Aldolase of Mytilus galloprovincialis, Lamarck, 1819: Gene structure, tissue specificity of expression level and activity.

In the present study, the structure of the fructose-1,6-bisphosphataldolase (FBA) gene in Mytilus galloprovincialis (Lamarck, 1819) was analyzed and its tissue specificity of expression level and activity was determined. A 1092 base pairs (bps) complete coding sequence of the FBA gene was assembled from M. galloprovincialis transcriptome. Only one gene encoding FBA (MgFBA) was identified in the M. galloprovincialis genome. The length of MgFBA was 363 amino acids with a molecular mass of 39.7 kDa. According to the amino acid residues, the detected MgFBA gene is a type I aldolase. The FBA gene in M. galloprovincialis had 7 exons; the maximum intron length was about 2.5 kbps. Intraspecific nucleotide diversity (15 mutations) between MgFBAs from the Mediterranean mussels and the Black Sea mussels (present study) was detected. All mutations were synonymous. Tissue specificity in FBA expression level and activity was established. No direct correlation between these functions was found. The highest level of FBA gene expression is found in muscle tissue. According to the phylogenetic analyses, FBA gene in invertebrates could be considered the ancestral gene of muscle type aldolase, which may explain the character of tissue-specific expression.

Genetic analysis and population genetic structure of hard-shelled mussel, Mytilus coruscus Gould 1861 (Mytiloida: Mytilidae) from the coasts of South Korea based on mitochondrial cytochrome oxidase (COI) gene sequences.

BACKGROUND: Mytilus coruscus Gould, 1861 is a mussel species in the family Mytilidae, native to the Northwest Pacific Ocean, ranging from the East China Sea, the Yellow Sea, and as far as the Peter the Great Gulf in the East Sea. In Korea, this species has been heavily exploited for nutrient-rich food resources and experienced severe reduction in their population. OBJECTIVE: The aim of this study was to investigate the genetic diversity and population structure and to provide baseline data to facilitate the conservation and sustainable use of the vulnerable species M. coruscus in South Korea. METHODS: Mitochondrial DNA (mtDNA) cytochrome c oxidase I (COI) sequences of 91 adult individuals from four islands and one coastal localities in South Korea were sequenced. We then compared genetic diversity and haplotype data with previously published Chinese wild populations. RESULTS: Mytilus coruscus populations on Korean coasts were found to exhibit high genetic diversity despite concerns regarding recent population reduction. A total of 42 haplotypes were defined by 56 polymorphic sites. High-level genetic diversity was observed on four Island sites (Hd = 0.906-0.955, π = 0.0068-0.0090). The other seashore site represented relatively lower genetic diversity (Hd = 0.529, π = 0.0011) and was genetically differentiated from the others. In a previous study, wild populations on the East China Sea exhibited similarly high genetic diversity as that observed in our study. Additionally, Chinese M. coruscus populations exhibit a distinct regional haplotype distribution pattern while sharing six haplotypes with Korean populations. CONCLUSIONS: The results of this study provide insights that further the current understanding regarding the evolution of M. coruscus species and provides comprehensive genetic data to facilitate the development of an effective conservation strategy.

Mitochondrial F1FO-ATPase and permeability transition pore response to sulfide in the midgut gland of Mytilus galloprovincialis.

The molecular mechanisms which rule the formation and opening of the mitochondrial permeability transition pore (mPTP), the lethal mechanism which permeabilizes mitochondria to water and solutes and drives the cell to death, are still unclear and particularly little investigated in invertebrates. Since Ca2+ increase in mitochondria is accompanied by mPTP opening and the participation of the mitochondrial F1FO-ATPase in the mPTP is increasingly sustained, the substitution of the natural cofactor Mg2+ by Ca2+ in the F1FO-ATPase activation has been involved in the mPTP mechanism. In mussel midgut gland mitochondria the similar kinetic properties of the Mg2+- or Ca2+-dependent F1FO-ATPase activities, namely the same affinity for ATP and bi-site activation kinetics by the ATP substrate, in spite of the higher enzyme activity and coupling efficiency of the Mg2+-dependent F1FO-ATPase, suggest that both enzyme activities are involved in the bioenergetic machinery. Other than being a mitochondrial poison and environmental contaminant, sulfide at low concentrations acts as gaseous mediator and can induce post-translational modifications of proteins. The sulfide donor NaHS, at micromolar concentrations, does not alter the two F1FO-ATPase activities, but desensitizes the mPTP to Ca2+ input. Unexpectedly, NaHS, under the conditions tested, points out a chemical refractoriness of both F1FO-ATPase activities and a failed relationship between the Ca2+-dependent F1FO-ATPase and the mPTP in mussels. The findings suggest that mPTP role and regulation may be different in different taxa and that the F1FO-ATPase insensitivity to NaHS may allow mussels to cope with environmental sulfide.

