Endowment lecture I Artemia Culture - University of Madras

STATUS OF ARTEMIA DIVERSITYIN INDIAN SUBCONTINENT :Biology, culture and conservation strategies.Prof. N. MUNUSWAMY, D. Sc.,Unit of Aquaculture & CryobiologyDepartment of ZoologyUniversity of MadrasGuindy CampusChennai – 600 025.Endowment lecture, Dept. of Zoology, Annamalai University 18 th March 2011

I. AQUACULTURE GROWTHWorldFish Model (CGIAR) 2020 Projections:• Due to domestic and international demand, aquaticproducts will be in severe short supply• Price of High value fish - to raise by 15 %• Price of low value fish - to raise by 4-6 %

Utilization80706 8.870 .472 .473.5 74.375.560Million tonnes504030202 4.513.42 3.213.520 .213 .720.213.72 0.513.720 .513 .7105.25.25.35.45.45.502004 2005 2006 2007 2008 2009YearHuman consumptionPopulation (billions)Non-food usesPer capita food fish supply (kg)

TOP TEN AQUACULTURE PRODUCERS (2000).Countries Native (mt) introduced (mt) Introduced /total (%)China 12,640,511 629,182 4.74India 1,717,453 253,989 12.82Bangladesh 278,000 226,000 44.84Indonesia 63,903 243,329 71.81USA 307,541 8,051 2.53Thailand 69,082 119,270 45.94Taiwan 60,082 58,922 49.51Philippines 14,523 85,622 76.61Brazil 12,379 82,465 86.95Myanmar 93,948 0 0.005

FISHERIES POTENTIAL RESOURCES IN INDIACoastlineExclusive Economic ZoneInshore area(

IndianFisheriesCapture fisheriesCulture fisheriesCultivable speciesFin fishesShell fishesMolluscansetc.,AquacultureMarineBrackish waterFreshwater

AquacultureSeedProduction(Hatcheries)Grow outCulture(Ponds)Processing&Marketing(Exports) Broodstock Inducedmaturation Spawning &Hatching Algal culture Larval rearing Seed productionInputsPathogen freeBroodersWaterAerationFeedsArtificialfeed(microEncapsulated/pellet)Livefeed(livingmicroOrganisms)

Types of Live food organismsPhytoplanktonChlorella sp.Chaetoceros sp.Skeletonema sp.Isochrysis sp.Tetraselmis sp.etc.,BranchiopodcrustaceanZooplanktonRotifersCopepodsCladoceronsMoina,etc.,ARTEMIA

ROLE OF ARTEMIA IN AQUACULTURE Size Nutritional valueProtein & lipid contentAmino acid levelFatty acid profile Nursery food for Shrimp,Prawn, Crab & Marine fishes Ornamental fish feed Ingredient for aquaculture feed Shrimp broodstock maturation

BRINE SHRIMPDIVERSITY OF ARTEMIA POPULATIONS IN INDIANSUBCONTINENT&SCOPE OF ARTEMIA CULTURE IN INDIA

Worldwide Distribution of ArtemiaWorldwide distribution and is discontinuousHabitat at 300 natural salt lakes in all the fivecontinentsBisexual and parthenogenetic strains are recordedAmong the bisexual strain, mainly six sibling specieshave been described so far

GEOGRAPHICAL DISTRIBUTION OF ARTEMIAPOPULATIONS

POSSIBLE VECTORS FOR ARTEMIA DISTRIBUTIONWater BirdsImproper disposal of the cystManMan-made inoculation

TAXONOMIC CONFUSION REGARDING ARTEMIASPECIATION First description of the brine shrimp by Schloesser, 1755 in UK Named as Cancer salinus by Linnaeus in 1758 Renamed as Artemia by Leach in 1819 Occurrence of morphotypes, mode of reproduction variousspecies were identified with various degrees of ploidy Now considered as complex of sibling species Initially, speciation was done by morphological characters andlater on by cytogenesis, allozyme studies and chromocentrenumbers, gene cloning, DNA sequencing and mtDNA analysis Based on these six bisexual strains and one parthenogeneticstrains were described

Bisexual strainsArtemia salina - Eurasia (now extinct)A. urmiana - IranA. franciscana - USAA. monica - CaliforniaA. tibetiana - TibetA. persimilis - ArgentinaArtemia spp. - KazakhstanParthenogenetic strainA. parthenogenetica - Asia

