Molecular Evidence for the Existence of Two Species of Marteilia in Europe

Marteilia refringens is one of the most significant pathogens of bivalve molluscs. Previous sequencing of the small subunit ribosomal RNA gene of M. refringens isolates derived from the infected mussels (Mytilus edulis and Mytilus galloprovinciallis) and the oyster (Ostrea edulis) in Europe did not reveal genetic polymorphisms despite indications from epizootiological data that distinct types may exist. We investigated the existence of polymorphisms in the internal transcribed spacer region of the ribosomal RNA genes. The sequences of this region proved to be clearly dimorphic among Marteilia from five sampling sites. The distribution of the two genetic types, named "O" and "M", appeared to be linked to the host species, oysters and mussels, respectively. We therefore support the recognition of two species of Marteilia in Europe and propose that the "O" type corresponds to M. refringens and the "M" type to M. maurini.     

Phylogenetic analysis of the small subunit ribosomal RNA of Marteilia refringens validates the existence of phylum Paramyxea (Desportes and Perkins, 1990)

Marteilia refringens is recognized as one of the most significant pathogens of bivalve molluscs. The nucleotide sequence of the small subunit ribosomal RNA gene of Marteilia refringens is used to elucidate the phylogenetic position of the phylum Paramyxea. Genomic DNA was extracted from sporangia of Marteilia, purified from infected blue mussels, Mytilus edulis, and flat oysters, Ostrea edulis. The sequences obtained from Marteilia species purified from both oysters and mussels were identical. The sequence identity was confirmed by in situ hybridization using a DNA probe targeted to a variable region of the ribosomal DNA. The small subunit ribosomal RNA gene sequence of M. refringens is very different from all known sequences of eukaryotic organisms, including those of myxosporeans and haplosporeans. Therefore, the phylum Paramyxea should continue to be recognized as an independent eukaryotic phylum.     

First record of Marteilia sp. in mussels Mytilus galloprovincialis in Croatia

Marteiliosis is a disease of molluscs caused by Marteilia refringens in Europe and M. sydneyi in Australia. During routine examination of cultured mussels Mytilus galloprovinciallis in the northern Adriatic, the occurrence of Marteilia sp. was recorded with a prevalence of 5%. This parasite was not detected in flat oysters reared in the same area. The affiliation of the detected parasite in M. galloprovinciallis was confirmed by in situ hybridization using a M. refringens probe, specific at the genus level. DNA of these infected mussels originating from the same area will be used to clarify the taxonomic position of this species within the genus Marteilia using a molecular approach.     

Infection dynamics of Marteilia refringens in flat oyster Ostrea edulis and copepod Paracartia grani in a claire pond of Marennes-Oléron Bay

The protozoan parasite Marteilia refringens has been partly responsible for the severe decrease in the production of the European flat oyster Ostrea edulis Linnaeus in France since the 1970s. The calanoid copepod Paracartia grani Sars was recently found to be a host for M. refringens in French shallow-water oyster ponds ('claires'). This study reconsidered M. refringens transmission dynamics in the light of this finding, taking into account not only oyster infection dynamics and environmental factors but also data concerning the copepod host. P. grani population dynamics in the claire under study revealed that this species is the dominant planktonic copepod in this confined ecosystem. During winter, M. refringens overwintered in O. edulis, with P. grani existing only as resting eggs in the sediment. The increase in temperature in spring controlled and synchronized both the release of M. refringens sporangia in the oyster feces, and the hatching of the benthic resting eggs of the copepod. Infection of oysters by M. refringens was limited to June, July and August, coinciding with (1) the highest temperature recorded in the claire, and (2) the highest abundance of P. grani. PCR detection of M. refringens in P. grani during the summer period was linked to the release of parasite sporangia by the oyster. Our results are supported by previous results on the effective transmission of this parasite from the oyster to the copepod.     

