Margot Doberva

Sophie Sanchez-Ferandin

Eve Toulza

Philippe Lebaron

Raphaël Lami

Diversity of quorum sensing autoinducer synthases in the Global Ocean Sampling metagenomic database

Keywords: Quorum sensing, Acyl-homoserine lactone synthases, Global Ocean Sampling, Environmental metagenomes

Quorum sensing (QS) is a cell-to-cell signalling pathway that allows bacteria to synchronize their genetic expression. It is mediated by autoinducers (AI), including (1) acyl-homoserine lactones (AHLs or AI-1), produced by Proteobacteria using AinS, LuxI and HdtS synthase families and (2) furanosyl-diester-borate (FDB or AI-2), produced by a large range of phylogenetically diverse bacteria and synthetized by the LuxS family. Few data have been collected about the presence and importance of QS in marine waters using culture independent methods. In this study, we examined the presence and the diversity of AI-1 and AI-2 synthases in the Global Ocean Sampling (GOS), a large metagenomic database, covering 68 stations across 3 oceans. We built 4 reference protein databases with maximal phylogenetic coverage containing all known AI synthase sequences to retrieve AI synthases sequences from the GOS metagenomes. We retrieved 29 environmental sequences affiliated to LuxI (synthesizing AI-1), 653 related to HdtS (AI-1), 31 related to LuxS (AI-2) and only one for AinS (AI-1). AI synthases sequences were found in the 3 oceans covered by the GOS cruise and spanned a large phylogenetic diversity. These data revealed a large number of new marine AI sequences, suggesting that QS based on AI-1 diffusion is a widespread mechanism in the marine environment.

INTRODUCTION

Quorum sensing (QS) is a cell-to-cell bacterial communication mechanism, allowing bacteria to sense their population density [START_REF] Nealson | Autoinduction of bacterial luciferase[END_REF] and coordinate their gene expression levels [START_REF] Bassler | How bacteria talk to each other: regulation of gene expression by quorum sensing[END_REF][START_REF] Fuqua | Listening in on bacteria: acyl-homoserine lactone signalling[END_REF] and physiological activities [START_REF] Miller | Quorum sensing in bacteria[END_REF]. To perform QSbased communication, bacteria produce, secrete and accumulate small hormone-like molecules called autoinducers (AI) in the nearby environment [START_REF] Fuqua | Quorum sensing in bacteria: the LuxR-LuxI family of cell densityresponsive transcriptional regulators[END_REF]. As cell density increases, the concentration of these AI increases. When a given threshold concentration is reached, targeted bacterial partners can respond to these compounds and modify their gene expression, and thus their behaviour and phenotype [START_REF] Withers | Quorum sensing as an integral component of gene regulatory networks in Gram-negative bacteria[END_REF][START_REF] Bassler | Small talk: cell-to-cell communication in bacteria[END_REF]. It has been shown that QS systems regulate and synchronize activities like biofilm production [START_REF] Parsek | Sociomicrobiology: the connections between quorum sensing and biofilms[END_REF][START_REF] Dickschat | Quorum sensing and bacterial biofilms[END_REF]), nodulation [START_REF] Cha | Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria[END_REF][START_REF] Loh | Quorum sensing in plant-associated bacteria[END_REF], bioluminescence [START_REF] Waters | QUORUM SENSING: cell-tocell communication in bacteria[END_REF], virulence factor production [START_REF] Smith | P. aeruginosa quorum-sensing systems and virulence[END_REF] and many others [START_REF] Diggle | Quorum sensing[END_REF]). The coordination of bacterial community activities conveys an ecological advantage to the population [START_REF] Case | AHL-driven quorum-sensing circuits: their frequency and function among the Proteobacteria[END_REF].

QS mechanisms are well described in medical or agronomic model bacterial strains [START_REF] Cha | Production of acyl-homoserine lactone quorum-sensing signals by gram-negative plant-associated bacteria[END_REF][START_REF] Loh | Quorum sensing in plant-associated bacteria[END_REF] known for their pathogenicity [START_REF] De Kievit | Bacterial quorum sensing in pathogenic relationships[END_REF] or bioengineering potential [START_REF] March | Quorum sensing and bacterial cross-talk in biotechnology[END_REF][START_REF] Brenner | Engineering microbial consortia: a new frontier in synthetic biology[END_REF]. By contrast, little attention has been paid to QS in the functioning of natural microbial communities, including in marine waters [START_REF] Decho | Quorum sensing in natural environments: emerging views from microbial mats[END_REF]. This is because bacterial densities in seawater (10 5 to 10 6 cells ml -1 ) are typically below the known thresholds that enable QS [START_REF] Mohamed | Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges[END_REF]. However, at a micrometer scale many ecological niches (such as organic matter particles or microalgal blooms) harbour bacterial concentrations compatible with QS [START_REF] Gram | Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow[END_REF][START_REF] Mohamed | Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges[END_REF]. In support of this hypothesis, the potential for QS in planktonic marine bacteria has been reported in many cultivated strains. These results were acquired either by directly detecting communication compounds or inferred by sequencing genes involved in QS in Proteobacteria [START_REF] Gram | Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow[END_REF][START_REF] Wagner-Döbler | Discovery of complex mixtures of novel longchain quorum sensing signals in free-living and hostassociated marine Alphaproteobacteria[END_REF].

The AI synthases are the key enzymes involved in AI production. It is now well established that the acylhomoserine lactone synthases (AHL or AI-1 synthases) are encoded by 3 groups of genes: luxI-like [START_REF] Engebrecht | Identification of genes and gene products necessary for bacterial bioluminescence[END_REF], ainS-like [START_REF] Gilson | AinS and a new family of autoinducer synthesis proteins[END_REF] and hdtS-like [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF][START_REF] Burton | Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt[END_REF][START_REF] Rivas | Second acyl homoserine lactone production system in the extreme acidophile Acidithiobacillus ferrooxidans[END_REF]. By contrast, luxS-like genes encode the (2S,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran-borate synthase or S-THMF-borate synthase or furanosyl diester borate synthase or (2R,4S)-2-methyl-2,3,3,4-tetrahydroxytetrahydrofuran synthase or R-THMF synthase, also known as the AI-2 synthases [START_REF] Xavier | LuxS quorum sensing: more than just a numbers game[END_REF][START_REF] Miller | Salmonella typhi mu rium recognizes a chemically distinct form of the bacterial quorum-sensing signal AI-2[END_REF]). The AI-1 synthases have been identified in cultivated marine strains from the groups Roseobacter and Vibrio [START_REF] Gram | Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow[END_REF][START_REF] Schaefer | Long-chain acyl-homoserine lactone quorum-sensing regulation of Rhodobacter capsulatus gene transfer agent production[END_REF][START_REF] Wagner-Döbler | Discovery of complex mixtures of novel longchain quorum sensing signals in free-living and hostassociated marine Alphaproteobacteria[END_REF]) and the AI-2 [START_REF] Chen | Structural identification of a bacterial quorum-sensing signal containing boron[END_REF] have been observed in the Vibrio genus [START_REF] Bassler | Cross-species induction of luminescence in the quorum-sensing bacterium Vibrio harveyi[END_REF][START_REF] Bassler | How bacteria talk to each other: regulation of gene expression by quorum sensing[END_REF]. By contrast, very little attention has been paid to the diversity of AI synthases in whole marine microbial communities. As only 1% of marine bacterial communities in the water column are readily cultivable by standard methods [START_REF] Amann | Phylogenetic identification and in situ detection of individual microbial cells without cultivation[END_REF]), an overview of potential QS mechanisms in the marine environment remains largely incomplete.

