Posts in Category: Education

Goodbye for Now

Elysia diomedea in front of Casa Caguama field station June 29, 2022.

This will probably be the last post for a while.  For starters, I will be retiring from teaching at the end of spring semester 2023.  Also, I think I have taken the project as far as I would like (but see below), and I have scurried back to the comfortable world of arthropod physiology.  I am so glad I got to know the many facets of Elysia, its behavior and biology, and it has been a fantastic opportunity to teach students about unusual organisms and the scientific process.

One of the last members of the brood hatched and raised last year.

It seems to be a good time to collect my thoughts and observations from the first seven or so years of the project.  Although I believe the field would benefit greatly from an up-to-date formal review, that is not my intention here.

Kleptoplasty is not Monolithic

When I started the project, I carried some misconceptions about the biology of Elysia.  The portrayal in the popular press at the time, and to some extent in the scientific literature, was that they were “crawling leaves” that had stolen the chloroplasts from their food plants and could therefore live indefinitely (or at least a very long time) with only light as an energy source.  My goal, in addition to using it as a teaching tool, was to understand the impact of kleptoplasty on the behavior and neurobiology of the slugs.

When I dug deeper, I discovered a lot of diversity, both between species and within a species based on which plant is the source of the chloroplasts.  For example, species can be divided into non-kleptoplastic, which digest all cellular contents immediately, short term kleptoplastic, which hold onto the chloroplasts, and long-term, which maintain functional chloroplasts for weeks or months (Christa et al., 2014).  However, even those species labeled as “long-term” vary widely in how long the chloroplasts are maintained, and this can further depend on the species of origin of the chloroplasts.

The only species that may be a true “crawling leaf” would be E. chlorotica.  Once they have obtained chloroplasts from the alga Vaucheria, they can support them for at least nine months (Green et al., 2000).  One could build a nice story that the function of kleptoplasty is to provide an energy source for this species.  Unfortunately, the story falls apart if one looks at other “long-term” kleptoplastic species, such as E. crispata or E. viridis.  Based on my own observations and those of others, these species will starve to death relatively quickly if not fed, despite the presence of actively photosynthesizing kleptoplasts.

Which brings us to the big question: Why do the slugs devote significant energy to separating chloroplasts from their food algae and maintaining them in a functional state?

The Function of Kleptoplasty: The Answer is Generally “Yes”

There have been many experiments performed to address hypotheses regarding the function of kleptoplasty in Elysia.  Some have been truly elegant, others not so much, but they have generated a lot of information and some insight regarding the benefits of stolen chloroplasts.  For me, one of the most important insights is that the chloroplasts are likely to provide multiple benefits to their hosts.

Here are some leading hypotheses:

Trophic Support: providing energy as nutrients or storage.

  • Carbon (sugar) production: Kleptoplasts continue to produce sugars and there is abundant evidence that that these reach the slugs’ cells (e.g., Cruz et al., 2020).
  • Storage tissue: Kleptoplasts act as a “living larder,” providing energy and nutrients during times of fasting (Laetz and Wagele, 2018).

Metabolic Support: Aiding cellular metabolism by either removing CO2 and other waste products, or producing O2.

  • Waste removal: Kleptoplasts can waste products from the slugs’ metabolism as substrates for energy production (Cruz et al., 2020).
  • O2 production: It has been demonstrated multiple times (e.g., Dionisio et al., 2018) that kleptoplastic slugs produce excess oxygen during photosynthesis.

Camouflage

  • Visual: The chloroplasts are responsible for the green color of Elysia species and undoubtedly conceal them from predators.
  • Chemical: metabolites from the kleptoplasts may help them to match the odors of their food plants and evade predators such as nudibranchs that hunt using their sense of smell (Gavagnin et al., 2000)

Chemical synthesis: Using biochemical pathways of the chloroplasts to make chemicals for the slug.

  • Egg production: Elysia provided with both food and light produce more eggs than those with only food (Shiroyama et al., 2020), and kleptoplasts have been shown to produce lipids that could support egg production.
  • Defense: E.rufescens uses the chemicals produced by kleptoplasts taken from Bryopsis algae for its own defense, with the added twist that the alga is using a bacterial symbiont to make the chemicals (Zan et al., 2019).

