Posts in Category: Slug Science
We Have Data!
It has definitely been a good week for the Solar Slug Project. Yesterday, all of the pieces for the barcoding project started to fall into place. As I described a few weeks ago, the idea is to use a sensitive method called polymerase chain reaction (PCR) to amplify DNA from chloroplasts within the slugs in order to figure out what they have been eating. Our first step was to extract and amplify DNA from potential food plants, just to be certain everything was working. Below, you can see Maryam and Haseeb, the two USG students who have been working on this project, diligently extracting DNA from Halimeda and Bryopsis samples.
Maryam and Haseeb extracting DNA from Algae. 3/25/16
After a few false starts, we got conditions to the point that everything appears to be working. One key change was switching to GE Healthcare’s “Ready-To-Go” beads as the source of polymerase, buffers, nucleotides, etc. The beads can be stored at room temperature (as opposed to being frozen, like other “master mixes”), which will make life a lot easier at the field station.
PCR products from 4/22/16, visualized with BioRad UView DNA stain.
The PCR products are the right size (about 600 base pairs), and there aren’t any extra bands. We used primers specific to Halimeda discoidea (“H primers”) for the products in the first four lanes, and primers for Bryopsis plumosa (“B primers”) for the last four. The species names, “Halimeda 1″ etc, indicate the following species:
Halimeda 1 (Presumed H. discoidea).
Halimeda species 1. Presumed H. discoidea. 3/25/16
The specimen of Halimeda 2 (H. incrassata) was a bit bleached, but yielded some nice DNA
Halimeda species 2, presumed H. incrassata. 3/25/16
Bryopsis “species 1” has a much finer structure.
Bryopsis species 1. Finer filaments. 3/25/16
Bryopsis 2 is stouter and longer. It is unlikely to be a different growth form of the same species, because they were cultured right next to each other. It’s a good opportunity to let the gene sequences unravel who’s who.
Bryopsis species 2. Coarser filaments. 3/25/16
The astute observer will notice that we got products from (almost) all species using both sets of primers. Overall, that’s a good thing, in that we can use these primers to amplify DNA from many species of algae, then submit the DNA for sequencing. On the other hand, we could use more stringent conditions (like a higher annealing temperature, for those who care about such details) to use primers to amplify DNA only from a particular species. For example, we could use Bryopsis primers on DNA extracted from slugs to ask specifically whether the animals contain Bryopsis DNA.
The quick summary is that we have worked out the details of methods needed to extract, amplify, and ultimately sequence DNA from chloroplasts. Haseeb and Maryam will be able to put together some nice reports about their work, and in Bahia de los Angeles this summer, we should be able to determine the species of algae that E. diomedea eats.
First Grant for the Solar Slugs!
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!
Ready to Extract
Qiagen DNeasy Plant Kit 3/21/16
Decisions have been made, orders have been placed, and materials have arrived. It turns out that we are treading a relatively well-worn path of DNA bar-coding. The goal for the semester is to extract, amplify and sequence the rbcL gene for the candidate food plants and the chloroplasts maintained by the slugs. Because rbcL encodes a component of the photosynthetic complex of the chloroplast, and the gene is found in the genome of the chloroplast (rather than the nucleus of the plant), the origin of the chloroplasts that the slugs are carrying can be identified on the basis of the sequence of the gene. Fortunately for us, the sequence of the rbcL gene has been studied by a lot of people. The chloroplasts of all plants have it, but it varies a little from one species to another. That variation can be used to examine how closely related different species are, or to determine if two populations that resemble one another are actually different species. Each species has a unique sequence or “bar code,” that can be used to identify it, and to distinguish it from other species.
Therefore, much of the work has been done for us. Kits, such as the one in the above photo, are readily available, procedures are largely worked out for amplifying the amount of DNA using polymerase chain reaction (although the primers for these algae may be a little tricky), and there are companies such as GeneWiz that provide a relatively inexpensive and simple resource for sequencing the resulting DNA. Makes a nice change after a career spent soldering and tweaking in order to perform experiments using arcane methods.
