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.     

Haplosporidiosis of the Pacific oyster, Crassostrea gigas.

Haplosporidan parasites were observed in 10/100 spat and 1/171 adult Pacific oysters, Crassostrea gigas, reared in Matsushima Bay, Japan. Eight of the infected spat contained mild to severe plasmodial infections. The multinucleated plasmodia were 6-12 microm x 7-15 microm and were associated with an infiltration of hemocytes that occurred throughout the vesicular connective tissues of all infected oysters. Two oysters, one adult and one spat, contained advanced sporogonic infections. These were characterized by the presence of sporocysts and immature and mature operculated spores that measured 5.6-6.0 microm x 6.0-8.0 microm and were found exclusively within the digestive tubule epithelium. Electron microscopic examination revealed that mature spores contained a hinge operculum, striated and layered wall, spherule, single nucleus, and haplosporosome formative regions. Parasite morphology and infection pattern closely resemble that of Haplosporidium nelsoni, a pathogen of American oysters (C. virginica).     

A tolloid homologue from the Pacific oyster Crassostrea gigas

The genes governing mesoderm specification have been extensively studied in vertebrates, arthropods and nematodes. The latter two phyla belong to the Ecdysozoan clade but little is understood of the role that these genes might play in the development of the other major protostomal clade, the Lophotrochozoa. As part of a wider project to analyze the functions associated with transforming growth factor beta superfamily members in Lophotrochozoa, we have cloned a gene encoding a tolloid homologue from the bivalve mollusc Crassostrea gigas. Tolloid is a key developmental protein that regulates the activity of bone morphogenetic proteins (BMPs). We have determined the intron-exon structure of the gene encoding C. gigas tolloid and have compared it with those of homologous genes from both protostomes and deuterostomes. In order to analyze the functionality of oyster tolloid the zebrafish embryo has been employed as a reporter organism and we show that over-expression of this protein results in the ventralization of zebrafish embryos at 24h post fertilization. The expression of the C. gigas tolloid gene during embryonic and larval development as well as in adult tissues is also explored.     

Chromosome inheritance in triploid Pacific oyster Crassostrea gigas Thunberg

Reproduction and chromosome inheritance in triploid Pacific oyster (Crassostrea gigas Thunberg) were studied in diploid female x triploid male (DT) and reciprocal (TD) crosses. Relative fecundity of triploid females was 13.4% of normal diploids. Cumulative survival from fertilized eggs to spat stage was 0.007% for DT crosses and 0.314% for TD crosses. Chromosome number analysis was conducted on surviving progeny from DT and TD crosses at 1 and 4 years of age. At Year 1, oysters from DT crosses consisted of 15% diploids (2n=20) and 85% aneuploids. In contrast, oysters from TD crosses consisted of 57.2% diploids, 30.9% triploids (3n=30) and only 11.9% aneuploids, suggesting that triploid females produced more euploid gametes and viable progeny than triploid males. Viable aneuploid chromosome numbers included 2n+1, 2n+2, 2n+3, 3n-2 and 3n-1. There was little change over time in the overall frequency of diploids, triploids and aneuploids. Among aneuploids, oysters with 2n+3 and 3n-2 chromosomes were observed at Year 1, but absent at Year 4. Triploid progeny were significantly larger than diploids by 79% in whole body weight and 98% in meat weight at 4 years of age. Aneuploids were significantly smaller than normal diploids. This study suggests that triploid Pacific oyster is not completely sterile and cannot offer complete containment of cultured populations.     

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.     

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.     

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.     

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.     

