Session 9: Coevolution
Transcript of Part 2: Termites and Their Symbiotic Gut Microbes
00:00:07.25 I'm Jared Leadbetter 00:00:09.08 and I'm at the California Institute of Technology. 00:00:12.07 And, for some 24 years now, 00:00:14.14 my research has sought to clarify the relationship 00:00:16.26 between termites and their hindgut microbes. 00:00:19.21 Now, of particular interest to me 00:00:22.22 is the metabolism of hydrogen 00:00:24.20 that is generated during this fermentation of wood, 00:00:27.11 but today I want to give you a broad overview 00:00:30.17 on microbial diversity 00:00:33.08 and on some of the essentials 00:00:35.22 of termite hindgut microbiology. 00:00:38.26 So, I want to talk about 00:00:41.07 biological diversity in general, 00:00:43.14 because many who are interested in biology 00:00:45.17 are missing, actually, some of the 00:00:48.21 full diversity that we see in the microbial world. 00:00:52.01 And, then I also want to comment about 00:00:54.15 how some of this diversity is surprisingly abundant 00:00:56.28 in certain areas of the world, 00:00:58.23 and that we must understand 00:01:01.11 what that diversity does and how it functions. 00:01:03.03 So, this will bring me to termites 00:01:05.17 and the symbiosis they form with their hindgut microbes, 00:01:08.14 and I want to introduce you to several major groups 00:01:11.10 of termite hindgut microbes, 00:01:13.17 the cellulose decomposing protozoa, 00:01:17.01 methane producing archaea, 00:01:18.27 and a group of abundant and unusual bacterial 00:01:21.27 called spirochetes. 00:01:24.12 I'm going to then touch on 00:01:26.26 how we can study different termites 00:01:29.06 and learn about different events that may have occurred 00:01:31.19 during this symbiosis in the past, 00:01:33.22 and then I'll give you some conclusions. 00:01:39.02 I wonder how many people who are watching this 00:01:41.11 have grown up thinking about three kingdoms. 00:01:45.10 Up until the 1960s, 00:01:47.01 I think most people grew up thinking 00:01:50.17 that there were animals, plants, and fungi, 00:01:52.24 and that maybe, depending on their education, 00:01:55.27 even up through current years, 00:01:59.22 most enthusiasts of biology 00:02:02.11 understood there to be between three and five kingdoms. 00:02:05.07 Maybe you had the bacteria as a fourth 00:02:08.29 and the protists as a fifth, 00:02:13.04 But, starting in the 1960s we had a revolution 00:02:16.25 in the study of the relationships between different organisms, 00:02:19.10 and started to realize that many things that we were seeing, 00:02:22.06 and also not seeing, 00:02:24.13 are very, very different from these three major groups. 00:02:27.12 So, for example, if you look at a key gene 00:02:30.10 that is present in all known organisms, 00:02:33.00 you can make comparisons between this gene 00:02:36.13 and from that infer 00:02:38.24 how those organisms are related to each other. 00:02:41.16 The thing I want to point out on this slide 00:02:44.13 is that you have the fungi 00:02:46.02 and the animals 00:02:48.02 and the plants, 00:02:50.16 and those are just three twigs on a branch 00:02:53.27 that has many other twigs. 00:02:56.03 Really? 00:02:58.10 If those three twigs and the length of those lines 00:03:01.11 denote evolutionary relationships, 00:03:04.27 then there are more than three kingdoms. 00:03:07.25 There are easily a half-dozen. 00:03:10.20 The other thing I want to point out here 00:03:13.04 is that if you think about "plant metabolism" 00:03:16.11 there are some organisms on this tree 00:03:19.14 which also carry out photosynthesis, 00:03:23.13 let's say the kelp or the red seaweed, 00:03:28.09 but those branches are actually 00:03:31.11 very different from the plants. 00:03:32.28 They are as distantly related to plants 00:03:35.08 as you and I are from plants, 00:03:37.15 and I think that's very important. 00:03:39.12 Also, when we talk about single-celled eukaryotes, like protozoa, 00:03:43.11 we realize, oh, 00:03:46.17 Paramecium and the protozoan Babesia 00:03:49.08 are actually two very different organisms, 00:03:51.21 again, as distantly related to each other 00:03:54.14 as we are from let's say the yeast 00:03:57.01 that you use to make beer and bread. 00:04:03.11 So, the number of kingdoms 00:04:08.16 or major divisions of life 00:04:11.01 already gets more complex than those three that we know. 00:04:15.13 The truth is actually much more complex than that, 00:04:17.22 because this is now just a snippet 00:04:20.27 of a branch of a much more complex tree. 00:04:23.19 You'll see that I have just blown up 00:04:26.14 this section of a much larger tree. 00:04:29.27 One of the things I want to point out on this slide 00:04:32.18 is that there are many, many branches on this, 00:04:34.22 easily 100 00:04:37.18 which are more distant from each other 00:04:40.22 than the distance between corn and animals, 00:04:44.12 and what that suggests, then, 00:04:46.13 is that we have a lot to learn about 00:04:51.24 the differences between these different groups. 