Marine Mussel Mytilus Galloprovincialis Research Articles

EFFECTS OF NUTRITION-MEDIATED LARVAL CONDITION ON JUVENILE PERFORMANCE IN A MARINE MUSSEL

The potential for long-lasting effects of larval experience on early juvenile stages is growing in recognition as an important aspect of the ecology of organisms with complex life cycles. For marine species, most studies of recruitment have focused on the numbers of settlers that arrive to a site, largely ignoring the potential variability in larval condition or quality and how such variability may influence the likelihood of recruitment success. In this study, I investigated the effect of larval nutritional history on early juvenile performance, and whether these effects are mediated by conditions in the juvenile habitat for the marine mussel Mytilus galloprovincialis. I raised larvae in the laboratory under different food concentrations. Larvae raised with the highest food concentrations were significantly larger in size at metamorphosis and had higher proportional lipid content at metamorphosis than those from lower food concentrations. Half of the newly metamorphosed juveniles were outplanted into intertidal and subtidal habitats for 2 wk. The other half were maintained in the laboratory at two different juvenile rations for up to 20 d. Juveniles raised with high larval food had higher juvenile growth rates and attained larger juvenile sizes than those from lower larval food in all cases. There was a significant positive relationship between size at metamorphosis and juvenile growth for juveniles maintained in the laboratory. Early juvenile growth rates did not change over time (between 10 and 20 d post-metamorphosis). Juveniles raised in higher juvenile rations had elevated growth rates and attained larger final sizes after 20 d than juveniles from low juvenile rations across all sizes at metamorphosis. For juveniles in the field, there was no effect of tidal height on growth for 2 wk post-metamorphosis. Field conditions were more favorable than laboratory conditions for juvenile growth, yet even these more favorable conditions did not compensate for the effects of larval nutritional history on early juvenile growth. Losses of juveniles from both the field and the laboratory were higher for those that had been raised in lower larval food conditions than for those that had been raised in higher larval food, suggesting that vulnerability to early juvenile mortality may also be affected by larval history.

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.

Effects of heavy metals on phospholipase C in gill and digestive gland of the marine mussel Mytilus galloprovincialis Lam

We studied the in vivo and in vitro effects of Hg 2+ and Cu 2+ on the activity of phospholipase C (PLC), specific for phosphatidylinositol 4,5-bisphosphate, in the mussel ( Mytilus galloprovincialis Lam). The enzyme activity was assayed in tissue homogenates from gills and digestive gland. The toxic effect of Hg 2+ appeared to be stronger than that of Cu 2+ both in vitro and in vivo, especially for the digestive gland. In in vitro tests, Hg 2+ was able to inhibit PLC activity when added directly to the reaction mixture. Conversely, Cu 2+ was effective only after preincubation, suggesting that the effect of the metal may be derived from lipid peroxidation due to Cu 2+-induced oxyradical production. Treatment of mussels with sublethal concentrations of Hg 2+ or Cu 2+ in vivo produced significant PLC inhibition after 1 or 4 days, respectively. A recovery was reached after 7 days of in vivo metal incubation. Data indicate that in mussel gills and digestive gland heavy metals impair PLC activity, thereby affecting IP 3-dependent Ca 2+ signaling.

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: A biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.

Seasonal variation of xanthine oxidoreductase activity in the digestive gland cells of the mussel Mytilus galloprovincialis: A biochemical, histochemical and immunochemical study

The activity and the tissue distribution of the oxygen radical producing enzyme xanthine oxidoreductase (XOR) were measured in the digestive gland of the common marine mussel Mytilus galloprovincialis Lmk along an annual cycle. No xanthine oxidase (XOX) activity could be measured, the enzyme only displaying xanthine dehydrogenase (XDH) activity in all the cases. This is interpreted as a mechanism to avoid the harmful effects of the oxygen radicals that would be produced by XOX during periods following anoxic conditions at low tide. The highest XDH activities coincided with the late spring/early summer months, the activity maxima being recorded from May to July. Histochemically XOR activity was very pronounced in duct and stomach epithelial cells as well as in the surrounding connective tissue and hemolymph vessels, the activity increasing towards the summer months. These seasonal variations in XDH or XOR activities are possibly linked to hormonal changes governing the reproductive cycle and to changes in food availability. The localization of the protein in the connective tissue lining the hemolymph vessels was confirmed immunohistochemically using a polyclonal antibody against rat liver protein that cross-reacted specifically with a polypeptide of 150 kDa of molecular mass in homogenates of the digestive gland. This polypeptide was linked to cytosolic fractions isolated by differential centrifugation from mussel digestive glands. In paraffin sections the antibody labeled the digestive cells of digestive tubules, as well as the connective tissue surrounding the hemolymph vessels, gonadal follicles, digestive epithelia and certain protozoan parasites. Taken together our results suggest that in the digestive gland of bivalve molluscs XOR is involved in the metabolism of purines and in the scavenging of oxygen free radicals.

