The ultrastructure of the apical organ of the Müller's larva of the tiger flatworm Prostheceraeus crozieri.

The tiger flatworm Prostheceraeus crozieri (Polycladida) develops via an eight-lobed, and three-eyed planktonic Müller's larva. This larva has an apical organ, ultrastructural details of which remain elusive due to a scarcity of studies. The evolution and possible homology of the polyclad larva with other spiralian larvae is still controversial. Here, we provide ultrastructural data and three-dimensional reconstructions of the apical organ of P. crozieri. The apical organ consists of an apical tuft complex and a dorso-apical tuft complex. The apical tuft complex features a central tuft of five long cilia, which emerge from four or five individual cells that are themselves encircled by two anchor cells. The necks of six multibranched gland cells are sandwiched between ciliated tuft cell bodies and anchor cells. The proximal parts of the ciliated cell bodies are in contact with the lateral brain neuropil via gap junctions. Located dorsally of the apical tuft complex, the dorso-apical tuft complex is characterized by several long cilia of sensory neurons, these emerge from an epidermal lumen and are closely associated with several gland cells that form a crescent apically around the dorsal anchor cell, and laterally touch the brain neuropil. Such ciliated sensory neurons emerging from a ciliated lumen are reminiscent of ampullary cells of mollusc and annelid larvae; a similar cell type can be found in the hoplonemertean decidula larva. We hypothesize that the ampullary-like cells and the tuft-forming sensory cells in the apical organs of these spiralian larvae could be homologous.

Structure and function of the digestive system in molluscs.

The phylum Mollusca is one of the largest and more diversified among metazoan phyla, comprising many thousand species living in ocean, freshwater and terrestrial ecosystems. Mollusc-feeding biology is highly diverse, including omnivorous grazers, herbivores, carnivorous scavengers and predators, and even some parasitic species. Consequently, their digestive system presents many adaptive variations. The digestive tract starting in the mouth consists of the buccal cavity, oesophagus, stomach and intestine ending in the anus. Several types of glands are associated, namely, oral and salivary glands, oesophageal glands, digestive gland and, in some cases, anal glands. The digestive gland is the largest and more important for digestion and nutrient absorption. The digestive system of each of the eight extant molluscan classes is reviewed, highlighting the most recent data available on histological, ultrastructural and functional aspects of tissues and cells involved in nutrient absorption, intracellular and extracellular digestion, with emphasis on glandular tissues.

An Antarctic flock under the Thorson's rule: Diversity and larval development of Antarctic Velutinidae (Mollusca: Gastropoda).

In most marine gastropods, the duration of the larval phase is a key feature, strongly influencing species distribution and persistence. Antarctic lineages, in agreement with Thorson's rule, generally show a short pelagic developmental phase (or lack it completely), with very few exceptions. Among them is the ascidian-feeding gastropod family Velutinidae, a quite understudied group. Based on a multilocus (COI, 16S, 28S and ITS2) dataset for 182 specimens collected in Antarctica and other regions worldwide, we investigated the actual Antarctic velutinid diversity, inferred their larval development, tested species genetic connectivity and produced a first phylogenetic framework of the family. We identified 15 Antarctic Molecular Operational Taxonomic Units (MOTUs), some of which represented undescribed species, which show two different types of larval shell, indicating different duration of the Pelagic Larval Phase (PLD). Antarctic velutinids stand as an independent lineage, sister to the rest of the family, with extensive hidden diversity likely produced by rapid radiation. Our phylogenetic framework indicates that this Antarctic flock underwent repeated events of pelagic phase shortening, in agreement with Thorson's rule, yielding species with restricted geographic ranges.

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.

Mollusk shell nacre ultrastructure correlates with environmental temperature and pressure.

