Last month we dove into the home culture and production of “greenwater”, which are the basic food item for enriching prey items that will feed our new fish larvae. So now that we have our phytoplankton growing from last month, let’s start in on the next step in the food chain for fish larvae.

Rotifer means “wheeled animals” and when viewed under a microscope these organisms appear to have wheels rotating about the tops of their heads. The phylum Rotifera consists of 3 classes, 120 genera and approximately 2000 described species and out of all these species only two brackish/saltwater species are commonly cultured for the aquaculture (Fig 1); Brachionus plicatilis ”L”strain and Brachionis rotundiform ”S” strain (Lubzens, 1987; Dhert et al., 1994). Brachionus rotifers are 200-350 mm holoplankton, which means they are permanent zooplankton residents which stay constantly adrift (Hagiwara,1985). As part of the zooplankton they help sustain a microscopic community that supports small fish, and allow them to eat and grow. An interesting fact about rotifers is that they are primarily  freshwater or brackish water organisms and are not a natural prey item for marine fish (Hagiwara, A., 1989). However they posses a number of beneficial features that allow us to utilize them for the culture of SW fish larvae. Such benefits are their small size that allows baby fish fry to fit them into their mouths; they have soft outer bodies and are highly digestible.  They move slowly and are easily followed and tracked by larvae; they stay suspended in the same water column like the fish fry and as such are in close proximity to the hungry fish larvae. Next, they are readily cultured in large numbers, rapidly reproduce, and when fed phytoplankton become enriched sufficiently to allow fish larvae to grow and develop. However, Rotifers themselves have little nutritional value – they act as “nutrient carriers” for transporting the high-value essential fatty acids (EPA and DHA) and other nutrients from the microalgae to the target species. Although you can feed rotifers a variety of items such as yeast, the rotifers will only be as nutritious as the feed they have ingested. An empty or poorly enriched (fed) rotifer will provide little value to your fish larvae.

Fig1 – commercial growout tanks of rotifers using large continuous batch culturing. Image courtesy of http://www.dec.ctu.edu.vn

So let’s start by understanding a little basic rotifer physiology and understand its feeding behavior to understand how we can utilize this microorganism to its fullest. Rotifers are built with a head, trunk, and foot (See figure and 2). The head and trunk combine to form the main axis of the body and are approximately 120-250 mm long. Rotifers are characterized by the presence of a ciliated organ on the head (called corona) that capture food, and a specialized pharynx (called mastax) with hard jaws (called trophi). The trunk contains a fluid filled stomach, and further down is a long tail like foot which is used to temporarily anchor the rotifer (Fig 2a). Of importance to us are beating cilia surrounding the head. These cilia circulate water, food, and nutrients toward the mouth opening. When the cilia detect a meal (usually phytoplankton in the 3-12 mm range), the trunk contracts to pull the mouth opening towards the food, the corona then surrounds the food item and if the food items is the correct size, the particle is crushed and passed into the stomach (Fig. 2b). This food response is repeated over and over within seconds, and this is how the energy demands of this creature are met (Fukusho, K., 1989). Therefore rotifer health and reproduction depends on how often they feed, how much energy they must put into finding and locating food, and how nutritious the meals are. Next we should understand how rotifers reproduce so we will be able to optimize our cultures. A single rotifer can become thousands of rotifers in a few days. Its primary mode of reproduction is called parthenogenesis, which is described as an asexual mode of reproduction. Essentially when environmental conditions are good, female rotifers produce up to 7 eggs simultaneously, without any genetic help from a male rotifer. These eggs are genetically identical, and will hatch to form new “daughter” rotifers with in 12-hrs. (Fig. 2c). By 18-hrs post hatching, the daughter rotifers begin to reproduce themselves, and egg production is maintained for up to a week or so. It is this rapid increase in rotifers that we as fish breeders find so useful for our home cultures. Nevertheless, you may have noted the statement “when conditions are good,” which implies that conditions can be bad, and in fact when conditions are not favorable or stressful rotifers convert to producing haplo-identical eggs, meaning only half the genetic material is placed into an egg. These halpo- identical offspring hatch as smaller nonfeeding male rotifers, which then fertilize the other haplo-identical eggs. These fully fertilized eggs develop a thick outer coating and are called cysts. Rotifer cysts can survive adverse conditions and remain dormant for years if required, and it’s these cysts (resting rotifers or rotifer cysts) that allow rotifers to be reestablished at a later time when conditions are favorable again. The main reason for describing the reproductive strategy for you is that dormant rotifer cysts can be used to restart rotifer cultures, or these cysts can be sent/shipped to other hobbyists so they can establish there own cultures. Also, many online stores will sell resting rotifers or rotifer cysts to start cultures.

