Sparrows, cavefish and fighting fungus

01 June 2020
Presented by Chris Smith
Production by Eva Higginbotham, Chris Smith.

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House sparrow

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This month we explore how genetic plasticity enables sparrows to live alongside us and fish to evolve rapidly to life in caves. We also hear why "Test! Test! Test!" is so critical to safe healthcare provision during the coronavirus pandemic, how a new technique can find drugs that boost the fungal killing power of fluconazole, and how changes in land use have knock-on effects on soil-dwelling invertebrates...

In this episode

House sparrow

00:36 - Sparrows: changing with the times

Genetic plasticity of sparrows may help them survive and adapt

Sparrows: changing with the times
Marty Martin, University of South Florida

With very few exceptions, wherever on Earth humans have set up home, sparrows have gone too, and this is a testimony to their incredible ability to adapt. And speaking with Chris Smith, evolutionary biologist Marty Martin from the University of South Florida, thinks it’s genetic plasticity that enables them to do this.

Marty - There are not too many species besides us, cockroaches, pigeons, a few species of rats and some mice that have so many successes in so many places and for about 20 years my group has been interested in trying to figure out why this species is able to do what so few other species seem able to do.

Chris - Can you tell us a bit about the origins of the humble sparrow? Where did it actually come from? How long has it been living alongside us?

Marty - It's been living with us for quite some time. Its origins, the oldest birds of which we have record, come from the Middle East. Many thousands of years ago it started to become quite popular in places where humans are active. There was some really nice work done in back in 2018 showing how the bird had managed to move out of the Middle East into Europe and as it moved its digestive capacity changed to be able to handle the diets it was more likely becoming exposed to. It's a granivorous species, it had always, you know, mostly focused on seeds and things. But as it became more commensal with human populations in Western Europe, it evolved to adjust to those populations.

Chris - Do we only find sparrows then where we find humans or do they actually live away from us under certain circumstances?

Marty - There are not too many places that we find sparrows without people anymore. But there are some exceptions that I find incredibly compelling. We have examples of sparrows living more than a mile underground brought there by miners and fed, you know, occasionally. So they're still in contact with people, but it's only when the miners are coming and going and bringing them food. And then there are some populations that are able to live in regions that are quite isolated from human populations. They tend to be favoring, scrubby sorts of habitats, mostly the edges of grasslands and such where their ancestors would have been. But by and large, the majority of populations now are living where people are.

Chris - And does that give us then a kind of model species to study to tell us how a species co-evolved with humans and adapts to humans? Can it tell us something about how we impact on nature but nature can mould itself around what we do?

Marty - It's becoming more and more popular as a model in that light. I should say that we wrote this paper for a special section on model organisms somewhat reluctantly because the idea of model to an evolutionary biologist, and I am one my PhD is evolutionary biology, it's always been a little bit of a difficult pill to swallow. But that said, this is a species that people have become interested in understanding how it has adjusted as we modify the environments by changing the habitats and sometimes adding more pollutants or pesticides or antibiotics or whatever it might be. There are more and more people interested in figuring out how the house sparrow deals with that as a proxy for how other, at least songbirds, would deal with that. But in a broader sense, it provides an opportunity for understanding two things. One that's more practical about as we change the planet, what types of habitats are wildlife most going to be able to occupy? And what are the mechanisms by which they do that? What has to evolve? What has to be adjusted behaviourally or physiologically for animals to thrive there? And that ends up being very interesting in a basic sense too, because those are ineffective mechanics of evolution. So how a population comes to get to new places, and maybe even ultimately how one species turns into two, has to do with the details by which individuals are moving and succeeding, breeding in new places.

Chris - What do we need to know about them, then? What do you think the big questions are for sparrows, but also birds more broadly that gets an evolutionary biologist like you interested?

Marty - My lab in particular works on something that I'm sure you know your audience has heard many times in the past, these molecular epigenetic mechanisms like DNA methylation. Without going into the details of how that works, for an animal to become bigger or smaller or faster or slower, that involves some level of gene expression and these little methylation marks, these methyl molecules sit on the DNA and influence how genes get expressed. What we're finding is this molecular epigenetic machinery is in a sense providing the species a plasticity that allows it to quickly adjust to new places. And that's compelling to me as an evolutionary biologist, again, because I'd like to know the details by which evolution proceeds, but I think it's really interesting too in the light of other species, especially when we think about how we're changing the climate, we're removing habitat and how will the species that are in those places adjust. Maybe there are some signatures in their genomes that'll help us to direct our management efforts. The sparrow being a guide for the conservation of native species is a pleasant thing to think about.

