Things were looking great. We had almost 20 slugs, protocols seemed to be working, and the students were becoming comfortable with all of the procedures.
It was time to get some Elysia chloroplast DNA from. Fortunately for the slugs, we did not need a lot of tissue. All we had to do was knock one out, and remove a piece of parapodium. As we showed before, it’s easy to paralyze a slug by soaking it in a magnesium chloride solution that matches the ionic strength (i.e., is isotonic with) of their bodily fluids. This solution rapidly enters their bodies and stops all neural signaling. After 15 minutes, the selected E. diomedea was relaxed and flat as a pancake.
After a quick snip, she was back in the tank, and roaming around within a few hours.
After that,it was time to extract the DNA. The crew got started, extracting DNA from the slimy slug piece, along with a fresh piece of Ulva. There was no time for PCR, but we did have a chance to do one more survey of the area in front of the station.
The conditions were not great, in that the water was somewhat cloudy and surgy by the time we got in. Nonetheless, we got a chance to explore and enjoy the sea life. We also found a few more slugs, which was definitely a bonus.
After that, it was time to pack up and get ready to be on the road. It was sad to be leaving the beautiful place and the people, but time, tides, and summer school wait for no one. We said our goodbyes after dinner. They continued the work for a few more weeks after I left, and I have been getting regular progress reports from Richy.
Hard to say goodbye to the slugs as well.
As always, we were up with the sun. We got on the road early, with tubes of DNA on ice.
The trip north was uneventful, and we arrived at the border in Mexicali on schedule. The wait at the border was about 1.5 hours, made somewhat less pleasant by the 112 degree F heat. We managed to get ourselves and the DNA across, and I was on my way home.
Summer classes started the day after I arrived back in Maryland, so it took a few days to find time to amplify the DNA we extracted in Bahia.It was worth it, though. Very nice bands for Elysia, Codium, and the second sample of Ulva. There were faint bands for the first sample as well, suggesting that the extraction was not a complete bust. With the DNA that was sent last week from the group, we now have a significant number of samples for sequencing, and, with luck, a nice story to tell. After the last round of sequencing did not produce usable data, I gave Macrogen a call. They have been amazing, and are in the process of troubleshooting the last samples I sent them. Keeping fingers crossed.
There was some sad news. The day after we left the station, temperatures shot up to a record 120 degrees F. With those kinds of temperatures, it was impossible to keep the holding tanks cool enough, and most of the slugs were lost. That was sad for the slugs, and meant that there would not be enough animals to finish the behavioral assays this year.
Nonetheless, as the Bahia program winds down this week, we can look back on a lots of success in terms of working out protocols, laying the groundwork for future population surveys, and acquiring DNA samples.
Having slugs meant that it was time to get to work on another part of the project, determining the light sensitivity of the little gals. The I-mazes built by Glass Cages were just right, and we were able to provide a range of light intensities using full-spectrum LED lamps. We first tested using Aplysia, so we could play with parameters a bit. Having only a few Elysia meant that actual experiments would have to wait until we found more.
Another goal of the project was to get a better sense of where Elysia were distributed in the bay. We knew they could be found in front of the station, and that Bertsch had found them at Punta la Gringa, but that was about it. Based on limited experience, the preferred habitat seemed to contain turfy coralline and green algae, along with bunches of Codium, but, again, this was based on a limited sample.
For this summer, we planned two surveys in the bay. In the first, we would spend a morning sampling areas east and south of the station. The second survey would be conducted north of the station, when the students go farther out and spend the night away from the station.
For the first day, we decided to explore two islands, Cabeza de Caballo and Gemelito Esta, along with a small inlet near El Rincon at the south of the bay.
Our first site was the north end of Cabeza, along the west side. There was considerable bird life along the rocks above the water, and we thought it would be worth finding out whether the higher nutrients from the guano supported more algae for the slugs. Of course, the nutrients could also support algae that the slugs don’t like, so we should be able to learn something either way.
Once we got into the water, we could see that the bottom was different from that around the station. Below the bird cliffs, there were heavy growths of brown algae, mostly Padina and Sargassum. The presence of these species did not automatically rule out Elysia, but the possibility of finding slugs 4 cm long in a foot or more of Padina was pretty remote.
