Session 9: Coevolution

00:00:08.00 My name is Margaret McFall-Ngai
00:00:09.14 and I'm a professor
00:00:11.14 and the Director of the Pacific Biosciences Research Center,
00:00:13.14 School of Ocean and Earth Science and Technology,
00:00:16.07 the University of Hawaii at Manoa.
00:00:18.12 I'd like to start my lecture here
00:00:19.28 with thanking the iBiology team
00:00:21.22 for this opportunity to speak to you today
00:00:24.24 about the field of symbiosis.
00:00:27.12 So, the field of symbiosis is about living together
00:00:29.22 -- it's host-microbial interactions
00:00:32.15 that I will be talking about specifically today.
00:00:34.27 And there's a bit of a revolution in biology.
00:00:38.23 Symbiosis and host-microbial interactions
00:00:41.09 are taking center stage in biology.
00:00:46.11 And I'm going to talk to you today about why this is happening now
00:00:49.04 and exactly why we're sort of in the center of a revolution.
00:00:52.09 So, I'm going to start by talking about
00:00:54.20 the words that we use to describe symbiosis.
00:00:57.11 So, this person right here is
00:00:59.29 Heinrich Anton de Bary
00:01:02.16 and this particular person
00:01:04.25 lived in the 19th century,
00:01:07.04 and at the end of the 19th century
00:01:08.15 he coined the term symbiosis.
00:01:11.21 And the definition that he gave to this term
00:01:13.24 was the living together of unlike organisms.
00:01:16.23 And this particular definition of symbiosis
00:01:21.00 has remained with the field
00:01:22.19 until the present day
00:01:24.20 -- it's a very, very, very general way,
00:01:27.18 but very useful way
00:01:29.24 to think about this particular phenomenon in biology.
00:01:34.00 So, that said,
00:01:37.07 symbiosis is a bit of a catch-all term
00:01:39.05 with no information
00:01:40.19 concerning the effect of fitness.
00:01:42.22 So, what I'm showing up here is
00:01:45.06 I'm showing a very classic symbiosis
00:01:46.21 that a lot of people know of
00:01:49.28 and a lot of people think about,
00:01:51.18 and this is the symbiosis between
00:01:53.09 a clownfish and an anemone,
00:01:55.08 and they live together, persistently,
00:01:57.19 for much of their lives,
00:01:59.07 and it's a classic symbiosis
00:02:01.27 that you can think about.
00:02:03.00 But like I said, the word itself
00:02:05.05 confers no... no...
00:02:09.22 it's a catch-all word with no information
00:02:11.22 concerning the effect on the fitness
00:02:13.19 of either partner.
00:02:14.28 And so first I want to describe
00:02:17.18 what fitness means.
00:02:18.20 So, fitness is an individual's reproductive success,
00:02:23.10 and that is the number of offspring
00:02:25.10 that an individual leaves to the next generation.
00:02:28.06 And so let's think about this idea
00:02:32.14 in terms of symbiosis.
00:02:33.23 So, when we talk about fitness,
00:02:35.23 when we're talking about symbiosis,
00:02:37.14 I think there are...
00:02:39.02 I'm going to consider that there are two partners
00:02:41.03 -- there's symbiont #1 and symbiont #2 --
00:02:42.17 and in a mutualism,
00:02:44.25 which is one of the types of symbiosis,
00:02:46.17 both partners will benefit,
00:02:49.12 and what that means is that both partners
00:02:51.07 will have more in the successive generations
00:02:57.02 from being a partner with the other.
00:02:58.11 And so in the case of the clownfish
00:03:00.17 and the anemone,
00:03:02.03 the clownfish gets a very safe place to live,
00:03:04.07 because the tentacles of the anemone
00:03:06.03 are harmful to other animals;
00:03:07.20 the fish, the clownfish,
00:03:10.28 has learned ways to avoid the danger
00:03:13.08 of living in the tentacles of this anemone;
00:03:15.23 and the anemone gains from having the fish there
00:03:20.25 because the fish releases ammonia
00:03:22.17 that is taken up by the anemone
00:03:24.16 and it's used in the energy production
00:03:28.01 of the anemone.
00:03:30.27 So, this is a true mutualism.
00:03:32.24 The next type of symbiosis I'm going to consider is commensalism.
00:03:36.13 In a commensalism,
00:03:38.14 one partner benefits and one partner is unharmed
00:03:41.19 -- there's no change in the fitness,
00:03:43.25 no change in the reproductive fitness.
00:03:46.29 So, what I'm showing here
00:03:48.25 is another example and this example, here,
00:03:51.05 is the shark/pilot fish example,
00:03:54.21 and in this particular case
00:03:58.15 what happens is is
00:04:01.25 the shark is a messy eater
00:04:03.21 and he eats fish and all sorts of various things
00:04:07.11 and food is released around him
00:04:10.20 and the pilot fish that are associating with the shark
00:04:14.24 take advantage of the fact that the shark is a messy eater.
00:04:18.22 In this case, the pilot fish
00:04:22.01 gained from living with the shark,
00:04:23.12 but it seems to have no effect on the shark.
00:04:26.02 And so in this case, it's very likely that
00:04:28.07 the pilot fish will leave more to the next generation
00:04:30.07 from living with the shark,
00:04:32.10 but the shark...
00:04:34.05 it won't affect the shark's fitness whatsoever.
00:04:36.05 The third example that I'm going to give
00:04:38.28 is the example of parasitism.
00:04:40.26 So, these are the three major types of symbiosis,
00:04:42.27 and in parasitism what happens is
00:04:46.15 one partner benefits and one partner is harmed.
00:04:48.22 So, I'm showing down here a tree,
00:04:51.10 and this particular tree trunk
00:04:53.28 has a large gall on it,
00:04:56.07 and the large gall is present
00:04:59.19 because there is a pathogen that is living,
00:05:05.00 a microbial pathogen is living inside this gall
00:05:06.04 and has created this large gall on this tree.
00:05:11.00 Now, the pathogen, that microorganism that's living in that gall,
00:05:14.06 benefits from living...
00:05:15.23 from parasitizing this tree.
00:05:17.29 The tree on the other hand
00:05:19.25 is harmed by this association,
00:05:21.10 so this is a classic parasitism.
00:05:23.24 So, one of the things I should mention is that
00:05:26.05 as the field has grown,
00:05:27.26 there's a little bit of misuse of these terms,
00:05:32.04 and this is one of the reasons
00:05:34.11 why I wanted to bring this up.
00:05:35.25 The term commensalism
00:05:38.22 is often used by the biomedical community,
00:05:40.13 in my mind and the mind of a lot of people who study symbiosis,
00:05:43.26 incorrectly.
00:05:45.07 And what they'll say is that
00:05:48.29 the microbes in your gut,
00:05:50.14 which I'm going to talk about a little bit...
00:05:51.24 the microbes in your gut are commensal,
00:05:54.11 and what this means is that
00:05:56.19 they have no effect on the host
00:05:58.24 -- they're benefiting but have no effect on the host.
00:06:01.14 But now we know that
00:06:03.29 they have a tremendous effect on the host
00:06:05.22 and are very often very important for our health,
00:06:10.01 and beneficial.
00:06:11.14 There are some in there that are likely commensal,
00:06:12.25 there are some in there that are likely
00:06:15.01 budding pathogens,
00:06:16.17 there are some in there that are likely,
00:06:18.00 or many in there, that are likely mutualists,
00:06:19.25 but we really can't categorize them,
00:06:22.14 so the very best thing to do in the instance
00:06:25.04 where you really don't understand
00:06:27.07 what type of symbiosis it is
00:06:29.19 is to just call it a symbiosis
00:06:31.19 and to call the partners a symbiont.
00:06:34.11 So, let's consider for a second
00:06:37.13 host-microbe symbioses.
00:06:38.29 So, what we're talking about is we're talking about
00:06:41.11 viruses, bacteria, protists
00:06:43.13 -- or single-celled eukaryotic cells --
00:06:45.02 and fungi, living with animals and plants, basically.
00:06:48.18 So, the question is,
00:06:51.02 is this a rarity?
00:06:52.21 And I have to say that when I started my career
00:06:54.09 working in this field,
00:06:56.23 back in the early 1980s,
00:06:58.12 this was considered a very unusual feature of symbiosis...
00:07:02.20 symbiosis was considered an unusual feature
00:07:04.19 in the biological world.
00:07:06.16 And in fact, if you look at a textbook,
00:07:09.03 even today,
00:07:11.00 you will see that symbiosis is covered,
00:07:13.16 but it's covered in only a few pages
00:07:15.10 of a 1200-page textbook.
00:07:17.13 It was and is today still considered a rarity,
00:07:20.15 but I hope you convince you that
00:07:23.00 it's not a rarity at all.
00:07:24.26 But why was it considered a rarity?
00:07:26.26 Well, we would find it...
00:07:28.29 historically, we were looking at
00:07:31.01 unusual situations in things like
00:07:34.14 the hydrothermal vent symbioses in the deep sea.
00:07:38.10 Down at 2000 meters, you know,
00:07:39.21 you see these smokers down at 2000 meters,
00:07:42.01 and these particular smokers
00:07:44.11 would have associated with them
00:07:46.16 these very large tube worms,
00:07:49.00 and these large tube worms
00:07:51.01 had in association with them,
00:07:53.29 bacteria, and these bacteria
00:07:55.28 allowed these particular tube worms
00:07:57.27 to live on the chemical energy
00:08:00.12 that was provided.
00:08:02.02 So that was a true symbiosis
00:08:04.16 that's been studied by lots and lots of people.
00:08:10.01 Bioluminescence is another kind of symbiosis
00:08:14.13 and very many organisms who are bioluminescent,
00:08:17.16 not all, but many organisms that are bioluminescent,
00:08:20.05 are bioluminescent because they harbor
00:08:22.18 luminous bacteria in association with the organism.
00:08:27.04 So, in the case of this anglerfish,
00:08:28.12 up here in the angle
00:08:30.15 is a pure culture of a particular bacterial species,
00:08:33.26 and that allows the anglerfish
00:08:36.21 to use that light in its predation.
00:08:40.09 And then, lastly,
00:08:42.10 I'm going to give the example of coral reefs.
00:08:44.24 Coral reefs would not form
00:08:47.04 if they did not have a very...
00:08:49.17 a mutualistic symbiosis
00:08:51.04 with a unicellular organism called
00:08:53.29 zooxanthellae.
00:08:55.06 And these zooxanthellae live inside of the cells of corals
00:08:58.03 and they provide them with the photosynthate.
00:09:00.29 So, these zooxanthellae
00:09:06.03 are capable of photosynthesis
00:09:08.00 and they translocate the photosynthate to the host.
00:09:10.26 And so the zooxanthellae get a place to live
00:09:14.02 and the host coral is given the photosynthate,
00:09:18.27 and so they both benefit.
00:09:20.07 So, in all the cases I'm showing on this slide,
00:09:22.13 these are mutualistic symbioses,
00:09:24.26 and they're ones that have been
00:09:28.05 studied for over 100 years...
00:09:31.18 actually, not this one,
00:09:33.00 this one they discovered in the late 1970s,
00:09:34.10 but the many, many symbioses
00:09:36.07 that are mutualistic like this,
00:09:38.05 and kind of unusual,
00:09:39.21 have been studied for decades.
00:09:41.12 But now we're finding out that
00:09:43.13 nearly all animals and plants
00:09:45.20 are likely to have beneficial symbioses.
00:09:47.28 So, this is a whole new area,
00:09:51.27 this is a whole new finding,
00:09:53.17 and this new information
00:09:56.23 is from the last two decades of work.
00:09:58.29 And so I'm just showing
00:10:01.25 a whole array of animals
00:10:04.00 that are now known to have
00:10:05.27 beneficial symbioses with microbes.
00:10:10.29 So, I want to just take one minute
00:10:13.10 to pay tribute to what I...
00:10:15.22 the person who I consider
00:10:17.23 the mother of the field of symbiosis,
00:10:19.22 and this is Lynn Margulis.
00:10:21.07 And Lynn Margulis lived 1938-2011,
00:10:23.27 and she was the person
00:10:26.12 who felt that symbiosis was a major driver
00:10:28.17 in the evolution of animals and plants,
00:10:30.19 and she was always way ahead of her time.
00:10:33.20 She became famous f
00:10:35.19 or the endosymbiotic theory
00:10:38.11 of the origin of the eukaryotic cell,
00:10:39.17 and that is to say that bacteria
00:10:41.05 became symbiotic with other bacteria
00:10:43.03 and made a more complex cell.
00:10:45.14 And she showed that at the end of the 1970s,
00:10:50.06 it was very, very controversial,
00:10:52.00 she was way ahead of her time and always was,
00:10:54.09 but she is actually the person
00:10:57.07 who had the vision to say
00:11:00.19 exactly what we're seeing today,
00:11:02.04 and that is that symbiosis
00:11:04.02 is a major thing in biology
00:11:05.16 and has likely been over evolutionary history.
00:11:10.01 So, like I said, nearly all animals and plants
00:11:13.01 are likely to have beneficial symbiosis
00:11:16.05 and this is new information
00:11:18.12 from the last two decades of work.
00:11:20.12 And so what I'm showing here
00:11:22.27 is I'm showing a person,
00:11:24.09 and this person in this artistic rendering
00:11:27.18 is completely covered by microbes.
00:11:30.09 And we are, from the minute we're born,
00:11:34.00 through our life,
00:11:36.25 we associate very intimately with the microbial world.
00:11:39.06 And so we know now that you have as many, if not more,
00:11:42.25 microbial cells than human cells in your body.
