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.
When Did I Earn Some Good Karma?
The colony had no business succeeding, or even surviving this fall, but somehow things have gone quite well.
As I posted a few months ago, Michael Middlebrooks was nice enough to send some freshly collected Bryopsis plumosa from Florida. After cleaning, it settled in and started to grow.
It had been over a year since the colony had been running, so I was out of the habit of performing routine chores, then classes started to ramp up, and it was difficult to keep up with maintenance. Flow from the CO2 cylinder stopped periodically, dosing with nutrients started too high, then dosers would run out, and I fell behind on water changes. As a result of neglect and nutrient imbalances, Ulva (sea lettuce, a non-desirable species) was absolutely thriving, but Bryopsis was struggling.
In late September I considered posting a sad entry about all the preparation resulting in yet another failure.
I was mentoring students in Slug Club (officially “Invertebrate Behavioral Physiology Research Seminar”), so there was no choice but to persevere. We would be performing experiments starting in late October, so it was time to order some new Elysia from KP Aquatics, and hope for the best.
I ordered 10 slugs, plus a generous collection of algae (from KP and Gulf Coast Ecosystems) to support the parents. The slugs arrived, looking pretty good (one was yellow and ultimately did not make it, but they had sent extras), and I split them between a tank in the lab and the Box of Slugs at home. All of the new algae were planted at home to avoid contaminating the lab cultures with any more undesirable species of plants, predators, or pathogens.
Elysia are durable creatures, and most settled in quickly.
One of the slugs at home laid eggs almost immediately. I collected them and set them up in a dish in the lab, thinking that there was a slight chance I would have enough B. plumosa to rear them.
Meantime, the semester did not lighten up, but I had developed a routine that kept the algae tanks cleaner and kept conditions relatively constant. There seemed to be some Bryopsis in the algae tanks, but they still seemed to be dominated by Ulva and Derbesia (a finer hair alga that Elysia do not seem to like).
One component that I added to the routine was cleaning out one of the algae tanks each week. The algae are supposed to be growing on tiles, so I am pulling out one of the tanks, rinsing the debris off the tiles with clean saltwater, and thoroughly scraping and scrubbing the tank. Bryopsis thrives in clean water with strong circulation, so keeping the tanks clean and the circulation vigorous should favor growth of Bryopsis over that of less desirable algae such as Ulva or Derbesia.
The eggs hatched right on schedule, but the veligers were not swimming particularly vigorously, and I expected the juveniles would probably succumb to bacterial or protozoan pathogens. At this stage, they need to have food algae to settle on and start eating, and I would normally pre-treat the algae with ivermectin to kill off potential predators, and rifampicin to reduce pathogens. The survival of the larvae was not a high priority, so I simply grabbed a glob of mixed algae from one of the algae tanks, rinsed adherent cyanobacteria and dinoflagellates off of it, and tossed it into the dish with the eggs. I fully expected failure and kept a later batch of eggs in reserve.
Imagine my surprise when I looked at the dish several days later and saw juveniles with chloroplasts in their diverticula. They had settled and started feeding! There were many more that were crawling around, looking healthy, but had not yet eaten.
Within days, I had hundreds of baby slugs that seemed to be healthy and feeding.
One thing that puzzles me is that they are very “sparkly.” In addition to the deep green of the chloroplasts in their bodies, they have a collection of iridescent little beady things, especially in their heads. I have seen such things in the parapodia of adults in the past, but do not remember the juveniles looking like this. Whether they are derived from diet or the slugs’ own metabolism is not clear, but they do not seem to be causing harm.
In the ensuing weeks, the baby slugs have gone through the usual routine of being moved into baking dishes, and then into their own 3-gallon tank where they will continue to grow. Despite being neglected, including being left for a week while I traveled to the west coast, there are still hundreds of them.
Fortunately, the number of labs interested in Elysia biology continues to grow, albeit slowly, so I will send most of them away in a few weeks.
There may be some lessons here:
My theory about algae growth seems to be supported, and the Bryopsis is taking over the tiles. There is still plenty of the other two species of algae, plus more cyanobacteria and dinoflagellates than I would like, but I think the algae cultures are moving in the right direction.
