Investigation of the loss of byssus in Mytilus galloprovincialis from mussel farms in the Adriatic Sea

A fungal infection has been found in the mussel Mytilus galloprovincialis from Adriatic Sea mussel farms. The infection ultimately results in the loss of the byssus, with serious consequences for mussel farming yield. The pathogen provokes the progressive destruction of the foot muscles, also damaging related structures such as the intra-organism part of the byssus apparatus, resulting in loss of the thread component. The affected health status of the animal is also sustained by modifications in the digestive gland structure, ranging from hyperactivity to extreme cell death in the tubula. At present, the identity of the harmful fungus is unknown.     

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.     

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.     

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.     

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.     

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.     

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.     

The effect of chlorpyrifos on protein content and acid DNase activity in the mussel,Mytilus galloprovincialis

We evaluated the effects of short-term exposure to an organophosphate pesticide chlorpyrifos on the digestive gland and gills of the mussel Mytilus galloprovincialis. We studied metabolic activity by quantifying protein content and physiological function responses using acid DNase activity. The increase in protein content was observed in both the target tissues of mussels exposed to 0.03 μg/L chlorpyrifos when compared with control mussels. The pattern of acid DNase activity in digestive gland and gills indicated a tissue-specific response, although the lowest concentration of chlorpyrifos caused changes in acid DNase activity in both tissues. In the digestive gland, the increase of acid DNase activity was observed in mussel exposed to 0.03 μg/L chlorpyrifos, followed by decrease up to 100 μg/L chlorpyrifos. Enzyme activity in the gills showed a dose response effect. The results support the use of acid DNase activity in the digestive gland as a sensitive response to an environmentally relevant range of pesticide concentrations. It may also indicate an effect on mussel physiological status.     

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.     

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.     

Metazoan parasite species in cultured mussel Mytilus galloprovincialis in the Thermaikos Gulf (North Aegean Sea, Greece)

This is the first study on parasites of cultured Mytilus galloprovincialis L. in Greek waters, and is based on samples collected every 2 to 3 mo between September 2000 and November 2001 at 3 stations in the Thermaikos Gulf. Each sample comprised 40 mussels. We found 4 metazoan parasite species: hydroid Eugymnanthea inquilina, gill turbellarian Urastoma cyprinae, trematode Proctoces maculatus and gut copepod Mytilicola intestinalis. Of 840 mussels examined, 406 (48.3%) mussels were harbouring hydroids of E. inquilina, 278 (33.1%) were infested with U. cyprinae, 94 (11.2%) were infested with M. intestinalis, and only 7 (0.8%) were infested with P. maculatus. The prevalence and intensity of these parasites were related to temperature and pollution. Mussels infested with these parasites had significantly lower condition indices than non-infested mussels; larger mussels were more often infested than smaller ones.     

Multilocus allozyme differentiation of populations of the mussel Mytilus galloprovincialis Lmk. from Moroccan coasts

Along the Moroccan coasts, the systematic status of Mytilus populations have been for a long time uncertain and confused, due to the use of unreliable morphometric criteria. In the present study, allozyme markers reveal the exclusive existence of M. galloprovincialis on Mediterranean and Atlantic coasts. Nei's genetic distances are low and reflect a high gene flow between Atlantic and Mediterranean populations. However, a significant multilocus discontinuity revealed by F-statistics separate southern Atlantic populations from Mediterranean and north Atlantic ones and could be explained by a gene flow breaking because of a larval dispersal decrease, due to a sea surface current direction change from Cap Ghir towards the Canaries archipelago, and probably by differential selection effects in these two geographic areas.     

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.     

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.     

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.     

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.     

Soluble and membrane-bound acetylcholinesterases in Mytilus galloprovincialis (Pelecypoda: Filibranchia) from the northern Adriatic sea

Three forms of acetylcholinesterase (AChE) were detected in samples of the bivalve mollusc Mytilus galloprovincialis collected in sites of the Adriatic sea. Apart from the origin of the mussels, two spontaneously soluble (SS) AChE occur in the hemolymph and represent about 80% of total activity, perhaps hydrolyzing metabolism-borne choline esters. These hydrophilic enzymes (forms A and B) copurified by affinity chromatography (procainamide-Sepharose gel) and were separated by sucrose gradient centrifugation. They are, respectively, a globular tetramer (11.0-12.0 S) and a dimer (6.0-7.0 S) of catalytic subunits. The third form, also purified from tissue extracts by the same affinity matrix, proved to be an amphiphilic globular dimer (7.0 S) with a phosphatidylinositol tail giving cell membrane insertion, detergent (Triton X-100, Brij 96) interaction and self-aggregation. Such an AChE is likely functional in cholinergic synapses. All three AChE forms show a good substrate specificity and are inactive on butyrylthiocholine. Studies with inhibitors showed low inhibition by eserine and paraoxon, especially on SS forms, high sensitivity to 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide (BW284c51) and no inhibition with propoxur and diisopropylfluorophosphate (DFP). The ChE forms in M. galloprovincialis are possibly encoded by different genes. Some kinetic features of these enzymes suggest a genetic polymorphism.     

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.