MM09 answer: Hoplonympha - loaded with bacteria

I'm really behind on the answers. I'll do the easier one first, so MM08 will be next.

Remember this myserious organism from a while ago? Johan got it: it's Hoplonympha, a parabasalian gut endosymbiont! (Opisthokont was also on the right track)

Hoplonympha. top: SEM of whole organism (F indicates flagella), the long strips are actually ectosymbiotic bacteria, as evident in the TEM cross section on the bottom. CM - cytoplasmic [inner] membrane, OM - outer membrane, SL - S-layer. Note that unlike in Streblomastix (an oxymonad), the host cell is substantially more convoluted. (Image from Ohkuma 2008 Tr Microbiol(free access), originally from Noda et al. 2005 Env Microbiol)

A Streblomastix wannabe. Although in a completely different clade. Not too surprising, considering the similarity of their habitats, that such a strong evolutionary convergence may occur. Note that unlike Streblo, this organism also seems to contain bacterial endosymbionts inside it. It's quite a jungle of symbiotic relationships there in the termite and cockroach guts!

What are those bacteria doing? For one thing, Parabaslians are anaerobes, containing highly derived relict mitochondria called hydrogenosomes -- which, as their name suggests, generate hydrogen gas. Bare hydrogen is a relatively rare commodity in nature, so there's plenty of bacteria that crave it for their own metabolic exercises. Many of the symbiotic bacteria are methanogens, and use the hydrogen gas in their methane production pathways.

The exact functions of some other bacteria in this bizzare and complex ecosystem aren't well understood (Ohkuma 2008 Tr Microbiol). For many obligate anaerobes, however, the gut of various animals became a rare haven from the oxygen pollution their ancestors have wrecked the environment with a couple billion years ago. In termites and wood-eating roaches you have the extra advantage of free poorly digested (by the host) carbon sources entering in the form of wood cellulose. It's a nice deal: you nibble on yummy cellulose and the host is happy with your excrement. Of course, as with any nice deal, a hungry horde of other creatures congregates around the fun. So we end up with something like this:

A sample of the complex interactions between the gut protists, bacteria and the host. For more info, read the source Ohkuma 2008 Tr Microbiol, a freely accessible pdf of which was found by Johan.

And it's likely only the beginning of the story. And yes, the cellulose digestion is predominantly done by the protists, not the bacteria. Apparently, removal of bacteria by antibiotics did not stop the cellulose digestion, whereas a removal of the gut protists wrecks it.

Since it's meaningless to look at organisms without at least considering their place in The Tree, Hoplonympha seem to form a sister clade to Eucomonympha, which together group cozily with the Trichonymphidae, with some peculiar Staurojoeninidae getting in the way:

You may recognise Trichonympha, Eucomonympha, and Cochlosoma in the Trichomonads. Trichonympha are NOT Trichomonads, but are Hypermastigotes. Just sayin'. Trichomonads tend to be a little less 'hyper' with their karyomastigont (nucleus + flagellar apparatus) multiplication. Turns out we're steadily building up quite a collection of Parabasalians here...we have these people to partly blame: (Carpenter, Chow & Keeling 2009 JEM)

There's a really cool Parabasalian with ectosymbiotic bacteria that act much like flagella, propelling the organism by beating in a synchronised fashion. This partly where Margulis gets her "spirochaete = flagellum" fantasies from, where spirochaetes mafically became attached to the proto-eukaryote and somehow became its flagellum. Which is obviously eukaryotic, and devoid of DNA, and not even barely spirochaete-like, but never mind. Or, as TC-S would say: the eukaryotic flagellum differs from a spirochaete "in every visible respect possible" for a subcellular structure. =D We'll look at this cool organism at some other time, so I'll leave you in suspense for now.

Before I finally shut up, there's a slightly annoying gap in our exploration of Hypermastigotes: While we've by now glanced at Trichonympha, Eucomonympha and Hoplonympha, what about this mysterious Staurojoenina thing between them? Guess what, it also has ectosymbiotic bacteria!

