Physical and biological determinants of the fabrication of Molluscan shell microstructures

Molluscs are grand masters in the fabrication of shells, because these are composed of the largest variety of microstructures found among invertebrates. Molluscan microstructures are highly ordered aggregates of either calcite or aragonite crystals with varied morphologies and three-dimensional arrangements. Classically, every aspect of the fabrication of microstructural aggregates is attributed to the action of proteins. There was, however, only direct evidence that the mineral phase, and indirect evidence that nucleation and the crystal shape, are determined by the types of soluble proteins. Some authors imply that crystal competition may also play a role. In addition, the fabrication of intergranular organic matrices typical of some microstructures (nacre, columnar prismatic) cannot have a protein-based explanation. Over the last decade I and collaborators have been applying a holistic view, based on analyzing and interpreting the features of both the organic (mantle, extrapallial space, periostracum, organic matrices) and inorganic (crystallite morphology, arrangement, and crystallography) components of the biomineralization system. By interpreting them on biophysical principles, we have accumulated evidence that, in addition to the activity of proteins, other mechanisms contribute in an essential way to the organization of molluscan microstructures. In particular, we have identified processes such as: (1) crystal nucleation on preformed membranes, (2) nucleation and growth of crystals between and within self-organized membranes, (3) active subcellular processes of contact recognition and deposition. In summary, besides the activity of organic macromolecules, physical (crystal competition, self-organization) and/or biological (direct cellular activity) processes may operate in the fabrication of microstructures. The balance between the physical and biological determinants varies among microstructures, with some being based exclusively on either physical or biological processes, and others having a mixed nature. Other calcifying invertebrates (e.g., corals, cirripeds, serpulids) secrete microstructures that are very similar to inorganic crystal aggregates, and only some brachiopods and, to a lesser extent, bryozoans may have secretory abilities comparable to those of molluscs. Here I provide a new perspective, which may allow microstructures to be understood in terms of evolutionary constraints, to compare the secretional abilities among taxa, and even to evaluate the probability of mimicking microstructures for the production of functional synthetic materials.AC received funding from Project CGL2013-48247-P and CGL2017-85118-P of the Spanish Ministerio de Economía y Competitividad (MINECO) and the Fondo Europeo de Desarrollo Regional (FEDER), from the Research Group RNM363 (Consejería de Economía, Innovación, Ciencia y Empleo of the Junta de Andalucía) and from the Unidad Científica de Excelencia UCE-PP2016-05 of the University of Granada

Physical and Biological Determinants of the Fabrication of Molluscan Shell Microstructures

Molluscs are grand masters in the fabrication of shells, because these are composed of the largest variety of microstructures found among invertebrates. Molluscan microstructures are highly ordered aggregates of either calcite or aragonite crystals with varied morphologies and three-dimensional arrangements. Classically, every aspect of the fabrication of microstructural aggregates is attributed to the action of proteins. There was, however, only direct evidence that the mineral phase, and indirect evidence that nucleation and the crystal shape, are determined by the types of soluble proteins. Some authors imply that crystal competition may also play a role. In addition, the fabrication of intergranular organic matrices typical of some microstructures (nacre, columnar prismatic) cannot have a protein-based explanation. Over the last decade I and collaborators have been applying a holistic view, based on analyzing and interpreting the features of both the organic (mantle, extrapallial space, periostracum, organic matrices) and inorganic (crystallite morphology, arrangement, and crystallography) components of the biomineralization system. By interpreting them on biophysical principles, we have accumulated evidence that, in addition to the activity of proteins, other mechanisms contribute in an essential way to the organization of molluscan microstructures. In particular, we have identified processes such as: (1) crystal nucleation on preformed membranes, (2) nucleation and growth of crystals between and within self-organized membranes, (3) active subcellular processes of contact recognition and deposition. In summary, besides the activity of organic macromolecules, physical (crystal competition, self-organization) and/or biological (direct cellular activity) processes may operate in the fabrication of microstructures. The balance between the physical and biological determinants varies among microstructures, with some being based exclusively on either physical or biological processes, and others having a mixed nature. Other calcifying invertebrates (e.g., corals, cirripeds, serpulids) secrete microstructures that are very similar to inorganic crystal aggregates, and only some brachiopods and, to a lesser extent, bryozoans may have secretory abilities comparable to those of molluscs. Here I provide a new perspective, which may allow microstructures to be understood in terms of evolutionary constraints, to compare the secretional abilities among taxa, and even to evaluate the probability of mimicking microstructures for the production of functional synthetic materials

