Criminally photosynthetic: Myrionecta, Dinophysis and stolen plastids

The microbial world is full of vicious beasts. Yes, much of microbial life is cute and cuddly in one way or another. But that doesn't stop many of them from making wolverines seem docile by comparison. There is an entire mafia out there built around...organ theft; including some multicellular players as well, in case you thought animals were saintly. Today we'll look at some famous thieving masterminds of the plastid black market, but keep in mind that there are many more fascinating relationships involving keeping entire organisms or their parts alive within the host, and vastly more oddities that have still escaped human attention (not hard to do, actually).

Let's start off the messy subject with a pretty diagram summarising the major plastid hoarding events of the [moderately] distant past:

Pac-Man!* Today all we need to do is appreciate the overall big picture: there were numerous symbiotic events and by about tertiary endosymbiosis, it gets messy. Not pictured are the cases of more-or-less transient kleptoplasty (plastid-theft), which would do serious harm to the readability and aesthetic qualities of this diagram. (Keeling 2004 Am J Bot; free access) For those keen on extra gory details of plastid endosymbiosis, see this recent review.
*If somebody were to make a game of Pac-Man: Endosymbiosis Edition...

Today's plastidial saga will involve an arduous journey from the cyanobacterium to the red algal endosymbiont of the cryptomonad, to the subsequent ingestion by a ciliate and a dinoflagellate. In fact, just keep in mind that the cryptomonad itself is the result of a hungry heterotroph getting a habit of devouring red algae and developing a case of terminal indigestion, ultimately gaining a plastid and plastid-targetting genes in its own nucleus. The cryptomonad in particular happens to be really awesome in another way: it actually still retains the original, eukaryotic, red algal nucleus of its former prey! That nucleus has been badly shrunk in the wash, and the genome is essentially on crack, but that's a long story for some other day.

Just so you get an idea of what a cryptomonad roughly looks like:

Cryptomonas. Note its very diminutive size. Source: Micro*scope.

We're about to move on to the sleazy thieving ciliates and dinoflagellates. But first, we must establish how kleptoplasty (lit. plastid theft) differs from endosymbiosis. To clarify, I use 'symbiosis' as a general term for an intimate interaction between two different species, including parasitism, mutualism and commensalism. Thus, an endosymbiont needn't feel the same way about the relationship as its host, and very often doesn't. Keep in mind that it is often not very obvious which exact category the symbiosis falls into, as nature doesn't particularly care for our naming fetish.

Endosymbiosis, in the context of organelles and other intracellular stuff, typically entails the complete engulfment of another organism by the cell. Once gene transfer occurs between the genomes of the two organisms, some declare the endosymbiont is now officially an organelle. The endosymbiont-organelle debate is old, stale and utterly pointless; thus, as I have declared in a previous post, I like to call plastids and mitochondria 'endosymbionts' and the more questionable cases, like Perkinsela, 'organelles'. That way, I can piss off just about everyone. Ha!

Then there is the much-awaited plastid theft, where only the plastid itself of the failed endosymbiont is retained, with the rest of it typically digested away. The katablepharid Hatena which Labrat wrote a wonderful post about (as well as Merry at Small Things Considered), is a striking case of kleptoplasty (and only discovered this past decade!). The intensity of kleptoplasty, as well as endosymbiosis, vary greatly from transient plastids (or endosymbionts) that are not essential to the host, to mostly permanent plastids or endosymbionts that are retained indefinitely, capable of reproducing on their own, and completely obligatory for the host's survival. This is nicely summarised in this diagram from a recent review on acquired photosynthesis by Stoeker et al 2009:

Two ways to get a plastid: 1) steal a plastid-bearing alga and lock it in your basement keep it alive within you (endosymbiosis); 2) mug the alga, steal its plastid and try to keep it alive yourself. Along the two paths lie multitudes of intermediate steps different in the persistence of the plastid (how long it lasts) and how dependent the host is upon it. (Stoecker et al. 2009 Aquat Microbiol Ecol)

In the endosymbiotic pathway, nucleomorphs (and the original plastidial prokaryotic genome) suggest the permanent associations we know among the 'normal' algae come from the endosymbiotic path, as there is evidence for whole host retention at some point. However, the data do not entirely rule out some independent secondary plastid acquisition via kleptoplasty rather than endosymbiosis. As for tertiary plastidial symbionts, it gets fun. The classic persistent cases like Kryptoperidinium tend to have a whole endosymbiont, nucleus and all, so the endosymbiotic pathway is also more likely, cut things like Dinophysis, on the other hand, are just weird.

Now, at last, our long-awaited thief: the ciliate Myrionecta rubra (=Mesodinium rubrum):

Myrionecta rubra (originally Mesodinium rubrum); c - cirri; ChC - chloroplast complexes; ECB - equatorial ciliary band (Taylor et al. 1969 Nature) Right: SEM of Myrionecta by Takayama Haruyoshi (more awesome micrographs here)

As you can see, this ciliate bears plastids - a rather non-ciliate activity. In fact, if you slice it up, you'll find that the plastids are very carefully arranged at the periphery:

N - cryptomonad nucleus; M - ciliate macronucleus (note the difference in chromatin organisation); note how the plastids are not only predominantly on the cell periphery but also tend to all face outward! (Oakley & Taylor 1978 Biosyst)

The ciliate captures a cryptophyte, takes its plastids -- along with the nucleomorphs, pyrenoids and other plastid-associated stuff, as well as cryptomonad mitochondria -- and packages them up in their own little compartments. Furthermore, the nucleus is also retained and consistently packaged in an entirely separate package from the plastids. Quite remarkably, the cryptomonad nucleus remains transcriptionally active! (Apparently, Elio beat me to it in 2007. Grrr) Presumably, maintaining an active host nucleus would help keep the plastids functional longer.

Oddly enough, I have difficulties finding anything on the exact process of crypto acquisition - I initially thought it just phagocytoses them, but a friend of mine studying weird plastid aquisition thinks they may actually employ myzocytosis - sucking out the contents of its prey through a 'straw', like many other alveolates do: this may explain the segregation and separate enveloping of the plastid and crypto nucleus. This would require Myrianecta to be quite fast and well-coordinated; the speed is there as it tends to jump instead of moving gradually (details here).


