cc  >> Nadia Dominguez: Thank you  for joining me for Wednesday  Nite at the Lab, at the  UW-Madison Biotechnology Center.  Tonight, we welcome Dr. Heidi  Goodrich-Blair, of the  Department of Bacteriology.  Heidi Goodrich-Blair received  her bachelor's degree in  biology, as well as her PhD  in molecular biology at the  University at Albany-SUNY.  After conducting her post  doctoral research at Harvard  Medical School, she joined the  Department of Bacteriology at  UW-Madison in 1997.  She is now a professor here at  UW.  She has dedicated herself to  conducting and promoting  microbial symbiosis research.  Along with Dr. Katrina Forest,  she wrote a successful cluster  hire proposal that led to the  recruitment of four faculty  members, who study beneficial  microbe-host associations.  Heidi directs the symbiosis  cluster, co-directs the NIH  training program, Microbes in  Health and Disease, and teaches  both a graduate level course in  microbial symbiosis and an  undergraduate course in  microbial genetics and molecular  machines.  Heidi's research focuses on the  molecular basis of symbiotic  associations between bacteria  and invertebrate animals, the  topic of tonight's seminar.  Please join me in welcoming  Dr. Heidi Goodrich-Blair to  Wednesday Nite at the Lab. 

 (applause)  >> Goodrich-Blair: All right,  thank you.  Can everybody hear me, if I'm  speaking into the microphone  correctly?  So, thank you, Nadia, for  inviting me to give a talk here  tonight, and thanks to all of  you.  I want to reiterate what Nadia  said, is that it's a beautiful  night out there, and I can't  believe that you guys chose to  be in here with me.  But hopefully, I'll make it  well worth your time.  And also, in the introduction, I  hope got across my passion for  microbial symbiosis research.  And what I'm showing up here on  the screen are a number of  systems in biology that are  being used as models to try and  understand microbial symbiosis  at the cellular level, at the  evolutionary level, any number  of scientific levels.  And what I want you to take away  from this slide, and appreciate  from this slide, is the great  diversity of organisms that are  on here, including where they  come from.  We've got marine organisms,  including corals,  hydrothermal vent tube worms.  We've got soil animals,  including earthworms, zebra  fish, hydra ants, insects, mice,  and of course, humans.  My favorite over here is my  niece. 

 And all of these plants and  animals are associated with  microbes.  In fact, there is no plant or  animal on the earth that does  not associate with a microbe at  some point in its life, probably  throughout its life.  And that includes us.  We have microbes on us and in  us.  And I know that there have been  some Wednesday Nite at the Lab  talks given by my colleague,  Katrina Forest, a couple years  ago, as a matter of fact.  And she commented on how we are  in fact more microbial than we  are human.  We have more microbial cells,  90% of our cells are microbial,  10% are human.  So, we really are a composite of  microbe and human cells.  And the same is true for every  organism on the earth.  So microbial symbiosis, or the  association of microbes with  plants and animals is a key  factor in all of biology. 

 And we would like to understand  how it works.  So, out of curiosity, how many  of you have considered symbiosis  to be mutualistic or beneficial?  Is that how you would define  symbiosis?  So, the majority of people.  What was that?  It's both, exactly.  So, I, working up into this,  before I became a microbial  symbiosis researcher, also  assumed that symbiosis was  primarily beneficial for both  partners in the relationship.  But in fact, that's not the  original definition that was put  forth by Anton de Bary.  So, Anton de Bary, in 1879, is  credited for the definition of  symbiosis, or coining the term  symbiosis in his book,  "The Phenomenon of Symbiosis."  And I have to admit, I've never  read this book, because I don't  read German.  It was translated into French,  but I don't read French, despite  five years of high school  French.  I just can't do it.  But his definition of it, did  not distinguish between whether  or not the association between  the two partners in a symbiosis  is going to be detrimental or  beneficial for either/or of the  partners.  So, symbiosis, this definition,  is the living together of unlike  organisms. 

 Period.  There's no other criterion  placed on these associations.  And of course, my particular  love is microbial symbiosis, and  that's what I'm going to focus  on.  But symbiosis can be between any  number of units, as long as it's  unlike organisms that are  entering into the symbiosis.  So just a little bit of  terminology that I wanted to put  out for a second.  I tend to use the term host for  the larger of two organisms in a  symbiosis.  And I tend to use symbiont for  the microbe in the symbiosis.  And occasionally, I'll say  symbions, which means both of  the organisms.  So, because Anton de Bary has  decided that symbiosis, and he's  the one that coined the term, so  he gets to decide, includes all  associations, whether or not the  outcome is beneficial or  detrimental for one or both of  the organisms.  In fact, symbiosis does  encompass everything from  beneficial associations, which  we call mutualisms, to  pathogenesis, which we're all  very well aware of. 

 And in fact, pathogenesis has  been the most studied of  symbiotic associations, for  obvious reasons.  We're very, very concerned about  microbes that cause disease, and  we want to stop them from  causing disease.  But in fact, the more common  relationship is one of  mutualism.  And it's now becoming  increasingly apparent to  scientists, to funding agencies,  to the general populous, who are  consuming probiotics, for  example, in large quantities,  that microbes can in fact be  beneficial.  And of course, it's been long  known in terms of beer making,  and wine making, and cheese  making, and all of those things,  that microbes can do beneficial  things.  All right, because mutualisms  have not been studied quite as  extensively as pathogenesis  models, we're sort of actually  behind in understanding  beneficial associations between  microbes and hosts.  But studying several model  systems that have been  developing, I would say over the  last 15 years, we're beginning  to understand much more about  it.  And in fact, we know now that  mutualistic microbial symbionts  can influence health in a number  of ways, including immunity and  defense.  And what I'm putting up here are  a couple of examples from labs  that are on campus.  These are people, Currie, Ane,  and McFfall-Ngai and Ruby, who  we've recruited through this  symbiosis cluster that was  mentioned at the introduction to  my talk. 

