Slugs of Bonaire 2019: Why So Blue?
Just back from a week diving in Bonaire. The focus was more on recreation than slugs, but we came across a few Elysia crispata along the way. As usual, we found the highest numbers in the more degraded sites to the north, but the slugs were always on long-dead coral regardless of where we found them.
For this post, I wanted to highlight the intense blue color of the slugs we found this year at Karpata, a site near the north end of the island. I have previously posted photos of blue slugs that have turned up at nearby sites, and a quick Google search will provide many more examples.
Elysia crispata at Karpata, Bonaire, 1/17/19. Note the patch of Dictyota algae to the lower right.
This seemed to be a common color pattern. Each photo in this post shows a different individual, but their colors are remarkably similar.
Even though the colors look downright artificial, I did little more than adjust the contrast of the photos. These slugs really look like this. I emphasize this point, because the range of colors of most species of Elysia is limited to shades of green.
Despite the striking appearance, the color pattern may provide camouflage. When the slugs were curled up and their rhinophores hidden, they did passable impressions of sponges. E. crispata are almost invariably scrunched up when we come across them on the reef, so this may be a successful way of hiding in plain sight.
The color of many Elysia species is derived from pigments taken from their food plants. For example, my E. crispata and E. clarki hatchlings have little color until they start feeding, and then take on the green color of the chloroplasts they sequester in their digestive diverticula. Costa et al, (2012), provide a nice example of this effect. They showed that E. timida could be either green or brown, depending on whether they were fed Acetabularia algae that had taken on different colors based on having been acclimated to low light (green) or high light (brown).
So what could be making these slugs so blue?
One possibility is that they are feeding on Dictyota, a brown alga that grows abundantly on the dead coral in this area. Although the Dictyota in the photo at the top of this post is not impressively blue, the alga is certainly capable of producing intensely blue color. Most species of Elysia feed on green, rather than brown algae, but it is not unheard of for them to branch out (e.g., Trowbridge et al., 2010). It is also possible that the slugs are consuming one of their more usual food plants that happen to be producing high levels of blue pigments. It would be interesting to take a small tissue sample and find out what the slugs have been eating.
It also brings up an interesting question regarding the coloration of Elysia in general. In addition to their green background, many species have distinctive patterns, such as the colorful markings on the parapodia and rhinophores of E. diomedea, below, or the colorful edges of the parapodia in E. clarki.
At a higher magnification, one can see concentrations of pigment spots, such as those shown below in the parapodium of E. clarki. Are these spots of concentrated pigment derived from their food plant, or are they synthesized by the slugs themselves?
As far as I can tell, there is no answer in the literature, but who knows what will turn up next.
Map of Elysia diomedea at Bahia de los Angeles
I have added a rudimentary map of the locations at which E. diomedea has been found during our fieldwork during the past few summers. At the moment, it provides a framework to which we can add more sightings as we turn up more slugs.
The short summary is that suitable habitat, consisting of rocky bottom with growths of Codium macroalgae, is distributed throughout the bay, and that the ability to find Elysia can vary wildly at the same site from one day to the next. With enough time and effort, I expect the little slugs would be found anywhere there is something to eat.
Back to Baby Food
Do they prefer the food of their infancy because it tastes better, is more nutrient-rich, or is easier to eat? Maybe they are just nostalgic for the food they ate when they were young.
Two-month-old Elysia clarki having an early morning crawl among the turtle grass and manatee grass in Box of Slugs 2. 9/29/18
A few of the youngsters from the most recent brood have moved in with their mom in the tank at home. They were weaned from Bryopsis plumosa to B. pennata once they were a few weeks old, and have been growing steadily.
For a few reasons, not all logically sound, I had assumed B. plumosa would be harder to culture. If an aquarist has a problem with Bryopsis, it is invariably B. pennata. Combined with the fact that B. plumosa is crucial for hatchling survival, and that I had to travel all the way to Tampa to get it, I figured I would have trouble keeping it going. As a consequence, I always shift young Elysia to B. pennata when they were ready to eat it.
Despite my preconceptions, B. plumosa is thriving at this point.
