Posts in Category: Education

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.

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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.

 

Wild Slugs: Sea of Cortez Edition (Part Two)

At this point, we had a lab set up, some algae had been collected, but no slugs were to be found.  Definitely need slugs.  Because Berstch had done almost all of his sampling at Punta la Gringa, at the north end of the bay, it seemed like a good idea to have a quick look up there.  I had never been there before, so I asked Drew to drive me up there during an afternoon lull in the action.

Labeled Map 2016

Bahia de los Angeles, showing a few sites relevant to this post and the next one.

It was a beautiful spot, with sand and smooth stones leading to the water, so it seemed worth bringing the students there on their next field research day.

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Punta la Gringa, looking seaward. 6/30/16

But first, it was time to do some molecular biology.  Despite the absence of Elysia, we had plenty of algae.  In order to know which plants the slugs are eating, we need to get DNA sequences from potential food plants, so we could make some progress by extracting DNA from the algae.  It also gives the students their first shot at working with real DNA.

The most likely food plants are Codium (dead man’s fingers), Ulva (sea lettuce) and Bryopsis (feather algae).  We have not found Bryopsis, but had plenty of the other two, so we set about grinding the plants up and separating the DNA from the rest of the stuff in the plant.

Ulva from shallows in front of field station. 7/1/16

Ulva from shallows in front of field station. 7/1/16

Codium, possibly simulans. Collected in front of field station and ready to be homogenized. 7/1/16.

Codium, possibly simulans. Collected in front of field station and ready to be homogenized. 7/1/16.

Considering the tight space, the students worked well together.  It is not easy to pipet stuff from one tube to the next, then wait for an incubation or for the centrifuge to run, then do more pipetting, and so on without going crazy from the heat.  Nonetheless they got the procedure finished in time for a trip to La Gringa before lunch.  Although we did not find any slugs, it was a very nice dive.

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Nancy, Allison and Rosalia extracting DNA.

During the next lab session, we took the extracted DNA and amplified it using PCR to make many, many more copies of our sequence of interest.  As before, we used primers specific for the rbcL gene, which is found in chloroplasts but not the nuclei of plants or animals.  We also included some controls to make sure the procedure worked.  First, we amplified DNA that had been extracted by Haseeb and Maryam at USG, and which we know has worked in the past.  When we ran the DNA on an agarose gel, to separate the DNA pieces by size, we also added DNA that had been amplified at USG, to be sure the apparatus was working and the dye showed the DNA.

The procedure worked, at least for Codium.  There was a visible band for Codium, as well as for the positive controls, so everything seemed to be working.  The lack of signal for Ulva could indicate that something went wrong with the extraction, or that the sample did not amplify.  Also, for some reason, the molecular weight markers did not show up at the left end of the gel.  Nonetheless, the result was very encouraging.

Agarose gel, showing bands for Codium collected at BLA, as well as positive controls from USG. 7/1/16

Agarose gel, showing bands for Codium collected at BLA, as well as positive controls from USG. 7/4/16

The weekend was upon us, which meant a break for the students from research, and an opportunity for the scientists to get ready for the next week.  Lots of details to deal with, getting protocols finalized, reagents tracked down, and field survey plans finalzied.

That Saturday, we went on a scorpion hunt, led as usual,by Drew.  Normally, the students start getting disappointed during the early part of the hike, because the scorpions wait a while before coming out.  This year, they were plentiful and out early.  Using flashlights with UV LEDs made them easy to see, because, for some as yet unknown reason, they fluoresce green under UV light.

Scorpion fluorescing under UV flashlight. Hills south and west of field station. 7/2/16

Scorpion fluorescing under UV flashlight. Hills south and west of field station. 7/2/16

Same scorpion, under normal light. Feeding on winged ant. 7/2/16

Same scorpion, under normal light. Feeding on winged ant. 7/2/16

Meantime, we still had exactly zero slugs.  I was beginning to feel a bit like Ahab in the obsessive pursuit of my little green nemeses.  So, on a beautiful Sunday morning, I decided to do yet another snorkel through the shallows to hunt through the algae.  The tide was especially low, so I started by just walking through the shallows, looking for slugs, while the mobulas jumped a short distance away.