 So these three labs have made a  number of contributions to our  understanding of how microbes  can influence human health, or  host health, rather.  Cameron Currie has shown that  ants, these are fungal farming  ants, and if you want to come  over to the Microbial Sciences  building, have any of you been  over there?  In the front foyer, there's a  place called the microbe place.  And inside, they have an ant  farm set up, showing how these  ants crawl down, they get  leaves, they bring them back to  their fungal garden.  You can actually look in and see  the fungal garden and watch the  ants doing their work.  So what Cameron has shown is  this wet, white waxy bloom  that's on the surface of the  ants is actually a bacterium.  And that bacterium produces an  antibiotic.  And that antibiotic kills a  fungal pathogen that can come in  and decimate that ant's fungal  garden.  So this is a symbiosis in which  the bacterium, we're not exactly  sure what the bacterium is  getting from the ant, but the  ant gets from the bacterium, a  specific antibiotic or a defense  mechanism against pathogens.  So the same is true of us.  We rely on our gut microbial  community for the development of  our immune system. 

 And without our gut microbial  community, we're much more  susceptible to pathogens.  That's one of the basins for  probiotics.  Microbes can also contribute to  nutrition.  So, Jean-Michel Ane studies the  leguminous plants.  And they're entering into  symbiosis with Rhizobium  bacteria that can fix nitrogen  for those plants.  What you're seeing here are  nodules that are on a root.  And within each nodule is a  whole big packet, if you will,  of bacteria.  And all they're doing is fixing  nitrogen and giving it to the  plant.  Then, the plant gives carbon  back to the symbiont.  And then Margaret McFall-Ngai  and Ned Ruby, I'm not sure, has  Margaret given a talk here?  So, they work on a squid  symbiosis with a bacterium. 

 And this is a bioluminescent  bacterium.  And what happens is, the squid  lives off the shores of Hawaii.  We actually recruited them away  from Hawaii, here to Madison.  So, you know, rock on.  Here they are.  They get to go back to Hawaii  every now and then, because they  have to collect squid from the  Hawaiian shores.  These squid don't live naturally  in Madison.  So these squid come out at night  to eat.  And because there's bottom  feeders, and if there's moon  light being cast down, they're  in shallow water, so the moon  light can cast a shadow on the  ground, and the night feeders  can come up and eat them.  So what they've evolved is a  symbiosis with bioluminescent  bacteria. 

 The squid can modulate how much  light is being emitted out of  their bottom to exactly match  the moonlight intensity, so they  don't cast a shadow and they  don't get eaten.  It's a very cool symbiosis.  So this is also potentially  defined as defense.  The other thing that happens in  this symbiosis is a very complex  series of events in which the  squid undergoes drastic  morphological changes and  developmental program that's  elicited by the initiation of  symbiosis with the bacterium.  So, in fact, bacteria and other  microbes can trigger cellular  responses in the host.  And the same thing happens,  again, in our gut.  We can see that our gut  microbiota increase the amount  of blood vessels that are formed  in the microvilli in our  intestine, for example.  So these are universal themes  among symbioses.  And they're being elucidated  using model systems, in these  cases, all invertebrate model  systems.  So, not a mouse among them. 

 Did I mention that I'm happy  to take questions throughout,  so feel free to interrupt.  (inaudible)  Cameron did speak about that?  Great.  It is a wonderful symbiosis.  Come, get a tour.  Apparently, they have a queen  that is nesting right at the  top, up at the lab.  I think it's fairly rare to be  able to see the queen.  But right now, they've got one  that you can see.  I probably shouldn't have said  that.  Now, hoards of people are going  to be going into their labs.  Call ahead. 

 I'm sure they'll let you in.  All right, so now I wanted to  turn a little bit to where the  microbes are on here.  I've shown you this waxy bloom  on the surface of these  bacteria, but it's actually a  little bit more complicated than  that.  So where do microbes live?  I've taken sort of a  human-centric approach to my  generic organism host here.  A little smiley face.  And I just wanted to get across,  a little bit in terms of  definitions.  So, microbes can exist on the  outside surfaces of their host,  just on top.  And when they do so, they're  called epibionts.  They can live inside the host,  but still on epithelial  surfaces, for example, in the  gut tract.  So this is open to the  environment. 

 They're constantly having an  influx of nutrients and  stresses.  They're constantly having to  fight being washed away because  of the flow going though these  epithelial cell surfaces.  But they are in fact not inside  cells.  They're interacting with  surfaces.  There's also a very common  phenomenon in which the microbes  enter into some specialized  pocket that exists inside the  host.  And these can be called anything  from ducts, glands, crypts,  receptacles, yadda, yadda,  they've got lots of different  names.  But basically, these are single  entrant, single exit openings  to the environment, and they  tend to be somewhat protected  from the environment.  And they tend to be very  specialized.  So these types of pockets, ducts  and glands, tend to have evolved  in order to promote association.  Well, I shouldn't have said it  that way, but they have evolved  and they do promote associations  with specific microbes. 

