One of my Slug Club students recently asked me whether it is really worth pursuing the study of obscure, squishy creatures in an era during which biological science seems obsessively focused on “translational” research to apply scientific discoveries to clinical problems.  My response was that, while translational research is unquestionably important, the study of the biologically weird is where we discover unexpected fundamental truths about the way organisms function.  This new insight often lays the groundwork for new areas of research, and can, in time, lead to improvements in people’s lives.

Which brings us to the paper at hand: Zan et al. (2019) “A microbial factory for defensive kahalalides in a tripartite marine symbiosis.”  Science 364: eaaw6732 (published online ahead of print).  This paper not only taught me something new about Elysia, but I discovered methods and biochemical pathways I had never heard of.

For those who do not want to bob and weave their way through the entire journey of discovery, here is the quick summary: The authors discovered a bacterium living within the species of Bryopsis algae upon which Elysia rufescens feeds. This bacterium is responsible for the production of a chemical compound called kahalalide F (hereafter “KF”).  The following observations are what make the story more interesting:

1. The bacterium makes KF by stitching together amino acids using a biochemical pathway very different from that used by animals and plants.
2. The bacterium has adapted to the comfortable life inside plant cells by getting rid of many of its genes. 
3. Although E. rufescens concentrates KF in its tissues to deter predators, it does not maintain the bacteria in its tissues.  This contrasts with the chloroplasts from Bryopsis, which the slug separates from the other cellular components and continue to photosynthesize.

If that sounds interesting to you, then read on.

It was very exciting to discover a paper about E. rufescens and its food plant Bryopsis, in the journal Science during my lunchtime reading this week.  Science is one of the two most-read scientific journals in the world, so any article must be splashy and important enough for a wide readership.  That a study about Elysia was deemed worthy of publishing there pleased me to no end.  To be honest, the paper is not really about E. rufescens, nor about Bryopsis, but about a bacterium that lives inside Bryopsis, but the alga and slug still play prominent roles.

A quick literature search for E. rufescens (or a peek at the E. rufescens page on this site), will show you that the species has been mostly studied because it contains a defensive molecule called Kahalalide F (KF).

Kahalalide F: A Complex Defensive Compound

At first glance, KF looks like an organic chemist’s nightmare (or dream, I suppose), but on closer inspection it is a peptide, i.e., a short string of amino acids. As described below, KF has a number of unusual features for a peptide. From an ecological perspective, Becerro and colleagues showed that KF is a strong deterrent to predation. KF has also been found to have anti-cancer activity, so there is a medical motivation to study it.

The source of KF was not completely clear. It was assumed to be produced by the Bryopsis species (not yet assigned a species name, so I will simply call it “Bryopsis“) on which E. rufescens feeds. The slugs not only avoid the toxic effects of KF, but they concentrate the compound in their tissues at a higher level than is found in Bryopsis. KF has a few weird chemical features that suggested to researchers that it might be made by microbes and not the alga. For example, there is a fatty acid stuck to one end of the molecule, and it contains a number of “non-proteogenic” amino acids, such as ornithine or dehydrobutyrine, which are features of compounds produced by bacteria.

To test their hypothesis that KF has a microbial origin, and to better understand how it is synthesized, the team used a wide variety of methods, including metagenomic, metatranscriptomic, evolutionary genomic, and biochemical analyses, along with some more traditional fluorescence microscopy. If all the “meta-” and “-omics” has your brain in a knot, don’t panic yet; I will do my best to explain the methods as we go.

First, they chemically extracted the Bryopsis from their collection location to be sure it contained KF. That being done, they extracted DNA from the Bryopsis and any microbes that happened to be in or on the algae when it was collected. Using high-throughput sequencing, which breaks up DNA into fragments and sequences the whole genome, they analyzed the ribosomal RNA from the sample. All organisms have ribosomes that enable them to synthesize proteins, each ribosome contains RNA that helps it function, and the ribosomal RNA from each species has a unique sequence. The Bryopsis sample contained RNA largely from three bacterial classes, one of which was the Flavobacteriia. Of the Flavobacterial sequences, one species comprised 74 to 92% of the sample, making it the most abundant species associated with Bryopsis. To jump ahead again, they ultimately found that this bacterium was a new species, that it was responsible for the synthesis of KF, and gave it the provisional name “candidatus Endobryopsis kahalalidefaciens” (candidate kahalalide-making from inside Bryopsis).