There is a plot twist to this story. A stroke of irony, or poetic justice, or karma if you're into such things. The thieving ciliate itself gets mugged...by a dinoflagellate!

At first glance, Dinophysis caudata is a normal photosynthetic dino, which isn't particularly surprising as roughly half of them are (most with their own plastids). Dinophyceans are quite trippy morphologically, which made it even more frustrating that Dinophysis appeared impossible to culture, despite being photosynthetic. For a while, no one could figure out what exactly was wrong with it. Turns out, its plastids aren't its own, and are rather cryptomonad-like. Great, so it kleptoplasties the cryptos, let's just grow it in a jar full of them! Again, no luck - for some reason, Dinophysis appeared incapable of ingesting the cryptomonads!

It was all rather perplexing until someone figured out the problem in the 2000's, publishing the first successful culturing attempt in 2006 (Park et al. 2006 Aquat Microbiol Ecol). Here's what was missing:

Dinophysis (the jug-like thing with a conspicuous flagellum) sucking the plastids out of Myrionecta, who's rolled up into a small, whimpering ball by this point. (Park et al. 2006 Aquat Microbiol Ecol)

Not only is Dinophysis caudata a stinkin' thief, but it can't even do the primary stealing itself - the dino requires Myrionecta to do all the dirty work of packaging up the plastids. But it gets messier. First, a summary of the plastid's plight:

Dinophysis ingests plastids from the ciliate Myrionecta, who in turn stole them from a cryptomonad. Who, if you recall, obtained it a long time ago as a red algal endosymbiont. Who, of course, obtained the original plastid as a cyanobacterial symbiont. I think it ends there though. That poor cyanobacterial genome has been through a lot! (Wisecaver & Hackett 2010 BMC Genomics)

Now, whether Dinophysis also bears proper plastids of its own is up to heated debate at the moment. It looks like I'm not the only one thoroughly confused by it, and sorting out this issues is slightly beyond the responsibilities of a mere blogger at the moment, so let's leave this part of the story explicitly vague. It seems like Dinophysis may somehow supplement its own stock with the stolen plastids, as it appears to have plastid-targetting genes in its own genome (Wisecaver & Hackett 2010 BMC Genomics). However, there are also cases of Dinophysis carrying plastids that appeared very non-cryptomonad, and most likely to be of dinoflagellate origin (Garcia-Cuetos et al. 2009 Harmful Algae).

The chaos is quite understandable: it is actually very difficult to determine the nature of a relationship between two organisms, especially on the microscopic scale, and especially when one is inside another. It's often hard to distinguish a permanent from a transient relationship, and a mutualistic from a parasitic one. While there is strong direct evidence that the dino sucks plastids out of Myrionecta, that does not necessarily mean all of its plastids originated there. Or that it lacks its own (though that would make sense). Or more importantly, that the various research teams are even looking at the same bloody organism! Speaking of which, Myrionecta and Dinophysis appear to be in a 'bit' of taxonomic mess too, so I'll just let the professionals fight it out amongst themselves.

While that's going on, one cannot help but wonder how many such 'unconventional' relationships there really are. Food webs are not as direct as people think, the once one peers a little further than the usual stereotyped interactions (predator, parasite, prey, producer, whatever), ecology actually becomes an interesting (admittedly, fascinating!) subject. On that note, I think we should really be careful when trying to force terrestrial and macroscopic ecological terms onto the microbial world -- and by careful, I think we should perhaps come up with a system specialised for microbial life from the very beginning. While we seldom see one animal rip out an organ of another and keep it alive for itself, organelle theft is actually not all that uncommon. Life on the cellular level is weird to us, and many traditional terms simply fail to describe it.

There's a whole black market of utterly bizarre microbial interactions out there. We are only scratching the surface.

References
Garcia-Cuetos, L., Moestrup, �., Hansen, P., & Daugbjerg, N. (2010). The toxic dinoflagellate Dinophysis acuminata harbors permanent chloroplasts of cryptomonad origin, not kleptochloroplasts Harmful Algae, 9 (1), 25-38 DOI: 10.1016/j.hal.2009.07.002

Johnson, M. (2010). The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles Photosynthesis Research DOI: 10.1007/s11120-010-9546-8

Johnson, M., Oldach, D., Delwiche, C., & Stoecker, D. (2007). Retention of transcriptionally active cryptophyte nuclei by the ciliate Myrionecta rubra Nature, 445 (7126), 426-428 DOI: 10.1038/nature05496

Keeling, P. (2004). Diversity and evolutionary history of plastids and their hosts American Journal of Botany, 91 (10), 1481-1493 DOI: 10.3732/ajb.91.10.1481

OAKLEY, B., & TAYLOR, F. (1978). Evidence for a new type of endosymbiotic organization in a population of the ciliate Mesodinium rubrum from British Columbia Biosystems, 10 (4), 361-369 DOI: 10.1016/0303-2647(78)90019-9

Park, M., Kim, S., Kim, H., Myung, G., Kang, Y., & Yih, W. (2006). First successful culture of the marine dinoflagellate Dinophysis acuminata Aquatic Microbial Ecology, 45, 101-106 DOI: 10.3354/ame045101

Stoecker, D., Johnson, M., deVargas, C., & Not, F. (2009). Acquired phototrophy in aquatic protists Aquatic Microbial Ecology, 57, 279-310 DOI: 10.3354/ame01340

TAYLOR, F., BLACKBOURN, D., & BLACKBOURN, J. (1969). Ultrastructure of the Chloroplasts and Associated Structures within the Marine Ciliate Mesodinium rubrum (Lohmann) Nature, 224 (5221), 819-821 DOI: 10.1038/224819a0

Wisecaver, J., & Hackett, J. (2010). Transcriptome analysis reveals nuclear-encoded proteins for the maintenance of temporary plastids in the dinoflagellate Dinophysis acuminata BMC Genomics, 11 (1) DOI: 10.1186/1471-2164-11-366

Sunday Protist - Euglyphids

I'm going to be lazy and leech off the Mystery Micrograph again. None of you saner people (non-protistgeeks) seem to have taken advantage of the massive handicap, and subsequent hint. Seriously, type in "testate amoebae" in Google image search, and it's on the first page! Perhaps I should do a tutorial on some methods of attacking those mystery images...

