Sunday Protist - Paramyxids: Nested parasites and introverted multicellularity

A while ago we peered into the lidded jar-like spores of Haplosporidia, memorable by their peculiar habbit of building up pressure and popping open the lid upon germination, much like a jack-in-the-box. But nastier, if you're an oyster. While digging around in obscure haplosporidian literature, we came across their lesser known close relatives, the Paramyxids, characterised by their pechant for sporulating 'inward' several times, in a strange genre of parasitism reminiscent of matryoshka dolls*. Not only do they undergo serial endogenous divisions, Paramyxids exhibit differentiation among those cell types as well. Instead of being multicellular by growing outward like normal organisms, Paramyxids have evolved some strange sort of 'introverted' multicellularity.

Judging from these hints of a rather complex life cycle, one can probably predict Paramyxids to be parasitic, like their Haplosporidian relatives. Parasites tend to love convoluted life cycle gymnastics, perhaps because it's a wonderful way to trick biologists into considering the various stages to be different species, or even higher taxa. Speaking of which, quite a bit of time was spend trying to sort out their damn phylogenetic position - their identity as fellow Cercozoan sisters of Haplosporidia was not unanimously agreed upon. Since that discussion involves lots of technical phylogeny- and taxonomy-related ramblings, I've shoved that for later, at the very bottom of the post. Feel free to ignore, although I do think it is interesting.

Introduction to the Spore-in-a-spore-in-a-spore-in-a-spore-...
There are four genera: Paramyxa, Marteilia, Paramarteilia and Marteilioides, although Feist et al. 2009 propose supressing the latter to Marteilia. So let's pretend it doesn't exist. The life cycle starts off with an amoeboid primary 'stem cell', which crawls around between host cells and later gives rise to a secondary cell inside it. This cell then undergoes equal mitosis, giving rise to a second secondary cell. This part is common between all Paramyxids. The secondary cell (sporont) then divides endogenously to form one or several spores, depending on the species. Those spores divide endogenously again several times, up to the sixth generation (so three times) in Paramyxoides nephtys. To start off, the 'simplest' is Paramarteilia, with the spores dividing once endogenously:

TEM of Paramarteilia canceri, a crab parasite. The organism on the right is in an earlier stage of development. The labeling conventions in the vast field of Paramyxidology are as such: C and N indicate cell/cytoplasm and nuclei, respectively, with the number indicating the generation of endogenous division. So N1 is the nucleus of the primary cell (C1), and the cell immediately internal is indicated as C2, etc. This gets annoyingly confusing when we get to Marteilia, Paramyxa and Paramyxoides. This organism is quite simple, relatively, with two secondary cells (C2) each holding two spores (C3), each of which, in turn, has a single internal cell (C4). (Feist et al. 2009 Folia Parasitol)

Paramyxids are topologically taxing, as the 'simple' Paramartelia already begins to suggest. It gets distinctly worse, as nicely summarised in these two diagrams:

Paramarteilia (1), Marteilia (2), Paramyxa (3) share a few common early stages, consisting of an amoeboid stem cell crawling between the host cells. Upon the first division, this stem cell produces a couple secondary cells inside it, which later give rise to a number of spores, which themselves have a number of cells within. The latter two traits are species-dependent. The figure on the left shows the development in Paramyxa paradoxa, to be read clockwise. The intelligent designer was definitely high out of his mind when he made these.(Desportes 1984 Origins of Life)

Paramyxoides sporulation: a cytological acid trip
Let's take Paramyxa (and Paramyxoides, which for our purposes can be considered more or less the same thing) for a bit of a cytological acid trip. Paramyxa crawls about as a primary cell, divides endogenously once to make one secondary cell, which then divides 'normally' to form a second secondary cell, each of which then divides endogenously to form four spore cells (tertiary), which then divids inward another three times to form a nested spore thing (fig 3 below, A and B). Meanwhile, towards maturity, each of the spores forms an external sac, because it doesn't have enough membranes yet.

To further enhance its elaborate membrane collection, the fourth and fifth cells (so second and third cells within the spore), form haplosporosomes. (Annoyingly, I can't quite figure out what the haplosporosomes do. Perhaps nobody really knows, judging from their consistent description as 'electron-dense vesicles', even in recent literature.) Then, the primary and secondary cells disintegrate, releasing the spores. By the way, we're inside the host cytoplasm at this point. Then, the extrasporal sac gets smaller, and striated projections are formed which bind the four spores together. Said striated projections cement together the four spores into a tetraspore complex (fig 3 below, D). What happens after? We don't talk about that. Actually, I don't think anyone knows...

I got really confused so I made this diagram:

EM of a Paramyxoides (parasite of a polychaete) spore from Larsson& Koie (2005), with an attempt at representing the whole organism. Note the six generations of endogenous cell budding involved in this process. Cells 4 and 5 exhibit the haplosporosomes characteristic of Ascetosporeans. The primary cell usually degrades early on in development, while the sporont disintegrates later to release the spores. The spores secrete an extracellular membrane, which later shrivels up and forms mysterious 'striated projections', whose function appears to be cementing the spores together. (Since I have no EM experience, trust my membrane tracing attempts at your own risk. It involved more imagination than anything else, really...)

Cellular invasion -- somebody needs to do some work on this so I can expand this section

So then I was excited to read about the Paramyxid cell invasion mechanism, and later compare it to that of microsporidia and apicomplexa. Alas, just as for the sister Haplosporidia, the literature lies silent about the exact journey of the paramyxid into the host cytoplasm. It's mentioned that the parasite develops in direct contact with the host cytoplasm (Larsson& Koie 2005), probably implying that there's no parasitophorous vacuole or any such thing. Thankfully. I have no idea how nature would put up with one more membrane...

Paramyxid life cycles: Question marks and dotted lines
To confuse ourselves further, I picked up a couple life cycle diagrams. Universal Law mandates that parasites must have life cycles from hell, so here's one of them:

Parasitic life cycles from hell, exhibit 1: Marteilioides chungmuensis (oyster parasite). Presumably, the dotted line means "And then it magically ends up inside the host cell" or something to that effect. It seems that the cell undergoes several developmental programs, and thus several different series of endogenous divisions, to make life interesting. Or maybe the same sequence but only executed to completion within the oocyte. Regardless, there is an 'extrasporogony' stage where the parasite endogenously replicates, after which it enters the gonads and magically forces its way into the oocyte, where sporulation happens. With question marks and dotted lines in-between. (Itoh et al. 2004 Int J Parasitol)

The oyster parasite Merteilia sydneyi (Kleeman et al. 2002) also has a life cycle from hell, also with an extrasporogony stage (incidently, it was first found there, and subsequently in Marteilioides). It seems that the gill epithelium may be a safe place for the parasite to proliferate. Once Marteilia reaches the gut epithelium, where it will eventually sporulate, it seems to undergo a few more rounds of non-sporogenic endogenous division, possibly to proliferate itself throughout the digestive system. In fact, this stage results in an exponential increase of parasitic cells, so efficient that nearly all the digestive tubules are infected. Once enough of the epithelial surface is covered, the parasites invade the host cells and form spores there. Which then...somehow...do something. Presumably, some membranes break somewhere in the process.

