Camille Détrée

François H Lallier

Arnaud Tanguy

Dr Jean Mary
email: jmary@sb-roscoff.fr

Identification and gene expression of multiple peptidoglycan recognition proteins (PGRPs) in the deep-sea mussel Bathymodiolus azoricus, involvement in symbiosis?

Keywords: Chemoautotrophic symbiosis, symbiont recognition, PRR, immune response, bacterial control

The relationship between the deep-sea mussel Bathymodiolus azoricus and its thiotrophic (SOX) and methanotrophic (MOX) symbionts has been ecologically and functionally well studied.

Endosymbiosis is common in deep-sea hydrothermal vent fauna, yet little is known about the molecular mechanisms underlying the regulation of interactions between host and symbionts. In this study we focused on a group of pattern recognition receptors (PRR), called PGRPs that are able to recognize the peptidoglycan of bacterial cell wall. We first characterised the different PGRPs isoforms in B. azoricus gills and identified five paralogs. Among them two displayed a signal peptide. Then, specific probes designed for each paralog were used to perform real-time PCR quantification in gills of individuals showing various bacterial content as a result of in situ experimental procedures. Overall we found a decrease of PGRPs expression when symbionts amount decreases, suggesting an implication of PGRPs in the regulation of symbionts in B. azoricus gills.

We therefore hypothesize that secreted proteins could act as cooperation signals to induce colonisation of symbiotic tissue while non-secreted proteins may regulate the density of endosymbionts within the gill tissue.

Introduction

Symbioses between invertebrates and bacteria are a common feature of chemosynthetic ecosystems [START_REF] Tunnicliffe | The biology of hydrothermal vents : ecology and evolution[END_REF][START_REF] Van Dover | Microorganisms as food resources at deep-sea hydrothermal vents[END_REF][START_REF] Dubilier | Symbiotic diversity in marine animals: the art of harnessing chemosynthesis[END_REF]. In particular, around most deepsea hydrothermal vents all over the world, bivalves with enlarged gills, a reduced digestive system and symbiotic sulphur-oxidizing and/or methane-oxidizing bacteria are dominant (Le Pennec et al.,

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M A N U S C R I P T ACCEPTED MANUSCRIPT Détrée et al. 2017 3 1990; [START_REF] Kyuno | Dispersal and differentiation of deep-sea mussels of the genus Bathymodiolus (Mytilidae, Bathymodiolinae)[END_REF][START_REF] Miyazaki | Evolutionary process of deep-sea Bathymodiolus mussels[END_REF]. Bathymodiolus azoricus, a mussel species from the Mid-Atlantic Ridge found at vent sites south of the Azores, acquires horizontally and hosts both types of intracellular gamma-proteobacterial symbionts within its specialised gill cells.