Sunrise over the islands. Taken from the staff house.

Sunrise over the islands. Taken from the staff house.

The snorkel itself was quite wonderful, slowly swimming back and forth from the front of the staff house to the south end of the Vermillion Sea field station, which had been used by the group some years ago.  As I swam slowly over the shallow bottom, I saw lots of algae, starfish, stingrays, corals, and many species of fishes.  I even found one cute little nudibranch.  I was however, beginning to despair of finding Elysia.

Stars in shallows. 7/3/16

Stars in shallows. 7/3/16

Stingray on sandy bottom. 7/3/16

Stingray on sandy bottom. 7/3/16

Gorgonian near Vermilion Sea Station. 7/3/16

Gorgonian near Vermilion Sea Station. 7/3/16

I also had to keep a close eye on the catch bag, because a small crowd of hungry puffers was following along, hoping to grab anything I might stir up.

Puffer, keeping an eye on me. 7/3/16

Puffers, keeping an eye on me. 7/3/16

After about 90 minutes, it was time to move on to other tasks.  We needed more algae-covered rocks for the station, so I put the mesh bag containing the little nudibranch into a bucket on the shore and proceeded to hunt around in the shallows for suitably-sized rocks with interesting algae.  When I looked at one patch of Codium, I saw what looked like some blue color among the uniform deep green.  Could it be?  A quick sweep of the hand sent a little Elysia flying through the water column.

I grabbed it, and gently held it while swimming toward the shore.  As I got out of the water, I looked in my hand, and it was gone!  I almost sobbed through my snorkel.  However, after many years as a research scientist, I am thoroughly accustomed to harsh disappointment, and went about my business collecting more rocks.  Fortunately for me, and for the project, there were three more of the little gals in separate clumps of Codium, and I was ready with the catch bag this time.  As can be seen in the photo below from a later hunt, the presence of Elysia is not always obvious.

Codium, concealing 3 - 4 E. diomedea.

Codium, concealing 3 – 4 E. diomedea.  Can you find all of them?

The drought had ended!  The captive Elysia adapted quickly to their new home.

Captive E. diomedea on Codium at station. 7/3/16

Captive E. diomedea on Codium at station. 7/3/16

E. diomedea exploring new home. 7/3/16

E. diomedea exploring new home. 7/3/16

Not bad.  We had the molecular biology working adequately, and we had slugs.  As often seems to be the case, finding one opens the door to finding more.

There was a lot more to do, though.  It was time to get serious about the slugs’ kleptoplast DNA, their responses to light, and their distribution in the bay.

 

Wild Slugs: Sea of Cortez Edition (Part One)

Has it really been that long?  Well, I’ve been busy.

The preparations of the last few months have now been tested in the field.  I am returning from the first two weeks of a five week field study in Bahia de los Angeles in Baja California.  As I think I posted earlier (apologies if I did not), the plan for the summer was to work with Ocean Discovery Institute on fleshing out some of the details of the life history and behavior of Elysia diomedea in Bahia de los Angeles.  Specifically, we want to know more about where they are found in the bay, what they eat, and how they respond to light. This information will help us to understand more about the role of kleptoplasty, along with the significance of the dramatic population fluctuations of the slugs documented previously by Hans Bertsch.

Even before we left, there were several challenges.  For example, for the project to get off the ground we needed permission from CONANP, the agency that oversees the marine reserve, to collect Elysia from the bay.  Months before the project began, we submitted an application for a permit to allow us to collect and study Elysia.  Unfortunately, that permit was rejected because we had not been clear about the relationship between the Elysia project and ongoing research in the bay islands.  Naturally, this caused us significant anxiety.  After a lot of work behind the scenes by folks at Ocean Discovery and by my very good friend Drew Talley at USD, our intentions were made clear, and we were given permission to go ahead with the project.

