WPT University Place Wednesday Nite at the Lab  Cameron Currie, "Ants, Agriculture and Antibiotics"  October 20, 2010 UP507    cc >> Tom Zinnen: Welcome, everybody, to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the University of Wisconsin-Madison Biotechnology Center. I also work for UW-Extension Cooperative Extension. And on behalf of the Wisconsin Public Television and the Wisconsin Alumni Association, thanks for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight I'm delighted to have Cameron Currie as the presenter. Cameron grew up in the Prairie Provinces of Canada, went to school at the University of Alberta. Got his bachelor's there and his masters. He then went to the University of Toronto, got his PhD, and did some post-doc work at the University of Texas at Austin. 

I'm sure that Texas and Alberta have a lot in common. [LAUGHTER] They have beef in common, that's for sure. And then you can tell us about other things! While I was at NSF recently, Cameron was one of the winners of the Presidential Early Career Award, which is the most prestigious presidential award that you can win as an early career person. I think he was one about 20 or 25 people total there. And it was a delight for me to see him win one of those awards and represent the University of Wisconsin. Tonight, Cameron will be talking about his work, which is some of the most elegant work on multiple layers of symbiosis that I'm aware of. And he will be talking about "Ants, Agriculture and Antibiotics." Please join me in welcoming Cameron Currie to Wednesday Nite at the Lab. [APPLAUSE] >> Cameron Currie: Thanks, Tom. It's great to be back at Wednesday Nite at the Lab. This is the second time I spoke. 

I think 2006 was the last time. Is that right, Tom, roughly? I'm curious, how many people were here last time I gave a talk at Wednesday Nite at the Lab? Three people. I'm not sure if that's good news or bad news. It's good news for the rest of you because you won't see any repeats of stuff you saw in 2006. It might be bad for me because clearly I didn't bring back the people who were here before. I like to start my talk with the most important slide, which is acknowledgements, for two reasons. First reason is the very important aspect of a lot of the work that I'm going to talk about today involves collaborations with diverse people from many different areas of science. And that's one of the aspects of working in the area symbiosis that I love is that I get to collaborate with scientists from all kinds of different areas. And to acknowledge current members of my lab and past members of my lab and funding, and so I like to acknowledge that a lot of this work has been a team effort that I'll talk about. And it's also always good to start with my acknowledgements because then I can just run my talk and not worry about timing as much. 

There's a practical aspect to it. I thought I'd start with a little bit of a explanation of why I'm a biologist and what I love about biology and what drives me. And I think the issue of diversity of life is a good place to start. How many people have read this classic book by E.O. Wilson, "The Diversity of Life"? So about two or three of you only. How many people were aware of it before I showed it right now? About a quarter of the room. And how many people actually know who E.O. Wilson is? Maybe two-thirds of the room, great. He wrote this great book and illustrated the importance of diversity. I assume everyone in this room recognizes the issue of preserving biodiversity, and the importance of biodiversity, and the critical aspect of it. But I think often people don't have an appreciation of what we mean when we say diversity of life. And that's an important aspect as well. 

I want to ask you two simple questions as an audience. How many species are there on the planet, roughly? And so does anyone have an idea of the number of species on the planet? >> Millions. >> Millions, billions. Who says millions? Maybe about a quarter or a third of the room. How many say billions? How many think less than millions? How many think more than billions? I don't know. So that's kind of an interesting answer. We have millions to billions, orders of magnitude difference. It illustrates the fact that we actually don't have any idea of the number of the species on the planet. There are roughly 1.7 million that have been named. But estimates range from 10 to 50 million, which I think is a vast underestimate as we'll get to in a minute. 

The other question I want to ask you is what is the most diverse group of organisms on the planet? Ants? Who said ants? Thank you. [LAUGHTER] No it's not right. There's only 10,000 species of ants, but I love ants, so I love that answer. So we have ants, bacteria, anything else? >> Insects. >> Insects, who thinks insects? Who thinks bacteria? Who thinks some other group that's not insects or bacteria? What do you think? >> Invertebrates. >> Invertebrates. So a broader group which includes insects. It's interesting, where we sit right now, there's nearly 1 million species of insects that are described. So arguably the most diverse group on the planet that has been documented is the insects. A little over 350,000 plants, over 100,000 fungi and just 5,500 described bacteria. 

This, though, I think is a vast underestimate of the real diversity of bacteria, I could go into lengths about that, but that's not necessarily appropriate for this talk today. But I think that there are arguably tens of millions of species of bacteria. One thing that kind of jumps out at me when I think about biodiversity is this issue of describing the biodiversity. So about 10,000 new species are described each year. If you think there's 10 million species on the planet, we're a thousand years away from having described all the species diversity. Which I think is fairly daunting. If you think that we're talking about tens of millions or billions, just documenting the diversity is a daunting task. So I think one of the cool aspects of this is the rich diversity of life. And so the way we tackle the issue of biodiversity and think about biodiversity is not actually going out and documenting it, but trying to understand what shapes diversity and what generates diversity. And often I start my talk with this quote from origin of species by Darwin which is one of his most famous quotes and I'll just read it to you. 

