Mushroom Volatile Compounds Mediate Avoidance Behavior in a Mycophagous Slug and Differential Responses in Drosophila Flies

Volatile organic compounds (VOCs) emitted by mushrooms may influence interactions with animal consumers, yet the roles of individual compounds and their mixtures remain poorly understood. This study examined odor-mediated behavioral responses of a mycophagous slug and three drosophilid species to mushroom-derived VOCs. Slug feeding avoidances were tested using 43 mushroom species, and tolerance to α-amanitin was evaluated to assess whether avoidance of toxic mushrooms was related to toxin sensitivity. Headspace volatiles from 22 mushroom species (48 samples) were analyzed using Gas Chromatography-Mass spectrometry (GC/MS), and behavioral assays were conducted to test responses of slugs and flies to selected VOCs and their mixtures. The slug showed complete avoidance only of Amanita pallidorosea and Russula subnigricans, and all individuals survived within the tested α-amanitin dose range, indicating that avoidance was not explained by toxin sensitivity. GC/MS detected 65 VOCs, and fragmented Amanita samples formed a distinct group characterized by sulfide-containing compounds. Dimethyl trisulfide induced strong avoidance in slugs, and repellency increased when compounds were combined. Among flies, avoidance responses were most pronounced in Drosophila melanogaster. These findings indicate that mushroom VOCs can function as chemical repellents and that behavioral sensitivity varies among consumers.