First record of carotenoid pigments and indications of unusual shell structure in chiton valves

Mollusc shells are typically made of calcite, aragonite or, in rare cases, vaterite (Weiner et al., 2009; Ehrlich, 2010). These minerals are interspersed with protein matrix and sometimes pigments (Comfort, 1949a; Hedegaard, Bardeau & Chateigner, 2006). Typically, the organic matrix is composed of β-chitin, silk-like proteins rich in alanine and glycine, and aspartic acid-rich proteins (Weiner & Traub, 1984; Addadi et al., 2006; Furuhashi et al., 2010). This matrix creates an environment isolated from the surrounding seawater that the mollusc can supersaturate with calcium carbonate. Mineral layers of the shell develop by the nucleation of crystals of calcium carbonate within the protein matrix (Weiner & Traub, 1984). The proteins of the organic matrices of bivalve and gastropod shells have been the most intensively studied. It has been shown that these proteins determine the growth of specific crystal polymorphs of calcium carbonate (Marin & Luquet, 2004; Furuhashi et al., 2010; Marin, Roy & Marie, 2012; Suzuki & Nagasawa, 2013). Pigments found within the shell are typically melanins and tetrapyrroles, specifically porphyrins and bilins (Comfort, 1949a, b, c; Nicholas & Comfort, 1949; Gysi, Chapman & Moshier, 1979; Cai et al., 2011). Comfort (1949b) identified porphyrins by observing their specific fluorescence in over 3,000 mollusc species. He also identified porphyrins using talc-adsorption chromatography in four pterioid bivalves, two caenogastropods and two vetigastropods (Comfort, 1949a). Nicholas & Comfort (1949) confirmed the presence of uroporphyrin in several mollusc shells using this method. In addition, Comfort (1949c) noted an unusual biliprotein initially suspected to be an indigoid. Cai et al. (2011) demonstrated a blue polyene in the shell of Haliotis discus by using a combination of UV-vis, infrared, NMR and ESI-mass spectroscopy. Using thinlayer chromatography and electrophoresis, Gysi et al. (1979) identified the pigments of Haliotis californiensis as bilipeptides. Raman spectroscopy can also be used to identify molluscan shell pigments. Raman spectroscopy is a nondestructive analytical technique that uses lasers to identify compounds present within an unknown sample. Raman microscopy combines Raman spectroscopy with optical microscopy, thus allowing Raman spectra to be obtained from precise locations within a sample. Using Raman spectroscopy, coloured pigments in a wide range of gastropods, bivalves and Nautilus have been demonstrated to be polyenes (Thompson et al., 2014), or specifically carotenoid polyenes (Barnard & de Waal, 2006; Hedegaard et al., 2006). The characteristics of Raman spectra are determined by the specific chemical bonds present in the analysed material. While the technique cannot conclusively identify the compounds present, identification is attempted by comparison of spectra with known standards. As in the case of shell proteins, work on shell pigments has focused on bivalves and gastropods. Reports on shell pigments from other classes of Mollusca are few (Matsuno, 2001; Barnard & de Waal, 2006; Hedegaard et al., 2006; Karampelas et al., 2009; Thompson et al., 2014) and entirely lacking for the clade Aculifera (Polyplacophora and Aplacophora; Kocot et al., 2011; Kocot, 2013). This lack is surprising in the case of chitons, which are common in the intertidal zone worldwide and include some species with a wide range of colour variation (Sigwart, 2016). Chitons have a shell structure that differs from that of other molluscs. The shell of a chiton is composed of eight overlapping aragonitic plates, or valves. Each valve has an outer organic layer (properiostracum) covering the shell and two primary mineral layers, the outer (dorsal) tegmentum and the inner (ventral) articulamentum. The articulamentum forms the bulk of the valve and is a composite prismatic layer (Connors et al., 2012). The dorsal tegmentum is also a prismatic layer which, uniquely in molluscan shells, incorporates sensory structures called aesthetes (Schwabe, 2010; Connors et al., 2012). These are composed of a canal system, with large and small surface pores (megalaesthetes and micraesthetes, respectively; Schwabe, 2010). Acanthopleura granulata also incorporates eye-like structures into the tegmentum that may have the ability to form rudimentary images and in which the pigment pheomelanin has been identified (Speiser et al., 2011; Li et al., 2015). In addition, pigments in the tissues of six chiton species from Japan have been shown to include 27 different carotenoids, although the extracellular pigments within the mineralized layers of chiton valves, or in girdle spicules, have not yet been studied (Tsushima, Maoka & Matsuno, 1989; Schwabe, 2010; Maoka, 2011; Speiser, Demartini & Oakley, 2014; Sigwart, 2016). Carotenoids were identified using Raman spectroscopy within the valves of eight chitons from New Zealand: Acanthochitona zelandica, Chiton glaucus, Ischnochiton maorianus, Leptochiton inquinatus, Notoplax violacea, Onithochiton neglectus, Sypharochiton pelliserpentis and S. sinclairi. Chitons were collected from the Otago Peninsula, South Island, New Zealand. At least three individuals of each of the eight species were collected and killed in 75% ethanol immediately after collection. The valves were dissected post-mortem and the soft tissues