Among the items that were absolutely necessary were aquaria for holding, observing and testing the slugs’ response to light.  In April, I had started working with a local company that builds custom aquaria to build two holding/observation tanks and two “I-mazes” for testing light preference.  By early June, the tanks had not materialized, and I was beginning to get nervous that missed deadlines and excuses would continue until time ran out.  I decided to go with a more experienced vendor, Glass Cages, and they got the tanks into production and had them air freighted to San Diego in plenty of time.  The whole process of working with them was pleasant and reassuring.

Tanks and I-Mazes from Glass Cages, freshly picked up from Southwest Air Freight. 6/23/16

Tanks and I-Mazes from Glass Cages, freshly picked up from Southwest Air Freight. 6/23/16

The other critical pieces for extracting and amplifying DNA are a microcentrifuge and a PCR thermal cycler.  Again, thanks to the persistence of Drew Talley, we borrowed an Eppendorf centrifuge from USD, and received the loan of a demonstration model thermal cycler from Thermo Fisher Scientific.

So, after months of planning, spending a semester making sure that the methods work, arranging for care of the system in Maryland while I was gone, and enjoying a roller coaster ride obtaining permissions and equipment, it was time to get into the field.

The trip down was slightly adventurous.  We traveled with the Ocean Discovery students, starting out at Hoover High School in City Heights.  This year, we took the eastern route, through Mexicali and San Felipe.  It was a little longer, but somewhat more scenic, and had a whole lot less traffic.  The road was not great in spots, and one of the vans developed a flat tire along the way.  After a little delay, we were back on our way, and arrived in the early evening.

Stopping to fix a flat between San Felipe and Laguna Chapala. 6/25/16

Stopping to fix a flat between San Felipe and Laguna Chapala. 6/25/16

Tire destroyed on way to BLA.

Tire destroyed on way to BLA.

As always, I was very happy to be there.  Its hard to think of a place that I would rather be.  The sea is beautiful and full of life, and the surrounding desert is spectacular in its own right.  Over the course of the two weeks at the station, I tried my best to savor the views, sounds and smells.

Town of Bahia de los Angeles from the field station.

Town of Bahia de los Angeles from the field station.

Naturally, I was eager to get started.  It was all I could do to sit still during review of procedures around the field station, because I was eager to collect slugs in order to be ready for the students’ upcoming projects.  Finally, I got into the water, and began to hunt for Elysia.  It was wonderful to be in the bay again, and there was lots to see.  The familiar zones of Padina, Codium, Ulva, and the many other algae on the rocks outside the station reminded me of where I thought I should look.  After about 1 ½ hours of unsuccessful searching, I headed back to the station to get ready for the rest of my day.

Small stingray over a big field of Padina in front of BLA station. Sargassum at lower right. 7/12/16

Small stingray over a big field of Padina in front of BLA station. Sargassum at lower right. 7/3/16

Codium in front of BLA station.

Codium in front of BLA station.

I got to meet my crew in person for the first time.  The five young women were full of energy, and ready to get going with the project.  The goals of the “Photobiology” group (I needed a somewhat official sounding name, sue me) will be to flesh out some basic biology of E. diomedea here in the bay.  As we did for E. clarki in Maryland, we want to extract DNA from E. diomedea, and compare the sequence of rbcL in kleptoplasts with those from potential food plants.  Also, we will be looking at light preferences, using “I-mazes,” which give the slugs a chance to select their favorite light intensity.  We are also hoping to have a chance to explore the bay, surveying for appropriate habitat and the presence of slugs.  Lots to do to get set up and get the students trained.

The Photobiology Crew. Richy, at left is the Directed Research Fellow. The students (from left): Crystal, Rosalia, Nancy, Allison and Susan.

The Photobiology Crew. Richy, at left is the Directed Research Fellow. The students (from left): Crystal, Rosalia, Nancy, Allison and Susan.

The hunt for Elysia continued during the morning of our first full research day.  In the past, the morning hours have been the most productive in terms of slug hunting, so I had planned several mornings during the first week for collection.  The crew was becoming very proficient in the water, and we hunted for about 90 minutes in the shallows in front of the station.   Sadly, despite our efforts, no Elysia were to be found.  After a quick cleanup, we headed for the classroom for a briefing on algal diversity, lab equipment and safety before lunch.  The students had other activities in the afternoon, which gave me the opportunity to continue setting up tanks and equipment.

