Solar Sea Slugs: my new project

[Keraxis]

I wish!  My understanding is that they tend to stay deeper than normal snorkeling depth, and are more around the islands than onshore.  There is a dive shop in town, and I hope to get a chance to go out with him next time I am down.

 

There were lots of other fish, though.  Cortez and king angels, wrasse, triggers, puffers, barracuda and more.  

I saw a few jawfish which i think were bluespot in about 20 feet of water in st. john.  I think it depends on the sand bed.

[mogurnda]

I saw a few jawfish which i think were bluespot in about 20 feet of water in st. john.  I think it depends on the sand bed.

Bluespotted jawfish are only in the Sea of Cortez.You can find pearly/yellowhead jawfish at just about any depth in the Gulf and Caribbean.

Edited July 20, 2014 by mogurnda

[mogurnda]

They're heeere!

 

Got the first shipment of 8 slugs from Carolina Biological.  They arrived on time, and were very perky during acclimation.  Will get some decent macros once I get the camera.  These are just with the phone.

 

 

Once in the tank, they immediately started looking around.

 

 

They quickly found the macroalgae.

 

 

Penicillus, yum!

 

[mogurnda]

The guys have been in the tank a few days, and seem to be having a great time.  I have learned a few more things about Elysia species along the way.

 

 

First, despite what the literature and field guides say, Elysia chlorotica is rarely, if ever in the Chesapeake.  Contacted Sidney Pierce, who used to study them when he was at UMd, and he said that they are rarely south of Cape Cod.  Even in the places in which they supposedly appeared, they have not been found in recent years.  Nor have they found their food algae, Vaucheria.  Oh well, so much for making a link to local ecology.

 

Second, the species that both Carolina and LiveAquaria sent was Elysia clarki, not diomedea or crispata.  The differences are a little more subtle (the spots are smaller, parapodia do not fuse in front like crispata), but they are definitely clarki.  Once I get one under a microscope, I will post photos.  Not a big deal, really, because clarki eats the same algae as crispata.

 

Third, they are active little guys.  They move around pretty constantly, exploring their little tank, and pausing to suck sap out of the macroalgae.  At this point, 6 of 12 of them are roving around.

 

 

 

As the papers say, they seem to be grazing on Penicillus.

 

 

But they also seem to munch on Udotea.  This new growth seems to be especially popular.

 

 

 

One last pic that I think is very cool.  Elysia species steal chloroplasts from their food plants, giving them a green color.  From a distance, the slugs just look green, with white spots.  Zooming in more closely, shows that the the color comes from little green spots, the chloroplasts themselves.  

 

[Keraxis]

Bluespotted jawfish are only in the Sea of Cortez.You can find pearly/yellowhead jawfish at just about any depth in the Gulf and Caribbean.

Your right.  It was a yellow head... they have really blue eyes though.  At 20 feet i was running out of air and they dont exactly wait for you to get a good look.  love the write up so far.  You may convince me to get a few.

[mogurnda]

Yeah, it can be hard to get a good look when they are so shy.

 

Now that I have learned more about the slugs, I think they would be great in a fuge, but awful in a display tank. The current and pumps would kill them pretty quickly.

 

They also are pretty leisurely about eating algae, so would not put a big dent in it. Very fun to watch, though.

[mogurnda]

Got the new microscope camera in, so I should be posting some anatomy photos by the end of the week.  

 

Meantime, more behavior.  They have a pretty steady daily routine.  When I get into the office in the morning, most are on the glass by the window, soaking up rays.  Most of the afternoon, they are foraging on their macroalgae.

 

Then they start climbing the walls in the afternoon...

 

 

...and flattening themselves out to soak up rays.

 

 

 

One more photo.  Just to give a sense of how simple their eyes are, take a look at that little black bit of smutz that the arrow is pointing to.  They are probably not able to do more than discriminate between dark and light.

 

Edited July 29, 2014 by mogurnda

[Origami]

I wonder if the pattern of chloroplasts remains stable in an individual, or if they're continuously replaced? What happens as light levels change? Curious creatures. They seem to have acclimated to their new surroundings, though. 

[mogurnda]

They are indeed doing well, to the point that I will need to get some more shaving brush to replace the ones they are ravaging.

