Elysia and related sacoglossans are beautiful and interesting, but their use of stolen chloroplasts puts them in a class above your average green sea slug. How the slugs accomplish this, and what function it serves, are coming into focus very slowly.

It has been known for a long time that kleptoplasts remain alive and functional in many Elysia species, but the idea that they can rely on the chloroplasts for all of their energy needs (the “crawling leaves” hypothesis) appears to be wrong, or at least overly simple.  The inadequacy of the crawling leaf theory got me thinking about a possible role for kleptoplasts as factories for making defensive chemicals.  There was support in the literature (e.g., Trench et al., 1972; Ireland and Scheuer, 1975), and I was interested enough to organize a student journal club on chemical ecology, and prepare experiments to test the connection between kleptoplasty and predation in Bahia de los Angeles in summer 2018.

To introduce the students to the journal club format, and to some important Elysia concepts, I presented the following paper by Baumgartner, Pavia and Toth from PLoS One, published in 2015.  The paper has restored some of my faith that kleptoplasty provides a substantial metabolic benefit to the host slug.

They performed a series of experiments to ask a straightforward question: Do kleptoplasts provide usable energy to E. viridis?

The main characters in Baumgartner et al., 2015. Codium (upper left), Cladophora (lower left), and Elysia viridis (right). Images of algae are from AglaeBase, and E. viridis is from Scottish Nudibranchs.

E. viridis‘ gets kleptoplasts from at least two of its natural food plants, Codium and Cladophora, and they differ in their viability in the slug.  Chloroplasts from Codium are highly functional, whereas those from Cladophora do not function well after being eaten.  This provides a natural experiment, in which one can compare the effects of light on the growth of slugs fed Codium (highly productive kleptoplasts) versus those fed Cladophora (little contribution from photosynthesis).  Importantly, the slugs in these experiments are not starved, so the authors are looking at the effects of photosynthesis over and above the energy and nutrients gained from feeding.

They tested the following hypotheses:

1. Slugs feeding on Codium under stronger light  will have higher growth efficiency (GE; defined below) and kleptoplasts will have higher relative electron transport rate (rETR) than those in low light.
2. There will be no difference between low- and high-light conditions in slugs feeding on Cladophora.
3. If there is a benefit from increased rETR, it will not correlate with other nutritional traits of the macroalgae.

E. viridis were divided into four groups: Those eating Codium,under high light (~100 μmol quanta m^-2 s^-1) or low light (~6 μmol quanta m^-2 s^-1); and those eating Cladophora in high or low light

In the first experiment, they examined the effect of illumination on growth of the slugs.  Rather than simply looking at growth rate, they used a measure called Growth Efficiency (GE), which calculated the amount of growth of the slugs as a function of how much the slugs ate.

Step 1: calculate slug Growth Rate (GR) = (M_end-M_before)/t, where M = mass and t = time

Step 2: calculate Consumption Rate (CR).  For this they needed to either measure the change in mass over time (Codium), or the number of damaged cells (Cladophora).  They had to use two different measures because the structures of the two species of algae posed different challenges.  They used algae kept without slugs as a control for the effect of time.

Step 3: Calculate GE = GR/CR, yielding the number of milligrams (mg) of slug growth per gram of algae consumed (Codium) or per 1000 cells consumed (Cladophora).  Because they were comparing the effect of light intensity on slugs fed the two algae, the different units used for consumption did not cause problems.

Figure 1A shows that slugs fed Codium show significantly better growth efficiency in high light than in low light.  Those feeding on Cladophora show no difference in two conditions.  The kleptoplasts from Codium contributed more to the growth of the slugs in bright light, which suggests that photosynthesis is producing energy that the slugs could use for growth.

However, they needed to be sure that the kelptoplasts from Codium were truly more functional than those from Cladophora, so they measured photosynthetic ability of chloroplasts and kleptoplasts using PAM fluorometry.  The absorbance and fluorescence of chlorophyll can be used to measure the health of chloroplasts, and to calculate the relative electron transport rate (rETR),  a measure of carbon fixation and therefore energy production.

Figure 2 shows that kleptoplasts from Codium responded to intense illumination with a strong increase in rETR, consistent with increased growth efficiency being caused by increased photosynthesis of healthy kleptoplasts.  Kleptoplasts from Cladophora had low rETR regardless of lighting, correlating well with the lack of effect of intense light on growth efficiency of E. viridis.  These observations support the idea that photosynthesis by kleptoplasts contributes to slug growth.

Just in case the increased growth rate of E. viridis was due to some other benefit provided by Codium, they measured nutritional quality of the two algal species.

In all cases, the nutritional value of Cladophora was as high or higher than that of Codium (Figure 3), so there was no evidence that Codium was intrinsically more nutritious.  Importantly, Panel 3N shows chloroplasts in Cladophora increased rETR in response to more intense light.  Therefore, the poor performance of Cladophora-derived kleptoplasts (Figure 2B) is likely to result from degradation inside the slug.

The evidence therefore supports the authors’ hypotheses, and they conclude that E. viridis derive measurable metabolic benefits from photosynthesizing kleptoplasts.  Whereas Slugs feeding on Cladophora receive nutrients by digesting the cellular contents (including the chloroplasts), those feeding on Codium get an extra boost from products produced by active, healthy kleptoplasts.

Overall, the experiments seemed well-executed and convincing, with the only caveat being that the slugs fed Codium and Cladophora were collected on those species and not randomly assigned to the groups.  There remains at least a formal possibility that the groups represent two populations of slugs that metabolize chloroplasts differently.

This brings us back to the the role of kleptoplasty in the biology of Elysia.  It could be:

• Increased energy available for growth (see paper above)
• Fixation of nitrogen into amino acids (Teugels et al., 2008; Journal Club in progress)
• Production of defensive chemicals (Trench et al., 1972; Ireland and Scheuer, 1975)
• Visual camouflage (they are green, no citation needed)
• Synthesis of chemicals needed for egg production (anecdotally, E. clarki appears to produce eggs when PAR is above 100)

We should bear in mind that parsimony may not equal truth, and there is no reason to believe that only one of these answers is correct for all species and life stages.