Bryopsis plumosa culture tank (surface is 10″ by 20″), stuffed to the top with algae. 9/14/18.
To the uninitiated, the B. plumosa tank would look like a mat of unruly glop. To an aficionado such as myself, it looks like an actively growing, unruly mat of precious food for hatchling Elysia. It is a “half-ten” aquarium: a ten-gallon tank, but only half the height (OK, so it is really only a 5-gallon tank), which provides a lot of surface but only a few inches of depth. The growth form is very different from B. pennata, which tends to be long and feathery. B. plumosa grows more like clumps of moss. I am concerned about the tank being taken over by B. pennata invading from elsewhere in the system, but so far so good on that front.
At the moment, the growing conditions are:
- Nutrient dosing at about 150:30:1 Carbon:Nitrogen:Phosphorus, plus Guillard’s F/2 at 2 ml/day (note this is the whole system, not just this tank).
- Circulation using a powerhead and rotating output (see Slug Safe Circulation). Trust me, it is in there somewhere.
- Lighting by an Evergrow S2 hydroponics light, about 6″ from surface.
To get to the point of this post, I had enough B. plumosa to throw some to the adults.
Elysia clarki eating Bryopsis plumosa in Box of Slugs 2. 9/26/18.
Unsurprisingly, the slugs ate it. I did not expect, however, that the largest female would rarely leave the clump of algae until it was completely consumed. She very much preferred the plumosa. I brought another clump home, and she is still sitting on it, along with one of her kids. The tank is full of B. pennata, at all levels, but the slugs stick right to the single clump of B. plumosa on the surface. It may be my imagination, but the big one seems larger and more colorful after a few weeks of eating B. plumosa.
Box of Slugs 2, with plenty of Bryopsis pennata among the turtle grass. 9/26/18
So, anecdotally, even grown up slugs prefer B. plumosa. Another thing to put on the list of things to test more rigorously. For now, one can speculate about why they seem to prefer it, and what cues (smell? texture?) draw the slugs to the algae.
Fat Babies Have No Pride
In the meantime, another brood has hatched and has started to grow. I am not sure why (I am not in any way a musical person), but when a new brood starts to eat, Lyle Lovett’s “Fat Babies” runs through my mind almost continuously. I have no idea whether the song has a subtle, subversive message (if so, I apologize for any offense), or whether it is simply about chubby infants not being proud.
Juvenile slug crawling among B. plumosa, with diverticula full of chloroplasts. 9/28/18
They are feeding and growing, and it looks like we’ll have several dozen ready for activities in the spring.
That’s OK. Who needs pride?
Bahia Adventures Part 5: Slugs Taste Bad (Again)
One of the goals of the Bahia field season was to look a little deeper at the interaction between kleptoplasty and chemical defense. The general idea went something like this:
- Kleptoplasts continue to photosynthesize after they take up residence in the slugs’ tissues.
- Slugs produce defensive chemicals that make them taste bad.
- Kleptoplasts could be the source of the defensive chemicals: is photosynthesis required to make them?
Based on this line of thinking, we hypothesized that slugs deprived of light should be less distasteful than those kept in bright light.
To put the hypothesis to the test, we set up two tanks that were nearly identical except for lighting. They were plumbed into the same sump and chiller, so their temperatures and chemistry were essentially identical, but one was illuminated by a high-intensity LED fixture to support photosynthesis (PAR ≥100 mol m−2s−1), while the other was shaded to reduce the light by 100-fold (PAR < 1 mol m−2).
Tanks ready for slugs. Each contains a nearly identical 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.
Once we obtained the permit and were allowed to collect, we randomly separated slugs into two tanks that contained roughly equal amounts of Codium on which they could feed. They were allowed to feed at will, because the goal was to test the role of photosynthesis in generating defensive chemicals, not the effect of starvation.
The original plan was to use mucus from experimental (unlit) and control (lit) slugs to make food cubes, then test which ones were eaten by fishes in the bay. However, based on experiments at USG, and the fact that we would not be able to extract enough mucus from our few dozen slugs, we decided to test the effects of tissue extracts from whole slugs. Surprisingly, the students were not as sad as we might have expected that they had to purée their pets.
