Zoological Studies 46(4): 379-388 (2007) Silica Biomineralization in the Radula of a Limpet Notoacmea schrenckii (Gastropoda: Acmaeidae) Tzu-En Hua and Chia-Wei Li* Institute of Molecular and Cellular Biology, College of Life Sciences, National Tsing Hua University, Hsinchu 300, Taiwan (Accepted November 6, 2006) Tzu-En Hua and Chia-Wei Li (2007) Silica biomineralization in the radula of a limpet Notoacmea schrenckii (Gastropoda: Acmaeidae). Zoological Studies 46(4): 379-388. The radulae of limpets are regarded as an ideal experimental material for studying biologically controlled mineral deposition, because they possess teeth in dif- ferent mineralization stages. The pattern of silica precipitation in the limpet, Notoacmea schrenckii (Gastropoda: Acmaeidae), was elucidated in this study using transmission electron microscopy (TEM), electron diffraction, energy-dispersive X-ray (EDX) analysis, and inductively coupled plasma mass spectrometry (ICP- MS). The ICP-MS elemental analysis showed that iron and silica both infiltrate into the radula in early stages of tooth development. Electron-dense granules in a nanometer size range were observed in ultrathin sections of tooth specimens in early mineral-deposition stage; electron diffraction analysis indicated that silica is the prima- ry component of these granules. TEM images revealed the intimate association between silica granules and the organic matrix, which implies that the organic matrix may take a more-active role in catalysis besides mere- ly functioning as a physical constraint during mineral deposition. Exposure of the tooth cusp to NH4F treatment and the appearance of silica spheres after the addition of silicate suggest that the organic molecules embedded within the minerals may assist silica precipitation. http://zoolstud.sinica.edu.tw/Journals/46.4/379.pdf Key words: Notoacmea schrenckii, Limpet radula, Silica deposition. Biominerals are the inorganic phases of fied to co-precipitate with amorphous silica during minerals found in biological systems, which are the cell wall-formation process (Kröger et al. 1997 distributed from microorganisms to the highest ani- 1999). These polypeptides are so tightly associat- mals and plants (Mann et al. 1989, Simkiss and ed with the amorphous silica that they can only be Wilbur 1989, Weiner and Lowenstam 1989, Mann extracted after dissolution of the cell wall in hydro- 2001). Although minerals composed of calcium gen fluoride (HF) to remove the silica. The HF- predominate, biominerals of barium, strontium, extractable polypeptides were named silaffins and iron, and silica are also well known. Biominerals were demonstrated to act as regulating molecules generally occur as oxides, hydroxides, phos- during biosilica formation in diatoms (Kröger et al. phates, and carbonates (Webb et al. 2001). 1999 2000 2001). Later studies showed that other Biosilicification, the biological formation of than the polyamine moieties, native silaffins are opal-like amorphous hydrated silica, occurs in a also heavily phosphorylated (Kröger et al. 2002). wide variety of organisms, including diatoms, Each serine residue is phosphorylated, and this sponges, mollusks, and higher plants (Voronkov et high level of phosphorylation is essential for , al. 1977, Lowenstam 1981, Simpson and Volcani silaffins ability to precipitate silica in ambient con- 1981). Among various organisms with the capabil- ditions as well as to regulate the activities of silica- ity to deposit silica, diatoms and sponges are the forming biomolecules (Kröger et al. 2002, Poulsen most thoroughly explored taxa. In the study of et al. 2003). A phase-separation model was pro- diatoms, species-specific polypeptides were identi- posed to account for the self-similar silica patterns *To whom correspondence and reprint requests should be addressed. E-mail:[email protected] 379 380 Zoological Studies 46(4): 379-388 (2007) observed in diatom cell walls based on the knowl- discovering the underlying principles of the edge obtained in the study of silaffin molecules microarchitecture within the mineralized tissues, (Sumper 2002). including the relationships between the inorganic Siliceous sponges deposit silica in needle-like phase and the organic matrix of protein or polysac- spicules that support the organism and provide charides. Although silica is known to play essen- defense against predation. Each spicule contains tial roles in tooth growth and maintaining radular a proteinaceous axial filament, later named sili- structural integrity (Mann et al. 1986), the process catein, which serves as a template or otherwise by which it infiltrates into the organic matrix and directs silica deposition (Shimizu et al. 1998, Uriz through what mechanism it is deposited remain et al. 2000). Protein