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.

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.

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

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.

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. 7/4/16

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

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.

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

Stingray on sandy bottom. 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.

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.  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

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.