Histological, elemental, and ultrastructural analysis of melanin in mantle of Pacific oyster (Crassostrea gigas).

Colorful shell of bivalve is mainly because of the biological pigments, of which melanin plays an important role in shell color formation. More and more studies focus on the genes function involved in melanin synthesis, but relatively few studies address the biochemical character and ultrastructure of melanin in bivalve from microscopic perspective. Here, we investigated the histological structure of mantle of Crassostrea gigas with orange shell color. Distribution of melanin in mantle was verified with histochemical staining. In addition, immunofluorescence technique showed that strongly positive signal of CgTYR was specific to the mantle margin, which is consistence with the location of brown granules in H&E staining. The further result of elementary composition of melanin displayed that metal Ca, Fe, and Zn were detected using scanning transmission electron microscope and energy dispersive spectroscopy mapping methods. Next, based on TEM observations, it was speculated that the series of cellular events leading to the formation and release of melanin. Melanocyte in the primary stage showed many mitochondria and rough endoplasmic reticulum as well as an extensive Golgi complex with numerous vesicles intermingled with melanosome. Subsequently, melanosome was expended and their hue gradually intensified, and Golgi complex and mitochondria were still observed in the cytoplasm. Finally, after melanosome was discharged into intercellular spaces, the disintegration of membranes in some cells, and severe cellular vacuolization. These data enrich the understanding of ultrastructural characteristic and formation of melanin in mantle of bivalve and pave the way for further investigating shell coloration at the cellular level.

A study of autophagy in hemocytes of the Pacific oyster, Crassostrea gigas.

Macroautophagy is a mechanism that is involved in various cellular processes, including cellular homeostasis and innate immunity. This pathway has been described in organisms ranging in complexity from yeasts to mammals, and recent results indicate that it occurs in the mantle of the Pacific oyster, Crassostrea gigas. However, the autophagy pathway has never been explored in the hemocytes of C. gigas, which are the main effectors of its immune system and thus play a key role in the defence of the Pacific oyster against pathogens. To investigate autophagy in oyster hemocytes, tools currently used to monitor this mechanism in mammals, including flow cytometry, fluorescent microscopy and transmission electron microscopy, were adapted and applied to the hemocytes of the Pacific oyster. Oysters were exposed for 24 and 48 h to either an autophagy inducer (carbamazepine, which increases the production of autophagosomes) or an autophagy inhibitor (ammonium chloride, which prevents the degradation of autophagosomes). Autophagy was monitored in fresh hemocytes withdrawn from the adductor muscles of oysters using a combination of the three aforementioned methods. We successfully labelled autophagosomes and observed them by flow cytometry and fluorescence microscopy, and then used electron microscopy to observe ultrastructural modifications related to autophagy, including the presence of double-membrane-bound vacuoles. Our results demonstrated that autophagy occurs in hemocytes of C. gigas and can be modulated by molecules known to modulate autophagy in other organisms. This study describes an integrated approach that can be applied to investigate autophagy in marine bivalves at the cellular level. Abbreviations: MAP1LC3: microtubule associated protein 1 light chain 3; MCA: multiple correspondence analysis; NH4Cl: ammonium chloride; PI: propidium iodide; TEM: transmission electron microscopy.

Gold nanoparticles ingested by oyster larvae are internalized by cells through an alimentary endocytic pathway.

The biological fate of nanoparticles (NPs) taken up by organisms from their environment is a crucial issue for assessing ecological hazard. Despite its importance, it has scarcely been addressed due to the technical difficulties of doing so in whole organism in vivo studies. Here, by using transmission electron microscopy and energy dispersive X-ray spectroscopy (TEM-EDS), we describe the key aspects that characterize the interaction between an aquatic organism of global ecological and economic importance, the early larval stage of the Japanese oyster (Crassostrea gigas), and model gold NPs dispersed in their environment. The small size of the model organism allowed for a high-throughput visualization of the subcellular distribution of NPs, providing a comprehensive and robust picture of the route of uptake, mechanism of cellular permeation, and the pathways of clearance counterbalancing bioaccumulation. We show that NPs are ingested by larvae and penetrate cells through alimentary pinocytic/phagocytic mechanisms. They undergo intracellular digestion and storage inside residual bodies, before excretion with feces or translocation to phagocytic coelomocytes of the visceral cavity for potential extrusion or further translocation. Our mechanistically-supported findings highlight the potential of oyster larvae and other organisms which feature intracellular digestion processes to be exposed to man-made NPs and thus any risks associated with their inherent toxicity.

