Identification of three cell populations from the shell gland of a bivalve mollusc.

The molluscan larval shell formation is a complicated process. There is evidence that the mantle of the primary larva (trochophore) contains functionally different cell populations with distinct gene expression profiles. However, it remains unclear how these cells are specified. In the present study, we identified three cell populations from the shell gland in earlier stages (gastrula) from the bivalve mollusc Crassostrea gigas. These cell populations were determined by analyzing the co-expression relationships among six potential shell formation (pSF) genes using two-color hybridization. The three cell populations, which we designated as SGCPs (shell gland cell populations), formed a concentric-circle pattern from outside to inside of the shell gland. SGCP I was located in the outer edge of the shell gland and the cells expressed pax2/5/8, gata2/3, and bmp2/4. SGCP II was located more internally and the cells expressed two engrailed genes. The last population, SGCP III, was located in the central region of the shell gland and the cells expressed lox4. Determination of the gene expression profiles of SGCPs would help trace their origins and fates and elucidate how these cell populations are specified. Moreover, potential roles of the SGCPs, e.g., development of sensory cells and shell biogenesis, are suggested. Our results reveal the internal organization of the embryonic shell gland at the molecular level and add to the knowledge of larval shell formation.

CgAATase with specific expression pattern can be used as a potential surface marker for oyster granulocytes.

Granulocytes are known as the main immunocompetent hemocytes that play important roles in the immune defense of oyster Crassostrea gigas. In the present study, an alcohol acyltransferase (designed as CgAATase) with specific expression pattern was identified from oyster C. gigas, and it could be employed as a potential marker for the isolation of oyster granulocytes. The open reading frame (ORF) of CgAATase was of 1431 bp, encoding a peptide of 476 amino acids with a typically conserved AATase domain. The mRNA transcripts of CgAATase were highest expressed in hemocytes, lower expressed in hepatopancreas, mantle, gonad, gill, ganglion, adductor muscle, and labial palp. The mRNA expression level of CgAATase in hemocytes was significantly up-regulated at 3-12 h and reached the highest level (27.40-fold compared to control group, p < 0.05) at 6 h after Vibrio splendidus stimulation. The total hemocytes were sorted as granulocytes, semi-granulocytes and agranulocytes by Percoll® density gradient centrifugation. CgAATase transcripts were dominantly observed in granulocytes, which was 8.26-fold (p < 0.05) and 2.80-fold (p < 0.05) of that in agranulocytes and semi-granulocytes, respectively. The monoclonal antibody against CgAATase was produced and employed for the isolation of granulocytes with the immunomagnetic bead. CgAATase protein was mainly detected on the cytomembrane of granulocytes. About 85.7 ±â€¯4.60% of the granulocytes were positive for CgAATase and they could be successfully separated by flow cytometry with immunomagnetic bead coated with anti-CgAATase monoclonal antibody, and 97.7 ±â€¯1.01% of the rest hemocytes (agranulocytes and semi-granulocytes) were negative for CgAATase. The isolated primary granulocytes could maintain cell activity for more than one week in vitro culture that exhibited numerous filopodia. These results collectively suggested that CgAATase was a potential marker of oyster granulocytes, and the granulocytes could be effectively isolated from total circulating hemocytes by immunomagnetic bead coated with the anti-CgAATase monoclonal antibody.

Molecular evolution and functional characterisation of insulin related peptides in molluscs: Contributions of Crassostrea gigas genomic and transcriptomic-wide screening.