Nacre, or mother-of-pearl, the tough, iridescent biomineral lining the inner side of some mollusk shells, has alternating biogenic aragonite (calcium carbonate, CaCO(3)) tablet layers and organic sheets. Nacre has been common in the shells of mollusks since the Ordovician (450 million years ago) and is abundant and well-preserved in the fossil record, e.g., in ammonites. Therefore, if any measurable physical aspect of the nacre structure was correlated with environmental temperatures, one could obtain a structural paleothermometer of ancient climates. Using X-ray absorption near-edge structure (XANES) spectroscopy, Photoelectron emission spectromicroscopy (PEEM), and X-ray linear dichroism we acquired polarization-dependent imaging contrast (PIC) maps of pristine nacre in cross-section. The new PIC-map data reveal that the nacre ultrastructure (nacre tablet width, thickness, and angle spread) is species-specific in at least eight mollusk species from completely different environments: Nautilus pompilius, Haliotis iris, Haliotis rufescens, Bathymodiolus azoricus, Atrina rigida, Lasmigona complanata, Pinctada margaritifera, and Mytilus californianus. Nacre species-specificity is interpreted as a result of adaptation to diverging environments. We found strong correlation between nacre crystal misorientations and environmental temperature, further supported by secondary ion mass spectrometry measurements of in situ δ(18)O in the nacre of one shell. This has far-reaching implications: nacre texture may be used as a paleothermometer of ancient climate, spanning 450 million years of Earth's history.

The neural origins of shell structure and pattern in aquatic mollusks.

We present a model to explain how the neurosecretory system of aquatic mollusks generates their diversity of shell structures and pigmentation patterns. The anatomical and physiological basis of this model sets it apart from other models used to explain shape and pattern. The model reproduces most known shell shapes and patterns and accurately predicts how the pattern alters in response to environmental disruption and subsequent repair. Finally, we connect the model to a larger class of neural models.

Nanoscale structure and mechanical behavior of growth lines in shell of abalone Haliotis gigantea.

In the natural world, bottom-up hierarchical construction of complex structures results in materials with remarkable properties. A well known example is the nacre of mollusk shells, commonly called "mother of pearl", whose excellent strength and toughness has been the subject of research for many decades. A significant discovery has been the presence of periodic layers called "growth lines". These are thin distinct layers within the bulk of the shell which form periodically, with their structure affected by environmental changes. Studies of their formation and behavior offer valuable insight into the architecture of seashells. In this work, the structure and mechanical behavior of growth lines in shells of abalone Haliotis gigantea were investigated using electron microscopy and nanoindentation. Growth lines form directly out of nacre into layers of blocks and irregular particles. In comparison to nacre, they have basic structures, form rapidly, and are harder, which suggest that they serve a protective role during lifecycle transitions. This exemplifies how natural structures are able to closely control growth architecture in order to form different structures for different functions, all from the same base materials.

Matrix-mediated biomineralization in marine mollusks: a combined transmission electron microscopy and focused ion beam approach.

The teeth of the marine mollusk Acanthopleura hirtosa are an excellent example of a complex, organic, matrix-mediated biomineral, with the fully mineralized teeth comprising layers of iron oxide and iron oxyhydroxide minerals around a calcium apatite core. To investigate the relationship between the various mineral layers and the organic matrix fibers on which they grew, sections have been prepared from specific features in the teeth at controlled orientations using focused ion beam processing. Compositional and microstructural details of heterophase interfaces, and the fate of the organic matrix fibers within the mineral layers, can then be analyzed by a range of transmission electron microscopy (TEM) techniques. Energy-filtered TEM highlights the interlocking nature of the various mineral phases, while high-angle annular dark-field scanning TEM imaging demonstrates that the organic matrix continues to exist in the fully mineralized teeth. These new insights into the structure of this complex biomaterial are an important step in understanding the relationship between its structural and physical properties and may help explain its high strength and crack-resistance behavior.

Characterization of the multilayered shell of a limpet, Lottia kogamogai (Mollusca: Patellogastropoda), using SEM-EBSD and FIB-TEM techniques.

The microstructure and its crystallographic aspect of the shell of a limpet, Lottiakogamogai, have been investigated, as the first step to clarify the mechanism of shell formation in limpet. The shell consists of five distinct layers stacked along the shell thickness direction. Transmission electron microscopy (TEM) with the focused ion beam (FIB) sample preparation technique was primarily adopted, as well as scanning electron microscopy (SEM) with electron back-scattered diffraction (EBSD). The five layers were termed as M+3, M+2, M+1, M, M-1 from the outside to the inside in previous works, where M means myostracum. The outmost M+3 layer consists of calcite with a "mosaic" structure; granular submicron sub-grains with small-angle grain boundaries often accompanying dislocation arrays. M+2 layer consists of flat prismatic aragonite crystals with a leaf-like cross section, stacked obliquely to the shell surface. It looks that the prismatic crystals are surrounded by organic sheets, forming a compartment structure. M+1 and M-1 layers adopt a crossed lamellar structure consisting of aragonite flat prisms with rectangular cross section. M layer has a prismatic structure of aragonite perpendicular to the shell surface and with irregular shaped cross sections. Distinct organic sheets were not observed between the crystals in M+1, M and M-1 layers. The {110} twins are common in all aragonite M+2, M+1, M and M-1 layers, with the twin boundaries parallel to the prisms. These results for the microstructure of each layer should be considered in the discussion of the formation mechanism of the limpet shell structure.