Fig 2a. Photomicrograph of a rotifer; photo courtesy of Dr. Elizabeth Walsh.

Rotifer Culture Requirements

So let’s start in with what required to culture rotifers at home.

Even though B. plicatilis can tolerate a broad range of specific gravities, we have a preference range, and a specific gravity between 1.007-1.014 is the optimum range for our home use (Fulks, 1991). Why do I say this? Mainly because we want to add rotifers to our fish larvae grow out tanks and any wide differences (anything greater than 0.007 units) between specific gravity of your rotifer culture and your larvae grow out tanks can result in osmotic shock to the rotifer culture. Rotifers that have undergone osmotic shock stop swimming and drop to the bottom of the tank. Motionless rotifers are not recognized as food. Wilkerson kept her rotifer cultures at no greater than 0.007 units different than her clownfish grow out tanks (Wilkerson, 1998). The reason we need to keep rotifers at a lower specific gravity than our grow-out tanks is that rotifers are more brackish water creatures than true saltwater ones, and rotifers reproduce faster at lower salinities.

Our next concern is pH and temperature. Rotifers can survive a wide pH and temperature range; however, we want to keep the rotifer culture somewhere close to a pH of 7.9. An old culture of rotifers will have a lower pH than a newly started culture and again we need to keep a watchful eye on keeping the rotifer culture conditions similar to our fish grow out tanks to avoid any shock. One major concern for hobbyists with rotifers is ammonia toxicity; rotifers are exquisitely sensitive to ammonia and therefore a hobbyist must balance between keeping the pH low enough to prevent any ammonia toxicity and keeping the pH within reasonable limits. According to Wilkerson, “worry about the ammonia first and pH later” (Wilkerson, 1998). The same goes with temperature: optimal conditions can be obtained between 68-880F. Cultures grown at higher temperatures grow faster than cooler temperature but with one caveat. Cultures grown at higher temperature are unstable and more prone to problems, because the nutrient balances are being produced/consumed at a greater rate. Again, try to match the temperature of your rotifer culture to that of your larvae tank; however, keeping a slightly lower temperature rotifer culture will ensure more stable conditions.

Feeding Rotifers

Rotifers are voracious animals – a single (L) strain will consume about 115,000 cells of Nannochloropsis each day. Assuming we are going to use our greenwater cultures to feed the rotifers, many aquaculture facilities recommend the best combination for feeding growing rotifers is 90% Nannochloropsis and 10% Tetraselmis (Lubzens, L., 1987). This is one of the alga’s we spoke of last month. “Nanno” or DTs phytoplankton is Nannochloropsis, which is a small green algae (2 mm) that has a very high lipid, protein, and calorie profile. Tetraselmis is a larger green algae that contains amino acids that stimulate feeding in marine animals. These alga will give your rotifers a very high nutritional and EPA profile. As a way to boost DHA levels it is recommended that we  use Isochrysis which contains 10% DHA as a percentage of lipids. DHA enrichment is done during the last 4-8 hours before feeding the rotifers to your animals. However, it has been my experience that if you were to use a single phytoplankton species, than Nannochloropsis is the best single algae for growing rotifers. Nevertheless, the addition of 10% Tetraselmis will result in slightly higher growth and fertility rates.