Quite shockingly(not!), Opisthokont got the last one. I agree with his statement that that was like shooting fish in a barrel, but easier since fish are actually difficult to shoot at from air due to refraction, etc. The organism behind the shell in the mystery micrograph was...

Euglypha!

(Wikipedia; Euglyphid test)

Euglypha morphologically belongs to the polyphyletic 'testate amoebae', but is phylogenetically quite distant from your garden variety test-building amoebozoans, like Arcella and Difflugia. Euglyphids are cercozoan rhizarians. Since those words likely mean nothing to the vast majority of the human population, here's a 'map' of Rhizaria in my Coelodiceras (Phaeodaria) post, and an overall tree of eukaryotes can be found here. They can often be found in moss samples, but are also present in soil and freshwater environments. Their test scales are made of silica, and preserve quite decently as fossils. Speaking of which, I apparently may have a slight fetish for unicellular microfossils:

(Javaux 2007 in Eukaryotic Membranes and Cytoskeleton: Origins and Evolution ed. Jékely; 10 - fossil; 11 - modern Euglyphid; 12 - VSM - 'vase-shaped microfossil' (micropaleontological equivalent of mycologists' LBM - 'little brown mushroom' ?) with holes possibly caused by predation; fossils ~750My old)

Fast forward 750My back to the present, the modern euglyphids are about as diverse as they are understudied:

(Lara et al. 2007 Protist; tree of Euglyphids)

Images of Euglyphid diversity, shamelessly stolen from the same paper:

(Lara et al. 2007 Protist; Euglyphid SEMs, scalebar 50um except for E,F,H, where it's 10um. A - Assulina; B- Placocista; C,D - Euglypha ciliata & compressa; E - Corythion; F - Trinema; H,G - Euglypha penardi)

Unfortunately, finding nice plates full of euglyphids is rather difficult, since until quite recently, they were lumped together with testate Amoebozoans. Also, since euglyphids fossilise, they seem to be mostly studied by paleontologists, who seem to have an 'interesting' relationship with systematics of the living. Where 'interesting' entails being at least a couple decades out of date. Well, they are millions of years in the past...

Paulinella can be argued to be particularly interesting - it is a case of an independent event of primary plastid endosymbiosis. Why this is interesting can be seen in this really nice overview:

(Keeling 2oo4 Am J Bot (free access); overview of plastid endosymbiosis - the 'Pacmen' are pretty awesome! Interestingly, if the Chromalveolate Hypothesis is correct, this would mean that Paulinella already had a plastid in its ancient past. However, it would've been a red algal plastid of a different cyanobacterial origin, not a Synechococcus-derived cyanelle)

Cyanelles are photosynthetic endosymbionts/organelles - they differ from plastids by retaining some prokaryotic features like the peptidoglycan wall. Among the conventional plastid-bearing algae, glaucophytes carry cyanelles from the primary cyanobacterial endosymbiotic event which eventually led to plant chloroplasts and most algal plastids. In sequence comparisons, Paulinella cyanelles branch with the cyanobacterium Synechococcus, and retain much of the gene order, suggesting a fairly recent endosymbiosis with Synechococcus (Yoon et al. 2006 Curr Biol). The authors predict a transfer of plastid division genes to the host nucleus; however, while the plastid genome has been sequenced (Nowack et al. 2008 Curr Biol), the nuclear genome is yet to be investigated, to my knowledge.

(Lauderborn 1895 (L) Melkonian (R) in Keeling & Archibald 2008 Curr Biol; Paulinella with cyanelles)

So are the Paulinella cyanelles to be considered as endosymbionts or organelles? As with the Perkinsela case discussed a while ago, who cares? Keeling & Archibald (2008) and Bodył et al. (2007) argue that the distinction between organelles and endosymbionts is too vague and fuzzy to obsess over, while Theissen & Martin (2006) seem to have nothing better to do. Well, to be fair, they argue that gene transfer to the host nucleus is the necessary rite of passage to become an organelle. But that is a rather arbitrary cut off, since you can also say that a complete disappearance of a certain class of genes, or the endosymbiont genome altogether, are necessary to be called an organelle. Alternatively, you could also argue that as soon as an endosymbiont spends its entire life cycle within the host cell, it's sufficient to be called an organelle. With lateral gene transfer turning out to be more ubiquitous than it first seemed, perhaps gene transfer isn't that significant of an event after all. Meanwhile, how about we just accept that nature doesn't particularly care for the artefacts of our reasoning -- ie. the obsessive-compulsive yearning to categorise the world around us -- and just enjoy the biology?

Nowack et al. (2008) avoid the whole Paulinella organelle vs. endosymbiont debate by settling for 'chromatophore'.

But just to annoy fellow cell biologists, I sometimes insist on referring to plastids and mitochondria as 'endosymbionts'. Speaking of which, from Theissen & Martin (2006):

"Calling the Paulinella endosymbiont a plastid or an organelle might make a story more exciting, but at the cost of scientific accuracy. Some proteobacterial endosymbionts of aphids have genomes smaller than those of some plastids 16 A. Nakabachi, A. Yamashita, H. Toh, H. Ishikawa, H.E. Dunbar, N.A. Moran and M. Hattori, The 160-kilobase genome of the bacterial endosymbiont Carsonella, Science 314 (2006), p. 267. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (86)[16]. Would anyone call those endosymbionts ‘mitochondria’? Hardly."

First of all, why 'mitochondria'?! Those are specifically defined as endosymbionts/organelles of that one alpha-proteobacterial endosymbiosis event ancestral to all known eukaryotes. Second of all, I would totally call them organelles. I see no problem with it. I guess I'm just special then, according to those guys. Actually, I'll go around intentionally calling them 'organelles' just to piss them off.