Chris: - Just sparrows that have the ability to do this or do all birds come with this enhanced ability to be genetically agile in this way and adapt in a more fluid way than say you or I could?

Marty - That's a great question, and in 10 years, I hope to have the answer for you. This field of, it's called ecological epigenetics, is a fairly young field. It's hard to do it in general, but it's especially difficult to do it with wild animals like sparrows for which we've only had a sequenced genome since 2017. It's to be expected that this process of DNA methylation exists in other species, but the propensity for species to use it differently maybe, you know, in the case of house sparrows, we think they have a lot of this stuff, what we call epigenetic potential, whether other species do is years of study yet to come.

A cave arch with light coming through the ceiling on the other side

06:36 - Cave fish evolution: how they did it in the dark

How long did it take cave fish to optimise to their environment?

Cave fish evolution: how they did it in the dark
Helena Bilandžija, Ruđer Bošković Institute

In some flooded caves, despite the cold, total darkness and paucity of food, some fish species nevertheless thrive. And they’re well adapted to their environment: for instance, they’ve lost their eyes, and their metabolism is very different from non-cave dwellers. So how long did it take these optimisations to appear? As she explains to Chris Smith, Helena Bilandžija wondered the same thing and was very surprised by the results of her study…

Helena - I was always fascinated by how these cave adapted animals ever colonized caves because we consider it as an extreme environment where you've got constant darkness and you don't have a lot of food. And you know, a transition from a surface environment to caves must be a difficult endeavor. And so there is this general model that explains how all of the ancestors of cave dwellers lived on the surface. From there, they entered the caves and through successive generations they acquired a series of these adaptations that allow them to survive in this extreme environment. Many, many different traits: morphological, physiological, there are neuro changes, behavioural changes. However, the surface ancestor who entered the caves didn't have any of these adaptations. And so how did it manage to survive? And so this was something that was always bugging me.

Chris - Over what sort of timescale do we think that the original species adapted to become a cave dwelling equivalent?

Helena - Well, that's the thing. It was always considered that it took millions of years, and in recent years a number of studies came out that suggest how some of these cave dwellers have originated very recently. And the species that we worked with, Astyanax mexicanus, is a wonderful model species because you have both the cave form and the surface form still present. So you can do a direct comparison. And recent studies have dated the cave fish lineage anywhere between 30,000 and 200,000 years ago. So this is a very short timeframe for the evolution of all these different traits.

Chris - And you said it bugged you, that you wanted to know how so many of these changes could be accrued in such a short space of time. So what did you do to find out?

Helena - We took the ancestral form of Astyanax, which is a surface fish, and we exposed them to conditions that we thought these ancestors had when they first entered the caves. And the only environmental cue that is present in all different subterranean habitats is actually darkness. So we took our surface fish and we put them in the dark and asked, how were you able to survive once you were flushed into the caves?

Chris - How long did they have to live in the dark for?

Helena - Our fish were in the dark anywhere between seven months and a couple of years.

Chris - So you've got a group of fish that you keep as controls presumably, and they're just having normal day and night cycle. And then you've got this group of fish, you're simulating cave dwelling, and you've abruptly put them into complete darkness for up to two years. My mind is boggling slightly about how you actually do the experiment. Then how do you keep fish in total darkness and still be a scientist who needs light to see? How did you study them?

Helena - We had a separate room in which we maintained complete darkness and when we needed to study them we would enter with dim red light so that we were able to see.

Chris - So you go into a room, which is like a photographic dark room from the good old days, take a small sample of the fish that have been living in the dark, and then you basically are just asking are these guys any different than the ones that are in the room next door with the normal light cycle.

Helena - Yes.

Chris - And when you do that, how quickly do you start to see changes, if any?

Helena - We started seeing changes already in the first generation of the fish. So we put our surface fish in the dark already a couple of days after they were spawned. And a couple of months later we looked at different traits and already we saw a series of traits being changed just by putting the fish in the dark. Higher fat content, higher starvation resistance, lower metabolic rate, different hormone levels, a series of traits that we didn't expect. And what was really remarkable was that the direction of these changes mimicked the cave fish phenotype.

Chris - The difference between the fish that have evolved over a long period of time in caves is that many of those adaptations are fixed genetically, are they? Whereas your fish change very rapidly to start adopting some of these characteristics, but they can't change their genetic information in such a short time. They must've just changed how they're using their genes.

Helena - Yes, the changes that we saw in the surface fish when you put it in the dark, were basically phenotypic changes and this is called phenotypic plasticity where you have a change in the phenotype without change in the genotype. However, these changes, these adaptations in cave fish are genetically imprinted and there must have been something called genetic assimilation that allowed these phenotypic changes to somehow get imprinted into the genotype of cave fish. And now they are irreversible because when you put cave fish back into the light, you don't get reverse of these phenotypes back to the surface fish. They are fixed in their genomes.

