Functional morphology of the glandular esophageal pouches of chitons (Mollusca, Polyplacophora).

The esophageal pouches of Chaetopleura angulata and Acanthochitona fascicularis were investigated using light and transmission electron microscopy. These pouches linked to the posterior region of the esophagus are known as sugar glands as they contain a fluid rich in polysaccharide digesting enzymes. They are the second largest glands in the digestive system of chitons, just after the digestive gland. In both species, the pouches contain a dense array of finger-shaped villi. The villi epithelium includes absorptive cells, basophilic secretory cells, mucus-secreting cells, and basal cells. Some absorptive cells were bordered by a dense cover of long microvilli, whereas other absorptive cells had short and sparse microvilli. Absorptive cells contain several lysosomes, mitochondria, peroxisomes, a few small Golgi stacks, some lipid droplets, and large amounts of glycogen. The basophilic secretory cells are characterized by the presence of many electron-dense vesicles, with a glycoprotein content, a large number of rough endoplasmic reticulum cisternae, and a highly developed Golgi apparatus. Mucus-secreting cells are characterized by large vesicles containing acid polysaccharides and wide Golgi stacks. Basal cells that were found at the base of the epithelium in contact with the basal lamina exhibit histological and ultrastructural features of enteroendocrine cells. We suggest that these glandular pouches are involved in extracellular and intracellular digestion, and accumulate lipid and glycogen reserves.

Phylogenetic Relationships Among Japanese Species of the Genus Ischnochiton (Polyplacophora: Ischnochitonidae), Including a New Species.

Seven (including one new) species of the polyplacophoran genus Ischnochiton (Ischnochitonidae) from the Pacific coast of Japan, namely, I. boninensis, I. comptus, I. manazuruensis, I. hakodadensis, I. hayamii sp. nov., I. paululus, and I. poppei, were investigated on the basis of DNA sequence analyses of COI, 16S rRNA, 18S rRNA, and 28S rRNA gene regions. For the latter four species, SEM observations were simultaneously carried out. A molecular phylogenetic tree based on the four gene regions for 18 chiton species indicated that the seven Japanese Ischnochiton species are polyphyletic and originated from two different clades. A haplotype network based on the COI gene region for the six Japanese Ischnochiton species, except I. hakodadensis, showed that the genetic distances among them were large. The SEM observations revealed that the denticles of the major lateral teeth in the seven Japanese Ischnochiton species were bicuspid, and an accessory process was only observed in the minor lateral teeth of I. hakodadensis. Ischnochiton hayamii sp. nov. cooccurs with I. boninensis, I. comptus, and I. manazuruensis at the two investigated localities, and was difficult to distinguish from other, similar species by naked eyes. However, these can be discriminated based on a combination of adult body size, girdle scales, and valve sculpturing in the lateral and central areas.

Valve microstructure and phylomineralogy of New Zealand chitons.

The microstructure and mineralogy of chiton valves has been largely ignored in the literature and only described in 29 species to date. Eight species: Acanthochitona zelandica, Notoplax violacea (Family Acanthochitonidae, Suborder Acanthochitonina, Order Chitonida), Chiton glaucus, Onithochiton neglectus, Sypharochiton spelliserpentis, Sypharochiton sinclairi (Family Chitonidae, Suborder, Chitonina, Order Chitonida), Ischnochiton maorianus (Family Ischnochitonidae, Suborder Chitonina, Order Chitonida), and Leptochiton inquinatus (Family Leptochitonidae, Suborder Lepidopleurina, Order Lepidopleurida) were collected from the Otago Peninsula, South Island, New Zealand. The valves of these chitons were analysed with X-ray diffractometry, Raman spectrometry, and Scanning Electron Micrography (SEM) to determine their mineralogy and microstructure. Both the XRD and Raman data show that the valves consisted solely of aragonite. The observed microstructures of the valves were complex, typically composed of four to seven sublayers, and varied among species. The dorsal layer, the tegmentum, of each species was granular and the ventral layer, the articulamentum, was predominately composed of a spherulitic sublayer, a crossed lamellar sublayer, and an acicular sublayer. The chitonids Sypharochiton pelliserpentis and S. sinclairi had the most complex microstructure layering with three crossed lamellar, two spherulitic sublayers, and a ventral acicular sublayer while the acanthochitonids Acanthochitona zelandica and Notoplax violacea as well as the ischnochitonid Ischnochiton maorianus had the simplest structure with one spherulitic, one crossed lamellar sublayer, and a ventral acicular sublayer. Terminal valves were less complex than intermediate valves and tended to be dominated by the crossed lamellar structure. The leptochitonid Leptochiton inquinatus generated a unique crossed lamellar sublayer different from the other analysed chitonids. Acanthochitona zelandica is the only analysed chitonid that utilizes two different crossed lamellar structures. Clearly, many of these properties do not reflect the currently recognized polyplacophoran phylogeny.

