Volatile organic compounds (VOCs) are low molecular mass organic compounds that easily evaporate at room temperature. Fungi produce diverse mixtures of VOCs, some of which may contribute to “sick building syndrome,” and which have been shown to be toxigenic in a variety of laboratory bioassays. We hypothesized that VOCs from medically important fungi might be similarly toxigenic and tested strains of Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Cryptococcus gattii, and Saccharomyces cerevisiae in a Drosophila melanogaster eclosion bioassay. Fungi were grown in a shared microhabitat with third instar larvae of D. melanogaster such that there was no physical contact between flies and fungi. As the flies went through metamorphosis, the numbers of larvae, pupae, and adults were counted daily for 15 days. After 8 days, ~80% of controls had eclosed into adults and after 15 days the controls yielded 96–97% eclosion. In contrast, eclosion rates at 8 days were below 70% for flies exposed to VOCs from six different A. fumigatus strains; the eclosion rate at 15 days was only 58% for flies exposed to VOCs from A. fumigatus strain SRRC 1607. When flies were grown in a shared atmosphere with VOCs from S. cerevisiae, after 15 days, 82% of flies had eclosed into adults. Exposure to the VOCs from the medically important yeasts Candida albicans, Cryptococcus neoformans, and Cryptococcus gattii caused significant delays in metamorphosis with eclosion rates of 58% for Candida albicans, 44% for Cryptococcus neoformans, and 56% for Cryptococcus gattii. Using gas chromatography-mass spectrometry, the VOCs from the most toxic and least toxic strains of A. fumigatus were assayed. The two most common VOCs produced by both strains were 1-octen-3-ol and isopentyl alcohol; however, these compounds were produced in 10-fold higher concentrations by the more toxic strain. Our research demonstrates that gas phase compounds emitted by fungal pathogens may have been overlooked as contributing to the pathogenicity of medically important fungi and therefore deserve more scrutiny by the medical mycology research community.

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Drosophila melanogaster is a useful model organism that offers essential insights into developmental and cellular processes shared with humans, which has been adapted for large scale analysis of medically important microbes and to test the toxicity of heavy metals, industrial solvents and other poisonous substances. We here give a brief review of the use of the Drosophila model in medical mycology, discuss the volatile organic compounds (VOCs) produced by the opportunistic human pathogen, Aspergillus fumigatus, and give a brief summary of what is known about the toxicity of some common fungal VOCs. Further, we discuss the use of VOC detection as an indirect indicator of fungal growth, including for early diagnosis of aspergillosis. Finally, we hypothesize that D. melanogaster has promise for investigating the role of VOCs synthesized by A. fumigatus as possible virulence factors.

Aspergillus fumigatus is a ubiquitous opportunistic pathogen. We have previously reported that volatile organic compounds (VOCs) produced by A. fumigatus cause delays in metamorphosis, morphological abnormalities, and death in a Drosophila melanogaster eclosion model. Here, we developed A. fumigatus deletion mutants with blocked oxylipin biosynthesis pathways (∆ppoABC) and then exposed the third instar larvae of D. melanogaster to a shared atmosphere with either A. fumigatus wild-type or oxylipin mutant cultures for 15 days. Fly larvae exposed to VOCs from wild-type A. fumigatus strains exhibited delays in metamorphosis and toxicity, while larvae exposed to VOCs from the ∆ppoABC mutant displayed fewer morphogenic delays and higher eclosion rates than the controls. In general, when fungi were pre-grown at 37 °C, the effects of the VOCs they produced were more pronounced than when they were pre-grown at 25 °C. GC–MS analysis revealed that the wild-type A. fumigatus Af293 produced more abundant VOCs at higher concentrations than the oxylipin-deficient strain Af293∆ppoABC did. The major VOCs detected from wild-type Af293 and its triple mutant included isopentyl alcohol, isobutyl alcohol, 2-methylbutanal, acetoin, and 1-octen-3-ol. Unexpectedly, compared to wild-type flies, the eclosion tests yielded far fewer differences in metamorphosis or viability when flies with immune-deficient genotypes were exposed to VOCs from either wild-type or ∆ppoABC oxylipin mutants. In particular, the toxigenic effects of Aspergillus VOCs were not observed in mutant flies deficient in the Toll (spz6) pathway. These data indicate that the innate immune system of Drosophila mediates the toxicity of fungal volatiles, especially via the Toll pathway.