Alternative strategies for the recombinant synthesis, DOPA modification and analysis of mussel foot proteins - A case study for Mefp-3 from Mytilus edulis.

Mussel foot proteins (Mfps) possess unique binding properties to various surfaces due to the presence of L-3,4-dihydroxyphenylalanine (DOPA). Mytilus edulis foot protein-3 (Mefp-3) is one of several proteins in the byssal adhesive plaque. Its localization at the plaque-substrate interface approved that Mefp-3 plays a key role in adhesion. Therefore, the protein is suitable for the development of innovative bio-based binders. However, recombinant Mfp-3s are mainly purified from inclusion bodies under denaturing conditions. Here, we describe a robust and reproducible protocol for obtaining soluble and tag-free Mefp-3 using the SUMO-fusion technology. Additionally, a microbial tyrosinase from Verrucomicrobium spinosum was used for the in vitro hydroxylation of peptide-bound tyrosines in Mefp-3 for the first time. The highly hydroxylated Mefp-3, confirmed by MALDI-TOF-MS, exhibited excellent adhesive properties comparable to a commercial glue. These results demonstrate a concerted and simplified high yield production process for recombinant soluble and tag-free Mfp3-based proteins with on demand DOPA modification.

Lysozyme Inhibitors as Tools for Lysozyme Profiling: Identification and Antibacterial Function of Lysozymes in the Hemolymph of the Blue Mussel.

Lysozymes are universal components of the innate immune system of animals that kill bacteria by hydrolyzing their main cell wall polymer, peptidoglycan. Three main families of lysozyme have been identified, designated as chicken (c)-, goose (g)- and invertebrate (i)-type. In response, bacteria have evolved specific protein inhibitors against each of the three lysozyme families. In this study, we developed a serial array of three affinity matrices functionalized with a c-, g-, and i-type inhibitors for lysozyme typing, i.e., to detect and differentiate lysozymes in fluids or extracts from animals. The tool was validated on the blue mussel (Mytilus edulis), whose genome carries multiple putative i-, g-, and c-type lysozyme genes. Hemolymph plasma of the animals was found to contain both i- and g-type, but not c-type lysozyme. Furthermore, hemolymph survival of Aeromonas hydrophila and E. coli strains lacking or overproducing the i- type or g-type lysozyme inhibitor, respectively, was analyzed to study the role of the two lysozymes in innate immunity. The results demonstrated an active role for the g-type lysozyme in the innate immunity of the blue mussel, but failed to show a contribution by the i-type lysozyme. Lysozyme profiling using inhibitor-based affinity chromatography will be a useful novel tool for studying animal innate immunity.

A Bivalve Biomineralization Toolbox.

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid-base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.

Integrated analysis of physiological, transcriptomics and metabolomics provides insights into detoxication disruption of PFOA exposure in Mytilus edulis.

Perfluorooctanoic acid (PFOA), a persistent environmental contaminant, resists environmental degradation and bioaccumulates in food chains. Lots of literatures have proved that PFOA exposure could disrupt detoxifying function in a variety of organisms, however, it still remained poorly known about this in mollusk. Here, we examined physiological, transcriptomic, and metabolomic responses to PFOA in Mytilus edulis, a model organism frequently used in studies of aquatic pollution. We aimed to characterize PFOA-induced stress responses and detoxification mechanisms. PFOA exposure significantly altered antioxidant enzyme activity levels and the abundances of lipid peroxidation products. In addition, transcriptomic analysis indicated that several genes associated with oxidative stress and detoxication were differentially expressed after PFOA exposure. In combination, transcriptomic and metabolomic analyses showed that PFOA exposure disturbed several metabolic processes in M. edulis, including the lipid metabolism, amino acid metabolism, and carbohydrate metabolism etc. Molecular examination and enzymes assay of PFOA-exposed M. edulis after a 7-day depuration period still did not recover to control levels. The Pathway enrichment analysis proved that several pathways related to detoxification, such as c-Jun N-terminal kinase (JNK) and p38-dependent mitogen-activated protein kinase (MAPK) pathway, Peroxisome proliferator-activated receptor γ (PPARγ) pathway etc, were obviously affected. The present work verifies firstly PFOA disruption to molluscan detoxification and identifies the key pathways to understand the molecular mechanisms thereof. This study provides new insights into the detoxication disruption invoked in response to PFOA exposure in M. edulis.

