Posts in Category: Slug Science

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.

Everything in Place?

The project seems to be where it needs to be.  Since the last post, I ordered new “degenerate” primers, which consist of a mix of almost all possible sequences that would match the rbcL gene of the algae of interest.  The folks at Midland Certified Reagent Co. were very helpful, and the primers arrived quickly.

As can be seen below, we get a much more robust signal from the presumed Avrainvillea with the new primers.  There was, however, no signal for Bryopsis species 1.  Because that batch of DNA consistently amplified in the past, I am going to attribute the failure to technical issues for the moment.

As was true for the previous run, the lane for Elysia clarki still had no signal.  The repeated failure to get a PCR product from this extract suggests that it is not a simple technical glitch.  The obvious culprit is mucus.  Even though we used a small amount of tissue, it produced copious mucus, some of which may have remained in the sample after purification.  The polysaccharides in mucus are expected to interfere with the PCR reaction, so that could be our problem.

Gel from 5/25/16

Gel from 5/25/16

 

Because his lab group has successfully extracted and sequenced DNA from Elysia species, I contacted Gregor Christa from Heinrich-Heine Universität Düsseldorf.  He suggested a very simple fix: dilute the DNA template.  It seemed too easy, but I performed another run yesterday, and, lo and behold, it worked! The undiluted DNA (& mucus) gave no signal, but the diluted DNA amplified nicely.  I guess the dilution reduced the concentration of mucus enough to allow the reaction to proceed, while the sensitivity of the PCR reaction allowed amplification of the diluted DNA.

3497_slug_DNA_060116_labeled

Everything seems to be on track.  The next step is to send some of our PCR products out for sequencing, hopefully within the next few days.

Journal Club: Why on Earth do they store chloroplasts?

The subject of this year’s Journal Club (Has it really been over a year?  Oh dear.)  is a paper by Gregor Christa and colleagues from back in 2013.

Christa 2014 front page

In this paper, the authors try to develop a scientifically rigorous explanation for long-term retention (LTR) of chloroplasts in Plakobranchus and Elysia.

Some species of marine slugs in the Plakobranchoidea, which includes the genera Elysia and Plakobranchus, can store chloroplasts for many months.  These are stored in branches of the digestive system that ramify throughout the slugs’ bodies.  Perhaps foreshadowing their conclusions, Christa et al. refer to this phenomenon as delayed digestion, rather than maintenance of the chloroplasts.

So what do the food-derived chloroplasts (“kleptoplasts”) do for these months?  In 1975*, Trench found that Elysia viridis could incorporate 14C from labeled CO2 into its tissues, and concluded that kleptoplasts retained the ability to generate sugars from light, water and CO2.  Based on this observation, he coined the term “leaves that crawl” to indicate that he believed the kleptoplasts generated energy for the slugs and made them partially or totally independent of the need for ingested food.  This idea took hold, both in the scientific literature and in the popular imagination.

There were a few complications in this story, however.

First, how are functional kleptoplasts maintained for such a long period outside of a plant cell? A functioning organelle needs a constant supply of new proteins to replace those that are degraded with time and use.  Although chloroplasts retain a small number of genes (including rbcL, which is important for the kleptoplast identification project), the vast majority of genes required for their continued function are located in the nucleus of plant itself.  That is a problem, because the plakobranchids rapidly digest the nuclei of the algae on which they feed.

The problem appeared to be solved, and in an interesting way, when reports appeared that suggested the slugs had incorporated algal genes into their own genomes, a phenomenon called “lateral gene transfer,” or “horizontal transfer of genes.”  Newly-synthesized  algal RNAs and proteins were identified in slugs (reviewed in Pierce et al., 2007).  However, the absence of algal gene products in eggs or newly settled slugs (Bhattacharya et al., 2013), and the relative scarcity of chloroplast RNAs and proteins in slugs, indicates that lateral transfer is unlikely to have occurred.  It seems unlikely that kleptoplasts could be self-sustaining in the long term.

Another issue is that animals are capable of incorporating small amounts of CO2 into organic molecules, through carboxylation reactions.  This being the case, how do we interpret Trench’s results of 14C incorporation into E. viridis?

Furthermore, starved plakobranchids (the shorthand term Christa et al. use for Elysia and Plakobranchus) do not stay fat and happy if they are provided with light.  They shrink, and change color from green to yellow, which indicates that they are breaking down their tissues and the kleptoplasts, in order to survive.

Back to the original question: what is the purpose of storing kleptoplasts?  Christa et al., asked two very basic questions about the role of kleptoplasts, using E. timida and Plakobranchus ocellatus.

