eLife Episode 10: Radiation, Anti-aphrodisiacs and Glowing Squid

31 March 2014
Presented by Chris Smith.

In this episode of the eLife podcast we hear about the mating habits of flies, radiation resistance in bacteria, how insects learned to smell, and the Hawaiian bobtail squid...

In this episode

00:41 - Vive la résistance

Single mutations in three genes can increase the ability of E. coli to survive ionizing radiation by a factor of 1000.

Vive la résistance
with Michael Cox, University of Wisconsin-Madison

Michael Cox from the University of Wisconsin Madison has been conducting an experiment into evolution. He has been speaking to Chris Smith about what was discovered... Radiation Resitant E. coli

Michael -   We wanted to understand what makes a cell resistant to ionising radiation.  So, ionising radiation is the kind of radiation that you get from x-rays.  It's the kind of radiation that causes enormous damage to cells.  It's important for a few reasons.  There are bacterial species and also some fungal species frankly that are very highly resistant to ionising radiation.  To give you an idea, ionising radiation is measured in units called gray.  A human being would be killed by having a dose of 3 to 5 Gray.  Bacterial species that we work with called Deinococcus radiodurans can survive 5,000 Gray with no lethality.  So, it's literally thousands of times more resistant than a human being would be.  A second issue is just understanding why ionising radiation causes cells to become sick - is it damage to their DNA? Is it damage to proteins in the cell?  What is it about ionising radiation that is most dangerous to a cell and causes radiation sickness?

Chris -   How have you pursued this?

Michael -   Well, we realise that we could let the cells tell us what was important basically.  You do that by doing an evolution experiment.  So basically, we took E. coli cells which are not particularly radiation resistant and we subjected them to high doses of ionising radiation, enough to kill 99.9% of the cells.  And then we grew up the survivors and then we did it again.  We went through 20 cycles of this selection.  And by the time we were finished, we had a culture of E. coli cells that was almost as resistant to ionising radiation as with Deinococcus.  We did that experiment four times and got the same result all four times and it was those populations of very resistant cells that we used to try to figure out what was important in making a cell resistant to ionising radiation.  The advantage of doing this experiment is you don't have to look through all the genes in the cell.  All you have to do is figure out which genes had changes in them.

Chris -   So, did you literally take the very  resistant coliforms that came out of the evolution experiment and just like for like compare the genomes?

Michael -   We took about 30 isolates from the various populations and obtained a complete genomic sequence of each isolate.  That told us where all the mutations were in which one of these isolates.  Now, a typical isolate has about 70 mutations and many of those mutations are irrelevant.  They're just neutral mutations that kind of appeared during the selection and don't really contribute to the phenotype.  So, the real job was to figure out which of the mutations were really important.  By this doing with four different populations, we could compare isolates from different populations and find patterns.  And so, we focused on genes that were targets of mutations over and over again in the different populations.  We were able to home-in on mutations that had a particular importance to this new capacity of the cells to resist ionising radiation.

Chris -   When you looked at the resistant forms, what changes had they adopted?

Michael -   In the population that we looked at, we were able to explain virtually the entire phenotype on the basis of three mutations and these mutations were all the genes that encode proteins that are involved in DNA repair.  In the case of this one population, the major change that occurred in the population was to improve its capacity to repair its DNA and explain virtually the entire phenotype.  Now, in other populations, there are mutations that we suspect are having a different kind of effect, making it more difficult for the proteins to become oxidatively damaged by the radiation.  So, in these other populations, some of the adaptations that are occurring have to do with protecting the cell from ionising radiation rather than repairing the DNA.  And so, this is an example of another way to make more resistant ionising radiation.

Chris -   What do you think the implications of this are?

Michael -   It's likely to give us a better understanding of what makes a cell particularly sick, what makes it susceptible to radiation poisoning.  I think damage to its proteins and damage to its DNA is clearly the major thing that happens to cells.  Another implication is, we should be able to generate eventually, radiation resistant bacteria that could potentially be used to give bioremediation and radiation waste dumps.  I think that there is potential to use some of these technology in useful ways.

