Marteilia pararefringens infections are more frequent than revealed by the Norwegian surveillance programme, highlighting the need for its improvement.

Norway had historically been considered free of marteiliosis in bivalves since the disease surveillance programme began in 1995. However, in 2016, Marteilia pararefringens, a protistan parasite of mussels Mytilus spp., was described in a heliothermic lagoon-a poll-previously used to produce flat oyster spat. To study whether the parasite was introduced, and possibly spread, via the historical flat oyster networks on the south and west coast, we sampled aquaculture polls that were part of different networks of farmers and wild, natural polls with no aquaculture activity. Additionally, we sampled mussel banks influenced by polls and sheltered bays that could have a similar environment to that of polls. We identified 7 sites with M. pararefringens-infected mussel populations: 5 were polls used in flat oyster production and 2 were in fjord areas with no known connection to any bivalve aquaculture. Prevalence ranged between 2 and 88%. At one site, Trysfjorden, we found M. pararefringens in atypical organs, including the gills, mantle, and intestine. Marteilia-like cells were also observed in the epithelium, lumen, and surrounding connective tissue of metanephridia and in the sinus of the anterior retractor muscle. Our results demonstrate that the parasite is more widespread than previously thought and is neither isolated to polls nor connected directly to aquaculture activity. Lastly, our findings highlight the need for an improved sampling strategy in surveillance programmes to detect marteiliosis in mussels.

Using marine mussels to assess the potential ecotoxicological effects of two different commercial microplastics.

Microplastics (MPs) in the aquatic environment pose a serious threat to biota, by being confounded with food. These effects occur in mussels which are filter-feeding organisms. Mussels from the genus Mytilus sp. were used to evaluate the ecotoxicological effects of two MPs, polypropylene (PP) and polyethylene terephthalate (PET), after 4 and 28-days. Measured individual endpoints were condition index and feeding rate; and sub-individual parameters, metabolism of phase I (CYP1A1, CYP1A2 and CYP3A4) and II (glutathione S-transferases - GSTs), and antioxidant defense (catalase - CAT). MPs decreased both condition index (CI) and feeding rate (FR). No alterations occurred in metabolic enzymes, suggesting that these MPs are not metabolized by these pathways. Furthermore, lack of alterations in GSTs and CAT activities suggests the absence of conjugation and oxidative stress. Overall, biochemical markers were not responsive, but non-enzymatic responses showed deleterious effects caused by these MPs, which may be of high ecological importance.

Using bait microalga as an oral delivery vehicle of antimicrobial peptide for controlling Vibrio infection in mussels.

In shellfish aquaculture, antibiotics are commonly used to address Vibrio infections. However, antibiotic abuse has increased the risk of environment pollution, which has also raised food safety concerns. Antimicrobial peptides (AMPs) are considered safe and sustainable alternatives to antibiotics. Hence, in this study, we aimed to develop a transgenic Tetraselmis subcordiformis line harboring AMP-PisL9K22WK for reducing the use of antibiotics in mussel aquaculture. Toward this, pisL9K22WK was assembled into nuclear expression vectors of T. subcordiformis. Post particle bombardment, several stable transgenic lines were selected after 6 months of herbicide resistance culture. Subsequently, Vibrio-infected mussels (Mytilus sp.) were orally fed transgenic T. subcordiformis to test the efficacy of this drug delivery system. The results showed that the transgenic line as an oral antimicrobial agent significantly improved the resistance of mussels to Vibrio. The growth rate of the mussels fed transgenic T. subcordiformis was considerably higher than that of mussels fed wild-type algae (10.35% versus 2.44%). In addition, the possibility of using the lyophilized powder of the transgenic line as drug delivery system was also evaluated; however, compared to that observed after feeding with live cells, the lyophilized powder did not improve the low growth rate caused by Vibrio infection, suggesting that fresh microalgae are more beneficial for the delivery of the PisL9K22WK to mussels than the lyophilized powder. In summary, this is a promising step toward the development of safe and environment-friendly antimicrobial baits.

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.

A case study of PAH contamination using blue mussels as a bioindicator in a small Greenlandic fishing harbor.