Marteilia sydneyi life cycle. See text. (Kleeman et al. 2002 Int J Parasitol)

The gut cell invasion and subsequent sporulation happens synchronously, en masse. This is also when the host finally launches an immune response, although a little too late. Parasites often employ the stealthy ninja tactics of multiplying rapidly somewhere quiet, where they go unnoticed, and then suddenly attacking in vast hordes and completely overwhelming the host. Kind of like the 14 year periodical cicadas overwhelm their predators, or how the malaria parasites lyse the host cells synchronously, causing the characteristic waves of fever.

The Kleeman et al. 2002 paper is the first study attempting to characterise the early developmental stages of a paramyxid oyster parasite. While sporulation has been glimpes at, we are only beginning to get an idea of this organism's fascinating life cycle. New techniques like in situ hybridisation are allowing researchers to finally trace the ellusive complex life cycles of many parasites, without having to rely on obscure morphological traits to identify the organism. A lot of the parasitic life cycles (as well as some free-living ones) turn out to be more complex than previously thought.

Summary and introverted multicellularity.
In case anyone is interested in the finer taxonomy of this group, here's the latest classification according to number of secondary cells, spores and number of cells within a spore.

(Feist et al. 2009 Folia Parasitol)

The coolest thing from all this? The whole idea of multicellularity happening endogenously rather than usual 'extroverted' way. In both cases, we have cellular differentiation happening by asymmetrical mitosis. In paramyxids, this mitosis is abnormal, ie the daughter cell ends up inside the mother. There's another group of organisms that do this. You may actually be familiar with them: flowering plants. Pollen formation involves endogenous budding as well, as you have a vegetative cell, which forms the pollen tube, and a generative cell, which in involved in fertilisation.

I'm perplexed by the cell biology of engogenous mitosis, and perhaps it may be similar to whatever happens in pollen formation. However, we may have enough material for today. In fact the following part isn't necessary, as it's a bit of a discussion about the phylogenetic position of the paramyxids. If you care, feel free to stay. We will be discussing a whole paragraph from the Book of Tom, which may be interesting if that's your 'thing'. So here you go, another obscure group of parasites has been brought to light, and hopefully not mangled too much in the process. Apparently, multicellularity can also happen 'introvertedly' -- who knew?

Phylogenetic Home: Snuggling with Haplosporidia in the land of Cercozoa

[Warning: We're going to increasingly bury ourselves deeper and deeper into technical phylogenetic details, random hypothesising, and readings from the Book of Tom. Proceed with caution. Emergency exit that way ---> ]

Naturally, back in the days of crown eukaryotes and the protozoan-metazoan divide, Paramyxids were especially confusing taxonomically. They seemed multicellular-ish, thereby tempting researchers to place them with something 'higher', like metazoan-y things. However, the presence of similar inclusions to those which the Haplosporidia are known for -- haplosporosomes -- alluded to their relationship even back in the 70's. (eg. this French paper compares various Haplosporidia with Marteilia) Sprague (1979 Mar Fish Rev) published a taxonomic paper wherein they were lumped with Haplosporidians under the phylum Ascetospora, described as the following:

Phylum IV. ASCETOSPORA ph. n. (ascet- Gr. asketos, curiously wrought. Refers to the strange and complex spore structure, recently revealed with the electron microscope.) Spore multicellular (or unicellular?), with one or more sporoplasms. without polar capsules or filaments; parasitic.(Sprague 1979 Mar Fish Rev)

Remember, this was back in the day when Protista was a massive taxonomic MESS consisting of random stuff clumped together based on superficial morphological appearances. Ultrastructural studies messed this up dramatically, but true hell happened once sequencing became more available. A bit of a disaster struck when it was decided that sequencing should be as easy as 1. grab ribosomal DNA 2. sequence 3. align 4. ??? 5. Profit. Well, things did get aligned, and a lot of the rDNA data was quite consistent. Unfortunately, in some cases, it was consistently wrong.

Paramyxids seem to have suffered from the same taxonomic affliction as microsporidia -- namely, extreme [intracellular] parasitism tends to drive genomes to do wonky things. Of course, wonky genomes tend to provide habitat for some rather non-conformist sequences for homologues of conservative conserved genes. Ribosomal DNA is no exception. As we've seen before, highly diverged sequences tend to group with each other due to Long Branch Attraction. You can probably guess where this is headed: Paramyxids were yet another ancient eukaryote, branching right after Microsporidia (Berthe et al. 2000). You'd think people would be a little more perplexed by the bulk of 'ancient' eukaryotes being parasites...of eukaryotes. Yeah.

After having spent a few hours earlier digging through some rather unhappy-looking trees to find what paramyxids actually are, I'll just settle for TC-S and Chao (2002, 2003) , who place them in "Ascetosporea" (Sprague 1979), sister to Haplosporidians. According to TC-S and Chao (2002, see fig.6), both ["excessively long-branch"]Paramyxids and Haplosporidia contain a characteristic Cercozoan SSU V6 terminal loop deletion. That and the haplosporosomes should be enough for out purposes. According to Tom, they seem to group with Phytomyxea, a clade of plant pathogens containing that nasty sworn enemy of the Brassicales, Plasmodiophora brassicae. He goes on to suggest a homology between Plasmodiophorid 'cored vesicles' and haplosporosomes, thereby forming a potential synapomorphy for a group he calls Endomyxa, a new subphylum of Cercozoa. Furthermore, he suggests chemically targetting those haplosporosomes/cored vesicles to treat some of the commercial afflictions their organisms cause. See, real men do all that within a single paragraph of one paper. Oh yeah.

I'm trying to figure out what those 'cored vesicles' are, and so far I just found these unconvincing things in fig8 here (if you're still here, and don't have access, 1. Holy crap you're awesome for hanging in there for so long! 2. It's your garden variety random vesicles. Nothing much to look at...also, I'm just rambling right now. Seriously, not missing out on anything =P). Oh, there's some interesting-looking 'osmiophilic bodies' in Polymyxa (fig 3). To be honest, a lot of things have mystery vesicles, so I'm a little of skeptical of claims like "oh hey, these random tiny blobs are homologous to those random tiny blobs!". Meh, he's right about 80% of the time, so let's pretend they are homologous, sure. See if I care.

