Photos and descriptions of sea slugs in their native habitats. May include some nearby residents.
Just back from a week diving in Bonaire. The focus was more on recreation than slugs, but we came across a few Elysia crispata along the way. As usual, we found the highest numbers in the more degraded sites to the north, but the slugs were always on long-dead coral regardless of where we found them.
For this post, I wanted to highlight the intense blue color of the slugs we found this year at Karpata, a site near the north end of the island. I have previously posted photos of blue slugs that have turned up at nearby sites, and a quick Google search will provide many more examples.
This seemed to be a common color pattern. Each photo in this post shows a different individual, but their colors are remarkably similar.
Even though the colors look downright artificial, I did little more than adjust the contrast of the photos. These slugs really look like this. I emphasize this point, because the range of colors of most species of Elysia is limited to shades of green.
Despite the striking appearance, the color pattern may provide camouflage. When the slugs were curled up and their rhinophores hidden, they did passable impressions of sponges. E. crispata are almost invariably scrunched up when we come across them on the reef, so this may be a successful way of hiding in plain sight.
The color of many Elysia species is derived from pigments taken from their food plants. For example, my E. crispata and E. clarki hatchlings have little color until they start feeding, and then take on the green color of the chloroplasts they sequester in their digestive diverticula. Costa et al, (2012), provide a nice example of this effect. They showed that E. timida could be either green or brown, depending on whether they were fed Acetabularia algae that had taken on different colors based on having been acclimated to low light (green) or high light (brown).
So what could be making these slugs so blue?
One possibility is that they are feeding on Dictyota, a brown alga that grows abundantly on the dead coral in this area. Although the Dictyota in the photo at the top of this post is not impressively blue, the alga is certainly capable of producing intensely blue color. Most species of Elysia feed on green, rather than brown algae, but it is not unheard of for them to branch out (e.g., Trowbridge et al., 2010). It is also possible that the slugs are consuming one of their more usual food plants that happen to be producing high levels of blue pigments. It would be interesting to take a small tissue sample and find out what the slugs have been eating.
It also brings up an interesting question regarding the coloration of Elysia in general. In addition to their green background, many species have distinctive patterns, such as the colorful markings on the parapodia and rhinophores of E. diomedea, below, or the colorful edges of the parapodia in E. clarki.
At a higher magnification, one can see concentrations of pigment spots, such as those shown below in the parapodium of E. clarki. Are these spots of concentrated pigment derived from their food plant, or are they synthesized by the slugs themselves?
As far as I can tell, there is no answer in the literature, but who knows what will turn up next.
I have added a rudimentary map of the locations at which E. diomedea has been found during our fieldwork during the past few summers. At the moment, it provides a framework to which we can add more sightings as we turn up more slugs.
The short summary is that suitable habitat, consisting of rocky bottom with growths of Codium macroalgae, is distributed throughout the bay, and that the ability to find Elysia can vary wildly at the same site from one day to the next. With enough time and effort, I expect the little slugs would be found anywhere there is something to eat.
One of the goals of the Bahia field season was to look a little deeper at the interaction between kleptoplasty and chemical defense. The general idea went something like this:
Based on this line of thinking, we hypothesized that slugs deprived of light should be less distasteful than those kept in bright light.
To put the hypothesis to the test, we set up two tanks that were nearly identical except for lighting. They were plumbed into the same sump and chiller, so their temperatures and chemistry were essentially identical, but one was illuminated by a high-intensity LED fixture to support photosynthesis (PAR ≥100 mol m−2s−1), while the other was shaded to reduce the light by 100-fold (PAR < 1 mol m−2).
Once we obtained the permit and were allowed to collect, we randomly separated slugs into two tanks that contained roughly equal amounts of Codium on which they could feed. They were allowed to feed at will, because the goal was to test the role of photosynthesis in generating defensive chemicals, not the effect of starvation.
