Integrated multi-omics analysis uncovers immune regulatory mechanisms in juvenile Procambarus clarkii during the Black May Disease epidemic period

Black May Disease (BMD), a highly lethal syndrome affecting Procambarus clarkii in late spring-early summer, poses threatens to aquaculture sustainability. To clarify the physiological and immune regulation in juvenile P. clarkii during the BMD epidemic period, we employed an integrated multi-omics approach, assessing biochemical indices, key immune pathways dynamics, and gut microbiota succession to explain disease susceptibility and progression. The results showed marked alterations in immune response and oxidative stress during the BMD epidemic period. The activities of acid phosphatase (ACP) and alkaline phosphatase (AKP) decline consistently, whereas those of lipase and amylase (AMS) increased gradually. Superoxide dismutase (SOD) activity and malondialdehyde (MDA) content peaked at stage S2 before subsequently decreased. Notably, key immune pathways—including the NOD-like receptor, PI3K-Akt, and lysosome pathways—were significantly activated from stages S2 to S3. By stage S4, these pathways showed reduced expression. At the same time, the gut microbiota clearly shifted, with the dominant phylum shifting from Proteobacteria to Firmicutes during the BMD epidemic period. Meanwhile opportunistic genera, such as Aeromonas and Acinetobacter declined, while probiotic-associated genera, including Lactobacillus and Bacillus, increased. Together, these patterns indicate coordinated immune regulation, microbial restructuring, and antioxidant responses that accompany a transition from acute immune activation to physiological homeostasis. In summary, this study elucidates adaptive mechanisms in juvenile P. clarkii during BMD and provides a conceptual basis for developing disease prevention and probiotic intervention strategies to mitigate BMD risk.