DISTRIBUTION OF ARTEMIA IN INDIAInitially, the occurrence of Artemia inIndia has been recorded from fourmajor habitats Vadala - Maharashtra(Kulkarni, 1953) Bhayander - Maharashtra(Ansari, 1982) Sambhar Lake - Rajasthan(Baid, 1959) Tuticorin - Tamil Nadu(Royan, 1970; Achari, 1971) Gulf of Kutch - Gujarat(Royan, 1979)After 1980, some of the saltpan areas of Tuticorin, Vedaranniyamand Kelambakkam, have been reported as an additional sources ofArtemia in Tamil Nadu -Petermariyan et al., 1983; Munuswamy,1996

DISTRIBUTION OF ARTEMIA POPULATIONS INSOUTH INDIACHENNAIKelambakkamBAY OFBENGALVEDARANYAMTUTICORINVeppalodaiThamaraikulamNAGERCOIL

MORPHOLOGICAL CHARACTERIZATIONMorphometric traits & structural variations• Total length – tl• Abdominal length – al• Distance between thecompound eyes – de• Eye diameter – ed• Head width - hw• Number of setae per furca – sf• Length of furca – lf• Width of 3 rd segment – ws• Width of ovisac – ow• Ration of abdominal length /total length (al / tl)-Hontoria, 1992-Leger et al., 1986Semi-diagrammatic representation of the adult female of Artemia

MORPHOMETRIC MEASURES OF SOUTH INDIANARTEMIA POPULATIONSMorphologicalTraits (mm)F -ValueP -ValueSFBStrainsKBMVRMTotal length225.820.000 **9.78 a(0. 58)13.26 b(1.27)16.65 c(0.63)Abdominal lengthWidth of headLength of 1 stantennaEye diameterDistancebetween eyes140.7297.88234.3228.8637.360.000 **0.000 **0.000**0.000**0.000**5.06 a(0.32)1.04 c(0.03)1.21 c(0.02)0.35 c(0.04)1.51 a(0.05)4.84 a(0.56)1.22 b(0.11)0.81 a(0.06)0.31 b(0.02)1.87 c(0.17)7.20 b(0.35)0.83 a(0.06)1.14 b(0.07)0.27 a(0.01)1.67 b(0.09)Note :1. ** denotes significant at 1 %level2. * denotes significant at 5% levelDifferent alphabet between straindenotes significant at 5 % levelWidth of ovisac313.160.000**1.38 a(0.12)2.62 b(0.19)1.48 a(0.14)Width of 3 rdsegment307.300.000**0.48 a(0.02)0.77 c(0.04)0.54 b(0.04)Fur cal length1.430.2490.27 a(0.02)0.26 a(0.04)0.28 a(0.02)No. of setae perfurcaRatio ofAbdominal/ Totallength67.7650.450.000**0.000**13.20c(0.68)51.79c(1.74)11.20 b(0.86)36.91 a(6.21)10.13 a(0.64)43.28 b(2.85)

FURCAL MORPHOLOGY OF SOUTH INDIANARTEMIA POPULATIONS

SCATTERPLOT OF INDIVIDUAL DISCRIMINANTFUNCTION SCORES IN ARTEMIA SAMPLESFROM SOUTH INDIA

EGG MORPHOLOGY OF SOUTH INDIAN STRAINS OF ARTEMIA

ParametersChennai(Kelambakkam)Tuticorin(Vepallodai)VedaraneyamThamaraikulamNagercoilPuthalamSize of the cystbefore hydration224.0 ± 12.53 201.5 ±18.0300.49 ± 0.14 266 ± 25 225 ± 9Size of the cystafterhydration234.5 ± 13.11213.5 ±16.5326.62± 13.11245.95 ±8.09257.83 ±8.16Salinity (%)(suitablerange)30 ± 530 ± 535 ± 530 ± 230 ± 2Hatching (%)9196816248HatchingEfficiency (n/g)1,12,0001,87,5001,67,5001,34,54097,920

CYST MEMBRANE PROTEIN POLYMORPHISMIN ARTEMIA POPULATIONS

CYST MEMBRANE PROTEIN POLYMORPHISM(Abatzopoulos et al., 1997) Dry cysts (1-3mg)DecapsulationStored at saturated NaCl at 4CPreparation of Embryonic MembraneIncubation of Decapsulated cysts in sterilized hatching medium* for 4 hrs.Harvest (Using 100µm filter)Homogenization(20 times of volume of ice cold double distilled water + 2.5mM PMSF)Centrifugation (300 rpm / 10min. – 4ºC)1. Discard supernatant2. Wash the pellet(Using lysis buffer – double distilled water + PMSF