(Hmmm, I wonder if there's anything interesting about the seemingly U-shaped nucleus in Plasmodiophora and the U-shaped sporoplasm nucleus noted in Paramyxa. Or if it's just some artefact. Or coincidence. Probably that. But still, someone wanna go check it out?)

I think I may be running out of steam now...

*[completely off-topic] (from first paragraph) While writing this, I managed to go from Paramyxids to the Novgorod Codex in about 20min. Yay Wikipedia! Ancient Russian is so damn hard to read, but I still find it amazing to be able to decipher some words and phrases, almost an entire milenium later! Interestingly, Slavic languages seem to have evolved slower than their Germanic counterparts -- not only is the language diversity in that family much lower (and mutual intelligibility quite a bit higher), but 1000 year old Slavic languages are easier for modern speakers to decipher than 1000 year old English texts for Anglophones (cf. Beowulf). How many mouse clicks am I from the socioeconomics of Fiji, and then succulents of New Mexico? Might even click our way to Higg's Boson if we keep this up...[/completely off-topic]

References
Cavalier-Smith, T., & Chao, E. (2003). Phylogeny of Choanozoa, Apusozoa, and Other Protozoa and Early Eukaryote Megaevolution Journal of Molecular Evolution, 56 (5), 540-563 DOI: 10.1007/s00239-002-2424-z

Desportes, I. (1984). The Paramyxea Levine 1979: An original example of evolution towards multicellularity Origins of Life, 13 (3-4), 343-352 DOI: 10.1007/BF00927182

Feist SW, Hine PM, Bateman KS, Stentiford GD, & Longshaw M (2009). Paramarteilia canceri sp. n. (Cercozoa) in the European edible crab (Cancer pagurus) with a proposal for the revision of the order Paramyxida Chatton, 1911. Folia parasitologica, 56 (2), 73-85 PMID: 19606783

ITOH, N. (2004). Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas International Journal for Parasitology, 34 (10), 1129-1135 DOI: 10.1016/j.ijpara.2004.06.001

Kleeman, S. (2002). Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis International Journal for Parasitology, 32 (6), 767-784 DOI: 10.1016/S0020-7519(02)00025-5

LARSSON IR, & KØIE M (2005). Ultrastructual Study and Description of Paramyxoides nephtys gen. n.,
sp. n. a Parasite of Nephtyscaeca(Fabricius, 1780) (Polychaeta: Nephtyidae) Acta Protozoologica, 44 (2), 175-187

SPRAGUE V (1979). Classification of the Haplosporidia Marine Fisheries Review, 41 (1-2), 40-44

"The anus was a prerequisite for intelligence" TC-S quote anthology 01

Still working on the Sunday Protist; it's the last week of classes, finals start next week, and I have to sort out some last minute stuff for the seminar we're running next term (finally got it officially up and running in terms of registration!). As you can see, I'm rather swamped here. Also, staying up till 4am working on the Sunday Protist last night was definitely not a splendid idea. I blame TC-S for distracting me with his elaborate hypotheses...

Speaking of which, I've started my very own personal collection of TC-S (Cavalier-Smith, for ye strangers) quotes. When you read most papers, there's just information being spat out and chewed up in more or less mechanistic technical language completely devoid of any human voice. Sometimes the monotony can be interrupted by a burst of "Holy fucking shit did they seriously just write that!? Who the flying fuck gave them a faculty position?" or "This sounds fishy". In contrast, Tom's stuff goes something like:

Looks interesting, let's do this! Ok, so this paper reviews...everything. *cringe* arggghhhh...dense...massive...life-changing hypothesis...crammed into...half a paragraph... OMG ROFLOLOLOL!!!! ...how could you postulate that? Eeeeek, shaky evidence there... dude, you just cited yourself like 10 times in a row!... omg, DEAD ON, YOU GET 'EM TOM! aaaahh....what is that supposed to mean... LOL!!! Wait, did he just write that in a research paper? You cheeky bastard you! ...wow, he reads paleogeology papers!? aaaah ANOTHER DENSE PARAGRAPH OF DOOM! o_O

And so on. See, it's never boring. Which is why despite being notoriously difficult, I actually find his papers easier to work through -- they appeal to my rather-easily dulled attention span. As soon as you begin drifting off somewhere, either you realise you actually needed to thoroughly understand every single sentence to follow along and get hopelessly lost, or you come across something like "The anus was a prerequisite for intelligence", and definitely pay more attention. Which is why he gets an entire label to himself here.

Here's some lines that made me chuckle:
"There was no spinach in the Archaean." (2006 Phil Transac R Soc B, p995 last paragraph)
"But it is not." (concise disagreement) (2006 PTRSB, p997 first paragraph)
"Assumptions that such a neomuran prokaryote existed for hundreds of millions of years are science fiction." (ouch!) (2006 PTRSB, p997 second last paragraph)
"Very likely this anal breakthrough ca. 550 GYr ago stimulated the long-puzzling Cambrian explosion." (2006 PTRSB, p998 second last paragraph)
"The anus was a prerequisite for intelligence; without it, heads and brains would not have evolved." (2006 PTRSB, p998 second last paragraph)
"I consider Brachiozoa, bivalves, Bryozoa [...] were not secondarily beheaded." (2006 PTRSB, p998 second last paragraph)
"Overstatements of gradualism asserting that megaevolution is simply the accumulation of large numbers of small mutations of the same kind as predominate at lower evolutionary levels are simply wrong and must be abandoned." [emphasis mine] (2006 PTRSB, p1000 first paragraph)
"[...] but have been partially forgotten and need restating each generation." (isn't that how science works?) (2006 PTRSB, p1000 last paragraph)
"When I spoke frequently on skeletal DNA over 20 years ago, a common reaction was why not make the nuclear skeleton of protein? One sometimes felt that one's critics would prefer any material but DNA - protein, chitin, even steel, or teflon." (and yes, he refers to himself as 'one' here...) (2005 Annals of Botany, p152 second paragraph)

And a line that makes me feel all warm, fuzzy and happy on the inside: "Understanding microbial megaevolution requires cell biology -- not just biochemistry." (2006 PTRSB, p1000 second last paragraph)

And 'one' must agree with this nice summary of how science academia the world works:

"[...] is not that we do not understand mutations well enough, but that the solution I have offered, and develop further here, is a synthesis drawing upon cell and molecular biology, population and evolutionary biology, developmental biology and ecology; evaluating such a complex synthesis is tough to a specialist in just one area. Population geneticists might prefer to be able to solve evolutionary problems strictly in their own terms and ignore detailed cell biology; cytologists might prefer to do the same and ignore abstruse population genetic arguments; molecular biologists might prefer to sequence DNA and ignore both the cell biology and population biology. But for a deeper understanding we must make the effort to combine the explanatory modes of all relevant disciplines." (2005 Annals of Botany, starting last line of p147)