Methanotrophic symbionts (MOX) use methane and fix its carbon using energy derived from its oxidation whereas sulphur-oxidizing or thiotrophic symbionts (SOX) oxidize hydrogen sulphide to generate energy and fix inorganic carbon into organic carbohydrates [START_REF] Cavanaugh | Evidence for methylotrophic symbionts in a hydrothermal vent mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge[END_REF][START_REF] Distel | Intracellular coexistence of methano-and thioautotrophic bacteria in a hydrothermal vent mussel[END_REF][START_REF] Fiala-Médioni | Ultrastructural, biochemical, and immunological characterization of two populations of the mytilid mussel Bathymodiolus azoricus from the Mid-Atlantic Ridge: evidence for a dual symbiosis[END_REF][START_REF] Duperron | A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia : Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge[END_REF]. These bacteria receive all metabolites necessary for chemoautotrophy from the surrounding seawatera mix between deep-sea water and hydrothermal fluidthrough the bivalve gills, which maintain a flux usually serving nutrition by filtration. This symbiosis is known to be plastic and highly dependent on environmental conditions [START_REF] Colaço | Nutritional relations of deep-sea hydrothermal fields at the Mid-Atlantic Ridge: a stable isotope approach[END_REF][START_REF] Fiala-Médioni | Ultrastructural, biochemical, and immunological characterization of two populations of the mytilid mussel Bathymodiolus azoricus from the Mid-Atlantic Ridge: evidence for a dual symbiosis[END_REF][START_REF] Salerno | Characterization of symbiont populations in life-history stages of mussels from chemosynthetic environments[END_REF][START_REF] Duperron | A dual symbiosis shared by two mussel species, Bathymodiolus azoricus and Bathymodiolus puteoserpentis (Bivalvia : Mytilidae), from hydrothermal vents along the northern Mid-Atlantic Ridge[END_REF][START_REF] Boutet | Conjugating effects of symbionts and environmental factors on gene expression in deep-sea hydrothermal vent mussels[END_REF]. Indeed the lack of hydrogen sulphur or methane can lead one or the other type of symbiont to take precedence on the other. Halary and co-authors showed that a high concentration of hydrogen sulphur, in mussels kept in aquaria, induces a higher level of sulphur-oxidizing symbionts in B. azoricus gills [START_REF] Halary | 3D FISH for the quantification of methane-and sulphur-oxidizing endosymbionts in bacteriocytes of the hydrothermal vent mussel Bathymodiolus azoricus[END_REF]. This flexibility is thought to be a host adaptation to cope with its heterogeneous and highly variable environment [START_REF] Boutet | Conjugating effects of symbionts and environmental factors on gene expression in deep-sea hydrothermal vent mussels[END_REF]. Despite the extensive research done on bathymodiolin symbiosis, the underlying molecular mechanisms that govern this symbiosis are largely unknown. Specifically, mechanisms involved in the interaction between host and symbiont at the cellular and/or molecular level have not been explored. To date, only a few studies have examined related question through microscopic investigations or gene expression [START_REF] Won | Environmental acquisition of thiotrophic endosymbionts by deep-sea mussels of the genus Bathymodiolus[END_REF][START_REF] Kádár | Experimentally induced endosymbiont loss and re-acquirement in the hydrothermal vent bivalve Bathymodiolus azoricus[END_REF][START_REF] Boutet | Conjugating effects of symbionts and environmental factors on gene expression in deep-sea hydrothermal vent mussels[END_REF][START_REF] Bettencourt | Site-related differences in gene expression and bacterial densities in the mussel Bathymodiolus azoricus from the Menez Gwen and Lucky Strike deep-sea hydrothermal vent sites[END_REF][START_REF] Barros | Post-capture immune gene expression studies in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus acclimatized to atmospheric pressure[END_REF]. As in other symbioses, B. azoricus may regulate its symbiont population through apoptosis mechanisms or immunological responses. These mechanisms imply that the host should be able to recognize its symbionts, differentiate them from other bacteria (pathogenic or not) and influence their proliferation. To further our understanding of the molecular mechanism used by bathymodiolin [START_REF] Dziarski | The peptidoglycan recognition proteins (PGRPs)[END_REF][START_REF] Guan | Peptidoglycan recognition proteins of the innate immune system[END_REF].

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PGRPs are innate immunity molecules that contain PGN-binding domain (around 165 amino acids in length) which enable them to recognize and bind the peptidoglycan (PGN) of bacterial cell-wall [START_REF] Yoshida | Purification of a peptidoglycan recognition protein from hemolymph of the silkworm, Bombyx mori[END_REF]. A minority of them, called catalytic-PGRPs, possess an amidase activity allowing them to hydrolyse the bond between MurNac and L-Ala in PGN [START_REF] Gelius | A mammalian peptidoglycan recognition protein with N-acetylmuramoyl-L-alanine amidase activity[END_REF][START_REF] Kim | Crystal structure of peptidoglycan recognition protein LB from Drosophila melanogaster[END_REF][START_REF] Wang | Human peptidoglycan recognition protein-L is an Nacetylmuramoyl-L-alanine amidase[END_REF][START_REF] Li | Zebrafish peptidoglycan recognition proteins are bactericidal amidases essential for defense against bacterial infections[END_REF]. PGRPs have been well studied in insects in which they are grouped into two classes -short (S) and long (L) ones -and can be intracellular, extracellular or transmembrane proteins [START_REF] Werner | A family of peptidoglycan recognition proteins in the fruit fly Drosophila melanogaster[END_REF]. These proteins are known to be involved in innate immunity in insects and more precisely in two NF-κB signalling pathways: Toll and Imd [START_REF] Leulier | The Drosophila immune system detects bacteria through specific peptidoglycan recognition[END_REF]. Indeed, in Drosophila, the secreted PGRP-SA and PGRP-SD recognize Lys-type PGN (Gram-positive bacteria), activate a protease that cleaves an extracellular cytokine-like protein (Späetzle) which in turn activates the Toll pathway, whereas the transmembrane PGRP-LC recognize DAP-type PGN (Gram-negative bacteria or bacillus) and triggers the Imd pathway [START_REF] Choe | Requirement for a peptidoglycan recognition protein (PGRP) in relish activation and antibacterial immune responses in Drosophila[END_REF][START_REF] Takehana | Peptidoglycan recognition protein (PGRP)-LE and PGRP-LC act synergistically in Drosophila immunity[END_REF][START_REF] Kaneko | PGRP-LC and PGRP-LE have essential yet distinct functions in the Drosophila immune response to monomeric DAP-type peptidoglycan[END_REF]. Both pathways lead to the production of anti-microbial peptides to fight against pathogenic bacteria.

