The blue mussel inside: 3D visualization and description of the vascular-related anatomy of Mytilus edulis to unravel hemolymph extraction.

The blue mussel Mytilus edulis is an intensely studied bivalve in biomonitoring programs worldwide. The lack of detailed descriptions of hemolymph-withdrawal protocols, particularly with regard to the place from where hemolymph could be perfused from, raises questions regarding the exact composition of aspirated hemolymph and does not exclude the possibility of contamination with other body-fluids. This study demonstrates the use of high resolution X-ray computed tomography and histology combined with 3D-reconstruction using AMIRA-software to visualize some important vascular-related anatomic structures of Mytilus edulis. Based on these images, different hemolymph extraction sites used in bivalve research were visualized and described, leading to new insights into hemolymph collection. Results show that hemolymph withdrawn from the posterior adductor muscle could be extracted from small spaces and fissures between the muscle fibers that are connected to at least one hemolymph supplying artery, more specifically the left posterior gastro-intestinal artery. Furthermore, 3D-reconstructions indicate that puncturing hemolymph from the pericard, anterior aorta, atria and ventricle in a non-invasive way should be possible. Hemolymph withdrawal from the heart is less straightforward and more prone to contamination from the pallial cavity. This study resulted simultaneously in a detailed description and visualization of the vascular-related anatomy of Mytilus edulis.

Hierarchically-structured metalloprotein composite coatings biofabricated from co-existing condensed liquid phases.

Complex hierarchical structure governs emergent properties in biopolymeric materials; yet, the material processing involved remains poorly understood. Here, we investigated the multi-scale structure and composition of the mussel byssus cuticle before, during and after formation to gain insight into the processing of this hard, yet extensible metal cross-linked protein composite. Our findings reveal that the granular substructure crucial to the cuticle's function as a wear-resistant coating of an extensible polymer fiber is pre-organized in condensed liquid phase secretory vesicles. These are phase-separated into DOPA-rich proto-granules enveloped in a sulfur-rich proto-matrix which fuses during secretion, forming the sub-structure of the cuticle. Metal ions are added subsequently in a site-specific way, with iron contained in the sulfur-rich matrix and vanadium coordinated by DOPA-catechol in the granule. We posit that this hierarchical structure self-organizes via phase separation of specific amphiphilic proteins within secretory vesicles, resulting in a meso-scale structuring that governs cuticle function.

Rapid self-assembly of complex biomolecular architectures during mussel byssus biofabrication.

Protein-based biogenic materials provide important inspiration for the development of high-performance polymers. The fibrous mussel byssus, for instance, exhibits exceptional wet adhesion, abrasion resistance, toughness and self-healing capacity-properties that arise from an intricate hierarchical organization formed in minutes from a fluid secretion of over 10 different protein precursors. However, a poor understanding of this dynamic biofabrication process has hindered effective translation of byssus design principles into synthetic materials. Here, we explore mussel byssus assembly in Mytilus edulis using a synergistic combination of histological staining and confocal Raman microspectroscopy, enabling in situ tracking of specific proteins during induced thread formation from soluble precursors to solid fibres. Our findings reveal critical insights into this complex biological manufacturing process, showing that protein precursors spontaneously self-assemble into complex architectures, while maturation proceeds in subsequent regulated steps. Beyond their biological importance, these findings may guide development of advanced materials with biomedical and industrial relevance.

Seawater acidification induced immune function changes of haemocytes in Mytilus edulis: a comparative study of CO2 and HCl enrichment.

The present study was performed to evaluate the effects of CO2- or HCl-induced seawater acidification (pH 7.7 or 7.1; control: pH 8.1) on haemocytes of Mytilus edulis, and the changes in the structure and immune function were investigated during a 21-day experiment. The results demonstrated that seawater acidification had little effect on the cellular mortality and granulocyte proportion but damaged the granulocyte ultrastructure. Phagocytosis of haemocytes was also significantly inhibited in a clearly concentration-dependent manner, demonstrating that the immune function was affected. Moreover, ROS production was significantly induced in both CO2 and HCl treatments, and four antioxidant components, GSH, GST, GR and GPx, had active responses to the acidification stress. Comparatively, CO2 had more severe destructive effects on haemocytes than HCl at the same pH level, indicating that CO2 stressed cells in other ways beyond the increasing H+ concentration. One possible explanation was that seawater acidification induced ROS overproduction, which damaged the ultrastructure of haemocytes and decreased phagocytosis.

First record of the green microalgae Coccomyxa sp. in blue mussel Mytilus edulis (L.) from the Lower St. Lawrence Estuary (Québec, Canada).

During autumn 2012 and spring 2013, blue mussels Mytilus edulis (L.) with strongly deformed (L-shaped) posterior shell margins and green spots in soft tissue (microalgae) were collected from intertidal zone along the south shore of the Lower St. Lawrence Estuary near Rimouski (Québec, Canada). Identification of algal cells infesting mussels as Coccomyxa sp. was confirmed by rRNA sequencing and HPLC pigment analysis. Flow cytometric analysis revealed the presence of algal cells in the hemolymph and extrapallial fluid in mussels with deformed and non-deformed shells; concentrations of algal cells were ranged from about 200mL(-1) in mussels with actually non-deformed shells to concentrations reaching up to 3.8×10(7)mL(-1) in mussels with heavily deformed ones. Chemical analyses of soft tissues led us to conclude that butyltin compounds and trace metals cannot be considered among factors responsible for the shell deformity observed. Using scanning electron microscopy, the biogenic nature of the erosion on the external shell surface and aragonitic lenses of prisms in the curvature zone of deformed shells (in sections) were recorded. The sequence of the green algae from M. edulis of the Lower St. Lawrence Estuary was closely related to Coccomyxa sp. infecting M. edulis from the Flensburg Fjord (North Sea) and Modiolus modiolus (L.) from the Vityaz Bay (Sea of Japan).