For the description below, whenever I write “we,” I really mean Ric and the students, because he was very much in charge of this project. Most of the below photos are his.
Zaira weighing carrageenan for a batch of food cubes. 7/3/18.
The process was as quirky as any of the other experiments we have done in Bahia. To make the gel base of the food cubes, we needed to dissolve carrageenan in water, which required heating the water to boiling. The easiest method is to microwave the water, but the only available microwave oven had a single working button (“popcorn”), which turned the microwave on (after a disconcerting delay) for 3.5 minutes. It took some practice and finesse to turn the machine off at the right time, but they managed.
Zaira stirring food and carrageenan mix. Dodgy microwave oven is at right, and some of the molecular crew (Lili, Dave, Elizabeth) are in the background. 7/3/18.
Fish food pellets were ground and added to the carrageenan solution to make the base food cubes. Mucus or tissue were then added to a portion of the food, depending on the experiment.
Food mixed and ready to pour. 7/3/18.
Once heated and thoroughly mixed, food was poured into silicone ice cube trays to make 1 cm cubes. Silicone O-rings, used to secure the food cubes to clips during experiments, were placed into the molds and held steady with acrylic rods.
Bennie setting up rings in food forms, getting ready to pour how food mix. 7/3/18.
Once the O-rings were in place, food was poured into the molds and evened out using a knife. After cooling for a bit, the whole assembly was stuffed into a ziploc bag and refrigerated until needed.
Food cubes poured and ready for use. Note acrylic rods to keep rings in place. 7/3/18.
At experiment time, food cubes were attached to monofilament lines to be anchored in the bay. The bottom end of the line was tied to small lead weights, while the top was held afloat by a 16-20 oz soda bottle. Food cubes were secured by O-rings to plastic safety pins tied to the line at regular intervals. Several bugs needed to be worked out. For example, we started with lightweight fishing line, but it had a tendency to break in the surge of the intertidal zone, causing loss of the whole bait line. We shifted to a much heavier gauge, which apparently scared the fish away, because all food cubes were present after 24 hours. Ultimately, we settled on something in between, and could start gathering real data.
Keyla, demonstrating system for placing food cubes in the bay. Plastic safety pins are tied to monofilament fishing line, which is suspended between the float in her right hand and the weights by her left foot. Food cubes are then clipped to safety pins via embedded O-rings. 7/13/18.
Once food cubes were secure, the lines were placed deep enough to keep them afloat at low tide.
Food cubes suspended in water column in bay. 7/13/18.
Lines were collected after a predetermined time (see below).
Collecting line of now-empty clips. 7/13/18.
For the first round of experiments, cubes were left in the bay for 24 hours. During this time, essentially all of the control cubes (i.e., without tissue or mucus) were eaten, as were most of the experimental cubes. Nonetheless, fish appeared to eat fewer cubes containing tissue from slugs kept in the light, compared to those kept in the dark.
Effect of food contents on consumption by fish in the bay. When collected, each line was given a score, with 1 = all cubes intact, and 3 = all cubes gone. In this experiment, all control cubes were missing (and presumed consumed) in 24 hours, as were cubes made from tissue prepared from slugs kept in the dark for one week (right most bar). Food made from slugs kept in light (second from right) had a lower score than the other groups, but the difference was not significant. Sample sizes: 18 control, 12 mucus, 12 lit, 12 dark.
Because there was concern that missing cubes may have fallen off rather than being eaten, they also tried leaving the cubes in the bay for only one hour. There was a slight decrease in consumption, but the overall result was the same: tissue from dimly-lit slugs was eaten more often than that from well-lit slugs. The students were running out of time for these experiments, so the results are preliminary and nothing is statistically significant.
Results of leaving the food lines in the bay for an hour. Food made from slugs kept in the light (middle bar) had a lower consumption score than those without slug tissue (left bar) or made from slugs kept in dim light (right bar). Sample sizes: 18 control, 24 lit, 24 dark.