Protozoan parasites in cultured mussels Mytilus galloprovincialis in the Thermaikos Gulf (north Aegean Sea, Greece)

The protozoans Ancistrum mytili (Oligohymenophorea: Ancistridae) and Marteilia refringens/maurini (Marteiliidea: Marteiliidae) were found parasitizing cultured mussels Mytilus galloprovincialis L. in the Thermaikos Gulf (north Aegean Sea, Greece). The former did not affect the condition index of infected mussels, in contrast to the latter, which did so and which also induced hemocyte infiltration in the affected digestive epithelium. The prevalence of both parasites was relatively high in a polluted area.     

Delta de l'Ebre is a natural bay model for Marteilia spp. (Paramyxea) dynamics and life-cycle studies

Marteilia spp. are paramyxean parasites that affect several bivalve species of economic interest, such as Ostrea edulis and Mytilus galloprovincialis. Certain aspects of Marteilia spp., such as their life cycle and host affinity and infection dynamics, still remain unknown. The 'Delta de l'Ebre' constitutes a natural model for the study of the life cycle of the parasite Marteilia, since uninfected mussels and flat oysters immersed in the bays can become infected. This, along with the geographical and ecological characteristics of the bays, make it a very interesting location to study the Marteilia life cycle. Preliminary results concerning marteiliosis, mainly in mussels, such as prevalence dynamics, infectious periods, host affinity and host intermediate candidates are reported in the present paper. This information will be required for further, more exhaustive, studies in the bays of the Ebre delta.     

Bonamia exitiosa (Haplosporidia) observed infecting the European flat oyster Ostrea edulis cultured on the Spanish Mediterranean coast

Bonamia exitiosa and Bonamia ostreae are parasites that reproduce within the haemocytes of several oyster species. In Europe, the host species is the flat oyster Ostrea edulis. The parasite B. ostreae has been responsible for mortalities since the late 1970s throughout the European Atlantic coast. B. exitiosa was first detected, in 2007, on this continent in flat oysters cultured in Galicia (NW Spain). Since then, the parasite has also been detected in France, Italy and the United Kingdom. The bays of the Ebro Delta in the south of Catalonia represent the main bivalve culture area in the Mediterranean coast of Spain. Previous information from the area includes reports of several flat oyster pathogens, including the notifiable parasite Marteilia refringens. However, the status with regard to Bonamia parasites was uncertain. In the present study, a Bonamia parasite was observed in flat oysters cultured in the Alfacs Bay of the Ebro Delta by histology and real-time PCR. PCR-RFLP and sequencing suggested the presence of B. exitiosa. Finally, phylogenetic analyses of the studied Bonamia isolates corroborated B. exitiosa infection. M. refringens was also observed in the same oyster batch, and co-infection with both parasites was also detected. This is the first detection of B. exitiosa, in Catalonia and the Spanish Mediterranean coast. The impact of the parasite on the Mediterranean flat oyster activity needs to be urgently addressed.     

Local adaptation and species segregation in two mussel (Mytilus edulis x Mytilus trossulus) hybrid zones

Few marine hybrid zones have been studied extensively, the major exception being the hybrid zone between the mussels Mytilus edulis and Mytilus galloprovincialis in southwestern Europe. Here, we focus on two less studied hybrid zones that also involve Mytilus spp.; Mytilus edulis and Mytilus trossulus are sympatric and hybridize on both western and eastern coasts of the Atlantic Ocean. We review the dynamics of hybridization in these two hybrid zones and evaluate the role of local adaptation for maintaining species boundaries. In Scandinavia, hybridization and gene introgression is so extensive that no individuals with pure M. trossulus genotypes have been found. However, M. trossulus alleles are maintained at high frequencies in the extremely low salinity Baltic Sea for some allozyme genes. A synthesis of reciprocal transplantation experiments between different salinity regimes shows that unlinked Gpi and Pgm alleles change frequency following transplantation, such that post-transplantation allelic composition resembles native populations found in the same salinity. These experiments provide strong evidence for salinity adaptation at Gpi and Pgm (or genes linked to them). In the Canadian Maritimes, pure M. edulis and M. trossulus individuals are abundant, and limited data suggest that M. edulis predominates in low salinity and sheltered conditions, whereas M. trossulus are more abundant on the wave-exposed open coasts. We suggest that these conflicting patterns of species segregation are, in part, caused by local adaptation of Scandinavian M. trossulus to the extremely low salinity Baltic Sea environment.     

