Patterns of admixture and introgression in a mosaic Mytilus galloprovincialis and Mytilus edulis hybrid zone in SW England.

The study of hybrid zones offers important insights into speciation. Earlier studies on hybrid populations of the marine mussel species Mytilus edulis and Mytilus galloprovincialis in SW England provided evidence of admixture but were constrained by the limited number of molecular markers available. We use 57 ancestry-informative SNPs, most of which have been mapped genetically, to provide evidence of distinctive differences between admixed populations in SW England and asymmetrical introgression from M. edulis to M. galloprovincialis. We combine the genetic study with analysis of phenotypic traits of potential ecological and adaptive significance. We demonstrate that hybrid individuals have brown mantle edges unlike the white or purple in the parental species, suggesting allelic or non-allelic genomic interactions. We report differences in gonad development stage between the species consistent with a prezygotic barrier between the species. By incorporating results from publications dating back to 1980, we confirm the long-term stability of the hybrid zone despite higher viability of M. galloprovincialis. This stability coincides with a dramatic change in temperature of UK coastal waters and suggests that these hybrid populations might be resisting the effects of global warming. However, a single SNP locus associated with the Notch transmembrane signalling protein shows a markedly different pattern of variation to the others and might be associated with adaptation of M. galloprovincialis to colder northern temperatures.

Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Genetic features of bivalve transmissible neoplasia in blue mussels from the Kola Bay (Barents Sea) suggest a recent trans-Arctic migration of the cancer lineages.

Ecology and biogeography of bivalve transmissible neoplasia (BTN) are underexplored due to its recent discovery and a challenging diagnostics. Blue mussels harbour two evolutionary lineages of BTN, MtrBTN1 and MtrBTN2, both derived from Mytilus trossulus. MtrBTN1 has been found only in M. trossulus from North Pacific. MtrBTN2 parasitizes different Mytilus spp. worldwide. BTN in M. trossulus in the Atlantic sector has never been studied. We looked for BTN in mussels from the Barents Sea using flow cytometry of cells, qPCR with primers specific to cancer-associated alleles and sequencing of mtDNA and nuclear loci. Both MtrBTN1 and MtrBTN2 were present in our material, though their prevalence was low (~0.4%). All cancers parasitized M. trossulus except one, MtrBTN1, which was found in a hybrid between M. trossulus and M. edulis. The mtDNA haplotypes found in both lineages were nearly identical to those known from the Northwest Pacific but not from elsewhere. Our results suggest that these two lineages may have arrived in the Barents Sea in recent decades with the maritime transport along the Northern Sea Route. A young evolutionary age of MtrBTN1 seems to indicate that it is an emerging disease in the process of niche expansion. Comparing the new and the published sequence data on tumour suppressor p53, we proved that the prevalence of BTN in mussels can reach epizootic levels. The finding of diverse recombinants between paternally and maternally inherited mtDNAs in somatic tissues of M. trossulus was an unexpected result of our study.

First isolation of Francisella halioticida strains from blue mussel (Mytilus edulis) in Normandy, France.

Mass mortality events affecting the blue mussels Mytilus edulis have been observed in France since 2014. The DNA of the bacterium Francisella halioticida, reported as pathogen of giant abalone (Haliotis gigantea) and Yesso scallop (Mizuhopecten yessoensis) has been detected recently in mussels from areas suffering mortalities. Isolation of this bacterium was attempted from individuals collected during mortality events. Identification was performed by 16S rRNA gene sequencing, real-time specific PCR and MALDI-ToF using spectra produced from the strain 8472-13A isolated from diseased Yesso scallop in Canada. Five isolates were identified as F. halioticida by real-time specific PCR and 16S rRNA sequencing. MALDI-ToF allowed the direct identification of four isolates (FR22a,b,c,d) which had 100% identity on the 16S rRNA gene with the known strains. On the other hand, one isolate (FR21) was not recognized by MALDI-ToF and had 99.9% identity on the 16S rRNA gene. The FR22 isolates showed difficult growth and required media optimization, which was not the case with the FR21 isolate. For these reasons, it was hypothesized that two type strains are present on French coasts, named FR21 and FR22. The FR21 isolate was selected for phenotypic analysis (growth curve, biochemical characteristics, electron microscopy), phylogenetic analysis and an experimental challenge. This isolate showed distinct differences compared to published F. halioticida strains, both at phenotypic and genotypic levels. Experimental infections of adult mussels led to 36% mortalities in 23 days following intramuscular injection with 3 × 107 CFU while a lower dose (3 × 103 CFU) did not lead to significant mortalities. In the context of this study, the strain FR21 was not virulent towards adult mussels.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid-base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Prevalence and polymorphism of a mussel transmissible cancer in Europe.

