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

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In 2014, a high and unusual mass mortality of mussels occurred in several important production areas along the French coasts of the Atlantic and English Channel. In the first quarter of 2016, mass mortalities hit farms on the west coast of the country once again. These heterogeneous mortality events elicited a multi-parametric study conducted during the 2017 mussel season in three sites in northern Brittany (Brest, Lannion and St. Brieuc). The objective was to assess the health status of these mussels, follow mortality and attempt to identify potential causes of the abnormal high mortality of farmed mussels in northern Brittany. Brest was the most affected site with 70% cumulative mortality, then Lannion with 40% and finally St. Brieuc with a normal value of 15%. We highlighted a temporal 'mortality window' that opened throughout the spring season, and concerned the sites affected by mortality of harmful parasites (including pathogenic bacteria), neoplasia, metal contamination, and tissue alterations. Likely, the combination of all these factors leads to a weakening of mussels that can cause death.
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High mortality of mussels in northern Brittany – Evaluation of the involve-
ment of pathogens, pathological conditions and pollutants
Maud Charles, Ismaël Bernard, Antonio Villalba, Elise Oden, Erika A.V.
Burioli, Gwenaël Allain, Suzanne Trancart, Valérie Bouchart, Maryline
Houssin
PII: S0022-2011(19)30115-6
DOI: https://doi.org/10.1016/j.jip.2019.107308
Reference: YJIPA 107308
To appear in: Journal of Invertebrate Pathology
Received Date: 23 April 2019
Revised Date: 11 December 2019
Accepted Date: 14 December 2019
Please cite this article as: Charles, M., Bernard, I., Villalba, A., Oden, E., Burioli, E.A.V., Allain, G., Trancart, S.,
Bouchart, V., Houssin, M., High mortality of mussels in northern Brittany Evaluation of the involvement of
pathogens, pathological conditions and pollutants, Journal of Invertebrate Pathology (2019), doi: https://doi.org/
10.1016/j.jip.2019.107308
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High mortality of mussels in northern Brittany – Evaluation of the involvement of pathogens,
pathological conditions and pollutants
Maud Charles1,2,*, Ismaël Bernard3, Antonio Villalba4,5,6, Elise Oden2, Erika A.V. Burioli2, Gwenaël Allain7,
Suzanne Trancart2, Valérie Bouchart2, Maryline Houssin1,2
1 Normandie Université, Université de Caen Normandie, FRE BOREA, CNRS-7208, IRD-207, MNHN, UPMC, UCN, Esplanade de la
Paix, 14032 Caen Cedex 4, France
2 LABÉO Frank Duncombe, 1 Route de Rosel, 14053 Caen Cedex 4, France
3 Eureka Modélisation, 22740 Lézardrieux, France
4 Centro de Investigacións Mariñas, Consellería do Mar (CIMA), Xunta de Galicia, 36620 Vilanova de Arousa, Spain
5 Departamento de Ciencias de la Vida, Universidad de Alcalá, 28871 Alcalá de Henares, Spain
6 Research Centre for Experimental Marine Biology and Biotechnology (PIE), University of the Basque Country (UPV/EHU), 48620
Plentzia, Basque Country, Spain
7 Armeria, 31B rue de la Concorde, 56670 Riantec, France
*Corresponding author: Maud Charles, LABÉO Frank Duncombe, Pôle Recherche, 1 Route de Rosel, 14053
Caen Cedex 4, France.
E-mail: maud.charles@outlook.fr ;
Abstract
In 2014, a high and unusual mass mortality of mussels occurred in several important production areas along the
French coasts of the Atlantic and English Channel. In the first quarter of 2016, mass mortalities hit farms on the
west coast of the country once again. These heterogeneous mortality events elicited a multi-parametric study
conducted during the 2017 mussel season in three sites in northern Brittany (Brest, Lannion and St. Brieuc). The
objective was to assess the health status of these mussels, follow mortality and attempt to identify potential causes
of the abnormal high mortality of farmed mussels in northern Brittany. Brest was the most affected site with 70%
cumulative mortality, then Lannion with 40% and finally St. Brieuc with a normal value of 15%. We highlighted
a temporal ‘mortality window’ that opened throughout the spring season, and concerned the sites affected by
mortality of harmful parasites (including pathogenic bacteria), neoplasia, metal contamination, and tissue
alterations. Likely, the combination of all these factors leads to a weakening of mussels that can cause death.