Insulin Related Peptides (IRPs) belong to the insulin superfamily and possess a typical structure with two chains, B and A, linked by disulphide bonds. As the sequence conservation is usually low between members, IRPs are classified according to the number and position of their disulphide bonds. In molluscan species, the first IRPs identified, named Molluscan Insulin-related Peptides (MIPs), exhibit four disulphide bonds. The genomic and transcriptomic data screening in the Pacific oyster Crassostrea gigas (Mollusc, Bivalvia) allowed us to identify six IRP sequences belonging to three structural groups. Cg-MIP1 to 4 have the typical structure of MIPs with four disulphide bonds. Cg-ILP has three disulphide bonds like vertebrate Insulin-Like Peptides (ILPs). The last one, Cg-MILP7 has a significant homology with Drosophila ILP7 (DILP7) associated with two additional cysteines allowing the formation of a fourth disulphide bond. The phylogenetic analysis points out that ILPs may be the most ancestral form. Moreover, it appears that ILP7 orthologs are probably anterior to lophotrochozoa and ecdysozoa segregation. In order to investigate the diversity of physiological functions of the oyster IRPs, we combine in silico expression data, qPCR measurements and in situ hybridization. The Cg-ilp transcript, mainly detected in the digestive gland and in the gonadal area, is potentially involved in the control of digestion and gametogenesis. The expression of Cg-mip4 is mainly associated with the larval development. The Cg-mip transcript shared by the Cg-MIP1, 2 and 3, is mainly expressed in visceral ganglia but its expression was also observed in the gonads of mature males. This pattern suggested the key roles of IRPs in the control of sexual reproduction in molluscan species.

Aneuploid progeny of the American oyster, Crassostrea virginica, produced by tetraploid × diploid crosses: another example of chromosome instability in polyploid oysters.

The commercial production of triploids, and the creation of tetraploid broodstock to support it, has become an important technique in aquaculture of the eastern oyster, Crassostrea virginica. Tetraploids are produced by cytogenetic manipulation of embryos and have been shown to undergo chromosome loss (to become a mosaic) with unknown consequences for breeding. Our objective was to determine the extent of aneuploidy in triploid progeny produced from both mosaic and non-mosaic tetraploids. Six families of triploids were produced using a single diploid female and crossed with three mosaic and non-mosaic tetraploid male oysters. A second set of crosses was performed with the reciprocals. Chromosome counts of the resultant embryos were tallied at 2-4 cell stage and as 6-hour(h)-old embryos. A significant level of aneuploidy was observed in 6-h-old embryos. For crosses using tetraploid males, aneuploidy ranged from 53% to 77% of observed metaphases, compared to 36% in the diploid control. For crosses using tetraploid females, 51%-71% of metaphases were aneuploidy versus 53% in the diploid control. We conclude that somatic chromosome loss may be a regular feature of early development in triploids, and perhaps polyploid oysters in general. Other aspects of chromosome loss in polyploid oysters are also discussed.

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.

The comprehensive immunomodulation of NeurimmiRs in haemocytes of oyster Crassostrea gigas after acetylcholine and norepinephrine stimulation.

BACKGROUND: Neural-endocrine-immune (NEI) system is a major modulation network among the nervous, endocrine and immune system and weights greatly in maintaining homeostasis of organisms during stress and infection. Some microRNAs are found interacting with NEI system (designated NeurimmiRs), addressing swift modulations on immune system. The oyster Crassostrea gigas, as an intertidal bivalve, has evolved a primary NEI system. However, the knowledge about NeurimmiRs in oysters remains largely unknown. RESULTS: Six small RNA libraries from haemocytes of oysters stimulated with acetylcholine (ACh) and norepinephrine (NE) were sequenced to identify neurotransmitter-responsive miRNAs and survey their immunomodulation roles. A total of 331 miRNAs (132 identified in the present study plus 199 identified previously) were subjected to expression analysis, and twenty-one and sixteen of them were found ACh- or NE-responsive, respectively (FDR < 0.05). Meanwhile, 21 miRNAs exhibited different expression pattern after ACh or NE stimulation. Consequently, 355 genes were predicted as putative targets of these neurotransmitter-responsive miRNAs in oyster. Through gene onthology analysis, multiple genes involved in death, immune system process and response to stimulus were annotated to be modulated by NeurimmiRs. Besides, a significant decrease in haemocyte phagocytosis and late-apoptosis or necrosis rate was observed after ACh and NE stimulation (p < 0.05) while early-apoptosis rate remained unchanged. CONCLUSIONS: A comprehensive immune-related network involving PRRs, intracellular receptors, signaling transducers and immune effectors was proposed to be modulated by ACh- and NE-responsive NeurimmiRs, which would be indispensable for oyster haemocytes to respond against stress and infection. Characterization of the NeurimmiRs would be an essential step to understand the NEI system of invertebrate and the adaptation mechanism of oyster.

