Of random rotifers and vicious amoebae

Still procrastinating with the Heterolobosea posts (no, I haven't forgotten). Real protistologists give me sorry looks when I mention being stuck writing about that rather obscure and messy group. I don't want to just do a taxonomic overview - I like to mention odds and ends about their cell biology as well. Sadly, the cell biology of most of those things is a sorry neglected mess. Even the eruptive pseudopodia that are quite characteristic of this group (although present in some amoebozoans as well) seem to not have been examined on a molecular level - I cannot find a single paper describing the cytoskeletal dynamics behind this peculiar mode of motility, and my sources point out a severe shortage of attention in that area. But I'm still scraping things together, so eventually something will appear...

Now for something completely different...since someone just sent me scouring piles of archaic metazoan 'mesozoan' literature to identify that damn bug (an orthonectid of some sort), I have a random metazoan of my own to show off a bit, from the latest pond microforay:

Rotifer. (Mine =P 40x* DIC) Note the contractile vacuole-like structure (actually its bladder, but appears to function in a very similar way. Another case of ultimate convergence?).

I can't quite key this one out, especially since this damn sucker is on its side. Probably the only reason I was able to capture a photo of it, since they tend to be quite hyperactive normally. This one appears to be immobilised by the cover glass. Perhaps it may be Aspelta or something from the Lepadellidae. Or might it be a Bdelloid? Perhaps a certain taxonomist could help out. Especially the one who made me browse orthonectids for the past few hours. ^^

*40x here, and onwards, refers to objective rather than magnification. We don't actually care about mag, it's the other properties of the optical system that matter much more when dealing with professional instruments. (Such as numerical aperture and field of view, focal length, focal depth, which type of immersion medium its calibrated for, etc) Technically, I should be writing down the NA and such, but we're not that meticulous crazy around here.
**Randomly found out there's a rotifer that lives inside colonies of Volvox, as a parasite...

Much of my resentment towards metazoa simply comes from them being overstudied and overemphasised, to the neglect of other phyla. That said, they are still interesting and still poorly understood lifeforms, especially among the spineless things. While representing but a mere sliver of life's overall diversity -- contrary to what our senses tell us -- metazoa are nevertheless, like all life, fascinating and weird. Metazoan multicellularity, as mundane and ubiquitous as it may seem to our rather biased eyes, is about as weird and insane as kinetoplastid kDNA editing, or the convoluted ciliate nuclear genomes, or the breathtakingly massive, and seemingly pointlessly so, nuclear genomes of dinoflagellates and some amoebozoa. I'm quite disappointed at the absense of the 'perspective of strangeness', if you will, that seems to dominate many animal-oriented classes (especially those catering to pre-meds. Must. not. rant.)

Interestingly, off all taxonomic and phylogenetic literature, I find metazoan papers to be the most difficult to follow* - we actually have common names to refer to quite a few animals metazoa. Furthermore, the obsession with ranked hierarchy is driving me insane - you DO NOT have to name every single possible grouping you can come up with, seriously! That makes searching for stuff an absolute disaster, as you have to look for the relevant subfamily, family, superfamily, suborder, order, superorder, hypersuperorder, subclass... etc. All named differently enough to not fit into one convenient Google (or PubMed, or WebofScience) search. And you have to find all of them. And they change. And your life becomes a living hell. Please stop, seriously! WHYYYYY!? Did TC-S write their taxonomies or something?! And they listened to ALL of it?! [/rant]

Another fascinating facet of biodiversity that seems to be thoroughly ignored is the interface between kindoms: the breathtaking diversity and strangeness of the possible relationships between the many corners of life. Of course we get the 'animals eat plants' story**, occasionally hear of animal-earing plants. Then we hear of fungi and bacteria as decomposers. If you hang out on the botany side of the Great Divide, you also hear of algae as the aquatic producers and fungi as mycorrhyzal symbionts (plant-fungal relationships are a fascinating world of awesome, by the way). But that's pretty much all ecology you ever get (outside of ecology programs anyways. I hope!), and frankly, it's pretty boring.

What about the vast 'multicultural' realm of the customs and ways of the parasites? What about the unexpected mutual endosymbioses, both of multicellular and unicellular things, on both multicellular and unicellular levels? What about multicellular predators, and parasites(!) of unicellular organisms? Or unicellular predators(!) of multicellular organisms? For a case of the latter, let's turn back to our rotifers. By the way, if you're desperate for a tree, or sexy SEMs of rotifer morphological features, this paper could come in handy(free access).

*Well, that and any prokaryotic literature. Bacteria are...complicated. Unlike Eukaryotes. ^_^
**A while ago while writing a biochem exam, I came across a question starting off with plant lipid synthesis. I got excited (OMG they're mentioning PLANTS!!!). Then I read on: "...which is then consumed by a herbivore." and wanted to throw something at whoever wrote that question. That pretty much sums up the biochemists' and zoologists' view of plants.

Quick aside: the border between parasite and predator is quite fuzzy and arbitrary, especially once you venture outside familiar grounds. Generally, people tend to call something a parasite when it feeds of something bigger than itself, and a predator when it feeds off something smaller.

Rotifers, like anything else out there, have no choice but to participate in the great web of things eating things. As is the case for many small pond organisms, their predators are plentiful. In fact, there's some rather embarassing ones. Including a relative of this slow thing:

(Mine again. Having my own photo stock would save me so much search and citation time. Of course, making my own stock might kind of negate that.)

Yeah, rotifers can get chomped on by Difflugia, of all things.

NOT mine this time. Han et al. 2008 Hydrobiologia. I wish I could see this live! See text for description.

The [planktonic thecate(!)] amoeba senses the thecate rotifer, quickly extends its pseudopodium along its length as if to 'measure' it, positions itself with its 'mouth' towards the rotifer's foot, somehow makes a hole in its protective jelly, and slurps in the rotifer's contents, while grasping the shell of its prey with the pseudopodia. All within 20min. Amazingly, this amoeba is not a rotifer specialist - it only eats them upon a rare chance encounter. Thus, the amoeba actually has complex behavioural patterns, enabling it to detect the type of prey, sense its shape and decide on an attack strategy.

