Journal Club: Kahalalide F, It Takes Three to Tango
One of my Slug Club students recently asked me whether it is really worth pursuing the study of obscure, squishy creatures in an era during which biological science seems obsessively focused on “translational” research to apply scientific discoveries to clinical problems. My response was that, while translational research is unquestionably important, the study of the biologically weird is where we discover unexpected fundamental truths about the way organisms function. This new insight often lays the groundwork for new areas of research, and can, in time, lead to improvements in people’s lives.
Which brings us to the paper at hand: Zan et al. (2019) “A microbial factory for defensive kahalalides in a tripartite marine symbiosis.” Science 364: eaaw6732 (published online ahead of print). This paper not only taught me something new about Elysia, but I discovered methods and biochemical pathways I had never heard of.
For those who do not want to bob and weave their way through the entire journey of discovery, here is the quick summary: The authors discovered a bacterium living within the species of Bryopsis algae upon which Elysia rufescens feeds. This bacterium is responsible for the production of a chemical compound called kahalalide F (hereafter “KF”). The following observations are what make the story more interesting:
- The bacterium makes KF by stitching together amino acids using a biochemical pathway very different from that used by animals and plants.
- The bacterium has adapted to the comfortable life inside plant cells by getting rid of many of its genes.
- Although E. rufescens concentrates KF in its tissues to deter predators, it does not maintain the bacteria in its tissues. This contrasts with the chloroplasts from Bryopsis, which the slug separates from the other cellular components and continue to photosynthesize.
If that sounds interesting to you, then read on.
It was very exciting to discover a paper about E. rufescens and its food plant Bryopsis, in the journal Science during my lunchtime reading this week. Science is one of the two most-read scientific journals in the world, so any article must be splashy and important enough for a wide readership. That a study about Elysia was deemed worthy of publishing there pleased me to no end. To be honest, the paper is not really about E. rufescens, nor about Bryopsis, but about a bacterium that lives inside Bryopsis, but the alga and slug still play prominent roles.
A quick literature search for E. rufescens (or a peek at the E. rufescens page on this site), will show you that the species has been mostly studied because it contains a defensive molecule called Kahalalide F (KF).
Kahalalide F: A Complex Defensive Compound
At first glance, KF looks like an organic chemist’s nightmare (or dream, I suppose), but on closer inspection it is a peptide, i.e., a short string of amino acids. As described below, KF has a number of unusual features for a peptide. From an ecological perspective, Becerro and colleagues showed that KF is a strong deterrent to predation. KF has also been found to have anti-cancer activity, so there is a medical motivation to study it.
The source of KF was not completely clear. It was assumed to be produced by the Bryopsis species (not yet assigned a species name, so I will simply call it “Bryopsis“) on which E. rufescens feeds. The slugs not only avoid the toxic effects of KF, but they concentrate the compound in their tissues at a higher level than is found in Bryopsis. KF has a few weird chemical features that suggested to researchers that it might be made by microbes and not the alga. For example, there is a fatty acid stuck to one end of the molecule, and it contains a number of “non-proteogenic” amino acids, such as ornithine or dehydrobutyrine, which are features of compounds produced by bacteria.
To test their hypothesis that KF has a microbial origin, and to better understand how it is synthesized, the team used a wide variety of methods, including metagenomic, metatranscriptomic, evolutionary genomic, and biochemical analyses, along with some more traditional fluorescence microscopy. If all the “meta-” and “-omics” has your brain in a knot, don’t panic yet; I will do my best to explain the methods as we go.
First, they chemically extracted the Bryopsis from their collection location to be sure it contained KF. That being done, they extracted DNA from the Bryopsis and any microbes that happened to be in or on the algae when it was collected. Using high-throughput sequencing, which breaks up DNA into fragments and sequences the whole genome, they analyzed the ribosomal RNA from the sample. All organisms have ribosomes that enable them to synthesize proteins, each ribosome contains RNA that helps it function, and the ribosomal RNA from each species has a unique sequence. The Bryopsis sample contained RNA largely from three bacterial classes, one of which was the Flavobacteriia. Of the Flavobacterial sequences, one species comprised 74 to 92% of the sample, making it the most abundant species associated with Bryopsis. To jump ahead again, they ultimately found that this bacterium was a new species, that it was responsible for the synthesis of KF, and gave it the provisional name “candidatus Endobryopsis kahalalidefaciens” (candidate kahalalide-making from inside Bryopsis).
How did they get there? First, they had to figure out the general way the peptide was made. If you are like me, and focus narrowly on multicellular creatures such as animals and plants, then you could be forgiven for thinking there is only one way to make a peptide, which is essentially a short protein. Turns out there are at least two.
RiPP or NRPS?
In the more canonical approach to making a protein, the cell uses a DNA sequence as a set of instructions to assemble a chain of amino acids. The DNA is read by an enzyme that makes (“transcribes”) messenger RNA (mRNA), and that mRNA is read (“translated”) by a ribosome, which sticks amino acids together in the correct order. Because some of the amino acids in KF are weird, they would have had to be modified after being assembled, so the authors termed this possibility “ribosomal peptide synthesized and posttranslationally modified peptide” pathway, or RiPP.
