McFall-Ngai (U.

Hawaii Manoa) 1: Living

together: The symbiosis of host-microbial
interactions
My name is Margaret McFall-Ngai and I'm a professor and the Director of the Pacific Biosciences Research Center, School of
Ocean and Earth Science and Technology, the University of Hawaii at Manoa. I'd like to start my lecture here with thanking the
iBiology team for this opportunity to speak to you today about the field of symbiosis. So, the field of symbiosis is about living
together -- it's host-microbial interactions that I will be talking about specifically today. And there's a bit of a revolution in
biology. Symbiosis and host-microbial interactions are taking center stage in biology. And I'm going to talk to you today about
why this is happening now and exactly why we're sort of in the center of a revolution. So, I'm going to start by talking about the
words that we use to describe symbiosis. So, this person right here is Heinrich Anton de Bary and this particular person lived in
the 19th century, and at the end of the 19th century he coined the term symbiosis. And the definition that he gave to this term
was the living together of unlike organisms. And this particular definition of symbiosis has remained with the field until the
present day -- it's a very, very, very general way, but very useful way to think about this particular phenomenon in biology. So,
that said, symbiosis is a bit of a catch-all term with no information concerning the effect of fitness. So, what I'm showing up here
is I'm showing a very classic symbiosis that a lot of people know of and a lot of people think about, and this is the symbiosis
between a clownfish and an anemone, and they live together, persistently, for much of their lives, and it's a classic symbiosis
that you can think about. But like I said, the word itself confers no... no… it's a catch-all word with no information concerning the
effect on the fitness of either partner. And so first I want to describe what fitness means. So, fitness is an individual's
reproductive success, and that is the number of offspring that an individual leaves to the next generation. And so let's think about
this idea in terms of symbiosis. So, when we talk about fitness, when we're talking about symbiosis, I think there are… I'm going
to consider that there are two partners -- there's symbiont #1 and symbiont #2 -- and in a mutualismwhich is one of the types
of symbiosis, both partners will benefit, and what that means is that both partners will have more in the successive
generations from being a partner with the other. And so in the case of the clownfish and the anemone, the clownfish gets a
very safe place to live, because the tentacles of the anemone are harmful to other animals; the fish, the clownfish, has
learned ways to avoid the danger of living in the tentacles of this anemone ; and the anemone gains from having the fish
there because the fish releases ammonia that is taken up by the anemone and it's used in the energy production of the
anemone. So, this is a true mutualism. The next type of symbiosis I'm going to consider is commensalism. In a commensalism,
one partner benefits and one partner is unharmed -- there's no change in the fitness, no change in the reproductive fitness.
So, what I'm showing here is another example and this example, here, is the shark/pilot fish example, and in this particular case
what happens is is the shark is a messy eater and he eats fish and all sorts of various things and food is released around
him and the pilot fish that are associating with the shark take advantage of the fact that the shark is a messy eater . In
this case, the pilot fish gained from living with the shark, but it seems to have no effect on the shark. And so in this case,
it's very likely that the pilot fish will leave more to the next generation from living with the shark, but the shark it won't affect the
shark's fitness whatsoever. The third example that I'm going to give is the example of parasitism. So, these are the three major
types of symbiosis, and in parasitism what happens is one partner benefits and one partner is harmed. So, I'm showing down
here a tree, and this particular tree trunk has a large gall on it, and the large gall is present because there is a pathogen that
is living, a microbial pathogen is living inside this gall and has created this large gall on this tree . Now, the pathogen.
that microorganism that's living in that gall, benefits from living… from parasitizing this tree. The tree on the other hand is
harmed by this association, so this is a classic parasitism. So, one of the things I should mention is that as the field has grown,
there's a little bit of misuse of these terms, and this is one of the reasons why I wanted to bring this up. The term commensalism
is often used by the biomedical community, in my mind and the mind of a lot of people who study symbiosis, incorrectly. And
what they'll say is that the microbes in your gut, which I'm going to talk about a little bit… the microbes in your gut are
commensal, and what this means is that they have no effect on the host -- they're benefiting but have no effect on the
host. But now we know that they have a tremendous effect on the host and are very often very important for our health, and
beneficial. There are some in there that are likely commensal, there are some in there that are likely budding pathogens, there
are some in there that are likely, or many in there, that are likely mutualists, but we really can't categorize them, so the very best
thing to do in the instance where you really don't understand what type of symbiosis it is is to just call it a symbiosis and to call
the partners a symbiont. So, let's consider for a second host-microbe symbioses. So, what we're talking about is we're talking
about viruses, bacteria, protists -- or single-celled eukaryotic cells -- and fungi, living with animals and plants, basically. So, the
question is, is this a rarity? And I have to say that when I started my career working in this field, back in the early 1980s, this was
considered a very unusual feature of symbiosis… symbiosis was considered an unusual feature in the biological world.
