The metallothionein genes of Mytilus galloprovincialis: genomic organization, tissue expression and evolution

Recently, increasing interest has been directed to the study of metallothioneins (MTs), which are small proteins that are able to bind metal ions. The induction of MT synthesis after exposure to metal or other environmental contaminants in a large number of aquatic invertebrates makes these proteins good biomarkers in water monitoring programs. Within bivalves, the species Mytilus galloprovincialis and Mytilus edulis represent model organisms for these types of studies, as well as for molecular studies regarding the expression and characterization of MT encoding genes. In the present paper, we focused on the genomic characterization, evolutionary, and tissue-expression analyses of the MT-10, MT-10 Intronless, and MT-20 genes in M. galloprovincialis. The comparison of the genomic sequences showed the presence of long nucleotide stretches within the introns of the MT genes that are conserved between M. galloprovincialis and M. edulis. These non-coding conserved sequences may contain regulatory motifs. Real-Time RT-PCR experiments revealed that, at the basal conditions, the MT-10 and MT-10 Intronless genes are expressed at levels considerably higher than the MT-20 gene, mainly in the digestive gland and gill tissue. The strong induction of the MT-20 gene expression detected in a field-collected sample is associated with the up-regulation of both the MT-10 and MT-10 Intronless genes. Evolutionary analysis revealed signals of localized positive selection that, together with the tissue-expression data, support a possible functional diversification between the MTs encoded by the MT-10 and MT-10 Intronless genes.     

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.     

Molecular characterization and function analysis of MT-10 and MT-20 metallothionein isoforms from Mytilus galloprovincialis

Structure and function of molluscan metallothioneins (MTs) are still poorly understood. The sea mussel Mytilus galloprovincialis displays two MT isoforms which differ in both primary sequences and physiological functions. MT-10 is the constitutive isoform, whereas MT-20 is mainly induced by cadmium (Cd). Both MTs were produced as recombinant proteins and showed identical Cd content and similar Cd-binding properties. Conversely, circular dichroism disclosed marked differences in the secondary conformations of the two Cd(7)-MTs. The possible relapses of these structural differences on protein stability and function were assessed. MT-10 presented a higher thermal stability and a more compact structure than MT-20, as it was inferred by absorption and emission spectroscopy studies. Moreover, the kinetics of Cd-release clearly indicated that MT-10 is much more sensitive to oxidation than is MT-20. The observed differences between MT-10 and MT-20 are discussed in terms of the different physiological roles exerted by the two isoforms in mussel.     

Adaptive gamete-recognition divergence in a hybridizing Mytilus population

Gamete-recognition proteins often evolve rapidly, but it is not known if their divergence occurs within species and corresponds with the evolution of reproductive isolation, or if divergence typically accumulates between already isolated lineages. We examined the evolution of a candidate gamete-recognition protein in several sympatric and allopatric populations of Mytilus blue mussels, species that hybridize in nature. Within a single species, Mytilus galloprovincialis, we found adaptive divergence of Lysin-M7, a sperm acrosomal protein that dissolves the egg vitelline envelope during fertilization. Mytilus galloprovincialis Lysin-M7 alleles group into two distinct clades (termed G and G(D)), and individual alleles in these clades are separated from each other by at least three and up to eleven amino-acid substitutions. Maximum-likelihood estimates of selective pressure (dN/dS =omega) implicate selection in the divergence between M. galloprovincialis Lysin-M7 clades, and within the G(D) clade. Exact tests of population differentiation indicate that the relative frequency of G and G(D) Lysin-M7 alleles differs significantly among M. galloprovincialis populations. Compared with allopatric Mediterranean samples, Lysin-M7 alleles in the G(D) clade are found at elevated frequency in samples from the East Atlantic and California, areas of secondary contact and hybridization between Mytilus species, and Australia, an area of unknown species composition. Adaptive divergence between the alleles most common in allopatry and those found at elevated frequency in samples from sympatry suggests that selection pressures acting in hybridizing populations, likely following Pleistocene secondary contact with M. edulis in the East Atlantic, drove the divergence of Lysin-M7 in M. galloprovincialis.     

