With the development of composite processing technology, three-dimensional braided composites have been widely used in automotive, aerospace and other high-tech industries. Due to specific yarn ...distribution in microstructure, three-dimensional braided composites have excellent mechanical properties. To further optimize or design their mechanical performance, it is important to accurately estimate the effective mechanical properties of braided performs with different braiding parameters such as braiding angle, pitch length and fiber volume fraction. To this end, the microstructure (i.e. yarn pattern) of braided perform generated by 1 × 1 four-step braiding process was first analyzed in detail with the three cell model, which was established from the simulation of braiding process. Secondly, multiphase finite element method was employed and introduced into asymptotic expansion homogenization framework to predict the equivalent elastic modulus and microscopic stress of three-dimensional braided composites. Finally, the calculated equivalent engineering elastic modulus was proved to be in good agreement with experimental measurements of braided composite samples. Thanks to the multi-scale approach, the microscopic stress, i.e., local stress state of the composite perform under macroscopic load can be obtained simultaneously, which can be used in further study of damage and failure behavior.
The molecules that mediate innate immunity are encoded by relatively few genes and exhibit broad specificity. Detailed annotation of the Pacific oyster (Crassostrea gigas) genome, a protostome ...invertebrate, reveals large-scale duplication and divergence of multigene families encoding molecules that effect innate immunity. Transcriptome analyses indicate dynamic and orchestrated specific expression of numerous innate immune genes in response to experimental challenge with pathogens, including bacteria, and a pathogenic virus. Variable expression of individual members of the multigene families encoding these genes also occurs during different types of abiotic stress (environmentally-equivalent conditions of temperature, salinity and desiccation). Multiple families of immune genes are responsive in concert to certain biotic and abiotic challenges. Individual members of expanded families of immune genes are differentially expressed under both biotic challenge and abiotic stress conditions. Members of the same families of innate immune molecules also are transcribed in developmental stage- and tissue-specific manners. An integrated, highly complex innate immune system that exhibits remarkable discriminatory properties and responses to different pathogens as well as environmental stress has arisen through the adaptive recruitment of tandem duplicated genes. The co-adaptive evolution of stress and innate immune responses appears to have an ancient origin in phylogeny.
Pacific oyster (Crassostrea gigas) distribute a steep gradient of environmental stress between intertidal and subtidal habits and provide insight into population-scale patterns and underlying ...processes of variation in physiological tolerance. In this study, 1-year-old-F1 oysters, collected from subtidal and intertidal habitats, were obtained after common garden experiment. Genetic differentiation and physiological responses under air exposure were examined to determine whether they had evolved into local adapted subpopulations. Mortality rate, anaerobic glycolysis metabolism, and energy status indicated that oyster had initiated metabolism depression and anaerobic glycolysis metabolism in both intertidal and subtidal oysters under air exposure. However, the subtidal oysters displayed the larger energy metabolism depressions and the earlier anaerobic glycolysis responses. This may indicate that subtidal oysters were more sensitives to hypoxia stress, which may lead the higher mortality rate under long term of air exposure. Based on a common garden experimental design, we propose that this diversification may have a genetic background. Overall, the clear differences between intertidal and subtidal oysters under air exposure have provided an important reference for their aquaculture and transportation used in commercial production.
•We found phenotypic variation between intertidal and subtidal oysters exposed to air.•Subtidal oysters were more sensitive to hypoxia than were intertidal oysters.•Subtidal oysters switched to anaerobic metabolism earlier than intertidal oysters.•HIF-1α regulates anaerobic metabolism via activation of PEPCK mRNA expression.•Diversification of intertidal and subtidal C. gigas may have a genetic background.
DNA methylation is one of the most important epigenetic modifications of eukaryotic genomes and is believed to play integral roles in diverse biological processes. Although DNA methylation has been ...well studied in mammals, data are limited in invertebrates, particularly Mollusca. The Pacific oyster Crassostrea gigas is an emerging genetic model for functional analysis of DNA methylation in Mollusca. Recent studies have shown that there is a positive association between methylation status and gene expression in C. gigas; however, whether this association exists at the exon level remains to be determined.
In this study, we characterized the genome-wide methylation pattern across two different tissues of C. gigas and found that methylated genes are expressed in more tissues and development stages than unmethylated genes. Furthermore, we found that different types of exons had different methylation levels, with the lowest methylation levels in the first exons, followed by the last exons, and the internal exons. We found that the exons included in the gene transcript contained significantly higher DNA methylation levels than skipped exons. We observed that the DNA methylation levels increased slowly after the start sites and end sites of exons seperately, and then decreased quickly towards the middle sites of exons. We also found that methylated exons were significantly longer than unmethylated exons.