To summarize several weeks of hard work, it looks like there is something to pursue. The sample sizes are small, but there is a consistent effect of slug tissue in reducing consumption by fishes, and this effect is reduced or eliminated by keeping slugs in the dark. Now that the bugs have been worked out, it will certainly be worth trying the experiment again if there is an opportunity to spend a few weeks making food and setting lines.
Thanks again to the USG Slug Club, who pioneered the foodmaking methods, and took care of the slug system while I was away.
Happy Fourth Birthday!
Baby Elysia, about three weeks old. Rhinophores well developed, diverticula full of chloroplasts from Bryopsis plumosa. 8/17/18
It seems appropriate that the site has reached an age at which kids are always asking “why, why, why?”
Looking back, the site is absolutely a reflection of my working style: make a rough plan, give it a shot, modify, repeat. Not the work of a perfectionist, and there are some posts and pages that make me cringe when I look back. Nonetheless, as a repository of pictures, information, and ideas, and a journal of my meandering process of discovery, the Solar Sea Slug Blog has exceeded expectations.
The past twelve months have been action-packed:
- The student Slug Club at USG explored chemical ecology. We spent fall semester doing a deep dive into the literature on Sacoglossan chemical ecology, learning a lot about the chemicals the slugs could be taking from food plants. In spring semester, we took the ideas into the lab by testing whether mucus and tissue from E. clarki could keep shrimp from eating food cubes.
- The experiments could not have been done without the availability of many slugs of different sizes. This leads into what was probably the most important breakthrough of the past year: rearing slugs from egg to adult. Over the years, the slugs have laid thousands of eggs, most of which hatched and then starved. This year, I discovered that the hatchlings will readily eat Bryopsis plumosa, and have ended up overwhelmed by babies. Knowing how to rear the hatchlings, and having a culture of B. plumosa on hand, is a huge leap forward. Now I can raise them as needed, at least in principle.
- The breakthrough in rearing of Elysia would not have happened with input from Skip Pierce and Mike Middlebrooks in Tampa. I had a great visit down there, and learned a ton about several species and the people who work on them. As a bonus, I brought back my first culture of B. plumosa to get the baby slugs started.
- We rediscovered a classic paper on Elysia neuroanatomy, recorded the the first action potentials in Elysia neurons. There is still some work to be done to get routine, stable recordings, but this is another big step in the right direction.
- All the stuff that happened in Bahia made for a great summer for the Solar Sea Slug Blog. We set up a lab, collected slugs and algae, did surveys, tested feeding methods, and obtained some useful DNA sequences from E. diomedea and their food algae. Stay tuned for the results of the feeding tests in the bay, and a map of where E. diomedea is found in the bay.
Self-indulgent reflections are not as interesting as sea slugs, so I have included some links to cool stuff below:
- A National Geographic article about the potential decline of Elysia chlorotica, possibly the most solar of the solar sea slugs. Thanks to Ric DeSantiago for forwarding the link.
- Here’s another on theft of stinging nematocysts by cute little nudibranchs. Thanks to Drew Talley.
- Because I like seahorses, especially small ones, here is a story about newly-described pygmy seahorses in Japan. Thanks to Joanna.
Bahia Adventures Part 4: Culmination But Not The End
The only thing better than a picture of a slug is a photo of a slug projected on a giant screen and admired by a bunch of people.
The Photobiology students presenting their results at the annual Report to the Community at the San Diego Museum of Natural History. 8/9/18. Photo Drew Talley
A few weeks ago, the Photobiology students presented at the Ocean Discovery Institute Report to the Community, held at the San Diego Museum of Natural History. This is the opportunity for the students to tell their parents, the community, supporters, and everyone associated with Ocean Discovery, about what they did this year in Bahia.
They started with the background and general questions that drove the research: what is kleptoplasty, and how can we learn more about the mechanisms and evolutionary benefits of the phenomenon? Because they had limited time on stage, they focused on the work we did collecting samples of slugs and algae for identifying food plants.
The punch line was satisfying. We found that the DNA sequence of the Codium algae collected in front of the station and that from the slugs’ tissue samples came from Codium decorticatum. I managed to get about 400 bases of sequence from the slugs and the algae, and it was about a 99% match to C. decorticatum.