On your next visit, you may find a photo of DNA bands on an agarose gel. Or maybe something else.
Things Have Been Happening
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.
Elysia clarki exploring broad-leaf algae 2/22/16
Elysia clarki discovering new crop of Bryopsis 2/22/16
E. clarki on Bryopsis 2/12/16
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.
Slugs among algae selection 2/12/16
That’s it for now. More soon, I hope.
Seems to be Working
As described in the last post, I was interested in improving the growth of the food plants. Among the many parameters to consider (spectrum and intensity of light, water flow, nutrients), I decided to systematically increase the nutrient levels by carefully dosing Guillard’s f/2 formula. I have been very pleased to see a significant improvement in color, growth rate and branchiness of the Bryopsis.
Previously, growth was unimpressive. The plants were pale and rangy, and I was becoming concerned that they would not ever grow enough to keep the slugs fed. In the photo below, the finer, fuzzier stuff is Derbesia, and the thicker strands are barely recognizable Bryopsis, which should be bushy and feathery. If you can’t figure out what Bryopsis should look like, it may help to read to the end of the post and then scroll back up here.
Bryopsis and Derbesia before dosing 11/16/15
Within a few days, the Bryopsis started looking better. In the photo below, an astute viewer might be able to see that the tips of the branches are beginning to become a darker green. With the added nutrients, cyanobacteria (the red film coating some of the plants) have also increased. With a little adjustment of the dosing, I hope to see less red and more green.
Stringy Bryopsis at start of dosing 11/18/15
In just over a week, growth is robust, color is a satisfying, deep green, and additional branches are starting to appear. At this point, I also changed the flow pattern a little to increase the current passing through this patch.
Bryopsis filling out 11/24/15
As of a few days ago, the plants are nice and green, and almost feathery. In addition, there are patches of new growth forming in areas of high flow, so the alga is spreading. At this point, NO3 levels are a little over 10 ppm, and PO4 is about 0.5. Over the next few weeks, I am going to try to bring the levels down a little bit to discourage cyanobacteria.
Bryopsis getting green and branchy 12/3/15.
So what have we learned? First, a more balanced approach to feeding the algae results in faster growth and better color. Without systematic removals and subsitutions of components, it’s hard to know which of the ingredients was limiting for growth (N, P, Mg, Mn, Fe…..?), but the mix has quickly done the job. Second, based on the quality of the growth in areas of high flow, Bryopsis seems to like a lot of current. At present, I do not know which species I have (I hope that will change by the end of next semester), but some are found in intertidal zones with significant surf.
With these lessons in mind, I will be converting the nursery tank into a Bryopsis cultivation tank to provide food for the hungry Elysia that will arrive in a month or so for the student research project.
Getting Ready for Science
The project has had the feel of watching grass grow lately, mostly because I have been spending a lot of time collecting and establishing potential food algae for the slugs, and then watching them grow. Nonetheless, there is beginning to be some motion.
For example, starting Spring semester (late January), I will have a couple of students starting some simple molecular experiments. Although the diet of Elysia clarki is well-characterized, that of the species we find in Baja California, E. diomedea, is still not known for certain. On the next trip to Bahia de los Angeles, I plan to collect some E. diomedea, and identify their food plants based on the DNA of the chloroplasts that they have stolen from their food. This kind of work is straightforward in a comfortable lab where one has access to liquid nitrogen and other luxuries. However, we will need to develop protocols that will work in the heat and limited resources of the field station in Mexico. So, we will use the semester to develop a protocol for extracting and purifying DNA that utilizes the simplest methods possible. Meantime, it will be necessary to put together reading lists on the biology of Elysia and methods of DNA extraction so that we can hit the ground running.
