Biomineralization of limpet teeth: a cryo-TEM study of the organic matrix and the onset of mineral deposition

The continuously growing limpet radula contains teeth at various stages of maturity and thus provides an excellent opportunity for studying the processes and mechanisms of their mineralization. We report here on our structural investigations of the pre-formed chitin matrix and the initial deposition and growth of goethite (α-FeOOH) crystals within the matrix. By using cryo-techniques, in which unstained sections of the teeth are examined in a frozen-hydrated state in a transmission electron microscope (TEM), we were able to characterize the process without introducing artifacts normally associated with the staining, dehydration, and embedding required for conventional TEM. The unmineralized matrix consists of relatively well ordered, densely packed arrays of chitin fibers, with only a few nanometers between adjacent fibers. There are clearly no pre-formed compartments that control goethite crystal size and shape; rather, crystals must push aside or engulf the fibers as they grow. By examining teeth nearly row-by-row around the onset of mineralization, we were able to image the first-formed mineral within the chitin matrix. These linear deposits of goethite appear to nucleate on the chitin fibers, which thus control the orientation of the crystals. Crystal growth, on the other hand, is apparently not influenced by the matrix, in contrast to many other biomineralization systems.     

小型腹足类齿舌的扫描电镜观察

介绍了应用扫描电镜观察小型腹足类齿舌的方法。描述了折叠萝卜螺和大脐圆扁螺齿舌带上齿片的排列方式,结果显示,两种螺齿舌的齿片排成许多模列,每一横列又包含多个齿片,齿片上缘或侧缘尖齿的数目和形态有差异。     

Structural and material design of mature mineralized radula teeth of Patella vulgata (gastropoda)

The molluscan radula is a cutting apparatus employed in feeding. It is a toothed ribbon located in the mouth cavity. This study focuses on the characterization, organization, and hardness of the various components in full grown, mineralized, radula teeth of the common limpet Patella vulgata. Three major components are identified: Fibrous organic material, acicular crystals of goethite (α-FeOOH), and silica (SiO₂). Each forms a coherent network intertwined with the other two. The goethite crystals lie parallel to the fibers in the organic framework. The orientations of fibers and crystals are described in detail. The hardness of the leading part, i.e., the part that leads in the cutting direction, is about twice the hardness in the trailing part. It is argued that this difference results from the greater compactness of the mineral matter in the leading part. The function of the tooth as a cutting device is discussed on the basis of the data presented in this study.     

Structural Organisation of the Cusps of the Radular Teeth of the Chiton Plaxiphora albida

Abstract The structure, morphology and organisation of the cusps of the major lateral radula teeth of the chiton Plaxiphora albida have been examined using light, transmission and scanning electron microscopy, together with energy dispersive X-ray analysis and Mössbauer spectroscopy. In this chiton species, both the anterior and posterior surfaces of the major lateral teeth are composed of magnetite, which is indicated to be non-stoichiometric and associated with some maghemite, together with small amounts of phosphorus and silicon. This outer layer surrounds an inner core region of the tooth, which only reaches the surface through a small window zone on the anterior surface and which contains large amounts of iron and phosphorus presumably in the form of iron(III) phosphate. The organic matrix, on which the teeth are constructed, consists of a zone of densely packed fine fibres at the surface of the tooth, underlain by larger fibres which become sparser deeper into the cusp. The core region is characterized by the presence of densely packed short fibres. In contrast to the situation found in most other species of chiton, large fibres of the organic matrix extend throughout the region of magnetite mineralization, leading to the suggestion that the matrix exerts more control over the mineralization of magnetite than has previously been thought.     

17种陆生贝类齿舌的扫描电镜观察及 分类学意义探讨

采用扫描电镜观察了3目10科12属17种陆生贝类的齿舌形态.结果 显示,17种陆生贝类齿舌的中央齿均为1列,侧齿12～218列不等,缘齿0～204列不等.中央齿依齿片上小齿数目分为单齿型、三齿型和多齿型;侧齿与缘齿的形态多样,侧齿齿片上小齿数1～6枚不等,缘齿齿片上小齿数1～10枚不等.结合以往报道的38种陆生贝类齿舌...     

