Metabolomics and lipidomics reveal the effects of the toxic dinoflagellate Alexandrium catenella on immune cells of the blue mussel, Mytilus edulis.

The increasing occurrence of harmful algal blooms, mostly of the dinoflagellate Alexandrium catenella in Canada, profoundly disrupts mussel aquaculture. These filter-feeding shellfish feed on A. catenella and accumulate paralytic shellfish toxins, such as saxitoxin, in tissues, making them unsafe for human consumption. Algal toxins also have detrimental effects upon several physiological functions in mussels, but particularly on the activity of hemocytes - the mussel immune cells. The objective of this work was to determine the effects of experimental exposure to A. catenella upon hemocyte metabolism and activity in the blue mussel, Mytilus edulis. To do so, mussels were exposed to cultures of the toxic dinoflagellate A. catenella for 120 h. The resulting mussel saxitoxin load had measurable effects upon survival of hemocytes and induced a stress response measured as increased ROS production. The neutral lipid fraction of mussel hemocytes decreased two-fold, suggesting a differential use of lipids. Metabolomic 1H nuclear magnetic resonance (NMR) analysis showed that A. catenella modified the energy metabolism of hemocytes as well as hemocyte osmolyte composition. The modified energy metabolism was reenforced by contrasting plasma metabolomes between control and exposed mussels, suggesting that the blue mussel may reduce feed assimilation when exposed to A. catenella.

Sensitivity to oil dispersants: Effects on the valve movements of the blue mussel Mytilus edulis and the giant scallop Placopecten magellanicus, in sub-arctic conditions.

In response to accidental oil spills at sea, chemical oil dispersants are utilized to limit negative impacts on nearby littoral zones. However, current evidence suggests that such dispersants may be toxic to aquatic organisms. Blue mussels (Mytilus edulis) and giant scallops (Placopecten magellanicus) were exposed to different environmentally relevant concentrations of oil dispersant and their behavioural responses were closely monitored using high frequency (10Hz) valvometry. Behavioural valve responses included rapid closures when oil dispersant was added to the experimental tanks. At higher concentrations, the mussels remained closed throughout the exposure period. The giant scallop displayed escape behaviours (clapping) prior to mortality, suggesting toxicity of the oil dispersant. Relationships between different behavioural indicators and oil dispersant concentrations were observed for both species, but with different trends. While scallops demonstrated positive correlations between gaping behaviours and dispersant concentration, mussels exhibited a concentration threshold beyond which the gaping behaviour was characteristic of longer closure periods. This study highlights behavioural response differences consistent with bivalve-specific biological traits: the continuous valve closure of an intertidal species, M. edulis, firmly attached to the substrate, and the escapement behaviours of a semi-mobile subtidal species, P. magellanicus. From these observations, it appears that valvometry could be used as a tool for environmental assessments.

Extraction Improvement of the Bioactive Blue-Green Pigment "Marennine" from Diatom Haslea ostrearia's Blue Water: A Solid-Phase Method Based on Graphitic Matrices.

The compound "marennine" is a blue-green pigment produced by the benthic microalgae Haslea ostrearia, with pathogenicity reduction activities against some bacteria and promising potential as a natural pigment in seafood industries. After decades of research, the chemical family of this compound still remains unclear, mainly because structural studies were impaired by the presence of co-extracted compounds in marennine isolates. To improve the purity of marennine extract, we developed a novel extraction method using a graphitic stationary phase, which provides various advantages over the previous procedure using tandem ultrafiltration. Our method is faster, more versatile, provides a better crude yield (66%, compared to 57% for ultrafiltration) and is amenable to upscaling with continuous photobioreactor cultivation. Our goal was to take advantage of the modulable surface properties of the graphitic matrix by optimizing its interactions with marennine. As such, the effects of organic modifiers, pH and reducing agents were studied. With this improvement on marennine purification, we achieved altogether the isolation of a fucoidan-related, sulfated polysaccharide from blue water. Characterization of the polysaccharides fraction suggests that roughly half of UV-absorbing compounds could be isolated from the marennine crude extracts. The identification of sulfated polysaccharides could be a major breakthrough for marennine purification, providing targeted isolation techniques. Likewise, the added value of Haslea ostrearia and the role of polysaccharides in previous marennine chemical characterization and bioactivity studies remain to be determined.

Whole cell solid-state NMR study of Chlamydomonas reinhardtii microalgae.

