The Sea Slug's Guide to Plastid Adoption

by Merry

A handsome E. chlorotica. Its rich green
pigmentation is courtesy of plastids captured
from its food source, the siphonaceous marine
alga Vaucheria litorea (seen in the background).
Source.

Having an intimate relationship with photosynthetic microbes is a widespread strategy adopted by numerous unicellular and multicellular organisms. Some eschew a committed relationship, and simply nab the plastids, sequestering them inside vacuoles where they continue to photosynthesize for a while. Previously we reported on a ciliate that captures the algal nuclei, as well, to support the plastids, and a flagellate that seems to in the process of converting their plastid into a well-mannered organelle. What about us metazoans? So far there is only one group known to practice this kleptoplasty—the sap-sucking sacoglossan sea slugs—and, so far, only one genus among them is known to have made this a long-term relationship.

The best-studied species here is Elysia chlorotica. These naked molluscs pass their quiet lives in salt marshes from Chesapeake Bay to Nova Scotia. Eggs laid each spring hatch into planktonic veliger larvae that home-in on the one particular species of algae that they use for food, Vaucheria litorea. The larvae attach to the algae where, within 24 hours, they metamorphose into hungry juvenile slugs that begin feeding. During winter months the slugs are inactive; with the arrival of spring and warmer water, the hermaphroditic adults lay their eggs and die soon thereafter.

The large cells of Vaucheria are coenocytic, each containing many nuclei and plastids. The slug slits open the cells with its radula (a rasp-like tongue), then slurps out the algal contents, plastids and all. (The movie below includes a stunning closeup view of a young slug's first meal.) The plastids are selectively taken up by specialized cells lining a portion of the gut and sequestered in vacuoles (by a mechanism as yet unknown). Here, the plastids keep on working. When given ¹⁴CO₂, the ¹⁴C is later found in various slug metabolites. Young slugs that have acquired their first plastids can complete their entire life cycle (~10 months) in aquaria without algae so long as they have light.

In these algae, more than 90% of the plastid proteins are encoded by nuclear genes. How do these algal plastids survive without support from the algal genome? This is an urgent matter as some of the proteins that make up the light-harvesting apparatus suffer ongoing damage and need to be replaced frequently. Researchers incubated algae and plastid-containing slugs with [³⁵S] methionine, then isolated their plastids. The plastids showed similar arrays of labeled proteins. Moreover, some of those plastid proteins synthesized in the slugs are synthesized on cytoplasmic ribosomes, not in the chloroplasts.

Subsequently, these researchers showed that the slug’s nuclear genome contains genes for at least three plastid proteins that are encoded in the algae’s nuclei. The slug also expresses several genes for the chlorophyll synthesis pathway and synthesizes chlorophyll a. These genes are present even in pre-hatched larvae that have never fed and do not contain plastids—strongly suggesting that the genes have been incorporated into the slug germline DNA and are vertically inherited. Another lab zeroed in on a gene from photosystem II (psbO) and found it in the DNA from E. chlorotica eggs—more evidence for germline transmission. Looks like this is an instance of horizontal gene transfer (HGT) between two eukaryotes, from the nuclear genome of the algae to that of the slug.

Portraits of E. chlorotica. (A) Free-swimming veliger larvae. The green coloring in the digestive gut is due to planktonic feeding, not plastid acquisition, at this stage. (B) The first meal of a metamorphosed juvenile. Plastid acquisition by feeding is required for continued development. (C) Young adult 5 days after first feeding. (D) Adult. (Bar = 100 µm in A, 500 µm in B-D). Source.

E. chlorotica is not the only "solar-powered" sea slug. E. clarki, found in the Florida Keys, maintains its plastids for up to 4 months. More accomodating than E. chlorotica, it acquires plastids from four different species of algae—sometimes housing plastids from two or more species in the same cell.

Mature icosahedral viruses with double envelopes budding
into cytoplasmic vacuoles in a hemocyte from a dying slug.
Bar = 0.5 µm. Source.