Rosalia drawing an example of a dichotomously branching alga. 6/29/16

Rosalia drawing an example of a dichotomously branching alga.

Our “molecular lab” is located in the garage, along with equipment for other Directed Research groups.  We share the space with a group studying ways of reducing bycatch of unwanted fish species and turtles, and another group that documents the flow of energy between the rich waters of the sea and the relatively barren land of the bay islands.  The space is a hive of activity at 7 am, when the other groups are rushing to get on boats.  After that the space is essentially ours until lunchtime.

Molecular Laboratory up and running in BLA station garage. Special thanks to USD for the centrifuge (left), and to Thermo Fisher for the loan of the PCR machine (right).

Molecular Laboratory up and running in BLA station garage. Special thanks to USD for the centrifuge (left), and to Thermo Fisher for the loan of the PCR machine (right).

The other part of our “lab” consists of the observation tanks.  These are in another part of the station containing the kitchen and computer lab.  The 16” cube tanks sit on a sturdy table, with circulation provided by air pumps, and lighting provided by morning sunlight supplemented by desk lamps with full-spectrum LED bulbs.  Once the slugs are in, the tanks hold slugs for DNA extraction and behavioral assays, with one being used solely for observation of the daily rhythms of undisturbed slugs.

Holding tanks at field station, with I-mazes in front. 6/29/16

Holding tanks at field station, with I-mazes in front. 6/29/16

Although we had not found any Elysia, at least a dozen small Aplysia rode into the tank with the plants.  This will actually be handy for comparison with the responses of Elysia to light.  Aplysia do not store chloroplasts, and might be expected to be repelled or indifferent to light.

Little Aplysia in holding tank. 6/29/16

Little Aplysia in holding tank. 6/29/16

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Small Aplysia in holding tank. 6/29/16

Day 2 was reserved for a field trip for the students.  It is supposed to be a non-work day, used to introduce the students to some aspect of the bay.  It started off great, with a visit to a sea lion colony, and up close encounters with very large fin whales.

Fin whale at north end of the bay. 6/28/16

Fin whale at north end of the bay. 6/28/16

We did squeeze in a little work, because part of the trip involved time for snorkeling at Coronadito island, at the far north end of the bay, and we were not explicitly banned from looking for Elysia.  Although we did not find any, it was useful to note and photograph the nature of the bottom, and the dominant algae species that were present.  Lots of Sargassum, some turfy coralline algae, but not a lot of large green algae.

Typical rocky bottom at Coronadito, with a trio of gobies. 6/28/16

Typical rocky bottom at Coronadito, with a trio of triplefins. 6/28/16

Susan next to a large mass of Sargassum. Coronadito 6/28/16

Susan next to a large mass of Sargassum. Coronadito 6/28/16

Fierce damselfish, Coronadito 6/28/16

Fierce damselfish, Coronadito 6/28/16

Algae community on rocky bottom. Coronadito, 6/28/16

Algae community on rocky bottom. Coronadito, 6/28/16

Photobiology group, plus a few others, at Coronadito. 6/28/16

Photobiology group, plus a few others, at Coronadito. 6/28/16

The days continued, with more briefings about identification of algae and molecular biology methods.  The big question was whether we would actually find any Elysia.  Fieldwork always requires some improvisation, but it’s a real challenge to improvise your way around the absence of your research subject.  Stay tuned.

Ready to Extract

Qiagen DNeasy Plant Kit 3/21/16

Qiagen DNeasy Plant Kit 3/21/16

Decisions have been made, orders have been placed, and materials have arrived.  It turns out that we are treading a relatively well-worn path of DNA bar-coding.  The goal for the semester is to extract, amplify and sequence the rbcL gene for the candidate food plants and the chloroplasts maintained by the slugs.  Because rbcL encodes a component of the photosynthetic complex of the chloroplast, and the gene is found in the genome of the chloroplast (rather than the nucleus of the plant), the origin of the chloroplasts that the slugs are carrying can be identified on the basis of the sequence of the gene.  Fortunately for us, the sequence of the rbcL gene has been studied by a lot of people.  The chloroplasts of all plants have it, but it varies a little from one species to another.  That variation can be used to examine how closely related different species are, or to determine if two populations that resemble one another are actually different species.  Each species has a unique sequence or “bar code,” that can be used to identify it, and to distinguish it from other species.

