DOI: 10.1021/cg901265x
Aragonite Prism-Nacre Interface in Freshwater Mussels Anodonta anatina (Linnaeus, 1758) and Anodonta cygnea (L. 1758)
2010, Vol. 10 344–347
Andy Freer,*,† Daniel Greenwood,‡ Peter Chung,‡ Claire L. Pannell,# and Maggie Cusack*,‡ †
Glasgow Biomedical Research Centre, University Place, Glasgow G12 8TA, U.K., Department of Geographical and Earth Sciences, University of Glasgow, Glasgow G12 8QQ, U.K., and # National Museums Scotland, National Museums Collection Centre, Edinburgh EH5 1JA, U.K. ‡
Received August 7, 2009; Revised Manuscript Received November 27, 2009
ABSTRACT: Electron backscatter diffraction is used to characterize the transition interface between aragonite prisms and nacre in two species of freshwater molluscs, Anodonta anatina (Linnaeus, 1758) and Anodonta cygnea (L. 1758). In the duck mussel, Anodonta anatina, the shell thickness is comprised mainly of nacre with only a thin outer prismatic layer, whereas in the swan mussel, A. cygnea, the shell comprises a greater thickness of aragonite prisms relative to the nacreous layer. In A. anatina, the prism-nacre interface is flat and featureless. In A. cygnea, the nacre below the base of the prisms is characterized by distinct concave footholds. In both species, the c-axis of aragonite nanogranules comprising the prisms is parallel with the prism length. This crystallographic orientation, with the c-axis perpendicular to the shell exterior, occurs in the nacreous layer and thus is conserved throughout the shell thickness. In A. cygnea alternate prisms have a crystallographic orientation with the c-axis deviating from the prism long axis by approximately 20 deg. The initial nacreous layer adopts the crystallographic orientation of the preceding prisms and subsequent nacreous laminae are oriented in register with the bulk nacre. This is consistent with the hypothesis that nacre evolved through horizontal partitioning of prisms.
Introduction Biogenic calcium carbonate is abundant in the marine environment where invertebrates such as brachiopods, corals, and molluscs produce protective calcium carbonate structures. Calcite and aragonite are the most common calcium carbonate polymorphs in the biosphere, and the apparent ease and ability of marine and freshwater organisms to produce diverse and highly functional biocomposite materials from the basic ingredient of CaCO3 has been the subject of much current research.1 This exquisite biological control is evident in the formation of well-defined microstructures in bivalve molluscs where layers of the two main polymorphs calcite and aragonite are elaborated sequentially to form an elegant and robust shell. The biological control, or guidance to form, may also be seen in the equally intricate sheaths that encase some biomineralized structures. In both pteriomorph and palaeoheterodont bivalves, this arrangement frequently occurs as prismatic columns and brick-like tablets of nacre. Anodonta anatina and A. cygnea bivalves (class Bivalvia; subclass Palaeoheterodonta; family Unionidae) are widely distributed in Europe, occurring as semi-infaunal burrowers in the bottoms of both lotic and lentic waters. The shell microstructure consists of a thin outer covering of periostracum underlain by an aragonite prismatic layer and an inner aragonite nacreous layer. In marine bivalves, these outer prisms are constructed from calcite2,3 with the exception of trigoniacean shells which have aragonite prisms.4 All freshwater bivalves studied to date have aragonite prisms5 including Anodonta woodiana.6 Molluscan prisms originate from spherulites in the inner surface of the periostracum,7-9 initially tempered by chemical *To whom correspondence should be addressed. (A.F.) E-mail: andy@ chem.gla.ac.uk. (M.C.) Tel: þ44 (0) 141 330 5491. E-mail: Maggie.Cusack@ ges.gla.ac.uk. pubs.acs.org/crystal
Published on Web 12/16/2009
crystal growth dynamics and space constraints.3,9-11 The prisms are enveloped by a hydrophobic organic network or sheath that takes a guiding role in the columnar prism assembly.12-16 Earlier work14 on the freshwater species Amblema plicata perplicata (Conrad, 1841) defined the organic sheath as prismatic bags where columnar prisms approximately 225-250 μm long were grown that also gave rise to specific areas where multiple prism layers occurred, coincident with the plicae (folds) of the undulating shell. In the bivalve mollusc Entodesma navicula what was previously thought to be a featureless homogeneous outer layer can now be described as a granular prismatic microstructure comprised of very short prisms about 