Differential susceptibility to a trematode parasite among genotypes of the Mytilus edulis/galloprovincialis complex

We show that parasitism by the trematode Prosorhynchus squamatus in parental and introgressed Mytilus edulis/galloprovincialis (Bivalvia) mussels occurs in individuals with a predominantly M. edulis genome. This result suggests that the restricted specificity of P. squamatus is dependent on genetic factor(s) present in M. edulis. Because of its strong pathogenic effects (i.e. total castration and possible death), this parasite may be a source of intense selection against M. edulis genomes when they are present in a site. As a consequence, it may favour the geographic extension of the M. galloprovincialis genome. Previous studies have indicated that, in hybrid zones, recombinant genotypes are more susceptible to parasitic infections than either parental genotype. We demonstrate that this is not the case for the M. edulis/M. galloprovincialis system, and that the parental genotype alone determines susceptibility.     

Electrophoretic investigation of systematic relationships in the marine mussels Modiolus modiolus L., Mytilus edulis L., and Mytilus galloprovincialis Lmk. (Mytilidae; Mollusca)

Genetic variation was assayed electrophoretically at 13–16 loci in Modiolus modiolus, Mytilus edulis, and Mytilus galloprovincialis. High genetic distance ( D ) values were observed between Modiolus modiolus and Mytilus edulis (1.516 ± 0.523) and between Modiolus modiolus and Mytilus galloprovincialis (1.564 ± 0.539), whereas the distance between Mytilus edulis and M. galloprovincialis (0.167 ± 0.118) was rather low. The systematic status ot Mytilus edulis and M. galloprovincialis is discussed in relation to these lindings and the genetic distance values are used to estimate divergence times which in turn are compared with paleontological estimates. The observations of high average heterozygosity in Modiolus modiolus, and high correlations of locus heterozygosity between taxa are discussed briefly.     

Genomic reticulation indicates mixed ancestry in Southern-Hemisphere Mytilus spp. mussels

Previous surveys of allozyme variation in smooth-shell Mytilus spp. mussels have reported the presence in the Southern Hemisphere of both Mytilus edulis and Mytilus galloprovincialis mussels. In the present study, nuclear DNA markers mac-1 and Glu-5 '/ Glu-3 ', both diagnostic for Northern-Hemisphere M. edulis and M. galloprovincialis , were used to further characterize the nuclear genomes of M. edulis from Kerguelen and M. galloprovincialis from Tasmania. Genomic reticulation was observed, with typical M. edulis allelomorphs fixed in both populations at locus mac-1 whereas, at locus Glu-5 '/ Glu-3 ', allelomorphs characteristic of M. galloprovincialis were present in Kerguelen and nearly fixed in Tasmania. Kerguelen mussels had a genome of mixed M. edulis and M. galloprovincialis ancestry without evidence of barriers to merging as shown by Hardy–Weinberg and linkage equilibrium. Tasmanian mussels possessed a predominantly M. galloprovincialis genomic background introgressed by M. edulis allelomorphs at locus mac-1 . Genetic drift superimposed on ancient hybridization and introgression may explain the genomic reticulation observed in both Kerguelen and Tasmanian mussels. There was no evidence of a recent introduction of Northern-Hemisphere M. galloprovincialis or M. edulis to Kerguelen or Tasmania.  © 2007 The Linnean Society of London, Biological Journal of the Linnean Society , 2007, 92 , 747–754.     

