Posts in Category: Slug Science
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.
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.
Wild Slugs: Sea of Cortez Edition (Part One)
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.
Tanks and I-Mazes from Glass Cages, freshly picked up from Southwest Air Freight. 6/23/16
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.
Stopping to fix a flat between San Felipe and Laguna Chapala. 6/25/16
Tire destroyed on way to BLA.
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.
Town of Bahia de los Angeles from the field station.
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.
Small stingray over a big field of Padina in front of BLA station. Sargassum at lower right. 7/3/16
Codium in front of BLA station.
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 Photobiology Crew. Richy, at left is the Directed Research Fellow. The students (from left): Crystal, Rosalia, Nancy, Allison and Susan.
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.
Rosalia drawing an example of a dichotomously branching alga.
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.
Molecular Laboratory up and running in BLA station garage. Special thanks to USD for the centrifuge (left), and to Thermo Fisher for the loan of the PCR machine (right).
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.
Holding tanks at field station, with I-mazes in front. 6/29/16
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.
Little Aplysia in holding tank. 6/29/16
Small Aplysia in holding tank. 6/29/16
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.
Fin whale at north end of the bay. 6/28/16
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.
Typical rocky bottom at Coronadito, with a trio of triplefins. 6/28/16
Susan next to a large mass of Sargassum. Coronadito 6/28/16
Fierce damselfish, Coronadito 6/28/16
Algae community on rocky bottom. Coronadito, 6/28/16
Photobiology group, plus a few others, at Coronadito. 6/28/16
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.
Everything in Place?
The project seems to be where it needs to be. Since the last post, I ordered new “degenerate” primers, which consist of a mix of almost all possible sequences that would match the rbcL gene of the algae of interest. The folks at Midland Certified Reagent Co. were very helpful, and the primers arrived quickly.
As can be seen below, we get a much more robust signal from the presumed Avrainvillea with the new primers. There was, however, no signal for Bryopsis species 1. Because that batch of DNA consistently amplified in the past, I am going to attribute the failure to technical issues for the moment.
As was true for the previous run, the lane for Elysia clarki still had no signal. The repeated failure to get a PCR product from this extract suggests that it is not a simple technical glitch. The obvious culprit is mucus. Even though we used a small amount of tissue, it produced copious mucus, some of which may have remained in the sample after purification. The polysaccharides in mucus are expected to interfere with the PCR reaction, so that could be our problem.
Gel from 5/25/16
Because his lab group has successfully extracted and sequenced DNA from Elysia species, I contacted Gregor Christa from Heinrich-Heine Universität Düsseldorf. He suggested a very simple fix: dilute the DNA template. It seemed too easy, but I performed another run yesterday, and, lo and behold, it worked! The undiluted DNA (& mucus) gave no signal, but the diluted DNA amplified nicely. I guess the dilution reduced the concentration of mucus enough to allow the reaction to proceed, while the sensitivity of the PCR reaction allowed amplification of the diluted DNA.
Everything seems to be on track. The next step is to send some of our PCR products out for sequencing, hopefully within the next few days.
Journal Club: Why on Earth do they store chloroplasts?
The subject of this year’s Journal Club (Has it really been over a year? Oh dear.) is a paper by Gregor Christa and colleagues from back in 2013.
In this paper, the authors try to develop a scientifically rigorous explanation for long-term retention (LTR) of chloroplasts in Plakobranchus and Elysia.
Some species of marine slugs in the Plakobranchoidea, which includes the genera Elysia and Plakobranchus, can store chloroplasts for many months. These are stored in branches of the digestive system that ramify throughout the slugs’ bodies. Perhaps foreshadowing their conclusions, Christa et al. refer to this phenomenon as delayed digestion, rather than maintenance of the chloroplasts.
So what do the food-derived chloroplasts (“kleptoplasts”) do for these months? In 1975*, Trench found that Elysia viridis could incorporate 14C from labeled CO2 into its tissues, and concluded that kleptoplasts retained the ability to generate sugars from light, water and CO2. Based on this observation, he coined the term “leaves that crawl” to indicate that he believed the kleptoplasts generated energy for the slugs and made them partially or totally independent of the need for ingested food. This idea took hold, both in the scientific literature and in the popular imagination.
There were a few complications in this story, however.
First, how are functional kleptoplasts maintained for such a long period outside of a plant cell? A functioning organelle needs a constant supply of new proteins to replace those that are degraded with time and use. Although chloroplasts retain a small number of genes (including rbcL, which is important for the kleptoplast identification project), the vast majority of genes required for their continued function are located in the nucleus of plant itself. That is a problem, because the plakobranchids rapidly digest the nuclei of the algae on which they feed.
The problem appeared to be solved, and in an interesting way, when reports appeared that suggested the slugs had incorporated algal genes into their own genomes, a phenomenon called “lateral gene transfer,” or “horizontal transfer of genes.” Newly-synthesized algal RNAs and proteins were identified in slugs (reviewed in Pierce et al., 2007). However, the absence of algal gene products in eggs or newly settled slugs (Bhattacharya et al., 2013), and the relative scarcity of chloroplast RNAs and proteins in slugs, indicates that lateral transfer is unlikely to have occurred. It seems unlikely that kleptoplasts could be self-sustaining in the long term.
Another issue is that animals are capable of incorporating small amounts of CO2 into organic molecules, through carboxylation reactions. This being the case, how do we interpret Trench’s results of 14C incorporation into E. viridis?
