Discovery of shikonin as a safe and potent oral candidate for the sustainable management of ichthyophthiriasis.

Ichthyophthirius multifiliis remains a devastating ciliate parasite in global aquaculture, yet sustainable therapies are critically scarce. While many plant-derived compounds display potent in vitro efficacy, their practical application is frequently hindered by the failure of immersion treatment to reach tissue-embedded parasites. To overcome this challenge, following a screening of 20 natural compounds, we identified shikonin (SK) as a highly promising orally bioavailable candidate with a novel mechanism. In vitro, SK demonstrated potent efficacy with EC50 values of 0.24, 0.37, and 4.05 mg/L against theronts, protomonts, and tomonts, respectively. Mechanistically, it acts primarily by inducing mitochondrial dysfunction and ATP depletion rather than non-specific membrane disruption. Crucially, driven by favorable in silico oral bioavailability predictions, we evaluated its in vivo potential via in-feed administration in goldfish (Carassius auratus). The results revealed that a 4-day oral regimen (80 mg/kg body weight) drastically reduced trophont burdens by 73.9%, profoundly suppressed tomont reproduction, and blocked secondary infection cycles. Furthermore, this dietary treatment effectively eliminated infection-induced tissue damage and restored host survival to 100%, compared to 57.1% in untreated controls. Coupled with a high safety margin (LD50 = 562.6 mg/kg, approximately 7-fold above the therapeutic dose), these findings highlight SK as a promising and safe oral phytomedicine for the sustainable management of ichthyophthiriasis.