Organization and Formation of the Crossed-Foliated Biomineral Microstructure of Limpet Shells

Abstract

To construct their shells, molluscs are able to produce a large array of calcified materials including granular, prismatic, lamellar, fibrous, foliated, and plywood-like microstructures. The latter includes an aragonitic (the crossed-lamellar) and a calcitic (the crossed-foliated) variety, whose modes of formation are particularly enigmatic. We studied the crossed-foliated calcitic layers secreted solely by members of the limpet family Patellidae using scanning and transmission electron microscopy and electron backscatter diffraction. From the exterior to the interior, the material becomes progressively organized into commarginal first-order lamellae, with second and third order lamellae dipping in opposite directions in alternating lamellae. At the same time, the crystallographic texture becomes stronger because each set of the first order lamellae develops a particular orientation for the c-axis, while both sets maintain common orientations for one {104} face (parallel to the growth surface) and one a-axis (perpendicular to the planes of the first order lamellae). Each first order lamella shows a progressive migration of its crystallographic axes with growth in order to adapt to the orientation of the set of first order lamellae to which it belongs. To explain the progressive organization of the material, we hypothesize that a secretional zebra pattern, mirrored by the first order lamellae on the shell growth surface, is developed on the shell-secreting mantle surface. Cells belonging to alternating stripes behave differently to determine the growth orientation of the laths composing the first order lamellae. In this way, we provide an explanation as to how plywood-like materials can be fabricated, which is based mainly on the activity of mantle cells.

Keywords: biomineralization; calcite; crystallography; material organization; molluscs; plywood structure.

Figure 1

General shell structure of the studied species of Patella. (a) Radial section through the shell of P. depressa, indicating the calcitic and aragonitic shell layers. Details of both can be seen in insets 1 and 2. (b) View of the interior of the shell of P. caerulea, showing the distribution of shell layers. The inset shows the commarginal distribution of first OLe. CCFL, ICFL: concentric crossed-foliated layer, irregular crossed-foliated layer; CL: crossed-lamellar.

Figure 2

Details of the crossed-foliated microstructure. (a) View of the internal shell surface of P. rustica. The alternating first OLe can be differentiated due to orientation contrast. They are irregular toward the margin (irregular crossed-foliated [ICF] layer, right side) and become commarginal toward the interior (concentric crossed-foliated [CCF] layer). The framed area is reproduced in black and white in Figure 8a. (b, c) Irregular distribution of first OLe of the ICF layer close to the margin of P. caerulea. Also, note the differences in shape and size. (d) Details of the third OL at the margin of P. caerulea. They are particularly thick and have coarsened edges. The inset is a detail. The rhombohedral surfaces looking toward the observer (r) are particularly smooth, compared to the lumpy aspect of the rest of the growth surfaces (ls). (e) Details of the third OLe of the ICF layer of P. caerulea in an area similar to those in b and c. They are identical to those of the CCF layer (e.g., i, j). (f) Distribution of first OLe on the internal shell surface of P. caerulea at the transition between the ICF layer and the CCF layer. (g) View of the CCF layer of P. rustica. The fracture on the right allows us to observe the changing inclinations of the second and third OLe in alternating first OLe. (h) Fracture through a first OL of P. rustica, showing the extent of the third OLe. (i) Detail of g (framed), at the contact between adjacent first OLe in P. rustica. Note slightly overlapping edges (oe) at the contact between second OLe dipping in opposite directions. Small arrows indicate initiating second OLe. (j) Detail of a similar contact in P. caerulea. Some overlapping edges (oe) and initiating second OLe (arrows) are indicated. (k) Two-tailed end of a first OL of the CCF layer of P. caerulea (central part of the image). (l) Close-up of a similar case in P. caerulea. Note irregularities at the very tips. (m, n) Two cases of divergence of first OLe in P. caerulea, beginning with a single third OL. In n, the divergence happens from a single spot. Arrows in b, c, i (large arrows), k, m, and n indicate the growth directions of the third OLe.

Figure 3

IPF maps and pole figures of the layers distinguished within the shells of Patellidae. (a) Radial section of P. depressa, with growth lines indicated (black dotted lines). (b) Commarginal section of P. caerulea. The red lines at the bottom of the image are the approximate orientations of the second OLe. CCFL, ICFL, TL: concentric cross-foliated, irregular cross-foliated, transitional layers. NG is the number of grains for each layer. The color triangle is the color key for orientations.

Figure 4

IPF maps, at different magnifications, done directly on the growth surface of P. depressa, together with the corresponding raw and contour pole figures (a, b). The 001 maxima are joined to their corresponding set of the first OLe with thin arrows. The overlapping 110 and 104 maxima are encircled with red ovals. Thick arrows indicate the growth directions of the third OLe. The color triangle is the color key for orientations.

Figure 5

Growth trajectories of the first OLe selected on the orientation maps of Figure 3. (a) P. depressa. Two first OLe per set have been selected. (b) P. caerulea. One first OL per set has been selected. Pole figures next to the orientation maps correspond to the rectangular area close to the growth surface. Positions of maxima for the two sets of first OLe are indicated with red, blue, and green crosses. Individual scatter pole figures for the selected lamellae are provided (color crosses indicate the positions of the maxima close to the growth surface for the corresponding set of first OLe). The trajectories (black arrows) are indicated both on the first OLe and on the magnified areas of the pole figures. These consist of two to three segments. The light blue arrows in both the lower map and its 001 pole figure (magnified area) indicate the high misorientation of the c-axis across the thickness of the first OL (∼30°). The color key for orientations is provided in Figure 3.

Figure 6

Views of the two lamellae prepared with FIB of the CCF layer of P. caerulea (a, b). The red ovals indicate the areas from which the SAED patterns (inserts) were taken. The boundaries between first OLe have been depicted with broken lines (based on dark field images, not shown). Due to the inclination of the laths (∼45°), the observed thickness is larger (by ∼1.4 times) than the actual thickness. The SAED patterns recorded from a and b indicate that the laths are seen along the [010] and [100] directions, respectively.

Figure 7

Crystallography of the CCF microstructure of Patellidae. (a) Correspondence between the distribution of maxima in idealized pole figures obtained on the growth surface (see Figure 4). (b) Distribution of crystallographic axes and faces of third OLe of alternating first OLe deduced from the idealized pole figures in (a). The lower right sketch is a side view of the lath of set 2. The {104} face looking toward the shell growth surface (in broken line) is not usually expressed. (c) Profiles of third OLe from different sources (color lines) and crystallographic faces, viewed along the a₂-axis.

Figure 8

Assimilation of the first OLe of the CCF layer to a zebra pattern and hypothesis for its formation. (a) Comparison of the distribution of first OLe on the growth surface of P. rustica and actual zebra skin patterns. The top image is a black-and-white image of the area framed in Figure 2a. The lower images have been taken from side views of actual specimens of zebra. (b) Model for the secretion of first OLe. The zebra pattern is developed on the shell-facing mantle surface (bottom part). The cell extensions are directed sideward toward the advancing crystals, in opposite directions in alternating stripes. The growth directions of laths are guided by the opposite flows of precursor particles toward their growth surfaces. The white polygons represent the outlines of the mantle cells. (c) Two TEM images of the mantle epithelium of P. caerulea at different magnifications, showing the cell microvilli. They seem to be discharging vesicles to the extrapallial space. Arrows point to intercellular boundaries.