Adult somatic progenitor cells and hematopoiesis in oysters.

Long-lived animals show a non-observable age-related decline in immune defense, which is provided by blood cells that derive from self-renewing stem cells. The oldest living animals are bivalves. Yet, the origin of hemocytes, the cells involved in innate immunity, is unknown in bivalves and current knowledge about mollusk adult somatic stem cells is scarce. Here we identify a population of adult somatic precursor cells and show their differentiation into hemocytes. Oyster gill contains an as yet unreported irregularly folded structure (IFS) with stem-like cells bathing into the hemolymph. BrdU labeling revealed that the stem-like cells in the gill epithelium and in the nearby hemolymph replicate DNA. Proliferation of this cell population was further evidenced by phosphorylated-histone H3 mitotic staining. Finally, these small cells, most abundant in the IFS epithelium, were found to be positive for the stemness marker Sox2. We provide evidence for hematopoiesis by showing that co-expression of Sox2 and Cu/Zn superoxide dismutase, a hemocyte-specific enzyme, does not occur in the gill epithelial cells but rather in the underlying tissues and vessels. We further confirm the hematopoietic features of these cells by the detection of Filamin, a protein specific for a sub-population of hemocytes, in large BrdU-labeled cells bathing into gill vessels. Altogether, our data show that progenitor cells differentiate into hemocytes in the gill, which suggests that hematopoiesis occurs in oyster gills.

Comparative study on the hemocytes of subtropical oysters Saccostrea kegaki (Torigoe & Inaba, 1981), Ostrea circumpicta (Pilsbry, 1904), and Hyotissa hyotis (Linnaeus, 1758) in Jeju Island, Korea: morphology and functional aspects.

We first characterized the morphology and immune-related activities of hemocytes in the subtropical oysters Saccostrea kegaki, Ostrea circumpicta, and Hyotissa hyotis using light microscopy and flow cytometry. Hemocytes of these three oyster species were classified into three main types: 1) granulocytes containing numerous granules in the cytoplasm, 2) hyalinocytes with no or fewer granules, and 3) blast-like cells characterized by the smallest size and very thin cytoplasm. The percentage of each hemocyte population was similar in all species; hyalinocytes were the most abundant cell in the hemolymph accounting for more than 59%, followed by granulocytes (23-31%) and blast-like cells (3-5%). The size of granulocytes of S. kegaki was smaller (P < 0.05) than those of O. circumpicta and H. hyotis. Light microscopy also allowed the description of vacuolated cells characterized by large vacuoles in the cytoplasm. Flow cytometry analysis confirmed that the granulocytes of the three oyster species were the major hemocytes engaged in cellular defense with the largest lysosome content, and the most active phagocytosis activity and oxidative activity, as was previously reported in several marine bivalves. Phagocytic activity was the lowest in S. kegaki hemocytes, and PMA-stimulated oxidative activity was the lowest in H. hyotis hemocytes. Our results provide the basic information of hemocytes population of three subtropical oysters for further investigations associated with various environmental disease stresses.

New insights from the oyster Crassostrea rhizophorae on bivalve circulating hemocytes.

Hemocytes are the first line of defense of the immune system in invertebrates, but despite their important role and enormous potential for the study of gene-environment relationships, research has been impeded by a lack of consensus on their classification. Here we used flow cytometry combined with histological procedures, histochemical reactions and transmission electron microscopy to characterize the hemocytes from the oyster Crassostrea rhizophorae. Transmission electron microscopy revealed remarkable morphological characteristics, such as the presence of membranous cisternae in all mature cells, regardless of size and granulation. Some granular cells contained many cytoplasmic granules that communicated with each other through a network of channels, a feature never previously described for hemocytes. The positive reactions for esterase and acid phosphatase also indicated the presence of mature cells of all sizes and granule contents. Flow cytometry revealed a clear separation in complexity between agranular and granular populations, which could not be differentiated by size, with cells ranging from 2.5 to 25 µm. Based on this evidence we suggest that, at least in C. rhizophorae, the different subpopulations of hemocytes may in reality be different stages of one type of cell, which accumulates granules and loses complexity (with no reduction in size) as it degranulates in the event of an environmental challenge.

Noradrenaline-functionalized hyperbranched fluoropolymer-poly(ethylene glycol) cross-linked networks as dual-mode, anti-biofouling coatings.

