Bryopsis 2: Thriving in the slug tanks

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.

10 gallon slug tank a few weeks after system revamp. Sponge-covered circulation pump visible at right, and a small clump of Bryopsis in rear center. 3/8/17.

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.

10 gallon slug tank, showing heavy growth of Bryopsis after a few months. Other algae are doing fine, but the Bryopsis is taking over. 5/9/17

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.

Parts of a slug-proof circulation system. The intake of the Maxi-Jet powerhead is covered by a strainer and a sponge, because a strainer alone will not prevent injury to slow, squishy, dumb slugs. The output goes through a Hydor Flo rotating deflector to provide surgy motion.

Assembled Elysia circulator. In the slug system, each one is anchored to the glass with a magnetic holder.

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.

New E. clarki, acclimating after arrival from KP Aquatics. 5/11/17

How to Grow Bryopsis

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.

E. clarki feeding on Bryopsis; the red alga, Botryocladia, is in the background.

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.

The whole gang enjoying the new batch of algae provided by a local retailer. 4/17/16.

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:

  • Levels of light seemed to be within the range I observed in tanks with steady outbreaks, so I left that alone.
  • Bryopsis always grows well in the areas with the strongest water flow (e.g., pump outlets).  I am not sure why, maybe it keeps other algae from settling on it, but I increased the flow to ridiculous levels in the 15-gallon primary culture tank by combining a power filter Aquaclear AC70), powerhead (Hydor 2450) and wavemaker (MaxSpect Gyre XF130) that were all rated for tanks 3-5 times the size.
  • I plunged back into the literature to try to find papers examining the nutrient requirements of benthic macroalgae, rather than phytoplankton.  After a little digging, I found that species related to Bryopsis demand much more carbon and nitrogen, and are not as dependent on phosphorous (e.g., Atkinson and Smith, 1983, Limnol. Oceanogr. 28:568; Pedersen and Borum, 1997, Mar. Ecol. Prog. Ser. 161:155).

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.

Bryopsis growing on eggcrate in 15 gallon algae culture tank. 3/2/17

By growing on plastic racks, Bryopsis cultures can be moved to slug tanks to be consumed, and then removed for regrowth.

Bryopsis-covered plastic rack in 20L slug tank. 3/23/17

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.

Three new Penicillus plants surrounding the faded parent taken from the wild. 4/11/17

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.

  • N: dosing KNO3 (13.9% N); stock = 80 grams/liter (g/l), diluted to 20 g/l; dosing 80 milliliters/day (ml/d); that equals 0.222 g/d of N, or 0.0159 moles/day
  • P: Dosing KH2PO4 (22.8% P); stock = 11.6 g/l; diluted to 1.45 g/l; dosing 50 ml/d; 0.0165 g/d or 0.00053 mol/d
  • C: from acetic acid: 5% solution (50 g/l); 48 ml/d; C = 40%; 0.96 g/d; 0.08 mol/d
  • K: from KNO3 AND KH2PO4:  0.64 g/d or 0.021 mol/d
  • Guillard’s F/2 trace element and vitamin solution: diluted 1:20, dosing 100 ml/d;
  • I have also started adding calcium hydroxide (CaOH2, kalkwasser) to the water used to make up for evaporation to maintain Ca.

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.

Dinoflagellates at low magnification. They can be seen as strings of brown dots surrounding the green algae branches. 4/11/17

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.

DInoflagellate from Bryopsis tank, 400X magnification. Gold color is from densely packed chloroplasts.  4/6/17.

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.

 

Journal Club: Chemical Camouflage and George Harrison

Welcome to the Roughly Annual Solar Sea Slug Journal Club.

Today’s paper came from the Proceeding of the National Academy of Sciences a few years ago (Rasher et al., 2015, Proc Natl Acad Sci 112:12110).  I came across it again when I was updating records for this site, and, because it is germane to one of my pet theories, it seemed perfectly suited for an extended discussion. You’ll see how George Harrison fits into the story later.  Yes, this post will meander a bit, but the fact that you are reading the Solar Sea Slug Blog suggests you may have some time on your hands.

