Rotifer, Brachionus Plicatilis, in Texas
Rotiferand Microalgae Culture Systems, Proceedings of a U.S. Asia Workshop.Honolulu, Hl, i99i. QcThe Oceanic Institute
Various Methods for the Culture of the Rotifer, Brachionus plicatilis, in Texas
C.R. Arnold and G.J. Holt The University of Texas at Austin Marine Science institute P.O. Box 1267 Port Aransas, Texas 78373 U.S.A.
ABSTRACT
Various metEodsfor culturingrotifers in bothindoor culture tanks and outdoor culture systems are discussed.The use of algaein combinationwith yeast and emulsified oil is discussed,and advantages of the various culture methods are also included.
1NTRODUCTIO N but the most common are single~lied algae suchas Tefraselmis,Otlorella, and1sochrysis. The rotifer, Brachionusplicatilis, is an Others includebaker's and torula yeast,emul- importantfood organismfor the first feeding sified oil enrichment, and artificial diets. stagesof larval marineanimals around the There are advantagesand disadvantagesto world. It is a cosmopolitaneuryhaline species, eachtype of culture. An algaediet helps thus it is very versatilein marine culture. preservegood water quality in the rotifer Pertinent literature citations are covered in culturesystem, but at theadded expense of Wohlschlaget al, 990!. Therotifer varies in maintainingalgae cultures. Yeast is a simple size dependingon strain and culture condi- andinexpensive source of food,but the nutri- tions, with adult sizeranging from 123to 315 tiona1quality of yeast-fedrotifers is deficient pm in length.This allows strains to becultured for manymarine organisms. To eliminatethis for a specific size Yufera 1982,Snell and problem,enrichment of yeast-fedrotifers has Carrillo 1984!. becomepopular Watanabeet al, 1983a!. Rotifers can be grown in seawaterin a wide rangeof salinities.Our studiesindicate 18ppt is the optimumsalinity for thestrain we METHODS use. The itiitial culture was obtained from the National Marine Fisheries Service Laborator- Stock cultures for inoculant or starter ies in La Jolla., California. Feed types vary, cultures are maintainedin 1 - 2-liter flasks. i 20 Arnold and Holt
Theseshould be keptin an areaseparate from 40/mlafter being rinsed three times through a the massculture tanks if possible,to prevent 60-pmfiltering cloth, to rid theculture of most contamination. Stock cultures can be main- contaminants.Beginning on Day 2, yeastis tainedon an algaldiet of Isochrysisgalbana fed at 1.5 g/10 rotifers and emulsified oil at at 24 - 25'C anda lightcycle of 12light:12 3 ml/106 rotifers daily until rotifer densit~ dark. Cultures should be restartedperi- reaches50/ml. Thenadd 1- 1.3g ofyeast/10 odically, at least every month or more, rotifers and 2 - 3 ml of emulsified oil/106 dependingon environmentalfactors. rotifers daily until a density of 100/ml is attained.Then feedyeast at 0.6 - 1.0 g/10 6 Culture Methods rotifers andemu1sified oil at 2 ml/10 rotifers until the density reaches150 - 200/ml, at The following methods havebeen used whichtime harvesting may begin Wohlschlag by the University of Texas Mariculture Pro- et al, 1990! Fig. I!. gram over the past ten years with varying To harvest, drain 20 - 25% of the tank degrees of success. Outdoor culture is limited volume through a 48-pmfiltering cloth to to the warmermonths of the year. Rotifers have been cultured in 1.8-m dia., round,flat-bottomed tanks that hold up to 1,800 liters of water; 140-liter conical tanks;160-liter clear, cylindrical fiberglass reinforced polymer sheet! tanks; 50-liter polyethylenebags; and 1.8-mdia., 3,200-liter round outdoor tanks. Tanks are sterilized beforeuse by additionof 2.5ppm bleach for 12- 24hours, rinsed and cleaned. They are then filled with filtered seawater and the salinity is adjusted with dechlorinated fresh water. Temperature is maintained at 24- 26 C exceptin theoutside tanks. All except the 140-liter conical and outside tanks have continuouslight supplied from light banks with 40-watt florescentbulbs aboveor beside thetanks. The conical tanks have no light other than room ceilinglights, andonly sunlightis used for the outside tanks.
Method casingbaker's yeast and ernuI- si fied oil Set up 1,800-liter round tank or conical tanksas describedabove. On Day 1, yeastis fed at 0.6 - 0.8 g/liter, emulsified oil at 1,0 Figure 1. Diagramof rotifer cultureusing ml/10 liters and rotifers are inoculated at 10- baker's yeast and emulsified fish oil. Rotifer Culture in Texas 121
collect rotifers. Refill tanks with filtered sea- water adjustedto a salinityof 18 ppt. Add yeastat0.6-0.8 f 10 rotifers aud emulsified oil at 2 - 3 mV10 rotifers. Repeatdaily until the rotifer population declines.
Method vsing a gae Isochrysis gai- bana!, yeast and emvisified oil Set up one 1,800-litertank as described above and inoculate with a 12-liter carboy of Jsochrysisgalbana 32,000 cells/ml! and medium .2 ml/liter of F/2 medium!. Methods for the carboy culture of 1. galbana and F/2 medium formulation are discussedby Treeceand Wohlschlag 990!. On the second andthird daysadd 0.1 mVliterof F/2 medium, and when the algaedensity reaches 132,000 cells/ml, inoculatewith l - 10 rotifers/ml. When the concentration of algae decreases, beginadding yeast at 50g/tank and emulsified oilat 1 -2 ml/10liters eachday. Whenrotifer densityreaches 100/ml or more,increase the daily ration of yeastand emulsified oil to 0.7 - 1.0 g/106 rotifers and2-3 ml/106 rotifers, respectively. Harvestingrotifers can begin whenthe densityreaches 200 rotifers/ml see Fig. 2!. Drain 15 - 25% of the tank/day. Repeatuntil the rotifer densitydrops. This culture method should maintain rotifer den- sities at 150 - 200/ml for about 30 days Wohlschlag et al. 1990!.
Methods using a/gae as the so/e nvtrient source When using algae, two methodscan be used. The algae can either be cultured sep- aratelyfrom the rotifers or both may be grown in the same container.