The animal care system is also evolving. As I described a while ago, I have been lucky enough to get hefty samples of a few varieties of hair algae such as Bryopsis and Derbesia from local aquarists. The trick has been to get enough growth to maintain a self-sustaining supply of food. Although filling a tank with algae, sticking it into a window, and dumping hefty amounts of ammonia and phosphate into it produced decent results, it was not very stable.
10 gallon tank full of Derbesia and Bryopsis, sitting in the sunlight.
Instead, I have added a 20 gallon tank devoted to growing algae to the slug culture system. The fancy hydroponics light gives a great spectrum for growth, but the strong red-pink quality of the light is not the most pleasing. There are a couple of species of hair algae, plus a tub of marine sand for macroalgae that need a substrate. To take advantage of the automated dosing of NO3, Ca and HCO3, it is plumbed into the rest of the system. Dosing of NO3 has already been increased a few times to keep up with the growth of the algae.
Elysia System 10/28/15.
In other news, the controller that currently controls the lights, temperature and pH will soon be replaced by a newer, cloud-based model. This will allow remote monitoring of temperature, pH, and power use, as well as sensing moisture under the tanks in case of leaks. It can send email alerts in case of equipment failure, and should help prevent loss of animals or damage to property.
…and They’ve Hatched
The eggs hatched last Monday (10/5/15), but I am finally getting around to posting. Assuming you have a little imagination, you should be able to see some little veligers whizzing around among the very wiggly embryos. The hatchery was not ready for them (they only took about 5 days to hatch), so I just added the veligers directly to the growout tank and hoped for the best. Whatever this species is, it could be very useful in the lab to have embryonic development done in less than a week.
Meantime, I have added some fresh Bryopsis from a local reef tank, so maybe the E. clarki will be in the mood to lay eggs soo.
Frilly Slug Going Home
There has been a frilly sacoglossan (Cyerce?) in the growout tank, which presumably rode in with the last batch of algae. In an effort to focus on E. clarki in the hatchery, and because there is probably another one of these guys remaining in Box of Slugs 2, she is getting moved home tonight.
Frilly sacoglossan 060115
Despite the utter failure of the environmental system at USG (temperature 28 – 30 degrees C over the past few days), the hatchery has muddled along. Even got the first small batch of eggs from the second generation of E. clarki. Thought it might be useful to start providing a sense of scale of these things.
First batch of eggs from the second generation.
Slug Makes New Species Top 10 List
The Washington Post reported that a species of photosynthetic nudibranch has made the SUNY Environmental Science and Forestry list of the Top 10 New Species of 2015. The field was large, about 18,000 species in all, but Phyllodesmium acanthorhinum made the list based on what the animals tell us about the evolution of the symbiosis between the slugs and the photosynthetic algae they host.
NEW SPECIES, PHYLLODESMIUM ACANTHORHINUM. PHOTOGRAPH: ROBERT BOLLAND
Like Elysia, species of Phyllodesmium steal the ability to perform photosynthesis from their food organisms and maintain the required components in sacs extending from the gut called digestive diverticula. There are some important differences, though. Unlike Elysia, Phyllodesmium is a true nudibranch, and it feeds on corals rather than macroalgae. Another important difference arises from the different biology of the algae that Elysia eat and the corals upon which Plyllodesmium feeds. Photosynthetic corals, such as Xenia, contain symbiotic algae (dinoflagellates, actually) called zooxanthellae, which provide the corals with most of their nutritional needs. When Phyllodesmium feeds on Xenia (or other coral species, depending on the species of Phyllodesmium), it steals the zooxanthellae and stores them in the diverticula. In this way, Phyllodesmium has it a bit easier, the stolen algae are autonomous cells, and the slugs do not need to worry about maintaining isolated chloroplasts.
