M Smits

S Artigaud

B Bernay

V Pichereau

L Bargelloni

Christine Paillard

A proteomic study of resistance to Brown Ring disease in the Manila clam, Ruditapes philippinarum

Keywords: Conceptualization, Methodology, Formal analysis, Writing -original draft Sébastien Artigaud : Formal analysis, Data curation, Validation, Writing -original Resources, Software, Formal analysis Vianney Pichereau : Data curation, Validation, Supervision, Writing -review Supervision, Funding acquisition, Resources

Marine mollusk aquaculture has more than doubled over the past twenty years, accounting for over 15% of total aquaculture production in 2016. Infectious disease is one of the main limiting factors to the development of mollusk aquaculture, and the difficulties inherent to combating pathogens through antibiotic therapies or disinfection have led to extensive research on host defense mechanisms and hostpathogen relationships. It has become increasingly clear that characterizing the functional profiles of response to a disease is an essential step in understanding resistance mechanisms and moving towards more effective disease control. The Manila clam, Ruditapes philippinarum, is a main cultured bivalve species of economic subject to Brown Ring Disease (BRD), an infection induced by the bacterium

Vibrio tapetis.

In this study, juvenile Manila clams were subjected to a 28-day controlled challenge with Vibrio tapetis, and visual and molecular diagnoses were carried out to distinguish two extreme phenotypes within the experimental clams: uninfected ("RES", resistant) and infected ("DIS", diseased) postchallenge. Total protein extractions were carried out for resistant and diseased clams, and proteins were identified using LC-MS/MS. Protein sequences were matched against a reference transcriptome of the Manila clam, and protein intensities based on label-free quantification were compared to reveal 49 significantly accumulated proteins in resistant and diseased clams. Proteins with known roles in pathogen recognition, lysosome trafficking, and various aspects of the energy metabolism were more abundant in diseased clams, whereas those with roles in redox homeostasis and protein recycling were more abundant in resistant clams.

Overall, the comparison of the proteomic profiles of resistant and diseased clams after a month-long controlled challenge to induce the onset of Brown Ring disease suggests that redox homeostasis and maintenance of protein structure by chaperone proteins may play important and interrelated roles in resistance to infection by Vibrio tapetis in the Manila clam.

INTRODUCTION

Mollusks represent over a fifth of the global aquaculture market, accounting for USD 29.2 billion in 2016 of which the most heavily traded species are oysters, clams, scallops, and mussels.

Originating from the Asian Pacific coast, the Manila clam, Ruditapes philippinarum has become the second major cultured bivalve in the world, with over 4.4 million tons per year produced worldwide [1]. This species was introduced to the French Atlantic coast for aquaculture diversification in the 1970s [START_REF] Flassch | Introduction in Europe, from 1972 to 1980, of the Japanese Manila clam[END_REF], and following a rapid increase in clam production, mortality events became increasingly frequent and severe, eventually leading to the closure of many clam production parks [START_REF] Zannella | Microbial diseases of bivalve mollusks: Infections, immunology and antimicrobial defense[END_REF]. The mass mortality episodes were subsequently associated to Brown Ring disease (BRD) [START_REF] Paillard | Brown ring disease in clams[END_REF][START_REF] Paillard | Etiologie de la maladie de l'anneau brun chez Tapes philippinarum: pathogénicité d'un Vibrio sp[END_REF], a chronic extra-pallial infection caused by Vibrio tapetis. After initial proliferation of the bacteria in the extra-pallial compartment, diseased clams manifest abnormal conchiolin deposits along the inner surface of the shell. In severe infections, the pathogen may cause lesions in the mantle and penetrate the hemolymph, in which case septicemia and death occur within 4-5 days [START_REF] Allam | Alterations in hemolymph and extrapallial fluid parameters in the Manila Clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis[END_REF][START_REF] Paillard | Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution[END_REF].

In France, BRD continues to negatively impact production and prevalence can reach 80 -100 % [START_REF] Paillard | Flye-Sainte-Marie, A theoretical individual-based model of Brown Ring Disease in Manila clams, Venerupis philippinarum[END_REF] along the Northern Atlantic coast. While the severity of the disease and the virulence of its etiological agent are known to be largely dependent on a number of environmental factors, namely temperature and salinity, bivalves have a number of sophisticated stress and immune response mechanisms, as well as a highly specific innate immune system on which they rely to directly combat infection [START_REF] Paillard | Effect of temperature on defense parameters in Manila clam Ruditapes philippinarum challenged with Vibrio tapetis[END_REF][START_REF] Reid | Salinity effects on immune parameters of Ruditapes philippinarum challenged with Vibrio tapetis[END_REF]. As marine bivalves lack an adaptive immune system, the innate genomic component of their immune system plays an essential role in mitigating the host response to pathogens.

Invertebrate innate immunity relies on a number of pathogen recognition factors that trigger signaling pathways involved in hemocyte recruitment, phagocytosis, and the production of a wide range of antimicrobial compounds for host defense. While resistance to infection in bivalves initially depends on the ability of mucosal interfaces to impede pathogen entrance into the host, circulating hemocytes from fluids (such as hemolymph and extra-pallial fluids) and bioactive molecules in the plasma are responsible for mediating the secondary host response through phagocytosis and direct bacterial neutralization by antimicrobial effectors [START_REF] Allam | Bivalve immunity and response to infections: Are we looking at the right place?[END_REF][START_REF] Allam | Defense factors in clam extrapallial fluids[END_REF]. Interestingly, clams have been shown to recover from BRD through shell repair processes, leading to the investigation of this resistant phenotype in several populations [START_REF] Trinkler | Resistance to Brown Ring Disease in the Manila clam, Ruditapes philippinarum: A study of selected stocks showing a recovery process by shell repair[END_REF]. Previous gene expression and transcriptomic studies on the Manila clam have led to the assembly of a transcriptome and have shed light on several factors such as pathogen recognition and killing, modulation of hemocyte cytoskeleton, regulation of apoptosis, and bio-mineralization that are likely to play key roles in the innate immune response against bacterial infections [START_REF] Paillard | Brown ring disease in clams[END_REF][START_REF] Paillard | Etiologie de la maladie de l'anneau brun chez Tapes philippinarum: pathogénicité d'un Vibrio sp[END_REF][START_REF] Allam | Alterations in hemolymph and extrapallial fluid parameters in the Manila Clam, Ruditapes philippinarum, challenged with the pathogen Vibrio tapetis[END_REF][START_REF] Paillard | Bacterial disease in marine bivalves, a review of recent studies: Trends and evolution[END_REF][START_REF] Paillard | Flye-Sainte-Marie, A theoretical individual-based model of Brown Ring Disease in Manila clams, Venerupis philippinarum[END_REF][START_REF] Jeffroy | Differential expression of genes involved in immunity and biomineralization during Brown Ring Disease development and shell repair in the Manila clam, Ruditapes philippinarum[END_REF][START_REF] Milan | Transcriptome sequencing and microarray development for the Manila clam , Ruditapes philippinarum : genomic tools for environmental monitoring[END_REF][START_REF] Lee | Immune response-related gene expression profile of a novel molluscan IκB protein member from Manila clam (Ruditapes philippinarum)[END_REF][START_REF] Menike | Manila clam, Ruditapes philippinarum Cathepsin D : Molecular analysis and immune response against brown ring disease causing Vibrio tapetis challenge[END_REF][START_REF] Allam | Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease[END_REF][START_REF] Brulle | Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge[END_REF]. The factors influencing virulence and highlighting the dynamics of the infection process leading to Brown Ring disease in the Manila clam are increasingly well described [START_REF] Paillard | Flye-Sainte-Marie, A theoretical individual-based model of Brown Ring Disease in Manila clams, Venerupis philippinarum[END_REF][START_REF] Richard | Immune responses of phenoloxidase and superoxide dismutase in the manila clam Venerupis philippinarum challenged with Vibrio tapetis -Part II: Combined effect of temperature and two V.tapetis strains[END_REF][START_REF] Richard | Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet[END_REF][START_REF] Marie | Proteomic Identification of Novel Proteins from the Calcifying Shell Matrix of the Manila Clam Venerupis Philippinarum[END_REF][START_REF] Paillard | An Ecological Approach To Understanding Host-Pathogen-Environment Interactions: the Case of Brown Ring Disease in Clams[END_REF], and it is has become clear that the interactions between host and pathogen during infection leads to a complex remodeling of the molecular framework of both organisms, highlighting the importance of understanding the changes in gene expression as well as those occurring on the proteomic level.