Fig 2b drawings of rotifer with stomach identified

How Often To Feed

The key to successfully maintaining a rotifer culture is keeping the right amount of algae in the water at all times. If you add too much algae it will not be eaten, resulting in fouling your water, and possibly causing bacterial contamination. If you add too little algae your animals will be hungry, and start the production of male rotifers and cyst formation. Rotifers need a consistent supply of “free” algae in the water at all times, allowing them to graze continuously. Due to their high metabolism, about 2-4 hours after they deplete all available phytoplanktons, they will be empty and beginning to starve which “shocks” their system and disrupts their reproduction.

Required Equipment List For Home Rotifer Culture

• Air pump.
• Air Hose — about 9 to 10 feet of 3/16″ flexible tubing.
• Gang Valve — the extra valve is to bleed off air saving wear and tear on the pump and give better bubble control in the culturing containers. A four container setup should have a 5 valve gang.
• Rigid 3/16-inch air tubing — enough to go from the bottom of your culturing container to about 2″ above lip of container.
• A culture container can be anything from a 5gal aquarium for continuous culture to 4-2L soda containers, or glass jars for batch culture.
• Salt water – This will be the culture water. 1.017 is a good specific gravity. 1/3 a cup of Salt to 1 Gallon of water will give a specific gravity in the above range.
• Hydrometer.
• Light. This can range from ambient lighting to a low powered light source for batch culture. For continuous culturing a light source which will keep phytoplanktons thriving; e.g., a 20 watt florescent or even a 75 watt lamp along with a timer set for 16 hours light to 8 hours dark is good. Ensure that the cultures don’t get to hot, about 800 F maximum.
• Set the valves on the gang valve to the opened position, and turn the pump on. Turn the bleed valve slowly towards the closed position until one or more of the air tubes start to bubble. Now tune the air valve so that there is a nice slow bubble stream in the culturing containers. The desired bubble rate is correct when the water just circulates: no more, no less.
  

  Fig2c drawing of rotifer with attached egg mass. Courtesy of http://www.aquatechgroup.com

Rotifer Culture Methods

For the home hobbyist, there are two basic rotifer culture methods: batch culture, and continuous culture. Batch culture produces dense cultures quickly (200+ rotifers/ml), but requires constant monitoring, and is more labor intensive. A benefit of batch culture is once the “batch” has reached optimum density you harvest it, and don’t have to worry about a potential “crash.” Batch culturing also allows you to clean the culture equipment between batches often preventing possible contamination. Continuous culture are much less work, and produce less rotifers (~100 rotifers/ml), but they often experience “crashing” – having all the rotifers die off in mass. If this is a hobbyist’s first attempt at raising rotifers batch culture is highly recommended.

Batch Culture: When using batch culture the hobbyist takes a clean culture container, filled with sterile seawater, with the water adjusted to match the pH and temperature of the starter culture. Optimal temperature is 20 – 30° C with a pH of 8.0. Phytoplankton or food substitutes (see below) are added to the container (a quarter cup of phytoplankton /2L soda bottle), and then cultures should be started by adding a minimum of 10-20-rotifers/ ml to minimize the possibility of a crash.

Continuous Culture: In continuous rotifer culture a larger container (usually a 5-30gal container) is filled with the same water quality as above. Rotifers are added 10-20 rotifer/ml to the container, and phytoplanktons are added to keep the culture a slightly green color. The rotifers multiply and a portion of their population are removed daily. In theory, a rotifer culture fed daily, will multiply continuously, and as long as some are harvested daily the culture should last indefinitely. In practical terms, most hobbyists will only need rotifers when they have fry hatching and therefore starting a new continuous culture at each hatching will ensure a plentiful supply. The problem is that an under-harvested or a poorly feed population of rotifers will pollute the culture water resulting in a rotifer crash.

Starting a Rotifer Culture

Rotifer cultures can be started from either live cultures or dormant cysts. Most beginning hobbyists will prefer to start with a living culture of rotifers and these can be ordered from the online sources listed below. (Author’s note: make sure you order saltwater rotifers NOT freshwater ones).

Fig3a. Home rotifer culture tanks. Shown here are the clownfish growout tanks of James Wiseman.

