SRAC Publication No. 701

VI October 2000 PR

Culture of Small Zooplankters for the Feeding of Larval Fish

Granvil D. Treece1 and D. Allen Davis2

In nature, zooplankton is one of is a euryhaline species, small and tion occurs below 35 ppt. Most the primary foods of larval fish. slow swimming, with good nutri- production facilities use 10 to 20 Two of the dominant zooplankton tional value. It is well suited to ppt salinity. Abrupt salinity groups are Rotifera (rotifers) and mass culture because it is prolific changes of more than 5 ppt can a sub-class of the Crustacea, and tolerates a wide variety of inhibit swimming or even cause Copepoda (copepods). These two environmental conditions. death, so acclimation should be groups are the preferred prey for Strain selection is important done slowly and carefully. shrimp and fish and are the live because reproduction rate, size Temperature, salinity and feed feeds used most often by cultur- and optimum culture conditions concentration all affect the growth ists. The intensive larval culture of (temperature and salinity) can all rate of rotifers, but temperature is most marine fish depends on a vary with different strains and the most critical factor. The opti- large supply of zooplankton. species. Some freshwater rotifer mum temperature for most strains Brachionus plicatilis (Fig.1a), is a variation can be seen in Figure 1b. is 28 to 32 oC (82.4 to 89.6 oF). small rotifer first developed as lar- Two of the best known strains of Above 28 oC, the salinity and size val fish food in Japan in the 1950s. brackishwater rotifers were of the strain are not very critical, Since then, many methods of cul- thought to be morphotypes of B. but the density of feed is very turing it have been developed. plicatilis, and were referred to as important. Below 26 to 28 oC More than 60 species of marine the “large” (L) and “small” (S) (78.8 to 82.4 oF), the bigger strains finfish are cultured using B. pli- types. Later it was found that tend to grow faster than the catilis as live food. This publica- these are two different species smaller ones. tion will concentrate on the cul- (L being B. plicatilis and S being Rotifers have broad nutritional ture and feeding of rotifers, but B. rotundiformis). Mean dry requirements that must be met to will include information on less weights are approximately 0.33 produce stable cultures. They are used zooplankton such as clado- microgram/rotifer for the L type planktonic filter feeders, feeding cerans (water fleas), copepods and and 0.22 microgram/rotifer for on organic particles brought to tintinnid ciliates. An important the S type. The size of the S type their mouths by the movements of larger zooplanktor used in aqua- is 126 to 172 micrometers accord- their coronas. The corona is a cili- culture is the Artemia (brine ing to one source, and 100 to 340 ated organ on the head region that shrimp), which is the subject of micrometers according to another. characterizes rotifers and is their SRAC publication 702. The L type is 183 to 233 microme- means of locomotion. Rotifers ters according to one source, and ingest many types of feed, includ- Rotifers 130 to 340 micrometers according ing bacteria, as long as the size of to another. Larval fish survive bet- B. plicatilis is the species used the particle is appropriate, so a ter with L-type rotifers, probably variety of food sources can be most commonly to feed larval fish because the larvae use less energy in hatcheries around the world. It used to rear rotifers. However, to feed on larger rotifers. rotifers cultured indoors often 1 Texas A&M University, Sea Grant College Rotifers may tolerate 1 to 97 ppt require vitamin B12 and vitamin A Program salinity, but optimum reproduc- supplements. 2Auburn University Saltwater Freshwater as “semi-continuous” or the com- bined “batch/semi-continuous a. b. technique.” Nutrient sources for culturing rotifers include baker’s yeast and emulsified oils; algae (Isochrysis galbana), yeast and emulsified oil; algae alone; bacteria alone; and outdoor culture using semi-pure or wild strains of algae. The high- est reproduction rate (21 offspring per female every week) has occurred when rotifers were fed a pure diet of Isochrysis galbana (Tahiti strain) and kept at a tem- perature of 20 to 21 oC (68 to Brachionus placatilis with eggs 69.8 oF). The optimum feeding Adult 100 to 300 microns (0.003 to 0.01 rate is 105 to 107 cells of the algae inches) in length Nannochloropsis oculata per indi- vidual rotifer, or 106 to 107 cells of baker’s yeast per individual rotifer. The normal concentration of rotifers is about 100 to 200 per ml, but often reaches more than 1,000 per ml with an adequate food supply. And if there is also a pure oxygen supply