Finally, PGRPs with amidase activity (PGRP-SC and PGRP-LB) can modulate the immune response to bacterial infection by inhibiting the activation of the IMD pathway [START_REF] Bischoff | Downregulation of the Drosophila immune response by peptidoglycan-recognition proteins SC1 and SC2[END_REF][START_REF] Mellroth | A scavenger function for a Drosophila peptidoglycan recognition protein[END_REF]Zaidman-Rémy et al., 2006). The key role of PGRPs in immunity and their high expression in gills of symbiotic bathymodiolin mussels such as B. azoricus or B. platifrons [START_REF] Bettencourt | Innate immunity in the deep sea hydrothermal vent mussel Bathymodiolus azoricus[END_REF][START_REF] Bettencourt | Site-related differences in gene expression and bacterial densities in the mussel Bathymodiolus azoricus from the Menez Gwen and Lucky Strike deep-sea hydrothermal vent sites[END_REF][START_REF] Martins | Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria[END_REF][START_REF] Wong | High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons[END_REF] Thereby, we observed that four of the PGRPs paralogs are down-expressed with the loss of symbionts suggesting a potential role of the corresponding protein in the maintenance of symbiotic relationship.

Materials and methods

Animal collection: field sampling, in situ experiments and quantification of symbionts

Deep-sea mussels Bathymodiolus azoricus were collected from one hydrothermal vent field (Lucky Strike) at the Montsegur site (37°17,286' N; 32°16,530' W; 1700 m depth) located on the Mid-Atlantic Ridge during the BioBaz and MoMARSAT cruises (July/August 2013). The sampling was done using the Remote Operating Vehicle (ROV), Victor 6000 on the Research Vessel "Pourquoi pas?". Around thirty mussels representing natural population were collected at the same time, and brought on board. Then gills were dissected, frozen in liquid nitrogen, and stored at -80°C until further analysis. This population correspond to the control of the in situ experiment that were previously described [START_REF] Détrée | Multiple I-type lysozymes in the hydrothermal vent mussel Bathymodiolus azoricus and their role in symbiotic plasticity[END_REF].

Briefly, between 15 and 30 mussels were put in several wire cages and 5 of these cages were placed a dozen meters away from the diffuse fluid emission. This condition was called "basalt". Cages were brought back on board in three steps: after 6 days on basalt (BT1), 20 days (BT2) and 27 days (BT3).

Moreover, after six days on basalt (BT1), two cages were translocated from basalt back to the mussel bed, at their original site (next to hydrothermal fluid) and brought back to the research vessel after respectively 14 days (called TR1.2) and 21 days (TR1.3) on mussel bed (Figure S1). Immediately

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after collection, gills were dissected, frozen in liquid nitrogen and stored at -80°C until further analysis. Then the whole DNA was extracted and a relative quantification of symbiont was performed by qPCR according to Boutet and co-authors [START_REF] Boutet | Conjugating effects of symbionts and environmental factors on gene expression in deep-sea hydrothermal vent mussels[END_REF] [START_REF] Webb | Comparative protein structure modeling using Modeller[END_REF]. The structures of the PGRP-LE, crystallized with the tracheal cytotoxin as substrate (pdb: 2CB3), isolated from Drosophila melanogaster, was used as template.

One hundred models were generated for each PGRPs and their quality assessed using the Modeller Objective Function parameter. The reliability of modelled structures was validated by Ramachandran

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plot analysis using PROCHECK (http://www.ebi.ac.uk/thornton-srv/databases/pdbsum/ Generate.html).

PGRPs expressions: RNA extraction and qPCR

The mRNA expression of PGRPs paralogs was analysed by real-time PCR. Total RNA was extracted from mussel gills by using Tri-Reagent (Sigma) according to the manufacturer's instructions. Both from the host was used as the internal PCR control (Table 1). We validated the RpL15 after having observed very low (less than 5%) variation of its expression in all samples (both experimental and field individuals) for gill tissue and three technical replicates were performed.

Statistical analysis

Data from quantitative real-time PCR experiments were analysed with a Kruskal-Wallis test GraphPad Prism, USA (www.graphpad.com) followed by a Dunn's Multicomparison Test Differences were considered statistically significant when p ≤ 0.05.