The paper is a very nice exploration of the interactions between herbivores and their food plants.  Up here on dry land, insects tend to specialize on particular food plants, and bugs and plants have evolved together in something of an arms race.  Insects use volatile chemicals produced by the plants to locate them, plants produce defensive chemicals to keep from being eaten and from being infected by insect-borne pathogens, insects develop resistance to the plant chemicals, and sometimes use them for their own defense, and so on.  The authors wondered if they could identify a similar web of interactions in the marine environment.

Elysia tuca in Box of Slugs 2.

The algae Halimeda incrassata would seem to be rather unpalatable.  It produces a collection of defensive chemicals, and is highly calcified, making it a crunchy, bad tasting mouthful.  Despite the defenses, Elysia tuca, a tiny and distinctively-patterned species, is commonly found on Halimeda.  The interaction between E. tuca and H. incrassata allowed the authors to ask how similar the relationship between a mollusc and a marine alga is to those of insects and terrestrial plants.

Halimeda incrassata, in Box of Slugs 2. Note the segmented appearance, which will be important in understanding Figures 3 and 4 of the paper. 3/12/17.

In order to compare the relationship between E. tuca and Halimeda to terrestrial plant-insect interactions, the study focused on five specific questions:
1)  Is E. tuca really a specialist? This is important for the development of an intimate plant-herbivore relationship.
2)  Does E. tuca find Halimeda based on chemical cues?
3)  What are the cues that E. tuca uses?
4)  What are the ecological consequences of E. tuca feeding on H. incrassata?
5)  Does Halimeda use counter-defenses to limit the damage inflicted by E. tuca.

With regard to E. tuca being a specialist, the answer was a pretty resounding “yes.”  They collected specimens of about 10 species of algae and marine plants at two sites, took them to the lab, and counted the numbers of E. tuca on each.  With a few exceptions (also in the genus Halimeda), E. tuca were only found on H. incrassata.  Further, when given the choice between many different algae species in the lab (Fig 1A, below), or three different species of Halimeda in the lab (Fig 1B), or in the field (Fig 1C) the slugs greatly preferred H. incrassata.  To test whether the slugs were following chemical cues, the experimenters soaked cotton balls in water that had held H. incrassata (Fig 1D), and found that E. tuca much preferred these to cotton balls soaked in plain seawater.  With regard to the questions posed by the paper, the results indicate that 1) E. tuca is a specialist, and 2) they find their host based on chemical cues.

Fig. 1. Elysia host preference. Number of trials in which an Elysia colonized one of 14 common seaweeds and seagrasses (n = 20) (A), three co-occurring seaweeds in the genus Halimeda (n = 20) (B and C), or a cotton ball laced with H. incrassata-conditioned seawater vs. seawater only (n = 40) (D), when offered in a still water arena (A, B, and D) or in the field (C). Choice was assessed after 2 h (A–C) or within a 5-min period (D). Results were analyzed by a Cochran’s Q (A–C) or Fisher’s exact (D) test. In A–C, different letters above bars indicate significant differences among seaweeds in terms of Elysia colonization frequency, as determined by Wilcoxon sign tests (corrected for multiple comparisons). AL, A. longicaulis; CC, Caulerpa cupressoides; CP, Caulerpa prolifera; CS, Caulerpa sertularioides; DC, Dictyosphaeria cavernosa; HI, H. incrassata; HM, H. monile; HO, H. opuntia; PC, Penicillus capitatus; PD, Penicillus dumetosus; RP, Rhipocephalus phoenix; SF, S. filiforme; TT, T. testudinum; US, Udotea sp

The next question regarded the identity of the chemical attractants from H. incrassata (which will be henceforth referred to as “Halimeda”).  Compounds were extracted from Halimeda with methanol, and the individual components of the extracts were separated as described in the supplementary methods.  Each fraction was tested for attractiveness to slugs using the cotton ball colonization assay described in Figure 1, above.  The first compound they described, 4-hydroxybenzoic acid (4-HBA; Figure 2A, left) is found in both “vegetative” Halimeda in the normal growing stage, and in “reproductive” Halimeda that are undergoing spawning events.   When 4-HBA was placed on cloth patches next to Halimeda, the plants were colonized by significantly more Elysia than to controls with cloth soaked in the solvent but no 4-HBA (Figure 2B, left panel).