Effects of intermittent hypoxia on cell survival and inflammatory responses in the intertidal marine bivalves Mytilus edulis and Crassostrea gigas.

Hypoxia is a major stressor in estuarine and coastal habitats, leading to adverse effects in aquatic organisms. Estuarine bivalves such as blue mussels (Mytilus edulis) and Pacific oysters (Crassostrea gigas) can survive periodic oxygen deficiency but the molecular mechanisms that underlie cellular injury during hypoxia-reoxygenation are not well understood. We examined the molecular markers of autophagy, apoptosis and inflammation during short-term (1 day) and long-term (6 days) hypoxia and post-hypoxic recovery (1 h) in mussels and oysters by measuring the lysosomal membrane stability, activity of a key autophagic enzyme (cathepsin D) and mRNA expression of the genes involved in the cellular survival and inflammation, including caspase 2, 3 and 8, Bcl-2, BAX, TGF-ß-activated kinase 1 (TAK1), nuclear factor kappa B1 (NF-κB) and NF-κB activating kinases IKKα and TBK1. Crassostrea gigas exhibited higher hypoxia tolerance, as well as blunted or delayed inflammatory and apoptotic response to hypoxia and reoxygenation as shown by the later onset and/or the lack of transcriptional activation of caspases, BAX and the inflammatory effector NF-κB, compared with M. edulis Long-term hypoxia resulted in upregulation of Bcl-2 in the oysters and mussels, implying activation of anti-apoptotic mechanisms. Our findings indicate the potential importance of the cell survival pathways in hypoxia tolerance of marine bivalves, and demonstrate the utility of the molecular markers of apoptosis and autophagy for the assessment of sublethal hypoxic stress in bivalve populations.

Preparation and effects on neuronal nutrition of plasmenylethonoamine and plasmanylcholine from the mussel Mytilus edulis.

Plasmenylethonoamine (pPE) and plasmanylcholine (aPC) are important phospholipid subclasses. Herein we explored optimum conditions for enzymatic purification and preparation of pPE and aPC from the mussel Mytilus edulis and bovine brain. Among them, pPE in Mytilus edulis PE was mainly p18:0-20:5 and p18:0-22:6, and its purity was 92.7%; aPC in PC was primarily a16:0-22:6 and a16:0-20:5, and aPC accounted for 90.2% of PC. We thereafter evaluated neurotrophic effects of Mytilus edulis pPE, aPC, and bovine brain pPE in a NGF-induced PC12 cell model. Morphologically, pPE and aPC could both promote differentiation, manifested in a significant increase in neurite length and number, due to increased expression of synaptophysin and growth protein GAP-43 in a dose-independent and structure-selective manner. Importantly, the effect on neuronal nutrition of pPE was better than aPC, and marine pPE was better than terrestrial pPE, which might be ascribed to vinyl-ether bond and differences in fatty acid composition.Abbreviations: AA: arachidonic acid; DHA: docosahexaenoic acid; EIC: extracted ion chromatogram; EPA: eicosapentanoic acid; GAP: growth-associated protein; HPLC: high-performance liquid chromatography; LC-MS/MS: liquid chromatography-tandem mass spectrometry; LPC: lyso-PC; LPE: lyso-PE; MS: mass spectrometry; NGF: nerve growth factor; PC: phosphatidylcholine; aPC: plasmanylcholine; PE: phosphatidylethanolamine; pPE: plasmenylethonoamine; PG: phosphoglycerols; PLs: phospholipids; PS: phosphoserines; TIC: total ion chromatogram.

