Posts in Category: General Slug Things

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.

Two of the new cohort of E. clarki from KP Aquatics. The slug on the left is very yellow, and is probably not going to survive, but they sent plenty to start the next generation of slugs. 10/16/21

Elysia are durable creatures, and most settled in quickly.

Elysia clarki from KP aquatics. Good size and color. Note the blue edges to the parapodia. 10/16/21

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.

Eggs laid by newly arrived E. clarki. These were collected and reared, resulting in the larvae and small slugs described below. 10/16/21

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.

Slugs at varying points of development, moving among the mixed algae. All have fed and contain chloroplasts in their guts, but the larger slugs show the beginnings of structures such as rhinophores and parapodia, while the smallest are much simpler. 11/15/21

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.

Juvenile Elysia, about a week after settling. Bumps in the nose show where the rhinophores will develop, and swellings are forming along the back where parapodia will appear. Note the smattering of sparkles, especially between the eyes. 11/15/21

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.

Two young Elysia clarki. Very small (1-2 mm), but already showing rhinophores and parapodia. Note the iridescent spots, especially around the head. 12/2/21

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.

Baby Elysia, indicated by arrows, feeding on a mix of Bryopsis (thicker fibers) and Derbesia (finer filaments). The field is 2-3 cm across, taking up only a very small part of a larger tank, so the total population is in the hundreds. 12/4/21
Bryopsis plumosa starting to thrive and take over the tiles on the bottom of an algae culture tank. Red objects are mostly cyanobacteria (“blue-green algae”) growing on the filamentous algae. 12/4/21

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.

Juvenile slug, now just visible with the naked eye, with clearly formed rhinophores (right end), parapodia, and green dots indicating chloroplasts. 12/3/21

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.

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.

Last photo of aging E. clarki. Parapodia almost entirely yellow. Photographed 2/15/20, last seen 2/20/20.

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.

E. clarki, hatched approximately 10/10/19, photographed 2/2/20. Note her healthy deep green coloration.

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.

First batch of eggs from young adult Elysia clarki, 2/22/20. Parents from eggs mass laid 9/24/19, hatched around 10/10/19.

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.

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?

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.

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 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.

Mini-Slugs

After passing the first hurdle, getting them to eat, everything should be easy, right?  Well, there are plenty of things that can go wrong.  Predators can sneak in with the food algae (you don’t have to be too big to eat a lot of hatchling slugs), and pathogens can decimate a whole brood.  Nonetheless, a few of the first batch have grown, sprouted rhinophores, extended parapodia, and look like teeny Elysia!

Miniature E. clarki, about 10 days after first meal. 2/5/18

They are currently living in a modified specimen container (like they use to catch fish at the pet store).  I drilled a few big holes in it, and covered them with fine nylon mesh to keep the babies in.  They get a constant, slow flow of UV-sterilized artificial seawater, and have a variety of algae to choose from.  They still seem to prefer Bryopsis plumosa for the moment, although they may be sneaking tastes of B. pennata.

Teeny E. clarki at about two weeks after first meal. 2/8/18.

It has been hard to get a proper measurement at this point, but they are about 2 mm long, are somewhat visible to the naked eye, and can be easily observed with a simple magnifying glass.  The photos above were shot with the macro setting of a digital point-and-shoot camera, so they are rapidly leaving the microscopic world!

Fingers crossed that they continue to grow.

Cool Solar Slug Videos

There is so much going on in the world of solar slugs!  I came across a couple of nice videos, and thought I’d share.

A broad collection of European slug scientists has launched a project to sequence the genome of Elysia timida.  That will be a wonderful resource for all of us who are interested in slug science.  The video below provides an explanation of the motivation, along with excellent general information.

The video below does a great job of describing the biology of E. viridis, with some beautiful footage.  Can you see the minor error?  (Hint: what is it eating?)