 Thank you all for coming. I was just telling Phil that the work I'm going to tell you about—oh, can everybody hear me? Good. I was just telling Phil that the work I'm going to tell you about today is something I had absolutely nothing to do with. I just found it very interesting, and when Shantel asked me to do a presentation, I thought, I'm going to talk about those ants. So that's why I'm telling you about this today. It's a little bit strange. So you're probably familiar with symbiosis, which is a relationship between two species that both of them benefit from, as opposed to parasitism, which only one partner benefits. For example, animals can't break down cellulose, so they can't get the sugar that is stored in cellulose, but a lot of herbivorous animals can digest cellulose by hosting bacteria or protozoans that do know how to break cellulose in their digestive tract, so they just carry them around. And so they give the symbionts a place to live, and the symbionts help to feed them. Another nice example is that there's a species of squid that has a light organ attached to its ink sac, and it doesn't produce the light itself. The light is actually produced by a bacterium, a vibrio, which comes in and colonizes the ink sac and makes the light, and then the light. It's a very small squid, and so the light helps to hide the squid in the dark oddly enough. So it doesn't cast, it doesn't look like it has a shadow. Yes, cellulose and starch, they have different kinds of glucose, actually, different glucose forms, but they are related. They are both polysaccharides. Okay, so symbiosis is just two species living together, and they may be so closely associated, like lichens, for example, that they can only survive together, but each species still retains something of its original identity. In endosymbiosis, you have one species, which is nearly always a prokaryote or some kind of bacterium, is inside the cells of the host species, not necessarily in all of their cells, but in at least some of their cells, and therefore becomes an essential part of the host and has to be transmitted to the progeny of that host organism. And probably one of the most well-known examples of endosymbiosis is in the origin of the eukaryotic cells, cells that have nuclei, and sometime during the development of that cell, two different kinds of bacteria started taking up residence inside those early nucleated cells, and one of them, aerobic bacteria, became mitochondria, which we use to produce a lot of our energy, and in some plant cells, chloroplasts, cyanobacteria, got into those cells and became chloroplasts, and so those cells are photosynthetic because of endosymbiosis. So in this case, they are in all of the cells. I mean, if you're a plant, you've got chloroplasts in nearly all of your cells, and if you're an animal, you've probably got mitochondria in all of your cells. So there's a bunch of insects that have endosymbia in them, and I've just listed four of them here. One of them is Drosophila, many, and there's a lot of different Drosophila species, and many of them carry an endosymbiote called Wolbachia, and there are different species of Wolbachia that are associated with different species of Drosophila. In aphids, there's an endosymbiote called Bushnera, and the psyllid insects, which are like teeny, tiny, cricket-like things, have a very small endosymbia called Carcinella, and in the carpenter ants that I'm going to talk about today have an endosymbian called Lufmania. And a lot of these relationships are still not fully understood. Wolbachia is a very successful endosymbia. It's not just endosophila. It's in a lot of different insects. It's also in some other, a lot of other invertebrates, especially different kinds of worms. It wasn't even discovered in insects until 1924 when it was founded Mosquitoes, and it was also found in Drosophila in 1965, and now there are a lot of known Wolbachia associations. What it does for the fruit fly is an entirely clear. It may protect Drosophila from other kinds of bacterial infections. Some species of Drosophila live longer when they have Wolbachia in them, but it can also lead to sterility or kill off the male progeny, so it's not entirely good for the Drosophila. A couple of cool things about Wolbachia is that Wolbachia is related to the Rickettsial ancestors of mitochondria, so I think that's kind of interesting. So we have a kind of young endosymbia that's related to a very old endosymbia. In one species of Drosophila, Drosophila on a Nasi, there's at least one full copy of the entire Wolbachia genome in the genome of the fly, and it's transcribed, and nobody knows what it's doing there and why it's transcribed or what it does for the fly, so there's still lots of mysteries. In the aphids, what the bushnera does is to produce some essential amino acids because the aphids eat this or sap suckers, and so they're not getting a lot of protein out of their diet, so they get their essential amino acids from the bushnera. A bushnera is from a totally different kind of prokaryote from Wolbachia. It's related to the endobacteria, like E. coli, but its genome is very, very