Staurojoenina. The things on its ass in the SEM are spirochaetes, while the rest of it is covered in rod-shaped bacteria, with some flagellar tufts towards the anterior. Also littered with endosymbionts. Kind of cute, but Eucomonympha and Trichonympha are fuzzier. (Stingl et al. 2004 Microbiol)

Now to write up MM08 (a really cool one), and you guys still need to figure out MM10!

References:

CARPENTER, K., CHOW, L., & KEELING, P. (2009). Morphology, Phylogeny, and Diversity of
(Parabasalia: Hypermastigida) of the Wood-Feeding Cockroach
Journal of Eukaryotic Microbiology, 56 (4), 305-313 DOI: 10.1111/j.1550-7408.2009.00406.x

Noda, S., Inoue, T., Hongoh, Y., Kawai, M., Nalepa, C., Vongkaluang, C., Kudo, T., & Ohkuma, M. (2006). Identification and characterization of ectosymbionts of distinct lineages in Bacteroidales attached to flagellated protists in the gut of termites and a wood-feeding cockroach Environmental Microbiology, 8 (1), 11-20 DOI: 10.1111/j.1462-2920.2005.00860.x

OHKUMA, M. (2008). Symbioses of flagellates and prokaryotes in the gut of lower termites Trends in Microbiology, 16 (7), 345-352 DOI: 10.1016/j.tim.2008.04.004

Stingl, U. (2004). Symbionts of the gut flagellate Staurojoenina sp. from Neotermes cubanus represent a novel, termite-associated lineage of Bacteroidales: description of 'Candidatus Vestibaculum illigatum' Microbiology, 150 (7), 2229-2235 DOI: 10.1099/mic.0.27135-0

Termite gut microforay - excavate time!

A couple days ago a local basal winged termite crossed my path. Then, like two microtubules approaching each other below the threshold angle, our paths zippered up together and the poor sucker had its parabasalian jewels strewn exposed on the slide. No worries, the termite lives on, albeit as a carbon source for other life...

Now I have rather limited experience working with gut endosymbionts (my second time), so I suck. Also, I didn't have the right solutions for keeping the denizens from exploding osmotically. So my images suck too. Kind of like this poor exploding trichonympha:

Actually, they seemed to be less exploded later when mounted in 5% glycerol (or perhaps they managed to die in the termite while I was working on the first sample, and experienced less osmotic shock that way somehow...) The glycerol may have helped out in the refractive index matching a bit, although perhaps I just magically came across better DIC settings later... (I need to stop speaking in strings of hypotheses. [fundie]I believe the glycerol helped my imaging, and I shall guard my faith until forever! Yeah, take that, 'evidence'! In other news, termite endosymbiosis is older than the earth itself. [/fundie])

First thing I saw was some mystery nematode: (was wiggling about way too much for a decent image) How do you key out nematodes anyway???

Top right: Some mystery...thing. Looks like an ex-organism, judging by the characteristic bubbles that tend to form when anaerobic protists die. Then again, those may well be artefacts of something else. Any ideas?
Bottom:Trichomitopsis! (it actually looks really cute when not completely mutilated)

(the scalebar on the second one is wrong; that would be about 12.5um, forgot to adjust for 40x settings...)
A slightly better shitty image of streblomastix; how many flagella can you count at the anterior end?

And fresh from the Crap on the Bottom of the Slide department, some mystery flagellates(?), or pieces of dirt sitting on top of bacteria (Or elongated pieces of crap). Hey, identifying whether crap is/was alive or not is kind of difficult sometimes! Although I'm pretty sure (B) is a real thing. (D) also looks kind of flagellated... any ideas?

Edit 18.08.09: With Opisthokont's help, possible IDs: A - hexamita?; B, E - monocercomonoides; C- a thing with an axostyle, or hexamita)

Then I had fun with the carnival ride that is a prism and and couple polarisers (aka DIC):

Trees in the autumn sky...

...roots on the autumn ground?

Shiny!