00:11:45.04 So, you have about 10^13 human cells
00:11:47.08 and somewhere between 10^13 and 10^14 bacteria
00:11:50.00 that live with you, persistently,
00:11:52.26 your whole life and confer health.
00:11:57.06 So, the question is,
00:11:58.15 why didn't we know about this?
00:12:00.16 Why wasn't this more obvious?
00:12:03.07 Well, it turns out that there was
00:12:05.19 a huge technical problem
00:12:07.06 that we've been able to overcome.
00:12:09.05 And so, the technical problem
00:12:11.02 was a difficulty in identifying
00:12:14.15 and classifying the microorganisms,
00:12:15.17 and why we couldn't do that
00:12:17.15 was because most of these organisms
00:12:19.19 were unculturable under laboratory conditions,
00:12:22.19 and so they're called viable but nonculturable.
00:12:26.00 In other words, they live,
00:12:27.22 but we just can't bring them into the lab
00:12:29.28 and study them the way that we
00:12:33.13 would want to.
00:12:34.18 So, it's about less than 1% of the bacteria
00:12:38.15 that live in association with animals are culturable,
00:12:40.22 so this was a huge technical problem.
00:12:43.12 And so... because
00:12:45.17 we just couldn't know who they were.
00:12:47.10 The other thing was that...
00:12:50.15 another technical problem was that
00:12:53.12 they are relatively featureless.
00:12:55.12 So, what I'm showing here
00:12:57.17 is I'm showing a set of
00:13:00.12 different types of shapes and so on and so forth
00:13:02.27 that you might see
00:13:04.19 in different kinds of bacteria.
00:13:06.00 And so you might compare and contrast that
00:13:10.15 with the morphologies that you see of
00:13:13.09 very, very many plants and animals.
00:13:15.28 I mean, you can really, very well,
00:13:19.12 tell the differences between plants and animals;
00:13:21.21 with microbes, it's not so easy.
00:13:23.25 So, they weren't culturable
00:13:26.08 and you couldn't... they didn't...
00:13:27.26 they were pretty well featureless,
00:13:29.11 so these were big problems.
00:13:31.12 So, even though they're featureless,
00:13:33.06 we began to visualize microorganisms
00:13:36.03 back with Anton van Leeuwenhoek.
00:13:38.06 So, this is Anton van Leeuwenhoek
00:13:40.11 with, you know, several hundred years ago,
00:13:42.08 but he was a guy who
00:13:44.23 developed the very first microscopes,
00:13:47.06 and so he was the first person
00:13:49.26 who allowed us to visualize microorganisms.
00:13:51.16 And what Anton van Leeuwenhoek did was
00:13:54.15 he took a swab
00:13:56.19 and swabbed the inside of his cheek
00:13:58.10 and then he applied that to his microscope,
00:14:01.16 and he was able to see, for the first time,
00:14:05.00 microorganisms.
00:14:06.23 So, not only was he the first person to see microorganisms,
00:14:08.21 but he was the first person to see microorganisms
00:14:10.26 that associate with humans,
00:14:12.08 and those were the microorganisms
00:14:14.16 inside of his own mouth.
00:14:17.07 So, then we fast-forward
00:14:19.29 to the electron microscope,
00:14:21.27 and the electron microscope
00:14:23.21 was invented several hundred years later,
00:14:25.21 in the late 1960s,
00:14:27.28 and the invention of the electron microscope
00:14:31.02 allowed us to see much more detail in microorganisms.
00:14:34.17 And this was actually the microscope
00:14:37.10 that allowed Lynn Margolis,
00:14:39.22 who I spoke about earlier,
00:14:41.06 to visualize the complexity of the eukaryotic cell
00:14:44.04 and to resolve the fact that,
00:14:46.12 in fact, the eukaryotic cell
00:14:49.07 was the result of a symbiotic association
00:14:51.22 between microorganisms,
00:14:54.22 or among microorganisms.
00:14:57.02 So, then fast-forward a little bit more...
00:14:59.14 although...
00:15:01.22 so, these two characters,
00:15:04.26 Carl Woese and Norm Pace.
00:15:06.27 So, Carl and Norm were doing molecular biology
00:15:10.05 of microorganisms
00:15:11.20 at the University of Illinois...
00:15:13.22 well, Carl was at the University of Illinois
00:15:15.15 and he working with Norm Pace,
00:15:17.08 and they introduced the use of gene sequences
00:15:20.01 to determine relationships in the biological world.
00:15:22.08 So, notice that this is 1977,
00:15:25.04 so it's around the same sort of time that people
00:15:27.25 are beginning to think about using the microscope,
00:15:29.20 the electron microscope,
00:15:31.11 as a mechanism by which to classify the biological world,
00:15:34.26 but this was a new instrument
00:15:36.22 that Carl Woese and Norm Pace
00:15:38.24 introduced to the community of biologists.
00:15:42.08 So, the instrument was the 16S ribosomal RNA gene.
00:15:46.02 It's a highly conserved marker gene,
00:15:48.19 and the reason why this gene could be used
00:15:50.17 was because it's conserved throughout evolutionary history
00:15:53.12 and so it gives you an idea of
00:15:56.24 changes that are very, very old,
00:15:58.12 and would allow you to
00:16:00.21 classify all of the biosphere by molecular methods.
00:16:04.22 And this is the phylogenetic tree
00:16:07.26 that came as a result of the analysis
00:16:10.14 of the biological world.
00:16:12.07 And so what you'll see is you'll see that
00:16:16.15 there are the bacteria...
00:16:17.25 there are three domains of life
00:16:19.09 -- the bacteria, the archaea, and the eukaryea --
00:16:21.26 and the eukaryea contain
00:16:25.12 the animals, plants, and fungi.
00:16:27.09 And so, look at... the animals, plants, and fungi
00:16:30.12 are just these three twigs
00:16:32.03 at the very top of the tree of eukaryea,
00:16:35.21 which is, you know, is showing us that
00:16:39.02 the vast diversity of the biological world
00:16:41.13 is invested in the archaea and the bacteria.
00:16:44.27 So this was a huge change in our world view.
00:16:49.03 So, how big was this change?
00:16:51.20 Well, let's go all the way back to Aristotle.
00:16:56.07 So, Aristotle was one of the first people
00:16:58.08 to classify the biological world,
00:16:59.28 and what Aristotle did was
00:17:01.25 he classified it based on what he could see.
00:17:04.01 So it's animals and plants, basically.
00:17:08.18 So then, you know, fast-forward, as I mentioned,
00:17:11.09 to Anton van Leeuwenhoek,
00:17:13.03 who saw animals, plants, and what he called animalcules.
00:17:16.15 And then, in the 1970s,
00:17:20.20 there was a worker named...
00:17:24.24 who used the electron microscope,
00:17:26.14 his name was Thomas Whittaker,
00:17:27.23 and he developed something called the five kingdom model,
00:17:30.07 and so the five kingdom model
00:17:32.15 had sort of the featureless bacteria and archaea,
00:17:34.22 down here at the base,
00:17:36.11 the protists above that,
00:17:38.07 and those are the eukaryotic cells,
00:17:40.07 and then up at the top he had
00:17:42.24 the fungi, animals, and plants.
00:17:47.20 So, Carl Woese and Pace,
00:17:50.07 looking at this in context with their sequencing,
00:17:54.10 what they had was these three domains.
00:17:57.23 And remember that all, all of this,
00:18:02.05 up here at the top,
00:18:03.24 all of this stuff up here at the top
00:18:05.13 is now invested in this group here.
00:18:09.23 So, it's a huge change
00:18:11.23 in the way we see the biological world.
00:18:13.17 So, what have we done?
00:18:15.12 We have moved from having visual analysis...
00:18:19.02 for a couple thousand years,
00:18:21.22 we looked at the biological world
00:18:23.20 and we classified it based on what we could see,
00:18:27.00 and now what Carl Woese and Norm Pace did
00:18:30.27 was they gave us molecular analysis.
00:18:34.26 And molecular analysis is the... are the genes,
00:18:37.15 and that allowed us to very correctly
00:18:40.12 organize the biological world
00:18:43.23 as it actually exists.
00:18:45.23 So, what we found, of course,
00:18:47.06 is that the biological world
00:18:50.09 is mainly microbial,
00:18:52.11 and the animals, plants, and fungi
00:18:54.02 are but a small patina
00:18:57.07 on the top of the microbial world.
00:18:59.21 So, what did this revolution mean for symbiosis?
00:19:04.21 So, what it meant was that we could identify microbes
00:19:10.09 and determine their relationships,
00:19:12.21 among them and between them,
00:19:14.06 even those that we not culturable.
00:19:15.15 We could extract the DNA from them and say,
00:19:18.04 what is the DNA telling us about who they are and what they are doing?
00:19:21.13 But at first, with Carl Woese in the very, very beginning,
00:19:25.14 sequencing was excruciatingly slow and expensive.
00:19:29.20 And then, around 2006,
00:19:32.10 something called next-generation sequencing
00:19:35.05 came onto the scene,
00:19:36.21 and this has been
00:19:38.22 the most important thing of the whole revolution.
00:19:42.21 And imagine... this was only 10 years ago.
00:19:45.19 So, what happened at this point
00:19:47.29 was that there was a technology-enabled transformation
00:19:51.24 in our ability to sequence quickly and cheaply.
00:19:55.01 So, what I'm showing in this graph
00:19:57.05 is I'm showing the cost of sequencing per megabase...
00:20:02.18 so, you see, in 2001,
00:20:04.14 it was up at about 6000 dollars a megabase,
00:20:07.23 and it followed Moore's Law
00:20:10.03 -- the doubling in computer power every two years --
00:20:15.09 it followed Moore's Law for...
00:20:17.05 until about 2006, down here...
00:20:19.16 until about 2006,
00:20:21.25 and then next-gen sequencing was invented.
00:20:25.25 And look at what happened.
00:20:28.01 It went down from...
00:20:30.01 the cost went down from 600 dollars a megabase
00:20:32.06 to 35 cents in less than 10 years,
00:20:35.19 and now it's down to 3.5 cents.
00:20:38.13 And so this was incredibly enabling
00:20:41.05 to the community of biologists.
00:20:43.05 In other words, lots and lots and lots of people
00:20:45.23 had the resources
00:20:47.17 to be able to characterize the microbial world,
00:20:49.25 and so people have gone all around the world
00:20:53.18 characterizing the microbial world.
00:20:56.10 So, a whole frontier opens...
00:20:58.05 a whole frontier.
00:21:00.10 We can learn, who are the microbial partners
00:21:02.25 of animals and plants?
00:21:04.28 What are they doing?
00:21:06.11 How are they doing it?
00:21:07.18 And what is there importance to health and disease?
00:21:10.18 And so when you're thinking about a symbiosis
00:21:12.28 you think that many...
00:21:15.07 most of them are established new each generation.
00:21:17.19 Not all kinds are,
00:21:19.17 some of them the symbionts are passed
00:21:21.17 in or on the egg,
00:21:22.25 but most of them are established anew each generation,
00:21:25.14 just after birth.
00:21:27.02 And so when a baby is born,
00:21:28.27 what happens is at soon as
00:21:32.01 it goes through the mother's birth canal
00:21:34.12 it begins to acquire its microorganisms.
00:21:36.17 Then there's a development,
00:21:38.08 a trajectory that's not unlike
00:21:41.10 what goes on in the maturation of a forest
00:21:43.22 -- there's a succession of organisms --
00:21:46.05 and in humans you get
00:21:49.09 a mature set of microorganisms
00:21:51.01 somewhere between the ages of two and four.
00:21:53.26 And then what happens is you become a stable association,
00:21:55.29 that changes some over life
00:21:59.22 with various changes and aging and so on,
00:22:02.22 but it's a fairly stable population.
00:22:06.19 So there's a trajectory
00:22:08.00 and we're learning a great deal
00:22:10.16 about how all of this works at this point,
00:22:14.04 but it's new data...
00:22:15.23 I mean, it's just the beginning of the field.
00:22:18.01 What have we learned so far?
00:22:19.28 What we have learned so far is that
00:22:23.01 for many, many, many animals, including humans,
00:22:25.26 the complexity is absolutely daunting.
00:22:28.22 And what do I mean by that?
00:22:30.17 So, let's take humans for example.
00:22:32.12 So, what I'm showing here is I'm showing
00:22:34.11 my favorite comedian, Woody Allen,
00:22:36.09 and I've been very generous with him
00:22:39.25 -- I'm making him 6 feet tall
00:22:42.05 or a little bit over --
00:22:44.03 and so... but his,
00:22:46.14 if you compare the ratio is his size to a microbe's size,
00:22:48.26 2 meters to 2 microns,
00:22:50.22 so he's a lot bigger than a microorganism.
00:22:53.24 However, if you look at the number of genes
00:22:57.21 that Woody Allen has
00:23:00.19 compared to the number of genes that the microbes
00:23:02.18 associated with his body have,
00:23:04.05 the ratio is about 1:1.
00:23:07.18 If you look at cell number,
00:23:09.11 it's thought to be somewhere between 1:1 and 1:10.
00:23:13.11 In other words, there are about 10 times as many
00:23:15.29 microbes associated with you,
00:23:17.12 and that number has just this last year
00:23:20.11 become a little bit controversial,
00:23:21.24 so it's somewhere between here and here,
00:23:24.00 but that is to say that there are
00:23:26.25 just as many microbes associated with you
00:23:28.12 as you have human cells.
00:23:30.01 Then, if you look at gene diversity,
00:23:32.25 because instead of just...
00:23:34.09 so, you know, all of your eukaryotic...