In addition, several studies focusing on Vibrio-induced expression of immune-related genes in R. philippinarum suggest a tendency towards a downregulation of the inflammatory response and an upregulation of genes related to homeostasis in this species, insisting on the importance of investigating the molecular mechanisms at play to identify markers of resistance [START_REF] Moreira | Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity[END_REF][START_REF] Moreira | Gene expression profile analysis of Manila clam (Ruditapes philippinarum) hemocytes after a Vibrio alginolyticus challenge using an immune-enriched oligo-microarray[END_REF]. The growing number of genomic and transcriptomic resources available for this host species have unveiled particularly high levels of polymorphism, a factor that may mitigate the observed functional variability in the immune response to pathogens [START_REF] Nie | Construction of a high-density genetic map and quantitative trait locus mapping in the Manila clam Ruditapes philippinarum[END_REF][START_REF] Ghiselli | The draft genome of Ruditapes philippinarum (the Manila clam), a promising model system for mitochondrial biology[END_REF], though to date there remains a significant knowledge gap surrounding the functional response of the Manila clam to infection with Vibrio tapetis, particularly in disease-resistant clams.

The present study aims to characterize the proteomic profiles of resistant and susceptible Manila clams following infection with Vibrio tapetis. By comparing the functional response in these two extreme phenotypes, we seek to shed light on the factors responsible for resistance to pathogens in this invertebrate species.

MATERIALS AND METHODS

Experimental design and sampling

Juvenile R. philippinarum (average shell length 12.37 mm) from a cohort of mixed families produced at the SATMAR hatchery (Marennes, France) were acclimated for 12 days in aerated seawater tanks at 14ºC. An experimental group (n=1200) was exposed to air for 8 hours at 14ºC to facilitate valve opening, then placed in a shallow tank and injected into the pallial cavity (without damaging the mantle epithelium) with 50 µL of V. tapetis suspension (strain CECT4600 T ) prepared in filter-sterilized sea water (FSSW) containing 8.2 x10 6 bacteria.mL -1 (4.1 x 10 5 bacteria injected per clam). A first control group (C1; n=300) was injected with 50 µL of FSSW, and a second control group (C2; n=300) received no treatment. All clams (injected as well as not injected) were then kept in dry conditions for at least 6 hours to ensure that the injected clams retained the fluid, then returned to separate tanks in a thermoregulated room at 14ºC with no food and constant aeration for four weeks, according to a standardized protocol established by Paillard & Maes [START_REF] Bidault | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams[END_REF]. As BRD is a chronic infection localized in the extra-pallial compartment, injection into the pallial cavity mimics the natural infection process, whereas injecting tissues results in rapid septicemia for V. tapetis injections, or tissue disruption in the case of sterile filtered sea water injection into the tissues. Dead clams, when they occurred, were recorded daily and removed from the tanks. The seawater remained unchanged throughout the duration of the experiment. After the four-week incubation period, all clams were sampled for whole weight, shell weight, shell length, and each clam was individually opened with a cleaned scalpel over a tube, allowing whole tissues and fluids to be collected together, after which the sample was flash frozen in liquid nitrogen and stored at -80ºC.

Diagnostic methods

For visual diagnosis, shells were observed under a binocular magnifier to identify and quantify the extent index of Brown Ring disease according to the classification system described by Paillard & Maes (1994). For molecular diagnosis, DNA from whole-body tissue and fluid samples was analyzed. Briefly, the samples were homogenized in a volume of phosphate buffer saline (PBS; pH = 7) based on tissue weight, for a final concentration of 0.25 mg/µL tissue in the buffer. Ceramic beads were added to each sample and mechanical tissue disruption was done using 2 cycles of 20 seconds beating (10 sec. pause) at room temperature at 6.5 m/sec on a FastPrep-24 benchtop homogenizer (MP-Bio). Total DNA extractions were carried out using 80 µL (eq. 20 mg) of the homogenate and the Nucleospin 96 Tissue Kit (Macherey-Nagel) according to the manufacturer's protocol with minor adaptations (see detailed protocol in supplementary file 1). The remaining homogenate was flash frozen and stored at -80ºC for subsequent protein extractions. PCR mix was prepared with 1 µL template DNA, 5 µL GoTaq G2 Flexi buffer, 0.15 µL GoTaq polymerase enzyme (1 U/µL), 0.5 µL dNTP mix (each 10 mM), 17.35 µL H 2 0, and 0.5 µL of forward and reverse primers specific to the virB4 gene of the V. tapetis strain CECT4600 T (final volume 25 µL, adapted from Bidault et al. [START_REF] Bidault | Development of a Taqman real-time PCR assay for rapid detection and quantification of Vibrio tapetis in extrapallial fluids of clams[END_REF]). Initial denaturation was done at 94ºC for 5 min, followed by 40 cycles of denaturation (94ºC), annealing (54ºC), and extension (72ºC) for 30 seconds each, and a final extension step at 72ºC for 3 min. PCR products were deposited on 1% agarose gel and electrophoresis was carried out at 110 V for 45 min. In total DNA samples containing V. tapetis DNA, a 173 bp amplicon was then visible by fluorescence.

Total protein extraction and digestion

Based on both visual and molecular diagnoses, samples were assigned to one of four categories representative of the disease kinetics: BRD-/PCR-(0); BRD-/PCR+ (1); BRD+/PCR-(2); BRD+/PCR+ (3). Total proteins were extracted from three samples from category 0 and three samples from category 3 (presenting the same extent index of conchiolin deposit according to Paillard & Maes [START_REF] Paillard | Brown ring disease in the Manila clam Ruditapes philippinarum: Establishment of a classification system[END_REF]), representing the extreme phenotypes hereafter referred to as "RES" (Category 0) and "DIS" (Category 3). Sample homogenates were defrosted on ice and 10 µL protease inhibitor mix (GE Healthcare) was added. After mixing by vortex, the samples were centrifuged at 15 000 x g for 10 min at 4ºC. The supernatant was transferred to a clean tube and proteins were quantified according to the Bradford method [START_REF] Bradford | A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF]. Based on protein concentration, the volume necessary for 50 µg of total proteins was transferred to a clean tube and volume was adjusted to 50 µL with an ammonium bicarbonate (AmBic) solution (100 mM). Samples were reduced with 5 µL dithiothreitol (10 mM) for 40 minutes at 56ºC, then alkylated with 10 µL iodoacetamide (20 mM) for 30 min in the dark. Protein digestion was carried out at 37ºC overnight with 10 µL trypsin buffer (0.1 µg/µL). After digestion, 5% formic acid was added and peptide samples were dehydrated using a SpeedVac™ concentrator.

LC-MS/MS analyses

Peptide quantification and identification was carried out through nano-LC MS/MS to allow for the comparison of the proteomic profiles of resistance to Brown Ring disease in the Manila clam.

Sample Preparation for Mass Spectrometry Analysis

For nano-LC fragmentation, peptide samples were first desalted and concentrated onto a µC18 Omix (Agilent) before analysis. The chromatography step was performed on a NanoElute (Bruker Daltonics) ultra-high pressure nano flow chromatography system. Peptides were concentrated onto a C18 pepmap 100 (5 mm x 300 µm i.d.) precolumn (Thermo Scientific) and separated at 50°C onto a Aurora reversed phase Reprosil column (25 cm x 75 µm i.d.) packed with 1.6 µm C18 coated porous silica beads (Ionopticks). Mobile phases consisted of 0.1% formic acid, 99.9% water (v/v) (A) and 0.1% formic acid in 99.9% ACN (v/v) (B). The nanoflow rate was set at 400 nl/min, and the gradient profile was as follows: from 2 to 15% B within 60 min, followed by an increase to 25% B within 30 min and further to 37% within 10 min, followed by a washing step at 95% B and reequilibration.

