Periodic Reporting for period 1 - CEREBRALfungi (CharactERizing the neuronal Effects of BehavioRAL-modifying proteins from the zombie FUNGI, Ophiocordyceps, using C. elegans)
Reporting period: 2023-08-01 to 2025-07-31
Ophiocordyceps unilateralis is a group of insect-infecting fungi known for their ability to manipulate behavior. They do so to help themselves spread easier to new hosts. In this way, they are reminiscent of the pathogens from various zombie movies that infect humans and cause them to behave in ways that spread the disease (e.g. biting). For this reason, these fungi are often referred to as "zombie fungi". Fungi in general are a well known source of many chemicals that can be used for therapeutic purposes. Antibiotics are a common example of fungal chemicals that can be used to keep people healthy. For that reason, our research team is interested in exploring this group of fungi, which is known to affect the nervous system of mammals, in hopes of finding new compounds that can be used to help with behavioral disorders. Understanding how these "zombie-making" fungi are able to affect the nervous system is thus an important step towards exploring if these chemicals have any therapeutic use. The behavioral effects caused by these fungi are well described in nature, particularly in ant-infecting Ophiocordyceps. Previous studies have shown that in zombie ants the fungus first disrupts the social behaviors of its host, reducing their communication with nestmates and causes them to leave the nest, abandoning their social roles. This is important for the fungus because it allows the parasite to avoid detection and potential destruction by nestmates. These zombie ants then exhibit a climbing behavior known as summit disease, where they climbing nearby vegetation to a higher point that aids in the fungus's growth and spore dispersal. Finally, the ant bites down onto the plant material at this elevated position, ensuring that it remains stuck to the plant after death when the fungus grows out of the ant from the back of the head and makes new spores to infect new ants. By causing these behavioral manipulations, the fungus is better able to spread its spores further using the wind. However, while the behaviors may be well known, the chemicals involved in these behavioral manipulations have been more difficult to uncover. This is partly due to the fact that these fungi are hard to work with in a laboratory setting, making many of the modern day genetic tools unavailable to them. For this reason, this project focused on exploring ways that other model organisms, like bacteria, yeast, and nematodes, that are much easier to work with, could be used to help explore the functions of compounds from Ophiocordyceps species, particularly small secreted proteins. Because this project incorporates so many different organisms, it spans many biological sub-disciplines, and thus, lends itself well to the international exchange of personnel, advanced training, and ideas. If we are able to use the molecular genetics tools available to these more easy to work with model organisms, then we can bypass the barriers of working directly with the fungi in lab and open the door for rapid study of new fungal compounds. This approach would not only make studying zombie fungi much easier, but it would also create tools that can be used by a lot of other types of scientists who are interested in exploring the neurobiology of other hard-to-work-with insects, such as those that cause damage to agricultural crops.
To accomplish our goal of establishing a new set of tools and protocols for the exploration of chemicals from hard-to-work-with fungi, a collaborative effort was formed between the developmental biology and the microbiology departments at Utrecht University. Members of the developmental biology department are experts in the imaging of nematode nervous systems, allowing us to visualize where fungal compounds wind up in the nervous system, and identify which neurons are affected. They also have experience performing a type of experiment with yeast that allows us to identify the molecular targets of small secreted proteins from the fungi. Together, these tools allowed us to identify what conserved neurological pathways were effected by fungal proteins during behavioral manipulation. Using these tools, we were able to identify a new kind of neuropeptide and describe how it interacts with the nervous system in nematodes. neuropeptides are very small proteins that interact with the nervous system. We found that this newly discovered neuropeptide binds to Scramblase proteins found in the neurons that play a role in muscle movement and smell, presumably interfering with the ants mobility and/or their ability to smell the pheromones used by other ants to communicate. These findings were therefore very helpful in informing our next steps to test how the protein affects the ants. Because we have evidence that motor neurons and olfactory neurons (the neurons involved in smell) are affected, we were able to set up behavior assays to test this further. To set up behavior assay with nematodes, our collaborative group further reached out to scientists at Wageningen University and the Vrije Universiteit Amsterdam to record nematode behavior and help create a software tool that could detect subtle differences in their movement. Using these new tools, we were able to detect a change in nematode behavior when exposed to this new neuropeptide. Confident that this neuropeptide affects the nervous system, we finally sought to test if this protein affects ants in the same way that we found it affected nematodes. This involved a series of behavioral experiments where the ants were either introduced to the neuropeptide by injection, or where the Scramblase targets of the protein were down regulated in the ants. We could then compare the results of the behavior tests, and if the tests show the same results, we could be confident that not only does the neuropeptide effect ant behavior, but that it does so by interfering with the proper function of Scramblase proteins. And this was precisely what we saw. Our behavior tests showed that if you expose these ants to this Ophiocordyceps neuropeptide, or if you inhibit the function of the Scramblase targets, ants communicate with one another less frequently and they are less sensitive to adverse smells like citronella. These findings are interesting for multiple reasons. First, our success with using model organisms as surrogates to study Ophiocordyceps proteins provides a new framework for other scientists to explore potential therapeutic or insecticidal compounds in other systems. Second, our findings suggest that Scramblases play a much more important role in neurons that previously thought, particularly in olfactory tissues. Finally, this work is the first to identify one of the many "effectors" used by Ophiocordyceps fungi to manipulate the behavior of ants.
This project achieved its goal of adopting molecular genetics tools from model systems to characterize the first Ophiocordyceps behavioral effector. In doing so, it provides a new pipeline for the exploration of biomolecules from other difficult-to-work-with systems as well. This pipeline involves the use of bioinformatic tools to identify secreted proteins, yeast for identification of protein targets, bacteria for transgenic production of fungal proteins, nematodes for the visualization of protein localization, identification of neurons involved, and preliminary behavior assays to inform future experiment. It also introduces new tools and techniques for studying the social behavior of ants. Furthermore, our findings support other recent works in mice and fruit flies that suggest Scramblase proteins play an important role in neuron function. This approach will be used in the future to further explore Ophiocordyceps proteins and their role in behavior manipulation.