Ratcheting up some splice leaders: a note on directionality

In the sea of eukaryotic genetic diversity also lurk different manners of doing day-to-day genome work itself. Ciliates run two nuclear genomes, trypanosome kinetoplasts contain a chainmail suit of RNA editing circles and dinoflagellates are just weird in every genome compartment they have. Their plastids contain tiny minicircles often containing but a single gene, capable of "rolling" transcription where the minicircle is much like a Mesopotamian cylindrical seal, leaving a concatenated repeated string of genes on the transcript. The mitochondria have linear genomes with short fragmented repeated chunks of important genes all over them. But the nuclear genome is the most fucked up: for one thing, dinoflagellates lack a few histones, and have enormous genomes stored in absolutely bizarre chromosomes. More importantly for our story: every single gene must be trans-spliced with a 'splice leader', a short sequence that attaches at the beginning of the mRNA transcript and brings to it the 3' cap necessary for transcription to work. Oddly enough, Euglenozoans like the trypanosomes and euglenids seem to have a very similar system, evolved entirely by chance* convergence (Lukes et al. 2009 PNAS goes over this remarkable convergence in more detail).

*Or perhaps something happened to both that made them prone to evolve this bizarre system.

Genomic quirks are not just interesting in their own right as some arcane oddities, but can reveal a great deal about the dynamics of genomes in general. The dinoflagellate splice leader system turns out to yield a very crisp illustration of the power of ratchets and the toll of reverse transcription on genomes.

To reiterate, every single nuclear gene transcript in a dinoflagellate must be spliced with the 3'cap-bearing 'splice leader', or else it simply won't work. This means that the dino is full of mature transcripts with splice leaders attached to the transcribed genes. Enter reverse transcriptases, which are prevalent in probably most, if not all, eukaryotic genomes, thanks to viruses and their partners in genomic parasitism crimes, transposons. When they're not busy moving transposons around and helping viruses move in, they reverse transcribe random gene transcripts for fun, that may then, on occasion, be successfully recombined back into the genome. This process probably doesn't happen [successfully] every day, but over thousands or millions of years (and countless individuals) is rampant enough to leave a noticeable trace in the genome.

So we have a load of transcripts floating around with an extra sequence stitched onto them from the splice leader. Do the reverse transcriptases care in the slightest? Of course not: to them, a ribonucleotide is a ribonucleotide, give or take some trace biophysical stuff that might make a couple people cringe at what I just said. (meaning, I wouldn't be surprised if there could be some slight but ultimately detectable biases there too) This means that splice leader, on occasion, actually makes its way back into the nuclear genome attached to the beginning of the gene.

However, this splice leader does not substitute for the usual splice leader trans-splicing, since the 3' cap must be added again, or else the transcript will not be translated. That now-nuclear gene-attached splice leader ends up being completely useless, and is able to gradually degrade into benign junk, provided it doesn't mess with the translation of the gene. What is really cool is that one can actually see this gradual degradation, as shown in Slamovits and Keeling 2008 Current Biol:

Mmmm, actual data! Note how the oldest SL piece closest to the gene (on the right) is the most degraded. (Slamovits & Keeling 2008 Curr Biol)

Once the unnecessary splice leader chunk becomes part of the gene, the gene gets transcribed and trans-spliced like any other – meaning it is once again susceptible to replaying that same process of reverse transcription, except this time it already has a relict sequence. It can acquire a second one on top of that. This explains how there can be several concatenated splice leader relics tagging along, like in the above figure.

Splice leader trans-splicing not necessarily promoting reverse transcription – only makes it easier to detect. In other words, it inadvertently makes for a wonderfully convenient system where you can actually track what happens to a gene after it gets reverse transcribed. Once the gene makes its new home, the old gene copy is still present and they generally would be functionally redundant, so the dual-copy state is extremely unstable as ultimately the loss of one of the copies will be tolerated. If the newly transcribed copy is lost, we never see it and thus don't talk about it in the first place. However, once the clean original is lost, only the gene with the crap from the splice leader remains, and reversal to the original state is so improbable it's practically impossible. In other words, this process is a wonderful example of an evolutionary ratchet.

Ratchets are interesting because they confer intrinsic directionality to a system, even in the absense of external pressures (like selection). The accumulation of splice leader junk in the dinoflagellate's genes isn't particularly healthy, nor is it particularly deleterious – it's effectively neutral. However, one can argue that we do have an example of bloated complexity here. Since you can't go back and lose chunks of splice leaders, this ratchet essentially ensures that left to its own devices, this aspect of genome complexity will increase on its own. At a certain point, there will probably be ever-increasing selection against accumulating further splice leaders, and those lineages that go too far will simply die off – the central tendency doesn't care, and the ratchet will keep on going regardless of what selection 'wants'.

