Photos and descriptions of sea slugs in their native habitats. May include some nearby residents.
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?
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.
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.
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.
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.
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.
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.
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:
There are likely to be more, better experiments, but the above provide a start.
Back from Bonaire, with a fresh puzzle.
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.
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.
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.
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.
What are these gals eating? The most prominent alga is Dictyota, a brown alga which, based on known feeding habits, is an unlikely food.
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?
As the summer winds down, it looks as though the project worked better than I had hoped. There is a lot left to do, so this is far from the end, but what a great beginning!
To remind you of the the primary goal of the summer’s project, we wanted to use the DNA contained in the slugs’ kleptoplasts to identify their primary food plant(s). The previous posts described how we worked out methods, collected slugs and candidate food algae, extracted the DNA, amplified the rbcL gene from the chloroplasts, and sent it off for sequencing.
The first sequence that came back from Macrogen did not look very good, which was disheartening. The chromatograms looked awful, and the sequence was gibberish, causing concern that our extractions or PCR reactions were contaminated.
Nonetheless, Paul Kim at Macrogen promised to optimize the reaction and sequencing conditions, and worked hard to provide interpretable data. Patience and persistence have finally paid off, and we can make some simple, declarative statements about the slugs and their food plants.
Statement 1: We obtained usable rbcL DNA sequence from Codium, Ulva and Elysia.
Statement 2: Elysia diomedea steals most, if not all of its kleptoplasts from Codium.
To flesh out these statements a bit:
From Bahia, we now have DNA sequence for Codium simulans and for Ulva. The Codium data is the first for the species. Although rbcL sequence for related species (such as C. isabelae) can be found in the NCBI database, there is currently nothing for C. simulans. We’re not sure which species of Ulva we used, although it is likely to be Ulva californica. In theory the DNA sequence could have told us which species it was, but the region of the rbcL gene that we amplified and sequenced is identical to that in many of the species in the database, so we would need to try another gene, or a different region of rbcL. An important lesson from this year’s work was that we need to preserve samples of the algae we sequenced.
The most exciting result was that we got sequence from E. diomedea kleptoplasts! Overall, we extracted DNA from two individual slugs at different times, and performed at least three separate PCR amplifications (both in BLA and at USG when I got back), and they all came back matching Codium! In retrospect, it is not a shock that slugs that we found in close association with Codium, and which spend a lot of their free time on Codium, actually eat Codium.
The figure above shows a small portion of the sequence, highlighting a few of the sites at which Elysia and Codium differ from Ulva. Overall, the DNA sequence from Elysia was 99% identical with that of Codium, and those few sites that differed appeared to be locations at which there was variation between individuals. Ulva showed about 81% identity to Codium and to kleptoplasts from Elysia.
Despite how it sounds, this is not a trivial result.
First off, Codium has been suspected, but never confirmed as a the food plant. Back in 1969, Trench and colleagues said that E. diomedea fed on green algae, possibly C. simulans, based on the chlorophylls found in the slugs and the morphology of the kleptoplasts, but their methods could not reliably distinguish between green algae species.
As a corollary, there is no evidence that they eat Ulva or Padina, despite being surrounded by them. We did not get rbcL sequence from Padina this year, but it is not closely related to Codium, and the sequence in the database for P. durvillei (the most common species in our study area) shows roughly 70% identity to that from Codium and E. diomedea. Had there been significant Padina or Ulva DNA in the slug sample, the presence of multiple divergent sequences are likely to have made interpreting the results impossible. In other words, we got lucky that there was one dominant species of kleptoplasts. Having sampled only two slugs, we can’t rule out other food plants. Another caveat is that the result shows that chloroplasts from Codium persist in the slugs’ tissues, but the slugs could be eating other species for which the chloroplasts do not last as long inside the slugs.
