Slug System Upgrade
The first version of the multi-tank slug system has served reasonably well, but it has had its limitations. The main problems have been the sprawl of equipment (note the tanks, dosers, auto-topoff, scattered about in the photo below), the low height of the shelves, which limits lighting options, and the cramped nature of the shelf unit, which makes maintaining or replacing tanks difficult. I also wanted a bigger sump, mostly to have a little more volume to prevent floods. To be honest, the photo makes it look even worse than it was, because I had moved a cabinet in preparation for the new system, leaving controllers and power supplies lying in a pile on a temporary shelf. Nonetheless, the system was long past due for an upgrade.
Old slug and algae system before upgrade. 2/25/17.
It took a few weeks to decide exactly how much space I could afford, and how to design the new shelves to accommodate existing tanks and allow flexibility in future configurations. I finally settled on a 60 X 16 inch footprint, which would accommodate the 15 and 20 gallon tanks on the top, plus a little extra space. It would be smaller than the space made available by the removal of one file cabinet and the old slug system, giving a little elbow room for maintenance and repair. I decided on 48 inch height. Enough for two shelves for tanks, and a bottom shelf for a sump. Three rows of tanks, a sump level, plus ample height for lights would just be too tall for me to reach easily. My experience with the current system has taught me that more tanks is not necessarily better, The second shelf would have room for a couple of 10-gallon tanks, or various combinations of 5- and 10-gallon tanks for smaller-scale experiments. The dosers and controllers would be on the bottom shelf with the sump, protecting them from splash, and making the system almost completely self-contained. By necessity, the chiller will have to be off to the side in order to move heat away from the tanks.
I decided to build the frame from 2X3 studs, and use 1/2″ plywood for the shelves. The studs should be plenty strong to support the 5 foot shelf, and 2X4s would be overkill and make the system that much heavier. The weight of the shelves is transmitted to the floor by 2X3s that run from the bottom of one shelf support to the top of the next. There is a second set of vertical 2X3s going all the way from the top shelf to the floor, providing more stability and support.
Shelves built 2/5/17
For paint, I chose a latex semi-gloss. I hope I don’t regret not using marine paint, but I am hoping that the primer plus three coats applied over the course of a week will be adequately waterproof. I tried to match the color of the walls in the office, but it turned out a bit more blue than I had intended. The piece of plywood on the back provides a surface for attaching controllers, power supplies, dosing pumps and drain brackets.
Shelves painted and ready for plumbing, 2/15/17.
In order to simplify moving tanks in and out, I installed a 2″ drain with 8 openings along the bottom shelf, and a 3/4″ supply pipe with barbed valves along the top shelf. Installing and removing a tank will be as simple as connecting a supply hose and placing a flexible drain hose from the tank into a drain opening.
Drains installed 2/18/17.
The new system in place, showing the supply valves 2/25/15.
Then came the hard work of moving everything over. After Joanna pointed out that the shelves would not fit in the Jetta, I reserved a U-haul pickup truck, and we moved it from home to the office. Then it was simply a matter of spending 10 or so hours reinstalling plumbing, draining and moving tanks, setting up lights and pumps, mounting dosers and controllers, fussing with details, and cleaning up the resulting mess.
Up and running, 2/26/17.
I am very happy with the results. My office is less cluttered, every aspect of the system is more accessible, the electronics are better protected from splash, and I don’t have to climb on a chair to work on the top tank. The Neptune Apex controller became a little buggy during the process, but I can again control and monitor the system remotely after a few reboots. The leak detection module is still not fully functional, so I am keeping fingers crossed that there will be no floods, large or small, until it is fixed.
One happy development is that the Bryopsis growing on the eggcrate in the 15-gallon tank (upper right in the photo above) has started to take off. Expect photos of that, plus a new shipment of marine plants, in the next day or so. Who knows, maybe there will once again be slugs in the Box of Slugs.
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.
Observations on Algae
Happy Holidays to all of you fans of slugs!