Aigae and rotiiers cuitUred separately Algae Isochrysisgalbana or Tetraselrnis Figure 2. Diagram of rotifer culture metho using algae, yeast and emulsifiedfish oil. chai
"fish emulsion", an organic fertilizer available to restart an increasein the daily count. Har- in liquid form Treeceand WohlscMag 1990!. vest as neededon a daily basis,but theculture Rotifers are cultured in separate1,800-liter mustbe harvestedas above if thedaily count tanks. All tanks receive continuous illumina- is thesame for two consecutivedays. tion from overheadlight banksand are we11 aerated.The culture room is keptat 24 + 2'C. Algae and rotifers cuitvred together Algae tanks are filled with seawater Fifty-liter polyethylenebags are used as which hasbeen filtered througha 1-pmfilter. culturecontainers. They are clampedand at- The salinity dependson the speciesof algae tachedto a support frame. IUumination is and the requirementof therotifer strain,but suppliedby 40-watt fluorescentlamps wall generally is in the range of 16 - 30 ppt. The light banks!. Aeration is supplementedwith tankis theninoculated with 100liters of algae C02 approximately50 standardcubic feet per stock 30,000 cells/ml!. If this muchalgae is hour!, injected every hour for 20 secondsto not available, lower the volume of seawaterin promotealgae growth, A valve is attachedto tank. It shouldtake three to fourdays to reach the top for aeration and seawateraddition. an algal density of 132,000 cells/ml. If a Bags are filled with filtered seawater, smaller volume is used, double the volume nutrientmedium is added F/2! andalgae is with filtered seawaterand fertilizer daily until inoculatedat 500ceHs/ml. Algae is grownfor a volume of 1,800 liters is reached. This threedays, and then rotifers are inoculatedat culturecan now be usedto feedrotifers by 10/ml. It shouldtake approximately four days draining50 - 60%of thetank volume daily to reachmaximum rotifer density. Densities andrefilling with filteredseawater and adding as high as 400 rotifers/ml have beenachieved fertilizer. This shouldbe donedaily evenif with this method.Rotifers are harvested by algae is not needed. drainingthe entire bag batchculture! and bags The rotifer culture tank is filIed with 900 are discarded after use Trotta 1981, Trotta litersof filteredseawater and 900 liters of algal 1983!. culture water. Rotifers are added, at least
1/ml, moreif available.When algae has been Outside culture consumed and the culture water becomes The culture of rotifers outdoors occurs clear, rotifers maybe harvested.This is ac- betweenMarch and November in south Texas. complishedby draining30 50% of the tank The tanks are covered with a 60% shade cloth volume. The densityshould be 100- 150/ml. during the hottest months of the summer to To maintain this rotifer count, refill the tank inaintain the temperature below 30'C. The with algalculture water and repeat daily until 3,200-liter tanks are filled with unfiltered rotifer population declines Arnold et al. seawaterand provided with aeration; the tank 1976!. is allowedto sit for two to threedays until the A modification of this method is to start algae bloom. It is then inoculated with rotifers with 300 - 400 liters of seawater and add 100 at 10/ml. Sixty gramsof torulayeast is added liters of algae. Inoculate with rotifers at 12 daily,beginning on the third day until harvest 15/ml. Add 100liters of algaedaily until the begins.There is daily monitoringof therotifer rotifer count is constantfor two days. It is population,and once the concentrationreaches necessary to harvest at least 40% of the tank 40/rnl,harvest begins. At thattime, 60 g of Rotifer Culture in Texas 123
yeastis addedtwice daily. Harvest up to 40% failures. However,there are certainproce- of thetank volume daily and refill thetank with dureswhich appear to helpmaintain a semi- seawater. The tank bottoms need to be continuousculture for an indefiniteperiod. vacuumedweekly to preventthe buildup of The more important onesare: sludgewhich can become anaerobic and cause ~ keepculture containers and water clean, the culture to crash. a control contaminantssuch as ciliates and bacteria, FeedingRotifers to LarvalFish a harvestdaily to maintainthe culture in growthphase this canbe ac- Larval red drum begin feeding approx- complishedby dailycounts and noting imatelythree days after hatching, when their thenumber of eggcarrying females, mouthparts develop earlier in hightempera- e andadd some algae daily even a small tures,later in low temperatures!.Rotifers are amount seems to help. fed at this time at a rateof 3 - 5 rotifers/ml untilLarger feed can be consumed Holt et al. 1981!. REFERENCES Due to the Lossin nutritional valuea few Arnold,C.R., J.L. LassweU,W.H. BaUey, T.D. Wil- hours after harvest,it is bestto feedrotifers liams andW.A. Fable.1976. Methods and techni- to fish at leasttwice a dayor wheneverrotifer quesfor spawning and rearing spotted seatrout in densitydrops below 3/ml Gatesoupeand thelaboratory. 30th Annual Conference of the Robin1981!. Anothermethod of keepingor SoutheasternAssociation of Gameand Fish Com- missioners. 13: 167-178. increasingthe nutritionaL value of therotifers Craig,S.R., G.J. Holt and C.R. Arnold. Submitted. is by enrichingthem. There are many methods The effectsof enriching live foodswith highly unsaturatedhtty acidson the growth and fatty acid of doing this. We haveevaluated algae, compositionof larval red drum Sciaenops ocel- baker'syeast and fish oii emulsionenrich- larus!Linnaeus. J. World Aquaculture Society. ment. The best growth was with algaeor Gatesoupe,F.J, and J.H. Robin. 1981. The dietary valuefor seabass larvae Dicenrrarchuslabrax! of baker'syeast plus an emulsion of menhaden therotifer Brachionus plicarilis fed with or without fish oil. The lowestgrowth ratesoccurred a laboratoryculturedalga. Aquaculture. 27:121- when larvae were fed rotifers which hadbeen 127. Holt,G.J., R. Godbout and C.R. Arnold. 1981. Effects culturedonly on baker's yeast Holt, in press!. of temperatureand salinity on egg hatching and Rotifers grown solelyon yeastare of low larvalsurvival of reddrum, Sciaenops oceIIara. nutritionalvalue because they have low levels Fish. Bull. 79 !:569-573. Holt,G J.In Press. Intensive culture of larval red drum: of ta3 HUFAs, comparedto algae-enriched Experitnentalstudies, J. WorldAquaculture rotifers Watanabeet al. 1983b,Craig et al., Society. submitted!. SneU,T.W. and K. CarriUo.1984. Body size variations amongstrains of therotifer Brachionaz plicariBs. Aquaculture.37:359-367. Treece,G.D. and N.S, Wohlschlag. 1990. Raising food CONCLUSIONS organismsfor intensivelarval culture: I. Algae. In: RedDrum Manual. TexasA&M SeaGrant CollegeProgram No. TAMU-SG~3, pp.57- Thereare many ways to culturerotifers, 64. but there does not seem to be a sure way to massproduce them without periodic culture 124 Arnold and Holt
Trotta, P. 1981.A simpleand inexpensive system for dietaryvalue of live foodsfor fish larvaeby feeding continuous monogenic mass culture of marine them on 3 highly unsaturatedfatty acidsand fat- micro algae. Aquaculture. 22:383-387. soluble vitamins. Bull. Jap. Soc. Sci. Fish. Trotta,P. 1983.An indoorsolution for massproduction 49 !; 471479. of the marineroti fer Brachionuspliconlis Muller Wohlschlag,N.S., L. Maotangand C.R. Arnold. 1990. fed oa the marinemicroalga Terraselniis suecica Raisingfood organismsfor intensivelarval culture: Butcher. AquacuituialEngineering. 2:93-100. II. Rotifers.In: Red Drum Aquaculture.Texas Watanabe,T., C. Kitajimaand S. Fujita.1983a. Nutri- ARM SeaGrant CollegeProgram No. TAMU-SG- tional valuesof live organismsused in Japanfor 90-603, p, 66-70. masspropagation of fish: A review. Aquaculture. Yufera, M. 1982. Morphometric charscterizabonof a 34: 115-143. small-sizedstrain of Brachionusplicarilis in cul- Watanabe, T T. Tamiya, A. Oka, M. Hirata, C. ture. Aquaculture. 27:5541. Kitajima and S. Fujita. 1983b.Improvement of Rotiferand Microalgae Culture Systems. Proceedings of a U.S. - Asia Workshop.Honolulu, HI, 199I. !The Oceanic Institute
Environmental Management for Mass Culture of the Rotifer, Brachionus plicatilis
IVlasachika Maeda National Research Institute of Aquaculture Nansei, Mie 516-01 JAPAN
and
Akinori Hino Faculty of Agriculture University of Tokyo Bunkyo, Tokyo 113 JAPAN
ABSTRACT
Bacteriaand protozoa are important biotic factors affecting the growth of culturedrotifers. The bacterial andprotozoan flora of rotifer cultures and their effect on rotifer growth were investigated. Thebacterial flora changedvery rapidly, but an equilibrium was struck between those microbes which were beneflcial torotifers and thosewhich were deleteriousto iotifer growth. Theciliated protozoan flora was composed mainly of Uronenia sp. and Zupiores sp, When Zuplotes sp. wasdominant, Uronenia sp. disappeared dueto conipetitian forbacterial food. Furthermore, thebacteria which coexistedwith Euplotes sp, strongly inhibited rotifer growth. We also found that ciliates could be removed effectivelywith a coarsevinyl filter or additionof thepredator Arreniia salina. A briefsummary of importantabiotic factors affecting cultured rotifers is also given.