Despite come across as a bit arrogant there (probably not his intent), he does have a great point. There is so little interaction and integration between the various clans and tribes of biology that one guy can go ahead and monopolize a nice chunk of an integrative field just like that. People complain, but what are they doing about the little issue of each department/discipline burying themselves into a nice comfy hole somewhere, never to venture outside and explore the world beyond. And then the occasional traveler ventures into a foreign burrow, only to be appalled at how horribly their own field gets abused there. Forget the monkeys, a ground squirrel can engage in better interdisciplinary integration =P

Anyway, enough for the first installment. I'm aware some of you guys also have nice TC-S quote collections -- feel free to contribute! There was one Opisthokont had that was oozing Tom-ness all over, can't find it at the moment... *nudge*

And this is definitely getting filed under ResearchBlogging =P

And the Neomuran eukaryogenesis post will be underway after I finish a couple more 70-page reviews of Everything. Well, actually, I should just start writing, probably. Although I did want to read some competing 50 page reviews of 'everything' by other people, although theirs are much more focused.

EDIT 18:26 03.12.09: If you think about it, the "anus was a prerequisite for intelligence" quote actually does make sense: The anus allowed Bilateria to evolve, since the input and output no longer had to be in the same place, thereby drastically altering the fundamental body plan. This freed up one end of the organism to specialise in obtaining food, which is aided by rather elaborate sensing systems. The mouth-containing head end was the focus of increasing cephalisation, eventually resulting in brains. Thus, in a way, the anus is responsible for allowing heads and brains to evolve. Just adding this in to point out that there IS good substance to that quirky TC-S quote, it's not just him going insane or anything (not this time anyway) ^_~

References:
CAVALIER-SMITH, T. (2005). Economy, Speed and Size Matter: Evolutionary Forces Driving Nuclear Genome Miniaturization and Expansion Annals of Botany, 95 (1), 147-175 DOI: 10.1093/aob/mci010

Cavalier-Smith, T. (2006). Cell evolution and Earth history: stasis and revolution Philosophical Transactions of the Royal Society B: Biological Sciences, 361 (1470), 969-1006 DOI: 10.1098/rstb.2006.1842

One more day for Open Lab 2009!

One more day left, submissions for the 2009 Open Lab anthology of science writing/blogging/etc due 01 Dec midnight, and by the looks of it, they really mean 30 Nov midnight. Use this form to submit a post and make Psi happy (and please read the instructions). Here's a message from Psi himself:

Come on, can you let him go emo on us?

While we're at it, if you have any time to spare, please nominate some posts for my fellow science bloggers at Catalogue of Organisms, The Mad Labrat, Musings of the Mad Biologist, Myrmecos, The Artful Amoeba, Small Things Considered, and everyone else I can't fit here (see blogroll to the right ---->) I think they would really appreciate it.

(And yes, Psi is male. The author of this blog is female. As any of you who have imaginary characters running around in your heads would know, they actually have lives of their own, and generally don't allow you to assume their identity entirely. It's complicated.)

EDIT 30.11.09: Sunday Protist will be a bit late, sorry. Busy and need to make it to 8am class tomorrow later today...

This is getting kinda boring...

~2.5 weeks and counting...

And before you tell me to feel lucky it ain't snowing, I actually ENJOY snow and sub-zero temperatures, thank you. 6 winters in Toronto and what drove me crazy there was actually the summer... Yeah, I love snowy winters and hate endless rain. So I came to Vancouver. Got a problem with that?

Heterolobosea II - 'Split Morphology Disorder': amoebo-flagellate transformation in Naegleria

Earlier, in Heterolobosea I, I promised brain-eating amoebae with a split morphology disorder. Having a bit of a morphology fetish, I find the latter topic fascinating, so bear with me as we get into some gory details of cell biology, which I strive to make at least somewhat readable to sane human beings. As always, please let me know if anything is unclear, or *gasp* inaccurate...

Fundamentals of cellular morphology
Most organisms strive to have some semblance of shape (including bacteria). To crudely simplify matters, in the style of biochemistry, cells are sphaerical double membraned lipid vesicles. Thus, by default, a cell 'wants' to be a round blob. That would be it's natural state.

But most cells are not round blobs. In fact, they can have some rather complex shapes like metazoan neurons, forams, parabasalians or the endlessly weird and sophisticated ciliates. The deeper you venture into the realm of protist diversity, the more awe-inspiring the cornucopia of variety that is cell morphology. Luckily, this vast variety also has some order to it, for much of it happens to fall into two fundamental 'genres' of cellular morphology: flagellates and amoebae*. Of course, as with anything in biology, there are exceptions, and things that have a comfortable niche in-between. Unicellular organisms also have this tendency to construct cortical of extracellular structures, which can have a large influence on their shape. But the key determinant of cellular morphology remains the cytoskeleton, and the composition of the cytoskeleton largely determines which of the two categories the cell allies to. The cytoskeleton is an intricate, often highly dynamic, complex system; it is a misconception to see cells as bags of cytoplasm with organelles floating about -- the innards of a cell are strictly regulated and usually connected to various cytoskeletal and membraneous elements. Things don't just float about aimless inside there.

*There's also a third type, the cyst, which is basically a lack of morphology (rounded up cell) usually with external protection of some sort. It often lacks any elaborate cytoskeletal organisation, both actin- and tubulin-based. Cysts are often used for mitosis or meiosis, as well as resting through periods of unfavourable conditions.

There are two main component systems of the cytoskeleton: actin and tubulin, ignoring the plethora of miscellaneous proteins that have been used for various structural jobs. You are likely familiar with microtubules as spindle fibres during mitosis. You may also be familiar with actin as a key player in cell motility and morphology in animal systems. Tubulin is also important for the flagellar apparatus -- we've yet to find one composed of actin (and probably with good reason). Actin is involved heavily in endomembrane trafficking within a cell, as well as endo- and exocytosis. If interested, this week's issue of Science has a nice overview of actin in morphogenesis and cell movement.

Their roles in morphogenesis, the formation of morphology (in this case, cellular), are much less clearly defined. Furthermore, it depends largely on the species -- plants, for example, rely very heavily upon microtubules for morphology, with actin being a minor player. Interestingly, plants also lack centrioles or basal bodies, except for in some gametes if I recall. Thus, the mitotic spindle in plants (and diatoms) forms entirely without centrioles. Yeast cells can also do mitosis without basal bodies if you surgically remove them. It seems like the characteristic centriole arrangement at mitosis is a way to ensure their inheritance in both daughter cells, rather than organising the spindle. More on this later, it will become relevant.

