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.