INTRODUCTION biotic factor, tendto be neglectedbecause of the difficulty in handling them. Also, in The culture environmentis composedof aquaculture,quite a fewpeople are only inter- two types of factors, abiotic and biotic. estedin high productionof fishes,despite the Abiotic factors, temperature, pH, dissolved significant role of live feedsin aquaculture oxygen, ammonium ion concentration,chemi- production. cal oxygen demand, etc. are generally regu- Even thoughthe cultivation of rotifers lated by exchangingwater, aerationand other seemsto work well, the nutritionalquality of meanS.On the Otherhand, IniCrOOrganiSmS,a rotifersused as feedvaries depending on their 126 Maeda and Hino
diet. This seriously affects the survival and IVIICROBIAL AND PROTOZOAN growth of fishes. Also, rotifers transmit SUCCESSION IN ROTIFER REARING pathogenic bacteria, mainly Vibrio spp., to WATER fish culture water Suzukiet al. 1990!. In fact, people have been realizing that microbes in When rotifers are cultured in an open rotifer rearing waterafTect not ordy the growth system,quite a few bacterialspecies are usual- of rotifers, but also the physiological and ly present.Some of them promote the growth hygienic conditions of cultured fishes. A of rotifers but others, if presentin high fre- rotifer culture program should be established quencies, might repress growth. Figure 1 with these points in mind. In this paper the showsthe successionof bacterial flora during meansby which bacteriaand protozoa affect the cultivation of rotifers in a 500-liter con- rotifer growth and possiblemethods to protect tainer. Rotifers and Nannochloropsisoeukua rotifers from the deleterious activities of were inoculated at 200 ind./ml and 5 x 10 microorganismsare described. Also, a brief cells/ml, respectively,and baker's yeast was summaryof importantabiotic factorsaffecting added regularly and maintained at 2 x 10 rotifer culture water is given. celIs/ml. The number of rotifers increased to 1,000ind./ml after three da~s, whereas bac- terial numbersclimbed to 10 colony forming units/ml Figs, 1 and2!. At the beginning of the experiment, Acinetobacter sp. dominated the bacterial population and Flavobacteriurnand Pseudo- monas spp. appeared together. In time, how- ever, the compositionof flora changed. The proportionof Pseudomonosincreased, becom- ing dominantafter nine hours Fig, 1!. Thus, the bacterial flora changedvery quickly. In Figure 2, ratios of Pseudomonasspecies are shown,from 24.1 to 93.0%, Among Psetdo- mortas groups, the strains NT-9, NT-10 and NT-11dominated. After ninehours, only two strains, NT-10 and NT-11, could be isolated. NT-10 inhibited the growth of rotifers, but NT-11 promotedtheir growth Table 1!. Although these data are not shown here, NT-10 supported the growth of the ciliated protozoaEuplotes sp, This mayexplain why large numbersof Euplotessp. suddenlyap- pearedin the aforementionedexperiment, reaching5,000 cells/liter after 72 hours Fig. Figure 1. Fiuctuati onsin the numbers and com- 2!, Thesedata suggest that the production of position of bacterial flora during the culture of rotifers. rotifers is assuredif an equilibriumis estab- Environmental Management of Rotifer Cultures 127
Figure2. Ratiosof Pseudomonasstrains among the Pseudomonas protozoaand rotjfers present in theexperiment shown in Figure1.
lished between the beneficial and deleterious nemaappeared on andaround dying rotifers, bacteria in rearing water. trying to invadethe bodies of weakenedin- In Figure l it canbe seenthat Flavobac- dividuals.In fact,approximately ten Uronema teriumsp. appearedonly on the first day. packedinside each dead rotifer. After multi- Table 2 shows how four generaof marine plyinginside these corpses, they exited and bacteriaaffected the growthaf rotifers. Al- eventuallybecame dominant over the attached though most, including Pseudomonas,Aci- forms Fig. 5!. Immediatelyafter the Euplotes netobacter,and Vibrio spp. seemedto support sp.population started increasing, the Uronema growth,Flavobacterium shows an inhibitory populationrapidly decreased, falling to zero effect. ManyFlavobacterium were isolated whenZuploses numbered about 100 cells/ml from Nannochloropsisrearing water Table Fig, 5!. This is becauseUronerna sp. was 1!. If rotifers do not grow well using Nan- outcompetedfor food mainlybacteria! Table nochloropsissp. as feed,it maybe dueto the 3!. Finally,although neither ciliate feeds on presenceof Flavobacterium. Nannochloropsis,they do competewith It is known that animals or plants can rotifers for yeast. promotethe growth of certainbeneficial bac- teria. This seems to be occurring between CHARACTERISTIC FEATURES OF Pseudomonas and rotifers. That is, bacteria which supportthe growthof rotiferseventual- BACTERIAL FLORA IN PROTOZOA ly dominatethe bacterialpopulation in culture AND ROTIFER CULTURE water. In rotifer rearing water, mainly two Table 4 shows the relative abundanceof speciesof ciliates were observed,Uronema bacterial strains isolated from rotifer and and Euplotes Figs. 3 and4!. At first, Uro- protozoan Euplotes sp.! cultures in whichan 128 Maeda and Hino
Table 1. Growthof rotiferscultured in the presenceof variousbacteria collected from Nannochloropsis and rotifer rearing water.
Fla.: Fkivobaderium,Aci.: hcirierobooer- 'orasc'iiagroup,Pse.: Pswdomonas-Al- Ieromonar-rficafigcrirs group, Vib.: Vibrio Tenrotifers were added to 10ml of seawater containing 200iig baker's yeast/ml with a bacterialsuain l0 9 cells/ntl!,and cultured for seven days in the dark at 25'C. Bacteria werc not addedin the controlexperiment.
equilibriumbetween B. plicatilisand Euplofes dominantin CultureI, effectivelysupported sp. wasnot established.In Culture I, densities thegrowth of rotifers Table5!. On theother of rotifers andZuplotes sp. were 240ind./ml hand, the dominant strains E-1 and E-2 in and 20 cells/ml, respectively.In CultureII CultureII stronglyinhibited the growth of they were zero and3 x 10 cells/ml, respec- rotifers Table5!. Sincesupernatant seawater tively. Euplofesoutcompeted the rotifers in fromboth cultures did notaffect the growth of Culture II. The strain R-l, which was rotifers, the deleterious or bene6cialeffects Environmental Management of Rotifer Cultures 129
Table 2. Average growth values of rotifers in the presence of four bacterial genera.
Figure 3. The ciliated protozoa Urortertta sp, 8arindicates f0 pm!. Experimentalconditions shown in Tablei.
were attributed to the bacterial strains them- selves. If, in situ, Euplotesnumbers increase in rotifer rearing water, theseinhibitory bac- terial strainsmight appearand repress rotifer growth
REMOVAL OF PROTOZOA FROM ROTlFER CULTURES Figure 4. The ciliated protozoa Euplotes sp. barindicates 10 pml. Free-swimming protozoa are divided into two groups, planktonic and psam- mophilic. Although the psammophilicproto- havior might aid in the removal of these zoa, which include Euptotesand Uronema, protozoa. can swim, they tend to crawl or slide alongthe surfaces of substrata. This characteristic be-
Table 3. Live feeds of the ciliatedprotozoa, Uronema sp. and Euplotessp., in rotifer cul- ture water,
+: utilized as feeds. not utiiized. : somehtrgcr Zuplotes sp. feed on Uroncmasp. 130 Maeda and Hino
Table4, Proportionsof the differentbacterial strains isolated from the rotiferMominant cul- ture Culturel! andEuplotes sp.-dominant culture Culture II!.
A 10-liter tank containingrotifers and shownin Figure 6, with increasingretention 3 Euplotes sp. was supplied with a 100-cm time, thenumber of protozoadecreased from container filled with thick, coarse filter 500 cells/ml to less than 100 ceHs/ml. Al- material pore sizeabout 5 mm!. Rotifercul- though protozoannumbers decreased, the ture water containing Zuplotes sp. was air maximumrotifer densitywas 100 ind./ml, the lifted, poured into the filter, andreturned to disadvantageof this method. therotifer tankby anoverflowing process. In We found that Artemia salina feeds on thiscirculating system, the density of rotifers Euplotesand Uronema. This fact should be was maintained at about 100 ind./ml. As
Table 5 . Growth of rotifers in the presence of bacterial strains in Table 4.
