Dinoflagelates. A disruptive treatment

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chema

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This article misses some important information. It states that the treatment was assayed in 11 aquaria. However there was no identification of the species or genus of dinoflagellates present in each tank. Therefore it is imposible to conclude that the treatment is valid for any dinoflagellate infestation.

Personally, I'm very skeptical about carbon dosing as a cure for dinoflagellate infestation. I have been dealing with amphidinium mixed with red ciano in my sand bed for almost two years. The infestation began while carbon dosing (vodka-acetic acid mixture). I kept the carbon dosing schedule for this entire period (between 0,04 and 0,07 ml/L) with no visible recession.

Now, I'm trying a mixed carbon dosing treatment (Tropic Marin Elimi-NP and the vodka-acetic acid mixture). It has not been planned for dinos eradication, but for other purposes. It has been one month now and the infestation does not recede.

The only thing that looked like some success was the addition of silicate. It din't affect much ciano, but the brown slime on the sand was a mixture of amphidinium and diatoms (three different species).

Also, I do not buy the common ground idea that low nutrients become the trigger for dinos. At least not always and definitely not in my case. My tank has always been around 5-15 ppm nitrate and 0,1-0,15 ppm phosphate. However, dinos came to stay.
 

Stigigemla

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The reason why it is so hard to defeat cyano is probably that there is so many species. That makes it unbelievable for me that one method should help in all cases.
In my case it didn't work. What it did was to slow down the growth so I had a chance to manually compete with it.
I think it is important to have a very small dose and dose at night.
If You use a dose high enough to reduce N and P there will always be some left in the day to feed the cyano.
 

Marshall53

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Could you keep us posted if the developments in your tank? Curious to see how it will develop specially on larger tanks were a larger initial dose is required. Have you got increased aeration to your system?
Yep. It looks better today. I have a big skimmer and good water agitation at top.
 

Dan_P

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THE PROBLEM

As is well known, dinoflagellate pests in the reef aquarium are perhaps the worst that we will face during the life of our system. These pests are quite difficult to eradicate because dinoflagellates have a great capacity for reproduction, propagation and survival. In addition, measures that are effective for one species are not equally valid for others. To complicate matters, aquarium history and environmental variables, e.g., existing nitrate and phosphate concentrations or, more specifically, the variation of these, play an extraordinarily relevant role.

Table 1 shows a list of measures, which alone or in combination, are commonly used by aquarists facing this problem. The table indicate the effectiveness, to the best of my knowledge and experience, of each of them. As we can see, none of the measures have a high degree of effectiveness in all cases, being necessary several of them, and a lot of patience, to reach a successful conclusion.

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Table 1. Common measures to fight dinos.

Fortunately, as we will see throughout this article, we have been able to verify that the gradual addition of an external source of organic carbon has a great effectiveness in solving the problem, most likely for all species of dinoflagellates. This treatment has been tested and verified during an experiment conducted in 11 reef aquariums over a period of 7 weeks, in the city of Madrid. In all cases the result has been the eradication of the pest. Let's see the details.

DINOFLAGELLATES. ¿WHAT ARE?

Dinoflagellates are small unicellular organisms belonging to the group of protists, which cannot be categorized as neither animals nor plants, since they have characteristics of both. They move by means of a rotary motion, using their flagella to propel themselves. For example, the well-known zooxanthellae that house corals, anemones and giant clams, are dinoflagellate protists, although they have given up their extensive mobility to have a safe environment within the tissues their host, sheltered from predators

Unwanted dinoflagellates in the aquarium are often are often classified as "algae" because they are photosynthetic protists, although many are mixotrophic organisms, capable of feeding also on simple organic matter. This dual feeding strategy contributes to their great capacity for survival. Many of them release very potent toxins into the water, e.g., Ostreopsis, Gambierdiscus and Prorocentrum species, which generate compounds similar to the palytoxins produced by Zoanthids. As an example, I will describe the life cycle of Osteropsis to understand what we are dealing with.