To start a culture from live rotifers (see Fig 3a):

• 1-quart of live rotifers-saltwater type
• 1-quart of water — clean, temp matched, correct pH, and specific gravity as the rotifer culture (1.017)
• 1-quart of greenwater from our last month’s cultures (or a food substitute-see below)

Rotifers are added to the smaller (2gal tank) while rotifers and greenwater are propagated in the larger 5gal tank.

Like last month, using 2L soda bottles are more than adequate for batch culture. However a 3-5gal aquarium is also fine for continuous culture. Start off by adding the saltwater and phytoplankton to the container, start your aeration (no airstones), again a slow rolling release of bubble is sufficient. Once this is set, add the rotifers 100-200/ml. You can add a light source to these cultures to keep the phytoplanktons happy and this will also slightly increase rotifer reproduction after a day or so. When the water starts to clear from green to light green, add greenwater to retain the same light green tint. Small frequent additions of greenwater are much better than a large addition of greenwater that might result in shocking your rotifers. Continue adding greenwater as the rotifers feed, until the culture reaches its maximum volume (2L). Once the desired volume is reached harvest the batch when the water starts to loose its slight green tint. For continuous culture remove 25% of the volume daily. As a precaution start a spare culture  — staggered in time — Just in case.

Starting a rotifer culture from resting cysts:

To start a rotifer culture from resting cysts you’ll need the following:

• 1 vial of rotifer cysts
• 3 oz of saltwater (1.007-1.014)
• Greenwater (or food substitute)

Resting rotifers are available from the online suppliers listed below, usually in a 1000-cyst vial. Simply add the cysts to the 3 0z of saltwater in a small flat container; aeration is not required to hydrate the cysts. Carefully observe this culture for 16-24hrs and once swimming rotifers are observed, they must be added to a culture of phytoplankton. If you do not observe any rotifers swimming after 24hrs add the emerging rotifer culture to greenwater. Often times the rotifers are difficult to see. To avoid culture shock it is always better to add greenwater to rotifers, and not the other way around. If you do plan on staring rotifer cultures from dormant cysts, ensure you plan your time line accordingly; expect to take about 15-30 days to bring these cultures up to speed. My personal experience with hatching rotifer from cysts was it was more difficult than establishing cultures from live samples; however, cysts are easily available and many aquaculture facilities prefer cysts
to live samples.

So how many rotifers do you need? A good starting point for feeding clownfish larvae is approximately 8 to 10 gallons of rotifers; however, this will depend on the number of hatching fry and the numbers of larvae you plan on rearing. Reproduction rates in rotifer cultures depend on how fast a culture recovers after harvesting. A healthy culture can triple daily, but a conservative estimate sees doubling once every three days. Therefore, plan accordingly.

Tips on improving a rotifer culture:

• Feed rates should be based on the actual density of rotifers in the system and care should be taken not to overfeed.
• If feeding continuously the drip rate should be monitored to avoid excess algae accumulation.
• If batch feeding, the culture tank should clear of algae before the next feeding to avoid excess algae accumulation.
• Any algae that is not consumed within 48 hours will degrade, increasing the level of ammonia and decrease the dissolved oxygen levels.
• Care should be taken to match the specific gravity of the rotifer tank to the specific gravity of the starter culture

Microalgae Ice Cubes  (Tip from Reed mariculture site):

• N. Oculuata can be frozen into small ice cubes that have the dual benefit of creating a pre-measured quantity of algae, and extends the shelf life of the algae. To make microalgae ice cubes simply pour the algae into cheap plastic ice cube trays (available at your local store) and store in the freezer overnight. When frozen, break the cubes out of the trays and store them in a plastic bag in your freezer. By measuring one of the cubes you will know the weight of all the cubes, simplifying your future feeding protocol. As needed, take 1 or more cubes from the freezer and dissolve them in a jar with water from your tank. If you have extra algae remaining, put it in your refrigerator where it will last for several days.