Centrifugation (300 rpm / 10min. – 4ºC)1 and 2Centrifugation (400 rpm / 10min. – 4ºC)1 and 2Centrifugation (1000 rpm / 10min. – 4ºC)1 and 2Examining under microscope for purity(Should be free from yolk granules)* Hatching medium – Sterilized sea water (30 ppt)Cyst membrane protein composition as a discriminant character in the genus Artemia (International study onArtemia IV) J.Mar.Biol.Asso.U.K. (1997) 77: 65-268

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RAPD - PCRCyst protein pattern (15% SDS PAGE).L1 : SFB (Bisexual)L2 : KBM (Bisexual)L3 : VRM (Parthenogenetic)MW : Medium range proteinmolecular weight marker

…aScanning electron micrographs of thehydrated cyst (5-10 hrs) of different strainsof Artemia,a. Artemia franciscana strainbb. Kelambakkam strain (Bisexual)cc. Vedaranniyam strain (A. parthenogenetica).• Smooth and granular surface• NO Significant variationNote: Scale bar 30 µm

TRANSMISSION ELECTRON MICROGRAPH OF KBM CYST

TRANSMISSION ELECTRON MICROGRAPHS OF SFB CYST

TRANSMISSION ELECTRON MICROGRAPHS OFVDM CYST

AL - Alveolar layer CL - Cortical layer FL - Fibrous layer ICM - Inner cuticularmembrane LB - Lipoid body N - Nucleus OCM - Outer cuticular membranePPR - Polygonal plate rim YP - Yolk platelet

POST EMBRYONIC DEVELOPMENTAL STAGES

POSTEMBRYONIC DEVELOPMENT OF PUTHALAMSTRAIN22 46 68 91 110 136 165 191 214 235 251 263 291 324 349 373 397 431 451 Hrs ofdevelop.1/2 3/4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Instars0 0+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 StagesT1 – T11G1 & G2A1 – A6- Thoracic segments- Genital segments- Abdominal segments- Segment formation- Thoracic limb bud formation- Maturation of thoracic limb bud- Complete maturation of genital segments- Complete maturation of abdominal segmentsT1T2T3T4T5T6T7T8T9T10T11G1G2A1A2A3A4A5A6

POSTEMBRYONIC DEVELOPMENT OF THAMARAIKULAM STRAIN23 48 71 95 119 143 159 182 206 232 269 298 320 344 378 396 420 449 472 Hrs ofdevelop.1/23/4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Instars0 0+ 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 StagesT1T2T3T4T5T6T7T8T9T10T11G1G2A1A2A3A4A5A6

COMPARISON OF POSTEMBRYONICDEVELOPMENT OF ARTEMIA STRAINSD15D14D13D12D11S13S12S11S10S9S12S11S10S9S8S13S12S11S10S10D16S14S13S14S8/S9S8S9D10D17S14S15S7S7S8D9D18S15S16S6S6S7D8D19S16S17S5S5S6D7D20S17S4S4S5D6S0 S0+ S1S0 S0+ S1S0 S0+ S1S2S2S2S3S3S3/S4D1D2D3D4D5- Puthalam - Thamaraikulam - Tuticorin - Final day/stage of developmentD1-D20 - Days of development S0-S17 - Stages of development

(A) Artemia parthenogenetica (B) Streptocephalus dichotomus (C) S. simplex (D) S. longimanus(E) S. echinus (F) S. spinifer (G) Branchinella kugenumaensis (H) B. maduraiensis (I) B. nalurensis.

MOLECULAR CHARACTERIZATION OF SOUTHINDIAN STRAINS OF ARTEMIA A preliminary study of mtDNA sequencing analysis of southIndian Artemia strainsGenetic variation using Random Amphilified Polymorphic DNA(RAPD) analysis and Restriction Fragment LengthPolymorphisms (RFLP) analysismt DNA sequencing and Phylogenetic Analysis

10 mg of dried cyst in 2 ml of eppendorf tubeSuspend 50 µl of sterile water and incubate 2 hr in room temperatureCentrifuge at 15000 rpm for 15 min.Add 600 µl of 2X CTB buffer and incubate this mixture at 60ºC for 60min.Centrifuge at 15000 rpm for 15 minCollect upper most layer in a fresh eppendorf tube to this addchloroform:isoamylalcohol(24:1)Centrifuge at 10000 rpm for 15 min.Collect top layer supernatant to this add 2volume ice cold ethanol (100%)DNA allowed to precipitate at -20 ºC for 2 hrCentrifuge at 15000 rpm for 15 min.Pellet was collected and washed with 70% ethanolAir dry the above pellet in room temperature and DNA was redissolved in 50 µl of TE buffer