The original plan was to use mucus from experimental (unlit) and control (lit) slugs to make food cubes, then test which ones were eaten by fishes in the bay. However, based on experiments at USG, and the fact that we would not be able to extract enough mucus from our few dozen slugs, we decided to test the effects of tissue extracts from whole slugs. Surprisingly, the students were not as sad as we might have expected that they had to purée their pets.
For the description below, whenever I write “we,” I really mean Ric and the students, because he was very much in charge of this project. Most of the below photos are his.
The process was as quirky as any of the other experiments we have done in Bahia. To make the gel base of the food cubes, we needed to dissolve carrageenan in water, which required heating the water to boiling. The easiest method is to microwave the water, but the only available microwave oven had a single working button (“popcorn”), which turned the microwave on (after a disconcerting delay) for 3.5 minutes. It took some practice and finesse to turn the machine off at the right time, but they managed.
Fish food pellets were ground and added to the carrageenan solution to make the base food cubes. Mucus or tissue were then added to a portion of the food, depending on the experiment.
Once heated and thoroughly mixed, food was poured into silicone ice cube trays to make 1 cm cubes. Silicone O-rings, used to secure the food cubes to clips during experiments, were placed into the molds and held steady with acrylic rods.
Once the O-rings were in place, food was poured into the molds and evened out using a knife. After cooling for a bit, the whole assembly was stuffed into a ziploc bag and refrigerated until needed.
At experiment time, food cubes were attached to monofilament lines to be anchored in the bay. The bottom end of the line was tied to small lead weights, while the top was held afloat by a 16-20 oz soda bottle. Food cubes were secured by O-rings to plastic safety pins tied to the line at regular intervals. Several bugs needed to be worked out. For example, we started with lightweight fishing line, but it had a tendency to break in the surge of the intertidal zone, causing loss of the whole bait line. We shifted to a much heavier gauge, which apparently scared the fish away, because all food cubes were present after 24 hours. Ultimately, we settled on something in between, and could start gathering real data.
Once food cubes were secure, the lines were placed deep enough to keep them afloat at low tide.
Lines were collected after a predetermined time (see below).
For the first round of experiments, cubes were left in the bay for 24 hours. During this time, essentially all of the control cubes (i.e., without tissue or mucus) were eaten, as were most of the experimental cubes. Nonetheless, fish appeared to eat fewer cubes containing tissue from slugs kept in the light, compared to those kept in the dark.
Because there was concern that missing cubes may have fallen off rather than being eaten, they also tried leaving the cubes in the bay for only one hour. There was a slight decrease in consumption, but the overall result was the same: tissue from dimly-lit slugs was eaten more often than that from well-lit slugs. The students were running out of time for these experiments, so the results are preliminary and nothing is statistically significant.
To summarize several weeks of hard work, it looks like there is something to pursue. The sample sizes are small, but there is a consistent effect of slug tissue in reducing consumption by fishes, and this effect is reduced or eliminated by keeping slugs in the dark. Now that the bugs have been worked out, it will certainly be worth trying the experiment again if there is an opportunity to spend a few weeks making food and setting lines.
Thanks again to the USG Slug Club, who pioneered the foodmaking methods, and took care of the slug system while I was away.
The only thing better than a picture of a slug is a photo of a slug projected on a giant screen and admired by a bunch of people.
A few weeks ago, the Photobiology students presented at the Ocean Discovery Institute Report to the Community, held at the San Diego Museum of Natural History. This is the opportunity for the students to tell their parents, the community, supporters, and everyone associated with Ocean Discovery, about what they did this year in Bahia.
They started with the background and general questions that drove the research: what is kleptoplasty, and how can we learn more about the mechanisms and evolutionary benefits of the phenomenon? Because they had limited time on stage, they focused on the work we did collecting samples of slugs and algae for identifying food plants.
The punch line was satisfying. We found that the DNA sequence of the Codium algae collected in front of the station and that from the slugs’ tissue samples came from Codium decorticatum. I managed to get about 400 bases of sequence from the slugs and the algae, and it was about a 99% match to C. decorticatum.
