The shell matrix of the european thorny oyster, Spondylus gaederopus: microstructural and molecular characterization.

Molluscs, the largest marine phylum, display extraordinary shell diversity and sophisticated biomineral architectures. However, mineral-associated biomolecules involved in biomineralization are still poorly characterised. We report the first comprehensive structural and biomolecular study of Spondylus gaederopus, a pectinoid bivalve with a peculiar shell texture. Used since prehistoric times, this is the best-known shell of Europe's cultural heritage. We find that Spondylus microstructure is very poor in mineral-bound organics, which are mostly intercrystalline and concentrated at the interface between structural layers. Using high-resolution liquid chromatography tandem mass spectrometry (LC-MS/MS) we characterized several shell protein fractions, isolated following different bleaching treatments. Several peptides were identified as well as six shell proteins, which display features and domains typically found in biomineralized tissues, including the prevalence of intrinsically disordered regions. It is very likely that these sequences only partially represent the full proteome of Spondylus, considering the lack of genomics data for this genus and the fact that most of the reconstructed peptides do not match with any known shell proteins, representing consequently lineage-specific sequences. This work sheds light onto the shell matrix involved in the biomineralization in spondylids. Our proteomics data suggest that Spondylus has evolved a shell-forming toolkit, distinct from that of other better studied pectinoids - fine-tuned to produce shell structures with high mechanical properties, while limited in organic content. This study therefore represents an important milestone for future studies on biomineralized skeletons and provides the first reference dataset for forthcoming molecular studies of Spondylus archaeological artifacts.

Bending and branching of calcite laths in the foliated microstructure of pectinoidean bivalves occurs at coherent crystal lattice orientation.

Foliated calcite is widely employed by some important pteriomorph bivalve groups as a construction material. It is made from calcite laths, which are inclined at a low angle to the internal shell surface, although their arrangement is different among the different groups. They are strictly ordered into folia in the anomiids, fully independent in scallops, and display an intermediate arrangement in oysters. Pectinids have particularly narrow laths characterized by their ability to change their growth direction by bending or winding, as well as to bifurcate and polyfurcate. Electron backscatter analysis indicates that the c-axes of laths are at a high, though variable, angle to the growth direction, and that the laths grow preferentially along the projection of an intermediate axis between two a-axes, although they can grow in any intermediate direction. Their main surfaces are not particular crystallographic faces. Analyses done directly on the lath surfaces demonstrate that, during the bending/branching events, all crystallographic axes remain invariant. The growth flexibility of pectinid laths makes them an excellent space-filling material, well suited to level off small irregularities of the shell growth surface. We hypothesize that the exceptional ability of laths to change their direction may be promoted by the mode of growth of biogenic calcite, from a precursor liquid phase induced by organic molecules.

Structure and distribution of chalky deposits in the Pacific oyster using x-ray computed tomography (CT).

Oysters are unusual among bivalves in that they possess chambers, often filled with soft, chalky calcite, that are irregularly scattered throughout the shell. Because the function of these so-called chalky deposits is still unclear, evaluating the growth and distribution of chalk is important for elucidating the ecological function of this unique shell trait. Specimens of the Pacific oyster Magallana gigas, an oyster well known for chalk expression, were grown in Bodega Harbor, Bodega Bay, CA. At the end of an 11 month growing period, specimens were culled and selected animals were submitted for x-ray computed-tomography imaging. Three-dimensional reconstructions of oyster shells were used to assess the overall distribution of chalk, and also to better understand the relationship between chalk and other structures within the shell. Results indicate that chalky deposits underly sculptural features on the shell exterior, such as external ridges and changes in growth direction, and also that there is a relationship between chalk formation and oyster processes of cementation. Overall, chalk is useful for a cementing lifestyle because it enables morphological plasticity needed to conform to irregular substrates, but also acts as a cheap building material to facilitate rapid growth.

Chlamydiae (with phages), mycoplasmas, and richettsiae in Chesapeake Bay bivalves.

Intracytoplasmic chlamydia-like organisms, some with phages, rickettsia-like organisms, and mycoplasma-like organisms have been found in clams and oysters from the Chesapeake Bay area by electron microscopy. None of these organisms have been previously detected in mollusks, nor have phages been previously observed in Chlamydia sp.

Early development of the Japanese spiny oyster (Saccostrea kegaki): characterization of some genetic markers.

The phylum Mollusca is one of the major groups of Lophotrochozoa. Although mollusks exhibit great morphological diversity, only a few comparative embryological studies have been performed on this group. In the present study, to begin understanding the molecular development of the diverse morphology among mollusks, we observed early embryogenesis in a bivalve, the Japanese spiny oyster, Saccostrea kegaki. Although several studies have begun to reveal the genetic machinery for early development in gastropods, very little molecular information is available on bivalve embryogenesis. Thus, as a step toward identifying tissue-specific gene markers, we sequenced about 100 cDNA clones picked randomly from a gastrula-stage cDNA library. This basic information on bivalve embryology will be useful for further studies on the development and evolution of mollusks.