Correlation of CYP1A1 induction, as measured by the P450 RGS biomarker assay, with high molecular weight PAHs in mussels deployed at various sites in San Diego Bay in 1993 and 1995

In 1993 collections of marine mussels ( Mytilus galloprovincialis) were deployed 1 m from the water surface at six sites in San Diego Bay for 88 days. A similar mussel deployment was conducted in 1995, except the animals were deployed 1 m off the bottom and only for 32 days. After recovery from the sites, tissue was extracted with dichloromethane and the solvent extracts analyzed for chemical contaminant content and the ability to produce CYP1A1 induction in a transgenic cell line (TV101L cells). The cells used in the assay (P450 RGS) are stably transfected with a plasmid containing firefly luciferase linked to human CYP1A1 promoter sequences. Induction (fold increase compared to control) was determined by luminometry 16 h after application of small volumes (2–10 μl) of solvent extracts to cultured cells. Small mussels deployed in the Naval Station (NAV) in 1993 exhibited very high bioaccumulation of polycyclic aromatic hydrocarbons (PAHs; 52 μg/g) and polychlorinated biphenyls (PCBs), in addition to very strong induction of CYP1A1 measured by reporter gene system (RGS) responses. Large mussels deployed at the NAV station in 1993 and intermediate-sized animals placed at three stations within the NAV station in 1995 accumulated 13–29 μg PAH/g and exhibited relatively high RGS responses. Correlation of RGS responses for all mussel samples to the measured PAH concentrations was 0.85 ( r 2). When the concentrations of seven specific PAHs found in the samples are converted to benzo[a]pyrene equivalents, from previously derived toxic equivalency factors (TEFs) for this test system, and compared to measured RGS responses, the correlations are approximately 0.9. The results of these studies indicate that the RGS biomarker can be used as a screening tool for detection of CYP1A1-inducing compounds in tissues, and an estimate of potential human health or ecological risk from ingestion of contaminated organisms. Positive RGS responses can be followed by detailed chemical analyses of PAHs and coplanar PCBs using the same extract.

The activity of multixenobiotic resistance mechanism determined by rhodamine B-efflux method as a biomarker of exposure

An important characteristic of multixenobiotic resistance (MXR) is its inducibility. The level of MXR-expression in field populations of aquatic organisms is directly proportional to the level of pollution. This property offered the possibility to use the MXR-activity as a biomarker of exposure. However, before application of MXR-activity as a relevant biomarker, direct demonstration of the dynamic of MXR-activity response to exposure to xenobiotics, as well as its response to cessation of exposure, are required to make plausible that idea. For these reasons we examined the dynamics of the rate of induction of MXR-activity in marine mussel Mytilus galloprovincialis during either experimental exposure to seawater spiked with 1 μM rhodamine 123 or with Diesel-2 oil (equivalent to 100 ppb of Kuwait oil), or in-the-field exposure to pollution. The rate of deinduction was followed in field-specimens during the recovery period after their transfer from the polluted to the unpolluted site. The level of expression of MXR-transporter in these experiments was assessed by measurement of the rate of accumulation, or the rate of release, of rhodamine B. The induction of MXR-activity was completed within four days, and its deinduction was completed within the same time. However, despite this promptness in response, it remains to explore in future research whether the high natural level of the MXR-activity in combination with a relatively weak rate of induction allows its plausible use as a biomarker of exposure to xenobiotics.

Excitotoxicity in CA1 neurons of the rat hippocampus induced by local application of glutamate to a dendritic region