 

The chloroplasts appear to be stable, and can last for months.  That begs the question of how the slugs can maintain chloroplasts, which need products of algal genes to keep going.  Studies a few years ago found evidence that algal genes had somehow found their way into the slug genome, a process termed "horizontal transfer."  It was controversial, and later work has indicated that the genetic material was from the algae the slugs had eaten, and was not in the slugs' genomes.  How the slugs can maintain the chloroplasts as long as they do is a subject of active research.

 

As far as light levels changing, one of the big benefits that animals have over plants is that they can move in order to optimize their exposure.  How they determine optimal exposure with their little brains is one of the things I want to find out.

 

One thing that is also becoming clear is that the amount of energy the slugs receive from photosynthesis probably does not contribute a lot to their metabolism.  In starved slugs, those for whom photosynthesis is blocked by darkness or drugs do not seem to get any skinnier compared to their brethren that are allowed to photosynthesize (very nice writeup here).  It may turn out that the chloroplasts simply act as stable energy stores, kind of like the way sailors used to store turtles in the hold for long voyages.

 

There is a lot of literature out there, and I am hoping to assemble a website bringing some of it together by the end of the summer.

[fry_school101]

Very interesting. Thanks for posting!

 

About how much shaving brush do they go through? Are you rotating them out like starfish for harlequin shrimp or just getting new ones?

 

What happens if you reverse your experiment and try maintaining them with a good food source but low light levels? If photosynthesis isn't contributing to their energy levels then they may not have any dependency at all.

 

I wonder if you could come up with an extracted version of the chloroplasts that they would consume and if it would be possible to dye it to see if they are swapping them out and at what rate.

[Keraxis]

Heres an article I read on a study of sea slugs and photosynthesis.  Kinda goes with what your doing. https://www.sciencenews.org/blog/wild-things/green-sea-slugs-aren%E2%80%99t-solar-powered-after-all.  It also half way answers fry_school101

Edited August 3, 2014 by Keraxis

[mogurnda]

Very interesting. Thanks for posting!

 

[i have added numbers]

 

1.  About how much shaving brush do they go through? Are you rotating them out like starfish for harlequin shrimp or just getting new ones?

 

2.  What happens if you reverse your experiment and try maintaining them with a good food source but low light levels? If photosynthesis isn't contributing to their energy levels then they may not have any dependency at all.

 

3.  I wonder if you could come up with an extracted version of the chloroplasts that they would consume and if it would be possible to dye it to see if they are swapping them out and at what rate.

1.  So far, they are fairly leisurely about consuming the algae, and have only managed to significantly drain one of them.  I have a new batch coming in this week, and it should spread the burden enough so that I will not need to rotate them.

 

2.  To my knowledge, this experiment has not been done.  Based on the current understanding (which will undoubtedly become more nuanced and complete in the future), one would expect they would be perfectly happy.  

 

3.  Very interesting idea.  There have been several experiments which have shown that the chloroplasts can be maintained in a fully functional state for up to 9 months (e.g., Green et al., 2000, Plant Physiol. 42:331) if the slugs are not allowed to feed, but (to my knowledge) nobody has done a labeling experiment to show whether the chloroplasts would be swapped out if the animals continued to feed.  

 

One issue that will definitely need to be addressed is that it is probably a mistake to lump all kleptoplastic slugs together.  Gould and colleagues were using Elsyia timida and Plakobranchus ocellatus when they showed that photosynthesis may not be all that important.  Rumpho and her colleagues have been using Elysia chlorotica, which appears to be a champion as far as the longevity of chloroplasts and photosynthetic performance.  The answers may not be the same for all of them.

 

 

 

Heres an article I read on a study of sea slugs and photosynthesis.  Kinda goes with what your doing. https://www.sciencen...wered-after-all.  It also half way answers fry_school101

Thanks!  It's a nice writeup of some of Gould's work.