This study constitutes the first genome-wide analysis to show an association between exon-level DNA methylation and mRNA expression in the oyster. Our findings suggest that exon-level DNA methylation may play a role in the construction of alternative splicing by positively influencing exon inclusion during transcription.
The Pacific oyster Crassostrea gigas is an important marine fishery resource, which contains high levels of glycogen that contributes to the flavor and the quality of the oyster. However, little is ...known about the molecular and chemical mechanisms underlying glycogen content differences in Pacific oysters. Using a homogeneous cultured Pacific oyster family, we explored these regulatory networks at the level of the metabolome and the transcriptome.
Oysters with the highest and lowest natural glycogen content were selected for differential transcriptome and metabolome analysis. We identified 1888 differentially-expressed genes, seventy-five differentially-abundant metabolites, which are part of twenty-seven signaling pathways that were enriched using an integrated analysis of the interaction between the differentially-expressed genes and the differentially-abundant metabolites. Based on these results, we found that a high expression of carnitine O-palmitoyltransferase 2 (CPT2), indicative of increased fatty acid degradation, is associated with a lower glycogen content. Together, a high level of expression of phosphoenolpyruvate carboxykinase (PEPCK), and high levels of glucogenic amino acids likely underlie the increased glycogen production in high-glycogen oysters. In addition, the higher levels of the glycolytic enzymes hexokinase (HK) and pyruvate kinase (PK), as well as of the TCA cycle enzymes malate dehydrogenase (MDH) and pyruvate carboxylase (PYC), imply that there is a concomitant up-regulation of energy metabolism in high-glycogen oysters. High-glycogen oysters also appeared to have an increased ability to cope with stress, since the levels of the antioxidant glutathione peroxidase enzyme 5 (GPX5) gene were also increased.
Our results suggest that amino acids and free fatty acids are closely related to glycogen content in oysters. In addition, oysters with a high glycogen content have a greater energy production capacity and a greater ability to cope with stress. These findings will not only provide insights into the molecular mechanisms underlying oyster quality, but also promote research into the molecular breeding of oysters.
Oyster is the worldwide aquaculture molluscan and evolves a complex immune defense system, with hemocytes as the major immune system for its host defense. However, the functional heterogeneity of ...hemocyte has not been characterized, which markedly hinders our understanding of its defense role. Here, we used the single-cell transcriptome profiling (scRNA-seq), which provides a high-resolution visual insight into its dynamics, to map the hemocyte and assess its heterogeneity in a molluscan oyster Crassostrea hongkongensis. By combining with the cell type specific RNA-seq, thirteen subpopulations belonging to granulocyte, semi-granulocyte, and hyalinocyte were revealed. The granulocytes mainly participated in immune response and autophagy process. Pseudo-temporal ordering of granulocytes identified two different cell-lineages. The hematopoietic transcription factors regulated networks controlling their differentiations were also identified. We further identified one subpopulation of granulocytes in immune activate states with the cell cycle and immune responsive genes expressions, which illustrated the functional heterogeneity of the same cell type. Collectively, our scRNA-seq analysis demonstrated the hemocytes diversity of molluscans. The results are important in our understanding of the immune defense evolution and functional differentiation of hemocytes in Phylum Mollusca.
•ScRNA-seq characterize the subtypes of hemocytes isolated from oysters.•Pseudo-temporal ordering of granulocytes revealed two lineages differentiation.•Hematopoietic transcription factors and regulatory networks were identified.•Functional heterogeneity of the granulocytes in oysters.
Epigenetic mechanisms such as DNA methylation have the potential to affect organism acclimatization and adaptation to environmental changes by influencing their phenotypic plasticity; however, little ...is known about the role of methylation in the adaptive phenotypic divergence of marine invertebrates. Therefore, in this study, a typical intertidal species, the Pacific oyster (Crassostrea gigas), was selected to investigate the epigenetic mechanism of phenotypic plasticity in marine invertebrates. Intertidal and subtidal oysters subjected to one-generation common garden experiments and exhibited phenotypic divergence were used. The methylation landscape of both groups of oysters was investigated under temperate and high temperature. The two tidal oysters exhibited divergent methylation patterns, regardless of the temperature, which was mainly original environment-induced. Intertidal samples exhibited significant hypomethylation and more plasticity of methylation in response to heat shock, while subtidal samples showed hypermethylation and less plasticity. Combined with RNA-seq data, a positive relationship between methylation and expression in gene bodies was detected on a genome-wide scale. In addition, approximately 11% and 7% of differentially expressed genes showed significant methylation variation under high temperatures in intertidal and subtidal samples, respectively. Genes related to apoptosis and organism development may be regulated by methylation in response to high temperature in intertidal oysters, whereas oxidation-reduction and ion homeostasis-related genes were involved in subtidal oysters. The results also suggest that DNA methylation mediates phenotypic divergence in oysters adapting to different environments. This study provides new insight into the epigenetic mechanisms underlying phenotypic plasticity in adaptation to rapid climate change in marine organisms.