This ratchet example is therefore an elegant case of evolutionary direction that's not particularly well explained by the central dogmas of Modern Synthesis or (neo)Darwinism, where selection is the force that crafts order and directionality, with mutation a mere passive provider of material to be molded. I will go into a deeper discussion of this in another post (there's a cool paper coming out soon), but I think it's worth briefly mentioning here too while we're at it. The "mutation" step (to which, I guess, this trans-splicing and reverse-transcription process can be awkwardly attached) here is what provides a drive, a push in a certain direction, and towards increasing complexity, no less (although that last detail is irrelevant). While selection is present and provides constraints (if both genes are lost, for example, the organism dies), it does not do the 'driving' or 'forcing' in this system. Very crudely put, selection here is the passive phenomenon, and mutation is at the wheel.

Another case of intrinsic directionality, but where reversal is allowed, is your garden variety directional bias – where proceeding in one direction is more probable than going backwards. A very basic example of that is if the replication machinery favours a certain type of nucleic acid – left to its own devices, the genome base composition would be skewed in that direction. Boundaries can also induce an apparent directionality, but in this case it's no longer intrinsic... that's, again, a topic for another day.

This idea was a part of the Mutationism theories in the early 20th century, which were a little extreme and perhaps premature, since mutation was far from being even marginally understood at the time. In the usual melodramatic manner characteristic of academia and the scientific community, the pendulum swung far to the opposite extreme, and Modern Synthesis was born. It became heresy to think that mutation itself can actively contribute to direction and order. The field became engulfed in a false dichotomy, where either selection or mutation can actively provide direction, with the modern folk siding with the former. That is a serious mistake and an unnecessary waste of great explanatory potential – you can go so much farther with selection, drift, mutation and recombination all at the wheel, each pulling with different magnitudes in various directions. Well, technically, you wouldn't if you were the thing being pulled – which resonates so well with the absense of 'ascension' or general active directionality in the evolutionary system as a whole. Evolution is a slow, painful, inefficient and rather stochastic process, partly because the cart is being pulled in so many ways.

(The latter part, concerning directional biases and Mutationism, is based on various publications and conversations with Arlin Stoltzfus and Dan McShea, whom I gratefully acknowledge. =D)

References:
McShea, D. (2001). The minor transitions in hierarchical evolution and the question of a directional bias Journal of Evolutionary Biology, 14 (3), 502-518 DOI: 10.1046/j.1420-9101.2001.00283.x

Slamovits, C., & Keeling, P. (2008). Widespread recycling of processed cDNAs in dinoflagellates Current Biology, 18 (13) DOI: 10.1016/j.cub.2008.04.054

Stoltzfus A (2006). Mutationism and the dual causation of evolutionary change. Evolution & development, 8 (3), 304-17 PMID: 16686641

"Just another ciliate" – importance of sexy descriptions

There are species descriptions, and then there are species descriptions. All too often, you come across a mention of some obscure but ridiculously cool-looking organism, with only a very scant description of what it looks like and what it does. Much less often, you can come across yet-another-new-species (usually of a ciliate), but a particularly nicely described one. Again, those super nice descriptions tend to be of ciliates, largely due to the likes of Wilhelm Foissner and his academic offspring. Descriptive detail can only make species more interesting, and eventually of great potential to be useful for science. (Conversely, many a taxon has been rendered invalid due to poor description)

A sexy description is also a great way to lure readers into noticing your otherwise garden variety new species. Case in point – I see this random IJSEM paper on a couple new marine ciliate Frontonia species – nothing too earth shattering. Being rather compulsive about skimming over any mention of a protist I see in the literature, I click. Being rather lazy and a shallow-minded picture-loving type, I head straight for the figures. Unexpectedly, they dazzle me with sexiness. Desperate for something easy to blog about for the next little while (impending interview, exams, end-of-term chaos, etc), I suddenly find your otherwise-routine new species description quite exciting and blog about it. Here, Frontonia mengi and F.magna get screentime largely thanks to their authors.

Some of us in science are that simple minded. If more people realised that and preyed upon our ilk with shiny pictures, think how much more presentable science as a whole would be!

(That said, no amount of gloss and shine can make your data more or less wrong. But it can, and does, dazzle some of us into overlooking a flaw or three...)

Actually, the above was just a long-winded elaborate excuse to post ciliate porn. Ah, check out the kineties on that ass!

Frontonia mengi. See text. (Fan et al. 2010 IJSEM)

Well, those were mostly just shots of its oral ciliature, but close enough. The root structures of the cilia are highlighted with silver nitrate and carbonate staining, yielding the pretty staining effect. a-c section through the 'mouth'; d shows the "membranelle" around the 'mouth'. e shows the area behind the mouth; arrowhead points to the cytopyge. 'Cytopyge'? Well, a cell's gotta get rid of its waste somehow, and ciliates actually have the cellular analogue of an asshole. Not the socially dysfunctional kind. So yeah, look at that ass. g shows detail of the cortex, h is the overall view of the ventral ciliature. At i, the rows of cilia "stitch together" at the 'anterior suture'. k shows the germline micronucleus (Mi) and somatic macronucleus (Ma).