instead of aeration, the number will reach Keratella spp. more than 10,000 individuals per ml. Concentrated Chlorella sp. also c. can be used for rotifer culture. No one food source contains all the nutrients required for the long- term culture of a species. Several food sources should be used for Figure 1. Rotifers cultures that are to be maintained for long periods of time. The nutritional value of rotifers tinuous and feedback culture for larval fish depends on the techniques evolved. Each system Larval culture with rotifers rotifers’ food source. Researchers has advantages and disadvan- have determined that highly tages. Batch culture is the most Rotifers usually are fed to fish lar- unsaturated fatty acids (HUFAs) reliable but the least efficient. vae as soon as the larvae have are essential for the survival and Semi-continuous is less reliable developed mouthparts. For larval growth of marine finfish larvae. than batch but more efficient; red drum (Sciaenops ocellatus), this Rotifer feeds containing DHA, however, it allows wastes to build will be on day 3 post-hatch. 22:6n-3, docosahexaenoic acid, up, which causes contamination. Rotifers are fed at a rate of three and EPA, 20:5n-3, eicosapen- Continuous cultures are the most to five rotifers per ml until larval taenoic acid, can be valuable, efficient and consistent but are fish can consume larger foods at with DHA the more essential for maintained under strictly defined about day 11 post-hatch. Larval marine fish larvae. Depending conditions and are almost always mullet (Mugil cephalus) require a upon their food source, rotifers “closed” and indoors, which lim- food density of 10 rotifers per ml, are about 52 to 59 percent protein, its the size and increases the cost when there are 25 to 50 larvae per up to 13 percent fat, and 3.1 per- of the operation. The feedback liter, through day 40. Once rotifers cent n-3 HUFA. system, developed in Japan, uses are harvested from the culture system food is often limited, so There are many methods of cul- wastes from rotifer culture (treat- ed by bacteria and the nutrients the nutritional value of rotifers turing rotifers. Some are low-den- decreases over time. It is best to sity and some high-density. An retrieved) as fertilizer for algae cultured in a separate tank. The feed them to fish at least twice a early method involved daily day, or replenish them whenever transfers of rotifers to fresh tanks Japanese consider this method the most efficient and reliable. The rotifer density drops below a des- of the same size after most of the ignated number per ml. For exam- algae were consumed. Following culture technique described in this publication is usually referred to ple, in red drum larval culture, this, batch, semi-continuous, con- replenishment should occur when rotifer density drops below 3 per gravity feed to rotifer tanks, umes may be harvested rou- ml. Since one fish larva can eat as or the algae can be pumped tinely by dropping to the 50 many as 1,900 rotifers per day, to rotifer tanks. Gravity feed percent level. Even if the from 13,300 to 57,000 rotifers are is preferred; it helps control rotifers are not needed in the needed to feed one fish larva contamination of algae tanks hatchery, the volume in the through this period (depending with rotifers. Rotifer tanks tank should be reduced, and upon fish species and rotifer size). are usually the same size rotifers discarded. Most producers estimate three (1,800 liters) as the algae 5) Drain-harvest rotifers for 1 times the amount of rotifers actu- tanks. Rotifer tanks must month unless a problem ally eaten (1,900 X 3 = 5,700 also have drains and harvest occurs such as a “crash” or rotifers per day) are fed per larva. baskets or nitex screen socks die-off. If this occurs, drain, Therefore, as many as 39,900 (48- to 60-micrometer mesh) clean, disinfect and restart rotifers (for a 7-day period) to to capture the rotifers. Both the tank. Restart the cultures 171,000 rotifers (for a 30-day peri- rotifer and algae tanks in clean tanks monthly. od) may be required to feed one should have aeration and Starter cultures of rotifers fish larva. Feeding too few rotifers illumination. should be maintained at low often results in slow growth and 2) A few days after inocula- densities and in a separate too much size variation; feeding tions the Tetrasalmis cultures facility. Densities of rotifers too many rotifers can cause the will turn a darker green and at harvest will vary, but the fish to ingest so much that assimi- cell densities will be about ranges to expect using this lation becomes a problem. 