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.

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 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.

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.

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.

Sydney rock oyster (Saccostrea glomerata) hemocytes: morphology and function.

In this study, three major hemocyte types were identified in the Sydney rock oyster. They were characterized primarily by light and electron microscopy based on the presence or absence of granules and nucleus to cytoplasm ratios. Hemoblast-like cells were the smallest cell type 4.0+/-0.4microm and comprised 15+/-3% of the hemocyte population. They had large nuclei and scanty basic cytoplasm. This cell type also had some endoplasmic reticuli and mitochondria. The second major type were hyalinocytes. Hyalinocytes represented 46+/-6% of all hemocytes. They were large cells (7.1+/-1.0microm) that had low nucleus:cytoplasm ratios and agranular basic or acidic cytoplasm. Hyalinocytes had the ability to phagocytose yeast cells and formed the core of hemocyte aggregates associated with agglutination. Four discrete sub-populations of hyalinocytes were identified. The third major cell type were the granulocytes, comprising 38+/-1% of the hemocyte population. These cells were large (9.3+/-0.3microm) and were characterized by cytoplasm containing many acidic or basic granules. Granulocytes were more phagocytic than hyalinocytes and they formed the inner layer of hemocytes during the encapsulation of fungal hyphae. Five discrete sub-populations of granulocytes were identified based on the types of granules in their cytoplasm. Flow cytometry showed that the hemocytes of rock oysters could be divided into between two and four major cell types based on their light scattering properties. The most common of the cell types identified by flow cytometry corresponded to hyalinocytes and granulocytes. Cytochemical assays showed that most enzymes associated with immunological activity were localized in granulocytes. Their granules contained acid phosphatase, peroxidase, phenoloxidase, superoxide and melanin. Hyalinocytes were positive only for acid phosphatase. All of these observations suggest that Sydney rock oysters have a broad variety of functionally specialized hemocytes, many of which are involved in host defense.

Histology, ultrastructure, and morphogenesis of a rickettsia-like organism causing disease in the oyster, Crassostrea ariakensis Gould.

Moribund specimens of the oyster, Crassostrea ariakensis Gould, aged 2-3 years were collected from Hailing Bay in Yangxi County of Guangdong Province from February to May and November to December in the years 2001, 2002, and 2003. A massive infection by an obligate intracellular prokaryote, specifically a rickettsia-like organism (RLO), was found. Here we report investigations of this RLO in the tissues of the oyster C. ariakensis Gould and describe the histology, ultrastructure, and morphogenesis of this pathogen in C. ariakensis Gould. Light microscopic observations of stained tissues revealed cytoplasmic inclusion bodies typical of prokaryote infection in about 87% (26/30) of the oysters. Most inclusions were observed in epithelial cells and connective tissues of the gill, mantle, and digestive gland of most of the infected oysters. The shape, size, and color of inclusions from different tissues were polymorphic. Electron microscopic examination of digestive gland, gill, and mantle tissues showed that the RLOs were intracytoplasmic. RLOs were often round, dumb-bell-shaped (undergoing binary fission), or occasionally rod-shaped and ranged from approximately 0.58 to 1.20microm in size. The organisms exhibited an ultrastructure characteristic of prokaryotic bacteria-like cells, including a trilaminar cell wall, electron-dense periplasmic ribosome zone, and a DNA nucleoid. Reproductive stages, including transverse binary fission, were observed by TEM. These stages were frequently observed within membrane-bound cytoplasmic vacuoles. Hexagonal phage-like particles in the cytoplasm of RLOs were also observed.

Hemocyte-mediated shell mineralization in the eastern oyster.

The growth of molluscan shell crystals is usually thought to be initiated from solution by extracellular organic matrix. We report a class of granulocytic hemocytes that may be directly involved in shell crystal production for oysters. On the basis of scanning electron microscopy (SEM) and x-ray microanalysis, these granulocytes contain calcium carbonate crystals, and they increase in abundance relative to other hemocytes following experimentally induced shell regeneration. Hemocytes are observed at the mineralization front using vital fluorescent staining and SEM. Some cells are observed releasing crystals that are subsequently remodeled, thereby at least augmenting matrix-mediated crystal-forming processes in this system.

Microtopography and antifouling properties of the shell surface of the bivalve molluscs Mytilus galloprovincialis and Pinctada imbricata.

Biofouling rapidly covers most submerged surfaces in the marine environment. However, some marine organisms remain clean despite strong fouling pressure. Potential physical inhibitors of fouling were investigated by comparing the thickness, cover, and microtopographic structure of the periostracum of two bivalve molluscs, the blue mussel, Mytilus galloprovincialis, and the pearl oyster, Pinctada imbricata. The cover and thickness of the periostracum were measured on four size classes of each species using histological and microscopic techniques. The periostracum of M. galloprovincialis was significantly thicker than that of P. imbricata and did not differ significantly between size classes. In contrast, the periostracum of P. imbricata decreased significantly with increasing size in both thickness and cover. The microtopography of the shell surface of both species was measured using atomic force microscopy (AFM) and scanning electron microscopy (SEM), which revealed a homogeneous ridged surface for M. galloprovincialis with a uniform distance of 1-2 microns between ridges with a mean depth of 1.5 microns. P. imbricata had a heterogeneous surface structure without a repeating structural pattern. To compare the potential antifouling properties of the shell surface the four size classes of both species were tested in fourteen-week field exposure trials. M. galloprovincialis was rarely fouled over the trial period with less than 10% of M. galloprovincialis shell across all size classes being fouled. In contrast, P. imbricata had significantly higher levels of fouling. Both the proportion of P. imbricata shells fouled and the density of fouling organisms were positively correlated with the age of the shell and the amount of intact periostracum. The relationship between the shell surface microtopography and the intensity of fouling is discussed.

[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).

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.

French scallops: a new host for ostreid herpesvirus-1.

Sporadic high mortalities were reported among larval French scallops (Pecten maximus). Electron microscopy of moribund larvae revealed particles with the characteristics of a herpesvirus in association with cellular lesions. PCR and DNA sequencing showed that the virus is a variant of ostreid herpesvirus-1 that has already been described in clams and oysters. This is the first description of a herpesvirus infection of a scallop species. The virus was transmitted successfully from an extract of infected scallop larvae to uninfected scallop or oyster (Crassostrea gigas) larvae, demonstrating that it is able to infect both species. Detection of viral DNA in asymptomatic adult scallops by in situ hybridisation indicates that the herpesvirus may have been transmitted from adults to larvae. It is notable that, unlike most herpesviruses, this virus has a wide host range reflected by its ability to infect several species of marine bivalve.

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.

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.