In vivo or whole-cell solid-state NMR is an emerging field which faces tremendous challenges. In most cases, cell biochemistry does not allow the labelling of specific molecules and an in vivo study is thus hindered by the inherent difficulty of identifying, among a formidable number of resonances, those arising from a given molecule. In this work we examined the possibility of studying, by solid-state NMR, the model organism Chlamydomonas reinhardtii fully and non-specifically 13C labelled. The extension of NMR-based dynamic filtering from one-dimensional to two-dimensional experiments enabled an enhanced selectivity which facilitated the assignment of cell constituents. The number of resonances detected with these robust and broadly applicable experiments appears to be surprisingly sparse. Various constituents, notably galactolipids abundant in organelle membranes, carbohydrates from the cell wall, and starch from storage grains could be unambiguously assigned. Moreover, the dominant crystal form of starch could be determined in situ. This work illustrates the feasibility and caveats of using solid-state NMR to study intact non-specifically 13C labelled micro-organisms.

Interspecies comparison of the mechanical properties and biochemical composition of byssal threads.

Several bivalve species produce byssus threads to provide attachment to substrates, with mechanical properties highly variable among species. Here, we examined the distal section of byssal threads produced by a range of bivalve species (Mytilus edulis, Mytilus trossulus, Mytilus galloprovincialis, Mytilus californianus, Pinna nobilis, Perna perna, Xenostrobus securis, Brachidontes solisianus and Isognomon bicolor) collected from different nearshore environments. Morphological and mechanical properties were measured, and biochemical analyses were performed. Multivariate redundancy analyses on mechanical properties revealed that byssal threads of M. californianus, M. galloprovincialis and P. nobilis have very distinct mechanical behaviours compared with the remaining species. Extensibility, strength and force were the main variables separating these species groups, which were highest for M. californianus and lowest for P. nobilis Furthermore, the analysis of the amino acid composition revealed that I. bicolor and P. nobilis threads are significantly different from the other species, suggesting a different underlying structural strategy. Determination of metal contents showed that the individual concentration of inorganic elements varies, but that the dominant elements are conserved between species. Altogether, this bivalve species comparison suggests some molecular bases for the biomechanical characteristics of byssal fibres that may reflect phylogenetic limitations.

Validation of trophic and anthropic underwater noise as settlement trigger in blue mussels.

Like the majority of benthic invertebrates, the blue mussel Mytilus edulis has a bentho-pelagic cycle with its larval settlement being a complex phenomenon involving numerous factors. Among these factors, underwater noise and pelagic trophic conditions have been weakly studied in previous researches. Under laboratory conditions, we tested the hypothesis that picoplankton assimilation by the pediveliger blue mussel larvae acts as a food cue that interacts with anthropic underwater sound to stimulate settlement. We used (13)C-labeling microalgae to validate the assimilation of different picoplankton species in the tissues of pediveliger larvae. Our results clearly confirm our hypothesis with a significant synergic effect of these two factors. However, only the picoeukaryotes strains assimilated by larvae stimulated the settlement, whereas the non-ingested picocyanobacteria did not. Similar positive responses were observed with underwater sound characterized by low frequency vessel noises. The combination of both factors (trophic and vessel noise) drastically increased the mussel settlement by an order of 4 compared to the control (without picoplankton and noise). Settlement levels ranged from 16.5 to 67% in 67 h.

Identification of lipid and saccharide constituents of whole microalgal cells by ¹³C solid-state NMR.

Microalgae are unicellular organisms in which plasma membrane is protected by a complex cell wall. The chemical nature of this barrier is important not only for taxonomic identification, but also for interactions with exogenous molecules such as contaminants. In this work, we have studied freshwater (Chlamydomonas reinhardtii) and marine (Pavlova lutheri and Nannochloropsis oculata) microalgae with different cell wall characteristics. C. reinhardtii is covered by a network of fibrils and glycoproteins, while P. lutheri is protected by small cellulose scales, and the picoplankton N. oculata by a rigid cellulose wall. The objective of this work was to determine to what extent the different components of these microorganisms (proteins, carbohydrates, lipids) can be distinguished by ¹³C solid-state NMR with an emphasis on isolating the signature of their cell walls and membrane lipid constituents. By using NMR experiments which select rigid or mobile zones, as well as ¹³C-enriched microalgal cells, we improved the spectral resolution and simplified the highly crowded spectra. Interspecies differences in cell wall constituents, storage sugars and membrane lipid compositions were thus evidenced. Carbohydrates from the cell walls could be distinguished from those incorporated into sugar reserves or glycolipids. Lipids from the plasmalemma and organelle membranes and from storage vacuoles could also be identified. This work establishes a basis for a complete characterization of phytoplankton cells by solid-state NMR.