There is, however, another way that bacteria make peptides, called non-ribosomal peptide synthesis, or NRPS. Unlike a ribosome, which can make any protein or peptide that can be encoded by an mRNA sequence, NRPS requires a collections of enzymes customized for each peptide. NPRS enzymes are composed of modules that assemble the amino acids, modify them as needed, add fatty acids, and so on. This was completely new to me, but there is apparently quite a lot known about it.
In the good old days, it might have taken a lifetime to identify the biochemical pathway that the bacterium uses to make KF. Now that researchers are capable of sequencing, assembling and analyzing whole genomes, it just takes a lot of really hard work. The researchers sequenced all of the genes from organisms associated with Bryopsis, looking for either:
- A gene that encodes the sequence of a predicted peptide, which could then be transcribed to mRNA and translated into a peptide. This would indicate that KF is made via RiPP.
- A cluster of genes that encode the number and type of modules that would be expected to synthesize KF, based on the sequences of known NRPS enzymes. Finding this would support NRPS as the source of KF.
They did not find a gene with the sequence predicted to encode the KF peptide, suggesting to them that KF was unlikely to be produced via RiPP.
KF Is Made Through NRPS
They combined the data they had from the high-throughput sequencing with a second method of sequencing that produced longer fragments of DNA sequence (“longer reads”), and assembled the entire genome of a bacterium. The genome contained multiple clusters encoding NRPS enzymes, one of which, which they called NRPS-8, had features expected to be required for KF synthesis:
- A predicted condensation domain, which would be needed to chemically bind the fatty acid chain (red in the diagram above) to the first amino acid.
- A total of 13 predicted NRPS modules, as would be expected for an enzymatic pathway that makes a peptide consisting of 13 amino acids.
- Eight of the NRPS modules contain domains that are expected to convert normal L-amino acids to D-amino acids (shown in blue in diagram).
Based on these features, the authors were confident that they had discovered the enzyme that produces KF, and therefore that KF is synthesized by the bacterium through the NRPS pathway.
Including NRPS-8, which the genome sequence of the bacterium contained genes encoding a total of 20 NRPS pathways. Intriguingly, previous work had extracted at least 15 kahalalides from Bryopsis and E.rufescens. When the authors compared the predicted NRPS pathways to the structures of the kahalalides, they could match eight of the “chemotypes” (chemical compounds with the same amino acid sequence, but differing in modifications) to bacterial NRPSs. They conclude that “Candidatus Endobryopsis kahalalidefaciens” (hereafter “cEK”) is responsible for at least nine, and probably all, of the kahalalides produced by Bryopsis.
The Bacterium Is Losing Genes
Based on the complete genome sequence, it was clear that cEK has lost some genes. When they compared its genome to a closely related, but free-living species, they found hundreds fewer genes. The missing genes encoded proteins that performed such functions as DNA repair, detoxification, chemotaxis (moving toward or away from chemical stimuli), adaptation to unusual conditions, and, notably, synthesis of amino acids. All of these would be needed for living in a cruel, variable world, but may not be necessary for a comfortable life associated with an algal cell that provides stability and nourishment.
The fact that the authors were unable to culture the bacterium separately from the alga supports the idea that it is dependent on Bryopsis for survival. Further, by labeling DNA specific to cEK, they showed that the bacterium is found inside the plant cells (see panel from Figure 4, below).
Figure 4D from Zan et al., showing the location of bacteria inside a cell of Bryopsis sp. Yellow dots indicate bacteria labeled with a probe specific for “Candidatus Endobryopsis kahalalidefaciens,” and are indicated by arrowheads labeled “cEK.” Other bacteria that do not contain the cEK marker, are labeled red, and are indicated by the arrowheads labeled “OB.” The cell wall of the alga is labeled blue.
Therefore, cEK appears to be an “endosymbiont,” an organism that lives symbiotically inside the cells of another, and is probably completely dependent on its host alga for survival. In exchange, the bacterium provides a collection of toxic compounds that reduce grazing on the alga. The importance of kahalalide production to the bacterium is underscored by the observation that a full 20% of the genome is devoted to genes devoted to NRPS pathways.
Slugs Do Not Host cEK
As a devotee of Elysia, you are familiar with kleptoplasty, the phenomenon by which the slugs take the chloroplasts from their algal food and maintain them in their bodies as an energy source. If the slugs have a propensity to keep useful cellular components in their bodies, one might ask whether they also store the cEK as a constant source of KF to deter predators from eating them.
The authors confirmed previous work that E. rufescens‘ tissue contains KF, and went on to look for evidence of cEK bacteria in the slugs’ bodies. Using highly sensitive methods to hunt for cEK DNA in the tissues, they found no evidence that the slugs contained significant populations of the bacteria. Attempts at labeling tissues with markers specific for cEK were also unsuccessful, despite positive labeling of other bacterial species. They conclude that, in contrast to their theft and use of algal chloroplasts, E. rufescens digest the bacteria fully when they suck the sap out of the Bryopsis cells.