The strategy of decorating antibiofouling hyperbranched fluoropolymer-poly(ethylene glycol) (HBFP-PEG) networks with a settlement sensory deterrent, noradrenaline (NA), and the results of biofouling assays are presented. This example of a dual-mode surface, which combines both passive and active modes of antibiofouling, works in synergy to improve the overall antibiofouling efficiency against barnacle cyprids. The HBFP-PEG polymer surface, prior to modification with NA, was analyzed by atomic force microscopy, and a significant distribution of topographical features was observed, with a nanoscopic roughness measurement of 110 ± 8 nm. NA attachment to the surface was probed by secondary ion mass spectrometry to quantify the extent of polymer chain-end substitution with NA, where a 3- to 4-fold increase in intensity for a fragment ion associated with NA was observed and 39% of the available sites for attachment were substituted. Cytoskeletal assays confirmed the activity of tethered NA on adhering oyster hemocytes. Settlement assays showed deterrence toward barnacle cyprid settlement, while not compromising the passive biofouling resistance of the surface. This robust strategy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling network.

A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE.

Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.

Immune parameters of QX-resistant and wild caught Saccostrea glomerata hemocytes in relation to Marteilia sydneyi infection.

Sydney rock oysters (SRO) Saccostrea glomerata suffer mass mortalities during summer and autumn as a result of infection by a protozoan parasite Marteilia sydneyi (QX disease). Mass selected disease resistant (QXR) lines have been used with some success in affected estuaries in recent years, with resistance attributed to oxidative defense systems. However, the role of hemocytes in resistance to QX by SRO has not been fully explored. In the present study, fifty QXR and fifty wild caught (WC) oysters were collected from a lease at Pimpama River during a QX outbreak in January 2011. Hemocytes characteristics (type, morphology) and functions (mortality, phagocytosis and oxidative activity) from both oyster lines were analyzed by flow cytometry in the context of infection intensity and parasite viability (determined histologically). Amongst the QXR oysters, 20% were diseased containing viable parasite, 74% had killed M. sydneyi and 6% were uninfected. In contrast, 86% of WC oysters were diseased, 2% had killed M. sydneyi and 12% were healthy. Significant differences in hemocyte number and physiology between the two oyster lines were found (ANOVA). Phagocytosis rate and the mean oxidative activity per cell were similar between both oyster lines. Higher numbers of infiltrating and circulating hemocytes, higher percentage of circulating granulocytes, their higher size and complexity in QXR oysters, and the production of reactive oxygen species were associated with the ability to kill the parasite. High abundance of M. sydneyi in the digestive tubule epithelium of both oyster lines implied inability to kill the parasite at the beginning of the infection. However, QXR oysters had the ability to kill M. sydneyi at the stage of sporangiosorae in the epithelium of digestive tubules. The similar phagocytic ability of hemocytes from both oyster lines, the size of the parasite at this infection stage, and its localization suggested that encapsulation is likely to be the main process involved in the eradication of M. sydneyi by QXR oysters.

Selection in the rapid evolution of gamete recognition proteins in marine invertebrates.

Animal fertilization is governed by the interaction (binding) of proteins on the surfaces of sperm and egg. In many examples presented herein, fertilization proteins evolve rapidly and show the signature of positive selection (adaptive evolution). This review describes the molecular evolution of fertilization proteins in sea urchins, abalone, and oysters, animals with external fertilization that broadcast their gametes into seawater. Theories regarding the selective forces responsible for the rapid evolution driven by positive selection seen in many fertilization proteins are discussed. This strong selection acting on divergence of interacting fertilization proteins might lead to prezygotic reproductive isolation and be a significant factor in the speciation process. Since only a fraction of all eggs are fertilized and only an infinitesimal fraction of male gametes succeed in fertilizing an egg, gametes are obviously a category of entities subjected to intense selection. It is curious that this is never mentioned in the literature dealing with selection, perhaps because we know so little about fitness differences among gametes. (Ernst Mayr, 1997).

Fullerene exposures with oysters: embryonic, adult, and cellular responses.