Omg, pseudo-darkfield! (no it's not)


The helical spiraly thing is a piece of xylem, methinks; termites eat wood right? Well, trichonympha eat wood inside the termite... so you get pieces of plant matter in them. And then you find random stomata floating about, which I'm pretty sure are not some weird contamination from my arabidopsis screening stuff (kinda reused the slide...)

Those things freaking haunt me, even when not doing research! Amazing how it survived intact...stomata can be quite fragile. Although perhaps that's specifically when you need them not to be...
Actually, wait, question: how the hell do stomata get in there if termites eat wood? While plants do have stomata on their stems, 1. termites don't munch on epidermal tissue all that muchl; 2. I doubt woody plants have any once bark is formed. Maybe it -is- some weird contamination from the previous sample...luckily, not actually doing any real science here.

Speaking of stomata, I has works to do. We're kind of like plant dentists - working on 'mouthes' all day! Especially with gene names like four lips(FLP), too many mouthes(TMM), speechless(SPCH), mute(MUTE), moustaches(MUS), etc. We also have YODA. Apparently somewhere in the Arabidopsis genome lies a regulatory gene SUPERMAN and its suppressor KRYPTONITE. We like to keep ourselves entertained...[/derail]

Let me know if you want more 'microforays'!

Sunday Protist - Streblomastix: intestinal torpedo-bearing sub

Taking a bit of a break from Rhizaria... haven't done Excavates in a while. From one obscure 'kingdom' to another... (although now I have this nagging feeling that I'm really neglecting unikonts - I haven't done amoebozoa or opisthokonts in ages...)

And the creature behind (or rather, containing) Mystery Micrograph #01 is...

(Leander & Keeling 2004 J Euk Microbiol; scalebar=5μm)
Streblomastix strix, an oxymonad. 2 - cross-section

Rosie more or less got this one ^.^ (I guess one can't really expect a sane person non-protistophile to randomly pass by and yell out OMG STREBLOMASTIX!!1!) It's a thing with several extremely long episymbiotic bacteria riding along it, kinda like torpedoes, so the 'eukaryote with bacterial symbionts' was right!

I won't go into detail about oxymonads, since Opisthokont knows a few orders of magnitude more about them than I can pretend to. Perhaps he'd like to contribute some. [/hint] Essentially they are an anaerobic, amitochondriate lineage of mostly termite/cockroach gut endosymbionts. For some reason, most of those gut endosymbiont protists tend to gather entire bacterial worlds around (and inside!) them. Perhaps due to the closed, small nature of the ecosystem, where there is time for various prokaryotes to coevolve with the protists and become closely associated without too much outside disturbance. These protists have been living with termites and cockroaches at least since the early Cretaceous (Poinar 2009 Parasit Vectors), so there's been plenty of time for entire phyla to be borne of this relationship.

Oh, apparently there's such thing as hunting around for termite endosymbionts in amber! Couldn't find any of Streblomastix, but here's a ~100 x 10⁶ year old relative, Dinenymphites:

(Poinar 2009 Parasites & Vectors; open access)

That paper is rather fascinating! And this is from someone who's not much of a paleontology nut. Anyway, back to Streblomastix.

An interesting question is, what are those bacteria doing? The cell structure seems adapted for episymbiosis, complete with specialised attachment sites. Sure enough, killing off the bacteria with antibiotics results in the eukaryote's imminent death (Leander & Keeling 2004 JEM). The specifics of the relationship still remain poorly understood, it seems. There's also even more elusive intracellular bacteria. Basically if you look at an animal or plant carefully enough, you'll find entire ecosystems of protists and prokaryotes. If you look at a protist carefully enough, chances are there'll be an entire bacterial ecosystem in its own right...but I digress.

Leander & Keeling 2004 J Euk Microbiol; m1-3 - three distinct morphotypes of bacteria, scalebar=0.5um; 16 - TEM cross section of a 'vane' tip with arrows showing the glycocalyx-like connections between bacteria and the host. 17 - posterior tip of Streblomastix showing the 'cupping' of the final bacterium.

