Gins & Genes: to catch a carcass
A listener has - kindly - sent in somewhere between ten and twenty dead flies. She's challenged us to do a high-tech fly autopsy: to sequence whatever DNA we could find, and figure out what the flies are. And what were they eating before they died? The results are in - and it's time for Gins & Genes...
Eva - Hi, I'm Eva Higginbotham and I'm a PhD student at the Department of Zoology.
Ed - Hi, I'm Ed Farnell. I'm a product support scientist at Illumina.
Matt - Hi there, I'm Matt Routledge. I'm an infectious disease doctor at Addenbrooke's hospital.
Phil - Welcome everybody. It's great to have you here for Gins & Genes. Will, what are we going to be drinking today?
Will - Today we're going to have a look at anty gin, which is a gin literally made from ants.
Phil - From ants.
Will - Yes. Certain ants have certain flavours about them. It's the formic acid that these ants use to both defend themselves and communicate, has a sort of lemongrassy flavour for us. I'll let you taste it and see what you think.
Eva - Ooh, yeah.
Ed - Okay. Yeah, yeah. Lemongrass, I guess.
Matt - Yeah. Wow. Yeah.
Phil - I don't like it.
Will - Well look, four out of five's not bad. So why don't you try it with the tonic as well?
Phil - Bottoms up everyone. So the reason we're all here: Matt, you work in the lab that had these flies, right?
Matt - Yeah. So I've got to admit, I've got slightly a vested interest in finding out what potentially I may have been inadvertently exposed to by these flies.
Phil - Was it a big infestation?
Matt - It wasn't as if we were batting them off all the time, but there were plenty. We have no idea why they got in. We don't know where they came from.
Phil - Okay. The field is open. Ed, we gave them to you. What did you do next?
Ed - I took one fly and basically dunked it in an extraction buffer to get the DNA out, but tried not to disturb the fly too much so we would just get the things on the outside. And then with another fly I mashed it up really well, which is quite disgusting, and then dissolved that all in lysis buffer. So we have this comparison of potentially what's in the fly and what's on the fly.
Phil - Did either of them work?
Ed - We were really lucky, I guess. Both of our extractions worked on the first attempt. One of the first interesting things is, we thought, we'd compare our different extraction methods and see what we have. So for the exterior-only fly, our fly that we dunked in the buffer, we found that was actually enriched for an epibiont signature - which is a word I did not know until today - but that means basically things that are on the outside of stuff. Which was really, really good! We found a signature for the outside of things on the outside of our fly. And we also find it didn't contain an insect intestinal signature. So we managed also not to get the outsides of our fly into our exterior extraction. And on the opposite side we found that the whole-mash fly had both the epibiont and the intestinal insect signatures.
Eva - If you were specifically looking at just gut bacteria, then the ideal scenario would be of course to dissect that out of many, many flies, and your hands would get really tired, and you would hate yourself by the end of it. But you'd want to dissect them all out and then just run your experiments on that and your extractions on that.
Ed - I found that my 15-year-old dissection skills weren't quite up to the job,
Eva - But it is really promising that you actually found your controls worked, right. You found that there were species of bacteria that should be on the outside, and they were there, and there are species of bacteria should be on the inside, and they were also there. So that's great.
Phil - So what fly did we give you?
Ed - So the flies that we had were Calliphora vicina. Common name for it is the urban bluebottle blowfly.
Phil - Calliphora vicina. I put the name to Zen Lewis from the University of Liverpool.
Zen - I don't know much about that, but I think it's one of the blowflies and I believe it's one of the species that's used in forensics to work out how long it's been since a body became a body. I'm not sure that anyone has examined what the core microbiota is, but I have read some work which suggested that because the flies feed on cadavers, they actually pick up bacteria from the cadavers that they eat.
Phil - This episode doesn’t get any less disgusting, does it? But at least this is promising news. If the flies pick up the things they eat, can the DNA sequencing figure out their last meal? Back to Gins & Genes.
Ed - When we compared the whole fly and the exterior fly samples, we actually found that chicken DNA and a species of fish were enriched in the sample that had come from the mashed up fly rather than the exterior, which might point us towards what they'd been feeding on.
Matt - Okay, right. So at least now we know what to go and look for. Probably some chicken wing or fish finger or something behind the radiator somewhere.
Phil - Who brings in chicken wings or fish fingers for packed lunch?
Matt - I don't know. I mean...
Phil - Is it you?
Matt - It's not me! No, no.
Phil - How big was the trace of chicken and fish in there?
