Molecular Systematics of Recent and Pleistocene Brachiopods

Enzyme linked immunosorbent assay (ELISA) of shell intracrystalline proteinaceous macromolecules has been applied to investigate the phylogenetic relationships among 53 living articulate brachiopods (Class Articulata), covering all the living superfamilies and most of the living families. One of the articulate superfamilies, the Cancellothyridoidea, has been investigated by a combined immunological and morphometric approach, with additional materials to include most of the living species of Terebratulina, which is one of the most prolific among the Cenozoic brachiopod genera. The immunological techniques have also been applied to the phylogenetic investigation of Pleistocene brachiopods, including 2 extinct species. Both living and fossil brachiopod shell intracrystalline macromolecules have been analysed using various biochemical techniques. Antisera prepared against 14 living and taxonomically-diverse species allowed ordinal and superfamilial discriminations within the class, and using the more specific pre-absorbed antisera, it was possible to obtain precise species-level information with taxonomic consistency. These experiments revealed that the examined living terebratulide families belonged to one of the following four groups (expressed in provisional superfamilial denotations), which were further clustered into three major groups (provisionally considered as subordinal rank) of a trichotomous relationship: (A) Cancellothyridoidea (short-looped): Cancellothyrididae, Chlidonophoridae; (B1) Terebratuloidea (short-looped): Terebratulidae, Dyscoliidae; (B2). 'Kraussinoidea' (long-looped): Kraussinidae, Megathyrididae, Macandreviidae, Ecnomiosidae; (C) Terebratelloidea (long-looped): Terebratellidae, Laqueidae, Dallinidae. These results were compared and intercalated with both morphological data and the fossil record reaching the following phylogenetic interpretation: The ancestors of the three groups radiated in the early Devonian, and each of those gave rise to the group A, B1, and C in the Mesozoic; in the Triassic another long-looped lineage (possibly the extinct Zeilleriidae) diverged from the short-looped B1 lineage, and this long-looped stock gave rise to the Kingenidae and group B2, probably by processes of neoteny. This scenario suggests that the long loop evolved at least twice independently in the Terebratulida, and also highlights the enigmatic origin of the Cancellothyridoidea. Among the Cancellothyridoidea, 21 living species and subspecies of Terebratulina were assigned into two major phylogenetic groups, which were further divided into 7 subgroups on the basis of the immunological, morphometric, and other data. Degrees of molecular variations between some Terebratulina species were comparable with those between families in other terebratulide superfamilies, suggesting the existence of deep branching events within the living Terebratulina. Fractionations of Terebratulina shell intracrystalline macromolecules by various liquid chromatography techniques revealed several discrete components, including at least one proteinaceous component, most of which were antigenic. Some of these components were revealed to have been preserved more or less intact in a Pleistocene sample, in terms of the molecular weight, amino acid composition, and antigenic properties. The fossil sample also contained degradation and condensation products. Immunological assays on Pleistocene materials from a series of horizons indicated a progressive decay of the macromolecules through time, but the novel serial antisera concentration method demonstrated that even 1 Myr old shells contained lineage-specific molecular information, which allowed family- to species-level phylogenetic reconstructions for the extinct terebratulide species, Kikaithyris hanzawai and an undescribed species of Terebratulina

Tuning of Calcite Crystallographic Orientation to Support Brachiopod Lophophore

Organisms exert exquisite control on mineral formation by tuning structural and material properties to meet functional requirements. Brachiopods are sessile marine organisms that filter feed via a large lophophore which is supported by a delicate calcite loop that grows from the inner surface of the shell. How does the loop support the weight of the large lophophore? Electron backscatter diffraction (EBSD) and nanoindentation analyses of the loop as it emerges from the shell of Laqueus rubellus reveal that calcite fiber crystallography generates asymmetry in the material properties of the structure. In the core of the emergent loop, the fibers are short and kernel‐like. Either side of the core, the long fibers have a different crystallographic orientation and resultant material properties. fibers on the anterior, load‐bearing side, are harder (H = 3.76 ± 0.24 GPa) and less stiff (E = 76.87 ± 4.87 GPa) than the posterior (H = 3.48 ± 0.31 GPa, E = 81.79 ± 5.33 GPa). As a consequence of the asymmetry in the material properties, the loop anterior may be more flexible under load. The brachiopod strategy of tuning crystallographic orientation to confer spatially determined material properties is attractive for additive manufacturing of synthetic materials that have complex heterogeneous material property requirements

