Skeptic Wonder

Free ImageJ Macro -- for citing images

2011-12-19T02:54:00.001-08:00

(of course all IJ macros and IJ itself are free...)

So I got sick of constantly clicking away just to resize an image and add some citation text to the bottom and then name the file that exact thing; in fact, it was a deterrent to blogging -- yes, I am *that* lazy, apparently. At 4 in the morning today, my latent inner yet-unexplored codemonkey decided it needed an ImageJ macro for doing that, and it needed it then, at 4 in the morning, the day before I fly for holidays, and therefore a very busy day. After a couple hours of failed negotiation attempts between brick walls and my head (and much profanity), I finally produced a piece of code that not only does something, but does what I want it to! Being an utter failure at anything technical (engineering aptitude turns out to not be particularly heritable, as my parents' experiment demonstrates), I get soooo excited when something I slap together out of copy+paste and single-finger typing on the keyboard actually works, that I absolutely have to share it with the world. Even if it's pathetically minor and useless. Shhh.

World, behold -- an ImageJ macro for adding citations to images!

// This macro creates citation (or other input) text by adding a 20p strip to the bottom of the image, aligned right
// Image is saved in Documents as the ref input text
// WARNING: saving several images with same citation input WILL result in overwrite!

ref=getString("Reference", "ref");
var width = getWidth();
var height = getHeight();
height=height+20;
run("Canvas Size...","width=&width height=&height position=Top-Center");

var textwidth=getStringWidth(ref);
var textmargin=width-(textwidth+5);
drawString(ref,textmargin, height-5);
var path="C:\\";
var name=path+" "+ref;
saveAs("Jpeg",name);

Hopefully this means I'll get back to regular blogging soon, as one more Gate of Laziness has been removed from the path. Now if only IJ could also upload and link those images...

Update!

2011-12-14T20:09:00.000-08:00

Neglect... so much neglect. Been swallowed up by my move to Indiana and settlement attempts. Haven't really been keeping up with my SciAm blogging either, but I do intend to return here and post the occasional snippet of something random and perhaps even technical, that I don't want to bother with in terms of translating to a human language. That was an awful sentence -- see what a lack of writing practice can do?

For the few who may care about personal life, well... being a full-time researcher takes up a lot of time, it turns out. Particularly when your experiments aren't really shining in glory or anything like that. And when your gentle introduction to a subject you despised all through undergrad happens to be a grad-level course you absolutely have to do well in. In other words, I've been thrown off into population genetics at the deep end. The course was great, actually, and think I learned *a lot*, but a little bit intensive to someone who merely a year ago defiantly ignored claims that evolution involved 'populations'.

In terms of research life, feels like I'm involved tangentially in enough projects to get away with not really doing anything in particular. In addition to maintaining some ciliate and diatom mutation accumulation lines (long, boring and painful multi-year project to ultimately measure the mutation rate and spectrum, which is actually very exciting as a final product!), I'm trying to learn the art of harnessing ciliate growth rates to be able to have them undergo autogamy (recreating their macronuclei, etc etc) at just the right times to gather RNA for sequence data (that is not my project), and figuring out imaging techniques for deciphering the identity of food vacuole bacteria that persist after longterm starvation for some inexplicable reason. In addition, also trying to start up new protist mutation accumulation lines to ultimately get a phylogenetically sounder sense of eukaryotic mutation rates.

As you can see, lots of trying and attempting and figuring stuff out, and not a whole lot of results and data, which gets frustrating after some time. But rumour has it that's not unusual when starting in a new lab.

Another shift was going from a protistology haven to being some sort of a sole regional expert on protist diversity, entirely for lack of anyone else in the field around here. It's rather alienating, and you can't argue with people about arcane topics in protist phylogeny and taxonomy as they'll just go with whatever you assert. Which renders argumentative assertion a lot less fun. On the other hand, there's an exciting challenge to convert locals to the dark side, and I'm trying to do anything I can in that department, mwahaha! After all, Indiana U used to be quite a bustling centre of protist research, back in the days of Tracy Sonneborn, his deciples and Paramecium genetics. A handful of us in this lab are all that's left of IU's proud protistology tradition...

So that's what I've been doing lately, leaving little productive time for blogging (but, of course, plenty of time for unproductive procrastination). Not easy getting started after a long break either...

Anyway, enough rambling, and onward with moar protists!

New paper on Constructive Neutral Evolution

2011-07-12T08:41:00.000-07:00

Don't have time to blog this right now (off to protistology conference today -- will bring back lots of goodies!), but there's a new review paper about CNE out a couple weeks ago. It's paywalled at some rather obscure journal that an institution as big as UBC doesn't have access to, so here's a pdf y'all can [hopefully] access!

Lukes et al. 2011 IUBMB Life: How a Neutral Evolutionary Ratchet Can Build Cellular Complexity

(original journal link)

See, if this was published in a proper (ie, open access) journal, I wouldn't have to do this. As taxpayers you all have the right to see this, outdated publishers be damned.

I'm taking off to ISoP in Seattle - I might attempt to live tweet parts of it (as @ocelloid), so do follow us on twitter using the #isop11 hashtag! And for those of you who are gonna be there... let's see if you can find me ;-) (spoiler alert: Psi is a pseudonym. Seriously! o.O)

Big Announcement: New blog -- The Ocelloid

2011-07-05T09:18:00.000-07:00

After several months of contained excitement and preparation, the embargo has been lifted and I can finally announce the unveiling of the new Scientific American blog network, of which I am honoured to be a small part at The Ocelloid, my new blog -- focusing, like this one, on protists and evolution, although with a stronger attempt at reaching the lay audience. I will continue blogging here at Skeptic Wonder as before, and since I already don't blog as frequently as I should, not much of a difference should be noticed. Basically, my goal is to have The Ocelloid more general audience friendly and introducing people to the protist world from a more superficial 'wow' angle, while Skeptic Wonder will be cater more to the current crowd that seems to consist mostly of people more qualified than I am about this stuff. It has been a bit awkward trying to reach both types of audiences from the same blog so I think this may work out well for everyone. I'll also keep more raw discussions here and The Ocelloid should be more polished up. We'll see where this goes. From time to time I'll cross-post between them but perhaps it's better to keep the recycling to a minimum.

Bora has an amazing detailed introduction to the SciAm network which discusses its purpose as well as awesome overviews of the individual blogs. The official launch press release is here, as well as a welcome post from the Editor-in-Chief and a post on The Observations. More once I get home, am out of town right now until tmr with crappy internet and no control over own time...

EDIT 23:30 05.07.11: Just to clarify things: I am keeping Skeptic Wonder and staying here at FoS as well! And there may be another change here coming up, for the better!

A Tree of Eukaryotes v1.3a

2011-06-30T21:24:00.000-07:00

Time for a new tree, finally. Some groups have been fixed and the diagram has moved from Powerpoint to a real vector art program (Illustrator), so hopefully it looks a bit nicer now and has slightly fewer glaring errors. Have yet to fix all issues, the biggest (and hardest) being the proportions taken up by the various groups -- the tree appears dominated by Excavates for some reason. Due to lack of convenient taxa for the heteroloboseans and euglenids, I expanded them to the genus level in some cases to attempt to capture some of the diversity better, but that screwed things up for the rest of the tree. Since fixing that would require some hardcore structural changes to the whole tree, I'll do that later, in the next edition (which will not take over a year to come out this time). Given some conferences coming up this summer, and that people have asked, I'll release what I have done now as v1.3a.

Enjoy! (And do complain if you spot anything awry...)

Previous versions and discussions, along with trees by other people, can be found here.

References

A shit ton (see image). But ResearchBlogging.org doesn't allow indexing 'shit ton', so I'm gonna be pathetically lazy and just cite this:

Keeling, P., Burger, G., Durnford, D., Lang, B., Lee, R., Pearlman, R., Roger, A., & Gray, M. (2005). The tree of eukaryotes Trends in Ecology & Evolution, 20 (12), 670-676 DOI: 10.1016/j.tree.2005.09.005

An update!

2011-06-21T15:10:00.000-07:00

No, not the annoying kind that secretly restarts your computer in the background because you just bought it and haven't gotten around to deactivating auto-update yet and told it to fuck off the last few times so it didn't pop up the window anymore because it was sad. Or the kind that Adobe's PDF reader mysteriously wants about four times a day. Just a very late bloggy kind.

Apologies for disappearing for a while there. Personal issues came up and didn't really feel like writing about science (or reading much about it for a while). Long story short, I'm may well be a failed scientist at this point (no grad school for me, yay), and the academic career is one of the few where once you fall off the track, it's practically impossible to get back on. And unlike in most other careers, the skills you acquire by that point are nontransferable anywhere else, meaning you're screwed, period. Add to that the worst economy since the Great Depression, and the party starts off with a bang. That said, I'll continue with my attempts to sneak past academia's fortifications under the cover of night, if no other reason than that banging my head against brick walls fucking arouses me.

Anyway, I'm getting back to blogging now. Should at least take advantage of the fact I still have a computer and internet; might be a bit harder to blog when unemployed and homeless ;-)

News
There are some exciting developments next month: one I can't tell you about yet as it's part of bigger news; the other is that I'll be going to a phycology-protistology meeting (PSA-ISoP) mid-July and will be officially blogging it! There's lots of awesome research going on in the area and I'm happy I'll be able to share some of it with you.

