LABÉO Maud Charles
email: maud.charles@outlook.fr

Ismaël Bernard

Antonio Villalba

Elise Oden

Erika A V Burioli

Gwenaël Allain

Suzanne Trancart

Valérie Bouchart

Maryline Houssin

Frank Duncombe

High mortality of mussels in northern Brittany -Evaluation of the involvement of pathogens, pathological conditions and pollutants

Keywords: Mytilus edulis, multi-parametric, neoplasia, Vibrio splendidus, Marteilia, hemocytic infiltration

 A multi-parametric approach was adopted to identify mortality causes in mussels.

 A 'mortality window' during the spring season was identified.

 Brest, Lannion and St. Brieuc (France) have different cumulative mussel mortality rates.

 Haemocytic infiltrations and Marteilia sp. are parameters linked to mortality.

 There was no specific significant involvement of disseminated neoplasia or Vibrio.

Introduction

Mussels play a key role in the aquatic environment and are considered to be important ecosystem engineers by providing a suitable habitat for a species rich community [START_REF] Buschbaum | Mytilid mussels: global habitat engineers in coastal sediments[END_REF]. Due to their intense filtration activity, they modify aquatic habitat, making it more suitable for their own species and other for organisms [START_REF] Borthagaray | Mussels as ecosystem engineers: Their contribution to species richness in a rocky littoral community[END_REF]. Mussels are also an important food source for many aquatic and terrestrial animals including humans, making them a product with high economic value. The blue mussel industry is important in Europe, a main producer of mussels globally, and France is second in Europe with a production of around 80,000 tons per year, most from aquaculture (FAO, 2018). In France, Brittany is a commercially important area for shellfish farming and is the second mussel producing region with 17,500 tons of Mytilus spp. produced in 2016 (CNC, 2016).

In 2014, France was affected by high and unusual mass mortality of mussels, both juveniles and adults, affecting several important production areas along the French coast of the Atlantic and English Channel. Production losses reached 50 -100% depending on the sites [START_REF] Béchemin | Episodes de mortalité massive de moules bleues observés en 2014 dans les Pertuis charentais[END_REF][START_REF] François | Bilan 2014 du réseau Repamo -Réseau national de surveillance de la santé des mollusques marins[END_REF][START_REF] Allain | Les mortalités de moules en 2014 et 2015 vues par les professionnels[END_REF][START_REF] Lupo | Bilan 2015 du dispositif national de surveillance de la santé des mollusques marins[END_REF].The phenomenon again affected farms on the west coast of France in the first quarter of 2016 (FAO, 2016). Unlike mortality events in Pacific oyster (Crassostrea gigas), very few massive moralities of Mytilus spp. occurred along the French coast until the winter of 2014 [START_REF] Brienne | Observations sur l'infestation des moules du pertuis breton par Mytilicola intestinalis Steuer[END_REF][START_REF] Blateau | Expériences de traitement des moules (M. edulis) de bouchots de la baie du Mont Saint-Michel parasitées par Mytilicola intestinalis -Septembre 1987 et[END_REF][START_REF] Guichard | Bilan 2010 du réseau Repamo -Réseau national de surveillance de la santé des mollusques marins[END_REF].

One of the proposed causes of mortality is pathogenic bacteria in the genus Vibrio [START_REF] Béchemin | Episodes de mortalité massive de moules bleues observés en 2014 dans les Pertuis charentais[END_REF]. Vibrios are among the major bacterial pathogens of marine organisms and have been identified as pathogens of several bivalve molluscs [START_REF] Beaz-Hidalgo | Diversity and pathogenecity of Vibrio species in cultured bivalve molluscs[END_REF][START_REF] Lemire | Populations, not clones, are the unit of Vibrio pathogenesis in naturally infected oysters[END_REF][START_REF] Eggermont | Isolation of Vibrionaceae from wild blue mussel (Mytilus edulis) adults and their impact on blue mussel larviculture[END_REF]. During mussel mortality events, different Vibrio splendidus strains were isolated from moribund mussels in France and were linked to mortality [START_REF] Travers | Bacterial diseases in marine bivalves[END_REF][START_REF] Ben Cheikh | First evidence for a Vibrio strain pathogenic to Mytilus edulis altering hemocyte immune capacities[END_REF]. Of the sites studied, the Bay of Brest, located at the extremity of the Brittany Peninsula, is one of the locations where mussels suffered massive mortality in 2014 and beyond. Estimated losses were 30% to 80%, depending on the year. Vibrio strains and pathogenic bacteria belonging to the Splendidus clade were identified and associated with these mortalities [START_REF] François | Bilan 2014 du réseau Repamo -Réseau national de surveillance de la santé des mollusques marins[END_REF][START_REF] Lupo | Bilan 2015 du dispositif national de surveillance de la santé des mollusques marins[END_REF]. Other researchers have suggested that the cause of mussel mortalities is disseminated neoplasia. This disease is well documented in marine bivalves all over the world [START_REF] Peters | Recent investigations on the disseminated sarcomas of marine bivalve molluscs[END_REF][START_REF] Elston | Disseminated neoplasia of bivalve mollusc[END_REF][START_REF] Landsberg | Neoplasia and biotoxins in bivalves: is there a connection[END_REF][START_REF] Carballal | Neoplastic diseases of marine bivalves[END_REF] and, in some bivalve species including the mussel Mytilus trossulus, spreads by transmission of cancerous cells between individuals [START_REF] Metzger | Horizontal Transmission of Clonal Cancer Cells Causes Leukemia in Soft-Shell Clams[END_REF][START_REF] Metzger | Widespread transmission of independent cancer lineages within multiple bivalve species[END_REF]. Some scientists observed chromosomal abnormalities associated with neoplasia in Mytilus trossulus [START_REF] González-Tizón | Polyploidy in a natural population of mussel, Mytilus trossulus[END_REF] while advanced neoplastic disease was associated with significant mortalities of Mytilus sp. [START_REF] Moore | Alternate pathogenesis of systemic neoplasia in the bivalve mollusc Mytilus[END_REF], including M. trossulus in Washington and Oregon (USA) and in British Columbia (Canada) [START_REF] Elston | Disseminated neoplasia of bivalve mollusc[END_REF]. In France, [START_REF] Benabdelmouna | The mass mortality of blue mussels (Mytilus spp.) from the Atlantic coast of France is associated with heavy genomic abnormalities as evidenced by flow cytometry[END_REF] and [START_REF] Benabdelmouna | Mortality investigation of Mytilus edulis and Mytilus galloprovincialis in France: An experimental survey under laboratory conditions[END_REF] presented evidence of the involvement of genomic abnormalities in mortality outbreaks in blue mussels and linked them to disseminated neoplasia. In addition, some cases of disseminated neoplasia in Mytilus edulis from Lannion (northern Brittany, France) were observed in mid-October 2016 by [START_REF] Burioli | First description of disseminated neoplasia in Mytilus edulis in northern Brittany (France) and development of a rapide diagnostic tool[END_REF] during a mortality event.

Following heterogeneous mortality events of mussels that occurred in Brittany in the past years [START_REF] Allain | Les mortalités de moules en 2014 et 2015 vues par les professionnels[END_REF][START_REF] Bernard | Mortalités des moules en Bretagne nord : bilan des connaissances[END_REF]), the Regional Shellfish Committee of North Brittany (CRCBN) set up mortality monitoring in 2017 with coupled samplings over a period of 8 months. The initial focus was on the detection of neoplasia and on the involvement of a specific strain of V. splendidus. Nevertheless, it is known that the magnitude of mortality in bivalves can be modulated by several biotic factors, including the presence of pathogens [START_REF] Carrasco | Pathogens and disease processes in marine molluscs[END_REF], and by abiotic factors such as chemical contaminants [START_REF] Coles | Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium[END_REF]Pipe & Coles, 1995;[START_REF] Dailianis | Environmental impact of anthropogenic activities: The use of mussels as a reliable tool for monitoring marine pollution[END_REF][START_REF] Moschino | Biomonitoring of a polluted coastal area (Bay of Muggia, Northern Adriatic Sea): A five-year study using transplanted mussels[END_REF], water temperature and animal conditioning [START_REF] Almada-Villela | The effects of temperature on the shell growth of young Mytilus edulis L[END_REF][START_REF] Seed | Population and community ecology of Mytilus[END_REF][START_REF] Gosling | The mussel Mytilus: ecology, physiology, genetics and culture[END_REF][START_REF] Gosling | Bivalve Molluscs: Biology, Ecology and Culture[END_REF]. In an exploratory process, a field study conducted in partnership with mussel farmers was conducted in situ, wherein analyses, including biometry (condition index), histopathological examination, determination of bacterial flora, detection of bivalve mollusc pathogens and measurement of trace elements in mussel tissues, were carried out in three different sites in northern Brittany. Our objective of this in situ study was to identify potential causes responsible for the abnormal high mortality of farmed mussels in northern Brittany.