Identification of Mytilus edulis genetic regulators during early development.

Understanding the mechanisms that enable growth and survival of an organism while driving it to the full range of its adaptation is fundamental to the issues of biodiversity and evolution, particularly regarding global climatic changes. Here we report the Illumina RNA-sequencing (RNA-seq) and de novo assembly of the blue mussel Mytilus edulis transcriptome during early development. This study is based on high-throughput data, which associates genome-wide differentially expressed transcript (DET) patterns with early activation of developmental processes. Approximately 50,383 high-quality contigs were assembled. Over 8000 transcripts were associated with functional proteins from public databases. Coding and non-coding genes served to design customized microarrays targeting every developmental stage, which encompass major transitions in tissue organization. Consequently, multi-processing pattern exploration protocols applied to 3633 DETs helped discover 12 unique coordinated eigengenes supposedly implicated in various physiological and morphological changes that larvae undergo during early development. Moreover, dynamic Bayesian networks (DBNs) provided key insights to understand stage-specific molecular mechanisms activated throughout ontogeny. In addition, delayed and contemporaneous interactions between DETs were coerced with 16 relevant regulators that interrelated in non-random genetic regulatory networks (GRNs). Genes associated with mechanisms of neural and muscular development have been characterized and further included in dynamic networks necessary in growth and functional morphology. This is the first large-scale study being dedicated to M. edulis throughout early ontogeny. Integration between RNA-seq and microarray data enabled a high-throughput exploration of hidden processes essential in growth and survival of microscopic mussel larvae. Our integrative approach will support a holistic understanding of systems biology and will help establish new links between environmental assessment and functional development of marine bivalves.

Integrative study of physiological changes associated with bacterial infection in Pacific oyster larvae.

BACKGROUND: Bacterial infections are common in bivalve larvae and can lead to significant mortality, notably in hatcheries. Numerous studies have identified the pathogenic bacteria involved in such mortalities, but physiological changes associated with pathogen exposure at larval stage are still poorly understood. In the present study, we used an integrative approach including physiological, enzymatic, biochemical, and molecular analyses to investigate changes in energy metabolism, lipid remodelling, cellular stress, and immune status of Crassostrea gigas larvae subjected to experimental infection with the pathogenic bacteria Vibrio coralliilyticus. FINDINGS: Our results showed that V. coralliilyticus exposure induced (1) limited but significant increase of larvae mortality compared with controls, (2) declined feeding activity, which resulted in energy status changes (i.e. reserve consumption, ß-oxidation, decline of metabolic rate), (3) fatty acid remodeling of polar lipids (changes in phosphatidylinositol and lysophosphatidylcholine composition`, non-methylene-interrupted fatty acids accumulation, lower content of major C20 polyunsaturated fatty acids as well as activation of desaturases, phospholipase and lipoxygenase), (4) activation of antioxidant defenses (catalase, superoxide dismutase, peroxiredoxin) and cytoprotective processes (heat shock protein 70, pernin), and (5) activation of the immune response (non-self recognition, NF-κκ signaling pathway, haematopoiesis, eiconosoids and lysophosphatidyl acid synthesis, inhibitor of metalloproteinase and antimicrobial peptides). CONCLUSION: Overall, our results allowed us to propose an integrative view of changes induced by a bacterial infection in Pacific oyster larvae, opening new perspectives on the response of marine bivalve larvae to infections.

A (2)H solid-state NMR study of the effect of antimicrobial agents on intact Escherichia coli without mutating.

Solid-state nuclear magnetic resonance (NMR) is a useful tool to probe the organization and dynamics of phospholipids in bilayers. The interactions of molecules with membranes are usually studied with model systems; however, the complex composition of biological membranes motivates such investigations on intact cells. We have thus developed a protocol to deuterate membrane phospholipids in Escherichia coli without mutating to facilitate (2)H solid-state NMR studies on intact bacteria. By exploiting the natural lipid biosynthesis pathway and using perdeuterated palmitic acid, our results show that 76% deuteration of the phospholipid fatty acid chains was attained. To verify the responsiveness of these membrane-deuterated E. coli, the effect of known antimicrobial agents was studied. (2)H solid-state NMR spectra combined to spectral moment analysis support the insertion of the antibiotic polymyxin B lipid tail in the bacterial membrane. The use of membrane-deuterated bacteria was shown to be important in cases where antibiotic action of molecules relies on the interaction with lipopolysaccharides. This is the case of fullerenol nanoparticles which showed a different effect on intact cells when compared to dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol membranes. Our results also suggest that membrane rigidification could play a role in the biocide activity of the detergent cetyltrimethyammonium chloride. Finally, the deuterated E. coli were used to verify the potential antibacterial effect of a marennine-like pigment produced by marine microalgae. We were able to detect a different perturbation of the bacteria membranes by intra- and extracellular forms of the pigment, thus providing valuable information on their action mechanism and suggesting structural differences.