Mass Spectrometry Analysis

MS experiments were carried out on a TIMS-TOF pro mass spectrometer (Bruker Daltonics) with a modified nano electrospray ion source (CaptiveSpray, Bruker Daltonics). The system was calibrated each week and mass precision was greater than 1 ppm. A 1400 spray voltage with a capillary temperature of 180°C was typically employed for ionizing. MS spectra were acquired in the positive mode in the mass range from 100 to 1700 m/z. In the experiments described here, the mass spectrometer was operated in PASEF mode with exclusion of single charged peptides. A number of 10 PASEF MS/MS scans was performed during 1.25 seconds from charge range 2-5.

Peptide Sequencing and Protein Precursor Identification

The fragmentation pattern was used to determine the sequence of the peptide. Database searching was performed using the Peaks X software. A custom database was used, consisting in the translated sequences of loci from the digestive gland transcriptome of the Manila clam (unpublished data; a FASTA file containing loci sequences for significantly differentially accumulated proteins can be sound in supplementary file 3, with the corresponding annotations in supplementary file 2). The variable modifications allowed were as follows: C-Carbamidomethyl, K-acetylation, methionine oxidation, and Deamidation (NQ). "Trypsin" was selected as Semispecific. Mass accuracy was set to 30 ppm and 0.05 Da for MS and MS/MS mode respectively. Data were filtering according to a FDR of 0.5% and the elimination of protein redundancy on the basis of proteins being evidenced by the same set or a subset of peptides.

Data analysis

Label-free quantitative data from Peaks X software were imported into Perseus in which statistical analyses were performed [START_REF] Tyanova | The Perseus computational platform for comprehensive analysis of (prote)omics data[END_REF]. Data were log2-transformed and only proteins identified in every sample of at least one of the conditions tested were kept for further analysis.

Data were then compared using a t-test between conditions "RES" and "DIS", a threshold of significance of 0.05 was applied, below which proteins were considered as statistically differentially accumulated.

RESULTS AND DISCUSSION

Identification of RES vs DIS individuals following experimental infection

A major goal of this study was to compare the proteomic changes in R. philippinarum individuals, from a single population, showing contrasted susceptibilities to BRD. Overall, a low mortality of 2.4 % was observed during the four-week incubation period, as it often observed when V. tapetis is injected into the pallial fluids. Moreover, this low mortality occurred mostly on days 5-6, as was previously described by authors which suggested this is due to septicemia following accidental injections of V. tapetis in the tissues [START_REF] Allam | Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and nonsusceptible bivalve species: I. Haemocyte changes following in vitro challenge[END_REF]. The highest mortality per group (5.5 %) occurred in group C1 (inoculated with FSSW), whereas group C2 (no treatment) and the experimental group (inoculated with V. tapetis) showed 1.8 % and 1.9 % total mortality, respectively, comparable to the results obtained in other BRD studies [START_REF] Trinkler | Resistance to Brown Ring Disease in the Manila clam, Ruditapes philippinarum: A study of selected stocks showing a recovery process by shell repair[END_REF]. Shell length (12.37±0.14 mm) and total weight (0.411±0.011 g) were measured for all individuals, and dual diagnosis was carried out for 430 experimental clams. Dual diagnosis of the experimental clams showed that 59 % showed varying degrees of conchiolin deposits, and 42 % were PCR-positive for the strain-specific virB4 173 bp amplicon. Overall, the experimental population was relatively evenly distributed in the four categories (Table 1). Control groups C1 (n=66) and C2 (n=49) showed conchiolin deposits in 1.5 % and 2 % of individuals, respectively, and none of the control samples were positive for molecular diagnosis.

The four diagnostic categories in which the experimental clams were placed represent four stages in the infection process as it occurs within the extra pallial compartment, summarized in Figure 1.

Clams from the two extreme phenotypes, i.e. category 0 (BRD-/PCR-) and category 3 (BRD+/PCR+), were chosen for the following proteomic study.

Differential shotgun proteomics of resistant (RES) vs diseased (DIS) clams

In all, we could identify 2093 proteins, of which 2021 were present in at least 2 out of 3 samples in one or both condition(s) (termed "RES" for category 0/resistant and "DIS" for category 3/diseased). Only proteins identified in both RES and DIS samples were retained for downstream analyses. Spectral counts were used to calculate the relative abundance of proteins. A Student Ttest was used to identify proteins for which abundance was significantly modified in either of the two categories, yielding a list of 102 proteins significantly more abundant in either RES or DIS clams (p-val < 0.05; a complete list of these proteins can be found in the supplementary file 2, and a FASTA file containing the corresponding sequences of the loci can be found in supplementary file 3). Of these, 49 proteins had a fold-change of at least 1.5: seventeen proteins were accumulated in the RES group and thirty-two proteins in the DIS group, four of which could not be characterized (C. gigas protein IDs: EKC23703, EKC34161, EKC41442, EKC37917). These 49 proteins are presented in Figure 2 and their annotations are further detailed in Table 2; they were functionally annotated by examining their associated COG categories, biological process GO terms, and literature review, and discussed below according to their potential roles in different aspects of Brown Ring disease, i.e. the "Immune response", "Energy production" and "Protein metabolism".

Table 1: Dual diagnosis through visual inspection of the inner surface of both valves (BRD+ or BRD-) and PCR

amplification of the virB4 gene region of 173 bp (PCR+ or PCR-) allows for distinction between 4 categories, ranging from CAT 0 (uninfected post-challenge, termed "RES") to CAT 3 (infected according to both diagnostic methods, termed "DIS"). The 430 samples tested show that 24 % and 26 % of samples fall in CAT 0 and CAT 3, respectively. The link between these categories and the kinetics of infection in the extra pallial compartment with Vibrio tapetis is described in Figure 1.

DIAGNOSTIC CATEGORIES:

Total ) and limit of detection (LOD) at 1x10 1 cells. "RES" clams are negative for both visual and molecular diagnosis. Bacteria enter the extrapallial compartment and become quantifiable at point (a), then proliferate rapidly (clams at this stage are termed "CAT 1" in our study), with highest concentrations generally observed around 7 DPI. The host then begins to trap the bacteria within the conchiolin deposits characteristic of Brown ring disease, thus leading to a decrease in the concentration of bacteria in the extrapallial compartment (clams at this stage are termed "CAT 2" in our study), represented by point (b). Clams can thus be positive for the visual diagnosis and negative for molecular diagnosis whilst the bacteria remain trapped against the inner surface of the shell, a process during which the host attempts to recalcify over the bacterial biofilm. In the case of "DIS" clams, conchiolin deposits are present but insufficient in limiting the pathogen, which will continue to proliferate (point (c), also termed "CAT 3" for this study) and reach high concentrations in the extrapallial compartment once more. The ongoing infection process in clams from the DIS group is supported by the presence of a number of proteins whose primary functions are associated with pathogen recognition and the subsequent triggering of signaling pathways, such as Galectin-9 (Gal9), 1-3-β-glucan-binding protein (BGBP), and Complement component C3 (C3). Proteins such as Galectins, which have previously been demonstrated as upregulated in the extra pallial fluids of V. tapetis-infected clams, and BGBP act as pattern recognition receptors (PRRs) by recognizing β-galactoside and lipopolysaccharide residues found on bacterial membranes [START_REF] Allam | Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease[END_REF][START_REF] Vasta | Structural, functional, and evolutionary aspects of galectins in aquatic mollusks: From a sweet tooth to the Trojan horse[END_REF][START_REF] Chaosomboon | Lipopolysaccharide-and β-1,3-glucan-binding protein from Fenneropenaeus merguiensis functions as a pattern recognition receptor with a broad specificity for diverse pathogens in the defense against microorganisms[END_REF]. This initiates the immune response by activating signaling pathways for chemotaxis, phagocytosis, and opsonization and induces antimicrobial peptide (AMP) synthesis through the prophenoloxidase and complement cascade systems [START_REF] Jayaraj | Isolation, purification and characterization of β-1,3-glucan binding protein from the plasma of marine mussel Perna viridis[END_REF][START_REF] Ricklin | Complement component C3 -The "Swiss Army Knife" of innate immunity and host defense[END_REF]. Interestingly, selective breeding for parasite resistance in the Sydney rock oyster found that resistance was directly related to the loss of a specific form of phenoloxidase enzymes, supporting the presence of this particular enzyme as a marker of susceptibility [START_REF] Raftos | Infectious microbial diseases and host defense responses in sydney rock oysters[END_REF][START_REF] Bezemer | Breeding for QX disease resistance negatively selects one form of the defensive enzyme, phenoloxidase, in Sydney rock oysters[END_REF]. The C3 protein identified in our dataset belongs to the complement cascade system, an essential component of the invertebrate immune response leading to the opsonization and lysis of pathogens [START_REF] Zhang | A novel C1q-domain-containing protein from Zhikong scallop Chlamys farreri with lipopolysaccharide binding activity[END_REF][START_REF] Gerdol | The C1q domain containing proteins of the Mediterranean mussel Mytilus galloprovincialis: A widespread and diverse family of immune-related molecules[END_REF][START_REF] Romero | An immuneenriched oligo-microarray analysis of gene expression in Manila clam (Venerupis philippinarum) haemocytes after a Perkinsus olseni challenge[END_REF][START_REF] Arivalagan | Insights from the Shell Proteome: Biomineralization to Adaptation[END_REF]. Upregulation of proteins associated to the complement system have previously been described in R. philippinarum through several transcriptomic studies investigating response to disease, highlighting the importance of this pathway in host response to