  1.  Are the kleptoplasts really involved in CO2 fixation?  To answer this, they compared incorporation of 14C in slugs that were kept in the light, in the dark, or in the light but treated with a drug that inhibits photosynthesis.  If the kleptoplasts are involved, then 14C incorporation will be higher in conditions that allow photosynthesis.
  2. Does the ability to photosynthesize reduce the rate of tissue loss during starvation?  If slugs are kept in the dark, or treated with drugs that inhibit photosynthesis, do they lose weight faster than slugs kept in bright light?

The answer to Question 1, was an emphatic “yes, 14CO2 incorporation is much higher in the light.”  As shown in Figure 2, below, both E. timida and P. ocellatus showed significant incorporation in the light at 60 and 120 minutes, but essentially none in the dark.  Incorporation was also significantly reduced by monolinuron, which blocks photosynthesis.

Christa_2013_fig2

So, it appears that the kleptoplasts fix carbon, which should provide energy to the slugs.  Does this keep the slugs from starving?

This brings us to question 2.  The authors examined two aspects of starvation to determine how the different conditions affected the ability to photosynthesize and maintenance of body weight.

First, they looked at what happens to chlorophyll function during starvation using Pulse Amplitude Modulated (PAM) Fluorimetry.  The authors suggest that those kept under more intense light declined more rapidly, but the sample sizes were small and the variation was large, so the strongest conclusion one can safely make is that quantum yield (their measure of photosynthetic function) decreased significantly with starvation.

Perhaps the most interesting result was that exposure to light made no difference in terms of weight loss.  Figure 4b, below, shows that all slugs, regardless of whether they were in light, darkness or treated with monolinuron, lost about the same amount of weight over 50 days.  Christa_2013_fig4b

Again, the sample sizes were tiny (two slugs each), so sweeping conclusions are not in order.  However, the shapes of the curves are intriguing.  The slugs in the light seemed to decline more rapidly than those in the dark, and the monolinuron treated slugs both showed a rapid decline followed by more gradual weight loss.  It would be interesting to know if there was interesting biology underlying these results (slugs burn through reserves faster in the light?), or whether they are quirks of a small sample.

Christa et al. conclude that the slugs are not photoautotrophic, i.e., they do not survive on light.  Instead they propose that the kleptoplasts are a food reserve.  This is certainly a plausible model, but others have not yet been ruled out.

For example, the authors suggest that the kleptoplasts could be required for the biosynthesis of compounds required for development or egg production.  One potentially interesting, and relatively straighforward, experiment would be to compare rates of egg laying between Elysia that are fed, but kept under high- and low-light conditions.  Given the metabolic demands of producing and provisioning egg masses, it would be interesting to see if kleptoplasty contributes to the process.

Another possibility is that the kleptoplasts help to “fatten up” the slugs before starvation.  In this scenario, slugs use both food and photosynthesis to fill storage tissues when times are good.  When food is scarce, or experimenters starve them, they use the kleptoplasts and stored tissue to produce energy and necessary metabolites.  This may be especially important for species that live on scattered or sparse food sources.  I don’t think anyone has done the math to determine how the energy estimated to be produced by photosynthesis (e.g., the amount of carbon fixed per unit time by the slugs in the light) compares with the metabolic demands of the slug.  It may be too small to keep a starving slug alive, but might help fatten up a feeding slug.

One final random thought regards the maintenance of kleptoplasts.  It is pretty clear that the slug does not have the genetic equipment to produce the proteins required for the upkeep of the chloroplasts.  However, plakobranchids (at least the E. clarki watching me here in my office) eat prodigious amounts of algae daily.  Would it be possible for ingested algal RNAs and proteins to find their way to the kleptoplasts?  It seems like a non-trivial problem to get the materials across a few membranes and into the plastids, but it would help to explain how chloroplasts can survive for months in cells that are lacking important equipment.

 

*note that all the references can be found in the full list of papers.

Kleptoplast Extraction

We made our first try at extrtacting and amplifying DNA from kleptoplasts.  It was relatively straightforward to get the tissue from the slug.  First, she was “relaxed” with isotonic MgCl2, which blocked synaptic transmission and paralyzed and anesthetized her.

Elysia clarki relaxed with 400 mM MgCl2. 4/27/16

Elysia clarki relaxed with 400 mM MgCl2. 4/27/16

Then, a small piece of parapodium was removed (see her left side, at bottom).  DNA was extracted using the same method as for the plants.

Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16

Elysia clarki with piece of parapodium removed for DNA extraction. 4/17/16

In case you were worried, she was fine the next day.

Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16

Elysia clarki one day after piece of parapodium removed. Hungry and looking for food. 4/17/16

We also tried a new species of algae, which I am calling Avrainvillea.  It may be Rhiphilia, I’m not completely certain.

Alga extracted 4/27/16.  Probably Avrainvillea.

