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Marine Velvet (Amyloodinium ocellatum)

Introduction
Marine velvet disease is caused by the Dinoflaggelate organism Amyloodinium ocellatum. Dinoflaggelates belong to the kingdom protista, which is an odd mix of organisms containing single celled organisms, such as Amoeba, and, of more interest to reef keepers, Cyryptocarrion irritans (the cause of marine white spot).

Amoeba and Cyryptocarrion fall into the groups within the protista that have animal characteristics. However, Amyloodinium ocellatum falls into a different group, which have both animal and plant characteristics. Indeed for many years Amyloodinium ocellatum was considered to be an alga. Being more plant than animal makes the treatment and management of this parasite particularly difficult for the marine aquarist and it often requires rather different treatment strategies to affect a cure.
Symptoms
Fish affected by this parasite often have a fine grey-gold dust-like covering of the parasite on the skin (Figure 1). The parasite usually starts to infect the fish near the base of the dorsal fin and spreads out across the body’s surface from this area. It is a classical symptom to see a saddle-like lesion develop on the skin. At its largest the parasite is only one tenth the size of Cyryptocarrion irritans (marine white spot) and this can make the disease hard to spot.

Figure 1 - Clownfish infected with Amyloodinium ocellatum (home2.pacific.net.ph/~sweetyummy42/disease.html)


Affected fish often present with rapid breathing because the gills often have very large numbers of the parasite present (Figure 2). They will also be flicking against rocks and have a hard-to-see dull grey-gold sheen across their skin. If the infection is allowed to progress the fish lose appetite and sometime gasp at the surface because of the gill damage. Towards the end stages of the infection the fish loose orientation and darken, often sitting listlessly on the bottom of the aquarium breathing rapidly. Death occurs by a combination of asphyxiation due to gill damage, physiological failure due to loss of tissue fluids and osmoregulatory imbalance.

Figure 2 - Electron micrograph of a gill infested with Amyloodinium ocellatum (round spheres) showing the high level of infection that can occur in gill tissue
Lifecycle
This parasite has a life cycle that has four stages (Figure 3).

When the parasite is on the fish it is called the trophont. The trophont sits on the skin’s surface and puts out small rhyzoids (root-like structures) into the skin of the host where it absorbs nutrients directly from the fishes’ skin. The trophont is quite small, being around 10 to 15µm in diameter when it settles on the skin, but growing to around 80µm as it matures. Depending on the temperature and strain of the parasite this stage can be completed in as little as 12 hours or as long as 4 days.

Once the parasite has built up its energy reserve sufficiently it drops off the host to form the tomont. The tomont lands on the sediment and begins to divide. After 48 to 96 hours (although it can take up to 20+ days) the tomont contains around 200 dinospores, increasing in size to around 1 mm. It then ruptures and releases the flagellated dinospores, which move upwards, attracted by light, and are ready to infect a fish. Dinospores can remain infective for up to 15 days, much longer than marine white spot, the disease it is most often confused with.

Although the life cycle is completed in 4 days on average, one problem with Amyloodinium ocellatum is that it can persist in the tank for a long period because both tomonts and dinospores can use photosynthesis as a means of obtaining nutrients. Thus the parasite is able to survive for long periods in tanks without a suitable host - we will discuss the implications of this later.

Figure 3 - Lifecycle of Amyloodinium ocellatum
Prevention
Amyloodinium ocellatum is a much more difficult disease to manage than marine white spot; however the advice in the previous article remains true. It is better to keep this parasite out of your display tank rather than to treat the occupants once infection has occurred: to this end we make no apology for repeating much of our earlier advice.

It is said prevention is better than a cure and this is very true of this parasite. By far the most effective way to keep this parasite from infecting your fish is to avoid introducing it into your display system in the first place. This is particularly true when you consider that the parasite is an obligatory pathogen (it needs a host to survive). So no susceptible animals in a system or successful treatments mean that the parasite can be successful eradicated from a display or quarantine system. This is unlike freshwater white spot where the cyst can survive for a long period off-fish. So the question is how do we avoid introducing the parasite in the first place? Well the following list may seem obvious but should always be followed:
  1. Buy from an LFS with a good reputation that you trust
  2. Quarantine your fish AND invertebrates
  3. Practice good bio-security between tanks, live feeds, visits to the LFS or helping with a friend’s system
  4. Use of dietary immunostimulants
  5. Ozone and UV
Let us consider each one in more detail:

1. Buy from an LFS with a good reputation that you trust
That statement may seem obvious but many a wipe-out has occurred from someone buying a “sympathy” fish, which is doing badly at a poor LFS, and adding it to their valuable display system without quarantine. As a rule of thumb you should get to know your LFS; ask about their health management regime (if they are OATA members they have access to OATA’s excellent document on bio-security and fish heath, which they would be wise to follow). They should be happy to tell you if they quarantine fish before sale, how long they do this for and if they use copper as a routine treatment in their fish systems (which is an excellent way of reducing the risk of this parasite).
The adage should be “if in doubt over a fish’s health leave it where it is”. Only if you are sure that there is little risk to it and your fish from your quarantine system should you buy it. But in reality, with a good LFS it has a good chance of recovery; with a bad LFS vote with your feet and hope they close down due a lack of trade from discerning reef keepers.

2. Quarantine your fish AND invertebrates
This is the most common way that parasites get into a system. Fish with a low level of infection or water from an invertebrate system that has infected fish in it are the major sources of bringing this parasite into a reef tank. Quarantine was designed for ships and refers to a period of 40 days and nights (very biblical). Usually this is sufficient for most mammalian diseases to appear and action to be taken. Fish being cold blooded 40 days may not be long enough.

Taking the maximum time reported for each stage, then the maximum time to complete the life cycle would be around 41 days. Although there are no published confidence intervals for marine velvet this is much longer than marine white spot where a maximum value of 71 days has been proposed to ensure that a fish is parasite free. To calculate a 95% confidence interval with a 50% safety margin, given a 41 day maximum life cycle, then a maximum quarantine period to provide a parasite free fish by isolation alone is around 117 days. So there is a figure for those of you who want a definitive answer to the “how long do I quarantine my fish for to make sure they are free of Amyloodinium ocellatum?” question. However, a confidence interval of 84% is achieved at 6 weeks and for many reef keepers 6 weeks plus a prophylactic treatment of copper is considered a gold standard.

Why quarantine my invertebrates? Well you can introduce trophonts, theronts or tomonts either with the water or on living rock or a coral’s skeleton. So a reef tank really needs two quarantine tanks - one for fish, where copper can be used, and one for invertebrates. In the invertebrate tank time is really the only guarantee of eradicating the parasite so we would recommend a quarantine period of 8-10 weeks or the use of a sentinel fish in the invert quarantine tank, which can then be treated in the fish quarantine tank should the need arise. Quarantining inverts also prevents the introduction of coral eating nudibranchs or Zooanthid eating pycnogonids which can be a huge advantage to a good reef tank where the invertebrates are as spectacular as the fish.

What should be in my quarantine tank?
Ideally the tank should be as large as possible have a simple filter (sponge filters are particularly good in this role) have minimal décor (bare bottom, no living rock etc) instead hiding places can be provided by plastic piping. For a fish system lighting should be subdued and it should have its own set of equipment (nets etc) which never go near the main tank. Most importantly you must know the volume of water very accurately to allow for the correct dose of medications to be administered.

3. Practice good bio-security between tanks, live feeds and visits to the LFS or helping with a friend’s system
Bio-security is the terminology that describes methods for preventing the spread of an organism from one system to another. Everyone, hopefully, washes their hands after going to the loo. Well reef keepers should do that before going into their tank. However, there it is a lot more to it than that; each tank should have its own nets, pipe-work, syphons etc. and they shouldn’t be swapped between tanks without disinfection. One of the best disinfectants I have used for fish tank equipment is Milton’s fluid and to use it just follow the instructions and soak all your equipment in it after use and wash thoroughly before drying. So our advice would be always wash and disinfect hands and equipment before using it on your tank. This is true if you are an angler, have loaned kit to a friend or have simply been helping them out and tinkering with their tank.

4. Use of dietary immunostimulants
One of the biggest recent advances in marine reef keeping is the use development of superb dry diets and the use of immunomodulating dry diets and sprays for enriching frozen foods. Unlike garlic and other crude terrestrial plant matter, where there is no evidence in the scientific literature that there is a benefit in fish, there is a huge amount of scientific evidence out there that immunostimulants help reduce the infection rate with fish pathogens (not just white spot). It is excellent to see that some manufacturers have decided to run with this and produce a range of excellent products aimed at the reef keeper. Immunostimulants work by up regulating the non-specific defence mechanisms of an animal thus preventing a pathogen establishing an infection or allowing an already infected animal to be in a position to rid itself of the pathogen more rapidly. However they can’t be fed all the time otherwise the animals become tolerant to their effect and most commercial regimes recommend two weeks on one type of immunostimulant followed by two weeks on a second immunostimulant then 4 weeks on the standard diet before returning to the immunostimulant diet.