Nano-structured biogenic calcite: a thermal and chemical approach to folia in oyster shell.

The thin sheets of calcite in oyster shell in Crassostrea gigas are termed folia and comprise much of the oyster shell. The folia are covered by a layer of discrete globules that has been proposed to consist of aggregations of an organic matrix and minerals. A continuous organic framework divides each tablet into nanograins. Their shape is globular with a mean extension from 30 to 40 nm. Chemical and thermal treatments to correlate between the organic matrix and the minerals are considered using spectrometers, thermal analyzers, and electron microscopes. After treatment, the nanograins of the foliar and organic matrix are clearly identified. The organic matrix plays a key role in the thermal stability and material properties of this biological composite. From analysis of the FT-IR results, it is identified that the organic matrix in folia is composed of proteins and polysaccharides.

Structure and crystallography of foliated and chalk shell microstructures of the oyster Magallana: the same materials grown under different conditions.

Oyster shells are mainly composed of layers of foliated microstructure and lenses of chalk, a highly porous, apparently poorly organized and mechanically weak material. We performed a structural and crystallographic study of both materials, paying attention to the transitions between them. The morphology and crystallography of the laths comprising both microstructures are similar. The main differences were, in general, crystallographic orientation and texture. Whereas the foliated microstructure has a moderate sheet texture, with a defined 001 maximum, the chalk has a much weaker sheet texture, with a defined 011 maximum. This is striking because of the much more disorganized aspect of the chalk. We hypothesize that part of the unanticipated order is inherited from the foliated microstructure by means of, possibly, [Formula: see text] twinning. Growth line distribution suggests that during chalk formation, the mantle separates from the previous shell several times faster than for the foliated material. A shortage of structural material causes the chalk to become highly porous and allows crystals to reorient at a high angle to the mantle surface, with which they continue to keep contact. In conclusion, both materials are structurally similar and the differences in orientation and aspect simply result from differences in growth conditions.

Spermatogenesis in the rock oyster, Saccostrea forskali (Gmelin, 1791).

Morphology of the differentiating spermatogenic cells of the rock oyster Saccostrea forskali (Bivalve: Ostreidae) was investigated by light and transmission electron microscopy. The testis is formed by several branching acini containing developing spermatogenic cells, classified into 7 stages based on nuclear characteristics, patterns of chromatin condensation and cytoplasmic contents. The spermatogonium is characterized by a euchromatic nucleus with a prominent nucleolus. The cytoplasm contains several round granulo-fibrillar dense bodies surrounded by numerous mitochondria. The round nucleus of the primary spermatocyte contains patches of electron-dense heterochromatin, numerous proacrosomal vesicles, ribosomes and mitochondria. The secondary spermatocytes contain a reticulated chromatin pattern and reduced number of proacrosomal vesicles. The early spermatids contain a small amount of euchromatin among dense patches of heterochromatin. A large single acrosomal vesicle is located in the posterior part of the cell. The middle spermatid is characterized by the migration of an acrosomal vesicle to the anterior part of the nucleus. The late spermatids contain highly condensed heterochromatin blocks and the acrosomal vesicle becomes cup-shaped and invaginated at the basal part. The spermatozoon contains a barrel-shaped head covered with the cup-like acrosome. At this stage, the subacrosomal space contains an axial rod in subacrosomal materials. Three to four transverse bands appear at the anterior region of the acrosome. The middle piece consists of spherical mitochondria surrounding the proximal and distal centrioles. The flagellum consists of 9+2 axonemal microtubule doublets surrounded by the plasma membrane. Our electron microscopic study of spermatogenesis in the S. forskali provides important new information on the mechanism of development of spermatogenesis of this species.

Structural and compositional characterization of the adhesive produced by reef building oysters.

Oysters have an impressive ability to overcome difficulties of life within the stressful intertidal zone. These shellfish produce an adhesive for attaching to each other and building protective reef communities. With their reefs often exceeding kilometers in length, oysters play a major role in balancing the health of coastal marine ecosystems. Few details are available to describe oyster adhesive composition or structure. Here several characterization methods were applied to describe the nature of this material. Microscopy studies indicated that the glue is comprised of organic fiber-like and sheet-like structures surrounded by an inorganic matrix. Phospholipids, cross-linking chemistry, and conjugated organics were found to differentiate this adhesive from the shell. Symbiosis in material synthesis could also be present, with oysters incorporating bacterial polysaccharides into their adhesive. Oyster glue shows that an organic-inorganic composite material can provide adhesion, a property especially important when constructing a marine ecosystem.