Improving Culture Performance

• There are several variables that can shock your rotifers, decreasing their growth and fertility rates. The most common are a change in temperature, pH, or specific gravity in the rotifer culture. Typically these “shocks” come from feeding large amounts of live algae but can also be caused by other factors.
• Phytoplanktons are plants and as such requires large amounts of plant fertilizers such as nitrates, phosphates, and iron. These are good for plants but somewhat toxic for animals. Microalgae cultures can have higher pH levels than rotifer cultures due to photosynthesis.
• For best growth and nutritional value algae are typically grown at 16-240C while rotifers are grown at 26-300C. When the algae culture that is full of fertilizers has a different pH, and a lower temperature is introduced into your rotifer culture, it “shocks” to the culture. This can cause weak animals to die, healthy animals to stop feeding for several hours, and females to stop egg production.
• The rotifer culture should be maintained at a pH around 8. At a pH lower than 7.2 or higher than 9 the culture will have significant mortality.
• ALWAYS KEEP YOUR ALGAE CULTURES ABOVE THE ROTIFER CULTURES or in a totally seperate area. Make sure any potential splashes always goes from the phytoplankton cultures towards the rotifer cultures, NEVER the reverse. It is better to potentially contaminate the rotifer cultures w/ algae than thealgae cultures w/ rotifers.
  

  Fig3b. A perfectly acceptable alternative to home culture of rotifers is purchasing greenwater + rotifers. Shown here is a gallon container of rotifers + greenwater (_N. Oculata_). (www.mountaincorals.com)

I will end this section by saying that a perfectly acceptable alternative to home culture of rotifers is to purchase greenwater and rotifer combinations (Fig 3b). Many on-line providers sell ready made mixtures containing 250-500 rotifers/ml plus greenwater in a quart to gallon size container. These cultures can be stored in cool areas for less than 1week before the rotifers exhaust the greenwater. If kept in the refrigerator the culture will last approximately 7-10 days. As mentioned last month when using commercial preparations the hobbyists trades convenience for expense.

Alternatives to live phytoplanktons

Can we raise fish larvae with out having to set up and maintain an algae culture system? Absolutely, more and more commercially produced substitutes are becoming available which will either supplement or potentially even replace live phytoplankton cultures.

The first alternative to live phytoplanktons is cryopreserved phytos. There are a few companies that currently supply concentrated phytoplanktons in commercial quantities. One such company is Inland seafarms Inc. (cryopreserved phytoplanktons can be purchased in hobbyist quantities from www.brineshrimpdirect.com). This company provides many different cryopreserved phytoplankton species along with their respective HUFA analysis. Next is Innovative aquaculture, this company provides monocultures of N. Oculata and Chaetoceros and mixed formulas of both. Due to the mixture formulas these products can be used as a nutritionally complete first diet. A third company is Reed Mariculture, this company supplies mono and mixed cultures of cryopreserved phytoplankton, and even provides a wonderful mixed of three phytoplankton called “Tahitian blend” which contain N. Oculata, T-Iso (Tahitian strain), and in proper ratios to ensure a wide distribution of HUFAs (www.brineshrimpdirect.com) sells Reed Mariculture’s cyropreserved phytoplanktons). A key feature about the cryopreserved phytoplanktons is there remarkable shelf life. According to company information, one can store these products at 40C for up to one yr w/o loss of nutritional value. When stored at –200C Inland claims a 2-yr shelf life. While these are manufacturer’s claims, ongoing studies are being performed to determine nutritional profiles after defined storage periods. Cryopreserved phytoplankton is an excellent alternative if you are in need of a large volume of greenwater. These products can be used for rotifer and nauplii cultures, with however one limitation. These products are dead phytoplanktons and as such contribute to water fouling immediate upon addition to the water. Therefore, be aware that use of these cryopreserved products will require you to do frequent water changes in your prey item cultures to maintain proper water
parameters.