Articulated Palaeozoic fossil with 17 plates greatly expands disparity of early chitons.

Modern chitons (Mollusca: Polyplacophora) possess a highly conserved skeleton of eight shell plates (valves) surrounded by spicules or scales, and fossil evidence suggests that the chiton skeleton has changed little since the first appearance of the class in the Late Cambrian period (about 500 million years before present, Myr bp). However, the Palaeozoic problematic taxon Multiplacophora, in spite of having a more complex skeleton, shares several derived characters with chitons. The enigmatic status of the Multiplacophora is due in part to the fact that its members had an exoskeleton of numerous calcium carbonate valves that usually separated after death. A new articulated specimen from the Carboniferous period (about 335 Myr bp) of Indiana reveals that multiplacophorans had a dorsal protective surface composed of head and tail valves, left and right columns of overlapping valves (five on each side), and a central zone of five smaller valves, all surrounded by an annulus of large spines. Here we describe and name the articulated specimen and present evidence that multiplacophorans were chitons. Thus the highly conserved body plan of living chitons belies the broad disparity of this clade during the Palaeozoic era.

Histochemical and ultrastructural characterization of the posterior esophagus of Bulla striata (Mollusca, Opisthobranchia).

The posterior esophagus of Bulla striata, running from the gizzard to the stomach, was investigated with light and electron microscopy to obtain new data for a comparative analysis of the digestive system in cephalaspidean opisthobranchs. In this species, the posterior esophagus can be divided into two regions. In the first, the epithelium is formed by columnar cells with apical microvilli embedded in a cuticle. Many epithelial and subepithelial secretory cells are present in this region. In both, electron-lucent secretory vesicles containing filaments and a peripheral round mass of secretory material fill the cytoplasm. These acid mucus-secreting cells may also contain a few dense secretory vesicles. In the second part of the posterior esophagus, the cuticle is absent and the epithelium is ciliated. In this region, epithelial cells may contain larger lipid droplets and glycogen reserves. Subepithelial secretory cells are not present, and in epithelial secretory cells the number of dense vesicles increases, but most secretory cells still contain some electron-lucent vesicles. These cells secrete a mixture of proteins and acid polysaccharides and should be considered seromucous. The secretory cells of the posterior esophagus are significantly different from those previously reported in the anterior esophagus of this herbivorous species.

Association of centrioles with the marginal band of a molluscan erythrocyte.

Continuous circumferential bundles of microtubules, or marginal bands (MBs), are best known as a prominent structural feature of all nonmammalian vertebrate erythrocytes and mammalian blood platelets. Since their discovery in the late 19th century, MBs have been thought to play a cellular morphogenetic role, but no cytological clues to the mechanism of MB biogenesis have been reported. In previous work we have established the presence of MBs in serveral invertebrate blood cell types, including amebocytes and coelomocytes of certain Arthropod species and erythrocytes of a Sipunculan. We report here the occurrence of MBs in erythrocytes of the ark Anadara transversa (Mollusca) and four closely related species. The MBs of these arks have a striking structural feature; each is physically associated with a pair of centrioles. The centrioles are identified as such on the basis of morphological criteria: size, cylindrical shape, right-angle orientation, pairing, and 9-triplet ultrastructure. This intimate association between centrioles and MBs suggests that centrioles may be MB-organizing centers and invites comparative investigation of their possible role in vertebrate erythrocyte and platelet morphogenesis.

Crystalline structure of sarcoplasmic reticulum from scallop.

Negatively stained sarcoplasmic reticulum from the scallop Placopecten magellanicus presented a variety of crystalline forms, the most common being tubular structures. These were characterized by paired rows of morphological units, spaced at approximately 120 A, running diagonally across the tubules. The orthogonal unit cell (120 X 55 A) contained two units, related by a twofold axis, which probably represented the part of the Ca2+-ATPase molecule projecting from the outer surface of the membrane.

