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Goodbye for Now

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.

Happy Fourth Birthday!

Baby Elysia, about three weeks old. Rhinophores well developed, diverticula full of chloroplasts from Bryopsis plumosa. 8/17/18

It seems appropriate that the site has reached an age at which kids are always asking “why, why, why?”

Looking back, the site is absolutely a reflection of my working style: make a rough plan, give it a shot, modify, repeat.  Not the work of a perfectionist, and there are some posts and pages that make me cringe when I look back.  Nonetheless, as a repository of pictures, information, and ideas, and a journal of my meandering process of discovery, the Solar Sea Slug Blog has exceeded expectations.

The past twelve months have been action-packed:

  • The student Slug Club at USG explored chemical ecology.  We spent fall semester doing a deep dive into the literature on Sacoglossan chemical ecology, learning a lot about the chemicals the slugs could be taking from food plants.  In spring semester, we took the ideas into the lab by testing whether mucus and tissue from E. clarki could keep shrimp from eating food cubes.
  • The experiments could not have been done without the availability of many slugs of different sizes. This leads into what was probably the most important breakthrough of the past year: rearing slugs from egg to adult.  Over the years, the slugs have laid thousands of eggs, most of which hatched and then starved.  This year, I discovered that the hatchlings will readily eat Bryopsis plumosa, and have ended up overwhelmed by babies.  Knowing how to rear the hatchlings, and having a culture of B. plumosa on hand, is a huge leap forward.  Now I can raise them as needed, at least in principle.
  • The breakthrough in rearing of Elysia would not have happened with input from Skip Pierce and Mike Middlebrooks in Tampa.  I had a great visit down there, and learned a ton about several species and the people who work on them.  As a bonus, I brought back my first culture of B. plumosa to get the baby slugs started.
  • We rediscovered a classic paper on Elysia neuroanatomy, recorded the the first action potentials in Elysia neurons.  There is still some work to be done to get routine, stable recordings, but this is another big step in the right direction.
  • All the stuff that happened in Bahia made for a great summer for the Solar Sea Slug Blog.  We set up a lab, collected slugs and algae, did surveys, tested feeding methods, and obtained some useful DNA sequences from E. diomedea and their food algae.  Stay tuned for the results of the feeding tests in the bay, and a map of where E. diomedea is found in the bay.

Self-indulgent reflections are not as interesting as sea slugs, so I have included some links to cool stuff below:

  • A National Geographic article about the potential decline of Elysia chlorotica, possibly the most solar of the solar sea slugs.  Thanks to Ric DeSantiago for forwarding the link.
  • Here’s another on theft of stinging nematocysts by cute little nudibranchs.  Thanks to Drew Talley.
  • Because I like seahorses, especially small ones, here is a story about newly-described pygmy seahorses in Japan.  Thanks to Joanna.

Updates for the New Year!

Happy 2018 to everyone from us at the Solar Sea Slug Blog.

Elysia zuleicae in Pierce Lab at the University of South Florida. 12/28/17

We’ve polished the place up a little for the new year, adding about 20 papers to the reference lists, and publishing species pages for E. papillosa and E. zuleicae.

Thank you all for your views and support.  Looking forward to more sluggy adventures in 2018.

So Much Happening!

Based on the absence of posts for the past few months, you can be forgiven for thinking the project is dead or dormant. In fact, the situation is just the opposite.  The semester has been so busy, there has not been time to write more than a few draft posts.  I envy people who have the time and energy both to do things and to write about them.

Bryopsis growth has continued strongly, producing about 150 to 250 grams per week of tasy slug food.  The slugs have responded by growing huge and laying eggs.

E. clarki in Box of Slugs 2.  Human finger for scale.  9/24/17

The E. clarki in the Box of Slugs 2 at home have produced a series of large egg masses (At least 12 so far, including the fresh batch below), providing many opportunities to test larval rearing ideas.  As has happened in the past, the eggs hatch, the veligers settle, but the baby slugs do not start eating.  I will finish the post describing our attempts, and a potential breakthrough, very soon.