Figure 5. Fluctuationsin the numbers of Uronemasp. and Euplotessp. in rotifer culture water. J.Attached form of Uronemasp. i:Ranktonicform of Uronemasp. J;Eup otes sp. :Experiment l. Expcrirncntal conditions shown in Table 1. -:Experiment ll. Environmental Management of Rotifer CvltureS 13l
FIgure 6. Decreasein the number of Euplotes sp. resulting from the use of a coarsevinyl fil- ter. employedfor theefficient removal of protozoa in water. The size of Artemia used in this work rangedfrom 1.2 - 7.0 mm in length Fig. 7! Figure 7. Various growth stages and sizes of and the concentration of Artemia was 1 Artemia salina from Heath 1924!. ind./ml; those of Zuplotes and Uronemaap- proximately10 ceUslml.As shownin Figures 8 and 9, Uronemasp. and Euplotes sp. were Nannochloropsisoctdata is eaten by consumed very quickly. Feeding rates for Paraphysomonassp., a flagellatedprotozoan large Artemia were higher than those of whichcan reduce a poplation ofN. oculata smaller sizes. When rotifers, Anemia and from 10 cells/ml to 10 cells/ml within a day. Zuplotessp. werecultured together, the num- Paraphysomonassp. grows well at low salt ber of Zuplotesdropped quickly, but rotifers concentrations,i.e. less than 30 ppt salt con- wereable to grow in this mixedculture Fig, centration. Yo prevent rapid growth of this 10!. protozoan,salinity shouldbe checkedcareful- ly and maintainedhigher than 30 ppt at all
Figure 8. Decreasein the number of Uronema Figure 9. Decrease in the number of Euplotes sp. when cultured with Artemia salina. sp. when cultured with Artemia salina. 132 Maeda and Hino
times.This flagellatecould also be reduced by normally rearedat 18ppt chlorinity normal meansof physicalstimulation, such as adding seawater!, too high for rotifers grown at 6- a waterfallto N. octdatarearing tanks. Details 10ppt. For thisreason, rotifers are generally are reported in M. Kanematsuet al. 989!. reared in normal seawater. Rotifers are very tolerant of dissolved oxygen DO! deficiencies. Under anoxic con- ABIOTIC FACTORS FOR ditions, 50% of a testpopulation of rotifers ENVIRONIVIENTAl IVIANAGENIENT survived for six hours. AH died after 12 hours. Oxygenconsumption rates for rotifers Abiotic factors,including water tempera- areapproximately 4 7 x 10 ml DO/ind./day ture,dissolved oxygen, pH, NH4+ concentra- at 20 - 25'C. Since large populationsof tions and chemicaloxygen demand COD!, bacteriaand protozoa consume more oxygen havebeen summarized thoroughly by Oka thanrotifers, aeration is necessaryfor rotifer 989! andSugimoto 989!. cultivation. Growth ratesof L- and S-typerotifers Rotifers grow within a pH range of 5- vary at differenttemperatures. S-type grow 10, but stablegrowth andhigh feedingrates fasterthan L-type rotifers above 25 C. On the on Namochloropsisor baker's yeastcan be other hand,below 25'C, thegrowth rates of obtainedat pH 7 - 8. L-type rotifers are higher. In fact, optimum According to Yu and Hirayama 986!, temperaturesfor the cultures of L- and S-type an acute toxicity test of NH>-N to rotifers rotifers are approximately25 and 30 C, showedthat the 24-hourLCSO was 17.0ppm respectively. Rotifer growth rate decreases at 23'C. Above2 ppm, however,the phys- greatly below 15'C, however, and once iological and reproductive states of rotifers rotifersare exposed to suchlow temperatures, were affected. it may be difficult for them to resumenormal OptimumCOD for rotifer productionis growth even if the temperatureis againraised generallywithin the rangeof 20 - 100ppm. above 25'C. Suddendecreases in the growth rate of The optimum chlorine concentrationfor rotifer populationsare frequentlyobserved in B, plicatilis is 6 - 10 ppt. Fish, however, are hatcheries.This mightbe caused by tempera- ture fluctuations,a feed deficiency,low quality feeds, and/or increased NE4 con- centrations and COD. In addition to these abioticfactors, the speciescomposition of the bacterialand protozoan flora canalso serious- ly affectthe growth of rotifers as mentioned above!.
Acknowiedgements Figure 10. Fiuctuetionsin the number of rotifers end Euplotes sp. cultured with and Part of this work was supportedby a without Arternia saiina. L. Number of rotifers.; grantfrom theMinistry of Mucation,Japan 3; Number of Eupfotes sp. Environmental Management of Rotifer Cultures 133
Grant No, 62860024!. We appreciate the A Live Feed-The Rotifer, BrachionusplicariDs, Kohseihsa-Kohseikaku,Tokyo. pp. 28-38. In helpful discussion renderedby ProfessorK. Japanese!. Hirayama,University of Nagasaki. Sugimoto,H. 1989. The reasonsof suddendecrease of rotifer production. In: K. Fukusho and K. Hirayama Eds.!. A Live Feed The Rotifer, Brachionusplicatilis. Kohseihsa-Kohseikaku, REFERENCES Tokyo. pp. 167-174. In Japanese!. Suzuki,K., K. Muroga,K. Nogamiand K, Maruyama, Heath, H. 1924. The externaI developmentof certain 1990. Bacterialflora of culturedswimming crab phyllopods.J. Morphol.38: 453-483. Portunus rrituberculatus!larvae. Fish Pathol. 25: Ksnematsu,M., M. Maeda,K. Yosedaand H. Yoneda. 29-36. 1989. Methods to repress the growth of a Nan- Yu, J.-P. and K. Hirayama. 1986. The effectof un- nochlompsis-grazingmicroflagellate. Nippon ionizedammonia on the populationgrowth of the Suisan Gakkaishi. 55: 1349-1352. In Japanese!. rotifer in mass culture. Bull. Japan. Soc. Sci. Fish. Oka, A, 1989. Environmental factorsof the growth of 52: 1509-1513. rotifer, In: K. Fukusho and K. Hirayama Eds.!. 134 Maeda and Hino Rotifcr and MicroaigacCulture Systcnis. proceedings of a U.S. Asia Workshop.Honolulu, Hl, l99i. !Thc Oceanic institute
An Overview of Live Feeds Production System Design in Taiwan
I-Chiu Liao Taiwan Fisheries Research Institute 199 Hou-Ih Road Keeiung TAIWAN
and
Mao-Sen Su and Huei-Meei Su Tungkang Marine Laboratory Taiwa n Fi s heries Research Institute Tungkang, Pingtung TAIWAN
ABSTRACT
Over fivebillion prawn and clam larvae and 150 million fish larvae were produced commercially in 1990 in Taiwan. The mainlive feedsused are tnicroaigae Skelerommacosrarrun, Isochrysis aff. galbana,Nan- nochloropsisocuhra, TerraselInis chai, and Chlorella sp,!, rotifersand natural plankton. In this paper,research effortson theproduction and use of theselive feedsin Taiwanare discussed and production facility designsaud operating proceduresare described.
INTRODUCTION the productionwas Romthree other penaeid speciesand 10 finfish species. Prawn larvae Commercial production of crustacean, areproduced mainly in southernTaiwan, par- molluscan and finfish larvae in 1990 in Taiwan ticularly in Kaohsiungand Pingtung Counties, was about 5.85 billion, 5 billion and 152 exceptfor P.j aponicuslarvae, one-fourth of million, respectively Table 1!. This produc- which are producedin northernTaiwan, in tion was mainly from Metapenaeusensis, the Ilan County, Hard clamlarvae are produced sand shrimp billion!; Penaeusjaponicus, exclusively in central Taiwan, in Yunlin the kuruma prawn billion!; Meretrix County.Finfish larvaeare produced mainly in lusoria, the hardclam billion!; andChanos southern Taiwan, in Kaohsiung and Pingtung ckanos,the milldish 30 mllion!. Therest of Counties,except for Plecoglossusaltivelis, the i36 Liao et af.
Table 1. Commercialproduction of crustacean,moi- fuscanand finfish larvaein 1 990in Taiwan Units: individuals!.