Therefore, much of the work has been done for us.  Kits, such as the one in the above photo, are readily available, procedures are largely worked out for amplifying the amount of DNA using polymerase chain reaction (although the primers for these algae may be a little tricky), and there are companies such as GeneWiz that provide a relatively inexpensive and simple resource for sequencing the resulting DNA.  Makes a nice change after a career spent soldering and tweaking in order to perform experiments using arcane methods.

On your next visit, you may find a photo of DNA bands on an agarose gel.  Or maybe something else.

Things Have Been Happening

It have been well over a month, so I suppose I have earned the title of World’s Worst Blogger.  It’s not for lack of news, though.  In fact, one of the reasons for the lack of posting is the abundance of activity.  In addition to the semester being in full swing, with the usual collection of lectures, exams and labs to attend to, the Elysia project has made some real strides in the past few months.  Today’s post will be a quick summary of the new beginnings, with the promise of more to come.

First off, this fall is the official start of student involvement in the Universities at Shady Grove (USG) Solar Slug Project.  Loyal followers of the blog will know that the USG project has been active for well over a year.  However, the primary goal of the project is to provide University of Maryland students here at USG with a research experience.  Now that the infrastructure is in place, there is now a squad of two undergraduate students who will be working on a small molecular biology project.  The main purpose of their work this semester is to develop a protocol for extracting chloroplast DNA that can be used to determine which species of plants the sea slugs are holding in their bodies.  We’ll start with a well-studied species, E. clarki, with a view to performing the same experiment on Elysia diomedea in Bahia this summer (see below).  The students have completed their first two assignments covering the basic biology of the system and the specifics of the methods they will be using.  We should be ordering reagents in a few days and doing some real biology next week.  Stay tuned for updates on their progress.

Another big news item is that solar slugs will be a significant part of the Ocean Discovery Institiute program in Bahia de los Angeles this summer.  One of the high points of my year is going down to Baja California to help them out with their work at the field station there.  This year, the slug project will expand to be a Directed Research project, involving a small group of students for the entire duration of their time at Bahia.  We will be performing experiments to identify the food plants and examine the activity patterns of Elysia diomedea.  Because large Elysia species are often found away from potential food plants, and because their method of feeding (sucking the sap) leaves no obvious bite marks, it can be difficult to know what they are actually eating.  Knowing what E. diomedea eats, and how much time it spends feeding, basking or hiding, should provide important insight into why it stores and maintains chloroplasts from its food plants.

For the semester’s experiments, we ordered some new E. clarki and have been fattening them up for experiments.  In addition to new Penicillus and Avrainvillea from collectors in the Keys, we are getting some nice Bryopsis and an unidentified species of broad-leafed algae in the half-ten algae growout tank.

Elysia clarki exploring broad-leaf algae 2/22/16

Elysia clarki exploring broad-leaf algae 2/22/16

3318_Eclarki_salad022216

Elysia clarki discovering new crop of Bryopsis 2/22/16

E. clarki on Bryopsis 2/12/16

E. clarki on Bryopsis 2/12/16

As you can see, it is still difficult to find the right nutrient balance that generates lush growth of the target algae without encouraging cyanobacteria (the red stuff), but we are moving closer all the time.

Slugs among algae selection 2/12/16

Slugs among algae selection 2/12/16

That’s it for now.  More soon, I hope.

Slug Makes New Species Top 10 List

The Washington Post reported that a species of photosynthetic nudibranch has made the SUNY Environmental Science and Forestry list of the Top 10 New Species of 2015.  The field was large, about 18,000 species in all, but Phyllodesmium acanthorhinum made the list based on what the animals tell us about the evolution of the symbiosis between  the slugs and the photosynthetic algae they host.