Short region (54 of >400 bases) of rbcL gene sequences derived in Bahia this summer to show similarities of our data (top two lines) with Codium decorticatum sequence in NCBI database. Elysia diomedea and Codium collected in Bahia were 99% identical to C. decorticatum. Closely related C. fragile only 91% identical (differences indicated by asterisks at bottom). Asterisk at top indicates difference between E. diomedea and Codium.
The figure above shows a short, representative stretch of DNA from the rbcL gene, from Elysia diomedea and the Codium species we extracted in Bahia (top two rows), along with sequences from two species of Codium from the National Center for Biotechnology Information (NCBI; bottom two rows). The Codium from Bahia matched C. decorticatum almost 100%, whereas the match was only about 91% for the very similar C. fragile.
There were, however, a few differences between the kleptoplast DNA from E. diomedea and C. decorticatum (e.g., asterisks above E. diomedea sequence). At this point, we don’t know whether this is due to variation within the species that the slugs are consuming, or the slugs are eating more than one species. We are still waiting for results from NextGen sequencing, which should be more informative and may clear up this puzzle.
A couple of notes about the algae. First, the Codium and slug sequences are identical to those we obtained in 2016. At the time, however, C. decorticatum had not been entered into the database, so all we knew was that we had a new species. The other observation is that the species of Codium we sampled (see photo of tissue here) is relatively low-growing and branchy, unlike the usual tall, sparsely branched appearance of C. decorticatum. There were specimens that looked like that (e.g., here), and it is likely that the appearance of the alga depends on the local conditions.
Most importantly, the students did a fantastic job of presenting. I was away visiting family, but I got multiple independent reports from people in the audience who talked about how strong and clear the students’ presentations were.
The Photobiology crew, as drawn by the ever-creative and wonderful Ric Desantiago. Pleas visit his site (Da Hood Scientist) to check out more of his observations and artwork.
I am very pleased and proud to have played a part in the students’ project this summer. Despite all of the up and downs and improvisation, it was a deeply satisfying experience. I am always amazed how a misanthrope such as myself can have such a great time packed like a sardine into the field station with so many people. Thanks to the students, the Ocean Discovery staff, Ric, Thiago, the visiting scientists, and everyone. Especially, thanks again to Dr Drew Talley, mi gemelo de otra madre, for making it possible.
Dopplegeezers at BLA station, 7/5/18
I still have a few posts left regarding the results of the feeding assays and slug surveys, and hope to have them ready in a few weeks’ time.
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.
Bahia Adventures 2018. Part 1
Another installment of adventure in science at Bahia de los Angeles on the Gulf of California. This year, I am once again working with students from Ocean Discovery Institute to exapand on what we have learned about feeding assays and DNA sequencing to develop more insight into the diet of Elysia diomedea, and how kleptoplast photosynthesis makes them taste bad.
Drew and I left San Diego with a truck full of stuff, including the tanks, sump and chiller for a slug setup, as well as a perfectly functioning PCR thermocycler from NIH surplus. Drove through Calexico to Mexicali, caught route 5 south through San Felipe. The road was largely good, but the unpaved section in the middle still not finished.
USD truck parked on the Mexicali side of the border. 6/20/18
We arrived in Bahia about 5:30, and stopped for the usual photo from the hills leading into town. When we arrived at the station, the setup crew was working away, although the station was mostly ready for business.
View of Bahia de Los Angeles from the hills above town. 6/20/18
We spent the rest of the evening starting to get organized. I also found out that we currently do not have a permit for collecting the slugs (all other activities have been approved), so it is not 100% clear that we will be able to do any work.
The next morning, I was eager to get started on assembling the lab space. It looked as though everything survived shipping from Maryland to San Diego and the drive to Bahia. We got some new tables, and strung some cords to safely manage electricity flow to the tanks, chiller, freezer, and other lab equipment.
Lab area ready to be set up. 6/21/18
Then we got to set up the tanks. All the plumbing I had assembled in Maryland connected nicely, but the connectors for the chiller were too small for the ¾” tubing used for the rest of the system. I could swear we double checked that. That meant we needed to head to “Home Depot,” the local construction supply lot. Fortunately, Ric, the Ocean Discovery fellow who is assisting me this summer speaks excellent Spanish, so he could easily explain what we were trying to do.
