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.

Getting Baby Slugs to Eat

Since the first batches of eggs were laid years ago, it has been a struggle to get the babies to make the jump from hatchling to juvenile slug.  The eggs hatch a few weeks after having been laid, and the veligers rapidly make the transition to cute baby slugs.

Elysia clarki hatchling on Cladophora (?). 11/1/17

The problem was that they almost never progressed to the point of looking like miniature adults, as in the photo below.

Very small E. clarki that will ultimately grow to adulthood in Box of Slugs 2.0. Note the chloroplasts (green dots) and the rhinophores.  2/24/15

Once they had rhinophores at the front end, and chloroplasts inside, as seen above, they grew quickly to adults.  The problem was that the hatchling slugs would never eat.  They would crawl around on the algae, and appeared to try to feed, but never ingested anything and ultimately starved.

It was hard to know what was wrong, because any number of factors might prevent them from eating.  Were there issues with environmental conditions or pathogens that were preventing them from feeding?  Was it the type of algae?  Adults devour Bryopsis and a number of other algae species quite happily, but maybe baby slugs do not eat the same species.  For example, E. timida adults eat Acetabularia exclusively, but the hatchlings only eat Cladophora.  So what do baby E. clarki eat?

As described in previous posts, the E. clarki in my home tank (“Box of Slugs 2.0”) have been producing eggs at a relatively constant rate of one egg mass per week.  It was getting depressing to watch broods hatch, then starve over and over again.  I wanted to order some Cladophora (if it works for E. timida, why not E. clarki?), but Hurricane Irma had wiped out collecting in Florida for the winter.  I did try a species of algae that had taken over a local aquarist’s tank and that looked a lot like Cladophora, but the babies were not interested.

The Chemical Ecology students shamed me into contacting Skip Pierce again, which turned out to be a very positive thing for a number of reasons. I was surprised to hear that they had no trouble getting the slugs to start eating, but they had never been able to get them to lay eggs on a regular basis.

After a few emails back and forth between me, Pierce, and his collaborator Mike Middlebrooks, we realized that I might have been using the wrong species of Bryopsis.  The students had sequenced the DNA of the species that I culture here, and it is unambiguously B. pennata.  I have been using it for the simple reason that it plagues local aquarists, and was therefore the species I could obtain with some regularity.  After a few years of tweaking, I have been able to grow it sustainably, and the adult slugs grow big and strong using it as an exclusive food source.

Nonetheless, the babies might prefer B. plumosa.  The Pierce lab tried feeding hatchling E. clarki with a long list of algae, but they would only eat B. plumosa or Derbesia tenuissima (Curtis et al., 2007).  They did not try B. pennata, and I was skeptical so the hatchlings would be so picky about two similar species of algae.  Skip and Mike were relatively certain that the hatchlings would survive if they were fed B. plumosa.  It is possible that the youngsters that had survived in my tank before had found some D. tenuissima, which is a common nuisance algae in marine aquaria.

However, feeding them B. plumosa was easier said than done.  To my knowledge, there is no retail source for B. plumosa, and Irma had made it difficult for Mike Middlebrooks to collect any for himself or for me.  I even tried to collect in on the Eastern Shore of Marlyand, because it had been reported growing on hard surfaces there.  The strong currents, and turbid, cold water of the Atlantic in November made that trip something of a fool’s errand.  There were several species of Ulva easily available, but no Bryopsis to be found.

Ulva at Ocean City. 11/11/17

I finally decided to invite myself to Tampa.  Even if we could not find B. plumosa, I would be able to see the setup at the Middlebrooks lab.  My entire career has been spent with lab-reared insects, not marine molluscs, so it seemed a good idea to visit a real Elysia lab.  As it turns out, I got to visit two Elysia labs, because the Pierce lab at USF and Mike’s lab at the University of Tampa are relatively close to one another.  I spent 3 days in Tampa, talking endlessly with Mike about all things sluggy, and getting caught up on a lot of detail.  There really is not better way to learn about a field than having long, unstructured conversations about details few other people care about.