The Pacific oyster, Crassostrea gigas, has developed special mechanisms to regulate its osmotic balance to adapt to fluctuations of salinities in coastal zones. To understand the oyster's euryhaline ...adaptation, we analyzed salt stress effectors metabolism pathways under different salinities (salt 5, 10, 15, 20, 25, 30 and 40 for 7 days) using transcriptome data, physiology experiment and quantitative real-time PCR.
Transcriptome data uncovered 189, 480, 207 and 80 marker genes for monitoring physiology status of oysters and the environment conditions. Three known salt stress effectors (involving ion channels, aquaporins and free amino acids) were examined. The analysis of ion channels and aquaporins indicated that 7 days long-term salt stress inhibited voltage-gated Na(+)/K(+) channel and aquaporin but increased calcium-activated K(+) channel and Ca(2+) channel. As the most important category of osmotic stress effector, we analyzed the oyster FAAs metabolism pathways (including taurine, glycine, alanine, beta-alanine, proline and arginine) and explained FAAs functional mechanism for oyster low salinity adaptation. FAAs metabolism key enzyme genes displayed expression differentiation in low salinity adapted individuals comparing with control which further indicated that FAAs played important roles for oyster salinity adaptation. A global metabolic pathway analysis (iPath) of oyster expanded genes displayed a co-expansion of FAAs metabolism in C. gigas compared with seven other species, suggesting oyster's powerful ability regarding FAAs metabolism, allowing it to adapt to fluctuating salinities, which may be one important mechanism underlying euryhaline adaption in oyster. Additionally, using transcriptome data analysis, we uncovered salt stress transduction networks in C. gigas.
Our results represented oyster salt stress effectors functional mechanisms under salt stress conditions and explained the expansion of FAAs metabolism pathways as the most important effectors for oyster euryhaline adaptation. This study was the first to explain oyster euryhaline adaptation at a genome-wide scale in C. gigas.
Single nucleotide polymorphisms (SNPs) are widely used in genetics and genomics research. The Pacific oyster (Crassostrea gigas) is an economically and ecologically important marine bivalve, and it ...possesses one of the highest levels of genomic DNA variation among animal species. Pacific oyster SNPs have been extensively investigated; however, the mechanisms by which these SNPs may be used in a high-throughput, transferable, and economical manner remain to be elucidated. Here, we constructed an oyster 190K SNP array using Affymetrix Axiom genotyping technology. We designed 190,420 SNPs on the chip; these SNPs were selected from 54 million SNPs identified through re-sequencing of 472 Pacific oysters collected in China, Japan, Korea, and Canada. Our genotyping results indicated that 133,984 (70.4%) SNPs were polymorphic and successfully converted on the chip. The SNPs were distributed evenly throughout the oyster genome, located in 3,595 scaffolds with a length of ~509.4 million; the average interval spacing was 4,210 bp. In addition, 111,158 SNPs were distributed in 21,050 coding genes, with an average of 5.3 SNPs per gene. In comparison with genotypes obtained through re-sequencing, ~69% of the converted SNPs had a concordance rate of >0.971; the mean concordance rate was 0.966. Evaluation based on genotypes of full-sib family individuals revealed that the average genotyping accuracy rate was 0.975. Carrying 133 K polymorphic SNPs, our oyster 190K SNP array is the first commercially available high-density SNP chip for mollusks, with the highest throughput. It represents a valuable tool for oyster genome-wide association studies, fine linkage mapping, and population genetics.
Next-generation sequencing (NGS) technology is being applied to an increasing number of non-model species and has been used as the primary approach for accurate genotyping in genetic and evolutionary ...studies. However, inferring genotypes from sequencing data is challenging, particularly for organisms with a high degree of heterozygosity. This is because genotype calls from sequencing data are often inaccurate due to low sequencing coverage, and if this is not accounted for, genotype uncertainty can lead to serious bias in downstream analyses, such as quantitative trait locus mapping and genome-wide association studies. Here, we used high-coverage reference data sets from Crassostrea gigas to simulate sequencing data with different coverage, and we evaluate the influence of genotype calling rate and accuracy as a function of coverage. Having initially identified the appropriate parameter settings for filtering to ensure genotype accuracy, we used two different single-nucleotide polymorphism (SNP) calling pipelines, single-sample and multi-sample. We found that a coverage of 15× was suitable for obtaining sufficient numbers of SNPs with high accuracy. Our work provides guidelines for the selection of sequence coverage when using NGS to investigate species with a high degree of heterozygosity and rapid decay of linkage disequilibrium.
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