small, and E. coli's genome is about 4.6 megabases, million bases, but the bushnera genome is only about 640,000, so it's much, much smaller, and that tends to happen in endosymbians. They tend to pass off genes or just either pass them off to their host or just lose them entirely. The carcinella endosemian is interesting because it's got the smallest known prokaryotic genome, only 174,000 bases, and it's practically, it's so small it's practically not a prokaryote anymore, it's practically an organelle, but what we're going to be looking at today is the blockmonia relationship to its carpenter ant host. Interesting thing about blockmonia is that it's probably, it's related, it's closest endosymbiotic relationship is butmera, and it may have been acquired by lateral transport from mealybugs, a similar endosemian in mealybugs. Okay, so carpenter ants. There are a lot of species of ants, about 12,000 species of ants, and about a thousand of them, a little over a thousand of them, are carpenter ants in the genus Canfinotus, and these carpenter ants live in tunnels that they make in wood but they don't, unlike termites, they don't eat the sawdust, so they just, you know, leave the sawdust, and they all have these obligate endosymbionts which produce some essential amino acids for them. So the ants provide shelter for the bacteria, and then they also transmit the bacteria to their offspring. So they're fairly typical endosymbionts in that respect, but there's a few other things that make them strange. Blockmonia was one of the very first endosymbionts to be described in an insect, although it wasn't identified initially as a bacteria, blockmon thought it was a fungus because it looks kind of like spaghetti inside the cells that it resides in, but later it was found to be bacterial, and then blockmon was honored by having it named for it. So the work that I want to talk about was done by some people at McGill University, Rafiki, Rajkumar, and Avuhe. And they were studying the relationship between blockmonia and its ant hosts, and they were looking at how the ant embryos developed and how they interacted with the bacteria, and the reference for the article is given there, and it's also in the references. So what they found was that the interaction between the ants and the blockmonia involved three important things. One of them is the germline, the cells that produce the germ cell, the eggs and sperm of the ant, maternal genes of the ant, which are active in the embryo, and then a group of genes called the hox genes. So I want to talk a little bit about each of those so that it all makes sense, sort of. So we'll start with the germline. General embryos develop from single cells, fertilized eggs, and these come from cells that are embryonic cells, which are called germline cells. And germline cells in all animals become cellularized, become separated off very, very early in development, and that's true of these ants. It's interesting that the germ cells develop before the gonads that they are eventually housed in, so they actually migrate into the gonads as the gonads form. So this is what the insect germline looks like. You have cytoplasm just on the very surface of the egg. So most of the egg is yolk material, but on the very surface of the egg is a very thin layer of cytoplasm, and on one end is what's called a polar cytoplasm, and this is a cytoplasm that's going to wind up in the germ cells. So this, I think this is meant to be the fertilized egg nucleus. So these nuclei divide, and they don't get partitioned into separate cells until fairly late. So at first the nuclei divide by themselves, and then they migrate into that surface cytoplasm. And in the very first cells that become actually cellularized, actually separate entities, are the primordial germ cells. So they develop before any other cell type even gets compartmentalized. So I just want to tell you something about the developmental sequence in insects. You've got several sets of genes that hacked in sequence. First you have maternal effect genes. What these do is set up the anterior and posterior axis of the embryo, the front from the back. And then you have a couple of sets of embryonic genes, and these, let me go back just one minute. These are actually maternal genes whose proteins are put into the egg. The gap genes and the peri-rule genes are genes that are active within the embryo itself. And they sequentially subdivide the embryo into smaller and smaller segments. So the gap genes divided up into big pieces, and then the peri-rule and the segment polarity genes divided up into smaller and smaller pieces until you have the separate segments. And these are the segments that becomes visible when you look at the caterpillar larvae or something like that. You can see the individual segments in it. And then finally the last genes, and these are also embryonic genes. The last genes to act are the hox genes, and these assign specific structures to this particular segment. So it decides where the leg is going to be, where the wing is going to be, where the eye is going to be, that sort of thing. And these are a subset, the hox genes