Development of Novel Microsatellite DNA Markers from the Pacific Oyster Crassostrea gigas

We document the potential of novel microsatellites as a genetic tool in furthering our understanding of the Crassostrea gigas genetic structure. From the microsatellite-enriched libraries we constructed, 123 repeat regions that had sufficient sequence information to design polymerase chain reaction primer sets were isolated. From these, 9 primer pairs were screened in a C. gigas population of 67 individuals to evaluate the genetic variability. All but 1 of the 9 loci showed allelic variation (number of alleles, 2–20; observed heterozygosity, 0.119–0.925; unbiased expected heterozygosity, 0.139–0.914). Considerable discrepancy of genotypic proportions from the Hardy-Weinberg equilibrium was observed at 1 locus with an apparent heterozygote deficiency. Several loci were successfully amplified in 3 other related species with the appropriate allele size: 6 loci in C. sikamea, 4 loci in C. ariakensis, and 5 loci in C. nippona.     

Effects of extender composition, cooling rate and freezing on the fertilisation viability of spermatozoa of the Pacific oyster (Crassostrea gigas)

The aim of this research was to optimise protocols for freezing spermatozoa of the Pacific oyster. All the phases of the cryopreservation procedure (choice of cryoprotectant, cooling, freezing, and thawing) were studied in relation to the species of spermatozoa to restore on thawing the morphological and physiological characteristics of fresh semen. The choice of type and concentration of cryoprotectant in which semen is incubated before freezing is fundamental for a successful cryopreservation: the cryoprotectants (dimethylsulfoxide--Me(2)SO, ethylene glycol--EG, propylene glycol-PG, and glycerol in concentrations between 5 and 15%) were tested for their toxicity on the semen exposed up to 30 min at +26 degrees C (room temperature) by evaluating its ability to fertilise and the embryo development to the regular D larval stage. The best cryoprotectants, Me(2)SO, EG, and PG 5, 10, and 15% respectively, were used for the pre-cooling (adaptation/cooling) tests. Two different adaptation/cooling procedures were tested: (A) from +26 degrees C to 0-2 degrees C (2.6 degrees C/min) and (B) at +26 degrees C for 15 min. Lastly, using the cryoprotectants and the adaptation procedure (B) that had given the best results in the preceding stages of the experiment, four cooling rates were tested: 6, 11, 16, and 21 degrees C/min. It was seen that the semen that was incubated with EG 10%, adapted at +26 degrees C for 15 min, and then cooled at a rate of 6 degrees C/min showed a percentage of regular D larvae on thawing comparable to that of fresh semen (p > 0.05).     

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.     

Fine structure of the early stages of spermatogenesis in the Pacific oyster, Crassostrea gigas (Mollusca, Bivalvia)

The aim of this study is to describe the early stages of spermatogenesis of the Pacific oyster Crassostrea gigas using both light and electron microscopy. The gonad is formed by gonadal tubules invaginated in a connective tissue constituting a storage tissue. Myoepithelial cells surround each gonadal tubule and are associated with an acellular matrix delimiting the outer part of the tubule, the inner part is composed by intragonadal somatic cells associated with germinal lineage. Two types of spermatogonia are identified, where type I spermatogonia (Spg I) are large, scarce and pale cells leaned against the base of the tubule (nuclear diameter: 5.5+/-0.5 microm). Type II spermatogonia (Spg II) are clustered and dark cells which appear smaller than type I (nuclear diameter: 4.3+/-0.3 microm). The aspect of nuage-like material in cytoplasm is described from pale spermatogonia to primary spermatocytes (nuclear diameter: pachytene 3.6+/-0.3 microm, diplotene 3.4+/-0.3 microm), while no structure related to a chromatoid body was observed in oyster spermatocytes and spermatids.     

