Transcriptome Analysis and CFEM Gene Overexpression in Metschnikowia bicuspidata Under Hemocyte and Iron Ion Stress

The "milky disease" in Chinese mitten crabs (<i>Eriocheir sinensis</i>), caused by <i>Metschnikowia bicuspidata</i>, poses significant threats to aquaculture, though its pathogenic mechanisms remain poorly understood. This study employs transcriptomic sequencing to analyze gene expression changes in <i>Metschnikowia bicuspidata</i> under hemocyte challenge, iron overload (1 mmol/mL), and combined stress, with functional validation through Common in Fungal Extracellular Membrane (CFEMgene) overexpression strains. Key findings reveal that (1) hemocyte challenge activated base excision repair (-log₁₀[P] = 7.58) and ribosome biogenesis pathways, indicating fungal adaptation through DNA repair and enhanced protein synthesis to counter host immune attacks (e.g., ROS-mediated damage). (2) Iron overload induced glutathione metabolism and pentose phosphate pathway enrichment, demonstrating mitigation of ferroptosis through NADPH/GSH antioxidant systems and autophagy/proteasome coordination. (3) Under combined stress, ribosome biogenesis (-log₁₀[P] = 1.3) and non-homologous end-joining pathways coordinated DNA repair with stress protein synthesis, complemented by vacuolar V-ATPase-mediated iron compartmentalization. (4) CFEM genes showed significant upregulation under hemocyte stress, with overexpression strains exhibiting enhanced biofilm formation (35% increased MTT cytotoxicity) and infectivity (40% higher infection rate), confirming CFEM domains mediate pathogenesis through iron homeostasis and virulence factor production. This work elucidates how <i>M. bicuspidata</i> employs metabolic reprogramming, oxidative stress responses, and CFEM-mediated iron regulation to establish infection, providing critical insights for developing targeted control strategies against milky disease.