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

Assorted link roundup

Some links from my epic procrastination foray:

Since I'm supposed to be cramming studying for my developmental biol lab exam (early chick embryology) this Monday (8am, ewww), NOVA's Guess the Embryo was quite fun! And amazingly difficult too... (the bat develops quite cutely, for some reason.)

An interesting application of DNA barcoding: testing sushi meat

Science standup comedy, via MolBio. On that note, lab cartoons, also via MolBio.

Forgot where I found this (was quite a while ago), but How to write consistently boring scientific literature (Sand-Jensen 2007 Oikos; free access) has 10 simple rules to ensure your scientific work never falls into the hands (or mind) of others.

CG animation of Aspergillus conidiophores via MycoRant. Fungal fruiting bodies seem very graceful there!

10 days left for 2009 Open Lab submissions, due 01 December at midnight.

And I guess that's it for now. Should probably get back to drawing embryos over and over and over again. Also, why is mammalian developmental biology literature so bloody hard to read? Plant people are kind enough to provide sufficient background for everyone, even zoologists, to understand what's going on. The mammalian guys assume you know everything already. Hello, some of us actually work with different systems, y'know?

It's kinda fun to be thrown in at the deep end of a completely foreign field though. Too bad we're expected to focus on only one paper for the class presentation thing, and I like to skim over a small pile of reviews before narrowing in on one topic. That seems to be quite discouraged, considering the time alotted to this endeavour. Maybe that's why I seem to get along quite well with TC-S papers (aka Megamonumental Reviews of Life, the Universe and Everything). FUZZBRAIN PRIDE!

When I take over the world, undegrad is going to be run so differently. Also, microbiology and protistology would be the most well-funded fields. Disciplinary tribalism will be banned, and everyone will work together and learn from each other's fields, even arts and science. Engineers will be banished to a deserted island in the middle of the Pacific somewhere. The steady state for a sane social model will be found, and societies will be engineered towards it, somehow. (all previously invented utopias failed to be steady states; this one will be different. Mwahaha) LOLcats will be displayed proudly and ceilingcat will be worshipped on Caturdays, along with FSM and various protist and bacterial demi-gods. And there will be NO bloody sweet meat. Evar. Sweet meat is a freaking abomination - how the HELL can anyone in their right mind pour honey over a delicious beef steak? That's just horrible. A crime against culinary arts, a grotesque malfunction of the human culinome. Seriously, new -ome coinages cannot begin to compare with the abomination that is sweetened meat*.

*Exceptions include: apples and cranberries in duck, turkey et al. And NOTHING ELSE!

And finally, the theft of forceps from microscopists will be a capital offense. I NEED THOSE FORCEPS FOR MY WORK, YOU %$@&$s.

I think I'm ready to quit procrastinating for a while...

Mystery Micrograph #08

Originally posted on 04 November 2009 4:20 AM
17.11.09: Bumping this up before it falls off the first page...
21.11.09: You guys have until tomorrow, unless someone says something. I actually have a reason to procrastinate this time... (waiting for a paper I had to order) So go, guess!

This time we have two organisms. Tell me what's going on here. And yes, you have to figure out both of them. Which is kind of necessary for this one anyway. Unlike some people(=P), I'm not very picky on very fine taxonomy (species, genera), so don't freak out!

(scalebar = 300um; to be referenced later)

Good luck and enjoy!

HINT 05.11.09: Unusual parasitism
08.11.09: Two kingdoms

Ciliate orgies and barnacles with twin penises

Like any other human beings on the planet, scientists too are enamoured with sex and genitalia. After all, procreation (self-replication) is the central theme in biology, and we tend to find it more fun when more than one individual is involved. Especially when these individuals differ anatomically into categories, in our case, two types, since that is what's most familiar to us. As far as I know, no lineage has evolved obligatory triple conjugations of three different mating types, although such a thing can be induced in the lab. But for now, let's have a look at a particularly unusual developmental glitch in an individual barnacle, and an even more surreal publication accompanying it:

An Individual Barnacle, Semibalanus balanoides, with Two Penises (Hoch & Yuen2009 J Crustacean Biol) [NOT SAFE FOR WORK due to images of genitalia. Unless you work in biology...]

I came across this while responding to someone's comment, and chuckled. Then I checked whether it really was a single individual case, and whether it really was published in a real journal. In 2009. Yes and yes. It was a rather entertaining read as well. Now, I would go off on a rant about some of their hypotheses and assumptions, but the type of work and the following accompanying note in the acknowledgements suggest this was actually an undergrad project, and I should be nice to my brethren:

"This work was partially supported by a Student Research Fellowship from the American Microscopical Society to [author] and a Crustacean Society Summer Research Fellowship to [author]."

"So, what did you do this summer?" "Found a barnacle with two penises, and you?"