Chris - Why is it a one way street then in the sense that, why is it easier for a non-cave dwelling fish to turn into a cave dwelling fish? But the adaptations can't be so readily undone?

Helena - It's because all these adaptations in cave fish are now genetically determined. There have been mutations in some of the genes that cause, I don't know, the loss of pigmentation or the increase in fat deposition and so on. So now, although there is still some plasticity in cave fish, we have discovered it never goes all the way back to the surface fish phenotype.

A team of doctors walking through a hospital corridor

13:30 - Screening healthcare workers for Covid-19

Some asymptomatic hospital staff are carrying coronavirus...

Screening healthcare workers for Covid-19
Michael Weekes, University of Cambridge

"Stay home, stay safe, protect the NHS" has been one of the UK government’s strategies to control the spread of coronavirus. But what about in hospitals themselves? Patients with the infection are of course being treated there, and staff need to come to work to look after them. It's, naturally, much harder to maintain social distancing in healthcare settings too. So how many staff are infected with the virus? Surprisingly, until Cambridge University Infectious Diseases consultant Mike Weekes and microbiologist Steve Baker set up a project to find out - by screening over a thousand healthcare workers at Addenbrooke's, the University of Cambridge teaching hospital -  no one knew. Speaking with Chris Smith, Mike Weekes first…

Mike - It's obviously a huge issue because staff within the hospital could transmit the infection to other staff and to patients, and actually hospitals could become their own individual hubs of transmission unless we do something about it. Staff wear PPE throughout the wards, but obviously people don't wear PPE in staff rooms; they don't wear PPE when they go to lunch, because you can't; and so there's the possibility that the virus could transmit. We got our infectious diseases team to visit different wards around the hospital, and take swabs from people's throats and noses, so that they could go to Steve's lab and we could see if they actually had coronavirus.

Chris - You got these samples from all over the hospital. How many samples did you get, Steve?

Steve - At the moment I think we've screened about two and a half to three thousand individuals in the hospital.

Chris - How long does this take?

Steve - The whole process from the point that we can receive the swabs, to we can report back - the quickest we've done it is about four hours. Usually we report back a result within 24 hours.

Chris - And Mike, you've looked at these staff members. What did you find?

Mike - It's really interesting. We actually found that three in a hundred people tested positive for coronavirus, and actually these people were split into three groups. The first is people who have no symptoms at all, and so they just have no idea they have the coronavirus. The second is a group of people who've had mild symptoms, like a bit of a cough, a sore throat, some have even lost their sense of smell; and they don't think that they have enough symptoms to warrant doing anything about, in general. The final group we found, actually, had had coronavirus a long time ago; and they'd had fever and a prolonged cough, the typical symptoms, and they appropriately self isolated at home and then came back to work when they were well. You can still test positive even though you shouldn't be infectious.

Chris - The fact that these people are not reporting symptoms is worrying though, isn't it? Because we're relying on symptoms to detect who we think might have it in society.

Mike - You're quite right. Unfortunately this is one of the features of coronavirus, that some people just have no symptoms; and some people's symptoms are so minor, they just don't think they should do anything about it. But we're really encouraging staff now to come and see us, so they can get tested if they have any symptoms.

Chris - Hospitals are seeking to control this by dividing the hospital up into areas that are red, they have patients in them with coronavirus as a diagnosis, confirmed; there are also areas of the hospital which are regarded as green, those people don't have coronavirus. If you look at the staff who work in those different areas, are there any differences in the likelihood that they've got coronavirus infection, because they're being exposed more in red areas? Do they catch it more?

Mike - We did actually find a significant difference between red and green areas. In red areas a greater proportion of staff actually do have coronavirus overall, but it might be because staff are getting it from patients. It might be staff getting it from staff, or it might even be that we'd sampled more of the red areas a bit earlier. We sampled over the course of three weeks, but as everyone had been on lockdown, there were fewer and fewer cases in the community. We can't make any firm conclusions from this, but what we can definitely say is that this needs to be studied a lot more.

Chris - What if you're asked by the hospital, "right? What do we do with these findings? What are your advice points?"

Mike - Test, test, test, and then test again. Because the kind of approaches we can apply in the community, like these contact tracing apps on iPhones, won't necessarily work in hospitals because so many people have contact with other people.

A line of differently-shaped pills.