Another important conclusion is that our methods actually worked. As a neurophysiologist setting up a molecular lab in a dusty, hot garage in an isolated location, there were no guarantees that we would get any usable data. In addition, we used degenerate primers for PCR, to amplify rbcL sequences from all potential algae species, counting on DNA sequencing to tell us which species were present. Our choice of Sanger sequencing, which is much less expensive but prone to problems if the amplified DNA comes from more than one species, could have also caused complications. Planning, persistence, and some luck all worked in our favor.
With these data in hand, there is lots more to do. To fill in some of the gaps discussed above, we need to sample from more slugs in more locations. At the same time, we need to more systematically collect specimens and DNA from algae at different sites around the bay, especially C. simulans. If we are going to generate DNA sequences, we may as well do it in such a way that we can add them to the database.
There is also a lot to be done to understand the big picture of kleptoplasty and how E. diomedea fits into the ecology of the bay. Because of delays in receiving equipment, we had very little time to prepare the behavioral experiments before we left Maryland. On top of that, the losses and stress caused to the slugs by the extreme heat this year, resulted in essentially no data regarding the slugs’ preferences for light. The I-mazes are build and ready, and we plan to add a chiller to the holding system, so procedures should be perfected before the next field season. We also still don’t know much about their environmental requirements. They eat Codium, and live on Codium, but do they have other requirements in terms of water movement, temperature, nutrients, or turbidity?
That the project worked can be chalked up to a lot of planning, hard work, and generosity on the part of a great group of people. At the risk of sounding like an Academy Award acceptance speech…
There would have been no Photobiology group without the “Angels,” Cristal, Rosalia, Nancy, Allison, and Susan. It was so much fun to watch them work and learn. They will be giving their presentation during the Report to the Community for Ocean Discovery this week, and it will be great.
Richy Alvarez, the intelligent and talented Directed Research Fellow, was another reason this project came together. There are so many big and small things that he did to make sure equipment was ready and that the students were prepared, I can’t thank him enough. Big thanks also to Thiago Lima, for generously taking time away from his postdoc at Scripps to work with the students in the field, and for giving advice on the project (he is an actual molecular biologist) along the way.
Huge thanks to all of the staff at Ocean Discovery Institute, especially Joel Barkan, who coordinated the process of turning the plan into a reality when I was 3,000 miles away. I can’t say enough good things about the support I received from everyone at Ocean Discovery, at all levels, and how easy it was to work so closely with so many people. Bahia de los Angeles is a magical place, but doing science there can be a hot, tiring affair. Working with this group makes the process so much more fun.
The experiments also required equipment. Some, like the PCR machine and centrifuge, were generously loaned (thanks ThermoFisher and USD!). Others, such as the tanks and DNA sequencing were purchased from vendors who went the extra mile to do things well and on time (Glasscages and Macrogen).
None of this could have happened without permission from the Comisión Nacional de Áreas Naturales Protegidas (CONANP), which administers the Biosphere Reserve at BLA, and the support of Jose Mercado, who owns and operates the Casa Caguama field station in BLA.
Finally, I owe an enormous debt to Drew Talley, my best friend for over 40 years. He introduced me to Bahia many years ago, and worked tirelessly this year to secure loans of equipment, permits, and who has been incredibly supportive of the development of this project. He has the right to call himself the Captain.
Things were looking great. We had almost 20 slugs, protocols seemed to be working, and the students were becoming comfortable with all of the procedures.
It was time to get some Elysia chloroplast DNA from. Fortunately for the slugs, we did not need a lot of tissue. All we had to do was knock one out, and remove a piece of parapodium. As we showed before, it’s easy to paralyze a slug by soaking it in a magnesium chloride solution that matches the ionic strength (i.e., is isotonic with) of their bodily fluids. This solution rapidly enters their bodies and stops all neural signaling. After 15 minutes, the selected E. diomedea was relaxed and flat as a pancake.
After a quick snip, she was back in the tank, and roaming around within a few hours.
After that,it was time to extract the DNA. The crew got started, extracting DNA from the slimy slug piece, along with a fresh piece of Ulva. There was no time for PCR, but we did have a chance to do one more survey of the area in front of the station.