In short, there is support for nearly every hypothesis that has been proposed.  Taking these results at face value, kleptoplasty can provide a range of benefits to the host slug.  Each species may then exploit a subset of the resources that would depend on its food plant, ecology and evolutionary history.

Where Now?

Re-review and re-think.  One crucial step in untangling the web of hypotheses would be a thorough re-review of the literature, emphasizing the biological diversity of kleptoplastic species (including Elysia and Plakobranchus) rather than pursuing a unified theory.  We already know that long-term kleptoplasty arose multiple times among sacoglossan species, so it is likely that the precise molecular mechanisms and metabolic benefits will vary across the group.  Given the size of the literature pool, a monograph may be more appropriate than a simple review article.

Molecular mechanisms.  A large amount of data has been already collected regarding possible functions of kleptoplasty, so it may be time to shift focus from “what is the benefit of kleptoplasty?” to “how does it work?”  The tools (if not the financial support) exist for rigorous examination of the ways in which chloroplasts are separated from the rest of the cellular contents of the algae, transported to the slugs’ tissues and maintained for months at a time.  Based on a career spent studying physiology, I fully expect that the diversity of function described above will be reflected in a diversity of cellular and molecular mechanisms.

There are some very talented people working on this line of inquiry, and I hope to have a chance to post about their discoveries in the future.

What about me?  The project has largely served its purpose.  Using the unusual biology of Elysia, several groups of students at USG and at Ocean Discovery Institute were introduced to topics such as PCR barcoding, chemical ecology, the neurobiology of behavior, and physiological ecology.  In the process, I was able to take deep dives into literature and develop experiments to test the hypotheses we developed.  Heck, I even learned how to culture E. crispata from egg to adult.  At some point, I will want to take the lessons I have posted on the Solar Slug site and turn them into something more accessible.

Coulometric respirometer measuring O2 consumption of scorpions.

For now, I have shut down the slug system, and have shifted back to arthropod physiology.  We have developed methods for measuring respiration in fruit flies, beetles, and scorpions, and it may be time to get to work popularizing the methods and results of that work.

Scorpion in respirometry chamber.

As long as E. crispata and its food, Bryopsis plumosa, are available, the project can always be restarted with a few emails.  So, this is not “goodbye” as much as “see you later.”

References:

Christa, G., Händeler, K., Kück, P., Vleugels, M., Franken, J., Karmeinski, D., Wägele, H. (2014) Phylogenetic evidence for multiple independent origins of functional kleptoplasty in Sacoglossa (Heterobranchia, Gastropoda). Organisms Diversity and Evolution, 15 (1), pp. 23-36

Cruz S, LeKieffre C, Cartaxana P, Hubas C, Thiney N, Jakobsen S, Escrig S, Jesus B, Kühl M, Calado R, Meibom A. (2020) Functional kleptoplasts intermediate incorporation of carbon and nitrogen in cells of the Sacoglossa sea slug Elysia viridis Sci Rep. 10: 10548.

Dionísio G, Faleiro F, Bispo R, Lopes AR, Cruz S, Paula JR, Repolho T, Calado R, Rosa R. (2018) Distinct Bleaching Resilience of Photosynthetic Plastid-Bearing Mollusks Under Thermal Stress and High CO(2) Conditions. Front Physiol. 9:1675.

Gavagnin M, Mollo E, Montanaro D, Ortea J, Cimino G (2000) Chemical studies of Caribbean sacoglossans: Dietary relationships with green algae and ecological implications. J Chem Ecol 26(7):1563–1578.

Green, B.J., Li, W.-Y., Manhart, J.R., Fox, T.C., Summer, E.J., Kennedy, R.A., Pierce, S.K., Rumpho, M.E. (2000) Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus. Plant Physiology, 124 (1), pp. 331-342.

Laetz EMJ, Wägele H. (2017) Chloroplast digestion and the development of functional kleptoplasty in juvenile Elysia timida (Risso, 1818) as compared to short-term and non-chloroplast-retaining sacoglossan slugs. PLoS One 12: e0182910.

Shiroyama H, Mitoh S, Ida TY, Yusa Y. (2020) Adaptive significance of light and food for a kleptoplastic sea slug: implications for photosynthesis. Oecologia 194: 455-463.