S ecies Estimated roduction C tustaceans 5,850,000,000 Metapenaeus ensis 3,000,000,000 Penaeus cJii nensis 50,000,000 Penaeusjaponicus 2,000,000,000 Penaeus monodon 300,000,000 Penaeuspenicillatus 500,000,000 Mollusc Meretrix lusoria 5,000,000,000 Finfishes 152,000,000 Acanthopagruslatus 3,000,000 Acanthopagrusschlegeli 5,000,000 Chanos chanos 130,000,000 Zpinephelusmalabaricus 2,000,000 Larescalcarifer 3,000,000 Lateolabraxj aponi cus 3,000,000 Lujtanus argentimaculatus 2,000,000 Pagrusmajor 2,000,000 Piecoglossusaltivelis 500,000 Sparussarba 3,000,000 Trachinotus blochii 900 000
ayu, which is produced in northern Taiwan CRUSTACEAN LARVAE Fig. I!, All prawn larvae are producedindoors andfed with microalgaeat thezoeal stage. Live Feeds Used and Their Mollusclarvae are alsoproduced indoors and Biologicat Characteristics fedonly microalgae before they settle, Most of thefinfish larvae are produced outdoors and Thefirst successin the artificial propaga- fed with naturalplankton. tion of Penaeusrnonodon larvae was obtained using Skeletonemacostatum as feed at the This paper summarizesresearch efforts zoealstage Liao et al. 1969a!. Skeletonema on bothrotifer and microalgal production and usein Taiwan.Live feeds production facility costaaunwas collected from KaohsiungHar- bor using 100-meshplankton netsand then designsand operating proceduresare also discussed. culturedusing the separate tank method Liao etal. 1983;Liao 1984!.Since then, larvae of severalpenaeid species have been propagated artificiallyusing the samefeeding regime Liao 1970!. Spirulinapiatensis combined System Design in Taiwan 137
120' l2l' 122' for the zoeal stage. %hen Tetraselmissp., Skeletonemacostanun and Artemia nauplii were fed to P. chinensis from Zt to P5, survival rates of 13.7 64.0% were obtained Tzeng et al. 1990!, Among these suitable microalgae, S. costatum was preferred be- causeit has more advantages,S. costatumis eurythermal and euryhaline, and its optimum 24' 24 temperature and salinity are between20 and 30'C and 15 and 30 ppt, respectively Su et al. 1990!. It grows very fast and can be concentrated in a net, Furthermore, its EPA eicosapentaenoicacid! content is higher 5- 23' 43%! than that of C. gracilis l9- 28%! and T. chai - 8%! Su et al. 1988!. Although its stock culture maintenanceis difficult Su et al. 1990!, inocula can be easilycollected from Kaohsiung Harbor. All these advantageshave 22' 22' resulted in the use of S. costatum in hatcheries in Taiwan almost exclusively. !20' Figure 1. Location of commercial hatcheriesin Skeletonema costa ttjm Production Taiwan and total production of prawn, clam, and finfish /arvae 990' Stock cultures Stock cultures for laboratory studiesare grown in 250-ml flasks containing100 ml F/2 with Skeletonema costatum was found suitable medium McLachlan 1973! and placed in a to feed the lysis stageof P.japonicus Tang growth chamber at 20'C and 500 lux 2L: 1977!. Spirulina platensis was also found 12D!. Subcultures are transferred every one suitable as feed for the mysis stage of P. to two weeks. Mass cultures are developed monodon Tsai 1980!. Later, commercially- stepwisein the following order: 250-ml flask produced S. platensis powder was used as ~ 1-liter fat flask ~ 15-liter circular glass supplementalfeed by commercialhatcheries. beaks ~ 500-liter circular FRP tank ~ 10-ton The effects of selected live feeds on the concrete pond. These cultures are agitated growth and survival rate of P. monodon larvae with an aerator with the exception of the was testedby Lei and Su 985!, The results 250-ml flask culture, which is manually showed Skeletonema costatum and rotifers to shaken occasionally. be the mostnutritious feeds. Isochrysisaff. Stock cultures for commercial hatcheries galbana,Chaetoceros gracilis and Tetraselmis are bought from suppliers which collect S. chui were found to be inferior to S. costatum costatumby boatwith 100-meshnets in Kaoh- and rotifers, while Dunaliellasp,, Chlorella siung Harbor. The microalgae are immedi- sp., and Spirtdina platensis were unsuitable ately packed in plastic bags 5 kgfbag! or 138 Liao et al.
Table 2. Enrichedseawater mediumfor Skeletonemacostatum in commercialhatcheries unit: 9/ton!.
Agricultural fertiTizer, Used occasiottaUy.
packed after subculture within 24 hours. The Fertilizers densityof S. costatumin the plastic bagsis The media available for S. costatum cul- suchthat two bagsare enoughto inoculateone ture havebeen described by Liaoet al. 983!. 10-tonconcrete pond. The suppliers began this At present,F/2 mediumis beingused for stock specializedbusiness in 1985,and the highest cultureat theTungkang Marine Laboratory demandon record was between 1986 and 1987 TML!, Taiwan Fisheries Research Institute at 300 bagsper day with an averageof 200 TFRI!. The reagentsused for commercial bagsper day. hatcheries are listed in Table 2. Fertilizers Skeletonema costatum is distributed in all suchas urea, ammonium sulfate,calcium su- coastal waters around Taiwan Huang et al. perphosphate,and potassiumchloride are 1986!. Before 1988, S. cosfatum could be major sourcesbecause they are cheapand collected year-round in Kaohsiung Harbor. suitablefor S. costatumgrowth, As shownin Its densitywas high duringthe rainy May to Table2, the quantityused varies greatly and June!and fall October to December!seasons, is dependenton who is preparingthem. This but low prior to the rainy season February to shows that both the quantity andquality of March!. This corroborated the findings of fertilizers are not very critical in the mass Liao 970!. However, after 1988, S. cos- culture of S. eostatum. tatum collection in KaohsiungHarbor became difficult becauseof pollution. Likewise, the Culture techniques mass culture of S. costanun encountered dif- Generally, S. costatum is cultured out- ficulties. doorsin rectangularconcrete ponds with 10- System Design in Taiwan
st ! for Skeletonema costate.
40 ton watervolume. Water depth in thepond beingsun-dried. Each pond is culturedfor is 1.5-2.0 m. Higherilluminance, over 5,000 threedays. One run, from start to finish,is lux saturatedilluminance!, was found to about10 days, comparable to theperiod from decreasethe growth rateand shorten the ex- hatchingto zoealstage of prawnlarvae, which ponentialperiod Su et al. 1990!.Increasing is also consistentwith the changein the size the culture water depthresults in decreased of S. cosranun. lightintensity, thus benefitting the culture by In culture, the diameter of S. costatum slowing the growth rateand extending the decreasesafter cell division, thus, the S, cos- exponentialperiod. tatum chainsbecome thinner and thinner. As To beginwith, twobags of stockculture the diameterbecomes smaller, the cell division from KaohsiungHarbor or anotherhatchery rateincreases, while theconcentrated biomass are inoculatedin one 10 - 20 ton pond. On the and the duration of the exponential period secondor third day,S, costatumis collected decrease.At thispoint, a cultureis considered with 100 meshnets or drainedinto cloth bags. unsuitable as an inoculum, Therefore, new The collectedrnicroalgae may be usedas an stocksare collectedfrom KaohsiungHarbor inoculumfor a new pond. The ratio of or nearbyhatcheries forthe next larval rearing inoculurnto culturevolume ranges from 1:20 run. to 1:100,depending on theconcentration of Thequality of S. cosrarumis very impor- theinoculum, the period of harvest,light and tant,and can be determinedby its colorand temperature,and on whois preparingthe smell. Light brownand briny-smeUing cells inoculum. The concentration of the culture at oftenappear in theexponential phase. These the start is about 100- 250 chains/mlor 1,500 cellshave a particularlyhigh EPA content Su - 4,000cells/ml. Time of inoculationis 0800 et al. 1988!. - 0900 h or 1500- 1600 h. After 30 hours at It is estimated that about one ton of least 12 hours!, one-fourth or one-fifth is microalgalculture is neededfor one10- harvestedto feedprawn larvae by drainingthe 15-tonlarval rearingpond. For example,two cultureinto cloth bagsfor oneor two days. 50-tonS. cosrarumcultures are sufficientto The optimumnumber of pondsis four. supplyeight 130-ton larval ponds inwhich 160 Table 3 lists the culture schedule. Every day, millionP. j aponicuslarvae are reared; and one of the four pondsis either readyfor three20-ton S. costaturncultures will supply culture, underculture, ready for harvestor 't 40 Liao et al.