Mike Middlebrooks and Skip Pierce, in the Pierce Lab at the University of South Florida. 12/28/17

As an added bonus, Mike took me to his collection site at Sand Key Park in Clearwater, where we found abundant, dense, happy growths of B. plumosa.  We collected several large Ziploc bags full of Bryopsis, along with some Cladophora for good measure.

Bryopsis plumosa (the deep green stuff on the edge of the rock) and other algae on Jetty at Sand Park Key, 12/28/17.

That left the question of how to get some of it to Maryland.  After some discussion of shipping options, I decided to quadruple bag a batch of it, wrap it in a wetsuit, and check it on the airplane.  It survived the night in the hotel before the flight, although it started to develop a slight smell of shore drift.

Ziploc bag full of B. pennata. Clump of Cladophora in foreground. 12/28/17

After the long journey in imperfect conditions, the algae looked pretty good.

B. plumosa in Box of Slugs 2.0. 12/29/17

The good news is that, as of last week, E. clarki hatchlings have been eating B. plumosa.  Yaaay!  They seemed to be drawn strongly to the algae, and within a few days the proof was in their guts.  The youngster in the photo below has algae in her pair of digestive diverticula that run along her sides.  She is still very young, possibly after her first meal, and not much bigger than the veliger larva next to her.

Hatchling, with food in digestive diverticula. Veliger larva at left. 1/28/18

The slug below is larger (although the scales are different), and has stuffed herself full.  There are also older animals that are starting to develop rhinophores and parapodia, but they were not positioned for photographs.  So, it looks like we are finally past the hurdle of the first meal.  Assuming they avoid the predators and pathogens in the system, we should soon see some miniature versions of the adults.

Elysia clarki hatchling, full of algal cytoplasm.  1/26/18

One problem solved, but it is still unclear why the Pierce and Middlebrooks labs can’t get their slugs to lay eggs.  What’s worse is that Irma seems to have wiped out the wild E. clarki for the moment.  The good news is that I carried a clutch of eggs to Tampa when I visited, and Skip Pierce let me know that they are feeding and growing.  Some Maryland slugs have made their way back home.

Just Some Eggs

Elysia clarki eggs in Box of Slugs 2. 1/16/18

This morning, one of the big Elysia clarki was coiled suspiciously on one of the Udotea .  Sure enough, she was laying another large egg mass.  There must be thousands of little embryos in there.  They have been laying about one mass per week, for quite some time, and the total count is at least 21 egg masses since September.  That is a lot of eggs for two slugs.

Keeping fingers crossed about getting the babies to eat, now that we have some new Bryopsis plumosa.

Updates for the New Year!

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

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

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

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

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?)

Journal Club: Maybe Kleptoplasty IS All About Energy

Elysia and related sacoglossans are beautiful and interesting, but their use of stolen chloroplasts puts them in a class above your average green sea slug. How the slugs accomplish this, and what function it serves, are coming into focus very slowly.

It has been known for a long time that kleptoplasts remain alive and functional in many Elysia species, but the idea that they can rely on the chloroplasts for all of their energy needs (the “crawling leaves” hypothesis) appears to be wrong, or at least overly simple.  The inadequacy of the crawling leaf theory got me thinking about a possible role for kleptoplasts as factories for making defensive chemicals.  There was support in the literature (e.g., Trench et al., 1972; Ireland and Scheuer, 1975), and I was interested enough to organize a student journal club on chemical ecology, and prepare experiments to test the connection between kleptoplasty and predation in Bahia de los Angeles in summer 2018.

To introduce the students to the journal club format, and to some important Elysia concepts, I presented the following paper by Baumgartner, Pavia and Toth from PLoS One, published in 2015.  The paper has restored some of my faith that kleptoplasty provides a substantial metabolic benefit to the host slug.

They performed a series of experiments to ask a straightforward question: Do kleptoplasts provide usable energy to E. viridis?

The main characters in Baumgartner et al., 2015. Codium (upper left), Cladophora (lower left), and Elysia viridis (right). Images of algae are from AglaeBase, and E. viridis is from Scottish Nudibranchs.

