L.R. Brooker

The junction zone: Initial site of mineralization in radula teeth of the chiton Cryptoplax striata (Mollusca: Polyplacophora)

Elemental composition and distribution in individual teeth of the whole radula of the chiton Cryptoplax striata were analyzed using energy‐dispersive spectroscopy. Both the element deposited and its position within the tooth vary according to the stage of mineralization. The initial site of mineralization is the junction zone, the region between the tooth cusp and base. In this region, the first element to be deposited is iron, followed by phosphorus and then calcium. Iron deposition next commences in the tooth cusp cap, where it proceeds rapidly, being virtually complete within 12 tooth rows. By contrast, mineralization in the core of the tooth cusp does not commence until well down the radula and consists initially of iron and phosphorus with the addition of a small amount of calcium 6 rows later. While mineralization in the tooth base commences early in radula development, it continues right through to the fully mature end of the radula. A number of minor elements are also found at various stages of mineralization. The data obtained have been used to construct a schematic of the progression of mineralization along the length of the radula.

Radula synthesis by three species of iron mineralizing molluscs: production rate and elemental demand

A cold-shock technique was used to determine radula production rates for the chitons Acanthopleura hirtosa and Plaxiphora albida, and for the limpet Patelloida alticostata, which replaced their radular teeth at rates of 0.40, 0.36 and 0.51 rows d-1, respectively. These rates are far slower than those determined previously for non-iron-mineralizing molluscs, suggesting that the improved working life of the teeth afforded by iron-mineralization acts to significantly reduce replacement rates. In addition, inductively coupled plasma-atomic emission spectroscopy has been used to determine the quantity of iron and other elements comprising the radula of each species. These data, used in conjunction with the radula production rates, reveal that A. hirtosa, Plaxiphora albida and Patelloida alticostata have daily radula mineralization requirements for iron of 3.06, 4.12 and 0.55 μg, respectively. Such information is vital for continuing studies related to the cellular delivery of ions and subsequent biomineralization of the tooth cusps in chitons and limpets.

Apatite Mineralization in Teeth of the Chiton Acanthopleura echinata

Abstract. Raman spectroscopy has been used to demonstrate, for the first time, that calcium mineralization in the core of the major lateral teeth of the chiton Acanthopleura echinata takes place as an ordered process, with crystalline carbonated apatite being the first mineral deposited. Deposition begins at the top of the tooth core, under the so-called tab region, progresses down the interior surface of the tab and lepidocrocite layer, and then extends outwards to the anterior surface. Mineralization is not initiated until the lepidocrocite layer has isolated the core of the tooth from the magnetite cap. The last region to be infiltrated is the anterior basal region of the tooth cusp, immediately above the junction zone. The junction zone is also a region of high ion density, as determined by energy dispersive spectroscopy (EDS) analysis, but we show here for the first time that it is free of mineral deposits, acting instead as a transfer and storage region.

A new biomineral identified in the cores of teeth from the chiton Plaxiphora albida

The hydrated iron(III) oxide limonite is reported for the first time as a biomineral. In situ laser Raman spectra of the tooth cores from major lateral teeth of the chiton Plaxiphora albida are compared with those of synthetic and mineral iron phosphates and iron oxides. Raman spectra measured on iron phosphate and iron oxide standard materials are shown to be easily distinguishable from one another. The central tooth cores of mature P. albida teeth do not show any evidence for the presence of a separate iron phosphate mineral. Rather, in each tooth a narrow band of the hydrated iron(III) oxide limonite is shown to separate the magnetite of the tooth surface from a central core region comprising both lepidocrocite and limonite. The high concentration of phosphorus in P. albida tooth cores, previously observed by energy dispersive spectroscopy, is not associated with a separate iron phosphate mineral, indicating that this element may be adsorbed onto the surface of the iron oxide minerals present. The failure to detect a separate iron(III) phosphate is discussed with reference to other chiton species that display high levels of iron and phosphorus in the cores of their mature major lateral teeth.

The chiton stylus canal: An element delivery pathway for tooth cusp biomineralization

A detailed investigation of the stylus canal situated within the iron mineralized major lateral teeth of the chiton Acanthopleura hirtosa was undertaken in conjunction with a row‐by‐row examination of cusp mineralization. The canal is shown to contain columnar epithelial tissue similar to that surrounding the mineralized cusps, including the presence of iron rich particles characteristic of the iron storage protein ferritin. Within the tooth core, a previously undescribed internal pathway or plume is evident above the stylus canal, between the junction zone and mineralizing posterior face of the cusp. Plume formation coincides with the appearance of iron in the superior epithelium and the onset of mineralization at tooth row 13. The plume persists during the delivery of phosphorous and calcium into the tooth core, and is the final region of the cusp to become mineralized. The presence of the stylus canal was confirmed in a further 18 chiton species, revealing that the canal is common to polyplacophoran molluscs. These new data strongly support the growing body of evidence highlighting the importance of the junction zone for tooth mineralization in chiton teeth, and indicate that the chemical and structural environment within the tooth cusp is under far greater biological control than previously considered. J. Morphol. 2009.