Also, tearing down the system, scrubbing and bleaching as much as possible appear to have been successful. Although Ulva and Derbesia are competing with the Bryopsis, and are not terribly desirable, Valonia (bubble algae), and Cladophora (Brillo® algae) were completely taking over the system.
I believe there may have been some simple, dumb luck involved as well.
Clean Start Fall 2021
I do not need to provide anyone with examples of how awful the Covid-19 pandemic has been. There have been a few bright spots, though, such as all the time we have been able to spend at home with our dogs.
One negative thing that had a slightly positive aspect was having to put the slug system on hold. I took the remaining slugs home, hoping that they would be the parents of the next generation. They did remarkably well for about a year living on the Bryopsis pennata that was growing in the reef and seagrass tanks.
Last slug from the last brood raised in the USG system. They came home last spring, and held out for over a year. 3/13/21
Over the course of the summer, the last one has faded and shrunk as she reached old age and lost the ability to eat algae and to maintain her chloroplasts.
After about 16 months, the last of the Elysia that grew up in the system is just about gone. As they fade away and use their tissues for energy, they get increasingly small and yellow. This one is about the size of a pea. 8/11/21.
After at least five generations, it is sad to say goodbye to the last of the progeny. Nonetheless, the lack of slugs meant that I could clean the USG system thoroughly.
During the years that I have been trying to culture Bryopsis, I have managed to introduce at least half a dozen invasive and undesirable species of algae, some of which were outcompeting the desired species. Valonia (bubble algae) and a coarse red alga were thriving under the same conditions as Bryopsis. Also, Bryopsis plumosa, the finer species that is a favorite of the hatchlings, was being overrun by B. pennata, which the babies will not eat. I had been trying to push the balance toward Bryopsis by manually removing the other species, or introducing grazers that prefer the nuisance species, but I was not winning. It was a wet mess.
Enter the pandemic. The system was unoccupied, allowing for a fresh start. Even if I will not be able to keep the system as an absolute monoculture, it would be worth getting rid of as many pests as possible, and maybe eliminate some of the bristleworms and flatworms that prey on the babies in the process.
Step one was to dismantle the system, scrub out the tanks, and clean as much of the plumbing as possible. Drainpipes, pumps, powerheads etc. were soaked in warm bleach solution, then scrubbed and left to dry.
Trying to eliminate at least some of the unwanted algae species by bleaching plumbing and equipment. 3/2/21
Once everything was clean and dry, it was reassembled, and left dry for a few months. Then it was filled with fresh water to flush out remaining debris and organisms, and to check for leaks.
Tanks back in place after scrubbing (center shelves). The tanks to the left contain crayfish for the neurobiology lab. 3/9/21
With the tanks in place and plumbing reassembled, I let it run for a couple of months to see what grew. The good news was that almost none of the species of pest algae has made a reappearance. There was little Ulva and some diatoms, but none of the worst characters had come back. It was time for new algae.
Elysia system filled with saltwater and circulating. There is a small amount of residual algae, but most species seem to have been eradicated. 3/25/21
This time around, my goal was as pure a culture of B. plumosa as possible. Hatchlings eat it, adults eat it, everybody eats it, so there is no need for other species. I contacted Michael Middlebrooks at the University of Tampa to find out if he would be able to send me a small amount as a starter.
At last, we found a time that he could send the algae and I would be here to receive it. It arrived last week, having survived near-record heat here in MD, and looked great.
Bryopsis freshly arrived from Florida. Ready for the first treatments to reduce predators, parasites and pathogens. Hopefully, I can get rid of some of the non-Bryopsis algae as well. 8/11/21
Under the microscope, you can see dark filaments full of green cytoplasm, indicating good health and growth potential.
Bryopsis plumosa, close up. Many of the filaments appear uniformly green, which is a good sign that much of the algae arrived healthy. 8/11/21
The wild intertidal zone is a complex environment, so there were other algae in the mix. There are a few species of red algae that grow closely with B. plumosa, often intertwined at the site of attachment to the rock.
