Posts By Dave

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.

Bahia II: Elysia diomedea Tastes Bad

As described in the previous post, I had very modest goals for this summer in Bahia.  Because I am getting more interested in the role of kleptoplasty in chemical defense, I thought it would be worth assessing the palatability of Elysia diomedea.  Some Elysia species are known to taste bad because of chemicals assimilated from their food plants (see, e.g., Rasher et al., described in this post).  E. diomedea is known to produce interesting derivatives of plant compounds (e.g., Ireland et al., 1978, J. Am. Chem. Soc. 100:1002), but, as far as I can tell, there is no evidence regarding the slugs’ palatability.

Fortunately for me, there is a relatively easy way to get a quick sense of their palatability.  When snorkeling at the field station, one is generally followed by a small parade of large bullseye puffers (Sphoeroides annulatus) waiting for tasty morsels to be stirred up.  What would happen if I dropped a slug in the water column and allowed the fish to eat it?  One might expect a puffer to eat anything.

After a day in the field, I had time for a snorkel, so it was a perfect opportunity.  After a short survey along the subtidal, I found a few Elysia in a small bunch of Codium (surprise!).  I pulled out this little beauty, apologized to her and carried her to the surface.

Elysia diomedea in the shallows in front of the Bahia de los Angeles field station. 5/23/17

If you click the link below for the short video (note: large-ish file), it is pretty clear that the puffer does not find the little Elysia to its liking.

Puffer spitting out Elysia diomedea. Bahia de los Angeles, 5/23/17

Not only one, but three puffers rejected the Elysia.  After the first spat out the slug, a second tried it, then a third.  In no case did one of the puffers as much as chew, they rejected it as soon as it was in their mouths.  Very good for the slug, and suggests that it may be something secreted in the mucus that repels the fish.  One might also conclude that puffers don’t learn from their friends, since each had to try it.

Based on one slug (but three puffers), we can tentatively conclude that E. diomedea tastes bad.  Are the bad tasting compounds derived from products made by the kleptoplasts?

Bahia de los Angeles 2017. Volume 1

Nature is perverse.

We’re back in Bahia de los Angeles, on the east coast of Baja California, Mexico. For the two previous years, I have had to suffer for a while before finding any Elysia diomedea.  It was rather nerve wracking, because I needed them for research projects each of those years.

Because of time constraints, my goals this year are to help my friend, Dr. Drew Talley, with his long term research, and to discuss plans for student Elysia projects in summer 2018 with Ocean Discovery Institute.

We had some complications at the border, because the Mexican authorities had some reservations about some equipment that was being used by one of the other research groups.  We were allowed to proceed after a few hours dealing with paperwork, but were delayed to the point that we had to stop along the way for the night.

We arrived without further incident, unloaded equipment and belongings, started setting up the station, had a great meal in town, we went to bed for the night.  It was amazing to be back under all the stars, listening to the ocean and the occasional breathing of a marine mammal.

We woke up to a classic sunrise, and soon we were on the islands, setting traps for insect surveys and savoring the bay and the scenery.

Heading back to boat after setting traps on Mitlan. 5/21/17

After returning to the station, we ran some errands, followed by a little open time to get in the water.  Although I may try a few extremely simple preliminary experiments, my work here does not depend on finding them.  Naturally, that means they were abundant in the shallows in front of the station. I found the first within five minutes, and saw at least six within the half hour allotted for the survey.

Elysia diomedea in front of field station. 5/21/17

They looked darker than the slugs we found last summer, but, as was true last summer, all were on or near Codium.

Keeping fingers crossed for a chance to test some ideas about chemical camouflage.

Bryopsis 2: Thriving in the slug tanks

Just when I thought I had it all figured out.

After struggling for way too long, I finally got reliable growth of Bryopsis in the algae culture tanks by providing strong lighting, balanced nutrients, along with very intense water movement.  Bryopsis grew well, although Derbesia also started to thrive and needed periodic removal, so I figured I was close to the magic formula.

