Preferential bivalent formation in tetraploid male of Pacific oyster Crassostrea gigas Thunberg

Artificially induced tetraploid Pacific oyster, Crassostrea gigas Thunberg, produces more aneuploid gametes than normal diploid one, although they showed a comparable fecundity to diploidy. The meiotic chromosome configuration of 3 tetraploid and 1 tetraploid/triploid mosaic males were analyzed through direct chromosome observation. A majority of metaphase I spermatocytes contained both bivalents and quadrivalents. The chromosome configuration of these males was characterized by preferential formation of bivalents to quadrivalents. Bivalents appeared in all spermatocytes and consisted of 86% of all chromosome aggregates. In comparison, quadrivalents occurred in 91% spermatocytes and consisted of only 12.6% of all chromosome aggregates. The mean bivalent frequency per spermatocyte varied between 14.4 and 17.2; while that of quadrivalents varied between 2.2 and 2.7. Most quadrivalents were tandemly chained(58%) or circled(39%). The total number of chromosome aggregates per spermatocyte ranged from 13 to 20 with an average of 17.6; while 18(16 bivalents and 2 quadrivalents) was the most frequent. Univalents and trivalents appeared in very low frequency. Aneuploid(hypotetraploid) spermatocytes were observed in a low frequency. The chromosome configuration of in the mosaic individual was similar to that of tetraploid individuals. The percentage of triploid spermatocytes(2%) of the mosaic individual was significantly lower(χ2 =30, P 0.01) than that of triploid cells(46%) in its somatic tissue.     

Response to selection for fast growth in the second generation of Pacific oyster (Crassostrea gigas)

Mass selection for fast growth was conducted in three Pacific oyster (Crassostrea gigas) stocks from China, Japan and Korea using previously established lines (CS1, JS1, and KS1). To determine whether continuous progress can be achieved by selection for growth, the progeny of three second-generation Pacific oyster lines was selected for shell height and evaluated via a 400-day farming experiment. When harvested at the end of the experiment, the selected crosses of CS2, JS2, and KS2 lines grew by 9.2%, 10.2% and 9.6% larger than the control crosses, respectively. During grow-out stage, the genetic gain of three selected lines was (10.2 ± 1.4)%, (10.4 ± 0.3)%, and (8.4 ± 1.6)%, respectively; and the corresponding realized heritability was 0.457 ± 0.143, 0.312 ± 0.071 and 0.332 ± 0.009, respectively. These results indicated that the selection for fast growth achieved steady progress in the second generation of oyster. Our work provides supportive evidence for the continuity of the Pacific oyster selective breeding program.     

Cytochemical characterization of yolk granule acid phosphatase during early development of the oyster Crassostrea gigas (Thunberg)

In this study,a cytochemical method and transmission electron microscopy was used to examine acid phosphatase activities of yolk granules throughout the early developmental stages of the Pacific oyster Crassostrea gigas. This study aimed to investigate the dynamic change of yolk granule acid phosphatase,and the mechanisms underlying its involvement in yolk degradation during the early developmental stages of molluscs. Three types of yolk granules(YGI,YGII,and YGIII) that differed in electron density and acid phosphatase reaction were identified in early cleavage,morula,blastula,gastrula,trochophore,and veliger stages. The morphological heterogeneities of the yolk granules were related to acid phosphatase activity and degrees of yolk degradation,indicating the association of acid phosphatase with yolk degradation in embryos and larvae of molluscs. Fusion of yolk granules was observed during embryogenesis and larval development of C. gigas. The fusion of YGI(free of acid phosphatase reaction) with YGII(rich in acid phosphatase reaction) could be the way by which yolk degradation is triggered.     