Materials and methods

Sampling sites and mussels

Three primary mussel production sites in northern Brittany were selected for this study: the bays of Brest (Finistère, France), Lannion (Côtes-d'Armor, France) and St. Brieuc (Côtesd'Armor, France) (Fig. 1). In each bay, one mussel farming company was involved in the study, representing approximately 8% of the mussel farmers in the Bay of St. Brieuc, approximately 25% of those located in the Bay of Brest, and the only farmer working in the Bay of Lannion. In each site, mussel farmers had introduced 7-mo-old mussels (23.51 ± 4.62 mm in length) in the fall 2016 (Sept./Oct.) for their 2017 production season. These mussels were used in this study and were monitored until September 2017. The mussels of Brest originated in La Plaine-sur-mer (Loire-Atlantique, France), those of Lannion in Pénestin (Morbihan, France) and those of St. Brieuc in Noirmoutier (Vendée, France). The maximum distance between the origination sites is 60 km. The aim of this field study was to monitor and analyse mussels that were representative of those cultivated by the mussel farmers in their area, which depended exclusively on where mussel farmers obtained the spat.

The identification of species was determined from 400 mussels (50 per date and per site) sampled only for this purpose (Table 1). The determination of the species was performed by genotyping on a set of ancestry informative SNPs [START_REF] Simon | Coadapted genomes and selection on hybrids: Fisher's geometric model explains a variety of empirical patterns[END_REF].

Mortality and temperature monitoring

In each site, 30 baskets of 100 mussels were prepared (Fig. 2A) and fixed on intertidal mussel stakes ("bouchot"; Fig. 2B) or subsurface longlines (for Lannion) at the beginning of the study. Every month, two baskets per site were collected and live mussels were counted for mortality monitoring. For Brest and St. Brieuc, in order to limit the time dedicated to counting during the tide, different baskets were counted each time; while for Lannion, because the tide is not a constraining factor, the same mussels were counted and put back into the baskets. Thus, each month, 200 mussels per site were counted to estimate mortality. Temperature sensors (HOBO Pendant ® ) were placed in the baskets containing mussels. These recorded the temperature around the animals (either water temperature or air temperature depending on tide) every 30 min.

Sampling for analysis

Sampling for analysis was carried out with different frequency among sites: February, April, May and September 2017 for Brest, and February and May 2017 for Lannion and St. Brieuc (Table 1). At each sampling, 50 mussels per site were collected from baskets for histology, and 20 additional mussels for biometry, molecular biology, bacteriological and chemical analyses (performed on the same animals). The collected mussels were transported in thermal bags to the laboratory and either analysed immediately or held in a refrigerator (4 °C) overnight and analysed the next day.

Biometry

All mussel samples were individually weighed (wet flesh and shell separately after drainage of intervalvar water) with a precision scale (± 0.01 g). A condition index (C.I.) was calculated as follows [START_REF] Davenport | A comparison of methods for the assessment of condition in the mussel (Mytilus edulis L.)[END_REF]:

Histological examination

For dissection, shells were opened by cutting the adductor muscle and the soft tissues were removed. An approximately 5 mm thick transverse section of mussel tissue containing mantle lobes, visceral mass (gut, digestive gland) and gills was excised, placed into histological cassettes and transferred to Davidson's fixative for 48 h before being transferred to 70% ethanol. Fixed tissues were then dehydrated through an ascending ethanol series and embedded in paraffin wax. Thick sections, 5 µm, were obtained using a rotary microtome and then stained with Harris' hematoxylin and eosin (HHE) [START_REF] Howard | Histological techniques for marine bivalve mollusks and crustaceans, 2nd edition[END_REF]. Histological sections were examined for all symbionts, including phoresis, commensalism, parasitism and mutualism [START_REF] Kinne | Diseases of marine animals[END_REF], and pathological conditions under light microscopy. For each sampling date indicated in Table 1, 50 histological slides corresponding to the 50 sampled mussels were examined.

Bacteriological analysis

Analysis was conducted to compare the bacterial flora of mussels from each of the three sites

Molecular analyses

The first step to determine the bacterial profile of the mussel batches consisted in discriminating between strains related to the V. splendidus clade (V. splendidus-related species) and other marine bacteria, as some vibrio strains belonging to this Splendidus clade are known to be pathogenic for molluscs. One TaqMan ® real-time PCR, targeting the 16S rRNA gene of V. splendidus-related strains (PCR1) [START_REF] Oden | Multilocus sequence analysis of Vibrio splendidus related-strains isolated from blue mussel Mytilus sp. during mortality events[END_REF], was carried out on a Smart Cycler ® (Cepheid, USA); the primers used for PCR1 were SpF1 5'ATCATGGCTCAGATTGAACG3' and SpR1 5'CAATGGTTATCCCCCACATC3' [START_REF] Nasfi | A European epidemiological survey of Vibrio splendidus clade shows unexplored diversity and massive exchange of virulence factors[END_REF] and the probe SpProbe 5'CCCATTAACGCACCCGAAGGATTG3'. The reaction volume of 25 µL contained 12.5 µL of Premix Ex Taq ® 2 X Takara ® (Lonza, Verviers, Belgium), 0.5 µL of each primer (20µM), 0.5 µL of probe (10 µM), 9 µL of purified water and 2 µL of extracted DNA (replaced with 2 µL of purified water in the negative control). The thermal cycling profile was 95°C for 10 s, followed by 40 cycles at 95 °C for 5 s and 62 °C for 30 s. When PCR1 was positive, a conventional PCR targeting the housekeeping genes mreB of V. splendidus-related strains (PCR2) was performed because it is the most discriminant gene for the identification of closely related strains among the Splendidus clade In parallel, the presence of known bivalve pathogens (Bonamia spp., Haplosporidium nelsoni, Marteilia sp., Mikrocytos mackini, Nocardia crassostreae, Ostreid Herpesvirus type 1 (OsHV-1), V. aestuarianus, V. tubiashii, V. harveyi, and V. splendidus) was investigated by PCR on DNA extracts from mussel tissues (Additional file 1).

Chemical analyses of flesh

The objective of chemical analyses was to assess if one site was more contaminated than others by one or more chemical compounds, which might explain why mortality was higher in one than in the others. For cost reasons, there were limitations in the number of samples that could be analysed. Because mortality began to increase in February, we chose to analyse and compare the three sites during that month rather than in May.

Presence of trace metal compounds (Pb, Hg, Cd, Co, Mo, Sn, Cr, Cu, Ni, Zn, Fe, Al, Mn and Ti) and non-metallic trace elements (As and Se) in mussel flesh was quantitatively determined by inductively coupled plasma-mass spectrometry (ICP-MS Triple Quad 8800, Agilent

Technologies, Santa Clara, CA, USA) after wet digestion through high-quality grade mineral acids (HNO 3 , Analpure ® , Analytika ® , Prague, Czech Republic) and oxidants (H2O2, Normapur ® ,VWR international, Radnor, Pennsylvanie, USA), following previously described protocols [START_REF] Squadrone | Presence of trace metals in aquaculture marine ecosystems of the northwestern Mediterranean Sea (Italy)[END_REF]. All analyses were calibrated against analytical standards.