Vitellogenin of Fujian oyster, Crassostrea angulata: Synthesized in the ovary and controlled by estradiol-17ß.

In this study, we cloned a full-length cDNA encoding vitellogenin (Vg) in the Fujian oyster Crassostrea angulata. The complete Vg cDNA consists of 5160 nucleotides with a long open reading frame encoding 1641 amino acid residues. The deduced amino acid sequence shared high similarity with the Vgs of other mollusc, fish, nematode and arthropod species, particularly in the N-terminal region. We analyzed the spatiotemporal expression of caVg transcripts by Real-time Quantitative PCR. In common with other mollusc Vgs, the caVg gene was expressed primarily in the ovary, and the levels were 348 and 177 times higher in maturation and ripeness stages (P<0.01), respectively, than in the partially spent stage. There was negligible expression in male oysters. In situ hybridization analysis further localized caVg mRNA to the follicle cells (also named auxiliary cells) surrounding the oocytes in the ovary. Moreover, in vivo waterborne exposure experiments in early gametogenesis oysters showed that estradiol-17ß (E2) administration resulted in a significant increase in caVg mRNA expression. We conclude that caVg is synthesized in the follicle cell surrounding the vitellogenic oocyte in C. angulata, and directly passed to oocytes through the extracellular space without mediation through hemolymph. Also, we hypothesize that this process is mediated by E2 in a dose dependent.

Pesticide residues, heavy metals, and DNA damage in sentinel oysters Crassostrea gigas from Sinaloa and Sonora, Mexico.

Pesticides and heavy metals were analyzed in sentinel Crassostrea gigas oysters placed in six aquaculture sites close to a contaminated agricultural region. Each site was sampled twice. Tests revealed the presence of organochlorine (OC) pesticides in the oysters at concentrations varying from 31.8 to 72.5 µg/kg for gamma-hexachlorocyclohexane (γ-HCH); from 1.2 to 3.1 µg/kg for dichlorodiphenyldichloroethylene (4,4-DDE); from 1.6 to 2.3 µg/kg for endosulfan I; and from 1.4 to 41.2 µg/kg for endosulfan II, as well as heavy metals in concentrations that exceeded Mexican tolerance levels (405.5 to 987.8 µg/g for zinc; 4.2 to 7.3 µg/g for cadmium; and 7.2 to 9.9 µg/g for lead). Significant levels of DNA damage in oyster hemocytes were also detected. There was a significant, positive correlation between genotoxic damage and concentration of nickel or the presence of endosulfan II. Cellular viability evaluated by cytotoxic analyses was found to be high at 80%. Marked inhibition in activity of acetylcholinesterase (AChE ) and induction of glutathione S-transferase (GST) activity was noted. Data demonstrated a significant relation between AChE activity inhibition and presence of endosulfan II, γ-HCH, copper, lead, and 4,4-DDE, as well as between AChE and GST activity at different sites.

Histological survey of Pacific oysters Crassostrea gigas (Thunberg) in Galicia (NW Spain).

In Galicia, there is an increasingly interest among representatives of the oyster industry in the development of Pacific oyster Crassostrea gigas culture. Nevertheless severe mortalities and emerging health problems in this species have been recently reported in European farming areas. A histological survey was performed from 2004 to 2009 to assess health status in both cultured and wild Galician oysters. Different symbiotic organisms and conditions were detected, including viral gametocytic hypertrophy (VGH) which is reported here for first time in Spanish coast. VGH, prokaryote-like colonies and ciliates were observed in oyster tissues without causing host damage. A haplosporidian infection, copepods inducing lesions and a cellular proliferative disorder were detected in some samples causing moderate host damage; their low prevalence suggests these parasites are not a threat for C. gigas in Galicia. None of the parasites detected is OIE (Office International des Epizooties: the World Organization for Animal Health) notifiable. Although the current study did not identify any pathogens or diseases of concern, it provides important prevalence baseline data for future health and epidemiological assessments needed to better understanding the existing and emerging health problems in this species.