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

Summary The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microseopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5–10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthognal texture of bundles of microfibrils that are thought to contain chitin. The pesence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

Mechanical wear of the gastropod radula: a scanning electron microscope study

The wear sustained by the teeth of the highly mineralized radula of Patella vulgata and the unmineralized radula of Agriolomax reticulatus has been studied with the scanning electron microscope. The unworn teeth of P. vulgata have tall pointed cusps and wear is first seen as a chipping of the tips then as abrasion. There is a well defined abraded surface giving the worn teeth a chisel shape. In cleaved teeth fibres ˜ 800 Å in diameter are observed parallel to the axis of the tooth. Evidence is presented for chemical etching and for the existence of a surface coat on the tooth. A. reticulatus teeth show wear over their whole surface. With the aid of the grooves in the jaw caused by the teeth the role of the jaw in flattening the radula and protracting and retracting the odontophore is confirmed. From the arrangement of the abraded surfaces on the teeth of P. vulgata it was deduced that the teeth rows are abraded one at a time while on a convex surface at or near the bending plane.     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

The snail Smaragdia bryanae (Neritopsina,Neritidae) is a specialist herbivore of the seagrass Halophila hawaiiana (Alismatidae,Hydrocharitaceae)

Abstract. The endemic Hawaiian gastropod Smaragdia bryanae is a specialized marine herbivore that uses the endemic seagrass Halophila hawaiiana as both food and habitat. These small neritids, their grazing scars, and their egg capsules are found year‐round on seagrass leaves, where they feed on protoplast contents released as the sharp outer‐lateral teeth of the snail's radula puncture leaf epidermal cells; the contents of these cells are likely swept into the mouth by the long, wispy cusps of the marginal teeth. Structural differences from the typical neritid radula include elongated outer‐lateral teeth with two sharply pointed cusps, delicate marginal teeth reduced in both size and number, and a compressed central section. Snails grazed on leaves of H. hawaiiana steadily in laboratory culture, and grew and reproduced on this diet. In laboratory choice experiments, snails did not graze the thalli of any of six macroalgal species growing near seagrass where snails were collected, and strongly preferred occupying seagrass. Seagrass samples from five field sites on Oahu and one on Maui showed from 30% to 94% of leaves damaged, with 11% of the total leaf standing area grazed. Snails are smaller (mean length 2.74±0.32 mm, mean width 2.15±0.17 mm, n=217) than the width of the leaves of H. hawaiiana (mean 3.24±1.26 mm, n=790). The snails associate constantly with their host, despite the scattered distribution, small patch size, and variability of the seagrass resource, demonstrated by a sevenfold range in the leaf area index (mean 1.11±0.61 cm² blade surface cm^?2, n=31) among samples. Damage on grazed leaves (mean 8.21±7.05 mm² per leaf, or 16.5% of leaf surface, n=511) is concentrated in the apical and central epithelia between the midrib and the marginal veins, where snails may access cells with thinner walls and few fibers. Details of the grazing interaction between these extant species in Hawai'i shed light on the ecological specialization of members of the genus Smaragdia to seagrasses over geological time.     

General morphology and ultrastructure of the radula of Testudinalia testudinalis (O. F. Müller, 1776) (Patellogastropoda,Gastropoda)

The radular morphology of the patellid species Testudinalia testudinalis (O. F. Müller, 1776) from the White Sea was studied using light, electron, and confocal microscopy. The radula is of the docoglossan type with four teeth per row and consisting of six zones. We characterize teeth formation in T. testidinalis as follows: one tooth is formed by numerous and extremely narrow odontoblasts through apocrine secretion; this initially formed tooth consists of numerous vesicles; the synthetic apparatus of the odontoblasts is localized in the apical and central parts of the cells throughout the cytoplasm and is penetrated by microtubules which are involved in the transport of the synthesized products to the apical part of the odontoblast; the newly formed teeth consist of unpolymerized chitin. Mitotic activity is located in the lateral parts of the formation zone. The first four rows contain an irregular arrangement of teeth, but the radular teeth are regularly arranged after the fifth row. The irregularly arranged teeth early on could be a consequence of the asynchronous formation of teeth and the distance between the odontoblasts and the membranoblasts. The morphological data obtained significantly expands our knowledge of the morphological diversity of the radula formation in Gastropoda.     

