Cadmium accumulation in tissues of the mussel Mytilus galloprovincialis

Studies have been made on cadmium accumulation in tissues of mussels kept within 20-60 days in water artificially enriched by Cd up to 20-100 micrograms/l. Irrespectively of cadmium concentration in the medium, its accumulation in tissues decreases in the following order: mid-gut gland, gills, gonads, mantle, adductor. Maximum concentration of Cd was found in the digestive tubuli of the mid-gut gland by X-ray microanalysis. The increase in S and, to a lower extent, P concentrations in these tubuli was also observed. It is suggested that the latter is due to immobilization of Cd by metal-binding proteins as well as to lyzosomal vesicles involved into detoxication of Cd. The increase in the external cadmium up to 100 micrograms/l did not affect the level of K, Ca and Mg in tissues of the mussel.     

Modulation of the chemiluminescence response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes

The influence of several factors on the chemiluminescence (CL) activity of haemocytes from the Mediterranean mussel (Mytilus galloprovincialis) was studied. Haemocytes were stimulated in vitro with different concentrations of zymosan, phorbol 12-myristate 13-acetate (PMA) and lipopolysaccharide (LPS) (adding superoxide dismutase, SOD, to the zymosan-stimulated haemocytes in order to test the specificity of the reaction). The in vitro effect of the clam pathogens Vibrio tapetis (bacteria) and a Perkinsus atlanticus-like protozoan tentatively named Pseudoperkinsus taapetis on the mussel haemocytes CL response was also assessed. To study the in vivo stimulation of haemocytes, mussels were inoculated with zymosan and the CL response of their haemocytes was subsequently measured. Zymosan added in vitro produced the highest CL response, although PMA also enhanced the CL emission and, in addition, increased the zymosan-stimulated CL. LPS and V. tapetis did not activate haemocytes. SOD significantly decreased the CL emission in zymosan-stimulated haemocytes. P. tapetis cells, as well as their extracellular products, inhibited the CL response to zymosan. Haemocytes from mussels injected with zymosan showed lower levels of stimulation than in vitro treated cells, and CL increased with time after injection.     

Functional and molecular immune response of Mediterranean mussel (Mytilus galloprovincialis) haemocytes against pathogen-associated molecular patterns and bacteria

The effect of live bacteria (Micrococcus lysodeikticus and Vibrio anguillarum), and PAMPs (poly I:C, zymosan, LPS, LTA and CpG) on the production of intermediate toxic radicals (respiratory burst activity and production of nitric oxide) and mytilin B, myticin C and lysozyme gene expression was studied in vivo and in vitro. In vitro, bacteria were able to modulate the haemocytes' respiratory burst activity, being significantly increased after 6 h of incubation. The effect of pathogen-associated molecular patterns (PAMPs) was also studied. Zymosan produced an increase of the PMA-mediated response but an inhibition of the zymosan-mediated response. A significant increase of nitric oxide production was found at all the sampled time points (1, 3 and 6 h) in comparison with controls on both, the Gram-positive and Gram-negative bacteria. The in vivo responses measured on haemocytes after M. lysodeikticus injection were faster than those induced by V. anguillarum. However, V. anguillarum induced stronger in vitro effects. Mytilin B, myticin C and lysozyme in vitro gene expression, occurred at short times after infection. The maximum in vitro expression was detected 3 h post-infection. The differences between M. lysodeikticus and V. anguillarum in different measured parameters may suggest that different signalling pathways might be involved. Moreover, among all assayed PAMPs, LPS elicited the highest response.     

Microflora of the Black Sea mussel Mytilus galloprovincialis L

The microbial cenosis in the mantle fluid and stomach of mussels is similar in its biochemical characteristics to the microflora of sea water.     

The mediterranean mussel Mytilus galloprovincialis Lmk. in Japan

Mussels ( Mytilus sp.) from Sanriku Bay, NE Honshu, Japan were examined using morphological characters and electrophoretically detectable enzyme polymorphisms. Using both sets of criteria, the mussels were identified as M. galloprovincialis , the mediterranean mussel. This confirms an earlier opinion, which was based on morphological criteria alone, that the mediterranean mussel occurs on the mainland coast of Japan. Investigation of some early Japanese literature suggests that mussels did not occur in this area earlier this century, and M. galloprovincialis may have been introduced to the region of Kobe, around 1930–1935. The present-day distribution of M. edulis and M. galloprovincialis in the Japanese archipelago may be explained by sea-surface temperatures in the region.     