Effects of hypoxia and reoxygenation on intermediary metabolite homeostasis of marine bivalves Mytilus edulis and Crassostrea gigas.

Marine benthic invertebrates are frequently exposed to fluctuating oxygen levels resulting in hypoxia-reoxygenation (H/R) stress in the intertidal, estuarine and shallow coastal habitats. H/R stress can strongly affect the organisms' physiological performance due to the negative shifts in bioenergetics and redox balance. H/R stress commonly leads to the depletion of energy substrates and accumulation of anaerobic end products, but the effects of H/R stress on the homeostasis of the intermediate nitrogenous compounds are not well understood. We studied the effects of the short-term and long-term hypoxia (1 and 6 days, respectively) and subsequent reoxygenation on the metabolite profiles of free amino acids (FAAs), as well as the intermediates of the urea cycle and purine metabolism in two species of hypoxia-tolerant intertidal bivalves, the blue mussels Mytilus edulis and the Pacific oysters Crassostrea gigas. Accumulation of succinate was assessed to determine the role of anaerobiosis in the metabolic responses to H/R stress. Our study showed that the more hypoxia-tolerant of the two studied species (C. gigas) had lower rate of succinate accumulation during hypoxia (indicating stronger metabolic rate suppression) and was better able to maintain the homeostasis of nitrogenous intermediates during H/R stress compared with the less hypoxia-tolerant M. edulis. Furthermore, analysis of the metabolite profiles indicate that the oysters maintain high levels of cytoprotective compounds (such as taurine and GABA), accumulate lower levels of potential prooxidants (such as succinate and hypoxanthine) and experience less damage to oxidation-prone thiol-containing amino acids such as cysteine, homocysteine and methionine during hypoxia and reoxygenation compared with the blue mussels. This study indicates a potentially important role of intermediate metabolite homeostasis in the tolerance to prolonged hypoxia and H/R stress in marine organisms and opens avenue for further testing of this hypothesis in a broader comparative framework.

PVC Does Not Influence Cadmium Uptake or Effects in the Mussel (Mytilus edulis).

Microplastics have become a global concern in recent years. In this study, we studied (i) whether the presence of polyvinyl chloride (PVC) microparticles may affect cadmium (Cd) uptake in mussel (Mytilus edulis); and (ii) the biological effects of PVC microparticles exposure alone or in combination with Cd. Significant Cd uptake in digestive gland was observed following Cd exposure. However, PVC did not significantly increase Cd uptake compared with Cd alone treatment. In terms of biological impacts, significantly lower neutral red retention (NRR) time and elevated expression of Metallothionein isoform 20-IV (MT-20) were observed in mussels exposed to Cd alone, or combined with microplastics, yet there was no significant difference between them. catalase (CAT) expression only showed a significant increase in mussels exposed to Cd alone. This work provides an insight into the relationship on resulting biological impacts between these two contaminants.

Fatty Acids Distribution in Seston, Tissues, and Faecal Pellets of Blue Mussels Mytilus edulis L.

The n-3 polyenoic fatty acids (phytoplankton origin) dominate in the fatty acid composition of seston, which is a food source for bivalves; this indicates the predominance of diatoms in the seston composition. The distribution of the main classes of lipids and their fatty acids in the tissues of blue mussels depends on the mollusk body size. As the body size of the mollusk increases, the ratio of membrane lipids decreases, and high-energy saturated and monounsaturated fatty acids accumulate in storage lipids. The bulk of the n-3 polyenoic acids consumed by mollusks in seston (i.e., their food) accumulates in the tissues of mollusks and is used for their metabolism, whereas the long-chain saturated fatty acids, oleic acid and the n-6 polyenoic acids, are excreted with faecal pellets.

