Gills Of Mytilus Galloprovincialis Research Articles

Difference in composition and functional analysis of bacterial communities between Mytilus galloprovincialis gills and surrounding water in a brackish inshore bay, analyzed by 16S rDNA multi-amplicon sequencing

BackgroundLagoons are important natural systems, with attractive favorable conditions for aquaculture production, such as shellfish cultivation. Despite their socio-economic relevance for human activity, information on the microbial diversity, community composition, and putative functions of gill-associated microbiota and seawater is still limited, particularly as regards the extent of specific taxa enrichment within the gills and the relative effects of the temporal and spatial variables. In this study, we used a 16S rDNA multi-amplicon sequencing approach using an Ion GeneStudio S5 System and a function prediction method (Functional Annotation of Prokaryotic Taxa (FAPROTAX), to inspect the springtime dynamics of microbial communities and their inferred metabolic features in an Adriatic lagoon (Po Delta, Italy).ResultsMussels and surrounding seawater were sampled in two rearing areas three times between April and June 2021. Sequencing results showed significant (p ≤ 0.05) differences in bacterial community composition and diversity between gills and seawater. Gills were dominated by the Methylobacterium-Methylorubrum and Burkholderia-Caballeronia-Paraburkholderia genera, while in seawater samples Izamaplasma, Planktomarina, and Candidatus Aquiluna were detected as being dominant. The microbiota composition did not differ significantly between the two rearing areas. The sampling time, although limited to a 3-month timeframe, instead revealed a structural variation of the bacterial profile both in gills and seawater for alpha and beta diversities respectively. The functional prediction analysis highlighted an overexpression of human gut-associated bacteria in relation to the season-related increase in seawater temperature.ConclusionsThese findings enhance our understanding of the differences between gill-associated and seawater microbiota composition and provide novel insights into the functions carried out by bacteria inhabiting these niches, as well as on the key host-symbiont relationships of bivalves in lagoon environments.

Effects of rare earth element samarium doped zinc oxide nanoparticles on Mytilus galloprovincialis (Lamarck, 1819): Filtration rates and histopathology

BackgroundDoping was reported to improve the photo catalytic performance, antioxidant, antibacterial and other biological properties of nanoparticles. While, improving the nanoparticle properties, doping could change toxicity profile to living organism. Hence, the aim of this work was to assess the effects of samarium doped zinc oxide nanoparticles (Sm doped ZnO NPs) on the edible mussel Mytilus galloprovincialis. MethodsSm doped ZnO nanoparticles were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM) and Fourier transform infrared spectroscopy (FTIR) techniques. 156 mussels were exposed during 7 days to a low, intermediate and high concentration of Sm doped ZnO NPs (0.5, 1 and 1.5 mg/L, respectively). The filtration rates were assessed after 1 and 2 h. Histopathological alterations were determined in gills, digestive glands and gonads using a quantitative analysis. ResultsThe filtration rates decreased in all individuals exposed to Sm doped ZnO NPs, a significant decrease was noted with the low and intermediate concentration (0.5 and 1 mg/L) of Sm doped ZnO NPs after 1 and 2 h, respectively. The histopathological index (Ih) estimated for gills, digestive glands and gonads showed differences depending on the organ and the nanoparticle concentration. The highest Ih were reported for digestive glands and female gonads exposed to the intermediate concentration (1 mg/L) of Sm doped ZnO NPs. As for gills and male gonads, the highest Ih were noted with the high concentration (1.5 mg/L) of Sm doped ZnO NPs. ConclusionResults from this study revealed the toxicity of Sm doped ZnO NPs in Mytilus galloprovincialis gills, digestive glands and gonads. The toxicity induced by this nanoparticle varies depending on the organ and the concentration.