06:13 - Buzz off: anti-aphrodisiacs in flies

Male flies rub chemicals called TAGs onto female flies during mating to make them less attractive to other males.

Buzz off: anti-aphrodisiacs in flies
with Joanne Yew, National University of Singapore

Pheromones are chemicals that can alter behaviour like mating.  Now, researchers have discovered the pheromone equivalent of an anti-aphrodisiac.  National University of Singapore scientist Joanne Yew found that male fruit flies impregnate a female fly with a cocktail of fats which puts other males off wanting to mate with her. Fly anti-aphrodisiac secretionShe has been speaking to Chris Smith...

Joanne -   Pheromones are chemicals that animals use to communicate with each other and we wanted to see what kinds of different chemicals are used by different species of fruit flies.  So, the reason that we might be able to see something that people hadn't seen before is because of a method we worked out a couple of years ago where we use laser to scan the surface of the insects.  And by doing so, we're able to detect different kinds of molecules then those kind of classical methods that are used to identify these molecules.

Chris -   So, that laser technique would enable you to study, not just what the insect itself is producing, but potentially, what another insect it's been knocking around with may have left on it.

Joanne -   Yes, that's right.  And so, by using a really small laser compared to the size of the insect, we get really good spatial specificity as to where these different molecules are expressed on the surface and also, where they get transferred, following some kind of behavioural interactions like mating.

Chris -   And that particular laser technique doesn't just tell you where on the insect.  It will actually give you the chemical identity of the substance, will it?

Joanne -   Yes.  Well, it gives you a good guess.  So, based on that guess, you know what is the elemental composition, what kinds of different atoms are there, and probably, number of double bonds they have.

Chris -   When you started doing these with these fruit flies, what did you see that stood out as clearly different or exceptional compared to what previously people had managed to spot using traditional techniques to study pheromones?

Joanne -   So, we had an expectation of the kinds of molecules you would see based on their mass.  So, we have a laundry list of things to look for, but what we saw that was novel was that in males only and not females, there are series of molecules that looked a lot heavier than what had been reported before.  And then when we looked at things like the elemental composition and number of double bonds, it seemed to fit characteristics of what we would expect from particular kind of fat.

Chris - So, these male flies are depositing onto the females, into the females' fats which appear to be linked to mating or influencing mating behaviour.

Joanne -   That's right, yes.

Chris -   So, what are these fats and how do they change the behaviour of the female, and when does the male put them on the female?

Joanne -   They're transferring a particular kind of fat called triglyceride.  This is a common kind of fat molecule and so, males express it in a region close to their genitals.  And so, when they mate with a female, they're transferring sperm as well as these particular fats.

Chris -   And the triglycerides go onto the female, but do they affect behaviour of the female or do they affect the behaviour of other males or both?

Joanne -   We looked at both aspects of that.  Females after they mate, they exhibit a series of rejection behaviours, where they really are not interested in mating anymore for some species.  And so, if a male tries to mate with them, she will kick them in the head and run away.  And so, we tried to see if we perfumed the female purely with these triglycerides and not have her mate with the male would cause her to do any of these behaviours.  But it doesn't seem to be the case.  So, it seems that it's more to tell other males to avoid this female.

Chris -   Let's just run over that.  The female that you coat in this material - in these triglycerides - does not appear to be less receptive to mating, but she's actively avoided by males that could potentially mate with her when this chemical is present.

Joanne -   Yes, that's right.

Chris -   Do you know how the males are picking it up or how they're responding to it?  If they do respond like this, why don't they get confused with their own smells and therefore, just go into a non-mating state?

Joanne -   Yes, that's a great question.  We speculate because it's a context-dependent.  And so, they're smelling this smell on top of the female's natural scent.  This whole bouquet of the female scent plus this added male fat is probably the signal that tells other males to avoid this female.