First record of the microsporidian parasite Steinhausia mytilovum in Mytilus sp. (Bivalvia: Mytilidae) from France

Steinhausia mytilovum is a globally distributed microsporidian parasite which infects the oocytes of the blue mussels Mytilus edulis and M. galloprovincialis. Despite the intensive monitoring effort made on mussel populations, the parasite has not previously been reported in France. We report herein on the occurrence of S. mytilovum in Mytilus sp. from 1 cultured and 2 natural populations on the northern coast of France, thus extending the parasite's known distribution northwards. We also report on the observation in 1989 of S. mytilovum in M. galloprovincialis from the Golfe de Fos area in the Mediterranean Sea (South of France). S. mytilovum was observed in the European hybrid zone between M. edulis and M. galloprovincialis, which therefore renders the exact taxonomic status of the infected hosts unknown. The prevalence of the parasite was low, which suggests that its effect on mussel populations was probably limited.     

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.     

Continuous culture of Perkinsus mediterraneus, a parasite of the European flat oyster Ostrea edulis, and characterization of its morphology, propagation, and extracellular proteins in vitro

Continuous in vitro cultures of Perkinsus mediterraneus were established from tissues of infected European flat oysters, Ostrea edulis. The parasite proliferated in protein-free medium and divided by schizogony in vitro. Cell morphology was similar to that observed for P. mediterraneus in tissues of naturally infected O. edulis and for other Perkinsus spp. cultured in vitro. Parasite cells enlarged approximately 8-fold when placed in alternative Ray's fluid thioglycollate medium, and stained black with Lugol's iodine solution, a response characteristic of Perkinsus spp. DNA sequences matched those determined previously for P. mediterraneus, and phylogenetic analyses on three different data sets indicated that this was a Perkinsus species with a close relationship to another recently described species, Perkinsus honshuensis. Parasite viability was high (>90%) in vitro, but the proliferation rate was low, with densities generally increasing 2-to-6-fold between subcultures at 6-wk intervals. Enzyme analysis of cell-free culture supernatants revealed protease-, esterase-, glycosidase-, lipase-, and phosphatase-like activities. Incubation with class-specific protease inhibitors showed that P. mediterraneus produced serine proteases, and eight proteolytic bands with molecular weights ranging from 34 to 79 kDa were detected in the supernatants by gelatin sodium dodecylsulfate-polyacrylamide gel electrophoresis.     

Pleistocene separation of mitochondrial lineages of Mytilus spp. mussels from Northern and Southern Hemispheres and strong genetic differentiation among southern populations

Smooth-shelled mussels, Mytilus spp., have an antitropical distribution. In the Northern Hemisphere, the M. edulis complex of species is composed of three genetically well delineated taxa: M. edulis, M. galloprovincialis and M. trossulus. In the Southern Hemisphere, morphological characters, allozymes and intron length polymorphisms suggest that Mytilus spp. populations from South America and Kerguelen Islands are related to M. edulis and those from Australasia to M. galloprovincialis. On the other hand, a phylogeny of the 16S rDNA mitochondrial locus demonstrates a clear distinctiveness of southern mussels and suggests that they are related to Mediterranean M. galloprovincialis. Here, we analysed the faster-evolving cytochrome oxidase subunit I locus. The divergence between haplotypes of populations from the two hemispheres was confirmed and was found to predate the divergence between haplotypes of northern M. edulis and M. galloprovincialis. In addition, strong genetic structure was detected among the southern samples, revealing three genetic entities that correspond to (1) South America and Kerguelen Island, (2) Tasmania, (3) New Zealand. Using the trans-Arctic interchange as a molecular clock calibration, we estimated the time since divergence of populations from the two hemispheres to be between 0.5 million years (MY) and 1.3 MY (average 0.84 MY). The contrasting patterns observed for the nuclear and the organelle genomes suggested two alternative, complex scenarios: two trans-equatorial migrations and the existence of differential barriers to mitochondrial and nuclear gene flow, or a single trans-equatorial migration and a view of the composition of the nuclear genome biased by taxonomic preconception.     