This study investigated the impact of local anthropogenic activity on the marine environment around the remote harbor of Qeqertarsuaq, West Greenland. Blue mussels (Mytilus sp.) were used as a bioindicator, and their physiological condition was found to decrease with increasing proximity to the harbor. Subsequently, the distribution of 19 polycyclic aromatic hydrocarbons (PAHs) and 9 groups of alkylated PAHs were measured in mussel and sediment samples. The highest values were found in a rocky collection area 15 m from a wooden pier frequented by small boats. A PAH source investigation, indicated a mixed source from light fuel oils and creosote used as boat coating. Finally, correlations between the mussels morphological condition and the PAH pollution were found to be significant for 4-, 5-, and 6-ring PAHs. In conclusion, the results indicate that pollution sources in harbors have significant effects on the local environment and should be considered in arctic conservation research.

Molecular response of a sub-antarctic population of the blue mussel (Mytilus edulis platensis) to a moderate thermal stress.

The Kerguelen Islands (49°26'S, 69°50'E) represent a unique environment due to their geographical isolation, which protects them from anthropogenic pollution. The ability of the endemic mussel, part of the Mytilus complex, to cope with moderate heat stress was explored using omic tools. Transcripts involved in six major metabolic functions were selected and the qRT-PCR data indicated mainly changes in aerobic and anaerobic energy metabolism and stress response. Proteomic comparisons revealed a typical stress response pattern with cytoskeleton modifications and elements suggesting increased energy metabolism. Results also suggest conservation of protein homeostasis by the long-lasting presence of HSP while a general decrease in transcription is observed. The overall findings are consistent with an adaptive response to moderate stresses in mussels in good physiological condition, i.e. living in a low-impact site, and with the literature concerning this model species. Therefore, local blue mussels could be advantageously integrated into biomonitoring strategies, especially in the context of Global Change.

Natural distribution of pure and hybrid Mytilus sp. along the south Mediterranean and North-east Atlantic coasts and sensitivity of D-larvae stages to temperature increases and metal pollution.

The distribution of the Mediterranean mussel Mytilus galloprovincialis extends more and more northwards in the Atlantic. Crossings are frequently observed with the blue mussel Mytilus edulis along the French and English coasts. The aim of this study is firstlyto identify the co-presence of M. galloprovincialis, M. edulis, and their hybrids in different sites of the Mediterranean and Atlantic coasts, and to provide insights for the thermal tolerance and toxicant susceptibility of Mytilus edulis, Mytilus galloprovincialis and their hybrids. Mussels were collected from the shore at 20 sampling sitesin Europe and Tunisia and identified using Me 15/16 primers targeting the adhesive protein gene sequence. Samples were screened for the presence of Mytilus edulis, Mytilus galloprovincialis, and hybrids alleles using PCR. To get more information on hybrids sensitivities to temperature and metals, freshly fertilized eggs of the two species and their hybrids were reared at four temperatures 18, 20, 22, and 24 °C and exposed to concentrations of Cu, Ag, and a mixture of both metals. Arrests of development and malformations were recorded after 48 h of exposure. The genotypic identification of the two species on 20 sites of the Mediterranean and Atlantic coasts carried out during this study confirms the presence of pure and hybrid species of mussel. Our results highlighted that hybrid larvae from a female of M. galloprovincialis are significantly more tolerant to temperature increases than pure larvae of M. galloprovincialis and pure and hybrid larvae of M. edulis. No significant interspecies-differences of sensitivity were noted for metal exposure alone. However, a co-exposure of larvae to both metal and high temperature highlighted the higher tolerance of hybrid larvae from a female of M. galloprovincialis to both stresses. The overall results could allow the prediction of the future evolution of mussel populations facing environmental changes.

Linking individual and population patterns of rocky-shore mussels.