The cells of Osteropsis ovata have a diameter at their largest part, about 40 micro meters, which, if we compare it to the diameter of a cyanobacteria cell (2-3 micro meters), gives us an idea of its relative size. Ostreopsis, like many other harmful dinoflagellates, secretes a large amount of mucus to build extracellular matrices, macroscopically characterized by the presence of strands and filaments, in which oxygen bubbles resulting from photosynthesis are trapped. These matrices settle on any existing substrate, sand, rocks, glasses, etc., suffocating the existing micro fauna.This can be observed, for example, when the aggregates formed on the substratum are siphoned off, revealing areas of sand with a dark gray color, indicating anaerobic bacterial activity, resulting from the depletion of oxygen produced by the layer of dinoflagellates settled on top. The extracellular matrices also serve to immobilize potential predators such as amphipods and copepods.

Ostreopsis and other noxious dinoflagellates have a life cycle characterized by the presence of cysts or resistance forms, which are formed as a result of the union of gametes, although also derived from asexual reproduction. When environmental conditions are not appropriate, for example, a shortage of nutrients or light, these cysts anchor to the substrate and remain "dormant" for months, until the situation improves.

This implies that when we use live rock in the aquarium, we are introducing these cysts as part of the resident micro fauna. It is important to note that the use of inert rock does not prevent the propagation of dinoflagellates, which are unintentionally introduced with the coral frags. In fact, aquaria started with inert rock are noted for a higher incidence and duration of this type of pests, because the lack of microorganism competition. I have been able to verify this point through microscope specimens and interviews with hobbyists. As an example, Figure 1 shows the progression of a dinoflagellate pests in an aquarium set up with 50% inert rock (left) and 50% live rock (right). The difference is remarkable.

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Figure 1. Progression of a dinoflagellate pest depending of life rock (right) or inert rock (left)

In the ocean, dinoflagellates produce toxic tides coinciding with periods of rising temperatures and dissolved inorganic nutrients. These blooms, are initiated when the cysts are activated and begin to divide, each cell generating two new individuals and so on. This reproduction generates an exponential pattern that subsequently stabilizes into a flat pattern. After several weeks the population density decreases until the bloom subsides. In the aquarium, the pest behaves in the same way, and can last for months.

DINOFLAGELLATES IN THE REEF AQUARIUM

Dinoflagellates are present in almost all aquaria, forming part of the food web and, therefore, contributing to the general mechanism of nutrient recycling, as they consume light, CO2, nitrate, phosphate, trace elements and dissolved organic matter. If the resident population is limited to a few small colonies and cysts, it does not pose much of a problem. However, it is absolutely necessary to avoid conditions that may favor a bloom, since, once started, it is quite difficult to control and eradicate.

The most favorable scenario is related to young aquaria with very low nitrate and phosphate concentrations, for example, below 0.02 phosphate and 2 ppm nitrate. There is also some probability that a bloom will be triggered when there is a fast reduction in phosphate concentration, which can happen, for example, after the use of lanthanum chloride or GFO. In all aquaria it is good practice to maintain a balanced equilibrium between the two nutrients.

So why, when there is too little nitrate and phosphate in the water, or there is a sudden reduction, is there a greater likelihood of triggering the pest? The answer is neither simple nor immediate, but there are some evidences that help us understand the problem. The key is not in the dinoflagellate itself, since a reduction in the concentrations of nutrients, both organic and inorganic, is not at all favorable to them. What happens is that other competing species, which, among other things, use nitrate and phosphate for food, get starved, and dinoflagellates take advantage of them.

BALANCE, MATURITY AND BIOLOGICAL NICHE

All microorganisms living in a reef aquarium, whether they are bacteria, protists (dinoflagellates, ciliates, coccolithophores, foraminifera, radiolarians), phytoplankton (diatoms, cyanobacteria) or zooplankton (amphipods, copepods) are in continuous competition for light, space or nutrients. They all exchange nutrients with the water. Each organism has a place where it spends most of its day, where it inhabits and performs its main primary functions: feeding, grow and reproduce. Every square cm of a marine aquarium is occupied by an infinite number of these tiny organisms, living in a constant equilibrium. This is what called a biological niche.

When the aquarium reaches a significant maturity (as a reference around 1 year after set-up), the stability of its environmental factors (light, water chemistry, nutrient flow, etc.), favors biodiversity in the populations of all microscopic species, since the greater the stability, the better the conditions for diversity. In this scenario it is more unlikely that one species to dominate over the rest and generate a pest, as most of them have everything they need.

