DOI: 10.1021/cg901263p
Alignment of Crystallographic c-Axis throughout the Four Distinct Microstructural Layers of the Oyster Crassostrea gigas
2010, Vol. 10 1243–1246
Joanne MacDonald,† Andy Freer,‡ and Maggie Cusack*,† †
Department of Geographical and Earth Science, Gregory Building, University of Glasgow, Lilybank Gardens, Glasgow G12 8QQ, U.K., and ‡Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, U.K. Received October 12, 2009; Revised Manuscript Received November 3, 2009
ABSTRACT: The superimposed layers of the true oyster shell have distinct morphology. The shells are mainly calcitic, comprising an outer prismatic region and inner foliated structure that is frequently interrupted by lenses of chalky calcitic deposits. Aragonite is restricted to the myostracum and ligament. Electron backscatter diffraction (EBSD) analysis has shown that despite the variations in structural morphology, the mineralized layers of the oyster shell maintain a single crystallographic orientation with the crystallographic c-axis orientated perpendicular to outer and inner shell surfaces. Varying crystal morphology, while maintaining crystallographic unity, may be an evolutionary trait that forms a crack-resistant shell with optimum strength and flexibility.
Introduction Biological systems exhibit exquisite control over biomineral production determining mineral polymorphs and microstructural layering sequences.1-3 This control results in biomineral structures with exceptional mechanical properties with the innate ability to perform a range of functions that facilitate the survival of the living organism. A number of recent studies have demonstrated that understanding the internal structure and crystallography of biological composites is extremely valuable in the inspiration and design of supertough crackresistant materials.4,5 The purely aragonitic structure of nacre is currently the most intensively studied bivalve shell microstructure due to its excellent mechanical properties4-7 compared with calcitic counterparts, such as folia, which is far weaker in terms of strength and hardness.6,8 The high tensile strength of nacre has allowed some bivalve species, with nacre as the dominant shell component, to evolve with very thin relatively flat shells.6 However, alternative designs occur with many of the most successful bivalves adopting shells composed mostly of calcite, which depend upon the microstructural arrangement and crystallographic orientation within the shell, rather than the outright strength offered by individual shell components.6 Oysters are among the most successful bivalves, first appearing in the geological record in the Triassic.9-11 This success may be attributed to the development of unique shell characteristics with the ability to reduce the effect of detrimental external impacts through crack deflection.5 Extant oysters of the order Ostreoida are composed predominantly of foliated calcite with aragonite restricted to the myostracum and ligament, where aragonite is preferentially selected as it serves better the function of attachment.12 This shows an evolutionary divergence from the shell structure of the earliest known oysters, which were probably nacreous.10,11 Early in the phylogenetic history of oysters, the microstructural arrangement of the shell was altered, resulting in the loss of the strongest structure, nacre,
and incorporation of the weaker shell structure, folia. Despite this replacement, oysters have maintained a crack-resistant shell.5 This study aims to define the relationship between the microstructure and crystallography of the oyster, Crassostrea gigas. The relationship between the microstructure and crystallography described here may offer a possible explanation and insight into how Nature can design extremely robust structures that comprise individually weaker components. Experimental Methods
*To whom correspondence should be addressed. E-mail: Maggie.Cusack@ ges.gla.ac.uk.