00:23:36.14 all the human cells are of a single genome,
00:23:39.04 but the microbial cells are of
00:23:42.01 thousands, or hundreds if not thousands, of genomes.
00:23:46.07 The difference in gene diversity is thought...
00:23:48.22 for every single human gene
00:23:50.09 there are 200 genes of microbes,
00:23:53.11 so the gene diversity of microorganisms
00:23:55.09 is very much greater.
00:23:57.18 So at this point Woody Allen
00:23:59.22 feels pretty insignificant.
00:24:01.28 What I'm showing here is I'm showing the power of animal model systems
00:24:04.06 for the study of complex characters,
00:24:06.01 and the example that I'm using
00:24:08.07 is developmental biology.
00:24:09.23 And in development biology,
00:24:11.18 what I'm showing here is I'm showing
00:24:13.11 a fertilized egg,
00:24:15.22 and that fertilized egg goes from 1 cell to 10^13 cells
00:24:18.23 and produces something like
00:24:21.02 the miracle of George Clooney, my favorite actor.
00:24:23.14 So, this has been, this has been...
00:24:28.13 this approach has been extremely successful
00:24:30.18 to understand developmental biology.
00:24:34.10 So, what we've been able to do
00:24:36.29 is to take model systems,
00:24:38.15 you know, experiments that evolution has done,
00:24:40.14 and ask how, in this simple model system,
00:24:43.14 a particular developmental phenomenon
00:24:47.11 has been solved.
00:24:49.09 And so what I'm showing is
00:24:51.16 I'm showing an array of model systems
00:24:53.12 and I'm showing ones in which
00:24:55.27 Nobel Prizes have been awarded,
00:24:57.16 and it turns out that 6 Nobel Prizes
00:24:59.28 in developmental biology
00:25:01.14 have been awarded since 1995 and 2007,
00:25:04.06 and all 6 of them went to
00:25:07.24 individuals working on models.
00:25:09.09 So models have been an extremely valuable way
00:25:12.28 to study very complex characters.
00:25:15.09 So, because symbiosis
00:25:17.23 is such a complex character,
00:25:19.18 we feel, those of us in the field
00:25:22.03 who study models,
00:25:23.27 feel that it's a really good way to try to understand symbiotic associations.
00:25:28.10 So, we are developing,
00:25:30.02 the field is developing some models for symbiosis,
00:25:32.17 we're exploiting nature's toolkit
00:25:34.18 and, as I said,
00:25:36.22 evolution has done some enlightening experiments.
00:25:39.17 So what I'm showing here is I'm showing
00:25:41.27 a variety of them,
00:25:43.06 and so there are
00:25:45.05 various nematode symbioses
00:25:47.01 and various other invertebrate symbioses,
00:25:51.19 and we even have, over at UC Berkeley,
00:25:54.19 Nicole King studying the choanoflagellate symbioses,
00:25:58.13 at the base...
00:26:00.05 thought to be the base of the animal kingdom.
00:26:02.26 And then up here some vertebrate models.
00:26:06.05 Now, the vertebrates,
00:26:07.24 all the vertebrates have complex associations,
00:26:09.29 they have large consortia associated with them,
00:26:13.10 and so these people studying over here
00:26:15.10 are really interested in studying germ-free animals,
00:26:18.17 and so I sort of look at them as
00:26:20.21 engineered models.
00:26:22.10 Over here, many of them are studying
00:26:24.19 natural models
00:26:26.16 and these, the invertebrates are
00:26:28.21 naturally very simple associations,
00:26:33.01 and so they lend themselves to looking at a natural situation
00:26:35.12 and how that...
00:26:37.12 how the bacterium and the host animal get together
00:26:40.22 and maintain a stable association.
00:26:43.22 So, I...
00:26:46.17 my lab works on this beautiful animal, here,
00:26:48.24 the bob tail squid.
00:26:51.14 This is not a giant's hand, this is my technician's hand.
00:26:54.08 A very small squid that's indigenous
00:26:56.21 to the Hawaiian archipelago,
00:26:58.25 and this is the symbiosis that I've been working on
00:27:02.10 and that I would like to tell you about
00:27:04.10 in my next lecture.
00:27:07.02 Thank you very much.

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.

00:07.2 My name is Jim Estes,
00:09.2 I am a professor of ecology and evolutionary biology
00:11.1 at the University of California in Santa Cruz,
00:13.1 and I'm very happy to be here today
00:16.0 to be talking to you about the ecological function
00:19.0 of apex predators in nature.
00:20.2 In Part I of my lecture,
00:22.2 I provided a conceptual foundation
00:24.2 for what the questions are
00:26.2 and how we have gone about trying to answer those questions.
00:28.3 In Part II, I'm going to give you
00:30.3 a particular case study.
00:32.1 I'm going to focus on sea otters and kelp forests,
00:34.1 and I'm doing this because
00:37.3 this is work that my lab been engaged in
00:39.2 for the last several decades.
00:43.1 So, this is an outline for Part II of the lecture.
00:46.1 Firstly, I'm going to explain or describe to you
00:50.1 the food webs, the key species.
00:52.1 Second, I'm going to tell you a little bit about the approach
00:55.0 that we have used to understanding the importance
00:58.1 of sea otters in these kelp forests ecosystems.
01:00.2 And then, lastly, I'm going to run through some of the findings
01:02.2 from the work that we have done.
01:05.2 The food web: sea otters are the apex predator
01:09.0 in many coastal kelp forest ecosystems
01:11.1 in the North Pacific.
01:13.3 Sea otters feed on sea urchins;
01:15.1 sea urchins feed on kelp;
01:17.0 kelp is what we call a foundation species,
01:18.3 that is, many other species in the ecosystem depend upon it,
01:22.0 either for habitat or for food or for primary production,
01:25.3 so many other species are linked into the ecosystem
01:28.3 by way of the kelps themselves.
01:32.0 The approach:
01:34.0 how have we gone about trying to understand
01:35.2 the importance of sea otters
01:37.1 in these coastal kelp forest ecosystems?
01:39.2 It's actually very simple.
01:41.1 We have used history as
01:44.2 a large natural experiment.
01:46.1 So, the blue line in this illustration
01:48.3 shows the range over which sea otters
01:51.2 historically have occurred in the North Pacific Ocean.
01:54.1 They were abundant across that range
01:57.1 until about the mid-1700s.
02:00.0 In the mid-1700s, the Pacific Maritime Fur Trade began
02:05.1 with the discovery of the New World by the Russians
02:08.1 and the discovery of vast fur resources,
02:11.1 and in particular sea otter resources, across the North Pacific,
02:13.2 and that began a long period of overexploitation
02:17.3 and extinction of sea otters throughout most of their range.
02:23.1 This illustrates the nature of the Pacific Maritime Fur Trade
02:27.1 and emphasizes that the Pacific Maritime Fur Trade
02:30.2 was the perturbation that we have used
02:33.0 to understand dynamic processes in this system.
02:36.2 The blue line is shown again on this slide,
02:38.3 that shows the historical range of sea otters,
02:41.0 and the little red dots are the locations
02:43.2 of known surviving colonies at the end of the fur trade,
02:48.1 which was over in the early part of the 20th century.
02:51.1 At the beginning of the fur trade,
02:53.0 there were probably at least a million sea otters
02:55.1 across the North Pacific Ocean, possibly many more.
02:58.1
03:00.2 By the end of the fur trade, there were well under a thousand,
03:03.1 and those few remaining animals occurred
03:06.1 in a couple of isolated colonies.
03:08.0 These colonies were protected by international treaty in 1912, 1913,
03:15.1 right around that time,
03:17.0 and they began to recover.
03:18.3 Subsequently, animals were taken from these recovered colonies
03:21.1 and they were relocated to other parts of the historical range
03:25.0 in an effort to repatriate the species
03:28.1 throughout its natural environment,
03:29.2 and that's shown by the green dots, here.
03:31.2 So, the red dots and the green dots
03:33.2 represent places that otters have been reintroduced,
03:36.1 and all of the places in between
03:38.2 are places where they once occurred but no longer did occur.
03:43.0 All we have done is simply compare the places where they occur
03:46.0 with the places that they don't occur,
03:47.3 and we have watched places that have become recolonized
03:50.2 and how those ecosystems have changed
03:53.0 as otter numbers have built up through time.
03:56.1 These are the locations where the work has been done:
03:58.3 the Aleutian archipelago,
04:00.2 Southeast Alaska and Vancouver Island,
04:03.1 and, as I mentioned before,
04:06.0 simply, what we have done, very simply,
04:08.0 is look at areas with and without sea otters within these large areas
04:11.2 and we have contrasted places through time
04:13.1 as the abundance of otters have waxed and waned.
04:17.2 So, what are the findings?
04:21.2 Very simply, if you look at places where otters are abundant,
04:24.1 what you see are abundant kelps
04:27.1 and relatively few sea urchins,
04:28.2 and if you go to nearby places where otters
04:31.0 once were abundant but are now gone,
04:32.2 you see abundant sea urchins and virtually no kelps,
04:36.1 as shown on the right here.
04:37.3 Amchitka island... these photographs were taken in the early 1970s...
04:41.3 Amchitka island was a place where otter populations
04:44.1 had recovered to what we think was their natural historical abundance.
04:47.1 Shemya island, which is several hundred miles
04:50.2 to the west of Amchitka island...
04:53.0 otters were exterminated on Shemya
04:54.3 and they had not repatriated or recovered on Shemya
04:58.0 at the time that these photographs were taken.
04:59.2 So, you can see visually here, very clearly and simply,
05:02.2 what the differences are between a system
05:05.2 with and without sea otters.
05:07.2 So, what's going on here?
05:08.2 What is going on here is what ecologists call
05:11.0 a trophic cascade.
05:12.1 That is, otters are having a top-down limiting effect on urchins,
05:16.1 urchins are having a top-down limiting effect on kelps.
05:20.2 By adding otters into the system,
05:22.3 it removes the urchins or reduces the urchins,
05:25.0 thus releasing the kelps from control by urchins,
05:29.0 so we have, in a system with otters, abundant kelp,
05:33.0 and we have, in a system without otters, abundant urchins
05:35.2 and relatively few kelps.
05:39.2 So, that's it, but what are some of the broader implications of this?
05:43.1 Well, the broader effects of this,
05:46.0 as I'm going to tell you about today,
05:48.1 largely spin off kelps and what happens
05:51.1 when we have systems with and without the kelp forests,
05:53.1 and I'm going to tell you about
05:55.2 three or four little vignettes of effects
05:58.1 that spin off of this trophic cascade
06:01.0 that are what we call indirect effects of the trophic cascade.
06:03.2 I'm going to tell you about how that is manifested in the abundance of fish.
06:07.0 I'm going to tell you about how it is manifested
06:10.1 in the behavior of various other consumers.
06:14.2 I'm going to tell you about how it influences
06:17.1 what we call the primary production of the coastal system.
06:21.0 And I'm going to tell you how that feeds back,
06:22.3 or has an influence,
06:24.2 on the physical environment through the sequestration of carbon dioxide
06:28.2 through the process of photosynthesis.
06:33.0 These are some data from a study
06:35.1 that my colleagues and I conducted several decades ago
06:38.3 in which we took baby mussels --
06:43.0 mussels are what we call filter feeders,
06:44.3 and these are young mussels
06:47.1 that were grown out at Friday Harbors Labs,
06:49.0 the University of Washington,
06:50.2 translocated to the Aleutian Islands,
06:52.1 and outplanted to islands with and without sea otters,
06:55.2 and thus to islands with and without abundant kelp forests.
06:57.2 And what you can see here is
07:00.1 the difference in growth rate over a one-year period,
07:02.3 the dark bar showing the growth rate
07:06.2 of these filter feeders in places where otters were abundant
07:08.3 and the light grey bar showing
07:11.2 comparable growth rates and comparable habitats
07:13.1 where otters are absent,
07:15.0 and what you can see from this is the growth rates of these filter feeders
07:17.1 are about twice as high where otters are abundant
07:20.0 compared with where they're absent.
07:22.1 Why is that?
07:23.1 It's simply because the primary production,
07:25.0 the abundance of autotrophs, these photosynthesizing kelps,
07:27.3 is much higher in systems with otters
07:30.0 than in systems without otters,
07:31.3 and therefore this consumer, the mussel in this case,
07:34.2 that is eating material
07:37.1 that is being provided by the primary producers,
07:39.2 grows more rapidly where sea otters are abundant compared with where they're absent.
07:43.2 These are data on the abundance of fish,
07:45.2 again, the dark bar showing information
07:48.1 from where sea otters are abundant,
07:50.1 the light bar showing comparable information
07:52.0 from places where they're absent,
07:53.3 and what you see from this is that
07:56.0 the measures that we have of fish abundance
07:58.1 indicate that they're almost an order of magnitude greater
08:01.0 where otters are abundant compared with where they're absent.
08:03.2 So, simply because these animals depend upon kelp
08:07.0 for habitat and food,
08:09.1 kelp is more abundant where otters are more abundant,
08:10.2 and thus fish are more abundant where otters are more abundant.
08:14.0 These are some data on the diet of
08:18.1 another species of consumer in these coastal ecosystems.
08:20.1 In this particular case,
08:21.2 a seagull, Glacous-winged Gull,
08:23.2 and let me just explain a little bit about what the data show.
08:27.0 In this particular panel, what I'm showing you is
08:30.1 the relative proportion of fish versus invertebrates
08:34.0 in the gulls' diet
08:36.0 between places where otters are abundant,
08:37.3 those are the black data, the black bars,
08:41.0 and places where otters are absent,
08:43.2 those are the light bars, the grey bars.