Effect of acclimatization on hemocyte functional characteristics of the Pacific oyster (Crassostrea gigas) and carpet shell clam (Ruditapes decussatus).

Most experimental procedures on molluscs are done after acclimatization of wild animals to lab conditions. Similarly, short-term acclimation is often unavoidable in a field survey when biological analysis cannot be done within the day of sample collection. However, acclimatization can affect the general physiological condition and particularly the immune cell responses of molluscs. Our aim was to study the changes in the hemocyte characteristics of the Pacific oyster Crassostrea gigas and the carpet shell clam Ruditapes decussatus acclimated 1 or 2 days under emersed conditions at 14 ± 1 °C and for 1, 2, 7, or 10 days to flowing seawater conditions (submerged) at 9 ± 1 °C, when compared to hemolymph withdrawn from organisms sampled in the field and immediately analyzed in the laboratory (unacclimated). The hemocyte characteristics assessed by flow cytometry were the total (THC) and differential hemocyte count, percentage of dead cells, phagocytosis, and reactive oxygen species (ROS) production. Dead hemocytes were lower in oysters acclimated both in emersed and submerged conditions (1%-5%) compared to those sampled in the field (7%). Compared to oysters, the percentage of dead hemocytes was lower in clams (0.4% vs. 1.1%) and showed a tendency to decrease during acclimatization in both emersed and submerged conditions. In comparison to organisms not acclimated, the phagocytosis of hemocytes decreased in both oysters and clams acclimated under submerged conditions, but was similar in those acclimated in emersed conditions. The ROS production remained stable in both oysters and clams acclimated in emersed conditions, whereas in submerged conditions ROS production did not change in both the hyalinocytes and granulocytes of oysters, but increased in clams. In oysters, the THC decreased when they were acclimated 1 and 2 days in submerged conditions and was mainly caused by a decrease in granulocytes, but the decrease in THC in oysters acclimated 2 days in emersed conditions was caused by a decrease in hyalinocytes and small agranular cells. In clams, the THC was significantly lower in comparison to those not acclimated, regardless of the conditions of the acclimatization. These findings demonstrate that hemocyte characteristics were differentially affected in both species by the tested conditions of acclimatization. The phagocytosis and ROS production in clams and phagocytosis in oysters were not different in those acclimated for 1 day under both conditions, i.e. emersed and submerged, and those sampled in the field (unacclimated). The THC was significantly affected by acclimatization conditions, so the differences between clams and oysters should be considered in studies where important concentrations of hemocytes are required. The difference in the immune response between both species could be related to their habitat (epifaunal vs. infaunal) and their ability of resilience to manipulation and adaptation to captivity. Our results suggest that functional characteristics of hemocytes should be analyzed in both oysters and clams during the first 1 or 2 days, preferably acclimated under emersed rather than submerged conditions.

Proliferating cell nuclear antigen in gonad and associated storage tissue of the Pacific oyster Crassostrea gigas: seasonal immunodetection and expression in laser microdissected tissues.