The Global Ocean Sampling (GOS) database is a large collection of marine metagenomes from 68 sampling sites. A total of 6.3 billion bp were Sanger sequenced from microplankton samples [START_REF] Rusch | The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific[END_REF]) from which the metaproteome (all coding sequences) was also predicted [START_REF] Yooseph | The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families[END_REF]). The GOS datasets mainly comprise sequences from the free-living fraction (FL) of bacterioplankton (0.1 to 0.8 µm pore size filter). Additionally, for 8 stations the particle-attached fraction (PA) (0.8 to 3 µm) has been sequenced (GS000, GS001, GS048, GS108, GS110, GS112, GS117 and GS122). The GOS offers a large snapshot of the diversity and functional potential of marine microbial communities and has been successfully explored, for example, for the potential to utilize selenium [START_REF] Zhang | Trends in selenium utilization in marine microbial world revealed through the analysis of the Global Ocean Sampling (GOS) project[END_REF] or to metabolize various chemical forms of iron [START_REF] Toulza | Analysis of the Global Ocean Sampling (GOS) project for trends in iron uptake by surface ocean microbes[END_REF].

In this study, we investigated the diversity patterns of QS AI synthases in the bacterial communities represented in the GOS metagenomes. We report the presence, the large diversity of both AI-1 and AI-2 synthase sequences and their distribution throughout the 3 oceans covered by the GOS expedition, and we highlight new sequences of AI synthases.

MATERIALS AND METHODS

Construction of reference databases for AI synthase proteins

We built a total of 4 databases, one for each fam ily of AI synthases: AinS-like, LuxI-like, HdtS-like and LuxSlike. Following the approach described in [START_REF] Toulza | Analysis of the Global Ocean Sampling (GOS) project for trends in iron uptake by surface ocean microbes[END_REF], we first selected protein sequences that have been functionally characterized in previous publications. To improve taxonomic coverage, we added to our reference databases additional sequences annotated as AI synthase from the GenBank non-redundant (NR) database (www.ncbi. nlm.nih.gov/). Each database was aligned using Clustal W [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF] in Jalview [START_REF] Waterhouse | Jalview version 2-a multiple sequence alignment editor and analysis workbench[END_REF] to discard redundant and partial se-quences. Our final AI databases comprised 569 LuxI, 30 AinS, 214 HdtS and 297 LuxS annotated protein sequences.

Screening for AI synthases in the GOS metagenome

The GOS expedition and its subsequent meta-genome analysis have been described previously (Rusch et al. 2007[START_REF] Yooseph | The Sorcerer II Global Ocean Sampling expedition: expanding the universe of protein families[END_REF]. Briefly, 200 l of seawater was collected at 68 different sampling stations and filtered onto 4 different successive filters with a porosity of 20, 3, 0.8 and 0.1 µm. In this work, we discarded 10 stations where no AI sequences were found (GS006, GS038-040, GS042-046, GS050). In our study, we also focused on both FL (0.1 to 0.8 µm) and PA (0.8 to 3 µm) fractions, which constitute the most complete sequence datasets in terms of sequencing effort. The GOS database contains sequences collected from various habitats within the Atlantic, Pacific and Indian Oceans. A total of 11 different habitat types were sampled, including open ocean (23 stations), coastal (22 stations), coral reefs (4 stations), estuary (2 stations) and 7 others [START_REF] Rusch | The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific[END_REF]. Environmental variables were also retrieved from [START_REF] Venter | Environmental genome shotgun sequencing of the Sargasso Sea[END_REF] and [START_REF] Rusch | The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific[END_REF].

The detection of AI-1 and AI-2 synthases from the GOS database (proteins predicted from reads) was conducted by sequence homology searches using the BLAST (Basic Local Alignment Search Tool) algorithm [START_REF] Altschul | Basic local alignment search tool[END_REF]) and our AI synthase databases as the query sequences. All our AI protein BLAST searches were performed using the CAMERA portal (http:// camera. crbs.u csd. edu/ projects/ details.p hp?id=CAM_PROJ_GOS) [START_REF] Sun | Community cyberinfrastructure for Advanced Microbial Ecology Research and Analysis: the CAMERA resource[END_REF]) using blastall default parameters (Seshadri et al. 2007) (including FL and PA fractions). A Reciprocal Best BLAST Hit (RBH) analysis (Moreno-Hagelsieb & Latimer 2008) was then performed on the sequences retrieved from the GOS dataset. Briefly, putative environmental AI synthases were searched against NR database and those GOS sequences with a RBH to the same annotated AI synthases were assigned to the gene and designated as most related to that taxon.

We also compared the distribution of AI environmental protein sequences in both the FL and PA fractions from the 8 GOS sampling stations for which these data are available. We pooled all the retrieved AI protein sequences from PA and FL fractions from all 8 sites, and clustered the environmental and annotated protein sequences using CD-HIT [START_REF] Li | Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences[END_REF][START_REF] Fu | CD-HIT: accelerated for clustering the next-generation sequencing data[END_REF]. The clustering parameters were 40% sequence identity and a word size equal to 2. The differences in the number of environmental sequences affiliated to HdtS between the FL and PA fractions were tested for significance using a Wilcoxon test with R (www.rproject.org).

Normalization of AI counts in GOS metagenomes to single copy genes

The number of sequences matching to AI synthases in the GOS was normalized to the number of RpoB protein sequences present in the sample. That gene is present in only a single copy in bacterial genomes and is not prone to horizontal transfer and thus is also a good taxonomic marker [START_REF] Case | Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies[END_REF]. Following the same procedure as described for the AI sequences, we built an RpoB protein reference databases with a large taxonomic coverage containing 1000 sequences. We finally reported the number of each family of AI environmental sequences related to our reference sequences relative to the number of RpoB protein sequences in the GOS dataset. This allowed normalization to the number of bacterial genomes in the dataset.

Phylogenetic analysis

Phylogenetic analyses were conducted for each targeted family of AI synthase. All environmental and annotated sequences were aligned using Clustal W [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF]) in MEGA v.5.1 software [START_REF] Tamura | MEGA5: Molecular Evolutionary Genetics Analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF], and alignments adjusted manually. Phylogenetic trees were constructed using both Neighbour-Joining (NJ) and Maximum Likelihood (ML) methods for each kind of AI synthase protein sequence alignments. NJ analyses were conducted with gamma correction and 1000 bootstrap replicates. Protein model selection was performed from each protein dataset, and phylogenetic analyses were then conducted with the selected models (WAG model for LuxI, HdtS and LuxS) using gamma correction and 100 bootstrap replicates. As we obtained similar topologies from the 2 methods, only those obtained by NJ are presented here, but when useful, percentage of bootstrap supports obtained by ML searches are also mentioned.