Keywords: Mytilus edulis; multi-parametric; neoplasia; Vibrio splendidus; Marteilia; hemocytic infiltration
1. 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 (Buschbaum et al., 2008). Due to their intense
filtration activity, they modify aquatic habitat, making it more suitable for their own species and other for
organisms (Borthagaray & Carranza, 2007). 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 (Béchemin et al., 2015; François et al., 2015; Allain
& Bernard, 2016; Lupo et al., 2016).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 (Brienne, 1964;
Blateau, 1989; Guichard et al., 2011).
One of the proposed causes of mortality is pathogenic bacteria in the genus Vibrio (Béchemin et al., 2015).
Vibrios are among the major bacterial pathogens of marine organisms and have been identified as pathogens of
several bivalve molluscs (Beaz-Hidalgo et al., 2010; Lemire et al., 2015; Eggermont et al., 2017). During mussel
mortality events, different Vibrio splendidus strains were isolated from moribund mussels in France and were
linked to mortality (Travers et al., 2015; Ben Cheikh et al., 2016). 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 (François et al.,
2015; Lupo et al., 2016). 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 (Peters, 1988; Elston et al.,
1992; Landsberg, 1996; Carballal et al., 2015) and, in some bivalve species including the mussel Mytilus
trossulus, spreads by transmission of cancerous cells between individuals (Metzger et al., 2015, 2016). Some
scientists observed chromosomal abnormalities associated with neoplasia in Mytilus trossulus (González-Tizón
et al., 2000) while advanced neoplastic disease was associated with significant mortalities of Mytilus sp. (Moore
et al., 1991), including M. trossulus in Washington and Oregon (USA) and in British Columbia (Canada) (Elston
et al., 1992). In France, Benabdelmouna & Ledu (2016) and Benabdelmouna et al. (2018) 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 Burioli et al. (2017) during a mortality event.
Following heterogeneous mortality events of mussels that occurred in Brittany in the past years (Allain &
Bernard, 2016; Bernard & Allain, 2017), 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 (Carrasco et al., 2015), and by abiotic factors such as chemical contaminants (Coles et al., 1995; Pipe
& Coles, 1995; Dailianis, 2010; Moschino et al., 2016), water temperature and animal conditioning (Almada-
Villela et al., 1982; Seed & Suchanek, 1992; Gosling, 1992, 2003). 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.
2. Materials and methods
2.1. 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 (Côtes-d’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 (Simon et al., 2018).
2.2. 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.
2. 3. Sa mpling 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.
2. 4. Bi ometry
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 (Davenport & Chen, 1987):



2.5. 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) (Howard et al., 2004). Histological sections were examined
for all symbionts, including phoresis, commensalism, parasitism and mutualism (Kinne, 1980), 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.
2. 6. Ba cteriological analysis
Analysis was conducted to compare the bacterial flora of mussels from each of the three sites on two different
dates (February and May). For each of the six batches, tissues of five live mussels (to avoid the emergence and
overexpression of opportunistic bacteria due to tissue degradation in dead animals) were pooled and mashed with
scalpels and 200 µL of Artificial Sterilized Sea Water (ASSW) was added to 50 mg of the homogenate. After
stirring and pulse centrifugation to pellet cellular debris, ten-fold dilutions of supernatant were made and 100 µL
of 1:100 and 1:10,000 dilutions were sown on Zobell marine agar (Agar and ASSW enriched with 0.5% peptone,
0.1% yeast extract, and 0.01% ferric phosphate, pH 7.6; Oppenheimer & ZoBell, 1952) and incubated for 48 h at
22 °C. For each pool, 20 predominant bacterial colonies with different phenotypes were re-isolated in order to
verify their purity. DNA extraction was performed by heating a colony placed in 250 µL of purified water
(DNA/nuclease free-water) for 10 min at 95 °C. Successful extraction was confirmed by DNA quantification
with NanoDropTM 2000c spectrophotometer (ThermoFisher ScientificTM, Waltham, MA USA). Then, strains
were characterized by molecular analysis (see below 2.6.1.).