PCR AMPLIFICATION REACTION PERFORMED WITHTOTAL VOLUME OF 50µL AND STANDARDIZED ASFOLLOWS FOR TAXONOMIC ANALYSIS OF SEQUENCING1. Template DNA – 2µl containing 20-40ng2. Primer (F)– 4µl (15ng)3. Primer (R)– 4µl (15ng)4. dNTPs – 3.5 µl l (2mM)5. 10x PCR buffer – 3.5 µl6. Taq DNA polymerase – 0.5 µl l (0.5U)7. Sterile water - 7.5 µlNote: A control sample without DNA included in eachamplification reaction.

PCR PROGRAMMES. No. Thermal Cycle1InitialDenaturation94˚C2 min.2 Denaturation 95˚C 1 min. 15 sec.3 Annealing 52˚C 0.45 sec.4 Extension 72˚C 2 min.5 Final Extension 72˚C 4 min.

PCR PERFORMED WITH TOTAL VOLUME OF 20µLAND STANDARDIZED AS FOLLOWS FORTAXONOMIC ANALYSIS OF RAPDS1. Template DNA – 2µl containing 20-40ng2. Primer – 3µl (15ng)3. dNTPs – 3.5 µl (2mM)4. 10x PCR buffer – 3.5 µl5. Taq DNA polymerase – 0.5 µl (0.5U)6. Sterile water - 7.5 µlNote: A control sample without DNA included in eachamplification reaction.

PCR PATTERN SHOWS THE AMPLIFICATION OF 1500bpPCR PRODUCT

The RAPD profiles produced by the method of Williamset al. (1990) and McClelland (1990)PRIMER SEQUENCESOPK-01CATTCGAGCOPK-02GTCTCCGCAOPK-03CCAGCTTAGOPK-04CCGCCCAAAOPK-05TCTGTCGAGOPK-06CACCTTTCCOPK-07AGCGAGCAAOPK-08GAACACTGGOPK-09CCCTACCGAOPK-10GTGCAACGTOPK-11AATGCCCCAOPK-12TGGCCCTCAOPK-13GGTTGTACCOPK-14CCCGCTACAOPK-15CTCCTGCCAOPK-16GAGCGTCGAOPK-17CCCAGCTGTOPK-18CCTAGTCGAOPK-19CACAGGCGGOPK-20GTGTCGCGA

THE ARROWS REPRESENT MULTIPLE COPIES OF A PRIMER.THE DIRECTION OF THE ARROW ALSO INDICATES THEDIRECTION IN WHICH DNA SYNTHESIS WILL OCCUR

RAPD PROGRAMME PERFORMED ANDSTANDARDIZED AS FOLLOWS FOR TAXONOMICANALYSIS OF RAPDSS. No. Thermal Cycle1InitialDenaturation94˚C2 Denaturation 95˚C3 Annealing 52˚C4 Extension 72˚C5 Final Extension 72˚C5 min.1 min.1 min.2 min.7 min.

RAPD - PATTERN SHOWING THE GENETICVARIATION OF DIFFERENT POPULATIONSPrimer –OPK - 07Lane 1. KKM strainLane 2. VDM strainLane 3. TCN strainLane 4. 1 kb LadderPrimer- OPK - 08Lane 5. KKM strainLane 6. VDM strainLane 7. TCN strainLane 8. 1 kb Ladder

MULTIPLE SEQUENCE ALLIGNMENT ARTEMIA STRAINS

PHYLOGRAM ANALYSIS

TREE VIEWAVERAGE DISTANCE TREENEIGHBOUR JOINING TREE

KBM STRAIN BLAST HITS

TCN STRAIN BLAST HITS

TKM STRAIN BLAST HITS

VDM STRAIN BLAST HITS

PHYLOGENETIC TREE VIEW

PHYLOGENETIC TREE VIEW

Secondary structure predicted for the 16S rDNA sequence of KBMstrain

MOUNTAIN GRAPH

Secondary structure predicted for the 16S rDNA sequence of TCNstrain

MOUNTAIN GRAPH

Secondary structure predicted for the 16S rDNA sequence of TKMstrain

MOUNTAIN GRAPH

Secondary structure predicted for the 16S rDNA sequence of VDMstrain

MOUNTAIN GRAPH

PHYLOGENETIC TREE VIEW

CONCLUSION ON PRESENT STATUS OF SOUTHINDIAN ARTEMIA STRAINS1980’sA. parthenogenetica &Bisexual populations1990’s2000’sA. parthenogeneticaA. franciscana (SFB/GSLpopulation)Mass levelproduction, improperdisposal2011’sA. franciscanaMorphological,biological andmolecular studiesmt DNA sequencing analysis clearly indicatesA. parthenogenetica NOW EXTINCT