NEW SPECIES, PHYLLODESMIUM ACANTHORHINUM.  PHOTOGRAPH: ROBERT BOLLAND

Like Elysia, species of Phyllodesmium steal the ability to perform photosynthesis from their food organisms and maintain the required components in sacs extending from the gut called digestive diverticula. There are some important differences, though.  Unlike Elysia, Phyllodesmium is a true nudibranch, and it feeds on corals rather than macroalgae.  Another important difference arises from the different biology of the algae that Elysia eat and the corals upon which Plyllodesmium feeds.  Photosynthetic corals, such as Xenia, contain symbiotic algae (dinoflagellates, actually) called zooxanthellae, which provide the corals with most of their nutritional needs.  When Phyllodesmium feeds on Xenia (or other coral species, depending on the species of Phyllodesmium), it steals the zooxanthellae and stores them in the diverticula.  In this way, Phyllodesmium has it a bit easier, the stolen algae are autonomous cells, and the slugs do not need to worry about maintaining isolated chloroplasts.

So how did this species end up in the top 10?  A recent paper describing Phyllodesmium acanthorhinum and analyzing the interrelationships of species within the genus (E. Moore and T.Gosliner, 2014, The Veliger 51:237) provides some new insight into how the ability to maintain zooxanthellae evolved within the group.  Earlier work had suggested that the branching of the diverticula, and their extension into the cerata (the frills on the back of the nudibranch) increases with the increased ability to sequester and maintain zooxanthellae.  In other words, species that simply digest the zooxanthellae have minimal branching, while those that maintain large collections of active zooxanthellae have more elaborate diverticula that branch deeply into the cerata.  Based on the descriptions of P. acanthorhinum and another species, P. undulatum, both of which are relatively less specialized for maintaining zooxanthellae, Moore and Gosliner provide additional support for this hypothesis.  Further, they suggest that the larger body sizes achieved by more derived species, i.e., those that are better able to maintain populations of zooxanthellae, result from the additional nutrients produced by the symbionts.

Once again, slugs find a way of hijacking photosynthesis from their food. Because Elysia and Phyllodesmium are only distantly related, and their biology and that of their food are so different, the two forms of theft-based photosynthesis must have evolved independently.  The similarities are striking, though.  It does make one wonder if there is some aspect of the biology of sea slugs that predisposes them to separate chloroplasts or entire zooxanthellae from their food and maintain them in digestive diverticula.

Journal Club: A Sea Slug’s Guide to Plastid Symbiosis

The Elysia literature is rich, varied, and growing constantly.  From time to time, I will highlight a recent paper that strikes my fancy.

The paper of the moment is a recent review by de Vries and his colleagues that, from my point of view, demonstrates how the field of kleptoplasty in sacoglossans is maturing as a growing number of researchers apply diverse methods and approaches. Although I will summarize some of the highlights below, it is worth reading this short, nicely written paper for yourself (de Vries et al., 2014, Acta Soc Bot Pol 83: 415-421)

deVries cover

This paper illustrates a larger point that I have had to learn repeatedly during my career: as much as possible, one needs to look at the data and the biology, unfiltered by the way you think it should work.  Almost invariably, one generates a mental model to organize one’s observations about a biological system.  This model forms the basis for additional experiments, which can potentially support the model, but which almost always show you how naïve and simple your initial model was.  The model is essential to focus one’s thinking, but any biological system is more complex (and therefore more interesting) than your limited human mind can imagine.  So, there comes a time to listen to what the biology is telling you, and to think very hard about the next generation of models.

The paper by deVries and colleagues highlights that the field is at a point where researchers can, and must, think more deeply about the mechanisms and functions of kleptoplasty.  They lay out a series of fundamental unanswered questions regarding the biology of solar sacoglossans.