Now for some delicious DIC:

Frontonia mengi. See text. (Fan et al. 2010 IJSEM)

Crisp DIC intoxicates me. The seductive allure of polarisation-derived faux-3D relief is nearly impossible to resist, especially when you have the fine complex cell of a ciliate. In fact, good DIC is often better than staining, since you don't have to fix (kill) anything. Unfortunately in the case of some larger ciliates, some degree of squishing must be done otherwise the sample is too damn thick for crisp DIC. I think the gist of microscopy can be summarised as the never-ending compromise between care of specimen and care of the optical setup. The most powerful microscopy generally requires total destruction of the specimen, whereas the most natural and undisturbed data can only be attained with simple techniques and weak optics. It's like the Heisenberg principle of microscopy: the more accurately you determine the state of your specimen, the more mangled your specimen gets.

I digress. In the above plate, a-e show general views of several individuals of F.mengi. Remember my rant a couple posts ago about the usefulness of depicting morphotypical (shape type) variation? I hope it is evident here how that can be useful. For example, if only figure a was published, one could be mislead to consider that large vacuole a characteristic feature of this particular ciliate species. The other four images, however, show that to be a feature of just that specimen instead (non-contractile vacuoles, in this case). Furthermore, the authors even invluded a table of morphometric data, measuring the body dimensions and some visible subcellular details (like numbers of kineties and nuclear size) of 23 individuals.

The arrow in 1b points to a contractile vacuole – one could just make out the channel leading to the cell's exterior for expelling its contents. f-g show sections of the mouth, live. h shows detail of the cell surface, the oral apparatus quite visible (as is the cytopyge). i details the cytopharyngeal rods, which are specialised structures this genus of ciliates employs to devour long strands of algae. The characteristically massive ciliate nuclei are visible in j – the arrow points to the macronucleus while the arrowhead points to the micronucleus. No staining necessary, fuck yah.

Frontonia, like many ciliates, is also armed and dangerous. The surface is loaded with extrusomes (k), which can fire leaving a trail, much like the cryptomonad ejectisomes (l). m and n show the contractile vacuole and its exit pore, respectively. The contractile vacuole is necessary for osmotic regulation, especially in freshwater species, and is somewhat analogous in function to our kidneys.

The second species, Frontonia magna, is also well-described. In these specimens, one can make out the algal filament and its constituents – particularly in b, e and f. Like F.menga, it's also loaded with extrusomes (h). I particularly like i, which shows the ciliature of the anterior suture. It's quite hawt.

Frontonia magna. See text. (Fan et al. 2010 IJSEM)

Of course, no description is properly complete (in my opinion) without drawings to accompany the micrographs. Drawings highlight the important features observed by the authors, and are useful in combining information gathered from multiple sections and imaging techniques in a convenient summary. Making an accessible visual summary of a huge pile of microscopy data is no easy task, and is very much an art.

Continuing with F.magna, a summarises the ventral view of a typical individual. b provides a sketch of the sutures, without the distracting detail. c shows the side view, along with the contractile vacuole. d shows the relative sizes and positions of the nuclei. e, again, emphasises variation – it shows the various ways a cell appears after overeating with algal filaments protruding all over the place. It's amazing how hard prey can try to make their predator look like an entirely new freaking domain of life, by stretching it out and colouring it in all sorts of funny ways. A similar phenomenon has been responsible for an entire mistaken genus, Ouramoeba, in the otherwise totally awesome Leidy 1874 work on amoebae. The algal prey is detailed in g, while h details the cilia around the oral apparatus.

Frontonia magna See text. (Fan et al. 2010 IJSEM)

Of course, no species description these days is complete without a healthy phylogeny, and Fan et al. got that covered too. I feel I've stolen more than enough figures already, so I'll just say their Frontonia spp. fit snugly within Peniculia, a group including the famous Paramecium, and the two species are sister to each other. There's also a composition of drawings from multiple sources for other members of this genus, so this paper is a nice current reference for Frontonia, if you ever wake up one morning needing one. Believe me, these cravings may strike at the oddest hour.

Anyway, I just thought these figures really deserve to see the light of day, and not just remain buried away in what will very soon be just the back issues of a microbial systematics journal. While some may look down on routine-seeming research like basic species descriptions for they do not provide a fancy high-level synthesis or anything, but ultimately, these fancy high-level syntheses are built on lower-ranking papers like these, and cannot exceed the quality of their constituents. It is primary 'basic' literature like this that forms the foundation of science; without species descriptions, without "yet another gene/genome/tree/whatever", there will be nothing to base the more glamorous studies on. This is why impact factor is a load of bullshit, and anyone whose hands itch to oppress "low impact" science should be kept the hell away from research funding strategies, for they obviously have no fucking clue how research works in the first place. Grrr. How can anyone vote against a species description as awesome as Fan et al. 2010 above?

