Punica granatum extract demonstrates antiparasitic effects against Caligus clemensi through in silico and in vitro studies.

The increasing resistance of aquatic ectoparasites, particularly Caligus clemensi, to synthetic antiparasitic agents highlights the need for exploration of alternative competing strategies in aquaculture. This study assessed the antiparasitic potential of Punica granatum (pomegranate) methanolic extract, focusing on its bioactive phytochemicals and their synergistic effects. Using gas chromatography‒mass spectrometry (GC‒MS) analysis conducted in this study, we identified a diverse range of compounds, including monoterpenes, aromatic hydrocarbons, and fatty acid derivatives, many of which are known for their neurotoxic, membrane-disrupting, and antimicrobial activities. Notable compounds, such as α-terpinene, γ-terpinene, δ-3-carene, and terpineol, were found to be major constituents of the extract. The presence of p-cymene and m-cymene in the extract may contribute synergistically to its antiparasitic activity by enhancing parasite cell membrane permeability and facilitating the uptake of other bioactive constituents. In addition, in silico molecular docking analysis of a representative pomegranate-derived compound (1-methyl-4-(1-methylethylidene)cyclohexene) revealed strong binding affinity to key detoxification enzymes such as glutathione S-transferase theta 1-1 (GSTT1) and cytochrome P450 3A24. These interactions suggest potential inhibitory effects, which may impair the parasite's detoxification pathways and metabolic resistance mechanisms. The observed antiparasitic activity is likely due to a novel multimodal mechanism involving neurotoxic, membrane-disruptive, and enzyme-inhibitory actions. P. granatum extract offers a sustainable, eco-friendly alternative to synthetic agents, with reduced resistance risk. This is the first report combining phytochemical profiling, molecular docking, and bioassays to demonstrate its efficacy against C. clemensi. These findings support its potential as a natural, broad-spectrum parasiticide for sustainable aquaculture and justify further in vivo and toxicological studies.