analysis of silicateins of sev- elusive. The radulae are especially attractive in eral different sponge species indicated that protein efforts to unravel silica deposition mechanisms sequences are conserved across sponge species, because they contain teeth at various mineraliza- and the sequences are similar to those of the tion stages from unmineralized soft tissue to fully cathepsin L subfamily of papain-like cysteine pro- mineralized complex teeth. Studies of such series teases (Shimizu et al. 1998, Cha et al. 1999). The of teeth at different mineralization stages may pro- silicatein gene has been cloned in some species, vide insights into the processes and mechanisms and the processes by which it coordinates and of mineral deposition. functions in biosilicification processes in sponges In this study, the limpet, Notoacmea have been unraveled (Krasko et al. 2000, Müller et schrenckii, was selected as the experimental al. 2003, Bavestrello et al. 2003, Werner et al. material. This particular species belongs to the 2003, Weaver and Morse 2003, Pozzolini et al. Phylum Mollusca, Class Gastropoda, Order 2005). Archaeogastropoda, and Family Acmaeidae. The In mollusks such as limpets and chitons, silica radulae of N. schrenckii can be segmented into 4 deposits are found in radulae, feeding organ of stages according to the extent of mineralization mollusks, where it is co-precipitated with iron in the based on our observations under stereomicro- construction of an efficient feeding apparatus scope. Stage I is composed of odontoblasts, from (Mann et al. 1986, Liddiard et al. 2004). Limpets which the growth of an organic framework of tooth are common organisms that can be found in inter- is initiated and no mineral deposition is occurred at tidal regions throughout the world. Previous stud- this stage. Cusps at the onset of mineral deposi- ies focusing on the radulae of the limpet species, tion turn light orange, and these rows of teeth are Patella vulgata (Runham and Thronton 1967, defined as in stage II. In stage III, the mineral Mann et al. 1986), demonstrated that limpet radu- deposition of teeth is almost complete, and the lae consist of a continuous series of teeth in vari- cusps in this stage are solid and firm; while in ous stages of mineralization, ranging from soft stage IV, mineral deposition of the teeth is com- unmineralized structures through fully mineralized plete and the teeth are ready to replace worn-out teeth, which make them ideal experimental organ- ones. isms to investigate the mechanisms of mineral In situ analysis utilizing electron microscopy deposition. Various elemental analyses carried out and inductively coupled plasma mass spectrome- on Patella vulgate (Jones et al. 1935, Lowenstam ter (ICP-MS) with the limpet N. schrenckii may pro- 1962, Runham et al. 1969, Grime et al. 1985, vide clues to deciphering the distribution of silica in Mann et al. 1986) revealed that the major teeth limpet teeth and a possible time frame for when sil- contain up to 12% ferric oxide in the form of ica takes part in radula maturation. Possible re- goethite (α-FeOOH), 7%-16% silica as hydrated deposition of amorphous silica spheres after treat- amorphous opal (SiO2·nH2O), and small amounts ment with ammonium fluoride following the addi- of calcium other than the organic components. tion of silicate suggests that the biomolecules Later studies concentrating on Patelloida alticosta- which induce silica deposition can be released ta, Patella peronii (Burford et al. 1986), and from the mineral and guide silica precipitation if Cellana toreuma (Lu et al. 1995) indicated that the sufficient ambient silicate is supplied. deposition of Fe is probably mediated by ferritin translocation. To help understand the molecular mechanisms controlling biosilicification, it is essen- MATERIALS AND METHODS tial to characterize the biomolecules encapsulated within biologically silicified structures. Studies of Collection of limpet specimens and isolation of these various minerals have been directed towards radulae Hua and Li -- Silica Biomineralization in Limpet Radulae 381 both cells and the radula were digested with 67% Notoacmea schrenckii specimens were col- HNO3 for 3 d. Both silica and iron contents in solu- lected at low tide from the intertidal zone of ble state were measured using a Perkin-Elmer Nanliao Fishing Port, Hsinchu, northwestern SCIEX ELAN 5000 inductively coupled plasma- Taiwan. Following collection, animals were placed mass spectrometer. in fresh seawater and immediately transported back to the laboratory. Radulae were dissected Transmission electron microscopy (TEM) out under a stereomicroscope and treated with 3% collagenase (Sigma) in 100 mM phosphate- Fresh radulae were dissected into 4 frag- buffered