Transmissible cancers are parasitic malignant cell lineages that have acquired the ability to infect new hosts from the same species, or sometimes related species. First described in dogs and Tasmanian devils, transmissible cancers were later discovered in some marine bivalves affected by a leukaemia-like disease. In Mytilus mussels, two lineages of bivalve transmissible neoplasia (BTN) have been described to date (MtrBTN1 and MtrBTN2), both of which emerged in a Mytilus trossulus founder individual. Here, we performed extensive screening of genetic chimerism, a hallmark of transmissible cancer, by genotyping 106 single nucleotide polymorphisms of 5,907 European Mytilus mussels. Genetic analysis allowed us to simultaneously obtain the genotype of hosts - Mytilus edulis, M. galloprovincialis or hybrids - and the genotype of tumours of heavily infected individuals. In addition, a subset of 222 individuals were systematically genotyped and analysed by histology to screen for possible nontransmissible cancers. We detected MtrBTN2 at low prevalence in M. edulis, and also in M. galloprovincialis and hybrids although at a much lower prevalence. No MtrBTN1 or new BTN were found, but eight individuals with nontransmissible neoplasia were observed at a single polluted site on the same sampling date. We observed a diversity of MtrBTN2 genotypes that appeared more introgressed or more ancestral than MtrBTN1 and reference healthy M. trossulus individuals. The observed polymorphism is probably due to somatic null alleles caused by structural variations or point mutations in primer-binding sites leading to enhanced detection of the host alleles. Despite low prevalence, two sublineages divergent by 10% fixed somatic null alleles and one nonsynonymous mtCOI (mitochondrial cytochrome oxidase I) substitution are cospreading in the same geographical area, suggesting a complex diversification of MtrBTN2 since its emergence and host species shift.

Transcriptomic analysis of shell repair and biomineralization in the blue mussel, Mytilus edulis.

BACKGROUND: Biomineralization by molluscs involves regulated deposition of calcium carbonate crystals within a protein framework to produce complex biocomposite structures. Effective biomineralization is a key trait for aquaculture, and animal resilience under future climate change. While many enzymes and structural proteins have been identified from the shell and in mantle tissue, understanding biomieralization is impeded by a lack of fundamental knowledge of the genes and pathways involved. In adult bivalves, shells are secreted by the mantle tissue during growth, maintenance and repair, with the repair process, in particular, amenable to experimental dissection at the transcriptomic level in individual animals. RESULTS: Gene expression dynamics were explored in the adult blue mussel, Mytilus edulis, during experimentally induced shell repair, using the two valves of each animal as a matched treatment-control pair. Gene expression was assessed using high-resolution RNA-Seq against a de novo assembled database of functionally annotated transcripts. A large number of differentially expressed transcripts were identified in the repair process. Analysis focused on genes encoding proteins and domains identified in shell biology, using a new database of proteins and domains previously implicated in biomineralization in mussels and other molluscs. The genes implicated in repair included many otherwise novel transcripts that encoded proteins with domains found in other shell matrix proteins, as well as genes previously associated with primary shell formation in larvae. Genes with roles in intracellular signalling and maintenance of membrane resting potential were among the loci implicated in the repair process. While haemocytes have been proposed to be actively involved in repair, no evidence was found for this in the M. edulis data. CONCLUSIONS: The shell repair experimental model and a newly developed shell protein domain database efficiently identified transcripts involved in M. edulis shell production. In particular, the matched pair analysis allowed factoring out of much of the inherent high level of variability between individual mussels. This snapshot of the damage repair process identified a large number of genes putatively involved in biomineralization from initial signalling, through calcium mobilization to shell construction, providing many novel transcripts for future in-depth functional analyses.