10 gallon slug tank a few weeks after system revamp. Sponge-covered circulation pump visible at right, and a small clump of Bryopsis in rear center. 3/8/17.

Imagine my surprise when Bryopsis started to thrive, largely without contamination by dinoflagellates or Derbesia, in the slug culture tanks.  In the 20 gallon long, the algae that was transferred on the plastic racks started to grow and spread, despite constant grazing from the resident E. clarki.  In the 10 gallon, a few scraps growing on some of the macroalgae grew to fill almost half the tank.

10 gallon slug tank, showing heavy growth of Bryopsis after a few months. Other algae are doing fine, but the Bryopsis is taking over. 5/9/17

This has made the single E. crispata very happy.  She has grown considerably, and her color is amazing.  Will post a photo when she comes out of the algae far enough to be photographed.

The tanks are all plumbed together, so they all receive the same nutrient input.  The slug tanks receive somewhat less light than the algae culture tanks.  The spectrum of the lights in the slug tanks is broader, with more green, yellow and UV, mostly because that is nicer for my eyes when I look at the tanks.  Finally, the circulation is considerably less intense.  The algae tanks are blasted with propeller pumps and wavemakers, while circulation to each of the slug tanks is only provided by a single Maxi-Jet 600 (600 liters per hour output), with the intake slug-proofed by a strainer and sponge, and the output directed through a Hydor Flo to provide swirling motion.

Parts of a slug-proof circulation system. The intake of the Maxi-Jet powerhead is covered by a strainer and a sponge, because a strainer alone will not prevent injury to slow, squishy, dumb slugs. The output goes through a Hydor Flo rotating deflector to provide surgy motion.

Assembled Elysia circulator. In the slug system, each one is anchored to the glass with a magnetic holder.

Because even these relatively small pumps give the slugs a bit of a wild ride, they are only turned on for 15 minutes of every hour.  Quick summary: despite my beliefs to the contrary, it is possible for Bryopsis to grow strongly with modest water movement.

Although I am pleased that there is now abundant food for the slugs, it does bother me that I still do not understand all of the factors affecting growth of Bryopsis.  Previously, Bryopsis struggled in conditions that were largely similar.  If I get a chance before leaving for Baja this week, I’ll make yet another deep dive into the system logs to determine which parameters may have changed.  The luxuriant growth has emboldened me to order a few more E. clarki, so that the colony can be going full steam by the end of the summer.

New E. clarki, acclimating after arrival from KP Aquatics. 5/11/17

How to Grow Bryopsis

To many, this post is akin to “how to give fleas to your dog,” or “how to grow ragweed.”  Bryopsis is a benthic (substrate-associated) genus of algae that most aquarists dread seeing in their tanks.  Bryopsis can thrive in reef aquaria, to the point that it can smother and kill corals.  Many methods of eradication have been explored, including near-toxic levels of magnesium, and the anti-fungal medication fluconazole.

E. clarki feeding on Bryopsis; the red alga, Botryocladia, is in the background.

On the other hand, I really want the stuff.  As far as I can tell, it is the favorite food of Elysia clarki.  E. clarki will eat other macroalgae, such as Penicillus, Avrainvillea or Halimeda, but, given the choice, they will go straight for Bryopsis and stay there.  Further, they have consistently produced eggs only at times when they have had unlimited availability of Bryopsis.  One of my primary goals is to maintain a self-sustaining colony of slugs, so I very much want a constant, adequate supply of algae.

I’d be perfectly happy if I could count on the outbreaks of local aquarists to supply my needs.  Unfortunately, sources have been spotty.  My primary source, Justin, who had a beautiful outbreak in a 500-gallon tank, unfortunately managed to get his infestation under control.  He swears the controlling factor for him is the level of phosphate; if PO4 levels creep up, he gets an outbreak.  Others have provided quality material as well, but not as consistently. Yet others have offered species that turn out not to be Bryopsis.  The take home lesson is that people who are trying to eradicate something are not the most dependable source.