A second alternative is a product called spray-dried cells of S_chizochytrium_ sp. Aquafuana marine Inc produces two enrichment products based on this concept. Algamac 2000 and RotiMac. Both AlgaMac and RotiMac can be used as direct food substitutes for rotifers and nauplii, or as a supplement to other foods. RotiMac has HUFA levels of 0.3% (EPA) and 19.6% (DHA) while AlgaMac has EPA levels of 0.6% and DHA 24.0%. RotiMac is actually sold as a food substitute for rotifers, whereas AlgaMac is considered as an enrichment product for rotifers and artemia. These products have found success in the shrimp aquaculture arena. One benefit of using these products is that unlike fish-oil based enrichment media RotiMac and AlgaMac can be used directly in your larval tank. Similar to using the other food substitutes, water fouling can be a serious problem, and one must keep a cautious eye on their use. The shelf life of these two products is listed as 9 months in a cool dry location.

A third food substitute is a fish oil based enrichment. These Selco/Selcon based medias (both liquid and dry forms; (Culture Selco, Roti-rich, Rich, Rich Advance, Algae Rich) are high in Omega-3 fatty acids and are particularly good for the final stages of rotifer and Artemia nauplii enrichment prior to feeding to your fish larvae. Approximately 4-24 hrs before you use the rotifers or nauplii add these Selcon/Selco type products to the water. After enrichment, collect the prey items and feed directly to your fish larvae, do not carry over Selco enriched water into the larvae tank; these products will quickly foul tank water. As with any enrichment, as soon as rotifers/nauplii clear their gut of any food item they are inadequate as a food item. Greenwater can aid in keeping rotifers/nauplii enriched over a period of time in the culture tanks, but additional care and maintenance will be required to prevent water quality issues in your grow out tanks when using these fish-oil based products. Interestingly, many manufacturers suggest that these products can be utilized as a direct microalgae replacement, and while they do provide adequate nutrients to the rotifers, they are best utilized when performing batch cultures. The main reason is that one can remove the fouled water between each batch, and clean the container to remove any oily film or residue.

A fourth option is to use Martin Moe’s V-8 technique. Initially described in “Breeding the Orchid Dottyback” (Moe 1997), this technique relies on the use of the product V-8 juice as a nutritional supplement. His description utilized a few mls of V-8 juice and selcon to boost levels in prey items. While this is a unique and interesting alternative, I’m not sure what the DHA or EPA levels of V-8, or the nutritional value of the rotifers that were fed V-8; however, Martin Moe was successful using this method and as such it should not be discounted. Again, as a reminder when using products like these, concern for water quality should be paramount, as they will quickly foul your water.

Interestingly, Rotifers can alternatively be cultured solely by being fed on bread yeast. A culture can be established which utilizes 2.5-20 gms of yeast /gal of tank water inoculated w/ 10-20 rotifers/ml. The bread yeast, dissolved in fresh water, is added twice daily at a ratio of 1 g of yeast to 106 rotifers. After 7 to 10 days, the rotifer density usually exceeds 100 individuals per milliliter. At this stage, they can be completely or partially harvested as feed for larvae.

Larval culture is possible without live phytoplanktons, but it may not be the best choice. Cryopreserved phytoplankton offer the closest alternative to live phytoplanktons, followed by the spray-dried products. Care must be exercised when using these products, as they will quickly foul small volumes of water. Lastly, if you do use a phytoplankton substitute consider adding an enrichment type substitute (like selcon) to ensure a well-balanced meal.

Hopefully after reading this column I have provided you with enough material and ideas to start your own home culture of both phytoplankton and rotifers. I will end by saying that some fish larvae are too small to consume rotifers as a first food. Therefore, we must consider food items smaller than rotifers as their first food. Fortunately, there are a number of marine organisms that are much smaller than rotifers and can be cultured at home in vast numbers. These are ciliates.

There are about 8,000 species in the Phylum Ciliophora. The name Ciliophora means “bearing eyelashes” and this is a good description of the tiny, short, whip shaped flagella that cover most species of ciliates. There are many species capable of living in the marine environment, both planktonic and benthic, and some, particularly in the genera Tintinnopsis and Euplotes, have potential as food organisms for very small fish larvae and perhaps invertebrates as well. Ciliates reproduce by mitotic division and so in the proper culture environment, reproduction can be rapid. One of the key features of any larval food organism is that it must be capable of rapid reproduction and must be able to sustain dense cultures in order to supply the quantity of food required to feed large numbers of larvae. Ciliates certainly fill these requirements. Other requirements, however, such as nutritive value and acceptability by larval fish as food organisms, are not as encouraging. Next month we will take an in-depth look at the techniques of rearing ciliates for fish culture and perhaps other aquaristic uses.