However, when a change occurs, the desired stability is disturbed. The organisms that used to occupy a niche may disappear, die, or simply migrate to find a better place. It is then that the "gap" they leave behind is quickly colonized by other opportunistic and fast-moving organisms. This is the case of unwanted dinoflagellates.

COMPETITION BETWEEN BACTERIA AND DINOFLAGELLATES. THE SURPRISING KEY TO THE SOLUTION.

As we can see, it is practically impossible to eliminate the existence of the different unwanted dinoflagellates in a reef aquarium, since they are introduced with the live rock, invertebrates or frags. The optimal situation is that their populations are limited through natural competition from other organisms such as phytoplankton, zooplankton, or bacteria. As is well known, one of the strategies used to eradicate a dinoflagellate pest caused by maintaining low nitrate and phosphate concentrations is precisely to increase those concentrations. However, the dinoflagellate uses nitrate and phosphate for feeding, so increasing these concentrations will favor it as well.

The rationale for this measure is that the new availability of nutrients will also favor competing organisms, being able to displace dinoflagellates. This strategy is colloquially referred to as "dirty warfare" and its rate of effectiveness is quite low, often favoring the expansion of other unwanted algae such as cyanobacteria or hair algae. However, this mechanism of feeding competing organisms works remarkably well with heterotrophic bacteria. These bacteria aggregate in biofilms within the aquarium substrate, on rocks and other available surfaces, feeding on organic carbon, nitrate, phosphate and trace elements.

It is important to note that all reef aquaria are usually limited in the amount of dissolved organic carbon available to bacteria (DOC), so such bacteria do not grow well until we provide an external carbon source. When adding organic carbon artificially, we get the heterotrophic bacterial communities to displace dinoflagellates.

As simple as this, the reader may ask? The answer is YES.

And why aren't hobbyists using this technique as a pest remediation? The answer is, as in many other cases, fear. The fear comes from the recommendation that all hobbyists follow not to let nitrate and phosphate concentrations fall to undetectable levels, precisely to avoid the risk of cyanobacteria and dinoflagellates blooms. However, in this essay we have verified that, when such low concentrations are the result of the gradual addition of organic carbon, there is no risk of dinoflagellate blooms, but rather, on the contrary, there is a significant regression of the of the existing population, until it disappears completely. As the reader can see, this treatment goes against all recommendations, but it works remarkably. Anyway, it is important to highlight that the risk of cyanobacteria bloom is still there, so we shall not let the inorganic nutrient concentration be reduced too much.

THE TREATMENT

Two brands of organic carbon additives were used in the study Xepta NP out and Red Sea NO3 : PO4-X. After some weeks we got to the conclusion of Xepta being faster in producing the dinoflagellates regression. Xepta NP out is composed of methanol, toluene, glucose, and acetic acid, while Red Sea NO3 : PO4-X contains methanol, ethanol and acetic acid. In the 11 aquaria where this treatment has been applied, the dinoflagellates have receded significantly with either Xepta or Red Sea.

The initial dosage was 1 ml of organic carbon per 100 liters of aquarium water, increasing to an additional 1ml/100 liter per week in cases where dinoflagellates are slow to recede. Up to 3 ml/100 liters have been used in some aquariums. It is important to perform the dosage when the aquarium lights have been turned off, in this way we avoid that the dinoflagellates from taking advantage of the organic carbon. Dose shall not exceed 3 ml/100 l of aquarium water, as we have not tested beyond.

Any sudden excess of organic carbon in aquarium water alters the equilibrium of the coral holobiont, as the heterotrophic bacteria that live within the coral tissues, receive an abnormal dose of food, which can be detrimental to the coral. It is very important that the dosage be as gradually as possible and, at any negative sign in fish, corals and invertebrates, reduce the dosage to half, maintain the treatment, and then increase it again some days after, once the aquarium has self-regulated.

Some side effects detected during the study are depicted below:

- Some corals and tridacnas that have closed more than usual.

- Cloudy water due to bacterial bloom and excessive growth of biofilms inside the return pump pipes.

