- Welcome, everyone, to Wednesday Nite @ the Lab. I'm Tom Zinnen. I work at the University of Wisconsin-Madison Biotechnology Center. I also work for the Division of Extension Wisconsin 4-H. And on behalf of those folks and our other co-organizers, PBS Wisconsin, the Wisconsin Alumni Association, and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite @ the Lab. We do this every Wednesday night, 50 times a year. Tonight, it's my pleasure to introduce to you Caitlin Calhoun. She was born in Madison and went to Memorial High School here. Then she went to the University of Wisconsin-Milwaukee and studied biological science and environmental conservation. She recently received her master's degree in bacteriology here at UW-Madison. Tonight, she's gonna speak with us about ants and antibiotics. Would you please join me in welcoming Caitlin Calhoun to Wednesday Nite @ the Lab?

- Thank you, Tom, for that wonderful introduction. I'm here to talk to you today about something I'm extremely passionate about, but I wanna be clear that it is about a lab that is not currently at the UW anymore and about a leafcutter ant colony that I managed for about eight years, but has since left the university. But the research here is ongoing and is extremely important. So today, I'm gonna talk to you about leafcutter ants. And you may have seen them or visited them at your local zoo or a museum or even at a university. So today, we're gonna talk about what it's like to care for these ants in a domestic setting. And we're also gonna talk about what it's like to witness them in the wild, and also how they're helping fight antimicrobial resistance. So if you had ever had a chance to visit the Microbe Place at the base of the Microbial Sciences building on the UW-Madison campus, there was a leafcutter ant colony there for about 15 years. The colony consisted of a foraging chamber, which we would fill with leaves that we would harvest locally and they would incorporate into their fungus garden. 

This outreach facility at the base of Microbe Place welcomed the public as well as lots of field trips and school groups, and was really beloved by everyone that worked in the building. When I was closing down the display colony, I had a graduate student come up to me and say that it was their meditation to come and watch the ants cut leaves every day and it helped them get through their PhD. And there were just many stories like this when we were taking apart the ant colony that came forward that were really heart-touching, were really touching. And so, like I said, there were many outreach events that these ants were a part of, and we would always be open for science expeditions, Wisconsin Science Festival and Darwin Days, among others. And we would participate in games and we'd get lots of ant-related questions, which was really lovely.

So right now, I'm gonna kind of bring you into what it was like to witness this ant colony at Microbe Place. And this is a video of leafcutter ants carrying bits of leaves from that foraging chamber that I was talking about. And what we would have to do as researchers is we would have to maintain these colonies, and they would cut about one full paper bag of leaves three days a week. So you can imagine that they would take a lot of leaves to maintain these colonies. And so during the summers, what we would have to do is we would have to go out and we'd have to collect leaves. And in this video, you can see that they're carrying little bits of these leaves that we would collect and they were taking them to their forging chambers in that cabinet that I showed before. And we always had a little bit of fun, like on holidays and things like that. And we would have them cut anything that we found. They particularly loved hibiscus flowers, and they could take one flower down and completely dismantle it in about a half an hour. And what you're seeing right now is a time-lapse video of them taking apart the hibiscus flower and incorporating that into their chambers. And it was kind of a treat for them. They didn't get flowers very often, but when they did, they would get very excited. [chuckling]

So as I discussed before, it would take about one paper bag full three times a week to feed these colonies. So as a lab, working in the Currie Lab, we would have to work tirelessly to make sure that during the summer, we had collected enough leaves so that we could maintain these colonies throughout the winter. So what we would do is we were lucky to have a partnership with the Lakeshore Preserve on campus, and we would get phone calls whenever they were trimming trees or whenever they needed maintenance done, they would have us come out. 
And what we would do is we would take all the maple and the oak leaves from whatever they were trimming and we would vacuum seal those and put them into a freezer for the storage over the winter. You can see here some photos from our leaf collecting days that we would do it as a lab, and we would have to pack probably between 300 and 400 bags every summer in order to make sure that we had enough leaves to feed the ants over the winter. And when we are collecting these leaves, you can imagine that we would get hitchhikers every once in a while. So this is my favorite story of the hitchhiker Charlotte, which we named this spider who lived in the forging chamber of the display colony. She lived there for a few months, and as you can see, she lived in harmony with the ants. She would feed on the little ants occasionally, but for the most part, they lived together in harmony. But at some point, we think that the ants detected her. And one day, we showed up and we were feeding the ants, and Charlotte was gone. But for a good few months, it was one of those things where students would come up and ask me and ask me if Charlotte was still there and how she was doing. And it was just a fun little anecdote whenever I would tell stories about the leafcutter ant colonies or how the ants could get along with other creatures, at least for a certain amount of time. 

