1 - Central Institute of Brackishwater Aquaculture

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 of Agricultural Research)141, MARSHALL'S ROAD, EGMORE, MADRAS - 600 008

Restricted CirculationPublished by.Dr. K. AlagarswamiDirectorCentral Institute of Brackishwater AquacuitureMadras-600 008.Edited By:Shri K. N. KrishnamurthyPrincipal ScientistCentral Institute of Brackishwater AquacultureMadras-600 008.Cover Photos:Top Le/r : Culture PondTop Rigtar : BiomassBorrotn Lefr : CystsBortor~r Riglat : NauplilPrinted el:Prints & Proofs. Madras-8.

PREFACEThe brine shrimp Arterttio is an inevitable component of operation ofshrimp and fish hatcheries all over the w oM The nauplii hatched from the cystsare used as live food of larval and postlarval stages of all species of shrimpand fish. While commercial cystproduction as an adjunct activity of 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 Central Institute of Brackishwater Aquacutlure initiated programmefor developing a techndogyforArrer?iio cystproduction in 1990at a pn'vate saltpanat Kelambakkam near Madras. In the midst of heay odds caused by unseasonalrains and floods which created problems for maintenance of salinity at therequired levels, Wo successful crops have been taken, one with the local(Kelambakkam) strain of Ancr?tio and the other with an imported strain Eftortshave also been made towards Arfcr~iiu biomass production which is valuableas an ingredient of 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 stagesof the cycle could be maintained through careful water management.The National Workshop on "Brackishwater Prawn Farming for HigherProduction" organised by the Ministry of Agriculture (Department ofAgricultureand Cooperation) at Bangalore in October 1989 recommended that this Instituteshould take up an R & D programme to develop a suitable technologyfor Ancrniu culture and cyst production. Implementing this recommendation,the Institute has been able to achieve successful results in the experimentalwork carried out so far. It is to be placed on record that the Department ofFisheries of Government of Ralasthan has taken up this technology for theirprogramme on Anct?riu cyst production and success has been reported.The present Bulletin is a result of the Institute'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 of stocking4.7 Water management4.8 Growth monitoring4.9 Harvesting techniques4.9.1 Biomass harvest4.9.2 Cyst harvest4.9.3 Storage of 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 of Artemia form an idealfood for the larvae of crustaceans and fishes, while Artemia juveniles and adults form suitablediet for prawnlfish juveniles. Induction of 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 ofaquaculture. Availability of Atremia cysts and biomass from natural sources is inadequate tomeet the growing demand. Culture of 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 offer excellent scope for carryingout Arfemia culture Most of 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 of 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 of antennules, antennae and stalked eyes are present in the head region. Thoraxhas eleven pairs of 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 ofthe 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 ofthe female, during copulation. Fertilization is achieved in the uterus. In the case of 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 of a brine shrimpis about 6 months. Approximately two weeks are required for the nauplius to attain adult stage.During this short period of development, nauplius of 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 of less than 50 microns in size. After attaining adult stage, the brine shrimp producesnauplii or cysts according to the prevailing conditions, at the rate of upto 300 per batch andcontinues to produce at an interval of 5-7 days throughout its Ilfe period depending upon theprevalence of required salinty conditions JFlow chart 1).

