Kadeem Gilbert: life in a death trap

Image Courtesy of Kadeem Gilbert

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We’ve heard a lot from Ulrike about the slippery sides of the pitcher plant that cause insects to fall into the bottom of the champagne flute, but what happens to them once they’ve fallen in? Dr Kadeem Gilbert is an ecologist and evolutionary biologist at Michegan State University who has been researching the pool of digestive juices. And it turns out that it’s not only a place where insects drown and get digested by the pitcher plant, it’s also home to a whole community of living things that are able to survive despite the harsh conditions, as I found out from Kadeem…

Sally: What is it like in the liquid bit at the bottom of a pitcher plant? What is happening in those juices at the bottom of the champagne flute we've just been learning about? 

Kadeem: Well, to start with, the plant is producing different digestive enzymes, including chitinases which break down the shells of insects, but also fungi are made out of chitin, so there might be some antifungal properties to those as well.

Kadeem: The pH level can vary widely between species, so some species are relatively moderate and moderate for a pitcher plant is between four and five. 

Sally: So what do we know that's around pH four or five? 

Kadeem: Four five is more like tomato juice. And six to seven you can see in a really old pitcher, which is basically water at that point.

Kadeem: The extreme goes to two and below. And below two is when you get closer to battery acid, if you actually reach pH one. 

Sally: Yeah, pH two is that about stomach acid? Is stomach acid a pH one?

Kadeem: Yes. Stomach acid is between one and two, in that region. 

Not so basic

Kadeem Gilbert measures the pH of a pitcher plant during one of his field trips. Image courtesy of Kadeem Gilbert

Sally: Far from my little champagne flute of acids and digestive enzymes being sterile as I thought it was.

Sally: You are telling me there's a load of stuff living in the liquid. So to start off with what is able to survive in that liquid?

Kadeem: Yeah, so it's definitely quite a paradox that they have these deadly traps, but life is pretty ubiquitous in that habitat as well. So there's specialised insects that live in there as larvae, different types of mosquitoes and flies, which are able to survive in there. And not to mention all of the different microbes.

Kadeem: You've got bacteria, you have some fungi, yeasts living in there, there's also algae. 

Sally: And how are they able to survive in this place that has evolved to kill things? 

Kadeem: Right! We know basically nothing about the physiology of the symbionts at this point. So it's really only in the past 10 years or so that we've gotten a picture of the diversity of microbes living in the pitcher plants.

Kadeem: So this has largely been using metabarcoding. So for bacteria, we use the 16S ribosomal region and 18S for Eukaryotic organisms. 

Sally: So you're looking at the genetic code that codes for ribosomes, the protein making bits? 

Kadeem: Right! It's a vital function so it can't mutate too quickly, but it evolves enough that we can at least separate different families of bacteria from one another.

Cheers!

(Genetics Unzipped does not recommend drinking from pitcher plants). Image courtesy of Kadeem Gilbert

Kadeem: Kind of a similar level of resolution for insects. But yeah, this just gives us a snapshot of all the DNA that's in the picture. 

Sally: And what does it look like to metabarcode a pitcher plant? I've got my plant in my pot or in the jungle. What do I do? 

Kadeem: Well, when you're in the field, you take a sterile pipette to suck up that pitcher fluid.

Kadeem: And one of the great things about this technique is that you don't necessarily even need to take the insects out of it because all of the DNA is dissolved in the fluid. Including things that may have been in the pitcher a week before you got there. 

Sally: So you've got your pipette full of dirty, insect pitcher juice.

Sally: In the tropical rainforest where nothing is clean and nothing is sterile, what do you do? 

Kadeem: Yeah, you just quickly transfer it into a sterile falcon tube and adding a type of preservative, but you try to get it back to a lab as quickly as we can. 

Sally: And then once they're back in the lab, is there a mystery black box you put it in and it prints out some results for you? Is that how Genetics works nowadays? Because I know it's changed since I lived.

Outstanding in his field

Some geneticists get to travel the world for their research. Image courtesy of Kadeem Gilbert.

Kadeem: You know, there's nice kits that you can use for doing various extraction protocols.

Sally: And then what comes back? What's on the piece of paper that you read off? 

Kadeem: You have so much data that you have to use bioinformatics pipelines to first, basically you read in the raw sequence data, you determine a threshold at which you want to assign your operational taxonomic units. 

Sally: That's a 'what is a species?' type thing. 

Kadeem: Right. We decide, say, if sequence is shared 97% similarity this is a species for your purposes. And then you can bend them in different ways.