[OcalaReefGirl]

That is amazing and really great info. The lettuce sea slugs that bred in my tank are doing great! I have about 30 of the original babies that are now fully grown! As much as I have watched them they seem to eat all kinds of algae with no particular favorite other that chaeto. Not sure why they bred or if they will breed again, but they are so amazing that I am always watching them. Will keep following along to see how I can better take care of my sea slugs    

[mogurnda]

That is amazing and really great info. The lettuce sea slugs that bred in my tank are doing great! I have about 30 of the original babies that are now fully grown! As much as I have watched them they seem to eat all kinds of algae with no particular favorite other that chaeto. Not sure why they bred or if they will breed again, but they are so amazing that I am always watching them. Will keep following along to see how I can better take care of my sea slugs    

Glad you're enjoying it.  It's very cool that you raised babies.  Because they have a very short (a few days), non-feeding planktonic stage, the larvae can do OK as long as they don't get sucked into pump intakes and such.  I do wonder what induces them to spawn.  The ones I dissected in Mexico were packed with eggs, but the few I have looked at in the tank here do not have a lot of mature eggs.  I don't know if it's seasonal, or they want some other form of algae.  That's one reason I have been hunting for other species, like Bryopsis.

 

 

From what I read, some species of Elysia specialize in one or a few species of algae.  E crispata and E clarki (which is the species I have) seem to be less picky.  Like you, I have seen these guys eat multiple types of algae.  My criteria for determining whether they eat something are pretty simple:

1.  Do they stick their faces in it?  When they are really excited, they even shove their rhinophores onto the plant (see last photo, post #28).

2.  Do they suck the sap out?  Their favorite food plants get white after enough of them have had their fill.

 

This Penicillus must have been especially tasty:

 

 

A few weeks ago, most of the slugs had piled onto one of the Halimeda.  I was worried that something was wrong, but then came back to the office a few days later and found that they had completely drained the plant of all juice.  Weird that they all decided it wa "Halimeda day."  

 

 

The rest of the plants still look normal, and the guys don't seem all that interested in them.

 

Based on my experience, they will eat just about anything green, like Penicillus, Bryopsis, Halimeda, Avrainvillea (looks like Udotea, mermaid's fan), and possibly Derbesia, and I expect the list will grow.  They seem to prefer Penicillus, which they spend a lot of time on, and Bryopsis.

[mogurnda]

The Elysia page is now up.  Content is slowly being added and edited.

 

I wrote an article about them a number of years ago.  Very cool creatures.

http://breedersregistry.org/maquaculture/elysia-crispata-sea-slug-spawning-in-a-100-gallon-reef-aquarium/

Speaking of which, Paul, I hope you don't mind that I added a link to your article in my "Popular Articles" section.

[mogurnda]

Well, they have finally done it.  First cluster of eggs, right where I can see them.  They weren't there Friday night when I left, and they were here Sunday morning when I came in.  

 

Stay tuned, maybe we'll see some little ones!

[YHSublime]

Cool! Love the patterns!

[gmerek2]

Are they reproducing successful?

[mogurnda]

Forgot this thread was still around.

 

Are they reproducing successful?

 

At this point, I have raised one generation from egg to adult, but want to do it more consistently and reliably.  I have been distracted with other aspects of the project for the past year or so, but am getting the pieces together.  It all seems to come down to food.  The adults need a constant supply of high-quality algae (mostly Bryopsis) to be in the mood to lay eggs, and the juveniles need top-quality algae (Bryopsis or maybe Derbesia) to begin feeding.  It's doable, but not trivial.  It should be a good species for aquaculture, because the larvae do not need to feed in the planktonic (veliger) stage.

[mogurnda]

Time for an update from the spring and summer.

 

 

Very busy times.  The goal for the summer was to determine which species of algae Elysia diomedea was eating at the field site in Bahia de los Angeles, Baja California. There are many reasons to study the little gals.  For example, they graze in very shallow water, so are very dependent on specific conditions of clarity and nutrients.  Also, they are kleptoplastic, meaning that they suck the sap out of their food algae, and digest everything but the chloroplasts.  They keep the chloroplasts, which keep performing photosynthesis, and it was thought for many years that the slugs themselves became photosynthetic, like corals with their zooxanthellae.  Turns out that is not true, so we have no idea why they steal chloroplasts.

 

 

Anyway, we can’t get very far if we don’t know what they eat.  Based on our observations and those of others, we figured it was probably Codium, Bryopsis, or some other filamentous green alga.  Others have suggested Padina, a brown alga which is highly abundant at sites where we find slugs, but which seems unlikely because all other Elysia species eat green algae.  Ulva is also present, so it is also a candidate.

 

 

Because of the way they feed, it’s hard to know if Elysia are eating something unless they have sucked all the sap out of a plant.  In the wild that’s not likely.  However, since they keep the chloroplasts, and the chloroplasts have their own DNA, we can extract the DNA from the slugs and compare it to candidate food algae.

