Ep 91: How stealth organs make super soldier ants (with Rajee Rajakumar)
What is eco-evo-devo? How can ants help us understand the evolution of development?
There are 20 quadrillion ants in the world, and they come in lots of different shapes and sizes. We even see big differences within colonies, like ants in the genus Pheidole which have different castes: workers, soldiers, and, in some species, super soldiers. Super soldiers are the muscle-y brutes of the ant world that grow huge heads to defend the colony and attack large food items, like other insects. This variation is all due to developmental plasticity – the same ant genotype produces distinct phenotypes depending on the environment of their early lives.
On this episode, we talk with Rajee Rajakumar, a professor at the University of Ottawa, who studies Pheidole ants to understand the interactions between their genes, their developmental environments, and their phenotypes. Rajee is also a HUGE ant fan! We talk with him about his 2018 paper in Nature about the mysterious organs that control these differences in development, and amazingly, how these organs could be socially regulated via pheromones.
Cover photo: Keating Shahmehri
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SPEAKERS
Art Woods, Rajendhran Rajakumar, Marty Martin
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AW: As you may know, ants are eusocial. This means that, in many species, related individuals live and work together in a single colony. Often, they sort themselves into different castes, and each caste carries out one or a few different tasks to support the whole group. Amazing!.
MM: We get it, you like ants!
AW: Castes also differ in size and shape. For example, some species in the genus Pheidole have evolved two worker castes: minor workers and soldiers. Minor workers look like your typical ant.
MM: The soldiers are bigger and have disproportionately large heads and mandibles, which they use to defend the colony and break up large food items. There’s even a handful of species within Pheidole that have an additional super soldier caste, which boast enormous heads.
AW: OK, how do you get different castes from what are basically the same eggs? It’s all thanks to developmental plasticity.
MM: Developing larvae sense and respond to local environmental cues – things like how much they get to eat and the kinds and amounts of pheromones put off by the workers tending to them – which then determine whether they grow into minor workers or soldiers.
AW: Rajee Rajakumar, a professor at the University of Ottawa, studies Pheidole ants and their ecological and evolutionary developmental biology–
MM: Eco-evo-devo, for short.
AW: –to better understand the interactions between development, genes, and environment. In a 2018 paper in Nature, Rajee and his team focused on the role of an unexpectedly important transient structure in soldier ant development.
MM: Here, we need a quick detour into insect development. Ants are part of a much larger group of insects – aw: called ‘the holometabolous insects’ – that undergo complete metamorphosis.
AW: In holometabolous species, the larval stage typically is specialized for feeding and growth while the adult stage is specialized for dispersal and reproduction.
MM: Not surprisingly, those two kinds of tasks can require very different morphologies – which is why larval Lepidoptera (AW: “Caterpillars”) look and act nothing like their corresponding adults (AW: “moths and butterflies”).
AW: OK, big question: where do adult structures come from during metamorphosis?
MM: The answer is…aw; drumroll….from ‘imaginal discs’ – multiple small groups of cells that are basically set aside during larval development but that grow rapidly during metamorphosis to take on the final adult form.
AW: There are imaginal discs for wings, for legs, for antennae, for mouthparts…etc.
MM: And side note – they’re called imaginal discs because an old term for ‘insect adult’ is ‘imago.’ So ‘imaginal discs’ are the groups of cells that will form the adult structures.
AW: Now back to Pheidole…
MM: In some developing larvae – such as the female and male reproductives – these discs will eventually develop into proper wings.
AW: But soldiers are wingless, and so after a brief period of time during development the wing discs disappear. The larvae of minor workers don’t grow wing discs at all.
AW: In this episode, we talk with Rajee about a clever set of experiments that he and colleagues did to figure out the roles of transient imaginal discs in developing castes of ants.
MM: I’m Marty Martin
AW: And I’m Art Woods
MM: And this is Big Biology.
[music break]
Art Woods 00:00
We're gonna dig into some details of this really awesome 2018 paper that you had in nature on ants. But before we do that, maybe just tell us a little bit about yourself and your field Eco-Evo-Devo, like what? What is that? And how did you get into it? And why? Why is that important?
Rajendhran Rajakumar 00:15
Basically, when I was an undergrad, I went to primarily undergrad teaching first university, a little bit more in the realm of the liberal arts type of comparison to r1 institutes in the US, for example, and I wanted to try to find some research opportunities during undergrad because I wanted to see science in action, as opposed to you know, memorizing in class. And, you know, I stumbled upon the Abouheif Lab, you have Abouheif Lab at McGill University, in Montreal, and another university in the city that I lived in. He was just starting his lab, and I went to go visit him. I didn't know what labs would really look like, let alone a brand-new lab, right? And as we all know, if you're starting a lab, you basically have pretty much nothing, just an empty room. But he had these Tupperware boxes on the shelves, and I was like, Okay, this is really weird. Like, that's not I'm expecting like cool equipment or whatever. and he's got these Tupperware boxes, I put my lunch in or something, right? And he put one down, and he opened it up. And inside was this like microcosm, this little mini-universe. That is what I soon found out was this little society that ants form in the form of colonies. And it wasn't just any ants as a species called Pheidole rhea, which, if you see a colony of these, they basically look like dinosaur ants, essentially, these crazy, huge ants related to work that we will talk about, hopefully, get to a little bit in the century later, where they have these workers and soldiers and super soldiers, like just phenotypic variation. What is this? I was a very mechanism-driven molecular cellular biologist and training in school, as like, This is crazy. These are the phenotypes that are possible, right? And so he sat me down and taught me two quick lessons that got me into Eco-Evo-Devo, which is first, these ants, although they look completely different. It's actually the same genome, the same genotypes that can give rise to these alternative phenotypes, and is through what's called developmental plasticity, how genes in an environment interact during development that can generate all these diverse phenotypic variants, all these variations on the theme of their phenotypes of their body plans and other traits you see amongst different individuals. And so in that one fell swoop, I learned that like, wow, development is that landscape that the genotypes traverse through to get to that phenotype and the genotype-phenotype map. And it's it's not a straight line. It's not like learning Mendelian genetics. And, you know, eye color and hair color and stuff you learned in genetics, it's more complicated, even more nuanced, is the fact that the environment can bias and direct and redirect the way cells and tissues and things during the build process actually go through. And in this case, on an organism level, rather than, say, determining what cell or organ or tissue become, it's on a whole individual of what has to become these workers and soldiers and super soldiers. So my first time visiting his lab. And this was back in 2000, the end of 2004. Okay, so when Eco-Evo-Devo wasn't even really a thing, you know, and I was in shock, extreme, amazing phenotypes. It's how the environment or ecology acts on development that can potentially give rise to many different evolutionary outcomes. So that's kind of my experience getting into it. In a nutshell.
