Comparative proteomic and phosphoproteomic analysis reveals differential heat response mechanism in two congeneric oyster species.

High-temperature stress caused by global climate change poses a significant threat to marine ectotherms. This study investigated the role of protein phosphorylation modifications in the molecular regulation network under heat stress in oysters, which are representative intertidal organisms that experience considerable temperature changes. Firstly, the study compared the extent of thermal damage between two congeneric oyster species, the relative heat-tolerant Crassostrea angulata (C. angulata) and heat-sensitive Crassostrea gigas (C. gigas), under sublethal temperature (37 °C) for 12 h, using various physiological and biochemical methods. Subsequently, the comparative proteomic and phosphoproteomic analyses revealed that high-temperature considerably regulated signal transduction, energy metabolism, protein synthesis, cell survival and apoptosis, and cytoskeleton remodeling through phosphorylation modifications of related receptors and kinases. Furthermore, the protein kinase A, mitogen-activated protein kinase 1, tyrosine-protein kinase Src, and serine/threonine kinase AKT, exhibiting differential phosphorylation modification patterns, were identified as hub regulators that may enhance glycolysis and TCA cycle to increase the energy supply, distribute protein synthesis, inhibit Caspase-dependent apoptosis activated by endogenous mitochondrial cytochrome release and maintain cytoskeletal stability, ultimately shaping the higher thermal resistance of C. angulata. This study represents the first investigation of protein phosphorylation dynamics in marine invertebrates under heat stress, reveals the molecular mechanisms underlying the differential thermal responses between two Crassostrea oysters at the phosphorylation level, and provides new insights into understanding phosphorylation-mediated molecular responses in marine organisms during environmental changes and predicting the adaptive potential in the context of global warming.

Experimental DNA Demethylation Reduces Expression Plasticity and Thermal Tolerance in Pacific Oysters.

Increasing seawater temperatures pose a great threat to marine organisms, especially those settled in fluctuating intertidal areas. DNA methylation, which can be induced by environmental variation, can influence gene expression and mediate phenotypic plasticity. However, the regulatory mechanisms of DNA methylation in gene expression-mediated adaptation to environmental stress have rarely been elucidated. In this study, DNA demethylation experiments were conducted on a typical intertidal species, the Pacific oyster (Crassostrea gigas), to determine the direct role of DNA methylation in regulating gene expression and adaptability under thermal stress. The global methylation level and the expression level of DNA methyltransferases (DNMT1, DNMT3a) showed an accordant variation trend under high temperatures, supporting that the genomic methylation status was catalyzed by DNMTs. DNA methylation inhibitor 5-Azacytidine (5-Aza) effectively inhibited DNA methylation level and decreased methylation plasticity at the 6th hour in thermal conditions. In total, 88 genes were identified as candidate DNA methylation-regulated thermal response genes; they exhibited reduced expression plasticity in response to heat stress, possibly caused by the decreased methylation plasticity. Post-heat shock, the thermal tolerance indicated by the survival curve was reduced when oysters were pretreated with 5-Aza, meaning that DNA demethylation negatively affected thermal adaptation in oysters. This study provides direct evidence for the crucial role of DNA methylation in mediating stress adaptation in marine invertebrates and contributes to the theoretical foundations underlying marine resource conservation and aquaculture.

Transgenerational effects of intertidal environment on physiological phenotypes and DNA methylation in Pacific oysters.

Climate change and intensifying human activity are posing serious threats to marine organisms. The fluctuating intertidal zone forms a miniature ecosystem of a rapidly changing environment for studying biological adaptation. Transgenerational plasticity (TGP), an evolutionary phenomenon in which parental experience influences offspring phenotypes, provides an avenue for adaptation, but the molecular mechanism was poorly understood in marine molluscs. In this study, wild Pacific oysters (Crassostrea gigas), which were collected from intertidal zones, were used to conduct two-generation breeding in a subtidal area combined with a heat shock experiment in the laboratory to investigate the intertidal environment-induced TGP under temperate subtidal condition and thermally exposed condition, respectively. We showed that TGP could influence the physiological phenotypes related to the status of oxidation and energy in non-stress-exposed subtidal offspring for at least two generations. Genomic DNA methylation exhibited heritable divergence between intertidal and subtidal oysters, and 1655 (or 42.83 %) differentially methylated genes (DMGs) in F0 were continuously reserved to F2, which may mediate physiological TGP by participating in biological processes including macromolecule metabolism, cellular responses to stress, and the positive regulation of molecular function, especially fatty acid metabolism. The intertidal experience also influenced the thermal plasticity of physiological phenotypes within and across generations. Totally, 320 (or 14.74 %) specific thermal response DMGs in the intertidal F0 generation were identified in F1 and F2, participating in pathways including carbohydrate, lipid, and energy metabolism, signal transduction, and the organismal immune system, which suggested transgenerational intertidal effect mediated by these genes could positively contribute to stress adaptation and had potential applications for aquaculture. This study demonstrates an epigenetic mechanism for TGP in stress adaptation in marine molluscs, and provides new avenues to improve the stress adaptation for marine resource conservation and aquaculture.