CONSERVATION STRATEGIES Establish cyst bank in all Artemia populations Study the hole DNA Sequencing and GenomeMapping and genetic relatedness to detectvariations in the populations, dominating in Indiansubcontinent Screen a potential and pure strains of Indian brineshrimp, Artemia populations for aquaculturepractices

ParametersChennai(Kelambakkam)RamarpathamVedaraneyamChinnaKodiyakaraiTuticorin(Vepallodai)ThamaraikulamNagercoilPuthalamSeasonsPost monsoonMonsoonMonsoonPost monsoonMonsoonMonsoonTemperature (°C)27.5 ± 4.5826.7 ± 3.2825.5 ± 2.6429.5 ± 5.5525 ± 227.00 ± 2Salinity (%o)96.00 ± 3.3867.5 ± 2.2655.5 ± 3.05101.00 ± 2.5011 ± 212.0 ± 0.10Dissolved Oxygen(mg/l)3.47 ± 0.624.50 ± 0.244.60 ± 0.163.30 ± 0.105.46 ± 0.235.90 ±0.30pH8.19 ± 0.038.50 ± 0.028.78 ± 0.049.00 ± 0.026.9 ± 0.027.20 ±0.58Nutrients:Inorganic Phosphate(µM)7.50 ± 0.026.53 ±0.056.35 ±0.356.66 ±0.766.70 ±0.027.40 ±0.56Inorganic Nitrate (µM)26.50 ±0.0221.18 ±0.2524.50 ±0.0228.6 ±2.0523.50 ±0.0331.50 ±0.30Inorganic Nitrite (µM)9.30±0.030.0311.68 ±0.0711.18 ±0.2514.47 ±1.4515.00 ±0.5616.35 ±0.35Reactive Silicate (µM)31.65 ±0.0630.75 ±0.0110.97 ±3.5026.66±1.0135.50 ±1.3539.50 ±0.80Total Ammonia (µg/l)1.20 ± 0.50.75 ±0.0010.15 ±0.0052.76 ±0.361.67 ±0.022.50 ±0.03

ParametersChennai(Kelambakkam)RamarpathamVedaraneyamChina KodiyakaraiTuticorin(Vepallodai)ThamaraikulamNagercoilPuthalamPopulation structureCystNaupliiSub adultAdultPPPPAPPPAPPPPPPPAAAAPAAACyst size (mean of50 cysts – µm)Size of hydratedcyst (1 hr)Mode ofreproductionNature of saltpans240.00 ± 15.00262.00 ± 13.10BisexualCrystallizerpond300.49 ± 0.14213.5 ± 16.50ParthenogeneticCrystallizerpond--ParthenogeneticCrystallizerpond201.5 ± 18.0213.5 ± 16.5BisexualCrystallizerpond266.00 ±25.0245.95 ±8.09ParthenogeneticCrystallizerpond225.00 ± 9.00257.83 ± 8.16ParthenogeneticCrystallizerpondA – AbsentP - Present

ARTEMIA CULTUREARTEMIA

WHY WE ARE CONCENTRATING ON ARTEMIA PRODUCTIONUSING INDIGENOUS STRAINArtemia demandin IndiaGlobal problemsArtemia sourcesin IndiaCulture potential In India-1.0 million ha cultureponds and tanksTo produce 1000 millionof shrimp post-larvae/yr.Requirement of ArtemiaCysts – 100 tons / yr.Importing cysts@ 56 Corers / yrAFI,2004A recent incidence inArtemia crisisEl Nino FactorSalinity - declineswater temp. - increasesAs a result- Poor quantity andquality of cysts &nauplii- cost increasesAvailability of saltpanareas of IndiaRecognized lands1.81 laks haUnder cultivation1.0 laks haReservoir tank10% (10000 ha)Culture production ofCysts 10 kg / ha / yr100 tons / yrSivagnanam,2005

ALGAL CULTURE MAINTENANCECulture speciesChlorella vulgaris , Cheatoceros calcitrons, Tetraselmis costatum &Isochrysis galbana

BIOENCAPSULATION

Processing: Artemia biomass are harvested andimmediately enriched at our holding facility usingan enrichment emulsion rich in essential fattyacids (HUFA’s). Following the enrichmentprocess, the Artemia biomass are rinsed,packaged, and blast-frozen to insure quality