RESULTS

Features of AI synthase protein reference databases

All our reference databases were built to have as broad a taxonomic coverage as possible, consisting of protein sequences assignable to AI synthases from the NR database at the time this study was conducted. The LuxI, LuxS and HdtS-like protein family databases had a large taxonomic coverage, which included the alpha-, beta-, gamma-and delta lineages of Proteobacteria. The LuxS protein reference database also contained annotated sequences from Spirochaetes, Bactero idales and the Gram-positive Firmicutes and Actino bacteria. The AinS family, limited to the Vibrionaceae, contained not only AinS but also LuxM and VanM synthases (AinS-like synthases). A total of 634 annotated protein sequences from the National Center for Biotechnology Information (NCBI) were affiliated to LuxI with a mean identity of 27% (number of conserved amino acids divided by total amino acids), 214 sequences to HdtS with a mean identity of 27%, 34% (Table 1). The mean length of these sequences was about 380 amino acids for AinS, 200 for LuxI, 250 for HdtS and 170 for LuxS.

Abundance and diversity of LuxI synthases in the GOS dataset

We retrieved 29 sequences related to LuxI AHL synthases in the GOS dataset. Those sequences were distributed over 10 stations of the GOS cruise (Table 1, Fig. 1) and appeared related to a large taxonomic diversity of AI-1 sequences in NCBI. These GOS sequences were aligned with annotated se-quences and phylogenetic reconstruction was per-formed (Fig. 2). All affiliated environmental sequences arose from Alphaproteobacteria (Fig. 2). A total of 19 environmental sequences (65%) matched with sequences belonging to Rhodobacterales (Rhodobacteraceae family), 2 (7%) with sequences belonging to Sphingomonadales (Sphingomonadaceae family) and 3 (14%) with sequences belonging to Rhizobiales (Bradyrhizobiaceae and Beijerinckiaceae families) (Fig. 2). These affiliations were supported by strong NJ bootstrap percentages (>99 for Rhodobacterales; 100 for Sphingomonadales; 97 and 100 for Rhizobiales; Fig. 2) and also in some cases by strong ML bootstrap percentages. Although well identified as AI-1 synthases by RBH, a large fraction of environmental sequences (14%) could not be unambiguously related to any annotated sequences.

LuxI sequences arising from PA fractions represented 10% of total retrieved LuxI sequences in the GOS dataset. These sequences were present at Stns GS048 and GS110. All these sequences appear enclosed in a larger phylogenetic group (defined with bootstraps values of 53/86 for NJ/ML, respectively; Fig. 2) comprising members of the Sphingomonodaceae family.

Abundance and diversity of HdtS synthases in the GOS dataset

A total of 653 environmental sequences related to HdtS were retrieved from the GOS meta-genomes (Table 1). Those sequences were found in all oceans crossed by the GOS expedition and in a large number of stations (57 different stations from a total of 58 sampled by the expedition) (Fig. 1). Collectively, all of these sequences were related to Alpha-, Beta-and Gammaproteobacteria. More precisely, a large fraction (19% or 127 sequences) of these sequences were related to the SAR11 group (Alphaproteobacteria, with bootstrap supports of 100 and 96 for NJ and ML, respectively), including its cultivated representative member Pelagibacter ubique (indicated by 'SAR11' sequences; Fig. 3). A lower fraction (3% or 21 se quences) of these sequences was related to the SAR116 group (Alphaproteobacteria, with bootstrap supports of 95 and 67 for NJ and ML, respectively; Fig. 3). Interestingly, one sequence was strongly associated to a sequence from Oceanicaulis (Rhodo-bacter a les), which belongs to the Hypho monadaceae family (bootstrap supports of 100 and 83 for NJ and ML, respectively). Among Gamma proteobacteria, a large fraction of sequences were related to the SAR86 group (23% or 153 sequences) with high bootstrap supports both in NJ and ML methods (100 and 62, respectively; Fig. 3). One sequence was related to Pseudomonadales, and a large fraction (21% or 140 sequences) appeared more closely related to other orders within Gamma proteo bacteria (Oceanospirillales, Chro matiales, Acidi th i-ob acillales and Triotrichales). Lastly, a significant number of seq uences appeared closely re lated to the Betaproteobacteria (represented by the Burkholderiales order, 19% or 126 sequences) and un classified Gammaproteobacteria (5% or 32 se quences) with relatively strong bootstrap supports (63 and 57 for NJ and ML, respectively; Fig. 3).

A few environmental sequences (19%) were recovered from the 8 sampling stations covering both the FL and PA fractions. Overall, a total of 63 sequences were recovered from the PA fraction. Interestingly, the 2 sequences GS051 FL and GS117 PA were more closely related to (NJ bootstrap support of 98), and found within, the SAR11 Alphaproteobacteria group. Six sequences from the PA fraction of the GS110 sampling site were recovered together and closely related to the Gammaproteobacteria, Francisella tularensis (NJ bootstrap support of 97; Fig. 3). To test whether there was any difference in the taxonomic composition between the FL and the PA fractions, we clustered all HdtS sequences using a threshold of 40% sequence identity, and found a total of 14 clusters (Table 2). As an example, cluster 6 contains environmental sequences related to SAR86 clade in the 2 fractions of the GOS Stns GS000, GS110 and GS112 (Table 2). In the environmental sequences, of which 11% were from the FL fraction and 15% from the PA fraction, we did not find any significant difference between the cluster affiliations of environmental sequences distributed in the 2 metagenome fractions (Wilcoxon test, p = 0.83).

Abundance and diversity of LuxS synthases in the GOS dataset

A total of 31 environmental sequences related to LuxS were detected in the GOS dataset at 3 sampling sites: GS000, GS013 and GS033 (Table 1, Fig. 1). All environmental sequences affiliated to LuxS were retrieved from the FL fractions. As previously noted, the LuxS family showed the widest taxonomic coverage compared to the other families. The environ mental sequences found were related to Gamma-proteobacteria (97% or 30 sequences, NJ and ML bootstraps support of 100 and 99%, respectively; Fig. 4) and to the Gram-positive bacteria from the Deinococcales (3% or 1 sequence, NJ and ML bootstraps support of 100 and 90%, respectively; Fig. 4). Among Gammaproteobacteria, 4 se quences (13%) group inside a clade found in both NJ and ML phylogenies, comprising sequences from Alishewanella and Rheinheimera species (ML bootstrap support of 92%; Fig. 4), but which were more closely related to Reinheimera (NJ and ML bootstraps support of 93 and 91%, respectively; Fig. 4). A large number of environmental sequences (65% or 20 sequences) seemed related to Shewanella species, although it was not well supported (except 3 GS000 FL se -quences with Shewanella frigidimarina, ML bootstrap support of 60%).

Diversity of AinS synthases in the GOS dataset

We retrieved only 1 sequence of AinS in the GOS dataset, located at Stn 148, in the Indian Ocean close to the Zanzibar coast in a fringing reef. The top high scoring BLAST pair was 'N-(3-hydroxybutanoyl)-Lhomoserine lactone synthase luxM' (WP_ 002541906.1) (70% sequence coverage and 90% identity) from Grimontia sp., a Vibrionaceae family member.

Correlation with environmental variables

For the LuxI group, we found no correlation with the environmental data. For HdtS family there was a positive correlation with metagenome size, and negative correlation between chlorophyll (Spearman ρ = -0.425, p = 0.002), nitrate (Spearman ρ = -0.553, p = 5.475 × 10 -5 ), silica concentration (Spearman ρ = -0.573801, p = 2.481 × 10 -5 ), primary production (Spearman ρ = -0.374, p = 0.01) and the number of environmental sequences related to HdtS. A link with LuxS-related environmental sequences and salinity was also observed (Spearman ρ = 0.341, p = 0.020). We did not find any correlation with the other environmental variables (temperature, iron concentration, habitat type) (Table 3).