The cytochemical and ultrastructural characteristics of phagocytes in the Pacific oyster Crassostrea gigas.

Phagocytes have been proved to play vital roles in the innate immune response. However, the cellular characteristics of phagocytes in invertebrates, especially in molluscs, remain largely unknown. In the present study, fluorescence activated cell sorting (FACS) was employed to sort the phagocytes from the non-phagocytic haemocytes of the Pacific oyster Crassostrea gigas. The cytochemical staining analysis revealed that phagocytes were positive staining for α-naphthyl acetate esterase and myeloperoxidase, while negative staining for toluidine blue and periodic acid-Schiff. The non-phagocytic haemocytes exhibited positive staining for periodic acid-Schiff, weak positive staining for toluidine blue, but negative staining for α-naphthyl acetate esterase and myeloperoxidase. In addition, phagocytes exhibited ultrastructural cellular features similar to those of macrophages, with large cell diameter, rough cell membrane and extended pseudopodia revealed by the scanning electron microscopy, while the non-phagocytic haemocytes exhibited small cell diameter, smooth cell surface and round spherical shape. Transmission electron microscopy further demonstrated that phagocytes were abundant of cytoplasmic bodies and mitochondria, while non-phagocytic haemocytes were characterized as the comparatively large cell nucleus with contorted and condensed heterochromatin adherent to the nuclear envelope. Moreover, compared with non-phagocytic haemocytes, phagocytes exhibited significantly higher levels of intracellular cytokines, including tumor necrosis factor, interferon-like protein and interleukin-17, and significantly higher abundance of lysosome and reactive oxygen species, which were of great importance to the activation of immune response and pathogen clearance. Taken together, these findings revealed the different cytochemical and ultrastructural features between phagocytes and non-phagocytic haemocytes in C. gigas, which would provide an important clue to investigate the mechanism of phagocytosis underlying the innate immune response.

Influences of developmental stages, protective additives and concentrations of cryoprotective agents on the cryopreservation of Pacific oyster (Crassostrea gigas) larvae.

BACKGROUND: The selection of the optimal developmental stage, appropriate cryoprotectants (CPAs) and their concentrations is important for successful cryopreservation. OBJECTIVE: To investigate the effects of developmental stage and CPA concentrations on the cryopreservation of Pacific oyster (Crassostrea gigas) larvae. MATERIALS AND METHODS: Seven developmental stages, various CPAs and concentrations were investigated for optimizing cryopreservation of Pacific oyster larvae. The morphologies of pre-frozen and frozen-thawed larvae were evaluated using scanning electron microscopy and transmission electron microscopy. RESULTS: The survival rate of frozen-thawed larvae increased with developmental stage; late umbo veligers exhibited a survival as high as 98.6%. The addition of 0.2 M or 0.5 M sucrose improved the survival of larvae, and 2.0 M ethylene glycol (EG) positively influenced the survival of frozen-thawed larvae. Moreover, the frozen-thawed larvae possessed irregularly arranged cilia and displayed a rough surface shell and a round-lumped cilium head. CONCLUSION: The findings indicate that the most desirable cryopreservation of Pacific oyster larvae may occur at any developmental stage except for the early trochophore stage. Sucrose at 0.2 M or 0.5 M and EG at 2.0 M or 2.5 M are appropriate cryoprotectant additives.

Larval and post-larval stages of Pacific oyster (Crassostrea gigas) are resistant to elevated CO2.