Amino Acid Composition, Antioxidant, and Cytoprotective Effect of Blue Mussel (Mytilus edulis) Hydrolysate through the Inhibition of Caspase-3 Activation in Oxidative Stress-Mediated Endothelial Cell Injury.

Enhanced oxidative stress plays a central role in promoting endothelial dysfunction, leading to the development of atherosclerosis. In this study, we investigated the protective effects of the hydrolysates derived from blue mussel (Mytilus edulis) against H2O2-mediated oxidative injury in human umbilical vein endothelial cells (HUVECs). The blue mussel hydrolysates were prepared by enzymatic hydrolysis with eight proteases, and blue mussel-α-chymotrypsin hydrolysate (BMCH) showed the highest antioxidant activities in DPPH radical scavenging, ABTS⁺ radical scavenging, and ORAC value compared to those of the other hydrolysates. BMCH also inhibited Cu2+-mediated low density lipoprotein (LDL) oxidation. Treatment of H2O2 resulted in the decreased HUVEC viability whereas pre-treatment with BMCH increased HUVEC viability and reduced reactive oxygen species (ROS) generation. BMCH pre-treatment increased cellular antioxidant capacities, including levels of glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) against H2O2-mediated oxidative stress in HUVECs. Flow cytometry and western blot analysis revealed that BMCH pre-treatment significantly reduced H2O2-mediated HUVEC apoptosis through inhibition of caspase-3 activation. Real-time-qPCR analysis showed that BMCH down-regulated expression of p53 and caspase-3 genes, as well as decreased the bax/bcl-2 ratio. Taken together, these results indicate that BMCH may be useful as functional food ingredients for protecting endothelial dysfunction or related disease.

Spatial and temporal impacts of the Skjervøy harbour diesel spill on native population of blue mussels: A sub-Arctic case study.

This work was designed to investigate biological impacts on blue mussels (Mytilus edulis spp) after being exposed to diesel spill. On December 2013, an 180,000-litre accidental acute diesel spill was reported in a small harbour of northern Norway (Skjervøy). In order to assess the biological effects on the wild population of blue mussels, bivalves were collected at three different locations: at the oil-spill spot, at the other side of the harbour (opposite the oil-spill area), and in an uncontaminated site. Body burden and seawater samples were collected from a few days up to five months after the diesel spill. Biomarkers of oxidative stress and immunological effects were assessed in the blue mussels digestive glands. Our findings reported significant modulation of GST (detoxification), SOD (antioxidant response) and MDA (lipid peroxidation) in bivalves exposed to diesel with a similar response at two and five months after the spill. Laccase-type enzyme also highlighted an important aspect in terms of biomarker response of the immune function. Overall, our study demonstrated that some biomarkers returned to basal levels a few months after the diesel spill. Consequently, it highlighted the usefulness of normalised tools and guidelines for biomonitoring strategies after a diesel spill.

Identification and In Silico Prediction of Anticoagulant Peptides from the Enzymatic Hydrolysates of Mytilus edulis Proteins.

Mytilus edulis is a typical marine bivalve mollusk. Many kinds of bioactive components with nutritional and pharmaceutical activities in Mytilus edulis were reported. In this study, eight different parts of Mytilus edulis tissues, i.e., the foot, byssus, pedal retractor muscle, mantle, gill, adductor muscle, viscera, and other parts, were separated and the proteins from these tissues were prepared. A total of 277 unique peptides from the hydrolysates of different proteins were identified by UPLC-Q-TOF-MS/MS, and the molecular weight distribution of the peptides in different tissues was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The bioactivity of the peptides was predicted through the Peptide Ranker database and molecular docking. Moreover, the peptides from the adductor muscle were chosen to do the active validation of anticoagulant activity. The active mechanism of three peptides from the adductor muscle, VQQELEDAEERADSAEGSLQK, RMEADIAAMQSDLDDALNGQR, and AAFLLGVNSNDLLK, were analyzed by Discovery Studio 2017, which also explained the anticoagulant activity of the hydrolysates of proteins from adductor muscle. This study optimized a screening and identification method of bioactive peptides from enzymatic hydrolysates of different tissues in Mytilus edulis.