To understand the processes involved in tissue remodeling associated with the seasonal reproductive cycle of the oyster Crassostrea gigas, we used immunodetection and expression measurements of proliferating cell nuclear antigen (PCNA). The expression of the PCNA gene was measured by real-time polymerase chain reaction in the whole gonadal area compared with laser microdissected gonad and storage tissue. Results underlined the advantage of the laser microdissection approach to detect expression, mainly for early stages of spermatogenesis. In the storage tissue, PCNA expression was reduced in the gonadal tubules, but immunolabeled hemocytes and vesicular cells were detected when the storage tissue was being restored. In the gonadal tubules, the PCNA gene was more highly expressed in males than in females. As soon as spermatogenesis was initiated, PCNA expression showed a high and constant level. In females, the expression level increased gradually until the ripe stage. The immunological approach established the involvement of peritubular cells in gonadal tubule expansion during early gametogenesis. In both sexes, gonial mitosis was immunodetected throughout the reproductive cycle. In males, the occurrence of two types of spermatogonia was ascertained by differential immunolabeling, and intragonadal somatic cell proliferation was noted. As expected, immunolabeling was never observed from stage II spermatocytes to spermatozoa. In females, positively stained cells were detected from oogonia to growing oocytes with various labeled intracellular locations.

Flow cytometry studies on the populations and immune parameters of the hemocytes of the Suminoe oyster, Crassostrea ariakensis.

The Suminoe oyster Crassostrea ariakensis has been attempted to be introduced in the Chesapeake Bay, USA, as an alternative to the eastern oyster Crassostrea virginica. Commercial production of Suminoe oysters is currently restricted due to the incomplete understanding of their biological, physiological and immunological nature. Accordingly, understanding immune system of C. ariakensis is crucial to prevent disease associated mortality and subsequent management of the Suminoe oyster. We investigated immunological activities and morphology of hemocytes of the Suminoe oyster using flow cytometry and light microscopy. Three types of hemocytes were identified in the hemolymph including hyalinocyte, granulocyte and blast-like cells. Hyalinocytes were the largest cells and the most abundant, while granulocytes were intermediate-size cell containing numerous granules in the cytoplasm. Blast-like cells were the smallest and least numerous. Flow cytometry revealed that the granulocytes are most active in the cell phagocytosis and spontaneous reactive oxygen species (ROS) production. The hyalinocytes also showed a certain level of the phagocytosis and oxidative activity but in a lesser extent than the granulocytes. In contrast, the blast-like cells did not show any phagocytosis or oxidative activity. The flow cytometry used in this study confirmed that as observed from other marine bivalves, the granulocytes are the main hemocytes involved in the cellular defence in the Suminoe oyster.

Analysis of genes isolated from plated hemocytes of the Pacific oyster, Crassostreas gigas.

A complementary deoxyribonucleic acid library was constructed from hemocytes of Crassostrea gigas that had been plated on poly-lysine plates for 24 h. From this library, 2,198 expressed sequence tags (ESTs) of greater than or equal to 100 bp were generated and analyzed. A large number of genes that potentially could be involved in the physiology of the oyster hemocyte were uncovered. They included proteins involved in cytoskeleton rearrangement, proteases and antiproteases, regulators of transcription and translation, cell death regulators, receptors and their associated protein factors, lectins, signal transduction proteins, and enzymes involved in eicosanoid and steroid synthesis and xenobiotic metabolism. Based on their relationship with innate immunity, the expression of selected genes was analyzed by quantitative polymerase chain reaction in gills from bacterial-challenged oysters. Several genes observed in the library were significantly upregulated by bacterial challenge including interleukin 17, astacin, cystatin B, the EP4 receptor for prostaglandin E, the ectodysplasin receptor, c-jun, and the p100 subunit of nuclear factor-kB. Using a similar approach, we have been analyzing the genes expressed in trout macrophages. While there are significant differences between the types of genes present in vertebrate macrophages compared with oyster hemocytes, there are some striking similarities including proteins involved in cytoskeletal rearrangement, proteases and antiproteases, and genes involved in certain signal transduction pathways underlying immune processes such as phagocytosis. Finally, C. virginica homologs of some of the C. gigas genes uncovered in the ESTs were obtained by aligning the ESTs reported here, against the assembled C. virginica ESTs at the National Center for Biotechnology Information.

First report of Perkinsus sp. infecting mangrove oysters Crassostrea rhizophorae from the Brazilian coast.