Response of fatty acids and lipid metabolism enzymes during accumulation, depuration and esterification of diarrhetic shellfish toxins in mussels (Mytilus galloprovincialis).

Bivalve mollusks accumulate diarrhetic shellfish toxins (DSTs) from toxigenic microalgae, thus posing a threat to human health by acting as a vector of toxins to consumers. In bivalves, free forms of DSTs can be esterified with fatty acids at the C-7 site to form acyl esters (DTX3), presumably a detoxification mechanism for bivalves. However, the effects of esterification of DSTs on fatty acid metabolism in mollusks remain poorly understood. In this study, mussels (Mytilus galloprovincialis) were fed the DST-producing dinoflagellate Prorocentrum lima for 10 days followed by an additional 10-days depuration in filtered seawater to track the variation in quantity and composition of DST acyl esters and fatty acids. A variety of esters of okadaic acid (OA) and dinophysistoxin-1 (DTX1) were mainly formed in the digestive gland (DG), although trace amounts of esters also appeared in muscle tissue. A large relative amount of OA (60%-84%) and DTX1 (80%-92%) was esterified to DTX3 in the visceral mass (referred to as digestive gland, DG), and the major ester acyl chains were C16:0, C16:1, C18:0, C18:1, C20:1 and C20:2. The DG and muscle tissues showed pronounced differences in fatty acid content and composition during both feeding and depuration periods. In the DG, fatty acid content gradually decreased in parallel with increasing accumulation and esterification of DSTs. The decline in fatty acids was accelerated during depuration without food. This reduction in the content of important polyunsaturated fatty acids, especially docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), would lead to a reduction in the nutritional value of mussels. Enzymes involved in lipid metabolism, including acetyl-coenzyme A carboxylase (ACC), fatty acid synthase (FAS), lipoprotein lipase (LPL) and hepatic lipase (HL), were actively involved in the metabolism of fatty acids in the DG, whereas their activities were weak in muscle tissue during the feeding period. This study helps to improve the understanding of interactions between the esterification of DSTs and fatty acid dynamics in bivalve mollusks.

Characterization of the main steps in first shell formation in Mytilus galloprovincialis: possible role of tyrosinase.

Bivalve biomineralization is a highly complex and organized process, involving several molecular components identified in adults and larval stages. However, information is still scarce on the ontogeny of the organic matrix before calcification occurs. In this work, first shell formation was investigated in the mussel Mytilus galloprovincialis. The time course of organic matrix and CaCO3 deposition were followed at close times post fertilization (24, 26, 29, 32, 48 h) by calcofluor and calcein staining, respectively. Both components showed an exponential trend in growth, with a delay between organic matrix and CaCO3 deposition. mRNA levels of genes involved in matrix deposition (chitin synthase; tyrosinase- TYR) and calcification (carbonic anhydrase; extrapallial protein) were quantified by qPCR at 24 and 48 hours post fertilization (hpf) with respect to eggs. All transcripts were upregulated across early development, with TYR showing highest mRNA levels from 24 hpf. TYR transcripts were closely associated with matrix deposition as shown by in situ hybridization. The involvement of tyrosinase activity was supported by data obtained with the enzyme inhibitor N-phenylthiourea. Our results underline the pivotal role of shell matrix in driving first CaCO3 deposition and the importance of tyrosinase in the formation of the first shell in M. galloprovincialis.