Conclusions
Once again, biology comes up with a story that is better than fiction. A bacterium infects an alga, takes up residence, and starts using an unusual biochemical pathway to produce nasty compounds that protect the alga from being eaten. Natural selection favors those algae that contain the toxins (and can tolerate them), so the bacteria are now found throughout the species, and possibly even more broadly. The sheltered existence inside the alga allows the bacterium to lose many of the genes it would normally rely on to survive in the outside world, eventually rendering it incapable of independent life. Elysia rufescens can tolerate the toxins produced by the bacteria, and develops mechanisms to concentrate the kahalalides in its tissues.
Do Byopsis pennata or B. plumosa, the favorite foods of E. clarki, contain cEK or a relative? I expect we will know soon enough.
Because the goal of this post was to bring attention to the original work, I have glossed over a huge amount of information regarding the methods used, the biochemistry of kahalalides, and the genomic structure of cEK. I would encourage anyone who is interested to get access to the original article at Science Magazine.
Slug Science Inches Forward
It has been a busy semester on several fronts, and the project has crawled forward a bit.
I am most interested in neurobiology and behavior, so we have moved from studying the chemical ecology of Elysia clarki to working out its behavioral response to external stimuli. The ultimate goal of figuring out how its nervous system encodes sensory input and motor output. Accomplishing this requires understanding of both the theoretical and technical aspects of molluscan neuroscience. We had three goals for Spring semester: 1) develop a stronger knowledge of the literature describing the behavior and nervous systems of opisthobranch molluscs, focusing mostly on nudibranchs and sea hares; 2) work out methods for reproducibly recording from slug neurons; 3) get a better sense of the slugs’ light preferences, in terms of spectrum and intensity.
USG Slug Club, 2019. From left: Josue, Nana, Marianne, Savana, Abigail, Cecilia, Dave. 4/12/19
The intrepid group of seniors and I got right to work.
For goal 1, we held a Slug Neurobiology journal club every Friday for the first 10 weeks of the semester. When we started, I was still pretty fuzzy on the details of the visual and nervous systems of opisthobranchs, and had only a vague idea of how they manage to crawl. I generated a list from multiple overlapping searches for papers describing the visual and motor systems of slugs, and Slug Club students and I presented about 18 papers, along with many more papers required for background. Despite nudibranchs being a diverse group, most papers described either navigation and swimming in Tritonia, or learning and memory in Hermissenda, with a few papers on Pleurobranchaea, Aplysia, and some snails thrown in for good measure. After our deep dive into the literature, we have a much better idea of mechanisms of light sensing, ciliary propulsion, and steering.
Goal 2 was to record from Elysia neurons. In spring 2018, we had found a good atlas of the Elysia nervous system, and had made a few recordings. However, the nervous system is surrounded by a tough sheath made of connective tissue, which makes it difficult to get delicate electrodes into the cells.
At this point, I needed a colleague to give me some pointers on getting the sheath off, or at least softening it up, but one can count the number of people recording from sea slug nervous systems on one hand. Fortunately, Paul Katz and his lab have extensive experience. Paul responded rapidly to my email, and gave me some excellent pointers. He was excited that there is another captive-bred sea slug being developed for neurobiology, and has been developing Berghia, a nudibranch that is also relatively easy to raise, as a neurobiological model system.
Berghia has some distinct advantages, such as a two month generation time (Elysia takes about 4 months), and surplus slugs can be sold to aquarists to control pest anemones. However, Berghia is much smaller and not kleptoplastic. Might be a good “normal” species to use for comparison with Elysia, though.
Even though the neurons that mediate the behavioral response to light are probably in the cerebro-pleural (which get inputs from the eyes) and pedal ganglia (which send outputs to the foot), I decided to start with the abdominal ganglion. It has a lot more big, pretty cells that should be easier to impale with a microelectrode, which increases the chance of success. With Paul’s advice, were able to get the sheath off and record from some large neurons in the abdominal ganglion.
Josue, Marianne and I practiced impaling neurons, and got some nice stable recordings. The neuron above fired action potentials at regular intervals and received a lot of synaptic input, indicated by the large bumps between the spikes. Because we currently know nothing about its connections, the significance of the neuron’s pattern of activity is unclear.
Another neuron was quiet at rest, but fired one or a few action potentials when stimulated with injected current. As with the previous cell, we know nothing about this neuron, but it provided an opportunity to practice techniques associated with current injection.
Time permitting, the next step is to record from neurons in the cerebro-pleural ganglion and find some that respond to light, and to look for others that control locomotion.
For goal 3, working out the spectra and intensities of light that Elysia prefer, you will have to stay tuned. The students just spent the past month gathering data, and should finish analyzing it within the next week or so.
Elysia clarki Eat Valonia, Too.
At least a little.