10 μm long.17 The transition from prisms to nacre is an interesting one, and it has been suggested that nacre evolved through simple horizontal partitioning of vertical prisms.18,19 Indeed, early observations of cephalopod shell ultrastructure noted that at the nacre-prism interface, the crystallites of both structures have the same dimensions and physical orientations.20 Marin (2008)21 recommended that crystallographic and biochemical analysis be carried out to assess this hypothesis. In this paper, we used electron backscatter diffraction (EBSD) to determine the crystallographic orientation at the prism-nacre interface in order to contribute to this discussion. Calcitic prisms have been extensively studied in terms of microstructure and organic content in Pinna nobilis L. 1758,15,22 Atrina rigida (Lightfoot, 1786),23 and Atrina serrata.24 Checa and others have also investigated the prism-nacre interface in several bivalves of the order Pterioida3 where the transition is from one polymorph (calcite) to another (aragonite), reporting that the calcite prisms appear monocrystalline although composed of crystallites.25 Thus, these calcitic prisms are examples of mesocrystals according to the terminology of C€ olfen,26 describing biomineral units composed of large numbers of nanogranules with uniform crystallographic orientation. r 2009 American Chemical Society
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The thick organic interprismatic envelope completely carpeted the terminal surface of the prisms before the nacre sheets began to form. In A. anatina and A. cygnea, the abrupt switch from one structure to another maintains the polymorph with a transition from aragonite prisms to aragonite nacre. This is in contrast to the calcite prism-aragonite nacre interface in the pearl oyster Pinctada margaritifera where there is a fibrous aragonite layer of approximately 50 μm creating an intermediate zone buffering prism from nacre.27 Experimental Section Materials. Shells of the genus Anodonta were analyzed, specifically A. anatina (Linnaeus, 1758) and A. cygnea (Linnaeus, 1758) donated by National Museums Scotland, Edinburgh. A. anatina (NMSZ.1974.36), collected in April 1931 from Gosford House, Aberlady, East Lothian, (56°000 29.7100 N 2°510 57.4700 W) measured length 103 mm width 59 mm. A. cygnea (NMSZ.1965.4), collected in April 1964 from Castle Loch, Lochmaben, Dumfriesshire (55°070 06.9700 N 3°250 51.2700 W), measuring 137 mm long 79 mm wide. The analyses were carried out using carefully cut and fractured sections that sampled a representative cross section of the shell in both valves for each species. Scanning Electron Microscopy (SEM) Analysis. Two cuts were made across the dorsoventral axis posterior to the midpoint producing a 10 mm strip that transverses from the exterior margin to the umbo. The sample was then fractured mechanically in three places. First, ∼10 mm from the exterior margin then ∼10 mm onward from the previous break and finally at the umbonal region ∼20 mm onward from the previous fracture. After fracture the shells were gold-coated using an AGAR Sputter Coater for 180 s (argon/ plasma coating system). SEM images were then captured using either a Cambridge S360 and Quanta FEI 200 field emission scanning electron microscope (FE-SEM). Electron Backscatter Diffraction (EBSD). To prepare A. anatina and A. cygnea for EBSD, the shells were set in Araldite resin blocks (epoxy/hardener ratio 5:1) to prevent splintering when cut. Strips 10 mm in width across the dorsoventral axis were cut at 400 r.p.m. on a Buehler Isomet low speed 11-1180 cutoff saw. The 10 mm strip was then divided into 10-12 sections (depending on shell dorsoventral width). The sections were orientated so that the face to be polished was facing downward before embedding in Araldite for a second time. This created two Araldite resin blocks containing cross sectional views across the entire dorsoventral axis. The Araldite blocks were then ground to produce a flat surface suitable for EBSD analysis. Samples were progressively polished through a series of grinding stages from 35 to 0.3 μm grit size before a final polish using noncrystallizing colloidal silica. The samples were mounted on aluminum stubs using silver Dag and coated with carbon to 35 nm thickness. EBSD patterns were captured on a Quanta FEI 200 field emission gun SEM equipped with a TSL EBSD system using OIM version 5 software to compare experimentally observed Kikuchi patterns with database derived patterns. The SEM was used in high vacuum mode for all scans at 20 kV spot size 4 