Posts in Category: Slug Science
First 100% Maryland Eggs
We have now come full cycle. Eggs from slugs collected in the Keys have hatched, and the little ones have settled, grown to adulthood and have now produced their own eggs. The slugs in Box of Slugs 2.0 have been producing eggs, but, because one wild-collected slug remains, I can’t be 100% sure who laid them.
This egg mass was laid in the 10-gallon growout tank at USG. All of the potential parents came from egg masses collected between 1/19/18 and 1/24/18, and started hatching between 1/31/18 and 2/6/18. So the maximum possible age of the parents is about 4 months, but they could have been a few weeks younger.
Egg mass in 10-gallon growout tank at USG. Found 5/27/18, but could have been deposited 5/26 or 5/27. Photograph 5/27/18.
Importantly, the eggs are almost 100% fertile. The embryos could be seen developing within a few days, and started to look like veligers in less than a week.
A small section of a developing egg mass from 5/27/18, approximately 8 days post deposition. Depending on the viewing angle, it is possible to see a faint velum extending from each side of the head (embryo viewed face-on), or the shell and head structures (side view). Rare unfertilized embryo marked with an asterisk. 6/4/18.
Because I will be away for much of the summer, I will not be able to rear these embryos. I am hoping for many more broods in the future.
Slugs Taste Bad II: Mucus vs Tissue
In the continuing quest to understand the benefits of kleptoplasty, the Slug Club students and I set up some experiments to determine whether Elysia mucus caused predators to avoid them, and, if so, which components. The logic behind the experiment was that 1) both the literature and my own observations indicated that slugs taste bad to predators; and 2) kleptoplasts produce organic compounds that appear in the mucus (Trench et al., 1972, Biol. Bull. 142:335); so maybe kleptoplasty plays a role in making defensive chemicals that keep fish and predators from eating them.
For these experiments, Zvi Kellman at IBBR was kind enough to provide some space for a predator setup. We got to set the system up, and not have to worry about vacating the space for the entire semester.
The first small hurdle was to find some predators. Because my preliminary experiments were done with fish, I had originally planned on testing fish from the same range as the E. clarki, maybe bluehead wrasse or small puffers. Then I was reminded of the paperwork for doing experiments with vertebrates, which is required even if we just watch what they eat and don’t do anything mean to them. Dreading the possibility that time would evaporate while I assembled forms and waited for replies, I became much more enthusiastic about invertebrate predators.
But which invertebrate predators? The primary requirements were that they were plausible natural predators, from the same geographic locations (i.e., sympatric), that we could obtain multiple individuals of the same species, and that they would not be too expensive or hard to keep. I was hoping to find some small, nasty crabs that local aquarists or shops had extracted from their tanks, but nobody seemed to have any available. Mike Middlebrooks had tried blue crabs, but they seemed rather large for our facilities. I finally settled on peppermint shrimp (Lysmata wurdemanni). They are sympatric, and are certainly willing to tear apart and eat other small, squishy invertebrates. KP Aquatics sells them in lots of 5, so I could set up multiple groups of them without going broke. Even if they don’t naturally prey on Elysia (nobody knows), they require no encouragement to eat, so we could at least test whether they are discouraged by slug slime.
We obtained a used 40 gallon breeder tank from a local aquarist (Thanks Rik), and moved in. We had a sink and a plentiful supply of purified water located a few feet away, so it was pretty easy to get set up.
Bench space at IBBR, with 40 breeder tank. 2/2/18
I siliconed in some acrylic dividers to have five independent sections. Each section had a drain leading to the sump, which had a skimmer and a small filter. The output of the return pump fed each section independently, and flow to each was adjusted with a ball valve. Each section also had a plastic plant and cave assembled from 1.5″ PVC fittings. At the beginning, each section had 4-5 shrimp.
Tank with dividers in place, just after adding water and salt. Each section drains to the 10 gallon sump on the right, with a protein skimmer and filter. Water is pumped back to shrimp enclosures, with flow controlled by ball valves that can be seen at the top of each section. 2/17/18
We messed around for a few weeks, then settled on an approach inspired by Becerro et al. (2001; J. Chem. Ecol. 27:2287). They made cubes based on powdered fish food and carrageenan, added potentially aversive compounds, and then placed them in the ocean to examine the effects on feeding by fish.
