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.     

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.     

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.     

Isolation of sperm bindin from the oyster (Crassostrea gigas)

A method was devised for isolating the insoluble content of the acrosome granule of sperm of the oyster Crassostrea gigas. The method involves the dissolution of the entire cell, except for the acrosome granule, in the detergent sodium lauroyl sarcosinate (sarcosy I). The isolated acrosome granule content is ring-shaped and is 84% protein by weight. SDS-polyacrylamide gel electro-phoresis of this material yields from 1 to 4 bands of 65,000; 53,000; 43,000 and 34,000 apparent molecular weight, all of which stain with the PAS reaction indicating the material is a glycoprotein. The 65,000 molecular weight component is always present, but the presence of the other three bands varies with each preparation. The isolated acrosome granules agglutinate formaldehyde-fixed oyster eggs. A trypsin-generated glycopeptide digest of oyster egg surfaces inhibits the agglutinin activity of the isolated acrosome granules. We propose that the acrosomal glyco-protein material is oyster sperm bindin which functions as the adhesive substance bonding the sperm to the egg during fertilizaion.     

太平洋牡蛎酪氨酸酶基因家族的系统发生分析

文章利用生物信息学方法对太平洋牡蛎(Crassostrea gigas Thunberg)酪氨酸酶基因家族的氨基酸序列特征、分类及系统发生进行了分析。结果表明, 太平洋牡蛎酪氨酸酶基因家族在进化过程中存在基因扩张现象, 其主要方式是基因重复。太平洋牡蛎酪氨酸酶可分为3种类型：分泌型 (Type A), 胞内型 (Type B)和具跨膜结构域型 (Type C)。根据太平洋牡蛎酪氨酸酶进化树分析, 发现Type A酪氨酸酶中, tyr18与其他Type A酪氨酸酶分化较大, 可能是较早分化出来的酪氨酸酶; Type B酪氨酸中的tyr2和tyr9以及Type C中的tyr8为较早分化出的酪氨酸酶。系统发生树分析发现太平洋牡蛎酪氨酸酶的聚类受酪氨酸酶类型以及基因位置的影响, 其分泌型酪氨酸酶首先与头足类分泌型酪氨酸酶聚在一起, 然后与线形动物门分泌型酪氨酸酶聚在一起, 与腔肠动物门分泌型酪氨酸酶分化明显。太平洋牡蛎胞内型酪氨酸酶自身分化较大, 总体上与线性动物门、其他软体动物胞内型酪氨酸酶聚为一支, 与扁形动物门、脊索动物门、腔肠动物门胞内型酪氨酸酶分化较大。太平洋牡蛎具跨膜结构域型酪氨酸酶与扁形动物门、环形动物门以及脊索动物门具跨膜结构域型酪氨酸酶分化明显, 与合浦珠母贝具跨膜结构域型酪氨酸酶聚为一支。这表明双壳类的Type C型酪氨酸酶与其他物种的同源酶的进化差异较大。文章首次探讨了太平洋牡蛎酪氨酸酶家族分类、分化及系统发生, 以期对太平洋牡蛎酪氨酸酶基因家族的理论研究和实际应用提供依据。     

Characterization of 79 microsatellite DNA markers in the Pacific oyster Crassostrea gigas

We characterized 79 microsatellite DNA markers, which were obtained from genomic libraries enriched for CA, GA, ATG and TAGA motif repeats, in the Pacific oyster Crassostrea gigas. For eight F₁ grandparents or great‐grandparents of mapping families, the average heterozygosity, 0.705, and average number of alleles per locus, 5.7, did not vary among motif‐repeat or motif‐complexity categories. Non‐amplifying polymerase chain reaction null alleles, which were confirmed by segregation in the mapping families, were detected at 41 (51.9%) of the 79 loci. Cross‐species amplifications from C. angulata, C. sikamea, C. ariakensis and C. virginica showed a precipitous decline with distance from the focal species C. gigas.     

Development, characterization, and inheritance of 113 novel EST-SSR markers in the Pacific oyster (Crassostrea gigas)

A total of 113 novel EST-derived simple sequence repeat (EST-SSR) markers were developed in the Pacific oyster (Crassotrea gigas). Polymorphisms of these markers were evaluated in a wild population of 30 individuals. The number of alleles per locus ranged from 2 to 27 with an average of 6.3, and the observed and expected heterozygosities varied from 0 to 0.9667 and from 0.0333 to 0.9701, respectively. Mendelian segregations were tested for 24 of the markers that were polymorphic in one family produced by single-pair mating. Null alleles were discovered at four loci. Nine tests of segregation ratios revealed significant departures from expected Mendelian ratios. As a useful addition to the collection of the microsatellites that are now available for C. gigas, these EST-SSR markers will help the advance in investigation of QTL mapping and genetic diversity in this species.     