Fosmid library construction and end sequences analysis of the Pacific oyster,Crassostrea gigas

The Pacific oyster (Crassostrea gigas) is globally distributed and is one of the most commercially and ecologically important marine organisms. However, little is known about the genome of this species. In this study, a C. gigas fosmid library was constructed that contains 459,936 clones with an average insert size of approximately 40 kb, representing 22.34-fold haploid genome equivalents. End sequencing generated 90,240 fosmid end sequences (FESs) with an average length of 384.27 base pairs (bp), covering approximately 2.58% of the Pacific oyster genome. The FESs were subsequently assembled and annotated, resulting in 6332 sequences with predicted open reading frames≥300 and 1,189,100 bp repeats. Furthermore, a total of 3200 microsatellite repeats were identified, and dinucleotide repeats were found to occur most abundantly, with AG and AAT being the most abundant repeat class of dinucleotides and trinucleotides. We also found that the repeat number was generally negatively proportional to the repeat element length. Microsatellites composition between the transcribed sequences and genomic sequences was shown to be different. Point mutations of microsatellite were non-random and underwent strong selection stress. Overall, a comprehensive sequence resource for the Pacific oyster was created, including annotated transposable elements, tandem repeats, protein coding sequences and microsatellites. These initial findings will serve as resources for further in-depth studies of physical mapping, gene discovery, microsatellite marker developing and evolution studies.     

Haplosporidiosis in the Pacific oyster Crassostrea gigas from the French Atlantic coast

Two cases of haplosporidian infection occurred during 1993 in Pacific oysters Crassostrea gigas from the French Atlantic coast. The localization and ultrastructure of the plasmodia are described. In situ hybridization of infected tissue sections was conducted with DNA probes for oyster-infecting haplosporidians. The Haplosporidium nelsoni-specific DNA probe MSX1347 hybridized with the C. gigas parasite, and the H. costale-specific probe SSO1318 did not hybridize. Total genomic DNA was extracted from the infected tissue sections for polymerase chain reaction (PCR) amplification of the haplosporidian. PCR amplifications with H. nelsoni-specific primers and with 'universal' actin primers did not yield the expected products of 573 and 700 bp, respectively. A series of primers was designed to amplify short regions of small subunit ribosomal DNA (SSU rDNA) from most haplosporidians. The primers encompass a highly variable region of the SSU rDNA and did not amplify oyster DNA. PCR amplification of the infected C. gigas genomic DNA with these primers yielded the expected-sized product from the primer pair targeting the shortest region (94 bp). This PCR product was sequenced and it was identical to the corresponding SSU rDNA region of H. nelsoni.     

Bioinformatic mining of EST-SSR loci in the Pacific oyster, Crassostrea gigas

A set of expressed sequence tag-simple sequence repeat (EST-SSR) markers of the Pacific oyster, Crassostrea gigas, was developed through bioinformatic mining of the GenBank public database. As of June 30, 2007, a total of 5132 EST sequences from GenBank were downloaded and screened for di-, tri- and tetra-nucleotide repeats, with criteria set at a minimum of 5, 4 and 4 repeats for the three categories of SSRs respectively. Seventeen polymorphic microsatellite markers were characterized. Allele numbers ranged from 3 to 10, and the observed and expected heterozygosity values varied from 0.125 to 0.770 and from 0.113 to 0.732 respectively. Eleven loci were at Hardy-Weinberg equilibrium (HWE); the other six loci showed significant departure from HWE (P < 0.01), suggesting possible presence of null alleles. Pairwise check of linkage disequilibrium (LD) indicated that 11 of 136 pairs of loci showed significant LD (P < 0.01), likely due to HWE present in single markers. Cross-species amplification was examined for five other Crassostrea species and reasonable results were obtained, promising usefulness of these markers in oyster genetics.     

Massive settlements of the Pacific oyster, Crassostrea gigas, in Scandinavia

The Pacific oyster (Crassostrea gigas) is an important aquaculture species world-wide. Due to its wide environmental tolerance and high growth rate, it has also become a successful invader in many areas, leading to major ecosystem changes. Low water temperatures were previously believed to restrict the establishment of Pacific oysters in Scandinavia. However, recent surveys reveal that the Pacific oyster is now established in many areas in Scandinavia. The biomass of oysters in the Danish Wadden Sea has increased dramatically between 2005 and 2007, large numbers were observed along the Swedish west coast from settlement in 2006, and in Norway, populations are established along the southwest coast to 60°N.     

