Posts having to do with the site
Based on the absence of posts for the past few months, you can be forgiven for thinking the project is dead or dormant. In fact, the situation is just the opposite. The semester has been so busy, there has not been time to write more than a few draft posts. I envy people who have the time and energy both to do things and to write about them.
Bryopsis growth has continued strongly, producing about 150 to 250 grams per week of tasy slug food. The slugs have responded by growing huge and laying eggs.
The E. clarki in the Box of Slugs 2 at home have produced a series of large egg masses (At least 12 so far, including the fresh batch below), providing many opportunities to test larval rearing ideas. As has happened in the past, the eggs hatch, the veligers settle, but the baby slugs do not start eating. I will finish the post describing our attempts, and a potential breakthrough, very soon.
The Chemical Ecology independent study project at USG was a big success. We went through a pile of articles about kleptoplasty, chemical defense, phototaxis, etc, which will result in at least a few Journal Club posts here. We did a little PCR, positively identifying some of the algae we have been working with (more posts to come!). The students also shamed me into contacting Skip Pierce and Mike Middlebrooks, about possible reasons the hatchlings haven’t been feeding, which may have led us to a solution.
As we start thinking about projects for next semester, I was getting a little despondent that none of the collectors have been able to provide E. clarki, since Irma struck the Keys. It was a huge relief when Blue Zoo Aquatics sent me a small group of very healthy looking E. crispata last week. Pricier than I am used to, but it’s just good to have them.
I look forward to finishing posts about all of the above, along with any surprises and breakthroughs we encounter.
The world seems to be in flux these days, but one can take a certain comfort in knowing that there are thousands of little green slugs sucking sap out of algae in shallow waters throughout the world.
Despite the travel craziness, the E. clarki and E. crispata at home have been having a nice summer. Now that the Bryopsis factory at Shady Grove is consistently producing algae, the slugs have been face down in food almost 24/7. The slugs are as big and colorful as I have ever seen.
This is looking like a great time for Elysia and slug science.
For example, at the Universities at Shady Grove, we’ll be starting a literature review on chemical ecology. A small group of students has agreed to join me in weekly journal clubs covering chemical ecology, algal secondary metabolites, plant herbivore interactions, kleptoplasty, and other topics related to Sacoglossans and their food plants.
The preparations for Bahia in 2018 are also moving steadily. We are identifying fudning, assembling equipment,and making detailed plans to extend the work on identifying algal food and defensive compounds of Elysia diomedea.
Check back for updates and insights as they develop.
Once again, I find myself apologizing to the hordes of Solar Slug fans for the long period of silence. After the frenzy and freedom of summer, it has been hard to find time to experiment, or even mess with the site, but I am hopeful that things will change now that the semester is tapering off. There has been a little news along the way.
A few weeks ago, the students participating in the Biology Honors Research Program at UM College Park invited me to give a seminar. Since my fly work is old and stale, it seemed like a good chance to talk about the beginnings of the Solar Slug project. As far as I could tell, the students found the ideas intriguing (golly, who wouldn’t?), and it was a great chance for me to assemble a seminar and impose some order on my thoughts.
Last month, we submitted a report to CONANP, who oversees the Biosphere Reserve in Bahia de los Angeles, about the past summer’s activities. A good chance to think hard about what we did, why it matters, and where it will lead. In related news, because of overwhelming time demands from the opening of their new Living Lab, Ocean Discovery Institute will not be working in Bahia in 2017. It is disappointing, but, at least in principle, will give me some time to put together more substantial funding for future years.
But let’s talk science. Several months ago, I acquired two old PCR machines from the surplus equipment program at the National Institutes of Health. It’s a great program, in which equipment that is no longer wanted by researchers at NIH can be acquired by educational institutions. The major caveat is that one can never be sure that the equipment is functional until it gets back to the lab and is tested. My students in the Cell Biology and Physiology lab course were running some PCR samples, so I thought it would be a good time to test out the new (old) machines in parallel with the very fancy PCR machine we use for student labs.
The thermocyclers are Applied BioSystems GeneAmp 9700s. In principle, they should do everything we need, plus they have a nice post-cycling chill cycle, so I can set them up and go home without worrying about the DNA sitting in the machine and degrading at room temperature. But do they work?
The choice of what to amplify was easy. Another Elysia fanatic, Susanne, had sent me a piece of parapodium from an E. diomedea that had an unfortunate encounter with a filter. Don’t fret, the slug survived, but she was nice enough to carefully preserve the tissue in ethanol, pack it, and ship it to USG.
The fragment sat forlorn for about a month. It was very exciting to be able to finally extract the DNA and see if we could amplify the rbcL region. I set about mashing and processing a small piece, and all looked well.
At the end, I had produced a tube of clear liquid. Was there DNA?