Effects of ifosfamide and cisplatin exposure combined with a climate change scenario on the transcriptome responses of the mussel Mytilus galloprovincialis

Coastal ecosystems are currently exposed to pollutants and climate change. Namely, the increasing consumption of antineoplastic drugs and their potential release to aquatic ecosystems are raising concerns. Nevertheless, information regarding the toxicity of these drugs towards non-target species is scarce, especially considering climate change scenarios. Ifosfamide (IF) and cisplatin (CDDP) are among the antineoplastics already detected in aquatic compartments and due to their mode of action (MoA) can negatively affect aquatic organisms. This study evaluates the transcription of 17 selected target genes related to the MoA of IF and CDDP in Mytilus galloprovincialis gills exposed to environmentally relevant and toxicological meaningful concentrations (IF – 10, 100, 500 ng/L; CDDP – 10, 100, 1000 ng/L), under an actual (17 °C) and predicted warming scenario (21 °C). Results showed an upregulation of the cyp4y1 gene when exposed to the highest concentrations of IF, regardless of the temperature. Both drugs upregulated genes related to DNA damage and apoptosis (p53, caspase 8 and gadd45), especially under warmer conditions. Increased temperature also downregulated genes related to stress and immune responses (krs and mydd88). Therefore, the present results showed a gene transcriptional response of mussels to increasing concentrations of antineoplastics and that warmer temperatures modulated those effects.

Multi-biomarker approach in Mytilus galloprovincialis and Ruditapes decussatus as a predictor of pelago-benthic responses after exposure to Benzo[a]Pyrene

This study evaluated the biomarker responses indicative of exposure to Benzo[a] Pyrene (B[a]P) in Mytilus galloprovincialis and Ruditapes decussatus. A significant increase of the total oxyradical scavenging capacity (TOSC) was observed after seven days of exposure to two concentrations of B[a]P (100 and 300 μg.L−1), in the digestive gland with the lowest concentration tested. The TOSC in the gills increased notably only after the exposure to 300 μg.L−1 of B[a]P. Interestingly, the superoxide dismutase (SOD) and catalase (CAT) activities in gills and digestive gland on one hand and glutathione S-transferase (GST) in gills in the other, were positively correlated with the concentration of B[a]P with a significant induction noticed at the highest concentration. In contrast, a significant increase of the GST activity was observed in the digestive gland following the exposure of bivalves to 100 μg.L−1. In pelagic (M. galloprovincialis) or benthic (R. decussatus) bivalves, the AChE activity decreased discernibly in digestive glands and gills with the increase of B[a]P concentrations as evidence of neurotoxic effects. In clams, the exposure to B[a]P was followed by a significant increase of Malondialdehyde level (MDA) in gills and digestive gland, this does not occur in gills of Mytilus galloprovincialis at the concentration of 100 μg.L−1. Overall, the results found seems to indicate that the mussel was more suitable as a predictor tool of toxicity of B[a]P.

İzmir Körfezi (Ege Denizi, Türkiye) genotoksisitenin değerlendirilmesinde biyobelirteç olarak Midye (Mytilus galloprovincialis) hemolenf, karaciğer ve solungaç hücrelerinde mikronükleus oluşumu

The sea has involved a large variety of environmental contaminants and plays a crucial role in aquatic ecosystems. The Izmir Bay, which has been rapidly polluted since the 1960s, was one of the intensely polluted areas in the Mediterranean. Organic materials, hydrocarbons, metals, and pathogenic organisms that are accumulated in the region, caused high pollution and threaten health and aesthetics. Because of this, the aim of this paper was focus on to investigations of the genetic damages in mussels in Izmir Bay. Investigations of mutagenic/carcinogenic potential have an advantage in genotoxicity studies because biomarker for pollution exposure in mussel is the early detection of possible long-term effects such as cancer. Therefore, genotoxicity was the focus of the biomarker investigations in mussel during the investigations. Thus, micronucleus tests were afforded to determine genetic damage in the haemolymph, liver, and gills of Mytilus galloprovincialis living in Izmir Bay (Western Coast of Turkey). In the present study, results showed that the frequency of MN was found at a high level in station 4 and station 5 where wastes from dockyard existed contributed to the high level of pollution. The recommendation is to standardize procedures for assessment of the toxic impact of pollutants at the cellular level in aquatic species by using micronucleus assays for biomonitoring of environmental pollution.