Amoeboid cells, as you may know, are primarily actin-based. In fact, amoebozoans tend to suck at internal microtubules, except for spindle formation at mitosis. They hate tubulin about as much as plants hate actin. Actin-based cells don't have to be amorphous -- they are still able obtain complex morphologies like that of neurons (although there may be some serious tubulin involvement in that. My knowledge of animal cell biol is rather pathetic). But there is a positive correlation between amoeboid-ness and actin-ness ('actinity'?). Turns out, the entire unikont clade, if it exists (ok, opisthokonts and amoebozoans), seems to really prefer actin over tubulin.

In contrast, flagellates are primarily tubulin-based -- of course, they still use actin for some intracellular work, but the shape depends largely on the whims of the 'tubes (microtubules, in the slang of our local plant cytoskeleton lab...). Perhaps not relying much on flagella in the amoeboid case allows them to lose so microtubule organisation pathways, thereby switching to actin; flagellates tend rely heavily on intact tubulin systems, and may thus be less prone to losing their structure. Also, if you're a flagellate, you kind of need shape for a modicum of streamlining -- try swimming around as a flattened reticulate blob of some sort! Keep in mind that life at that scale is very different -- viscosity becomes a crucial factor when considering unicellular motility. Perhaps being hydrodynamic isn't even as important as simply retaining shape. Otherwise you'd be like a blob of molasses trying to swim through a sea of maple syrup. Not gonna get very far.

Whatever the reason, amoeboid cells tend to have a predominantly actin-based cytoskeleton; whereas flagellates have a penchant for tubulin. Of course, not all organisms are decisive enough to make this committment, so we've got amoeboflagellates in the middle:

But your conventional amoeboflagellates are only the beginning -- plenty of cells, especially Cercozoans for some reason, fancy transitioning between being more amoeboid or more flagellate. But few cells actually dispense with flagella and basal bodies altogether, only to form them anew when special conditions arise.

As we've seen earlier, de novo centriole formation was until recent considered fairly impossible (ignoring the organism we're about to prod at). As we all know, there must be a reason for the guaranteed inheritance of centrioles at mitosis, and they must be hard to form. After all, tubulin nucleation doesn't happen randomly very often, and new microtubules seldom start without some sort of 'seed' (gamma-tubulin), as the tubulin has to form a ring prior to growing into a tube, which isn't likely to happen on its own. (Actin, on the other hand, is only two monomers wide, and can form spontaneously relatively easily) Thus, for basal bodies to pop out of nowhere is also rather unlikely, but, as we're about to see (and as several more recent experiments show in yeasts and animals), de novo centriole formation can and does happen.

Naegleria and Tetramitus: Heteroloboseans with 'split morphology disorder'
Several paragraphs into our journey, we've yet to see any Heteroloboseans. Let's change that. Meet Naegleria, famous to medical biologists as a brain-eating opportunist, and to real biologists as the master of de novo flagellar creation:

Naegleria gruberi. (by Jonckheere at ToLWeb)

And before someone accuses me of focusing on biomedically relevant organisms, Tetramitus, another Heterolobosean, does it too, and has more flagella to oogle at:

Dramatic transformation of Tetramitus between amoeboid and flagellate forms. (Outka & Kluss 1967 JCB)

In fact, while I failed to find a nice picture of Naegleria's flagellate form, that of Tetramitus reveals its marvelous complexity. Keep in mind this thing was a 'formless' amoeba a few hours before:

Quite a complex structure to be formed within a 4h period! Note that while the organelle positions in amoeboid cells are fairly flexible, they become fixed in the flagellate form. It even has subpellicular microtubule bundles, a cytostome, and the oral groove characteristic of Excavates. This cell is unquestionably worthy of being called a flagellate, so this really is a complete transformation between two fundamentally different cell morphologies. (Outka & Kluss 1967 JCB)

Of course, heteroloboseans are not the only group to claim amoebo-flagellate transformations; other predominantly flagellate groups like the parabasalia also contain organisms with a bit of a split personality morphology disorder: Gigantomonas herculea, for example, is a termite endosymbiotic Trichomonad that can also switch between amoeboid and thoroughly flagellate forms (Brugerolle 2005 Acta Protozool.). However, unlike Naegleria, Tetramitus et al., they still retain basal bodies during the amoeboid stage.

Of Naegleria's culinary preferences
But because of Naegleria's pesky little habit to occasionally get into human brains and eat them (oops!), it gets a lot more research attention (Which, in the world of Heterolobosean biology, doesn't really mean very much) It should be noted that while there is some waves of 'Naegleraphobia' out there, incidences of human infection are very very VERY rare (small handful of cases a year; some years without any cases whatsoever). Unfortunately, it is almost universally fatal, but so are many much more ubiquitous diseases. But that's enough excuse for the media to put up a scary article on Naegleria every once in a blue moon, often accompanied by an image of... Amoeba proteus or Chaos sp. Hey, at least they're still eukaryotes!

Before we continue on with our hard core cell biology, let's clarify Naegleria is not a parasite, but rather an opportunist that seems to not mind the 37 degree heat of the human body. It gets into the human brain through the nose, generally from swimming in warm water. I'm not sure if it even makes it past the brain - it may eat the brain, kill its host, and end there, as the likelihood of ending up in a suitable environment after invading the brain is pretty low. So Naegleria's gastronomic inclinations may well just be an accident, albeit a rather costly one to the few humans it infects.

Ok, that exceeded my dose of biomed tolerance for the next three months. I often add "-clinical -patient -pathology" when searching for articles online; that's how much I avoid medicine. I'm a cruel, evil person, I know =P

Amoeboid to flagellate transformation
So here's an overview of Naegleria's life cycle. It consists of an eating and mitotically able amoeboid form (interestingly, mitosis happens without basal bodies), a flagellate form induced by addition of water (where swimming is favoured over crawling). Presumably, it can still feed in its flagellate form, considering it bothers to construct an elaborate oral groove and cytostome. The third form is a resting cyst, which it forms and hibernates in when times are bad. Thus, Naegleria's life cycle actually involves all three fundamental cell types, which is why it's such a wonderful system for studying the transformations between them (and cellular differentiation.)

Naegleria's principal morphology is amoeboid, which is the stage for mitosis. Flagellation happens upon addition of water, whereas encystment is often a response to unsuitable living conditions. Note that mitosis happens without basal bodies. (based on this figure, in turn based on Fulton 1970 in Methods in Cell Physiology; also, don't pay any attention to the portrayal of the flagellar root appartus...)

I'm probably trying to get through way too much in one post, so I'll unfortunately have to do an injustice to the gory details of the cell and molecular biology behind this process, and just give a skimpy overview.