So now that we've talked a little bit about the domesticated ants, I wanna take a step back and talk about these fungus-farming ants and what they're doing with these leaves, and talk about, you know, what you would look like or what this would look like in the wild if you were to witness them in Central or South America, where they're native. This is a beautiful illustration by a former Currie Lab undergrad named Julia Buskirk. And you can see here that this is, like, a depiction of a leafcutter ant colony where a bunch of ants carrying leaves, but that's just the tip of the iceberg. Underneath the ground, you're gonna see their actual fungus chambers and their dump chambers, where they're gonna be holding that food that they need to survive. So here, you're looking at a family tree of the leafcutter or of the fungus-farming ants, or it's also called a phylogeny. There are 15 genera or 300 species of fungus farming ants, and they have been farming for about 60 to 80 million years. The lower agriculture fungus farmers depicted here in tan and in pink, they farm a fungus that is a lot smaller and shaped a bit different than the higher agriculture fungus-farming ants in purple. With the tan Cyphomyrmex, they actually farm a yeast, which is similar to what you're using to make bread or beer. They farm a type of yeast. And then Apterostigma, another lower agriculture fungus-farming ant farms a coral fungus which is gonna be found, instead of underground, it's gonna be found on the undersides of leaves or on tree trunks instead of under the ground, like I said before. 

And then the higher agriculture fungus-farming ants are really interesting. With the Trachymyrmex and the Sericomyrmex, they're gonna have really small colonies where they're only gonna have, like, one fungus chamber, whereas you've got the Atta and Acromyrmex, which are gonna be those really large colonies similar and also the kind that we had in the display colony at the UW. So what's really interesting about the fungus of the higher agriculture fungus-farming ants is that the fungus that they farm is only found with the ants. So the fungus that they farm cannot grow in any other environment besides in the fungus chambers that the ants tend to. Whereas with the Cyphomyrmex and Apterostigma, their fungus can actually grow outside and be found free living in the environment. So an example of what, so the higher agriculture, but smaller fungus-farming ants like the Trachymyrmex, the Sericomyrmex, you're gonna find only one fungus chamber, and their whole colony is gonna live in that one fungus chamber. Whereas if you look at the higher attines, which are the Atta and Acromyrmex, this is just an example of what the fungus would look like out of one chamber, but they're gonna have numerous chambers in one colony. And because of that, they're gonna have a lot of more complex social structure as well, which we'll talk about in a minute.

So if you're traveling to Central and South America, one of the things you'll notice is that these leafcutter ants make these little highways, and they can actually indent the soil and affect the vegetation around them because they're using these paths so much. So this is a video where you have a lovely tracking shot as if you were from the point of view of an ant carrying a piece of leaf in these highways that they form. And you can see them carrying little bits of vegetation and looks like some seed pods, but they're really beautiful to see when you're walking around in Central or South America. And when these ants are going to be climbing up these trees and actually trimming these little pieces of leaf off, which you'll notice is that their mandibles are razor-sharp, and the ants are gonna be using surgeon-like precision to be cutting these pieces to incorporate into their fungus gardens. And after they've cut the piece of leaf off and they carry the leaf in their mandibles, that's the equivalent of a 150-pound person carrying a car. So these leaf pieces that they're carrying are gonna be about 50 times their weight. So not only are these ants going to be cutting pieces of leaves and just stripping trees, stripping plants, but they're gonna be doing it extremely fast because there's gonna be so many of them. And they are also the dominant herbivore in the neotropical forest environment, meaning that they cut more vegetation in Central and South America than any of the deer or the pigs. They're gonna be accounting for about 25% of all herbivory on a daily basis.