E. clarki egg mass in Box of Slugs 2.  12/7/17

The Chemical Ecology independent study project at USG was a big success.  We went through a pile of articles about kleptoplasty, chemical defense, phototaxis, etc, which will result in at least a few Journal Club posts here.  We did a little PCR, positively identifying some of the algae we have been working with (more posts to come!).  The students also shamed me into contacting Skip Pierce and Mike Middlebrooks, about possible reasons the hatchlings haven’t been feeding, which may have led us to a solution.

As we start thinking about projects for next semester, I was getting a little despondent that none of the collectors have been able to provide E. clarki, since Irma struck the Keys.  It was a huge relief when Blue Zoo Aquatics sent me a small group of very healthy looking E. crispata last week.  Pricier than I am used to, but it’s just good to have them.

I look forward to finishing posts about all of the above, along with any surprises and breakthroughs we encounter.

Elysia Abides

The world seems to be in flux these days, but one can take a certain comfort in knowing that there are thousands of little green slugs sucking sap out of algae in shallow waters throughout the world.

Despite the travel craziness, the E. clarki and E. crispata at home have been having a nice summer.  Now that the Bryopsis factory at Shady Grove is consistently producing algae, the slugs have been face down in food almost 24/7.  The slugs are as big and colorful as I have ever seen.

Elysia clarki, Box of Slugs 2. 8/27/17

Elysia clarki on Coral, Box of Slugs 2. 8/27/17

This is looking like a great time for Elysia and slug science.

For example, at the Universities at Shady Grove, we’ll be starting a literature review on chemical ecology.  A small group of students has agreed to join me in weekly journal clubs covering chemical ecology, algal secondary metabolites, plant herbivore interactions, kleptoplasty, and other topics related to Sacoglossans and their food plants.

The preparations for Bahia in 2018 are also moving steadily.  We are identifying fudning, assembling equipment,and making detailed plans to extend the work on identifying algal food and defensive compounds of Elysia diomedea.

Check back for updates and insights as they develop.

Testing New (Old) Machines

Once again, I find myself apologizing to the hordes of Solar Slug fans for the long period of silence.  After the frenzy and freedom of summer, it has been hard to find time to experiment, or even mess with the site, but I am hopeful that things will change now that the semester is tapering off.  There has been a little news along the way.

A few weeks ago, the students participating in the Biology Honors Research Program at UM College Park invited me to give a seminar.  Since my fly work is old and stale, it seemed like a good chance to talk about the beginnings of the Solar Slug project.  As far as I could tell, the students found the ideas intriguing (golly, who wouldn’t?), and it was a great chance for me to assemble a seminar and impose some order on my thoughts.

honors-seminar-f2016-ver102216

Last month, we submitted a report to CONANP, who oversees the Biosphere Reserve in Bahia de los Angeles, about the past summer’s activities.  A good chance to think hard about what we did, why it matters, and where it will lead.  In related news, because of overwhelming time demands from the opening of their new Living Lab, Ocean Discovery Institute will not be working in Bahia in 2017.  It is disappointing, but, at least in principle, will give me some time to put together more substantial funding for future years.

But let’s talk science.  Several months ago, I acquired two old PCR machines from the surplus equipment program at the National Institutes of Health.  It’s a great program, in which equipment that is no longer wanted by researchers at NIH can be acquired by educational institutions.  The major caveat is that one can never be sure that the equipment is functional until it gets back to the lab and is tested.  My students in the Cell Biology and Physiology lab course were running some PCR samples, so I thought it would be a good time to test out the new (old) machines in parallel with the very fancy PCR machine we use for student labs.

The thermocyclers are Applied BioSystems GeneAmp 9700s.  In principle, they should do everything we need, plus they have a nice post-cycling chill cycle, so I can set them up and go home without worrying about the DNA sitting in the machine and degrading at room temperature.  But do they work?