The transitiona between the principal cell morphologies involve a regulation of actin and tubulin monomer levels - or the amount and concentration for the 'building blocks' for each morphology. In fact, the reason many organism encyst for mitosis (or at least reduce their flagellar length) is to redirect tubulin from the cytoskeleton and flagellum to form the mitotic spindle. Otherwise, you'd have to synthesise a bunch of new tubulin which would soon become useless. If you look at some images of Naegleria amoebae undergoing mitosis, you'd notice the cells are much rounder than normal.

Naturally, there's also a regulation of proteins involved in cytoskeletal organisation for each system (and the interactions between them), but that doesn't seem to be studied at all in Naegleria. We do know that the commitment to flagellation involves a rapid increase of tubulin production, since the amoeboid form has barely any.

A quick caveat: a common misconception is that flagella are microtubular things that 'stick out' of the cell, and are on the 'outside' -- flagella are bound by the cytoplasmioc plasma membrane, so the tubulin is quite accessible to regulation. To give you an idea of how a flagellum grows, have a look at these TEMs from Naegleria:

Note that it's still surrounded by the plasma membrane. There's even a little knob at the growing end. (Dingle & Fulton 1966 JCB)

Naegleria takes ~120min at 25C to transition from an amoeba to a flagellate. The earliest event in this process is the initiation of tubulin synthesis, followed by basal body assembly 30min later. Cells round up as actin filaments depolymerise, and basal bodies begin to sprout flagella at about the hour mark. In another 20min, the tubulin cytoskeleton is molded into the proper flagellar body shape, with flagella reaching full length at ~110-120min.

See text. Actin vs. tubulin concentrations are relative, and do not imply either reaches zero at either end, although the amoeboid tubulin concentrations are very low, whereas actin levels remain more or less constant. (After Fulton & Walsh 1980 JCB; free access)

The data above was gathered rather painfully via drug experiments -- you can target actin and tubulin for various forms of disruption (as well as protein synthesis), and determine at which timepoints actin or tubulin is the most crucial for a proper differentiation. Now we have sleek antibodies and GFP and genetic tools of all sorts (they had many of the techniques before, but they has become much cheaper, easier and higher quality over the years. Perhaps at an expense of the quality of science itself, since you can cover up sloppy work with sexy pictures quite a bit easier...), so we can actually see the changes in actin and tubulin throughout the transformation process:

Immunostaining of the cytoskeleton during flagellar transformation. red - actin; green - tubulin. Yellow - co-localisation of actin and tubulin. Note that these are different cells, as they had to be fixed (killed) for immunostaining. I would love to see a life cell timelapse of this process!* Note how the flagellate morphology is 'molded' by the longitudinal microtubule bundles. (Walsh 2006 Eur J Cell Biol)

*Doesn't look like Naegleria has had its genome sequenced yet. And yet we're about to sequence 10K vertebrate genomes. Would they mind sparing us a couple slots? But once it happens, transgenic lines and away we go with sexy timelapse. In 4D. *drools* (4D hyperstacks are really hard to analyse, so despite sounding sexy, the scientific value of some of their applications is debatable)

Hopefully I've convinced you at least that Naegleria would make an interesting model for studying cell differentiation and transitions in morphology, as well as cytoskeletal dynamics. While the field is still miniscule, at least its non-clinical component, there is work being done right now on investigating the regulation of cytoskeletal development, such as this paper on the localisation of microtubule-related mRNAs. But not much.

It's actually kind of worrisome: the generation of protist cell biologists from the 60's and 70's is now retiring, while the younger researchers in protistology are predominantly molecular biologists. They're useful, sure, but I insist that there's more to an organism than its genome. I know a few molecular biologists who agree, but they still do genomics work! They say there's wonderful opportunity in investigating protist cell biology in the near future, but if the older cell biologist retire before they can pass on their knowledge and skills and wisdom, are we going to now be set back a good decade or two, wasting our time to make the same mistakes and learn what has already been learned? Fancy equipment cannot replace experience and insight, nor can insight be written into a paper.

But anyway, I'd just like to point out again the humans, yeast and Arabidopsis are not the best organisms to study all biological processes - they're good for some things, and not quite so nice for others. This is where we should use a wide diversity of models to understand processes that are more accessible there - such as flagellar transformation in Heteroloboseans. I'm not aware of any other system that involves such a complete transition between three fundamental cell types, even with de novo basal body formation! Seriously, what more can you ask for? (let's hope it has a 'nice' genome...)

Apologies to the less cell-biology-oriented portion of our audience. But at least we've got pretty pictures! =P

References
Dingle AD, & Fulton C (1966). Development of the flagellar apparatus of Naegleria. The Journal of cell biology, 31 (1), 43-54 PMID: 5971974

Fulton C, & Walsh C (1980). Cell differentiation and flagellar elongation in Naegleria gruberi. Dependence on transcription and translation. The Journal of cell biology, 85 (2), 346-60 PMID: 6154711

González-Robles, A., Cristóbal-Ramos, A., González-Lázaro, M., Omaña-Molina, M., & Martínez-Palomo, A. (2009). Naegleria fowleri: Light and electron microscopy study of mitosis Experimental Parasitology, 122 (3), 212-217 DOI: 10.1016/j.exppara.2009.03.016

Outka DE, & Kluss BC (1967). The ameba-to-flagellate transformation in Tetramitus rostratus. II. Microtubular morphogenesis. The Journal of cell biology, 35 (2), 323-46 PMID: 4861775

WALSH, C. (2007). The role of actin, actomyosin and microtubules in defining cell shape during the differentiation of Naegleria amebae into flagellates European Journal of Cell Biology, 86 (2), 85-98 DOI: 10.1016/j.ejcb.2006.10.003

Happy 150th, Origin!

Science is a rather peculiar activity. Upon solving a particular problem, many more scientific questions arise from that. There is no end -- any elucidation tends to result in more and more work to do. Which is why we'd be eternally employable, were it not for a rather anti-scientific attitude in the general public (partly our doing). To quote a nice tidbit from a protistology lecture: "People in science get famous for creating problems". An example that went along with that was the discovery of microbial life by van Leeuwenhoek -- with the help of a series of microscopes, he unlocked a Pandora's box worth of questions, eventually leading to the birth of a fairly major field -- microbiology. He created some serious problems: How could life exist on such a small scale? How many more microorganisms are there? Where do they live? What do they eat? How do they eat? and [much later] May microorganisms be in fact responsible for some human diseases? Leeuwenhoek discovered a whole new world, perhaps even the closest thing we'll have to aliens within our lifetimes.