The unique fibrilar to platy nanoand microstructure of twinned rotaliid foraminiferal shell calcite

Open Access funding enabled and organized by Projekt DEALGerman Research Council (Grant No. GR9/1234 SCHM/930/11-2)

Anisotropy of Mechanical Properties of Pinctada margaritifera Mollusk Shell

The research was co-financed by the European Union from the resources of the European Social Fund (Project No.WND-POWR.03.02.00-00-I043/16), the DAAD program. A.C. acknowledges project CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovaciónfor funding.The mechanical properties such as compressive strength and nanohardness were investigated for Pinctada margaritifera mollusk shells. The compressive strength was evaluated through a uniaxial static compression test performed along the load directions parallel and perpendicular to the shell axis, respectively, while the hardness and Young modulus were measured using nanoindentation. In order to observe the crack propagation, for the first time for such material, the in-situ X-ray microscopy (nano-XCT) imaging (together with 3D reconstruction based on the acquired images) during the indentation tests was performed. The results were compared with these obtained during the micro-indentation test done with the help of conventional Vickers indenter and subsequent scanning electron microscopy observations. The results revealed that the cracks formed during the indentation start to propagate in the calcite prism until they reach a ductile organic matrix where most of them are stopped. The obtained results confirm a strong anisotropy of both crack propagation and the mechanical strength caused by the formation of the prismatic structure in the outer layer of P. margaritifera shell.The research was co-financed by the European Union from resources of the European Social Fund (Project No.WND-POWR.03.02.00-00-I043/16)

Ribs of Pinna nobilis shell induce unexpected microstructural changes that provide unique mechanical properties

The authors thank Mr Łukasz Niedzielski from Keyence International for the possibility of performing microstructural analysis using a VHX- 7000 digital microscope. Thanks to Damian Sapijaszka who collected the P. nobilis specimen for scientific research. The work was supported by the Polish National Agency for Academic Exchange (grant PPI/APM/ 2018/1/00049/U/001) and the National Science Center (grant UMO- 2018/29/B/ST8/02200). MS was supported by the European Union from the resources of the European Social Fund (Project No.WNDPOWR. 03.02.00-00-I043/16). AGC was funded by project CGL2017- 85118-P of the Spanish Ministerio de Ciencia e Innovaci´on. Jos´e Rafael García March (Universidad Cat´olica de Valencia, Spain) provided the images of Pinna nobilis of Fig. A1.The reinforcement function of shell ribs depends not only on their vaulted morphology but also on their microstructure. They are part of the outer layer which, in the case of the Pinna nobilis bivalve, is built from almost monocrystalline calcitic prisms, always oriented perpendicular to the growth surfaces. Originally, prisms and their c-axes follow the radii of rib curvature, becoming oblique to the shell thickness direction. Later, prisms bend to reach the nacre layer perpendicularly, but their c-axes retain the initial orientation. Calcite grains form nonrandom boundaries. Most often, three twin disorientations arise, with two of them observed for the first time. Nano-indentation and impact tests demonstrate that the oblique orientation of c-axes significantly improves the hardness and fracture toughness of prisms. Moreover, compression tests reveal that the rib area achieves a unique strength of 700 MPa. The detection of the specific microstructure formed to toughen the shell is novel.Polish National Agency for Academic Exchange (grant PPI/APM/ 2018/1/00049/U/001)National Science Center (grant UMO- 2018/29/B/ST8/02200)European Social Fund (Project No.WNDPOWR. 03.02.00-00-I043/16)CGL2017- 85118-P of the Spanish Ministerio de Ciencia e Innovaci´o