Individual traits and population parameters can be used as proxies of processes taking place within a range of scales, thus improving the way we can evaluate species response to environmental variability. In intertidal rocky shores, patterns at the within-site scale, i.e., between centimeters to hundreds of meters, are important for understanding the population response into these highly variable environments. Here, we studied a rocky-shore mussel population at the within-site spatial scale (1) to test how intertidal height and orientation of the shore affect individual traits and population parameters, (2) to infer the link between individual and population level features, and (3) to explore the upscaling mechanisms driving population structure and processes. We analyzed the patterns of six population parameters: density, biomass, crowding, median individual size, recruitment and mortality rate, and four individual traits: growth rate, spawning phenology, size and condition index. Crowding was defined as the degree of overlapping of individuals within a given area, for which we created a "crowding index". Mussels were studied along the intertidal height gradient in two rocky shores with contrasted orientation at one site over a full year. Our results showed a significant effect of intertidal height and shore orientation on most of individual traits and population parameters studied. In contrast, biomass contained in a full covered surface did not vary in space nor in time. This pattern likely results from relatively constant crowding and a trade-off between median individuals' size and density. We hypothesize that growth, mortality and recruitment rates may all play roles in the stability of the crowding structure of mussel aggregations. Variation in spawning phenology between the two shores in the study site was also observed, suggesting different temporal dynamics of microclimate conditions. Interestingly, despite the different population size distribution between the two shores, our estimates indicate similar potential reproductive output. We hypothesize that the structure of the patches would tend to maintain or carry a maximum of biomass due to trade-offs between density and size while maintaining and maximizing the reproductive output. The patterns of spatial variability of individual traits and population parameters in our study site suggest that heterogeneous within-site conditions influence variation in individual performance and population processes. These results provide insights about the relationship between individual traits and how these relationships make patterns at the population level emerge. They provide baseline information necessary to improve models of metapopulation with spatially explicit processes.

Growth and behaviour of blue mussels, a re-emerging polar resident, follow a strong annual rhythm shaped by the extreme high Arctic light regime.

Polar regions are currently warming at a rate above the global average. One issue of concern is the consequences on biodiversity in relation to the Northward latitudinal shift in distribution of temperate species. In the present study, lasting almost two years, we examined two phenological traits, i.e. the shell growth and behavioural rhythm of a recently re-established species in the high Arctic, the blue mussel Mytilus sp. We compared this with a native species, the Islandic scallop Chlamys islandica. We show marked differences in the examined traits between the two species. In Mytilus sp., a clear annual pattern of shell growth strongly correlated to the valve behaviour rhythmicity, whereas C. islandica exhibited a shell growth pattern with a total absence of annual rhythmicity of behaviour. The shell growth was highly correlated to the photoperiod for the mussels but weaker for the scallops. The water temperature cycle was a very weak parameter to anticipate the phenology traits of both species. This study shows that the new resident in the high Arctic, Mytilus sp., is a highly adaptive species, and therefore a promising bioindicator to study the consequences of biodiversity changes due to global warming.

First ecotoxicological characterization of paraffin microparticles: a biomarker approach in a marine suspension-feeder, Mytilus sp.

Microplastics are one of the main environmental pollutants in marine ecosystems, and their presence in seawater is a consequence of the widespread use of plastic materials in modern commodities. This wide usage of plastics includes the employment of microspheres in common personal care products, which end up being ultimately released into the aquatic compartment. Known ecotoxicological effects of microplastics favoured the search for technologically viable and environmentally safer alternatives, such as paraffin wax microparticles, whose ecotoxicological risks have not been entirely characterized. To address this gap, the present study exposed mussels (Mytilus sp.) for 96 h to three densities (5 mg/L, 20 mg/L and 80 mg/L) of four size ranges (100-300 µm, 300-500 µm, 400-850 µm, and 800-1200 µm) of paraffin wax particles. Toxicological endpoints were the activities of four enzymes involved in key cellular processes, including antioxidant defence (catalase (CAT), glutathione reductase (GRed) and glutathione peroxidase (GPx)) and phase II metabolism (glutathione S-transferases (GSTs)), as well as lipid content and fatty acid profiles of the digestive gland. Significant interactions between the presence/absence of food and paraffin particle density were found, as food sometimes worked as a confounding factor in the analysed biomarkers. Despite this uncertainty, some overall patterns emerged. In general, smaller paraffin wax particles (100-300 µm) caused little effects on the activity of the four enzymes tested, whereas larger particles (800-1200 µm) caused significant effects on almost all biomarkers. CAT activity was enhanced in animals exposed to larger paraffin particles, whilst GPx activity was depressed; GRed activity was not affected by the exposure to paraffin particles. The activity of GSTs was enhanced, but only in one tested condition. No effects were observed in terms of the total lipid content and fatty acids of exposed animals. Overall, data obtained in this work suggest that, at densities of paraffin wax particles comparable to the levels found in the environment for microplastic beads, no toxicity is expected to occur in the tested mussel species, by measuring the here-assessed toxicological endpoints.

Molecular mechanisms underlying the effects of temperature increase on Mytilus sp. and their hybrids at early larval stages.