Red macroalgae mixed in with Bryopsis. The green “fingers” to the left are part of a healthy growth of Bryopsis plumosa. Unfortunately, the red stuff likes to grow in and among it. 8/11/21
I removed as much of the non-Bryopsis algae as possible, and will try to be vigilant about removing whatever crops up as the new algae take hold in the tanks.
Meantime, the algae are settling in and growing. It is remarkable how quickly Bryopsis will find a place to anchor itself and start growing.
Filaments of Bryopsis plumosa extending from a clump that arrived on 8/12/21. Photo 8/17/21
Healthy Bryopsis plumosa growing on a powerhead. 8/17/21
Some algae are making small adherent spots on the tiles that I added as a substrate, along with the glass and silicone sealant. At this point, it is impossible to know whether it is a species I want or a pest, but let’s keep our fingers crossed.
Dots and threads of small unidentified algae stuck to ceramic tiles. 8/17/21
If all goes well, it will be time to add some slugs in a month or so.
Journal Club: Don’t Quit While You’re A Head
Once again, Elysia makes a splash with its unusual biology. This week, it’s an observation that at least two species of Elysia can shed their entire bodies, and regrow them. This was described in the journal Current Biology (Curr. Biol. 31: R215-R240, 2021). Because it was so strange, the paper also received attention in the New York Times and Science Magazine.
The authors Sayaka Mitoh and Yoichi Yusa, discovered that slugs in their captive-reared colony of Elysia cf. marginatus (the “cf” apparently being due to uncertainty about identities of members of a species complex) would spontaneously shed their whole bodies from time to time (Figure 1A, below). They also observed this phenomenon in a field-collected Elysia cf. marginata and in part of a cohort of wild-collected Elysia atroviridis. The process of shedding a body part, termed autotomy, is well-known among animals, and has been observed before in sacoglossan species related to Elysia that release body parts to avoid predation. Autotomy on this scale is rare, though, and had never been observed in molluscs.
Mitoh and Yusa Figure 1A. The head is at the top, and the body is below. Arrow indicates the heart.
The pattern was similar for most: the head separated at a line that the authors referred to as a “breakage plane” (Figure 1I, below).
Mitoh and Yusa Figure 1I. The presumed autotomy plane is indicated by a dashed white line.
The wound healed up, and a new body was regenerated over the course of a few weeks (Figure 1 E through H, below).
Mito and Yusa Figure 1E through H. Elysia atroviridis autotomizes its body (E and F), then regenerates a new one (G and H).
The authors emphasized that the heart, easily visible on the dorsal surface, remained with the body, and they believed that most of the organs were also lost. Unfortunately, it was a brief communication, so no anatomical data were provided regarding what exactly was left behind. Because of the weird development of gastropod molluscs like Elysia, in which there is a twisting of the embryonic body, much of the digestive and reproductive systems end up in the head, so it will be interesting to see in future studies what exactly remains with the head. They did note that many of the severed heads began to feed within hours of losing their bodies, so some capacity for digestion must have remained.
In any case, it is remarkable that the head could survive at all, much less generate a whole new body. The authors speculate that kleptoplasty, the famous ability of Elysia species to retain chloroplasts from their food plants and use them to generate energy, could be an important factor in the survival of the head. The diverticula that contain chloroplasts are certainly found in the head (as shown in the image below of a baby Elysia crispata), so this seems plausible.
Baby Elysia, about three weeks old. Rhinophores well developed, diverticula, including those in the head, are full of chloroplasts from Bryopsis plumosa. 8/17/18
Why do the slugs do it? It certainly stresses them, as indicated by the fact that all slugs older than 480 days died after autotomy. It is not likely due to predation, because it is too slow to prevent being eaten, and because simulating predation by pinching the slugs did not cause autotomy. It is more likely a way to eliminate diseased or parastized tissues. Among the field-collected Elysia atroviridis, the only ones that performed autotomy were those containing a parasitic copepod. Curiously, the minority of parasitized animals autotomized their bodies, while many more gradually dissolved the affected parts through a process called autolysis. When one combines this observation with the fact that lab-reared (and presumably unparasitized) Elysia marginata performed autotomy, it appears that much about the phenomenon remains to be understood.
