EMeyer

Hi everyone, Since some of you have had your aquariums' microbiomes tested more than once now, you have probably noticed differences and wondered why. While we're thinking about changes in the microbiome I wanted to remind you of this classic paper on the effects of large water changes in aquariums. The authors found that Microbial diversity increased substantially following a large water change (90%) The groups enriched after a water change include both Cenarchaeaceae (ammonia oxidizing Archaea) and Vibrionaceae (a group with lots of pathogens). (These specific effects probably depend on what community you start with before the change) The authors emphasize that microbial communities are destabilized by disturbances, leads to a succession effects like those that happen after clear cutting a forest (just much more quickly!) This is why I've always advised clients to sample before doing any of their daily maintenance. But its worth thinking about more broadly. Even if you didnt change anything in your system between sample 1 and sample 2, even normal maintenance events may affect the community for a while. The authors suggest the changes are unlikely to affect the health of the inhabitants, but I'm not so ready to assume that tripling the ammonia oxidizing microbes and tripling Vibrio are neutral effects. They just werent measured here. And the study makes me wonder about automated low volume water changes vs infrequent large ones. I didnt capture that info in the survey. I wonder, for those of you who were tested, what are your water change practices and how do those affect the stability of your microbiomes?

Yeah, thats definitely the safest answer. I do plan to make a standardized diversity score (based on resampling to a standardized sequencing depth), so we can (within the PNWMAS group) compare our round 1 and 2 samples diversity using that metric. Directly comparing the reports could be misleading. Although as I described above in many ways these results are consistent.

The best answer is sampling was so different we should not compare them on the basis of raw number detected. This is one of the reasons I was so hesitant to adjust the sampling method, but felt it was worth it in the end. I am working on a standardized diversity score that will make it possible to compare the diversity numbers themselves. With that said, putting aside the diversity numbers themselves, the community barplots are directly comparable. I've showed before that taking two samples side by side from a single tank produces nearly identical results, so we know the measurement itself is reproducible. Your samples had plenty of DNA and sequenced well, so I have no reason to doubt the new community profile or the old one. Lacking any other information I have to conclude the community has changed and am also curious why. I do note that in many has it hasnt: You consistently have detectable levels of AOB and NOB in both samples, something many of us envy. You consistently have low levels of cyano, which in both cases includes Ulvophyceae. You consistently have no fish pathogens (found in 1/8 of tanks) or coral pathogens (found in 1/10 from the first round, zero client samples from the second) Many of the core families are consistently similar in your tank to the typical tank, e.g. in both samples, these families match the abundance in the average tank: Rhodobacteraceae, Vibrionaceae, Pseudoalteromonadaceae, Oceanospirillaceae, Hyphomicrobiaceae, Cenarchaeaceae, and Bacteriovoracaceae. That is a lot of agreement! (In comparing these plots, I note that I did change the color scheme slightly in the updated version, sorry about that. I'm trying to minimize changes to make these comparisons less confusing) It looks to me like what has happened is a proliferation of Alteromonadaceae and a reduction in Flavobacteriaceae and Pelagibacteraceae. Since the latter two are the most abundant in the typical reef tank (and in your first sample), and Flavobacteriaceae is one of the more diverse groups, these changes alone could account for a lot of what we're seeing in your scores. The high levels of Alteromonadaceae in your sample are almost entirely a single type, a bacterium that has not been identified beyond the family level. It showed up in many of the tanks surveyed, but was absent from most. A few had this same exact bug at high levels. Its present in all 4 of my home tanks at relatively low levels, for example. I can't find examples of this exact bug in previous environmental samples, although other members of the family are widespread in marine environments https://obis.org/taxon/393035 I include this just to say its not odd to have it present, but interesting to have it bloom like this in a few tanks. Your water conditions appear to have very stable during this time, in terms of what we typically measure. I wonder if there were other unmeasured changes. By any chance have you done any ICP during this period?

As you discuss this with various LFS it'd be great to ask about wholesalers who can special order it, too. For example at QM the Tonga Nano Branch Rock is not in inventory right now but is still shown on the special order page, so it might be available as it was earlier this year. I don't know if other wholesalers could special order other live rock not currently in stock, and only those with accounts can easily see what various wholesalers have to offer. (I have a hard enough time getting wholesalers to even respond to emails.) What helped me in discussions with suppliers was emphasizing (as you pointed out) that what I was looking for was rubble from high quality natural live rock.. it didnt have to be big and pretty.