"It is interesting to contemplate a tangled bank, clothed with many plants of many kinds, with birds singing on the bushes, with various insects flitting about and with worms crawling through the damp earth." And so this passage illustrates to me kind of the complexity of species interactions that go on in natural communities. And then Darwin goes on to say, "And to reflect that these elaborately constructed forms, so different from each other, and dependent upon each other in so complex a manner, have all been produced by laws acting around us." And the law that Darwin is kind of hinting is evolution, obviously, and specifically the process of co-evolution. Species interactions shape diversity of life and are major shapers, have a major impact on shaping diversity of life. Within the area of species interaction, the work that we do in my lab is focused, as Tom indicated, on symbiosis, and arguably symbiotic associations shape all levels of biological organization. From the functioning of individual cells, in terms of things like chloroplasts and mitochondria that are endosymbiotic, symbionts, to even major evolutionary transitions like the origin of plants on land, which is one of the major transitions in the history of life, is thought to be facilitated by a symbiotic association with fungi. Microbes play major roles in aiding animal digestion. Symbiosis also includes antagonistic interactions including parasites which are major players in natural systems. And one of the important things that I think people are starting to recognize is the importance of symbiosis in human biology. And one of the things we know now is that there's an order of magnitude more bacterial cells in the human body than human cells. And so one of my colleagues that works in symbiosis, Dr. Margaret McFall-Ngai, often mentions the issue of looking at your neighbor and recognize that your neighbor is only 10% human and 90% bacterial kind of puts it in perspective. 

[LAUGHTER] Yes, you're mostly bacteria. The symbiosis that we work on mostly in my lab involves this association between ants and fungi. And this is an obligate mutualism. The ants can't survive without the fungus, and the fungus can't survive without the ants. And I'll go into a lot of detail about this association. The benefits to the fungus in this kind of mutually beneficial symbiosis is the queens disperse the fungus from parent to offspring nest. The workers provide substrate for growth of the fungus and provide the protection to the fungus. In exchange, the fungus serves as a primary food source for the ant colony. And so you can look here on this illustration here, this little swelling here is a hyphal swelling that we call gongylidia which is full of nutrients that the larvae and the queens feed on within the colony. So the ants grow fungus for food. 

So this is an equivalent of a like an agricultural association. How many people have seen fungus growing ants or leaf cutting ants before? We have about, people are not very proud of seeing leaf cutting ants. Come on, let's put your hands up. [INAUDIBLE] I will get to Microbe Place, for sure. How many people have seen leaf cutting ants on campus? How many people have seen leaf cutting ants not on campus, somewhere else? Good, good. So leaf cutting ants, which, as I'll get to later, we have a colony in the Microbe Place, are members of a tribe of ants that grow fungus for food. But not all fungus growing ants are leaf cutting ants. And there's a lot more diversity, as I'll get into. But this is a map showing the distribution of fungus growing ants. Here you can see that they occur from the southern parts of Argentina up into actually the Pine Barrens in New Jersey. 

They're only present in the new world. And we think the origin of the an fungus mutualism dates back to about 50 million years ago. So this is an ancient association between ants and fungi. As I indicated, there's a lot of diversity in the ants. There's not a single species of ants that grow fungus for food, but there's over 200 described species of ants. Here's an evolutionary tree, a phylogenetic tree representing the evolutionary history of fungus growing ants. And you can see there's a lot of diversity in the association. There are the most diversity of fungus growing ants actually occur in cryptic colonies that are difficult to see. This is actually a colony that I found in bamboo, in a broken piece of bamboo. This species here in the genus Cyphomyrmex, that opening to that nest is about the size of the face of a quarter. 

So very cryptic and small. And then you have the culmination of fungus growing in ants occurring in the leaf cutting ants which cut leaves and use fresh leaf material for growing their fungus garden. >> How do they grow fungus from ants? How do the ants grow fungus? >> So I'll get to that. The question is how to the ants grow fungus, and that is a great question. And I'll get to that and I'll actually show video of that in a second. The most impressive fungus growing ants are the leaf cutters, as I indicated. And here's nice illustration of the ants cutting leaves, and then you can actually see the ants forming a trail. But I think the still images don't really do justice to this impressive behavior. So I have some videos for you to watch. Here's a leaf cutting ant cutting a leaf. 

One thing to look for is their mandibles are serrated to increase the efficiency of cutting which makes it much more effective at cutting leaf fragments but it also makes them much more effective at biting of humans and graduate students who are digging into nests. [LAUGHTER] Once they've cut the fragment, they hoist the fragment over their head and they form these trails in the new world tropics. This is a trail of leaf cutting ants. This video was taken by Jared Scott, a grad students in my lab, in Panama. And you can get a sense of the scale of the number of ants that forage for leaves. And so an individual colony of ants can actually have tens of thousands of workers out foraging for leaf fragments. >> Is that normal time? >> That's the normal time. That's the pace that they go at. So that kind of illustrates a little bit of the scale of leaf cutting ants but not necessarily getting a fantastic sense of the true scale, which I think I'll show here. This is a Google Earth image from 1.85 kilometers above Paraguay. 