Evidence of selective mortality in favour of the Mytilus galloprovincialis Lmk phenotype in British mussel populations

Samples of mussels ( Mytilus ) were collected from 17 localities within hybrid zones of Mytilus edulis and Mytilus galloprovincialis in south-west and north-east England. The study of two polymorphic allozyme loci ( esterase-D and octopine dehydrogenase ), which are partially diagnostic for the two forms of mussel, reveal the existence of widespread length-dependent allele frequency variation. Larger mussels tend to have a higher frequency of alleles characteristically at high frequency in Mytilus galloprovincialis. Also at a given shell length galloprovincialis alleles have a higher frequency higher up the shore. Computer simulation is used to demonstrate that length-dependent variation may be generated not only by differential mortality but also by differential growth and in models including or excluding immigration. Evidence supports the hypothesis that selective mortality acting in favour of the galloprovincialis phenotype within hybrid populations in Britain is balanced by immigration of the more abundant Mytilus edulis.     

An RFLP assay to determine if Mytilus galloprovincialis Lmk. (Mytilidae; Bivalvia) is of Northern or Southern hemisphere origin

Mytilus galloprovincialis is one of three smooth shelled blue mussel species belonging to the Mytilus edulis species complex. Naturally occurring and introduced populations of M. galloprovincialis are widely distributed throughout many regions of the globe. Mytilus galloprovincialis includes morphologically indistinguishable Northern and Southern hemisphere mtDNA lineages that have been separated for ～1 my. To distinguish recently introduced Northern M. galloprovincialis from resident Southern M. galloprovincialis in New Zealand, we developed a 16s rRNA RFLP assay. We compared RFLP assignments of 178 mussels with those generated from a 16s rRNA sequence-estimated phylogeny. All mussels were correctly assigned by the RFLP to their sequence-based phylogenetic placement. This assay allows the rapid identification of Northern and Southern hemisphere M. galloprovincialis and will provide an important tool for monitoring human mediated introductions of otherwise cryptic lineages.     

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.     

Effect of diesel fuel pollution on the lipid composition of some wide-spread Black Sea algae and invertebrates

Two green algae (Ulva rigida and Cladophora coelothrix), the mussel Mytilus galloprovincialis and the snail Rapana thomasiana from the Bulgarian Black Sea shore have been treated with diesel fuel (100mg l(-1)) in an aquarium with sea-water for three days. The lipids and their fatty acid changes have been examined. Significant changes have been observed mainly in the polar lipids and in the saturation of the fatty acids. These changes appeared to be bigger in the evolutionary less advanced species from both groups of marine organisms--algae and invertebrates (Ulva rigida and Mytilus galloprovincialis respectively). The data obtained could be used for a biomonitoring of the pollution.     

Transfection of spermatozoa in bivalve molluscs using naked DNA

Spermatozoa from the bivalve molluscs Mytilus galloprovincialis, Mytilus chilensis and Chamelea gallina were transfected in vitro using the p-GeneGrip construct, which encodes green fluorescent protein. The efficiency of transfection after brief incubation was assessed by fluorescence and confocal laser microscopy, and was about 58.5-70.01% in the species used. The foreign gene was principally located in the sperm nuclei, as demonstrated by laser confocal serial sections. In some spermatozoa, mitochondria, which are grouped in the base of the nucleus, also appeared to be transfected. Polymerase chain reaction and Southern blot analyses suggested that the foreign DNA had been integrated into the nuclear genome in Mytilus galloprovincialis spermatozoa. This simple method for spermatozoon transfection in molluscs of commercial interest could have biotechnological applications.     

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.     

Geographic variation and positive selection on M7 lysin, an acrosomal sperm protein in mussels (Mytilus spp.)