Mytilus galloprovincialis CYP1A-like mRNAs reveal closer proximity of mytilid CYP1A to the eumetazoan CYP2 family.

Due to the role of Cytochrome P450, Family 1, Subfamily A (CYP1A) in the detoxification of many polycyclic aromatic hydrocarbons (PAHs), there has been an effort to characterise the gene and the products from its expression in organisms that are relevant for biomonitoring and toxicity testing procedures. Nonetheless, the existence of functional homologues in aquatic invertebrates is not entirely consensual, especially in bivalve molluscs, which pose as one of the most important models for aquatic toxicologists, especially mytilids. After isolation and sequencing of CYP1A-like mRNA from the Mediterranean mussel, Mytilus galloprovincialis, phylogenetics incorporating homologues from molluscs and other eumetazoans, vertebrates included, yielded notorious similarity to sequences belonging to the CYP2 Family. Altogether, the findings further indicate that CYP1A-like CYPs may be absent in bivalves, in lieu of Families CYP2, 3 and 4, suggesting caution when interpreting data from common biomarkers of exposure to aromatic hydrocarbons that have been developed for CYP1A activity and expression in higher deuterostomes.

Two novel CYP3A isoforms in marine mussel Mytilus coruscus: Identification and response to cadmium and benzo[a]pyrene.

CYP3A enzymes play a crucial role in metabolic clearance of a variety of xenobiotics. However, their genetic information and function remain unclear in molluscs. In the present study, two novel CYP3A genes i.e. McCYP3A-1 and McCYP3A-2 were identified and characterized from the thick shell mussel Mytilus coruscus, and their tissue distribution as well as the response to cadmium (Cd) and benzo[a]pyrene (B[α]P) exposure were addressed using real time quantitative RT-PCR (qRT-PCR) and erythromycin N-demethylase (ERND) assay. McCYP3A-1 and McCYP3A-2 possess typically domains of CYP family such as helix-C, helix-I, helix-K, PERF and the heme binding domain as well as the characteristic domains of CYP3s including six SRS motifs. McCYP3A-1 and McCYP3A-2 transcripts were constitutively expressed in all examined tissues with high expression level in digestive glands, hepatopancreas and gonads. Upon B[α]P exposure, McCYP3A-1 and McCYP3A-2 mRNA expression in digestive glands showed a pattern of up-regulation followed by down-regulation, while under Cd exposure, showed a time-dependent induction profile. In addition, ERND activity, generally used as an indicator of CYP3, increased in a time-dependent manner after exposure to Cd and B[α]P. These results collectively indicated that McCYP3A-1 and McCYP3A-2 are CYP3A family member and may play a potential role in metabolic clearance of xenobiotics. Meanwhile, the current results may provide some baseline data to support McCYP3A-1 and McCYP3A-2 as candidate biomarkers for monitoring of PAHs and heavy metal pollution.

Effects of Dissolved Organic Carbon on Copper Toxicity to Embryos of Mytilus galloprovincialis as Measured by Diffusive Gradient in Thin Films.

Diffusive gradient in thin films (DGT) potentially better quantifies bioavailable copper (Cu) in seawater. Laboratory exposure of DGTs and Mytilus galloprovincialis embryos at varying concentrations of dissolved organic carbon and Cu were performed to resolve the degree to which mimicry of toxicity buffering occurs in passive sampler quantification. The results provide preliminary median effect concentrations (EC50s) ranging from 4.8 to 11.5 µg/L as CDGT Cu over the span of 0.896 to 8.36 mg/L DOC. Environ Toxicol Chem 2019;00:1-6. Published 2019 Wiley Periodicals, Inc. on behalf of SETAC. This article is a US government work and, as such, is in the public domain in the United States of America.

Baseline levels of antioxidant activities in Mytilus galloprovincialis along the coast of Cape Town, South Africa.