Naturally, you are all wondering whether I have observed this phenomenon in Elysia crispata in the lab here. So far, no. If they could shed their bodies, I expect they might have done so when some of the tanks were infested with protozoans and the slugs were being eaten away. Nonetheless, it may still occur under some conditions.
It makes you wonder how the French Revolution may have been different if the royalty had been Elysia.
Coronavirus 2020: Breeding In Isolation
Along with most of the working world, the Solar Slug team was summarily ejected from the lab on March 19, 2020. I have been allowed to return occasionally to perform maintenance on the tanks, but was otherwise expected to stay home and continue teaching and other work remotely. The next generation of slugs had been laying eggs, but I would not be able to devote any form of loving attention to the offspring.
As a devoted follower of the blog, you will remember that it took several years to work out conditions for rearing baby slugs. The hatchlings need to be protected from pathogens and predators, and must have access to a clean supply of Bryopsis plumosa in order to take their first meals.
Some of the baby slugs did not get the memo. A few weeks after we began isolation, I saw a few tiny slugs in one of the tanks. It was a bit of a relief, because I had begun to wonder if I would be able to rear the next generation before the adults became too old to produce eggs.
- Baby slug, reared without effort. Note the large cluster of Valonia at the right of the photo. 4/14/20
The photo below gives a better sense of scale. The youngster, probably already a few weeks old, is less than a centimeter, and is dwarfed by one of the parents. It is impossible to be certain whether it is feeding on the red, filamentous algae, or finding green algae in there somewhere. Their overall color makes it clear that they are both full of green chloroplasts.
As I mentioned above, hatchling Elysia can be delicate, and are very particular about their first meals. If Bryopsis plumosa is not available, then they will starve. During the period of neglect, Bryopsis pennata (excellent food for the adults) has been growing well. Other algae, such as Ulva and Valonia (bubble algae) have also been thriving, along with the unidentified filamentous red algae. Although I have not seen it, there very well may be adequate growth of B. plumosa hidden among the other algae.
Early mortality must have been pretty high, because there are a few dozen youngsters (rather than hundreds) cruising the tank. That is more than enough slugs to keep the colony going for the year, but would not be adequate for experiments. Fortunately/unfortunately the campus is unlikely to return to normal by the fall, so student Slug Club will probably be postponed. Meantime, the new kids will grow into the next generation of parents.
Progression of Generations
A matriarch fades away, but the next generation reaches maturity.
At this point, it is a familiar cycle. They start out as a members of an egg mass, with thousands of their siblings. After they hatch, a lucky few dozen or maybe a hundred settle down and feed, selected mostly on the basis of luck. Once they start feeding, they grow rapidly, and a smaller, luckier group gets to go home to Box of Slugs 2. These will be allowed to get large and produce the next generation. And so on.
The previous generation has now come to an end. Several slugs from a brood laid in early January 2019 were placed in the tank on May 1.
Matriarch in her prime. July 1, 2019.
After growing to the usual big size, and laying many clutches of eggs over several months, the last of the group started to fade in late January 2020.
Elysia clarki at one year old. Hatched about 1/25/19, photographed 2/2/20. Note yellow parapodia and faded green body.
The biology underlying the change is unknown, at least to me, but I assume that the slugs fail to maintain or replenish their chloroplasts for some reason. Regardless of the cause, it seems to be a one-way street, and the old Elysia become more yellow, shrink, and start to look unhealthy.
Even in her last days, the old female appeared to feed on the available Bryopsis alongside her daughters, but she was unable to either absorb or process the food. Within a few days, she had disappeared. When a body is almost all water, it does not last long after death. She lived for over a year, which is not close to the record of two years reported by Pierce and colleagues, but is a pretty good life for a sea slug.
Fortunately, life also involves renewals. Over the summer and fall, the slugs produced thousands of eggs, and some of the offspring have matured to produce eggs of their own.