The conditions were not great, in that the water was somewhat cloudy and surgy by the time we got in. Nonetheless, we got a chance to explore and enjoy the sea life. We also found a few more slugs, which was definitely a bonus.
After that, it was time to pack up and get ready to be on the road. It was sad to be leaving the beautiful place and the people, but time, tides, and summer school wait for no one. We said our goodbyes after dinner. They continued the work for a few more weeks after I left, and I have been getting regular progress reports from Richy.
Hard to say goodbye to the slugs as well.
As always, we were up with the sun. We got on the road early, with tubes of DNA on ice.
The trip north was uneventful, and we arrived at the border in Mexicali on schedule. The wait at the border was about 1.5 hours, made somewhat less pleasant by the 112 degree F heat. We managed to get ourselves and the DNA across, and I was on my way home.
Summer classes started the day after I arrived back in Maryland, so it took a few days to find time to amplify the DNA we extracted in Bahia.It was worth it, though. Very nice bands for Elysia, Codium, and the second sample of Ulva. There were faint bands for the first sample as well, suggesting that the extraction was not a complete bust. With the DNA that was sent last week from the group, we now have a significant number of samples for sequencing, and, with luck, a nice story to tell. After the last round of sequencing did not produce usable data, I gave Macrogen a call. They have been amazing, and are in the process of troubleshooting the last samples I sent them. Keeping fingers crossed.
There was some sad news. The day after we left the station, temperatures shot up to a record 120 degrees F. With those kinds of temperatures, it was impossible to keep the holding tanks cool enough, and most of the slugs were lost. That was sad for the slugs, and meant that there would not be enough animals to finish the behavioral assays this year.
Nonetheless, as the Bahia program winds down this week, we can look back on a lots of success in terms of working out protocols, laying the groundwork for future population surveys, and acquiring DNA samples.
Having slugs meant that it was time to get to work on another part of the project, determining the light sensitivity of the little gals. The I-mazes built by Glass Cages were just right, and we were able to provide a range of light intensities using full-spectrum LED lamps. We first tested using Aplysia, so we could play with parameters a bit. Having only a few Elysia meant that actual experiments would have to wait until we found more.
Another goal of the project was to get a better sense of where Elysia were distributed in the bay. We knew they could be found in front of the station, and that Bertsch had found them at Punta la Gringa, but that was about it. Based on limited experience, the preferred habitat seemed to contain turfy coralline and green algae, along with bunches of Codium, but, again, this was based on a limited sample.
For this summer, we planned two surveys in the bay. In the first, we would spend a morning sampling areas east and south of the station. The second survey would be conducted north of the station, when the students go farther out and spend the night away from the station.
For the first day, we decided to explore two islands, Cabeza de Caballo and Gemelito Esta, along with a small inlet near El Rincon at the south of the bay.
Our first site was the north end of Cabeza, along the west side. There was considerable bird life along the rocks above the water, and we thought it would be worth finding out whether the higher nutrients from the guano supported more algae for the slugs. Of course, the nutrients could also support algae that the slugs don’t like, so we should be able to learn something either way.
Once we got into the water, we could see that the bottom was different from that around the station. Below the bird cliffs, there were heavy growths of brown algae, mostly Padina and Sargassum. The presence of these species did not automatically rule out Elysia, but the possibility of finding slugs 4 cm long in a foot or more of Padina was pretty remote.
The tide also happened to be very low, so the best slug habitat may have been above or near the water line. However, exploration of the shallows did not turn up anything in the way of Codium or slugs
Farther south, things opened up a bit, and there was more bare rock among the brown algae.
There was even a little Codium. No Elysia visible, though.
The snorkel itself was awesome. Lots of different species of fish, often quite large. We were even visited by a school of Jacks, zooming by for a quick look.
After taking a few more photos for documentation, it was time to move on to the next site, Gemelito Este. As can be seen in the photo below, there is plenty of guano on the island, which suggests a lot of nutrient input.
The bottom seemed more conducive to Elysia, however. Plenty of Padina, but also significant patches of coralline and green algae.