The whole gang enjoying the new batch of algae provided by a local retailer. 4/17/16.

How hard can the stuff be to grow?  It is a pest, after all.  Like any aquatic plant, it needs light, nutrients, and circulation.  It gets a little more difficult when you try to provide for the plants’ requirements on a consistent basis.  As far as I can tell (and PLEASE email or comment if I am wrong), nobody has published specific requirements of Bryopsis for PAR (photosynthetically-available radiation), macronutrients (e.g., carbon, nitrogen, phosphorous), or micronutrients (like iron, copper and cobalt).  When marine aquarists have outbreaks, much of the nutrient load is derived from fish food (well, really fish poop), which is not measured.  In fact, most reef aquaria don’t have Bryopsis outbreaks, which suggests that the unlucky few have managed to combine all the right ingredients.  For a constant supply of Bryopsis, I need to provide for all of its needs.

If I wanted to grow phytoplankton, the single-cell, free-floating relatives of Bryopsis, I would be in luck.  Because of the important role of phytoplankton in our oceans, and because there is a growing industry using them to generate hydrocarbons from sunlight, growing conditions have been explored and published at length.  The most popular formula is Guillard’s F/2, so named because it consists of his formula F diluted to 50% (Guillard & Ryther, 1962, Can. J. Microbiol. 8:229), is readily available through suppliers such as Florida Aqua Farms, or even Ebay.  The formula provides a balanced blend of nitrogen and phosphorous (with an option to add silicon if you want to grow diatoms), along with a mix of trace elements and vitamins.  Mix it up, add a starter of the phytoplankton of your choice, bubble in some CO2 as a carbon source, and you’ll soon have a thriving culture of green water.

I started dosing the Florida Aqua Farms version (“Plant Fuel Too”) almost a year and a half ago, using the complete dry mix (minus silicates).  The Bryopsis started to look darker and happier pretty quickly.  Perhaps unsurprisingly, there was also a phyoplankton bloom that turned the water green and generated a layer of green scum on the surface of the water.  Also, the added nutrients seemed to increase levels of nuisances like cyanobacteria (a.k.a., red slime algae). That would have all been tolerable, but the biggest sticking point was that Bryopsis would go through cycles of boom and bust, which is not conducive to long-term management of a slug colony.  I needed to get the right balance of nutrients that would get the Bryopsis to grow maximally and out-compete the species I don’t want.

With all of the possible factors (e.g., light, circulation, temperature, nutrients) to be within the right ranges, there were a lot of variables to play with:

  • Levels of light seemed to be within the range I observed in tanks with steady outbreaks, so I left that alone.
  • Bryopsis always grows well in the areas with the strongest water flow (e.g., pump outlets).  I am not sure why, maybe it keeps other algae from settling on it, but I increased the flow to ridiculous levels in the 15-gallon primary culture tank by combining a power filter Aquaclear AC70), powerhead (Hydor 2450) and wavemaker (MaxSpect Gyre XF130) that were all rated for tanks 3-5 times the size.
  • I plunged back into the literature to try to find papers examining the nutrient requirements of benthic macroalgae, rather than phytoplankton.  After a little digging, I found that species related to Bryopsis demand much more carbon and nitrogen, and are not as dependent on phosphorous (e.g., Atkinson and Smith, 1983, Limnol. Oceanogr. 28:568; Pedersen and Borum, 1997, Mar. Ecol. Prog. Ser. 161:155).

In order to separate the variables a bit better, I started using Florida Aqua Farms original “Plant Fuel,” which contains all of the trace elements and vitamins, but no nitrogen or phosphate. That would allow me to play with levels of trace elements, N, and P separately. I also added a source of carbon.  Because white vinegar is cheap, always available, and is readily broken down into CO2 by bacteria, it seemed like a good choice.