Many protist predators are actually quite picky, sifting through the various detritus they come across and deciding on what to engulf. Combine that with eyespots and ocelli (Warnowiid dino camera eyes!), along with other oddities of the unicellular world, and we've got ourselves a whole field of Protist Behavioural Biology.

As we look more and more carefully at the unicellular world, it becomes increasingly more apparent that cells can 'think' -- not in the woo-ey "OMG feelings and consciousness!!1!" kind of way (in fact, I HATE that shit) -- but in terms of processing information from their environment in a rather complex way. Doesn't seem like much is known about cellular 'molecular intelligence' (or 'molecular instinct' - the line between canonical behaviour and genetic/biochemical pathways is quite blurry, it seems), and the field is dominated by junk thinking at the moment. And computer scientists, who don't seem to be bothered by biological reality. But hopefully as more and more results pile up suggesting some sort of 'cellular intelligence*', some fundamentally interesting stuff may come out of that research.

*I feel I may end up hating myself for using that term, but I can't think of anything better at the moment...sadly, too many good words get hijacked by sloppy thinking.

If you ever find yourself randly shrinked to the micron scale some day: Stay the fuck AWAY from amoebae. They may look slow and stupid and oozy, but they're out to fucking get you. They vicious. They don't care what unicellular or multicellular phylum you happen to be a proud member of: if they can hug you, they WILL squish you with nasty enzymes. Or make holes in you. They're freaking SCARY! o_O

Same goes for forams and "radiolaria", by the way. Actually, on the micron scale, if something doesn't eat you from the outside, chances are, it's patiently waiting to devour you from the inside. The microbial world tends to be quite innovative. And we haven't even gotten to the lethal veil of pallium...

Starting to feel good about your size yet?

As much as I'm obsessed with the microscopic world, it feels much safer to be a mere observer, rather than a participant. There's some truly terrifying monsters in those waters!

References
Han, B., Wang, T., Lin, Q., & Dumont, H. (2007). Carnivory and active hunting by the planktonic testate amoeba Difflugia tuberspinifera Hydrobiologia, 596 (1), 197-201 DOI: 10.1007/s10750-007-9096-z

Riemann, O., Kieneke, A., & Ahlrichs, W. (2009). Phylogeny of Dicranophoridae (Rotifera: Monogononta) - a maximum parsimony analysis based on morphological characters Journal of Zoological Systematics and Evolutionary Research, 47 (1), 61-76 DOI: 10.1111/j.1439-0469.2008.00482.x

Could this be a foram?

Edit: No. Turns out to be a pollen grain. Thanks Chris!
(pollen morphology is quite fascinating and breathtakingly diverse, btw! Plant sex is weird...)

Found this in a marine benthic sample this past spring, never got around to cropping it:

(40x DIC; obsessive-compulsive optical sectioning alert...)

Could this be some sort of foraminiferan? Reminds me of something Globigerina-oid, but if I recall it's planktonic, not benthic - although those things are quite capable of 'littering' once they die. Any thoughts?

(can provide a higher res image if interested)

Assorted musings on barcoding

Speaking of Guelph (see prev post), Paul Hebert, the Canadian barcode god (or so I'm told), gave a talk today about...well, barcoding life. Overall, it was a good talk, and I can see why he's in charge of a bunch of stuff -- his talks are quite convincing. At least he's convinced me not to automatically fear/resent barcoding. Not that anyone asked for my opinion on these matters, but this is my little corner of the internet so I'll ramble some thoughts about it. (yes, ramble. You've been warned.)

I have a few traditional taxonomist friends who have painted a pretty negative image of it for me; there's even a mycology grad student who does quite a bit of barcoding herself and has come to be quite skeptical of it. Furthermore, being fascinating by the biology of things rather than their mere existence, all those billions of species don't exist as far as I'm concerned until they've been actually studied. And no, 600bp of a conserved gene sequence does not qualify.

The value of being able to identify something in the field without having to look at the 'length of the spine on the 3rd tarsus', to [badly] paraphrase Hebert, is clearly there. His dream of a handheld barcoding device perhaps could be especially useful to people who actually do fieldwork (wait, there's biology outside the lab? WTF?), although how you can 'read' a DNA sequence sufficiently quickly still eludes me. Don't you still have to wait for crap to get amplified? Would be awesome if they have some way of doing really quick (and cheap) PCR, that could come in quite handy even for the model organism crowd. I don't necessarily enjoy waiting 2h to genotype some crap (oh, and another hour before that to extract gDNA). But I digress. This barcoding technology stuff does sound awesome.

However, I often have some skepticism towards awesome-sounding revolutionary technology -- too often people get carried away in the hype, and start attempting to use machinery to replace human intellect. That can be dangerous. Some tasks appear more mechanical than they really are, and it seems taxonomic identification and description is definitely among them. Most reasonable barcoding proponents don't seem to dismiss traditional taxonomy, but there is the fear that public opinion and funding agencies might not feel the same way. And as much as we love to reduce everything within our reach to digitised strings of characters, we still have to interact with the physical world.

Hopefully, the barcoding 'revolution' (that word is automatically associated with TC-S for me now, for some reason...) would be used in combination with the insight, knowledge and intelligence (ie talent) of traditional taxonomists, rather than attempting to replace them. Molecular phylogeny is substantially more reliable than anything morphology-based, as anyone who's wandered around in the protist kingdom would know*. But that doesn't mean morphological evolution is now to be ignored - on the contrary, molecular biology has made it much more exciting now that you could see how the organisms are related, and then investigate their morphology.

Another thing that annoys me is this whole 'species counting' business. You know, when someone spits out "There are 10 000 species of blah in blah", and the media picks up on it and we end up with "OMG, there are 20 000[sic] species of blah in [incorrect] blah!!!one! We are doomed/saved/awesome/cured of cancer[pick one]!" Seriously, who cares? Does it really matter whether there's 10 000 reproductively isolated clusters of something or 15 000 or 100 of them? Does counting them reveal anything profound? The majority of life on earth doesn't particularly care much about reproductive isolation! And among the few sorry exceptions where reproductive isolation becomes somewhat important (metazoa mostly; plants turn out to be a little more promiscuous), all species-counting can say is perhaps something interesting about...evolutionary dynamics of reproductive isolation. Furthermore, this ignores intraspecific variation, which is important and seemingly ignored by many evolutionary ecologists. And then we wonder why people get confused about where variation are. They see diversity as being composed of heavily discrete units, which may perhaps be confusing when trying to understand evolution itself.