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. II. Localization of intracellular calcium and its translocation during mechanical activity.

The localization of Ca-accumulating structures in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia and their role in the contraction-relaxation cycle were studied by fixing the LBWM fibers at rest and during mechanical response to 400 mM K or to 10(-4)--10(-3) M acetylcholine in a 1% OsO4 solution containing 2% K pyroantimonate. In the resting fibers, electron-opaque pyroantimonate precipitate was mostly localized at the peripheral structures, i.e., along the inner surface of the plasma membrane, at the membrane of the surface tubules, and at the sarcoplasmic reticulum. In the fibers fixed during mechanical activity, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. Electron-probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate may serve as a measure of Ca localization. These results are in accord with the view that, in the LBWM, the Ca stored in the peripheral structures is released into the myoplasm to activate the contractile mechanism.

The basal apparatus. Mass isolation from the molluscan ciliated gill epithelium and a preliminary characterization of striated rootlets.

The basal apparatus, consisting of an array of interconnected basal bodies bearing bifurcating striated rootlets encompassing a nucleus, has been isolated from hypertonically deciliated columnar gill epithelial cells of the bay scallop Aequipecten irradians through gentle lysis with Triton X-100. The rootlets, 8-10 mum in length, were not easily preserved with conventional electron microscope fixatives, suggesting that the extent of their contribution to cellular architecture has been somewhat underestimated, even though Englemann described many of the structural details of the basal apparatus in 1880. The striated rootlets were soluble at high but not at low pH, in 2 M solutions of sodium azide and potassium thiocyanate but not sodium or potassium chloride, in 1% deoxycholate but not digitonin, and in the denaturing solvents 6 M guanidine-HC1, 8 M urea, and 1% sodium dodecylsulfate at 100 degrees C. The protein found consistently when rootlets were solubilized migrated on SDS-polyacrylamide gels as a closely spaced doublet with apparent molecular weights of 230,000 and 250,000 daltons. This unique protein, distinct from tropocollagen or various muscle components, has been named ankyrin because of the rootlet's anchor-like function in the cell.

Effect of Ca2+ on the dimeric structure of scallop sarcoplasmic reticulum.

Scallop sarcoplasmic reticulum (SR), visualized in situ by freeze-fracture and deep-etching, is characterized by long tubes displaying crystalline arrays of Ca2+-ATPase dimer ribbons, resembling those observed in isolated SR vesicles. The orderly arrangement of the Ca2+-ATPase molecules is well preserved in muscle bundles permeabilized with saponin. Treatment with saponin, however, is not needed to isolate SR vesicles displaying a crystalline surface structure. Omission of ATP from the isolation procedure of SR vesicles does not alter the dimeric organization of the Ca2+-ATPase, although the overall appearance of the tubes seems to be affected: the edges of the vesicles are scalloped and the individual Ca2+-ATPase molecules are not clearly defined. The effect of Ca2+ on isolated scallop SR vesicles was investigated by correlating the enzymatic activity and calcium-binding properties of the Ca2+-ATPase with the surface structure of the vesicles, as revealed by electron microscopy. The dimeric organization of the membrane is preserved at Ca2+ concentrations where the Ca2+ binds to the high affinity sites (half-maximum saturation at pCa approximately 7.0 with a Hill coefficient of 2.1) and the Ca2+-ATPase is activated (half-maximum activation at pCa approximately 6.8 with a Hill coefficient of 1.84). Higher Ca2+ concentrations disrupt the crystalline surface array of the SR tubes, both in the presence and absence of ATP. We discuss here whether the Ca2+-ATPase dimer identified as a structural unit of the SR membrane represents the Ca2+ pump in the membrane.

Reconstitution of ciliary membranes containing tubulin.