The very nice owner was pessimistic that he would have the right adaptor, but he found one. Then Ric told him we needed two, and his pessimism increased. He miraculously managed to produce another. However, he had no hose for the connection, so he gave us directions to the other, larger supply lot that we had never heard of. It was indeed larger, and they had lots of hose. We bought 3 meters, to have some extra for a siphon hose, and were on our way.
Tanks set up , plumbed and ready for water. 6/21/18
The tank was quickly assembled, and we hauled buckets of sea water to fill up the two tanks and sump. The water was brown from a dinoflagellate bloom, and looked kind of yucky. We hoped the skimmer woul clear it out. Once the tanks were full, we turned the main pump on for a test and cleaned up the splashed water so that we could look for leaks. It all seemed just right, but water kept on pooling on the floor. Sure enough, there was a crack on the bottom of the sump, presumably from shipping. After some discussion and back and forth, Ric and I drove back to Home Depot to get adhesives for a possible fix. The guy was amused that we were back, and showed us what he had. We bought the multi-purpose adhesive and some silicone sealer just to be sure.
It was time for the first snorkel. I put on my wetsuit and headed into the water in front of the staff house. The door for charging the camera opened promptly after I entered the water and that was the end of that. Oy. Nonetheless I looked at a lot of Codium, and was grunted at and bitten by many very aggressive damsels. Most importantly, I found about 4 small slugs up in front of the house, and called it a day.
Small Elysia diomedea in bay in front of station. 6/23/18
I borrowed Drew’s nearly identical camera for a snorkel on the next day, and went out with Ric in the same area. The visibility was still crummy, but I managed to find a small slug on Codium after a bit, and brought both up to show Ric. Soon enough, he was finding his own, and we found quite a few, taking some good photos. Without a permit, however, the slugs will stay in the bay for now.
Ric with yet another slug. 6/23/18
Elysia diomedea in Bahia. 6/23/18
The lab was set up, there were plentiful slugs in front of the station, and the students would be arriving that night, so it looks pretty good for a successful time in the field.
Lab just about ready to go, with slug tanks (left) extraction table (middle) and PCR table (right). 6/23/18
In Bahia, there is always some drama. In this case, it appeared in the form of permits. It turned out that all of the group’s collecting activities, which included an enormous range of fishes, crustaceans, anemones, and molluscs, were approved, with the exception of my project. For some reason, maybe something I wrote set off alarm bells, the collection of Elysia diomedea and marine algae required approval from a different agency.
Stay tuned…
Elysia Neuroanatomy: Old becomes new again!
Ah, the Good Old Days, when “curiosity-driven” science wasn’t naughty. Of course, one might not want to get too nostalgic when the woman whose work is the subject of this post is referred to as “Miss Lillian Russell” in the running head of the paper. Apparently, gender and marital status were considered relevant in a scientific publication 90 years ago.
The primary goal of the Solar Sea Slug project is to study the neurobiological specializations of photosynthetic molluscs. In order to chart the input and output pathways, we need a good roadmap of the central nervous system (CNS) and the nerves that connect it with the rest of the animal. I had hoped that someone had generated at least a crude diagram, and had found some drawings of the nervous systems of Elysia viridis (Huber, 1993, J. Moll. Stud. 59:381) and E. crispata (Gascoigne, 1972, Trans. R. Soc. Edinburgh 69:137), but had not found much more. I fully expected to map the peripheral nerves by myself, and we had even done some preliminary experiments with fluorescent labels for axons for Slug Club during spring semester.
As is often the case, the breakthrough came as I was burrowing through the references from one of the older papers. Huber’s 1993 paper reviews the structure of the CNS of a wide range of gastropod molluscs, and includes a nice diagram of the central nervous system of E. viridis, but provides no information about the periphery. I had skimmed the section describing the image, but had mostly focused on the figure itself. He made a passing reference to earlier work by Russell (1929, Proc. Zool. Soc. Lond. 14:197), so, for the sake of completeness, I ordered the paper through inter-library loan, making it clear that I would like them to include any photographic plates, which are often in a section separate from the main body of the paper.