132,000 cells per ml. Then technique are 100 to 150 For most marine finfish species gravity flow or pump algae per ml. being reared indoors, the weaning to the rotifer tank and The health of the rotifers will pri- of larvae from live rotifers and replace the algae volume marily be determined by the Artemia to dry food should begin with clean, sterilized seawa- availability of an adequate food well in advance of the transfor- ter and nutrients. In the supply. Hence, algae should be mation from larvae to juveniles. rotifer tank, place a stock supplied in slight excess. In gener- This transition might be timed to culture of rotifers in the al, the cultures should not be take 3 days or as long as 2 weeks, algae (at least 1 rotifer per cleared of algae in less than 24 but should be done gradually. ml). Note: The larger the hours (i.e., after replacing algae; Food particles should be the stock culture, the faster the the culture should have a rich largest that can be swallowed eas- desired numbers of rotifers color that will clear to a lighter ily by the fish (one-fourth to one- will be reached. color in not less than 24 hours). half of mouth width). Starter feeds 3) After several days the algae Rotifer numbers and health should contain 50 to 60 percent numbers should be obvious- should be checked daily. Using a high quality protein. As an exam- ly decreasing (water looks dissecting microscope, a sample of ple, in the past red drum larvae clearer) and the rotifer num- the rotifers should be observed for were generally fed rotifers from bers increasing. Start drain- swimming speed (fast is good, day 3 post-hatch to day 11, harvesting the rotifer tank slow is bad), gut fill (well packed Artemia nauplii from day 11 to 21, into the mesh sock or har- gut that is easy to see indicates and then weaned onto dry feeds. vest screen until approxi- good feeding; little or no food More recent protocols include the mately 30 to 50 percent of indicates poor food densities, an co-feeding of micro-particulate the culture tank is drained. undesirable species of algae, or larval diets starting at day 5. Replace the drained volume contamination), percentage of Although live zooplankton are with algae culture. Initially, rotifers with eggs (the more eggs still used, dependence on them as the collected rotifers can be the better the culture), and num- the sole nutrient source has been placed back into the culture ber of egg sacks carried (one indi- significantly reduced, and the container. However, once the cates an adequate culture, two or need to wean the animals from desired density is reached more a very healthy culture). live foods is eliminated. (about 100 to 150 per ml) Most problems with rotifer cul- about half of the rotifers will tures are caused by an inadequate Production examples have to be harvested each supply of algae because of poor Batch/semi-continuous culture day. algae culture techniques, under- sizing of algae production, or an of rotifers fed algae 4) Continue harvesting or dis- carding rotifers and refilling inability of the culturist to match 1) Culture Tetraselmis chuii in the rotifer culture tank with the rotifer populations with the 1.8-ton (1,800-liter or 475- new algae culture daily. algae supply. The latter is general- gallon) circular, fiberglass Volumes harvested from the ly a result of not discarding excess tanks. The elevated fiber- rotifer tank may vary rotifers. glass tanks should be according to demands of the equipped with drains that hatchery; or standard vol- Batch/semi-continuous culture 4) Once rotifer densities are 200 seem to work well in a wide range using mixed feeds per ml, drain-harvest by of densities, prepared feeds gener- draining 30 to 50 percent of ally work best in super-intensive Even though zooplankton are the tank volume daily and batch production systems (more generally considered good food capturing the rotifers in a 48- than 100 rotifers per ml), which sources, they can be deficient in to 60-micrometer mesh net. are harder to manage over long several essential nutrients, espe- Repeat until rotifer density periods of time. Because they are cially the n-3 highly unsaturated drops. This culture method not live products, they do not stay fatty acids (n-3 HUFAs) required should maintain rotifer den- suspended without considerable for good growth and development sities at 150 to 200/ml for aeration. It should be noted that of marine fish larvae. This is one about 30 days. using batch cultures and intensive of the primary disadvantages of Emulsified oil is a mixture of sea- feeding regimes, an initial starter rotifers, especially if they are culture of 100 rotifers per ml can grown on a food source that is not water, fish oil, and egg yolk at a ratio of 100 ml: 5 ml: 1g, with the reach densities of 1,300 per ml in 6 rich in HUFAs. Because a variety to 7 days. Although such densities of factors