Marty Martin 03:09
Do you think that Eco-Evo-Devo is the end? Are we going to have another modifier of some sort? Because development fine. Evolution, Evo-Devo was all the rage when I was a graduate student, which was just a little bit earlier than, you know, 2004, you were talking about? What Eco-Evo-Devo. I've never been able to get my head around the difference of Eco-Evo-Devo and Evo-Devo. How are they different? Are they importantly, different? It sounds like it.
Rajendhran Rajakumar 03:33
Well, so here's the thing to me, Mary Jane West-Eberhard's magnum opus that she wrote in... I don't remember exactly if it's 2005 or 2003.
Marty Martin 03:42
Developmental plasticity and evolution? Is that the one?
Rajendhran Rajakumar 03:44
Exactly developmental plasticity and evolution.
Marty Martin 03:46
That's one of my favorite books. So yeah.
Rajendhran Rajakumar 03:48
and to me, I actually, I don't care if it sounds biased, but I'll be as bold to say that we'll look back 50 years from now. And it'll be second only to the origin of species to the way we think about evolution and how it unfolds. And you know, if you look at it citation rate, it's a book about developmental evolution of plasticity, and yet, its citation levels is like, say, Coin and Ore's classic book, you know, in more mainstream Eco-Evo-Devo spheres. And I think it's really only a matter of time. And it's, again, is because it's this really key missing link about the role that the environment not just as a selector, not just as playing a role in say, natural selection, but a role in generating variation. So it's in both, it's coming from both those directions. So through development plasticity, you see that during development, you can have the environment interact with developmental processes, and give rise to variations in phenotype that can be raw materials for natural selection to act upon. And so in many ways, I feel that her work and work that preceded it, that inspired it and work that as follow it. I think that role of the environment and how an introduction for genes and with cells and hormone production and epigenetic mechanisms during development and in adulthood in your physiological processes that really helped connect the dots of how you go from a mechanistic "how" science that is developmental genetics to developmental biology to a more holistic larger scale "why" science like as to quote, you know, Ernst Mayr kind of thing. I think it's just that how, who you're talking to and finding a common wavelength, the language. So when you think about variation, and to me, that's one of my favorite words in biology... variation. And if you're talking about, say variation of phenotypes, on one side of the coin, you can think about adaptive variation that can be selected upon in an evolutionary context. But on the other side of the coin, we can think about it in a maladaptive context, not just in evolutionary biology, but in a biomedical context as well. You think about the development mechanisms, physiological mechanisms, and molecular cell biology, all of these levels of regulation, so many things can be tinkered with or perturbed, can lead to... Yes, adaptive variation that can be selected upon for evolutionary outcomes, but also can be the basis for disease and countless diseases, that we complex diseases, we have
Marty Martin 05:53
Plasticity at the heart of everything. So I'm completely on board with that book. I don't know if I'd go so far to say it's second to On the Origin of Species, but not that I have an replacement, I just have to... I would have to think a little bit harder about it. But um, there's a conventional question with these sorts of things. What are the key empirical discoveries that substantiate the platform that Mary Jane has in that book? I mean, what what are the data that ground the sort of new ways of thinking about Eco-Evo-Devo and evolution in general?
Rajendhran Rajakumar 06:26
I mean, you know, I guess it depends on how you come at it even going beyond just say, talking about classical experiments or anything like that. It's just whether you're, say, a plant researcher, and you know, for instance, Plant Biologists have been the pioneers of plasticity work way long before people working with animals. I mean, just because, you know, you go back to early 20th century, amazing plant biologist studying how, you know, in different climates, that same species of plants are different phenotypes, you'd see a rise depending on to say they're at the top of the mountain or mid level of a mountain or at the bottom, different temperatures, different elevations. And, you know, of course, some plants, they can't just pull up the roots and go walk around and, you know, evade things and deal with, you know, limited resources and, and adapt, right like we can, and so they're highly plastic, and lots of plant research over the last century, have demonstrated that like, they might have genotypes when they emerge, but they can interact with their environment in very extraordinary ways. Now, that's true for all kinds of animal research, that has also unfolded. And I think that, you know, the discovery or our findings of how along mechanistic levels, how hormones, and non genetic processes like epigenetic mechanisms, including histone modifications, micro RNA biology, DNA methylation, so on and so forth. These are non genetic processes, chemical modifications, that could be mediators of the environment, to translate a changing environment and how an organism adapts to that environment. You know, you can just pick various different examples across different systems to show that like, yes, you know, plasticity is important. I mean, if you go into retrospective 2020, you know, when Darwin was looking at ants, and its origin of species and his extended larger drops of his extended book, that was the Origin of Species was supposed to be the abstract for once upon a time. When he's talking about ants, and he's looking at army ants and other species of ants, he looked at a colony and see how different the individuals are within the same colony,and in the same family, so to speak. And he was he'd actually say, in his writings that this, it almost seems potentially as like a fatal flaw to my theory of natural selection, because how could you how could it be that you have a sterile cast, the worker cast propagate, you know, all of these different phenotypes, compared to say, the reproductive cast that look completely different, they look as different from each other, and I was just different species, but different general, a different subfamily, so on and so forth. And you know, like, fast forward, you know, it's due to plasticity mechanisms, whether it's hormones and development, epigenetic mechanisms and other things. And I think it's just findings that we've had over time of how you can have single genotypes. And you know, the starting point in an experiment for geneticists, we tried to like, deal with, you know, control only one variable at a time and make sure the environment stable? Well, when you do take genotypes, and you do vary in the environment, and see different outcomes in plants and animals, the writing's on the wall, you know, that plasticity is actually very universal feature, you know, the organism a world, and it's fascinating and super exciting to study and could have everything from evolutionary to biomedical relevance.
