Field of Science

Sunday Protist - Paramyxids: Nested parasites and introverted multicellularity

ResearchBlogging.orgA while ago we peered into the lidded jar-like spores of Haplosporidia, memorable by their peculiar habbit of building up pressure and popping open the lid upon germination, much like a jack-in-the-box. But nastier, if you're an oyster. While digging around in obscure haplosporidian literature, we came across their lesser known close relatives, the Paramyxids, characterised by their pechant for sporulating 'inward' several times, in a strange genre of parasitism reminiscent of matryoshka dolls*. Not only do they undergo serial endogenous divisions, Paramyxids exhibit differentiation among those cell types as well. Instead of being multicellular by growing outward like normal organisms, Paramyxids have evolved some strange sort of 'introverted' multicellularity.

Judging from these hints of a rather complex life cycle, one can probably predict Paramyxids to be parasitic, like their Haplosporidian relatives. Parasites tend to love convoluted life cycle gymnastics, perhaps because it's a wonderful way to trick biologists into considering the various stages to be different species, or even higher taxa. Speaking of which, quite a bit of time was spend trying to sort out their damn phylogenetic position - their identity as fellow Cercozoan sisters of Haplosporidia was not unanimously agreed upon. Since that discussion involves lots of technical phylogeny- and taxonomy-related ramblings, I've shoved that for later, at the very bottom of the post. Feel free to ignore, although I do think it is interesting.

Introduction to the Spore-in-a-spore-in-a-spore-in-a-spore-...
There are four genera: Paramyxa, Marteilia, Paramarteilia and Marteilioides, although Feist et al. 2009 propose supressing the latter to Marteilia. So let's pretend it doesn't exist. The life cycle starts off with an amoeboid primary 'stem cell', which crawls around between host cells and later gives rise to a secondary cell inside it. This cell then undergoes equal mitosis, giving rise to a second secondary cell. This part is common between all Paramyxids. The secondary cell (sporont) then divides endogenously to form one or several spores, depending on the species. Those spores divide endogenously again several times, up to the sixth generation (so three times) in Paramyxoides nephtys. To start off, the 'simplest' is Paramarteilia, with the spores dividing once endogenously:
TEM of Paramarteilia canceri, a crab parasite. The organism on the right is in an earlier stage of development. The labeling conventions in the vast field of Paramyxidology are as such: C and N indicate cell/cytoplasm and nuclei, respectively, with the number indicating the generation of endogenous division. So N1 is the nucleus of the primary cell (C1), and the cell immediately internal is indicated as C2, etc. This gets annoyingly confusing when we get to Marteilia, Paramyxa and Paramyxoides. This organism is quite simple, relatively, with two secondary cells (C2) each holding two spores (C3), each of which, in turn, has a single internal cell (C4). (Feist et al. 2009 Folia Parasitol)

Paramyxids are topologically taxing, as the 'simple' Paramartelia already begins to suggest. It gets distinctly worse, as nicely summarised in these two diagrams:

Paramarteilia (1), Marteilia (2), Paramyxa (3) share a few common early stages, consisting of an amoeboid stem cell crawling between the host cells. Upon the first division, this stem cell produces a couple secondary cells inside it, which later give rise to a number of spores, which themselves have a number of cells within. The latter two traits are species-dependent. The figure on the left shows the development in Paramyxa paradoxa, to be read clockwise. The intelligent designer was definitely high out of his mind when he made these.(Desportes 1984 Origins of Life)

Paramyxoides sporulation: a cytological acid trip
Let's take Paramyxa (and Paramyxoides, which for our purposes can be considered more or less the same thing) for a bit of a cytological acid trip. Paramyxa crawls about as a primary cell, divides endogenously once to make one secondary cell, which then divides 'normally' to form a second secondary cell, each of which then divides endogenously to form four spore cells (tertiary), which then divids inward another three times to form a nested spore thing (fig 3 below, A and B). Meanwhile, towards maturity, each of the spores forms an external sac, because it doesn't have enough membranes yet.