Samples of C. gigas were obtained from an oyster farm located at the head of Loch Fyne, Scotland (56.2708° N, 4.9211° W). The soft tissues were removed, and valves were separated and cleaned under running water. Microstructural Analysis. Both valves of two samples were prepared for microstructural analysis. The hinge region was removed by sawing parallel to the hinge axis at a ventral distance of approximately 1 cm from the beak. A radial section was cut from the shell along the dorsal-ventral axis immediately adjacent to the posterior and anterior sides of the muscle scar. This strip of shell was then fractured approximately every 1 cm along the long axis to produce a series of blocks. Each fractured block was glued onto an aluminum stub with an orientation such that the samples are viewed in cross section and coated in gold. Secondary electron and backscatter electron images were produced using a FEI Quanta 200F Environmental scanning electron microscope (SEM) in high vacuum mode with spot size 4, aperture 4, and an accelerating voltage of 20 kV. Electron Backscatter Diffraction (EBSD) Analysis. Both valves of one sample of C. gigas were prepared for crystallographic analysis. Valves were individually mounted in resin blocks (Epoxycure 5:1) and left to cure for 24 h before being cut along the dorsal-ventral axis on adjacent sides of the adductor muscle scar and then further sectioned along the long axis as described above. Sections were polished, coated with carbon, and mounted on stubs with silver paint. All EBSD analyses were carried out using a FEI Quanta 200F Environmental SEM with TSL OIM 5.2 data collection software. The SEM was operated in high vacuum at 20 kV with spot size 4 and aperture 4. EBSD data were analyzed, and maps were produced through OIM Analysis v4 software. Data is partitioned through a two-stage cleanup procedure so that only grains of confidence index (CI) g 0.2 are displayed in the final data set.
r 2009 American Chemical Society
Published on Web 11/19/2009
pubs.acs.org/crystal
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Figure 1. Microstructure and crystallography of C. gigas shell: (A) schematic diagram showing microstructural layering sequence within shell; (B) backscatter image of the myostracum; (C) backscatter image of the foliated structure (folia); (D) backscatter image of the chalky structure; (E) MIX image (secondary electron and backscatter electron) of the prismatic region; (F, G) crystallographic orientation map color keys for calcite and aragonite, respectively, with reference to the normal direction; (H-K) electron backscatter diffraction maps of crystallographic orientation normal to view according to color key (F, G) for myostracum, folia, chalk, and prismatic regions, respectively (scale bars are 5, 25, 45, and 25 μm, respectively); (L) pole figure showing crystallographic orientation of aragonite crystals in myostracum in reference to {001} plane; (M) pole figure showing crystallographic orientation of calcite crystals in folia in reference to {0001} plane; (N) pole figure showing crystallographic orientation of calcite crystals in chalky structure in reference to {0001} plane; (O) pole figure showing crystallographic orientation of calcite crystals in prismatic region in reference to {0001} plane with colors according to those in panels H-K.
Results Microstructure. Four principle mineralized microstructural types comprise both right and left valves of C. gigas (Figure 1A). The myostracum comprises well-developed, simple aragonitic prisms, orientated with the long axis of the prisms perpendicular to the inner shell surface. Myostracal prisms are fine, needle-like, closely packed first-order units with no observable interprismatic organic sheaths (Figure 1B). Myostracal prisms are overlaid by foliated calcite (folia), which accounts for the majority of the shell
thickness. The folia are composed, for the most part, of long, tabular laths or blades joined laterally to form a series of thin sheets of folia, which are stacked vertically to form the foliated microstructure (Figure 1C). Sheets of laths are highly organized and exist as a series of clusters of alternating orientation. Clusters of foliated sheets are referred to as having type 1 or type 2 orientation where type 1 are orientated with the long axis of the lath parallel to outer shell surface, while type 2 are orientated with the long axis of the laths up to 45° from parallel to outer shell.5 The bulk of the
