A Transcription Factor-based Framework for Monitoring Stress Status in Oysters: Applications in Conservation and Restoration.

Developing effective molecular indicators to monitor stress status of coastal species is a top priority due to the impacts of climate change. However, the complexity of stress responses, which are regulated by multiple genes, limits the effectiveness of single-gene approaches in accurately reflecting stress status. Transcription factors (TFs) are promising candidates for comprehensively assessing stress responses, as they regulate numerous stress-responsive genes. In this study, we present a framework for identifying TF indicators that reflect the stress status of Pacific oysters (Crassostrea gigas). Specifically, oysters were exposed to high, medium, and low tide conditions to assess the physiological responses of oysters to tidal-induced stress. Enrichment analysis of differentially accessible chromatin peaks derived from assay for transposase-accessible chromatin sequencing (ATAC-seq) identified several key TFs. Among these, CCCTC-binding factor (Cg-CTCF) and MYB proto-oncogene A (Cg-MYBA) were significantly upregulated under tidal-induced stress and occupied critical positions in regulatory networks, as indicated by RNA-seq. RNA interference experiments confirmed that both genes contribute to enhancing survival under heat stress, a major stressor affecting oysters. Additionally, field experiments demonstrated significant upregulation of these genes under natural stress conditions, suggesting their potential as indicators for oyster reef management. To our knowledge, this is the first study on the coastal invertebrate to combine ATAC/RNA-seq with in vivo TF knockdown and field validation to propose TFs as practical stress indicators. We advocate for the broad application of our framework to explore TFs as molecular indicators of health status in marine organisms, thereby enabling informed strategies for conservation management.