This is a savannah. Can everyone see those white dots on the surface? Those white dots are not crop circles but are in fact active nests of leaf cutting ants. So you can actually see them from the upper atmosphere. And here is an excavated nest of a mature nest of leaf cutting ants. This nest can be composed of 10 million workers, live 10 to 15 years and defoliate a mature eucalyptus tree overnight, just a single colony. So an incredible super-organism, if you will. The success of leaf cutting ants is through their association with cultivating fungus for food. There is a single queen in a leaf cutting ant nest, as illustrated here. This is the head of the queen. This is the thorax and the abdomen. 

And these are actually workers in this ant colony that are actually tending and caring for the queen. So a single queen in a mature nest of leaf cutting ants, she can lay 60 million eggs in her life span. She stores sperm from the nuptial flight when she mates, when she first is produced and goes on her nuptial flight, she stores sperm from males, and she'll store that sperm for 10 to 15 years, and she is never mated again, which is an impressive feat. So leaf cutting ants are an amazing kind of example of mutualism in the success of symbiosis as they have major impacts on the ecosystems in which they occur. And as I indicated there's a lot of diversity of leaf cutting ants. So now I want to talk a little bit about what got me into studying leaf cutting ants other than the kind of charismatic aspect of their behavior. And what got me interested is when I was looking for PhD projects I was familiar with the mutualism between ants, fungus growing ants and the fungus they grow for food. And this paper was published by Chapela, et al. in the Journal of Science. And in this paper they compared the evolutionary history in the ants with the evolution history of the fungus the ants culture. 

And so just like there's diversity in the ants, there's diversity in the fungus the ants culture. And so as the ants diversified, their fungal cultivars diversified. Which is illustrated by the similarity in their deep relationships as you go up the phylogeny of the ants, you have corresponding divergence of the fungal cultivar that corresponds to the ants. So ants are specialized in specific groups of fungi and the fungi have diversified, their fungal crops have diversified over the 50 million years of evolutionary history of the association. So one of the other things that really fascinated me about the system was, it was reported the ants were so good at agriculture they maintained their garden free of disease. And at the same time the speculation by these authors was that the ants had been clonally propagating their cultivar for millions of years. So in human agriculture when we, and they maintain them in monocultures. So in human agriculture when we maintain genetically homogeneous crops in large scale monocultures, what's the major issue? >> Disease. >> Disease, right. So the prediction is if these ants have been growing fungus for food for tens of millions of years, clonally propagating their cultivar and maintaining them in monocultures, they should have major problems with disease. 

But at the same time the thought was the ants maintain their garden free of disease. So my PhD was really addressing two questions. First, are the fungal garden host of virulent parasites, is there disease present that people have actually overlooked and not found? And then second, how do the ants in the cultivar combine to deal with disease? And so the thought I had was maybe there's pathogens in the system that are exploiting the association that just have not been found. Or maybe the ants and their fungi have discovered mechanisms for dealing with disease that are novel and are very effective. And, in fact, I discovered both. I discovered presence of a specialized parasite that exploits the ant fungus garden. This is another fungus. This is a fungus that attacks the ant's fungus, it's called escovopsis. This is what it looks like in culture. And this is what its micromorphology would look like. 

And to illustrate the impact that this pathogen can have on the system, this is a healthy nest here on your left. And this is from the same species of ants, one that's been completely overgrown by the parasite in the lab and completely devastated. So the parasite can have a major impact on the health of leaf cutting ants. The parasite, as I indicated, is only found associated with fungus growing ant colonies. And I won't show you the data but we've showed it's actually co-evolved with the ants and their fungi and have diversified as well. So how do the ants deal with pathogens in their fungus garden? That was a lot of work that I did as a PhD student, it's work that continues in my lab here at the University of Wisconsin. And the first mechanism is a behavior we call fungus grooming. This is work that Alison Stewart and myself did, a fellow grad student at the University of Toronto. And what you can see and what you should look for here is the worker actually uses her antennae to touch and sense the presence of the pathogen in the fungus garden. And then she runs her mouth parts over the fungus, which is this white fluffy material here, and actually cleans it and laps up the spores. 