The tide also happened to be very low, so the best slug habitat may have been above or near the water line. However, exploration of the shallows did not turn up anything in the way of Codium or slugs
Farther south, things opened up a bit, and there was more bare rock among the brown algae.
There was even a little Codium. No Elysia visible, though.
The snorkel itself was awesome. Lots of different species of fish, often quite large. We were even visited by a school of Jacks, zooming by for a quick look.
After taking a few more photos for documentation, it was time to move on to the next site, Gemelito Este. As can be seen in the photo below, there is plenty of guano on the island, which suggests a lot of nutrient input.
The bottom seemed more conducive to Elysia, however. Plenty of Padina, but also significant patches of coralline and green algae.
We even found some snail or slug eggs. Not from Elysia, but a good sign that molluscs were about.
No Elysia at Gemelito Este, either. Undeterred, we continued southward to an inlet on the mainland, just north of El Rincon. The bottom looked very promising, with lots of coralline, green algae, and Codium.
Toward the end, we were hunting among the clumps of Codium and other algae, and Nancy kicked up a little Elysia. Another data point supporting our ideas about appropriate slug habitat.
Perhaps as a reward for the students’ hard work, a couple of whale sharks swam by the boat. Ricardo maneuvered the boat perfectly, to allow the students to have a quick swim with one of the sharks. Very much a high point for all.
The following day was another field trip, which included another period for snorkeling. This time, it was a small island outside the bay, Isla Pescador. No harm in looking around, right?
Unfortunately, the site is a bit more exposed to wave action, and the surge on this day made it difficult to do too much slug hunting. The bottom looked promising, though.
The following day, the Spatial Subsidy group had their field trip. They snorkeled at a different site, and brought back 11 (yes, eleven) more Elysia for us. It seemed like things were really getting started.
At this point, we had a lab set up, some algae had been collected, but no slugs were to be found. Definitely need slugs. Because Berstch had done almost all of his sampling at Punta la Gringa, at the north end of the bay, it seemed like a good idea to have a quick look up there. I had never been there before, so I asked Drew to drive me up there during an afternoon lull in the action.
It was a beautiful spot, with sand and smooth stones leading to the water, so it seemed worth bringing the students there on their next field research day.
But first, it was time to do some molecular biology. Despite the absence of Elysia, we had plenty of algae. In order to know which plants the slugs are eating, we need to get DNA sequences from potential food plants, so we could make some progress by extracting DNA from the algae. It also gives the students their first shot at working with real DNA.
The most likely food plants are Codium (dead man’s fingers), Ulva (sea lettuce) and Bryopsis (feather algae). We have not found Bryopsis, but had plenty of the other two, so we set about grinding the plants up and separating the DNA from the rest of the stuff in the plant.
Considering the tight space, the students worked well together. It is not easy to pipet stuff from one tube to the next, then wait for an incubation or for the centrifuge to run, then do more pipetting, and so on without going crazy from the heat. Nonetheless they got the procedure finished in time for a trip to La Gringa before lunch. Although we did not find any slugs, it was a very nice dive.
During the next lab session, we took the extracted DNA and amplified it using PCR to make many, many more copies of our sequence of interest. As before, we used primers specific for the rbcL gene, which is found in chloroplasts but not the nuclei of plants or animals. We also included some controls to make sure the procedure worked. First, we amplified DNA that had been extracted by Haseeb and Maryam at USG, and which we know has worked in the past. When we ran the DNA on an agarose gel, to separate the DNA pieces by size, we also added DNA that had been amplified at USG, to be sure the apparatus was working and the dye showed the DNA.
The procedure worked, at least for Codium. There was a visible band for Codium, as well as for the positive controls, so everything seemed to be working. The lack of signal for Ulva could indicate that something went wrong with the extraction, or that the sample did not amplify. Also, for some reason, the molecular weight markers did not show up at the left end of the gel. Nonetheless, the result was very encouraging.
The weekend was upon us, which meant a break for the students from research, and an opportunity for the scientists to get ready for the next week. Lots of details to deal with, getting protocols finalized, reagents tracked down, and field survey plans finalzied.