By adjusting the relative input of all four ingredients (C, N, P and trace elements), combined with very high flow in the algae culture tank, I was able to get significantly better growth.  The final improvement was something that I had not expected to have such an impact: rebuilding the system.  Within about a week of the big move, the algae was thriving on the racks of plastic eggcrate I was using as a substrate.  I suspect that the 50% water change and improvements to circulation in the new system played a large part in the improved growth.

Bryopsis growing on eggcrate in 15 gallon algae culture tank. 3/2/17

By growing on plastic racks, Bryopsis cultures can be moved to slug tanks to be consumed, and then removed for regrowth.

Bryopsis-covered plastic rack in 20L slug tank. 3/23/17

So we’re making forward progress.  We are controlling the growth of food algae to the point where it seems to be sustainable.  As a side benefit, the other macroalgae are also thriving.  For example, the three deep green Penicillus in the photo below are new sprouts from the ratty old plant in the center.

Three new Penicillus plants surrounding the faded parent taken from the wild. 4/11/17

At the moment (and likely to change) the system receives major elements at the (molar) ratio of 150:30:1 C:N:P.  This is roughly in the range reported for other macroalgae, and does not support much growth of cyanobacteria or phytoplankton.  In other words, it looks like I am providing nutrients at about the rate at which the are being consumed by the desired algae.

  • N: dosing KNO3 (13.9% N); stock = 80 grams/liter (g/l), diluted to 20 g/l; dosing 80 milliliters/day (ml/d); that equals 0.222 g/d of N, or 0.0159 moles/day
  • P: Dosing KH2PO4 (22.8% P); stock = 11.6 g/l; diluted to 1.45 g/l; dosing 50 ml/d; 0.0165 g/d or 0.00053 mol/d
  • C: from acetic acid: 5% solution (50 g/l); 48 ml/d; C = 40%; 0.96 g/d; 0.08 mol/d
  • K: from KNO3 AND KH2PO4:  0.64 g/d or 0.021 mol/d
  • Guillard’s F/2 trace element and vitamin solution: diluted 1:20, dosing 100 ml/d;
  • I have also started adding calcium hydroxide (CaOH2, kalkwasser) to the water used to make up for evaporation to maintain Ca.

There is still plenty of room for improvement.  The priority for the moment is eliminating or reducing dinoflagellates in the algae culture tanks.  They do not seem to cause too much trouble in the slug tanks, but they form a slimy film on the Bryopsis in the culture tanks, presumably reducing growth, competing for nutrients, and producing toxins.

Dinoflagellates at low magnification. They can be seen as strings of brown dots surrounding the green algae branches. 4/11/17

At higher magnification they are kind of pretty.  Based on the shape, size and general appearance, it looks a lot like Ostreopsis ovata (Faust and Gulledge, 2002, Contr. U.S. Natl. Herb. 42: 1-144).  Like many dinoflagellates, O. ovata produces toxins, some of which are toxic to mice (Nakajima et al., 1981, Okinawa Bull. Jpn. Soc, Sci. Fish. 47: 1029).  The effects on slugs or other invertebrates is not known.

DInoflagellate from Bryopsis tank, 400X magnification. Gold color is from densely packed chloroplasts.  4/6/17.

At the moment, I have reduced the photoperiod (the time the lights are on), and am rinsing the Bryopsis racks in clean artificial seawater periodically to reduce the population.  At some point, it might be necessary to use harsher methods, such as antibiotics (metronidazole seems to affect dinoflagellates somewhat specifically) or starting a clean culture of Bryopsis and preventing entry of other species using UV sterilization.

For now, there is plentiful food for the slugs, which is a step forward.


Journal Club: Chemical Camouflage and George Harrison

Welcome to the Roughly Annual Solar Sea Slug Journal Club.

Today’s paper came from the Proceeding of the National Academy of Sciences a few years ago (Rasher et al., 2015, Proc Natl Acad Sci 112:12110).  I came across it again when I was updating records for this site, and, because it is germane to one of my pet theories, it seemed perfectly suited for an extended discussion. You’ll see how George Harrison fits into the story later.  Yes, this post will meander a bit, but the fact that you are reading the Solar Sea Slug Blog suggests you may have some time on your hands.