As often happens, I kept too many babies from a recent brood, and they rapidly consumed the large growth of Bryopsis in their tank., Algae production was a little slow in the system, so I chose to reduce their ration. I did not exactly starve them, but there was not always Bryopsis in the tank. There was , however, quite a bit of the pest algae Valonia, known as “bubble algae,” because it grows as clusters of large unicellular vesicles. Although I periodically have tried to remove it, it was thriving in the tank with the young slugs.
Elysia clarki, about 3 cm long, with empty Valonia bubbles. A large, empty bubble can be seen by the slug’s tail. 2/26/19
The slugs appeared to be feeding on the algae, and some of the bubbles, which are normally intense green, became clear. Although it would require DNA sequence analysis of the slugs’ kleptoplasts to be certain, the circumstantial evidence indicates that they are sucking sap from the algae. It makes sense, because Valonia bubbles are large single cells, which would allow a slug to have a big meal with just one puncture.
A recent paper by Mike Middlebrooks and collaborators (Biol. Bull 236:88, 2019) demonstrates that Elysia crispata (with which E. clarki is most probably synonymous) eats a wide variety of algae in the wild. In the aquarium, it looks like we can add one more species.
The good news is that the slugs seem to be wiling to make use of Valonia when necessary. Unfortunately for anyone with an outbreak, the slugs consume them so slowly that there is little chance they would eradicate an infestation.
Slugs of Bonaire 2019: Why So Blue?
Just back from a week diving in Bonaire. The focus was more on recreation than slugs, but we came across a few Elysia crispata along the way. As usual, we found the highest numbers in the more degraded sites to the north, but the slugs were always on long-dead coral regardless of where we found them.
For this post, I wanted to highlight the intense blue color of the slugs we found this year at Karpata, a site near the north end of the island. I have previously posted photos of blue slugs that have turned up at nearby sites, and a quick Google search will provide many more examples.
Elysia crispata at Karpata, Bonaire, 1/17/19. Note the patch of Dictyota algae to the lower right.
This seemed to be a common color pattern. Each photo in this post shows a different individual, but their colors are remarkably similar.
Even though the colors look downright artificial, I did little more than adjust the contrast of the photos. These slugs really look like this. I emphasize this point, because the range of colors of most species of Elysia is limited to shades of green.
Despite the striking appearance, the color pattern may provide camouflage. When the slugs were curled up and their rhinophores hidden, they did passable impressions of sponges. E. crispata are almost invariably scrunched up when we come across them on the reef, so this may be a successful way of hiding in plain sight.
The color of many Elysia species is derived from pigments taken from their food plants. For example, my E. crispata and E. clarki hatchlings have little color until they start feeding, and then take on the green color of the chloroplasts they sequester in their digestive diverticula. Costa et al, (2012), provide a nice example of this effect. They showed that E. timida could be either green or brown, depending on whether they were fed Acetabularia algae that had taken on different colors based on having been acclimated to low light (green) or high light (brown).
So what could be making these slugs so blue?
One possibility is that they are feeding on Dictyota, a brown alga that grows abundantly on the dead coral in this area. Although the Dictyota in the photo at the top of this post is not impressively blue, the alga is certainly capable of producing intensely blue color. Most species of Elysia feed on green, rather than brown algae, but it is not unheard of for them to branch out (e.g., Trowbridge et al., 2010). It is also possible that the slugs are consuming one of their more usual food plants that happen to be producing high levels of blue pigments. It would be interesting to take a small tissue sample and find out what the slugs have been eating.
It also brings up an interesting question regarding the coloration of Elysia in general. In addition to their green background, many species have distinctive patterns, such as the colorful markings on the parapodia and rhinophores of E. diomedea, below, or the colorful edges of the parapodia in E. clarki.
At a higher magnification, one can see concentrations of pigment spots, such as those shown below in the parapodium of E. clarki. Are these spots of concentrated pigment derived from their food plant, or are they synthesized by the slugs themselves?
As far as I can tell, there is no answer in the literature, but who knows what will turn up next.
Map of Elysia diomedea at Bahia de los Angeles
I have added a rudimentary map of the locations at which E. diomedea has been found during our fieldwork during the past few summers. At the moment, it provides a framework to which we can add more sightings as we turn up more slugs.
The short summary is that suitable habitat, consisting of rocky bottom with growths of Codium macroalgae, is distributed throughout the bay, and that the ability to find Elysia can vary wildly at the same site from one day to the next. With enough time and effort, I expect the little slugs would be found anywhere there is something to eat.
Back to Baby Food
Do they prefer the food of their infancy because it tastes better, is more nutrient-rich, or is easier to eat? Maybe they are just nostalgic for the food they ate when they were young.
Two-month-old Elysia clarki having an early morning crawl among the turtle grass and manatee grass in Box of Slugs 2. 9/29/18
A few of the youngsters from the most recent brood have moved in with their mom in the tank at home. They were weaned from Bryopsis plumosa to B. pennata once they were a few weeks old, and have been growing steadily.