E. viridis‘ gets kleptoplasts from at least two of its natural food plants, Codium and Cladophora, and they differ in their viability in the slug.  Chloroplasts from Codium are highly functional, whereas those from Cladophora do not function well after being eaten.  This provides a natural experiment, in which one can compare the effects of light on the growth of slugs fed Codium (highly productive kleptoplasts) versus those fed Cladophora (little contribution from photosynthesis).  Importantly, the slugs in these experiments are not starved, so the authors are looking at the effects of photosynthesis over and above the energy and nutrients gained from feeding.

They tested the following hypotheses:

  1. Slugs feeding on Codium under stronger light  will have higher growth efficiency (GE; defined below) and kleptoplasts will have higher relative electron transport rate (rETR) than those in low light.
  2. There will be no difference between low- and high-light conditions in slugs feeding on Cladophora.
  3. If there is a benefit from increased rETR, it will not correlate with other nutritional traits of the macroalgae.

E. viridis were divided into four groups: Those eating Codium,under high light (~100 μmol quanta m-2 s-1) or low light (~6 μmol quanta m-2 s-1); and those eating Cladophora in high or low light

In the first experiment, they examined the effect of illumination on growth of the slugs.  Rather than simply looking at growth rate, they used a measure called Growth Efficiency (GE), which calculated the amount of growth of the slugs as a function of how much the slugs ate.

Step 1: calculate slug Growth Rate (GR) = (Mend-Mbefore)/t, where M = mass and t = time

Step 2: calculate Consumption Rate (CR).  For this they needed to either measure the change in mass over time (Codium), or the number of damaged cells (Cladophora).  They had to use two different measures because the structures of the two species of algae posed different challenges.  They used algae kept without slugs as a control for the effect of time.

Step 3: Calculate GE = GR/CR, yielding the number of milligrams (mg) of slug growth per gram of algae consumed (Codium) or per 1000 cells consumed (Cladophora).  Because they were comparing the effect of light intensity on slugs fed the two algae, the different units used for consumption did not cause problems.

Figure 1A shows that slugs fed Codium show significantly better growth efficiency in high light than in low light.  Those feeding on Cladophora show no difference in two conditions.  The kleptoplasts from Codium contributed more to the growth of the slugs in bright light, which suggests that photosynthesis is producing energy that the slugs could use for growth.

However, they needed to be sure that the kelptoplasts from Codium were truly more functional than those from Cladophora, so they measured photosynthetic ability of chloroplasts and kleptoplasts using PAM fluorometry.  The absorbance and fluorescence of chlorophyll can be used to measure the health of chloroplasts, and to calculate the relative electron transport rate (rETR),  a measure of carbon fixation and therefore energy production.

Figure 2 shows that kleptoplasts from Codium responded to intense illumination with a strong increase in rETR, consistent with increased growth efficiency being caused by increased photosynthesis of healthy kleptoplasts.  Kleptoplasts from Cladophora had low rETR regardless of lighting, correlating well with the lack of effect of intense light on growth efficiency of E. viridis.  These observations support the idea that photosynthesis by kleptoplasts contributes to slug growth.

Just in case the increased growth rate of E. viridis was due to some other benefit provided by Codium, they measured nutritional quality of the two algal species.

In all cases, the nutritional value of Cladophora was as high or higher than that of Codium (Figure 3), so there was no evidence that Codium was intrinsically more nutritious.  Importantly, Panel 3N shows chloroplasts in Cladophora increased rETR in response to more intense light.  Therefore, the poor performance of Cladophora-derived kleptoplasts (Figure 2B) is likely to result from degradation inside the slug.

The evidence therefore supports the authors’ hypotheses, and they conclude that E. viridis derive measurable metabolic benefits from photosynthesizing kleptoplasts.  Whereas Slugs feeding on Cladophora receive nutrients by digesting the cellular contents (including the chloroplasts), those feeding on Codium get an extra boost from products produced by active, healthy kleptoplasts.

Overall, the experiments seemed well-executed and convincing, with the only caveat being that the slugs fed Codium and Cladophora were collected on those species and not randomly assigned to the groups.  There remains at least a formal possibility that the groups represent two populations of slugs that metabolize chloroplasts differently.