Art Woods 09:24
So you're sort of anticipating my next question here. But I want to turn more solidly now to ants, and to ask about what you view as the utility of ants for answering some of these big, Eco-Evo-Devo questions. And I get it that there's also just a lot of passion for ants here. And I, myself do a lot of insect physiology, so I get it, but like in terms of like, thinking about ants as the system for answering some of these big questions, why are they good?
Rajendhran Rajakumar 09:52
Oh, yeah. Okay. Well, so first off, disclaimer, I'm obsessed with ants! So some of my things that I might say right now might be a little bit biased about to justify why they're a great model for Eco-Evo-Devo. But, you know, just keep that in mind... To zoom out a bit... ants as an organism are incredible. You know, the paper and proceedings of National Academy of Science just came out saying that there's something like 20 quadrillion ants on this planet, right? It's that means that there's like, for every one human, there's 2,500,000 ants, approximately. So yeah, it's crazy. So when you're a kid, and you're like taking a magnifying glass and burning an ant, or something like that, well, guess what, there's another 2,249,999 waiting for you!
Marty Martin 10:34
And they're coming for you.
Rajendhran Rajakumar 10:35
Exactly. In your dreams, they're haunting you in your dreams. So you know, they're everywhere. They're ecologically dominant and incredible, fascinating. They've independently evolved societies, like we have. You know, just people have studied them over the centuries. You know, they see ourselves many ways and you know, when you study them long enough, I see in humans ant things and you kind of like, ANThropomorphize everything that you see them to eventually. Yeah, sorry dad joke!
Marty Martin 10:59
You really you just got a gold star from Art, that's his favorite.
Rajendhran Rajakumar 11:02
And so well, you know, they're everywhere. And I was telling my class the other day, and my microbiology classes, students that they're in pretty much every continent, except for Antarctica. And then one student raised her hand and heckle me and is like, well, that's really funny. I was like, why? Well, it's called Antarctica, you'd think you'd have ants there. And I was like, alright, I'll keep that one. I'm putting that one in my bucket. I'm keeping that for my kids or something. But anyway, so they're, they're everywhere. They're super awesome. There's 15,000 plus species around the world, and every one of our species have their own story of our own beautiful life history. There are own fascinating characteristics. But then what what is it? What is it so awesome about that? Well, there's two major hallmarks I feel that are worth noting. One is obviously the cooperation you see within their societies that they form. As I said, they've independently evolved sociality in an even extreme form where some individuals, they divide reproductive labor, some individuals are cast that are meant to reproduce, and workers, worker caste, so the Queen's exempt, for example, in the worker caste that are predominantly sterile, and do non reproductive tasks that were foraging for food, taking care of the youngest one, and so forth. And so cooperation that you see an optimized amongst the cast in those colonies, that's a really important thing that potentially has enhanced or enabled that diversification and awesomeness ecologically speaking. But then the other thing is, what is developmental plasticity? How do you make all of those specialized morphological castes that you see within the colony? and it's that their developmental process is very sensitive to the environment and environmental cues, abiotic and biotic cues. Such that through during development, the same gene and the same genotype can experience different environmental cues at certain optima can go on to become one caste or another.
Art Woods 12:44
So they're insects. So they're awesome as a given... but what you're saying of the insects, they're sort of the masters of translating environmental and social cues into plasticity that gives different forms within the colony.
Rajendhran Rajakumar 12:59
Well, I'm going to refrain from saying to the mastering in case, someone else will, will feel that's a little subjective. But I will say this, they are a fantastic system to study gene by environment interactions, if you want to know how development and your genes and your genome can unfold in a dynamic way, in response to environmental variation. This is a fantastic system, because you know, when you think about like, say cell differentiation, you have a stem cell, and it can go on to differentiate into cell type A or cell type B, and they look really different. They behave functionally different in a tissue, so on and so forth. And there's different growth, environmental cues within your body growth factors, so on and so forth, that can stimulate that... Well, this is a whole new level in the ant world. These aren't cells differentiated, determining and differentiating. These are whole individuals, these are castes that are determining and differentiating, these aren't cells going down what's called Washington's landscape of being pulled through the developmental process to have different phenotypic outcomes. This is a whole individuals that are experiencing the developmental landscape and exposed to environmental variables and becoming a queen that is completely different than soldiers and workers, for example,
Marty Martin 14:00
So Rajee, I mean, I don't mean to be antagonistic, but to me, the body plan of ants among the insects, and even if you think about things like extreme metamorphosis, in the case of say butterflies and moths, like that is a whole other amazing circus act of plasticity, that if you look at the ants... I mean fine, these giant super soldiers, that's all cool, but it's an embellishment on a theme. Right? So my argument would be, I don't know, my perspective, my naive backbone bias would be the ants are a simpler version of plasticity. Does it even make sense?
Rajendhran Rajakumar 14:36
uh, I guess, I guess it depends if so when I when I'm thinking about plasticity, I'm thinking about variations on the adult body plans. So if you're looking at butterflies, you know of a given species. Yeah, there are seasonal morphs, and they're beautiful, you know, you see the different wing morphs and how they deal with the different surfaces of the wings to deal with sexual selection and actual sexual and simultaneously fantastic systems, But uh, in terms of adult forms, and in terms of the same genotype giving rise to different phenotypes. If you look at an ant colony, yes, there are some ant species where all the worker ants look identical. You know, if you look at the common sidewalk ant, like Tetramorium immigrans, where they are having these ant wars whenever the seasons are changing, you see a bunch of ants like killing each other and your sidewalk. Yeah, they look like ant and you're like, okay, cool, and you keep walking along. But if you see some of the ants I have seen whether the leafcutter ants or army ants or truck draw ants? Just amazing, extreme elaborations, as you say, so it's how far they push those elaborations on the theme. And the fact is, you see what is the body plan? You see what is the theme they're elaborating upon? And so you can have that be your ground state that you're trying to investigate how have they elaborated? and you can look across many species in a high resolution comparative framework to see how have you transition from in subtle ways to more elaborate ways to varying those morphological phenotypes, and then try to trace it to what are the developmental and molecular mechanisms that have facilitated those transitions? And we're just talking about morphologies, like people study, and it's also why it's the ecological context. There are ants that like form their own agricultural systems, there are ants have their own sewer system, every ant colony has their own cemeteries that they do for quarantining there recently dead. Their chemical communication challenges or linguistics, you know, in terms of the way we've evolved means to communicate with each other, I can go on and on about how cool ants are as an organism!