are a subset of genes called homeotic genes. These all contain a common 180-base homeobox sequence, which encodes a particular protein which is probably DNA binding, and that's called the homeo domain. So the hox genes, an important thing to remember here, is maternal effect genes act very early, and the hox genes act very late in development. So that's the way it usually works. All right, so this is a picture of the hox genes because they have some other peculiar features. These are the hox genes in Drosophila because the Drosophila hox genes have been studied quite a lot, and a lot of the names refer to their structures in Drosophila. So these are the hox genes, and what's interesting about them is that not only do they decide what goes where, they are ordered on the chromosome in the same relative positions as the body regions that they determine. So the map of the hox genes is the map of the embryo. So for example, this labial gene produces mouthparts, where it tells the segment to produce mouthparts. Whereas this abdominal B gene, down to the other end, produces the posterior abdominal segments. So they not only are mapped in structural order, they're also mapped in activation order. So the first genes that are active are the genes at the anterior end of the embryo, and the last genes that act are the genes at the posterior end of the embryo. And the two hox genes we're going to be interested in are these two here, UBX and abdominal A. UBX is for ultravide thorax. So these two genes have a role to play in the interaction between blocmonia and the ants. And this just lists the different hox genes. It tells you what the names are. And I just thought it would be interesting for you to see what the hox genes look like in humans, because humans have four sets that have the same relationship to the anterior posterior axis of the body that they do in Drosophila. And they have four sets because of whole genome duplication during the evolution of the vertebrae. Okay, so I just want to review the relative activity times for the maternal effect genes which are the first genes to act and the hox genes which act much later generally. Okay, so what the McGill researchers discovered when they were studying the genes that are active in these developing ant embryos were some very odd things. And the first odd thing, or one odd thing, was that there were two hox genes that were expressed very early. They were expressed in those posterior germ cell cytoplasm. And those were maternal genes, maternal hox genes that were active in the germ line. So those weren't from the embryonic genome, they were from the maternal genome and the proteins had been parked by the mom in the egg. So these genes are not only acting much earlier than they would normally act, they've been modified so that they are activating the germ line genes. So they're now doing something weird that these hox genes don't do in other animals. The other weirdness was that the organization of the embryo in these ants is different than it is in other insects. First, the posterior germ cell region wasn't used to produce the gametes. Most of the endosymbians collected in that posterior germ cell region and were used to produce bactericytes, and the bactericytes carry the endosymbians into the developing gut where they became active in digestion and in producing amino acids. The embryo region, the part of the early embryo that became the embryo was displaced from the anterior end of the egg, and three more germ cell regions are produced in these ants. These aren't found in most other insects. So we've got weird hox genes and we've got weird germ cell genes. So this is a picture of the bactericytes, and you can't really quite see the bucamania in there, but the bacteria are inside these cells. The bactericytes are developed from what normally would have been primordial germ cells, but these are bactericytes, and instead of going into the gonad, they go to the guts where they make these essential amino acids. So to try to figure out what was going on in these ants, what the investigators did was they looked at the embryos of carpenter ants, they compared their genetic activity to that of a lot of other related insect species, and they asked several questions. They asked, where is the germ line? They asked, where are the maternal hox genes active, or are the maternal hox genes active? Do they have endosymbionts, and if they do, what kind do they have? Where is the embryo developed within that egg, and which of the germ lines produces the gametes, and what are the others up to? So that's what they're looking at, and looking at dozens and dozens of different species. And this is just a summary of what they found. Okay, so what's typical of most insects is this picture that you see here. There is one germ line zone, it's in the posterior, that is going to become the germ cells, and the germ cells, as the embryo begins to develop, will then migrate into the embryo and snuggle up into the gonads. So this is your typical insect. Now some of the relationship, or some of the relations of blockmonia, that we're not blockmonia, remember there's a thousand