00:04:53.28 So, for instance, everything 00:04:56.12 that is lying outside of this circle is a microbe, 00:05:00.02 the single-celled organisms 00:05:01.19 which are smaller than you can see with your naked eye. 00:05:04.11 So, for as much as we can appreciate biological diversity 00:05:06.19 that you can see, 00:05:08.17 the true diversity of life 00:05:10.22 is beyond the resolution of the human eye 00:05:12.12 and we have to use other methods to really 00:05:14.02 understand how it works. 00:05:16.14 The second thing I want to point out 00:05:18.23 is how different the way of living is 00:05:22.15 for corn, or a plant, 00:05:24.20 and ourselves, 00:05:27.05 or from a yeast that's used to make bread and beer. 00:05:29.28 If the length of those lines, 00:05:31.25 which is comparatively short, 00:05:33.28 and the differences of these organisms is so great, 00:05:38.06 imagine the possible differences 00:05:41.13 in the ways that these organisms live. 00:05:44.08 So, we are potentially really missing out, 00:05:46.28 not just on the diversity 00:05:49.17 in terms of how things are related, 00:05:51.16 but also missing out on the diversity 00:05:53.25 of what organisms are actually doing in the environment. 00:05:56.10 And so, if we're to understand the environment, 00:05:58.16 we really have to learn more about 00:06:00.22 what these other organisms are doing. 00:06:04.27 Let's come back to this tree. 00:06:07.06 Let's come back to this organism kelp. 00:06:09.23 I want to illustrate another point: 00:06:11.25 it's not just that there are many, many organisms out there 00:06:14.25 which are different from the organisms we're most familiar with, 00:06:17.04 but in some environments those organisms are present 00:06:21.15 and very, very abundant. 00:06:23.14 Take the kelp - 00:06:25.29 you can find kelp forests off the coast of California, 00:06:29.02 and those kelp are performing 00:06:31.21 what we might call "plant metabolism". 00:06:33.25 So, they are the primary producers in those environments, 00:06:36.06 but, keep in mind, they're not plants. 00:06:39.03 So, the story of these coastal ecosystems 00:06:43.03 is in a large part driven 00:06:45.12 by an organism that's very different from a plant, 00:06:47.20 and so if we want to understand those coastal ecosystems 00:06:50.10 part of the story is understanding 00:06:53.02 the biology of kelp 00:06:54.20 and understanding in what ways they are similar 00:06:57.03 and different to the terrestrial, 00:06:59.05 or land plants that we study. 00:07:03.24 Now, I want to shift from the oceans 00:07:06.02 to my own research, which is study on 00:07:08.25 termites and their hindgut microbes, 00:07:11.00 and I want to point out that the termite hindgut 00:07:14.02 is an environment. 00:07:16.02 It happens to be an environment that lives 00:07:18.17 in a small insect, 00:07:20.15 but we can compare and contrast that environment 00:07:22.16 with, for instance, 00:07:24.24 a very rich marine environment like the Sargasso Sea. 00:07:28.03 There are certain reasons why 00:07:30.16 you might want to study a small environment 00:07:31.14 like in a termite. 00:07:33.00 The Sargasso Sea is a wonderful and amazing place, 00:07:34.28 and very important to study, but it's a thousand kilometers across, 00:07:38.07 and there's only one of the them. 00:07:41.03 The termite hindgut is only about one cubic millimeter in volume, 00:07:46.01 and yet it contains hundreds and hundreds 00:07:48.13 of microbes that you find no where else in nature. 00:07:51.08 Just if you were to take the top millimeter 00:07:53.14 of the Sargasso Sea, 00:07:55.22 the volume of that across those thousands of kilometers 00:07:59.19 is 19 orders of magnitude greater 00:08:01.27 than the volume of that one termite. 00:08:03.21 So, we can actually bring a termite into the laboratory 00:08:05.28 and be able to study an entire environment. 00:08:09.25 The hindgut... it's tiny yet complex... 00:08:12.18 many hundreds of species 00:08:14.20 and some of those species are yet unstudied, 00:08:17.13 so it is still bewildering complex, 00:08:20.11 and it's well-bounded. 00:08:22.12 We know that the gut lining 00:08:23.00 and the outside of the termite 00:08:24.27 are where you might define the boundaries of that system. 00:08:27.26 The Sargasso Sea is wonderful, 00:08:29.25 but there's only one of them, 00:08:31.29 and its boundaries are a little bit user-defined. 00:08:34.03 We think that it's, you know, 00:08:35.23 the currents that are intersecting here and there 00:08:37.25 are what bound that region, 00:08:39.13 whereas in the insect it's very well-bounded. 00:08:42.00 And, of course, the termite is available 00:08:44.09 in large number of replicates, 00:08:46.04 so we can, in a laboratory, 00:08:48.22 have that one environment that's tiny and well-bounded 00:08:51.10 replicated in termite, after termite, after termite. 