Caterpillars use different strategies to protect themselves from their enemies; many are camouflaged, while others use their bright colors as warning signals, have stinging hairs or secrete toxic substances, some even take threatening postures. Scientists at the Max Planck Institute for Chemical Ecology have now discovered a previously unknown protective mechanism: Tobacco hornworm larvae can exhale a small fraction of nicotine they ingest as they feed on tobacco leaves. To do so, they transfer some of the nicotine they ingest into their hemolymph (insect blood) from which a “defensive halitosis” is created that repels a major predator. These insights were made possible by combining molecular techniques with a natural history approach in field experiments in the native habitat of the study organisms.

For their field experiments, researchers planted tobacco plants that were deficient in producing nicotine. In addition, they used a plant-mediated RNAi technique to silence a cytochrome P450 enzyme in the midgut of tobacco hornworm larvae which is usually activated by nicotine ingested when the larvae feed on tobacco leaves. The scientists then observed what happened to caterpillars feeding on nicotine-deficient plants in order to compare their fate with those caterpillars that had ingested nicotine but lacked the active catalyzer for the toxin in their midgut.

The function of the cytochrome P450 proved hard to reveal by laboratory-based experiments, but then the researchers received unexpected support from a wolf spider Camptocosa parallela. Surprisingly, the nocturnal predator preferably preyed not only on larvae that fed on nicotine-free leaves, but also preyed on their cytochrome P450-silenced conspecifics that were deficient in their response to nicotine in the food. The gene must therefore have played an important role in a spider defense mechanism that usually excludes the spider from the list of Manduca sexta’s enemies.

Further analysis revealed that the enzyme plays a role in transporting the ingested nicotine from midgut to the hemolymph which allows the nicotine to be exhaled out the spiracles, the nose-equivalents of the caterpillars. Caterpillars exhale a small fraction of this nicotine. And this functions as an anti-spider signal. Other predators of Manduca sexta, such as bugs or antlions, seem to be completely insensitive to this defensive halitosis.

Nicotine, the defensive substance in their host plant, is too toxic for the larvae to sequester. Most of it is excreted. That the larvae repurposes only a tiny amount of the toxin for their own defense in order to ward off spiders with a kind of toxic halitosis came as a surprise for the scientists. “This case of toxic breath as a defense is unique,” says Ian Baldwin. The example of the wolf spider illustrates the importance of combining molecular biology and natural history to understand the function of genes at the level of the organism. Scientists from the Department of Molecular Ecology headed by Baldwin pioneer this approach and call it an unbiased, “ask the ecosystem” approach. “Nature is our most important teacher,” Baldwin emphasizes. “Nature is the arbitrator of who survives. Elucidation of gene functions is only possible if you study organisms in their native environment − including all the unknowns of the wild.”

Content: MPI press release (modified).