18:35 - Killing fungi by boosting fluconazole

Combining treatments to increase sensitivity to anti-fungal drugs

Killing fungi by boosting fluconazole
Jessica Brown, University of Utah

Now to a potential strategy to boost the fungus-killing power of the commonly-used drug fluconazole. Jessica Brown has been looking for chemicals that can sensitise fungi to the effects of this drug. She’s began with another agent that has the effect she wants but isn’t suitable for clinical use, and she’s used that to select for test fungi that can highlight other potential compounds. As she explained to Chris Smith, the strategy has also revealed some potential drug combination “no-nos” that can actually cut the effectiveness of fluconazole... 

Jessica - Our goal was to improve the treatment of fungal infections called cryptococcal meningitis. And this is when an infection caused by the fungus cryptococcus neoformans spreads from the initial site in the lungs and then disseminates to places such as the brain. And there are relatively few drugs, fewer than there are for antibiotics, because the cellular structure of the pathogen is very similar to our cellular structure. What functionally this means for cryptococcus is that the treatment itself takes about a year. Patients receive high doses of an oral drug called fluconazole. And the problem with fluconazole is that it is what's called a fungistatic drug. So it will inhibit fungal cell growth. That'll cause the cells to become static, but it won't kill the cells, which means that if they're a solid organ transplant patient, they basically are on these drugs for life.

Chris - So what can you do to improve things?

Jessica - So we wanted to improve this by finding drugs that increase the activity of fluconazole, such that it will better kill a fungus and we can ideally eventually shorten these treatment times. So what we've done is we've identified drugs that amplify the activity of fluconazole. And in our case at least one of the drug pairs we found convert fluconazole from static to cidal.

Chris - Do you know how it does it?

Jessica - So our best hypothesis about how these drugs work is that they target a pathway that interacts with the pathway targeted by fluconazole such that when you inhibit two pathways simultaneously, you're then able to kill the cell instead of just causing it to grow more slowly or stop growing.

Chris - So how did you go about, in the first instance, finding these agents that could do this? What was the approach?

Jessica - What we did is we took fluconazole and we took a second drug that is known to show this interaction with fluconazole that we want, the synergistic interaction where they amplify each other's activities. The problem with the second drug is that it's an immunosuppressant. So we can't really use this to treat patients, but we can use it as a training drug and use its properties to try to identify other drugs that show similar properties. So what we did is we treated fungal cells that were either what's called wild type, a standard healthy fungal cell, or carries a mutation in a subset of genes. We then grew those fungal cells in the presence of these two drugs and we identified a subset of mutants that showed a certain response to each of these two drugs. Now these two drugs have very different targets within the fungal cell, and so normally genes whose mutants would show a response to fluconazole would not show a response to the second drug. However, we noticed that with just a couple of mutants, they've responded to both the fluconazole and the second drug. And so we then used those mutants in a much larger screen to identify additional drugs that showed that same response.

Chris - So you don't know what the changes that's happened in these cells, you just know that by some mechanism there is something in these cells that means that in the presence of both fluconazole and the other agent, these cells will die. And so you can now use them as almost a marker. So if we come along with another drug, they're going to be exquisitely sensitive to another drug that will work in the same way as the other agent that wasn't suitable enabling you to find other agents that will potentiate the action of the fluconazole.

Jessica - Exactly. And what this means is that because we are looking for things that show a hypersensitivity for anything that potentiates fluconazole, we can then use this in a very large scale screen. We screened a library of molecules that were already approved by the FDA to treat other diseases. And the logic behind this was that because they're approved for other indications, they can be used off label by physicians who think that this is an appropriate form of treatment. And thus they don't require expensive clinical trials and can get into the clinic much more quickly. And what we found is that for our most promising combination, we did see a considerable present killing after 24 hours of treatment.

Chris - And out of interest, what is that promising compound that you've identified?

Jessica - So one of our favourites is a drug called dicyclomine and this inhibits G protein coupled receptors. It's what's called orally bioavailable, which means it can be taken as a pill. You don't need to be hospitalized to receive IV treatment. And it's normally used to treat irritable bowel syndrome, it relaxes muscles. But in fungi we find that it acts very strongly in combination with fluconazole. And when we infect mice with a fungus, let that infection progress until the mice have a brain infection, and then start treating with them with fluconazole plus dicyclomine we see an over twofold increase in the time to death for these mice compared to just treating with fluconazole alone.

Chris - Is that agent an easy bedfellow with the kinds of drugs that we will also be using in the sorts of patients that you're seeking to treat though? Because some drug-drug interactions preclude the use of some combinations that we would love to use, but they remain out of scope. So will it work in that way?

