A break down of our latest BMC Evolutionary Biology paper

Lab Note #3
Sep 19, 2014

As promised, in this lab note I will break down our latest paper, published open access in BMC Evolutionary Biology, for you. Since it appeared online last month it has already been accessed 7863 times! Something that would never have happened if the paper were published behind a pay wall. Open access is really paying off. Add to this the many media outlets that have reported about it and a recommendation on F1000 Prime, and quite some people across the globe have at least picked up something about the work we have done!

Here you can find 3 of my favorite write-ups:

Turns Out the U.S. Has Its Very Own Species of Ant-Zombifying Fungus

Zombie Fungus Enslaves Only Its Favorite Ant Brains

Zombifying Fungus Recognizes the Brain of its Favorite Host

The paper starts with us introducing that the fungi of the complex Ophiocordyceps unilateralis aren’t just found in the tropics. The US has its very own species as well! The species that we use in this paper is one that we have found in South Carolina. In fact, citizen scientist Kim Fleming did. Kim is a mathematics teacher who loves to go for walks in the woods behind her house with her camera to photograph all kinds of critters. As such, she discovered the zombie ants in her very own back yard! Since then she has been working together with our lab and we get to visit her yearly to do our fieldwork. And because of her invaluable work, she is also an author on our published paper. Here are two of the many beautiful pictures she has taken:

[Camponotus americanus and Camponotus castaneus infected with Ophiocordyceps unilateralis, credit: Kim Fleming]

While doing our fieldwork we realized that 2 Carpenter ant species in the area are infected and manipulated to bite by Ophiocordyceps but another, very frequently encountered species, is not. Of course this can have many reasons: maybe these ants never encounter the spores, or the fungal spores are simply not able to infect these ants. But since we came up with a way to infect ants in the lab by injecting them we were able to test if these environmental factors would be the (only) barrier that prevents this fungus from infecting all Carpenter ant species in the area.

What we found after injecting was quite remarkable: the fungus was able to kill all three ant species as a result of the infection, but only manipulated the 2 that it is able to manipulate in nature as well. So, even when we take away all the environmental factors and make sure infection takes place, the non-host species is still not manipulated! In fact, the fungus was not even able to grow out of the ant cadaver after death, as is seen for the natural host species. Behavioural manipulation therefore seems to be rather species-specific.

Here’s a figure to recap this quickly for you, before we move on to the more complicated molecular biology stuff:

[Experimental set up used for the infection experiments, showing that the non-host ant species tested cannot be manipulated while the natural hosts are. C. castaneus photo credit: Roel Fleuren. C. americanus and C. pennsylvanicus photo's © Alex Wild,used by permission. Naturally infected C. castaneus and C. americanus photo credit: Kim Fleming]

So, it thus appears that the species-specificity of the manipulation is at least partly due to the interaction of the fungus with the host while growing inside the ants’ body. Now, brain manipulation is most likely achieved by the fungus secreting bioactive compounds onto the ants’ brains. Therefore, as a next step, we let Ophiocordyceps interact with the brains of different ant species. To do this, we kept ant brains alive in “jars” and threw in the fungus. To measure what the fungus would secrete as a reaction to these different species brains, we then harvested the medium in which they were kept and measured all the compounds that were in there. These compounds were compared to control samples in which we only had the fungus, only had the ant brains, and only had the medium to be able to tell which compounds were there because the fungus secreted them to affect the different brains it was presented with.

[The "brain in the jar" experiment. The inserted picture shows you what the drawing of the ant brains and the fungus look like in real life]

Then, we compared the results we got from the different species interactions to see if the fungus reacted the same way or differently to them. What we found was that apparently Ophiocordyceps is able to recognize that it is dealing with a different ant brain because most of the compounds it would secrete were different for the various interactions. This could explain why we don’t find every old ant species turned into zombie ants. The parasite-host interaction to achieve control of the host is apparently a very specific one.

[Video of a still alive Camponotus castaneus ant infected in the lab with O. unilateralis, recorded during manipulated biting behavior]

Now, last but not least, we have tried to identify what the candidate compounds are that we have found. This is a very difficult process at the moment, so most of them stay undiscovered for now, but we have been able to identify 2: guanidinobutyric acid (GBA) and sphingosine. When searching the literature for both of these, they appear to be involved in neurological diseases. This makes them interesting candidates as ants infected with Ophiocordyceps have some sort of neuro-disease as well (as their brains tell them to go bite a leaf instead of forage for food!). But, it also shows us the potential of discovering that this fungus indeed secretes some very interesting neuromodulators that might be good candidates for novel drug discovery.

[Mass spectra of the 2 candidate compounds we have identified. To verify if the identification was indeed correct we bought standards of the compounds. The top is what we have found in the fungal secretion samples and the mirror images are the standards of those compounds we have used to show the spectra are indeed identical.]

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