The explosive trinitrotoluene (TNT) induces gene expression of carbonyl reductase in the blue mussel (Mytilus spp.): a new promising biomarker for sea dumped war relicts?

Millions of tons of all kind of munitions, including mines, bombs and torpedoes have been dumped after World War II in the marine environment and do now pose a new threat to the seas worldwide. Beside the acute risk of unwanted detonation, there is a chronic risk of contamination, because the metal vessels corrode and the toxic and carcinogenic explosives (trinitrotoluene (TNT) and metabolites) leak into the environment. While the mechanism of toxicity and carcinogenicity of TNT and its derivatives occurs through its capability of inducing oxidative stress in the target biota, we had the idea if TNT can induce the gene expression of carbonyl reductase in blue mussels. Carbonyl reductases are members of the short-chain dehydrogenase/reductase (SDR) superfamily. They metabolize xenobiotics bearing carbonyl functions, but also endogenous signal molecules such as steroid hormones, prostaglandins, biogenic amines, as well as sugar and lipid peroxidation derived reactive carbonyls, the latter providing a defence mechanism against oxidative stress and reactive oxygen species (ROS). Here, we identified and cloned the gene coding for carbonyl reductase from the blue mussel Mytilus spp. by a bioinformatics approach. In both laboratory and field studies, we could show that TNT induces a strong and concentration-dependent induction of gene expression of carbonyl reductase in the blue mussel. Carbonyl reductase may thus serve as a biomarker for TNT exposure on a molecular level which is useful to detect TNT contaminations in the environment and to perform a risk assessment both for the ecosphere and the human seafood consumer.

Doubly Uniparental Inheritance of mtDNA: An Unappreciated Defiance of a General Rule.

We recount the basic observations about doubly uniparental inheritance (DUI) of mtDNA in bivalvian mollusks with an emphasis on those that were obtained from work in Mytilus and appeared after the review by Zouros (Evol Biol 40:1-31, 2013). Using this information, we present a new model about DUI that is a revised version of previously suggested models. The model can be summarized as follows. A Mytilus female either provides its eggs with the "masculinizing" factor S and the "sperm mitochondria binding" factor Z, or it does not. This property of the female is determined by two nuclear genes, S and Z, that are always in the on/on or the off/off phase. In fertilized eggs without factors S and Z the embryo develops into a female and the sperm mitochondria are randomly dispersed among cells following development. In fertilized eggs with factors S and Z, the first factor causes the cell to become eventually sperm and the second causes the sperm mitochondria to aggregate and anchor to the nuclear membrane by binding to a specific motif of the sperm-derived mtDNA. Factors S and Z are continuously co-synthesized and co-localized in the cell line from the egg to the sperm. The sperm mitochondria of the aggregate escape the mechanism that eliminates the cell's mitochondria before the formation of the sperm. The rescued mitochondria are subsequently packed into five mega-mitochondria in the sperm and are delivered in the egg.

Ralstonia eutropha, containing high poly-ß-hydroxybutyrate levels, regulates the immune response in mussel larvae challenged with Vibrio coralliilyticus.

Marine invertebrates rely mainly on innate immune mechanisms that include both humoral and cellular responses. Antimicrobial peptides (AMPs), lysozyme and phenoloxidase activity, are important components of the innate immune defense system in marine invertebrates. They provide an immediate and rapid response to invading microorganisms. The impact of amorphous poly-ß-hydroxybutyrate (PHB-A) (1 mg PHB-A L-1) on gene expression of the AMPs mytimycin, mytilinB, defensin and the hydrolytic enzyme lysozyme in infected blue mussel larvae was investigated during "in vivo" challenge tests with Vibrio coralliilyticus (105 CFU mL-1). RNAs were isolated from mussel larvae tissue, and AMPs were quantified by q-PCR using the 18srRNA gene as a housekeeping gene. Our data demonstrated that AMPs genes had a tendency to be upregulated in challenged mussel larvae, and the strongest expression was observed from 24 h post-exposure onwards. The presence of both PHB-A and the pathogen stimulated the APMs gene expression, however no significant differences were noticed between treatments or between exposure time to the pathogen V. coralliilyticus. Looking at the phenoloxidase activity in the infected mussels, it was observed that the addition of PHB-A significantly increased the activity.