The average pH of surface oceans has decreased by 0.1 unit since industrialization and is expected to decrease by another 0.3-0.7 units before the year 2300 due to the absorption of anthropogenic CO2. This human-caused pH change is posing serious threats and challenges to the Pacific oyster (Crassostrea gigas), especially to their larval stages. Our knowledge of the effect of reduced pH on C. gigas larvae presently relies presumptively on four short-term (<4 days) survival and growth studies. Using multiple physiological measurements and life stages, the effects of long-term (40 days) exposure to pH 8.1, 7.7 and 7.4 on larval shell growth, metamorphosis, respiration and filtration rates at the time of metamorphosis, along with the juvenile shell growth and structure of the C. gigas, were examined in this study. The mean survival and growth rates were not affected by pH. The metabolic, feeding and metamorphosis rates of pediveliger larvae were similar, between pH 8.1 and 7.7. The pediveligers at pH 7.4 showed reduced weight-specific metabolic and filtration rates, yet were able to sustain a more rapid post-settlement growth rate. However, no evidence suggested that low pH treatments resulted in alterations to the shell ultrastructures (SEM images) or elemental compositions (i.e., Mg/Ca and Sr/Ca ratios). Thus, larval and post-larval forms of the C. gigas in the Yellow Sea are probably resistant to elevated CO2 and decreased near-future pH scenarios. The pre-adapted ability to resist a wide range of decreased pH may provide C. gigas with the necessary tolerance to withstand rapid pH changes over the coming century.

Shell thickening and chambering in the oyster Crassostrea gigas: natural and anthropogenic influence of tributyltin contamination.

Abnormal thickening and chambering in Crassostrea gigas oysters have been adopted for many years as bioindicators of available tributyltin (TBT) in coastal waters. Nevertheless, since natural causes can also induce the formation of multiple chambers, a field study and laboratory experimentation has been conducted with 72 examples of C. gigas in successive culture media. This work has enabled differences to be established between natural fine sediment-induced characteristics and the influence of TBT on the shells. External shell deformities have been assessed using three biometric indices, shell thickness index, weight index and volume index. Internal differences have been observed in longitudinal sections of the shell: retraction of growth, stagnation of the adductor muscle scar and thinning of the chambers in the TBT-polluted shell secretion. A new index, the opening chambers index, has been proposed, with a value of less than 1 in the TBT-polluted environment and greater than 1 in shells secreted in an unpolluted production site. These conclusions should be borne in mind when C. gigas is used in biomonitoring programmes.

Intragonadal somatic cells (ISCs) in the male oyster Crassostrea gigas: morphology and contribution in germinal epithelium structure.

The ultrastructure of somatic cells present in gonadal tubules in male oyster Crassostrea gigas was investigated. These cells, named Intragonadal Somatic Cells (ISCs) have a great role in the organization of the germinal epithelium in the gonad. Immunological detection of α-tubulin tyrosine illustrates their association in columns from the basis to the lumen of the tubule, stabilized by numerous adhesive junctions. This somatic intragonadal organization delimited some different groups of germ cells along the tubule walls. In early stages of gonad development, numerous phagolysosomes were observed in the cytoplasm of ISCs indicating that these cells have in this species an essential role in the removal of waste sperm in the tubules. Variations of lipids droplets content in the cytoplasm of ISCs were also noticed along the spermatogenesis course. ISCs also present some mitochondria with tubullo-lamellar cristae.

Mechanical characteristics and morphological effect of complex crossed structure in biomaterials: fracture mechanics and microstructure of chalky layer in oyster shell.

The complex crossed structures with a polymorph of calcite, termed a chalky layer, which make up much of the shell of an oyster, are composed of flames and leaflets. Two layers, folia and the chalky layer in the giant Pacific oyster (Crassostrea gigas) were examined using SEM (scanning electron microscope), micro-area-XRD (X-ray diffraction) and FT-IR (Fourier transform infrared spectrometer) to determine their morphologies and component characteristics. The chalky layer was also tested using microindentation to assess its mechanical properties, and a microcrack was generated to study the fracture mechanism of the chalky layer. From an analysis of the secondary protein structure, it was shown that the ordered structures of the two layers, α-helix and ß-structure, are similar but that the unordered structures are different. Moreover, the foliated rods at the interface of the chalky layer play a key role in the crystal growth of the chalky layers. Comparing the morphology and the preferred orientation of foliated laths, the advantages of the relatively high density and low hardness of the chalky layer have interesting implications regarding the development of sophisticated complex composites.