2. 7. Mo lecular 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) (Oden et al., 2016), was carried out on a
Smart Cycler® (Cepheid, USA); the primers used for PCR1 were SpF1 5’ATCATGGCTCAGATTGAACG3’
and SpR1 5’CAATGGTTATCCCCCACATC3’ (Nasfi et al., 2015) 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 (Oden et al., 2016). The primer pair for PCR2 was mreB-F
5’CTGGTGCTCGYGAGGTTTAC3’ and mreB-R 5’CCRTTYTCTGAKATATCAGAAGC3′. For colonies not
identified as members of the Splendidus clade (PCR1 negative), another conventional PCR targeting the 16S
rRNA gene (PCR3) (Burioli et al., 2018) was done; the primer pair for PCR3 was 16S27-F
5’AGAGTTTGATCMTGGCTCAG3’ and 16S1492-R 5’ACCTTGTTACGACTTCAC3’. For the conventional
PCR, typical 25-µL reaction mixtures contained 12.5 µL of Premix Ex Taq® 2 X Takara® (Lonza, Verviers,
Belgium), 0.5 µL of each primer (20 µM), 9.5 µL of purified water and 2 µL of DNA template (replaced with 2
µL of purified water in the negative control). Conventional PCR amplifications were carried out in a T100TM
Thermal Cycler (Bio-Rad, France) and the thermal program was as follows: 10 s at 95°C; 30 cycles for 10 s at
95°C, 30 s at 55°C, 40 s at 72°C and a final extension of 3 min at 72°C. PCR products were then analysed with
QIAxcel® Advanced System (Qiagen, Courtaboeuf, France) and those with the expected size were sent to
Eurofins MWG Operon (Ebersberg, Germany) to be purified and sequenced. Species were identified using the
National Center for Biotechnology Information (NCBI) Basic Local Alignement Search Tool (BLAST) with
individual 16S rRNA sequences, and mreB sequences were aligned using a multiple sequence alignment Multiple
Sequence Comparison by Log-Expectation (MUSCLE). Phylogenetic analyses were performed in MEGA7
(Kumar et al.,, 2016) using the Neighbor Joining method (Tamura et al, 2013) and the maximum composite
likelihood model with a bootstrap of 1000 replications; sequences of the mreB gene from 44 different reference
bacterial strains from the Splendidus clade were used (see 2.1. in Oden et al., 2016). V. aestuarianus 02/041, V.
ordalii 12B09 and V. penaeicida AQ115, were provided from the Genomic of Vibrio Research Department
(CNRS Roscoff, France) and used as the Splendidus clade outgroup.
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).
2. 8. Ch emical analyses of fle sh
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 (HNO3, Analpure®, Analytika® , Prague, Czech Republic) and oxidants
(H2O2, Normapur®,VWR international, Radnor, Pennsylvanie, USA), following previously described protocols
(Squadrone et al., 2016). 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.
2.9. Data analysis
2. 9.1. Half- stock index
To compare mortality phenomena over time, the half-stock index (Bernard et al., 2018) 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 N0 the population at the
beginning of the study, then: N(t) = N0e λt. Between two successive counts in ti-1 and ti, 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: d0.5 = 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).
2. 9.2 S tatis tical 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.r-project.org/) and ‘dplyr’, ‘tidyr’ and ‘ggplot2’
packages.
3. Results
3. 1. Mu ssel species, temperat ure m onitoring and mortality
For species identification, a dataset of 81 common markers was obtained (for more details, see Simon et al, 2019),
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.
3. 2. Bi ometry
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).
3.3. 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%). 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. Figure 8B illustrated 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. 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 Marteilia –equal 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.
