Molecular cloning of calnexin and calreticulin in the Pacific oyster Crassostrea gigas and its expression in response to air exposure

Calnexin (CNX) and calreticulin (CRT) are endoplasmic reticulum (ER) chaperones. CNX is a type I transmembrane protein and CRT is a soluble CNX homologue. In the ER, CNX and CRT are important for Ca(2+) homeostasis and protein maturation. Here, we describe the full-length cDNA of the first mollusk CNX (cgCNX) and a second mollusk CRT (cgCRT) from the oyster Crassostrea gigas. CgCNX, containing 3255bp, was composed of a 1764bp open reading frame (ORF) that encodes a 588-amino acid protein. CgCRT, containing 1727bp, was composed of a 1242bp ORF that encodes a 414-amino acid protein. CgCNX and cgCRT contains an N-terminal 21- and 16-amino acid sequence, respectively, which is characteristic of a signal sequence. At the C-terminus, cgCRT also contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif suggesting that cgCRT localizes in the ER. Northern blot analysis showed that both cgCNX and cgCRT mRNAs are induced by air exposure. The expression patterns of cgCNX mRNA differed from those of cgCRT during air exposure. This suggests that these two molecular chaperones have different roles in the response to air exposure.     

Survival of Pacific oyster, Crassostrea gigas, oocytes in relation to intracellular ice formation

The effect of IIF in Pacific oyster oocytes was studied using cryo and transmission electron microscopy (TEM). The viability of oocytes at each step of a published cryopreservation protocol was assessed in an initial experiment. Two major viability losses were identified; one when oocytes were cooled to −35 °C and the other when oocytes were plunged in liquid nitrogen. Although the cryomicroscope showed no evidence of IIF in oocytes cooled with this protocol, TEM revealed that these oocytes contained ice crystals and were at two developmental stages when frozen, prophase and metaphase I. To reduce IIF, the effect of seven cooling programmes involving cooling to −35 or −60 °C at 0.1 or 0.3 °C min⁻¹ and holding for 0 or 30 min at −35 or −60 °C was evaluated on post-thaw fertilization rate of oocytes. Regardless of the cooling rate or holding time, the fertilization rate of oocytes cooled to −60 °C was significantly lower than that of oocytes cooled to −35 °C. The overall results indicated that observations of IIF obtained from cryomicroscopy are limited to detection of larger amounts of ice within the cells. Although the amount of cellular ice may have been reduced by one of the programmes, fertilization was reduced significantly; suggesting that there is no correlation between the presence of intracellular ice and post-thaw fertilization rate. Therefore, oyster oocytes may be more susceptible to the effect of high solute concentrations and cell shrinkage than intracellular ice under the studied conditions.     

High genetic load in the Pacific oyster Crassostrea gigas

The causes of inbreeding depression and the converse phenomenon of heterosis or hybrid vigor remain poorly understood despite their scientific and agricultural importance. In bivalve molluscs, related phenomena, marker-associated heterosis and distortion of marker segregation ratios, have been widely reported over the past 25 years. A large load of deleterious recessive mutations could explain both phenomena, according to the dominance hypothesis of heterosis. Using inbred lines derived from a natural population of Pacific oysters and classical crossbreeding experiments, we compare the segregation ratios of microsatellite DNA markers at 6 hr and 2-3 months postfertilization in F(2) or F(3) hybrid families. We find evidence for strong and widespread selection against identical-by-descent marker homozygotes. The marker segregation data, when fit to models of selection against linked deleterious recessive mutations and extrapolated to the whole genome, suggest that the wild founders of inbred lines carried a minimum of 8-14 highly deleterious recessive mutations. This evidence for a high genetic load strongly supports the dominance theory of heterosis and inbreeding depression and establishes the oyster as an animal model for understanding the genetic and physiological causes of these economically important phenomena.     

Characterization of genes involved in ceramide metabolism in the Pacific oyster (Crassostrea gigas)

ABSTRACT: BACKGROUND: The lipid signaling molecule, ceramide, is a key component of the vertebrate stress response, however, there is limited information concerning its role in invertebrate species. In order to identify genes involved in ceramide metabolism in bivalve molluscs, Pacific oyster genomic resources were examined for genes associated with ceramide metabolism and signaling. RESULTS: Several genes were identified including full-length sequences characterized for serine palmitoyltransferase-1, 3-ketodihydrosphingosine reductase, acid ceramidase, and ceramide glucosyltransferase. Genes involved in ceramide synthesis and metabolism are conserved across taxa in both form and function. Expression analysis as assessed by quantitative PCR indicated all genes were expressed at high levels in gill tissue. The role of the ceramide pathway genes in the invertebrate stress response was also explored by measuring expression levels in adult oysters exposed to Vibrio vulnificus. Two genes demonstrated increased expression during the bacterial challenge: a gene involved in hydrolytic breakdown of ceramide (acid ceramidase) and a gene involved in de novo generation of ceramide (3-ketodihydrosphingosine reductase), suggesting a possible role of ceramide in the invertebrate stress and immune responses. CONCLUSIONS: In silico and laboratory results support that Pacific oysters have the basic components of the ceramide metabolism pathway. These results also indicate that ceramide may have analogous functions in vertebrates and invertebrates. The gene expression pattern of acid ceramidase and 3-kethodihydrosphingosine reductase in response to bacterial exposure especially supports that ceramide and sphingolipid metabolism may be involved in the oyster's stress and/or immune responses.     

