シロアリの腸内微生物（Zootermopsis Angusticollis）からDNAを抽出し、腸内微生物の可視化

Here, your reaction to this environment isn't, isn't unique. Most people, even non-scientists are, are fairly blown away.Yeah. By the, The diversity and complexity.

Hi, my name is Eric Matson and I'm a postdoc in the Department of Environmental and Science Engineering here at California Institute of Technology. My work under the direction of Professor Jared Ledbetter focuses on the microbial interactions and diversity of species that inhabit the termite hind gut. In particular, we're interested in some of the biochemical pathways that go on and especially are interested in the process of ace agenesis, whereby these microbes consume hydrogen and CO2 gases and convert them to acetate, which is a key nutrient of the termite host's energy metabolism.

Myself and others in the laboratory have developed targeted primer sets to be able to go in and assess key enzymes in this pathway. But in order to do so, requires us to generate clean DNA samples of the community community, DNA, in order to use for targets for this process. So today I wanted to demonstrate how we dissect the termite species that we're talking about, extract the DNA from the hind gut and then purify it and for use later for investigations.

Today we're going to focus on one species of termite, in particular called Zo Opsis Nevadensis. This is a California damp wood termite collected locally here, and it's a rather large species, which makes it pretty easy to do the dissections and extractions from. Okay, so today I've already prepared some of the supplies that we'll need for the procedure.

One of the first things we need is, is a certain amount of ice to, to chill down the, the insects that will be di dissecting. I've also pre chilled some TE buffer, and this contains a compound that will help stabilize the DNA once it's released from the cells in here. I've got sterile vials preloaded with zircon zirconia silicate beads.

These are 0.1 millimeter beads and there's about half a gram or so in here. This is what we'll actually be doing, mechanical shearing. And then I have a couple of reagents involved in the lysis and purification procedure.

Here I've got a bottle containing 1%polyvinyl PolyOne or PVPP in te buffer. The PVPP is important for removing humic acids from the DNA preps, which can inhibit our PCR analysis of the results. And then I have some sodium ECCO sulfate, which is a surfactant that we'll use to help extract and, and disrupt the cells.

And then for doing the dissection of the termites, all we need is some sterile forceps. These are fairly fine tipped and that's what we'll be actually using to remove the gut tracks from. And finally, to do the mechanical shearing, we have a bee beating device here that spins at, at, at very high RPMs and it, along with the zir zirconia silicate beads, will do the mechanical disruption of the cells.

So after we've collected the termites in the field, we bring them back here to the lab. Most of the California damp wood termites we work with are very prone to desiccation. So one of the advancements that we've made in this lab is to incorporate the use of humidity controlled chambers, such as these 10 gallon fish tanks.

Inside here, we collect the individual colonies and keep them separated in these, in these boxes. And at the base of the tank are basically containers that have different salt solutions. This particular one contains a solution of potassium phosphate, which keeps the relative humidity around 95 to 96%which is perfect.

And, and with those conditions, we've had the termites actually breed in captivity though, when we want to go and actually run an experiment, we collect fresh from the field. But in this box you can see some of the wood that they feed on. This is just Ponderosa or Jeffrey Pine that we collect at the, at the site of the, the termite nest.

And there are a number of different termite casts, including the, the male and female reproductives. Most of the, the colony is made up of worker termites with the number of, of soldier casts as well. Interestingly, the soldiers have mouth parts mandibles that are designed for defending the colony rather than for eating the wood.

And so they depend on the worker termites to feed them as well. And so you can see in this box both workers and soldier termites as well as some young that have recently hatched from, from eggs. Well in captivity here.

So I've, so I've selected five worker termites from the, the group that I just showed you. And I've started by chilling them on ice here. To get ready for the dissection, I'm gonna use these forceps and work with the aid of a dissecting scope, and hopefully you'll be able to see the large gut volume that we'll be able to remove from, from each of these individuals.

Just flip the termite on its back and with one forcep, you hold the head end and with the other, all you do is pinch. Just at the very tip of the abdomen here. And with one pole, you should see the gut tract being removed from the organism.Okay.

And you can see the large volume, roughly 30%40%of the weight of the insect is actually, is actually contained within that environment there. And you can see that it's divided into several sections. We've got the four gut and then and the midgut, and then this hind gut punch, which is the very large chamber that contains most of the, most of the microbial community that carries out the fermentations.

So here I've removed the guts of, of five worker termites. And now the next step is to stabilize the nucleic acids in that are contained within the community here in a solution of ice cold te buffer. At this point, they can be frozen at minus 20 degrees Celsius if you're working in the field.

Or we can continue straight with the, the extraction procedure. But all of the cells and, and, and DNA material is contained still within the gut. And I'm just gonna put them down into 100 microliters of te buffer.

