Supplemental Materials from Balancing selection for aflatoxin in <i>Aspergillus flavus</i> is maintained through interference competition with and fungivory by insects

The role of microbial secondary metabolites in the ecology of the organisms that produce them remains poorly understood. Variation in aflatoxin production by <i>Aspergillus flavus</i> is maintained by balancing selection, but the ecological function and impact on fungal fitness of this compound are unknown. We hypothesize that balancing selection for aflatoxin production in <i>A. flavus</i> is driven by interaction with insects. To test this, we competed naturally occurring aflatoxigenic and non-aflatoxigenic fungal isolates against <i>Drosophila</i> larvae on medium containing 0–2000 ppb aflatoxin, using quantitative PCR to quantify <i>A. flavus</i> DNA as a proxy for fungal fitness. The addition of aflatoxin across this range resulted in a 26-fold increase in fungal fitness. With no added toxin, aflatoxigenic isolates caused higher mortality of <i>Drosophila</i> larvae and had slightly higher fitness than non-aflatoxigenic isolates. Additionally, aflatoxin production increased an average of 1.5-fold in the presence of a single larva and nearly threefold when the fungus was mechanically damaged. We argue that the role of aflatoxin in protection from fungivory is inextricably linked to its role in interference competition. Our results provide the first clear evidence of a fitness advantage conferred to <i>A. flavus</i> by aflatoxin when interacting with insects.