This study investigated the cytotoxic, oxidative and genotoxic effects of two commonly used imidazolium ionic liquids (ILs), [bmim][BF4] (1-butyl-3-methylimidazolium) and [omim][BF4] (1-methyl-3-octylimidazolium tetrafluoroborate), on the marine mussel Mytilus galloprovincialis, as well as whether acetone could mediate their toxic profile. In this context, mussels were firstly exposed to different concentrations of [bmim][BF4] or [omim][BF4], with or without the presence of acetone (at a final concentration of 0.06% v/v), for a period of 96 h, in order to determine the concentration that causes 50% mussel mortality (LC50 values) in each case. Thereafter, mussels were exposed to sub- and non-lethal concentrations of ILs for investigating their ability to cause lysosomal membrane impairment (with the use of neutral red retention assay/NRRT), superoxide anion and lipid peroxidation byproduct (malondialdehyde/MDA) formation, as well as DNA damage and formation of nuclear abnormalities in hemocytes. The results showed that [omim][BF4] was more toxic than [bmim][BF4] in all cases, while the presence of acetone resulted in a slight attenuation of its toxicity. The different toxic behavior of ILs was further revealed by the significantly lower levels of NRRT values observed in [omim][BF4]-treated mussels, compared to those occurring in [bmim][BF4] in all cases. Similarly, [bmim][BF4]-mediated oxidative and genotoxic effects were observed only in the highest concentration tested (10 mg L−1), while [omim][BF4]-mediated effects were enhanced at lower concentrations (0.01–0.05 mg L−1). Overall, the present study showed that [bmim][BF4] and [omim][BF4] could induce not only lethal but also nonlethal effects on mussel M. galloprovincialis. The extent of [bmim][BF4] and/or [omim][BF4]-mediated effects could be ascribed to the length of each IL alkyl chain, as well as to their lipophilicity. Moreover, the role of acetone on the obtained toxic effects of the specific ILs was reported for the first time, giving evidence for its interaction with the ILs and the modulation of their toxicity.

Inhibitors of the multixenobiotic resistance mechanism in natural waters: In vivo demonstration of their effects

AbstractXenobiotics in water that can inhibit the multixenobiotic resistance (MXR) defense mechanism in water organisms, the chemosensitizers, may cause severe ecotoxicological effects. We determined their concentrations in polluted and unpolluted river waters and seawaters using different methods. Here we present the method that enables direct determination of MXR‐inhibitory potential in natural waters. This method measures the modulating potential of river water or seawater on the rate of rhodamine B (RB) accumulation in gills of a freshwater clam (Dreissena polymorpha) or a marine mussel (Mytilus galloprovincialis), respectively. The sensitivity of the method with RB even enabled the determination of MXR inhibitors by measuring their potential to modulate the rate of efflux of RB from gills of mussels. The concentrations of MXR inhibitors found by these methods (expressed in μM of verapamil‐equivalents) were higher in natural waters from polluted rivers or in natural seawaters from polluted marine sites than in natural waters from unpolluted rivers or in natural seawaters from unpolluted sites. Polluted waters enhanced accumulation or decreased efflux rate of RB, a good substrate of P‐glycoprotein, demonstrating that the complex mixture of chemicals present in polluted waters contains MXR‐inhibiting potential. In addition, the efflux version of the method with RB has the clear advantage of not requiring that organisms be killed and so allows repeated use of the same individuals. In addition, it is the simplest and the most reliable method for measuring the activity of MXR in these organisms.

Expression of multixenobiotic resistance mechanism in a marine mussel Mytilus galloprovincialis as a biomarker of exposure to polluted environments

The gills of mussel Mytilus galloprovincialis express a multixenobiotic resistance mechanism (MXRM) mediated by P-glycoprotein activity, as evidenced by the rate of (G- 3H) vincristine accumulation and its sensitivity to verapamil, as well as by the direct immunochemical detection of P-glycoprotein by Western blotting analysis. The MXRM is induced at polluted sites: gills from mussels living at polluted sites accumulate less vincristine, the vincristine accumulation is less sensitive to verapamil, and in most cases express higher levels of P-glycoprotein. The state of induction of functional activity of MXRM is proportional to the level of pollution, as demonstrated at sites along the well defined pollution gradient in the aquatorium of Rovinj, Northern Adriatic. Since the immunological expression of P-glycoprotein may be compromised by high interindividual differences, season, and some as yet unknown factors, it could not be used as a single marker of exposure. However, the rate of vincristine accumulation and its sensitivity to verapamil offer a new, informatively powerful, molecular biomarker of exposure.

Reversion of the multixenobiotic resistance mechanism in gills of a marine mussel Mytilus galloprovincialis by a model inhibitor and environmental modulators of P170-glycoprotein

In gills of the marine mussel Mytilus galloprovincialis there is a P-glycoprotein-mediated multixenobiotic resistance mechanism (MXRM) which is similar to the multidrug resistance (MDR) mechanism in tumor cells resistant to cytotoxic drugs. This mechanism in mussel gills could be inhibited by verapamil, a known competitive inhibitor of P-glycoprotein, as measured by the enhanced accumulation of (G- 3H)vincristine (VCR). The activity of MXRM is indicated to be regulated in mussel by protein kinase C (PKC), since the PKC-specific inhibitor, bisindolylmaleimide, inhibits its activity. Similar inhibitory effects were exerted by a water-extract of Diesel-2 oil, or xenobiotics from the XAD-2 concentrates of river water or river sediment pore-water. MXRM was elevated in mussel specimens living at, or transplanted to, a polluted site, as demonstrated by the striking decrease in the rate of VCR accumulation. These results indicate that MXRM may be used as a biomarker of exposure to pollutants, and stress the importance of a new type of hazardous xenobiotic — the ‘chemosensitizers’ of MXRM.