- Increased export rate from the skimmer. Mostly bacterio plankton and remains of dead organisms.

For cases where there is a large number of dinoflagellates, prior to start the treatment, it is convenient to siphon them out without water change. To do this, a sock is used to retain the dinoflagellates while the siphoned water is returned to the sump. As a consequence of the addition of organic carbon, nitrate and phosphate concentrations will drop significantly, even to the point of being undetectable. We do not want this to happened, because organic carbon addition favors the growth of unwanted cyanobacteria.

ACTION MECHANIMS

The biological mechanism underlying is the combined effect of two factors that cause the dinoflagellate populations to be reduced.

1- Bacteria "steal" food from the dinoflagellates. Due to the addition of organic carbon, the heterotrophic bacteria that are housed in the sand, the rocks and other surfaces of the aquarium, start to reproduce in an extraordinary way, removing nitrate, phosphate and trace elements from the water. They form biofilms that spread on all available surfaces, displacing dinoflagellates.

2- Bacteria directly inhibit the growth of dinoflagellate cells through allelopathy (chemical warfare). The survival strategy of bacteria, protists and phytoplankton, includes a line of defense, which consists of releasing chemical compounds toxic to other species. For example, as a result of copepod predation on diatoms, the algae produce polyunsaturated aldehydes (PUAS), which are noxious to copepods and some phytoplankton species, such as urchin larvae. These PUAS are also produced by other phytoplankton species, such as cyanobacteria, cryptophytes, primnesiophytes, and synurophytes. In addition, PUAS have a potent inhibitory effect on the growth of the dinoflagellate Ostreopsis ovata, altering its DNA structure and disrupting its development.

Figures 2 to show the evolution of 2 of the of the 11 aquaria where the essay was carried out. The addition of organic carbon, allows the heterotrophic bacterial communities to reproduce and generate their allelopathy compounds, which inhibit the growth of dinoflagellates. In the following scientific study: https://www.frontiersin.org/articles/10.3389/fmars.2015.00100/full, it is explained how the dinoflagellate Alexandrium tamarense is inhibited by allelopathy compounds generated by marine bacteria Brevibacterium, Thalassobius, Alteromonoas, Rhodobacteracea, Pseudoalteromonas, Vibrio and Halomonas. According to the article, there is a regression of the cells of Alexandrium tamarense after 10 min, 30 min and 24 h, because of the introduction of the bacteria. The aforementioned study has been kindly shared by Postdoctoral Researcher. Mrs. Esther Garcés, who is a scientific authority in the field of dinoflagellates.

Aquarium no. 1.

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Aquarium no. 2.

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We can see, therefore, that many organisms are capable of interfering with the growth of others through competition for space and nutrients, but also directly through allelopathy. In an aquarium, where the volume is so extraordinarily limited compared to nature, these chemical warfare compounds are of decisive importance. For example, soft corals emit steroids, diterpenes and sesquiterpenes. which are poisonous to many animals, including hard corals. Anemones also make use of allelopathy in the aquarium, which is proven when, after the introduction of new specimens, resident anemones that were thriving without major problems, begin to undergo swelling cycles, that sometimes lead to their death.

CONCLUSIONS

In the aquarium water, when there is a shortage of, whether nitrate, phosphate, trace elements or organic matter, many of the organisms in the food web, will not be able to complete their metabolic processes and, therefore, to emit their allelopathic compounds or even compete for light and space. This is the perfect scenario for dinoflagellates to spread, as they will not be inhibited by allelopathic compounds and have the capability of growing with ultra-low nutrient concentrations. Dinoflagellates begin to reproduce and spread over all available surfaces, using their flagella, with extraordinary mobility.

Using an external source of organic carbon, heterotrophic bacteria reproduce in an exponential rate, grouping together in biofilms that cover all available surfaces: sand, rocks, pumps, aquarium and sump walls, etc. These biofilms "suffocate" and "intoxicate" dinoflagellates, conveniently deactivating them.

One of the most important conclusions of the test carried out in the 11 aquariums is the following: maintaining very low or undetectable concentrations of nitrate and phosphate in a reef tank, does not pose a risk for the emergence of dinoflagellates, as long as it is a consequence of the addition of an external source of organic carbon. It is important to highlight that the risk of cyanobacteria is still there, as they are favored by organic carbon and not affected by the chemical warfare compounds released by the heterotrophic bacteria.