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.     

Development,inheritance and evaluation of 55 novel single nucleotide polymorphism markers for parentage assignment in the Pacific oyster (Crassostrea gigas)

Expressed sequence tags (ESTs) are essential to ascertain gene function, but also to identify polymorphic gene-associated single nucleotide polymorphisms (SNPs) (type I markers) suitable for map construction and population analysis in the Pacific oyster (Crassostrea gigas). In this study, a total of 48,769 putative SNPs were detected from 46,171 ESTs of the Pacific oyster. Fifty-five gene-derived SNPs were isolated and characterized by means of high resolution melting analysis. The observed and expected heterozygosities ranged from 0.063–0.563 and 0.091–0.448, with an average of 0.284 and 0.279, respectively. The SNPs were tested on 6 families of C. gigas for examination of inheritance mode of SNPs. One hundred and thirty-two tests of segregation ratios at 48 loci revealed 30 (22 %) significant departures from expected Mendelian ratios, but no null allele was detected. The power of these SNPs in parentage assignment was evaluated, and the real data demonstrated that 17 % of all real offspring were unambiguously assigned parents with 30 SNPs, and 100 % of the offspring were correctly allocated to their parents when 40 or more SNPs were used. The results obtained in this study suggest that gene-derived SNPs will complement the currently available microsatellite markers and may be useful for comparative mapping, marker-assisted selection and evolutionary studies.     

A study of diagnostic methods for Marteilioides chungmuensis infections in the Pacific oyster Crassostrea gigas

The eggs of the Pacific oyster, Crassostraea gigas, become infertile when infected by the parasite Marteilioides chungmuensis. Histologically, M. chungmuensis infects the oyster oocyte cytoplasm, and the ovaries take on a "lumpy" appearance once infected, which lowers commercial value of the oyster. This has a negative economic impact on oyster farms in South Korea and Japan. In this study, we compared traditional diagnostic methods (histology) with two molecular-based methods (polymerase chain reaction [PCR] amplification and in situ hybridization [ISH]) to identify M. chungmuensis-infected oysters. The efficacy of PCR and ISH to identify M. chungmuensis-infected oysters was compared to that of routine histology in 100 oysters. Thirty infections were identified using PCR and 16 using histology, whereas 31 infections were identified using ISH. The ISH and PCR assays were more sensitive compared to using histology with standard epidemiological methods. We strongly recommend that early parasitic invasion should be monitored with PCR/ISH methodologies as a basis for developing effective diagnostic techniques to identify M. chungmuensis-infected oysters.     

Potential of genomic selection for improvement of resistance to ostreid herpesvirus in Pacific oyster (Crassostrea gigas)

In genomic selection (GS), genome-wide SNP markers are used to generate genomic estimated breeding values for selection candidates. The application of GS in shellfish looks promising and has the potential to help in dealing with one of the main issues currently affecting Pacific oyster production worldwide, which is the ‘summer mortality syndrome’. This causes periodic mass mortality in farms worldwide and has mainly been attributed to a specific variant of the ostreid herpesvirus (OsHV-1). In the current study, we evaluated the potential of genomic selection for host resistance to OsHV-1 in Pacific oysters, and compared it with pedigree-based approaches. An OsHV-1 disease challenge was performed using an immersion-based virus exposure treatment for oysters for 7 days. A total of 768 samples were genotyped using the medium-density SNP array for oysters. A GWAS was performed for the survival trait using a GBLUP approach in blupf 90 software. Heritability ranged from 0.25 ± 0.05 to 0.37 ± 0.05 (mean ± SE) based on pedigree and genomic information respectively. Genomic prediction was more accurate than pedigree prediction, and SNP density reduction had little impact on prediction accuracy until marker densities dropped below approximately 500 SNPs. This demonstrates the potential for GS in Pacific oyster breeding programmes, and importantly, demonstrates that a low number of SNPs might suffice to obtain accurate genomic estimated breeding values, thus potentially making the implementation of GS more cost effective.     

Analysis of the effects of Perkinsus marinus proteases on plasma proteins of the Eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas).

