock protein @ RNA polymerase sigma factor RpoH @ @ @ Nitric-oxide reductase 1.7.2.5 @ @ @ NO-inducible flavohemoprotein @ Choline ABC transporter substrate-binding protein @ Choline dehydrogenase 1.1.99.1 @ Dimethylglycine demethylation protein DgcA @ @ @ Glycine betaine/L-proline ABC transporter ATP-binding protein ProV @ ProQ/FinO family protein @ Transcriptional regulator BetI @ @ Signal transduction Symmetrical bis(5-nucleosyl)-tetraphosphatase @ SOS response

Transcriptional repressor LexA @ Stringent starvation protein A @ RNA polymerase sigma factor RpoS @ @ @ Bifunctional (p)ppGpp synthetase/guanosine-3,5-bis @ @ GTP diphosphokinase RelA @ @ @ RNA polymerase-binding protein DksA @ @ @ Sugar phosphate stress

Methylglyoxal synthase @

Toxin/antitoxin Toxin/antitoxin system @ @ Sulfur compound metabolism Cysteine degradation D-cysteine desulfhydrase 4.4.1.15 @ @ Sulfate adenylyltransferase subunit CysD 2.7.7.4 @ @ HTH-type transcriptional regulator CysB @ @ Sulfite oxidation Sulfite dehydrogenase 1.8. 6). This suggests that sulfur cycling is a property of the colonizing core microbiota as a whole, and not of a single genus. A similar association between Arcobacter and Marinobacter species has been described in biogeochemical cycling, specifically in the environment of natural gas wells [57]. We believe that such interdependence and cooperation within microbial communities allows these communities to meet metabolic requirements and mount an efficient stress response, which are key determinants of their success. We further believe that this helps to explain the conserved pathobiota structure associated with POMS across distinct environments and oyster genetic backgrounds.

Supplementary Materials

Supplementary Supplementary Table 6: List of the overexpressed (enriched) proteins in the seven main genera found to colonize diseased oysters. A protein was considered enriched when it had a log2(EF) ≥ 1 with a pvalue ≤ 0.05 (Student's t-test) in at least two conditions.

Supplementary Conflict of interest statement. There are no conflicts of interest. This manuscript represents original results, and has not been submitted elsewhere for publication.


Bertrand Trilling

Sinara Vijayan

Cecile Goupil

Elio Kedisseh

Antoine Letouzey

Pierre Alain Barraud

Jean Luc Faucheron

Gaëlle Fiard

Dr Sandrine Voros
email: sandrine.voros@univ-grenoble-alpes.fr

Enhanced Laparoscopic Vision improves detection of intraoperative adverse events during laparoscopy

Keywords:

Background: The absence of visibility of the entire surgical scene in laparoscopic surgery can lead to unforeseen intraoperative complications. An Enhanced Laparoscopy Vision System (ELViS) was developed to eliminate the blind spots of the traditional endoscope by providing a broad view of the surgical scene from a distance, thanks to two additional images. This study assessed whether the broad view provided by the Enhanced Laparoscopic Vision (ELV) helped the surgeon to detect and react to an unexpected intraoperative adverse event (simulated hemorrhage) occurring while performing a standard task.

Methods: While participants were performing a standard task (sorting pins) on a dry lab laparoscopic simulator with or without ELV, a simulated bleeding (LED lighting) was randomly triggered. Perprocedure metrics were recorded and surgeons' feedback gathered at the end of the session.

Results: Thirteen Senior surgeons participated. Mean response time was significantly reduced when using ELV, with a similar number of simulated bleeding events between both modalities. All surgeons agreed that ELV could be helpful and constitutes an acceptable increase in cognitive load.

Conclusion:

In a dry lab setup, compared to traditional endoscopy, the broader field of view provided by ELV improved outcomes when dealing with unforeseen complications like bleeding.