ENRICHED ARTEMIAWhat is bio-enrichmentof Artemia nauplii?Artemia are non-selective, continuous filter feeders. Anything that is theright particle size (10-50 microns) can be taken into the gut of the liveArtemia. Thus, Artemia can essentially deliver food components ornutrients to your fish through several hours of bioencapsulation orbioenrichment.Are there commercially available bioenrichment formulas available onthe market?Yes, there are several bioenrichment formulas available providing HighlyUnsaturated Fatty Acids (HUFA) in the form of EPA and DHA. Thesebioenrichment formulas are primarily available through aquaculturesupply companies for enriching live rotifers and Artemia for marineshrimp and fish hatcheries.

What exactly are in these bio-enrichment formulas?These high HUFA emulsion formulas are essentially emulsified fish oils(cod or menhaden oil). These formulas also contain antioxidants aspreservatives and may be fortified with vitamins.How can I make my own fish oil emulsion?Purchase cod liver or menhaden oil and find a source of lecithin(powdered or liquid). A 3-5% inclusion of lecithin to the fish oil is all youneed. Add some water to the fish oil and lecithin and blend (kitchenblender) for around 30 seconds. Feed supplements, such as Naturose(astaxanthin), spirulina, or stabilized vitamin C, can be added to theemulsion as well. The fish oil emulsion can be stored in a refrigerator forseveral weeks.How many hours should I enrich the Artemia nauplii for?The Artemia nauplii must molt into the Instar II stage before filterfeeding can begin. This occurs around 30 hours after stocking the hatchcone. A minimum of 12 hours of enrichment time is required for theenrichment formula to absorb into the intestinal tract and tissues.

What density should I use during enrichment?Approximately one gram/quart of cysts or 50% of therecommended stocking density is used when enriching babybrine shrimp. This lower stocking density is used because ofthe reduced dissolved oxygen levels due to the fish oilemulsion. Strong aeration is recommended.Can I enrich the Artemia nauplii in the same water the naupliiwere hatched in?Definitely not! The nauplii should be harvested, washed, andrestocked in a clean conical tank with new salt water.

CAN I ENRICH ADULT ARTEMIA BIOMASS?Yes. Live adult Artemia are much more efficient at filter feeding thanArtemia nauplii and only require 1-2 hours to enrich. The higher the watertemperature, the faster the adult Artemia will pack it's gut with theemulsion. A density of 2000 - 3000 adults per quart to one gram ofenrichment formula is recommended. Once the adults are fully enriched,feed them to your fish or shrimp immediately as they will continue feedingand emptying their gut of what they ingested. Otherwise, freeze theenriched biomass immediately for later feedings

CYSTS HATCHING TECHNIQUES Hydration Decapsulation Incubation Hatching Harvesting Nauplii-- Tamuru et al., 2000

HYDRATION•Take known quantity of Artemia dry cystsIn a conical shape container containingLow saline water (15-25ppt)•Provide vicarious aeration•Keep for 30 minutes•Observe under microscope•Cysts should be in a spherical shapeSize oftheCysts250µHydrated cystsDry cysts

DECAPSULATIONDisinfection for microbesRemoval of external layerImprove the hatching efficiencyTake 5-10gms hydrate for 30 minutesCollect (filter) with 100 micro hand netHydrated cystsChorion layerTransfer into 500ml measuring cylinderAdd 15ml of Decapsulating solution with85 ml of sea waterDecapsulationsolutionProvide vicarious aerationKeep for 5-15minutesWash with running fresh water & incubateSea water for 24 hours.Decapsulated cysts

HATCHINGQuality assessment of Artemia cystsHatching Percentage (HP) :(nos. of nauplius/100 nos. of cysts)Hatching Efficiency (HE) :(nos. of nauplii/ gram of Cysts)Hatching Rate (HR) :(Hatching period with in time)Hatching tanks

Harvesting and feeding / culture /further assessmentAfter complete hatching,Stop the aerationCover the surface of the tankAllow them to settle on bottomCollect the nauplii with 150µhand netWash with fresh sea waterDisinfect with 5-10 ppm formalin

Essential nutrientsENRICHMENTEnriched RotifersCrab larvaPigmentsAfter6hrs.Enriched ArtemianaupliiFish larvaEnrichingnaupliiShrimp larvaAntibioticsStuffed gutProbiotics