3. 4. Mo lecular analyses
3. 4.1. Bacte rial 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 February, between four (Brest & Lannion)
and five (St. Brieuc) different species per site were present among the 20 strains isolated compared to seven
(Brest & St. Brieuc) and nine (Lannion) different species in May. However, despite this difference in diversity,
two bacteria remained dominant: V. splendidus and Pseudoalteromonas sp. V. splendidus prevalence in February
was 42% in Brest, 35% in Lannion and 80% in St. Brieuc; in May, prevalence was 30% in Brest, 0% in Lannion
and 23% in St. Brieuc. In February Pseudoalteromonas sp. prevalence was 21% in Brest, 55% in Lannion and
5% in St. Brieuc; while in May, prevalence was 45% in Brest, 42% in Lannion and 29% in St. Brieuc. Among
the 30 strains of V. splendidus isolated in the three February batches –7 in Lannion, 15 in St. Brieuc and 8 in
Brest– all had a different mreB sequence, except for three strains found in St. Brieuc (1) and in Brest (2), which
had an identical sequence. When comparing bacterial profiles with the occurrence of mortalities, no difference
appeared; V. splendidus was found in high abundance in almost all sites, as was Pseudoalteromonas sp. to a lesser
extent, with or without mortalities.
3. 4.2. Prese nce o r absence of know n pat hogen s of bivalve moll uscs
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.
3. 5. Ch emical analyses of fle sh
Table 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.
4. 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.
4. 1. Te mperature, conditionin g and mortality
Mytilus edulis is known to resist extreme temperatures (Aarset, 1982; Seed & Suchanek, 1992; Almada-
Villela et al., 1982), 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 (Benabdelmouna et al., 2018;
Dégremont et al., 2019). 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 (Gosling, 2003). 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 (Najdek
& Sapunar, 1987). Mussel growth is primarily influenced by the reproductive stage and food availability
(Gosling, 1992) but growth capacities also have been linked to site and animal origin (Dickie et al., 1984). 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, Prou and Goulletquer (2002) 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 (Cloern, 1996) and mussels undergo long-time starvation periods (Harbach & Palm, 2018). Mytilus sp. can
handle longer food restriction periods and maintain its shell size by using energy from its own tissue (Dare &
Edwards, 1975; Riisgård et al., 2014). 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.
4. 2 Mussel symbionts
Villalba et al. (1997) 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.
Figueras et al. (1991) 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 (Fichi et al. 2018). 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., Brienne (1964) and Blateau (1989) 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 (Davey,
1989; Robledo et al., 1994). 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 Brienne (1964) and Blateau (1989).
Lauckner (1983) 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 (Robledo et al., 1994; Laruelle et al., 2002). Bakhmet et al. (2017) found a metabolic
level supported by significant growth deficiency in parasitized M. edulis. This also was observed in Normandy
(France) by Le Breton & Lubet (1992), as well as a variation in parasitism over time as we observed in Brest, and
an increase in mortality between January and July.
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 (Villalba et al. 1993; Villalba et al. 1997; Fuentes et al., 1995; Arzul
et al., 2014). 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 (Villalba et al., 1993; Garcia et
al., 2005). 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 (Grizel & Tigé, 1973). 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 (Grizel, 1985). 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 (Villalba et al., 1993). 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 (Kerr et al. 2018). 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.
4.3 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 (Cuevas et al., 2015). Cuevas et al. (2015)
showed that mussels from the most impacted sites (by metallurgic and shipyard activities) endured the most
significant deleterious effects showing inflammation. Also, Sheir & Handy (2010) established a potential link
between hemocytic infiltration and lesions with the presence of xenobiotics. Lowe & Moore (1979) 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 (Lauckner, 1983; 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. Villalba et al. (2001) 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 (Elston et al.,
1988; Ciocan & Sunila, 2005). Because hemocytes are cells with a key role in many physiological functions
(digestion, excretion, nutrition, defense mechanisms; Cheng, 1984; Fisher, 1986, 1988), 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 (Kent et al., 1989). Mass mortalities of various bivalve species have been associated
with disseminated neoplasia (see reviews by Elston et al., 1992; Carballal et al., 2015). In France, Benabdelmouna
& Ledu (2016) 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,
Benabdelmouna et al. (2018) 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. Elston et al. (1988) 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.
4.4 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 (Dailianis, 2010). It is known that anthropogenic compounds and heavy metals play
roles in defence capacities of bivalve molluscs and could increase susceptibility to disease (Coles et al., 1995;
Pipe & Coles, 1995; Morley, 2010). In addition, the effects of environmental contaminants sometimes correspond
to a direct toxic action on tissues or cells (Gagnaire et al., 2004).