08:45.1 And what you can see is that when otters are abundant
08:47.3 these gulls feed almost entirely on fish;
08:49.2 when otters are lost from the system
08:52.0 they forgo feeding on fish to feed on the now more abundant invertebrates.
08:57.1 These are some comparable data
08:59.2 on the diet of another consumer in the system,
09:01.1 in this particular case, the bald eagle.
09:03.1 And what you can see here again,
09:05.1 by looking at places with and without otters,
09:07.1 is that eagles eat a roughly even mix of
09:11.2 marine mammals, seabirds, and fish
09:14.2 when otters are abundant,
09:15.3 and when otters are lost from the system
09:17.2 the proportion of marine mammals
09:20.1 and the proportion of fish in their diet goes down,
09:21.3 and the abundance of seabirds goes way up.
09:24.0 So, the overall composition of the major things that they feed on
09:28.1 is linked to the effects of sea otters on this coastal ecosystem.
09:32.2 Lastly, I want to tell you a little bit about
09:36.0 some recent work that we've done on the potential sequestration effect
09:39.0 of this trophic cascade,
09:40.2 that is, the enhancement of primary producers by sea otters,
09:43.3 on atmospheric carbon dioxide.
09:46.0 And why would we think that to be an interesting thing to look at?
09:49.1 Because carbon dioxide is the material that fuels photosynthesis.
09:52.3 If you have a plant species in the system that's more abundant,
09:57.1 one might expect that the rate of photosynthesis
09:58.3 is going to be higher,
10:00.2 and therefore the drawdown of carbon dioxide from the environment
10:03.3 and the surrounding oceans is going to be greater.
10:05.2 So, when we look at this, we find that in fact
10:08.1 the sea otter effect is substantial,
10:10.1 that sea otters are responsible...
10:13.2 a system with sea otters will draw down
10:16.0 about 10% of the overlying carbon dioxide in the atmosphere,
10:19.3 compared to a system without sea otters.
10:22.1 Or, if we put this in a slightly different context
10:24.2 and asked the question,
10:26.2 how much of the increase in atmospheric carbon dioxide
10:29.2 that has followed the onset of the industrial revolution
10:33.1 might this accommodate?
10:36.1 It's about half.
10:38.1 We can put this into dollar terms,
10:40.2 because carbon is something that's been valued
10:43.1 on what we carbon exchanges or carbon markets,
10:45.2 and when we do that, and these data are based on
10:48.1 the value of carbon in the European carbon market
10:51.3 as of 2012,
10:53.2 we see that, in the areas that I've been working,
10:56.2 the standing biomass effect of sea otters on kelp
11:00.1 is potentially worth somewhere between 200 and 400 million dollars.
11:04.1 And the potential sequestration effect of that,
11:07.2 that is, the amount of that carbon that's being put into the bank,
11:10.3 and in this case the bank is the deep sea...
11:13.1 carbon is translocated in some cases to the deep sea...
11:16.2 depending upon how much of that carbon that's fixed by the kelp
11:21.1 goes into the deep sea,
11:23.1 that value may range anywhere up to almost a billion dollars.
11:27.2 So, now I want to change gears
11:29.2 and talk about evolutionary consequences of these species interactions,
11:32.3 and why would I do that?
11:34.1 Because evolution is a consequence of natural selection,
11:37.3 and natural selection is going to be affected by
11:42.2 the strength of interspecies interactions,
11:44.1 and we see strong species interactions in this system
11:46.3 that are a consequence of these apex predators,
11:49.1 in this particular case a sea otter.
11:53.1 So, I'm going to focus on one particular part of this trophic cascade
11:56.2 that I've told you about,
11:58.2 and that is the interaction between the herbivores and the plants,
12:01.1 and how is it that sea otters may have influenced
12:04.2 the evolutionary dynamics between these herbivores and plants.
12:09.2 We can see from this illustration,
12:11.2 and from what I've told you before,
12:13.2 that in systems with sea otters
12:16.2 the strength of the interaction between the herbivores,
12:18.2 that is the sea urchins in this case,
12:21.2 and the kelps is going to be very weak,
12:24.2 and when otters are lost from the system
12:27.2 the strength of that interaction is going to increase substantially.
12:30.2 So, how might we approach the question of,
12:33.1 what were the evolutionary consequences of this interaction?
12:36.1 The way my colleagues and I have done this
12:38.2 is simply by looking elsewhere in the world,
12:40.3 where there is a kelp forest that evolved in the absence of sea otters,
12:44.2 so we chose to do this in the
12:48.0 southwestern temperate Pacific
12:51.1 in the area of Australia and New Zealand,
12:53.1 which has a very physically similar system to the North Pacific
12:56.2 in that it's a cold water system that has fleshy macroalgae
12:59.1 that are very similar to kelps,
13:00.3 but it lacks a predator on the sea urchins in that system,
13:04.2 and the other grazers, like sea otters.
13:07.2 So, the first thing that my colleagues and I did
13:11.1 to try to get some sense of the changes in the strength
13:13.3 of plant-herbivore interactions between these various systems
13:17.3 is that we measured the rate of tissue loss
13:21.2 in fleshy macroalgae on the sea floor to herbivory,
13:24.3 and the way that we did this was we simply took a kelp plant,
13:27.1 we put it on the sea floor,
13:29.0 we stuck another kelp plant next to it in a cage
13:31.2 from which the herbivores were excluded,
13:33.1 and we contrasted the rate of tissue loss over 24 hours
13:37.2 between these experimental and control,
13:40.1 caged and uncaged, treatments.
13:42.2 The data that you see here are the relative differences
13:45.3 between grazing intensity in different parts of the world.
13:48.1 So, on the very far right
13:51.0 you'll see data from Shemya Island,
13:53.1 which I told you before is a place where otters are absent in the North Pacific,
13:57.1 and what you can see there is that the rate of grazing,
13:59.2 as indicated by the black bar, is very high.
14:03.1 If you go over to the far left-hand side of this graph,
14:07.1 what you will see are comparable data done from the exact same sort of experimental protocol
14:11.1 at Amchitka Island, where otters are abundant,
14:13.3 and there what you see is that the intensity of grazing
14:16.2 is virtually zero.
14:18.3 And then if you go to Australia or New Zealand,
14:21.0 in this particular case these are data from
14:23.2 four different marine reserves in New Zealand,
14:26.1 what you see is something that was very exciting to us,
14:28.1 and that is that the intensity of grazing
14:32.1 is much less than it is in systems
14:35.0 where sea otters are absent in the North Pacific,
14:36.2 but much higher than it is in systems
14:40.0 where sea otters are present in the North Pacific.
14:42.0 In other words, the intensity of herbivory
14:44.1 in these southern hemisphere kelp forest systems
14:46.2 is clearly higher than it is in natural systems in the North Pacific,
14:50.1 where sea otters are present.
14:52.1 That led us to believe that there would be
14:55.1 some sort of coevolutionary response in the dynamics
14:58.2 between the herbivores and the plants in this system.
15:02.2 What might we expect to see as a consequence of that?
15:05.1 Well, we knew going into this that
15:08.0 the most likely way in which marine plants
15:10.3 are able to defend themselves,
15:12.2 or the most well-known way in which marine plants are known to defend themselves against herbivores,
15:17.3 is through chemical defenses.
15:19.2 And in the case of brown algae,
15:22.0 the kelps and the things that are related to them,
15:24.0 the common group of compounds that does this
15:27.1 are compounds called phlorotannins,
15:29.0 and I've drawn the molecular structure here of one of those phlorotannins.
15:33.2 There are many other molecules and, categorically,
15:36.3 they seem to pretty much act the same way
15:39.2 in terms of the way that they affect herbivores in those systems.
15:43.0 So, this is an illustration that shows
15:45.2 the composition of the various different plant species
15:49.2 in the northern hemisphere,
15:51.0 in the panel on the top,
15:52.3 and the southern hemisphere,
15:54.3 in the panel on the bottom,
15:56.2 and what it shows is the proportion of dry weight of these plants
16:00.3 that is composed of phlorotannins,
16:03.1 and what you see, in contrast in these two different areas of the world,
16:06.1 is that the composition of the plants in terms of the phlorotannin concentrations
16:09.3 is radically different between the North Pacific, where they're very uncommon,
16:14.0 and the South Pacific, where they're very abundant.
16:16.2 So, the overall average percent dry weight
16:20.1 of phlorotannins in southern hemisphere kelp species
16:23.0 is about 10%;
16:25.0 in the northern hemisphere it's less than 1%.
16:28.1 The next question that my colleagues and I asked was,
16:31.1 how are the herbivores in these two different parts of the world
16:35.0 reacting to these phlorotannins?
16:37.2 Now, this was a little bit tricky because
16:40.2 there are lots of other differences between the plants,
16:42.2 and so it wasn't simply a matter of looking at rates of grazing.
16:45.1 What we had to do was isolate the compounds
16:48.0 and then subject the herbivores to
16:52.1 diets that varied only in the composition of these secondary compounds.
16:55.3 So, what we did is we took a green algae called Ulva
17:00.2 that every known herbivore in the marine environment likes to eat,
17:03.1 it's very poorly defended and very nutritious,
17:05.2 we freeze-dried this stuff, we ground it up,
17:08.2 and we put it into little agar discs,
17:10.2 and that's what you see here.
17:12.0 So, these agar discs are then the grazing model
17:15.1 that we exposed to the various herbivores.
17:17.2 We were then able to manipulate the concentration of phlorotannins
17:21.2 s in these discs
17:24.1 and thereby look at how the phlorotannins, in isolation of everything else,
17:27.2 was influencing the grazing rate of the herbivores.
17:31.0 And all we did was simply build these discs
17:33.3 using different phlorotannin concentrations
17:35.2 from different plants
17:38.2 and then look at how herbivores in the northern and southern hemisphere
17:41.1 responded to varying concentrations of phlorotannins.
17:45.1 And here's an illustration that shows the results.
17:48.1 So, what you see here is a whole bunch of data,
17:51.0 it's fairly complicated in terms of there's a lot of material here,
17:53.2 but keep in mind that the big 'NS' values
17:57.1 are indicative of no effect of the phlorotannins,
18:00.0 and the little stars indicate that there was a significant deterrent effect.
18:04.3 So, all of the data on the left side of the vertical line in the middle, here,
18:09.1 are data from herbivores that came from the northern hemisphere,
18:13.0 and all the information you see on the right hand side of the panel
18:16.1 is from herbivores from the southern hemisphere.
18:20.1 And you can immediately see from this that
18:23.2 regardless of whether the phlorotannins came from southern hemisphere algae
18:27.2 or northern hemisphere algae,
18:29.1 they were deterrent to northern hemisphere herbivores
18:32.0 but largely undeterrent or nondeterrent
18:34.2 to southern hemisphere herbivores.
18:36.2 So, I've just encapsulated this in a simplification of that illustration
18:41.3 to show that in the North Pacific
18:44.1 we see a strong deterrence effect of phlorotannins,
18:48.3 regardless of where the phlorotannins are from,
18:52.1 whether they come from the northern hemisphere or the southern hemisphere,
18:54.1 and in Australasia we see either a weak deterrence or no deterrence effect
18:58.2 regardless of where the phlorotannins were from.
19:01.2 So, based on all of what I have told you,
19:03.3 we have come to this view of the coevolutionary dynamic consequences
19:09.0 of sea otters in the North Pacific.
19:11.0 In the southern hemisphere,
19:13.3 we have a two trophic level system,
19:15.3 we don't have an ecological analogue of the sea otter.
19:18.2 As a consequence, the intensity of herbivory on the plants is high.
19:22.2 As a consequence of that,
19:25.0 the plants appear to have evolved defenses in the form of high concentrations of phlorotannins,
19:29.0 and as a consequence of those high concentrations of phlorotannins
19:32.1 the herbivores, in turn, have evolved resistance.
19:35.2 So, we've had a strong coevolution
19:38.2 of defense and resistance
19:40.2 in the south hemisphere kelp forests.
19:42.3 In the northern hemisphere, we've had a third trophic level
19:45.3 in the form of the sea otter.
19:47.2 The sea otter reduces the herbivore,
19:49.3 thus it breaks this coevolutionary arms race,
19:52.1 and as a consequence what we see are
19:55.2 really poorly defended plants
19:58.1 and we see herbivores that have not developed an ability
20:01.0 to resist those defenses because they've never had to.
20:06.2 So, I've made the argument that there has actually been
20:10.0 important coevolutionary going on in this system
20:12.1 that's a consequence of an apex predator.
20:14.2 What are some of the ecological spinoffs
20:16.2 or other effects that this might have had
20:18.3 on other species and patterns in the system?
20:21.1 I'm going to spend a little bit of time telling you about that now.
20:25.1 So, this is an illustration that shows
20:28.1 the co-occurrence of the abundance of plants,
20:31.1 that is, kelps, and herbivores,
20:33.3 that is, urchins,
20:36.0 in both the northern hemisphere and the southern hemisphere.
20:38.1 So, the panel on the top are
20:41.2 data from Southeast Alaska in the northern hemisphere.
20:43.1 The panel on the bottom are data from New Zealand.
20:47.1 In the upper panel, the filled symbols
20:50.1 are data from places where sea otters are abundant;
20:53.0 the open symbols are data from places where sea otters are absent.
20:57.2 And what you can see in the northern hemisphere is that
21:00.1 when sea otters are present
21:02.1 the abundance of sea urchins is very low
21:04.2 and the abundance of kelps is high,
21:06.2 and when sea otters are present...
21:09.0 or, I'm sorry, absent,
21:11.2 the abundance of kelps is very low
21:13.2 and the abundance of sea urchins is very high.