Microscopy Reddit Community - /r/microscopy
Every once in a while a stack of undeciphered micrographs appears before someone's conscience, and every once in a while a resolution of this issue is attempted by approaching yours truly. I'm still a novice to the realm of the small, and usually fail to identify creatures (or artefacts) in question, leaving behind a trail of disappointment and pristine befuddlement. Forwarding those images to friends and colleagues would be awkward, since those people have enough on their plate to begin with. In short, would be nice to have a centralised place where people could share images and others could voluntarily look them over and comment on them. Micro*scope/EOL is a nice image repository, but generally the images there are of good quality and are finished products; furthermore, I still don't know how to work the interface there despite having access privileges. What would be great is if people could host images wherever they like, and then link to them in a centralised place for discussion where anyone could participate. In other words, Reddit.

There already was a microscopy subreddit (a Reddit community), but it was largely inactive and abandoned. Anyway, I'm now a moderator there, and would like to develop it into a community where micrographs of all sorts can be shared and discussed, with emphasis on microbial organisms (but sliced up macrobes welcome too). Creating an account is really easy, as is submitting a link (just make sure it goes to /r/microscopy and not some other area of reddit). We need participants though, so if you have any neglected mystery images, please post them, and if you're in the mood to browse micrographs from time to time, feel free to stop by! Just keep in mind anyone can see the subreddit including the images, so careful with potentially publication-worthy data...

Hope to see you there!

Random link
There's a really awesome Russian underwater macrophotography blog I came across a while ago that you should all know about. The photos are stunning, mainly of pretty tiny inverts in the White Sea in northern Russia (and plenty of shots of Northern Lights and white nights and all that).

Sticky proteins, complexity drama and selection's blind eye

2011-05-21T08:03:00.000-07:00

*For your entertainment, rejected titles:
[Sticky proteins and complex relationships]
[(protein) Relationship drama: promiscuous proteins in small populations]
[Not all is good that sticks: non-adaptive complexity gain through compensatory protein adhesion]
[Man, I suck at titles]

NB: This post can be considered as part 2.5 of my In defense of constructive neutral evolution series; also recommended for some background are part 1, discussing selection, drift and Neutral Theory, and part 2, discussing Constructive Neutral Evolution; to answer a popular question, part 3 *will* materialise ~~eventually~~ once I get off my ass and write it.

Constructive neutral evolution is one mechanism of complexity increase without any associated increase in fitness – or, in other words, non-adaptive complexity gain. Basically, a random interaction between two proteins can lead to a fixed dependency if this interaction compensates for a mutation that was otherwise lethal – termed 'pressuppression'. In this way, previously unnecessary dependencies accumulate to make a very bulky, bureaucratic system that essentially does the same thing. We've all seen it in our institutions, and evolution is about as efficient.

Now, one bottleneck in this model is waiting for proteins to actually interact. Proteins are quite sticky and non-specific by nature, but usually not too much as that can be quite deleterious. Piling up a bunch of proteins on each other has a non-negligible chance of interfering with their function, and one would expect for chance interactions to not be excessively promiscuous, although those who have done regulatory genetics and protein work are probably aware just how annoyingly non-specific some of the protein binding can get. Luckily, there is now a possibly mechanism boosting these chance interactions, and thus alleviating that particular bottleneck in the Constructive Neutral Evolution process, rapidly accelerating complexification and protein network obfuscation to the extent where the interaction map looks like a web; not a finely organised web of an orb-weaver but rather one of those clumpy webs that are a clusterfuck of stickiness and silk. Enter this week's Fernández and Lynch 2011 Nature paper, from here onwards referred to as "the paper".

Protein 'stickiness' can be enhanced by biochemical means. Proteins vary in stability, and themselves come in populations – generally, most are in the optimal conformation that is presumably functional, but some individuals are messed up. This happens well past the sequence and folding errors, and some perfectly 'normal' proteins can be in a suboptimal state at any given time. Clearly, this affects the overall efficiency of the protein – even if it's enzymatically awesome, the overall 'protein' as we biologists understand it (sans population aspect) would decline in efficiency if a large chunk of its population is in a misfolded state.

One aspect that pushes around the proportion of the protein in the 'right' conformation is how well it plays with water. It shouldn't be too surprising that hydrophobic regions induce instability. What was new to me, but perhaps old news to those who actually understood chemistry, is that the exposure of the polar(hydrophilic) protein backbone to water also has a destabilising effect – and not only that, but often more significant than that of exposed hydrophobic regions! This may seem counterintuitive – doesn't water like hydrophilic regions? And there lies our problem.

Water molecules are attracted to polar groups, and the amino acid backbone is quite polar. This means little water molecules wander in towards the backbone and form hydrogen bonds with it. The problem is twofold: first of all, the protein, like all molecules, likes to 'jiggle'. The more it can jiggle in its given conformation, the more favourable that conformation is thermodynamically since its satisfied by more states. Entropy, etc. (now we're *really* entering territory I know nothing about, since my phys chem experience is locked away by PTSD...). Hooking up this backbone with water molecules reduces its 'jiggle' room, and makes it less thermodynamically stable – making change to other conformations more probable, therefore possibly leading to more errors in the protein population.

Secondly, as detailed further in the paper, water likes to hang out with more of itself. Water molecules are happiest in foursomes, sharing four hydrogen bonds with their neighbours. When a creepy protein backbone emerges and lures an unsuspecting water molecule away into the protein's murky depths, the water molecule cannot form as many bonds with its fellows (or as many hydrogen bonds, period), and is really sad and lonely. Or, in proper terms, the system becomes less stable, since thermodynamics will favour an arrangement where these water molecules are all happily coordinated with each other, and not being molested in a corner by an amino acid polar group. In other words, exposing the polar backbone (Solvent-Accessible Backbone Hydrogen Bonds, SABHBs in the paper) to water induces what is called Protein-Water Interfacial Tension (PWIT).

One way this tension can be released and the backbone exposure ('coded for' by genes, by the way) can be compensated for is if a random other protein (or more of its own kind) are recruited to cover that exposed backbone. This would help stabilise the protein conformation, and allow this potentially deleterious drawback to be tolerated (and get fixed in the population). Ultimately, the second (and third, etc) protein can become exapted for something useful, although just an eventual dependency is good enough to make sure these proteins stick together permanently. The crazy web of interactions gets crazier.

Fernández & Lynch's fig1a suffices perfectly but I like making diagrams, so I made one anyway. See text.

(Disclaimer: I'm horrible at chemistry, this may all have been thoroughly wrong...read the paper.)

Now I'm about the last person to willingly blog about biochemistry, and this seems to have little only a distant relevance to evolution, particularly the non-adaptive kind that fascinates yours truly. It will make sense in a bit. Recall from a few seconds ago (hey, already difficult for some of us) that protein instability leads to reduced protein efficiency. This reduction is generally tolerated, however, until it's bad enough to have a higher chance of being removed. Recall from [what should be] introductory population genetics that selection acts probabilistically, with true slightly deleterious mutations have a lesser, but still significant, chance of fixation than strongly deleterious mutations, which selection has a higher chance of taking care of before drift quietly fixes it. (more detail in older post here) Since proteins are, quite unsurprisingly, also governed by fundamental principles of population genetics, drift becomes involved there too.

As populations get smaller, drift becomes a more dominant force relative to selection, and the window of 'effectively neutral' mutations – slightly beneficial and slightly deleterious, but unlikely to be dealt with by selection – increases. More mess is tolerated. This means more protein inefficiencies are allowed to fix in the population, those induced by backbone exposure among them. Since there are now more proteins that are no longer happy with themselves (or, rather, have an increased Protein-Water Interfacial Tension), they are more likely to stick together for biochemical stability. And here Constructive Neutral Evolution can come in too, allowing further deleterious mutations that are now presuppressed by the recruited proteins. In a way, this greases the presuppression process, rather than competing with it as this BBC news piece made Ford Doolittle appear to suggest.

Now, this is all great in theory, but is there any real data in support of this? For one thing, there is a clear increase of interactome (set of all interactions in an organism) complexity correlating with decrease in effective population size, suggesting a link between lax selection and accumulating complexity. Furthermore, the proteins in organisms of these smaller populations have more blistering backbone exposures to water. Supporting the relationship with population size further yet with the advantage of more phylogenetically independent events (but less interactome data), bacterial intracellular endosymbionts consistently exhibit higher protein backbone exposure (hydration) than their free-living counterparts. Selection appears to disfavour not only polar backbone exposure (also described as 'poorly wrapped proteins' in the paper), but once again, the rise of interaction complexity as a whole. (Fernández and Lynch 2011 Nature, in case you somehow managed to miss that)

Obviously I like this paper because it adds another mechanism to the arsenal of evolutionary processes happening independently of adaptation. But moreover, I don't think one can find too many examples of biochemistry mixed with population genetics. You hardly find cell and developmental biologists thinking about population genetics, and perhaps many biochemists have never even been exposed to such a subject. When fields that should never come that close together do, some really nice explosions of insight can occur (my sad attempt at chemical metaphors). We really need to talk to other more, and maybe even wander over to other departments from time to time. It's sometimes (often) frustrating to communicate with those strange ones from afar, but just like ethnic xenophobia, its interdisciplinary counterpart must also be overcome.