Oysters are an ecologically important group of filter-feeders, and a valuable toxicology model for characterizing the potential impacts of nanoparticles to marine organisms. Fullerene (C60) exposure studies with oysters, Crassostrea virginica, were conducted with a variety of biological levels, e.g., developmental studies with embryos, whole organism exposures with adults, and isolated hepatopancreas cells. Significant effects on embryonic development and lysosomal destabilization were observed at concentrations as low as 10 ppb. Moreover, based on our extensive experience with the lysosomal assay, the lysosomal destabilization rates at fullerene concentrations > or = 100 ppb were regarded as biologically significant as they are associated with reproductive failure. Interestingly, there was no significant increase in lipid peroxidation levels in hepatopancreas tissues. Oyster hepatopancreas tissues are composed of lysosomal rich cells, and confocal microscopy studies indicated thatthe fullerene particles readily accumulated inside hepatopancreas cells within 4 h. Fullerene aggregates tended to be localized and concentrated into lysosomes. The microscopic work in conjunction with the lysosomal function assays supports the premise that endocytotic and lysosomal pathways may be major targets of fullerenes and other nanoparticles. Nanoparticles that affect normal lysosomal and autophagic processes may contribute to long-term, chronic problems for individual health as well as ecosystem health.

In vitro effects of noradrenaline on Sydney rock oyster (Saccostrea glomerata) hemocytes.

Our prior work has shown that the catecholamine hormone, noradrenaline, mediates environmental stress responses in Sydney rock oysters, resulting in impaired immunological function. In the current study, we tested the cellular basis of this stress response. Hemocytes were exposed to noradrenaline in vitro before cell morphology and viability were analyzed. Noradrenaline was shown to induce apoptotic markers, including the loss of mitochondrial membrane potential, DNA fragmentation and plasma membrane blebbing. F-actin appeared to play an important role in the changes observed in hemocytes, being concentrated mostly in the plasma membrane blebs of noradrenaline-treated hemocytes. This may explain why hemocyte adhesion and pseudopodia formation were inhibited by noradrenaline. Cellular dysfunction induced by norarenaline mainly affected the hyalinocyte sub-population of hemocytes, whilst the other major cell type, granulocytes, remained unaffected. Given that hyalinocytes are important immunological effectors, the results of this study help to explain why immunosuppression accompanies noradrenaline-mediated stress responses in oysters.

Morphological and morphometric aspects of Crassostrea rhizophorae (Guilding, 1828) oocytes in three stages of the gonadal cycle / Aspectos Morfológicos y Morfométricos de Oocitos de Crassostrea rhizophorae (Guilding, 1828) en Tres Fases del Ciclo Gonadal

Crassostrea rhizophorae (Guilding, 1828) es una de las especies de ostras tropicales cultivadas en la costa brasilera, que presenta alto valor comercial, no existiendo información sobre las características gonadales, durante las fases reproductivas de esta especie. El objetivo de este trabajo fue analizar la morfología de los ovocitos y de los folículos gonadales de Crassostrea rhizophorae, en tres fases del ciclo gonadal. Analizando los cortes histológicos de las gónadas y considerando los ovocitos que presentaban núcleo y nucléolo evidentes, fueron determinadas tres fases del ciclo gonadal: gametogénesis inicial, crecimiento y maduración. En la fase de gametogénesis inicial, el diámetro de los folículos y de los ovocitos era de 180,29 (+ - 41,91) e 18,66 (+ - 6,85) µm, respectivamente; también identificamos gran cantidad de tejido conjuntivo interfolicular y un número mayor de ovocitos previtelogénicos. En la fase de crecimiento, el diámetro de los folículos y de los ovocitos era de 218,02 (+ - 43,19) y 25,92 (+ - 9,94) µm, respectivamente. Este fase fue caracterizada por una pequeña cantidad de tejido conjuntivo interfolicular y predominio de ovocitos vitelogénicos. En la fase de maduración, el diámetro de los folículos y de los ovocitos era de 298,16 ( + - 99,24) y 35,27 (+ - 6,2) µm, respectivamente, existiendo gran número de ovocitos maduros. De esta manera, concluimos que durante la implantación del cultivo, Crassostrea rhizophorae tolera la influencia de los factores intrínsecos y extrínsecos y no presenta alteraciones significativas en su actividad reproductiva.