The paper is a very nice exploration of the interactions between herbivores and their food plants.  Up here on dry land, insects tend to specialize on particular food plants, and bugs and plants have evolved together in something of an arms race.  Insects use volatile chemicals produced by the plants to locate them, plants produce defensive chemicals to keep from being eaten and from being infected by insect-borne pathogens, insects develop resistance to the plant chemicals, and sometimes use them for their own defense, and so on.  The authors wondered if they could identify a similar web of interactions in the marine environment.

Elysia tuca in Box of Slugs 2.

The algae Halimeda incrassata would seem to be rather unpalatable.  It produces a collection of defensive chemicals, and is highly calcified, making it a crunchy, bad tasting mouthful.  Despite the defenses, Elysia tuca, a tiny and distinctively-patterned species, is commonly found on Halimeda.  The interaction between E. tuca and H. incrassata allowed the authors to ask how similar the relationship between a mollusc and a marine alga is to those of insects and terrestrial plants.

Halimeda incrassata, in Box of Slugs 2. Note the segmented appearance, which will be important in understanding Figures 3 and 4 of the paper. 3/12/17.

In order to compare the relationship between E. tuca and Halimeda to terrestrial plant-insect interactions, the study focused on five specific questions:
1)  Is E. tuca really a specialist? This is important for the development of an intimate plant-herbivore relationship.
2)  Does E. tuca find Halimeda based on chemical cues?
3)  What are the cues that E. tuca uses?
4)  What are the ecological consequences of E. tuca feeding on H. incrassata?
5)  Does Halimeda use counter-defenses to limit the damage inflicted by E. tuca.

With regard to E. tuca being a specialist, the answer was a pretty resounding “yes.”  They collected specimens of about 10 species of algae and marine plants at two sites, took them to the lab, and counted the numbers of E. tuca on each.  With a few exceptions (also in the genus Halimeda), E. tuca were only found on H. incrassata.  Further, when given the choice between many different algae species in the lab (Fig 1A, below), or three different species of Halimeda in the lab (Fig 1B), or in the field (Fig 1C) the slugs greatly preferred H. incrassata.  To test whether the slugs were following chemical cues, the experimenters soaked cotton balls in water that had held H. incrassata (Fig 1D), and found that E. tuca much preferred these to cotton balls soaked in plain seawater.  With regard to the questions posed by the paper, the results indicate that 1) E. tuca is a specialist, and 2) they find their host based on chemical cues.

Fig. 1. Elysia host preference. Number of trials in which an Elysia colonized one of 14 common seaweeds and seagrasses (n = 20) (A), three co-occurring seaweeds in the genus Halimeda (n = 20) (B and C), or a cotton ball laced with H. incrassata-conditioned seawater vs. seawater only (n = 40) (D), when offered in a still water arena (A, B, and D) or in the field (C). Choice was assessed after 2 h (A–C) or within a 5-min period (D). Results were analyzed by a Cochran’s Q (A–C) or Fisher’s exact (D) test. In A–C, different letters above bars indicate significant differences among seaweeds in terms of Elysia colonization frequency, as determined by Wilcoxon sign tests (corrected for multiple comparisons). AL, A. longicaulis; CC, Caulerpa cupressoides; CP, Caulerpa prolifera; CS, Caulerpa sertularioides; DC, Dictyosphaeria cavernosa; HI, H. incrassata; HM, H. monile; HO, H. opuntia; PC, Penicillus capitatus; PD, Penicillus dumetosus; RP, Rhipocephalus phoenix; SF, S. filiforme; TT, T. testudinum; US, Udotea sp