Zan J, Li Z, Tianero MD, Davis J, Hill RT, Donia MS. (2019) A microbial factory for defensive kahalalides in a tripartite marine symbiosis. Science. 364: eaaw6732.

New Space!

After a long, tiring semester, we have moved into a new location in a shiny new building.

The Biomedical Sciences and Engineering building opened to great hoopla in November.  Local and state bigwigs participated in the ribbon cutting, but, more importantly, so did some Biological Sciences students.

 

Official opening of the new building. Dignitaries include Stew Edelestein, the Executive Director of USG (center in dark suit), Larry Hogan, Maryland Governor (second from right), Marc Elrich, County Executive (Left of Stew), and Mikal Abraha, Associated Students President and Biological Sciences senior (3rd from left).

During all the hubbub, the students in the Cell Biology Lab course were going full speed in their new cell culture facility upstairs. A few even made their way into a Washington Post story about the event.

Although the building was officially open, it has token a while for it to be truly ready for use. Even now, there are contractors coming and going to put the finishing touches on the structure, and some necessities, such as ice machines are on the way.

Nonetheless, we made the move to our new space last week. It has taken months of preparation to have the spaces ready for the equipment, and the equipment ready for the spaces. We have been running Cell Biology, Neurobiology, and Physics labs all in the same room, so it will be luxurious to have two large, well laid-out lab spaces and associated preparation areas.

Neurobiology Lab Space, before moving all the equipment in. Great layout, and point exhausts in case we ever work with toxic fumes.

The most important space for the slug project is the new “preserved specimen” room. Someone must have decided that we would be dissecting cadavers, so we have a prep room devoted to dead things, and point exhausts over the lab benches to ventilate fumes from preservatives.

Since we have no plans to store pickled carcasses, the preserved specimen room will make an excellent “live specimen” room. The room is separate from the rest of the lab, so animals can be kept away from chemicals, and it has marine grade shelving perfect for aquaria.

Marine grade steel shelves in the live animal room.
Sink and counter in live animal room.

On top of that, it has a floor sink for washing tanks and other equipment.

Floor Sink in animal room, Perfect for cleaning tanks and equipment.

It took about two full days to set up the plumbing, to move the slug and algae tanks, and to get the control system set back up. Big thanks to Paul, Kevin, and the rest of the IT crew for helping me to get the controller connected to the local network.

The Elysia and Bryopsis system in place. Slug tanks at top, algae tanks in middle, and sump and chiller at bottom. December 24, 2019.

The wiring is still a bit messy, but that can wait until I get my office and the two labs unpacked. Meantime, there are about a dozen slugs enjoying their new home.

Elysia and Bryopsis in the new animal room.

The slugs will soon be joined by the earthworms, crickets, and crayfish for the Neurobiology Lab course.

Posts will probably continue to be sparse for a while. Elysia is still a wonderful system for teaching neurobiology, and I expect some of the students to use them for projects this semester. In the longer term, I am excited about developing multi-unit recording methods to study the activity many nerve cells at a time during sensory processing. However, that will be on hold for a little while while I work on a few other things.

Slug Science Inches Forward

It has been a busy semester on several fronts, and the project has crawled forward a bit.

I am most interested in neurobiology and behavior, so we have moved from studying the chemical ecology of Elysia clarki to working out its behavioral response to external stimuli.  The ultimate goal of figuring out how its nervous system encodes sensory input and motor output.  Accomplishing this requires understanding of both the theoretical and technical aspects of molluscan neuroscience.  We had three goals for Spring semester: 1) develop a stronger knowledge of the literature describing the behavior and nervous systems of opisthobranch molluscs, focusing mostly on nudibranchs and sea hares; 2) work out methods for reproducibly recording from slug neurons; 3) get a better sense of the slugs’ light preferences, in terms of spectrum and intensity.

USG Slug Club, 2019. From left: Josue, Nana, Marianne, Savana, Abigail, Cecilia, Dave.  4/12/19

The intrepid group of seniors and I got right to work. 