The reproductive stage of Halimeda is significantly more attractive than the vegetative stage, in part because the reproductive cells (gametes) are a rich source of nutrients.  When patches soaked in extract from reproductive plants were placed next to Halimeda plants, they attracted more than twice as many slugs as those from vegetative plants (Figure 2B, right).  This led the authors to identify halimedatetraacetate (HTA; Figure 2A, right) a chemical compound enriched in reproductive Halimeda.  It was known that HTA deters feeding on Halimeda by other species, and that E. tuca sequesters HTA in its tissues.  The authors went on to show that an extract from E. tuca that contained HTA deterred feeding by predatory wrasse.

This brings us to question #4, what are the ecological consequences of E. tuca grazing on Halimeda?  Surprisingly, the effects of such tiny slugs are significant.  The fact that the slugs feed on reproductive structures (which have the highest HTA content) is expected to substantially reduce the plants’ fecundity.  Further, when the authors manipulated the numbers of Elysia on plants in the field, those with more slugs showed less growth (Figure 3A) and more branch loss (Figure 3B).  Placing E. tuca in enclosures on branches (Figure 3D) also caused more branch loss compared with enclosures with no slugs.  So E. tuca can cause significant damage to Halimeda.  Because H. incrassata aids the development of seagrass beds, and generates the majority of carbonate sediments (a.k.a., nice white sand) in those areas, the authors suggest that grazing by E. tuca can have ecosystem-wide consequences.

How can a small slug that sucks sap cause such dramatic loss of plant tissue?  One hypothesis is that the plant self-amputates segments that have been fed upon by Elysia.  The model Rasher et al. propose is that the plants are trying to avoid the introduction of pathogens by the slugs by sacrificing segments.  After culturing fungi from the slugs’ radullae, which they use to pierce the plants’ tissues, they tested one fungal species they referred to as Et-2.  Halimeda innoculated with the fungus dropped segments above the injection site (Figure 4A).  Injection of a fungus that is a pathogen of other species did not have the same effect (Figure 4B).  The data are therefore consistent with the hypothesis that loss of segments is a defensive strategy in response to feeding by E. tuca, suggesting that the answer to question #5 is also yes.

The authors conclude that the answer to all of the questions they posed is “yes,” and that marine plant herbivore interaction described above strongly resembles those in terrestrial ecosystems, despite more than 400 million years of separation between the participating species.

At some point, this paper got me thinking of a potential alternative function for kleptoplasty.  Shall we meander our way there?

By the end of last summer, I was finding most of the prevailing theories regarding kleptoplasty to be rather unsatisfying.  While not every aspect of biology must have a function, kleptoplasty has costs that must be offset.  It takes energy to segregate and store the chloroplasts, and they must be protected from the immune system.  Plus, the animals that are active in the sunlight are exposed to predation and damage from UV light.  Despite these costs, Elysia is a very successful genus, with species found worldwide in the shallows of tropical and temperate seas.  Therefore kelptoplasty must provide a significant benefit.

So, what good is kleptoplasty?  If you buy the arguments presented by deVries et al, photosynthesis by kleptoplasts do not supply a significant portion of the animals’ energy needs.  Is the energy produced by photosynthesis used to make starch or fat for use during lean times?  Maybe.  Could the kleptoplasts be a “living larder,” being digested when food is scarce?  The animals certainly become pale when they are starved, suggesting the kleptoplasts are being broken down, but why not just digest them at the time they are eaten and turn them into fat like the rest of us do?

One idea is that the kleptoplasts are merely used as camouflage.  In the case of E. diomedea, which spends a lot of its time hidden in its food plant, this seems sensible.  Not so much for E. crispata, which is easily visible against hard bottom reefs, which are generally not very green.  Further, it seems like there are other, less complicated ways of making or storing green pigment to match one’s surroundings.  However, let’s hold that thought for a minute.

Aside from making carbohydrate from sunlight and being green, chloroplasts produce important precursors for many biochemicals (e.g., Gould et al., 2008, Ann. Rev. Plant Biol. 59:491).  These could be used by the slugs for the synthesis of fats or essential aromatic amino acids for their own nutrition, or to be used for their prodigious production of eggs.  Given that there is absolutely no data regarding the role of photosynthesis in egg production by Elysia, this remains an attractive hypothesis.