Introduction

Minimally invasive surgery using laparoscopy represented a breakthrough in abdominal surgery at the end of the 20 th century, and is now considered the gold standard for many urological, digestive, and gynecologic interventions. Compared to standard laparotomy, laparoscopy improves recovery time and cosmesis, while reducing pain, overall complications, and length of hospital stay [START_REF] Keus | Laparoscopic versus open cholecystectomy for patients with symptomatic cholecystolithiasis[END_REF][START_REF] Beck | Robotic, Laparoscopic, or Open Hysterectomy: Surgical Outcomes by Approach in Endometrial Cancer[END_REF][START_REF] Ratti | Laparoscopic vs Open Surgery for Colorectal Liver Metastases[END_REF], without altering the quality of the surgery [START_REF] Bonjer | A Randomized Trial of Laparoscopic versus Open Surgery for Rectal Cancer[END_REF]. Laparoscopy can be used to treat many diseases. However, during the intervention, the surgeon has limited access and a restricted view of the surgical scene. Indeed, the endoscope inserted through the small incision magnifies the details of the dissection to allow more precise dissection of the organ of interest, but visibility of the surrounding abdominal cavity is lost. This loss of vision can be problematic, especially as surgical instruments can inadvertently encounter other structures. Blind spots can thus result in intraoperative complications such as bowel perforation [START_REF] Van Der Voort | Bowel injury as a complication of laparoscopy[END_REF] or hemorrhage [START_REF] Kaushik | Bleeding complications in laparoscopic cholecystectomy: Incidence, mechanisms, prevention and management[END_REF]7] that may have severe consequences if not dealt with during the procedure. In addition, the lack of visibility of the surroundings may lead to the surgeon and their assistant losing the orientation of the endoscope with respect to the surgical horizon. In this event, the surgeon's cognitive workload is increased [START_REF] Law | NASA-Task Load Index Differentiates Surgical Approach: Opportunities for Improvement in Colon and Rectal Surgery[END_REF] as is the risk of adverse events which may affect post-operative outcome [START_REF] Garcia-Martinez | Automatic detection of surgical haemorrhage using computer vision[END_REF].

Various approaches have been explored to improve the surgeon's awareness of the surroundings of the surgical scene, including panomorph lenses [START_REF] Roulet | 360° endoscopy using panomorph lens technology[END_REF], optical prisms [START_REF] Kobayashi | A Wide-Angle View Endoscope System Using Wedge Prisms[END_REF], enhanced multiple lenses [START_REF] Tseng | A Flexible FOV Capsule Endoscope Design Based on Compound Lens[END_REF], and panoramic views [START_REF] Naya | Usefulness of panoramic views for novice surgeons doing retroperitoneal laparoscopic nephrectomy: Panoramic views for novice surgeons[END_REF]. Alternatively, some groups have proposed increasing the field of view using distributed vision systems (either with multiple individual cameras or two to three stereoscopy systems) [START_REF] Tamadazte | Enhanced vision system for laparoscopic surgery[END_REF][START_REF] Cao | Comparison of a supplemental wide field of view versus a single field of view with zoom on performance in minimally invasive surgery[END_REF][START_REF] Sumi | A prospective study of the safety and usefulness of a new miniature wide-angle camera: the "BirdView camera system[END_REF][START_REF] Arain | A randomized comparison of laparoscopic, magnetically anchored, and flexible endoscopic cameras in performance and workload between laparoscopic and single-incision surgery[END_REF][START_REF] Kim | Large-Field-of-View Visualization Utilizing Multiple Miniaturized Cameras for Laparoscopic Surgery[END_REF].

In our laboratory, based on preliminary work by Tamadazte et al. [START_REF] Tamadazte | Enhanced vision system for laparoscopic surgery[END_REF][START_REF] Tamadazte | Multi-view vision system for laparoscopy surgery[END_REF], we developed an Enhanced Laparoscopy Vision System (ELViS) to overcome the blind spots inherent to laparoscopic surgery. The system is a camera trocar that has been "augmented" by two miniature cameras. After insertion of the ELViS into the abdominal cavity, the two miniaturized cameras are deployed on either side of the trocar, and a standard endoscope is inserted inside its hollow shaft (Figure 1). Since the additional cameras are placed on the abdominal wall, they provide a perspective from a distance, while the surgery is performed mainly using the traditional endoscopic view zoomed on the organ of interest. With the two additional side cameras, the ELViS continuously provides a full view of the surgical scene, like in laparotomy, to help eliminate the blind spots inherent to laparoscopy.