The limit of quantitation was 0.02 mg/kg for elements analysed by ICP-MS. All concentrations are given on a wet weight (w. w.) basis.

Data analysis

Half-stock index

To compare mortality phenomena over time, the half-stock index [START_REF] Bernard | Bilan de l'observatoire des mortalités de moules en Bretagne Nord pour la saison 2016-2017 et premiers éléments sur les organismes pathogènes présents[END_REF] is more suitable than using a cumulative mortality rate since it allows visualization of what is happening at a given time. Indeed, to compare different mortality episodes, it is preferable to consider the exponential aspect of population decrease. Thus, if λ is the mortality rate, supposed constant N(t) the population at t time, and N 0 the population at the beginning of the study, then:

N(t) = N 0 e-λt .
Between two successive counts in t i-1 and t i , there is only one λ which describes these two points:

To obtain a more tangible index, the time (in days) needed to halve the population was used:

d 0.5 = ln (2) / λ.
Therefore, this index corresponds to the number of days it takes to halve the population with the measured mortality rate, in a very similar way to half-life of radioactive elements. The lower this index, the higher the mortality rate and decrease of the stock. In the absence of mortality, this index tends towards infinity. For a better graphical representation, a maximum value was assigned to this index equal to 1500 (which corresponds to a loss of 15% over 1 year).

Statistical analysis

The half-stock index has been used to analyse and compare instantaneous mortality with different sampling intervals. It has also been used as the variable response to link the variation of mortality with the prevalence of pathological observations. This relationship was tested using a Spearman correlation test. Temperatures presented here have been smoothed in order to represent annual variation using generalized additive models with smoothing term with a moving window corresponding to one month.

Regarding biometry data, replicates were averaged, and the values were tested for normality (Shapiro). Then, a first ANOVA was used to compare the C.I. of Brest mussels at the four sampling dates, and a second to compare the C.I. of mussels from the three sites on both dates (February and March). When a significant difference was obtained, paired comparisons by Tukey's post hoc test were performed to identify which C.I. were different from the others.

Statistical significance was accepted for *p < 0.05. The association between the C.I. and observed mortality was evaluated using a Pearson correlation test. Statistical analyses and graphical representations were performed by using R software, version 3.5.1 (https://www.rproject.org/) and 'dplyr', 'tidyr' and 'ggplot2' packages.

Results

Mussel species, temperature monitoring and mortality

For species identification, a dataset of 81 common markers was obtained (for more details, see [START_REF] Simon | Replicated anthropogenic hybridisations reveal parallel patterns of admixture in marine mussels[END_REF], which allowed identification of all mussels as the species M. edulis.

Regarding thermal profiles (Fig. 3), Lannion appears to be a rather temperate site compared to those of Brest and St. Brieuc, which showed wider temperature variation, especially higher temperatures in summer. From February to August, the temperature of Lannion rose from 9 to 18 °C, that of St. Brieuc from 9 to 20 °C and that of Brest from 8.5 to 20 °C.

The first abnormal mortalities were observed at the very end of winter and early spring 2017 (Fig. 3) in the most western site. Indeed, Brest was the most affected site as it suffered continuous mortality as illustrated by the half-stock index until mid-summer (Fig. 3B). The site had nearly 70% cumulative mortality at the end of the study (Fig. 3A). Lannion was less severely affected than Brest -but more than St. Brieuc-with a progressive decreasing trend in the half-stock index leading to a final cumulative mortality of almost 40%. St. Brieuc experienced some sporadic mortality peaks, but not considered abnormal. At the end of the study, a cumulative mortality of about 15% was obtained, which was a typical value for institutions (Council Directive 95/70/EC abrogated by 2006/88/CE) and mussel farmers.

Furthermore, mortality decreased significantly or even stopped in mid-summer when temperatures exceeded 18 °C.

Biometry

When the C.I. of Brest mussels at the four sampling dates between February and September 2017 were compared, despite an upward trend in the April index, no significant difference was observed (ANOVA, p > 0.05) (Fig. 4). When the C.I. of mussels from the three sites on both dates (February and May 2017) were compared, no significant differences was observed in February but in May, the C.I. of mussels from Lannion were significantly higher than those of Brest mussels (ANOVA, p < 0.05 and Tukey's post hoc test) (Fig. 5). There was no significant difference between the two C.I. in February and in May for the same site and no significant link appeared between observed mortality and C.I. (Pearson correlation, p > 0.05).

Histological examination

Histopathological examination showed the occurrence of various symbionts and pathological conditions. Regarding symbionts, the different stages characterising parasites of the genus Marteilia were observed in the stomach and digestive diverticula epithelia (Fig. 6A), from primary cells enclosing some secondary cells in the stomach and digestive diverticula epithelium to fully developed stages in digestive diverticula, in which secondary cells included tertiary cells and had refringent granules in their cytoplasms. Occasionally, early stages of Marteilia sp. were observed in the gills, associated with heavy inflammatory response. Two types of ciliates were also observed, one type resembling Ancistrum mytili in the gills (Fig. 6B) and intracellular ciliates in the epithelium of digestive tubules (Fig. 6C).

Trematode sporocysts enclosing developing cercariae were observed in the connective tissue of the mantle and the visceral mass (Fig. 6D). Mytilicola sp. copepods were found in the intestinal lumen (Fig. 6E). Regarding other pathological conditions, cases of heavy hemocytic infiltration of the connective tissue of different organs were detected, but without any identifiable inducing agent (Fig. 7A). The occurrence of large masses of hemocytes, mostly granulocytes, surrounded by several layers of flattened, epithelioid cells, were also observed in the connective tissue of various organs (Fig. 7B). This type of inflammatory structure is usually called a granulocytoma and its occurrence was not linked to any detectable agent.

Finally, cases of disseminated neoplasia, characterised by the infiltration of the connective tissue by abnormally large cells that also proliferate through the circulatory system (Fig. 7C)

were observed. The nucleus of the abnormal cells was significantly larger than that of normal cells, with at least one patent nucleolus, showing frequent mitotic figures that indicated a high division ratio (Fig. 7D). The prevalence of symbionts and pathological conditions in each site is shown in Fig. 8. Regarding those with potentially the most serious consequences (granulocytomas, hemocytic infiltration, disseminated neoplasia, trematode sporocysts and Marteilia), Brest was the most affected site (Fig. 8A). Indeed, it was the only site in which 4 out of 5 serious pathological conditions occurred in February (granulocytomas: 2.9%; hemocytic infiltration: 14.3%; trematode sporocysts: 11.4% and Marteilia: 22.9%) and in May (granulocytomas: 4.1%; hemocytic infiltration: 26.5%; trematode sporocysts: 2.0% and Marteilia: 22.5%). It was also the only site affected with Marteilia sp., which occurred over the entire study period and had a prevalence peak in April with 36% of individuals infected (Fig. 8B). In February, Lannion and St. Brieuc were affected by granulocytomas with similar prevalence (6.7% and 6.5%, respectively), twice that of Brest, and twice as many individuals

showed trematode sporocysts at St. Brieuc (6.5%) than at Lannion (3.3%

Molecular analyses

Bacterial profiles

Following the identification of the 120 isolated bacterial strains from tissue culture, it was noted that bacterial diversity was relatively higher in May than in February (Fig. 10). In

Presence or absence of known pathogens of bivalve molluscs

Among the four pools of DNA extracts from tissues of each of the nine batches we investigated presence of 10 known pathogens, Bonamia spp., Haplosporidium nelsoni, Marteilia sp., Mikrocytos mackini, Nocardia crassostreae, OsHV-1, V. aestuarianus, V.

tubiashii, V. harveyi, and V. splendidus belonging to Splendidus cluster. V. splendidus was detected in all the batches from each site, while Marteilia sp. was recorded in all batches from Brest (from February to September), which confirmed the histological results. All the other TaqMan ® real-time PCR results were negative.

