A transcription factor-based framework for monitoring stress status in oysters: applications in conservation and restoration Running title: Transcription factors-based monitoring of oyster health

Abstract 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 ecologically relevant TF indicators that reflect the stress status of oysters. Specifically, Pacific oysters (Crassostrea gigas) 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 in oysters. Among these, Brother of Regulator of Imprinted Sites (BORIS) and myeloblastosis 73 (MYB73) 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 abiotic 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 the best of our knowledge, this is the first study to use TF indicators for assessing stress in coastal species. 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.