I recently had an epiphany about how to grow Bryopsis more efficiently. In retrospect, it was pretty obvious, and I wonder why it took 2 years to get to this point.
It seemed as though culturing adequate Bryopsis was under control. However, there’s nothing like 6+ weeks of travel to turn things upside-down. I had hired a service to come in and keep things going while I was away, but their primary task was to prevent biological meltdown, or worse, a big salty disaster that would have me forever on the naughty list of the facilities people. I am happy to say, there were no smelly or wet disasters.
Unfortunately, I was not there to give the algae cultures the kind of attention they need, and by the time I was back in the office, the system was overrun with Derbesia, and the slugs had devoured the Bryopsis that I had left for them. The 20-gallon slug tank and both algae culture tanks were full of felty, green hair algae. In retrospect, I should have taken photos, but I was more focused on cleaning up the mess and getting ready to teach a summer class.
Over a week or so, I pulled out the algae tanks off the system, cleaned out at least two pounds of green glop, salvaged the remaining Bryopsis, and set the tanks back up. Somewhere along the line, I came across a few posts about “algae reactors,” cylindrical chambers with water flowing through and some sort of light source. My first thought was “maybe I should buy one of those things.” Then I remembered that I had two media reactors sitting idly in the basement. They are clear cylinders, designed to have water flow through them, which is exactly what I wanted.
The amount of Bryopsis remaining was so small that it didn’t seem worthwhile to have two algae tanks. Instead, I shut down the 10-gallon tank and stuck some of the remaining algae into a reactor. The reactor was hooked up to one of the valves, and connected to the drain, and sat under the grow light where the 10-gallon tank had been.
Within a week or so, it became clear that the experiment was working, so I added the second reactor. I had enough Bryopsis to harvest some for the slugs, and the culture in the first reactor had seeded a sponge that I could use to start the second reactor.
It was working. Time to make things a little less clumsy. I built a rack from 3/4″ PVC, setting the reactors at an angle to optimize the connections to the input valves and drains.
Things went so well, I begged a few unused reactors from local aquarists. This one is from Alan (of unidentified algae fame), and I have one more waiting in the wings.
At this point, it was clear that my former method of rearing Bryopsis in aquaria was not very efficient. Raising Bryopsis in reactors allows me to play with growth parameters, like flow and nutrients, much more easily. Further, keeping multiple separate cultures will make it much easier to eradicate unwanted algae. At some point, it should be straightforward to maintain cultures free of unwanted algae and invertebrate pests (I am all for biodiversity, except when it eats slug larvae) by UV sterilizing the water going into the chambers.
As far as I can tell, this was a successful experiment, so I converted the 15 gallon algae tank into the second slug tank, shutting down the 10-gallon slug tank.
When I walked in today, the system had two slug tanks (top and middle left), and one remaining algae tank (top right)
Five hours of cleaning and rearranging later, there were two slug tanks and three algae reactors. In the process, the system now has two fewer circulation pumps and one fewer light fixture.
In the future, you can expect a few more reactors. Now it’s time to play with parameters to maximize algae growth. Maybe we’ll finally see some consistent egg production from the slugs.
Just when I thought I had it all figured out.
After struggling for way too long, I finally got reliable growth of Bryopsis in the algae culture tanks by providing strong lighting, balanced nutrients, along with very intense water movement. Bryopsis grew well, although Derbesia also started to thrive and needed periodic removal, so I figured I was close to the magic formula.
Imagine my surprise when Bryopsis started to thrive, largely without contamination by dinoflagellates or Derbesia, in the slug culture tanks. In the 20 gallon long, the algae that was transferred on the plastic racks started to grow and spread, despite constant grazing from the resident E. clarki. In the 10 gallon, a few scraps growing on some of the macroalgae grew to fill almost half the tank.
This has made the single E. crispata very happy. She has grown considerably, and her color is amazing. Will post a photo when she comes out of the algae far enough to be photographed.
The tanks are all plumbed together, so they all receive the same nutrient input. The slug tanks receive somewhat less light than the algae culture tanks. The spectrum of the lights in the slug tanks is broader, with more green, yellow and UV, mostly because that is nicer for my eyes when I look at the tanks. Finally, the circulation is considerably less intense. The algae tanks are blasted with propeller pumps and wavemakers, while circulation to each of the slug tanks is only provided by a single Maxi-Jet 600 (600 liters per hour output), with the intake slug-proofed by a strainer and sponge, and the output directed through a Hydor Flo to provide swirling motion.