The figure above shows a short, representative stretch of DNA from the rbcL gene, from Elysia diomedea and the Codium species we extracted in Bahia (top two rows), along with sequences from two species of Codium from the National Center for Biotechnology Information (NCBI; bottom two rows). The Codium from Bahia matched C. decorticatum almost 100%, whereas the match was only about 91% for the very similar C. fragile.
There were, however, a few differences between the kleptoplast DNA from E. diomedea and C. decorticatum (e.g., asterisks above E. diomedea sequence). At this point, we don’t know whether this is due to variation within the species that the slugs are consuming, or the slugs are eating more than one species. We are still waiting for results from NextGen sequencing, which should be more informative and may clear up this puzzle.
A couple of notes about the algae. First, the Codium and slug sequences are identical to those we obtained in 2016. At the time, however, C. decorticatum had not been entered into the database, so all we knew was that we had a new species. The other observation is that the species of Codium we sampled (see photo of tissue here) is relatively low-growing and branchy, unlike the usual tall, sparsely branched appearance of C. decorticatum. There were specimens that looked like that (e.g., here), and it is likely that the appearance of the alga depends on the local conditions.
Most importantly, the students did a fantastic job of presenting. I was away visiting family, but I got multiple independent reports from people in the audience who talked about how strong and clear the students’ presentations were.
I am very pleased and proud to have played a part in the students’ project this summer. Despite all of the up and downs and improvisation, it was a deeply satisfying experience. I am always amazed how a misanthrope such as myself can have such a great time packed like a sardine into the field station with so many people. Thanks to the students, the Ocean Discovery staff, Ric, Thiago, the visiting scientists, and everyone. Especially, thanks again to Dr Drew Talley, mi gemelo de otra madre, for making it possible.
I still have a few posts left regarding the results of the feeding assays and slug surveys, and hope to have them ready in a few weeks’ time.
It was nerve-wracking not to have the permit. Without permission, we could gather neither algae nor slugs, so experiments were at a standstill. Fortunately, we could get started on a few things, like surveying different sites for slugs, and optimizing conditions for the DNA experiments.
Snorkeling in front of the station allowed us an extended period to observe the animals and their habitat. It was also fun, and gave the students lots of practice at slug hunting. It was a good year for Elysia at the station, and we saw plenty . All the more frustrating that we could not collect them.
Finding them is never trivial, however. Below is a medium-small slug with a finger for scale. It would be a lot easier if they got as big as E. clarki, but I have never seen them larger than 5 cm or so.
There was a lot of Codium, and multiple other species of green algae that could potentially interest the slugs. I even found a small patch of Bryopsis, which is reputed to be a favorite of captive E. diomedea.
We also had the chance to play with the new PCR primers I designed for use in NextGen sequencing. I had not had much of a chance to test them before I left Maryland, so we took some time in the morning to set up a reaction using DNA I had brought for use as a positive control.
The results were encouraging, but imperfect. The PCR products had two bands, instead of one, suggesting the conditions needed to be modified to amplify only the rbcL gene.
The following day, we set off for a slug survey at Playa La Gringa, a beach area near the north end of the bay. It is a beautiful spot for a snorkel, and has a lot of potential for slug hunting. Once there, we suited up and got ready.
They were soon in the water, exploring and enjoying. This part of the beach receives cold water directly from the Gulf, so the students were happy to have their wetsuits.
The rocky bottom looked promising, with a wide range of mixed algae species.
There were plenty of small treasures, such as urchins, sponges, and hydroids, along with a profusion of fishes.
Codium was also plentiful, in at least two growth forms (or species, not sure), which might indicate the presence of Elysia.
Toward the south end of the beach, the rocks were relatively bare, with very little green algae visible. The brown alga, Padina, was still abundant.
Despite intensive searching by many eyes, we did not find any Elysia anywhere along the beach. This does not mean that the slugs are not there, but they certainly did not make their presence known.
At this point, Ric and I had been improvising, or exercising “adaptive management,” for many days, and were running out of tricks. I had requested that the crew in Maryland send some more DNA samples, but they would not arrive until the following week. Although despair is way too strong a word, there was a profound sense of “now what?”