Reference
Fan, X., Chen, X., Song, W., Al-Rasheid, K., & Warren, A. (2010). Two new marine Frontonia species, F. mengi spec. nov. and F. magna spec. nov. (Protozoa; Ciliophora), with notes on their phylogeny based on SSU rRNA gene sequence data INTERNATIONAL JOURNAL OF SYSTEMATIC AND EVOLUTIONARY MICROBIOLOGY DOI: 10.1099/ijs.0.024794-0

Sunday Protist -- Blue Mats of the deep sea: Folliculinopsis

Far, far away, in the land of eternal darkness along the base of the deep sea hydrothermal vents of the Juan de Fuca Ridge lie stretches of surface covered by 'blue mats'.

(Kouris et al. 2007 Mar Ecol)

These blue mats are produced by yet another tube-forming denizen of the hydrothermal vents. To non-tube-dwellers like us they may even look vaguely reminiscent of the much more famous giant tube worms, and the concept is quite similar up until that point.

However, if you look inside a tube with its live host, something distinctly non-annelid peers out:

This creature is, in fact, a ciliate - a relative of the elegant Folliculina (referred to in the good ol' days as the "bottle-animalcule"), Folliculinopsis sp., a heterotrich like the giant Stentor:

Folliculinopsis. The two long 'wings' or 'ears' sticking out are its peristomal lobes, which can be seen in the preceding SEM. (Ji et al. 2004 J Ocean Univ China)

Folliculinopsis is host to countless bacterial symbionts; in fact so lushly the bacteria thrive on it that one can barely see the ciliate beneath them! Presumably, these bacteria may be involved in chemical defense, protection from the rather toxic surrounding environment or assist in metabolism. Symbiosis with prokaryotes seems to be fairly common for eukaryotes living awkward (extreme) environments, in large part because prokaryotes are simply amazing at biochemistry unlike their metabolically-challenged nucleated counterparts.

SEMs and TEM of symbiotic bacteria on Folliculinopsis sp. The lorica is covered mostly with filamentous bacteria (top left) whereas the surface of the ciliate is entirely covered with coccoid and rod-shaped episymbionts (bottom two SEMs). Moreover, the inside of the ciliate is full of bacteria-containing vacuoles, as seen in the TEM (near the cortex). (Kouris et al. 2007 Mar Ecol)

In another folliculinid, Eufolliculina, the surface of the peristomal lobes has a peculiar feature: short membrane-covered pins at the base of each cilium. Mulisch (1991 Cell Tissue Res) proposes these pins may act as sensory organelles, perhaps to transmit oriented mechanical stimuli. The cilia have a swelling at the level of the pin, filled with peculiar granular particles with potential involvement in calcium regulation (as you may recall from intro-level physiology, Ca²⁺ is quite popular in signaling systems). Similar cilium-pin complexes have also been found in other folliculinids, suggesting it may be a shared feature.

Cilia with sensory pegs at the base (arrows). (Mulisch 1991 Cell Tissue Res)

The cilium-peg complex reminds me of sensory hairs or sensilla on insects. Mulisch relates it to the hydrozoan cnidocil in the cnidocyst, or the stereocilia (microvili) at the base of the kinocilium in vertebrate sensory hair bundles. Perhaps this is yet another instance of ultimate convergence, as there is ultimately a finite number of ways particular functions can be performed, and evolution's random walks are bound to chance upon some more than once.

The biology of protist sensory mechansims and overall behaviour is still vast, mysterious, murky territory desperately in need of serious investigation. Unicellular organisms have complex behaviours just like multicellular ones, and are no more 'mere automatic responders to stimuli' than we are (due to our cumbersome complexity, much more random noise tends to creep in; perhaps where creativity comes from...); somehow, without a brain or even a nervous system, many unicellular organisms are nevertheless quite capable of performing complex behaviours in response to various stimuli.

This topic was quite popular in the early 20th century, but seems to have been largely abandoned today (in unicellular organisms). Considering the volumes of papers published daily on cell motility in tissue cultures, would it be too much to ask for some investigation of more intelligent cell types, ie. those that also act as entire organisms? Surely a ciliate must be much more fascinating to work with than some confused helpless cells ripped out of context in some suspension? There's enough work to do in this corner of science to keep us busy for many more years to come...!

On that note, the sun is rising. I should respond to the stimulus. By sleeping... (spent a few more hours scratching my head over some potential centrohelids...freaking gaps in the literature are really annoying, especially when you can't access half of it as it lies under piles of dust in some obscure obsolete journals that have been forgotten for the past five decades or so. Fun times.

