[34cygni]

Happy New Year, everyone.

Sorry for disappearing, but it wasn't for lack of interest in the subject matter -- rather the opposite, actually. I've been off doing more homework, starting with an interlibrary loan request for a book on the evolutionary history of marine primary producers. After I read it, I spent some time following up on some of the papers cited in it that looked interesting, but which weren't particularly relevant to the topic at hand (this guy is my new hero). Having fallen well behind the thread by then, I hit a runtime problem: the longer I was off doing my own thing, the more there was to catch up with.

Let's start with skimmate dosing. Some good questions have been asked since I last popped up and I'll try to answer a few of them along the way, but that's the obvious old business, and addressing it gives me an avenue to lead you guys into what's going to be a very lengthy science lecture about dinos, corals, bacteria, and algae in general... I apologize up front for the length of this write-up, which is cosmically longer and more science-y than my earlier posts on this topic -- so much so that I'm going to have to break it up into several separate posts because RC has a 25,000 character limit. Yikes! But as I said before, wrestling with O. ovata brings us about as close to the cutting edge of science as reefers are ever likely to get, and investigating this over the last few months proved to be the necessary motivation and correct angle of attack to at last bring my mental model of how reefs function into the 21st Century. I like to regard my experience of this hobby as playing "catch up with the scientists", and I found all the cool scientists hanging out in a Venn diagram where evolutionary biology intersects with the microbial loop and biogeochemical cycling. It's a party. You guys should totally check it out, and this is my best attempt to sketch out a map that will get you there.

Long story short (and vastly oversimplified) the bacteria associated with toxic dinoflagellates can kill corals, and the bacteria associated with healthy corals and other macrofauna can kill toxic dinos, including O. ovata.

I doubt this is a happy coincidence. It looks like an evolved defense against benthic dinoflagellates. Corals have lived alongside dinos for at least 228 million years, when the first dinoflagellate cysts show up in the fossil record, so obviously they've had lots of time to work the problem. That's undoubtedly what Montireef stumbled onto when he dumped skimmate into his tank: healthy corals release mucus loaded with coral-friendly bacteria; coral mucus is colloidal and its chemical backbone is a polypeptide, making it highly skimmable; the coral-friendly bacteria weaponized Montireef's skimmate and knocked back his dino bloom.

So if you are sufficiently geeky to ride this ride, go pee, maybe swing by the fridge, and settle in. Software updates always take a while, but BONUS!!! Along the way: sponges. I know -- exciting, right?

Okay, everybody ready? Off we go...

Originally Posted by DNA

Yesterday morning I started to add the content of my skimmer back to the tank after having sat there for a week.
I drained the wet part from the skimmer in 3 doses 3 hours apart to make sure it was not too much of a shock to the fish.
I can't say I like the smell of sulfur in the morning, but it didn't seem to have any effect on the fishes.

Sulfide is generated by anaerobic bacterial respiration. Under the "millions of microscopic rafts of colloidal organic carbon" theory of probiotic skimmate dosing, the presence of anaerobes would reduce the potential effectiveness of skimmate dosing against a dino bloom because they'd die (or, if aerotolerant, go dormant) as soon as they hit oxygenated water.

Originally Posted by DNA

Since corals are showing a little color it could be that the corals are hosting a new type of dinos.

I suppose it's possible, but it seems unlikely that corals of different species would all accept new symbiotes at the same time, and the dinos Montireef reported finding in his skimmate, oxyrrhis marina, would not be of any use to corals because they're heterotrophic and from the wrong genus. In fact, they're a well known "out group" in genetic reconstructions of dinoflagellate family trees and are thought to be a basal lineage that predates the evolution of mixotrophic dinos.

12/30/2014, 10:41 AM #557
Montireef
Yes, my system was too ULNS and noticed important bacterial growth in the skimmate so I dumped the whole cup. Corals got very happy and extended polyps.

Did you see anything like that, DNA? Polyp extension is often a reaction to nighttime hypoxia in the wild, but in our tanks, that's typically feeding behavior. That would suggest there's food in the water, which would be a simpler explanation for improved color than swapping in a new species of dinos. And we know corals eat bacteria...

Originally Posted by The Coral Probiotic Hypothesis

Three of the most abundant organic compounds in the sea, cellulose, agar and chitin, are degraded by bacteria but not by eukaryotes. After the bacteria degrade these compounds and multiply, some of these bacteria may serve as food for the coral animal. Thus, coral bacteria can allow corals to obtain energy from a complex mixture of polymers. In addition to having an enormous genetic potential to produce degradative enzymes, the relative amounts of different coral degradative bacteria can change rapidly as the nutrient source changes.

