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.

Molecular modularity and asymmetry of the molluscan mantle revealed by a gene expression atlas.

Background: Conchiferan molluscs construct a biocalcified shell that likely supported much of their evolutionary success. However, beyond broad proteomic and transcriptomic surveys of molluscan shells and the shell-forming mantle tissue, little is known of the spatial and ontogenetic regulation of shell fabrication. In addition, most efforts have been focused on species that deposit nacre, which is at odds with the majority of conchiferan species that fabricate shells using a crossed-lamellar microstructure, sensu lato. Results: By combining proteomic and transcriptomic sequencing with in situ hybridization we have identified a suite of gene products associated with the production of the crossed-lamellar shell in Lymnaea stagnalis. With this spatial expression data we are able to generate novel hypotheses of how the adult mantle tissue coordinates the deposition of the calcified shell. These hypotheses include functional roles for unusual and otherwise difficult-to-study proteins such as those containing repetitive low-complexity domains. The spatial expression readouts of shell-forming genes also reveal cryptic patterns of asymmetry and modularity in the shell-forming cells of larvae and adult mantle tissue. Conclusions: This molecular modularity of the shell-forming mantle tissue hints at intimate associations between structure, function, and evolvability and may provide an elegant explanation for the evolutionary success of the second largest phylum among the Metazoa.

Complex hierarchical microstructures of Cambrian mollusk Pelagiella: insight into early biomineralization and evolution.

Shell microstructure and mineralogy of Pelagiella madianensis Zhou & Xiao, 1984, a globally distributed Cambrian micromollusk, are investigated based on abundant and extraordinarily well-preserved specimens from Xinji Formation, Longxian, Shaanxi, North China. Five types of aragonitic microstructures have been recognized. The lamello-fibrillar microstructure, previously known from Pelagiella, constructs the outermost shell layer, while the remaining four types are reported here for the first time in this genus. They include fibrous foliated, foliated aragonite, crossed foliated lamellar and isolated tablets. The animal constructs these five types of microstructures to build its shell in a complex hierarchical pattern with four orders: crystallite columns, laths, folia and lamellae. These findings demonstrate that the capability of building complex shell microstructures had already evolved by the Cambrian explosion. In addition, this work shows that early aragonitic shells were constructed with fibers, laths, folia and isolated tablets, indicating increased controls over biomineralization by the animal.

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.

Nanoscale assembly processes revealed in the nacroprismatic transition zone of Pinna nobilis mollusc shells.

Intricate biomineralization processes in molluscs engineer hierarchical structures with meso-, nano- and atomic architectures that give the final composite material exceptional mechanical strength and optical iridescence on the macroscale. This multiscale biological assembly inspires new synthetic routes to complex materials. Our investigation of the prism-nacre interface reveals nanoscale details governing the onset of nacre formation using high-resolution scanning transmission electron microscopy. A wedge-polishing technique provides unprecedented, large-area specimens required to span the entire interface. Within this region, we find a transition from nanofibrillar aggregation to irregular early-nacre layers, to well-ordered mature nacre suggesting the assembly process is driven by aggregation of nanoparticles (∼50-80 nm) within an organic matrix that arrange in fibre-like polycrystalline configurations. The particle number increases successively and, when critical packing is reached, they merge into early-nacre platelets. These results give new insights into nacre formation and particle-accretion mechanisms that may be common to many calcareous biominerals.

Towards high-performance bioinspired composites.

Biological composites have evolved elaborate hierarchical structures to achieve outstanding mechanical properties using weak but readily available building blocks. Combining the underlying design principles of such biological materials with the rich chemistry accessible in synthetic systems may enable the creation of artificial composites with unprecedented properties and functionalities. This bioinspired approach requires identification, understanding, and quantification of natural design principles and their replication in synthetic materials, taking into account the intrinsic properties of the stronger artificial building blocks and the boundary conditions of engineering applications. In this progress report, the scientific and technological questions that have to be addressed to achieve this goal are highlighted, and examples of recent research efforts to tackle them are presented. These include the local characterization of the heterogeneous architecture of biological materials, the investigation of structure-function relationships to help unveil natural design principles, and the development of synthetic processing routes that can potentially be used to implement some of these principles in synthetic materials. The importance of replicating the design principles of biological materials rather than their structure per se is highlighted, and possible directions for further progress in this fascinating, interdisciplinary field are discussed.

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.

Development of the excretory system in the polyplacophoran mollusc, Lepidochitona corrugata: the protonephridium.

A single pair of protonephridia is the typical larval excretory organ of molluscs. Their presence in postlarval developmental stages was discovered only recently. We found that the protonephridia of the polyplacophoran mollusc, Lepidochitona corrugata, achieve their most elaborate differentiation and become largest during the postlarval period. This study describes the protonephridia of L. corrugata using light and electron microscopy and interactive three-dimensional visualization. We focus on the postlarval developmental period, in which the protonephridia consist of three parts: the terminal part with the ultrafiltration sites at the distal end, the voluminous protonephridial kidney, and the efferent nephroduct leading to the nephropore. The ultrafiltration sites show filtration slits between regularly arranged thin pedicles. The ciliary flame originates from both the terminal cell and the duct cells of the terminal portion. The efferent duct also shows ciliation. The most conspicuous structures, the protonephridial kidneys, are voluminous swellings composed of reabsorptive cells ("nephrocytes"). These cells exhibit strong vacuolization and an infolding system increasing the basal surface. The protonephridial kidneys, previously not reported at such a level of organization in molluscs, strikingly resemble (metanephridial) kidneys of adult molluscan excretory systems.

