Posts in Category: Slugs in Nature

Photos and descriptions of sea slugs in their native habitats. May include some nearby residents.

Cool Solar Slug Videos

There is so much going on in the world of solar slugs!  I came across a couple of nice videos, and thought I’d share.

A broad collection of European slug scientists has launched a project to sequence the genome of Elysia timida.  That will be a wonderful resource for all of us who are interested in slug science.  The video below provides an explanation of the motivation, along with excellent general information.

The video below does a great job of describing the biology of E. viridis, with some beautiful footage.  Can you see the minor error?  (Hint: what is it eating?)

Slugs of Madagascar

Every 5 or 6 years, we end up exploring someplace a little more exotic.  This year, we decided to go to Madagascar, and we arranged for a driver and accommodations for about 10 days of overland travel and saw some incredible people, landscapes, and wildlife.  We’re still sorting through the photos of lemurs, chameleons, villages, and vistas, and it is likely to take some time.

Of course, you don’t come to this web site for the lemurs.

Because we were traveling all the way to an island in the tropical Indian Ocean, I pushed for a stretch of diving at the end.  When we were planning the trip, I asked our tour operator, Cactus Tours (an excellent Madagascar-based company) to arrange for some diving at the island of Nosy Be, at the north end of Madagascar.  We ended up spending a few days in Nosy Be, and had three days and two night of diving on a sailing catamaran.

After bouncing around in a four-wheel drive for over a week, it was pure luxury to be on the boat. For just the two of us, there was a driver, cook, and a very experienced and knowledgeable dive master, all arranged through Madavoile Cruises. We had six excellent dives, and were completely blown away by the diversity of corals, fish and invertebrates.

Our dive guide, Nicolas, figured out pretty quickly that I wanted to see nudibranchs and sea slugs, and he did not disappoint.   The photos below were taken with an Olympus Tough TG-4 camera, which is rated to 50 feet without a housing.  Because the housing was clumsy, we took the unhoused camera as deep as 65 feet, which worked just fine despite its increasingly strident warnings. Identifications are based largely on a digital version of Gosliner et al’s “Nudibranch and Sea Slug Identification”  and the Sea Slugs from Reunion Island Web site, which is an excellent reference for the southwest Indian Ocean.  Please let me know if you believe a species to be misidentified.

Phyllidia varicosa, Sugarloaf Island, 6/30/17


Phyllidia sp., maybe P. marindica, Sugarloaf Island, 6/30/17.


Phyllidiella zeylanica, North Nosy Tanikely, 6/29/17.


Phyllidiella zeylanica, Ankazo Reef, 6/30/17.


Phyllidiella pustulosa, Sugarloaf Island, 6/30/17.


Phyllidia ocellata, Sugarloaf Island, 6/30/17.


Doriprismatica paladentata, Ankazo Reef, 6/30/17.


Glossodoris pallida, Sugarloaf Island, 6/30/17.


Goniobranchus geometricus, Ankazo Reef, 6/30/17.

Although we saw plenty of Caulerpa, Halimeda and other macroalgae, and looked very hard for Elysia, we came up empty.  At Nosy Sakatia, we spent our last morning snorkeling with the turtles before we dove, and I was pleased to see a wide expanse of turtle grass (Thalassia) and manatee grass (Syringodium), which had some excellent growths of Halimeda incrassata.

Halimeda incrassata, among other algae and seagrasses, Nosy Sakatia, 7/1/17.

Unfortunately, I did not get to spend hours searching the seagrass beds.  Watching the biggest green turtles I had ever seen graze right in front of me was a nice consolation.  It is hard to get a sense of just how big these monsters are from a photograph.

Huge green turtle grazing at Sakatia, 7/1/17.

I did find one sacoglossan, Plakobranchus ocellatus, on the reef at Sakatia Arch.  Unfortunately, the camera housing I was using for the dive had fogged, so it is a lousy photo.  Just not the trip for sap-sucking slugs, I guess.

Plakobranchus ocellatus, Sakatia Arch, 7/1/17.

In addition to the plentiful slugs, there were quite a few flatworms pretending to be nudibranchs. All were in the genus Pseudoceros, and all were found in Humann and Deloach’s Reef Creature Identification book, which has an impressive section on flatworms.

Pseudoceros tristriatus, Ankazo Reef, 6/30/17.


Pseudoceros lindae, Ankazo Reef, 6/30/17


Pseudoceros bifurcus, Sugarloaf Island, 6/30/17.