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valve comprises sheets of folia of type 1 orientation, which are interrupted by thin clusters of type 2 orientated folia. Lenses of soft, porous, white, chalky calcitic structure occur randomly, interrupting the foliated microstructure of both valves. These chalky deposits form lenticular or blisterlike bodies of various sizes. Chalky deposits comprise extremely thin, smooth, platy calcite flakes or blades of various sizes. Within the chalky microstructure, it is possible to define a set of first-order main blades orientated perpendicular to the inner surface of the valves, from which smaller leaf-like microstructures (termed leaflets) branch at varying angles (Figure 1D). Leaflets interlock with each other, leaving a large amount of void space between them. The outermost mineralized layer of the shell is known as the calcitic prismatic region (CPR). This microstructure comprises firstorder simple calcitic prisms orientated with the long axis of the prisms perpendicular to outer shell surface (Figure 1E). The CPR varies significantly from the foliated structure to which it binds to the inner shell surface. Most noticeable is the greater size of the prisms compared with the laths. Prisms of the CPR typically range from 22 to 190 μm in height and 5 to 37 μm in width, in contrast the laths of the folia, which range from 0.1 to 0.6 μm in height and 0.9 to 3 μm in width with variable lengths. Crystallographic Orientation. EBSD crystallographic analysis carried out at several locations within each microstructural domain reveals that, despite variation in crystal morphology and mineral polymorph, crystallographic orientation remains uniform throughout the shell. EBSD maps relative to inner and outer shell surface with superimposed crystal models show the same crystal orientation for all microstructures, with the c-axis orientated perpendicular to the inner and outer shell surface (Figure 1F-I). Pole figures representing the {0001} crystal plane of calcite and {001} crystal plane of aragonite show for all microstructures one single maxima perpendicular to the {0001} plane of calcite and {001} plane of aragonite with lateral variation of up to 25° reaching a maximum of 42° in the folia (Figure 1J-M). Examination of the foliated structure at several locations, including areas where laths of both type 1 and type 2 orientation are present, shows no change in crystallography. This implies that the difference in the orientation of type 1 and type 2 laths is a morphological characteristic, which alters the in-plane orientation of the laths while maintaining uniform orientation of the crystallographic c-axis. Discussion Like most bivalves, the valves of Crassostrea gigas are complex biomineral structures comprising CaCO3 crystals embedded within an organic framework. The shells comprise both calcite and aragonite polymorphs with minor amounts of the organic component, traditionally termed conchiolin.9 Throughout the thickness of the valves, crystals display varied morphologies and structural arrangements creating a series of superimposed layers.9,12,13 In agreement with previous studies of C. virginica9,12,14 four principle mineralized microstructural groups have been identified in both valves of C. gigas. Previous studies into the ultrastructure of C. virginica identified calcitic prisms as the outermost mineralized layer of both right and left valves.12 However, for other species including C. gigas and Ostrea edulis, it is reported that the calcitic prismatic region is restricted to the right valve and does not appear in the left.8,15,16 In the C. gigas samples analyzed in this study,
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calcitic prisms form the outermost mineralized layer of both right and left valves. These prisms are, however, considerably less conspicuous in the left compared with the right. The CPR of both valves consists of parallel, columnar, closely packed, first-order calcitic prisms, which are delineated from each other by relatively thick organic walls9,12,14-16 and orientated with the long axis of the prism perpendicular to outer shell surface. In agreement with Checa et al.,15,16 EBSD data indicates that calcitic prisms are monocrystalline orientated with the c-axis perpendicular to shell surface (parallel with the long axis of the prism) with individual prisms rotated around the c-axis alignment. Orientation of the c-axis coincident with orientation of the prisms is not unique to the oyster and has been recorded in other bivalves such as the wholly aragonitic Entodesma navicula.17 Harper et al.17 proposed that this morphological-crystallographic relationship in the prismatic region of E. navicula, together with a high quantity of organic matrix, provides the shell with flexibility that enhances the ability of the shell to withstand impact. It is proposed that the presence of comparable characteristics in the prismatic region of C. gigas may serve a similar function in minimizing the effect of impact by adding flexibility to the shell ultrastructure.16 Foliated calcite occupies the bulk of both right and left valves of C. gigas comprising blades or laths of calcite arranged laterally to form sheets. Laths within a single sheet have identical orientation which may be of type 1 (parallel to growth surface) or type 2 (up to 45° from type 1).5 The majority of the folia show a type 1 orientation with fine clusters of type 2 orientated as sheets. EBSD analyses of the folia show that lath in-plane orientation does not affect the orientation of the crystallographic c-axis, which remains orientated perpendicular to the inner and outer shell surface. Variation in lath in-plane orientation must therefore be around the a- and b-axes of the calcite crystals, while the c-axis remains constant with only one possible orientation.18 The calcitic c-axis of the folia demonstrates identical orientation to that of the overlying