The Molecular Evolution of the Pif Family Proteins in Various Species of Mollusks

Various novel proteins have been identified from many kinds of mollusk shells. Although such matrix proteins are believed to play important roles in the calcium carbonate crystal formation of shells, no common proteins that interact with calcium carbonate or that are involved in the molecular mechanisms behind shell formation have been identified. Pif consists of two proteins, Pif 80 and Pif 97, which are encoded by a single mRNA. Pif 80 was identified as a key acidic protein that regulates the formation of the nacreous layer in Pinctada fucata, while Pif 97 has von Willebrand factor type A (VWA) and chitin-binding domains. In this study, we identified Pif homologues from Pinctada margaritifera, Pinctada maxima, Pteria penguin, Mytilus galloprovincialis, and in the genome database of Lottia gigantea in order to compare their primary protein sequences. The VWA and chitin-binding domains are conserved in all Pif 97 homologues, whereas the amino acid sequences of the Pif 80 regions differ markedly among the species. Sequence alignment revealed the presence of a novel significantly conserved sequence between the chitin-binding domain and the C-terminus of Pif 97. Further examination of the Pif 80 regions suggested that they share a sequence that is similar to the laminin G domain. These results indicate that all Pif molecules in bivalves and gastropods may be derived from a common ancestral gene. These comparisons may shed light on the correlation between molecular evolution and morphology in mollusk shell microstructure.     

A Bivalve Biomineralization Toolbox

Mollusc shells are a result of the deposition of crystalline and amorphous calcite catalyzed by enzymes and shell matrix proteins (SMP). Developing a detailed understanding of bivalve mollusc biomineralization pathways is complicated not only by the multiplicity of shell forms and microstructures in this class, but also by the evolution of associated proteins by domain co-option and domain shuffling. In spite of this, a minimal biomineralization toolbox comprising proteins and protein domains critical for shell production across species has been identified. Using a matched pair design to reduce experimental noise from inter-individual variation, combined with damage-repair experiments and a database of biomineralization SMPs derived from published works, proteins were identified that are likely to be involved in shell calcification. Eighteen new, shared proteins likely to be involved in the processes related to the calcification of shells were identified by the analysis of genes expressed during repair in Crassostrea gigas, Mytilus edulis, and Pecten maximus. Genes involved in ion transport were also identified as potentially involved in calcification either via the maintenance of cell acid–base balance or transport of critical ions to the extrapallial space, the site of shell assembly. These data expand the number of candidate biomineralization proteins in bivalve molluscs for future functional studies and define a minimal functional protein domain set required to produce solid microstructures from soluble calcium carbonate. This is important for understanding molluscan shell evolution, the likely impacts of environmental change on biomineralization processes, materials science, and biomimicry research.     

Tyrosinase localization in mollusc shells

In molluscan shellfish, pigmentation is frequently observed in the calcified shell, but the molecular basis of this process is not understood. Here, we report two tyrosinase proteins (Pfty1 and Pfty2) found in the prismatic shell layer of the pearl oyster Pinctada fucata; this layer is recognized as the pigmented region in P. fucata. The protein sequences were deduced from the corresponding cDNAs and confirmed by MALDI-TOF/TOF analysis. The sequences suggest that both tyrosinases have two copper-binding sites in similar N-terminal domains that are homologous to tyrosinases of cephalopods and hemocyanins of gastropods. In turn, this suggests that bivalve tyrosinases are evolved from a common ancestral copper-binding protein in the mollusc. Pfty1 and Pfty2 were specifically expressed in the mantle, and their expression in the mantle is different from each other, suggesting that these tyrosinases have distinctive roles in melanogenesis in shells.     

The structure of mollusc larval shells formed in the presence of the chitin synthase inhibitor Nikkomycin Z

Background  

Chitin self-assembly provides a dynamic extracellular biomineralization interface. The insoluble matrix of larval shells of the marine bivalve mollusc Mytilus galloprovincialis consists of chitinous material that is distributed and structured in relation to characteristic shell features. Mollusc shell chitin is synthesized via a complex transmembrane chitin synthase with an intracellular myosin motor domain.     