A variety of Mytilus inhibitory peptides in the ABRM of Mytilus edulis: isolation and characterization.

1. Five species of Mytilus inhibitory peptides, MIP1-5, were isolated from acetone extracts of the anterior byssus retractor muscle (ABRM) of Mytilus edulis. MIP1 and MIP2 were shown to be S2-MIP and A2-MIP, respectively, first isolated from the pedal ganglia of the animal. 2. All the five peptides had a common C-terminal structure of -Pro-Xaa-Phe-Val-NH2, which was shown to be important for their biological activity. 3. The five MIPs showed similar inhibitory effects on contractions of the ABRM but did not affect catch tension and its relaxation. 4. In addition to the MIPs, catch-relaxing peptide (CARP) was also found in the ABRM.     

Growth factor from the tissues of the marine mollusk Mytilus edulis]

The absence of specific stimulators of cell proliferation, i.e. of growth factors, is one of the causes of failure in long-term cultivation of the marine invertebrate cells. In search for such stimulators in tissues of marine invertebrates, we succeeded in discovering in some tissues a stimulator of cell proliferation, similar to EGF, with a comparatively high content of it in tissues of Mytilus edulis. The similarity of the obtained factor with EGF was shown by the substitution of 125I-EGF, connected with EGF receptors on the surface of A431 cells, with analysed extracts, as well as by the ability of this extract to induce the internalization of EGF-receptor complexes. The fraction of acid/ethanol extract of M.edulis stimulating the cell proliferation in resting mouse fibroblasts Swiss 3T3, was obtained in the same conditions as EGF did, using reversed phase chromatography. Thus, the factor from tissues of M.edulis may belong to the family of EGF-like factors.     

Bivalve immunity: comparisons between the marine mussel (Mytilus edulis), the edible cockle (Cerastoderma edule) and the razor-shell (Ensis siliqua)

Much of the current knowledge concerning bivalve immunology and immunotoxicology has come from studies on the mussel genus, Mytilus, or from the American oyster, Crassostrea virginica. Following a major oil spill, it was observed that the marine mussel, Mytilus edulis, underwent significant immunosuppression but no oil-induced mortalities, while in contrast, mass mortalities were noted in the edible cockle, Cerastoderma edule, and the razor-shell, Ensis siliqua. A study comparing immune cells and functions in these three species was initiated (i) to assess whether M. edulis was a representative model species and (ii) to provide baseline data on immunity in two common species, which had previously received little or no attention in this respect. While all three species shared similar cell types, their lectin-binding and enzyme cytochemistry differed considerably. M. edulis had significantly different proportions of haemocytes binding with the lectins concanavalin A, wheatgerm agglutinin and Helix pomatia agglutinin and stained positive for eight enzymes, compared with only five in C. edule and three in E. siliqua. In terms of immune function, M. edulis haemocytes were much more active in phagocytosis and superoxide generation than haemocytes of the other two species. The results show that immune cells and functions differed extensively in these three closely related species, with M. edulis showing a much higher level of immunological vigour that may be linked to its considerable resilience to adverse environmental conditions. This suggests that M. edulis may not be particularly representative of the bivalves in terms of immune reactivity and that a wider range of species should be included in studies of molluscan immunotoxicology.     

Identification and characterization of highly expressed proteins in sperm cells of the marine mussel Mytilus edulis

Proteomic analysis on sperm has been restricted to only a few model organisms. We present here a 2DE PAGE proteome map of sperm cells from a nonmodel organism, the marine mussel Mytilus edulis, a free-spawning marine invertebrate with external fertilization. Ninety-six protein spots showing high expression were selected and of these 77 were successfully identified by nESI-MS analysis. Many of the identifications are relevant to sperm cell physiology and mtDNA functioning. The results and proteomics approach used are discussed in relation to their potential for advancing understanding of the unusual system of mtDNA inheritance described in Mytilus spp., and for the testing of evolutionary hypotheses pertaining to the role of fertilization in the speciation process.