References:
Ji, D., Lin, X., & Song, W. (2004). Complementary notes on a ‘well-known’ marine heterotrichous ciliate, Folliculinopsis producta (Wright, 1859) Frauré-Fremiet, 1936 (Protozoa, ciliophora) Journal of Ocean University of China, 3 (1), 65-69 DOI: 10.1007/s11802-004-0011-1

Kouris, A., Kim Juniper, S., Frébourg, G., & Gaill, F. (2007). Protozoan?bacterial symbiosis in a deep-sea hydrothermal vent folliculinid ciliate (Folliculinopsis sp.) from the Juan de Fuca Ridge Marine Ecology, 28 (1), 63-71 DOI: 10.1111/j.1439-0485.2006.00118.x

Mulisch, M. (1991). Ultrastructure and membrane topography of special ciliary organelles in the ciliate Eufolliculina uhligi (Protozoa) Cell and Tissue Research, 265 (1), 145-150 DOI: 10.1007/BF00318148

Sunday Protist -- Tachyblaston: A suctorian parasite of suctorians

[it's totally still Sunday in someone's mind somewhere...right?]

Reading old protistology books can be quite a frustrating exercise: image you come across a really cool-looking organism, try to follow up on what happened to it since, and discover it's only been written up once in the distant past and neglected ever since. This happens to a very annoying percentage of organisms described in those older books (newer books tend to forget the phantom and near-phantom species). Now this organism in particular at least has a very detailed source behind it, but alas! ...in German. I saw it in Grell's (1973) Protozoology, and the original description comes from... Grell 1950 . The former I have an English copy of, the latter I do not. So don't expect much detail.

Ecologists often lump microorganisms together as 'decomposers' (at least in undergrad courses); those of us living in a different scale of things beg to differ. From an intro ecology text, you get the idea that ecology somehow ceases to happen once you reach a certain size or phylum, and everything's just a part of this amorphous blob that exists to recycle nutrients so that the rest of us can live on. Shockingly enough, this amorphous blob has a whole ecosystem of its own, complete with predators and photosynthesisers and those who do both, as well as parasites and mutualist endosymbionts and saprophytes, etc. They interact with each other in ways not in the slightest less interesting than fluffy animals. In the microscopic world, cells become bodies that, just like ours, can get hunted, infected or benefited by some other organism. Or host a pile of commensals (who do exist, by the way, by similar arguments that Nearly Neutral Theory employs for mutations)

*Zoological ecologists also tend to treat plants as 'those things that exist for animals to eat', which annoys the hell out of anyone dealing with plants. On the first day of ecology the instructor causally mentioned that 'plants don't do much in the way of behaviour', and thus the course will largely ignore them. I expressed disagreement after class, noting there is little fundamentally different between a plant biochemical response leading to, say, discharge of toxins or some regulatory change, and an animal biochemical response leading to observable [to our eye] mechanical change. Yeah, this is why I have difficulty talking to the more 'traditional' biologists sometimes...but that is completely off-topic.

Remember how crabs can sometimes be covered in sea anemonies? Many smaller crustaceans can often be covered in organisms superficially resembling miniature sea anemonies - namely, Suctorians - highly derived (=weird) ciliates covered in miniature tentacles. Suctorians also reproduce by budding, as opposed to conventional symmetrical mitosis employed by the canonical ciliate. Just like sea anemonies and other cnidarians, suctorians also have stalked and swarming forms, like the polyp vs. medusa destinction in the former. Which is quite unsurprising, really, as aquatic sessile organisms usually use specialised free-swimming forms to spread. But still another cool bit of ultimate convergence discussed a couple posts ago.

Top: A copepod covered in suctorians; an SEM of Ephelota gemmipara from the copepod. (Fernandez-Leborans et al. 2005 J Nat Hist) Bottom: Ephelota superba, suctorian episymbiont of Antarctic krill. Quite reminiscent of an anthozoan. (Stankovic et al. 2002 Polar Biol)

Now, imagine a microscopic sea anemone being parasitised by another. I'm not sure whether there are any cnidarian parasites of other cnidarians (wouldn't be too surprised), so the analogy stops around here. The awesome does not, however: parasites are never truly simple. Tachyblaston's infancy consists of finding an Ephelota, attaching itself and piercing the cell membrane to leech off the cytoplasm. Over time, the entire cell can become filled with parasites. During this stage, the parasite buds to produce swarmers.

Tachyblaston invading Ephelota cell body. Right: Tachyblaston budding. (Grell 1950 Z.Protistenk)

Afterwards, the swarmers swim around and attach themselves to an Ephelota stalk, where they themselves form a stalked cup structure. There the parasite buds multiple times, yielding a cup full of Tachyblaston, which is subsequently emptied as the buds (this time with a single thick tentacle, according to Martin 1909) evacuate and crawl up the stalk toward the main cell body of Ephelota to infect it and start the cycle over.

Left: Swarmers. The stage that actually sort of looks like a ciliate... Middle: Full 'cup' of Tachyblaston in stalked stage. Right: Empty cup after all (Grell 1950 Z.Protistenk)

To summarise Tachyblaston's life cycle, the cell-penetrating parasites of the Ephelota cell body bud to form swarmers, which, in addition to reminding us of suctorians' ciliate leanings, find another Ephelota and attach themselves to the stalk, forming a cup which they fill up by budding again, finally releasing single-tentacled forms that crawl up the stalk to the next victim. How's that for unicellular organisms having 'primitive' differentiation capabilities?