influence the nutritional addition of vitamin mix at 0.5% weight/volume of oil mixture. are desirable, the cultures are quality of the rotifer, most produc- much harder to manage and tion systems now use several food Vitamin E is also added at 0.1% weight/volume of oil mixture. require careful attention to water sources to enhance the nutritional quality and feeding regimes. content of live feeds. This mixture usually is a cod liver or menhaden oil, raw chicken egg Follow these steps to culture yolk, vitamin E (Tocopherol), and Copepods, Cladocerans, rotifers on algae, baker’s yeast a vitamin mix (AIN Vitamin and Tintinnid ciliates as and oil emulsion: Mixture 76®). The mixture is live feed 1) Follow the steps above for blended for 2 minutes in a blender growing rotifers with algae. and then stored in a refrigerator Copepods are common zooplank- Hold a rotifer starter/back- up to 1 week. The oil adds essen- ton both in freshwater and in up culture at lower densities tial fatty acids and vitamins not brackishwater. They are natural (100 per ml) in green water found in yeast. The eggs can be feeds for larvae and juveniles of and use it to initiate the purchased at a grocery store. The many finfish and crustaceans cultures as previously oil, vitamin E and vitamin mix- (Figs. 2a and 2b). In the wild, described. ture can be purchased from ICN most marine larvae feed on cope- pod eggs and nauplii during the 2) After algae is depleted for Nutritional Biochemicals, first few weeks of life. Because the first time in the rotifer Cleveland, Ohio. The menhaden some species of copepods have tank, stop feeding algae. oil is produced by Zapata-Haynie very small larvae (a necessity for Instead, add the following Corp., Reedville, Virginia. Dry some larval fish species) and can two products daily: baker’s baker’s yeast can be obtained have very high levels of HUFAs yeast at 0.5 g/10 liters and from wholesale grocery compa- and other essential nutrients, they oil emulsion (see makeup nies or most grocery stores. Some are an excellent food source for below) at 1 to 2 ml/10 liters. researchers and commercial pro- first-feeding larvae. In fact, a The remaining volume can ducers choose not to mix their number of marine larval fish can- be replaced with clean sea- own oils, but prefer to purchase not be reared using rotifers as the water or de-chlorinated tap commercial enrichment products. first feed but have been reared on water. Lowering the salinity There are also a variety of pre- either laboratory reared or wild to 16 to 18 ppt in the rotifer pared rotifer feeds that can be caught copepod nauplii. Research tank can be beneficial and used as a replacement for the with several species, such as the may improve growth after yeast. turbot and red snapper, has the rotifers are no longer Commercial enrichments and shown that when offered mixed being fed algae. Discard rotifer feeds are available from plankton diets, young larvae con- water when the rotifers are companies such as Aquafauna sume more copepod nauplii than harvested so they can adjust Biomarine Inc. ( California), rotifers and prefer copepod nau- rapidly to the higher salinity Sander’s Brine shrimp Co. (Utah) plii because of the differences in in the fish larvae environ- and Inve Aquaculture, Inc. (Utah). size and swimming patterns of ment. Algae pastes or concentrates are the two prey types. Consequently, also available (Reed Mariculture 3) Once rotifer population den- there is considerable interest in Inc., California). The algae is sity reaches 100 per ml, the use of copepods as feed grown under controlled condi- increase this daily yeast and sources for small marine larval tions, concentrated using a cream- oil emulsion level to 0.7 to fish. 1.0 g yeast per million separator, then preserved and Copepods are cylindrical with a rotifers and 2 to 3 ml oil packaged. These products can be trunk comprised of 10 segments, emulsion per million rotifers. refrigerated for 1 month or frozen for more than a year. Although consisting of head, thorax and the concentrated algae products abdomen. Adult copepods range from 0.5 to 5.0 mm. The larval (Tisbe and Tigriopus spp.), and which gives them an advantage stages consist of six naupliar and cyclopoids (see Fig. 2a for shape over the rotifers. Copepods can six copepodite stages. The main differences). also eat detritus. They differ from suborders of copepods found in Herbivorous copepods are pri- Artemia (brine shrimp) and brackishwater are calanoids marily filter feeders and typically rotifers in that they do not repro- (Acartia, Calanus and feed on very small particles. But duce asexually. Copepods mate Pseudocalanus spp.), harpacticoids they can feed on larger particles, after maturing and the female