Inheritance mode of microsatellite loci and their use for kinship analysis in the Pacific oyster （Crassostrea gigas）

Five full-sib families of the Pacific oyster(Crassostrea gigas) larvae were used to study the mode of inheritance at eight microsatellite loci,and the feasibility of these markers for kinship estimate was also examined.All eight microsatellite loci were compatible with Mendelian inheritance.Neither evidence of sex-linked barriers to transmission nor evidence of major barriers to fertilization between gametes from the parents was shown.Three of the eight loci showed the presence of null alleles in four families,demonstrating the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies.Although the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance,offspring from five full-sib families were unambiguously discriminated in the neighbor-joining dendrogram.This result indicates that the microsatellite markers may be capable of discriminating between related and unrelated oyster larvae in the absence of pedigree information,and is applicable to the investigation of the effective number of parents contributing to the hatchery population of the Pacific oyster.     

Iodothyronine deiodinase gene analysis of the Pacific oyster Crassostrea gigas reveals possible conservation of thyroid hormone feedback regulation mechanism in mollusks

Iodothyronine deiodinase catalyzes the initiation and termination of thyroid hormones(THs) effects, and plays a central role in the regulation of thyroid hormone level in vertebrates. In non-chordate invertebrates, only one deiodinase has been identified in the scallop C hlamys farreri. Here, two deiodinases were cloned in the Pacific oyster C rassostrea gigas( Cg Dx and C g Dy). The characteristic in-frame TGA codons and selenocysteine insertion sequence elements in the oyster deiodinase c DNAs supported the activity of them. Furthermore, seven orthologs of deiodinases were found by a tblastn search in the mollusk Lottia gigantea and the annelid C apitella teleta. A phylogenetic analysis revealed that the deiodinase gene originated from an common ancestor and a clade-specific gene duplication occurred independently during the differentiation of the mollusk, annelid, and vertebrate lineages. The distinct spatiotemporal expression patterns implied functional divergence of the two deiodinases. The expression of C g Dx and Cg Dy was influenced by L-thyroxine T4, and putative thyroid hormone responsive elements were found in their promoters, which suggested that the oyster deiodinases were feedback regulated by TH. Epinephrine stimulated the expression level of C g Dx and Cg Dy, suggesting an interaction effect between different hormones. This study provides the first evidence for the existence of a conserved TH feedback regulation mechanism in mollusks, providing insights into TH evolution.     

Inheritance of 15 microsatellites in the Pacific oyster Crassostrea gigas： segregation and null allele identification for linkage analysis

Microsatellites were screened in a backcross family of the Pacific oyster, Crassostrea gigas. Fifteen microsatellite loci were distinguishable and polymorphic with 6 types of allele-combinations. Null alleles were detected in 46.7% of loci, accounting for 11.7% of the total alleles. Four loci did not segregate in Mendelian Ratios. Three linkage groups were identified among 7 of the 15 segregating loci. Fluorescence-based automated capillary electrophoresis (ABI 310 Genetic Analyzer) that used to detect the microsatellite loci, has been proved a fast, precise, and reliable method in microsatellite genotyping.     

Pigment Distribution and Secretion in the Mantle of the Pacific Oyster(Crassostrea gigas)

The color of Mollusca shells is one of the most important attributes to consumers. At the cellular level, black color is mainly from the melanin produced by melanocytes. The melanosome is a specialized membrane-bound organelle that is involved in melanin synthesis, storage, and transportation. How the complex pigmentation process in the Crassostrea gigas is established remains an open question. The objectives of this studies are to examine the morphological characteristics of melanosomes or mela...     

Linkage Disequilibrium in Wild and Cultured Populations of Pacific Oyster(Crassostrea gigas)

Linkage disequilibrium(LD) can be applied for mapping the actual genes responsible for variation of economically important traits through association mapping.The feasibility and efficacy of association studies are strongly dependent on the extent of LD which determines the number and density of markers in the studied population,as well as the experimental design for an association analysis.In this study,we first characterized the extent of LD in a wild population and a cultured mass-selected line of Pacific oyster(Crassostrea gigas).A total of 88 wild and 96 cultured individuals were selected to assess the level of genome-wide LD with 53 microsatellites,respectively.For syntenic marker pairs,no significant association was observed in the wild population;however,three significant associations occurred in the cultured population,and the significant LD extended up to 12.7 c M,indicating that strong artificial selection is a key force for substantial increase of genome-wide LD in cultured population.The difference of LD between wild and cultured populations showed that association studies in Pacific oyster can be achieved with reasonable marker densities at a relatively low cost by choosing an association mapping population.Furthermore,the frequent occurrence of LD between non-syntenic loci and rare alleles encourages the joint application of linkage analysis and LD mapping when mapping genes in oyster.The information on the linkage disequilibrium in the cultured population is useful for future association mapping in oyster.     