In [START_REF] Tamadazte | Multi-view vision system for laparoscopy surgery[END_REF], we focused on presenting the ELViS' design and a preliminary validation of its usability in a dry lab setting. With this study, our objective was to focus on another validation aspect by exploring the potential clinical added-value of the system, based on pre-clinical evaluations. We hypothesized that the Enhanced Laparoscopic Vision (ELV) provided by the two additional cameras in the ELViS could improve the surgeons' awareness of the surgical scene and how they manage adverse intraoperative events such as bleeding. The aim of the study presented here was to assess whether providing an ELV of the laparoscopic scene could improve the capacity of experienced surgeons to detect and deal with unexpected intraoperative lateral stimuli in a specifically-designed dry lab experiment.

Material and Methods

Study setting

Abdominal training test bench

We searched the surgical literature for a test bench that would meet our needs for the tests to be performed. Unfortunately, no appropriate test bench was found, as standard laparoscopic trainers allow the surgeon to learn to perform tasks with laparoscopic instruments. These trainers are designed to be easy to transport or store (cubic shape) but in no case represent the dimensions of an inflated abdomen in real surgeries. Wet benches simulating hemorrhage were an alternative that we considered, but the available models [START_REF] Xiao | An ex vivo liver training model continuously perfused to simulate bleeding for suture skills involved in laparoscopic liver resection: development and validity[END_REF][START_REF] Lee | High Fidelity Simulation Based Team Training in Urology: A Preliminary Interdisciplinary Study of Technical and Nontechnical Skills in Laparoscopic Complications Management[END_REF] did not seem easy to implement.

Faced with this lack of appropriate options, we decided to build our own testbench (Figure 2) which consisted of a curved opaque plexiglass sheet (55 cm*30 cm) attached to a metal structure (50 cm*30 cm) and provided a trocar entry-point. The curve in the plexiglass sheet simulated the inflated abdomen, and the surgical scene length corresponded to that of an inflated human abdomen (~25 cm).

A 10-mm trocar for the endoscope and two 5-mm trocars for insertion of instruments were inserted through small holes in the plexiglass sheet. The exercise board described below was placed on the test bench, under the plexiglass sheet, and the video feed from the three cameras (left camera, endoscope, and right camera) were displayed on three screens. All three screens were placed at the same height. The setup was the same for each participant.

A simplified version of the ELViS, where the system was maintained in a deployed position, was used to ensure an identical setup for all experiments. A fixed-position plastic casing was designed with the same dimensions as that of the camera "head" of the ELViS (Figure 2). Two mini-CMOS Raspberry Pi "Spy" cameras (https://www.adafruit.com/product/1937, Vertical angle: 62.2°, Horizontal angle: 48.8°

and resolution 3280 × 2464 pixels) were inserted through this casing, they had the same viewing angle and spacing as the ELViS. The casing was designed to ensure that the cameras were positioned with the same orientation and center-to-center distance as in the actual ELViS. Each camera was controlled by a Raspberry Pi mini-computer for real-time video-stream acquisition. A third Raspberry Pi camera was fixed to the proximal tip of a tube inserted through the endoscope trocar, to provide the endoscopic feed.

The Raspberry Pi is a small, low-cost computer that allowed the easy, real-time recording of the three video feeds (endoscope, lateral cameras providing the ELV) for the experiments. The system was then fixed by screws to the endoscope trocar close to the plexiglass sheet: the endoscope could be inserted through the trocar as usual and move independently, while the ELV cameras' orientation was maintained similar to that of the endoscope since it was rigidly fixed to the trocar. The ELV system clearly provided a wider view of the surgical scene, especially when the endoscope was zoomed in (Figure 3).

