Monthly Archives: May 2016

Journal Club: Why on Earth do they store chloroplasts?

The subject of this year’s Journal Club (Has it really been over a year?  Oh dear.)  is a paper by Gregor Christa and colleagues from back in 2013.

Christa 2014 front page

In this paper, the authors try to develop a scientifically rigorous explanation for long-term retention (LTR) of chloroplasts in Plakobranchus and Elysia.

Some species of marine slugs in the Plakobranchoidea, which includes the genera Elysia and Plakobranchus, can store chloroplasts for many months.  These are stored in branches of the digestive system that ramify throughout the slugs’ bodies.  Perhaps foreshadowing their conclusions, Christa et al. refer to this phenomenon as delayed digestion, rather than maintenance of the chloroplasts.

So what do the food-derived chloroplasts (“kleptoplasts”) do for these months?  In 1975*, Trench found that Elysia viridis could incorporate 14C from labeled CO2 into its tissues, and concluded that kleptoplasts retained the ability to generate sugars from light, water and CO2.  Based on this observation, he coined the term “leaves that crawl” to indicate that he believed the kleptoplasts generated energy for the slugs and made them partially or totally independent of the need for ingested food.  This idea took hold, both in the scientific literature and in the popular imagination.

There were a few complications in this story, however.

First, how are functional kleptoplasts maintained for such a long period outside of a plant cell? A functioning organelle needs a constant supply of new proteins to replace those that are degraded with time and use.  Although chloroplasts retain a small number of genes (including rbcL, which is important for the kleptoplast identification project), the vast majority of genes required for their continued function are located in the nucleus of plant itself.  That is a problem, because the plakobranchids rapidly digest the nuclei of the algae on which they feed.

The problem appeared to be solved, and in an interesting way, when reports appeared that suggested the slugs had incorporated algal genes into their own genomes, a phenomenon called “lateral gene transfer,” or “horizontal transfer of genes.”  Newly-synthesized  algal RNAs and proteins were identified in slugs (reviewed in Pierce et al., 2007).  However, the absence of algal gene products in eggs or newly settled slugs (Bhattacharya et al., 2013), and the relative scarcity of chloroplast RNAs and proteins in slugs, indicates that lateral transfer is unlikely to have occurred.  It seems unlikely that kleptoplasts could be self-sustaining in the long term.

Another issue is that animals are capable of incorporating small amounts of CO2 into organic molecules, through carboxylation reactions.  This being the case, how do we interpret Trench’s results of 14C incorporation into E. viridis?

Furthermore, starved plakobranchids (the shorthand term Christa et al. use for Elysia and Plakobranchus) do not stay fat and happy if they are provided with light.  They shrink, and change color from green to yellow, which indicates that they are breaking down their tissues and the kleptoplasts, in order to survive.

Back to the original question: what is the purpose of storing kleptoplasts?  Christa et al., asked two very basic questions about the role of kleptoplasts, using E. timida and Plakobranchus ocellatus.

  1.  Are the kleptoplasts really involved in CO2 fixation?  To answer this, they compared incorporation of 14C in slugs that were kept in the light, in the dark, or in the light but treated with a drug that inhibits photosynthesis.  If the kleptoplasts are involved, then 14C incorporation will be higher in conditions that allow photosynthesis.
  2. Does the ability to photosynthesize reduce the rate of tissue loss during starvation?  If slugs are kept in the dark, or treated with drugs that inhibit photosynthesis, do they lose weight faster than slugs kept in bright light?

The answer to Question 1, was an emphatic “yes, 14CO2 incorporation is much higher in the light.”  As shown in Figure 2, below, both E. timida and P. ocellatus showed significant incorporation in the light at 60 and 120 minutes, but essentially none in the dark.  Incorporation was also significantly reduced by monolinuron, which blocks photosynthesis.


So, it appears that the kleptoplasts fix carbon, which should provide energy to the slugs.  Does this keep the slugs from starving?

This brings us to question 2.  The authors examined two aspects of starvation to determine how the different conditions affected the ability to photosynthesize and maintenance of body weight.