We employed two in vitro buffer systems to determine the potential pathogenic effects of Perkinsus marinus serine proteases on the plasma proteins of the eastern oyster (Crassostrea virginica) and the Pacific oyster (Crassostrea gigas). Specifically, this study characterized the oyster plasma protein targets of P. marinus proteases. Additionally, protease-specific inhibitory activity was revealed upon comparison of artificial (PBS) and endogenous (plasma-based) diluents employed during protease digestions. It was found that a C. virginica plasma protein of approximately 35 kDa was eliminated when a standard buffer (PBS) was used as a diluent; however, this protein was preserved when a low-molecular-weight, plasma-based, diluent was used. The results strongly indicate that low-molecular-weight inhibitors of P. marinus proteases are present in oyster plasma. A control (nonparasitic) serine protease, alpha-chymotrypsin, was employed to ascertain the specificity of the protease inhibitors. Although alpha-chymotrypsin possesses ample proteolytic activity for C. virginica plasma proteins, the anti-proteases could specifically inhibit only P. marinus proteases. Such specificity of anti-protease activity is not uncommon among low-molecular-weight serine proteases. The hemolymph target protein was isolated by 2D electrophoresis and isoelectrically isolated for further characterization by N-terminal amino acid sequencing.     

Comparison of quantitative gonad maturation scales in a temperate oyster (Crassostrea gigas) and a sub-tropical oyster (Crassostrea corteziensis)

Quantitative scales to evaluate maturity of female gonads were compared for a temperate oyster, Crassostrea gigas and a tropical oyster, Crassostrea corteziensis grown in the same locality. C. gigas had well-defined maturation and spawning periods and a resting phase in winter; in C. corteziensis mature individuals occurred most of the year and there were several spawning peaks. Of the quantitative scales used here, average oocyte diameter and gonad coverage area, much used for C. gigas, and ovary maturity index, less used, were inadequate to distinguish the reproductive pattern of C. corteziensis, since they both skewed the degree of maturation in vitellogenic stages in favor of C. gigas. Maximum oocyte diameter and maximum cytoplasm area were different among species at vitellogenic stages and also in previtellogenic stages. Nucleus:cytoplasm ratio was significantly different in previtellogenic and spawned stages between C. gigas and C. corteziensis. The Index of gonadal development was skewed in favor of C. gigas in postvitellogenic stage. The only scale that was comparable between species was reproductive potential, but it also was one of the most laborious. Other ordinal scales commonly used, such as a visual external evaluation of the gonad, only classified correctly a quarter of the stages.     

Exploiting EST databases for the development and characterization of EST-SSRs in the Pacific oyster (Crassostrea gigas)

A total of 147 microsatellite-containing expressed sequence tags (ESTs) (3.63%) were detected from 4053 ESTs of the Pacific oyster (Crassostrea gigas) in GenBank. The average density of simple sequence repeats (SSRs) was 1 per 8.25 kb of EST after redundancy elimination. Dinucleotide repeat motifs appeared to be the most abundant type. Sixteen new polymorphic EST-SSRs were developed. The number of alleles per locus varied from 3 to 12, with an average of 5.9 alleles per locus. Marker transferability was tested on 2 other Crassostrea species, and 14 loci gave successful amplifications in both species. Twenty EST-SSRs were tested on 3 families of C. gigas for examination of inheritance mode of EST-SSRs. Thirty-five tests of segregation ratios revealed 5 significant departures from expected Mendelian ratios, 4 of which confirmed Mendelian expectations when accounting for the presence of null alleles. Null alleles were detected at 3 loci (15.0%) of the 20 loci, and the frequency of null alleles at EST-SSRs was lower than that in genomic SSRs in C. gigas. The results obtained in this study suggest that C. gigas EST-SSRs will complement the currently available genomic SSR markers and may be useful for comparative mapping, marker-assisted selection, and evolutionary studies.     

Spatio-temporal variations in biological performances and summer mortality of the Pacific oyster Crassostrea gigas in Normandy (France)

Mortality and biological performances of half-grown Crassostrea gigas were studied from spring 2000 to autumn 2001 at six instrumented stations located in two areas (Gefosse and Grandcamp) of the Bay of Veys (Normandy). Shell and meat growth, condition indexes and a macroscopic maturity index were determined on oysters deployed at the six stations in order to assess spatial variability in the influence of environmental conditions. In addition, histological and biochemical analyses were performed in order to determine the sex and establish the reproductive cycle (at all six sites) and the biochemical composition (at four stations). The data set including monthly mean temperatures and data provided by examination of 2,837 oysters were analysed by Principal Component Analysis and a Hierarchical Ascending Clustering which resulted in the formation of four clusters. The highest station on the shoreline belonged to a cluster characterized notably by low total weight due to a short immersion/feeding period, whereas all other stations belonged to another single cluster. Nevertheless, various biological differences were found between these stations, e.g. the reproductive cycle was generally synchronized throughout the bay but some differences relative to spawning occurrence were observed. In 2000, oyster mortality was higher at Gefosse than at Grandcamp, the latter being a more marine area. In 2001, oyster mortalities were significantly higher and all stations were strongly affected. In the Bay of Veys, oyster biological performances and mortality thus showed spatio-temporal variations which were worthy to be discussed.