That's not to say variation isn't important - it is, and it's definitely fascinating. But it does not do it much justice to simplify everything down to 'species', which then become treated as solid units of variation, rather than a subset thereof. It's far more complex than a mere absense of interbreeding.

Barcoding may be quite interesting for exploring how asexual vs. sexual (and facultatively sexual) organisms tend to cluster, how this clustering differs among various phyla, habitats, etc. Hebert did mention plans to pursue some of those directions, and it will be interesting to see what turns up.

Lastly, they seem to be using a single gene for barcoding. I think that's might even be what 'barcoding' would mean, strictly speaking. This is worrisome. Single gene phylogenies are CREEPY and SCARY o_O. I wouldn't touch one with a 10 foot pole anymore. Of course they picked something that [they think] is extremely conserved and constant (yet flexible enough to allow for variation at their desired resolution) - a commonly-shared mitochondrial gene (by the sound of it, Cox1 perhaps). They say it's conserved and reliable. Awesome - that's what they said about SSU rDNA. How did that turn out?

Bits of freaking FUNGI ended up as basal to all Eukaryotes, along with diplomonads, parabasalia and archamoebae, which have little to do with each other. Oh, and they still print this tree (which was quite a breakthrough at the time, to be fair; but things have changed in the last couple of decades...) Why? Some lineages experience faster rates of evolution than others, and this leads to much greater sequence divergence from its neighbours, which often screws things up in the alignment algorithms, resulting in Long Branch Attraction - things that diverged a shitload relative to everything else tend to cluster together. Some normally stable genes under certain circumstances can go haywire. Single gene trees a fucking dangerous. So are multigene trees, but we haven't got much else going for us...

Basically, the problems that plague SSU alignments can also fuck with barcoding. Especially once you wander outside the small comfy familiar home we call Metazoa...

Of course, everyone knows that, but I wouldn't be surprised if people suddenly started worshipping (and publishing) single gene trees based on barcode sequences. Faith in the scientific community seems to be inversely proportional to experience with it...

So yeah, it's exciting, but we must also be cautious. I guess that would be my [rather unhelpful] answer to pretty much everything...

Do you guys have any thoughts?

*Morphological classification led to almost all non-photosynthetic basal stramenopiles/heterokonts being mistaken for something considerably different: Labyrinthulids, oomycetes and Blastocystis were considered to be fungi, opalinids were 'ciliates', actinophryids were 'heliozoa', and there's even a genus called Pseudobodo (a bicosoecid) that was mistaken for...bodonids! Molecular phylogeny continues to churn out one surprise after another!

Microbial Art

Several clans of geeks profs and their students put together an awesome collection of art done on plates with bacterial and fungal cultures - go check it out at MicrobialArt.com!

Of course, this one had to be one of my favourites:

(Gregory Lab; exhibit #12)

Perhaps 'tis about time I start plating my bacteria 'creatively'...

Mystery Micrograph #07

Alright, this one should be a little bit easier.

To be referenced later; scalebar = 2um

Good luck!

---

HINT (29.10.09):


(Source: Keeling et al 2005 TrEE; do I even need to keep citing this anymore? Can we be on familiar terms and refer to it as 'that tree' yet? =P )

It's somewhere in there... I'm so helpful it hurts, I know. Mwahaha.

Answer to MM#06 - Cochlosoma: a peculiar gut denizen

Christopher Taylor just got last week's Mystery Micrograph - it's Cochlosoma anatis, a trichomonad parasite of turkeys. It's sort of related to Pentatrichomonas (Hampl et al. 2006 Int J Sys & Evol Microbiol), although the support for that seems rather weak.

(Cooper et al. 1994 Avian Diseases; SEM of Cochlosoma anatis; Bar = 3um)

It reminds me of diplomonads (eg. Giardia) with its flat shape and adhesive sucker disc appendage - most likely a good adaptation for the intestinal environment. The ventral side is even more diplomonad-esque:

(Cooper et al. 1994 Avian Diseases; SEM of Cochlosoma anatis on intestinal mucosa(dorsal view), note the marks left by the parasite's 'suckers' in the vili; Bar = 5um)

Also, both are 'amitochondriate' anaerobes; trichomonads (and all the other parabasalians) have hydrogenosomes, which are ultra-reduced mitochondria with a pathway for producing hydrogen gas. Diplomonads and retortamonads lack hydrogenosomes, and have really taken the whole Mitochondria Lite business to the extreme - they have tiny non-descript mitosomes which are basically kept around for one metabolic purpose: FeS cluster metabolism*.

I digress, but that's pretty much all I have to say about Cochlosoma. Besides its peculiar shape, it seems to be your garden variety trichomonad with a case of Giardia-envy. Although who knows, these things are also grotesquely understudied...

Brace yourselves for the next mystery micrograph, which shall be posted SOON! done

*Thus far no eukaryote has been found without at least that part of the mitochondrial metabolic pathways left behind. Kreb's/TCA cycle can be quite 'easily' dispensed with or greatly reduced, despite it being the more 'famous' part of mitochondrial metabolism. Microsporidia (intracellular parasitic fungi) have the most reduced mitochondria. Turns out that their mitochondria lack ATP production altogether. The ATP is stolen from the host cell, and actually imported to the mitochondrion! (Tsaousis et al. Nature 2008)

Cooper, G., Shivaprasad, H., Bickford, A., Nordhausen, R., Munn, R., & Jeffrey, J. (1995). Enteritis in Turkeys Associated with an Unusual Flagellated Protozoan (Cochlosoma anatis) Avian Diseases, 39 (1) DOI: 10.2307/1592001

Hampl, V. (2006). Affiliation of Cochlosoma to trichomonads confirmed by phylogenetic analysis of the small-subunit rRNA gene and a new family concept of the order Trichomonadida INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY, 56 (1), 305-312 DOI: 10.1099/ijs.0.63754-0

Tsaousis, A., Kunji, E., Goldberg, A., Lucocq, J., Hirt, R., & Embley, T. (2008). A novel route for ATP acquisition by the remnant mitochondria of Encephalitozoon cuniculi Nature, 453 (7194), 553-556 DOI: 10.1038/nature06903

Mystery Micrograph hint

Last week's MM is getting covered in cobwebs and accumulating a thicket of tumbleweeds at the edges. With a chorus of crickets.