Because even these relatively small pumps give the slugs a bit of a wild ride, they are only turned on for 15 minutes of every hour. Quick summary: despite my beliefs to the contrary, it is possible for Bryopsis to grow strongly with modest water movement.
Although I am pleased that there is now abundant food for the slugs, it does bother me that I still do not understand all of the factors affecting growth of Bryopsis. Previously, Bryopsis struggled in conditions that were largely similar. If I get a chance before leaving for Baja this week, I’ll make yet another deep dive into the system logs to determine which parameters may have changed. The luxuriant growth has emboldened me to order a few more E. clarki, so that the colony can be going full steam by the end of the summer.
To many, this post is akin to “how to give fleas to your dog,” or “how to grow ragweed.” Bryopsis is a benthic (substrate-associated) genus of algae that most aquarists dread seeing in their tanks. Bryopsis can thrive in reef aquaria, to the point that it can smother and kill corals. Many methods of eradication have been explored, including near-toxic levels of magnesium, and the anti-fungal medication fluconazole.
On the other hand, I really want the stuff. As far as I can tell, it is the favorite food of Elysia clarki. E. clarki will eat other macroalgae, such as Penicillus, Avrainvillea or Halimeda, but, given the choice, they will go straight for Bryopsis and stay there. Further, they have consistently produced eggs only at times when they have had unlimited availability of Bryopsis. One of my primary goals is to maintain a self-sustaining colony of slugs, so I very much want a constant, adequate supply of algae.
I’d be perfectly happy if I could count on the outbreaks of local aquarists to supply my needs. Unfortunately, sources have been spotty. My primary source, Justin, who had a beautiful outbreak in a 500-gallon tank, unfortunately managed to get his infestation under control. He swears the controlling factor for him is the level of phosphate; if PO4 levels creep up, he gets an outbreak. Others have provided quality material as well, but not as consistently. Yet others have offered species that turn out not to be Bryopsis. The take home lesson is that people who are trying to eradicate something are not the most dependable source.
How hard can the stuff be to grow? It is a pest, after all. Like any aquatic plant, it needs light, nutrients, and circulation. It gets a little more difficult when you try to provide for the plants’ requirements on a consistent basis. As far as I can tell (and PLEASE email or comment if I am wrong), nobody has published specific requirements of Bryopsis for PAR (photosynthetically-available radiation), macronutrients (e.g., carbon, nitrogen, phosphorous), or micronutrients (like iron, copper and cobalt). When marine aquarists have outbreaks, much of the nutrient load is derived from fish food (well, really fish poop), which is not measured. In fact, most reef aquaria don’t have Bryopsis outbreaks, which suggests that the unlucky few have managed to combine all the right ingredients. For a constant supply of Bryopsis, I need to provide for all of its needs.
If I wanted to grow phytoplankton, the single-cell, free-floating relatives of Bryopsis, I would be in luck. Because of the important role of phytoplankton in our oceans, and because there is a growing industry using them to generate hydrocarbons from sunlight, growing conditions have been explored and published at length. The most popular formula is Guillard’s F/2, so named because it consists of his formula F diluted to 50% (Guillard & Ryther, 1962, Can. J. Microbiol. 8:229), is readily available through suppliers such as Florida Aqua Farms, or even Ebay. The formula provides a balanced blend of nitrogen and phosphorous (with an option to add silicon if you want to grow diatoms), along with a mix of trace elements and vitamins. Mix it up, add a starter of the phytoplankton of your choice, bubble in some CO2 as a carbon source, and you’ll soon have a thriving culture of green water.
I started dosing the Florida Aqua Farms version (“Plant Fuel Too”) almost a year and a half ago, using the complete dry mix (minus silicates). The Bryopsis started to look darker and happier pretty quickly. Perhaps unsurprisingly, there was also a phyoplankton bloom that turned the water green and generated a layer of green scum on the surface of the water. Also, the added nutrients seemed to increase levels of nuisances like cyanobacteria (a.k.a., red slime algae). That would have all been tolerable, but the biggest sticking point was that Bryopsis would go through cycles of boom and bust, which is not conducive to long-term management of a slug colony. I needed to get the right balance of nutrients that would get the Bryopsis to grow maximally and out-compete the species I don’t want.
With all of the possible factors (e.g., light, circulation, temperature, nutrients) to be within the right ranges, there were a lot of variables to play with:
In order to separate the variables a bit better, I started using Florida Aqua Farms original “Plant Fuel,” which contains all of the trace elements and vitamins, but no nitrogen or phosphate. That would allow me to play with levels of trace elements, N, and P separately. I also added a source of carbon. Because white vinegar is cheap, always available, and is readily broken down into CO2 by bacteria, it seemed like a good choice.
