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.

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.

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.

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.

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.

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.

Acute exposure to TiO2 nanoparticles produces minimal apparent effects on oyster, Crassostrea virginica (Gmelin), hemocytes.

The response of oyster (Crassostrea virginica) hemocytes was studied following exposure to anatase nanoparticles (ca. 7.4nm), surface-coated rutile nanocomposites (UV-Titan M212, ca. 86nm) and bulk titanium dioxide (TiO2) particles (anatase and rutile crystalline forms; 0.4-0.5µm). Hemocytes were collected from oysters and exposed to one of the four particle types at concentrations of 0.1, 0.5, and 1.0mg/L under dark and environmentally-relevant light conditions for periods of two and four hours. Hemocyte mortality, phagocytosis, and reactive oxygen species (ROS) production were then evaluated using flow-cytometric assays. Bulk and nanoparticulate TiO2 had little effect on viability of oyster hemocytes or on production of ROS. Significant changes in phagocytosis occurred after exposure to anatase nanoparticles for 4h under dark conditions, and UV-Titan for 2h under light conditions. Results demonstrate that TiO2 particles (bulk or nanoscale) produce minimal effects on hemocyte biomarkers examined following acute, in vitro exposures.

The neuroendocrine immunomodulatory axis-like pathway mediated by circulating haemocytes in pacific oyster Crassostrea gigas.

The neuroendocrine-immune (NEI) regulatory network is a complex system, which plays an indispensable role in the immunity of host. In this study, a neuroendocrine immunomodulatory axis (NIA)-like pathway mediated by the nervous system and haemocytes was characterized in the oyster Crassostrea gigas Once invaded pathogen was recognized by the host, the nervous system would temporally release neurotransmitters to modulate the immune response. Instead of acting passively, oyster haemocytes were able to mediate neuronal immunomodulation promptly by controlling the expression of specific neurotransmitter receptors on cell surface and modulating their binding sensitivities, thus regulating intracellular concentration of Ca2+ This neural immunomodulation mediated by the nervous system and haemocytes could influence cellular immunity in oyster by affecting mRNA expression level of TNF genes, and humoral immunity by affecting the activities of key immune-related enzymes. In summary, though simple in structure, the 'nervous-haemocyte' NIA-like pathway regulates both cellular and humoral immunity in oyster, meaning a world to the effective immune regulation of the NEI network.

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.

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.

Physiological and cellular responses of oysters (Crassostrea hongkongensis) in a multimetal-contaminated estuary.

The Pearl River estuary, southern China, suffers from multiple sources of metal contamination as a result of the rapid industrial development in the region; but the biological impacts of contamination remain unknown. In the present study, a euryhaline oyster, Crassostrea hongkongensis, was collected from different sites of the Pearl River estuary; and various physiological (heart rate, alkaline phosphatase as homeostatic regulation, and glycogen as energy reserve) and cytological (lysosomal membrane stability) biomarkers were quantified to assess this species as a potential bioindicator of metal pollution in contaminated areas. Large variations of metal accumulation levels in the oysters were documented, especially for copper (Cu), zinc (Zn), cadmium (Cd), chromium, and nickel (Ni). Among these metals, the authors demonstrated significant correlations between the digestive gland metal accumulation of Cu, Zn, and Ni and the cellular homeostasis (alkaline phosphatase) and glycogen reserves. Heart rate was positively correlated with Cd but negatively correlated with Cu and Zn concentrations in the gills. Lysosomal membrane stability was significantly inhibited at the most contaminated sites but had no relationship with the accumulated metal concentrations. These measurements indicate that multimetal contamination in the Pearl River estuary impacts the physiological and cytological performance of oysters. Environ Toxicol Chem 2016;35:2577-2586. © 2016 SETAC.

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.

Abcb1 gene expression pattern and function of copper detoxification in Fujian oyster, Crassostrea angulata.