Applied Biosystems PCR machine from NIH surplus. This is Machine 1, for which the temperature was within one degree C of the programmed value. 11/4/16

Applied Biosystems 9700 PCR machine from NIH surplus. This is Machine 1, for which the temperature was within one degree C of the programmed value. 11/4/16

The choice of what to amplify was easy.  Another Elysia fanatic, Susanne, had sent me a piece of parapodium from an E. diomedea that had an unfortunate encounter with a filter.  Don’t fret, the slug survived, but she was nice enough to carefully preserve the tissue in ethanol, pack it, and ship it to USG.

Fragment of parapodium (in vial) carefully packed and shipped from Austin.

Fragment of parapodium (in vial, center) carefully packed and shipped from Austin.

The fragment sat forlorn for about a month.  It was very exciting to be able to finally extract the DNA and see if we could amplify the rbcL region.  I set about mashing and processing a small piece, and all looked well.

Small piece of E. diomedea parapodium before extraction. 11/2/16

Small piece of E. diomedea parapodium before extraction. 11/2/16

At the end, I had produced a tube of clear liquid.  Was there DNA?

DNA extracted from E. diomedea. 11/2/16.

DNA extracted from E. diomedea. 11/2/16.

I amplified DNA from E. diomedea, a control sample from BioRad (to make sure the machines functioned at all), and some DNA I had extracted from an Avrainvillea plant in the slug system.  During the thermal cycling, I used a thermocouple probe to check the temperatures of the machines.  Machine 1 was just about perfect, whereas Machine 2 was way too warm during the cooler parts of the cycle, and I expected poor results.

Results of first test of new PCR machines. + control" is commercially prepared DNA from BioRad, containing a mix of DNA with and without an ALU insert in the PV92 region of the human genome. There should be bands at 941 and 641 base pairs. E. diomedea and Avrainvillea DNA were extracted a few days before. 11/4/16

Results of first test of new PCR machines. + control” is commercially prepared DNA from BioRad, containing a mix of DNA with and without an ALU insert in the PV92 region of the human genome. There should be bands at 941 and 641 base pairs.
E. diomedea and Avrainvillea DNA were extracted a few days before. 11/4/16

The control samples worked in both machines, which was somewhat surprising based on the temperature measurements.  I guess you can get away with a lot if you start with really clean DNA and well-established primers.  The only sample from the new extracts that worked was the E. diomedea DNA in machine 2.  I expect we can get things to work better if I reduce the DNA concentration, but it is puzzling that a sample in the less reliable machine worked better.  Nonetheless, I now have some DNA I can send off for sequencing when there’s a little time.

Happy Birthday to us!

Today marks the 2nd birthday of the Solar Sea Slug Blog.  It started as a place to curate information about Elysia, and to track the progress of our modest project.  Since then, it has taken on a life of its own, and morphed into a place to curate information about Elysia, and to track the progress of our modest project.  My how we have grown!

I suppose the concept of this site as a journal of my work with Elysia (a “web log,” so to speak) is rather old-school in this age of tweets and Instragram.  Fortunately, I am at a place in life where I can play the geezer card, using the excuse that I simply don’t have the mental bandwidth to generate content at that rate.  Despite all the nice messages that people send me to help improve my SEO, I actually worry more about getting too much traffic than too little.  To paraphrase Tim Curry, “going viral would be too much responsibility for me.”

The list of possible tweaks and improvements gets longer, not shorter, and the site will continue to improve incrementally.  There are things I would like to simplify, such as how to leave a comment, or the process of updating reference lists.  Then there is the slow work of fleshing out the species pages.  It would be great to host the subset of articles that are open-access as well.  Rome wasn’t built in a day, and Romulus didn’t have a full time job or dogs to walk, so we’ll have to be patient.

Thank you, dear reader, for checking in from time to time.  Although it is unlikely you will find links to cat videos (but never say never), I hope you will stop by to find out more about amazing and abundant little molluscs, their biology, and the people who are working to understand them.

First Grant for the Solar Slugs!

We just got some great news, that we have received the first outside funding for the Solar Slug project!  The Washington Area Marine Aquarist Society has awarded the project $500 to get work started on slug neuroanatomy.  The general idea is to stain baby slugs for acetylated tubulin, which will stain axons, and therefore tell us where the nerves are.  This will be crucial in working out how the slugs process information about their environment.  Because the slugs are essentially pancakes with everything embedded in tissue (rather than having nice hollow spaces like people or insects), it has been difficult to do this kind of thing using standard dissection methods.

