1 - Central Institute of Brackishwater Aquaculture
1 - Central Institute of Brackishwater Aquaculture
1 - Central Institute of Brackishwater Aquaculture
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TECHNOLOGY FOR ARTEMIACYST ANDBIOMASS PRODUCTIONClBA BULLETIN NO. 4JANUARY 1995wlraIC'ARd;;it4mm?.mqmsglcem(* @ qmVflBG)i.w, wh k, m, m -
TECHNOLOGY FOR ARTEMIA CYST ANDBIOMASS PRODUCTIONS. KULASEKARAPANDIAN, S. SRINIVASAGAM, P. RAVICHANDRANAND K.O. JOSEPHClBA BULLETIN NO. 4JANUARY 1995wiwICA RCENTRAL INSTITUTE OF BRACKISHWATER AQUACULTURE(Indian Council <strong>of</strong> Agricultural Research)141, MARSHALL'S ROAD, EGMORE, MADRAS - 600 008
Restricted CirculationPublished by.Dr. K. AlagarswamiDirector<strong>Central</strong> <strong>Institute</strong> <strong>of</strong> <strong>Brackishwater</strong> AquacuitureMadras-600 008.Edited By:Shri K. N. KrishnamurthyPrincipal Scientist<strong>Central</strong> <strong>Institute</strong> <strong>of</strong> <strong>Brackishwater</strong> <strong>Aquaculture</strong>Madras-600 008.Cover Photos:Top Le/r : Culture PondTop Rigtar : BiomassBorrotn Lefr : CystsBortor~r Riglat : NauplilPrinted el:Prints & Pro<strong>of</strong>s. Madras-8.
PREFACEThe brine shrimp Arterttio is an inevitable component <strong>of</strong> operation <strong>of</strong>shrimp and fish hatcheries all over the w oM The nauplii hatched from the cystsare used as live food <strong>of</strong> larval and postlarval stages <strong>of</strong> all species <strong>of</strong> shrimpand fish. While commercial cystproduction as an adjunct activity <strong>of</strong> salt industryor as an independent production system has been established in somecountries such as U.S.A and Thailand, lndia has no such commercial productionsystem as yet. Hence, the shrimp hatcheries in lndia have to rely onimported Artetttia cyst.The <strong>Central</strong> <strong>Institute</strong> <strong>of</strong> <strong>Brackishwater</strong> Aquacutlure initiated programmefor developing a techndogyforArrer?iio cystproduction in 1990at a pn'vate saltpanat Kelambakkam near Madras. In the midst <strong>of</strong> heay odds caused by unseasonalrains and floods which created problems for maintenance <strong>of</strong> salinity at therequired levels, Wo successful crops have been taken, one with the local(Kelambakkam) strain <strong>of</strong> Ancr?tio and the other with an imported strain Eftortshave also been made towards Arfcr~iiu biomass production which is valuableas an ingredient <strong>of</strong> shrimp maturation diet. Thus, a technology base has nowbecome available for Ano?liu cyst and biomass production in the country.Production results would improve, if required salinity levels for different stages<strong>of</strong> the cycle could be maintained through careful water management.The National Workshop on "<strong>Brackishwater</strong> Prawn Farming for HigherProduction" organised by the Ministry <strong>of</strong> Agriculture (Department <strong>of</strong>Agricultureand Cooperation) at Bangalore in October 1989 recommended that this <strong>Institute</strong>should take up an R & D programme to develop a suitable technologyfor Ancrniu culture and cyst production. Implementing this recommendation,the <strong>Institute</strong> has been able to achieve successful results in the experimentalwork carried out so far. It is to be placed on record that the Department <strong>of</strong>Fisheries <strong>of</strong> Government <strong>of</strong> Ralasthan has taken up this technology for theirprogramme on Anct?riu cyst production and success has been reported.The present Bulletin is a result <strong>of</strong> the <strong>Institute</strong>'s work on Anernio cystand biomass production and I would like to compliment Dr. S. Kulasekarapandian,Shri S. Srinivasagam, Dr. P. Ravichandran and Dr. K. 0. Joseph, scientistsfor its preparation and Shri K, N. Krishnamurthy, Principal Scientist for itsediting. The oblect is to disseminate the available technology to theentrepreneurs who would be interested in it and also to encourage the saltpanowners to consider it as an integrated system which goes well with theiroperation and would bring in additional income. I would like to record ourgratitude to Shri K. Manappan for providing facilities to our scientists for thiswork at his saltpan at Kelambakkam.Madras - 82 1.1995K. ALGARSWAMIDIRECTOR