By adjusting the relative input of all four ingredients (C, N, P and trace elements), combined with very high flow in the algae culture tank, I was able to get significantly better growth. The final improvement was something that I had not expected to have such an impact: rebuilding the system. Within about a week of the big move, the algae was thriving on the racks of plastic eggcrate I was using as a substrate. I suspect that the 50% water change and improvements to circulation in the new system played a large part in the improved growth.
By growing on plastic racks, Bryopsis cultures can be moved to slug tanks to be consumed, and then removed for regrowth.
So we’re making forward progress. We are controlling the growth of food algae to the point where it seems to be sustainable. As a side benefit, the other macroalgae are also thriving. For example, the three deep green Penicillus in the photo below are new sprouts from the ratty old plant in the center.
At the moment (and likely to change) the system receives major elements at the (molar) ratio of 150:30:1 C:N:P. This is roughly in the range reported for other macroalgae, and does not support much growth of cyanobacteria or phytoplankton. In other words, it looks like I am providing nutrients at about the rate at which the are being consumed by the desired algae.
There is still plenty of room for improvement. The priority for the moment is eliminating or reducing dinoflagellates in the algae culture tanks. They do not seem to cause too much trouble in the slug tanks, but they form a slimy film on the Bryopsis in the culture tanks, presumably reducing growth, competing for nutrients, and producing toxins.
At higher magnification they are kind of pretty. Based on the shape, size and general appearance, it looks a lot like Ostreopsis ovata (Faust and Gulledge, 2002, Contr. U.S. Natl. Herb. 42: 1-144). Like many dinoflagellates, O. ovata produces toxins, some of which are toxic to mice (Nakajima et al., 1981, Okinawa Bull. Jpn. Soc, Sci. Fish. 47: 1029). The effects on slugs or other invertebrates is not known.
At the moment, I have reduced the photoperiod (the time the lights are on), and am rinsing the Bryopsis racks in clean artificial seawater periodically to reduce the population. At some point, it might be necessary to use harsher methods, such as antibiotics (metronidazole seems to affect dinoflagellates somewhat specifically) or starting a clean culture of Bryopsis and preventing entry of other species using UV sterilization.
For now, there is plentiful food for the slugs, which is a step forward.
The first version of the multi-tank slug system has served reasonably well, but it has had its limitations. The main problems have been the sprawl of equipment (note the tanks, dosers, auto-topoff, scattered about in the photo below), the low height of the shelves, which limits lighting options, and the cramped nature of the shelf unit, which makes maintaining or replacing tanks difficult. I also wanted a bigger sump, mostly to have a little more volume to prevent floods. To be honest, the photo makes it look even worse than it was, because I had moved a cabinet in preparation for the new system, leaving controllers and power supplies lying in a pile on a temporary shelf. Nonetheless, the system was long past due for an upgrade.
It took a few weeks to decide exactly how much space I could afford, and how to design the new shelves to accommodate existing tanks and allow flexibility in future configurations. I finally settled on a 60 X 16 inch footprint, which would accommodate the 15 and 20 gallon tanks on the top, plus a little extra space. It would be smaller than the space made available by the removal of one file cabinet and the old slug system, giving a little elbow room for maintenance and repair. I decided on 48 inch height. Enough for two shelves for tanks, and a bottom shelf for a sump. Three rows of tanks, a sump level, plus ample height for lights would just be too tall for me to reach easily. My experience with the current system has taught me that more tanks is not necessarily better, The second shelf would have room for a couple of 10-gallon tanks, or various combinations of 5- and 10-gallon tanks for smaller-scale experiments. The dosers and controllers would be on the bottom shelf with the sump, protecting them from splash, and making the system almost completely self-contained. By necessity, the chiller will have to be off to the side in order to move heat away from the tanks.
I decided to build the frame from 2X3 studs, and use 1/2″ plywood for the shelves. The studs should be plenty strong to support the 5 foot shelf, and 2X4s would be overkill and make the system that much heavier. The weight of the shelves is transmitted to the floor by 2X3s that run from the bottom of one shelf support to the top of the next. There is a second set of vertical 2X3s going all the way from the top shelf to the floor, providing more stability and support.
For paint, I chose a latex semi-gloss. I hope I don’t regret not using marine paint, but I am hoping that the primer plus three coats applied over the course of a week will be adequately waterproof. I tried to match the color of the walls in the office, but it turned out a bit more blue than I had intended. The piece of plywood on the back provides a surface for attaching controllers, power supplies, dosing pumps and drain brackets.
