Truffles are prized as food, while millipedes release secretions to defend themselves. Yet the same molecule, dimethyl sulfide (DMS), has been detected in these seemingly unrelated organisms.
This connection comes not from one experiment directly comparing them, but from bringing together two original studies with different aims. One asked what cues truffle-hunting dogs use. The other examined compounds initially emitted by living millipedes and the effects of some of them on cells. Together, they also illustrate the difference between sharing a molecule and sharing an overall aroma.
Looking for a common truffle cue
The 2022 truffle study sought chemical cues that allow dogs to find different truffle species. Researchers analyzed four species, the white truffle Tuber magnatum, the black truffle T. melanosporum, the summer truffle T. aestivum, and T. indicum, along with commercial truffle oil.
Samples were thinly sliced on the day they arrived. A 0.5 g portion was placed in a sealed container and held at 40°C for 15 minutes, after which the headspace gas was analyzed by gas chromatography–mass spectrometry. Compounds were identified mainly by comparison with a mass-spectral database.
DMS was detected in all four species. Its share of the overall signal differed between species, however, as did the combinations of other compounds. Notable compounds included 2,4-dithiapentane in white truffles, and 2-butanone and 2-butanol in summer truffles.
These proportions are based on analytical peak areas. Because some compounds smell strong even in small amounts, the figures cannot be treated as equivalent to contributions to aroma. Commercial oil contained some compounds shared with fresh samples and others that differed. Even a sample labeled as black truffle showed a prominent compound considered characteristic of white truffles.
Nineteen dogs responded frequently to DMS and DMDS
Using their analyses and the literature, researchers selected 11 compounds for the dog experiment. Each was dissolved in white oil at 25 mg/L. Together with a white-oil-only control and truffle oil, the resulting 13 samples were sent to 19 farms in the United States, Spain, and Germany.
The farmers soaked sponges with the samples, buried them approximately 5 cm underground, and recorded whether trained dogs responded to them as truffles. Most dogs usually searched for black truffles. Samples were tested one at a time in a fixed order.
Sixteen of the 19 dogs responded to DMS and 16 to dimethyl disulfide (DMDS). Sixteen also responded to truffle oil, with more dogs responding to these samples than to the other individual compounds. DMDS had not been detected in the study’s instrumental analysis of truffles; it was included as a candidate on the basis of earlier research.
However, six dogs also responded to white oil alone. The authors discuss possible influences including owners’ footprints and odors, weather, and training history. Given the fixed presentation order and differences between farm environments, DMS and DMDS are promising candidates, but were not established as the only cues the dogs use.
Living millipedes also emitted DMS
The 2021 millipede study focused on compounds initially released into the air, which conventional analysis involving solvent extraction of the body can have difficulty capturing. It examined two species collected in Okinawa, Oxidus gracilis and Chamberlinius hualienensis.
Live animals without adhering plant debris were placed in containers at room temperature for 40 minutes. A 100 mL sample of headspace gas was then concentrated in three stages and analyzed. Animals were pooled by species: four males and three females for O. gracilis, and two males and seven females for C. hualienensis.
DMS was detected in both species. Monoterpenes including α-pinene, β-pinene, and limonene, as well as furans, were also found. Some identifications were confirmed against standards, while others were tentative database identifications. This revealed a different set of compounds from the phenols and quinones prominent in conventional solvent extracts.
The study’s central discovery was not DMS alone. An important finding was the presence of monoterpenes also found in plants among the early emissions of living millipedes. The authors discuss the possibility that millipedes accumulate plant-derived compounds by eating leaf litter and use them for defense. However, the study did not directly track transfer from food to secretory glands or test whether the compounds repel predators.
A separate experiment examined effects on cells
The millipede study also applied standards of detected compounds to mouse-derived macrophage-like cells and rat-derived PC12 cells. Cells were exposed for 17 hours at 50–200 µg/mL, and researchers measured viability and the activity of caspases 3/7, which are associated with cell death.
Some monoterpenes, including α-pinene, β-pinene, and 3-carene, reduced viability, and increases in caspase activity were also observed. The authors describe this as cell death involving apoptosis.
These findings help inform discussion of the biological activity of compounds in the secretions. Because the substances were added directly to cultured cells, the results cannot simply be extrapolated to smelling millipedes or eating truffles. Nor does the shared presence of DMS establish that truffles are dangerous.
The same molecule can have different companions and roles
In the truffle study, DMS was a candidate cue supporting dogs’ searches. In the millipede study, it was one of a range of initially emitted compounds. Considering the two together shows that a characteristic odor cue for one organism need not be a molecule found exclusively in that organism.
Which compounds are released, in what amounts, and alongside what others? What has the organism smelling them learned, and how does it respond? Even when a molecular name is shared, the overall aroma and its role need to be understood within these combinations.
Summary
Separate studies detected DMS in four truffle species and two species of living millipedes. Truffle-hunting dogs frequently responded to DMS and DMDS, suggesting their importance as search cues. Responses to white oil alone, however, show that the conditions determining dogs’ responses need further investigation.
The millipede study established the presence of monoterpenes in early emissions and effects of some compounds on cultured cells. Beginning with the unexpected places where the same molecule can be found, these studies show how understanding odors involves both combinations of compounds and responses from the organisms receiving them.
The truffle study was funded by Landwirtschaftliche Rentenbank through a German government special-purpose fund. At the time of the study, one coauthor was affiliated with sglux SolGel Technologies. No separate funding statement was found for the millipede study. The authors of both papers declared no conflicts of interest.
References
Epping R, Bliesener L, Weiss T, Koch M. Marker Substances in the Aroma of Truffles. Molecules. 2022;27:5169. Original research article
Taira J, Tamashiro M, Naka K, Gakiya S, Taira K. Initial defensive secretory compounds emitted from the live millipede and the induction of apoptotic cell death. Scientific Reports. 2021;11:8222. Original research article
