Expression of several domains of twitchin and myorod in the ontogeny of the mussel <Emphasis Type=Italic>Mytilus trossulus</Emphasis>

The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of “adult” muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the “larval” isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.     

Expression of thick filament proteins during ontogenesis of the mussel Mytilus trossulus (Mollusca: Bivalvia)

The appearance of thick filament proteins organized into supramolecular complexes was studied by SDS-PAGE and Western-blot analysis at different developmental stages of the mussel Mytilus trossulus. Paramyosin appeared at the egg stage, while twitchin and myorod appeared at the blastula stage (12 h after fertilization). In addition, RT-PCR analysis showed that the twitchin genes were expressed starting from the blastula stage. Thus, the proteins forming thick filaments of the contractile apparatus of mussel muscles are expressed long before the formation of the first well-organized muscle system of the veliger larvae (55 h). Further, the ratios actin/myosin heavy chain (MHC) and paramyosin/MHC at the veliger stage (96 h) distinctly differed from those in the adult mussel.     

Complete sequences of mitochondrial genomes from the Baltic mussel Mytilus trossulus

Marine mussels Mytilus possess two mitochondrial (mt) genomes, which undergo doubly uniparental inheritance (DUI). Female (F) and male (M) genomes are usually highly diverged at the sequence level. Both genomes contain the same set of metazoan genes (for 12 proteins, 2 rRNAs and 23 tRNAs), both lack the atp8 gene and have two tRNAs for methionine. However, recently recombination between those variants has been reported. Both original F and M mt genomes of M. trossulus were replaced by M. edulis mtDNA in the Baltic populations. Highly diverged M genome occurs rarely in the Baltic mussels. Full sequences of the M genome identified in males (sperm) and F genome in females (eggs) were obtained. Both genomes were diverged by 24% in nucleotide sequence, but had similar nucleotide composition and codon usage bias. Constant domain (CD) of the control region (CR), the tRNA and rRNA genes were the most conserved. The most diverged was the variable domain 1 (VD1) of the control region. The F genome was longer than M by 147 bp. and the main difference was localised in the VD1 region. No recombination was observed in whole mtDNA of both studied variants. Nuclear mitochondrial pseudogenes (numts) have not been found by hybridisation with probes complementary to several fragments of the Baltic M. trossulus mtDNA.     

Polyploidy in a natural population of mussel, Mytilus trossulus.

We have analyzed natural polyploidy in a population of Mytilus trossulus from Vancouver Island (British Columbia, Canada) by means of cytogenetic techniques. Results obtained are the first reporting on this type of numerical chromosome aberrations in mussels.     

Evidence for recombination of mtDNA in the marine mussel Mytilus trossulus from the Baltic

A number of studies have claimed that recombination occurs in animal mtDNA, although this evidence is controversial. Ladoukakis and Zouros (2001) provided strong evidence for mtDNA recombination in the COIII gene in gonadal tissue in the marine mussel Mytilus galloprovincialis from the Black Sea. The recombinant molecules they reported had not however become established in the population from which experimental animals were sampled. In the present study, we provide further evidence of the generality of mtDNA recombination in Mytilus by reporting recombinant mtDNA molecules in a related mussel species, Mytilus trossulus, from the Baltic. The mtDNA region studied begins in the 16S rRNA gene and terminates in the cytochrome b gene and includes a major noncoding region that may be analogous to the D-loop region observed in other animals. Many bivalve species, including some Mytilus species, are unusual in that they have two mtDNA genomes, one of which is inherited maternally (F genome) the other inherited paternally (M genome). Two recombinant variants reported in the present study have population frequencies of 5% and 36% and appear to be mosaic for F-like and M-like sequences. However, both variants have the noncoding region from the M genome, and both are transmitted to sperm like the M genome. We speculate that acquisition of the noncoding region by the recombinant molecules has conferred a paternal role on mtDNA genomes that otherwise resemble the F genome in sequence.     

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.     