Jessica - So that we haven't tested yet. I haven't seen data suggesting that interactions for dicyclomine are a major problem. This is a very important question though, because part of what we found in this study is critical antagonistic interactions with fluconazole, where antagonistic interactions are those that decrease the efficacy of a drug of interest.

Chris - And that was going to be my next question, which is did you discover the reverse effect, which is some drugs which actually we hadn't realized are really very bad for the ability of fluconazole to poison fungi and therefore we ought to avoid them in our patient population at all costs?

Jessica - Yes, we did. And this is a particular problem for some of our patient populations. And so what we found was that a subset of very commonly used antibiotics decreases the efficacy of fluconazole very substantially. And since these antibiotics are those that are frequently used to treat staphylococcus infections, this could be a problem for our patient population.

Hands holding soil

Insect diversity: when humans move in
Andrew Dopheide, Manaaki Whenua Landcare Research

When humans move into an environment, we inevitably change it: trees are felled, brush is cleared, crops are planted and non-native animals are reared. The soil underfoot is of course still there, but what are the consequences of these land-use changes for the insects and other invertebrates that live underfoot? The answer is that their diversity shrinks too. Chris Smith spoke to Auckland-based ecologist Andrew Dopheide…

Andrew - We used a technique called DNA meta-barcoding. We use DNA sequencing to identify lots of different insects and other small animals in a very efficient and large scale way. The team of researchers visited 75 sites throughout New Zealand, each of which was in a different land use type such as agriculture or horticulture or forest and collected a number of soil cores. The invertebrates got separated out and then were taken into the lab and analysed.

Chris - Presumably you grind this lot up and then just say, right, what DNA sequences are in here and then compare it to a database.

Andrew - Ah yes that's right.

Chris - To what extent is this quantitative? Cause I can see how that would enable you to tick things off as you discover them: I've got the genetic sequence of this, I've got the genetic sequence of that, but how does that relate to numbers? Can you tell roughly how many of any given thing is there?

Andrew - Not exactly. We use a technique called PCR to copy or amplify the bits of DNA that we're interested in. The amount of DNA we get from different organisms is quite variable and therefore doesn't accurately reflect its abundance.

Chris - So it's reasonably good for working out the broad spectrum of what is there, but it's not going to tell you very much about how much of any particular thing is there, that would take a different kind of study to elicit that information.

Andrew - Yes, that's right. However, if you have preexisting information about how a particular organism, how its abundance works, you can actually do that.

Chris - And what were you comparing then? Were you just saying, well I'm going to compare the forest to this area, which has had animals on it. And what are you actually then comparing those things to, what's the gold standard?

Andrew - We compared five different land use types. So at the one extreme we've got native forest, forest that's always been here, relatively undegraded and unimpacted. Then we've got pine forest, which has been planted for forestry purposes. Two different levels of agriculture, so one of them is quite low intensity, just tussock grass, few sheep, that sort of thing. Another is more high intensity agriculture, so just a monoculture of grass and lots of fertiliser inputs. And then at the other end we've got perennial crop land, which is basically horticulture, fruit and nut orchards primarily. We figured that the native forest sites represented a relatively natural state for New Zealand. So by comparing all the other land uses to the natural forest, that gives us a measure of how things have changed as a result of agriculture.

Chris - And how have they changed?

Andrew - We observed a general decline in the number of species as you go from natural forest to grassland to cropland. Each of our native forest sites had quite a unique set of species that weren't found anywhere else. But as we looked at grassland sites and horticulture sites, we found that the communities of animals became more and more similar everywhere. So it goes from native forest having very distinct and unique sets of organisms or animals to agriculture, just having the same things everywhere.

Chris - And do you know why that might be happening? What do you think the mechanism is?

Andrew - It's probably due to factors such as removal of trees and replacement with grass. Really just simplification of the habitat. So there's fewer places for different animals to live, fewer food sources. And also you've got these chemicals which are designed to suppress these same animals as well, all in order to make a more friendly environment for things such as cows and sheep. But at the same time you're removing the habitat for all the smaller invertebrates.

Chris - It sounds pretty worrying, doesn't it? Is this a permanent thing? If you were to revert the environment back to what it would have been originally, do you get your species back?

Andrew - That's something we do not know, but I think it would be worth trying.

Chris - And what might be the consequences of not having these species there if you lose these animals? Obviously a loss is a loss and that's sad on so many levels, but what are the more direct consequences of not having some of these species there?

Andrew - All these species of animals have a role in their ecosystem. All together, they're contributing to the fertility and the stability and the health of the soil basically. So if you remove these invertebrates, you're left with a soil community which may be unstable and less resilient to environmental stress, such as drought or climate change.

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