Evaluation of remodeling and geometry on the biomechanical properties of nacreous bivalve shells

The supports provided by the PIA ANILLOS ACT project No. 172037 of the Chilean Council for Research and Technology (ANID -Ex CONICYT), Millennium Science Initiative Program -ICN2019_015 (SECOS), and the Projects CGL2017-85118-P of the Spanish Ministerio de Ciencia e Innovacion, and the Research Group RNM363 of the Junta de Andalucia. In addition, we thank the support provided by DICYT from the Universidad de Santiago de Chile and by ANID PfCHA/DOCTORADO BECAS CHILE/2019 -CEL00011051.Mollusks have developed a broad diversity of shelled structures to protect against challenges imposed by biological interactions(e.g., predation) and constraints (e.g., pCO2-induced ocean acidification and wave-forces). Although the study of shell biomechanical properties with nacreous microstructure has provided understanding about the role of shell integrity and functionality on mollusk performance and survival, there are no studies, to our knowledge, that delve into the variability of these properties during the mollusk ontogeny, between both shells of bivalves or across the shell length. In this study, using as a model the intertidal mussel Perumytilus purpuratus to obtain, for the first time, the mechanical properties of its shells with nacreous microstructure; we perform uniaxial compression tests oriented in three orthogonal axes corresponding to the orthotropic directions of the shell material behavior (thickness, longitudinal, and transversal). Thus, we evaluated whether the shell material’s stress and strain strength and elastic modulus showed differences in mechanical behavior in mussels of different sizes, between valves, and across the shell length. Our results showed that the biomechanical properties of the material building the P. purpuratus shells are symmetrical in both valves and homogeneous across the shell length. However, uniaxial compression tests performed across the shell thickness showed that biomechanical performance depends on the shell size (aging); and that mechanical properties such as the elastic modulus, maximum stress, and strain become degraded during ontogeny. SEM observations evidenced that compression induced a tortuous fracture with a delamination effect on the aragonite mineralogical structure of the shell. Findings suggest that P. purpuratus may become vulnerable to durophagous predators and wave forces in older stages, with implications in mussel beds ecology and biodiversity of intertidal habitats.PIA ANILLOS ACT project of Chilean Council for Research and Technology (ANID -Ex CONICYT) 172037Millennium Science Initiative Program ICN2019_015Instituto de Salud Carlos III Spanish Government CGL2017-85118-PJunta de Andalucia RNM363DICYT from the Universidad de Santiago de ChileANID PfCHA/DOCTORADO BECAS CHILE/2019 CEL0001105

Shell microstructure and its inheritan ce in the calcit ic helcionellid Mackinnonia

Mackinnonia davidi from the Cambrian (Series 2) of Australia has a prismatic outer shell layer and, as newly described here, a calcitic semi-nacre inner layer. The pattern is the same as in stenothecids such as Mellopegma, providing more evidence for a strong phylogenetic signal in the shell microstructure of Cambrian molluscs. In addition, calcite now appears to have been common in helcionellids and other molluscs during the early and middle Cambrian, with many species exhibiting foliated calcite. This is surprising given the dominance of aragonite in molluscs, both modern and from post-Cambrian fossil deposits with exceptional shell microstructure preservation, including localities from the Ordovician of the Cincinnati region, USA.The work was funded by a Marie Curie Postdoctoral Fellowship (IIF 301668) from the European Commission. This research is a contributio on to IGCP Project 591