Presence of and stress-related changes in urocortin-like molecules in neurons and immune cells from the mussel Mytilus galloprovincialis

The distribution of urocortin (UCN)-like material is investigated in the bivalve mollusc Mytilus galloprovincialis. Immunocytochemical data demonstrate that UCN-like molecules are present in ganglionic neurons, microglial cells and immunocytes. Moreover, a co-localization of UCN- and corticotrophin-releasing hormone (CRH)-like molecules is found in microglial cells and in immunocytes, but not in neurons. Following high salinity-stress experiments, immunoreactivity for UCN and CRH increased in ganglionic neurons and immunocytes. Our findings extend the number of molecules potentially used by molluscan immunocytes to confront stress situations and strengthen the idea of functional conservation of stress-related molecules during evolution.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10?? M epinephrine. The concentration that inhibited metamorphosis by 50% (IC??) for chlorpromazine and amitriptyline was 1.6 x 10?? M and 6.6 x 10?? M, respectively. Idazoxan was less effective with an IC?? of 4.4 x 1013 M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K+ was not inhibited by 10?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

Insights into the innate immunity of the Mediterranean mussel Mytilus galloprovincialis

Background

The Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity.

Results

The MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1.

Conclusions

The genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.     

Larval metamorphosis of the mussel Mytilus galloprovincialis Lamarck, 1819 in response to neurotransmitter blockers and tetraethylammonium

The metamorphic response of pediveliger larvae of Mytilus galloprovincialis to the neurotransmitter blockers chlorpromazine, amitriptyline, rauwolscine, idazoxan, atenolol and butoxamine, and to tetraethylammonium chloride (TEA) was investigated through a series of bioassays. Chlorpromazine, amitriptyline and idazoxin inhibited larval metamorphosis induced by 10^?4 M epinephrine. The concentration that inhibited metamorphosis by 50% (IC₅₀) for chlorpromazine and amitriptyline was 1.6 × 10^?6 M and 6.6 × 10^?5 M, respectively. Idazoxan was less effective with an IC₅₀ of 4.4 × 10¹³ M. Moreover, these three inhibitors showed no toxicity at any of the concentrations tested. The larval metamorphic response to K⁺ was not inhibited by 10^?3 M tetraethylammonium chloride after 96 h. Thus, the neurotransmitter blockers chlorpromazine and amitriptyline are inhibitors of larval metamorphosis, and will be useful tools for antifouling studies.     

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.     

Biosynthesis and role in osmoregulation of glycine-betaine in the Mediterranean mussel Mytilus galloprovincialis LMK

Quaternary bases, for example glycine-betaine, are difficult to quantify in biological materials because of a lack of specificity. However, nuclear magnetic resonance (NMR) can determine quaternary bases even in the presence of high water concentrations. Using NMR concentrations of glycine-betaine, the posterior adductor muscle of the Mediterranean mussel Mytilus galloprovincialis were measured up to 256 micromole/g dry weight. These concentrations were related to external salinity concentrations. The biosynthesis of glycine-betaine was demonstrated in M. galloprovincialis from the precursor (14)C choline.     

The IL-2 receptor in hemocytes of the sea mussel Mytilus galloprovincialis Lmk

In the hemolymph of the sea mussel Mytilus galloprovincialis, two different cell types have been found. Rounded (RH) cells display a nucleus that is very large in relation to the cell size; spread (SH) cells have an expanded cytoplasm and multiple granules. We determined by flow cytometry that only the SH cell types express three interleukin-2 receptor (IL-2R) subunits. Mussel IL-2R alpha and IL-2R beta subunits display a molecular weight similar to those in vertebrates tissues, whereas mussel IL-2R gamma is smaller than that in vertebrates. Both lipopolysaccharide and IL-2 induce IL-2R alpha expression, and such induction depends on the concentration of both agonists.     