We even found some snail or slug eggs. Not from Elysia, but a good sign that molluscs were about.
No Elysia at Gemelito Este, either. Undeterred, we continued southward to an inlet on the mainland, just north of El Rincon. The bottom looked very promising, with lots of coralline, green algae, and Codium.
Toward the end, we were hunting among the clumps of Codium and other algae, and Nancy kicked up a little Elysia. Another data point supporting our ideas about appropriate slug habitat.
Perhaps as a reward for the students’ hard work, a couple of whale sharks swam by the boat. Ricardo maneuvered the boat perfectly, to allow the students to have a quick swim with one of the sharks. Very much a high point for all.
The following day was another field trip, which included another period for snorkeling. This time, it was a small island outside the bay, Isla Pescador. No harm in looking around, right?
Unfortunately, the site is a bit more exposed to wave action, and the surge on this day made it difficult to do too much slug hunting. The bottom looked promising, though.
The following day, the Spatial Subsidy group had their field trip. They snorkeled at a different site, and brought back 11 (yes, eleven) more Elysia for us. It seemed like things were really getting started.
At this point, we had a lab set up, some algae had been collected, but no slugs were to be found. Definitely need slugs. Because Berstch had done almost all of his sampling at Punta la Gringa, at the north end of the bay, it seemed like a good idea to have a quick look up there. I had never been there before, so I asked Drew to drive me up there during an afternoon lull in the action.
It was a beautiful spot, with sand and smooth stones leading to the water, so it seemed worth bringing the students there on their next field research day.
But first, it was time to do some molecular biology. Despite the absence of Elysia, we had plenty of algae. In order to know which plants the slugs are eating, we need to get DNA sequences from potential food plants, so we could make some progress by extracting DNA from the algae. It also gives the students their first shot at working with real DNA.
The most likely food plants are Codium (dead man’s fingers), Ulva (sea lettuce) and Bryopsis (feather algae). We have not found Bryopsis, but had plenty of the other two, so we set about grinding the plants up and separating the DNA from the rest of the stuff in the plant.
Considering the tight space, the students worked well together. It is not easy to pipet stuff from one tube to the next, then wait for an incubation or for the centrifuge to run, then do more pipetting, and so on without going crazy from the heat. Nonetheless they got the procedure finished in time for a trip to La Gringa before lunch. Although we did not find any slugs, it was a very nice dive.
During the next lab session, we took the extracted DNA and amplified it using PCR to make many, many more copies of our sequence of interest. As before, we used primers specific for the rbcL gene, which is found in chloroplasts but not the nuclei of plants or animals. We also included some controls to make sure the procedure worked. First, we amplified DNA that had been extracted by Haseeb and Maryam at USG, and which we know has worked in the past. When we ran the DNA on an agarose gel, to separate the DNA pieces by size, we also added DNA that had been amplified at USG, to be sure the apparatus was working and the dye showed the DNA.
The procedure worked, at least for Codium. There was a visible band for Codium, as well as for the positive controls, so everything seemed to be working. The lack of signal for Ulva could indicate that something went wrong with the extraction, or that the sample did not amplify. Also, for some reason, the molecular weight markers did not show up at the left end of the gel. Nonetheless, the result was very encouraging.
The weekend was upon us, which meant a break for the students from research, and an opportunity for the scientists to get ready for the next week. Lots of details to deal with, getting protocols finalized, reagents tracked down, and field survey plans finalzied.
That Saturday, we went on a scorpion hunt, led as usual,by Drew. Normally, the students start getting disappointed during the early part of the hike, because the scorpions wait a while before coming out. This year, they were plentiful and out early. Using flashlights with UV LEDs made them easy to see, because, for some as yet unknown reason, they fluoresce green under UV light.
Meantime, we still had exactly zero slugs. I was beginning to feel a bit like Ahab in the obsessive pursuit of my little green nemeses. So, on a beautiful Sunday morning, I decided to do yet another snorkel through the shallows to hunt through the algae. The tide was especially low, so I started by just walking through the shallows, looking for slugs, while the mobulas jumped a short distance away.
