Picoeucaryot alga infecting blue mussel Mytilus edulis in southern Norway

During summer 2001, blue mussels Mytilus edulis with abnormal shell growth were collected near Krager?, southern Norway. The mussels had green spots in their mantle tissues, mainly posteriorly and ventrally, and in the adductor muscle. Mussels from 4 sites had a prevalence of green spots varying from 2 to 71% that correlated well with shell deformities. Histological examination revealed the presence of round or ovoid algae, 0.9 to 1.5 x 1.2 to 2.4 microm, free within haemocytes and in the lesions, characterised by an inflammatory response and the presence of cellular debris. The alga contain a relatively large nucleus, 1 chloroplast and 1 mitochondrion. Size and morphology suggest that the alga might be a picoeucaryot green alga. Infection of mussel tissues appears to start in the posterior mantle edge, near the siphons, and spread anterior-ventrally in the mantle connective and storage tissues-occasionally spots were also found in the gonad follicles. Large infected areas were also observed in sinuses within the adductor muscle. Only mussels that were 3 yr old or more were infected. Deformations apparently resulted from years of continuous shell formation by a contracted, partly deformed mantle. Most deformed mussels had eroded shells, allowing some light penetration through the exposed, thin nacre. Young, thin-shelled mussels were not infected. The present work suggests that the alga has, at least partially, a parasitic relationship with the mussels, and is associated with pathological alterations in mussel tissues.     

Multiple invertebrate lysozymes in blue mussel (Mytilus edulis)

Initial analyses of lysozyme activities in individual blue mussels Mytilus edulis indicated variations in features of activity from the crystalline style to the remaining body parts (the soft body). Two separate larger scale lysozyme isolations were performed employing extracts from 1000 styles and 50 soft bodies, respectively. The soft body origin contained one, or one major, lysozyme that was purified to homogeneity. This 13 kDa protein, designated bm-lysozyme, was sequence-analysed and found to represent the product of a recently published invertebrate-type lysozyme gene from M. edulis. Three additional lysozymes were isolated from the style extract and one of them was fully purified. All four lysozymes showed different profiles of enzymatic features such as responses to pH, ionic strengths and divalent cations. From the results and the profound differences demonstrated we believe that the observed multiple forms of lysozyme activities in blue mussel reflect multiple genes instead of individual lysozyme variants and that the lysozymes serve different functions in the blue mussel.     

Peroxisomal proteomic approach for protein profiling in blue mussels (Mytilus edulis) exposed to crude oil

Peroxisomal proteomic protein profiles of exposure to marine pollution have been recently introduced in biomonitoring experiments. However, laboratory experiments to study the independent effect of common pollutants are needed to define a minimal protein expression signature (PES) of exposure to a specific pollutant. The aim of this study was to obtain PESs in blue mussels (Mytilus edulis) exposed to two different crude oil mixtures for future application in biomonitoring areas affected by oil spills. In the study, peroxisome-enriched fractions from digestive gland of M. edulis (L., 1758) were analysed by two-dimensional fluorescence difference electrophoresis (DIGE) and mass spectrometry (MS) after 3 weeks of exposure to crude oil mixtures: crude oil or crude oil spiked with alkylated phenols (AP) and extra polycyclic aromatic hydrocarbons (PAH) in a laboratory flow-through system. A minimal PES composed by 13 protein spots and unique PESs of exposure to the two different mixtures were identified. A total of 22 spots from the two-dimensional maps that had shown a significant increase or decrease in abundance in each of the exposed groups exposed were analysed. The hierarchical clustering analysis succeeded in discriminating the exposed groups from the control groups based on the unique PES. The PESs obtained were consistent with protein patterns obtained in previous field experiments. The results suggest that the protein profiles obtained by peroxisomal proteomics could be used to assess oil exposure in marine pollution assessments.     

Peroxisomal proteomic approach for protein profiling in blue mussels (Mytilus edulis) exposed to crude oil

Peroxisomal proteomic protein profiles of exposure to marine pollution have been recently introduced in biomonitoring experiments. However, laboratory experiments to study the independent effect of common pollutants are needed to define a minimal protein expression signature (PES) of exposure to a specific pollutant. The aim of this study was to obtain PESs in blue mussels (Mytilus edulis) exposed to two different crude oil mixtures for future application in biomonitoring areas affected by oil spills. In the study, peroxisome-enriched fractions from digestive gland of M. edulis (L., 1758) were analysed by two-dimensional fluorescence difference electrophoresis (DIGE) and mass spectrometry (MS) after 3 weeks of exposure to crude oil mixtures: crude oil or crude oil spiked with alkylated phenols (AP) and extra polycyclic aromatic hydrocarbons (PAH) in a laboratory flow-through system. A minimal PES composed by 13 protein spots and unique PESs of exposure to the two different mixtures were identified. A total of 22 spots from the two-dimensional maps that had shown a significant increase or decrease in abundance in each of the exposed groups exposed were analysed. The hierarchical clustering analysis succeeded in discriminating the exposed groups from the control groups based on the unique PES. The PESs obtained were consistent with protein patterns obtained in previous field experiments. The results suggest that the protein profiles obtained by peroxisomal proteomics could be used to assess oil exposure in marine pollution assessments.     