Marty Martin 16:20
Okay, we we can tell you like ants.
Rajendhran Rajakumar 16:23
Yeah. So you add all that beauty, you add all the beauty of the organism beyond just their morphology. It's a wonderful contexts to to, you know, study your organism, because you know, you might change morphology in a lab, you tinker with things, and all of a sudden, you make their jobs different, or their eyes different, or their heads different. But then you can be talking to an animal behavior person. And they're like, Oh, how do they behave? Do they behave differently? Do behaviors couple or get decoupled with these morphological variations that you're inducing a lab, where you see in the wild, natural variation? And all of a sudden, you start connecting to beyond the morphology in a more extended phenotype context. And that more colorful life history context becomes something that is directly relevant to developmental plasticity research that I'm doing.
Art Woods 17:07
You know, it sounds like a lot of this stems from and maybe is dependent on the fact of the BU social and colonies acting like super organisms. And you, you sort of made the allegory between individuals in an ant colony and cells in a body. And you know, the same way the stem cell can become many different phenotypes of cells in our own body, you can get individuals becoming very many different kinds of individuals. And so in a sense, it seems like you're leveraging this idea that you're getting extreme plasticity from the same genotype over and over, because you're getting this diversification of body forms within this super organism. And yet, somehow, because they're individual ants, you can deduce, you know, interesting rules about how forms come to be and how allometrys or mechanistic basis of the underlying allometrys. Is that a fair way to put that?
Rajendhran Rajakumar 17:54
Yeah, I actually see that as even better explanation of what I wanted to say! You hit a very key buzzword, the superorganism. And yes, you know, you brought up butterflies and contrasting. So those effectively cells within the colony, right, it's like the colony behaves like an organism. So just to take a step back. So the queens are basically the individuals that are now taking on the reproductive roles of the colony. And so some species that can make hundreds of progeny a day, and in their lifetime, they can make millions. And the reason for that is that, as opposed to the workers that are starting predominantly sterile, and live for the range of say, months, the queen can live on the scale of decades. So we're not just talking about an order of magnitude of lifespan or longevity differences. We're talking about orders of magnitude of difference. And again, same genotype different phenotype outcomes. So if you're saying now, you're an extension of developmental plasticity, interested in longevity, and health span, lifespan stuff, or, or reproductive differences, these are individuals specialized in a colony, like so specialized in our bodies that act as one organism where the queen is like the germline, and the workers are like the soma, the somatic tissue that differentiates.
Marty Martin 19:05
So Rajee, you're probably the best guy to ask this question to something that's been in the back of my head for him, probably since I was in high school, the number of cell types and the typical vertebrate, because thats my bias is on the order of like 200, something like that, right? What's the maximum number of castes in the most differentiated insect? Because it's not even close? Right? Talking about orders of magnitude? Why is it that within bodies, you get so many cell types and than we get castes? Picking on your plasticity argument, sorry!
Rajendhran Rajakumar 19:35
No actually, that is a that's a very nice perspective to take in terms of thinking, because you know what, for me, one thing about developmental plasticity and Morpho space, phenotypic space, right and how much you can explore. One notion that I've really like to think about sometimes is what is you know, one way to think about is like forbidden phenotypes essentially, like what what are constraints?What is possible and what isn't possible, both experimentally and in the natural world? Right. And yeah, it's true. That is quite a distinction, especially for that analogy. And of course, I am way of phrasing as an analogy, although if you push me to limit, I can make it almost sound like I'm thinking there, one to one. So to come to ants, I'm not a leaf cutter specialist, but I remember hearing someone say on the order of sort of, like 16, specialized, you know, subcaste of worker of the worker caste system.
Marty Martin 20:21
Wow. Okay.
Rajendhran Rajakumar 20:22
And that's the other thing is that in ants, it's not just morphological castes, but also the behavioral caste side of things. So you can have individuals that look identical, but they really do form essentially different behavioral castes within the colony. They really carry out... they specialize in specific tasks that have to be done, jobs in the day in the colony. So if you multiply morphological castes by behavioral castes, and some of them even argue, have gone on to argue like physiological costs, which is a whole other kind of view. Technically speaking, there's a lot of diversity. Is it on the order that you were describing in our human body the way you know, the cells that we have? Maybe not... But then again, we've studied the human body for a long time as humans investigating humans. And you know, the more we investigate ants, the more we find the subtleties, the specialized cell types, so to speak, a specialized caste.
Marty Martin 21:08
Right? Well, and I think that criticity is a good way to think about it because it could be the morphology is the most conspicuous... That's, you know, naturalists were the first biologists, so maybe as we dig more into even, you know, the regulation of epigenetic marks, there could be diversity in who can differentiate best, fastest, most efficiently, reversibility, all of this kind of stuff. A lot of more latent plasticity that we did, we can necessarily see
Rajendhran Rajakumar 21:31
Just one more extension! Food for thought... Extending the super organism, is that not just how you see them carry out tasks within the colony, but also how they communicate with each other. So there's obviously the classical, our understanding of chemical communication, right? That these cells in the colonies, figuratively speaking, these ants are communicating with each other. But recently, in the last five/six years, a colleague of mine, Adria Leboeuf, has discovered that actually, ants communicate in a way that we never expected, so they basically communicate through social fluids where they use their mouths to trophallaxis, transmit fluids between adult-adult and larvae to adult and adult to larvae kind of thing. And in those fluids, which we thought were just nutrients, you know, that they're feeding each other, maybe information from where they just where kind of thing. It turns out that there's metabolites, proteins, hormones, micro RNAs, and countless other things in this social fluid, as we're, she's calling. And so it's like an it's like a circulatory system that's actually interconnecting this society. So yes, it's becoming more and more of an organism as, as we even as I even thought, you know, starting off, it's pretty crazy.