and aperture size 4. The EBSD scans were carried out with a step size of 0.2 μm at a working distance of 10-14 mm. The final scans were partitioned to remove points with a confidence index (CI) of less than 0.1 for the nacre scans, while CI partitioning could not be applied to the prism analysis. Demineralization. Four demineralized samples were prepared, two each from A. anatina and A. cygnea. All four samples were cut from the anterior margin using a high-speed rotary saw. In each of the two valves, cuts were made to produce sections 10 mm 20 mm shell thickness (1-3 mm). One sample from each species of A. anatina and A. cygnea had the periostracum removed by treatment with 0.2% w/v active chlorine in the form of diluted sodium hypochlorite (NaOCl) for one week (that from A. anatina took two days extra due to the increased thickness of the periostracum compared with A. cygnea). All four samples (i.e., with and without NaOCl treatment) were then placed in 50 mL beakers with 20 mL of 1 M acetic acid at room temperature (20 °C) and left for one week to completely demineralize. The samples were washed in deionized, purified water
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(Millipore) for 2 min each before being dried on glass slides for 2 days at room temperature (20 °C). The four samples were cleaned using compressed air before gold coating for 180 s using an AGAR Sputter Coater.
Results and Discussion Shell Microstructure. The exterior of the shells of A. anatina and A. cygnea are quite different; A. anatina has a rough exterior and A. cygnea has a thin, almost translucent, smooth shell. The shell of A. anatina is much thicker (2.3-4.6 mm) than that of A. cygnea (0.8-1.6 mm). In both cases, the thicker hinge region has been omitted from these measurements. The periostracum is noticeably thicker in A. anatina than in A. cygnea and the ratio of prism to nacre thickness is also different in the two species. A. anatina has a relatively consistent prism to nacre ratio throughout the shell, with typical values of 0.7:3.8 mm in the exterior region and 1.1:3.9 mm at the interior. At the external margins, A. cygnea has a higher ratio of prism to nacre, 230:140 μm respectively. In the more central region of the shell, the prism layer height remains relatively constant at about 230 μm and the nacre layer is thicker with typical prism to nacre values of 220:850 μm. This difference in shell microstructure is further seen in the relative width of the prisms, where A. anatina has broader prisms in the range 30-80 μm and A. cygnea has prisms that are 25-55 μm wide. X-ray diffraction (XRD) analysis (not shown) of powdered shell confirms that in both A. anatina and A. cygnea, like all other the palaeoheterodont bivalves studied to date, the prisms are aragonite. The composition of the shells of the two species is fairly uniform with aragonite prisms underlain by nacre. Differences, however, occur in the relative ratios of prismatic to nacreous layer thicknesses. The dominance of prisms at the margin of A. cygnea shells may explain the shells apparent fragility at the gape where pieces are easily broken from this very thin area. However, this sharp thin edge has high tensile strength and is probably an adaptation for A. cygnea’s mode of life in soft, muddy substrate. Although both species often co-occur, they exhibit different habitat preferences: A. cygnea prefers quiet bodies of water such as slow moving rivers, canals, lakes, and reservoirs with muddy but not oozy bottoms, avoiding gravelly or rocky substrates. A. anatina is much more common in flowing water with a sandy substrate. Thinner shells also permit more rapid burrowing in environments where the formation of a thick shell to avoid abrasion is unnecessary.28 Aragonite Prism-Nacre Interface. Another noticeable difference between the two shell ultrastructures is seen at the margin between prism and nacre. In A. anatina, this interface is flat and almost featureless (Figure 1A). In A. cygnea, the interface is clearly defined by uniform, distinct concave indentations in the nacre approximately 20-50 μm across that are the “footholds” of the prisms (Figure 1B,C). Demineralized shell reveals the organic sheaths in which the prisms are formed (Figure 2A). In Figure 2B nanogranules are evident inside the organic casing. SEM analyses of the fracture sections highlight the nanogranular composition of the prisms (Figure 2C,D). The aragonite columnar prisms are assembled from a cadre of nanogranules as described in earlier work20 that externally appear as a single structure (Figure 2C).