So once they've taken their leaves and they've incorporated it into their fungus garden, this is a close-up look of what that fungus garden is gonna look like. And what you can see here is that there are many different layers to this one fungus chamber. On the top, you can see that it's a very light gray, but, like, with some darker marbling. 
And that is gonna be where that new leaf material is gonna be incorporated in. And then as the fungus starts to break down that leaf matter, it's gonna turn a different color. And each one of those little pockets is gonna house, like, this nutrient-rich nodules called gongylidia, which is what the ants are actually gonna be eating. And they're gonna be like any other garden crop. They're gonna be constantly weeding out dead pieces and taking out any material that isn't gonna be productive or healthy for the fungus, so they're gonna be constantly weeding this garden. To see a few examples of what the fungus garden looks like from those other ant species that I talked about. On the left here, this is an example of that coral fungus from that Apterostigma ant. And you can see that it's kind of looks like a triangular, almost palace, and there's a tiny opening at the bottom, and that's where they're gonna enter and exit. But the whole inside of that is gonna be hollowish, and that's where their whole colony is gonna be living. And again, that's gonna be on the underside of a tree or underside of a branch. 

Where this picture was taken, it was kind of on, like, an overhang on the side of a road, like where something had been dug out. There were a bunch in tree roots, where they were hanging really delicately. And then on the top right, that's a beautiful example of a fungus garden from either a Trachymyrmex or a Sericomyrmex, where you have that one chamber underground, and that, again, is gonna be where the entire colony is gonna be living. And then finally, another close-up look of that one fungus chamber from an Atta colony that we've talked about, which, again, is just gonna be one of many, and we're gonna go into how big those can get in a minute. And then I just wanted to share with you, which I think that the fact that these ants can cultivate yeast is just fascinating and they look like, as you can see, the close-up on this image, those little, like, granular grains of rice are gonna be the yeast that they form, or that they farm. And then here's a awesome video of Dr. Charlotte Francoeur and graduate student Silver Ceballos in Costa Rica, collecting a Cyphomyrmex colony. And you can see that it was on the side of a tree, and when they kind of lift down the leaf, you can see those rice grains, and that's gonna be that yeast cultivar that we had talked about. So now that we've talked about the different types of fungus in the fungus growing phylogeny, I wanna now specifically focus on the Atta and the large and complex social structures that come with having such a large and complex colony.

So to get an idea of how large these colonies are gonna be, there's an image here on the left, and all of those little white, like, light dots are colonies. So this image is actually taken from space. So these colonies can be so massive that you could actually potentially see them from space, but scientists didn't really understand how big they were. So in order to figure that out, they ended up finding a ant colony. And this is an image of the ant colony before they poured in the cement. You can see that there's multiple entrance holes for the ants, and then there's some cows there for some scale. And so they poured about ten tons of concrete into this colony over three days. And they discovered a colony that was about eight meters inside or 50 square meters, which is just giant. So here's a image of after they had excavated that colony after the cement was poured in, and they ended up excavating about 40 tons of soil, which is the equivalent of, like, billions of ant loads of, like, ants individually carrying out little pieces to excavate this colony. And so on a human scale, that would be equivalent to us traveling one kilometer or 0. 62 miles every time we would take a load of dirt out to excavate this colony. 

And to kind of orient yourself here, all those little nodules are gonna be fungus chambers. So the building of this colony would be the human equivalent of building the Great Wall of China. So with each of those nodules being a fungus chamber, I think the largest Atta ant colony that's been excavated like this had something like 1,500 fungus chambers. So, which is very different from those lower agriculture colonies that we were talking about that only have one fungus chamber. So when you have that many fungus chambers and this colony is just, you know, colony so large that it can be seen from space, you need to develop strategies in order to maintain the health of that colony. And the attine ants have developed a very complex social structure, where not only do they have, they have different castes of workers that have different roles in their colonies.

So what you're looking at now is an image of the different sizes. And you can see that the soldier is quite massive compared to the minima worker standing right next to it. But there's a whole range of workers that have different roles in order to maintain this colony. So the soldier is the largest, and they're gonna be very protective of the ants. 
So if you're walking down a little ant highway and you come across, you're gonna see that they are on the outside. They're gonna be flanking all of the foragers and they're gonna be protecting the foragers to make sure that they can get their leaf pieces to the fungus gardens safely. And then as you get smaller, you'll get all the way down to the minima workers. Those workers are gonna be the ones that are gonna be weeding out the fungus garden or they're gonna be taking care of the queen, which is arguably the most important job because she is the one that is supplying this colony with workers. And then you have the other foragers and different sizes in between, and those are either gonna be helping cut the leaves or they're also gonna be tending the gardens, or they're gonna be in charge of tending to the eggs and the pupae and making sure that they hatch okay. So each one of these ants is determined at egg size, depending on nutrition, of what size they will be. So if the colony needs more soldiers, the pupae will be fed more and then they'll get larger and they'll become soldiers. Or if they need more minima workers, then they'll be fed less and they'll end up developing smaller, and then they'll be tending to the garden or tending to the queen.