Morphology of the larval shell of three oyster species of the genus Crassostrea Sacco, 1897 (Bivalvia: Ostreidae).

In this study we describe the morphology of the larval shell of three oyster species of Crassostrea genus. Two species, C. rhizophorae and C. brasiliana, are native to the Brazilian coast, and C. gigas is an introduced species. Samples of laboratory reared larvae, obtained through artificial fertilisation, were collected at intervals during the cultivation process for analysis using Scanning Electron Microscopy (SEM). Prodissoconch morphology was observed in relation to the presence, position, form and number of teeth in the three larval stages: D-shaped larva, umbo larva and pediveliger. Characteristic of D-shaped larvae of C. rhizophorae was the total absence of teeth in the provinculum area while C. brasiliana and C. gigas had two anterior and two posterior teeth in each valve. In the umbo larval phase, the three species had the same number of teeth in each valve: two posterior and two anterior teeth in the right valve and three posterior and three anterior in the left valve. In the pediveliger stage the three species could be differentiated by the number of anterior teeth of the right valve: C. rhizophorae had two teeth, C. brasiliana one tooth and C. gigas three teeth.

Morphology of the larval shell of three oyster species of the genus Crassostrea Sacco, 1897 (Bivalvia: Ostreidae) / Morfologia de conchas larvais de três espécies de ostras do gênero Crassostrea Sacco, 1897 (Bivalvia: Ostreidae)

In this study we describe the morphology of the larval shell of three oyster species of Crassostrea genus. Two species, C. rhizophorae and C. brasiliana, are native to the Brazilian coast, and C. gigas is an introduced species. Samples of laboratory reared larvae, obtained through artificial fertilisation, were collected at intervals during the cultivation process for analysis using Scanning Electron Microscopy (SEM). Prodissoconch morphology was observed in relation to the presence, position, form and number of teeth in the three larval stages: D-shaped larva, umbo larva and pediveliger. Characteristic of D-shaped larvae of C. rhizophorae was the total absence of teeth in the provinculum area while C. brasiliana and C. gigas had two anterior and two posterior teeth in each valve. In the umbo larval phase, the three species had the same number of teeth in each valve: two posterior and two anterior teeth in the right valve and three posterior and three anterior in the left valve. In the pediveliger stage the three species could be differentiated by the number of anterior teeth of the right valve: C. rhizophorae had two teeth, C. brasiliana one tooth and C. gigas three teeth.

Neste estudo, foi descrita a morfologia das conchas larvais de três espécies de ostras do gênero Crassostrea. Duas espécies, C. rhizophorae e C. brasiliana, são nativas da costa brasileira e C. gigas é uma espécie introduzida. Amostras de larvas produzidas em laboratório através de fertilização artificial foram coletadas em intervalos durante o processo de cultivo para análises dos número de dentes nos três estágios larvais: larva D, umbo e pediveliger. As larvas D de C. rhizophorae caracterizaram-se pela total ausência de dentes na área do provinculum, enquanto C. brasiliana e C. gigas apresentaram dois dentes anteriores e dois posteriores em cada valva. Na fase umbo, as três espécies apresentaram o mesmo número de dentes em cada valva: dois dentes anteriores e dois posteriores na valva direita; e três dentes posteriores e três anteriores na valva esquerda. Na fase pediveliger, as três espécies se diferenciaram pelo número de dentes anteriores na valva direita: C. rhizophorae apresentou dois dentes; C. brasiliana, um dente; e C. gigas, três dentes.

Microtexture of larval shell of oyster, Crassostrea nippona: a FIB-TEM study.