CPR implying a direct relationship between the CPR and the folia. Previous studies have identified that the prisms of the CPR are composed of calcitic laths identical to those of the foliated layer16,19 and this, together with identical crystallographic orientation, suggests that the foliated microstructure is derived from the calcitic prisms.13,19,20 The derivation of the foliated microstructure from the CPR facilitated a dramatic change in the microstructural arrangement of the oyster shell,19 which resulted in the replacement of nacre and the evolution of a predominantly calcitic shell. However, the foliated layers contain very little organic matrix compared with the overlying CPR and, as a consequence, have been shown to be weak in most mechanical tests.6 It is assumed that to make up for this the orientation of the foliated laths is altered from type 1 to type 2 enabling the shell to withstand impact by crack deflection.5 Schmahl et al.21 report that, like the folia of the oyster, the secondary fibrous layer of the brachiopod (order Terebratulida) is composed of calcitic fibers orientated parallel with the shell surface while the crystallographic c-axis is perpendicular to the shell surface. Calcite crystals preferentially cleave along the {104} plane. By orientation of the {0001} plane perpendicular to the outer shell surface, the {104} plane is not coincident with the shell surface and thus delamination by cleavage is avoided. This morphological-crystallographic arrangement in the terebratulide brachiopod, which is analogous to that of the foliated structure of the oyster, provides optimum resistance
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to fracture.21 Within the oyster shell folia, the crystallographic c-axis is orientated consistently with that of the CPR and therefore perpendicular with the outer shell surface and long axis of the laths. It is proposed that through this arrangement the shell maintains a degree of strength as a first line of defense against impact. Altering lath orientation may supply the shell with flexibility and provide deflection in the event of cracking by inhibiting the progress of any cleavage failure through adjacent folia. The foliated layer of the oyster shell is frequently interrupted by lenses of chalky material. Chalky lenses are composed of blades of calcite orientated perpendicular to inner and outer shell surfaces with smaller leaflets branching from these blades at various angles and interlocking around pore space. Lenses are normal shell structures12 that occur randomly throughout both valves. EBSD analysis again shows that the crystallographic c-axis of the chalky structure is orientated perpendicular to inner and outer shell surfaces, coincident with the morphological orientation of the main blades of chalk and identical to the crystallographic orientation of surrounding folia. The position of the chalk lenses within the folia and the presence of identical crystallographic orientation suggests that chalky calcite evolved from foliated calcite,12 in a similar manner to the folia developing from the CPR. Further evidence of this is the continuity between the laths of the folia and the main blades of the chalk.22 Organic matter is present in the chalky shell structure in a higher proportion than that of the surrounding folia (ref 12 and references therein) but less than that of the CPR. Orientation of the c-axis coincident with orientation of the main blades together with the presence of organic matter suggests that the function of the chalky lenses may be similar to that of the CPR and comparable to the prismatic region of E. navicula,17 enhancing the ability of the shell to absorb impact. Similarly, Taylor and Layman6 suggest that the chalky lenses of the oyster serve a similar function to the holes incorporated into bone,23 which act as “crack stoppers”. The inclusion of chalky lenses within oyster shell folia may further enhance the crack resistance of the oyster shell by providing another defense against crack propagation. Conclusions Oysters have evolved with a unique shell structure and arrangement that has ensured the survival and diversification of the order since the Triassic. Despite the replacement of nacre as the main shell component with folia, which exhibits inferior mechanical properties,6 oysters have maintained a shell with high crack resistance.5,6 This crack resistance may be attributed to the morphological-crystallographic relationship within the shell. Both valves are composed of a series of superimposed layers with distinct morphology yet identical
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crystallographic orientation. It is proposed that crystallographic unity throughout the shell may provide a degree of strength while morphological variation provides flexibility and crack deflection. Acknowledgment. Research is funded by a BBSRC DTA award, which is gratefully acknowledged. We also thank Peter Chung for his assistance with SEM and EBSD analysis as well as John Gilleece and Nick Kamenos for assistance with sample preparation. Thanks also to the Loch Fyne Oyster Farm for providing samples used in this study.
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