Cis- and Trans-variations of Stearoyl-CoA Desaturase Provide New Insights into the Mechanisms of Diverged Pattern of Phenotypic Plasticity for Temperature Adaptation in Two Congeneric Oyster Species.

The evolution of phenotypic plasticity plays an essential role in adaptive responses to climate change; however, its regulatory mechanisms in marine organisms which exhibit high phenotypic plasticity still remain poorly understood. The temperature-responsive trait oleic acid content and its major gene stearoyl-CoA desaturase (Scd) expression have diverged in two allopatric congeneric oyster species, cold-adapted Crassostrea gigas and warm-adapted Crassostrea angulata. In this study, genetic and molecular methods were used to characterize fatty acid desaturation and membrane fluidity regulated by oyster Scd. Sixteen causative single-nucleotide polymorphisms (SNPs) were identified in the promoter/cis-region of the Scd between wild C. gigas and C. angulata. Further functional experiments showed that an SNP (g.-333C [C. gigas allele] >T [C. angulata allele]) may influence Scd transcription by creating/disrupting the binding motif of the positive trans-factor Y-box factor in C. gigas/C. angulata, which mediates the higher/lower constitutive expression of Scd in C. gigas/C. angulata. Additionally, the positive trans-factor sterol-regulatory element-binding proteins (Srebp) were identified to specifically bind to the promoter of Scd in both species, and were downregulated during cold stress in C. gigas compared to upregulated in C. angulata. This partly explains the relatively lower environmental sensitivity (plasticity) of Scd in C. gigas. This study serves as an experimental case to reveal that both cis- and trans-variations shape the diverged pattern of phenotypic plasticity, which provides new insights into the formation of adaptive traits and the prediction of the adaptive potential of marine organisms to future climate change.

Genome-Wide Association Analysis of Heat Tolerance in F2 Progeny from the Hybridization between Two Congeneric Oyster Species.

As the world's largest farmed marine animal, oysters have enormous economic and ecological value. However, mass summer mortality caused by high temperature poses a significant threat to the oyster industry. To investigate the molecular mechanisms underlying heat adaptation and improve the heat tolerance ability in the oyster, we conducted genome-wide association analysis (GWAS) analysis on the F2 generation derived from the hybridization of relatively heat-tolerant Crassostrea angulata ♀ and heat-sensitive Crassostrea gigas ♂, which are the dominant cultured species in southern and northern China, respectively. Acute heat stress experiment (semi-lethal temperature 42 °C) demonstrated that the F2 population showed differentiation in heat tolerance, leading to extremely differentiated individuals (approximately 20% of individuals die within the first four days with 10% survival after 14 days). Genome resequencing and GWAS of the two divergent groups had identified 18 significant SNPs associated with heat tolerance, with 26 candidate genes located near these SNPs. Eleven candidate genes that may associate with the thermal resistance were identified, which were classified into five categories: temperature sensor (Trpm2), transcriptional factor (Gata3), protein ubiquitination (Ube2h, Usp50, Uchl3), heat shock subfamily (Dnajc17, Dnaja1), and transporters (Slc16a9, Slc16a14, Slc16a9, Slc16a2). The expressional differentiation of the above genes between C. gigas and C. angulata under sublethal temperature (37 °C) further supports their crucial role in coping with high temperature. Our results will contribute to understanding the molecular mechanisms underlying heat tolerance, and provide genetic markers for heat-resistance breeding in the oyster industry.

Construction and analysis of the chromosome-level haplotype-resolved genomes of two Crassostrea oyster congeners: Crassostrea angulata and Crassostrea gigas.