Antioxidant activities in Mytilus galloprovincialis were determined from samples collected at Scarborough, Hout Bay, Green Point, Milnerton and Bloubergstrand in Cape Town, South Africa. Antioxidant enzyme activity was determined by measuring CAT, SOD and GSH. The total antioxidant capacity was measured using FRAP and ORAC, while the content and lipid peroxidation marker levels of CDs and TBARS. Antioxidant activities and responses in mussels varied between sites with significant correlations for 85% and 71% of all antioxidant measurements made for Fe and Zn, respectively. The oxidative stress results reported here are novel for the region and indicated that mussels in Cape Town do not have (relatively) high levels of antioxidant activities as a result of exposure to metals. The research undertaken suggests that antioxidant responses was an appropriate biomarker of exposure to metals but more environmental parameters should be considered when interpreting antioxidant responses in the natural environment.

Preliminary results on the uptake and biochemical response to water-exposure of Tamiflu® (oseltamivir phosphate) in two marine bivalves.

Tamiflu® (oseltamivir phosphate, OST) is an antiviral drug used for the pandemic treatment of avian influenza but few data are available regarding its toxicity. It should be noted that acute adverse responses are not likely to occur due to low environmental presence of this drug. Nonetheless, water concentration levels of this compound may reach the µg/L range under influenza episodes. Bivalves are reliable sentinels of chemical exposure due to their low metabolism; however, biotransformation of drugs does occur in these aquatic invertebrates. Two species of bivalves, namely mussels Mytilus galloprovincialis and clams Ruditapes philippinarum, were exposed for 48 h to 100 µg/L OST. Hemolymph from control and treated bivalves was withdrawn and the presence of OST and its metabolite oseltamivir carboxylate (OST-C) determined by LC-MS/MS. Gills and digestive gland were excised from control and exposed bivalves and carboxylesterase (CE) activities measured using different substrates. In addition, antioxidant defences and lipid peroxidation levels were determined. Higher metabolism of OST seemed to occur in mussels, since both OST and OST-C were found in hemolymph, whereas in clams only the parent compound was detected. In contrast, biomarker responses were more evident in exposed clams which indicate that this species may be considered as more sensitive to OST exposure. CE-related activities successfully reflected OST exposure, with substrates 1-naphthyl acetate (1NA) and 1-naphthyl butyrate (1NB) displaying the highest sensitivity in the two bivalve species. Data thus indicate the usefulness of CE-related activities as biomarkers for OST exposure in bivalves.

Multibiomarker biomonitoring approach using three bivalve species in the Ebro Delta (Catalonia, Spain).

Bivalves have proved to be useful bioindicators for environmental pollution. In the present study, mussels (Mytilus galloprovincialis), cockles (Cerastoderma edule), and razor shells (Solen marginatus) were collected in the Ebro Delta, an extensive area devoted to rice farming and affected by pesticide pollution, from April to July, the heaviest rice field treatment period. Possible effects of pollution were assessed through biochemical markers (carboxylesterase (CE), antioxidant and neurotoxicity-related enzymes, and lipid peroxidation levels). Data on environmental variables, bivalve reproductive condition, and presence of organic pollutants, marine phycotoxins, pathogens, or histopathological conditions in bivalve's tissues were also evaluated. Although the bioaccumulated pesticides did not explain the patterns observed for biochemical responses, the obtained results point to an effect of environmental pesticide pollution on enzymatic markers, with a prominent contribution of CE to such changes. Mussels and razor shells provided a more sensitive biochemical response to pollution than cockles. Environmental variables, bivalve reproductive condition, and marine phycotoxins did not seem to have a relevant effect on the biomarkers assessed.

A novel biomarker for marine environmental pollution of CAT from Mytilus coruscus.