However, an insight I thought was particularly brilliant was that chloroplasts synthesize isopentyl diphosphate (IPP), a precursor to a wide range of things, such as chlorophylls and terpenes. Some of these compounds are expected to be smelly, and, in principle, make the slugs smell like their food.  Some of the chemicals may also taste bad, rendering the soft, slow animals less palatable.

Predators of Elysia are expected to include nudibranchs, which are largely blind and find their food by smell, or fish, many of which find their prey by sight.  If the chloroplasts were pumping out chemicals that gave the slugs a smell of their food, it would make it much more difficult to find them by scent.  One bonus is that the green color of the kleptoplasts will also make it more difficult for visual predators, such as fish, to find the slugs.  On top of that, any noxious taste would protect the slugs from predators, regardless of their hunting methods.  Overall, this model seemed to have fewer caveats than any of the others.

I thought I had come up with this idea on my own.  Then I rediscovered the above paper by Rasher et al. while I was updating a saved search in the Scopus database.  PNAS is my Wednesday lunchtime reading, and I am sure that I was excited to come across a paper about Elysia, so I am certain that I read it when it came out.  I am saddened by the fact that I forgot that I had read the paper, and assume that the paper got me thinking about kleptoplasty and chemical camouflage.

Have you figured out the connection to George Harrison yet?  You have to be getting on in years or love music trivia to remember, but he produced a popular song “My Sweet Lord,” during his post-Beatles solo career.  He ran into some legal trouble when the Chiffons’ record label sued him for appropriating the melody from their highly popular “He’s So Fine.”  If you go online and listen to both of them, it won’t take you long to think “dang, he stole their melody.”  Harrison admitted that he was very familiar with the melody, and the judge ruled that he had committed “subconscious plagiarism.”  In the same way, I had no memory of even having read the Rasher paper when I was formulating ideas and researching the biosynthetic capabilities of chloroplasts.  I just thought I was being terribly clever.  Nonetheless, it is highly likely that the paper was somewhere in the recesses of my mind during the process.  Are there any truly new ideas?

But, more importantly, how does one test this?  The first step is to make some predictions, and here are a few possibilities:

  1. Comparison of compounds produced by food plants, normal Elysia, and Elysia in which photosynthesis is blocked will show compounds produced by plants and normal slugs that are absent or significantly less abundant when photosynthesis is blocked.  All we need to do is make some extracts and find a potential collaborator who is willing to perform some gas chromatography-mass spectroscopy on a small budget.
  2. Elysia in which photosynthesis has been blocked will be easier to detect or more attractive to predators.  Placing a predatory mollusc or fish in a Y-maze with a choice between an arm containing a control and one containing  a photosynthesis-blocked Elysia might do the trick.  There are plenty of hungry wrasses that we could use for the fish test, either in the lab or in Bahia.  As far as predatory slugs, Navanax or Roboastra are good candidate predators for E. diomedea in Baja California.

There are likely to be more, better experiments, but the above provide a start.

 

 

 

 

Slug System Upgrade

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.

Old slug and algae system before upgrade. 2/25/17.

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.

Shelves built 2/5/17

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.

Shelves painted and ready for plumbing, 2/15/17.

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.

Drains installed 2/18/17.

The new system in place, showing the supply valves 2/25/15.

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.

Up and running, 2/26/17.

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.

What does Elysia crispata do on the reef?

Back from Bonaire, with a fresh puzzle.

Elysia crispata at The Cliff in Bonaire. January 11, 2017.

In research, as in life, there are things that don’t make sense.  Often these things make enough sense that you ignore them, choosing to focus on other mysteries.  One such little small, nagging issue is the question of what draws Elysia crispata to hard-bottom coral reefs, which lack obvious growth of green algae known to be their food.  Based on observations of many years, the slugs are not in transit, most are just sitting there.