Dual transcriptomics reveals co-evolutionary mechanisms of intestinal parasite infections in blue mussels Mytilus edulis.

On theoretical grounds, antagonistic co-evolution between hosts and their parasites should be a widespread phenomenon but only received little empirical support so far. Consequently, the underlying molecular mechanisms and evolutionary steps remain elusive, especially in nonmodel systems. Here, we utilized the natural history of invasive parasites to document the molecular underpinnings of co-evolutionary trajectories. We applied a dual-species transcriptomics approach to experimental cross-infections of blue mussel Mytilus edulis hosts and their invasive parasitic copepods Mytilicola intestinalis from two invasion fronts in the Wadden Sea. We identified differentially regulated genes from an experimental infection contrast for hosts (infected vs. control) and a sympatry contrast (sympatric vs. allopatric combinations) for both hosts and parasites. The damage incurred by Mytilicola infection and the following immune response of the host were mainly reflected in cell division processes, wound healing, apoptosis and the production of reactive oxygen species (ROS). Furthermore, the functional coupling of host and parasite sympatry contrasts revealed the concerted regulation of chitin digestion by a Chitotriosidase 1 homolog in hosts with several cuticle proteins in the parasite. Together with the coupled regulation of ROS producers and antagonists, these genes represent candidates that mediate the different evolutionary trajectories within the parasite's invasion. The host-parasite combination-specific coupling of these effector mechanisms suggests that underlying recognition mechanisms create specificity and local adaptation. In this way, our study demonstrates the use of invasive species' natural history to elucidate molecular mechanisms of host-parasite co-evolution in the wild.

Linking genotype to phenotype in a changing ocean: inferring the genomic architecture of a blue mussel stress response with genome-wide association.

A key component to understanding the evolutionary response to a changing climate is linking underlying genetic variation to phenotypic variation in stress response. Here, we use a genome-wide association approach (GWAS) to understand the genetic architecture of calcification rates under simulated climate stress. We take advantage of the genomic gradient across the blue mussel hybrid zone (Mytilus edulis and Mytilus trossulus) in the Gulf of Maine (GOM) to link genetic variation with variance in calcification rates in response to simulated climate change. Falling calcium carbonate saturation states are predicted to negatively impact many marine organisms that build calcium carbonate shells - like blue mussels. We sampled wild mussels and measured net calcification phenotypes after exposing mussels to a 'climate change' common garden, where we raised temperature by 3°C, decreased pH by 0.2 units and limited food supply by filtering out planktonic particles >5 µm, compared to ambient GOM conditions in the summer. This climate change exposure greatly increased phenotypic variation in net calcification rates compared to ambient conditions. We then used regression models to link the phenotypic variation with over 170 000 single nucleotide polymorphism loci (SNPs) generated by genotype by sequencing to identify genomic locations associated with calcification phenotype, and estimate heritability and architecture of the trait. We identified at least one of potentially 2-10 genomic regions responsible for 30% of the phenotypic variation in calcification rates that are potential targets of natural selection by climate change. Our simulations suggest a power of 13.7% with our study's average effective sample size of 118 individuals and rare alleles, but a power of >90% when effective sample size is 900.

Single and repetitive microplastics exposures induce immune system modulation and homeostasis alteration in the edible mussel Mytilus galloprovincialis.

Seashore invertebrates such as mussels are exposed to multiple bouts of pollution related to human activities. Plastic debris originating from land-based activities are a concerning issue as they may be fragmented in smaller pieces (microplastics, < 5 mm diameter) which have an excellent potential for uptake by a large variety of animals. Here, we set out to explore the whole transcriptome profiling of Mytilus galloprovincialis associated with temporal variability of microplastics concentrations. Mussels were submitted to (i) a single 18 days-exposure to a concentration of microplastics found during pollution events (4.6 E+5 polyethylene microbeads L-1), (ii) a recovery period to investigate the reversibility of microplastics effects and (iii) a repeated exposure to microplastics to evidence acclimation to microplastics pollution events. Overall, 18 days-exposure to microplastics was mostly associated with disruption of mussel global homeostasis resulting in the production of stress and immune-related proteins and as a consequence, a diminution of energy allocated to growth. During the recovery period, a contrasting response was observed with the activation of apoptotic processes and the up-regulation of immune-receptors and stress-related proteins (glutathione peroxidase, hsp70) in mussels previously exposed to microplastics. These divergent responses, suggest that the establishment of compensatory mechanism as an attempt to recover, is not sufficient to counteract physiological stress induced by the first exposure. Finally, the differences observed in gene expression between single and repeated exposures to microplastics suggest, under the experimental conditions tested, that mussels may be able to establish a stress-memory upon microplastics exposure.