Phylogenetic implications and diagenetic stability of macromolecules from pleistocene and recent shells of Mercenaria mercenaria (mollusca,bivalvia)

The phylogeny and diagenesis of Pleistocene and Recent bivalves were studied immunologically by use of a conventional antiserum elicited against an EDTA‐soluble macromolecular extract from shells of the modern bivalve mollusc Mercenaria mercenaria. ELISA tests of the antiserum with shell fragments of a wide range of modern bivalves gave taxonomically significant results. The antiserum reacted with palaeoheterodonts and heterodonts but not with representatives of other bivalve subclasses. This phylogenetic reactivity was also apparent in tests with fossil shells, although the specificity and overall strength of the reaction were both reduced. Absorption of the antiserum with etched shell powders of various (palaeo)heterodonts yielded more specific antibody preparations.

Investigations of shell matrix diagenesis, using the anti‐Mercenaria serum, demonstrated that small amounts of original determinants could be detected even in fossils over one million years old. The reactivity of the serum with extracts of fossil Mercenaria decreased with sample age. The relationship between serum reactivity and the degree of amino acid racemization was almost linear. Clearly, the various determinants to which antibodies were elicited were being destroyed at different rates.     

Predatory shell drilling by two species of Austroginella (Gastropoda: Marginellidae)

Predatory shell drilling of bivalve mollusc shells is reported for the gastropods Austroginella johnstoni and A. muscaria from south-eastern Australia. This is the first record of this feeding behaviour in the family Marginellidae. The drill holes are circular and paraboloid, with a small inner penetration hole. The corroded nature of the aragonite crystals within the drill holes suggests a chemical dissolution drilling mechanism. No obvious accessory boring organ was located. The gastropods have subepithelial gland cells in the proboscis, a pair of small salivary glands and a large foregut gland. The latter has a duct bypassing the valve of Leiblein and joining the anterior oesophagus.     

Early Shell Formation During Molluscan Embryogenesis, with New Studies on the Surf Clam, Spisula solidissima

Shell formation in molluscs begins early in embryogenesis duringsome stage of archenteron formation. Ultrastructural informationon early formation of external shells is available from onlya few bivalves and gastropods. Secretion of the very first shellmaterial by shell field epithelial cells is preceded by an invaginationof the dorsal ectoderm in the region of the shell field. A centuryago, this invagination was termed the "shell gland." As a secretoryfunction for this invagination has not yet been demonstratedand as the term "shell gland" has taken on various meaningsin the literature, the invagination will be referred to as theshell field invagination. The opening into the shell field invaginationseems to be circular in gastropods and elongate in bivalves.Accordingly initial organic shell material seems to form a ringin gastropods and a saddle in bivalves. As in adult molluscs,shells of pre-metamorphic molluscs are composed of both organicand inorganiccomponents. Ultrastructural data from bivalvesand gastropods indicate that the initial organic shell materialis secreted just outside the shell field invagination (acrossthe pore). Initial inorganic shell materials have not been localizednor their pathway traced into or through any pre-metamorphicmolluscs. New SEM and TEM data show that the invagination inthe bivalve Spisula solidissima is composed of a wide outerregion and very narrow inner region with the first shell materialforming at the junction between the two. This is unlike ultrastructuraldata available for other species. Many sections give the falseimpressions that: 1) the shell field invagination is closedto the outside and, 2) that the first organic shell materiallines the innermost region of the invagination. It is not clearwhether the cells of the outer invagination in this speciesare shell field cells. It is suggested that they are not.     

Comparison of aragonitic molluscan shell proteins

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.     

The distribution of chitin in larval shells of the bivalve mollusk Mytilus galloprovincialis

The insoluble matrix of larval shells of the marine bivalve mollusk Mytilus galloprovincialis is investigated by confocal laser scanning microscopy using a GFP fusion protein with a chitin-binding domain for labeling of chitinous structures. We show that chitinous material is present in the larval shell, presumably as a chitin-protein complex. We further show that the structure of the chitinous material changes with the development of the larvae. We conclude from the presence of characteristic chitinous structures in certain shell regions that chitin fulfills an important function in the formation and functionality of larval bivalve shells.     

Aragonite shells are more ancient than calcite ones in bivalves: new evidence based on omics

Two calcium carbonate crystal polymorphs, aragonite and calcite, are the main inorganic components of mollusk shells. Some fossil evidences suggest that aragonite shell is more ancient than calcite shell for the Bivalvia. But, the molecular biology evidence for the above deduction is absent. In this study, we searched for homologs of bivalve aragonite-related and calcite-related shell proteins in the oyster genome, and found that no homologs of calcite-related shell protein but some homologs of aragonite-related shell proteins in the oyster genome. We explained the results as the new evidence to support that aragonite shells are more ancient than calcite shells in bivalves combined the published biogeological and seawater chemistry data.     