Art Woods 22:42
So I think this is a good segue into your paper, this 2018 paper in Nature, which examined the sort of origins of some really interesting morphologies, and different castes of minor workers, soldiers and super soldiers, and then these just astounding developmental origins of those of those morphologies. So maybe let's just get into it with a little bit of terminology for so maybe just describe what what's a minor worker? What's a soldier and what's a super soldier?
Rajendhran Rajakumar 23:12
Right? So Pheidole is the group of the genus of ants that papers predominantly focused on. This is a hybrid diverse genus, there's over 1000 species globally, it's unlike most other genera in the animal world in that regard. And of those 1000 plus species, a major hallmark is the evolution of a soldier cast. So this is a morphologically specialized caste, also behaviorally speaking, as well where its uses its disproportionately large head with special soldiers specific muscles in its head, and really large mandibles to both attack predators and competitors. And also to process food. Like, there's some species of Pheidole that that are seed farmers, for example. Minor worker caste, they're smaller, they look a little bit more typical ant-wise in terms of body proportions, and they forage for food and nurse the young, just as examples of things they do. Now, the super soldier caste is something where you know, there's over 1000 species, less than 1% of them actually. So about eight, potentially nine, now, species have an additional caste, where their heads are just humongous for their body size. They're huge again, allometric really disproportionate to their body even compared to the soldiers and then the soldiers, the workers, and so on and so forth. So they are their own very discreet cast within the morphous space of how their heads for example, scale to their body size. There's still very little known about how they're specialized in colonies in the wild. One of our colleagues, Ming Huang, who was actually an author of earlier work. Tpaper published in Science, where we actually first describe the evolution and development of super soldiers. He had observed in the wild that in the species Pheidole obtusospinosa, which has the super soldier caste, they actually live in the desert and as opposed to the usual ant colony where you see, you know, an anthill, and a hole at the top... I'm talking about it in a cartoonish way, but that's really how we kind of imagined them. These ants live in like rock crevices in the desert areas and these more open, vulnerable entrances to the colony. And they also live amongst what are called army ants that can come divide and conquer, destroy everything around them resources and other insects. And so you think they need soldiers, but in fact, they have what are called super soldiers were when they're under attack by and are detected by scout army ants coming to the entrance of the colony, the workers and other individuals in the colony will go into retreat into the colony. And then they'll recruit all of these super soldiers to the nest entrance, and then they bend all of their gigantic Super Soldier heads together like a Spartan shield, essentially, and block the army ants from coming in. And then they disband, start attacking the army ants rubbing their abdomens on the floor to disrupt, you know, chemical cues and drills and stuff with the army. And so they're in like, complete disarray from a war logistic kind of point of view. And then they reformed the shield again, like a Spartan shield, and they keep going through this until the army ants are freaked out leave. And so they have this very fascinating coupling between this very extreme morphology of their head and body sizing proportionality, and the behaviors that they use to carry out this like tank like head and how they use them as a group.
Marty Martin 26:12
So they don't use that head as sort of I mean, in a soldier sense... It's a little bit miss representative, it's more of a defense than attack, or is it also attack? I mean, presumably, they've got giant mandibles too.
Rajendhran Rajakumar 26:23
Yeah, exactly. So that's like a novel observation, in a defense sense as you're describing. But yes, they definitely use their mandibles to attack as well. I've worked with, you know, maybe 30 ish different species of Pheidole and keeping colonies in the lab over the years. And, you know, they each have their own little nuances and everything. But if you give them some fresh food, you'll see the workers come find the food, then they'll go back to their, their colony tubes, or however you haven't set up in your lab environment. And they'll recruit soldiers to come attack the food, process the food, use their mandibles, if it, say is a live insect, use the mandibles to attack... Say its a cricket, for example, the workers will come to the pull all the legs to the sides, basically to like, hold it down. And then the soldiers will come, you know, waltzing in, after all, the hard work has been done by the minor workers and basically start you know, using their giant mandible process. Yeah, yeah, cool.
Art Woods 27:14
Well, let's Let's move now to some of the developmental events that underlie the origin of these castes, especially the soldier and the super soldier caste. So a lot of this hinges on these things called wing discs, which are an important sort of developmental thing inside insects that go through a complete metamorphosis. So maybe just tell us what are these discs and what specifically is a winged disc?
Rajendhran Rajakumar 27:37
Okay, so holometabolous insects, which undergo as you alluded to complete metamorphosis from a larval form to an adult form. They have what are called imaginal tissues that are ultimately powdered and grown into the adult structures that they have. So they're these populations of cells that communicate with each other and form an axes you know, what will become the proximal distal aspects of the appendage or organ, dorsal ventral, anterior posterior, so on and so forth, just like our limbs are patterned, for example. But once they're done growing and patterning, they'll undergo metamorphosis, and then you'll see the adult structure come out on the other end of metamorphosis. So you'll have say imaginal discs, or imaginal tissues, these populations themselves, they can give rise to see the wings of in these insects of say, the winged queens, for example, and can give rise to you know, the legs of these insects, their eyes, their antenna, so on and so forth. So they're very important populations of cells that form in the larval stage, that basically pattern and grow into the adult appendages, for example.
Art Woods 28:38
So a lot of adult insects that are holometabolous, have wings, and those wings come from wing discs, why are those so important in Ants?
Rajendhran Rajakumar 28:46
Well, so there's a couple of ways to get at it. But one thing that's really striking about ants, is that at the origin of ants, you have a winged caste and a wingless caste, right? So all ants all 13,000 plus species of ants, all the worker caste, it's universally wingless. That's a universal property of ants. So the origin of what is called wing polyphenism, or the ability for that wing imaginal tissue that you're mentioning, to either develop into a wing in the adult, or to either not be developed at all or be halted or something along the development process to give a wingless worker, individual and adult form... is a hallmark to the entire origin of ants, right? And there's some explanations of why that can be beneficial, you know, prevents workers from getting wings and dispersing and flying and starting their own thing. And increases you know, density within the colony, constraints to keep them in, it's more social interactions and go on from there like a springboard to kind of catapult them to evolving more social connections interactions amongst each other in a colony. So that weightlessness is a huge distinction in caste in Ants. So with like Pheidole, for example, there's the group that the paper that you're mentioning is focused on and a lot of my work is focused on. You have the winged queen, for example, and then you have the wingless caste. Typically, a wingless worker, but in this case, there's wingless minor workers and wingless soldier caste. So, wing differences is a huge thing in ant world. And those tissues develop properly into wings in the queen. But there's a bit of more nuance and mystery when it comes to understanding why workers and soldiers are wingless.