For a few reasons, not all logically sound, I had assumed B. plumosa would be harder to culture. If an aquarist has a problem with Bryopsis, it is invariably B. pennata. Combined with the fact that B. plumosa is crucial for hatchling survival, and that I had to travel all the way to Tampa to get it, I figured I would have trouble keeping it going. As a consequence, I always shift young Elysia to B. pennata when they were ready to eat it.
Despite my preconceptions, B. plumosa is thriving at this point.
Bryopsis plumosa culture tank (surface is 10″ by 20″), stuffed to the top with algae. 9/14/18.
To the uninitiated, the B. plumosa tank would look like a mat of unruly glop. To an aficionado such as myself, it looks like an actively growing, unruly mat of precious food for hatchling Elysia. It is a “half-ten” aquarium: a ten-gallon tank, but only half the height (OK, so it is really only a 5-gallon tank), which provides a lot of surface but only a few inches of depth. The growth form is very different from B. pennata, which tends to be long and feathery. B. plumosa grows more like clumps of moss. I am concerned about the tank being taken over by B. pennata invading from elsewhere in the system, but so far so good on that front.
At the moment, the growing conditions are:
- Nutrient dosing at about 150:30:1 Carbon:Nitrogen:Phosphorus, plus Guillard’s F/2 at 2 ml/day (note this is the whole system, not just this tank).
- Circulation using a powerhead and rotating output (see Slug Safe Circulation). Trust me, it is in there somewhere.
- Lighting by an Evergrow S2 hydroponics light, about 6″ from surface.
To get to the point of this post, I had enough B. plumosa to throw some to the adults.
Elysia clarki eating Bryopsis plumosa in Box of Slugs 2. 9/26/18.
Unsurprisingly, the slugs ate it. I did not expect, however, that the largest female would rarely leave the clump of algae until it was completely consumed. She very much preferred the plumosa. I brought another clump home, and she is still sitting on it, along with one of her kids. The tank is full of B. pennata, at all levels, but the slugs stick right to the single clump of B. plumosa on the surface. It may be my imagination, but the big one seems larger and more colorful after a few weeks of eating B. plumosa.
Box of Slugs 2, with plenty of Bryopsis pennata among the turtle grass. 9/26/18
So, anecdotally, even grown up slugs prefer B. plumosa. Another thing to put on the list of things to test more rigorously. For now, one can speculate about why they seem to prefer it, and what cues (smell? texture?) draw the slugs to the algae.
Fat Babies Have No Pride
In the meantime, another brood has hatched and has started to grow. I am not sure why (I am not in any way a musical person), but when a new brood starts to eat, Lyle Lovett’s “Fat Babies” runs through my mind almost continuously. I have no idea whether the song has a subtle, subversive message (if so, I apologize for any offense), or whether it is simply about chubby infants not being proud.
Juvenile slug crawling among B. plumosa, with diverticula full of chloroplasts. 9/28/18
They are feeding and growing, and it looks like we’ll have several dozen ready for activities in the spring.
That’s OK. Who needs pride?
Bahia Adventures Part 5: Slugs Taste Bad (Again)
One of the goals of the Bahia field season was to look a little deeper at the interaction between kleptoplasty and chemical defense. The general idea went something like this:
- Kleptoplasts continue to photosynthesize after they take up residence in the slugs’ tissues.
- Slugs produce defensive chemicals that make them taste bad.
- Kleptoplasts could be the source of the defensive chemicals: is photosynthesis required to make them?
Based on this line of thinking, we hypothesized that slugs deprived of light should be less distasteful than those kept in bright light.
To put the hypothesis to the test, we set up two tanks that were nearly identical except for lighting. They were plumbed into the same sump and chiller, so their temperatures and chemistry were essentially identical, but one was illuminated by a high-intensity LED fixture to support photosynthesis (PAR ≥100 mol m−2s−1), while the other was shaded to reduce the light by 100-fold (PAR < 1 mol m−2).
Tanks ready for slugs. Each contains a nearly identical collection of rocks and algae. The tank on the right is illuminated by a high-output LED fixture, while the one on the left is shaded by black felt, resulting in a 100-fold difference in intensity. 6/30/18.
Once we obtained the permit and were allowed to collect, we randomly separated slugs into two tanks that contained roughly equal amounts of Codium on which they could feed. They were allowed to feed at will, because the goal was to test the role of photosynthesis in generating defensive chemicals, not the effect of starvation.
The original plan was to use mucus from experimental (unlit) and control (lit) slugs to make food cubes, then test which ones were eaten by fishes in the bay. However, based on experiments at USG, and the fact that we would not be able to extract enough mucus from our few dozen slugs, we decided to test the effects of tissue extracts from whole slugs. Surprisingly, the students were not as sad as we might have expected that they had to purée their pets.
For the description below, whenever I write “we,” I really mean Ric and the students, because he was very much in charge of this project. Most of the below photos are his.
Zaira weighing carrageenan for a batch of food cubes. 7/3/18.
The process was as quirky as any of the other experiments we have done in Bahia. To make the gel base of the food cubes, we needed to dissolve carrageenan in water, which required heating the water to boiling. The easiest method is to microwave the water, but the only available microwave oven had a single working button (“popcorn”), which turned the microwave on (after a disconcerting delay) for 3.5 minutes. It took some practice and finesse to turn the machine off at the right time, but they managed.