 

 

The plan was to set up a small molecular lab at the field station, extract DNA, amplify the chloroplast sequence using polymerase chain reaction (PCR), and take the PCR product back to the states for sequencing. 

 

 

Rather than working out the procedures in a dirty, hot garage 12 hours from nowhere, I talked a couple of my UMd students into testing the protocols and reagents here in Maryland.  They spent much of spring semester extracting DNA from plants and slugs, and we had everything worked out using a closely related species, Elysia clarki, along with a couple of species of Bryopsis and Halimeda.

 

 

 

 

Here's our experimental subject, with a divot on her parapodium from where we sampled the tissue.  If you ever want to knock a marine mollusc out, isotonic (400 mM) MgCl₂ does the job.

 

[mogurnda]

Looked like we were ready to take it on the road.  Originally, I had planned to grab some samples in Bahia de los Angeles (BLA), while working loosely with Ocean Discovery Institute, and do the extraction back in the lab at UMd.  Somehow, we decided to make it a project for Ocean Discovery students to do the extraction and PCR in the field.  That made it more interesting, but increased the chances of a train wreck.  Well, what the heck.

 

There were quite a few sleepless nights, such as when it was not clear whether export or collecting permits would be available, and when the local company that was supposed to be building my holding tanks and acrylic “I-mazes” had still not delivered a week before I was supposed to leave. Fortunately, we got all the permissions, and I learned that Glass Cages is able to build and ship custom acrylic tanks at a moment’s notice.  Anyway, after months of planning, buying equipment and reagents, and tracking down equipment that we could borrow and use in Mexico, we were off. 

 

My students, “Dave’s Angels,” were great.  Very enthusiastic, and very smart.  I was also lucky to get Richy, a fellow from UCSB, to help out with logistics and with managing the students.  You can see the Glasscages holding tanks through the windows at right.

 

 

We got the lab set up, and all was well, except that I found zero slugs in the first week.  We could get started on the algae extractions, but would need slugs at some point.

 

 

Lots of good snorkeling, though, while we looked for Elysia. 

 

 

 

 

The puffers by the station were too tame.  They would eat anything you stirred up, which could mean the quick loss of a specimen.  

 

 

As I said, Padina is abundant there, as is Sargassum.

 

 

Below is one of our survey sites at the island Gemelito Este.

 

 

Plus, we got to see the usual whales, dolphins and sea lions when we were out on the bay.

 

 

Will we find slugs?  Stay tuned?

Edited September 3, 2016 by mogurnda

[mogurnda]

Of course we found slugs.  As always seems to be the case, finding the first one is the hard part.  

 

On a day without fieldwork or lab work, I got up at the usual time, had my coffee and got ready to go.

 

 

After a few hours sweeping back and forth over the shallows, I saw an odd color in the Codium.  A slug, yaay!  As you can see in the photo below, it's not easy to see the gals.  On a clear morning, it's not so bad, but it can be a real challenge when the surge kicks up.  There are at least three, maybe four slugs in the Codium below.

 

 

 

They went into the holding tanks for behavioral observation and later DNA sampling.  Note the little Aplysia to her left.  We had a few dozen ride in with some algae we collected.

 

 

We knocked one of them out, and snipped off a piece of parapodium.

 

 

The team extracted and amplified the DNA, I took some of the samples back across the border for sequencing.

 

After cleaning up the samples to get rid of enzymes and whatnot, Macrogen USA (conveniently located in Rockville) returned some very nice sequence.

 

Below is an alignment of a short stretch of the gene of interest (rbcL), for Elysia and two species of algae we collected.  The arrows indicate the sites at which the slug sequence differs from Ulva.  The DNA from the slug kleptoplasts is essentially identical to Codium.  Maybe not surprising, since they are always found near Codium, and often nestled among its branches.  Still, it needed to be confirmed.  Along the way, we got the first DNA sequence for the local species, Codium simulans.

 

 

Short summary: the DNA work went better than we could have expected.  We were up against a lot, but it all came together.

 

We still have a lot to do, like replicating the results in more individuals and curating the C. simulans samples, plus moving forward on behavioral work and field surveys.  Next summer's work should be enough to get the initial DNA analysis published, and we can make a start on the other parts of the project. 