A pair of young slugs from a brood laid on 9/24/19 has produced their first clutch of eggs. As is usually the case, the first egg mass is smallish, maybe a few hundred eggs, but the size will rapidly increase. Production of the first eggs at four months of age is also consistent with previous observations.
So we are, once again, coming full circle. I need to sit down at some point and count how many generations have passed since the first hatchlings survived and grew into baby slugs, but it is satisfying that the group can keep itself going.
New Space!
After a long, tiring semester, we have moved into a new location in a shiny new building.
The Biomedical Sciences and Engineering building opened to great hoopla in November. Local and state bigwigs participated in the ribbon cutting, but, more importantly, so did some Biological Sciences students.
During all the hubbub, the students in the Cell Biology Lab course were going full speed in their new cell culture facility upstairs. A few even made their way into a Washington Post story about the event.
Although the building was officially open, it has token a while for it to be truly ready for use. Even now, there are contractors coming and going to put the finishing touches on the structure, and some necessities, such as ice machines are on the way.
Nonetheless, we made the move to our new space last week. It has taken months of preparation to have the spaces ready for the equipment, and the equipment ready for the spaces. We have been running Cell Biology, Neurobiology, and Physics labs all in the same room, so it will be luxurious to have two large, well laid-out lab spaces and associated preparation areas.
The most important space for the slug project is the new “preserved specimen” room. Someone must have decided that we would be dissecting cadavers, so we have a prep room devoted to dead things, and point exhausts over the lab benches to ventilate fumes from preservatives.
Since we have no plans to store pickled carcasses, the preserved specimen room will make an excellent “live specimen” room. The room is separate from the rest of the lab, so animals can be kept away from chemicals, and it has marine grade shelving perfect for aquaria.
On top of that, it has a floor sink for washing tanks and other equipment.
It took about two full days to set up the plumbing, to move the slug and algae tanks, and to get the control system set back up. Big thanks to Paul, Kevin, and the rest of the IT crew for helping me to get the controller connected to the local network.
The wiring is still a bit messy, but that can wait until I get my office and the two labs unpacked. Meantime, there are about a dozen slugs enjoying their new home.
The slugs will soon be joined by the earthworms, crickets, and crayfish for the Neurobiology Lab course.
Posts will probably continue to be sparse for a while. Elysia is still a wonderful system for teaching neurobiology, and I expect some of the students to use them for projects this semester. In the longer term, I am excited about developing multi-unit recording methods to study the activity many nerve cells at a time during sensory processing. However, that will be on hold for a little while while I work on a few other things.
Journal Club: Kahalalide F, It Takes Three to Tango
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:
- The bacterium makes KF by stitching together amino acids using a biochemical pathway very different from that used by animals and plants.
- The bacterium has adapted to the comfortable life inside plant cells by getting rid of many of its genes.
- 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:
- 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.
- 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:
- 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.
- A total of 13 predicted NRPS modules, as would be expected for an enzymatic pathway that makes a peptide consisting of 13 amino acids.
- 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.
Slug Science Inches Forward
It has been a busy semester on several fronts, and the project has crawled forward a bit.
I am most interested in neurobiology and behavior, so we have moved from studying the chemical ecology of Elysia clarki to working out its behavioral response to external stimuli. The ultimate goal of figuring out how its nervous system encodes sensory input and motor output. Accomplishing this requires understanding of both the theoretical and technical aspects of molluscan neuroscience. We had three goals for Spring semester: 1) develop a stronger knowledge of the literature describing the behavior and nervous systems of opisthobranch molluscs, focusing mostly on nudibranchs and sea hares; 2) work out methods for reproducibly recording from slug neurons; 3) get a better sense of the slugs’ light preferences, in terms of spectrum and intensity.
USG Slug Club, 2019. From left: Josue, Nana, Marianne, Savana, Abigail, Cecilia, Dave. 4/12/19
The intrepid group of seniors and I got right to work.
