Microstructural characterization of inner shell layers in the freshwater bivalve Anodonta cygnea.
Article Type: Report
Subject: Bivalvia (Research)
Bivalvia (Physiological aspects)
Shells (Research)
Authors: Lopes-Lima, Manuel
Rocha, Antonio
Goncalves, Fernando
Andrade, Jose
Machado, Jorge
Pub Date: 12/01/2010
Publication: Name: Journal of Shellfish Research Publisher: National Shellfisheries Association, Inc. Audience: Academic Format: Magazine/Journal Subject: Biological sciences; Zoology and wildlife conservation Copyright: COPYRIGHT 2010 National Shellfisheries Association, Inc. ISSN: 0730-8000
Issue: Date: Dec, 2010 Source Volume: 29 Source Issue: 4
Topic: Event Code: 310 Science & research
Geographic: Geographic Scope: Portugal Geographic Code: 4EUPR Portugal
Accession Number: 247523215
Full Text: ABSTRACT The shell microstructure of freshwater bivalve Anodonta cygnea was observed from the ventral margin toward the intrapallial regions by scanning electronic microscopy during the spring/summer period. The three main structure--the periostracum, prismatic, and nacreous layers--were visualized in a sequential path from the ventral margin toward the intrapallial regions. Although with always the same calcium carbonate polymorph (aragonite), A. cygnea presented composites of aragonite crystals embedded within an organic matrix displaying varied morphologies and structural arrangements. The prisms of the prismatic shell layer are composed of single-crystal fibers radiating from spheruliths, whereas in the nacreous layer the structure is similar to that of a brick wall, with tablets in each layer offset with respect to those in the layers above and below them. From the shell ventral margin, the last nacreous lamina formed exhibit crystals with rounded forms, followed by rhombic and hexagonal shapes toward the interior with irregular microstructure. There are gradual intermediate forms between these distinct shapes. This study offers a description of the inner calcareous mineralized layers of A. cygnea and will be useful for future comparative studies in several research areas such as phylogenetic evolution, ecotoxicology, and the influence of environmental and physicochemical factors on the shell formation process in freshwater bivalves.

KEY WORDS: scanning electronic microscopy, bivalve, shell structure, nacre, calcification, Anodonta cygnea


The skeletons produced by molluscs to protect and support their soft bodies are organomineral composites that exhibit a high degree of order (Carter 1990). These minerals are generally composed of calcium carbonate in the form of calcite and/or aragonite and are embedded within an organic matrix. The crystals display varied morphologies and structural arrangements and are described as having different microstructures. In most cases, the shells are constructed of two or more superimposed layers with different microstructures, which may even be composed of different calcium carbonate polymorphs (i.e., calcite or aragonite). The polymorphic phase, morphology, and orientation of individual crystals may be defined and controlled either by specific proteins of the shell organic matrix or cellular remodeling (Falini et al. 1995, Belcher et al. 1996, Mount et al. 2004) or physical-chemical conditions in the biomineralization microenvironment. Because different species have specific shell microstructures, it is generally believed to be genetically directed (Addadi & Weiner 1992), but the physical-chemical characteristics of the organic matrix may play a significant role in the resulting microstructure and even in the polymorphism of the minerals in vitro (Takeuchi et al. 2008).

Unionid shells are characterized by an outer periostracum, an intermediate aragonite prismatic layer, and an inner nacreous layer. These mineralized structures may have mechanical properties far superior to each of the constituent components. For instance, mother-of-pearl or nacre is more than 1,000 times tougher than the chemically precipitated counterpart, aragonite (Jackson et al. 1988). The characteristics of mollusc shell microstructure may be used to determine the phylogenetic evolution and to specify the stratigraphic age of geological formations. For a precise description of shell microstructures, scientists have relied on scanning electron microscopy (Hedegaard 1990, Machado et al. 1991, Hedegaard 1997, Moura et al. 2000a).

The focus of this study was to perform a detailed topographical and morphological description by scanning electron microscopy on the microstructure and crystal growth habit over the entire inner surface of the shell of the freshwater bivalve Anodonta cygnea from the edge to the intrapallial regions.