Vibrio pathogens [START_REF] Allam | Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease[END_REF][START_REF] Moreira | Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity[END_REF]. The significantly high abundance of proteins specifically involved in pathogen recognition in the DIS group supports a strong acute response to the ongoing infection with V. tapetis in these animals.

Interestingly, the RES clam group showed high abundance of Ras GTPase-activating-like protein 1 (IQGAP1), a pathway-activating protein that is implicated in a number of immune-associated functions. Namely, during infection by microbial pathogens that target the host microtubule network, similar to the way in which V. tapetis inhibits pseudopod formation in the hemocytes of R. philippinarum, IQGAP1 has been shown to bind to and modulate the activity of proteins involved in bacterial invasion, ultimately interacting with the actin cytoskeleton [START_REF] Brulle | Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge[END_REF][START_REF] Kim | IQGAP1 in microbial pathogenesis: Targeting the actin cytoskeleton[END_REF].

Pathogen-associated lysosomal activity

Other proteins highly abundant in the DIS group are involved in immune response through antimicrobial and lysosomal activity. The putative serine carboxypeptidase (CPLV) and the serine protease Ovochymase (OVCH1) identified in the DIS group are known to have antibacterial activity and can be involved in proteolytic digestion of lysosomal components [START_REF] Harris | A vitellogenic-like carboxypeptidase expressed by human macrophages is localized in endoplasmic reticulum and membrane ruffles[END_REF][START_REF] Gao | Ovochymase in amphioxus Branchiostoma belcheri is an ovary-specific trypsin-like serine protease with an antibacterial activity[END_REF]. As lysosomes contain the hydrolytic enzymes necessary for degradation of cellular components as well as encapsulated pathogenic agents, it is also interesting to note the higher abundance of Ependymin-related protein 1 (EPDR1) in DIS samples, which has been suggested

to function as a lysosomal activator protein, and Palmitoyl-protein thioesterase-1 (PPT1), which is associated with lysosomal degradation of proteins [START_REF] Wei | Crystal structures of human lysosomal EPDR1 reveal homology with the superfamily of bacterial lipoprotein transporters[END_REF][START_REF] Bannan | The drosophila protein palmitoylome characterizing palmitoyl-thioesterases and DHHC palmitoyl-transferases[END_REF]. An uncharacterized protein in the DIS clam group belonging to the carbohydrate esterase 4 (CE4) family may also play a role in degrading phagocytosed bacteria as well as inhibiting biofilm formation, as certain enzymes of the CE4 family, whose main function is to de-acetylate polysaccharides, specifically degrade the essential peptidoglycan polymers of bacterial cell walls [START_REF] Balomenou | Polysaccharide Deacetylases: New Antibacterial Drug Targets[END_REF]. In addition, active intracellular membrane trafficking (ex. phagosomes) in DIS clams is suggested by the high abundance of proteins such as the Ras-related protein RAB43, which regulates vesicular movement following immune system activation by microbial infections, and a vacuolar-sorting protein VPS35, which directs transmembrane cargo proteins to the lysosomal degradation pathway [START_REF] Pei | Immune regulation of Rab proteins expression and intracellular transport[END_REF][START_REF] Linhart | Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson's disease mutant of Leucine-rich repeat kinase 2 (LRRK2)[END_REF].

Cytoskeleton-associated immune response

The cytoskeleton is a network of filaments that plays an essential role in certain aspects of immunity through cell structure maintenance, transport, phagocytosis, and communication between cellular components. As such, it is also known to be one of the targets of invading pathogens such as Perkinsus olseni and V. tapetis in the Manila clam [START_REF] Brulle | Transcriptomic analysis of Ruditapes philippinarum hemocytes reveals cytoskeleton disruption after in vitro Vibrio tapetis challenge[END_REF][START_REF] Fernández-Boo | Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni[END_REF][START_REF] Rahmani | Transcriptomic analysis of clam extra pallial fluids reveals immunity and cytoskeleton alterations in the first week of brown ring disease development[END_REF]. Interestingly, a dynactin subunit (DCTN2) and a sarcoplasmic calcium-binding protein (SCP) were highly abundant in DIS clams, both of which play a role in cytoskeleton function. DCTN2 is part of a dynein/dynactin complex which coordinates the microtubule movement of vacuoles towards lysosomes and plays an important role in the biogenesis and transportation of pathogencontaining vacuoles in rabbit cells infected by obligate intracellular bacteria [START_REF] Ortiz Flores | The role of microtubules and the dynein/ dynactin motor complex of host cells in the biogenesis of the Coxiella burnetii-containing vacuole[END_REF][START_REF] Gill | Dynactin, a conserved, ubiquitously expressed component of an activator of vesicle motility mediated by cytoplasmic dynein[END_REF]. This may reflect the internalization of V. tapetis by hemocytes in the case of DIS clams, activating microtubule motors such as the dynein/dynactin complex in an attempt to fuse bacteria-containing vacuoles with lysosomal membranes. SCPs, which are known to interact with the cytoskeleton by regulating the calcium balance, have been shown to be upregulated in Manila clam hemocytes in response to both parasitic and bacterial infections [START_REF] Allam | Transcriptional changes in Manila clam (Ruditapes philippinarum) in response to Brown Ring Disease[END_REF][START_REF] Marie | Proteomic Identification of Novel Proteins from the Calcifying Shell Matrix of the Manila Clam Venerupis Philippinarum[END_REF][START_REF] Fernández-Boo | Protein expression profiling in haemocytes and plasma of the Manila clam Ruditapes philippinarum in response to infection with Perkinsus olseni[END_REF]. Bacteria of the genus Vibrio have been suggested to inhibit intracellular trafficking and the fusion of bacteriacontaining phagosomes with lysosomes in order to avoid neutralization, a relatively common immune-evasion technique seen in a number of pathologies [START_REF] Flannagan | Antimicrobial mechanisms of phagocytes and bacterial evasion strategies[END_REF]. In this light, the elevated abundance of lysosome-associated proteins in clams of the DIS group may reflect an attempt to counter the bacteria's inhibitory processes.