21:15.2 And in this graph, which we call a state-space diagram,
21:19.0 which shows the abundance of two co-occurring species
21:20.2 in relation to one another,
21:23.0 all the data points occur at the perimeter,
21:25.1 that is, they occur in this sort of hyperbolic relationship
21:27.2 along both of the two axes,
21:29.1 but you never see any place in the North Pacific
21:32.2 where both urchins and kelps co-occur in high abundance,
21:37.2 or very few, and in these particular sites where we sampled, none.
21:40.3 If you go to New Zealand and make the same measurements,
21:43.2 you see a radically different pattern in terms of
21:46.2 the way in which herbivores and plants live together,
21:48.2 and you can see this from the symbols in this illustration.
21:51.3 The symbols are not important,
21:54.0 all that's really important for you to note here
21:56.0 is that this state-space diagram is populated extensively
21:59.2 by points where both herbivores and plants are abundant.
22:03.1 So, this, we believe, is one of the ecological spinoffs
22:06.3 of this coevolutionary arms race that I told you about
22:10.2 that is driven by the existence of sea otters in the North Pacific Ocean.
22:15.1 What about other species?
22:17.0 How might they have been affected?
22:19.1 We aren't terribly sure of this,
22:22.0 but one example that is very intriguing has to do with
22:25.0 what we call the Hydrodamaline sirenians.
22:27.0 So, the Hydrodamaline sirenians
22:29.2 are a lineage of manatee-like or dugong-like creatures
22:33.1 that are fairly closely related to tropical dugongs
22:36.3 in the tropical Pacific.
22:38.2 So, dugongs, as you may know,
22:40.3 as sea grass feeders, and they live in the tropics,
22:43.0 but with the cooling of the poles,
22:46.1 what happened was that a lineage of that family of dugong and sirenians
22:51.1 formed what are called the Hydrodamalines,
22:53.2 and the Hydrodamalines radiated into the North Pacific
22:56.1 and they became kelp feeders.
22:58.1 And what's interesting about this particular radiation of mammals
23:02.0 is that the only place in the world that it occurred
23:05.1 is in the North Pacific,
23:06.3 and the only place in the world where we have flora
23:09.0 that seems to be good for herbivores to eat
23:11.1 is also in the North Pacific.
23:13.2 So, we have imagined, and the argument can be made,
23:16.0 that the evolution of the Steller sea cow was, in fact,
23:19.0 a consequence of the fact that sea otters occurred in this system
23:21.2 and created a food resource that made it possible for them to do this.
23:26.2 Another group that's interesting in this context are the abalones.
23:29.0 The abalones are an old group of gastropod mollusks,
23:33.1 and here are pictures of abalones,
23:35.1 and one of the most interesting thing about abalones
23:37.2 is the tremendous variation in maximum body size across species.
23:42.2 So, you see that here in this illustration.
23:44.0 The figure on the left is a tropical abalone
23:46.2 or a warm water abalone,
23:48.2 Haliotis varia,
23:50.3 and the part of the abalone you see on the right
23:53.0 is a cold water abalone from the North Pacific,
23:55.1 this is the red abalone, Haliotis rufescens,
23:58.3 and you see the radical different in body size.
24:00.3 So, the question is,
24:04.1 how might the evolution of the food resource of abalones,
24:07.1 which are kelps,
24:09.1 influence their body size and the evolution of maximum body size?
24:12.1 And you can see that in this illustration.
24:14.1 So, what this illustration shows is
24:19.1 information on the extant (currently living) abalone faunas
24:23.1 from all the different oceans of the world,
24:26.1 and the data on the maximum shell length
24:29.0 or the maximum body size from these different faunas.
24:31.1 And what you can see is that in Australia, New Zealand,
24:35.3 the Indo-Pacific, the Mediterranean,
24:37.3 South Africa, and everywhere,
24:40.1 the maximum body size of abalones is substantially less
24:43.1 than it is in the North Pacific,
24:46.3 and we believe that this has quite a bit to do with the fact that
24:51.1 the food resources that these abalones have evolved
24:54.1 feeding on is a very nutritious food resource
24:57.2 and that, again, is a consequence of the existence of the sea otters in the system.
25:01.2 So, to wrap up, let me recap
25:04.0 what I have told you about sea otters in kelp forests.
25:06.0 I've told you something about the approach that we have used,
25:08.2 that my colleagues and I have used in understanding the dynamics of this system
25:13.0 and in particular how sea otters fit into those dynamic processes.
25:16.1 We have done that by first modularizing the food web,
25:19.1 focusing on species that seemed to matter
25:22.0 so far as the sea otter is concerned,
25:23.3 and then we have used a perturbational analysis
25:26.1 and in this particular case we've used the North Pacific Maritime Fur Trade
25:29.3 to manipulate the abundance of otters in the system
25:32.1 and to observe dynamic consequences
25:35.1 of their ecological interactions by doing that.
25:38.1 The general findings are that
25:41.1 we have discovered what we call a trophic cascade,
25:43.1 that is, an interaction that starts high in the food web
25:47.2 with this apex predator, the sea otter,
25:49.2 and flows downward through sea urchins and kelp,
25:52.1 that this trophic cascade has what I call serpentine influences
25:56.3 on many other ecological processes and species in the system,
26:00.2 I've given you an overview of a few of those,
26:03.2 and that these strong ecological interactions
26:05.2 have led to natural selection and evolution.
26:10.2 I should acknowledge both my collaborators
26:13.3 and some of the support that we have received for this work over the years.
26:17.1 My collaborators are posted on the left
26:19.3 and the major funding sources for the work that we've done
26:23.1 are listed on the right.
26:25.1 Thank you for your attention and I hope you'll come back and join us for Part III,
26:27.2 which will be an exploration of other large species of apex predators
26:31.1 and other ecosystems.

00:00:13.05 My name is Ian Baldwin and I'm delighted, here, to be presenting Part 2 in a three-part
00:00:18.15 story on how to study the plant ecological interactions in the genomics era.
00:00:24.18 I'm a Director of the Max Planck Institute for Chemical Ecology.
00:00:28.16 And in Part 2, here, I'll be talking about Nicotiana attenuata, the plant that is right
00:00:35.12 here, it's ability to be able to respond to attack from a nicotine-tolerant herbivore.
00:00:42.12 And I just want to remind you this is Part 2 of a three-part series and in the third
00:00:48.04 part I'll be talking about the plant's perspective on sex, seeds, and microbes.
00:00:54.24 In Part 1, I talked about how the Max Planck Institute for Chemical Ecology came about
00:01:03.01 and how it fits into the rich history of the field of plant-herbivore interactions, and
00:01:06.24 how Ernst Stahl, in 1888, really started the field.
00:01:11.19 I also talked about the process of training genome enabled field biologists and how to...
00:01:18.05 how they are trying to phytomorphize themselves and understand what plants are doing with
00:01:23.00 this incredible chemical prowess that they have, and how they use those chemicals to
00:01:28.10 solve ecological problems.
00:01:30.09 I also introduced the "ask the ecosystem" approach, which combines both field and laboratory
00:01:36.02 studies of transgenic plants, and introduced the important process of silencing genes to
00:01:43.15 understand their function at a Darwinian level in an organismic context.
00:01:55.05 These field experiments are conducted with these genetically modified plants in their
00:01:59.16 native habitat in a nature preserve in the southwestern deserts of the United States,
00:02:05.14 in Utah, in a collaboration with Brigham Young University.
00:02:10.04 What I want to do here in Part 2 is talk about this particular interaction that's unfolding
00:02:15.24 to you right here.
00:02:17.22 This is an interaction of the plant that we work, Nicotiana attenuata, and the hawk moth,
00:02:25.24 Manduca sexta and Manduca quinquemaculata.
00:02:28.23 It's a remarkable interaction filmed here in fast motion, fortunately enough, by the
00:02:35.03 team of Volker Arzt from the movie Kluge Pflanzen, and they were so kind for letting us use their
00:02:40.09 outtakes.
00:02:41.09 This is a remarkable interaction because the plant is chock-a-block full of one of the
00:02:47.01 most toxic compounds for human beings, and for almost any animal with a neuromuscular
00:02:52.17 junction, namely, nicotine.
00:02:54.11 Now, many of us have had an addictive relationship with nicotine as smokers, but if any smoker
00:03:01.23 had ever tried to eat a Nicotiana plant you'd realize just how poisonous this plant is.
00:03:08.21 Nicotine poisons the neuromuscular junction, the acetylcholine receptor called the nicotinic
00:03:15.07 acetylcholine receptor, and that receptor mediates how muscles move.
00:03:21.03 Now, if you were a plant and you wanted to design a chemical defense which would poison
00:03:28.09 animals that moved with muscles, this would be an ideal defense compound to produce.
00:03:33.17 And this is exactly what Nicotiana attenuata and some of the other tobacco plants have
00:03:38.08 done -- they've evolved this molecule.
00:03:40.07 Now, this molecule evolved from two primary metabolic pathways, the NAD pathway and the
00:03:45.21 polyamine pathway, that produced the two rings that both contain a nitrogen and then they're
00:03:50.16 fused together to form the molecule... the molecule nicotine.
00:03:55.11 Nicotine is synthesized, as I said, from these two primary pathways, and its biosynthesis
00:04:00.02 has been worked out by a number of researchers over time.
00:04:03.07 But what is more recent is its understanding of the evolutionary history of this biosynthetic
00:04:08.08 pathway.
00:04:09.08 And this was done recently by Shuqing Xu in our department and a number of his colleagues
00:04:15.17 in the informatics group that is involved in assembling the genome of Nicotiana attenuata,
00:04:21.03 which is currently under review.
00:04:22.13 And what Shuqing Xu and colleagues found out was that umm... all of the genes that are
00:04:28.02 involved in nicotine biosynthesis are genes that are part of a whole genome triplication
00:04:35.13 event that happened with the Solanaceae, namely, all the plants that are of the group of plants
00:04:42.03 that are called solanaceous plants: potatoes, tomatoes, eggplant.
00:04:48.06 All went through a genome triplication event.
00:04:50.22 Those extra copies of the genes were therefore given the evolutionary privilege to be able
00:04:56.02 to be combined in novel things other than their primary metabolic pathways.
00:05:00.09 And potatoes and tomatoes and tobacco all produced nicotine, but tomatoes and potatoes
00:05:06.24 produce them at much lower levels -- about three orders of magnitude lower than tobacco
00:05:11.12 plants.
00:05:12.20 Tobacco plants' remarkable ability to produce enormous quantities of nicotine, to really
00:05:17.21 make it defensive, and smokeable, has to do with the ability of the plant to have corralled
00:05:25.11 the biosynthesis of those pathways into the roots, and to have fused the two rings in
00:05:30.16 a very efficient way, and funnel a lot of reduced nitrogen into the biosynthetic pathway.
00:05:36.08 That's described in this paper that is currently under review.
00:05:39.24 Now, nicotine biosynthesis can be inhibited by silencing a single gene.
00:05:45.14 This gene, here, putrescine methyltransferase, which we have silenced by RNAi and been able
00:05:51.13 to produce plants that are relatively nicotine-free.
00:05:55.01 And when you make a plant that's relatively nicotine-free and you take it back out into
00:05:59.06 the native habitat and plant it in some natural habitats, you realize just how effective this
00:06:04.00 defense is.
00:06:05.00 Because every deer, every rabbit, every gopher in the neighborhood finds out about it, and
00:06:11.11 here's an example of a gopher that's coming up, has dug a special tunnel up underneath
00:06:16.13 this nicotine-free plant and is pulling it down to its burrows.
00:06:20.00 So, without nicotine, the plants become quite defenseless and are stripped bare of their...
00:06:27.13 of their phloem by rabbits and other mammal... mammalian browsers them, and usually don't
00:06:32.17 last very long.
00:06:34.16 Now, Manduca sexta, which was gobbling, devouring those plants in that first video that I showed
00:06:39.21 you, is able to do it because it is... well, it basically holds the world's record for
00:06:45.14 nicotine tolerance.
00:06:47.00 If you compare the LD50 -- the lethal dose at which 50% of an experimental population
00:06:52.15 dies -- you realize that even the most hardcore, [unknown], carton-a-day smoking human being
00:07:00.23 still has an LD50 that is 750 times lower than that of Manduca sexta, which is about
00:07:08.18 1500 milligrams per kilogram that it's able to tolerate.
00:07:12.11 Now, it's been known since the '60s that Manduca sexta's tolerance of nicotine is based on
00:07:19.11 a physiology that allows it to excrete all the nicotine that it ingests without any apparent
00:07:26.04 metabolism at... or any apparent effect on its nervous system.
00:07:30.22 How it does that is still very much an active area of discovery, but, as we look at a caterpillar
00:07:38.05 eating a plant, we've been interested in asking the caterpillar, transcriptomically, what
00:07:44.10 is it doing inside of its gut to be able to handle those many human doses of lethal doses
00:07:50.22 of nicotine that it's ingesting almost on an hourly basis.
00:07:55.00 And when you ask the caterpillar, transcriptomically, there is consistently one cytochrome P450
00:08:02.09 which is constantly being regulated in direct proportion to the amount of nicotine that
00:08:06.00 is ingested by the caterpillar.
00:08:07.07 And this is a cytochrome P450 with a long complicated name called 6B46.
00:08:14.11 And you can see it regulates at a high level when it's eating nicotine-containing plant
00:08:18.15 and downregulates when it's eating nicotine-free plants.