Now that the adults are getting fat on recent batches of Bryopsis, I hope to soon see some eggs, and then grow some small slugs for staining.  It may be a while, but I have a year to spend the money.  Keeping fingers crossed that a little support may attract more, espcecially if we start to get some nice preliminary data.

Thank you WAMAS!

Things Have Been Happening

It have been well over a month, so I suppose I have earned the title of World’s Worst Blogger.  It’s not for lack of news, though.  In fact, one of the reasons for the lack of posting is the abundance of activity.  In addition to the semester being in full swing, with the usual collection of lectures, exams and labs to attend to, the Elysia project has made some real strides in the past few months.  Today’s post will be a quick summary of the new beginnings, with the promise of more to come.

First off, this fall is the official start of student involvement in the Universities at Shady Grove (USG) Solar Slug Project.  Loyal followers of the blog will know that the USG project has been active for well over a year.  However, the primary goal of the project is to provide University of Maryland students here at USG with a research experience.  Now that the infrastructure is in place, there is now a squad of two undergraduate students who will be working on a small molecular biology project.  The main purpose of their work this semester is to develop a protocol for extracting chloroplast DNA that can be used to determine which species of plants the sea slugs are holding in their bodies.  We’ll start with a well-studied species, E. clarki, with a view to performing the same experiment on Elysia diomedea in Bahia this summer (see below).  The students have completed their first two assignments covering the basic biology of the system and the specifics of the methods they will be using.  We should be ordering reagents in a few days and doing some real biology next week.  Stay tuned for updates on their progress.

Another big news item is that solar slugs will be a significant part of the Ocean Discovery Institiute program in Bahia de los Angeles this summer.  One of the high points of my year is going down to Baja California to help them out with their work at the field station there.  This year, the slug project will expand to be a Directed Research project, involving a small group of students for the entire duration of their time at Bahia.  We will be performing experiments to identify the food plants and examine the activity patterns of Elysia diomedea.  Because large Elysia species are often found away from potential food plants, and because their method of feeding (sucking the sap) leaves no obvious bite marks, it can be difficult to know what they are actually eating.  Knowing what E. diomedea eats, and how much time it spends feeding, basking or hiding, should provide important insight into why it stores and maintains chloroplasts from its food plants.

For the semester’s experiments, we ordered some new E. clarki and have been fattening them up for experiments.  In addition to new Penicillus and Avrainvillea from collectors in the Keys, we are getting some nice Bryopsis and an unidentified species of broad-leafed algae in the half-ten algae growout tank.

Elysia clarki exploring broad-leaf algae 2/22/16

Elysia clarki exploring broad-leaf algae 2/22/16

3318_Eclarki_salad022216

Elysia clarki discovering new crop of Bryopsis 2/22/16

E. clarki on Bryopsis 2/12/16

E. clarki on Bryopsis 2/12/16

As you can see, it is still difficult to find the right nutrient balance that generates lush growth of the target algae without encouraging cyanobacteria (the red stuff), but we are moving closer all the time.

Slugs among algae selection 2/12/16

Slugs among algae selection 2/12/16

That’s it for now.  More soon, I hope.

Site Reorganization

In an effort to make navigation a bit more sensible, I have started making a few changes.  There is now a top-level page devoted to slug husbandry, Slugkeeping, which contains information (from this Web site and others) regarding how to keep and breed Elysia, as well as sources of slugs, food plants, and hardware.  The new Slug Science page collects the links to the labs and scientific papers that make up the world of Elysia research.  There is also a link to the occasional Solar Slug Journal Clubs that examine current papers in more detail.

Keep an eye out for more updates in the near future. Also, please let me know about links that may have been broken in the process of rearrangement.

In the meantime, here is a photo of a presumed E. papillosa that rode in on the recent batch of plants from KP Aquatics.

Elysia papillosa, June 2015

Elysia papillosa, June 2015