Habitat values for artificial oyster (Crassostrea ariakensis) reefs compared with natural shallow-water habitats in Changjiang River estuary

studies have compared oyster reefs with adjacent natural shallow-water habitats.Here the resident benthic macroinvertebrate communities in an artificial oyster(Crassostrea ariakensis) reef and in adjacent natural estuarine shallow-water habitats(salt marsh,intertidal mudflat,and subtidal soft bottom) in the Changjiang(Yangtze) River estuary were described.The mean total densities and biomass,Margalef’s species richness,Pielou’s evenness and Shannon-Weaver biodiversity indices of the resident benthic macroinvertebrate communities differed significantly among the habitats.Significantly higher densities and biomass of benthic macroinvertebrates occurred in the oyster reef compared with the other three habitats.Ordination plots showed a clear separation in benthic macroinvertebrate communities among the four habitat types.The results demonstrated that the artificial oyster reef supported distinct and unique benthic communities,playing an important role in the complex estuarine habitat by supplying prey resources and contributing to biodiversity.In addition,the results suggested that the oyster reef had been restored successfully.     

Cloning and expression patterns of two Smad genes during embryonic development and shell formation of the Pacific oyster Crassostrea gigas

Increasing evidence indicates that transforming growth factor β(TGF-P) signaling pathways play many important roles in the early development of mollusks.However,limited information is known concerning their detailed mechanisms.Here,we describe the identification,cloning and characterization of two Smad genes,the key components of TGF-P signaling pathways,from the Pacific oyster Crassostrea gigas.Sequence analysis of the two genes,designated as cgi-smad1/5/8 and cgi-smad4,revealed conserved functional characteristics.The two genes were widely expressed in embryos and larvae,suggesting multiple roles in the early development of C.gigas.The mRNA of the two genes aggregated in the D quadrant and cgi-smad4 was highly expressed on the dorsal side of the gastrula,indicating that TGF-P signaling pathways may be involved in dorsoventral patterning in C.gigas.Furthermore,high expression levels of the two genes in the shell fields of embryos at different stages suggested important roles for TGF-P signaling pathways in particular phases of shell development,including the formation of the initial shell field and the biomineralization of larval shells.The results of this study provide fundamental support for elucidating how TGF-P signaling pathways participate in the early development of bivalve mollusks,and suggest that further work is warranted to this end.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Area inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster(Crassostrea gigas) and blood cockle(Arca inflata Reeve).The larvae were reared at pH 8.2(control),7.9,7.6,or 7.3beginning 30 min or 24 h post fertilization.Exposure to lower pH during early embryonic development inhibited larval shell formation in both species.Compared with the control,larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3.Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3.However,when exposure was delayed until 24 h post fertilization,shell formation was only inhibited in blood cockle larvae reared at pH 7.3.Thus,the early embryonic stages were more sensitive to acidified conditions.Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves.Although the effects appear subtle,they may accumulate and lead to subsequent issues during later larval development.     

The potential of ocean acidification on suppressing larval development in the Pacific oyster Crassostrea gigas and blood cockle Arca inflata Reeve

We evaluated the effect of pH on larval development in larval Pacific oyster （Crassostrea gigas） and blood cockle （Arca inflata Reeve）. The larvae were reared at pH 8.2 （control）, 7.9, 7.6, or 7.3 beginning 30 min or 24 h post fertilization. Exposure to lower pH during early embryonic development inhibited larval shell formation in both species. Compared with the control, larvae took longer to reach the D-veliger stage when reared under pH 7.6 and 7.3. Exposure to lower pH immediately after fertilization resulted in significantly delayed shell formation in the Pacific oyster larvae at pH 7.3 and blood cockle larvae at pH 7.6 and 7.3. However, when exposure was delayed until 24 h post fertilization, shell formation was only inhibited in blood cockle larvae reared at pH 7.3. Thus, the early embryonic stages were more sensitive to acidified conditions. Our results suggest that ocean acidification will have an adverse effect on embryonic development in bivalves. Although the effects appear subtle, they may accumulate and lead to subsequent issues during later larval development.     