First, they looked at what happens to chlorophyll function during starvation using Pulse Amplitude Modulated (PAM) Fluorimetry.  The authors suggest that those kept under more intense light declined more rapidly, but the sample sizes were small and the variation was large, so the strongest conclusion one can safely make is that quantum yield (their measure of photosynthetic function) decreased significantly with starvation.

Perhaps the most interesting result was that exposure to light made no difference in terms of weight loss.  Figure 4b, below, shows that all slugs, regardless of whether they were in light, darkness or treated with monolinuron, lost about the same amount of weight over 50 days.  Christa_2013_fig4b

Again, the sample sizes were tiny (two slugs each), so sweeping conclusions are not in order.  However, the shapes of the curves are intriguing.  The slugs in the light seemed to decline more rapidly than those in the dark, and the monolinuron treated slugs both showed a rapid decline followed by more gradual weight loss.  It would be interesting to know if there was interesting biology underlying these results (slugs burn through reserves faster in the light?), or whether they are quirks of a small sample.

Christa et al. conclude that the slugs are not photoautotrophic, i.e., they do not survive on light.  Instead they propose that the kleptoplasts are a food reserve.  This is certainly a plausible model, but others have not yet been ruled out.

For example, the authors suggest that the kleptoplasts could be required for the biosynthesis of compounds required for development or egg production.  One potentially interesting, and relatively straighforward, experiment would be to compare rates of egg laying between Elysia that are fed, but kept under high- and low-light conditions.  Given the metabolic demands of producing and provisioning egg masses, it would be interesting to see if kleptoplasty contributes to the process.

Another possibility is that the kleptoplasts help to “fatten up” the slugs before starvation.  In this scenario, slugs use both food and photosynthesis to fill storage tissues when times are good.  When food is scarce, or experimenters starve them, they use the kleptoplasts and stored tissue to produce energy and necessary metabolites.  This may be especially important for species that live on scattered or sparse food sources.  I don’t think anyone has done the math to determine how the energy estimated to be produced by photosynthesis (e.g., the amount of carbon fixed per unit time by the slugs in the light) compares with the metabolic demands of the slug.  It may be too small to keep a starving slug alive, but might help fatten up a feeding slug.

One final random thought regards the maintenance of kleptoplasts.  It is pretty clear that the slug does not have the genetic equipment to produce the proteins required for the upkeep of the chloroplasts.  However, plakobranchids (at least the E. clarki watching me here in my office) eat prodigious amounts of algae daily.  Would it be possible for ingested algal RNAs and proteins to find their way to the kleptoplasts?  It seems like a non-trivial problem to get the materials across a few membranes and into the plastids, but it would help to explain how chloroplasts can survive for months in cells that are lacking important equipment.


*note that all the references can be found in the full list of papers.

Kleptoplast Extraction

We made our first try at extrtacting and amplifying DNA from kleptoplasts.  It was relatively straightforward to get the tissue from the slug.  First, she was “relaxed” with isotonic MgCl2, which blocked synaptic transmission and paralyzed and anesthetized her.

Elysia clarki relaxed with 400 mM MgCl2. 4/27/16

Elysia clarki relaxed with 400 mM MgCl2. 4/27/16

Then, a small piece of parapodium was removed (see her left side, at bottom).  DNA was extracted using the same method as for the plants.

Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16

Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16

In case you were worried, she was fine the next day.

Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16

Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16

We also tried a new species of algae, which I am calling Avrainvillea.  It may be Rhiphilia, I’m not completely certain.

Alga extracted 4/27/16.  Probably Avrainvillea.

Alga extracted 4/27/16. Probably Avrainvillea.

Unfortunately, the amplification was not a success this time around.  The positive control samples, DNA extracted from Bryopsis during previous sessions, worked well.  “Avrainvillea” showed a much weaker signal, and there was no signal from the slug extract.

Results from week of 4/25/16

Results from week of 4/25/16

A number of things may have gone wrong.  The slug sample was extremely slimy with mucus, and those polysaccharides could have interfered with extraction or amplification.  Also, the Bryopsis-specific primers may not have annealed adequately with the DNA from Avrainvillea and whatever is in the slug.

Now that things are calming down after the semester, I am going to try again, making a few changes.  Most importantly, I have ordered “degenerate” primers, which contain a mix of sequences that will complement just about any algal sequence.  Still waiting on tips regarding the slime.