-----
Figure 2a annoyed me a little as it ignored phylogenetic relationships, which is a big no-no when comparing properties of taxa. The figure is technically fine, especially since there aren't any correlation analyses there, but it's hard to discount phylogenetic history as being the cause behind the correlation of the traits without actually the characters on a tree. Anyway, since I like playing with data and running statistical analyses on things, especially when I didn't actually have to go through the pain of obtaining the data myself, I mapped some characters (interactome complexity from fig2a) on a phylogeny:

Unfortunately, even the most basic statistical operations become an epic headache when trees are involved, and very quickly things become painfully complicated, for the human as well as the computer. Especially when you're handed a dataset of mixed categorical and continuous characters, as I learned the hard way last night. After fighting Mesquite for a good many hours, I finally had to resort to extracting the Ne*µ (effective pop size * mutation rate; roughly put, both lead to increased selection efficiency) estimates from Lynch & Conery 2003 – relying on an intersection of two datasets meant that our taxon sampling was quite sad by the end of this enterprise. Anyway, I ran a pairwise comparison test (Maddison 1999 J Theor Biol) on the data, which probably isn't the best thing ever, but I got something resembling significance: p = 0.019. Depending on how statistically noisy your field is, you may even deem this acceptable. In any case, not too bad given my crude (and somewhat clueless) analysis and limited taxon sampling:

Moral of the story: the inverse correlation between interactome complexity and effective population size is unlikely to be a mere artefact of shared phylogenetic history. In other words, Fernández & Lynch's hypothesis stands strong.

I mostly did this because I thought it'd take a couple hours max. If hours meant days, that wasn't too far off... but hey, I learned something!

Acknowledgments: thanks to Lucas Brouwers for helping me wade through the heavy biochemical stuff, and to Mike Lynch for explaining the key idea of the paper a while earlier. Otherwise I would've probably been too daunted to even read it, let alone blog about it...
Oh, and my Twitter people for random phylogenetics advice ;-)

Reference
Fernández, A., & Lynch, M. (2011). Non-adaptive origins of interactome complexity Nature DOI: 10.1038/nature09992

[will add some supplementary refs once I return to internet on Monday...]

Ratcheting up some splice leaders: a note on directionality

2011-05-17T05:08:00.000-07:00

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

Protistology Q&A on Reddit + gratuitous ciliate video

2011-05-09T03:17:00.000-07:00

Earlier today I felt like procrastinating a little and posted a protistology IAmA thread on Reddit (basically threads where the opening poster answers questions, titles formatted like this: I Am A XYZ, Ask Me Anything). I expected a couple questions before the thread disappears forever into the obscurity of the great internet graveyard. Shockingly enough, apparently people actually care about science or something because I was typing away non-stop at a blizzard of questions for a few hours, until now. It was quite inspiring to see the type of questions people can come up with (even the basic badly-worded ones show that at least people care enough to ask), and learned a few things along the way too. Anyway, I'll come back to answer more stuff tomorrow, but here's the thread for the curious. Feel free to stop by and ask stuff! I find that sometimes the blog comment area can be a bit intimidating if you feel you have a dumb question, so you don't ask anything. Reddit dilutes that effect, and is quite a bit more anonymous.

Protistology Q&A on Reddit

And now to randomly show off a random Haptorian ciliate – meet Litonotus, a vicious predator armed with terrifying toxicysts, which you can see as long narrow things in its cytoplasm. Also note the two prominent macronuclei visible as clear-ish round areas in the cell. Litonotus is cool and all, but the bastard preys on creatures like Euplotes, which are kind of adorable (imagine Litonotus eats kittens...that's how bad it is). Nature is red in ~~tooth~~ cytostome and ~~claw~~ cilium indeed...

MolBiol Carnival #10: Assays, cyanobacteria and metabolism regulation

2011-05-07T21:57:00.000-07:00

Welcome to the 10th edition of the MolBiol Carnival!

Apologies for the delay – am behind on pretty much everything and frantically trying to tie up loose ends of my degree, fun times. Also, it's kinda awkward to write up a carnival post with only THREE submissions – you guys really need to submit more and/or write more MolBiol posts!

It seems molecular biology doesn't get blogged about specifically as much as evolution and diversity – perhaps because molecular biologists are usually busy troubleshooting their PCRs and RNA work for weeks on end, and have little time left over to write. In fact, judging from recent woes experienced by some of my lab buddies, I'm beginning to doubt the existence of RNA and believe it may all be a giant elaborate hoax invented to enslave more grad students. Have any of you ever *seen* RNA? That's what I thought...

This month we have a very biochemical (post-translational, if you will) MolBiol Carnival featuring enzyme spec, cyanobacterial biofuel precursors and some sweet diastereomer metabolism regulation.

Enzyme Assay
Christopher Dieni at BitesizeBio has a nice write-up on measuring enzyme kinetics using UV spectrophotometry, complete with procedure, tips and troubleshooting – the kind of thing you wish accompanied every assay you've been assaulted by. Not being anything close to a biochemist, I had no idea you could actually observe enzyme action using something as simple as a spec, so this is quite cool!

Cyanobacteria and biofuel production
With growing concerns with using land plants for biofuels (for one thing, kind of odd to use food to power cars when not everyone has enough of it...), increasing attention has been turned towards algae eukaryotic and not. For one thing, algae are already quite good at photosynthesising and are vastly more abundant than plants, and arguably have the largest contribution to global photosynthesis – not surprising given the earth's surface is 70% ocean. Michael Scott Long at a NASW.org blog explains recent developments in genetic engineering and domestication of cyanobacteria for fatty acid production.

Diastereomers and regulation of metabolism
Stereoisomers are the beginning chemistry student's worst nightmare – they're so similar and easy to mix up, particularly if you're like me and can't tell left from right to begin with. However, a bacterium (rather, its enzymes) would have little trouble with the stereochemistry portion of a intro biochem class – to them, stereoisomers are day and night (and other things). Glucose and galactose are 'close enough' to each other for a biochem student, but a flipped arrangement at just a single stereocentre is enough to require a whole new set of enzymes and drastic changes in the pathway. E.coli prefers glucose, but can also process galactose (compromising its growth rate) by embellishing its metabolic pathways a little – the products of galactose digestion are sent to the tricarboxylic acid cycle via the glycoxylate shunt. Becky Ward at It Takes 30 discusses how sugar type availability affects the transcriptional regulation of this glycoxylate shunt, among other things, featuring a galactose-loving mutant.

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This was fun. Wish there were more submissions – having to write up random blog posts forces me to revisit forgotten subjects and explore new ones: I'd never brave a post on metabolic regulation on my own! By not submitting, y'all are having a deleterious effect on my education... ;-)

The next edition will be hosted by our resident microbiologist @labratting at Lab Rat, and she better get more than three submissions... come on, we do so much molecular biology in almost every field of biology! Write 'er up, dammit!

Protists gone motile! (and a Euglenid metaboly video)

2011-05-02T01:07:00.000-07:00

So I caved and got me a Youtube account, partly inspired by a comment in the previous post. Accumulated half a metric shit ton of random protist videos by now, and compressing them for Bloggers crappy video sharing system would take way too much time, and I barely have the time to grab stills and post them here. So finally there's a suitable outlet for my raw video data – maybe someday when I'm not going completely insane and falling behind on a million things I didn't really have time for in the first place, I may put together a properly edited video. But don't hold your breath for it...

We have some pretty awesome microscopy and video equipment in the lab, and I'm lucky to have a PI nice enough not to mind some of us using it to fuck around with random samples in the middle of the night. I hope it may help bring the microbial world a little closer to you, and add a whole new dimension of time to our protists.

Let's start off with some euglenid metaboly, since it's quite hard to talk about without seeing it. Actually, the true reason is that it's about the first thing my cursor landed on when I opened my pile of videos for file conversion. But just as we ascribe purpose to evolutionary happenings, we can likewise ascribe purpose to my selection here ;-)

Since I'm lazy and behind on about a million things (to the point where I must mention it twice), just gonna copy the short description I wrote for this bug on the YouTube page. Enjoy!

This is a heterotrophic euglenid, perhaps a Peranema sp., exhibiting metaboly in all its splendour. The cell might be slightly squashed or otherwise damaged, keeping the flagellate conveniently in one place. The clear vesicle near the base of the flagellum that grows and shrinks is the contractile vacuole, the flagellate's analogue of the animal secretory system. At the tail end are refractile starch granules used to store energy.

Metaboly is a form of cell movement that is most famously exemplified by ciliates, but also known in some other flagellates. It appears to be caused by the specific arrangement of microtubule (cell skeleton) bundles at the cell periphery, and greatly enhanced by the 'armour plates' of the euglenid surface, which is lined with long pellicle strips going from the flagellar insertion all the way to the tip of the 'tail' -- as the cell twists about, the strips slide against each other and result in this movement. Euglenids with fused pellicle strips, like Phacus, are incapable of metaboly. The function of this movement is unknown, and there may not be any in particular.

The hairy thing next to the euglenid is a badly mangled ciliate.

Freshwater, Apr 2011, Vancouver

And please let me know if you have any requests, comments or suggestions for these videos. I'm new to the world of moving pictures (instead I see videos as image sequences, like any proper cell biologist ought to...), so I'm in an even greater state than usual in not knowing what the hell I'm doing.

Marine Microforay – foram and a thecofilosean party

2011-04-27T03:09:00.000-07:00

Apologies for disappearing for a while – had an interview, finals and then my arm decided to temporarily rediscover RSI-like symptoms just when I had a term paper to write, so I had to lay off extraneous typing for a while. Then I realised just how much of my life depends on typing, and losing that ability would not only make me worthless and unemployable, but also unable to communicate with many of my friends who happen to be inconveniently dispersed around the globe. So yeah, I should probably stop casually dismissing ergonomics about now...as should you, if you haven't already!

I've accumulated another batch of microscopic findings, this time from marine samples. By the looks of it, I might be moving to the Midwest soon, and thus be deprived of my ocean (and mountains *sob*), so I figured that focusing on marine protists while I have the chance would be a good idea. Swampy pondwater is available pretty much anywhere anyway.