Exercise board

The exercise was designed to study how surgeons responded to an unexpected adverse event, without impacting the time to complete a complex task or the patients' safety. During the exercise, a luminous stimulus was triggered on the side of the surgical scene, to simulate an unexpected event, such as bleeding, in a blind spot for standard laparoscopic vision. The luminous stimulus migrated progressively to the center of the field, eventually becoming visible in the periphery of the working field (endoscope view). The surgeons were asked to react to this ("bleeding") event while continuing to perform a standard sorting task. They were asked to give equal priority to performing the task and controlling the bleeding and. The test bench was fully presented before starting the experiment.

The surgical scene in the simulator was composed of three plates located in the center, with two breadboards to the left and right of the plates (Figure 4). The primary task was to sort 16 multi-colored pins (4 pins x 4 colors) placed in the central plate into the two lateral plates, with two colors for each plate (4 pins x 2 colors). The breadboards had five low-intensity red LEDs each which simulated bleeding by lighting up. The surgeon could extinguish the LEDs by grasping a metallic plate placed between the two breadboards at the extremity of the board. The first sequence of LED illumination was triggered randomly between 2 and 10 s after the surgeon started to perform the task. Each of the 5 LEDs on one side would light successively from the side to the center of the surgical scene and remained lit until the surgeon extinguished the LED ribbon by grasping the metal plate. This luminous stimulus aimed to simulate a bleeding coming from outside the surgical scene and progressively gaining the center. Once the "bleeding" was stopped, the waiting time was randomized (<20 s) before the next round of stimulus was triggered. The bleeding simulation and data/video recording was programmed in Python (https://www.python.org/) on one of the three Raspberry Pis used for video capture. Figure 4 illustrates the setup for the test. A video showing the feeds from the three cameras on the display screens is provided as supplementary material.

Population and Experimental protocol

A cohort of experienced laparoscopic surgeons (Digestive surgeons, Urologists and Gynecologists)

working at Grenoble Alpes University Hospital were recruited for the study. All surgeons were senior surgeons familiar with laparoscopic techniques (>100 laparoscopic procedures as operating surgeon).

Each experimental session included only one participant and always started with a presentation of the ELViS project and the manipulation of the ELViS prototype under development, so that the participant would better understand its potential added-value when performing live surgery. The experimental setup and the exercise board were thoroughly described, emphasizing how the visual information from each camera was displayed. The task to be performed and the means to stop the LED signal during the exercise was explained to participants. For all exercises performed during the session, the same senior laparoscopic resident (CG) held the endoscope and provided an image centered on the surgical instruments. To allow participants to familiarize themselves with the three-screen display and the use of ELV, a peg-transfer exercise was performed with the three images displayed, and repeated (at least three times) until the participant felt comfortable with the setup.

Participants were randomized to start the exercise with or without ELV. In the "without ELV" setting, the side screens were turned off so that only the video feed from the endoscope was displayed. After completing the first test, the exercise was performed in the second condition (with ELV if the first round was with a standard endoscope or vice versa). Total time to complete the exercise and average time for the surgeons to respond to bleeding (LED stimulus) in the exercise were recorded throughout the experimental sessions. The average observable motion of the endoscope in the video feed was also measured using Optical flow (Gunner Farneback's state-of-the-art algorithm [START_REF] Farnebäck | Two-Frame Motion Estimation Based on Polynomial Expansion[END_REF], from the OpenCV computer vision library [START_REF] Bradski | {The OpenCV Library}[END_REF]). We used the endoscopic view to determine the average optical flow over the whole duration of the exercise. Although this value is approximate due to the incompletely static nature of the scene (the surgeon's instruments are moving), the average amplitude of displacement of the endoscope, when the endoscope was used alone, or when using the ELV. The data was analyzed to determine whether ELV altered how the assistant handled the endoscope in any way.