Bivalves use anti-oxidative enzyme systems to defend themselves against excessive reactive oxygen species, which are often catalyzed by environmental pollution. As a key member of anti-oxidative enzyme family, catalase plays a crucial role in scavenging the high level of reactive oxygen species to protect organisms against various oxidative stresses. In this study, a catalase homologue was identified from Mytilus coruscus (named McCAT, KX957929). The open reading frame of McCAT was 1844bp with a 5' untranslated region of 341bp and a 3' untranslated region of 927bp. The deduced amino acid sequence was 512 residues in length with theoretical pI/MW 8.02/57.91kDa. BLASTn and phylogenetic analyses strongly suggested that it was a member of catalase, also known as CAT family for its conserved catalytic site motif and proximal heme-ligand signature motif. Real-time fluorescence quantitative PCR showed that constitutive expression of McCAT was occurred, with increasing order in mantle, adductor, gill, hemocyte, gonad and hepatopancreas. It was observed that bacterial infection and heavy metals stimulation up-regulated McCAT mRNA expression in hepatopancreas with time-dependent manners. The maximum expression appeared at 8h after pathogenic bacteria injecting, with 15-fold in Vibrio parahemolyticus and 60-fold in Aeromonas hydrophila than that of 0h. The highest point of McCAT mRNA appeared at different times for exposure to heavy metals with copper at day 5 (0.1mg/L 30-fold, 0.5mg/L 15-fold, 1.5mg/L 6-fold) and plumbum at day 3 (3.0mg/L 20-fold). The enzymatic activity analysis found that McCAT activity in the gill of M. coruscus was affected by heavy metals concentration. The results suggested that McCAT plays a significant role in antioxidation and the expression of McCAT can be used as a biomarker for detection of marine environmental pollution.

Acute exposure to sunscreen containing titanium induces an adaptive response and oxidative stress in Mytillus galloprovincialis.

The use of sunscreens to protect against ultraviolet radiation exposure progressively increases as result of a greater awareness of the people and the greater arrival of tourists. The components of these creams can end up in the waters affecting coastal species. Mediterranean mussels (Mytillus galloprovincialis) were subjected to an acute exposure of a sunscreen with TiO2 in their composition during 24h. The low and medium concentrations used in the assays contained a concentration of TiO2 in the range of values found in coastal waters of the Balearic Islands. Titanium and metallothionein concentrations were progressively increasing in gills with the sunscreen concentration in a dose-dependent manner. The activities of the antioxidant enzymes and the detoxification glutathione s-transferase evidenced a hormetic shape response with increased activities at lower sunscreen concentrations, a response that was abolished at the highest concentration. In accordance with these enzyme activities, the levels of malondialdehyde, as a marker of lipid peroxidation, were significantly elevated by the higher sunscreen concentrations. Acetylcholinesterase activity maintained control activities except for the highest sunscreen concentration, where a significant decrease was evidenced. In conclusion, the treatment of mussels with a sunscreen containing TiO2 in the range of Balearic coastal waters induces an adaptive response that is overcome by the highest concentration. Follow-up biomonitoring studies are necessary to control the concentration of sunscreen compound in coastal waters such as titanium since they can induce oxidative stress to affected organisms.

Effect of Environmental Pollutant Mixtures on Acid DNase Activity in Mussel Mytilus galloprovincialis: Ex Situ and In Situ Study.

Acid DNase activity in marine organisms displays a sensitive response to pollutants. In the present study, ex situ effect of toxic seawater organic extracts (Rijeka, Vranjic, Ploce) and in situ impact of environmental factors at referent (St. Andrew) and a polluted site (ACI Rovinj) on acid DNase in the digestive gland and gills of mussels was investigated. In the digestive gland, enzyme activity was related to potential toxicity of seawater organic extracts. Seawater organic extracts showed an increase of the enzyme activity in digestive gland following exposure. In in situ study, polluted environment affected mussel's acid DNase activity in digestive gland, when compared to gills. Acid DNase activity in the digestive gland expressed significant difference between the referent and polluted site, particularly from early spring through summer. Both ex situ and in situ studies displayed more sensitive acid DNase response to the presence of toxic mixtures in the digestive gland.