CONTENTS1. INTRODUCTION2. BIOLOGY OF THE BRINE SHRIMP3. DISTRIBUTION OF BRINE SHRIMP IN INDIA4. BRINE SHRIMP CULTURE IN SOLAR SALTPAN IN INDIA4.1 Present status4.2 Culture site4.3 Pond preparation4.4 Water intake4.5 Fertilization4.6 Stocking4.6.1 Strain selection4.6.2 Rate and time <strong>of</strong> stocking4.7 Water management4.8 Growth monitoring4.9 Harvesting techniques4.9.1 Biomass harvest4.9.2 Cyst harvest4.9.3 Storage <strong>of</strong> harvested products4.9.3.1 Biomass4.9.3.2 cpf5. RATE OF PRODUCTION
1. INTRODUCTIONThe brine shrimp, Artemia, is a crustacean which thrives in hypersaline waters and isdistributed worldwide. Decapsulated cysts and freshly hatched nauplii <strong>of</strong> Artemia form an idealfood for the larvae <strong>of</strong> crustaceans and fishes, while Artemia juveniles and adults form suitablediet for prawnlfish juveniles. Induction <strong>of</strong> maturity in broodstock prawns can be achieved if theyare fed with reproductively active adult Artemia. Thus, demand for Artemia especially for itsnauplii, in other words, cysts, continues to increase with the development and progress <strong>of</strong>aquaculture. Availability <strong>of</strong> Atremia cysts and biomass from natural sources is inadequate tomeet the growing demand. Culture <strong>of</strong> Anemia is the only alternative to fulfill the demand andhence it has gained importance. In India, all along the coastline, a vast area is under saltproduction. During dry season, these man-made solar saltpans <strong>of</strong>fer excellent scope for carryingout Arfemia culture Most <strong>of</strong> the infrastructure required for Artemia production is alreadyavailable in these saltpans. Anemia will form a valuable byproduct to salt-farmers.2. BIOLOGY OF THE BRINE SHRIMPThe genus Artemia is comprised <strong>of</strong> both bisexual and parthenogenetic strains. The latter arefound in Asia and Europe and have been commonly designated as Artemia parthenogenetica, eventhough they have important differences in ploidy level and isoenzyme pattern. Bisexual strains areclassified into six sibling species which are. A. saiina, A. tunisiana, A. franciscana , A, monica , A.persimilis and A. urmiana. Among these, A, salina is extinct now. The adult Arfemia measuresabout 10 mm in total length. However, in some polyploid parthenogenetic strains, It grows upto20 mm in length. Arfemia has an elongated body which can be divided into head, thorax andabdomen. A pair <strong>of</strong> antennules, antennae and stalked eyes are present in the head region. Thoraxhas eleven pairs <strong>of</strong> appendages, known as thoracopods. The abdomen ends in a furca, coveredwith spines (Fig. 1). In the females, the antennules and antennae are sensory in function. Theantennae are developed into a clasper, forming a secondary sexual character in the males <strong>of</strong>the bisexual strain (Plate 1 A 8 C). The female has paired ovaries and oviducts. The eggs,liberated from ovaries, will pass through the oviducts and reach the unpaired ovisac (or uterus)(Plate 1 8). In bisexual strains, male has penis by which the sperm is transferred into uterus <strong>of</strong>the female, during copulation. Fertilization is achieved in the uterus. In the case <strong>of</strong> parthenogeneticstrains, embryonic development starts directly as soon as the eggs reach theuterus. The embryos develop into nauplii or are coated with shell to form cysts, in the uterus, asper the prevailing environmental conditions. The liberated cysts are 200-300 microns in size whilethe newly emerged nauplii measure 400-500 microns in length. The life span <strong>of</strong> a brine shrimpis about 6 months. Approximately two weeks are required for the nauplius to attain adult stage.During this short period <strong>of</strong> development, nauplius <strong>of</strong> 0.4-0.5 mm in length and 0.002 mg in weightgrows into an adult ranging 8-10 mm in lengthand 1 mg in weight. During development, thoracicappendages are differentiated in relation to their function as locomotory, respiratory and filterfeeding appendages (Plate 1 D). Artemia are non- selective filter feeders and feed on particulatematters <strong>of</strong> less than 50 microns in size. After attaining adult stage, the brine shrimp producesnauplii or cysts according to the prevailing conditions, at the rate <strong>of</strong> upto 300 per batch andcontinues to produce at an interval <strong>of</strong> 5-7 days throughout its Ilfe period depending upon theprevalence <strong>of</strong> required salinty conditions JFlow chart 1).