Marty Martin 30:20
So I think this gene called vestigial, comes into play here. But can you connect those dots for me because like you, you were referred earlier to this allometric kind of thing. And on the show many times we've talked about allometry. So I think a lot of listeners will be familiar with that. But I couldn't quite wrap my head around how you end up with spatial variation and expression of this gene, leading to these allometrys. What's coordinating the location of expression.
Rajendhran Rajakumar 30:45
So basically, what you have here is these two endpoint phenotypes being winged or being wingless, right. And if you go back over a century, there was the first descriptions of when you dissect open the larvae of individuals that would grow up into a worker caste, for example, they have what are rudimentary wing imaginal disc. So there are these tissues, these populations themselves, they're in the homologous location of where wingness would form, say in the queen, developing a queen larvae. But when they go through metamorphosis, they don't become a wing. So you have these rudimentary structures that transiently developed and then disappear. And so now bring it to you brought up the gene, vestigial. So what we wanted to do, was along with some other several pieces of evidence that we can maybe touch on later, that basically got us to this point, cutting to the chase, that maybe these rudimentary structures that start to develop, and then go away, maybe they might have more of a role than we once thought... Because one key thing that we noticed is that you see the correlation of the elaboration of the growth of this transient tissue happening a lot wherever you see the independent evolution of an ant group that has a soldier caste. And so we thought, okay, well, let's, let's go about trying to test the function of this tissue. And now coming to your the gene that you mentioned vestigial. So this is a gene that's known from the flight-related traits. We know one of the first genes that Thomas Hunt Morgan, one of the fathers of genetics, as we know it today, vestigal was one of the earlier mutants that he was describing. The fascinating thing about that is that unlike say, you know, different pigmentation of eyes, for example, it literally has no wings, its wings are gone. It's like a flightless fly essentially, right. And fast forward many decades, lots of research has gone into understanding what that that gene does, and its role in the cell molecular or genetic context. And so basically, people have found that it's both necessary and sufficient for initiating and carrying out wing development. And so what we wanted to do is, maybe that's a target, because we wanted to see is there a way that we can molecularly ablate or destroy that rudimentary tissue in the developing soldier that has these really big, rudimentary structure that grows and proliferate really fast and then dies and goes away? To target it specifically without affecting any other tissue at the same time? So first, we saw that its expression is unique to the rudimentary wing discs, in the same way, it's unique to the Queen's wing disc.
Art Woods 32:56
So specifically, you used RNA interference to interfere with the expression of vestigal. So can you give us like a primer on RNAi?
Rajendhran Rajakumar 33:09
Absolutely. So there's, there's a few ways to to to use an RNA interference approach. But basically, our cells have machinery that process RNA molecules into micro RNAs and sRNA molecules, from plants to humans. Essentially, it's this machinery that can, instead of turning RNA molecules, or messenger RNA molecules into transcripts into proteins, through translation, can actually be used to have their own ability to regulate gene activity, and so on and so forth. Basically, in a synthetic world, in the molecular biologists toolkit world, you can hijack that system essentially inactivated by injecting into tissues, double-stranded RNA. So our cells don't typically have double-stranded RNA that float around and aren't targeted by some surveillance system, like the one I was just describing for micronase. And so if you inject double-stranded RNA of a fragment, that is that gene vestigial, the cells machinery of the ants will detect that, chop it up into smaller fragments, and process it such that they become basically little molecular bullets that can go target the transcript of vestigal that is being transcribed by wing cells, and that developing larvae and lead it to its degradation and prevents it from being translated.
Art Woods 34:20
So you inject this RNAi, you're interfering with this vestigal and what happens to the wing disc?
Rajendhran Rajakumar 34:25
Yeah, so we micro inject them larvae at a key stage where soldiers are just starting soldier development, we can see that they're just barely, if not the same size as the biggest larvae that would be come a minor worker. But you can look at other characteristics of larvae see, oh, it's still developing, like distribution of fat cells and many other things that color the gut of the larvae and several other markers, morphological, visual, invisible markers that we can use to say, oh, it's not just starting the soldier development pathway. And as that's happening, it's just initiating now, the development of this rudimentary wing disc because unlike the Queen where she's starting to grow that wing tissue right from the beginning of larval development, soldiers only start doing that in their last instar. If they produce a high level of a key growth hormone called juvenile hormone, and if it passes that threshold, they'll go on to soldier development. And so we targeted this very precise stage to say like, Okay, if we tried to destroy this tissue, perturb this tissue, right as a starting to get going, what might happen? And so we microinjected larvae at that stage. And then we let them go through metamorphosis. And when looked at them, and tried to see what might happen, what phenotypes might arise.
Marty Martin 35:30
So what I think is the coolest part of this paper there many elements, but my absolute favorite part is back to the super organism idea that the regulation of this whole phenomenon has... it's a socially mediated process, right? It's either through nutrition and the juvenile hormone that you just mentioned, or what I thought was even more compelling, pheromonal influences, right? So can you talk a little bit about those two scenarios?