00:08:54.13 So, we can start to do some comparative studies 00:08:56.08 and perturbation studies 00:08:58.07 which are just not possible with a large environment 00:09:00.25 like the Sargasso Sea, 00:09:02.13 for which there's only one. 00:09:07.06 So, I study a very particular termite 00:09:09.14 that we find in ponderosa pine 00:09:11.18 that's fallen in the Angeles National Forest 00:09:14.02 of southern California, 00:09:15.19 and here is one of these ponderosa pines 00:09:18.10 and two of my former students, 00:09:20.25 who have peeled off some of the bark from this log, 00:09:22.28 which has been on the ground for probably five or ten years, 00:09:25.15 and if you look a little bit more closely 00:09:27.24 you can see that just on the underside of that bark 00:09:29.18 are a number of termites 00:09:31.19 of different, what we call, morphological castes. 00:09:34.02 These ones with the dark mandibles are actually soldiers. 00:09:36.29 Rather than eating wood, 00:09:38.21 they have big mandibles that they can use to attack, 00:09:41.01 for instance, another termite or an invading ant. 00:09:45.22 So, this is termite that we study, for the most part, 00:09:49.01 in my laboratory. 00:09:50.18 It's the Dampwood Termite, Zootermopsis nevadensis, 00:09:54.17 and it's about a centimeter in length. 00:09:56.09 It's one of the larger termites that you'll find on Earth. 00:09:58.27 Now, this is what we call a worker, 00:10:02.24 and from another specimen I've extracted the hindgut tract, 00:10:07.29 and that is shown here. 00:10:10.18 And, what you'll observe is that 00:10:13.17 there is a long tubular region, 00:10:15.18 which is somewhat analogous to our small intestine, 00:10:18.24 and then you have this hindgut paunch, 00:10:22.10 which is somewhat analogous 00:10:25.07 to our large intestine, 00:10:27.02 and it's in this paunch 00:10:29.22 that really you find a lot of things that excite microbiologists. 00:10:33.04 You find a density of microbes 00:10:35.08 you find nowhere else in nature, 00:10:37.12 and they represent all three domains of life. 00:10:40.04 Earlier on that slide, 00:10:42.06 I'd shown you that the tree of life, 00:10:44.16 where you actually have three major subgroups, 00:10:47.05 and those are the archaea, 00:10:49.00 the bacteria, 00:10:50.19 and the eukaryotes. 00:10:52.09 You find single-celled relatives of all three of those groups, 00:10:56.06 comprising hundreds of species, 00:10:58.07 in this hindgut paunch. 00:11:00.19 So, before I tell you about termite gut microbes, 00:11:03.04 I want to tell you a little bit more about termites. 00:11:05.17 There are about 3000 species of termites on Earth. 00:11:10.24 Termites are related to cockroaches 00:11:13.24 and to the mantids, like the praying mantis, 00:11:15.19 and they're actually, although insects, 00:11:18.05 quite distantly related to ants, wasps, and bees, 00:11:22.02 which are also social. 00:11:24.06 So, this is an example of where 00:11:27.06 society, or sociality in insects, 00:11:29.12 has arisen in two different groups of insects 00:11:31.08 which are very different from each other. 00:11:33.19 So, termites, and those 3,000 species of them, 00:11:37.07 can be subgrouped into several different families, 00:11:41.11 and their closest relative in the insect world 00:11:45.04 is what we call the Wood Roach, 00:11:47.04 which is a non-social but wood-feeing insect 00:11:49.22 that you find in the Carolinas 00:11:52.19 and the Pacific Northwest 00:11:54.22 and in parts of China. 00:11:56.26 And, many of the microbiology... 00:12:00.02 the features of the microbiology in the Wood Roach 00:12:02.22 are actually shared with the termites, 00:12:06.13 and so it's thought that many of the 00:12:09.07 microbial processes that arose 00:12:11.28 arose in the last common ancestor of this roach and termites. 00:12:16.08 Now, the termite that I study, Zootermopsis, 00:12:19.07 belongs to this one groups in the Termopsidae. 00:12:21.22 So, over time, we can start to ask, 00:12:24.12 are any of the patterns that we see here 00:12:27.18 present in some of these other groups? 00:12:31.09 Now, if you extract the gut from Zootermopsis, 00:12:34.03 or another termite, 00:12:36.08 and you take a cross-section through that gut, 00:12:38.20 what you'll see is that the insect tissue itself 00:12:40.25 is actually very, very thin. 00:12:43.05 It's only about 10 microns in diameter, 00:12:46.05 so 1% of a millimeter in thickness. 00:12:49.14 The bulk volume of that is comprised by the gut contents, 00:12:53.10 and what you see here, these larger objects, 00:12:56.05 are single-celled eukaryotes called protozoa. 00:12:59.20 Now, historically, until the mid-1990s, 00:13:03.01 this region, this environment 00:13:05.17 was thought to be completely anoxic, 00:13:07.23 so, devoid of oxygen. 00:13:09.20 Many of the microbes that you find 00:13:11.18 in the core regions of this gut are poisoned by oxygen. 00:13:15.12 Really? 00:13:16.21 Here you have this insect, 00:13:18.08 which is living in the aerobic world 00:13:20.09 and wandering around on its six legs 00:13:23.00 in a piece of wood or on a piece of wood, 00:13:27.15 containing microbes which are poisoned by oxygen. 00:13:31.09 The story is actually even more complex, 00:13:33.13 because it turns out, 00:13:35.13 through studies that were performed 00:13:37.05 by Andreas Brune and John Breznak in the mid-1990s, 00:13:40.19 the oxygen actually diffuses across the insect gut wall 00:13:45.03 and then is consumed by biological processes 00:13:48.07 in the periphery of that hindgut, 00:13:50.28 and it's those biological processes in the periphery 00:13:54.07 which lead to the lack of oxygen in the core, 00:13:56.20 which protects some of those oxygen-sensitive microorganisms. 