Okay, before I actually begin the extraction of the, the gut samples, I need to prepare the buffer that I'm gonna be using to disrupt the cells and tissues in. So to this tube that I've already got about half a gram of zircon beads, I'm gonna add PVPP 700 microliters of this. This is the 1%in te buffer and 50 microliters of a 20%solution of SDS.

It's important to resuspend the PVPP before you add it since it's insoluble in aqueous buffers. At this point, we're ready to add the phenol to the sample, so I'm gonna use a pipette to add about 500 microliters or so.Okay. And with that, we're ready to begin the extraction.Yeah.

With the phenol added, we're ready to add the gut sample that we dissected from the termite earlier, so it helps to macerate the tissues just a little bit. It tends to make the transfer somewhat easier. And now with the phenol added, we're ready to begin the mechanical disruption with the bead beading device.

The technique we're gonna use is three cycles at full speed, 30 seconds, followed by 30 seconds on ice. It's important to incorporate the step on ice to cool down the sample because it tends to heat up because of friction. So I'll just insert the sample into the bead beading device here, And I will go for 30 seconds.Okay.

At the end of the procedure, you should see that most of the tissue has been homogenized. There should be very few clumps left, and you'll see that it's, it's got kind of a sudsy appearance probably because of the SDS in there. At this point, we're ready to centrifuge and then begin the actual extraction purification of the DNA itself from this sample.

Now that we've mechanically disrupted the cells, we're gonna centrifuge out the insoluble material. We'll do this by putting in the centrifuge and using 8, 000 times gravity for one minute. And now what we expect to see is most of the soluble material collected near the bottom of the tube.

And what we're interested in is the aqueous solution at the top. And this is what we will actually add to the KaiGen DNA extraction kit following the procedure for crude lysate. Now that we've got our, our sample centrifuge, we're gonna take 200 microliters per preparation of the supernatant, and we're going to extract the DNA just using A-D-N-E-Z KaiGen kit using the protocol indicated for crude lysate.

Now we've got the 200 microliters of gut sample, and I'm going to proceed using the kyogen d easy kit using the protocol indicated for crude lysate. Whenever we do a gut extraction, it's always a good idea to check the status of the gut contents. So we do that by dissecting a termite and just making sure that it's representative of things that we've seen in the past.

We'll actually do that visually by looking at the cells in this microbial community. Just at the very tip of the abdomen here. And with one pole, you should see the gut tract.

So this is just a, a buffered salt solution that matches very closely the composition of the actual gut fluid of the termite. And I'm just gonna macerate this up a little bit and then I'll add it to the microscope slide and we'll have a look. Okay, now we're ready to look at that into the microscope.

Yeah, that looks really good. Termite gut. There are representatives of all three recognized domains of life.

There are eukaryotic protozoa, there are a meth organisms, and then there are also bacteria. And our lab is predominantly concerned with the bacterial population in this environment. Namely the SPI kes in this particular type of termite spire keets are, are among the most dominant of the bacterial members of the community.

And our current research shows that in fact they do engage to a large extent in CO2 reductive a ketogenesis. So these, these are just various different types of, of protozoa, and they're thought to participate, namely in the, in the, in the, the primary breakdown of, of wood. So they're thought to contain xin AEs and cellulase that, that allow their enzymes access to these wood polymers.

And, and then they, they ferment those into mono sugar monomers and, and, and polymers that are further broken down by other members of the community. And these protozoa also produce acetate themselves, but, but one of their byproducts is hydrogen and CO2 and this hydrogen and CO2 again is, is what's the used or consumed by the aceto populations in this environment. And here you can just see a swarm of, of protozoa organisms probably right next to a very large species of, of trick and infa there.

It's one of the largest organisms in, in the termite gut. As you go down and you actually look at areas that don't contain these large protozoa cells, you'll see that the background is this sort of interstitial fluid is, is also loaded with, with spi, keets and other bacterial cells. And so, you know, the complexity doesn't get any less even in those spaces.

One of the things that I should point out is that the typical volume of the hind gut punch, such as the one that we looked at here, is anywhere from two to five microliters depending on the, the size of the termite. And so the wet mount prep that I've made here really represents at least a one to 20 dilution of, of that environment. And so it's actually these species are, are packed in much, much more densely in the actual intact gut than, than what's represented here.

So that gives you some idea of, of the di the density as well as the diversity of life in this, in this environment. So today I've shown you how we isolate the DNA from the termite gut with these purified samples. Now we're going to be able to apply the specific targeted primer sets.

Namely, we're gonna be looking at genes for form a dehydrogenase, carbon monoxide dehydrogenase, and formal tetra hydro folate synthesis, which are three of the primer sets that our lab has developed. We're interested in studying the diversity and specific sequence characteristics of a number of these different genes for this environment. So that'll involve PCR amplifying the different genes, cloning them, and sequencing them and building gene inventories.

Based on the results, we can learn something about the nature of some of these interactions and specifically how these pathways are constructed in these different microorganisms through this process.