DISCUSSION

Our study revealed the presence of AI-1 and 2 synthases, key enzymes in QS pathways, within the GOS predicted metaproteome. To date, QS genes have only been directly found in cultured marine bacteria [START_REF] Engebrecht | Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri[END_REF][START_REF] Gilson | AinS and a new family of autoinducer synthesis proteins[END_REF][START_REF] Gram | Possible quorum sensing in marine snow bacteria: production of acylated homoserine lactones by Roseobacter strains isolated from marine snow[END_REF][START_REF] Wagner-Döbler | Discovery of complex mixtures of novel longchain quorum sensing signals in free-living and hostassociated marine Alphaproteobacteria[END_REF] or indirectly inferred from AHL production and genome sequence annotation [START_REF] Mohamed | Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges[END_REF][START_REF] Cuadrado-Silva | Detection of quorum sensing systems of bacteria isolated from fouled marine organisms[END_REF], Doberva et al. 2014a,b). By contrast, no previous studies have examined the presence and diversity of those genes in the marine water column using culture-independent approaches.

We detected a large phylogenetic diversity of AI synthases in the GOS metagenomic libraries. However, it has not been possible to affiliate a large fraction of the retrieved sequences at a fine phylogenetic scale, as only a few marine cultivated strains harbour AI synthase genes that would allow a more accurate taxonomic association. At a higher phylogenetic level, the AI sequences retrieved from the GOS were affiliated to many different groups of bacteria. LuxI environmental sequences were mainly related to Rhodobacterales [START_REF] Cude | Acyl-homoserine lactone-based quorum sensing in the Roseobacter clade: complex cellto-cell communication controls multiple physiologies[END_REF], Shingomonadales and Rhizobiales, while HdtS sequences were mainly related to SAR11 and Pseudomonadales. These results must be very carefully interpreted, as it has been demonstrated that the phylogenetic signal in AI sequences may be affected by large horizontal transfer between bacterial groups [START_REF] Gray | The evolution of bacterial LuxI and LuxR quorum sensing regulators[END_REF][START_REF] Lerat | The evolutionary history of quorum-sensing systems in bacteria[END_REF]). However, our data clearly increase the currently known diversity of AI se quences. This high percentage of AI sequences without close relatives from known species in the GOS datasets clearly demonstrates that QS potential is present in a much wider diversity of marine bacteria than previously suspected from cultivable marine bacteria.

We may have underestimated the extent of AI diversity present in the GOS databases. Since our approach was based on sequence homology, we retained only the environmental sequences that were close to known and annotated sequences in databases to avoid false positives. Because of the high stringency of the search criteria used in this study (evalue 10 -50 for first BLAST and 10 -20 for RBH), it is possible that divergent AI environmental sequences were missed. This means that all AI sequences we can detect are dependent on the number and diversity of annotated sequences currently available in the NCBI NR database. The diversity of AI synthases is probably much greater than detectable by similarity searching. Despite this, the number of new AI se quences detected in this study was high, even though total diversity is still probably largely underestimated.

Another recurrent bias to assess when working with metagenomic datasets is the potential functional diversity of annotated proteins (Uchiyama & Miya zaki 2009[START_REF] Prakash | Functional assignment of meta genomic data: challenges and applications[END_REF]. Our work indicates that the majority of AI-1 synthases in the GOS dataset are related to the hdtS gene family. HdtS is a member of the lysophosphatidic acid acyltransferase family [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF] and harbours a dual functionality: acylation of lysophosphatidic acid [START_REF] Cullinane | Identification of two lysophosphatidic acid acyltransferase genes with overlapping function in Pseudomonas fluorescens[END_REF]) and AHL synthesis [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF][START_REF] Churchill | Structural basis of acylhomoserine lactone-dependent signaling[END_REF]). The production of AHL based on HdtS has been well demonstrated experimentally in Pseudomonas fluorescens [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF], Acidithiobacillus ferooxidans [START_REF] Rivas | Second acyl homoserine lactone production system in the extreme acidophile Acidithiobacillus ferrooxidans[END_REF]) and Nitrosomonas europea [START_REF] Burton | Identification of acyl-homoserine lactone signal molecules produced by Nitrosomonas europaea strain Schmidt[END_REF]. When expressed in Escherichia coli, HdtS protein enabled the production of 3-OH-C14:1-AHL, C10AHL and C6-AHL [START_REF] Laue | The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase[END_REF]. One possible mechanism is that HdtS could transfer acyl chains from acyl-ACP or acyl-CoA to S-adenosylmethionine to generate AHLs [START_REF] Cullinane | Identification of two lysophosphatidic acid acyltransferase genes with overlapping function in Pseudomonas fluorescens[END_REF]. Although the experimental evidence accumulated from the strains mentioned above indicates a strong link with AHL production, the large diversity of HdtS found in the GOS metagenomic libraries highlights the need to further confirm a role of diverse HdtS-like proteins in QS.

Our results did not reveal any major difference in AI diversity for HdtS between the FL (0.1-0.8 µm) and PA (0.8-3 µm) fractions. It is commonly thought that bacteria in sea-water occur in low densities and thus AHLs molecules produced by a cell would quickly be diluted before reaching a receiving cell, hampering QS [START_REF] Hmelo | Kinetic constraints on acylated homoserine lactone-based quorum sensing in marine environments[END_REF]. Thus, a recurrent hypothesis is that QS in marine communities may have more chance to occur in particleattached conditions where bacteria could be concentrated, such as on the surface or inside a particle of sinking organic matter [START_REF] Mohamed | Diversity and quorum-sensing signal production of Proteobacteria associated with marine sponges[END_REF][START_REF] Hmelo | Possible influence of bacterial quorum sensing on the hydrolysis of sinking particulate organic carbon in marine environments[END_REF]. The absence of a significant difference (Wilcoxon test, p = 0.83) in HdtS diversity distribution between PA and FL fractions is not surprising as marine bacterial groups can present both FL and PA lifestyles [START_REF] Ghiglione | Diel and seasonal variations in abundance, activity, and community structure of particleattached and free-living bacteria in NW Mediterranean Sea[END_REF][START_REF] Crespo | Taxonomic composition of the particleattached and free-living bacterial assemblages in the Northwest Mediterranean Sea analyzed by pyrosequencing of the 16S rRNA[END_REF]. As the presence of QS genes is similar between PA and FL microenvironments, future studies should focus on detecting differential expression of these genes. Interestingly, recent work conducted on Dinoroseo bacter shibae tends to support this hypothesis [START_REF] Patzelt | You are what you talk: quorum sensing induces individual morphologies and cell division modes in Dino roseo bacter shibae[END_REF].

The LuxI and HdtS AI-1 synthase sequences re trieved in this study were detected in metagenomic libraries from the Atlantic, Pacific and Indian Oceans covered by the GOS. This result highlights that AI synthases were present in a wide range of marine environmental conditions, including open and coastal oceans, coral reefs and salt marshes. This observation supports the hypothesis that QS mechanisms may be widespread in many marine environments, and opens the wider question of the potential role of QS in marine prokaryotic species. Furthermore, the negative correlation between environmental se-quences related to HdtS and environmental variables linked to coastal marine habitats suggests that HdtS was more prevalent in bacteria living in the open ocean.