Ed - So it wasn't very high. And I think this sort of comes around to some of the caveats of our experiment. So we would have needed another the fly that we'd taken from a completely different environment, and have also extracted that, and that would have controlled for any DNA that was in our environment when we were doing the extraction in our labs, or indeed just giving us a background of what's normal in a fly.
Phil - Plus do you know how long ago necessarily the fly would have had to have eaten the fish or chicken?
Eva - The flies that I know about are actually a little bit different from the flies we're talking about here. So I work on Drosophila melanogaster, which is a fruit fly, and they're quite a lot smaller I think than the blowflies you're talking about. But if fruit flies are anything to go by, they actually have a quite complex digestive process. Their stomach is split into a midgut, a hind gut, and a foregut; but the midgut itself is split into five sections as well, and actually each of those have their own version of a microbiome and bacterial signature. I don't know if that means that if a fly ate a little bit of chicken, it's going to hang around for a long time; but it does go to show that actually we think of these insects as being really, really simple, but they have in some ways a more complex digestive process than we do.
Phil - Did you find anything else in there that might've been hiding or living inside their digestive system?
Ed - We were actually really surprised that there weren't more bacteria. We kind of expected flies... you think of them as being fairly disgusting, living in rubbish dumps; actually the proportion of bacterial DNA was really, really low within the samples. But we did find a couple... one of the most abundant bacteria found was actually Cryptosporidium muris. That can cause diarrhoea in immune-compromised individuals. So that's really not nice, and was there in actually a fairly decent quantity.
Matt - In clinical practice, Cryptosporidium we generally think of as a gut pathogen, and we quite commonly pick it up when we take stool samples from patients. And in the main it doesn't cause anyone any significant harm. It's only usually patients who've got HIV, or they're on kind of immunosuppressive therapies, that we worry about. Cryptosporidium.
Phil - So this might be something common to a lot of flies that live around people?
Matt - Yeah, I would have thought so. I mean, it's probably quite common for most people to have some Cryptosporidium infection at some point in their lives. And for most people it's pretty probably asymptomatic, and they don't notice any symptoms.
Phil - Okay. What's next?
Ed - We then also went looking for some familiar-sounding bacteria, things that we might know of and be more familiar with. So we did find some Salmonella in there, which we all know can be quite unpleasant causes vomiting. And we also found Pseudomonas aeruginosa, which is a really horrible bacteria that can affect wounds, and it's really unpleasant.
Phil - What do you say to that Matt? Are those bacteria that you're familiar with, and what do they do?
Matt - Salmonella is probably the one that most people have heard of. And there's lots of different types of Salmonella. The most common thing that Salmonella causes is like a gastroenteritis, particularly associated with eating under-cooked eggs. People think that the actual egg itself is infected with the Salmonella, but in actual fact the Salmonella sits on the outside of the egg; because as I'm sure some of your listeners are aware, birds have a shared cloaca, where both their GI tract and their reproductive tract drain into. So when the chicken's passing the egg it gets contaminated with the Salmonella that lives in the gut, and so the Salmonella is all sitting on the outside of the egg. And so the moment you crack it, you inoculate the egg with the Salmonella. And if it's not cooked properly, you eat it, it gives you gastroenteritis. Pseudomonas aeruginosa, so that's an interesting bug. It forms biofilms on things, so it's incredibly sticky. It's a big problem in intensive care units. It's also a bug that's incredibly naturally resistant to lots of commonly used antibiotics. So I'm slightly worried that I may have been exposed to now Salmonella and Pseudomonas aeruginosa.
Phil - Anything else from the results?
Ed - We did a bit of a sanity check and we went down out list looking at things that we thought sounded reasonable, or that we recognised the names of. And actually what we found was: the levels of these two bacteria in the flies were lower than bald eagle, wolf, and minke whale. So we suspect that there could well be some false positives in there.
Phil - What has this fly been eating!
Ed - So this is a bit of a cautionary tale about using metagenomics. You're always... the method uses some reasonably complex bioinformatics to try and fit things, you're looking at very small fragments of DNA and trying to align them against wider genomes. And you want to do it with enough stringency so you don't call too many false positives, weird things like bald eagle and minke whale, but without causing, you know, without losing too much data. So I think this is a reflection of how far we've got down the list, you know, before we get to something weird.
Phil - Right. Good sequencing job. What are you expecting next Ed?
Ed - I have no idea. I think it'd be really interesting to know what else people would like to see sequenced. We have these sequencets waiting for experiments to be done. So let's see what we can find.
Phil - Right, good job everyone. Your work is done. Let's have a drink.