Monarch butterflies are notoriously choosy when picking out a spot to lay their eggs. Of all the plants in the world, only milkweeds pass muster. But monarchs sick with a debilitating protozoan parasite are pickier still. Butterflies infected with Ophryocystis elektroscirrha (OE) flit from plant to plant, searching for those with enough cardenolides—a toxic steroid—to reduce infection in their offspring. Now, researchers have found that how much cardenolide a plant stores may depend on an unlikely ally: soil fungi.

"We’ve known that ecological systems are complex… but the order of complexity just keeps getting larger," says Kabir Peay, a soil microbial ecologist at Stanford University who wasn’t involved in the study. "It was surprising to see that interactions underground between these fungi and the plant can have effects that cascade across four biological kingdoms with pretty significant impacts on the health of the butterflies."

Scientists have long known that arbuscular mycorrhizal fungi are crucial to most of the world’s terrestrial plants. The microscopic organisms, whose intricate network of filaments lace through the soil, deliver nitrogen and phosphorus to plants in exchange for sugar. Without the help of these mycorrhizal allies, it’s possible that plants might not have colonized land some 400 to 500 million years ago.

What wasn’t known, however, was whether the fungi might influence disease transmission among organisms like the monarch. In the wild, monarch caterpillars contract OE when they eat parasite spores left behind by their infected mothers. The more spores—which reproduce inside the growing caterpillar—the more damage they do. But, according to other research, cardenolides seem to reduce the number of spores these caterpillars carry later on in life.

To find out if mycorrhizal fungi played a role in milkweed cardenolide levels—and possibly monarch health—the researchers grew six species of milkweed that naturally produce different amounts of the medicinal cardenolide toxins. Some of the plants were grown with no mycorrhizal fungi, some with low levels of the fungus, and still others with high levels.

The scientists then fed the leaves to two groups of monarch caterpillars; one healthy and the other infected with OE spores. The caterpillars were then left alone to pupate and turn into butterflies, with the researchers recording how long each insect survived.

The results illustrate the complexity of the four-way relationship among the butterfly, the milkweed, the fungi, and the parasite, the researchers report today in Proceedings of the Royal Academy B. The results varied highly based on milkweed species and on the density of fungi in plant roots. In some milkweed species, the fungi did increase the levels of medicinal cardinolides in the plant’s leaves. But that didn’t necessarily translate into a health benefit for butterflies infected with the parasite. That’s because the soil fungi also appear to influence how much of another compound—the nutrient phosphorous—occurred in plant leaves. And the changes in phosphorous, at times, appear to cancel out other potentially beneficial changes in leaf chemistry.

"It was striking to us that we actually found an effect in the first place," says co-author Leiling Tao, a postdoctoral researcher in disease ecology at Emory University in Atlanta. "It was even more surprising that the different milkweed species can have such different responses to the fungi."

Their work shows that animal self-medication doesn’t rely merely on the plants that are used, explains co-author and chemical ecologist Mark Hunter of the University of Michigan in Ann Arbor. "We need to know something about the associated soil communities, too. That’s challenging because our understanding of those systems isn’t nearly as developed."

The finding has potential implications for popular monarch conservation efforts, spurred on by an alarming drop in monarch numbers at their overwintering sites in Mexico. In the United States, those efforts have focused on planting milkweed in habitats decimated by the spread of herbicide-resistant crops. But scientists recently discovered that planting a nonnative, tropical milkweed species actually increases OE infections among monarchs in some places.

Next for the researchers is figuring out how exactly the infected monarchs know which milkweed species—and fungal communities—are most beneficial to them. One possible explanation is that fungi-induced changes in the cocktail of volatile organic compounds produced by milkweed may generate a distinctive smell, drawing monarchs. For the moment, however, whether or not the butterflies can actually detect those changes is unknown, "but it would be surprising if they couldn’t," Hunter says.