Posts in Category: Box of Slugs
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.
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.
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.
Back to Baby Food
Do they prefer the food of their infancy because it tastes better, is more nutrient-rich, or is easier to eat? Maybe they are just nostalgic for the food they ate when they were young.
Two-month-old Elysia clarki having an early morning crawl among the turtle grass and manatee grass in Box of Slugs 2. 9/29/18
A few of the youngsters from the most recent brood have moved in with their mom in the tank at home. They were weaned from Bryopsis plumosa to B. pennata once they were a few weeks old, and have been growing steadily.
For a few reasons, not all logically sound, I had assumed B. plumosa would be harder to culture. If an aquarist has a problem with Bryopsis, it is invariably B. pennata. Combined with the fact that B. plumosa is crucial for hatchling survival, and that I had to travel all the way to Tampa to get it, I figured I would have trouble keeping it going. As a consequence, I always shift young Elysia to B. pennata when they were ready to eat it.
Despite my preconceptions, B. plumosa is thriving at this point.
Bryopsis plumosa culture tank (surface is 10″ by 20″), stuffed to the top with algae. 9/14/18.
To the uninitiated, the B. plumosa tank would look like a mat of unruly glop. To an aficionado such as myself, it looks like an actively growing, unruly mat of precious food for hatchling Elysia. It is a “half-ten” aquarium: a ten-gallon tank, but only half the height (OK, so it is really only a 5-gallon tank), which provides a lot of surface but only a few inches of depth. The growth form is very different from B. pennata, which tends to be long and feathery. B. plumosa grows more like clumps of moss. I am concerned about the tank being taken over by B. pennata invading from elsewhere in the system, but so far so good on that front.
At the moment, the growing conditions are:
- Nutrient dosing at about 150:30:1 Carbon:Nitrogen:Phosphorus, plus Guillard’s F/2 at 2 ml/day (note this is the whole system, not just this tank).
- Circulation using a powerhead and rotating output (see Slug Safe Circulation). Trust me, it is in there somewhere.
- Lighting by an Evergrow S2 hydroponics light, about 6″ from surface.
To get to the point of this post, I had enough B. plumosa to throw some to the adults.
Elysia clarki eating Bryopsis plumosa in Box of Slugs 2. 9/26/18.
Unsurprisingly, the slugs ate it. I did not expect, however, that the largest female would rarely leave the clump of algae until it was completely consumed. She very much preferred the plumosa. I brought another clump home, and she is still sitting on it, along with one of her kids. The tank is full of B. pennata, at all levels, but the slugs stick right to the single clump of B. plumosa on the surface. It may be my imagination, but the big one seems larger and more colorful after a few weeks of eating B. plumosa.
Box of Slugs 2, with plenty of Bryopsis pennata among the turtle grass. 9/26/18
So, anecdotally, even grown up slugs prefer B. plumosa. Another thing to put on the list of things to test more rigorously. For now, one can speculate about why they seem to prefer it, and what cues (smell? texture?) draw the slugs to the algae.
Fat Babies Have No Pride
In the meantime, another brood has hatched and has started to grow. I am not sure why (I am not in any way a musical person), but when a new brood starts to eat, Lyle Lovett’s “Fat Babies” runs through my mind almost continuously. I have no idea whether the song has a subtle, subversive message (if so, I apologize for any offense), or whether it is simply about chubby infants not being proud.
Juvenile slug crawling among B. plumosa, with diverticula full of chloroplasts. 9/28/18
They are feeding and growing, and it looks like we’ll have several dozen ready for activities in the spring.
That’s OK. Who needs pride?
First 100% Maryland Eggs
We have now come full cycle. Eggs from slugs collected in the Keys have hatched, and the little ones have settled, grown to adulthood and have now produced their own eggs. The slugs in Box of Slugs 2.0 have been producing eggs, but, because one wild-collected slug remains, I can’t be 100% sure who laid them.
This egg mass was laid in the 10-gallon growout tank at USG. All of the potential parents came from egg masses collected between 1/19/18 and 1/24/18, and started hatching between 1/31/18 and 2/6/18. So the maximum possible age of the parents is about 4 months, but they could have been a few weeks younger.
Egg mass in 10-gallon growout tank at USG. Found 5/27/18, but could have been deposited 5/26 or 5/27. Photograph 5/27/18.
Importantly, the eggs are almost 100% fertile. The embryos could be seen developing within a few days, and started to look like veligers in less than a week.
A small section of a developing egg mass from 5/27/18, approximately 8 days post deposition. Depending on the viewing angle, it is possible to see a faint velum extending from each side of the head (embryo viewed face-on), or the shell and head structures (side view). Rare unfertilized embryo marked with an asterisk. 6/4/18.