The next question regarded the identity of the chemical attractants from H. incrassata (which will be henceforth referred to as “Halimeda”).  Compounds were extracted from Halimeda with methanol, and the individual components of the extracts were separated as described in the supplementary methods.  Each fraction was tested for attractiveness to slugs using the cotton ball colonization assay described in Figure 1, above.  The first compound they described, 4-hydroxybenzoic acid (4-HBA; Figure 2A, left) is found in both “vegetative” Halimeda in the normal growing stage, and in “reproductive” Halimeda that are undergoing spawning events.   When 4-HBA was placed on cloth patches next to Halimeda, the plants were colonized by significantly more Elysia than to controls with cloth soaked in the solvent but no 4-HBA (Figure 2B, left panel).

The reproductive stage of Halimeda is significantly more attractive than the vegetative stage, in part because the reproductive cells (gametes) are a rich source of nutrients.  When patches soaked in extract from reproductive plants were placed next to Halimeda plants, they attracted more than twice as many slugs as those from vegetative plants (Figure 2B, right).  This led the authors to identify halimedatetraacetate (HTA; Figure 2A, right) a chemical compound enriched in reproductive Halimeda.  It was known that HTA deters feeding on Halimeda by other species, and that E. tuca sequesters HTA in its tissues.  The authors went on to show that an extract from E. tuca that contained HTA deterred feeding by predatory wrasse.

This brings us to question #4, what are the ecological consequences of E. tuca grazing on Halimeda?  Surprisingly, the effects of such tiny slugs are significant.  The fact that the slugs feed on reproductive structures (which have the highest HTA content) is expected to substantially reduce the plants’ fecundity.  Further, when the authors manipulated the numbers of Elysia on plants in the field, those with more slugs showed less growth (Figure 3A) and more branch loss (Figure 3B).  Placing E. tuca in enclosures on branches (Figure 3D) also caused more branch loss compared with enclosures with no slugs.  So E. tuca can cause significant damage to Halimeda.  Because H. incrassata aids the development of seagrass beds, and generates the majority of carbonate sediments (a.k.a., nice white sand) in those areas, the authors suggest that grazing by E. tuca can have ecosystem-wide consequences.

How can a small slug that sucks sap cause such dramatic loss of plant tissue?  One hypothesis is that the plant self-amputates segments that have been fed upon by Elysia.  The model Rasher et al. propose is that the plants are trying to avoid the introduction of pathogens by the slugs by sacrificing segments.  After culturing fungi from the slugs’ radullae, which they use to pierce the plants’ tissues, they tested one fungal species they referred to as Et-2.  Halimeda innoculated with the fungus dropped segments above the injection site (Figure 4A).  Injection of a fungus that is a pathogen of other species did not have the same effect (Figure 4B).  The data are therefore consistent with the hypothesis that loss of segments is a defensive strategy in response to feeding by E. tuca, suggesting that the answer to question #5 is also yes.

The authors conclude that the answer to all of the questions they posed is “yes,” and that marine plant herbivore interaction described above strongly resembles those in terrestrial ecosystems, despite more than 400 million years of separation between the participating species.

At some point, this paper got me thinking of a potential alternative function for kleptoplasty.  Shall we meander our way there?

By the end of last summer, I was finding most of the prevailing theories regarding kleptoplasty to be rather unsatisfying.  While not every aspect of biology must have a function, kleptoplasty has costs that must be offset.  It takes energy to segregate and store the chloroplasts, and they must be protected from the immune system.  Plus, the animals that are active in the sunlight are exposed to predation and damage from UV light.  Despite these costs, Elysia is a very successful genus, with species found worldwide in the shallows of tropical and temperate seas.  Therefore kelptoplasty must provide a significant benefit.

So, what good is kleptoplasty?  If you buy the arguments presented by deVries et al, photosynthesis by kleptoplasts do not supply a significant portion of the animals’ energy needs.  Is the energy produced by photosynthesis used to make starch or fat for use during lean times?  Maybe.  Could the kleptoplasts be a “living larder,” being digested when food is scarce?  The animals certainly become pale when they are starved, suggesting the kleptoplasts are being broken down, but why not just digest them at the time they are eaten and turn them into fat like the rest of us do?