And these workers will actually accumulate the spores in a structure in their head called the infrabuccal pocket until they get a large pallet in there, and then they'll actually discard the spores into their dump. So it's a hygiene almost like a vacuuming behavior that's very important in preventing the establishment of infection. And one of the things that we were very excited about is E.O. Wilson, who I mentioned earlier in my talk, leaf cutting ants are his favorite, one of his favorite groups of ants, and he's actually done a lot of the work on the behaviors involved with growing fungus gardens in leaf cutting ants. And he actually overlooked this behavior and never noticed it. And the reason is, is this behavior is very rare in the absence of infection but when you experimentally infect the nest, the workers abandon other tasks and start doing fungus grooming. So it's a very important behavior which is specific to infection. Yes, question? >> It seems to me to be a waste management item and somebody characterized the ants as an absolute necessity for waste management. >> So the question is it seems like that's a waste management. And there is waste management aspects to this and I'll get to, specifically, and even better example of waste management in my next slide, which is even more, this behavior is even more of a link to waste management. And this behavior is called weeding. 

And so if the ants are not able to prevent the establishment of infection and an infection establishes, workers actually go and they rip out parts of the fungus garden. So did you see that worker actually collect that piece of fungus in her mandibles? And she'll actually carry that out and discard it. I'll just show that last little bit again.  So watch for the ripping there of the fungus out of the fungus garden. And so she'll actually remove the infected parts from the fungus garden and discard it. And so it's a second layer of defense against a pathogen. If infection establishes, they'll remove it behaviorally. So behavioral defenses are very important in the success of leaf cutting ants, specifically dealing with their specialized parasite escovopsis. But another mechanism of defense has to do with actinobacteria. And one of the things that I discovered as a PhD student is that the ants have this whitish coating on their bodies that is an actinobacterium. 

And this actually had been noticed by others but had been thought to be a wax produced by the ants themselves. Does anyone know what actinobacteria are well known for? Tom. >> They used to be called actinomycetes. >> Actinomycetes, yeah. Actinomycetes are actinobacteria. >> And they aid with antibiotic production. >> Antibiotic production. So how many people here have taken an antibiotic in their life? How many people have taken Streptomycin, for example? Quite of a few of you. So Streptomycin is an antibiotic that is derived from bacteria in the genus Streptomyces, and Streptomyces is a genus of actinobacteria. And the majority of antibiotics that we use pharmaceutically are derived from this individual genus of bacteria called Streptomyces. 

And it's been incredibly important over the history of human medicine in terms of development of antibiotics. And so we get our antibiotics from bacteria and fungi but our major source are actinobacteria. And so it was actually a bit of a joke at the time, I thought the ants evolve the ability to grow fungus for food millions of years before humans did, maybe they also discovered, through evolution, the benefit of associating with actinobacteria for production of antibiotics. And, in fact, it was a bit of a laugh, I had a laugh with my PhD advisor. We thought that that would be a little bit outrageous. But in fact that's what the evidence indicates from my work on my PhD and work we've been doing here at the University of Wisconsin. So this is an illustration of the actinobacteria that can completely cover the surface of this ant. What I found as a PhD student is the queens carry the bacteria from parent to offspring nest, which is what we call vertical transmission. This is a form of transmission that is typical of mutualistic beneficial associations. The ants carry their fungus because they need their fungus. 

They also carry the bacteria, indicating there's a role for the bacteria. I also found the bacteria occurs in specific locations on the ants. And I'll show you more details on that in a minute. But then what I did was I grew the bacteria on nutrient media here and I challenged it with the pathogen escovopsis, this specialized parasite of the fungus garden, and I found this potent zone of inhibition. Evidence indicating that the bacteria on the ants are producing antibiotics that are capable of inhibiting their garden parasite. Subsequent to this, we've done a number of studies where we've actually done experimental manipulations when we removed the bacteria and we infect nests, and we've shown that in fact experimentally that the bacteria on the ants are helping the colony deal with the parasite escovopsis. In addition, working with natural product chemists at Harvard Medical School in Jon Clardy's lab, we've characterized one of the compounds responsible for this activity, named Dentigerumycin. This is a new antibiotic to science, and we published that last year in Nature Chemical Biology. And we're working a lot on looking at chemically characterizing the antibiotics that are produced. So we have this association between ants that grow fungus for food, as I indicated it's an obligate mutualism, the ants can't survive without the fungus, the fungus can't survive without the ants. 

You have specialized parasites called escovopsis that are only known from the system that exploit the ant fungus gardens. The equivalent of an agricultural pathogen. And to deal with escovopsis, the ants have specialized behavioral defenses, but they also maintain bacteria on their bodies that produce antibiotics that help mediate the health of the fungus garden. So I want to tell three kind of newer stories. That was largely work that I did as a PhD student. I want to tell three newer stories of things that we've been doing more recently. The first is providing evidence that the fungus growing ants have been associating with these antibiotic producing bacteria for millions of years, like they have with their fungus garden. Then I want to talk a little bit about other microbial symbionts in the system that we've recently discovered in this association. And I want to end with discussion about whether other insects associate with antibiotic producing bacteria to deal with disease. And so in terms of evidence of the role or the association between the ants and the actinobacteria, I'm going to show you three lines of evidence. 