Alga extracted 4/27/16. Probably Avrainvillea.

Unfortunately, the amplification was not a success this time around.  The positive control samples, DNA extracted from Bryopsis during previous sessions, worked well.  “Avrainvillea” showed a much weaker signal, and there was no signal from the slug extract.

Results from week of 4/25/16

Results from week of 4/25/16

A number of things may have gone wrong.  The slug sample was extremely slimy with mucus, and those polysaccharides could have interfered with extraction or amplification.  Also, the Bryopsis-specific primers may not have annealed adequately with the DNA from Avrainvillea and whatever is in the slug.

Now that things are calming down after the semester, I am going to try again, making a few changes.  Most importantly, I have ordered “degenerate” primers, which contain a mix of sequences that will complement just about any algal sequence.  Still waiting on tips regarding the slime.

We Have Data!

It has definitely been a good week for the Solar Slug Project.  Yesterday, all of the pieces for the barcoding project started to fall into place.  As I described a few weeks ago, the idea is to use a sensitive method called polymerase chain reaction (PCR) to amplify DNA from chloroplasts within the slugs in order to figure out what they have been eating.  Our first step was to extract and amplify DNA from potential food plants, just to be certain everything was working.  Below, you can see Maryam and Haseeb, the two USG students who have been working on this project, diligently extracting DNA from Halimeda and Bryopsis samples.

Maryam and Haseeb extracting DNA from Algae. 3/25/16

Maryam and Haseeb extracting DNA from Algae. 3/25/16

After a few false starts, we got conditions to the point that everything appears to be working.  One key change was switching to GE Healthcare’s “Ready-To-Go” beads as the source of polymerase, buffers, nucleotides, etc.  The beads can be stored at room temperature (as opposed to being frozen, like other “master mixes”), which will make life a lot easier at the field station.

PCR products from 4/22/16, visualized with BioRad UView DNA stain.

PCR products from 4/22/16, visualized with BioRad UView DNA stain.

The PCR products are the right size (about 600 base pairs), and there aren’t any extra bands.  We used primers specific to Halimeda discoidea (“H primers”) for the products in the first four lanes, and primers for Bryopsis plumosa (“B primers”) for the last four.  The species names, “Halimeda 1″ etc, indicate the following species:

Halimeda 1 (Presumed H. discoidea).

Halimeda species 1. Presumed H. discoidea. 3/25/16

Halimeda species 1. Presumed H. discoidea. 3/25/16

The specimen of Halimeda 2 (H. incrassata) was a bit bleached, but yielded some nice  DNA

Halimeda species 2, presumed H. incrassata. 3/25/16

Halimeda species 2, presumed H. incrassata. 3/25/16

Bryopsis “species 1” has a much finer structure.

Bryopsis species 1. Finer filaments. 3/25/16

Bryopsis species 1. Finer filaments. 3/25/16

Bryopsis 2 is stouter and longer.  It is unlikely to be a different growth form of the same species, because they were cultured right next to each other.  It’s a good opportunity to let the gene sequences unravel who’s who.

Bryopsis species 2. Coarser filaments. 3/25/16

Bryopsis species 2. Coarser filaments. 3/25/16

The astute observer will notice that we got products from (almost) all species using both sets of primers.  Overall, that’s a good thing, in that we can use these primers to amplify DNA from many species of algae, then submit the DNA for sequencing.  On the other hand, we could use more stringent conditions (like a higher annealing temperature, for those who care about such details) to use primers to  amplify DNA only from a particular species.  For example, we could use Bryopsis primers on DNA extracted from slugs to ask specifically whether the animals contain Bryopsis DNA.

The quick summary is that we have worked out the details of methods needed to extract, amplify, and ultimately sequence DNA from chloroplasts.  Haseeb and Maryam will be able to put together some nice reports about their work, and in Bahia de los Angeles this summer, we should be able to determine the species of algae that E. diomedea eats.

First Grant for the Solar Slugs!

We just got some great news, that we have received the first outside funding for the Solar Slug project!  The Washington Area Marine Aquarist Society has awarded the project $500 to get work started on slug neuroanatomy.  The general idea is to stain baby slugs for acetylated tubulin, which will stain axons, and therefore tell us where the nerves are.  This will be crucial in working out how the slugs process information about their environment.  Because the slugs are essentially pancakes with everything embedded in tissue (rather than having nice hollow spaces like people or insects), it has been difficult to do this kind of thing using standard dissection methods.

Now that the adults are getting fat on recent batches of Bryopsis, I hope to soon see some eggs, and then grow some small slugs for staining.  It may be a while, but I have a year to spend the money.  Keeping fingers crossed that a little support may attract more, espcecially if we start to get some nice preliminary data.

Thank you WAMAS!

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.