That Saturday, we went on a scorpion hunt, led as usual,by Drew. Normally, the students start getting disappointed during the early part of the hike, because the scorpions wait a while before coming out. This year, they were plentiful and out early. Using flashlights with UV LEDs made them easy to see, because, for some as yet unknown reason, they fluoresce green under UV light.
Meantime, we still had exactly zero slugs. I was beginning to feel a bit like Ahab in the obsessive pursuit of my little green nemeses. So, on a beautiful Sunday morning, I decided to do yet another snorkel through the shallows to hunt through the algae. The tide was especially low, so I started by just walking through the shallows, looking for slugs, while the mobulas jumped a short distance away.
The snorkel itself was quite wonderful, slowly swimming back and forth from the front of the staff house to the south end of the Vermillion Sea field station, which had been used by the group some years ago. As I swam slowly over the shallow bottom, I saw lots of algae, starfish, stingrays, corals, and many species of fishes. I even found one cute little nudibranch. I was however, beginning to despair of finding Elysia.
I also had to keep a close eye on the catch bag, because a small crowd of hungry puffers was following along, hoping to grab anything I might stir up.
After about 90 minutes, it was time to move on to other tasks. We needed more algae-covered rocks for the station, so I put the mesh bag containing the little nudibranch into a bucket on the shore and proceeded to hunt around in the shallows for suitably-sized rocks with interesting algae. When I looked at one patch of Codium, I saw what looked like some blue color among the uniform deep green. Could it be? A quick sweep of the hand sent a little Elysia flying through the water column.
I grabbed it, and gently held it while swimming toward the shore. As I got out of the water, I looked in my hand, and it was gone! I almost sobbed through my snorkel. However, after many years as a research scientist, I am thoroughly accustomed to harsh disappointment, and went about my business collecting more rocks. Fortunately for me, and for the project, there were three more of the little gals in separate clumps of Codium, and I was ready with the catch bag this time. As can be seen in the photo below from a later hunt, the presence of Elysia is not always obvious.
The drought had ended! The captive Elysia adapted quickly to their new home.
Not bad. We had the molecular biology working adequately, and we had slugs. As often seems to be the case, finding one opens the door to finding more.
There was a lot more to do, though. It was time to get serious about the slugs’ kleptoplast DNA, their responses to light, and their distribution in the bay.
Has it really been that long? Well, I’ve been busy.
The preparations of the last few months have now been tested in the field. I am returning from the first two weeks of a five week field study in Bahia de los Angeles in Baja California. As I think I posted earlier (apologies if I did not), the plan for the summer was to work with Ocean Discovery Institute on fleshing out some of the details of the life history and behavior of Elysia diomedea in Bahia de los Angeles. Specifically, we want to know more about where they are found in the bay, what they eat, and how they respond to light. This information will help us to understand more about the role of kleptoplasty, along with the significance of the dramatic population fluctuations of the slugs documented previously by Hans Bertsch.
Even before we left, there were several challenges. For example, for the project to get off the ground we needed permission from CONANP, the agency that oversees the marine reserve, to collect Elysia from the bay. Months before the project began, we submitted an application for a permit to allow us to collect and study Elysia. Unfortunately, that permit was rejected because we had not been clear about the relationship between the Elysia project and ongoing research in the bay islands. Naturally, this caused us significant anxiety. After a lot of work behind the scenes by folks at Ocean Discovery and by my very good friend Drew Talley at USD, our intentions were made clear, and we were given permission to go ahead with the project.
Among the items that were absolutely necessary were aquaria for holding, observing and testing the slugs’ response to light. In April, I had started working with a local company that builds custom aquaria to build two holding/observation tanks and two “I-mazes” for testing light preference. By early June, the tanks had not materialized, and I was beginning to get nervous that missed deadlines and excuses would continue until time ran out. I decided to go with a more experienced vendor, Glass Cages, and they got the tanks into production and had them air freighted to San Diego in plenty of time. The whole process of working with them was pleasant and reassuring.
The other critical pieces for extracting and amplifying DNA are a microcentrifuge and a PCR thermal cycler. Again, thanks to the persistence of Drew Talley, we borrowed an Eppendorf centrifuge from USD, and received the loan of a demonstration model thermal cycler from Thermo Fisher Scientific.