Chemical analyses of flesh

Discussion

An in situ study combining various analytical procedures was performed to explore causes of mussel mass mortality. The experimental design for field work involved assuming the culture procedures and using the mussel batches of the professional mussel farmers, taking advantage of their activity to avoid any element or circumstance different from the common practice that could distort the usual mussel performance through on-growing. This approach provided an accurate representation of what was occurring on the farms and was chosen as a first exploration assuming that more refined experimental designs eventually may be needed while taking advantage of the results of this first approach.

Temperature, conditioning and mortality

Mytilus edulis is known to resist extreme temperatures [START_REF] Aarset | Freezing tolerance in intertidal invertebrates (a review)[END_REF][START_REF] Seed | Population and community ecology of Mytilus[END_REF][START_REF] Almada-Villela | The effects of temperature on the shell growth of young Mytilus edulis L[END_REF], however, the thermal profiles observed in our sites were not extreme or unusual. The narrower temperature range observed in Lannion was probably due to the farming method because mussels on subsurface longlines are always submerged and undergo less marked temperature variation than mussels on intertidal stakes.

Results showed that a 'mortality window' opened in all sites during the spring season; similar observations also have been reported recently in France by other researchers [START_REF] Benabdelmouna | Mortality investigation of Mytilus edulis and Mytilus galloprovincialis in France: An experimental survey under laboratory conditions[END_REF][START_REF] Dégremont | Response to selection for increasing resistance to the spring mortality outbreaks in Mytilus edulis occurring in France since 2014[END_REF]. This period corresponded to the spawning season of mussels. Gonad development usually begins in October/November, and by the end of the winter the gonads are ripe [START_REF] Gosling | Bivalve Molluscs: Biology, Ecology and Culture[END_REF]. All energy accumulated during the quiescent period is used to fuel gametogenesis and finally spawning; thus, when mussels spawn, they are in poor condition during the remainder of the spring period, with low glycogen content [START_REF] Najdek | Total and methyl-mercury content in bivalves, Mytilus galloprovincialis Lamarck and Ostrea edulis Linnaeus: Relationship of biochemical composition and body size[END_REF]. Mussel growth is primarily influenced by the reproductive stage and food availability [START_REF] Gosling | The mussel Mytilus: ecology, physiology, genetics and culture[END_REF] but growth capacities also have been linked to site and animal origin [START_REF] Dickie | Influences of Stock and Site on Growth and Mortality in the Blue Mussel (Mytilus edulis)[END_REF]. The higher C.I. of mussels from Lannion in May could be explained by (i) their better growth capacities due to their origin, (ii) the greater trophic richness of the site, and (iii) the subtidal farming -which allows them continuous access to trophic resources. Indeed, [START_REF] Prou | The French Mussel Industry: Present Status and Perspectives[END_REF] showed that mussels grown on longlines had higher growth performance than mussels grown on bouchot. However, this did not prevent mortality. Primary production, and thus food availability, are reduced during winter periods when seawater temperatures are below 10°C [START_REF] Cloern | Phytoplankton bloom dynamics in coastal ecosystems: A review with some general lessons from sustained investigation of San Francisco Bay, California[END_REF] and mussels undergo long-time starvation periods [START_REF] Harbach | Development of general condition and flesh water content of long-time starved Mytilus edulis-like under experimental conditions[END_REF]. Mytilus sp. can handle longer food restriction periods and maintain its shell size by using energy from its own tissue [START_REF] Dare | Seasonal changes in flesh weight and biochemical composition of mussels (Mytilus edulis L.) in the Conwy Estuary, North Wales[END_REF][START_REF] Riisgård | Allometric equations for maximum filtration rate in blue mussels Mytilus edulis and importance of condition index[END_REF]. However, no link between C.I. of mussels and mortalities obtained in site was found.

These various data and observations clearly illustrate that this transition period between winter and spring -corresponding to the spawning period-is stressful for mussels and decisive in their survival. Thus, spawning stress could contribute to mussel weakening and to an increased mortality rate to some extent. However, considering the very similar C.I.

observed between the different sites studied -between those with high mortality and those with low mortality-this parameter alone cannot explain abnormal mortality. [START_REF] Villalba | Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the Rías of Galicia (NW Spain)[END_REF] classified mussel symbionts in three groups according to their pathogenicity. Among those identified in this study, ciliates are in the first group with unnoticeable pathogenic effects, Mytilicola sp. are in the second group that may damage the host, but are not lethal, and Marteilia sp. and trematodes are in the third group with potentially lethal effects. [START_REF] Figueras | Diseases and parasites of rafted mussels (Mytilus galloprovincialis Lmk): preliminary results[END_REF] observed that, even at the highest densities of infestation, ciliates caused no detectable tissue damage in gills or in digestive tubules. More recent detailed studies of the intracellular ciliates in digestive tubules reported similar conclusions [START_REF] Fichi | Characterisation of the intracellular protozoan MPX in Scottish mussels, Mytilus edulis Linnaeus, 1758[END_REF]. Our results are consistent with those reports, no associated inflammation or abnormal mortality was detected in St. Brieuc, the site most affected by ciliates. Regarding Mytilicola sp., [START_REF] Brienne | Observations sur l'infestation des moules du pertuis breton par Mytilicola intestinalis Steuer[END_REF] and [START_REF] Blateau | Expériences de traitement des moules (M. edulis) de bouchots de la baie du Mont Saint-Michel parasitées par Mytilicola intestinalis -Septembre 1987 et[END_REF] observed weight loss in French mussels infested with this copepod at the end of winter and early spring (breeding period); the mortality it caused was related to the decrease in mollusc vitality. Nevertheless, in most cases, despite a high infestation intensity reported in English and Spanish mussels, it is evident that the host population can sustain the infestation indefinitely [START_REF] Davey | Mytilicola intestinalis (Copepoda: Cyclopoida): A ten year survey of infested mussels in a Cornish Estuary, 1978-1988[END_REF][START_REF] Robledo | Mytilicola intestinalis and Proctoeces maculatus in mussel (Mytilus galloprovincialis Lmk.) beds in Spain[END_REF]. Again, our results were consistent because the highest prevalence of this copepod was recorded in St. Brieuc and no abnormal mortalities were observed over the period at this site; the mortality peak observed in May could be attributed to weakness due to the breeding season, which could be intensified by the presence of Mytilicola sp. as observed by [START_REF] Brienne | Observations sur l'infestation des moules du pertuis breton par Mytilicola intestinalis Steuer[END_REF] and [START_REF] Blateau | Expériences de traitement des moules (M. edulis) de bouchots de la baie du Mont Saint-Michel parasitées par Mytilicola intestinalis -Septembre 1987 et[END_REF]. [START_REF] Lauckner | Diseases of Mollusca: Bivalvia[END_REF] found that digenetic Trematoda were the most frequent and important metazoan parasites of bivalves. Indeed, trematode sporocysts and metacercaria were capable of causing a wide range of harmful consequences in their hosts (lesions, compression of tissues, castration, and deep weakness) that can lead to death when infestation is heavy [START_REF] Robledo | Mytilicola intestinalis and Proctoeces maculatus in mussel (Mytilus galloprovincialis Lmk.) beds in Spain[END_REF][START_REF] Laruelle | Histological analysis of trematodes in Dreissena polymorpha: their location, pathogenicity, and distinguishing morphological characteristics[END_REF]. [START_REF] Bakhmet | Effect of infection with Metacercariae of Himasthla elongata (Trematoda: Echinostomatidae) on cardiac activity and growth rate in blue mussels (Mytilus edulis) in situ[END_REF] found a metabolic level supported by significant growth deficiency in parasitized M. edulis. This also was observed in Normandy (France) by Le [START_REF] Breton | Resultats d'une intervention sur une parasitose a Proctoeces maculatus (Trematoda, Digenea) affectant la mytiliculture de l'ouest Cotentin[END_REF], as well as a variation in parasitism over time as we observed in Brest, and an increase in mortality between January and July.