Crassostrea rhizophorae (Guilding, 1828) is one of the tropical species of oysters cultivated on the Brazilian shore. Despite its high commercial value, there is no information on the gonadal characteristics during the reproductive stages of this species. The objective of this work was to analyze the morphology and morphometry of Crassostrea rhizophorae oocytes and follicles in three stages of the gonadal cycle. Were analized histological sections of gonads considering that the oocytes presented visible nuclei and nucleoli, it was observed three gonadal cycle stages: early gametogenesis, growth and maturation. In the early gametogenesis stage, follicles and oocytes presented diameters of 180.29 (± 41.91) and 18.66 (± 6.85) µm, respectively, and presence of large amount of connective tissue and previtellogenic oocyte. In the growth stage, follicles and oocytes presented diameters of 218.02 (± 43.19) and 25.92 (± 9.94) µm, respectively, this stage was characterized by a small amount of interfollicular connective tissue with vitellogenic oocyte predominate. In the maturation stage, follicles and oocytes presented diameters of 298.16 (± 99.24) and 35.27 (± 6.2) µm, respectively, and presence of large number of mature oocytes. We concluded that during culture Crassostrea rhizophorae tolerates the influence of intrinsic and extrinsic factors and does not undergo significant changes in reproductive activity.

Improved chromatographic separation of thio-arsenic compounds by reversed-phase high performance liquid chromatography-inductively coupled plasma mass spectrometry.

A new group of arsenic species, thio-arsenicals, have recently been reported in several natural samples such as molluscs, algae, and urine. These compounds are the sulfur analogues of oxo-arsenicals, a large group of naturally-occurring compounds, whereby the arsinoyl (As=O) group is substituted by an arsinothioyl group (As=S). The most common separation technique for oxo-arsenicals is anion-exchange HPLC with polymer-based columns, but under these conditions the thio-arsenicals show strong retention, resulting in unacceptably long analysis times and broad peaks. We report the development of a reversed-phase HPLC method, with ICPMS detection, which allows separation of the known thio-arsenicals within 15 min with significantly improved peak shapes. The detection limit is about 0.6 microg As/L based on 10 microL injection volume. Further, we have applied the method to the identification and quantification of thio-arsenic species in two standard reference materials, BCR 710 oyster tissue and NIES 18 human urine.

Detection of human adenoviruses and enteroviruses in Korean oysters using cell culture, integrated cell culture-PCR, and direct PCR.

Oysters are known to be carriers of food-born diseases, but research on viruses in Korean oysters is scarce despite its importance for public health. We therefore tested oysters cultivated in Goheung, Seosan, Chungmu, and Tongyeong, for viral contamination using cell culture and integrated cell culture PCR (ICC-PCR) with Buffalo green monkey kidney (BGMK) and human lung epithelial (A549) cells. Additional screens via PCR, amplifying viral nucleic acids extracted from oysters supplemented our analysis. Our methods found 23.6%, 50.9%, and 89.1% of all oysters to be positive for adenoviruses when cell culture, ICC-PCR, and direct PCR, respectively, was used to conduct the screen. The same methodology identified enteroviruses in 5.45%, 30.9%, and 10.9% of all cases. Most of the detected enteroviruses (81.3%) were similar to poliovirus type 1; the remainder resembled coxsackievirus type A1. A homology search with the adenoviral sequences revealed similarities to adenovirus subgenera C (type 2, 5, and 6), D (type 44), and F (enteric type 40 and 41). Adenovirus-positive samples were more abundant in A549 cells (47.3%) than in BGMK cells (18.2%), while the reverse was true for enteroviruses (21.8% vs. 14.5%). Our data demonstrate that Korean oysters are heavily contaminated with enteric viruses, which is readily detectable via ICC-PCR using a combination of A549 and BGMK cells.

Systematic factor optimization for sperm cryopreservation of tetraploid Pacific oysters, Crassostrea gigas.

The availability of tetraploid Pacific oysters provides a unique opportunity for comparative studies of sperm cryopreservation between diploids and tetraploids. In parallel to studies with sperm from diploid oysters, this study reports systematic factor optimization for sperm cryopreservation of tetraploid oysters. Specifically, this study evaluated the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), and straw size. Similar to sperm from diploids, the optimal cooling rate was 5 degrees C/min to -30 degrees C, followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations showed that a combination of the cryoprotectants 6% polyethylene glycol/4% propylene glycol and 6% polyethylene glycol/4% dimethyl sulfoxide yielded consistently high post-thaw motility. A long equilibration (60 min) yielded higher percent fertilization, and confirmed that extended equilibration could be beneficial when low concentrations of cryoprotectant are used. There was no significant difference in post-thaw motility between straw sizes of 0.25 and 0.5 mL. Despite low post-thaw fertilization (<10%) in general for sperm from tetraploids, optimized protocols in the present study effectively retained post-thaw motility for sperm from tetraploid oysters. This study confirmed that sperm from tetraploid Pacific oysters were more negatively affected by cryopreservation than were those of diploids. One possible explanation is that sperm from these two ploidies are different in their plasma membrane properties (e.g., structure, permeability, and elasticity), and the plasma membrane of sperm from tetraploids is more sensitive to cryopreservation effects. The fact that combinations of non-permeating and permeating cryoprotectants improved post-thaw motility in sperm from tetraploids provided presumptive evidence for this interpretation.