So as you can see, the queen is not on this slide 'cause she gets her own slide 'cause she's very special. So one thing you will notice, the first thing you'll notice is that she is massive and she looks very different from the other ants. So she does not have wings. She is about three times the size of the ants or all the worker ants. And her job is going to be birthing those worker ants. From when she forms the colony, she is gonna have all of her every needs. . . All of her needs are taken care of by the minima workers. So she's gonna be groomed, she's gonna be fed, and then all of those eggs that she births are gonna be taken by those workers and they're gonna be put in the most nutrient-rich parts of that fungus garden and fed only the nicest fungus to make sure that they develop correctly. But she doesn't start out this way. So she starts out with wings. There are two seasons in the neotropics. There's the dry season and the wet season. And the dry season is when the leaves are gonna be a little bit more brittle and not as nutrient-packed. And that is gonna signal to a leafcutter ant colony that resources could be limited. And when that happens, the queen of that colony is going to start producing something called virgin queens and alates. Alates are gonna be the male ants.

So here's a depiction of what a virgin queen would look like inside the colony. You can see that she's a little bit lighter-colored and she has wings. And then once the wet season comes to the neotropics, that's gonna signal to this ant colony that resources are gonna be abundant and that this would be the best time for these new queens and alates to leave the nest and potentially start their own colonies. But before they leave, the queens are gonna have this little pocket underneath their chin, and they're gonna take a piece of that fungus from their original fungus garden and they're gonna put and stuff it in that pocket. And then they're going to leave the nest by the thousands and they are going to mate as many times as possible with those male ants that are also gonna be all leaving the nests at this same time. And they're gonna mate, you know, as much as they can. And then that will be the only time that the queen will ever need to mate. And then the males will end up dying and they will provide a very nutrient-rich source for the rest of the ecosystem. And the queen will end up ripping off of her wings and then taking that little bit of fungus fragment that she had collected from her original colony and adding that to her wings. And then that's gonna be the start of her new fungus chamber. So she's gonna dig down and start her fungus chamber with the wings as that substrate. And then from there, she's gonna start birthing eggs, which will lead to her first workers, which will then tend the fungus garden. And then from there, they will just continue to grow and grow as the environment allows.

So you can see here, this is a video of the queen carrying a bit of fragment of fungus, and she's gonna be searching for that fungus chamber that she's gonna crawl into. So in a domestic setting, I know that a fungus-farming ant colony is really healthy if I can't see the queen at all because a very healthy queen is gonna be in the middle of that fungus chamber, and she is gonna be covered with other small minima ants, being taken care of. And so if I don't see her, I know that that colony is healthy. And when we go out and collect these research colonies to take back to the lab, we don't have to take the entire colony. So the most important part of the colony is gonna be the queen, a little bit of fungus, and then a few workers. So on the left here, this is what it looks like when you collect ant colonies in the field. What you do is you kind of, you can grab a Petri dish or you grab a little container and you put some cotton balls in there with some moisture to make sure that the fungus garden doesn't dry out. And then you try to find the queen. And then you put in some fungus garden and some workers and a little food. And I say you try to find the queen because in a colony that has multiple chambers, it really is the luck of the draw. You don't know which chamber the queen's gonna be in. [chuckles] And it makes it kind of fun for digging, fun or just hard work.

And then once the colony is taken back to the lab, you can then build up that fungus garden again. And you can do that by feeding them a lot of leaves and also by making sure that their colonies are well-moisturized and kept clean. So here's a video of me cleaning a leafcutter, one of those experimental colonies that I've taken care of for many years. And you can see, this is one of my favorite colonies. She's yelling at me. And these ants are practicing something called stridulation. And I just wanna invite you to look at that white underneath their chin 'cause that's gonna be something we're gonna talk about next. But that stridulation is something that insects do. And in this case, the colony is producing a warning because I am invading their space and they don't want me to be there. 
[ants chittering]