My recommendation is to always maintain detectable concentrations of both nitrate and phosphate, so that there is a small amount of "leftover" nutrients available for all the organisms, avoiding the growth limitation due to lack of nutrients. During the dosing of organic carbon, it is very important to go progressively, monitoring the status of fish and corals to stop or decrease the dosage at any negative sign. It is necessary to be attentive to fish breathing, water cloudiness and possible symptoms of STN in corals.
I have to thank you again for writing this article.

Allelopathy is a subject that I have ignored until you mentioned it as a possible mechanism. A Google search has provided much information about allelopathy that occurs in marine environments. Unfortunately, at the level that we observe our systems, competition for nutrients v allelopathy may not be distinguishable. But knowing that bacteria, dinoflagellates and cyanobacteria can use allelopathy will help me design algae studies to look for allelopathy.

Dan
 

chris_pull

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Anyone been trying this? I bought the NOPOX but I'm nervous to start...corals have been growing fine despite the dinos (LCA), but sand and rock is smothered in dino biofilm, and has been since about December. I'm thinking about getting my nutrients quite high to begin with so there's less chance of them bottoming out.
 

GuppyHJD

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Thanks for a really interesting article, it certainly sounds promising. After a good 8 months of trying to eradicate large cell Amphidinium through phyto and silicate dosing (neither of which are having much impact) you've definitely piqued my interest! I have some questions that I'd really like to know more about.

I'm curious about the statement that you should not let nutrients bottom out whilst carbon dosing. Did you actively prevent this during the weeks you were treating for dinos? Did you monitor daily and stop dosing if they got too low, or do you let them get low/to zero during the treatment, then increase them?

Like others have said, the solution could be that you're using bacteria to physically outcompete the dinos (i.e. pushing them out of their ecological niche with a bacterial biofilm) or starving them out by using bacteria to consume all available nutrients, and essentially resetting the microbiome for "good" microbes to colonise.

Once dinos have been eradicated, did you continue carbon dosing or stop and return to normal nutrient levels?

Thanks so much in advance!
Chris - interested in your progress. I have LCA now and have only started the battle.
 

GuppyHJD

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Anyone been trying this? I bought the NOPOX but I'm nervous to start...corals have been growing fine despite the dinos (LCA), but sand and rock is smothered in dino biofilm, and has been since about December. I'm thinking about getting my nutrients quite high to begin with so there's less chance of them bottoming out.
Chris, I was dosing NOPOX when I got LCA. I had my nitrates near 30 and my phosphates around 1.0. I wanted to drop both and started NOPOX. The phosphates hit .01 and nitrates 15.9 and LCA took off.
Now dosing silicates and NEOPHOS daily trying to get phosphates up - so far still getting a zero reading on Salifert Phosphate test.
 

Mike konesky

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Anyone been trying this? I bought the NOPOX but I'm nervous to start...corals have been growing fine despite the dinos (LCA), but sand and rock is smothered in dino biofilm, and has been since about December. I'm thinking about getting my nutrients quite high to begin with so there's less chance of them bottoming out.
Day 4 of half dose with NoPox (.5ml/100l). Will bump dose after a week. No visible changes. No changes in N (16) or P (.08). Silicate at 1-1.2ppm and still slowly raising to 2ppm. Also using 10ml mb7 nightly. Will update with progress.
 

GuppyHJD

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(Tank is 120g, tank started 18 months ago, using dead rock) I was using NOPOX to lower my phosphates from 1.2 to .05 (goal), but got .02. I think I learned HANNA ULR margin for error likely got me to 0.0.

When I mentioned I was using NOPOX an expert in the industry (over 30 yrs), advised me to switch to a different carbon source. His recommendation was something without methanol. I have been looking at vinegar, vodka or Brightwell Biofuel.
Before I start, I need to get the phosphates up. Currently I am dosing silicates ( I have LCA dinos). 20ml of MBacter7 daily. I am dosing 30ml of Brightwell NeoPhos and am still testing 0.00 (Salifert). First two weeks were 20ml.