Successful cryopreservation of Pacific oyster (Crassostrea gigas) oocytes.

Protocols for cryopreservation of sperm and oocytes would provide the ultimate control over parental crosses in selective breeding programmes. Sperm freezing is routine for many species, but oocyte freezing remains problematic, with virtually zero success in aquatic species to date. This paper describes the development of a successful protocol for cryopreserving high concentrations of Pacific oyster (Crassostrea gigas) oocytes. Ethylene glycol (10%) and dimethyl sulfoxide (15%) were found to be the most effective cryoprotectants resulting in post-thaw fertilization rates of 51.0+/-8.0 and 45.1+/-8.3%, respectively. Propylene glycol was less effective and methanol resulted in zero fertilization post-thaw. The use of Milli-Q water rather than seawater as a base medium significantly improved fertilization (20.4+/-3.0 and 8.7+/-2.2%, respectively) as did the inclusion of a 5 min isothermal hold at -10 or -12 degrees C (35.9+/-5.0 and 31.9+/-4.6%, respectively). The optimal cooling rate post-hold was 0.3 degrees C min(-1), with virtually zero post-thaw fertilization with cooling rates of 3 and 6 degrees C min(-1). Using an optimized protocol, post-thaw fertilization rates for oocytes from eight individual females ranged from 0.8 to 74.5% and D-larval yields from 0.1 to 30.1%. For three individuals, larvae were reared through to spat. Development of D-larvae to eyed larvae and spat was similar for larvae produced from unfrozen (24.8+/-4.1% developed to eyed larvae and 16.5+/-3.2% to spat) and cryopreserved (28.4+/-0.6 and 18.7+/-0.5%, respectively) oocytes. The ability to cryopreserve large quantities of oyster oocytes represents a major advance in cryobiology and selective breeding.

Systematic factor optimization for cryopreservation of shipped sperm samples of diploid Pacific Oysters, Crassostrea gigas.

Despite some 26 published reports addressing oyster sperm cryopreservation, systematic factor optimization is lacking, and sperm cryopreservation has not yet found application in aquaculture on a commercial scale. In this study, the effects of cooling rate, single or combined cryoprotectants at various concentrations, equilibration time (exposure to cryoprotectant), straw size, and cooling method were evaluated for protocol optimization of shipped sperm samples from diploid oysters. Evaluation of cooling rates revealed an optimal rate of 5 degrees C/min to -30 degrees C followed by cooling at 45 degrees C/min to -80 degrees C before plunging into liquid nitrogen. Screening of single or combined cryoprotectants at various concentrations suggested that a low concentration (2%) of polyethylene glycol (FW 200) was effective in retaining post-thaw motility and fertilizing capability when combined with permeating cryoprotetcants such as dimethyl sulfoxide (DMSO), methanol (MeOH), and propylene glycol (P-glycol). However, polyethylene glycol alone was not as effective as MeOH, DMSO, and P-glycol when using the same methods. The highest post-thaw motility (70%) and percent fertilization (98%) were obtained for samples cryopreserved with 6% MeOH. However, this does not exclude other cryoprotectants such as DMSO or P-glycol identified as effective agents in other studies. There was no significant difference in post-thaw motility between straw sizes of 0.25- and 0.5-ml. Equilibration time (exposure to cryoprotectant) of 60 min could be beneficial when the cryoprotectant concentration is low and solution is added in a step-wise fashion at low temperature. Differences in post-thaw sperm quality (e.g., motility or percent fertilization) among individual males were evident in this research. As a consequence, a generalized classification describing males with different tolerances (broad, intermediate, and narrow) to cryopreservation was developed. This classification could be applied to strain or species differences in tolerances to the cryopreservation process. The present study demonstrated that oyster sperm could be collected and shipped chilled to another facility for cryopreservation, and that it could be shipped back to the hatchery for fertilization performed at a production scale yielding live larvae with >90% fertilization. Given the existence of facilities for commercial-scale cryopreservation of dairy bull sperm, the methods developed in the present study for oysters provide a template for the potential commercialization of cryopreserved sperm in aquatic species.