To further enhance its elaborate membrane collection, the fourth and fifth cells (so second and third cells within the spore), form haplosporosomes. (Annoyingly, I can't quite figure out what the haplosporosomes do. Perhaps nobody really knows, judging from their consistent description as 'electron-dense vesicles', even in recent literature.) Then, the primary and secondary cells disintegrate, releasing the spores. By the way, we're inside the host cytoplasm at this point. Then, the extrasporal sac gets smaller, and striated projections are formed which bind the four spores together. Said striated projections cement together the four spores into a tetraspore complex (fig 3 below, D). What happens after? We don't talk about that. Actually, I don't think anyone knows...

I got really confused so I made this diagram:
EM of a Paramyxoides (parasite of a polychaete) spore from Larsson& Koie (2005), with an attempt at representing the whole organism. Note the six generations of endogenous cell budding involved in this process. Cells 4 and 5 exhibit the haplosporosomes characteristic of Ascetosporeans. The primary cell usually degrades early on in development, while the sporont disintegrates later to release the spores. The spores secrete an extracellular membrane, which later shrivels up and forms mysterious 'striated projections', whose function appears to be cementing the spores together. (Since I have no EM experience, trust my membrane tracing attempts at your own risk. It involved more imagination than anything else, really...)

Cellular invasion -- somebody needs to do some work on this so I can expand this section
So then I was excited to read about the Paramyxid cell invasion mechanism, and later compare it to that of microsporidia and apicomplexa. Alas, just as for the sister Haplosporidia, the literature lies silent about the exact journey of the paramyxid into the host cytoplasm. It's mentioned that the parasite develops in direct contact with the host cytoplasm (Larsson& Koie 2005), probably implying that there's no parasitophorous vacuole or any such thing. Thankfully. I have no idea how nature would put up with one more membrane...

Paramyxid life cycles: Question marks and dotted lines
To confuse ourselves further, I picked up a couple life cycle diagrams. Universal Law mandates that parasites must have life cycles from hell, so here's one of them:

Parasitic life cycles from hell, exhibit 1: Marteilioides chungmuensis (oyster parasite). Presumably, the dotted line means "And then it magically ends up inside the host cell" or something to that effect. It seems that the cell undergoes several developmental programs, and thus several different series of endogenous divisions, to make life interesting. Or maybe the same sequence but only executed to completion within the oocyte. Regardless, there is an 'extrasporogony' stage where the parasite endogenously replicates, after which it enters the gonads and magically forces its way into the oocyte, where sporulation happens. With question marks and dotted lines in-between. (Itoh et al. 2004 Int J Parasitol)

The oyster parasite Merteilia sydneyi (Kleeman et al. 2002) also has a life cycle from hell, also with an extrasporogony stage (incidently, it was first found there, and subsequently in Marteilioides). It seems that the gill epithelium may be a safe place for the parasite to proliferate. Once Marteilia reaches the gut epithelium, where it will eventually sporulate, it seems to undergo a few more rounds of non-sporogenic endogenous division, possibly to proliferate itself throughout the digestive system. In fact, this stage results in an exponential increase of parasitic cells, so efficient that nearly all the digestive tubules are infected. Once enough of the epithelial surface is covered, the parasites invade the host cells and form spores there. Which then...somehow...do something. Presumably, some membranes break somewhere in the process.

Marteilia sydneyi life cycle. See text. (Kleeman et al. 2002 Int J Parasitol)

The gut cell invasion and subsequent sporulation happens synchronously, en masse. This is also when the host finally launches an immune response, although a little too late. Parasites often employ the stealthy ninja tactics of multiplying rapidly somewhere quiet, where they go unnoticed, and then suddenly attacking in vast hordes and completely overwhelming the host. Kind of like the 14 year periodical cicadas overwhelm their predators, or how the malaria parasites lyse the host cells synchronously, causing the characteristic waves of fever.

The Kleeman et al. 2002 paper is the first study attempting to characterise the early developmental stages of a paramyxid oyster parasite. While sporulation has been glimpes at, we are only beginning to get an idea of this organism's fascinating life cycle. New techniques like in situ hybridisation are allowing researchers to finally trace the ellusive complex life cycles of many parasites, without having to rely on obscure morphological traits to identify the organism. A lot of the parasitic life cycles (as well as some free-living ones) turn out to be more complex than previously thought.