So mitochondria and cyanobacterial plastids = endosymbionts; Paulinella cyanelles and Wolbachia bacterial endosymbionts = organelles. I think that pisses off just about everyone. My job here is done. =D


I hope this post has helped cast some familiarity upon yet another obscure group of Rhizarians. There's probably volumes more to say about them, but [insert rant about biomed crushing real biology here], so they're as understudied as the rest of Rhizaria. And much of the rest of Eukarya. Who knows how much biology weirdness lurks behind some of those obscure taxon names. Perhaps that 'Obscurius obscura' may hold the perfect properties to help us sort out some biological connundrum or other. Like what ciliate genomic madness has done for the discovery of telomeres (Blackburn & Gall 1978) and telomerase (Greider & Blackburn 1985). Come on guys, let's do something!

What if each lab was to take on a (culturable) obscure organism as a small side project? Even the big biomed labs... if each scientist played with a random organism long enough, perhaps we could unearth a freaking Pandora's box-worth of discoveries and new research directions? And the mixing of disciplines would do wonders to our overall understanding of biology! This may even be *gasp* slightly more efficient than 10 labs staring at one protein and barricading their labs from rampant scoopage... but I'll stop here with my heretic thoughts.


I have several posts in the works at the moment, among them one on origins of eukaryotes and a series on Heterolobosea, which Christopher Taylor challenged be to blog about. But first, a couple midterms, a talk I'm giving on Tuesday which I have yet to start working on, and a whole wad of research- and seminar course-related stuff. Learning seems to happen predominantly outside of class, which I find rather debilitating to grades. So multitasking must happen, and generally the class-related stuff gets cut. Which is bad. There's a dilemma between developing your mind/knowledge and getting decent marks - the former is essential for any reasonable progress in research/other professions, the latter is necessary to actually get anywhere after graduating. And they conflict with each other. Yay. [/rant]

And I seriously intended this post to be like "Hi, this is Euglypha. It is pretty. Gotta go. Bye." Cannot resist the allure of journal surfing at odd hours of the night...

References
BODYL, A., MACKIEWICZ, P., & STILLER, J. (2007). The intracellular cyanobacteria of Paulinella chromatophora: endosymbionts or organelles? Trends in Microbiology, 15 (7), 295-296 DOI: 10.1016/j.tim.2007.05.002

Javaux EJ (2007). The early eukaryotic fossil record. Advances in experimental medicine and biology, 607, 1-19 PMID: 17977455

Keeling, P. (2004). Diversity and evolutionary history of plastids and their hosts American Journal of Botany, 91 (10), 1481-1493 DOI: 10.3732/ajb.91.10.1481

KEELING, P., & ARCHIBALD, J. (2008). Organelle Evolution: What's in a Name? Current Biology, 18 (8) DOI: 10.1016/j.cub.2008.02.065

LARA, E., HEGER, T., MITCHELL, E., MEISTERFELD, R., & EKELUND, F. (2007). SSU rRNA Reveals a Sequential Increase in Shell Complexity Among the Euglyphid Testate Amoebae (Rhizaria: Euglyphida) Protist, 158 (2), 229-237 DOI: 10.1016/j.protis.2006.11.006

NOWACK, E., MELKONIAN, M., & GLOCKNER, G. (2008). Chromatophore Genome Sequence of Paulinella Sheds Light on Acquisition of Photosynthesis by Eukaryotes Current Biology, 18 (6), 410-418 DOI: 10.1016/j.cub.2008.02.051

THEISSEN, U., & MARTIN, W. (2006). The difference between organelles and endosymbionts Current Biology, 16 (24) DOI: 10.1016/j.cub.2006.11.020

Yoon, H., Reyes-Prieto, A., Melkonian, M., & Bhattacharya, D. (2006). Minimal plastid genome evolution in the Paulinella endosymbiont Current Biology, 16 (17) DOI: 10.1016/j.cub.2006.08.018

Sunday Protist - Perkinsela: Life as an organelle

We've all heard of the primary endosymbiosis of bacteria that eventually became mitochondria* and plastids, on two separate occasions (three if you count Paulinella plastid origin). Some have heard of secondary, and maybe even tertiary, plastid endosymbiosis (eg. brown algae with red algal plastids). There's a fascinating case of tertiary endosymbiosis where an entire diatom inhabiting a dino (Kryptoperidinium), etc. Another interesting phenomenon is the endosymbiosis resulting in other essential 'organelles', eg. Polynucleobacter in Euplotes(Görtiz 2006 in Prokaryotes 1:364-402). While plastids have been transferred about the tree several times, secondary endosymbiosis of mitochondria or whole non-photosynthetic eukaryotes seems to be extremely rare. Thus, the following case of an endosymbiosis of a kinetoplastid by an amoeba I find to be rather interesting.

*Well, there's still remnants of a crackpot adherence to the autogenous model of mitochondrial origin...

Meet Perkinsela (formerly Perkinsiella; Dyková et al. 2008b), an endosymbiont of amoebae that took until Hollande 1980 to be recognised as an organism rather than organelle! (although Grell 1973 Protozoology (p.363) does suggest a link to the endosymbiosis theory that was just becoming established at that time). Here's the amoebozoan host Neoparamoeba with an arrow pointing to Perkinsela:

(Eva Dyková, Tolweb Perkinsiella page)

This endosymbiont's life cycle has become completely confined within the host cell, as it is perpetrated along with nuclei upon host cell division. It is often found in a strange 'bipolar' form, with nuclei opposite of each other across the massive mitochondrion (which contains the kinetoplast - a dense disk of mitochondrial DNA unique to Kinetoplastids, which include Trypanosomes, the cause of African Sleeping Sickness), and in close association with the host nucleus:

(Dyková et al. 2003 Eur J Protistol; 8 shows Neoparamoeba with its endosymbiont (NN - host nucleus, K - kinetoplast (mitochondrial DNA), n - Perkinsela nucleus; 9 - Perkinsela itself. Note the two nuclei across the kinetoplast from each other (c- cytoplasm))

The nature of this endosymbiotic relationship remains unknown, although it seems to be mutualistic as the host and the endosymbiont both die without each other (Dyková et al. 2008b).