A questionnaire was administered at the end of the experiment (Appendix) to assess subjective parameters such as how well the experiment reflected real-life surgery and to record surgeons' feedback and impressions of ELV and the ELViS. Three display arrangements were described orally to the participants: a three-screen horizontal interface like in the experimental setup, a two-screen horizontal interface (one for the endoscope view and one with the views from the two lateral cameras), and a twoscreen vertical interface (one for the endoscope view and one with the views from the two lateral cameras). The idea of the panoramic view was described but not presented. Participants were asked which screen interface they thought would be most suitable, and if they had any suggestions as to how to improve the display setting.

Compliance with ethical standards

All participants signed a consent form for participation in the study and a confidentiality agreement. No ethical approval was necessary according to current French legislation, since the study did not involve interventions on live animals or humans. The authors have declared any potential conflicts of interest.

Data analysis

Data is presented as mean and standard deviation. As the data did not follow a normal distribution, Wilcoxon's tests were used to compare the average response time to bleeding, the total time taken to complete the task, the level of movement of the endoscope with or without the ELViS, and the mean number of times the LED stimulus was controlled by the surgeon. Statistical analyses were performed using IBM SPSS Statistics v26 (IBM Corp., USA).

Raw data are available upon request to the corresponding authors.

Results

Participants

Thirteen surgeons participated in the experiment and were assisted by the same senior resident. They had a mean post-residency experience of 7.7 years (1-30). All but two participants were right-handed.

Eight of the participants were senior consultants (Professor of Surgery or Attending), and the other five were Surgical Fellows with at least 1 year of practice (1-4 years). The areas of expertise of the surgeons were as follows: Digestive surgery (n=8), Gynecology (n=3), and Urology (n=2). On average, participants performed 4.4 (3-6) laparoscopic surgeries per week.

Bench metrics

Table 1 summarizes the results relating to the time required to complete the task, the time before the surgeon responded to a bleeding event, the movement of the endoscope by the assistant (average optical flow throughout the exercise), and the number of times the surgeon stopped the simulated bleeding.

The mean time to respond to bleeding was significantly lower using ELV compared to conventional endoscopy (6.41 s vs 11.36 s, p:0.007). The number of simulated bleeding events dealt with during the exercise was similar in both modalities. All bleeding events were dealt with by surgeons. Overall, the time to complete the task was not statistically different whether ELV was used or not (138 s vs 128 s, p:022). Two participants took more time to complete the task with ELV than with the endoscope alone, but the minimum time to complete the task was measured for a participant using ELV (78 s). There was no difference in mean normalized camera movement with or without ELV, thus the distance covered by the camera was similar: The person in charge of displacing the laparoscope (CG) did not seem to be influenced in her management by the presence or absence of ELV.

Questionnaire

Answers from the questionnaire are summarized in Table 2. Based on these answers, 76% (10/13) of participants fully agreed that the ELV setting did not interfere with the surgical procedure. All participants agreed that the exercise designed for the purpose of this study had constraints (display, bleeding management) similar to real-life surgery (13/13). All surgeons declared they would accept to use the ELViS in clinical practice if it took less than 10 min to set up, but two declared they would only use the ELViS for long and difficult surgeries. The benefits of ELV indicated by the participants were: elimination of blind spots (instrument insertion, blind instruments manipulated by surgical assistant), early detection of adverse events, overall safety improvement, help with endoscope orientation for the assistant, assistance with display difficulties (such as grasping failure or bowel displacement), and other complex tasks. The main concerns expressed in relation to using the ELViS were price, duration of setup, learning curve for display use, and size of the equipment that had to be added to the existing laparoscopic screen. The screen setup with three horizontal screens was the preferred display configuration (10/13 participants). A small side screen located to the right of the laparoscopic image displaying the two images from the lateral cameras was also considered a good alternative by 2 participants. One participant preferred the panoramic view.

Discussion

This study aimed to determine whether providing additional vision (ELV) to the surgeon during laparoscopic surgery allowed them to detect unexpected events on a bench test simulating a bleeding event. The results, based on reaction times following onset of the simulated bleeding, indicate that detection was improved without affecting the time required to complete a complex task. Surgeons' feedback through a questionnaire indicated that the device was associated with an acceptable cognitive load.