So now that we've talked about leafcutter ants and what it's like for them to live in the wild, I want to switch gears a little bit here and start talking about the anti and ant symbiosis. So as we've discussed, the leafcutter ants cut the leaves and they feed those leaves to their fungus garden, which is a monoculture that they farm. And with any monoculture, you. . . And any sort of interrelated plant or fungal cultivar, there's going to be pathogens that are gonna be opportunistic, that are gonna wanna feed on that plant. And the fungus garden is no different. And the opportunistic fungal pathogen that really loves to infiltrate leafcutter ant colonies is called Escovopsis here, noted with the skull. So this opportunistic fungus that likes to kill the ant fungus will just decimate leafcutter ant colonies. So the ants have developed a defense in order to protect their livelihood, which is this fungus garden, from this invading pathogen. And that is called Pseudonocardia, which is a type of actinobacteria. And if you remember from the slide before, I was asking you to watch the little white underneath the chin in these ants. And that's because that is where the Pseudonocardia grows and lives predominantly.

And so this Pseudonocardia is very interesting because it acts as a natural pesticide, protecting the fungal cultivar against this pathogen called Escovopsis. So when you're looking at a leafcutter ant colony in the lab, or even if you've dug out a leafcutter ant colony in the wild, what you'll notice is that there is something that is, like, this white, powdery substance or it looks like a white, powdery substance on either the backs of these ants that are living in the fungus garden or underneath, like if you lift up an ant and you turn it over, there's, like, gonna be white that's going on underneath their chin. And so this substance is the Pseudonocardia that I mentioned before. And you can see in this image, we call them sheep ants. And you can see that this one has a beautiful blanket of Pseudonocardia on its back. And so the Pseudonocardia is really interesting because the ants will vertically transmit this bacteria from their bodies to the eggs and to the pupae, and they will specifically groom and take it off their backs onto this pupae. And then from there, the ants will acquire this bacteria; in fact, to the point where the ants have developed these crypts or these little, like, holes and structures in their exoskeleton that particularly house this bacteria. 

So we know that this is a symbiotic relationship that these ants have acquired that is useful for the protection of their fungus garden, and that they're passing it on. So the ants are very particular about their fungus garden. And as you can see here, there's this powdery substance that you can see that's, like, this brownish in this video. And the ant here is detecting that it is not supposed to be there, that it is Escovopsis, and that they need to weed it out. So they will practice taking that Pseudonocardia off their body and rubbing it onto their fungus garden. They'll take the Pseudonocardia as they munch up the leaf material and incorporate it into the fungus garden. These are the types of things that they'll do in order to transmit that bacteria into their fungus garden to protect it from this Escovopsis. And on top of that, they are gonna be continually weeding, like we said before, with the minima workers. Their entire job is gonna be looking and detecting this Escovopsis and making sure that it doesn't invade the colony, or it doesn't invade the fungus garden.

So the actinobacterial symbionts or that Pseudonocardia that I'm talking about is visible on the workers' bodies and underneath their chin; it's called a propleural plate. And it is apparent on most of the phylogenetic diversity of fungus-growing ants. And I said before, to summarize, it is vertically transplanted from parent to offspring and then to the nest. And it occurs in these specialized locations in their exoskeleton. And so scientists, and particularly Dr. Cameron Currie has been able to isolate that Pseudonocardia from the ant and grow it in a Petri dish. And you can see here, I said from that propleural plate underneath their chin, we are able to scrape off that Pseudonocardia and then grow it onto a plate. And then from there, we let that Pseudonocardia grow for, like, two weeks. And then we were also able to isolate Escovopsis and also put that onto the same plate. And so when the Pseudonocardia has grown on the plate and the Escovopsis is put on the same plate, the Pseudonocardia does not allow that Escovopsis to grow. So you can see here, there's a wonderful zone of inhibition is what we call it when the area in between the bacteria and the fungus can't grow. So that bacteria is inhibiting the growth of that Escovopsis. So we know that the bacteria is producing some sort of chemical compound that is not allowing this fungus to grow.

So as scientists and researchers, we're really interested in figuring out what that is. And we know that the ants have been farming fungus for millions of years. And so this system is extremely complex. And so we know that they're using this chemical response as a defense against this fungal pathogen. But that isn't the only instance of insects using bacteria and the chemical compounds that they produce as a defense. So another really great example of that is the southern pine beetle, which uses a chemical compound produced by an actinobacteria, and the compound is called mycangimycin. And that also mediates and protects their offspring from an invading fungus. And then there's also another example of this in honey bees with a compound called piceamycin, which is also coming from, again, an actinobacteria. And this one protects the honey bees against this really bad infection called foulbrood. So we know that these insects are using these bacteria in order to protect their colonies from invading pathogens. And so this led to a group of researchers at the UW to look at this and think about, how can this be applied to human health?