The endosymbiont is a sister group to Ichthyobodo, and even contains the splice leader sequences characteristic of Euglenozoa (the larger containing group of kinetoplastids, diplonemids and euglenids (remember Euglena?)) (Dyková et al. 2003; 2008b). Here's a tree to orient yourselves: (because everyone knows what Jakobids and Diplonemids are...feel free to go here for the bigger picture ^.^)

(Simpson et al. 2006 Trends Parasitol.; family tree of creatures with 'hockey puck' mitochondrial DNA...the intelligent designer was definitely tripping out on some serious stuff when he made this clade ^.^)

(Let's just say Neoparamoeba is an Amoebozoan. I have no desire to sort out Amoebozoan taxonomy at this hour, as it's a fucking mess. I have three trees before me from various periods, and the burning urge to rip all my hair out is a little too much. Seriously, Amoebozoa are just fucked up, as morphology-based classification failed more abysmally than usual there. It's hard to determine morphological features of something so...amoeboid ^^. I challenge a certain taxonomist who reads this to blog about their phylogeny! Have fun =P)

Interstingly, both Neoparamoeba and Ichthyobodo are fish gill parasites. While Neoparamoeba is an opportunistic parasite (Young et al. 2007) (ie it can also live freely; a more vicious example of opportunistic parasitism is Naegleria, which is harmless until it accidentally gets into a brain - it happens to love neural tissue!), Ichthyobodo is an obligatory ectoparasite. ('ectoparasite' means it attaches to the surface of the host cell to drain it of its 'juices', instead of going completely inside).

Fish gills seem to be rather fertile ground for parasites of all levels of devotion; for the chances of passing by one when you live in water are pretty good. It seems like the long-term close association between Neoparamoeba and Ichthyobodo parasitising off the same host has led to this intimate endosymbiosis - would be interesting to know the approximate timescale of the divergence between Perkinsela and Ichthyobodo, to see how long it can take for such relationships to evolve.

Here's the Neoparamoeba opportunist in action:

(Lovy et al. 2007 Vet Pathol; A - amoeba, E - fish epithelial layer; bar = 3um)

To summarise what I'm talking about:

(M - mitochondrion with kinetoplast; N - nucleus; HN - host (Neoparamoeba) nucleus)

If we were to analyse the Perkinsela genome, it would likely show signs of substantial genome reduction, due to it being no longer necessary to keep the entire set (depending how old the relationship is, of course). What would be even more exciting is if gene transfer to the host nucleus has already occurred! Perhaps the mitochondrion-targetting genes may go first; as far as I know, whether host-to-endosymbiont-nucleus targetting genes exist is still poorly understood. There are cases of host-to-endosymbiont-plastid targetting (dinoflagellates Karenia, Karlodinium...), however; and endosymbiont mitochondria tend to disappear rather early in endosymbiosis, so it's surprising to find it so prominent here.

Which makes one wonder...perhaps the host is keeping the endosymbiont for its mitochondrion? The cytoplasm is extremely reduced, so that the cell appears to be little more besides a nucleus or two and a kinetoplast. Could the kinetoplastid mitochondrion be capable of something the Amoebozoan one is not, that also happened to be useful for the amoeba? Doesn't seem very likely, but who knows... perhaps the ancestral Perkinsela was engulfed by the predatory Neoparamoeba as prey, and led to the mitochondrial analogue to kleptoplasty ('stealing of plastids' from prey practised by some predatory protists; sometimes they'll keep photosynthetic (algal) cells around for their plastids until they die - could be how cyanobacterial endosymbiosis first started)?

Or is Perkinsela just a really good parasite, successful to the point of no longer needing to even try, enjoying its free ride along with the host? This doesn't explain why Neoparamoeba dies without it, though. I guess all it would take is for the host to lose a gene or two essential for producing something that is made and exported by the endosymbiont/parasite; thereby fixing a dependency upon it. But I'm just rambling at this point...

There seems to be no mention of basal bodies/centrioles in Perkinsela ultrastructure studies; this worries me. kDNA replication is molecular cell biology on potent hyperhallucinogenic acid, and is a susbtantial topic best left for another day. In Trypanosomes, the final steps of kinetoplast replications require a system of fibrils attached to the flagellar root; the mitochondrion is tightly associated with the basal bodies (Liu et al. 2005 Trends Parasitol). If Perkinsela evolved from a 'stuck' amastigote kinetoplastid (ie. one in a non-flagellar stage of its life cycle, although that doesn't seem to happen in modern Ichthyobodo...), it could still retain a pair of centrioles, devoid of flagella. However, those should be fairly visible in EM.

I'm have this nagging feeling that I'm not making much sense anymore... >_> To wrap this up, there's also potential endosymbiotic association between the amoebozoan Thecamoeba and a labyrinthulid species. The labys seem to be able to proliferate at will without destroying the host, thereby seeming rather non-parasitic at the moment (Dyková et al. 2008a) Interesting...

Microbial diversity is amazing as is, but as soon as you start treating a cell as a potential ecosystem in its own right, the hidden universe of intracellular parasites and symbionts is overwhelming. This is where those popular charts showing the majority of biodiversity as invertebrates are just abusrd - each and every one of them is a possible ecosystem for microbial life, both bacterial and eukaryotic, and each and every cell thereof is yet another niche. And every protist is a possible ecosystem for some other protists, or prokaryote. Sometimes, those relationships persist and develop, and, on occasion, blur the line between organism and organelle.

So I wonder: is Perkinsela now an 'organelle'?

(This is why you should support basic biological research in addition to biomed; one cannot tackle cancer before understanding how single cells work in the first place. The 'higher' biology lies in the fundamentals, not select, limited cases like humans or mice...)

