Re:Live Cultures!

Pete Giwojna

Dear Suzanne:

I don’t know what species they may be but you are correct — the calanoid copepods are planktonic or free-swimming and the application they are best suited for is feeding seahorse fry since they are so small and do indeed remain in the water column.

The care sheet for the copepods is available online at the following URL; it explains how to acclimate your culture of copepods but all it says about culturing them is that Ocean Rider recommends feeding them with their microalgae (i.e., CHAGRA):

Click here: – Seahorse, Sea Life, Marine Life, Aquafarm Sales, Feeds and Accessories – Copepods

I’ve never cultured the OR calanoid copepods, but for whatever it’s worth, this is how I usually maintain my copepod cultures, Suzanne:

Culturing Phytoplankton & Copepods

Seahorse fry alter their diet as they grow (Vincent, 1990). This may be due to the fact that they change microhabits as they develop (e.g., when pelagic fry complete their planktonic stage and begin to feed at the bottom as they begin orienting to the substrate). Or it may simply be due to the fact that they become better hunters and perfect their feeding skills as they grow, thus enabling them to tackle larger, more active prey (Vincent, 1990). Whatever the cause, one good way to keep up with the fry’s changing dietary requirements is by providing them with cultured copepods at progressively later stages of development.

Step 1: Providing Marine Microalgae (Phytoplankton).

Marine microalgae or phytoplankton is available from many sources. It can be cultured at home, and if you have a green thumb and are experienced with such greenwater cultures, that may be your best option. However, home culturing may not be for everyone. Greenwater cultures can be tricky to maintain. They are easily contaminated and are prone to "crashing" suddenly and unpredictably, which can have dire consequences if you are relying on the phytoplankton to provide food for your seahorse fry.

Alas, I am one of those unfortunates who cannot seem to maintain a decent greenwater culture for any length of time no matter what I try. Consequently, I now much prefer to obtain live marine phytoplankton from other sources rather than attempting to culture my own. Commercially available phytoplankton tends to be more concentrated than homegrown cultures as a rule, and I find purchasing it to be far more convenient, efficient, and productive. Given my repeated failures and the time I spent for naught on my own greenwater cultures, I’m certain that buying live phytoplankton is more economical for me in the long run as well. If you are inexperienced with greenwater culture or simply lack the time to culture your own, I recommend buying your live phytoplankton instead (see the Resources page for suppliers). Whichever source you decide to use, home grown or store bought, make sure you use it strictly according to instructions to prevent contamination and spoilage of the phytoplankton.

The type of phytoplankton or microalgae you use is not that crucial. Chlorella is one of the most popular microalgae used in mariculture (Wilkerson, 1995), but Dunaliella also works extremely well and is recommended by Dr. Amanda Vincent (Vincent, 1995c), an authority on the breeding habits of seahorses. Serious breeders often use a mixture of different types of phytoplankton to feed copepods or rotifers, rather than a microalgae monoculture, with the goal of enhancing the nutritional profile of the ‘pods or rotis as much as possible (David Warland, pers. com.).

There is a great deal of merit to that approach, but in the past, maintaining separate cultures of different species of microalgae was beyond the capabilities of most home hobbyists, myself included. I prefer to keep things simple and I have always used Nannochlroposis as the phytoplankton I feed to copepods, both because it produces good results and because it is commercially available from a number of sources. To simplify things all the further, I purchase my Nannochlroposis in quantity as needed, rather than struggling with phytoplankton cultures.

The product I like best at the moment for this now includes a concentrated mixture of live marine phytoplankton (two species of Nannochlroposis, N. oculata and N. salina, as well as a Chlorella sp.) in every bottle (DT’s Live Marine Phytoplankton, 2003). That makes it a simple matter to provide my ‘pods with a diversified diet to maximize their nutritional value as fry food — I just unscrew the cap on the bottle and pour the requisite amount of this phytoplankton mixture into my culture tank whenever it’s losing its greenish tinge, and I’m in business (DT’s Live Marine Phytoplankton, 2003)! No muss, no fuss. Quick, easy and effective — just the way I like it!

Step 2: Culturing Zooplankton (copepods and/or rotifers).

