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.
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:
There are likely to be more, better experiments, but the above provide a start.
The first version of the multi-tank slug system has served reasonably well, but it has had its limitations. The main problems have been the sprawl of equipment (note the tanks, dosers, auto-topoff, scattered about in the photo below), the low height of the shelves, which limits lighting options, and the cramped nature of the shelf unit, which makes maintaining or replacing tanks difficult. I also wanted a bigger sump, mostly to have a little more volume to prevent floods. To be honest, the photo makes it look even worse than it was, because I had moved a cabinet in preparation for the new system, leaving controllers and power supplies lying in a pile on a temporary shelf. Nonetheless, the system was long past due for an upgrade.
It took a few weeks to decide exactly how much space I could afford, and how to design the new shelves to accommodate existing tanks and allow flexibility in future configurations. I finally settled on a 60 X 16 inch footprint, which would accommodate the 15 and 20 gallon tanks on the top, plus a little extra space. It would be smaller than the space made available by the removal of one file cabinet and the old slug system, giving a little elbow room for maintenance and repair. I decided on 48 inch height. Enough for two shelves for tanks, and a bottom shelf for a sump. Three rows of tanks, a sump level, plus ample height for lights would just be too tall for me to reach easily. My experience with the current system has taught me that more tanks is not necessarily better, The second shelf would have room for a couple of 10-gallon tanks, or various combinations of 5- and 10-gallon tanks for smaller-scale experiments. The dosers and controllers would be on the bottom shelf with the sump, protecting them from splash, and making the system almost completely self-contained. By necessity, the chiller will have to be off to the side in order to move heat away from the tanks.
I decided to build the frame from 2X3 studs, and use 1/2″ plywood for the shelves. The studs should be plenty strong to support the 5 foot shelf, and 2X4s would be overkill and make the system that much heavier. The weight of the shelves is transmitted to the floor by 2X3s that run from the bottom of one shelf support to the top of the next. There is a second set of vertical 2X3s going all the way from the top shelf to the floor, providing more stability and support.
For paint, I chose a latex semi-gloss. I hope I don’t regret not using marine paint, but I am hoping that the primer plus three coats applied over the course of a week will be adequately waterproof. I tried to match the color of the walls in the office, but it turned out a bit more blue than I had intended. The piece of plywood on the back provides a surface for attaching controllers, power supplies, dosing pumps and drain brackets.
In order to simplify moving tanks in and out, I installed a 2″ drain with 8 openings along the bottom shelf, and a 3/4″ supply pipe with barbed valves along the top shelf. Installing and removing a tank will be as simple as connecting a supply hose and placing a flexible drain hose from the tank into a drain opening.
Then came the hard work of moving everything over. After Joanna pointed out that the shelves would not fit in the Jetta, I reserved a U-haul pickup truck, and we moved it from home to the office. Then it was simply a matter of spending 10 or so hours reinstalling plumbing, draining and moving tanks, setting up lights and pumps, mounting dosers and controllers, fussing with details, and cleaning up the resulting mess.
I am very happy with the results. My office is less cluttered, every aspect of the system is more accessible, the electronics are better protected from splash, and I don’t have to climb on a chair to work on the top tank. The Neptune Apex controller became a little buggy during the process, but I can again control and monitor the system remotely after a few reboots. The leak detection module is still not fully functional, so I am keeping fingers crossed that there will be no floods, large or small, until it is fixed.
One happy development is that the Bryopsis growing on the eggcrate in the 15-gallon tank (upper right in the photo above) has started to take off. Expect photos of that, plus a new shipment of marine plants, in the next day or so. Who knows, maybe there will once again be slugs in the Box of Slugs.
Back from Bonaire, with a fresh puzzle.
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.
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?
As I mentioned in the previous post, sometimes it is not so easy to identify an alga. In this case, it is a species that bloomed spectacularly when a local reefkeeper set up a new tank. The rock had been thoroughly cleaned and bleached, and no corals or fish had been added, so Alan did not expect the growth of nuisance algae. He was rather surprised to see a rapid, spectacular bloom of long, furry green algae.
At first we thought is might be Bryopsis (yay!), so it seemed worth trying to feed to the slugs. Once I saw and felt it, it was clearly something else. It was soft, like Derbesia, but longer and had branches that extended radially (like a bottle brush) from the main stem. Bryopsis feels coarser, and the branches extend in a single plane (like a fan). So, it was not one of the usual suspects. Nonetheless, it was worth throwing some into a tank to test whether the gals would eat it. They did not immediately plunge into it, as they would have for Bryopsis, but they seemed to find it palatable enough. Note the fine structure of the branches in the photos below.