Previous studies have revealed a wide phylo-genetic distribution of the LuxS group among Proteobacteria, including the marine bacteria genera Vibrio [START_REF] Bassler | How bacteria talk to each other: regulation of gene expression by quorum sensing[END_REF] and Shewanella [START_REF] Bodor | Potential for luxS related signalling in marine bacteria and production of autoinducer-2 in the genus Shewanella[END_REF], as well as within the Firmicutes, Actinobacteria, Bacteroidales, Deinococcales (Deinococcus) and Spirochaetes [START_REF] Schauder | The LuxS family of bacterial autoinducers: biosynthesis of a novel quorum-sensing signal molecule[END_REF][START_REF] Lerat | The evolutionary history of quorum-sensing systems in bacteria[END_REF]. The involvement of LuxS in QS remains a matter of debate (see [START_REF] Rezzonico | Lack of genomic evidence of AI2 receptors suggests a non-quorum sensing role for luxS in most bacteria[END_REF][START_REF] Rezzonico | Lack of genomic evidence of AI2 receptors suggests a non-quorum sensing role for luxS in most bacteria[END_REF][START_REF] Platt | What's in a name? The semantics of quorum sensing[END_REF][START_REF] Platt | What's in a name? The semantics of quorum sensing[END_REF]references therein). However, many studies also report LuxSbased synthesized molecules as the initiators of a bacterial Esperanto [START_REF] Winans | Bacterial Esperanto[END_REF], i.e. allowing interspecies communication between bacteria. However, our results show a low abundance and a narrow taxonomic coverage of LuxS sequences retrieved from the GOS dataset. This may be due to the lifestyle of bacteria harbouring the AI-2 synthases that are frequently found in association with large organisms (Dworkin & Falkow 2006[START_REF] Bodor | Potential for luxS related signalling in marine bacteria and production of autoinducer-2 in the genus Shewanella[END_REF]) and thus may be undetectable with the protocols employed by the GOS cruise. Similarly, AinS AI synthases were very poorly represented in the GOS dataset. This result is perhaps not surprising as these AI synthases are known in only a few members of the Vibrionaceae [START_REF] Milton | The LuxM homologue VanM from Vibrio anguillarum directs the synthesis of N-(3-hydroxyhexanoyl)homoserine lactone and Nhexanoylhomoserine lactone[END_REF], and therefore the low detection rate could be due to the small search database size. Interestingly, [START_REF] Rusch | The Sorcerer II Global Ocean Sampling Expedition: Northwest Atlantic through Eastern Tropical Pacific[END_REF] also did not detect any member of the Vibrionaceae family in the GOS dataset based on 16S rRNA and rpoB gene analysis. This makes it more likely that the low number of AinS detected was due to the low sequence coverage of the GOS metagenomes being unable to detect rare members of the community such as Vibrionaceae. Again, these biases probably underestimated the real extent of marine AI diversity, which we have extended with this study.

Overall, our work has revealed a large number of new AI sequences. Our results show that diverse AI synthase genes are present in the marine environment and that many are related to presently uncultivated bacteria. It appears that AI synthases are diverse and present in all oceans crossed by the GOS expedition, supporting the hypothesis that QS cellto-cell bacterial communication systems are widespread in marine waters. Thus, this study opens the door to the wider questions about the conditions under which expression of these genes occurs and the potential role of QS in marine bacteria and ecosystems.

ACKNOWLEDGMENTS

We thank Olivier Naret for his help in reference database constitution. We thank Sheree Yau for ther advice on the revised manuscript version. We are grateful to the CNRS-UPMC ABiMS bioinformatics platform (http://a bims. sb-roscoff.fr) for providing computational re sources and support. This project was supported by an Emergence UPMC grant. The authors declare no conflict of interest.


Bernard Quinio
email: bernard.quinio@parisnanterre.fr

Nicolas Boulbes

Pauline De Pechpeyrou

Baptiste Kotras

Use cases of virtual reality to visualize a database how useful is VR for archaeology researchers?

We present here an original Virtual Reality (VR) application for navigating an archaeological database. The Activity Theory is drawn on to describe the use of the immersive tool, which was developed in the context of an archaeological research project. Based on the observation of researchers interacting with the tool and their verbal expression, we show that the efficiency of technology use and activities depends on the removal of technical obstacles but also on the interaction of three components: the subject, the objective and the tool. The activities, which seem the most relevant produce results that affect one or more of the three components. It seems that activity theory is a relevant tool to analyze the use of this kind of technology, VR, to visualize a database. Finally, we present lines of thought for digital humanities.

Introduction

In this communication, we present the analysis of the uses of a virtual reality tool created for viewing an archaeological database for academic purposes. The tool being studied was developed as part of the ANR Schopper project 1 which is drawing to a close [START_REF] Grégoire | De la fouille à la reconstitution des environnements et des comportements préhistoriques : l'outil de recherche SCHOPPER, un système immersif en aide à la formulation d'hypothèses scientifiques[END_REF]. We worked on this project since its beginning in a context of action research as both actor and observer. This project covers several aspects (AI, Immersion, Virtual Reality (VR), and Project Management) around the prehistoric site of Caune de l'Arago and its valley, but we only deal here with the use of VR, which is designed to view a database of more than 400,000 archaeological objects from excavations.

What is the real use of such a technical device, Virtual Reality, to visualize a database in the context of archaeological research? To answer this question, the authors of this communication carried out a cross analysis of the technological uses based on the observations of the archaeologists in charge of the project.

The Digital Humanities can be defined as the incorporation of information technologies into SSH (social sciences and humanities) research [START_REF] Besson | Le temps des humanités digitales[END_REF]. This incorporation goes beyond simple use to address the study of the impact of these technologies on SSH research methods. Digital humanities can also be presented as an interdisciplinary study of the use of socio-technical tools and social science methods [START_REF] Desfriches-Doria | What is Digital Humanities' identity in interdisciplinary practices? An experiment with digital tools for visualizing the francophone DH network[END_REF]. Digital Humanities deal with the usage of technology to create new tools, which may affect the process of collaboration among scholars, and provide the functionality to visualize data across time and space [START_REF] Trace | Information management in the humanities: Scholarly processes, tools, and the construction of personal collections[END_REF]. The question that drives this research therefore seems to be in symbiosis with the rationale of the digital humanities.

Virtual Reality is used in archaeology on many projects, but it generally serves to immerse a researcher or the public in a reconstructed environment [START_REF] Cassidy | A Virtual Reality Platform for Analyzing Remote Archaeological Sites[END_REF]. In the field of digital culture, technology like VR used to digitally reconstruct objects from the past; to give access to cultural resources and to digitally represent cultural artifacts [START_REF] Rosner | The digitization of cultural practices[END_REF]. In the Schopper project, the objective is quite different. It is to immerse the archaeologist in a database for research's purpose. By using an original approach based on the Activity Theory, already employed in several research projects, we reveal the first uses of VR and their developments. We try to identify the determinants of a relevant use of VR for the visualization of data and to analyze the impacts of the technology on the work of archaeologists at individual and collective level. We also make comparison with more classical tools of Digital Humanities. The communication is structured in the following way. We first present the Schopper project and describe the tool concerned. Then we briefly set out the Activity Theory and the principles of its use in the context of this research. Finally, we present the methodology employed and the results we obtained before discussing them in relation with digital humanities.