Rajendhran Rajakumar 35:53
Yes, I'd love to. So the result of that RNAi injection, and targeting a tissue is that when you dissect open the larvae, that disc is gone. Okay? So it affected it. And when you look at the metamorphosed individuals, those soldier destined, individuals, their heads and body sizes are completely perturbed. So you completely affect the head to body allometry scaling properties. And we were shocked by that, that this is the property that got you know, the hallmark of this whole group, a soldier caste that has this disproportionate head size, to the body size. We perturb it by messing with this tissue we thought was completely useless for 150 years since it's been described, right? We thought that was super cool, that it can potentially control the allometry and the development of soldiers. Now, coming to what you just mentioned, when we saw that and we demonstrated for other methods, we also physicically cauterize that tissue, physically ablated it like, you know, you go into dermatologists office and they like, you know, you got a mole, you got to zap the same kind of method, although of course with a really precise probe. And we found the same thing. So now coming to pheromones, as you said, when we saw this when we scratch your heads, like what are other instances where you have the regulation of soldier castes? Yes, we are very familiar with nutritional control of production juvenile hormone that crosses a threshold that can make the larvae develop into soldiers, right. But in the 1980s, Diana Wheeler, Fred Nijhout, did pioneering work in species of Pheidole , like the one we worked with. They tested this idea that it had a longer history in the ant literature, where if you have these pheidole ants and you have, they're under attack and there's competitors, you generate more soldiers in the colony. And so you increase the ratio of soldiers to workers in the colonies. Typically, there's like five to 10%, that are soldiers to the 90 95% that are minor workers. They're very costly to make soldiers. They're hyper specialized. So you want to keep this ratio, this social demography ratio, when you produce a lot of soldiers to deal with competitors, predators, so on and so forth. Over time, when they're gone, when that skew is gone, you want to recalibrate the colony so that they have the homeostatic level of ratio, that's default ratio. And I use homeostasis again, as a, as a word to allude to that super organismal regulation, the colony acting like an organism, right? Except it's social demography, instead of like homeostatically regulating differentiated cell types or something. And so they basically suppress developing larvae from becoming future soldiers. So that now the next gen that are overlapping generations with each other will contribute to the workforce so that they're not all you know, this high level of soldier they're being produced. And they did experiments that basically demonstrated that there must be some kind of pheromone that is actually inhibiting when there's high density of soldiers present. They're producing a soldier inhibitory pheromone that blocks the development of future soldiers. Were thinking like, Oh, my goodness, like, how cool would it be... if we actually replicated those social demography, racial changes in the lab? and see does it affect that rudimentary wing disc in developing soldiers, something that we now know is a developmental mechanism that influences what caste you become. And so that's what we did, we raised larvae that were soldier destined with different proportions of soldiers, so say 100% Soldiers raising them, versus, say, 100% minor workers as two extremes, for example. When you dissect the adults, as Dinah Wheeler and Fred Nijhout, had described, were smaller, although we now describe their allometry they did affect the allometry scaling and everything and yes, it did, and it blocks soilder development. And we dissected open the larvae and it affected their wing disc in the exact same way that our RNAi and our ablation experiments were doing. And so it really seems that as you said, there are both positive and negative social interactions that generate positive nutritional for example, cues that can produce hormones like juvenile hormone to get them to go on to become soldiers, but also negative social interactions that through inhibitory pharamones that can suppress individuals from becoming soldiers to again keep this fine balance and that ratio that social demography
Marty Martin 39:39
Wow it's such an amazingly cool system... that's that's really neat. It's nice that you connected all those dots at multiple levels of organization too... it's impressive system that way.
Art Woods 39:56
Let me stop you're in summarize this as I understand it at the moment and I just won I want to say how bizarre I think this is. So we have a couple of casts of soldier ants, and minor workers and their morphologies are controlled by to a large degree wing discs. They don't have wings, but they have these sort of wings discs during development. And so what we have is almost like, we've been using this word vestigial to refer to a gene, but it's almost like we have a vestigial organ. This winged disc that arises during development, does something really profound and then disappears and never makes a wink. So so how amazing is that?
Rajendhran Rajakumar 40:36
So basically, its acting as the signaling hub. So the one thing I didn't mention when we're talking about imaginal discs, is they have like our organs during development, ways to communicate with other organs to ensure the development which at times can be asynchronous, how they're growing, and patterning are all in check at the end of the development so that when you are born, or when you go into metamorphosis, we're talking about insects, all your organs are ready to go, things are coordinated. And so basically, this tissue is sending off some kind of signals. And there are precedents for signals like that in imaginal discs in fly research, for example, there are, there's an insulin like peptide that can be secreted by this tissue, that can influence developmental timing and how long development happens in soldiers that develop for longer so on and so forth, in ants. And so future work is definitely set on trying to figure out what are the signals that these tissues that is transiently developing structure that appears, pops up and then goes away, just disappears? What signals it might be emitting to the developing head and other soldiers specific morphologies to influence its growth to coordinate its growth, along with the growth of the whole system. So there are there are clues based on the fly work, but it's really is truly a Pandora's box, there's a lot to be studied about, what is the signal? How is that signal received? And how does that influence the receiving tissue? It is super fascinating that this structure we thought was totally useless. Not only is it governing the development of the key phenotype of soldier caste in this group, but it's doing it in this crazy interorgan way interorgan signaling way, when it's in that transient developmental process. And somehow it's able to interpret the social environment through inhibitory pheromones, which is, by the way, a whole other crazy element of this paper. And you know, when we were trying to figure out the title of this paper, we had a hard time because there were a lot of major unexpected findings that we had as we're going through this roller coaster of a ride. Yeah, but that's,
Marty Martin 42:21
Yeah, but that's a good problem. Right? If you have so many cool results, you have many alternative titles, I would like to have that problem.
Rajendhran Rajakumar 42:27
Yes, I'm with you.
Marty Martin 42:29
So Rajee, thank you so much for the chat. We're becoming a little bit sensitive to your time. So we want to broaden out a little bit and then give you some space to sort of hit things that you haven't done yet. But one more sort of big picture question you write at the end of the paper, a couple of lines that I thought were so punchy. And you know, I love that kind of provocative getting people in their systems to think about these kinds of things. You say, first, that rudimentary organs might acquire novel regulatory function to facilitate adaptive evolution. And then you allude to similarly that these organs are storing some sort of ancestral developmental potential. Do you have examples in other systems where we know that this is the case, something that's unrelated to wing discs altogether? Maybe something that's not even in an insect?