Evolution of the molluscan body plan: the case of the anterior adductor muscle of bivalves

The separated shell plates with the rearranged musculature (adductor muscle) is a novelty for bivalves. Despite its importance in the bivalve bodyplan, the development of the anterior adductor muscle remains unresolved. In this study, we investigate the myogenesis of the bivalve species Septifer virgatus to reveal the developmental origin of the larval muscles in bivalves, focusing on the anterior adductor muscle. We observed that larval retractor muscles are differentiated from the ectomesoderm in bivalves, and that the anterior adductor muscles are derived from primordial larval retractor muscles via segregation of the myoblast during the veliger larval stage. Through the comparative study of myogenesis in bivalves and its related taxa, gastropods, we found that both species possess myoblasts that emerge bilaterally and later meet dorsally. We hypothesize that these myoblasts, which are a major component of the main larval retractor in limpets, are homologous to the anterior adductor muscle in bivalves. These observations imply that the anterior adductor muscle of bivalves evolved as a novel muscle by modifying the attachment sites of an existing muscle.     

淡水贝类贝壳多层构造形成研究

对几种淡水贝（包括蚌、螺）进行形态及组织学观察，并通过实验方法重现贝壳三种物质，即：角质、棱柱质、珍珠质的生成过程，结果表明：外套膜外表皮细胞是由相同类型细胞组成，这些相同细胞在不同的作用条件下形成贝壳多层构造。     

Biotic interactions revealed by macroborings in arctic bivalve molluscs

The presence of organisms whose bodies have low preservation potential may be deduced by searching for the traces produced by them. The addition of predatory gastropods and soft-bodied epizoans to Quaternary marine faunas dominated by bivalves was facilitated by an examination of borings in bivalve shells. Borings attributed to predatory gastropods (ichnogenus Oichnus ) were observed in shells of Astarte spp., Hiatella arctica and Macoma calcarea. Astarte, Hiatella and Macoma were preyed upon in preference to other members of a diverse suspension-feeding bivalve community. Borings attributed to epizoans (ichnogenus Cautostrepsis ) were observed in bivalve shells (Astarte spp. Hiatella arctica ), calcareous algae and limestone clasts. Biotic interactions revealed by trace fossils are employed, for the first time, to reconstruct the trophic structure of arctic Quaternary marine benthic faunas. ▭ Arctic molluscs, palaeoecology, Oichnus, Caulostrepsis.     

In situ chemical speciation of sulfur in calcitic biominerals and the simple prism concept

The microstructure and composition of two mollusc shells were investigated using a combination of light microscopy, SEM, EPMA, and XANES. The shells of Pinna and Pinctada are composed of calcite prisms separated by organic walls. The prismatic units of Pinna are monocrystalline, and those of Pinctada are polycrystalline with internal organic radial membranes. High-spatial-resolution XANES maps for the different S species across adjacent prisms show that sulfate is the principal component in both the intraprismatic organic matrices and the outer membranes. Additionally, these maps confirm that the inner structures of the prismatic units are different for both genera. In many ways, the prisms of Pinna and Pinctada are different and invalidate the "simple prism" concept.     

Mid-Neolithic Exploitation of Mollusks in the Guanzhong Basin of Northwestern China: Preliminary Results

Mollusk remains are abundant in archaeological sites in the Guanzhong Basin of Northwestern China, providing good opportunities for investigations into the use of mollusks by prehistoric humans. Here we report on freshwater gastropod and bivalve mollusks covering the time interval from about 5600 to 4500 cal. yrs BP from sites of Mid-Late Neolithic age. They are identified as Cipangopaludina chinensis and Unio douglasiae, both of which are currently food for humans. The shells are well preserved and have no signs of abrasion. They are all freshwater gastropods and bivalves found in pits without water-reworked deposits and have modern representatives which can be observed in rivers, reservoirs, and paddy fields in the studied region. Mollusk shells were frequently recovered in association with mammal bones, lithic artifacts, and pottery. These lines of evidence indicate that the mollusks are the remains of prehistoric meals. The mollusk shells were likely discarded into the pits by prehistoric humans after the flesh was eaten. However, these mollusk remains may not have been staple food since they are not found in large quantities. Mollusk shell tools and ornaments are also observed. Shell tools include shell knives, shell reaphooks and arrowheads, whereas shell ornaments are composed of pendants and loops. All the shell tools and ornaments are made of bivalve mollusks and do not occur in large numbers. The finding of these freshwater mollusk remains supports the view that the middle Holocene climate in the Guanzhong Basin may have been warm and moist, which was probably favorable to freshwater mollusks growing and developing in the region.     