The Staining of Radulae

A review of the various methods of staining and mounting radulae is given. Normally the radula should be extracted with 0.5 to 1% sodium hydroxide solution, and the associated tissues removed before staining. Two staining methods are recommended for facilitating the interpretation of radulae. Newly formed teeth and the bases of older ones are well stained by saturated aqueous chlorazol black E (up to 10 minutes). A more uniformly stained specimen) in which the cusps of all but the young teeth are alone stained, may be obtained by using the “oxidation-dahlia technic”. The radula is oxidized in N/10 potassium permanganate solution until black and subsequently decolorized in saturated aqueous oxalic acid. It is then stained in 0.1% aqueous dahlia (Hofmann's violet), the staining time varying from 10 to 30 minutes, according to the material. It is then dehydrated and passed through xylene and clove oil into Canada balsam. Other mountants may be employed but glycerin jelly is only recommended for the rapid examination of unstained radulae. Several other staining methods are mentioned, and general precautions to be observed while mounting are discussed.     

红条毛肤石鳖齿舌主侧齿中铁还原酶分布及与生物矿化的关系

以红条毛肤石鳖Acanthochiton rubrolineatus(Lischke)齿舌为材料,通过切片和酶组织化学技术,在光镜和电镜下对齿舌主侧齿的微结构及高铁还原酶的存在进行观察,从微观角度了解齿舌主侧齿齿尖的矿化机理。结果显示,成熟主侧齿由齿尖和齿基组成。齿尖结构由外至内分为三层,最外层为磁铁矿层,前后齿面磁铁矿层的厚度不等,后齿面约50μm,前齿面约5-10μm。向内依次为棕红色的纤铁矿层,厚约10μm,及略显黄色的有机基质层,有机基质层占据着齿尖内部的大部分结构。高分辨透射电镜下显示磁铁矿由条状四氧化三铁颗粒组成,长约2-3μm,宽约100-150nm。齿舌的矿化是一个连续过程,不同部段处于不同的矿化阶段,齿舌囊上皮细胞沿囊腔分布,并形成齿片。未矿化的新生主侧齿齿尖中存在由有机基质构成的网状结构。随矿化的进行,有机基质内出现矿物颗粒。初始矿化的齿尖外表面有一个细胞微突层,微突的另一端为囊上皮细胞,矿物质经由微突层达齿尖并沉积于有机基质中,齿尖随之矿化并成熟。初始矿化齿尖的外围有大量的三价铁化物颗粒,稍成熟的齿尖外围同时还出现二价铁化物。新生或初始矿化主侧齿齿尖外围的囊上皮细胞中有大量球形类似于铁蛋白聚集体的内容物,直径0.6-0.8μm,球体由膜包围。齿舌囊上皮组织中存在三价高铁还原酶,此酶分布于上皮细胞的膜表面,可能与齿尖表面磁铁矿的生成有一定的关系。       

Electron microscopic localization of chitin using colloidal gold labelled with wheat germ agglutinin

The lectin wheat germ agglutinin (WGA) has a binding site which is able to bind a sequence of three N-acetyl-glucosamine residues. Therefore, it has a very strong affinity for the polymers of this sugar, especially chitin. Colloidal gold can be labelled with WGA and used as a specific electron-dense marker for the electron-microscopic localization of chitin. The specificity of the WGA-gold binding can be checked by competitive inhibition with 5-10 mM triacetyl chitotriose. The reliability of this method was tested in three species. In the formation zone of the radula of the snail, Biomphalaria glabrata Say, chitin or chitin precursors were localized in vesicles of the odontoblasts, outside the extremely long microvilli of odontoblasts and in the newly formed teeth. The inner peritrophic envelope of the earwig, Forficula auricularia L., is characterized by an orthogonal texture of bundles of microfibrils that are thought to contain chitin. The presence of chitin was proved using the present method. In the peritrophic membranes of the blowfly, Calliphora erythrocephala Meigen, it was possible to differentiate between chitin and glycoproteins which have N-acetylglucosamine residues.     