species of those. So some of these other insects did have a new germ line zone down here, and for some reason this new germ line was participating in producing the germ line. Some of these species had other endosymbionts, but some of them had no endosymbionts. In these species, the maternal abdominal A and UBX were expressed in both of these germ line zones. So the second zone probably was, or may have been active in producing the germ line, but we don't exactly know, they don't exactly know what that second zone is for in those insects. Okay, third stage is the acquisition of the blockmonia endosymbion. This occurred just judging by which species have it and which species don't have it about 51 million years ago, and as the blockmonia came in and started messing with the germ line, the embryo moved anteriorly, moved out of the way of the blockmonia, or apparently looks like it's getting out of the way of the blockmonia. And there's a third germ line zone up at the anterior end. So this combination is seen in a few carpenter ant species, but mostly what you see in the carpenter ants is this situation in which you have now got the original germ line zone, the two new zones, and then a fourth zone, which is called number two here. I know that's confusing, but that's the way they number it. And this fourth zone is now the real germ line. So this is where the eggs and sperm for the next generation are going to develop. And these cells also pick up a few blockmonia from this sort of decoy germ line zone, which makes the bacteria sites. So these all become bacteria sites. These become gametes, but they also pick up just a few of the bacteria so that they can transport them into the next generation. So very complicated. And these cells here become active in the midline of the embryo, and these seem to help the bacteria sites move into the gut. So they all have jobs to do that they didn't have before. So this is just a summary of that history. So here's a date line up here. So this is about the time, about here, where blockmonia gets into the ant eggs and becomes a kind of permanent endosymbion. That's an enormous amount of work that they were doing. So a couple of questions here then. What are those maternal hox genes, UVX and abdominal A, doing? So one of the things they looked for was, you know, where are those two genes active? And they found that the abdominal A messenger RNAs localize in germ line zones one and three. Those were those, that number three was the first extra one to be produced. So abdominal A activates that one or goes into that one. And UVX is in all four germ line zones. And so they asked the question, well, what happens if we inactivate UVX or those, all of the UVX and abdominal A genes by using interfering RNA. So interfering RNA is, pairs up with the RNAs and keeps them from being transcribed and doing the normal thing. So this will specifically inactivate those two genes. So what happened when they inactivated abdominal A was the germ line genes didn't get expressed. The buccalmonia didn't get incorporated, the bactericides didn't form, and they didn't get into, and blocking UVX led to misplacement of the zone two germ cells. And they didn't get into that region. So those didn't get into the gonads. They don't into the embryo. So blocking those genes totally screws up the embryonic germ line. So they also say, well, what happens if we take away the endosemia using antibiotics? And you can do this in Drosophila. If you do this in Drosophila to get rid of the Wolbachia, the Drosophila will be just fine. If you do that to the ants, if you do that to campinotus by using antibiotics to get rid of the endosymbionts, half the embryos don't develop at all. And the ones that do develop don't have effective germ lines. The germ lines aren't acting normally. So they're basically sterile. So the endosymbionts seem to activate genes in the embryo, including the hawks genes. And the hawks genes from the ants have been modified to activate the ant germ line. So those two things have sort of become interactive, which is very, very strange, but it's what happens in this blochamania and it's been worked out over the last 51 million years or before. OK, so I just want to make a couple of other comments about blochamania. As I mentioned before, if you take it out of the Drosophila, the Drosophila is going to be fine. I mean, it doesn't actually have to have it, although the Wolbachia does produce seminal acids in the gut of the insect. However, Wolbachia has also found some nematodes and it is essential to those. So it's possible that it's been in the nematodes longer than it's been in the insects because the Wolbachia genome in the nematodes, these worms, is much smaller than it is in Drosophila, in the flies. OK, so at some point in future time, maybe it will become essential to Drosophila, but it isn't yet. So blochamania, on the other hand, is absolutely essential to the survival of all carpenter ants. And so it is effectively part of this insect species because it transmits it from one generation to another, and it interacts with the genes of the host cell. And this is the references