Research into psychedelic compounds is in resurgence because of the exciting potential for their use in the treatment of psychiatric and mental health disorders. Despite this revival, remarkably little is known about their evolution. One of the most intriguing psychedelic compounds is psilocybin, the compound found in 'magic' mushrooms and used in ritual ceremonies in North America for generations. Associated with agaricomycete fungi across eight distantly related genera, psilocybin acts in a similar way to the neurotransmitter serotonin, yet how and why natural selection favoured its biosynthesis remains unclear. Given the resemblance to serotonin, a highly conserved neurotransmitter across invertebrates and vertebrates, modulation of invertebrate behaviour for defence is a likely explanation, but neither this nor alternative hypotheses have ever been formally tested. Here, we show thatlarvae exposed to extracts frommushrooms exhibit reduced survival, pupation rates, and inhibited locomotion. Adults exposed during development show reduced thorax and wing size, along with small but significant deviations from perfect bilateral symmetry in wing venation, indicating developmental stress. However, mutants lacking the serotonin receptor that mediates psilocybin's effects in humans (5HT2A) showed the same response toextracts as wild-type flies. Furthermore, DNA metabarcoding revealed that althoughdemonstrates a distinct invertebrate community compared to most other grassland fungi, it overlapped with the non-psychedelic species. This study provides a crucial first step toward understanding the evolutionary role of psilocybin-producing fungi and provides a grounding for future research into the molecular mechanisms, ecological interactions and evolutionary origins of psychedelic compounds in nature. Drosophila Psilocybe Psilocybe Psilocybe semilanceata Mycena epipterygia