Because I will be away for much of the summer, I will not be able to rear these embryos. I am hoping for many more broods in the future.
Smothered in Slugs Part 2: Thinning the herd
If you are squeamish, you may want to skip this post.
As described in the previous post, I somehow ended up with hundreds more Elysia than expected. All well and good, but even after reducing the numbers by a few hundred, I simply could not raise enough food to keep up with the appetites of all those hungry slugs. After pulling out the primary growout tank and removing the exhausted algae, I could have a clear view of the population.
A few hundred young slugs from rearing tank. There were not supposed to be so many. 4/29/18.
Oy! I had already set aside a small number in another tank for physiology experiments and the next generation of parents, so I did not have an immediate use for all of these. I was faced with a tough choice: cull some of them, or let them all die slowly of starvation. It gave me some sympathy for Thanos.
I set aside about 30, which seemed sustainable. The rest were placed first into a net to extract mucus for future use. After setting aside the mucus, the slugs were then anesthetized and placed into a small (1.5 cup) food processor.
Anesthetized Elysia in 1.5 cup food processor. 4/29/18.
The job was done quickly. I froze the tissue for future use.
125 ml of slug puree in food processor. 4/29/18.
I don’t feel great about having pureed these guys, but one of the responsibilities of maintaining a captive invertebrate colony is getting the numbers right.
The remaining slugs are feeding and growing vigorously.
Smothered in Slugs, Part 1
After devoting so many hours to learning how to feed and rear slugs, I suppose I can’t complain about the current situation.
Baby slugs filling 10 gallon tank. 3/21/18
I am drowning in Elysia.
In the past month, we have shipped well over a hundred baby E. clarki (plus a few dozen E. crispata) back to their homeland in Florida, gave another dozen to local aquarists, fixed at least a few hundred for anatomical studies, and yet there seem to be hundreds more. They are destroying Bryopsis as fast as I can feed it to them, and I had to cull another few hundred last week to keep the rest from starving. I honestly did not think I had that many babies growing in the system.
I will keep the remaining slugs for the next few weeks, because several groups of students have proposed using them for their independent projects in Neurobiology Lab class. It will be very exciting to see what the students can accomplish. We have also been extracting mucus from groups of slugs, for use in feeding assays (soon to be the subject of another post, I hope). Finally, I am holding onto some of the smallest for another round of staining (yet another upcoming post) and predation assays (yet, yet another upcoming post).
When I resorted to buying E. crispata collected in Haiti last fall, because I was not able to obtain E. clarki from the Keys, I would never have dreamed that there would be such a turnaround. We now have enough slugs of all sizes to do any kind of experiment we can imagine.
Elysia clarki eggs, from 2nd generation parents. Box of Slugs 2.0, 4/1/18.
Not only that, there are no longer any mysterious gaps in the life cycle, from egg to veliger to hatchling to adult to egg. The offspring from the first brood have become reproductively mature, so we are getting eggs from slugs that grew up here in Maryland. As a result, I have put together a page about how to culture E. clarki.
Mother and daughter dozing in the early morning. Box of Slugs 2.0, 3/31/18.
There will undoubtedly be challenges ahead, but developing a self-sustaining colony was one of the major goals of the Elysia project. Now the fun can begin.
Stay tuned for updates on Elysia anatomy, making food with mucus, predation assays, and take a look at the details of how I ended up with several hundred baby slugs.
Growing Up Fast
In only a few weeks, the first batch has gone from barely visible (see the previous post) to nearly adult.
A week ago, it was time to move the four survivors into tanks with the grownups. They had been weaned from Bryopsis plumosa to B. pennata, and were big enough to avoid being eaten by most of the worms and amphipods that inhabit the boxes of slugs. As far as I can tell, they all survived once their rhinophores and parapodia were fully developed, so they are sturdy little gals.
This is the best photo I could get last week of a youngster exploring her new world in the home tank. I found her egg mass on 12/30/17, moved her to the USG system, grew her up, and now she’s home! She’s a little over 1 cm, I would guess, and mom (dad?) towers over her. Elysia look about the same whether they are happy, sad, scared, excited, angry, or bored, so I am not sure if the parent slug looks proud.
Baby Elysia clarki, below mom’s head, in Box of Slugs 2.0. 2/18/18.
After only a week of stuffing herself full of Bryopsis, she has nearly doubled in size. Still dwarfed by mom, but on her way to adulthood.
Young Elysia clarki, with her mother. Box of Slugs 2.0, 2/25/18.
As the older of her parents becomes paler and slowly slides into senescence, it will soon be time for the little ones to take over as matriarchs. We’ll see how long it takes before they produce their first eggs.
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