I amplified DNA from E. diomedea, a control sample from BioRad (to make sure the machines functioned at all), and some DNA I had extracted from an Avrainvillea plant in the slug system. During the thermal cycling, I used a thermocouple probe to check the temperatures of the machines. Machine 1 was just about perfect, whereas Machine 2 was way too warm during the cooler parts of the cycle, and I expected poor results.
The control samples worked in both machines, which was somewhat surprising based on the temperature measurements. I guess you can get away with a lot if you start with really clean DNA and well-established primers. The only sample from the new extracts that worked was the E. diomedea DNA in machine 2. I expect we can get things to work better if I reduce the DNA concentration, but it is puzzling that a sample in the less reliable machine worked better. Nonetheless, I now have some DNA I can send off for sequencing when there’s a little time.
Today marks the 2nd birthday of the Solar Sea Slug Blog. It started as a place to curate information about Elysia, and to track the progress of our modest project. Since then, it has taken on a life of its own, and morphed into a place to curate information about Elysia, and to track the progress of our modest project. My how we have grown!
I suppose the concept of this site as a journal of my work with Elysia (a “web log,” so to speak) is rather old-school in this age of tweets and Instragram. Fortunately, I am at a place in life where I can play the geezer card, using the excuse that I simply don’t have the mental bandwidth to generate content at that rate. Despite all the nice messages that people send me to help improve my SEO, I actually worry more about getting too much traffic than too little. To paraphrase Tim Curry, “going viral would be too much responsibility for me.”
The list of possible tweaks and improvements gets longer, not shorter, and the site will continue to improve incrementally. There are things I would like to simplify, such as how to leave a comment, or the process of updating reference lists. Then there is the slow work of fleshing out the species pages. It would be great to host the subset of articles that are open-access as well. Rome wasn’t built in a day, and Romulus didn’t have a full time job or dogs to walk, so we’ll have to be patient.
Thank you, dear reader, for checking in from time to time. Although it is unlikely you will find links to cat videos (but never say never), I hope you will stop by to find out more about amazing and abundant little molluscs, their biology, and the people who are working to understand them.
We just got some great news, that we have received the first outside funding for the Solar Slug project! The Washington Area Marine Aquarist Society has awarded the project $500 to get work started on slug neuroanatomy. The general idea is to stain baby slugs for acetylated tubulin, which will stain axons, and therefore tell us where the nerves are. This will be crucial in working out how the slugs process information about their environment. Because the slugs are essentially pancakes with everything embedded in tissue (rather than having nice hollow spaces like people or insects), it has been difficult to do this kind of thing using standard dissection methods.
Now that the adults are getting fat on recent batches of Bryopsis, I hope to soon see some eggs, and then grow some small slugs for staining. It may be a while, but I have a year to spend the money. Keeping fingers crossed that a little support may attract more, espcecially if we start to get some nice preliminary data.
Thank you WAMAS!
It have been well over a month, so I suppose I have earned the title of World’s Worst Blogger. It’s not for lack of news, though. In fact, one of the reasons for the lack of posting is the abundance of activity. In addition to the semester being in full swing, with the usual collection of lectures, exams and labs to attend to, the Elysia project has made some real strides in the past few months. Today’s post will be a quick summary of the new beginnings, with the promise of more to come.
First off, this fall is the official start of student involvement in the Universities at Shady Grove (USG) Solar Slug Project. Loyal followers of the blog will know that the USG project has been active for well over a year. However, the primary goal of the project is to provide University of Maryland students here at USG with a research experience. Now that the infrastructure is in place, there is now a squad of two undergraduate students who will be working on a small molecular biology project. The main purpose of their work this semester is to develop a protocol for extracting chloroplast DNA that can be used to determine which species of plants the sea slugs are holding in their bodies. We’ll start with a well-studied species, E. clarki, with a view to performing the same experiment on Elysia diomedea in Bahia this summer (see below). The students have completed their first two assignments covering the basic biology of the system and the specifics of the methods they will be using. We should be ordering reagents in a few days and doing some real biology next week. Stay tuned for updates on their progress.
Another big news item is that solar slugs will be a significant part of the Ocean Discovery Institiute program in Bahia de los Angeles this summer. One of the high points of my year is going down to Baja California to help them out with their work at the field station there. This year, the slug project will expand to be a Directed Research project, involving a small group of students for the entire duration of their time at Bahia. We will be performing experiments to identify the food plants and examine the activity patterns of Elysia diomedea. Because large Elysia species are often found away from potential food plants, and because their method of feeding (sucking the sap) leaves no obvious bite marks, it can be difficult to know what they are actually eating. Knowing what E. diomedea eats, and how much time it spends feeding, basking or hiding, should provide important insight into why it stores and maintains chloroplasts from its food plants.