The proline-rich myticalins from Mytilus galloprovincialis display a membrane-permeabilizing antimicrobial mode of action

Bivalve mollusks are continuously exposed to potentially pathogenic microorganisms living in the marine environment. Not surprisingly, these filter-feeders developed a robust innate immunity to protect themselves, which includes a broad panel of antimicrobial peptides. Among these, myticalins represent a recently discovered family of linear cationic peptides expressed in the gills of Mytilus galloprovincialis. Even though myticalins and insect and mammalian proline-rich antimicrobial peptides (PrAMPs) share a similar amino acid composition, we here show that none of the tested mussel peptides use a non-lytic mode of action relying on the bacterial transporter SbmA. On the other hand, all the tested myticalins perturbed and permeabilized the membranes of E. coli BW25113, as shown by flow-cytometry and atomic force microscopy. Circular dichroism spectra revealed that most myticalins did not adopt recognizable secondary structures in the presence of amphipathic environments, such as biological membranes. To explore possible uses of myticalins for biotech, we assessed their biocompatibility with a human cell line. Non-negligible cytotoxic effects displayed by myticalins indicate that their optimization would be required before their further use as lead compounds in the development of new antibiotics.

Microplastic intake and enzymatic responses in Mytilus galloprovincialis reared at the vicinities of an aquaculture station

Aquaculture is a potential source of microplastics (MPs) that could be strong stressors for marine organisms. In this study, we evaluated the effects of MPs derived from aquaculture in antioxidant defences and oxidative stress markers in gills of Mytilus galloprovincialis. Mussels were distributed in three areas with different impacts: inside aquaculture cages, Control 1 (located inside Andratx harbour) and Control 2 (located in a no-anthropized area). Samples were obtained along three different time periods in May (T0), July (T60) and in September (T120). At each sampling period, mussels’ biometric measurements were taken, and tissue samples were kept frozen for biochemical determinations and to determine the intake of MPs. An increase in MPs intake was detected throughout the study, and this increase was significantly higher in samples from the aquaculture cages. Similarly, antioxidant enzyme activities (catalase, superoxide dismutase, glutathione reductase and glutathione peroxidase) were significantly higher in samples from cages at T120. Additionally, a similar tendency was observed in glutathione-s-transferase, with a higher activity in the aquaculture cages at T60 and T120. Malondialdehyde and carbonyl protein derivates as a marker of oxidative damage were also measured and samples from aquaculture cages presented higher oxidative stress markers, mainly in T120. In conclusion, living in environments exposed to aquaculture activities at sea may imply a higher intake of MPs which in turn might cause an antioxidant response in M. galloprovincialis which is not enough to avoid oxidative damage.

Hypoxia exerts oxidative stress and changes in expression of antioxidant enzyme genes in gills of Mytilus galloprovincialis (Lamarck, 1819)

ABSTRACT Bivalve molluscs are widespread in coastal environments, where they are subjected to a wide range of environmental fluctuations, which can represent a significant impact to their antioxidant status. Hypoxia is a global problem affecting the world’s shallow waters, which is forecast to increase in coastal areas due to anthropogenic pressure and global warming. In this work, the effects of 24 h exposure to hypoxia (0.2 mg O2 l−1) on antioxidant parameters of Mytilus galloprovincialis were investigated. Functional parameters of gill samples (the number of dead cells and intracellular reactive oxygen species concentration) were measured by flow cytometry, and the expression level of antioxidant enzyme genes (CAT, GST, SOD) as well as NADH dehydrogenase subunit 2 were analysed using transcriptome analysis. Gills responded to oxygen depletion by an increased total number of intracellular reactive oxygen species, which was accompanied with high levels of mortality of cells (10 times higher compared with control). Significant increases of Mn-superoxide dismutase, glutathione S-transferase and NADH dehydrogenase subunit 2 gene expression levels were observed. Hypoxia did not cause changes in the expression of CAT. The results indicate an overall shift in antioxidant parameters in gill tissue that may represent a certain level of oxidative stress in mussels and decreased capacity of the antioxidant system to maintain redox homeostasis after exposure to hypoxia.

