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.     

Metallothionein concentration in sponges (Spongia officinalis) as a biomarker of metal contamination

The synthesis of metallothioneins (MTs) is often induced when organisms are exposed to heavy metals in the field. They are among the major "specific" biomarkers identified to date. With a view to include MTs in biomonitoring programs, the organisms most commonly studied are bivalves. Sponges present most of the characteristics researched in bioindicators of pollution and consequently have been proposed to constitute a "Sponge Watch Program". The detection of large quantities of metals in sponges suggests the existence of detoxification systems and indeed, the presence of metallothionein-like proteins (MTLPs) has been reported in two different species of sponges. In Spongia officinalis, the present study has demonstrated the presence of compounds exhibiting most of the characteristics of MTs: cytosolic, heat-stable, with apparent molecular mass of 4 to 15 kDa and binding (at least) Ag, Cu and Zn. Specimens have been collected along the French Mediterranean coast from three sites differing by their degree of contamination. Relationships between MTLP and metal concentrations have been established. For copper, mercury and zinc, the correlations were significantly positive.     

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.     

Histopathological indices and inflammatory response in the digestive gland of the mussel Mytilus galloprovincialis as biomarker of immunotoxicity to silver nanoparticles

Background: Histopathological assessments approaches in bivalves have become an important tool in environmental toxicology. This study seeks to develop a quantitative histopathological index (Ih) and inflammation score as biomarkers in the aim to assess the health status of nanoparticles exposed mussels.

Methods: Digestive gland hematoxylin and eosin (H&;E) stained sections from Mytilus galloprovincialis were assessed after in vivo exposure (for 3, 6 and 12?h) to silver nanoparticles (Ag-NPs?Results: Silver nanoparticles clearly induced histopathological alterations in digestive gland (maximum inflammation 2.75 with AgNP?p?Ih with AgNP?p?Ih were recorded after uptake routes were blockade: AgNP?p?Conclusions: Histopathological assessments showed to be promising tool in nanotoxicity which seems to depend on nanoparticles size, exposure time and interestingly to uptake routes. It was not clear: is it the length of exposure or the size of particles is more impactful.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Fine structure of Minchinia sp., a haplosporidan infecting the mussel Mytilus galloprovincialis L.

The fine structure of the mature spore of a haplosporidan found in the mussel Mytilus galloprovincialis L. is described. The mature spore (10 × 5 µm) is operculate and characterised by the presence of epispore cytoplasm that is prolongated into two bipolar extensions or 'tails' of 80–100 µm in length. The extensions are supported by bundles of microtubule-like structures which are not found in continuity with the wall. Due to the presence of episporal extensions and because the present species differs in some morphological characters from other haplosporidans assigned to the genus Minchinia Labbé, the parasite of the mussel has been placed in this genus as Minchinia sp.     

A mitochondrial genome with a reversed transmission route in the Mediterranean mussel Mytilus galloprovincialis

Species of the marine mussel genus Mytilus are known to contain two mitochondrial genomes, one transmitted maternally (the F genome) and the other paternally (the M genome). The two genomes have diverged by more than 20% in DNA sequence. Here we present the complete sequence of a third genome, genome C, which we found in the sperm of a Mytilus galloprovincialis male. The coding part of the new genome resembles in sequence the F genome, from which it differs by about 2% on average, but differs from the M genome by as much as the F from the M. Its major control region (CR) is more than three times larger than that of the F or the M genome and consists of repeated sequence domains of the CR of the M genome flanked by domains of the CR of the F genome. We present a sequence of events that reconstruct most parsimoniously the derivation of the C genome from the F and M genomes. The sequence consists of a duplication of CR elements of the M genome and subsequent insertion of these tandemly repeated elements in the F genome by recombination. The fact that the C genome was found as the only mitochondrial genome in the sperm of the male from which it was extracted suggests that it is transmitted paternally.     

Selective activation of carcinogenic aromatic amines to bacterial mutagens in the marine mussel Mytilus galloprovincialis

The postmitochondrial fraction of the marine mussel Mytilus galloprovincialis digestive gland activates selectively precarcinogenic aromatic amines, but not precarcinogenic benzo[a]pyrene, to Salmonella typhimurium TA 98 mutagens. This activation potential is NADPH-dependent, is not inducible by exposure to Diesel 2 oil and a polluted environment, and is inhibited by methimazole. The characteristics of this activation potential are consistent with the recent finding of the presence of FAD-containing-, and lack of cytochrome P-450 dependent-, monooxygenase activity in Mytilus edulis. The presence of such selective potential in marine invertebrate(s) may bring new insight into our understanding of the fate and the effects of carcinogens in the marine environment.     

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.     

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.