Elysia diomedea in front of Casa Caguama field station June 29, 2022.

This will probably be the last post for a while.  For starters, I will be retiring from teaching at the end of spring semester 2023.  Also, I think I have taken the project as far as I would like (but see below), and I have scurried back to the comfortable world of arthropod physiology.  I am so glad I got to know the many facets of Elysia, its behavior and biology, and it has been a fantastic opportunity to teach students about unusual organisms and the scientific process.

One of the last members of the brood hatched and raised last year.

It seems to be a good time to collect my thoughts and observations from the first seven or so years of the project.  Although I believe the field would benefit greatly from an up-to-date formal review, that is not my intention here.

Kleptoplasty is not Monolithic

When I started the project, I carried some misconceptions about the biology of Elysia.  The portrayal in the popular press at the time, and to some extent in the scientific literature, was that they were “crawling leaves” that had stolen the chloroplasts from their food plants and could therefore live indefinitely (or at least a very long time) with only light as an energy source.  My goal, in addition to using it as a teaching tool, was to understand the impact of kleptoplasty on the behavior and neurobiology of the slugs.

When I dug deeper, I discovered a lot of diversity, both between species and within a species based on which plant is the source of the chloroplasts.  For example, species can be divided into non-kleptoplastic, which digest all cellular contents immediately, short term kleptoplastic, which hold onto the chloroplasts, and long-term, which maintain functional chloroplasts for weeks or months (Christa et al., 2014).  However, even those species labeled as “long-term” vary widely in how long the chloroplasts are maintained, and this can further depend on the species of origin of the chloroplasts.

The only species that may be a true “crawling leaf” would be E. chlorotica.  Once they have obtained chloroplasts from the alga Vaucheria, they can support them for at least nine months (Green et al., 2000).  One could build a nice story that the function of kleptoplasty is to provide an energy source for this species.  Unfortunately, the story falls apart if one looks at other “long-term” kleptoplastic species, such as E. crispata or E. viridis.  Based on my own observations and those of others, these species will starve to death relatively quickly if not fed, despite the presence of actively photosynthesizing kleptoplasts.

Which brings us to the big question: Why do the slugs devote significant energy to separating chloroplasts from their food algae and maintaining them in a functional state?

The Function of Kleptoplasty: The Answer is Generally “Yes”

There have been many experiments performed to address hypotheses regarding the function of kleptoplasty in Elysia.  Some have been truly elegant, others not so much, but they have generated a lot of information and some insight regarding the benefits of stolen chloroplasts.  For me, one of the most important insights is that the chloroplasts are likely to provide multiple benefits to their hosts.

Here are some leading hypotheses:

Trophic Support: providing energy as nutrients or storage.

• Carbon (sugar) production: Kleptoplasts continue to produce sugars and there is abundant evidence that that these reach the slugs’ cells (e.g., Cruz et al., 2020).
• Storage tissue: Kleptoplasts act as a “living larder,” providing energy and nutrients during times of fasting (Laetz and Wagele, 2018).

Metabolic Support: Aiding cellular metabolism by either removing CO₂ and other waste products, or producing O₂.

• Waste removal: Kleptoplasts can waste products from the slugs’ metabolism as substrates for energy production (Cruz et al., 2020).
• O₂ production: It has been demonstrated multiple times (e.g., Dionisio et al., 2018) that kleptoplastic slugs produce excess oxygen during photosynthesis.

Camouflage

• Visual: The chloroplasts are responsible for the green color of Elysia species and undoubtedly conceal them from predators.
• Chemical: metabolites from the kleptoplasts may help them to match the odors of their food plants and evade predators such as nudibranchs that hunt using their sense of smell (Gavagnin et al., 2000)

Chemical synthesis: Using biochemical pathways of the chloroplasts to make chemicals for the slug.

• Egg production: Elysia provided with both food and light produce more eggs than those with only food (Shiroyama et al., 2020), and kleptoplasts have been shown to produce lipids that could support egg production.
• Defense: E.rufescens uses the chemicals produced by kleptoplasts taken from Bryopsis algae for its own defense, with the added twist that the alga is using a bacterial symbiont to make the chemicals (Zan et al., 2019).

In short, there is support for nearly every hypothesis that has been proposed.  Taking these results at face value, kleptoplasty can provide a range of benefits to the host slug.  Each species may then exploit a subset of the resources that would depend on its food plant, ecology and evolutionary history.

