| Since biogenic magnetite was first found in the teeth of chitons (Polyplacophora, Mollusca), the radular teeth have been good materials for biomineralization research, particularly for iron mineralization, as massive iron biomierals including magnetite nanoscale particles are deposited in the major lateral teeth, and that magnetic nanoparticles have been widely applied to many fields. The Acanthochiton rubrolineatus Lischke (1873) is one familiar chiton species which lives in the rocky intertidal zones in China. In this article, the process and mechanism of biomineralization of cusp of the major lateral tooth, together with some physical, chemical and material characteristics determinations of the radula were undertaken detailed investigation using a combination of light microscopy, scanning and transmission electron microscopy, superconducting quantum interference device magnetometer, powder X-ray diffraction and Fourier transform infrared spectroscopy. At first, in the morphology aspects, a holistic approach has been adopted that encompasses observations over a range of spatial scales, from whole radula mineralization process to those occurring within individual tooth cusps at various stages of development, also observation of microstructure of magnetite crystals in the magnetite layer. Secondly, in the magnetism aspects, the initial magnetization curves of radular segments and tooth cusps at various stages of development were measured, the hysteresis loops of radular segments were also measured, as a consequence of measurement, the contents of magnetite within the cups and radular segments were calculated; in addition, in the materials science aspects, microhardness of tooth cusps at various stages of development was determined. Thirdly, in the histochemistry and immunohistology aspects, the locations of chiton ferritin and ferric iron reductase in radular sac epithelia were identified. Finally, in the biochemistry aspects, the ferritin was isolated and purified.Investigation results showed that chiton A.rubrolineatus has a length of 1.5cm and width of 2—3mm tongue-like radula which bears about 60 transverse rows of teeth. Among these teeth, two lines of major lateral teeth that are obviously distinguished from others by shape and color distribute along the length of the radula. Major lateral tooth consists of two parts, cusp and tooth base, which are different from each other in shape, and the two parts connect by a joint zone. Cusp presents tricuspid shape, while tooth base presents long handle shape. According to mineralization degree, the radula can be divided into four stages of development (I-IV segments), and the cusps of major lateral teeth at various stages appear different colors range from white, red brown to shinny black, indicating the process of radula mineralization. Accompanying mineralization development, the cusp undergoes substantial changes in composition and framework. New formed cusp shows white color and no minerals are deposited in; while incipient mineralizing cusp shows red-brown color and minerals starts to be deposited in; for mature cusp, it shows shiny-black color and lots of minerals are deposited in.The formation of new teeth is taken place at the terminal end of radular sac, at which the cells of so-called sac odontoblasts distribute regularly within the sac in three dimensional organization, and they are of strong ability of cell division and secretory functions, cells at different regions of sac perform various roles, some are responsible for formation of radular membrane, some for formation of new teeth, and some for secretion of functional enzymes. Within newly formed cusp, massive organic microfibrils constitute mesh-like framework, in the structure there are no minerals; within incipient mineralizing cusp, organic fibrous filaments represent bundle forms, mineral granules starts to be biomineralized inside the fibres; with development of biomineralization, more of minerals granules are accumulated into the organic matrix and results in high biomierlization of mature cusp. Cusp mineralization has two pathways, namely internal and external pathways. By the external pathway, the superior sac epithelia surrounding the cusp via microvillus-like structure may unceasingly transport organic materials and mineral elements into the cusp; by the internal pathway, the inferior sac epithelia in the stylus canal of tooth base can provide materials via a comb-like structure occurring below the joint zone between the cusp and tooth base. Radular sac epithelium contains numerous membrane bound iron-containing aggregates, these aggregate exhibit various statuses in morphology, some of them are fully filled, and some of them are partly filled; in general, aggregates are oval shaped, and on average are about～600nm in diameter. Immunohistology reaction indicates the iron-containing granules are ferritin nature. Ferric iron reductase exists in membrane of the epithelium, which possibly plays important roles in ironbiomineralization of cusp, also may be responsible for the transformation of magnetite from other phases of iron minerals.The mature cusp is about～250μm in length (vertical axis),～150μm in width (horizontal axis) and～100μm in thickness (longitudinal axis); while tooth base is approximately～400μm in length. Cusp may be divides into three layers in structure, the magnetite (Fe3O4) layer (outer layer), is in different thickness on the posterior and anterior surface respectively, and partial magnetite in the layer transforms to maghemite (γ-Fe2O3) as magnetic as magnetite by oxidation. On the posterior surface, the maximum thickness of magnetite layer reaches approximately～20μm, on the contrary, the maximum thickness is only approximately～5μm; Inside the magnetite layer, magnetite present in a form of lath-shaped pieces that are several microns in length and about～100nm in width, these pieces arrange parallel to the vertical axis direction of cusp and are composed of many magnetite crystals, magnetite crystals vary in size, and mean size is approximately～52nm in diameter, crystals exhibit single, twinned and polycrystalline forms, fault phenomenon also occurs between the crystals, the d-spacing of (111) and (022) in single crystal is～0.48nm and 0.29nm respectively. Adjacent to the magnetite layer is lepidocrocite layer (α-Fe2O3), which is about～1μm in thickness and appears in brick red color; the core mainly is organic matrix, about～80μm thick, in which a few of calcium minerals and so on are mineralized. The tooth base deposits little minerals compared to the cusp, main composition is ofα-chitin in nature. Inside the tooth base, there is a long stylus canal that is about～50μm in diameter, and one apex of the canal directly reaches the site just below the joint zone; two openings are situated on the two ends of the tooth base respectively. For II-IV segments of radula, the saturated magnetization value is 6.5emu/g, 15.1emu/g and 14.0emu/g respectively; while for cusps, the saturated magnetization value is 68.6emu/g, 79.2emu/g and 76.0emu/g respectively. As a result, the magnetite content calculated within each of radula segments (II-IV segments) is 6.83%, 16.39% and 14.71% by dry weight, for the whole radula, magnetite content arrives at approximately 13% by dry weight, 57% by minerals weight, being equal to approximately 1011-12 magnetite crystals, approximately 89.04% iron (13%/14.60%) presents in the magnetite (or other magnetic material) form. Therefore, the value of microhardness of cusps of corresponding segments is 124.38 kg/mm2, 473.04 kg/mm2, 646.80 kg/mm2 respectively, taking account of the cusps within II segment are not fully mature, so the mean value of microhardness of cusps of III-IV segments as whole is calculated to be 559.92 kg/mm2. The chiton ferritin has been isolated and purified from radula sac to electrophoretic purity by ion-exchange chromatography and electrophoresis, and the native ferritin contains 1000 Fe atoms per molecule of protein and considerable amounts of phosphate (Fe/P =30). The molecule size is 8.2 (±1.0) nm. The ferritin has Mr of 300kDa, pI of 4.2-5.3 and is composed of two subunits Mr 25.4kDa and Mr 34.8kDa. Conclusion is that biomineralization of the cusp of major lateral tooth of chiton A. rubrolineatus is a progressive process which develops temporally and spatially, and mineralization has two pathways, ferric iron reductase and ferritin occurs in radular sac epithelia, they are important for the mineralization. In addition, magnetite content within radula is large, and magnetite presents in crystalline form. Therefore, the biomineralization causes the cusps are of magnetic, and have somemechanical hardness. |
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