[Diversity and microstructure of quitons (Mollusca: Polyplacophora) from the Caribbean of Costa Rica]. / Diversidad y microestructura de quitones (Mollusca: Polyplacophora) del Caribe de Costa Rica.

Diversity and microstructure of quitons (Mollusca: Polyplacophora) from the Caribbean of Costa Rica. The polyplacophorans of the coral reef on the Caribbean coast of Costa Rica have been insufficiently studied. The examination of coral rubble accumulated in the shallow sublitoral waters on four collection stations in Provincia Limón revealed a higher diversity of chitons than was documented. From the country eight species were previously known: Ischnochiton erythronotus (C.B. Adams 1845); Ischnoplax pectinata (Sowerby 1840); Stenoplax boogii (Haddon 1886); S. purpurascens (C.B. Adams 1845); Acanthopleura granulata (Gmelin 1791); Chiton marmoratus Gmelin 1791; C. tuberculatus Linnaeus 1758 and Acanthochitona rhodea (Pilsbry 1893). This study added five more species that are reported here for the first time: Callistochiton portobelensis Ferreira 1976; Ischnochiton kaasi Ferreira 1987; I. pseudovirgatus Kaas 1972; Acanthochitona balesae Abbott 1954 and Cryptoconchus floridanus (Dall 1889).

A chiton uses aragonite lenses to form images.

Hundreds of ocelli are embedded in the dorsal shell plates of certain chitons. These ocelli each contain a pigment layer, retina, and lens, but it is unknown whether they provide chitons with spatial vision. It is also unclear whether chiton lenses are made from proteins, like nearly all biological lenses, or from some other material. Electron probe X-ray microanalysis and X-ray diffraction revealed that the chiton Acanthopleura granulata has the first aragonite lenses ever discovered. We found that these lenses allow A. granulata's ocelli to function as small camera eyes with an angular resolution of about 9°-12°. Animals responded to the sudden appearance of black, overhead circles with an angular size of 9°, but not to equivalent, uniform decreases in the downwelling irradiance. Our behavioral estimates of angular resolution were consistent with estimates derived from focal length and receptor spacing within the A. granulata eye. Behavioral trials further indicated that A. granulata's eyes provide the same angular resolution in both air and water. We propose that one of the two refractive indices of the birefringent chiton lens places a focused image on the retina in air, whereas the other does so in water.

Animal eyes: defending the coat of mail.

The eyes on the backs of molluscs known as chitons are shadow and motion detectors, the lenses of which are made of birefringent aragonite. These provide a focus both in and out of water.

Diversidad y microestructura de quitones (Mollusca: Polyplacophora) del Caribe de Costa Rica

Diversity and microstructure of quitons (Mollusca: Polyplacophora) from the Caribbean of Costa Rica. The polyplacophorans of the coral reef on the Caribbean coast of Costa Rica have been insufficiently studied. The examination of coral rubble accumulated in the shallow sublitoral waters on four collection stations in Provincia Limón revealed a higher diversity of chitons than was documented. From the country eight species were previously known: Ischnochiton erythronotus (C.B. Adams 1845); Ischnoplax pectinata (Sowerby 1840); Stenoplax boogii (Haddon 1886); S. purpurascens (C.B. Adams 1845); Acanthopleura granulate (Gmelin 1791); Chiton marmoratus Gmelin 1791; C. tuberculatus Linnaeus 1758 and Acanthochitona rhodea (Pilsbry 1893). This study added five more species that are reported here for the first time: Callistochiton portobelensis Ferreira 1976; Ischnochiton kaasi Ferreira 1987; I. pseudovirgatus Kaas 1972; Acanthochitona balesae Abbott 1954 and Cryptoconchus floridanus (Dall 1889). Rev. Biol. Trop. 59 (1): 129-136. Epub 2011 March 01.

Los poliplacóforos asociados a los arrecifes de coral en la costa caribeña de Costa Rica han sido poco estudiados. El examen del cascajo de coral acumulado en el sublitoral somero, en cuatro estaciones de colección, localizadas en la Provincia de Limón reveló una diversidad de quitones mayor a la documentada. Anteriormente se habían registrado ocho especies para el Caribe costaricense: Ischnochiton erythronotus (C.B. Adams, 1845); Ischnoplax pectinata (Sowerby 1840); Stenoplax boogii (Haddon, 1886); S. purpurascens (C.B. Adams, 1845); Acanthopleura granulata (Gmelin, 1791); Chiton marmoratus Gmelin, 1791; C. tuberculatus Linnaeus, 1758; Acanthochitona rhodea (Pilsbry, 1893). Otras cinco se registran aquí por primera vez: Callistochiton portobelensis Ferreira 1976; Ischnochiton kaasi Ferreira 1987; I. pseudovirgatus Kaas 1972; Acanthochitona balesae Abbott 1954; Cryptoconchus floridanus (Dall 1889).