00:08:22.02 So, to understand what this particular gene was doing in the caterpillar and why it was
00:08:28.04 being up-regulated every time the caterpillar ate a high-nicotine-containing plant, two
00:08:33.13 scientists in the department, Pavan Kuma and Sagar Pandi, designed a procedure that allowed
00:08:44.06 the study of this particular gene to occur in the natural environment of both the insect
00:08:48.21 and the plant.
00:08:50.03 And what they did was they took that gene, made a double-stranded contract, which is
00:08:53.14 depicted here in yellow in the plant, transferred it into the plant so that was consistently
00:08:58.10 expressing this double-stranded piece of the gene that they wanted to silence, and then
00:09:03.23 they planted it out in Utah and let free-ranging caterpillars feed on them.
00:09:09.16 And, in that process of feeding on these particular plants, the caterpillar ingests double-stranded
00:09:15.04 and then the gene in the caterpillar gets silenced.
00:09:18.19 And in those genes silence caterpillars they were able to understand the function of that
00:09:24.22 particular cytochrome P450, which is up-regulated during the defense process.
00:09:31.08 And what they discovered was really remarkable, but let me show... first show you some data
00:09:35.04 on just how effective this plant-mediated RNAi process is.
00:09:40.08 Here on the y axis is the transcript levels for the particular gene that they're looking
00:09:44.21 at in the various tissues of the caterpillar.
00:09:47.15 And I want you to focus particularly on the midgut, which shows that caterpillars eating
00:09:54.18 nicotine-containing plants have very high levels of that transcript.
00:09:58.14 But if the caterpillars are feeding on a nicotine-free plant, the transcript levels are quite low.
00:10:04.07 But if the caterpillars are feeding on one of these PMRi...
00:10:08.03 PMRi plants that are expressing a double-stranded construct of that cytochrome P450, and those
00:10:14.15 plants contain normal high levels of nicotine, you would expect the transcript levels to
00:10:19.16 be this high, but instead they're that low.
00:10:22.21 And they're that low because the gene is being silenced by that plant... by the plant's food,
00:10:31.01 and the caterpillars are ingesting that gene and that RNAi process is happening in basically
00:10:36.19 free-living, free-ranging caterpillars in the field.
00:10:39.15 It's a remarkable experimental tool that allows us to study plant-insect interactions in nature
00:10:45.04 using genetic tools to manipulate not just the plant, but also the insects that are feeding
00:10:50.01 on the plant.
00:10:51.04 Now, what's remarkable about this story is that it was actually a wolf spider occurring
00:10:56.04 in the natural habitat of the plant that told us the function of this particular gene in
00:11:03.12 the caterpillar.
00:11:05.09 And now I'm going to show you a series of videos and here's a video of a wolf spider
00:11:09.14 attacking a nicotine-free plant and you can see from that video that it just gobbled it
00:11:14.21 up.
00:11:15.21 So, if the caterpillar is feeding on a nicotine-free plant it has no nicotine in it and the wolf
00:11:21.05 spider finds it as food.
00:11:23.21 Now, here is, in the next video, a spider attacking a caterpillar that is fed on one
00:11:30.01 of these PMRi plants.
00:11:31.14 Now, remember those have are full of nicotine but they are silencing this particular gene
00:11:36.17 in the caterpillar.
00:11:37.18 And you can see from this video that the caterpillar is attacked and eaten as if it was nicotine-free,
00:11:44.06 and this was discovered by the two scientists who had placed caterpillars on plants out
00:11:51.04 in the field, having them feeding on these particular plants that silence the gene in
00:11:55.05 the caterpillar, and all the caterpillars disappeared at night.
00:11:58.12 And the wolf spider hunts at nighttime and that's how they found the wolf spider.
00:12:02.18 Now, here is the key moment, the key observation that allowed them to understand what was going
00:12:08.09 on, because in the next video here is a spider attacking a nicotine-containing plant, that's
00:12:17.01 a normal empty vector wild-type plant, and you can see all it did was go up and palpitate
00:12:22.21 the spider... the caterpillar and then it immediately backed away.
00:12:26.06 And what was going on in that palpitation, that little moment when the caterpillar being
00:12:31.07 assessed by the spider and the spider decided, oh...
00:12:33.15 I'm not gonna eat this, was that the caterpillar was, through its spiracles... caterpillars
00:12:40.16 have 17 spiracles, they are basically the lungs of the caterpillar... caterpillars have
00:12:46.06 all these tubes and that's how they exchange air... and through the spiracle the caterpillar
00:12:51.01 is puffing out a load of nicotine into the face of the caterpillar... into the face of
00:12:57.06 the attacking spider.
00:12:58.06 And that's why the attacking spider jumped away.
00:13:01.11 And what this gene is doing is mediating that process, in a way that we don't really understand
00:13:06.08 biochemically, allowing the caterpillar to basically divert a lot of... some portion
00:13:11.13 of that massive amount of nicotine that's flowing through its gut, that it's excreting
00:13:15.02 out, but then it moves it into the spiracles and it uses it defensively when a spider comes
00:13:20.03 up and says, are you good food?, and the caterpillar then just puffs out this thing of nicotine
00:13:23.23 and repels it, okay?
00:13:26.10 So, that shows you that, actually, the caterpillar, even though it's excreting most of its nicotine,
00:13:32.16 is using it defensively, it's co-opting just a small fraction of what's going through its
00:13:36.19 gut for its own defensive purposes.
00:13:38.09 But, now, what I'm going to tell you, for the rest of this talk, is what happens when
00:13:43.20 the plant recognizes that it's being attacked by that particular nicotine-tolerant caterpillar.
00:13:50.21 Because that recognition process results in six changes in the plant that all involve
00:13:58.07 how the plant deals with a caterpillar that has broken through one of its major defenses
00:14:04.00 and has to figure out something else to do with this guy that's going to eat it, and
00:14:08.18 that's going to make lunch of it.
00:14:10.08 And that recognition process starts right here.
00:14:13.15 And if you look right at that cut leaf edge, there, you can see a little bit of green slimy
00:14:18.10 stuff that the caterpillar is leaving on the edge of the leaf.
00:14:21.19 Now, it turns out it's not doing that intentionally, that's just part of the eating process, it's
00:14:25.13 part of its oral secretions, that's part of the process of masticating the leaves to be
00:14:29.06 able to digest it, but in those oral secretions are a group of compounds that are called fatty
00:14:35.07 acid amino acid conjugates.
00:14:36.24 FACs is what we call them, and the structures of those FACs are right here.
00:14:41.11 They're very simple molecules -- they're just fatty acids esterified to amino acids.
00:14:45.16 There's two of them, there's five fatty acids, and they make basically eight different structures,
00:14:50.04 and those eight structures are what the plant uses to say, aha I'm being attacked by Manduca
00:14:57.24 sexta and I know that it's nicotine resistant in some way or another.
00:15:00.23 And those are...
00:15:01.23 I'm anthropomorphizing but that's basically the message.
00:15:04.20 Now, what I'm going to do... this is, by the way... this fatty acid amino acid conjugates
00:15:09.06 were discovered by Rayko Halitschke in his thesis and published back in 2001.
00:15:13.14 What I'm going to do now is to take you through those six layers of defense, avoidance, and
00:15:19.24 tolerance that the plant goes through when it recognizes this... that it's being attacked
00:15:27.01 by this... this particular caterpillar.
00:15:31.00 And those six layers are both an up and down regulation of direct defenses, a bunch of
00:15:35.23 indirect defenses, an interaction between indirect and direct defenses, tolerance responses,
00:15:41.07 and avoidance responses.
00:15:43.02 So, follow with me and we're going to go through this remarkable journal... journey of what
00:15:47.23 happens to the plant as it reorganizes its metabolism, physiology, to deal with the fact
00:15:54.19 that it's got a predator that it really has to deal with.
00:15:58.11 Okay.
00:15:59.11 Now, first I want to talk a little bit about the recognition process.
00:16:02.15 So, umm... we've been able to, because we have these synthetic fatty acid amino acid
00:16:08.23 conjugates, we have the elicitors... we're able to start the interaction between plant
00:16:14.05 and its responses without having to have a caterpillar.
00:16:17.06 So, we simply just take a pattern wheel and we add these oral secretions to spit to the
00:16:21.10 leaves, to the holes that are made in leaves with the pattern wheel, and that elicits a
00:16:25.05 very complicated set of signaling responses.
00:16:28.14 We haven't identified the elicitor... the... the receptor yet for the elicitor.
00:16:32.03 We know the elicitor -- those are the FACs, the receptor is unknown, but that that elicits
00:16:38.12 a very complicated signaling network that involves MAP kinases, SIP and WIP kinases,
00:16:43.19 the jasmonate signaling cascade, and a lot of modulation of that jasmonate signaling
00:16:50.11 cascade through other kinases, the activation of CDP kinases as well, and the perception
00:16:59.04 by other receptors, LecRK, that it basically involves a regulation of jasmonate signaling.
00:17:06.05 And, because caterpillars do not brush their mandibles when they eat a plant, they also
00:17:11.14 contain bacteria and other sorts of bacterial signals, and the plant has to make sure that
00:17:16.18 it's activating a jasmonate signaling cascade and not a salicylate signaling cascade, so
00:17:22.17 all of this signaling has to do with being able to make sure that the caterpillar doesn't
00:17:28.09 fake out the plant with its bacterial signals, but rather generates a nice clean jasmonate
00:17:35.07 response, which activates five out of the six layers that I'm now going to talk to you
00:17:39.10 about.
00:17:41.03 Now, that was a lot of work, and that work was done by some remarkable group leaders
00:17:48.00 and a remarkable number of talented students that I wish I could talk about in greater
00:17:52.16 detail -- but here are their pictures.
00:17:55.13 It also illustrates another important message that I want to bring up in this talk and that
00:17:59.17 is that interplay between mechanism and function, that if you understand the details by which
00:18:06.00 these responses come about, you have the very tools that you can manipulate genetically
00:18:11.13 to be able to create plants that are not able to show the response, and all of those steps
00:18:17.00 in those signaling pathways have been very useful tools to allow us to be able to manipulate
00:18:22.23 some aspects of these six responses in different combinations, and test them functionally in
00:18:27.15 the field, in the actual habitat in which the plant evolved.
00:18:31.23 Now, let me go through the six responses.
00:18:34.00 The first response was the up- and down-regulation of these, what we call, direct defenses.
00:18:39.07 Now, direct defenses basically can be categorized in two groups.
00:18:43.05 They're either toxins, things that poison animals that eat plants, without poisoning
00:18:49.10 the plant too much, and are specifically targeted against the things that are different between
00:18:54.15 animals and plants, like nervous systems; plants don't have a nervous system, so it's
00:18:58.19 really easy for plants to make nervous system poisons that are not toxic to them, but are
00:19:04.18 very toxic to the animals that want to eat them.
00:19:07.01 So, in addition to toxins, there's also another type of direct defense that are called digestibility
00:19:12.08 reducers.
00:19:13.08 They're basically interfering with the main reason why a caterpillar wants to eat a plant
00:19:18.11 in the first place, which is to turn caterpillar protein... plant protein into caterpillar
00:19:23.23 protein, to turn caterpillar... plant energy substances like glucose and sucrose and starch
00:19:30.02 into energy substances that the caterpillar can use.
00:19:33.11 So, that digestibility process can be interfered with lots of different ways.
00:19:40.00 Interfering with all the steps of ingestion and digestion... for example, there are protease
00:19:45.06 inhibitors we're going to talk a little bit about, there are tannins and amylase inhibitors
00:19:48.11 that are basically affecting the digestive enzymes that take apart plant proteins and
00:19:52.21 starches, and make them available to be uptake... taken up by the guts of caterpillars.
00:19:57.24 But there's also abrasives, things that wear down the mandibles and the teeth of the herbivores,
00:20:02.20 because, you know, if an herbivore doesn't have a pair of teeth, a set of mandibles or
00:20:08.18 a set of teeth, it can't chew a plant.
00:20:10.24 And plants fill themselves with silica and other sorts abrasives that just wear down
00:20:15.18 the teeth.
00:20:16.18 And there is no easier way to starve an ungulate than to wear out its teeth, and plants do
00:20:22.17 that all the time.
00:20:23.17 Now, I just want to talk about the down-regulation, as well as the up-regulation, because the
00:20:28.09 first thing that happens when those FACs are recognized by the plant is the plant has an
00:20:34.02 ethylene burst and shuts down the very gene that we silenced to make a nicotine-free plant.
00:20:38.24 And that's in fact the reason why we did it, because we learned from the caterpillar how
00:20:43.17 it was shutting down nicotine biosynthesis in the plant.
00:20:48.02 And it's very clear, now, that since the caterpillar is co-opting a certain portion of the nicotine
00:20:53.13 for its own defense, the plant is most likely down regulating its nicotine production so
00:20:59.00 that the plant... so the caterpillar can't co-opt the extra nicotine it produces.
00:21:02.23 If it was a deer or a rabbit producing... doing the damage rather than a Manduca sexta
00:21:08.14 larvae, nicotine production would be operated 5- or 6-fold and the... you know, the plant
00:21:14.17 would become even more full of nicotine than it already is, so that a single leaf would
00:21:20.00 have the same amount of nicotine in it as half a carton of [unknown] cigarettes.
00:21:24.13 So, that massive up-regulation process is just basically stopped and the... the plant
00:21:30.01 is down-regulating nicotine production when it knows that it's being attacked by a nicotine-resistant
00:21:34.10 caterpillar.
00:21:35.10 Okay.
00:21:36.10 Then it produces a whole bunch of other types of compounds, many of which we had no idea
00:21:41.06 what they did.
00:21:42.06 And I just want to talk, just briefly, about a group of compounds called diterpene glycosides.