3. DISTRIBUTION OF BRINE SHRIMP IN INDIAIn India . only parthenogenetic strain <strong>of</strong> Memia has been repwted to be presznt as naturalpopulation in the Sambhar and Didwana lakes in Rajasthan and saltpans in the Gulf <strong>of</strong> Kutch,Gujarat, Vadala in Bombay, Veppalodai near Tuticorin, Karsewar Island <strong>of</strong>f Tuticorin, Kelambakkamnear Madras and Vedaranyam near Muthupet lagoon. Among these places, Sambhar andDidwana lakes are inland and the others are in the coastal belt. It is interesting to note that theseplaces <strong>of</strong> occurrence <strong>of</strong> Artemia are located in the migratory route <strong>of</strong> flamingoes, which suggestthat the birds are the possible agentslor the distribution <strong>of</strong> the same parthenogenetic strain indifferent oarts <strong>of</strong> India4. BRINE SHRIMP CULTURE IN SOLAR SALTPAN IN INDIA4.1. Present statusThe National institute <strong>of</strong> Oceanography. Goa undertook a project on Anemia culture during1979 -1981. at Bharath Salt Chemicds Industries Ltd , Mundra, Gujarat. A pond <strong>of</strong> half a hectare areawas inoculated with nauplii, hatchdout from 5W g cysts <strong>of</strong> San Francisco Bay strain in January1979. By April 1979, nearly 15 kg cysts were harvested Similar results were obtained in thesubsequent year also Tata Chemicals Ltd . Mithapur. Gujarat achieved a production <strong>of</strong> 0.924 kgihadry cysts in 212 days by stockirig @ 1 nauplius (Gujarat strain) per litre The pond was fertilizedbefore and during the culture with ra& cow dung By stocking San Francisco Bay strain <strong>of</strong> Artemia,a production <strong>of</strong> 22 kg dry cysts in 6 months was obtained in Jamnagar, GujaratThe <strong>Central</strong> <strong>Institute</strong> <strong>of</strong> <strong>Brackishwater</strong> <strong>Aquaculture</strong> conducts experiments on Artemla cystproduction at Kelambakkam saltpan. In one experiment, local strain <strong>of</strong> parthenogenetic Arterniawas stocked at the rate <strong>of</strong> 16 naupliillitre The pond was fertilized with inorganic fertilizers only.The salinity was maintained at 70 - 200 ppt. This experiment yielded a production <strong>of</strong> 21.32 kgdry cystslhafl99 culture days4.2. Culture riteSolar saltpans have primary reservoirs, evaporation ponds and crystallizer beds. Water isallowed to flow from the primary reservoirs to a series <strong>of</strong> evaporation ponds alter retention for ashort duratlon in each pond. During this operation, salinity increases due to evaporation and atthe end, water becomes brine which wlll be allowed to settle as sodium chloride in crystaliizersTo enhance the rate <strong>of</strong> evaporation, the evaporation ponds are generally kept shallow. Thesalinity ranges from 30 ppt to 240 ppt in these evaporation ponds. Gemia citurk can be carrlmout in the evaporation ponds having 70 - 200 ppt salinity with slight modifications These pondsshould retaln atieast 70 - 100 cm water which helps to keep the temperature well below tne lethallevel <strong>of</strong> 40°C This can be achieved either by pond excavat~on or by increasina the dvke heiahtThe water source should be free from pollution. Water level should be maintained in the pondat all times.