I want to clarify the meaning of the color codes to be sure the results aren't concerning anyone needlessly. I dont have, and I dont think anyone has, evidence for what is the threshold between a good and bad microbiome. I am purely comparing each of our tanks to the rest. Red = most tanks (75%-100%) are more diverse than yours Yellow = 50%-75% of tanks are more diverse than yours Green = your tank is more diverse than at least 50% of tanks So yellow is the lower half of the typical range, not an extreme outlier. 50%, the line between yellow and green, would be the typical tank (the median diversity). 2 of my 4 home tanks are in the yellow (151,156), my oldest (5 y) is in the green (201), and my newest is in the red (72). None of these were started with good live rock. If a group buy for some real live rock (coral rubble from the ocean) happens I am in! Here is my thinking about diversity -- diversity is just a proxy for having the right microbes. We don't know exactly what each bug does, but most of us agree there are many beneficial roles for microbes. So if most of the tanks are more diverse than mine and my tank has problems, its likely I need some of what they've got. If my tank's doing great, I've probably got the microbes that perform the important roles. -- I also like to emphasize that its not just about diversity. Look, if I come over and drop a tube of Aquarickettsia rohweri, Vibrio coralliilyticus, and Vibrio alginolyticus in your tank, BAM! I've just increased the diversity. But I don't think anyone imagines the tank would be healthier I think as much about balance as diversity. My home tanks' balance scores range from 0.32-0.4, reasonable scores close to the median (0.3). But I see systematic excesses of Alteromonadaceae in 3 of them, deficits of Pelagibacteraceae in 3 of them, and deficits of Flavobacteraceae in all 4. So for most of my tanks, addressing the balance is a higher than the diversity per se. I'm still in if we can get our hands on some good live rock though!

I dont wanna reveal the microbiome of anyone's products without their consent, but I'll just say you can't spell "Fiji saltwater live rock" without "live rock a" Still grumpy about that purple cement

I've tested one of the products. I concluded that while it had the advertised effects of quickly metabolizing DON, it had basically no impact on the microbial community. I'll write up those results soon. We need more tests, but tentatively, my working model is that these products have very short lived effects on the microbiome and that using them for making a persistent change to the community (an off-label application, for most or all) is unlikely to make any difference. But this is testable, and I'm considering experiments on this subject for the upcoming round. For now, Live Rock is what I have found evidence for. Here is what good live rock vs bad live rock vs dry rock does in the first month of a tank's life. (Each symbol is the average of duplicate tanks. Dashed lines indicate 10th and 50th percentiles from my initial survey of 20 PNWMAS tanks). My home tanks also came up kind of low on diversity. One of them I'm running a nutrient experiment on, so I'm not gonna do anything else to it. But I'd be interested in adding some new live rock to the others. Maybe a group buy? I havent tested enough live rock to say this with confidence, but my gut feeling is that the good live rock in my study (above) was good because it was real coral rubble. The bad live rock was Walt Smith purple-dyed (i.e. concrete), that was covered in some of the most beautiful live rock life I've ever seen (which all promptly died). I still get grumpy when I think about how dense that stuff was when I broke it open. Fake live rock that looked so real. Lets find some nice, imported coral rubble that has stayed wet since it was in the ocean. Thats what I've found to rapidly promote a healthy microbiome. Maybe one of our PNWMAS retailers could set up a group buy for some nice live rock? The good stuff above was from Quality Marine. I could look into setting up an order through them if we don't have other options.

Hi everyone, I am pushing results out today, you'll be notified by email. Since the new (and future) samples include both water and biofilm, I'll caution you that comparing them with results from the previous "water only" samples may lead to some head scratching. I'm happy to discuss details but on a practical level, I think its something of a moot point because I can't imagine dropping the biofilm samples in the future. If you don't get a notification email by end of day it might mean your sample is not logged on the website. So if anyone sent me a sample but never logged it online, now would be a good time to do it! I'll post soon with some discussion of this round.

Finally fixed my ad GIFs so they appear to work on mobile too.

In terms of my tests, as long as you are dosing one of the three carbon sources I mentioned, it will be useful. In terms of whether your tank needs or would benefit from carbon dosing I don't have any special insights. I will say that I don't regularly carbon dose, since I don't understand how people achieve such lovely high nitrate levels in the first place. I'm really excited when I see a nitrate reading above 2 ppm, I struggle to get detectable nitrates more than I struggle to keep them low. IMO the presence of algae is proof the tank has enough N and P, but we want N and P for the corals anyway. I know its a contentious subject, but IMO nuisance algae arent caused by too much nutrients if your measurable NO3 is low, they're caused by not enough grazers. For my experiments (one of which I'm running on my main display) I will be dosing NO3 along with the carbon in order to avoid bottoming out nitrates. If PO4 drops too low I will add that too. My goal is not nutrient reduction, its promoting the right microbes.