150 years ago one has witnessed a milestone in the discovery of yet another alien world of questions, parts of which are luckily quite visible to the naked eye. While the microbial world remained hidden by barriers of scale, this world was perhaps hindered by a barrier in perspectives; after all, entering this world required, in the words of one of its critics, "a strange inversion of reasoning" (MacKenzie 1868 qtd in Dennett 2009). The mischievous creator of problems at this milestone was Charles Darwin. What is truly wonderful about evolutionary theory is its broad applicability throughout various corners of disciplines, from biology to linguistics to the humanities to engineering, computer sciences and beyond. Like a fractal, it is at some level so simple, yet upon further examination, there is no end to its complexity. Seldom do theories surface with such breadth and profundity.

Doubtlessly, The Origin was an important work, skillfully blending a breathtaking variety of naturalistic observations and Malthus' idea of natural selection to create a central, unifying theory adressing the origins and causes of the diverse living world. He established that heredity, variability and selection* were the key principles of an evolving system. Of course, there were problems, such as his rather primitive understanding of the mechanisms of heredity. But overall, it was a quite an important catalyst in the creation of a new world of questions: namely evolutionary biology, and eventually other applications of the theory. 150 years ago, this very day.

*Let's not get into THAT argument here...

However, perhaps we give Darwin a little too much credit, elevating him to something akin to a deity of evolutionary biology. Or at least some very powerful spirit. It almost feels as if evolutionary theory stopped in its tracks after Darwin. Thing is, ideas are seldom generated. They come from somewhere -- usually by being blended from other ideas (recombination, anyone?). He happened to listen to the right people, at the right place, at the right time. And was also a wonderful writer and populariser of his own ideas, which is key to spreading any idea. In fact, there was plenty of work done before him that was just waiting to be materialised into some foundational book. Read more about this in this level-headed approach to Darwin by John Wilkins at Evolving Thoughts.

Headlines such as "Darwin was wrong: Scientists find X" are particularly annoying. Ok, so what? Of course Darwin was wrong on most things, just like the rest of us! Especially since science has progressed a little in the last 150 years. Is it really headline-worthy material? Why don't we ever say "Wallace was wrong" or "Newton was wrong", by the way? And, on that note, why not "Psi was wrong"? =D Darwin is not the Holy Guardian of Evolutionary Theory! He missed a lot of things, mainly because he lived over a century ago! Besides, evolutionary biology has yet to be complete, and other applications of evolutionary theory are still young.

So what are some frontier areas of evolutionary questions? Of course there's still piles and piles of questions in biology - we barely understand a thing still. But it's happening. Now we have a decent idea of how characters may be inherited, at least genetic ones anyway. Cortical and cytoplasmic inheritance are still poorly understood, especially in fascinating cases like the heritable ciliary row inversion case in Paramecium (Beisson & Sonneborn 1965 PNAS, free access), where surgically altering cortical organisation results in it actually being inherited further and further, despite a lack of genomic alterations. The mechanisms behind this peculiar case are only beginning to be understood.

Another 'Wild West' of evolutionary thinking is in the humanities. Linguistics is an example of a particularly successful application of the theory, emphasised perhaps by the appearance of an evolutionary linguistics paper in Nature Rev. Genet. this summer. It's a pretty nice place to get a review in, thus evolutionary linguistics is finally accepted as a field, despite being practically banned until Pinker & Bloom 1990. Cultural evolution is a bit of a bigger warzone, although I think it too will follow the path of linguistics and find evolutionary modeling very useful and insightful. Unfortunately, many scholars in the humanities seem to have some rabid aversion to anything science related. To the point of quickly wrapping up the conversation and moving away upon finding out your affiliations. Linguists are much closer to the canonical 'natural' sciences (what is unnatural about language or culture or psychology is beyond me). Although even in linguistics they've got Chomsky, who viciously opposes any materialistic explanation of language and its origins, for reasons that seem to escape everyone. Don't get me started on Chomsky...

Incidently, I'm co-directing a student-directed seminar on the applications of evolutionary theory outside biology. It should be plenty of fun! It's a very exciting time for exploring these topics, for people are finally beginning to share wisdom between conventionally separated fields. One of these wisdoms that has great potential to deepen our understanding of the world around us, is evolutionary theory.

Anyway, here's to Darwin's Origin and all the subsequent developments in evolutionary theory! *toast*

Some more random links on the anniversary:
Darwin 200 Nature specials (may require subscription)

T. Ryan Gregory has a collection of 19th century Darwin caricatures over at Evolverzone. It's quite entertaining.

Greg Laden has a celebratory reflective post here.


By the way, this has been in my head all day: Happy Monkey!

Mystery Micrograph #09

Mystery Micrograph #8 has been solved -- congrats to Mark Patterson! It was a nematode parasitising a foram, and I'll write it properly sometime soon -- waiting for a paper to arrive via interlibrary loan. From Russia. Paid for by you-the-kind-taxpayer. I love university...

Now for the next one. Top image scalebar = 10um, bottom image (TEM) scalebar = 0.1um, inset scalebar = 0.2um. You only need to identify the protist in this one [/hint]

(to be referenced later)

Good luck, and have fun! Someone here might really like this one =P

Sunday Protist - Litostomatea: rumen ciliates with incredible morphology

Scary lab exam in 9h, so this one will be another 'protist appreciation' compilation rather than anything particularly informative. Today this maltreatment will be inflicted upon some poor ciliates, which deserve much more than just a simple appreciation post. Ciliates ARE the higher eukaryotes, the most advanced and awesome organisms on earth. What god created in his/her/its image wasn't the ugly naked humans, it was, in fact, Spirotrich ciliates. Everything else is just basal offshoots, failed experiments. This is an unbiased, objective opinion with piles upon piles of supporting evidence. 'xcuse my word choice, but ciliates are fucking awesome.

Complex shapes in multicellular species aren't particularly amazing, as intricate self-organising properties can arise in populations even without any attemps at coordination (eg. [some?] bacterial swarming). A thing with 10¹³ own cells exhibiting complex morphology isn't all that amazing, especially as many of the architectural principles are simply repeated over and over again. What IS truly amazing, is when a unicellular organism can achieve complex shapes* like these rumen denizens:

Some horse rumen gut Litostomatea from Stueder-Kypke (2007 EJP) The thick black thing in the drawings is the macronucleus (MAC).