Overview of the whole life cycle of Tachyblaston. Oh the tentacles... (Grell 1950 Z.Protistenk)

Tachyblaston's original description by Martin 1909:380 J Cell Sci can be found here. The parasite was very distinctive due to a major refringent particle of unknown origin or function present within each Tachyblaston cell. The genus name reflects the extraordinary speed with which the parasite epidemic can sweep over an entire population of Ephelota, which end up a decimated forest of bare stalks. Creepy.

And last but not least, here's an obligatory tree to orient ourselves phylogenetically:

Tachyblaston and Ephelota are both suctorians in Phyllopharyngea, which contains some other bizarre (and somewhat obscure) creatures like Chonotrichs. (Gong et al. 2008 JEM)

PS: Blogging about ciliates is very difficult. They are too damn distracting - you start reading about one and come across ten others you suddenly must look up, and so on. About as bad as Wikipedia. Actually, since looking these things up is now actually relevant to my day job, the distractions get worse as I feel compelled to write down and follow anything potentially related to work. Just in case. Apparently, sort of using blogger as a reference manager... hence the exploding drafts folder. Sigh.

References:
Fernandez-Leborans, G., Freeman, M., Gabilondo, R., & Sommerville, C. (2005). Marine protozoan epibionts on the copepod Lepeophtheirus salmonis , parasite of the Atlantic salmon Journal of Natural History, 39 (8), 587-596 DOI: 10.1080/00222930400001525

GONG, J., GAO, S., ROBERTS, D., AL-RASHEID, K., & SONG, W. (2008).
n. sp. (Ciliophora, Phyllopharyngea, Cyrtophoria): Morphological Description and Phylogenetic Analyses Based on SSU rRNA and Group I Intron Sequences
Journal of Eukaryotic Microbiology, 55 (6), 492-500 DOI: 10.1111/j.1550-7408.2008.00350.x

Grell, K. (1950). Der Generationswechsel des parasitischen Suktors Tachyblaston ephelotensis Martin Zeitschrift f�r Parasitenkunde, 14 (5) DOI: 10.1007/BF00260027

Martin, CH (1909). Some Observations on Acinetaria: Part I.—The " Tinctin-kbrper " of Acinetaria and the Conjugation of Acineta papillifera. Quarterly journal of microscopical science, 53 (2), 351-389

Sunday Protist -- Gymnodinium catenatum: Like beads on a string

I'm going to be genuinely lazy today. Seriously cannot go off on a 3h literature journey looking at random protists with an invert zool final happening so very soon (Monday). Even though the instructor is actually a protistologist. Maybe his final will let me ramble about choanoflagellates somewhere...

Today we have some random dinoflagellates; well, not as much random as rather 'typical' for the group, although with an interesting tendency to form long chains, which is not so typical. Meet Gymnodinium catenatum, a concatenated naked dinoflagellate:

Chains of dinos! Source; more dino and algal bloom pictures here.

Gymnodinium is photosynthetic, and has an unfortunate side effect of producing chemicals humans and other animals find toxic. Despite the occasional public mania on the topic, not all dinoflagellates do this, and those who do didn't really mean to, and would perhaps even write a letter of apology were they capable of and interested in such a thing. Also, algal blooms have been happening for millions of years and are not a 'new' thing by any means. Just that nobody 'noticed' until we came alone and started writing about it. That said, harmful algal blooms are an important research topic, but probably the efforts should be directed to finding antidotes and other medical solutions to the problem, and educating the public instead of causing panic, rather than naive attempts at eradicating blooms altogether. But that's just my entirely non-expert opinion. Yeah, I personally don't find that topic particularly fascinating, and am kind of annoyed by the majority of dinoflagellate papers seeming to be about their toxicity and blooms instead of their biology and evolutionary history. Meh.

Seriously, look how cute dinos are! There's quite a few extreme oddities in the group as well, and I'll leave them for later. Previously, I've touched on the morphologically awesome dinophysis, ferocious palium-feeding Protoperidinium that eats things bigger than itself, and Ceratium which grows plastid-filled 'fingers' during the day and retracts the plastids and fingers during the night. I still haven't touched on the really weird stuff though! It shall happen eventually...

Ok, back to dreading finals and life in general. Just two weeks. I can do this...I think.

Sunday Protist -- Trichotokara nothriae: Guitar-shaped gregarine

This post turned into a bit of a hodgepodge of various gregarine-related trivia. Proceed with caution.

Gregarines are a group of apicomplexans (='Sporozoa', a vastly diverse group famous for the malarial parasite Plasmodium and the behaviour-altering Toxoplasma) characterised by a monoxenous (single host) lifestyle that is quite different from that of other 'apis'. Christopher Taylor wrote a nice post about them here.