Successful fertilization in free-spawning marine organisms depends on the interactions between genes expressed on the surfaces of eggs and sperm. Positive selection frequently characterizes the molecular evolution of such genes, raising the possibility that some common deterministic process drives the evolution of gamete recognition genes and may even be important for understanding the evolution of prezygotic isolation and speciation in the marine realm. One hypothesis is that gamete recognition genes are subject to selection for prezygotic isolation, namely, reinforcement. In a previous study, positive selection on the gene coding for the acrosomal sperm protein M7 lysin was demonstrated among allopatric populations of mussels in the Mytilus edulis species group (M. edulis, Mytilus galloprovincialis, and Mytilus trossulus). Here, we expand sampling to include M7 lysin haplotypes from populations where mussel species are sympatric and hybridize to determine whether there is a pattern of reproductive character displacement (RCD), which would be consistent with reinforcement driving selection on this gene. We do not detect a strong pattern of RCD; neither are there unique haplotypes in sympatry nor is there consistently greater population structure in comparisons involving sympatric populations. One distinct group of haplotypes, however, is strongly affected by natural selection, and this group of haplotypes is found within M. galloprovincialis populations throughout the Northern Hemisphere concurrent with haplotypes common to M. galloprovincialis and M. edulis. We suggest that balancing selection, perhaps resulting from sexual conflicts between sperm and eggs, maintains old allelic diversity within M. galloprovincialis.     

Genetic connections between some species of Mytilidae (Mollusca: Bivalvia) from the northern part of the Pacific Ocean

In 1978 and 1999, seven and eight species of Mytilidae (Mollusca: Bivalvia) were analyzed using gel electrophoresis. Mean heterozygosity per individual (Hobs and Hexp) and genetic distances (Rogers' DR, Nei's DN, and others) were estimated for 21 and 24 allozyme loci. Mytilus modiolus had the highest variation among the species examined. Genetic distances were lowest for the M. trossulus-M. galloprovincialis species pair: DR = 0.147, DN = 0.078. Overall, five species of the genera Mytilus and Crenomytilus were genetically closer to each other (DR = 0.147, DN = 0.078) than to the remaining three species of this group (DR = 0.807, DN = 2.243). The relationships among the species were examined using cluster analysis and parsimony methods. The densest clusters in the dendrograms consisted of (1) M. trossulus and M. galloprovincialis and (2) M. coruscus, M. californianus, and M. grayanus. These two clusters form a larger cluster (3), which comprises all representatives of the nominal genus Mutilus and C. grayanus. The Mytilus-Crenomytilus cluster is consecutively joined by Adula falcatoides, Mytilus modiolus, and Septifer keenae. According to Nei's genetic distances DN, the time of divergence between M. trossulus and M. galloprovincialis is 0.8-1.6 Myr; between M. californianus and C. grayanus, it is approximately 9 Myr; and between M. coruscus and the latter pair, it is 13 Myr before present. Two representatives of the Mytilus ex gr. edulis complex diverged from the Mytilus-Crenomytilus group of large-size Pacific species about 20 Myr ago. These results are in good agreement with paleontological data and indicate a relatively recent origin of the Mytilus ex gr. edulis complex. The results obtained can be used in systematics and phylogeny of modern Mytilidae.     

Mitochondrial contribution to metabolic changes in the digestive gland of Mytilus galloprovincialis during anaerobiosis

The activities of several enzymes and the levels of metabolites have been measured in the digestive gland of Mytilus galloprovincialis exposed to increasing hypoxia from 7 to 168 hr. A sharp decrease of pyruvate kinase was observed after 7 hr. The anoxic enzyme showed increased Km for phosphoenolpyruvate and decreased apparent Ki for alanine. Glyoxalase I was constant after up to 72 hr of exposure and then decreased. Phosphoenolpyruvate carboxykinase and alanopine dehydrogenase decreased. The metabolites alanine and succinate increased with hypoxia time, whereas D- and L-lactic and aspartic acids were undetectable and constant respectively. Mitochondrial formation of pyruvate from D-lactate was demonstrated in intact mitochondria isolated from the digestive gland of Mytilus galloprovincialis. The significance of the observed enzyme and metabolite changes in hypoxia is discussed in comparison with other invertebrate organisms. The role of mitochondria in the overall adaptive strategy of Mytilus galloprovincialis is discussed. J. Exp. Zool. 286:107-113, 2000.     

Comparative analysis of different satellite DNAs in four Mytilus species.