Esterase activity (EA), total oxidant status (TOS) and total antioxidant capacity (TAC) in gills of Mytilus galloprovincialis exposed to pollutants: Analytical validation and effects evaluation by single and mixed heavy metal exposure

The aims of the present study were to optimize and validate methods for esterase activity (EA), total oxidant status (TOS) and total antioxidant capacity (TAC) determination in mussel' gills, and to establish the relationships between these biomarkers and Pb, Cd and Cu pollution, in single form and ternary mixture. Two different buffers for sample homogenization, the need of ultracentrifugation, and analytical validation were evaluated. Coefficients of variation, when buffer without additives and ultracentrifugation were used, were <15%, and recovery were 97%–109% in all cases. The EA response tends to decrease with treatments, TOS decreased significantly in Cd and ternary groups, while TAC tended to increase in treatments with Pb, Cd and ternary groups. In conclusion, the methods for EA, TOS and TAC measurements in gills of mussel were precise and accurate and could be interesting resources in biomonitoring programmes.

Is gene transcription in mussel gills altered after exposure to Ag nanoparticles?

Nanotechnology is a rapid field of development with the enhancement of the production of different types of nanoparticles (NPs) applied in several industrial and commercial applications which increase the risk of their presence in the aquatic environment. Ag NPs have a wide application in everyday life products. However, there is concern about the exposure effects on aquatic organisms to these NPs. Therefore, this study aims to assess gene transcription alterations in mussels Mytilus galloprovincialis gills exposed for 2 weeks to Ag NPs (42 ± 10 nm, 10 μg.L(-1)). The genes were selected based on previous biomarkers and proteomic results and included superoxide dismutase (SOD), catalase (CAT), glutathione transferase (GST), caspase 3/7-1 (CAS), cathepsin L (CATH), heat-shock protein 70 (HSP 70), cytochrome P450 4YA (CYP 4YA), the elongation factor (EF1), actin and α- tubulin. No significant changes in gene transcription profiles were observed after exposure of M. galloprovincialis to Ag NPs for 15 days. The lack of significant gene transcription responses is in light with previous results obtained for mussels exposed to these NPs and may be related to the fact that enzyme kinetics and relative abundance of proteins (increase of antioxidant enzymes and metalllothioneins (MTs) with the time of exposure) do not always directly reflect their relative mRNA levels. Nevertheless, their overall expression maintenance may signify that, at end of the exposure period (15 days), the transcription of the respective genes is no longer required, pointing out to a possible adaptation effect to nanoparticles or due to the levels of Ag NPs accumulated in this tissue at this exposure time. This study highlights that gene transcription application and role as an additional and/or alternative end point approach is important to understand the mode of action of these emergent contaminants in aquatic organisms. However, in future studies, the time window needs to be adjusted, as genes are likely to respond earlier to the nanoparticle exposure.

Titanium dioxide nanoparticles modulate the toxicological response to cadmium in the gills of Mytilus galloprovincialis

We investigated the influence of titanium dioxide nanoparticles (nano-TiO2) on the response to cadmium in the gills of the marine mussel Mytilus galloprovincialis in terms of accumulation and toxicity. Mussels were in vivo exposed to nano-TiO2, CdCl2, alone and in combination. Several cellular biomarkers were investigated in gills: ABC transport proteins and metallothioneins at gene/protein (abcb1, abcc-like and mt-20) and functional level, GST activity, NO production and DNA damage (Comet assay). Accumulation of total Cd and titanium in gills as in whole soft tissue was also investigated. Significant responses to Cd exposure were observed in mussel gills as up-regulation of abcb1 and mt-20 gene transcription, increases in total MT content, P-gp efflux and GST activity, DNA damage and NO production. Nano-TiO2 alone increased P-gp efflux activity and NO production. When combined with Cd, nano-TiO2 reduced the metal-induced effects by significantly lowering abcb1 gene transcription, GST activity, and DNA damage, whereas, additive effects were observed on NO production. A lower concentration of Cd was observed in the gills upon co-exposure, whereas, Ti levels were unaffected. A competitive effect in uptake/accumulation of nano-TiO2 and Cd seems to occur in gills. A confirmation is given by the observed absence of adsorption of Cd onto nano-TiO2 in sea water media.