Since we've established it's a trichomonad, we've narrowed it down to this tree:

(Noda et al. 2009 Mol Phylogenet Evol; a parabasalian phylogeny. It's somewhere in there. It's even mentioned. /massive hint)

I do realise this one is even more obscure/diffucult than usual.

Sunday Protist - Nucleariids

While exploring the various corners of the protistan world, I've been neglecting our close relatives - the Opisthokonts. Let's quickly remedy the situation.

A couple weekends ago I had some pond water on hand, and it turned out to be quite productive. I was on a bit of a heliozoan and amoeba spree when I encountered these things:

At first it seemed like a 'heliozoan'*, but wasn't quite round enough. Then I noticed filopodia. Heliozoa with filopodia? Nah. But it didn't quite qualify for your typical amoebozoan either, so I was rather confused. To make it even more fun, some of them had spicules sticking out (see the optical section through the top in the rightmost image above). Then I started seeing similar things without spicules, and they seemed to be related:

So I spent an hour or so trying to figure this one out**. They turned out to be Nucleariids, a group of filose amoebae basal to fungi. The top one appears to be Rabdiophrys, which has been, in fact, confused with or considred as heliozoa; the bottom one may well be Nuclearia itself - some mixed images of Nuclearia and Rabdiophrys can be found here.

Nucleariids are filose amoebae, meaning they produce long thin thread-like pseudopodia without internal microtubule bundles (which would be axopodia, like those of 'heliozoa'). They are on the fungal side of the great cauldron of Miscellaneous Opisthokonts sometimes called 'Choanozoa' by TC-S. Other times, he seems to reserve Choanozoa for those on the animal side of opisthokonts. Yet other times, he seems to fail to piss off cladists and actually use monophyletic terms. Which one of those is 'in season' likely depends on the monsoon patterns in Bangladesh. Or the temperature fluctuations on one of Jupiter's moons. Further research needs to be done. Materials include ethanol and acid, if I recall. Anyway, here's a damn tree already:

(Ruiz-Trillo et al. 2007 Trends Genet; Opisthokonta - our fellow ass-tailed relatives)

The Choanozoa/Misc Opisthokonts actually tend to be insignificant-looking amoeboid things most of the time, except for Choanoflagellates, which are these really cute lorica-building flagellates with a cone of microvili surrounding the flagellum on their asses. Speaking of which, opisthokont means 'posterior flagellum', or, less pretentiously, ass-tail. Some fungi (chytrids) have flagellated motile spores, and their flagellum happens to be on the posterior relative to the cell's swimming direction. As mentioned earlier, in most eukaryotes the flagellum performs a pulling action, whereas the opisthokont flagellum pushes the cell. This actually poses some problems for filter-feeding organisms, which use flagella for propelling food particles towards their 'mouth', and may be part of the reason some Choanoflagellates started aggregating into colonies - to stop themselves from moving away from their prey when using flagella.

Here's another specimen of putative Rabdiophrys:

And yet another:

I can keep going:

Seriously, I've got A LOT of those guys!

Switching to Nuclearia now:

Note the absense of spicules:

Hey, it could be worse: I also have a freaking pile of non-descript random amoeboid things. SMALL non-descript random amoebae.

There's a 'cellular' slime mould that turns out to be among the nucleariids: Fonticula (Brown et al. 2009 MBE; advance publication). The poor thing has been lumped with everything from Acrasids to cellular slime moulds, and subsequently neglected for a couple decades. This is the first documented case of slime mould aggregation in opisthokonts, which may contribute a thing or two to the evolution of fungal and metazoan multicellularity. (off topic note: ciliates can aggregate too!)

So now we've finally covered an opisthokont. Phew. That was bugging me.

* Just FYI, heliozoa are not a real group - they're united by their sun-like morphology, axopodia and nothing else...
** Being too impatient to use dichotomous keys (which also simply fail to exist for some organisms), I use a combination of papers, websites and Google image search to find stuff. Basically, you find something that lists a bunch of organisms in the vicinity of what you think it might be, and then look to see if any of the pictures might match. If it's something so obscure that even Google is unaware of its existence, you have to sift through ancient forlorn journal articles sometimes, but usually it doesn't take that long to cross another name off the list.

If completely stumped, I'll just start googling random morphological descriptions in both scholar and image search, until hitting something familiar. As random and haphazard and unprofessional as this method is, it's actually much more effective than figuring out dichotomous keys, in my opinion. Especially when you're unfamiliar with the structural terminology. And especially when such a thing doesn't even exist for your group of organisms, as is too often the case for protists... [/hint for Mystery Micrographs]

Brown, M., Spiegel, F., & Silberman, J. (2009). Phylogeny of the "forgotten" cellular slime mold, Fonticula alba, reveals a key evolutionary branch within Opisthokonta Molecular Biology and Evolution DOI: 10.1093/molbev/msp185

RUIZTRILLO, I., BURGER, G., HOLLAND, P., KING, N., LANG, B., ROGER, A., & GRAY, M. (2007). The origins of multicellularity: a multi-taxon genome initiative Trends in Genetics, 23 (3), 113-118 DOI: 10.1016/j.tig.2007.01.005

Blogging Ban (exam ahead...)

I've been placed by someone on a blogging ban until Thu evening. Thus, I must blog it. Delicious ironing is delicious. But just fyi, there'll be a 2 day posting blackout. Tragic, I know. To help soothe the traumatising shock, a random piece of weird research before I 'take off':

Dominance, Politics, and Physiology: Voters' Testosterone Changes on the Night of the 2008 United States Presidential Election (PLoS ONE 2009; open access)

Oh, speaking of which, yay Open Access Week!