 In each case, these are called  extracellular symbionts.  They're existing in close  association with host cells, or  near host cells, but they're not  within host cells.  In contrast, you have  endosymbionts.  They're the ones that are  existing inside host cells.  And there are a number of  examples of those.  As far as I know, humans do not  have any examples of  endosymbionts that live inside  our human cells.  Not in terms of bacterial  symbionts.  So I just wanted to show a  couple examples of these.  Laxus oneistus is a marine  nematode.  It lives in the ocean. 

 And what it does is to migrate  between the oxygen layers and  the sulfide layers of the ocean.  And what it's doing is carrying  its symbionts, these are sulfur  oxidizing bacteria.  And what they can do is to  oxidize sulfur.  So the nematode takes them, they  pick up their sulfur, then it  takes them to where the oxygen  is.  And then they can have the  oxygen to be able to oxidize the  sulfur.  And the bacteria produce carbon,  fix carbon and energy from the  oxidation of sulfur and provide  that to the nematode.  Meanwhile, the nematode is  swimming around, giving the  bacterium the optimal  environment to do that  physiology.  Down here, you might have seen  from Cameron Currie's talk, if  you were here for that, this is  the crypt.  So that white, waxy bloom that  you see on the surface, actually  is covering over.  These bacteria are sitting  inside this crypt that's  underneath the surface of the  ant. 

 And then the bacteria grow up  and presumably, they're getting  nutrients from the ant from  inside this crypt, but I don't  think they know the answer to  that question yet.  And then finally, this is the  mouse gut.  And what you're seeing here is a  microvilli, and then the  epithelium.  This square here is getting  magnified over here.  And then this square here is  magnified here.  And then this square here, you  can actually finally see a  bacterium that's sitting inside  this mucus gimmish that's  actually covering over the  intestinal surface.  So sometimes they're not really  having any kind of intimate  association with the host cell.  Sometimes they're embedded in a  matrix that's covering over  those host cells.  So, given all of these  different, diverse places that  microbes can colonize, can we  say anything about how hosts and  microbes actually communicate  with each other to develop these  kinds of associations.  How is it that we recruit  certain types of bacteria to  live on our skin surface?  Or how is it that some types of  microbes can colonize our  intestinal surfaces?  How is it that we recognize each  other, hosts and microbes?  So there are some common themes  that have been coming out,  primarily from the work in  pathogenesis. 

 But it looks as though the  systems in mutualisms are  looking very similar.  And what the basis for this  recognition is, is molecular  patterns, and then pattern  recognition.  I'll just show you a couple of  schematic diagrams to show that.  So, molecular patterns tend to  be repeating units of some kind  of molecule, a sugar or a  protein.  And they are part of the  organism's cell structure.  And they're an integral part of  the organism's cell structure.  So as an example, bacteria have  what's known as peptidoglycan  surrounding them.  And this peptidoglycan, which is  a repeating unit of sugars and  peptides, provides structure to  those cells.  And all bacterial cells, or  almost all, have peptidoglycan.  So any host organism that can  detect peptidoglycan would be  able to recognize that there is  a microbe in its vicinity,  because those cells, animal  cells and plant cells don't have  that kind of peptidoglycan  repeating unit anywhere around  them. 

 So patterns, repeating sugars  and glycans, and you can have  all sorts of different  combinations, as well, so you  can have specificity in terms of  what types of patterns you have  on the surfaces of things.  And then, recognizing those can  be proteins that are on the  surface of the other organism  associating in the symbiosis.  And that can be a very specific  molecule that can recognize that  specific pattern.  So, it's not rocket science.  It's like enzyme substrate  recognition.  It's just that now we're talking  about repeated molecules on the  surface being recognized by  specific proteins or other  molecules on the surface of the  other organism.  That's just one example.  These pattern molecules and  receptors is one way in which  you can get communication  between a host and a microbe.  Signaling molecules, diffusible  signaling molecules.  So for example, bacteria can  secrete small molecules. 

 And those small molecules can  diffuse over to the host cell,  get taken up, and that can cause  some kind of communication to  occur.  Bacteria have the ability to  inject molecules, including  proteins, into host cells.  They have a machine that's  called the Type 3 secretion  machinery.  And what this does is, it's real  needle-like.  It looks like a syringe.  And it gets built from the  inside of the cell, the  bacterial cell, to the outside  of the bacterial cell.  It continues to be built.  And it can be inserted into the  membrane of a host cell.  And then, punched through that  syringe are certain proteins.  Those proteins then end up in  the host cell, and they can go  and change host cell gene  expression and cause a response. 

 That's one of the ways that they  can communicate with each other.  And then of course, there's  other types of adherence  mechanisms, this repeated sugar  or protein unit and receptor  molecule type of adhesion.  Regardless, these are all very  common languages that are being  used for communication between  host cells and microbial cells.  Given that there's this set  language, it's not surprising  perhaps to know that these same  units are being used in both  mutualisms and in pathogenesis  associations, or pathogenic  associations.  So it's not clear, currently,  what is it that distinguishes a  relationship or an association  that's going to be detrimental  for one of the hosts, or for one  of the partners in the  association, versus one that's  going to be beneficial.  They're using the same  communication mechanisms.  So why is it that sometimes it  turns out really well, and other  times it doesn't turn out  very well.  And that is still a fundamental  question in microbial symbiosis,  and is one of the things that  I'm interested in understanding  using the model system that I  work on.  So with that, I'm going to turn  to tell you about the biology of  the model system that I work on.  I work on a bacterium called  Xenorhabdus nematophila, is the  one that I usually use. 