Actually, a winged termite (Zootermopsis!) had the great misfortune of appearing before my face yesterday. This is a sample of the result:

Yes, the shittiest picture of Streblomastix in the history of termite gut microscopy. I tried to get the flagella in focus, but the whole cell is pretty mangled. The Trichonymphas turned out better, but barely... Sigh, I really need Trager medium... and access to a camera that is not too slow even for plants. I guess that means no colour CCD, since those tend to need a lot of light, and therefore a much longer exposure. Although there must be fast colour cameras too...

(still must redeem self:

Trichonympha. Happy? More termite gut imagery to follow later...)

Ok, it's getting late, I think I'm getting a cold of sorts (I wish it'd make up its mind already, whether it feels like showing up or not. And please, let it be finished before I visit the parents. Mothers + cold = overreaction x 1000!!!) and I'm just gonna have to leave this post in a half-sucky state. Did any of it make sense?

As for the next major topic, it'll be the origins of Eukarya and the Neomuran Hypothesis, with plenty of famously clear and comprehensible diagrams by the God of Protistology. Also, there's a recent Science article by Carl Zimmer that awaits a bit of gentle shredding. Actually, I'll wait until Rosie's reply to his sex article is published next week =D But prior to Neomura, there is a massive rant in store. Evolutionary directionality is GOING DOWN! Mwahaha...

And as for ye who foolishly voted 'cheese!' in the poll - watch out! It's coming...
---
LEANDER, B., & KEELING, P. (2004). Symbiotic Innovation in the Oxymonad Streblomastix strix The Journal of Eukaryotic Microbiology, 51 (3), 291-300 DOI: 10.1111/j.1550-7408.2004.tb00569.x

Poinar GO (2009). Description of an early Cretaceous termite (Isoptera: Kalotermitidae) and its associated intestinal protozoa, with comments on their co-evolution Parasites and Vectors, 2 : 10.1186/1756-3305-2-12

"Sunday" Protist - Trichonympha returns!

Finally published today: Extreme Trichonympha sexiness:
(Carpenter, Chow and Keeling 2009. Morphology, Phylogeny, and Diversity of Trichonympha (Parabasalia: Hypermastigida) of the Wood-Feeding Cockroach Cryptocercus punctulatus. J Euk Microbiol 56:305-313
I stole these while the manuscript was in advance online publication, before the images were shrunk and butchered to fit print quality:




The little rod shaped things in 12-15 are some bacteria on the posterior end of the cell. 26-28 - after removing the anterior 'cap' (operculum). Scale bar is 10um in #2, for size comparison.

Trichonympha is this giant and utterly adorable wood-eating gut endosymbiont of early-branching dictyoptera (cockroaches and termites). Sadly, it's anaerobic and thereby difficult to play with unless you have a steady supply of termites going on in your lab, like those guys do. As for the wood roach from which these particular critters come from, it has to be ordered. This roach also has the amazing Saccinobaculus...Trichonympha can also be found in basal termites; we're lucky to have some native ones here in Vancouver (alas, devoid of Saccinobaculus =( )

It seems like cockroaches and termites formed endosymbiotic relationships with the protists before the two diverged - both groups have endosymbionts in their basal lineages, and lose them later on. The protistan endosymbiont diversity is wonderful: you have the aforementioned and much beloved 'snake-in-a-bag' (Saccinobaculus; can you tell I'm obsessed yet?) and fellow oxymonad companions like Streblomastix - a long cell with 'docking' for even longer episymbiont bacteria on it; Trichomitopsis and its protruding axostyle when it curls up into a ball; accompanied by loads of symbiont and parasitic bacteria.

The wood-eating dictyoptera require endosymbionts to digest cellulose, since we metazoans suck at it. The more derived termites can get by with bacteria it seems; but interestingly the protists are actually doing the digesting themselves in the basal termites - killing off the gut bacteria does not prevent the termite from being able to digest the wood, if I recall correctly from class... (was a while ago since we sliced up some termites and cockroaches). Either way, you end up with this complex society with protists of all sorts with bacterial endo- and episymbionts, as well as free-living forms. I find it amazing how this system survived locked inside the termite/roach guts for millions of years; it would not survive without them!