Ummm...and here's a random amoeba of sorts:


Ok, I'm outta here. Feel free to drop off hints about how to learn and understand information cram for a biochem midterm. That's 50% of your final grade, and 100% memorisation. Of medical material. In a supposively 'general' biol course. Stuffed full of "[blank] kinase kinase" (via MolBio Res. Highlights). I'm so excited!

[random literature foray]
I was about to type "why the fuck should I ever care about the urea cycle if I don't ever intend to work on animals!?". Then I did a quick google search. HOLY SHIT plants do the urea cycle too, although more of it seems to happen in the mitochondrion. Diatoms have the complete thing, even though they don't really need to export nitrogen waste (this implying it has other metabolic functions). And holy crap, Plasmodium seems to perhaps be capable of it too. Apparently, Trypanosomes seem to lack it in complete form, as well as Tetrahymena(although it has the components). Among bacteria, Helicobacter(epsilon-proteobacteria) does urea, as do actinomycetes and a planctomycete relative; they seem to use it for carbonic acid synthesis.

It seems the process is actually universal to life; however, this doesn't rule out a transfer of the pathway to eukaryotes from the alpha-proteobacterium-derived mitochondrion. While mapping out a [very hypothetical] metabolic map of the proto-mitochondrion based on comparisons with alpha-proteobacterial genes, these guys found an absense of the urea cycle, implying it may have been derived from the host eukaryote or something else; however, the evidence doesn't seem to be too overwhelming. Perhaps when a host-->mitochondrial transport system evolved, some eukaryotic versions could have 'ousted' the bacterial ones, somehow. Or maybe the mitochondrial system outcompeted the native eukaryotic system, or the eukaryotic version simply degraded first, or partially degraded first (parts of the pathway still happen in the cytosol in many eukaryotes, it seems).
[/random literature foray]

Anyway, I should probably get back to mindless cramming. No one really gives you marks for randomly prowling the literature around midnight, even if it does make the subject much more exciting and memorable. In the process of briefly and very shallowly digging around here, I sort of figured out the pathway (because I had to), learned random cool shit about the diversity of this pathway and bits and pieces about its evolution (diversity and evolution are two things that all biologists should get aroused by, no?), and made myself a mini-index of potentially useful references in the preceding paragraphs. And made this seem relevant for myself, even for a little while - which stimulates the memory and allows for a modicum of comprehension of the material!

You know, the modern university hasn't gone very far from its monastic origins... we still must sit there in rudimentary conditions (poverty and hunger) memorising sacred scriptures and recanting verses in a foreign (and still latinate!) language...


Meanwhile, this needs to be resolved by you guys. I await in suspense. Feel free to ask questions, etc.

Mystery Micrograph #06

It's that time again!

Bar (barely visible - bottom right) = 3um, SEM; to be referenced later

This one is demonically hard, so open to everyone. And their PIs.
We really desperately need some outbreeding here, so please someone NOT affiliated (past or present) with a certain dep't get it!

Due Sunday sometime. Beer reward* until too many hints have been dispensed (which is at my discretion =P)

*That is, if/when it is able to be collected, and if/when I'm actually able to afford it. Alternatively, you could hire me and then the beer would be very easy to obtain. That's always an option. Srsly.

If it helps at all, an epic description of this creature:

"Looks a little like Giardia put through some medieval torture device. Or that thing on the Muppet Show that rearranged people's faces: "Here, I'll just grab your adhesive disc and stretch your posterior end over the top of that, and maybe chop off a couple of flagella, oh, and a funky divetty thing in the middle of the disc would look really neat too...."
Kind of a Mr-Potato-Head approach to protist ID."

- Opisthokont

Sunday Protist - Diplonemids: Metaboly without a pellicle and the dawn of kDNA?

Took you guys a while to get the past Mystery Micrograph, which gave me ample excuses to procrastinate with last week's Sunday Protist. Of course, no one noticed, so it's all good, right? Opisthokont finally got it after every single other discicristate lineage has been eliminated, and grotesquely revealing hints have been given away. Johan guessed their sister clade, Kinetoplastida. More importantly, we need fresh blood on this blog, and thus far, the Mystery Micrograph winners have been an incestuous group, all linked in the present or past with a single department. Srsly. So half the win goes to Johan for getting sort of close-ish enough, as well as being somebody I don't actually know outside the blogosphere...

So the correct answer for MM#05 was...

Rhynchopus, a diplonemid. (Roy et al. 2007 JEM) Note the odd writhing movement in the timelapse (1-8) as well as absense of flagella in most figures. This group is very obscure - check out Table 1 in the above mentioned publication if you can, and marvel at the sea of question marks regarding the various diplonemids...

That's right, yet another obscure protist, Rhynchopus, a genus of diplonemids. And everyone knows what those are, right? *crickets* Well, hang in there and find out! Feel free to point and laugh any time you see 'Rhyncopus' - apparently, I have serious issues spelling that one... (although it's definitely not as bad as Barleeiidae) Speaking of taxonomy, Diplonema was formerly known as 'Isonema', in case anyone gets the urge to scour ancient protistology literature...

Cell Structure
I assumed that the absense of flagella in the SEM was due to them falling off due to stress or just being generally shitty specimens for EM prep. Turns out, that's actually how they roll - flagella are only visible in swarmer cells produced in hungry (starved) cultures. The flatness is also a characteristic attribute of this cell, not so much a dehydration artefact. (Roy et al. 2007 JEM). The cell contains a large flagellar apparatus and a cytostome (cell mouth), generally oriented parallel to each other, with a lip protruding outside the opening:

Drawings of Rhynchopus cell structure (Roy et al. 2007 JEM). Hopefully this makes the above paragraph make a little more sense...

Not much is really known about the diplonemid nuclear genome organisation, although the chromosomes appear to be permanently condensed, just like in the euglenids. Weird chromosome structure is often correlated with genomes on crack, so one can only wonder what kind of oddities could be concealed in their chromatin. However, the nuclear genome does seem to contain a splice leader gene (Sturm et al. 2001 JEM), a characteristic feature of kinetoplastids, which are famous for their polycistronic primary mRNA transcripts - they have several genes following a single promotor, which are then broken up by trans-splicing 5' cap-containing splice leaders before each gene. Rumour has it, euglenids might also be capable of splice leader trans-splicing, so this may well be a general euglenozoan trait.