As you can see, they're quite diverse and...weird. These Litostomateans live in digestive systems of various animals, mainly rumens of cows, sheep, horses and the like. They're microaerobic (since there's still trace amounts of oxygen in the gut), and some seem to even have hydrogenosomes (modified mitochondria). There's also some interesting flat ones found in Australian marsupials, with a weird mesh pattern on the left and right sides:

Macropodinium; also contains endosymbiotic bacteria just under the cilia. (Cameron & Donoghue 2002 EJP)

A brief tour of the anatomy of an arbitrary Litostomatean ciliate is in order. Let's oogle in awe at the complex morphology of Eudiplodinium, from Furness & Butler 1985 JEM**:

Let's start with osmotic regulation, the ciliate equivalent of excretory system, if you will. The two blobs labelled CV are contractive vacuoles, which tend to be common (if not obligatory) among freshwater organisms. I'd imagine the rumen would qualify as closer to freshwater than marine, so that's probably why Eudiplodinium has them. The contractile vacuole itself is a complicated structure, consisting of ducts (which drain the excess water, somehow), a central 'bladder' equivalent with a channel leading outside, and subcellular 'muscle' equivalents responsible for vacuole contraction.

Then we have the germline micronucleus(MI) and somatic macronucleus(MN) (the substantially bigger one); I've explained this recently towards the bottom of this post, and I'm being really lazy right now. Go read about it there, or in the introduction of this report. Or this post from the Catalogue of Organisms.

The DZS and AZS are dorsal and adoral zones of syncilia, respectively, where clumps of cilia like those in the first figure appear. The adoral zone happens to be near the peristome (mouth), and may thus likely be involved in feeding. The dorsal zone could be involved in motility perhaps? The paper seems to assume the reader knows this stuff, as it's more of a cytomorphological description; although to be honest, I haven't actually read the entire thing.

Speaking of the peristome, next we have a subcellular analogue of a digestive system - the cytoalimentary system - consisting of a ridiculously complicated cytopharynx (mouth and throat, if you will) leading to the endocytoplasm, where the prey or food particles are packaged into vacuoles and digested alive. And the secreted out the cytoproct, or 'cell anus', if you will. In fact, here's what the menacing cytopharynx looks like in detail (again, from Furness & Butler 1985 JEM):

The structures marked by L at the top are the cellular equivalent of lips. Another point for ultimate convergence. 'Primitive organisms' my ass.

The cortex, or 'skin' of the organism is no less menacing structurally. Especially around the cilia. First off, ciliates belong to Alveolata, which are characterised by having alveolae, or small membranous sacs just underneath the plasma membrane. These structures are often modified to contain protein secretions building up structures like armour plates. Probably have other functions too, I just don't know very much about them at this point. Now when you add rows and rows of cilia to that, you get a convoluted maze of basal bodies, the cytoskeleton and various fibrillar and endomembrane networks 'servicing' the entanglement of cilia. I found a really nice example for a relative of Eudiplodinium, Epidinium, from Furness & Butler 1983 JEM:

I should probably get back to cramming studying soon, so let's just leave it at that: It's complicated. If only cell structures had Facebook relationship statuses...

You've probably had enough of Litostomatea for now, if such a thing is possible. So we'll wrap up here. As for their phylogenetic neighbourhood, here's my simplified version of Lynn 2003 Eur J Prot diagram (branching depth not to scale):

Litostomatea are the third from top. As a tiny sample of how wonderful ciliates can get: Spirotrichs include 'walking' ciliates like Euplotes and Stylonychia, some of which are famous for 'scrambled genes' (see figs 3b,c of this report); Colpodea which include the giant Bursaria(really nice gallery, btw); Heterotrichs, incl. the giant (1-2mm!) trumpet-shaped Stentor*; Oligohymenophorea with the familiar Paramecium and Tetrahymena(NSFW); Armophorea with the amitochondriate Nyctotherus; Phyllopharyngea with Chilodonella and suctorians like Ephelota with its really neat branching MAC; and lastly, amitosis-lacking Karyorelictids with Tracheloraphis and Loxodes. Overall diversity summed up nicely in this striking picture. Eventually I may explore some of these in detail, as what I've done here is rather offensive.

Hopefully you are slowly nearing Enlightenment, wherein you will realise that ciliates are the higher eukaryotes, and the awesomest organisms on the planet. To supplement this process of personal spiritual growth, I recommend slapping some pond water or soil samples on a slide, and watching various ciliates running around, in the case of Hypotrichs -- literally. All hail our ciliate overlords! May your macronucleus forever be pure and contain a complete set of genes. On that note, be careful about conjugation -- procrastinate too long and you'd have to undergo autogamy. And nobody wants that.

*A certain Parabasalia fanatic asserts that hypermastigotes have the most sophisticated cell structure -- he could not be more wrong. Hypermastigotes merely took karyomastigotes and multiplied them over and over again in a spiral -- overwhelming in terms of flagellar number, but architecturally quite simple. Ciliates have a sophisticated assymetrical non-repeating cortical organisation, for one thing, and are much more complex on the genomic level as well. Parabasalians don't even have proper mitochondria, for crying out loud! I mean, even dinos are more sophisticated than Parabasalia. Dinos are the second highest eukaryotes. Are we happy now? (there's a war between ciliatologists and people who study dinos, and clearly ciliates are winning as they actually have a name for people studying them. Dinologist? Dinoflagellatologist? Ewww. See, clearly our side is winning. Dinos are just "free-living apicomplexans". Case closed.)

**JEM website: how I hate you for displaying the online publication date in a very prominent location on the abstract page for old articles, rather than their original publication date. Oh how often I get led astray, sometimes even confused, reading something from the long gone past thinking it was published last year. Sometimes I get excited thinking people still do quality cytology work as opposed to aligning sequences all day. I even get a glimmer of hope for humanity. And then I check the paper itself. And become heartbroken. That's just cruel.

References
CAMERON, S. (2002). The ultrastructure of and revised diagnosis of the Macropodiniidae (Litostomatea: Trichostomatia) European Journal of Protistology, 38 (2), 179-194 DOI: 10.1078/0932-4739-00861

FURNESS, D., & BUTLER, R. (1983). The Cytology of Sheep Rumen Ciliates. I. Ultrastructure of Epidinium caudatum Crawley The Journal of Eukaryotic Microbiology, 30 (4), 676-687 DOI: 10.1111/j.1550-7408.1983.tb05343.x

Furness, D., & Butler, R. (1985). The Cytology of Sheep Rumen Ciliates. II. Ultrastructure of Eudiplodinium maggii The Journal of Eukaryotic Microbiology, 32 (1), 205-214 DOI: 10.1111/j.1550-7408.1985.tb03041.x

LYNN, D. (2003). Morphology or molecules: How do we identify the major lineages of ciliates (Phylum Ciliophora) European Journal of Protistology, 39 (4), 356-364 DOI: 10.1078/0932-4739-00004

STRUDERKYPKE, M., KORNILOVA, O., & LYNN, D. (2007). Phylogeny of trichostome ciliates (Ciliophora, Litostomatea) endosymbiotic in the Yakut horse (Equus caballus) European Journal of Protistology, 43 (4), 319-328 DOI: 10.1016/j.ejop.2007.06.005