Left-right asymmetric expression of dpp in the mantle of gastropods correlates with asymmetric shell coiling

Background: Various shapes of gastropod shells have evolved ever since the Cambrian. Although theoretical analyses of morphogenesis exist, the molecular basis of shell development remains unclear. We compared expression patterns of the decapentaplegic (dpp) gene in the shell gland and mantle tissues at various developmental stages between coiled-shell and non-coiled-shell gastropods. Results: We analyzed the expression patterns of dpp for the two limpets Patella vulgata and Nipponacmea fuscoviridis, and for the dextral wild-type and sinistral mutant lineage of the pond snail Lymnaea stagnalis. The limpets had symmetric expression patterns of dpp throughout ontogeny, whereas in the pond snail, the results indicated asymmetric and mirror image patterns between the dextral and sinistral lineages. Conclusion: We hypothesize that Dpp induces mantle expansion, and the presence of a left/right asymmetric gradient of the Dpp protein causes the formation of a coiled shell. Our results provide a molecular explanation for shell, coiling including new insights into expression patterns in post-embryonic development, which should aid in understanding how various shell shapes are formed and have evolved in the gastropods.ArticleEVODEVO. 4:15 (2013)journal articl

Sclerite formation in the hydrothermal-vent “scaly-foot” gastropod — possible control of iron sulfide biomineralization by the animal

A gastropod from a deep-sea hydrothermal field at the Rodriguez triple junction, Indian Ocean, has scale-shaped structures, called sclerites, mineralized with iron sulfides on its foot. No other organisms are known to produce a skeleton consisting of iron sulfides. To investigate whether iron sulfide mineralization is mediated by the gastropod for the function of the sclerites, we performed a detailed physical and chemical characterization. Nanostructural characterization of the iron sulfide sclerites reveals that the iron sulfide minerals pyrite (FeS2) and greigite (Fe3S4) form with unique crystal habits inside and outside of the organic matrix, respectively. The magnetic properties of the sclerites, which are mostly consistent with those predicted from their nanostructual features, are not optimized for magnetoreception and instead support use of the magnetic minerals as structural elements. The mechanical performance of the sclerites is superior to that of other biominerals used in the vent environment for predation as well as protection from predation. These characteristics, as well as the co-occurrence of brachyuran crabs, support the inference that the mineralization of iron sulfides might be controlled by the gastropod to harden the sclerites for protection from predators. Sulfur and iron isotopic analyses indicate that sulfur and iron in the sclerites originate from hydrothermal fluids rather than from bacterial metabolites, and that iron supply is unlikely to be regulated by the gastropod for iron sulfide mineralization. We propose that the gastropod may control iron sulfide mineralization by modulating the internal concentrations of reduced sulfur compounds

Evolution of Epidermal Growth Factor (EGF)-like and Zona Pellucida Domains Containing Shell Matrix Proteins in Mollusks

Several types of shell matrix proteins (SMPs) have been identified in molluskan shells. Their diversity is the consequence of various molecular processes, including domain shuffling and gene duplication. However, the evolutionary origin of most SMPs remains unclear. In this study, we investigated the evolutionary process EGF-like and zona pellucida (ZP) domains containing SMPs. Two types of the proteins (EGF-like protein (EGFL) and EGF-like and ZP domains containing protein (EGFZP)) were found in the pearl oyster, Pinctada fucata. In contrast, only EGFZP was identified in the gastropods. Phylogenetic analysis and genomic arrangement studies showed that EGFL and EGFZP formed a clade in bivalves, and their encoding genes were localized in tandem repeats on the same scaffold. In P. fucata, EGFL genes were expressed in the outer part of mantle epithelial cells are related to the calcitic shell formation. However, in both P. fucata and the limpet Nipponacmea fuscoviridis, EGFZP genes were expressed in the inner part of the mantle epithelial cells are related to aragonitic shell formation. Furthermore, our analysis showed that in P. fucata, the ZP domain interacts with eight SMPs that have various functions in the nacreous shell mineralization. The data suggest that the ZP domain can interact with other SMPs, and EGFL evolution in pterimorph bivalves represents an example of neo-functionalization that involves the acquisition of a novel protein through gene duplication