Comparation of Heavy Metals and Selenium Contents in The Digestive Gland and Gills of Mytilus galloprovincialis (Lamarck, 1819) Caught in Izmir Bay (Turkey)

Objective: The aim of this study was to evaluate heavy metals (Cr, Fe, Ni, Cu, Zn, As, Cd, Hg, Pb) and selenium levels in digestive gland and gill tissues of wild Mediterranean mussel Mytilus galloprovincialis (Lamarck, 1819) collected from four locations in Izmir Bay. Methods: A total of 180 samples of the wild Mediterranean mussel M. galloprovincialis with shell length of 50-60 mm were collected from four locations. Inductively coupled plasmamass spectroscopy was used to determine heavy metals and selenium concentrations in mussel samples after microwave digestion process. Results: The highest values (mg kg-1 dry weight) obtained for digestive gland and gills were 0.17 and 0.15 for Cr, 28.62 and 29.49 for Fe, 0.25 and 0.29 for Ni, 2.53 and 1.78 for Cu, 18.52 and 22.03 for Zn, 1.26 and 1.08 for As, 0.04 and 0.04 for Cd, 0.02 and 0.02 for Hg, 0.19 and 0.16 for Pb, 0.40 and 0.48 for Se, respectively. Statistically significant differences among digestive gland and gills (p<0.05) were found for Cr, Ni, Zn, As, Se and Cd. In general, the

DNA damage and transcriptional changes in the gills of mytilus galloprovincialis exposed to nanomolar doses of combined metal salts (Cd, Cu, Hg).

Aiming at an integrated and mechanistic view of the early biological effects of selected metals in the marine sentinel organism Mytilus galloprovincialis, we exposed mussels for 48 hours to 50, 100 and 200 nM solutions of equimolar Cd, Cu and Hg salts and measured cytological and molecular biomarkers in parallel. Focusing on the mussel gills, first target of toxic water contaminants and actively proliferating tissue, we detected significant dose-related increases of cells with micronuclei and other nuclear abnormalities in the treated mussels, with differences in the bioconcentration of the three metals determined in the mussel flesh by atomic absorption spectrometry. Gene expression profiles, determined in the same individual gills in parallel, revealed some transcriptional changes at the 50 nM dose, and substantial increases of differentially expressed genes at the 100 and 200 nM doses, with roughly similar amounts of up- and down-regulated genes. The functional annotation of gill transcripts with consistent expression trends and significantly altered at least in one dose point disclosed the complexity of the induced cell response. The most evident transcriptional changes concerned protein synthesis and turnover, ion homeostasis, cell cycle regulation and apoptosis, and intracellular trafficking (transcript sequences denoting heat shock proteins, metal binding thioneins, sequestosome 1 and proteasome subunits, and GADD45 exemplify up-regulated genes while transcript sequences denoting actin, tubulins and the apoptosis inhibitor 1 exemplify down-regulated genes). Overall, nanomolar doses of co-occurring free metal ions have induced significant structural and functional changes in the mussel gills: the intensity of response to the stimulus measured in laboratory supports the additional validation of molecular markers of metal exposure to be used in Mussel Watch programs.

Effects of sublethal, environmentally relevant concentrations of hexavalent chromium in the gills of Mytilus galloprovincialis