From time to time, you can be lucky enough to find a foram shell in the sediments around here. Live forams can be found too, but much more rarely – I have a couple, but still need to process the videos. This is not a snail:

Foram. Wreck Beach. 20x obj, DIC except for last image, which is in phase.

To save loading time, the rest are below the fold.

A pennate diatom of some sort, on its side. The girdle, where the two valves ('shells') meet, can be seen along the middle.

40x obj, DIC

A flagellate of some sort. Has a strange groove from which flagella appear to emerge (see bottom second from left). Looks like some sort of thecofilosean/cryomonad like Protaspis, but might be some heterotrophic euglenid for all I know.

A marine Thecamoeba-like thing: (as suggested by ridges on the surface)

The partial collar and flagellar pocket suggest euglenidness. Entosiphon sp, perhaps? Eats diatoms. Short flagella perplex me though; I vaguely recall having searched long and hard for their continuation, but they appeared truly short despite that.

And another individual, presumably of the same species or closely related.

This might be a cercomonad that ate something colourful and refractile. I recall it moved in a rather cercomonad-y fashion. Glides like hell.

An obvious thecofilosean. Verrucomonas or Protaspis; leaning towards Verrucomonas based on position of the massive nucleus (see top right) and how intensely the ventral groove bisects the cell; not sure how reliable those characters are. Looks like fig 1n in Chantangsi et al. 2010 Mol Phyl Evol, which is Verrucomonas longifila (also fig 1h and 3h in Chantangsi & Leander 2010 IJSEM). Can't say much else except that it's a gliding flagellate with a tendency to protrude fine pseudopodia from the groove along its bottom. Thecofiloseans aren't particularly overstudied or anything...

Another thecofilosean with colourful refractile inclusions – probably food-related (characteristic groove evident in rightmost image).

A Lecythium-like creature. Lecythium is a cercozoan organic-test-bearing amoeba with thin filose pseudopods. Plenty found in freshwater too.

A...thing. Appears to have colourful food vacuoles or something. In the top right image there's a barely visible groove edge of some sort along the right edge, with a kink too. Anyone offended if I write this off as a marine cercozoan, eg. cryomonad of some sort?

Metromonas simplex. It anchors to the substrate with the curly end of its flagellum and sways back and forth rhythmically, for hours on end. Back and forth, back and forth. Like a metronome, hence its name. Apparently it's got nothing better to do. Intend to upload some videos eventually... it's really cool! Uncertain affinity at this point, despite how quickly these guys are found everywhere after leaving some benthic seawater in a dish. Thinking I could quickly yoink a couple off the dish and PCR them to resolve the issue with little effort, I very quickly found out why no one's ever bothered and succeeded: these things are practically impossibly to pry off the surface! I think they've invented superglue or something...

Part of a decaying arthropod. Spherical things might be parasites/cysts, or some animal cell type I'm totally oblivious too. Wish there was an easy way to learn what the various cell types look like in arthropods – most introductory literature is tissue-oriented. I'm perplexed by the surface of the spherical things visible in the top right and bottom left images.

20x obj, DIC

The rest are from a different marine sample, captured by video camera.

This ciliate appears to be related to Paramecium based on the shape of its oral region, as well as the rectangular grid-like arrangement of the cortex. In the bottom middle image, the macronucleus is apparent as a clear-ish area in the cytoplasm. I think the top right features a contractile vacuole with its channel towards the left end.

Top two: 20x obj; bottom: 40x. DIC

Heterotrophic euglenid, or a cercozoan of some sort. Probably the former as there seem to be visible edges of pellicle strips in the top right image. The flagellar pocket also points towards a euglenid identity, though the flagella are a bit short and on the thin side. Could be out of focus though.

Discocelis saleuta, a common marine flagellate of uncertain affinity. The disc shape is very characteristic and cool, would probably make an interesting SEM subject. Apparently the cell edge can be lined with extrusomes (Vørs 1988 ref'd in Walker et al. 2011 Parasitol S1:88); given the flagellate is a mere 5µm, it's hard to imagine how those could even fit in there.

That was probably way too many images...

Adorable velvet worm composite

2011-04-12T06:41:00.000-07:00

Too busy to write a proper post these days, but just happened across a cool Current Biology quick guide to onychophorans(=velvet worms), with a pretty picture showing their diversity (and really pretty textile-like patterns):

Australian onychophorans (Blaxter & Sunnucks 2011 Curr Biol)

The guide itself is quite interesting, recommend reading it if you have time. To entice you, they talk about the diversity of breeding behaviours found in onychophorans:

"Some are fully live-bearing (viviparous), with well-developed placenta-like, extra-embryonic structures that attach to the mother's uterine wall and nourishes growing embryos until the birth of sequences of self-reliant, mini velvet worms."

Onychophorans are way cooler than arthropods ;p

And they can be social with dominant/submissive behaviours, which I talked about in an earlier post (which happens to be one of my most visited, probably because it's not about protists =( ).

And with that, there may or may not be a surprise while I'm away, so stay tuned. In any case, I should engage in some form of proper blogging sometime after the 20th... (finals, shoot me)

Personal army of diplomonads (doodle)

2011-04-08T05:46:00.000-07:00

Accidentally discovered the Symbols tool in Illustrator, and had a little too much fun creating a personal army of swirling multi-coloured diplomonads:
They really remind me of diodes. Incidentally, they can also invade diagrams and make them barely legible:
Aren't you glad I haven't gotten around to making cartoon-y spiders and cockroaches yet?

In other news, I'm rather swamped for the next week and a half (as if I wasn't before), as laws of the universe mandate that right between classes and finals not only do you end up with a [potentially awesome] trip across the continent but a particular obscure somewhat rare flagellate you've been searching for throughout the past 5 months or so randomly decides to announce itself unexpectedly. Not only are protists sentient and exceptionally intelligent, the sly little bastards are also evil as fuck.

I do have a couple posts in the making, but don't guarantee anything until after the 20th (this includes replying to comments and emails too)...

May this round of finals be my last...! For this degree anyway...

RQ#03 Is there really a non-natural selection?

2011-04-06T02:02:00.000-07:00

I haven't done a random question in a while. This is the third one, apparently.

A grocery store still life, primarily Brassica oleracae

Lately I've been involved in some fairly theoretically discussions about evolution, which tend to push one to pay more attention to terminological precision. ~~Or get very confused~~ And get very confused regardless. Additionally, I hang around some biologists with minority opinions on certain aspects of evolution, and ultimately end up talking about evolution differently, to the point of using different words or same words differently. The usual side effects of specialisation. This becomes particularly evident in heated argument with someone outside your tribe – you start speaking slightly different dialects, if you will. Of course, where there's variation, there's opportunity to pick the variant that suits you better. Ideally, that has something to do with accuracy, since we are, hopefully, still attempting to do science and what-not.

Let's start with the easier of the usage and terminology discrepancies – the term 'natural selection'. Is it useful or does the simpler 'selection' make it redundant? I tend to drop the 'natural' part; laziness and word limits may help, but I think there may be valid theoretical or philosophical merit in doing so:

1. 'Natural selection' was initially proposed in contrast to 'artificial selection', which was used as an effective pedagogical/explanatory move. It got the point across, particularly in an age when humans were unquestionably special and distinct from the natural world. Nowadays, few scientists would seriously make a distinction between human and non-human nature in the context of biology, and thus there really is no artificial selection per se. 'Artificial selection' is 'natural selection' performed by humans to pressure their organisms towards traits the humans find favourable. In this case, the humans are part of the environment, playing a similar role to predators, except they breed the variants they like instead of instantly culling them. With no need for an 'artificial selection', is there still a need for 'natural selection', since there no longer is a valid contrast?

2. 'Natural selection' is often equated with adaptation. This isn't to say 'selection' by itself isn't, but 'natural selection' is the variant used most often in popular writing, some of which can be careless and inconsistent with its terminology. While presumably many of the authors do truly understand that selection and adaptation are different things, adaptationism has led some to consider the difference irrelevant. If adaptation is the sole phenomenon responsible for all the observable or cool things in biology, does it really matter if it's used interchangeably with natural selection? When a term is learned and frequently used incorrectly, it is extremely difficult to fix even in an individual, let alone a population. While 'natural selection' is not meant to be conflated with adaptation, it is, and has thus been tainted.

3. Use of 'natural selection' implies that phenomena like sexual selection and kin selection are somehow distinct, or special. These are secondary phenomena, special cases or manifestations of selection. That is, sex and kin selection are subtypes of 'natural selection' and do not lie on equal hierarchical level as it may first seem. While most of the scientific community has no problems understanding this, it is perhaps not the clearest delineation of the terms for the general public or students. This way, we can also keep 'artificial selection' to refer to domestication (although I don't see the necessity in doing so) without it contrasting with the 'natural' kind.

4. This is the least important point, but rather a more personal one. I dislike Darwin-worship; I'm not a 'Darwinian' (nor a "Neo-Darwinian), don't know what that means and frankly don't consider this question relevant now, over a century after Darwin's death. While history of science is indeed fascinating and undeniably worthwhile to learn about, we shouldn't trap ourselves in our history. In fact, I think equating evolution with Darwinism is a bit offensive to all the hard work and frustration of subsequent researchers that have contributed to the field – do they not matter? They work for evolution, not Darwin. 'Natural selection' has been too often tightly associated with 'Darwinism', and often plays a part in Darwin-worship. In other words, the term has acquired some baggage; mind you, not through Darwin but rather through his fervent supporters afterwards.