Bahia II: Elysia diomedea Tastes Bad

As described in the previous post, I had very modest goals for this summer in Bahia.  Because I am getting more interested in the role of kleptoplasty in chemical defense, I thought it would be worth assessing the palatability of Elysia diomedea.  Some Elysia species are known to taste bad because of chemicals assimilated from their food plants (see, e.g., Rasher et al., described in this post).  E. diomedea is known to produce interesting derivatives of plant compounds (e.g., Ireland et al., 1978, J. Am. Chem. Soc. 100:1002), but, as far as I can tell, there is no evidence regarding the slugs’ palatability.

Fortunately for me, there is a relatively easy way to get a quick sense of their palatability.  When snorkeling at the field station, one is generally followed by a small parade of large bullseye puffers (Sphoeroides annulatus) waiting for tasty morsels to be stirred up.  What would happen if I dropped a slug in the water column and allowed the fish to eat it?  One might expect a puffer to eat anything.

After a day in the field, I had time for a snorkel, so it was a perfect opportunity.  After a short survey along the subtidal, I found a few Elysia in a small bunch of Codium (surprise!).  I pulled out this little beauty, apologized to her and carried her to the surface.

Elysia diomedea in the shallows in front of the Bahia de los Angeles field station. 5/23/17

If you click the link below for the short video (note: large-ish file), it is pretty clear that the puffer does not find the little Elysia to its liking.

Puffer spitting out Elysia diomedea. Bahia de los Angeles, 5/23/17

Not only one, but three puffers rejected the Elysia.  After the first spat out the slug, a second tried it, then a third.  In no case did one of the puffers as much as chew, they rejected it as soon as it was in their mouths.  Very good for the slug, and suggests that it may be something secreted in the mucus that repels the fish.  One might also conclude that puffers don’t learn from their friends, since each had to try it.

Based on one slug (but three puffers), we can tentatively conclude that E. diomedea tastes bad.  Are the bad tasting compounds derived from products made by the kleptoplasts?

Bahia de los Angeles 2017. Volume 1

Nature is perverse.

We’re back in Bahia de los Angeles, on the east coast of Baja California, Mexico. For the two previous years, I have had to suffer for a while before finding any Elysia diomedea.  It was rather nerve wracking, because I needed them for research projects each of those years.

Because of time constraints, my goals this year are to help my friend, Dr. Drew Talley, with his long term research, and to discuss plans for student Elysia projects in summer 2018 with Ocean Discovery Institute.

We had some complications at the border, because the Mexican authorities had some reservations about some equipment that was being used by one of the other research groups.  We were allowed to proceed after a few hours dealing with paperwork, but were delayed to the point that we had to stop along the way for the night.

We arrived without further incident, unloaded equipment and belongings, started setting up the station, had a great meal in town, we went to bed for the night.  It was amazing to be back under all the stars, listening to the ocean and the occasional breathing of a marine mammal.

We woke up to a classic sunrise, and soon we were on the islands, setting traps for insect surveys and savoring the bay and the scenery.

Heading back to boat after setting traps on Mitlan. 5/21/17

After returning to the station, we ran some errands, followed by a little open time to get in the water.  Although I may try a few extremely simple preliminary experiments, my work here does not depend on finding them.  Naturally, that means they were abundant in the shallows in front of the station. I found the first within five minutes, and saw at least six within the half hour allotted for the survey.

Elysia diomedea in front of field station. 5/21/17

They looked darker than the slugs we found last summer, but, as was true last summer, all were on or near Codium.

Keeping fingers crossed for a chance to test some ideas about chemical camouflage.

Journal Club: Chemical Camouflage and George Harrison

Welcome to the Roughly Annual Solar Sea Slug Journal Club.

Today’s paper came from the Proceeding of the National Academy of Sciences a few years ago (Rasher et al., 2015, Proc Natl Acad Sci 112:12110).  I came across it again when I was updating records for this site, and, because it is germane to one of my pet theories, it seemed perfectly suited for an extended discussion. You’ll see how George Harrison fits into the story later.  Yes, this post will meander a bit, but the fact that you are reading the Solar Sea Slug Blog suggests you may have some time on your hands.

The paper is a very nice exploration of the interactions between herbivores and their food plants.  Up here on dry land, insects tend to specialize on particular food plants, and bugs and plants have evolved together in something of an arms race.  Insects use volatile chemicals produced by the plants to locate them, plants produce defensive chemicals to keep from being eaten and from being infected by insect-borne pathogens, insects develop resistance to the plant chemicals, and sometimes use them for their own defense, and so on.  The authors wondered if they could identify a similar web of interactions in the marine environment.

Elysia tuca in Box of Slugs 2.