In order to simplify moving tanks in and out, I installed a 2″ drain with 8 openings along the bottom shelf, and a 3/4″ supply pipe with barbed valves along the top shelf. Installing and removing a tank will be as simple as connecting a supply hose and placing a flexible drain hose from the tank into a drain opening.
Then came the hard work of moving everything over. After Joanna pointed out that the shelves would not fit in the Jetta, I reserved a U-haul pickup truck, and we moved it from home to the office. Then it was simply a matter of spending 10 or so hours reinstalling plumbing, draining and moving tanks, setting up lights and pumps, mounting dosers and controllers, fussing with details, and cleaning up the resulting mess.
I am very happy with the results. My office is less cluttered, every aspect of the system is more accessible, the electronics are better protected from splash, and I don’t have to climb on a chair to work on the top tank. The Neptune Apex controller became a little buggy during the process, but I can again control and monitor the system remotely after a few reboots. The leak detection module is still not fully functional, so I am keeping fingers crossed that there will be no floods, large or small, until it is fixed.
One happy development is that the Bryopsis growing on the eggcrate in the 15-gallon tank (upper right in the photo above) has started to take off. Expect photos of that, plus a new shipment of marine plants, in the next day or so. Who knows, maybe there will once again be slugs in the Box of Slugs.
As I mentioned in the previous post, sometimes it is not so easy to identify an alga. In this case, it is a species that bloomed spectacularly when a local reefkeeper set up a new tank. The rock had been thoroughly cleaned and bleached, and no corals or fish had been added, so Alan did not expect the growth of nuisance algae. He was rather surprised to see a rapid, spectacular bloom of long, furry green algae.
At first we thought is might be Bryopsis (yay!), so it seemed worth trying to feed to the slugs. Once I saw and felt it, it was clearly something else. It was soft, like Derbesia, but longer and had branches that extended radially (like a bottle brush) from the main stem. Bryopsis feels coarser, and the branches extend in a single plane (like a fan). So, it was not one of the usual suspects. Nonetheless, it was worth throwing some into a tank to test whether the gals would eat it. They did not immediately plunge into it, as they would have for Bryopsis, but they seemed to find it palatable enough. Note the fine structure of the branches in the photos below.
The plant has some characteristics of the order Bryopsidales, such as the lack of clear cellularization. It looks like the plant is made up of a continuous, single cell.
I thought a quick look at the DNA sequence would clear things up, but that was not the case. The closest match, Acrosiphonia, with 88% sequence identity. That’s not a very good match, and even though it looks somewhat like Acrosiphonia, the unidentified alga lacks several key features, such as the hooks on the branches (which cause mature plants to develop a dreadlocked appearance) and clear cellularization of Acrosiphonia. Plus, Acrosiphonia is a cold water species, unlikely to thrive in a warm reef aquarium.
The closest visual match so far is Trichosolen, which does have warm water species. The only species with rbcL sequence in the database (T. myura) is only an 86% match for DNA, so it’s probably not the one either.
By way of comparison, the usual pest algae (various species of Bryopsis and Derbesia) were only 82% – 83% identical, so we can at least rule out the possibility that it is an oddball species of one of those.
The hunt continues for a match. Not very satisfying, but some days are like that.
Happy Holidays to all of you fans of slugs!
Although the site and the project are devoted to adorable molluscs, we would be nowhere without algae. These days, I spend more time and resources trying to acquire, grow, and identify algae than I do attending to Elysia. It should not be a surprise, given the outsize role of algae in the biology of the slugs, but, until this project was underway, I had never given a lot of thought to the care and diversity of algae. Subsequent posts will describe some of the progress in algae care, but today we’ll focus on some systematics and molecular biology.
The plant in the photo above has been nagging at me for well over a year. I can’t remember exactly how it came up, but KP Aquatics mentioned that they had a species of algae they called “spongy sea pansy,” which was like Udotea, but larger and squishier. They were quite a bit taller than Udotea, grew in clumps, and were indeed quite spongy. Their biology is somewhat different from other algae in the order, in that the thallus (the body of the alga) dies back periodically, and a new one grows from the rhizoid (the rootlike part). In my experience, species like Udotea or Penicillus send out runners that produce new thalli, and the old ones just die off.
I have been referring to them as Avrainvillea, because they fit the description reasonably well, but had never done the hard work of verifying that it was not a similarly squishy genus, such as Rhipilia or Cladocephalus.
A real phycologist (algae specialist) would have probably started with a good microscope and species key. I took the molecular route, since I was already using PCR to amplify DNA from the rbcL gene in a few other species, and sending it off for sequencing.