Hi all, As I'm running promotions on other platforms I cant forget PNWMAS! Out of thanks for your help with building the initial DB, I'm putting a standing 20% discount on all testing services for PNWMAS members. This stacks with any other promotions or coupons. To get your discount, send an email to elimeyer@aquabiomics before placing your order to receive your coupon code. -Eli

I've noticed the same, I am a huge fan of glass lids for this reason. Keep the heat and water inside the tank! People always bring up gas exchange but in a tank with a sump and a skimmer I am sure there is more than enough gas exchange.

I should add that I plan to dose nitrates along with the carbon. Its is a good point -- carbon on its own will reduce nitrates to very low levels. As for nuisance algae, IMO the right grazers are a big part of the picture. Twice I've seen algae erupt in an otherwise clean tank, right after a tang died...

Cool, which carbon source do you want to test? I should say up front I do not currently carbon dose - I struggle to keep any nitrates in my systems and sometimes have to dose nitrates. So I'm not proposing these experiments as an advocate of carbon dosing. But since it is a tool many use to control nitrates, that relies specifically on bacterial activity, I need to test what it does to the microbiome.

Hi everyone, In my next batch, I will have room for a few additional samples and I'm once again turning to PNWMAS for this. But I'm afraid since I have actual paid clients now, I don't have room for just any samples, this time I'm being picky. I will be running carbon dosing experiments in my lab, and would like to compare these with similar experiments in established reef tanks. If you don't want to run experiments on your own reef tank I totally get it, no pressure. But in fact, many of us are running experiments like this all the time as we try new products or discontinue their use. If you were already planning on this, maybe you'd be interested in participating. Here is what it takes to participate. You must Have an established reef tank (≥6 months) Have not been carbon dosing for at least the past month Be willing to start daily carbon dosing for at least two weeks Be willing to sample your tank before dosing and again after 2 weeks Be willing to measure nitrate levels every 2-3 days throughout the experiment and share your data with me I'm testing three different carbon sources: vinegar, ethanol, and a carbon supplement I've developed. My homebrewed carbon supplement is a mixture of complex carbohydrates designed specifically to simulate the composition of DOC in natural reef waters. Its made of natural, food-grade ingredients derived from algae and crustaceans. (I'd be happy to discuss more details in person or on the phone if anyone is curious). I am conducting safety tests beginning this week so I'll have data demonstrating its safety before I ask you to try it. I will be looking for 1 or at most 2 people to test each of these (vinegar, vodka, or my homebrew DOC). If no one wants to, that is OK, but I figured its worth a shot. Anyone want to run an experiment with me?

Thanks to everyone who contributed samples in this round. I finished the batch yesterday and am sending them for sequencing today. Its possible we'll have results by end of week, but in any case not much longer. I've sent them to a different facility this time that promises quick turnaround instead of the 5+ weeks we had last time! If you didn't get your sample back to me in time, no worries. Send it at your convenience and if I have room in the next batch I will fit it in there.

Thanks for posting. It was nice of Ed to do this, hopefully it answers questions someone had!

I've started a thread at R2R with some additional thoughts on this, motivated by the reading I've been doing on this bug. https://www.reef2reef.com/threads/a-recently-discovered-parasitic-bacterium-causes-disease-and-impaired-growth-in-corals.660413/ Here are the key things I want to highlight: It occurs in about 1/3 of wild corals sampled worldwide. It occurs in many genera that we keep as hobbyists, including Acropora, Montipora, Seriatopora, Stylophora, and Goniopora. It also occurs in anemones (including Aiptasia, as if you needed another reason to hate Aiptasia!) and Palythoa It causes White Band Disease in Acropora cervicornis. The pathogen can be transmitted by coral-eating snails, which also serve as a reservoir for the bacterium. The pathogen can also be transmitted through the water to injured corals. As someone who frags corals, this caught my attention. (Most interestingly of all) the parasite normally makes up a small part of infected corals' microbiomes, but nitrate enrichment leads to a bloom of A. rohweri and impaired coral growth in infected corals. Researchers hypothesize it directly steals ATP and amino acids from the host cells, sapping the host's energy. -Eli

The presence is sediment samples may very well reflect its presence in micro invertebrates rather than a truly free living stage. The study is open access, available at https://www.nature.com/articles/s41396-019-0482-0.pdf As for species distributions, I'm embarrassed to say I scoured the supplementary info before I realized its shown right in Figure 7. Here it is - the corals and non corals where it is found, arranged in hobbyist terms. This is not all of them, just some of those commonly in the hobby. SPS - Acropora, Montipora, Pocillopora, Seriatopora, Stylophora, Porites LPS - Pavona, Turbinaria, Galaxea, Acathastrea, Cyphastrea, Favites, Platygyra, Leptastrea Soft corals - Rhodactis, Sinularia, Sarcophyton, Palythoa Anemones - Heteractis, Aipasia Its friggin everywhere. ... honestly, based on the ridiculously huge divergence times between these groups, I have to conclude this is as widely distributed in Cnidarians as Wolbachia is in Ecdysozoans, and potentially as important for understanding the biology of the group. This was a good time to get into aquarium microbiology, I think.