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
(Sarmiento et al., 2011). Among all the metallic elements that Moschino et al., (2016) 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 (Depledge, 1994; Dailianis, 2010), and it was shown that mussels
have lower defense mechanisms against metal oxidative challenge and toxicity than oysters. Indeed, Funes et al.
(2006) 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, Viarengo et al. (1991) and Petrović et al., (2004) 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) (Belin et al., 2013). 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 (Auffret, 1983).
Lacroix et al. (2015) 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. Lacroix et al. (2017) 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.
4.5 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 (Yang et al., 2013; Li et al., 2014). On the other hand, Venkateswaran & Dohmoto (2000)
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 (Gram &
Huss, 1996; Madigan et al., 2014). Also, Lokmer & Wegner (2015) 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 (Thompson et al., 2004). 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, Béchemin et al. (2015) 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
(Kwan & Bolch, 2015). 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 (Thompson et al., 2004).
Knowledge of their role as animal pathogens and their mechanisms of action in pathogenesis has been limited.
Indeed, Bruto et al. (2018) 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. Dare (1976) 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 (Dickie et al., 1984; Mallet et al., 1987; Dégremont
et al., 2019).
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 (Bierne et al., 2003;
Fly et al., 2015; Michalek et al., 2016, Simon et al, 2019).
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 De Lorgeril
et al. (2018), 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.
5. 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.
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.
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Figure 1: Location of the three sampling sites: the bays of Brest, Lannion and St. Brieuc.
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)
Table 1. Information on study sites, sampling dates and number of mussels used for each analysis.
Sampling date
Site
Latitude
Longitude
Farming
method
Feb.
Apr. May
Sept.
Species
identification
Histological
examination
Other analyses (biometry,
bacteriological, molecular,
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
(total=200)
50 per date
(total=200)
20 per date (total=80)
Bay of Lannion
48°44'44.8"N
3°35'24.9"W
Longlines
24/02/2017
17/05/2017
50 per date
(total=100)
50 per date
(total=100)
20 per date (total=40)
Bay of St. Brieuc
48°33'07.6"N
2°38'55.6"W
Bouchot
27/02/2017
28/05/2017
50 per date
(total=100)
50 per date
(total=100)
20 per date (total=40)
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: 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: 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).
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 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: 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: 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: 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: Bacterial profiles in samples from the 3 bays (Lannion, Brest, St. Brieuc) collected in February and
May 2017.
Table 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
Graphical abstract
Highlights:
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.
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
digestive gland was subjected to DNA extraction using a QIAamp DNA minikit® (Qiagen,
Courtaboeuf, France) following the manufacturer’s protocol for blood or body fluids, except
for elution performed in 60 μL Qiagen elution buffer AE. The quality of the extracted DNA
was checked with NanoDropTM 2000c spectrophotometer (ThermoFisher ScientificTM,
Waltham, MA USA). Within the same batch, the individual extracts were pooled in groups of
5 to have 4 replicates per batch for each PCR done. The known bivalve pathogens investigated
by TaqMan® real-time PCR and classical PCR were: 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 (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
Target gene
Oligo-nucleotides sequences (5’-3’)
References
Bonamia sp.
28S rRNA
Fw TCCCTGCCCTTTGTACACA
Rv CTCTTATCCACCTAATTCACCTCAG
Pr TxR-CGCCCGTCGCTTCTACCGATT-BHQ2
Present paper
Haplosporidium nelsoni
SrRNA
Fw CACGCGCGCTACAATGT
Rv CGAGATTACCCGGCCTTCT
Pr FAM-CACGCAACGAGTTCAACCTTGCC-BHQ1
Present paper
Marteilia sp.