 There's a quiz at the end!  You have to be able to name all  of the organisms that I'm  talking about!  Get out your pencils!  Get out your notes!  So Xenorhabdus bacteria, they're  shown here in the middle.  Each one of these little purple  rods, this has been stained with  a dye called crystal violet.  So each one of these purple rods  is a single bacteria cell.  And they're called gram negative  cells.  They've got an outer membrane  and an inner membrane.  They're a lot like E. coli,  basically, but they have a few  interesting differences that  make us love them even more than  E. coli as a model system.  So one of the things that we are  really quite taken with for  these bacteria is that they  associate with invertebrate  animal hosts, and with one of  their invertebrate animal hosts,  steinernema nematodes, they have  a mutually beneficial  association.  But with another animal host,  insects, larval stage insects of  a wide variety, mostly in the  lepidoptera, they have a  pathogenic association.  So they're lethal to insects,  really potent insect pathogens.  So here's this one bacterium  that associates with two  different animals. 

 And depending on which animal  it's associating with, it's  either really, really nice, or  it's really, really not nice.  And so, we're trying to use this  to understand how it is that it  changes its behavior depending  on which animal it's associating  with.  How does it know which animal  it's associating with?  And how do the animals respond  differently to this single  bacterium?  Okay, so for the rest of my  talk, I'm going to concentrate  on these organisms.  There's finally the title of my  talk.  Mutualism and Pathogenesis: How  Can One Bacterium Do It All?  And what I'm going to do is give  you a description of the life  cycle, so we understand how  these three organisms fit in  together.  And I'm going to show you some  of the experimental methods that  we can use in the lab to try and  study these questions of  microbial symbiosis.  And I'll try to leave you with a   sense if what some of the  current questions are that we're  trying to address.  So as with any life cycle, you  have to start somewhere.  So I'm going to start here, with  a steinernema nematode.  And it's a particular stage of  the nematode that we call the  infective juvenile stage, or IJ,  I will refer to it as well. 

 So this infective juvenile stage  lives in the soil.  It's the only form of the  nematode that is free-living.  All of the other forms exist in  side insects, as I'll show you  in a minute.  So this is a free-living hunter,  basically.  What it's doing is it's trying  to find an insect host that it's  going to be able to infect.  And it's got a couple of  features that make it very  interesting.  One of the features is this  outer cuticle that I think you  might be able to see here.  And this is a very tough  cuticle.  It's a sheath that protects the  nematode from UV stress, for  example, desiccation, other  stresses that it might be  encountering in the soil.  So it's quite resistant to  stress. 

 For those of you who know  about C. elegans, a big model  system for nematodes.  This is not C. elegans.  But this is a lot like  a C. elegans dauer larvae.  It's got a lot of the same  features as a dauer larva.  It's non-feeding, so it has  a -- plug in its mouth.  It's anus, which is over here,  is closed, so I didn't show you.  Here's the tail.  Here's the head.  A closed mouth, closed anus.  No one feeding. 

 Nothing going in.  Nothing going out.  It's got this cuticle around it.  What it's doing is living off of  lipid reserves in its intestinal  cells.  And you can see these paired  intestinal cells going all the  way down.  So it's just living off of  reserves, looking for an insect  host to infect.  When it finds an insect host  that it's going to infect, one  of the things that it needs to  have with it is its weapon.  And its weapon is Xenorhabdus  bacteria.  So it's carrying with it,  Xenorhabdus bacteria that it's  going to use to kill the insect  host that it infects.  And it carries those bacteria  here in a special pocket. 

 What I'm showing you here is  somewhere on the order of 200  individual bacteria cells that  are sitting in the lumen, so the  space in between two epithelial  cells of the nematode.  So these two cells, sort of form  this pocket, these symbionts,  these bacteria, are  extracellular.  They're not sitting inside  cells.  They're just sitting in between  two cells.  So you can see one nematode cell  here, and you can see the other  one down here.  Here is a stained nucleus of one  of those cells.  And it's in the anterior portion  of the intestine, sitting right  where that box is showing you.  So this population of bacteria  is, physiologically, we're not  sure in what state.  So obviously, they must be  alive.  Whether they're just sitting in  stasis, we haven't figured that  out yet. 

 But everybody is searching the  soil for an insect host to  infect.  Now we're going to turn to my  little schematic movie.  It's not really a movie.  I have to actually push it  through.  But this is my schematic diagram  to take you through the life  cycle.  So the nematode, carrying its  bacterial population, which I've  shown here in a little green  dot, is cruising around in the  soil.  And there are two different  types of nematode behaviors.  There's the cruisers and there's   the ambushers.  The one that we work with mostly  is an ambusher.  The cruisers waste a lot of  energy, but they're much more  likely to find an insect host to  infect. 

 The ambushers kind of sit and  wait.  They're not using a whole bunch  of energy.  But they're not as likely to  find an insect host in as timely  a manner.  So these guys can sit around in  the soil for up to six months, I  think, is the longest recorded  time.  They can over-winter.  So they're very resistant.  They can live a long time on  their lipid reserves.  What they're looking for is the  unsuspecting insect larvae to  either come by or to happen  upon.  They have a behavior called  nictation.  They're curling around in the  soil. 

