Autophagy-related responses in Pacific oyster Crassostrea gigas exposed to phenanthrene: Association with a putative E3 ubiquitin ligase-NF-κB regulatory axis

Phenanthrene (PHE) is a common marine pollutant, but whether it induces autophagy-related responses in bivalves and how such responses contribute to cellular adaptation under pollutant stress remain incompletely understood. Here, using multimodal imaging, ultrastructural analysis, proteomics, and gene-expression assays, we found evidence consistent with the engagement of an E3 ubiquitin ligase-NF-κB-autophagy regulatory axis in hemocytes of the Pacific oyster Crassostrea gigas following phenanthrene exposure. Confocal laser scanning microscopy, Cyto-ID flow-cytometry screening, and transmission electron microscopy revealed increased autophagy-related signals and the presence of autophagic vesicle-like structures in PHE-exposed hemocytes. The proteomics results showed that protein levels of HUWE1, TRIM36, and ATG7 were significantly upregulated. The expression of axis-related genes was significantly upregulated after PHE exposure, while CAPE-mediated inhibition of NF-κB attenuated the PHE-associated induction of LC3 and p62/SQSTM1 transcripts, supporting the involvement of NF-κB signaling in the autophagy-related transcriptional response. Similar upregulation of axis-related transcripts in RAW264.7 macrophages supported a comparable direction of transcriptional response, but did not establish conserved pathway activation or causality. Together with changes in hemocyte number, apoptosis rate, phagocytic capacity, and ROS levels, these findings are consistent with a cellular adaptation response under PHE stress. This study provides integrated evidence for autophagy-associated stress responses in Pacific oysters exposed to phenanthrene and offers a mechanistic framework for future validation of pollutant tolerance in marine bivalves.