5. Ozone and UV
Both ozone and UV can be very useful tools in the fight against this parasite. UV kills the parasite by damaging its genetic material while Ozone disrupts cell membranes. Both methods require pumping of water out of the aquarium through either a UV lamp system or into a protein skimmer that has ozone injected into it. Used correctly and at sufficiently high enough dose both ozone and UV are very effective in removing trophonts and theronts from the water column and hence reducing the infective pressure during an infection or preventing an infection establishing itself.
Treatments
Once Marine velvet has broken out in a system all the susceptible fish will have the parasite and, even if they may have no signs, all the fish in that system will need treating. Otherwise, on re-introduction of the most susceptible species, the parasite will breakout again from the asymptomatic carrier fish that have remained in the main tank.

Treatments for marine velvet generally fall into three categories: chemical, environmental and experimental.

Probably because this disease is often misdiagnosed as something else, such as Brooklynella hostilis or marine white spot, it hasn’t attracted a lot of grey literature cures unlike marine white spot. Although Vitamin C (ascorbic acid) therapy has been suggested as a “reef safe” cure, it is a bit of a long shot to say the least.

However, the following words of warning should be heeded before chemical treatment of a reef tank is attempted. (Source: Simon Garratt, posted on UltimateReef)
“Modern reef keeping methods have now moved way beyond the days when LR was considered a simple source of bacteria, and filtration. In modern systems the 'critter' population regularly feature as a major player in the functioning of the tank. As such, any treatment should consider the impact it will have on the tanks ecosystem. 'Reef safe' chemicals have often only been tested with the most commonly kept 'show' inverts. Many have not been tested nor claim to be safe with the multitude of background life that 'modern' reef keeping deems as beneficial, and form a substantial part of a modern reef eco-system, especially those containing sand beds etc. The use of 'chemicals' designed to kill parasites can have disastrous consequences in more diverse systems as they will kill off these beneficial animals as well as the parasite. As a rule of thumb Keep chemicals out of the reeftank and carry out any treatments in a specialised quarantine tank. Whilst it’s perfectly acceptable and correct to deem the survival of the fish as a priority, it shouldn’t be at the detriment of the rest of the system, and certainly not to the degree it jeopardises the systems stability.”

Chemical treatments

a) Copper
Copper based treatments are easy to use and readily available from LFS in either ionic or chelated forms: chelated forms have a longer ˝ life in the aquarium, otherwise there is no difference between them because the active ingredient, copper ions, is the same. The two important things to remember about copper are that it will kill all invertebrates, and so cannot be used in a reef tank, and that you must get the dose right. If the dose is too low then the treatment will be ineffective, too high and you will poison your fish. I recommend that all copper treatments are carried out in a quarantine tank away from your invertebrates and that the levels of copper are monitored daily with a copper test kit to make sure the dose of copper falls into the therapeutic range of 0.2 to 0.3ppm for 7 to 10 days. Once a tank has been treated with copper it can be problematic reintroducing invertebrates because copper forms complexes with carbonates in the aquarium and these can leach back into the water with time causing copper toxicity to the invertebrates.

For this parasite, this isn’t the drug of choice. Being mostly plant it has much better copper detoxification methods than other protista that have more animal characteristics, such as Cryptocarion. So to cure this parasite you need to use longer exposure to copper in the range of 0.2 to 0.3ppm (10 to14 days) or higher copper doses. Data from the research community indicates that the median dose required to cure this parasite is 0.37ppm, however, this is getting close to the upper limit of copper toxicity for some of the more delicate fish species (circa 0.4ppm). Indeed, there is one report of a strain of Amyloodinium ocellatum requiring a dose of 1.2ppm to affect a cure.

b) Chloroquine diphosphate
Chloroquine diphosphate was reported by Noga & Levy (1995) as an effective and safe treatment for Amyloodinium ocellatum. In this study a dose between 5 to10 mg/l cured the infection in ten days. However, this drug isn’t available in the UK from an LFS and has to be purchased (probably ordered) from a pharmacy. Although safe for fish it is very toxic to macro and micro algae and some invertebrates (in particular corals and anemones) so treatment should be done in a quarantine tank.

c) Formalin
Cheap, cheerful and effective: sounds perfect, so what’s the catch? Well it’s toxic, carcinogenic and an irritant. It is, however, a uselful treatment for Amyloodinium ocellatum. It can be purchased readily from your chemist and some off-the-shelf cures contain it or a related chemical (paraformaldehyde or gluteraldehyde) so a read of the labels or data sheets of some products is essential if you want to use it.