Hexavalent chromium Cr(VI) is an important contaminant released from both domestic and industrial effluents, and represents the predominant chemical form of the metal in aquatic ecosystems. In the marine bivalve Mytilus galloprovincialis exposure to non-toxic, environmentally relevant concentrations of Cr(VI) was shown to modulate functional parameters and gene expression in both the digestive gland and hemocytes. In this work, the effects of exposure to Cr(VI) (0.1–1–10μgL−1animal−1 for 96h) in mussel gills were investigated. Gill morphology and immunolocalization of GSH-transferase (GST), of components involved in cholinergic (AChE and ChAT), adrenergic (TH) and serotoninergic (5-HT3 receptor) systems, regulating gill motility, were evaluated. Total glutathione content, activities of GSH-related enzymes (glutathione reductase – GSR, GST), of catalase, and of key glycolytic enzymes (phosphofructokinase – PFK and pyruvate kinase – PK) were determined. Moreover, mRNA expression of selected Mytilus genes (GST-π, metallothionein isoforms MT10 and MT20, HSP70 and 5-HT receptor) was assessed by RT-q-PCR. Cr(VI) exposure induced progressive changes in gill morphology and in immunoreactivity to components involved in neurotransmission that were particularly evident at the highest concentration tested, and associated with large metal accumulation. Cr(VI) increased the activities of GST and GSR, and total glutathione content to a different extent at different metal concentrations, this suggesting Cr(VI) detoxication/reduction at the site of metal entry. Cr(VI) exposure also increased the activity of glycolytic enzymes, indicating modulation of carbohydrate metabolism. Significant changes in transcription of different genes were observed. In particular, the mRNA level for the 5-HTR was increased, whereas both decreases and increases were observed for GST-π, MT10, MT20 and HSP70 mRNAs, showing sex- and concentration-related differences. The results demonstrate that Cr(VI) significantly affected functional and molecular parameters in mussel gills, and indicate that this tissue represents the major target of exposure to environmentally relevant concentrations of the metal.

Induction of apoptosis in mussel Mytilus galloprovincialis gills by model cytotoxic agents

Apoptosis signaling pathway was investigated in the marine mussel Mytilus galloprovincialis exposed to various stressors. Analyses were performed in mussels exposed to two major pollutants of the aquatic environment: tributyltin and the water soluble fraction of diesel oil, for 1 h and animals were then maintained in sea water for a recovery period of 6 and 24 h. Apoptosis was evaluated at several levels of the cell signaling cascade by measuring Bcl-xS expression, caspase-3 activity and DNA damage (Fast micromethod(®) and TUNEL techniques). H(2)O(2) was used as a control of apoptosis induction for validation of the assays. Results showed an induction of Bcl-xS expression, a protein implicated in apoptosis, after 1 h exposure to all concentrations of chemicals. Moreover, in the same manner, apoptotic DNA damage was induced with all chemicals tested. Besides, caspase 3 activity was detected after 1 h exposure to low doses of TBT and diesel oil while the high concentrations induced this protein after 6 h. The achieved data were also correlated with our previous study, demonstrating an induction of the mitogen-activated protein kinase (MAPK) activity in the mussel M. galloprovincialis exposed to the same conditions. In conclusion, this study was one of the first characterizing the MAP kinase cell signaling pathway leading to apoptosis in the mussel M. galloprovincialis exposed to chemicals. It showed for the first time that the Bcl-xS protein was present in these mussels as in other species and played a role in apoptosis mediation. Moreover, the main originality of this work was that it showed that two apoptotic pathways might be present in the mussel: a caspase 3-dependent and a caspase 3-independent pathways.

Non-steroidal anti-inflammatory drug (NSAID) ibuprofen distresses antioxidant defense system in mussel Mytilus galloprovincialis gills

Active pharmaceutical ingredients (APIs) are presently considered an emergent class of environmental contaminants. Ibuprofen (IBU) is one of the most applied non-steroidal anti-inflammatory drugs (NSAIDs) in the world. Several authors report the occurrence of IBU in influents and effluents of waste water treatment plants (WWTPs), surface, river and public tap water in numerous countries. However, very little is known about the risks and chronic effects of IBU exposure in non-target organisms. This approach undertakes the assessment of several oxidative stress biomarkers responses through the analysis of antioxidant enzymes activities (superoxide dismutase – SOD, catalase – CAT, glutathione S-transferase – GST, glutathione reductase – GR) and lipid peroxidation (LPO) levels in sentinel species mussel Mytilus galloprovincialis gills exposed for 2 weeks to an environmental realistic concentration of IBU. Results clearly show the significant induction and positive correlation between SOD activity and LPO in exposed gills, concomitant to an antioxidant defense depletion of CAT, GR and GST compared to controls. The integration of all biomarkers in mussels’ gills separates non- and exposed groups supporting the breakdown of the redox defense system and IBU's pro-oxidant action. Further studies are needed to test possible endocrine disruption effects in mussels’ reproduction fitness as IBU is involved on prostaglandins biosynthesis inhibition.