Results

Characterisation and phylogenetic position of Ba-PGRPs

The analysis of the EST database of Bathymodiolus azoricus revealed the existence of five paralogs of PGRPs, referred to as Ba-PGRP 1 to 5. The five PGRPs are encoded with paralogous genes and contain from 191 to 435 residues (Figure S2, Table 2). Ba-PGRPs 2 to 5 are short (S) and contain one PGRP domain whereas Ba-PGRP 1 is a long one (L) with also only one PGRP domain in its Cterminal part. Ba-PGRP 3 and 5 possess a predicted signal peptide in their N-terminal part indicating that these two paralogs are possibly secreted into the extracellular space where they may recognize and bind PGN. Sequence analysis suggest that Ba-PGRP 2 and 5 recognize specifically DAP-type peptidoglycan residues responsible for this binding selectivity being conserved (Gly 89, Trp 90 and Arg 109 in Drosophila melanogaster PGRP LB) (Figure 1) [START_REF] Lim | Structural basis for preferential recognition of diaminopimelic acid-type peptidoglycan by a subset of peptidoglycan recognition proteins[END_REF][START_REF] Swaminathan | Dual strategies for peptidoglycan discrimination by peptidoglycan recognition proteins (PGRPs)[END_REF]. For Ba-PGRP 1 and 3 the arginine and tryptophan residue are conserved and the third position is variable, while for the Ba-PGRP 4 only the arginine residue is conserved. This suggests that Ba-PGRP 1, 3 and 4 may recognize DAP-type PGN with lower affinity, or other PGN domains. In the case of the four short paralogs (Ba-PGRP 2 to 5), the amino acid residues involved in amidase activity (N-acetylmuramoyl-L-alanine amidase) are conserved: Zn 2+ -binding site (His 59, Tyr 95, His 169 and Cys 177) and Thr175 (in Dm-PGRP LB) [START_REF] Kim | Crystal structure of peptidoglycan recognition protein LB from Drosophila melanogaster[END_REF]. In the long paralog, Ba-PGRP1, the cysteine residue (Cys 177 in Dm-PGRP LB) is replaced by a glycine. This kind of mutation was already observed in human, leading to a loss of the amidase activity [START_REF] Michel | Drosophila Toll is activated by Grampositive bacteria through a circulating peptidoglycan recognition protein[END_REF]Wang et ), but also in Drosophila leading to an abolishment of its ability to activate the Toll pathway [START_REF] Wang | Human peptidoglycan recognition protein-L is an Nacetylmuramoyl-L-alanine amidase[END_REF]. Sequence alignment suggests that the four short paralogs, Ba-PGRPs 2 to 5 contain one disulphide bridge (Figure 1). The structure of the complex between Ba-PGRP 2 and the tracheal cytotoxin (TCT) was predicted by homology modelling (Figure S3). Ba-PGRP 2 in the 3D

Relative expression of Ba-PGRPs in natural population and in situ experiments

The relative expression of the five Ba-PGRPs was measured in gill tissue by using qPCR in different conditions, corresponding to different symbiotic state (Table 3). First, the relative expression of each paralog in gills of individuals from natural population was investigated (Figure 3). Results showed that Ba-PGRP 3 and 5 have a significantly higher expression in gills than Ba-PGRP 1, 2 and 4. From this first analysis, it appears that Ba-PGRP 3 and 5 are the two paralogs that are particularly expressed in gills. Gills are directly exposed to the environment due to the water flux they entertain and, because of the main function of PGRPs it is possible that these two paralogs may be implicated in the response to environmental pathogens and/or in the regulation of symbiosis.

To go further we investigated the relative expression of the 5 paralogs in individuals translocated away from the venting area and maintained on basalt for 6, 20 and 27 days (i.e. conditions BT1, BT2 and BT3) and compared it to the relative expression in natural population individuals (Figure 4).

Note that, mussels translocated to basalt have lost partially (BT1) or almost completely (BT2 and BT3) their symbionts (Table 3). Interestingly the 4 paralogs Ba-PGRP 1, 2, 3 and 4 have the same

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expression pattern in BT1 and BT2. Indeed, their relative expression decreased significantly in BT2 (compared to natural population) i.e when mussels were facing a massive loss of symbiont. In BT3 the down-regulation of Ba-PGRP 2 and 3 remains significantly different from the natural population while in the case of Ba-PGRP 4 a slight up-regulated in BT3 was observed. The expression pattern of Ba-PGRP 5 is similar to that of Ba-PGRP 4, but not significantly different to the natural population. Thus, a massive lost of symbionts (BT2) seems to induce a significant down-regulation of the expression of four Ba-PGRPs paralogs.

The analysis of the relative expression of the five paralogs in individuals that were translocated away from venting area during 6 days, and then re-exposed to hydrothermal fluid in their original mussel bed (TR1.2 and TR1.3) (Figure 4) show no significant differences for 4 paralogs (Ba-PGRP 1, 2, 3, 5), while Ba-PGRP 4 is significantly down-regulated in TR1.2 and TR1.3 compared to natural population. Comparison between the control of translocation (i.e. BT1) and TR1.2 and TR1.3 show no significant effects of a return to mussel bed on paralogs expressions. However, the expression of Ba-PGRP 3 and 5 seems to increase after a return to mussel bed while the expression of Ba-PGRP seems to diminish.