The best way to use this chemical is as a long term bath or to combine it with a freshwater dip (see environmental treatments).

For a long term bath, add 25ppm to your quarantine tank (it’s toxic to some invertebrates and algae, including most coralline algae species, so cannot be used in a reef situation) and this dose repeated every day for 10 days. Remember liquid formalin (which is how you will get it from the chemists) is 37 to 40% formaldehyde and you want 25ppm so you need to add 0.0625ml formalin per litre rather than 0.02ml to get the correct dose.

You can also use it as a short term dip in seawater at 200 to250ppm for 1 hour or combined with a freshwater dip at 200 to 250ppm for 3-5 minutes. For both dips the treatment regime should be carried out on days 1, 2, 3, 5, 7 & 11 followed by 4 to 6 weeks of observation in a quarantine tank.

NB formalin dips are a stressful process and are not advised for very delicate or very ill fish.

As this chemical is very toxic I would recommend that appropriate protective clothing is worn such as gloves and safety glasses and use it in a well ventilated place.

d) Acreflavin
In my opinion to Acreflavin is one of the most under-used treatments available to marine fish keepers. It has a broad range of effect, being effective against protozoans, bacterial infections and external fungal diseases. It is as “reef safe” as any other “reef safe” treatment and is easily obtained. It can be bought in several formulations from the LFS but make sure it isn’t combined with malachite green or methylene blue which have toxicity issues in marine systems.

It is effective at a concentration of 6 ppm against Amyloodinium ocellatum (Paperna, 1984) and this dose should be added to the aquarium on days 1, 2, 3, 5, 7, & 11. Your skimmer should be turned off and any activated charcoal removed. It dyes the water a greenish yellow colour which will change the light spectrum reaching your corals (if you decide to use it in a reef tank, personally I believe all medications are best kept out of a reef aquarium and used in a quarantine tank) and this colour is a bit of a pig to get rid of, but after treatment turning your skimmer back on and adding activated charcoal helps remove the coloration (as do water changes).

e) Hydrogen peroxide
More often used for crustacean ectoparasites, like sea lice, hydrogen peroxide can be very efficacious in treating Amyloodinium ocellatum. It is administered as a bath of 100ppm hydrogen peroxide in seawater for 30 minutes. This treatment regime should be carried out on days 1, 2, 3, 7 & 11 followed by 4 to 6 weeks of observation in a quarantine tank.

NB Hydrogen peroxide dips are a stressful process and are not advised for very delicate or very ill fish.

Environmental Treatments

a) Hyposalinity
Hyposalinty is often quoted as a good and safe way for treating Amyloodinium ocellatum in a reef tank. Unfortunately Amyloodinium ocellatum has a natural salinity range 3 to 45 ppt (Noga, 2000). The lower end of which has a SG of around 1.005, far to dilute to support marine fish and invertebrates. So if you DON’T want to cure your outbreak of marine velvet then by all means use hyposalinity therapy.

b) Freshwater dips
In freshwater the parasite drops off the skin of the fish very rapidly (leaving a small wound, which is important as we shall see later). Noga (2000) and Noga & Levy (1995) both reported that a single freshwater dip would remove 80-90% of the parasite. However, if the fish are simply returned to the tank where the outbreak occurred then they will just become re-infected. For this treatment to be effective then (like all the dip treatments discussed in this article) the fish should be kept in a quarantine tank and the main display tank kept fish free for 8 to 12 weeks. Once in the quarantine tank the treatment should be repeated on days 1, 2, 3, 5, 7 & 11 followed by 4 to 6 weeks of observation in a quarantine tank.

Remember those little wounds the parasite leave? Unfortunately, there can be thousands of them on an infected fish and those wounds can cause the fish to lose body fluids to the environment. This is exacerbated in freshwater so remember that a heavily infected fish could easily become physiologically stressed with this treatment method.

One of the most important things about a freshwater dip is that it will buy you some time to start another treatment or to set up a quarantine tank as even a very badly infected fish can lose most of its parasite in a few minutes and improve quite dramatically.