Cloning, expression and purification of the α-carbonic anhydrase from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

We cloned, expressed, purified, and determined the kinetic constants of the recombinant α-carbonic anhydrase (rec-MgaCA) identified in the mantle tissue of the bivalve Mediterranean mussel, Mytilus galloprovincialis. In metazoans, the α-CA family is largely represented and plays a pivotal role in the deposition of calcium carbonate biominerals. Our results demonstrated that rec-MgaCA was a monomer with an apparent molecular weight of about 32 kDa. Moreover, the determined kinetic parameters for the CO2 hydration reaction were kcat = 4.2 × 105 s-1 and kcat/Km of 3.5 × 107 M-1 ×s-1. Curiously, the rec-MgaCA showed a very similar kinetic and acetazolamide inhibition features when compared to those of the native enzyme (MgaCA), which has a molecular weight of 50 kDa. Analysing the SDS-PAGE, the protonography, and the kinetic analysis performed on the native and recombinant enzyme, we hypothesised that probably the native MgaCA is a multidomain protein with a single CA domain at the N-terminus of the protein. This hypothesis is corroborated by the existence in mollusks of multidomain proteins with a hydratase activity. Among these proteins, nacrein is an example of α-CA multidomain proteins characterised by a single CA domain at the N-terminus part of the entire protein.

The role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes in the [omim][BF4]-mediated toxic mode of action in mussel hemocytes.

The present study investigates the role of phosphatidylinositol-3-OH-kinase (PI3-kinase) and respiratory burst enzymes, NADPH oxidase and NO synthase, in the 1-methyl-3-octylimidazolium tetrafluoroborate ([omim][BF4])-mediated toxic mode of action in mussel hemocytes. Specifically, cell viability (using the neutral red uptake assay) was primarily tested in hemocytes treated with different concentrations of [omim][BF4] (0.1-10 mg L-1) and thereafter [omim][BF4]-mediated oxidative (in terms of superoxide anions/O2- and nitric oxide/NO generation, as well as the enhancement of lipid peroxidation by-products, in terms of malondialdehyde/MDA) and genotoxic (in terms of DNA damage) effects were determined in hemocytes treated with 1 mg L-1 [omim][BF4]. Moreover, in order to investigate, even indirectly and non-entirely specific, the role of PI3-kinase, NADPH oxidase and NO synthase, the [omim][BF4]-mediated effects were also investigated in hemocytes pre-incubated with wortmannin (50 nM), diphenyleneiodonium chloride (DPI 10 µM) and NG-nitro-l-arginine methyl ester (l-NAME 10 µM), respectively. The results showed that [omim][BF4] ability to enhance O2-, NO, MDA and DNA damage, via its interaction with cellular membranes, was significantly attenuated in the presence of each inhibitor in almost all cases. The current findings revealed for the first time that certain signaling molecules, such as PI3-kinase, as well as respiratory burst enzymes activation, such as NADPH oxidase and NO synthase, could merely attribute to the [omim][BF4]-mediated mode of action, thus enriching our knowledge for the molecular mechanisms of ILs toxicity.

Monitoring of organic pollutants in marine environment by semipermeable membrane devices and mussels: accumulation and biochemical responses.

This study involves the monitoring of organic pollutants using transplanted mussels (Mytilus galloprovincialis) as bioindicator organisms and semipermeable membrane devices (SPMDs) as passive samplers. Mussels and SPMDs were deployed to marinas, shipyards and shipbreaking yards on the coastal area of Turkey and retrieved after 60 days. Polycyclic aromatic hydrocarbons (PAH), polychlorinated biphenyls (PCB) and organochlorine pesticide (OCP) compounds were analysed with high-resolution GC-MS. Total PAH concentrations in SPMDs and mussels ranged from 200 to 4740 ng g sampler-1 and from 7.0 to 1130 ng g-1 in wet weight (ww). PCB and OCP concentrations in SPMDs changed between 0.04-200 and 4.0-26 ng g sampler-1, respectively. The highest PCB (190 ng g-1 ww) and OCP (200 ng g-1 ww) concentrations in mussels were measured at shipyard stations. A strong correlation was observed between the PAH and PCB concentrations in SPMDs and mussels. Enzyme assays (acetylcholinesterase, ethoxyresorufin-O-deethylase, glutathione S-transferase, glutathion reductase and carboxylesterase activities) were performed as biomarkers to reveal the effects of pollution on the mussels. There was no clear relationship found between the enzyme levels and the pollutant concentrations in mussels. Integrated biomarker responses were calculated to interpret the overall effect of pollutants.