5. Population geneticists seem perfectly happy with just 'selection'. They're the ones who actually study the mechanisms of this stuff, so if it works for them, perhaps it should be adequate for the rest of us?

I don't mean to nitpick on words and 'mere semantics', but given the difficulty of conveying ideas to those outside your field and the general public, any site of potential confusion is worth trimming if we can. Those on the writing end are also prone to sloppiness and mistakes, so we too are susceptible to the confusion potential. That said, 'natural selection' has stuck around for this long – perhaps there is a beneficial reason I missed out on? This is an honest question – I've never really been formally trained in evolutionary biology save for a basic first year level, and may thus miss large chunks of theory. As I mentioned before, I'm being 'brought up' in some minority circles of evolutionary thought.

Why should we still use 'natural selection'?
Your turn. Just be gentle with the philosophy – I'm rather slow at following complicated abstract theoretical discussions, which is why I do experimental science ;-)

"Just another ciliate" – importance of sexy descriptions

2011-04-04T04:20:00.000-07:00

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

Dermamoeba – Having your coat and eating it too

2011-03-28T03:27:00.000-07:00

We've been neglecting the micro-squishies lately (filose amoebae ain't proper squishies – too many fine protrusions in the way). Amoebozoa is a eukaryotic supergroup comprised of predominantly lobose amoebae, meaning their pseudopods are rounded and not fine and pointy (like those in the preceding post's organism – Filoreta). Aside from the test-bearing Arcellinids, amoebozoans tend to be naked amoebae ('gymnamoebae'), like the well-known Amoeba proteus, often erroneously referred to as a 'primitive', 'simple' or 'ancient' organism. "Naked amoeba" is a bit of a misnomer – while they don't lug rocks and heavy dishware around like testate amoebae, they generally carry some sort of cover, as most cells do. Gymnamoebae just pack light. Some, like Cochliopodium, dress themselves in intricate scales, while others, like many Vannellids, are covered in thin, pointy glycostyles. Dermamoeba, in turn, wears a thick, heavy coat.

5-8 Dermamoeba going about its business (n – nucleus, cv – contractile vacuole). 9 – Dermamoeba lounging about in cysts (c) upon devouring some algae (chain-forming diatom or some Trebonema-like thing). Nom nom nom. (Smirnov et al. 2011 EJP)

Dermamoeba's fine coat consists of thick bi-layered glycocalyx (a covering of fluffy sugar-proteins), sometimes with additional 'dense matter' lining the cell membrane. Upon encystation, an extra layer, the cell wall, is formed, but the contraption is thick enough without it already, at about half a micron.

EM sections through the intense Dermamoeba cell coat. m – cell membrane, gl – glycocalyx, adm – 'arrangement of dense material' (ie, "we don't know"). The glycocalyx often forms pretty patterns when sectioned. (15 is part of a Golgi body) (Smirnov et al. 2011 EJP)

This thick coat poses some problems of its own. Amoebae eat by engulfing prey with their pseudopods – and this involves some degree of nudity and cell membrane exposure. Half a micron of glycocalyx wouldn't be particularly flexible, and and not much fun to digest. Dermamoeba has to nibble on its coat before the meal. Upon contacting prey (typically algae), the amoeba forms a concave food cup around it, from the centre of which the cell coat gradually disappears. As the food cup deepens, the prey is pulled in to meet its doom via thick bundles of actin microfilaments spanning much of the cell – another unusual feature of this process. The prey is consequently engulfed for eventual digestion. As a result, the prey-containing vacuole has no glycocalyx for the amoeba to choke on (or rather, presumably, waste energy digesting).

Diagram of Dermamoeba's unusual feeding procedure. After the algal prey (al) is contacted by the amoeba (am), the glycocalyx (gl) is digested and the prey is drawn in by thick actin microfilament bundles (mf). The resulting food vacuole (fv) is conveniently devoid of coat material. (Smirnov et al. 2011 EJP)

And here the food cup is 'live', or was before some electron microscopist brutally murdered it in osmic acid and sliced it up:

EM sections through prey (al) being engulfed by the amoeba (am). Note the disappearance of the glycocalyx (gl) at the centre of the invagination. (Smirnov et al. 2011 EJP)

How do some of the other coat-bearing amoebae get around their irremovable clothing? Without going into much detail (amoebozoan surface coverings are really cool...), the glycostyle-bearing Pellita simple sticks small 'subpseudopodia' through it for both moving about and feeding. In fact, some propose that the glycostyles may help it move by reducing the surface area in contact with the substrate – keeping the sticky cell membrane away on stilts.

Top left: Pellita walking on stilts of glycostyles (depicted at the right). Bottom: extruding sub-feet across stilts for feeding. (Smirnov & Kudryavtsev 2005 EJP)

I'm decidedly avoiding amoebozoan systematics here. Christopher Taylor did a nice overview of it at the Catalogue of Organisms a while back, but keep in mind that some of the groups did jump around since then, and the phylogenies are in the works. Maybe if more people cared, the taxonomy could be resolved sooner...

PS: My committee* has voted to remove "Sunday Protist" from Sunday Protist titles, since:
a) They seldom come out on Sundays anyway (lulz); and
b) Takes up too much valuable headline real estate. Since we bloggers are supposedly playing pseudo-journalists or something, might as well play it right... ;-)
(and c) Structure and I aren't the best of friends.)

* Given how inefficient my brain is at accomplishing anything, I've concluded it can only be composed of a close neural approximation of a committee. Explains the indecisiveness as well. Probably requires a double majority to pass any major decisions, and hence is about as effective as the Californian government. Without the sovereign debt crisis, fortunately.

References
SMIRNOV, A., & KUDRYAVTSEV, A. (2005). Pellitidae n. fam. (Lobosea, Gymnamoebia) – a new family, accommodating two amoebae with an unusual cell coat and an original mode of locomotion, n.g., n.sp. and comb. nov European Journal of Protistology, 41 (4), 257-267 DOI: 10.1016/j.ejop.2005.05.002

Smirnov AV, Bedjagina OM, & Goodkov AV (2011). Dermamoeba algensis n. sp. (Amoebozoa, Dermamoebidae) – An algivorous lobose amoeba with complex cell coat and unusual feeding mode European Journal of Protistology : 10.1016/j.ejop.2010.12.002

Reticulose amoeba: cells can be fine nets too

2011-03-24T03:39:00.000-07:00

Again, the protist kingdom is a special paradise for a cell biologist: as soon as one steps outside the plant and animal kingdoms (and yeast), diversity of cellular forms and structures explodes beyond reason. Cells can also take the shape of a fine net with no obvious cell body proper:

Cover slip floated ~ 1wk on marine sample from intertidal silt at Stanley Park. (40x obj, DIC and phase, resp.)
EDIT: Confirmed Filoreta.

Almost overlooked it thinking it was just slide gunk. Amoebae suffer all too often from that fate – apparently Parvamoeba, one of the most common and ubiquitous amoebae, was only described in the early 90's (Rogerson 1993 EJP) because it was tiny and no one noticed.

Could be something like Filoreta sp. (Rhizarian), but something feels off about it. Filoreta doesn't seem to stretch cytoplasm between filopodia like this specimen does. Maybe it's more like the amoebozoan Corallomyxa and Stereomyxa, or stramenopile Leukarachnion. Then again, amoebae are notoriously dynamic in their morphology. Something that's a far bigger issue in the microbial world is the necessity of getting a sense of the morphotype range of a species; one specimen doesn't quite cut it as it does for animal taxonomy.

In fact, perhaps instead of the ridiculious (for us) ICZN and ICBN requirements for submission of material for curation (many species neither like being cultured nor preserve all that well on a slide), for microbial species there should be a requirement for additional images of different specimens, if possible, to try to capture some of the morphological range. But then again, I'm not a taxonomist, so what do I know.

Right, midterm... (hey, at least I procrastinate productively!)

Mystery Micrograph #27

2011-03-23T04:27:00.000-07:00

Busy week here, so have a mystery micrograph:

To be referenced later. Won't reveal the scale yet.

Kekeke. *evil grin*
Anyway, I have a "mid"term, almost two weeks before the term ends. Took the art of neglecting coursework to a whole new dimension this term – incredibly difficult to give a fuck in term 2 of year 5. Lots of catching up to do...

Trypanosomatid plastids and uninentional scientific comedy

2011-03-17T00:58:00.000-07:00

One need not read past the abstract:

"It is usually assumed that the trypanosomatid plastid shared a common origin with that of euglenids, but Δ4 desaturase phylogenies suggest that it could have originated via an independent, tertiary endosymbiosis involving a haptophyte alga. It is also possible that ancestors of the Trypanosomatidae initially possessed a primary plastid that later was replaced by a secondary or tertiary plastid." – Bodyl et al 2010 J Parasitol (pdf)

I could go on for many, many pages about the implausibility of most entirely unnecessary serial plastid symbiosis theories; I could go on for pages yet on how little a single gene phylogeny means these days; I could go over the typical first few lectures on phylogenetic reconstruction and the fundamental principle of parsimony. But instead, I've quickly thrown together a diagram highlighting the KEY problem with Bodyl et al.'s hypothesis:

Taxa in black – non-photosynthetic and non-plastid-bearing.

Trypanosomes don't have plastids.

Or any reason to suspect they might.

*to be fair, they are (I hope) talking about a plastid in their ancestry, but those things are seldom lost completely due to inevitable strong dependencies.