Summary and introverted multicellularity.
In case anyone is interested in the finer taxonomy of this group, here's the latest classification according to number of secondary cells, spores and number of cells within a spore.
The coolest thing from all this? The whole idea of multicellularity happening endogenously rather than usual 'extroverted' way. In both cases, we have cellular differentiation happening by asymmetrical mitosis. In paramyxids, this mitosis is abnormal, ie the daughter cell ends up inside the mother. There's another group of organisms that do this. You may actually be familiar with them: flowering plants. Pollen formation involves endogenous budding as well, as you have a vegetative cell, which forms the pollen tube, and a generative cell, which in involved in fertilisation.

I'm perplexed by the cell biology of engogenous mitosis, and perhaps it may be similar to whatever happens in pollen formation. However, we may have enough material for today. In fact the following part isn't necessary, as it's a bit of a discussion about the phylogenetic position of the paramyxids. If you care, feel free to stay. We will be discussing a whole paragraph from the Book of Tom, which may be interesting if that's your 'thing'. So here you go, another obscure group of parasites has been brought to light, and hopefully not mangled too much in the process. Apparently, multicellularity can also happen 'introvertedly' -- who knew?

Phylogenetic Home: Snuggling with Haplosporidia in the land of Cercozoa
[Warning: We're going to increasingly bury ourselves deeper and deeper into technical phylogenetic details, random hypothesising, and readings from the Book of Tom. Proceed with caution. Emergency exit that way ---> ]
Naturally, back in the days of crown eukaryotes and the protozoan-metazoan divide, Paramyxids were especially confusing taxonomically. They seemed multicellular-ish, thereby tempting researchers to place them with something 'higher', like metazoan-y things. However, the presence of similar inclusions to those which the Haplosporidia are known for -- haplosporosomes -- alluded to their relationship even back in the 70's. (eg. this French paper compares various Haplosporidia with Marteilia) Sprague (1979 Mar Fish Rev) published a taxonomic paper wherein they were lumped with Haplosporidians under the phylum Ascetospora, described as the following:
Phylum IV. ASCETOSPORA ph. n. (ascet- Gr. asketos, curiously wrought. Refers to the strange and complex spore structure, recently revealed with the electron microscope.) Spore multicellular (or unicellular?), with one or more sporoplasms. without polar capsules or filaments; parasitic.(Sprague 1979 Mar Fish Rev)
Remember, this was back in the day when Protista was a massive taxonomic MESS consisting of random stuff clumped together based on superficial morphological appearances. Ultrastructural studies messed this up dramatically, but true hell happened once sequencing became more available. A bit of a disaster struck when it was decided that sequencing should be as easy as 1. grab ribosomal DNA 2. sequence 3. align 4. ??? 5. Profit. Well, things did get aligned, and a lot of the rDNA data was quite consistent. Unfortunately, in some cases, it was consistently wrong.

Paramyxids seem to have suffered from the same taxonomic affliction as microsporidia -- namely, extreme [intracellular] parasitism tends to drive genomes to do wonky things. Of course, wonky genomes tend to provide habitat for some rather non-conformist sequences for homologues of conservative conserved genes. Ribosomal DNA is no exception. As we've seen before, highly diverged sequences tend to group with each other due to Long Branch Attraction. You can probably guess where this is headed: Paramyxids were yet another ancient eukaryote, branching right after Microsporidia (Berthe et al. 2000). You'd think people would be a little more perplexed by the bulk of 'ancient' eukaryotes being parasites...of eukaryotes. Yeah.

After having spent a few hours earlier digging through some rather unhappy-looking trees to find what paramyxids actually are, I'll just settle for TC-S and Chao (2002, 2003) , who place them in "Ascetosporea" (Sprague 1979), sister to Haplosporidians. According to TC-S and Chao (2002, see fig.6), both ["excessively long-branch"]Paramyxids and Haplosporidia contain a characteristic Cercozoan SSU V6 terminal loop deletion. That and the haplosporosomes should be enough for out purposes. According to Tom, they seem to group with Phytomyxea, a clade of plant pathogens containing that nasty sworn enemy of the Brassicales, Plasmodiophora brassicae. He goes on to suggest a homology between Plasmodiophorid 'cored vesicles' and haplosporosomes, thereby forming a potential synapomorphy for a group he calls Endomyxa, a new subphylum of Cercozoa. Furthermore, he suggests chemically targetting those haplosporosomes/cored vesicles to treat some of the commercial afflictions their organisms cause. See, real men do all that within a single paragraph of one paper. Oh yeah.