And in fact, if you look at a textbook, even today, you will see that symbiosis is covered, but it's covered in only a few pages of a
1200-page textbook. It was and is today still considered a rarity, but I hope you convince you that it's not a rarity at all. But why
was it considered a rarity? Well, we would find it… historically, we were looking at unusual situations in things like the
hydrothermal vent symbioses in the deep sea. Down at 2000 meters, you know, you see these smokers down at 2000 meters,
and these particular smokers would have associated with them these very large tube worms, and these large tube worm had in
association with them, bacteria, and these bacteria allowed these particular tube worms to live on the chemical energy that was
provided. So that was a true symbiosis that's been studied by lots and lots of people. Bioluminescence is another kind of
symbiosis and very many organisms who are bioluminescent, not all, but many organisms that are bioluminescent, are
bioluminescent because they harbor luminous bacteria in association with the organism . So, in the case of this anglerfish, up
here in the angle is a pure culture of a particular bacterial species, and that allows the anglerfish to use that light in its predation.
And then, lastly, I'm going to give the example of coral reefs. Coral reefs would not form if they did not have a very...a
mutualistic symbiosis with a unicellular organism called zooxanthellae. And these zooxanthellae live inside of the cells of corals
and they provide them with the photosynthate. So, these zooxanthellae are capable of photosynthesis and they translocate
the photosynthate to the host. And so the zooxanthellae get a place to live and the host coral is given the photosynthate, and
so they both benefit. So, in all the cases I'm showing on this slide, these are mutualistic symbioses, and they're ones that have
been studied for over 100 years… actually, not this one, this one they discovered in the late 1970s, but the many, many
symbioses that are mutualistic like this, and kind of unusual, have been studied for decades. But now we're finding out that nearly
all animals and plants are likely to have beneficial symbioses. So, this is a whole new area, this is a whole new finding, and this
new information is from the last two decades of work. And so I'm just showing a whole array of animals that are now known to
have beneficial symbioses with microbes. So, I want to just take one minute to pay tribute to what I… the person who I consider
the mother of the field of symbiosis, and this is Lynn Margulis. And Lynn Margulis lived 1938-2011, and she was the person who
felt that symbiosis was a major driver in the evolution of animals and plants , and she was always way ahead of her time.
She became famous for the endosymbiotic theory of the origin of the eukaryotic cell, and that is to say that bacteria
became symbiotic with other bacteria and made a more complex cell. And she showed that at the end of the 1970s, it was
very, very controversial, she was way ahead of her time and always was, but she is actually the person who had the vision to say
exactly what we're seeing today, and that is that symbiosis is a major thing in biology and has likely been over evolutionary
history. So, like I said, nearly all animals and plants are likely to have beneficial symbiosis and this is new information from the
last two decades of work. And so what I'm showing here is I'm showing a person, and this person in this artistic rendering is
completely covered by microbes. And we are, from the minute we're born, through our life, we associate very intimately with the
microbial world. And so we know now that you have as many, if not more, microbial cells than human cells in your body. So, you
have about 10^13 human cells and somewhere between 10^13 and 10^14 bacteria that live with you, persistently, your whole
life and confer health. So, the question is, why didn't we know about this? Why wasn't this more obvious? Well, it turns out that
there was a huge technical problem that we've been able to overcome. And so, the technical problem was a difficulty in
identifying and classifying the microorganisms, and why we couldn't do that was because most of these organisms were
unculturable under laboratory conditions, and so they're called viable but nonculturable. In other words, they live, but we just
can't bring them into the lab and study them the way that we would want to. So, it's about less than 1% of the bacteria that live in
association with animals are culturable, so this was a huge technical problem. And so... because we just couldn't know who they
were. The other thing was that… another technical problem was that they are relatively featureless. So, what I'm showing here
is I'm showing a set of different types of shapes and so on and so forth that you might see in different kinds of bacteria. And so
you might compare and contrast that with the morphologies that you see of very, very many plants and animals. I mean, you can
really, very well, tell the differences between plants and animals; with microbes, it's not so easy. So, they weren't culturable and
you couldn't... they didn't… they were pretty well featureless, so these were big problems. So, even though they're featureless,