00:14:00.29 So, not only do you find organisms 00:14:03.03 which are unique to the termite gut habitat, 00:14:06.01 that you find nowhere else on Earth, 00:14:07.28 but many of these are poisoned by oxygen 00:14:10.04 and are very sensitive to desiccation. 00:14:12.29 So, their life outside the termite is very, very limited. 00:14:18.20 So, what you have 00:14:22.01 are microbes that are very dependent on their host 00:14:24.14 and, because of their processes, 00:14:26.10 which allow the host to derive nutrition from wood, 00:14:29.10 makes the host very dependent on their microbes. 00:14:33.25 And, when a termite emerges from its egg, 00:14:37.01 it doesn't have these microbes in its gut. 00:14:39.01 It's fed those microbes by other of its littermates 00:14:43.02 or by its parents, 00:14:45.06 and if those microbes don't take hold 00:14:48.04 that termite will fail, 00:14:51.21 and if that termite fails 00:14:54.25 those microbes will also fail. 00:14:57.15 So, when we look at one termite 00:14:59.14 that's walking around on a piece of wood today, 00:15:02.05 we are looking at over 100 million years 00:15:04.26 of having this microbial community 00:15:07.16 passed from one termite to the next termite to the next termite, 00:15:11.14 generation to generation to generation. 00:15:14.04 It's quite a remarkable story 00:15:17.04 of a journey that's been taken 00:15:19.04 between many, many organisms together. 00:15:22.26 So, if we go and look at a little bit of a higher magnification 00:15:26.08 of what's inside the gut, 00:15:29.13 this is what we call a DIC image 00:15:34.04 using a microscope 00:15:36.02 of some of the larger protozoal cells. 00:15:37.29 These are about 60 microns in length, 00:15:40.23 so roughly 20 of these laid end to end to end 00:15:43.27 would be a millimeter of length, 00:15:46.05 and some of these are the primary agents 00:15:48.08 of wood degradation in this termite. 00:15:50.02 You also see some smaller cells 00:15:52.26 which are also protozoa. 00:15:55.13 So, these are single-celled eukaryotes, 00:15:58.26 of which there are about a dozen 00:16:00.27 in this one termite 00:16:02.23 that you find nowhere else in nature, 00:16:04.14 and their closest relatives are in other termites. 00:16:08.04 There are some interesting associations 00:16:10.03 that you find between these protozoa and certain bacteria. 00:16:13.23 For instance, if you look at the surface of one of these, 00:16:16.15 at higher magnification, 00:16:18.11 you'll see that those protozoa 00:16:19.28 are covered with long lines of grooves, 00:16:23.03 and in those grooves you see these little black objects? 00:16:26.15 Those are bacteria. 00:16:29.09 So, the surface of that single cell of eukaryote 00:16:32.23 is arrayed with a very regular group 00:16:36.16 of a very specific bacterium 00:16:39.19 and, without knowing much more about, 00:16:42.07 the notion you might have is that 00:16:44.19 there is something that that protozoan 00:16:47.01 is getting from that bacterium and vice versa. 00:16:50.22 So, there are associations between the microbes 00:16:52.29 that live in the gut, 00:16:54.23 and there are associations with those microbes and their host. 00:16:58.04 So, there are many levels of biological interactions 00:17:00.06 which are occurring in this environment. 00:17:03.03 Another example of a protozoan that has a bacterial association 00:17:10.16 is this organism, which is called Streblomastix strix. 00:17:13.22 The eukaryote, the single-celled protozoan, 00:17:17.10 is actually very, very slender 00:17:19.04 and running through the center of this. 00:17:21.06 That protozoan is covered with a blanket, or a coat, 00:17:25.06 of long, thin bacterial cells 00:17:28.07 that are creating those ridges that you see. 00:17:31.05 We know very little about the interaction 00:17:33.06 between this protozoan and the bacteria 00:17:35.09 and what they're doing for each other, 00:17:37.22 but clearly it's a very specific and interesting interaction. 00:17:42.06 There are many cases of protozoa 00:17:44.27 and the bacteria that occur in these environments, 00:17:47.11 of which we have much more to learn in the future. 00:17:52.04 Now, there's an opening line to a book 00:17:54.26 by the biophysicist Howard Berg. 00:17:57.20 His book is called "Random Walks in Biology", 00:18:00.23 and the opening lines 00:18:03.11 are that biology is wet and dynamic. 00:18:06.10 What does that mean? 00:18:08.01 I've just showed you these still pictures, 00:18:10.02 but I think that the still pictures 00:18:12.12 don't do this environment justice. 00:18:14.25 Really, when you're looking at this environment live, 00:18:17.24 this is now some of that gut fluid 00:18:19.23 which has been diluted. 