340-ton larval ponds in which 2 - 3 million/30 gae. The best microalgal species for the tons P. rnonodon larvae are reared, swimming larvae is I. aff. galbana, with Tetraselmissp. next;but specieswith thick cell Problems and prospects walls are not recommended Chen 1984!. On Heavy metalslike copper and cadmium, the other hand, Yang and Ting 985! found which are found in seawaterand absorbedby I'. aff. galbanaand Chlorella sp. to be the best S. coslatwn in high concentrations, are sus- feeds for the growth of purple clam larvae, pected to be one of the factors causing the with TerraselInissp. and Spirulinaplatensis mortality of P. monodonlarvae in 1986.Thus, coming in next. They found Chaetoceros formulated feedswere developedto replaceS, gracilisunsuitable. Isochrysis aff. galbanahas costatum. At present, S. costatum is used in been used solely for the massproduction of combination with formulated feedsfor prawn the swimming larvae of hard clam, but its larvae at the zoeal stage, This has resultedin nutritive value as feed for juveniles was in- the decreaseduse of S. costarvm. However, ferior to preparedfeed Hon 1990!. the nutritional and operationaladvantages of Isochrysisaff. galbana wasintroduced to this live feed makeit superior to other feeds, Taiwan from the Tahiti-basedAQUACOP in including formulated feeds Liao et al. 1988!. 1980. Its stock cultures are maintained in A major problem in S. costanunculture TML, and distributed to hatcheriesin Taiwan. is the short exponentialgrowth phase and its A study by Kao 986! on the infIuence of tendency to perish after only a short culture nutrients, salinity and pH on the growth of I. period. Frequentsubcultures are thus neces- aff. galbana showed that it was euryhaline sary to maintain the microalgaein good con- with an optimum salinity of 10 - 25 ppt. dition. A stock culture supply center is, therefore,necessary since Kaohsiung Harbor Isochrysis aff. galbana Production has become so polluted, allowing other microalgaeto graduallydisplace S. costatum. Thefollowing production system is being adopted in TFRI's Taishi Branch.
MOLLUSCAN LARVAE Medivm and water filtration The medium used for I. aff. galbana Uve Feeds Used and Their cultureis Walne medium Walne 1966! Table Biological Characteristics 4!. Reagent-gradeingredients are used for stock and indoor mass culture while industrial More than 13 molluscan species have chemicals are used for outdoor mass culture. been successfully cultured in tidal lands, es- Vitamins are not added to outdoor cultures. tuaries, and shallow seasalong the westcoast Seawater is treated as follows: of Taiwan. Artificial propagation has been ~ Ceramic filter ~ autoclave ~ stock cul- successfulfor Meretrix lusoria Chen 1984!; ture Hiaacla diphos, the purple clam Lai 1984!; a Storagetower ~ filtration tank 0 cm Tapesvariegata, the Manila clam Yen 1985!; dia. x 200cm, 25-p.mnet filter! ~ 2-pm and Anadara granosa, the bloom clam Tsai filter cartridge ~ ceramic filter ~ indoor 1986!. All the larvae were fed with microal- culture System Design in Taiwan
Table 4. Formula of Walne medium Walne 1966!.
~ e Acidifywith sufficient concentrated HClto get a cia sotutiott;Acidify to pH 4.5 before autoclttving.
~ Local seawater~ settling pond ~ first glass!. Its southernwall is fitted with glass, filtration pond 0 x 12 x 2,5 m ! ~ while its northern wall has a mirror to increase reservoir x 200 tons! ~ second filtra- illumination. tion pond~ storagetower ~ outdoorml- Stock cultures of 2.5-liter plateflasks are ture. keptin a 14C waterbath-t~pe rectangular glasstank ,200 x 45x 30cm ! anthe second Culture facilities floor. The glass tank is placedon top of a wooden desk and located at the south end of Culture rooms the room. Fourteen60-liter rectangularglass Two separate40-m 2.3 x 3.4 m ! tanks 5 l x 30 w x 30 d cm, 0 5-cmthick rooms Fig. 2! locatedon the first andsecond glass!are situated at thenorth end of theroom. floor of the algalculture center of thebranch Thetemperature for stockculture is regu- are used for indoor culture. Installed on the lated at 14'C usingcool water. The culture first floor are 22 350-liter rectangular glass room temperatureis maintainedat 25'C with tanks 20 l x 45 w x 75 d cm, 0.8-cm thick an air-conditioner. 142 Liao et al.
2.5-liter stock cultures, and cultured for four to sevendays. The tankis thendrained by gravity and used as the inoculum for one 350-liter tank. The 350-liter tanksare thefirst in the culture procedureto be providedwith aeration, One 700-liter outdoorconical tank is inoculatedfrom a fourto seven-day-old350- liter culture.Afterwards, every four to seven days, a larger pond is inoculated from a smallerpond in thefollowing order: 700 liters ~ 2.5 tons ~5 tons ~ 10 tons. Eachtime, 3 - 5 tonsof 1. aff. galbana culture is pumpedto one 400-ton indoor clam Figure 2. Schematic diagramof Taishi Larvaepond to maintainthe microalgal density Branch 's culture rooms. at 1,000cells/ml, Too high a densityinhibits larva1growth. Larval density is 2 - 10ind /ml. Naturaland artificial lightsprovide il- lumination.Artificial light is providedby 40- Problems and prospects wattfluorescent lamps installed on the ceiling Few problemsare encounteredin indoor culture. On theother hand, outdoor cultures or wall. The lainps are controlled automat- icallyduring the light period, that is, theLamps experiencefrequent problems in the summer dueto contaminationwith protozoa or toohigh turn on if thelight is inadequateand off if there is enoughnatural light. Thelight period lasts temperatures. These problems also occur duringthe rainy season or whenthe change in 12 hours,and the light intensityfor stock water temperature exceeds 5'C. cultures, 60-liter tank and 350-liter tank cul- Outdoorcommercial pond culture, par- tures are 1,000- 1,500, 5,000, and 6,000- ticular1yI. aff. galbanaculture, encounters 7,000lux, respectively. problemsfrequently. Thus, naturaLLy~cur- Culture ponds ring microalgaein growoutponds are instead usedto feedthe swimming hrvae. The method Outdoorculture tanksand ponds are lo- usedis asfollows: first, prawnor fishpond cated on the southern side of the culture waterand brown or greenwater from nearby rooms. Twenty-two 700-liter conical fiber- pondsare filtered through a 25-pmnet. They glasstanks, 20 2.5-ton,LS 5-ton and 14 10-ton arethen either transported by motorvehicle or concrete ponds are used either for the mass througha pipeline,and used directly or in- cultureof l. aff. galbanaor Nannochloropsis directlyto feedclam larvae. If indirectlyfed, ocuhua, or both. filtrates are provided as inoculum for outdoor pond culture, which are enrichedwith chemi- Culture techniques caland organic fertilizers. Organicfertilizers Microalgalculture is routinelyconducted includefish meaL,fish soluble and prepared using the batch technique. One 60-liter food. The unstablesupply of suitablenatural microalgal tank is inoculatedwith four to six phytoplankton,however, is themain problem System Design in Taiwan 143
when using this method. Selection of more seasons,and enough heat during the winter environmentally resistant and nutritious and spring seasonsin Taiwan. Otherwise, microalgae like S. cosratum in local water bigger strains and low salinity are the best would be a solution. Also, formulated feeds choicesif heatersare not usedduring the cold are currently being developedto replacelive season. Recently, TML has collected more feeds. strains from natural watersand ponds, and the selectionof strainswith highergrowth ratesis in progress.This study hopesto obtain suitab1e FlNFlSH LARVAE strainswith optimum sizes. Microalgae,baker's yeast,yeast powder, Live Feeds Used and Their prepared feeds, and chicken droppings are Biological Characteristics used to feed rotifers, but the best growth is obtained with microalgae. Microalgae Since 1968, when the first successin the Chlorella sp., Tetraselmischui, N, oculata, artificial propagationof grey mullet Mugil and l. aff, galbana! are all suitabIe to feed cephalus!fry was achievedby Liao et al. rotifers. The rotifers grow best, however, 969b, Liao et al. 1972!, many finfish fry when fed T. chui. The HUFA highly un- have been producedcommercially Table 1!. saturatedfatty acid! content of rotifers varies The feedingregimes used are almostthe same. a greatdeal and is relatedto its feed, The EPA Routinely,as thelarvae grow, artificially-fer- content of rotifers fed N. ocular is the highest, tilized oyster eggs, rotifers, copepods,Ar- about 15 - 20% of total fatty acid; with I. aff. remianauplii, and preparedfood are given galbanaor T, chui, EPA contentis low, about successively.Green water is also usedto feed 5 - 10%; and with Chloeella sp, or yeast, very the zooplankton and to stabilize the water low, nearly0% H.M. Su, unpublisheddata!. quality. Nanseiochloropsis ocul' was provided Most strains of Brachionus plicarilis by theNational Research Institute of Aquacul- found in aquacultureponds in Taiwan are of ture tN$DA!,Japan and introduced to Taiwan the S-type and range from 100 - 305 pm in 1987. At present, most hatcheriesuse this Temperature, salinity and feed concentration speciesand consider it to be thebest nutritive all affect the growth rate of the L- and S-type feed for rotifers. However, it is very difficult strains. Of thesefactors, temperatureis the to culture in southern Taiwan, where most most critical. The most suitable temperatures hatcheriesare found, during summer due to for both strains are between 28 and 32'C. high temperaturesand contaminationwith Above28'C, thesalinity and size of thestrain protozoa.Contaminated cultures turn brown arenot very critical, but thedensity of feedis and then perish. Treatments,such as adding very important. However, below 28'C, the sodiumhypochlorite or increasingsalinity as bigger strains 83 - 233 pm in length, mean reportedby Kanematsuet al. 989! did not = 210 +3 pm! grow faster than the sma11er help. Techniquesfor repressingor diminish- ones 26 - 172 pm, mean = 143 +3 p.m!. ing protozoancontamination must be devel- Decreasing salinity 0 - 30 ppt! increasesthe oped to stabilize productionof N, oculata. growth rate of both strains. Therefore, enough Aside from these, culture temperature must food must be provided during the warm not exceed30 C. Chlorella sp. occurs often Liao et al.