Viral gametocytic hypertrophy caused by a papova-like virus infection in the Pacific oyster Crassostrea gigas in Korea.

During a routine survey of the Pacific oyster Crassostrea gigas in Tongyoung (previously Chungmu) on the southern coast of Korea, basophilic inclusions were observed in the gonadal tissues. They were detected from March to May at a prevalence rate of 3.3 to 7.1%. The inclusion bodies were Feulgen-positive and stained orange-red with phloxine tartrazine. Electron microscopic observation revealed non-enveloped, icosahedral particles 40 to 45 nm in diameter. These morphological characteristics resemble those of papova virus-like inclusions previously described from Pacific and eastern (American) oysters C. virginica in North America. Although many mitochondrial bodies and intact sperm cells were observed around the inclusion body, no host reaction, such as hemocytic infiltration, was detected.

Hemocyte-mediated shell mineralization in the eastern oyster.

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

Insulin-like system and growth regulation in the Pacific oyster Crassostrea gigas: hrIGF-1 effect on protein synthesis of mantle edge cells and expression of an homologous insulin receptor-related receptor.

The involvement of molecules belonging to the insulin/IGF family in regulation of growth has been investigated in the Pacific oyster Crassostrea gigas. In vitro biological effects of human recombinant IGF-1 (hrIGF-1) on mantle edge cells, involved in oyster shell and soft body growth, were studied over an annual cycle. In mantle edge cells hrIGF-1 stimulates protein synthesis of 56+/-5.1% over basal for 10(-10) M in September with in addition a clear dose-effect corresponding to the highest shell growth period, and 57.5+/-3.45% over basal for 10(-11) M in March and 51+/-5.4% over basal for 10(-10) M in April corresponding to the period of mantle growth. These insulin-like effects were associated with the expression of a recently identified C. gigas insulin receptor-related receptor (CIR) in mantle edge cells as demonstrated by RT-PCR. Moreover, in situ hybridisation (ISH) confirmed this expression at the level of the inner and outer epithelia involved in mantle growth and shell formation.

MAPK is involved in metaphase I arrest in oyster and mussel oocytes.

Oocytes of Crassostrea gigas and Mytilus galloprovincialis are arrested in metaphase I when they are spawned and ready to be fertilized. To investigate the role of MAP kinase in maintaining metaphase I arrest, oocytes were exposed to the MEK inhibitor U0126, and the effects on chromosome behavior and MAPK activity were examined by bisbenzimide staining and in immunoblots with anti-phospho MAPK antibodies. Following treatment with 50 microM U0126, active MAPK was undetectable and oocytes resumed meiosis, forming enlarged polar bodies and undergoing chromosome decondensation. Prophase stage oyster oocytes maturing spontaneously in seawater completed germinal vesicle breakdown in the presence of U0126, but failed to arrest in metaphase I, and also formed polar bodies and underwent chromosome decondensation. Treatment of oyster oocytes with the protein synthesis inhibitor, emetine (500 microM), also caused them to resume meiosis, although substantial MAPK activity remained. Levels of phospho-MEK also decreased during emetine treatment. 35 S-methionine incorporation in emetine treated oocytes was reduced to only 5% of control values. These data show that, while active MAPK is necessary to maintain metaphase I arrest, other proteins are also required.

Measurement of Crassostrea gigas hemocyte oxidative metabolism by flow cytometry and the inhibiting capacity of pathogenic vibrios.

A flow cytometric method to measure the production of oxidative metabolism products was adapted for use with Crassostrea gigas hemocytes. The method is based upon the oxidation, by hydrogen peroxide (H2O2), of intracellular 2',7'-dichlorofluorescin (DCFH) to green-fluorescent dichlorofluorescein. Activation of the respiratory burst (RB) was tested using phorbol myristate acetate with no success. By contrast, activation by zymosan particles increased oxidation of DCFH in C. gigas hemocytes, mainly granulocytes, and optimization tests showed a good response with 20 zymosan particles per hemocyte. Anti-aggregant solution, used to prevent hemocytes from clumping during bleeding, inhibited the RB activity measured by DCFH oxidation. The flow cytometric method developed during this work was used to evaluate the DCFH oxidation-inhibiting capacity of four strains of vibrio bacteria, known or suspected to be pathogenic for bivalves.