Chris -   Now, you've looked just in these insects in this first instance, but do you think that this whole concept - for want of a better phrase - an anti-aphrodisiac, do you think this applies in other animals too and dare I say, even in humans?

Joanne -   I think so.  I mean, I know so because there's actually a large body of literature showing this phenomenon in other insects.  In humans, it is difficult to say especially in terms of chemical signals, but it benefits both the male and the female to have some kind of signal indicating a period where the female is not interested where males won't be successful.  So, it seems reasonable that it would be a conserved mechanism.

12:06 - How squid and vibrio bacteria talk

The Hawaiian bobtail squid and V. fischeri bacteria use a chemical conversation to establish a close working relationship...

How squid and vibrio bacteria talk
with Caitlin Brennan, Harvard

There is a unique partnership between a small 3-cm long squid called Euprymna scolopes and a bacterium called Vibrio fischeri.  In return for sugars and amino acids, the bacteria inhabit specialised organs on the squid where they pump out light, providing the animal with camouflage.  But how do these microbes talk with their hosts?  Caitlin Brennan has found that molecules from the sheaths around the flagellae that they use to propel themselves along are the critical component. She spoke to Chris Smith... Hawaiian Bobtail Squid (jpg)

Caitlin -   We're really interested in how bacteria are moving around and using a flagellum to swim through different media and different liquids, and how this behaviour was important for interaction with host.  To do this, we were using the squid-Vibrio mutualism, which is a model of host-microbe interactions and is used to look at issues of host specificity.

Chris -   So, what did the squid do with the Vibrio bacteria?

Caitlin -   The objective of the mutualism is to provide light for the squid, which is for counter-illumination which is a camouflaging mechanism and Vibrio fischeri is the only bacterium that's able to enter the specialised organ, grow there and provide the light that the squid needs for this behaviour.

Chris -   So, how did you use that system to understand what the flagella does normally in relation to its host?

Caitlin -   So because of the specificity between one bacterium and one organism, we're able to probe various specific questions in terms of the conversations between these two organisms.  In a way, we're not able to in a more complex environment like the many, many microbes that are found in the human gut.  That conversation is very, very complex because there are so many players.

Chris -   So, do you think then there's some kind of signal coming out of the bacterium which is altering the behaviour of the squid and in order to achieve this mutualistic association?

Caitlin -   So, we've shown in the past that there are specific signals put out by the bacteria that are really actually common signals to many bacteria.  So, these include things like LPS or lipopolysaccharide and parts of peptidoglycan.  These are surface molecules that are found on many, many bacteria, not just Vibrio fischeri.  But what's unique about these signals is where they're expressed by Vibrio fischeri and where this squid is being exposed to them.  They're being exposed to these signal molecules at a place only Vibrio fischeri is able to access.  Exploiting this relationship to sense when Vibrio fischeri is there.

Chris -   When you study this interaction, what is this telling you about that chemical conversation?

Caitlin -   So, it's telling us a lot about whether these molecules are the same molecules that are often causing really reactive host responses and a pathogenic relationship.  If this was happening in your gut by the wrong bacteria, you'd be very sick and you'd have an inflammation.  But here, it's part of the natural progression of the interaction between Vibrio fischeri and the squid.

Chris -   So, how did you actually study this?  What did you do in order to unpick this?

Caitlin -   So, we used three different strains - one bacterium that produces a flagellum and is able to swim, another that produces a flagellum, but can't actually rotate this flagellum so it can't swim, and one that doesn't produce a flagellum at all.  Using these three different strains, we're able to pull apart what we'd actually do to the production of a flagellum and what we'd actually do to being able to swim to rotate that flagellum.  What we found was that it didn't matter whether Vibrio fischeri made a flagellum.  If it couldn't swim and if it couldn't turn this flagellum then it was unable to cause this host respond.  So, we looked a little more closely at the flagellum of Vibrio fischeri and it's known that it's covered in a sheath.  This particular flagellar sheath is a continuation of the rest of the outer membrane of the bacterium.  And so, what this is largely made of is lipopolysaccharide.  And so, we wondered whether being unable to turn this prevented release of this molecule in a way that stimulated the host response by wild type Vibrio fischeri.

