Breaking the long-standing morphological paradigm: Individual prisms in the pearl oyster shell grow perpendicular to the c-axis of calcite

Abstract

Cross-sections of calcitic prismatic layers in mollusk shells, cut perpendicular to growth direction, reveal well-defined polygonal shapes of individual “grains” clearly visible by light and electron microscopy. For several kinds of shells, it was shown that the average number of edges in an individual prism approaches six during the growth process. Taking into account the rhombohedral symmetry of calcite, often presented in hexagonal axes, all this led to the long-standing opinion that calcitic prisms grow along the c-axis of calcite. In this paper, using X-ray diffraction and electron backscatter diffraction (EBSD), we unambiguously show that calcitic prisms in pearl oyster Pinctada margaritifera predominantly grow perpendicular to the c-axis. The obtained results imply that the hexagon-like habitus of growing crystallites may be not necessarily connected to calcite crystallography and, therefore, other factors should be taken into consideration. We analyze this phenomenon by comparing the organic contents in Pinctada margaritifera and Pinna nobilis shells, the later revealing regular growth of calcitic prisms along the c-axis.

Introduction

Mollusk shells are a mixture of mineral (calcium carbonate, CaCO₃) and organic components (Hérissant, 1766, De Bournon, 1808), which act in synergy producing different structures or layers. Two polymorphs of CaCO₃, aragonite and/or calcite, are used as building blocks. The aragonitic crossed-lamellar layer is the main architecture in Bivalvia and Gastropoda, but the most studied arrangement is rather rare nacro-prismatic one. In nacro-prismatic shells, the prisms are built of either calcite or aragonite (Boggild, 1930). The large-size and nearly honeycomb morphology of the prismatic blocks of Pinna were already noticed by Carpenter, 1844, Bowerbank, 1844. Pinna, Atrina and Pinctada are tightly connected from a taxonomical point of view: they all are Pteriomorphia. In the three taxa, it is relatively easy to separate nacre and prisms, a helpful feature for detailed analyses.

The shapes of the calcitic prisms in these three taxa are simple: elongated columns with polygonal transverse sections. Based on these observations, it is often assumed that their structure, composition and properties are similar. In particular, these calcitic prisms have been defined as “simple prisms”, despite Watabe and Wada (1956), using transverse thin sections of Pinctada martensi in polarized light, found that “each prism extinguishes in several smaller blocks” (see also Taylor et al., 1969). Later on (Taylor, 1973), it was stated that in the simple prismatic structure, “the crystallographic c axis is generally parallel to the long axis of the prisms”. The shape of the prisms of Pinna and Atrina is in accordance with the usual assertion for abiotic calcite: “prismatic crystals parallel to the c axis are length-fast” (Gribble and Hall, 2012). In Pinna, frequently used as a model of prismatic growth, the calcite c-axis is normal to the layer’s surface, i.e. nearly parallel to the long morphological axis of the polygonal prisms (Chateigner et al., 2002). This assertion is now regularly taken as granted when being applied to pteriomorph shells (Checa et al., 2005, MacDonald et al., 2010, Suzuki et al., 2017). There is a general agreement about the nearly single-crystalline structure of the prisms in Pinna and Atrina (Wada, 1961, Cuif and Raguideau, 1982, Dauphin et al., 2003), though careful high-resolution synchrotron X-ray diffraction measurements revealed small misorientation angles (about 1.5°, as maximum) within individual prisms of Pinna nobilis (Metzger et al., 2014).

More serious discrepancies exist in the literature dedicated to the pearl oyster Pinctada. Using thin sections examined in crossed-polarized light, Wada, 1956, Watabe and Wada, 1956 have shown diverse extinction patterns within the prisms of Pinctada. They reported that the c-axes are “oriented nearly perpendicular to the shell surfaces”. This orientation relationship was confirmed by Wada (1961). However, Checa et al. (2013), using electron backscatter diffraction (EBSD), found some misorientation angles (up to 20°) between the c-axis and the normal to the shell surface in the prismatic layer of Pinctada margaritifera. Even larger misorientation angles (up to 50°) were found in the prisms of Pinctada fucata by Gilbert et al. (2011) using X-ray absorption spectroscopy.

In post-larval shells of Pinctada margaritifera, the very first prisms have single-crystalline characteristics (Baronnet et al., 2008). The prism morphology in P. fucata was investigated in both transversal and longitudinal sections by SEM imaging and EBSD analyses (Okumura et al., 2010). The crystalline orientations in the sectors of a prism are similar, but not identical. According to Okumura et al. (2013), the differences between the prisms of Atrina (nearly single-crystalline) and Pinctada (polycrystalline) could be linked to the spatial distribution of the intra-crystalline organic matrix. In the growing zone of the shell of Pinctada, Suzuki et al. (2013) have observed that the “platelet calcite crystals immediately after the nucleation” are single-crystalline, and that the c-axis is “almost parallel to the growth direction of prismatic crystals”. In P. margaritifera, there is a main feature in the growth of the prisms: the young prisms of an adult shell or the prisms in a post-larval shell are nearly single-crystalline, whereas the adult prisms are more polycrystalline. The change from single- to poly-crystalline behavior is visible in thin sections examined in polarized light, and also on the outer longitudinal surface of the prisms (Checa et al., 2013, Checa et al., 2016, Cuif et al., 2014).

Up-to-now, all published data show that the prisms of Pinna (or Atrina) are nearly single-crystalline throughout the life span of the animal, and that the c-axis practically coincides with the prisms’ growth direction. Moreover, it was recently shown that the average number of edges in individual prism in Pinna nobilis (and some other shells) during its growth tends to six (Bayerlein et al., 2014, Zöllner et al., 2017). This is interpreted as the consequence of the classical two-dimensional grain coarsening scenario (Mullins, 1956), which is thermodynamically favorable (Smith, 1964). On the contrary, the literature dedicated to the calcitic prismatic layer of the pearl oyster is controversial. The co-existence of single- and polycrystalline prismatic units seems to be unique among the mollusc shells. Moreover, the orientation of the c-axis is not yet well established.

In this study, we therefore examine the structure, mineralogy, composition and growth of the prisms in juvenile shells of the pearl oyster, Pinctada margaritifera, and their subsequent evolution in adult shells. The focus is on the orientation of the calcite c-axis with respect to the growth direction. In addition, we quantitatively analyze the difference between “single-crystalline” and “polycrystalline” prisms in terms of preferred orientation.