Apoptotic processes

Apoptosis of host immune cells during an infection can represent one of the ultimate defense strategies against invading pathogens, whereby the host cell is sacrificed so as to eliminate the internalized pathogenic agent [START_REF] Romero | The complexity of apoptotic cell death in mollusks: An update[END_REF]. Induction of apoptosis has been suggested to be one of the mechanisms of survival put in place by resistant oysters, Ostrea edulis, perhaps in response to anti-apoptotic mechanisms that are a known survival strategy for some intracellular pathogens, such as the protozoan parasite Bonamia ostreae [START_REF] Morga | New insights in flat oyster Ostrea edulis resistance against the parasite Bonamia ostreae[END_REF]. Clams from the RES group in our study demonstrated higher abundances of Endophilin B1 (Bif-1), a protein associated with the formation of pores in the outer mitochondrial membrane that leads to apoptosis through the caspase pathway [START_REF] Morga | New insights in flat oyster Ostrea edulis resistance against the parasite Bonamia ostreae[END_REF][START_REF] Pawlowski | Bax-induced apoptotic cell death[END_REF][START_REF] Takahashi | Loss of Bif-1 Suppresses Bax/Bak Conformational Change and Mitochondrial Apoptosis[END_REF]. In contrast, DIS clams showed high abundance of adenosine deaminase (CECR1) a protein that, while primarily associated with the purine metabolism, also plays an important role in reducing the concentration of the toxic derivatives of adenosine and deoxyadenosine to protect cells from apoptosis [START_REF] Mutsuzaki | Influence of knee immobilization on chondrocyte apoptosis and histological features of the anterior cruciate ligament insertion and articular cartilage in rabbits[END_REF][START_REF] Semerjyan | Effects of Mg2+, Cd2+, Cu2+ Low Concentrations and Immobilization Stress on the Activity of Adenosine Deaminase in Different Organs of Rabbits[END_REF]. A serine/threonine protein phosphatase PPP3CC, also more abundant in DIS clams, bears close resemblance with the protein phosphatase 3 catalytic subunit beta, PPP3CB, which was recently suggested to promote cell-proliferation and may play an anti-apoptotic role in tumorous human kidney cells [START_REF] Chen | PPP3CB Inhibits Migration of G401 Cells via Regulating Epithelial-to-Mesenchymal Transition and Promotes G401 Cells Growth[END_REF]. While apoptosis is a complex process that can benefit immune defense, anti-apoptosis is also a mechanism by which the host may maintain cellular functions and continue combatting infection, though seemingly at a cost given the relatively high abundance of proteins involved in energy metabolism in the DIS clam group (see part 3.4).

Redox homoeostasis

Host production of reactive oxygen species (ROS) and reactive nitrogen species (RNS) is a known mechanism of defense and pathogen neutralization, though this process requires a delicate balance as the accumulation of ROS/RNS can also lead to oxidative damage to the host [START_REF] Anderson | Reactive oxygen species and antimicrobial defenses of invertebrates: A bivalve model[END_REF].

Glutathione peroxidase (GPX3), a member of the cellular antioxidant system, was more abundant in the DIS clam group. Interestingly, a previous studies on BRD-infected Manila clams reported a decrease in enzymatic activity of GPX at 7 dpi, though the study did not measure enzyme abundance nor activity at 30 dpi [START_REF] Richard | Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet[END_REF]. Oysters subjected to bacterial infection, however, have shown an upregulation of glutathione peroxidase gene expression in pathogen-challenged larvae, suggesting that GPX may be an indicator of oxidative stress that may have been occurring in DIS clams from our study as a result of the ongoing antibacterial processes discussed in part 3.3.2 [START_REF] Richard | Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet[END_REF][START_REF] Genard | Integrative Study of Physiological Changes Associated with Bacterial Infection in Pacific Oyster Larvae[END_REF].

The RES clam group was characterized by a higher abundance of proteins more or less directly associated with ROS/RNS production and scavenging. Thioredoxin domain-containing protein 5 (TXNDC5) is a member of the protein disulfide isomerase family whose expression has been associated with oxidative stress and cellular pathology [START_REF] Chawsheen | A critical role of the thioredoxin domain containing protein 5 (TXNDC5) in redox homeostasis and cancer development[END_REF][START_REF] Horna-Terrón | TXNDC5, a newly discovered disulfide isomerase with a key role in cell physiology and pathology[END_REF]. At least two shorter-term (max 7 days) studies have investigated the role of thioredoxin in response to V. tapetis, pointing towards an increase in activity of thioredoxin in V. tapetis-challenged clams compared to controls or to resistant clams, thus the higher abundance of thioredoxin observed in RES clams of this study may reflect a possible reversal of this mechanism in the case of long-term resistance (30 days) [START_REF] Richard | Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet[END_REF][START_REF] Umasuthan | Mitochondrial thioredoxin-2 from Manila clam (Ruditapes philippinarum) is a potent antioxidant enzyme involved in antibacterial response[END_REF]. Similarly, xanthine oxidoreductase (XOR), an enzyme implicated in the purine metabolism, has previously been associated with a number of innate immunity processes including redox homeostasis through the production of ROS and RNS by xanthine oxidase (XO), as well as indirect free radical sequestration by xanthine dehydrogenase (XD) through the synthesis the antioxidant uric acid [START_REF] Anderson | Reactive oxygen species and antimicrobial defenses of invertebrates: A bivalve model[END_REF][START_REF] Liu | Construction of a highdensity, high-quality genetic map of cultivated lotus (Nelumbo nucifera) using next-generation sequencing[END_REF][START_REF] Vorbach | Xanthine oxidoreductase is central to the evolution and function of the innate immune system[END_REF][START_REF] Glantzounis | Uric Acid and Oxidative Stress[END_REF]. RES clams also presented a significantly higher abundance of two Angiotensin-converting enzymes (ACE), which were recently demonstrated to induce superoxide production and thereby enhance bactericidal activity in mouse neutrophils [START_REF] Khan | Angiotensin-converting enzyme enhances the oxidative response and bactericidal activity of neutrophils[END_REF], and class-III alcohol dehydrogenase (ADH3). ADH3 uses the reducing power glutathione to eliminate the organic compounds formaldehyde and S-nitrosothiols (SNO), ultimately limiting their decomposition into the RNS nitric oxide [START_REF] Di Meo | Role of ROS and RNS Sources in Physiological and Pathological Conditions[END_REF][START_REF] Zhou | Synthesis and nitric oxide releasing properties of novel fluoro S-nitrosothiols[END_REF]. Nitric oxide is a known toxic agent produced by immune cells of invertebrates for pathogen defense, and NO production by R.

philippinarum in response to V. tapetis has been correlated to hemocyte rounding and pseudopod loss [START_REF] Jeffroy | Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacterium Vibrio tapetis[END_REF], highlighting the possible importance of NO-sequestration by proteins such as ADH3 in order to counter the negative effects of this compound on the host.

These findings support previous hypotheses suggesting that ROS/RNS production by hemocytes may represent an alternative anti-bacterial response against pathogens such as V. tapetis, which is known to evade host immune responses such as phagocytosis by inhibiting pseudopod formation [START_REF] Jeffroy | Involvement of nitric oxide in the in vitro interaction between Manila clam, Ruditapes philippinarum, hemocytes and the bacterium Vibrio tapetis[END_REF][START_REF] Tafalla | Production of nitric oxide by mussel (Mytilus galloprovincialis) hemocytes and effect of exogenous nitric oxide on phagocytic functions[END_REF]. In addition, the higher abundance of the redox homeostasis-associated enzymes RES clams supports findings from previous studies indicating that resistance may be associated with a greater ability to balance ROS/RNS production and scavenging, allowing them to rapidly neutralize pathogens before they are able to colonize the host while simultaneously protecting host cells from oxidative damage, a dual process previously suggested using enzyme activity assays in the Manila clam exposed to V. tapetis [START_REF] Richard | Metabolic responses of clam Ruditapes philippinarum exposed to its pathogen Vibrio tapetis in relation to diet[END_REF].

PROCESSES ASSOCIATED WITH ENERGY PRODUCTION

Proteins potentially associated with energy production through carbohydrate and amino acid catabolism represent the second largest group in our dataset, most of which (10 out of 12) are more abundant in the DIS clam group. This section groups together the proteins implicated in the degradation of glycogen and amino acids that generate essential metabolites for the tricarboxylic acid (TCA) cycle, leading to the production of high-energy electron donors such as NADH which can integrate the electron transport chain (Figure 3).