Oysters are considered hyper-accumulators of Cu, but the molecular mechanism by which they maintain Cu cell homeostasis is still unclear. ATP-binding cassette protein subfamily B member 1 (ABCB1, P-glycoprotein) can transport a variety of substrates across the cell membrane in aquatic animals. In this study, to provide insight into the roles of ABCB1 in resistance against Cu in oysters, complete cDNA of abcb1 gene in Crassostrea angulata was cloned and analyzed. The complete sequence of C. angulata ABCB1 showed high identity to ABCB1 from other bivalves and contained some classical motifs of ABCB transport proteins. Abcb1 was mainly expressed in the apical epithelial cell of gills and epithelia of mantles. Abcb1 expression and Cu accumulation were also studied in control oysters and oysters exposed to Cu (30, 100, 300 µg/L Cu, 1-15 days). Cu accumulation in the gill and mantle were measured after abcb1 gene interference. The complete sequence of C. angulata ABCB1 showed high identity to ABCB1 from other bivalves and contained some classical motifs of ABCB transport proteins. The mRNA transcript of abcb1 showed hypersensitivity to Cu exposure. A concentration-dependent highest abcb1 mRNA level (up to 5.61-fold to the control) in the gill and mantle existed across all Cu exposure concentrations after 3 days of Cu exposure. The gill and mantle Cu concentration were significantly higher after the abcb1 mRNA interference. According to these results, it is here speculated that ABCB1 may underlie cell protection against Cu in C. angulata.

Differential gene transcription, biochemical responses, and cytotoxicity assessment in Pacific oyster Crassostrea gigas exposed to ibuprofen.

Pharmaceuticals, such as anti-inflammatory nonsteroidal drugs, are frequently detected in aquatic ecosystems. Studies about the effects of these substances in nontarget organisms, such as bivalves, are relevant. The aim of this study was to evaluate the effects on antioxidant status caused by ibuprofen (IBU) in oysters Crassostrea gigas exposed for 1, 4, and 7 days at concentrations 1 and 100 µg L(-1). Levels of IBU in tissues of oysters, as well as cell viability of hemocytes, were measured. The transcription of cytochrome P450 genes (CYP2AU2, CYP356A1, CYP3071A1, CYP30C1), glutathione S-transferase isoforms (GST-ω-like and GST-π-like), cyclooxygenase-like (COX-like), fatty acid binding protein-like (FABP-like), caspase-like, heat shock protein-like (HSP70-like), catalase-like (CAT-like), and the activity of catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), and glutathione S-transferase (GST) were also evaluated in the gills of oysters. The highest levels of IBU were observed in animals exposed to 100 µg L(-1). A significant upregulation of CYP2AU1, CYP356A1, CYP3071A1, GST-ω-like, GST-π-like, COX-like, and FABP-like was observed in oysters exposed to IBU under different experimental conditions. Oysters exposed to 1 µg L(-1) for 7 days showed a significantly higher transcription of CYP2AU2, CYP356A1, CYP3071A1, GST-ω-like, and GST-π-like but lower GR activity. In conclusion, C. gigas exposed to environmentally relevant concentrations of IBU (1 µg L(-1)) exhibited increased transcription of certain genes and alterations on antioxidant and auxiliary enzymes, which could, in the the long term, cause damages to exposed organisms.

Transcriptomic profiling of gametogenesis in triploid Pacific Oysters Crassostrea gigas: towards an understanding of partial sterility associated with triploidy.

BACKGROUND: Triploidy can occur in many animal species but is often lethal. Among invertebrates, amphibians and fishes, triploids are viable although often sterile or infertile. Most triploids of the Pacific oyster Crassostrea gigas are almost sterile (named "3nß") yet a low but significant proportion show an advanced gametogenesis (named "3nα"). These oysters thus constitute an interesting model to study the effect of triploidy on germ cell development. We used microarrays to compare the gonad transcriptomes of diploid 2n and the abovementioned triploid 3nß and 3nα male and female oysters throughout gametogenesis. RESULTS: All triploids displayed an upregulation of genes related to DNA repair and apoptosis and a downregulation of genes associated with cell division. The comparison of 3nα and 3nß transcriptomes with 2n revealed the likely involvement of a cell cycle checkpoint during mitosis in the successful but delayed development of gonads in 3nα individuals. In contrast, a disruption of sex differentiation mechanisms may explain the sterility of 3nß individuals with 3nß females expressing male-specific genes and 3nß males expressing female-specific genes. CONCLUSIONS: The disruption of sex differentiation and mitosis may be responsible for the impaired gametogenesis of triploid Pacific oysters. The function of the numerous candidate genes identified in our study should now be studied in detail in order to elucidate their role in sex determination, mitosis/meiosis control, pachytene cell cycle checkpoint, and the control of DNA repair/apoptosis.