The algae Halimeda incrassata would seem to be rather unpalatable.  It produces a collection of defensive chemicals, and is highly calcified, making it a crunchy, bad tasting mouthful.  Despite the defenses, Elysia tuca, a tiny and distinctively-patterned species, is commonly found on Halimeda.  The interaction between E. tuca and H. incrassata allowed the authors to ask how similar the relationship between a mollusc and a marine alga is to those of insects and terrestrial plants.

Halimeda incrassata, in Box of Slugs 2. Note the segmented appearance, which will be important in understanding Figures 3 and 4 of the paper. 3/12/17.

In order to compare the relationship between E. tuca and Halimeda to terrestrial plant-insect interactions, the study focused on five specific questions:
1)  Is E. tuca really a specialist? This is important for the development of an intimate plant-herbivore relationship.
2)  Does E. tuca find Halimeda based on chemical cues?
3)  What are the cues that E. tuca uses?
4)  What are the ecological consequences of E. tuca feeding on H. incrassata?
5)  Does Halimeda use counter-defenses to limit the damage inflicted by E. tuca.

With regard to E. tuca being a specialist, the answer was a pretty resounding “yes.”  They collected specimens of about 10 species of algae and marine plants at two sites, took them to the lab, and counted the numbers of E. tuca on each.  With a few exceptions (also in the genus Halimeda), E. tuca were only found on H. incrassata.  Further, when given the choice between many different algae species in the lab (Fig 1A, below), or three different species of Halimeda in the lab (Fig 1B), or in the field (Fig 1C) the slugs greatly preferred H. incrassata.  To test whether the slugs were following chemical cues, the experimenters soaked cotton balls in water that had held H. incrassata (Fig 1D), and found that E. tuca much preferred these to cotton balls soaked in plain seawater.  With regard to the questions posed by the paper, the results indicate that 1) E. tuca is a specialist, and 2) they find their host based on chemical cues.

Fig. 1. Elysia host preference. Number of trials in which an Elysia colonized one of 14 common seaweeds and seagrasses (n = 20) (A), three co-occurring seaweeds in the genus Halimeda (n = 20) (B and C), or a cotton ball laced with H. incrassata-conditioned seawater vs. seawater only (n = 40) (D), when offered in a still water arena (A, B, and D) or in the field (C). Choice was assessed after 2 h (A–C) or within a 5-min period (D). Results were analyzed by a Cochran’s Q (A–C) or Fisher’s exact (D) test. In A–C, different letters above bars indicate significant differences among seaweeds in terms of Elysia colonization frequency, as determined by Wilcoxon sign tests (corrected for multiple comparisons). AL, A. longicaulis; CC, Caulerpa cupressoides; CP, Caulerpa prolifera; CS, Caulerpa sertularioides; DC, Dictyosphaeria cavernosa; HI, H. incrassata; HM, H. monile; HO, H. opuntia; PC, Penicillus capitatus; PD, Penicillus dumetosus; RP, Rhipocephalus phoenix; SF, S. filiforme; TT, T. testudinum; US, Udotea sp

The next question regarded the identity of the chemical attractants from H. incrassata (which will be henceforth referred to as “Halimeda”).  Compounds were extracted from Halimeda with methanol, and the individual components of the extracts were separated as described in the supplementary methods.  Each fraction was tested for attractiveness to slugs using the cotton ball colonization assay described in Figure 1, above.  The first compound they described, 4-hydroxybenzoic acid (4-HBA; Figure 2A, left) is found in both “vegetative” Halimeda in the normal growing stage, and in “reproductive” Halimeda that are undergoing spawning events.   When 4-HBA was placed on cloth patches next to Halimeda, the plants were colonized by significantly more Elysia than to controls with cloth soaked in the solvent but no 4-HBA (Figure 2B, left panel).

The reproductive stage of Halimeda is significantly more attractive than the vegetative stage, in part because the reproductive cells (gametes) are a rich source of nutrients.  When patches soaked in extract from reproductive plants were placed next to Halimeda plants, they attracted more than twice as many slugs as those from vegetative plants (Figure 2B, right).  This led the authors to identify halimedatetraacetate (HTA; Figure 2A, right) a chemical compound enriched in reproductive Halimeda.  It was known that HTA deters feeding on Halimeda by other species, and that E. tuca sequesters HTA in its tissues.  The authors went on to show that an extract from E. tuca that contained HTA deterred feeding by predatory wrasse.