Amino acid degradation for energy production

Propionyl-CoA carboxylase beta chain (PCCB) and Methylglutaconyl-CoA hydratase (AUH), enzymes involved in the degradation of branched-chain amino acids (BCAAs) such as leucine, were significantly more abundant in DIS and RES clams, respectively. Demand for BCAAs as energy metabolites or for the synthesis of immune-related molecules has been demonstrated to increase during disease [START_REF] Calder | Branched-Chain Amino Acids and Immunity[END_REF][START_REF] Wongkittichote | Propionyl-CoA carboxylase -A review[END_REF]. Decreased levels of a number of other amino acids during Vibrio infection in mussels have also been reported, suggesting that the significantly higher abundance of Alanine aminotransferase (GPT2), Aspartate aminotransferase (GOT1), and Excitatory aminoacid transporter-1 (SLC1A1) in DIS clams may also be associated with the degradation and/or conversion of amino acids into metabolites such as pyruvate, oxaloacetate, and α-ketoglutarate for the TCA cycle [START_REF] Nguyen | Metabolomics Study of Immune Responses of New Zealand Greenshell TM Mussels (Perna canaliculus) Infected with Pathogenic Vibrio sp[END_REF][START_REF] Taber | A simple experiment demonstrating the allosteric regulation of yeast pyruvate kinase[END_REF][START_REF] Van Waarde | Biochemistry of non-protein nitrogenous compounds in fish including the use of amino acids for anaerobic energy production[END_REF][START_REF] Kanai | The glutamate/neutral amino acid transporter family SLC1: Molecular, physiological and pharmacological aspects[END_REF].

Carbohydrate metabolism and TCA cycle

Our dataset showed a high abundance of proteins linking glycolysis with the TCA cycle for the aerobic production of ATP such as Pyruvate dehydrogenase (PDH) E1 subunit (alpha type III) in RES clams, as well as Glycogen debranching enzyme (AGL) and Phosphoglucomutase-1 (PGM1) in DIS clams, which may reflect the degradation of carbohydrates for the production of pyruvate, namely in the DIS clam group [START_REF] Patel | The pyruvate dehydrogenase complexes: Structure-based function and regulation[END_REF]. This group is also characterized by a greater number of proteins associated with various aspects of the immune response, a process known to be energetically demanding [START_REF] Brokordt | Reproduction immunity trade-off in a mollusk: Hemocyte energy metabolism underlies cellular and molecular immune responses[END_REF]. More importantly, weight loss, decreased glycogen reserves, and condition index, which are indicative of energy imbalance and poor health, have previously been attributed to the negative impact of BRD on energy balance in the Manila clam [START_REF] Paillard | Flye-Sainte-Marie, A theoretical individual-based model of Brown Ring Disease in Manila clams, Venerupis philippinarum[END_REF][START_REF] Flye-Sainte-Marie | An ecophysiological approach of Brown Ring Disease in the Manila clam, Ruditapes philippinarum: experiments and modelling[END_REF][START_REF] Goulletquer | A bibliography of the Manila Clam Tapes philippinarum[END_REF].

Malate dehydrogenase (MDH1), which participates in the TCA cycle by oxidizing malate to form oxaloacetate, was also more abundant in DIS clams, supporting the hypothesis of increased carbohydrate degradation for energy production. Our dataset also indicated a significantly higher abundance of the mitochondrial Phosphoenolpyruvate carboxykinase (PEPCK-M) in the DIS group, which is generally associated with catalyzing the irreversible conversion of oxaloacetate to phosphoenolpyruvate (PEP) for gluconeogenesis. While this appears to contradict the hypothesis of glycogen and glucose degradation, recent studies have demonstrated that overexpression of PEPCK-M (as opposed to the cytosolic form PEPCK-C) may play a role in antiviral immunity in insects, and the accumulation of PEP in infected ticks was suggested to be an antibacterial mechanism against the bacterial pathogen Anaplasma phagocytophilum [START_REF] Guo | Phosphoenolpyruvate carboxykinase is involved in antiviral immunity against Bombyx mori nucleopolyhedrovirus[END_REF][START_REF] Cabezas-Cruz | Ixodes scapularis Tick Cells Control Anaplasma phagocytophilum Infection by Increasing the Synthesis of Phosphoenolpyruvate from Tyrosine[END_REF].

Electron transport chain

The end products of the TCA cycle ultimately convey their electrons to the electron transport chain (ETC), composed of molecules within the inner membrane of the mitochondria, including the essential proteins ubiquinone (coenzyme Q10) and ATP synthase. In the DIS group, the high abundance of 3-demethylubiquinone-9 3-O-methyltransferase, which participates in the final step of ubiquinone synthesis, suggests that there is a high demand for electron acceptor molecules which increase the proton gradient in the inter membrane space of the mitochondria [START_REF] Sokolov | Effects of hypoxia-reoxygenation stress on mitochondrial proteome and bioenergetics of the hypoxia-tolerant marine bivalve Crassostrea gigas[END_REF]. Overall, proteins associated with the energy metabolism, including ATP synthase subunits and ETCassociated proteins, as well as proteins associated with energy production from both amino acids and sugars, are more abundant in the DIS group than in the RES group, reflecting a high energy demand likely as a result of the active immune response described previously.

PROTEIN METABOLISM

Seven highly abundant proteins in the dataset were associated with various aspects of protein metabolism, namely proteolysis, transport, and chaperones.

Proteolysis

Proteolytic processes are represented in our dataset by the proteins cullin-associated NEDD8dissociated protein 1 (CAND1) and subunit 4 of the 26S proteasome complex (PSMC1) in the RES group, and by the 26S proteasome non-ATPase regulatory subunit 7 (PSMD7) in the DIS group (Table 2). In eukaryotic cells, proteolysis, or the degradation of proteins and recycling of their components, is a process mediated by the conjugation of polyubiquitin chains to proteins which are then recognized by the 26S proteasome complex, a multi-subunit enzyme responsible for proteolysis [START_REF] Pickart | Proteasomes and their kin: Proteases in the machine age[END_REF]. CAND1 binds to unneddylated CUL1, one of the three major components of an E3 ubiquitin ligase playing an essential role in protein degradation by regulating ligase ubiquitination [START_REF] Zheng | CAND1 Binds to Unneddylated CUL1 and Regulates the Formation of SCF Ubiquitin E3 Ligase Complex[END_REF]. Interestingly, ubiquitin ligase complexes were found to be one of the targets of pathogenic bacteria during infection, whereby inhibiting factors produced by pathogens may be able to effectively bind to CUL1, preventing it from correctly forming the ligase complex [START_REF] Jubelin | Pathogenic bacteria target NEDD8-conjugated cullins to hijack host-cell signaling pathways[END_REF]. PSMC1 coordinates substrate recruitment and translocation into the proteolytic chamber of the proteasome, and is essential for rapid proteolysis [START_REF] Rosenzweig | The central unit within the 19S regulatory particle of the proteasome[END_REF]. Similarly, PSMD7 is another component of the proteasome important in mediating the recognition of polyubiquitin chains and cleavage of ubiquitin from degraded proteins [START_REF] Shi | PSMD7 downregulation induces apoptosis and suppresses tumorigenesis of esophageal squamous cell carcinoma via the mTOR/p70S6K pathway[END_REF]. In a previous geneexpression study of P. olseni-infected Manila clams, proteasome subunits were found to be downregulated in diseased animals, indicating decreased proteolytic activity [START_REF] Romero | An immuneenriched oligo-microarray analysis of gene expression in Manila clam (Venerupis philippinarum) haemocytes after a Perkinsus olseni challenge[END_REF]. In that respect, the elevated abundance of CAND1 and PSMC1 in RES clams may indicate a trend towards ubiquitin tagging of damaged proteins and more active protein degradation in RES clams than in DIS clams.

High proteolytic activity ion RES clams may be linked to the digestion of phagocytosed and neutralized bacteria, and may also reflect the presence of a pro-apoptotic protein in this group. In addition, it may be possible that RES clams were able to sustain the cellular functions necessary for effective elimination of the pathogen, after which proteins damaged by oxidative stress during the immune response are degraded.

Transport, folding, and chaperone functions

Our dataset also contained proteins associated with various aspects of protein synthesis, including protein transport (Table 2). Titin (TTN), a large structural protein thought to function as a scaffold protein, has been shown to interact with actin and filamin, proteins of the cytoskeleton implicated in the movement of cellular components and proteins [START_REF] González-Morales | Filamin actin-binding and titin-binding fulfill distinct functions in Z-disc cohesion[END_REF]. In vertebrate striated muscle, TTN was specifically shown to recruit E3 ubiquitin-ligase [START_REF] Mrosek | Molecular determinants for the recruitment of the ubiquitin-ligase MuRF-1 onto M-line titin[END_REF], thus its high abundance in RES clams may reflect transport associated with proteolysis, consistent with the fact that this group also showed higher abundance of CAND1 and a proteasome subunit. Another protein implicated in protein transport is 14-3-3 epsilon (YWHAE), a binding protein suggested to play a role in protein transport to the secretory pathway, namely through interaction with GTPase-activating proteins such as IQGAP1 (see 3.3.3

Cytoskeleton-associated immune response), also found highly abundant in RES clams [START_REF] Mrowiec | Schwappach, 14-3-3 Proteins in membrane protein transport[END_REF].