For goal 1, we held a Slug Neurobiology journal club every Friday for the first 10 weeks of the semester.  When we started, I was still pretty fuzzy on the details of the visual and nervous systems of opisthobranchs, and had only a vague idea of how they manage to crawl.  I generated a list from multiple overlapping searches for papers describing the visual and motor systems of slugs, and Slug Club students and I presented about 18 papers, along with many more papers required for background.  Despite nudibranchs being a diverse group, most papers described either navigation and swimming in Tritonia, or learning and memory in Hermissenda, with a few papers on Pleurobranchaea, Aplysia, and some snails thrown in for good measure.  After our deep dive into the literature, we have a much better idea of mechanisms of light sensing, ciliary propulsion, and steering. 

Goal 2 was to record from Elysia neurons.  In spring 2018, we had found a good atlas of the Elysia nervous system, and had made a few recordings.  However, the nervous system  is surrounded by a tough sheath made of connective tissue, which makes it difficult to get delicate electrodes into the cells. 

At this point, I needed a colleague to give me some pointers on getting the sheath off, or at least softening it up, but one can count the number of people recording from sea slug nervous systems on one hand.  Fortunately, Paul Katz and his lab have extensive experience.  Paul responded rapidly to my email, and gave me some excellent pointers.  He was excited that there is another captive-bred sea slug being developed for neurobiology, and has been developing Berghia, a nudibranch that is also relatively easy to raise, as a neurobiological model system.  

Berghia nudibranch, from the Berghia Brain Project.

Berghia has some distinct advantages, such as a two month generation time (Elysia takes about 4 months), and surplus slugs can be sold to aquarists to control pest anemones. However, Berghia is much smaller and not kleptoplastic.  Might be a good “normal” species to use for comparison with Elysia, though.  

Even though the neurons that mediate the behavioral response to light are probably in the cerebro-pleural (which get inputs from the eyes) and pedal ganglia (which send outputs to the foot), I decided to start with the abdominal ganglion. It has a lot more big, pretty cells that should be easier to impale with a microelectrode, which increases the chance of success. With Paul’s advice, were able to get the sheath off and record from some large neurons in the abdominal ganglion. 

Spontaneous activity in a neuron in the abdominal ganglion. Large action potentials, reaching about 50 mV, along with large postsynaptic potentials (bumps between action potentials). 4/8/19

Josue, Marianne and I practiced impaling neurons, and got some nice stable recordings. The neuron above fired action potentials at regular intervals and received a lot of synaptic input, indicated by the large bumps between the spikes. Because we currently know nothing about its connections, the significance of the neuron’s pattern of activity is unclear.

Response of an abdominal ganglion neuron to a +1 nA current pule. The neuron fires a single action potential; the spikes at the beginning and end of the current pulse are due to the capacitance of the electrode. 4/8/19.

Another neuron was quiet at rest, but fired one or a few action potentials when stimulated with injected current. As with the previous cell, we know nothing about this neuron, but it provided an opportunity to practice techniques associated with current injection.

Time permitting, the next step is to record from neurons in the cerebro-pleural ganglion and find some that respond to light, and to look for others that control locomotion.

For goal 3, working out the spectra and intensities of light that Elysia prefer, you will have to stay tuned. The students just spent the past month gathering data, and should finish analyzing it within the next week or so.

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 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.

Slugs Taste Bad II: Mucus vs Tissue

In the continuing quest to understand the benefits of kleptoplasty, the Slug Club students and I set up some experiments to determine whether Elysia mucus caused predators to avoid them, and, if so, which components.  The logic behind the experiment was that 1) both the literature and my own observations indicated that slugs taste bad to predators; and 2) kleptoplasts produce organic compounds that appear in the mucus (Trench et al., 1972, Biol. Bull. 142:335); so maybe kleptoplasty plays a role in making defensive chemicals that keep fish and predators from eating them.

For these experiments, Zvi Kellman at IBBR was kind enough to provide some space for a predator setup.  We got to set the system up, and not have to worry about vacating the space for the entire semester.

The first small hurdle was to find some predators.  Because my preliminary experiments were done with fish, I had originally planned on testing fish from the same range as the E. clarki, maybe bluehead wrasse or small puffers. Then I was reminded of the paperwork for doing experiments with vertebrates, which is required even if we just watch what they eat and don’t do anything mean to them.  Dreading the possibility that time would evaporate while I assembled forms and waited for replies, I became much more enthusiastic about invertebrate predators.