 They'll lift themselves up on  their tail, and they'll start  waving around like this, trying  to get a gradient of air for  their sensory organs that are on  either side.  So, they're waving around trying  to sense changes in the chemical  cues that might be in the air.  One of the chemical cues that  they can sense is the carbon  dioxide that's being emitted by  animals in the vicinity.  So there are movies in existence  of this nictation behavior.  And if you're watching one of  these nematodes do this, and you  blow a puff of air on it, it'll  kind of pause.  Then it'll wait and do that, but  it doesn't do anything, except  maybe go back down to the ground  and crawl around a bit more.  But if you put an insect inside  a Pasteur pipette and then you  blow a puff of insect emitted  air onto this nematode, it curls  up on itself and it jumps.  So these have a jumping behavior  that I think is fascinating.  >> How big are these nematodes?  >> Goodrich-Blair: They're about  15 microns by about 150 microns.  They're teeny tiny. 

 The camera is zoomed in on this  thing.  The scientists that were working  on this, this is Harry Kaya in  California, had like a flea  circus kind of thing.  They would set up petri dishes  and they'd blow puffs of insect  air on the petri dish and see  how far the different nematodes  could jump.  It was like this little contest,  three-ring circus thing that  they had going on.  Okay, so it recognizes this  carbon dioxide cue.  It jumps on top of the insect.  It enters the insect through  natural openings, mouth,  sphericals, anus.  And once inside, bores a hole  into the blood system of the  insect.  So insects are like walking  guts.  They've got a gut, an outer  cuticle and everything in  between is basically their blood  system. 

 And they're movement is what  makes the blood move around.  They don't have any circulatory  system to speak of.  So it's this blood system that  is the site of infection.  Once the nematode loses the --  plug that's blocking up its  mouth, then it starts ingesting  the insect's blood.  And nematodes will ingest  anything.  But the ingestion of the insect  blood actually triggers release  of the bacteria.  So the bacteria get released by  defecation, that I'm showing  here, if you can see the green  dot.  So, now we're going to consider  this a single bacterial cell.  I know we went from 200 to one,  being represented by the little  green dot.  Once they're inside the blood  system, the bacteria also like  insect blood as a nutrient  source. 

 There's glucose in there.  There's amino acids in there.  There's lots of other stuff in  there that's pretty stressful.  But pretty much as soon as they  get access to this as a nutrient  source, they will begin to grow  and divide.  So they're going to start to  reproduce in numbers as soon as  they hit the insect's blood.  The insects are not completely  helpless.  In fact, they have immune  systems.  And I'll get into it a little  later, but they have innate  immune systems that are much  like being in systems that you  and I have, except not adaptive  immunity, like you and I have.  So this innate immune system  consists of molecules that can  insert into the membranes of  bacteria and cause them to lyse.  It includes cells that can  either phagocytose bacteria or  modulate around them from these  masses around them and just wall  them off from the rest of the  tissue. 

 And those cells can also target  larger organisms, such as a  nematode.  So as soon as these guys get in  there, the insect's immune  response is right there, and  they're trying to get rid of  these nematodes and these  bacteria.  But to no avail.  The nematode and the bacteria,  together, counteract the immune  system.  They get rid of it.  They don't have to worry about  that immune system anymore.  So, once the immune system is  gone and wasted, the bacteria  produce toxins that kill the  insect very rapidly, 24-48  hours.  And in fact, very few bacterial  cells are sufficient to be able  to kill an insect, as few as  five colony-forming units of  bacteria.  It's a very low infectious dose.  So, once the insect is dead, now  it's a really suitable nutrient  source for the nematode and the  bacterium. 

 I just want to make one  statement here.  And that is that this is one  level of the symbiosis.  The symbiosis between these  nematodes and these bacteria is  multifaceted.  There's a number of layers and  levels that they're helping each  other.  One level is that the nematode  can't efficiently kill insects  on its own.  It might be able to very, very  infrequently.  And if the insect were  immuno-compromised, for example.  But they're not very good insect  pathogens.  They really need these bacteria  to be able to efficiently kill  the insect.  But the bacteria are not lethal  on their own. 

 They have to be in the  bloodstream to be lethal.  You can't smear some of these  bacteria on the outside of an  insect and get death.  They can't invade on their own.  So this is the symbiosis.  The nematode carries the  bacterium in.  The bacterium kills the insect  for the nematode.  Okay, so once the insect is  dead, the nematode develops  through its other life stages,  all the other life stages.  It develops into adults that  mate and lay eggs.  The eggs don't have any bacteria  closely associated with them.  They hatch into juvenile stages  that molt between four different  juvenile stages. 

 So this is what I would call  the vegetative cycle  of the nematode.  They're using the nutrients  in the insect to reproduce.  Meanwhile, the bacteria are also  reproducing.  In fact, the bacteria are  necessary for this reproductive  cycle in ways that we don't  necessarily understand very  well.  But it's thought that bacteria  produce enzymes that help  degrade the insect cadaver and  convert it into a usable  nutrient source for the  nematodes.  But we do know that the bacteria  are necessary for this stage.  So the nematode can go through  two to three rounds of this  developmental cycle in a single  insect cadaver, depending on how  big that cadaver is.  So we're talking eight days,  approximately, inside a single  insect, going through multiple  rounds.  And then food is going to run  out.  There's going to be lots and  lots of progeny nematodes  swimming around inside this  insect, and I'll show you that  in a minute. 