Main patterns of radula formation and ontogeny in Gastropoda

Gastropoda is morphologically highly variable and broadly distributed group of mollusks. Due to the high morphological and functional diversity of the feeding apparatus gastropods follow a broad range of feeding strategies: from detritivory to highly specialized predation. The feeding apparatus includes the buccal armaments: jaw(s) and radula. The radula comprises a chitinous ribbon with teeth arranged in transverse and longitudinal rows. A unique characteristic of the radula is its continuous renewal during the entire life of a mollusk. The teeth and the membrane are continuously synthesized in the blind end of the radular sac and are shifted forward to the working zone, while the teeth harden and are mineralized on the way. Despite the similarity of the general mechanism of the radula formation in gastropods, some phylogenetically determined features can be identified in different phylogenetic lineages. These mainly concern shape, size, and number of the odontoblasts forming a single tooth. The radular morphology depends on the shape of the formation zone and the morphology of the subradular epithelium. The radula first appears at the pre- and posttorsional veliger stages as an invagination of the buccal epithelium of the larval anterior gut. The larval radular sac is lined with uniform undifferentiated cells. Each major phylogenetic lineage is characterized by a specific larval radula type. Thus, the docoglossan radula of Patellogastropoda is characterized by initially three and then five teeth in a transverse row. The larval rhipidoglossan radula has seven teeth in a row with differentiation into central, lateral, and marginal teeth and later is transformed into the adult radula morphology by the addition of lateral and especially marginal teeth. The taenioglossan radula of Caenogastropoda is nearly immediately formed in adult configuration with seven teeth in a row.     

OsCERK1 and OsRLCK176 play important roles in peptidoglycan and chitin signaling in rice innate immunity

Microbe‐associated molecular pattern (MAMP)‐triggered immunity plays critical roles in the basal resistance defense response in plants. Chitin and peptidoglycan (PGN) are major molecular patterns for fungi and bacteria, respectively. Two rice (Oryza sativa) lysin motif‐containing proteins, OsLYP4 and OsLYP6, function as receptors that sense bacterial PGN and fungal chitin. These membrane receptors, which lack intracellular kinase domains, likely contain another component for transmembrane immune signal transduction. Here, we demonstrate that the rice LysM receptor‐like kinase OsCERK1, a key component of the chitin elicitor signaling pathway, also plays an important role in PGN‐triggered immunity in rice. Silencing of OsCERK1 suppressed PGN‐induced (and chitin‐induced) immunity responses, including reactive oxygen species generation, defense gene expression, and callose deposition, indicating that OsCERK1 is essential for both PGN and chitin signaling initiated by OsLYP4 and OsLYP6. OsLYP4 associated with OsLYP6 and the rice chitin receptor chitin oligosaccharide elicitor‐binding protein (CEBiP) in the absence of PGN or chitin, and treatment with PGN or chitin led to their disassociation in vivo. OsCERK1 associated with OsLYP4 or OsLYP6 when induced by PGN but it associated with OsLYP4, OsLYP6, or CEBiP under chitin treatment, suggesting the presence of different patterns of ligand‐induced heterooligomeric receptor complexes. Furthermore, the receptor‐like cytoplasmic kinase OsRLCK176 functions downstream of OsCERK1 in the PGN and chitin signaling pathways, suggesting that these MAMPs share overlapping intracellular signaling components. Therefore, OsCERK1 plays dual roles in PGN and chitin signaling in rice innate immunity and as an adaptor involved in signal transduction at the plasma membrane in conjunction with OsLYP4 and OsLYP6.     

Activity profiling of platelets by chemical proteomics

Chemical proteomics or activity based proteomics is a functional proteomics technology where molecular probes are used to target a selective group of functionally related proteins. Its emergence has enabled specific targeting of subproteomes, overcoming the limitations in dynamic range of traditional large‐scale proteomics experiments. Using a chemical proteomics strategy, we attempt to differentially profile the nucleotide‐binding proteome of active and resting platelets. We apply an affinity chromatography protocol using immobilized adenosine triphosphate, cyclic adenosine monophosphate, and cyclic guanosine monophosphate. The specificity of the immobilized nucleotides was demonstrated by competitive assays and by immunoblotting. LC coupled MS/MS was applied to identify the proteins recovered by our chemical proteomics strategy. When compared to a standard set of platelet lysate proteins, we confirmed that enrichment for nucleotide‐binding proteins was indeed taking place. Finally, by employing label‐free MS‐based comparative quantification, we found a small number of platelet proteins that show statistically significant difference between the active and resting nucleotide‐binding proteome.     