2 Mussel symbionts

No significant statistical link was established for Marteilia sp., but the p-value close to 0.05 should be highlighted as it was entirely influenced by results from Brest. The presence of Marteilia sp. only in the most affected site should not be neglected, especially because it not only severely harms flat oysters, Ostrea edulis, but also M. edulis and M. galloprovincialis [START_REF] Villalba | Marteiliasis affecting cultured mussels Mytilus galloprovincialis of Galicia (NW Spain). I. Etiology, phases of the infection, and temporal and spatial variability in prevalence[END_REF][START_REF] Villalba | Symbionts and diseases of farmed mussels Mytilus galloprovincialis throughout the culture process in the Rías of Galicia (NW Spain)[END_REF][START_REF] Fuentes | Effects of stock and culture environment on infections by Marteilia refringens and Mytilicola intestinalis in the mussel Mytilus galloprovincialis cultured in Galicia (NW Spain)[END_REF][START_REF] Arzul | Contribution to the understanding of the cycle of the protozoan parasite Marteilia refringens[END_REF]. Indeed, several studies have shown that high mortality rates observed in certain mussel populations in Spain and France were positively correlated with the presence of this parasite [START_REF] Villalba | Marteiliasis affecting cultured mussels Mytilus galloprovincialis of Galicia (NW Spain). I. Etiology, phases of the infection, and temporal and spatial variability in prevalence[END_REF][START_REF] Garcia | Bilan 2004 du réseau REPAMO -Réseau national de surveillance zoosanitaire des mollusques marins[END_REF]. Marteiliosis leads to the arrest of growth due to the degradation of digestive cells, loss of glycogen in tissues and resulting in considerable weight loss of the mollusc [START_REF] Grizel | La maladie de la glande digestive d'Ostrea edulis Linne[END_REF]. This disease could explain the lower C.I. in Brest.

Marteiliosis also causes a general weakening of the host, promoting opportunistic parasite development (bacteria, ciliates) that could cause secondary infestations [START_REF] Grizel | Etude des récentes epizooties de l'huître plate Ostrea edulis Linne et de leur impact sur l'ostreiculture bretonne[END_REF]. For Brest mussels, no significant changes were observed between the C.I. for weight calculated in February and those calculated in September. This is not typical because mussels should be fleshy at the end of summer and require accumulated energy reserves for gonadal development and winter survival. Previous studies in flat oysters showed that mortality due to Marteilia sp. was observed just after the breeding period during which the animal consumes a lot of energy. Marteiliosis also is known to cause hemocytic infiltration and occasionally granulocytomas, which cause the destruction of the pathogen but also of the host tissues [START_REF] Villalba | Marteiliasis affecting cultured mussels Mytilus galloprovincialis of Galicia (NW Spain). I. Etiology, phases of the infection, and temporal and spatial variability in prevalence[END_REF]. These effects were observed in Brest. The drastic decrease in prevalence observed in Brest between May and September could be explained by death of infected mussels. Histological examination does not allow discriminating between the congeneric species Marteilia refringens and M. pararefringens; although both species have been detected in mussels, M. pararefringens is more frequently reported [START_REF] Kerr | Marteilia refringens and Marteilia pararefringens sp. nov. are distinct parasites of bivalves and have different European distributions[END_REF].

The infected samples analysed with the real time PCR procedure showed infection with Marteilia sp.

Each of the symbionts and parasites detected in the study have a varying degree of negative effect on their hosts. A single non-lethal parasite may cause some damage but not death, but the cumulative effect of different non-lethal parasites may have an impact on the survival of the host. This is particularly true during a harsh season and at a key stage in the life cycle of the mussel. It is clear from our data that Marteilia was one of the major factors contributing to mortalities in Brest.

Lesions and alteration: hemocytic infiltration, granulocytomas and neoplasia

Some cases of hemocytic infiltration (positively correlated with mortality) and granulocytomas were not linked to a specific pathogen. It should be noted that abnormal is the most frequent alteration and this is considered to be an important biomarker of lesion and inflammation in bivalves [START_REF] Cuevas | Development of histopathological indices in the digestive gland and gonad of mussels: Integration with contamination levels and effects of confounding factors[END_REF]. [START_REF] Cuevas | Development of histopathological indices in the digestive gland and gonad of mussels: Integration with contamination levels and effects of confounding factors[END_REF] showed that mussels from the most impacted sites (by metallurgic and shipyard activities) endured the most significant deleterious effects showing inflammation. Also, [START_REF] Sheir | Tissue Injury and Cellular Immune Responses to Cadmium Chloride Exposure in the Common Mussel Mytilus edulis: Modulation by Lipopolysaccharide[END_REF] established a potential link between hemocytic infiltration and lesions with the presence of xenobiotics.

Lowe & [START_REF] Lowe | The cytology and occurrence of granulocytomas in mussels[END_REF] stated that granulocytomas reflect symptoms of long-term exposure to contaminants; the same observation was made about a potential correlation between neoplasia and water pollution because neoplastic disorders have been reported in bivalves collected from polluted areas [START_REF] Lauckner | Diseases of Mollusca: Bivalvia[END_REF]ICES, 2017).

In our study, pollution alone cannot explain the presence of granulocytomas. In May, they were more frequent in Lannion, the site with the lowest concentrations of chemical compounds. In addition, the highest prevalence of granulocytomas in Brest were observed in April and September while in these two months, the records of trace elements showed lower concentrations, more specially for lead. Finally, the prevalence of granulocytomas recorded in St. Brieuc was close to that of the other two sites and hemocytic infiltration and neoplasia were not recorded, while the concentration of all trace elements, except for lead, was higher in this site than in the others. [START_REF] Villalba | Disseminated neoplasia and large foci indicating heavy haemocytic infiltration in cockles Cerastoderma edule from Galicia (NW Spain)[END_REF] reported association of cockle, Cerastoderma edule, mortality with lesions resulting from heavy inflammatory reaction without a clearly identified cause, and with disseminated neoplasia. We observed inflammations with heavy hemocytic infiltrations correlated with mortality in Brest and Lannion, but not always associated with a specific cause. Disseminated neoplasia is a progressive disease insofar as neoplastic cells proliferate and replace normal hemocytes in circulation and could lead to death [START_REF] Elston | Progression, lethality and remission of hemic neoplasia in the bay mussel Mytilus edulis[END_REF][START_REF] Ciocan | Disseminated neoplasia in blue mussels, Mytilus galloprovincialis, from the Black Sea, Romania[END_REF]. Because hemocytes are cells with a key role in many physiological functions (digestion, excretion, nutrition, defense mechanisms; [START_REF] Cheng | A classification of molluscan hemocytes based on functional evidences[END_REF][START_REF] Fisher | Structure and Functions of Oyster Hemocytes[END_REF][START_REF] Fisher | Environmental influence on host response: Environmental influence on bivalve hemocyte function[END_REF], heavily affected mussels have reduced abilities and usually die (ICES, 2017). Indeed, mussels affected by disseminated neoplasia had, after the decrease in the number of normal circulating hemocytes, a weakened defense system and showed reduced bacterial clearance [START_REF] Kent | Impaired defense mechanisms in bay mussels, Mytilus edulis, with hemic neoplasia[END_REF]. Mass mortalities of various bivalve species have been associated with disseminated neoplasia (see reviews by [START_REF] Elston | Disseminated neoplasia of bivalve mollusc[END_REF][START_REF] Carballal | Neoplastic diseases of marine bivalves[END_REF]. In France, [START_REF] Benabdelmouna | The mass mortality of blue mussels (Mytilus spp.) from the Atlantic coast of France is associated with heavy genomic abnormalities as evidenced by flow cytometry[END_REF] observed that genomic abnormalities, namely aneuploidy of the hemolymph cells, were significantly correlated with Mytilus sp. mortalities observed in 2015 in the French Atlantic coast. Furthermore, [START_REF] Benabdelmouna | Mortality investigation of Mytilus edulis and Mytilus galloprovincialis in France: An experimental survey under laboratory conditions[END_REF] linked the occurrence of a significant percentage of aneuploid hemolymph cells to disseminated neoplasia and concluded that this disease could be viewed as a major cause of morbidity and mortality for French mussels. [START_REF] Elston | Progression, lethality and remission of hemic neoplasia in the bay mussel Mytilus edulis[END_REF] followed the evolution of this disease over 4 months in M. trossulus; they reported a relatively long dynamic progression of the disease in the early stages (2-3 months) and then a quick evolution when disseminated neoplasia leads to rapid death. This is consistent with the mortality dynamics observed in our study and the absence, or possibly undetected early-stage cases of disseminated neoplasia in February. A relatively high prevalence of 18% was observed in Brest in April and therefore most certainly contributed to a percentage of mortality during this period and/or may also have been an aggravating factor with an already present pathology; but abnormal mortalities began in February with no sign of disseminated neoplasia being observed at that time. Lannion presents a similar pattern; in May few cases were found with a prevalence of 2.4% but no signs in February. We hypothesize that if histological observations on Lannion mussels had been made in April, a higher rate of neoplastic mussels would have been observed and may have played a role in the observed mortality percentage.