Apicomplexa are alveolates along with ciliates and dinoflagellates; you can find them on the left side of this tree . The apicomplexan phylogeny is a complete mess at the moment; the old coccidian-haematozoan-gregarine divisions aren't too well-supported and the relationships of stuff within them are even murkier. As an aside, many apis have an 'apicoplast', or a relic plastid of red algal origin -- their ancestors were once photosynthetic! In fact, a paraphyletic group of organisms basal to apicomplexa (Chromera et al.) are currently photosynthetic, further supporting the photosynthetic ancestry of these weird mostly-intracellular parasites, most of whom rarely ever see the light of day!

Gregarines are typically invertebrate parasites and unlike other apicomplexans, tend to spend most of their lives extracellularly; in fact, their cellular penetration consists of attaching themselves to a cell via the mucron (holdfast-like structure). You can read more about their biology and life cycle on their ToLWeb page. (also a review in Tr Parasitol: Leander 2007) If you want to see some for yourself, kidnapping and slicing up an earthworm is an easy way to do so: Monocystis is a parasite of earthworm seminal vesicles (feeds on sperm), and a rather abundant one. It may actually be quite easy to find various apicomplexan parasites in insects -- it is estimated that most of them may have an api specialised in parasitising them, which hints at the total apicomplexan diversity being something outrageously vast! Such a project would also be a good excuse to learn insect anatomy, which I find to be quite complicated.

Right, you wanted to see a new genus of guitar-shaped gregarines:

Trichotokara from the intestine of an onuphid tubeworm. a-e: trophozoites (feeding forms). M - mucron ('holdfast'), CB - cell body. Arrow - junction between mucron and cell body, which can be seen extending further into the mucron in (e; arrowheads). f: gamonts in syzygy, or gregarine sex. Scalebars: a-e 10um; f 25um. (Rueckert & Leander 2010 J Invert Pathol)

By the way, if anyone asks you for a six-letter word in English 'devoid of any vowels', keep 'syzygy' in mind. Technically it does have vowels, as any phonologist would tell you, but most people insist on equating letters with sounds, and y 'is not a vowel'. Regardless, it's still a really awesome word. Syzygy!

More gregarine awesomeness. Note how the cell surface seems to strive for increased surface area, especially in the mucron which gets inserted into a cell:

SEM of Trichotokara. b - close-up of hair-like projections of the mucron. c - junction between mucron and cell body. d - folds along the cell body. Scalebars: a - 10um; b-d - 1um. (Rueckert & Leander 2010 J Invert Pathol)

This gives me an excuse to mention a paper on proximate vs. ultimate convergence by the senior author on the above gregarine paper: Leander 2008 JEM (free access). Among several other examples of ultimate convergence between multicellular and unicellular organisms inhabiting similar environments, gregarines and nematodes are compared in terms of their structural organisation. While nematodes have longitudinal muscles just beneath the elastic epidermis, gregarines have subpellicular bands of longitudinal microtubules running just underneath the elastic cortex (although used differently -- see gliding motility below). Curiously, in both cases the result is a sinusoidal (wiggling) pattern of movement. Additionally, tapeworm and Haplozoon (dinoflagellate) surface morphology are noted to be similar (covered with microvili), for the obvious purpose of increasing surface area. It's probably not much of a stretch to add gregarine surface structure to that list. (see Leander et al. 2003 J Parasitol for more gregarine surfaces)

Interesting case of structural ultimate convergence between nematodes and gregarines. Purple - bands of muscle and microtubules, respectively. Blue - elastic epidermis and tri-layered cortex, respectively. The three cortical layers consist of the plasma membrane at the very surface, with two alveolar membranes immediately below. (Leander 2008 JEM)

Before we proceed to a digression on apicomplexan motility, oblicatory phylogeny of Trichotokara and relatives. Note its extremely diverged SSU sequence resulting in a hellishly long branch:

ML tree of SSU rDNA sequences. Probably wouldn't trust its exact placement among the gregarines just yet... (Rueckert & Leander 2010 J Invert Pathol)

Apicomplexans are generally aflagellate in their trophic stage (I say 'generally' just in case...) -- their motility is an interesting topic, as they can't exactly extrude pseudopodia either. Nor do they have any spirochaetes doing the work for them as in Mixotricha, nor do they wildly thrash about an internal bundle of microtubules like Saccinobaculus. So how do they do it? Just like pennate diatoms: by gliding motility.