Discussion

Numerous studies have focused on the mechanisms of symbiosis from its physiology, to the nutrient exchange or to the specific molecular communication between host and symbionts. Indeed, the ability of the host to choose between symbiotic bacteria and other bacteria, especially in symbiosis with horizontal transmission, connote the presence of a very efficient system of recognition from both host and symbionts as was shown for the well-documented association between Rhizobium/leguminous plant [START_REF] Cooper | Early interactions between legumes and rhizobia : disclosing complexity in a molecular dialogue[END_REF].

In this study, we investigated the putative role of a family of protein, PGRPs, in the symbiotic association between the deep-sea mussel Bathymodiolus azoricus and its symbionts. Paralogs of PGRPs were identified from B. azoricus transcriptome and their expression was measured by qPCR in gills of individuals hosting different amount of symbionts. Thus, the expression of PGRPs in natural population individuals (average amount of 800 U) was compared with individuals that experimented a partial loss of symbionts (between 370 and 70 U), and individuals that experimented a partial loss of symbionts and were brought back to mussel bed (between 350 and 400 U) [START_REF] Détrée | Multiple I-type lysozymes in the hydrothermal vent mussel Bathymodiolus azoricus and their role in symbiotic plasticity[END_REF].

PGRPs have been highlighted in several bivalves' species (e.g. mussels, scallops, oysters, razor clams) and proved to play a key role in the defence against pathogenic bacteria [START_REF] Itoh | Distribution of multiple peptidoglycan recognition proteins in the tissues of Pacific oyster, Crassostrea gigas[END_REF][START_REF] Martins | Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria[END_REF][START_REF] Ni | Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819)[END_REF][START_REF] Wei | Molecular cloning and mRNA expression of two peptidoglycan recognition protein (PGRP) genes from mollusk Solen grandis[END_REF]. Grippingly, only three paralogs of PGRPs were reported in mytilids (Mytilus galloprovinciallis), among which two are predicted to be bound to the membrane and one to be secreted [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF] while in a cold seep symbiotic mussel eleven transcripts matching PGRP sequence were identified. These last can be clustered in three groups according to their sequence which suggest the presence in this specie of three paralogs of PGRPs with at least one predicted to have a signal peptide [START_REF] Wong | High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons[END_REF]. In our study, five paralogs of PGRPs in Bathymodiolus azoricus' gills were identified. Among these, no membrane-bound paralogs were identified but two secreted paralogs were reported (Ba-PGRP 3 and Ba-PGRP 5). A previous study on Bathymodiolus azoricus transcriptome highlighted only one paralogs of PGRP [START_REF] Bettencourt | Highthroughput sequencing and analysis of the gill tissue transcriptome from the deep-sea hydrothermal vent mussel Bathymodiolus azoricus[END_REF] that corresponds to the Ba-PGRP 3 in the present one.

The transcriptome used by Bettencourt's group in their studies have been obtained from gills while our data were obtained from gills, mantle and digestive gland. As, in our study, three paralogs (Ba-PGRP 1, 2 and 4) have a very low expression in gills of individuals from natural population, it is likely that their identification has been possible by the sequencing of three different tissues including gills. Nevertheless, the absence of membrane-bound PGRP in the five paralogs we identified suggest that the list of reported PGRP in this study might still not be completely exhaustive. However, the presence in Bathymodiolus azoricus' gills of two secreted proteins instead of one in other non-

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ACCEPTED MANUSCRIPT Détrée et al. 2017 symbiotic mussels may suggest that one of these proteins might be link to the presence of symbiont.

Numerous studies, including one on B. azoricus focused on PGRP's expression after an exposure to pathogenic bacteria. galloprovincialis, the only PGRP paralog identified in B. azoricus was not responding to these pathogenic bacteria [START_REF] Martins | Comparative study of immune responses in the deep-sea hydrothermal vent mussel Bathymodiolus azoricus and the shallow-water mussel Mytilus galloprovincialis challenged with Vibrio bacteria[END_REF]. The same research group performed another challenge of B. azoricus with, this time, Vibrio parahaemolytics, flavobacterium and seawater as control.

Surprisingly, they observed an important up-regulation of PGRP (Ba-PGRP 3) only 24h after a seawater challenge [START_REF] Bettencourt | Site-related differences in gene expression and bacterial densities in the mussel Bathymodiolus azoricus from the Menez Gwen and Lucky Strike deep-sea hydrothermal vent sites[END_REF] while no response was observed with bacterial challenge, suggesting that the paralog reported was not involved in the defence against these bacteria but may be responding to the presence of others in seawater.

