Mechanistic elucidation of Esculetin's anti-WSSV activity and its application potential in sustainable aquaculture

White Spot Syndrome Virus (WSSV) poses a major challenge to crustacean aquaculture, causing White Spot Disease (WSD) and significant economic losses worldwide. Despite its high infectivity, effective control strategies for WSSV are still lacking. In this study, 30 medicinal plants were screened, and C. fraxini was identified as a strong inhibitor of WSSV proliferation. Its active compound, esculetin, demonstrated potent antiviral activity, achieving 98.56 % inhibition at 80 mg/kg. Further analysis showed that esculetin inhibits WSSV replication in a dose-dependent manner, reduces viral transcription, and improves the resilience of infected crayfish. In both prophylactic and therapeutic treatments, as well as cohabitation assays, esculetin significantly decreased pathogen loads and prevented inter-host transmission, underscoring its potential as an effective disease control strategy. Esculetin works through multiple mechanisms: it suppresses viral genes involved in WSSV transcription, disrupts the virus's ability to manipulate host immune signaling, and interferes with its replication cycle. Additionally, esculetin modulates key enzymes, stabilizes apoptotic pathways, and mitigates oxidative stress and inflammation caused by infection. By restoring proteostasis and boosting host defense, esculetin ultimately enhances crayfish resistance to WSSV. These findings shed light on the pharmacological properties of esculetin and provide a strong rationale for its development as a potential agent to control WSSV outbreaks, supporting the sustainability of crustacean aquaculture.