Protozoan parasites of the genus Perkinsus are considered important pathogens responsible for mass mortalities in several mollusk species worldwide. In the present study we describe for the first time a parasite of the genus Perkinsus infecting the mangrove oyster Crassostrea rhizophorae from the Brazilian coast. Prevalence of this parasite was low in the Pacoti River estuary (Ceará, northeast Brazil) and absent in oysters from southern Brazil. Oyster gill and rectum tissues incubated in Ray's fluid thioglycollate medium (RFTM) revealed the presence of spherical hypnospores (5 to 55 microm diam.). Histological analysis showed the occurrence of typical signet-ring trophozoites and schizonts (3 to 6 microm diam.) infecting connective tissues of several organs and digestive epithelia. PCR assays specific to the genus Perkinsus, followed by cloning and sequencing of the internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid (rRNA) gene complex, confirmed a close phylogenetic relationship between Brazilian Perkinsus sp. and P. beihaiensis infecting Chinese oysters.

Autophagy in nutrient storage cells of the Pacific oyster, Crassostrea gigas.

In oysters, nutrients are stored in a special type of cells referred to as vesicular-connective tissue cells (VCT-cells). These cells accumulate and provide nutrient to satisfy various needs of the organism, including gametogenesis. During the annual reproductive cycle, VCT-cells pass through a series of changes in their morphology associated with nutrients mobilization for developing germ cells. The results presented here show an approximately 33-35% increase in the number of autophagic vesicles in cytoplasm of VCT-cells in the gonadal area of C. gigas during the stage of active gametogenesis as compared to the resting stage of reproductive cycle. No destruction of VCT-cells due to autophagy or any other factors was observed, both in males and females. Our results indicate that autophagy does increase in VCT-cells of C. gigas and plays a certain role in nutrient mobilization from these cells.

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.

Differential responses in the biotransformation systems of the oyster Crassostrea gasar (Adanson, 1757) elicited by pyrene and fluorene: molecular, biochemical and histological approach - Part I.

Polycyclic aromatic hydrocarbons (PAHs) are among the main contaminants in aquatic environments. PAHs can affect organisms due to their carcinogenic, mutagenic and/or teratogenic characteristics. Depending on the PAHs, concentration, and period of exposure, biological damage can occur leading to histopathologic alterations. This study aimed to evaluate the molecular, biochemical and histological responses of the oyster Crassostrea gasar exposed to pyrene (0.25 and 0.5 µM) and fluorene (0.6 and 1.2 µM), after exposure for 24 and 96 h. Concentrations of both PAHs were quantified in the water and in oyster tissues. Transcript levels of phase I (CYP3475C1, CYP2-like, CYP2AU1 and CYP356A) and phase II (GSTO-like, MGST-like and SULT-like) biotransformation-related genes and the activities of ethoxyresorufin-O-deethylase (EROD), total and microsomal glutathione S-transferase (GST and MGST) were evaluated in the gills. Also, histological changes and localization of mRNA transcripts CYP2AU1 in gills, mantle, and digestive diverticula were evaluated. Both PAHs accumulated in oyster tissues. Pyrene half-life in water was significantly lower than fluorene. Transcript levels of all genes were higher in oysters exposed to of pyrene 0.5 µM (24 h). Only CYP2AU1 gene was up-regulated by fluorene exposure. EROD and MGST activities were higher in oysters exposed to pyrene. Tubular atrophy in the digestive diverticula and an increased number of mucous cells in the mantle were observed in oysters exposed to pyrene. CYP2AU1 transcripts were observed in different tissues of pyrene-exposed oysters. A significant correlation was observed between tubular atrophy and the CYP2AU1 hybridization signal in oysters exposed to pyrene, suggesting the sensibility of the species to this PAH. These results suggest an important role of biotransformation-related genes and enzymes and tissue alterations associated to pyrene metabolism but not fluorene. In addition, it reinforces the role of CYP2AU1 gene in the biotransformation process of PAHs in the gills of C. gasar.