And this is where the Antimicrobial Discovery Project was born. So the ants inspired Antimicrobial Discovery Project that uses. . . That maybe humans can use, and what they've learned from these insect systems to help us protect ourselves from other invading pathogens that make us sick. And on that note, I want to just take a step back for a minute and talk about, well, where do most antimicrobials or antibiotics come from? Well, most of them come from the soil. So about 90% of all antimicrobials that are used in clinical settings such as hospitals come from actinobacteria that we've been talking about, and with 70% of those coming from a specific genus. And antibiotic resistance is a huge issue facing the human world right now, and around the globe. So right here, you're looking at a timeline of antibiotic discovery and the onset of resistance. So with the first antibiotics being discovered in about the 1920s. And then you can see here that there hasn't been a new class of antibiotics discovered since, like, 1987. But during that time period, there has been a huge uptick and increase in antibiotic resistance.

So there's been a decline in the discovery of new antibiotics while we have this major increase in antibiotic-resistant infections. So the need for these new antibiotics is really strong. So with that comes the Antimicrobial Resistance Project. And where that leads, where that comes from for me is I was able to lead some research and field work trips in order to collect as many insects as possible and isolating their bacteria to see whether or not we could discover new antimicrobial compounds that could be used in a clinical setting, potentially to combat antibiotic resistance. So over the five years of the Antimicrobial Discovery grant, I traveled to all of the locations you see here on the map. These purple dots are the locations that I traveled to and the amount of insects that I collected. So you can see that my favorite trips were to Alaska and to Hawaii, but all over the United States was really wonderful. And from here, you can see how many insects I collected was variable depending on the location. But on average, I collected about 400 from each field work trip. And they spanned many different hosts. So the most was collecting from either ants, bees, and wasps, or moths and butterflies. And then we also collected some beetles and some other insects and some flies. But we also covered not only tropical environments, but temperate environments and arctic and subtropical.

So once we had all of these hosts, we needed to see whether or not they had interesting actinobacteria that could be used in a clinical setting potentially. So we designed an experiment to test these bacteria that we had grown from these insect hosts against 24 clinically-relevant environmental and human-associated microbes. So this is an example of my first round of tests after these bacteria were isolated into pure culture. So we would test them on 12-well plates, where the experimental strain or the strain that I collected from the host would be struck onto the left side. And then they were allowed to grow for seven days. And then on the right side, we would put our pathogen of interest or our pathogen that could be harmful to humans. And then from there, we let them incubate together for seven days. And then after that seven days, we were able to see whether or not our experimental strain could inhibit that clinical strain. And so I like to call them my little cage matches. And this plate up here's a great example of a wide array of results that we would get.

So on the top left corner, you can see that there's still a dot there. So we needed to make sure that we inoculated each well. So we'd make sure to dot into the auger to make sure that we inoculated that well. But you can see that there's no bacterial growth around that dot. So we know that there is complete inhibition there, and we would then assign that to a three or the most inhibition. And then that orange, beautiful orange bacteria dot, you can see that there's a clear cutoff. And then if, you know, thinking back to a few previous slides when I discussed zones of inhibition, that zone, so the bacteria of the, the human-associated bacteria is able to grow only on the right side of that zone. And then because the experimental strain is producing something that's not allowing it to grow any further than that. And so that would be considered a two. And then if you look at the bottom right well, you can see. . . Zeros and ones are kind of hard to tell, so it basically just would mean whether or not there's no inhibition, and we would always have a control plate with just the pathogen or just the human-associated strain grown on it, and we would be able to compare whether or not there was no inhibition or slight inhibition. 