References
DYKOVA, I. (2003). -like endosymbionts of spp., relatives of the kinetoplastid European Journal of Protistology, 39 (1), 37-52 DOI: 10.1078/0932-4739-00901

DYKOVA, I., FIALA, I., DVORAKOVA, H., & PECKOVA, H. (2008). Living together: The marine amoeba Thecamoeba hilla Schaeffer, 1926 and its endosymbiont Labyrinthula sp. European Journal of Protistology, 44 (4), 308-316 DOI: 10.1016/j.ejop.2008.04.001

DYKOVA, I., FIALA, I., & PECKOVA, H. (2008). Neoparamoeba spp. and their eukaryotic endosymbionts similar to Perkinsela amoebae (Hollande, 1980): Coevolution demonstrated by SSU rRNA gene phylogenies European Journal of Protistology, 44 (4), 269-277 DOI: 10.1016/j.ejop.2008.01.004

Liu, B., Liu, Y., Motyka, S., Agbo, E., & Englund, P. (2005). Fellowship of the rings: the replication of kinetoplast DNA Trends in Parasitology, 21 (8), 363-369 DOI: 10.1016/j.pt.2005.06.008

Lovy J, Becker JA, Speare DJ, Wadowska DW, Wright GM, & Powell MD (2007). Ultrastructural examination of the host cellular response in the gills of Atlantic salmon, Salmo salar, with amoebic gill disease. Veterinary pathology, 44 (5), 663-71 PMID: 17846238

SIMPSON, A., STEVENS, J., & LUKES, J. (2006). The evolution and diversity of kinetoplastid flagellates Trends in Parasitology, 22 (4), 168-174 DOI: 10.1016/j.pt.2006.02.006

YOUNG, N., CROSBIE, P., ADAMS, M., NOWAK, B., & MORRISON, R. (2007). Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)☆ International Journal for Parasitology, 37 (13), 1469-1481 DOI: 10.1016/j.ijpara.2007.04.018

Two rants on endosymbiosis

First off, quick point: Can we please stop using headlines like "Darwin was wrong about [x]"!? Srsly, big deal, some dead dude from the 19th century was wrong about something. Well shit. Evolutionary biology as we know it is now fundamentally flawed. Because Darwin didn't have PCR or fancy sequencers or GFP-tagged whatever. Tragic. What about all the shit Mendel was wrong about? Are we gonna ignore him now? /rant #01

Sometimes when writing up a post on something, I come across 'interesting' sites and papers. I mention my reaction in brackets, and find more crap to rant about. I then move the contents of the brackets to a footnote and unleash an off topic at the bottom of the post. But sometimes, this rant would be -really- off topic, and would be rather distracting if it becomes longer than the post itself. Today, we have come across one such case.

Warm-up - (excessive endosymbiosis)
Before embarking on a little journey of "Did he seriously just write that/get a faculty position/get a degree", a warm up paragraph from this week's Nature:

"In the former, the peptidoglycan layer is sandwiched between the outer and inner membranes, so that it surrounds the inner membrane: in contrast, in the latter there is no inner membrane, and the peptidoglycan layer, located outside the cell, surrounds the outer membrane." (Lake 2009 Nature) (via Catalogue of Organisms, who beat me to it, grrr XP)

That, my friends, is a wonderful example of epic semantics and topology fail. What he's talking about is that double membraned bacteria in question have cytosol-Inner Membrane(IM) - murein wall - Outer Membrane(OM) - outside. Single membraned bacteria have a cytosol-OM-murein wall-outside arrangement [sic]. Ie, somehow OM-M became IM-M-OM, raising the question of how the outer membrane end up on the other side of the murein. Thus, Lake invoked endosymbiosis to explain this 'conundrum' - an OM-M endosymbiont entered another OM-M prokaryote, and the endosymbiont OM became the inner membrane, while the host lost its murein wall. Very complicated stuff.

It may be quite evident at this point what the real 'conundrum' is there. Instead of comparing biochemical properties of the membranes to each other, he compared their relative positions. There is a very interesting topological property where if you have an double membrane and lose the outer layer, the former 'inner membrane' becomes the outer one. I think I may be onto something here...anyone wanna collaborate on a Nature paper? Any mathematicians out there wanna contribute a proof?

So how could someone who's probably a decent scientist fall for something like that? In fact, this seems common as soon as you put the 'hype' into 'hyp[e]othesis' - in this case, the guy seemed desparate for endosymbiosis, to the point of overlooking this very simple point in semantics. The reviewers and editors were no better - they too were getting carried away with the endosymbiosis hype (of course, they've still got ways to go to reach Margulis levels thereof...) For some reason, the fact that there's only one confirmed case of prokaryote-prokaryote endosymbiosis in the literature seems to worry no one...

(Coming from the TC-S camp of eukaryotic evolution, it was probably the double membrane state that was ancestral, with the loss of the outer membrane leading to what TC-S calls 'negibacteria', which eventually gave rise to us Neomurans. Even if you propose that single membraned bacteria came first, there's still no need for endosymbiosis, for they could have perhaps devised a way to form the outer membrane on their own. That would still be more parsimmonious, and more likely, than Lake's hypothesis above...) /rant #02

The big rant - (insufficient endosymbiosis)
We have some major endosymbiosis people in our department, so I never really came across any skeptics of mitochondrial endosymbiosis. The endosymbiotic theory of mitochondrial and plastid origins is pretty much beyond dispute these days, and the evidence is simply overwhelming. However, there always has to be someone to blow against the wind gale. Very rarely, they happen to be right; but even in those cases, their argument tends to be well-reasoned and supported by at least some data from the start. The other 99% of crackpots remain just that - timeless testament to our innate irrationality.

There's some guy who seems mildly annoyed by mitochondrial endosymbiosis. To the point of dedicating an entire website to the topic: http://www.origin-of-mitochondria.net/

So there's a whole page 'critiquing' the endosymbiotic mitochondrial origin theory. I know this is only half a step up from bashing creationists, but it got me a little irritated. Not because I feel threatened, but because it seems to be so easy to get employed as a crackpot, and I'm a little envious of their capabilites. See, if you actually try to abide by proper reason and the scientific method and all that crap, you'll be dirt poor and socially marginalised for the rest of your life. Thus, I shall enjoy one of the few advantages we do get - the feeling of intellectual superiority as you rip into some crackpot's drivel with a barrage of citations and proper data. It's a sport.

"The extensive gene transfer that is needed in the endosymbiotic theory would wreak havoc in a complex genome since frequent insertion of random pieces of mitochondrial DNA would disrupt existing functions."