Homoepitaxial meso- and microscale crystal co-orientation and organic matrix network structure in Mytilus edulis nacre and calcite.

New developments in high-resolution, low accelaration voltage electron backscatter diffraction (EBSD) enable us to resolve and quantify the co-orientation of nanocrystals constituting biological carbonate crystals with a scan step resolution of 125 nm. This allows the investigation of internal structures in carbonate tablets and tower biocrystals in the nacre of mollusc shells, and it provides details on the calcite-aragonite polymorph interface in bivalves. Within the aragonite tablets of Mytilus edulis nacre we find a mesoscale crystallographic mosaic structure with a misorientation distribution of 2° full width at half maximum. Selective etching techniques with critical point drying reveal an organic matrix network inside the nacre tablets. The size scales of the visible aragonite tablet subunits and nanoparticles correspond to those of the open pore system in the organic matrix network. We further observe by EBSD that crystal co-orientation spans over tablet boundaries and forms composite crystal units of up to 20 stacked co-oriented tablets (tower crystals). Statistical evaluation of the misorientation data gives a probability distribution of grain boundary misorientations with two maxima: a dominant peak for very-small-angle grain boundaries and a small maximum near 64°, the latter corresponding to {110} twinning orientations. However, the related twin boundaries are typically the membrane-lined {001} flat faces of the tablets and not {110} twin walls within tablets. We attribute this specific pattern of misorientation distribution to growth by particle accretion and subsequent semicoherent homoepitaxial crystallization. The semicoherent crystallization percolates between the tablets through mineral bridges and across matrix membranes surrounding the tablets. In the "prismatic" calcite layer crystallographic co-orientation of the prisms reaches over more than 50 micrometers.

Food supply and seawater pCO2 impact calcification and internal shell dissolution in the blue mussel Mytilus edulis.

Progressive ocean acidification due to anthropogenic CO(2) emissions will alter marine ecosystem processes. Calcifying organisms might be particularly vulnerable to these alterations in the speciation of the marine carbonate system. While previous research efforts have mainly focused on external dissolution of shells in seawater under saturated with respect to calcium carbonate, the internal shell interface might be more vulnerable to acidification. In the case of the blue mussel Mytilus edulis, high body fluid pCO(2) causes low pH and low carbonate concentrations in the extrapallial fluid, which is in direct contact with the inner shell surface. In order to test whether elevated seawater pCO(2) impacts calcification and inner shell surface integrity we exposed Baltic M. edulis to four different seawater pCO(2) (39, 142, 240, 405 Pa) and two food algae (310-350 cells mL(-1) vs. 1600-2000 cells mL(-1)) concentrations for a period of seven weeks during winter (5°C). We found that low food algae concentrations and high pCO(2) values each significantly decreased shell length growth. Internal shell surface corrosion of nacreous ( = aragonite) layers was documented via stereomicroscopy and SEM at the two highest pCO(2) treatments in the high food group, while it was found in all treatments in the low food group. Both factors, food and pCO(2), significantly influenced the magnitude of inner shell surface dissolution. Our findings illustrate for the first time that integrity of inner shell surfaces is tightly coupled to the animals' energy budget under conditions of CO(2) stress. It is likely that under food limited conditions, energy is allocated to more vital processes (e.g. somatic mass maintenance) instead of shell conservation. It is evident from our results that mussels exert significant biological control over the structural integrity of their inner shell surfaces.

Colloidal complexed silver and silver nanoparticles in extrapallial fluid of Mytilus edulis.

Metal transport in mollusk extrapallial fluid (EPF) that acts as a "bridge" between soft tissues and shell has surprisingly received little attention until now. Using ultrafiltration and radiotracer techniques we determined silver concentrations and speciation in the EPF of the blue mussel Mytilus edulis after short-term uptake and depuration laboratory experiments. Radiolabelled silver ((¹¹°m)Ag) was used in dissolved or nanoparticulate phases (AgNPs < 40 nm), with a similar low Ag concentration (total radioactive and cold Ag ~0.7 µg/L) in a way that mussels could uptake radiotracers only from seawater. Our results indicated that silver nanoparticles were transported to the EPF of blue mussels at a level similar to the Ag ionic form. Bulk activity of radiolabelled silver in the EPF represented only up to 7% of the bulk activity measured in the whole mussels. The EPF extracted from mussels exposed to both treatments exhibited an Ag colloidal complexed form based on EPF ultrafiltration through a 3 kDa filter. This original study brings new insights to internal circulation of nanoparticles in living organisms and contributes to the international effort in studying the potential impacts of engineered nanomaterials on marine bivalves which play an essential role in coastal ecosystems, and are important contributors to human food supply from the sea.

A comparative study of the anti-settlement properties of mytilid shells.

Marine organisms have evolved defence mechanisms to prevent epibiosis. This study investigated the anti-settlement properties of natural periostracal microtopographies of two mytilid species, Mytilus edulis (from North, Baltic and White Seas) and Perna perna (from the SW Atlantic). Resin replicas of shells were exposed to cyprids of the barnacle Semibalanus balanoides. Replicas with intact isotropic microtopographies and smooth controls were much less fouled than roughened anisotropic surfaces. This indicates that in both M. edulis and P. perna the periostracum possesses a generic anti-settlement property, at least against S. balanoides cyprids, which is not regionally adapted. Such a potential globally effective anti-settlement mechanism possibly contributes to the invasive success of Mytilidae.