 That triggers an alternate  developmental pathway toward the  infective juvenile stage again.  Now, things are running out.  It's time to leave the insect.  We've got to go find a new  insect host.  And what we need to do before we  go is to take our weapon with  us.  So now we need to re-associate  in that very special pocket in  this non-feeding infective  juvenile stage, before leaving  the insect host.  That's what we call  re-association.  So once the bacteria are safely  inside that intestinal pocket,  the nematode progeny will emerge  out of the insect to go look for  a new insect host.  All those other stages get left  behind.  It's only the infective juvenile  that will migrate away from the  insect cadaver. 

 Were there any questions about  the life cycle?  >> Just from the nematode-- is  it sexual reproduction?  >> Goodrich-Blair: Does the  nematode require sexual  reproduction?  Yes.  >> So there has to be more than  one.  >> Goodrich-Blair: There has to  be more than nematode that  infects an insect.  And it's not clear right now  whether or not there's a founder  nematode, for example the male  might be the colonizing sex.  There's been some indication  that that's true, where the male  will be the first to infect and  set up shop, and that that will  recruit species-specific females  to also enter the insect  cadaver.  Yes?  (inaudible)  So that when it infected it,  what happens to it?  What has happened to it, is that  it's molted from this infective  juvenile stage into a full-stage  juvenile.  And then it develops into an  adult, either a male or a female  adult.  And so it's the progeny of that  initial colonizing nematode  that are now leaving.  (inaudible)  The adults die  within the cadaver.  Yes, as a matter of fact,  there's this really gross thing,  and I saw this for the first  time when I was pregnant, and it  was really, really icky. 

 And it's called endotokia  matricida, which means I think  development within the mother,  or eating the mother.  It's icky.  A lot of times, the eggs will  not be laid, and they will hatch  inside the mother.  And they'll develop, using the  mother as the nutrient source.  Yeah, not something I would wish  upon my worst enemy.  Any other questions?  Okay, all right.  So now I'm just going to show  you a couple of pictures of  this.  I'm not sure if you can see it  with the lighting.  What we have up here on the left  is a waxworm.  You fisherpeople out there might  recognize this from your local  bait shop, which is where we get  them from. 

 We use waxworms, or Galleria  mellonella, as our propagation  insect.  It's not very hardy.  It has a wimpy immune system.  So they're really good for  actually making lots of  nematodes.  So what we do is take some of  these living waxworms and we put  them on a filter that's got some  infective juveniles distributed  on it.  They'll infect, kill and start  to reproduce.  And what you're seeing here are  dead cadavers that are about  eight to ten days old.  So, they're spent.  And the nematodes are  hightailing it out of these  spent insect cadavers, and  getting out of town.  That's what these streamers are,  just loads and loads of  nematodes that are emerging out  of these insects. 

 And they're attracted to water.  They're trying to get away, in  part from ammonia that has built  up inside the cadaver.  They'll migrate toward water, so  what we do is we put these petri  dishes with all these dead  insect cadavers into a larger  petri dish that has water.  They'll crawl up the side of the  petri dish, and they'll  self-harvest themselves.  I don't think you can see it,  but there's all these little  white specks that are in the  water.  Those are individual nematodes.  And they can sit there in the  water for months and months and  months.  So we just siphon them out.  We put them into tissue culture  flasks and that's our nematode  stock for however long we need  it, until we re-do the  infection.  So this is just a microscope  image of these nematodes that  have been colonized by  Xenorhabdus bacteria that's  expressing the green  fluorescent protein. 

 Because these nematodes are  transparent, it's very easy to  see the bacteria.  You can see them as these little  green dots.  And I'll show you some close-ups  of those in a minute.  But basically, this is how they  look when they're sitting in  the water.  Just to show you  another icky close-up--  You know, if you get to hear  about squid, they're very cute.  And I can't compete with,  really, ants or squid.  But I have the gross factor.  Those of you who like horror  movies, I'm the symbiosis for  you.  Okay, so if you blow this kind  of picture up, if you're just  looking at just one portion of  this insect, looking at it  up-close.  This is not the same insect, but  this is what the nematodes look  like when they're crawling out  of this insect host into the  water. 

 So there's loads of them that  come out of here.  You can get 500,000 individual  infective juveniles emerging out  of a single insect cadaver.  So we get a lot.  Any other questions?  Yeah?  >> Are these nematodes in the  soil around here?  >> Goodrich-Blair: These  nematodes are in the soil around  here, yes.  Yes, they can feed off of any of  the insects.  >> And we can't see them because  they're so small?  >> Goodrich-Blair: They are very  small.  You can see them, again, in this  petri dish, right?  They're just little white  specks.  They're smaller than an eyelash.  So they're visible to the naked  eye, but in a soil sample, you  wouldn't be able to pick them  out.  >> So, they're right around  here?  >> Goodrich-Blair: These  nematodes are everywhere. 

 They have really no geographic  limitation, as far as they're  found at all sorts of altitudes,  all sorts of soil types.  They require a certain amount of  moisture, so they don't do very  well in arid environments.  They over-winter and the cold  does not harm them.  They probably burrow down a  little bit deeper, and they'll  just sit and wait for things to  start happening.  (inaudible)  So, they are used as  a biological control agent.  And with different efficacy,  depending on the study.  But in fact, there are companies  that are marketing these  nematodes.  There's one in Iowa.  I can't remember the name of it.  I probably shouldn't say it  anyway. 