Copepods – saltwater Cladocerans (water fleas) – freshwater

a. c.

HARPACTICOID

Egged female

Daphnia

CALANOID Daphnia CYCLOPOID

Copepods – freshwater b.

Macrothrix

Cyclops Diaptomus Figure 2. Copepods and Cladocerans produces 250 to 750 fertilized Tigriopus and Acartia for rearing viduals per ml could be main- eggs (rotifers produce 15 to 25 per fish larvae approximately 7mm in tained on Tetraselmis chui after female). The copepod lifespan is length. U.S. researchers com- maximum density was attained 40 to 50 days (5 to 12 days for pared the growth and biochemical (for general culture). In Thailand, rotifers), and it has a longer gen- composition of mahi-mahi culturists are growing eration time (1 to 3 days for the (Coryphaena hippurus) larvae that Diaphanosoma on Chlorella sp. In rotifer and 7 to 12 days for the were fed brine shrimp, rotifers 1998, researchers at SEAFDEC in copepod). and the copepod Euterpina acu- the Philippines successfully used Unlike the rotifer, copepods are tifrons, cultured in 700-liter tanks. Diaphanosoma as an Artemia sub- more difficult to culture on a Larvae fed copepods survived stitute for Barramundi larvae commercial basis. Only a few better under stressful conditions. (Lates calcarifer). species of copepods, such as A system for the mass culture of a Other cladocerans considered Tigriopus japonicus, have been benthic marine harpacticoid cope- promising species are Evandne mass cultured successfully. Even pod, described by Sun and tergestina, Penilia avirostris and this technique employs the com- Fleeger (1995), should be useful Podon polyphemoides. The cladocer- bination of rotifer culture and the for aquaculture. an Moina macrocopa has been used use of baker’s yeast or omega-3 Other copepods considered to be in Southeast Asia as feed for sea yeast as feed. Unfortunately, the promising species for mass cul- bass fry immediately after wean- amount of yeast used to produce ture are Acartia clausi, A. lon- ing from Artemia and prior to the copepod and rotifer combina- giremis, Eurytemora pacifica, feeding minced fish flesh. During tion outdoors is fairly high. There Euterpina acutifrons, Oithona brevi- this period, sea bass, being a are outdoor production systems cornis, O. similis, Pseudodiaptomus catadromous species (moving into that can produce large numbers inopinus, P. marinus, Microsetella freshwater for a portion of its life of copepods; however, these sys- norvegica and Sinocalanus tenellus. cycle), may be reared at lower salinities and fed freshwater zoo- tems are very inefficient in terms Cladocerans or water fleas (Fig. plankton. This practice is not of number of copepods per liter 2c), such as Daphnia magna, have commonly used or proven to be of culture water. Considerable been cultured as live food using viable on a commercial scale. A work needs to be done on culture techniques similar to those related cladocera, Moina salina, and harvest techniques before described for rotifers. Many labo- has been used in finfish culture in copepods become as widely used ratories use Daphnia as the inver- Spain. as rotifers. tebrate of choice to conduct toxici- One interesting advantage of ty tests because it is easy to cul- Tintinnid ciliates are consumed by copepods is that under appropri- ture and maintain in the laborato- larval fish and crustaceans in the ate conditions some species will ry. Cladocerans are mainly fresh- wild and are considered promis- produce a resting egg similar to water zooplankters; most do not ing candidates for mass produc- that of Artemia. So once commer- tolerate salinities higher than 3 tion. However, since the technolo- cial techniques are developed, ppt., and are generally not found gy for mass production of rotifers copepod eggs could be collected in brackishwater. One exception is is well established and micropar- in large numbers and stored for Diaphanosoma celebensis ticulated diets are being co-fed months, like Artemia (brine (=aspinosum). In Asia there is a with rotifers or have been devel- shrimp) and rotifer cysts. growing use of this species. This oped to partially substitute for Photoperiod and temperature is a saline-tolerant (1- to 42-ppt) live food, the role of copepods, largely determine