MicroRNAs, as one of the post-transcriptional regulation of genes, play an important role in the development process, cell differentiation and immune defense. The sea cucumber Apostichopus japonicus is an important cold-water species, known for its excellent nutritional and economic value, which usually encounters heat stress that affects its growth and leads to significant economic losses. However, there are few studies about the effect of miRNAs on heat stress in sea cucumbers. In this study, high-throughput sequencing was used to analyze miRNA expression in the body wall of sea cucumber between the control group (CS) and the heat stress group (HS). A total of 403 known miRNAs and 75 novel miRNAs were identified, of which 13 miRNAs were identified as significantly differentially expressed miRNAs (DEMs) in response to heat stress. A total of 16,563 target genes of DEMs were predicted, and 101 inversely correlated target genes that were potentially regulated by miRNAs in response to heat stress of sea cucumbers were obtained. Based on these results, miRNA-mRNA regulatory networks were constructed. The expression results of high-throughput sequencing were validated in nine DEMs and four differentially expressed genes (DEGs) by quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, pathway enrichment of target genes suggested that several important regulatory pathways may play an important role in the heat stress process of sea cucumber, including ubiquitin-mediated proteolysis, notch single pathway and endocytosis. These results will provide basic data for future studies in miRNA regulation and molecular adaptive mechanisms of sea cucumbers under heat stress.

DNA methylation is an important epigenetic modification that regulates many biological processes. The sea cucumber Apostichopus japonicus often suffers from heat stress that affects its growth and leads to significant economic losses. In this study, the mRNA expression patterns and DNA methylation characteristics in the body wall of A. japonicus under heat stress were analyzed by whole-genome bisulfite sequencing (WGBS) and transcriptome sequencing (RNA-seq). We found that CpG was the main DNA methylation type, and heat stress caused a significant increase in the overall methylation level and methylation rate, especially in the intergenic region of the A. japonicus genome. In total, 1,409 differentially expressed genes (DEGs) and 17,927 differentially methylated genes (DMGs) were obtained by RNA-seq and WGBS, respectively. Association analysis between DNA methylation and transcription identified 569 negatively correlated genes in both DMGs and DEGs, which indicated that DNA methylation affects on transcriptional regulation in response to heat stress. These negatively correlated genes were significantly enriched in pathways related to energy metabolism and immunoregulation, such as the thyroid hormone signaling pathway, renin secretion, notch signaling pathway and microRNAs in cancer. In addition, potential key genes, including heat shock protein (hsp70), calcium-activated chloride channel regulator 1(clca1), and tenascin R (tnr), were obtained and their expression and methylation were preliminarily verified. The results provide a new perspective for epigenetic and transcriptomic studies of A. japonicus response to heat stress, and provide a reference for breeding sea cucumbers resistant to high temperatures.