Cloning and mRNA expression of antioxidant enzymes in the Pacific oyster, Crassostrea gigas in response to cadmium exposure

Cadmium (Cd) is one of the most toxic heavy metal pollutants in the aquatic environment and can induce the formation of reactive oxygen species (ROS) that cause oxidative stress. In present study, we cloned catalase (CAT) and glutathione peroxidase (GPX) cDNA, and investigated its time- and dose-related effects of three Cd concentrations (0.01, 0.05 or 0.1 ppm) on mRNA levels of antioxidant enzymes (superoxide dismutase (SOD), CAT, GPX) in the gill and changes enzyme levels in the hemolymph of the Pacific oyster, Crassostrea gigas. The cDNA indentified encoded proteins of 516 and 244 amino acids corresponding to CAT and GPX, respectively. BLAST analysis from other species indicated that the residues essential to the enzymatic function of CAT and GPX proteins of C. gigas are highly conserved. Cd treatment significantly increased antioxidant enzyme mRNA expression in the gill in a time- and dose-dependent manner. The mRNA expression at 0.1 ppm Cd concentration increased up to 3 days (CAT, GPX) or 7 days (SOD) and then decreased by 7 days (CAT, GPX) or 11 days (SOD). Aspartate aminotransferase, alanine amintransferase and hydrogen peroxide (H(2)O(2)) concentrations levels increased significantly with exposure to 0.05 or 0.1 ppm Cd for 7 days. These results suggest that antioxidant enzymes play important roles in the physiological changes related to metabolism and cell protection that occur in Pacific oysters exposed to Cd.     

Metabolic adjustments in the oyster Crassostrea gigas according to oxygen level and temperature

The purpose of this study was to examine the responses of the oyster Crassostrea gigas to oxygen levels at subcellular and whole organism levels. Two experiments were carried out. The first experiment was designed to measure the clearance and oxygen consumption rates of oysters exposed at different concentrations of oxygen at 15, 20 and 25°C for 20 h. The goal of this first part was to estimate the hypoxic threshold for oysters below which their metabolism shifts towards anaerobiosis, by estimating the oxygen critical point (PcO₂) at 15, 20 and 25°C. The second experiment was carried out to evaluate the metabolic adaptations to hypoxia for 20 days at three temperatures: 12, 15 and 20°C. The metabolic pathways were characterized by the measurement of the enzymes pyruvate kinase (PK) and phosphoenolpyruvate carboxykinase (PEPCK), the alanine and succinate content and the adenylate energy charge. Respiratory chain functioning was estimated by the measurement of the activity of the electron transport system (ETS). The values of PcO₂ were 3.02±0.15, 3.43±0.20 and 3.28±0.24 mg O₂ l^-1 at 15, 20 and 25°C, respectively. In whole oysters, hypoxia involved the inhibition of PK whatever the temperature, but PEPCK was not stimulated. Succinate accumulated significantly only at 12°C and alanine at 12 and 15°C. A negative relationship between the PK activity and the alanine content was only found in hypoxic oysters. Finally, hypoxia increased significantly the activity of ETS. With high PcO₂ values, the metabolic depression occurred quickly, showing that oysters had a low capacity to regulate their respiration when oxygen availability is reduced, particularly in the summer.     

Xenobiotic biotransformation in the Pacific oyster (Crassostrea gigas)

1. Oyster visceral mass and gill tissues possessed measurable flavin-containing monooxygenase (FMO) activity. 2. FMO activity was confirmed in visceral mass microsomes by oxygen uptake experiments utilizing various nitrogen and sulfur-containing chemicals along with measurement of N,N-dimethylaniline (DMA) N-oxidase and methimazole oxidation activities. DMA N-oxidase and methimazole oxidation activities also were present in gill microsomes. 3. Excluding oyster gill methimazole oxidation, there were no consistent seasonal differences in FMO activity in oyster gill or visceral mass microsomes. 4. Although lacking spectral evidence for cytochrome P-450, a peak at 418 nm was observed along with NADPH-cytochrome c reductase activity in visceral mass and gill microsomes suggesting the presence of a denatured cytochrome P-450 system. 5. NADPH-independent benzo(a)pyrene hydroxylase (BPH) activity was observed in both oyster visceral mass and gill microsomes suggesting a co-oxidation pathway possibly involving a one electron transfer of oxygen from a lipid hydroperoxide.     