Chris -   What is the effective bottom line here then?  So, the bugs use their flagella to release or dispense LPS and that the host is responding to that LPS and in the case of the squid, for beneficial outcome.

Caitlin -   This flagellar sheath is something that's been very poorly understood.  I found a review at one point during my graduate work that I believe was from 1981.  It was the only review there's ever been about a flagella sheath.  And every single question they posed remains unanswered to this day.  People have been assuming that the function of this sheath is to really prevent an immune response to the flagellum subunit.  We just haven't had the tools to probe that in any bacteria that has a flagella sheath.  This includes several important human pathogens like Helicobacter pylori and Vibrio cholerae.  What we were able to do was use the squid-Vibrio symbiosis to highlight something that's potentially common to all these bacteria with sheath flagella and maybe important in how those bacteria are interacting with their respective hosts as well.

17:31 - Flight drove insect olfaction evolution

Up in the air: why flight drove evolution of insect olfaction

Flight drove insect olfaction evolution
with Christine Missbach, Max Planck Institute for Chemical Ecology, Jena

Insect olfaction evolutionHow did insects come by their sense of smell? They actually have two different smell systems - an ancient one that they share with their crustacean cousins like crabs and a newer one that they've evolved separately.  Chris Smith spoke to Christine Missbach...

Christine -   Insects mainly use two receptive families.  One, the family of so-called insect olfactory receptors - these are specific to the insects.  The closest relatives of the insects, the crabs, do not have this kind of receptors.  It's the hypothesis that insect olfactory receptors have lost when the insect had to transition from water to land, and detect airborne chemicals.

Chris -   So, how did you try to understand whether that was the case that as soon as the insects left the water and came on to land, they responded by producing a new class of olfactory receptors?

Christine -   Most studies on the insect olfactory receptors were done in flying insects.  So, nobody has ever looked into taxa that are older, for example, the jumping bristletails or the firebrats.  And by comparing the results we got from those two higher insects or the closely related crustaceans, we hope to get new insights into the evolution of this insect olfactory genes.

Chris -   So, how did you study those insect species which are these more primitive non-flying insects to find out how they respond to smells?

Christine -   First of all, of course, we had to show that this insect have a functional olfactory system.  Using electrophysiology, the insects have very long antenna that are covered by small cuticular structures called sensilla and the neurons are inside the sensilla and we can contact these sensilla and record from the neurons inside the sensilla.

Chris -   So, you were making electrical recordings from the olfactory apparatus of these insects and presenting them with different chemicals to see if they all responded and if they responded in the same way to those chemicals across the different classes of insects you studied.

Christine -   Yeah.  First of all, we can use this technique to show that they can smell and also, try to categorise the different neurons in a sensilla and count also the number of types of neurons that are present.

Chris -   And that will presumably give you a handle on that evolutionary timeline because presenting these chemicals to the different groups of insects, I presume what you're able to do is to show that with evolutionary time, the smelling ability becomes more diverse.

Christine -   Yes.  First of all, the numbers of neurons we identified for this primitive insect were much smaller and also, rather broadly tuned.

Chris -   So, did you then, having identified the responses to the chemicals go to the genome and find the genes corresponding to these receptors?

Christine -   We used RNA-Seq, looking which genes are active in the olfactory tissue and then searched into this data for the receptors.

Chris -   So, putting all of the findings together, what do you conclude?

Christine -   We think the insect type of olfactory receptor is not an adaption to the terrestrial conditions and the detection of volatile, air-borne chemicals but evolved later during the evolution of insects.  We think that they could be an adaption to the insect fly because during flight, also, much higher temporal resolution and higher sensitivity is necessary.

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