Integrated m6A RNA methylation and transcriptomic analysis of Apostichopus japonicus under combined high-temperature and hypoxia stress

Abstract

Background: Global climate change has significantly increased environmental stress in marine ecosystems, with rising sea surface temperatures and declining dissolved oxygen (DO) levels. These stressors pose critical challenges to aquaculture, particularly for Apostichopus japonicus, an economically significant species in China. A. japonicus is highly sensitive to combined high-temperature and hypoxia stress, which disrupts physiological processes, suppresses immune responses, and increases mortality. While epigenetic mechanisms such as N6-methyladenosine (m6A) RNA modifications are known to regulate stress adaptation, their role under dual stressors in A. japonicus remains poorly understood.

Results: This study integrates m6A methylation sequencing (MeRIP-seq) and transcriptomic analysis (RNA-seq) to investigate molecular responses in A. japonicus under combined high-temperature (32 °C) and hypoxia (DO = 2 mg/L). Results show that approximately 90% of genes had 1-3 m6A peaks, with single peaks being the most frequent (∼ 60%). Genes with m6A modifications exhibited varying expression levels, with some showing significantly higher expression, suggesting a complex relationship between m6A methylation and stress-responsive gene expression. GO and KEGG enrichment analyses revealed that m6A-modified genes regulate pathways associated with oxidative stress, protein homeostasis, and energy metabolism, such as the PI3K-Akt and MAPK signaling pathways. Key stress-responsive genes, including HSP70, NOX5, and SLC7A11, exhibited dynamic m6A methylation changes, highlighting their roles in redox homeostasis and cellular resilience. Comparative analysis across experimental groups revealed distinct molecular responses to hypoxia, high-temperature stress, and their combination, with combined stress inducing more pronounced changes in m6A methylation and gene expression.

Conclusion: In this study, we explored the central regulatory role of m6A RNA methylation in the response of A. japonicus to the dual environmental stress of high-temperature and hypoxia. The findings show that m6A modification regulates the expression of key genes, allowing A. japonicus to effectively adapt to harsh environmental conditions. This study not only provides an important new perspective on the molecular stress recovery mechanism of marine invertebrates in the face of complex environmental stress, but it also provides theoretical support for aquaculture practice, assisting in the development of more stress-resistant aquaculture systems to deal with the severe challenges posed by global climate change.

Keywords: Apostichopus japonicus; High-temperature stress; Hypoxia stress; Stress adaptation mechanisms; m6A RNA methylation.

Fig. 1

Illustrates the experimental design and the general distribution patterns of m6A peaks under different stress conditions in A. japonicus. (A) Represents the experimental treatment groups, detailing the environmental stress conditions applied, including variations in temperature and dissolved oxygen levels. (B) Summarizes the total number of m6A peaks identified in each group, reflecting the overall methylation landscape under each condition. (C) Shows a Venn diagram displaying shared and unique m6A peaks among the different treatment groups, while (D) Categorizes the proportion of genes containing different numbers of m6A peaks. (E) Visualizes the distribution of m6A peaks across transcript regions, including 3’UTR, 5’UTR, CDS, start codon, and stop codon, emphasizing functional localization patterns. (F) Depicts the density distribution of m6A peaks along transcripts, highlighting positional preferences and regions with higher methylation density. (G) Identifies consensus motifs associated with m6A peaks, revealing sequence-specific regulatory features linked to stress adaptation. (H) and (I) present Venn diagrams comparing shared and unique m6A peaks and m6A-associated genes across group pairs, respectively. Finally, (J) displays the volcano plot illustrating the differential m6A peaks between groups and their associated genes

Fig. 2

Demonstrates the functional enrichment analysis of genes associated with differential m6A peaks across stress conditions. (A) GO annotation results for biological processes (BP), molecular functions (MF), and cellular components (CC) of genes associated with differential m6A peaks. (B) KEGG pathway enrichment analysis of m6A peak-associated genes

Fig. 3

Visualizes transcriptional differences and functional characteristics of DEGs under varying stress conditions. (A) PCA plot shows distinct clustering of the three experimental groups. (B) Venn diagram summarizes shared and unique DEGs among the groups. (C) Bar plot displays the number of upregulated and downregulated DEGs in each pairwise comparison. (D) Volcano plots illustrate the magnitude and significance of transcriptional changes, identifying key stress-responsive genes. (E) GO annotation categorizes DEGs by biological functions. (F) KEGG pathway analysis highlights pathways involved in stress adaptation

Fig. 4

Integrated Analysis of m6A Methylation and mRNA Expression. (A) Displays the expression levels of genes with and without m6A modifications. (B) Venn diagram shows the overlap between differentially m6A-methylated genes and DEGs. (C) Categorizes genes into four groups based on changes in m6A methylation and expression levels, reflecting diverse regulatory responses. (D) Functional enrichment analysis identifies key biological processes and pathways associated with genes showing coordinated changes in methylation and expression. (E) Presents representative genes involved in stress adaptation, emphasizing their roles in cellular responses