It was at that point that the intensive lobbying by Ocean Discovery paid off, and the permit finally came through. The group could collect slugs and algae, and start work on the planned experiments.
Another installment of adventure in science at Bahia de los Angeles on the Gulf of California. This year, I am once again working with students from Ocean Discovery Institute to exapand on what we have learned about feeding assays and DNA sequencing to develop more insight into the diet of Elysia diomedea, and how kleptoplast photosynthesis makes them taste bad.
Drew and I left San Diego with a truck full of stuff, including the tanks, sump and chiller for a slug setup, as well as a perfectly functioning PCR thermocycler from NIH surplus. Drove through Calexico to Mexicali, caught route 5 south through San Felipe. The road was largely good, but the unpaved section in the middle still not finished.
We arrived in Bahia about 5:30, and stopped for the usual photo from the hills leading into town. When we arrived at the station, the setup crew was working away, although the station was mostly ready for business.
We spent the rest of the evening starting to get organized. I also found out that we currently do not have a permit for collecting the slugs (all other activities have been approved), so it is not 100% clear that we will be able to do any work.
The next morning, I was eager to get started on assembling the lab space. It looked as though everything survived shipping from Maryland to San Diego and the drive to Bahia. We got some new tables, and strung some cords to safely manage electricity flow to the tanks, chiller, freezer, and other lab equipment.
Then we got to set up the tanks. All the plumbing I had assembled in Maryland connected nicely, but the connectors for the chiller were too small for the ¾” tubing used for the rest of the system. I could swear we double checked that. That meant we needed to head to “Home Depot,” the local construction supply lot. Fortunately, Ric, the Ocean Discovery fellow who is assisting me this summer speaks excellent Spanish, so he could easily explain what we were trying to do.
The very nice owner was pessimistic that he would have the right adaptor, but he found one. Then Ric told him we needed two, and his pessimism increased. He miraculously managed to produce another. However, he had no hose for the connection, so he gave us directions to the other, larger supply lot that we had never heard of. It was indeed larger, and they had lots of hose. We bought 3 meters, to have some extra for a siphon hose, and were on our way.
The tank was quickly assembled, and we hauled buckets of sea water to fill up the two tanks and sump. The water was brown from a dinoflagellate bloom, and looked kind of yucky. We hoped the skimmer woul clear it out. Once the tanks were full, we turned the main pump on for a test and cleaned up the splashed water so that we could look for leaks. It all seemed just right, but water kept on pooling on the floor. Sure enough, there was a crack on the bottom of the sump, presumably from shipping. After some discussion and back and forth, Ric and I drove back to Home Depot to get adhesives for a possible fix. The guy was amused that we were back, and showed us what he had. We bought the multi-purpose adhesive and some silicone sealer just to be sure.
It was time for the first snorkel. I put on my wetsuit and headed into the water in front of the staff house. The door for charging the camera opened promptly after I entered the water and that was the end of that. Oy. Nonetheless I looked at a lot of Codium, and was grunted at and bitten by many very aggressive damsels. Most importantly, I found about 4 small slugs up in front of the house, and called it a day.
I borrowed Drew’s nearly identical camera for a snorkel on the next day, and went out with Ric in the same area. The visibility was still crummy, but I managed to find a small slug on Codium after a bit, and brought both up to show Ric. Soon enough, he was finding his own, and we found quite a few, taking some good photos. Without a permit, however, the slugs will stay in the bay for now.
The lab was set up, there were plentiful slugs in front of the station, and the students would be arriving that night, so it looks pretty good for a successful time in the field.
In Bahia, there is always some drama. In this case, it appeared in the form of permits. It turned out that all of the group’s collecting activities, which included an enormous range of fishes, crustaceans, anemones, and molluscs, were approved, with the exception of my project. For some reason, maybe something I wrote set off alarm bells, the collection of Elysia diomedea and marine algae required approval from a different agency.
Stay tuned…
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?)
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.
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.
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.
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.
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.
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.
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.
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?
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