One of the main issues during laparoscopic surgery is the existence of "blind spots". At times, the surgeon loses sight of their instruments, and the instruments inserted and controlled by the surgical assistant. Inexperienced assistants can easily damage bowel tissue or tear organs as they attempt to orient their instruments appropriately. Undetected bowel injury may occur in 0.01-0.08% of laparoscopic procedures, and can have dramatic effects on the post-operative course [START_REF] Francis | EAES classification of intraoperative adverse events in laparoscopic surgery[END_REF][START_REF] Bishoff | Laparoscopic Bowel Injury: incidence and clinical presentation[END_REF][START_REF] Picerno | Bowel injury in robotic gynecologic surgery: risk factors and management options. A systematic review[END_REF]. ELV improved the mean time to stop simulated bleeding, and most participants indicated that they would use the device in the operating room.

Our laparoscopic simulator of unexpected adverse events was designed specifically for this study. To our knowledge, no existing simulator of adverse events during laparoscopic interventions has been described previously, except for wet lab simulators. These simulators consisted of a mock organ perfused with liquid that leaks in the event of injury, and are used to train for hemorrhage control [START_REF] Xiao | An ex vivo liver training model continuously perfused to simulate bleeding for suture skills involved in laparoscopic liver resection: development and validity[END_REF][START_REF] Lee | High Fidelity Simulation Based Team Training in Urology: A Preliminary Interdisciplinary Study of Technical and Nontechnical Skills in Laparoscopic Complications Management[END_REF].

In this case, the hemorrhage occurs on the organ on which the operator is working. One of the purposes of ELV was to be able to detect an adverse event (such as bleeding) early, even when it occurs in a blind spot for traditional laparoscopy. The wet simulators described in the literature were not appropriate to assess this aspect of adverse laparoscopic events. In addition, they represent a cumbersome experimental setup. For the purposes of our research, we developed a new dry lab laparoscopic simulator to represent an adverse event, in this case a hemorrhage starting laterally and moving progressively into the surgical area.

The study presented here has several limitations. Our laparoscopic simulator was designed specifically for this study to assess unexpected events occurring in blind spots during laparoscopic surgery and was not validated before use. Nevertheless, the participants declared that the constraints during the exercise were similar to those encountered in real surgery. In addition, our results might be underpowered because of the size of the study population (13 expert surgeons). Most of the expert surgeons in the institution agreed to participate in the study, but only 13 surgeons managed to fit the experiment into their schedule during the study period. Finally, the duration of the intervention to be performed was much shorter than a real-life surgical intervention. The full session lasted approximately 1 hour from the presentation of the ELViS project, to the actual experiment and gathering responses to the questionnaire.

Other groups have also attempted to improve vision for surgeons performing laparoscopic surgery. For example, Naya et al. [START_REF] Naya | Usefulness of panoramic views for novice surgeons doing retroperitoneal laparoscopic nephrectomy: Panoramic views for novice surgeons[END_REF] studied how a broad field of view helped novice surgeons performing nephrectomy by retroperitoneoscopy. They obtained a panoramic view by interlinking subsequent video frames acquired from the endoscope feed and displayed the image on an auxiliary screen. While the surgery was performed, the surgical scene changed with the dissection and mobilization of anatomical structures. The surgeon could then obtain a new panoramic view by starting a new iteration. Resident surgeons using the panoramic view performed the whole procedure (212 min vs 254 min, p=0.0489) and ligation of renal artery (69 min vs 106 min, p=0.0016) faster and with less blood loss (87 mL vs 334 mL, p=0.0005). However, the time from ligation of the renal artery to cutting of the renal vein, and the time after cutting the renal vein were not significantly different. ELV provides a real-time image of the surgical surroundings, which is not the case for the system developed by Naya et al., where the panoramic view must be refreshed at the surgeon's request. However, because the exercises in our study were much shorter than the average duration of real surgery (a few minutes compared to one or more hours), ELV had no impact on speed of surgery. Longer experiments would be needed to assess this aspect, although we expect that ELV could help reduce the duration of surgery for long surgeries involving complex tasks.