we began to visualize microorganisms back with Anton van Leeuwenhoek. So, this is Anton van Leeuwenhoek with, you know,
several hundred years ago, but he was a guy who developed the very first microscopes, and so he was the first person who
allowed us to visualize microorganisms. And what Anton van Leeuwenhoek did was he took a swab and swabbed the inside of
his cheek and then he applied that to his microscope, and he was able to see, for the first time, microorganisms. So, not only was
he the first person to see microorganisms, but he was the first person to see microorganisms that associate with humans,
and those were the microorganisms inside of his own mouth. So, then we fast-forward to the electron microscope, and the
electron microscope was invented several hundred years later, in the late 1960s, and the invention of the electron microscope
allowed us to see much more detail in microorganisms. And this was actually the microscope that allowed Lynn Margolis, who I
spoke about earlier, to visualize the complexity of the eukaryotic cell and to resolve the fact that, in fact, the eukaryotic
cell was the result of a symbiotic association between microorganisms, or among microorganisms. So, then fast-forward
a little bit more… although… so, these two characters, Carl Woese and Norm Pace. So, Carl and Norm were doing molecular
biology of microorganisms at the University of Illinois… well, Carl was at the University of Illinois and he working with Norm Pace,
and they introduced the use of gene sequences to determine relationships in the biological world . So, notice that this is
1977, so it's around the same sort of time that people are beginning to think about using the microscope, the electron
microscope, as a mechanism by which to classify the biological world, but this was a new instrument that Carl Woese and Norm
Pace introduced to the community of biologists. So, the instrument was the 16S ribosomal RNA gene. It's a highly conserved
marker gene, and the reason why this gene could be used was because it's conserved throughout evolutionary history and so it
gives you an idea of changes that are very, very old, and would allow you to classify all of the biosphere by molecular methods.
And this is the phylogenetic tree that came as a result of the analysis of the biological world. And so what you'll see is you'll see
that there are the bacteria… there are three domains of life -- the bacteria, the archaea, and the eukaryea -- and the eukaryea
contain the animals, plants, and fungi. And so, look at... the animals, plants, and fungi are just these three twigs at the very top of
the tree of eukaryea, which is, you know, is showing us that the vast diversity of the biological world is invested in the archaea
and the bacteria. So this was a huge change in our world view. So, how big was this change? Well, let's go all the way back to
Aristotle. So, Aristotle was one of the first people to classify the biological world , and what Aristotle did was he classified it
based on what he could see. So it's animals and plants, basically. So then, you know, fast-forward, as I mentioned, to Anton van
Leeuwenhoek, who saw animals, plants, and what he called animalcules. And then, in the 1970s, there was a worker named…
who used the electron microscope, his name was Thomas Whittaker, and he developed something called the five kingdom
model, and so the five kingdom model had sort of the featureless bacteria and archaea, down here at the base, the protists
above that, and those are the eukaryotic cells, and then up at the top he had the fungi, animals, and plants. So, Carl Woese and
Pace, looking at this in context with their sequencing, what they had was these three domains. And remember that all, all of this,
up here at the top, all of this stuff up here at the top is now invested in this group here. So, it's a huge change in the way we see
the biological world. So, what have we done? We have moved from having visual analysis… for a couple thousand years, we
looked at the biological world and we classified it based on what we could see, and now what Carl Woese and Norm Pace did
was they gave us molecular analysis. And molecular analysis is the... are the genes that allowed us to very correctly
organize the biological world as it actually exists. So, what we found, of course, is that the biological world is mainly
microbial, and the animals, plants, and fungi are but a small patina on the top of the microbial world. So, what did this revolution
mean for symbiosis? So, what it meant was that we could identify microbes and determine their relationships, among them
and between them, even those that we not culturable. We could extract the DNA from them and say, what is the DNA telling
us about who they are and what they are doing? But at first, with Carl Woese in the very, very beginning, sequencing was
excruciatingly slow and expensive. And then, around 2006, something called next-generation sequencing came onto the
scene, and this has been the most important thing of the whole revolution. And imagine... this was only 10 years ago. So, what
happened at this point was that there was a technology-enabled transformation in our ability to sequence quickly and cheaply.
