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The main component of the shell is calcium carbonate, formed by the deposition of crystals of this salt in an organic matrix composed of polysaccharide β‐chitin, a hydrophobic silk protein and a complex assemblage of hydrophilic proteins, many rich in aspartic acid (Addadi et al. 2006). The mineral component represents more than 95% of the shell weight, while the organic matrix represents 1–5%. Despite this, the organic component increases the fracture toughness of the shell by two or three orders of magnitude (Kamat et al. 2000). Three layers make up the shell: (1) a thin outer periostracum of conchiolin, a tough insoluble protein forming the organic matrix of the shell, within which calcium carbonate is deposited – this layer is often a different colour to the underlying shell layers; (2) a middle prismatic layer composed of fibres of aragonite or calcite – both crystalline forms of calcium carbonate (Figure 2.2); and (3) an inner nacreous layer composed of tablets of aragonite arranged in layers separated by sheets of an organic matrix of elastic biopolymers composed of chitin and silk‐like proteins – the nacre is either of a dull texture or iridescent mother‐of‐pearl, depending on the species.

Figure 2.2 Calcitic fibrous layers of adult mytilids. (a–d) Fibrous layer of Bathymodiolus azoricus. (a) General view showing high irregularity in size of fibres. (b–d) Details of fibres. The arrows point to (b) newly formed, (c) twisting or (b,d) splitting fibres. (e,f) Choromytilus chorus. (e) General view of the layer, showing high regularity in size and distribution. (f) Detail of the same layer close to the shell margin. The arrow points to bending fibres. (g,h) Mytilus edulis platensis (now accepted as M. platensis by World Register of Marine Species, WoRMS; www.marinespecies.org). General view and detail of the fibrous layer. Note the even orientation of rhombohedral faces in (h). (i) Mytilus californianus. Fibres are characterised by irregular outlines and endings. The arrow indicates a bent fibre.

Source: From Checa et al. (2014). Reproduced with permission from Schweizerbart Science Publishers.

A range of techniques, such as scanning electron microscopy (SEM), transmission electron microscopy (TEM), X‐ray diffraction (XRD), electron backscatter diffraction (EBSD) and atomic force microscopy (AFM), have played an important role in clarifying the microstructure of the different shell layers (Chateigner et al. 2000; Furuhashi et al. 2009; Checa et al. 2014; Nakamura Filho et al. 2014), while analytical methods such as secondary ion mass spectrometry (SIMS; Shirai et al. 2008), electron probe microanalysis (EPMA; Jacob et al. 2008) and laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS; Jacob et al. 2008) have been used to elucidate chemical composition of the shell in several species. In addition, the NanoSIMS microprobe allows trace element imaging and quantification in bivalve shells (Shirai et al. 2008).

The construction of the shell begins very early in larval development. An area of the ectoderm thickens in the dorsal region of the developing embryo. The area invaginates to become a shell gland, which forms a groove, which eventually becomes the future ligament between the two shell valves (Marin & Luquet 2004). The peripheral cells of the shell gland produce an extracellular lamella, the future periostracum, which will serve as a scaffold for the developing shell. Subsequently, the shell gland everts and the shell field spreads by flattening of the cells and mitotic divisions, thus becoming the calcifying mantle. Between the periostracum and the cells of the shell field, primary mineralisation takes place. The first larval shell is the prodissoconch I stage, followed by the prodissoconch II stage and then the dissoconch stage after metamorphosis (details in Chapter 5). At the prodissoconch I stage, the mineral produced is usually amorphous calcium carbonate, followed by either aragonite or calcite at prodissoconch II (Marin & Luquet 2004). The sequence can vary. For example, in the oyster, Ostrea edulis, the first mineral deposited is calcite, followed by aragonite at the prodissoconch II stage; at the dissoconch stage, the fraction of calcite rapidly increases and that of aragonite decreases (Medaković et al. 1997). By the time the juvenile stage is reached, the shell is heavily calcified and has different pigmentation and more conspicuous sculpturing than the larval shell.

Shell formation in juveniles and adults involves three separate elements: the mantle and its outer epithelium, the periostracum and the interface between the outer epithelium, the periostracum and the growing shell (Marin et al. 2012). Distinct areas of the outer epithelium secrete prismatic and nacre layers. Also, the epithelium contains membrane pumps and channels for extruding the inorganic precursors of calcium carbonate such as calcium (Ca²⁺) and bicarbonate (HCO₃ ⁻). Calcium and bicarbonate ions are taken up from food, from water filtration activity or by passive diffusion over the body surface; bicarbonate may also come from hydration of metabolic carbon dioxide.

The outer mantle epithelium is not in direct contact with the shell but is separated from it by the minute fluid‐filled extrapallial space (ES). Calcium and bicarbonate ions are transported in the haemolymph to the calcifying epithelium, where they are stored as granules and pumped into the ES when needed. In addition to these precursor ions, the fluid contains other inorganic ions, minor elements, proteins and glycosaminoglycans (GAGs) (Marin et al. 2012 and references therein). As the extrapallial fluid is supersaturated, macromolecules, especially acidic proteins and GAGs, maintain calcium in solution by inhibiting the precipitation of calcium carbonate and by allowing it to precipitate where needed.

The edge of the mantle has three folds (Figure 2.3). The ridge between the outer and middle fold is called the periostracal groove; here, specialised cells secrete the periostracum and prismatic layers, while the inner nacreous layer is secreted by the general mantle surface. The myostracum is a very thin mineral layer covering the nacre, where muscles attach to the shell. The shell grows in circumference by the addition of material from the edge of the mantle and grows in thickness by deposition from the general mantle surface. The functions of the periostracum are to act as a first support for calcium carbonate crystals, to protect the shell and to serve as a seal of the ES in such a way that supersaturation conditions – a prerequisite for crystal formation – can be attained (Marin & Luquet 2004). In adults, the periostracum is often much reduced due to mechanical abrasion, fouling organisms, parasites or disease.

Figure 2.3 Schematic representation of the mytilid shell margin.

Source: After Taylor et al. (1969). From Génio et al. (2012). Reproduced with permission from Elsevier.

While the organic fraction (matrix) of the shell is small (1–5%), it is comprised of a mixture of extracellular macromolecules that play an important role in the mineralisation process. A wide variety of shell matrix proteins (SMPs) have been identified in molluscs, including mussels, but to date their specific functions have not been elucidated (Marin et al. 2012; Gao et al. 2015; Yarra et al. 2016). However, several candidate genes coding for nacreous and prismatic layer proteins have been identified in bivalves (Inoue et al. 2010; Jackson et al. 2010; Kinoshita et al. 2011; Hüning et al. 2016; Bjärnmark et al. 2016 and references therein). A wide range of enzyme proteins are expressed during the formation of the shell, including carbonic anhydrase, alkaline phosphatase, DOPA‐oxidase (tyrosinase)/peroxidase and chitin synthase. After proteins, polysaccharides are the next most important component of the organic matrix; for example, chitin is a long‐chain insoluble polymer that plays a key role by defining the interlamellar matrix between nacre tablets (Addadi et al. 2006). Soluble acidic polysaccharides, many of which are bound to protein, are also present in the matrix, but their characterisation in mussels is still in its infancy.