Does the membrane pacemaker theory of metabolism explain the size dependence of metabolic rate in marine mussels?

According to the membrane pacemaker theory of metabolism (MPT), allometric scaling of metabolic rate in animals is determined by the composition of cellular and mitochondrial membranes, which changes with body size in a predictable manner. MPT has been elaborated from interspecific comparisons in mammals. It projects that the degree of unsaturation of membrane phospholipids decreases in larger organisms, thereby lowering ion permeability of the membranes and making cellular, and thus whole-animal metabolism more efficient. Here, we tested the applicability of the MPT to a marine ectotherm, the mussel Mytilus edulis at the intraspecific level. We determined effects of body mass on whole-organism, tissue and cellular oxygen consumption rates, on heart rate, metabolic enzyme activities and on the lipid composition of membranes. In line with allometric patterns, the organismal functions and processes such as heart rate, whole-animal respiration rate and phospholipid contents showed a mass-dependent decline. However, the allometry of tissue and cellular respiration and activity of metabolic enzymes was poor; fatty acid unsaturation of membrane phospholipids of gill tissue was independent of animal size. It is thus conceivable that most of the metabolic allometry observed at the organismal level is determined by systemic functions. These whole-organism patterns may be supported by energy savings associated with growing cell size but not by structural changes in membranes. Overall, the set of processes contributing to metabolic allometry in ectotherms may differ from that operative in mammals and birds, with a reduced involvement of the mechanisms proposed by the MPT.

Functional and molecular responses of the blue mussel Mytilus edulis' hemocytes exposed to cadmium - An in vitro model and transcriptomic approach.

The bivalve mollusk, Mytilus edulis, is used as a sentinel species in several monitoring programs due to its ability to bio-accumulate contaminants. Its immune system consists of hemocytes and humoral components, which constitute the main part of the hemolymph. The present study is aimed at understanding the effects of Cd on the differentially expressed genes involved in the phagocytosis of M. edulis' hemocytes. Our approach focuses on an in vitro model by exposing hemocytes to different concentrations of Cd ranging from 10-9 M to 10-3 M. Phagocytosis and cell viability as functional markers were measured using flow cytometry. The molecular mechanisms regulated by Cd were investigated using RNA-seq and DGE analysis. Results showed that viability and phagocytosis of hemocytes exposed to 10-3 M of Cd were significantly decreased after 21 h of exposure. RNA sequencing data showed that 1112 transcripts (out of 352,976 contigs) were differentially regulated by the highest concentration of Cd. Among these identified transcripts, 1028 and 84 were up and down-regulated respectively. The induction of super oxide dismutase (SOD), glutathion-s-transferase (GST), cytochrome P450 2C8 (CYP2C8), multidrug resistance protein (MRP1) and heat shock protein 70 (HSP70) suggests that Cd can regulate key molecular mechanisms. In addition, several toll-like receptors (TLR) as well as genes involved in phagocytosis (actin and CDC42) and apoptosis (caspase 8 and XIAP/IAP) were induced by Cd. Thus, our model highlights the effect of Cd on the phagocytic function of M. edulis' hemocytes along with the regulation of gene expression involved in innate immunity, detoxification and apoptosis. Further investigations need to be pursued to unravel the effects of Cd on the molecular mechanisms identified in this study.

Impact of ocean acidification on antimicrobial activity in gills of the blue mussel (Mytilus edulis).