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.

The identification and characteristics of immune-related microRNAs in haemocytes of oyster Crassostrea gigas.

BACKGROUND: MicroRNAs (miRNAs) represent a class of small ncRNAs that repress gene expression on the post-transcriptional level by the degradation or translation inhibition of target mRNA. METHODOLOGY: Three small RNA libraries from oyster haemocytes were sequenced on the Illumina platform to investigate the latent immunomodulation of miRNAs after bacteria challenge and heat stress. Totally, 10,498,663, 8,588,606 and 9,679,663 high-quality reads were obtained in the control, bacteria and bacteria+heat library, respectively, from which 199 oyster miRNAs including 71 known and 128 novel ones were identified. Among these miRNAs, 6 known and 23 novel ones were predicted to possess more than one precursor-coding region, and cgi-miR-10a, cgi-miR-184b, cgi-miR-100, cgi-miR-1984 and cgi-miR-67a were observed to be the most abundant miRNAs in the control library. The expression levels of 22 miRNAs in the bacteria library were significantly higher than those in the control library, while there were another 33 miRNAs whose expression levels were significantly lower than that in the control library. Meanwhile, the expression levels of 65 miRNAs in the bacteria+heat library changed significantly compared to those in the bacteria library. The target genes of these differentially expressed miRNAs were annotated, and they fell in immune and stress-related GO terms including antioxidant, cell killing, death, immune system process, and response to stimulus. Furthermore, there were 42 differentially expressed miRNAs detected in both control/bacteria and bacteria/bacteria+heat comparisons, among which 9 miRNAs displayed the identical pattern in the two comparisons, and the expression alterations of 8 miRNAs were confirmed using quantitative RT-PCR. CONCLUSIONS: These results indicated collectively that immune challenge could induce the expression of immune-related miRNAs, which might modulate the immune response such as redox reaction, phagocytosis and apoptosis, and the expression of some immune-related miRNAs could be also regulated by heat stress to improve the environmental adaption of oyster.

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.

Ecological relevance of Sentinels' biomarker responses: a multi-level approach.

In response to the need for more sensitive and rapid indicators of environmental quality, sublethal effects on the lowest levels of biological organization have been investigated. The ecological relevance of these responses assumes a prevailing role to assure effectiveness as indicator of ecological status. This study aimed to investigate the linkages between biomarker responses of caged bivalves and descriptive parameters of macrobenthic community structure. For this purpose a multi-level environmental assessment of marine and estuarine zones was performed in São Paulo coast, Brazil. Multivariate analysis was applied to identify linkages between biological responses and ecological indices, as well as to characterizing the studied stations. Individuals of the marine mussel Perna perna caged along Santos Bay showed signs of oxidative stress, lysosomal membrane destabilization, histological alterations and reduced embryonic development. The estuarine oyster Crassostrea rhizophorae caged along Santos Port Channel showed alterations on biotransformation enzymes and antioxidant system, DNA damage and lysosomal membrane destabilization. The benthic community analysis showed reduced richness and diversity in the same areas of the Santos bay and estuary where biomarker responses were altered. Our results revealed that xenobiotics are inducing physiological stress, which may lead to changes of the benthic community structure and deterioration of the ecological status over time. Integrating biomarker responses and ecological indexes improved certainty that alterations found at community level could be related to xenobiotic as stressors, which was very useful to improve the discriminatory power of the environmental assessment.