00:21:47.03 This is some work done by a PhD student who's just finishing enough, Sven Heiling, and he's
00:21:51.06 done some beautiful analytical work characterizing these molecules that were basically unknown.
00:21:56.15 There were 46 of them in Nicotiana attenuata and they are basically produced in the chloroplast
00:22:05.23 by what's called the MEP pathway and the DOX pathway, to produce a basic backbone diterpene
00:22:13.02 structure, and that backbone diterpene structure is depicted there.
00:22:17.10 It's hydroxylated and then sent out to the plant and decorated further by enzymes that
00:22:24.11 add different types of sugars to them -- I'll talk about that a little bit later.
00:22:29.12 But, because this is a secondary metabolic pathway, the main enzyme that's involved there,
00:22:35.22 this NaGGPPS that is highlighted in bold, there, also has three copies, because of that
00:22:43.12 trip... the genome duplication event, and, if you silence the one that's dedicated for
00:22:48.04 the production of these pathways, you can completely take out the whole biosynthetic
00:22:52.03 pathway by one gene silencing step.
00:22:54.20 So, by silencing that particular gene, we're able to make DTG-free plants and if you fed
00:23:01.07 them to caterpillars you can see that the caterpillars basically were able to increase
00:23:06.19 their growth rate almost fivefold when they're feeding on these DTG-free plants.
00:23:11.19 So, even though we had no idea that these were toxic or defensive when we looked at
00:23:17.07 the structures and figured out their structures, when we silence them and produce plants that
00:23:21.05 were where DTG-free the caterpillars told us that, oh... this is really a pretty nasty
00:23:26.17 defense compound.
00:23:28.18 And what Sven has been able to do is to identify all the different enzymes that are involved
00:23:33.22 in decorating them with sugars of various sorts, glucose and rhamnose, here, and then
00:23:40.18 they are malonated in addition, and that's what generates all those 48 different...
00:23:43.19 48 different structures.
00:23:44.20 Now, it turns out that if you look at the... the poop of a caterpillar, the frass that
00:23:50.04 comes out the busi... the other end of the caterpillar after it's eating leaves, Spoorthi
00:23:54.15 Poreddy, who is a PhD student who just finished up, along with Sven and Jianciai Li, have
00:23:59.21 been discovering that there's a very interesting dynamic that's going on in the caterpillar's
00:24:06.13 gut as it's trying to remove particular sugar groups from these DTGs, in a way so as not
00:24:13.07 to expose the toxic backbone, which is toxic to the plant also, but also not remove all
00:24:18.00 of them, which produces other toxic compounds.
00:24:21.07 So, this is a story that is ongoing, we're going... we're still working on it, but there's
00:24:25.22 this wonderful digestive duet that's occurring as the caterpillar is removing certain sugar
00:24:31.03 molecules and putting them back on, and putting other molecules back on to protect it and
00:24:36.02 detoxify this molecule as it goes through -- an example of direct defenses.
00:24:41.03 Now, I want to switch to indirect defenses.
00:24:45.05 Now, indirect defenses are based on a concept that probably every politician knows.
00:24:51.06 Now, here's the basic scenario.
00:24:53.22 Here's the plant.
00:24:54.22 And the plan is attacked by Manduca sexta, which is its enemy, right?
00:24:59.02 Now, Manduca sexta is, in turn, attacked by other predators that are all depicted here,
00:25:04.22 there are six of them right here, and they of course are the predators of the herbivore.
00:25:11.11 Now, anyone knows that the enemy of your enemy is your friend.
00:25:17.02 And that is the basis of how indirect defenses work.
00:25:22.09 Indirect defenses, in contrast to the direct defenses, are signals or traits that the plant
00:25:29.24 produces that help predators or parasitoids find and feed on the herbivores that are feeding
00:25:37.02 on them.
00:25:38.23 And that's what that indirect defense looks... how that works.
00:25:41.23 Now, the way it works in Nicotiana attenuata is that, when Manduca sexta begins to feed
00:25:47.06 on an attenuata plant, the plant recognizes it from those FACs that are in the caterpillar's
00:25:52.07 spit and it activates a series of transcription factors, and it activates the production of
00:25:57.05 a beautiful, volatile bouquet, like a Chanel No. 5 that's released not just from the attack
00:26:02.14 leaf but the entire plant.
00:26:04.15 And it's basically just producing this signal that includes a number of molecules, the most
00:26:10.00 important of which is a sesquiterpene called trans-alpha-bergamotene, and trans-alpha-bergamotene
00:26:15.22 attracts this little predator that's down here called Geocoris pallens, a little predator
00:26:20.13 that lives around in the soil on the plant, and it's basically listening, smelling in
00:26:25.06 the air, and when it senses that molecule it knows there's a caterpillar feeding on
00:26:29.17 a plant somewhere.
00:26:30.23 But that little Geocoris also needs local information.
00:26:34.18 Once it arrives on a plant, the plant is big, the caterpillar could be anywhere in the plant,
00:26:39.07 and it utilizes other compounds like these green leafy volatiles on the top there, and
00:26:43.12 particular... particularly the change in a double bond in those green leafy volatiles
00:26:47.23 that gives it local information and allows the Geocoris to be able to localize where
00:26:52.22 on the plant that particular caterpillar is feeding.
00:26:55.07 And, when it gets there to the caterpillar, it just plunges its beak inside the caterpillar
00:27:00.17 and sucks it out and it does that many times.
00:27:04.03 And so what this process is just like calling the police.
00:27:08.12 It doesn't have to do anything more than simply provide accurate, honest information about
00:27:14.20 where a caterpillar is feeding on it, how it's being attacked, and then the predators
00:27:19.05 take it from there.
00:27:21.10 It's a wonderful evolutionarily stable way of dealing with defense because the evolutionary...
00:27:26.19 coevolutionary loop between plant and herbivore is broken by this predator link.
00:27:33.04 Now, we discovered that thanks to the brilliance, really, of the graduate student in the group,
00:27:38.23 Andre Kessler, who is now professor at Cornell, and he invented a predation assay that allowed
00:27:44.04 us to monitor the behavior of this predator in the field under natural conditions.
00:27:48.13 And the predation assay was beautifully simple.
00:27:51.00 He simply just glued eggs of this Manduca onto the bottom of leaves and used those eggs
00:27:59.03 as a monitor for whether or not the predator had come up to the plant.
00:28:02.24 The predator is a very skittish predator.
00:28:05.16 It's called the big eyed bug... it has big eyes, it pays attention to a lot of things,
00:28:10.03 you can't walk up and see, it runs away... and so you need an indirect way to know whether
00:28:14.06 or not it's been around.
00:28:15.21 And yet, when the predator feeds, you can see that it sucks out the egg and it leaves
00:28:19.23 the egg in a nice state behind, and by gluing eggs onto the plant you can see how many predators
00:28:27.06 have come up and visited the plant.
00:28:29.01 And that predation assay had allowed us to be able to work out the transcription factors
00:28:32.17 that regulate volatile production, which volatiles are important, the long- and short-distance
00:28:36.12 signals, all the details of this particular process.
00:28:39.14 Now, it turns out that these indirect defenses don't work alone; they work in synergy with
00:28:46.09 the direct defenses.
00:28:48.04 So, when the cater... when the caterpillar attacks a plant and it causes the plant to
00:28:52.16 produce this wonderful volatile bouquet that is functioning as an alarm call, bringing
00:28:57.24 in predators from long distances away, that will then attack the caterpillars, there are
00:29:03.05 other things going on too, namely that the plant is also producing compounds that are
00:29:08.24 interfering with the digestive process.
00:29:11.06 And these are the protease inhibitors that Jorge Zavala worked on, and the protease inhibitors...
00:29:16.00 here's a seven-domain protease inhibitor... and what they do is they inter... interact
00:29:20.16 with the digestive enzymes of the caterpillar's gut and keeps the caterpillar from digesting,
00:29:25.06 which means that the caterpillar can eat and eat and eat but it doesn't grow, because it's
00:29:28.15 not getting the nutrients.
00:29:30.03 Now, when a caterpillar goes through the stages from being small to large, it becomes pretty
00:29:36.01 immune to this predator, because it's a bratwurst-sized caterpillar at the end and it pretty much
00:29:41.00 can thumb its nose at this little predator who is trying to attack it.
00:29:44.21 But if the plant keeps the caterpillar in a nice, small, vulnerable stage longer, the
00:29:49.20 indirect defense of the predator works much better.
00:29:52.14 So, it's the synergy between direct and indirect defenses that really helps to bruise the...
00:29:58.02 lower the population of caterpillars.
00:30:01.05 Now, there's another type of synergy that occurs as well, and this is depicted very
00:30:05.08 nicely in some videos by Mary Schuman, who is pretending to be a Geocoris predator, sticking
00:30:10.22 a little blue pin up the butt of the caterpillar.
00:30:13.02 And you can see, on a caterpillar that's feeding on a wild-type plant, a wild-type plant that's
00:30:17.11 full of defenses, it's behaving pretty sluggishly -- it's not moving at all when she's poking
00:30:23.17 it, she picks it up with the forceps, it doesn't do any wagging, it just hangs there limp like
00:30:28.01 a doornail.
00:30:29.02 Now, remember this caterpillar is spending a lot of metabolic energy detoxifying the
00:30:34.13 defenses that are in the leaves, the direct defenses.
00:30:39.01 And it doesn't have a whole lot of energy to fight back when it's attacked by predators.
00:30:45.00 Compare that when Mary tries to poke a caterpillar that's feeding on a protease-inhibitor-free
00:30:50.21 plant -- it's got plenty of energy.
00:30:52.11 It's banging around, it's thrashing, and it's defending itself quite well.
00:30:56.24 And that's another example of the synergy between direct and indirect defenses, is that
00:31:04.00 caterpillars that are feeding on toxic plants are lethargic.
00:31:06.15 They are having to spend a lot of energy detoxifying all those metabolites that are going through
00:31:11.22 them, and that slows them down and makes them much more vulnerable to their predators.
00:31:18.00 We so frequently forget because we eat defenseless plants in our normal food supply, we made
00:31:24.09 them defenseless through our agricultural practices, that we forget that eating native
00:31:28.11 plants that are full of chemicals is actually hard, metabolically demanding work.
00:31:34.24 Now, there's another type of direct defense... indirect defense that I want to tell you about.
00:31:39.08 And that's an indirect defense that occurs in the trichomes, which are these little hairs
00:31:43.03 on the surface of the leaves, and you can see as a little droplet appearing here from
00:31:47.11 this magnification of a trichome on the surface of an attenuata leaf.
00:31:50.22 Now, in the trichome is... is a particular type of compound called an acylsugar.
00:31:55.18 Now, acylsugars were thought to be direct defenses, toxins, and there's a good bit of
00:32:01.00 evidence that they are sticky substances that catch insects and sort of tie them up.
00:32:06.13 But... and this was actually first worked on by Alexander Weinhold in the group, and
00:32:11.23 Alexander characterized the structures of these things, and that these acylsugars basically
00:32:17.02 consists of a sucrose molecule and then on each of the hydroxyl groups of the sucrose
00:32:21.10 molecule is esterified a small, short-chain fatty acid.
00:32:26.03 Here are the characteristics of these short... short-chain fatty acids, and these short-chain
00:32:31.06 fatty acids have the smell of baby barf. they're sort of an unpleasant smell and that's the
00:32:36.15 reason why Alexander actually started the project in the beginning, because he had to
00:32:40.08 take care of the caterpillar colony, and he always thought that the caterpillars smelled
00:32:44.15 fairly bad, and... and noticed that when they were feeding on these leaves they were of
00:32:51.03 course eating acylsugars.
00:32:53.02 And when we took these plants to the field... took plants to the field and noticed what
00:32:57.03 caterpillars did when they first hatch out of their egg, we noticed that these... these
00:33:01.05 acylsugars are not defensive at all, they're in fact the first meal of a caterpillar.
00:33:05.11 A caterpillar hatches out of its egg and it begins to lick these... these tops like they're
00:33:10.07 little lollipops, and they get their first meal, and, in the process of getting that
00:33:15.00 first meal, they end up getting a body odor.
00:33:19.00 And the body odor comes from eating those acylsugars and having those fatty acid groups
00:33:24.16 deesterify and come off the body.
00:33:27.09 And so the caterpillar begins to smell of those baby barf fatty acids that are esterified
00:33:33.12 to those sugars.
00:33:34.13 Now, we were very interested to know whether or not smelling attracted the attention of
00:33:39.11 predators that were on the plants.
00:33:41.04 And so we looked at all the predators that occur on plants and none of them cared about
00:33:45.03 this... these baby barf smells -- they didn't seem to respond more to caterpillars that
00:33:48.23 were scented or non-scented.
00:33:50.02 So, we investigated some more.
00:33:52.08 But it turns out that there was another thing that these compounds did to a caterpillar's
00:33:58.21 body odor.
00:33:59.24 Not only did it change the body odor, but it also changed the smell of its poop.
00:34:04.24 There was a caterpillar just pooping there.
00:34:07.03 And poop, when it happens, when it falls, usually falls according to the laws of gravity.
00:34:16.03 It falls down.
00:34:17.03 It doesn't always hit the fan as... as the metaphor goes.
00:34:20.20 And the caterpillar, when it poops, produces a smelly, fresh, redolent poop that falls
00:34:27.14 directly on the ground, and this is Utah where the ground is hot, it's frequently 50 degrees,
00:34:32.16 and those are short-chain fatty acids, so they immediately volatilize, and after five
00:34:36.20 minutes or so they become scent-less and they no longer have that smell.