Zaira stirring food and carrageenan mix. Dodgy microwave oven is at right, and some of the molecular crew (Lili, Dave, Elizabeth) are in the background. 7/3/18.
Fish food pellets were ground and added to the carrageenan solution to make the base food cubes. Mucus or tissue were then added to a portion of the food, depending on the experiment.
Food mixed and ready to pour. 7/3/18.
Once heated and thoroughly mixed, food was poured into silicone ice cube trays to make 1 cm cubes. Silicone O-rings, used to secure the food cubes to clips during experiments, were placed into the molds and held steady with acrylic rods.
Bennie setting up rings in food forms, getting ready to pour how food mix. 7/3/18.
Once the O-rings were in place, food was poured into the molds and evened out using a knife. After cooling for a bit, the whole assembly was stuffed into a ziploc bag and refrigerated until needed.
Food cubes poured and ready for use. Note acrylic rods to keep rings in place. 7/3/18.
At experiment time, food cubes were attached to monofilament lines to be anchored in the bay. The bottom end of the line was tied to small lead weights, while the top was held afloat by a 16-20 oz soda bottle. Food cubes were secured by O-rings to plastic safety pins tied to the line at regular intervals. Several bugs needed to be worked out. For example, we started with lightweight fishing line, but it had a tendency to break in the surge of the intertidal zone, causing loss of the whole bait line. We shifted to a much heavier gauge, which apparently scared the fish away, because all food cubes were present after 24 hours. Ultimately, we settled on something in between, and could start gathering real data.
Keyla, demonstrating system for placing food cubes in the bay. Plastic safety pins are tied to monofilament fishing line, which is suspended between the float in her right hand and the weights by her left foot. Food cubes are then clipped to safety pins via embedded O-rings. 7/13/18.
Once food cubes were secure, the lines were placed deep enough to keep them afloat at low tide.
Food cubes suspended in water column in bay. 7/13/18.
Lines were collected after a predetermined time (see below).
Collecting line of now-empty clips. 7/13/18.
For the first round of experiments, cubes were left in the bay for 24 hours. During this time, essentially all of the control cubes (i.e., without tissue or mucus) were eaten, as were most of the experimental cubes. Nonetheless, fish appeared to eat fewer cubes containing tissue from slugs kept in the light, compared to those kept in the dark.
Effect of food contents on consumption by fish in the bay. When collected, each line was given a score, with 1 = all cubes intact, and 3 = all cubes gone. In this experiment, all control cubes were missing (and presumed consumed) in 24 hours, as were cubes made from tissue prepared from slugs kept in the dark for one week (right most bar). Food made from slugs kept in light (second from right) had a lower score than the other groups, but the difference was not significant. Sample sizes: 18 control, 12 mucus, 12 lit, 12 dark.
Because there was concern that missing cubes may have fallen off rather than being eaten, they also tried leaving the cubes in the bay for only one hour. There was a slight decrease in consumption, but the overall result was the same: tissue from dimly-lit slugs was eaten more often than that from well-lit slugs. The students were running out of time for these experiments, so the results are preliminary and nothing is statistically significant.
Results of leaving the food lines in the bay for an hour. Food made from slugs kept in the light (middle bar) had a lower consumption score than those without slug tissue (left bar) or made from slugs kept in dim light (right bar). Sample sizes: 18 control, 24 lit, 24 dark.
To summarize several weeks of hard work, it looks like there is something to pursue. The sample sizes are small, but there is a consistent effect of slug tissue in reducing consumption by fishes, and this effect is reduced or eliminated by keeping slugs in the dark. Now that the bugs have been worked out, it will certainly be worth trying the experiment again if there is an opportunity to spend a few weeks making food and setting lines.
Thanks again to the USG Slug Club, who pioneered the foodmaking methods, and took care of the slug system while I was away.
Happy Fourth Birthday!
Baby Elysia, about three weeks old. Rhinophores well developed, diverticula full of chloroplasts from Bryopsis plumosa. 8/17/18
It seems appropriate that the site has reached an age at which kids are always asking “why, why, why?”
Looking back, the site is absolutely a reflection of my working style: make a rough plan, give it a shot, modify, repeat. Not the work of a perfectionist, and there are some posts and pages that make me cringe when I look back. Nonetheless, as a repository of pictures, information, and ideas, and a journal of my meandering process of discovery, the Solar Sea Slug Blog has exceeded expectations.
The past twelve months have been action-packed:
- The student Slug Club at USG explored chemical ecology. We spent fall semester doing a deep dive into the literature on Sacoglossan chemical ecology, learning a lot about the chemicals the slugs could be taking from food plants. In spring semester, we took the ideas into the lab by testing whether mucus and tissue from E. clarki could keep shrimp from eating food cubes.