Other devices have also been developed to enhance laparoscopic vision. Roulet et al. [START_REF] Roulet | 360° endoscopy using panomorph lens technology[END_REF] proposed the use of a panomorph lens at the tip of the endoscope to provide a high definition 180° wide-angle view.

In addition to problems with lens manufacturing, the screen display from this type of lens may seem unfamiliar as the panomorph view includes distortion. In the display setting proposed, the surgeon could attenuate distortion in the region of interest without moving the endoscope. Kobayashi et al. [START_REF] Kobayashi | A Wide-Angle View Endoscope System Using Wedge Prisms[END_REF] developed a double prism automated endoscopic system that could provide a wider image by shifting the focus to the edge of the standard endoscope. However, the dimensions of the system were incompatible with insertion through a standard laparoscopic trocar.

The person manipulating the laparoscope (surgical assistant) must provide the appropriate point of view for the surgeon and display the surgical scene when required during surgery. One of the roles of the assistant is to align the endoscope by rotating the laparoscope to focus the narrow field of view correctly with respect to the background scene, and misalignments could adversely affect performance, especially for junior surgeons [START_REF] Rhee | The effects of viewing axis on laparoscopic performance: a comparison of non-expert and expert laparoscopic surgeons[END_REF]. The ELViS could indirectly enhance learning of laparoscopic surgery by helping the surgical assistant. When asked, most surgeons felt that ELViS could represent a significant aid to the assistant. In our study, we saw that the ELViS did not affect how the assistant handled the endoscope in terms of total movement distance measured for this camera. In future experiments, emphasis will be placed on assessing the viewing axis.

Conclusion

The results presented in this paper describe the positive impact of providing additional visual information to surgeons for the early detection of an adverse event in a dry lab simulation, without affecting the time to complete a task. Use of the ELViS was considered to improve some aspects of the quality of surgery. Surgeons mentioned that they would prefer to use the ELViS for long and difficult surgery out of concern for the cost of the device. We plan to perform further studies to bring the ELViS into a clinical setting. The device helps manage bleeding during the procedure 6 7 0 0

The device helps handle obstacles better than during typical laparoscopic surgery 6 7 0 0

The device helped the assistant to be more effective during the experiment

4 7 2 0
This device will contribute to patient safety during surgery 5 8 0 0

Table 2. Summary of responses for each item of the questionnaire.

Figure 1 .BFigure 2 .

 12 Figure 1. A. ELViS prototype in the folded position. The device is an enhanced trocar that is inserted through a 15-mm port and deployed inside the abdomen. The micro cameras with integrated LED placed in the camera module are connected to the computer via USB connectivity. B. ELViS prototype inserted through the upper plate of a laparoscopic simulator and deployed. The side cameras are placed as far as possible from the surgical scene to provide a wide view.

Figure 3 .

 3 Figure3. The endoscope can be moved vertically, and the tip of the endoscope can be approached or withdrawn from the surgical scene to zoom in or out. A. When distance between the tip of the endoscope and the surgical scene measures 13 cm, the endoscope camera provides a 125-mm horizontal field of view. B. When the distance between the tip of the endoscope and the surgical scene measures 6 cm, the endoscope camera provides a 62.5-mm horizontal field of view. The ELViS gives a 225-mm horizontal field of view whatever the endoscope's position.

Figure 4 .

 4 Figure 4. Exercise board designed to simulate bleeding during an intervention. 1) The two white breadboards (5 LEDs each) placed laterally.2) The three central plates into which the 16 multi-colored pins were to be sorted. 3) The metal plate in the center between the two breadboards was the switch used to stop the simulated bleeding.

Table 1 .

 1 Time-to-completion, response time, number of interruptions, and mean normalized movement of the endoscope during simulated interventions.