Nanoscale chemical tomography of buried organic-inorganic interfaces in the chiton tooth.

Biological organisms possess an unparalleled ability to control the structure and properties of mineralized tissues. They are able, for example, to guide the formation of smoothly curving single crystals or tough, lightweight, self-repairing skeletal elements. In many biominerals, an organic matrix interacts with the mineral as it forms, controls its morphology and polymorph, and is occluded during mineralization. The remarkable functional properties of the resulting composites-such as outstanding fracture toughness and wear resistance-can be attributed to buried organic-inorganic interfaces at multiple hierarchical levels. Analysing and controlling such interfaces at the nanometre length scale is critical also in emerging organic electronic and photovoltaic hybrid materials. However, elucidating the structural and chemical complexity of buried organic-inorganic interfaces presents a challenge to state-of-the-art imaging techniques. Here we show that pulsed-laser atom-probe tomography reveals three-dimensional chemical maps of organic fibres with a diameter of 5-10 nm in the surrounding nano-crystalline magnetite (Fe(3)O(4)) mineral in the tooth of a marine mollusc, the chiton Chaetopleura apiculata. Remarkably, most fibres co-localize with either sodium or magnesium. Furthermore, clustering of these cations in the fibre indicates a structural level of hierarchy previously undetected. Our results demonstrate that in the chiton tooth, individual organic fibres have different chemical compositions, and therefore probably different functional roles in controlling fibre formation and matrix-mineral interactions. Atom-probe tomography is able to detect this chemical/structural heterogeneity by virtue of its high three-dimensional spatial resolution and sensitivity across the periodic table. We anticipate that the quantitative analysis and visualization of nanometre-scale interfaces by laser-pulsed atom-probe tomography will contribute greatly to our understanding not only of biominerals (such as bone, dentine and enamel), but also of synthetic organic-inorganic composites.

Characterization of biominerals in the radula teeth of the chiton, Acanthopleura hirtosa.

Understanding biomineralization processes provides a route to the formation of novel biomimetic materials with potential applications in fields from medicine to materials engineering. The teeth of chitons (marine molluscs) represent an excellent example of a composite biomineralized structure, comprising variable layers of iron oxide, iron oxyhydroxide and apatite. Previous studies of fully mineralized teeth using X-ray diffraction, Raman spectroscopy and scanning electron microscopy (SEM) have hinted at the underlying microstructure, but have lacked the resolution to provide vital information on fine scale structure, particularly at interfaces. While transmission electron microscopy (TEM) is capable of providing this information, difficulties in producing suitable samples from the hard, complex biocomposite have hindered progress. To overcome this problem we have used focused ion beam (FIB) processing to prepare precisely oriented sections across interfaces in fully mineralized teeth. In particular, the composite structure is found to be more complex than previously reported, with additional phases (goethite and amorphous apatite) and interface detail observed. This combination of FIB processing and TEM analysis has enabled us to investigate the structural and compositional properties of this complex biocomposite at higher resolution than previously reported and has the potential to significantly enhance future studies of biomineralization in these animals.

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.

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.

On fertilization in Chaetopleura apiculata and selected Chitonida.

Early events of fertilization are described in Chaetopleura apiculata and other selected Chitonida. C. apiculata egg hulls are elaborated into multi-branched spines with interlocking polygonal bases. Around the perimeter of each base are a series of open pores, ranging in size from 0.1-0.5 microm, which permit sperm direct access to the vitelline layer. In Callochitonidae (Chitonida) even larger pores occur in egg jelly coats, but this is considered to be the plesiomorphic condition, found also in Lepidopleurida such as Deshayesiella curvata. Other Chitonina, such as Rhyssoplax tulipa and Acanthopleura granulata, have a continuous outer dense layer that lacks pores and must be digested by penetrating sperm. Fertilization in Chitonida is unique and involves injection of chromatin into the egg via a narrow tubular nuclear extension that appears to exclude other sperm organelles, including mitochondria, centrioles, and flagellum. New evidence from studies of fertilization in Mopalia muscosa (Chitonida: Acanthochitonina) supports this hypothesis. This type of fertilization implies maternal inheritance of both mitochondria and centrioles, which is highly unusual, because in most animals one sperm centriole assists movements of pronuclei and regulates organization of the mitotic spindle. This mechanism of fertilization is defined by a series of apomorphic characters that unify the order Chitonida.