4.3. Pond prepantionThe pond has to be dried and exposed to sun for a period <strong>of</strong> 7-10 days. Soil pH <strong>of</strong> 7.0 -7.4 is suitable for the culture <strong>of</strong> Artemia. The sol has to be treated with lime at the rate <strong>of</strong> 1000kglha, n its p~ is below 7.0.4.4. Watw intakeWater with 70 - 75 ppt salinity has to be taken Initially as predators and competitors haveless chance <strong>of</strong> survival in this salinity. However, the intake water has to be screened to preventthe entry <strong>of</strong> predators. Water which is mildly alkaline in reaction with a pH <strong>of</strong> 8 is suitable. Toimprove the quality <strong>of</strong> water and to raise the pH, lime can be applied.4.5. FertilizationThe pond may be fertilized either with inorganic or organic manure or both in order topromote the natural food production. Chicken droppings are found to be a suitable organicmanure while urea, superphosphate and diammonium phosphate are the inorganic fertilizersused during Anemia culture. Apart from an initial basal dose, regular fertilization is carried outat fortnightly intervals depending on the natural food production in the pond4.6. Stocking4.6.1. Strain relectionSan Francisco Bay strain <strong>of</strong> Arfemia gives rise to 428 - 431 microns sized nauplius, whichis a suitable sized prey to early larval stages <strong>of</strong> crustaceans and fishes. Because <strong>of</strong> its small sizednauplii, San Francisco Bay strain has a ready market. This is in contrast to the locally availableparthenogenetic strain which has 442 - 520 microns sized nauplius. These large sized nauplllare preferrable to feed the later stages such as prawn postlarvae and fish fry. Because <strong>of</strong> thesmall sized nauplii, San Francisco Bay strain <strong>of</strong> Arfemia sp. is preferred as suitable candidatestrain for culture. The culture pond should be devoid <strong>of</strong> any natural population. If the saltpan hasa natural population <strong>of</strong> the local strain, stocking is preferred with the existing local strain whichhas the prospects <strong>of</strong> better survival. Further, introduction <strong>of</strong> exotic strain, along with the existingone, will result in the imbalance and subsequent dominance <strong>of</strong> any one <strong>of</strong> them. This conditionsometimes may lead to the disappearence <strong>of</strong> the local strain.4.6.2. Rate and time <strong>of</strong> stockingStocking has to be done at naupiiar stage and the stocking density should be 40 naupliiper litre <strong>of</strong> water in the culture pond. Quantum <strong>of</strong> nauplii, required for stocking, has to be workedout from the water quantity available in the pond. Based on the hatching efficiency <strong>of</strong> the cystand the number <strong>of</strong> nauplii required, the amount <strong>of</strong> cysts to be kept for hatching is calculated.The cysts will hatch out within 20-36 hours (for San Francisco Bay strain) or 36 .48 hours (forpanhenogenetic strains) and the hatched nauplii have to be stocked in the culture pond duringlate evening or eady hours <strong>of</strong> the day.
Salinity d water should be maintained around 100-120 ppt during the first two months <strong>of</strong>the culture period in order to maintain ovwiviparity and thereby population multiplication. Duringthe thlrd c4ture-month, oviparity is induced: pr&iing salinity stress is one <strong>of</strong> the methods toInduce wiparlty. This Is achieved by raising the salinity <strong>of</strong> the pond water to 150 ppt and aboveeither by evaporation or by pumping in high saline water. During the last two months <strong>of</strong> theculture, sallnlty has to be maintained between 150 ppt and 200 ppt for the continuation <strong>of</strong> cystproduction.4.8. Growth monitoringGrowth o! the population has to be monitored by collecting data on the populationcomposition. For this purpose, the population samples have to be collected and analysedperiodically after grouping them as nauplii, juveniles and adults. Overall production status <strong>of</strong>the stock can be understood by observing the presence <strong>of</strong> naupliilcysts and shell gland in thebroodsac. For example, presence <strong>of</strong> only adults reflects the status <strong>of</strong> no recruitment. It ispossible to find out whetherthe population is in growth phaseor in stationaryphase by observingthe reproductive status <strong>of</strong> the adults, which can be found out by observing the presence <strong>of</strong>nauplillcysts and shell gland in the broodsac Determination <strong>of</strong> popuiation density throughsampling procedure will not help inview <strong>of</strong> strong heterogenic distribution <strong>of</strong> Anemia. Minimummaximumwatertemperature, rainfall, salinity levelsand water turbidity haveto bedaily recorded.These data are necessary to monitor the population structure by adjusting the intensity <strong>of</strong>fertilization, water management and harvest.4.9. Harvesting techniques4.9.1. Blomass harvestBiomass can be harvested when the population density reaches above 200 adults I litre. Adip