It's one of nature's most disturbing horror stories: a fungus takes control of a living ant and uses it to spread its spores through the colony. But now there's a strange new wrinkle to the story, as a new study led by Penn State University has found that the fungus makes these "zombie ants" without directly infecting the brains of its hosts.

Officially, the parasite is known as Ophiocordyceps unilateralis sensu lato, but it's commonly known as the zombie ant fungus. When it infects its target – carpenter ant workers – the fungus drives the insect against its will to the underside of a leaf or a stick. Once there, the ant is forced to clamp its mandibles to the leaf, where it eventually dies and gives the parasite the perfect place to grow. Over four to 10 days, a stalk grows out of the dead ant's head, releasing spores down to the ground below to infect other ants and repeat the cycle anew.

This ruthless life cycle has been well studied over more than a century, but the complicated mechanics behind how the fungus controls the ant weren't fully understood. And that's where the Penn State study comes in.

"To better understand how such microbial parasites control animal behavior, we looked at cell-level interactions between the parasite and its carpenter-ant host at a crucial moment in the parasite's lifecycle — when the manipulated host fixes itself permanently to vegetation by its mandibles," says Maridel Fredericksen, lead author of the study. "The fungus is known to secrete tissue-specific metabolites and cause changes in host gene expression as well as atrophy in the mandible muscles of its ant host. The altered host behavior is an extended phenotype of the microbial parasite's genes being expressed through the body of its host. But it's unknown how the fungus coordinates these effects to manipulate the host's behavior."

To gain a better understanding of the processes involved, the researchers first infected ants with either the zombie ant fungus or another fungus, to try to uncover which cell-level effects are unique to the former. Then, they built 3D images using scanning electron microscopy to see where the fungal cells were in the ants' bodies, and in what concentrations.

To peer inside the ants and see the interactions between parasite and host, the team took 50-nanometer slices of tissue, imaged them, and stacked them again to make incredibly high resolution 3D images. Artificial intelligence and machine learning algorithms were then employed to analyze the images and differentiate between ant and fungus cells.

These images revealed that the fungus cells had spread throughout the ants' bodies, showing up in the head, thorax, abdomen and legs of the insects. The cells form a 3D network to communicate and take control of the ant, and were present in such large numbers that the researchers declared that "in essence, these manipulated animals were a fungus in ants' clothing."

Interestingly, there were no fungal cells in the ants' brains at all, although they did cluster around the outside of the brain, where they can influence the insects' behavior chemically.

"Normally in animals, behavior is controlled by the brain sending signals to the muscles, but our results suggest that the parasite is controlling host behavior peripherally," says David Hughes, senior author of the study. "Almost like a puppeteer pulls the strings to make a marionette move, the fungus controls the ant's muscles to manipulate the host's legs and mandibles."

A 3D reconstruction of an ant mandible muscle (red) surrounded by fungal cells (yellow)(Credit: Hughes Laboratory / Penn State)

Infecting the brain seems like a shortcut to taking control of the insect, but the researchers believe that showing restraint in that department helps the fungus keep the ant alive until it serves its ghastly purpose.

"We hypothesize that the fungus may be preserving the brain so the host can survive until it performs its final biting behavior – that critical moment for fungal reproduction," says Hughes. "But we need to conduct additional research to determine the brain's role and how much control the fungus exercises over it."

The research was published in the journal Proceedings of the National Academy of Sciences.

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