Monthly Archives: July 2018
Bahia Adventures. Part 3: The Late Middle
Elysia diomedea in field station tank. 6/30/18
Having the permits meant that we could get straight to work. The first order of business was to get the tanks ready and find some slugs. As a happy coincidence, the tides were very low, allowing us to explore the tide pools for interesting organisms. Even better, we could collect rocks and plants for the slugs tanks by simply picking them up and putting them into a bucket, rather than having to dive down to get them. We quickly had the tanks ready for sluggy inhabitants.
Tanks ready for slugs. Each contains a small collection of rocks and algae. The tank on the right is illuminated by a high-output LED fixture, while the one on the left is shaded by black felt, resulting in a 100-fold difference in intensity. 6/30/18.
The tanks were also ready for experiments. One of the hypotheses we wanted to test was that photosynthesis by the slugs’ kleptoplasts contributes to the presence of bad tasting compounds in their mucus and/or tissues. To test this hypothesis, half of the slugs would be kept in the dark for a week, while the others would be live under lighting adequate for photosynthesis. For the experiment, one tank would be lit by strong LED lighting, with photosynthetically active radiation (PAR) above 100 µmol photons per square meter per second (plenty for photosynthesis), while the other received less than 1% of that amount. The tanks were connected to the same life support system and had similar amounts of algae and rocks, so the conditions in the two tanks were nearly identical.
The plants and rocks were an excellent start, but our luck was even better. We managed to find seven relatively large Elysia in the tide pools just in front of the station. Even though I have not found Elysia in the same locations during hundreds of hours of snorkeling, they were present in abundance during low tide. It looked as thought the forces controlling the bay were smiling upon us after thumbing their noses at us for a week.
The slugs settled in well, exploring their new habitat and lounging on the algae.
Elysia diomedea in tank at BLA station. 6/30/18.
Elysia diomedea exploring rocks in new home. 6/30/18.
There was even reproductive activity. As can be seen below, slugs appeared to court each other.
Elysia diomedea courting in a tank at the BLA station. 7/2/18.
They deposited multiple egg masses. The masses contained thousands of tiny white eggs without extra-embryonic yolk, which is consistent with what others have observed for the species (see e.g., the Sea Slug Forum) .
Egg mass of Elysia diomedea. 7/4/18.
Egg mass of Elysia diomedea in BLA station tank. 7/3/18.
Although, I did not have a camera with sufficient resolution to show the details of the masses, I would agree with others that E. diomedea embryos are smaller than those of E. clarki and E. crispata. The relatively small size of the embryos, and resulting smaller amount of yolk, means that E. diomedea are probably “planktotrophic,” hatching earlier and feeding on plankton in order to finish development. Larger embryos, such as those of E. clarki and E. crispata, are “lecithotrophic” living on plentiful yolk stores until it is time for the veligers to settle and start feeding on Bryopsis.
Having slugs and algae also gave us the chance to do some DNA extraction. The students took small samples from a couple of slugs, and some local algae, then extracted the DNA using the DNeasy Plant kit from Qiagen.
Codium specimen. A small sample was removed for DNA extraction and amplification. 7/3/18.
Unidentified green alga. DNA was sampled for identification. 7/3/18.
After a few rounds of incubations and separations, the students had generated tubes of clear liquid that presumably held DNA.
Elizabeth, Maria, Keyla, and Lily holding tubes of DNA extracts. 7/3/18.
Meantime, there was lots of other stuff going on. Ric had been leading the other half of the group in troubleshooting and starting the feeding experiments. Because we wanted to test the palatability of the tissues from slugs kept in the dark, we needed to modify the feeding assay we used in tanks at USG for use with fish in the bay. It seemed simple enough to hang food cubes on fishing line that is anchored by lead weights at one end and held in the water column by a float at the other. Figuring out the right thickness of fishing line, and how best to secure the lines to the weights and floats, required a good bit of trial and error.
The students were also working on their scientific presentation skills. In one exercise, Ric had them give short summaries of the work while standing in the bay, in order to have them project their voices in a noisy, distracting environment.
Students giving talks while standing in the bay.
7/2/18.