The snorkel itself was quite wonderful, slowly swimming back and forth from the front of the staff house to the south end of the Vermillion Sea field station, which had been used by the group some years ago. As I swam slowly over the shallow bottom, I saw lots of algae, starfish, stingrays, corals, and many species of fishes. I even found one cute little nudibranch. I was however, beginning to despair of finding Elysia.
I also had to keep a close eye on the catch bag, because a small crowd of hungry puffers was following along, hoping to grab anything I might stir up.
After about 90 minutes, it was time to move on to other tasks. We needed more algae-covered rocks for the station, so I put the mesh bag containing the little nudibranch into a bucket on the shore and proceeded to hunt around in the shallows for suitably-sized rocks with interesting algae. When I looked at one patch of Codium, I saw what looked like some blue color among the uniform deep green. Could it be? A quick sweep of the hand sent a little Elysia flying through the water column.
I grabbed it, and gently held it while swimming toward the shore. As I got out of the water, I looked in my hand, and it was gone! I almost sobbed through my snorkel. However, after many years as a research scientist, I am thoroughly accustomed to harsh disappointment, and went about my business collecting more rocks. Fortunately for me, and for the project, there were three more of the little gals in separate clumps of Codium, and I was ready with the catch bag this time. As can be seen in the photo below from a later hunt, the presence of Elysia is not always obvious.
The drought had ended! The captive Elysia adapted quickly to their new home.
Not bad. We had the molecular biology working adequately, and we had slugs. As often seems to be the case, finding one opens the door to finding more.
There was a lot more to do, though. It was time to get serious about the slugs’ kleptoplast DNA, their responses to light, and their distribution in the bay.
Has it really been that long? Well, I’ve been busy.
The preparations of the last few months have now been tested in the field. I am returning from the first two weeks of a five week field study in Bahia de los Angeles in Baja California. As I think I posted earlier (apologies if I did not), the plan for the summer was to work with Ocean Discovery Institute on fleshing out some of the details of the life history and behavior of Elysia diomedea in Bahia de los Angeles. Specifically, we want to know more about where they are found in the bay, what they eat, and how they respond to light. This information will help us to understand more about the role of kleptoplasty, along with the significance of the dramatic population fluctuations of the slugs documented previously by Hans Bertsch.
Even before we left, there were several challenges. For example, for the project to get off the ground we needed permission from CONANP, the agency that oversees the marine reserve, to collect Elysia from the bay. Months before the project began, we submitted an application for a permit to allow us to collect and study Elysia. Unfortunately, that permit was rejected because we had not been clear about the relationship between the Elysia project and ongoing research in the bay islands. Naturally, this caused us significant anxiety. After a lot of work behind the scenes by folks at Ocean Discovery and by my very good friend Drew Talley at USD, our intentions were made clear, and we were given permission to go ahead with the project.
Among the items that were absolutely necessary were aquaria for holding, observing and testing the slugs’ response to light. In April, I had started working with a local company that builds custom aquaria to build two holding/observation tanks and two “I-mazes” for testing light preference. By early June, the tanks had not materialized, and I was beginning to get nervous that missed deadlines and excuses would continue until time ran out. I decided to go with a more experienced vendor, Glass Cages, and they got the tanks into production and had them air freighted to San Diego in plenty of time. The whole process of working with them was pleasant and reassuring.
The other critical pieces for extracting and amplifying DNA are a microcentrifuge and a PCR thermal cycler. Again, thanks to the persistence of Drew Talley, we borrowed an Eppendorf centrifuge from USD, and received the loan of a demonstration model thermal cycler from Thermo Fisher Scientific.
So, after months of planning, spending a semester making sure that the methods work, arranging for care of the system in Maryland while I was gone, and enjoying a roller coaster ride obtaining permissions and equipment, it was time to get into the field.