Table5. Ponds'capacity, initial and final larval density, and survival rate of Snfishlarvae reared indoors and outdoors
Species Water Eggs stocked Initial Final Survival volume No. Density N o. Density No. Density rate tons! x1,000! No./ton! x1,000! No./ton! x1,000! No./ton! %!
Cbanos chanos 2,000 180 600 30 Epinephelus 14 50 3,500 16 1,143 ealabaricus 15 190 12,666 180 12,000 10 666 305.7 300 1,200 4,000 720 2,400 20 67 2,8
475 600 1,263 40 84 6.7
840 3,600 4,285 2,160 2,571 40 48 2.2
canthopagrus 15 150 10,000 17 1,113 11.1 schlegeli 25 370 15,000 120 5,000 30
1,000 3,000 3,000 50 50 1.7
canthopagrus 25 370 15,000 40 1,600 10 latus 45 2,190 48,666 1,660 36,888 20 444 1.4
400 1,100 2,750 250 625 22 475 2,000 4,210 50 105 2.5
in brackishwaterponds in Taiwan.However, other zooplankton in Taiwan basedon the thesix strainsisolated have a verylow EPA larval rearing methodsused. There are two content almost 0%!, and their nutritional basicmethods. With the intensivemethod, value for rotifers is inferior to ¹ oculata's. high densitiesof larvae 0 - 50 ind./!iter!are Therefore,Chlorella sp, cultureis now rare. rearedindoors in concreteponds; microalgae, Tetraselmis chui was obtained in 1983 rotifers, andArtemia nauplii are providedas fromthe microalgae section of thePhilippine- feeds.The other is theextensive method, that basedSoutheast Asian Fisheries Development is, low densitiesof larvae I - 4 ind./liter!are Center SEAFDEC!. Althoughits EPA con- rearedoutdoors in earth-bottomponds; natured tent - 8%! is also lower than that of ¹ p1ankton supplemented with rotifers and oculata, its nutritive value for rotifers is copepodsare the main live feeds.The ponds' higher. Mass cultures of T. chui are usedas capacityfor larval rearing, the initial and final feed for rotifer cultures. larval density, and survival rateachieved with these two methodsare summarize in Tab1e5. Zooplankton Production To satisfy the heavydemand for live feeds, intensivemass production of microalgaeand Various designsand procedureshave rotifers are performed. To simulateand main- beendeveloped for producingrotifers and tain the ecosystem,natural plankton are raised System Design in Taiwan
Table 6. Six differentsystems used for the culture of rnicroalgae,rotifers and fish larvaein some indoor and outdoor larval rearing ponds unit: tons!.
Capacityratio of microalgae:rotifers.fishlarval culture ponds. to provide diversified naturallive feedsfor smallercapacity of the microalgal ponds. In larvae reared outdoors. CaseIII, experimentsand trials were designed to determinethe optimumdesign system for Culture systems rotiferproduction. Case IV is a modelsystem Examplesof six differentsystems used to designedto be testedin the future.The best culturemicroalgae, rotifers and fish larvaein set for microalgal culture is six pondsin a someindoor and outdoor larval rearingponds group;for rotifer culture, four pondsin a are listed in Table 6. In Case I, there are no group. In CasesV and VI, microalgaeare exclusive rotifer ponds. Instead, microalgal provided from prawn and finfish growout ponds contaminatedwith rotifers are usedfor ponds.Only greenpond water is used. Thus, rotifer culture. Otherwise,naturally occurring manyponds are needed. Bloomsleading to rotifers are collectedfor culture in microalgal greenwater can easily occur and be sustained ponds and harvestedas feedafter two or three in prawnponds. The main speciesfound in days. On the other hand, microalgal water is greenwater includeSynechocystis pevalekii, addeddaily to larvalponds to feedthe rotifers ChroococcuscohaererLr, Chlorella vulgaris, and stabilize water quality. Therefore, more Oocystisborgei and Ankistrodesmuscon- microalgalponds are needed.In Case0, al- volutus. Besidesthese, flagellates, diatoms, though larvae are rearedin clear water and no protozoa,rotifers and copepods also occur and microalgalwater is addedfor larvalponds, the could be used as live feeds for larvae. Facilities rotifer yields are often deficientdue to the for rotifer culture are eithervery limited or not 146 Liao et aj.
Table 7. Fertilizers for microalgal culture unit: g/ton!.
C: Chlarella sp.; N: Hannochloropsisocalata; T; Tctraschnischai available. In extreme cases, surface water sodium thiosulphate.Light for indoor culture containing a naturalplankton bloom is directly is provided by fluorescent lamps ,000- introducedinto larvalponds, and copepods are 5,000 lux!; natural light is used for outdoor provided at the late stageof larval rearing. culture. Temperature is maintainedat 25- 30'C for indoor culture. For outdoorculture, Culture techniques for rni croalgae temperaturechanges naturally. In other re- Table 7 shows the fertilizers used in searchfacilities and commercial hatcheries, microalgal culture and the speciescultivated. the sameoutdoor proceduresare used. They are almost the samein quality but vary There are no definite methods to follow in quantity due to local water and weather for exploiting green water naturally occurring conditions. Microalgal culture is routinely in prawn or finfish ponds. By experience, performed according to the batch technique, good quality green water often occurs during and fertilizers are providedat the beginningof the middle part of the prawn growout cycle. the culture. In TML, stockcultures in 250-ml On the other hand, raw feeds seem to be flasks, 1,000-ml fiat flask cultures, and 15- superior to preparedfeed in promotingblooms liter glass beaker cultures are performed in- in the finfish ponds. Furthermore,the culture doors. Fifteen-liter subcultures are transferred of high-priced finfishes is more lucrative. every four to five days, and 15-liter cultures Therefore, 1,000 - 5,000 carnivorous fishes are provided as inoculum for the 200-liter like grouper, sea perch and porgy are cul- outdoor culture tanks, From 200 liters, the tivatedin one 0.2-hapond to provide the green cultures are expandedstepwise from 200 liters water. Then, this greenwater is pumpedvia a ~700 liters ~ 3,000 liters ~ 10,000 liters. The pipe to larval rearing ponds to stabilize the inoculated ratio is 1:2 - 1:5. Culture periods water quality and to feed zooplankton. Some for each step last three to five days according of thesepipelines are more than 2 km long. to requirementsand culture conditions. Seawater for indoor culture is sterilized Culture techniques for roti fers by autoclave,while seawaterfor outdoorcul- For small ponds below 20 tons!, batch ture is treated by settling, addition of 10 ppm techniquesare adopted. At the start, three sodium hypochloride, and neutralized with partsof 20-pptseawater are added to onepart System Design in Taiwan 147
ionus plicatilis.