The Schopper project and the use of VR for the database

The ANR Schopper project brings together four partners whose expertise is pooled to develop innovative technological solutions. The aim of the project is to produce a simulator which will allow us to validate the behaviors of prehistoric man in a reconstructed immersive environment. The project initiator, CERP, is a prehistoric archaeology laboratory.

It brings its scientific expertise, and is in charge of the excavation of the Caune de l'Arago. One author of this paper is from the CERP. Craft-ai is a startup specializing in artificial intelligence.Immersion Tools contributes its expertise in virtual reality: they developed the virtual reality application which we present in this communication. The role of CEROS, of which three authors of this communication are members, was to analyze the interactions between the CERP researchers and the tool developed: AI simulation, the use of VR for the database and the use of 3D immersion in the valley. We only deal with the use of VR for the database here.

The Caune de l'Arago (South of France) located between the east of Pyrenees Mountains and the Mediterranean Sea is a major cave site from the Lower Paleolithic (figure 1). It owes its fame to the importance of its filling, covering 600,000 years of history and several climatic cycles, its archaeological richness and its numerous human remains including the famous skull of the "Man of Tautavel" [START_REF] Lumley De | Caune de l'Arago Tome VI. Tautavel-en-Roussillon, Pyrénées-Orientales, France : Individualisation des unités archéostratigraphiques[END_REF] [START_REF] Perrenoud | Diversité des occupations humaines en contexte glaciaire à la Caune de l'Arago[END_REF]. Since 1967, each archaeological object (animal bones, lithic remains or industries, stone ...) extracted from the site has been numbered, drawn to scale, and entered in an excavation book with the identity of the square (or "zone") and the layer where it was found, its nature, its spatial coordinates (XYZ), its orientation, its pendage (inclination) and its dimensions. It is then recorded in the laboratory in a SQL database "Palaeontological and Prehistoric Material" developed in the course of the 1980s [START_REF] Pois | Habitats préhistoriques au Paléolithique inférieur : étude de l'ensemble stratigraphique II de la Caune de l'Arago (Tautavel, Pyrénées-Orientales, France). Approche informatique du mode de vie de l'Homme de Tautavel [Prehistoric habitat of lower paleolithic. Study of II stratigraphic unit at the Arago Cave (Tautavel, Eastern Pyrenees)[END_REF]. From the first information entered, linked to the discovery in the field, CERP researchers complete the digital file of each object in their specialty according to a well-established codification (fauna and industry connected databases with numerous data fields). For example, the palaeontologist specifies if possible the anatomical determination of bone/teeth and the species to which it belongs, the age/sex of the animal; he codifies its fragmentation, the state of surface (alteration, color, presence of cut marks. . . ).

In the figure 1, you can see on the left a longitudinal section of the cave showing the stratigraphical sequence with the main levels (red capital letters) and plan of the excavation (divided into 1 m squares) and on the right, one photography of the cave, the filling and detail of the level L, 540,000 years (photos c Denis Dainat, EPCC-CERP, Tautavel). To date, the Caune de l'Arago database contains more than 500,000 objects, including 400,000 coordinated objects corresponding to at least 55 archaeological levels [START_REF] Lumley De | Caune de l'Arago Tome VI. Tautavel-en-Roussillon, Pyrénées-Orientales, France : Individualisation des unités archéostratigraphiques[END_REF] [START_REF] Perrenoud | Diversité des occupations humaines en contexte glaciaire à la Caune de l'Arago[END_REF]. Prehistoric men frequented the Caune de l'Arago between 690,000 years and 70,000 years and an archaeological unit (or archaeological unit) corresponds to a level of occupation of the cave at a given time (Figure 1).

The archaeologists have queried this database since the 1980s with tools, which have evolved over time. These queries relate especially to the extraction of specific quantitative data, the study of the spatial distribution of objects, the refitting (finding the joints between elements extracted from the same lithic block or fragments from the same bone, like a puzzle) and defining the occupation levels of the cave. Before Schopper, the results obtained were only tables or figures and simple inert viewing modes in 2D or 3D of the different levels and the objects. With the Virtual Reality tool that has been developed, the researchers can now view the results of their requests through the VR headset and interact with these results; they are immersed in the information they need. Using its coordinates noted at the time of excavation, each object is replaced in the cave, which has been fully scanned for the project. The objects are represented by parallelepipeds whose dimensions, spatial position and orientation/pendage correspond exactly to the measurements made on the field. The color code used in the excavation identify the nature of the object (ex : blue for industries, yellow for bones, red for stones...). Equipped with a headset and two controllers, the researcher can select an object with the aid of a "laser". An "object" information sheet then appears, which contains its main features and indicates the availability (or not) of a 3D scan or a photo which can then be viewed. A small interface also gives access to various options (measurement tool, teleportation movement mode, grid of the squares, go back to initial point).

The display can be viewed horizontally by level, or vertically by square excavated. The principle is to launch a request via the Boolean operators in the main fields of the database by selecting the desired items with the laser and moving through the different menus visible in the headset (see Figure 2). The numerous data fields of the fauna and industry databases are also accessible. The results of the request are displayed, and the researcher can then move through the cloud of objects with the controllers or by moving physically. The different objects can be viewed essentially by turning the head with the headset then by selecting the desired object with the controller laser. It can also measure distances between objects. The display of levels in Figure 1 is not flat, as geological movement has distorted them between the observed time of their deposit and the excavation. The layers are defined at the excavation but the precise delimitation of a level and the allocation of a particular object to a level (archaeostratigraphic individualization) require precise analyses and comparisons between different archaeological disciplines [START_REF] Lumley De | Caune de l'Arago Tome VI. Tautavel-en-Roussillon, Pyrénées-Orientales, France : Individualisation des unités archéostratigraphiques[END_REF]; these are two important research questions for the archaeologists.

At the beginning of the project, the researchers' needs were not well defined and focused on the representation of the cave. After some months of work, the project focused on viewing data in the cave. It is an ongoing project with frequent interactions between developers of VR and archaeologists. After each session of tests, all the bugs and the need for further functionality were sent to the developers.

The Activity Theory for studying the use of VR

The adoption and appropriation of digital technologies has been studied in numerous disciplines. In this communication, we have chosen to draw on the Activity Theory which has already been used in the study of digital technologies [2] [13]. The result of work by Soviet psychologists at the beginning of the 20th century, mainly Leontiev and Vykotski, the Activity Theory offers an alternative to the strictly behaviorist and cognitivist approaches. We refer the interested reader to reference [START_REF] Engeström | Théorie de l'Activité et Management[END_REF]. The main elements of the Activity Theory have been represented in the form of schemas, with titles sometimes badly translated from Russian to English, and then from English to French. We represent activity under a form which seems to us on the one hand, more faithful to the initial conception and on the other hand more valuable for the analysis of activities. The activity must not be represented in the form of a triangle but in that of a triad, the central point of Figure 3 and the inseparable conjunction of three elements, the subject, the objective and the tool. The Actors is the human subject considered in their environment. The Objective is the purpose, the raison d'être of the activity for the actor, the motivation which drives the actor, whatever the restrictions which may be placed on them and which they do or do not take account of. The objective is the element of the triad, which gives meaning and a mindset to the activity. In other words, an activity without objective becomes a simple operation. The tool, the most composite element of the three points of the triad, comprises first of all the technical part. But it is also composed of rules which are derived from the history of the actor and his community of belonging. These rules define what the actor can or must do with the tool.