Genetic Variation Assessed with Microsatellites in Mass Selection Lines of the Pacific Oyster(Crassostrea gigas) in China

Four successive mass selection lines of the Pacific oyster, Crassostrea gigas, selected for faster growth in breeding programs in China were examined at ten polymorphic microsatellite loci to assess the level of allelic diversity and estimate the effective population size. These data were compared with those of their base population. The results showed that the genetic variation of the four generations were maintained at high levels with an average allelic richness of 18.8–20.6, and a mean expected heterozygosity of 0.902–0.921. They were not reduced compared with those of their base population. Estimated effective population sizes based on temporal variances in microsatellite frequencies were smaller to that of sex ratio-corrected broodstock count estimates. Using a relatively large number of broodstock and keeping an equal sex ratio in the broodstock each generation may have contributed to retaining the original genetic diversity and maintaining relatively large effective population size. The results obtained in this study showed that the genetic variation was not affected greatly by mass selection progress and high genetic variation still existed in the mass selection lines, suggesting that there is still potential for increasing the gains in future generations of C. gigas. The present study provided important information for future genetic improvement by selective breeding, and for the design of suitable management guidelines for genetic breeding of C. gigas.     

Genome-wide survey and analysis of microsatellites in the Pacific oyster genome: abundance,distribution, and potential for marker development

Microsatellites are a ubiquitous component of the eukaryote genome and constitute one of the most popular sources of molecular markers for genetic studies. However, no data are currently available regarding microsatellites across the entire genome in oysters, despite their importance to the aquaculture industry. We present the fi rst genome-wide investigation of microsatellites in the Pacifi c oyster Crassostrea gigas by analysis of the complete genome, resequencing, and expression data. The Pacifi c oyster genome is rich in microsatellites. A total of 604 653 repeats were identifi ed, in average of one locus per 815 base pairs(bp). A total of 12 836 genes had coding repeats, and 7 332 were expressed normally, including genes with a wide range of molecular functions. Compared with 20 different species of animals, microsatellites in the oyster genome typically exhibited 1) an intermediate overall frequency; 2) relatively uniform contents of(A)n and(C)n repeats and abundant long(C)n repeats(≥24 bp); 3) large average length of(AG)n repeats; and 4) scarcity of trinucleotide repeats. The microsatellite-fl anking regions exhibited a high degree of polymorphism with a heterozygosity rate of around 2.0%, but there was no correlation between heterozygosity and microsatellite abundance. A total of 19 462 polymorphic microsatellites were discovered, and dinucleotide repeats were the most active, with over 26% of loci found to harbor allelic variations. In all, 7 451 loci with high potential for marker development were identifi ed. Better knowledge of the microsatellites in the oyster genome will provide information for the future design of a wide range of molecular markers and contribute to further advancements in the fi eld of oyster genetics, particularly for molecular-based selection and breeding.     

Seasonal variation of the glycogen enzyme activity in diploid and triploid Pacific oyster gonad during sexual maturation

The glycogen content and the activities of two key enzymes in glycogen metabolism, glycogen phosphorylase and gly- cogen synthetase, in the gonad of diploid and triploid Pacific oysters (Crassostrea gigas) were compared during maturation. The glycogen content in the gonad of diploids decreased with gametogenesis (by 85.7%), but the glycogen content in the gonad of trip- loids did not vary significantly. Activity of glycogen phosphorylase (GP) in the gonad of diploids decreased with gametogenesis (by 55.5%), while GP activity of triploids did not vary significantly during maturation. Activity of glycogen synthetase (GS) in the gonad of diploids increased slightly with gametogenesis, reaching a peak in June. Activity of GS declined sharply from June to July, which might be due to gonad spawning. GS activity of triploid oysters in spawning time (July and August) was significantly higher than that in other months, which might be explained with a ‘compensating’ mechanism for the higher glycogen content in triploids.     