Diplonemids are interesting from an evolutionary perspective. While sharing some distinctive features with many euglenids (metaboly, condensed nuclear chromatin, cytopharynx structure), they are actually basal to kinetoplastids, with the euglenids basal to both kinetoplastids AND diplonemids. Together, they form the Euglenozoa*:

(Simpson et al. 2004 Protist; tree of Euglenozoa; note diplonemids branching basal to kinetoplastids, and not to euglenids.)

* By the way, Euglenozoa = large group of excavates containing kinetoplastids (eg. Trypanosomes), euglenids (eg. Euglena) and diplonemids (subject of this post). We like to recycle fragments of Latin lexicon wherever possible.

Metaboly
Many euglenids share a characteristic form of motility called 'metaboly', which is essentially a writhing motion of a cell, where it twists and turns like mad, moving a blog of cytoplasm from one end to the other. Euglenids are covered with protein pellicle strips arranged longitudinally from one end of the cell to the other. Among many euglenids, those strips have a very complex structure, which enables them to slide against each other and allow metaboly to occur. Beneath each stript is a bundle of microtubules. (This page pretty much has nearly everything you ever wanted to know about Euglenids.)

[rant]One anoying thing about Euglenid biologists (that is, people studying euglenids, not euglenids who have chosen a questionable career path...) is their referring to the pellicle strips as 'cytoskeleton', which the rest of us reserve strictly for microtubules, actin filaments and intermediate fibres. As cool as your strips might be, they do not consist of actin, 'tubes OR intermediate fibres, and are therefore NOT cytoskeleton. Otherwise anyone outside 'Euglenology' becomes very confused. Thanks. [/rant]

Anyway, you may wonder why we're rambling about Euglenids all of the sudden. Thing is, diplonemids also have metaboly-like motility. Except that they lack pellicle strips altogether. However, they still have the longitudinal bundles of microtubules lining the cortex of the cell:

TEM across membrane showing the densely packed microtubule bundles just underneath the membrane (Roy et al. 2007 JEM)

The 'function' or significance of euglenoid metaboly is still unknown. Now, it is often said that the euglenid pellicle strips 'cause' the euglenoid motion (metaboly) (eg. Leander et al. 2001 Evol, although they get more careful in later publications). The secondary fusion of pelicle strips, eg. as in Phacus, inhibits metaboly from happening, but that's unrelated to the acquisition of metaboly in the first place. As we've seen in the topmost figure, diplonemids too seem to exhibit metaboly, but no pellicle stripts. This would make them a good model for examining the mechanisms of metaboly, as well as its potential adaptive value, if any exists.

Furthermore, diplonemids are basal to kinetoplastids, and if they share a trait with euglenids, one wonders if kinetoplastids have retained it, as it would be more parsimonious to assume their common ancestor must've had it as well. Kinetoplastids, at least their Trypanosome representatives, also have pellicular microtubules (Woods et al. 1989 J Cell Sci), and some Bodonids have even been observed to excibit metaboly-like motion (Swale 1973 Biol J Linn Soc, and references made to Cryptobia helicis, eg. in Vickerman 1977 J Protozool, although I can't seem to access the original C.helicis account by Kozloff 1948 J Morphol), although it is uncertain if the pellucular 'tubes have anything to do with it.

Some ciliates have also been described to undergo metaboly-like motion, although the similarity between them is uncertain. If more organisms do actually exhibit something similar to euglenoid motion, then perhaps it's not so special after all. It could merely be enhanced by the pellicle strips providing extra rigidity to the longitudinal 'ridges' formed by the microtubules. Thus, there may well be no function of metaboly - it may just be an artefact of having fairly rigid structures lining your cell from head to toe, which in turn would have evolved for entirely different reasons (or lack thereof).

So here's a mini project for someone with lots of time and stray grant money lying about:
- determine the mechanisms of metaboly in both the pellicle-bearing euglenids and 'naked' diplonemids; likely, it has something to do with the organisation and dynamics of pellicular microtubules. Use [MT-disrupting] drugs, they're good for science.
- examine other eukaryotes with some sort of pellicular MT bundles, and see how they roll move about.
- look over older accounts of 'metaboly-like' motion in non-euglenids, and verify them. Check if anything is known about their cytoskeletal organisation.
- my guess would be, the much-noted euglenoid motility might actually be just an epiphenomenon of euglenid cytoskeletal and pellicular structures, which are shaped like that for other reasons. It likely has no function*. Ie, perhaps it's slightly...overrated!

*There have been observations of eukaryovores tending to be more likely to exhibit metaboly than bacteriovores, perhaps due to larger prey sizes, although, as pointed out in the aforementioned link, this correlation is rather fuzzy; and, even in the case of there being such a relationship, the presence or absense of metaboly could be a secondary feature resulting from different requirements of the cytoskeleton itself in eukaryo- and bacteriophagy. That was quite possibly the worst sentence in the history of English.

Mitochondrial Genomes
Diplonemid mitochondria are arranged in a cortical network pattern, and are devoid of a kinetoplast (characteristic of their sister clade)(Roy et al. 2007 JEM). At first glance, the mitochondrial genome seems mundanely circular (and unlinked):

(Marande et al. 2005 Euk Cell; Diplonema circular mitochondrial DNA in TEM)

However, those circles are about ~7Kb in size, which is small for mitochondrial DNA. (Marande et al. 2005). This is starting to get a little weirder. Diplonemids also seem to have flattened, rather than discoidal, mitochondrial cristae - quite unusual for a discicristate. Furthermore, some genes seem to be fragmented and distributed over multiple chromosomes. This is starting to get quite interesting. So why would we expect weird mitochondrial genomes in diplonemids anyway?

Because among the neighbouring kinetoplastids, they happen to be on crack:

(Simpson et al. 2002 MBE; evolution of Trypanosome 'chainmail' genomes and its relatives.)

I won't go into detail here, but let's just say Trypanosome MtDNA organisation is a major piece of supportive evidence for the 7th Day LSD theory of intelligent design, wherein the intelligent designer got fucking high on acid and designed the protists on his day off. I've alluded to it elsewhere, as it is a substantial part of my personal faith, and therefore true. I should go form a church around my revelation...