The first has to do with this specific location on the ants of the bacteria that I mentioned. And this is a head of an ant. This is the ventral view, the under view. These two white patches are the actinobacteria. So when the bacteria is not all over the body of the ant, is typically occurs on this location, which is almost the equivalent of an ant's neck if ants had necks. And so this is an ant on the fungus garden. And so one of the things I asked myself was why is it occurring on that specific location? And so what I did was I actually bleached off the bacteria off the ant, and what I discovered is that they have large crypts that are specialized for housing the bacteria. And so this is a close up with the bacteria removed of that plate on the ant. And on each side they have these paired structures. And then with working with an expert, we sectioned through this plate. 

Here's the cuticle, the exoskeleton of the ant. The dark purple here is the bacteria on the ants. And this on this inner surface there's glands here. Can everyone see that? Should I turn down the lights slightly? Is that okay, Tom? Can people see that or no? I can bring it down just a bit. So underneath the cuticle, that's much better, so here this is the cuticle here of the ant. The dark purple is the bacteria. This is the inside of the ant, and there's paired glands and the glands are connected to the channel or to the crypt by channels. So the ants are modified, morphologically in terms of having crypts and also physiologically in terms of having glands, presumably to produce the nutrient source for the growth of the bacteria. And if we can keep the lights down just for the next slide as well, that'd be great. So the modifications in the ants supports and ancient association that these ants have been maintaining bacteria for a long period of time because of the extent of it. 

Can everyone see the little white dots that completely cover this ant? Put up your hand. Can you see it at the back? Yeah. Does everyone agree that this ant is covered in these white dots? Yeah. Well, we sectioned through this ant and what we discovered is each one of those little white dots is actually an opening to a crypt or a flask embedded into the cuticle of this ant full of the antibiotic producing bacteria. So if you look at this section of this ant, this ant is completely outlined by crypts that are full of the antibiotic producing bacteria. Arguably suggesting these ants are walking pharmaceutical factories. And under each one of these crypts there's a channel and a gland cell as well. Some major modification in the ants for maintaining these bacteria supporting the importance of the bacteria for the success of this system of these ants and also supporting that it's a long-term association. The next piece, if this doesn't convince you I hope the next piece of evidence does. Working with Ted Schultz, who's an expert on fungus growing ants and leaf cutting ants, he's a taxonomist who works on the phylogenetics and taxonomy of these ants, he had ants from Dominican amber in his desk. 

When we were in the field we were talking he was telling me he had these so I convinced him to come and visit me and bring his ants in amber so we could look for the bacteria. And we found the presence of the bacteria on this specimen. This is Dominican amber. It dates back 20 million years. So the presence of the bacteria on the ants in the Dominican amber indicates the ants have been associating with the bacteria for at least 20 million years. So I think the lights could be raised a little bit now. So the last piece of evidence I just want to go over very quickly is that this is work that Matias Cafaro, a former post-doc in my lab, directed, and it involves kind of studying the evolutionary history of the bacteria. So we've selected strains from across the diversity of fungus growing ants, and we've sequenced multiple genes from each of these bacteria and we generated what we call a phylogenetic tree or a tree that represents the evolutionary history of the bacteria. And so on your right here is just that phylogeny, and the colored taxa in these boxes represent bacteria associated with the ants. And the main point of this is indicating that there's a lot of diversity in the bacteria that are associated with ants, suggesting these bacteria have diversified in association with the ants and supporting it. 

And so that is the main conclusion, and the other main point of this is that there also is evidence that there are switches between ant colonies of bacteria over evolutionary time. And there's complex kind of co-evolutionary association going on between the ants and the bacteria over their evolutionary history. So I hope those three pieces of lines of evidence support, convince you that the ants have been associating with these antibiotic producing bacteria for probably likely from the earliest origin of the mutualism and may date back as many as 50 million years. Yes? >> Where did they get the bacteria? Does the bacteria live anywhere else? >> So that's a great question. The question is where do they get the bacteria? And that's a very difficult question to answer. Relatives of these bacteria can occur in soil, can occur in associated with plants, we've found them associated with other insects. But the queens, when they go on their nuptial flights, also are covered with the bacteria. And so the assumption is that the major way they get the bacteria is from their parent nests is they carry the bacteria with them from parent to offspring nest. And so it is vertical transition. How did the bacteria enter the association is something that we're not sure of and is something that we're very interested in and are working on but we don't have any idea yet. 