Dual Gene Repertoires for Larval and Adult Shells Reveal Molecules Essential for Molluscan Shell Formation

Molluscan shells, mainly composed of calcium carbonate, also contain organic components such as proteins and polysaccharides. Shell organic matrices construct frameworks of shell structures and regulate crystallization processes during shell formation. To date, a number of shell matrix proteins (SMPs) have been identified, and their functions in shell formation have been studied. However, previous studies focused only on SMPs extracted from adult shells, secreted after metamorphosis. Using proteomic analyses combined with genomic and transcriptomic analyses, we have identified 31 SMPs from larval shells of the pearl oyster, Pinctada fucata, and 111 from the Pacific oyster, Crassostrea gigas. Larval SMPs are almost entirely different from those of adults in both species. RNA-seq data also confirm that gene expression profiles for larval and adult shell formation are nearly completely different. Therefore, bivalves have two repertoires of SMP genes to construct larval and adult shells. Despite considerable differences in larval and adult SMPs, some functional domains are shared by both SMP repertoires. Conserved domains include von Willebrand factor type A (VWA), chitin-binding (CB), carbonic anhydrase (CA), and acidic domains. These conserved domains are thought to play crucial roles in shell formation. Furthermore, a comprehensive survey of animal genomes revealed that the CA and VWA-CB domain-containing protein families expanded in molluscs after their separation from other Lophotrochozoan linages such as the Brachiopoda. After gene expansion, some family members were co-opted for molluscan SMPs that may have triggered to develop mineralized shells from ancestral, nonmineralized chitinous exoskeletons

Putative Neural Network Within an Olfactory Sensory Unit for Nestmate and Non-nestmate Discrimination in the Japanese Carpenter Ant: The Ultra-structures and Mathematical Simulation

Ants are known to use a colony-specific blend of cuticular hydrocarbons (CHCs) as a pheromone to discriminate between nestmates and non-nestmates and the CHCs were sensed in the basiconic type of antennal sensilla (S. basiconica). To investigate the functional design of this type of antennal sensilla, we observed the ultra-structures at 2D and 3D in the Japanese carpenter ant, Camponotus japonicus, using a serial block-face scanning electron microscope (SBF-SEM), and conventional and high-voltage transmission electron microscopes. Based on the serial images of 352 cross sections of SBF-SEM, we reconstructed a 3D model of the sensillum revealing that each S. basiconica houses > 100 unbranched dendritic processes, which extend from the same number of olfactory receptor neurons (ORNs). The dendritic processes had characteristic beaded-structures and formed a twisted bundle within the sensillum. At the “beads,” the cell membranes of the processes were closely adjacent in the interdigitated profiles, suggesting functional interactions via gap junctions (GJs). Immunohistochemistry with anti-innexin (invertebrate GJ protein) antisera revealed positive labeling in the antennae of C. japonicus. Innexin 3, one of the five antennal innexin subtypes, was detected as a dotted signal within the S. basiconica as a sensory organ for nestmate recognition. These morphological results suggest that ORNs form an electrical network via GJs between dendritic processes. We were unable to functionally certify the electric connections in an olfactory sensory unit comprising such multiple ORNs; however, with the aid of simulation of a mathematical model, we examined the putative function of this novel chemosensory information network, which possibly contributes to the distinct discrimination of colony-specific blends of CHCs or other odor detection