Determination of isotopic labeling of proteins by precursor ion scanning liquid chromatography/tandem mass spectrometry of derivatized amino acids applied to nuclear magnetic resonance studies.

RATIONALE: A method has been developed for the quantitation of isotopic labeling of proteins using liquid chromatography/tandem mass spectrometry (LC/MS/MS) for the application of protein nuclear magnetic resonance (NMR) studies. NMR relies on specific isotopic nuclei, such as (13)C and (15)N, for detection and, therefore, isotopic labeling is an important sample preparation step prior to in-depth structural characterization of proteins. The goal of this study was to develop a robust quantitative assay for assessing isotopic labeling in proteins while retaining information on the extent of labeling for individual amino acids. METHODS: Complete digestion of proteins by acid hydrolysis was followed by derivatization of free amino acids with 6-aminoquinolyl N-hydroxysuccinimidyl carbamate (AQC) forming derivatives having identical MS/MS fragmentation behavior. Precursor ion scanning on a hybrid quadrupole-linear ion trap platform was used for amino acid analysis and determining isotopic labeling of proteins. RESULTS: Using a set of isotope-labeled amino acid standards mixed with their unlabeled counterparts, the method was validated for accurately measuring % isotopic contribution. We then applied the method for determining the (13)C isotopic content of algal proteins during a feeding study using (13)C(6)-glucose- or (13)C-bicarbonate-supplemented culture media as well as the level of labeling in mussel byssal threads obtained after feeding with labeled algae. CONCLUSIONS: This method is ideally suited for assessing the extent of protein labeling prior to NMR studies, where the isotopic labeling is a determining factor in the quality of resulting protein spectra, and can be applied to a multitude of different biological samples.

Expression of candidate genes related to metabolism, immunity and cellular stress during massive mortality in the American oyster Crassostrea virginica larvae in relation to biochemical and physiological parameters.

Quantification of mRNA of genes related to metabolism, immunity and cellular stress was examined in relation to a massive mortality event during the culture of American oyster larvae, Crassostrea virginica which was probably, in regard to previous microbiological analysis, induced by Vibrio infection. To document molecular changes associated with the mortality event, mRNA levels were compared to biochemical and physiological data, previously described in a companion paper. Among the 18 genes studied, comparatively to the antibiotic control, 10 showed a lower relative gene expression when the massive mortality occurred. Six of them are presumed to be related to metabolism, corroborating the metabolic depression associated with the mortality event suggested by biochemical and physiological analyses. Relationships between the regulation of antioxidant enzyme activities, lipid peroxidation, and the mRNA abundance of genes linked to oxidative stress, cytoprotection, and immune response are also discussed. Finally, we observed an increase in the transcript abundance of two genes involved in apoptosis and cell regulation simultaneously with mortality, suggesting that these processes might be linked.

High swimming and metabolic activity in the deep-sea eel Synaphobranchus kaupii revealed by integrated in situ and in vitro measurements.

Several complementary studies were undertaken on a single species of deep-sea fish (the eel Synaphobranchus kaupii) within a small temporal and spatial range. In situ experiments on swimming and foraging behaviour, muscle performance, and metabolic rate were performed in the Porcupine Seabight, northeast Atlantic, alongside measurements of temperature and current regime. Deep-water trawling was used to collect eels for studies of animal distribution and for anatomical and biochemical analyses, including white muscle citrate synthase (CS), lactate dehydrogenase (LDH), malate dehydrogenase (MDH), and pyruvate kinase (PK) activities. Synaphobranchus kaupii demonstrated whole-animal swimming speeds similar to those of other active deep-sea fish such as Antimora rostrata. Metabolic rates were an order of magnitude higher (31.6 mL kg(-1) h(-1)) than those recorded in other deep-sea scavenging fish. Activities of CS, LDH, MDH, and PK were higher than expected, and all scaled negatively with body mass, indicating a general decrease in muscle energy supply with fish growth. Despite this apparent constraint, observed in situ burst or routine swimming performances scaled in a similar fashion to other studied species. The higher-than-expected metabolic rates and activity levels, and the unusual scaling relationships of both aerobic and anaerobic metabolism enzymes in white muscle, probably reflect the changes in habitat and feeding ecology experienced during ontogeny in this bathyal species.