An activity always has a result: a delivered statement, an object created or modified, a tool used or modified. This result can have an effect: on the actor, on the tool, on the objective, on another person and consequently, on other activities. Any representation, in the form of a schema, of a dynamic or even a continuous concept, as an activity is, is limiting, see figure 3. Therefore, in order to represent the effects of the result on the different elements of the activity, we place the result at the center of it, the double arrows signaling the dynamic, actions and effects, of the activity. There are as many triads as there are many actors concerned by an activity, even if each one carries out the same tasks, it is therefore essential to keep the actor as part of the study model. In organizations, numerous activities are complementary, they interact with each other, these activities are collective or joint [START_REF] Lorino | Concevoir l'activité collective conjointe : l'enquête dialogique. Étude de cas sur la sécurité dans l'industrie du bâtiment[END_REF] which can be represented in the form of a network. In this project we focus on individual use, but activities by the same actor can be linked to each other and form a network of individual activities. The network of activities, individual or collective, is in perpetual evolution, as the objectives changes, the tools change and become more complex and the actors learn. This evolution must be able to be traced by an analysis of the activity network. Finally, the Activity Theory approach underlines the importance of contradictions and tensions between and within activities [START_REF] Hasan | An activity-based model of collective knowledge[END_REF]. These tensions are the main causes of developments and enrichment of activities. How should a relevant activity be defined in a digital field? Two avenues are proposed [START_REF] Quinio | la recherche sur les univers virtuels, une proposition de démarche fondée sur l'activité[END_REF]. Firstly, an activity presents a potential for value if the triad is enriched by the effects of its result:

1. on the actor who acquires one or more new skills (which we call internalization); 3. on the objective which evolves and allows other activities to be considered.

Secondly, the value created must be able, in one form or another, to be transferred or exported into the real world of work, in our case archaeological research. This is possible when the enrichment of the activity is implanted in the tool and can therefore subsequently be used or reused by the same or another subject or when the skill acquired by internalization is transferable via the researcher subject to another research activity. [START_REF] Clement | Connecting theory and practice in digital humanities information work[END_REF] The research methodology and the results obtained

Methodology

Two of the authors of this communication have been working on this project since it began in January 2017. The two others joined the project at the end of 2019. Eight archaeological researchers out of eleven on the CERP team tested the technical tool on two occasions: the first time in May and November 2019, the second in March 2020. Each use was fully filmed (duration between 30 and 60 minutes). As soon as the first session ended, the researcher concerned recorded his free impressions in an observation notebook. The recordings of each test were studied and discussed by the three researchers, then they were fully transcribed in text. In addition to these observations, we conducted individual interviews with each researcher after the second round of testing. These interviews were also fully transcribed.

We applied an inductive qualitative approach to the study of the materials collected [START_REF] Miles | Analyse des données qualitatives[END_REF]. We chose to code via NVIVO 12 all the VR tests on the database which all followed an identical protocol; this represents more than 150 pages of transcriptions and more than thirteen hours of video recordings. The other materials (interviews, observation notebook, emails and meeting reports) were used for triangulation purposes. A large coding grid of 48 codes was devised by three researchers using an inductive and iterative approach then crosstested on several tests. The purpose of this grid was to capture all of the events occurring in the tests outside of a theoretical presupposition (see Appendix with the two sessions and the number of codes for each). In the end, the 16 tests (2 for each of the 8 researchers who carried out these tests) were coded and gave rise to around 1,500 codes which were analyzed.

The eight researchers who carried out the tests came from separate archaeological disciplines and have different characteristics (gender, wearing of glasses, familiarity or not with video game controllers, etc.) which could have an impact on the use of a VR headset. They have all been working on the site for several years and all know very well the cave, the excavation methods applied and the recording and entering of requests in the database.

During the first test session (Session 1 in the verbatim records), the version of the tool still presented several technical problems which made it difficult to use. On a second version of the tool (session 2), several months later and with a second version of the tool, the technical problems were still numerous but less blocking. In both sessions, the tests were supervised by one of the VR designers and/or two CERP researchers who were very familiar with the VR application; they were named Coaches 1 to 3 and their words are also recorded in the verbatim records. In the analyses below, we reproduce the verbatim records directly from the coding made in Nvivo.

The results obtained -verbatim are in italics

Activities depend on the actor; we must therefore observe the significant differences in use in accordance with the subjects with similar objectives and tools. In other words, without taking into account the characteristics of the actor you cannot understand the activity in depth. We have a small test group with heterogeneous characteristics, but we knew each researcher and this knowing seems to be necessary to understand the usage and the difficulties met by each of them. Researcher 1 is a woman specializing in the study of animals (palaeontologist). Like the majority of researchers, she knows the site well and has been partly trained on site. She is one of the first to try out the VR headset. She looks for one of the objects she knows and has a 3D scan. She is satisfied that she has found it and that she can observe the scan.

(Session 1) I'm going to look at that there. Hee hee [very proud of herself ), I know where my stuff is, I do. [She stoops to identify the object she wishes to select. The object actually is one of the objects which has a scan].

The technical defects of the tool impede use in the sense that they break the triad by preventing interaction between actor / objective / tool and therefore the creation of a result. Researcher 5 is a geologist but above all he is the one who knows the cave the best and who is the current excavation manager. He observes the objects with their angles of inclination. He notices a large object which interests him a lot, but the tool does not allow him to find out what it is. The activity is stopped.

(Session 2) Researcher 5. That's pretty! [He closes the object window] The bear skull has an enormous slope. You get the impression that, especially towards the cave wall, there are objects which have a slope that . . . it's as if the material had moved. That's definitely odd. When you move. . . And there on the surface, I'm trying to see. . . [He turns his head from left to right; spying an object] Oh wow, that thing! What is that monster! E8-465 [the information sheet appears -465 is the number of the object founded in the square E8] What do I do to find out? I can't click on it. Coach 3. No, there's no fauna option, nothing. We just have that information there. Researcher 5. So, I can't find out what E8-465 is.

This need of jump between the ground level and the object is very similar to those between close and distant reading for text. "Close reading retains the ability to read the source text without dissolving its structure, distant reading does the exact opposite" [START_REF] Jänicke | Visual Text Analysis in Digital Humanities: Visual Text Analysis in Digital Humanities[END_REF].

The discontinuity problems prevent the smooth sequencing of activities. What we refer to as discontinuity is a change of interface or a step backwards which stops the sequencing of uses and is detrimental to enrichment.

An activity can provoke emotions in the actor and so have an impact on them. We observed several clearly expressed emotions. But it is not clear whether these emotions lead to or will lead later to other activities or a change in research practices. Researcher 11 studies man-made tools. Like all his colleagues, he is very familiar with the site which he has excavated for a long time. In the VR he sees a collection of objects which he is very familiar with and expresses his admiration through his words and his movements, but we do not see at this time the consequences of this admiration.

(Session 1) What I was saying, what I find extraordinary is that when I do that [he leans to get "underneath" the column, which he is looking upwards at], I am in the objects layer, wow!

The literature underlines the importance of tensions and frictions in activities (difficulties or surprising results) which are sources of activity enrichment. One example of what we call frictions is given below. For us friction means a discrepancy between what is seen in the VR and what the researcher knows. In this case we can clearly observe the beginning of a sequence of individual activities. Researcher 9 is perfectly familiar with the 151 human remains found on the site. He asks for them to be displayed and expresses his amazement at the relative positioning of one of these objects and this gives him the idea to dig at this point.

