ľ�ϸ���Ӱ��

Timing is essential for the zombie ant fungus Ophiocordyceps. When this parasite takes over the behavior of its host, an ant, it also seems to manipulate its biological clock. This way, the parasite makes sure that the ant does not return to its nest, and that it climbs up in a plant at exactly the right moment. Today, a about rhythm-manipulation by parasites written by Utrecht ľ�ϸ���Ӱ�� researchers Joana Dopp and Charissa de Bekker was published in the scientific journal npj Biological Timing and Sleep. Dopp also recently received an EMBO Fellowship to find out more on exactly how the the zombie ant parasite adjusts its host’s clock.

Ants infected with the fungus Ophiocordyceps camponoti-floridani display striking behavioral changes. Usually, these ants are nocturnal: they search for food during the night and return to their nest during the day. But once infected, the ants become active day and night and never return to their nest. Dopp: “They abandon their colony. This way, the other ants cannot detect that the ant is infected.”

The parasite needs the ant to stay alive a little while longer. Eventually, the infected ant climbs up a plant, bites into it and dies. The fungus then digests the ant from within, and when the time is right, a fungal fruiting body emerges from the ant’s body. From this mushroom-like structure, the fungal spores are then spread on the forest floor, to be picked up by the next unsuspecting ant.

Dopp: “The climbing of the infected ant is timed. In nature, it happens around solar noon, when the sun is at its highest position in the sky. This way, the fungus is able to make sure the ant climbs up to the right spot, with just the right amount of sunlight.”

Clock-related genes

A “biological clock” is the internal system that helps organisms keep track of time and coordinate their daily rhythm. The central biological clock of animals, including ants, is located in specific brain cells. It is regulated by genes and influenced by a variety of internal and external factors, such as light, temperature and the age of the organism. Previous research that looked at gene expression, which genes were “turned on” and actively producing proteins, during behaviour manipulation has found that infection by the parasite affects the expression of genes that are related to the biological clock of the ants.

“Besides the behavioral changes that we see in the ant, this is another piece of evidence that the ant’s clock plays an important role in the manipulation by the parasite,” Dopp says.

We will see how the changes in the genes affect the behaviour of the flies. Will it also make them hyperactive, or perhaps change their behaviour in different ways?

Single cell approach

With the EMBO Fellowship, the next two years Dopp is setting out to take a closer look at what exactly goes on in the brains of infected ants. She is using a single cell approach, which enables her to see what is happening in individual brain cells and to pinpoint what regions in the ant’s brain are affected by the parasite.

“This technology is actually really cool,” Dopp says. “We take a brain of an ant, either infected or uninfected, and mix it up. We then load this mixture of brain cells onto a chip, on which each individual cell is encapsulated by an oil droplet. This setup allows us to map the mRNA present in each cell separately. mRNA molecules carry the instructions from genes to make proteins, so by analyzing them, we can see which genes are active in each cell.

Some of these active genes, not necessarily related to the biological clock, help us identify which part of the brain each cell came from. By comparing the gene activity in infected versus uninfected ants, we can then build a brain atlas that shows how infection by the parasite affects the expression of genes in different brain regions.”

Visual validation

This brain atlas will result in a set of genes that might play a role in the clock-manipulation of the parasite. Dopp: “First, I plan to validate these genes using microscopy. I will design probes, molecules that bind only to a specific mRNA sequence inside cells. Under a microscope, thanks to these probes I can then visually inspect where exactly in the brain a gene of interest is active in manipulated and healthy ants.”

Thanks to technological advances, we can apply tools, such as single-cell RNA sequencing and microscopy to any organism, allowing us to study fascinating biological phenomena in nature, such as zombie behaviour.

Back to flies

Next, Dopp plans to return to the animals she worked with during her PhD: fruit flies. Dopp: “To try to mimic what the parasite is doing, we can silence (“turn off”) or activate our candidate genes in specific cells in flies. Unfortunately, we do not have the tools to do this in the ants themselves. But both ants and flies are insects and many biological mechanisms are similar. We will then see how the changes in the genes affect the behaviour of the flies. Will it also make them hyperactive, or perhaps change their behaviour in different ways?”

Ecological relevance

Fruit flies are model organisms, which means they have been extensively studied for a longer time and many tools are available to study them. That is different for the ants she is working with now. “The fly research field is focused on doing research under very controlled conditions,” Dopp says. “But I kind of missed working on ecologically relevant questions about natural populations.”

According to Dopp, now is a good time to shift the focus to non-model organisms. “Thanks to technological advances, we can apply tools, such as single-cell RNA sequencing and microscopy to any organism, allowing us to study fascinating biological phenomena in nature, such as zombie behaviour.”