Basically, the intelligent designer spent too much time watching fantasy movies set in medieval Europe (before humans were even created), and was wondering if it would be possible to replicate various pieces of arms and armour using DNA molecules. You would do it too if you were bored, high and almighty. So this very intelligent designer created 'chainmail' genomes in Trypanosomes. Not only that, but he was also apparenly bored with the canonical DNA -> pre-mRNA -> splicing ->translation pathway, and decided to spice it up with some RNA editing. Why bother producing proper mRNA from the start when you can make shit mRNA and then fix it with a whole bunch of guideRNAs. And then laugh at biologists trying to come up with elaborate adaptive explanation for the evolution of this nonsense. Hahaa!

Basically, big circles code precursor mRNA, which is then fixed up and debugged by 'gRNA' from little circles. This process requires upwards of about 500 proteins to work. Other eukaryotes just use RNA polymerase. Efficiency at its best. Yeah.

Interestingly, in the Euglenids, Petalomonas is suspected of having mitochnodrial RNA editing on crack possibly even exceeding that of Trypanosomes! Unfortunately, the understanding of Euglenid mitochondrial genomes seems to be a mess at the moment, but they do have potential to turn out to be weird. (Roy et al. 2007 Protist).

So what about those fragmented genes hanging out on different chromosomes? Marande et al. (2005) suggest they may be the beginnings of gRNAs. They seem to be significantly larger than Trypanosome gRNAs, and thus may be merely fragments that get trans-spliced together. But often where there is trans-splicing of gene fragments, there may also be RNA editing (eg. dinoflagellate mitochondrial DNA). The diplonemid mitochondrial genome is actually relatively massive (~56 chromosomes, 360Kb total), and turns out that most mitochondrial genomes actually only contain one type of circular plasmid; among the few exceptions are the trypanosomes and Amoebodinium (all from Marande et al. 2005). Hmmm. Thus, while little is still known (or published anyway) about the Diplonemid mitochondrial genomes, they seem to be quite promising for studying the early evolution of kDNA architecture.

If euglenids have similar madness too, one can only wonder why Heterolobosean mitochondrial genomes may reveal. Perhaps the origins of the evolutionary absurdity known as kinetoplast DNA might actually lie earlier on in the discicristates?

There seem to be multitudes of similarities among the euglenozoa, now that I look at it again. And the sister Heterolobosea are too poorly known genetically (or even developmentally) to make any conclusions or generalisations there. But it's quite fascinating to watch the vast and diverse evolutionary history of eukaryotes slowly come together, piece by piece, into a giant tapestry of evolutionary stories. Our intelligent designer on LSD had no idea how much sense he would inadvertently make amid the nonsense...


PS: Please feel free to point out any defects/inaccuracies -- I've been staring at this review post far too long to notice anything!

References:
Leander, B., Witek, R., & Farmer, M. (2001). TRENDS IN THE EVOLUTION OF THE EUGLENID PELLICLE Evolution, 55 (11) DOI: 10.1554/0014-3820(2001)055[2215:TITEOT]2.0.CO;2

Marande, W., Lukes, J., & Burger, G. (2005). Unique Mitochondrial Genome Structure in Diplonemids, the Sister Group of Kinetoplastids Eukaryotic Cell, 4 (6), 1137-1146 DOI: 10.1128/EC.4.6.1137-1146.2005

ROY, J., FAKTOROVÁ, D., BENADA, O., LUKEŠ, J., & BURGER, G. (2007). Description of Rhynchopus euleeides n. sp. (Diplonemea), a Free-Living Marine Euglenozoan The Journal of Eukaryotic Microbiology, 54 (2), 137-145 DOI: 10.1111/j.1550-7408.2007.00244.x

Roy, J., Faktorová, D., Lukeš, J., & Burger, G. (2007). Unusual Mitochondrial Genome Structures throughout the Euglenozoa Protist, 158 (3), 385-396 DOI: 10.1016/j.protis.2007.03.002

Simpson AG, Lukes J, & Roger AJ (2002). The evolutionary history of kinetoplastids and their kinetoplasts. Molecular biology and evolution, 19 (12), 2071-83 PMID: 12446799

SIMPSON, A. (2004). Early Evolution within Kinetoplastids (Euglenozoa), and the Late Emergence of Trypanosomatids Protist, 155 (4), 407-422 DOI: 10.1078/1434461042650389

STURM, N., MASLOV, D., GRISARD, E., & CAMPBELL, D. (2001). Diplonema spp. Possess Spliced Leader RNA Genes Similar to the Kinetoplastida The Journal of Eukaryotic Microbiology, 48 (3), 325-331 DOI: 10.1111/j.1550-7408.2001.tb00321.x

SWALE, E. (1973). A study of the colourless flagellate Rhynchomonas nasuta (Stokes) Klebs Biological Journal of the Linnean Society, 5 (3), 255-264 DOI: 10.1111/j.1095-8312.1973.tb00705.x

VICKERMAN, K. (1977). DNA Throughout the Single Mitochondrion of a Kinetoplastid Flagellate: Observations on the Ultrastructure of Cryptobia vaginalis (Hesse, 1910) The Journal of Eukaryotic Microbiology, 24 (2), 221-233 DOI: 10.1111/j.1550-7408.1977.tb00970.x

Heterolobosea I: Eruptive amoebae, a preview

So I've been challenged to write about Heterolobosea. It all began with my slightly cruel and potentially foolish challenging of our friendly neighbourhood taxonomist to write up a series of posts on Amoebozoa. I was rather confused about the Chaos chaos in the Amoebozoan kingdom, so the sensible thing to do was making someone else sort it out for me ^_~ And sort them out he did, with the following fascinating and informative posts that I urge you all to read:

General Amoebozoa: Putting the Formless in Formation.
Breviata anathema: TAFKAMI; TAFKAMI Walks.
Tubulinea: The Paragons of Amoeboids; Amoeba: Much Wierder than You Think.
Discosea: Keeping a Low Profile.
Mycetozoa: The Diversity of Slime Moulds.
Various Amoebozoa incertae sedis ('Variosea'): Amoebozoan Oddments.
Archamoebae: The Apogee (or Nadir) of Amoebozoan Evolution.