Seasonal expression patterns of clock-associated genes in the blue mussel Mytilus edulis

Environmental cues allow organisms to synchronise their internal biological rhythms with external environmental cycles. These rhythms are regulated on a molecular level by oscillating interactions between clock genes and their proteins. Light is a particularly relevant environmental cue, provisioning daily information via light/dark cycles as well as seasonal information via day-length (photoperiod). Despite the ecological and commercial importance of bivalves, little is known about the interactions comprising their molecular clock mechanism. This study investigates the link between the annual seasonal progression and reproductive development in the blue mussel (Mytilus edulis), using mRNA expression patterns of clock-associated genes: Clock, Cry1¸ ARNT, Timeout-like, ROR/HR3 and aaNAT, in the gonads of both sexes, sampled over three daily time-points on a tidal beach during the winter and summer solstices. Significant differences in mRNA expression levels, including some seasonal differences at comparable time-points, were detected for all genes with the exception of Timeout-like. These differences occurred seasonally within sex (Clock, Cry1, ROR/HR3), seasonally between sexes (Clock, Cry1, ARNT, ROR/HR3, aaNAT) and daily between sexes (Cry1), although no significant daily differences were detected in summer or winter for either sex for any of the genes. This study reveals that clock-associated genes show seasonal responses in this species of bivalve. Understanding the mechanisms by which environmental cues drive biological rhythms is critical to understanding the seasonal sensitivity of this keystone species to environmental changes.     

AFLP-based genetic linkage maps of the blue mussel (Mytilus edulis)

We report the construction of the first genetic linkage map in the blue mussel, Mytilus edulis. AFLP markers were used in 86 full-sib progeny from a controlled pair mating, applying a double pseudo-test cross strategy. Thirty-six primer pairs generated 2354 peaks, of which 791 (33.6%) were polymorphic in the mapping family. Among those, 341 segregated through the female parent, 296 through the male parent (type 1:1) and 154 through both parents (type 3:1). Chi-square goodness-of-fit tests revealed that 71% and 73% of type 1:1 and 3:1 markers respectively segregated according to Mendelian inheritance. Sex-specific linkage maps were built with mapmaker 3.0 software. The female framework map consisted of 121 markers ordered into 14 linkage groups, spanning 862.8 cM, with an average marker spacing of 8.0 cM. The male framework map consisted of 116 markers ordered into 14 linkage groups, spanning 825.2 cM, with an average marker spacing of 8.09 cM. Genome coverage was estimated to be 76.7% and 75.9% for the female and male framework maps respectively, rising to 85.8% (female) and 86.2% (male) when associated markers were included. Twelve probable homologous linkage group pairs were identified and a consensus map was built for nine of these homologous pairs based on multiple and parallel linkages of 3:1 markers, spanning 816 cM, with joinmap 4.0 software.     

Nuclear and cytosolic distribution of metallothionein in the blue mussel Mytilus edulis L

Four tissues from the blue mussel, Mytilus edulis L., were examined for the presence of nuclear metallothionein (MT), and the nuclear:cytosolic (N:C) MT ratios and nuclear MT:DNA ratios investigated. Gill, digestive gland, gonad and posterior adductor muscle tissues were dissected, homogenized and subjected to differential centrifugation in order to isolate the nuclear and cytosolic fractions, which were then analyzed for MT and DNA. MT was present in all samples of the nuclear fractions from all four tissues. The nuclear MT concentration was either lower or the same as the cytosolic MT concentration from the same tissue. The mean N:C MT ratio of the digestive gland was significantly lower than that of the gill. The mean nuclear MT:DNA ratio of the digestive gland was significantly higher than that of the gill and posterior adductor muscle. In addition to being the first report of nuclear MT in bivalves, we showed that N:C MT ratios and nuclear MT:DNA ratios differ among tissues of the same organism. This raises important questions concerning the regulation of nuclear MT concentrations and the role of nuclear MT in metal regulation and DNA protection.     