Slug Club Spring 2018, making food cubes. From left: Kathleen, Claudia and Elyson. 4/18/18.
The students took the lead in working out how best to make carrageenan based food. They figured out how to get the carrageenan dissolved, when to add the powdered food (Zoplan freeze dried zooplankton by Two Little Fishies), and the best way to add mucus or slug tissue to experimental cubes. The hot liquid food was poured into silicone ice cube trays to make as many 1 cm cubes as needed. Silicone O-rings, used later to secure the food cubes in the shrimp tanks, were held in place with plastic rods during pouring.
Ready to pour food. Silicone O-rings, threaded onto plastic rods, are placed into compartments of ice cube tray.
The food cubes were then placed in the shrimp enclosures. Each was secured by its O-ring to a plastic paperclip tied to a monofilament line suspended vertically in water column. Each line had multiple possible attachment sites. The shrimp were not shy, and rapidly moved in to explore the potential food source.
Testing food assay system. Food cubes are secured by O-rings to plastic paper clips that were tied to monofilament line. Line is weighted by PVC elbow at bottom, hooked to input hose above. Shrimp rapidly approach and sample the cubes. 3/28/18.
Experiment 1 tested to effect of mucus on feeding. Two cubes, one control and one experimental, were placed in each section. The control cubes were made with powdered food, carrageenan and artificial seawater (ASW); the ASW was replaced by mucus in the experimental cubes.
Mucus dripping from annoyed slugs in dip net. 4/9/18.
Collecting the mucus was not trivial. Even though the slugs readily secrete mucus when annoyed (such as when crowded into a dip net), the sticky mucus is not easy to separate from the slugs. Ultimately, we just let it drip into a beaker for 30 minutes or so, then returned the irritated by unharmed slugs to their tank. The mucus was then mixed into the food, and the unused portion frozen for later use.
Detail of food cube, showing embedded red silicone O-ring connected to plastic paper clip (yellow). 3/28/18.
To control for effects of position in the tank, food cubes from both groups were placed at different levels. We recorded the weight before adding to the tanks, and the position in the tank, then left the cubes with the shrimp for 24 hours. They were collected, and each was weighed again. The amount consumed was then calculated.
The results from the first round of experiments were underwhelming, but informative. The shrimp were unequivocally indifferent to the presence of mucus in the food. The graph below shows that shrimp consumed about 0.5 grams per day of food, regardless of whether it contained mucus.
Experiment 1: Effect of mucus on feeding by shrimp (means and standard errors). Control food cubes contained 10 ml ASW, mucus cubes had 10 ml of mucus from Elysia clarki. High food content (5g/90 ml). Sample sizes are 25 cubes for each group.
So, shrimp do not seem to mind mucus in their food.
The next step was to determine whether Elysia even taste bad to shrimp. Peppermint shrimp do, in fact, eat a number of nasty things, including the anemone Aiptasia. We had predicated our experiments on the idea that Elysia contain compounds the shrimp would find distasteful, but we never actually tested this idea. It was time for a “Friday Afternoon Experiment,” in which one tries a quick and dirty test to see what happens. In this case, we threw some baby Elysia to the shrimp.
Shrimp devouring baby slug during first trial with live Elysia. 3/26/18
The results were horrifying and illuminating. Shrimp rapidly grabbed, dismembered and ate the baby slugs. However, they left fragments of uneaten slugs, which gave a glimmer of hope that they did not find them to be the most palatable meals. We repeated the experiment twice more, with a week in between, and the shrimp consumed less at each session, as if they had learned not to eat them, but consistently attacked and tore at the slugs.
Although brutal, there are some lessons here.
First, shrimp should definitely be included in the list of possible predators. Naive shrimp had no qualms about eating Elysia tissue, and they would be well placed to hunt down small mollusks during their nightly forays to rummage the reef.
Second, the damaged slugs often recovered. The usual examples of toxic or unpalatable species (think monarch butterflies) are often killed when they are sampled by predators. Kin might benefit from future behavior of the predator, but the sampled individual is out of luck. In this case, the slug may be able to limp away after having been tasted and rejected, and benefit directly from containing aversive compounds. This may also explain why Elysia lack the bright warning coloration often associated with unpalatable prey. Maybe the first line of defense is to be inconspicuous, and, if that fails, expect to be spat out after being tasted.