Because I was testing new PCR machines, I had set up three independent reactions, and the results were the same. The screenshot below shows the results of a BLAST search for one of the sequences through the NCBI database, with the closest match at the top. The second best, with 98% of the nucleotides being identical, is Avrainvillea nigricans. The best match (99%) is to an “uncultured Ulvophyceae” clone from a study by Christa, Gould, Wagele, and their collaborators. If I read the entry correctly, the sequence is from kleptoplasts extracted from Costasiella, a Caribbean slug that feeds on…did you guess…Avrainvillea. To provide a little context, Cladocephalus and Rhipilia, the genera that were possible candidates based on appearance, were only 93% and 82% identical, respectively.
That is a pretty clear-cut result. It looks like Avrainvillea, it is squishy like Avrainvillea, and its DNA is essentially an exact match for Avrainvillea nigricans. It is Avrainvillea.
As you’ll see in the next post, the results aren’t always so easy to interpret.
In the “one reefer’s pestilence is a slug’s tasty snack” department, Marcos from Exotic Reef Creations sent over a batch of rock and zoanthids and live rock that were covered with lush Bryopsis growth. Although the dedicated algae tanks have helped keep the supply up, I was very excited to receive the new infusion. The gals are over the moon about it. The photos below show the whole gang hunkered down in feeding posture. They have been face down in it for almost 24 hours now.
The goal is to have them clear the algae so the corals can go back home. It will be interesting to see how long that takes.
Meantime, the molecular experiments are moving along. More soon.
It is time to get the system up to full capacity. The last component, a half-height 10-gallon tank (yes, that’s really a 5-gallon tank for the mathematically inclined) is now in operation. Originally designed to be a nursery, it seemed better suited to use as a Bryopsis culturing tank. I have been pleased with growth rates under the Evergrow S2 hydropnics light, so I picked up another one from Exotic Reef Creations. So there are now two slug tanks and two algae tanks for the upcoming project. With the red light from the hydroponics lights and the green from the algae, one might be tempted to think it looks a little like Christmas around here.
Here’s a better view of the new algae tank, with eggcrate to acts as a substrate and a small Hydor propeller pump to provide current. Several of the original samples of Bryopsis had started growing in fellow aquarists coral propagation systems, and were provided conveniently mounted on “frag plugs” onto which fragments of coral colonies are normally glued to enable them to grow. In this case, the growth of the algae on the plugs allowed easy and stable placement in the eggcrate.
There have been a few other improvements as well. In November, I got the new Neptune Systems Apex controller hooked up. Although it is capable of much more, it is hooked up to monitor and log energy use, system pH, water temperature, air temperature (in case of failure of environmental controls in the building), and moisture on the floor. The unit controls all of the lights, pumps, chiller, and heater, and can, for example shut down the pumps automatically in case of a spill being detected. Further, the system is completely accessible through a cloud-based interface, which allows control while I am away from the office.
Along with keeping an eye on the system when I am away, the Apex provides a log of some parameters. For example, when all of the lights were on at the same time of day, pH fluctuated significantly. The upper plot in the graph show detail of pH rising from 8.1 in the early morning to 8.5 when the lights went out at the end of the day. Because CO2, which reduces pH through the formation of carbonic acid, is consumed during photosynthesis when the lights are on and builds up at night, it is not surprising that pH steadily rises during the day.
Although it did not seem to cause the plants or animals much stress, it seemed worth trying to reduce the fluctuation. Rather than having all lights on at the same time of day, I set the lights on the algae growth tank to be on during the night. In this way, photosynthesis would be relatively constant in the system over the course of the day, with the potential benefits of stabilizing pH and keeping consumption of nutrients relatively steady. I was pleasantly surprised to see the pH fluctuation reduced to between 8.25 and 8.35. Frankly, the livestock did not seem as excited as I was.
Finally, I added a small protein skimmer (a.k.a., foam fractionator) to remove microalgae and organic material from the water column. Some might argue that suspended material serves as a source of nutrients for the plants, but in my experience desirable algae and plants tend to outcompete nuisance algae when a skimmer is used. None of the the nutrients provided by the dosing system are removed by skimming, so the skimmer just removes the compounds that are not under my control. With the height restrictions of the sump, I went with a Reef Octopus BH50, which is designed for small systems. It has been easy to set up and adjust, and extracts significant amounts of smelly material every day.
For the next few weeks, we’ll be watching algae grow. After the holidays, it will be time to order up a batch of slugs for the next round of Slug Science!