ITS1
Fw CACACTACTCTTCGCTTTCGAT
Rv GACTACCCGTGCCGAACA
Pr Cy3-TCGCAAACAGGAAGCGGCTCTC-BHQ1
Present paper
Mikrocytos mackini
28S rRNA
Fw GGTGGCCGAATGACGTAGT
Rv GCCTATGACAGCACGAAGCA
Pr Cy5-CCGCTTCGGCGTGCAGTCTC-BHQ2
Present paper
Nocardia crassostreae
16S-23S rNA ITS
Fw CCTCGATACCGCCGAAGAA
Rv CAACACACCCGCATCAAA
Carrasco et al. (2013)
OsHV-1
B region
Fw GTCGCATCTTTGGATTTAACAA
Rv ACTGGGATCCGACTGACAAC
Pr TxR-TGCCCCTGTCATCTTGAGGTATAGACAATC-BHQ2
Martenot et al. (2010)
V. aestuarianus
dnaJ
Fw GTATGAAATTTTAACTGACCCACAA
Rv CAATTTCTTTCGAACAACCAC
Pr TxR-TGGTAGCGCAGACTTCGGCGAC-BHQ2
Saulnier et al. (2009)
V. tubiashii
vtpA
vth
Fw GGTACGGACTATCCGGGATT
Rv TTCACCGCTGAGTTGTTCAT
Pr Cy3-ATCGTCGATAAATCAGGCACAACCTGT-BHQ1
Fw CGGTTGATATTCGCGTCAA
Rv GTGTGAAACCCTGCGAAGTA
Pr Cy5-TATCACAGATGCGCTCGGTTCAGTC-BHQ2
Present paper
Present paper
V. harveyi
16S rRNA
Fw CGAGCGGAAACGAGTTATCTG
Rv CTCACCAACTAGCTAATCCCACCTA
Pr TxR-CCGCATAATACCTACGGGTCAAAGAGGG-BHQ2
Present paper
V. splendidus
(Splendidus cluster)
toxR
Fw AGCAGCGGCTGAAATTGCA
Rv GGCCGCAGTTGGTGTTGTT
Pr FAM-CAATGACTGAAGCTGTCGAGCCC-BHQ1
Oden et al. (2018)
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Human-mediated transport creates secondary contacts between genetically differentiated lineages, bringing new opportunities for gene exchange. When similar introductions occur in different places, they provide informally replicated experiments for studying hybridisation. We here examined 4279 Mytilus mussels, sampled in Europe and genotyped with 77 ancestry informative markers. We identified a type of introduced mussels, called ‘dock mussels’, associated with port habitats and displaying a particular genetic signal of admixture between M. edulis and the Mediterranean lineage of M. galloprovincialis . These mussels exhibit similarities in their ancestry compositions, regardless of the local native genetic backgrounds and the distance separating colonised ports. We observed fine-scale genetic shifts at the port entrance, at scales below natural dispersal distance. Such sharp clines do not fit with migration-selection tension zone models, and instead suggest habitat choice and early stage adaptation to the port environment, possibly coupled with connectivity barriers. Variations in the spread and admixture patterns of dock mussels seem to be influenced by the local native genetic backgrounds encountered. We next examined departures from the average admixture rate at different loci, and compared human-mediated admixture events, to naturally admixed populations and experimental crosses. When the same M. galloprovincialis background was involved, positive correlations in the departures of loci across locations were found; but when different backgrounds were involved, no or negative correlations were observed. While some observed positive correlations might be best explained by a shared history and saltatory colonisation, others are likely produced by parallel selective events. Altogether, genome-wide effect of admixture seems repeatable, and more dependent on genetic background than environmental context. Our results pave the way towards further genomic analyses of admixture, and monitoring of the spread of dock mussels both at large and fine spacial scales.
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We present the latest version of the Molecular Evolutionary Genetics Analysis (MEGA) software, which contains many sophisticated methods and tools for phylogenomics and phylomedicine. In this major upgrade, MEGA has been optimized for use on 64-bit computing systems for analyzing bigger datasets. Researchers can now explore and analyze tens of thousands of sequences in MEGA. The new version also provides an advanced wizard for building timetrees and includes a new functionality to automatically predict gene duplication events in gene family trees. The 64-bit MEGA is made available in two interfaces: graphical and command line. The graphical user interface (GUI) is a native Microsoft Windows application that can also be used on Mac OSX. The command line MEGA is available as native applications for Windows, Linux, and Mac OSX. They are intended for use in high-throughput and scripted analysis. Both versions are available from www.megasoftware.net free of charge.