In fact, unlike apicomplexans, trypanosomes are nested firmly within a completely non-photosynthetic phylum in a predominantly non-photosynthetic subgroup of an almost-exclusively non-photosynthetic supergroup. Furthermore, the many possible phylogenies of euglenid evolution overwhelmingly support a single symbiotic event; character evolution supports this further, in one of the few cases where there's actually little room for dispute. Endosymbiosis may not be excessively rare, but it ain't common either, particularly in a full-fledged form involving vast transfer of plastid genes to the nucleus AND mechanisms of plastid targeting of the synthesised proteins. Too many an ambitious biologist completely forget about targeting, or that there's actual cell biology happening around their beloved gene sequences.

For a properly scientific and civil demolition of an earlier iteration of this ridiculous idea, see Leander 2004 Tr Microbiol (pdf). That smell of something burning? No need to worry – probably just coming from the link.

Lastly, as ridiculous as this hypothesis is and as amusing as it is that this actually survived peer review (no offense to J Parasitol, but phylogenies and evolution do not seem to be their strong point based on some other cases...), I fully support it being published. It is the excessive censorship of atypical theories rather than sketchy papers that "stiffles [scientific] thought"...

(Note: I would've submitted this to the high IF Journal of Are You Fucking Kidding, but I'm out of hard liquor and would thereby fail the author instructions...)

References
Bodył, A., Mackiewicz, P., & Milanowski, R. (2010). Did Trypanosomatid Parasites Contain a Eukaryotic Alga–Derived Plastid in Their Evolutionary Past? Journal of Parasitology, 96 (2), 465-475 DOI: 10.1645/GE-1810.1

LEANDER, B. (2004). Did trypanosomatid parasites have photosynthetic ancestors? Trends in Microbiology, 12 (6), 251-258 DOI: 10.1016/j.tim.2004.04.001

Out in the field: freshwater microforay picture dump

2011-03-16T15:50:00.000-07:00

I've probably accumulated about 10-20GB of protist pics by now. And a couple DV tapes' worth of video. Still got some work to do before I can catch up with my 80GB of Arabidopsis epidermis pictures (mostly of all sorts of mutilated stomata), but this is for fun rather than data, and thus accumulates much slower. Most of them are crap or uninteresting by now, but the others might as well get dumped here as raw data from 'field work'. The wonderful thing about microscopy is that the more you know, the more new things you observe, and the more interesting it gets. Eg. once you're no longer distracted by trying to identify unknown things, you pay more attention to behaviour. Anyway, I'll dump the photos in random installments here and there, hope there's at least something interesting for you from time to time.

To begin, a fuzzy ciliate of sorts. Prominent contractile vacuole, and I think in the left image I think you can make out its macronucleus or two. Can't see the mouth so IDing it is a bit difficult.

[to shave off some page loading time, the rest is below the jump (if it works)]

This one's got a big mouth. Seems to be Lembadion sp., an oligohymenophoran ciliate. Guessing the yellowish particles are prey or food storage of some sort.

Epalxellid of sorts, perhaps of the genus Epalxella itself:

What appeared to be a developmentally-'unique' double stentor; or one in the process of division that remained attached. Actually, may well be division, didn't think they'd keep filtering away in the midst of cytokinesis:

Goniomonas, the non-photosynthetic sister group to plastid-bearing cryptomonads:

Diplophrys, a stramenopile with a conspicuous lipid(?) droplet and very fine filopodia.

View of surface pellicle strips on some photosynthetic euglenid:

Some sort of glissomonad, I think. Looks hatchet/pike-like, would totally name it something silly like Hatchetomonas were it undescribed...

Some filose amoeba with seemingly eruptive lobopodia in addition to thin filopodia. Can nucleariids (amoeboid sister group to fungi) do this? Though the prominent nucleus at its centre points towards nucleariidness:

An amoeba with branching filopodia. Quite vacuolated. Fucked if I know what it is. Some sort of vampyrellid? (eg vaguely like this)

Nucleariid like the previously mentioned Rhabdiophrys? Or vampyrellid? Damn, identifying amoebae was way easier when I knew less of them...

Some small round thing that liked to wrap its [attached-to-substrate] flagellum around itself. Ummm, that sort of made sense, right? Don't think I'm allowed to ever write species descriptions for some reason...

"You really like amoebae" I was told the other week. Yes I do, and FYI, someone's chlorarachniophytes are amoebae too. All that said, I remain abysmal at identifying the buggers; this, my friends, is some filosey amoeba of sorts. Yup.

The following is from an anoxic rotting leaf sample (same as in the previous Trimastix post) – a very long and narrow cell, appears to be a euglenid based on how it moves the free flagellum (tip-heavy action); it's attached to the glass with the other flagellum. Guessing it's a Heteronema sp.; appears similar to H.acutissimum, but the latter is marine and for euglenids, the marine vs. freshwater distinction is generally considered sufficient to be a different species.

And now I'm off too look at more samples. Will take a while to go through my stockpile of protist pics already, but must still get more...

Sunday Protist – Trimastix marina

2011-03-15T03:20:00.000-07:00

Before we begin, two things about [current] Trimastix marina – it has four flagella (not three) and is found in freshwater. The taxonomic author, Saville-Kent, is a bit notorious for some rather sketchy descriptions, and Trimastix is one of his 'trophies'. That said, it may be that Kent did actually see a three-flagellated and/or marine thing like this and it just hasn't been found or published yet. But for the time being, feel free to point and laugh at the double misnomer.

This past fall I dumped a bunch of leaves in a dish and kept them wet for a while. Turns out, the abundance and diversity of microbes and meiofauna thriving in that pile of dead leaves in your yard is quite amazing – all sorts of ciliates, myxomycetes (slime moulds), tardigrades, rotifers, springtails, flagellates, amoebae – you name it. Some of this world can be seen with a simple dissecting scope; it helps to put a coverslip or some other piece of glass on the wet leaves to see better. This coverslip is also great for investigating what lives on the surface of the rotting leaves. The other impressive detail was how quickly the leaf tissues rot away, after a couple months leaving little more than the bare skeleton of the vascular system. Dead leaves are the whale falls of the terrestrial microbiome.

Rotting tissues tend to have relatively low oxygen concentrations, and thus host some unique organisms. Among them was this peculiar flagellate that simply screamed "EXCAVATE" at the top of its lungs, but I couldn't quite figure out what it was:

Trimastix marina. The cell body is about 25-30µm, with a prominent anterior flagellum sticking out in front, and three smaller flagella trailing behind. The nucleus is the little blob at the very anterior tip of the cell, in front of a large circular food vacuole. At the very posterior tip is the contractile vacuole characteristic of freshwater things in general. Along the side of the cell is an exceptionally conspicuous groove, through which one of the recurrent flagella runs – a characteristic feature of excavates. Anoxic, leaf litter moistened with ample water for a couple of weeks. 40x obj, DIC

The part that screamed "EXCAVATE" at me was the distinctive groove (namesake of the supergroup) along the side of the cell. You can often discern it in other excavates like Jakobids, Retortamonads and Carpediemonas-like organisms (CLOs; hey, it beats "Clade B"...), but here you don't even have to look hard. Curiously, the closely related oxymonads (see Streblo, Saccinobaculus) seem to have lost the groove, but that's another story.

Overview of 'basic' excavate cell types. Trimastix marina is the very distinctive one in the bottom middle. There's something distinctive and cute about its thick anterior flagellum and the way it moves. (Simpson et al. 2002 JEM)

Thus far, Trimastix may seem like your garden variety peculiar flagellate. But there's something universally eukaryotic you might have difficulty finding – a proper mitochondrion.

I mentioned earlier the sample was somewhat anoxic. It wasn't irrelevant, because I've never seen anything like this critter in regular pondwater or well-aerated soil. Like many of its excavate relatives, Trimastix has lost the necessity to maintain the elaborate complexity of aerobic pathways and their accompanying structures, like cristae. Furthermore, it lacks a mitochondrial genome. This led to the conclusion that Trimastix lacks anything mitochondrial altogether, and may have diverged prior to mitochondrial endosymbiosis – a perfectly reasonable assumption given the data at the time. This landed Trimastix (along with the better-known sister Oxymonads) a position in then-subkingdom/phylum Archezoa (Cavalier-Smith 1983)
[NB: Archezoa = 'beginning/early animals', not ArchaEzoa, which would be 'ancient animals'. He seems particular about that.]

Trimastix wasn't a major player in the Archezoa Hypothesis (wherein 'amitochondriate' lineages are contemporary representatives of pre-endosymbiotic eukaryotes) since it's rather obscure, but was still a piece of the puzzle. Eventually, better phylogenetic techniques and improved taxon sampling destroyed the Archezoa Hypothesis, particularly as mitochondrial genes and derived organelles (such as mitosomes and hydrogenosomes) were found. Trimastix's mitochondrial genes were found later than those of other anaerobes, perhaps owing to its obscurity – but they're there: mitochondrion-targetting genes in the nuclear genome (Hampl et al. 2008 PLoS ONE). Furthermore, the aftermath of mitochondrial reduction looks like a generic double-membrane bound blob in electron micrographs (Hampl & Simpson 2008 in Hydrogenosomes and Mitosomes: Mitochondria of Anaerobic Eukaryotes) – no wonder it was so hard to find!