Comparison of fatty acid profiles of enriched and Non-enriched ofMoina, Rotifer & ArtemiaMoinaRotiferArtemiaFattyacidsNon-enrichedEnrichedwithSelcoNon-enrichedEnrichedwithAlgamacNon-enrichedEnrichedwithMicrofeast14 : 016 : 016 : 1n-718 : 018 : 1n-918 : 2n-618 : 3n-318 : 4n-320 : 1n-920 : 4n-620 : 5n-322 : 1n-111122 : 6n-30.030.431.360.221.770.110.040.010.010.160.07----0.321.290.860.261.180.080.030.030.050.21--0.370.750.170.890.050.210.481.122.640.170.290.050.11----0.180.920.240.270.671.540.930.000.250.080.52--0.420.080.920.330.301.250.401.900.200.030.080.44----0.141.850.350.911.770.814.970.690.080.190.59--0.25Total 4.22 5.42 6.25 6.34 5.97 12.60(Values are expressed in mg/100 mg dry weight).Tamaru et al., 2000

USE OF ARTEMIA IN DIFFERENT FORMS FORAQUACULTURE PRACTICESArtemia Decapsulated cysts : Directly using for Crab and Fish larvaeJust hatched nauplii : Shrimp, Fish and all cultivable speciesII stage nauplii : Using for enrichment and feedingSub-adult : Feeding for post-larval stagesAdult : Nursery rearing: Used as ingredient for different typesformulated feed for shrimp, fish and poultryArtemia by-products are using in shrimp and fish hatcheriesDecapsulatedembryosArtemiaflakeArtemiapasteArtemiacake(pellet)FrozenArtemiaArtemiabiomass

Enrichment ProcessProcess of bioencapsulationDHA Molecule

BioassayFeeding regimeShrimp larvalstagesRearingperiod(days)Normalpractice(no. ofnauplii/ml)RecommendedScheduled(No. ofnauplii/mlUtilizationof importedcysts (%)Renovationwith localavailablecysts (%)Mysis - IMysis - IIMysis - III1110.25nos/ml0.25nos/ml0.5nos/ml0.25nos/ml0.25nos/ml0.5nos/ml*20 %* 20 %Post-larvaePL 1 – PL552 – 5nos./ml2 – 5 nos./ml1.5 – 2 nos./ml*50 %*20 %**30 %PL6 – PL 10PL11 – PL 15PL 16 – PL 205552 – 3nos./ml+Artificialfeed2 – 5nos./ml3 – 6nos./ml6 –10nos./ml*30 %**40 %* Imported cysts nauplii ** Indigenous cysts nauplii

Colouration of fishes fed with different diets(A) fed wit artificial diet (B) fed with live feed diets

PRAWN LARVAE FEEDING ON ENRICHED ARTEMIANAUPLII

LAB TO LAND PROGRAMMEUPSCALLING OF ARTEMIA BIOMASSAND CYST PRODUCTION IN MAN-MADE MADESALTERNS OFF KELAMBAKKAM

Develop and Standardize a suitable technique for massproduction of Artemia biomass and cyst in man-madesaltpans of Kelambakkam using indigenous strain – anoutdoor culture practice Annual production Quality assessment of biomass and cyst harvested Low cost benefit to Aqua farmers/ End users

Meeting with Scientists, Entrepreneurs, Deputy salt commissioner & Saltcommissionerwith entrepreneurs (Jun 19, 09)with Deputy salt commissioner (Jul 28, 09)with Salt Commissioner,Govt. of India (Aug 17, 09)

A Typical lay-out of Kelambakkam salt worksSource: S.K. Subbiah Pillay & sonsBRINE PIT

Satellite image of the saltpanEvaporation pondSalt harvestedCrystallizers

SALT PRODUCTIONUSA: 475 Lakh tonnesChina : 370India : 15512%3%OthersAP15%TNGJ70%

SITE SELECTION Ponds, nearby evaporation pond and pumpingstation were selected Bunds were strong enough to prevent leakage Facilities were made to pump low & high saline waterbased on requirement Bottom of the pond was Heavy clay + less sand Drainage facility were also setup to remove excesswater Perimeter were fixed with bamboo, based on winddirection, to facilitate cyst collection

MODIFICATIONS DONE IN PRE-EXISTING EXISTING POND Water was completely drained from selected pondand allowed to dry for a week Barrier nets were fixed on the inlet and outlet of thepond Flow through system setup to maintain desired waterdepth Evaporation ponds were connected to theexperimental pond

CONSTRUCTION OF EXPERIMENTALPONDFishingDryingBarrier netsOverflow systemCyst harvesting

