JOURNAL OF MORPHOLOGY 270:588–600 (2009) The Chiton Stylus Canal: An Element Delivery Pathway for Tooth Cusp Biomineralization Jeremy A. Shaw,1* David J. Macey,2 Lesley R. Brooker,3 Edward J. Stockdale,1 Martin Saunders,1 and Peta L. Clode1 1Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley, WA 6009, Australia 2School of Biological Sciences and Biotechnology, Murdoch University, Murdoch, WA 6150, Australia 3Faculty of Science, Health and Education, University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia ABSTRACT A detailed investigation of the stylus canal termed the radula; an elongated ribbon of repeat- situated within the iron mineralized major lateral teeth ing tooth rows that moves progressively toward of the chiton Acanthopleura hirtosa was undertaken in the mouth in a manner analogous to a production conjunction with a row-by-row examination of cusp line. Each tooth row consists of 17 teeth, 16 of mineralization. The canal is shown to contain columnar which are paired either side of a single central epithelial tissue similar to that surrounding the miner- alized cusps, including the presence of iron rich par- tooth. The major laterals (also referred to as sec- ticles characteristic of the iron storage protein ferritin. ond laterals) are the only tooth pair that are min- Within the tooth core, a previously undescribed inter- eralized using iron and calcium. Initially, the teeth nal pathway or plume is evident above the stylus canal, form at the posterior end of the radula, where between the junction zone and mineralizing posterior odontoblast cells construct an extracellular organic face of the cusp. Plume formation coincides with the matrix, composed primarily of the polysaccharide appearance of iron in the superior epithelium and the a-chitin (Evans et al., 1990). This matrix acts as a onset of mineralization at tooth row 13. The plume per- scaffold that dictates the overall structure of the sists during the delivery of phosphorous and calcium teeth and plays an important functional role as into the tooth core, and is the final region of the cusp to they progress from the unmineralized to the min- become mineralized. The presence of the stylus canal was confirmed in a further 18 chiton species, revealing eralized state. that the canal is common to polyplacophoran molluscs. One of the most intriguing and well-studied These new data strongly support the growing body of aspects of tooth formation in chitons is the highly evidence highlighting the importance of the junction ordered internal structure of each tooth cusp, zone for tooth mineralization in chiton teeth, and indi- where various mineral types are separated into cate that the chemical and structural environment architecturally discrete regions (see Fig. 1). The within the tooth cusp is under far greater biological composite structure of the teeth provides a number control than previously considered. J. Morphol. 270: of functional benefits that prolong the working life 588–600, 2009. Ó 2008 Wiley-Liss, Inc. of each cusp, including shock absorbance, minimiz- ing crack propagation, and the ability to self sharpen KEY WORDS: SEM; X-ray microanalysis; EDS; TEM; EFTEM; microwave; iron; magnetite; mollusk; radula (van der Wal et al., 2000). Although tooth formation in chitons represents a classic example of extracellu- lar matrix-mediated biomineralization, the mecha- nisms involved in the controlled formation of these INTRODUCTION discrete mineral phases are not well understood. Chitons (Mollusca: Polyplacophora) utilize a During development, the major lateral tooth range of iron and calcium-based minerals to cusps are surrounded by superior epithelial tissue harden their teeth, which allows them to graze (cusp epithelium), which is thought to be the main upon algae growing on, or within, hard substrates (Lowenstam, 1962). In particular, the iron oxide Contract grant sponsor: Australian Research Council Discovery magnetite is used to structurally reinforce the pos- Grant; Contract grant number: DP0559858. terior surface of each major lateral tooth cusp. De- spite this hardening process, the abrasive nature *Correspondence to: Jeremy A. Shaw, CMCA (M010), The Univer- of their feeding activities quickly wears down the sity of Western Australia, 35 Stirling Highway, Crawley, WA 6009, teeth, making it necessary for them to be continu- Australia. E-mail: [email protected] ally replaced (Runham and Thornton, 1967; Shaw Published online 23 December 2008 in et al., 2008a). The organ involved in the synthesis, Wiley InterScience (www.interscience.wiley.com) development, and eventual loss of the teeth is DOI: 10.1002/jmor.10705 Ó 2008 WILEY-LISS, INC. THE STYLUS CANAL IN CHITONS 589 scribed. The presence of this structure was first reported by Nesson and Lowenstam (1985), how- ever no suggestions were raised as to its possible function. An investigation of the major lateral tooth bases of the chiton Acanthopleura hirtosa was under- taken to determine both the overall morphology of the stylus