Development of Genomic Microsatellite Multiplex PCR Using Dye-Labeled Universal Primer and Its Validation in Pedigree Analysis of Pacific Oyster(Crassostrea gigas)

There is an increasing requirement for traceability of aquaculture products, both for consumer protection and for food safety. There are high error rates in the conventional traceability systems depending on physical labels. Genetic traceability technique depending on DNA-based tracking system can overcome this problem. Genealogy information is essential for genetic traceability, and microsatellite DNA marker is a good choice for pedigree analysis. As increasing genotyping throughput of microsatellites, microsatellite multiplex PCR has become a fast and cost-effective technique. As a commercially important cultured aquatic species, Pacific oyster Crassostrea gigas has the highest global production. The objective of this study was to develop microsatellite multiplex PCR panels with dye-labeled universal primer for pedigree analysis in C. gigas, and these multiplex PCRs were validated using 12 full-sib families with known pedigrees. Here we developed six informative multiplex PCRs using 18 genomic microsatellites in C. gigas. Each multiplex panel contained a single universal primer M13(-21) used as a tail on each locus-specific forward primer and a single universal primer M13(-21) labeled with fluorophores. The polymorphisms of the markers were moderate, with an average of 10.3 alleles per locus and average polymorphic information content of 0.740. The observed heterozygosity per locus ranged from 0.492 to 0.822. Cervus simulations revealed that the six panels would still be of great value when massive families were analysed. Pedigree analysis of real offspring demonstrated that 100% of the offspring were unambiguously allocated to their parents when two multiplex PCRs were used. The six sets of multiplex PCRs can be an important tool for tracing cultured individuals, population genetic analysis, and selective breeding program in C. gigas.     

Linkage disequilibrium in wild and cultured populations of Pacific oyster (<Emphasis Type="Italic">Crassostrea gigas</Emphasis>)

Linkage disequilibrium (LD) can be applied for mapping the actual genes responsible for variation of economically important traits through association mapping. The feasibility and efficacy of association studies are strongly dependent on the extent of LD which determines the number and density of markers in the studied population, as well as the experimental design for an association analysis. In this study, we first characterized the extent of LD in a wild population and a cultured mass-selected line of Pacific oyster (Crassostrea gigas). A total of 88 wild and 96 cultured individuals were selected to assess the level of genome-wide LD with 53 microsatellites, respectively. For syntenic marker pairs, no significant association was observed in the wild population; however, three significant associations occurred in the cultured population, and the significant LD extended up to 12.7 cM, indicating that strong artificial selection is a key force for substantial increase of genome-wide LD in cultured population. The difference of LD between wild and cultured populations showed that association studies in Pacific oyster can be achieved with reasonable marker densities at a relatively low cost by choosing an association mapping population. Furthermore, the frequent occurrence of LD between non-syntenic loci and rare alleles encourages the joint application of linkage analysis and LD mapping when mapping genes in oyster. The information on the linkage disequilibrium in the cultured population is useful for future association mapping in oyster.     

Inheritance mode of microsatellite loci and their use for kinship analysis in the Pacific oyster （Crassostrea gigas）

Five full-sib families of the Pacific oyster （Crassostrea gigas） larvae were used to study the mode of inheritance at eight microsatellite loci, and the feasibility of these markers for kinship estimate was also examined. All eight microsatellite loci were compatible with Mendelian inheritance. Neither evidence of sex-linked barriers to transmission nor evidence of major barriers to fertilization between gametes from the parents was shown. Three of the eight loci showed the presence of null alleles in four families, demonstrating the need to conduct comprehensive species-specific inheritance studies for microsatellite loci used in population genetic studies. Although the null allele heterozygotes were considered as homozygotes in the calculation of genetic distance, offspring from five full-sib families were unambiguously discriminated in the neighbor-joining dendrogram. This result indicates that the microsatellite markers may be capable of discriminating between related and unrelated oyster larvae in the absence of pedigree information, and is applicable to the investigation of the effective number of parents contributing to the hatchery population of the Pacific oyster.