We report the characterization of three satellite DNAs in four species of mussel: Mytilus edulis, Mytilus galloprovincialis, Mytilus trossulus, and Mytilus californianus. The monomers of the Apa I satellite DNAs were 173, 161, and 166 bp long. These satellite monomers were used to construct phylogenetic trees to infer relationships among these species. The topologies obtained clearly indicate that M. californianus is the most divergent species with respect to the other three. Furthermore, localization of satellite DNAs on metaphase chromosomes was performed using fluorescent in situ hybridization (FISH). Fluorescent signals revealed a different organization and distribution of these three satellite DNAs.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

Two events are responsible for an insertion in a paternally inherited mitochondrial genome of the mussel Mytilus galloprovincialis

Frequent nonhomologous recombination has been previously postulated to explain the 1045-bp insertion in one mitochondrial sperm-transmitted haplotype of Mytilus galloprovincialis. Such recombination would lead to the disruption of gene order and so the existence of a specific mechanism for maintaining the same gene order in both mitochondrial genomes of Mytilus has been proposed. Here the simpler explanation of the observed structure, involving a tandem duplication and a deletion, is presented. Their occasional occurrence in Mytilus mtDNA proves the similarity, not the difference, between animals with and without DUI.     

Phosphorylation events catalyzed by major cell signaling proteins differ in response to thermal and osmotic stress among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels

Sharp environmental gradients encountered within the intertidal zone have driven the evolution of physiological adaptations that allow its inhabitants to maintain cellular function in the presence of fluctuating abiotic factors. These adaptations are mediated by gene-regulatory networks that, despite their inherent complexity, must remain evolvable and capable of responding to different selection pressures associated with specific ecological niches. Phosphorylation events catalyzed by cell-signaling enzymes represent a parsimonious mechanism to integrate new functional or regulatory properties into these gene-regulatory networks. In this study, proteins phosphorylated on consensus sequences for protein kinases A, B, and C; cyclin-dependent kinases; and mitogen-activated protein kinases, as well as the abundance of phosphorylated stress-activated protein kinase (phospho-SAPK/JNK), were quantified in order to ascertain whether phosphorylation events are divergent among native (Mytilus californianus and Mytilus trossulus) and invasive (Mytilus galloprovincialis) species of mussels that differ in their tolerance toward environmental stress. Abundances of phosphorylated substrate proteins for each of the major signaling proteins that were investigated, as well as the abundance of phospho-SAPK/JNK, differed both within and between species during thermal and osmotic stress. These data suggest that modulating protein function via phosphorylation may be an important mechanism to integrate novel properties into stress-regulatory networks. In turn, differential phosphorylation during environmental stress may contribute to species-specific tolerances toward abiotic stress, interspecies dynamics, and biogeographic patterns in Mytilus congeners.     

Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea

Seasonal variations of six mussel (Mytilus galloprovincialis) biomarkers at two sites in the Mediterranean Sea were compared with physiological indices (condition, growth and gonad maturation), environmental parameters (temperature, salinity and turbidity), and chemical contamination levels. The basal levels of acetylcholinesterase (AChE), DNA adducts, benzo[a]pyrene hydroxylase (BPH), heat-shock proteins (HSP70), metallothioneins (MT) and P-glycoprotein (P-gp)-mediated multixenobiotic resistance (MXR) were estimated as early warning signals in caged mussels sampled at Carteau (native site) and La Fourcade (transplantation site) over a 2-year period. The Carteau and La Fourcade mussels have specific chemical contamination profiles but a similar range of values. For example, both are highly contaminated by heavy metals (201 and 258.4 mg kg(-1) dw, respectively) and considered as moderately impacted for polychlorinated biphenyls (PCBs) and polycyclic aromatic hydrocarbons (PAHs). However, contamination levels at Carteau are twice as high for PAHs (101.5 mg g(-1) dw) and PCBs (90.2 mg g(-1) dw) than La Fourcade. The seasonal contamination trend at Carteau showed six-fold higher levels of pyrolytic pollutants in winter. Although few tissue lesions were detected in individuals studied at either site, greater parasitic infestation was observed at Carteau. The results of findings from the two Mediterranean pilot studies support the adaptability of transplanted mussels to be used as biomarkers and to establish physiological endpoints for chemical contaminant exposure.