This brings us back to the the role of kleptoplasty in the biology of Elysia.  It could be:

  • Increased energy available for growth (see paper above)
  • Fixation of nitrogen into amino acids (Teugels et al., 2008; Journal Club in progress)
  • Production of defensive chemicals (Trench et al., 1972; Ireland and Scheuer, 1975)
  • Visual camouflage (they are green, no citation needed)
  • Synthesis of chemicals needed for egg production (anecdotally, E. clarki appears to produce eggs when PAR is above 100)

We should bear in mind that parsimony may not equal truth, and there is no reason to believe that only one of these answers is correct for all species and life stages.

So Much Happening!

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

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

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

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

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

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

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

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

Elysia Abides

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

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

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

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

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

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

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

Check back for updates and insights as they develop.

Culturing Bryopsis (Part Trois)

I recently had an epiphany about how to grow Bryopsis more efficiently.  In retrospect, it was pretty obvious, and I wonder why it took 2 years to get to this point.

It seemed as though culturing adequate Bryopsis was under control.  However, there’s nothing like 6+ weeks of travel to turn things upside-down.  I had hired a service to come in and keep things going while I was away, but their primary task was to prevent biological meltdown, or worse, a big salty disaster that would have me forever on the naughty list of the facilities people.  I am happy to say, there were no smelly or wet disasters.

Unfortunately, I was not there to give the algae cultures the kind of attention they need, and by the time I was back in the office, the system was overrun with Derbesia, and the slugs had devoured the Bryopsis that I had left for them.  The 20-gallon slug tank and both algae culture tanks were full of felty, green hair algae.  In retrospect, I should have taken photos, but I was more focused on cleaning up the mess and getting ready to teach a summer class.

Over a week or so, I pulled out the algae tanks off the system, cleaned out at least two pounds of green glop, salvaged the remaining Bryopsis, and set the tanks  back up.   Somewhere along the line, I came across a few posts about “algae reactors,”  cylindrical chambers with water flowing through and some sort of light source.  My first thought was “maybe I should buy one of those things.”  Then I remembered that I had two media reactors sitting idly in the basement.  They are clear cylinders, designed to have water flow through them, which is exactly what I wanted.

The amount of Bryopsis remaining was so small that it didn’t seem worthwhile to have two algae tanks.  Instead, I shut down the 10-gallon tank and stuck some of the remaining algae into a reactor. The reactor was hooked up to one of the valves, and connected to the drain, and sat under the grow light where the 10-gallon tank had been.

Two Little Fishies media reactor, with a small wad of Bryopsis held in place by eggcrate. 7/12/17

Within a week or so, it became clear that the experiment was working, so I added the second reactor.  I had enough Bryopsis to harvest some for the slugs, and the culture in the first reactor had seeded a sponge that I could use to start the second reactor.

Second reactor, with a sponge seeded from the Bryopsis in the first. 7/21/17

It was working.  Time to make things a little less clumsy.  I built a rack from 3/4″ PVC, setting the reactors at an angle to optimize the connections to the input valves and drains.

Bryopsis reactors, in their improved configuration. 7/26/17.

Things went so well, I begged a few unused reactors from local aquarists.  This one is from Alan (of unidentified algae fame), and I have one more waiting in the wings.

Three reactors. 8/1/17.

At this point, it was clear that my former method of rearing Bryopsis in aquaria was not very efficient.  Raising Bryopsis in reactors allows me to play with growth parameters, like flow and nutrients, much more easily.  Further, keeping multiple separate cultures will make it much easier to eradicate unwanted algae.  At some point, it should be straightforward to maintain cultures free of unwanted algae and invertebrate pests (I am all for biodiversity, except when it eats slug larvae) by UV sterilizing the water going into the chambers.

As far as I can tell, this was a successful experiment, so I converted the 15 gallon algae tank into the second slug tank, shutting down the 10-gallon slug tank.

When I walked in today, the system had two slug tanks (top and middle left), and one remaining algae tank (top right)

Elysia system before rearrangement. 8/5/17.

Five hours of cleaning and rearranging later, there were two slug tanks and three algae reactors.  In the process, the system now has two fewer circulation pumps and one fewer light fixture.

Current slug system arrangement. Two tanks for slugs and and their food on top, three reactors in the middle, and room to expand. 8/5/17.