Unfortunately, two weeks had passed, which meant that it was time for me to leave the students, the great people from Ocean Discovery, my friend Drew, the bay, and the slugs, and return to Maryland. The students were doing great, the assays had started to work (although the first round of PCR amplification of the DNA extracts was not successful), Ric had everything well in hand, and there would be a real molecular biologist arriving in a few days to act as my substitute.
I watched my last sunrise at Bahia for the season.
Sunrise over the bay.
I grabbed some of the DNA, packed my things, said my goodbyes, and headed north to San Diego.
Slug Life! The Photobiology group on my last morning. From left: Keyla, Bennie, Zaira, Me, Maria, Ric, Lily, Diana, Melanie, Elizabeth. 7/6/18.
At this point, we were about halfway through the field season. After all the hard work and adaptive management, will there be results?
Bahia Adventures 2018. Part 2: the Middle
It was nerve-wracking not to have the permit. Without permission, we could gather neither algae nor slugs, so experiments were at a standstill. Fortunately, we could get started on a few things, like surveying different sites for slugs, and optimizing conditions for the DNA experiments.
Snorkeling in front of the station allowed us an extended period to observe the animals and their habitat. It was also fun, and gave the students lots of practice at slug hunting. It was a good year for Elysia at the station, and we saw plenty . All the more frustrating that we could not collect them.
Elysia diomedea in front of field station. 6/26/18
Finding them is never trivial, however. Below is a medium-small slug with a finger for scale. It would be a lot easier if they got as big as E. clarki, but I have never seen them larger than 5 cm or so.
Elysia diomedea on human finger. BLA field station 6/23/18.
There was a lot of Codium, and multiple other species of green algae that could potentially interest the slugs. I even found a small patch of Bryopsis, which is reputed to be a favorite of captive E. diomedea.
Bryopsis species, probably B. pennata. In front of BLA field station 6/26/18.
We also had the chance to play with the new PCR primers I designed for use in NextGen sequencing. I had not had much of a chance to test them before I left Maryland, so we took some time in the morning to set up a reaction using DNA I had brought for use as a positive control.
Results of playing with parameters for PCR amplification, using DNA from the alga Avrainvillea nigricans extracted in Maryland. First lane is ladder, which failed for some reason. The rest of the lanes contain DNA amplified using varying amounts of algal DNA and primers. 6/29/18.
The results were encouraging, but imperfect. The PCR products had two bands, instead of one, suggesting the conditions needed to be modified to amplify only the rbcL gene.
The following day, we set off for a slug survey at Playa La Gringa, a beach area near the north end of the bay. It is a beautiful spot for a snorkel, and has a lot of potential for slug hunting. Once there, we suited up and got ready.
The Photobiology Research Group at Playa la Gringa. From left: Maria, Bennie, Lily, DIana, Elizabeth, Zaira, Keyla, Melanie. 6/29/18
They were soon in the water, exploring and enjoying. This part of the beach receives cold water directly from the Gulf, so the students were happy to have their wetsuits.
Students in the water at La Gringa. 6/29/18.
The rocky bottom looked promising, with a wide range of mixed algae species.
Many species of green, red, and brown algae on rocks at Playa La Gringa. 6/29/18.
Detail of rocky bottom, showing green, red, and brown algae, along with small gorgonian corals. 6/29/18
There were plenty of small treasures, such as urchins, sponges, and hydroids, along with a profusion of fishes.
Hydroids among the algae. Playa La Gringa. 6/29/18.
Codium was also plentiful, in at least two growth forms (or species, not sure), which might indicate the presence of Elysia.
Large growths of Codium at Playa La Gringa. 6/29/18.
Codium with sparser, longer branches. Playa La Gringa, 6/29/18.
Toward the south end of the beach, the rocks were relatively bare, with very little green algae visible. The brown alga, Padina, was still abundant.
Area to the southeast with heavy growth of Padina, but no visible green algae. 6/29/18.
Despite intensive searching by many eyes, we did not find any Elysia anywhere along the beach. This does not mean that the slugs are not there, but they certainly did not make their presence known.
At this point, Ric and I had been improvising, or exercising “adaptive management,” for many days, and were running out of tricks. I had requested that the crew in Maryland send some more DNA samples, but they would not arrive until the following week. Although despair is way too strong a word, there was a profound sense of “now what?”
It was at that point that the intensive lobbying by Ocean Discovery paid off, and the permit finally came through. The group could collect slugs and algae, and start work on the planned experiments.
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