In vitro interaction of Perkinsus marinus merozoites with eastern and Pacific oyster hemocytes.

This study compared hemocyte responses of eastern and Pacific oysters to Perkinsus marinus, in vitro. Except for the percentage of hemocytes associated with P. marinus there was little or no significant difference between eastern and Pacific oysters with regard to their hemocytic response to P. marinus. In phagocytosis assays, merozoites were bound to all hemocyte types but in unequal proportions, unlike zymosan which was found predominantly associated with granulocytes. The number of merozoites enlarging in Ray's fluid thioglycollate medium after incubation with hemocytes in plasma for one day was significantly lower than after incubation in plasma alone in both oyster species. Electron microscopy or merozoites indicated that the parasites were rapidly phagocytosed and that some of the merozoites showed signs of degeneration in less than 12 h. The results suggest that limited intracellular killing of P. marinus had occurred, but was probably not mediated by oxygen metabolites, since no increase in chemiluminescence was observed when hemocytes of either eastern or Pacific oysters were exposed to merozoites.

Herpes-like virus detection in infected Crassostrea gigas spat using DIG-labelled probes.

An in situ hybridization protocol for detecting the herpes-like virus which infects French Pacific oysters, Crassostrea gigas, was developed. Two DNA probes were synthesized by incorporation of digoxigenin 11-dUTP during PCR. Two oyster herpes-like virus specific primer pairs, A5/A6 and C1/C6, were used. Both DIG-labelled probes were able to detect 50 pg of herpes-like virus PCR amplified DNA in Southern blot hybridizations. The probes hybridized with viral DNA in paraffin sections of infected C. gigas spat. No non-specific binding was observed. The ability of the defined in situ hybridization technique to diagnose herpes-like virus infections in oysters was compared with light and transmission electron microscopy techniques in infected and non-infected spat. In situ hybridization assays were also conducted on paraffin sections to determine virus distribution within the host and to study the pathogenesis infection. In situ hybridization confirmed that the expression pattern of the herpes-like virus was restricted to connective tissues as described previously by light and transmission electron microscopy. However, this technique also allowed the detection of viral DNA in the oyster nervous system. Some labelled cells were observed in the visceral ganglion of infected oyster spat.

Influences of subzero thermal acclimation on mitochondrial membrane composition of temperate zone marine bivalve mollusks.

The phospholipid and phospholipid fatty acid composition of gill mitochondrial membranes from two temperate zone marine bivalve mollusks, the quahog, Mercenaria mercenaria, and the American oyster, Crassostrea virginica, were examined after acclimation to 12 and -1 degree C. Cardiolipin (CL) was the only phospholipid with proportions altered upon acclimation to -1 degree C, increasing 188% in the mitochondrial membranes of M. mercenaria. Although the ratio of bilayer stabilizing to destabilizing lipids is frequently associated with cold acclimation in ectothermic species, no change was found in this ratio in either of the species. Polyunsaturated fatty acids (PUFA) were found only to increase in C. virginica with cold acclimation, with total n-3 PUFA increasing in the phospholipid phosphatidylethanolamine, total n-6 PUFA increasing in CL, and total PUFA increasing in phosphatidylinositol. Monounsaturated fatty acids, not PUFA, were found to have increased in M. mercenaria, with 18:1 n-9 increasing by 150% in CL, and 20:1 increasing in both CL and phosphatidylcholine, by 146 and 192%, respectively. These manipulations of membrane phospholipid and fatty acid composition may represent an attempt by these species to help maintain membrane function at low temperatures.

Nucleation and growth of calcite on native versus pyrolyzed oyster shell folia.

The thin sheets of calcite, termed folia, that make up much of the shell of an oyster are covered by a layer of discrete globules that has been proposed to consist of agglomerations of protein and mineral. Foliar fragments, treated at 475 degrees C for 36 h to remove organic matter, were imaged by atomic force microscopy (AFM) as crystals grew on the foliar surfaces in artificial seawater at calcite supersaturations up to 52-fold. Crystals were also viewed later by scanning electron microscopy. After pyrolysis, the foliar globules persisted only as fragile remnants that were quickly washed away during AFM imaging, revealing an underlying morphology on the foliar laths of a tightly packed continuum of nanometer-scale protrusions. At intermediate supersaturations, crystal formation was seen immediately almost everywhere on these surfaces, each crystal having the same distinctive shape and orientation, even at the outset with crystals as small as a few nanometers. In contrast, nucleation did not occur readily on non-pyrolyzed foliar surfaces, and the crystals that did grow, although slowly at intermediate supersaturations, had irregular shapes. Possible crystallographic features of foliar laths are considered on the basis of the morphology of ectopic crystals and the atomic patterns of various surfaces. A model for foliar lath formation is presented that includes cycles of pulsed secretion of shell protein, removal of the protein from the mineralizing solution upon binding to mineral, and mineral growth at relatively high supersaturation over a time frame of about 1 h for each turn of the cycle.