Damn, I wish my summers were as exciting. They usually get spent in a dark room staring at blue [DAPI] dots all day. Sometimes I surgically rape a tiny flower with a pair forceps. Other times I drown my poor seedlings in nasty cytotoxins, and wonder why how they die. This guy got to measure barnacle genitalia. And infer about its sex life.

Here are the two penises in all their glory:

Lemme paraphrase all the scientific lingo: "OMG, TWO penises!" (Hoch & Yuen 2009 J Crustacean Biol)

So why barnacles, of all things? Thing is, upon reaching maturity, they glue themselves to the rock. And become stuck there. And then they get horny, but they can't go out. Kind of like that basement-dwelling 4chan crawling internet loser nerd stereotype. With one key difference: in the barnacle case, size does matter. A lot. Maybe that's not such a key difference, and we won't go there in this polite company (LOL!), but the barnacles can do it without ever going out. All they need is really loooooong penises, long enough to reach the next mother's basement-dwelling geek barnacle. And those beyond it. You can see how this particular sticky situation can lead to evolutionary peddling of male organ enlargment solutions*.

*My horrible, horrible mind is trying to imagine what molecular spam would look like... alas, my imagination does not stretch as far as barnacle penises If only they were immersed in some serious horizontal gene transfer...

They've proposed that at least one of the penises is fully functional, as the sole nearby barnacle has been fertilised. Then they wonder whether this was a genetic or developmental accident. Well, when your sample size is...ONE...it becomes very difficult to separate the two. Especially when you terminate the sole specimen by chopping its penises off. A regularly inherited twin penis trait would be rather unlikely; it's quite improbable for a genetic change to result in the doubling of an entire organ in the metazoan version of multicellularity. So it's likely just a chance developmental glitch. I've seen a photo of a tulip with half its leaf converted to a petal, but only one tulip and a single leaf -- another example of an entertaining, but biologically unimportant, developmental glitch.

So, to summarise:

Lab slave: Holy shit guys, this one has two penises!
PI: OMG, let's see if we can publish that!
Slave: LOL nice joke, buddy!
PI: No, seriously!
[...]
Reviewer: So what's the scientific significance of this work?
Authors: It has two penises, lol XD!
Reviewer: *giggle* Ah what the hell, we need our [juvenile] entertainment too! *accept*

So what is their actual stated conclusion?

"The result is significant because it shows that the mating ability of the barnacle is resilient to developmental instability and able to overcome extreme departure from normal morphology."

For a sample size of ONE. Remember guys, this isn't a mutant line or anything, this is ONE single freak case. But they had to write something ^_~ Still amused by this getting published though...

And now for another anomaly:

Ciliate orgies
Ciliates are obligate sexual organisms. If they don't have sex within a certain number of generations (~50 for Paramecium), their somatic nucleus basically rots away. Thing is, they actually have two different nuclei - one the pass on, and one they use (ie transcriptionally active). The transcriptionally active 'macronucleus' (MAC) is essentially a giant bag of linear plasmids, sometimes having upwards of ~9000 copies of a single unigenomic, or just really short, chromosome. A problem with having so many chromosomes is evident at mitosis -- how do you attach a bundle of microtubules to every single chromosome of the thousands there are? Well, you just...don't.

Ciliate MACs undergo a special form of nuclear division termed 'amitosis', where the nucleus is more or less pinched in half (ciliates do closed mitosis where the nuclear membrane remains intact the whole time), more or less evenly. Sort of. This is roughly compensated for by some rather crazy DNA replication regulation stuff, but eventually the organism may start losing genes. (how it corrects for the ever-changing gene copy numbers is beyond me...*)

This is were sex comes in -- the ciliate exchanges its germline 'pronuclei' (haploid gametes, if you will) with that/those of its partner (in some species, it gets complicated...), and makes a new MAC. Actually, the making of a new MAC heavily depends on the preceding one, which gets destroyed just as the new one is formed, yet its information can still influence it...basically, a paradise for anyone obsessing over epigenetics! In fact, I've done an essay on that stuff for a class: Ciliate genome rearrangements pdf (not my best writing as I spent way too much time reading stuff and not enough time constructing sensible sentences...)

*Hmmm, just thought of this: overexpression lines might be a problem in a ciliate model, no? It may well be that just like what seems to be happening with the polycistronic gene situation in trypanosomes, may be also happening here: namely -- highly emphasised post-transcriptional and post-translational regulation of gene expression levels rather than direct regulation of the promoter... thus would mean that driving a gene with a highly expressed promoter might be compensated for by some pathways specific to regulating that gene (or its class), thereby screwing with your overexpression attempts. Any thoughts?