Genomic abnormalities affecting mussels (Mytilus edulis-galloprovincialis) in France are related to ongoing neoplastic processes, evidenced by dual flow cytometry and cell monolayer analyses.

In the context of the abnormal mass mortality of mussels in France since 2014, Flow CytoMetry (FCM) was used in 2015 and 2016 to study the DNA content and cell cycle characteristics of hemic circulating cells collected from 2000 mussels. The mussels were sampled from 12 wild and cultivated blue mussels stocks distributed along the French Atlantic coast from the south Brittany to Pertuis Charentais areas. During these surveys, various genetic abnormalities were frequently detected, and ploidy characteristics revealed contrasting profiles that corresponded to respective contrasting sanitary status, i.e. healthy mussels with high cytogenetic quality (HCQ) versus diseased mussels with low cytogenetic quality (LCQ). In the present work, FCM and hemocytology cell monolayer techniques were combined in order to determine the putative causes of the observed genetic abnormalities that were significantly associated with mortality levels. FCM and cell monolayer approaches permitted the definition of new threshold values delimiting HCQ mussels from LCQ ones. FCM histograms of mussels from the HCQ group showed one single or a largely dominant population of diploid (2n) nuclei and a large majority of normal hemocytes. Hemolymph cell-monolayer analyses showed predominantly acidophil granulocytes characterized by nuclei of normal size and a large cytoplasm with numerous granulations. In contrast, FCM histograms for the LCQ group showed, in addition to the normal diploid (2n) nuclei, populations of nuclei that displayed aneuploidy patterns in a broad ploidy range, including diploid-triploid (2-3n), tetraploid-pentaploid (4-5n) and heptaploid-octaploid levels (7-8n). The corresponding hemolymph cell-monolayer showed cellular features characteristic of disseminated neoplasia disease with frequent abnormal anaplastic cells that exhibited noticeable numbers of mitotic figures with both normal and aberrant chromosomes segregation patterns. These neoplastic cells were a rounded shape with a reduced, granulation-free cytoplasm and large (11-12 µm) to very large (up to 21 µm) round or ovoid nuclei that correspond to the 4-5n and 7-8n nuclei previously detected by FCM analyses. These characteristics suggest that the genetic abnormalities detected by means of FCM were related to an ongoing neoplastic process that is affecting blue mussels in France, at least since the onset in 2014 of the mortality that heavily impacted French blue mussels stocks.

Functional and molecular responses of the blue mussel Mytilus edulis' hemocytes exposed to cadmium - An in vitro model and transcriptomic approach.

The bivalve mollusk, Mytilus edulis, is used as a sentinel species in several monitoring programs due to its ability to bio-accumulate contaminants. Its immune system consists of hemocytes and humoral components, which constitute the main part of the hemolymph. The present study is aimed at understanding the effects of Cd on the differentially expressed genes involved in the phagocytosis of M. edulis' hemocytes. Our approach focuses on an in vitro model by exposing hemocytes to different concentrations of Cd ranging from 10-9 M to 10-3 M. Phagocytosis and cell viability as functional markers were measured using flow cytometry. The molecular mechanisms regulated by Cd were investigated using RNA-seq and DGE analysis. Results showed that viability and phagocytosis of hemocytes exposed to 10-3 M of Cd were significantly decreased after 21 h of exposure. RNA sequencing data showed that 1112 transcripts (out of 352,976 contigs) were differentially regulated by the highest concentration of Cd. Among these identified transcripts, 1028 and 84 were up and down-regulated respectively. The induction of super oxide dismutase (SOD), glutathion-s-transferase (GST), cytochrome P450 2C8 (CYP2C8), multidrug resistance protein (MRP1) and heat shock protein 70 (HSP70) suggests that Cd can regulate key molecular mechanisms. In addition, several toll-like receptors (TLR) as well as genes involved in phagocytosis (actin and CDC42) and apoptosis (caspase 8 and XIAP/IAP) were induced by Cd. Thus, our model highlights the effect of Cd on the phagocytic function of M. edulis' hemocytes along with the regulation of gene expression involved in innate immunity, detoxification and apoptosis. Further investigations need to be pursued to unravel the effects of Cd on the molecular mechanisms identified in this study.

