The “milky disease” in Chinese mitten crabs (Eriocheir sinensis), caused by Metschnikowia bicuspidata, poses significant threats to aquaculture, though its pathogenic mechanisms remain poorly understood. This study employs transcriptomic sequencing to analyze gene expression changes in Metschnikowia bicuspidata 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 M. bicuspidata 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.

“Milky disease” of the Chinese mitten crab (Eriocheir sinensis), caused by Metschnikowia bicuspidata, leads to substantial economic losses. Despite extensive research on its pathogenesis, the genetic basis of host resistance and underlying regulatory mechanisms remain unclear, limiting the development of disease-resistant varieties. This study aimed to (1) evaluate resistance differences among 10 E. sinensis families (five highly resistant and five sensitive) via artificial challenge; (2) identify disease-resistance-associated genetic loci using genome-wide association analysis (GWAS). Our findings revealed that the F05 family exhibited the strongest resistance, with a mortality rate of only 3% and a molting rate of 73%, accompanied by superior growth performance. Whole-genome resequencing identified 10,161,545 high-quality SNPs, and GWAS detected 767 loci significantly associated with disease-resistance traits, among which two pleiotropic SNPs (Chr46:18395778 and Chr1:20680490) were simultaneously associated with both “dead or not” and “qPCR fixed amount of fungi”, reflecting their functional relevance in regulating survival and pathogen load. Notably, we propose for the first time that E. sinensis achieves resistance by inducing M. bicuspidata into a viable but nonculturable (VBNC) state, in which the fungal cells remain metabolically active but cannot form colonies on conventional culture media. This study establishes a multidimensional resistance evaluation system, clarifies the genetic basis and novel mechanism of resistance, and provides valuable molecular markers for marker-assisted breeding. The findings contribute to reducing disease-related losses and promoting sustainable development of the E. sinensis aquaculture industry.

Frequent outbreaks of bacterial and fungal diseases in aquaculture cause severe economic losses, making biological control using probiotics a crucial strategy. Bacillus velezensis synthesizes diverse antimicrobial secondary metabolites; however, its biocontrol potential is tightly regulated by environmental signals such as cultivation time. In this study, we investigated the effect of growing time on the antifungal activity of B. velezensis GHZJ-1, isolated from an aquatic environment. It was found that GHZJ-1 shows obvious antifungal activity against the aquatic pathogen Metschnikowia bicuspidata upon 48 h growth but no such activity for 24 h via the agar-diffusion method. We further compared the transcriptomes of GHZJ-1 cells collected at 24 h and 48 h through RNA-Seq. Our results revealed that compared with 24 h, 1821 genes were differentially expressed at 48 h, with 903 upregulated and 918 downregulated. Downregulated genes were enriched in primary metabolic pathways (e.g., ribosome assembly and carbon metabolism), whereas upregulated genes were enriched in secondary metabolite biosynthesis and transmembrane transport. Importantly, 35 upregulated genes directly associated with antimicrobial activity were identified, notably including the ones encoding core elements of a large polyketide synthase (e.g., pksN, log₂FC = 2.24), the petrobactin siderophore system (highest log₂FC = 3.39), and various antimicrobial peptide export systems. Furthermore, the degU gene was activated at 48 h. These data suggest that facing continuously increased environmental stress over time, GHZJ-1 undergoes global transcriptional reprogramming and resource reallocation, downregulating basal metabolism to construct a synergistic antagonistic system coupling chemical defense with nutritional competition. This study elucidates the time-dependent mechanism for antifungal activity in GHZJ-1, providing a molecular theoretical basis for its green biocontrol application in aquaculture diseases caused by fungi.