Epigenetic Regulation In The Adaptation Of Intertidal Environment In Pacific Oyster

The intertidal zone is the confluence area of the ocean and the land.It can be affected by a variety of environmental factors from the ocean,land and atmosphere,resulting in a highly variable and susceptible condition.The marine organisms that naturally inhabit in the intertidal zone have to cope with the challenges caused by tides and environmental changes due to direct or indirect human activities.As the representative organisms in the intertidal zone,oysters with typical biological characteristics have important ecological and economic values,and they are also suffering from the decline of natural resources and mass mortality that induced by high-temperature environments.Studying the intertidal adaptation mechanism of oysters not only has important theoretical significance for understanding the response mechanism of marine organisms to environmental changes,but also has practical significance for solving the industrial problem of large-scale oyster deaths in summer.Genetic variation and phenotypic plasticity are two important forms of adaptive evolution.The previous studies have shown that phenotypic plasticity plays an important role in the environmental adaptation process of oysters and other marine hyperthermic animals,and epigenetic modifications such as DNA methylation are important mechanisms that can regulate phenotypic plasticity and mediate rapidly environmental responses.The external characteristics of oysters can be easily affected by its growth environment.For example,intertidal oysters generally have smaller sizes,irregular shell shapes,and harder shells compared with subtidal oysters.In addition,the first generation of intertidal oysters and subtidal oysters cultured in the common environment shows phenotypic differentiation in morphological traits,resistance capabilities,and nutrient content.However,knowledge about the internal mechanism of the phenotypic differentiation of oysters derived from intertidal and subtidal zone remains unknown.It is not clear whether the differentiation between intertidal and subtidal oysters results from the genetic variation or the phenotypic plasticity caused by the difference in environment,whether or how much the differentiation can be inherited across generations.The underlying mechanism remains to be explored.In order to solve the above questions,Pacific oyster（Crassostrea gigas）derived from intertidal and subtidal area were used in this study to explore the epigenetic regulation mechanism in the adaptation of the intertidal environment.The parents and offspring were analyzed together,including environments,phenotypes,population genetic structure,gene expression,and epigenetic modification,especially DNA methylation was focused on.The specific content and results are as follows:1.The direct effect of intertidal environment on Pacific oysterThe temperatures of the intertidal and subtidal zone at Laoting,Hebei province showed that intertidal area exhibited higher variability in temperature and had more high temperature（>30°C）days.Phenotypes,population genetic structure and genomic DNA methylation were compared between wild oysters collected from intertidal and subtidal zone.It was showed that there were divergences between the two tidal oysters in stress-related phenotypes,including mortality subjected to heat stress,respiratory rate and physiological indices,with intertidal oysters having higher adaptive capacity than subtidal oysters.There was also differentiation between the two populations in genomic DNA methylation,with differently methylated genes（DMGs）were enriched in fatty acid metabolism,GTPase activity regulation and cell adhesion.No population genetic divergence was detected using Reduced-Representation Sequencing,implying that the epigenetic differentiation could be mainly induced by intertidal environment.2.The inheritance of environment-induced epigeneticsBreeding was performed on oysters wildly collected from intertidal and subtidal areas to generate the first-generation offspring.The offspring were reared in the common subtidal region and were used to investigate the inheritance of DNA methylation induced by intertidal environment.The results of genomic methylation sequencing showed that there was epigenetic differentiation between F₁of intertidal and subtidal oysters,and the differentiation had not correlation with genetic divergence,which supported the conclusion that the epigenetic divergence was mainly induced by intertidal environment rather than genetic divergence.About 41.52%DMGs in parents transmitted to the next generation,and 68.66%of them have consistent regulating trends in the two generations.In addition,F₁ was exposed to high temperature to investigate the epigenetic response of oysters to high temperature.The results showed that in the response to high temperature stress,the methylation level of the intertidal oyster genome showed downward-regulated trend and had high plasticity,while the subtidal oyster exhibited opposite pattern.Genes which potentially regulated by DNA methylation were further identified by integrating methylation data and RNA-seq data,and 67 and 55 genes were identified in intertidal and subtidal oysters,respectively,which provided candidate genes for future functional validation of DNA methylation.3.The stability of inheritance of environment-induced epigeneticOysters wildly collected from intertidal and subtidal area were subjected to two-year breeding to generate F₁ and F₂,and the three generations were analyzed together to explore the inheritance of environment-induced phenotypic variation and DNA methylation.The transgenerational effect was detected on phenotypes and DNA methylation.Phenotypic and epigenetic differentiations between intertidal and subtidal oysters over three generations were revealed,including mortality after heat shock,physiological indices and genomic methylation,which implied that the phenotypic and epigenetic divergence can be stably transmitted over two generations.Furthermore,320heat responsive DMGs that inherited over the three generations were identified by detecting the response to heat stress on the three-generation oysters.And these genes were participated in the processes of energy metabolism,signal transduction and apoptosis.4.The regulatory mechanism of DNA methylation on phenotypeThe inhibitor of DNA methyltransferase 5-Aza was used in adult oysters to reduce the genomic methylation level in oyster.RNA-seq and phenotypes were detected to explore the effects of DNA methylation on gene expression and phenotypes.The depression effect of 5-Aza was firstly detected in oysters.It was showed that the depression effect was dose-dependence within a certain concentration range.RNA-seq was performed for the most depressed group,with 246 differently expressed genes（DEGs）identified.These genes were enriched in the process of superoxide metabolism and ATP synthesis.Phenotypic measurement also showed divergence of the activity of superoxide dismutase and ATPase in the experimental samples and control samples.In addition,high temperature conditions were set on the basis of inhibitor treatment,resulting in a higher mortality in the methylation-suppressed group.And 88 DEGs related to high temperature stress were identified,which can participate in the processes of carbohydrate and amino acid metabolism,signal transduction,apoptosis and autophagy,and immunity.