This brings us to question #4, what are the ecological consequences of E. tuca grazing on Halimeda?  Surprisingly, the effects of such tiny slugs are significant.  The fact that the slugs feed on reproductive structures (which have the highest HTA content) is expected to substantially reduce the plants’ fecundity.  Further, when the authors manipulated the numbers of Elysia on plants in the field, those with more slugs showed less growth (Figure 3A) and more branch loss (Figure 3B).  Placing E. tuca in enclosures on branches (Figure 3D) also caused more branch loss compared with enclosures with no slugs.  So E. tuca can cause significant damage to Halimeda.  Because H. incrassata aids the development of seagrass beds, and generates the majority of carbonate sediments (a.k.a., nice white sand) in those areas, the authors suggest that grazing by E. tuca can have ecosystem-wide consequences.

How can a small slug that sucks sap cause such dramatic loss of plant tissue?  One hypothesis is that the plant self-amputates segments that have been fed upon by Elysia.  The model Rasher et al. propose is that the plants are trying to avoid the introduction of pathogens by the slugs by sacrificing segments.  After culturing fungi from the slugs’ radullae, which they use to pierce the plants’ tissues, they tested one fungal species they referred to as Et-2.  Halimeda innoculated with the fungus dropped segments above the injection site (Figure 4A).  Injection of a fungus that is a pathogen of other species did not have the same effect (Figure 4B).  The data are therefore consistent with the hypothesis that loss of segments is a defensive strategy in response to feeding by E. tuca, suggesting that the answer to question #5 is also yes.

The authors conclude that the answer to all of the questions they posed is “yes,” and that marine plant herbivore interaction described above strongly resembles those in terrestrial ecosystems, despite more than 400 million years of separation between the participating species.

At some point, this paper got me thinking of a potential alternative function for kleptoplasty.  Shall we meander our way there?

By the end of last summer, I was finding most of the prevailing theories regarding kleptoplasty to be rather unsatisfying.  While not every aspect of biology must have a function, kleptoplasty has costs that must be offset.  It takes energy to segregate and store the chloroplasts, and they must be protected from the immune system.  Plus, the animals that are active in the sunlight are exposed to predation and damage from UV light.  Despite these costs, Elysia is a very successful genus, with species found worldwide in the shallows of tropical and temperate seas.  Therefore kelptoplasty must provide a significant benefit.

So, what good is kleptoplasty?  If you buy the arguments presented by deVries et al, photosynthesis by kleptoplasts do not supply a significant portion of the animals’ energy needs.  Is the energy produced by photosynthesis used to make starch or fat for use during lean times?  Maybe.  Could the kleptoplasts be a “living larder,” being digested when food is scarce?  The animals certainly become pale when they are starved, suggesting the kleptoplasts are being broken down, but why not just digest them at the time they are eaten and turn them into fat like the rest of us do?

One idea is that the kleptoplasts are merely used as camouflage.  In the case of E. diomedea, which spends a lot of its time hidden in its food plant, this seems sensible.  Not so much for E. crispata, which is easily visible against hard bottom reefs, which are generally not very green.  Further, it seems like there are other, less complicated ways of making or storing green pigment to match one’s surroundings.  However, let’s hold that thought for a minute.

Aside from making carbohydrate from sunlight and being green, chloroplasts produce important precursors for many biochemicals (e.g., Gould et al., 2008, Ann. Rev. Plant Biol. 59:491).  These could be used by the slugs for the synthesis of fats or essential aromatic amino acids for their own nutrition, or to be used for their prodigious production of eggs.  Given that there is absolutely no data regarding the role of photosynthesis in egg production by Elysia, this remains an attractive hypothesis.

However, an insight I thought was particularly brilliant was that chloroplasts synthesize isopentyl diphosphate (IPP), a precursor to a wide range of things, such as chlorophylls and terpenes. Some of these compounds are expected to be smelly, and, in principle, make the slugs smell like their food.  Some of the chemicals may also taste bad, rendering the soft, slow animals less palatable.

Predators of Elysia are expected to include nudibranchs, which are largely blind and find their food by smell, or fish, many of which find their prey by sight.  If the chloroplasts were pumping out chemicals that gave the slugs a smell of their food, it would make it much more difficult to find them by scent.  One bonus is that the green color of the kleptoplasts will also make it more difficult for visual predators, such as fish, to find the slugs.  On top of that, any noxious taste would protect the slugs from predators, regardless of their hunting methods.  Overall, this model seemed to have fewer caveats than any of the others.

I thought I had come up with this idea on my own.  Then I rediscovered the above paper by Rasher et al. while I was updating a saved search in the Scopus database.  PNAS is my Wednesday lunchtime reading, and I am sure that I was excited to come across a paper about Elysia, so I am certain that I read it when it came out.  I am saddened by the fact that I forgot that I had read the paper, and assume that the paper got me thinking about kleptoplasty and chemical camouflage.