We showed that inflammation and hemocytic infiltration were correlated with mortality, but it was not always possible to link them to an identified cause. In Brest, pollution could have a role (see below) but for Lannion, the cause(s) remain unknown.

Chemical contamination

First, and in relation to what has been previously discussed, when food availability is low and competition between individuals is high, mussels increase their levels of water uptake through their gills. This leads to congestion and accumulation of useless substances in their tissues, including pollutants, which could result in increase of mortality rates [START_REF] Dailianis | Environmental impact of anthropogenic activities: The use of mussels as a reliable tool for monitoring marine pollution[END_REF]. It is known that anthropogenic compounds and heavy metals play roles in defence capacities of bivalve molluscs and could increase susceptibility to disease [START_REF] Coles | Alteration of the immune response of the common marine mussel Mytilus edulis resulting from exposure to cadmium[END_REF]Pipe & Coles, 1995;[START_REF] Morley | Interactive effects of infectious diseases and pollution in aquatic molluscs[END_REF]. In addition, the effects of environmental contaminants sometimes correspond to a direct toxic action on tissues or cells [START_REF] Gagnaire | In vitro effects of cadmium and mercury on Pacific oyster, Crassostrea gigas (Thunberg), haemocytes[END_REF].

Some of the elements analyzed, Hg, As, Cd, Pb and Sn, have no known biological function and, except for Sn, are included in the list of the ten chemicals of major public health concern as part of International Programme on Chemical Safety (World Health Organisation, 2010). These elements are natural trace components of the aquatic environment, but their levels increase due to agricultural, industrial and mining activities. Even low metal concentrations may threaten the health of aquatic and terrestrial organisms, humans included [START_REF] Sarmiento | Toxicity and potential risk assessment of a river polluted by acid mine drainage in the Iberian Pyrite Belt (SW Spain)[END_REF]. Among all the metallic elements that [START_REF] Moschino | Biomonitoring of a polluted coastal area (Bay of Muggia, Northern Adriatic Sea): A five-year study using transplanted mussels[END_REF] have measured in mussel tissues (As, Cd, Cr, Pb, Al, Fe, Hg, Cu, Ni, Zn) during an in situ study over several years, a positive correlation between mussel mortality rate and Pb, Fe and Al was observed. Furthermore, the average concentrations they measured in the mussel tissues for these three elements were comparable with ours. It should also be noted that for Fe and Al, relatively similar high concentrations were found in St. Brieuc and Brest, while for lead, only mussels from Brest were above the threshold. Several biomarkers representative of the health status of the aquatic environment have been identified in mussels [START_REF] Depledge | The role of biomarkers in environmental assessment (2). Invertebrates[END_REF][START_REF] Dailianis | Environmental impact of anthropogenic activities: The use of mussels as a reliable tool for monitoring marine pollution[END_REF], and it was shown that mussels have lower defense mechanisms against metal oxidative challenge and toxicity than oysters. Indeed, [START_REF] Funes | Ecotoxicological effects of metal pollution in two mollusc species from the Spanish South Atlantic littoral[END_REF] have shown that activities of antioxidant enzymes are insufficient (compared to those observed in the Pacific oyster, C. gigas), which means that mussels are not sufficiently protected from the oxidative stress associated with metal pollution. In addition, [START_REF] Viarengo | Seasonal variations in the antioxidant defence systems and lipid peroxidation of the digestive gland of mussels[END_REF] and [START_REF] Petrović | Seasonal variations of physiological and cellular biomarkers and their use in the biomonitoring of north adriatic coastal waters (Croatia)[END_REF] observed seasonal variations in antioxidant responses related to physiological processes and showed inhibition of these defense mechanisms in winter related to the reproductive period and gonad resorption.

From the Chemical Contamination Observation Network of Ifremer (ROCCH), the Bay of Brest is known to be one of the sites most affected by lead contamination on the French coast for many decades (the first most contaminated on the west side of France) [START_REF] Belin | Qualité du Milieu Marin Littoral-Synthèse Nationale de la -Surveillance[END_REF]. According to these authors and local authorities (com. pers.), the high Pb concentrations found in Brest mussels are mainly explained by the presence of silver lead mines around the Aulne river (surface area: 1842 km²) several kilometers upstream (at Poullaouën and Huelgoat, Finistère, France). The river provides more than 63% of the bay's freshwater supply [START_REF] Auffret | Dynamique sédimentaire de la Marge Continentale Celtique[END_REF]. [START_REF] Lacroix | Active and passive biomonitoring suggest metabolic adaptation in blue mussels (Mytilus spp.) chronically exposed to a moderate contamination in Brest harbor (France)[END_REF] observed differences in physiological responses to contaminants between native Brest Bay and imported mussels. Non-natives were more sensitive and showed more sensitive biomarker responses. Therefore, it can be supposed that mussels caught in a more open and less contaminated marine area will be strongly impacted and stressed when they arrive in a more polluted area. [START_REF] Lacroix | Seasonal monitoring of blue mussel (Mytilus spp.) populations in a harbor area: A focus on responses to environmental factors and chronic contamination[END_REF] also observed an altered physiological state, early spawning, in mussels in a polluted area (Bay of Brest) compared to those on the Atlantic coast of Brittany (under oceanic influence) suggesting that their health is compromised at this period. Therefore, the pollution in Brest is an additional deleterious factor to others previously discussed and could be an explanation for the heavy hemocytic infiltrations not found to be linked to a specific pathogen. However, this is probably not the case for Lannion, an unpolluted site.

Bacterial profiles

Species belonging to the genus Pseudoalteromonas are widely distributed in marine environments globally. Some studies have shown a positive role of Pseudoalteromonas sp.

biofilms in the settlement of mussel, Mytilus coruscus larvae [START_REF] Yang | Larval settlement and metamorphosis of the mussel Mytilus coruscus in response to monospecific bacterial biofilms[END_REF][START_REF] Li | Effects of bacterial biofilms on settlement of plantigrades of the mussel Mytilus coruscus[END_REF]. On the other hand, [START_REF] Venkateswaran | Pseudoalteromonas peptidolytica sp. nov., a novel marine mussel-thread-degrading bacterium isolated from the Sea of Japan[END_REF] identified a species of Pseudoalteromonas which play a natural antifouling role due to the products it excretes, which prevent fixation of mussel byssal threads. However, no pathogenic or lethal role has been demonstrated by this species on bivalves. Species of the genus Pseudoalteromonas and

Vibrio are part of the normal microflora of temperate water marine animals and their proportion increases during the spoilage of the bivalves [START_REF] Gram | Microbiological spoilage of fish and fish products[END_REF][START_REF] Madigan | A microbial spoilage profile of half shell Pacific oysters (Crassostrea gigas) and Sydney rock oysters (Saccostrea glomerata)[END_REF]. Also, [START_REF] Lokmer | Hemolymph microbiome of Pacific oysters in response to temperature, temperature stress and infection[END_REF] showed that host-associated microbial communities are linked to abiotic and biotic factors.