They proposed a new species (rhoweri), genus (Aquarickettsia) and family (Midichloriaceae) for this bug. Within the order Rickettsiales, as you inferred. (Side note, see what they did there? Midichlorians.) I agree, Chloramphenicol and Doxycycline are on the list to try. They work for some other diseases in the rickettsiales. I read some suggestions that rifampicin and tetracyclin may be less harsh but all 4 are on the list. Most of these endosymbiotic bacteria are difficult or impossible to culture out of their hosts, but this bug is common in sediment too.Perhaps it is culturable. My hope is to get some infected coral, and infect an experimental tank or two with it. I have one wholesaler account, if you have some wholesaler contacts lets talk! Maybe we could split an order? I think wild imported corals are our best shot at getting our hands on this bug. since we have no data on its prevalence in tank corals but a big dataset showing its in 1/3 of wild corals... I have noticed odd behavior with wild zoas too (i.e. they sometimes have to adjust to captivity before they start growing explosively like tank raised zoas). I'm gonna check some of these too. If it grows in zoas it'd be trivial to culture, and since zoas are essentially immortal I'd rather grow it in them than acros. The study found it in multiple non-coral Cnidarians but I havent looked into which ones yet.

Good question. Its found in lots of corals (51 different genera so far) but the best evidence is in Acropora. So SPS would be the most interesting. But really, anything that is also available in an aquacultured form would be useful. I have some wild vs cultured zoas I'm going to test and wouldnt mind adding to the sample.

I want to follow up again in terms of possible treatments. This parasite is part of a newly proposed species, genus, and family... so we have to rely on information from close relatives. Fortunately the evolutionary relationships are pretty clear -- this is a close relative of the family Anaplasmataceae, which includes lots of other intracellular symbionts, parasites and pathogens. Because these cause a variety of diseases in animals and humans, there is some information on their sensitivity to antibiotics. I don't see anything terribly specific (i.e. these treatments will also kill some other bacteria). This makes them more suitable as dips rather than whole tank treatments. But several show potential, including some antibiotics available without a prescription if one is creative with sources (rifampicin and tetracyclin). If these can clear Wolbachia from an insect or nematode they should be able to clear Aquarickettsia from corals... easy enough to test once I find an infected coral...

I've read parts of the R2R threads discussing this product but it would be interesting to get to talk to him in person. My two big questions: Has he published his theory anywhere? And what is in his product? I can find lots and lots of papers linking bacteria in the genus Vibrio with tissue lysis diseases in corals. They don't call it RTN, they have a variety of names like white syndrome. Working with advice from some microbiologists at OSU, a student of mine was able to repeat published methods for causing RTN using a cultured strain Vibrio. Having seen these effects in person, I can say -- thats RTN, in terms of the symptoms. I find it entirely plausible that there are multiple causes of tissue lysis in corals... maybe not all RTN has the same cause. But in terms of published studies, it seems to me the weight of the evidence points to one or more bacterial pathogens. But I remain open to data showing otherwise!

This is an odd one! Is anyone importing corals? I would like to take a quick, non-invasive swab of them on arrival if so (pre-dip), to test their microbiomes. I am chasing a newly discovered parasite, which is present in 1/3 of corals worldwide. I am testing the hypothesis that this contributes to the observation that wild corals are not as hardy in captivity as aquacultured corals. We've detected it in some tanks in captivity. Now I want to know, is it more abundant in wild corals than tank-raised? (see this discussion for more details) If anyone is importing wild colonies these days, please get in touch... Thanks!

Yeah, I've read a couple times and there is a lot more I havent digested yet, and I havent even touched the supplemental info yet. A few details I didnt see highlighted previously: The parasite A. rhoweri is found in 1/3 of corals sampled worldwide, in all oceans. It is also highly abundant in sand and reef sediments. It also occurs in other invertebrates. In corals, it is a true intracellular symbiont like the insects' Wolbachia. Its abundance is conclusively linked to nutrient enrichment (NH4, NO3, PO4). I didn't see any description of aquarium populations in their paper, but may have missed it. I think we may be the first to know that this is a thing for corals in reef tanks. I say again, I want to compare wild corals with aquacultured corals now! What if this contributes to the challenges of keeping freshly imported wild colonies? What if its because they, like so many wild corals, have this parasite and when placed in the high nutrient environment of a reef tank, the parasite kicks in?