Dynamics of sheet nacre formation in bivalves

Abstract

Formation of nacre (mother-of-pearl) is a biomineralization process of fundamental scientific as well as industrial importance. However, the dynamics of the formation process is still not understood. Here, we use scanning electron microscopy and high spatial resolution ion microprobe depth-profiling to image the full three-dimensional distribution of organic materials around individual tablets in the top-most layer of forming nacre in bivalves. Nacre formation proceeds by lateral, symmetric growth of individual tablets mediated by a growth-ring rich in organics, in which aragonite crystallizes from amorphous precursors. The pivotal role in nacre formation played by the growth-ring structure documented in this study adds further complexity to a highly dynamical biomineralization process.

Introduction

Nacre, or mother-of-pearl, is an aragonite-dominated structure produced by bivalves, gastropods, and cephalopods as an internal coating of their otherwise calcitic shells. Because of its highly organized internal structure, chemical complexity, mechanical properties and optical effects, which create a characteristic and beautiful lustre, the formation of nacre is among the best-studied examples of calcium carbonate biomineralization (Wada, 1961, Bevelander and Nakahara, 1969, Mutvei, 1979, Lowenstam, 1981, Levi-Kalisman et al., 2001, Currey, 2005, Snow and Pring, 2005, Addadi et al., 2006). The high-strength mechanical properties of nacre and the fact that the world pearl jewellery represents a global market worth about 5 billions US$ annually (Anon, 2006) have prompted efforts to mimic nacre formation by industrial processes (Addadi et al., 2006). Such efforts require a high level of understanding of the biochemistry and the growth dynamics of nacre formation in nature.

Among the nacre-forming organisms, great diversity is observed with regard to the three-dimensional arrangement of this highly organized structure (Mutvei, 1979). This structural diversity implies substantial variation in the process of nacre formation among different organisms. Here we focus on nacre structure and formation in the pearl-producing bivalve Pinctada margaritifera. Fig. 1 shows the characteristic brick-and-mortar structure of the nacreous layer in P. margaritifera. The primary structural component of mature nacre is a pseudohexagonal tablet, about 0.5–1 micrometer (μm) thick and about 5–10 μm in width, consisting primarily of aragonite: a polymorph of CaCO₃. At the nanometer (nm) length scale, the aragonite component inside individual tablets is embedded in a crystallographically oriented foam-like structure of intra-crystalline organic materials in which the mean size of individual aragonite domains is around 50 nm, (Rousseau et al., 2005a, Stolarski and Mazur, 2005). The crystallographic properties of nacre are that of single crystal aragonite with the c-axis oriented perpendicular to the plane defined by individual tablets. Locally, the a and b axes in the plane of the tablet layers show long-range (i.e. mm-scale) orientation but can also be rotated differently (up to about 45°) around the c-axis in different parts of the shell (Wada, 1958, Weiner and Addadi, 1997, Caseiro, 1995, Checa and Rodriguez-Navarro, 2005, Rousseau et al., 2005a, Checa et al., 2006). It is thought that crystallographic orientations are defined by the inter-lamellar, foam-like network of organic material and transferred from tablet to tablet by ‘bridges’ of similar material (Rousseau et al., 2005a). Checa and Rodriguez-Navarro, 2005, Checa et al., 2006 have suggested a model for how crystal orientation is established in sheet nacre, in which the b-axis becomes gradually aligned with the radial nacre growth direction. In any case, mature tablets are organized in layers in which each tablet is in physical contact with its neighbour, only separated by a nm thick, inter-tabular matrix consisting primarily of aragonite-nucleating proteins and carboxylates, (Addadi et al., 2006, Nudelman et al., 2006, Dauphin et al., 2008). Individual layers of tablets are separated by a 50–100 nm layer consisting mainly of chitin and organic material (Levi-Kalisman et al., 2001). This is the inter-lamellar matrix. Several layers of nacre tablets form at the same time across a growth front, with subsequent layers arranged as ‘stair-cases’ in which each step is typically 10–15 μm wide, (Rousseau et al., 2005b). In this way, up to 3–4 layers of nacre tablets are formed per day (Caseiro, 1995). It has been established that nacre tablet formation takes place in the space defined by the two-top layers of inter-lamellar matrix (Bevelander and Nakahara, 1969). We refer to the layer of organics that separates the animal from the growing nacre as the ‘membrane’. This highly cross-linked protein-rich membrane (Nudelman et al., 2008) is in direct contact with the epithelial cell-structure of the overlying organism, which is referred to as the ‘mantle’ (Fig. 1). In the space beneath the membrane, tablets growth proceeds from nucleation sites, which are complex structures consisting of different layers of organic molecules (Nudelman et al., 2006). The distribution of nucleation sites is dictated by the geometry of the underlying layer of tablets, each nucleation site is often situated above a triple-junction of underlying tablets (Rousseau et al., 2005b). The mantle delivers specific organic molecules (including proteinaceous materials – the so-called ‘silk-phase’) and carbonate components to the closed compartment between the membrane and the first inter-lamellar matrix-layer, which was the top membrane during the formation of the previous layer of nacre tablets (Bevelander and Nakahara, 1969). It has been speculated that growth of individual nacre tablets occur by nucleation and crystallization of amorphous calcium carbonate (ACC) (Towe and Hamilton, 1968). However, because our knowledge about the formation of the nacre structure is largely based on studies of mature nacre structures, little is known about the growth dynamics of individual tablets. The small dimensions of the closed compartment, in which nacre formation takes place (Bevelander and Nakahara, 1969), and the tight physical relationship between the growing nacre structure and the mantle render direct studies difficult. Here, we circumvent these difficulties with a simple preparation technique and by applying new ion microprobe technology to the study of developing nacre tablets prepared with a minimum of alteration of inter-lamellar and inter-tablet organics.