Although the site and the project are devoted to adorable molluscs, we would be nowhere without algae. These days, I spend more time and resources trying to acquire, grow, and identify algae than I do attending to Elysia. It should not be a surprise, given the outsize role of algae in the biology of the slugs, but, until this project was underway, I had never given a lot of thought to the care and diversity of algae. Subsequent posts will describe some of the progress in algae care, but today we’ll focus on some systematics and molecular biology.
“Spongy sea pansy” in 20 gallon sandy-bottom tank in office system. 12/22/16.
The plant in the photo above has been nagging at me for well over a year. I can’t remember exactly how it came up, but KP Aquatics mentioned that they had a species of algae they called “spongy sea pansy,” which was like Udotea, but larger and squishier. They were quite a bit taller than Udotea, grew in clumps, and were indeed quite spongy. Their biology is somewhat different from other algae in the order, in that the thallus (the body of the alga) dies back periodically, and a new one grows from the rhizoid (the rootlike part). In my experience, species like Udotea or Penicillus send out runners that produce new thalli, and the old ones just die off.
Slugs mating on Udotea 10/30/14
New growth from rhizoid of Avrainvillea, aka “spongy sea pansy” 11/16/15
I have been referring to them as Avrainvillea, because they fit the description reasonably well, but had never done the hard work of verifying that it was not a similarly squishy genus, such as Rhipilia or Cladocephalus.
A real phycologist (algae specialist) would have probably started with a good microscope and species key. I took the molecular route, since I was already using PCR to amplify DNA from the rbcL gene in a few other species, and sending it off for sequencing.
Because I was testing new PCR machines, I had set up three independent reactions, and the results were the same. The screenshot below shows the results of a BLAST search for one of the sequences through the NCBI database, with the closest match at the top. The second best, with 98% of the nucleotides being identical, is Avrainvillea nigricans. The best match (99%) is to an “uncultured Ulvophyceae” clone from a study by Christa, Gould, Wagele, and their collaborators. If I read the entry correctly, the sequence is from kleptoplasts extracted from Costasiella, a Caribbean slug that feeds on…did you guess…Avrainvillea. To provide a little context, Cladocephalus and Rhipilia, the genera that were possible candidates based on appearance, were only 93% and 82% identical, respectively.
That is a pretty clear-cut result. It looks like Avrainvillea, it is squishy like Avrainvillea, and its DNA is essentially an exact match for Avrainvillea nigricans. It is Avrainvillea.
As you’ll see in the next post, the results aren’t always so easy to interpret.
Testing New (Old) Machines
Once again, I find myself apologizing to the hordes of Solar Slug fans for the long period of silence. After the frenzy and freedom of summer, it has been hard to find time to experiment, or even mess with the site, but I am hopeful that things will change now that the semester is tapering off. There has been a little news along the way.
A few weeks ago, the students participating in the Biology Honors Research Program at UM College Park invited me to give a seminar. Since my fly work is old and stale, it seemed like a good chance to talk about the beginnings of the Solar Slug project. As far as I could tell, the students found the ideas intriguing (golly, who wouldn’t?), and it was a great chance for me to assemble a seminar and impose some order on my thoughts.
Last month, we submitted a report to CONANP, who oversees the Biosphere Reserve in Bahia de los Angeles, about the past summer’s activities. A good chance to think hard about what we did, why it matters, and where it will lead. In related news, because of overwhelming time demands from the opening of their new Living Lab, Ocean Discovery Institute will not be working in Bahia in 2017. It is disappointing, but, at least in principle, will give me some time to put together more substantial funding for future years.
But let’s talk science. Several months ago, I acquired two old PCR machines from the surplus equipment program at the National Institutes of Health. It’s a great program, in which equipment that is no longer wanted by researchers at NIH can be acquired by educational institutions. The major caveat is that one can never be sure that the equipment is functional until it gets back to the lab and is tested. My students in the Cell Biology and Physiology lab course were running some PCR samples, so I thought it would be a good time to test out the new (old) machines in parallel with the very fancy PCR machine we use for student labs.
The thermocyclers are Applied BioSystems GeneAmp 9700s. In principle, they should do everything we need, plus they have a nice post-cycling chill cycle, so I can set them up and go home without worrying about the DNA sitting in the machine and degrading at room temperature. But do they work?