[Origami]

Neat work, Dave. So now you have an idea of what this local species eats. Could there still be some variation in preferred diet in the region? The samples that you collected were collected off of Codium, so it's not terribly surprising that the chloroplasts were found to be from Codium. Not finding a mix of different DNA, though, suggests a preference of the individuals in your sample, I guess. Hey, if they don't keep the chloroplasts for nutrition, what's the reason for keeping them intact? Is that a question you'll try to address? 

[mogurnda]

Thanks, Tom.  I feel like we have more loose ends than data, but I guess that's a good thing.  You ask some very good questions.

 

 

Could there still be some variation in preferred diet in the region? The samples that you collected were collected off of Codium, so it's not terribly surprising that the chloroplasts were found to be from Codium. 

Entirely possible.  We surveyed about 8 spots in the bay, and the only places we found the slugs also had Codium, but the proof would be in the DNA.  Part of the plan for next summer is to take samples from more slugs at more sites.  

 

 

 

Not finding a mix of different DNA, though, suggests a preference of the individuals in your sample, I guess.

It could mean they prefer to eat Codium, or that they preferentially keep the chloroplasts from Codium.  In my office tanks, the Elysia clarki spent almost all of their time face down in Bryopsis, but the one good sequence we got last spring was from Halimeda incrassata, suggesting that the slugs were sneaking off to eat Halimeda, or that Halimeda chloroplasts that they do eat last longer.  Other people have shown that some species of choloplasts last longer in the slugs as well.  More work to be done.

 

 

 

Hey, if they don't keep the chloroplasts for nutrition, what's the reason for keeping them intact? Is that a question you'll try to address? 

That's the $64 question.  It is clear that the chloroplasts produce energy, but nobody has done the calculation to figure out what portion of their metabolism could be supplemented by photosynthesis.  I'd love to be able to do the measurements and calculations for that, but not just yet.  

 

They have to do something for the slug.  There is a metabolic cost to segregating and storing the chloroplasts, so there has to be some advantage to make it worthwhile.  The abundance of Elysia worldwide suggests that the lifestyle is advantageous.  There are several viable hypotheses:

1. They serve as an energy store.  Although photosynthesis does not contribute a lot to the slugs' energy budget, the chloroplasts themselves may be burned for energy when times get hard.  
2. They help the slugs generate and store energy when times are good.  Photosynthesis may not be enough to prevent starvation, but the little bit of extra energy may help the slugs increase their fat stores.  
3. Products of the chloroplasts enhance the production of eggs.  Once they get going, these gals (actually, hermaphrodites) produce a lot of eggs, and nobody has tested the role of photosynthesis or photosynthetic products on reproductive success.
4. Visual camouflage.  What better way to match your food plant?  Seems a very metabolically expensive way to be green, but that doesn't mean it's not the case.
5. Chemical camouflage.  This is my current favorite.  Chloroplasts produce a lot of chemicals, and these could help the slug smell like their food.  Based on other molluscs, I expect that their major predators are nudibranchs, which are essentially blind and hunt by smell.  I am still working on ways to test this in the field and in the lab.  The idea would go something like: a) what eats the slugs; b) does blocking photosynthesis in the slugs make it easier for predators to find them.  
6. Chemical defense.  Similar to the above, but the chloroplasts make the slugs taste bad to predators.  

As I said, option 5 is the hypothesis which has the fewest flaws, at least in my mind.  At the moment, we are all digesting what we learned this summer, taking care of business, and starting to think about next year.  With the molecular tools and the info from the surveys, we are in an excellent position for next year.  

 

A few more photos for fun:

 

The group, surveying and having fun.

 

Scorpion, under UV

 

Normal light.  One of the best things about coming home was not having to worry about scorpions or rattlesnakes on the way to the bathroom.

[Origami]

Fascinating.

 

I liken your description of #1 to our fat cells.

 

#2 brings up the question, do these slugs have discernible fat cells? If so, #1 would seem to make less sense unless there was some reason to favor two separate strategies for energy storage.

 

#3 sounds interesting.

 

#5 - I like your test approach. 

 

If the chloroplasts were being retained to produce energy and they required energy to harvest and maintain, what would happen if they were plunged into prolonged darkeness? Would they expel (or consume) the chloroplasts since they become an energy sink? Would the chloroplasts simply die?