So you have this four-part association, these ants associating with three microbial symbionts, two fungi and a bacterium in this complex, what we call, quadripartite association. This complexity of associations is really fascinating, but one of the things that we're interested in is complexity and so we ask the question are there other associations, other microbial symbionts in the system? And the presence of two more suggests that there may be even more. So I'm going to tell you about one of them, and this has to do with analogy with human agriculture. One of the major issues with human agriculture is the need for fertilizer. This has become a bigger issue with larger scale agriculture. But historically, nitrogen was integrated into human agriculture over human history through cultivating plants that have bacterial associates or nitrogen fixing bacteria in their root nodules such as soy bean. So plants that have nitrogen fixing bacteria have been important sources of nitrogen in human agriculture history and arguably have been critical for the development of human agriculture until the advent of fertilizer. So we ask the question, do the ants have bacteria that fix nitrogen that fertilize their system? And this is work that Adrian Pinto conducting in my lab, a former PhD student who is now a professor at the University of Costa Rica. And Adrian conducted this work and basically the idea and the premise of this work is that ultimately the major input of nutrient into the system is through leaf material. And we know that in the carbon/nitrogen ratio of leaf material to insect chitin and tissue is that insects that feed on plant material are nitrogen limited. 

What that means is that there is a lower amount of nitrogen per carbon in plant material than what insects need for development. And so if you're an insect and you feed on plant material, you're nitrogen limited. Is there clear? Okay. I didn't explain it exactly right. So this would suggest that leaf cutting ants should be nitrogen limited. But we also know that the ants have evolved to be incredibly energetically efficient. So it didn't really make a lot of sense to us that they would waste carbon out the back end of the system and not supplement nitrogen into their fungus garden. So the first thing that Adrian did was ask the question, are leaf cutting ant's colonies nitrogen limited? So what he did is he calculated the percent nitrogen content for different components of leaf cutting ant nests. So this is percent nitrogen content for leaf material, fungus garden, ant workers, ant brood, whole ant colony and refuse dump. And so if leaf cutting ants were nitrogen limited, we'd expect there would be a decrease in nitrogen content as you move through the system. 

So the input is the leaves of the fungus garden actually has an increase in nitrogen. Obviously we already know that ants have a higher nitrogen content ratio than the leaf material. But what's interesting is that even by the back end the refuse dump from this garden material discarded, you have an enrichment or an increase content of nitrogen. So that supports the idea that nitrogen is not limited as would be predicted. But it doesn't actually provide any evidence that nitrogen is being integrated into the system through bacteria that fix nitrogen. And the reason for that is that they could be supplementing nitrogen or they could be metabolizing off extra carbon. And so the definitive test for determining the presence of nitrogen fixation is called acetylene reduction activity. And this is based on the nitrogenase ability to produce a triple bond and acetylene to produce ethylene. And so this is considered the functional test of nitrogen fixation. And so what Adrian did is he put ants or fungus garden or brood or leaf material into an air tight container. 

He injected acetylene. He let it incubate over time. And then he extracted the atmosphere here to look for the production of ethylene. And so if he shows production of ethylene that indicates that nitrogen is being fixed by bacteria within that system. And here is his data. This is acetylene reduction activity by different components in the system. Leaf material, fungus garden, ant workers and ant brood. And you can see that there's significant acetylene reduction activity present in the fungus garden of leaf cutting ants. And we had insignificant amounts in the leaves, the ant workers and the ant brood. One of the things we found surprising about this is that nitrogen fixation occurs in termites and is well known it termites and occurs inside the guts of termites. 

And so we actually predicted that we would see nitrogen fixation occurring, if we found it that it would be in the ant workers themselves. So we were actually surprised to find it in the fungus garden. But then we started thinking about the system a little bit differently. Termites have gut microbes, they consume wood, they've got microbes that help break down the wood material to provide nutrients for the termite. The ants actually collect leaf material integrated in their fungus garden and their fungus produces these hyphal swellings for nutrients. So the digestive system of the ant is actually the fungus garden. And so that then made, under that kind of thinking, the presence of nitrogen fixation in the fungus garden makes sense. So the discovery of nitrogen fixation in the fungus garden and bacteria there was very exciting, but it actually doesn't provide concrete, suggestive that the ants have nitrogen fixing bacteria for their benefit as a mutualism, but it's not conclusive because there could be bacteria present in the fungus garden that are fixing nitrogen for their own benefit and exploiting the ant fungus garden. So the work that Adrian did next on this system is to actually try to show fixed nitrogen bacteria, the nitrogen fixed by bacteria in the fungus garden ended up being incorporated into the ants themselves to show that there is a benefit. So he developed this experimental setup where he used this apparatus he constructed. 

And there's two chambers. The chamber on the left with this white block and this leaf material was the foraging chamber of the ant. On the right is a fungus garden that he put in here. And so then he basically took a subcolony and he split it in half and he put half in one apparatus and another half in another apparatus. And so he had a control apparatus here set up and a treatment here. And so he basically withdraw the atmosphere in his control and replaced it with 14N in his control. In his treatment, he replaced the atmosphere with 15N. So this is a stable isotope of nitrogen. The benefit of using the stable isotope of nitrogen is we can actually detect the integration of the stable isotope as it passes through the system. And so working on stable isotope and utilizing a stable isotope like this is very beneficial for this type of work. 