These stories leave us wondering just how this particular HGT might have occurred. It's a long way from the nucleus of an alga being digested in the gut of a sea slug to the nuclear DNA in the slug's germ cells. No definite answers yet, but some suspect that an endogenous retrovirus may have played a role. Evidence? First, a brief digression back to the slug life cycle. The entire adult population dies synchronously each spring. Slugs in lab aquaria die at the same time as those in the field, regardless of the time of year when they had been collected.(How’s that for timing?) TEM of dying slugs, but not of younger slugs, revealed viruses in their cells. The enveloped icosahedral virions resemble those of known retroviruses; most tellingly, their bloom is associated with a hundred-fold increase in reverse transcriptase activity. Smaller capsids are also seen in the plastids.

Are these 'retroviruses" killing the slugs? Some of the researchers don't think so. Many cells in the dying slugs show characteristics of apoptosis, suggesting programmed cell death. The viruses might maintain a latent infection and then reactivate when the slug's defenses have been compromised. Did these viruses have something to do with the algal→slug gene transfer? Maybe. Retroviruses are a feasible vector to ferry the algal genes to the slug genome. Would be nice to have the complete genome sequence for this virus so we could see if algal genes are on board. Meanwhile, it is early summer in the marshes and another generation of juvenile slugs have gone green.

Pierce, S., Curtis, N., & Schwartz, J. (2010). Chlorophyll a synthesis by an animal using transferred algal nuclear genes Symbiosis, 49 (3), 121-131 DOI: 10.1007/s13199-009-0044-8

Rumpho ME, Worful JM, Lee J, Kannan K, Tyler MS, Bhattacharya D, Moustafa A, & Manhart JR (2008). Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proceedings of the National Academy of Sciences of the United States of America, 105 (46), 17867-71 PMID: 19004808

Comments

[Mark O. Martin]

And the craziness keeps on keeping on...

http://www.nature.com/news/2010/100730/full/news.2010.384.html

Symbiogenesis fever. Catch it!

[Juliet Pendray]

Thank you for checking Merry,

Below is the base reference that took me on a hunt for more info on certain algicolous marine fungi, but I'm almost certain I found something concrete and more current, discussing Elysia species browsing preferentially on fungi-weakened algae. The reference was a bit off topic for me at the time, presumably why I don't recall this well.

Kohlmeyer/Kohlmeyer "Marine Mycology: The Higher Fungi" 1979 page 64 "Didymosphaeria danica ... restricted to cystocarps of Chondrus crispus ... Often holes can be observed in the tips of C. Crispus where cystocarps had been located. Either infected algal fruiting bodies fall out after senescence of the fungus or marine animals feed preferentially on the parasitized tissues."

But yes if I find a better clue - or the material itself if I'm lucky - I will send it along to you.

Yours in curiosity,
Juliet

[Zwirko]

Are there are any documented experiments where chloroplasts have been transferred into mammalian cells in tissue culture? I've been trying to find an example of such a study, without much success. I realise that this wouldn't make photosynthetic animal cells for a whole bunch of reasons, but I was wondering what would actually happen and has it been studied? For example, how long would the chloroplast survive, would it be targeted for degradation, how long does it remain photosynthetic, how long do its remains hang around in the cell, would it be toxic to the cell etc etc?

Anybody know if such a study has been done before?

Merry replies:

I do not know of any such studies, but perhaps some of our readers do. Meanwhile, thought-provoking questions to ponder. Thanks, Zwirko.

[Juliet Pendray]

Intriguing article, intriguing critters - thank you for writing and posting this!

I recall, hopefully correctly, that there is a fungal connection to Elysia's story, something about the slugs preferentially browsing on areas algae colonized by fungi (or possibly a specific fungus). Unfortunately I can't remember what the implications were of that.
Did this come up in your research by any chance?

Thanks!
Juliet

Merry replies:

Thanks for your comment, Juliet. In my reading, I had not run into anything about sea slugs wanting a fungus served with their algae. I just did a search through the research papers I used as my sources and found no mention of fungi, and some of those papers go into quite a bit of detail about the natural history of E. chlorotica. Terrestrial slugs, on the other hand, do feed on fungi, and fungi have been in the news of late for their role in horizontal gene transfer, but to aphids. If you come up with more clues, please pass them on to me.