We will be using standard 10-gallon glass aquaria as our batch culture tanks. It’s a good idea to run at least 2 such tanks simultaneously; that way, if one of the cultures falters, the other tank can pick up the slack and you won’t miss a beat. Depending on how many seahorse fry you are rearing, you many need to operate several such tanks to assure you will be producing sufficient food for them all.

Fill each of these culture tanks slightly less than half full with synthetic saltwater, adjust the salinity of the culture tank to match the salinity of your nursery tanks, and maintain the pH at 7.9 or below (Rhodes, 2003). This will assure that the copepods (or rotifers) we are culturing do not experience any salinity shock when we feed them to our seahorse fry. No heater is necessary — the cultures will do just fine at room temperature (24C-28C is optimum). Provide very low aeration (Rhodes, 2003). Airstones are unnecessary — a naked bubbler stem is sufficient. Adjust the airflow so it produces a slow, steady stream of coarse air bubbles (slow enough so that you can count the individual bubbles). Ambient room lighting is adequate or you may provide low wattage fluorescent lighting if you prefer.

Add enough greenwater (either commercially produced phytoplankton you’ve purchased or your own homegrown microalgae) to tinge the culture tanks green, and you’re ready to start culturing copepods. All that remains at this point is to "seed" the culture tanks with copepods. Add a starter culture of marine copepods to each tank, acclimating the ‘pods if necessary exactly as you would acclimate a new aquarium fish. They will do the rest.

To nurture the copepods, simply maintain a nice green tint to the culture water by adding more phytoplankton whenever the water in the tanks begins to clear in color. (Be conservative with these phyto-feedings. One dose of phytoplankton every 7-10 days is generally adequate, depending on production and your copepod harvest rates; Rhodes, 2003.) The ‘pod population in the culture tanks will double every 2-3 days, depending on the temperature and how well they are fed (Rhodes, 2003), and as soon as the population builds up sufficiently, we can begin to harvest copepods to feed to our seahorse fry. When you begin to notice numbers of copepods gathering on the tank glass, that’s a good indication that their population density can support daily harvesting.

The best way to harvest copepod nauplii is to strain the desired amount from the culture tank using a 35-micron sieve and then rinse or backwash the strainer in the nursery tank (Rhodes, 2003). Alternate which culture tank you harvest the copepods from for each feeding in order to avoid depleting the ‘pod population too much in any given tank.

Periodically, it will be necessary to restart the copepod culture tanks to filter out the detritus that accumulates on the bottom. This is typically done every month or two (Rhodes, 2003) and is a surprisingly simple process. Just siphon out the water from the culture tank, straining the water in the process in order to retain the copepods. A 125 -micron sieve works well for a strainer. That size mesh will retain all the reproductive adults you need to restart your culture (Rhodes, 2003). It’s a good idea to use a small diameter siphon at first, being careful to suck up as little of the detritus as possible since it will clog up your strainer and your goal at this point is to recover as many copepods as you can. Once you’ve strained out most of the ‘pods, backwash them into container of clean saltwater and set them aside to seed the culture tank after you’ve finished cleaning it. Having saved as many pods as possible, switch to a larger siphon and drain the culture tank completely, removing all of the accumulated detritus. Fill the tank half way with freshly mixed saltwater you’ve prepared in advance and adjust the aeration. Then return the copepods you strained out previously and add enough concentrated phytoplankton to tinge the water green, and your culture is ready to begin producing again. If you restart your culture tanks on alternate months, one or more of them will be in full production at all times, and you can keep a thriving copepod population going indefinitely in this manner.

If you so desire, rotifers can be cultured in exactly the same manner. The only difference is that the 10-gallon culture tanks should each be seeded with a quart of live rotifers initially (Giwojna, Jan. 1997). When necessary, add enough concentrated phytoplankton or greenwater to keep the rotifer culture tanks slightly green. As long as the rotifers are being fed algae, about 25% of the rotifer cultures can be harvested each day to feed to your seahorse fry (Wilkerson, 1995). Try to keep more than one rotifer culture going at all times in case of crashes, and be sure to keep the bottom of the culture tanks scrupulously clean (Giwojna, Jan. 1997).