Changes in the lipid composition of embryonic cells of the mussel Mytilus trossulus during cryopreservation

A comparative analysis of the lipid composition of embryonic cells of the mussel Mytilus trossulus prior to, and after, cryopreservation in liquid nitrogen was carried out in the presence of two types of cryoprotectors: 1) dimethylsulfoxide and trehalose; and 2) dimethylsulfoxide, trehalose, total lipid extract from the mussel Crenomytilus grayanus, alpha-tocopherol and ascorbic acid. It was found that not only the cell viability but also the fatty acid composition of cell lipids after cryopreservation depend on the composition of cryoprotectors used. The content of saturated fatty acids, monoenic and polyenic fatty acids, the omega 3/omega 6 ratio, and the index of nonsaturation in the fatty acid composition of lipids was shown to change remarkably after cryopreservation. Possible reasons of these changes are discussed.     

Development of the larval muscle system in the mussel Mytilus trossulus (Mollusca,Bivalvia)

In the present study we examined muscle development throughout the complete larval cycle of the bivalve mollusc, Mytilus trossulus. An immunofluorescence technique and laser scanning confocal microscopy were used in order to study the organization of the muscle proteins (myosin, paramyosin, twitchin, and actin) and some neurotransmitters. The appearance of the muscle bundles lagged behind their nervous supply: the neuronal elements developed slightly earlier (by 2 h) than the muscle cells. The pioneer muscle cells forming a prototroch muscle ring were observed in a completed trochophore. We documented a well‐organized muscle system that consisted of the muscle ring transforming into three pairs of velar striated retractors in the early veliger. The striations were positive for all muscle proteins tested. Distribution of FMRFamide and serotonin (5‐HT) immunocytochemical staining relative to the muscle ring differed significantly: 5‐HT‐immunioreactive cells were situated in the center of the striated muscle ring, while Phe‐Met‐Arg‐Phe‐NH2 neuropeptide FMRFamid immunoreactive fibers were located in a distal part of this ring. Our data showed clearly that the muscle proteins and the neurotransmitters were co‐expressed in a coordinated fashion in a continuum during the early stages of the mussel development. Our study provides the first strong evidence that mussel larval metamorphosis is accompanied by a massive reorganization of striated muscles, followed by the development of smooth muscles capable of catch‐contraction.     

Development of the muscle system and contractile activity in the mussel Mytilus trossulus (Mollusca, Bivalvia)

The development of contractile apparatus was subjected to comparative analysis during ontogenesis of the mussel Mytilus trossulus. Indirect immunofluorescence with the polyclonal antibody against mussel twitchin, a protein of thick filaments, and florescent phalloidin as a marker of filamentous cell actin were used to monitor changes in the developing muscle system at different larval stages. The first definitive muscle structures were found at the late trochophore stage (36 h after fertilization) and starting from the midveliger stage (96 h), striated muscles, which are never present in adult mussels, were distinctly seen. The striated muscle periodicity was 1.25 microm in both mussle larvae and adult scallop. The contractile activities of veliger and adult muscles were measured using an electronic signal-processing videosystem. This work is the first complex study of morphological, biochemical, and physiological characteristics of the muscle system in the larvae and adult mollusks.     

Changes in the level of lectins in the mantle of the mussel Mytilus trossulus in response to anthropogenic contaminants

The lectin activity in an extract from the mantle of the mussel Mytilus trossulus was tested for the first time. Using the method of the hemagglutination inhibition assay, it was shown that lectins were Gal/GalNAc-specific and best agglutinated with rabbit erythrocytes. The influence of foreign compounds on the lectin level in the M. trossulus mantle was examined. Upon cadmium acetate exposure, the level of lectin activity exhibited phasic alterations and depended on the contaminant concentration or the time of exposure. During exposure of mussels to a synthetic detergent or diesel fuel, changes in lectin contents were dependent on the time of exposure.     

Comparative study of microbial communities from cultured and natural population of the mussel Mytilus trossulus in Peter the Great bay

The 525 strains of heterotrophic bacteria isolated from natural and cultured populations of the mussel Mytilus trossulus and the surrounding seawater were identified to a genus level on the basis of phenotypic analysis and the fatty acid composition of cell lipids. Gram-negative isolates were dominated by six genera of the family Enterobacteriaceae and by the genera Pseudoalteromonas, Vibrio, Photobacterium, Cytophaga/Flavobacterium/Bacteroides, Pseudomonas, and Moraxella, Gram-positive isolates were mainly represented by the genus Streptomyces. The taxonomic compositions of natural and cultured populations of the mussel M. trossulus in Peter the Great Bay were similar.     