So, after months of planning, spending a semester making sure that the methods work, arranging for care of the system in Maryland while I was gone, and enjoying a roller coaster ride obtaining permissions and equipment, it was time to get into the field.
The trip down was slightly adventurous. We traveled with the Ocean Discovery students, starting out at Hoover High School in City Heights. This year, we took the eastern route, through Mexicali and San Felipe. It was a little longer, but somewhat more scenic, and had a whole lot less traffic. The road was not great in spots, and one of the vans developed a flat tire along the way. After a little delay, we were back on our way, and arrived in the early evening.
As always, I was very happy to be there. Its hard to think of a place that I would rather be. The sea is beautiful and full of life, and the surrounding desert is spectacular in its own right. Over the course of the two weeks at the station, I tried my best to savor the views, sounds and smells.
Naturally, I was eager to get started. It was all I could do to sit still during review of procedures around the field station, because I was eager to collect slugs in order to be ready for the students’ upcoming projects. Finally, I got into the water, and began to hunt for Elysia. It was wonderful to be in the bay again, and there was lots to see. The familiar zones of Padina, Codium, Ulva, and the many other algae on the rocks outside the station reminded me of where I thought I should look. After about 1 ½ hours of unsuccessful searching, I headed back to the station to get ready for the rest of my day.
I got to meet my crew in person for the first time. The five young women were full of energy, and ready to get going with the project. The goals of the “Photobiology” group (I needed a somewhat official sounding name, sue me) will be to flesh out some basic biology of E. diomedea here in the bay. As we did for E. clarki in Maryland, we want to extract DNA from E. diomedea, and compare the sequence of rbcL in kleptoplasts with those from potential food plants. Also, we will be looking at light preferences, using “I-mazes,” which give the slugs a chance to select their favorite light intensity. We are also hoping to have a chance to explore the bay, surveying for appropriate habitat and the presence of slugs. Lots to do to get set up and get the students trained.
The hunt for Elysia continued during the morning of our first full research day. In the past, the morning hours have been the most productive in terms of slug hunting, so I had planned several mornings during the first week for collection. The crew was becoming very proficient in the water, and we hunted for about 90 minutes in the shallows in front of the station. Sadly, despite our efforts, no Elysia were to be found. After a quick cleanup, we headed for the classroom for a briefing on algal diversity, lab equipment and safety before lunch. The students had other activities in the afternoon, which gave me the opportunity to continue setting up tanks and equipment.
Our “molecular lab” is located in the garage, along with equipment for other Directed Research groups. We share the space with a group studying ways of reducing bycatch of unwanted fish species and turtles, and another group that documents the flow of energy between the rich waters of the sea and the relatively barren land of the bay islands. The space is a hive of activity at 7 am, when the other groups are rushing to get on boats. After that the space is essentially ours until lunchtime.
The other part of our “lab” consists of the observation tanks. These are in another part of the station containing the kitchen and computer lab. The 16” cube tanks sit on a sturdy table, with circulation provided by air pumps, and lighting provided by morning sunlight supplemented by desk lamps with full-spectrum LED bulbs. Once the slugs are in, the tanks hold slugs for DNA extraction and behavioral assays, with one being used solely for observation of the daily rhythms of undisturbed slugs.
Although we had not found any Elysia, at least a dozen small Aplysia rode into the tank with the plants. This will actually be handy for comparison with the responses of Elysia to light. Aplysia do not store chloroplasts, and might be expected to be repelled or indifferent to light.
Day 2 was reserved for a field trip for the students. It is supposed to be a non-work day, used to introduce the students to some aspect of the bay. It started off great, with a visit to a sea lion colony, and up close encounters with very large fin whales.
We did squeeze in a little work, because part of the trip involved time for snorkeling at Coronadito island, at the far north end of the bay, and we were not explicitly banned from looking for Elysia. Although we did not find any, it was useful to note and photograph the nature of the bottom, and the dominant algae species that were present. Lots of Sargassum, some turfy coralline algae, but not a lot of large green algae.
The days continued, with more briefings about identification of algae and molecular biology methods. The big question was whether we would actually find any Elysia. Fieldwork always requires some improvisation, but it’s a real challenge to improvise your way around the absence of your research subject. Stay tuned.