Have you figured out the connection to George Harrison yet?  You have to be getting on in years or love music trivia to remember, but he produced a popular song “My Sweet Lord,” during his post-Beatles solo career.  He ran into some legal trouble when the Chiffons’ record label sued him for appropriating the melody from their highly popular “He’s So Fine.”  If you go online and listen to both of them, it won’t take you long to think “dang, he stole their melody.”  Harrison admitted that he was very familiar with the melody, and the judge ruled that he had committed “subconscious plagiarism.”  In the same way, I had no memory of even having read the Rasher paper when I was formulating ideas and researching the biosynthetic capabilities of chloroplasts.  I just thought I was being terribly clever.  Nonetheless, it is highly likely that the paper was somewhere in the recesses of my mind during the process.  Are there any truly new ideas?

But, more importantly, how does one test this?  The first step is to make some predictions, and here are a few possibilities:

  1. Comparison of compounds produced by food plants, normal Elysia, and Elysia in which photosynthesis is blocked will show compounds produced by plants and normal slugs that are absent or significantly less abundant when photosynthesis is blocked.  All we need to do is make some extracts and find a potential collaborator who is willing to perform some gas chromatography-mass spectroscopy on a small budget.
  2. Elysia in which photosynthesis has been blocked will be easier to detect or more attractive to predators.  Placing a predatory mollusc or fish in a Y-maze with a choice between an arm containing a control and one containing  a photosynthesis-blocked Elysia might do the trick.  There are plenty of hungry wrasses that we could use for the fish test, either in the lab or in Bahia.  As far as predatory slugs, Navanax or Roboastra are good candidate predators for E. diomedea in Baja California.

There are likely to be more, better experiments, but the above provide a start.





What does Elysia crispata do on the reef?

Back from Bonaire, with a fresh puzzle.

Elysia crispata at The Cliff in Bonaire. January 11, 2017.

In research, as in life, there are things that don’t make sense.  Often these things make enough sense that you ignore them, choosing to focus on other mysteries.  One such little small, nagging issue is the question of what draws Elysia crispata to hard-bottom coral reefs, which lack obvious growth of green algae known to be their food.  Based on observations of many years, the slugs are not in transit, most are just sitting there.

Lettuce slug, with some Dictyota and red turfy algae, but not much in the way of greens. The Cliff, 1/11/17

My knowledge of the habits of Elysia in the wild is far from encyclopedic, but the species I know best have hearty appetites and stay close to their food.  E. diomedea are found on or near Codium in Bahia de los Angeles, and E. clarki spend most of their time face down in their food in aquaria.  This tends to hold true in the literature as well.  For example, E. tuca is generally found on its favorite food, Halimeda incrassata (Rasher et al., 2015, PNAS 112: 12110).  As a counter example, Middlebrooks et al. (2014) found that E. clarki were often found at sites that contained few or no specimens of their food plants (Penicillus, Halimeda, Bryopsis) determined via DNA barcoding.

Two slugs, in typical habitat. The Cliff, January 11, 2017.

In any case, I think I am justified in being puzzled by the lack of an obvious food source on the reef.  The photos in this post are all from a single dive at The Cliff, a site in the north-ish part of Bonaire.  We found maybe a dozen slugs, most in the face-down posture, which makes them look like large blobs of colorful frosting on the rocks.  The area had a lot of dead coral, which possibly serves as a substrate for the growth of food algae.  However, there were no obvious growths of green algae anywhere nearby, although algae such as Halimeda and Caulerpa are plentiful in mangroves on the island.

E. crispata, with a mix of turfy algae, including possible green filamentous species. The Cliff, January 11, 2017

Rather than snap a few photos of the more photogenic slugs, I thought it might be useful to document as many of the slugs as I could, with emphasis on the substrate.  Honestly, what you see is what you get; there are no large clumps of Bryopsis or Halimeda hiding around the corner.

Two E. crispata, surrounded by short, sparse turf. The Cliff, 1/11/17

What are these gals eating?  The most prominent alga is Dictyota, a brown alga which, based on known feeding habits, is an unlikely food.

E. clarki, surrounded by the flat leaves of Dictyota. No green algae in sight. The Cliff, January 11, 2017.

Are they grazing on the little strands of green algae that can be seen if one expands the photos and looks really hard?  Is this a late life stage that does not feed as much?  Are E. crispata truly crawling leaves, getting their energy from photosynthesis?  Is the much lighter color of E. crispata, compared to related species, like E. clarki and E. diomedea, a clue?

Slug from the beginning of the post, with wider field.




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.


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.


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.


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.


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


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.


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.


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.