Setting tools for the early assessment of the quality of thawed Pacific oyster (Crassostrea gigas) D-larvae

Parameters used to assess the survival of larvae after cryopreservation generally misestimate the damages that prevent larval development. The objectives of the present study were to 1) define the reliability of the survival rate, assessed at 2 and 7 days post fertilization, to estimate Pacific oyster larval quality after thawing, and 2) select complementary tools allowing an early and reliable estimation of their quality. Oyster larvae were reared for 25 h after fertilization at 19 °C and cryopreserved at early D-stage. Then, thawed larvae were incubated in 2-L beakers. At 2 days post fertilization, the survival rate of thawed Pacific oyster larvae was lower than that of fresh larvae for only one experiment (Experiment 3) among the four identical experiments carried out in this work (Experiments 1-4). By contrast, the survival of thawed larvae, as assessed 7 days after fertilization, was lower than that of fresh larvae for the four experiments. These results confirm that the quality of thawed larvae is lower than that of fresh larvae and that the survival rate, estimated 2 days post fertilization, is not adapted to a reliable estimation of the subsequent development ability of thawed larvae. Then, complementary parameters were tested at 2 days: the movement characteristics (Experiments 1 and 2) and the morphologic features (Experiments 3 and 4) of thawed larvae. Compared to values observed on fresh larvae, the percentage of thawed motile larvae was different for only one experiment (Experiment 2) of the two. Compared to control, a reduced Average Path Velocity (VAP) of larvae (determined at the D-larval stage using a CASA-Computer Assisted Sperm Analysis-system) was observed after thawing for both experiments (Experiments 1 and 2), suggesting the ability of larval movement velocity to assess the decrease of the quality of thawed oyster larvae. Using an ASMA (Automated Sperm Morphology Analysis) device, a lower area of thawed larvae was observed, compared to control and for the two experiments (Experiments 3 and 4). By contrast, the Crofton perimeter of thawed larvae was lower than that of control larvae for only one experiment (Experiment 3) and no significant difference of circularity between fresh and thawed larvae was recorded for Experiments 3 and 4. In conclusion, changes in the movement velocity (assessed by CASA) and in the area (measured by ASMA) of D-larvae allow an early and reliable estimation of the quality of thawed Pacific oyster larvae.     

Commercial-scale sperm cryopreservation of diploid and tetraploid Pacific oysters, Crassostrea gigas

Cryopreservation of sperm from tetraploid organisms (the possession of four chromosome sets) is essentially unexplored. This is the first cryopreservation study to address sperm from tetraploid Pacific oysters, Crassostrea gigas, and addresses the commercial production of triploid oysters (three chromosome sets). Initial motility, refrigerated storage of undiluted sperm, osmolality of extender solutions, sperm concentrations, equilibration time, and cryoprotectants of propylene glycol and dimethyl sulfoxide were evaluated with sperm from diploid and tetraploid oysters. Unlike most teleost fishes, in which the duration of active motility is typically brief, the motility of sperm from oysters lasts for hours. The present study showed that responses to treatment effects by sperm from tetraploids were different from diploids. The majority of tetraploid experiments resulted in less than 10% motility after thawing and less than 5% fertilization. The highest fertilization obtained for thawed sperm was 96% for sperm from diploid oysters and 28% for sperm from tetraploid oysters. Differential responses to treatments by sperm from tetraploid and diploid oysters may be due to differences in gonadal development. However, the use of cryopreserved sperm from tetraploid Pacific oysters produced 100% triploid offspring by fertilization of eggs from diploid females as determined by flow cytometry of larvae. This study demonstrates that sperm from tetraploid oysters can be collected, frozen, and stored for production of triploid offspring.     

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.     

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.     

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.     