00:18:21.19 It's even more densely packed than this inside the termite, 00:18:24.02 but if you dilute that fluid and put it on a microscope slide 00:18:26.22 you see some of these protozoa 00:18:29.15 and coursing unamongst them, 00:18:31.15 lots of bacteria which are moving so quickly 00:18:33.22 you can barely focus on them. 00:18:36.12 I can just look at this forever, 00:18:38.26 and you can look at it using different types of microscopy 00:18:43.02 to show different details on some of these cells. 00:18:46.02 The point here is that 00:18:48.11 when I go to work every morning 00:18:50.04 I go to work in what I call a miniature Alice in Wonderland. 00:18:52.23 That it is, just from a naturalist's standpoint, 00:18:55.13 a very wonderful and diverse place 00:18:58.00 that begs lots of questions. 00:19:01.27 So, what is the interaction 00:19:03.29 that termites have with their gut microbes? 00:19:07.10 I want to give you an overview 00:19:09.08 of some of the major things 00:19:11.15 that we've learned over about the last 100 years 00:19:14.00 on the association between 00:19:16.19 the insect and its gut microbes. 00:19:19.00 Now, microbes have a huge challenge in life. 00:19:21.26 The challenge is, how do you eat something 00:19:24.02 larger than your head? 00:19:26.06 If you are the size of 00:19:29.18 one thousandths of a millimeter, 00:19:32.02 how do you gain access to nutrients 00:19:34.19 in a 2x4 or in a big log? 00:19:37.12 So, you have a really wonderful association 00:19:40.01 with an insect that has jaws and grinding mandibles, 00:19:43.12 which are very, very hard, 00:19:45.01 that can then take a large block of wood 00:19:47.09 and grind it into really small particles, 00:19:50.07 and then bring them into the gut 00:19:52.03 in a very controlled and wonderful environment 00:19:54.08 in which these microbes can thrive. 00:19:56.22 Now, in the hindgut, these protozoa that I showed you 00:20:01.03 have enzymes of their own, 00:20:03.02 and enzymes that they recruit from the insect, 00:20:05.14 to start breaking down the polysaccharides in wood... 00:20:08.18 the cellulose and another component 00:20:10.23 which we call xylan or the hemicellulose... 00:20:14.18 and these protozoa perform a very unusual fermentation. 00:20:17.22 It's a fermentation that differs 00:20:20.00 from the one that you use to make sauerkraut 00:20:22.02 or that you use to make beer and wine. 00:20:25.00 What those protozoa do 00:20:27.02 is they break down the hexoses in cellulose, 00:20:29.28 primarily to acetate, 00:20:32.00 so, neutralized vinegar, 00:20:35.05 and that acetate builds up in the hindgut of the termite 00:20:37.22 and is absorbed by the insect. 00:20:39.29 So, the insect is actually absorbing the acetate 00:20:43.21 and using it as its biofuel. 00:20:46.01 It is the source of carbon for the insect 00:20:48.19 and the source of energy for the insect. 00:20:53.00 Now, those protozoa 00:20:55.07 also produce hydrogen gas, 00:20:57.21 so think the 1930s, 00:20:59.27 this classic picture of the Hindenburg blimp 00:21:01.26 over New Jersey, blowing up in fire. 00:21:04.10 It was filled with hydrogen. 00:21:06.01 There's a lot of energy in hydrogen. 00:21:07.21 It's not just combustible; 00:21:09.24 it's an energy source that can be used by different microorganisms. 00:21:13.04 So, in the termite, 00:21:15.10 and in many environments which are non-marine 00:21:17.16 and devoid of oxygen, 00:21:19.17 hydrogen and CO2 is converted into methane 00:21:23.08 by a group of organisms called methanogenic archaea, 00:21:26.29 and this methane is emitted by the insect. 00:21:30.00 It is sort of lost calories. 00:21:31.24 So, as we know, we can burn methane, 00:21:33.24 we use it as a fuel, 00:21:35.24 and that methane which is emitted by the insect, then, 00:21:38.22 is a fuel, a potential energy source, 00:21:40.21 which is lost from the system. 00:21:43.07 So, we can use a form of microscopy 00:21:46.07 to observe these methanogenic archaea in the termite. 00:21:50.07 Several years ago, I was trying to find out, 00:21:54.07 where are those archaea present in the system? 00:21:56.09 And what I learned is that 00:21:59.06 they are colonizing the gut wall of many... 00:22:03.21 inside the gut wall of many termites. 00:22:05.18 So, if you dissect out the gut, 00:22:07.29 you cut open the gut, you wash away all the contents, 00:22:11.05 and you sort of open that up 00:22:13.00 and look at the internal surface of it, 00:22:15.09 you can look for a type of fluorescence 00:22:19.08 called F420 fluorescence. 00:22:21.20 These organisms that make methane contain a vitamin, 00:22:24.04 and when you shine UV light on that vitamin 00:22:26.13 the vitamin turns blue. 00:22:28.16 And so, with a proper microscope, 00:22:30.29 you can start to see a number of different cell types, 00:22:33.18 which are blue, 00:22:35.18 that live on the inside of this gut wall. 00:22:38.12 And, in this image you see that there are 00:22:40.13 three different morphologies of organisms. 00:22:42.10 Now, I'm somebody who likes food, 00:22:43.28 so I like to say that this one long one 00:22:45.29 looks like long, blue spaghetti, 00:22:47.25 the curves rods look like basmati rice, 00:22:49.28 and some of these straight rods look like regular rice. 00:22:53.06 Now, termites emit up to 4% of global methane every year. 00:22:57.03 So, by studying these organisms in their environment, 00:23:00.00 and also culturing them and bringing them into the lab, 00:23:02.18 we can put a face on the process, 00:23:06.00 at a single microbial cell level, 00:23:08.19 for actually a very significant source of global methane... 