BACKGROUND: The Portuguese oyster Crassostrea angulata and the Pacific oyster C. gigas are two major Crassostrea species that are naturally distributed along the Northwest Pacific coast and possess great ecological and economic value. Here, we report the construction and comparative analysis of the chromosome-level haplotype-resolved genomes of the two oyster congeners. FINDINGS: Based on a trio-binning strategy, the PacBio high-fidelity and Illumina Hi-C reads of the offspring of the hybrid cross C. angulata (♂) × C. gigas (♀) were partitioned and independently assembled to construct two chromosome-level fully phased genomes. The assembly size (contig N50 size, BUSCO completeness) of the two genomes were 582.4 M (12.8 M, 99.1%) and 606.4 M (5.46 M, 98.9%) for C. angulata and C. gigas, respectively, ranking at the top of mollusk genomes with high contiguity and integrity. The general features of the two genomes were highly similar, and 15,475 highly conserved ortholog gene pairs shared identical gene structures and similar genomic locations. Highly similar sequences can be primarily identified in the coding regions, whereas most noncoding regions and introns of genes in the same ortholog group contain substantial small genomic and/or structural variations. Based on population resequencing analysis, a total of 2,756 species-specific single-nucleotide polymorphisms and 1,088 genes possibly under selection were identified. CONCLUSIONS: This is the first report of trio-binned fully phased chromosome-level genomes in marine invertebrates. The study provides fundamental resources for the research on mollusk genetics, comparative genomics, and molecular evolution.

Transcriptomics Analysis and Re-sequencing Reveal the Mechanism Underlying the Thermotolerance of an Artificial Selection Population of the Pacific Oyster.

The Pacific oyster is a globally important aquaculture species inhabiting the intertidal environment, which experiences great temperature variation. Mass deaths in the summer pose a major challenge for the oyster industry. We initiated an artificial selection breeding program in 2017 using acute heat shock treatments of the parents to select for thermotolerance in oysters. In this study, we compared the respiration rate, summer survival rate, gene expression, and gene structure of F2 selected oysters and non-selected wild oysters. A transcriptional analysis revealed global divergence between the selected and control groups at the larval stage, including 4764 differentially expressed genes, among which 79 genes were heat-responsive genes. Five heat shock proteins were enriched, and four of the six genes (five heat stock genes in the enriched GO terms and KEGG pathways and BAG4) were differentially expressed in 1-year-old oysters. Integration of the transcriptomic and re-sequencing data of the selected and the control groups revealed 1090 genes that differentiated in both gene structure and expression. Two SNPs (single nucleotide polymorphism) that may mediate the expression of CGI_10022585 and CGI_10024709 were validated. In addition, the respiration rate of 1-year-old oysters varied significantly between the selected group and the control group at room temperature (20°C). And the summer survival rate of the selected population was significantly improved. This study not only shows that artificial selection has a significant effect on the gene structure and expression of oysters, but it also helps reveal the mechanism underlying their tolerance of high temperature as well as the ability of oysters to adapt to climate change.

Divergence and plasticity shape adaptive potential of the Pacific oyster.

The interplay between divergence and phenotypic plasticity is critical to our understanding of a species' adaptive potential under rapid climate changes. We investigated divergence and plasticity in natural populations of the Pacific oyster Crassostrea gigas with a congeneric oyster Crassostrea angulata from southern China used as an outgroup. Genome re-sequencing of 371 oysters revealed unexpected genetic divergence in a small area that coincided with phenotypic divergence in growth, physiology, heat tolerance and gene expression across environmental gradients. These findings suggest that selection and local adaptation are pervasive and, together with limited gene flow, influence population structure. Genes showing sequence differentiation between populations also diverged in transcriptional response to heat stress. Plasticity in gene expression is positively correlated with evolved divergence, indicating that plasticity is adaptive and favoured by organisms under dynamic environments. Divergence in heat tolerance-partly through acetylation-mediated energy depression-implies differentiation in adaptive potential. Trade-offs between growth and survival may play an important role in local adaptation of oysters and other marine invertebrates.

Draft genome and SNPs associated with carotenoid accumulation in adductor muscles of bay scallop (Argopecten irradians).

Carotenoids are commonly deposited in the gonads of marine bivalves but rarely in their adductor muscles. An orange-adductor variant was identified in our breeding program for the bay scallop Argopecten irradians. In the present study, bay scallop genome survey sequencing was conducted, followed by genotyping by sequencing (GBS)-based case-control association analysis in a selfing family that exhibited segregation in adductor color. K-mer analysis (K=17) revealed that the bay scallop genome is about 990 Mb in length. De novo assembly produced 217,310 scaffold sequences, which provided 72.1% coverage of the whole genome and covered 72,187 transcripts, thereby yielding the most informative sequence resource for bay scallop to date. The average carotenoid content of the orange-adductor progenies was significantly higher than that of the white-adductor progenies. Thus, 20 individuals of each subgroup were sampled for case-control analysis. As many as 15,224 heterozygous loci were identified in the parent, among which 9280 were genotyped in at least 10 individuals of each of the two sub-groups. Association analysis indicated that 126 SNPs were associated with carotenoid accumulation in the adductor muscle and that 88 of these were significantly enriched on 28 scaffolds (FDR controlled P < 0.05). The SNPs and genes located on these scaffolds can serve as valuable candidates for further research into the mechanisms by which marine bivalves accumulate carotenoids in their adductor muscles.