Uhhh...heard of transposons, by any chance? I'm sure those are a few orders of magnitude more plentiful and more violent than the occasional piece of mitochondrial DNA. Yet they still...happen. And genomes have generally been able to deal with it. Random gene insertions do disrupt functions, but then you've got a few million other genomes to take their place! Isn't evolution awesome?

"Most pictures in textbooks of mitochondria resemble bacteria, but in reality, mitochondria form a dynamic network of interconnecting tubules (reticulum)"

May we introduce you to bacteria that don't look like 'bacteria'? Say hi to Streptomyces and Planctomyces, for example.

I'm just making shit up hypothesising here, but it seems like the stereoptypical bacterial morphology may be limited by the fact that it swims. There are certain shapes optimal for a flagellate lifestyle, and netlike/mycelial/branching forms are not among them. Once a bacterium has become commited to living exclusively in the intracellular environment, it no longer needs to be hydrodynamic, and can start taking on whatever other form it likes. I'd imagine that >800my is plenty of time for drastic morphological changes, considering eukaryotes managed it quite well.

"It is said that mitochondria, like bacteria, divide by fission, but the mechanisms are completely different and mitochondria use mainly components of unique eukaryotic origin."

Ever heard of intracellular parasites? An intracellular lifestyle does some weird shit in terms of intense reduction - microsporidia (fungi which shoot their cytoplasm into the host cell, where it takes over and lives off the host's resources, until forming new spores) have highly reduced genomes that have been harsh to introns due to space limitations(eg. 13 introns in an entire genome (E.cuniculi)), as well as a great purge of proteins for nucleotide+amino acid biosynthesis (Keeling & Slamovits 2005 Curr Op Genet Dev)* This makes sense - you don't have to make your own amino acids if you can just steal them from the host! So any degeneration and subsequent loss of previously essential genes is now tolerated, and thereby bound to happen.

*Ok, when you come across a paper with the following introductory paragraph, you just have to read it:
"At the bottom of the rabbit hole, Alice found a bottle labeled, ‘‘Drink Me’’. When she did, Alice shrank to a perfectly functioning, ten-inch miniature of herself. In reality, shrinking can be more difficult than simply drinking a potion, because the component parts of many systems are not themselves shrinkable, and so the system fails to function properly. In the world of eukaryotic nuclear genomes this is probably true, despite the fact that they vary in size by factors of hundreds of thousands (Figure 1), much more than all of Alice’s many transformations combined." (Keeling & Slamovits 2005 Curr Op Genet Dev; free access)
Classical studies geek really shows here...
(I'm quite bothered by the desolate desert around Rhizaria in fig.1 =( )

Similarly, the early mitochondrion no longer desperately needed to maintain its own division machinery, which eventually became transferred over to the host or lost. In a way, it has been able to hijack the host cell to take care of its own division. (so who's 'enslaving' whom here?) Through extreme evolutionary 'laziness', some lineages have been able to lose all genomic DNA entirely, and reduce to tiny membrane bound compartments essentially for specific parts of the host's metabolism. They basically 'disolved' into the host over time! (of course, de Roos' theory would probably claim those lineages as an ancestral state, eventually increasing in complexity. Too bad phylogeny king of stands in the way. Oops.) Just because an organism isn't capable of free life now doesn't mean it ancestrally wasn't either. Again, parasites support that very well. de Roos seems to have fallen for the 'evolution aims to gain complexity' misconception, and had difficluties with it 'going backwards', as it often likes to.

"So, although we see some characteristics that are shared between mitochondria and bacteria, we see many more examples where mitochondria are actually quite different."

Yeah, shit diverges over 850my. Just because they're 'different' doesn't mean they can't share a common origin, even a fairly recent one. Again, microsporidia were considered to be very ancient due to their apparent 'absense' of mitochondria and a highly reduced structure (wiser people were a bit alarmed by the latter detail; parasitism is almost universally a secondary trait (looking back far enough, it always is; first life must have been free living, otherwise we get the chicken-and-egg problem)). Turns out, they're fungi, like the mold in your fridge. We're not well-equiped mentally to deal with such timescales, but a lot can happen in just a few million years.

Thus, as long as we do not have a clear picture of the last common ancestor and its relationship with eukaryotes, it will be difficult to interpret gene similarity as evidence for the endosymbiotic theory.

This is where parsimony helps. Sure phylogeny is fallible (again, see microsporidia), but if an endosymbiont and a free living organism share a significantly large chunk of genes, it takes a lot less explanation and hand waving to invoke endosymbiosis than to craft elaborate hypotheses of weird massive lateral gene transfer stuff. That alone doesn't make it right, but definitely much more probable. And we're really working with probabilities here to begin with.

The mitochondrial genes could be derived from transposable elements, plastids or viruses and could come from either the nuclear genome or a bacterial genome.

'Domestication' of transposons is not as easy or probably as we may like it to be. Also, much of this would have to happen between the proto-eukaryote and the last common ancestor of most eukaryotes alive today, which is an epic question mark at the moment, although it does seem like that time period may not have been that long after all ('short' paper here: Cavalier-Smith 2006 Phil Trans R Soc B; free access). Cell structure, on the other hand, seems to be more malleable than large-scale gene organisation. Also, has there been at least one case of genes randomly congregating into a de novo genome in a random compartment? That would be quite ridiculously unlikely! How did the replication and maintenance machinery get in there then?

And plastid origin of mitochondrial genes? Ok, maybe once or twice that could, in theory, happen (has it?), but we're talking about mitochondrial genes in primarily plastid-less organisms! Does this guy propose a plastid endosymbiosis as ancestral to all eukaryotic lineages with mitochondrial genomes!? He seriously needs to explore something a little outside his metazoa. He needs to take one good look at a proper tree of eukaryotes (one without the 'crown eukaryote' abomination, kthx), and read a TC-S paper or two on eukaryogenesis. Or perhaps we should cross him with Margulis, and the result would have an intermediate phenotype, and perhaps even be a decent scientist!

Intermediates exist in the form of hydrogenosomes and mitosomes from amitochondriate primitive eukaryotes.