How did they get there? First, they had to figure out the general way the peptide was made. If you are like me, and focus narrowly on multicellular creatures such as animals and plants, then you could be forgiven for thinking there is only one way to make a peptide, which is essentially a short protein. Turns out there are at least two.

RiPP or NRPS?

In the more canonical approach to making a protein, the cell uses a DNA sequence as a set of instructions to assemble a chain of amino acids. The DNA is read by an enzyme that makes (“transcribes”) messenger RNA (mRNA), and that mRNA is read (“translated”) by a ribosome, which sticks amino acids together in the correct order. Because some of the amino acids in KF are weird, they would have had to be modified after being assembled, so the authors termed this possibility “ribosomal peptide synthesized and posttranslationally modified peptide” pathway, or RiPP.

There is, however, another way that bacteria make peptides, called non-ribosomal peptide synthesis, or NRPS. Unlike a ribosome, which can make any protein or peptide that can be encoded by an mRNA sequence, NRPS requires a collections of enzymes customized for each peptide. NPRS enzymes are composed of modules that assemble the amino acids, modify them as needed, add fatty acids, and so on. This was completely new to me, but there is apparently quite a lot known about it.

In the good old days, it might have taken a lifetime to identify the biochemical pathway that the bacterium uses to make KF. Now that researchers are capable of sequencing, assembling and analyzing whole genomes, it just takes a lot of really hard work. The researchers sequenced all of the genes from organisms associated with Bryopsis, looking for either:

1. A gene that encodes the sequence of a predicted peptide, which could then be transcribed to mRNA and translated into a peptide. This would indicate that KF is made via RiPP.
2. A cluster of genes that encode the number and type of modules that would be expected to synthesize KF, based on the sequences of known NRPS enzymes. Finding this would support NRPS as the source of KF.

They did not find a gene with the sequence predicted to encode the KF peptide, suggesting to them that KF was unlikely to be produced via RiPP.

KF Is Made Through NRPS

They combined the data they had from the high-throughput sequencing with a second method of sequencing that produced longer fragments of DNA sequence (“longer reads”), and assembled the entire genome of a bacterium. The genome contained multiple clusters encoding NRPS enzymes, one of which, which they called NRPS-8, had features expected to be required for KF synthesis:

1. A predicted condensation domain, which would be needed to chemically bind the fatty acid chain (red in the diagram above) to the first amino acid.
2. A total of 13 predicted NRPS modules, as would be expected for an enzymatic pathway that makes a peptide consisting of 13 amino acids.
3. Eight of the NRPS modules contain domains that are expected to convert normal L-amino acids to D-amino acids (shown in blue in diagram).

Based on these features, the authors were confident that they had discovered the enzyme that produces KF, and therefore that KF is synthesized by the bacterium through the NRPS pathway.

Including NRPS-8, which the genome sequence of the bacterium contained genes encoding a total of 20 NRPS pathways. Intriguingly, previous work had extracted at least 15 kahalalides from Bryopsis and E.rufescens. When the authors compared the predicted NRPS pathways to the structures of the kahalalides, they could match eight of the “chemotypes” (chemical compounds with the same amino acid sequence, but differing in modifications) to bacterial NRPSs. They conclude that “Candidatus Endobryopsis kahalalidefaciens” (hereafter “cEK”) is responsible for at least nine, and probably all, of the kahalalides produced by Bryopsis.

The Bacterium Is Losing Genes

Based on the complete genome sequence, it was clear that cEK has lost some genes. When they compared its genome to a closely related, but free-living species, they found hundreds fewer genes. The missing genes encoded proteins that performed such functions as DNA repair, detoxification, chemotaxis (moving toward or away from chemical stimuli), adaptation to unusual conditions, and, notably, synthesis of amino acids. All of these would be needed for living in a cruel, variable world, but may not be necessary for a comfortable life associated with an algal cell that provides stability and nourishment.