...so it's not much of a stretch to conclude that corals would be interested in bits of colloidal organic carbon with bacteria growing on them. That's pretty much what their mucus is, actually.

BTW -- fun science fact: nitrogen is required for the synthesis of chitin (poly-N-acetyl-D-glucosamine), the protein used to form the exoskeletons of, among other things, pods. And corals eat pods.

Originally Posted by DNA

The results are in.
After 5 days the dinos are just going on with their daily lives as usual.
If fact I've got slightly more of them right now than last 12 months.

This means we can't say that recycled skimmate will help with a dino problem.
We also can't say it's useless until several others try this out.

Well, that's very disappointing. I suggest trying again with fresh skimmate, as that's what Montireef indicated he used.

Originally Posted by Quiet_Ivy

Day 3 adding week-old skimmate collected in a jar back to my tank.

Good news: Got here this morning and Whoah! 90% of the cyano is gone!

Bad news: big dino outbreak, I see the harder brown circular spots on the glass and some have developed strings since yesterday morning. Hermit crab is ok. Corals ok except for my hammer which sucked all its polyps in and looks very cranky.

That correlates with DNA's experience, so it looks like good data to me... Week old skimmate makes dinos worse.

Since both you and DNA reported dino growth, the simplest explanation for these failures is that week-old skimmate is dino food, either indirectly by providing organic carbon to feed the heterotrophic bacteria the dinos are farming, directly by providing bacteria to feed the dinos themselves, or both at the same time. Your dinos were fruitful and multiplied, and they ate all the cyano.

To think about how this went wrong, I needed information, so something I had been avoiding had to be faced up to: I knew from the beginning that I should've looked into the known bacterial associates of O. lenticularis to see what they had in common, but I also knew this would be rather time consuming and only relevant to O. ovata to the extent that I could get away with reasoning by inference, so I had no idea if it would be time well spent or not... But with DNA and Quiet_Ivy reporting failure, I was curious to find out if there were any bacteria that would do well in anoxic or hypoxic skimmate, so I bit the bullet.

And sure enough, there was a pattern. It looked like O. lenticularis favors a class of bacteria called gamma-proteobacteria, many of which are facultative anaerobes, but Old School lab techniques are biased towards detecting gamma-proteobacteria because they generally grow readily on agar in petri dishes, so that could be an artifact. Happily, a more recent paper that examined the microbiome of two strains of O. lenticularis using genetic tools capable of detecting bacteria that can't be cultured confirmed that these dinos consort with gamma-proteobacteria, and more intriguingly, they may have an obligate association with another, entirely unrelated species of bacteria.

Originally Posted by Bacteria Associated with Toxic Clonal Cultures of the Dinoflagellate Ostreopsis lenticularis

O. lenticularis, as well as other toxic dinoflagellates, have been reported to have bacterial species associated to them. Aeromonas, Alteromonas, Bacillus, Cytophaga, Flavobacterium, Moraxella, Pseudomonas, Roseobacter, and Vibrio are the bacterial genera most frequently associated with toxic dinoflagellates. ...

A total of 127 sequences (62 sequences from clone no. 302 and 65 from clone no. 303) were generated and analyzed phylogenetically...these clusters were grouped within two major bacterial clades that included: Proteobacteria (alpha and gamma) and the CFB complex. Previous studies have shown that bacterial communities associated with different toxic dinoflagellates are also restricted to these two bacterial phyla. From this analysis, we found that both O. lenticularis clones have nine bacterial species associated to them, two of which where common to both dinoflagellate clones. These bacterial species were studied further because they may represent organisms persistently and specifically associated to the dinoflagellate. The first bacterial species, represented by clusters 302T-1 and 303T-9 (100% homology), belongs to the CFB complex (referred to as CFB 302T-1 from now on) and is the most predominant organism constituting 51% and 47% of the sequences from clone no. 302 and clone no. 303, respectively. The second organism, a gamma-Proteobacterium represented by clusters 302T-9 and 303T-2 (100% homology), constitutes 19% and 15% of the sequences from clone no. 302 and clone no. 303, respectively.