For goal 1, we held a Slug Neurobiology journal club every Friday for the first 10 weeks of the semester. When we started, I was still pretty fuzzy on the details of the visual and nervous systems of opisthobranchs, and had only a vague idea of how they manage to crawl. I generated a list from multiple overlapping searches for papers describing the visual and motor systems of slugs, and Slug Club students and I presented about 18 papers, along with many more papers required for background. Despite nudibranchs being a diverse group, most papers described either navigation and swimming in Tritonia, or learning and memory in Hermissenda, with a few papers on Pleurobranchaea, Aplysia, and some snails thrown in for good measure. After our deep dive into the literature, we have a much better idea of mechanisms of light sensing, ciliary propulsion, and steering.
Goal 2 was to record from Elysia neurons. In spring 2018, we had found a good atlas of the Elysia nervous system, and had made a few recordings. However, the nervous system is surrounded by a tough sheath made of connective tissue, which makes it difficult to get delicate electrodes into the cells.
At this point, I needed a colleague to give me some pointers on getting the sheath off, or at least softening it up, but one can count the number of people recording from sea slug nervous systems on one hand. Fortunately, Paul Katz and his lab have extensive experience. Paul responded rapidly to my email, and gave me some excellent pointers. He was excited that there is another captive-bred sea slug being developed for neurobiology, and has been developing Berghia, a nudibranch that is also relatively easy to raise, as a neurobiological model system.
Berghia has some distinct advantages, such as a two month generation time (Elysia takes about 4 months), and surplus slugs can be sold to aquarists to control pest anemones. However, Berghia is much smaller and not kleptoplastic. Might be a good “normal” species to use for comparison with Elysia, though.
Even though the neurons that mediate the behavioral response to light are probably in the cerebro-pleural (which get inputs from the eyes) and pedal ganglia (which send outputs to the foot), I decided to start with the abdominal ganglion. It has a lot more big, pretty cells that should be easier to impale with a microelectrode, which increases the chance of success. With Paul’s advice, were able to get the sheath off and record from some large neurons in the abdominal ganglion.
Josue, Marianne and I practiced impaling neurons, and got some nice stable recordings. The neuron above fired action potentials at regular intervals and received a lot of synaptic input, indicated by the large bumps between the spikes. Because we currently know nothing about its connections, the significance of the neuron’s pattern of activity is unclear.
Another neuron was quiet at rest, but fired one or a few action potentials when stimulated with injected current. As with the previous cell, we know nothing about this neuron, but it provided an opportunity to practice techniques associated with current injection.
Time permitting, the next step is to record from neurons in the cerebro-pleural ganglion and find some that respond to light, and to look for others that control locomotion.
For goal 3, working out the spectra and intensities of light that Elysia prefer, you will have to stay tuned. The students just spent the past month gathering data, and should finish analyzing it within the next week or so.
Elysia clarki Eat Valonia, Too.
At least a little.
As often happens, I kept too many babies from a recent brood, and they rapidly consumed the large growth of Bryopsis in their tank., Algae production was a little slow in the system, so I chose to reduce their ration. I did not exactly starve them, but there was not always Bryopsis in the tank. There was , however, quite a bit of the pest algae Valonia, known as “bubble algae,” because it grows as clusters of large unicellular vesicles. Although I periodically have tried to remove it, it was thriving in the tank with the young slugs.
Elysia clarki, about 3 cm long, with empty Valonia bubbles. A large, empty bubble can be seen by the slug’s tail. 2/26/19
The slugs appeared to be feeding on the algae, and some of the bubbles, which are normally intense green, became clear. Although it would require DNA sequence analysis of the slugs’ kleptoplasts to be certain, the circumstantial evidence indicates that they are sucking sap from the algae. It makes sense, because Valonia bubbles are large single cells, which would allow a slug to have a big meal with just one puncture.
A recent paper by Mike Middlebrooks and collaborators (Biol. Bull 236:88, 2019) demonstrates that Elysia crispata (with which E. clarki is most probably synonymous) eats a wide variety of algae in the wild. In the aquarium, it looks like we can add one more species.
The good news is that the slugs seem to be wiling to make use of Valonia when necessary. Unfortunately for anyone with an outbreak, the slugs consume them so slowly that there is little chance they would eradicate an infestation.
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