And so the idea is in the treatment we'd find bacteria would fix the nitrogen, the nitrogen would be N15 instead of N14 and we could track that N15 passage from the bacteria into the fungus garden into the ants.  Does that make sense? Okay. And this is his results. This is delta value, which is the ratio of N15 to N14 by different components of the system including fungus garden, worker ants, and ant brood. The white bar represents the control and the dark bar represents the treatment where he enriched for N15. And so what you can see here is in the fungus garden there's a significant enrichment of N15 in the fungus garden in the treatment compared to the control. And the only way that can be increased is through bacteria fixing the nitrogen integrating in the fungus garden. Then he also found significant integration in the ant worker themselves. There's a significant increase in the delta value in the treatment compared to the control, supporting that this fixed nitrogen in this system is getting integrated in the ant and supporting that it's a mutualism. And so he published his paper in the Journal of Science last year, and one of the things he also did was document the presence of nitrogen fixing bacteria in the genus Pantoea and Klibsiella. 

And he did a lot more work than I showed you in that brief five slides. So we now know that the ants and their fungus do not occur in isolation but have a parasite of the fungus garden and actinobacteria. So there's a four-part association. And now we've shown the presence of nitrogen fixing bacteria that appear to fertilize this system. And I don't have time to present it but we've actually discovered another parasite of this system that actually exploits the ant bacterial mutualism. This is work that Ainslie Little, a former PhD student in my lab, conducted. So you have this complex six-part association, and it illustrates the complexity of symbiotic associations. The last story I want to tell is these discoveries in the ant system, do they translate into other systems or are these types of associations present in other systems? And specifically I want to talk about do other insects engage in symbiotic association with antibiotic producing bacteria. And I'm going to show you a little bit of the justification of why we think this is likely. Pathogens are important agents of selection, and all organisms have serious problems with pathogens. 

As we talked about actinobacteria potent producers of antimicrobials that would benefit associations. And the last point is over the history of life symbiotic associations have formed between diverse groups of hosts with specific lineages bacteria in many different cases. So that would be a prediction. The last point is that work in Europe had discovered, in 2005, the presence of Streptomyces actinobacteria in the antennae of bee wolves that appear to benefit the bees. So in addition to the ants there's another published example. >> What's a bee wolf? >> Bee wolf is a solitary wasp that digs into the soils and lays its larvae. And so it's like a mud dauber. Do you know what a...? So we've been working on, we're interested in complex symbiosis. So this is my one connection to a Canada slide for Tom. And we're interested in complex symbioses. 

And one of the symbioses that is well known is well study is bark beetles. And bark beetles have major landscape impacts. This is a slide of the outbreak in red here of mountain pine beetle between 1959 and 2002. And I think this area is probably the equivalent of the state of Wisconsin in terms of the outbreak and impact. And these beetles killed trees on landscape scales. Is every one familiar with mountain pine beetle? And so they have devastating impacts. One of the things most people aren't aware of is that mountain pine beetles associate with fungi that are presumed to provide some nutritional benefit and may help the beetles overcome the tree defenses. And so bark beetles associate with fungi, and this type of association might be present. We didn't start out working among pine beetles, although we are working on this now. This is work with Aaron Adams and Ken Raffa from the Department of Entomology. 

But I started working on this with Kier Klepzig. This is work that Jared Scott in my lab directed, and it was on the southern pine beetle system which is very similar to mountain pine beetle. The nice thing about the southern pine beetle system is it's well known to have a beneficial fungus called Entomoc corticium. This is a fungus that helps with the nutrition for the larvae of these beetles. And they're also known to have an antagonistic fungus, Ophiostoma minus. And so it's like the ant system in that the beetles depend on a beneficial fungus and they're harmed by an antagonistic fungus. And so the prediction would be if there's an actinobacteria present in the system that they would produce antibiotics that inhibit the antagonistic fungus but not the beneficial fungus, and this would provide a benefit for the beetle. And that's, in fact, what we found. What Jared did was he went in and he isolated from southern pine beetle. Can we bring the lights down one more time just for this illustration? This is a larva of a southern pine beetle in a gallery. 

And what we found was when we zoomed in here the fungus was along the wall but in between the wall and the beetle we found actinobacteria growing. And, in fact, when you zoom in up close you can see the actinobacteria growing directly in connection with the fungus. And so what Jared did was he did extensive isolation and he found, in fact, actinobacteria in the genus Streptomyces were commonly associated with southern pine beetle. You can bring up the lights again. So as I indicated the prediction would be if there's actinobacteria present in this system that were benefited the beetles that it would inhibit the antagonist but not strongly inhibit the beneficial fungus. And that's in fact what Jared found. This is the Streptomyces, the bacterium from the galleries of southern pine beetle. And you can see these potent zones of inhibition indicating that these bacteria are producing an antibiotic that is inhibiting the growth of this fungus. And you can see here the beneficial fungus of the beetles is only partially inhibited. So there's much stronger inhibition of the antagonist. 