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Infectious diseases are mostly explored using reductionist approaches despite repeated evidence showing them to be strongly influenced by numerous interacting host and environmental factors. Many diseases with a complex aetiology therefore remain misunderstood. By developing a holistic approach to tackle the complexity of interactions, we decipher the complex intra-host interactions underlying Pacific oyster mortality syndrome affecting juveniles of Crassostrea gigas, the main oyster species exploited worldwide. Using experimental infections reproducing the natural route of infection and combining thorough molecular analyses of oyster families with contrasted susceptibilities, we demonstrate that the disease is caused by multiple infection with an initial and necessary step of infection of oyster haemocytes by the Ostreid herpesvirus OsHV-1 µVar. Viral replication leads to the host entering an immune-compromised state, evolving towards subsequent bacteraemia by opportunistic bacteria. We propose the application of our integrative approach to decipher other multifactorial diseases that affect non-model species worldwide.
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Natural selection plays a variety of roles in hybridization, speciation, and admixture. Most research has focused on two extreme cases: crosses between closely related inbred lines, where hybrids are fitter than their parents, or crosses between effectively isolated species, where hybrids suffer severe breakdown. But many natural populations must fall into intermediate regimes, with multiple types of gene interaction, and these are more difficult to study. Here, we develop a simple fitness landscape model, and show that it naturally interpolates between previous modeling approaches, which were designed for the extreme cases, and invoke either mildly deleterious recessives, or discrete hybrid incompatibilities. Our model yields several new predictions, which we test with genomic data from Mytilus mussels, and published data from plants (Zea, Populus, and Senecio) and animals (Mus, Teleogryllus, and Drosophila). The predictions are generally supported, and the model explains a number of surprising empirical patterns. Our approach enables novel and complementary uses of genome‐wide datasets, which do not depend on identifying outlier loci, or “speciation genes” with anomalous effects. Given its simplicity and flexibility, and its predictive successes with a wide range of data, the approach should be readily extendable to other outstanding questions in the study of hybridization.
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Il s'agit du second rapport technique sur les mortalités de moules pour le CRC Bretagne Nord.
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The acute course of disease in young oysters infected by OsHV-1 and the rapid tissue degradation often preclude histological examination of specimens collected during outbreaks in field. Herein, live spat originated from two geographical areas were sampled just at the onset of a mortality event that occurred in Normandy (France) in June 2016. The lesions, associated with high OsHV-1 DNA quantities, were characterized by severe and diffuse haemocytosis mainly involving blast-like cells, myocyte degeneration and large, irregularly shaped degenerate eosinophilic cells in the connective tissue. The herpesvirus was identified by negative staining TEM and real-time PCR. Sequencing of the C region and ORFs 42/43 confirmed that the variants met the definition of OsHV-1 μVar. We sequenced 30 other ORFs in twenty OsHV-1-positive individuals and compared them to the μVar specimens isolated between 2009 and 2011. The ORFs encoding putative membrane proteins showed the highest number of variations. Seven different genotypes were identified, confirming the presence of relevant genetic diversity. Phylogenetic analysis provided evidence for a well-separated μVar new group, with an evolutionary divergence estimated at 0.0013 from the other μVar variants. The geographical distribution of these newly described variants and their effective virulence should be investigated in future.
Poster
Neoplasia is a pathological process that consists in an uncontrolled and irreversible proliferation of atypical cells. The presence of two predominant types of malignant neoplasia has been confirmed in various marine bivalve species: a germinoma and a "disseminated neoplasia", also called "haemic neoplasia", characterised by the proliferation of abnormal circulating cells of unknown origin. Between October 2015 and October 2016, a stock of blue mussels, Mytilus edulis, farmed in longline systems in Lannion (Northern Brittany, France) and originating from Pénestin (Southern Brittany, France) has been affected by a sequence of mortality events, resulting in a cumulative mortality rate of about 70%. During one of these events occurred in mid-October 2016, individuals from the batch affected by mortalities and individuals from a second batch of the species Mytilus galloprovincialis, farmed in the same locality but arising from natural collection in Southern Brittany and unaffected by the phenomenon, were collected. Five live individuals from each batch were analysed by gross and histological examination. A second step consisted in the development of a rapid non-invasive diagnostic tool based on cytological examination of haemolymph.