For the semester’s experiments, we ordered some new E. clarki and have been fattening them up for experiments. In addition to new Penicillus and Avrainvillea from collectors in the Keys, we are getting some nice Bryopsis and an unidentified species of broad-leafed algae in the half-ten algae growout tank.
As you can see, it is still difficult to find the right nutrient balance that generates lush growth of the target algae without encouraging cyanobacteria (the red stuff), but we are moving closer all the time.
That’s it for now. More soon, I hope.
In an effort to make navigation a bit more sensible, I have started making a few changes. There is now a top-level page devoted to slug husbandry, Slugkeeping, which contains information (from this Web site and others) regarding how to keep and breed Elysia, as well as sources of slugs, food plants, and hardware. The new Slug Science page collects the links to the labs and scientific papers that make up the world of Elysia research. There is also a link to the occasional Solar Slug Journal Clubs that examine current papers in more detail.
Keep an eye out for more updates in the near future. Also, please let me know about links that may have been broken in the process of rearrangement.
In the meantime, here is a photo of a presumed E. papillosa that rode in on the recent batch of plants from KP Aquatics.
Apologies for the lack of excitement here lately. The plans for the hatchery have all come together, and now I am waiting for the components to arrive. Meantime, the next generation of broodstock is maturing, and new papers have been added to the scientific literature pages. It continues to be an exciting time to be interested in Elysia, and I recommend having a look at the most recent papers in the kleptoplasty section.
At the end of Part 1, I had discovered Elysia diomedea, and the seed was planted about diverting possible future research toward marine organisms.
A lot of changes were occurring before and during 2012. The event that probably had the most impact was the death of my long-time mentor and Lab Chief, Howard Nash, in 2011. Aside from the emotional consequences of such a loss, there were also the practical details associated with the closure of the lab. Papers needed to be finished, and I needed to chart the next stage of my career.
In retrospect, it was a great opportunity to ask myself what I would do if I could do anything I wanted. Well, almost anything. Astronaut or dinosaur hunter, for example, were pretty much off the table. In the end, the direction that most excited me was teaching at the university level, hopefully with the possibility of sneaking in a little research with the undergraduates.
As luck would have it, a lecturer position in physiology and neurobiology (my two areas of expertise) opened at the Universities at Shady Grove campus of the University of Maryland College Park. I was pleased, relieved, and amazed to be offered the position, and then overwhelmed with the amount of work that it took to do a credible job teaching undergraduate lecture and lab courses. In the back of my mind, though, I continued thinking about involve students in studying an interesting and relevant question.
One of the first questions regarded which organism to study. I had spent the previous 20 years or so studying Drosophila, which is a marvelous organism with an enormous experimental toolkit. It is also extremely small (not so good for most aspects of neurophysiology) and is being studied by close to a gazillion people. Hard to find a niche for a shoestring operation in that melee.
I had become excited about Nematostella, a little mud-dwelling anemone that is becoming an increasingly popular model organism for the study of development, genetics, and evolution of cnidarians. These animals have a simple nervous system that may resemble that of our earliest multicellular ancestors. It seemed like studying the activity and connections in the nervous system could provide fundamental insight into how nervous systems evolved. They are easy to keep, and the Nematostella community is very enthusiastic and supportive. Recording electrical activity from the teeny neurons in the Nematostella nervous system, however, was somewhat more ambitious than was practical with the available resources.
Elysia should have been the obvious choice from the beginning. They are sizable molluscs, and molluscs are known for large, easily identifiable neurons that are accessible to relatively simple recording techniques. There was even a small literature regarding neurobiology of E. chlorotica. Furthermore, E. chlorotica had been reported in the Chesapeake Bay, so it seemed like an excellent way of making a connection between work in the lab on campus and the local ecology.
A plan was forming: Behavior and ecology with E. diomedea in Bahia, physiology and ecology with E. chlorotica in Maryland.
After finishing the 2014 spring semester at UM, I was again lucky enough to be able to join Ocean Discovery at Bahia de los Angeles. Professional and personal obligations left me with less time for planning than I would have liked, but I had developed two questions for the trip. First, is E. diomedea attracted to light, as one might expect of a photosynthetic organism? This would lay the groundwork for more detailed experiments on spectral preference and neural circuitry. Second, what does the slug eat? Surprisingly, that was not (and is still not) known. Almost all other Elysia species eat green macroalgae, but E. diomedea was rumored to feed upon Padina, a brown alga with which the slug is commonly associated.
The plan was simple: collect a small group of E. diomedea as soon as possible, and perform behavioral assays. This was to be followed by a workshop in which I would enlist the students to help extract the chlorophylls from the slugs and perform paper chromatography to separate the pigments and compare them to those of local algae species. I brought my electrophysiology kit, along with some LEDs and a controller for phototaxis assays, and managed (with a lot of help from Dr Talley and several trips to Dixieline Hardware), to collect the solvents, tubes, paper and other items required for chromatography.
