LC-MS and molecular networking-based identification of bioactive compounds in termite fungus combs

Fungus-farming termites (subfamily Macrotermitinae) maintain an ancient mutualistic association with Termitomyces fungi, resulting in the formation of specialized biostructures known as fungus combs. These complex matrices are essential for colony nutrition and are traditionally used in ethnomedicine, yet their chemical composition and bioactive potential remain insufficiently characterized. Here, we investigated the antibacterial activity and metabolomic profiles of 13 fungus combs associated with four termite species collected from different regions of Senegal. A total of 78 fractions were obtained through sequential liquid–liquid extraction and analysed using UHPLC-HRMS/MS combined with feature-based molecular networking and in silico annotation tools. Antibacterial activity was assessed against Micrococcus luteus. Ethyl acetate and dichloromethane fractions exhibited the strongest antibacterial activity, with inhibition zones exceeding 25 mm in extracts derived primarily from Macrotermes bellicosus and Macrotermes subhyalinus combs. Molecular networking revealed a chemically diverse metabolome dominated by terpenoid-related compounds, polyketides, alkaloids, and some amino acids and peptides. Several annotated metabolites, including macrolides potentially associated with actinobacterial symbionts, were enriched in the most active fractions in positive electrospray ionization modes (ESI+). Overall, our findings demonstrate that termite-associated fungus combs represent chemically complex and biologically active environments. This integrative metabolomics approach provides new insights into the ecological mechanisms supporting Termitomyces monoculture and highlights fungus combs as promising reservoirs of bioactive natural products.