巢式甲基化特异性PCR检测肺癌病人WIF-1基因启动子区异常甲基化

为了检测肺癌患者血浆中WIF-I基因启动子区的甲基化状态,收集肺癌患者及健康对照者的血浆标本,采用巢式甲基化特异性PCR（nMSP）法检测WIF-I基因启动子区甲基化状态,并与普通甲基化特异性PCR（MSP）法进行了比较,结果在58例肺癌患者血浆样品中经nMSP法发现20例WIF-I基因启动子的过甲基化,用MSP法只检出10例,有吸烟史组WIF-1基因的甲基化率高于无吸烟史组（P〈0．05）．而20例正常对照血浆中都未检测到形胆J基因启动子的过甲基化;表明利用巢式MSP（nMSP）法检测外周血血浆中WIF-1基因启动子的甲基化,可为非损伤性筛选和早期诊断肺癌提供有价值的信息．     

Relationship between Marteilioides chungmuensis infection and reproduction in the Pacific oyster, Crassostrea gigas

Marteilioides chungmuensis, a protozoan paramyxean parasite, infects the oocytes of the Pacific oyster, Crassostrea gigas. The effects of infection on the reproductive cycle of C. gigas were investigated over two consecutive years at Okayama Prefecture, Japan. In male oysters, gonadal development began during February/March, maturity was achieved in June and spawning activity extended from July to September. In November and December, male oysters were not seen, probably because their gonads regressed to connective tissue and they transformed into undifferentiated oysters. By contrast, female oysters, in which parasite spore formation occurred, were still carrying oocytes until the following March and the spawning process of female oysters took 5 months longer than that of males in epizootic areas. The prevalence of M. chungmuensis infection increased from July to September, when most female oysters had their spawning period, and declined from October to the following April when oysters were at the spent stage. The prevalence of infection increased again in May of the following year and high prevalence was observed in the following July. When prevalence was compared between oysters of different age classes, higher prevalence was detected in older than in younger oysters. Histological examination showed that infected oysters produced oocytes continuously and spawned repeatedly from October to March, during which period healthy oysters were reproductively inactive. Parasites can infect the oocytes of infected oysters throughout the longer spawning period. These observations suggest that M. chungmuensis extends the reproductive period of infected oysters for its own reproductive benefit.     

Detection of Puccinia striiformis in Latently Infected Wheat Leaves by Nested Polymerase Chain Reaction

Stripe rust, caused by Puccinia striiformis f. sp. tritici ( Pst ), is one of the most devastating wheat diseases worldwide, especially in temperate regions with cool moist weather conditions. The identification of the pathogen in infected plants based on morphological or physiological criteria before sporulation is labour-intensive and time-consuming. To accelerate and simplify the process of detection, a nested Polymerase Chain Reaction (PCR) assay was developed for specific and sensitive detection of Pst . Specific primers Psta-Psts were designed according to a genome-specific sequence of Pst . In nested PCR, with a 10-fold dilution series of template DNA, the detection limit was 2 pg DNA in the first PCR with the primers Psta-Psts. The second round PCR was then performed using amplified products from the first PCR as the template and Nesta-Nests as the primers. An amplification signal was detectable even when only 2 fg of P. striiformis f. sp. tritici DNA was used as the template in nested PCR. With nested PCR, the sensitivity of detection was enhanced 1000 fold. Using extracts from stripe rust-infected wheat leaves, the fungus could be determined in the leaves before symptom appearance. The assay provides a rapid and sensitive method for detection of P. striiformis f. sp. tritici in latently infected leaves of overwintering wheat plants.     

Sterol composition of dark-grown Isochrysis galbana and its implication in the seed production of Pacific oyster, Crassostrea gigas

Isochrysis galbana was cultured heterotrophicallywith glucose and mannose as a potential food for larval Pacific oyster,Crassostrea gigas. The food was evaluated in terms offeeding experiments and changes in sterol composition. The larvae showed delayedgrowth and higher mortality compared with ones fed light-grown material, with asignificant difference in mortality from day 8. Light-grown I.galbana contained three major sterols (24-oxocholesterol acetate,ergost-5-en-3-ol, and cholest-5-en-24-1,3-(acetyloxy)-,3-ol) and twominor sterols (24-methylcholesta-5,22-dien-3-ol and24-methylcholest-5-en-3-ol). The sterol content decreased markedly aftertransfer to dark culture, especially in two of the major sterols,24-oxocholesterol acetate and ergost-5-en-3-ol. The other major sterol,cholest-5-en-24-1,3-(acetyloxy)-,3-ol, fall to about 50% of theautotrophic control by day 12. These results indicate that heterotrophicallygrown I. galbana is not a favorable alternative to normallygrown material for larval C. gigas culture as far as sterolcomposition is concerned.     