The initial formation and subsequent development of larval shells in marine bivalve, Crassostrea nippona were investigated using the FIB-TEM technique. Fourteen hours after fertilization (the trochophore stage), larvae form an incipient shell of 100-150nm thick with a columnar contrast. Selected-area electron diffraction analysis showed a single-crystal aragonite pattern with the c-axis perpendicular to the shell surface. Plan-view TEM analysis suggested that the shell contains high density of {110} twins, which are the origin of the columnar contrast in the cross-sectional images. 72h after fertilization (the veliger stage), the shell grows up to 1.2-1.4mum thick accompanying an additional granular layer between the preexisting layer and embryo to form a distinctive two-layer structure. The granular layer is also composed of aragonite crystals sharing their c-axes perpendicular to the shell surface, but the crystals are arranged with a flexible rotation around the c-axes and not restricted solely to the {110} twin relation. No evidence to suggest the existence of amorphous calcium carbonate (ACC) was found through the observation. The well-regulated crystallographic properties found in the present sample imply initial shell formation probably via a direct deposition of crystalline aragonite.

Detection of phenoloxidase activity in early stages of the Pacific oyster Crassostrea gigas (Thunberg).

The presence of phenoloxidase (PO) activity was detected in different developmental stages of the Pacific oyster, Crassostrea gigas. A significant reduction in PO activity was observed from the 6h embryo stage to the day 11 larvae by spectrophotometry. A progressive increase was also observed from the day 13 larvae right through to the juvenile stage. The microscopy studies with '6h embryo' and adult samples confirmed the presence of PO activity. Various modulators of PO activity were used to study the triggering of pro-phenoloxidase (proPO) activating system of C. gigas but also to confirm the exact nature of the monitored activity. The enzyme activation mechanisms appear to differ with the developmental stage: bacterial lipopolysaccharides constitute an early elicitor of the proPO-PO system, whereas a purified trypsin triggers proPO-PO system in C. gigas spat. Phenoloxidase activity was totally suppressed by PO-specific inhibitors such as beta-2-mercaptoethanol, sodium diethyldithiocarbonate and tropolone. This study demonstrated the selective response of PO-like activity by different elicitors and suggested that proPO-PO activating system, which is supposed to play an important function in non-self recognition and host immune reactions in oyster, is expressed early in the Pacific oyster, C. gigas.

TEM immunocytochemistry of a 48 kDa MW organic matrix phosphoprotein produced in the mantle epithelial cells of the Eastern oyster (Crassostrea virginica).

Immunohistochemical TEM of Eastern oyster (Crassostrea virginica) mantle epithelial cells using a polyclonal antibody to a gel purified 48 kDa MW oyster shell phosphoprotein revealed that it is phosphorylated in the Golgi, packaged into secretory vesicles and subsequently exocytosed across the apical membrane of specialized cells. These phosphoprotein producing cells are concentrated along the mantle side facing the shell, in the region of the outer mantle lobe. A layer of calcium enriched immuno-reactive mucous is associated with the apical microvilli of these cells. The 48 kDa phosphoprotein forms a component of the fibrous organic matrix and appears to be involved in calcium supply thus enabling crystal growth at the mineralization front.

Multibiomarker assessment of three Brazilian estuaries using oysters as bioindicators.

Oysters have been largely employed as bioindicators of environmental quality in biomonitoring studies. Crassostrea rhizophorae was selected to evaluate the health status of three estuarine areas impacted by anthropogenic activities along the Brazilian coast, in three estuarine complexes, ranging in latitude from 7 to 25 degrees S. In each estuary three sites were sampled in Winter and in Summer: a site considered as reference, and two sites next to contamination sources. Condition index was similar at all sites and estuaries, with the highest values found for Itamaracá oysters in Summer. Necrosis, hyperplasia, mucocyte hypertrophy and fusion of ordinary filaments were the main histopathological lesions observed. Muscle cholinesterase activity was overall similar, but with a strong seasonal effect. Inhibition or activation of branchial total ATPase and Na,K-ATPase activities at the contaminated sites was observed. The health status of these estuarine areas is quite similar, and the combined use of biomarkers is recommended.

The correlation between organic matrices and biominerals (myostracal prism and folia) of the adult oyster shell, Crassostrea gigas.