Most of the protists are anaerobic and lack conventional mitochondria, instead carrying highly reduced relics as mitosomes or hydrogenosomes; the latter produce hydrogen gas as a byproduct of their metabolic pathways. It was once thought those organisms were primarily amitochondriate, thus shoving them to the base of the eukaryotic tree, also known as the
Archezoan Hypothesis, put forth by Tom Cavalier-Smith; later this hypothesis was rejected as relics of ancient mitochondrial gene transfers were found in some of the host nuclei and the evidence accumulating for one of the prime archaezoans, microsporidia, being found to branch smack in the middle of fungi (beginnings of demise of Archaezoa discussed in Keeling 1998 BioEssays; free access).

It's 4am and I should stop procrastinating with my assignment... but here ya go. Aren't protists so cute and awesome? ^.^

CARPENTER, K., CHOW, L., & KEELING, P. (2009). Morphology, Phylogeny, and Diversity of Trichonympha(Parabasalia: Hypermastigida) of the Wood-Feeding Cockroach Cryptocercus punctulatus
Journal of Eukaryotic Microbiology, 56 (4), 305-313 DOI: 10.1111/j.1550-7408.2009.00406.x

Sunday(?) Protist - Eucomonympha

Internet broken at home, and also incredibly busy: lots of research stuff, final this Friday, another summer course just started, and unofficially auditing a microscopy course that's happening right now. Basically, back to chaos as usual.

So out of laziness, a cute Eucomonympha to oogle at:

(Carpenter & Keeling 2007 J Euk Microbiol)

Cockroach gut endosymbiont, eats wood, anaerobe. Scale bars 20um, so this thing is quite humongous. Surface consists of multitudes of flagella interspersed with spirochaete ectosymbionts, and some other bacteria. I'd love to do cell biology on this thing, but that creator made those things anaerobic to inconvenience our attempts to deny his existence. What a sly bastard!

Ok, they're making me ditch protists for the next little while... the cruelty! =(

There's also a recently accepted (but still in advance publication) paper full of absolutely drool-worthy Trichonympha SEMs by the same guys... will save for later.

Delicious squashed cockroach guts

Done finals for the term. Have more in June, so it doesn't seem like the end really...

And a certain Dictyopteran-endosymbiont-obsessed faculty in our department is indirectly responsible for the following 'study break':

I'm sitting there, cramming biochem, getting some studying done. Along comes a cockroach (I've spotted at least three species of cockroaches in our building already). I instinctively catch it and key it out, to subsequently determine it's phylogenetic position without the cockroaches. It was Supella longipalpa, sadly not a basal roach, and thus unlikely to host the Parabasalian zoo I was looking for (eg. Trichonympha). Further literature searches revealed only bacterial gut flora, and parasitoid wasp larvae. However, anything trumps biochem, so I decided to go have a look.

As expected, not much excitement in the way of gut flora. A couple flagellated-seeming tiny things (a couple microns) -- possibly bacteria, possibly some obscure excavate -- couldn't quite tell. Couldn't take pictures of those as our camera is too slow even for us plant people. But then I discovered the awesome that is insect tissue under polarised light:





Microscopy is truly the closest thing we got to exploring foreign universes...


Surreal landscapes...


This one reminds me of Southern Ontario fields in the winter:

(you can see why cockroaches are in order Dictyoptera - net-wing.

More Martian tripiness:


And can anybody tell me what those greenish round things are?


I really need to read up on insect anatomy... have no idea what I'm looking at most of the time. Not to mention that it's all squashed to begin with...

Sadly, as expected, no cool Parabasalian freaks found in Supella longipalpa hindgut this time. K, next cockroach!