In fact, you can even maintain a dual culture of copepods and rotifers in the same tank if you wish. But you must avoid cross-contamination of your culture tanks with brine shrimp at all costs! Newly hatched brine shrimp (Artemia nauplii) are considerably larger than either copepods or rotifers, and the Artemia will happily fed on them as well as the phytoplankton. So if any brine shrimp ever find their way into your culture tanks, you will very shortly thereafter be culturing Artemia instead of ‘pods or rotis, leaving you with nothing but live food that’s too large for pelagic fry to eat.

Harpacticoid copepods such as Nitokra lacustris go through 6 naupliar stages as they grow, followed by 6 copepodite stages, before they become reproductive adults. They range in size from 45 microns (smaller than rotifers) up to 270 microns as full-sized adults. The many different stages of development copepods undergo is actually a blessing for the aquarist since it makes it possible to provide progressively larger ‘pods to the seahorse fry as they grow simply by using sieves with different sized mesh to harvest them. For instance, a 35-micron sieve will gather up even the smallest copepod nauplii for newborn fry, while a 125-micron will collect only adult-sized pods for older fry and juveniles, leaving the smaller ‘pods behind to develop further. An 80-micron sieve will take intermediate-size ‘pods along with the adults.

And here are some good copepod culture instructions from other sources:

Species Nitokra lacustris (Harpacticoid)
Motility Planktonic (in the water column)
Size 40 – 270 microns
Application Larval and Adult Fish
Larval and Adult Seahorses
Zooplankton feeding corals
Wide aperture filter feeders

Life Cycle Eggs ->
Nauplii ->
Copepodites (Juveniles) ->
Adults – Females bearing egg sacs
Reproduction The females only need to mate once in order to have 3 to5 sacs of eggs with 35 eggs each.
How long can a culture be maintained? Indefinitely if treated correctly
What is the doubling time? 2-3 days, depending upon temperature and feed
Culturing Technique
In batch cultures use very low aeration. Treat water with chlorine or filters (to 1 micron) before using. The water should be at 30 ppt salinity and 24 to 28 degrees Celsius for optimum growth. Add copepods, and 100,000 to 200,000 cells algae concentrate (recommend Thalasiosira, Pavlova, Isochrysis or Tetraselmis). Add more algae every 9 to 10 days, depending on level of productivity. It is important to be conservative with the feed.
Periodically (every one to two months) filter and restart the cultures to remove detritus. Filter for small juveniles with 35 micron sieve and adults with 125 micron sieve. Use smaller stages for fish feed, retrieve females with egg sacs for restarting cultures.
Restart cultures with females bearing egg sacs. (About 10/L if you have low numbers, 50 to 100/L will result in faster population growth.)
This formula works for every size container, used in batch culture (meaning no recirculation). It may be possible to increase population growth by removing ciliates by filtration – this is not known yet.
These copepods are about the same size as rotifers which means that they will be hard to see with the naked eye. This product is only recommended for customers that are interested in culturing copepods – not for customers that need an immediate feed source.

Dear Hobbyist:

The following is the standard
procedure for culturing algae and copepods. I use a 5-gallon plastic water
bottle for the algae culture and a 30-galloon plastic trash can for the
copepods. The temperature range is 75 to 85 F and the salinity is 1.022. Air
pump and air stones are used in both for circulation.
By the way, you can enrich newly hatched Artemia with this algae culture.
A. Algae culture
Microscopic, single-celled algae (phytoplankton) is cultured for use as a
food for filter feeders such as the Tridacna clam and various corals, and
for feeding microinvertebrates such as brine shrimp (Artemia), rotifers, and
copepods. Microinvertebrates are used for feeding larval and juvenile fishes
and corals. We culture several families of phytoplankton; the techniques for
their culture are almost identical. The species we use are Tetraselmis
1) Clean the containers. Bottles should be acid cleaned and flushed. Large
vats should be scrubbed lightly and rinsed out. The object here is to remove
most of the surface-bound organic matter so that the chlorine sterilization
(next step) is more effective. Be sure to fill acid cleaned bottles
completely with tap water to remove acid vapors.
2) Fill containers with seawater, and add 1.0 ml of Clorox (5.25 % sodium
hypochlorite) for every litre of seawater. Let the containers sit unaerated
away from strong light overnight. If water is needed sooner, add 5 ml of
Clorox per litre and let stand at least 2 hours. The object here is to
maintain a chlorine residual of > 10 ppm overnight. Residual chlorine is
affected by organic load, so more will be needed in dirtier water. A
swimming pool test kit is adequate for measuring residual. Strong light and
aeration will disperse chlorine and reduce its strength.
3) Neutralize the chlorinated seawater by adding 1.0 ml of sodium
thiosulfate solution (1N, 250 g Na2S2O3-5H2O per litre of distilled water)
for every 4.0 ml of Clorox that was added earlier. Neutralization is
complete when water is mixed (i.e. turn on air). Airstones are not
recommended for algae cultures because they take too long to clean. A
plastic pipette or piece of lead on an airhose will do. If airstones are
used, soak them in full-strength Clorox between batches and rinse them
thoroughly before use.
4) Add nutrients to the sterile seawater. Our "F/2" nutrient stocks
(Guillard, 1975) are made up at 500 times final concentration. Thus, 2 ml of
nutrient should be added to each litre of sterile seawater. Nutrients are
kept in a plastic bottle in the refrigerator. Normally, 2L bottles get 4 ml
of F/2; 100L buckets get 200 ml of F/2, etc.
Alternate nutrients: Put 2 level tablespoons Miracid in 300ml tap water.
Store this in any clean, capped plastic bottle at room temperature. Use 1ml
of this blue solution for every liter of algae for dense TE growth. This is
"F/1" strength, and works very well with outdoor Tetraselmis in partial
shade. It may not work for all species. (Stockly’s Aquariums method)
5) Add algae inoculant. It is best to check microscopically and record cell
counts and health occasionally. You can usually tell by color if a 2L
culture is healthy and dense enough to use as inoculant for 100, 200, or
600L cultures. Note the date, inoculant source, inoculant quantity, and
algal species on a piece of tape (or directly on glass bottles) whenever a
new culture is started. This procedure will help you to determine how well a
culture is doing, and is good for inventory control.
C. Copepods
The harpacticoid copepod, Euterpina acutifrons, is a nearly ideal food for
larval marine fishes due to its size, trophic ecology, nutritional value,
culturability, and (most importantly) acceptability by pelagic marine fish
larvae. There are few literature references to successful copepod culture.
Suggested readings include Theilacker and Kimball (1984); and Zurlini et aL
(1978). Culture techniques are easy, using the following suggestions.
1) Do not use an algae that gets too slimy and settles heavily. You will
want to aerate sufficiently to suspend the algae, but not so much that you
interupt sexual coupling. Slimy surfaces will trap nauplii. Chaetoceros
gracilis (4 x 4 x 5um) works well. So does TE. You may get faster growth and
a higher fecundity if a dinoflagellate or other flagellated green
phytoplankter is present. Normal growth rates at 25oC are 10-15 % per day
(up to 100 fold increase in 8 days), with harvest densities of 20 to 50
adults copepods per ml.
2) Partial shading helps, if cultures are outdoors. Keep the cultures in a
growth phase, and change them over to a clean container every few weeks.
3) Inoculate with 1-10% of your harvest. I like to keep the density above 1
per ml so I can count them with a 1 ml. pipette, but they will grow fast at
densities of 1 per litre. Algal densities of 5 x l0e4 to 2 x lOe5 cells per
ml will give good growth rates. You can approximate these densities as
visibilities of 7 to 10 cm.
4) Do not let rotifers enter the system. They will usually outproduce the
copepods. It is difficult to keep these two zooplankters separate with
screens because their sizes overlap. If you do get a rotifer takeover,
isolate 10-100 gravid female copepods and start over in 2 to 40L of new
medium (check them in a microscope to make sure there are no rotifers).
5) There are 6 naupliar stages, and 6 copepodite stages, including the
adult. Size is 50 x 50 x 70um (N1) to 150 x 175 x 700um(C6). I use a 37um
screen to harvest N1 & N2, and a 100um screen for copepodites. Generation
time is about 8 to 11 days under best conditions, at temperatures of
24-26oC. If you stock your rearing container with all sizes of copepods, new
nauplii will be produced by the adults to replace those consumed by the fish
Good Luck,
Bill Stokely

The calanoid copepods may go through slightly different larval stages but I believe they should respond well to the same basic culture techniques.

Best of luck with your live food cultures, Suzanne!

Happy Trails!
Pete Giwojna

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