Pharmacological induction of larval settlement and metamorphosis in the blue mussel Mytilus edulis L

The blue mussel Mytilus edulis L. is an important aquaculture and fouling species in northern seas. Although the general role of chemical cues for settlement of larvae of the blue mussel has been proposed, few studies have focused on induction of settlement and metamorphosis by pharmacological agents. In this study, the induction of larval settlement of the blue mussel by pharmacological compounds was investigated through a series of laboratory experiments with an aim of identifying artificial cues for laboratory bioassay systems in fouling and antifouling research. Gamma-aminobutiric acid (GABA), dihydroxyphenyl L-alanine (DOPA), isobutyl methylxanthine (IBMX) and acetylcholine chloride (ACH) at 10(-7)-10(-2) M as well as KCl at 10-40 mM K+ in excess of the level in normal seawater were tested for their inductive effect on larval settlement. In filtered seawater (FSW) < 9% of the larvae settled after 48 h. Elevated K+ and GABA levels had no effect on larval settlement and metamorphosis. DOPA at 10(-5) M and IBMX at 10(-6)-10(-4) M induced 41-83% larval settlement and ACH at 10(-7)-10(-5) M induced < 40% larval settlement. While the highest settlement rates were observed after 48 h exposure to the chemical, most of the larvae settled within 24 h. Compounds at concentrations of 10(-3)-10(-2) M were either toxic to larvae or retarded the growth of the post-larvae shell. Juveniles resulting from induction by lower concentrations of chemicals had a very high survival rate, completed metamorphosis and grew as well as the juveniles that metamorphosed spontaneously. IBMX at 10(-6)-10(-4) M and L-DOPA at 10(-5) M are effective agents for induction of settlement and metamorphosis for future studies using juvenile M. edulis.     

Protection against suspended sand: the function of the branchial membrane in the blue mussel Mytilus edulis

Blue mussels (Mytilus edulis) living in estuaries have to cope with varying concentrations of suspended sand. Sand flowing through the inhalant siphons comes into the infrabranchial chamber. The inhalant siphon can be partially closed by the branchial membrane. As a result the inward flow decreases, and suspended sand sinks and can be eliminated. Experiments with mussels from three ecologically different locations showed about the same response of the branchial membrane on contact with suspended sand. The presence and function of the branchial membrane appears to be an adaptation of mussels to their estuarine environment.     

Microbial biomass and activity in the vicinity of a mussel bed built up by the blue mussel Mytilus edulis

Our study focuses on the impact of the biosedimentary system mussel bed (Mytilus edulis) on the spatial variability of benthic microbial biomass and activity in relation to organic deposits. We sampled a transect of six stations from the muddy mussel bed towards a reference station in the sandflat in monthly intervals for 1 year. The sediment grain size fraction <63 μm and the total organic carbon (TOC) concentration decreased significantly towards the sandflat. Bacterial numbers and total microbial biomass (total adenylates) showed a high spatial variability and were not correlated to increasing distance from the mussel bed. However, a significant relationship with the TOC concentration was found. In contrast, the energetic status (adenylate energy charge) of the microbial community in the mussel bed was significantly lower than in the sandflat. A principal component analysis of the substrate-utilization pattern revealed clear differences between the microbial communities in the mussel bed and in the sandflat. Our results indicate that the sandflat may be dominated by a relatively specialized benthic microbial community with an increased efficiency in utilizing organic carbon sources. As a disadvantage, however, such r-strategists are only able to meet environmental changes within a comparatively narrow range. Benthic microbial communities in the vicinity of an M. edulis mussel bed, in contrast, are dominated by relative generalists with a greater physiological capacity to buffer discrete environmental changes. Such K-strategists show a lack of specialization which generally means a reduced efficiency in utilizing a particular resource. Received: 2 May 1999 / Received in revised form: 22 November 1999 / Accepted: 6 December 1999     

Winter growth of the blue mussel Mytilus edulis L.: importance of stock and site

To investigate the influence of genetics and environment on the growth of Mytilus edulis at low temperatures, we transplanted juveniles from 11 stocks to 10 sites along the coast of Nova Scotia, Canada. For each stock at each site, the mean change in shell length, dry shell weight, dry tissue weight and viability from early November 1985 to mid-April 1986 were documented. Stock and site differences were important in explaining the variance in shell growth (length and weight) but site alone accounted for most of the variation in tissue growth. Tissue growth was higher at ice-free sites than at ice-covered sites, but shell growth was similar among sites. We speculate that tissue growth was food-limited at those sites where ice-coverage delayed the onset of the spring phytoplankton bloom.     

Proteome modifications of blue mussel (Mytilus edulis L.) gills as an effect of water pollution

The discharge of chemicals such as oil associated or not with derived products constitutes a real threat for the environment. We report here the differential expression of the blue mussel (Mytilus edulis) gill proteins corresponding to two contaminated environmental conditions: crude oil and offshore produced water. In order to evaluate and understand contaminants, effects and adaptive response of these organisms, we identified proteins using MS. The latter can be grouped into three main classes: proteins involved in the cellular structure, in metabolism, and in defence proteins.     