  1. How do the slugs sort the kleptoplasts from the stuff that gets digested? When sacoglossans feed, they pierce the wall of an algal cell with a specialized radullar tooth, and suck out the contents.  The extracted material contains mostly stuff the slugs will digest immediately (e.g., cytoplasm, nuclei, mitochondria), but also chloroplasts.  How does a slug’s digestive system handle the material, with chloroplasts segregated and moved from digestive organelles to the cytoplasm, where photosynthesis can continue?
  2. How are stolen plastids maintained? Despite initial reports of horizontal transfer of genes from the genomes of the plants to those of the slugs, more recent experiments indicate that this is not the case. So how are the kleptoplasts maintained in a functional state without an algal genome (remember, the nucleus was digested) to direct the synthesis of the structural proteins and enzymes needed to replace those that are constantly degraded?  Perhaps the kleptoplasts carry the capability with them, but the details remain mysterious.
  3. What is special about the biology of slug and algal species that participate in long-term retention (LTR) of chloroplasts for up to months at a time. There are seven species of sacoglossans known to be LTR slugs.  These seven slugs are not monophyletic (i.e., are not derived from a common ancestor), and vary in the details of their diets.  Some, like E. chlorotica, specialize on a single food plant, whereas others (e.g., E. clarki) feed on multiple species.  In some cases, LTR species feed on the same food plants as short-term retention (STR) species, which maintain plastids for only a few days or weeks (e.g., E. clarki vs. E. papillosa).  The slugs themselves, therefore must have some specializations to enable LTR.  However, in a given LTR species, retention times vary for kleptoplasts of different algal species.  These observations indicate that both the slugs and the algae have specializations, completely unknown at this point, that enable long-term survival of chloroplasts inside LTR slugs’ cells.
  4. Possibly the most fundamental question regards the functions of stolen chloroplasts: what good are kleptoplasts anyway? Multiple experiments have demonstrated that photosynthesis alone cannot support slug survival in the long term, and it is currently unclear whether starch produced by photosynthesizing kleptoplasts even enters the slugs’ cytoplasm. One hypothesis is that the plastids serve as “living larders,” being digested as needed by the slugs during periods of starvation. Kleptoplasts may also produce biochemicals needed by the slugs. For example it has been suggested that juveniles may depend on kleptoplast-derived lipids during their development.  It is surprising that the function of long-term kleptoplasty remains mysterious so many years after its discovery.
  5. I wanted to end with an issue that the authors touched on briefly, that of photobehavior. Experiments have been performed examining phototaxis and parapodial extension in response to light, with the interpretation of the data being strongly influenced by the assumption that the behaviors support photosynthesis in some way. This issue is of particular interest to me, and, although a full discussion of the literature must await a future post, a lot of the data are not really consistent with the behaviors serving to optimize photosynthesis.  Like, why aren’t slugs most active in mid-day, or why do they seem to be most affected by wavelengths in the middle part of the spectrum that are nearly useless for photosynthesis?  From my point of view, some hard thinking about experimental design and interpretation are in order.

The paper ends with a quote from Ed Yong that echoes my introduction to this blog post, but is much more eloquent: “Science is about resisting the easy pull of conclusions.  It’s about testing stories that seem like they should be right to see if they actually are right.”

As the study of kleptoplasty evolves from “gosh, wow, a photosynthetic slug” to a more complex and interesting view of the animals’ biology, the questions become more focused and more sophisticated.  It will be fun to watch, and with a little time and effort, participate in the process.

Slugs on NPR!

Terry Gosliner of the California Academy of Sciences was on NPR’s Science Friday this afternoon, talking about…SEA SLUGS!

Mostly he talked about the northward movement of the Hopkins Rose nudibranch (below), and what that tells us about warming temperatures in the Pacific.

Photo by Gary McDonald, posted on FeaturedCreature.com

Of course, he could not help but mention Solar Sea slugs and kleptoplasty.  I mean, who can spend an entire interview talking about a spiky pink abomination when you can talk about green beauties like Elysia?  It was a bit disappointing that he referred to Elysia as a nudibranch (we all know that they are not), and implied that they derived as much benefit from their chloroplasts as corals do from their zooxanthellae.  Nevertheless, any radio show about sea slugs is a good radio show.

More information and audio can be found on the Science Friday web site.