Hyperthermia-induced Hsp70 and MT20 transcriptional upregulation are mediated by p38-MAPK and JNKs inMytilus galloprovincialis(Lamarck); a pro-survival response

In the present study we investigated the signal transduction cascades triggered by acute thermal stress in Mytilus galloprovincialis gills. This particular species has been reported to exhibit a significant tolerance to high temperatures; thus, it was intriguing to examine the molecular mechanisms responsible for this extraordinary trait. In particular, exposure to 30 degrees C was found to cause a significant and sustained stimulation of p38-MAPK phosphorylation while the activation profile of JNKs was transient and relatively moderate. We also observed that hyperthermia induced apoptosis as a delayed response, with both MAPK subfamilies rapidly translocating to the nucleus. The phosphorylation of cJun, ATF2 and NFkappaB was detected next. Using selective inhibitors, phosphorylation of these transcription factors was established to be dependent on p38-MAPK or JNKs. Subsequently, potential changes in gene expression were assessed. In this context, hyperthermia resulted in the transcriptional upregulation of Hsp70 and MT20 genes with a widely known salutary effect, preserving mussel fitness and performance under adverse environmental conditions. Interestingly, p38-MAPK and JNKs were found to mediate the hyperthermia-induced Hsp70 and MT20 upregulation as well as the delayed induction of apoptosis under the interventions studied. Overall this is, to our knowledge, the first time that an insight into the compensatory survival ;programme' initiated in Mytilus galloprovincialis gills, contributing to this organism's exceptional tolerance to thermal stress, has been gained. In particular, we provide evidence demonstrating the principal role of p38-MAPK and JNKs in transducing the stress signal via mobilization of specific transcription factors and the transcriptional upregulation of cytoprotective genes.

Expression of metallothionein mRNAs by in situ hybridization in the gills of Mytilus galloprovincialis, from natural polluted environments

Metallothioneins (MTs), metal-inducible proteins, are crucial proteins for the regulation of essential metals, and are transcriptionally induced in all organisms by certain heavy metals, oxidative stress and inflammation. The gills represent an organ of uptake and loss of metals in which different mechanisms are present controlling the functions directly involved in the maintenance of homeostasis. In this study, the morphological and histomorphological aspects of branchial epithelium in Mytilus galloprovincialis from polluted environment (Faro swamp, Messina, Italy) have been investigated. The reverse transcriptase-polymerase chain reaction (PCR) has been used to isolate complementary DNA of both MT isoforms present from RNA extracted from mussel gills. The respective mRNAs on histological sections have been visualized by in situ hybridization. These methods showed that MT-10 mRNA is expressed at the basal level. In contrast, the MT-20 expression level was very low under basal conditions, while its mRNA increased dramatically in individuals collected in Faro. The presence of acid mucocytes and MTs in the gills may be considered a further defensive mechanism also related to the significantly higher concentration of Cd, Pb and Cr found in gills of M. galloprovincialis from Faro than specimens from the reference site (Goro). The results obtained show that, in stressed mussels, the defensive processes increase to maintain the normal functions of the organs more exposed to the action of polluted substances.

Adenylyl cyclase activity and its modulation in the gills of Mytilus galloprovincialis exposed to Cr 6+ and Cu 2+