Effect of thermal stress on protein expression in the mussel Mytilus galloprovincialis Lmk

The exposure of organisms to stressing agents may affect the level and pattern of protein expression. Certain proteins with an important role in protein homeostasis and in the tolerance to stress, known as stress proteins, are especially affected. Different tissues and cells show a range of sensitivities to stress, depending on the habitat to which organisms have adapted. The response of different tissues and cells from the mussel Mytilus galloprovincialis Lmk. to heat shock has been studied in this work using different exposure times and temperatures. During the assays, protein expression was observed to vary depending on the tissue studied, the temperature or the exposure time used. But maybe the most prominent thing is the different response obtained from the cultured haemocytes and those freshly obtained from stressed mussels, which makes us think that the extraction procedure is the main cause of the response of non-cultured cells, although the haemolymph may contain components that modulate haemocyte response.     

Genetic variability of the marine mussel Mytilus galloprovincialis assessed using two-dimensional electrophoresis

Two-dimensional electrophoresis (2-DE) has been used to measure the degree of genetic variability of the marine mussel Mytilus galloprovincialis. Genetic polymorphisms were detected in 33 of a total of 86 polypeptides scored among the most abundant proteins from foot samples in 38 individuals. Estimates of average heterozygosity were 0.101+/-0.018 and 0.114+/-0.021 in a natural and a cultured population, respectively, from the NW of the Iberian Peninsula. These are the highest estimates of average heterozygosity reported by 2-DE in an animal species to date. We consider that these data throw open the question of the level of genetic variability detectable by two-dimensional electrophoresis. Multilocus genotype data were used to infer haplotypic frequencies by means of the EM algorithm in order to detect linkage disequilibrium between loci coding abundant proteins. Significant associations were found in 22.7% of the 406 two-locus pairs analysed. Also, clusters of loci in which all pairwise combinations exhibit statistically significant associations were detected and physical linkage between some of these loci is postulated from the linkage disequilibrium data.     

Proteomic approach to probe for larval proteins of the mussel Mytilus galloprovincialis

A proteomic approach was used to search for larval proteins specific to the mussel Mytilus galloprovincialis from Galicia in northwest Spain. The study included both a comparative analysis, through two-dimensional electrophoresis, of protein expression maps of the larvae of the mussel and of 5 abundant and commercially important bivalve species from the region (Ostrea edulis, Cerastoderma edule, Pecten maximus, Tapes decussatus, and Venarupis pullastra) and subsequent mass spectrometric analysis of some of the protein spots. A total of 18 spots were selected and isolated from gels of M. galloprovincialis larvae. From their relative position on the electrophoresis gels, 6 of these were clearly exclusive to the mussel species. However, it was not clear whether the other spots were shared by other species. To overcome this ambiguity, first an analysis using matrix assisted laser desorption ionization with time-of-flight (MALDI-TOF) was conducted on the 6 spots of Mytilus that could possibly be shared with only one species. The peptide mass fingerprinting was completely different for the proteins compared. This result confirmed that the 6 proteins were exclusively mussel proteins, but demonstrated the utility of this approach when working with species that are poorly represented at the protein level in databases.     

Glucuronides in mussel Mytilus galloprovincialis as a possible biomonitor of environmental carcinogens

1. The in vitro incubation of mussel digestive gland with 1 mM aminofluorene resulted in the formation of glucuronides that (a) became mutagenic with carp liver S9, and (b) liberated S9-dependent mutagenic aglucones after beta-glucuronidase treatment. 2. Natural populations of mussels from unpolluted and polluted sites, as well as mussels exposed to 3 ppm of aminofluorene or to used engine oil, did not accumulate detectable amounts of premutagens, mutagens, or mutagenic glucuronides/aglucones either in digestive gland tissue or in shell-cavity water. 3. The mutagenicity testing of mussel's glucuronides/aglucones does not seem to be useful as a biomonitor of environmental carcinogens.     

