To cope with environmental stresses, organisms often adopt a memory response upon primary stress exposure to facilitate a quicker and/or stronger reaction to recurring stresses. Sulfide is a common environmental pollutant; however, some organisms have successfully colonized sulfur-rich environments. Whether stress memory plays important role in sulfide stress adaptation remains unclear. In this study, to determine whether Urechis unicinctus, a sulfur-tolerant organism, retains the memory of previous sulfide stress, we simulated a repetitive sulfide stress/recovery system. The results showed that the tolerance of U. unicinctus to sulfide stress was significantly increased after priming with 50 µM sulfide. Further, transcriptional memory genes (TMGs) involved in regulating sulfide stress memory were identified, classified according to their expression patterns, and functionally analyzed. TMGs involved in sulfide metabolism, sugar metabolism, and protein homeostasis pathway showed an enhanced response, whereas those related to DNA repair pathway demonstrated a modified response pattern. Subsequently, functional analysis was performed on the key genes including FoxO and HSPA12-6, which identified through transcriptome analysis. 
    On the other hand, understanding the mechanisms underlying stress memory formation is crucial to enhance adaptive strategies. DNA methylation, a common epigenetic marker, is involved in various biological processes, and has been linked to environmental stress. However, its role in sulfide stress memory formation remains unclear. In our study, we performed a combined analysis of DNA methylation and transcriptome data to determine whether DNA methylation is involved in sulfide stress memory. We found that elevated levels of DNA methylation under repetitive sulfide stress regulated gene expression and resulted in enhanced sulfide stress tolerance in U. unicinctus, a phenomenon verified using DNA methylase inhibitors. Transcriptional memory can be induced in genes related to oxidative stress, regulation of autophagy, and maintenance of protein homeostasis by altering the level of DNA methylation to facilitate sulfide stress acclimation. Further, functional analysis was conducted on the key gene HIF involved in DNA methylation regulation.
    In conclusion，our study indicated that U. unicinctus retains memory of sulfide stress priming, DNA methylation regulates stress memory and mediates plasticity during acclimation to repeated sulfide stress in U. unicinctus.

 

Aquatic animals; Environmental stress; Sulfide; Stress memory; Molecular mechanism