Developmental dynamics of myogenesis in Pacific oyster Crassostrea gigas.

Pacific oyster (Crassostrea gigas) is a sessile bivalve living in the intertidal zone. It has become an attractive model for developmental studies due to its metamorphic transition from a mobile planktonic larvae to a sessile adults. To determine the effect of metamorphosis on muscle development in oyster larvae, we characterized myogenesis during larval development and metamorphosis by phalloidin staining which labels filamentous actin filaments. Our data revealed a dynamic pattern of myogenesis during embryonic and larval development. It appears that simple "U-shaped" muscle ring first developed at the trochophore stage. This was followed by a more complex musculature including an anterior adductor, velum ventral retractors at the veliger stage, and the addition of posterior adductors and foot retractors at the veliger and pediveliger stages. During metamorphosis, muscle structures in the anterior adductor, velum retractors and ventral retractors were degenerated. At the same time, mantle and gill musculature appeared and became the primary muscle system in juveniles together with the posterior adductor. In addition, indirect immunofluorescence with the monoclonal antibody against C. gigas muscle proteins (myosin heavy chains (MYHC) and α-actinin) were used to monitor changes in the developing musculature at different larval stages. The immunofluorescence staining results of muscle proteins were consistent with phalloidin staining. The expression locations of two muscle proteins were similar and mainly located in larval velum retractor and adductor muscle. The α-actinin expression positions were located in Z-lined of velum striated retractors. Data from these studies provide a comprehensive description of myogenesis in C. gigas embryos and larvae. Moreover, our data showed that metamorphosis has a significant impact on remolding the musculature after transition from a mobile planktonic larvae to a sessile mollusk, associated with certain muscle group degradation.

Visualization of shell matrix proteins in hemocytes and tissues of the Eastern oyster, Crassostrea virginica.

The tissues of the oyster were examined for the presence of shell matrix proteins (SMPs) using a combination of Western, proteomic, and epi-fluorescent microscopy techniques. SMP, including 48 and 55 kDa phosphoproteins, was detected in the epithelial cells of mantle, gill, heart, and adductor muscle and linings of arteries and veins. The 48 kDa SMP circulates continuously within the hemolymph, and is present in the immune system hemocytes. It appears to be secreted from hemocytes on induction of shell repair. We suggest that the 48 and 55 kDa proteins are multifunctional and bridge the process of soft tissue repair and shell formation by mediating cellular activities during immune response as well as interacting with the mineral phase during deposition.

Identification and tissue expression analysis of C-type lectin and galectin in the Pacific oyster, Crassostrea gigas.

As an initial step in the functional analysis of lectins in the Pacific oyster, Crassostrea gigas, we attempted to obtain the full coding sequences of C. gigas lectins and conduct tissue expression analyses. To obtain lectin genes quickly, we identified C. gigas expressed sequence tags that coded for lectins in GenBank, and selected three encoding partial sequences of C-type lectin 1 (CgCLec-1), galectin (CgGal) and fucolectin. We obtained full open reading frames of CgCLec-1 and CgGal cDNAs by RACE-PCR. CgCLec-1 is a typical C-type lectin with a signal peptide and C-type lectin domain. CgCLec-1 mRNA was expressed only in specialized basophilic cells involved with digestive enzyme secretion in the digestive gland, suggesting that CgCLec-1 is secreted into the lumen of the digestive diverticula. CgGal is a prototype galectin with a single galactose-binding domain that was expressed in all of the tissues examined. As suggested for vertebrate galectin-1 (prototype galectin), CgGal may function in general cell activities such as cell adhesion. Fucolectin in C. gigas was expressed specifically in the gonads, indicating a possible function in gonadal development. CgCLec-1 and CgGal expression in hemocytes was not upregulated after injecting Vibrio tubiashii into adductor muscle, suggesting that bacterial infection does not induce synthesis of these lectins. Of the three lectins examined, CgCLec-1 is an interesting target for future investigations of innate immunity in the digestive system of C. gigas.