So how did this species end up in the top 10? A recent paper describing Phyllodesmium acanthorhinum and analyzing the interrelationships of species within the genus (E. Moore and T.Gosliner, 2014, The Veliger 51:237) provides some new insight into how the ability to maintain zooxanthellae evolved within the group. Earlier work had suggested that the branching of the diverticula, and their extension into the cerata (the frills on the back of the nudibranch) increases with the increased ability to sequester and maintain zooxanthellae. In other words, species that simply digest the zooxanthellae have minimal branching, while those that maintain large collections of active zooxanthellae have more elaborate diverticula that branch deeply into the cerata. Based on the descriptions of P. acanthorhinum and another species, P. undulatum, both of which are relatively less specialized for maintaining zooxanthellae, Moore and Gosliner provide additional support for this hypothesis. Further, they suggest that the larger body sizes achieved by more derived species, i.e., those that are better able to maintain populations of zooxanthellae, result from the additional nutrients produced by the symbionts.
Once again, slugs find a way of hijacking photosynthesis from their food. Because Elysia and Phyllodesmium are only distantly related, and their biology and that of their food are so different, the two forms of theft-based photosynthesis must have evolved independently. The similarities are striking, though. It does make one wonder if there is some aspect of the biology of sea slugs that predisposes them to separate chloroplasts or entire zooxanthellae from their food and maintain them in digestive diverticula.
Hatchery in Progress
As described a while back, all steps in the culturing process seems to be going pretty well, except for one bottleneck. The adult broodstock is (are?) happy to lay eggs, the eggs hatch consistently, and the veligers settle. However, they will not develop much farther after settling in a controlled environment. Oddly, the settled veligers will develop if left on their own in a large tank full of algae. Although I have now reared E. clarki from egg to adult, it is not really possible to plan experiments based on when slugs may or may not decide to develop in a display aquarium. A more systematic approach was needed.
The hatchery is an attempt at making the juveniles happier during and after settling. Egg masses will still develop in glass crystallization dishes, but they will be placed in the new setup just before hatching. There were a few issues that may have impeded development, and they should be addressed by the new setup.
The tank is an acrylic “half-ten” from Glasscages.com. It is essentially a half-height 10-gallon tank (10″ W X 20″ L X 6″H). I had originally planned on using a standard 10 gallon, but it became clear that it would be clumsy and result in a lot of wasted space.
Acrylic “Half-Ten” tank from Glass Cages. 5/19/15
One problem that arises with free-swimming veliger larvae is that they are positively phototactic (attracted to light). This may not be a problem in the open sea or a large aquarium, but in the small dishes I was using for hatching, it meant that they would swim to the surface and promptly get stuck in the air-water interface. This would leave little rafts of veligers on the surface. These floaty veligers were capable of settling, so it was not a complete disaster, but it could not be good for them. Some authors (e.g., Dionisio et al, 2013) go so far as suggest rearing them in the dark. My solution is to illuminate from below. Marineland makes a nice little submersible LED light that can be used to keep the veligers swimming downward. Below are views of a prototype hatching chamber (2″ PVC pipe, with 50 micron nylon mesh to retain the veligers and larvae), showing the light coming from underneath.
Light from below, with prototype hatching chamber. Front view. 5/19/15
Light from below, with prototype hatching chamber. 5/19/15
Secondly, the presence of a relatively large food plant (large enough to be certain all of the little slugs can climb on) might have altered water chemistry, either through the process of photosynthesis (raising pH, e.g.) or by releasing chemicals that inhibit the slugs’ feeding or development. Continuously recirculating ASW (artificial seawater) from the larger system through their hatching chambers should reduce or eliminate this problem. In order to keep voracious invertebrates from entering the chambers, ASW will pass through a UV sterilizer before being distributed in the hatchery.
The manifold for distributing the ASW to the chambers is made from a few PVC pipe fittings. Once the cement has cured, it will be drilled to accommodate valves to control the flow to each chamber.
Components of the manifold. 5/19/15
Manifold, assembled but not drilled. 5/19/15
Naturally, water arriving in the tank needs to leave, so I drilled it and added a bulkhead to drain to the sump.
Tank drilled and bulkhead installed for drain. 5/19/15
Once the manifold is finished, and the UV unit arrives, it will be ready to hook up and accommodate the next available brood. Stay tuned.
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