In the future, you can expect a few more reactors.  Now it’s time to play with parameters to maximize algae growth.  Maybe we’ll finally see some consistent egg production from the slugs.

Slugs of Madagascar

Every 5 or 6 years, we end up exploring someplace a little more exotic.  This year, we decided to go to Madagascar, and we arranged for a driver and accommodations for about 10 days of overland travel and saw some incredible people, landscapes, and wildlife.  We’re still sorting through the photos of lemurs, chameleons, villages, and vistas, and it is likely to take some time.

Of course, you don’t come to this web site for the lemurs.

Because we were traveling all the way to an island in the tropical Indian Ocean, I pushed for a stretch of diving at the end.  When we were planning the trip, I asked our tour operator, Cactus Tours (an excellent Madagascar-based company) to arrange for some diving at the island of Nosy Be, at the north end of Madagascar.  We ended up spending a few days in Nosy Be, and had three days and two night of diving on a sailing catamaran.

After bouncing around in a four-wheel drive for over a week, it was pure luxury to be on the boat. For just the two of us, there was a driver, cook, and a very experienced and knowledgeable dive master, all arranged through Madavoile Cruises. We had six excellent dives, and were completely blown away by the diversity of corals, fish and invertebrates.

Our dive guide, Nicolas, figured out pretty quickly that I wanted to see nudibranchs and sea slugs, and he did not disappoint.   The photos below were taken with an Olympus Tough TG-4 camera, which is rated to 50 feet without a housing.  Because the housing was clumsy, we took the unhoused camera as deep as 65 feet, which worked just fine despite its increasingly strident warnings. Identifications are based largely on a digital version of Gosliner et al’s “Nudibranch and Sea Slug Identification”  and the Sea Slugs from Reunion Island Web site, which is an excellent reference for the southwest Indian Ocean.  Please let me know if you believe a species to be misidentified.

Phyllidia varicosa, Sugarloaf Island, 6/30/17

 

Phyllidia sp., maybe P. marindica, Sugarloaf Island, 6/30/17.

 

Phyllidiella zeylanica, North Nosy Tanikely, 6/29/17.

 

Phyllidiella zeylanica, Ankazo Reef, 6/30/17.

 

Phyllidiella pustulosa, Sugarloaf Island, 6/30/17.

 

Phyllidia ocellata, Sugarloaf Island, 6/30/17.

 

Doriprismatica paladentata, Ankazo Reef, 6/30/17.

 

Glossodoris pallida, Sugarloaf Island, 6/30/17.

 

Goniobranchus geometricus, Ankazo Reef, 6/30/17.

Although we saw plenty of Caulerpa, Halimeda and other macroalgae, and looked very hard for Elysia, we came up empty.  At Nosy Sakatia, we spent our last morning snorkeling with the turtles before we dove, and I was pleased to see a wide expanse of turtle grass (Thalassia) and manatee grass (Syringodium), which had some excellent growths of Halimeda incrassata.

Halimeda incrassata, among other algae and seagrasses, Nosy Sakatia, 7/1/17.

Unfortunately, I did not get to spend hours searching the seagrass beds.  Watching the biggest green turtles I had ever seen graze right in front of me was a nice consolation.  It is hard to get a sense of just how big these monsters are from a photograph.

Huge green turtle grazing at Sakatia, 7/1/17.

I did find one sacoglossan, Plakobranchus ocellatus, on the reef at Sakatia Arch.  Unfortunately, the camera housing I was using for the dive had fogged, so it is a lousy photo.  Just not the trip for sap-sucking slugs, I guess.

Plakobranchus ocellatus, Sakatia Arch, 7/1/17.

In addition to the plentiful slugs, there were quite a few flatworms pretending to be nudibranchs. All were in the genus Pseudoceros, and all were found in Humann and Deloach’s Reef Creature Identification book, which has an impressive section on flatworms.

Pseudoceros tristriatus, Ankazo Reef, 6/30/17.

 

Pseudoceros lindae, Ankazo Reef, 6/30/17

 

Pseudoceros bifurcus, Sugarloaf Island, 6/30/17.