- The experiments could not have been done without the availability of many slugs of different sizes. This leads into what was probably the most important breakthrough of the past year: rearing slugs from egg to adult. Over the years, the slugs have laid thousands of eggs, most of which hatched and then starved. This year, I discovered that the hatchlings will readily eat Bryopsis plumosa, and have ended up overwhelmed by babies. Knowing how to rear the hatchlings, and having a culture of B. plumosa on hand, is a huge leap forward. Now I can raise them as needed, at least in principle.
- The breakthrough in rearing of Elysia would not have happened with input from Skip Pierce and Mike Middlebrooks in Tampa. I had a great visit down there, and learned a ton about several species and the people who work on them. As a bonus, I brought back my first culture of B. plumosa to get the baby slugs started.
- We rediscovered a classic paper on Elysia neuroanatomy, recorded the the first action potentials in Elysia neurons. There is still some work to be done to get routine, stable recordings, but this is another big step in the right direction.
- All the stuff that happened in Bahia made for a great summer for the Solar Sea Slug Blog. We set up a lab, collected slugs and algae, did surveys, tested feeding methods, and obtained some useful DNA sequences from E. diomedea and their food algae. Stay tuned for the results of the feeding tests in the bay, and a map of where E. diomedea is found in the bay.
Self-indulgent reflections are not as interesting as sea slugs, so I have included some links to cool stuff below:
- A National Geographic article about the potential decline of Elysia chlorotica, possibly the most solar of the solar sea slugs. Thanks to Ric DeSantiago for forwarding the link.
- Here’s another on theft of stinging nematocysts by cute little nudibranchs. Thanks to Drew Talley.
- Because I like seahorses, especially small ones, here is a story about newly-described pygmy seahorses in Japan. Thanks to Joanna.
Bahia Adventures Part 4: Culmination But Not The End
The only thing better than a picture of a slug is a photo of a slug projected on a giant screen and admired by a bunch of people.
The Photobiology students presenting their results at the annual Report to the Community at the San Diego Museum of Natural History. 8/9/18. Photo Drew Talley
A few weeks ago, the Photobiology students presented at the Ocean Discovery Institute Report to the Community, held at the San Diego Museum of Natural History. This is the opportunity for the students to tell their parents, the community, supporters, and everyone associated with Ocean Discovery, about what they did this year in Bahia.
They started with the background and general questions that drove the research: what is kleptoplasty, and how can we learn more about the mechanisms and evolutionary benefits of the phenomenon? Because they had limited time on stage, they focused on the work we did collecting samples of slugs and algae for identifying food plants.
The punch line was satisfying. We found that the DNA sequence of the Codium algae collected in front of the station and that from the slugs’ tissue samples came from Codium decorticatum. I managed to get about 400 bases of sequence from the slugs and the algae, and it was about a 99% match to C. decorticatum.
Short region (54 of >400 bases) of rbcL gene sequences derived in Bahia this summer to show similarities of our data (top two lines) with Codium decorticatum sequence in NCBI database. Elysia diomedea and Codium collected in Bahia were 99% identical to C. decorticatum. Closely related C. fragile only 91% identical (differences indicated by asterisks at bottom). Asterisk at top indicates difference between E. diomedea and Codium.
The figure above shows a short, representative stretch of DNA from the rbcL gene, from Elysia diomedea and the Codium species we extracted in Bahia (top two rows), along with sequences from two species of Codium from the National Center for Biotechnology Information (NCBI; bottom two rows). The Codium from Bahia matched C. decorticatum almost 100%, whereas the match was only about 91% for the very similar C. fragile.
There were, however, a few differences between the kleptoplast DNA from E. diomedea and C. decorticatum (e.g., asterisks above E. diomedea sequence). At this point, we don’t know whether this is due to variation within the species that the slugs are consuming, or the slugs are eating more than one species. We are still waiting for results from NextGen sequencing, which should be more informative and may clear up this puzzle.
A couple of notes about the algae. First, the Codium and slug sequences are identical to those we obtained in 2016. At the time, however, C. decorticatum had not been entered into the database, so all we knew was that we had a new species. The other observation is that the species of Codium we sampled (see photo of tissue here) is relatively low-growing and branchy, unlike the usual tall, sparsely branched appearance of C. decorticatum. There were specimens that looked like that (e.g., here), and it is likely that the appearance of the alga depends on the local conditions.
Most importantly, the students did a fantastic job of presenting. I was away visiting family, but I got multiple independent reports from people in the audience who talked about how strong and clear the students’ presentations were.
The Photobiology crew, as drawn by the ever-creative and wonderful Ric Desantiago. Pleas visit his site (Da Hood Scientist) to check out more of his observations and artwork.
I am very pleased and proud to have played a part in the students’ project this summer. Despite all of the up and downs and improvisation, it was a deeply satisfying experience. I am always amazed how a misanthrope such as myself can have such a great time packed like a sardine into the field station with so many people. Thanks to the students, the Ocean Discovery staff, Ric, Thiago, the visiting scientists, and everyone. Especially, thanks again to Dr Drew Talley, mi gemelo de otra madre, for making it possible.