I'm trying to figure out what those 'cored vesicles' are, and so far I just found these unconvincing things in fig8 here (if you're still here, and don't have access, 1. Holy crap you're awesome for hanging in there for so long! 2. It's your garden variety random vesicles. Nothing much to look at...also, I'm just rambling right now. Seriously, not missing out on anything =P). Oh, there's some interesting-looking 'osmiophilic bodies' in Polymyxa (fig 3). To be honest, a lot of things have mystery vesicles, so I'm a little of skeptical of claims like "oh hey, these random tiny blobs are homologous to those random tiny blobs!". Meh, he's right about 80% of the time, so let's pretend they are homologous, sure. See if I care.

(Hmmm, I wonder if there's anything interesting about the seemingly U-shaped nucleus in Plasmodiophora and the U-shaped sporoplasm nucleus noted in Paramyxa. Or if it's just some artefact. Or coincidence. Probably that. But still, someone wanna go check it out?)


I think I may be running out of steam now...

*[completely off-topic] (from first paragraph) While writing this, I managed to go from Paramyxids to the Novgorod Codex in about 20min. Yay Wikipedia! Ancient Russian is so damn hard to read, but I still find it amazing to be able to decipher some words and phrases, almost an entire milenium later! Interestingly, Slavic languages seem to have evolved slower than their Germanic counterparts -- not only is the language diversity in that family much lower (and mutual intelligibility quite a bit higher), but 1000 year old Slavic languages are easier for modern speakers to decipher than 1000 year old English texts for Anglophones (cf. Beowulf). How many mouse clicks am I from the socioeconomics of Fiji, and then succulents of New Mexico? Might even click our way to Higg's Boson if we keep this up...[/completely off-topic]


References
Cavalier-Smith, T., & Chao, E. (2003). Phylogeny of Choanozoa, Apusozoa, and Other Protozoa and Early Eukaryote Megaevolution Journal of Molecular Evolution, 56 (5), 540-563 DOI: 10.1007/s00239-002-2424-z

Desportes, I. (1984). The Paramyxea Levine 1979: An original example of evolution towards multicellularity Origins of Life, 13 (3-4), 343-352 DOI: 10.1007/BF00927182

Feist SW, Hine PM, Bateman KS, Stentiford GD, & Longshaw M (2009). Paramarteilia canceri sp. n. (Cercozoa) in the European edible crab (Cancer pagurus) with a proposal for the revision of the order Paramyxida Chatton, 1911. Folia parasitologica, 56 (2), 73-85 PMID: 19606783

ITOH, N. (2004). Early developmental stages of a protozoan parasite, Marteilioides chungmuensis (Paramyxea), the causative agent of the ovary enlargement disease in the Pacific oyster, Crassostrea gigas International Journal for Parasitology, 34 (10), 1129-1135 DOI: 10.1016/j.ijpara.2004.06.001

Kleeman, S. (2002). Detection of the initial infective stages of the protozoan parasite Marteilia sydneyi in Saccostrea glomerata and their development through to sporogenesis International Journal for Parasitology, 32 (6), 767-784 DOI: 10.1016/S0020-7519(02)00025-5

LARSSON IR, & KØIE M (2005). Ultrastructual Study and Description of Paramyxoides nephtys gen. n.,
sp. n. a Parasite of Nephtyscaeca(Fabricius, 1780) (Polychaeta: Nephtyidae) Acta Protozoologica, 44 (2), 175-187


SPRAGUE V (1979). Classification of the Haplosporidia Marine Fisheries Review, 41 (1-2), 40-44

4 comments:

  1. Impressive indeed!

    Reading 1000-year-old texts is easier in German than in English, but not that much either. The Slavic languages haven't undergone any major vowel or consonant shifts during that time, that's the main difference… other features have changed, for instance the tense system got reworked (differently in different languages) while it's superficially still identical in English and German.

    ReplyDelete
  2. But... What's the AdAPtiVe PURPOSE of that????

    ReplyDelete
  3. You're right, from now on, such questions shall be written in spam format. Especially in academic literature ^_^

    ReplyDelete

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