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Figure 1. Secondary electron images of fracture sections of prism-nacre interface in A. anatina and A. cygnea. (A) Flat interface between prisms and nacre in A. anatina. (B) Interface between prisms and nacre in A. cygnea with concave indentations evident. Black box indicates region presented at higher resolution in C. Scale bars = 200, 100, and 5 μm, respectively.
Figure 2. Organic casings and nanogranular composition of prisms in A. cygnea. (A) Organic casings of A. cygnea prisms. (B) Organic content of partially demineralized prisms. (C) Fractured prism revealing nanogranular composition of prism structure. (D) High magnification image of prism nanogranules in C. Scale bars=50, 1, 20, and 2 μm, respectively.
Crystallography of the Aragonite Interface. EBSD analyses on the prisms of A. anatina (Figure 3) shows that the nanocrystals making up the prisms have near identical orientation with the c-axis parallel with the prism length, pointing to the shell exterior analogous to the nanogranular tablets of nacre (Figure 4). In both species, the overall crystallographic orientation follows the well-established pattern where the c-axis of nacre is perpendicular to the nacre-prism interface for example ref 6. This crystallographic orientation is manifest in views parallel to the prism-nacre interface where the a- and b-axes of aragonite are shown in green and blue (Figures 3C and 4B). EBSD analyses of A. anatina serves to demonstrate that the prisms, although composed of nanogranules, embedded in an organic matrix are consistent with the concept of mesocrystals (Figure 3). However, the distribution of points on the pole figure (Figure 4E) indicates a greater variance than would occur in a single crystal. It is, however, the interface between the columnar aragonite prisms and the brick-like nacre that holds the interest. We have used EBSD
Figure 3. Electron backscatter diffraction (EBSD) of A. anatina prisms. (A) Secondary electron image of polished section of A. anatina prism. (B) Diffraction intensity map of area outlined in A. (C) Crystallographic orientation map with crystallographic plane normal to field of view according to color key in Figure 4C. (D) Pole figure indicating that the c-axis of calcite (001) is parallel with the prism length and perpendicular to shell exterior which is to the top of A-C. Scale bars = 15, 20, and 20 μm, respectively, in A-C.
to elucidate if there is a change in the crystallographic orientation at this transition point. In both species, there is an almost perfect continuum in crystallographic orientation across the interface from prism to nacre as shown for A. cygnea (Figure 4B). In A. cygnea, there is a small angular difference of approximately 20° between alternate prisms which is evident in the color difference in Figure 4B. This alternating prism orientation is seen across a wide area of prisms (not shown). Where this alternate prism crystallographic orientation occurs with the c-axis being 20 deg from perpendicular (pink shade in Figure 4B) this crystallographic alignment occurs in the first-formed nacre (up to 5 μm thick). The crystallographic orientation of the nacre in this region adjusts very quickly (within a few micrometers) to the overall bulk nacre orientation (Figure 4B).
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Figure 4. Electron backscatter diffraction (EBSD) of A. cygnea prisms and nacre. (A) Diffraction intensity map of prisms and nacre. (B) Crystallographic orientation map of A with crystallographic plane normal to field of view according to the color key in C. (D-H) Pole figures indicating the orientation of the c-axis of aragonite (001) in the entire area presented in B; (D), blue-green prisms (E), blue-green nacre (F), pink prisms (G), and pink nacre (H). Scale bars = 35 μm.
Conclusion Although the overall shell structure is common to both species, there are differences in the prism dimensions and in the interface between prisms and nacre, with A. cygnea having well-defined footholds. These structural differences have no effect on crystallographic orientation, with the aragonite c-axis parallel to the prism length. This is consistent with the overall bulk crystallographic orientation of nacre, where the c-axis is perpendicular with the shell exterior. Thus, throughout the shell thickness, from outermost prism to innermost nacre, the crystallographic orientation of the c-axis is highly conserved. This supports the hypothesis that nacre arose from horizontal partitioning of prisms. This is further supported by the observation that, in A. cygnea, where alternate prisms have slight deviations from the perpendicular, this crystallographic orientation is adopted in the nacreous layer for the first few micrometers before rapid adjustment to conform to the bulk nacre crystallographic orientation.
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