Rajendhran Rajakumar 43:09
So in terms of the ancestral development potential part, the second part of your question is, so it links back to a science paper from 2012 that we did, where we showed that the super soldier caste has not only independently evolved, but actually is likely an ancient feature of the group of Pheidole, you know, of over 1000... Its independence kind of flickered this recurrent phenotype across to have a super soldier caste, and they have more elaborate wing discs and and I won't get into the details, but it seems like basically, by reactivating this latent Super Soldier developmental potential or pathway, you're able to potentially reactivate those phenotypes in the wild and then have natural selection act upon it. And that could explain the re-evolution of this phenotype across the group. And at the time, we were talking about the level of hormones juvenile hormone is very important for making a soldier versus a worker. But we found that actually, there's an additional switch to make a soldier versus a super soldier, and that you can actually reactivate that dormant switch in species that don't have super soldiers, you can actually have very special time in development, leading development, you can reactivate a super soldier phenotype and species that don't have them. And that was actually inspired by findings we had in the wild in Long Island where we found in a species that that the Abouheif Lab has studied for years, that doesn't have super soldier castes. We found these anomalies that were these massive soldiers. We looked at them more carefully in the lab and look like what we thought were these huge anomalies in terms of their head and body size. They will just like the species there's the super soldier so they're like super soldier like anomalies. And we've developed a system to reactivate in every species that we treat it with hormone, we're able to reactivate this dormant ancestral developmental potential to make a super soldier phenotype. And now fast forward to 2018 shedding completely new light on the 2012 work is that when we did those reactivations we would actually reactivate the wing imaginal disc to be super soldier like wing imaginal disc too. It's only now in 2018, in this Nature paper that we realized, like, Man, wing discs have had this rule, and Pheidole obtusospinosa were they still just have just one pair of wing rudimentary tissue, the super soldiers have two pairs, and they're way more elaborate in growth. And that might be the way that not just how the hormones can actually instill and initiate the growth of that tissue to lead to that elaborate allometrys. So that's like ancestral developmental potentials in ants, how you can reactivate it with these rudimentary structures right. Now, more generally, there are already examples of rudiments across plants and animals. You know, if we're talking about humans, for example, you can think about the webbings that happen in traditional webbings that have in between our digits in the development of our hands and feet. You can think about the tails that we have, you know, during fetal development that regresses, you can think about out notochord. So the notochord gives rise to the spinal vertebral body in us, but in many other vertebrates, it actually persists but in us it's kind of a vestige but it actually signals the formation of the neural tube. Now, what's really fascinating in this context, is it really pushes the limit of what how those signals can be. So in that case, it's it's really neighboring tissues, it's like, pair chronotype signaling. In our case, it's like hormonal inter organ that are distant, right? It's an endocrine endocrinological scale of signaling. And so it really opens the door. All of these rudiments, i described to you in humans, do these tissues actually have properties to make factors that can signal more and more longer distant ranges to influence the development of other organs and other tissues? I think that's a very important uncharted question to answer in the future. So there's lots of rudiments I can go on and on about how many rudiments and vestiges and some little bits that carry on in the adult life see spurs and pythons and other things that you see in adults structures that are a little bit remnants of an ancestor. But that very elaborate developmental process that before it goes away in development, what is it do? I think now, they might play novel regulatory roles for us to now investigate further,
Art Woods 46:54
It feels like these these rudiments, even if in some lineages, they give rise to full fledged morphological phenotypes in juveniles or adults, that maybe in all lineages, they're retained, because they assume these sort of important developmental signaling roles, so they become sort of irreplaceable, right? They can't be gotten rid of, Is that Is that a fair way to walk?
Rajendhran Rajakumar 47:17
So that's a new possibility to entertain, like, how, how much can you really lose something and you know, when you think of a loss of characteristics, loss of eyes, loss of limbs, you know, whether, say you're talking about snakes, or lizards that have lost their pentadactyl growth plan of their hand, other other appendages or their loss of limbs completely? How much do they really lose the underlying mechanisms and tissues and everything? Yeah, they might completely degenerate them, they might retain some, well, before retaining some of those development processes. Do we even think that mattered at all, or it's just an idiosyncratic process of development? Well, now it's a new perspective, maybe they might have some regulatory involvement. And that might explain why they persist. And more importantly, it could explain how you can have a phenotype whether it's through plants and animals, that is a recurrent phenotype across phylogeny that flickers, essentially, you know, and you're thinking like... Well, is it you're reinventing the wheel every time or in this case, you're activating, it's being held dormant through these other regulatory potential uses, transiently during development, and you can reactivate and elaborate it more so that you can basically re-elicit or reactivate that dormant phenotypic potential to make adult structures that were long lost in an ancient ancestor.
Marty Martin 48:25
Well, it seems like we need to update the dictionary definitions of vestigial. I mean, this is a completely different connotation than I think we usually mean with vestigal. Well. Hey, Rajee, thank you so much for being on. The last question that we posed to guests is just to sort of give you the floor. And is there anything that you wanted to make sure to bring up that we didn't give you the chance to say.
Rajendhran Rajakumar 48:43
Oh, well, first off, thank you so much for having me on your show. It's quite an honor. You've had amazing guests over over the past episodes that I look to as heroes in the field and inspired my work. I guess you hit on all the key spots of that work that we were talking about today. I think really the future there's a lot of amazing promises more questions than answers that are generated by this work. A lot of it was unexpected, which is beautiful about research. And I think serendipity is something that, you know, don't be shocked and surprised by when it comes to you in a negative way. Embrace it. But again, you know, ants are awesome, everyone should study ants. I'll just plug that at the end. If you want to make ants bigger, smaller, like atman or you know, give them you know, super soldiers like Captain America and they're super soldier program and the Avengers. Come join an ant lab like mine. And you'll we'll do all kinds of cool, Frankenstein experimental biology and tie it to the natural history of these animals.
Marty Martin 49:34
Well, when you work that out, we'll have you back on you can let us know about your Ant Man, super soldiers. That'd be pretty fantastic. Rajee, thank you so much. It was really great talking to you.
Rajendhran Rajakumar 49:41
The pleasure is mine. Thank you guys. Take care.
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AW: Thanks to the College of Public Health at the University of South Florida, the College of Humanities and Sciences at the University of Montana, and the National Science Foundation for support.
MM: Music on the episode is from Podington Bear and Tieren Costello.