IRON BIOMINERALS IN MEDICINE AND THE ENVIRONMENT

The nature and function of iron biominerals, particularly ferrihydrite (5Fe2O3·9H2O) and goethite (α-FeOOH), in medicine and the environment are reviewed through three case studies: the tissue iron deposits formed in the iron overload associated with the genetic disease of thalassemia, a medical condition of global significance; the tissue iron deposits formed in the liver of the endangered tropical marine mammal, the dugong Dugong dugon; the granules formed in the tissue of the freshwater mussel Velusunio angasi.

The Chiton Radula: A Unique Model for Biomineralization Studies

Chitons (Mollusca: Polyplacophora) are slow moving, bilaterally symmetrical and dorsoventrally flattened molluscs that are commonly found on hard substrata in intertidal regions of coastlines around the world (Kaas & Jones, 1998). All species are characterized by a series of eight dorsal, articulating shell plates or valves, which may be embedded, to varying degrees, in a fleshy, muscular girdle (Kaas & Jones, 1998) (Figure 1). Approximately 750 living species are known, and while intertidal regions are home to the majority of chitons, a number of species can be found at depths of up to 8000m where they feed on detrital material (Kaas & Jones, 1998).

Ultrastructure of the epithelial cells associated with tooth biomineralization in the chiton Acanthopleura hirtosa

The cusp epithelium is a specialized branch of the superior epithelium that surrounds the developing teeth of chitons and is responsible for delivering the elements required for the formation of biominerals within the major lateral teeth. These biominerals are deposited within specific regions of the tooth in sequence, making it possible to conduct a row by row examination of cell development in the cusp epithelium as the teeth progress from the unmineralized to the mineralized state. Cusp epithelium from the chiton Acanthopleura hirtosa was prepared using conventional chemical and microwave assisted tissue processing, for observation by light microscopy, conventional transmission electron microscopy (TEM) and energy filtered TEM. The onset of iron mineralization within the teeth, initiated at row 13, is associated with a number of dramatic changes in the ultrastructure of the apical cusp cell epithelium. Specifically, the presence of ferritin containing siderosomes, the position and number of mitochondria, and the structure of the cell microvilli are each linked to aspects of the mineralization process. These changes in tissue development are discussed in context with their influence over the physiological conditions within both the cells and extracellular compartment of the tooth at the onset of iron mineralization.

Methods of sample preparation of radula epithelial tissue in chitons (Mollusca: Polyplacophora)*

Abstract A glutaraldehyde fixative developed for preserving the radula superior epithelium of the adult chiton Acanthopleura hirtosa (Blainville, 1825), was used in conjunction with conventional and microwave-assisted sample processing to produce high quality tissue preservation for light and electron microscopy. In addition, high-pressure freezing (HPF) and cryo-substitution were used to fix the radula tissue of juvenile specimens. Microwave-assisted fixation was preferred to conventional bench-top techniques due to the superior preservation of fine cell structure together with reduced processing times and chemical exposure. Although restricted to very small (<200 μm) samples, the quality of juvenile radulae processed by HPF was excellent. The improvements in tissue preservation using microwave and cryo-preservation techniques are therefore critical for obtaining accurate ultrastructural information on the radula in marine molluscs. In particular, these findings highlight additional processing options available for the study of cellular structures in biomineralizing tissues.

Tooth use and wear in three iron-biomineralizing mollusc species

Chitons and limpets harden their teeth with biominerals in order to scrape algae from hard rock surfaces. To elucidate relationships between tooth structure and function, light and electron microscopy were used to examine naturally worn teeth in three species of mollusc with iron-mineralized teeth and to analyze the grazing marks left by members of these species feeding on wax. For the two chiton species, teeth wore down progressively from the medial to the lateral edge of the cusp, while for the limpet, wear was more evenly distributed across the edges of each cusp. In chitons, this pattern of wear matched the medially biased morphology of the cusps in their protracted position and relates to what is known about the mineral composition and substructure of the teeth. The patterns of progressive tooth wear for each of these species, together with the distinct grazing marks left by each species on the wax substrate, indicate that the teeth are designed to remain functionally effective for as long as possible, and have proved to be a valuable means of rationalizing the internal architecture of the teeth at a range of spatial scales. This information is critical for ongoing studies aimed at understanding the interactions between the organic matrix and mineral components of these teeth.