 So they market them with things  like, "our bugs kill grubs."  They have formulations.  You can either put them into  sprayers and spray them on your  crops, or they've got these  sponges that you can just put in  various places on the ground and  they'll emerge out of those  sponges and go and hunt.  It's most efficacious, as I  understand it, in smaller garden  settings or fruit farms, places  where the moisture content is  good enough.  You can't really put these  things onto a corn field.  They just aren't going to  survive that well.  The toxins that are produced by  the Xenorhabdus bacteria, that  do kill the insect, have been  cloned.  And in fact, genetically  modified plants have been  created to express these toxins.  And it does protect against  feeding insects.  So, it's been useful in a  different way as well, in terms  of the molecules that are used  to kill insects.  >> Are these commercial  preparations, are they likely to  hurt beneficial insects?  >> Goodrich-Blair: Are the  commercial preparations likely  to harm beneficial insects?  That's a good question. 

 And I'm not sure I'm entirely  qualified to answer it, because  I don't know the species that  are being produced by some of  these companies.  For the most part, in  laboratories, you can get these  nematodes to kill any insect  that you put them on.  >> I was thinking of the  Xenorhabdus.  >> Goodrich-Blair: Oh, the  bacteria.  The bacterium itself is not  pathogenic to insects without  being carried in by a nematode.  So, we don't find the bacteria  free-living in the soil.  They're only carried inside the  nematode.  Now, it's something I should've  said, as well, in terms of the  basis of the symbiosis.  What the bacteria are getting  out of this, we think, is a safe  environment.  So, they're never anywhere  except inside the nematode or  inside the insect. 

 And then back into the nematode,  back into the insect.  You know, it's hard to say.  The absence of evidence  is not evidence of absence.  So we can't necessarily say  that these bacteria aren't  in the soil free-living,  but we've never found them.  And there's no evidence  that they do exist.  And in fact, they're a little  bit wimpy, even in the  laboratory.  We have to treat them  kind of nicely.  They don't like stress  very much.  (inaudible)  The bacteria are the primary  killing agent, yes.  So it varies depending on  species of nematode, which is  something that these biological  control companies are very  interested in understanding for  different species of nematodes,  how much do they rely on their  bacterial symbionts. 

 So it does vary.  But for the most part, the  nematodes are not robust insect  pathogens on their own.  Any others?  Okay, so how am I doing with  time?  Should I be finishing things up?  Just keep going for a little  while?  You can, you know, drag me off  the stage.  All right, so my lab has been  studying pretty much every  aspect of this symbiosis, with  these three stages sort of being  the general ones that we've been  looking at.  So, we haven't necessarily  looked very much at longevity of  nematodes in the soil.  We're really mostly interested  in the symbiotic aspects inside  the insect cadaver.  So we're interested in how the  bacteria deal with the immune  system of the insect, and how  they kill the insect.  How do they know that they're  inside an insect?  We're interested in what  contributions they make to  nematode reproduction.  And we're interested in the  molecules and the specificity  that allows these bacteria and  only these bacteria to  re-associate with the nematode,  even though there's lots of  other microbes that have managed  to get into this insect cadaver,  despite the best efforts of  other bacteria to get in there,  they can't colonize these  nematodes.  So what is it that allows this  bacterium to associate  specifically with this nematode  host?  Some of the tools that we have  available to us to study these  different aspects of the  symbiosis are what I'm going to  go over now. 

 And I'm going to start just with  the pathogenesis aspects of  Xenorhabdus.  So, I've shown you, really, how  we can use insects to propagate  nematodes.  What I'm showing you here is  Manduca sexta, the tobacco  hornworm.  And we use this particular  insect in the lab as our  pathogenesis model, because it  does have a robust immune  response.  So, you know, Xenorhabdus really  has to work hard to kill Manduca  sexta.  It doesn't go down easily.  So for us, that's a good model  for trying to figure out how  Xenorhabdus does that.  So we can recapitulate an  infection inside the lab, where  the nematode carries its  bacterial symbiont into the  insect.  But we can also just get rid of  the nematode entirely, and just  work on bacterial pathogenesis.  How we do that is just to grow  the bacteria in liquid culture  in the laboratory. 

 They'll grow in a minimal  medium, and they'll grow in a  rich medium with lots of  nutrient sources.  We can dilute that, so we know  how many cells approximately,  per mil, we have in our solution  of bacteria.  And then we can take a certain  number, 100 colony forming  units, or 100 cells, 1,000  cells, 10,000 cells, and we can  inject that inoculum into ten  different insects.  And as long as we directly  inject the bacteria into the  blood system of the insect,  Xenorhabdus can do its thing.  So the bacteria just need to get  there.  And then they can be insect  pathogens.  We can completely bypass the  nematode, essentially as a  vector into the insect.  So, this is what one of those  assays looks like.  These are ten Manduca sexta  larvae.  We get the larvae as eggs. 

 We rear them on a wheat germ  diet until they get to the  fourth instar.  Then we inject using a  micro-syringe.  And in this case, these ten  Manduca were all injected with  the same basic inoculum of  Xenorhabdus bacteria, and eight  of them died.  And two of them didn't.  So we would call that 80%  mortality, 20% survival.  This is just a percent survival  assay that we can do.  And it's pretty easy to tell the  dead insects from the live  insects, because the live ones  still keep eating, and the dead  ones look floppy.  As a matter of fact, there is  a gene in Xenorhabdus  that has been named  "makes caterpillars floppy"  or MCF.  (laughter)  Yep, I wish I had discovered it,  but I didn't.  I wish I had the creativity to  name it. 