Sadly, Drew was unable to travel down to Bahia that year. That cost me the opportunity to spend time with a friend and colleague, and it meant that he would not be able to supervise the students studying the movement of energy from the sea to the islands (“spatial subsidy,” a.k.a. “Energy Transfer”). Although I was far from a perfect substitute, I had spent a few summers observing, and had an adequate theoretical overview of the project, so I offered to help out with the students and staff for the few weeks I was there. (Be patient, this will eventually be relevant to the slug project.)
The silver lining was that I was able to spend every morning out on the islands with the students. On the trip out, there was something exciting to see almost every morning. Dolphins, whales, sea lions and whale sharks all greeted us at one time or another.
Plus, the islands themselves were spectacular, and it was invigorating just to be on them.
So I got to spend mornings pretending to be a biological oceanographer. Afternoons were filled with a variety of tasks, but I tried to spend as much time as possible snorkeling in pursuit of Elysia. (See, I told you we would return to sea slugs.). Based on how quickly I had found them the previous summer, I fully expected to have a small pile of them in short order. Strangely, though, there seemed to be none to be found. I combed areas heavily grown with Padina (the potential food plant mentioned above), other areas with Codium (“dead man’s fingers,” another potential food plant), rocky areas with turf algae, but found no slugs. I looked in the shallows, and as deep as was practical with a snorkel and a few weights. Nothing.
After almost a week, it became rather distressing. Reviewing Hans Bertsch’s work on the distribution of opisthobranchs in Bahia de los Angeles, I noticed that there had been a steady decline during the years of his study, which worried me a bit. Were conditions deteriorating in some way, so that E. diomedea was increasingly scarce in the bay? Also, he had not mentioned collecting as far south as we were, so maybe conditions were not as good at our field station. But I had found them the previous year…
The breakthrough came on a day when the students were working at the station, so we did not have to get on a boat to the islands first thing in the morning. With a little free time, it seemed like a great opportunity for more slug hunting, even if my optimism was starting to fade. I was pleasantly surprised to find one of the little guys within a relatively short period. So surprised, in fact, that I had forgotten to bring any sort of container, which led to a comical juggling act of carrying a small, squishy creature back to shore in large clumsy hands. Given their apparent rarity, the little gal was extremely valuable, and losing her would have been a huge blow. She made it back to shore, and got her own spacious plastic tub with an airstone for circulation. For good measure, I added a small rock with a collection of possible food plants. Below is a photo of her, with a ruler to provide the appearance of scientific rigor.
The following morning was also available for snorkeling, and I promptly collected three more Elysia. Although it might have been coincidence, finding four animals during a relatively short period of collecting in the morning, compared to finding zero after several hours of hunting in the exact same area during afternoons, suggests that it was the time of day, rather than the location or long-term trends, that was the important factor in success.
In retrospect, it makes sense that the slugs were most exposed in the morning. My working model when I started the project was that “solar sea slugs” would be strongly dependent on light, and maximize their exposure by lolling about in the afternoon sun. Recent work indicates that, despite the presence of kleptoplasts, the slugs depend almost entirely on feeding for their energy and nutrition. My captive slugs seem to be (note that this is fully anecdotal at the moment) at their most active, especially regarding egg laying and general exploration, in the evening and early morning. The rest of their time is mostly spent face down in their food plants, like so many squishy, aquatic cows.
The remaining time allowed for a few experiments, including a quick and dirty look at phototaxis and spectral preference. Unsurprisingly, they are attracted to light, and appear to prefer the long wavelengths, such as the orange shown below. The experiment was pretty crude, but the results match what had previously been shown for other species.
This almost closes the genesis of the Elysia project, and gets us just about to the beginning of the Solar Sea Slug Blog. I left Bahia knowing much more about the behavior and anatomy of Elysia than when I had arrived, and realized that the little slugs were much more complex than would be expected of “crawling leaves.”
Back in Maryland, I was ready to start hunting for local E. chlorotica in the Chesapeake. An email exchange with Dr. Sidney Pierce, now Professor Emeritus at the University of South Florida quickly disabused me of the idea. In his opinion, reports of that species, and its food plant, here are dubious.
Perhaps it was just as well. E. clarki is easily available and easy to fatten up on nuisance algae, and E. papillosa may be smaller, but appears to have similarly broad preferences and a rapid generation time (which will be the subject of a future post).
Greetings from Bonaire. Hoped to do some live blogging, but the new computer is down. Many exciting things to see here, as always, and we have some wonderful new photos of E.crispata to share when we can upload them.
Meantime, here is one of the new E. clarki that arrived in Box of Slugs 1.0 last week. Will bring real camera back to office next week for better photos.