Here, we aimed to investigate potential effects of ocean acidification on antimicrobial peptide (AMP) activity in the gills of Mytilus edulis, as gills are directly facing seawater and the changing pH (predicted to be reduced from ∼8.1 to ∼7.7 by 2100). The AMP activity of gill and haemocyte extracts was compared at pH 6.0, 7.7 and 8.1, with a radial diffusion assay against Escherichia coli. The activity of the gill extracts was not affected by pH, while it was significantly reduced with increasing pH in the haemocyte extracts. Gill extracts were also tested against different species of Vibrio (V. parahaemolyticus, V. tubiashii, V. splendidus, V. alginolyticus) at pH 7.7 and 8.1. The metabolic activity of the bacteria decreased by ∼65-90%, depending on species of bacteria, but was, as in the radial diffusion assay, not affected by pH. The results indicated that AMPs from gills are efficient in a broad pH-range. However, when mussels were pre-exposed for pH 7.7 for four month the gill extracts presented significantly lower inhibit of bacterial growth. A full in-depth proteome investigation of gill extracts, using LC-Orbitrap MS/MS technique, showed that among previously described AMPs from haemocytes of Mytilus, myticin A was found up-regulated in response to lipopolysaccharide, 3 h post injection. Sporadic occurrence of other immune related peptides/proteins also pointed to a rapid response (0.5-3 h p.i.). Altogether, our results indicate that the gills of blue mussels constitute an important first line defence adapted to act at the pH of seawater. The antimicrobial activity of the gills is however modulated when mussels are under the pressure of ocean acidification, which may give future advantages for invading pathogens.

Toxic dinoflagellates and Vibrio spp. act independently in bivalve larvae.

Harmful algal blooms (HABs) and marine pathogens - like Vibrio spp. - are increasingly common due to climate change. These stressors affect the growth, viability and development of bivalve larvae. Little is known, however, about the potential for interactions between these two concurrent stressors. While some mixed exposures have been performed with adult bivalves, no such work has been done with larvae which are generally more sensitive. This study examines whether dinoflagellates and bacteria may interactively affect the viability and immunological resilience of blue mussel Mytilus edulis larvae. Embryos were exposed to environmentally relevant concentrations (100, 500, 2500 & 12,500 cells ml(-1)) of a dinoflagellate (Alexandrium minutum, Alexandrium ostenfeldii, Karenia mikimotoi, Protoceratium reticulatum, Prorocentrum cordatum, P. lima or P. micans), a known pathogen (Vibrio coralliilyticus/neptunius-like isolate or Vibrio splendidus; 10(5) CFU ml(-1)), or both. After five days of exposure, significant (p < 0.05) adverse effects on larval viability and larval development were found for all dinoflagellates (except P. cordatum) and V. splendidus. Yet, despite the individual effect of each stressor, no significant interactions were found between the pathogens and harmful algae. The larval viability and the phenoloxidase innate immune system responded independently to each stressor. This independence may be related to a differential timing of the effects of HABs and pathogens.

Validation of the flooding dose technique to determine fractional rates of protein synthesis in a model bivalve species, the blue mussel (Mytilus edulis L.).

For the first time, use of the flooding dose technique using (3)H-Phenylalanine is validated for measuring whole-animal and tissue-specific rates of protein synthesis in the blue mussel Mytilus edulis (61mm shell length; 4.0g fresh body mass). Following injection, the phenylalanine-specific radioactivities in the gill, mantle and whole-animal free pools were elevated within one hour and remained elevated and stable for up to 6h following injection of (3)H-phenylalanine into the posterior adductor muscle. Incorporation of (3)H-phenylalanine into body protein was linear over time following injection and the non-significant intercepts for the regressions suggested incorporation into body protein occurred rapidly after injection. These results validate the technique for measuring rates of protein synthesis in mussels. There were no differences in the calculated rates following 1-6h incubation in gill, mantle or whole-animal and fractional rates of protein synthesis from the combined time course data were 9.5±0.8%d(-1) for the gill, 2.5±0.3%d(-1) for the mantle and 2.6±0.3%d(-1) for the whole-animal, respectively (mean values±SEM). The whole-animal absolute rate of protein synthesis was calculated as 18.9±0.6mg protein day(-1). The use of this technique in measuring one of the major components of maintenance metabolism and growth will provide a valuable and convenient tool in furthering our understanding of the protein metabolism and energetics of this keystone marine invertebrate and its ability to adjust and respond to fluctuations, such as that expected as a result of climate change.