Lettuce slug, with some Dictyota and red turfy algae, but not much in the way of greens. The Cliff, 1/11/17

My knowledge of the habits of Elysia in the wild is far from encyclopedic, but the species I know best have hearty appetites and stay close to their food.  E. diomedea are found on or near Codium in Bahia de los Angeles, and E. clarki spend most of their time face down in their food in aquaria.  This tends to hold true in the literature as well.  For example, E. tuca is generally found on its favorite food, Halimeda incrassata (Rasher et al., 2015, PNAS 112: 12110).  As a counter example, Middlebrooks et al. (2014) found that E. clarki were often found at sites that contained few or no specimens of their food plants (Penicillus, Halimeda, Bryopsis) determined via DNA barcoding.

Two slugs, in typical habitat. The Cliff, January 11, 2017.

In any case, I think I am justified in being puzzled by the lack of an obvious food source on the reef.  The photos in this post are all from a single dive at The Cliff, a site in the north-ish part of Bonaire.  We found maybe a dozen slugs, most in the face-down posture, which makes them look like large blobs of colorful frosting on the rocks.  The area had a lot of dead coral, which possibly serves as a substrate for the growth of food algae.  However, there were no obvious growths of green algae anywhere nearby, although algae such as Halimeda and Caulerpa are plentiful in mangroves on the island.

E. crispata, with a mix of turfy algae, including possible green filamentous species. The Cliff, January 11, 2017

Rather than snap a few photos of the more photogenic slugs, I thought it might be useful to document as many of the slugs as I could, with emphasis on the substrate.  Honestly, what you see is what you get; there are no large clumps of Bryopsis or Halimeda hiding around the corner.

Two E. crispata, surrounded by short, sparse turf. The Cliff, 1/11/17

What are these gals eating?  The most prominent alga is Dictyota, a brown alga which, based on known feeding habits, is an unlikely food.

E. clarki, surrounded by the flat leaves of Dictyota. No green algae in sight. The Cliff, January 11, 2017.

Are they grazing on the little strands of green algae that can be seen if one expands the photos and looks really hard?  Is this a late life stage that does not feed as much?  Are E. crispata truly crawling leaves, getting their energy from photosynthesis?  Is the much lighter color of E. crispata, compared to related species, like E. clarki and E. diomedea, a clue?

Slug from the beginning of the post, with wider field.

 

 

 

Mystery Alga

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.

Elysia clarki grazing on unknown species of alga. 11/20/16

Alan’s algae, view of whole plant. 12/27/16.

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.

Tip of branch. Scale bar = 2 mm. 12/27/16.

Stalk, showing absence of cellular divisions.  The little round bumps on the branches are reproductive structures.  Scale bar = 1 mm. 12/27/16

Acrosiphonia spinescens from Algaebase, showing cellular divisions and hook-like branches. © Ignacio Bárbara

Branch tip of Alan’s alga. Scale = 1 mm. 12/27/16.

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.

Observations on Algae

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.

“Spongy sea pansy” in 20 gallon sandy-bottom tank in office system. 12/22/16.

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.

Slugs mating on Udotea 10/30/14

New growth from rhizoid of Avrainvillea, aka “spongy sea pansy” 11/16/15

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.

Testing New (Old) Machines

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.

honors-seminar-f2016-ver102216

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?

Applied Biosystems PCR machine from NIH surplus. This is Machine 1, for which the temperature was within one degree C of the programmed value. 11/4/16

Applied Biosystems 9700 PCR machine from NIH surplus. This is Machine 1, for which the temperature was within one degree C of the programmed value. 11/4/16

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.

Fragment of parapodium (in vial) carefully packed and shipped from Austin.

Fragment of parapodium (in vial, center) carefully packed and shipped from Austin.

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.

Small piece of E. diomedea parapodium before extraction. 11/2/16

Small piece of E. diomedea parapodium before extraction. 11/2/16

At the end, I had produced a tube of clear liquid.  Was there DNA?

DNA extracted from E. diomedea. 11/2/16.

DNA extracted from E. diomedea. 11/2/16.

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.

Results of first test of new PCR machines. + control" is commercially prepared DNA from BioRad, containing a mix of DNA with and without an ALU insert in the PV92 region of the human genome. There should be bands at 941 and 641 base pairs. E. diomedea and Avrainvillea DNA were extracted a few days before. 11/4/16

Results of first test of new PCR machines. + control” is commercially prepared DNA from BioRad, containing a mix of DNA with and without an ALU insert in the PV92 region of the human genome. There should be bands at 941 and 641 base pairs.
E. diomedea and Avrainvillea DNA were extracted a few days before. 11/4/16

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.