00:23:12.17 not the most significant source of global methane, 00:23:15.25 but a small but significant source. 00:23:19.28 I like to show this slide, 00:23:23.01 of a paper mache cow, 00:23:25.01 because I knew that when I first got to Caltech 00:23:27.00 I was having some impact on undergraduate life there, 00:23:30.02 because when I talked about termites 00:23:32.22 and processes that occur in a cow 00:23:34.22 the next Caltech Ditch Day, 00:23:36.26 that occurs every spring, 00:23:39.06 the students had made this large paper mache cow 00:23:41.17 and filled it with chocolate pudding, 00:23:44.08 Easter grass, oatmeal, 00:23:47.06 and Easter eggs that were filled with clues 00:23:49.12 on where the students should go to their next puzzle. 00:23:53.21 And, you can see here, 00:23:55.23 the students trying to find those eggs. 00:23:57.28 The point I want to make here is that 00:24:01.11 cows lose about 20% of their electrons in their food 00:24:05.05 as methane. 00:24:07.06 It's a huge waste of energy. 00:24:09.09 And, although termites contain methanogens, 00:24:11.19 and emit methane, 00:24:13.25 it's only a very small amount of this hydrogen and CO2 00:24:16.25 that is lost to the system as methane. 00:24:19.11 On a global scale, it's significant, 00:24:21.22 but, actually, on a global scale, 00:24:23.22 the methane emission by termites 00:24:25.09 would be much more significant 00:24:28.03 if this hydrogen and CO2 was not being consumed 00:24:30.21 by a different group of organisms 00:24:33.14 which we call CO2-reducing homoacetogens. 00:24:36.17 So, many termites contain microbes 00:24:38.28 that completely push these methane organisms 00:24:41.19 out of the picture, 00:24:43.22 or push 90% of them out of the picture. 00:24:47.11 So, many termites will take... 00:24:49.20 have microbes that convert hydrogen and CO2 into acetate, 00:24:52.19 and this acetate then goes into that pool in the gut 00:24:56.05 and is absorbed by the insect. 00:24:58.10 So, up to a third to a fifth 00:25:01.01 of the acetate which is used as the biofuel 00:25:03.22 by these insects 00:25:05.27 is derived from carbon dioxide and hydrogen 00:25:09.02 by way of the activity of those protozoa, 00:25:11.11 and by way of the activity 00:25:14.00 of these organisms here. 00:25:16.02 So, I have long been interested 00:25:18.00 in the interaction between organisms competing 00:25:20.24 for this hydrogen and CO2 00:25:22.26 that make acetate and that make methane 00:25:25.26 from those... 00:25:28.02 to understand how they compete with each other, 00:25:30.01 how has this process come to pass in termites, 00:25:33.25 why doesn't it occur in the cow rumen, 00:25:35.29 and how do these hydrogen consumers 00:25:38.06 interact with the organisms which are producing the hydrogen, 00:25:40.18 the protozoa. 00:25:44.16 So, CO2-reductive acetogenesis 00:25:47.20 is a bacterial activity. 00:25:51.27 The process involves 00:25:54.13 the fixation of two molecules of carbon dioxide... 00:25:58.11 one, two... 00:26:00.26 with four molecules of hydrogen... 00:26:03.20 one, two, three, four... 00:26:08.09 and in the process, those two carbons are joined 00:26:11.07 and reduced to form the acetate, 00:26:13.15 and this metabolism 00:26:15.28 actually yields energy for the bacteria 00:26:17.24 which are performing it, 00:26:19.22 in addition to yielding the acetate 00:26:21.10 which can be used by the insect. 00:26:22.10 So, it's a mutually beneficial metabolism 00:26:26.08 that takes hydrogen produced during this fermentation, 00:26:28.18 turns it into additional fuel for the insect, 00:26:30.25 meanwhile supporting the activity of the bacteria that perform it. 00:26:35.22 But, for years, 00:26:37.19 we did not have a very good understanding 00:26:40.03 about what bacteria in the termite 00:26:42.19 are actually catalyzing this process. 00:26:44.09 We had some ideas, but, over the years, 00:26:47.06 we've been trying to learn more. 00:26:49.10 Now, if you look in the hindguts of termites 00:26:51.23 you'll even see, on some of these protozoa, 00:26:55.05 that there are very abundant spiral-shaped organisms, 00:26:57.24 which can be attached to the protozoa 00:26:59.28 and that can also be seen 00:27:03.10 living and swimming amongst the protozoa, 00:27:06.03 and in most termites these organisms we call spirochetes 00:27:09.10 are some of the more abundant bacteria 00:27:11.11 that you'll see swimming in and amongst these protozoa. 00:27:14.28 If you go to another portion of the gut, 00:27:17.09 maybe you'll see that there are even more 00:27:19.14 of these spiral-shaped organisms. 00:27:21.13 So, starting in the 1990s, 00:27:23.17 scientists at Michigan State and in Germany 00:27:27.02 discovered that these spirochetes 00:27:29.19 are actually very closely related to Treponema pallidum. 00:27:33.28 That's one of the most famous organisms in microbiology. 