The relative sensitivity of sperm, eggs and embryos to copper in the blue mussel (Mytilus trossulus)

Copper, an essential element, is toxic at elevated concentrations, and as a result of anthropogenic activities is becoming increasingly prevalent in marine environments. In this study, we examined the effects of copper on early life stages of the blue mussel, Mytilus trossulus. We assessed the impacts of increasing copper concentrations on embryo development, egg viability, sperm fertilization capacity and, in particular, on sperm swimming speed using computer-assisted sperm analysis. Sensitivity to copper followed the pattern: embryos > sperm > eggs. A dramatic increase in abnormal embryo development was observed following exposure to copper concentrations exceeding 10 microg/L. Sperm swimming speeds decreased significantly when exposed to 100 microg/L of copper, but lower doses did not influence sperm swimming speed. Copper exposure (at any tested concentration) did not affect sperm flagellum length, or alter egg viability. Based on our results, we suggest that exposure of sperm to copper may interfere with mitochondrial activity, which reduces sperm swimming speed during the extended duration of sperm motility in blue mussel.     

Interspecies transfer of female mitochondrial DNA is coupled with role-reversals and departure from neutrality in the mussel Mytilus trossulus.

Mussels of the genus Mytilus have distinct and highly diverged male and female mitochondrial DNA (mtDNA) genomes with separate routes of inheritance. Previous studies of European populations of Mytilus trossulus demonstrated that 33% of males are heteroplasmic for a second mtDNA genome of increased length and that hybridization with Mytilus edulis does not block mtDNA introgression, in contrast to reports for American populations. Here, we demonstrate that the female mtDNA type of M. edulis has replaced the resident female mtDNA type of European M. trossulus. This is supported by COIII sequence data indicating that the female mtDNA of European M. trossulus is very similar to that of M. edulis and that in phylogenetic trees, the mtDNAs of these two species cluster together but separately from American M. trossulus sequences, the latter not being disturbed by introgressive hybridization. We also provide evidence that the mtDNA genome of increased length found in heteroplasmic males of European M. trossulus derives from a recent partition of an introgressed M. edulis female type into the male route of transmission. Neutrality tests reveal that European populations of M. trossulus display an excess of replacement polymorphism within the female mtDNA type with respect to conspecific American populations, as well as a significant excess of rare variants, of a similar magnitude to those previously reported for the invading European M. edulis mtDNA. Results are consistent with a nearly neutral model of molecular evolution and suggest that selection acting on European M. trossulus mtDNA is largely independent of the nuclear genetic background.     

A connective tissue tumor in the mussel Mytilus trossulus from a polluted region of Nakhodka Bay, the Sea of Japan

A tumor was found for the first time in a mussel Mytilus trossulus from a heavily polluted area of Nakhodka Bay, Sea of Japan. Tumor cells were found in the connective tissue of different organs and also in gill vessels and hemal sinuses of the visceral mass. They were both attached and diffuse. The tumor was at an advanced stage, replacing the normal connective tissue cells, and formed nodes. The tumor cells were polymorphic, with a high nucleocytoplasmic ratio, and had a prominent nucleolus. The size of their nuclei was three to five times that of the nuclei of agranular hemocytes. The mitotic activity of the tumor cells was more than an order of magnitude higher than in the normal cells: the mean mitotic index was 1.4 +/- 0.5%, ranging from 0.97 to 2.3% in different organs. The mitotic indices in the connective tissue cells of three normal mussels were 0, 0, and 0.12%. A significant proportion (up to 78%) of the mitotic cells were at metaphase. The frequency of abnormal mitoses was 17%. Metaphases with displaced (often multiple) chromosomes constituted 71% of abnormal mitoses; anaphases, 8%; and tri- and tetrapolar mitoses, 11%. The tumor described is similar to diffuse sarcomatoid diseases of mussels from other geographical regions.     