the production water flea in the 400- to 800- cladocerans and tintinnid ciliates of copepod resting eggs. Labora- micrometer range that has been is not as important. tory production of these eggs is successfully cultured in backyard possible, but has not yet proved hatcheries. Biomasses of up to 1 Overview to be economically feasible. It is kg in 1 cubic meter of water every hoped that using copepods as a 3 days have been reached. To be Finfish producers are concerned food source can improve the cul- effective as a replacement, the with improving the quality, quan- ture of a variety of species, such organism must be enriched before tity and cost effectiveness of their as the red drum, by reducing the it is fed. This enrichment is live feed production facilities. size variability and mortality. accomplished with a source of Many of them now supplement cultures with omega yeast, vita- The use of copepods, especially DHA, but usually not one with an oil emulsion base because of mins (E, D, C and B12), marine the harpacticoids (Fig. 2a), is well oils or other HUFA sources, and documented in marine fish cul- gill and water fouling problems. vitamin B12-producing bacteria to ture. Researchers have reared Schizochytrium (a spray-dried or drum-dried algae developed by improve feed quality. Today, live copepods in vessels of 100 liters feeds for fish larvae are being (26 gallons) and 450 liters (118 Omega-Tec, Inc.), is the most common enrichment agent used improved by adjusting their bio- gallons) and reported that the chemistry through controlling system provides 250,000 nauplii in Thailand for Diaphanosoma. Researchers have found that their diet and supplementing the per day. The Japanese have rou- cultures with microencapsulated tinely cultured the copepods mean densities of 72 to 100 indi- feeds or emulsified oils. While Japan. In the past it was thought Suggested reading algae and rotifers are the most that fish larvae have low concen- widely used live food items, their trations of digestive enzymes until Davis, C. 1955. The Marine and use is not without problems and they reach approximately 6 mm; it Freshwater Plankton. Michigan limitations. Rotifer and copepod was also thought that they are State University Press, East cultures are subject to collapse or unable to digest inert feeds. Lansing, Michigan. “crash.” Producers are finding Evidence is accumulating to sup- Hyman, L.H. 1951. The new species of live food organ- port the idea that properly formu- Invertebrates: Acanthocephala, isms better suited for specific cul- lated diets are digested and pro- Aschelminthes and Ectoprocts, ture situations. Larvae of some vide a controlled way of deliver- Vol. III. McGraw-Hill, New York, species (e.g., angelfish, butterfly- ing nutrients to larvae. For many New York. fish, damselfish, parrotfish) have commercial species, the co-feeding Fulks, W. and Main, K.L. 1991. small mouths and might require of live and artificial feeds during Rotifer and Microalgae Culture prey smaller than rotifers. the larval stages is recommended. Systems. The Oceanic Institute, Dinoflagellates such as Microencapsulated diets do have Honolulu, Hawaii. Gymnodinium sp., ciliates such as one very positive attribute—they Euplotes sp., and the nauplii of are an alternative way to adminis- Sun, B. and Fleeger, J.W. 1995. many copepods are in the size ter vaccines and therapeutic Sustained mass culture of range suitable for those fish lar- agents to larvae. Even though the Amphiascoides atopus, a marine vae. Larvae of oysters, barnacles large-scale, intensive production harpacticoid copepod, in a recir- and sea urchins have also been of microalgae and rotifers is culating system. Aquaculture 136: used, but are not as reliable in expensive and often unreliable, 313-321. quantity and quality. Advances the production of live food organ- have also been made in the area of isms continues to be a very impor- formulated feeds, especially in tant first step in aquaculture. The work reported in this publication was supported in part by the Southern Regional Aquaculture Center through Grant No. 97-38500-4124 from the United States Department of Agriculture, Cooperative States Research, Education, and Extension Service.