00:27:37.03 It's what causes syphilis. 00:27:39.11 Actually, all these bacteria in the termite 00:27:42.16 are species that belong to the same genus 00:27:46.13 as the agent of syphilis, 00:27:48.13 and yet you always find these organisms 00:27:51.12 present in happy and healthy termites. 00:27:53.28 But we didn't know what they did because, like syphilis, 00:27:57.13 they had never been cultured in vitro. 00:27:59.28 First observed in the 1860s, 00:28:01.21 over a century went by 00:28:03.26 before we had actually learned about what any of these do, 00:28:06.07 and I would still argue that we are still in our infancy 00:28:09.04 of understanding what the full breadth of the different roles 00:28:11.25 of the hundred or more species of spirochetes 00:28:14.12 that you can see in an individual termite hindgut. 00:28:18.21 But, a number of years ago, 00:28:20.27 I really endeavored for a very long period of time 00:28:23.04 to try to coax one or two of these species 00:28:26.11 into laboratory culture so that we could ask what they do, 00:28:29.12 and I had an idea: 00:28:31.19 maybe some of these are these acetogens 00:28:33.12 that can take hydrogen and CO2 00:28:35.07 and make acetate. 00:28:37.01 The problem with that is that activity 00:28:39.05 was not known to occur in any spirochete, 00:28:42.08 and none of these spirochetes from the termite 00:28:44.07 had been cultured. 00:28:46.01 So, it's an idea, but what you really need to do 00:28:47.24 is get one of these into the laboratory and ask it, 00:28:50.22 are you capable of doing that? 00:28:52.08 And, if not, what do you do? 00:28:55.20 So, what is a spirochete? 00:28:57.25 This is a political cartoon from the early 70s, 00:29:00.29 and will sort of shoot right over the heads 00:29:04.02 of almost all of us, 00:29:05.27 but I still include it because it's a little bit 00:29:08.21 of American history that Richard Nixon's first vice president, 00:29:11.23 Spiro T. Agnew, 00:29:14.15 sort of had to leave office for some misdealings that he had, 00:29:19.16 and this was before even the Watergate scandal blew up. 00:29:22.25 So, when I say spirochete, 00:29:24.26 I'm not talking about a spirochete, 00:29:27.21 but a different organism, 00:29:30.15 and these are bacteria 00:29:33.17 that have a very unusual body plan. 00:29:35.28 So, many bacteria can swim 00:29:38.04 and they have flagella that extend into the extracellular milieu 00:29:42.10 and act like propellers, 00:29:44.25 but spirochetes have flagella 00:29:46.17 that extend not out of the cell, 00:29:49.14 but out past the first membrane, 00:29:52.21 but lie in between the inner and the outer membrane 00:29:55.03 of the bacterial cell, 00:29:58.27 and actually will wrap around the cell, okay? 00:30:02.12 If you look at a cross-section, you can see what I mean. 00:30:05.24 These are the flagella that lie 00:30:08.06 in between the inner and the outer membrane, 00:30:10.04 and when those flagella turn 00:30:12.05 the entire cell becomes a propeller, 00:30:14.12 as opposed to being attached to the propeller, 00:30:17.01 and spirochetes are known to be able to move 00:30:19.11 into very thick, viscous environments, 00:30:22.02 and are sort of the world record holders in the microbial world 00:30:25.07 for being able to wiggle into really thick and tight places. 00:30:29.12 And, all the organisms that have this body plan 00:30:31.10 are also related to each other, 00:30:33.09 so it's both a related group 00:30:35.19 by genetics and by their body plan. 00:30:40.01 So, these are microscopic images 00:30:42.06 of the first termite gut spirochete that was isolated. 00:30:45.29 We call this organism Treponema primitia, 00:30:49.01 and the first thing we wanted to ask 00:30:51.21 was whether it was really a spirochete, 00:30:54.06 and what I want to point out here is that 00:30:56.09 if you look at it with a whole-cell negative stain 00:30:58.05 by transmission electron microscopy, 00:31:00.05 or a thin section, 00:31:02.02 it has these hallmark flagella 00:31:05.18 that are lying in between the inner and the outer membrane. 00:31:08.23 Now, the second thing we learned about Treponema primitia 00:31:11.28 is that it is actually a hydrogen+CO2 acetogen. 00:31:15.11 Hydrogen stimulated its growth 00:31:18.01 and it consumed hydrogen and made acetate 00:31:21.18 in the expected four hydrogen to one acetate stoichiometry. 00:31:25.26 You could also grow this organism 00:31:27.27 under radioactive carbon dioxide, 00:31:29.29 and when you do that 00:31:32.07 it generates radioactive acetate, 00:31:34.21 so it's fixing CO2 into organic carbon, 00:31:39.02 and when you ask, 00:31:41.19 are both carbon positions of acetate labeled? 00:31:44.14 They were. 00:31:46.10 And lastly, there are enzymes associated with this pathway, 00:31:49.17 and this organism exhibits them all. 00:31:52.03 So, it is a bonafide hydrogen/CO2 acetogen 00:31:55.12 and, although a close relative 00:31:57.07 of the organism that causes syphilis, 00:31:59.08 this organism actually plays a key role 00:32:01.17 in the fermentation of food in the termite, 00:32:04.03 and in taking some nutritional value of that wood 00:32:07.04 and passing it back on to the termite. 