The organic membrane at the interface between the myostracum (aragonite) and folia (calcite) was obtained and identified as a chitin-like macromolecule after chemical treatment. It was confirmed that the organic membrane was faced to the crystal (001) plane of the myostracum and folia. The secondary structures in soluble protein of the myostracum and the folia of oyster shell were identified as a great quantity of beta-structure. The acidic amino acids, Asp and Glu, to play a role as templates on shell formation were confirmed in the myostracum and the folia.

Fixation methods can produce misleading artifacts in sperm cell ultrastructure of diploid and tetraploid Pacific oysters, Crassostrea gigas.

Spermatozoa from diploid and tetraploid Pacific oysters (Crassostrea gigas) were examined after anisotonic fixation. Morphological anomalies, such as membrane rupture, detached tails, and the formation of tail vesicles (typically associated with damage attributable to procedures such as cryopreservation) were observed; the Mantel-Haenszel Chi-square test indicated a strong association between the anomalies and fixative osmolality (P<0.001). The present study also indicated that media in a range of 800 to 1,086 mOsm/kg could be assumed to be functionally isotonic to Pacific oysters, and osmolalities below or above this caused severe cell damage. For example, the maximum volume of flagella obtained after hypotonic fixation was approximately twice the volume of the flagella in isotonic fixation. Sperm cell flagellar volumes after hypertonic fixation (1,110 mOsm/kg) were 32% smaller than those in isotonic fixation, and sperm heads were 25% smaller. Although the damage associated with anisotonic fixation was evident in all parts of the sperm cells, the most vulnerable locations were the plasma membrane and flagellum motor apparatus. The formation of tail vesicles after hypotonic fixation was also examined. Because of water uptake, oyster sperm became swollen in hypotonic fixative, and bending or coiling of the axoneme within the tail vesicles led to the appearance of multiple axonemal structures in cross sections when observed by transmission electron microscopy. This phenomenon might be generally misinterpreted as the presence of double tails. This and other fixation artifacts can lead to the misinterpretation of damage caused by cryopreservation in ultrastructure studies of sperm of aquatic species, especially those in marine species.

Effects of acclimation temperature and cadmium exposure on cellular energy budgets in the marine mollusk Crassostrea virginica: linking cellular and mitochondrial responses.

In order to understand the role of metabolic regulation in environmental stress tolerance, a comprehensive analysis of demand-side effects (i.e. changes in energy demands for basal maintenance) and supply-side effects (i.e. metabolic capacity to provide ATP to cover the energy demand) of environmental stressors is required. We have studied the effects of temperature (12, 20 and 28 degrees C) and exposure to a trace metal, cadmium (50 microg l(-1)), on the cellular energy budget of a model marine poikilotherm, Crassostrea virginica (eastern oysters), using oxygen demand for ATP turnover, protein synthesis, mitochondrial proton leak and non-mitochondrial respiration in isolated gill and hepatopancreas cells as demand-side endpoints and mitochondrial oxidation capacity, abundance and fractional volume as supply-side endpoints. Cadmium exposure and high acclimation temperatures resulted in a strong increase of oxygen demand in gill and hepatopancreas cells of oysters. Cd-induced increases in cellular energy demand were significant at 12 and 20 degrees C but not at 28 degrees C, possibly indicating a metabolic capacity limitation at the highest temperature. Elevated cellular demand in cells from Cd-exposed oysters was associated with a 2-6-fold increase in protein synthesis and, at cold acclimation temperatures, with a 1.5-fold elevated mitochondrial proton leak. Cellular aerobic capacity, as indicated by mitochondrial oxidation capacity, abundance and volume, did not increase in parallel to compensate for the elevated energy demand. Mitochondrial oxidation capacity was reduced in 28 degrees C-acclimated oysters, and mitochondrial abundance decreased in Cd-exposed oysters, with a stronger decrease (by 20-24%) in warm-acclimated oysters compared with cold-acclimated ones (by 8-13%). These data provide a mechanistic basis for synergism between temperature and cadmium stress on metabolism of marine poikilotherms. Exposure to combined temperature and cadmium stress may result in a strong energy deficiency due to the elevated energy demand on one hand and a reduced mitochondrial capacity to cover this demand on the other hand, which may have important implications for surviving seasonally and/or globally elevated temperatures in polluted estuaries.