Cost of selective feeding by the blue mussel (Mytilus trossulus) as measured by respiration and ammonia excretion rates

The metabolic cost as measured by respiration and ammonia excretion rates associated with a selective as compared to a non-selective feeding behaviour was determined for the blue mussel, Mytilus trossulus. Mussels were challenged with four environmentally relevant seston matrices of different quality and quantity, which were known to evoke either a sorting response (i.e., selective feeding) where organic-rich particles were selected over inorganic particles as compared to no sorting (i.e., non-selective) where either inorganic or organic particles were ingested by the bivalve. Seston matrices were prepared by mixing known quantities of silt and algae such that the following extent of feeding responses would occur; no pre-sorting of ingested material, (1) no algae+50 mg silt l⁻¹, (2) 150×10⁶ cells l⁻¹ of algae+no silt, and, where pre-sorting of ingested material occurred, (3) 20×10⁶ cells l⁻¹ of algae and 20 mg l⁻¹ of silt, and (4) 150×10⁶ cells l⁻¹ of algae and 50 mg l ¹ of silt. A control, which represented basal metabolism consisting of mussels exposed only to filtered seawater (0.45 μm), was included for a total of five treatments. Mussel respiration and ammonia excretion rates were independent of whether mussels were pre-sorting or not sorting ingested material. Of the four matrices, only rates determined for conditions of maximum seston quality and quantity where pre-sorting of the seston occurred were significantly different from control mussels (p<0.05, ANOVA). Estimates of net energy used for feeding, where net energy is total energy intake (food) minus energy expenditure (energy lost through respiration and excreta as measured by ammonia excretion rates), indicated that feeding, whether selective or non-selective required only 0.92% of net energy intake. Hence, mussels appear to be highly adapted to a dynamic food environment with negligible costs associated with the feeding process, even when significant pre-selection of organic-rich particles occurs.     

Within- and between-patch variability of predation intensity on the mussel Mytilus trossulus Gould on a rocky intertidal shore in Oregon, USA

Variability of predation intensity is an important cause of spatial differences of community structure and organization in the intertidal rocky shore. Field experiments were conducted to evaluate the within- and between-patch variability of the effects of different types of predators (small invertebrates and birds) on Mytilus trossulus Gould, which occupies an intermediate position in the competitive hierarchy among sessile organisms in disturbance patches within a California mussel (Mytilus californianus Conrad) bed community on the central Oregon Coast. Predation by birds did not significantly affect the mortality of M. trossulus. On the contrary, predation by small invertebrate whelks (Nucella spp.) had a significant effect on M. trossulus mortality. Predation by whelks also caused between- and within-patch variability of mortality of M. trossulus. Within patches, M. trossulus mortality at patch margins was significantly higher than at patch centers only when invertebrate predators were present. Wave exposure did not cause between-patch variability of predation intensity. Between-and within-patch variability of predation intensity may be caused by the variability of supply of whelks from the surrounding mussel mat. The movement of predators between patches and surrounding matrices may play an important role in the patch dynamics of these communities.     

Allozyme Loci Selection in the Pacific Mussel Mytilus trossulus under Stressful Conditions

The allozyme variability for 15 loci in two samples of the Pacific mussel Mytilus trossulus collected from a single giant cluster was investigated using the method of gel electrophoresis. One sample was subjected to short-term anaerobic stress and then to a longer aerobic stress, leading to the death of about 85% of the individuals. At some of the loci, significant differences in the genotypic and allele frequencies were found between the samples. The results are suggestive of the differential survival of mussels with different genotypes and alleles at some of the surveyed loci under stress. Our data are in agreement with the hypothesis of the adaptive significance of allozyme polymorphism.     

Stress and immune response in the mussel Mytilus galloprovincialis

The present study investigates the effects on immune-related parameters of various stress factors (air exposure, mechanical stress, high temperature and extreme salinity conditions) faced by the bivalve mollusc Mytilus galloprovincialis during marketing procedures. We observed that some stress typologies increase phagocytosis and the number of circulating immunocytes, while others can modify immunocyte response towards a further perturbation, i.e. the marine algal toxin yessotoxin. Our results suggest that non-lethal stress can be counteracted for sometime by increasing the level of some defence parameters. Moreover, our data indicate that fishing and transport procedures could interfere with mussel immunosurveillance.