To carry out a freshwater dip:
  1. Take some freshwater (RO is best) and heat it up to the same temperature as the tank: I find the best thing is to float a plastic ice cream tub full of freshwater in the tank until the temperature equalises.
  2. Adjust the pH of the freshwater to match the tank using a commercial pH buffer.
  3. Catch the fish and pop it into the freshwater bath, watch the fish carefully and be prepared to remove it if it becomes very distressed. Normally 3 minutes in a freshwater bath will dislodge most parasites; this can be extended to five minutes. I would not leave the fish any longer than five minutes in a freshwater bath.
  4. Catch the fish and put it back into the tank. Do not pour the freshwater back in the tank as this may introduce the parasites back into the display tank.
NB A freshwater dip is a pretty stressful process and is not advisable for very delicate or very ill fish.

c) Low light & water changes
Amyloodinium ocellatum is a photosynthetic organism so a useful supporting strategy to any of these treatments is to keep the tank under a very low light regime just actincs or moonlight LEDS are perfect. It won’t cure the disease but can buy you some time until natural immunity takes over (see below) or you can set up a quarantine tank. If you also carry out 25% water changes each day you will also reduce the parasite load in the tank: again it won’t cure the disease but will buy you some time.

However, the low light will be detrimental to corals etc. But in the quarantine tank situation low light is very beneficial as it is not only detrimental to Amyloodinium ocellatum but will help reduce the fish’s stress levels as well.

Experimental treatments

Having read this far you may think “damn, this is a tough disease and the remedies aren’t that effective, or difficult to use”, and to some extent you would be right. However, recent research on malaria (another protista which has some plant-like characteristics in its genome) suggests that some herbicides have tremendous potential for treating this pathogen. Again these treatments may be some way off and wouldn’t necessarily be suitable for a reef tank. However, they may, at last, provide a simple and effective way of controlling this pathogen.
Natural Immunity
As with many infectious diseases the host’s immune system can play an important role in recovery and provide protection in case of future exposure to the same pathogen. This is usually achieved by the host developing an antibody response or cell mediated immunity. These articles are too short to go into any depth concerning the functioning of the immune system of fish. However, I would like to give a brief outline of the immune system’s function as it is important in explaining why some fish in a tank do not succumb to a pathogen.

Antibodies (http://en.wikipedia.org/wiki/Antibodies)

Antibodies are proteins produced by the B cells of the immune system which bind specifically to antigens (an antigen is a molecule usually found on a pathogen’s surface). They damage the pathogen either directly by inhibiting its function or secondarily by activating another series of proteins called the compliment cascade. Fish, although they only have one class of antibody, IgM, have very good antibody responses which can provide life-long protection against disease. This is why most farmed fish and some commercially-bred reef fish are vaccinated against common pathogens. After exposure to the pathogen or vaccine, full immunity takes about 6 weeks to develop so, provided the fish can be kept alive during this critical period of infection, there is a good chance that the anybodies, which circulate in the blood or mucus, will neutralise any further encounters with that pathogen.

Cell mediated immunity (http://en.wikipedia.org/wiki/Cell-mediated_immunity)

Again, following exposure to a pathogen, groups of specialised immune cells, such as natural killer cells, CD8 & CD4 cells, granulocytes and macrophages swing into action to regulate the immune response. They damage the pathogen either by ingesting it (macrophages) or by discharging enzymes and free radicals next to the pathogen to kill it. The most obvious thing you can see when a cell mediated response has occurred is inflammation and pus.

Once an animal has become immune to the pathogen, either through exposure or vaccination, when exposure to that pathogen happens again the immune system can respond more quickly, preventing the pathogen establishing itself. This why you often see a new fish in a tank developing a disease the other fish recovered from some weeks before: the new fish has never seen that disease before and succumbs to any residual pathogens while the surviving inhabitants remain uninfected. This is why at least 6 weeks between restocking is advised after any disease outbreak has finished, ensuring that the risk of any pathogen remaining in the tank is minimal.
Conclusion
Sadly, Amyloodinium ocellatum is possibly the toughest parasitic disease you can get in a reef tank and untreated you can expect 80 to 90% losses. Quarantine is the only way to be sure of not introducing it. When it comes to treatments on the whole they have to be carried out in a quarantine tank as virtually all of the effective medications are very detrimental to invertebrates.


One of a series of articles written for UltimateReef by fish health professionals.
Published on May 1st, 2007 at UltimateReef.com

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