Proteomic Strategy for Identifying Mollusc Shell Proteins Using Mild Chemical Degradation and Trypsin Digestion of Insoluble Organic Shell Matrix: A Pilot Study on Haliotis tuberculata

A successful strategy for the identification of shell proteins is based on proteomic analyses where soluble and insoluble fractions isolated from organic shell matrix are digested with trypsin with the aim of generating peptides, which are used to identify novel shell proteins contained in databases. However, using trypsin as a sole degradative agent is limited by the enzyme's cleavage specificity and is dependent upon the occurrence of lysine and arginine in the shell protein sequence. To bypass this limitation, we investigated the ability of trifluoroacetic acid (TFA), a low-specificity chemical degradative agent, to generate clusters of analyzable peptides from organic shell matrix, suitable for database annotation. Acetic acid-insoluble fractions from Haliotis tuberculata shell were processed by trypsin followed by TFA digestion. The hydrolysates were used to annotate an expressed sequence tag library constructed from the mantle tissue of Haliotis asinina, a tropical abalone species. The characterization of sequences with repeat motifs featured in some of the shell matrix proteins benefited from TFA-induced serial cutting, which can result in peptide ladder series. Using the degradative specificities of TFA and trypsin, we were able to identify five novel shell proteins. This pilot study indicates that a mild chemical digestion of organic shell matrix combined with trypsin generates peptides suitable for proteomic analysis for better characterization of mollusc shell matrix proteins.     

Evolutionary trends within bivalve prey of chesapeake group naticid gastropods

Five genera of Miocene bivalves evolved antipredatory adaptations in response to predation by drilling naticid gastropods. I examined the evolution of two traits affecting predator‐prey interaction, prey shell thickness (TH) and internal volume (IV). Thickness controls predation costs by determining drilling time, and internal volume influences the benefit derived by the predator.

Internal volume showed no consistent pattern of temporal change among the taxa studied. IV fluctuated nondirectionally during the history of most genera, though both increasing and decreasing trends occurred within species ofAstarte. In contrast, all five genera exhibited significant thickness increases (from 8–157%) during the three‐million‐year interval. Both gradual intraspecific and interspecific directional changes occurred. Taxa with the greatest predation intensities displayed the most change, suggesting that predation selected for the thickness increases. Increased thickness apparently reduced predation; a significant negative correlation between TH and predation intensity occurred within four of the bivalve genera. Improvement of predator capabilities apparently did not keep pace with increased antipredatory morphologic adaptations over the interval studied.     

Bryozoans as taphonomic engineers,with examples from the Upper Ordovician (Katian) of Midwestern North America

A combination of encrusting calcitic bryozoans and early seafloor dissolution of aragonitic shells recorded in the Cincinnatian Series of the upper Midwest of North America allowed the preservation of abundant moulds of mollusc fossils bioimmured beneath the attachment surfaces of the bryozoans. We here call this preservational process ‘bryoimmuration’, defined as a bryozoan‐mediated subset of bioimmuration. The bryozoans moulded very fine details of the mollusc shells, usually with more accuracy than inorganic sediment moulds. Most of the bryozoans are heterotrypid trepostomes with robust low‐Mg calcite skeletons. The molluscs are primarily bivalves, gastropods, nautiloids and monoplacophorans with their originally aragonitic shells now dissolved. Many of the encrusting bryozoans are so thin and broad that they give the illusion of calcitic mollusc shells clinging to the moulds. Some molluscs in the Cincinnatian, especially monoplacophorans and epifaunal bivalves, would be poorly known if they had not been bryoimmured. Unlike internal and external moulds in sediment, bryoimmured fossils could be transported and thus record aragonitic faunas in taphonomic assemblages (e.g. storm beds) in which they would otherwise be rare or absent. In addition, bryoimmurations of aragonitic shells often reveal the ecological succession of encrustation on the shells by exposing the earliest encrusters and borings that were later overgrown. Bryoimmuration was common during the Late Ordovician because the calcite sea at the time quickly dissolved aragonitic shells on the seafloor before final burial, and large calcitic bryozoans very commonly used molluscs as substrates. Bryoimmuration is an important taphonomic process for preserving aragonitic faunas, and it reveals critical information about sclerobiont palaeoecology. Several Cincinnatian mollusc holotypes are bryoimmured specimens. Bryozoans involved in bryoimmuration enhance the preservation of aragonitic fauna and thus act as taphonomic engineers.