(Session 1) What is this [??] human remains which are on the wrong side? [Indicating an object] Ah, you see, I had already forgotten that there was one there, which is all on its own here; I'd really like to check what it is.

Enrichment of the tool is requested by the researchers who understand clearly that it is necessary in order to export the results of the activity into their research work. They ask for example to be able to save their researcher profile with the requests already made and also to have the option of taking the results from the tool and putting them into their publications. Researcher 4 is a woman specializing in the study of pollens (palynologist). She is very involved overall in the project as viewing vegetation in a reconstructed environment is essential. She considers uses linked to the publication of the article which she is currently working on. She took a measurement between two objects (coprolites that contain pollens) which she could use for a publication or a course.

(Session 2) Researcher 4. Can we save all the measurements like that, yes or no? Coach 2. I'm talking about requests. Saving measurements that would surprise me. Researcher 4. Because if I need that to illustrate an article, or a PowerPoint. Coach 2. There, you have to insert a screenshot, something like that. We had asked for that right at the beginning. Even to record it as well.

We go back to Researcher 9 who is thinking about his future publications and has clearly understood the value of saving requests in order to reuse them later.

( We observed some activities which produced a result which really enriched the actors by internalization. In Session 2, Researcher 3 asked to use the VR tool to confirm one of her archaeological intuitions. It was the first direct request for use and was over quickly (20 minutes whereas the tests lasted for around one hour). Researcher 3 is an experienced palaeontologist and taphonomist. Here, the contribution of the tool was directly linked to its three-dimensional nature which allows you to see objects directly in order to check that they belong to the same level. As soon as she has her result, she wants to stop the test and go back to her researcher's work.

(Session 2) Researcher 3. Finally, the representation of the bison really is on one and the same level. You see, I thought that anyway they'd be spread over a greater depth, because seeing the size of the bones and what we dug up... there and there, we have already excavated at least three separate levels. My bisons are very much on the same level. That's very interesting. Well, that's good, that's what I wanted to see. What do I press now [in order to quit]?

Discussion

The studied virtual reality tool allows you to walk through a virtual cave, which is an archaeological site. We observe from the evolution between the two test sessions that the correction of technical problems is a prerequisite for real uses and that mastering the tool is not obvious and requires time and repetition in handling. The tool is not finalized and many requests go back and forth between developers and archaeologists.

What can we learn from this case study for digital humanities? First, academics must be directly involved in the design and development processes, which will thus create powerful tools adapted to their needs, see digital humanities manifesto [START_REF] Julien | Manifeste pour des humanités numériques 2.0[END_REF]. In our project, developers of VR cannot understand alone all the complexity of the archaeologists needs and their evolution.

Second, the activity theory seems to be a good way to study technology's use in Human Science especially for research work. This theory implies to take into account the characteristics of each actor (individual level) and to connect the activities between them (collective level). We think that these frequent jumps between these two classical views of technology's use are the best way to improve knowledge in this field. The activity theory also may deal with the rules, which must be integrated in the tools. For example, in archaeology, the positioning of found objects obeys strict rules that must be transferred inside the digital tool. In Schopper, when done badly, objects float in the air or become embedded in the walls of the cave. So VR is a good tool for data curation [START_REF] Clement | Connecting theory and practice in digital humanities information work[END_REF] and it hadn't been anticipated at all. Finally, activity theory offers a way of knowing whether a technological use can create value. This will be the case if the tool can be enriched by the actor or if the actor is enriched by the tool. For example, a researcher use the VR to check the spatial repartition and position of several new lithic refitting (reassembly of different parts of an object) discovered at the laboratory. This required several actions in the tools: selection, visualization and distance measurements, information of depth which made it possible to identify a problem on one reassembly, which had been done without the tool by a young researcher. With activity theory, we can follow these value-creating activities in and out of the technical tool and see their sequence.

Third, in our case even though the use of VR is an individual matter, some researchers begin to see the links between the use of VR and collective research work. We find Researcher 5 again; this begins here with a friction and is extended by ideas for collective action. He observes a group of objects whose pendage, (angle of inclination), seems to him to be to be totally illogical. He deduces from this that some information is incorrect and envisions reviewing the collective excavation procedures on the site. Usually he thinks that the tools can help the team to understand the "geometry" of a level.

(Session 2) Researcher 5: [Still crouching and immersed, he arrives at a zone with only blue and purple objects] Here we see that they are [the objects] in all directions. You can really see they have been scattered around, eh! That's incredible! When we say "it's in situ, it's in situ", perhaps we'll need to look again at them, eh, because with regard to the slopes, we perceive much more than it is in every way. Did we make mistakes with the excavation?

So we see in this example a clear link between individual usage of technology and collective work. We also have some verbatim about "the cave according to others" (see appendix) which show that researcher takes into account the interests of other team members. The collective face is present in an individual use of technology.

Fourth, the use of VR to visualize a database has points in common with more traditional tools used in digital humanities. We have presented above the comparison with text analysis tools: close reading and distant reading. The identification by the VR of the structure (distant reading) will allow comparisons with two other archaeological sites (one in Spain and the other in Korea) with the details of some particular object (close reading). We have also noticed a strong demand from archaeologists to be able to color the results of their queries. This need is clearly identified in history field [START_REF] Suri | Spatial mediations in historical understanding: GIS and epistemic practices of history[END_REF] where a choropleth map can be considered as a data-rich visual primary source and different choropleth map layers can be considered a visual secondary source. Indeed, for archaeologist, the relative positioning of objects is linked to precise research questions (refitting, spatial repartition, individualization). And for this need, most users ask to have the possibility of "coloring" queries such as, for example, displaying with different colors the remains of deer and reindeer or two types of raw materials from the same level. It is indeed the basis of archaeostratigraphic distinction in addition to refitting to delimit a level. Researcher 10 is very concerned by this point and she clearly asks for this modification of the tools for studying two levels, K and L, which are very closed.

(Session 2) Researcher 10: Yes but what will interest me, is to know if K and L will have different colors. Coach 2. Not exactly. Researcher10: Otherwise, you cannot distinguish between the two. Coach3. This is exactly what we asked for. Exactly. . . have a small palette of colors to identify different requests.

Finally, with the use of VR we can mix factual information captured by tools (data) with the emotions of users. We feel that this mix may be interesting for the research work but we cannot prove it. Our work shows limitations (small number of tests in a very specific field) but we hope that the identified areas of reflection will be promising for research in digital humanities.

Figure 1 :

 1 Figure 1: The Caune de l'Arago

Figure 2 :

 2 Figure 2: Example of visualisation of a level in VR

Figure 3 :

 3 Figure 3: The activity triad

2 .

 2 on the tool which is enriched by a new technical functionality, a new content or a new formal or informal rule;

  2) Coach 2. After all, logically, when you have recorded your requests, you can access them directly. Researcher 9. Yes, so for the first time, I'll go and prepare all my stuff . . . Coach2. That's it, because you'll have your personal account. Researcher 9. But could I name my requests? Coach 2. Of course. Researcher 9. So, I'll have my "fibula" request, my "femur" request, and my "femur plus fibula" request. Coach 2. Exactly. Researcher 9. That's very good.

Appendix : Codes and their definition S1 is the first session of test and the number in the column measures the number of verbatim associated with the code of the line.