Any recommendations on carbon source?
Other thoughts? ANY HELP is welcome.
 

jrmailo

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(Tank is 120g, tank started 18 months ago, using dead rock) I was using NOPOX to lower my phosphates from 1.2 to .05 (goal), but got .02. I think I learned HANNA ULR margin for error likely got me to 0.0.

When I mentioned I was using NOPOX an expert in the industry (over 30 yrs), advised me to switch to a different carbon source. His recommendation was something without methanol. I have been looking at vinegar, vodka or Brightwell Biofuel.
Before I start, I need to get the phosphates up. Currently I am dosing silicates ( I have LCA dinos). 20ml of MBacter7 daily. I am dosing 30ml of Brightwell NeoPhos and am still testing 0.00 (Salifert). First two weeks were 20ml.

Any recommendations on carbon source?
Other thoughts? ANY HELP is welcome.
I used to dose absolut vodka. Worked great. Switched to cheap generic vodka. Still working great. The brand doesn’t matter. Just do not dose any thing with flavor infused.

Other dose vinegar. Some dose vodka AND vinegar.
 

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For those fighting dino, hang in there. I know it’s frustrating. It might take time, but you will eventually beat them.

I recommend anyone dealing with dinos to stop dosing amino acids or other supplements.

Some products (some brand of bacteria in a bottles/sludge remover/live rocks enhancer/etc) that are advertised beat/control dino and/or increase biodiversity had actually made my outbreak significantly worse. So be mindful of what you are dosing and go slow until you see that it at least is not doing more harm than good.

I would also recommend you to be careful with excessive feeding. This was how I went from small patches of Dino (ostreopsis + prorocentrum) to full blown out break when I was trying to get my small regal angel to start eating.

As others had mentioned, UV is an effective mean to controlling ostreopsis. I was able to knock them back by 90% with this method alone. UV is significantly less effective when it came to benthic sp. of dinos but I could halt their growth by reducing my feeding. In both instances, they never truly went away until I started dosing organic C and really aggressively control my nutrients.

While my experience might be anecdotal, my tank went from where 80% of the surface were covered by dino to clean white sand and rocks with the methods mentioned above.
 

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chris_pull

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Chris - interested in your progress. I have LCA now and have only started the battle.
What progress? :grinning-face-with-sweat: To be honest, I don't think there's really a fix for LCA. I've been dosing silicates and a five-species mix of phyto since January and it's not made any difference. Things did seem to improve a bit when my nitrates were higher, around 20 ppm, but that could be anecdotal. Nitrate is currently about 10 and the tank is blanketed in them, so I'm going to walk it back up. I've also been dosing various bacteria and AF reef mud, but nothing is breaking through. If I could I think I would add a sand sifting goby to help break up the dino matts in the sand (but I don't have a lid, can't easily add one because of hang-on equipment), as stirring the sand is the only thing that at least visually looks better (and my hope is makes it easier for other microbes to colonise), but then the "experts" say not to disturb it. I'm very exasperated with the whole thing honestly, and the idea of temporarily removing the sandbed is starting to seem more and more like a good one! I'm also wondering if people who have beaten LCA have actually done so through the prescribed treatments or whether it went away on its own, as it's not uncommon for people to say it took 6-12 months to "treat" them. I really think LCA is the worst thing that can happen to a reef tank, as the other dinos can be knocked out fairly easily with UV. I'd like to see the major additive companies investing heavily into R&D to try and find a solution to them.
 

vtecintegra

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I'm very exasperated with the whole thing honestly, and the idea of temporarily removing the sandbed is starting to seem more and more like a good one!
LCA. I siphoned the sand out after months of them killing corals and wrecking the tank. They were especially bad in the sand. After three months they finally completely disappeared and I put sand back in.
 

vtecintegra

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It all started by bottoming nitrate and phosphate. Tried lights out-no effect. And dosed nitrate and phosphate for a few months with minimal effect. 75% were in the sand, so I finally siphoned it out. Was nasty with Dinos. In the mean time, I added a lot of coral which should have brought in some other microbes to compete. After three months, any patches of Dinos on the rock disappeared. Then I added sand back.
 
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