Increased gonad ratio in the blue mussel, Mytilus edulis, exposed to starfish predators

The presence of invertebrate predators can induce morphological, physiological, and behavioural changes in sessile marine prey species. In this study, I investigated whether the risk of predation can alter the reproductive effort in the blue mussel Mytilus edulis. In the field, I cultured blue mussels in the presence and in the absence of the starfish, Asterias rubens, using enclosures with two compartments. Two experiments were performed with field periods of 19 and 30 days, respectively. After the field exposure, the mussels were examined for weight of flesh and gonad tissue. In both experiments, the starfish-exposed mussels developed a significantly larger gonad to flesh ratio, compared with that of the unexposed control mussels. After 30 days, the exposed mussels had also lost flesh weight significantly, probably due to spawning. These changes suggest that M. edulis accelerates the gonad development and/or increases its reproductive effort in the presence of predators. Predator-inducible adjustment of life-history characters should be a beneficial trait for this sessile species, that typically encounters a high and variable predation pressure. This revised version was published online in August 2006 with corrections to the Cover Date.     

Uptake of arsenic-betaines by the mussel Mytilus edulis

Mussels (Mytilus edulis) were exposed to trimethyl(carboxymethyl)arsonium bromide (arsenobetaine, C-1 betaine), trimethyl(2-carboxyethyl)arsonium bromide (C-2 betaine), or trimethyl(3-carboxypropyl)arsonium bromide (C-3 betaine). Arsenic was accumulated by the mussels in all cases but the efficiency of uptake decreased with the number of methylene units in the carboxyalkyl group. Arsenobetaine (C-1 betaine) was the most readily accumulated, followed by the C-2 betaine (70% as efficient as arsenobetaine) and the C-3 betaine (∼7%). Chromatographic analysis (HPLC-ICPMS) of extracts of the mussels demonstrated that the arsenic compounds were accumulated uncahanged. A 46-day depuration period which followed exposure did not significantly reduce the arsenic concentration in any of the three groups. Comparison with previous data on accumulation of arsenic compounds by M. edulis indicates that uptake may be influenced by the presence of a quaternary arsonium group and the zwitterionic nature of the arsenic-betaines.     

Differential segregation patterns of sperm mitochondria in embryos of the blue mussel (Mytilus edulis)

In Mytilus, females carry predominantly maternal mitochondrial DNA (mtDNA) but males carry maternal mtDNA in their somatic tissues and paternal mtDNA in their gonads. This phenomenon, known as doubly uniparental inheritance (DUI) of mtDNA, presents a major departure from the uniparental transmission of organelle genomes. Eggs of Mytilus edulis from females that produce exclusively daughters and from females that produce mostly sons were fertilized with sperm stained with MitoTracker Green FM, allowing observation of sperm mitochondria in the embryo by epifluorescent and confocal microscopy. In embryos from females that produce only daughters, sperm mitochondria are randomly dispersed among blastomeres. In embryos from females that produce mostly sons, sperm mitochondria tend to aggregate and end up in one blastomere in the two- and four-cell stages. We postulate that the aggregate eventually ends up in the first germ cells, thus accounting for the presence of paternal mtDNA in the male gonad. This is the first evidence for different behaviors of sperm mitochondria in developing embryos that may explain the tight linkage between gender and inheritance of paternal mitochondrial DNA in species with DUI.     

Microbiological quality of blue mussels (Mytilus edulis) in Nunavik, Quebec: a pilot study

This pilot study was aimed at documenting the presence of fecal indicators and enteric pathogens in blue mussels (Mytilus edulis) from 6 communities in Nunavik, Quebec. One to four 2?kg samples of mussels were collected at low tide in each community. Samples were investigated by enumeration methods for the fecal indicators enterococci, Escherichia coli, F-specific coliphages, Clostridium perfringens, and by molecular identification for the pathogens norovirus, Salmonella spp., Campylobacter jejuni, Campylobacter coli, and Campylobacter lari, verocytotoxin-producing E.?coli (particularly serovar O157:H7), Shigella spp., and Yersinia enterocolitica. In 5 communities, the presence of Giardia duodenalis and Cryptosporidium spp. was also tested by microscopy and molecular methods and that of Toxoplasma gondii was tested by molecular methods. Apart from small quantities of Clostridium perfringens in 2 samples, no bacterial or viral pathogens were detected in the mussels. Toxoplasma gondii was also not detected. However, G.?duodenalis and Cryptosporidium spp. were present in 18% and 73% of the samples investigated for these pathogens, respectively. When considering the indicators and the viral and bacterial pathogens investigated, the mussels examined were of good microbiological quality, but considering the presence of potentially zoonotic protozoa, it should be recommended that consumers cook the molluscs well before eating them.