So, before I get too carried away with this, ciliates need sex. Furthermore, I'll argue that sex was a prerequisite for such a ridiculous genomic system to evolve in the first place -- frequent sex allowed their genomes to get loose like that, for it could be easily compensated for by more sex. Perhaps this is what fundies fear under 'sex addiction'? In a way, sex does a wonderful job at screwing up otherwise perfectly self-sufficient organisms. So yeah, they're right, sex is a sin. Remember the poor barnacles? Divine punishment.

On the topic of impure deviations and dirty sex, ciliates have been observed having orgies. Like the more familiar fungi, ciliates too have multiple mating types, and avoid breeding with their own. If you have a culture with, say, three mating types, you may, occasionally, see orgies of three (rarely four) organisms. Even when such orgies do occur, most often they feature a pair of ciliates conjugating, while the third kind of sits there sadly and has sex with itself. Literally (autogamy)*. (Chen 1940 PNAS)

The big solid black things are MACs, the things undergoing meiosis are the smaller germline nuclei, which are about to be exchanged. Don't you feel sorry for the poor little guy who got left out? =( (Chen 1940 PNAS)

However, sometimes they can arrange themselves into a circle and pass around the pronuclei in a circular fashion. A true triple conjugation where all the participants can get their fair share:

Wheee - a circular orgy! I suspect there may be enough material for a ciliate Kama Sutra out there... (Preparata & Nanney 1977 Chromosoma)

They also had ways to induce massive group copulation (I guess also orgies, to some extent) using a sex-inducing mystery fluid; which caused massive autogamy sprees in populations of a single mating type -- Chen (1945) proposes it was actually a 'killer', toxic substance secreted by bacterial endosymbionts which render their host immune, and kill off everything else (I don't think they knew about the endosymbionts yet at the time). Basically, their defense response to the threat of death seems to be...having sex.

I think I've just ruined ciliates for you too. You're welcome ^_^

Note: Nice picture of ciliate triple conjugation on its way when I finally scan this Russian paper I 'had' to order through the library...

*When it gets too old and still solitary and mateless, the ciliate can also cure that by having sex with itself. Ciliate microbial sex life would be an awesome topic for a popular book...

References
Chen, T. (1940). Conjugation of Three Animals in Paramecium bursaria Proceedings of the National Academy of Sciences, 26 (4), 231-238 DOI: 10.1073/pnas.26.4.231

Chen, T. (1945). Induction of Conjugation in Paramecium bursaria Among Animals of One Mating Type by Fluid from Another Mating Type Proceedings of the National Academy of Sciences, 31 (12), 404-410 DOI: 10.1073/pnas.31.12.404

Hoch, J., & Yuen, B. (2009). An Individual Barnacle, Semibalanus balanoides, with Two Penises Journal of Crustacean Biology, 29 (1) DOI: 10.1651/08-3037.1

Preparata RM, & Nanney DL (1977). Cytogenetics of triplet conjugation in Tetrahymena: origin of haploid and triploid clones. Chromosoma, 60 (1), 49-57 PMID: 870290

Rule 34: Proteins

(alpha-helix, beta-barrel)
The reader is now encouraged to go out and find real examples of protein Rule 34 in action. There must be some suggestive protein interactions out there somewhere! And we shall never view biochem the same way again...

Inspired by this comment...


PS: In light of Jonathan Eisen's 'competition', shall we hereby coin 'pornome'?

How about genomeome, a set of genomes? "The genomeome of this phylum suggests..."
And then genomeomeome "A comparative analysis of genomeomes in multiple phyla led to the assembly of a larger genomeomeome..." I can keep going. But now we're getting off-topic...

Pond Microforay encore part 2 - Amorphous amoebae of ambiguous affinity

Part 1 featuring both familiar and mysterious types of flagellates found here.

These suckers are amoeboid and potentially amoeboflagellate, thereby leaving me rather clueless with regards to their identity. Help would be much appreciated! And I know some of you real protistologists read this. You're not as anonymous as you think =P So go help me ID some of this stuff, so we can have more excuses to learn (and blog) about obscure organisms. Please? ^_^

A few tiny blobs with crap (pseudopodia) oozing out:

Seriously, what the fuck are these things!?

Amoebozoan (most likely) with what appear to be acanthopodia. Probably not a cercozoan, right?

Another random small amoeba...

And another. Was trying to stalk it to see it eat, but no success.


I'd ramble off some stories about them, but again, I have no idea what they are. Probably could make up some cool 'factoids', but that's not really why we're here, is it? As appealing as academic fanfiction seems in principle...

I know I have some comments to respond to here, as well as VERY LONG OVERDUE posts. Ummm, they're in preparation, seriously. Unfortunately, there's some more high-profile 'submissions' queued up as well, such as lab exams (labelling crap in chick embryos o_O) and multitudes of assignments, and an eternally data-craving PI (aren't they all?).

Looking forward to tomorrow evening though: Carl Zimmer's talk on evolution of disease!