The fact that the authors were unable to culture the bacterium separately from the alga supports the idea that it is dependent on Bryopsis for survival. Further, by labeling DNA specific to cEK, they showed that the bacterium is found inside the plant cells (see panel from Figure 4, below).

Figure 4D from Zan et al., showing the location of bacteria inside a cell of Bryopsis sp. Yellow dots indicate bacteria labeled with a probe specific for “Candidatus Endobryopsis kahalalidefaciens,” and are indicated by arrowheads labeled “cEK.” Other bacteria that do not contain the cEK marker, are labeled red, and are indicated by the arrowheads labeled “OB.” The cell wall of the alga is labeled blue.

Therefore, cEK appears to be an “endosymbiont,” an organism that lives symbiotically inside the cells of another, and is probably completely dependent on its host alga for survival. In exchange, the bacterium provides a collection of toxic compounds that reduce grazing on the alga. The importance of kahalalide production to the bacterium is underscored by the observation that a full 20% of the genome is devoted to genes devoted to NRPS pathways.

Slugs Do Not Host cEK

As a devotee of Elysia, you are familiar with kleptoplasty, the phenomenon by which the slugs take the chloroplasts from their algal food and maintain them in their bodies as an energy source. If the slugs have a propensity to keep useful cellular components in their bodies, one might ask whether they also store the cEK as a constant source of KF to deter predators from eating them.

The authors confirmed previous work that E. rufescens‘ tissue contains KF, and went on to look for evidence of cEK bacteria in the slugs’ bodies. Using highly sensitive methods to hunt for cEK DNA in the tissues, they found no evidence that the slugs contained significant populations of the bacteria. Attempts at labeling tissues with markers specific for cEK were also unsuccessful, despite positive labeling of other bacterial species. They conclude that, in contrast to their theft and use of algal chloroplasts, E. rufescens digest the bacteria fully when they suck the sap out of the Bryopsis cells.

Conclusions

Once again, biology comes up with a story that is better than fiction. A bacterium infects an alga, takes up residence, and starts using an unusual biochemical pathway to produce nasty compounds that protect the alga from being eaten. Natural selection favors those algae that contain the toxins (and can tolerate them), so the bacteria are now found throughout the species, and possibly even more broadly. The sheltered existence inside the alga allows the bacterium to lose many of the genes it would normally rely on to survive in the outside world, eventually rendering it incapable of independent life. Elysia rufescens can tolerate the toxins produced by the bacteria, and develops mechanisms to concentrate the kahalalides in its tissues.

Do Byopsis pennata or B. plumosa, the favorite foods of E. clarki, contain cEK or a relative? I expect we will know soon enough.

Because the goal of this post was to bring attention to the original work, I have glossed over a huge amount of information regarding the methods used, the biochemistry of kahalalides, and the genomic structure of cEK. I would encourage anyone who is interested to get access to the original article at Science Magazine.

Ah, the Good Old Days, when “curiosity-driven” science wasn’t naughty.  Of course, one might not want to get too nostalgic when the woman whose work is the subject of this post is referred to as “Miss Lillian Russell” in the running head of the paper.  Apparently, gender and marital status were considered relevant in a scientific publication 90 years ago.

The primary goal of the Solar Sea Slug project is to study the neurobiological specializations of photosynthetic molluscs.  In order to chart the input and output pathways, we need a good roadmap of the central nervous system (CNS) and the nerves that connect it with the rest of the animal.  I had hoped that someone had generated at least a crude diagram, and had found some drawings of the nervous systems of Elysia viridis (Huber, 1993, J. Moll. Stud. 59:381) and E. crispata (Gascoigne, 1972, Trans. R. Soc. Edinburgh 69:137), but had not found much more.  I fully expected to map the peripheral nerves by myself, and we had even done some preliminary experiments with fluorescent labels for axons for Slug Club during spring semester.

