Dermamoeba – Having your coat and eating it too

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

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

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

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

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)]

Sunday Protist – Trimastix marina

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

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!