In our study, we analysed the expression of the five paralogs of PGRP in natural population and in individuals depleted of part of their symbionts. The high expression of the secreted paralogs Ba-PGRP 3 and 5 in natural population followed by their down-regulation in individuals that lost part of their symbionts raised our awareness and led to two main hypotheses. Due to the key role of PGRPs in the immune response of bivalves and invertebrates in general [START_REF] Gerdol | An updated molecular basis for mussel immunity[END_REF], we can suggest (i) that these two secreted paralogs are highly expressed in gills to defend the organism against pathogenic bacteria. As gills are one of the first barriers between the host and environment, the host to prevent an infection could produce Ba-PGRP 3 and 5. Upon infection, the amidase activity of these paralogs could weaken the bacteria cell wall having, in synergy with others innate immune response actors such as lysozymes, a putative bactericidal effect. As no membrane-bound PGRP were reported, the possibility of triggering an immune cascade is less likely. Thus, the loss of

A C C E P T E D M A N U S C R I P T ACCEPTED MANUSCRIPT Détrée et al. 2017
13 symbionts, may lead to a concomitant loss of energy supply (as symbiont content is small after 21 and 27 days) that will induce a diminution of energy allocated to the defence against pathogens and may lead to the down-regulation of these PGRPs. However, PGRPs have been shown to be highly involved in symbiotic relationships. In several symbioses such as the association between the squid Euprymna scolopes and Vibrio fischeri or the cold seep mussel Bathymodiolus platifrons with its MOX bacteria, the expression of PGRPs have been shown to be higher in symbiotic tissue or bacteriocytes [START_REF] Anselme | Host PGRP gene expression and bacterial release in endosymbiosis of the weevil Sitophilus zeamais[END_REF][START_REF] Nyholm | Expression and putative function of innate immunity genes under in situ conditions in the symbiotic hydrothermal vent tubeworm Ridgeia piscesae[END_REF][START_REF] Wong | High-throughput transcriptome sequencing of the cold seep mussel Bathymodiolus platifrons[END_REF]. Some hypothesis followed these observations, with authors suggesting the putative role of PGRP in the acquisition and regulation of symbionts and proposed PGRP as a candidate to study immunomodulation. In Bathymodiolus azoricus' symbiosis few informations are available so far. Nevertheless, gill colonisation by endosymbiotic bacteria seems to be a continuous process throughout mussels lifetime [START_REF] Wentrup | Forever competent: deep-sea bivalves are colonized by their chemosynthetic symbionts throughout their lifetime[END_REF], suggesting a necessity for the host to produce constantly a signal of cooperation to indicate the correct location for the colonisation of future symbionts as it has been suggest in the association between the weevil Sitophilus zeamais and its symbionts [START_REF] Reynolds | Immune function keeps endosymbionts under control[END_REF]. Therefore we may postulate that (ii) Ba-PGRP 3 and 5 would rather be used as a signal of cooperation in natural population to acquire constantly new symbionts. Thus, on bare basalt, where less free-living bacteria are observed the cooperation signal would be down regulated. This hypothesis is supported by the up-regulation of the two secreted paralogs when mussels were brought back to mussel bed. However as the down-regulation is significant after a loss of symbiont only for Ba-PGRP 3 we can suppose that only this paralogs is involve in this process. This hypothesis may seem more likely as there is a growing amount of evidence that mussels are able to digest there symbionts when the later are in excess, having as a consequence access to enough energy to produce an immune response [START_REF] Détrée | Multiple I-type lysozymes in the hydrothermal vent mussel Bathymodiolus azoricus and their role in symbiotic plasticity[END_REF][START_REF] Fiala-Médioni | Ultrastructural, biochemical, and immunological characterization of two populations of the mytilid mussel Bathymodiolus azoricus from the Mid-Atlantic Ridge: evidence for a dual symbiosis[END_REF]. More, symbiotic bacteria (SOX) in deep-sea mussels have been shown to be able to defend host against A C C E P T E D M A N U S C R I P T pathogens [START_REF] Sayavedra | Abundant toxin-related genes in the genomes of beneficial symbionts from deep-sea hydrothermal vent mussels[END_REF]. Thus it seems more likely that when the amount of symbionts is low, the host would be able to take over and protect itself.

In the case of other PGRPs paralogs, their down-regulation correlated with the loss of symbionts can also suggest their implication in the regulation of symbiosis. Nonetheless their low expression in natural population may suggest that they would rather be involve in the defence against pathogenic bacteria. Thus, we suggest that the expression of these paralogs may be inducible upon infection but that the basal expression stay low not to interfere with symbiotic bacteria.