Biochemical characterization of the native α-carbonic anhydrase purified from the mantle of the Mediterranean mussel, Mytilus galloprovincialis.

A α-carbonic anhydrase (CA, EC 4.2.1.1) has been purified and characterized biochemically from the mollusk Mytilus galloprovincialis. As in most mollusks, this α-CA is involved in the biomineralization processes leading to the precipitation of calcium carbonate in the mussel shell. The new enzyme had a molecular weight of 50 kDa, which is roughly two times higher than that of a monomeric α-class enzyme. Thus, Mytilus galloprovincialis α-CA is either a dimer, or similar to the Tridacna gigas CA described earlier, may have two different CA domains in its polypeptide chain. The Mytilus galloprovincialis α-CA sequence contained the three His residues acting as zinc ligands and the gate-keeper residues present in all α-CAs (Glu106-Thr199), but had a Lys in position 64 and not a His as proton shuttling residue, being thus similar to the human isoform hCA III. This probably explains the relatively low catalytic activity of Mytilus galloprovincialis α-CA, with the following kinetic parameters for the CO2 hydration reaction: kcat = 4.1 × 105 s-1 and kcat/Km of 3.6 × 107 M-1 × s-1. The enzyme activity was poorly inhibited by the sulfonamide acetazolamide, with a KI of 380 nM. This study is one of the few describing in detail the biochemical characterization of a molluskan CA and may be useful for understanding in detail the phylogeny of these enzymes, their role in biocalcification processes and their potential use in the biomimetic capture of the CO2.

Antioxidant response of the hard shelled mussel Mytilus coruscus exposed to reduced pH and oxygen concentration.

Ocean acidification (OA) and hypoxic events are increasing worldwide problems, their interactive effects have not been well clarified, although their co-occurrence is prevalent. The East China Sea (the Yangtze River estuary area) suffers from not only coastal hypoxia but also pH fluctuation, representing an ideal study site to explore the combined effect of OA and hypoxia on marine bivalves. We experimentally evaluated the antioxidant response of the mussel Mytilus coruscus exposed to three pH levels (8.1, 7.7 and 7.3) at two dissolved oxygen (DO) levels (2.0mgL-1 and 6.0mgL-1) for 72h. Activities of superoxide dismutase, catalase, glutathione peroxidase, acid phosphatase, and alkaline phosphatase and levels of malondialdehyde were measured in gills and hemolymph. All enzymatic activities in hemolymph and gills followed a similar pattern throughout the experiment duration. Generally, low DO showed greater effects on enzyme activities than elevated CO2. Significant interactions between DO, pH and time were only observed at superoxide dismutase and catalase in both tissues. PCA revealed positive relationships between most enzyme activities in both gills and hemolymph with the exception of alkaline phosphatase activity and the level of malondialdehyde in the hemolymph. Overall, our results suggested that decreased pH and low DO induced similar antioxidant responses in the hard shelled mussel, and showed an additive effect on most enzyme activities. The evaluation of multiple environmental stressors, a more realistic scenario than single ones, is crucial to predict the effect of future global changes on coastal species and our results supply some insights on the potential combined effects of reduced pH and DO on marine bivalves.

Assessing the Effects of Amoxicillin on Antioxidant Enzyme Activities, Lipid Peroxidation and Protein Carbonyl Content in the Clam Ruditapes philippinarum and the Mussel Mytilus galloprovincialis.

In this study, we evaluated the capability of amoxicillin (AMX)-one of the most widely used antibiotics worldwide-to induce oxidative stress in both gills and digestive gland from two bivalve species, the clam Ruditapes philippinarum and the mussel Mytilus galloprovincialis. Superoxide dismutase (SOD) and catalase (CAT) activities, as well as the lipid peroxidation levels (LPO) and protein carbonyl content (PCC), were measured in bivalves exposed to 100, 200 and 400 µg AMX/L for 1, 3 and 7 days. The results obtained demonstrated that AMX affected slightly biomarker responses of molluscs.