Applied Biosystems 9700 PCR machine from NIH surplus. This is Machine 1, for which the temperature was within one degree C of the programmed value. 11/4/16
The choice of what to amplify was easy. Another Elysia fanatic, Susanne, had sent me a piece of parapodium from an E. diomedea that had an unfortunate encounter with a filter. Don’t fret, the slug survived, but she was nice enough to carefully preserve the tissue in ethanol, pack it, and ship it to USG.
Fragment of parapodium (in vial, center) carefully packed and shipped from Austin.
The fragment sat forlorn for about a month. It was very exciting to be able to finally extract the DNA and see if we could amplify the rbcL region. I set about mashing and processing a small piece, and all looked well.
Small piece of E. diomedea parapodium before extraction. 11/2/16
At the end, I had produced a tube of clear liquid. Was there DNA?
DNA extracted from E. diomedea. 11/2/16.
I amplified DNA from E. diomedea, a control sample from BioRad (to make sure the machines functioned at all), and some DNA I had extracted from an Avrainvillea plant in the slug system. During the thermal cycling, I used a thermocouple probe to check the temperatures of the machines. Machine 1 was just about perfect, whereas Machine 2 was way too warm during the cooler parts of the cycle, and I expected poor results.
Results of first test of new PCR machines. + control” is commercially prepared DNA from BioRad, containing a mix of DNA with and without an ALU insert in the PV92 region of the human genome. There should be bands at 941 and 641 base pairs.
E. diomedea and Avrainvillea DNA were extracted a few days before. 11/4/16
The control samples worked in both machines, which was somewhat surprising based on the temperature measurements. I guess you can get away with a lot if you start with really clean DNA and well-established primers. The only sample from the new extracts that worked was the E. diomedea DNA in machine 2. I expect we can get things to work better if I reduce the DNA concentration, but it is puzzling that a sample in the less reliable machine worked better. Nonetheless, I now have some DNA I can send off for sequencing when there’s a little time.
Happy Birthday to us!
Today marks the 2nd birthday of the Solar Sea Slug Blog. It started as a place to curate information about Elysia, and to track the progress of our modest project. Since then, it has taken on a life of its own, and morphed into a place to curate information about Elysia, and to track the progress of our modest project. My how we have grown!
I suppose the concept of this site as a journal of my work with Elysia (a “web log,” so to speak) is rather old-school in this age of tweets and Instragram. Fortunately, I am at a place in life where I can play the geezer card, using the excuse that I simply don’t have the mental bandwidth to generate content at that rate. Despite all the nice messages that people send me to help improve my SEO, I actually worry more about getting too much traffic than too little. To paraphrase Tim Curry, “going viral would be too much responsibility for me.”
The list of possible tweaks and improvements gets longer, not shorter, and the site will continue to improve incrementally. There are things I would like to simplify, such as how to leave a comment, or the process of updating reference lists. Then there is the slow work of fleshing out the species pages. It would be great to host the subset of articles that are open-access as well. Rome wasn’t built in a day, and Romulus didn’t have a full time job or dogs to walk, so we’ll have to be patient.
Thank you, dear reader, for checking in from time to time. Although it is unlikely you will find links to cat videos (but never say never), I hope you will stop by to find out more about amazing and abundant little molluscs, their biology, and the people who are working to understand them.
Wild Slugs: Sea of Cortez Edition (Conclusion?)
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.
Lousy chromatogram from Sanger sequencing. Note multiple possible bases (different colors) at each site. Uninterpretable.
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.
Codium sequence from Macrogen. Note a few sites showing more than one possible base, presumably polymorphisms. 8/10/16
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.
Portion of rbcL sequences extracted from Codium simulans (top), Elysia diomedea (middle) and Ulva sp. (bottom). Sites at which Ulva differs from both Codium and Elysia are indicated by arrows.
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.
E. Diomedea on Codium in tank at BLA station
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…
Photobiology Group. Cristal, Rosalia, Nancy, Richy, Allison, Susan & Thiago.
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.
There would have been no time to work out procedures once we arrived in Bahia, so Maryam and Haseeb’s experiments and troubleshooting at USG were crucial.