When I downloaded the paper about a day later, I was pleasantly shocked. As part of a study of the taxonomy of the ascoglossans (= sacoglossans), Lillian Russell had performed a thorough, detailed analysis of the nervous system, nerves, and internal anatomy of E. viridis and a nudibranch, Aeolidia papillosa. She provided an excellent description of the fusion of about seven ganglia to produce the nerve ring that comprises the CNS, along with multiple excellent drawings of the CNS, the peripheral nerves and internal organs of E. viridis.
Plate 4 of Russell (1929). Detailed map of the ganglionic origins and terminations of peripheral nerves that innervate the body wall.
It is hard to know which image is my favorite, but I am very fond of the drawing below, which shows the ganglia in the head, the nerves that originate from them, and the terminations of the nerves in sensory organs such as the eyes and rhinophores.
Russell (1929) plate 1. Detail of the head, including the nervous system and internal organs. Highlights include the eyes (labeled as such), the cerebral ganglia (labeled 1 and 8a), and the rhinophorial nerve (labeled n. 1).
The drawing below, of the CNS and nerves removed from the animal, is also quite impressive, showing the individual ganglia that make up the CNS, and the origins of the many nerves that carry sensory information in, and motor commands out.
Russell (1929) plate 3. Isolated central nervous system and nerves. The ganglia that comprise the CNS can be seen clearly with the esophagus removed.
Aside from publishing a similar description of E. clarki (which probably only differs in slight details), and leaving a note in her will to let me know about it, I can’t imagine how she could have done any more to help me out. Although I had intended to do the work myself, anatomical description of this quality requires considerable patience and artistic skill. I am not very patient, nor am I a good artist. Thanks to Russell’s hard work, I am free to do my behavioral experiments and electrical recordings, and be in my happy place.
So, why is this useful? In a general sense, it gives a starting place for finding neurons and circuits that generate behavior. If you want to know where sensory information goes in the nervous system, finding the nerve that carries the axons from the relevant sense organ or part of the body is a good start. Same goes for finding the motor neurons that cause muscles in a certain part of the body to contract. As a specific example, if we are trying to find how information about light gets to the brain, we first need to find the nerves that connect to the eyes or (hypothetically) other sense organs that detect light or photosynthetic activity. Then we can record monitor impulse activity in the nerves, or from the specific neurons with axons in those nerves.
This is a nice reminder about a few things.
- Possibly most importantly, we truly are standing on the shoulders of others as we try to move science forward. I hesitate to say “on the shoulders of giants,” because, for all I know, Lillian Russell was of average height.
- You really never know when something you did will be useful to another scientist. Ninety years ago (90!), nobody knew about kleptoplasty, and the study was carried out because nervous system structure was considered to be a good marker for evolutionary relationships. It is not reasonable to argue that every experiment that can be imagined must be performed, but during this time in which research needs to be either “translational” or “transformative” in order to be funded, it would be nice to make a tiny bit of room for work that is simply of high quality.
- It may be necessary to dig deep into the literature to avoid reinventing the wheel. Many of us are guilty of looking over the figures of a paper and skimming the text, rather than reading every word. Time is short and papers can be long. Nonetheless, some of the most relevant information to people in the field is in the body of the text, rather than in figures or tables. Who knows what else I will find as I work my way through the old literature.
The Solar Sea Slug project has progressed by leaps during the past half year or so. In addition to finding the roadmap described above, and establishing a self-sustaining colony of E. clarki, some of the students in the Neurobiology Lab course helped me to work out procedures for recording from neurons in the CNS. There are still a few improvements to be made, such as more easily getting the electrode through the sheath that surrounds the ganglia, but we have a little real data. Below is an action potential that was stimulated by injecting 1 nA of current into a large neuron in the abdominal ganglion (Pierce’s “Parker Cell?”).
Action potential recorded from a large neuron in abdominal ganglion of Elysia clarki. 4/19/18
How are we doing?
Self-sustaining culture:
Neuroanatomical roadmap:
Dissection techniques:
First intracellular recording:
This project is really starting to take off.
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