The genus Vibrio includes a ubiquitous, diverse and abundant temperate coastal marine bacterial community [START_REF] Thompson | Diversity and Dynamics of a North Atlantic Coastal Vibrio Community[END_REF]. The species of the genus Vibrio show a very high diversity; more than 110 species have been identified, each with different relationships with their hosts, ranging from symbiosis to significant pathogenicity (Travers et al., 2015). Among those that appear to be pathogenic to bivalves, species belonging to the Splendidus clade are systematically highlighted. For example, [START_REF] Béchemin | Episodes de mortalité massive de moules bleues observés en 2014 dans les Pertuis charentais[END_REF] identified V. splendidus-related species in moribund mussels during mussel mass mortality outbreaks in summer 2014 in France; these isolates appeared to be capable of inducing mortality under laboratory conditions. Also, Ben Cheikh et al. (2016,2017) observed that a pathogenic strain of Vibrio splendidus clade inhibits the immune response in M. edulis by altering hemocyte function and viability and causes hemocytic infiltration and granulocytomas. The high genotype diversity of the bacteria belonging to the Splendidus clade and the dynamic nature of microbial communities complicates considerably efforts to elucidate the role of V. splendidus clade bacteria in vibriosis [START_REF] Kwan | Genetic diversity of culturable Vibrio in an Australian blue mussel Mytilus galloprovincialis hatchery[END_REF]. In addition, Vibrio studies are culture dependent and specialists themselves admit that they do not know if the dynamics they observed in bacteria populations reflect physiological changes in a viable but non-cultivable state or fluctuations in density with environmental parameters [START_REF] Thompson | Diversity and Dynamics of a North Atlantic Coastal Vibrio Community[END_REF]. Knowledge of their role as animal pathogens and their mechanisms of action in pathogenesis has been limited. Indeed, [START_REF] Bruto | Ancestral gene acquisition as the key to virulence potential in environmental Vibrio populations[END_REF] recently showed that, within the Splendidus clade, virulence represents an ancestral trait, but it has been lost from several populations. They identified two loci necessary for virulence and can now associate virulence in bivalves with one or many specific V. splendidus strains. Thus, simply finding some V. splendidus strains in mussels is no longer sufficient to incriminate them during a mortality episode. Accordingly, since V. splendidus was found in all sites, no matter the mortality rate, assessing whether these two virulence genes are present or not in our isolates would be highly interesting.

To explain abnormal mortalities, to identify a 'biotic disease' with one identifiable lethal pathogen (virus, bacteria or parasite) is desired, but there is evidence that surrounding conditions (abiotic factors like pollution, temperature and seasons), as well as the presence of multiple pathogens and parasites affecting the condition of animals are inseparable and cannot be considered individually. Because many factors could be involved, it is complicated to understand precisely mortality factors that have very high inter-site and inter-annual variability. Some factors can lead to gradual changes that progressively disrupt animal homeostasis while others can cause acute mortality. [START_REF] Dare | Settlement, growth and production of the mussel, Mytilus edulis L[END_REF] concluded that mortality in mussel populations resulted from an interaction between several physical and biological factors. Similarly, Lokmer & Wegener (2015) argued that, in addition to the presence of pathogens, environmental factors have a strong impact on disease efficacy and mortality. In addition, it seems that genetics could also have an impact on mortality [START_REF] Dickie | Influences of Stock and Site on Growth and Mortality in the Blue Mussel (Mytilus edulis)[END_REF][START_REF] Mallet | Mortality Variations in Natural Populations of the Blue Mussel, Mytilus edulis[END_REF][START_REF] Dégremont | Response to selection for increasing resistance to the spring mortality outbreaks in Mytilus edulis occurring in France since 2014[END_REF].

Our experimental design, using different mussel stocks corresponding to different geographic sources in the different experimental sites, did not allow evaluating the influence of the genetic background on the mussel mortality, that is to say if mussel stocks from different geographic sources would show different susceptibility to mortality outbreaks.

Mussels on the French Atlantic coast have complex genetic structure [START_REF] Bierne | Introgression patterns in the mosaic hybrid zone between Mytilus edulis and M. galloprovincialis[END_REF][START_REF] Fly | Physiology and biogeography: The response of European mussels (Mytilus spp.) to climate change[END_REF][START_REF] Michalek | Mytilus hybridisation and impact on aquaculture: A minireview[END_REF][START_REF] Simon | Replicated anthropogenic hybridisations reveal parallel patterns of admixture in marine mussels[END_REF].

Obviously, the possibility of missing an unknown and yet undetectable or uncultivable pathogen should not be excluded. Furthermore, a complex 'pathosystem' between two or more pathogens, as shown by [START_REF] De Lorgeril | Immunesuppression by OsHV-1 viral infection causes fatal bacteraemia in Pacific oysters[END_REF], may also exist and would require an integrative and holistic approach to be understood as recommended by these authors in the case of multi-factorial diseases.

Conclusion

This multi-parametric field study, carried out on commercially cultivated mussels, was a first approach to identifying potential causes of mortality in northern Brittany. We completed a first sorting and identified important factors for future experimental studies. In Brest, the presence of Marteilia, inflammatory lesions and pollution, with additional weakening factors such as the breeding period, explains part of the mortalities. In Lannion, no relevant parasites or pollution were found, although heavy inflammatory lesions were observed; thus, further research is required to explain those hemocytic infiltrations and to determine the cause(s) of the mortality observed in this site.

Figure 1 :

 1 Figure 1: Location of the three sampling sites: the bays of Brest, Lannion and St. Brieuc.

Figure 2 :

 2 Figure 2: Pictures of (A) the preparation of the baskets each containing 100 mussels and (B) the fixing of the baskets on the intertidal mussel stakes ("bouchot"). Photo credit: Regional Shellfish Committee of North Brittany (CRCBN)

Figure 3 :

 3 Figure 3: Variation of temperature (broken line) and: (A) cumulative mortality (%) observed over the duration of the study based on monthly mortality monitoring (continuous line with dots); (B) half-stock indices (days) calculated monthly to visualize the occurrence of mortalities for each month (line with dots). The vertical black lines correspond to the dates of sampling for analyses.

Figure 4 :

 4 Figure 4: Evolution of the Condition Index (C.I.) of the Brest mussels on four dates (February, April, May and September 2017). Values are means ± SEM (ANOVA, p > 0.05).

Figure 5 :

 5 Figure 5: Comparison of the Condition Indices (C.I.) of mussels from the 3 sites (Brest, Lannion, St. Brieuc) on both dates (February and May 2017). Values are means ± SEM (ANOVA, *p < 0.05 and Tukey's post hoc test).

  . The prevalence of the symbionts and other pathological conditions showed an inverse relationship with the half-stock index. When testing the link between the prevalence of the different pathological conditions and the half-stock index (Spearman correlation test; Fig.9), only hemocytic infiltration was significantly positively correlated to the intensity of mortality (p* < 0.05). Nevertheless, the p-value of Marteiliaequal to 0.08-was influenced by its absence in Lannion and St. Brieuc; in Brest, the higher the prevalence of Marteilia, the lower the half-stock index.

Figure 6 :

 6 Figure 6: Micrographs of histological sections showing symbionts found in Mytilus edulis. A: section of the digestive gland showing different developing stages of Marteilia sp. (arrows) in the epithelium of digestive tubules. B: section through the gills showing a cell of the ciliate Ancistrum mytili (arrow). C: section through the digestive gland showing various intracytoplasmic ciliates (arrows) in the epithelium of a digestive tubule. D: section through the mantle showing trematode sporocysts (arrows) enclosing cercariae. E: section through the visceral mass showing a copepod Mytilicola sp. (star) in the intestinal lumen (L).