that this one right here, this is the first article that I saw and I thought, oh, that's really interesting. So and then the rest of it is just some of the other information. So I have not been watching the chat because I can't keep track of it while I'm talking. Are there any questions? Oh, one thing I wanted to mention about ants, which you may or may not know, ants are kind of like bees in that the male ants don't have daddies. The male ants develop only from a single set of chromosomes or use just a single set of chromosomes that they get from their moms. So probably nearly all of the embryos that these guys were looking at were females that had arisen from fertilized eggs. So the fertilized eggs become females depending on what they eat, they may or may not become queens. The unfertilized eggs, of which there are not very many, become males. I'm pretty sure those are circular genomes because they're prokaryotic. Do you mean the blochamania genomes? Right, Phil? Yeah. Back in the 70s, I've got to do a little bit of stuff with a postdoc person with some messing around with the circular DNA. Yes, they do. They do. Any particular group of ants is going to be nearly all females and nearly all workers. The only, the worker ants have ovaries and they have eggs. But they don't ever get them fertilized. So they do have gametes. They just don't use them. I mentioned those other insects. And let's see, there are other endosymbionts. I think the most starling examples of endosymbionts are the ones that are not the ones that produce the mitochondria and the chloroplast. In fact, the chloroplasts have kind of an interesting story because most of the, most plant chloroplasts come from prokaryotes, from cyanobacteria. But there are some unicellular green algae that are eukaryotes. And those have become incorporated in the endosymbionts. And those have become incorporated into some other kinds of cells. And some of the, some of the protists, like euglena, I think, actually got its chloroplasts from a green algae rather than from a cyanobacterium. Yeah, the very, the very, the very first, a new queen, a new queen, when the queen mates, and this is true bees too, I think, when the queen mate, she's probably going to mate once, not necessarily with just a single male, but she's going to mate once. She's going to store all the sperm cells and then as she will then release those from wherever she keeps them, and I'm not exactly sure where that is, but she releases those every time she produces a new set of eggs. So mostly those eggs are fertilized. And her first litter is always a bunch of females because she's going to need some worker ants to help take care of her. So she makes a bunch of those. And in most cases, the males don't get produced until later until she's about to, you know, give off to another queen. I think the thing I found the most interesting was that early action of the hawks genes, because the hawks genes are found in so many different organisms and they generally act after all those other embryonic genes, and in this case, they're acting very early. Those two hawks genes, that are the maternal hawk genes, are acting very early, which means that what they do has been changed. I mean, their job has been modified, and I found that totally fascinating. And of course, that has happened in other hawks genes over evolutionary time because the hawks genes and, you know, humans don't do exactly the same thing as the hawks genes in, say, ants. Although in many cases, you can substitute a human gene, a human hawk gene for an insect hawk gene, and it will do what it's supposed to do in the insect. So there's quite a lot of similarity among those genes. In fact, you can still tell which one is which. You notice that they were, let me back up and show you the color coding on them. I should have started from the other end. I just wanted to show you the human hawks genes and the fruit fly hawks genes. Here they are. Okay, so you see the color coding. So all of these pale ones are labial genes. And there have been some additional genes added in the human genome. For example, this one isn't in fruit flies, but it is in the human genome and possibly some other mammalian genomes as well. It's not just in human. So all mammals have these four sets of hawks genes. Yes, hawks have hawks. You said, why might that be? And I'm not quite sure what you mean, Phil. Oh, it's hard to say why something happens. It's more like how, but if Hawks genes are activated early. Is there an advantage or was this just some sort of mutation that was retained? I think it has to do with bilateral symmetry because he's in all these Hawks genes are in all bilaterally symmetrical animals. So they're in earthworms. They're in flies. They're in chickens. They're pretty much the same Hawks genes. And in most vertebrates, you've got these four sets because of the whole genome duplication very early in vertebrate evolution. There are some other genes like I think some of the myosins that there are also four copies of. But because maybe because the Hawks genes are active during development, more of them have been retained. Oh, that's an