Calcite fibre formation in modern brachiopod shells

The fibrous calcite layer of modern brachiopod shells is a hybrid composite material and forms a substantial part of the hard tissue. We investigated how cells of the outer mantle epithelium (OME) secrete calcite material and generate the characteristic fibre morphology and composite microstructure of the shell. We employed AFM, FE-SEM, and TEM imaging of embedded/etched, chemically fixed/ decalcified and high-pressure frozen/freeze substituted samples. Calcite fibres are secreted by outer mantle epithelium (OME) cells. Biometric analysis of TEM micrographs indicates that about 50% of these cells are attached via hemidesmosomes to an extracellular organic membrane present at the proximal, convex surface of the fibres. At these sites, mineral secretion is not active. Instead, ion transport from OME cells to developing fibres occurs at regions of closest contact between cells and fibres, however only at sites where the extracellular membrane at the proximal fibre surface is not developed yet. Fibre formation requires the cooperation of several adjacent OME cells. It is a spatially and temporally changing process comprising of detachment of OME cells from the extracellular organic membrane, mineral secretion at detachment sites, termination of secretion with formation of the extracellular organic membrane, and attachment of cells via hemidesmosomes to this membrane.This is a BASE-LINE Earth project supported by the European Union’s Horizon 2020 research and innovation program under grant agreement No. 643084. This is publication nr. 159 of Huinay Scientific Field Station

Structure and crystallography of foliated and chalk shell microstructures of the oyster Magallana: the same materials grown under different conditions.

Oyster shells are mainly composed of layers of foliated microstructure and lenses of chalk, a highly porous, apparently poorly organized and mechanically weak material. We performed a structural and crystallographic study of both materials, paying attention to the transitions between them. The morphology and crystallography of the laths comprising both microstructures are similar. The main differences were, in general, crystallographic orientation and texture. Whereas the foliated microstructure has a moderate sheet texture, with a defined 001 maximum, the chalk has a much weaker sheet texture, with a defined 011 maximum. This is striking because of the much more disorganized aspect of the chalk. We hypothesize that part of the unanticipated order is inherited from the foliated microstructure by means of, possibly, [Formula: see text] twinning. Growth line distribution suggests that during chalk formation, the mantle separates from the previous shell several times faster than for the foliated material. A shortage of structural material causes the chalk to become highly porous and allows crystals to reorient at a high angle to the mantle surface, with which they continue to keep contact. In conclusion, both materials are structurally similar and the differences in orientation and aspect simply result from differences in growth conditions

Crystallographic control of the fabrication of an extremely sophisticated shell surface microornament in the glass scallop Catillopecten

The external surface microornament of the glass scallops Catillopecten natalyae and malyutinae is made by calcitic spiny projections consisting of a stem that later divides into three equally spaced and inclined branches (here called aerials). C. natalyae contains larger and smaller aerials, whereas C. malyutinae only secreted aerials of the second type. A remarkable feature is that aerials within each type are fairly similar in size and shape and highly co-oriented, thus constituting a most sophisticated microornament. We demonstrate that aerials are single crystals whose morphology is strongly controlled by the crystallography, with the stem being parallel to the c-axis of calcite, and the branches extending along the edges of the {104} calcite rhombohedron. They grow epitaxially onto the foliated prisms of the outer shell layer. The co-orientation of the prisms explains that of the aerials. We have developed a model in which every aerial grows within a periostracal pouch. When this pouch reaches the growth margin, the mantle initiates the production of the aerial. Nevertheless, later growth of the aerial is remote, i.e. far from the contact with the mantle. We show how such an extremely sophisticated microornament has a morphology and co-orientation which are determined by crystal growth.Instituto de Salud Carlos III Spanish Government CGL2017-85118-P PID2020-116660GB-I00Unidad Cientifica de Excelencia of the University of Granada UCE-PP2016-05Junta de Andalucia RNM363Ministry of Science, ICT & Future Planning, Republic of Korea 13.1902.21.001 075-15-2020-79