canal and the nature of the cells located within the canal. In addition, the major lateral teeth from a number of other chiton species were examined to determine whether the canal is a fea- ture common to this class of molluscs. To elucidate links between the junction zone and tooth cusp mineralization, a detailed row-by-row investigation of mineral deposition within the major lateral teeth was also conducted. In this article, evidence is provided that strongly supports the involvement of the stylus canal in tooth cusp mineralization and its role in element transfer between the junc- tion zone and tooth core. MATERIALS AND METHODS The Stylus Canal Adult specimens of the chiton Acanthopleura hirtosa (Blain- ville, 1825) were collected from Woodman Point, Perth, on Aus- tralia’s south-west coast (Lat. 328S, Long. 1168E). For investiga- tions of gross stylus canal morphology, excised radulae were cleaned in 2% sodium hypochlorite (5 min) and then rinsed in distilled water to remove the epithelial tissue attached to the teeth. Whole radulae, radula segments, individual major lateral teeth, and fractured teeth were either imaged on a dissecting Fig. 1. Back-scattered SEM micrograph of a longitudinal sec- microscope (Olympus, SZH10) fitted with a digital camera tion through a major lateral tooth from Acanthopleura hirtosa, (Olympus, DP10) or processed further for scanning electron mi- highlighting the overall internal structure of a mature tooth croscopy (SEM). For SEM, radulae were fixed for at least 12 h including the tooth base or stylus (ts), junction zone (jz), and in 2.5% glutaraldehyde buffered in seawater before being dehy- tooth cusp (tc). In addition, the cusp is further divided into dis- drated through a graded series of ethanol followed by amyl ace- tinct mineral regions, which include magnetite (m), lepidocro- tate. Samples were critical point dried with CO2, mounted on cite (l), and apatite (a). p, posterior surface. Scale bar 5 50 lm. aluminum stubs using double-sided carbon tape, and then coated with 30 nm gold before SEM imaging (Philips, XL 20) at 10 kV. For the superior epithelium and stylus canal tissue, radulae route of delivery for elements involved in the min- were dissected and then processed using a microwave-assisted eralization process. However, there is a growing protocol as described in Shaw et al. (2008b). Briefly, radulae body of evidence to suggest that the supply of ele- were excised from freshly collected specimens and then immedi- ments for tooth mineralization in chitons is ately bathed in 2.5% glutaraldehyde buffered in 0.1 M phos- 21 derived from two sources (Macey and Brooker, phate at pH 7.2 (osmotic pressure adjusted to 900 mmol kg using sucrose) before further fixation, dehydration, and infiltra- 1996; Brooker et al., 2003). The first is the direct tion in a laboratory microwave (Pelco, Biowave1). Once poly- cellular transport of elements stored within the merized, resin blocks were oriented to provide transverse and cusp epithelium, while the second is via a pool of longitudinal sections of the stylus canal, which were cut to a elements that accumulate at an interface between thickness of 1 lm and 100 nm for light microscopy (LM) and transmission electron microscopy (TEM), respectively. For LM, the tooth cusp and base, termed the junction zone sections mounted on glass slides were stained with 1% Methyl- (see Fig. 1). To date, no attempts have been made ene Blue 11% Azur II (aqueous) for 20 s and imaged on an opti- to ascertain how elements are delivered and selec- cal microscope (Olympus, BX51) fitted with a digital camera tively stored at the junction zone or how they are (Olympus, DP70). For TEM, sections mounted on formvar- subsequently delivered into the tooth cusps. filmed copper grids were double stained with uranyl nitrate (10 min) and Sato’s lead citrate (10 min) (Hanaichi et al., 1986) and Despite the wealth of literature available on imaged on a TEM (JEOL, 2000FX) with plate film at 80 kV. tooth cusp mineralization, very little work has For energy-filtered TEM imaging and analysis, unstained sec- been conducted on the major lateral tooth bases tions (100 nm thick) were mounted on copper grids and ana- (also referred to as the tooth stylus in many ana- lyzed at 200 kV in a TEM (JEOL, 2100), fitted with a Gatan Imaging Filter (GIF, Tridiem). Bright-field TEM images were tomical manuscripts). In particular, the role of a taken before obtaining elemental maps for iron, which were curious tube-like tissue filled cavity within the acquired using the iron M-edge and generated using the con- major lateral tooth stylus has remained unde- ventional three-window method (see, Brydson, 2001). Two pre- Journal of Morphology 590 J.A. SHAW ET AL. TABLE 1. Chiton species in which the major lateral tooth bases were investigated for the presence of the stylus canal Species Order Lepidopleurida Suborder Lepidopleurina Collection site Lat Long Family Leptochitonidae Leptochiton liratus (Adams and Angas, 1864) Cottesloe Reef, WA.