The adenylyl cyclase (AC)/cAMP system regulates a large number of physiological functions in bivalve mussels, although its basal properties and the potential effects of environmental pollutants are scarcely studied. We characterized some properties of AC and measured both the enzyme activity and the cAMP levels in the gills of the filter-feeding sea mussel Mytilus galloprovincialis. Basal AC activity was 5.6 ± 0.8 pmol cAMP 10 min −1 mg protein −1 and showed a K m value of 0.82 ± 0.06 mM for ATP in the presence of 5 mM Mg 2+. It was stimulated up to 2.5- and 3.5-fold by 5-HT and GTPγS, respectively. Similarly to what was found in other bivalves, forskolin is a poor activator that reached significant stimulation only at 100 μM. Both basal and 5-HT-stimulated AC activity were significantly increased in the gills of mussels exposed for 7 days in aquaria to Cr 6+ (10 ng/l) and Cu 2+ (5 μg/l). The cAMP content of the gill under these conditions was also significantly higher than in control animals. In vitro exposure of gill membrane preparations to Cr 6+ and Cu 2+ induced a bimodal effect. Cu 2+ significantly stimulated AC activity at nanomolar concentrations, but a strong inhibition was displayed in the micromolar range. A similar bell-shaped curve was obtained in the presence of Cr 6+, with maximal AC stimulation at 10 −8 M and inhibition at 10 −5 M. Overall, these data suggest that the mussel AC/cAMP system can be affected with different patterns by heavy metals. AC activity is strongly affected by acute exposure to heavy metals in vitro, probably through a direct interaction of the pollutants with the enzyme molecule, while AC activity and cAMP content increase in organisms exposed for 7 days in vivo, probably as a defense response to acclimate the physiological functions to the environmental challenge.

DNA integrity determination in marine invertebrates by Fast Micromethod ®

This study was focused toward the adaptation of the previously developed Fast Micromethod ® for DNA damage determination to marine invertebrates for the establishment of biomonitoring assessment. The Fast Micromethod ® detects DNA damage (strand breaks, alkali-labile sites and incomplete excision repair) and determines DNA integrity in cell suspensions or tissue homogenates in single microplates. The procedure is based on the ability of the specific fluorochrome dye PicoGreen ® to preferentially interact with high integrity DNA molecules, dsDNA, in the presence of ssDNA and proteins in high alkaline medium, thereby allowing direct fluorometric measurements of dsDNA denaturation without sample handling and stepwise DNA separations. The results presented herein describe the influence of the DNA amount and the pH of the denaturation media on slopes of the kinetic denaturation curves and calculated strand scission factors (SSFs). The optimal amount of DNA in Mytilus galloprovincialis gills homogenate was found to be 100 ng ml −1 and the greatest differences in DNA unwinding kinetics (slopes and SSF values) were reached at pH 11.5. The induction of DNA damage and loss of DNA integrity was measured in native DNA isolated from cotton-spinner Holothuria tubulosa, marine sponge Suberites domuncula cells and mussel M. galloprovincialis gills homogenate. DNA damage and loss of DNA integrity were detected after induction by different doses of (γ-rays, generated by 137 Cs 1800 Ci; 0–500 rad in marine sponge S. domuncula cells up to SSF×(−1) values 0.082±0.012 for the highest radiation dose). Analysis by chemical xenobiotics based on the in vitro action of bleomycin (bleomycin–Fe(II) complex 0–50 or 0–83 μg ml −1 (μM)) with native DNA from cotton-spinner H. tubulosa and mussel M. galloprovincialis gills homogenate yielded values of 0.537±0.072 and 0.130±0.018, respectively. In vivo experiments with mussel M. galloprovincialis gills homogenate by 4-nitroquinoline- N-oxide (NQO; 0–1 μg g −1 NQO mussel) and bezo[a]pyrene (B[a]P; 0–20 μg g −1 B[a]P mussel) indicated SSF×(−1) values of 0.121±0.016 and 0.090±0.007, respectively, for the highest applied doses of chemical xenobiotics. The analytical technique described here allows simple and fast analysis of DNA integrity, requires very short time for multiple analyses (less than 3 h) and even less than 100 ng DNA per single well (50 ng DNA isolated from cotton-spinner, 12,500 sponge cells or about 10 mg of mussel gills homogenate) in a microplate. This makes the Fast Micromethod ® applicable for the measurement of DNA integrity of small samples for genotoxicity assessment (biomonitoring), the effects of genotoxins on lower marine taxa or sessile invertebrates in marine environment (e.g. sponges, mussels) and the estimation of directional changes and harmful effects in the ecosystem.