Furthermore, starved plakobranchids (the shorthand term Christa et al. use for Elysia and Plakobranchus) do not stay fat and happy if they are provided with light. They shrink, and change color from green to yellow, which indicates that they are breaking down their tissues and the kleptoplasts, in order to survive.
Back to the original question: what is the purpose of storing kleptoplasts? Christa et al., asked two very basic questions about the role of kleptoplasts, using E. timida and Plakobranchus ocellatus.
- Are the kleptoplasts really involved in CO2 fixation? To answer this, they compared incorporation of 14C in slugs that were kept in the light, in the dark, or in the light but treated with a drug that inhibits photosynthesis. If the kleptoplasts are involved, then 14C incorporation will be higher in conditions that allow photosynthesis.
- Does the ability to photosynthesize reduce the rate of tissue loss during starvation? If slugs are kept in the dark, or treated with drugs that inhibit photosynthesis, do they lose weight faster than slugs kept in bright light?
The answer to Question 1, was an emphatic “yes, 14CO2 incorporation is much higher in the light.” As shown in Figure 2, below, both E. timida and P. ocellatus showed significant incorporation in the light at 60 and 120 minutes, but essentially none in the dark. Incorporation was also significantly reduced by monolinuron, which blocks photosynthesis.
So, it appears that the kleptoplasts fix carbon, which should provide energy to the slugs. Does this keep the slugs from starving?
This brings us to question 2. The authors examined two aspects of starvation to determine how the different conditions affected the ability to photosynthesize and maintenance of body weight.
First, they looked at what happens to chlorophyll function during starvation using Pulse Amplitude Modulated (PAM) Fluorimetry. The authors suggest that those kept under more intense light declined more rapidly, but the sample sizes were small and the variation was large, so the strongest conclusion one can safely make is that quantum yield (their measure of photosynthetic function) decreased significantly with starvation.
Perhaps the most interesting result was that exposure to light made no difference in terms of weight loss. Figure 4b, below, shows that all slugs, regardless of whether they were in light, darkness or treated with monolinuron, lost about the same amount of weight over 50 days. 
Again, the sample sizes were tiny (two slugs each), so sweeping conclusions are not in order. However, the shapes of the curves are intriguing. The slugs in the light seemed to decline more rapidly than those in the dark, and the monolinuron treated slugs both showed a rapid decline followed by more gradual weight loss. It would be interesting to know if there was interesting biology underlying these results (slugs burn through reserves faster in the light?), or whether they are quirks of a small sample.
Christa et al. conclude that the slugs are not photoautotrophic, i.e., they do not survive on light. Instead they propose that the kleptoplasts are a food reserve. This is certainly a plausible model, but others have not yet been ruled out.
For example, the authors suggest that the kleptoplasts could be required for the biosynthesis of compounds required for development or egg production. One potentially interesting, and relatively straighforward, experiment would be to compare rates of egg laying between Elysia that are fed, but kept under high- and low-light conditions. Given the metabolic demands of producing and provisioning egg masses, it would be interesting to see if kleptoplasty contributes to the process.
Another possibility is that the kleptoplasts help to “fatten up” the slugs before starvation. In this scenario, slugs use both food and photosynthesis to fill storage tissues when times are good. When food is scarce, or experimenters starve them, they use the kleptoplasts and stored tissue to produce energy and necessary metabolites. This may be especially important for species that live on scattered or sparse food sources. I don’t think anyone has done the math to determine how the energy estimated to be produced by photosynthesis (e.g., the amount of carbon fixed per unit time by the slugs in the light) compares with the metabolic demands of the slug. It may be too small to keep a starving slug alive, but might help fatten up a feeding slug.
One final random thought regards the maintenance of kleptoplasts. It is pretty clear that the slug does not have the genetic equipment to produce the proteins required for the upkeep of the chloroplasts. However, plakobranchids (at least the E. clarki watching me here in my office) eat prodigious amounts of algae daily. Would it be possible for ingested algal RNAs and proteins to find their way to the kleptoplasts? It seems like a non-trivial problem to get the materials across a few membranes and into the plastids, but it would help to explain how chloroplasts can survive for months in cells that are lacking important equipment.
*note that all the references can be found in the full list of papers.
Kleptoplast Extraction
We made our first try at extrtacting and amplifying DNA from kleptoplasts. It was relatively straightforward to get the tissue from the slug. First, she was “relaxed” with isotonic MgCl2, which blocked synaptic transmission and paralyzed and anesthetized her.
Elysia clarki relaxed with 400 mM MgCl2. 4/27/16
Then, a small piece of parapodium was removed (see her left side, at bottom). DNA was extracted using the same method as for the plants.
Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16
In case you were worried, she was fine the next day.
Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16
We also tried a new species of algae, which I am calling Avrainvillea. It may be Rhiphilia, I’m not completely certain.
Alga extracted 4/27/16. Probably Avrainvillea.
Unfortunately, the amplification was not a success this time around. The positive control samples, DNA extracted from Bryopsis during previous sessions, worked well. “Avrainvillea” showed a much weaker signal, and there was no signal from the slug extract.
Results from week of 4/25/16
A number of things may have gone wrong. The slug sample was extremely slimy with mucus, and those polysaccharides could have interfered with extraction or amplification. Also, the Bryopsis-specific primers may not have annealed adequately with the DNA from Avrainvillea and whatever is in the slug.
Now that things are calming down after the semester, I am going to try again, making a few changes. Most importantly, I have ordered “degenerate” primers, which contain a mix of sequences that will complement just about any algal sequence. Still waiting on tips regarding the slime.
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