Experiment 2 was based on a suggestion by one of the students: if shrimp don’t mind mucus, what about the slugs’ tissues?
We anesthetized slugs, and worked out how to produce tissue puree. We quickly learned that a standard mortar and pestle has little effect on tissue is both slimy and rubbery. Chopping with a razor blade was better, and produced adequate tissue for the first round of experiments. Later, I had to cull a large number, and used a food processor to make a tissue homogenate.
Experiment 2: Effect of mucus and slug puree on feeding. Medium food content (2.5 g/90 ml). 20 cubes per group.
The results were consistent with our expectations. Mucus alone had little or no effect on feeding. Slug tissue appeared to reduce feeding by more than 35%. However, the results were not statistically significant, possibly due to variability between groups of shrimp.
We accidentally performed an additional experiment on the effect of food concentration on the rate of feeding and the aversive effect of slug tissue. For one batch of food, we ran out of powdered food, and used less than half the normal amount.
Experiment 3: Effect of mucus and slug puree on feeding. Low food concentration (1.16 g/90 ml). 12 cubes per data point.
The shrimp ate less overall, and the effect of including slug tissue appeared to be stronger, reducing feeding by more than half. As above, the results were not statistically significant, presumably due to the small sample and variability between sections. Nonetheless, balancing the attractiveness of food versus the distastefulness of slug tissue needs to be considered for future experiments.
Relationship between amount of food cubes eaten (means and standard errors) and concentration of food powder. All groups show a modest increase in consumption when cubes have more food content. Cubes containing slug tissue (green line) appear to be less palatable at both food concentrations tested.
For the moment, we can tentatively conclude that mucus from Elysia has no impact on feeding by peppermint shrimp, but that slug tissue inhibits feeding.
Because Mike Middlebrooks tried similar experiments on blue crabs with adult slugs, the last experiment of the semester was to put full grown Elysia into the shrimp enclosures. Normally approach food rapidly, but were very cautious. When slugs dropped right in front, picked with pincers, but did not tear at the slugs.
In the future, there are several improvements that could help to sharpen the results.
One issue that arose was the change in shrimp populations in the sections over time. A few shrimp were lost (the absence of carcasses suggests cannibalism) over the three months of the experiment, yielding a final range number of 2-5 shrimp per section, which resulted in considerable inter-section variation in food consumed in a 24 hour period. Doing a periodic census and adjusting populations will presumably reduce variability.
Another possible confounding factor as that some cubes appeared to be torn apart and left on the bottom, and were therefore scored as eaten when they may have only been consumed partially. Using video analysis of time spent feeding on each type of cube would help distinguish between destruction and consumption of food.
One other issue was the growth of algae during the semester. Despite relatively low light in the lab, protein skimming and periodic water changes, the addition of food supported algae growth. Given the omnivorous nature of the shrimp, the algae may have served as an alternative food source. Giving the enclosures a quick scrub during weekly maintenance would force the shrimp to focus on the food cubes.
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.
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:
- 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.
- There will be no difference between low- and high-light conditions in slugs feeding on Cladophora.
- 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.
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?
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:
- 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.
- 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.
What does Elysia crispata do on the reef?
Back from Bonaire, with a fresh puzzle.
Elysia crispata at The Cliff in Bonaire. January 11, 2017.
In research, as in life, there are things that don’t make sense. Often these things make enough sense that you ignore them, choosing to focus on other mysteries. One such little small, nagging issue is the question of what draws Elysia crispata to hard-bottom coral reefs, which lack obvious growth of green algae known to be their food. Based on observations of many years, the slugs are not in transit, most are just sitting there.
Lettuce slug, with some Dictyota and red turfy algae, but not much in the way of greens. The Cliff, 1/11/17
My knowledge of the habits of Elysia in the wild is far from encyclopedic, but the species I know best have hearty appetites and stay close to their food. E. diomedea are found on or near Codium in Bahia de los Angeles, and E. clarki spend most of their time face down in their food in aquaria. This tends to hold true in the literature as well. For example, E. tuca is generally found on its favorite food, Halimeda incrassata (Rasher et al., 2015, PNAS 112: 12110). As a counter example, Middlebrooks et al. (2014) found that E. clarki were often found at sites that contained few or no specimens of their food plants (Penicillus, Halimeda, Bryopsis) determined via DNA barcoding.