The persistent and specific association of these bacterial species was further tested... The results show that the gamma-Proteobacterium was not present in the new O. lenticularis clones. ... The 16S rRNA gene from CFB 302T-1 did not amplify from total bacterial DNA isolated from a clonal culture of a free living dinoflagellate Cochlodinium polykricoides also established from southwestern Puerto Rico. Considering that CFB 302T-1 was present in clonal cultures established a decade after the original clones used in this study and that it was not present in C. polykricoides, we conclude that this organism has a persistent and apparently specific association with O. lenticularis. ...

These two 16S rRNA sequences share 99% identity to each other, but were only 95% similar to the closest GenBank match (AM040105), suggesting that both these clusters represent a new VBNC [viable but non-culturable] bacterial species belonging to the genus Bacteroides

This is puzzling... Bacteroides are found in anoxic marine sediments, but they're generally regarded as anaerobic gut bacteria -- or that, at any rate, is clearly where most of the grant money is ATM. They do have a tendency to produce useful chemicals like vitamins B-12 and K that make them potential symbiotes, and it has been speculated that endosymbiotic bacteria are supplying dinoflagellates with B-12, but bacteroides are not what one would expect to find in association with a primary producer that releases oxygen when the sun shines. The author seems to be aware of this, as the "family tree" published in that paper emphasizes a more tenuous connection between CFB 302T-1 and bacteria from the genus cytophaga (a 90% hit, below the 93% similarity that's taken to indicate two bacteria are probably from the same genus), which consists of aerobic bacteria that fill a similar ecological niche as bacteroides. Indeed, in the original version of this paper (a master's thesis from 2006) the bacteria are unambiguously described as a cytophaga species -- the 95% hit on AM040105 is acknowledged but otherwise ignored. Reading between the lines, I can't help but suspect that the student's faculty advisor was similarly suspicious of the hit on AM040105: CFB 302T-1 can't really be a bacteroides! Hmmm... Best go with the second-best match, instead.

But on the other hand, an obligate association with anaerobic bacteria might make sense for benthic dinos, and it would explain why sulfurous skimmate is dino chow... After all, in the absence of water-pumping infauna like worms and shrimp that toxic benthic dinos would drive off or kill, typically only the top 5-10 mm (call it a quarter to half an inch) of reef sands in the wild are oxygenated; and it's well known that diatoms and other single-celled organisms on tidal flats migrate as much as several centimeters into the sand at low tide to protect themselves from UV light and dessication, so it may be that strong swimmers like dinos routinely dive into hypoxic or anoxic sand in search of a meal. In fact, bacteroides are capable of breaking down and consuming the ostis' cellulose armor, so cultivating an aerotolerant bacteroides that goes dormant in the presence of oxygen would be a good way for ostis to keep their food from eating them while they're swimming around on the nightly hunt or looking for someplace to establish a new bacteria farm.

Additionally, dinoflagellates are unique among oxygenic photosynthesizers in that the vast majority of mixotrophic dinos use form II rubisco, and that version of the 4 billion year old enzymatic flywheel that primary producers use to fix carbon is associated with anoxygenic photosynthesis and photoheterotrophy. Dinos stole the genes for it from bacteria and made it work, presumably because it's fast, and being able to rapidly fix carbon is obviously a useful trait for a primary producer. In fact, it has been argued that stealing genes from bacteria is dinos' raison d'etre -- their nuclei store DNA in a weird way that's not quite eukaryotic and not quite prokaryotic, so much so that they were hypothesized decades ago to be descended from an ancient "missing link" organism halfway between bacteria and eukaryotes, but that didn't pan out. Apparently, the reason dinos store their DNA this way is to make it easier to insert bacterial DNA into their own genomes. Stealing and hoarding DNA would explain why dinos have oversized genomes, and they're so freakishly good at it that they made form II rubisco work where it has no business being: inside a eukaryotic, oxygenic primary producer. If they can do that, I wouldn't put it past benthic dinos to have acquired genes that help them survive foraging expeditions in anoxic environments, so maybe O. lenticularis really does have an obligate association with a bacteroides species.

Google Scholar couldn't find many useful papers about benthic bacteroides in marine sediments and reef sands. They're part of the normal benthic anaerobic community, but most of the current research into bacteroides in the marine environment concerns sewage outflows from coastal cities and fecal bacteria contaminating beaches and near-shore waters, which BTW ostreopsis dinos are negatively correlated with in the Mediterranean... If ostis actually do heart bacteroides, it's apparently an aerotolerant marine species that they're into, not an intestinal species we're flushing into the oceans.