But which invertebrate predators?  The primary requirements were that they were plausible natural predators, from the same geographic locations (i.e., sympatric), that we could obtain multiple individuals of the same species, and that they would not be too expensive or hard to keep.  I was hoping to find some small, nasty crabs that local aquarists or shops had extracted from their tanks, but nobody seemed to have any available.  Mike Middlebrooks had tried blue crabs, but they seemed rather large for our facilities.  I finally settled on peppermint shrimp (Lysmata wurdemanni).  They are sympatric, and are certainly willing to tear apart and eat other small, squishy invertebrates.  KP Aquatics sells them in lots of 5, so I could set up multiple groups of them without going broke.  Even if they don’t naturally prey on Elysia (nobody knows), they require no encouragement to eat, so we could at least test whether they are discouraged by slug slime.

We obtained a used 40 gallon breeder tank from a local aquarist (Thanks Rik), and moved in.  We had a sink and a plentiful supply of purified water located a few feet away, so it was pretty easy to get set up.

Bench space at IBBR, with 40 breeder tank. 2/2/18

I siliconed in some acrylic dividers to have five independent sections.  Each section had a drain leading to the sump, which had a  skimmer and a small filter.  The output of the return pump fed each section independently, and flow to each was adjusted with a ball valve.  Each section also had a plastic plant and cave assembled from 1.5″ PVC fittings.  At the beginning, each section had 4-5 shrimp.

Tank with dividers in place, just after adding water and salt. Each section drains to the 10 gallon sump on the right, with a protein skimmer and filter. Water is pumped back to shrimp enclosures, with flow controlled by ball valves that can be seen at the top of each section. 2/17/18

We messed around for a few weeks, then settled on an approach inspired by Becerro et al. (2001; J. Chem. Ecol. 27:2287).  They made cubes based on powdered fish food and carrageenan, added potentially aversive compounds, and then placed them in the ocean to examine the effects on feeding by fish.

Slug Club Spring 2018, making food cubes. From left: Kathleen, Claudia and Elyson. 4/18/18.

The students took the lead in working out how best to make carrageenan based food.  They figured out how to get the carrageenan dissolved, when to add the powdered food (Zoplan freeze dried zooplankton by Two Little Fishies), and the best way to add mucus or slug tissue to experimental cubes.  The hot liquid food was poured into silicone ice cube trays to make as many 1 cm cubes as needed.  Silicone O-rings, used later to secure the food cubes in the shrimp tanks, were held in place with plastic rods during pouring.

Ready to pour food. Silicone O-rings, threaded onto plastic rods, are placed into compartments of ice cube tray.

The food cubes were then placed in the shrimp enclosures.  Each was secured by its O-ring to a plastic paperclip tied to a monofilament line suspended vertically in water column.  Each line had multiple possible attachment sites.  The shrimp were not shy, and rapidly moved in to explore the potential food source.

Testing food assay system. Food cubes are secured by O-rings to plastic paper clips that were tied to monofilament line. Line is weighted by PVC elbow at bottom, hooked to input hose above.  Shrimp rapidly approach and sample the cubes. 3/28/18.

Experiment 1 tested to effect of mucus on feeding.  Two cubes, one control and one experimental, were placed in each section.  The control cubes were made with powdered food, carrageenan and artificial seawater (ASW); the ASW was replaced by mucus in the experimental cubes.

Mucus dripping from annoyed slugs in dip net. 4/9/18.

Collecting the mucus was not trivial.  Even though the slugs readily secrete mucus when annoyed (such as when crowded into a dip net), the sticky mucus is not easy to separate from the slugs.  Ultimately, we just let it drip into a beaker for 30 minutes or so, then returned the irritated by unharmed slugs to their tank. The mucus was then mixed into the food, and the unused portion frozen for later use.

Detail of food cube, showing embedded red silicone O-ring connected to plastic paper clip (yellow). 3/28/18.

To control for effects of position in the tank, food cubes from both groups were placed at different levels.  We recorded the weight before adding to the tanks, and the position in the tank, then left the cubes with the shrimp for 24 hours.  They were collected, and each was weighed again. The amount consumed was then calculated.