Baseline and oxidative DNA damage in marine invertebrates.

Anthropogenic pollutants produce oxidative stress in marine organisms, directly or following generation of reactive oxygen species (ROS), potentially resulting in increased accumulation of DNA strand breaks quantified. The aim of this study is to quantify baseline levels of DNA strand breaks in marine species from four phyla and to assess relative sensitivity to oxidative stress as well as ability to recover. DNA strand breaks were determined using a formamidopyrimidine DNA glycosylase (Fpg)-amended comet assay in circulating cells from blue mussel (Mytilus edulis), shore crab (Carcinus maenas), sea star (Asterias rubens), and vase tunicate (Ciona intestinalis). Lymphocytes from Atlantic cod (Gadus morhua) were used as a reference. In addition to immediate analysis, cells from all species were exposed ex vivo to two concentrations of hydrogen peroxide (H2O2) at 25 or 250 µM prior to assay. Mean baseline DNA strand breaks were highest for cells from sea star (34%) followed by crab (25%), mussel (22%), tunicate (17%), and cod (14%). Circulating cells from invertebrates were markedly more sensitive to oxidative stress compared to cod lymphocytes. DNA strand breaks exceeded 80% for sea star, crab, and mussel cells following exposure to the lowest H2O2 concentration. There was no recovery for cells from any species following 1 hr in buffer. This study provides an in-depth analysis of DNA integrity for ecologically important species representing 4 phyla. Data indicate that circulating cells from invertebrates are more sensitive to oxidative stress than cells from fish as evidenced by DNA strand breaks. Future studies need to address the extent to which DNA strand breaks may exert consequences for body maintenance costs in marine invertebrates.

Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis).

Here, we aimed to investigate potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, as gills are directly facing seawater and the changing pH (predicted to be reduced from ∼8.1 to ∼7.7 by 2100). The AMP activity of gill and haemocyte extracts was compared at pH 6.0, 7.7 and 8.1, with a radial diffusion assay against Escherichia coli. The activity of the gill extracts was not affected by pH, while it was significantly reduced with increasing pH in the haemocyte extracts. Gill extracts were also tested against different species of Vibrio (V. parahaemolyticus, V. tubiashii, V. splendidus, V. alginolyticus) at pH 7.7 and 8.1. The metabolic activity of the bacteria decreased by ∼65-90%, depending on species of bacteria, but was, as in the radial diffusion assay, not affected by pH. The results indicated that AMPs from gills are efficient in a broad pH-range. However, when mussels were pre-exposed for pH 7.7 for four month the gill extracts presented significantly lower inhibit of bacterial growth. A full in-depth proteome investigation of gill extracts, using LC-Orbitrap MS/MS technique, showed that among previously described AMPs from haemocytes of Mytilus, myticin A was found up-regulated in response to lipopolysaccharide, 3 h post injection. Sporadic occurrence of other immune related peptides/proteins also pointed to a rapid response (0.5-3 h p.i.). Altogether, our results indicate that the gills of blue mussels constitute an important first line defence adapted to act at the pH of seawater. The antimicrobial activity of the gills is however modulated when mussels are under the pressure of ocean acidification, which may give future advantages for invading pathogens.

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.