A Complementary Method for Production of Tetraploid Crassostrea gigas Using Crosses Between Diploids and Tetraploids with Cytochalasin B Treatments

We present a new method to produce tetraploid Crassostrea gigas by cytochalasin B inhibition of polar body 2 expulsion in diploid females crossed with tetraploid males. This offers a means of direct introgression of genetic characters from selected diploid to tetraploid lines, avoiding a triploid step. Offspring larval ploidy shifted over time and depended on size, with tetraploids more frequent among the smaller larvae and triploids among the large. Viable tetraploids were found at 4 and 6 months, indicating the technique was successful. The possibility that gynogenesis occurred was tested by microsatellite analysis to confirm the presence of paternally inherited alleles. These were present in all animals of the 2n × 4n + CB (female first) cross. However, a 4n × 2n + CB cross produced triploids, including some gynogens. Our method illustrates for the first time that diploid C. gigas eggs, if selected for large size, can give viable tetraploid offspring.     

Pollutant effects on Pacific oyster, Crassostrea gigas (Thunberg), hemocytes: Screening of 23 molecules using flow cytometry

The shellfish industry is an important economic activity in France, occurring mostly in estuarine zones subject to pollution due to anthropogenic activities. The harmful effects of pollutants on species inhabiting these estuarine zones are not well known. Among marine species, bivalve mollusks---particularly Pacific oyster, Crassostrea gigas---may serve a model of interest. The species is sedentary and filter-feeding, which favors bioaccumulation of pollutants in their tissues. Oysters may be suitable for studies on disturbance by pollutants of physiological activities, among which defense mechanisms are poorly documented in bivalves. In this study, effects of pollutants on hemocyte functions were monitored in Pacific oyster, C. gigas. Hemocytes were exposed in vitro to selected pollutants. The strategy for investigating the effects of pollutants on hemocyte functions is based on several biomarkers, which is more relevant than that of published papers based on single-endpoint experiments. Pollutants belonging to the most important groups of xenobiotics (PAHs, PCBs, and pesticides) were selected and their effect on hemocyte activities was analyzed using flow cytometry. Twenty-three pollutants were tested and eight of them showed significant modulation of hemocyte activities. PAHs and PCB 77 induced a decrease of hemocyte activity after an incubation periods of 4 and 24 h at 200 μmol/L. Three pesticides (2,4D, paraoxon, and chlorothalonil) modulated hemocyte activities. A mixture of eight pesticides also decreased phagocytotic activity. This study is one of the first to investigate the effects of so many pollutants on hemocyte functions at the same time and therefore allows a real comparison of different pollutant effects     

A Nested Polymerase Chain Reaction Protocol for in planta Detection of Fusicladium eriobotryae,Causal Agent of Loquat Scab

Scab caused by the fungus Fusicladium eriobotryae is the most serious disease affecting loquat in Spain. Isolation of F. eriobotryae from infected tissue on culture media can be difficult due to its slow growth. A polymerase chain reaction (PCR)‐based protocol was developed for F. eriobotryae‐specific identification from pure culture or infected loquat tissues. The primer set was designed in the elongation factor 1‐α gene (EF1‐α), and specificity and sensitivity for single and nested PCR were validated. The nested PCR assay resulted in 100% positive detection of F. eriobotryae in naturally and artificially infected tissues. This protocol can be useful for routine diagnosis, disease monitoring programmes and epidemiological research.