SEA WATER FLOW SYSTEM IN KELAMBAKKAMSALTPAN Pumping stationStorage pond - IStorage pond -II

AERIAL VIEW OF ARTEMIA CULTURE PONDSPond 2Pond 11 & 2 Experimental Ponds

ARTEMIA MASS CULTURE PRACTICES ATKELAMBAKKAM SALT PONDSOverview of experimental culture systemExperimental site: SKMS Salt Works KelambakkamCulture system : Experimental & Fertilization ponds (F1 & F2)Pond Area : Pond 1= (69m x 108m) = 7452 m 2Pond 2= (61m x 108m) = 6588 m 2Experimental periodStocking density: 320 days: 60 nauplii/ l

OUTCOME OF EXPERIMENTAL TRIALSCulture done during summer - Experimental resultsExperimentalControlCulture period (days) 96 96Turbidity (cm) 15-205-32Cyst harvested (kg) 22.92 8.82Biomass count (no./L ) 798 483(adults only)

EXPERIMENTAL RESULTSSeasonsCultureFeed usedWaterCystBiomassHatchingPeriod(Days)Turbidity(cm)harvested(kg DW)harvested(kg) %Monsoon 96 Algae 15-2036.48 11.9973 72 - 10(Aug - Nov, 09)Summer 120(Mar - Jun, 10)Rice bran,Algae 17-25 22.92 7.0346 80 - 72Monsoon 86(Aug - Nov, 10)Rice bran,Algae 15-2010.54 nil 10TOTAL 302 69.94 19.0319

Photographs showing field research laboratory andequipments inSKMS saltworks at Kelambakkam

EQUIPMENTS

Artemia Culture in salt pondsCyst harvesting placeArtemia Biomass

CYST COLLECTION AND PROCESSING

Artemia cyst dryingPacked Artemia Biomass & CystHarvested Artemia BiomassProcessed & Packed cyst

OUTDOOR CULTURE OF ARTEMIA –BIOMASS COLLECTION AND PROCESSING

VALIDATION REPORT ON THE CYST ANDADULT BIOMASS Vacuum packed cysts given to various hatcheries M/s. Matsyafed Prawn Hatchery (Govt. of Kerala), Kannur M/s. Sri Devi Priyanka Hatchery, ChennaiFEED BACK REPORTSCyst qualityBiomass- As much as 72% hatching was encountered– Given as feed aquarium hobbyists in Chennai &Marakkanam- They appreciated as clean, good and qualityfeed to ornamental fishes and is readily acceptable

VALIDATION REPORT FROM PRAWN HATCHERY

Upscaling of Artemia production inKelambakkam saltpanDescriptionCONCLUSIONSParametersHarvestable productPond AreaWater DepthCulture period0.5 ha40-45 45 cm300 days~70kg (DW) of cystFeedSalinitySpirulina Sp., DunaliellaSp., Rice bran etc80-110 (sudden changeto >150ppt)15-20cmand> 1000kg (WW) ofBiomassWater turbidity

InvestmentCOST BENEFIT RATIO(per annum)Pond preparation & maintenance = 20,000.00Labour charges (2x250/day) = 1,80,000.00Technical assistant (20,000/m) = 2,40,000.00Electrical charges & other expenses = 1,50,000.00TOTAL EXPENSES = 5,90,000.00SalesCyst (1000/kg x 140) = 1,40,000.00Biomass (350/kg x 2000) = 7,00,000.00TOTAL INCOME = 8,40,000.00Financial gain (per hector per annum) = 2,50,000.00

My sincere thanks to.......................• The authority of Annamalai University• The authority of Annamalai University• Prof. Selvi Sabhanayagam &• Faculty members of Zoology Department,Annamalai Unievrsity

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Shanmugasundaram, G.K., Ramasubramanian, V. & Munuswamy, N. (1996). α-tocopherol in Artemia cysts : A report. AquacutlureInternational.,(Netherlands) 4:377-378. Sugumar, V. & Munuswamy, N. (2006). Ultrastructure of cyst cell andunderlying membranes of three strains of the brine shrimp Artemia(Branchiopoda : Anostraca) from the south India. Microscopy Research andTechnique. (USA) 69 (1212):957-959. Sugumar, V. & Munuswamy, N. (2006). Interpopulation studies on the cystmembrane proteins of South Indian strains of Artemia. Journal of MarineBiological Association. (India). 86: 1097-1100. Munuswamy, N. (2005). Fairy shrimps as live food in Aquaculture. Aquafeeds,.(USA).2 (1) (Feedware LLC)