Seasonal changes of nucleotides in mussel (Mytilus galloprovincialis) mantle tissue

Seasonal variations of nucleotides in Mytilus galloprovincialis mantle tissue were analyzed. Separation and quantification was achieved by reversed-phase high-performance liquid chromatography. Total nucleotides show a pronounced seasonal variation with maximum and minimum values in autumn and spring, respectively. Adenine nucleotides accounted for the major part in spring and summer, guanosine and cytidine nucleotides in winter; uridine nucleotides were relatively constant throughout the year. Their inverse variation suggests inter-conversion among them and the maintenance of the potential cell energy in winter by other triphosphate nucleotides different from ATP. These results reflect environmental and nutritional conditions, and also the reserves and gametogenic cycles taking place in M. galloprovincialis mantle tissue.     

Biochemical properties of metalloproteinases from the hemolymph of the mussel Mytilus galloprovincialis Lam

The expression of matrix metalloproteinases (MMP) with gelatinase activity was found in the whole hemolymph of the marine mussel Mytilus galloprovincialis Lam. Cleavage activity was specific for gelatin; very little activity towards human type-IV collagen, and no activity for cold fish gelatin, casein or bovine serum albumin were detected. EDTA and 1,10-phenanthroline were inhibitory, suggesting that mussel MMPs require divalent cations for their proteolytic activity; in fact, the presence of exogenously added divalent ions significantly protected the MMPs from inhibition. No inhibition was detected with serine or cysteine proteinase inhibitors. The specific vertebrate inhibitors as well as the classical vertebrate activator of MMPs were without effect, whereas sulphydryl reducing agents had a strong inhibitory effect. Mussel MMPs showed an exponential curve of thermal-dependent decay that was not protected by the presence of metal ions. Overall the results indicate both similarities and differences between invertebrate and vertebrate gelatinases, providing information for understanding the biological role of these ancient proteinases.     

Metallothionein concentration in the mussel Mytilus galloprovincialis as a biomarker of response to metal contamination: validation in the field

Mussels were translocated from a shell-fish breeding area (Sète, on the French Mediterranean coast) to sites exposed to trace element inputs in April 2000. They were recovered 3 months later. Whole soft tissues from all of the sites (n = 97) were analysed for arsenic, cadmium, chromium, copper, mercury, nickel, lead and zinc. Metallothioneins (MTs) were also measured in the digestive gland and in the remaining tissues (allowing calculation of whole soft tissue concentrations) at 22 of the 97 sites. MT concentrations in the digestive gland and the whole soft tissues were strongly correlated. The condition index varied with food availability at different sites. This did not influenced MT concentrations in the whole soft tissues, whereas the condition index was negatively correlated to trace element concentrations. A model is proposed to minimize this influence of condition. Metal concentrations adjusted using this model showed significant correlations with MT levels for those metals (cadmium, copper, nickel and zinc) that are known to bind to this protein, with the exception of mercury. Even in moderately contaminated sites, measurement of the MT level in the soft tissues of mussels was generally able to discriminate between different levels of contamination, allowing the use of a simplified procedure compared with dissection of the digestive gland. It is recommended to avoid translocation and sampling during the reproductive period, which is well documented for commercial species such as Mytilus sp.     

Tissue distribution of neutral deoxyribonuclease (DNase) activity in the mussel Mytilus galloprovincialis

The presence of neutral DNase activity in bivalves is reported for the first time. The enzyme activity in four tissues of the mussel Mytilus galloprovincialis was analyzed by three different methods (i) specific denaturating SDS-PAGE zymogram, (ii) sensitive single radial enzyme diffusion (SRED) assay and (iii) rapid and sensitive fluorimetric determination of DNase activity with PicoGreen. The fluorimetric assay was rapid and sensitive enough for determination of hydrolytic activity of dsDNA in mussel hepatopancreas, adductor, gills and mantle. Maximal activity in all mussel tissue extracts was obtained in the presence of Ca(2+) and Mg(2+) at pH 7.0 with dsDNA as substrate. The neutral DNase activity in mussel tissue decreases in order hepatopancreas, mantle>gills>adductor. The enzyme activity displays interindividual variability in particular tissue as well as variability among tissues within one specimen. In the hepatopancreas one to three distinct proteins expressing neutral, Ca(2+), Mg(2+)-dependent, DNase activity were detected by denaturating SDS-PAGE zymogram. This heterogeneity of neutral nucleases involved in DNA hydrolysis in hepatopancreas could reflect interindividual variability in mussel food utilization and nutrient requirement.