The plant has some characteristics of the order Bryopsidales, such as the lack of clear cellularization. It looks like the plant is made up of a continuous, single cell.
Stalk, showing absence of cellular divisions. The little round bumps on the branches are reproductive structures. Scale bar = 1 mm. 12/27/16
Acrosiphonia spinescens from Algaebase, showing cellular divisions and hook-like branches. © Ignacio Bárbara
I thought a quick look at the DNA sequence would clear things up, but that was not the case. The closest match, Acrosiphonia, with 88% sequence identity. That’s not a very good match, and even though it looks somewhat like Acrosiphonia, the unidentified alga lacks several key features, such as the hooks on the branches (which cause mature plants to develop a dreadlocked appearance) and clear cellularization of Acrosiphonia. Plus, Acrosiphonia is a cold water species, unlikely to thrive in a warm reef aquarium.
The closest visual match so far is Trichosolen, which does have warm water species. The only species with rbcL sequence in the database (T. myura) is only an 86% match for DNA, so it’s probably not the one either.
By way of comparison, the usual pest algae (various species of Bryopsis and Derbesia) were only 82% – 83% identical, so we can at least rule out the possibility that it is an oddball species of one of those.
The hunt continues for a match. Not very satisfying, but some days are like that.
Happy Holidays to all of you fans of slugs!
Although the site and the project are devoted to adorable molluscs, we would be nowhere without algae. These days, I spend more time and resources trying to acquire, grow, and identify algae than I do attending to Elysia. It should not be a surprise, given the outsize role of algae in the biology of the slugs, but, until this project was underway, I had never given a lot of thought to the care and diversity of algae. Subsequent posts will describe some of the progress in algae care, but today we’ll focus on some systematics and molecular biology.
The plant in the photo above has been nagging at me for well over a year. I can’t remember exactly how it came up, but KP Aquatics mentioned that they had a species of algae they called “spongy sea pansy,” which was like Udotea, but larger and squishier. They were quite a bit taller than Udotea, grew in clumps, and were indeed quite spongy. Their biology is somewhat different from other algae in the order, in that the thallus (the body of the alga) dies back periodically, and a new one grows from the rhizoid (the rootlike part). In my experience, species like Udotea or Penicillus send out runners that produce new thalli, and the old ones just die off.
I have been referring to them as Avrainvillea, because they fit the description reasonably well, but had never done the hard work of verifying that it was not a similarly squishy genus, such as Rhipilia or Cladocephalus.
A real phycologist (algae specialist) would have probably started with a good microscope and species key. I took the molecular route, since I was already using PCR to amplify DNA from the rbcL gene in a few other species, and sending it off for sequencing.
Because I was testing new PCR machines, I had set up three independent reactions, and the results were the same. The screenshot below shows the results of a BLAST search for one of the sequences through the NCBI database, with the closest match at the top. The second best, with 98% of the nucleotides being identical, is Avrainvillea nigricans. The best match (99%) is to an “uncultured Ulvophyceae” clone from a study by Christa, Gould, Wagele, and their collaborators. If I read the entry correctly, the sequence is from kleptoplasts extracted from Costasiella, a Caribbean slug that feeds on…did you guess…Avrainvillea. To provide a little context, Cladocephalus and Rhipilia, the genera that were possible candidates based on appearance, were only 93% and 82% identical, respectively.
That is a pretty clear-cut result. It looks like Avrainvillea, it is squishy like Avrainvillea, and its DNA is essentially an exact match for Avrainvillea nigricans. It is Avrainvillea.
As you’ll see in the next post, the results aren’t always so easy to interpret.
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. 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 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.
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…
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.
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.
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.
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.
After a quick snip, she was back in the tank, and roaming around within a few hours.
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.
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.
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
Hard to say goodbye to the slugs as well.
As always, we were up with the sun. We got on the road early, with tubes of DNA on ice.
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.
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.
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.
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.
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.
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.
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.
There was even a little Codium. No Elysia visible, though.
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.
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.
The bottom seemed more conducive to Elysia, however. Plenty of Padina, but also significant patches of coralline and green algae.
We even found some snail or slug eggs. Not from Elysia, but a good sign that molluscs were about.
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.
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.
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?
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.
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