GenBank is open the public -- our tax dollars at work! -- so I searched for AM040105 and found that it's an uncultured Bacteroidetes first detected in the sands of tidal flats in the North Sea. The paper in which it was described is behind a paywall, but the abstract is available and points towards "the Cytophaga/Flavobacterium group" aka the Cytophaga-Flavobacterium-Bacteroides (CFB) group. The sands AM040105 were found in are described as well oxygenated, but on the other hand, aerotolerant anaerobes tied into the sulfur cycle were detected in significant numbers -- maybe there are aerotolerant bacteroides present, as well, or maybe there are anoxic microenvironments in biofilms growing on buried detritus where bacteroides can thrive.

So not much help there, but we know AM040105 is a marine Bacteroidetes, and so is CFB 302T-1 as the CFB group is now the phylum Bacteroidetes. To have narrowed it down even that much is useful.

Originally Posted by Ecology of marine Bacteroidetes: a comparative genomics approach

Members of the phylum Bacteroidetes are the most abundant group of bacteria in the ocean after Proteobacteria and Cyanobacteria. They account for a significant fraction of marine bacterioplankton especially in coastal areas, where they represent between 10% and 30% of the total bacterial counts. They are globally distributed in a variety of marine environments such as coastal, offshore, sediments and hydrothermal vents.

The better known members of the Bacteroidetes are specialized in processing polymeric organic matter, particularly in the mammalian gut (for example, Bacteroides spp.) or in soils (Cytophaga). In aquatic habitats, Bacteroidetes are abundant during and following algal blooms, showing a preference for consuming polymers rather than monomers. In the oceans, the main lifestyle of Bacteroidetes is assumed to be attachment to particles and degradation of polymers. ... Thus, Bacteroidetes likely have a different life strategy to that of other marine bacteria such as Alphaproteobacteria and Cyanobacteria. The latter are photoautotrophs, while marine Alphaproteobacteria (at least the most abundant ones) are aerobic heterotrophs that preferentially use monomers and live suspended in the water column. If the preference of Bacteroidetes for polymers and an existence attached to surfaces could be confirmed, their role in the carbon cycle of the oceans would be complementary to that of the other two groups. ...

The number of peptidases and GHs [glycoside hydrolases, enzymes that break down high molecular weight polysaccharides like cellulose] increased with the size of the genome in all bacteria. Most Bacteroidetes had more of these enzymes than the average bacterium, irrespectively of the genome size. This is one of the major observations showing the dedicated role of marine Bacteroidetes as polymer degraders. ...

A striking observation was that marine Bacteroidetes had many more peptidases than GHs. This was not the case for the non-marine Bacteroidetes examined. This strongly suggests a specialization of marine Bacteroidetes on the degradation of proteins, which is consistent with experimental studies using microautoradiography. ...

These indices show that not only do these bacteria have more peptidases than GHs, but that there is a larger diversity of the former. Thus, the conclusion that protein degradation is the main speciality of marine Bacteroidetes is robust.

So regardless of whether the obligate association O. lenticularis has is with an anaerobic bacteroides or an aerobic cytophaga or from some other genus entirely, we know it's a marine Bacteroidetes and thus have a good idea of what ecological niche it fills.

The assertion that proteobacteria are planktonic and Bacteroidetes colonize detritus isn't wrong but should not be taken as gospel. Bacteroidetes have an unusually large number of genes for making adhesion proteins that facilitate sticking to stuff which indicates that this is an important aspect of their biology, but they're also found among the plankton, and proteobacteria have genes for adhesion proteins, too, and are found growing on marine snow. Similarly, proteobacteria tend to consume labile organic carbon and Bacteroidetes specialize in breaking down and consuming very large organic molecules, but there are proteobacteria that can consume high molecular weight polysaccharides like cellulose, lignin, and chitin, and Bacteroidetes can consume the simple sugars that polysaccharides are made from (for example, cellulose is made from polymerized glucose -- hence "polysaccharide").

The observation that Bacteroidetes play a complementary role in the marine carbon cycle to cyano and proteobacteria is very interesting, as Bacteroidetes and proteobacteria normally dominate communities of marine heterotrophic bacteria. Cyano makes organic carbon, and diazotrophic cyano is protein-rich because it can fix nitrogen (...broadly speaking, nitrogen consumption indicates protein synthesis and growth, while phosphorous consumption indicates the synthesis of genetic material and reproduction), while proteobacteria and Bacteroidetes respectively specialize in metabolizing labile organic carbon and recalcitrant organic carbon -- or as we hobbyists would see it, that's a primary producer and a CUC. So that raises an interesting question about the bacterial community around O. lenticularis: Is there a similar bacterial CUC associated with dinos?

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