 But you know, it was another lab  that did that.  They're very floppy.  They turn a different color.  They really get sick.  There's more than just death.  We can monitor the progression  of death.  We can see that they have  diarrhea.  They throw up.  They stop eating.  They sweat, so we think they  might run a fever. 

 And as a matter of fact, if we  put them at a higher  temperature, they can clear the  infection.  So, running a fever is something  else that we might be able to  look at eventually, using this  as a model.  Not all bacteria are created  equal with regard to their  ability to kill insects.  What I'm showing you here is a  survival curve.  The experiment done just the way  I showed you, of ten insects  that were injected with  Salmonella enterica, which for  us is a nasty bacterium.  But for an insect, not so much.  We've injected them with 10,000  cells of Salmonella.  And all of the insects survived.  So these guys had, we've  injected up to a million cells,  directly into the blood stream,  and these guys recovered, no  problem.  It's not just having bacteria in  your blood that's causing death  in this case. 

 What I'm showing you over here  is a single insect that we  monitored over time using a  machine called the in vivo  imaging system.  And in this case, the bacteria  were expressing green  fluorescent protein.  And when that green fluorescent  protein is present and apparent,  it reads red on this heat map.  If it's not present, it's  reading down here on the heat  map.  So what you can see is these are  basically very cool insects.  The bacteria are not growing.  They're not taking over this  insect over time.  So this is just a way for us to  monitor an infection in a  different way.  So, why is it that Manduca can  survive being injected with  10,000 bacterial cells?  I've already told you that they  have an immune response.  And that's comprised of a  cellular response of cells that  can eat bacteria, that can  wall-off bacteria. 

 There is a humoral response, as  well.  These are small molecules that  can punch holes in the bacterial  membranes, or create reactive  oxygen species that cause damage  and kill cells eventually.  So the way this immune response  gets triggered is back to the  beginning of my talk, when I was  talking about those molecular  patterns that are on the  surfaces of bacterial cells,  such as peptidoglycan or lipid  polysaccharide.  These are recognized by host  cells as foreign and bad.  And what they do is to turn on,  they have a signal response  cascade.  They recognize that something is  there and they cause a whole  bunch of things to happen until  they turn on all of the things  that are necessary to cause  these cells to start abrogating,  cells to start eating bacteria,  turn on the production of these  antimicrobial peptides, turn on  the production of reactive  oxygen species.  So that's all that's down here.  This type of signal transduction  cascade, as we call it,  is conserved.  So humans have it.  Mammals have it. 

 Plants have it.  Invertebrates have it.  Everybody's got it.  We're able to, from a very long  evolutionary history, recognize  microbes and respond to them  with an immune response.  So in fact Manduca sexta is  among those organisms that can  do this.  And what I'm showing you here is  a Northern blot.  So what we're looking at are  transcript levels.  These are RNA in the signal  transduction cascade, where you  have DNA, you make RNA, and  you're going to make protein.  This is looking at the RNA for  antimicrobial peptides.  Transcripts that are going to  make that. 

 Attacin and cecropin are two  types of antimicrobial peptides.  Then, actin is just our control.  What you can see is if we inject  insects with PBS, there you get  some actin.  You don't have a lot of  cecropin.  You don't have a lot of attacin.  But if you inject with  Salmonella enterica, actin looks  about the same, but now you're  getting a really robust amount  of these antimicrobial peptides.  So injection with bacteria  causes Manduca to induce immune  response.  This is true also of the  cellular response.  We can do other measures for  that.  I'm just not showing them here. 

 So why is Xenorhabdus different?  Here's our survival curve for  Xenorhabdus.  It can kill insects very  rapidly.  And then we're looking at our in  vivo imaging system.  You can see that by about 30  hours, this particular insect is  completely overwhelmed by  Xenorhabdus bacteria.  It's showing very hot on our  heat map.  Why is it that Xenorhabdus can  do this?  One reason is that it's able to  suppress the immune response of  Manduca.  So this is just that same blot  that I was showing you before.  Here's PBS.  Here's our induced Salmonella  insects.  And now, if we inject with  Xenorhabdus, we don't get an  induction of the antimicrobial  peptide response. 

 In fact, it looks just like it  does when we inject with just a  buffer solution.  So we think that this is not--  One way to look at this is that  Xenorhabdus is just invisible,  right?  Maybe it's masking its surface,  and it's hiding out from the  immune system, and that's why  it's not inducing anything.  But if we co-inject--  There are two reasons we don't  think that's true.  If we co-inject Salmonella with  Xenorhabdus, we don't get an  immune response.  So Xenorhabdus is able to shut  down, even a response that we  know can be induced.  And the second is that if we  heat-kill Xenorhabdus, leaving  the surface intact, but they're  dead, now we can induce an  immune response.  So they've got the surfaces  capable of eliciting an immune  response.  They're just shutting it down.  So our current question is,  trying to identify what  bacterial factors are allowing  Xenorhabdus to suppress insect  immunity.  And we think that it has to be  at this stage. 

 After the bacteria have been  recognized by the pattern  recognition receptors, but  before this branch of humoral  and cellular responses, for the  reasons that I just told you.  We know that they can in fact be  recognized.  But it's unlikely that they're  independently inhibiting each of  these branches separately.  So we'd like to define what  branch of the immune response is  being inhibited, and how  Xenorhabdus is inhibiting it.  But that's an open question  right now.  I think I am running out of time  now.  So, with that, I think I will  thank you for your attention.  And I'll take any more  questions.  (applause)