interesting question there gone. The genes, the Hawks genes in the Hawks genes that produce the appendages, the arms and legs, they are active kind of in the same. The same order. So the posterior Hawks genes are, well, you can see the color color coding here. You got three sets of Hawks genes that are active in the same order. This gene, this gene, that gene are active going starting from the proximal region of the limb to the distal region. So it's kind of like the anterior posterior axis except it's the part that's near the midline and furthest from the midline. So these are acting to produce the different parts of, you know, the upper and the lower leg in the feet or the hands. So I think that geometry I think is very, very interesting. So, oh, so in answer to Sisi's question, yeah, possibly. I think that it might have, might have led to some muscular changes that would have led to bipedal movement. If you look at the nobos, I don't know if you've ever watched the nobos, but those are the, sometimes called the pygmy chimps, although they're not that much smaller. If you look at the nobos, they are quite bipedal. They walk around on their back legs quite a lot. Even though their arms are still quite long and they're still essentially quadrupeds, but they stand up and hold things. I've got a picture of a bonobo. I took it to San Diego Zoo that standing up holding a stick. It looks for all the world like, you know, he's holding a spear or something. Yes, we're quite close to the chimpanzees. In fact, the bonobos evolved after we did. We split off before the bonobos did from the chimpanzees. Too bad we didn't split off, split off after. He might have been less aggressive. Yes, the, the upright posture is definitely a defining character of humans. And I think, I think the, the voice boxes to the changes in the lyrics. Oh, well, I, I never met a homo erectus, but I think I would call it human. They're certainly included with the hominids. Oh, barrigan. Okay. That'll be fun. Yes, I think the, I think the ability to disagree might, might have been refined with the development of language. Well, it's in the genus Homo. Homo erectus. That's a good question. Maybe whales. Actually, the bonobos are pretty nice to each other. Yes, some of the hox gene mutations are very strange. Antenopedia, for example, puts legs instead of the antenna. So it's got feet on its head. And the proboscopy, I think has feet on its nose. Possibly. I mean, there are a lot of gene mutations. I think one of the important ones is the language gene. And I don't know if that gene is homeotic. I'll have to check that out. Oh, you know, it might be it's Foxp2. It might be a homeotic gene, but it's, I don't think it's a hox gene. Well, the, the, they picked up the Blocomani about 51 million years ago by comparison with the other insect species. So is that early relative to insects? Insects have been around for maybe 400 million years. So I don't, I don't think it's early in insect evolution or, well, that one is an early insect. There may be some that are, that are early. Bushner, for example, has such a small genome. It's probably been around for a while. Oh, elephants are sentient. I think so. Somebody was talking about elephants, maybe here might have been here somewhere about experiments that have been done with elephants. Putting, you know, dots on their faces and they will feel around with their trunks if you show it to them in a mirror. So they know it's them. They're, they're looking at a reflection and they know there's something up there that's not normally there. So there's, I think there's a lot of sentience in that. Yes, supposable thumbs are, I think, what got us in all the trouble that we get into the fact that we're tool users. And tool, tool builders, you know, that we're so manipulative of our environment. Yes, there you go. Although there, there, there are some other. Well, the, the chimps, the chimps are tool-using too. A little bit. And some birds. The Darwin's cactus finches are tool users. They use the, the spines to dig, dig stuff out. Well, did I miss any questions? Oh, well, the endosymbiosis that produced the eukaryotic cells occurred pretty early. The pro, the eukaryotes were a couple of billion years after the earliest of the prokaryotes. So, but two billion years is pretty old. And there are, there are a lot of, a lot of symbiotic relationships among groups of prokaryotes that, you know, bacteria that hang out together and help each other out. In fact, probably that kind of association was the precursor to the development of eukaryotic cells. And predators tend to have eyes in front of their head. Oh, I can, I'll send that to Chantal. The slides, Alva. I'm sorry, I meant to do that before, but I'll go ahead and do it. Well, I'm glad you found it interesting. I, I've enjoyed the opportunity to dig into it a little bit more. Well, thank you, Phil. Yes, I should do this more often. It's been a while. Oh, welcome, Rosa. I hope you'll come back. Oh, good. I'm, thanks for saying that tag. Sometimes I mumble. Well, I will send those slides to Chantal. And so they'll be available. And thanks again for coming.