Adult freshwater clams--A, cygnea (Unionidae)--were collected during the spring/summer from their natural environment of Mira Lagoon in the north of Portugal (40[degrees]27'14.4" N, 08[degrees]48'03.1" W) and kept in aerated and dechlorinated water tanks for 48 h. The animals (n = 20) were considered healthy and statistically valid for inclusion in this study if they closed their valves when disturbed and if the inner surface of the shell was smooth and shiny after removal of the mantle.

Shell preparation for scanning electron microscopy consisted of 4 consecutive fragments, each approximately 1 x 1 cm square, cut from the shell edge, cut perpendicular to the growth lines using a dental drill (Fig. 1). The shell pieces were then carefully brushed and rinsed with deionized water. We assessed the morphology of crystals on the inner shell surface of the external prismatic and internal nacreous layers of the shell by scanning electron microscopy. Shell pieces were gold-coated (FINE-COAT Ion sputter JFC-1100) and glued to aluminum stubs for scanning electron microscopic observations using the JEOL JSM-35C scanning electron microscope operated at 25 kV.


Shell Sample Analysis

Scanning electron microscopic-acquired images of the bi-valve shell, organized by sections that correspond to the shell fragments labeled (Fig. 1A-D) from the ventral margin to the pallial line, allowed us to obtain a detailed description from the border toward the inner regions of the shell.


The images are representative of the normal morphological features and microstructural characteristics of A. cygnea, from the shell border (Plates 1 and 2, 1.2A-1.20B), through the pallial line (Plate 2, 1.21C), to the interior regions (Plate 2, 1.22C-1.24D). Plate 1, 1.1A shows 2 periostracal pellicles that bear the growth lines: the internal pellicle folded over the forming prismatic layer. In Plate 1, 1.2A-1.4A the aragonite prismatic layer presents a typical polygonal arrangement, with prisms of very different sizes, resulting in part from joined columnar structures evidenced by a magnified image in Plate 1, 1.5A. These columns grow within organic membranous structures (Plate 1, 1.6A-1.9A). Subsequent mineral growth is in the form of a transition zone (Plate 1, 1.10A), revealing the simultaneous formation of 3 or 4 lamellae of nacre crystals, which corresponds to the beginning of the nacreous layer. At this point the nacre is generally composed by rounded, flat, unconnected crystals growing laterally and fusing to form a thin lamella (Plate 1, 1.11A, 1.12A). Toward the shell interior, these crystals start to lose their rounded shape and appear to become rhombic in habit (Plate 2, 1.13B-1.15B). Regular and polygonal crystal shapes, with hexagonal habits and coexisting with rhombic shapes, are observed toward the sinuous pallial line (Plate 2, 1.16B-1.18B). The next sequential sample has predominantly rhombic crystals (Plate, 1.19B, 1.20B). Closer to the pallial line (Plate 2, 1.21C), both in the anterior and posterior regions, a nacreous layer with densely packed crystals of undefined shape and with high organic matrix content is observed. This feature reveals a poorly defined crystal microstructure (Plate 2, 1.22C). The pallial line is represented by the dark line in the shell interior (Plate 2, 1.21C). A great amount of organic matter is observed in this region, decreasing toward more interior regions. Inward of the pallial line it is possible to observe nacreous layers composed of crystalline lamina with undefined and irregular microstructures seen during this seasonal period (Plate 2, 1.23D, 1.24D).


The calcification of the shell is a regulated and complex process, in which calcium carbonate is enmeshed in an organic matrix consisting mainly of soluble and insoluble compounds (Crenshaw 1972, Wilbur & Manyak 1984, Moura et al. 2000b). Both organic and inorganic components of the shell must be secreted or transported by the mantle and hemocytes (Mount et al. 2004) into the extrapallial fluid, which is in contact the shell surface in bivalves. The biologically formed crystals of the shell are generally all of uniform size, have oriented crystallographic axes, and adopt sizes and shapes ("crystal habits") quite different from those found in their nonbiological counterparts. These properties indicate that the crystals form under well-controlled cell-mediated conditions.