00:32:10.06 Now, we have been studying this organism 00:32:12.22 for almost 20 years now, 00:32:14.21 and one of the things that we've done 00:32:16.28 is really looked at its genes for this pathway, 00:32:19.10 and used our study of these genes, in red, 00:32:23.15 to do comparative studies in other termites 00:32:26.07 and also in this particular termite and ask, 00:32:29.29 can we learn if this is the only acetogen, 00:32:33.18 or if there are other species, 00:32:35.13 and who are those other species? 00:32:37.25 So, we can take an approach, now, 00:32:39.28 where we can take a look at the diversity 00:32:42.16 of these genes for this pathway in this one termite, 00:32:45.23 but also in members of these two other major subgroups 00:32:48.29 of the termite line of descent, 00:32:51.11 and also in the Wood Roach. 00:32:53.11 And so, we've been learning a lot 00:32:55.16 about the diversity of organisms 00:32:57.18 that can carry out that metabolism 00:32:59.24 in a diversity of different species 00:33:02.25 and actually major subgroupings of insects that eat wood. 00:33:07.25 Now, we've also been able 00:33:11.29 to isolate a second spirochete from Zootermopsis. 00:33:15.04 This one we call Treponema azotonutricium. 00:33:18.28 Now, this organism 00:33:21.06 plays a very different role in the symbiosis 00:33:23.08 with the termite and its hindgut microbes. 00:33:26.01 So, if you think about it, 00:33:28.02 wood is not only tough to eat, 00:33:30.23 it's not a very good source of protein. 00:33:33.10 You know, at best, it's like a potato, right? 00:33:36.29 It's got a lot of polysaccharide, 00:33:38.23 a lot of carbs, 00:33:40.25 but not a lot of nitrogen. 00:33:43.00 So, you can be degrading that wood 00:33:45.06 and providing the calories to the host, 00:33:48.01 but that's only one of the hosts major problems in life. 00:33:50.26 The other problem is to make protein, 00:33:54.04 and so if you ask what's going to 00:33:56.08 limit the ability of this insect that's eating a block of wood, 00:33:58.27 or your home, 00:34:00.25 what's going to limit its proliferation, 00:34:03.15 part of the story is on protein. 00:34:06.10 So, it turns out that some 35 years ago 00:34:10.06 John Breznak, at Michigan State University, 00:34:13.10 discovered that termites contain microbes 00:34:15.25 that can take atmospheric nitrogen, 00:34:17.25 which is bathing all of us in the atmosphere all around us, 00:34:21.01 and can take that and turn it into protein, 00:34:24.09 that can then be fed to the insect. 00:34:27.18 And, this particular spirochete, 00:34:29.26 when we got it into culture in the laboratory, 00:34:32.19 we could show can do that same activity. 00:34:35.28 So, this is one of the organisms that we say 00:34:38.17 can exhibit diazotrophic growth. 00:34:40.18 It can grow with N2 gas 00:34:43.11 as its sole source of eventual protein, 00:34:46.08 and it shows several activities 00:34:48.17 which are associated with that activity, 00:34:51.04 and therefore it's playing a role 00:34:53.13 in taking a very abundant but unusable source of nitrogen 00:34:56.01 around the insect 00:34:57.25 and actually feeding the insect protein-level nitrogen. 00:35:01.29 I'll mention too that this particular organism 00:35:04.29 is unlike the first. 00:35:06.24 It's not an organism that can consume hydrogen 00:35:10.20 and fix CO2 into acetate. 00:35:13.07 It actually degrades sugars 00:35:15.14 and produces hydrogen that it can feed to the other spirochete. 00:35:18.19 So, it plays different roles in this symbiosis. 00:35:23.05 So, I've talked to you about some protozoa, 00:35:28.13 about some methanogenic archaea, 00:35:32.20 and about just some of the many, many bacteria 00:35:35.09 that you can find in a termite. 00:35:38.02 There are many other stories I could tell you, 00:35:40.05 but I want to leave off with my talk 00:35:41.27 by just pointing out that 00:35:46.06 we've talked about these three major groups in this environment. 00:35:49.15 That environment is dominated 00:35:51.11 by members of diverse groups 00:35:53.11 which are very different from the host itself. 00:35:55.28 So, just like the kelp forests, 00:35:57.23 these are environments 00:35:59.28 which are dominated by genetic groups 00:36:02.01 and groups performing physiologies 00:36:03.26 which are very different from sort of the paradigms of biology, 00:36:07.01 and that we learn a lot by studying them. 00:36:09.27 So, I think with that, 00:36:11.15 I'll close this introductory talk 00:36:14.08 on termite gut microbiology. 00:36:16.15 There are many, many general aspects 00:36:18.14 that we could discuss, 00:36:20.00 and of course can go into many other aspects 00:36:22.08 in great detail. 00:36:23.20 And so, I'd like to point out that 00:36:25.28 there have been many research groups and scientists 00:36:28.07 that have been working on termites for well over a century, 00:36:30.25 and I've tried to encapsulate some of their findings, 00:36:33.00 as well as the findings of my own laboratory, 00:36:35.21 into the presentation that I have given you today. 00:36:38.16 Thank you very much.