If anyone's got some spare Cryptocercus lying about, feel free to send 'er over! My current clip of Saccinobaculus sucks arse, and that organism is way too cool for sucky videos.

Sunday Protist -- Saccinobaculus

If you're a unicellular organism, chances are you might want motility at some point or another. Being able to move helps escape predators, find food, find better sunlight access if you're photosynthetic, find a partner for some nice quick steamy sex; as well as entertaining easily-amused cell biologists.

Cell motility tends to fall into two (crudely-defined) broad categories:
1. Amoeboid -- cell extends pseudopodia ("false feet") and pulls itself into a particular direction along a surface.
2. Flagellate -- cell uses whip-like flagella to propell itself through a fluid. The flagella can be short and numerous, as in the cillia of a Paramecium, or long and few in number. You'd expect the flagellum to originate in the back of the cell, pushing it forward. Our sperm do that. However, interestingly enough, the majority of eukaryotic life voted against posterior flagella. Animals and their closest relatives, fungi, form a group called opisthokonts -- Greek for "posterior tail". Almost every other eukaryotic organism has anterior flagella -- and at least a couple of them. We're just weird.

There's also adhesive-based motility, where the cell is like a rock climber -- it anchors itself to the surface, uses cytoskeletal motors to move ahead, and then abandons the anchor. Diatoms and the malarial parasite use such technique.

However, there's something even weirder. (this is Protista -- there ALWAYS is something weirder!)

Imagine you're floating around in fluid, and you just can't be bothered to whip things around on the outside. Nor do you feel like protruding pseudopodia along a surface, like some lowly amoeba. Nor do you want to associate yourself with the brown algae because you're racist like that. Or rather, imagine that evolution does weird things. It really does...

What would you do?

This creature decided it wants to be a snake-in-a-bag:


(source)

Meet Saccinobaculus, an anaerobic resident of the gut of the wood-eating cockroach Cryptocercus. The stiking feature in the middle is the axostyle -- a think bundle of microtubules (cytoskeletal structural elements) that functions in a similar way to the muscle tissue... of a snake in a bag, which is what the name really means! This axostyle is highly motile and wriggles around inside the cell, causing it to move. This seems to be a rather inefficient mode of motility, but the creature hasn't gone extinct yet, so it can't be that bad.

But you have to see it in action:



I searched all over the internet for a movie of a Saccinobaculus moving about. Unfortunately, I failed to find any. Either my search skills suck or there just isn't any publically available out there yet. So I'll have to make do with a crappy clip I shot in class. Appologies for the shitty quality, and half-deteriorated specimen. As soon as I have legitimate access to a professional scope with a decent camera, and acceptable specimens, I'd try to fix this...

So next time you catch a harmless snake and throw it in a plastic bag, for whatever awkwardly surreal reason, be sure to remember that there's an organism that moves like that for a living. And please don't hurt the snake -- they're beautiful animals!


This advice is serious: If you have access to a microscope, and some wood-eating roaches, or Zootermopsis termites nearby, please remove the gut of one and examine it under the microscope. You'll be amazed by what you find! Unfortunately, the organisms tend to die quickly upon contact with air, so you can't really culture them for your enjoyment...

Sunday Protist -- Trichonympha

(http://www.microscopy-uk.org.uk/mag/artmar03/rhtermite.html)

Trichonympha

Termite gut symbiont, 200-300 microns long (huge for a protist). On the inside, bottom half, are pieces of wood -- termite gut protists digest cellulose for the termite, who feeds on the metabolic byproducts. The round thing in the centre is the nucleus (with permanently condensed chromosomes), and rows upon rows of flagella. Being a parabasalian, this creature lacks mitochondria like ours -- instead, they have been reduced to hydrogenosomes, which produce hydrogen gas.

The termite gut is a lush ecosystem FULL of cool things, more of which will be posted later. You also have episymbiotic bacteria covering some of the symbiotic protists, resulting in an ecosystem-inside-an-ecosystem type of environment.

Hard to resist the compelling urge to start slicing open random insect guts in hopes of finding protists...