A common mode of crystal growth in these tissues is through the coordinated formation of an organic structural framework (the "organic matrix") with embedded crystalline nuclei that subsequently grow and mature into specific crystal polymorphs (Machado et al. 1990, Machado et al. 1991, Nudelman et al. 2006, Nudelman et al. 2007, Furuhashi et al. 2009). The regulation of crystal growth is accomplished in part by an array of matrix macromolecules, many of which are synthesized by specific cells. From these observations it is accurate to say that the mechanism of biomineralization in molluscs is essentially a cellular process, although it is under the influence of environmental constraints, such as the physical-chemical parameters of the water. Additionally, shell calcification is under cellular control so, any factor that affects the outer mantle epithelium cellular metabolism may also affect the natural shell morphology structure. These factors may not only be variations on the physical parameters of the water, such as pH, temperature, hydrostatic pressure, and the ionic composition of the water, but also the presence of several pollutants known to affect the biomineralization process, such as tributyltin diflubenzuron and heavy metals (Machado et al. 1989, Machado et al. 1990, Moura et al. 2000b). Therefore, it is essential to know the regular and normal crystal morphology and microstructure of the shells inner layer, because it is the last to be deposited. The hard tissues of bivalves contain considerable information about their own growth history, including the varying condition of mineralization and environmental stress or disease (Okoshi & Sato-Okoshi 1996). This information is preserved as structural, morphological, and chemical changes within the shell layers, and may be good indicators of changes in environmental conditions.


In this study a morphological description of the inner layer shells of A. cygnea was carried out to determine the normal pattern of the microstructure and crystal habit from the ventral margin of the shell to the interior shell surface near the pallial line. From the prismatic (the intermediate) layer, which can be seen in the edge of the shell, it was possible to observe a polygonal arrangement with joined columnar prisms of varying sizes. These observations were noted by Checa (2000) and Vancolen and Verrecchia (2008), who stated that, during periostracum formation, the entire periostracum reflects at the shell margin where the middle periostracum becomes vacuolated and forms a cavernous structure called the antrum. These cavities are filled with calcium carbonate (aragonite) crystals perpendicularly to the outer periostracum and separated by a conchiolin membrane. Following a transect from the margin toward the umbo, there is a crystal transition zone between the prismatic and nacreous layers with 34 simultaneous lamellae of nacre crystals layered on top of the prismatic layer. This layering is in agreement with that noted by Checa and Rodriguez-Navarro (2001), who described the characteristics of the transition zone as the nacreous tablets adopting the crystallographic orientation of the most distal fibers of the prisms of the prismatic layer, which act as epitaxial mineral substrates. From this transitory zone, the nacreous layer is initially composed of rounded, flat, unconnected crystals growing laterally and fusing to form a thin, confluent lamella. These crystals start to lose their rounded shape and exhibit a tendency to become rhombic in habit, as we saw in the area of the pallial line. It has already been observed (Checa & Rodriguez-Navarro 2001) that as the nacreous layer grows thicker, the orientation of crystals deteriorates. We observed this lack of crystal orientation in the current study. In the interior regions near the pallial line, both in the anterior and posterior regions, a nacreous layer with densely packed crystals of undefined shape and embedded in a high organic matrix content was observed. However, according Machado et al. (1991) the shell crystals in the intrapallial regions can show different habits according the seasonal periods. In fact, because our observations were noted during spring/summer, the crystals exhibited irregular and unformed habits, which agrees with previous observations on this period (Machado et al. 1991). Although exhibiting unformed habits, the shell nacreous layer of A. cygnea forms in an aragonite crystalline system (Machado et al. 1991).


In conclusion, the normal ultrastructure of the shell inner layer of A. cygnea was established and may be used in future comparative studies on the influence of toxins and other environmental parameters on the shell formation process of the freshwater bivalve A. cygnea.


We were supported by Fundacao para a Ciencia e Tecnologia (FCT).


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(1) ICBAS--Abel Salazar Institute of Biomedical Sciences, University of Porto, Lg Prof Abel Salazar, 2, 4099-003, Porto, Portugal; (2) CIIMAR--Interdisciplinary Centre of Marine and Environmental Research, University of Porto, Rua dos Brag, as, 289, 4050-123, Porto, Portugal

* E-mail: jmachado@icbas.up.pt
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