The present work aims to investigate the effects of water temperature increase on Mytilus galloprovincilis and Mytilus edulis pure larvae (PG, PE) and their hybrids (HFG, HFE). D-larvae were maintained at 18 °C or exposed to a higher temperature of 22 °C for 48 h. Initially, Embryotoxicity test was evaluated. Second, a transcriptomic analysis using a recently developed microarray platform was applied to determine the main biological processes involved in early life stages responses to temperature increase. Finally, an immunofluorescence investigation was performed to bridge the gap between transcriptomic regulation and the real changes at cellular/tissue levels. Embryotoxicity test revealed a higher sensitivity of M. edulis (PE) D-larvae as well as hybrids from females M. edulis (HFE) to temperature increase, with the highest rate of larval malformations. Transcriptomic results indicated a lack of an adequate heat shock protein (Hsp) response in PE and HFE larvae (the high expression was observed in PG larvae); the differential expression of gene involved in translation, energy metabolism and oxidative stress response may contribute to explain the observed complex alterations in the studied conditions. As revealed by immunohistochemistry, cytoskeleton proteins changes associated with a drastic decrease of Histidine-Rich Glycoprotein (HRG) may elucidate the larval abnormalities in shell development observed for PE and HFE larvae. Overall, the results indicate that each type of pure larva (PG and PE) and their respective female hybrid (HFG and HFE) react similarly to the temperature increase. Our data should be carefully considered in view of the water temperature increase in marine ecosystems and especially for the mussel's species in confluence zones.

Biomarkers of general stress in mussels as common indicators for marine biomonitoring programmes in Europe: The ICON experience.

This study investigated whether general stress biomarkers in mussels can be applied as common first-tier biomarkers in regional biomonitoring programmes in the North Sea (including Iceland) and western Mediterranean Sea. Stress on Stress (SoS) and lysosomal membrane stability (LMS) biomarkers were analysed in resident mussels (Mytilus sp.) from 8 coastal sites and in transplanted mussels (Mytilus galloprovincialis) from two Spanish Mediterranean coastal sites. The assessment of results, as input to pollution monitoring strategies, was performed jointly for LMS and SoS data from the two regions. Contaminant body burden of the mussels was compared with biomarker results. The results demonstrated that these two general and non-expensive stress biomarkers in mussel can be applied throughout European waters, providing a cost-effective and harmonised approach to screen contaminant-related biological effects within the framework of wide-scale pollution biomonitoring programmes, such as that proposed by the European Union, i.e. the Marine Strategy Framework Directive.

A sensory and nutritional comparison of mussels (Mytilus sp.) produced in NW Iberia and in the Armona offshore production area (Algarve, Portugal).

A biometric, nutritional and sensory analysis of raw and cooked mussels comparing Mytilus sp. from the north-west coast of Portugal and Spain (Minho and Galicia, respectively) and the new offshore production site of Armona (Algarve, south Portugal) was carried out. In addition, multiple factorial analysis was performed to explore potential relationships between sensory attributes and nutritional content properties between the different mussels. Results showed that, at similar times of sale, biometrics of mussels from Armona and Vigo were similar and bigger than the remaining. Nonetheless, despite some similarities in proximate composition, mussels presented differences in lipid classes, fatty acid content and free amino acids profiles. These differences were not fully reflected in the sensory assessment by the panel, which were able to distinguish different production sites in raw specimens but displayed problems in discrimination these in cooked mussels. Some nutritional components were related to specific sensory sensations.

Copper bioavailability and toxicity to Mytilus galloprovincialis in Shelter Island Yacht Basin, San Diego, CA.

The bioavailability and toxicity of copper (Cu) in Shelter Island Yacht Basin (SIYB), San Diego, CA, USA, was assessed with simultaneous toxicological, chemical, and modeling approaches. Toxicological measurements included laboratory toxicity testing with Mytilus galloprovincialis (Mediterranean mussel) embryos added to both site water (ambient) and site water spiked with multiple Cu concentrations. Chemical assessment of ambient samples included total and dissolved Cu concentrations, and Cu complexation capacity measurements. Modeling was based on chemical speciation and predictions of bioavailability and toxicity using a marine Biotic Ligand Model (BLM). Cumulatively, these methods assessed the natural buffering capacity of Cu in SIYB during singular wet and dry season sampling events. Overall, the three approaches suggested negligible bioavailability, and isolated observed or predicted toxicity, despite an observed gradient of increasing Cu concentration, both horizontally and vertically within the water body, exceeding current water quality criteria for saltwater.