				Endoscope only	ELV	p-value
	Time	to	complete	138.15 (26.88)	128.54 (37.80)	0.22
	exercise (in seconds)
	Mean reaction time to	11.37 (8.10)	6.41 (3.06)	0.007
	bleeding (in seconds)
	Mean	normalized	0.11 (0.03)	0.12 (0.03)	0.12
	endoscope movement by
	assistant (movement over
	time)
	Mean	no.	of	6.6	5.8	0.43
	interruptions to control
	simulated bleeding

Acknowledgments

The authors would like to thank the participants: Dr Francois Istasse, Dr Mathieu Jolivet, Dr Edouard Girard, Dr Pierre Yves Sage, Pr Jean Alexandre Long, Dr Nicolas Taton, Dr Julio Abba, Dr Fabian Reche, Dr Fabien Stenard, Pr Mircea Chirica, Dr Camille Micoud, and Dr Sophie Gobillot. They would also like to thank the Centre d'Investigation Clinique -Innovation Technologique of Grenoble Alpes University Hospital and Maighread Gallagher-Gambarelli (TWS Editing) for Editorial support.

Funding: This work was funded in part by the French National Research Agency (Agence Nationale de la Recherche) as part of the DEPORRA2 project under grant ANR-14-CE17-0009-01 and the Investments for the Future (Investissements d'Avenir) program (Labex CAMI) under grant ANR-11-LABX-0004. Other costs were covered by recurrent institutional funding (CHU Grenoble, INSERM).

Highlights vision system developed for use during laparoscopic surgery.

During our ELViS project, two Distributed Laparoscopy devices [START_REF] Sumi | A prospective study of the safety and usefulness of a new miniature wide-angle camera: the "BirdView camera system[END_REF][START_REF] Rivas-Blanco | Global vision system in laparoscopy[END_REF] were reported and tested successfully on animals and humans. Duration of procedure was not compared to a control group. Both systems are attached to the abdominal wall, providing a different point of view to that of a traditional endoscope. To avoid the cognitive load required to combine images from very different points of view, we chose to design an "augmented trocar" with miniature cameras to provide a similar axis of vision to that of the endoscope for the overall view of the surgical scene.

Kim et al. [START_REF] Kim | Large-Field-of-View Visualization Utilizing Multiple Miniaturized Cameras for Laparoscopic Surgery[END_REF] presented a Trocar Camera Assembly which concept of use was similar to our ELViS prototype, with deployment of additional cameras after insertion of an enhanced trocar. Their prototype deployed four small cameras, and images were displayed on a screen and merged with the endoscope image. The field of view using the Trocar Camera Assembly was increased by 133% compared to a standard 10-mm endoscope. Modifications will be needed before it can be used on animals or humans as the foldable camera holders suffer from the same fragility as Tamadazte's early prototype [START_REF] Tamadazte | Multi-view vision system for laparoscopy surgery[END_REF]. The authors chose to merge the different images in a single display using stitching techniques. However, laparoscopic images are not optimally-adapted for stitching: in a real clinical setup, parallax issues would be encountered, resulting in phantom instruments and reconstruction inconsistencies. Thus, these types of display would be difficult to use in a clinical setting. In our system, we overcame this issue by choosing to have as few overlaps as possible between the lateral images and the endoscope.

Display of additional information during surgery might be challenging since the cognitive burden during surgery is already high. Adding two screens to the surgeon's view could become cumbersome.

Understanding the views provided by the ELViS on the lateral screens seemed easier thanks to the small overlap of corresponding landmarks on the endoscope and the ELViS screens. When asked, none of the participants indicated that the three-screen setup was a hindrance while performing the exercise. Some participants proposed an enhanced screen setup with a small side screen located to the right of the laparoscopic image, displaying the two images from the lateral cameras. This setup would add to the ergonomic advantages and convenience, as only one additional screen would be brought into the operating room. However, this type of display would considerably reduce the size of the images from the side cameras.

Supporting information captions:

Appendix "Questionnaire": questionnaire submitted to the participating surgeons for the qualitative assessment of the Enhanced Laparoscopic Vision system Video "ELV to improve laparoscopy": video showing the exercise performed for the evaluation, through the 3-screen interface.