Hey, let's pull a little prank! How about we introduce him to Blastocystis and the ciliate Nyctotherus with mitochondria-like organelles (Stechmann et al. 2008 Curr Biol)? Actually, the table in that page, if you can access it, is a powerful demonstration of the dangers of relying on a single morphological for reconstructing evolutionary history. Essentially, if you follow organelle complexity, you'll get something like: Microsporidia, Giardia, Trichomonas, Nyctotherus, Blastocystis, and us. Let's draw that as a tree, mentally (let microsporidia be basal to the rest). So far so good. Ok, let's pull out a certain tree I tend to [ab]use a lot:

(Keeling et al 2005 trends ecol evol)
(I went for quite a while without pulling that out! Did you notice? See, self-restraint works sometimes! Until it doesn't...)

Let's do a little exercise. Grab a mental marker, and let's find Microsporidia. It will be among the opisthokonts, close to chytrids and zygomycetes. Done? Great, now find Giardia. It's a Diplomonad, close to Malawimonas in the Excavates. Trichomonas is a trichomonad, close to hypermastigotes (remember Trichonympha?), again in the Excavates. Now head over to the Chromalveolates, the alveolate neibourhood, for Nyctotherus, a ciliate. And then go down to the Stramenopiles, where you'll find Blastocystis between Actinophryids ('heliozoans') and Bolidophytes. And then point at pretty much everything else. And now look at our single-trait tree, which was built keeping de Roos' hypothesis in mind. So...how'd that go? I think someone needs to read up on basic eukaryote diversity before making shit up about the origins thereof...

See, while both de Roos and Cavalier-Smith like to make up grand hypotheses that tend to contradict the mainstream theories, Cavalier-Smith is actually good at it. He thoroughly reads astounding volumes of literature, formulates rational, testible hypothesis that make sense, and backs off his theories when evidence definitively proves them wrong (as with Archaezoa). de Roos has ways to go to even dream of such level.

And finally,

"In order for an evolutionary theory to be considered a scientific fact or a valid scientific theory, there are some basic requirements. First, it is necessary to have a reasonably detailed mechanism that explains the basic steps in the endosymbiotic scenario. [done] Second, this mechanism should be placed in the context of current Darwinian evolutionary theory and should contain no fundamental problems or falsifications[huh...?]. Third, a substantial body of empirical evidence that directly supports this scenario should be present.[nope, no evidence whatsoever... I know of a lab where people just sit around on their asses all day because there's simply no data in that field. Also, they don't publish any ridiculous number of papers, thereby making us cell biologists very jealous.] Fourth, no credible or logically sound alternatives should exist[huh? Since when is that a requirement for a valid theory?]. If these criteria are not met, the endosymbiotic theory cannot be considered to be a scientific fact that has been proven beyond reasonable doubt. Remarkably, the endosymbiotic theory fails all points." [bolded edits mine]

Yeah, to all my friends working on endosymbiosis: IT IS A LIE! OH NOES!!1!

Seriously, how can people argue that bullshit, and SOMEHOW be employed in biology?! This guy is apparently an actual biologist (although more of a biochem/bioinformatics background; As a devoted cell biologist, I have an obligation to hate them a little...you see, the academic community has ascended far beyond the primordial practices of stone age tribalism.) After a brief search, I found another interesting abstract, although we don't have access to this paper (and I can't be bothered to ILL it):

"Current theories about the origin of the eukaryotic cell all assume that during evolution a prokaryotic cell acquired a nucleus. Here, it is shown that a scenario in which the nucleus acquired a plasma membrane is inherently less complex because existing interfaces remain intact during evolution. Using this scenario, the evolution to the first eukaryotic cell can be modeled in three steps, based on the self-assembly of cellular membranes by lipid-protein interactions. First, the inclusion of chromosomes in a nuclear membrane is mediated by interactions between laminar proteins and lipid vesicles. Second, the formation of a primitive endoplasmic reticulum, or exomembrane, is induced by the expression of intrinsic membrane proteins. Third, a plasma membrane is formed by fusion of exomembrane vesicles on the cytoskeletal protein scaffold. All three self-assembly processes occur both in vivo and in vitro. This new model provides a gradual Darwinistic evolutionary model of the origins of the eukaryotic cell and suggests an inherent ability of an ancestral, primitive genome to induce its own inclusion in a membrane." (de Roos 2006 Artificial Life; emphasis mine)

Huh? Umm...this...just...like...errr...no! NO! Does not compute! AAAAAH! My eyes! I can feel my brain liquifying and oozing out of all sorts of orifices! See why the computery bioinformatics folk must be kept away from any mention of an actual organism? (ok, admittedly, some can manage it well, but that doesn't mean I shouldn't stereotype for personal fun =P )

I'll fix this sometime within the coming week, and 'translate' for you a real hypothesis on eukaryotic origins.

>Psi Wavefunction casts lvl10 TC-S Attack on lvl8 Crackpot for 500 damage.
Lvl5 Crackpot sustains 500 damage; HP 130/630
>Crackpot uses Copy Attack to cast Psi's lvl10 TC-S attack.
Psi sustains 0 damage due to TC-S Immunity.
>Psi casts lvl40 HAHAPWNEDLULz! on Crackpot for 1000 damage.
>Crackpot defeated!
*cue Final Fantasy victory music
>YOU gain 2000XP

Now back to working on this week's Sunday Protist ^.^

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

KEELING, P., & SLAMOVITS, C. (2005). Causes and effects of nuclear genome reduction Current Opinion in Genetics & Development, 15 (6), 601-608 DOI: 10.1016/j.gde.2005.09.003

Lake, J. (2009). Evidence for an early prokaryotic endosymbiosis Nature, 460 (7258), 967-971 DOI: 10.1038/nature08183

STECHMANN, A., HAMBLIN, K., PEREZBROCAL, V., GASTON, D., RICHMOND, G., VANDERGIEZEN, M., CLARK, C., & ROGER, A. (2008). Organelles in Blastocystis that Blur the Distinction between Mitochondria and Hydrogenosomes Current Biology, 18 (8), 580-585 DOI: 10.1016/j.cub.2008.03.037