The trip down was slightly adventurous. We traveled with the Ocean Discovery students, starting out at Hoover High School in City Heights. This year, we took the eastern route, through Mexicali and San Felipe. It was a little longer, but somewhat more scenic, and had a whole lot less traffic. The road was not great in spots, and one of the vans developed a flat tire along the way. After a little delay, we were back on our way, and arrived in the early evening.
As always, I was very happy to be there. Its hard to think of a place that I would rather be. The sea is beautiful and full of life, and the surrounding desert is spectacular in its own right. Over the course of the two weeks at the station, I tried my best to savor the views, sounds and smells.
Naturally, I was eager to get started. It was all I could do to sit still during review of procedures around the field station, because I was eager to collect slugs in order to be ready for the students’ upcoming projects. Finally, I got into the water, and began to hunt for Elysia. It was wonderful to be in the bay again, and there was lots to see. The familiar zones of Padina, Codium, Ulva, and the many other algae on the rocks outside the station reminded me of where I thought I should look. After about 1 ½ hours of unsuccessful searching, I headed back to the station to get ready for the rest of my day.
I got to meet my crew in person for the first time. The five young women were full of energy, and ready to get going with the project. The goals of the “Photobiology” group (I needed a somewhat official sounding name, sue me) will be to flesh out some basic biology of E. diomedea here in the bay. As we did for E. clarki in Maryland, we want to extract DNA from E. diomedea, and compare the sequence of rbcL in kleptoplasts with those from potential food plants. Also, we will be looking at light preferences, using “I-mazes,” which give the slugs a chance to select their favorite light intensity. We are also hoping to have a chance to explore the bay, surveying for appropriate habitat and the presence of slugs. Lots to do to get set up and get the students trained.
The hunt for Elysia continued during the morning of our first full research day. In the past, the morning hours have been the most productive in terms of slug hunting, so I had planned several mornings during the first week for collection. The crew was becoming very proficient in the water, and we hunted for about 90 minutes in the shallows in front of the station. Sadly, despite our efforts, no Elysia were to be found. After a quick cleanup, we headed for the classroom for a briefing on algal diversity, lab equipment and safety before lunch. The students had other activities in the afternoon, which gave me the opportunity to continue setting up tanks and equipment.
Our “molecular lab” is located in the garage, along with equipment for other Directed Research groups. We share the space with a group studying ways of reducing bycatch of unwanted fish species and turtles, and another group that documents the flow of energy between the rich waters of the sea and the relatively barren land of the bay islands. The space is a hive of activity at 7 am, when the other groups are rushing to get on boats. After that the space is essentially ours until lunchtime.
The other part of our “lab” consists of the observation tanks. These are in another part of the station containing the kitchen and computer lab. The 16” cube tanks sit on a sturdy table, with circulation provided by air pumps, and lighting provided by morning sunlight supplemented by desk lamps with full-spectrum LED bulbs. Once the slugs are in, the tanks hold slugs for DNA extraction and behavioral assays, with one being used solely for observation of the daily rhythms of undisturbed slugs.
Although we had not found any Elysia, at least a dozen small Aplysia rode into the tank with the plants. This will actually be handy for comparison with the responses of Elysia to light. Aplysia do not store chloroplasts, and might be expected to be repelled or indifferent to light.
Day 2 was reserved for a field trip for the students. It is supposed to be a non-work day, used to introduce the students to some aspect of the bay. It started off great, with a visit to a sea lion colony, and up close encounters with very large fin whales.
We did squeeze in a little work, because part of the trip involved time for snorkeling at Coronadito island, at the far north end of the bay, and we were not explicitly banned from looking for Elysia. Although we did not find any, it was useful to note and photograph the nature of the bottom, and the dominant algae species that were present. Lots of Sargassum, some turfy coralline algae, but not a lot of large green algae.
The days continued, with more briefings about identification of algae and molecular biology methods. The big question was whether we would actually find any Elysia. Fieldwork always requires some improvisation, but it’s a real challenge to improvise your way around the absence of your research subject. Stay tuned.