DIS clams showed a high abundance of FK506 binding protein 14 (FKBP14), which is thought to accelerate protein folding as it belongs to a family of peptidyl-prolyl cis-trans isomerases that play a role in the folding of newly synthesized proteins [START_REF] Shaw | Peptidyl-prolyl isomerases: a new twist to transcription[END_REF]. The higher abundance of this protein in the DIS group may reflect an increase in the synthesis of immune-related proteins, which were also more abundant in DIS clams. Furthermore, it is also interesting to note that this family of proteins (also called immunophilins) has been shown to inhibit early establishment and intracellular infection by bacteria [START_REF] Bella | Peptidyl-prolyl cis-trans isomerases (immunophilins) and their roles in parasite biochemistry, host-parasite interaction and antiparasitic drug action[END_REF]. Though not directly implicated in protein metabolism, the DIS group also presented high abundance of an Abhydrolase domain-containing protein associated with biosynthesis of phosphatidylcholine, an essential class of membrane phospholipids, possibly reflecting an increase in membrane synthesis due to the internalization of bacteria in phagosomes [START_REF] Van Meer | Membrane lipids: where they are and how they behave[END_REF].

Heat-shock protein 90 (HSP90), a chaperone protein that plays a crucial role in protecting protein structure in response to stress conditions, was highly abundant in RES clams. Due to its interaction with the major histocompatibility complex (MHC) and the antigen processing pathways in vertebrates [START_REF] Imai | Heat shock protein 90 (HSP90) contributes to cytosolic translocation of extracellular antigen for crosspresentation by dendritic cells[END_REF], HSP90 has been suggested to play a role in the innate immune system response and resistance to infection in invertebrates [START_REF] Sung | Heat Shock Proteins and Disease Control in Aquatic Organisms[END_REF]. As HSP synthesis is promoted by protein denaturation, the trend towards proteolytic activity in RES clams discussed in part 3.5.1 may be partially responsible for activating HSP synthesis. That said, the higher abundance of HSP90 in RES clams may also indicate that resistant clams have a lower threshold for protein denaturation and thus perhaps more rapidly activate the synthesis of protective chaperone proteins, granting them an advantage over DIS clams when it comes to cellular protection.

OTHER

The transcriptional activator protein Pur-alpha (PURA), a protein involved in controlling DNA replication and gene transcription processes [START_REF] Darbinian | Association of Purα and E2F-1 suppresses transcriptional activity of E2F-1[END_REF][START_REF] Rocher | 2-DE Mapping of the Blue Mussel Gill Proteome: The Usual Suspects Revisited[END_REF], was highly abundant in the DIS group, while a Poly (rc)-binding protein 3 (PCBP3) associated with negative regulation of transcription [START_REF] Kang | Novel dual-binding function of a poly (C)-binding protein 3, transcriptional factor which binds the double-strand and single-stranded DNA sequence[END_REF] was highly abundant in the RES group [START_REF] Kang | Novel dual-binding function of a poly (C)-binding protein 3, transcriptional factor which binds the double-strand and single-stranded DNA sequence[END_REF].

Proteins associated with translation include RPL27A, a structural component of the 60S ribosome subunit whose upregulation has previously been reported in white-spot infected shrimp and in hypoxiastressed oysters [START_REF] Wongpanya | Analysis of gene expression in haemocytes of shrimp Penaeus monodon challenged with white Spot syndrome virus by cDNA microarray[END_REF][START_REF] David | Response of the Pacific oyster Crassostrea gigas to hypoxia exposure under experimental conditions[END_REF], and asparaginyl-tRNA synthetase (NARS), whose primary function in translation is to catalyze the attachment of asparagine to its corresponding tRNA.

Finally, four significantly abundant proteins were uncharacterized; three were highly abundant in the DIS group and one in the RES group. showing concentration of V. tapetis in extrapallial fluids (cells.mL -1 ) and limit of detection (LOD) at 1x10 1 cells. "RES" clams are negative for both visual and molecular diagnosis. Bacteria enter the extrapallial compartment and become quantifiable at point (a), then proliferate rapidly (clams at this stage are termed "CAT 1" in our study), with highest concentrations generally observed around 7 DPI. The host then begins to trap the bacteria within the conchiolin deposits characteristic of Brown ring disease, thus leading to a decrease in the concentration of bacteria in the extrapallial compartment (clams at this stage are termed "CAT 2" in our study), represented by point (b). Clams can thus be positive for the visual diagnosis and negative for molecular diagnosis whilst the bacteria remain trapped against the inner surface of the shell, a process during which the host attempts to recalcify over the bacterial biofilm. In the case of "DIS" clams, conchiolin deposits are present but insufficient in limiting the pathogen, which will continue to proliferate (point (c), also termed "CAT 3" for this study) and reach high concentrations in the extrapallial compartment once more.

Figure 1 :

 1 Figure 1: Schematic representation of the kinetics of Brown ring disease development during 30 days post injection (DPI) in a controlled challenge, adapted from Bidault et al. 2015, showing concentration of V. tapetis in extrapallial fluids (cells.mL -1) and limit of detection (LOD) at 1x10 1 cells. "RES" clams are negative for both visual and molecular diagnosis. Bacteria enter the extrapallial compartment and become quantifiable at point (a), then proliferate rapidly (clams at this stage are termed "CAT 1" in our study), with highest concentrations generally observed around 7 DPI. The host then begins to trap the bacteria within the conchiolin deposits characteristic of Brown ring disease, thus leading to a decrease in the concentration of bacteria in the extrapallial compartment (clams at this stage are termed "CAT 2" in our study), represented by point (b). Clams can thus be positive for the visual diagnosis and negative for molecular diagnosis whilst the bacteria remain trapped against the inner surface of the shell, a process during which the host attempts to recalcify over the bacterial biofilm. In the case of "DIS" clams, conchiolin deposits are present but insufficient in limiting the pathogen, which will continue to proliferate (point (c), also termed "CAT 3" for this study) and reach high concentrations in the extrapallial compartment once more.

Figure 2 :

 2 Figure 2: Number of proteins per functional group (i.e. "immune response", "energy production", "protein metabolism") that were more abundant in DIS clams (dark bars) and in RES clams (light bars).

Figure 3 :

 3 Figure 3: Schematic representation of energy production and the carbohydrate and amino acid metabolisms, including glycolysis, the TCA cycle, amino-acid degradation pathways, and the electron transport chain. The processes in which highly abundant proteins in DIS (orange) and RES (green) clams are implicated are annotated with the abbreviated protein name. Glycogen debranching enzyme (AGL), Phosphoglucomutase-1 (PGM1), and Phosphoenolpyruvate carboxykinase (PEPCK) are associated to the carbohydrate metabolism (both glycolysis and gluconeogenesis). Alanine aminotransferase (GPT2), Propionyl-CoA carboxylase beta chain (PCCB), Methylglutaconyl-CoA hydratase (AUH), Aspartate aminotransferase (GOT1), and Excitatory amino-acid transporter-1 (SLC1A1) all participate in the degradation of amino acids that can play a role in replenishing metabolites of the TCA cycle. Malate dehydrogenase 1 (MDH1) is an essential enzyme of the TCA cycle, the high-energy products of which are then shuttles to the electron transport chain where proteins such as 3-demethylubiquinone-9 3-methyltransferase (REBM) and ATP synthase subunit alpha (ATP5A1) participate in the production of ATP.