[Gonadic cycle of the American oyster Crassostrea virginica (Lamellibranchia: Ostreidae) in Mecoacán, Tabasco, México]. / Ciclo gonádico del ostión americano Crassostrea virginica (Lamellibranchia: Ostreidae) en Mecoacán, Tabasco, México.

The American oyster Crassostrea virginica is exploited along Gulf of Mexico. This resource represents a job source and incomes for fishermen. In Mexico the production is supported by Tabasco state, the first producer. However, the mexican landings of this bivalve had been dropped about 40% last ten years. By 1999, Tabasco presents a unique ban season fishery of oysters. This season was based in evaluation of gonadal development by visual observations of color and texture, larvae and seeds abundance. In 2000, the government set up two ban seasons in terms of evaluation of populations every year, without gonadal analysis. In general, exists different spawning seasons for American oyster, in accordance with the environmental conditions. It is necessary to establish a ban season according to reproductive cycle. This study presents the gonadic cycle for an oyster population of Mecoacin lagoon along a year. It was defined five phases of the gonad development: resting, gametogenesis, mature or ripe, spawn and post spawn. Gametogenesis is present all the year, except December. The spawning activity was detected all year, except July and August. The ripe phase presented maximum values in August and December. It was proposed a modification of the ban seasons from April 15 - May 30, and September 15 - October 30 to March 15 - May 15 and September 1 - October 30, respectively, according to gonadic cycle obtained in this work. This modification would to avoid capture mature organisms (ready to spawn).

Comparative ultrastructural study of spermatozoa in some oyster species from the Asian-Pacific Coast.

Sperm organization in the oysters Crassostrea gigas, Crassostrea nippona, Crassostrea cf. rivularis and Saccostrea cf. mordax inhabiting Asian Pacific coast was studied. The spermatozoa of all studied species had a number of common morphological characters such as a cup-like acrosome with heterogeneous matrix on its top, an axial rod in the subacrosomal space, a barrel-shaped nucleus, four mitochondria in the midpiece, pericentriolar complexes, and a 9+2-organized flagellum. The spermatozoa of C. cf. rivularis differed from the other species by having cytoplasm processes in the midpiece region. Such structures have never been described in the Ostreidae. Additionally, each species could be identified by the shape and size of sperm compartments (acrosome, nucleus, anterior nuclear fossa). The most significant interspecific difference was found in the size of an anterior nuclear fossa. The smallest anterior nuclear fossa was found in C. cf. rivularis (about 0.24 µm in length reaching about 22% of the nuclear length) while the biggest in C. gigas from the Sea of Japan (about 0.53 µm in length reaching about 46% of the nuclear length). The spermatozoa of C. gigas collected from the Sea of Japan and Taiwan Strait differed significantly in almost all the studied parameters. Since sperm morphology has been successfully used for species differentiation, this suggests the existence of two species rather than two populations. The data obtained indicate the species-specific difference in the sperm ultrastructure within the Ostreidae, which may be identified both ultrastructurally and morphometrically.

New approach for fabrication of folded-structure SiO2 using oyster shell.

Inspired by the phenomenological differences of layers in oyster shell and the morphological mimicry of SiO(2) thin film, a folded-structure SiO(2) was created by simple spray deposition system. The folded-structure SiO(2) was analyzed by scanning electron microscopy, energy dispersive spectrometer and microindentation. At the molecular level, the chalky and the folia were assembled and determined through biomineralization based on the differences of soluble protein in layers. At the macro-scale, the granular SiO(2) particles deposited at the surface of shell layers or Ca(OH)(2) and grew into thin film, thus leading to mimic the morphology of substrate.

Bacterial diversity and structural changes of oyster shell during 1-year storage.

We examined the biodiversity of bacteria associated with oyster-shell waste during a 1-year storage period using 16S ribosomal DNA analysis. Temperature variation and structural changes of oyster shell were observed during storage. Initial and final temperatures were at 16-17 degrees C, but a high temperature of about 60 degrees C was recorded after approximately 6 months of storage. The crystal structure and nanograin of the oyster shell surface were sharp and large in size initially and became gradually blunter and smaller over time. Phylogenetic analysis revealed that Firmicutes were dominant in the oyster-shell waste initially, during the high-temperature stage, and after 1 year of storage (making up >65% of the biodiversity at all three sampling times). Bacillus licheniformis was presumed as the predominate Firmicutes present. These bacteria are likely to have important roles in the biodegradation of oyster shell.