So, what I'm showing in this graph is I'm showing the cost of sequencing per megabase… so, you see, in 2001, it was up at
about 6000 dollars a megabase, and it followed Moore's Law -- the doubling in computer power every two years -- it followed
Moore's Law for… until about 2006, down here… until about 2006, and then next-gen sequencing was invented. And look at
what happened. It went down from… the cost went down from 600 dollars a megabase to 35 cents in less than 10 years ,
and now it's down to 3.5 cents. And so this was incredibly enabling to the community of biologists. In other words, lots and lots
and lots of people had the resources to be able to characterize the microbial world, and so people have gone all around the world
characterizing the microbial world. So, a whole frontier opens… a whole frontier. We can learn, who are the microbial partners of
animals and plants? What are they doing? How are they doing it? And what is there importance to health and disease? And so
when you're thinking about a symbiosis you think that many… most of them are established new each generation. Not all kinds
are, some of them the symbionts are passed in or on the egg, but most of them are established anew each generation just after
birth. And so when a baby is born, what happens is at soon as it goes through the mother's birth canal it begins to acquire its
microorganisms. Then there's a development, a trajectory that's not unlike what goes on in the maturation of a forest -- there's a
succession of organisms -- and in humans you get
a mature set of microorganisms somewhere between the ages of two and four. And then what happens is you become a stable
association, that changes some over life with various changes and aging and so on, but it's a fairly stable population. So
there's a trajectory and we're learning a great deal about how all of this works at this point, but it's new data… I mean, it's just the
beginning of the field. What have we learned so far? What we have learned so far is that for many, many, many animals,
including humans, the complexity is absolutely daunting. And what do I mean by that? So, let's take humans for example. So,
what I'm showing here is I'm showing my favorite comedian, Woody Allen, and I've been very generous with him -- I'm making
him 6 feet tall or a little bit over -- and so... but his, if you compare the ratio is his size to a microbe's size, 2 meters to 2 microns,
so he's a lot bigger than a microorganism. However, if you look at the number of genes that Woody Allen has compared to the
number of genes that the microbes associated with his body have, the ratio is about 1:1. If you look at cell number, it's thought to
be somewhere between 1:1 and 1:10. In other words, there are about 10 times as many microbes associated with you, and
that number has just this last year become a little bit controversial, so it's somewhere between here and here, but that is to say
that there are just as many microbes associated with you as you have human cells. Then, if you look at gene diversity,
because instead of just… so, you know, all of your eukaryotic… all the human cells are of a single genome, but the microbial
cells are of thousands, or hundreds if not thousands, of genomes . The difference in gene diversity is thought… for every single
human gene there are 200 genes of microbes, so the gene diversity of microorganisms is very much greater. So at this point
Woody Allen feels pretty insignificant. What I'm showing here is I'm showing the power of animal model systems for the study
of complex characters, and the example that I'm using is developmental biology. And in development biology, what I'm showing
here is I'm showing a fertilized egg, and that fertilized egg goes from 1 cell to 10^13 cells and produces something like the
miracle of George Clooney, my favorite actor. So, this has been, this has been… this approach has been extremely successful to
understand developmental biology. So, what we've been able to do is to take model systems, you know, experiments that
evolution has done, and ask how, in this simple model system, a particular developmental phenomenon has been solved. And so
what I'm showing is I'm showing an array of model systems and I'm showing ones in which Nobel Prizes have been awarded,
and it turns out that 6 Nobel Prizes in developmental biology have been awarded since 1995 and 2007 , and all 6 of them
went to individuals working on models. So models have been an extremely valuable way to study very complex characters.
So, because symbiosis is such a complex character, we feel, those of us in the field who study models, feel that it's a really good
way to try to understand symbiotic associations. So, we are developing, the field is developing some models for symbiosis, we're
exploiting nature's toolkit and, as I said, evolution has done some enlightening experiments. So what I'm showing here is I'm
showing a variety of them, and so there are various nematode symbioses and various other invertebrate symbioses, and we
even have, over at UC Berkeley, Nicole King studying the choanoflagellate symbioses, at the base… thought to be the base of
the animal kingdom. And then up here some vertebrate models. Now, the vertebrates, all the vertebrates have complex
associations, they have large consortia associated with them, and so these people studying over here are really interested in
studying germ-free animals, and so I sort of look at them as engineered models. Over here, many of them are studying natural
models and these, the invertebrates are naturally very simple associations, and so they lend themselves to looking at a natural
situation and how that… how the bacterium and the host animal get together and maintain a stable association. So, I… my lab
works on this beautiful animal, here, the bob tail squid. This is not a giant's hand, this is my technician's hand. A very small
squid that's indigenous to the Hawaiian archipelago, and this is the symbiosis that I've been working on and that I would like
to tell you about in my next lecture. Thank you very much.