Happy Birthday to us!

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.

Wild Slugs: Sea of Cortez Edition (Conclusion?)

As the summer winds down, it looks as though the project worked better than I had hoped.  There is a lot left to do, so this is far from the end, but what a great beginning!

To remind you of the the primary goal of the summer’s project, we wanted to use the DNA contained in the slugs’ kleptoplasts to identify their primary food plant(s).  The previous posts described how we worked out methods, collected slugs and candidate food algae, extracted the DNA, amplified the rbcL gene from the chloroplasts, and sent it off for sequencing.

The first sequence that came back from Macrogen did not look very good, which was disheartening.  The chromatograms looked awful, and the sequence was gibberish, causing concern that our extractions or PCR reactions were contaminated.

badchromatogram

Lousy chromatogram from Sanger sequencing. Note multiple possible bases (different colors) at each site. Uninterpretable.

Nonetheless, Paul Kim at Macrogen promised to optimize the reaction and sequencing conditions, and worked hard to provide interpretable data. Patience and persistence have finally paid off, and we can make some simple, declarative statements about the slugs and their food plants.

Codium sequence from Macrogen. 8/10/16

Codium sequence from Macrogen. Note a few sites showing more than one possible base, presumably polymorphisms.  8/10/16

Statement 1: We obtained usable rbcL DNA sequence from Codium, Ulva and Elysia.

Statement 2: Elysia diomedea steals most, if not all of its kleptoplasts from Codium.

To flesh out these statements a bit:

From Bahia, we now have DNA sequence for Codium simulans and for Ulva.  The Codium data is the first for the species.  Although rbcL sequence for related species (such as C. isabelae) can be found in the NCBI database, there is currently nothing for C. simulans.  We’re not sure which species of Ulva we used, although it is likely to be Ulva californica.  In theory the DNA sequence could have told us which species it was, but the region of the rbcL gene that we amplified and sequenced is identical to that in many of the species in the database, so we would need to try another gene, or a different region of rbcL.  An important lesson from this year’s work was that we need to preserve samples of the algae we sequenced.

The most exciting result was that we got sequence from E. diomedea kleptoplasts!  Overall, we extracted DNA from two individual slugs at different times, and performed at least three separate PCR amplifications (both in BLA and at USG when I got back), and they all came back matching Codium!  In retrospect, it is not a shock that slugs that we found in close association with Codium, and which spend a lot of their free time on Codium, actually eat Codium.  

Portion of rbcL sequences extracted from Codium simulans (top), Elysia diomedea (middle) and Ulva sp. (bottom). Sites at which Ulva differs from both Codium and Elsysia are indicated by arrows.

Portion of rbcL sequences extracted from Codium simulans (top), Elysia diomedea (middle) and Ulva sp. (bottom). Sites at which Ulva differs from both Codium and Elysia are indicated by arrows.

The figure above shows a small portion of the sequence, highlighting a few of the sites at which Elysia and Codium differ from Ulva.  Overall, the DNA sequence from Elysia was 99% identical with that of Codium, and those few sites that differed appeared to be locations at which there was variation between individuals.  Ulva showed about 81% identity to Codium and to kleptoplasts from Elysia.

E. Diomedea on Codium in tank at BLA station

E. Diomedea on Codium in tank at BLA station

Despite how it sounds, this is not a trivial result.

First off, Codium has been suspected, but never confirmed as a the food plant. Back in 1969, Trench and colleagues said that E. diomedea fed on green algae, possibly C. simulans, based on the chlorophylls found in the slugs and the morphology of the kleptoplasts, but their methods could not reliably distinguish between green algae species.