We, again, worked with Jon Clardy's group at Harvard Medical School, who's a natural product chemist, to get at the antibiotics that are being produced. And his post-doc, Dong-Chan Oh, determined the structure. This is --peroxide that's new to science, a new antibiotic to science. It's named after the structure the beetles have for maintaining their fungus. Due to time, I won't go into too much detail, but this is fluorescent units which is growth of the fungus by the concentration of --, and you can see there's much stronger inhibition of the antagonist than the beneficial fungus. And so the support with the compound that the antibiotic that's being produced is benefiting the beetle. The last indication of that these types of associations may be widespread is work that Kirk Grubbs, a grad student in my lab, is doing on honeybees. And I think everyone here is probably aware that there is concern over the health of honeybees, and in the popular media there's talk about colony collapse disorder. Kirk's work is looking at the microbiome or the microbial symbionts of honeybee colonies and whether there's beneficial bacteria present in colonies that mediate the health of the colony in the sense of providing potential protection and other benefits. Specifically were interested in whether there's actinobacteria that produce antibiotics that might mediate the health as well. 

And just very quickly, what we've found is we've isolated actinobacteria again of Streptomyces and then when we challenged this Streptomyces with Paenibacillus larvae, which is the major bacterial disease impacting honeybees, we get potent zones of inhibition. And so the actinobacteria in the honeybee colonies are producing an antibiotic that inhibit the major bacterial pathogen of honeybees. We've actually shown that this activity is fairly specialized to this pathogen. The one thing we have not and are working on is establishing that there's enough abundance of this Streptomyces, this bacterium in colonies to be having a major impact on the health of nests. But we're very excited about this work. I want end with just one or two slides and just talk a little bit about the applied aspect of this work. And this is a little bit of a dated slide, but it still shows the trend quite well. This is discoveries per year of new drugs and new antibiotics. And so after the kind of emergence of antibiotic discovery and the development of the antibiotic age we had a major increase in the number of antibiotics being discovered. But then you can see that starting in the mid to late '80s we have a significant drop off of the discovery of new antibiotics. 

Does anyone have any idea why this is the case? Yeah? >> The market. You're going to take an antibiotic maybe once every five years. The drug companies are going to go for pills that you take daily. They're not going to put the investment in the research for the antibiotic. >> So there's a major decrease in investment by pharmaceutical companies on discovery of antibiotics partly because of other drugs making more money. Anything else? >> Extinction of species. Less diversity. >> She said extinction of species and less diversity. That's probably not influencing the pharmaceutical companies but is probably a major issue that we need to think about for sure. And I'll come back to that. 

Anyone else? Yeah? >> The human being became resistant to antibiotics. >> So she said that antibiotic resistance has become a major issue. So that is a major factor. One of the things that pharmaceutical companies have discovered is that their drugs become obsolete fairly quickly because of emergence of resistance. The last point that no one has jumped on is that the discovery of antibiotics has become much more difficult. And part of the reason we have this major increase here is because there was no antibiotics that previously had been discovered. And so what happened is in the late 1980s and 1990s when you went and looked for new antibiotics, what pharmaceutical companies were finding was the same antibiotics that have already been discovered. And discovery of new antibiotics was much more difficult. One estimate is that you might need to look at 10,000 different strains of actinobacteria to discover a new antibiotic, which is a massive undertaking. And this is for the Streptomyces the major producers of antibiotics will work on. 

So one of the outcomes of the work that I did was the possibility of what we call drugs from bugs of bugs. This idea that these bacteria associated with insects could lead to new drugs. And I've talked about the ant system where we're discovered stromycin. I've talked about the beetle system where we discovered another one. And I talked with the bee system where we discovered Apinimycin. And I can't show the structure because of IP issues. But those are three novel antibiotics that we discovered from three different strains of our system. And we've actually discovered five novel antibiotics or insect associated bacteria from about a collection of about 25 or 30 strains that we've tested. And so comparative finding an antibiotic from one out of 10,000 strains we're finding an antibiotic from one out of five. So this is an opportunity to discover new antibiotics. 

We've actually had some indication these have a promising activity. One has high activity against malaria. And another has actually passed an anticancer screen at the National Institutes of Health. So these are promising drug leads. So just in conclusion, here's a little cartoon of ants that have bacteria that produce what's called a death ray to inhibit a pathogen of their system. I hope I've illustrated that leaf cutting ants are interesting organisms to study from a complexity of association perspective and illustrate the importance of symbiotic associations. I hope I've shown you that these kinds of associations are likely widespread, and I ended with this issue of kind of the idea of pure research of today has the potential of being applied research of tomorrow. And the work I did was not funded to discover new antibiotics but that was a outcome of support for pure science and so this is a plug for the importance of pure science. The last thing I just want to put up here is that this is our live web cam URL. So check out our web cam which is going to our leaf cutting ant nest that is maintained in the microbe place in the microbial science building. 

And next time, if you've not seen it, next time you're in the microbial science building neighborhood then please go in. It's in the first floor, and we're very proud of our new educational outreach exhibit. Thank you. [APPLAUSE]