One idea is that the kleptoplasts are merely used as camouflage.  In the case of E. diomedea, which spends a lot of its time hidden in its food plant, this seems sensible.  Not so much for E. crispata, which is easily visible against hard bottom reefs, which are generally not very green.  Further, it seems like there are other, less complicated ways of making or storing green pigment to match one’s surroundings.  However, let’s hold that thought for a minute.

Aside from making carbohydrate from sunlight and being green, chloroplasts produce important precursors for many biochemicals (e.g., Gould et al., 2008, Ann. Rev. Plant Biol. 59:491).  These could be used by the slugs for the synthesis of fats or essential aromatic amino acids for their own nutrition, or to be used for their prodigious production of eggs.  Given that there is absolutely no data regarding the role of photosynthesis in egg production by Elysia, this remains an attractive hypothesis.

However, an insight I thought was particularly brilliant was that chloroplasts synthesize isopentyl diphosphate (IPP), a precursor to a wide range of things, such as chlorophylls and terpenes. Some of these compounds are expected to be smelly, and, in principle, make the slugs smell like their food.  Some of the chemicals may also taste bad, rendering the soft, slow animals less palatable.

Predators of Elysia are expected to include nudibranchs, which are largely blind and find their food by smell, or fish, many of which find their prey by sight.  If the chloroplasts were pumping out chemicals that gave the slugs a smell of their food, it would make it much more difficult to find them by scent.  One bonus is that the green color of the kleptoplasts will also make it more difficult for visual predators, such as fish, to find the slugs.  On top of that, any noxious taste would protect the slugs from predators, regardless of their hunting methods.  Overall, this model seemed to have fewer caveats than any of the others.

I thought I had come up with this idea on my own.  Then I rediscovered the above paper by Rasher et al. while I was updating a saved search in the Scopus database.  PNAS is my Wednesday lunchtime reading, and I am sure that I was excited to come across a paper about Elysia, so I am certain that I read it when it came out.  I am saddened by the fact that I forgot that I had read the paper, and assume that the paper got me thinking about kleptoplasty and chemical camouflage.

Have you figured out the connection to George Harrison yet?  You have to be getting on in years or love music trivia to remember, but he produced a popular song “My Sweet Lord,” during his post-Beatles solo career.  He ran into some legal trouble when the Chiffons’ record label sued him for appropriating the melody from their highly popular “He’s So Fine.”  If you go online and listen to both of them, it won’t take you long to think “dang, he stole their melody.”  Harrison admitted that he was very familiar with the melody, and the judge ruled that he had committed “subconscious plagiarism.”  In the same way, I had no memory of even having read the Rasher paper when I was formulating ideas and researching the biosynthetic capabilities of chloroplasts.  I just thought I was being terribly clever.  Nonetheless, it is highly likely that the paper was somewhere in the recesses of my mind during the process.  Are there any truly new ideas?

But, more importantly, how does one test this?  The first step is to make some predictions, and here are a few possibilities:

  1. Comparison of compounds produced by food plants, normal Elysia, and Elysia in which photosynthesis is blocked will show compounds produced by plants and normal slugs that are absent or significantly less abundant when photosynthesis is blocked.  All we need to do is make some extracts and find a potential collaborator who is willing to perform some gas chromatography-mass spectroscopy on a small budget.
  2. Elysia in which photosynthesis has been blocked will be easier to detect or more attractive to predators.  Placing a predatory mollusc or fish in a Y-maze with a choice between an arm containing a control and one containing  a photosynthesis-blocked Elysia might do the trick.  There are plenty of hungry wrasses that we could use for the fish test, either in the lab or in Bahia.  As far as predatory slugs, Navanax or Roboastra are good candidate predators for E. diomedea in Baja California.

There are likely to be more, better experiments, but the above provide a start.