Membranes from the gill cilia of the mollusc Aequipecten irradians may be solubilized readily with Nonidet P-40. When the detergent is removed from the solution by adsorption to polystyrene beads, the proteins of the extract remain soluble. However, when the solution is frozen and thawed, nearly all of the proteins reassociate to form membrane vesicles, recruiting lipids from the medium. The membranes equilibrate as a narrow band (d = 1.167 g/cm3) upon sucrose density gradient centrifugation. The lipid composition of reconstituted membranes (1:2 cholesterol:phospholipids) closely resembles that of the original extract, as does the protein content (45%). Ciliary calmodulin is the major extract protein that does not associate with the reconstituted membrane, even in the presence of 1 mM calcium ions, suggesting that it is a soluble matrix component. The major protein of reconstituted vesicles is membrane tubulin, shown previously to differ hydrophobically from axonemal tubulin. The tubulin is tightly associated with the membrane since extraction with 1 mM iodide or thiocyanate leaves a vesicle fraction whose protein composition and bouyant density are unchanged. Subjecting the detergent-free membrane extract to a freeze-thaw cycle in the presence of elasmobranch brain tubulin or forming membranes by warming the extract in the presence of polymerization-competent tubulin yields a membrane fraction with little incorporated brain tubulin. This suggests that ciliary membrane tubulin specifically associates with lipids, whereas brain tubulin preferentially forms microtubules.

Ultrastructural and physiological studies on the longitudinal body wall muscle of Dolabella auricularia. I. Mechanical response and ultrastructure.

The physiological properties of mechanical response and the ultrastructure in the longitudinal body wall muscle (LBWM) of the opisthobranch mollusc Dolabella auricularia were studied to obtain information about excitation-contraction coupling in somatic smooth muscles responsible for smooth and slow body movement of molluscans. The contracture tension produced by 400 mM K was not affected by Mn ions (5--10 mM) and low pH (up to 4.0), but was reduced by procaine (2 mM). The K-contracture tension was not readily eliminated in a Ca-free solution containing ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA). A large contracture tension was also produced by rapid cooling of the surrounding fluid from 20 degrees to 5 degrees--3 degrees C even when the preparation showed no mechanical response to 400 mM K after prolonged (more than 2 h) soaking in the Ca-free solution. These results indicate that the LBWM fibers contain a large amount of intracellularly stored Ca which can be effectively released by membrane depolarization. The fibers were connected with each other, forming the gap junctions, the desmosomes, and the intermediate junctions. The sarcoplasmic reticulum (SR) consisted of vesicular and tubular elements, and was mostly located near the fiber surface. The plasma membrane showed marked tubular invaginations of 600-800 A in diameter, with many branches (surface tubules), extending inwards for approximately 2 micron. These surface tubules were closely apposed to the SR, and the bridgelike structures analogous to those in the triadic junction of vertebrate skeletal muscle were observed in the space between the surface tubules and the SR. It is suggested that the influence of membrane depolarization is transmitted inwards along the surface tubules to cause the release of Ca from the SR.

Microtubule-membrane interactions in cilia. II. Photochemical cross-linking of bridge structures and the identification of a membrane-associated dynein-like ATPase.

Photochemical cross-linking of both Tetrahymena and Aequipecten ciliary membrane proteins with the lipophilic reagent 4,4'-dithiobisphenylazide links together a high molecular weight dynein-like ATPase, membrane tubulin, and at least two other proteins. Electron microscopy of detergent-extracted cilia reveals that the cross-linked complex remains attached to the outer-doublet microtubules by a microtubule-membrane bridge. Cleavage of the reagent's disulfide bond releases the bridge-membrane complex and the dynein-like membrane-associated ATPase. Electron microscopy was used to ensure that the dynein-like protein did not result from the solubilization of the dynein arms attached to the outer-doublet microtubules. The dynein-like protein has been isolated using sucrose gradients and is similar to axonemal dynein with respect to its sedimentation characteristics nucleotide specificity, and divalent cation requirements. Photochemical cross-linking of ciliary membrane porteins in vivo results initially in the modification of ciliary beat and, eventually, in the cessation of ciliary movement. These results suggest that a dynein-like ATPase comprises the bridge which links the ciliary membrane to the outer-doublet microtubules and that this bridge is involved in the modulation of normal ciliary movement.

Correlation of the orientation of stacked aragonite platelets in nacre and their connection via mineral bridges.

In this work, we studied the correlation of the orientation of stacked aragonite platelets of Haliotis laevigata nacre, using selected area diffraction (SAD) in transmission electron microscopy (TEM). From the position of the center of Laue circle (COLC) within the diffraction patterns the tilt angles of the investigated platelets relatively to a reference platelet (oriented in zone axis) are determined. The strong correlation of the platelets supports the existence of mineral bridges, which connect the stacked platelets and enable a transfer of the platelet orientation during growth. Electron tomography and subsequent reconstruction of the obtained data yield information about the shape of the mineral bridges. The crystalline structure of the material within the mineral bridges was investigated by high resolution TEM (HRTEM).