Two slugs, in typical habitat. The Cliff, January 11, 2017.
In any case, I think I am justified in being puzzled by the lack of an obvious food source on the reef. The photos in this post are all from a single dive at The Cliff, a site in the north-ish part of Bonaire. We found maybe a dozen slugs, most in the face-down posture, which makes them look like large blobs of colorful frosting on the rocks. The area had a lot of dead coral, which possibly serves as a substrate for the growth of food algae. However, there were no obvious growths of green algae anywhere nearby, although algae such as Halimeda and Caulerpa are plentiful in mangroves on the island.
E. crispata, with a mix of turfy algae, including possible green filamentous species. The Cliff, January 11, 2017
Rather than snap a few photos of the more photogenic slugs, I thought it might be useful to document as many of the slugs as I could, with emphasis on the substrate. Honestly, what you see is what you get; there are no large clumps of Bryopsis or Halimeda hiding around the corner.
Two E. crispata, surrounded by short, sparse turf. The Cliff, 1/11/17
What are these gals eating? The most prominent alga is Dictyota, a brown alga which, based on known feeding habits, is an unlikely food.
E. clarki, surrounded by the flat leaves of Dictyota. No green algae in sight. The Cliff, January 11, 2017.
Are they grazing on the little strands of green algae that can be seen if one expands the photos and looks really hard? Is this a late life stage that does not feed as much? Are E. crispata truly crawling leaves, getting their energy from photosynthesis? Is the much lighter color of E. crispata, compared to related species, like E. clarki and E. diomedea, a clue?
Slug from the beginning of the post, with wider field.
Mystery Alga
As I mentioned in the previous post, sometimes it is not so easy to identify an alga. In this case, it is a species that bloomed spectacularly when a local reefkeeper set up a new tank. The rock had been thoroughly cleaned and bleached, and no corals or fish had been added, so Alan did not expect the growth of nuisance algae. He was rather surprised to see a rapid, spectacular bloom of long, furry green algae.
At first we thought is might be Bryopsis (yay!), so it seemed worth trying to feed to the slugs. Once I saw and felt it, it was clearly something else. It was soft, like Derbesia, but longer and had branches that extended radially (like a bottle brush) from the main stem. Bryopsis feels coarser, and the branches extend in a single plane (like a fan). So, it was not one of the usual suspects. Nonetheless, it was worth throwing some into a tank to test whether the gals would eat it. They did not immediately plunge into it, as they would have for Bryopsis, but they seemed to find it palatable enough. Note the fine structure of the branches in the photos below.
Elysia clarki grazing on unknown species of alga. 11/20/16
Alan’s algae, view of whole plant. 12/27/16.
The plant has some characteristics of the order Bryopsidales, such as the lack of clear cellularization. It looks like the plant is made up of a continuous, single cell.
Tip of branch. Scale bar = 2 mm. 12/27/16.
Stalk, showing absence of cellular divisions. The little round bumps on the branches are reproductive structures. Scale bar = 1 mm. 12/27/16
Acrosiphonia spinescens from Algaebase, showing cellular divisions and hook-like branches. © Ignacio Bárbara
Branch tip of Alan’s alga. Scale = 1 mm. 12/27/16.
I thought a quick look at the DNA sequence would clear things up, but that was not the case. The closest match, Acrosiphonia, with 88% sequence identity. That’s not a very good match, and even though it looks somewhat like Acrosiphonia, the unidentified alga lacks several key features, such as the hooks on the branches (which cause mature plants to develop a dreadlocked appearance) and clear cellularization of Acrosiphonia. Plus, Acrosiphonia is a cold water species, unlikely to thrive in a warm reef aquarium.
The closest visual match so far is Trichosolen, which does have warm water species. The only species with rbcL sequence in the database (T. myura) is only an 86% match for DNA, so it’s probably not the one either.
By way of comparison, the usual pest algae (various species of Bryopsis and Derbesia) were only 82% – 83% identical, so we can at least rule out the possibility that it is an oddball species of one of those.
The hunt continues for a match. Not very satisfying, but some days are like that.
Recent Comments