All that's left of Trimastix's mitochondrion, as the eons of anaerobic existence devoured its need to maintain one. It is uncertain whether it produces hydrogen gas – which would make it a hydrogenosome rather than a mitosome – though at least some of the necessary genes seem to be present in the nuclear genome. (Hampl & Simpson 2008)

As an aside, there's no known case yet of a reduced mitochondrion that simply disappeared – in addition to aerobic respiration, eukaryotes have also become dependent upon it for some other vital metabolic pathways, such as those involving the Fe-S cluster. In fact, in at least one species of microsporidia, ATP is imported into the mitochondrial relic in order to keep the key metabolic pathways running. (I vaguely recall having written about this before, somewhere...)

Lastly, Trimastix is host to some lateral gene transfer for its glycolytic pathway – it appears to have picked up and replaced at least four of the eukaryotic genes with bacterial versions (Stechmann et al. 2006 BMC Evol Biol). There was a discussion somewhere on the blogosphere lately (Coyne's blog, IIRC) about the relative importance of LGT – it sure as hell does happen in eukaryotes as well, though not crazy enough to wreak havoc on the phylogenies.

And the rain hasn't stopped yet. But I can't skip a second night of sleep... as much as I'd love to keep blogging about stuff.

References
Hampl, V., Silberman, J., Stechmann, A., Diaz-Triviño, S., Johnson, P., & Roger, A. (2008). Genetic Evidence for a Mitochondriate Ancestry in the ‘Amitochondriate’ Flagellate Trimastix pyriformis PLoS ONE, 3 (1) DOI: 10.1371/journal.pone.0001383

Hampl, V, & Simpson, AGB (2008). Possible Mitochondria-Related Organelles in Poorly-Studied “Amitochondriate” Eukaryotes HYDROGENOSOMES AND MITOSOMES: MITOCHONDRIA OF ANAEROBIC EUKARYOTES DOI: 10.1007/7171_2007_107

SIMPSON, A., RADEK, R., DACKS, J., & O'KELLY, C. (2002). How Oxymonads Lost Their Groove: An Ultrastructural Comparison of Monocercomonoides and Excavate Taxa The Journal of Eukaryotic Microbiology, 49 (3), 239-248 DOI: 10.1111/j.1550-7408.2002.tb00529.x

Stechmann, A., Baumgartner, M., Silberman, J., & Roger, A. (2006). The glycolytic pathway of Trimastix pyriformis is an evolutionary mosaic BMC Evolutionary Biology, 6 (1) DOI: 10.1186/1471-2148-6-101

Cryptomonads: solar-powered armoured battleships

2011-03-02T02:32:00.000-08:00

I've been 'scoping around some pond water lately and came across some relatively big cryptomonads (g. Cryptomonas, I think). Cryptos aren't all that rare, but most of them whirl about rather hyperactively, rendering them as troublesome photo subjects. This specimen, on the other hand, had a convenient habit of pausing every once in a while to have its picture taken. Finally, I have my own cryptomonad shots!

Cryptomonas(?) sp. The cell is about ~30µm long, pretty big for a cryptomonad. On its right side the cryptomonad has a furrow – or, in some species, an tube-like gullet – lined with ejectisomes (particularly visible in the top right image). The vesicle at the anterior tip of the cell is its contractile vacuole. Refractile stuff is the starch granules. 40x objective, DIC

Despite their small size and superficially generic algal appearance, cryptomonads do have quite a few awesome bits about them. From an evolutionary standpoint, they have pretty damn awesome plastids – products of secondary endosymbiosis of red algae, complete with a shrunken relict nucleus ("nucleomorph") of the red algal ex-host! The plastids also have four membranes, complicating the delivery of plastid-targetting proteins from the cryptomonad host nucleus. But I'll save that story for some other time, and instead keep it superficial. Literally: it has ejectile things lining its surface, and who doesn't like the idea of a microscopic solar-powered hyperactive battleship?

Prior to embarking on some battle scenes, lets look around the ship's anatomy a little bit ~~mostly as an excuse to show off a diagram~~. At its fore we have a pair of flagella, lined with little hairs – also a characteristic of many Alveolates and Stramenopiles, with whom Cryptomonads might share the secondary red algal symbiosis event with. Much of the cell is occupied with a single plastid, making the fucker a bit difficult to diagram. In all his/her/its infinite wisdom, the designer apparently failed to take into consideration the future pains of this student attempting to tame the wild beast that is Illustrator while drawing this cell. Asshole. Besides the plastid, there's also a single mitochondrion and a bunch of other small crap that a eukaryote ought to have. The plastid's outermost (fourth) membrane is contiguous with the endoplasmic reticulum system, presumably homologous to the original digestive vesicle that enveloped the 'enslaved'* red alga. The third membrane derives from the red algal cell membrane, whereas the inner pair are the usual plastid membranes. Pop quiz: where would you expect to find the relict endosymbiont's nucleus (the nucleomorph)? (Answer at the bottom of the post, or in the diagram if you're so inclined to 'cheat' ;p)

*Google [scholar] "Cavalier-Smith" and "enslaved". When he likes certain words, he really likes them.

Back to the surface. The cryptomonad surface is quite complex, consisting of an inner and surface periplast layers separated by the cell membrane. Sometimes the surface layer can be be covered in scales, sometimes fibrous matter. This periplast is perforated with pores for ejectisomes, much like battlements on a warship. Ejectisomes themselves consist of coiled proteinaceous ribbons that extend forcefully upon firing.

Cryptomonad periplast. IPC – inner periplast layer, PM – plasma membrane, S – scales (of the surface periplast layer). On the right is a freeze fracture EM of the plasma membrane, which shows imprints of the surface scales (vaguely hexagonal) and pores for ejectisomes (E). In other words, the surface of an armoured warship with battlements. (Brett & Wetherbee 1986 Protoplasma)

Ejectisomes – more generally, extrusomes – are not all that unusual in the protist world. Many ciliates are loaded with menacing trichocysts and green algae like Pyramimonas are not afraid to fire similar structures either. Some bacterial endo- and episymbionts also bear similar coiled structures, but that's a topic for some other day as well. Extrusomes can also be used more locally to glue prey to the organism – if you, upon finding yourself shrunk to microns, accidentally bump into a frail-looking centrohelid heliozoan, be afraid. Be very afraid. It will smother you in adhesive proteins from the extrusomes lining its fragile-looking axopodia and devour you alive and possibly paralysed.

Ejectisomes in cryptomonads and their non-photosynthetic close relatives, katablepharids. Pyramimonas is only distantly related, and probably evolved its ejectisomes completely independently. (Kugrens et al. 1994 Protoplasma: nice review on protist ejectisomes in general, excluding ciliates)

One of the poor cryptomonads got stuck as my slide was drying out, and in its agony, released an explosion of ejectisomes. As any other biologist excessively attached to their subjects, I hate seeing protists die; at least this one didn't die in vain but gave us a nice demonstration. Extrusome firing often accompanies stress in protists that have them, drying out definitely qualifying. The following images are quite graphic, and not for the faint of heart. At least because the image quality is seriously compromised by a random layer of air between the coverslip and the specimen covered with remnants of water – a total chaos of refraction indexes.

Lysed cryptomonad on a dried out slide, surrounded fired ejectisomes. The fibrils around the cryptomonad remains are the uncoiled ribbons propelling the ejectisomes (refractile granules seen well in phase contrast, bottom images). 40x obj, DIC and PC.

While the cryptomonad may use its ejectisomes for hunting (most photosynthetic unicellular protists tend to be predators as well), perhaps they play a larger role in defense. Partly in stabbing its own predators, but additionally in a way that's quite counterintuitive to large creatures like us – sudden movement.

You might notice there isn't really much projectile action per se happening at the microbial scale. The firing is closer to an extrusion of a structure rather than freely propelling it a far distance. Furthermore, unlike an actual battleship, the cryptomonad can stop and turn almost instantaneously, and doesn't have much inertia. There is a reason for that, and it lies in the physics of fluid dynamics, a topic few of us outside biophysical biology concern ourselves with. Luckily, Purcell took care of that for us in his rather interesting 1977 paper, "Life at low Reynold's Number*" – turns out, the effect of viscosity on the behaviour of an object depends on its size, and water from a microorganism's perspective is a very different substance than what it is to us. In fact, it helps to imagine that microbial creatures live in honey or molasses – while water's viscosity doesn't actually change, it acts on µm-size things in a manner somewhat similar to how highly viscous fluids would act on things of our scale. Biophysics is quite a bit different at that scale, and different strategies are required in dealing with it.

*Reynold's number = proportion between object's velocity*size*[fluid density] and the fluid's viscosity)

In highly viscous fluids, coasting is not really an option. Things stop as soon as the driving force ceases to be applied, as anyone who's paddled a canoe across a lake of molasses would know (Bostonians from the early 1900's, perhaps?). This is why you don't really see stiff fins on bacteria or single-celled eukaryotes, at least not for motility itself. There are many ways to use a flagellum – a topic deserving of its own post – the beating strategy requiring it to be flexible at the right times. More importantly to our topic, you can't realistically give something enough force for it to keep moving like a bullet, so shooting things is out of question. Instead, the projectile must keep being pushed, usually by something unfolding or unraveling – in the case of the cryptomonad, a coiled protein ribbon. Cryptomonad artillery is perhaps more similar to harpoons than cannons.

This means a fired ejectisome can be used to essentially "push off" in the opposite direction, providing the organism with a sudden, drastic movement it wouldn't be able to obtain by flapping its flagella. The armoury of a threatened cryptomonad may be more important in providing it with rapid escape than damaging its pursuers. The microbial art of war is seldom discussed in non-enzymatic terms, but it is too a diverse and fascinating area, peppered with counterintuitive surprises. Life, and war, are indeed very different at low Reynold's numbers.