As is often the case, the breakthrough came as I was burrowing through the references from one of the older papers.  Huber’s 1993 paper reviews the structure of the CNS of a wide range of gastropod molluscs, and includes a nice diagram of the central nervous system of E. viridis, but provides no information about the periphery.  I had skimmed the section describing the image, but had mostly focused on the figure itself.  He made a passing reference to earlier work by Russell (1929, Proc. Zool. Soc. Lond. 14:197), so, for the sake of completeness, I ordered the paper through inter-library loan, making it clear that I would like them to include any photographic plates, which are often in a section separate from the main body of the paper.

When I downloaded the paper about a day later, I was pleasantly shocked.  As part of a study of the taxonomy of the ascoglossans (= sacoglossans), Lillian Russell had performed a thorough, detailed analysis of the nervous system, nerves, and internal anatomy of E. viridis and a nudibranch, Aeolidia papillosa. She provided an excellent description of the fusion of about seven ganglia to produce the nerve ring that comprises the CNS, along with multiple excellent drawings of the CNS, the peripheral nerves and internal organs of E. viridis.

It is hard to know which image is my favorite, but I am very fond of the drawing below, which shows the ganglia in the head, the nerves that originate from them, and the terminations of the nerves in sensory organs such as the eyes and rhinophores.

The drawing below, of the CNS and nerves removed from the animal, is also quite impressive, showing the individual ganglia that make up the CNS, and the origins of the many nerves that carry sensory information in, and motor commands out.

Aside from publishing a similar description of E. clarki (which probably only differs in slight details), and leaving a note in her will to let me know about it, I can’t imagine how she could have done any more to help me out.  Although I had intended to do the work myself, anatomical description of this quality requires considerable patience and artistic skill.  I am not very patient, nor am I a good artist.  Thanks to Russell’s hard work, I am free to do my behavioral experiments and electrical recordings, and be in my happy place.

So, why is this useful?  In a general sense, it gives a starting place for finding neurons and circuits that generate behavior.  If you want to know where sensory information goes in the nervous system, finding the nerve that carries the axons from the relevant sense organ or part of the body is a good start.  Same goes for finding the motor neurons that cause muscles in a certain part of the body to contract.  As a specific example, if we are trying to find how information about light gets to the brain, we first need to find the nerves that connect to the eyes or (hypothetically) other sense organs that detect light or photosynthetic activity. Then we can record monitor impulse activity in the nerves, or from the specific neurons with axons in those nerves.

This is a nice reminder about a few things.

1. Possibly most importantly, we truly are standing on the shoulders of others as we try to move science forward.  I hesitate to say “on the shoulders of giants,” because, for all I know, Lillian Russell was of average height.
2. You really never know when something you did will be useful to another scientist.  Ninety years ago (90!), nobody knew about kleptoplasty, and the study was carried out because nervous system structure was considered to be a good marker for evolutionary relationships.  It is not reasonable to argue that every experiment that can be imagined must be performed, but during this time in which research needs to be either “translational” or “transformative” in order to be funded, it would be nice to make a tiny bit of room for work that is simply of high quality.
3. It may be necessary to dig deep into the literature to avoid reinventing the wheel.  Many of us are guilty of looking over the figures of a paper and skimming the text, rather than reading every word.  Time is short and papers can be long.   Nonetheless, some of the most relevant information to people in the field is in the body of the text, rather than in figures or tables.  Who knows what else I will find as I work my way through the old literature.

The Solar Sea Slug project has progressed by leaps during the past half year or so.  In addition to finding the roadmap described above, and establishing a self-sustaining colony of E. clarki, some of the students in the Neurobiology Lab course helped me to work out procedures for recording from neurons in the CNS.  There are still a few improvements  to be made, such as more easily getting the electrode through the sheath that surrounds the ganglia, but we have a little real data.  Below is an action potential that was stimulated by injecting 1 nA of current into a large neuron in the abdominal ganglion (Pierce’s “Parker Cell?”).

How are we doing?

Self-sustaining culture: 

Neuroanatomical roadmap:

Dissection techniques: 

First intracellular recording:

This project is really starting to take off.

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?

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.

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.

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.  

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.