Dopplegeezers at BLA station, 7/5/18
I still have a few posts left regarding the results of the feeding assays and slug surveys, and hope to have them ready in a few weeks’ time.
Bahia Adventures. Part 3: The Late Middle
Elysia diomedea in field station tank. 6/30/18
Having the permits meant that we could get straight to work. The first order of business was to get the tanks ready and find some slugs. As a happy coincidence, the tides were very low, allowing us to explore the tide pools for interesting organisms. Even better, we could collect rocks and plants for the slugs tanks by simply picking them up and putting them into a bucket, rather than having to dive down to get them. We quickly had the tanks ready for sluggy inhabitants.
Tanks ready for slugs. Each contains a small collection of rocks and algae. The tank on the right is illuminated by a high-output LED fixture, while the one on the left is shaded by black felt, resulting in a 100-fold difference in intensity. 6/30/18.
The tanks were also ready for experiments. One of the hypotheses we wanted to test was that photosynthesis by the slugs’ kleptoplasts contributes to the presence of bad tasting compounds in their mucus and/or tissues. To test this hypothesis, half of the slugs would be kept in the dark for a week, while the others would be live under lighting adequate for photosynthesis. For the experiment, one tank would be lit by strong LED lighting, with photosynthetically active radiation (PAR) above 100 µmol photons per square meter per second (plenty for photosynthesis), while the other received less than 1% of that amount. The tanks were connected to the same life support system and had similar amounts of algae and rocks, so the conditions in the two tanks were nearly identical.
The plants and rocks were an excellent start, but our luck was even better. We managed to find seven relatively large Elysia in the tide pools just in front of the station. Even though I have not found Elysia in the same locations during hundreds of hours of snorkeling, they were present in abundance during low tide. It looked as thought the forces controlling the bay were smiling upon us after thumbing their noses at us for a week.
The slugs settled in well, exploring their new habitat and lounging on the algae.
Elysia diomedea in tank at BLA station. 6/30/18.
Elysia diomedea exploring rocks in new home. 6/30/18.
There was even reproductive activity. As can be seen below, slugs appeared to court each other.
Elysia diomedea courting in a tank at the BLA station. 7/2/18.
They deposited multiple egg masses. The masses contained thousands of tiny white eggs without extra-embryonic yolk, which is consistent with what others have observed for the species (see e.g., the Sea Slug Forum) .
Egg mass of Elysia diomedea. 7/4/18.
Egg mass of Elysia diomedea in BLA station tank. 7/3/18.
Although, I did not have a camera with sufficient resolution to show the details of the masses, I would agree with others that E. diomedea embryos are smaller than those of E. clarki and E. crispata. The relatively small size of the embryos, and resulting smaller amount of yolk, means that E. diomedea are probably “planktotrophic,” hatching earlier and feeding on plankton in order to finish development. Larger embryos, such as those of E. clarki and E. crispata, are “lecithotrophic” living on plentiful yolk stores until it is time for the veligers to settle and start feeding on Bryopsis.
Having slugs and algae also gave us the chance to do some DNA extraction. The students took small samples from a couple of slugs, and some local algae, then extracted the DNA using the DNeasy Plant kit from Qiagen.
Codium specimen. A small sample was removed for DNA extraction and amplification. 7/3/18.
Unidentified green alga. DNA was sampled for identification. 7/3/18.
After a few rounds of incubations and separations, the students had generated tubes of clear liquid that presumably held DNA.
Elizabeth, Maria, Keyla, and Lily holding tubes of DNA extracts. 7/3/18.
Meantime, there was lots of other stuff going on. Ric had been leading the other half of the group in troubleshooting and starting the feeding experiments. Because we wanted to test the palatability of the tissues from slugs kept in the dark, we needed to modify the feeding assay we used in tanks at USG for use with fish in the bay. It seemed simple enough to hang food cubes on fishing line that is anchored by lead weights at one end and held in the water column by a float at the other. Figuring out the right thickness of fishing line, and how best to secure the lines to the weights and floats, required a good bit of trial and error.
The students were also working on their scientific presentation skills. In one exercise, Ric had them give short summaries of the work while standing in the bay, in order to have them project their voices in a noisy, distracting environment.
Students giving talks while standing in the bay.
7/2/18.
Unfortunately, two weeks had passed, which meant that it was time for me to leave the students, the great people from Ocean Discovery, my friend Drew, the bay, and the slugs, and return to Maryland. The students were doing great, the assays had started to work (although the first round of PCR amplification of the DNA extracts was not successful), Ric had everything well in hand, and there would be a real molecular biologist arriving in a few days to act as my substitute.
I watched my last sunrise at Bahia for the season.
Sunrise over the bay.
I grabbed some of the DNA, packed my things, said my goodbyes, and headed north to San Diego.
Slug Life! The Photobiology group on my last morning. From left: Keyla, Bennie, Zaira, Me, Maria, Ric, Lily, Diana, Melanie, Elizabeth. 7/6/18.
At this point, we were about halfway through the field season. After all the hard work and adaptive management, will there be results?
Recent Comments