References
Brett, S., & Wetherbee, R. (1986). A comparative study of periplast structure inCryptomonas cryophila andC. ovata (Cryptophyceae) Protoplasma, 131 (1), 23-31 DOI: 10.1007/BF01281684

Kugrens, P., Lee, R., & Corliss, J. (1994). Ultrastructure, biogenesis, and functions of extrusive organelles in selected non-ciliate protists Protoplasma, 181 (1-4), 164-190 DOI: 10.1007/BF01666394

Purcell, E. (1977). Life at low Reynolds number American Journal of Physics, 45 (1) DOI: 10.1119/1.10903

Answer to the nucleomorph scavenger hunt: between the third (red algal) and second (plastid outer) membranes. The nucleus was originally in the cytoplasm, within the red algal cell membrane and outside the plastid. Oh, and if you want real topological clusterfuck, may I recommend the tertiary endosymbiosis in Kryptoperidinium – also try to count the genomes!

Sunday Protist – Gromia: beautiful predatory grapes of the sea

2011-02-20T21:09:00.000-08:00

And we're back. The protists and I, that is. Well, the protists never quite went anywhere but you know what I mean...

You may have heard of Gromia a couple years ago when it hit the news by leaving tracks on the ocean floor resembling Ediacaran trace fossils (tracks). Or perhaps not; I tend to get overly excited the one time a year some protist makes the news. The giant (3cm) track-leaving Gromia in question sounded even cooler as it came from the great deep sea; other species of Gromia are in fact quite content with the more familiar shallow waters as well, crawling on the holdfasts of kelp in addition to thriving in the ice cold polar waters of McMurdo Sound (Antarctica), where the vibrantly colourful first specimen below comes from:

Gromia, from various locales including Antarctica (A), Madeira in the Atlantic (B) and Guam (C). Big and colourful, what's not to like there? Scalebars: A - 1mm; B - 0.5mm; C - 0.1mm (Burki et al. 2002 Protist)

For some time, there has been considerable confusion between Gromia and the cruelly similarly-named foraminiferan Allogromia, bad enough to warrant a Nature paper (Hedley 1958). At first glance, they do appear somewhat similar: a sizeable grape-like blob of a test surrounded by a mass of fine pseudopodia. While foram pseudopodia form a rather elaborate and extensive network of doom and terror for anything they come in contact with, Gromia uses more modest non-fusing alternative of pseudopodia. Thus, prior to molecular data, it was often considered a sort of a precursor to the more grown-up real forams, and assumed to have a rather simple test. They could hardly be more wrong, both with the assumed relation to forams and the simplicity of its test.

Typically (using that word rather loosely), a protist test consists of the plasma membrane covered by some sort of an organic matrix (often sugary proteins and protein-y sugars), followed by the deposited structural material, be it agglutinated bits of rock from the environment (often carefully and specifically selected) or secreted calcium carbonate, siliceous scales or something else entirely. To my knowledge, the process of selecting material for the test in agglutinating species, and formation of the test in general, is still quite poorly understood. There is some understanding of how diatoms and some coccolithophorids build their extracellular wonders, but most amoebae have been largely ignored, even the more 'famous' representatives like the forams, euglyphids and arcellinids. The Allogromia mentioned earlier has the organic (non-calcified, non-agglutinated) test characteristic of Allogromiids at large, who form a vast paraphyletic sea of diversity from which the more popular lineages arise – the Protista of the foram world, if you will.

Gromia also carries an organic test, hence the confusion with Allogromiids. But its test turns out to be a bit more elaborate, with an inner lining consisting of up to ten layers of odd honeycomb membranes, the whole thickness of the structure penetrated by multitudes of pores. The test surface is sometimes covered by attached bacteria (Aranda da Silva & Gooday 2009 DSR II). Furthermore, rather than simply a hole in the wall, its opening is surrounded by a complicated oral capsule which acts as a valve or a trap door: when the pseudopodia are withdrawn, the opening closes. One seldom thinks of movable structural parts on the microscopic level, but here you go:

The structure of Gromia's test and oral capsule, in that order. The figure on the right shows a pseudopodium gradually protruding through the closed aperture. (Hedley & Bertaud 1962 J Protozool; Mazei & Tsiganov 2006 in Presnovodniye Rakoviye Amyobi (in Rus.))

The pores make the test surface look quite pretty in reflected light:

The giant deep sea Gromia sphaerica; note the complex test surface structure with prominent perforations. (Matz et al. 2008 Curr Biol)

To make these mysterious 'grapes' of the sea even sexier, they are known to have sex. Upon conjugation, flagellated gametes are exchanged between the parents, producing amoeboid diploid swarmers that ultimately form a new test and complete the cycle. The parental shell and all the work that went into building one is abandoned in this process. Both forams and gromiids are 'mortal' in our sense – they spend a period of time building a body that eventually becomes abandoned by the next generation. Organisms like many flagellates, for example, are somewhat 'immortal' in that the cell is never abandoned between generations, but rather split up and shared by mostly clonal offspring. Extra structural complexity often bears the curse of losing 'immortality'.

Gromia's life cycle. (Arnold 1966 J Protozool)

Gromia shares some strangeness with giant deep sea Xenophyophores (forams): they like to live in their own excrement. This may sound disturbing, but they're still fairly microscopic, so their shit is of a rather more chemical character. Curiously, both Xenophyophores and Gromia tend to lean toward the upper end of the protist size range, perhaps partly aided by their inability or lack of desire to part with their faeces – these faecal pellets, so-called stercomata, appear to play a structural role in the Xenophyophores, and may well contribute to structure in Gromiids as well. Waste accumulation is not too grave a problem for these organisms due to their habit of generating clouds of swarmers that ditch the parental shell forever.

As alluded to earlier, gromiids are fairly distantly related to forams, and definitely evolved their elaborate test independently. For some time, gromiids were thought to be closely related to filose amoebae with shells, ie cercozoan euglyphids and such. Turns out, while not too close to the euglyphids, Gromia is a cercozoan (Burki et al. 2002 Protist), and tends towards the endomyxean side with the plant parasite phytomyxids, vicious vampyrellid amoebae and the ornate haplosporidia. In other words, things that look little like them, aside from a tendency to form thin pseudopodia.

Gromiids probably have a bigger story to tell, as environmental sequence data reveal swaths of cryptic diversity, and new species are still being described from the deep sea as well as polar waters (eg. Rothe et al. 2009 Zool J Linn Soc; Gooday & Bowser 2004 Protist; Rothe et al. 2011 Polar Biol; and Aranda da Silva et al. 2006 Mar Biol for diversity porn). As for their ecological niche, the gromiids seem to play a similar role to allogromiids and other forams – versatile predators preying on algae and anything else that gets caught in their feet.

Aww, they even grow on trees! Gromia schmoozing with a tree-shaped foram, Pelosina, possibly in some sort of a symbiotic relationship. (Gooday & Bowser 2004 Protist)

Hopefully back to more regular blogging now. Quality of writing is at the mercy of my irritating writer's block, you have been warned...

References
Aranda da Silva, A., & Gooday, A. (2009). Large organic-walled Protista (Gromia) in the Arabian Sea: Density, diversity, distribution and ecology Deep Sea Research Part II: Topical Studies in Oceanography, 56 (6-7), 422-433 DOI: 10.1016/j.dsr2.2008.12.027

Silva, A., Pawlowski, J., & Gooday, A. (2005). High diversity of deep-sea Gromia from the Arabian Sea revealed by small subunit rDNA sequence analysis Marine Biology, 148 (4), 769-777 DOI: 10.1007/s00227-005-0071-9

ARNOLD, Z. (1966). Observations on the Sexual Generation of Gromia oviformis Dujardin The Journal of Eukaryotic Microbiology, 13 (1), 23-27 DOI: 10.1111/j.1550-7408.1966.tb01863.x

Zach M. Arnold (1952). Structure and Paleontological Significance of the Oral Apparatus of the Foraminiferoid Gromia oviformis Dujardin Journal of Paleontology, 26 (5), 829-831

BURKI, F. (2002). Phylogenetic Position of Dujardin inferred from Nuclear-Encoded Small Subunit Ribosomal DNA Protist, 153 (3), 251-260 DOI: 10.1078/1434-4610-00102

[Fuck this, the browser crashed TWICE and I'm not finding all the links again. Not tonight anyway. URLs are in the post.]

Eight supergroups on a table

2011-02-14T03:45:00.000-08:00

Back, after a bit of a distraction and an irritating bout of writer's block (how do you get rid of those things, seriously?). While I go write up a post for the long-interrupted Sunday Protist series, have a protisty doodle. My friends have this makeshift coffee table from two pieces of wood, which serves as a canvas for ~~procrastination~~ painting and such; anyway, if you supply me with permission to doodle on something, it invariably ends up protisty (or anime characters, or a frightening mix of the two), so here are eight supergroups on a table: (flash gets a a sharper image but the colours get screwed up)

Watercolour, pencil and [lab] marker on painted wood. Really fun to do watercolours on top of paint – you're not sneered at by warped wet paper, and it's very easy to wash off mistakes.

The cast:
Archaeplastida – Acetabularia
Alveolata – dinoflagellate (eg. Protoperidinium)
Stramenopila – Chaetoceros
Rhizaria – Gromia
"Hacrobia" – centrohelid "heliozoan"
Amoebozoa – tubulinid amoeba
Opisthokonta – choanoflagellate with a chitinous basket
Excavata – photosynthetic euglenid

(Accuracy not guaranteed as I was too lazy to use references)