Since 2014, France's blue mussel industry has been facing heavy mortality outbreaks (90-100%) affecting both juveniles and adults. This report presents evidence of heavy genomic abnormalities associated with mortality outbreaks in blue mussels, Mytilus edulis-galloprovincialis, from the Atlantic coast of France. In this study, ploidy characteristics of hemic cells were investigated using Flow CytoMetry (FCM), revealing an unusual, broad continuum of ploidy distribution from hypodiploidy to tetraploidy. FCM was additionally used to evaluate, at individual and populations levels, different thresholds of genomic abnormality (GA%) using the percentage of non-diploid nuclei. Individual mussels were considered to be abnormal when more than 10% of hemocytes in S-G2/M phase were present. At the population level, a threshold of 6% for the mean intensity of the abnormality is proposed, which means in the population, more than 6% of individual mussels have to present with more than 10% of their hemocytes in S-G2/M phase. GA% was found to be significantly predictive of the final mortality. Based on the established thresholds, only two mussel stocks analyzed in this study were considered to have good cytogenetic quality, while all other stocks appeared to be affected. FCM offers a very powerful tool to help manage current blue mussel mortality in France. We also believe that annual and extensive determination of cytogenetic quality of wild and cultivated mussel beds along with exclusive use of FCM-qualified mussel seeds should be a priority.

Proteomic analysis of eggs from Mytilus edulis females differing in mitochondrial DNA transmission mode.

Many bivalves have an unusual mechanism of mitochondrial DNA (mtDNA) inheritance called doubly uniparental inheritance (DUI) in which distinctly different genomes are inherited through the female (F genome) and male (M genome) lineages. In fertilized eggs that will develop into male embryos, the sperm mitochondria remain in an aggregation, which is believed to be delivered to the primordial germ cells and passed to the next generation through the sperm. In fertilized eggs that will develop into female embryos, the sperm mitochondria are dispersed throughout the developing embryo and make little if any contribution to the next generation. The frequency of embryos with the aggregated or dispersed mitochondrial type varies among females. Previous models of DUI have predicted that maternal nuclear factors cause molecular differences among unfertilized eggs from females producing embryos with predominantly dispersed or aggregated mitochondria. We test this hypothesis using females of each of the two types from a natural population. We have found small, yet detectable, differences of the predicted type at the proteome level. We also provide evidence that eggs of females giving the dispersed pattern have consistently lower expression for different proteasome subunits than eggs of females giving the aggregated pattern. These results, combined with those of an earlier study in which we used hatchery lines of Mytilus, and with a transcriptomic study in a clam that has the DUI system of mtDNA transmission, reinforce the hypothesis that the ubiquitin-proteasome system plays a key role in the mechanism of DUI and sex determination in bivalves. We also report that eggs of females giving the dispersed pattern have higher expression for arginine kinase and enolase, enzymes involved in energy production, whereas ferritin, which is involved in iron homeostasis, has lower expression. We discuss these results in the context of genetic models for DUI and suggest experimental methods for further understanding the role of these proteins in DUI.

The development and implementation of a method using blue mussels (Mytilus spp.) as biosentinels of Cryptosporidium spp. and Toxoplasma gondii contamination in marine aquatic environments.

Surveillance monitoring for microbial water quality typically involves collecting single discrete grab samples for analyzing only one contaminant. While informative, current approaches suffer from poor recoveries and only provide a limited snapshot of the microbial contaminants only at the time of collection. To overcome these limitations, bivalves have been proposed as effective biosentinels of water quality particularly for their ability to efficiently concentrate and retain microbial contaminants for long periods of time. In this study, we examined the use of indigenous blue mussels (Mytilus spp.) as biosentinels to monitor for the presence of Toxoplasma gondii and Cryptosporidium water. An efficient method to extract oocyst DNA from various mussel tissues followed by PCR-based detection of these pathogens was developed, which resulted in the detection down to 10 oocysts. This method was then used to conduct a small survey in Point Lobos and Morro Bay, California to determine prevalence T. gondii and Cryptosporidium. Results revealed that mussels from Morro Bay were contaminated with T. gondii (33 %), while mussels from Point Lobos were contaminated with T. gondii (54 %) and Cryptosporidium (26.9 %) oocysts. Phylogenetic analysis using the SSU rRNA gene identified two novel Cryptosporidium parvum-like genotypes. Overall, this study demonstrated the application of using native California Mytilus spp. as biosentinels for pathogen contamination along the central California shorelines. More importantly, T. gondii and Cryptosporidium were found at higher prevalence rates in Morro Bay and in Point Lobos, an area not previously reported to be contaminated with these pathogens.