net or dragnet having a cod end with 200 micron mesh size, can be used to harvest thebiomass. The net should be emptied at an interval <strong>of</strong> every 15 minutes to avoid the death <strong>of</strong> theaccumulated Anemia in the cod end. To harvest the biomass, 800 micron sized hand sieves arealso used. Biomass harvesting can be continued until the density <strong>of</strong> the population reduces to100 aduitsjitre.4.9.2. Cyst harvestThe population can be induced towards cyst production by slowly Increasing the salinityto 150 ppt and above and maintaining il below 200 ppt by careful water management. Between200 ppt and 250 ppt, though Artemia survives, It will not have energy for cyst production andbeyond 250 ppt, it will become weak and die. The liberated cysts will float. They will be driienby wind towards the shore <strong>of</strong> the pond, where they accumulate (Plate 2 A). Cysts, thusaccumulated, must be harvested every day. otherwise, they will get dried UD on the shore andcarried away by wind. Further, they have to face repeated hydf@& and dehydration which willlead to non-viability. To avoid contamination, the cysts have to be hawesled when thev are inthe water surface. To ensure this, cyst-barriers should be installed in the pond, dose to shoreline.4
4.9.3. Stonge <strong>of</strong> harvested productsThe harvested biomass is cleaned by washing with freshwater Wch will eliminate theadhering salt. Then, the live biomass has to be spread out in thin layers in plastic bagslice trays.Subsequently, it has to be frozen at - 25OC in a quick freezer.4.9.3.2. CystThe harvested cysts can be stored temporarily in saturated brine. For long periods <strong>of</strong>storage, they are to be cleaned, processed and dehydrated. Cdlected cysts may be contaminatedwith dead Memia, algae, debris, etc., This debris should be removed to ensure thepurity <strong>of</strong> the cysts. While cleaning, different mesh-sized sieves are used to remove the correspondingsued impurities and cleaned cysts wjll be cdlected with 100 micron sieve. Thecontaminants, which are equal in size with the cysts, are removed by the biphase floatationtechnique in whichcysts are initially suspended in brlne. In brine solution, cystsand lighter debrlsfloat while heavy dirt particles sink. The floating cysts are cdlected with 100 micron net. Theyare resuspended for 10 - 15 minutes in freshwater/seawater to remove the lighter debris andnon-viable cysts which float. Senled, viable cysts are collected and dried. The moisture content<strong>of</strong> the cyst should be less than 10%. Dried cysts are packed in tlns or in polyethylene bags eitherunder vacuum or with nitrogen (Plate 2 B).5. RATE OF PRODUCTIONA production <strong>of</strong> about 30 kg <strong>of</strong> dry Anemia cysts and 200 kg live biomass/ha/5 months canbe achieved on an average and the cost and returns analysis Is given in Annexure 1.6. ACKNOWLEDGEMENTSWe are greatful to Dr. K AJagarswami, Director, <strong>Central</strong> <strong>Institute</strong> d <strong>Brackishwater</strong> <strong>Aquaculture</strong>,Madras for his guidance a@ encouragement during the course <strong>of</strong> the development <strong>of</strong> thetechnology and to Shri K N. Krishnamurthy, Principal Sclentist, for crltlcal reading <strong>of</strong> themanuscript. We thank Shri M. Kathirvel, Scientlst for photographs.
projection d economics d culturing Memia in 1 ha pond IS gben below:ANNEXURE : 1ACAPITAL COSTCoetdl haiandRaising the bund to I .5 mEquipment : 5 HP Diesel pump with accessoriesTdal6. OPERATIONAL COST FOR ONE CROP OF FIVE hWNMSCyst for inoculumFertilizersPower & DleselMaintenanceManpowerPacklng chargesAnnual interest on capital @ 18%TotalOperational cost for 2 crops in an yearC. SALE PROCEEDSSale <strong>of</strong> 60 kg <strong>of</strong> cysts in 2 crops @ Rs.I250/kgSale <strong>of</strong>400 kg Biomass in 2 crops @RS. a/kgTotal sale proceedsD. PROFIT from a 1 ha pond in an year ( c - )
C.I.B.A. BULLETIN No. 4 PLATE 1A. ~dult male; B. Adult female; C. Clasper <strong>of</strong> male; D. Juveniles
I.B.A. BULL ETlN No. 4 PLATE 2A Accumulation <strong>of</strong> floating cysts1B Packed cysts
JuvenileJILalenaupliusHalchi ngL 1Fig 1. Life cycle <strong>of</strong> the brine shrimp Artemia sp. A. Antennule; B. Antenna;C. Compound eye; D Thoracopods; E. Uterus; F. Eggs; G. AWomen;H. Caudalfurca; I. Mandible, J. Medianeye; K Developlngthoracopods.
FLOW CHART 1\ Cyst (200-300 microns in size)Water temperature-24-48 hrs for hatching in less26 - 30°C; than 70 ppt salinity; InitialpH above 8.0. hydration ;Aeration 10-20 litre airlminute;Light minimum 1000 lux.E 1 stage~mbrelia stageIFreshly hatched nauplii7 1One day dd naupliiINon-selective;filter feeder;feeding with paniculatefood <strong>of</strong> less than 50 micronsin size(bacteria,planktonicalgae,etc.,).About 2 weeks; Predator freemedium;Under optimal cultureconditions.Adult (about 10 rnm in total length)I upto 1 ~ ppt ) salinity 150-250 ppt salinity 250-350 p t salinity(favourable conditions) (unfavourabie conditions) P11Ovoviviparltyrelease <strong>of</strong> 60-300I naupliUadJt/ about 5 days1Oviparity(60-300 cysts1 adult1about 5 days)Dying phase