For the uninitiated, a nice intro to amoebae; keep in mind that the ones there are amoebozoans (except for page 3 - those appear to be Cercozoans). Before we set of on an amoeboid adventure, a quick clarification: the colloquial 'amoeba' refers to the blobby cell shape, which is called 'amoeboid'; amoeboid cells exist in many distantantly-related taxa, the larger and more famous of which is Amoebozoa. To make matters even more confusing, Amoeba is a genus of amoebae within Amoebozoa. In short, Amoeba != amoebozoan != amoeboid cell type. Amoeboid cells can actually be quite diverse:

(Leidy 1879 Fresh-Water Rhizopods of North America; some amoeboid diversity, mostly amoebozoans and cercozoans. Amoebae were formerly classified under Sarcodina and Rhizopoda, a dumping ground for organisms with various forms of pseudopodia.

Amoebae of the Rhizarian (incl. Cercozoa) and Amoebozoan 'kingdoms' are summarised and organised (phylogenetically) in Pawlowski & Burki (2009 JEM); we shall, however, move on to explore yet another island of amoebae in the eukaryotic kingdom: the eruptive Heterolobosea!

Heterolobosea are discicristate excavates, meaning they are members of what may be a monophyletic grouping, Excavata, with paddle-shaped mitochondrial cristae. What this means I will explain later on. They're alternatively refered to as Percolozoa, until Cavalier-Smith came along and bundled them up with the 'ciliatoid' Stephanopogon under the name Heterolobosea (Page & Blanton 1985 Protistologica), which was used before to describe the Percolozoa minus Stephanopogon... Well, sort of. Thanks to Cavalier-Smith, the story is substantially more complicated, as per usual. For our purposes, let's pretend it never happened...

Let's take another look at their phylogenetic neibourhood, and then move on to actually discussing what they are, lest I begin to look like a taxonomist =P

(Lara et al. 2006 JEM; placement of heterolobosea among the excavates and beyond. They are among discicristates, between Jakobids and Euglenozoans)

Heteroloboseans have unusual (non-canonical) Golgi bodies, as well as unique mitochondrial structure (and in some cases, hydrogenosomes), and are generally pretty weird in terms of cell structure. I will go into that in grater detail in a later post, but to clear up the confusion around 'paddle-shaped' cristae: there are several shape types for mitochondrial cristae, or folds of the inner membrane that go into the inner matrix. We opisthokonts have flattened cristae, while the dominant form seems to be tubular. The shape of the cristae seems to be fairly conserved - allowing us to categorise organisms based on that character - thus resulting in a whole group partly held together by their discoidal cristae.

So what have we got among the Heteroloboseans?

• extremophiles - both heat-loving thermophiles and salt-loving halophiles
• anaerobes- including some mostly-amitochondriate members
• an organism once considered a proto-ciliate; far from amoeboid, it has incredible structural complexity mimicking Hypotrich ciliates, to some extent.
• an amoeboflagellate that can change between amoeboid and flagellate stages (two fundamentally different cell types) within a couple of hours, with de novo centriole formation
• brain-eating amoebae
• slime moulds: aggregation, stalk, fruiting body and all.

Some sample Heteroloboseans: 1 - Stephanopogon (Wikimedia commons); 2 - Percolomonas (Wikimedia); 3,4 - Tetramitus (Robinson et al. 2006 Eur J Protistol)

And a glimpse of the family structure:

(Park et al. 2008 Protist; a Heterolobosean phylogeny. Letters represent cell types present: A - amoeboid, F - flagellate, ? - this group needs loving care and attention...)

Overall, Heterolobosea are a fairly obscure and understudied group, save for one genus that happens to be an occasional menace to humans. In which case, we suddenly start to care a lot.

So why do I insist on calling them 'eruptive'? Stay tuned for the next episode of Heterolobosea - Vahlkampfiidae: The attack of eruptive brain-eating amoebae with split personality disorder. Scary...

*Off topic, but while looking up Vampyrella, came across this neat little page, wherein we have amoebae boring holes in fungal spores and feeding on them!

References:
CAVALIER-SMITH, T., & NIKOLAEV, S. (2008). The Zooflagellates Stephanopogon and are Percolomonas a Clade (Class Percolatea: Phylum Percolozoa)
Journal of Eukaryotic Microbiology, 55 (6), 501-509 DOI: 10.1111/j.1550-7408.2008.00356.x

LARA, E., CHATZINOTAS, A., & SIMPSON, A. (2006). Andalucia (n. gen.)-the Deepest Branch Within Jakobids (Jakobida; Excavata), Based on Morphological and Molecular Study of a New Flagellate from Soil The Journal of Eukaryotic Microbiology, 53 (2), 112-120 DOI: 10.1111/j.1550-7408.2005.00081.x

NIKOLAEV, S., MYLNIKOV, A., BERNEY, C., FAHRNI, J., PAWLOWSKI, J., ALESHIN, V., & PETROV, N. (2004). Molecular Phylogenetic Analysis Places Percolomonas cosmopolites within Heterolobosea: Evolutionary Implications The Journal of Eukaryotic Microbiology, 51 (5), 575-581 DOI: 10.1111/j.1550-7408.2004.tb00294.x

Park, J., Simpson, A., Brown, S., & Cho, B. (2009). Ultrastructure and Molecular Phylogeny of two Heterolobosean Amoebae, Euplaesiobystra hypersalinica gen. et sp. nov. and Tulamoeba peronaphora gen. et sp. nov., Isolated from an Extremely Hypersaline Habitat Protist, 160 (2), 265-283 DOI: 10.1016/j.protis.2008.10.002

PAWLOWSKI, J., & BURKI, F. (2009). Untangling the Phylogeny of Amoeboid Protists Journal of Eukaryotic Microbiology, 56 (1), 16-25 DOI: 10.1111/j.1550-7408.2008.00379.x

ROBINSON, B., DEJONCKHEERE, J., & DOBSON, P. (2007). Two new Tetramitus species (Heterolobosea, Vahlkampfiidae) from cold aquatic environments European Journal of Protistology, 43 (1), 1-7 DOI: 10.1016/j.ejop.2006.08.001