00:34:41.01 But for five minutes, when the fresh poop has fallen on the ground, it's providing beautiful
00:34:46.01 information to a whole other group of predators.
00:34:49.23 And those are the predators that are walking along in the ground, the lizards and the ants,
00:34:54.19 and it turns out that the lizards and the ants use that volatile information to know
00:34:59.07 that, oops, there's a caterpillar above them, they can just climb up the plant.
00:35:03.03 And you can take fresh frass and dried frass, or you can just isolate the... that... that...
00:35:09.07 those fatty acids and make your own little perfume, which will be available in duty-free
00:35:13.19 shops soon, and call it the scent of the caterpillar, and you can spray it on the ground and spray
00:35:17.16 it on sticks in front of ant nests, and the ants will just come charging up after you've
00:35:21.23 sprayed them, looking for caterpillars.
00:35:24.06 And so, in the end, these trichomes may well be the first meal for the caterpillar, and
00:35:32.05 they're delicious, sugary lollipops, but in the process of scenting their bodies and scenting
00:35:38.10 their frass, they actually turn out to be evil lollipops because they tag them for predation.
00:35:43.16 And that's just another example of how a plant is utilizing indirect defenses to protect
00:35:49.22 themselves.
00:35:51.00 They have very clever ways of bringing in predators.
00:35:54.07 And that was the fourth layer.
00:35:55.23 Now, I'm going to go to the fifth layer, now, and the fifth layer activated by those fatty
00:36:02.06 acid amino acid conjugates that are in the caterpillar's spit.
00:36:05.07 In the fifth layer is a layer of tolerance responses that the plant activates.
00:36:10.15 We had talked earlier in Part 1 about how a plant is a growth machine, fixing carbon
00:36:15.23 dioxide, taking that carbon dioxide, making a whole bunch of metabolites for growth, reproduction,
00:36:21.03 storage, and defense, but at the same time it's also possible to use it to make the plant
00:36:28.10 more tolerant of herbivore attack.
00:36:31.07 Now, until this work, the whole tolerance thing was pretty much a trait-less concept,
00:36:36.01 something that you could look at in populations of plants, but not something you could really
00:36:40.02 nail down to a particular trait.
00:36:42.05 And here we've been able to nail it down to a particular trait.
00:36:45.06 And it came, again, from a field observation.
00:36:47.09 The field observation was that caterpillar-attacked plants, after they plants had senesced and
00:36:53.04 dried out, and then there was another rain, they frequently reflowered -- they produced
00:36:58.00 new flowers after a rain -- but the plants that were not attacked by caterpillars didn't
00:37:02.15 do this reflowering.
00:37:03.16 So, that was an interesting observation.
00:37:05.12 And you sort of wondered, where did these caterpillar-attacked plants get the resources
00:37:09.14 to reflower?
00:37:10.20 This is an annual plant; it should have shut down life, made all the flowers that it could've,
00:37:14.22 and senesced and called it quits.
00:37:16.07 But that's not what they were doing.
00:37:17.21 And I think the answer comes in the life history of the caterpillars that feed on them.
00:37:23.20 Caterpillars go through two stages.
00:37:25.08 They have the eating machine stage, which is depicted right here, where Manduca...
00:37:29.24 Manduca sexta is simply just a larvae trying to consume as much plant material as possible,
00:37:33.18 but then it pupates and molts into this beautiful moth, and it becomes a sex machine.
00:37:39.21 And, as a sex machine, it's no longer eating the caterpillar... eating the plant anymore.
00:37:44.09 And that means that the caterpillar is out of its... out of the concerns of the plant,
00:37:49.13 and the plant, if it had waited and stored resources somewhere else, it was able to reflower
00:37:55.05 and start that whole process over again without having the tissues being lost.
00:37:58.24 And this is what is happening.
00:38:00.19 When... and this is work done by Jens Schwachtje, and his PhD project, and he discovered that
00:38:06.24 the FACs in caterpillar's spit, they elicit a bunkering of photoassimilates into the roots.
00:38:13.20 Now, a plant is a growth machine, right?
00:38:15.22 It's assimilating carbon dioxide from the air and, normally, it would be fixing those...
00:38:20.24 that carbon dioxide into sucrose and sending it from source leaves up to sink leaves to
00:38:25.17 grow more leaf area to make more of a growth machine -- that's what plants normally do.
00:38:30.00 But if they're making more of a growth machine, they're also making more leaves for the caterpillar
00:38:34.03 to eat, grrr...
00:38:35.03 So, you need to stop that process.
00:38:37.12 And when you have a caterpillar on the plant, or you put FACs on a plant, and it doesn't
00:38:42.18 matter where on the plant, the plant, instead of taking that fixed CO2 and sending it up
00:38:48.07 to young sink leaves, it bunkers it down below ground.
00:38:51.15 And it... and Jens was able to show this with some beautiful experiments in collaboration
00:38:56.04 with the Phytosphere Julich, which has a synchrotron, is able to make C-11 carbon dioxide.
00:39:01.09 C-11 has a half-life of 15 minutes, so you have to be right close to the synchrotron
00:39:05.14 -- you can't ship it very far -- and it allows you to look at very short-term partitioning
00:39:10.21 of carbon in a plant after it's fixed and where is it moving and where it moves it around.
00:39:15.14 And here's just some of the data from Jens' work.
00:39:17.23 He was able to show that... up is transport of C-11-labeled CO2 into young leaves, and
00:39:26.03 you can see that it goes... when you just wound and water a plant... and you treat the
00:39:31.14 wounds with water... the fixed carbon dioxide goes up the plant, but if you add spit to
00:39:36.18 the wound it goes down.
00:39:39.08 And it's the specific FACs in that spit that cause it to go down.
00:39:45.13 And he was also able to show that there's a particular subunit of a SnRK kinase which
00:39:49.24 is regulating that.
00:39:51.01 This is this GAL83 subunit that is down-regulated by the FACs.
00:39:55.06 And that's sort of the... the master sink-source regulator, the genetic element that... that
00:40:01.21 is causing this response.
00:40:04.15 And that bunkering, having put that carbon down below ground into the roots, allows the
00:40:09.13 plant to reflower, make bigger flowers, after the caterpillar has gone.
00:40:14.21 So, in many ways this level, this response, this number five, is a man... is the kind
00:40:21.12 of response that Mahatma Gandhi would have against a predator.
00:40:25.17 You just sort of lay low and let it go by, and don't engage in a fight, but just regrow
00:40:32.18 and be able to start again.
00:40:36.17 Okay.
00:40:37.24 The sixth layer and the last layer is probably the most intriguing layer.
00:40:42.19 It's a type of avoidance of this herbivore and it's an avoidance response that has...
00:40:50.11 has to deal with a fairly common natural history problem that... that all organisms have.
00:40:55.16 And that is that some of their interactions are with good guys and some of them with bad
00:40:59.00 guys, and sometimes the good guy and the bad guy are a part of the same genome.
00:41:03.00 So, this moth is a good guy -- it's a pollinator for the plant -- but it lays eggs that are
00:41:09.05 bad guys, that grow into little herbivores that sometimes turn into very big herbivores,
00:41:13.12 that are very disastrous for the plant.
00:41:16.00 And the sixth response has to do with... with dealing with this herbivore by dealing with
00:41:22.07 its mother, its pollinator.
00:41:24.21 Now, I told you in session 1 that this is a plant that attracts that pollinator by producing
00:41:32.23 a compound called benzylacetone, which is depicted up there above the flower, and...
00:41:37.16 and what Danny Kessler discovered is that when the moth is attracted by that particular
00:41:44.22 structure of benzylacetone, not only is it attracted because of the nectar, it nectars
00:41:50.09 and then it oviposits.
00:41:51.17 So, nectaring and ovaposition are linked processes; the more they get nectared by and more visited
00:41:58.16 by this pollinator, the more eggs show up on the plant.
00:42:02.08 The eggs of course turn into herbivores and therefore the more pollination services you
00:42:06.20 get, you might end up getting more herbivores, if the other types of defenses I've talked
00:42:11.12 about earlier aren't effective in cleaning out those herbivores and getting rid of them.
00:42:16.13 Now, we were able to silence benzylacetone production and when we do that we know that,
00:42:21.23 if the plant is not producing benzylacetone, it's pretty much ignored in terms of pollinator
00:42:27.19 activity, and also ovaposition activity by the moth.
00:42:32.05 And Danny Kessler, who is a remarkable photographer but also a remarkable observer of natural
00:42:39.06 history, noticed that attacked plants, when you looked at this... let's do it again at
00:42:43.18 this day night transition... that the plants were beginning to produce a different type
00:42:49.18 of flower after they were attacked.
00:42:52.07 They were producing their normal night flowers, but then they started, when they were attacked,
00:42:55.18 producing a different type of flower that was really only opening in the morning.
00:42:59.24 Now, here is the difference between the morning-open flower on the bottom and the night-open flower
00:43:05.12 at the top.
00:43:06.21 The normal flower is the night-open flower, the one here.
00:43:10.18 And you can see that it opens up in the first night open, and it opens and scents and attracts
00:43:15.22 the moth, and then it closes a little bit for the day, and then opens again and attracts
00:43:19.24 the moth again for the second night.
00:43:22.04 The morning-open flower stays closed that first night.
00:43:25.22 It doesn't scent.
00:43:27.04 And it doesn't attract any moths.
00:43:29.04 And then it opens up just slightly in the next morning, and it attracts a different
00:43:35.13 pollinator, and this is the pollinator -- a hummingbird.
00:43:39.10 And the hummingbird has a very nice characteristic that it lays hummingbird eggs, not caterpillar
00:43:46.08 eggs.
00:43:47.12 And by switching its sexual system to a different pollinator, asking for a different type of
00:43:53.20 postman to bring gametes to you, the plant has basically solved its herbivore problem.
00:44:00.21 And that's pretty remarkable.
00:44:03.07 So, what I've done is told you about all of these changes that occur in the plant when
00:44:10.10 it perceives these compounds here that are in the spit of the plant... the spit of the
00:44:15.13 caterpillar as it chews on... on the plant, and elicits this very complex defense avoidance
00:44:20.14 and tolerance responses.
00:44:22.10 And what I've also told you, I hope... there's basically three messages in behind this remarkable
00:44:28.22 transition that occurs in the plants when it sees these spit factors.
00:44:33.04 The first, of course, is that direct defense is not the only way of coping with herbivores,
00:44:37.10 and most of our agricultural practices dealing with protecting our crop lands have to do
00:44:42.01 with direct defenses -- insecticides that directly kill the crop press... the crop pests.
00:44:48.10 Now, as we've learned from this story, there's many other ways of dealing with your herbivore.
00:44:53.14 And we should be thinking about how to incorporate some of those many other ways into our cropping
00:44:58.11 systems, because some of them may well be much more evolutionary stable than just using
00:45:02.22 direct defenses alone.
00:45:05.10 The second main take-home message that I want to get from this is this interplay of the
00:45:09.08 importance of knowing mechanism so that you can use mechanisms to be able to manipulate
00:45:14.20 function.
00:45:16.12 And when you can manipulate function you can begin to ask, in an unbiased way, what is
00:45:21.03 actually happening in nature between plants and insects, and all the other interactors.
00:45:26.23 And the third main message I want you to get from this is that you can observe an awful
00:45:32.02 lot by just watching.
00:45:33.15 Now, this little tautology is something from Yogi Berra, but I think it applies so cogently
00:45:40.14 to biology today, because we don't teach our students how to watch, particularly not natural
00:45:49.06 interactions, anymore.
00:45:50.06 This is not part of our biological training programs.
00:45:53.08 And so much of the innovation that I've just shown you comes from simple natural history
00:46:00.11 observations.
00:46:01.23 Okay.
00:46:03.11 So, in the third part... that was the end of the second part, in the third part I'm
00:46:08.02 going to talk about seeds, sex, and microbes.
00:46:11.08 In Part 1, I told you that this is a plant that chases fires, it produces seeds that
00:46:16.13 have to live in the seed bank for hundreds of years before the next fire comes along,
00:46:21.09 and I'm going to be talking about how it uses sex to get the best genetic material, to be
00:46:25.24 able to survive that long-time period of... as it waits for the next germination event,
00:46:33.08 and also how it recruits microbes when it does decide to... to... to germinate in opportunistic
00:46:39.08 mutualisms to help protect it against all sorts of stresses that you could hardly predict
00:46:43.14 if you had been in the seed bank for hundreds of years.
00:46:46.07 So, I want to thank you for your attention, but I particularly want to thank both the
00:46:51.04 funding organizations that make this work possible, the long-term, patient funding of
00:46:56.00 the Max Planck Society, and the grants we received from these wonderful agencies that
00:47:01.02 are so unbureaucratic in their administration, and really promote curiosity-driven science
00:47:06.12 in the best way possible.
00:47:07.23 I want to thank the folks at Brigham Young University, particularly Dr. Larry StClair,
00:47:12.22 Ken Packard, and Heriberto Madrigal, that make this wonderful interaction with that
00:47:17.23 remarkable University work, and allow us to use their Lytle Ranch Preserve as a laboratory
00:47:24.09 to study, for the site of these field interactions.
00:47:27.01 And I want to talk...
00:47:28.01 I want to thank all the people who have provided the stunning pictures and movies, the talented
00:47:34.13 photographers and... and folks in the group who have helped support and make some of these
00:47:39.07 slides.
00:47:40.07 And, particularly, Erna Buffie and Volker Arzt, who are really masters of translating
00:47:47.22 science, making beautiful movies, and allowed us to use many of their outtakes from their
00:47:53.03 movies in this presentation.
00:47:55.08 And, you, for your attention.