So after testing many of these, about 1,162 insect-associated bacterial isolates, 186 plant isolates, and 263 soil isolates, we were able to get some results. [chuckles] And the key takeaway from this slide here is that the insect-associated actinomyces or actinobacteria were significantly inhibiting those gram-negative and gram-positive, all those human-associated pathogens more than microbes that were isolated from plants or microbes that are isolated from soil. So this bioactivity was calculated by calculating the average fraction inhibition of the target isolate. So each of the clinical and environmental pathogens were taken against the target isolate. And so if the strain had inhibited all the target pathogens, it would get a one. So the y-axis is gonna be the average number of pathogens that experimental or producer strain inhibited. So you can see that the blue bars are significantly higher than the soil or the plants. So this was extremely exciting because when learning about the ants and learning about these other insects, we knew that these bacteria that were associated with these specific, in these specific systems were potent against these pathogens in their own systems, but we weren't sure whether or not they could be useful against human-associated bacteria. So this is very promising results.

And then to go a step further, we had discussed earlier about the Cyphomyrmex ant, which, again, from the picture earlier. So from here, we were able to look at one specific bacteria isolated from the Cyphomyrmex ant here. And we tested it again against not only Escovopsis, as we had discussed before, but also against the specific fungal pathogen that has really become a huge problem in hospitals today, which is called candida albicans. And so in the center here, this is a different type of assay than you saw before with the two bacteria growing together. On these two plates, what you're seeing is, so after we have grown the bacteria on the plate, the bacteria itself can produce many different types of chemical compounds. And so from there, we need to figure out which chemical compound is producing that activity that we want. So from here, we go through a chemical fractionation process, and we are able to isolate out all those compounds that could be active against that bacterial strain or that target strain that we're interested in. And so down here in the center, you can see those beautiful zones of clearing on this plate. So this plate is a plate that has grown completely with candida albicans, and then we have dotted our specific chemical compounds to determine whether or not those compounds are potent against this bacteria. And those zones are showing that we saw multiple compounds that were very active against that target. And then the same thing above, that yellow is gonna be Escovopsis and those zones of clearing are gonna be where those chemical compounds were placed. And then they are producing something that is not allowing the Escovopsis to grow in those areas.

And then going a step farther, now that we've isolated those chemical compounds, we were able to take those chemical compounds and put them into mice. So this graph here with the mouse head on it is showing where we had took different concentrations of that chemical compound and we have infected mice with that target, candida albicans. And what you can see here is that it reduced the amount of infection within the ants versus the control. So the control had stated a high level with candida albicans in the mouse's system, but then at those two different concentrations, we were able to decrease the amount of candida albicans in that mouse. And it was not toxic either, meaning it did not kill the mouse. Finally, that really long chemical compound up at the top there with the "three" label next to it, that is what we were able to identify that compound that was being produced by that bacteria. And we named it Cyphomycin after the Cyphomyrmex ant that it was isolated from. So this project, we looked through thousands of bacterial isolates that produced, you know, thousands of compounds and we were able to identify a new antifungal compound isolated from that Cyphomyrmex ant, which was very exciting. So these ants are not only a part of this highly-evolved system, but because they've been living in this in concert for so long, we as humans are able, or as scientists are able to research and explore those deep connections and then utilize them for our own gain. 

And there are many highly-evolved, complex bacterial symbiotic relationships with insects that have yet to be explored. And we also have thousands upon thousands of strains that we still have yet to test. And the research here at the UW is ongoing to find those chemical compounds that could be useful to combat that antibiotic resistance. And I will say that this is step one in a multi-year process. So just because we were able to find a novel antifungal, there will be many years of testing before that ends up on the shelves from a pharmaceutical company. But this is one step forward into maybe finding those new antibiotic classes, or those new antibiotics that are more potent than the ones that we are currently using, that are more targeted even, that we could use to combat antimicrobial resistance.

So on that note, I wanna thank Dr. Cameron Currie and all the Currie Lab members, past and present. This research would not be possible without the many graduate students and postdocs that worked on it, as well as all of the undergraduates that helped during the summers collecting leaves and caring for these leafcutter ant colonies so meticulously throughout the years. Also to acknowledge that this is a massive interdisciplinary grant across campus where many people are working together, many teams are working together to process these thousands of isolates and the chemical compounds that they're producing, and also using them to try and combat this really major problem of antimicrobial resistance. And I hope that my talk today gave you some exciting knowledge about leafcutter ants as well as about how they might be very, very small, but these systems, these intricate systems can be very useful in a broader context and also help solve global issues that are facing our society. And I'd also hope that if you see an ant on a sidewalk, you might look at it with some curiosity and think about what it means to be an ant, and maybe step over it instead of on it.