As a corollary, there is no evidence that they eat Ulva or Padina, despite being surrounded by them.  We did not get rbcL sequence from Padina this year, but it is not closely related to Codium, and the sequence in the database for P. durvillei (the most common species in our study area) shows roughly 70% identity to that from Codium and E. diomedea.  Had there been significant Padina or Ulva DNA in the slug sample, the presence of multiple divergent sequences are likely to have made interpreting the results impossible.  In other words, we got lucky that there was one dominant species of kleptoplasts.   Having sampled only two slugs, we can’t rule out other food plants.  Another caveat is that the result shows that chloroplasts from Codium persist in the slugs’ tissues, but the slugs could be eating other species for which the chloroplasts do not last as long inside the slugs.

Another important conclusion is that our methods actually worked.  As a neurophysiologist setting up a molecular lab in a dusty, hot garage in an isolated location, there were no guarantees that we would get any usable data.  In addition, we used degenerate primers for PCR, to amplify rbcL sequences from all potential algae species, counting on DNA sequencing to tell us which species were present.  Our choice of Sanger sequencing, which is much less expensive but prone to problems if the amplified DNA comes from more than one species, could have also caused complications.  Planning, persistence, and some luck all worked in our favor.

With these data in hand, there is lots more to do.  To fill in some of the gaps discussed above, we need to sample from more slugs in more locations.  At the same time, we need to more systematically collect specimens and DNA from algae at different sites around the bay, especially C. simulans.  If we are going to generate DNA sequences, we may as well do it in such a way that we can add them to the database.

There is also a lot to be done to understand the big picture of kleptoplasty and how E. diomedea fits into the ecology of the bay.  Because of delays in receiving equipment, we had very little time to prepare the behavioral experiments before we left Maryland.  On top of that, the losses and stress caused to the slugs by the extreme heat this year, resulted in essentially no data regarding the slugs’ preferences for light.  The I-mazes are build and ready, and we plan to add a chiller to the holding system, so procedures should be perfected before the next field season.  We also still don’t know much about their environmental requirements.  They eat Codium, and live on Codium, but do they have other requirements in terms of water movement, temperature, nutrients, or turbidity?

That the project worked can be chalked up to a lot of planning, hard work, and generosity on the part of a great group of people.  At the risk of sounding like an Academy Award acceptance speech…

Photobiology Group. Cristal, Rosalia, Nancy, Richy, Allison, Susan & Thiago.

Photobiology Group. Cristal, Rosalia, Nancy, Richy, Allison, Susan & Thiago.

There would have been no Photobiology group without the “Angels,” Cristal, Rosalia, Nancy, Allison, and Susan.  It was so much fun to watch them work and learn.  They will be giving their presentation during the Report to the Community for Ocean Discovery this week, and it will be great.

Richy Alvarez, the intelligent and talented Directed Research Fellow, was another reason this project came together.  There are so many big and small things that he did to make sure equipment was ready and that the students were prepared, I can’t thank him enough.  Big thanks also to Thiago Lima, for generously taking time away from his postdoc at Scripps to work with the students in the field, and for giving advice on the project (he is an actual molecular biologist) along the way.

Huge thanks to all of the staff at Ocean Discovery Institute, especially Joel Barkan, who coordinated the process of turning the plan into a reality when I was 3,000 miles away.  I can’t say enough good things about the support I received from everyone at Ocean Discovery, at all levels, and how easy it was to work so closely with so many people.  Bahia de los Angeles is a magical place, but doing science there can be a hot, tiring affair.  Working with this group makes the process so much more fun.

There would have been no time to work out procedures once we arrived in Bahia, so Maryam and Haseeb’s experiments and troubleshooting at USG were crucial.

The experiments also required equipment.  Some, like the PCR machine and centrifuge, were generously loaned (thanks ThermoFisher and USD!).  Others, such as the tanks and DNA sequencing were purchased from vendors who went the extra mile to do things well and on time (Glasscages and Macrogen).

None of this could have happened without permission from the Comisión Nacional de Áreas Naturales Protegidas (CONANP), which administers the Biosphere Reserve at BLA, and the support of Jose Mercado, who owns and operates the Casa Caguama field station in BLA.

3715_Drew070116

Dr Drew Talley, BLA staff office 7/1/16

Finally, I owe an enormous debt to Drew Talley, my best friend for over 40 years.  He introduced me to Bahia many years ago, and worked tirelessly this year to secure loans of equipment, permits, and who has been incredibly supportive of the development of this project.  He has the right to call himself the Captain.