Kadeem: And then you get your table where you have OTU 5 4 8 9 blah, blah, blah, whatever. 

Sally: So it doesn't give you, you've got this group of fungi here, you've got this. It just gives you what DNA sequences are out the end. 

Kadeem: Yeah. So first you assign your OTUs and then afterwards you use BLAST or some sort of search program to say, 'Oh, what is this OTU closest to?'

Sally: So you use genetic Google to convert your sequences into like actual living thing names. 

Kadeem: Right, exactly. And then you get varying success with different OTUs. Some sequences are ambiguous and you could just say, 'Oh, this is a bacteria, but we don't know why.' 

Sally: Well, yeah, presumably hundreds of them are just unknown to science.

Sally: You're looking for all living things in a rainforest, there is absolutely no way we know what every species is living in a rainforest. 

Life in the death trap

Some insect larvae are able to survive and thrive inside the acidic, digestive juices of a pitcher plant. Image courtesy of Kadeem Gilbert.

Kadeem: Right, exactly. So for the insects we see that we'd be able to identify more of the insects to a finer resolution. But as you say for bacteria there's a high likelihood that something in there that's truly just unknown to science. 

Sally: But you must have an idea of how many species or these groupings of living things are living within a pitch plant, even if you can't name what the individuals are. So how many are we talking? There's a dozen bacterial species? 

Kadeem: Way more. 

Sally: There's a hundred bacterial species? What are we talking? 

Kadeem: More! More along the order of magnitude of in the thousands. 

Sally: Okay, so there's quite a lot of stuff that you are finding in these pitcher plants, but that's just finding the DNA of it and presumably the things that fell in the pitcher and died are still going to leave their DNA in the pitcher plant. So how on earth do you work out which of these operational taxonomic units, which of these species were living inside the digestive acid juices and which ones had just fallen in and have been eaten by the plant? 

Kadeem: Very true. It's difficult and maybe not actually possible to distinguish fully with this approach. So I have some ongoing work using a transcriptomic approach. So you can look at RNA and then, RNA is very short lived, so if you're finding an RNA signature of a microorganism, then the chances are it's still alive. 

Sally: So thinking from the plant perspective, you briefly mentioned that they, just like we delegate to our gut microbes a lot of our digestion tasks, that the pitcher plants might be delegating some of their digestion to the stuff living in it.

Sally: Which is wild. I mean, it kind of makes sense when you think like, well of course they're gonna do that. Evolution loves doing that kind of thing. But also I'm like, that's so weird. A plant having a living gut, micro flora, how does that work? What is it that the bacteria and the microbes are able to do that the plant can't? What are they getting out of the relationship? 

Kadeem: Yes, so I'll start by referencing the North American pitcher plants, the species that you can find around Michigan and throughout the Northeast. It has very mildly acidic fluid and doesn't produce a lot of its own enzymes. Well, there's some controversy of that. It may produce no enzymes or may produce just very little enzymes. So in that case, it's relying entirely on bacteria and midge larvae to break down food for it. 

Kadeem: So aquatic insects, their waste, it comes out as ammonia. But ammonia is a particularly assimilable form for a plant.

Kadeem: So by converting the dead insects into ammonia, then that can be uptaken much more quickly by the plant. But it is complicated though because as you allude to the, as far as evolution is concerned, if you can get as much out of it as you want without concerning the other organisms around you then you will. 

Sally: And thinking of the pitcher like the gut, are there good microbes and bad microbes to colonise a pitcher from the plant's perspective?

Kadeem: Yeah, again, there's much to be done in terms of the actual fitness benefits and costs of the specific microbes in there. I can note one observation I made in one study where within one greenhouse, some species had pitchers with a very brightly colored fluid, like a bright neon green or neon orange, just very striking colouration in the pitcher fluid. I thought that maybe, you know, maybe there's a certain microbe colonising them that's responsible for the colour, but that doesn't seem to be the case here. So other studies that had noticed colouration like this, that actually did the chemistry, it seems likely that the colour is by this other compound droserone. Which is induced by insects and has an antifungal property. And then what I saw with my data is the coloured pitcher fluid had more fungus gnat DNA because then there were still fungus snaps getting into the greenhouse. But there were five specific fungal OTUs that were reduced in abundance in just the coloured pitcher fluid.

Kadeem: So, that droserone may not be a broad acting fungicide, but some fungi are very actively destroyed in these particular pitchers producing this compound. Yeah, a next step would be, can we isolate these and then see do these steal more nutrients from the pitcher, and would that be a reason for producing the antifungal?