Layers of Symbiosis – Visualizing the Termite Hindgut Microbial Community

Jared Leadbetter takes us for a nature walk through the diversity of life resident in the termite hindgut - a microenvironment containing 250 different species found nowhere else on Earth. Jared reveals that the symbiosis exhibited by this system is multi-layered and involves not only a relationship between the termite and its gut inhabitants, but also involves a complex web of symbiosis among the gut microbes themselves.

So I sometimes tell people that, that For me, this is a little bit like being an ecotourist in a miniature Alice in Wonderland because I, I just look at this and I just think it's a wonderful nature walk even before you do any, any other type of research on the system. Well, there are different ways of looking at these Microbes by light microscopy. We have with this microscope the capacity to do dark field microscopy where cells will appear white or be outlined in white with a dark background.

We can do phase contrast microscopy where cells or edges of cells will be dark on a gray background. We can do regular light microscopy where you don't see much of anything unless the sample is colored. But you'll get the real, the real color of the sample.

So if your sample is green or red, you may actually see that in the cells. We also have the capacity to do something called DIC microscopy. So difference interference contrast, which as opposed to giving you contrast in terms of either white and black or, or sort of dark and gray, it gives you more of a lunar landscape type of edge contrast in the sense of you look at the moon with binoculars and it's all gray, but you will, when you have focus it, you'll start to see edges very nicely.

So I've set this right now to look at the sample using this DIC. What that does, it gives you surface textures of cells and internal structures of cells if they're large and if they have those structures much more cleanly. Whereas under a different type of microscopy, the whole cell may appear gray On a, on a light background or white on a black background here, you'll start to see textures on the actual cell or within the actual cell itself.

And for many of the protozoa that we have, the larger eukaryotes that we have, that will allow you to see not only internal structures in the cell, but also perhaps particles of wood. And so here right now we're looking at two gut protozoa. These belong to the genus opsis.

These are cellulose degrading protozoa. And right now with this one that's sitting right in the center here, I can adjust this a little bit to bring out some of this, this edge contrast. It's a little bit hit or miss.Okay.

Oh, this is looking gorgeous. For instance, this is one of these very large trichomonas related protozoans and it's starting to bleb out. It's starting to get ill.

And that's what some of these little blebs out the side are. You can use this DIC to really get an optical section. So I'm just gonna go in from the surface.

You start to see the surface come into focus. It looks like it's covered in these long lines, long grooves. And now I'm going into the cell and now I'm going through the other side.

And it allows you, if you do this on the posterior of the cell, you can see the nucleus. So I'm coming up, I'm looking at the surface, now I see the nucleus, and now I start to see the other side. What I can do is I can compare is phase contrast, it looks good.

This is this edge contrast. It also looks good and it's just a matter of taste. And for some of the thinner cells, this phase contrast Looks better.

Actually we see two protosome Here. One is this one in the center with these little frills and the other one, which looks like a bundle of sticks. And this really brings out this DIC imaging really brings that out.

And there's some adjustments you can make. It's a little bit like cooking. You can do it to taste, which is you look at your sample and you just make the adjustments to just bring out in that particular sample some of the textures of the cells.

It's almost empirically determined. So now I'm just gonna try and find a few other objects worth looking at. This is looking on dark field.

Now where the biological material appears bright white on a black background here we see large trifa with the smaller fascist protozoan stroma. STRs are zooming in around the looking like slender little almost worms, which are shooting around these larger cells. So those are these smaller protozoa.

So there are quite a few different species of protozoa that you find in this environment that are different sizes. You have these very large trifa, you have these medium-sized tri opsis, and then you have these very long thin, slender bacteria coated stroma STRs. So we can see some of these trichy opsis and stromas.

St stroma STRs is the bundle of sticks. Tri opsis is this beautiful little organism with these little frills that run around the cell very quickly and the larger trifa. This is tri opsis.

This is the only termite gut protozoan that's ever been cultured that in studied in vitro and about three or four PA papers on this protosome 20 years ago. And then the culture was lost and no one has ever been able to culture it again. But this studies on this organism established that protozoa will ferment cellulose and xylan, which are the pento and hexoses polymers and wood, and produces products acetate hydrogen and CO2.

I'm just gonna zoom around a little bit. So you got some of these spi keets. There's a nice DIC image of API keet right there in the center.

And you can see it's coils. Nice regular coil. I see that moving.

Oh, it's, it's, it's still alive, but it's, some of these have been damaged a little bit by the oxygen. Mm.Because many of these organisms are poisoned by oxygen. Sometimes you make wet mounts and they work out and sometimes they don't.

But Actually, this one's a real Butte. I'm just gonna look at this protozoan Here. There are two or three of them here.

There are these long slender protozoa that look as if they're striped. And you really see that with the DIC when you look at them. And I always say they're fascist protozoa.

And what do I mean by that? You know, the Latin term fasis is the word that describes this bundle of sticks on the back of a dime. And it's the, the word that really also describes e pluribus unum, you know, and many, there's one, and this I call the fascist protosome because the proteasome itself is actually exceedingly thin.

And the very little that we know about it, studies have shown that this protosome is covered with a coat of thin bacteria, long, slender bacteria. So it's a long stick like protozoan eukaryote that's covered with a coat of long hair-like bacteria here, you see some of these hairs are actually starting to fray a little bit and come off of it. But you'll see a lot of these.

And functionally we have no idea what they do. So this is an example of, it's nice to look at. It's inspiring in the sense of, it tells me there are a lot of these protozoans in here.

We can look at the phylogeny of the host and the cells. We know what the protozoan is and we know what the, the species that attach to it. But we don't know what either the protozoan or those bacteria do.

What is their physiology? What do they eat? What do they make?

How do they interact with the other microbes? How do these products impact the other microbes or the host? And so we functionally don't know where they fit into the puzzle.

Presumably all the cells that we're seeing here that are abundant are symbiance en engaged in the mutualism. That is their beneficial microbes that really are given something to the system and or to the host. But in this case, the devil's in the details.

And we actually can't say, can't say with any certainty. And I actually don't have a real hypothesis about what those organisms do. I mean, it's interesting because the fascist protozoan are covered in bacterial themselves.

So it's like symbiosis upon symbiosis layers. And whereas some of the other protozoa, you can see that they've engulfed wood particles into the cytoplasm or or into the cell. These, there's no evidence that they're engulfing wood particles.

So it's not at, you can't even really say, oh, they're degrading wood. I don't know that they're degrading wood at all. And and more possibly they're positioning these bacteria somewhere in the guts of the bacteria can do something and perhaps feed the protozoan examples of other symbiosis within symbiosis.

If we look at this trica nifa, this larger protozoan that we're looking at here is sort of a squashed trifa. And we can see that it's has these long striations that go from anterior to posterior. But if we look a little bit more closely, we'll see these little towards the front end of it, these little black dots.

Those are also bacteria. And this, this protozoan has both ecto and endo symbiance. It is covered with a layer of bacteria on its surface, but also it contains bacteria inside the cell.

And again, we don't have any understanding of what the physiology of those bacteria on the surface or on the inside are. We have some sense that these protozoa ferment, cellulose and xylan, but we don't know where these bacteria fit into the picture. So we're missing something fundamental.

So I love the system because there's lots of diversity and there's lots of action, and we've learned a lot. But actually I think we know much less than we Know more.