Benzo(a)pyrene hydroxylase activity in the marine mussel Mytilus galloprovincialis: a potential marker of contamination by polycyclic aromatic hydrocarbon-type compounds

The aim of this work is to optimize and standardize the incubation conditions necessary to determine benzo(a)pyrene hydroxylase (BPH) activity in mussel microsomes. Evidence is presented that the reaction is dependent on NADPH and linear with time, at least up to the incubation time used. To measure BPH activity with the highest efficiency, the concentrations of benzo(a)pyrene (70 μM) microsomal proteins (0–75 mg/0–8 ml) and NADPH (0.74 mM), and the incubation time (10 min) were optimized to avoid plateauing of the activity due to a lack of substrate or NADPH. The best compromise between signal/noise ratio and linearity of the reaction with time was considered. BPH induction in mussels treated with 3-methylcholanthrene or exposed to field contamination by polycyclic aromatic hydrocarbons (PAH)-type compounds demonstrated the ability of this technique to be a potential marker of exposure to PAH type compound.

DNA damage in marine mussel Mytilus galloprovincialis as a biomarker of environmental contamination

DNA damage in marine mussel Mytilus galloprovincialis as a biomarker of environmental contamination

DNA damage in marine mussel Mytilus galloprovincialis as a biomarker of environmental contamination

DNA damage in marine mussel Mytilus galloprovincialis as a biomarker of environmental contamination

Effects and fate of sediment-sorbed linear alkylbenzene sulphonate (LAS) on the bivalve mollusc mytilus galloprovincialis Lmk

The toxicity of LAS on aquatic animals has been evaluated in particular relation to its concentration in water, although it is well known that sediments play an important role as repositories and sources of many contaminants, including surfactants. The aim of this study was to evaluate the effects of sediment-sorbed LAS on the marine mussel Mytilus galloprovincialis exposed to sediments drugged with 132 mg/kg dry weight of LAS mixtures. Four experiments were performed and some physiological parameters (filtration, oxygen uptake, nitrogen excretion) were measured on control and treated animals. In order to study uptake and release of sediment-sorbed LAS by mussels, quantification of weight, percentage of organic matter, LAS concentration and LAS homologue distribution on faeces were determined. Results showed no significant differences in physiological responses of treated mussels compared to controls, and the absence of a toxic action of LAS contaminated sediments. Moreover one notable aspect was that higher LAS concentrations were found in faeces than in uningested sediments recovered from water. This fact is tentatively explained either as the result of decreased desorption in the mussel intestinal tract with respect to circulating water, or as enrichment in fine particles by mussel filter feeding activity.

Biochemical characterization of a phosphatidylinositol 4,5-bisphosphate-specific phospholipase C activity in gills and digestive gland of the marine mussel Mytilus galloprovincialis lam

1. 1. Polyphosphoinositide-specific phosphodiesterase (phospholipase C, PLC) activity against phosphatidylinositol 4,5-bisphosphate, present in gill and digestive gland homogenates of mussel ( Mytilus galloprovincialis Lam.), has been biochemically characterized. 2. 2. The enzyme was strictly modulated by free calcium ion concentration in both tissues and maximally activated at 10 −5 M Ca 2+ (19 ± 4 and 11 ± 2 nmol phosphatidylinositol 4,5-bisphosphate hydrolysed/min/mg of protein for gill and digestive gland PLC, respectively, at 19°C). Optimum pH at 10 −5 M Ca 2+ was around 7.0 in both cases. The Ca 2+-stimulated PLC activity showed high specificity for PIP 2; the K Ma for PIP 2 were 150 and 170 μM for the gills and digestive gland, respectively. 3. 3. Good substrate dispersion was obtained in the presence of sodium deoxycholate; the concentration routinely used in the assay (0.08%) produced a 9-fold activation of both gill and digestive gland PLC, consistent with previous reports. 4. 4. The possible biochemical and physiological role of the enzyme in mussel tissues is discussed.