3. DISTRIBUTION OF BRINE SHRIMP IN INDIAIn India . only parthenogenetic strain of Memia has been repwted to be presznt as naturalpopulation in the Sambhar and Didwana lakes in Rajasthan and saltpans in the Gulf of Kutch,Gujarat, Vadala in Bombay, Veppalodai near Tuticorin, Karsewar Island off 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 of occurrence of Artemia are located in the migratory route of flamingoes, which suggestthat the birds are the possible agentslor the distribution of the same parthenogenetic strain indifferent oarts of India4. BRINE SHRIMP CULTURE IN SOLAR SALTPAN IN INDIA4.1. Present statusThe National institute of Oceanography. Goa undertook a project on Anemia culture during1979 -1981. at Bharath Salt Chemicds Industries Ltd , Mundra, Gujarat. A pond of half a hectare areawas inoculated with nauplii, hatchdout from 5W g cysts of 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 of 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 of Artemia,a production of 22 kg dry cysts in 6 months was obtained in Jamnagar, GujaratThe Central Institute of Brackishwater Aquaculture conducts experiments on Artemla cystproduction at Kelambakkam saltpan. In one experiment, local strain of parthenogenetic Arterniawas stocked at the rate of 16 naupliillitre The pond was fertilized with inorganic fertilizers only.The salinity was maintained at 70 - 200 ppt. This experiment yielded a production of 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 of 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 of 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 of 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 of 7-10 days. Soil pH of 7.0 -7.4 is suitable for the culture of Artemia. The sol has to be treated with lime at the rate of 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 of survival in this salinity. However, the intake water has to be screened to preventthe entry of predators. Water which is mildly alkaline in reaction with a pH of 8 is suitable. Toimprove the quality of 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 of Arfemia gives rise to 428 - 431 microns sized nauplius, whichis a suitable sized prey to early larval stages of crustaceans and fishes. Because of 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 of thesmall sized nauplii, San Francisco Bay strain of Arfemia sp. is preferred as suitable candidatestrain for culture. The culture pond should be devoid of any natural population. If the saltpan hasa natural population of the local strain, stocking is preferred with the existing local strain whichhas the prospects of better survival. Further, introduction of exotic strain, along with the existingone, will result in the imbalance and subsequent dominance of any one of them. This conditionsometimes may lead to the disappearence of the local strain.4.6.2. Rate and time of stockingStocking has to be done at naupiiar stage and the stocking density should be 40 naupliiper litre of water in the culture pond. Quantum of nauplii, required for stocking, has to be workedout from the water quantity available in the pond. Based on the hatching efficiency of the cystand the number of nauplii required, the amount of 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 of the day.

Salinity d water should be maintained around 100-120 ppt during the first two months ofthe culture period in order to maintain ovwiviparity and thereby population multiplication. Duringthe thlrd c4ture-month, oviparity is induced: pr&iing salinity stress is one of the methods toInduce wiparlty. This Is achieved by raising the salinity of the pond water to 150 ppt and aboveeither by evaporation or by pumping in high saline water. During the last two months of theculture, sallnlty has to be maintained between 150 ppt and 200 ppt for the continuation of 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 ofthe stock can be understood by observing the presence of naupliilcysts and shell gland in thebroodsac. For example, presence of only adults reflects the status of no recruitment. It ispossible to find out whetherthe population is in growth phaseor in stationaryphase by observingthe reproductive status of the adults, which can be found out by observing the presence ofnauplillcysts and shell gland in the broodsac Determination of popuiation density throughsampling procedure will not help inview of strong heterogenic distribution of Anemia. Minimummaximumwatertemperature, rainfall, salinity levelsand water turbidity haveto bedaily recorded.These data are necessary to monitor the population structure by adjusting the intensity offertilization, 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 of every 15 minutes to avoid the death of 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 of 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 of 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 of 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 ofstorage, 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 of 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 contentof 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 of about 30 kg of 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, Central Institute d Brackishwater Aquaculture,Madras for his guidance a@ encouragement during the course of the development of thetechnology and to Shri K N. Krishnamurthy, Principal Sclentist, for crltlcal reading of 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 of 60 kg of cysts in 2 crops @ Rs.I250/kgSale of400 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 of male; D. Juveniles

I.B.A. BULL ETlN No. 4 PLATE 2A Accumulation of floating cysts1B Packed cysts

JuvenileJILalenaupliusHalchi ngL 1Fig 1. Life cycle of 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 of 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 of 60-300I naupliUadJt/ about 5 days1Oviparity(60-300 cysts1 adult1about 5 days)Dying phase