Micro- and nanometric characterization of the celestite skeleton of acantharian species (Radiolaria, Rhizaria).

We clarified the specific micrometric arrangement and nanometric structure of the radiolarian crystalline spines that are not a simple single crystal. A body of the celestite (SrSO4) skeleton of acantharian Acanthometra cf. multispina (Acanthometridae) composed of 20 radial spines having four blades was characterized using microfocus X-ray computed tomography. The regular arrangement of three types of spines was clarified with the connection of the blades around the root of each spine. The surface of the spines was covered with a chitin-based organic membrane to prevent from dissolution in seawater. In the nanometric scale, the mesocrystalline structure that consists of nanoscale grains having distorted single-crystal nature was revealed using scanning- and transmission electron microscopies, electron diffraction, and Raman spectroscopy. The acantharian skeletons have a crystallographically controlled architecture that is covered with a protective organic membrane. These facts are important for penetrating the nature of biogenic minerals.

The structural origins of brittle star arm kinematics: An integrated tomographic, additive manufacturing, and parametric modeling-based approach.

Brittle stars are known for the high flexibility of their arms, a characteristic required for locomotion, food grasping, and for holding onto a great diversity of substrates. Their high agility is facilitated by the numerous discrete skeletal elements (ossicles) running through the center of each arm and embedded in the skin. While much has been learned regarding the structural diversity of these ossicles, which are important characters for taxonomic purposes, their impact on the arms' range of motion, by contrast, is poorly understood. In the present study, we set out to investigate how ossicle morphology and skeletal organization affect the flexibility of brittle star arms. Here, we present the results of an in-depth analysis of three brittle star species (Ophioplocus esmarki, Ophiopteris papillosa, and Ophiothrix spiculata), chosen for their different ranges of motion, as well as spine size and orientation. Using an integrated approach that combines behavioral studies with parametric modeling, additive manufacturing, micro-computed tomography, scanning electron microscopy, and finite element simulations, we present a high-throughput workflow that provides a fundamental understanding of 3D structure-kinematic relationships in brittle star skeletal systems.

Avenues for Investigating the Neural Crest and Its Derivatives in Non-model (Unconventional) Vertebrates: A Craniofacial Skeleton Perspective.

One of the early, profound insights regarding the biology of the neural crest was the observation of its contribution to the skeletal structures of the cranium and jaws. The critical nature of these structures made the comparative analysis of the cranial neural crest and its derived structures essential investigative aims toward our understanding of the development and evolution of vertebrates and vertebrate-specific structures. Though classically applied to a relatively wide range of taxa in the nineteenth and early twentieth centuries, the application of traditional methodologies for complex comparative developmental and anatomical analyses subsequently become more limited by their time-consuming nature, resource scarcity, and a greater emphasis on the genetic and molecular regulation of patterning and morphogenesis in a select number of tractable model organisms. Recently, however, this trend has been reversed, and the value of genetic and molecular-based questions applied to non-model (unconventional) vertebrate organisms has been re-appreciated. This is particularly true of comparative investigations of cranial neural crest biology. Herein, we present methodologies for the analysis of the cranial neural crest and its structural derivatives employable in modern investigations of both model and unconventional vertebrate organisms.

Morphology, shape variation and movement of skeletal elements in starfish (Asterias rubens).

Starfish (order: Asteroidea) possess a complex endoskeleton composed of thousands of calcareous ossicles. These ossicles are embedded in a body wall mostly consisting of a complex collagen fiber array. The combination of soft and hard tissue provides a challenge for detailed morphological and histological studies. As a consequence, very little is known about the general biomechanics of echinoderm endoskeletons and the possible role of ossicle shape in enabling or limiting skeletal movements. In this study, we used high-resolution X-ray microscopy to investigate individual ossicle shape in unprecedented detail. Our results show the variation of ossicle shape within ossicles of marginal, reticular and carinal type. Based on these results we propose an additional classification to categorize ossicles not only by shape but also by function into 'connecting' and 'node' ossicles. We also used soft tissue staining with phosphotungstic acid successfully and were able to visualize the ossicle ultrastructure at 2-µm resolution. We also identified two new joint types in the aboral skeleton (groove-on-groove joint) and between adambulacral ossicles (ball-and-socket joint). To demonstrate the possibilities of micro-computed tomographic methods in analyzing the biomechanics of echinoderm skeletons we exemplarily quantified changes in ossicle orientation for a bent ray for ambulacral ossicles. This study provides a first step for future biomechanical studies focusing on the interaction of ossicles and soft tissues during ray movements.