Pathogenicity of the protozoan parasite Marteilioides chungmuensis in the Pacific oyster Crassostrea gigas

Marteilioides chungmuensis is an ovarian parasite that causes nodule-like structures to appear on the gonads of female Pacific oysters, Crassostrea gigas. It is known that the prevalence of infection increases in summer and decreases from autumn to spring. To investigate the decrease in prevalence of infection and pathogenicity of the parasite, a biopsy method was developed to detect infected oysters, which were then monitored to calculate the mortality rate. Mortality of infected oysters was recorded monthly and changes in reproductive development observed histologically. Compared with control groups, a significant difference in mortality was observed in infected oysters in September and October. Histological observations showed that infected oysters produced oocytes continuously, even in autumn when healthy oysters were reproductively inactive. This prolonged spawning activity of infected oysters resulted in nutritional wasting and mortality. From December onwards, however, almost all infected oysters survived, though the infection persisted. Infection intensity decreased gradually from December. Histological observations revealed that, in winter, infected oysters released infected and uninfected oocytes through the genital canal. The gonad subsequently degenerated and was replaced with connective tissue, as in normal, healthy spent oysters. The results revealed that prevalence of infection decreased from September to May. It is hypothesised that the decline in prevalence within the epizootic area in autumn occurred because infected oysters died and that the winter decrease was due to recovery from infection.     

Linkage maps of microsatellite DNA markers for the Pacific oyster Crassostrea gigas

We constructed male and female consensus linkage maps for the Pacific oyster Crassostrea gigas, using a total of 102 microsatellite DNA markers typed in 11-day-old larvae from three families. We identified 11 and 12 linkage groups in the male and female consensus maps, respectively. Alignment of these separate maps, however, suggests 10 linkage groups, which agrees with the haploid chromosome number. The male linkage map comprises 88 loci and spans 616.1 cM, while the female map comprises 86 loci and spans 770.5 cM. The male and the female maps share 74 loci; 2 markers remain unlinked. The estimated coverages for the consensus linkage maps are 79% for the male and 70-75% for the female, on the basis of two estimates of genome length. Ninety-five percent of the genome is expected to lie within 16 and 21 cM of markers on the male and female maps, respectively, while 95% of simulated minimum distances to the male and female maps are within 10.1 and 13.6 cM, respectively. Females have significantly more recombination than males, across 118 pairs of linked markers in common to the parents of the three families. Significant differences in recombination and orders of markers are also evident among same-sex parents of different families as well as sibling parents of opposite sex. These observations suggest that polymorphism for chromosomal rearrangements may exist in natural populations, which could have profound implications for interpreting the evolutionary genetics of the oyster. These are the first linkage maps for a bivalve mollusc that use microsatellite DNA markers, which should enable them to be transferred to other families and to be useful for further genetic analyses such as QTL mapping.     

First isolation of Nocardia crassostreae from Pacific oyster Crassostrea gigas in Europe

In summer 2006 an extensive mortality of Pacific oysters Crassostrea gigas occurred in Lake Grevelingen, the Netherlands. A sample of Pacific oysters was investigated for the presence of shellfish pathogens as potential causes of the mortality. Yellow-green lesions were observed in several oysters upon clinical inspection. Histopathology showed that 6 out of 36 oysters had a suspected bacterial infection, including 4 Nocardia-like infections. Two bacterial species, Vibrio aestuarianus and Nocardia crassostreae, were isolated from haemolymph samples and identified using PCR and sequencing of the 16S rRNA gene. This is the first isolation of N. crassostreae from shellfish in European waters. The near full-length 16S rRNA sequence of this Dutch Nocardia sp. isolate was identical to other known N. crassostreae isolates from the west coast of North America. The primary cause of oyster mortality was thought to be the physiological stress from environmental conditions, including prolonged high water temperatures and low oxygen levels. The multiple bacterial species isolated from the diseased Pacific oysters may have been a secondary cause.