Concluding remarks

Overall our results show a high correlation between the loss of symbionts and the down-regulation of

Genes

Primers sequence 5'-3'

RpL15

For 5'-TATGGTAAACCTAAGACACAAGGAGT-3' Rev 5'-TGGAATGGATCAATCAAAATGATTTC-3' Ba-PGRP 1

For 5'-GTCTTCATACATCATACTGCCATGAGTTA-3' Rev 5'-CCATTATAGAAACGCTACTGCAACGTC-3' Ba-PGRP 2

For 5'-GCTGTGATGATGCATGTAGCAGCCCCATC-3' Rev 5'-CTTGTATGGCACTCTGCATCAGTACAACC-3' Ba-PGRP 3

For 5'-ATGATATGGGCTATTCTTCCGCTCGTTGC-3' Rev 5'-TAATTGTAACACCGGAACAATCACCGGT-3' Ba-PGRP 4

For 5'-GCCTTAAGTGTGACATCCACCCGACG-3' Rev 5'-AAGCATGCCGGTAGCCACTCTACA-3' Ba-PGRP 5

For 5'-ACAGGGTCTCACACACCACAGCTGGGTGTCC-3' Rev 5'-GTACATCCACCCGACGCCCCATGATGGAT-3' (Mytilus galloprovincialis PGRP-3), CL8290, and CL2060, PGRPs from Bathymodiolus platifrons (Wong et al., 2016), Bt Bathymodiolus thermophilus.

Legends

  makes them interesting candidates for studying mechanisms underlying symbiosis. In this study we propose to look at the implication of PGRPs in the initiation, breakdown or maintenance of the association between B. A C C E P T E D M A N U S C R I P T azoricus and SOX/MOX bacteria. To do so, we characterised PGRPs paralogs present in the gills of B. azoricus. Then, after the induction of a gradual loss of symbiont in mussels during in situ experiments, we analysed expression of the different PGRPs in gills of individuals with few or a very few symbionts and compared these expressions with those of individuals from natural population.

  quantity and quality of RNA were assessed via UV absorbance (OD260/280/230) using a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Delaware, USA). Two μg of total RNA were reverse transcripted using M-MLV reverse transcriptase (Promega), an anchor-oligo(dT) primer (5′-CGCTCTAGAACTAGTGGATCT-3′) and random hexamers (Promega). A volume of 2 μL of each diluted reverse transcription product (1:200) was subjected to real-time PCR in a final volume of 5 μL containing 40 nM of each specific primer and 2×Lightcycler®480 SYBR Green I Master mix (Roche Diagnostics, Mannheim Germany). The amplification was carried out as follows: initial enzyme activation at 95°C for 15 min, then 45 cycles at 95°C for 10 sec and 60°C for 30 sec. A dissociation curve was generated and PCR efficiency was estimated for each primer pair. All primer pairs tested generated a single peak in the dissociation curve and a PCR efficiency of 95 to 100%.Relative expression of each gene (fold change) was calculated according to the comparative Ct method using the formula: Ratio = 2 -ΔCt (with ΔCtgeneX = CtGeneX-CtRpL15) for the comparison of PGRPs expression in natural population, while the formula used for the in situ experiment was Ratio = 2 -ΔΔCt (with ΔCt geneX = Ct GeneX -Ct RiboL15 and ΔΔCt = (ΔCt geneX -meanΔCt geneX ). The later formula was chosen to standardize the levels of gene expression to a mean of zero and consequently give the same weight to all response variables. A fragment of ribosomal protein L15 gene (RpL15)

  model has three main α-helices and one central β-sheet composed of six β-strands and the TCT occupies the potential PGN-binding cleft. The phylogenetic tree of mollusc PGRPs show that the different Ba-PGRPs are clustered with those of other Mytilidae, B. thermophilus, B. platifrons as well as M. galloprovincialis in agreement with the classical taxonomy (Figure 2). Moreover, their distribution within five distinct clades support the fact that these PGRPs sequences are actual paralogs.

  PGRPs paralogs in the gills of the deep-sea mussel Bathymodiolus azoricus. In such a specific and intimate association, the stability and homeostasis of the holobiont are thought to require a tight regulation through complex molecular mechanisms. An important speculation from this work is that PGRP might be involve in the regulation of symbionts through the constant acquisition of SOX and MOX bacteria. Although the correlation between the amount of symbiont and expression of PGRP is clear, their key role in immunity prevent us from assigning a specific role to these proteins in gills of B. azoricus. Future investigations should focus on a functional role of PGRPs in the deep-sea mussel by analysing the putative interaction between these five paralogs and microbe associated molecular pattern (MAMPs) to confirm or not our main speculation. this article. Camille Détrée was supported by a PhD grant from Région Bretagne (ARED APROSBAZ) and UPMC.

Figure 1 .