  of the fastest-growing sectors of aquaculture worldwide, mollusk production continues to suffer significant losses due in part to the impact of infectious diseases. The study of proteomic profiles offers the possibility of better understanding the complex functional mechanisms at play during host response to disease, and may shed light on factors associated with resistance to disease.The aim of the present study was to investigate the proteomic profiles of resistance to controlled infection with Vibrio tapetis, the etiological agent of Brown Ring Disease, in the Manila clam, Ruditapes philippinarum. The comparison of proteomic profiles of two extreme phenotypes (RES and DIS) observed in juvenile Manila clams shows a number of functional differences in highly abundant proteins implicated in the immune response-associated processes, energy production, and protein metabolism. Twice as many significantly abundant proteins associated with the immune response were accumulated in the DIS group compared to the RES group, reflecting the ongoing infection as established by the presence of both visual and molecular signs of disease. That said, the function of immune-associated proteins in the RES group was almost consistently associated with redox homeostasis, whereas in DIS group the abundant proteins were mostly involved in pathogen recognition, signaling, and neutralization. This may suggest that disease resistant clams are better equipped to manage ROS production and scavenging in order to simultaneously eliminate the pathogen and protect host cellular components from oxidative stress. Protein degradation as well as protection by chaperones were another process highly represented in resistant clams, with degradation possibly as a result of successful elimination of the pathogen which may nonetheless have left a number of cellular components damaged by oxidative stress. The fact that only resistant clams showed significantly high abundance of a chaperone-associated protein suggests that this may be an important factor of resistance to disease. In contrast, diseased clams showed a higher abundance of proteins involved in protein synthesis and functional modifications, possibly in response to activation by the immune system in order to continue fighting infection. Both immune response and protein synthesis are energy demanding processes, which is further supported by the presence of proteins involved in glycolysis, TCA cycle, and the electron transport chain. Overall, the comparison of the proteomic profiles of resistant and sick clams suggests that redox homeostasis and maintenance of protein structure by chaperone proteins may play important and interrelated roles in resistance to infection by Vibrio tapetis in the Manila clam.

Figure 1 :

 1 Figure 1: Schematic representation of the kinetics of Brown ring disease development during 30 days post injection (DPI) in a controlled challenge, adapted from Bidault et al. 2015,showing concentration of V. tapetis in extrapallial fluids (cells.mL -1 ) and limit of detection (LOD) at 1x10 1 cells. "RES" clams are negative for both visual and molecular diagnosis. Bacteria enter the extrapallial compartment and become quantifiable at point (a), then proliferate rapidly (clams at this stage are termed "CAT 1" in our study), with highest concentrations generally observed around 7 DPI. The host then begins to trap the bacteria within the conchiolin deposits characteristic of Brown ring disease, thus leading to a decrease in the concentration of bacteria in the extrapallial compartment (clams at this stage are termed "CAT 2" in our study), represented by point (b). Clams can thus be positive for the visual diagnosis and negative for molecular diagnosis whilst the bacteria remain trapped against the inner surface of the shell, a process during which the host attempts to recalcify over the bacterial biofilm. In the case of "DIS" clams, conchiolin deposits are present but insufficient in limiting the pathogen, which will continue to proliferate (point (c), also termed "CAT 3" for this study) and reach high concentrations in the extrapallial compartment once more.

Figure 2 :

 2 Figure 2: Number of proteins per functional group (i.e. "immune response", "energy production", "protein metabolism") that were more abundant in DIS clams (dark bars) and in RES clams (light bars).

Figure 3 :

 3 Figure3: Schematic representation of energy production and the carbohydrate and amino acid metabolisms, including glycolysis, the TCA cycle, amino-acid degradation pathways, and the electron transport chain. The processes in which highly abundant proteins in DIS (orange) and RES (green) clams are implicated are annotated with the abbreviated protein name. Glycogen debranching enzyme (AGL), Phosphoglucomutase-1 (PGM1), and Phosphoenolpyruvate carboxykinase (PEPCK) are associated to the carbohydrate metabolism (both glycolysis and gluconeogenesis). Alanine aminotransferase (GPT2), Propionyl-CoA carboxylase beta chain (PCCB), Methylglutaconyl-CoA hydratase (AUH), Aspartate aminotransferase (GOT1), and Excitatory amino-acid transporter-1 (SLC1A1) all participate in the degradation of amino acids that can play a role in replenishing metabolites of the TCA cycle. Malate dehydrogenase 1 (MDH1) is an essential enzyme of the TCA cycle, the high-energy products of which are then shuttles to the electron transport chain where proteins such as 3-demethylubiquinone-9 3-methyltransferase (REBM) and ATP synthase subunit alpha (ATP5A1) participate in the production of ATP.

Table 2 :

 2 Proteins significantly abundant (p-val < 0.05) in DIS and RES clams, with fold-change > 1.5 ("group ratio"). Based on Clusters of Orthogous Groups (COG), Biological Process GO terms, and literature review regarding the roles of these proteins in the context of disease, the proteins are grouped into three main functional roles described in the discussion; "Immune response", "Energy production", and "Protein metabolism".

	DIS Intensity	RES Intensity	Group Ratio	Student's T-test p-value	Unique peptides	R. philippinarum locus ID	C. gigas protein ID	Protein name	COG C. gigas gene description	Biological process GO term	Functional role discussed
												Immune
		948	1.51:1.00	0.002	3	Locus 6868673	EKC40501	LGALS9	W	Galectin-9	GO:0006954 inflammatory response	response -pathogen
												recognition
											GO:0002752 cell surface	Immune
		421	2.97:1.00	0.041	1	Locus 5288240	EKC31577	BGBP	G	Beta-1,3-glucan-binding protein 1	pattern recognition receptor signaling	response -pathogen
											pathway	recognition
	384	244	1.57:1.00	0.026	1	Locus 4334179	EKC24393	C3	O	Complement C3	GO:0006955 immune response	Immune response -signalling
	647	1770	2.73:1.00	0.005	3	Locus 615620	EKC23268	IQGAP1	Z	Ras GTPase-activating-like protein	GO:1903829 positive regulation of cellular protein localization	Immune response -cytoskeleton
											GO:0006888 endoplasmic	Immune
		832	1.53:1.00	0.025	3	Locus 2955238	EKC27178	DCTN2	Z	Dynactin subunit 2	reticulum to Golgi vesicle-	response -
											mediated transport	cytoskeleton
	14900	4540	3.29:1.00	0.018	9	Locus 7235177	EKC29122	SCP	S	Sarcoplasmic calcium-binding protein	GO:0051480 regulation of cytosolic calcium ion concentration	Immune response -cytoskeleton
		2010	1.59:1.00	0.025	4	Locus 2215912	EKC27269	CPVL	O	Putative serine carboxypeptidase CPVL	GO:0051603 proteolysis involved in cellular protein catabolic process	Immune response -lysosomal
	910	486	1.87:1.00	0.005	1	Locus 2384090	EKC25290	EPDR1	S	ependymin related 1	GO:0007160 cell-matrix adhesion	Immune response -lysosomal
												Immune
	903	446	2.03:1.00	0.045	1	Locus 2964637	EKC26355	OVCH1	O	Ovochymase-1	GO:0006508 proteolysis	response -
												lysosomal
		531	5.02:1.00	0.029	2	Locus 3062641 ENSDARP00000139466*	PPT1	S	palmitoyl-protein thioesterase 1	GO:0007042 lysosomal lumen acidification	Immune response -lysosomal
	680	374	1.82:1.00	0.014	1	Locus 665720	EKC31469	RAB43	U	Ras-related protein Rab-43	GO:0090382 phagosome maturation	Immune response -vesicles

Ratios are presented as RES:DIS for those more abundant in RES clams, and as DIS:RES for those more abundant in DIS clams. * Danio rerio protein ID and protein description 3.3. IMMUNE RESPONSE-ASSOCIATED PROTEINS 3.3.1. Pathogen recognition and immune-pathway activation

ACKNOWLEDGMENTS

The authors would like to acknowledge the "investment for the future" programs LabexMER (ANR-10-LABX-19) and ISblue (ANR-17-EURE-0015) for funding international joint PhD agreements and proteomic analyses, the EU Horizon2020 project VIVALDI (grant agreement N°678589) for providing funding and biological material. The authors also would like to thank Fabrizio Ghiselli and Mariangela Ianello from the University of Bologna, and Massimo Milan and Luca Bargelloni from the University of Padova, for providing the full annotated and assembled transcriptome of the Manila clam digestive gland used for protein identification.