+ Concentrationunknown. microalgalculture and inoculated with rotifers volumeof rnicroalgalwater is addeddaily or havinga highgrowth rate. The initial rotifer onceevery two to threedays. The harvest densityis 30 - 50 ind./ml.One-third to one- densityis usually50 60 ind./ml.Each pond fifth part of microalgalwater combined with is used for one month or more. baker'syeast Table8, columnsI andII! is The organicwastes used to feedrotifers provideddaily fromthe second or thirdday to are listedin Table8 columnsIII, IV, V, VI!. theend. After five to sevendays, rotifers are Generally,naturally-occurring green water or harvested and used as feed or to inoculate a. brackish water is introduced into rotifer new culture. The operationis thenrepeated. ponds. Chickendroppings, fish meat,pow- The harvest densities exceed 100 ind./ml. deredyeast, or preparedTilapia feed are Salinity is adjustedat 25 ppt, andheaters are provided,as described in Table8, to supply used in winter to maintain water temperature organicdetritus, bacteria and microalgae to at26 - 28'C. Light andpH arenot controlled. rotifers. Usually,rotifers are inoculated,but For bigger ponds more than 20 tons! in some hatcheriesno inoculations are used. semi-continuoustechniques are adopted.At After four to 15days, rotifers are harvested, thebeginning, microalgae is grownin a rotifer with densitiesbelow 50 ind./ml. Yields are pond, then the rotifers are inocuhted when hard to predict andusually vary. The opera- microalgalcell densityreaches 10 cells/ml tion,however, is easy.Natural plankton and X ocular or Chlorellasp.! or 10 cells/ml artificial feedsare usedto reducethe needfor T. chai!. Baker's yeastis provided as sup- rotifers. Therefore, this production technique plementalfeed. One-thirdto one-fifthof the is practicedby mostcommercial hatcheries in rotifer culture is harvested and an equal Taiwan. 148 LIao et aL
Problems and prospects type continuous massculture system with a The critical problems encounteredin in- capacityof more than one ton must be under- tensiverotifer cultureare thedifficulty of taken to determine its technical and economic maintaininga continuoussupply of microalgae feasibility in Taiwan. and low temperatureduring winter when Taiwan has severalindigenous microal- heatersare not used. Microalgalshortages gal speciesand rotifer strains. Likewise, occurredoften in summerdue to hightempera- several exotic speciesand strains have also tures and protozoan contamination. There- beenintroduced. A one-stopculture collection fore, temperature-controlledculture systems andresearch center must, therefore, be estab- for microalgae in summer and rotifers in lished. winter must be designed. To decreasecon- On the other hand, in extensive larval tamination,the water used for massmicroalgal rearingsystems, natural blooms of greenalgae culturemust be sterilized more thoroughly, for andplankton are difficult to controland very example, by UV light or ozoneafter filtration unstable. Based on the commercial hatcheries' in series with 25-, 5-, 1-, and0.45-pm filters. experience, a total of 10 ha of ponds are Inocula for outdoor cultures must also be neededto supply sufficient microalgaefor restartedfrom the indoorculture once every 0.5-hafish larval ponds. If optimumcondi- one or two weeks. tionscouId be maintained,the pond area could In addition,Nannochloropsis sp. must be be greatly reduced.Therefore, ways to culturedin greenhouseswhere temperature is managethe growout pond water to stabilize kept below 30'C. Nannochloropsissp. is the supply of live feedsfor fish larvaemust be thereforeused only for secondaryenrichment investigatedin the future. of rotifers andmaintenance of themicroalgal density in larval rearing ponds. The selection of high temperature-tolerantspecies may help CONC LUS lONS abatethis problem. Tecraselmissp. can be cultured as feed for rotifers becauseit is less Most production systemsfor live feeds sensitiveto environmentalstress. However, developedin Taiwan are extensive, and the the rotifers mustbe secondarilyenriched with operations simple. There are severalreasons Nannochloropsissp. dueto thelow EPA con- for these,namely: tent of Tetraselmissp. ~ culturespecies often vary a greatdeal, Furthermore, the labor-intensivework of thus, constructioncosts of specialized inoculation,culturing, harvest, and washing facilitiesfor live feedsare expensive; mustbe decreasedby adoptingautomated sys- ~ manpoweris limited. Likewise, tems. To stabilizethe waterparameters and specialistson live feedsproduction are production of rotifers, a continuousculture few.The operation of extensivesystems system may be an alternative. It has more is simpleand easy to handle.It is also advantagesthan the batch culture system and availableanytime. Furthermore, 20 ha canbe automated,thereby greatly decreasing canbe handledby oneperson; labor. However, the initial investment and s most owners of commercial hatcheries operatingcosts are higherand more special- havebeen practicing the growout of ized skills are needed.The designof a proto- prawns,finfishes, and molluscs. They System Design in iaiwan 149
are familiar with the traditional techni- studies to the commercial scale will be the ques,and thus,know how to enrichpond main topic to be studied. water with natural plankton; The automatedand mechanizedproduc- a the weather in Taiwan is suitable for the tion of rotifersin Japanis intendedto stabilize diverse assemblageof plankton which the rotifer supplyand replacerepetitious tasks. occur naturally in ponds. Aquaculture This systemis expensive,and is desirableonly pondsare distributedalmost everywhere, where there is limited spacefor aquaculture thus, selectedplankton are easily ob- and the weather is unfavorablefor naturally- tained; occurringplankton. Introducing this intensive a providing severalspecies of plankton systemwholly into Taiwan inight not be prac- might be morebeneficial to the larvae tical and economical. On the other hand, re- than some monospecificdiets, suchas search efforts on formulated diets to replace green algae, rotifers, andAnemia live feeds for prawns, molluscs and finfish nauplii. The first successin the mass larvae are ongoing, and already, several suc- productionof milkfishlarvae and the lar- cesseshave been reported for diets for some val production of over 150 million for thepast severalyears attests to thisfact; penaeidand finfish species.We hopea com- pleteformulated diet will bedeveloped so that ~ massproduction of larvaeis easilyestab- only the simpleand cheaperproduction sys- lished in somespecies like milkfish and seabreams Table 1!. Thus, using the in- tems for live feeds will need to be used. tensivesystem would only addto over- production, Acknowledgments However, there are somedisadvantages. First, the extensive systemis easily affected by natural phenomenasuch as typhoonsand Many thanks are due Dr. H.C. Chen, National Taiwan University; Messrs. C.H. long periods of rain or low temperatures. Second, many finfish or prawn ponds are Wu, Y.D. Hon, Y.H. Chou, M.Y. Leu, needed to provide green or brown water. Taishi Branch, TFRI; J.L. Lie, Tainan Third, the volume of the larval pond must be Branch, TFRI; K.J. Lin, Penghu Branch, large enough to stabilize water quality and TFRI; A.J. Chang, C.C. Chen, M.Y. Cheng, temperature.Fourth, there are frequentdis- C.H, Hsieh,W.F. Hung,P.W. Leu,L.T. Lin, K.C. Lin, C.C. Peng, L.C. Tseng, C.S. Wu, ease outbreaks. There is a need, therefore, to refinethe productionsystems of live feedsand J.J. Wu, ownersof commercialhatcheries; AQUACOP, SEAFDEC, and NRIA, par- larvae. On the other hand, several successes ticularly Dr. M. Okauchi; and J.V.A. A. obtained with indoor larval rearing trials for Nunez, for providing either valuable data, grouper in 1990 increased the estimated culture stocks, or assistancein the preparation figures for the intensive production of live of this manuscript. feeds for some hatcheries. Therefore, com- mercial-scale intensive culture systems are expected to be developedand adoptedin the future. How to bring the successof laboratory 150 Liao et aL
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