Reproductive patterns of the Pacific oyster Crassostrea gigas in France

Summary

In France, national management programs focus research on understanding reproductive factors in Crassostrea gigas to confront problems of the oyster industry. However, little information has been documented in which reproductive patterns include sexual changes. The reproductive cycle of oysters at three sites of the Atlantic coast of France was examined from 1996 to 1998, and the seasonal variations in oocyte size-frequencies, and sex ratio were described. The results showed a synchronism within the population concerning reproductive behavior. Young oocytes are generated after spawning and show no apparent changes during winter. Growth of oocytes begins in spring and cells reach maturity in April-May and are ready for a single spawning season in June-July. Oocytes that were not released during spawning are reabsorbed within the gonad. The significant difference between sites is that spawning occurred 1 month later in the southern area. A modal analysis showed that oocyte populations in the sample individuals are primordially bimodal, but with polymodal occurrences in June-July, in some cases. Irregular alternative sexuality was detected at all sites, and hermaphrodites appear to be a transition phase that allows changes from male to female during early spring. Previous observations, together with the study of the development of oocyte cohorts over time, permit a hypothetical model concerning the kinetics of gametogenesis in C. gigas. The model suggests that primary oocytes are generated from energy supplied from degenerating, as well as young oocytes that do not reach the mature stage within the gonad during autumn-winter. It seems that, during vitellogenesis, there is disintegration of smaller cells coupled with transfer of energy to the larger oocytes, which continue to grow and mature.     

Polymorphism of metallothionein genes in the Pacific oyster Crassostrea gigas as a biomarker of response to metal exposure.

Quantification of metallothioneins (MTs) is classically associated with a cellular response to heavy metal contamination and is used in the monitoring of disturbed ecosystems. Despite the characterization of several MT genes in marine bivalves, only a few genetic studies have used MT genes as potential biomarkers of pollution. The aim of this study was to assess whether MT gene polymorphism could be used to monitor exposure of the Pacific oyster Crassostrea gigas to heavy metals and to develop specific genetic markers for population genetic studies in relation to environmental stress. The polymorphism of two exons of the C. gigas MT gene CgMT1 were studied using polymerase chain reaction single-strand conformation polymorphism (PCR-SSCP) in both field populations exposed to various metals concentrations and in experimentally exposed populations. High frequencies of two SSCP types in exons 2 and 3 of the CgMT1 gene have found to be significantly associated with tolerance to metals in experimental and field oyster populations. The use of MT1 gene polymorphism in C. gigas as in the present study should therefore be of high ecological relevance. In conclusion, the analysis of the types in these two CgMT1 gene exons, which can confer a greater tolerance to heavy metals, can constitute a good biomarker of effect of the presence of heavy metals in ecosystems.     

Ovarian and sperm regulatory peptides regulate ovulation in the oyster Crassostrea gigas

For more than six decades, several studies have shown that genital products to entering the mantle cavity via the incurrent siphon, initiate in oyster, strong and rhythmic contractions of the adductor muscle (AM). In order to characterize the regulatory peptides capable of triggering AM contractions, we focused on the identification of putative myotropic peptides from genital products. Two experimental approaches were developed. The first one, based on a mass spectrometry screening of the male genital products, led to the identification of the tetrapeptide APGWamide. This neuropeptide was also detected in the seminal secretions of the cephalopod Sepia officinalis. In this species, APGWamide is directly involved in the oocyte transport. In Crassostrea, in vitro bioassay demonstrated that APGWamide modulates the AM contractions that insure the release of oocytes in the external medium. Exposure of oysters to a physiological concentration of APGWamide triggered repetitive shell closures. The second experimental approach was based on the monitoring of HPLC purification by a myotropic bioassay using the cuttlefish oviduct contractions as a target. The successive purification steps of the acidic extraction of ovaries from mature female oysters, led to the characterization of the hexapeptide PIESVD. When applied to mature female oysters, this peptide triggered the increase of shell closure frequency. The activity of these two regulatory peptides is the first experimental evidence of a peptidergic control of egg-laying in oyster. APGWamide and PIESVD could be used, in commercial and experimental hatcheries, for the identification of mature females to be selected for in vitro fertilization.     