Seasonality and toxins effects on oxidative/nitrosative metabolism in digestive glands of the bivalve Mytilus edulis platensis.

The hypothesis presented here is that oxidative and/or nitrosative metabolism in the bivalve Mytilus edulis platensis is altered by the presence of planktonic toxins. Digestive glands (DG) were isolated from specimens collected in the Argentinean Sea during summer, winter and spring (in the presence of harmful planktonic toxins). The labile iron pool content was not significantly different in DG from animals collected in summer and winter, but was 2.3-fold increased in samples from spring compared to summer collected mollusks. The 2',7' dichlorofluorescein diacetate (DCFH-DA) oxidation, ascorbyl radical/ascorbate and lipid radical/α-tocopherol content ratios showed no significant differences between samples collected in winter and summer. However, spring collected samples showed significantly higher DCFH-DA oxidation rate and oxidative ratios in comparison to DG from mollusks collected in summer. Superoxide dismutase activity decreased by 75% in winter, and 93% in spring, compared to samples collected in summer. Glutathione S-transferase activity decreased by 89% in winter, and 30% in spring, compared to samples collected in summer. Catalase activity in winter animals increased by 3.8-fold in comparison to summer values, with no differences between spring and summer collected mollusks. Nitrite plus nitrate content was not significantly different among samples collected in the three seasons, but nitric oxide content was 8.5- and 2.7-fold higher in samples from winter and spring collected mollusks than values obtained in summer, respectively. These results showed the lack of effects of climatic changes on the integrative oxidative indexes; however, under exposure to toxins, both oxidative and nitrosative metabolisms were affected.

CONTENT OF 70 kDa STRESS PROTEIN DURING ACCLIMATION OF MUSSEL MYTILUS EDULIS L. TO LOW SALINITY.

The paper deals with the dynamics of stress-proteins content in gill epithelium of bivalved mollusks Mytilus edulis in the course of their phenotypic adaptation to the changes of environmental salinity. It has been shown that in low salinity (10 ‰) causing the work of «isolating reflex¼, the induction of stress proteins from HSP70 family comes on the 5th day of the acclimation. Parallel measurements of the mussels extravisceral liquid osmolarity have shown that the signal for stress-protein induction in these mollusks is the osmolarity of their inner medium, but not the salinity of marine water outside.

Shotgun analysis of the marine mussel Mytilus edulis hemolymph proteome and mapping the innate immunity elements.

The marine mussel innate immunity provides protection to pathogen invasion and inflammation. In this regard, the mussel hemolymph takes a main role in the animal innate response. Despite the importance of this body fluid in determining the physiological condition of the animal, little is known about the molecular mechanisms underlying the cellular and humoral responses. In this work, we have applied a MS (nano-LC-MS/MS) strategy integrating genomic and transcriptomic data with the aim to: (i) identify the main protein functional groups that characterize hemolymph and (ii) to map the elements of innate immunity in the marine mussel Mytilus edulis hemolymph proteome. After sample analysis and first protein identification based on MS/MS data comparison, proteins with unknown functions were annotated with blast using public database (nrNCBI) information. Overall 595 hemolymph proteins were identified with high confidence and annotated. These proteins encompass primary cellular metabolic processes: energy production and metabolism of biomolecules, as well as processes related to oxidative stress defence, xenobiotic detoxification, drug metabolism, and immune response. A group of proteins was identified with putative immune effector, receptor, and signaling functions in M. edulis. Data are available via ProteomeXchange with identifier PXD001951 (http://proteomecentral.proteomexchange.org/dataset/PXD001951).