Figure 7 :

 7 Figure 7: Micrographs of histological sections showing pathological conditions of Mytilus edulis. A: section through the visceral mass showing heavy hemocytic infiltration (star) of the connective tissue. B: section through the digestive gland showing granulocytomas consisting of large masses of hemocytes (stars) surrounded by layers of flattened, epithelioid cells (arrowheads). C: section through the visceral mass showing the connective tissue heavily infiltrated with masses of neoplastic cells (star). D: Higher magnification of the previous micrograph showing abundant neoplastic cells (arrows) in a hemolymph sinus: a cell in mitotic process is pointed out with an arrowhead.

Figure 8 :

 8 Figure 8: Prevalence (%) of the most remarkable histopathological observations: granulocytomas (GRA), inflammation/haemocytic infiltration (INF), disseminated neoplasia (NEO), trematode sporocysts enclosing cercariae (TRE), Marteilia sp. (MAR), Mytilicola sp.(MYT) and ciliates (CIL) identified (A) in the 3 bays (Brest, Lannion, St. Brieuc) on both dates (February and May 2017); (B) in Brest on four dates (February, April, May and September 2017).

Figure 9 :

 9 Figure 9: Link between the prevalence (%) of symbionts and histopathological conditions and the half-stock index (days) for each site: Brest (letter x), Lannion (plus sign) and St. Brieuc (open circle). INF: haemocytic infiltration; GRA: granulocytomas; TRE: trematode sporocysts enclosing cercariae; NEO: disseminated neoplasia; MAR: Marteilia sp.; MYT: Mytilicola sp.; CIL: ciliates.

Figure 10 :

 10 Figure 10: Bacterial profiles in samples from the 3 bays (Lannion, Brest, St. Brieuc) collected in February and May 2017.

Table 1 .

 1 Information on study sites, sampling dates and number of mussels used for each analysis.

					Sampling date			Other analyses (biometry,
			Farming					Species	Histological
	Site	Latitude	Longitude						bacteriological, molecular,
			method	Feb.	Apr.	May	Sept.	identification	examination
									and chemical analyses)
	Bay of Brest	48°20'22.5"N	4°19'48.4"W Bouchot	27/02/2017 24/04/2017 30/05/2017 20/09/2017	50 per date	50 per date	20 per date (total=80)
								(total=200)	(total=200)
	Bay of Lannion	48°44'44.8"N	3°35'24.9"W Longlines	24/02/2017		17/05/2017		50 per date	50 per date	20 per date (total=40)
								(total=100)	(total=100)
	Bay of St. Brieuc	48°33'07.6"N	2°38'55.6"W Bouchot	27/02/2017		28/05/2017		50 per date	50 per date
								(total=100)	(total=100)

  ). In May, a low percentage of Lannion mussels had disseminated neoplasia (2.4%), while it was not detected in St. Brieuc. It is important to note that St. Brieuc had no sign of hemocytic infiltration, neither in February nor May. In contrast, St. Brieuc was the most affected by two less damaging symbionts, ciliates and Mytilicola sp., both in February and May. The prevalence of hemocytic infiltration increased in Brest from February to May, while it remained almost constant at Lannion. Figure8Billustrated these observations for Brest; indeed, there was an increase in hemocytic infiltration and Marteilia between February and April, then a gradual decrease from April to September. Furthermore, 18% of mussels from Brest showed disseminated neoplasia in April, while it was not found in February or May, and its prevalence was 2% in September. In Brest, the prevalence of most symbionts and the other pathological were highest in April

Table 2

 2

	shows trace element concentrations in the mussel tissues and the maximum level
	(ML) set by the Commission Regulation ((EC) No. 1881/2006 amended by (EC) No.
	629/2008 and (EC) No. 420/2011) for regulated chemical compounds in the relevant
	foodstuffs category of bivalve molluscs. Comparing the three sites in February, mussels from
	St. Brieuc had concentrations of chromium (Cr), molybdenum (Mo), nickel (Ni) and titanium

(Ti) overall 2-4x higher than those of Brest and Lannion. The Lannion site was distinguished by iron (Fe) and aluminum (Al) concentrations that were more than 2x lower than the other two sites. In contrast, lead (Pb) concentration was highest in Brest, more than 5x higher than that of the mussels from Lannion and St. Brieuc, and exceeding the ML. Regarding temporal variation of chemical compounds in Brest mussels, Pb concentration was similar in February and April with a twofold decrease in September; Fe and Al concentrations showed noticeable decrease over time. The highest concentrations were found in winter and a general decrease was observed throughout the study period.

Table 2 .

 2 Trace element concentrations (mg/kg w.w.) in mussel tissues collected from three bays in northern Brittany: Brest (BRE), Lannion (LAN) and St. Brieuc (STB), in February 2017, and in Brest in April and September 2017. Limit of detection: < 0.002 mg/kg). ML: Maximum Level set by the Commission Regulation (EC) No. 1881/2006 amended by (EC) No. 629/2008 and (EC) No. 420/2011.

			February		April	September
	Chemical compounds	STB	LAN	BRE	BRE	BRE
	Cd (ML: 1.0)	0.07	0.07	0.13	0.14	0.10
	Hg (ML: 0.5)	0.01	0.02	0.04	0.03	0.02
	Pb (ML: 1.5)	0.32	0.23	1.62	1.54	0.70
	Co	0.15	0.09	0.21	0.12	0.07
	Cu	1.42	1.35	1.43	1.36	1.59
	Cr	3.80	1.49	0.99	0.54	0.09
	Fe	175.98 62.28 157.48	85.65	34.10
	Mn	3.15	1.79	2.69	1.81	1.22
	Mo	0.84	0.37	0.23	1.32	0.14
	Ni	2.31	1.00	0.65	0.49	0.12
	Zn	13.7	15.84	15.85	18.73	12.32
	Ti	5.90	1.57	1.91	0.95	0.38
	Sn	<0.02 <0.02	<0.02	<0.02	0.19
	Al	173.11 65.85 143.72	69.09	22.70
	Se	1.28	1.48	1.39	1.04	0.85
	As	3.16	3.99	3.15	6.42	3.41

Acknowledgements

The authors thank Yann Deydier (CRCBN) for technical support in the collection of mussels, María J. Brianes, María I. Meléndez and Elena Penas for their technical assistance with histology (CIMA), and Patrick Céron for design of the map. Many thanks to Annette Byrne for the proofreading of English and Dr. D'Rego for the finishing touches.

The Regional Shellfish Committee of North Brittany (CRCBN), throught the European Maritime and Fisheries Fund (EMFF), and the laboratory LABÉO financially supported this study. Maud Charles received co-funding from the Normandy region and from the laboratory LABÉO Frank Duncombe.

Additional file 1: Supplementary Materials and Methods

PCR screening for bivalve mollusc known pathogens

For each mussel, a pool of minced tissues from foot, adductor muscle, gills, mantle, and Herpesvirus type 1 (OsHV-1), V. aestuarianus, V. tubiashii, V. harveyi, and V. splendidus (the true species V. splendidus of the Splendidus clade named here Splendidus cluster) (Table S1).

Amplification reactions were performed in a total volume of 25 μL using a SmartCycler ® (Cepheid, USA). Each reaction contained 12,5 μL of a Premix Ex Taq ® 2 X Takara ® (Lonza, Verviers, Belgium), 9 μL of purified water, 2 μL of DNA sample (replaced with 2 µL of purified water in the negative control) and 0.5 μL of each primer (20 μM) and probe (10 μM) or SYBR Green (for N. crassostreae). Each assay included negative and positive control reactions. The thermal cycle profile consisted of 95 °C for 10 s followed by 40 cycles of 95 °C for 5 s and 60° for 30 sec (or 62 °C for V. splendidus or 64 °C for V. harveyi). Beforehand, for each real-time PCR developed for this study, the inclusivity and exclusivity were tested.

Moreover, in order to exclude false-positive results, when a signal was obtained, the amplicon was sent to Eurofins MWG Operon (Ebersberg, Germany) to be purified and subsequently sequenced; only then, after using the NCBI BLAST, confirmation of the targeted pathogen could be made.

Table S1. Target genes, primers (forward (Fw) and reverse (Rv)) and probe (Pr) used for detection of known bivalve molluscs pathogens.

Name of pathogens