Where Now?

Re-review and re-think.  One crucial step in untangling the web of hypotheses would be a thorough re-review of the literature, emphasizing the biological diversity of kleptoplastic species (including Elysia and Plakobranchus) rather than pursuing a unified theory.  We already know that long-term kleptoplasty arose multiple times among sacoglossan species, so it is likely that the precise molecular mechanisms and metabolic benefits will vary across the group.  Given the size of the literature pool, a monograph may be more appropriate than a simple review article.

Molecular mechanisms.  A large amount of data has been already collected regarding possible functions of kleptoplasty, so it may be time to shift focus from “what is the benefit of kleptoplasty?” to “how does it work?”  The tools (if not the financial support) exist for rigorous examination of the ways in which chloroplasts are separated from the rest of the cellular contents of the algae, transported to the slugs’ tissues and maintained for months at a time.  Based on a career spent studying physiology, I fully expect that the diversity of function described above will be reflected in a diversity of cellular and molecular mechanisms.

There are some very talented people working on this line of inquiry, and I hope to have a chance to post about their discoveries in the future.

What about me?  The project has largely served its purpose.  Using the unusual biology of Elysia, several groups of students at USG and at Ocean Discovery Institute were introduced to topics such as PCR barcoding, chemical ecology, the neurobiology of behavior, and physiological ecology.  In the process, I was able to take deep dives into literature and develop experiments to test the hypotheses we developed.  Heck, I even learned how to culture E. crispata from egg to adult.  At some point, I will want to take the lessons I have posted on the Solar Slug site and turn them into something more accessible.

Coulometric respirometer measuring O2 consumption of scorpions.

For now, I have shut down the slug system, and have shifted back to arthropod physiology.  We have developed methods for measuring respiration in fruit flies, beetles, and scorpions, and it may be time to get to work popularizing the methods and results of that work.

Scorpion in respirometry chamber.

As long as E. crispata and its food, Bryopsis plumosa, are available, the project can always be restarted with a few emails.  So, this is not “goodbye” as much as “see you later.”

References:

Christa, G., Händeler, K., Kück, P., Vleugels, M., Franken, J., Karmeinski, D., Wägele, H. (2014) Phylogenetic evidence for multiple independent origins of functional kleptoplasty in Sacoglossa (Heterobranchia, Gastropoda). Organisms Diversity and Evolution, 15 (1), pp. 23-36

Cruz S, LeKieffre C, Cartaxana P, Hubas C, Thiney N, Jakobsen S, Escrig S, Jesus B, Kühl M, Calado R, Meibom A. (2020) Functional kleptoplasts intermediate incorporation of carbon and nitrogen in cells of the Sacoglossa sea slug Elysia viridis Sci Rep. 10: 10548.

Dionísio G, Faleiro F, Bispo R, Lopes AR, Cruz S, Paula JR, Repolho T, Calado R, Rosa R. (2018) Distinct Bleaching Resilience of Photosynthetic Plastid-Bearing Mollusks Under Thermal Stress and High CO(2) Conditions. Front Physiol. 9:1675.

Gavagnin M, Mollo E, Montanaro D, Ortea J, Cimino G (2000) Chemical studies of Caribbean sacoglossans: Dietary relationships with green algae and ecological implications. J Chem Ecol 26(7):1563–1578.

Green, B.J., Li, W.-Y., Manhart, J.R., Fox, T.C., Summer, E.J., Kennedy, R.A., Pierce, S.K., Rumpho, M.E. (2000) Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus. Plant Physiology, 124 (1), pp. 331-342.

Laetz EMJ, Wägele H. (2017) Chloroplast digestion and the development of functional kleptoplasty in juvenile Elysia timida (Risso, 1818) as compared to short-term and non-chloroplast-retaining sacoglossan slugs. PLoS One 12: e0182910.

Shiroyama H, Mitoh S, Ida TY, Yusa Y. (2020) Adaptive significance of light and food for a kleptoplastic sea slug: implications for photosynthesis. Oecologia 194: 455-463.

Zan J, Li Z, Tianero MD, Davis J, Hill RT, Donia MS. (2019) A microbial factory for defensive kahalalides in a tripartite marine symbiosis. Science. 364: eaaw6732.