The results from the first round of experiments were underwhelming, but informative.  The shrimp were unequivocally indifferent to the presence of mucus in the food.  The graph below shows that shrimp consumed about 0.5 grams per day of food, regardless of whether it contained mucus.

Experiment 1: Effect of mucus on feeding by shrimp (means and standard errors). Control food cubes contained 10 ml ASW, mucus cubes had 10 ml of mucus from Elysia clarki.  High food content (5g/90 ml).  Sample sizes are 25 cubes for each group.

So, shrimp do not seem to mind mucus in their food.

The next step was to determine whether Elysia even taste bad to shrimp.  Peppermint shrimp do, in fact, eat a number of nasty things, including the anemone Aiptasia.  We had predicated our experiments on the idea that Elysia contain compounds the shrimp would find distasteful, but we never actually tested this idea.  It was time for a “Friday Afternoon Experiment,” in which one tries a quick and dirty test to see what happens.  In this case, we threw some baby Elysia to the shrimp.

Shrimp devouring baby slug during first trial with live Elysia. 3/26/18

The results were horrifying and illuminating.  Shrimp rapidly grabbed, dismembered and ate the baby slugs.  However, they left fragments of uneaten slugs, which gave a glimmer of hope that they did not find them to be the most palatable meals.  We repeated the experiment twice more, with a week in between, and the shrimp consumed less at each session, as if they had learned not to eat them, but consistently attacked and tore at the slugs.

Although brutal, there are some lessons here.

First, shrimp should definitely be included in the list of possible predators.  Naive shrimp had no qualms about eating Elysia tissue, and they would be well placed to hunt down small mollusks during their nightly forays to rummage the reef.

Second, the damaged slugs often recovered.  The usual examples of toxic or unpalatable species (think monarch butterflies) are often killed when they are sampled by predators.  Kin might benefit from future behavior of the predator, but the sampled individual is out of luck.  In this case, the slug may be able to limp away after having been tasted and rejected, and benefit directly from containing aversive compounds.  This may also explain why Elysia lack the bright warning coloration often associated with unpalatable prey.  Maybe the first line of defense is to be inconspicuous, and, if that fails, expect to be spat out after being tasted.

Experiment 2 was based on a suggestion by one of the students: if shrimp don’t mind mucus, what about the slugs’ tissues?

We anesthetized slugs, and worked out how to produce tissue puree.  We quickly learned that a standard mortar and pestle has little effect on tissue is both slimy and rubbery.  Chopping with a razor blade was better, and produced adequate tissue for the first round of experiments.  Later, I had to cull a large number, and used a food processor to make a tissue homogenate.

Experiment 2: Effect of mucus and slug puree on feeding. Medium food content (2.5 g/90 ml).  20 cubes per group.

The results were consistent with our expectations.  Mucus alone had little or no effect on feeding.  Slug tissue appeared to reduce feeding by more than 35%.  However, the results were not statistically significant, possibly due to variability between groups of shrimp.

We accidentally performed an additional experiment on the effect of food concentration on the rate of feeding and the aversive effect of slug tissue.  For one batch of food, we ran out of powdered food, and used less than half the normal amount.

Experiment 3: Effect of mucus and slug puree on feeding. Low food concentration (1.16 g/90 ml).  12 cubes per data point.

The shrimp ate less overall, and the effect of including slug tissue appeared to be stronger, reducing feeding by more than half.  As above, the results were not statistically significant, presumably due to the small sample and variability between sections.  Nonetheless, balancing the attractiveness of food versus the distastefulness of slug tissue needs to be considered for future experiments.

Relationship between amount of food cubes eaten (means and standard errors) and concentration of food powder. All groups show a modest increase in consumption when cubes have more food content. Cubes containing slug tissue (green line) appear to be less palatable at both food concentrations tested.

For the moment, we can tentatively conclude that mucus from Elysia has no impact on feeding by peppermint shrimp, but that slug tissue inhibits feeding.