Online sources of rotifers and rotifer culture information

• Brine shrimp direct: (online source of Reed Mariculture’s cryopreserved phytoplanktons)
• http://www.instant-algae.com: (source for Inland seafarms and reed mariculture phytoplanktons)
• Reed maricultures site on rotifers
• Carolina Biological Supply, 2700york rd Burlington, NC 27215 800-334-5551 (source for  phytoplankton starter cultures,  culture vessels, nutient mediums)
• Florida Aqua Farms Inc, 33418 Old Saint Joe Road Dade City, FL 33525, Phone: 352-567-0226, (source for f/2, phytoplankton and rotifer cultures, measuring dip sticks, reading material)
• http://www.soest.hawaii.edu/SEAGRANT/Makai/dec00/3.html, A scientific abstract on the population of rotifers grown under various conditions.
• Sach’s basic notes on home culture
• http://www.athiel.com/lib5/rotifers.htm: interesting article about outdoor culture of large ponds of rotifers.
• http://dmc.utep.edu/rotifer/: UTexas El Paseo website “the rotifer systematics database”
• http://www.nies.go.jp/chiiki1/protoz/morpho/rotifera/r-brach1.htm, Online scientific reference of the phyla rotifera
• http://216.25.82.220/Rotifer.PDF, Online scientific article on mass culture of rotifers.
• http://www.biosynthesis.co.uk/rotifers1.htm, UK based company which sells everything you need to start your own cultures
• http://www.northcoastmarines.com/PLANKTONESV.htm: a background page on EVS spray-dried phyoplanktons
• http://www.echonet.org/shopsite_sc/store/html/PlanktonCultureManual.html: a online store which sells the plankton culture manual
• Online source for Rotimac: http://www.aquafauna.com/Diets-Rotimac.htm
• http://dmoz.org/Science/Biology/Flora_and_Fauna/Animalia/Rotifera/: link page full of excellent rotifer sites

References

1. Fukusho, K., 1989. Biology and mass production of the rotifer, Brachionus plicatilis. Int. J. Aq. Fish. Technol., 1:232-240.
2. Fulks, W., K. L. Main, 1991. Rotifer and Microalgae Culture Systems. The Oceanic Inst., Honolulu, USA. 364pp.
3. Hagiwara, A., 1989. Recent studies on the rotifer Brachionus plicatilis as a live food for the larval rearing of marine fish. La mer 27:116-121.
4. Hagiwara, A., A. Hino, 1989. Effect of incubation and preservation on resting egg hatching and mixis in the derived clones of the rotifer Brachionus plicatilis. Hydrobiologia 186/187:415-421.
5. Hagiwara, A., A. Hino, R. Hirano, 1985. Studies on the appearance of floating fertilized eggs in the rotifer Brachionus plicatilis. The Aquaculture 32(4): 207-212.
6. Hagiwara, A., C. S. Lee, 1991. Resting egg formation of the L- and S-type rotifer Brachionus plicatilis under different water temperature. Nippon Suisan Gakkaishi 57(9):1645-1650.
7. Lubzens, L., 1987. Raising rotifers for use in aquaculture. Hydrobiologia 147:245-255.
8. Pourriot, R., T. W. Snell, 1983. Resting eggs in rotifers. Hydrobiologia 104:213-224.
9. Snell, T. W., K. Carrillo, 1984. Body size variation among strains of the rotifer Brachionus plicatilis. Aquaculture 37:359-367.
10. Wilkerson, J.,  Clownfishes, Microcosm Limited; ISBN: 1890087041; June 1998.
11. http://www.echonet.org/shopsite_sc/store/html/PlanktonCultureManual.html