 1 Figure 1. Multiple alignment of the Ba-PGRPs domain. The black and grey regions indicate positions where amino acid residues are conserved or highly similar, respectively. *: Residues implicated in the DAP-type PGN selectivity. ◆: Zn 2+ binding site, ❍: residues contributing to amidase activity, s: cysteine residues implicated in the disulphide bridge. Dm-PGRP LB: Drosophila melanogaster PGRP LB (access number Uniprot Q8INK6).

Figure 2 .

 2 Figure 2. Phylogenetic analysis of PGRPs among molluscs The distance tree was obtained by neighbor joining with 1000 bootstrap resampling of the data set (value below the branches, Seaview 4.5.4). The scale bare refers to 10% sequence variation. Red boxes indicate the Bathymodiolus azoricus paralogus PGRPs. The UniProt accession no. and the species are as follows : B2DEU5 (Crassostrea gigas PGRP-S2), B2DEU3 (Crassostrea gigas

Figure 3 .

 3 Figure 3. Relative expression of Ba-PGRPs paralogs in B. azoricus gills in natural population. Bars shows the expression of each paralog compared with the reference gene RpL15 according to comparative Ct method using the formula: Relative expression= 2 -ΔCt (with ΔCt geneX = Ct GeneX -Ct RiboL15 ) a: significantly different from Ba-PGRP 5 and b: significantly different from Ba-PGRP 3. (p < 0.05), (Kruskal-Wallis test, Dunns post test), n=10.

Figure 4 .

 4 Figure 4. Relative expression of PGRPs paralogs in B. azoricus gills after symbiont loss and translocation. Relative expression level of mRNA (Ratio) is indicated according to comparative Ct method using the formula 2 -ΔΔCt , with ΔCt geneX = Ct GeneX -Ct RiboL15 and ΔΔCt = (ΔCt geneX -meanΔCt geneX ) and measured in natural population (n=10), BT1 (n=15), BT2 (n=19), BT3 (n=22), TR1.2 (n=10) and TR1.3 (n=11). * Significantly different from Nat. population, # significantly different from BT2, (p<0.05), (Kruskal-Wallis test, Dunns post test).

  mussels to interact with their symbionts, we conducted in situ experiments and focused on a family of PRRs (Pattern Recognition Receptors): PeptidoGlycan Recognitions Proteins (PGRPs). PGRPs are present in most animals including insects, echinoderms, molluscs and vertebrates, but absent in some other metazoan such as nematodes

  Martins and co-authors focused on the response of Mytilus galloprovinciallis and B. azoricus' immune related receptors and effectors in response to the injection of different Vibrio strains. Interestingly while M. galloprovinciallis' PGRP is up-regulated 12 and 24 hours postinjection, very little effects are observed on B. azoricus' PGRP (Ba-PGRP 3) which basal expression in the control is similar to the one of M. galloprovincialis. Thus, on the contrary of M.

Table 1 . Sequence of primers used for real-time PCR amplification

 1 Zaidman-Rémy, A., Hervé, M., Poidevin, M., Pili-Floury, S.,Kim, M.S., Blanot, D., Oh, B.H., Ueda, R., Mengin-Lecreulx, D., Lemaitre, B., 2006. The Drosophila amidase PGRP-LB modulates the

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Table 2 . Molecular properties of the five PGRPs paralogs of Bathymodiolus azoricus.

 2

	Name	Length	Signal	MW (Da)*	Theoretical pI*	Potential
			peptide			amidase activity
	Ba-PGRP 1	431	No	49367	6.55	No
	Ba-PGRP 2	195	No	22274.9	6.35	Yes
	Ba-PGRP 3	235	1-17	25983.1/24158.7	9.89/9.89	Yes
	Ba-PGRP 4	223	No	24795.4	6.66	Yes
	Ba-PGRP 5	265	1-17	28555.9/26764.7	7.7/8.05	Yes
	*Molecular weight and pI were determined with or without the signal peptide

Table 3 : Relative quantification of SOX symbionts in the different in situ experimental conditions

 3 (from Détrée et al., 2016).

		Experimental conditions	SOX relative	Standard deviation
			quantification (U)
	Natural	/	805	400
	population
	BT1	6 days on basalt	374	175
	BT2	20 days on basalt	79	32
	BT3	27 days on basalt	73	18
	TR1.2	6 days on basalt, brought	358	122
		back to mussel bed for 14
		days
	TR1.3	6 days on basalt, brought	407	125
		back to mussel bed for 21
		days

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Acknowledgements

We thank the crew and pilots of the NO Pourquoi Pas? and the ROV Victor 6000 for their assistance and technical support, as well as the chief scientist Blandin Jerome during the MomarSAT 2013 cruise. We also, want to thank the Laboratory of Biotechnology and Aquatic Genomics, Interdisciplinary Center for Aquaculture Research (INCAR) for the time allowed for the writing of

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