ULTRASTRUCTURE OF THE RADULA APPARATUS IN SOME SPECIES OF APLACOPHORAN MOLLUSCS

Radula morphology, tooth formation and chitin localization areinvestigated in four genera of Solenogas-tres and one genusof Caudofoveata using transmission electron microscopy. Radulateeth are secreted by groups of odontoblasts in the radula sheath.Tooth shape is determined by the arrangement of the odontoblastsand preformed by microvilli. Chitin is produced in the odontoblastsand is present in the teeth as well as in the basal materialof Solenogastres and in the radula membrane of Caudofoveata.Because of its development pattern and its ultrastructural morphologythe basal material is confirmed as being identical with theradula membrane. Mitotic activity in the basal epithelium isthought to support the forward movement of the radula. (Received 18 April 1991; accepted 12 December 1991)     

The mineralization and hardness of the radular teeth of the limpet Patella vulgata L.

Summary A study of the Patella vulgata radula has been made using: the scanning electron microscope in its normal and compositional contrast modes of operation, the electron microprobe analyser, ion etching with argon ions and microhardness testing.Only iron, silicon and small amounts of sulphur were detected in the radula. The teeth can be subdivided into a cusp, a junctional area where the cusp is joined to the base, and the base which is embedded in the radular membrane. From a study of longitudinal vertical and transverse sections of the mature teeth it was found that the cusp could be subdivided into a posterior iron-rich area (44–51% Fe, 1–6% Si) and an anterior silicon-rich area (22–30% Fe, 27–32% Si). The junctional zone consisted of a poorly mineralised layer at its border with the cusp and an iron-rich layer where it joined the base. The upper part of the base (5% Fe, 16% Si) could be clearly differentiated from the silicon-rich anterior and lower parts of the base (3–4% Fe, 28–35% Si). No minerals were detected in the membrane. The changes in the mineral content of the teeth cusps along the length of the radula were studied. Iron appeared in the cusps at the 25th row and the concentration increased to 28% at the 50th row. The iron was here evenly distributed throughout the cusp. Silicon appeared in the anterior part of the cusp at the 50th row and as it increased in concentration so the iron was displaced, and at the same time the concentration of iron increased in the posterior part of the cusp. Mineralization appeared to be complete by the 150th row.The teeth cusps appear to consist of 800 Å fibres grouped into 1 thick bundles and the tooth appears to be covered by a thin enamel-like layer. It is suggested that the fibres contain the silicon-rich phase and the matrix the iron-rich phase.The significance of the arrangement of the fibres and the distribution of the minerals are discussed with relation to the function of the teeth.We wish to thank Mr. A. Rees and Mr. A. Davies for their technical assistance; Prof. Lewis and Dr. James for the use of the Electron Microprobe; and the S.R.C. for their financial support.     

The radula of the Late Cretaceous scaphitid ammonite Rhaeboceras halli (Meek and Hayden, 1856)

Abstract: Radular teeth occur between the jaws in two specimens of the Late Cretaceous scaphitid ammonite Rhaeboceras halli (Meek and Hayden, 1856) from the Western Interior of the United States. The detailed morphology of the teeth has been revealed by propagation phase contrast X‐ray synchrotron microtomography. Each row of the radula of R. halli consists of a total of seven teeth (a central rachidian, two pairs of lateral and one pair of marginal teeth), as in other known ammonoid radulae, although the central tooth could not be confirmed in the specimens examined. The lateral teeth are multicuspid and robust, and the marginal teeth are long (4.6 mm) and slender. In overall morphology, the heterodont and ctenoglossan radula of R. halli is similar that of Jurassic and Cretaceous ammonites with the same aptychus‐type lower jaw, that is, the Aptychophora. This discovery reveals the range of variation in radular morphology, which could be related to ecological or phylogenetic factors. It also invalidates the hypothesis that the hook‐like structures in R. halli previously described are radular elements.