Evidence for a multixenobiotic resistance mechanism in the mussel Mytilus galloprovincialis

Membrane vesicles isolated from the gills, mantle, and digestive gland of the marine mussel Mytilus galloprovincialis possess a potential similar to that involved in multidnig resistance (MDR) described in resistant tumor cell lines: they bind 2-acetylaminofluorene (AAF) in saturable and trypsin- and verapamil-sensitive manner. Cytosolic fractions from mussel tissue homogenates reveal a relatively high glulathione- S-transferase (GST) and glutathione-peroxidase (GSH-Px) activities with substrate affinities similar to the isoenzyme of anionic GST and its intrinsic GSH-Px involved in MDR. In mussel these biochemical elements of the MDR mechanism seem to be inherent, rather than induced, since they were found in specimens living in the pristine aquarium of Limski Kanal, Northern Adriatic. The enhancement in the accumulation of AAF in laboratory exposure experiments in the presence of verapamil indicates that the MDR-like mechanism functions in mussel also in vivo. The recognition that the MDR-like mechanism is operative in mussel may explain the relative resistance of this species to pollution. The knowledge that the presence of one xenobiotic may block the pumping out, and hence accelerating accumulation, of other(s), may help us to understand and interpret our present and past data on different parameters obtained with this most exploited indicator organism.

Induction of micronuclei by mitomycin C and colchicine in the marine mussel Mytilus galloprovincialis

The frequencies of micronuclei (MNi) in the gill cells of the mussel Mytilus galloprovincialis were determined over a long period (up to 28 days) following a 48-h treatment with colchicine. The frequency of MNi at the end of treatment was significantly higher than in controls and 24 h later it had increased even more. After this period, the frequency of MNi rapidly declined until a plateau level was reached on day 2–3, which was significantly higher than the control baseline level, and persisted until the end of the experiment (28th day). In the same cell system we previously reported a persistence of an increased frequency of MNi after treatment with mitomycin C (MMC) (Majone et al., 1987). In order to establish the origin of MNi, the difference between their size distribution in MMC- and colchicine-treated animals was determined at the end of treatment as well as during the plateau phase. The difference was statistically significant ( P < 0.001) at the end of treatment, the MNi induced by MMC being smaller than those induced by colchicine. However, the difference during the plateau phase was not statistically significant. Human CREST antikinetochore fluorescent antibodies reacted with chromosome centromeres of Mytilus and were applied to gill cells at the end of a 48-h treatment with MMC or colchicine. About 60% of the MNi induced by colchicine but only 30% of those produced by MMC reacted positively with the fluorescent antibodies. This result indicates that the majority of MNi observed at the end of a 48-h treatment with MMC or colchicine originate, respectively, from acentric chromosome fragments and from whole lagging chromosomes.

Frequencies of micronuclei detected on Mytilus galloprovincialis by different staining techniques after treatment with zinc chloride

The frequencies of micronuclei induced by ZnCl 2 and detected on the gill tissue of the marine mussel Mytilus galloprovincialis with different staining techniques (acridine orange, gallocyanin chromallum, Feulgen, Giemsa) were compared. At least in the used system, the Feulgen and gallocyanin chromallum methods gave a frequency of micronuclei significantly lower than that obtained with the acridine orange and Giemsa techniques. No significant difference between the frequencies obtained with acridine orange and Giemsa was shown. So, though the acridine orange is surely the method which provides the more reliable data, in environmental screening works the Giemsa technique may be more suitable for its simplicity.

Preliminary data on PAH metabolism in the marine mussel Mytilus galloprovincialis from Arcachon Bay, France

Oxidation of polycyclic aromatic hydrocarbons (PAH) and epoxide-conjugation capacities were examined from mussels, Mytilus galloprovincialis, collected from a “clean” site at the entrance to Arcachon Bay (south-west France) in 1984, using subcellular preparations from whole bodies and digestive glands. The existence of low-cytochrome P-450-dependent oxygenase activities [cytochrome P-450 content, NADPH cytochrome c reductase and benzopyrene monooxygenase (BaPMO) activities] was confirmed, as well as the presence of a comparatively important capacity for epoxide conjugation [epoxide hydrolase (EH), glutathione S epoxide-transferase (GST) activities and total cytosolic-glutathione content]. The optimal incubation temperatures for the individual in vitro enzyme activities in the mussel (25°C for BaPMO, 31°C for EH and GST) were lower than that required for optimal mammalian enzyme activities. Except for cytochrome P-450 content, there was no significant difference in enzyme activities recorded in whole-body and digestive gland fractions.