Biofilm Development and Stage‐Specific Multi‐Omics Analysis of Metschnikowia bicuspidata Associated With Milky Disease in the Eriocheir sinensis

ABSTRACT Metschnikowia bicuspidata , the causative agent of milky disease in the Chinese mitten crab ( Eriocheir sinensis ), is an important aquatic pathogenic yeast in freshwater aquaculture. However, the developmental characteristics and molecular regulation of its biofilm formation remain poorly understood. In this study, biofilm development of M. bicuspidata LNES0119 was characterized using scanning electron microscopy, confocal laser scanning microscopy and crystal violet quantification, and stage‐specific molecular changes were investigated by integrated transcriptomic and untargeted metabolomic analyses. The results showed that M. bicuspidata formed a typical biofilm within approximately 16 h and underwent four developmental stages: adhesion (0–4 h), proliferation (4–8 h), maturation (8–10 h) and dispersal (10–16 h). Biofilm formation was promoted in Sabouraud dextrose broth, at an initial inoculum density of 1 × 10 8 CFU/mL, 37°C, pH 7.0 and 5‰ NaCl. Transcriptomic analysis revealed progressive increases in differentially expressed genes across developmental stages, whereas metabolomic profiling identified pronounced stage‐dependent metabolic remodelling, with the largest number of differential metabolites detected during maturation. Functional enrichment analysis showed that early biofilm development was associated with membrane‐related processes, transmembrane transport, glycolysis, oxidative phosphorylation and ribosome‐associated pathways, whereas carbon metabolism was more prominent during maturation and secondary metabolism, together with fructose/mannose metabolism, changed markedly during dispersal. These findings establish a stage‐based framework for biofilm development in M. bicuspidata and provide insight into the molecular basis of biofilm‐associated adaptation in this pathogen.