Because Mike Middlebrooks tried similar experiments on blue crabs with adult slugs, the last experiment of the semester was to put full grown Elysia into the shrimp enclosures.  Normally approach food rapidly, but were very cautious.  When slugs dropped right in front, picked with pincers, but did not tear at the slugs.

In the future, there are several improvements that could help to sharpen the results.

One issue that arose was the change in shrimp populations in the sections over time.  A few shrimp were lost (the absence of carcasses suggests cannibalism) over the three months of the experiment, yielding a final range number of 2-5 shrimp per section, which resulted in considerable inter-section variation in food consumed in a 24 hour period.  Doing a periodic census and adjusting populations will presumably reduce variability.

Another possible confounding factor as that some cubes appeared to be torn apart and left on the bottom, and were therefore scored as eaten when they may have only been consumed partially.  Using video analysis of time spent feeding on each type of cube would help distinguish between destruction and consumption of food.

One other issue was the growth of algae during the semester.  Despite relatively low light in the lab, protein skimming and periodic water changes, the addition of food supported algae growth.  Given the omnivorous nature of the shrimp, the algae may have served as an alternative food source. Giving the enclosures a quick scrub during weekly maintenance would force the shrimp to focus on the food cubes.

Smothered in Slugs, Part 1

After devoting so many hours to learning how to feed and rear slugs, I suppose I can’t complain about the current situation.

Baby slugs filling 10 gallon tank. 3/21/18

I am drowning in Elysia.

In the past month, we have shipped well over a hundred baby E. clarki (plus a few dozen E. crispata) back to their homeland in Florida, gave another dozen to local aquarists, fixed at least a few hundred for anatomical studies, and yet there seem to be hundreds more.  They are destroying Bryopsis as fast as I can feed it to them, and I had to cull another few hundred last week to keep the rest from starving.  I honestly did not think I had that many babies growing in the system.

I will keep the remaining slugs for the next few weeks, because several groups of students have proposed using them for their independent projects in Neurobiology Lab class.  It will be very exciting to see what the students can accomplish.  We have also been extracting mucus from groups of slugs, for use in feeding assays (soon to be the subject of another post, I hope).  Finally, I am holding onto some of the smallest for another round of staining (yet another upcoming post) and predation assays (yet, yet another upcoming post).

When I resorted to buying E. crispata collected in Haiti last fall, because I was not able to obtain E. clarki from the Keys, I would never have dreamed that there would be such a turnaround. We now have enough slugs of all sizes to do any kind of experiment we can imagine.

Elysia clarki eggs, from 2nd generation parents. Box of Slugs 2.0, 4/1/18.

Not only that, there are no longer any mysterious gaps in the life cycle, from egg to veliger to hatchling to adult to egg.  The offspring from the first brood have become reproductively mature, so we are getting eggs from slugs that grew up here in Maryland.  As a result, I have put together a page about how to culture E. clarki.

Mother and daughter dozing in the early morning. Box of Slugs 2.0, 3/31/18.

There will undoubtedly be challenges ahead, but developing a self-sustaining colony was one of the major goals of the Elysia project.  Now the fun can begin.

Stay tuned for updates on Elysia anatomy, making food with mucus, predation assays, and take a look at the details of how I ended up with several hundred baby slugs.

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.

badchromatogram

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. 8/10/16

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 Elsysia are indicated by arrows.

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

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.

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.

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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

Some of the captive slugs, 7/8/16

E. diomedea and Aplysia in holding tank.

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

Elysia diomedea, relaxed and ready for surgery. 7/7/16

E. diomedea, with small piece of parapodium removed. 7/8/16

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. 7/8/16

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

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

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

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

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

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

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.

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.

3801_ImazeAplysia070416

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.

The islands Cabeza de Caballo (left, large island) and Gemelito Oeste. Gemelito Este is behind Gemelito Oeste.

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.

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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

Sargassum and Padina, northwestern part of Cabeza de Caballo. 7/5/16

Heavy Padina growth, 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

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

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

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

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

Bottom at Gemelito Este. Mixed Padina and other algae. 7/5/16

Large Oyster, covered in turf algae. Gemelito Este, 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

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

Green turfy algae north and east of El Rincon. 7/5/16

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Algae near El Rincon. Cladophora or something related? 7/5/16

Codium near El Rincon. 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

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

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.