First evidence of laccase activity in the Pacific oyster Crassostrea gigas

Phenoloxidases (POs) are a family of enzymes including tyrosinases, catecholases and laccases, which play an important role in immune defence mechanisms in various invertebrates. The aim of this study was to thoroughly identify the PO-like activity present in the hemolymph of the Pacific oyster Crassostrea gigas, by using different substrates (i.e. dopamine and p-phenylenediamine, PPD) and different PO inhibitors. In order to go deeper in this analysis, we considered separately plasma and hemocyte lysate supernatant (HLS). In crude plasma, oxygraphic assays confirmed the presence of true oxidase activities. Moreover, the involvement of peroxidase(s) was excluded. In contrast to other molluscs, no tyrosinase-like activity was detected. With dopamine as substrate, PO-like activity was inhibited by the PO inhibitors tropolone, phenylthiourea (PTU), salicylhydroxamic acid and diethyldithio-carbamic acid, by a specific inhibitor of tyrosinases and catecholases, i.e. 4-hexylresorcinol (4-HR), and by a specific inhibitor of laccases, i.e. cetyltrimethylammonium bromide (CTAB). With PPD as substrate, PO-like activity was inhibited by PTU and CTAB. In precipitated protein fractions from plasma, and with dopamine and PPD as substrates, PTU and 4-HR, and PTU and CTAB inhibited PO-like activity, respectively. In precipitated protein fractions from hemocyte lysate supernatant, PTU and CTAB inhibited PO-like activity, independently of the substrate. Taken together, these results suggest the presence of both catecholase- and laccase-like activities in plasma, and the presence of a laccase-like activity in HLS. To the best of our knowledge, this is the first time that a laccase-like activity is identified in a mollusc by using specific substrates and inhibitors for laccase, opening new perspectives for studying the implication of this enzyme in immune defence mechanisms of molluscs of high economic value such as C. gigas.     

Assessment of female gamete quality in the Pacific oyster Crassostrea gigas

Summary

The possible relationship between certain oocyte and embryo characteristics and larvae viability was investigated with reference to the following aspects: (1) morphological—oocyte diameter and shape; (2) cytological—overall ultrastructure and membrane integrity; (3) biochemical—content of lipids, proteins and carbohydrates; and (4) physiological—respiration. The rate of survival and incidence of abnormality were estimated 24 h after fertilization. The first results showed that 80–90% of oocytes were cytologically viable before fertilization. Eighty to 90% of oocytes are apparently viable before fertilization on the basis of staining with Trypan blue, but this parameter shows little correlation with larval viability. However, Trypan blue staining is of value in allowing the recognition of oocytes with damaged membranes. Respiration was measured for unfertilized oocytes 5 min after stripping, after 6 h, and for 3-h embryos. Positive correlations were found between the O₂-consumption of embryos and both the rate of fertilization and the hatching rate of 24-h larvae. In contrast, no correlation was found between hatching parameters and the O₂-consumption of unfertilized oocytes. These results suggest that embryos possess quality indicators, relating to metabolic characteristics, which can be quantified more easily than those of oocytes.     

Ontogenetic variations of hydrolytic enzymes in the Pacific oyster Crassostrea gigas

Occurrence and level of hydrolytic enzymatic activity (proteases, glycosidases, phosphatases, lipases, and esterases) were studied in oocytes, larvae, juveniles, and adult haemolymph of the Pacific oyster Crassostrea gigas. Samples were obtained as oocyte lysate supernatant, larval homogenate supernatant, juvenile homogenate supernatant, haemocyte lysate supernatant, and plasma. The presence of enzymes was demonstrated by colorimetric and lysoplate assay techniques. Between stages, significant differences in enzymatic activity determined by the colorimetric technique were found. Higher levels of enzymatic activity were found in the adult stage. Lysozyme-like activity was not found in oocytes, but was present in larvae, juveniles, and adults. In larvae, the highest lysozyme-like activity was in 3-d larvae. Juveniles had a 48-fold higher level of lysozyme-like activity, compared with 20-h larvae and was six-fold higher compared with 3-d larvae. In adults, lysozyme-like activity had a five-fold higher level in haemocyte lysate supernatant compared with plasma and was 98-fold higher compared with 20-h larvae. As determined with the API ZYM kit, 19 hydrolytic enzymatic activities were present, in oocytes, larvae, juveniles, and adult haemolymph of C. gigas. The presence of important lysozyme-like activity was confirmed from trochophora larvae (20 h) to adult stages.     

Branchial acid-base transfers in the euryhaline oyster toadfish during exposure to dilute sea water

Sea water dilution (to 5% and 1% of sea water) induced a reversible, plasma metabolic acidosis in a euryhaline teleost (the oyster toadfish, Opsanus tau ). Net H+ transfer between the fish and the water decreased in both low salinity exposure (LSE) groups during the first week of exposure and ΔH⁺ returned to normal after ∼10 days of 5% LSE, but not during 1% LSE.