While sharing some basic similarities with diatom gliding, the apicomplexan variant has an unrelated origin and is quite different. One annoying thing (to us) about alveolates is their alveolae, or little membranous sacs just underneath the plasma membrane. In apicomplexan cell biology literature, this is called the Inner Membrane Complex. Prior to explaining why this detail is particularly annoying, first let's go over the crude basics of gliding motility: First, you need something to anchor to the substrate. This material is usually discarded, leaving behind a trail of 'slime', if you will. Then, you need an adaptor protein [complex] that attaches to the anchoring substance and crosses the plasma membrane. This adaptor must have a way of reaching a cytoskeletal element, usually actin via myosins (eg. see Molino & Wetherbee 2008 Biofouling; also, that journal title is very WTF...). The problem (again, mostly for researchers, and students...) with apicomplexa is their tendency to have the Inner Membrane Complex in the middle of that. This means the mechanism looks roughly like this:

Complicated motility. (Soldati & Meissner 2003 Curr Op Cell Biol)

Or, in the language of Nature Reviews:

Cell biology: Always more fun with extra gene/protein names thrown in, especially those irrelevant to the point. Shall we look up some protein structures while we're at it?
Ignore the target cell part -- a similar process happens along other surfaces too. If I recall, the model with intra-IMC proteins reaching across between actin and microtubule systems is actually more up-to-date; the "rolling IMC conveyor belt" model was outdated. Don't quote me on this though! (Baum et al. 2006 Nature Rev Microbiol)

Remember cramming the molecular biology of amoeboid motion? Isn't it almost a good thing that traditional cell biology courses are so phylogenetically impoverished? So many things are much more complex than animal cells, so we actually get the easy (and [arguably] boring) option. In a nutshell, you have something like this:

anchor-adhesin-actin-myosin-[interamembranous particles?]-subpellicular microtubules

The myosins move to the opposite of cell motility (and actin polymerisation), thereby pushing the pellicular microtubule skeleton in the right direction. Look at the Soldati & Meissner figure again to see why. It's a rather convoluted process just to get a cell moving! Of course, that complexity is more of a problem for cell biologists than the organism, considering how abundant and efficient apicomplexans tend to be.

Another aside: Apicomplexans, as well as numerous other organisms, are capable of a cell divison process known as palintomy: they can undergo several rounds of mitosis without cytokinesis, resulting in multinucleate cells (helps to not have open mitosis), and then simultaneously undergo cytokinesis for each of those nuclei (cellularisation). In gregarines, this looks vaguely like budding, as the nuclei tend to congregate near the cortex during this process (Kuriyama et al. 2005 Cell Motility Cytosk). Drosophila embryos do something similar, so palintomy isn't that unusual, but still pretty cool.

Back to gregarines, there are some more species that seem to be on a morphological acid trip. Some of them have been described only once and never noted again, which makes me sad:

Aikinetocystis singularis. I really want an SEM of that! Too bad it's from an obscure burmese earthworm... (Gates 1926 Biol Bulletin)

So if you like finding new species and genera and describing them, may I recommend apicomplexan diversity. It's taking a while for entrail-hungry parasitologists to go through all the various invertebrate (and vertebrate) parasites out there, so there's still plenty of room for work. If there is truly one species of apicomplexa for roughly each species of insects (and other animals), that pie chart of diversity showing most life as insects (and protists but a tiny splinter) is truly laughable:

LOL. Simply hilarious! Looks like the "global biodiversity assessment" team was a bit short on microbiologists... (at least they admit to not knowing much bacterial diversity; at least they put 'protozoa' and 'algae' in quotation marks...) (Purvis & Hector 2000 Nature)

All hail microbial parasites -- the bane of biodiversity research!

References
Baum, J., Papenfuss, A., Baum, B., Speed, T., & Cowman, A. (2006). Regulation of apicomplexan actin-based motility Nature Reviews Microbiology, 4 (8), 621-628 DOI: 10.1038/nrmicro1465

G. E. Gates (1926). Preliminary Note on a New Protozoan Parasite of Earthworms of the Genus Eutyphœus Biological Bulletin, 51 (6), 400-404

LEANDER, B. (2008). Marine gregarines: evolutionary prelude to the apicomplexan radiation? Trends in Parasitology, 24 (2), 60-67 DOI: 10.1016/j.pt.2007.11.005

LEANDER, B. (2008). A Hierarchical View of Convergent Evolution in Microbial Eukaryotes Journal of Eukaryotic Microbiology, 55 (2), 59-68 DOI: 10.1111/j.1550-7408.2008.00308.x

Molino, P., & Wetherbee, R. (2008). The biology of biofouling diatoms and their role in the development of microbial slimes Biofouling, 24 (5), 365-379 DOI: 10.1080/08927010802254583

Purvis, A., & Hector, A. (2000). Getting the measure of biodiversity Nature, 405 (6783), 212-219 DOI: 10.1038/35012221

Rueckert, S., & Leander, B. (2010). Description of Trichotokara nothriae n. gen. et sp. (Apicomplexa, Lecudinidae) – an intestinal gregarine of Nothria conchylega (Polychaeta, Onuphidae) Journal of Invertebrate Pathology DOI: 10.1016/j.jip.2010.03.005

Soldati, D., & Meissner, M. (2004). Toxoplasma as a novel system for motility Current Opinion in Cell Biology, 16 (1), 32-40 DOI: 10.1016/j.ceb.2003.11.013