We evaluated the individual and combined effects of salinity and alkalinity on gill Na⁺/K⁺-ATPase enzyme activity, plasma ion concentration, and osmotic pressure inLuciobarbus capito. Increasing salinity concentrations (5, 8, 11, and 14 g/L) were associated with an initial increase and then decrease inL. capitogill Na⁺/K⁺-ATPase activity. Activity was affected by the difference between internal and external Na⁺ion concentrations and osmotic pressure (P<0.05). Both plasma ion (Na⁺, K⁺, and Cl⁻) concentration and osmotic pressure increased significantly (P<0.05). An increase in alkalinity (15, 30, 45, and 60 mM) caused a significant increase in plasma K⁺and urea nitrogen concentrations (P<0.05) but had no effect on either plasma osmotic pressure or gill filament ATPase activity. In the two-factor experiment, the saline-alkaline interaction caused a significant increase in plasma ion (Na⁺, Cl⁻, and urea nitrogen) and osmotic pressure (P<0.05). Variance analysis revealed that salinity, alkalinity, and their interaction significantly affected osmotic pressure, with salinity being most affected, followed by alkalinity, and their interaction. Gill filament ATPase activity increased at first and then decreased; peak values were observed in the orthogonal experiment group at a salinity of 8 g/L and alkalinity of 30 mM.

The outbreak of white spot syndrome (WSS) is a looming challenge, due to dramatic losses to the crustacean aquaculture industry. However, at present, there are no prophylactic or therapeutic means to control this infectious viral disease. Here, we screened fifteen medicinal plants for their inhibitory activity on the white spot syndrome virus (WSSV), using red swamp crayfish (Procambarus clarkii) as a model species. The results showed that the crude extracts of Pinellia ternata (Thunb.) Breit. had the highest inhibitory effect (91.59%, 100 mg/kg) on WSSV proliferation, and its main component, beta-sitosterol, showed a much higher activity (95.79%, 50 mg/kg). Further, beta-sitosterol potently reduced (p < 0.01) viral loads and viral gene transcription levels in a concentration-dependent fashion, and significantly promoted the survival rate of WSSV-challenged crayfish (57.14%, 50 mg/kg). The co-incubation assay indicated that beta-sitosterol did not influence the infectivity of WSSV particles. Both pre- and post-treatment of beta-sitosterol exerted a significant inhibitory effect (p < 0.01) on the viral load in vivo. Mechanistically, beta-sitosterol not only interfered with the expression of viral genes (immediate early gene 1, ie1; DNA polymerase, DNApol) that are important in initiating WSSV transcription, but it also attenuated the hijacking of innate immune signaling pathways (Toll, IMD, and JAK/STAT pathways) by viral genes to block WSSV replication. Moreover, the expression of several antiviral immune, antioxidant, pro-inflammatory, and apoptosis-related genes changed significantly in beta-sitosterol-treated crayfish. Beta-sitosterol is a potent WSSV inhibitor and has the potential to be developed as an effective anti-WSSV agent against a WSS outbreak in crustacean aquaculture.