Elemental composition and ultrafine structure of the skeleton in shell-bearing protists-A case study of phaeodarians and radiolarians.

Cross-sections were prepared by ultramicrotome (UM) and focused ion beam (FIB) system in order to examine the skeletal structure of ecologically and geologically important shell-bearing protists: phaeodarians and radiolarians. The elemental composition of the skeleton was clarified by the energy dispersive X-ray spectroscopy, suggesting that the skeletons of both groups are mainly made of amorphous silica (SiO2·nH2O) with other minor elements (Na, Mg, Al, Cl, K, Ca and Fe) and that these two groups have similar elemental composition, compared with other siliceous organisms (diatoms and sponges). However, the structural difference among the two groups was confirmed: phaeodarian skeletons are porous, unlike radiolarians with solid skeletons. It was also revealed that the phaeodarian skeleton contains concentric layered structure with spaces, presumably related to the ontogenetic skeleton formation. The distinction in the skeletal ultrafine structure (porous/solid and non-dense/dense) would reflect the ecological difference among the two groups and could be an effective criterion to determine whether microfossils belong to Radiolaria or Phaeodaria. The UM and FIB combined method presented in this study could be a useful approach to examine the chemical and structural characteristics of unculturable and/or rare microorganisms.

Biochemical characterization of the skeletal matrix of the massive coral, Porites australiensis - The saccharide moieties and their localization.

To construct calcium carbonate skeletons of sophisticated architecture, scleractinian corals secrete an extracellular skeletal organic matrix (SOM) from aboral ectodermal cells. The SOM, which is composed of proteins, saccharides, and lipids, performs functions critical for skeleton formation. Even though polysaccharides constitute the major component of the SOM, its contribution to coral skeleton formation is poorly understood. To this end, we analyzed the SOM of the massive colonial coral, Porites australiensis, the skeleton of which has drawn great research interest because it records environmental conditions throughout the life of the colony. The coral skeleton was extensively cleaned, decalcified with acetic acid, and organic fractions were separated based on solubility. These fractions were analyzed using various techniques, including SDS-PAGE, FT-IR, in vitro crystallization, CHNS analysis, chromatography analysis of monosaccharide and enzyme-linked lectin assay (ELLA). We confirmed the acidic nature of SOM and the presence of sulphate, which is thought to initiate CaCO3 crystallization. In order to analyze glycan structures, we performed ELLA on the soluble SOM for the first time and found that it exhibits strong specificity to Datura stramonium lectin (DSL). Furthermore, using biotinylated DSL with anti-biotin antibody conjugated to nanogold, in situ localization of DSL-binding polysaccharides in the P. australiensis skeleton was performed. Signals were distributed on the surfaces of fiber-like crystals of the skeleton, suggesting that polysaccharides may modulate crystal shape. Our study emphasizes the importance of sugar moieties in biomineralization of scleractinian corals.

Ultrastructural, material and crystallographic description of endophytic masses - A possible damage response in shark and ray tessellated calcified cartilage.

The cartilaginous endoskeletons of elasmobranchs (sharks and rays) are reinforced superficially by minute, mineralized tiles, called tesserae. Unlike the bony skeletons of other vertebrates, elasmobranch skeletons have limited healing capability and their tissues' mechanisms for avoiding damage or managing it when it does occur are largely unknown. Here we describe an aberrant type of mineralized elasmobranch skeletal tissue called endophytic masses (EPMs), which grow into the uncalcified cartilage of the skeleton, but exhibit a strikingly different morphology compared to tesserae and other elasmobranch calcified tissues. We use materials and biological tissue characterization techniques, including computed tomography, electron and light microscopy, X-ray and Raman spectroscopy and histology to characterize the morphology, ultrastructure and chemical composition of tesserae-associated EPMs in different elasmobranch species. EPMs appear to develop between and in intimate association with tesserae, but lack the lines of periodic growth and varying mineral density characteristic of tesserae. EPMs are mineral-dominated (high mineral and low organic content), comprised of birefringent bundles of large calcium phosphate crystals (likely brushite) aligned end to end in long strings. Both tesserae and EPMs appear to develop in a type-2 collagen-based matrix, but in contrast to tesserae, all chondrocytes embedded or in contact with EPMs are dead and mineralized. The differences outlined between EPMs and tesserae demonstrate them to be distinct tissues. We discuss several possible reasons for EPM development, including tissue reinforcement, repair, and disruptions of mineralization processes, within the context of elasmobranch skeletal biology as well as damage responses of other vertebrate mineralized tissues.