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.

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.

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.

Interactive 3D volume rendering in biomedical publications.

We present three examples of interactive, 3D volume rendering models embedded in a PDF publication. The examples are drawn from three different morphological methods - confocal microscopy, serial sectioning and microcomputed tomography - performed on members of the phylum Mollusca. A description of the entire technical procedure from specimen preparation to embedding of the visual model including 3D labels in the document is provided. For comparison, volume rendering with standard visualization software, and surface rendering incorporated in the 3D PDF figures, are provided. The principal advantages and disadvantages of the techniques and models are discussed. Volume rendering for serial sections is relatively work-intensive, while confocal data have limitations in terms of 3D presentation. Volume renderings are normally downsampled in resolution to achieve a reasonable PDF file size, however intentional information is largely retained. We conclude that volume rendering of 3D data sets is a valuable technique and should become standard in PDF versions of biomedical publications.

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.

Tooth use and wear in three iron-biomineralizing mollusc species.

Chitons and limpets harden their teeth with biominerals in order to scrape algae from hard rock surfaces. To elucidate relationships between tooth structure and function, light and electron microscopy were used to examine naturally worn teeth in three species of mollusc with iron-mineralized teeth and to analyze the grazing marks left by members of these species feeding on wax. For the two chiton species, teeth wore down progressively from the medial to the lateral edge of the cusp, while for the limpet, wear was more evenly distributed across the edges of each cusp. In chitons, this pattern of wear matched the medially biased morphology of the cusps in their protracted position and relates to what is known about the mineral composition and substructure of the teeth. The patterns of progressive tooth wear for each of these species, together with the distinct grazing marks left by each species on the wax substrate, indicate that the teeth are designed to remain functionally effective for as long as possible, and have proved to be a valuable means of rationalizing the internal architecture of the teeth at a range of spatial scales. This information is critical for ongoing studies aimed at understanding the interactions between the organic matrix and mineral components of these teeth.

The ultrastructure and histology of the perinotal epidermis and defensive glands of two species of Onchidella (Gastropoda: Pulmonata).

Histology and electron microscopy were used to describe and compare the structure of the perinotal epidermis and defensive glands of two species of shell-less marine Systellommatophora, Onchidella capensis and Onchidella hildae (Onchidiidae). The notum of both species is composed of a layer of epithelial and goblet cells covered by a multi-layered cuticle. Large perinotal multi-cellular glands, that produce thick white sticky mucus when irritated, are located within the sub-epidermal tissue. The glands are composed of several types of large secretory cell filled with products that stain for acidic, sulphated and neutral mucins, and some irregularly shaped support cells that surround a central lumen. The products of the secretory cells are produced by organelles that are basal in position. The entire gland is surrounded by a well-developed capsule of smooth muscle and collagen, and in addition smooth muscle surrounds the cells within the glands. Based on the size of the gland cells, their staining properties, and the appearance of their stored secretions at the transmission electron microscope level, five different types of secretory cells were identified in O. capensis and four in O. hildae. The products of these cells, which are released by holocrine secretion, presumably mix in the lumen of the duct as they are forced out by contraction of the smooth muscle. The structural similarity of these glands to those of siphonariids, suggest that they have a common ancestry.

Studies on molluscan shells: contributions from microscopic and analytical methods.

Molluscan shells have always attracted the interest of researchers, from biologists to physicists, from paleontologists to materials scientists. Much information is available at present, on the elaborate architecture of the shell, regarding the various Mollusc classes. The crystallographic characterization of the different shell layers, as well as their physical and chemical properties have been the subject of several investigations. In addition, many researches have addressed the characterization of the biological component of the shell and the role it plays in the hard exoskeleton assembly, that is, the biomineralization process. All these topics have seen great advances in the last two or three decades, expanding our knowledge on the shell properties, in terms of structure, functions and composition. This involved the use of a range of specialized and modern techniques, integrating microscopic methods with biochemistry, molecular biology procedures and spectroscopy. However, the factors governing synthesis of a specific crystalline carbonate phase in any particular layer of the shell and the interplay between organic and inorganic components during the biomineral assembly are still not widely known. This present survey deals with microstructural aspects of molluscan shells, as disclosed through use of scanning electron microscopy and related analytical methods (microanalysis, X-ray diffraction, electron diffraction and infrared spectroscopy). These already published data provide relevant information on shells and also contribute for better understanding the biomineralization process.

[Cytoplasmic syncytial interneuronal connection in molluscs].

The absolute criteria developed by the authors have been presented; they allow revealing cytoplasmic syncytial connections between processes of nerve cells in vivo and in vitro at the light microscopy level by using classical methods and time lapse videoshooting in the phase contrast. With aid of electron microscopy, metastable membrane contacts and their perforations, cytoplasmic syncytial interneuronal pores, and fusion of nerve processes are demonstrated. In the culture of isolated molluscan neurons, the process of formation of syncytial connection between processes of the same neuron or of different neurons is reproduced. Processes of one neuron, which have syncytial connection with another neuron, are shown to remain viable after death of its neuronal soma. The cytoplasmic varicosities formed on processes of one neuron are able to overcome the place of syncytial contact with processes of another neuron and to move to the body of the latter. A hypothesis is put forward that the cytoplasmic syncytial connection between nerve processes is formed under the condition of the absence of their glial sheaths.

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.