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.
Dr Drew Talley, BLA staff office 7/1/16
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.
Wild Elysia: Sea of Cortez Edition (Part Four)
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.
Some of the captive slugs, 7/8/16
E. diomedea and Aplysia in holding tank.
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.
Elysia diomedea, relaxed and ready for surgery. 7/7/16
E. diomedea, with small piece of parapodium removed. 7/8/16
After a quick snip, she was back in the tank, and roaming around within a few hours.
Elysia diomedea, the day after surgery. Note the missing piece of parapodium on her right side. 7/8/16
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.
Susan, Rosalia & Nancy, ready for a slug survey at the station. 7/8/16
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.
Gorgonian in shallows near BLA station. 7/8/16
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.
The photobiology crew: Bottom row: Allison, Rosalia, Susan, Nancy; Top row: Crystal, me, Richy. 7/8/16
Hard to say goodbye to the slugs as well.
Captive E. diomedea crawling on Colpomenia, BLA station 7/8/16
As always, we were up with the sun. We got on the road early, with tubes of DNA on ice.
Sunrise on departure day. 7/9/16
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.
Road to Mexicali. 7/9/16
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.
PCR products from extractions at BLA.
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.
Wild Slugs: Cortez Edition (Part Three)
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.
Testing I-maze with Aplysia. 7/4/16
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.
Cabeza de Caballo (left, large island) and Gemelito Oeste (small white island to the far right), from BLA station. Gemelito Este is behind Gemelito Oeste.
Photobiology group on way to Cabeza. Ricardo, the driver, was awesome at following the group as we drifted in the water. 7/5/16
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.
Sargassum and Padina, northwestern part of Cabeza de Caballo. 7/5/16
Heavy Padina growth, Cabeza de Caballo, 7/5/16
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.
Rocky area, Cabeza de Caballo. 7/5/16
There was even a little Codium. No Elysia visible, though.
Codium, at about 15 feet in rocky area of Cabeza de Caballo. 7/5/16
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.
Jacks visiting the shallows at Cabeza de Caballo. 7/5/16
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.
East side of Gemelito Este. 7/5/16
The bottom seemed more conducive to Elysia, however. Plenty of Padina, but also significant patches of coralline and green algae.
Bottom at Gemelito Este. Mixed Padina and other algae. 7/5/16
Large Oyster, covered in turf algae. Gemelito Este, 7/5/16
We even found some snail or slug eggs. Not from Elysia, but a good sign that molluscs were about.
Snail or slug eggs on Padina. Gemelito Este 7/5/16
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.
Green turfy algae north and east of El Rincon. 7/5/16
Algae near El Rincon. Cladophora or something related? 7/5/16
Codium near El Rincon. 7/5/16
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.
Whale shark approaching the boat. Near El Rincon, 7/5/16
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?
Crew entering water at Isla Pescador. 7/6/16
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.
Wild Slugs: Sea of Cortez Edition (Part Two)
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.
Bahia de los Angeles, showing a few sites relevant to this post and the next one.
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.
Punta la Gringa, looking seaward. 6/30/16
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.
Ulva from shallows in front of field station. 7/1/16
Codium, possibly simulans. Collected in front of field station and ready to be homogenized. 7/1/16.
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.
Nancy, Allison and Rosalia extracting DNA.
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.
Agarose gel, showing bands for Codium collected at BLA, as well as positive controls from USG. 7/4/16
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.
Scorpion fluorescing under UV flashlight. Hills south and west of field station. 7/2/16
Same scorpion, under normal light. Feeding on winged ant. 7/2/16
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.
Sunrise over the islands. Taken from the staff house.
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.
Stars in shallows. 7/3/16
Stingray on sandy bottom. 7/3/16
Gorgonian near Vermilion Sea Station. 7/3/16
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.
Puffers, keeping an eye on me. 7/3/16
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.
Codium, concealing 3 – 4 E. diomedea. Can you find all of them?
The drought had ended! The captive Elysia adapted quickly to their new home.
Captive E. diomedea on Codium at station. 7/3/16
E. diomedea exploring new home. 7/3/16
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.
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